Necropsy Guide - Rodents and The Rabbit-CRC Press (1988) Feldman, Donald B. - Seely, John C

 

NECROPSY GUIDE: Rodents and the Rabbit DoNALD B. FELDMAN, D.V.M. Manager

Animal Research Facility Research Triangle Institute Research Triangle Park, North Carolina

JoHN C . SEELY, D.V.M. veterinary Pathologist

PATHCqinc. Research Triangle Park, North Carolina

Boca Raton

CRCPress London New York Washington, D.C.

 

Library Librar y o f  Co n g re ss s s C ataloging-i ataloging-in n -P ublication

D ata

Feldman, Don Donaa ld B. Necropsy guide. Bibliography: p . I n cludes index. ISBN 0-8493-493 0-8493-4934-6 4-6

1. R o d e n t s -A - A u to p sy . 2. R a b b its- A u to p sy . I. Seely, John C. II. Title Title.. SF997.5.R64F SF997.5.R64 F 45 1988 636'.9323 636'.932 3

87-20956 C IP

T h is book contains contains   information obta obtaii ned from authent authentii c and highly reg regaa rded sources. Re Rep p rinted m aterial is is   quoted with permission, and sources and  sources are indica indica ted. A wide varie varietty of references are  are   listed. Reasonable Reasonable  efforts have been been   made to publish reliable data aan n d information, bu butt the author and tth h e publisher cann o t assume respons responsii bility for the validity valid ity of all materials  materials   or for the c onsequences of th eir use. Neitherr   this book nor any Neithe any   part may be repr repr oduced or transm itted in any form o r by any means, electronic or mech mec h anical, including inclu ding photocopyin photocopying, g, microfilming, a nd recording, or   by any informati informat ion storage or retrieval retrieval system, wit wi t hout prior permission in wr wrii ting from the pub lisher. The consent o f CRC Press LLC  does not extend tto o  copy ing for gene general ral distribution, fo forr promotion, for creating c reating new works, works, or for resale resal e . Specific permiss permis s ion must be obtai obta i ned in writing from fro m CRC Press LL C   for such copying copying . Direct al a l l inquiries to CRC   Press LLC, 200 2000 0   N.W. Corporate Corporate   Blvd., Boca Raton Rato n , Florida 33431. Tr a demark demark   Notice: Product or corpo corporate rate names may be trademarks or or   registered tradem arks, and are used us ed only for identification and  and   explanation, witho with o ut intent to infrin infrin ge.

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9  0

 

PREFACE Each o f our veterinary specialities, laboratory animal medicine and pathology, respectively, requires that we either perform necropsies or supervise the performance o f necropsies. With regard to the latter, this would include teaching necropsy methodology to novice prosectors. In the past, we have had to rely upon various anatomy texts and dissection guides for individual species. These books emphasized the anatomical approac approach h to animal dissection rather than an approach necessary for the identification and collection of lesions encountered at necropsy. Therefore, Therefore , we perceived the value o f having a single volume describing necropsy methodology for those species most commonly used in biomedical research. Donald B. Feldman John Curtis Seely

 

ACKNOW LEDGM ENTS and   Tria n g le Institute (D r. Clarence E. Cook) and  in d ebted to the R esearch Trian The a uthors are ind enco u ragement. sup port and encou Hildebrandt) t)   for their sup PaulK K . Hildebrand PAT HCO, In I n c. (Dr. Paul Ph a rD r. Linda Ziem ba (Stuart Pha Jennifer   Colette (PAT H CO, Inc.); Dr thank   Ms. Jennifer  W e also thank  their   assistance. (Patholog y Associates, In c.) for their  Shel d on (Pathology maceuticaa ls), and Dr. W inslow Sheld maceutic (R es earch Frank ie Tutor (Res expresse d   to Mrs. Frank appreciat io n is expressed Special ackn ack n owledgment a nd appreciatio many   is manuscrip manuscriptt   and its many this in  the typing of  th patien ce   and skill in  fo r her patience Triang Trian g le Institute) for revisions. Jacqu e lyn S to ckstill, Jacque ou r lives, Beth Sto specia l people in our so m e very special th is book to som W e dedicate th Underwood. Samanth a   Underwood. Seely,, and Samantha Seely

 

THE AUTHORS D o n ald B. Feldm a n , D . V . M . ,  ,   is manager manager  of  the animal re s earch facility facilit y at the Researc Resea rc h   Triangle Ins Insti titt ute, Research Resear ch   Triangle Par Par k , North Caro Ca ro lina. He re c ei eived ved his B.S . degree from the th e University o f  Illinois in 19 1 9 51, his M.S . d egree from D ePaul Univers Unive rsity ity in 1956,  1956,   a nd   his D.V.M. D.V.M . degree from   the the University Universit y of  Illinois C ol lege of Vete Veteri rinary nary Medicine Medici ne   in 1961. After Afte r six years of practice in co mpanion anim an im al medicine, medicine , he served a  a   tw two-year o-year residen reside n c y in laborato laborat o r y animal m ed icine at the U n iversity of M ichigan. In 19 1969 69 he joine d the  the   National Ins Ins titute o f E n vi ronmental He H e alth Sciences Science s (NIH), Resear Rese arch ch Triangle Triangle   Park, North Caro Ca ro lina, as veteri vete rin n ary medical  medical   officer. Dr. Fel F eldman dman becam e certified as a  Diplomate  D iplomate in the   American C o llege of L a bo ratory Anim a l Medicine in 1971. In 198 19 8 0 Dr. Feldm an   as sumed his pre p re sent position. position . His research  research   interests interests are par p ar asitology, exp e xp erimental surg su rg ery, the develo deve lo p ment o f ne w techniques for for   laboratory an imals, and col c ol laborative eff e ffo o rts in the de v el o pment o f b io medical devic dev ic e s for human  human   medicine. Dr. Dr. F eldman is the he   author of o v e r

25 publica publicati tio o ns.

J o hn C urtis Se Se e ly, D.V.M.,  D.V.M.,   is currently currently   a Veterinary Pat P at hologist with with PAT HCO, IIN N C ., Research T ri angle Park,  Park,   North Carolina. Carolin a.   Dr. Seely See ly  received his  his  A .B. and D.V . M. degrees fro m the Unive Univers rs ity of Missou Missouri ri   in 1971 and 19 75, respective respecti ve ly. He comple comp le ted postdoctor postdoct or ate training at the Walter Ree R eed d Institute of R esearch in com c om parative path pat h o logy. He is a D iplomate of th thee American C o lle llege ge o f Veter Vet er i nary Patholog ists. Pathologis ts. D r. S r.  S eely's pro profe s sional affiliat affili at i ons include the  Am erican erican Ve Ve terinary Medic Med ical al Association Associatio n , t he American America n College of V e terinary Patho Pat ho logists, the S o ciety of T ox ic icologic ologic Patho Path o lo gists, The Am e rican Assoc Associa ia tion for Labor Lab or atory Anim al Science, the  the   International nternational   Academy of P at h ology, and the th e North Caro Car o l ina Society of  Toxicology Toxicology.. His major int i nt erests include includ e toxicologic pat p at hology and di a gnostic ultrast ultra st ructural patho path o l ogy.

H e also se rves as a con co n tributing tr ibuting edito edit o r for pathology patholog y in L ab Anim al. al . He is an avid av id saltwater  saltwater

fishin fish ing g sportsman sportsman..

 

TABLE OF CONTENTS Chapter 1 Th Thee Necropsy Necr opsy in General Gener al . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 Chapter 2 Necropsy o f the Rat (Rattus norvegicus) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23 Chapter 3 Necropsy o f the Mouse (Mus musculus) .... . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 51 Chapter 4 Necropsy o f the Hamster (Mesocricetus auratus) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 77 Chapter 5 Necropsy o f the Guinea Pig (Cavia porcellus) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 105 Chapter 6 Necropsy o f the Rabbit (Oryctolagus cuniculus) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 133 Index . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . .... . . . . . . . . . . . . .. . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . .... . . . . . . . . . . 161

 

1 Chapter 1

THE NECROPSY IN GENERAL TABLE OF CONTENTS I.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

II.

Euthanasia . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 A. Rodents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 B. The Rabbit . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4

III.

Materials and Instruments. Instru ments. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5

IV.

Topographical Topographic al Anatomy and Necropsy Terminology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8 A. Topographical Topograp hical Anatomy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8 B. Necropsy Terminology Terminolog y . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8

V.

Artifacts and Postmortem Changes. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .1. . . . . . . . A. Artifacts . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11 B. Postmortem Changes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12

VI.

Fixatives and Fixation Techniques Technique s . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14 A. Fixatives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14 B. Fixation Techniques . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16

VII.

Special Considerations Considerat ions for Diagnostic and Toxicologic Necropsies . . . . . . . . . . . . . . 17 A. Diagnostic Necropsies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17 B. Toxicologic Toxicolog ic Necropsies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19

References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20

 

2

Necropsy Guide: Rodents and the Rabbit

I. INTRODUCTION

This book is intended to be a guide to the step-by-step necropsy of commonly used laboratory rodents and the rabbit. The objective is to present, mainly pictorially, a systematic approach to routine dissection and examination of animals for the purpose of identifying obvious abnormalities abnormal ities and for for collecting tissues. The necropsy technique described herein is just one of a variety of approaches and the pathologist may desire to modify some procedures. Moreover, the experimental protocol may dictate how, and in what order, certain organs need to be collected during necropsy. Although the method of dissection in this text is basically the same from species to species, multiple species have been incorporated into a single volume in order to point out important morphological differences between species. Rodents and rabbits are frequently used in areas of biomedical research that rely upon a pathologist to evaluate the effect of test agents on the mammalian body. Careful postmortem examination and collection of tissues from animals on study is essential. In many cases the dissection is performed by a technician under the direction of a pathologist. The technician must be trained to perform a careful, thorough examination and to recognize visible (gross) abnormalities. It is therefore important to develop a necropsy technique that is consistent from animal to animal so that tissues are not overlooked. The dissection must be conducted in a manner that will not crush, tear, or otherwise distort the normal architecture of the tissues. A necropsy guide would not be complete without a discussion of related subjects such as euthanasia; materials and instruments; topographical anatomy and necropsy terminology; artifacts and postmortem changes; fixatives and fixation techniques; and finally special considerations for diagnostic and toxicologic necropsies.

II. EUTHANASIA

Research animals must be killed humanely, in a manner that evokes minimal pain and stress. This is termed euthanasia. Aside from from the humane aspect of euthanasia, it is important that the method employed does not traumatize or cause artifacts in those tissues required for histopathologic examination. Thus, the method of euthanasia is of concern to the pathologist and should be recorded on the necropsy form.

A. Rodents Overexposure to carbon dioxide is often considered to be the method of choice for euthanasia of mice, rats, hamsters, and guinea pigs. Carbon dioxide has depressant and anesthetic effects on an animal before inducing fatal anoxia. 1 Most important is that C 0 2 does not distort cellular architecture except for small hemorrhages in the lungs (Figure 1) which tend to disappear when the lungs are infused with fixative 2 (Figure 2). Any closed system allowing the rapid buildup of carbon dioxide can be used, such as a chamber connected to a carbon dioxide tank with a regulator valve (Figure 3). Since C 0 2 is heavier than air it is important to allow sufficient time for the C 0 2 to dispel the air in the container. Dry ice can also be used as a source of C 0 2 provided that the animals do not come in contact with the ice. As an alternative alternati ve to C 0 2 , an overdosage of sodium pentobarbital can be administered by

 

3

Figure 2.

Figure I.

Figure 3.

rapid intravenous injection. Death is almost instantaneous and the tissues are unaffected except for slight congestion of the pulmonary blood vessels. 2 Skill must be developed with this technique because the veins in rodents are very small and difficult to enter cleanly even with a small gauge hypodermic needle. I f he drug is accidentally deposited outside of the vein (perivascularly) it may be absorbed too slowly to kill the animal.

 

4

Necropsy Guide: Rodents and the Rabbit

Figure 4.

Intraperitoneal or intrathoracic injection of a euthanasia agent is not recommended because of artifacts produced on the surface of the viscera. For example, sodium pentobarbital administered intraperitoneally causes subcapsular vacuolization (Figure 4, arrow) of the liver. For histological purposes cervical dislocation and decapitation are not acceptable methods of rodent euthanasia because of the trauma inflicted to the tissues of the head and neck. Nitrogen (N 2   , methoxyflurane, halothane, ether, and chloroform have been used for rodent euthanasia but there are important disadvantages to using these agents. Nitrogen may induce convulsions and vocalization in an animal, thus it is esthetically objectionable. In addition, some geographical areas legally prohibit the use of N 2 for euthanasia. 1 Methoxyflurane and halothane are relatively expensive and may be toxic to humans subjected to repeated exposure; chloroform is a hepatotoxin and a suspected carcinogen; ether is highly flammable and explosive.

B. The Rabbit The method of choice for euthanasia of rabbits when tissues need to be examined histologically is an overdose of sodium pentobarbital injected into the marginal ear vein (Figure 5). Death occurs rapidly and tissues are not adversely affected except for enlargement of the spleen resulting from blood congestion. There also is slight congestion of the pulmonary blood vessels. T -61 ® administered intravenously may be substituted for sodium pentobarbital but the dosage should shoul d not exceed 0. 3 m€/kg body weight, otherwise pulmonary artifacts are induced. 3 pentobarbital nor T-61 ®should be injected directly into the As with rodents, neither sodium pentobarbital abdominal or thoracic cavities because they induce artifacts on the surface of the viscera. I f injectable drugs are contraindicated for euthanasia because of the nature of the experiment, rabbits can be killed by overexposure to carbon dioxide. This method, although painless, is less desirable in the rabbit because some animals struggle before lapsing into unconsciousness. Rapid intravenous injection of air into the marginal ear vein of unanesthetized rabbits and

 

5

F igure 5 

thanasia f or eu euthanasia a nd s hould not be e m pl oye d for consider e d i nhum a ne and h '' ar aree considere also the ''r a b b i t punc punch

of r abbits.

II I . MA T E R I A L S A N D I N S T R U M E N T S L ab theirr clothing a nd   person. Lab advi sed to protect thei necro psiess are advised iduals perform per form ing necropsie I ndiv ndividuals glovess should protective   rubber glove surgica l s crub suits are r e c om m e n d e d   and protective oratory coats c oats   or surgica recom m e nde d. gla sses   are also recom wo rn. Safety glasses a lw ays be worn. th e o p erator from   e n c l osure that will protect the performe d in a vented en shou ld b e performed N e c ro psies should adequa tely lighted, sho uld be adequately solvents.  T he area should fixatives   an d  solvents.  noxious fu m es given o f f b y fixatives  desira ble for is   h ig hly desirable illuminated d m agnifier is cal lamps.  lamps.   An An   illuminate surgical erably by o n e   or m or e surgi preferably pref operator to to fill out the area   fo r  the operator b e a clean area  ns. There sh o uld also be ng small lesio l esions. o b s ervi erving necropsy   fo rm . equip m e nt. ents   and equip nt space to to   sto st o re instrum ents suf ficient op s y l aboratory m u s t have sufficie T he ne c r ops followin g processed. T h e following to   be be   processed. sto rage   of specim e n s   waiting to T he re r e sh ould be sh e lv ing for storage ry: lab oratory: a   n ecropsy laborato t hat comprise a  is a list o f b as ic items that

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6 

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8

Necropsy Guide: Rodents and the Rabbit

IV. TOPOGRAPHICAL ANATOMY AND NECROPSY TERMINOLOGY A. Topographical Anatomy Topographical anatomy is a method of indicating in descriptive terms the relative positions of various parts of the body. In this book the terminology applies to four-legged animals assuming a standing position. Dorsal or s u p e r i o r - toward the animal's back; away from the ground. Dorsal also refers to the surface of the forelimb or hindlimb opposite the surface that contacts the ground. Ventral or i n f e r i o r - toward the abdomen; in the direction of the ground. Anteri Ant erior or or cran crania iall - to towa ward rd th thee head head.. Po Post ster erio iorr or cau cauda dall - to towa ward rd th thee tai tail. l. M e d i a l - toward the center of the body. L a t e r a l - away from the center of the body. P r o x i m a l - closer to the long axis of the body, e.g., toward the origin of an appendage. D i s t a l - away from the axis of the body, e.g., toward the unattached end of an appendage. Su Supe perf rfic icia iall - towa toward rd the the sur surfa face ce of the body. Deep De ep - away away fr from om th thee sur surfa face ce of the body. V o l a r - the surface of the forelimb that contacts the ground. P l a n t a r - the surface of the hindlimb that contacts the ground. C e r v i c a l - relating to the neck, also referring to the cervix. T h o r a c i c - relating to chest or thorax. L u m b a r - relating to the loins. S a c r a l - relating to the sacrum. A x i l l a r y - relating to the axilla or underarm. I n g u i n a l - relating to the groin.

Planes of Dissection (Figure 9) Median p l a n e - dividing a bilaterally symmetrical animal into right and left halves. Sagittal p l a n e - a plane parallel to the median plane. Transverse p l a n e - perpendicular to the median plane. planeFrontal to the median and transverse planes, i.e., dividing the body into dorsalperpendicular and ventral halves. Coron oronaal pla plane ne - sam same as frontal plane, but usually referring to the head.

B. Necropsy Terminology To many individuals the language of pathology is filled with lengthy words that are often difficult to pronounce, let alone understand. It is true that many of the terms used in medicine and pathology are highly specialized and, like a foreign language, take years of study to master. However, pathologic terminology associated with gross necropsies can be simplified due to its descriptive practicality. During the gross necropsy the prosector or pathologist must record precisely what is observed, yet remain deliberately vague, allowing the scientific diagnosis to be made during the subsequent microscopic evaluation. For example, a grossly apparent hemangiosarcoma (malignant vascular neoplasm) in the spleen of a rat would be appropriately described as a spherical mass, 0.5 em in diameter, solid, and dark red on its cut surface. None o f the above descriptive terms used in the preceding example is highly scientific, yet together they accurately describe the gross appearance of a splenic hemangiosarcoma. Likewise, specific terms such as hemorrhage or congestion should be avoided.

 

9 Transverse

Plane

Sagittal Plane

Figure 9.

These color changes can better be described as diffuse redness or focal foca l redness. Moreover, Moreove r, if the above color changes were due to postmortem degeneration (to be discussed later), the pathologic significance would be different had the terms hemorrhage or congestion been used. The objective of a gross necropsy is to precisely describe any abnormality according to its size, color, shape, texture, location, severity, and number, if applicable. 4 •5 The term lesion is used frequently to denote a tissue change arising from any one of a wide variety of disease conditions. A lesion may be produced by an infectious agent, toxicity, neoplasia, or an inherent genetic or age-related change. Included in this category would also be any other pathologic abnormality that does not have an obvious cause. Some lesions are very characteristic, e.g., hydronephrosis, and may be used by experienced prosectors, but if there is any question in the mind of the prosector, it is always best to describe the lesion according to the criteria previously mentioned. The term neoplasia neoplas ia should not be recorded during the g gross ross necropsy because this diagnosis ultimately needs to be determined microscopically. The term tumor, although commonly equated with neoplasia also should not be used because tumor, simply defined, means a swelling". In describing neoplasms or growths, the terms mass or nodule seem best suited and both enjoy wide acceptance. I f all pathologists or laboratories used similar styles or terminology it would simplify the gross necropsy. Unfortunately, this is not the case. Some pathologists prefer a freehand style while others are more dogmatic in their approach. In addition, some laboratories have devised forms which guide prosectors through this descriptive exercise. In this age of automation, computers often contain preloaded terminology which is selected where appropriate. The terms and comments below are offered merely as guidelines. 6 1. Lesions Abscess

Laceration

 

10

Necropsy Guide: Rodents and the Rabbit Adhesions Alopecia Calculi Crust Cyst Deformity Dilatation Enlarged Fluid Focus Foreign body Fracture

Lesions, NOS* Mass Nodule Obstruction Parasite Perforation Pigment Prolapse Rupture Small Thick Thin

The location of a lesion should always be cited. 2. Size and Number Lesions should be measured in centimeters and in three dimensions, if possible. Spherical lesions are best measured in diameter. Flat lesions should be measured in two dimensions, e.g., 3 X 4 em. Diffuse changes in a tissue may be referred to as enlarged or smaller depending upon the change. Multiple masses or nodules should be counted up to five, thereafter thereaft er reporting them greater great er tha than n five. Terms such as focal, multifocal, or diffuse can be used as well. 3. Color Blue Brown Black Clear Cloudy Dark Green Gray

Mottled Opaque Pale Pink Red Tan White Yellow

of colors may also be used to describe in-between color changes (i.e., paleCombinations yellow, yellowish-brown).

4. Shape Compressed Confluent Cystic Depressed Flat Irregular Linear Lobulated Oval

*

NOS

=

not otherwise specified.

Nodular Papillary Pitted Punctate Raised Round Smooth Spherical Tubular

 

11

Symmetry is very important in the consideration of tissue alteration. Paired organs, for instance, can be compared to one another for detection of subtle changes. The effect on one tissue due to the presence of a lesion within another should always be investigated. For example, if a pituitary gland is greatly enlarged, it could cause significant compression of the overlying brain tissue. 5. Texture (Consistency) Brittle Caseous Firm Friable Gelatinous

Gritty Hard Rubbery Soft Watery

The texture or consistency of a lesion is most revealing when it is cut open. The opened surface is usually referred to as the cut sur surface face ". The terms terms homogeneous or heterogeneous are often used in conjunction with the appearance of the cut surface. 6. Severity Minimal Mild

Moderate Marked

7. Weight/Volume I f an experiment or study protocol calls for organ weights or fluid volumes to be measured,

weights should be recorded in grams, and volumes in milliliters. Organs need to be weighed fresh, and in a consistent manner if statistical evaluations are to be meaningful. Studies have been conducted in comparing fresh vs. fixed organ weights, as well as the effects of exsanguination on organ weights. In general, as one would expect, these variables do influence organ weights. Fixed organ weights may be a valid alternative to fresh organ weights and may actually help to avoid necropsy-induced artifacts. 7 Organs from rodents found dead should not be weighed because of uncontrollable variables. 8

V. ARTIFACTS AND POSTMORTEM CHANGES A. Artifacts As stated previously, the function of the gr-oss necropsy is to identify lesions present in the animal and to collect tissue in an orderly fashion for subsequent microscopic evaluation. A properly conducted necropsy reduces the number of artifacts, which are alterations induced in tissues and, as their name would indicate, represent artificial changes. Artifacts can be produced by a number of means including excessive tissue manipulation, improper moistening fluids, and poor fixation. In general, experienced pathologists can quickly recognize artifacts produced at the time of necropsy; however, there are some artifacts which can mimic true lesions, and thus may be confusing. The elimination of artifacts during the necropsy greatly enhances the pathologist's ability to evaluate tissue lesions at the microscopic level. I t is unfortunate that many necropsy prosectors never see the final result of their efforts. A number of artifacts could be eliminated if pathologists would take a few moments to give some instructional feedback to the prosectors. It has often been expressed that the quality o f the the necropsy is a result of o f well-trained well-trained and properly supervised technicians. 9 · 10 One of the most common problems encountered during the necropsy is the production of artifacts caused by poor tissue handling. The squeezing of tissues by simple digital pressure

 

12

thee R a b bit dentss a n d   th psy G ui d e : R o dent ecropsy N ecro

F igure 11.

Fi gure 1 0.

o n , som e  ditio by   p ro s e c to rs .   In a d diti injurr ed by  be  inju u e c an   be  tissu resh   tiss w ay s   tha t   fresh  ex p e r im e n t In a  an n ex su es .   In  tis issu n c tu re  t  pun  a lso  c ru s h , te ar, o r  pu  for n e c ro p s y c a n  al o f  th e  i n str u m e n ts u s e d  for rc ep s forc th e t hy ro i d g la n d w i th sm oo th   fo on   on e   sid e   of  the u si ng a   n orm a l   rat, g e n t le   p re s su re   on as   ture   was hitecc ture 10). T h e n o rm a l th y ro id   a rc hite  ex te n s iv e   his t o l o g i c in j u r y ( F i gu re   10). p r o d u c e d ra th e r  ex

is j u s t o n e

of  m a n y

io n tratio thee in f il trat d is r u p te d to th e p o int o f re s e m b l in g   tis s ue   d eg e n e ra ti o n (b l a c k ar ro w )   w ith   th te d in   llecte thee   th yr oi d w a s c o llec (wh it e   arr o w ). T h e c o n tr a l a te ra l s id e o f th of  in f la m m a to r y   c ell s   (wh ). 11). the g la n d ( F i g ur e 11 not c o m p re ss   the a m a n n e r t h a t d id   not thee   co n n e c ti v e an  or g an   by gr a s p in g th is  a lw ay s a d v is a b le   to a v oi d d ir e c t c o n ta c t   with   an  t is  alinee   be   avo id e d b y u si ng p h y s io lo g ic a l s alin also   c an   be t is su e   su r ro u n d in g th a t o r g a n .   Art i fa c ts   also otic  e q u il ib r iu m . osm otic to   k ee p t is s u e s m o is t, t h e re b y   ma i n ta in in g   the p r o p e r   osm in s te a d  o f  w a ter to tio o n w ill b e tiss u e fi x at io n. F ix a ti v e s   and   fix a ti ts   ca n   a lso   ari s e fr o m   in a d e q u a te   tiss ifacts A rt ifac d i s c u s s e d la te r   in th i s c h a p te r.  

B . P o s t m o r t e m   C ha n g e s lled   ke   pla c e im m e d ia t e l y c a lled to   ta take  a  po s tm o rt e m   c ha n g e   b eg in s to W h e n   an a n im a l   die s  a  irst   pro c e ss si c p ro c e ss e s.   Th e   first basi ress u lt of  tw o   ba ysis o r s e l f - d i g e s t i o n   wh ic h   is the   re utolysis a utol  do w n  n orm al  c ell u la r c o m p o n e n t s.    re le a s e of i n tr in si c  t iss u e e n z y m e s th a t b e g in  to b r e a k  do is is   th thee re org an s . lly y   pre s e n t in m a ny   org flor or a   nor m a ll rom m  the   ba c t e r ia l   fl  pro c e ss is fe r m e n ta tion   fro T h e s e c o n d  pro

sev e ra l m e an s. F ix a ti o n w ill in hi b i t o r sl o w   ti ss u e be  m in im iz e d   th r o u g h   sev A u to ly s is   c an   be  inte rv a l b e tw e e n th e   tim e   o f d e a th   an d   tis s u e   the r e fo re , r e d u c in g the   inte atic   re a c t io n s;   the e n z y m atic as   in the   ca s e of  s p o n ta n e o u s   be   c o n tr o ll e d   as fi xa ti o n is a d v a n t a g e o u s .   Th i s c a n n o t a l w a y s   be w s a ut ol y si s. F r e e z in g low de a th s. A n im a ls f o u nd   de a d  sh o u l d b e   re fri g e r a t e d b e ca u s e c o o li n g  s lo is  to b e p e r fo rm e d b e c a u s e    ex a m i n a t io n is  do n e if  f u r th e r m i c r o sc o p ic  ex th thee c a r c a s s s h o u ld n o t b e  do ic icee   c ry s t als c a u s e d is ru p ti o n

of

c e l lu la r   arc h it e c tu r e .

P o s tm o r te m  c h a n g e s   va r y w ith it h th e   le n g t h of tim ti m e b e tw e e n de a th   and and   ti tisss ue fi xati xatio o n, th thee 5 In  a d d it io n , teriaa .   In ue   b ac teri tissue c a u s e  o f d e a th, te m p e ratu r e , b o d y c o n d it io n   and th e p re se n c e o f tiss and th e   it h   liv e r,   e ye ,   k id n e y , p a n c re a s ,   and d i ff e re n t o r g a n s   va ry   in t h e ir d e g re e  o f a u to ly si s   with

 

13

Figure 12.

intestinal tract undergoing autolysis more rapidly than other tissues. Advanced autolysis causes several recognizable changes as well as being malodorous (an occupational hazard of prosectors and pathologists). During marked autolysis, tissues begin to soften and are characterized by discolorations which are mainly due to the breakdown of hemoglobin released from lysed red blood cells. In addition, the gastrointestinal tract may be markedly distended because of gas production through bacterial fermentation. The accumulation of gas may be so severe as to rupture portions of the gastrointestinal tract or to cause pale areas in adjacent tissues merely from the pressure exerted during this phenomenon. The degree of autolysis resulting from an extended postmortem interval is demonstrated in Figures 12 and 13. In Figure 12 a segment of jejunum was fixed 10 min after death, whereas in Figure 13 an adjacent segment of jejunum was fixed 8 hr after death. Both segments were held at room temperature and subsequently fixed in 10 neutral buffered formalin. The normal microscopic appearance o f the intestinal lining may be appreciated in the normal section (Figure 12) while in the autolytic section (Figure 13) the lining tissue is disrupted and o f questionable diagnostic quality. Other events associated with postmortem degeneration include muscle stiffening (rigor mortis), gravitational settling of blood to the down side of the animal, and the yellowishbrown discoloration o f tissues next to the gallbladder from the seepage of bile. 5 • 11 A certain degree of postmortem change is inevitable in many cases but usually it does not interfere significantly with the pathologist's ability to evaluate the tissue section. Advanced autolysis may preclude any worthwhile microscopic evaluation. Moreover, if too many tissues in a given study are autolyzed, the pathology data could be jeopardized and the statistical analysis could be compromised.

 

14

N e c ro p s y G u jd e: R o d e n ts a n d t he   Ra b b it  

igure   13. F igure

F in a ll y , s o m e   in e x p e ri e n c e d in d iv id u a ls ha ve b ee n   fo o le d o r   e mb a rr a s s e d   n ific ificaa n c e   to p o s tm o rt e m   c ha n g e . ign p a th o lo g ic   s ig

a sc ri b in g

V I .   F IX A T I V E S   AN D F I X A T I O N T E C H N I Q U E S A . F ixati ixativ v es in   step ep in first rst st the   suc c e ss o f  an y n e c r o p s y . F ix at i on   is the fi A d e q u a t e f ix atio n   is c ru c ia l to the  is   to s ta b il iz e   the m o le c u la r tion on is g ic a l se c ti o n s   and   its c h ie f   fun c ti tolog th e p r e p a ra ti o n o f h is tolo  fix ix a ti o n ,   pro t e i n s a r e d e n a tu re d , c r o s s -l in k e d ,  pro tein s . D u r in g  f s tr u c t u re  o f  ti s s u e s , e s p e c ia ll y  pro inss o lu b l e . L i pi d s o r c a rb o h y d ra te s   may   or m a y n o t be   pre se rv e d d e p e n d in g o n a n d re n d e r e d   in thee r e n z y m a tic and   o th re   to a rr e st   a uto ly s is   and ativee s a re n a l fu n c ti o n s o f  f ix ativ ition th e   fix a ti ve .  12   A d d itio issuee s thee   tissu tiss su es ; a nd   to h a r de n   th ind e x of  ti in  the th e   re fra c ti v e   ind to   b rin g o u t d i ff e re n c e s   in  a ctiv it y ; to tivess ar e   no t  wit h o ut t h eir p ro b le m s . fo r s u b s e q u e n t h i s to lo g ic   pre p a ra t io n . 13 H o w e v e r,   fi xa tive tive,, so m e st ru c tu re s m a y S o m e   s hri n k a g e o r sw e l li n g m a y o c c u r a nd , d e p e n d i n g   on th e   fi xa tive it h st a in in g   pro c e d u r e s . terfee r in g   with no t b e p r e s e r v e d , t hu s i n terf  fix ix a ti ve s   tion  is p ro p o s e d . M a n y  f tivess is g o v e r n e d by   wh a te v e r e x a m in a tion T h e s e le c ti o n of  fi x a tive g no s ti c iag io ns .   3 • 1 4 F o r r o utin e   d ia m itat itatio lim ti e s a nd   li its o w n  sp e c i a l q u a li tie a re a v a il a b le e a c h   ha v i n g   its tive ve o f c h o ic e . thee   fix a ti x ic o lo g ic   re s e a rc h   10%   n e ut ra l   bu ff e re d   fo rm a li n   (N B F ) is th tox a nd   to

Z e n k e r 's fi x a t iv e   is a   go o d fi x a t iv e   for ey es bu t it de s tr o ys   red b lo o d c e l ls , th u s r e s tric tricti ti n g an  alc o h o li c fi x a t iv e , is us ed to  x a ti v e ,   an  fix se   fo forr b o n e   ma rr o w   p re s e rv a t io n . C a r n o y 's   fi its  its   use  ti ss u e e o s in o p h il i a w h e n an   un d e s ir a b l e i n c re a s e  in o v e r a ll tiss  gly c o g e n   bu t im p a rt s an  d e m o n s tr a te  gly

 

15

Figure 14.

Figure 15.

tissues are stained with hematoxylin and eosin. The same staining problem may be observed with Bouin's fixative. 15 As previously stated, NBF is the preferred fixative for most routine studies and is the most common fixative used in pathology. 16 Formalin is an excellent fixative and penetrates tissue up to 1 mrn/hr. The amount of NBF, as with other fixatives, should be 10 to 20 times the amount of tissue to be fixed, but usually tissue no thicker than 5 mm should be fixed if thorough fixation fixation is to be guaranteed. NBF is a mixture of formaldehyde in water, and is used as a 10 solution by volume, buffered to a neutral pH. Formaldehyde is highly toxic and safety precautions should be taken during its use. I t has been associated with irritation of the eyes, nose, and throat; headaches; dizziness; and allergic reactions. 17 • 18 Formaldehyde, a known mutagen, also causes damage to DNA and has been shown to produce nasal tumors in rats during inhalation studies. 19 In a Danish study, humans with an occupational risk of formaldehyde had an increased risk of nasal cancer. 20 Based on all the information, the National Toxicology Program regards formaldehyde as posing a carcinogenic risk to humans. 21 Precautions needed to prevent any unnecessary exposure to formaldehyde include adequate ventilation, the use of rubber gloves, and keeping containers sealed as much as possible. Using formaldehyde under exhaust hoods should be maximized. There are few tissue artifacts associated with the use of NBF. Formalin has been known to cause a consistent artifactual detachment of the retina in rat eyes. This can be avoided by using Zenkers, or a comparable fixative where the eye is a potential target organ. Although tissues are usually fixed by immersion, special care must be taken during intratracheal perfusion of the lungs. The normal histologic appearance of lung tissue is characterized by airways (alveoli) lined by delicate cells where the transfer of oxygen and carbon dioxide takes place (Figure 14). Poor perfusion can result in alveolar walls that appear thicker than normal (Figure 15). This could be mistaken for pneumonia or pathologic-induced airway collapse. The lung sections in the figures were from the same animal where one side was perfused normally and the other side allowed to collapse without perfusion. In addition to injecting formalin into the trachea with a syringe, techniques are available for intratracheal perfusion under controlled pressure which eliminates the occasional occasional undesirable effect of

 

21 26. Cline, J, M. and Maronpot, R. R., Variations in the histologic distribution of rat bone marrow cells with respect to age and anatomic site, Toxicol. Pathol., 13, 349, 1985. 27. Brown, B. A., Hematology: Principles Principles and Procedures Procedures,, 4th ed., Lea & Febiger, Philadelphia, 1984. chap. 2. 28. Ziemba, L., personal communication, 1986. Gerarde, e, H. W., A method for the quantitative collection of femoral marrow in small laboratory animals, 29. Gerard Arch. Ind. Health, 13, 331, 1956. F.,, Vali Valigorsky gorsky,, J, M., M. , Jones, R. T., Mergner, W. J,, J, , Garci Garcia, a, J, H., and Cowley, R. A., 30. Trump, B. F. The application of electron microscopy and cellular biochemistry to the autopsy, autopsy, Hu Hum. m. Pathol., 6, 499, 31. 32. 33. 34. 35. 36. 37. 38. 39.

1975. Sobel, H. J, and Marquet, E., Usefulness of electron microscopy in the diagnosis of tumors, Pathol. Res. Pract Pract., ., 167, 22, 1980. Johannessen, Johanne ssen, J, V., Oppedal, Oppedal, B. R., Ormos, J,, Gould, V. E., and Sobel, H. J,, Electron microscopy and the autops autopsy, y, Invest. Cell Pathol Pathol., ., 2, 239, 1979. Mackay, B. and Srigley, J, R., Diagnostic electron microscopy: a review of current applications, Lab. Manage., Manag e., 14, 639, 1983. Johannessen, J, V., Use of paraffin material in in electron microscopy, Pathol. Ann., Ann ., 12, 189, 1977. McDowell, E. M. and Trump, B. F., Histological fixatives suitable for diagnostic light and electron microscopy, microsc opy, Arch. Pathol. Lab. Med., 100, 405, 1976. Sheldon, W., personal communication, 1986. Benirschke, Benirsch ke, K ., Garner, F. M., and Jones, T. C., Eds., Pathology o f Laboratory Animals, Vols. Vols. I and 2, Springer-Verlag, Berlin, 1978. Menegus, M. A., Diagnostic virology, in Textbook o f Human Virology, Belshe, R. B., Ed., PSG Pub ., Littleton, Mass., 1984, chap. 7. Washington, J, A. and Wilson, W. R., Collection and handling of specimens, in Laboratory Procedures

in Clinical Microbiology, 2nd ed., Washington, J. A., Ed Ed., ., Springer-Verlag, Berlin, Berlin, 985, chap. I. 40. Finegold, S.M. and Baron, E. J,, Bailey and Scott's Diagnostic Microbiology, 7th ed., C. V. Mosby, St. Louis, Mo., 986, chap chap.. 6. 6. Hildebrandt, ndt, P. K., Importance of uncut lesions and animal identity, in Managing and Conduct and Data 41. Hildebra Quality o f Toxicology Studies, Hoover, B. K., Baldwin, J. K., Uelner, A. F., Whitmire, C. E., Davies, C. L., and Bristol, D. W., Eds., Princeton Scientific Pub ., Princeton, N.J., 1986, 257. 42. Black, H. E., A manager's view of the musts in a quality necropsy, in Managing and Conduct and Data Quality o f Toxicology Studies, Hoover, B. K., Baldwin, J. K., Uelner, A. F., Whitmire, C. E., Davies, C. L., and Bristol, D. W. W.,, Eds., Princeton Scient Scientific ific Pub ., Princeton Princeton,, N.J ., 986, 249. Highman,, B., and Konvicka, A. J,, Advances in automation for experimental pathology, 43. Frith, C. H., Highman Lab. Anim. Sci. Sci.,, 26, 171, 1976. 44. Reuber, M . D., Necropsy of animals for scientific research, Clin. Toxicol., 0, I l l , 1977. 45. Townes, J,, Federal regulations: an overview, Lab La b Anim Anim., ., 9, 16, 1980. 46. DHEW, Food and Drug Administrat Administration ion (FDA), Nonclinical Laboratory Studies, Good Laboratory Practice Regulations, Fed. Reg., 43, 59986, 1978.

 

23 Chapter 2

NECROPSY OF THE RAT (Rattus norvegicus) Step 1. Step 2. Step 3.

Step 4. a.

b.

Note: Step 5.

Check animal's identification number. Record animal's weight. Verify animal's sex according to characteristics below. Male (Figure 16)

Female (Figure 17)

Long distance between anus and genital opening Nipples inconspicu inconspicuous ous Pronounced scrotum

Short distance between anus and genital opening Conspicuous nipples

External examination. Observe entire body surface and orifices for abnormalities. Feel (palpate) the entire body for superficial swellings beginning from the head and proceeding posteriorly to the neck, chest, abdomen, and appendages. Palpate for enlarged organs or masses within the abdomen. All tissues and organs should be examined in situ before being dissected from the body. Collection of the eye, Harderian gland, intraorbital lacrimal gland, exorbital lacrimal gland, parotid salivary gland, and Zymbal's gland.

Autolytic changes occur early in the eye, therefore it should be collected first, along with neighboring glands on the side of the head. a.

b. c.

d. e. f.

Step 6. a.

Lay the rat on its its side and make a skin incision from above and and just jus t in front of the eye to the convergence of the upper and lower jaws just posterior to the base of the ear. Reflect the skin ventrally. Press a small curved forceps forceps deeply into the orbit and elevate the globe. Cut the tough fibrous tissue beneath the forceps with small scissors (Figure 18) 18) and remove the eye, along with the large, light-yellow Harderian gland which almost completely surrounds the globe within the bony orbit (Figure 19). The Harderian gland in both sexes of the rat secretes porphyrin, a pigment which accounts for the red color of nasal and/or ocular discharges. The small light brown colored intraorbital lacrimal gland lies in the lateral comer of the eye superficial to the Harderian gland and may adhere to the Harderian gland when the eye is excised. Identify the discrete, flat, brown exorbital lacrimal gland which is located just jus t posterior and ventral to the eye (Figure 20). Remov e and fix the more diffuse, pink parotid salivary gland which is located ventral to and just behind the base of the ear (Figure 20). Cut the cartilaginous base of the ear close to the head and identify the whitishyellow glandular tissue at the entrance to the bony ear canal. This is the largest of multiple Zymbal glands (auditory sebaceous glands) (Figure 21). Others are present just beneath the epithelium of the ear canal near the tympanic membrane. These glands are best visualized in decalcified skull sections cut through the junction o f the middle ear and external ear canal. Collection of superficial masses. Remove Remov e any abnormal enlargement enlargeme nt by cutting completely around it, being careful

 

24

Necropsy Guide: Rodents and the Rabbit

Figure 16.

b.

Step 7. a.

b.

c.

d.

e.

Figure 17.

to include overlying skin, if possible, and at least some normal tissue surrounding the mass. Scrape the hair away from the skin because hair dulls the microtome knife which is used to section tissues in the preparation of slides. Divide large excised excise d masses into 0.30.3 - to 0.5-cm slices with a sharp scalpel and place in fixative. Masses smaller than 0.5 em can be fixed fixed undisturbed. If the mass has invaded bone try to expose as much of the abnormal tissue to the fixative without destroying the architecture of the surrounding normal structures. Allow tissue to fix at least 48 hr. The bone must be decalcified before the specimen is sectioned. If there is some question as to the removal of a mass, consult the pathologist. Collection Collecti on of cervical lymph nodes, submaxillary and sublingual salivary glands, mammary glands, and preputial (or clitoral) glands. Lay the rat on its its back on a dissecting board. board. (Rats usually do not need to be pinned down.) Wetting the surface of the body with alcohol helps prevent loose hair from falling into the incisions. Stretch the skin in opposite directions with with the thumb and forefinger and make a midline skin incision extending from the lower jaw to the vulva, or scrotum, respectively. Stretching the skin helps to prevent accidentally cutting into deeper structures. While exerting gentle outward traction on the skin with forceps, carefully incise the loose subcutaneous tissue and retract the skin on either side of the incision (Figure 22). Grasp the loose connective tissue just posterior to one of the paired submaxillary glands and bluntly blunt ly dissect out the above gland together wi with th the sublingual salivary gland and cervical lymph nodes (Figure 23). Place the tissues in fixative. In female animals locate a nipple on the reflected abdominal skin and identify the whitish-pink mammary tissue beneath the skin (Figure 24). (Mammary glands are most obvious in lactating rats and may extend from the inguinal area to the neck [Figure 25]). Scrape the hair away from the nipple and cut a dime-sized circular area out of the skin including the nipple and underlying tissue.

 

25

F igure 18.

Figure 19

Figure   20.

F igure 21 21..

 

26

Necropsy Guide: Rodents and the Rabbit

Figure 22. Figure 23.

Figure 25. Figure 24.

 

27

Figure 26.

f.

Step 8.

Figure 27.

The clitoral glands are paired flat, flat, light pink masses masses of tissu tissuee located located just anterior to the vulva (Figure 26). Collect the gland(s) by grasping the surrounding tissue with fine forceps and by cutting the underlying connective tissue with fine fine scisso scissors. rs. Corresponding structures in the male rat are the preputial glands which are located on either side of the preputial opening (Figure 27). Collection of the male or female reproductive tracts and urinary bladder.

Male

a. b.

c.

d. e. f.

g.

Grasp Gras p the most posterior poster ior part of the abdominal wall and cut into the abdominal cavity just above the base of the penis (Figure 28). Cut the body wall on both sides close to the reflected skin being careful not to cut into any organs. Extend the incisions anteriorly to the rib cage and then reflect the cut portion of the abdominal wall over the chest (Figure 29). Pull both testicles out of the scrotum and sever the fibrous connection (gubernaculum testis) between the tail of the epididymis and the scrotum (Figure 30). Sever the vas deferens (Figure 31). Place the testicle with attached epididymis (Figure 32) into fixative. Repeat the above procedure to collect the second testicle. Reflect the intestines anteriorly to reveal the urinary bladder, prostate gland, and seminal vesicles and coagulating glands (Figures 33 and 35). Insert one blade of a dissecting scissors under the center of the pubis just above the base of the penis and cut through the cartilaginous connection (pubic symphysis) (Figure 33). Separating the bones will reveal the underlying urethra and rectum (Figure 34). Dissect Disse ct the skin away from the penis. Grasp the end of the penis with forceps and while pulling gently upward carefully dissect the connective tissue around

 

28

Necropsy Guide: Rodents and the Rabbit

Figure 28.

Figure 29.

Figure 30.

Figure 31.

 

29

Figure 32.

Figure 33.

Figure 34.

Figure 36. Figure 35.

 

Necropsy Guide: Rodents and the Rabbit

30

h.

Female a. b.

c.

d.

e.

Step 9.

a.

b. c.

d.

e.

the urethra and beneath the prostate gland. Place the excised male reproductive tract with attached urinary bladder (Figure 35) onto a moistened paper towel. Place a loose ligature around the neck of the urinary bladder. Express most of the urine out of the bladder if is is distended and then tighten the ligature. Replace about half the volume by injecting formalin into the lumen using a small gauge needle (Figure 36). Caution must be taken not to inject fixative into the wall of the bladder. Fix the reproductive organs and urinary bladder. Note: as an alternative, the urinary bladder may be injected in situ before the reproductive tract is dissected from the body.

Grasp the most posterior part of the abdominal wall and cut into the abdominal cavity just above the vulva (Figure 37). Cut the body wall on both sides close to the reflected skin being careful not to cut into any organs. Extend the incisions anteriorly to the rib cage and then reflect the cut portion of the abdominal wall over the chest (Figure 38). Insert one blade of a dissecting scissors under the center of the pubis and cut through the cartilaginous connection (pubic symphysis) (Figure 39). Separating the bones will reveal the underlying vagina and rectum. Cut the skin between the vulva and anus (Figure 40) and dissect free the posterior end of the vagina being careful not to cut into the rectum. Grasp the end of the vagina and while gently pulling upward sever the film-like connective tissue between the vagina and the rectum (Figure 41). Do not cut away the urinary bladder. Continue dissecting anteriorly to the area of the cervix and then cut the mesentery supporting the uterine horns up to the ovaries (Figure 42). Cut the supporting tissue between the ovaries and kidneys and transfer the entire reproductive tract (Figure 43) onto a paper towel moistened with saline. Trim excessive fat from the uterine horns and ovaries. Place a loose ligature around the neck of the urinary bladder. Express most of the urine out of the bladder if it is distended and then tighten the ligature. Replace about half the volume by injecting formalin into the lumen using a small gauge needle (Figure 44). Caution must be taken not to inject fixative into the wall of the bladder. Fix the reproductive organs and urinary bladder. Note: As an alternative, the urinary bladder may be injected in situ before the reproductive tract is dissected from the body. Collection of the spleen, pancreas, stomach, duodenum, jejunum, ileum, cecum, colon, rectum, and mesenteric lymph nodes. Gently grasp the greater curvature of the stomach with serrated forceps, pull down, and carefully sever the filmy membrane between the stomach and the caudate lobe of the liver (Figure 45). Sever the esophagus approximately 0.5 em above the stomach (Figure 46). This will noticeably loosen the stomach from its attachments. Grasp the esophagus with fine forceps close to the stomach and while maintaining tension, sever the bile duct and remaining connective tissue between the liver and the stomach (Figure 47). Maintain tension and sever the mesentery between the intestines and the dorsal body wall (Figure 48) but do not cut the large blood vessels in this area. This should free Cut the spleen the entire gutthe (Figure except for moistened its termination at the anus. around and the anus and lay gut on49) a paper towel with saline. The intestinal tract is most efficiently examined by stretching it to its full length.

 

31  31  

re 37. Figure Figu

Figure 38.

Figure 39.

Figure 40. 40 .

 

3 

G uide uide:: Rode Roden n t s and   the R a b bit

Figure Figu re 4 1

Fi g ur e   42

F ig ure 43

F igure igure   44  

 

33

Figure 4 45 5.

Figure 47.

F igure 46.

Figure 48.

 

Necropsy Guide: Rodents and the Rabbit

34

f.

g.

h.

Step 10. a.

b.

c.

Sever the mesentery which binds the coils of the intestine (Figure 50). The mesenteric lymph nodes should remain imbedded in the mesentery close to the ileocecal junction (Figure 51). Once the mesenteric attachments are severed the entire gut may be laid out "serpentine-fashion" (Figure 52). Collect the spleen and a portion of the diffuse, pink pancreas by cutting between the spleen and stomach (Figure 53). The stomach may be prepared for fixation by either an open or closed method, depending upon the experimental protocol. i. Closed fixation traps the stomach contents. Tighten a ligature around the duodenum close to the stomach and then tie a loose ligature around the esophagus. Inject approximately 5.0 cc of fixative into the stump of the esophagus using a balltip feeding needle or other blunt tipped needle and then tighten the ligature (Figure 54). Free the stomach by cutting the duodenum distal to the ligature. ii. The open method of fixation is used if the lining of the stomach needs to be examined. Cut the stomach open from the pylorus to the nonglandular region (Figure 55). Flush out the stomach contents before fixing the organ. The intestinal tract also may be collected by an open or closed method. i. Closed intestinal fixation is the process of inserting a blunt needle into the duodenum and injecting sufficient formalin to fix the entire length of the gut. The intestinal contents may be expelled into a beaker. Sections of the intestinal tract may be collected by placing a ligature at each end of the desired section and cutting behind each ligature. I f necessar necessary, y, each segment can be further injected with fixative using a fine gauge hypodermic needle, but the intestine should not be overdistended. I t is desirable to include a section of small intestine containing aggregates of lymphoid tissue called Peyer's patches (Figure 56). Note: additional pancreatic tissue may be collected with the proximal part of the duodenum. n. Open intestinal fixation is the process of inserting one blade of a small blunt scissors into the duodenal opening and then cutting open the entire length of intestinal tract. Desired sections of the gut may be excised, and placed onto a piece of cardboard mucosal side up, then placed into fixative. Collection of the liver, kidneys, and adrenal glands. The rat liver can be excised intact by grasping the tough connective tissue under the median lobe with tissue forceps (Figure 57) and while exerting tension posteriorly, severing the esophagus and blood vessels passing through the diaphragm (Figure 58). Cut any remaining connections to free the liver. Fix the liver intact or sectioned as specified. Note: the rat does not possess a gallbladder. Once the liver is removed, the kidneys and adrenal glands are easily examined (Figure 59). The ureters may be difficult to identify unless they are distended. The right adrenal gland is located slightly anterior to the left adrenal gland and lies closer to the vena cava. Adrenal glands of female rats are larger than those of male rats. I f the adrenals are to be collected and/or weighed apart from the kidneys, grasp the adrenal artery and vein close to each gland with fine forceps, undercut the fat medial to the forceps (Figure 59), and lift out the gland. Trim the fat away from each gland on a moistened paper towel. I f he adrenals are to remain with the kidneys, grasp the left renal artery and vein with theto forceps close the60). kidney and sever tension both vessels a fine medial the forceps (Fitogure (Figure Exert upward on the with kidney with scissors forcep forcepss and bluntly dissect the kidney away from its bed o f perirenal fat. Continue dissecting anteriorly to include the adrenal gland with its surrounding fat. Free

 

35

Figure 49.

Figure 50.

Figure 51.

re 52. Figure Figu

 

36

bbit   Necropsy   G uide: Roden ts a nd the Ra bbit

Figure 53,

F igure 55,

Figure 54,

Figure 56,

 

37

re 57. Figure Figu

Figure 58.

Fi g ure 59.

Figure 60.

finee scissors scissors.. G rasp the k id n ey with aa   ad renal with with   fin an d attached attached   adrenal the kidney kidney   and

d.

m oist oistened ened gauze gauz e sp onge and b is ect it long longitud itudinally inally with  with   a sharp scalpel. scalp el. Th e n ey   (Figure 61). 61).   th the e right kid kidn ft k idney from le dinall cut distin disti n g u ishes the left longitu dina e. B isect the rig r ig h t kidney kidney ey as abov above. glan d  and right kidn C o llect the right r ight   adrenal glan re 6 1). (Figure id n ey in  in  slide slide   se ctions (Figu kid distinguish inguish it from   the left k ansversely ersely to dist tr ansv

 

bit Rabbit Rodentt s and the Rab uide:   Roden N e c ropsy G uide:

38 

re 61. 61 . Figure Figu

11.  S te p   11.  a.

ra l   juncti juncti o n). oc hond hondra (costoc rib (cost Co l le l e c tion of  the s te rnum   and rib o id   xipho rib b s an d xiph fro o m th e last ri wall   away fr te d flap fla p of ab d o m in al wall eflecte C u t th e r eflec er n u m .   ster pr o ce ss of the st

b.

cu t throu thr ou gh m ,   an d  cut ternum the   sternu ag m u n d e r the  iaphrag sl it the  the   diaphr ro cess, cess,   slit p h oid p pro xip G rasp rasp   the xi re   Figure sides   close to   the st e rnum   (Figu ribs  on bo th sides io n   of th e   ribs  portio ginouss port cartil a ginou t h e   cartil to   cut c ar eful to firstt  rib be ing car ough   the firs n   anteri anteri o rly th r ough ncision 62 ) . E xten d each   incisio en sion an d w ard tten upw ). M ai n ta in up gu re 63 63). in s (Fi (Figu vein n e n t jug u l ar ve m edia ediall to   th e  p ro m i ne c o n tinue tinue   cuttin utting g the m u scles scles   on eithe eith e r   side  side  o f th e   trache racheaa up to  to   the low lo w er ja w e t he st e rn u m ste   re rem m o v a w  a n d lo wer j   to  to   the ched  a tt a ched 64) . S e v e r th e m u sc les att (Fig u r e 64). a nd   o p en e d trache acheaa ( Figu re 64) and h o f the  the   tr length fu ll lengt cles.   The full w ith atta at ta ched n e c k m u s cles.

c.

t. point. ed   at this this poin reveal vealed be   re F i gure 65 ) sh ou ld be  th o ra cic ccaa v ity ((F reveall the to   revea thee stern ste rn u m to isect   th then   bisect th e   stern stern u m an d then from om the issue   fr xcess   tissue Trim   excess alcifiee d   u m m u s t  b e dec alcifi  sternu T he  stern igure   66). The re d patch pat ch e s o f b one m a rrow (F igure  colore r ed  colo on. fixatio afterr fixati afte

d.

rib b   ca ge. S o m e ribs m a y n g   the ri eadin m a nu ally s pr eadi st a l m u sc les o n tercost cuttin g   the in terco by   cuttin o ne   rib by pro c e ss. S el ect one re d in th e proc racture b e f ractu ap p r oxim at e ly 0 .5 e m a b ove nail il cutt cu tt er app a   na eitherr side an d th en cut th e   rib w ith a  eithe in o u s a nd   tilagin c a r tilag thee ju n c tion of the ca rking   th m ent m a rking largem o w   th e  white en large elo a n d   b el E nlarg e the c h e s t o p e ning

thee ster6 ) . As w ith th 66 ti o n) (F ig ure 6 al  junc  juncti ondral rib   (costo ch ondr b o n y segm en ts o f t he rib fix at ion. lc i fied af t er fixat decalc b e deca n u m , th e rib m ust be S tep 12 12..

E x a m i n ati a ti o n o f th e oral ora l cavit cavity y. on   both thee base bas e of th e e ar on  th e m ou th to th o n   from  from   the c o m e r of the ncisio ski n i ncisi M a k e a skin rig h t and le f t ve ring th e righ seve dible)) by se lo wer ja w   (m an dible ra t. L o o s en   th e  lower si d es of t he rat. h neath t h e   j a w   b eneat an d th en   by cu tt ing th alpel   and re   67) w i th a sc alpel (Figure uscless (Figu seter   m uscle m a s seter

 

39

Figure 62.

Figure 63.

Figure 64.

Figure 65.

 

40

Necropsy Guide: Guide: Rodents and the Rabbit

Figure 66.

Step 13. a.

b.

c.

d.

with a bone cutter (Figure 68). Pull the jaw downward and examine the surface of the tongue, the teeth and the rest of the oral cavity (Figure 69). Collection Collecti on of the tongue, thyroid glands, parathyroid glands, larynx, esophagus, trachea, thymus, heart, lungs, and mediastinal and bronchial lymph nodes. Cut the remainder of the muscles that hold the jaw in place until the mandible is freed from the body. Grasp the front of the mandible and pull downward while severing the soft palate and any remaining pharyngeal attachments behind the esophagus (Figure 70). Continue dissecting the trachea and esophagus together away from the body; at the level of the first ribs carefully cut the attachments to the dorsal chest wall (Figure 71). Continue cutting all remaining attachments of the lungs down to the diaphragm until the trachea, heart, lungs, and accompanying structures can be freed (Figure 72). Place the tissues on a moistened paper towel. Just below the larynx, identify the reddish-brown thyroid glands (Figure 72). The isthmus of the thyroid may be seen crossing the trachea ventrally. The parathyroid glands are small, spherical white masses imbedded in the anterolateral poles of each thyroid gland. These are better visualized with a magnifying lens or a dissecting microscope. Examine Exami ne the mediastinal mediastina l lymph nodes located ventral to the trachea at the level of the thymus (Figure 73). The bronchial lymph nodes are situated at the bifurcation of the trachea. These nodes are quite small and sometimes difficult to identify. Insert Inser t one blade of a fine scissors into the pharyngeal opening of the esophagus. Cut open and examine the entire length of the esophagus (Figure 74).

 

4 1  

 

42

Necropsy Guide: Rodents and the Rabbit

Figure 71.

Figure 70.

Figure 72.

Figure 73.

 

43

e.

f.

g. h. 1.

Step 14. a. b. c.

d.

Place a loose ligature around the trachea 2 to 3 mm below the thyroid glands. Fill a 10-cc syringe with fixative and attach a 20-gauge needle. Insert the needle into the opening of the trachea (glottis) and snug the ligature around the needle. Slowly inject sufficient fixative to enlarge all lobes of the lungs to a point corresponding to full inflation (Figure 75). (The margins of the lobes should remain sharp.) Do not overdistend the lungs. Note that the left lung consists of a single lobe as in the mouse and hamster. Withdraw the needle and tighten the ligature. Sever the trachea and esophagus transversely below the level of the thyroid glands (Figure 76) and fix the mandible with attached tongue, larynx, and thyroid/ parathyroid glands. Cut the heart and thymus away from the the lungs lungs and place the the lungs lungs into fixative. Dissect Diss ect the thymus (Figure 77) from the base of the heart and fix. Section the heart from the base to the apex incorporating the aorta (Figure 78). Collection Collect ion of the brain, pituitary gland, and turbinate bones. Tu Tum m the rat over onto its ventral side side and and saturate the fur on the head with alcohol. Make an incision from between the eyes to behind the head and reflect the skin away from the cranium (Figure 79). Bend the the rat' ra t'ss head head down and cut through the dorsal muscles of the neck until the spinal cord can be seen entering the skull (Figure 80). Reflect the temporal muscles on either side of the cranium (Figure 81). Insert one blade of a small bone cutter into the opening of the skull (foramen magnum) and place the other blade just above the opening of the ear canal (Figure

e. f.

g.

h.

1.

Step 15.

82). Cut the bone between the blades being careful not to penetrate the brain. Repeat on the other side of the skull. Extend Exten d the bone incisions anteriorly to the level of the nose (Figure 83), cutting superficially just above the zygomatic arch (Figure 81). Lift the posterior cranium upward away from the head exposing the cerebellum and most of the cerebral hemispheres (Figure 84). I f present, remove the tough membrane (dura mater) that surrounds the brain (Figure 84). Remnants of the dura usually need to be removed from the junction of the cerebral hemispheres and cerebellum. Carefully dissect away the frontal bones, exposing the remainder of the brain, including the olfactory lobes. (Figures 85 and 86). Sever the olfactory olfacto ry lobes (Figure 86) and then tilt the head upward and backward. Sever the optic nerves (Figure 87). At this point the brain should begin to fall backward exposing the remaining cranial nerve attachments. Carefully cut any remaining attachments (Figure 88) and then sever the brain stem (medulla oblongata) posterior to the cerebellum, freeing the brain (Figures 89 and 90). Handle the brain carefully and place it into fixative. fixative. Examine the pituitary pituitar y gland (Figure 91) which is firmly seated in the hypophyseal fossa and covered by a delicate layer of dura mater. Allow the pituitary gland to be fixed with the base of the skull so that the gland is not lost. I f the pituitary gland needs to be removed for weighing etc. it can be removed with small, sharp scissors or scooped out with a small flat spatula (Figure 91) after severing the dura on either side of the gland. Remove the skin over the nose and upper jaw and place the entire skull in fixative. The nasal passages (turbinate bones) and middle ears are normally sectioned after the skull is decalcified. Collection Collec tion of the sciatic nerve and adjacent muscle. The sciatic nerve is located somewhat parallel and posterior to the femur in the thigh. Make a skin incision parallel to the femur along the medial side of the

 

N

G

 

45

Figure 79.

Figure 80.

Figure 81.

Figure 82.

 

46

N ecropsy G uide uide:: Rodents Rodents   a n d  the Rabb Rabbit it

Figure 83.

Fi g u re 85.

Figurr e 84. Figu

Figure 86.

 

47

Fi gure 87.

re 89. Figure Figu

Figure 88.

Figure 90.

 

48

Necropsy Guide: Rodents and the Rabbit

Figure 91.

Step 16.

Step 17.

thigh. Separate the skin and bluntly dissect through the muscle on the posterior one third of the leg (Figure 92). Careful deep dissection will expose the white fibers o f the nerve running down the leg (Figure 93). Cut out approximately 1 em of nerve and include adjacent muscle. Careful dissection and handliing of the nerve is very important. Collection of the proximal femur with bone marrow. Bone marrow may be collected from the femur as well as from the sternum. Dissect the muscles away from the femur as far up as the hip joint. Cut through the hip joi joint nt with bone cutters and free free the proximal portion of the the femur. Cut the distal femur approximately half way down the thigh (Figure 94). Remove and fix the bone. Collection of the vertebral column and spinal cord. I t is tedious and too time consuming to dissect out the entire spinal cord from the bony vertebral column for routine necropsies. The simplest and most acceptable method of collection is to cut away as much muscle as possible from around the vertebral column and then to cut out segments of the spinal cord (cervical, thoracic, lumbar) by severing the vertebral column at intervals using bone forceps. The crushing action of the forceps will distort the spinal cord, therefore it is important for the tissue trimmer to cut out only the central portion of each segment after the bone has been decalcified.

 

4

 

50

N e c ro r o p s y Guide: R o d ents and the th e R abbit

SUGGESTED READING 1. B i v e n ,   S . W., Crawf Cra wf o rd, P. M., and Br e wer, N. M., Morphology, in T he Laboratory R at, Vol. I,

B ak er , H. J., Lindsey Lindsey,, J. R., and Weisb Wei sb roth, S. H., E d s .,   Academic Pres Press, s,   New York, 19 1979 79 , chap. 4. 2. C hi asson, R. B., L a boratory Anato m y of the White R at , 4th ed., Wm. Wm.   C. Brown Co., D u buque, Iowa, 1980. 3 . Gr eene, E. C. ,  A n a t o m y o f th thee R at., Hafner Pr Pres es s , New York, re repr pr inted 1970. 4 . H a f e z , E. S. E. , Reproduction   and Breeding Te Tech ch niques for Labo Labora ra tory Animals, L e a &   Febiger, Ph Phil ilaadelphia, 19 70. 5 . H e b e l , R. and St romber romberg, g, H . W., A n a to t o m y o f he Labora Laborato to ry Rat, William s &   Wilkins, Balt Baltim im ore, 1976. 6 . O l d s ,   R . J . and Olds, J .   R ., Color Atlas Atla s o f he Rat, John   W iley & Sons, Ne w York, 1979. 7. Wa ynforth ynforth,, H. B., Experimental a n d   Surgical Techn Techniq iq ue in the Rat, A c ademic Press, N e w York, 1980.

 

52

thee  R a bbi t Guide : R o de nt s a nd   th N ecro psy Guide:

Figure 95.

Figuree 96. Figur

Figure 97. 97.  

Fi Fig g ure 98.

 

53

Step 7. a.

b.

c.

d.

e.

without destroying the architecture of the surrounding normal structures. Allow tissue to fix at least 48 hr. The bone must be decalcified before the specimen is sectioned. I f there is some question as to the removal of a mass, consult the pathologist. Collection Collec tion of cervical lymph nodes, submaxillary and sublingual salivary glands, mammary glands, and preputial (or clitoral) glands. Lay the mouse on its back on a dissecting board and pin down the legs. Wetting the surface of the body with alcohol helps prevent loose hair from falling into the incisions. Stretch Stret ch the skin in opposite directions with the thumb and forefinger and make a midline skin incision extending from the lower jaw to the vulva, or scrotum, respectively. Stretching the skin helps to prevent accidentally cutting into deeper structures. While exerting exerti ng gentle outward traction on the the skin with forceps, carefully incise the loose subcutaneous tissue (Figure 99) and retract the skin on either side of the incision (Figure 100). Grasp the loose connective connecti ve tissue just posterior to one of the paired submaxillary glands and bluntly dissect di ssect out the above gland together with tthe he sublingual salivary gland and cervical lymph nodes (Figure 101). Place the tissues in fixative. In female animals locate a nipple on the reflected abdominal skin and identify the whitish-pink whitish -pink mammary tissue beneath the skin (Figure 102). (Mammary glands are most obvious in lactating mice and may extend from the inguinal area to the neck [Figure 103].) Scrape the hair away from the nipple and cut a dime-sized circular area out of the skin including the nipple and underlying tissue.

f.

Step 8.

The clitoral glands are small, paired, flat, yellow masses of tissue located just anterior to the vulva (Figure 104). Both glands can be collected together by grasping the surrounding tissue with fine forceps and freeing the glands by cutting the underlying connective tissue with fine scissors. Corresponding structures in the male mouse are the preputial glands. These relatively large, flat, pale yellow glands are located on either side of the preputial opening (Figure 105). Collection Collec tion of the male or female reproductive tracts and urinary bladder.

Male

a. b.

c.

d. e. f.

g.

Grasp the most posterior part of the abdominal wall and cut into the abdominal cavity just above the base o f the penis (Figure 106). 106). Cut the body wall on both sides close to the reflected skin, being careful not to cut into any organs. Extend the incisions anteriorly to the rib cage and then reflect the cut portion of the abdominal wall over the chest (Figure 107). Pull both testicles out of the scrotum and sever the fibrous connection (gubernaculum testis) between the tail of the epididymis and the scrotum (Figure 108). Sever the vas deferens (Figure 109). 109). Place the testicle with attached epididymis (Figure 110) into fixative. Repeat the above procedure to collect collec t the second testicle. Reflect the intestines anteriorly to reveal the urinary bladder, prostate gland, seminal vesicles, and coagulating glands. Insert one blade of a small scissors under the center of the pubis just above the base of the penis and cut through the cartilaginous connection (pubic symphysis) (Figure 111). Separating the bones will reveal the underlying urethra and rectum (Figure 112). Dissect Dissec t the skin away from the penis. Grasp the end of the penis with fine forceps and while pulling gently upward carefully dissect the connective tissue around

 

54

Necr opsy op sy Guide: Ro Rod d e n ts and the R q b bit

Figure 99.

Figure 1 00.

Figuree Figur

Ol.

Figure 1 02.

 

 

 

56

Rab b it Rode n ts   and the Rab G uide: Roden

F igure 106.

Figure 107. 107.  

Fi g ure 108.

Figure

09.

 

57

Fi gu r e

10

F igu re 11 112. 2.

F igur iguree I l l  

Fi g ure   11 113. 3.

 

58

Necropsy Guide: Rodents Roden ts and the Rabbit

Figure 114.

h.

the urethra and beneath the prostate gland. Place the excised male reproductive tract with attached urinary bladder (Figure 113) onto a moistened paper towel. Place a loose ligature around the neck of the urinary bladder. Express most of the urine out of the bladder if it is distended and then tighten the ligature. Replace about half the volume by injecting formalin into the lumen using a small gauge needle (Figure 114). Caution must be taken not to inject fixative into the wall of the bladder. bladder . Fix the reproductive reproduct ive organs and urinary bladder. Because of the small size of the urinary bladder in mice, slide sections may reveal a track caused by the path of the needle. Note: as an alternative, the urinary bladder may be injected in situ before the reproductive tract is dissected from the body.

Female a.

b.

c.

d.

Grasp the most posterior poster ior part of the abdominal wall and cut into the abdominal cavity just above the vulva (Figure 115). Cut the body wall on both sides close to the reflected skin, being careful not to cut into any organs. Extend the incisions anteriorly to the rib cage and then reflect the cut portion of the abdominal wall over the chest. Insert one blade of a small scissors under the center of the pubis and cut the cartilaginous connection (pubic symphysis) (Figure 116). Separating the bones will reveal the underlying vagina and rectum. Cut the skin between the vulva and anus (Figure 117) and dissect free the posterior end of the vagina, being careful not to cut into the rectum. Grasp the end of the vagina with fine forceps and while gently pulling upward sever the film-like connective tissue between the vagina and the rectum (Figure 118). Do not cut away the urinary bladder. Continue dissecting anteriorly to the area of the cervix and then cut along the mesentery supporting the uterine horns up to the ovaries.

 

59

Figure 115.

Figure ll6.

Figure ll7.

Figure liS.

 

an d the Rabbit Gui d e:   Rodents an N e cropsy Guid

60

F ig ure 119.

Figure 1 20.

tran sf e r the entire kid n e ys and transf o v a ries and kidn tissue   b etween the ov Cut the su p p o rting tissue  salin e. Trim moiste n e d with saline. pape r to wel moisten o nto a paper tra c t (Figure I re p ro ductive trac ovar ie s . h or ns and ovarie ex c e ss ive fat from   the uterine hor e.

urina ry bladder. E x p r ess most o f ne ck of the urinary Place a lo o se ligature ar o und the neck ligature . R eplace tight en the ligature. distended  a nd then tighten if  it is distended a t he  urine out o f th e bladder if  gau g e us in g   a small gau into   th thee lumen usin injecting   formalin into  about h a lf the volum e by injecting  needle (Fig (F ig ure 120). C au tion must be  be   ta taken ken not to i n je ject ct fixative int i nt o   the wall o f k " caused "track ctions may rev r ev eal a "trac slide   se sections male   mouse, slide  t he   bladder. A s w ith the male  Notee : urin ar y   bladder. Not o rg a ns and urinar ne ed le. Fix the re p roductive org by the  the   p ath of the need befo re the reproinjected in situ before ay   be injected uri na r y bladder m ay altern a t ive, the urina as an alterna

Step 9. a.  a. 

b.

thee   body. d u ctiv e tract is d is s ected from th cec u m , duo d e num, jejunu m , ileum, cecu pancreas , st omach, duod sp le e n, pancreas, Collecc ti o n of the sple Colle n od es. mese n te ric lymph nod colon, rectu rect u m , and mesen forc ep s, pull omach with  with   serrated forcep stomach curv ature of th e   st G e ntly grasp th e   greater curv omach and  and   th thee the   st stomach membran e b etween the  fi lm y membrane carefull y s ever the film down , a nd carefully caudate lobe lob e of the liver (F igure 121). 122 ). T his stomach ( Figure 122). abov e   the stomach approximately   0.5 em abov esophag u s   approximately S e v e r the esophagu

c.

attachm en ts. fro m   its attachmen will notice noti ce a bly loosen th e   stomach fro while   maintaining stom ac h and while  cl o se   to the stomac Grasp the eso es o p hagus with fin e forceps clo the liver tiss ue   between the c o n nective tissue an d remaining co th e bile duct and te n si o n, sever the

d.

(Figu re   123). and the st o mach (Figure dorsaa l intestines   and the dors betwe en   the intestines and s ever the m es e ntery between Maintaa in   tension and Maint ssels in this  this   area. This blood   ve vessels th e large blood  b ut do not cut the body wall (F ( F i gure 124) but

 

62

the   R a bbit n ts an d the e: R ode nts Guide: psy Guid ecropsy N ecro

21 121 Fi g ure 1

2 122 Fi Fig g ure 12

re 123 123   Figure Figu

re 124 124   Figure Figu

 

6 

F ig u r e   125 

F ig u re   1 2 6 

F ig u r e   1 27  

F ig u r e   12 8  

 

Necropsy Guide: Rodents and the Rabbit

64

Figure 129.

Figure 130.

vein with the forceps close to the kidney and sever both vessels with a fine scissors medial to the forceps (Figure 135). Exert upward tension on the kidney with forceps and bluntly dissect the kidney away from its bed of perirenal fat. Continue dissecting anteriorly to include the adrenal gland with its surrounding fat fat.. Free the kidney and attached adrenals with fine fine scissors. Grasp the kidney with a moistened gauze sponge and bisect it longitudinally with a sharp scalpel. The longitudinal cut distinguishes the left kidney from the right kidney (Figure d. Step 11. a. b.

c.

136). Collect the right adrenal gland and right kidney as above. Bisect the right kidney transversely to distinguish it from the left kidney (Figure 136). Collection Collecti on of the sternum and ribs (costochondral junction). Cut the reflected fla flap p o f abdominal wall away from the last ribs and xiphoid process o f the sternum. Grasp the the xiphoid process, slit the diaphram under the sternum, and cut through the cartilaginous portion o f the ribs on both sides close to the sternum (Figure 137). Extend each incision anteriorly through the first rib being careful to cut medial to the prominent jugular veins. Maintain upward tension and continue cutting the muscles on either side of the trachea up to the lower lowe r ja jaw w (Figure 138). Sever the muscles attached to the lower jaw and remove the sternum with attached neck muscles. The full length of the trachea and opened thoracic cavity should be revealed this point. Trim excessattissue from the the sternum and then bisect the sternum to reveal the red-colored patches of bone marrow (Figure 139). The sternum must be decalcified after fixation.

 

65

Fig ure

32.. 32

Figure Figu re 131.

33. Figure 1 133.

13 4. F igure 134.

 

Necropsy Guide: Rodents and the Rabbit

66

Figure 135.

d.

Step 12.

Step 13. a.

b.

Figure 136.

Enlarge Enlarg e the the chest ches t opening by manually spreading the rib cage. Some ribs ribs may be fractured in the process. Cut out several ribs together including the white enlargements marking the junction of the cartilaginous and bony segments of the ribs (costochondral junction) (Figure 139). As with the sternum the ribs must be decalcified after fixation. Examination Exami nation of the oral cavity. Insert a small scissors into the comer of the mouth and cut through the skin, masseter muscle, and lower jaw (mandible) (Figure 140). Repeat on the opposite side. Pull the jaw downward and examine the surface of the tongue, the teeth, and the rest of the oral cavity (Figure 141). Collection Collecti on o f the tongue, thyroid glands, parathyroid glands, larynx, esophagus, trachea, heart, thymus, lungs, mediastinal and bronchial lymph nodes. Free all attachments of the mandible to the body but do not cut away the tongue at this point. Grasp the mandible with small forceps and pull downward while severing the soft palate and any remaining pharyngeal attachments behind the esophagus with sharp scissors (Figure 142). Continue dissecting the trachea and esophagus together away from the body and at the level of the first ribs carefully cut the attachments to the dorsal chest wall (Figure 143). Continue cutting the remaining attachments of the lungs as far as the diaphragm until the trachea, heart, lungs, and accompanying structures are freed (Figure 144). Place the tissues on a moistened paper towel. Just below the larynx, identify the reddish-brown thyroid glands (Figure 144). The isthmus of the thyroid gland is difficult to visualize in the mouse. The parathyroid glands are spherical white masses imbedded in the anterolateral poles of each thyroid gland. The parathyroid glands are very small in the mouse and are better visualized with a magnifying lens or a dissecting microscope.

 

 

6 7  

 

68

Necropsy Guide: Rodents and the the Rabbit

Figure 140.

Figure 141.

c.

d. e.

f.

g. h. i. Step 14. a.

b.

Attempt to identify the mediastinal and bronchial lymph nodes. These nodes are particularly small in the mouse and difficult to locate unless they are enlarged. The mediastinal lymph nodes are located ventral to the trachea at the level o f the thymus. The bronchial lymph nodes are situated at the bifurcation of the trachea. Insert one blade of a fine scissors into the pharyngeal opening of the esophagus. Cut open and examine the entire length of the esophagus (Figure 145). Place a loose ligature around the trachea 1 to 2 mm below the thyroid glands. Fill a 1.0-cc syringe with fixative and attach a 25-gauge needle. Insert the needle into the opening o f the trachea (glottis) and snug the ligature around the needle. Slowly inject sufficient fixative to enlarge all lobes o f the lungs to a point corresponding to full inflation (Figure 146). (The margins of the lobes should remain sharp.) Do not overdistend the lungs. Note that the left lung consists o f a single lobe as in the rat and hamster. Withdraw the needle and tighten the ligature. Cut the trachea and esophagus transversely below the level o f the thyroids (Figure 147) and fix the mandible with attached tongue, larynx, and thyroid/parathyroid glands. Cut the heart and thymus away from the lungs and place the lungs into fixative. Dissect the thymus from the base of the heart (Figure 148) and fix. Section the heart from the base to the apex incorporating the aorta (149). Collection o f the brain, pituitary gland, and turbinate bones. Turn the mouse over onto its ventral side and saturate the fur on the head with alcohol. Make an incision from between the eyes to behind the head and reflect the skin away from the cranium (Figure 150).

 

69 69  

Figure   142 Figure

43 143 Fi g ure 1

F igure 144

145   Figuree 145 Figur

 

70

Necrop Necro p sy   Guide: Ro Rod d en ts and the R a b bit

Figure   146. Figure

Fiigure 147. F

Figure 148.

c. d.

F i gure 149.

Ben d th e m ous e' s  Bend s   he ad down an a n d c ut through throug h th e dorsal m u sc les of the n ec k u n til the spinal spi nal cord c ord can be b e se en entering enterin g the  the   skull (Figure (Figure   151). Insert one blade of a s mall mall   pointed scisso scissors rs into the  the   o p ening o f tth h e sk ull (foram e n m ag num) and cut the bone jus j us t above a bove the open opening ing o f tthe he   e a r  canal (Figu (Figu re   152)

e.

being carefu ca refull not to pe netr netrate ate the brain. br ain. Repeat on  on   th thee  other side  side  of th thee skull. Ext end the bone incis Extend incisions ions anteriorly anter iorly   to the level lev el of the nose  nose   (F igure 153). 153) . C u t

f.

ac ross the fronta fr ontall bones coveri co vering ng the olfacto olf actory ry lobes (Figu ( Figure re 154). Lift the  the   p o sterior craniu cran iu m   upward aw aw ay   from the head head,, exposing  exposing   th thee  cerebellum cerebellum and cere cerebral bral hemisphe hemi spheres res (Figur (Figuree 15 4). I f p re sent sent,, remove the t he t ough mem bran e th at surround surroun d s the brain (dur (duraa mater). R em nants of the dura may  may   n ee eed d to be r em ove ov e d f rom the juncti ju nctio o n o f the cereb c erebral ral hemispher hemis pheres es and cerebel cer ebellum. lum.

 

71

Figure 150.

Fi gure 152.

 

12 12  

cropsy sy Guide: Guide:   R o dents and the R abbit V ecrop

Fi g ure 151.

Fi gure 153.

Figuree 154. Figur

Figure 155 155..

Figure 156.

Figuree 157. Figur

 

73

g.

tilt  the head ssorss and then   tilt  scissor 155)   w ith fine sci olfactory ctory lobe lobess (F igure 155) Sever the olfa u p w ard and bac b ac k ward. Sev e r the optic ner n erv v es (Figure 15 6 ). At this  this   p oint the al nerve attach at tach-c ranial th e re maining crani backwa r d e xposing the brain   sh ould begin to fall backwar then   sever the (Fig ure 57) and then att ac h ments (Figure any   re m aining attac ments. Care Car e fu lly cut any  posteriorr to the cer eb e llum tra nsversely at a  level posterio ulla oblongata  oblongata)) transversely b r ain stem (med ulla place  it caref ully   and place  (Figu re s 158 and 15 9 ). Handle the brain carefully freeing th e   brain (Figu in to fixative. fixative.  

h.

ull so skull ixed   with the b as e of the sk re 1 60) to be ffixed y g land (Figu (Figure pituitary Allo w t he pituitar gland d is not lost. lo st. that the glan

i.

thee en tire skull in fi x ative. se a nd upper ja w a nd place th in o ver the no nose R e m ove the sk skin afterr ally   sectioned afte arss are norm ally a nd middle e middle  e ar (turbinate binate bones) bon es) and Th e nas n as a l passages (tur

15.. Step   15

ecalcified.. the skull  skull   is decalcified mus cle. and  ad jacent muscle. sci atic   nerve and  Collec tion   o f the sciatic Collection sterior ior to the fe m ur in the ar a llel and po poster d some w ha t p par nervee is locate located T he sciatic nerv thig h . Make a skin sk in incision parall pa rallel el to the ffem em u r along th thee m edial side  side   of the muscle p osterior to the ly d issect throu thro u g h   the muscle bluntly s ki n   and blunt thigh. S ep arate the ski fibers rs of  the expose the white fibe d eep dissection disse ction   will expose Care ful deep fe mur (Figure (Figu re 1 61). Careful tely   0 . 5 e m of n e rve  approximately u re   162). Cut Cut   ou t approxima w n the leg (Fig (Figu dow n er v e r unning do and   h andling of the n erve is ve r y Careful   di ssection and  and includ inclu d e adjacent m us c le. Careful  im p ortant.

Step   16. Step

roximal ximal fem u r w ith bone m ar row. ion o f th e   p ro Collect Co llection fro o m the sternum . m the femur as w ell as fr collected llected fro from arrow w may be be   co Bo n e m arro th rough thee h ip joint. C ut through uscles es away from fr om the femur as f ar up as th D is sect the m uscl istall portion ion o f the fe m ur (Figure 16 3 ). Cut the d ista th e proximal port ip jo int freeing freeing   the the h hip

em o ve the bon e fo r fixation thee t high and rrem roximately mately hal halff w ay down th femur app approxi tion.. decalcification a nd decalcifica 17.. S te p 17

rd. an d spinal co cord. rtebral ral column column   and tion   of the ve verteb Collection Collec ntiree spinal cord co rd from the an d time con conss u m ing to d is s ec t out the e ntir I t   is tedious tedious   and ble acceptable simplest   an d  most accepta necropsies.. T h e simplest  rout ine necropsies b o n y v ertebral co lu m n for routine thee aroun d th usclee as possible possib le f rom around ay as much m uscl n is  is   to cut aw away collection m e t ho h o d o f collectio thee   spinal cord cord (cervical, column mn and th en to cut ou t se gments of th vertebral colu eps. als using u sing bone forc bone  forceps. lu m n at interv intervals he v ertebral co colu oracic, ic, lumbar lumbar)) by  severing tthe th orac it is will ll distort the t he spinal cord , th erefore, it forceps   wi ng action of t he forceps  Th e c rushi r ushing onl y the centr centraa l p ortion of ea ch segment m e r to cut ou outt only the  tissue trim trimm im portan t ffo o r the tissue d. decal cified h as b een decalcifie a f ter the bone has

 

 

7 5  

 

76

Necropsy Guide: Rodents and the Rabbit

SUGGESTED READING I. Cook, M., The Anatomy o f the Laboratory Mouse, Academic Press, New York, 1965. 2. Cook, M. J,, Anatomy, in The Mouse in Biomedical Research, Foster, H. L., Small, J. D., and Fox, J. G., Eds., Academic Press, New York, 1983, chap. 7. 3. Green, E. L., Biology o f the Laboratory Mouse, McGraw-Hill, New York, 1966. 4. Hafez, E. S. E., Reproduction and Breeding Techniques in Laboratory Animals, Lea & Febiger, Philadelphia, 1970.

 

77 Chapter 4

NECROPSY OF THE HAMSTER (Mesocricetus auratus) Step I. Step 2. Step 3.

Step 4. a. b.

Note: Step 5.

Check animal's identification number. Record animal's weight. Verify animal's sex according to characteristics below. Male (Figure 164)

Female (Figure 165)

Long distance between anus and genital opening Nipples inconspicuous Pronounced scrotum

Short distance between anus and genital opening Conspicuous nipples, especially in lactating hamsters (Figure 171)

External examination. Observe entire body surface and orifices for abnormalities. Feel (palpate) the entire body for superficial swellings beginning from the head and proceeding posteriorly to the neck, chest, abdomen, and appendages. Palpate for enlarged organs or masses within the abdomen. All tissues and organs should be examined in situ before being dissected from the body. Collection of the eye, Harderian gland, intraorbital lacrimal gland, exorbital lacrimal gland, and parotid salivary gland.

Autolytic changes occur early in the eye, therefore it should be collected first, along with neighboring glands on the side of the head. a.

b. c.

d. e.

Step 6. a.

b.

Lay the hamster on its side and make a skin incision from above and just in front of the eye to the convergence of the upper and lower jaws just posterior to the base of the ear. Reflect the skin ventrally. Press a small curved forceps forceps deeply into the orbit and elevate the globe. Cut the tough fibrous tissue tissue beneath the forceps with small scissors (Figure 166) 166) and remove the eye, along with the large, light-yellow Harderian gland which almost completely surrounds the globe within the bony orbit (Figure 167). In female hamsters the Harderian gland contains pigment granules, but these are absent in the male. The small reddish-colored intraorbital lacrimal gland lies in the lateral comer com er of the eye superficial to the Harderian gland and may adhere to the Harderian gland when the eye is excised. Identify the discrete, discret e, pinkish-colored pinkish-col ored exorbital lacrimal gland which is located just posterior and ventral to the eye (Figure 167). Remove and fix the more diffuse, pink parotid salivary gland which is located ventral to and just behind the base of the ear (Figure 167). The parotid is usually covered with superficial fat. Collection Collec tion o f superficial masses. Remove any abnormal enlargement by cutting completely around it, being careful carefu l to include overlying skin, i f possible, and at least some normal tissue surrounding the mass. Scrape the hair away from the skin because hair dulls the microtome knife which is used to section tissues in the preparation of slides. Divide large excised masses into 0.3- to 0.5-cm 0.5- cm slices slices with a sharp scalpel and

 

78

N ecro ecrop p s y G u id e: Rod Ro d ents an a n d the R abbit abbit  

Figure Figu re 1 64

Fi g ure 165

Fi g ure 166

F igur iguree 16 167 7

 

80

bb it ents   a n d  the Ra bbit uide:: R o d ents sy G uide cropsy N e crop

168   Figuree 168 Figur

Figure   169

igure   170 F igure

F igure 171

 

81

re 172 172 Figure Figu

Fi g ure 173

urethra   and erlying   urethra un d erlying reveal  the und es will  will   reveal bones rating  the bon Sep a rating ure 180 18 0 ). Sepa (Figure physii s) (Fig phys

181).  (Figure   181). rectum   (Figure rectum g.

orceps   peni s   with f orceps en d of th e penis Grasp   the en penis.   Grasp th e   penis. skin   away f rom the thee skin D i s sect th ar o und ti s sue aro connecc tive tis the   conne diss e ct the  carefulll y disse pward  careful g ently u pward p u lling gently and w hile pu oductiv ve rep r oducti exc i sed m a le repr gland.   Place t he exci p rostate   gland. beneatt h the prostate and   benea uret h ra and  th e ureth th e   large N ote the to wel. Note mois tened p aper to onto   a mois blad d er onto urinar y bladd tractt   with a ttache d   urinar trac ure (Figure gla n d (Fig pros t ate glan anterio r prost th e   anterio jectionss of the pr o jection la te ral pro les and  and   the late vesicles seminaa l vesic semin

h.

1 8 2). E xpress   most o f   bla dder. Express urin ary bladder. th e urinary ne ck of the around   the ne gature   around l i gature P lace a loose li igature.. The tight e n the l igature the n tighte blad d er i f it  is d is t ended a nd then o u t of th e bladd the u rine ou olume   by h a lf the v olume a b out ha Re p lace ab clou d y. Rep e specialll y cloud m a y be especial urine o f h am s ters ma Caut io n 183 ). Cautio (Figu re 183) need l e (Figure gau g e needl usi n g a sm all gaug lum en usin into  the lum  formaa lin into the njecting g  form i njectin uctive    reprod d uctive he  repro ladder..  F ix tth wall o f the b ladder fixativ e into t he wall inje c t fixativ not to injec ta k en not  m u s t be tak ma y   b e blad d er may urinar y bladd th e   urinar lternatt ive, the Note: as an  an   a lterna ladder..   Note: ur inary b ladder organss   and ur organ body.   th e   body. diss ected f rom the trac t   is diss oductiv v e tract rep r oducti in jected in situ b efore t he repr

F e m a le a. b

c.

d.

G r asp th thee   m os t  t  poster posterii or part  part  o f th e   abdom i nal wall wa ll and c ut into  into   the abdom abdom ina in a l 184 ). (Fig u re 184). vul v a (Figu cavitt y just a bove t he vulv cavi no t   to car e ful not be in g care s kin bein ref l ected skin c l ose to t he refl o n   both s ides cl th e   body wall on C ut the reflect   then  reflect a n d then  rib  cage an to  the rib  anter i orly to  ncision n s anteri s. Exte Ext e nd the i ncisio  organs. an y  organ c u t  into any (F igure 1 85). ov e r the c hest (Figure abdo m inal w all ove porti o n of th e abdom the cu t portio cu t   th e   pubis a nd cut center   of the the   center un d er the  g sciss scis s ors und ssecting blade  o f a d i ssectin on e   blade  In s ert one Sepa r ating 186 ) . Separ (Fig u re 186) physiss ) (Figu sy m physi tion (pu (p u bic sym connection cartilaginous  connec the   cartilaginous throu thro u gh the  g vagin vagi n a and r ectum .   underlying reveal   the underlyin wil l  reveal the bon bo n es will  posterr ior the  poste issect  free the and  dissect 18 7) and d (F i gure 18 he vulv vul v a and a nus (Fi tween t  the b e tween the   skin be C ut the  Grasp   the en d  o f th e   ctum.   Grasp r e ctum. in t o the re to  cut int carefull   not to  being  carefu vagina   being  en d   o f th e   vagina

 

82

Necropsy Guide: Rodents and the Rabbit

Figure 174.

Figure 175.

Figure 176.

Figure 177.

 

83

Figure 178.

Figure 179.

Figure 180.

Figure 181.

 

84

nd the Rabbit Rabb it Rodents  and Necropsy G u i de: Rodents a

re 182. Figure Figu

83. Figure 1 183.

tiss u e c o n nective tissu t he film-like co upw ard sever the ge ntly   pulling upw vagina an d while gently

thee   urinary Do   no t  cut away th (Fig ur e   188). Do  th e r ectum (Figur b etween the v a gina and the cu t the cerv ix   and then cu a re a o f the cervix ant er i orly to the are Continue   d issecting anter blad d e r . Continue  tissue   su p p o rting tissue  ovarie s . Cut the sup horns   up to up to the ovarie th e u terine horns  mesentery   supporting mesentery su pporting the between the  the   ovaries and kid k id n eys and tran tra n s fer the entire entir e r eproductive eproductive   tr act (Figure from the  the  u terine excessi v e fat from saline. T rim excessiv moisten ed  with saline. pape r t o wel moistened 189) 18 9) onto a paper

e.

horns aand nd ovaries. Exp re ss most of urinary   bladder. Expre neck   of the urinary  aro u nd the neck Place a loo lo o se   ligature aro tighten  th thee ligature. R e place a nd   then tighten  th e u rine out of t he  bladder if it is distended and ng a small g a uge sing into   the lumen u si inject in g   formalin into vol u m e by injectin ab o u t half the volu into o   the wall of   ken not to in je ct fixative int be   ta taken needle (Fig (F ig u re 190). C au t ion must be  bla d d e r. Note: as an altera nd urinary blad reproductiv ctivee organs and Fi x the reprodu t he bladder. Fix t ra c t b e i njected in s itu before the re p roductive tra b la dder may be native,, th e urinary bla native bod y . dissected   from from the body is dissected

St ep   9. a.

b.

c.

d.

stomach , d uodenum, jjee ju num, ileum , c ecum, pan c re as, stomach, thee spleen, panc C o llection of th ctum, and m e senteric lym ph   nodes. colon,   re colon, rectum, fo rc eps, pull with   serrated fo thee   stomach with greater   curvature cu rvature of th Gently grasp gra sp the greater  and   th thee left the s tomach and  membr an e   between the the   filmy filmy membran care fu l ly sever the  d o w n, and carefu (Fig u re   191). lobe of  th e liver (Figu (Figure   192). This st o m ach (Figure  e m above the sto appro x im ately 0.5 em eso p h agus approx S ever the esop hments. attachments. lo o se n the stom ac h   from its attac will   n oticeably loo will  maintain in g   omach and w h ile maintainin the  st stomach force ps   close to the   esophagus   w ith fine forceps Grasp th e  esophagus betwe en   the liver connective t issue between remain in g   connective duct and remainin tension, seve sev e r   the bile duct an d   the the stomach (F igure 193). d or sal intes tiness and the dor me sentery betw e e n the intestine a nd   sever the me Mainta Main ta in tension and th is area. This blood v essels in this thee   large blood igure 194) b u t do not cut th body wall  wall  (F (Figure th e a nus. ter m i nation at the tire gut excep exc ep t for its term the   en entire s pl een and the  sh o u l d free the spl moistened   with saline. to w e l moistened  gut on a paper tow and   lay the gut Cut arou aro u n d   the anus and

 

85

Figure 184.

Figure 185.

Figure 186.

Figure 187.

 

86

Necropsy Guide: Rodents and the Rabbit

Figure 188.

Figure 189.

Figure 190.

 

87

Figure 191.

Figure 192.

Figure 193.

Figuree 194. Figur

 

88

Necropsy Guide: Rodents and the Rabbit e.

f.

g.

h.

The intestinal tract is most efficiently examined by stretching it to its full length. Sever the mesentery which binds the coils of the intestine (Figure 195). The mesenteric lymph nodes should remain imbedded in the mesentery close to the ileocecal junction (Figure 196). Once the mesenteric attachments are severed the entire gut may be laid out "serpentine-fashion" (Figure 197). Collect the spleen and a portion of the diffuse, pink pancreas by cutting between the spleen and stomach (Figure 198). The stomach may be prepared for fixation by either an open or closed method, depending upon the experimental protocol. 1. Closed fixation traps the stomach contents. Tighten a ligature around the duodenum close to the stomach and then tie a loose ligature around the esophagus. Inject approximately 5.0 cc of fixative into the stump of the esophagus using a balltip feeding needle (Figure 199) or other blunt tipped needle and then tighten the ligature. Free the stomach by cutting the duodenum distal to the ligature. n. The open method of fixation is used if the lining of the stomach needs to be examined. Cut the stomach open from the pylorus through the extensive nonglandular region (Figure 200). Flush out the stomach contents before fixing the organ. The intestinal tract also may be collected by an open or closed method. The small intestine of the hamster is relatively short compared to the mouse and the rat, whereas, the colon is considerably longer in the hamster. Closed intestinal fixation is the process of inserting a blunt needle into the i. duodenum and injecting sufficient formalin to fix the entire length of the gut. The intestinal contents may be expelled into a beaker. Sections of the

intestinal tract may be collected by placing a ligature at each end of the desired section and cutting behind each ligature. If necessary, each segment can be further injected with fixative using a fine gauge hypodermic needle,

Step 10. a.

b.

c.

but the intestine should not be over-distended. I t is desirable to include a section o f small intestine containing aggregates of lymphoid tissue called Peyer's patches (Figure 201). Note: additional pancreatic tissue may be collected with the proximal part of the duodenum. n. Open intestinal fixation is the process of inserting one blade of a small blunt scissors into the duodenal opening and then cutting open the entire length of intestinal tract. Desired sections of the gut may be excised, placed onto a piece of cardboard mucosal surface up, and then placed into fixative. Collection Collec tion o f the liver, gallbladder, gallbl adder, kidneys, and adrenal adrenal glands. The hamster liver can be excised intact by grasping the tough connective connecti ve tissue under the median lobe with tissue forceps (Figure 202) and while exerting tension posteriorly, severing the esophagus and blood vessels passing through the diaphragm (Figure 203). Fix the liver intact or sectioned as specified. Once the liver is removed, the kidneys and adrenal glands are easily examined (Figure 204). The ureters may be difficult to identify unless they are distended. The adrenal glands in hamsters lie close to the anterior poles of the kidneys and are distinctly darker than those of the rat or mouse. The adrenals of male hamsters are larger than those of the female. I f the adrenals are to be collected and/or weighed apart from the kidneys, grasp the adrenal artery and vein close to each gland with fine forceps (Figure 204), undercut the fat medial to the forceps, and lift out the gland. Trim the fat away from each gland on a moistened paper towel. I f he adrenals are to remain with the kidneys, grasp the left renal artery and vein with the forceps close to the kidney and sever both vessels with a fine scissors

 

89 

Figurr e 195. Figu

196..  Fi g ure 196

Figurr e 197. Figu

Figurr e 198. Figu

o n on the k id ne y   w ith ensio xert   u pw ar d t ensi (Figuree 2 0 5 ) . E xert ceps   (Figur m ed ial to the fo rceps

d. S te p 1 1. a

forcep s an d b luntl forcep luntly y d isse issect ct the k id n ey a w a y from fro m its bed b ed o f p e rire riren n al  fat.  fat.   C ontin ntinu ue fa t. F re e   o u n d in g fat. surro d w ith its surr al glan gland drenal the   adren iorly   to to   in c lu d e the  ting   ante an te r iorly d i ssec ssecting y with with   a idney rasp   the the k idne o rs . G rasp cisso finee s ciss re n al w ith   fin h ed   ad adre ttach n ey   and an d a ttac the th e   k id idn sc al p el ( Fi g ure it h a s h a rp scal n a lly w ith itudin and d b i se c t it lo n g itudi isten n ed   g auze sp o n g e   an m o iste y. idney rightt k idne idne y   fr om the righ left   k idne al cut d is ting u is hes the left tudinal 2 0 6). T h e lo n g i tudin y idney right k idne kid n e y as ab o ve. B i sect th e   right d a nd r ig h t kidn al glan gland drenal right a dren C o l lect th e   right 20 6). gure   206). slidee se ct i ons (F igure left   ki dne y   in slid h it from fro m the left stinguis guish sv e rsely rsely   to d i stin transv tran on ). nctio stoc h o n d ra l ju ncti thee   st ern u m a nd   rib (c o stoch ti o n of th C ollec ollecti hoid   rib s and x ip hoid fro m the la s t ribs d f lap o f ab d o m inal w a ll a w a y from reflecte ected C u t the refl  the he   st steern u m . p ro ce s s o f  t

 

9

N e o y G u d R

s a h R

 

9 1  

 

Necropsy Guide: Rodents and the Rabbit

92

Figure 205.

b.

Figure 206.

Grasp the xiphoid process, slit the diaphragm under the sternum, and cut through the cartilaginous portion of the ribs on both sides close to the sternum (Figure 207). Extend each incision anteriorly through the first rib being careful to cut medial to the prominent jugular veins (Figure 207). Maintain upward tension and

continue cutting the muscles on either side of the trachea up to the lower jaw. Sever the muscles attached to the lower jaw and remove the sternum with attached

c.

d.

Step 12.

Step 13. a.

neck muscles. The full length of the trachea and opened thoracic cavity (Figure 208) should be revealed at this point. Trim excess tissue from from the sternum and and then bisect th thee sternum to reveal reveal the red-colored patches of bone marrow (Figure 209). The sternum must be decalcified after fixation. Enlarge the chest ches t opening by manually spreading the rib cage. Some rib ribss may be fractured in the process. Select one rib by cutting the intercostal muscles on either side and then cut the rib with a nail cutter approximately 0.5 em above and below the white enlargement marking the junction of the cartilaginous and bony segments of the rib (costochondral junction) (Figure 209). As with the sternum, the rib must be decalcified after fixation. Examination of the oral cavity. Make a skin incision from the comer of the mouth to the base of the ear on both of the hamster. Loosen the lower jaw (mandible) by severing the right and sidesmasseter left muscles (Figure 210) with a scalpel and then by cutting the jaw beneath with a bone cutter (Figure 211). Pull the jaw downward and examine the surface of the tongue, the teeth, and the rest of the oral cavity (Figure 212). Note the openings to the cheek pouches which are located lateral to the upper incisor teeth (Figure 212). The cheek pouches are easily everted for examination (Figure 213). Collection Collect ion of the tongue, thyroid glands, parathyroid glands, larynx, esophagus, trachea, thymus, heart, lungs, and mediastinal and bronchial lymph nodes. Cut the remainder of the muscles that hold the jaw in place until the mandible

 

9  

 

94  94  

cropsy sy Guide: Guide:   R o dents and the R abbit N e crop

Figure 210.

Figure 211. 211.  

Fi g ure 212.

Figure 213.

 

96

 

it  a n d  the Rabb Rabbit uide:: Rodents Rodents a N e cropsy G uide

Figure 214 214..

Figure 215.

Figure 216.

Figure 217. 217.  

97

g.

h.

 

Figure 218.

Figure 219.

Figure 220.

Figure 221.

Sever Seve r the olfactory lobes (Figure (Figure 229) and and then then tilt the head upward upward and backward. Sever the optic nerves (Figure 230). At this point the brain should begin to fall backward exposing the remaining cranial nerve attachments. Carefully cut any remaining attachments (Figure 231) and then sever the brain stem (medulla oblongata) posterior to the cerebellum freeing the brain (Figures 232 and 233). Handle the brain carefully and place it into fixative. Examine the pituitary gland (Figure 234) which is firmly seated seated in the hypophyseal hypophyse al fossa and covered by a delicate layer of dura mater. Allow the pituitary gland to be fixed with the base of the skull so that the gland is not lost. I f the pituitary gland needs to be removed for weighing etc. it can be removed with small, sharp scissors or scooped out with a small flat spatula (Figure 234) after severing the dura on either side o f the gland.

98

Necropsy Guide: Rodents and the Rabbit

Figure 222.

Figure 223.

Figure 224.

 

Figure 225.

99

 

Figure   226. Figure

Fi gure 227.

F i gure 22 228. 8.

Figure Figu re 229.

100

 

N e cropsy Guide: Gu ide:   Rodents and the Rabbit  Rabbit  

Fi g ure 230.

Figure 231.

Figuree   232. Figur

Figuree   233. Figur

101

Figure 234.

i.

Step 15.

Step 16.

Step 17.

Remove the skin over the nose and upper jaw and place the entire skull in fixative. The nasal passages (turbinate bones) and middle ears are normally sectioned after the skull is decalcified. Collection Collect ion of the sciatic nerve and adjacent muscle. The sciatic nerve is located somewhat parallel and posterior to the femur in the thigh. Make a skin incision parallel to the femur along the medial side of the thigh. Separate the skin and bluntly dissect through the muscle on the posterior one third of the leg (Figure 235). Careful deep dissection will expose the white fibers o f the nerve running down the leg (Figure 236). Cut out approximately 1 em of nerve and include adjacent muscle. Careful dissection and handling of the nerve is very important. Collection of the proximal femur with bone marrow. Bone marrow may be collected from the femur as well as from the sternum. Dissect the muscles away from the femur as far up as the hip joint. Cut through the hip joint with bone cutters and free the proximal portion of the femur (Figure 237). Cut the distal femur approximately half way down the thigh. Remove and fix the bone. Collection Collec tion of the vertebral column and spinal cord. I t is tedious and too time consuming to dissect out the entire spinal cord from the bony vertebral column for routine necropsies. The simplest simpl est and most acceptable method of collection is to cut away as much muscle as possible from around the vertebral column and then to cut out segments of the spinal cord (cervical, thoracic, lumbar) by severing the vertebral column at intervals using bone forceps. The crushing action of the forceps will distort the spinal cord, therefore it is important for the tissue trimmer to cut out only the central portion of each segment after the bone has been decalcified.

 

102

N e c r o p s y G u id e

thee R a b b i t ents   a n d th R od ents

Fi g ure 235

re 2 37 Figure Figu

 

iguree 23 6 F igur

gure   23 8   Fi gure

103   103

Figure   240. Figure

Figurr e 239. Figu

S te p   18.

Step 1 9.

gla n ds. fla nk glan Collecc tion o f the flank Colle (Figure   msterss   (Figure h a mster gl a nds of  male ha  sebacee ous gla mented d  sebac pi g mente darkly pig  glandss  are darkly flan k  gland T h e flank ove   r e m ove  advisab b le to re is   advisa ination n , it is exa m inatio forr exam col l ected fo to   be coll tissu e is to 23 8 ). f  h is tissue ising tth h em. excising befo o re exc gla n ds bef the fu furr   over t he glan a t. n of b r own f at llection C o llectio ce r vical ve n tral cer in   the ven sp ace in ubcuticc ular space fro m   the s ubcuti collect ed from be   collect B r o w n fat m ay be  24 0 ). (Fig ure 240 blad es (Figure should d er blades the   shoul betwe en the  from   betwe 239)   or from (Figur e 239)  region regio n   (Figur

S U G G E S T E D   R E A D IN G   Phila-Febigee r, Phila orat01y 1y A ni m als, Le a   &   Febig  for La b orat0 chniques ques for g T e chni re eding oduction tion   a n d  B reedin l. H afez, E . S. E . Repr oduc ,   1970. delphia, delphia Research arch , H o ffman,  a n d   Use Use   in M ed i cal Rese Its   B iology io logy a lden   H am st e r, Its  in  Th e G o lden 2 M a g a lh a e s ,   H. A n atomy, in  mes, Ames, niversity   Press, A University agalhaee s, H., E ds., Iow a   State U n son, P. F., and M agalha Robin R. A ., Robi

chap..   6 9 68, chap

 

106

Necropsy Guide: Rodents and the Rabbit

' Figure 241.

a.

b.

c.

d.

e.

f.

Step 8.

 

Figure 242.

Lay the guinea pig ondown.) its back on a dissecting pigs usually do not need to be pinned Wetting the surfaceboard. of the(Guinea body with alcohol helps prevent loose hair from falling into the incisions. Stretch Stret ch the skin in opposite opposi te directions direct ions with with the thumb and forefinger foref inger and make a midline skin incision extending from the lower jaw to the vulva, or scrotum, respectively. Stretching the skin helps to prevent accidentally cutting into deeper structures. While exerting gentle outward traction on the skin with forceps, carefully incise the loose subcutaneous tissue (Figure 246) and retract the skin on either side of the incision (Figure 247). Carefully dissect the fat and connective tissue away from the anterior point of the sternum. Bluntly dissect through the superficial muscle in the neck and extend the incision anteriorly for 1 to 2 em. Retract the muscle laterally to expose the light pink thymus which appears as two oval structures on either side of the midline (Figure 248). The thymus in the guinea pig is extrathoracic, whereas in the mouse, rat, hamster, and rabbit, the thymus is intrathoracic. Grasp the connective tissue next to either lobe of the thymus, undercut the thymus, and place it into fixative. The salivary glands are located anterior and deep to the thymus. Grasp the loose connective tissue just posterior to one of the paired submaxillary glands and bluntly dissect out the above gland together with the sublingual salivary gland and cervical lymph nodes (Figure 249). Place the tissues in fixative. In female guinea pigs locate one of the two nipples on the reflected abdominal skin in the inguinal region and identify the light yellow mammary tissue beneath the skin (Figure 247). These glands are most obvious in breeding females. Scrape the hair away from the nipple and cut a dime-sized circular area out of the skin including the nipple and underlying tissue. Collection o f the male or female reproductive tracts and urinary bladder.

1   7  

 

10 8

Rod e n ts and the R a b bit Necrops y Guide: Rode

Figure 246.

Figuree 247. Figur

Figure 248.

Figure 249.

 

110

Guide:   R o dents and the R abbit Guide:

Figure 250.

gure 251. Fi Figure

Figure 252.

Figure 253 253..

 

111

Figure 254.

Figure 255.

Figure 256.

 

1 12

Rabbit   the   Rabbit dents a nd the  Rodents Gu i de: Ro Necr op o p s y Gui

Figure 257.

re 2 58. Figure Figu

placee the ligat lig at u re. R e plac ti g hten hten   the disten en d ed and tig is   dist rine   o ut of th e b l a d d e r i f i t is th e u rine small all g a u g e using g a   sm in to the lu m en usin n g form al in into injecti jectin thee   v o lu m e by   in abou t h alf th wall   of   fi xativ ativee in to t he wall ject   fix be  taken taken n ot to in ject n m u st st   be  ution (F iguree 2 64). C a utio n e edle edle   (Figur er .  N ote: ote: as an a ltery b ladd ladder inary n s   an and d  u rinar v e orga organ uctiv thee   re pro d ucti r. Fix th adder. th e   b ladde d b ef o re the r e p r o d u ctiv e igated cted   and an d l igate injected ri n ary b la d d e r m ay be inje uri n ativ ativee , the u St ep 9. a.

b.  b.   c.

cted   fr om t he b od y. tr a ct is  d i ss e cted cec u m , ile u m , cecu duo d e n u m , je ju n u m , ileu reas,, st o m a c h , duod C olle ollecc ti o n o f th e sp le e n , p a n c reas s. h node nodes. ic lymp lymph nteric and m es e nter c o lo n , r ec t u m ,   and p s,  s,   p ull d f orce orcep rated at u re o f the st o m ac h w ith se r rate ter curv curvat greater G e n t ly g ra s p th e grea lly y  seve  severr the film y m em b ra n e b et w ee n th e stom ac h an d the refull d o w n , an d   c arefu liverr (F ig u re 2 6 5) . te lobe lobe   of the live q u adra adrate This   u re   2 6 6). This (Figu sto m a c h (Fig the   sto te l y 1 . 0 e m ab ov e the  ximate S e v e r  the e so p h a g u s   a p pro xima its   at tac h m en ts. from   its thee  st om ac h from b ly   lo ose n   th iceab w ill n o t icea g ining st om a ch a n d   w hile m a i n ta inin ith h   forcep forcepss c lo s e to the   sto th e e so p h a g u s w it G ra s p the th e  li ver issuee b e tw e en   the ectivee t issu rem ai n in g co n n ectiv io n , s e v e r   th e  b ile d u ct   an d  rem tensio tens (Figu r e 2 6 7). sto o m a ch   (Figu an d   th e  st

d

d o r sal tiness an d the do ntestine th e i ntes ry betw ee n the entery M a in ta i n te n si o n a nd   se ve r the m es ente large bl o od v es sels in this ar e a. T h is   the   large do   not c u t the  b o d y w al l   (F ig u r e 26 8) b u t   do tion   p t for its term in a tion excep (Figu r e 26 9) exce sh o u ld   fr ee the s p le en a nd   the en tire gu t  (Figu isten n ed   w ith tow el   m oiste on  a pap e r towel anus a nd   lay the   g u t  on  the   anus aro u n d the  an us. C u t arou a t the  the   anus. sa li n e.

 

113

Figure 259.

Figure 260.

Figure 261.

Figure 262.

 

114

Necropsy Guide: Rodents and the Rabbit

Figure 263.

e.

Figure 264.

The intestinal tract is most efficiently examined by stretching it to its full length. Sever the mesentery which binds the coils of the intestine (Figure 270). The mesenteric lymph nodes should remain imbedded in the mesentery close to the

ileocecal junction (Figure 271). Once the mesenteric attachments are severed the entire gut may be laid out "serpentine-fashion" (Figure 272). f.

g.

h.

Collect the spleen and a portion of the diffuse, pink pancreas by cutting between the spleen and stomach (Figure 273). The guinea pig stomach is totally glandular, unlike that of the mouse, rat, and hamster. It is relatively large and thin-walled. Because of its size, the stomach is usually not infused with fixative, but is examined after being opened completely (Figure 274). Portions of the fundus and pylorus are routinely collected for sectioning. The intestinal tract also may be collected by an open or "closed" method. Note that the guinea pig has both a long small intestine and a long colon. The cecum is relatively larger than that of the mouse, rat and hamster. i. Closed intestinal fixation is the process of inserting a blunt needle into the duodenum and injecting sufficient formalin to fix the entire length of the gut. The intestinal contents may be expelled into a beaker. Sections of the

ii.

of the intestinal tract and maycutting be collected placing a ligature at each endsegment desired section behind by each ligature. I f necessary, each can be further injected with fixative using a fine gauge hypodermic needle, but the intestine should not be over-distended. I t is desirable to include a section of small intestine containing aggregates of lymphoid tissue called Peyer's patches (Figure 275). Note: additional pancreatic tissue may be collected with the proximal part of the duodenum. Open intestinal fixation is the process of inserting one blade of a small blunt scissors into the duodenal opening and then cutting open the entire length of intestinal tract. Desired sections of the gut may be excised, placed onto a piece of cardboard mucosal surface up and then placed into fixative.

 

115

Figure 265.

Figure 266.

Figure 267.

Figure 268.

 

116

Necropsy Guide: Rodents and the Rabbit

Figure 269.

Figure 270.

Figure 271.

 

 

Figure 272.

118

Necropsy Guide: Rodents and the Rabbit

Step 10. a.

b.

c.

e. Step 11. a. b.

c. d.

Step 12.

Step 13.

 

Collection of the liver, gallbladder, kidneys, and adrenal glands. The guinea pig liver has a number of divisions but can be excised intact. Grasp the tough connective tissue in the region of the bile duct with a toothed tissue forceps (Figure 276) and while exerting tension posteriorly, sever the esophagus and blood vessels passing through the diaphragm (Figure 277). Cut any remaining connections to free the liver. Fix the liver intact or sectioned as specified. Once the liver is removed, the kidneys, ureters, and adrenal glands are more easily examined (Figure 278). Note that the adrenal glands in the guinea pig are proportionately large. right located anterior to left adrenal gland and The lies on theadrenal surface gland of theisvena cava.slightly If the adrenals arethe to be collected and/or weighed apart from the kidneys, grasp the left adrenal artery and vein with fine forceps close to the gland, undercut the fat medial to the forceps (Figure 278), and lift out the gland. It is difficult to dissect the right adrenal gland away from the vena cava without nicking the vein. Place the glands on a moistened paper towel and trim away excessive fat. I f the adrenals are to remain with the kidneys, grasp the left renal artery and vein with the forceps close to the kidney and sever both vessels with a fine scissors medial to the forceps (Figure 279). Exert upward tension on the kidney with forceps and bluntly blun tly dissect the kidney away from from its bed of perirenal fat. Continue Continu e dissecting dissecti ng anteriorly anterior ly to include the adrenal gland with with its surrounding fat. Free the kidney and attached adrenal with fine scissors. Bisect the left kidney longitudinally with a sharp scalpel (Figure 280). The longitudinal cut distinguishes the left kidney from the right kidney. Collect Collec t the right adrenal gland and right kidney as above. Bisect the right kidney transversely to distinguish it from the left kidney in slide sections (Figure 280). Collection of the sternum and rib (costochondral junction). Cut the reflected flap of abdominal wall away from the last ribs and xiphoid process of the sternum. Grasp the xiphoid process, process , slit the diaphragm under the sternum, and cut through the cartilaginous portion of the ribs on both sides close to the sternum (Figure 281). Extend each incision anteriorly through the first rib being careful to cut medial to the jugular veins. Maintain upward tension and continue cutting the muscles on either side of the trachea up to the lower jaw. Sever the muscles attached to the lower jaw and remove the sternum with attached neck muscles. The full length of the trachea should be revealed at this point (Figure 282). Trim excess tissue from from the the sternum and then bisect the sternum to reveal the red-colored patches of bone marrow (Figure 283). The sternum must be decalcified after fixation. Enlarge the chest opening b by y manually spreading the rib cage. Some rib ribss may be fractured in the process. Select one rib by cutting the intercostal muscles on either side and then cut the rib with a nail cutter approximately 0.5 em above and below the white enlargement marking the junction of the cartilaginous and bony segments of the rib (costochondral junction) (Figure 283). As with the sternum, the rib must be decalcified after fixation. Examination of the oral cavity. Make a skin incision from the comer of the mouth to the base of the ear on both sides of the guinea pig. Loosen the lower jaw (mandible) by severing the right and left masseter muscles (Figure 284) with a scalpel and then by cutting the jaw beneath with a bone cutter (Figure 285). Pull the jaw downward and examine the surface of the tongue, the teeth, and the rest of the oral cavity (Figure 286). Collection of the tongue, thyroid glands, parathyroid glands, larynx, esophagus, trachea, heart, lungs, and mediastinal (tracheobronchial) lymph nodes. \

1

 

Necropsy Guide: Rodents and the Rabbit

L20

Figure 279.

a.

Figure 280.

Cut the remainder of the muscles that hold the jaw in place until the mandible is freed from the body. Grasp the front of the mandible and pull downward while severing the soft palate and any remaining pharyngeal attachments behind the esophagus (Figure 287). Continue dissecting the trachea and esophagus together

of

b.

c. d. e.

f.

away thechest bodywall and (Figure at the level the first cutting ribs carefully cut the attachments to the from dorsal 288). Continue all remaining attachments of the lungs down to the diaphragm until the trachea, heart, lungs, and accompanying structures can be freed (Figure 289). Place the tissues on a moistened paper towel. Just below the the larynx, identify the reddish-colored thyroid glands (Figure 289). The parathyroid glands are small spherical, white masses imbedded in the anterolateral and posterolateral poles of each thyroid gland. These are better visualized with a magnifying lens or a dissecting microscope. Examine the mediastinal (tracheobronchial) lymph nodes (Figure (Figure 290). Insert one blade of a fine scissors into the pharyngeal opening of the esophagus. Cut open and examine the entire length of the esophagus (Figure 291). Place a loose ligature around the trachea 2 to 3 mm below the thyroid glands. Fill a 20-cc syringe with fixative and attach a 16-gauge needle. Insert the needle into the opening of the trachea (glottis) and snug the ligature around the needle. Slowly inject sufficient fixative to enlarge all lobes of the lungs to a point corresponding to full inflation (Figure 292). (The margins of the lobes should remain sharp.) Do not overdistend the lungs. Note that both lungs of the guinea pig are multi-lobed in contrast to the mouse, rat, and hamster which possess a single left lobe. Withdraw the needle and tighten the ligature. Sever the trachea trache a and esophagus transversely below the level of the thyroid glands (Figure 293) and fix the mandible with attached tongue, larynx, and thyroid/ parathyroid glands.

 

121

Figure 281.

Figure 282.

Figure 283.

g. h. Step 14. a.

Cut the the heart hea rt away from the mediastinum and place the the lungs into fixative. fixati ve. Section the heart from the base to the apex incorporating the aorta (Figures 294 and 295). Collection Collect ion o f the brain, pituitary gland, and turbinate bones. Tum the guinea pig over onto onto its its ventral ventral side side and and saturate the fur on the head with alcohol.

 

1

N e o y G u d R

s a h R

 

  2 3  

 

124

Necropsy Guide: Rqdents and the Rabbit

Figure 290.

Figure 292.

Figure 291.

b. c.

d.

Make an incision incisio n from between the eyes to behind the head and and reflect the skin away from the cranium (Figure 296). Bend the guinea guine a pi pig' g'ss head down and cut through the dorsal muscles of the neck until the spinal cord can be seen entering the skull (Figure 297). The temporal muscles are not well developed in the guinea pig, so those muscles do not have to be reflected. Insert one blade of a small bone cutter into tj:J e opening of the skull (foramen magnum) magnu m) and place the other ot her blade just above the op ni~g or the ear canal (Figure

 

125

Figure 293.

Figure 294.

e. f.

Figure 295.

298). Cut the bone between the blades, being careful not to penetrate the brain. Repeat on the other side of the skull. Extend Exten d the bone incisions anteriorly to the level of the nose, cutting superficially just above the zygomatic arch (Figure 299). Lift the posterior cranium upward away from the head exposing the cerebellum

 

126

Necropsy Guide: Rodents and th thee Rabbit

Figure 296.

Figure 297.

Figure 298.

g.

Figure 299.

and most of the cerebral hemispheres (Figure 300). Carefully dissect away the frontal bones, exposing the remainder of the brain, including the olfactory lobes (Figure 301). I f present, remove the tough membrane that surrounds the brain (dura mater, Figure 302). Remnants of the dura may need to be removed from the junction o f the cerebral hemispheres and cerebellum. Sever the olfactory lobes (Figure (Figure 303) and then tilt the head upward and backward. Sever the optic nerves (Figure 304). At this point the brain should begin to fall backward exposing the remaining cranial nerve attachments. Carefully cut any

 

127

Figure 300.

Figure 301.

Figure 302.

Figure 303.

 

1

N e o y G u d R

s a h R

 

129

Figure 307.

Figure 308.

remaining attachments (Figure 305) and then sever the brain stem (medulla oblongata) posterior to the cerebellum (Figure 306) freeing the brain (Figure 307). h.

Handle the brain carefully and place it into fixative. Examine the pituitary gland (Figure 308) which is firmly firmly seated seated in the hypophyseal hypophyseal fossa and covered by a delicate layer of dura mater. Allow the pituitary gland to be fixed with the base of the skull so that the gland is not lost. I f he pituitary

 

1

N e o y G u d R

s a h R

 

131

gland needs to be removed for weighing etc. , it can be removed with small, sharp scissors or scooped out with a small flat spatula after severing the dura on either side o f the gland. i. Remove Remov e the skin over the nose and upper jaw and place the entire skull in fixative. The nasal passages (turbinate bones) and middle ears are normally sectioned after the skull is decalcified. Step 15. Collection Collec tion of the sciatic nerve and adjacent muscle. The sciatic nerve is located somewhat parallel and posterior to the femur in the thigh. Make a skin incision parallel parallel to the femur along the medial side of the thigh. Separate the skin and bluntly dissect through the muscle on the posterior one third o f the leg (Figure 309). Careful deep dissection will expose the white fibers of the nerve running down the leg (Figure 310). Cut out approximately 1 em of nerve and include adjacent muscle. Careful dissection and handling of the nerve is very important. Step 16. Collection Collec tion o f the proximal femur with bone marrow.

Step 17.

Bone marrow may be collected from the femur as well as from the sternum. Dissect the muscles away from the femur as far up as the hip joint. Cut through the hip joint with bone cutters and free the proximal portion of the femur. Cut the distal femur approximately halfway down the thigh (Figure 311). Remove and fix the bone. Collection Collec tion o f the vertebral column and spinal cord. I t is tedious and too time consuming to dissect out the entire spinal cord from the bony vertebral column for routine necropsies. The simplest and most acceptable method o f collection is to cut away as much muscle as possible from around the vertebral column and then to cut out segments of the spinal cord (cervical, thoracic, lumbar) by severing the vertebral column at intervals using bone forceps. The crushing action of the forceps will distort the spinal cord, therefore it is important for the tissue trimmer to cut out only the central portion o f each segment after the bone has been decalcified.

SUGGESTED READING 1. Cooper, G. and Schiller, A. L., Anatomy o f he Guinea Pig, Harvard University Press, Cambridge, Mass., 1975. 2. Hafez, E. S. E., Reproduction and Breeding Techniques for Laboratory Animals, Lea & Febiger, Philadelphia, 1970. 3. Wagner, J. E. and Manning, P. J,, The Biology o f he Guinea Pig, Academic Press, New York, 1976, chap. 6.

 

133 Chapter 6

NECROPSY OF THE RABBIT (Oryctolagus cuniculus) Step 1. Step 2. Step 3.

Check animal's identification number. Record animal's weight. Verify animal's sex according to characteristics below. Male (Figure 312) Round genital opening Dewlap not prominent (Figure 313) Nipples inconspicuous Pronounced testes

Step 4. a. b.

Note: Step 5.

Female (Figure 314) Oval genital opening Pronounced dewlap (Figure 315) Conspicuous nipples especially in lactating rabbits (Figure 323)

External examination. Observe entire body surface and orifices for abnormalities. Feel (palpate) the entire body for superficial swellings beginning from the head and proceeding posteriorly to the neck, chest, abdomen, and appendages. Palpate for enlarged organs or masses within the abdomen. All tissues and organs should be examined in situ before being dissected from the body. Collection of the eye, Harderian gland, lacrimal gland, and parotid salivary gland.

Autolytic changes occur early in the eye, therefore it should be collected first, along with neighboring glands on the side of the head. a.

Lay the the rabbit on its side and and make a skin incision from above and just ju st in front

of the eye to the convergence of the upper and lower jaws just posterior to the base of the ear. Reflect the skin ventrally.

b. c.

d.

Step 6. a.

b.

Press a small curved forceps deeply into the orbit and elevate the globe. Cut the tough fibrous tissue tissue around the orbit (Figure 316) and dissect out the eye and the glands lying within the orbit. The large, light-yellow Harderian gland is located anteroventrally, whereas the smaller light-brown lacrimal gland lies posteroventrally to the globe (Figure 317). Remove and fix the more diffuse, light-brown parotid salivary gland which is located ventral to and just behind the base of the ear (Figure 317). The parotid gland is usually covered with superficial fat. Collection Collec tion of superficial masses. Remove any abnormal enlargement enlarge ment by cutting completely around it, being careful to include overlying skin, if possible, and at least some normal tissue surrounding the mass. Scrape the hair away from the skin because hair dulls the microtome knife which is used to section tissues in the preparation of slides. Divide large excised masses into 0.30.3 - to 0.5-cm slices with a sharp scalpel and place in fixative. Masses smaller than 0.5 em can be fixed undisturbed. I f the mass has invaded bone try to expose as much of the abnormal tissue to the fixative without destroying the architecture of the surrounding normal structures. Allow tissue to fix at least 48 hr. The bone must be decalcifie decalcified d before the specimen is sectioned. I f there is some question as to the removal of a mass, consult the pathologist.

 

134

Necropsy Guide: Rodents Roden ts and the Rabbit

Figure 312.

Figure 313.

Figure 314.

Figure 315.

 

135

Figure 316.

Step 7. a.

Figure 317.

Collection of cervical lymph node, submaxillary salivary gland, and mammary gland. Lay the rabbit on its back and wet the ventral surface of the surface of the body

b.

c.

d.

with alcohol. This helps to prevent loose hair from falling into the incisions. Stretch the skin in opposite directions with the thumb and forefinger and make a midline skin incision extending from the lower jaw to the vulva, or testes, respectively. Stretching the skin helps to prevent accidentally cutting into deeper structures. While exerting gentle outward traction on the skin with forceps, carefully incise the subcutaneous tissue (Figure 318) and retract the skin on either side of the incision (Figure 319). Incising the pectoral muscles (Figure 320) at this point will facilitate opening the thoracic cavity later. Dissect away the loose connective tissue at the angle o f the lower jaw. Excise and fix a cervical lymph node and the red-colored submaxillary salivary gland (Figure 321). Note: the sublingual salivary gland in the rabbit lies deep to the submaxillary gland and rests against the medial surface of the mandible. Because of its deep location and its relatively small size, the sublingual gland is not

e.

Step 8.

routinely collected. In female animals locate a nipple on the reflected abdominal skin and identify the whitish-yellow mammary tissue beneath the skin (Figure 322). These glands are most obvious in lactating rabbits (Figure 323). Scrape the hair away from the nipple and cut a dime-sized circular area out of the skin including the nipple and underlying tissue. Collection o f the male or female reproductive tracts, and urinary bladder.

 

136

Necropsy Guide: Rodents and the Rabbit

Figure 318.

Figure 319.

Figure 320.

Figure 321.

Male a. b.

Grasp the most posterior part of the abdominal wall and cut into the abdominal cavity just above the base of the penis and testes (Figure 324). Cut the body wall on both sides close to to the reflected reflecte d skin being careful not to cut into any organs. Extend the incisions anteriorly to the rib cage and then reflect the cut portion of the abdominal wall over the chest (Figure 325).

 

137

Figure 322.

c.

d.

Figure 323.

Pull both testicles out of the scrotum and sever the fibrous connection (gubernaculum testis) between the tail of the epididymis and the scrotum (Figure 326). Sever the vas deferens (Figure 327). Place the testicle with attached epididymis (Figure 328) into fixative. Repeat the above procedure to collect the second testicle.

e.

f.

g.

Female a.

b.

c.

Reflect the intestines anteriorly. Express most of the urine out o f the bladder. Note the cloudy urine which is caused by the abundance o f crystals. Dissect the connective tissue from around the base of the penis. Insert one blade o f a bone cutting forceps under the center of the pubis just above the base of the penis and cut through the cartilaginous connection (pubic symphysis) (Figure 329). Manually separate the pubic bones to reveal the underlying urethra and rectum. Dissect the skin away from the penis. Grasp the end o f the penis with forceps and while pulling gently upward carefully dissect away the connective tissue and muscle around the urethra and beneath the urinary bladder (Figure 330). This should free the male reproductive reprodu ctive tract (Figure 331) with at attached tached urinary bladder. Place the tissues onto a moistened paper towel. Open the urinary bladder completely and examine the lining. Fix the reproductive reproductiv e tract with attached urinary bladder.

Grasp the most posterior poster ior part of the abdominal wall and cut into the abdominal cavity just above the vulva (Figure 332). Cut the body wall on both sides sides close to to the reflected skin skin being careful not to cut into any organs. Extend the incisions anteriorly to the rib cage and then reflect the cut portion o f the abdominal wall over the chest. Reflect the intestines anteriorly (Figure 333). Express most of the urine out of the bladder. Note the cloudy urine which is caused by the abundance o f crystals.

 

138

N e c r opsy Guide Guide:: R odents and and   th thee  Rabbit

Figuree 324. Figur

Fi g ure 325.  325. 

Fi gure 326 326..

Figure Figu re 327.

 

139

8. Figure 32 328.

Figure 329 329..

Fi g ure 330.

gure 331. Fi Figure

 

Necropsy Guide: Rodents and the Rabbit

140

d.

Insert one blade of a bone cutting forceps under the center of the pubis just above the vulva, cut through the cartilaginous connection (pubic symphysis) (Figure

e.

334), manually Cut theand skin skin between separate the the vulvathe andbones. anus (Figure 335) and dissect free the posterior end of the vagina being careful not to cut into the rectum. Grasp the end of the vagina and while gently pulling upward sever the film-like connective tissue between the vagina and the rectum (Figure 336). Continue dissecting anteriorly to the area of the cervix and then cut the mesentery supporting the uterine horns (Figure 337) up to the ovaries. Cut the supporting tissue between the ovaries and kidneys and transfer the entire reproductive tract with attached urinary bladder (Figure 338) onto a paper towel moistened with saline. Trim excessive fat from the uterine horns and ovaries. Open the urinary bladder completely and examine the lining. Fix the reproductive tract with attached urinary bladder. Note: one may select to open the entire reproductive tract for closer examination. Collection o f the spleen, pancreas, stomach, duodenum, jejunum, ileum, cecum, sacculus rotundus, colon, rectum, and mesenteric lymph nodes. Carefully sever the filmy filmy membrane membrane between the stomach and the quadrate lobe o f the liver (Figure 339). Sever the esophagus approximately 1.0 em above the stomach (Figure 340). This will noticeably loosen the stomach from its attachments. Grasp the esophagus with with forceps forceps close close to the stomach and while maintaining tension, sever the bile duct and remaining connective tissues between the liver and the stomach (Figure 341). Maintain tension and sever the mesentery between the intestines and the dorsal body wall (Figure 342) but do not cut the large blood vessels in this area. This should free the spleen and the entire gut except for its termination at the anus. Cut around the anus and lay the gut on a paper towel moistened with saline. The intestinal tract is most efficiently examined by stretching it to its full length.

f.

Step 9. a. b. c.

d.

e.

f.

g.

h.

Carefully sever the mesentery which binds the coils of the intestine (Figure 343). The mesenteric lymph nodes should remain imbedded in the mesentery close to the ileocecal junction (Figure 344). Once the mesenteric attachments are severed the entire gut may be laid out "serpentine-fashion" (Figure 345). Collect the spleen and a portion of the diffuse, pink pancreas by cutting between the spleen and stomach (Figure 346). The rabbit stomach, similar to the guinea pig, does not have a nonglandular region as found in the mouse, rat, and hamster. The stomach is a large, thin-walled organ, thus easily ruptured during necropsy. In addition, the stomach of the rabbit undergoes autolysis rapidly and may have ruptured spontaneously by the time the necropsy is performed. Cut the stomach open completely from the pylorus to the fundus and examine the lining. Excise samples of fundus and pylorus for sectioning (Figure 347). The intestinal tract of the rabbit generally is opened as it is being examined. Note the relatively long duodenum, jejunum, and ileum (Figure 345). Identify the sacculus rotundus, a round, yellowish structure located at the the junction junct ion of the ileum and the cecum. The yellow coloration is due to a concentration of lymphoid tissue. The large dark-green cecum narrows into the yellowish appendix which also is composed largely of lymphoid tissue. The partially sacculated large intestine leads from the cecum to the rectum. Samples are routinely collected from each of the above intestinal regions by flattening them onto a piece of cardboard mucosal surface up and then placed into fixative. Collect a section of small intestine containing an aggregate of lymphoid follicles (Peyer's patch) (Figure 348).

 

141

igure   332 F igure

Fi gure 3 33

F igure 334

35 ure 3 33 Figure Fig

 

  4 2  

N e o y G u d R o s a h R a

 

143

Figure 339.

Figure 340.

Figure 341.

Figure 342.

 

14 4

N e c r o p s y   G u i d e R o d e n ts a n d th e R a bbit bbit  

Figu Fi gu r e 3 43 .

F ig u re   34 345. 5.

Fi g ur e   344 .  

F ig ur e   34 346. 6.

 

145

Figure 347.

Step 10. a.

b.

c.

d. Step 11. a. b.

intestine containing an aggregate of lymphoid follicles (Peyer's patch) (Figure 348). Collection o f the liver, gallbladder, kidneys, and adrenal glands. The rabbit liver can be excised intact by grasping grasping the the tough tough connective tissue underr the median lobe with tissue forceps unde forceps (Figure 349) and while exerting tension posteriorly, severing the esophagus and blood vessels passing through the diaphragm (Figure 350). Cut any remaining connections to free the liver. Open and examine the gallbladder gallbladd er (Figure 351). Fix the liver intact or sectioned as specified. Once the liver is removed, the kidneys kidneys and adrenal glands are easily examined (Figure 352). The ureters also are easily identifiable in the rabbit. The adrenal glands lie some distance away from the kidneys, thus are collected separately (Figure 352). Dissect the adrenals free of surrounding fat and bisect them lengthwise before fixation. Note the dark central medulla and the light outer cortex (Figure 353). To collect the kidneys grasp the the left renal artery and vein with with forceps forceps close to the kidney and sever seve r both vessels with a fine fine scissors medial to to the forceps (Figure 354). Exert upward tension and bluntly dissect the kidney away from its ped o f perirenal fat. Grasp the kidney with a moistened gauze sponge and bisect it longitudinally with a sharp scalpel (Figure 355). The longitudinal cut distinguishes the left kidney from the right kidney. Collect the right kidney as above. Bisect the right kidney transversely to distinguish it from the left kidney in slide sections. (Figure 355). Collection o f the sternum and rib (costochondral junction). Cut the reflected flap of abdominal wall away from the last ribs and xiphoid process of the sternum. Dissect away the pectoral muscles covering the chest. Grasp the xiphoid process, slit the diaphragm under the sternum, and cut through through

 

 46 

Figure 348.

N e c r o p sy   Gu id e:  R o d e n t s  a  an n d  t h e   Ra b b it  

Figu Fi gu r e 34 9

Fi gu r e 35 1

F ig ur e   3 50  

Fi gu re   35 2

 

147

Figure 353.

Figure 355.

Figure 354.

c.

d.

Step 12.

Step 13.

a.

of the ribs on both sides close to the sternum. Extend the cartilaginous portionthrough each incision anteriorly the first rib being careful to cut medial to the prominent jugular veins (Figure 356). Exert upward tension and continue cutting the muscles on either side of the trachea up to the lower jaw. Sever the muscles attached to the lower jaw and remove the sternum with attached neck muscles. The full length of the trachea should be revealed at this point (Figure 357). Trim excess tissue from the sternum and then bisect the sternum ~o reveal the red-colored patches of bone marrow (Figure 358). The sternum must be decalcified after fixation. Enlarge the chest opening by manually spreading the rib cage. Some ribs may be fractured in the process. Select one rib by cutting the intercostal muscles on either side and then cut the rib with a nail cutter approximately 1.0 em above and below the white enlargement marking the junction of the cartilaginous and bony segments of the rib (costochondral junction) (Figure 359). As with the sternum, the rib must be decalcified after fixation. Examination of the oral cavity. Make a skin incision from the comer of the mouth to the base of the ear on both sides of the rabbit. Loosen the lower jaw (mandible) by severing the right righ t and left masseter muscles (Figure 360) with a scalpel and then by cutting the jaw beneath with a bone cutter (Figure 361). Pull the jaw downward and examine the surface of the tongue, the teeth, and the rest of the oral cavity (Figure 362). Collection of fue tongue, thyroid glands, parathyroid glands, larynx, esophagus, ( trachea, thymus, heart, lungs, and mediastinal and bronchial lymph nodes. Cut the remainder of the muscles that hold the jaw in place until the mandible is freed from the body. Grasp the front of the mandible and pull downward while severing the soft so ft palate and any remaining pharyngeal attachments behind the esophagus (Figure 363). Continue dissecting the trachea and esophagus together

away from the body and at the level of the first ribs carefully cut the attachments to the dorsal chest wall (Figure 364). Continue cutting all remaining attachments

 

148

Necropsy Guide: Rodents and the Rabbit

Figure 356.

Figure 357.

Figure 358.

Figure 359.

of the lungs down to the diaphragm until the trachea, heart, lungs, and accom-

b.

panying structures can be freed (Figure 365). Place the tissues on a moistened paper towel. Just below the larynx, identify the dark-pink thyroid glands (Figure 365). The parathyroid glands are small spherical, pink masses which may be imbedded in the posterolateral poles of the thyroid glands or else located close to the thyroid gland. Themicroscope. parathyroid glands are better visualized with a magnifying lens or a dissecting

 

1 4 9  

 

150

Necropsy Guide: Rodents and the Rabbit

Figure 363.

Figure 364.

Figure 365.

Figure 366.

 

1

N e o y G u d R

s a h R

 

153

Figure 370.

Figure 371.

h.

Step 15.

to dissect away the bone in order to expose the gland. Cut the bone at each lateral point (Figure 384) and then posteriorly (Figure 385). Carefully lift the bone away from the gland. The pituitary may be left in place or it can be also dissected free with fine scissors (Figure 386) or needles. Remove all remaining skin over the nose and upper ja jaw w and place the the entire skull in fixative. The nasal passages (turbinate bones) and middle ears are normally sectioned after the skull is decalcified. Collection of the sciatic nerve and adjacent muscle. The sciatic nerve is located somewhat parallel and posterior to the femur in the thigh. Make a skin incision parallel to the femur along the medial side of the thigh. Separate the skin and bluntly dissect through the muscle on the posterior one third of the leg (Figure 387). Careful deep dissection will expose the white fibers of the nerve running down the leg (Figure 388). Cut out approximately 1 em of nerve and include adjacent muscle. Careful dissection and handling of the nerve are very important.

 

154

Necropsy Guide: Rodents and the Rabbit

Figure 372.

Figure 373.

Figure 374.

Figure 375.

 

155

Pigure 376.

Figure 378.

Step 16.

Figure 377.

Figure 379.

Collection of the proximal femur with bone marrow. Bone marrow may be collected from the femur as well as from the sternum. Dissect the muscles away from the femur as far up as the hip joint. Cut through the joi joint ntmur withapproximately bone cutters and free proximal portion of the femu femur. r. Cut the hip distal fe femur half hal f free way waythe down the thigh (Figure 389). Remove and fix the bone.

 

156

N e cr opsy Guide: Guide:   Rodents and the  the   Rabbit

 

Figure Figu re 380.

Figure   381. Figure

Figure Figu re 382.

Figure 383.

  5 7  

 

1

N e o y G u d R

s a h R

 

159  159  

St e p 17 .

ord. and   spinal ccord. rtebral al colum n and  ction   of the ve vertebr Collection Colle

co rd from e ntire spinal spin al cord con suming to d is sect out the entire I t  is  tediouss an d too tim e consuming is  tediou ostt acand   m os sies. The  The   si m plest and  r outine necrop ne cropsies.  column for routine the bony b ony vertebral vert ebral column muscl e as possible from collection is to cu t away as m u ch muscle le m ethod of   collection ceptable ceptab spi nal cord th e spinal se g m e nts of the then   to c ut out seg vertebral colum col um n and then  ar ound the  the   vertebral us ing interva ls using vertebral colum colu m n at intervals the   vertebral lu mbar) by se v ering the  rvical, l, thoracic, thorac ic, lumbar) (c e rvica cord, therefore therefo re spinal   cord, action n may distort dis tort the spinal  saw. T h e cutting actio bone forceps fo rceps   or a saw. po rtion   of each only  the central portion tis sue trimmer to cu t   out only  por tant for the tissue it it   is im portant nt. al cord co rd segme segment. spinal spin

SU GGESTE D READING  ersity sity of Toronto P ress, Toronto, Univer thee Rabbit, Univ I. C r a igie, E. H., L ab oratory Guide to the Anatomy o f th C an a da, 1966. Febigee r, Philation and Breedin g  Techniques for Laboratory Anim als, Lea & Febig Reproduction 2. H a f e z , E .   S. E., Reproduc delphia, 1 970. York,, 1979. bbit, Academic P ress, New York Rabbit, 3. Kaplan, H . M. and T i m m o n s , E . H. ,  The Ra ology and the bi bio he   anatomy, physio physiology 4. K o z m a , C., M a c klin, W ., C u m m i n s , L . M., a n d   M a u e r , R. , T he an d Lab oratory Rabbit, W eisbroth, S. H .,   Flatt, R. E., an B iology o f the Laboratory chemistry of the  the   rabbit, in The Biology 1974.. Press,   New York, 1974 K r ause, A. L., E ds .,   Academic Press, Rabbit, Wm. C. Brow n Co., Dubuque, Io wa, 1977. Anato m y o f the Rabbit, 5. Mc L a u g h l i n , C. A ., Laboratory Anatom R abbit, Dover Pub Wells,,   T. A. G ., The Rabbit, Pu b   ., New York, 19 19 68. 6. Wells

 

161 INDEX

of rat, 43

A

Cerebral hemispheres of guinea pig, 126 of hamster, 95 of mouse, 70 of rat, 43 Cervix, 4 of guinea pig, I 09

Adrenal glands of guinea pig, 118 of hamster, 8 8 - 8 9 of mouse, 61, 64 of rabbit, 145 of rat, 34, 37 Anatomy, topographical, 8 Animals, identity of, 19 Appendix of rabbit, 140 Artifacts, 2, 4, 11-12, 1 4 - 1 6 Autolysis, 1 2 -1 4 , see Postmortem changes Autopsy, 17

of hamst hamster, er, 84 of mouse, 58 of rabbit, 140 of rat, 30 Cheek pouches of hamster, 92 Chloroform, as euthanasia agent, 4 Clitoral glands, see Preputial glands Closed system, for carbon dioxide, 2 Clothing, protective, 5 Coagulating glands, 27, 53, 79 Collection of samples for culture, 18 Colon of guinea pig, 112, 114 of hamster, 84 of mouse, 60 of rabbit, 140

B Bacteria, identification of, 18 Bile duct of guinea pig, 112 of hamster, 84 of mouse, 60 of rabbit, 140 of rat, 30 Bone marrow, 14, 16 of guinea pig, 118, 129 of hamster, 92, 101 of mouse, 64, 73 of rabbit, 1 147, 47, 55 of rat, 38, 48 Bouin's fixa fixative tive,, 15 Brain of guinea pig, 121-129 of hamster, 95 of mouse, 68 of rabbit, 51 of rat, 43 Brown fat of hamster, 79, 103

of rat, rat, 30 Color, 9, 10, 13 Congestion, 8 - 9 Consistency of lesion, reporting of, II Costachondral junction, see Ribs Cranium of guinea pig, 124 of hamst hamster, er, 95 of mouse, 68 of rabbit, 51 of rat, 43 Culture, collection of samples for, 18

D Decapitation, 4

c Carbon dioxide, overexposure to as euthanasia

Degeneration, postmortem, 9, 1 2 - 1 4 Denaturation of proteins during fixation, 14 Detection of ectoparasites, 18 Dewlap, of rabbit, 133 Diagnostic necropsies, 1 7 - 1 9 Diaphragm of guinea pig, 118 of hamster, 88 of mouse, 61 of rabbit, 145 of rat, 34 Disease surveillance programs, 18 Dissection, planes of, 8 - 9 Duodenum of guinea pig, 112 of hamster, 84

method, 2, 5 Carcinogenicity testing, 19 Carcinogenic risk, 15 Carnoy's fixative, 1 4 - 1 5 Cavia porcellus, 105 Cecum of guinea pig, 112 of hamster, 84 of mouse, 60 of rabbit, 140 of rat, 30 Cerebellum of guinea pig, 125-126 of hamster, 95 of mouse, 70 of rabbit, 51

of mouse, 60 of rabbit, 140

 

162

Necropsy Guide: Rodents and the Rabbit

ofrat, 30

Dura mater

techniques of, 16---17 Fixatives, 1 4 - 1 6

of guinea pig, 126 of hamster, 95 of mouse, 70 of rabbit, 51 of rat, 43

E Ear canal of hamster, 95 of mouse, 70 of rabbit, 51 of rat, 43 Ear, middle of guinea pig, 129 of hamster, I0 I of mouse, 73 of rabbit, 53 of rat, 43 Ectoparasites, detection of, 18 Electron microscopy, 17 Epididymis of guinea pig, 109 of hamster, 79 of mouse, 53 of rabbit, 137 of rat, 27

Esophagus of guinea pig, 118-120 of hamster, 92, 95 of mouse, 66, 68 of rabbit, 147, 147, 51 of rat, 40, 43 Ether, as euthanasia agent, 4 Euthanasia, 2 - 5 External examination of guinea pig, I 05 of hamster, 77 of mouse, 51 of rabbit, 133 of rat, 23 Eye, 16 of guinea pig, l 05 of hamster, 77 of mouse, 51 of rabbit, 133 ofrat, 23

Flank glands, of hamster, I 03 Fluid accumulations, 11, 18 Foramen magnum of guinea pig, 124 of hamster, 95 of mouse, 70 of rabbit, 51 of rat, 43 Forestomach, small lesions in, 17 Formaldehyde, 15 Formalin, 15, 17, see also Neutral buffered formalin Freezing, 12, 18 Frontal bones of guinea pig, 126 of hamster, hamster, 95 of mouse, 70 of rat, 43 Fungi, identification of, 18

G Gallbladder of guinea pig, 118 of hamster, 88 of mouse, 61 of rabbit, 145 Glottis of guinea pig, 120 of hamster, 95 of mouse, 68 of rat, 43 GLP, see Good Laboratory Practices Glutaraldehyde, 17 Good Laboratory Practices (GLPs), 19 Gubernaculum testis of guinea pig, I09 of hamster, 79 of mouse, 53 of rabbit, 137 of rat, 27 Guinea pig

euthanasia of, 2--4 necropsy of, 105-131

H

F Femur, 16 of guinea pig, 129 of hamster, I0 I of mouse, 73 of rabbit, 55 of rat, 43, 48 Fixation inadequate, 1 1 - 1 2 open and closed, 34, 61, 88, 114

Halothane, as euthanasia agent, 4 Hamster euthanasia of, 2--4 necropsy of, 7 7 -1 0 3 Harderian gland of guinea pig, 105 of hamster, 77 of mouse, 51 ofrabbit, 133 ofrat, 23 Heart of guinea pig, 118-121 of hamster, 92, 95

 

163  163   of  mouse, 66, 68

of m o use, 66, 66 , 68

of ra bbit, 14 7

of rabbi t, 14 7 rabbit,

of ra t , 40, 43

of rat, 40, 40 , 4 3

ription   of, 8 ngiosarcoma, rcoma, desc description H e m a ngiosa

19    17,   19 Lesion, Lesi on, 9 - 1 1 , 17,

a nd eosin, 15 Hemato xylin and

Liver

Hemorrhag e,

8

rtifacts   in, 4 a rtifacts

9 

H e morrhages,   lung, caused by c arbon dioxide, 2

of g uinea pig, 118 of ha m ster, 8 8 of m o u s e, e , 61

I

o f rabbit, 145 of  rat, 34

Ileum of gu inea pig, pi g, 112

Lungs

o f hamster hamster,, 84

of guinea   pig, 118- 12 0

o f mouse. 60 60  

of h amster amster,, 92, 95

of  rabbit, 140

hemorr hages by C 0 2 , 2  

of rat, rat,   30

of mouse , 66 of  rabbit, 147

f ixation, 1 5 -1 7 I m m ersion e rsion fixation, in trathoracic,   4 ion, intraperitonea in traperitoneall or intrathoracic, Injection, Inject for   necropsies, necropsies, Instrume nts for  Instruments

5

7

of  rat, 40 L y m p h   nodes ical cervical cerv

sites, 18 In t estinal para parasites,

of guine a pig, l 05

In testinal trac tractt   14  of g uinea pig, I 14

of h a mster, 79

of hamste hamster, r, 88

o f mouse, 53

of mouse mouse,, 61

o f rabbit, 135 of  rat, 24

bit, 140 14 0 of rab rabbit,

diastinal al and bron chial m e diastin

34   o f rat, 30, 34  1, 77, ex  orbital lacrimal g lands, 23, 5 51, lacr imal glands, Intraorbital and ex

of guinea   pig, 118- 120   of h amste amster, r, 92, 95

105, 133 n, 4 rathoracic cic injectio injection, raperitoneal oneal or int intrathora In t raperit

of mouse , 66, 68 68 151 1 ofrabbi t, 147, 15

J 

of rat, 40 m esente ric of guin ea pig, 112

Je j unum g, 112  112   o f  guinea pi pig,

of hams ter, 84

of ha m ster, 84

of m ou se, 60

of mous mouse, e, 60

of rabbit rabbit,, 140

it, 140  140   o f rabb rabbit,

o f r at, 30, 34

of rat, 30 30 ns, 38,  38,  64, 92, 118 118,, 147 Jugular vei veins,

K 

M glands M a m m a r y   glands ig, 1 0 5- 106 of  guinea p pig,

aldehyde, de, 17 ' s paraform aldehy K a m o v s k y 's

9 o f hamster, 7 79

Kidneys   Kidneys

of m ouse, 53

11 8 of guinea pig, 118

of rabbit rabbit,, 135

of  hamster, 8 8 - 8 9

of ra t , 24

o f m ouse, 61, 64

Mandible

5 of rabb rabbit, it, 14 145

1188 of guinea pig, 11

of ra t, 34, 37 rat,

of  hamster, 9 2 of m o use, 66

L  orbitall intraorbital bital and ex orbita Lacrim al glands, intraor

of guin guinea ea p ig, l 05  05 

of ra bbit, 1 47 of r at, 38 Mass, 9 M a sses

of h amster amster,, 77

reporting repo rting of, l 0  

51    o f m o use, 51

ion of collection s uperfic ial, collect

133   o f rabbit, 133

05   f or guin ea pig, l 05

of r at, 23

amster, r, 7 7 - 7 9 for h hamste

L a rynx

se, 5 1 - 5 3 for mou mouse,

of guinea guinea   pig, 118- 120

for rabbit, rabbit,   1 33

of h amste amster, r, 92, 95

fo r rat, 2 3 - 2 4

 

164

Necropsy Guide: Rodents and the Rabbit

Mesentery of guinea pig, 109, 112

of guinea pig, 109 of hamster, 84

of hamster, 84

of mouse, 58

of mouse, 61

of rabbit, 140

of rabbit, 140

of rat, 30

of rat, 34

Mesocricetus auratus, 77 Methanol fixation, 16 Methoxyflurane, as euthanasia agent, 4 Microorganisms, identification of, 18 Mites, detection of, 18 Moistening fluids, artifacts caused by, 1 1 - 1 2 Mouse euthanasia of, 2---4 necropsy of, 5 1 - 7 5 Muscles masseter of guinea pig, 118 of hamster, 92 of mouse, 66 of rabbit, 147 of rat, 38 temporal of hamster, 95 of rat, 43 Mus musculus, 51

N NBF, see Neutral buffered formalin Necropsy, 8 - 1 1 , 17 Neoplasia, 9 Neutral buffered formalin (NBF}, 14---15 Nitrogen, as euthanasia agent, 4 Nodule, 9 Nodules, reporting rep orting of, I0

0 Olfactory lobes of guinea pig, 126 of hamster, 95, 97 of mouse, 70, 73

p Palate, soft of guinea pig, 120 of hamster, 95 of mouse, 66 of rabbit, 147 of rat, 40

Pancreas of guinea pig, 112 of hamster, 84, 88 of mouse, 60 of rabbit, rabbit, 140 of rat, 30, 34 Parasites, detection of, 18 Parathyroid glands of guinea pig, 118-120 of hamster, 92, 95 of mouse, 66, 68 of rabbit, 147 of rat, 40, 43 Penis of guinea pig, pig, I09 of hamster, 79 of mouse, mouse, 53 of rabbit, 136 of rat, 27 Perfusion fixation, 1 5 - 1 7 Peyer's patches, 34, 61, 88, 114 Physiological saline as moistening fluid, 12 Pituitary gland of guinea pig, 121-129 of hamster, 95, 97 of mouse, 68, 73 of rabbit, rabbit, 151-153 of rat, 43 Planes of dissection, 8 - 9 Porphyrin secretion, 51 Postmortem changes, 9, 12-14, 16 Preputial glands, 24-27, 53, 79 Prostate gland of guine guineaa pig, I09 of hamster, 79, 81 of mouse, 53, 58 of rat, 27, 30 Protective clothing, 5 Proteins, denaturation of during fixation, 14 Pubic symphysis of guinea pig, I09 of hamster, 7 9 -8 1 of mouse, 53, 58 ofrabbit, 137, 140 of rat, 27, 30

of rabbit, 51 of rat, 43

Optic nerves of guinea pig, 126 of hamster, 97 of mouse, 73 of rabbit, 51 of rat, 43 Oral cavity of guinea pig, 118 of hamster, 92 of mouse, 66 of rabbit, 14 7 of rat, 38, 40 Organ perfusion, 16 Organ weight, II Oryctolagus cuniculus, 133 Ovaries

Pulmonary artifacts, 2, 4, 1 5 - 1 6

 

165

Q

zygomatic, of guinea pig, 105