Lab Report Y.docx

MEDSCI 206 –  206 – Laboratory Laboratory 2 “Human nerve conduction velocity”

William Lin 6737564

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Date: 17  August 2015

Experiment: Human nerve conduction velocity measurements Condu cted by  : William Lin, Samuel Yu, Gagan Joshi, Kelly Cudmore (A7) Aims: Part Part 1 

- Record, and interpret EMG as from a range of voluntary contractions (FDM) - Measure and calculate conduction velocities for ulnar and median nerve by measuring Compound muscle action potential at sites (FDM and APL respectively) as well as stimulating at respective sites Part Part 2 

- Analyse and interpret both normal and abnormal nerve conduction measurements and thereby proposing potential neuropathies. Introduction:

In mature mammals, extrafusal muscles are innervated by single alpha-motor neurons (lower motor neurons). Because the number of available neuronal axons are in large deficit compared to the muscular tissue present in the body, neurons often branch-out to form multiple synaptic connection with muscle fibres to attain a large surface area for electrical stimulation (Purves, 2008). 2008) . The term “Motor unit” defines both the alpha motor neurons that forms the neuromuscular junction (via its axon terminal) as well as the muscle fibres which that particular neuron innervates (Purves, 2008). In the human body, there are various classes of motor unit, ranging from S (Slow) motor units, FF (Fast-fatigable) motor units and an intermediate class known as FR (Fast-fatigue resistant) motor units (Purves, 2008).  Neurons, otherwise also known as Nerve cells, are specialized tissue which act as the electrical wiring of the body, its ability to conduct electrical signal allows for it to propagate and transmit information from the higher centres to the lower target organs (Purves, 2008). The lower motor neurons (alpha-motor) which forms the neuromuscular junction with muscle fibres are modulated through synaptic interaction (at the level of the ventral horn) with upper motor neurons which project from the cerebral cortex etc. (Purves, 2008). The two nerves which are focused on in the laboratory session are the ulnar nerve and the Median nerve which all derive from branches of the brachial plexus (Martini, Timmons & Tallitsch, 2012). The ulnar nerve originates as one of the major nerve divisions from the medial cord and contributes in both sensory and motor functions. The median nerve like the ulnar nerve, also serves in both sensory and motor functions and is formed from collectively the musculocutaneous nerve and the medial cord (Martini, Timmons & Tallitsch, 2012). When a neurone is stimulated by some form of electrical electr ical activity, it results in a membrane depolarization which leads to an influx of Ca2+ ions via voltage-gated calcium channels. This influx of calcium ions, causes vesicles containing the neurotransmitter ACh to dock with  proteins that primes its fusion. Once the vesicles fuse, its contents are released into the synaptic cleft where it binds to the nicotinic receptors at the post-synaptic density which causes an excitatory end-plate current. (Boron & Boulpaep, 2012) Similar to neurones, muscle fibres also have the ability to conduct electrical signal in order to

MEDSCI 206 – Laboratory 2 “Human nerve conduction velocity”

William Lin 6737564

 produce fine and coordinated contractions. As a result, electrical current that is generated by a group of muscle fibres will propagate through the fluid-filled tissue and captured at the surface of the skin by recording electrodes; this method which electrical activity of voluntary contraction is known as EMG (Electromyography) (Reaz, Hussain & Mohd-Yasin, 2006). EMG implements a non-invasive method because electrodes are placed at the level of the skin and records a composite of electrical activity generated by motor pools. CMAP, which stands for compound muscle action potential is a compound electrical signal produced by muscles when recruited via electrical stimulation (neuronal or external). Because it measures action potential across a motor neuron pool, it is a “graded response” rather than an individual motor unit which produces a “all or none response” (The Compound Action Potential (CAP) Of the Frog’s Sciatic Nerve, 2005). On a clinical level, CMAP and EMG’s are often coupled to produce a more three dimensional perspective on the nature of the neuropathy as well a s to distinguish a severity and type of disorder. Comprehensive interpretation of the data allows one to localize the nature of the neuropathology i.e. Non-focal lesion or Focal lesion and allows for better diagnosis for pathological events e.g. Carpal Tunnel Syndrome (Hopkinsmedicine.org, 2015). Those CMAP measurements allow for conduction velocity to be calculated, it is often affected by personal factors as well as anthropometric factors such as age, sex, height etc. (Stetson, Albers, Silverstein & Wolfe, 1992) M ethods

1A.

-Set up the DS7 Stimulator (as shown in page 41 of Laboratory manual). It should be advised that if the red fault light comes, please notify experienced personnel e.g. Demonstrator or Technician -To locate the digiti minimi muscle, place load down on to subject’s little finger (5 th digit) and ask him/her to contract against this load. Once the muscle belly had been located, clean the region of the digiti minimi muscle with alcohol and rub it gently with the sand paper  provided -Make the following electrode placements as per below: (Ref. Diagram in page 43) - Active (negative) electrode to be placed over the FDM muscle (lateral-ventral side near the funny bone region) - Reference (positive) electrode to be placed distally to the 5th metacarpal-phalangeal joints on the anterior side of the subject’s little finger (5th digit) - Ground electrode to be placed on the back of the subject’s hand above the region of the wrist-joint -In a fixed time frame of 10 seconds (as calibrated per page 41), the is asked to perform a gradual progression of muscular actions starting from resting and then to minimal, moderate and sustained maximal contraction. -It is advised that data should be saved prior to moving on to the next part (1B) 1B.

-LabChart settings were readjusted to that described in page 44 -The Ground and reference electrode placements were kept as per part 1A, however there is an addition of stimulation electrode which is held by either the subject or other group

MEDSCI 206 – Laboratory 2 “Human nerve conduction velocity”

William Lin 6737564

member at the region of the medial epicondyle of the humerus. - Ensure that the anode (red, positive electrode) was more proximal along the length of the arm and not placed near the median nerve and that the cathode (black, negative electrode) lies on top of the ulnar nerve - Starting from 5mA of stimulus protocol, increase the stimulus parameter in intervals of 5mA until a first visible signal is evident on the LabChart recording; take a note of this current - Go down (decrease) in smaller intervals from this current (that produced the first signals) in smaller intervals (e.g. 1mA) until you lose your signal again; the current just before losing your signal is the threshold potential - Now continue to implement a stimulus protocol of 5 mA intervals until you get no more increase in CMAP amplitude (make sure at least 5 increasing stimulus all produce same amplitudes); this is your supramaximal threshold - For the wrist, the stimulating electrode is now moved to the anterior, medial side of the wrist (same side which the elbow was stimulated at) and stimulate the wrist with the already measured supramaximal threshold potential (in our case 75 mA).

MEDSCI 206 – Laboratory 2 “Human nerve conduction velocity”

William Lin 6737564

Part 1C.

- Recording electrode placement as follows - Negative electrode over muscle belly of APL –  Right hand - Positive electrode positioned distally to the metacarpal-phalangeal joint - The grounding electrode should be maintained in the position it had conformed to in the  previous experiment (Part 1B) - Stimulating electrode is NOW placed on the median nerve which located on the posterior surface of the arm i.e. it is located slightly above the elbow - For the wrist-stimulation, the electrode should be placed between the tendons of the FCU and PL - Follow similar procedures described in part 1B of measuring the threshold and supramaximal stimulus as well as the wrist-level stimulation (NOTE: Only difference is stimulating electrode placement) Conduction Velocities were calculated as per follows: - Latency was the time taken from the initial stimulus artefact to the CMAP amplitude spike i.e. initial positive deflection - Difference in latency was calculated LatencyElbow –  LatencyWrist - Distance between the elbow and the wrist was measured by marking the position of the stimulating electrode placements and measuring the distance between - Conduction velocity = distance/difference in latency - v = d/t - v = 0.22/0.0051 = 43.17 ms -1 (Ulnar nerve)

MEDSCI 206 – Laboratory 2 “Human nerve conduction velocity”

William Lin 6737564

Results

Muscle at rest

Medium contraction

Minimal contraction

Sustained maximal contraction

   )    V   m    (   e   g   a    t    l   o    V

Time (s)

 Figure 1A. EMG of different levels of voluntary contraction of the flexor digiti minimi (FDM) –  (Group A6, 2015) Figure 1. Shows a range of voluntary contractions that were made over the course of 10 seconds. When the Muscle (FDM) is in its resting (basal) tone, there is very minimal fluctuation in amplitude signals; as the tone of muscle activity is increased (from Minimal to Maximal) there is an evident increase in amplitude (~ 1mV at Minimal contraction compared to ~ 4mV at maximal contraction) accompanied with an increased frequency of the spikes.

   )    V   m    (   e   g   a    t    l   o    V

Time (ms)

 Figure 2. CMAP of Ulnar nerve with varying stimulus parameters (mA) at the Elbow Joint Figure 2 shows a graded response in terms of the CMAP of the Ulnar nerve when stimulated at increasing stimuli parameters. A pre-trigger of 10 ms was used and the CMAP of the Ulnar nerve was recorded for a fixed duration of 50 ms. First CMAP

MEDSCI 206 – Laboratory 2 “Human nerve conduction velocity”

William Lin 6737564

Time (ms)

 Figure 3. CMAP for Ulnar nerve at its supramaximal threshold (75 mA) stimulated at both the elbow and wrist Figure 3. When stimulated at both the elbow and the wrist joint, an evident delay was observed in that of the CMAP resulted from the elbow joint stimulation compared to that resulting from the wrist stimulation. A latency of 0.0051s was calculated from the two supramaximal deflections; the distance between the elbow and wrist electrode  placement was measured to be that of 0.22m (22 cm) and from the above data, a conduction velocity of 43.17 ms -1 was calculated

   )    V   m    (   e   g   a    t    l   o    V

MEDSCI 206 – Laboratory 2 “Human nerve conduction velocity”

William Lin 6737564

Time (ms)

 Figure 4. CMAP of median nerve with varying stimulus parameters (mA) at the Elbow Joint Figure 4 shows a graded response with increasing current stimulation at the median nerve at the level of the Elbow joint. Similar to Figure 2, a 10 ms delay was implemented; the CMAP si gnal was measured over a period of 50 ms. Sub-threshold stimulus (5 mA  –  40 mA) produced no visible signal on the LabChart programme. Stimulus parameters were made in 5 mA intervals, and the threshold stimulus was identified at 45 mA. From 45 mA, a graded increase is evident until 90 mA (Supramaximal stimulus) to which further stimuli produced no further increase in the CMAP signal    )    V   m    (   e   g   a    t    l   o    V

MEDSCI 206 – Laboratory 2 “Human nerve conduction velocity”

William Lin 6737564

   )    V   m    (   e   g   a    t    l   o    V

Time (ms)

 Figure 5. CMAP for Median nerve at its supramaximal threshold (90 mA) stimulated at both the elbow and wrist Figure 5. When the CMAP signals from supramaximal stimulation was recorded for the Median nerve at the elbow and

MEDSCI 206 – Laboratory 2 “Human nerve conduction velocity”

William Lin 6737564

Table 1. Class data of conduction velocities for both ulnar and median nerves acquired from different  subjects Group

Ulnar Nerve

Median Nerve

A1

33.6

45.5

A3

43.9

59.5

A4

50.2

45

A5

61.86

63.96

A6

63.6

53.1

A7

43.1

62.2

A8

70.3

38.21

A9

42.1

31.9

A10

62.5

54

B2

37.5

70.7

B3

51.9

48.9

B4

40.3

26.3

B6

48.81

44.4

B7

60.23

52.68

50.70714286

49.73928571

Average

Table 1. Shows that conduction velocities for the two nerves (ulnar and median) show variations  between different human subjects. The range of conduction velocities for the ulnar nerve between individuals is 33.6 ms -1 to 70.3 ms -1 and the range for the median nerve is 31.9 ms -1 to 70.7 ms -1. Some individuals show a faster conduction velocity for the ulnar nerve (and others vice versa), as a result no obvious trend can be identified between the two nerves. Despite individual variation, average values (ref. to table, averages row) suggest that the two nerves were quite similar in their conduction velocities; 50.7 ms -1 (ulnar) and 49.7 ms -1 (median). Not all groups had inputted their values resulting an incomplete series of values.

MEDSCI 206 – Laboratory 2 “Human nerve conduction velocity”

William Lin 6737564

Discussion:

Muscle fibres are specialized contractile fibres which respond to neural stimulation from the higher centre (Brain or spinal cord) causing in an influx of ion into its sarcoplasm and ultimately results in the depolarization of its local electrical field (Purves, 2008); this depolarized electrical field then propagates through fluid-composed tissue and is measured at the level of the skin (Reaz, Hussain & Mohd-Yasin, 2006) . The recording method implemented in the lab is that of a non-invasive method i.e. mounted to the skin of the subject, as a result the signal produced in figure 1. Illustrates a composite of all the local action potentials produced by the muscular tissue under the region which the electrodes were placed. During voluntary contraction, electrical signal is transmitted from conduit of neuronal pathway from the Primary motor cortex via alpha motor neurons to the muscle fibre targets (Purves, 2008). Evident from figure 1, an increase in the voluntary contraction generated a larger amplitude as well as higher frequency in the EMG signals. This observation can be explained with the fact that more motor units are recruited in the process and therefore resulting in a greater degree of depolarization of the local muscle fibres required for the action  (Reaz, Hussain & Mohd-Yasin, 2006) . Because the recording method records the composite action potential of the motor units, depolarization of individual muscle fibres occur at ra ndom intervals and thereby resulting in an increased frequency i.e. more electrical activity is packed within a given time frame. (Reaz, Hussain & Mohd-Yasin, 2006)  Despite a fairly obvious trend of increased amplitude and frequency with increasing voluntary contraction, there is still evident (but minimal) electrical activity even during the muscles resting stage; this may be due to multiple factors but appropriate assumptions are the basal tone of Gamma motor neurons which helps to maintain a degree of tautness and therefore calibrates the sensitivity of the muscle spindles (Purves, 2008). Another  potential cause is due to the electrical noise of the surrounding which may also be picked up on the EMG recording. (Chowdhury et al., 2013) The CMAP curves were of a biphasic nature, this is because the recording electrodes consist of both a positive and negative component; when CAP passes through the proximal electrode it generates an upward deflection and vice versa  (The Compound Action Potential (CAP) Of the Frog’s Sciatic Nerve, 2005). Figures 2 and 4 illustrate the CMAP which is recorded upon differential stimulation parameters (mA) at the ulnar nerve (elbow) and the median nerve (elbow) respectively. The curves that are seen in these CMAP recordings show that of a graded response i.e. increase stimulus parameter will result in a larger amplitude in signal. CMAP signals differ from singular muscular action potential (“All or none -response) in the sense that it records the action potential generated by a local composite of motor units. The signal i s graded because of the differential stimulus threshold of motor neurons; the larger neuronal axons are more easily depolarized (excitable) than the smaller motor axons. When stimuli  parameter increases, more axons surpass this threshold potential and thereby recruiting a larger number of motor units  (The Compound Action Potential (CAP) Of the Frog’s Sciatic Nerve , 2005). This idea can be similarly superimposed to the regulation of muscular force i.e. when stimuli  parameter increased, the subject demonstrated a greater amount of force generated due to an increased number of motor units recruited; this systematic relationship is regarded as the size  principle (Purves, 2008). In both figures 2 and 4, it is evident that below a certain stimulus current, there is no definitive CMAP signal. In the CMAP recording for the ulnar nerve (elbow), stimuli below 23 mA generated no signal, and in the recording for the median nerve (elbow), stimuli below 45 mA generated no signal. The stimuli current to which a CMAP is  produced is known as the threshold potential. Threshold potential defines a point to which stimulation will result in greater sodium influx (causing depolarization) compared to  potassium out-flux (repolarization) (Purves, 2008) and thereby causing propagation of action  potential across the muscular tissue to produce the compound action potential. As well as a

MEDSCI 206 – Laboratory 2 “Human nerve conduction velocity”

William Lin 6737564

threshold stimulus, supramaximal stimulus currents were also recorded for both the ulnar and median nerve (elbow, ref. figure 2 and 4) at 75 mA and 90 mA respectively. Supramaximal stimulation exists because there is a finite number of motor units recruited and further stimulation past this point will have an uncorrelated relationship to motor units recruited. (Campbell, n.d.)

Figures 3 and 5 illustrates the supramaximal stimulation of the ulnar and median nerve at  both the elbow and wrist joint respectively. In figure 3. The conduction velocity is calculated to be 43.17 ms-1 and 61.162 ms-1 in figure 5. The conduction velocity at these both sites all fell within the normal range of conduction velocities (51-75 ms-1 for ulnar nerve conduction and 49-74 ms-1 for median nerve). However, when we compare these conduction velocities values, it is evident in our subject that the conduction velocity for the median nerve (61.2 ms1) was much higher than that of the ulnar nerve (43.2 ms-1). This difference in conduction velocity could be attributed to the fact that it has been proposed that the median nerve is generally larger in diameter compared to that of the ulnar nerve  (Kundu, Harreby & Jensen, n.d.) ; a larger diameter of the axon will result in a decrease of internal resistance to current and increase passive current flow (Purves, 2008) . Furthermore, other factor which may attribute to the increased conduction velocity of the median nerve is a notion regarding neuromuscular  perisynaptic Schwann cell activity in regulating myelination and plasticity of motor neurons as a result of increased muscle memory etc (Auld & Robitaille, 2003) . In figure 3, the amplitude of CMAP signal between ulnar nerve at the elbow and wrist suggested that the CMAP signal at the wrist was lower in amplitude to that in the elbow. This observation is most likely due to the fact that electrodes are placed quite distal to the nerve such that only more terminal nerves may be stimulated (Martini, Timmons & Tallitsch, 2012) . However, in figure 5, an opposite observation was made such that the wrist CMAP signal of the median nerve was larger than that at the elbow. This could be due to human error i.e. subject may have been moving when recordings were made and therefore adding electrical noise to the signal recording. When looking at the class data, average values of 50.7 ms-1 (Ulnar nerve) and 49.7 ms-1 (Median nerve) were calculated (ref. table 1); although these values all fall within the normal ranges of nerve conduction velocity (ref. Part B), they all situate at the lower limits of their  physiological normal range. A Factor which has a positive relationship to nerve conduction velocity is temperature; an increase in temperature affects to reduce the temperature dependent velocity and thus increases conduction velocity  (Waxman, 1980) . Factors which have a general negative correlation to conduction velocity are: Hand factors e.g. index finger circumference, Height (decrease in 0.5 m/s per inch increase in height) and sex, which is not valid in this experiment because subjects are all of similar age (18-20). (Stetson, Albers, Silverstein & Wolfe, 1992)

Variables applicable to the Nerve conduction experiment can be narrowed to some of the following: There may be variation in experiment protocol between different groups e.g. Some groups may have had another member (other than the subject themselves) holding the stimulating electrode; this is more likely to reduce a degree of error when recording as the subject may move when muscles contract therefore resulting in additional electrical noise. There is also an undeniable factor of individual variation; as mentioned in the above  paragraph, different personal factors and anthropometric factors will ultimately affect the results of the nerve conduction velocity. Time limit on certain groups would have meant that some groups did not have time to repeat measurements and therefore leaving a possibility that results may have been inaccurate but no further time was allowed to make repeated measurements. Some errors which may have resulted from these variables are the differential  placement of electrode between each recording, which will ultimately affect the region of stimulation to a certain extent. Furthermore, some subjects may feel anxious or nervous

MEDSCI 206 – Laboratory 2 “Human nerve conduction velocity”

William Lin 6737564

during the experiment and therefore perspiration is a normal response; however this means that the electrodes are of a higher tendency to fall off and therefore replacement will result in a slightly different position each time this occurs.

Conclusion:

The findings from experiments 1A, B and C demonstrate the muscle’s bi -functional characteristic i.e. it is able to both conduct electrical activity as well as generating contractile tension as seen in the EMG. A graded response was evident in the CMAP signals for both the ulnar nerve and median nerve recording and this therefore satisfies the pre-established Size  principle theory relating muscle recruitment to force generated (and therefore electrical activity of motor neuron pool). Conduction velocities that were calculated for our group fell under the normal physiologically prescribed ranges of value but a closer comparison to the average class data allowed us to understand the degree of variability that may arise in a non pathological state due to factors such as human errors, personal and anthropometric factors as well as a potential flaw in the experiment protocols.

MEDSCI 206 – Laboratory 2 “Human nerve conduction velocity”

William Lin 6737564

References

Auld, D., & Robitaille, R. (2003). Perisynaptic Schwann Cells at the Neuromuscular Junction:  Nerve- and Activity-Dependent Contributions to Synaptic Efficacy, Plasticity, and Reinnervation. The Neuroscientist, 9(2), 144-157. doi:10.1177/1073858403252229

Boron, W., & Boulpaep, E. (2012). Medical Physiology, 2e Updated Edition. London: Elsevier Health Sciences.

Campbell, W. Essentials of electrodiagnostic medicine.

Chowdhury, R., Reaz, M., Ali, M., Bakar, A., Chellappan, K., & Chang, T. (2013). Surface Electromyography Signal Processing and Classificati on Techniques. Sensors, 13(9), 1243112466. doi:10.3390/s130912431

Han, B., Lin, O., & Isherwood, G. (2015). Human nerve conduction velocity (p. Results: Figure).

Hopkinsmedicine.org,. (2015). Nerve Conduction Studies | Johns Hopkins Medicine Healt h Library. Retrieved 23 August 2015, from http://www.hopkinsmedicine.org/healthlibrary/test_procedures/neurological/nerve_conductio n_velocity_ncv_92,P07657/

Kundu, A., Harreby, K., & Jensen, W. Comparison of median and ulnar nerve morphology of Danish landrace pigs and Göttingen mini pigs, 1-4.

Martini, F., Timmons, M., & Tallitsch, R. (2012). Human anatomy. Boston: Pearson Benjamin Cummings.

Purves, D. (2008). Neuroscience. Sunderland, Mass.: Sinauer.

Reaz, M., Hussain, M., & Mohd-Yasin, F. (2006). Techniques of EMG signal analysis: detection, processing, classification and applications (Correction). Biol. Proced. Online, 8(1), 163-163. doi:10.1251/bpo124

MEDSCI 206 – Laboratory 2 “Human nerve conduction velocity”

William Lin 6737564

Stetson, D., Albers, J., Silverstein, B., & Wolfe, R. (1992). Effects of age, sex, and anthropometric factors on nerve conduction measures. Muscle & Nerve, 15(10), 1095-1104. doi:10.1002/mus.880151007

The Compound Action Potential (CAP) Of the Frog’s Sciatic Nerve. (2005) (1st ed., pp. 1 17). Montreal.

Waxman, S. (1980). Determinants of conduction velocity in myelinated nerve fibers. Muscle & Nerve, 3(2), 141-150. doi:10.1002/mus.880030207