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ABSTRACT

The Illinois Junior Academy of ScienceThis form/paper may not be taken without IJAS authorization.

CATEGORY : Health Science STATE REGION # 6

SCHOOL : Adlai E. Stevenson High School IJAS SCHOOL # 6092

CITY/ZIP : Lincolnshire, 60069 SCHOOL PHONE # (847) 415-4000SPONSOR: Mrs. Palffy

MARK ONE: EXPERIMENTAL INVESTIGATION

NAME OF SCIENTIST: Monica Muthaiya GRADE : 12th

* If this project is awarded a monetary prize, the check will be written in this scientist's name, and it will be his/her responsibility to distribute the prize money equally among all participating scientists.

PROJECT TITLE: The Effect of Stretch Injury on Cross Sectional Areas of the Tibial and Peroneal Divisions of the Sciatic Nerve.

Purpose: The purpose of this study is to compare the histological differences and cross-sectional area between the peroneal and tibial divisions of the sciatic nerve with or without manual stretching using fresh cadaver material. Nerve injury occurs in 1-2% of patients who undergo total joint arthroplasty. Injury to the peroneal division of the sciatic nerve is most common, being involved in nearly 80% of the cases. This experiment seeks out to find out why injury to the peroneal division, as opposed to the tibial division is the most common.

Procedure: Peroneal and tibial nerves were harvested bilaterally from 10 adult cadavers from a prone position. The sciatic nerve dissected free from surrounding tissue, including the peroneal and tibial nerves. The peroneal and tibial nerves sectioned into 2 samples, each 60 mm in length. A 25mm section in middle was isolated, also distal and proximal control samples taken. The proximal sections were stretched, embedded in paraffin, and stained w/Masson’s trichrome stain (distinguishes connective tissue from other components). The nerves examined under light microscopy and digital images were collected. These images were then calibrated and uploaded to ImageJ software. The nerves were analyzed for overall and individual fascicular cross sectional area and extent of roundness. The thickness of perineurium was measured at these 3 locations of the fascicle in the subsample and compared to their areas. Statistical analysis performed using SPSS software according to Friedman test, keeping right and left samples separate.

Conclusion: In conclusion, by examining the areas of the fascicles in each division of the sciatic nerve of fresh human cadaver nerves, there seems to be a pattern developing. Based on Dr. Amirouche’s current research and paper and my findings, there seems to be several histological differences between the tibial and the peroneal nerves. As already mentioned, the tibial nerve is much larger than the peroneal nerve, meaning that there are more fascicles in the tibial nerve. This numerical difference in fascicles and the excess amount of extra fascicular connective tissue explains why the area of fascicles in the tibial nerves were higher than in comparison to the area of fascicles in the peroneal nerves. We can conclude that the elliptical shape of the tibial nerves following stretch offers protection, but from this study, we cannot conclude why. This is not fully clear, but Dr. Amirouche’s study and my study do offer some understanding as to the differences between the two main nerves, their connective tissue similarities and differences, and other aspects which may eventually help to explain why the peroneal nerve is more at risk than the tibial nerve following the moderate stretch associated with hip surgery.

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While conducting this experiment, I did not handle any human cadaver parts. I worked in an office-like setting at the University of Illinois at Chicago and used the collected data from Dr. Amirouche and his fellow professors and physicians to conduct my experiment.

Safety concerns during these experiments mostly revolved around the use of the mice, and precautions taken included wearing of masks, gloves, and lab coats when handling mice and performing dissection.

SafetySafety concerns during these experiments mostly revolved around the use of the mice, and precautions taken included wearing of masks, gloves, and lab coats when handling mice and performing dissection.SafetySafety concerns during these experiments mostly revolved around the use of the mice, and precautions taken included wearing of masks, gloves, and lab coats when handling mice and performing dissection.SafetySafety concerns during these experiments mostly revolved around the use of the mice, and precautions taken included wearing of masks, gloves, and lab coats when handling mice and performing dissection.

SafetySafety concerns during these experiments mostly revolved around the use of the mice, and precautions taken included wearing of masks, gloves, and lab coats when handling mice and performing dissection.

SIGNED _________________________________________________Student Exhibitor(s) SIGNED _________________________________________________

Sponsor * *As a sponsor, I assume all responsibilities related to this project.

Muthaiya

SAFETY SHEET

The Illinois Junior Academy of Science

Directions: The student is asked to read this introduction carefully, fill out the bottom of this sheet, and sign it. The science teacher and/or advisor must sign in the indicated space.

Safety and the Student: Experimentation or design may involve an element of risk or injury to the student, test subjects and to others. Recognition of such hazards and provision for adequate control measures are joint responsibilities of the student and the sponsor. Some of the more common risks encountered in research are those of electrical shock, infection from pathogenic organisms, uncontrolled reactions of incompatible chemicals, eye injury from materials or procedures, and fire in apparatus or work area. Countering these hazards and others with suitable controls is an integral part of good scientific research.

In the box below, list the principal hazards associated with your project, if any, and what specific precautions you have used as safeguards. Be sure to read the entire section in the Policy and Procedure Manual of the Illinois Junior Academy of Science entitled "Safety Guidelines for Experimentation" before completing this form.

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The Effect of Stretch Injury on Cross Sectional Areas of the Tibial and Peroneal Divisions of the Sciatic Nerve

Monica Muthaiya, Adlai E. Stevenson High School, Grade 12

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Table of Contents

Page 1: Abstract

Page 2: Safety Sheet

Page 3: Title Page

Page 5: Acknowledgements

Page 6 - 7: Purpose and Hypothesis

Pages 8 - 12: Review of Literature

Page 13 – 14: Materials and Procedure

Page 15 – 17: Results

Page 18 - 21: Conclusions

Page 22 - 23: References

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Acknowledgements

I would like to thank my mentor for the duration of this experiment, Dr. Amirouche and the faculty of Department of Bioengineering at the University of Illinois at Chicago. Dr.

Amirouche allowed me to conduct research in his laboratory and I would not have gained this valuable experience or have this project to showcase if it wasn’t for him. The medical

and Ph.D. students in the laboratory also offered support and advice each day. In addition, I would like to thank Mr. Erdmann and Ms. Willock, the S.P.A.R.K. (Science Professionals of America as Resource Knowledge) coordinators, for providing me with

the means necessary in earning school credit for my research and the opportunity to showcase my work at the annual symposium.

I would also like to thank my sponsor, Mrs. Palffy, for taking time out of her busy schedule to help me with my project. She was always very patient and helpful. I am very thankful to have such a wonderful sponsor. I would also like to thank my parents, who

were encouraging of doing research at the University and offered constructive criticism.

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Purpose:

The purpose of this study is to compare the histological differences and

cross-sectional area between the peroneal and tibial divisions of the sciatic nerve

with or without manual stretching using fresh cadaver material. Nerve injury occurs

in 1-2% of patients who undergo total joint arthroplasty. Injury to the peroneal

division of the sciatic nerve is most common, being involved in nearly 80% of the

cases. This experiment seeks out to find out why injury to the peroneal division, as

opposed to the tibial division is the most common.

There have been very few experiments investigating the sciatic nerve

divisions in respect to nerve injury, through arthroplasty. With the conclusions of

this experiment, there will be support to see how the two divisions vary

histologically. Physicians and researchers will be able to use the results of the

experiment to understand how exactly the divisions differ and why one is more

easily damageable than the other, as described in the conclusion.

Hypothesis:

If tibial and peroneal divisions of the sciatic nerve are histologically compared, there

will be histological differences between the two since the peroneal nerve is less

protected and more prone to injury.

If tibial and peroneal divisions of the sciatic nerve are histologically compared, the

peroneal nerve will have a smaller area of axons, since it is less protected and more

prone to injury in comparison to the sciatic division.

Independent Variable: Division of Sciatic Nerve (Tibial or Peroneal)

Dependent Variable: Cross-Sectional Area of Sciatic Nerve Division and Histological

Differences.

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Constants: Temperature and environment cadavers were kept in, size of sectioned

of piece of nerve, program (ImageJ) and tool (Elliptical) used to analyze cross-

sectional areas.

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Review of Literature

Nerve injury tends to occur in 1-2% of patients who undergo total joint

arthroplasty (De Hart, 1999). Total joint arthroplasty literally means the "surgical

repair of a joint.” It is a procedure that is conducted to restore function to joints

after damage by arthritis or other trauma and illnesses. The goal of arthroplasty is

not only to restore the function of the joints, but also to reduce pain and swelling of

the surrounding areas (HeeMD, n.d.).

- an orthopedic surgery where the articular surface of a musculoskeletal joint is replaced, remodeled, or realigned by osteotomy or some other procedure.

Out of the 1-2% of patients who are injured from total joint arthroplasty,

injury to the peroneal division of the sciatic nerve is most common and is involved

in nearly 80% of the cases (Schmalzried, 1997). It is also concluded that the risk of

nerve palsy, in regards to total hip replacement, is typically higher for female

compared to male patients. The risk is also higher for individuals with a diagnosis of

developmental dysplasia and in patients undergoing revision surgery. Revision

surgery is when a surgeon must operate shortly after operating a surgery on a

patient recently (Farcy & Schwab, n.d.) and developmental dysplasia is when there

is an abnormality, often present at birth and in females, of the way that the hip joint

develops (Patient.co.uk., n.d.). The risk of nerve palsy in association with total knee

replacement is also increased for patients with valgus deformity, flexion contracture

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greater than 15 degrees, epidural anesthesia, use of pneumatic tourniquets, and for

those undergoing revision surgery (Park, 2013).

In a majority of cases, the origin of the palsy is unknown. Many nerve injuries

can be classified as neurapraxia, or the focal conduction block across the zone of

nerve injury. Conduction within the nerve, both proximal and distal to the lesion,

remains intact and the continuity of all structures including the axon is preserved.

Because peripheral nerves are sensitive to compression, unrecognized compression

may play a role in these cases and may be a large indicator as to how physicians can

prevent nerve injury to the sciatic nerve divisions.

The peroneal nerve is found on the outside part of the lower knee and is

responsible for transmitting impulses to and from the leg, foot, and toes. When

damaged, the muscles controlled by the peroneal nerve tend to lose sensation,

strength, and control. This leads to a condition called foot drop, which is essentially

the inability to raise the foot upwards (Peroneal Nerve Injury Information - The

Mount Sinai Hospital, n.d.). Trauma to the peroneal nerve can occur from knee

injury, a broken leg bone, ankle injuries, or surgery to the knee or leg, which will be

the focus of this experiment.

Causes of Damage/Discomfort:The common peroneal nerve is the most commonly injured nerve in the leg,

which is often attributed to its superficial location where it courses laterally around

the neck of the fibula. Since most injuries involve the peroneal division of the sciatic

nerve, it is important to be familiar with the gross anatomy. The common peroneal

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nerve is derived from L4, L5, S1, S2 as a part of the sciatic nerve. It travels to the

posterior component, supplies the short head of the biceps femoris in the thigh,

crosses posterior to the lateral head of the gastrocnemius, and becomes

subcutaneous behind the fibular head. It penetrates the posterior intermuscular

septum, and becomes closely opposed to the periosteum of the proximal fibula

(Wood, 1991). The superficial branch controls the muscles of the lateral

compartment of the leg, which function primarily to evert, or turn outward, the foot.

The deep peroneal nerve controls the anterior compartment of the leg, whose

muscles mainly act as dorsiflexors of the foot and toes. Dorsiflexors essentially

control the upward movement or extension of feet, toes, hands, or fingers.

The tibial nerve also originates from the sciatic nerve, with contributions

from L4, L5, S1, S2, and S3. It splits in the distal thigh and then passes through the

popliteal fossa. It then runs under the arch of the soleus and continues distally on its

undersurface (Wood, 1991). When the tibial nerve is damaged, one may have a loss

of plantarflexion of the foot, loss of flexion of the toes, and weakened inversion of

the foot. The tibial nerve is considerably larger than the peroneal nerve and has a

more distal distribution in the extremity with fewer points of connective tissue

tethering. This is the main reason for why physicians and researchers believe that

the tibial nerve is prone to less nerve damage during arthroplasty and other

surgeries. Dr. Gustafson noted the course of sciatic nerve fascicles to their muscle

targets in three fixed cadavers stained with hematoxylin and eosin (Gustafson,

2012). His work allowed researchers, such as Dr. Farid Amirouche, at the University

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of Illinois at Chicago’s Department of Bioengineering, to look into the histological

differences between the tibial and peroneal divisions of the sciatic nerve.

The prognosis for neurologic recovery is related primarily to the degree of

nerve damage. Complete recovery occurs in approximately 41% while another 44%

have only a mild deficit. Approximately 15% will have weakness that limits

ambulation and/or persistent dysesthesia (Schmalzried, 1997). Patients with some

motor function immediately after surgery and those who recover some motor

function within approximately 2 weeks of surgery have had a good prognosis for

recovery. It is unclear, however, as to why the peroneal division is more prone to

injury compared to the tibial division apart from the obvious structural difference.

Some of the literature suggests that the reason may be due to the subcutaneous

location of the peroneal nerve (King, 2008). The purpose of my study is to compare

the histological differences between the peroneal and tibial divisions of the sciatic

nerve with or without manual stretching using fresh cadaver material. I

hypothesized that since the peroneal nerve is more prone to injury, that there may

be histological differences between the peroneal nerve and tibial nerve, which is

more protected.

When comparing data results, one can use the t-test. The t-test is a statistical

test that is used to determine if there is a significant difference between the mean or

average scores of two groups. The t-test essentially does two things. Primarily, it

determines if the means are sufficiently different from each other to say that they

belong to two distinct groups. This is done by getting the average score of each

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group, and then getting the difference of the two means. The t-test also takes into

account the variability in scores of the two groups. This is called the standard error,

which simply answers the question: "How far is each score from the group mean?"

Thus, if the p value is greater than 0.05, we accept the null hypothesis and conclude

that the 2 means are actually the same. We have to conclude that the independent

variable did not have an effect on the dependent variable. The means are not

statistically different. If the p value is less than 0.05, then we reject the null

hypothesis and conclude that the 2 means are indeed different from each other and

the data is statistically significant. We can go on to conclude that, if all other factors

are the controlled in the experiment, then the independent variable has had an

effect on the dependent variable.

** ADD INFO ABOUT DR.AMIROUCHE’S RESEARCH, IMAGEJ PROGRAM, FINAL

STATISTICS PROGRAM USED (friedman test?)**

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Materials:

ImageJ software (National Institutes of Health public domain)

10 peroneal nerves from fresh human cadavers

10 tibial nerves from fresh human cadavers

1 scalpel

2-0 nylon sutures

Karnovsky’s Fixative (5% glutaraldehyde + 4% formaldehyde in 0.1M

cacodylate buffer)

Masson’s trichrome stain

Paraffin

Procedure:

1. The peroneal and tibial nerves were harvested bilaterally from 10 fresh

human cadavers. Any available adult patient was included; exclusion criteria

included patients with abnormal anatomy or history of nervous system

pathology or injury.

2. Dissection was completed with the cadavers in a prone position. After skin

incision, the dissection continued deep between the bodies of the hamstrings

(semitendinosus and semimembranosus) and biceps femoris.

3. The sciatic nerve was visualized and dissected free from the surrounding

tissue. The sciatic nerve was freed proximally to the gluteus maximus, the

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peroneal nerve was freed to its course around the fibular head, and the tibial

nerve was freed to its course under the arch of the soleus muscle. At this

midthigh location, branching is minimal. The proximal and distal poles the

nerves were transected with a scalpel and the nerves were freed from the

body.

4. The peroneal and tibial nerves were each sectioned into two samples, each

being approximately 60mm in length.

5. A 25mm section in the middle of each nerve sample was isolated and tied off

with 2-0 nylon sutures, which were circumferentially wrapped around the

nerve and secured with square knots. The distal control sample was

suspended from a frame, with the sutures lightly secured to the frame, at its

native length and placed in Karnovsky’s Fixative (5% glutaraldehyde + 4%

formaldehyde in 0.1M cacodylate buffer).

6. The proximal sample was tied with its sutures to a frame and stretched to

20-25% of its native length and placed in the fixative.

7. After 1 week in fixative, each nerve was washed 3 times in 0.1M-cacodylate

buffer at 7-day intervals.

8. The nerves were then embedded in paraffin, transversely sectioned and

stained with Masson’s trichrome stain (which distinguishes the connective

tissue from other components).

9. Each nerve was examined under light microscopy and digital images were

collected.

10. These images were calibrated and uploaded into ImageJ software (National

Institutes of Health public domain). Each nerve was analyzed for its overall

and individual fascicular cross sectional area and the extent of roundness [(4

x area) / (pi x diameter2)].

11. The thickness of the perineurium was measured at three locations of the

fascicle in a subsample, and compared to the respective fascicular area by

dividing these values.

12. Statistical analysis was performed using SPSS software according to the

Friedman test, keeping the left and right-sided samples separate. The sample

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size was relatively small and the data set did not have a normal distribution,

so a non-parametric analysis was used. Significance was at an alpha level of <

0.05%.

Results

*Nerve areas were calculated in mm2*

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Conclusion

To summarize, there are at least three main issues that point to the reason

why the tibial nerve has greater protection compared to the peroneal nerve against

stretch injury following hip surgery:  size, shape and distribution.

To begin, as a larger nerve with more fascicles, the tibial division of the

sciatic nerve has a greater proportion of the nerve occupied by extrafascicular

epineurial tissue, which is predominantly adipose tissue. Type I collagen of the

epineurium contributes most to tensile strength and protection, which may account

for this. There are also distinct differences between the epineurium and

perineurium’s mechanical properties and protection features against injury. Some

researchers, such as Brushart believe that the epineurium is best designed to

accommodate nerve stretch (Brushart, 2011).

Others, however, believe that the tensile strength and elasticity of a nerve

trunk depends more on the perineurium (Sunderland, 1965). It is currently believed

that a nerve, no matter its depth, with adipose tissue, and thus, cushioning, may

have an advantage over mechanical forces distributed to the delicate axons residing

in the nerve interior. A limitation on experiments intended to study this concerns

the fact that the sciatic nerves of rats or other similar species are not suitable

models for this tissue distribution, because they lack a similar amount of adipose

tissue as present in humans (Kerns 2008). The number of fascicles may directly

correlate with the forces evenly distributed on the tibial nerve.

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Dr. Amirouche’s experiment is the first study to recognize that the shape of

human nerve fascicles is predominantly oval, and becomes more so following

stretch. The first stage in his experiment was measuring the cross-sectional areas

with a circular tool, but when I went back to find the areas with an elliptical tool, it

was noted that this was a more efficient and accurate method of capturing the cross-

sectional area. The oval shape is most likely related to the observation that both the

endoneurial and epineurial connective tissues are tightly bound to the intermediate

perineurial layer, which is very cellular and compact. The nerve also lays in a

flattened plane between muscle layers along its course down the extremity.

Lastly, the anatomical distribution of the two nerves in the thigh might

contribute to the protection of the tibial nerve. Stress on an elongate tissue

dissipates according to length. The peroneal nerve contains a shorter course with

fewer muscle targets and has points easily susceptible to damage due to short,

abrupt tethering; its course is more superficial and lateral around the head of the

fibula as it enters the anterior compartment of the leg. Upon stretch, this may be the

greatest point of compression directly upon the underlying axons. It is assumed that

stretching of the nerve will affect both the blood supply and the axons directly

either by compression or by disruption of the axons themselves (Lundborg, 2004).

The connective tissues at all three levels afford an extensive level of protection

against axonal damage. The location and grouping of axons traveling along the

distal-to-proximal course of a nerve in the extremity can vary through a given nerve

fascicle or between fascicles. This was once thought to be intraneural chaos based

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on Sunderland’s plexus formation, but now is viewed as more orderly with partial

localization (Brushart 2011, p. 19).

Classification of fascicular patterns has large clinical significance and

applications if one repairs transected nerves with epineurial or fascicular repair. A

surgeon’s consideration of repair depends upon level of injury, the relative amounts

of fascicular and epineurial tissue and timing of surgery. Dr. Lundborg concluded

that the pathophysiology of nerve stretch is similar to nerve compression and large

nerves with a small amount of epineurium are more vulnerable (Lundborg, 20014).

Nerve roots at the proximal level lack these connective tissue coverings and are

particularly at risk to avulsion injuries. Dr. Amirouche’s findings and my findings

suggest that at more peripheral locations the internal epineurium between fascicles

is also an important consideration.

Both the studies conducted by Dr. Amirouche and I certainly have many

limitations and errors. It would be ideal to have more samples and be able to

examine more than just the mechanical properties of the nerve. For this all to occur,

there must be extensive nerve lengths and this would not be able to include intrinsic

control specimens. Also, the left and right sides of the human nerves cannot be

assumed to be equal and the sections of the nerves that were taken were

approximates, not exact measurements, as that is fairly difficult to measure due to

the ovular shape of the nerves. When a nerve stretches, it is believed that the

perineurium is the first to break. However, a break in the axon would have more

functional consequences, as indicated by the study. Further studies in Dr.

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Amirouche’s laboratory may explore the functional, as opposed to structural,

consequences of the study.

In conclusion Dr. Amirouche’s and my histological study provide a solid basis for

comparing the specific fascicular patterns for the two main nerves of the lower

extremity of interest to orthopaedic surgeons. Several differences between the tibial

versus the peroneal may relate to the specifically of sparing afforded to the tibial

nerve. The most obvious of these distinctions is the large size of the tibial nerve with

many more fascicles than the peroneal nerve. The size factor includes both the

number of fascicles and the amount of extra fascicular connective tissue. It is not yet

clear how the significant shape change seen in the tibial nerve following stretch,

from a more circular shape to a more oval shape, confers protection. The present

study does provide some new insights into differences between the two main

nerves, their connective tissue similarities and differences and other aspects which

may help to explain why the peroneal nerve is more at risk than the tibial nerve

following the moderate stretch associated with hip surgery. Such insights, i.e.

“handle with care”, can serve as precautions in surgical indications in order to

prevent sciatic nerve damage, and specifically, to the peroneal division.

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References

Brushart, T.M. 2011, Nerve Repair, (New York: Oxford University Press).

 DeHart et al. Nerve Injuries in Total Hip Arthroplasty. Am Acad Orthop Surg March 1999 vol. 7 no. 2 101-111.

Farcy, J., & Schwab, F. (n.d.). Revision Surgery. Retrieved January 22, 2015, from http://www.orthospine.com/index.php/home-mainmenu-1/32

Gustafson,  K.J., Grinberg, Y., Joseph, S., and Triolo, R.J., 2012, Human distal sciatic nerve fascicular anatomy: Implications for ankle control using nerve-cuff electrodes., J. Rehabil. Res. Dev., 49(2): 309-322.

HeeMD. (n.d.). Retrieved January 22, 2015, from http://www.heemd.com/library/?id=58&t=pr/

ImageJ Features. (n.d.). Retrieved January 22, 2015, from http://imagej.nih.gov/ij/features.html

Kerns JM. The microstructure of peripheral nerves. Tech. Regional Anesth Pain Management 2008 Vol.12, 127-133.

King JC. Peroneal neuropathy. In: Frontera WR, Silver JK, Rizzo TD, eds. Essentials of Physical Medicine and Rehabilitation: Musculoskeletal disorders, pain and rehabilitation.2nd ed. Philadelphia, Pa: Saunders Elsevier; 2008:chap 66.

Millesi, H. and Terzis, J.K., 1984, Nomenclature in peripheral nerve surgery, Clin. Plast. Surg., 11: 3-8.

Park et al. Common peroneal nerve palsy following total knee arthroplasty: prognostic factors and course of recovery. J Arthroplasty 2013 Oct;28(9):1538-42.

Peroneal Nerve Injury Information - The Mount Sinai Hospital. (n.d.). Retrieved January 22, 2015, from http://www.mountsinai.org/patient-care/health-library/diseases-and-conditions/peroneal-nerve-injury

Scherer SS. Nodes, paranodes, and incisures: from form to function. Ann NY Acad Sci. 1999 Sep Vol.14, no.883, 131-142.

 Schmalzried et al. Update on nerve palsy associated with total hip replacement. Clin Orthop Rel Res 1997 Nov;(344):188-206.

Sunderland, S., 1965, The connective tissue of peripheral nerves. Brain, 88: 841-854.

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Watson J, Gonzalez M, Romero A, Kerns J. Neuromas of the hand and upper extremity. J Hand Surg Am. 2010 Mar Vol.35, no.3, 499-510. JMK – Is there a place in the paper to cite this study?

Wood et al. Peroneal nerve repair. Surgical results. Clin Orthop Rel Res 1991 Jun;(267):206-10.

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