REANIMATION OF A DENERVATED MUSCLE USING UPPER
MOTONEURON INJURED LOWER MOTONEURONS IN SPINAL
CORD INJURY PATIENTS: A RAT MODEL
by
SREENATH NARAYAN
Submitted in partial fulfillment of the requirements
For the degree of Master of Engineering
Thesis Advisor: Dr. Robert F. Kirsch
Department of Biomedical Engineering
CASE WESTERN RESERVE UNIVERSITY
January, 2006 CASE WESTERN RESERVE UNIVERSITY
SCHOOL OF GRADUATE STUDIES
We hereby approve the dissertation of
______
candidate for the Master of Engineering degree *.
(signed)______(chair of the committee)
______
______
______
______
______
(date) ______
*We also certify that written approval has been obtained for any proprietary material contained therein. Contents
1 Introduction 6
2 Background 9
2.1 Injury to Peripheral Nerves ...... 9
2.2 Current Techniques ...... 9
2.2.1 Orthoses ...... 9
2.2.2 Tendon Transfers ...... 10
2.2.3 Peripheral Nerve Rerouting ...... 11
2.2.4 Direct Muscle Neurotization ...... 12
2.3 Related Experiments ...... 13
2.3.1 Regrowth of Nerves Under Active Control Through an Empty Epineu-
ral Sheath ...... 13
2.3.2 Effect of Conduction Block ...... 14
2.3.3 Diaphragmatic Reanimation ...... 14
3 Methods and Materials 15
3.1 SCI and Denervation ...... 16
3.2 Nerve Transfer ...... 17
3.3 Post Chronic Surgery Care ...... 19
3.4 Force Analysis ...... 20
3.4.1 Muscle Characterization (Pulse Width Modulation) ...... 20
3.4.2 Spinal Cord Injury Completeness Testing ...... 22
1 3.4.3 Assurance of Graft Area Conduction ...... 23
4 Results 24
4.1 Animal Care ...... 24
4.2 Pulse Width Modulation ...... 26
4.3 Spinal Cord Injury Verification ...... 30
4.4 Assurance of Graft Area Conduction ...... 32
5 Discussion 34
5.1 Pulse Width Modulation ...... 35
5.2 Spinal Cord Injury ...... 36
5.3 Assurance of Nerve Graft ...... 37
Acknowledgments 38
References 38
2 List of Tables
1 Summary of Force Data ...... 31
3 List of Figures
1 Three Types of Nerves in SCI ...... 6
2 Picture of Exposed Spinal Cord ...... 17
3 Picture of Sciatic Nerve and its Branches ...... 18
4 Diagram of Procedure 1 ...... 19
5 Diagram of Procedure 2 ...... 20
6 Force Measurement Jig ...... 21
7 Raw Muscle Force Data ...... 26
8 Length-Tension Curve ...... 27
9 Pulse Width-Force Curves ...... 28
10 Spinal Cord Lesion Verification ...... 30
11 Graft Area Conduction Efficiency ...... 33
4 Abstract
Reanimation of a Denervated Muscle Using
Upper Motoneuron Injured Lower Motoneurons in
Spinal Cord Injury Patients: A Rat Model
Abstract
by
SREENATH NARAYAN
This project aims to show that a denervated muscle can be reanimated following spinal cord injury using upper motoneuron injured lower motoneurons regrowing through non-electrically excitable nerves. The newly reanimated muscle can then be stimulated with command signals from an electrical stimulation system. A rat model was created with a spinal cord injury and a transection of the tibial nerve to create the denervation. One week post injury, the upper motoneuron injured peroneal nerve was transferred to the sheath of the tibial nerve.
About five weeks after that, force analysis showed significant regrowth. The same force analysis was performed on both the experimental side, which had undergone the surgical procedures outlined above, and the contralateral side, which previously had not undergone any peripheral surgical procedures. The maximum experimental side gastrocnemius force was approximately 50% that of contralateral side.
5 1 Introduction
There are approximately 11,000 new cases of spinal cord injury (SCI) each year [38].
Tetraplegia, in both the complete and incomplete form, accounts for 56.4% of all SCI [37,
38], and results in total or partial paralysis of the body from the neck down [48]. Often, tetraplegia means that the muscles of the shoulder are not under voluntary control, which lends itself to techniques such as Functional Electrical Stimulation (FES) to restore limited mobility [34].
In SCI, three types of motoneuron conditions exist, as illustrated in figure 1: those that have intact upper motoneurons (UMN) and intact lower motoneurons (LMN) (part a), those
Figure 1: Illustration of the three types of nerves that can be found in a spinal cord injury patient: (a) Type 1 Nerve - Intact UMN and LMN. (b) Type 2 Nerve - Damaged UMN and intact LMN. Target muscle is paralyzed but innervated; nerve is electrically excitable. (c)
Type 3 Nerve - Damaged LMN cell body. Target muscle is paralyzed and denervated; nerve is not electrically excitable.
6 that have intact LMN but do not receive signals from the brain due to damage to the UMN
(part b) [46], and those that are dead, which have sustained damage to the cell body of the
LMN (part c) [24, 34, 43]. Neurons with intact LMNs that lead to paralyzed muscles are referred to here as ”type 2 nerves”, whereas those with damage to the LMN cell body that lead to denervated muscles are called ”type 3 nerves”.
FES can restore limited mobility to a SCI patient by artificially eliciting limited muscle contractions, which is done by stimulating the LMN of a paralyzed muscle, which serves as a conduit for the stimulation signal [1, 3, 31, 34]. However, if the cell body of the
LMN is damaged due to the SCI, it will no longer conduct an action potential toward the neuromuscular junction [1, 34]. Furthermore, the neuromuscular junction degenerates [31], and muscle contractions cannot be elicited through any type of nerve stimulation [1, 31,
34]. It is possible, through direct stimulation of the muscle, to immediately generate an attenuated amount of force [22], and to eventually restore the electrical conditions of the muscle membrane to normal conditions [30, 31]. However, the muscle force generation is usually not sufficient to for functional applications [34], unless a large amount of current [22] is used to individually excite each single muscle fiber [31], which is not desirable. The project at hand aims to address the problem of denervation by attempting to regrow intact and electrically excitable LMNs through the non-electrically excitable sheath left behind after denervation.
C4 through C6 level injuries account for a combined 39.4% of spinal cord injuries, while the rest of the injuries are almost evenly spread over the rest of the levels [37]. It is at these levels of the spinal cord that the majority of the LMNs that lead to the muscles of the shoulder and many of those that lead to the muscles of the elbow (especially the
7 biceps) emerge from the spinal cord [34], meaning that these muscles are often at least partially denervated [34, 42]. Peckham, et al., suggested that LMN injury could reduce the effectiveness of FES recipients with C4 level SCI [34, 42]. This claim was supported by
Doerr and Long, who found that three out of four patients with C4 injury had damage to the
LMNs of the biceps [11, 34]. Though the proposed technique can be used with denervation concomitant with all levels of SCI, the frequency of injury to the muscles of the shoulder and elbow makes a technique that can address denervation of these muscles especially attractive, which is why they were chosen to be the primary target of this research.
Denervation is a generally pervasive problem in SCI, so other solutions, such as direct motor neurotization [41], tendon transfers [27] and voluntary nerve rerouting [55] have been proposed. However, the muscles of the elbow and the shoulder are are not conducive to many of these techniques due to reasons that will discussed here in more detail. For example, when the suprascapular nerve has suffered damage, many of these previous proposed methods may not be of benefit to the patient, but one of the intercostal nerves, which have been used in the past as donors [28, 55], could be used to reanimate the infraspinatus and supraspinatus muscles. Therefore, if the proposed technique proves suitable for restoring mobility, it will help further the goal of FES in restoring muscle function.
8 2 Background
2.1 Injury to Peripheral Nerves
When a peripheral nerve is injured, it undergoes Wallerian Degeneration [32], a phenom- ena that is seen regardless of the mechanism of the injury [33]. Several events are indicative of Wallerian degeneration: the axoplasm undergoing disruption, fragmentation of neural tubules and neurofilaments leading to a loss of longitudinal orientation, the mitochondria becoming swollen, discontinuities developing in the axolemma, and proliferating Schwann cells and macrophages further degrading the axon [6, 33, 36]. Since these symptoms are seen regardless of the mechanism of the injury [33], the distal portion of a peripheral nerve that is simply transected can model a peripheral nerve that has sustained damage to its cell body.
2.2 Current Techniques
Currently, there are four major methods for overcoming the deficits caused by denervation in SCI patients. Each of these will be discussed in some detail in the following sections.
2.2.1 Orthoses
Orthoses are devices that provide stability for joints [34]. Generally, this means that the orthosis provides support while remnant voluntary function or FES provides the minimal power necessary to navigate the workspace [23]. However, the benefit of these devices is often limited for SCI patients with denervation because there is no way to power the limbs.
Since orthoses have been required in the past to provide the necessary limb support [23, 34], one possible solution to denervation might be to combine the orthotic approach with the
9 approach presented in this experiment. For example, when the elbow muscles are denervated, a possible treatment might be to support the weight of the arm with an orthosis while the patient performs tasks using an FES system, which is able to stimulate its target muscles because of the therapy for denervation that is proposed in this experiment.
2.2.2 Tendon Transfers
The first technique to create muscle function when a muscle becomes denervated is not neurological, but rather orthopedic. In the presence of muscles that are similarly oriented and elicit similar movements to a denervated muscle, a tendon transfer can be performed to make a non-denervated muscle perform the function of the denervated one [27]. This technique can be used regardless of whether the denervation is caused by SCI [27], or by brachial plexus root avulsion [7].
Surgically, the tendon of the denervated muscle is usually transected. The tendon of the donor muscle is also transected, and the proximal portion of this muscle is attached to the distal portion of the denervated muscle tendon, such that the donor muscle performs the function of the denervated muscle. Although the donor muscle can be either a muscle still under voluntary control or one that is paralyzed and is stimulated using techniques such as Functional Electrical Stimulation (FES), the procedure is often performed with muscles under voluntary control [27].
In the shoulder, the use of this technique is hampered by the lack of proper donor muscles, which are especially rare because so few muscles remain under voluntary control in cervical
SCI. Transfers involving the levator scapulae, trapezius, sternocleidomastoid and latissimus dorsi muscles do not yield satisfactory results [7]. Furthermore, the muscles of the shoulder
10 fan out and attach in many places, adding to the difficulty of selecting the donor muscle(s).
Since the recipient muscles also attach in multiple places, multiple tendon transfers must be performed to fully replace the function of the denervated muscle, adding even further to the burden of donor muscle selection. Due to these limitations on tendon transfers, nerve transfers, where a single donor nerve can be transferred to a single recipient nerve proximal to the branching, offer an opportunity for better restoration of function [7].
2.2.3 Peripheral Nerve Rerouting
Nerve transfers seem to be the treatment of choice for shoulder muscle denervation [7].
However, as with tendon transfers, the problem lies in selecting a proper donor. In the treatment of avulsed brachial plexus injury, it is common to transfer a donor nerve that is under voluntary control to the recipient nerve. In this case, several donor nerves are available: the phrenic [17, 50], intercostal [35], and contralateral limb nerves [7, 18].
Peripheral Nerve Rerouting (PNR) is a type of of nerve transfer where the donor nerve is under voluntary control (type 1), and therefore has its roots above the SCI [5, 55](see
figure 1). The advantage of this technique is that there is no other required surgical pro- cedure beyond the transfer. No other equipment needs to be implanted and tested, since the command signals are carried from the brain, through the donor nerve, into the recipient nerve, and to the muscle with no intermediate intervention.
However, several disadvantages make PNR undesirable. Although it is has been some- what successful, it is not optimal due to a lack of suitable donors in the shoulder and elbow.
A value judgment must be made in selecting a donor nerve to sacrifice, and it is generally not worth the loss of already restricted voluntary movement for the partial restoration of a
11 different motion. Even if a suitable donor nerve were to be found, the patient then must be retrained to use the nerve to control its new destination muscle [44].
2.2.4 Direct Muscle Neurotization
The final major technique for treating the problem of denervation is Direct Muscle Neu- rotization (DMN) [41], also called Motor Nerve Transplantation [16]. DMN attempts to address the poor results of most other nerve transfer techniques, which are largely due to misdirected growth at the suture site [16] or neuron death [9], by delivering intact nerve
fibers directly to the target muscle [16]. However, once again, this technique is difficult to use in the shoulder due to extensive branching.
The idea of DMN is to transfer a neuromuscular junction from a non-denervated muscle to the belly of a denervated one. The donor neuromuscular junction can come from two sources: either a paralyzed muscle, or one under voluntary control. If the donor muscle is under voluntary control, the motor endplate zone is simply harvested from the donor muscle and transposed into the recipient muscle [16]. If, on the other hand, the donor muscle is paralyzed, the donor neuromuscular junction must be given a command source. One method of note to provide this control is to combine DMN with end-to-side neurorrhaphy [41]. Here, the donor nerve, including the neuromuscular junction and a section of the axon, is surgically removed. End-to-side neurorrhaphy is used to attach the donor nerve to a type 1 (see figure 1) nerve, while the donor neuromuscular junction, including a small piece of the donor muscle, is implanted into the belly of the recipient muscle [41]. In this way, the command nerve is able to control a muscle that it previously did not control. However, DMN is an impractical option for the muscles of the shoulder, due to the complexity of the procedure and the lack
12 of donor nerves that is multiplied by the extensive branching found in the nerves of the shoulder.
2.3 Related Experiments
A combination of several prior studies indicates that the proposed technique will be successful. For example, the proposed conduit has been tested with regrowth of nerves under voluntary control [26]. In a different study, a nerve that was not conducting signal, similar to the donor nerve in the proposed technique, was able to regrow [40]. Finally, a study has shown the technique to work in the diaphram [28].
2.3.1 Regrowth of Nerves Under Active Control Through an Empty Epineural
Sheath
A study from 2001 tested the feasibility of a nerve under active voluntary control to regrow through an empty epineural sheath. The study showed that the nerve would regrow to the same extent regardless of whether an empty epineural sheath or an immediately transferred section of nerve was used [26].
Similar procedures are used in patients with upper extremity injuries. For example, serious injury to the musculocutaneous and axillary nerves, which is often present with injuries resulting from vehicle accidents, can be treated with a neighboring accessory nerve, phrenic nerve, suprascapular nerve or intercostal nerve, any of which can be sutured to the end of the recipient nerve [8]. Within 1 to 2 years following the procedure, the muscle power returns to a reasonable functional level [8]. A similar treatment is presently available for brachial plexus injury (BPI) [7].
13 This study indicates that it is possible for a healthy nerve to regrow down a path left by a dead nerve [8, 26, 54].
2.3.2 Effect of Conduction Block
In 1977, a study was done in baboons to show that blocking conduction of signals through a nerve had no effect on its ability to regenerate after a crush injury, which leaves the sheath of the nerve intact [32, 51]. A tourniquet tied around the knee was used to create an upstrem conduction block [12, 40, 51]. Wallerian degeneration is almost absent in the blocked axons [40], meaning that excitability and conduction velocity are maintained distal to the point of blocking [51]. A nerve crush was performed on the same nerve as conduction block at the ankle on both the experimental side and the control side, which did not receive the tourniquet conduction block.
The conclusion of this experiment was that prolonged conduction block does not affect the capacity of blocked nerve fibers to regenerate after a distal crush injury [51]. However, since the axons are left intact, albeit inactive, any chemical transport mechanisms along the axon are left undisturbed. Therefore, while this experiment shows that upstream electrical activity is not required for axonal generatation, it does not show that an axon will regenerate without any upstream activity, electrical, chemical or otherwise. So, this experiment [51] cannot serve a true model for the conditions present in SCI.
2.3.3 Diaphragmatic Reanimation
An experiment similar to the one described in this report was performed in patients with
C3 to C5 level SCI. A type 2 intercostal nerve was transferred to a type 3 (see figure 1)
14 phrenic nerve, to be used with a diaphragmatic pacemaker. Three months were allowed for axonal regeneration. The results showed that the regrowth occurred to the extent where the patient could survive and even tolerate using solely the pacemaker with no external pressure ventilators [28]. However, the study does not mention quantitative measurements of the extent of the regeneration beyond tidal volumes, end CO2 values and patient comfort [28].
The capacity of a reanimated muscle to produce force must be known if this technique is to be used in the limb, especially shoulder, muscles.
3 Methods and Materials
To test the hypothesis that has been put forth in this document, a multi-stage procedure is necessary, first to induce the experimental conditions (SCI and denervation), then to perform the graft, and finally, to test the regeneration. An animal model was created to explore this phenomena.
Since this project was a feasibility study toward the development of a clinical treatment, the validity of the animal model did not have to exactly reflect human physiology. Any mammal would be sufficient, since Wallerian degeneration is the same in all mammals [33].
Consequently, the best animal to use for this model would be the least expensive mammal, given that the nerves in the animal are large enough to manipulate and transfer. Fischer rats from Charles River were chosen for the model because they fit this criteria. However, since rat spinal cords often maintain some remnant function [25, 36, 48] and sometimes possess limited regenerative capabilities [45, 47, 52], the completeness of the spinal lesion must be tested at the end of the experiment.
15 Data about similar experimental conditions is sparse, so an estimation about the results was difficult to make, meaning that statistical predictions of the necessary sample size were also difficult to make. Previous studies in the field [4, 20, 41] have used sample sizes of 6-12.
Since this was a feasibility study, the necessary sample size was judged to be about the same for this experiment. All experiments were done with the approval of Institutional Animal
Care and Use Committee at Case Western Reserve University.
3.1 SCI and Denervation
The first of the two survival surgeries was used to create the SCI and to create the
mg denervation model. Under an anesthetic mixture of Ketamine (50 − 70 kg ), Xylazine (10 −
mg mg 14 kg ) and Acepromazine (5 − 7 kg ), the spinal cord was exposed at around T7-T11 through a laminectomy, as shown in figure 2. Approximately .1cc Marcaine was injected directly into the cord to decrease the immediate effect of the SCI, which in turn helped increase the survival rate. Using a specially modified forceps in conjunction with the controlled crush contusion method developed at Acorda Therapeutics [13], the spinal cord was crushed for
30 seconds.
Since it can be difficult to exactly control the area of a spinal cord lesion such that it leads to the death of a particular LMN, the non-electrically excitable (type 3) nerve was not modeled as shown in figure 1. Instead, the left tibial nerve was exposed, using a previously published anatomical description [41], as shown in figure 3, and was transected. The proximal portion was buried into a neighboring muscle, while the distal portion was left to die and turn into the model of the type 3 nerve. A summary of the first procedure can be seen in
16 Figure 2: Spinal cord is exposed in preparation for controlled crush contusion injury. Mar- caine is injected into the cord, before specially modified forceps are inserted around it. Spinal cord is squeezed with forceps for 30 seconds to perform the crush.
figure 4.
3.2 Nerve Transfer
One week separated the creation of the experimental conditions and the nerve transfer to emulate the conditions found in clinical settings. One week is sufficient for the spinal shock responses to fade, and for the Wallerian degeneration of the tibial nerve, which takes at least
79-81 hours [15, 19] (type 3 nerve, according to figure 1) to reach its steady state. Both of these conditions are desirable in an animal model, as both are found in clinical conditions.
A compressive, crush injury, like the one used to create the SCI in this case, usually results
17 Figure 3: Sciatic nerve and its branches are exposed - both tibial and peroneal nerves can be seen. To create a denervation model, the tibial nerve is transected. The proximal stump is buried into a neighboring nerve while the distal stump is left to become a non-electrically excitable nerve (see figure 1).
in a central cavitation with axons persisting mainly along the perimeter of the cord [25, 36,
48]. The primary mechanical injury is usually augmented by delayed secondary mechanisms such as ischemia and free radical induced peroxidation of cell membranes [36]. The acute responses to SCI occur within hours to days following the injury [10]. So, by the second procedure, the spinal cord should have settled into its steady state, reducing the possibility of interference from the acute responses.
The leg lesion site was reopened under the same anesthesia cocktail. The bulb of the distal
18 Figure 4: Diagram of the first surgical procedure: gray boxes represent lesions. SCI is inflicted and denervation model is created.
stump of the tibial (type 3, non-electrically excitable) nerve was cut. Then, the peroneal nerve was transected and the proximal portion, which is a type 2, electrically excitable, nerve, was sutured (end-to-end) to the distal portion of the tibial nerve. A summary of this second procedure can be seen in figure 5.
3.3 Post Chronic Surgery Care
mg At the end of each chronic surgical procedure, Gentamyacin (antibiotic) (.5 100g ) was given subcutaneously for one week, 5−10cc of lukewarm Lactated Ringer’s solution (isotonic saline solution) was given intraperitoneally or subcutaneously as needed, and Buprenex (analgesic)
mg (.01 − .05 100g ) was given subcutaneously as needed.
19 Figure 5: Diagram of the second surgical procedure: transfer of the peroneal nerve to the distal, non-electrically excitable stump of the tibial nerve.
3.4 Force Analysis
3.4.1 Muscle Characterization (Pulse Width Modulation)
The final surgery was a non-survival data-collection procedure. The gastrocnemius, to which the tibial nerve leads, was exposed in its entirety on the control side of the animal
(the side where no surgeries had previously been performed), and was attached to a force transducer (ELFS-T3E-50N-/R, manufactured by Entran) with a force resolution of about
.0023N. An active length-tension curve was created by measuring the force response of the muscle to constant stimulation parameters, using either a bipolar (rats 16-17) or tripolar
(rats 20-22) hook electrode, when it was stretched to different lengths. To keep the effect of variations in electrode placement to a minimum, the electrode first positioned to optimize force generation, and then was held in place with a soldering clamp. The muscle force was quantified at each length by visually estimating the magnitude the peaks generated by the stimulations at each length. The muscle was left pulled to the length where the maximum
20 Figure 6: Anesthetized rat placed in the force measurement jig. A clamp holds the leg bone while the tendon of the target muscle is attached to a force transducer (left, foreground of the picture). The nerve is stimulated, and the resulting muscle contraction is measured by the force transducer.
force was produced. Then, the tibial nerve was stimulated with various frequencies, pulse widths, and amplitudes of current to develop a picture of the strength of the gastrocnemius muscle.
A similar procedure was performed on the experimental side by stimulating the peroneal nerve above the transfer site, such that the signal would travel initially down the peroneal nerve, through the tibial nerve, and then to the target muscle, which was once again the
21 gastrocnemius. The force that was produced on this side of the animal was normalized to the force produced on the control side, which allowed the experiment to control for muscle atrophy and physiological differences between individual rats.
3.4.2 Spinal Cord Injury Completeness Testing
Since the crush injury only causes Wallerian degeneration with minimal damage to the basement membrane [32], and since the rat spinal cord has shown some limited regenerative abilities [52] and remnant function [36], we tested the extent of the SCI to ensure that it had affected the experimental muscles. Presently, there are two major methods of testing the completeness of a spinal cord injury; both somatosensory evoked potential (SEP) or direct motor pathway stimulation are available [39]. SEP is the predominant technique, but we believe direct motor stimulation to be both more convenient and functionally relevant for the outcomes of this experiment, because somatosensory monitoring may be insensitive to motor tract injury [21]. A third alternative for testing the completeness of the SCI was to reopen the thoracic lesion and directly stimulate the spinal cord, but the animals most likely would not have been able to tolerate or survive this procedure.
The reinnervation of interest in this experiment is part of the motor tract. So, the most important characteristic of the SCI is not that it is complete, but rather that there is no path for a signal to travel from the brain to the donor peroneal nerve. Since this connection would exist through a motor pathway, not a sensory pathway [21, 39], direct motor pathway stimulation was chosen.
In surgical settings, direct motor stimulation evoked potentials are usually monitored on the caudal spinal cord, on a peripheral nerve, or on the muscle [39]. If electrical measurements
22 taken on the muscles are valid measures of SCI, force measurements of those same muscles are just as valid. Since force, not electrical, measurements are being used in other parts of the experiment, the marginal cost of measuring the force resulting from direct motor stimulation evoked potentials was much lower than measuring electrical signals.
The SCI in this experiment was tested by stimulating the spinal cord and measuring the amount of force generated in the gastrocnemius as a result. Two 25 gauge, 1.5 inch syringe needles were cut from their plastic holders. Both were placed as close together as possible, with at least one inside the spinal cord, below the level of the SCI. Approximately 3mA of current were passed between the makeshift electrodes, and the resulting force was recorded.
Then, the two needles were placed above the level of the SCI, with at least one within the spinal cord. Once again, with 3mA stimulations were passed between the electrodes, while the resulting force was recorded.
3.4.3 Assurance of Graft Area Conduction
A simple test was performed to ensure that the stimulation signal was being carried to the target muscle through the grafted nerve, and not through some unknown side pathway.
Conduction failure in the distal portion of a nerve after transection does not fail until Walle- rian degeneration prevents conduction of signals [15, 29]. However, if stimulated proximally to the transection, the nerve has no way of effecting a muscle response.
A 2 Hz supramaximal pulse was applied to the grafted nerve; the resulting muscle force was measured. The nerve was cut proximally to the graft, and immediately thereafter, the distal stump was stimulated with the same current; the muscle force was measured. Finally, the nerve was stimulated proximally to the transection and the muscle force was measured.
23 If the force when stimulating distal to the transection matched the force with an intact nerve, and if the force when stimulating proximal to transection was zero, the conclusion can be drawn that stimulation signal was, in fact, being transmitted through the grafted nerve.
4 Results
4.1 Animal Care
A high mortality rate has claimed many of the 24 animals that have gone through this procedure. The expected rate of mortality following a spinalization procedure is commonly thought to be about 50%. However, probably due to the inclusion of a second surgical procedure, bladder trauma, and overall stress on the animals, the experiment has seen an 80% mortality rate. Many of these casualties appear to be due to a digestive system depression accompanied by a lack of ability to to urinate, which pressurizes the bladder.
The rate of mortality directly resulting from the procedures was about 50%. The first six
mg rats were anesthetized with Nembutal (Sodium Pentobarbital, 3 100g ). Two of these died on the operating table, and an additional one died the first night. During the first week post-
SCI, the animals never seemed to recover from the anesthesia, and two more died before the end of the week. Necropsies showed severe digestive depression and discoloration of the spleen, liver, and heart. One had extensive internal bleeding. The other had a full, packed secum with pervasive intestinal gas. Both were given Buprenex and fluids twice daily, at which times the bladders were manually expressed. Both bladders showed signs of trauma; one rat had a peritoneum full of a clear fluid that could be either water or leaking
24 urine. The one remaining animal underwent the second procedure with rodent cocktail, as described above, without problem. However, at the terminal procedure, the animal was unable to survive another dose of Nembutal, and died before yielding any data. For this reason, rodent cocktail was used in later trials.
We have found less aggressive care to be more successful. Initially, the bladder was emptied, and intraperitoneal fluids were given twice daily. In necropsies of animals that had died shortly after a surgical procedure, severe bladder trauma was found, accompanied by the occasional leak. When the animals were simply left to urinate on their own, they did not seem to have any problems doing so past the first couple of days post spinal cord injury, when reflexive bladder control returned [36]. Therefore, in later trials, while the animals were still closely monitored to ensure they were voiding on their own, urination was not aggressively initiated by a team member. Fluids, instead of being given intraperitoneally, were given subcutaneously.
Better rates of animal survival were also found with less aggressive analgesia. Initially, a dose of Buprenex was to be given daily for 2-3 days. However, we found that the drug depressed the digestive system, possibly leading to some of the digestive ailments that were seen. When the pain killer was not given, or only given once, the animals did not seem to be in any additional pain, so in later trials, it was only given as needed.
Surprisingly, there were almost no problems with mutilation, even when the animals were housed two per cage. A single episode of self-mutilation was seen in an animal that did not have a complete spinal cord injury.
25 Rat 16 0.14
0.12
0.1
0.08
0.06 Muscle Force (N)
0.04
0.02
0 0 100 200 300 400 500 600 700 800 900 1000 Time (ms)
Figure 7: Raw time trial data for Rat 16, pulse width modulation. The figure shows two trials from the experimental pulse width-force set, each at a different pulse width. The difference between and maximum and minimum points of each trial was taken to be force generated by the muscle at the given pulse width. This maximum force is plotted in figure 9 as a function of pulse width.
4.2 Pulse Width Modulation
Figure 9 shows the maximum force that the gastrocnemius produced at varying pulse widths and a constant pulse amplitude. The pulse amplitude was a supramaximal pulse at the base pulse width (either 50 or 100 µs). Since the amplitude of the pulse was chosen to be the supramaximal pulse at either 50 or 100 µs pulse widths, the maximum contraction
26 Rat22: Experimental Side Length−Tension Curve 0.06
0.05
0.04
0.03
Muscle Force (N) 0.02
0.01
0 7.8 7.9 8 8.1 8.2 8.3 8.4 Length (cm)
Figure 8: Experimental side active length-tension curve. The muscle was pulled to different lengths, and was stimulated with constant parameters at each point. The maximum muscle force was measured at each point as described in figure 7. The length at which the muscle was able to produce the most force (length corresponding to the peak force in this plot) was used in all of the other trials.
of the muscle was reached at this point. Longer pulse widths did not result in larger muscle contractions.
The pulse width-force plots of figure 9 were constructed from raw data that is excerpted in
figure 7. The voltage output of the force transducer was sampled and stored. The offset was removed, and the voltages were converted to force using a multiplying factor derived from
27 Rat 16: Pulse Width−Force Curves
0.4
0.2
0 Control Side Rat 17: Pulse Width−Force Curves Experimental Side
0.2
0.1
0
Rat 20: Pulse Width−Force Curves 0.2
0.1
Muscle Force (N) 0
Rat 21: Pulse Width−Force Curves 0.2
0.1
0
Rat 22: Pulse Width−Force Curves 0.2
0.1
0 0 50 100 150 200 250 300 350 400 450 500 Pulse Width (us)
Figure 9: Pulse width-force curves for tibial muscle on both the control and reanimated side.
The pulse width was varied at a constant frequency (2Hz) and pulse amplitude (about 1mA.
The maximum force that the muscle produced was recorded. Each panel shows the curves for a different animal. The relative forces of the control and experimental sides at maximum pulse width were used to evaluate the extent of regeneration, as shown in table 1.
a calibration trial, where a known weight was suspended from the transducer. The data displayed in figure 7 resulted. The difference between the maximum and minimum force
28 during each trial was taken to be force production of the muscle given a set of stimulation parameters. By varying the pulse width while keeping the other stimulation parameters constant, muscle force as a function of pulse width was plotted in figure 9.
Muscle force varies with the tension under which the muscle is placed, so a constant baseline had to be chosen. Figure 8 shows an active length-tension curve for an experimental side gastrocnemius. The force production capability of the muscle was measured, from data similar to figure 7, as the length to which the muscle was pulled was varied. The length at which the muscle was able to produce the largest force was used as the constant length for the other trials.
The extent of the regeneration, as shown in table 1 is calculated from the data that is presented in figure 9. The muscle force did not generally grow as the pulse width grew to be longer than the base pulse width, which is the pulse width at which the supramaximal amplitude was determined. Therefore, the value of the force at these longer pulse widths was taken to be the maximum force that the muscle could produce. The values presented in table 1 are the average heights of the peaks in a single, long pulse width trial. The calculated standard deviations of the heights of these peaks are also presented in table 1, although most of these values are smaller than the resolution of the force transducer. The ratio of the maximum force of the experimental gastrocnemius to the maximum force of the control gastrocnemius is presented as the extent of the regeneration. On average, stimulation of the experimental side peroneal (type 2, see figure 1) nerve yielded about 50% of the force that was produced by stimulation of the control side tibial nerve.
The experimental side gastrocnemius was fairly consistent between animals in the mag- nitude of force that it could produce, at an average of .1N. However, the control side
29 gastrocnemius, while producing a larger amount of force (.2N), was more variable between animals.
Rat 20: Spinal Cord Lesion 0.04 Below Lesion 0.035 Above Lesion
0.03
0.025
0.02
0.015 Muscle Force (N) 0.01
0.005
0 0 200 400 600 800 1000 Time (ms)
Figure 10: Needles were placed in the spinal cord to verify the completeness of the lesion.
When the cord was stimulated caudal to the lesion, a gastrocnemius muscle twitch resulted.
When it was stimulated rostral to the lesion, intercostal muscle twitching was found, but with no effect on the gastrocnemius muscle.
4.3 Spinal Cord Injury Verification
Direct motor stimulation of the spinal cord through percutaneous electrodes confirmed the integrity of the SCI, which needs to be shown to support the conclusions of this experi-
30 Trial Experimental Control
Rat Length End Mass Side Side Expected
ID (Weeks) (kg) (N) (N) Ratio SCI Route
Ave St.Dev. Ave St.Dev
16 4 .270 .117 .002 .481 .003 .243 Yes Yes
17 5 .265 .145 .003 .207 .001 .701 Yes Yes
20 5 .333 .045 .002 .094 .001 .474 Yes Yes
21 6 .311 .050 .001 .115 .001 .434 Yes Yes
22 6 .310 .053 .001 .100 .001 .541 Yes Yes
Ave. 5.5 .291 .082 .997 .479 N/A N/A
Table 1: Summary of rat strength data. Maximum forces on both the control and experimental side (averaged over a single, long pulse width trial) of every rat are shown. The ratio of experimental side maximal force to control side maximal force, which represents the extent of regeneration controlled for the size of the animal and variations between individuals, is also shown. The standard deviations presented here are calculated from variations in peak heights in a single trial, and were found to be smaller than the minimum resolution of the force transducer.
31 ment. The method was able to qualitatively evaluate the SCI.
Figure 10 shows traces of the forces generated by a representative rat when stimulated both above and below the SCI. As the figure shows, a small force is generated in the gas- trocnemius - with the amplitude being unpredictable - when the cord is stimulated below the SCI; but, when a stimulus is applied above the SCI, no such force is generated.
We found that to evoke a response from the spinal cord, both poles of the bipolar stim- ulator had to be inside the cord. The needles were first placed in the spinal cord below the level of the SCI. Many of the muscles of the lower body contracted noticeably but not vigorously. The gastrocnemius was observed for contraction to minimize the chance that the measured force was an artifact of the whole body contraction.
When the needles were placed above the SCI, once again with both poles inside of the cord, a twitching of the entire upper body was observed. However, there did not seem to be any transmission of signal past the zone of injury, which was supported by the force mea- surement of the gastrocnemius. Due to inherent difficulty of placing percutaneous electrodes inside of the spinal cord, the contraction of upper body muscles was used as a check of the method.
4.4 Assurance of Graft Area Conduction
The final portion of the procedure served to show that the signal that was thought to be transmitted through the grafted nerve was actually being transmitted as such. We found that the signal was transmitted exclusively through the predicted conduit.
Figure 11 shows the results of the conduction test. Stimulation with the experimental
32 Rat 20:Graft Area Conduction Efficiency 0.04 Intact graft 0.035 Peroneal cut, stimulated distally Peroneal cut, stimulated proximally 0.03
0.025
0.02
0.015 Muscle Force (N)
0.01
0.005
0 0 200 400 600 800 1000 Time (ms)
Figure 11: A test was performed to ensure that the stimulation signal was being carried by the nerve through the graft. Muscle contraction was found when the nerve was stimulated.
When it was transected and stimulated distal to the transection, muscle contraction of similar amplitude was found. However, when the nerve was stimulated proximal to the transection, the muscle did not react.
nerve intact shows a contraction of the muscle, as was seen throughout the trial. After the nerve was transected, stimulation proximal to the transection showed no generated force in the gastrocnemius. However, when the stimulated distal to the transection, a twitch was produced. Usually, the force response to this stimulation was similar in amplitude to the
33 response seen prior to the transection. The apparent phase shift seen in the plots is not significant to the interpretation of the results, as this shift appears due to differences in stimulus timing between trials.
5 Discussion
The purpose of this experiment was to see if it was feasible to use a paralyzed nerve in conjunction with an electronic means of stimulation to address the prevalent problem of motoneuron death and denervation in spinal cord injury (SCI). From the preliminary results that we have gathered, we have found that the technique is feasible. The implication of the experiment is that motor nerves can reanimate muscles without cortical control.
The major assumption that we have made in the design of this experiment is to follow the dogma that peripheral nerves regenerate at a rate of about 1mm per day. Given that the peroneal nerve would have to regrow over a distance of about 1cm, allowing four to six weeks for the regeneration period should, theoretically, be plenty of time. However, in past studies, this theoretical rate has found to be a bad guideline [28]. This might be a mitigating factor of our results - if the nerve had not yet completed its regeneration when we tested it, the results will show lower forces than are possible. Future studies should include systematic variation of the length of the trials to show the effect of allowing longer periods of time for regeneration.
After further experimentation, if this technique proves to be feasible in the restoration of LMN functionality, it would be of use to patients with high level cervical SCI who are unable to receive the benefit of a FES system. Before the technique is developed to that stage,
34 however, further testing is needed in animals that more closely mimic human conditions, to estimate how we:ll the rat model will transfer to humans. If those animal tests are successful, they should be followed by testing in humans, to show that the technique can be used with benefit in clinical settings.
5.1 Pulse Width Modulation
The maximum force that can be produced by a gastrocnemius muscle of a .35−.50kg rat is about 550mN [14]. The rats that were used in this experiment were smaller than those used by Gardiner, et al [14]; so, accounting for the lower-limb atrophy caused by the SCI, the magnitude of the control-side gastrocnemius forces (average of 180mN seem reasonable.
The experimental side muscle forces were always a fraction of the control side forces.
One limitation on the ability of the peroneal nerve to restore complete function to the gastrocnemius is the size difference between the tibial and peroneal nerves. The cross- sectional area of the peroneal nerve is about half the cross-sectional area of the tibial nerve, meaning that the axon number mismatch between the two nerves limits the possible extent of the regeneration. In other words, even if every axon in the peroneal nerve was able to reinnervate the gastrocnemius, the restored force would only be about half as much as the control side force.
Complete regeneration is generally not expected, since failure to reinnervate with a suf-
ficient number of fibers results in poor recovery [53]. Furthermore, failure to reinnervate a muscle before motor endplate atrophy, which occurs during the week in between the first two procedures [15, 19], also results in poor recovery [53]. Other similar experiments have
35 yielded force restoration of 30% - 50% [2, 49, 50], so the 50% regeneration seen in this ex- periment seems reasonable. The fact that the recovery found in this experiment is at the higher end of that range could be attributed to the fact that the tibial nerve transection was performed only about 1 − 2cm from the target gastrocnemius muscle. When the transfer is performed at such close proximity to the muscle, the reinnervation occurs to a greater extent [9], because the time required for the regeneration is smaller, meaning that neuronal death is less prevalent. However, this might be infeasible in humans, possibly limiting the direct applicability of the model to humans.
5.2 Spinal Cord Injury
Unpublished work at Acorda Therapeutics [13] shows the efficacy of the controlled crush contusion as a means to reliably create SCI. However, the rats in this experiment showed signs of some limited spinal function, such as the ability to urinate without assistance, or to slightly move the hindlegs while walking. The extent and regions of this ability varied between rats. The phenomena could be attributed to one of two factors, either remnant bridges of function connecting regions above and below the lesion [25, 36, 48], or remnant spinal reflexes. The results of the spinal function test imply that all apparent spinal activity must be attributed to the latter, due to the absence of conduction across the lesion. Rat
19 was an aberration because it seemed to not have received a SCI at all. While the other animals were able to reflexively move their feet without significant force generation, rat 19 was able to stand on its hindlegs, as non-injured rats are able. Direct motor stimulation showed almost no difference in the force generated by the gastrocnemius between when the
36 spinal cord was stimulated above and below the anticipated level of SCI. This lends further support to the ability of our test to qualitatively judge SCI.
The direct motor stimulation method that we have used to evaluate our SCI, at this stage, cannot be used as a quantitative measure of an SCI, but only as a qualitative measure. The amplitude of of gastrocnemius force found when the spinal cord was stimulated varied greatly from animal to animal. This variation could be due to many factors, including placement of the needles within the spinal cord, health of the caudal spinal cord, and age post-injury. The test is, however, able to show whether or not a connection exists. The low force magnitude seen in the gastrocnemius with direct motor stimulation of the spinal cord can most likely be attributed to the factors described above. Since the gastrocnemius contraction was verified by visual inspection when testing the SCI, we are confident that measured forces are due to the contraction of this target muscle. The effect of a body twitch on the output of the force transducer was very small, as can be seen in trails where the upper body underwent large twitches, yielding almost no change in the output.
5.3 Assurance of Nerve Graft
This procedure was performed to show that the stimulations were reaching the muscle by the desired route, through the graft. The results show that nearly all of the stimulation was traveling as thought.
Immediately following transection, the distal stump of a nerve responds as the nerve did prior to the transection [15, 29]. If the signal is traveling through the graft as thought, a stimulus proximal to the transection should produce no muscle response, whereas a stimulus
37 distal to the transection should produce the same response as a stimulus prior to the tran- section. If this condition is met, there exists no pathway other than the anticipated one for the conduction of the stimulus.
One salient feature of these trials is that the magnitude of the force is smaller during these trials than it is during the construction of the pulse width-force curves. Since chronologically, this test was performed after all of the other tests, due to the necessity of destroying the grafted nerve, a large portion of this decrease in muscle response can be attributed to nerve and muscle fatigue. Therefore, this tests serves most reliably as a qualitative test to show whether the large majority of the stimulation signal was carried by the grafted nerve, rather than a quantitative test to determine how much of the stimulation signal was carried by this nerve.
Acknowledgments
The author would like to acknowledge the funding for this project (N01-NS-1-2333), com- mittee members Dr. Robert Kirsch, Dr. Harry Hoyen and Dr. Kevin Kilgore, the assistance provided by Dr. Niloy Bhadra, Dr. Douglas Miles, Dr. Dawn Taylor, and Katharine Polasek.
Dr. Michael Zimber at Acorda Therapeutics, the veterinary staff at the Neural Engineering
Center of Case Western Reserve University, specifically Dr. Mark Jamba and Tina Goetz, and finally, Dr. Kenneth Gustafson and Dr. Dominique Durand of the Neural Engineering
Center of Case Western Reserve University also provided assistance as needed.
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46