Embryonic Cord Transplants in Peripheral Nerve Restore Skeletal Muscle Function

Christine K. Thomas,1 Daniel E. Erb,2 Robert M. Grumbles,1 and Richard P. Bunge1

 1The Miami Project to Cure Paralysis and Department of Neurological Surgery, University of Miami School of Medicine, Miami 33136; and  2Division of Physical Therapy and Department of Orthopedics and Rehabilitation, University of Miami School of Medicine, Miami, Florida 33124


    ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Thomas, Christine K., Daniel E. Erb, Robert M. Grumbles, and Richard P. Bunge. Embryonic Cord Transplants in Peripheral Nerve Restore Skeletal Muscle Function. J. Neurophysiol. 84: 591-595, 2000. The rapid atrophy of skeletal muscle after denervation severely compromises efforts to restore muscle function. We have transplanted embryonic day 14-15 (E14-E15) ventral spinal cord cells into adult Fischer rat tibial nerve stump to provide neurons for reinnervation. Our aim was to evaluate medial gastrocnemius reinnervation physiologically because this transplant strategy will only be effective if the reinnervated muscle contracts, generates sufficient force to induce joint movement, and is fatigue resistant enough to shorten repeatedly. Twelve weeks posttransplantation, brief duration electrical stimuli applied to the transplants induced medial gastrocnemius contractions that were strong enough to produce ankle movement in 4 of 12 rats (33%). The force of these four "low-threshold" reinnervated muscles and control muscles declined only gradually during five hours of intermittent, supramaximal stimulation and without depression of EMG potential area, which is strong evidence of functional neuromuscular junctions and fatigue resistant muscles. Sectioning of the medial gastrocnemius nerves confirmed that these contractions were innervation dependent. Weakness in low-threshold reinnervated muscles (8% control force) related to incomplete reinnervation, reductions in muscle fiber size, specific tension, and/or the presence of nonfunctional neuromuscular junctions. Muscle reinnervation achieved using this novel transplantation strategy may salvage completely denervated muscle and may provide the potential to evoke limb movement when injury or disease precludes or delays peripheral axon regeneration.


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

When denervated muscles have no opportunity to be reinnervated from regenerating peripheral axons or when reinnervation is delayed, as often occurs with central or peripheral nervous system injuries and diseases (Dyck et al. 1993; Fu and Gordon 1995), there is no effective therapy to reverse progressive muscle atrophy. Stimulation of denervated muscle to reduce atrophy (Schmalbruch et al. 1991) or to produce limb movement is impractical because of the large currents needed to excite muscle directly (Mortimer 1981). Early intervention is preferable if denervated muscles are to be salvaged.

Our approach to restore muscle function is to transplant E14-E15 ventral spinal cord cells into nearby peripheral nerve. Motoneurons present in the transplant survive and regenerate axons that grow along the peripheral nerve to form junctions with denervated muscle fibers (Erb et al. 1993). The objective of this study was to evaluate medial gastrocnemius reinnervation physiologically because this transplantation strategy will only be effective if the reinnervated muscle contracts, develops adequate force to produce limb movement, and is fatigue-resistant enough to shorten repeatedly.


    METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

All procedures adhered to the National Institutes of Health guidelines and were approved by the University of Miami.

Cell transplantation

As described previously (Erb et al. 1993), E14-E15 ventral spinal cord was dissected from inbred pregnant Fischer rats and dissociated for transplantation into 12 adult Fischer rats [weight: 171 ± 3 (SE) g; Fig. 1A]. Each host rat was anesthetized (halothane, 2% nitrous oxide). Ligatures were placed on the left tibial nerve 5-14 mm proximal to its entry into medial gastrocnemius to prevent cell escape. Cells (5-7 × 106, 10-15 µl volume) were injected into the nerve 2 mm distal to the ligatures. The tibial nerve was cut proximal to the ligatures and the proximal stump sewn into hip adductors to prevent peripheral axon reinnervation. Three "surgical control" rats (176 ± 3 g) underwent the same surgery but had only medium injected into the tibial nerve, while three naive rats were controls (168 ± 2 g).



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Fig. 1. A: experimental and surgical control rats underwent denervation of the left tibial nerve and then transplantation of E14-E15 ventral spinal cord cells or media, respectively, into the distal tibial nerve stump. Physiological and anatomical assessments of medial gastrocnemius reinnervation were made 12 wk after transplantation. Control rats were subjected to a final acute experiment only. Twitch forces from (B) control, (C) low-threshold reinnervated, and (D) high-threshold reinnervated muscle. E: final low-threshold reinnervated muscle twitch EMG (top) and force produced by both muscle nerves (----), after severance of one nerve branch (- - - -), and then both branches (... . .). F: voltage-force data from B-D, respectively (+, black-triangle, ; 100-µs duration pulses: triangle , open circle ).

Physiological analysis

Twelve weeks posttransplantation, rats were anesthetized with sodium pentobarbital (40 mg/kg i.p., then intravenously as needed) (Thomas et al. 1999). In experimental rats, we verified that the proximal nerve stump remained in hip muscles and freed the transplant and tibial nerve from all surrounding tissues. In controls, the left hindlimb was denervated except for the nerve to medial gastrocnemius.

During physiological recordings, the rat lay prone on a heating pad. The knee and ankle were clamped. The medial gastrocnemius tendon was tied to a transducer to record isometric force at optimal muscle length. Intramuscular EMG were recorded with two bared wires inserted longitudinally. The transplant or sciatic nerve was laid across two silver stimulation electrodes. Surrounding skin was used to form a pool that was filled with mineral oil maintained at 37°C.

Force thresholds and force-voltage recruitment data were obtained by delivering >= 50 µs duration pulses at 1 Hz and in 0.2- or 1-V steps until no further force increments occurred. Thereafter, twice-threshold stimuli and 50 µs duration pulses were used. Stimuli at 50 Hz for 1 s were used to evaluate muscle strength. Fatigue was induced with 13 pulses at 40 Hz every second for 2 min (Burke et al. 1973). These intermittent stimuli were then delivered for 5 hr to deplete muscle glycogen.

EMG and force were sampled online (Thomas et al. 1999). Measurements included area of the first phase of the EMG potentials, determined by isoelectric crossings; peak twitch and tetanic forces. Fatigue index (final to initial 40-Hz force ratio) was calculated.

Tissue analysis

Muscles were frozen in isopentane cooled in liquid nitrogen. Cross-sections (15-20 µm) were stained for glycogen (periodic acid Schiff stain) (Pearse 1961). Fiber cross-sectional-areas were measured from two each of low threshold reinnervated, high-threshold reinnervated, surgical control, and control muscles [800 fibers/muscle; high-threshold reinnervated muscles only shortened when strong stimuli (>= 40 V) were applied to their transplants]. Assessments made in low threshold reinnervated and control muscles included the following: 1) glycogen-depleted fiber number; 2) fiber numbers (whole muscle area/mean fiber area); 3) motor unit numbers from distinct force steps with increments in stimulus intensity [in controls, large diameter (>7 µm) myelinated axons were counted, with one-half of the fibers considered as motor]; and 4) innervation ratio (glycogen-depleted fiber number/motor unit number), without adjustment for possible multiple innervation of muscle fibers.

The transplant and muscle nerves were fixed and embedded in Epon araldite (Thomas and Westling 1995). Cross-sections (1 µm) were stained with toluidine blue. In nerves, myelinated axons were counted, maximum and minimum diameters measured, and mean diameters calculated.

Statistics

Mean ± SE are given. Differences between mean values for different groups of rats were tested using analysis of variance (ANOVA; P < 0.05).


    RESULTS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Muscle reinnervation

Figure 1, B-D, shows twitch forces evoked by stimulating the sciatic nerve of a typical control rat and the transplant of two representative experimental rats with 50-µs duration stimuli at increasing intensity. Force was evoked in the control muscle with 2.4 V (Fig. 1, B and F; +). Threshold was 3.8 V in one experimental muscle. With stimulus intensity increases, seven distinct but variable-sized twitches were evoked, each increment representing activation of another motor unit (Fig. 1, C and F; black-triangle). In contrast, 40 V were required to evoke any force in the other experimental muscle (Fig. 1, D and F; ). Even with 80 V, this evoked force was weak, suggesting excitation of thinly myelinated axons.

Four of 12 muscles (33%), termed low-threshold reinnervated muscles, generated force when their transplants were stimulated with 50-µs duration, weak stimuli (4.2 ± 0.7 V). Sectioning the medial gastrocnemius nerves confirmed that these muscle contractions depended on exciting axons that grew from the transplant. Figure 1E shows that the final EMG and force of one low-threshold reinnervated muscle were reduced after severance of one nerve branch and eliminated when both branches were cut.

The other eight experimental muscles behaved like the muscle shown in Fig. 1, D and F (). Even though their thresholds were reduced with longer duration pulses (>= 100 µs), typical force gradations were weak (Fig. 1F; open circle ). These muscles were called high-threshold reinnervated because they only shortened when strong stimuli were applied to their transplants (>= 40 V). These transplants were not studied further as selective muscle activation is unlikely with such stimuli.

In animals with no cells transplanted into the tibial nerve, strong transplant stimulation (150 V, 10-ms pulses) evoked no contractions, suggesting that all surgical control muscles remained denervated.

Strength and fatigue resistance

Mean low-threshold reinnervated muscle force was significantly lower than mean control force (Fig. 2A; 322 ± 159 vs. 4093 ± 111 mN). Reinnervated muscle contractions averaged 8% control strength, and prior to tendon detachment, induced ankle movement.



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Fig. 2. Mean tetanic force (A) and fatigue index (B) (n = 3; one experimental rat died before tetanic force measurements). C and E: low-threshold reinnervated and (D) control muscle forces evoked by 13 pulses at 40 Hz after the stimulation times indicated. F: first of 13 EMG potentials evoked with the force in E. G-I: toluidine blue-stained cross-section of nerve to control (G), low-threshold reinnervated (H), and surgical control (I) muscle.

Fatigue resistance was comparable for low-threshold reinnervated and control muscles (Fig. 2B; 0.46 ± 0.12 vs. 0.22 ± 0.02), although control force always declined to at least 24% initial in 2 min, whereas the force of two reinnervated muscles declined to <50% initial (Fig. 2, C and D). Even with 5 hr of stimulation both low-threshold reinnervated (Fig. 2E) and control muscle force declined only gradually and without depression of EMG potential area. Reductions in EMG amplitude were offset by increases in potential duration (Fig. 2F), probably attributable to muscle conduction velocity declines (Bigland-Ritchie 1981).

Muscle weakness

Weakness in low-threshold reinnervated muscles may result from incomplete reinnervation, reductions in muscle fiber size, reductions in muscle fiber number, and/or specific tension (force/cross-sectional area). Functional reinnervation was determined by the percentage of glycogen-depleted muscle fibers. All control fibers were pale and devoid of glycogen (Fig. 3A) whereas only 27 ± 8% of low-threshold reinnervated muscle fibers were glycogen-depleted. These glycogen-depleted fibers were grouped (Fig. 3B), a characteristic of reinnervated motor units (Karpati and Engel 1968). Glycogen-retaining fibers must be chronically denervated and/or have nonfunctional neuromuscular junctions, features that contribute to muscle weakness.



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Fig. 3. A and B: cross-section of PAS-stained muscle. C-F: representative muscle fiber cross-sectional areas and means from 2 each of the muscles indicated.

Strength also depends on muscle fiber size. The mean cross-sectional area of glycogen-depleted fibers in low-threshold reinnervated muscles (Fig. 3C) was significantly larger than fibers in high-threshold reinnervated muscles (Fig. 3D) and denervated muscle fibers from surgical controls (Fig. 3E). These data suggest that functional innervation prevented some muscle deterioration. Fiber size was significantly reduced in low-threshold reinnervated muscles compared with controls however (Fig. 3F), consistent with observed decreases in whole muscle strength (Fig. 2A), cross-sectional area (11 ± 2 vs. 46 ± 4 mm2), and weight (103 ± 17 vs. 487 ± 17 mg). The strength decline (8% control) exceeded the reductions in cross-sectional area (24% control), suggesting that lower specific tension also contributed to low-threshold reinnervated muscle weakness. Muscle degeneration was not evident because fiber numbers were similar in low-threshold reinnervated and control muscles (9046 ± 2790 vs. 13,093 ± 507). Some muscle weakness was also counteracted by significant increases in the mean number of muscle fibers innervated by each regenerating axon (263 ± 25) compared with control values (177 ± 7).

Axon regeneration

Too few regenerating axons may have limited reinnervation (Fig. 2, G-I). Compared with control motor units (74 ± 5), nerves to low-threshold and high-threshold reinnervated muscles (n = 4) had significantly fewer myelinated axons (45 ± 14 and 35 ± 27, respectively). Nerves to muscles of surgical control rats contained no myelinated axons. Axon regeneration into muscle nerves itself was not indicative of transplant success or muscle function however. All transplants contained large multipolar cells with central nuclei; similar cells in other transplants were immunoreactive to antibodies against nonphosphorylated neurofilaments, suggesting that they were motoneurons (Erb et al. 1993). Transplants of surgical controls contained no motoneuron-like cells. Numbers and diameters of myelinated axons were also similar in nerves to low-threshold and high-threshold reinnervated muscles. Thus the functional discrepancies between these reinnervated muscles (Fig. 1) probably also reflect differences in the formation of fully functional neuromuscular junctions.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

The need to salvage denervated muscle from deterioration has long been recognized in cases of peripheral nerve injury. Far less attention has been given to developing ways to rescue muscle that is denervated by the motoneuron death known to occur after trauma or diseases, even though these situations can preclude muscle reinnervation by regenerating peripheral axons. Our low-threshold reinnervated muscles shortened repeatedly for hours without EMG decrements, which is evidence of secure neuromuscular junctions and fatigue resistant muscles. These contractions were also strong enough to induce ankle joint movement, demonstrating the feasibility of using E14-E15 ventral spinal cord transplants in peripheral nerve to restore some muscle function.

Our data also emphasize the importance of physiological analysis to evaluate functional reinnervation. There were distinct differences in the electrical excitability of low-threshold and high-threshold reinnervated muscles (Fig. 1) even though the corresponding peripheral nerves contained myelinated axons that were similar in number and caliber. Many studies have used peripheral nerve as a conducive conduit for axon growth (e.g., Richardson et al. 1980; Sieradzan and Vrbová 1989), but transplant function is not always assessed. We have provided convincing evidence that the low-threshold reinnervated muscle contractions were innervation-dependent because muscle signals were eliminated after nerve severance (Fig. 1E).

Another distinguishing feature was our attempt to define the functional limits of the reinnervation. Maximal stimulation of low-threshold reinnervated muscles evoked 8% control force, which was enough to produce ankle movement. Clinically, only 9% of control strength is needed for human triceps brachii muscles weakened by spinal cord injury to shorten against gravity (Needham-Shropshire et al. 1997). Furthermore, our low-threshold reinnervated muscles were as resilient as control muscles (Fig. 2). Each muscle type shortened for 5 hr without electrical failure. This maintenance of EMG potential area suggests that stimulation reliably excited the membranes of similar populations of muscle fibers repeatedly, strong evidence of secure, functional innervation and fatigue resistant muscle. The converse, excessive fatigability, is characteristic in various neuromuscular disorders (McComas et al. 1995).

Nevertheless, improvements in reinnervation are needed using this transplantation strategy. More reinnervated muscles must shorten in response to weak currents, at strengths sufficient to induce joint movements and without compromise of fatigue resistance. From a clinical perspective, locally placed transplants are useful to restore innervation quickly, reducing atrophy that occurs while regenerating peripheral axons grow the considerable distances often needed to reach denervated muscle. In cases where denervation is induced by death of all motoneurons in a pool, reinnervation from substitute motoneurons may prevent muscle degeneration. Restoring muscle innervation, even though it is no longer connected to spinal motoneurons provides the potential for the muscles to be excited artificially by electrical stimuli to produce simple behaviors. Furthermore, if proximal axons were permitted and able to synapse onto the transplanted cells, the reinnervated muscles may be receptive to renewed activity from regenerating central or peripheral axons, restoring voluntary muscle control.


    ACKNOWLEDGMENTS

We thank Dr. Richard Bunge, our valued mentor and colleague who urged us, "Let's go forward." We also thank Drs. Mary Bartlett-Bunge, Blair Calancie, Naomi Kleitman, and Patrick Wood for critical review of the manuscript.

This research was funded by the Hollfelder Foundation, National Institute of Neurological Disorders and Stroke Grant NS-39098 to C. K. Thomas, and The Miami Project to Cure Paralysis.

Present address of D. E. Erb: Dept. of Physical Therapy, Duke University Medical Center, Box 3965, Durham, NC 27710.


    FOOTNOTES

Address for reprint requests: C. K. Thomas, The Miami Project to Cure Paralysis, University of Miami School of Medicine, 1600 NW 10th Ave. (R-48), Miami, FL 33136 (E-mail: cthomas{at}miamiproject.med.miami.edu).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received 13 January 2000; accepted in final form 5 April 2000.


    REFERENCES
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

0022-3077/00 $5.00 Copyright © 2000 The American Physiological Society




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