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. Author manuscript; available in PMC: 2014 Apr 1.
Published in final edited form as: Plast Reconstr Surg. 2013 Apr;131(4):499e–511e. doi: 10.1097/PRS.0b013e31828275b7

Nerve Regeneration in Rat Limb Allografts: Evaluation of Acute Rejection Rescue

Ying Yan 1, Matthew R MacEwan 2, Daniel A Hunter 1, Scott Farber, Piyaraj Newton, Thomas H Tung 1, Susan E Mackinnon 1, Philip J Johnson 1
PMCID: PMC3613760  NIHMSID: NIHMS435294  PMID: 23542267

Abstract

Background

Successful nerve regeneration is critical to the functional success of composite tissue allografts (CTA). The present study was designed to characterize the effect of acute rejection on nerve regeneration and functional recovery in the setting of orthotopic limb transplantation.

Methods

A rat orthotopic limb transplantation model was used to evaluate the effects of acute rejection on nerve regeneration and motor recovery. Continuous administration of FK506 (Full suppression), administration of FK506 for the first 8 of 12 weeks (Late rejection), or delayed administration of FK506 / dexamethasone following noticeable rejection (Early rejection) was used to preclude or induce rejection following limb transplantation. Twelve weeks postoperatively, nerve regeneration was assessed via histomorphometric analysis of explanted sciatic nerve, and motor recovery was assessed via evoked muscle force measurement in extensor digitorum longus (EDL) muscle.

Results

A single episode of acute rejection that occurs immediately or late after reconstruction does not significantly alter the number of regenerating axonal fibers. Acute rejection occurring late after reconstruction adversely affects EDL muscle function in CTA.

Conclusion

Collected data reinforces that adequate immunosuppressant administration in cases of allogeneic limb transplantation ensures levels of nerve regeneration and motor functional recovery equivalent to that of syngeneic transplants. Prompt rescue following acute rejection was further demonstrated not to significantly affect nerve regeneration and functional recovery post-operatively. However, instances of acute rejection that occur late after reconstruction affect graft function. In total, the present study begins to characterize the effect of immunosuppression regimens on nerve regeneration and motor recovery in the setting of CTA.

Keywords: Composite Tissue Allograft, acute rejection, rat, nerve regeneration

Introduction

Since the first successful hand transplantation in September 1998, 1 almost 70 procedures have been performed worldwide2, 3. One of the primary concerns of hand-recipients remains the totality of functional recovery. Unlike visceral organ transplants, composite tissue transplants (CTA), such as hand transplants, require successful regeneration of nerve components to gain use of the grafted tissues.

Robust reinnervation of graft motor and sensory targets is necessary for intrinsic muscle function and sensory input in CTA. Unlike peripheral nerve regeneration in autologous tissue, nerve regeneration in CTA is susceptible to immune rejection. Previous studies have demonstrated that immune rejection can negatively affect regeneration in a nerve allograft4–7. Nerve regeneration in autologous tissue is highly dependent on Schwann cell (SC) de-differentiation and proliferation to stimulate and maintain axonal regeneration8–13. Following successful regeneration, SCs secret factors and myelinate axons that promote neuronal survival and allow for efficient transmission of information via saltatory conduction. Thus, the absence of SCs following injury prevents regeneration14–16 and the loss of SCs after successful regeneration eliminates end organ function through conduction block9. Significant loss of distal donor SCs to rejection either early after reconstruction or at chronic time points following neurologic recovery could significantly impair function of CTAs.

The current study evaluates the effect of acute rejection on nerve regeneration and functional recovery in the setting of CTA. A rat orthotopic limb transplantation model was employed, and animals were administered various regimens of Tacrolimus (FK506) designed to modulate the extent of acute rejection post-operatively17, 18. Histomorphometric and functional assessments of experimental groups demonstrated that a single episode of acute rejection that occurs early (5 days) after CTA does not affect establishment of graft function in CTA. However, acute rejection occurring late (8 weeks) after reconstruction does affect maintenance of graft function in a rodent model.

Material and Methods

Animal Care

Animal studies were performed in accordance with the NIH Guide for the Care and Use of Laboratory Animals under experimental protocols approved by the Animal Studies Committee at Washington University in St. Louis. Animals were housed in a central animal care facility with access to standard rodent chow and water ad libitum. Post-operatively, all animals were evaluated daily for clinical signs of distress and graft rejection. Criteria used to diagnose rejection were similar to those previously described for rodent CTA models. 19–24 Acute rejection was quantitatively graded as: 0=no rejection, 1=edema, 2=erythema, 3=epidermolysis/desquamation, 4=necrosis and 5=mummification based on previously established guidelines25. Occurrence of edema and erythema was considered the start of acute rejection. 12 weeks postoperatively was defined as the end time point.

Experimental Design

Twenty five male Lewis (RT11) rats (200–250 g) (Charles River Laboratories, Wilmington, MA) were randomized into four experimental groups containing 4 to 9 animals per group (Table 1). Animals in all allogeneic groups underwent allogeneic hind limb transplantation in which Lewis rats served as graft recipients and Brown Norway (BN, RT1n) rats served as graft donors. Animals undergoing syngeneic orthotopic hind limb transplantation (SYN, n = 5) served as a positive control. A group of animals underwent allogeneic orthotopic hind limb transplantation followed by continuous FK506 (LC Laboratories, Woburn, MA USA, Cat. No: F-4900) administration (2mg/kg, s.c., daily) to prevent acute rejection (ALLO-NR, n = 4). An additional group underwent allogeneic limb transplantation followed by one episode of acute rejection due to delayed FK506 administration (ALLO-ER, n = 7) to simulate the effect of a single episode of rejection early after reconstruction on regeneration and ultimate CTA function. Immunosuppression was withheld until animals demonstrated Grade 2 acute rejection in grafted limbs (Figure 1), at which time FK506 (2mg/kg, s.c.) and Dex (2mg/kg, s.c., Sigma, St. Louis, MO) were administrated to reverse rejection. Upon resolution of acute rejection, dexamethasone was withdrawn and FK506 treatment was continued for the duration of the study (12 weeks). To evaluate the presence of immune reactive cells within the nerve during rejection, an additional 3 animals were added to ALLO-ER and were sacrificed before and shortly after rescue. The final experimental group underwent allogeneic limb transplantation followed by late acute rejection (ALLO-LR, n = 9) to determine the effect of late episodes of acute rejection on the maintenance of CTA function. FK506 administration was initiated immediately after allogeneic limb transplant and continued for 8 weeks post-operatively, at which time FK506 was withdrawn. Upon evidence of Grade 2 acute rejection in grafted limbs animals were rescued with FK506 and Dex, as described above.

Table 1.

Groups and rejection rescue

Group Rat Number Rats showed rejection Sign Rejection Sign Days of rejection-post operation Days of rejection-post FK506 withdrawal
Syngeneic transplant (SYN) 5 0
Allogeneic-No Rejection (ALLO-NR) 4 0
Allogeneic-Early Rejection (ALLO-ER) 7 7 Edema & Erythema 4.9±1.2
Allogeneic-Late Rejection (ALLO-LR) 9 7 Erythema 19.1±4.3
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Rescue: Treatment with FK506 (2mg/kg) and Dexamethasone (2mg/kg). Then maintain FK506 until endpoint (12 weeks).

Figure 1.

Figure 1

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Visual identification of allograft rejection. Left panel (A and C): Acute rejection episodes were identified by the presence of erythema and edema in the transplanted limb. Right panel (B and D): The grafted limb appears normal 24 hrs following rescue with FK506 and Dex injection.

Surgical Procedures

Orthotopic hind-limb transplantation was performed from BN to Lewis rats and from Lewis to Lewis rats under allogeneic and syngeneic scenarios, respectively 26, 27. Anesthesia was induced and maintained via isoflurane gas (2%, IH). Right hind limbs of donor and recipient animals, including bone, muscle, femoral vessels, sciatic nerve, saphenous nerve, and skin, were amputated at the mid-femoral level. Donor hind limbs were orthotopically transplanted onto recipients. Osteosynthesis was achieved using an 18-gauge needle as an intramedullary rod. Musculature was re-approximated by groups and secured using 6-0 Vicryl suture (Ethicon, Somerville, NJ). Donor femoral artery and vein were microsurgically anastomosed end-to-end to recipient femoral artery and vein using 10-0 nylon suture. Animals were administered 5ml saline via intraperitoneal injection prior to releasing vessel clamps. Following re-established blood flow, host and donor sciatic and saphenous nerve stumps were approximated with epineurial neurorrhaphy using 10-0 nylon suture. Skin incisions were then closed with 4-0 nylon suture. Post-operatively, animals were maintained on a warming pad overnight for observation prior to returning to the animal care facility.

Muscle Force Measurement

Twelve weeks post-operatively, functional recovery was assessed by measuring evoked motor response in donor EDL muscle. Animals were anesthetized via subcutaneous injection of ketamine (75 mg/kg; Fort Dodge Animal Health, Fort Dodge, IA) and dexmedetomidine (0.5 mg/kg; Pfizer Animal Health, Exton, PA) prior to immobilization in an automated functional assessment station (FASt System, Red Rock Laboratories, St. Louis, MO)28–30. Distal tendons of the EDL muscle were exposed and fixed to a 5 N load cell. Cathodic, monophasic electrical impulses (duration=200 ms, frequency = single–200 Hz, burst width = 300 ms, amplitude = 0–1000 μA) were applied to the host sciatic nerve proximal to the point of coaptation via silver wire electrodes, while resulting force production in the EDL was recorded using data acquisition software as described previously (RRL V.1.0, Red Rock Laboratories) 28–30. Values were compared to measurements taken from 4 unoperative, healthy Lewis rats. Following muscle force measurement, repaired sciatic nerves (recipient /donor complex) were harvested for histomorphometric analysis.

Histomorphometric Analysis

Repaired sciatic nerves (recipient / donor complexes) were harvested and processed in 3% glutaraldehyde (Polysciences Inc., Warrington, PA) and 1% osmium tetroxide and embedded in Araldite 502 (Polysciences, Inc., Warrington, PA) as described previously31. Ultra-thin (< 1um) cross-sections were obtained from donor nerve distal to the site of coaptation using an Ultramicrotome, stained with toluidine blue, and evaluated under light microscopy. Further analyses of microscopic images were performed via automated digital image analysis system linked to custom histomorphometry software, 31 which was utilized to quantify total nerve fiber counts. An observer blinded to the experimental groups performed all analyses and obtained all measurements.

Immunohistochemical Staining with anti-S100

Immunohistochemical Staining with anti-S100 (Dako, UK) was used to evaluate the presence of SCs in the donor nerve of CTA during episodes of rejection. Distal explanted nerves were fixed with 4% (v/v) paraformaldehyde (Sigma, MO) overnight and were then transfer to 30% sucrose for cryoprotection. The nerves were cryosectioned (25μm) and then transferred onto glides slides. The sections were incubated with blocking buffer that consisted 2% (v/v) Triton X-100, 20% (v/v) goat serum and 5%BSA in PBS for 1 h. Slides were then incubated with primary antibodies rabbit anti-S100 (1:500, Dako, UK) for overnight. The next day slides were incubated with secondary antibody (1:500, Alexa Fluor goat anti-rabbit 488, Invitrogen, USA) at room temperature in the dark for 1 h. Repeated washing steps were done after secondary antibody incubation. The slides were then mounted with Vectashield with DAPI (Vector Labs, UK) and examined under fluorescence microscope.

Statistical Analysis

Statistical analyses were performed using Statistica statistical software (StatSoft, Inc.). One-way analysis of variance (ANOVA) was performed to evaluate the differences between experimental groups. Post-hoc Student-Newman-Keuls test was used to isolate significant differences between groups with correction for multiple comparisons. Statistical significance was established at p < 0.05. All results are reported as mean ± standard deviation.

Results

Acute rejection and rescue

All transplanted limbs demonstrated some degree of edema, which generally resolved within the first week post-operatively. Syngeneic transplants (SYN) showed no sign of rejection. Allografts with continuous administration of FK506 (ALLO-NR) showed no signs of rejection. In allograft subjected to an early episode of immune rejection (ALLO-ER), withholding FK506 administration resulted in visible erythema/edema of the grafted limb at an average of 4.9 days post-operatively (Table 1). Administration of FK506 and Dex resulted in noted recovery within 24 hours of rescue (Figure 1). 25 Following acute rescue and continued FK506 administration, grafted limbs survived without additional signs of rejection. In allografts subjected to a late episode of immune rejection (ALLO-LR), animals were treated with FK506 for 8 weeks postoperatively. After withdrawing FK506, seven of nine animals demonstrated signs of rejection appearing at an average of 19.1 days after FK506 withdrawal. Administration of one bolus of FK506 and Dex to animals resolved symptoms of rejection. Interestingly, two animals in Group D showed no sign of rejection long after discontinuation of FK506 (Table 1, Figure 2). Only animals that exhibited outward signs of rejection where included in further data analysis.

Figure 2.

Figure 2

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A) A representative image from ALLO-NR demonstrates the normal growth new hair and nails in the grafted limb at the end time point of 12 weeks post transplantation. B) Removal of the hair demonstrated normal healing of the suture line and muscle tone of donor graft.

Quantitative nerve histomorphometry

Nerve regeneration was assessed via histomorphometric analysis of explanted sciatic nerve. Nerve sections from donor nerve tissue distal to the site of coaptation were analyzed in all groups. SYN demonstrated robust nerve regeneration in the setting of syngeneic transplantation (Figure 3A). ALLO-NR, ALLO-ER, and ALLO-NR groups demonstrated similar regenerative nerve tissue in the settings of allogeneic transplant with and without early and late acute rejection. In comparison to SYN, sections taken from ALLO-NR, ALLO-ER, and ALLO-NR groups demonstrated qualitatively smaller diameter nerve fibers and larger amount of degenerative debris (Figure 3B, 3C, and 3D). Macrophage/monocyte infiltration was not observed in all samples denoting an absence of immune rejection at the time of harvest.

Figure 3.

Figure 3

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Representative histograms for nerve fibers from each experimental group. A) SYN group demonstrates robust regeneration with normal mature myelinated nerve architecture at 12 weeks post-transplantation. Within the allograft groups (ALLO-NR, ALLO-ER, and ALLO-LR) a similar extent of axonal regeneration was observed. Compared with SYN, the allograft groups appeared to demonstrate smaller fibers and increases in degenerative debris. However, for all groups other than the ALLO-LR these differences where not quantitatively significant. All allograft groups demonstrated a complete lack of immune cell related infiltration demonstrating the absence of host immune response at the time of sacrifice.

The number of myelinated nerve fibers and the fiber width within repaired sciatic nerve tissue was used to quantify the degree of nerve regeneration following transplantation. The presence of numerous myelinated nerve fibers demonstrated robust nerve regeneration in all experimental groups. No statistically significance differences in the number of myelinated fibers were noted between experimental groups (Figure 4A). The ALLO-LR group demonstrated a significant decrease in average fiber width compared to all other groups (p<0.05). The fiber width is a measure of the combined axon fiber and myelin diameter.

Figure 4.

Figure 4

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Histomorphometric analysis of total fiber counts (upper panel) and fiber width (lower panel) for each experimental group. A) No significant differences were found in the total number of regenerated myelinated nerve fibers. B) Quantification of the fiber width in each group revealed significant differences between the ALLO-LR compared to every other experimental group. (Error bars denote standard deviation, * indicates p<0.05).

Functional recovery in grafted EDL muscles

Twelve weeks post-operatively, motor recovery was assessed via evoked tetanic muscle force measurement in transplanted donor EDL. ALLO-ER group demonstrated the largest degree of functional recovery, ~86% (23.4±4.1 N/cm2) compared with unoperative control (100%, 27.3±1.7 N/cm2). The other groups followed with SYN group ~80% (21.8±10.1 N/cm2) in syngeneic graft, ALLO-NR group with 76% (20.9±3.4 N/cm2) in no rejection allogeneic graft, and ALLO-LR group with ~40% (11.0±8.5 N/cm2) in allogeneic graft with late rejection (Figure 5). The ALLO-LR group exhibited significantly less EDL function when compared to unoperative control and ALLO-NR (p<0.05).

Figure 5.

Figure 5

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Evoked muscle force measurement of extensor digitorum longus (EDL) muscle at 12 weeks post transplantation. Transplanted limbs from all experimental groups except ALLO-LR recovered muscle function in the EDL to a level not statistically different from a cohort of healthy unoperated controls (UN-OP). The tetanic specific muscle force comparison of the ALLO-LR group was significantly lower in comparison to ALLO-NR and UN-OP. (Error bars represent standard deviation, * indicates p<0.05).

Immunohistochemical Staining with anti-S100

Immunohistochemical staining with anti-S100 was used to evaluate differences in SCs population in the explanted sciatic nerve in both early and late rejection groups. The macrophage/monocyte infiltrations in explanted nerve before and after rescue treatment were also evaluated by histological analysis. Qualitatively, the expressions of S-100 in CTA subjected to early episodes of rejection did not show any difference between early rejection animals with and without rescue (Figure 7A and B). Additionally, histological analysis showed an absence of immune infiltrate in early rejection groups (data not shown). However, evaluation of histological samples of the distal nerve obtained from the ALLO-LR group demonstrated decreased myelination and an abundance of thinly myelinated large caliber fibers (Figure 7C and D).

Figure 7.

Figure 7

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Images of immunohistochemical staining with anti-S100 and electro microscopy analysis. A) Immunohistochemical staining of harvested nerve from donor CTA after acute rejection without rescue. S100 staining for SCs is shown in green and nuclear staining with DAPI shown in blue. B) The same immunohistochemical staining of harvested nerve from donor CTA after acute rejection with rescue intervention. Again SCs are shown in green (S100) and nuclear staining in blue (DAPR). Histological micrographs of distal donor nerve sections from CTA in the ALLO-LR group are shown at 40x (C) and 400x (D). An abundance of fibers can be observed but these fibers are associated with a qualitative decrease in myelination. Histological micrographs of the EDL muscle after acute rejection showed normal micro architecture in both low (x40, E) and high power magnification (x100, F).

Discussion

Peripheral nerve regeneration is essential for long term CTA utility. Successful nerve regeneration and function is dependent on SCs. In traditional nerve transplantation, donor SCs in peripheral nerve allografts stimulate host axonal regeneration and facilitate chronic nerve function, allowing for successful repair of significant segmental nerve injuries (Figure 6A)32–34. However, donor SCs are susceptible to immune rejection, and we have demonstrated that the loss of donor SCs in an allograft is compensated for by the migration of host SCs into nerve allografts35–38. In a nerve allograft under complete immunosuppression, host SC migration into the graft is minimal because of the maintained presence of donor SCs10. Removal of immunosuppression results in rejection and loss of donor SCs in the graft, which stimulates host SC migration into the graft10. Migration of distal stump SCs is the predominant source of replacement host SC to a nerve allograft39 with only minor contributions from the proximal stump. Thus, in a nerve allograft, where rejection is a permanent threat, host SCs replace lost donor SCs, and support continued axonal regeneration and maintenance of long term nerve function.

Figure 6.

Figure 6

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SC migration in nerve grafting. The proliferation and migration of SCs in vivo following nerve grafting is stimulated by the loss of SCs in an adjacent location along the nerve. A) In nerve allograft, lost donor SCs (Blue) due to immune rejection are replaced by host SCs (yellow) that migrate from both the proximal and distal coaptation sites. However, a more substantial contribution of replacement SCs is provided from the distal nerve stump. Complete loss of donor SCs in a nerve allograft leads to complete replacement by host SCs and restoration of function. B) In CTA, lost donor SCs are replaced by proximal host SCs only (I). Early episodes of acute rejection likely stimulate proximal host SCs migration into the nerve (II). Under complete immune suppression, the regenerating host axons reach the donor muscle target and are myelinated by donor SCs (III). In the event of unexpected late episodes of acute rejection, loss of donor SCs can result in the loss of neurological function and conduction block unless host SCs can migrate sufficient distances to re-myelinate the exposed host axons (IV). Note that in all situations it is the host axons that regenerate towards the distal motor/sensory targets.

CTA procedures involving grafted nerve tissue represent a unique corollary to studies of long-term nerve allograft viability (Figure 6B). Specifically, the distal stump population of host SCs does not exist in the paradigm of CTA. In a CTA donor SCs are the major source of support for regenerating axons and for long term maintenance of neurologic function. In the event of an unforeseen episode of acute rejection, a common occurrence in CTA, replacement of loss donor SCs by host SCs may be limited. The present study was designed to characterize the effect of episodes of acute rejection on nerve regeneration and CTA motor function in the setting of orthotopic limb transplantation.

In the absence of immunosuppression, initial signs of CTA rejection (erythema) in rodents have been observed 4 to 6 days following transplantation25. In the present study, withholding FK-506 resulted in similar symptoms (Figure 1). During the early episode of rejection (ALLO-ER), rescue of transplanted limbs was effective, with visible recovery noted within 24 hours (Figure 1). Additionally, histological analysis of nerve tissue after 12 weeks demonstrated an absence of immune infiltrate, inflammation and cellular debris that is characteristic of immunologic rejection of the nerve5, 7. The present findings are consistent with a previous report that the combination of cyclosporine (CsA) and Dex are effective in rescue interventions25.

In contrast to the consistent early rejection seen in ALLO-ER, 77.8% of the animals from ALLO-LR were observed to develop signs of rejection following late discontinuation of FK506. Of those that rejected, the average onset of symptoms occurred 19.1 days after FK506 withdrawal (Table 1). The remaining two animals in ALLO-LR did not show any outward or histological signs of rejection through the remainder of the study (Figure 2). The bone component of the CTA used in the present study contains vascularized bone marrow, and limb transplantation constitutes donor bone marrow transplantation. The engraftment of donor marrow facilitates rapid population of bone marrow-derived stem cells into the recipient lymphoid organs40, 41. Engraftment of certain fractions of donor bone marrow have been demonstrated to produce chimerism and variant levels of graft tolerance42. Over the 8 weeks of FK506 administration in ALLO-LR, it is therefore hypothesized that the combination of FK506 and vascularized bone marrow provided a means of inducing bone marrow-derived chimerism in lymphoid tissues within the animals preventing late rejection. The present hypothesis offers a possible explanation for the animals in the late rejection group that did not develop signs of rejection well after FK506 had been withdrawn.

Twelve weeks post-operatively, the affect of acute rejection on nerve regeneration was assessed via histological and histomorphometric analysis of explanted donor sciatic nerve. Prior experimental research, as well as clinical experience, has demonstrated the ability of FK506 to improve nerve regeneration across many different models of nerve injury and repair5, 43–50. In all studies, FK506 was used as an immunosuppressant to prevent rejection and for rescue intervention. Within the present study, histological data demonstrated that all groups experienced robust nerve regeneration post-operatively. Given the time point for histomorphometric evaluation (12 weeks), differences in axonal regeneration due to FK-506 were not expected49. The absence of statistically significant differences between experimental groups indicates multiple important considerations. First, effective immunosuppression following allotransplantation ensures an equal capability for functional recovery compared to syngeneic transplants. Second, prompt rescue following a single early or late episode of acute rejection effectively reverses clinical signs of rejection.

Evoked force production in donor EDL muscle was measured in all animals and provides a unique measure of functional reinnervation of muscle fibers by regenerating axons51, 52. Specific tetanic force measurements in all allogeneic transplantation groups showed similar functional recovery compared to syngeneic transplant controls. The ALLO-LR group, however, demonstrated significant differences when compared to ALLO-NR and the unoperated controls. Collected data indicates that a single early episode of acute rejection followed by prompt rescue did not interfere with functional reinnervation of the donor end-organ muscle. It is likely that rejection and loss of donor SCs early after graft transplantation is compensated for by host SCs present in the proximal stump and thus had no discernible effect on regeneration. While early acute rejection demonstrated no significant effect on regeneration or graft function, previously we have shown in nerve allografts that the duration of the rejection event affects the possibility of nerve rescue and a similar window of rejection exposure likely exists for CTA5. Qualitatively, S-100 expression in the graft did not demonstrate a significant change in response to rejection. However, in the absence of prompt rescue, it is possible that early episodes of acute rejection could prevent successful regeneration.

Average specific tetanic force in the allogeneic late rejection group (ALLO-LR) was significantly decreased when compared to allografts experiencing no rejection (ALLO-NR) and the unoperated control. Within the late stage rejection group, seven of the nine animals demonstrated outward signs of rejection after FK506 withdrawal. The seven animals that underwent rejection had a significantly lower specific force value when compared to the animals that did not demonstrate noticeable signs of rejection after FK506 removal (11.0 N/cm2 in rats with rejection/rescue v.s. 24.39N/cm2 in rats without any rejection sign, p<0.05). The decrease suggests that episodes of acute rejection post transplantation have a detrimental effect on graft function at late time points following reconstruction.

The significant loss of muscle function observed in the animals from ALLO-LR could be a result of immunological related effects on the nerve or the muscle. Previously, we have demonstrated and have discussed above that donor SCs are susceptible to immune rejection10. Loss of or damage to donor SCs in the CTA could cause loss of signal transduction in the innervating nerve via conduction block. The result of graded loss of SCs (i.e. not all the donor SCs) would result in sub maximal recruitment of muscle fibers and thus a decrease in the maximum force generated by the end organ muscle. Immunohistochemical staining of SCs in the distal nerve of the late rejection group showed no qualitative differences in intensity of staining. However, analysis of the fiber width demonstrated a significant decrease in the fiber width of the ALLO-LR groups when compared to all other groups. Fiber width is a measure of axon fiber and myelin diameter and the decrease may indicate some SC related pathology in the ALLO-LR group. Similarly, immunogenic reaction in the donor muscle could have affected our endpoint evaluation. The loss of donor muscle fibers due to immune rejection may have affected the ability of the muscle to generate force. Although, histological evaluation of the muscle from both rejections groups demonstrated no overt adverse effects to the donor muscle following episodes of graft rejection.

The ability of CTA graft function to recover from late episodes of acute rejection was not evaluated. In the groups subjected to early episodes of acute rejection (ALLO-ER), the rejection occurred mostly within the first week after surgery and was immediately followed by rescue intervention. The animals in that group recovered for ~79.5 days following rescue. This recovery period provided additional time for both donor and recipient SCs to recover from the immune assault. In contrast, the animal in the ALLO-LR group on average recovered for only 8.9 days following rejection before muscle evaluation. Any damage to SCs or neurologic function may not have had time to recover. It is possible, given sufficient time that EDL muscle function would return to baseline levels.

Conclusions

In summary, variable FK506 administration was utilized to preclude and induce rejection following limb transplantation in a rodent model. Prompt rescue after one episode of acute rejection was effective at restoring capacities for both nerve regeneration and functional recovery post-operatively. The present study also indicates that with proper immunosuppressant administration, nerve regeneration and motor functional recovery in allergenic transplanted animals can achieve equivalent outcomes to that of syngeneic control. Furthermore, the results suggest that episodes of acute rejection that occur chronically after reconstruction negatively affect the function of the graft despite previously successful nerve regeneration and establishment of function.

Table 2.

The recovery time after rescue intervention

Group Animals The day of rescue after surgery Days for recovery after rescue Average (days for recovery)
ALLO-ER ER-1 6 78 79.5±1.3
ER-2 4 80
ER-3 3 81
ER-4 5 79
ALLO-LR LR-1 80 4 8.86±4.3
LR-2 79 5
LR-3 76 8
LR-4 68 16
LR-5 72 12
LR-6 73 11
LR-7 78 6
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Note: in late rejection group, animals were treated with FK506 for 8 weeks, the end time points were 12 weeks

Acknowledgments

This study was supported by National Institutes of Health, NINDS grants (R01NS033406) and partly by National Endowment for Plastic Surgery under the Plastic Surgery Educational Foundation (NEPS07-07). The authors have no financial interest in the publication of this work.

Footnotes

A portion of this work was presented at the 2011 Plastic Surgery Research Council meeting in Louisville, Kentucky.

FD - None of the authors has a financial interest in any of the products, devices, or drugs mentioned in this manuscript.

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