Basement membrane component changes in nerve allografts and isografts

This study describes immunocytochemical changes in laminin, which is an integral basement membrane (BM) component, during axonal regeneration through antigenic nerve allografts and nonantigenic nerve isografts. In normal rat nerve, laminin was localized in the BM of Schwann cells and the perineurium. During nerve allograft rejection, the perineurium and Schwann cells disappeared. However, the Schwann cell BMs persisted and became distorted and collapsed. In isografted nerves, the perineurium and Schwann cells were present, and only a few Schwann cell BMs appeared to be distorted; however, the staining for laminin was faint, indicating a possible BM breakdown. A new BM appeared as small rings around the Schwann cells after they had become associated with regenerated axons. Because only a limited axonal regeneration occurred in allografts as compared to isografts, it is concluded that the viable Schwann cells, and their BM architecture, are essential for regeneration through long nerve grafts.     

Immunohistochemical assessment of rat nerve isografts and immunosuppressed allografts

Objective: Autologous peripheral nerve grafts are commonly used clinically as a treatment for peripheral nerve injuries. However, in research using an autologous graft is not always feasible due to loss of function, which in many cases is assessed to determine the efficacy of the peripheral nerve graft. In addition, using allografts for research require the use of an immunosuppressant, which creates unwanted side effects and another variable within the experiment that can affect regeneration. The objective of this study was to analyze graft rejection in peripheral nerve grafts and the effects of cyclosporine A (CSA) on axonal regeneration.

Methods: Peripheral nerve grafts in inbred Lewis rats were compared with Sprague-Dawley (SD) rats to assess graft rejection, CSA side effects, immune responses, and regenerative capability. Macrophages and CD8+ cells were labeled to determine graft rejection, and neurofilaments were labeled to determine axonal regeneration.

Results: SD rats without CSA had significantly more macrophages and CD8+ cells compared to Lewis autografts, Lewis isografts, and SD allografts treated with CSA. Lewis autografts, Lewis isografts, and SD autografts had significantly more regenerated axons than SD rat allografts. Moreover, allografts in immunosuppressed SD rats had significantly less axons than Lewis rat autograft and isografts.

Discussion: Autografts have long been the gold standard for treating major nerve injuries and these data suggest that even though CSA is effective at reducing graft rejection, axon regeneration is still superior in autografts versus immunosuppressed allografts.     

The fate of cryopreserved nerve isografts and allografts in normal and immunosuppressed rats

Donor Schwann cells, perineurial cells, and vasculature are known to survive in grafts of peripheral nerve. In the present study, we attempted to cryopreserve nerve to determine whether these cellular components of nerve would survive after transplantation and support host axonal regeneration through the graft. Four-centimeter lengths of peroneal nerves were removed from inbred adult American Cancer Institute (ACI) rats and placed into vials that contained a cryoprotective mixture of dimethyl sulfoxide and formamide (DF) at room temperature. Each vial with nerves in DF was cooled at a rate of 1–1.5°C/minute down to –40°C at which point the vials were plunged into liquid nitrogen at –196°C. After 5 weeks of storage, the nerves were thawed and DF removed. Some of the cryopreserved-thawed ACI nerves were transplanted as isografts into the legs of ACI rats. Other ACI nerves were used as allografts and inserted into immunologically normal Fischer (FR) rats that were untreated or were immunosuppressed with the drug Cyclosporin A (Cy-A). At surgery, only one end of the nerve graft was joined to the cut proximal end of the peroneal nerve of the host. The cellular elements of ACI grafts were present at 5 weeks in grafts removed from ACI rats and FR rats treated with Cy-A. Non-immunosuppressed FR rats rejected ACI nerves as did FR rats in whom Cy-A was stopped after 5 weeks of treatment. All surviving ACI grafts underwent Wallerian degeneration and consisted of columns of Schwann cells, which in their proximal portion were associated with regenerating host axons. The donor perineurial sheath and vasculature were also present in surviving grafts. ACI isografts only were examined 20 weeks postoperatively. All normal tissue components survived in these older grafts and contained regenerated and myelinated host axons throughout their 4 cm lengths. These results demonstrated that the cellular elements of nerve can be cryopreserved, and after transplantation, survive and function. Because nerves survived after prolonged cryopreservation, it seems feasible to establish a nerve bank from which grafts can be withdrawn to repair gaps in injured nerves. However, cryopreserved nerves used as allografts remain immunogenic and require immunosuppression for their survival. Published in 1993 by Wiley-Liss, Inc.     

A quantitative developmental study of the peripheral nerve lipid composition during myelinogenesis in normal and trembler mice

The quantitative evolution of 10 polar lipids was examined in the sciatic nerves of normal and trembler mice between the ages of 3 days and 60 days. In normal nerves, the polar lipids accumulated slowly until the age of 9 days. A period of rapid accumulation then took place until 18 days of age, after which the phospholipids plateaued, while the glycolipid content continued to increase at a slower rate. The results obtained for the sciatic nerves of trembler mice show that the accumulation of all the polar lipids studied, except phosphatidylcholine and hydroxysulfatides, is abnormal from the earliest stages of postnatal development, and strongly support the view that the primary disorder in the trembler peripheral nervous system is one of dysmyelination. With the exception of cardiolipin, all the lipids in the trembler nerves stopped accumulating at the age of 18 days. The cerebrosides were the lipids the most affected severely at all ages.     

Metabolic alterations of endoneurial lipids in developing trembler nerve

In vitro incorporation of [14C]acetate into desheathed sciatic nerve (endoneurium) was studied in developing normal and mutant trembler mice. The total uptake of [14C]acetate peaked 6 days after birth and decreased thereafter for both normal and trembler mice. Substantially less [14C]acetate (approximately 50% of normal) was incorporated in trembler mouse as early as 3 days after birth. In 3 and 6 day-old trembler mice, proportionately less [14C]acetate was incorporated into phospholipids and cerebrosides and more was incorporated into cholesterol. From 9 days, however, less [14C]acetate was incorporated into cholesterol of trembler compared to normal nerve. At later times (greater than or equal to 20 days), the proportion of [14C]acetate in both phospholipids and cerebrosides of trembler nerve was increased above that of controls. Throughout development, the relative incorporation of [14C]acetate into triacylglycerol and cholesteryl esters was about two times higher in trembler than in normal mice. In normal nerve, the 14C-label in free fatty acids increased (4-25%) progressively with age. In contrast, this incorporation in trembler nerves remained constantly low (less than 7%). Morphologically, the nerves of trembler mouse are markedly hypomyelinated. The abnormal myelin sheath undergoes cycles of breakdown and partial regeneration. Thus, the biochemical profile of extended active but perturbed lipid metabolism may provide a basis for the morphologic findings of continued active but ineffective myelination in trembler peripheral nerve.     

Improved myelination in nerve grafts from the leucodystrophic twitcher into trembler mice: Evidence for enzyme replacement

The possibility of the treatment of globoid cell leucodystrophy in the twitcher mouse by enzyme replacement was investigated using nerve grafts from affected animals into trembler hosts. The trembler mouse has no known enzyme deficiency but its peripheral nerves are hypomyelinated due to a Schwann cell abnormality and this defect represents a marker used in the present study to exclude the possibility of migration of Schwann cells from the host into the graft. Twitcher grafts were examined after periods ranging from 1 to 4 months. At all stages myelin sheaths were well formed and did not show signs of degeneration. Moreover the interstitial oedema, characteristic of the twitcher nerve, was greatly diminished in amount and no globoid cells were seen. These results were compared with previous studies done in vivo and in vitro in other types of lysosomal storage disease. We concluded that the improvement of the conditions of the myelin in the transplant is possibly due to enzyme replacement from the host.     

Alteration of sulfatide synthesis in control and trembler mice during wallerian degeneration and remyelination

Sulfatide synthesis from sulfate is much greater in the peripheral nerves of the Trembler mouse. After nerve transection, during Wallerian degeneration, this synthesis rate drops down very rapidly in both normal and Trembler mice. Twenty-four hours after permanent transection, the rate of synthesis is reduced by 80% in the mutant and 50% in the normal mouse. Four days after transection, the synthesis rate in the Trembler is only 9% of that observed in intact nerves and 21% of that in the intact nerves of normal animals. After 5 d the synthesis remains constant. Thus, enhanced synthesis of sulfatides in the Trembler mouse is probably not caused by Wallerian degeneration. After crush of the sciatic nerve, the synthesis rate decreases very rapidly in the normal mouse as it does after permanent transection. But during regeneration, from the 7th day, it rises dramatically and 14 d after crush, a 2.5-fold increase in the synthesis rate is observed, compared to that in the contralateral control nerve. This synthesis rate returns to normal 1 mo after crush. In the Trembler, the synthesis decreases for 2 d after crush and increases from then on, eventually reaching the value of the contralateral control Trembler nerve within 2 mo. In the mutant there is no prominent peak of sulfatide synthesis during regeneration.     

Prolonged cold-preservation of nerve allografts

The goal of this study was to determine the effect of varying durations of cold-preservation on the immunogenicity of nerve allografts and their subsequent ability to facilitate neuroregeneration across a short nerve gap. Allografts preserved for 1, 4, and 7 weeks were compared to untreated allografts and isografts. There was a shift from an interferon-gamma-producing cellular response (untreated allografts) to an absence of response (7-week cold-preserved allografts and isografts). There were no detectable alloantibodies by flow cytometry. Histomorphometry distal to the graft showed robust regeneration in the isograft and 7-week cold-preserved groups when compared to the untreated allograft group. Increasing duration of cold-preservation diminished the cellular immune response. This cold-preservation does not preclude subsequent nerve regeneration across a short nerve graft. Prolonged cold-preservation of nerve allograft tissue could serve as a means to produce unlimited graft material for use in peripheral nerve reconstruction.     

Rat aorta isografts possess nerve regeneration-promoting properties in silicone Y chambers

Recent investigations using synthetic entubation materials have demonstrated that nerve regeneration across a gap requires peripheral nervous tissue as a distal component within the chamber. Regeneration fails if the distal end of the regeneration chamber remains open to the environment, or is filled with tendon or skin. These results are contrary to those of Weiss and Taylor (1944) who used homologous aorta as a chamber material and found that regeneration was successful across a gap regardless of the distal tissue, even in the branches of the aorta that were closed or open. In the present report, silicone Y chambers were implanted with the distal branch remaining open, or filled with pieces of nerve, tendon, or isologous aorta. A homogenous fibrin matrix formed in all the chamber branches by 1 week postimplantation. At 4 weeks, regeneration across a 10-mm gap failed in the chamber branches that were open or filled with tendon. However, regeneration was successful in those branches containing aorta or nerve. These experiments strongly support the hypothesis that aorta tissue, like peripheral nervous tissue, is a source of regeneration-promoting materials. The results provide a probable explanation for and a reconciliation of the discrepancies between the earlier observations of Weiss and Taylor and those of more recent investigators.     

Acellular nerve allografts in peripheral nerve regeneration: a comparative study

Introduction: Processed nerve allografts offer a promising alternative to nerve autografts in the surgical management of peripheral nerve injuries where short deficits exist. Methods: Three established models of acellular nerve allograft (cold‐preserved, detergent‐processed, and AxoGen‐processed nerve allografts) were compared with nerve isografts and silicone nerve guidance conduits in a 14‐mm rat sciatic nerve defect. Results: All acellular nerve grafts were superior to silicone nerve conduits in support of nerve regeneration. Detergent‐processed allografts were similar to isografts at 6 weeks postoperatively, whereas AxoGen‐processed and cold‐preserved allografts supported significantly fewer regenerating nerve fibers. Measurement of muscle force confirmed that detergent‐processed allografts promoted isograft‐equivalent levels of motor recovery 16 weeks postoperatively. All acellular allografts promoted greater amounts of motor recovery compared with silicone conduits. Conclusion: These findings provide evidence that differential processing for removal of cellular constituents in preparing acellular nerve allografts affects recovery in vivo. Muscle Nerve, 2011     

Effects of experimental diabetes on axonal and Schwann cell changes in sciatic nerve isografts

A reduced ability to regenerate peripheral axons may be partly responsible for diabetic neuropathy. The source of the impairment has not been narrowed down to axonal or Schwann cell failure. We used nerve grafts from control or diabetic donor rats transplanted into control or diabetic hosts to pursue this differential diagnosis. An isograft between the left sciatic nerves of inbred Lewis rats was performed 8 weeks after STZ treatment and on age-matched controls. The nerve exchanges were control-control, control-diabetic, diabetic-control and diabetic-diabetic. At postsurgical day 14, nerves were excised and analysed for levels of axonal markers, total and phosphorylated neurofilament, and Schwann cell receptors, ErbB2 and p75(NTR), using immunohistochemistry and Western blotting. The aim was to measure ingress of axonal markers into the graft and judge the appropriateness of Schwann cell phenotype changes. Transfer of nerve from diabetic to control rats resulted in a doubling in neurofilament, both phosphorylated and nonphosphorylated (both P<0.05). ErbB2 was decreased in grafts from diabetic rats (53% of control, P<0.05) and p75(NTR) levels were increased in both types of graft in diabetic rats (to 300-400% of controls, P<0.05). Schwann cells in diabetic nerve grafts showed receptor levels more similar to controls when placed into a normal environment and the converse also appeared to hold. TUNEL staining revealed increased apoptosis in diabetic nerve distal to the graft. The data show that alterations in Schwann cell phenotype in diabetes are reversed by transfer to control rats and develop in normal nerve after transfer to a diabetic host.     

Myogenic cell replication in minced skeletal muscle isografts of Swiss and BALBc mice

The onset and pattern of muscle precursor replication in minced skeletal muscle isografts were examined autoradiographically in 22 BALBc and 23 Swiss mice. Tritiated thymidine was injected into mice at various times after grafting to label replicating muscle precursors and regenerated muscle isografts removed 14 days after grafting when labeled precursors had fused into myotubes. Analysis of labeled myotube nuclei in regenerated minced isografts showed a difference between the onset of DNA synthesis in muscle precursors of the two strains, although precursor proliferation peaked around day 5 and was greatly reduced by day 8 in both strains. The onset and duration of muscle precursor replication was similar to that seen in isografts of intact whole muscles: however, precursor proliferation was delayed and protracted compared with injured muscles. This autoradiographic model enables quantitative investigations of muscle precursor behavior in vivo, with clinical potential for improving muscle regeneration after severe injury, transplantation, and some myopathies.     

Functional and immunological peculiarities of peripheral nerve allografts

This review addresses the accumulating evidence that live(not decellularized)allogeneic peripheral nerves are functionally and immunologically peculiar in comparison with many other transplanted allogeneic tissues.This is relevant because live peripheral nerve allografts are very effective at promoting recovery after segmental peripheral nerve injury via axonal regeneration and axon fusion.Understanding the immunological peculiarities of peripheral nerve allografts may also be of interest to the field of transplantation in general.Three topics are addressed:The first discusses peripheral nerve injury and the potential utility of peripheral nerve allografts for bridging segmental peripheral nerve defects via axon fusion and axon regeneration.The second reviews evidence that peripheral nerve allografts elicit a more gradual and less severe host immune response allowing for prolonged survival and function of allogeneic peripheral nerve cells and structures.Lastly,potential mechanisms that may account for the immunological differences of peripheral nerve allografts are discussed.     

Conditioned enhancement of skin allografts in mice

Healthy A/J mice grafted with either BALB/c or C57BL/6 tail skin routinely reject these grafts with a mean survival time (MST) of 12-14 days. Low dose cyclophosphamide, Cy (50 mg/kg) on the day of engraftment can enhance survival of both grafts (MST 17-20 days). If mice are given three weekly intravenous injections of BALB/c peripheral blood prior to grafting, specific enhancement of BALB/c but not C57BL/6 grafts results (MST 18 and 12 days, respectively). Mice given several ip treatments with Cy in association with a novel taste (saccharin, Sacc) in their drinking water also show a conditioned immunosuppression if subsequently exposed to Sacc alone. Such mice given BALB/c or C57BL/6 skin grafts and re-exposed to Sacc also show prolonged survival of these skin allografts (MST 16-17 days). If conditioned mice are also treated, by pretransplant donor-specific transfusion, to develop a state of specific suppression of allograft immunity, then subsequent grafting with BALB/c or C57BL/6 grafts coupled with re-exposure to Sacc lead to a further prolonged survival of grafts specifically in the BALB/c mice (MST 29 days).     

Optic axons regenerate into sciatic nerve isografts only in the presence of Schwann cells

Optic axons regenerate into normal but not acellular peripheral nerve (PN) grafts. The first axons penetrate the PN graft before 5 days and grow inside the basal lamina tubes amongst the Schwann cells. By 30 days, 4% of the surviving retinal ganglion cells (RGC) regenerate axons for at least 10 mm into the PN graft. Laminin rich basal lamina tubes persist in the acellular PN transplants but only a few axons penetrate the most proximal parts of the tubes by 5 days and none grow farther into the graft by 30 days. RGC counts demonstrate that 34% of the normal RGC population survive 30 days after anastomosing a normal PN to the transected optic nerve. After anastomosing acellular PN grafts, 25% of RGCs survive compared with 10% after optic nerve section. These findings demonstrate that laminin does not promote regeneration of axons and that Schwann cells play the primary role of offering trophic support and even a substrate for growth. RGC survival is also enhanced by PN grafts even when Schwann cells are absent. This latter result suggests that RGC survival is promoted by a trophic substance released from axons and/or Schwann cells in the PN grafts which survives the thawing/freezing procedure (used to kill the Schwann cells) and is active in the grafts in the immediate post operative period.     

An ultrastructural comparison of peripheral nerve allografts and autografts

Summary There is a marked difference in the cellular response of the host to peripheral nerve allografts and autografts. The response elicited by allografts is characterised by invasion of tissue with lymphocytes, plasma cells and activated macrophages. These cells disrupt the nerve architecture, and cause rupture and consequent compression of the neurolemmal tubes which are the essential conduit element of a nerve graft.By contrast, in the autograft, regeneration follows the initial process of Wallerian degeneration without the complication of immune reaction. The Schwann cells and macrophages rapidly remove the myelin and axons from the neurolemmal tubes of the donor nerve; new axonal sprouts are then able to traverse these channels and reach the periphery in large numbers. If this process is delayed, the neurolemmal tubes in the distal nerve segment are compressed by surrounding collagen which limits the size of regenerating axons.     

Ganglioside promotes the bridging of sciatic nerve defects in cryopreserved peripheral nerve allografts

Previous studies have shown that exogenous gangliosides promote nervous system regeneration and synapse formation.In this study,10 mm sciatic nerve segments from New Zealand rabbits were thawed from cryopreservation and were used for the repair of left sciatic nerve defects through allograft bridging.Three days later,1 m L ganglioside solution(1 g/L) was subcutaneously injected into the right hind leg of rabbits.Compared with non-injected rats,muscle wet weight ratio was increased at 2–12 weeks after modeling.The quantity of myelinated fibers in regenerated sciatic nerve,myelin thickness and fiber diameter were elevated at 4–12 weeks after modeling.Sciatic nerve potential amplitude and conduction velocity were raised at 8 and 12 weeks,while conduction latencies were decreased at 12 weeks.Experimental findings indicate that ganglioside can promote the regeneration of sciatic nerve defects after repair with cryopreserved peripheral nerve allografts.     

The vascularization pattern of acellular nerve allografts after nerve repair in Sprague-Dawley rats

Objective: We have demonstrated that angiogenesis in acellular nerve allografts (ANAs) can promote neuroregeneration. The present study aimed to investigate the microvascular regeneration pattern of ANAs in Sprague-Dawley (SD) rats.

Methods: Sixty male SD rats were randomly divided into an autologous group and a rat acellular nerve allograft group (rANA), and 10-mm sciatic nerve defects were induced in these rats. On the 7th, 14th and 21st days after surgery, systemic perfusion with Evans Blue (EB) or lead oxide was performed on the rats through carotid intubation. Samples were then collected for gross observation, and the microvessels in the nerves were reconstructed through microscopic CT scans using MIMICS software. The vascular volume fraction (VF, %) and microvessel growth rate (V, mm/d) in both groups were then measured, and 1 month after surgery, NF-200 staining was performed to observe and compare the growth condition of the axons.

Results: Early post-operative perfusion with gelatin/EB showed EB permeation around the acellular nerve. Perfusion with gelatin/lead oxide showed that the blood vessels had grown into the allograft from both ends 7 days after the operation. Fourteen days after the operation, the microvessel growth rate of the autologous group was faster than that of the rANA group (0.39 ± 0.17 mm/d vs. 0.26 ± 0.14 mm/d, p < 0.05), and the vascular VF was also higher than that of the rANA group (8.92% ± 1.54% vs. 6.31% ± 1.21%, p < 0.05). Twenty-one days after the operation, the blood vessels at both ends of the allograft had connected to form a microvessel network. The growth rate was not significantly different between the two groups; however, the vascular VF of the autologous group was higher than that of the rANA group (12.18% ± 2.27% vs. 9.92% ± 0.84%, p < 0.05). One month after the operation, the NF-200 fluorescence (IOD) in the autologous group significantly increased compared with that of the rANA group (540,278 ± 17,424 vs. 473,310 ± 14,636, respectively, p < 0.05), suggesting that the results of the repair after nerve injury were significantly better in the autologous group than in the rANA group.

Conclusion: Both the autologous nerve and ANAs rely on the permeation of tissue fluids to supply nutrients during the early stage, and microvessel growth mainly starts at both ends of the graft and enters the graft along the long axis. Compared to ANAs, the growth speed of revascularization in autologous nerve grafts was faster, leading to a better outcome in the autologous nerve group.     

Long term evaluation of experimental median nerve repair by frozen and fresh nerve autografts,allografts and allografts repopulated by autologous Schwann cells

The authors used different kinds of peripheral nerve grafts to reconstruct a terminal branch of the brachial plexus (the median nerve) gap of adult Sprague-Dawley rats, including fresh or frozen autografts and allografts from Norway rats. They also performed acellular allograft repopulation by autogenous Schwann cells, to improve the environment for nerve regeneration. Three, six, nine and twelve months after grafting, rats underwent histological assessment (muscle, nerve and spinal cord) and simple functional assessment by the grasping test. Initially, the functional recovery of frozen grafts was lower than fresh graft recovery, but twelve months after surgery it was similar for both types of graft.