Piyaraj Newton

Limited regeneration in long acellular nerve allografts is associated with increased Schwann cell senescence

Repair of large nerve defects with acellular nerve allografts (ANAs) is an appealing alternative to autografting and allotransplantation. ANAs have been shown to be similar to autografts in supporting axonal regeneration across short gaps, but fail in larger defects due to a poorly-understood mechanism. ANAs depend on proliferating Schwann cells (SCs) from host tissue to support axonal regeneration. Populating longer ANAs places a greater proliferative demand on host SCs that may stress host SCs, resulting in senescence. In this study, we investigated axonal regeneration across increasing isograft and ANA lengths. We also evaluated the presence of senescent SCs within both graft types. A sciatic nerve graft model in rats was used to evaluate regeneration across increasing isograft (~autograft) and ANA lengths (20, 40, and 60 mm). Axonal regeneration and functional recovery decreased with increased graft length and the performance of the isograft was superior to ANAs at all lengths. Transgenic Thy1-GFP rats and qRT-PCR demonstrated that failure of the regenerating axonal front in ANAs was associated with increased levels of senescence related markers in the graft (senescence associated β-galactosidase, p16(INK4A), and IL6). Lastly, electron microscopy (EM) was used to qualitatively assess senescence-associated changes in chromatin of SCs in each graft type. EM demonstrated an increase in the presence of SCs with abnormal chromatin in isografts and ANAs of increasing graft length. These results are the first to suggest that SC senescence plays a role in limited axonal regeneration across nerve grafts of increasing gap lengths.

Nerve endoneurial microstructure facilitates uniform distribution of regenerative fibers: a post hoc comparison of midgraft nerve fiber densities.

Despite their inferiority to nerve autograft, clinical alternatives are commonly used for reconstruction of peripheral nerve injuries because of their convenient off-the-shelf availability. Previously, our group compared isografts with NeuraGen(®) (Integra, Plainsboro, NJ) nerve guides, which are a commercially available type I collagen conduit and processed rat allografts comparable to Avance(®) (AxoGen, Alachua, FL) human decellularized allograft product. From this study, qualitative observations were made of distinct differences in the pattern of regenerating fibers within conduits, acellular allografts, and isografts. In the current post hoc analysis, these observations were quantified. Using nerve density, we statistically compared the differential pattern of regenerating axon fibers within grafts and conduit. The conduits exhibited a consistent decrease in midgraft density when compared with the isograft and acellularized allografts at two gap lengths (14 mm and 28 mm) and time points (12 and 22 weeks). The decrease in density was accompanied by clustered distribution of nerve fibers in conduits, which contrasted the evenly distributed regeneration seen in processed allografts and isografts. We hypothesize that the lack of endoneurial microstructure of conduits results in the clustering regenerating fibers, and that the presence of microstructure in the acellularized allograft and isografts facilitates even distribution of regenerating fibers.

NERVE ALLOGRAFTS SUPPLEMENTED WITH SCHWANN CELLS OVEREXPRESSING GLIAL-CELL-LINE-DERIVED NEUROTROPHIC FACTOR

We sought to determine whether supplementation of acellular nerve allografts (ANAs) with Schwann cells overexpressing GDNF (G‐SCs) would enhance functional recovery after peripheral nerve injury.

A transgenic rat expressing green fluorescent protein (GFP) in peripheral nerves provides a new hindlimb model for the study of nerve injury and regeneration

BACKGROUND In order to evaluate nerve regeneration in clinically relevant hindlimb surgical paradigms not feasible in fluorescent mice models, we developed a rat that expresses green fluorescent protein (GFP) in neural tissue. METHODS Transgenic Sprague-Dawley rat lines were created using pronuclear injection of a transgene expressing GFP under the control of the thy1 gene. Nerves were imaged under fluorescence microscopy and muscles were imaged with confocal microscopy to determine GFP expression following sciatic nerve crush, transection and direct suturing, and transection followed by repair with a nerve isograft from nonexpressing littermates. RESULTS In each surgical paradigm, fluorescence microscopy demonstrated the loss and reappearance of fluorescence with regeneration of axons following injury. Nerve regeneration was confirmed with imaging of Wallerian degeneration followed by reinnervation of extensor digitorum longus (EDL) muscle motor endplates using confocal microscopy. CONCLUSION The generation of a novel transgenic rat model expressing GFP in neural tissue allows in vivo imaging of nerve regeneration and visualization of motor endplate reinnervation. This rat provides a new model for studying peripheral nerve injury and regeneration over surgically relevant distances.

A systematic evaluation of Schwann cell injection into acellular cold-preserved nerve grafts.

Peripheral nerve regeneration after injury depends on environmental cues and trophic support. Schwann cells (SCs) secrete trophic factors that promote neuronal survival and help guide axons during regeneration. The addition of SCs to acellular nerve grafts is a promising strategy for enhancing peripheral nerve regeneration; however, inconsistencies in seeding parameters have led to varying results. The current work sought to establish a systematic approach to seeding SCs in cold-preserved acellular nerve grafts. Studies were undertaken to (1) determine the needle gauge for optimal cell survival and minimal epineurial disruption during injection, (2) track the seeded SCs using a commercially available dye, and (3) evaluate the seeding efficiency of SCs in nerve grafts. It was determined that seeding with a 27-gauge needle resulted in the highest viability of SCs with the least damage to the epineurium. In addition, Qtracker(®) dye, a commercially available quantum dot nanocrystal, was used to label SCs prior to transplantation, which allowed visualization of the seeded SCs in nerve grafts. Finally, stereological methods were used to evaluate the seeding efficiency of SCs in nerve grafts immediately after injection and following a 1- or 3-day in vitro incubation in SC growth media. Using a systematic approach, the best needle gauge and a suitable dye for SC visualization in acellular nerve grafts were identified. Seeding efficiency in these grafts was also determined. The findings will lead to improvements ability to assess injection of cells (including SCs) for use with acellular nerve grafts to promote nerve regeneration.

A NIR dye for development of peripheral nerve targeted probes

Current imaging modalities lack the ability to quickly assess and classify nerve injury for predicting favourable versus unfavourable healing outcomes, which could minimize episodes of chronic pain and loss of function by allowing for early intervention. Thus, the development of a technique to noninvasively assess peripheral nerve damage is of critical importance. While the development of nerve specific near infrared (NIR) molecular probes capable of such diagnostics constitutes our long term goal, initial studies to identify a NIR dye for constructing such a probe are required. We have evaluated the properties of a novel highly hydrophilic and functionalizable polymethine dye, and its more hydrophobic analogue indocyanine green, within the sciatic nerve of rats following intra-nerve injection. The reporting ability of both dyes at critical depths for nerve imaging, the importance of hydrophilicity on dye transport through nervous tissue, and their toxicity - or lack thereof - to the neural environment have been evaluated. The results suggest that the novel NIR dye is an appropriate fluorescent reporter for use in designing nerve-specific optical molecular probes for non-invasive diagnosis and classification of nerve injury.

Nerve regeneration in rat limb allografts: evaluation of acute rejection rescue.

Background: Successful nerve regeneration is critical to the functional success of composite tissue allografts. 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 by means of histomorphometric analysis of explanted sciatic nerve, and motor recovery was assessed by means of evoked muscle force measurement in extensor digitorum longus 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 extensor digitorum longus muscle function in composite tissue allografts. Conclusions: Collected data reinforce 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 postoperatively. 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 composite tissue allografts.

Viral transduction of primary Schwann cells using a Cre‐lox system to regulate GDNF expression

Glial cell‐line‐derived neurotrophic factor (GDNF) is a potent neurotrophic factor known to enhance motor nerve regeneration following its delivery. However, recent studies have determined that extended GDNF delivery to regenerating axons can entrap motor axons at the site of GDNF delivery. This entrapment leads to reduced motor axons available to reinnervate muscle. To address this issue, we designed a cell‐based GDNF expression system that can temporally regulate protein expression using an inducible gene excision mechanism to prevent entrapment at the site of expression. To design this system for regulation of GDNF expression, we transduced two lentiviral vectors, one containing a constitutively active GDNF transgene flanked by two loxP sites, and the other containing a tetracycline‐inducible cre transgene along with its constitutively active transactivator, into Schwann cells (SCs). These SCs over‐express GDNF, but expression can be suppressed through the administration of tetracycline family antibiotics, such as doxycycline. The engineered SCs produced significantly more GDNF as compared to untransduced controls, as measured by enzyme‐linked immunosorbent assay (ELISA). Following doxycycline treatment, these SCs produced significantly lower levels of GDNF and induced less neurite extension as compared to untreated SCs. Engineered SCs treated with doxycycline showed a marked increase in Cre recombinase expression, as visualized by immunohistochemistry (IHC), providing evidence of a mechanism for the observed changes in GDNF expression levels and biological activity. This cell‐based GDNF expression system could have potential for future in vivo studies to provide a temporally controlled GDNF source to promote axon growth. Biotechnol. Bioeng. 2014;111: 1886–1894.

Robust Axonal Regeneration in a Mouse Vascularized Composite Allotransplant Model Undergoing Delayed Tissue Rejection.

Background: Nerve regeneration in vascularized composite allotransplantation (VCA) is not well understood. Allogeneic transplant models experience complete loss of nerve tissue and axonal regeneration without immunosuppressive therapy. The purpose of this study was to determine the impact of incomplete immunosuppression on nerve regeneration. Methods: In this study, transgenic mice (4 groups in total) with endogenous fluorescent protein expression in axons (Thy1-YFP) and Schwann cells (S100-GFP) were used to evaluate axonal regeneration and Schwann cell (SC) migration in orthotopic-limb VCA models with incomplete immunosuppression using Tacrolimus (FK506). Survival and complication rates were assessed to determine the extent of tissue rejection. Nerve regeneration was assessed using serial imaging of axonal progression and SC migration and viability. Histomorphometry quantified the extent of axonal regeneration. Results: Incomplete immunosuppression with FK506 resulted in delayed rejection of skin, muscle, tendon, and bone in the transplanted limb. In contrast, the nerve demonstrated robust axonal regeneration and SC viability based on strong fluorescent protein expression by SCs and axons in transgenic donors and recipients. Total myelinated axon numbers measured at 8 weeks were comparable in all VCA groups and not statistically different from the syngeneic donor control group. Conclusions: Our data suggest that nerve and SCs are much weaker antigens compared with skin, muscle, tendon, and bone in VCA. To our knowledge, this study is the first to prove the weak antigenicity of nerve tissue in the orthotopic VCA mouse model.

Hyaluronic acid/carboxymethyl cellulose directly applied to transected nerve decreases axonal outgrowth

Neuroma management is an unresolved problem. Biomaterials to limit unwanted axonal growth could be a tool to manage neuroma. Hyaluronic acid/carboxymethyl cellulose (HA/CMC) is an antiadhesive, biodegradable material that is nontoxic to nerve. The purpose of this study was to evaluate the efficacy of this biomaterial to limit axonal growth. Rats received a sciatic nerve transection and repair with a short conduit (5 mm) containing HA/CMC, fibrin, or nothing (empty conduit). In another study, nerve was transected and either left undisturbed or wrapped with HA/CMC around the proximal and distal ends. In a final study, nerve was transected and repaired with an HA/CMC wrap. Four weeks following the procedures, nerves were harvested and assessed using histomorphometry to measure axonal regeneration. Axonal regeneration following transection was significantly inhibited by direct axonal contact with HA/CMC, whether within a conduit or wrapped around the transected proximal nerve end. Axonal regeneration following epineurial repair was not affected by HA/CMC wrapped around nerve, demonstrating axonal growth inhibition due to direct contact of regenerating axons with HA/CMC. These studies demonstrate the efficacy of HA/CMC to limit axonal outgrowth by contact with regenerating axons. HA/CMC barriers may prove to be a tool to prevent neuroma formation by inhibiting axonal growth.