Peripheral Nerve Grafts Research Articles

Post-Injury Delivery of rAAV2-CNTF Combined with Short-Term Pharmacotherapy Is Neuroprotective and Promotes Extensive Axonal Regeneration after Optic Nerve Trauma

Recombinant adeno-associated viral (rAAV) vectors expressing neurotrophic genes reduce neuronal death and promote axonal regeneration in central nervous system (CNS) injury models. Currently, however, use of rAAV to treat clinical neurotrauma is problematic because there is a delay in the onset of transgene expression. Using the adult rat retina and optic nerve (ON), we have tested whether rAAV gene therapy administered at the time of injury combined with short-term pharmacotherapy has synergistic effects that enhance neuronal survival and regeneration. The ON was transected and a 1.5 cm segment of autologous peripheral nerve (PN) was grafted onto the cut end. At this time, bicistronic rAAV2 encoding ciliary neurotrophic factor (CNTF) and green fluorescent protein (rAAV2-CNTF-GFP) was injected into the injured eye. To provide interim support for axotomized retinal ganglion cells (RGCs) during vector integration and therapeutic transgene expression, rCNTF protein and a cyclic adenosine monophosphate (cAMP) analogue (CPT-cAMP) were injected intravitreally 3 and 10 days postoperatively. For comparison, another rAAV2-CNTF-GFP group received two intravitreal saline injections 3 and 10 days after the PN-ON surgery. A further PN graft group received only postoperative intravitreal injections of rCNTF plus CPT-cAMP. After 4 weeks, regenerating RGCs were retrogradely labelled by applying fluorogold to the distal end of each PN graft. Compared to saline-injected animals, both RGC survival and axonal regrowth were significantly higher in the rCNTF and CPT-cAMP injected rAAV2-CNTF-GFP group; approximately one third of the RGC population survived axotomy, and 27% of these regrew an axon. These values were also higher than those obtained in rats that received only rCNTF plus CPT-cAMP injections. Therefore, we show for the first time that rAAV-mediated gene delivery at the time of, or just after, neurotrauma is most successful when combined with temporary post-injury trophic support, and is potentially a viable treatment strategy for patients after acute CNS injury.

Methadone keeps people alive

We have shown previously that intraocular elevation of cAMP using the cAMP analog 8-(4-chlorophenylthio)-cAMP (CPT-cAMP) failed to promote axonal regeneration of axotomized adult retinal ganglion cells (RGCs) into peripheral nerve (PN) grafts but significantly potentiated ciliary neurotrophic factor (CNTF)-induced axonal regeneration. Using the PN graft model, we now examine the mechanisms underlying spontaneous and CNTF/CPT-cAMP-induced neuronal survival and axonal regrowth. We found that blockade of the cAMP pathway executor protein kinase A (PKA) using the cell-permeable inhibitor KT5720 did not affect spontaneous survival and axonal regeneration but essentially abolished the CNTF/CPT-cAMP-induced RGC survival and axonal regeneration. Blockade of CNTF signaling pathways such as phosphotidylinositol 3-kinase (PI3K)/akt by 2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one (LY294002), mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK) by 2-(2-diamino-3-methoxyphenyl-4H-1-benzopyran-4-one (PD98059), or Janus kinase (JAK)/signal transducer and activators of transcription (STAT3) by tyrphostin AG490 also blocked the CNTF/CPT-cAMP-dependent survival and regeneration effects. PKA activity assay and Western blots showed that KT5720, LY294002, and PD98059 almost completely inhibited PKA, PI3K/akt, and MAPK/ERK signal transduction, respectively, whereas AG490 substantially decreased JAK/STAT3 signal transduction. Intraocular injection of CPT-cAMP resulted in a small PKA-dependent increase in CNTF receptor α mRNA expression in the retinas, an effect that may facilitate CNTF action on survival and axonal regeneration. Surprisingly, in the absence of CNTF/CPT-cAMP, LY294002, PD98059, and AG490, but not KT5720, significantly enhanced spontaneous RGC survival, suggesting differential roles of these pathways in RGC survival under different conditions. Our data suggest that CNTF/CPT-cAMP-induced RGC survival and axonal regeneration are a result of multiple pathway actions, with PKA as an essential component, but that these pathways can function in an antagonistic manner under different conditions.

Peripheral nerve graft with immunosuppression modifies gene expression in axotomized CNS neurons

Adult central nervous system (CNS) neurons do not regenerate severed axons unaided but may regenerate axons into apposed predegenerated peripheral nerve grafts (PNGs). We examined gene expression by using microarray technology in laser-dissected lateral vestibular (LV) neurons whose axons were severed by a lateral hemisection at C3 (HX) and in lateral vestibular nucleus (LVN) neurons that were hemisected at C3 and that received immunosuppression with cyclosporine A (CsA) and a predegenerated PNG (termed I-PNG) into the lesion site. The results provide an expression analysis of temporal changes that occur in LVN neurons in nonregenerative and potentially regenerative states and over a period of 42 days. Axotomy alone resulted in a prolonged change in regulation of probe sets, with more being upregulated than downregulated. Apposition of a PNG with immunosuppression muted gene expression overall. Axotomized neurons (HX) upregulated genes commonly associated with axonal growth, whereas axotomized neurons whose axons were apposed to the PNG showed diminished expression of many of these genes but greater expression of genes related to energy production. The results suggest that axotomized LVN neurons express many genes thought to be associated with regeneration to a greater extent than LVN neurons that are apposed to a PNG. Thus the LVN neurons remain in a regenerative state following axotomy but the conditions provided by the I-PNG appear to be neuroprotective, preserving or enhancing mitochondrial activity, which may provide required energy for regeneration. We speculate that the graft also enables sufficient axonal synthesis of cytoskeletal components to allow axonal growth without marked increase in expression of genes normally associated with regeneration.

Regulation of chondroitin sulphate proteoglycan and reactive gliosis after spinal cord transection: effects of peripheral nerve graft and fibroblast growth factor 1

The combined treatment of peripheral nerve (PN) graft and fibroblast growth factor (FGF)-1 for spinal cord injury produces functional recovery, but how it affects injury events is still unknown. This project studied the effect of PN graft and FGF-1 on white matter degeneration following spinal cord injury. Rats were divided into four groups: (i) complete spinal cord transection and T8 segment removed; the remaining three groups underwent transection followed by (ii) PN grafting; (iii) supply of exogenous FGF-1; and (iv) PN grafting plus FGF-1 treatment. Chondroitin sulphate proteoglycan (CSPG) deposition, astrocytes and macrophage activation, cavity size, and calcitonin gene-related peptide and synaptophysin immunoreactivity were compared. Peripheral nerve grafting increased CSPG levels compared to transection surgery alone. This CSPG was associated with the proximity to the PN graft. FGF-1 reduced CSPG deposition in grafted animals regardless of the proximity to the graft. The CSPG reduction was accompanied by reduced GFAP expression and macrophage activation. The amount of CSPG with dissociated glycosaminoglycan did not differ between groups. FGF-1 in Schwann cell-astrocyte coculture did not reduce CSPG deposition. Furthermore, the PN graft increased the calcitonin gene-related peptide immunoreactivity and altered the distribution of synaptophysin-positive axons. Peripheral nerve graft supported sensory re-innervation and partial protection of the grey matter, but up-regulated CSPG in the graft-stump junction compared to non-grafted rats. The reduction of CSPG was caused by FGF-1-PN synergy, and did not involve dissociation of CSPG or the suppression of a general immune response.

Axonal tracing of the normal and regenerating visual pathway of mouse, rat, frog, and fish using manganese-enhanced MRI (MEMRI)

To assess optic nerve (ON) regeneration after injury by applying manganese-enhanced MRI (MEMRI) in a study of comparative physiology between nonregenerating rat and mouse species and regenerating frog and fish species. The normal visual projections of rats, mice, frogs, and fish was visualized by intravitreal MnCl(2) injection followed by MRI. Rats and mice with ON crush (ONC) were divided into nonregenerating (ONC only), and regenerating animals with peripheral nerve graft (ONC+PNG; rats) or lens injury (ONC+LI; mice) and monitored by MEMRI at 1 and 20 days post-lesion (dpl). Frog and fish with ON transection (ONT) were monitored by MEMRI up to 6 months postlesion (mpl). Signal intensity profiles of the Mn(2+)-enhanced ON were consistent with ON regeneration in the ONC+PNG and ONC+LI rat and mice groups, respectively, compared with the nonregenerating ONC groups. Furthermore, signal intensity profiles of the Mn(2+)-enhanced ON obtained between 1 mpl and 6 mpl in the fish and frog groups, respectively, were consistent with spontaneous, complete ON regeneration. Taken together, these results demonstrate that MEMRI is a viable method for serial, in vivo monitoring of normal, induced, and spontaneously regenerating optic nerve axons in different species.

Functional characterization of optimized acellular peripheral nerve graft in a rat sciatic nerve injury model

Objectives: Acellular grafts are a viable option for use in nerve reconstruction surgeries. Recently, our lab created a novel optimized decellularization procedure that removes immunological material while leaving the majority of the extracellular matrix structure intact. The optimized acellular (OA) graft has been shown to elicit an immune response equal to or less than that elicited by the isograft, the analog of the autograft in the rat model. We investigated the performance of the OA graft to provide functional recovery in a long-term study.Methods: We performed a long-term functional regeneration evaluation study using the sciatic functional index to quantify recovery of Lewis rats at regular time intervals for up to 52 weeks after graft implantation following 1 cm sciatic nerve resection. OA grafts were compared against other decellularized methods (Sondell treatment and thermal decellularization), as well as the isograft and primary neurorrhaphy.Results: The OA graft supported comparable functional recovery to the isograft and superior regeneration to thermal and Sondell decellularization methods. Furthermore, the OA graft promoted early recovery to a greater degree compared to acellular grafts obtained using either the thermal or the Sondell methods.Discussion: Equivalent functional recovery to the isograft suggests that the OA nerve graft may be a future clinical alternative to the current autologous tissue graft.

Potential of an Electric Prosthesis for Dynamic Facial Reanimation

Chronic facial paralysis is a devastating condition with severe functional and emotional consequences. The current surgical armamentarium permits the predictable reestablishment of a protective blink as well as good resting symmetry. Yet the ultimate goal of symmetric, spontaneous emotional expression remains elusive despite significant progress in the areas of peripheral nerve grafting and free tissue transfer. This commentary explores the possibility of an implantable electrical prosthesis for facial reanimation. It reviews animal studies supporting this concept as well as recent human data suggesting that such an implant could rescue denervated facial musculature, thus overcoming a major hurdle for existing reanimation techniques.

A Long Peripheral Nerve Autograft Model in the Sheep Forelimb

Autologous nerve grafts remain the only proven means of bridging lengthy gaps in peripheral nerve. However, there is very little literature on a reliable long (> 5 cm) nerve autograft animal model. To establish a reproducible long nerve gap and autograft animal model that is clinically relevant but not cost prohibitive. The extent of nerve regeneration and electrophysiological recovery after segmental repair of a long nerve defect was evaluated with a sheep model. Thirteen Suffolk sheep were used. An 18-cm segment of radial sensory nerve was harvested from the forelimb, trimmed, divided into 2 equal segments of 7 cm each, and microsurgically repaired to a surgically created defect of 5 cm in the median nerve within the same forelimb. Electrophysiological studies were performed on 6 sheep at 6 months and 6 sheep at 9 months. Samples of the grafted segments were obtained for histology, immunohistochemistry, and morphometric analyses. Electric studies were also performed on an uninjured median nerve of a control animal in tissue that was similarly harvested and processed. At 6 and 9 months, all sheep had recordable robust nerve action potentials. Nerve conduction velocity and amplitude were slightly decreased compared with control, but the difference was statistically insignificant. Histomorphometric assessment demonstrated that the autografts contained a large number of regenerating axons through graft fascicles in all animals. The median nerve in the sheep forelimb is a reproducible and reliable model for assessing regeneration through long peripheral nerve grafts.

Nerve Grafting for Spinal Cord Injury in Cats: Are We Close to Translational Research?

Nerve Grafting for Spinal Cord Injury in Cats: Are We Close to Translational Research?

Acid Fibroblast Growth Factor and Peripheral Nerve Grafts Regulate Th2 Cytokine Expression, Macrophage Activation, Polyamine Synthesis, and Neurotrophin Expression in Transected Rat Spinal Cords

Spinal cord injury elicits an inflammatory response that recruits macrophages to the injured spinal cord. Quantitative real-time PCR results have shown that a repair strategy combining peripheral nerve grafts with acidic fibroblast growth factor (aFGF) induced higher interleukin-4 (IL-4), IL-10, and IL-13 levels in the graft areas of rat spinal cords compared with transected spinal cords at 10 and 14 d. This led to higher arginase I-positive alternatively activated macrophage (M2 macrophage) responses. The gene expression of several enzymes involved in polyamine biosynthesis pathways was also upregulated in the graft areas of repaired spinal cords. The treatment induced a twofold upregulation of polyamine levels at 14 d, as confirmed by HPLC. Polyamines are important for the repair process, as demonstrated by the observation that treatment with inhibitors of arginase I and ornithine decarboxylase attenuates the functional recoveries of repaired rats. After 14 d, the treatment also induced the expression of neurotrophin nerve growth factor (NGF) and brain-derived neurotrophic factor (BDNF), as well as M2 macrophages within grafted nerves expressing BDNF. IL-4 was upregulated in the injury sites of transected rats that received aFGF alone compared with those that received nerve grafts alone at 10 d. Conversely, nerve graft treatment induced NGF and BDNF expression at 14 d. Macrophages expressing polyamines and BDNF may benefit axonal regeneration at 14 d. These results indicate that aFGF and nerve grafts regulate different macrophage responses, and M2 macrophages may play an important role in axonal regeneration after spinal cord injury in rats.

Olfactory enseathing glia enhances reentry of axons into the brain from peripheral nerve grafts bridging the substantia nigra with the striatum

Reconstruction of lost axonal pathways in the central nervous system (CNS) is possible with the use of peripheral nerve grafts (PNG). However, these permit the entry of axons, while their reentry back into the CNS is compromised. Olfactory enseathing glia (OEG) may permit this reentry of axons if cografted with PNG. We compared the number of tyrosine-hydroxylase positive (TH+) fibers reinnervating PNGs and crossing the graft–striatum interface in PNG placed between the substantia nigra and the striatum in rats receiving both PNG and OEGs and animals receiving PNG only. More TH fibers were seen inside the grafts when OEG was cografted. Although the number of fibers decreases along the graft's length, this effect is less severe when OEG is present. TH+ fibers are seen crossing the PNG–striatum interface in the OEG group. This is correlated with a higher synaptic density at the striatum near the graft when OEG is co-grafted. While these results must be replicated in animal models of Parkinsonism, their implications may apply both to the treatment of Parkinson's disease and to other pathologies, such as spinal cord lesions, where regeneration of long axonal pathways is necessary.

Peripheral nerve grafts support regeneration after spinal cord injury.

Traumatic insults to the spinal cord induce both immediate mechanical damage and subsequent tissue degeneration leading to a substantial physiological, biochemical, and functional reorganization of the spinal cord. Various spinal cord injury (SCI) models have shown the adaptive potential of the spinal cord and its limitations in the case of total or partial absence of supraspinal influence. Meaningful recovery of function after SCI will most likely result from a combination of therapeutic strategies, including neural tissue transplants, exogenous neurotrophic factors, elimination of inhibitory molecules, functional sensorimotor training, and/or electrical stimulation of paralyzed muscles or spinal circuits. Peripheral nerve grafts provide a growth-permissive substratum and local neurotrophic factors to enhance the regenerative effort of axotomized neurons when grafted into the site of injury. Regenerating axons can be directed via the peripheral nerve graft toward an appropriate target, but they fail to extend beyond the distal graft-host interface because of the deposition of growth inhibitors at the site of SCI. One method to facilitate the emergence of axons from a graft into the spinal cord is to digest the chondroitin sulfate proteoglycans that are associated with a glial scar. Importantly, regenerating axons that do exit the graft are capable of forming functional synaptic contacts. These results have been demonstrated in acute injury models in rats and cats and after a chronic injury in rats and have important implications for our continuing efforts to promote structural and functional repair after SCI.

Scientific assessment on the outcome of clinical application with peripheral nerve graft materials

Objective To discuss how to assess scientifically on the outcome of clinical application with peripheral nerve graft materials. Methods All Pubmed database from 1990 to 2010 were retrieved,and searched the English literatures about the application with peripheral nerve graft materials. The literatures consisted of original clinical research and review excluding animal experiments, repetitive research and irrelevant literatures. The clinical trials data of U.S. was also our target. The information about the safety and effectiveness of peripheral nerve graft materials and related statistical problems were discussed. Results Totally 1578 literatures were identified. Following reading titles and abstracts, we excluded some irrelevant articles. Finally 31 literatures and 2 issue of clinical research from clinical trial data of U.S. were included. After analysis on the literatures, we gained the following results: a remarkable degree of homogeneity among patients can be formed by setting the inclusion and exclusion criteria. For the assessment of proper digital nerve repair, Macknnon- Dellon evaluation is commonly applied, but for the composite nerve, BMRC evaluation is the main method and electromyography can be used as a secondary choice. The safety of peripheral nerve graft materials cannot be evaluated throughout one's life according to the current level of science and technology. It should be evaluated by long-term clinical observation. Randomized clinical trials with random grouping was a gold standard for clinical trials with a good balance and strong comparability. However, non-randomized controlled trials also have an important value. Conclusion It is impossible to make all affected factors homogeneity in a limited timespace conditions of clinical trial. However, we can try our best to keep factors homogeneity to maximum degree by setting the inclusion and exclusion criteria. The scientific assessment of outcome of peripheral nerve repair can be carried out with reasonable and internationally recognized nerve function evaluation methods, strict follow-up time and statistics programme meeting the clinical requirement. Key words: Peripheral nerve; Graft materials; Nerve function evaluation; Literatures assessment

Preserving Peripheral Nerves in Desiccating Conditions With a Mixture of Carbon Dioxide and Oxygen Gases

Preserving Peripheral Nerves in Desiccating Conditions With a Mixture of Carbon Dioxide and Oxygen Gases

Long-term morphometric stabilization of regenerating retinal ganglion cells in the adult rat

Adult retinal ganglion cells (RGCs) can regenerate their cut axons within peripheral nerve grafts used to replace the distal optic nerve stump. We examined the long-term stabilization of RGCs by guiding their regenerating axons into different termination areas. The optic nerve (ON) of adult rats was completely cut intraorbitally and its ocular stump was connected with different visual target areas (cortex, midbrain) or with non-visual areas (e.g. muscle). Control groups consisted of blind ending graft and ON cut without graft. The function of the retina was regularly examined by electroretinography. At one, six and nine months postsurgery RGCs were retrogradely labelled with 4-(4-(didecylamino)styryl)-N-methylpyridinium iodide and examined morphometrically. Regenerating RGCs were categorized into three major classes representing the morphological types I, II and III. Our data show that regenerating RGCs remain stable up to nine months after grafting at the ON, although the numbers of axons are low, that is less than 1%, and this number is not significantly effected by reconnection with targets. However, there are significant quantitatively and morphometrically assessable differences between the experimental groups depending on the tissue the RGCs are connected with visual targets. Regenerating RGCs show the highest stability in morphology if reconnected with visual target tissue. Adult RGCs of the rat can be reconnected with visual centers using a peripheral nerve graft. This reconnection stabilizes the cells at morphological and the retina at functional levels for a long period of time, although it does not significantly increase cell survival.

Review of "Scar-Busting Chondroitinase With Peripheral Nerve Grafting Promotes Axonal Regeneration in Chronic Spinal Cord Injury"

Review of "Scar-Busting Chondroitinase With Peripheral Nerve Grafting Promotes Axonal Regeneration in Chronic Spinal Cord Injury"

Negative impact of rAAV2 mediated expression of SOCS3 on the regeneration of adult retinal ganglion cell axons

Intravitreal injections of recombinant ciliary neurotrophic factor (rCNTF) protect adult rat retinal ganglion cells (RGCs) after injury and stimulate regeneration, an effect enhanced by co-injection with a cAMP analogue (CPT-cAMP). This effect is partly mediated by PKA and associated signaling pathways, but CPT-cAMP also moderates upregulation of suppressor of cytokine signaling (SOCS) pathways after rCNTF injection, which will also enhance the responsiveness of RGCs to this and perhaps other cytokines. We now report that intravitreal injections of CPT-cAMP do not potentiate RGC axonal regeneration when CNTF is expressed via an adeno-associated viral vector (rAAV2), and concomitantly we show that increases in retinal SOCS mRNA expression are less when CNTF is delivered using the vector. We also directly tested the impact of elevated SOCS3 expression on the survival and regeneration of injured adult RGCs by injecting a bicistronic rAAV2-SOCS3-GFP vector into the vitreous of eyes in rats with a peripheral nerve graft sutured onto the cut optic nerve. Overexpression of SOCS3 resulted in an overall reduction in axonal regrowth and almost complete regeneration failure of RGCs transduced with the rAAV2-SOCS3-GFP vector. Furthermore, rAAV2-mediated expression of SOCS3 abolished the normally neurotrophic effects elicited by intravitreal rCNTF injections. In summary, CNTF delivery to the retina using viral vectors may be more effective than bolus rCNTF injections because the gene therapy approach has a less pronounced effect on neuron-intrinsic SOCS repressor pathways. Our new gain of function data using rAAV2-SOCS3-GFP demonstrate the negative impact of enhanced SOCS3 expression on the regenerative potential of mature CNS neurons.

In Vivo Evaluation of Polysialic Acid as Part of Tissue-Engineered Nerve Transplants

With the aim to develop new biomaterials for peripheral nerve grafts, the current study used bioidentical polysialic acid (polySia) as complement in synthetic conduits. polySia provides an important guidance cue during nervous system development and regeneration. First in vivo results on the use of cell-free and Schwann cell-containing synthetic peripheral nerve grafts complemented with soluble exogenous K1-polySia are presented. Reconstructing 10 mm rat sciatic nerve gaps, K1-polySia complementation significantly improved structural nerve regeneration in comparison to cell-free and K1-polySia-free grafts. Subsequently, long nerve gaps (13 mm) were reconstructed by Schwann cell transplants plus K1-polySia and compared to nerve autotransplantation. Structural but also functional regeneration could be observed using K1-polySia transplants; however, autotransplantation was still significantly more successful. Overall, the current study demonstrates that exogenous K1-polySia has no negative but rather regeneration promoting effects. This is important novel evidence on the applicability of exogenous polySia in vivo. Further studies are required to develop solid three-dimensional polySia-based scaffolds for nerve tissue engineering. Biocompatible and assessable biodegrading materials will ensure long-lasting presence of polySia to allow its applicability and prolonged efficacy in the slow regenerating scenario of human peripheral nerve reconstruction.

Poster 378: Nerve Regeneration and Improvement of Bladder Function in Complete Spinal Cord Transected Rats Treated With a Peripheral Nerve Graft, Acidic Fibroblast Growth Factor, and Chondroitinase ABC

Poster 378: Nerve Regeneration and Improvement of Bladder Function in Complete Spinal Cord Transected Rats Treated With a Peripheral Nerve Graft, Acidic Fibroblast Growth Factor, and Chondroitinase ABC

A model for ex vivo spinal cord segment culture—A tool for analysis of injury repair strategies

Most spinal cord injury research is undertaken using in vivo animal models but the extensive care associated with spinalised animals, inherent variability between animals, and complex surgeries makes alternative models especially valuable. Here we present a novel ex vivo model that enables culture of intact post-natal spinal cord segments for up to five days and the assessment of peripheral nerve grafting repair, enhanced with connexin43 antisense oligodeoxynucleotides (Cx43 AsODN), in this model. Down-regulating Cx43 expression with Cx43 AsODN in cultured spinal cord segments prevents cell death and inhibits inflammation spreading from the site of injury to neighbouring tissue, hence maintaining culture viability. Reduction in segment swelling and improvement in neuron survival were evident after Cx43 AsODN treatment. Furthermore, the combination of Cx43 AsODN with peripheral nerve graft implants into cultured spinal cords promoted axon sprouting from the spinal cord into the peripheral nerve graft. This ex vivo spinal cord segment culture model provides a valuable addition to tools currently available for spinal cord injury research.