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The use of acellular nerve allografts (ANAs) to reconstruct long nerve gaps (>3 cm) is associated with limited axon regeneration. To understand why ANA length might limit regeneration, we focused on identifying differences in the regenerative and vascular microenvironment that develop within ANAs based on their length. A rat sciatic nerve gap model was repaired with either short (2 cm) or long (4 cm) ANAs, and histomorphometry was used to measure myelinated axon regeneration and blood vessel morphology at various timepoints (2-, 4- and 8-weeks). Both groups demonstrated robust axonal regeneration within the proximal graft region, which continued across the mid-distal graft of short ANAs as time progressed. By 8 weeks, long ANAs had limited regeneration across the ANA and into the distal nerve (98 vs. 7583 axons in short ANAs). Interestingly, blood vessels within the mid-distal graft of long ANAs underwent morphological changes characteristic of an inflammatory pathology by 8 weeks post surgery. Gene expression analysis revealed an increased expression of pro-inflammatory cytokines within the mid-distal graft region of long vs. short ANAs, which coincided with pathological changes in blood vessels. Our data show evidence of limited axonal regeneration and the development of a pro-inflammatory environment within long ANAs.

Background: Clinical outcomes following nerve injury repair can be inadequate. Pulsed-current electrical stimulation (ES) is a therapeutic method that facilitates functional recovery by accelerating axon regeneration. However, current clinical ES protocols involve the application of ES for 60 minutes during surgery, which can increase operative complexity and time. Shorter ES protocols could be a strategy to facilitate broader clinical adoption. The purpose of the present study was to determine if a 10-minute ES protocol could improve outcomes., Methods: C57BL/6J mice were randomized to 3 groups: no ES, 10 minutes of ES, and 60 minutes of ES. In all groups, the sciatic nerve was transected and repaired, and, in the latter 2 groups, ES was applied after repair. Postoperatively, changes to gene expression from dorsal root ganglia were measured after 24 hours. The number of motoneurons regenerating axons was determined by retrograde labeling at 7 days. Histomorphological analyses of the nerve were performed at 14 days. Function was evaluated serially with use of behavioral tests up to 56 days postoperatively, and relative muscle weight was evaluated., Results: Compared with the no-ES group, both ES groups demonstrated increased regeneration-associated gene expression within dorsal root ganglia. The 10-minute and 60-minute ES groups demonstrated accelerated axon regeneration compared with the no-ES group based on increased numbers of labeled motoneurons regenerating axons (mean difference, 202.0 [95% confidence interval (CI), 17.5 to 386.5] and 219.4 [95% CI, 34.9 to 403.9], respectively) and myelinated axon counts (mean difference, 559.3 [95% CI, 241.1 to 877.5] and 339.4 [95% CI, 21.2 to 657.6], respectively). The 10-minute and 60-minute ES groups had improved behavioral recovery, including on grid-walking analysis, compared with the no-ES group (mean difference, 11.9% [95% CI, 3.8% to 20.0%] and 10.9% [95% CI, 2.9% to 19.0%], respectively). There was no difference between the ES groups in measured outcomes., Conclusions: A 10-minute ES protocol accelerated axon regeneration and facilitated functional recovery., Clinical Relevance: The brief (10-minute) ES protocol provided similar benefits to the 60-minute protocol in an acute sciatic nerve transection/repair mice model and merits further studies., Competing Interests: Disclosure: The Disclosure of Potential Conflicts of Interest forms are provided with the online version of the article (http://links.lww.com/JBJS/G615)., (Copyright © 2021 by The Journal of Bone and Joint Surgery, Incorporated.)

Background: Treatments to manage painful neuroma are needed. An operative strategy that isolates and controls chaotic axonal growth could prevent neuroma. Using long acellular nerve allograft to "cap" damaged nerve could control axonal regeneration and, in turn, regulate upstream gene expression patterns., Methods: Rat sciatic nerve was transected, and the distal nerve end was reversed and ligated to generate a model end-neuroma. Three groups were used to assess their effects immediately following this nerve injury: no treatment (control), traction neurectomy, or 5-cm acellular nerve allograft cap attached to the proximal nerve. Regeneration of axons from the injured nerve was assessed over 5 months and paired with concurrent measurements of gene expression from upstream affected dorsal root ganglia., Results: Both control and traction neurectomy groups demonstrated uncontrolled axon regeneration revealed using Thy1-GFP rat axon imaging and histomorphometric measures of regenerated axons within the most terminal region of regenerated tissue. The acellular nerve allograft group arrested axons within the acellular nerve allograft, where no axons reached the most terminal region even after 5 months. At 5 months, gene expression associated with regeneration and pain sensitization, including Bdnf, cfos, and Gal, was decreased within dorsal root ganglia obtained from the acellular nerve allograft group compared to control or traction neurectomy group dorsal root ganglia., Conclusions: Long acellular nerve allografts to cap a severed nerve arrested axon regeneration within the acellular nerve allograft. This growth arrest corresponded with changes in regenerative and pain-related genes upstream. Acellular nerve allografts may be useful for surgical intervention of neuroma., Competing Interests: Disclosure:Dr. Matthew D. Wood has been the recipient of sponsored research agreements from Checkpoint Surgical, Inc., and has consulted for Foundry Therapeutics, LLC, and The Foundry, LLC. No personal compensation was provided. None of the other authors has any conflicts of interest to disclose., (Copyright © 2021 by the American Society of Plastic Surgeons.)

Acellular nerve allografts (ANAs) are increasingly used to repair nerve gaps following injuries. However, these nerve scaffolds have yet to surpass the regenerative capabilities of cellular nerve autografts; improved understanding of their regenerative mechanisms could improve design. Due to their acellular nature, both angiogenesis and diverse cell recruitment is necessary to repopulate these scaffolds to promote functional regeneration. We determined the contribution of angiogenesis to initial cellular repopulation of ANAs used to repair nerve gaps, as well as the signaling that drives a significant portion of this angiogenesis. Wild-type (WT) mice with nerve gaps repaired using ANAs that were treated with an inhibitor of VEGF receptor signaling severely impaired angiogenesis within ANAs, as well as hampered cell repopulation and axon extension into ANAs. Similarly, systemic depletion of hematogenous-derived macrophages, but not neutrophils, in these mice models severely impeded angiogenesis and subsequent nerve regeneration across ANAs suggesting hematogenous-derived macrophages were major contributors to angiogenesis within ANAs. This finding was reinforced using CCR2 knockout (KO) models. As macrophages represented the majority of CCR2 expressing cells, a CCR2 deficiency impaired angiogenesis and subsequent nerve regeneration across ANAs. Furthermore, an essential role for CCL2 during nerve regeneration across ANAs was identified, as nerves repaired using ANAs had reduced angiogenesis and subsequent nerve regeneration in CCL2 KO vs WT mice. Our data demonstrate the CCL2/CCR2 axis is important for macrophage recruitment, which promotes angiogenesis, cell repopulation, and subsequent nerve regeneration and recovery across ANAs used to repair nerve gaps., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Background: Stereology and histomorphometry are widely used by investigators to quantify nerve characteristics in normal and pathological states, including nerve injury and regeneration. While these methods of analysis are complementary, no study to date has systematically compared both approaches in peripheral nerve. This study investigated the reliability of design-based stereology versus semi-automated binary imaging histomorphometry for assessing healthy peripheral nerve characteristics., New Method: Stereological analysis was compared to histomorphometry with binary image analysis on uninjured sciatic nerves to determine nerve fiber number, nerve area, neural density, and fiber distribution., Results: Sciatic nerves were harvested from 6 male Lewis rats, aged 8-12 weeks for comprehensive analysis of 6 nerve specimens. From each animal, sciatic nerve specimens were fixed, stained, and sectioned for analysis by light and electron microscopy. Both histomorphometry and stereological peripheral nerve analyses were performed on all specimens by two blinded and independent investigators who quantified nerve fiber count, fiber width, density, and related distribution parameters., Comparison With Existing Methods: Histomorphometry and stereological analysis provided similar outcomes in nerve fiber number and total nerve area. However, the light microscopy, but not electron microscopy, stereological analysis yielded higher nerve fiber area compared to histomorphometry or manual measurement., Conclusion: Both methods measure similar fiber number and overall nerve fiber area; however, stereology with light microscopy quantified higher fiber area. Histomorphometry optimizes throughput and comprehensive analysis but requires user thresholding., Competing Interests: Declaration of Competing Interest None (no disclosures), (Copyright © 2020 Elsevier B.V. All rights reserved.)

Repair of traumatic nerve injuries can require graft material to bridge the defect. The use of alternatives to bridge the defect, such as acellular nerve allografts (ANAs), is becoming more common and desired. Although ANAs support axon regeneration across short defects (<3 cm), axon regeneration across longer defects (>3 cm) is limited. It is unclear why alternatives, including ANAs, are functionally limited by length. After repairing Lewis rat nerve defects using short (2 cm) or long (4 cm) ANAs, we showed that long ANAs have severely reduced axon regeneration across the grafts and contain Schwann cells with a unique phenotype. But additionally, we found that long ANAs have disrupted angiogenesis and altered leukocyte infiltration compared to short ANAs as early as 2 weeks after repair. In particular, long ANAs contained fewer T cells compared to short ANAs. These outcomes were accompanied with reduced expression of select cytokines, including IFN-γ and IL-4, within long versus short ANAs. T cells within ANAs did not express elevated levels of IL-4, but expressed elevated levels of IFN-γ. We also directly assessed the contribution of T cells to regeneration across nerve grafts using athymic rats. Interestingly, T cell deficiency had minimal impact on axon regeneration across nerve defects repaired using isografts. Conversely, T cell deficiency reduced axon regeneration across nerve defects repaired using ANAs. Our data demonstrate that T cells contribute to nerve regeneration across ANAs and suggest that reduced T cells accumulation within long ANAs could contribute to limiting axon regeneration across these long ANAs., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Background: Nerve grafting with an autograft is considered the gold standard. However, the functional outcomes of long (>3 cm) nerve autografting are often poor. The authors hypothesized that a factor contributing to these outcomes is the graft microenvironment, where long compared to short autografts support axon regeneration to different extents., Methods: A rat sciatic nerve defect model was used to compare regeneration in short (2 cm) and long (6 cm) isografts. Axon regeneration and cell populations within grafts were assessed using histology, retrograde labeling of neurons regenerating axons, immunohistochemistry, quantitative reverse transcriptase polymerase chain reaction, and electron microscopy at 4 and/or 8 weeks., Results: At 8 weeks, for distances of both 1 and 2 cm from the proximal coaptation (equivalent regenerative distance), long isografts had reduced numbers of regenerated fibers compared with short isografts. Similarly, the number of motoneurons regenerating axons was reduced in the presence of long isografts compared with short isografts. Considering the regenerative microenvironments between short and long isografts, cell densities and general populations within both short and long isografts were similar. However, long isografts had significantly greater expression of senescence markers, which included senescence-associated β-galactosidase, p21, and p16, and distinct chromatin changes within Schwann cells., Conclusions: This study shows that axon regeneration is reduced in long compared with short isografts, where long isografts contained an environment with an increased accumulation of senescent markers. Although autografts are considered the gold standard for grafting, these results demonstrate that we must continue to strive for improvements in the autograft regenerative environment.

Providing temporally regulated glial cell line-derived neurotrophic factor (GDNF) to injured nerve can promote robust axon regeneration. However, it is poorly understood why providing highly elevated levels of GDNF to nerve can lead to axon entrapment in the zone containing elevated GDNF. This limited understanding represents an obstacle to the translation of GDNF therapies to treat nerve injuries clinically. Here, we investigated how transgenic Schwann cells (SCs) overexpressing GDNF-IRES-DsRed impact nerve regeneration. Cultured primary SCs were transduced with lentiviruses (GDNF-overexpressing transgenic SCs), one of which provides the capability to express high levels of GDNF and regulate temporal GDNF expression. These SC groups were transplanted into acellular nerve allografts (ANAs) bridging a 14 mm rat sciatic nerve defect. GDNF-overexpressing transgenic SCs expressing GDNF for as little as 1 week decreased axon regeneration across ANAs and caused extensive extracellular matrix (ECM) remodeling. To determine whether additional gene expression changes beyond GDNF transgene expression occurred in GDNF-overexpressing transgenic SCs, microarray analysis of GDNF-overexpressing transgenic SCs compared to untreated SCs was performed. Microarray analysis revealed a set of common genes regulated in transgenic SC groups expressing high levels of GDNF compared to untreated SCs. A co-culture model of GDNF-overexpressing transgenic SCs with fibroblasts (FBs) revealed differential FB ECM-related gene expression compared to untreated SCs. These data suggest a component of axon entrapment is independent of GDNF's impact on axons. Biotechnol. Bioeng. 2017;114: 2121-2130. © 2017 Wiley Periodicals, Inc., (© 2017 Wiley Periodicals, Inc.)

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. © 2015 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 105B: 568-574, 2017., (© 2015 Wiley Periodicals, Inc.)

Purpose: To evaluate the regenerative effect of the additional suture line when using either isografts (ISOs) or acellular nerve allografts (ANAs) placed end-to-end to span a short gap in a rat model., Methods: Rat sciatic nerves were transected and repaired with 2-cm nerve grafts (ISO or ANA). The grafts were 2 cm in length or a 1-cm segment was connected end-to-end to a 1-cm segment to yield a 2-cm length. At 8 weeks, extensor digitorum longus (EDL) muscle force and mass were measured. Nerves were harvested for histomorphometry. In a separate parallel study, the nerves were harvested 2 weeks following graft implantation to assess gene expression changes., Results: All grafts demonstrated regeneration across the 2-cm segment(s). The additional suture line did not result in statistical differences in the number of myelinated nerve fibers that reached the distal nerve. However, when the graft types were compared, there was a significant decrease in nerve fibers in the ANA groups. The EDL muscle mass was significantly greater by using nerve ISOs compared with ANAs, regardless of an additional suture line, but there were no statistical differences noted in EDL muscle force. Gene expression analysis did not differ owing to an additional suture line., Conclusions: Minimal axonal loss and no functional deficits were identified with an additional suture line in this rodent short nerve gap model., Clinical Relevance: Placing nerve grafts in series is a viable option for treating short nerve gaps; however, the use of autografts remains preferable over the use of ANAs., (Copyright © 2016 American Society for Surgery of the Hand. Published by Elsevier Inc. All rights reserved.)

In contrast to adult rat nerve injury models, neonatal sciatic nerve crush leads to massive motor and sensory neuron death. Death of these neurons results from both the loss of functional contact between the nerve terminals and their targets, and the inability of immature Schwann cells in the distal stump of the injured nerve to sustain regeneration. However, current dogma holds that little to no motoneuron death occurs in response to nerve crush at postnatal day 5 (P5). The purpose of the current study was to fully characterize the extent of motor and sensory neuronal death and functional recovery following sciatic nerve crush at mid-thigh level in rats at postnatal days 3-30 (P3-P30), and then compare this to adult injured animals. Following nerve crush at P3, motoneuron numbers were reduced to 35% of that of naïve uninjured animals. Animals in the P5 and P7 group also displayed statistically fewer motoneurons than naïve animals. Animals that were injured at P30 or earlier displayed statistically lower sensory neuron counts in the dorsal root ganglion than naïve controls. Surprisingly, complete behavioral recovery was observed exclusively in the P30 and adult injured groups. Similar results were observed in muscle twitch/tetanic force analysis, motor unit number estimation and wet muscle weights. Rats in both the P5 and P7 injury groups displayed significant neuronal death and impaired functional recovery following injury, challenging current dogma and suggesting that severe deficits persist following nerve injury during this early postnatal developmental period. These findings have important implications concerning the timing of neonatal nerve injury in rats., (© 2015 S. Karger AG, Basel.)

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., (Copyright © 2013 Elsevier Inc. All rights reserved.)

The majority of bioengineering strategies to promote peripheral nerve regeneration after injury have focused on therapies to bridge large nerve defects while fewer therapies are being developed to treat other nerve injuries, such as nerve transection. We constructed delivery systems using fibrin gels containing either free GDNF or polylactide-glycolic acid (PLGA) microspheres with GDNF to treat delayed nerve repair, where ELISA verified GDNF release. We determined the formulation of microspheres containing GDNF that optimized nerve regeneration and functional recovery in a rat model of delayed nerve repair. Experimental groups underwent delayed nerve repair and treatment with GDNF microspheres in fibrin glue at the repair site or control treatments (empty microspheres or free GDNF without microspheres). Contractile muscle force, muscle mass, and MUNE were measured 12 weeks following treatment, where GDNF microspheres (2 weeks formulation) were superior compared to either no GDNF or short-term release of free GDNF to nerve. Nerve histology distal to the repair site demonstrated increased axon counts and fiber diameters due to GDNF microspheres (2 weeks formulation). GDNF microspheres partially reversed the deleterious effects of chronic nerve injury, and recovery was slightly favored with the 2 weeks formulation compared to the 4 weeks formulation., (Copyright © 2012 Wiley Periodicals, Inc.)

Glial-derived neurotrophic factor (GDNF) and nerve growth factor (NGF) have both been shown to enhance peripheral nerve regeneration following injury and target different neuronal populations. The delivery of either growth factor at the site of injury may, therefore, result in quantitative differences in motor nerve regeneration and functional recovery. In this study we evaluated the effect of affinity-based delivery of GDNF or NGF from fibrin-filled nerve guidance conduits (NGCs) on motor nerve regeneration and functional recovery in a 13 mm rat sciatic nerve defect. Seven experimental groups were evaluated consisting of GDNF or NGF and the affinity-based delivery system (DS) within NGCs, control groups excluding the DS and/or growth factor, and nerve isografts. Groups with growth factor in the conduit demonstrated equivalent or superior performance in behavioral tests and relative muscle mass measurements compared to isografts at 12 weeks. Additionally, groups with GDNF demonstrated greater specific twitch and tetanic force production in extensor digitorum longus (EDL) muscle than the isograft control, while groups with NGF produced demonstrated similar force production compared to the isograft control. Assessment of motor axon regeneration by retrograde labeling further revealed that the number of ventral horn neurons regenerating across NGCs containing GDNF and NGF DS was similar to the isograft group and these counts were greater than the groups without growth factor. Overall, the GDNF DS group demonstrated superior functional recovery and equivalent motor nerve regeneration compared to the isograft control, suggesting it has potential as a treatment for motor nerve injury.

The controlled delivery of nerve growth factor (NGF) to the peripheral nervous system has been shown to enhance nerve regeneration following injury, although the effect of release rate has not been previously studied with an affinity-based delivery system (DS). The goal of this research was to determine if the binding site affinity of the DS affected nerve regeneration in vivo using nerve guidance conduits (NGCs) in a 13-mm rat sciatic nerve defect. These DSs consisted of bi-domain peptides that varied in heparin-binding affinity, heparin and NGF, which binds to heparin with moderate affinity. Eight experimental groups were evaluated consisting of NGF with DS, control groups excluding one or more components of the DS within silicone conduits and nerve isografts. Nerves were harvested 6 weeks after treatment for analysis by histomorphometry. These DSs with NGF resulted in a higher frequency of nerve regeneration compared to control groups and were similar to the nerve isograft group in measures of nerve fiber density and percent neural tissue, but not in total nerve fiber count. In addition, these DSs with NGF contained a significantly greater percentage of larger diameter nerve fibers, suggesting more mature regenerating nerve content. While there were no differences in nerve regeneration due to varying peptide affinity with these DSs, their use with NGF enhanced peripheral nerve regeneration through a NGC across a critical nerve gap.

Glial-derived neurotrophic factor (GDNF) promotes both sensory and motor neuron survival. The delivery of GDNF to the peripheral nervous system has been shown to enhance regeneration following injury. In this study, we evaluated the effect of affinity-based delivery of GDNF from a fibrin matrix in a nerve guidance conduit on nerve regeneration in a 13 mm rat sciatic nerve defect. Seven experimental groups were evaluated which received GDNF or nerve growth factor (NGF) with the delivery system within the conduit, control groups excluding one or more components of the delivery system, and nerve isografts. Nerves were harvested 6 weeks after treatment for analysis by histomorphometry and electron microscopy. The use of the delivery system (DS) with either GDNF or NGF resulted in a higher frequency of nerve regeneration vs. control groups, as evidenced by a neural structure spanning the 13 mm gap. The GDNF DS and NGF DS groups were also similar to the nerve isograft group in measures of nerve fiber density, percent neural tissue and myelinated area measurements, but not in terms of total fiber counts. In addition, both groups contained a significantly greater percentage of larger diameter fibers, with GDNF DS having the largest in comparison to all groups, suggesting more mature neural content. The delivery of GDNF via the affinity-based delivery system can enhance peripheral nerve regeneration through a silicone conduit across a critical nerve gap and offers insight into potential future alternatives to the treatment of peripheral nerve injuries.

Summary: Animal models of nerve compression, crush, and transection injuries of peripheral nerves have been subject to extensive study in order to understand the mechanisms of injury and axon regeneration and to investigate methods to promote axon regeneration and improve functional outcomes following nerve injury. Six outcome measures of regenerative success including axon and neuron counts, muscle and motor unit contractile forces, and behavior are reviewed in the context of nerve injury types, crush, transection and nerve repair by direct coaptation, or transection and repair via a nerve graft or conduit. The measures are evaluated for sciatic, tibial, common peroneal, femoral, single nerve branches such as the soleus nerve, and facial nerves. Their validity is discussed in the context of study objectives and the nerve branch. The case is made that outcome measures of axon counts and muscle contractile forces may be valid during the early phases of axon regeneration when regenerating sprouts emerge asynchronously from the proximal nerve stump and regenerate towards their denervated targets. However, care must be taken especially when experimental interventions differentially affect how many neurons regenerate axons and the number of axons per neuron that sprout from the proximal nerve stumps. Examples of erroneous conclusions are given to illustrate the need for researchers to ensure that the appropriate outcome measures are used in the evaluation of the success of peripheral nerve regeneration. [Copyright &y& Elsevier]

The controlled delivery of nerve growth factor (NGF) to the peripheral nervous system has been shown to enhance nerve regeneration following injury, although the effect of release rate has not been previously studied with an affinity-based delivery system (DS). The goal of this research was to determine if the binding site affinity of the DS affected nerve regeneration in vivo using nerve guidance conduits (NGCs) in a 13-mm rat sciatic nerve defect. These DSs consisted of bi-domain peptides that varied in heparin-binding affinity, heparin and NGF, which binds to heparin with moderate affinity. Eight experimental groups were evaluated consisting of NGF with DS, control groups excluding one or more components of the DS within silicone conduits and nerve isografts. Nerves were harvested 6 weeks after treatment for analysis by histomorphometry. These DSs with NGF resulted in a higher frequency of nerve regeneration compared to control groups and were similar to the nerve isograft group in measures of nerve fiber density and percent neural tissue, but not in total nerve fiber count. In addition, these DSs with NGF contained a significantly greater percentage of larger diameter nerve fibers, suggesting more mature regenerating nerve content. While there were no differences in nerve regeneration due to varying peptide affinity with these DSs, their use with NGF enhanced peripheral nerve regeneration through a NGC across a critical nerve gap. [ABSTRACT FROM AUTHOR]

Absract Peripheral nerve injury evokes a complex cascade of chemical reactions including generation of molecular radicals. Conversely, the reactions within nerve induced by stress are difficult to directly detect or measure to establish causality. Monitoring these reactions in vivo would enable deeper understanding of the nature of the injury and healing processes. Here, we utilized near-infrared fluorescence molecular probes delivered via intra-neural injection technique to enable live, in vivo imaging of tissue response associated with nerve injury and stress. These initially quenched fluorescent probes featured specific sensitivity to hydroxyl radicals and become fluorescent upon encountering reactive oxygen species (ROS). Intraneurally delivered probes demonstrated rapid activation in injured rat sciatic nerve but minimal activation in normal, uninjured nerve. In addition, these probes reported activation within sciatic nerves of living rats after a stress caused by a pinprick stimulus to the abdomen. This imaging approach was more sensitive to detecting changes within nerves due to the induced stress than other techniques to evaluate cellular and molecular changes. Specifically, neither histological analysis of the sciatic nerves, nor the expression of pain and stress associated genes in dorsal root ganglia could provide statistically significant differences between the control and stressed groups. Overall, the results demonstrate a novel imaging approach to measure ROS in addition to the impact of ROS within nerve in live animals. [ABSTRACT FROM AUTHOR]