Total Axon Research Articles

Abstract 88: Local Delivery of FK506 to a Mouse Sciatic Nerve Injury Increases Nerve Regeneration Outcomes

PURPOSE: In the clinic, there are limited treatment strategies for the repair of peripheral nerve injuries, and the majority of off-the-shelf nerve repair devices lack added functionality. Direct repair is among the most commonly performed nerve repair procedures and presently there is no clinical therapeutic available that improves outcomes after direct repair. The objective of this study is to assess the efficacy of local, controlled delivery of FK506 to improve nerve regeneration outcomes in the setting of nerve transection injury and repair. METHODS: Solvent casting techniques were used to embed FK506 into poly(lactide-co-caprolactone) (PLC) films for the creation of a nerve wrap – drug delivery system that is capable of an extended, localized release of FK506 to injured nerves. In vitro drug rate of release testing and drug bioactivity assays were performed to determine the FK506 release profile and to verify release FK506 bioactivity. A mouse sciatic transection and direct-suture repair model was used to evaluate the effect local, controlled FK506 delivery could have on nerve regeneration outcomes greater than direct-suture repair alone. Animals were sacrificed at 6 weeks for electrophysiological assessment, determination of gastrocnemius muscle mass recovery, and sciatic nerve histological evaluation. RESULTS: A linear release (R2=0.991) of FK506 for 30+ days was observed for two FK506 loading concentrations (0.01 % and 0.05% w/w of FK506/PLC). The released FK506 matched control FK506 levels of neurite extension in the dorsal root ganglion assay. Relative gastrocnemius muscle weight, relative foot EMG, and total number of axons distal of the repair site were significantly greater in mice treated with the 0.01% FK506 nerve wrap than the non-drug groups. For the 0.05% nerve wraps, the total axon count distal to the repair site was found to be significantly greater, but no significant differences were observed for the other metrics. CONCLUSION: Results of this study indicate that extended, localized delivery of FK506 to a mouse sciatic transection and direct repair can improve nerve regeneration outcomes in a sciatic nerve transection and repair model. A dose-dependent response in the nerve regeneration metrics was observed, indicating there is an optimal dose range for local delivery of FK506.

Neurofilament-histomorphometry comparison in the evaluation of unmyelinated axon regeneration following peripheral nerve injury: An alternative to electron microscopy

BackgroundCurrently, assessment of unmyelinated axon regeneration is limited to electron microscopy (EM), which is expensive, time consuming and not universally available. This study presents a protocol to estimate the number of unmyelinated axons in a regenerating peripheral nerve without the need for electron microscopy. New methodThe common peroneal nerve of Sprague-Dawley rats was transected, repaired and regenerated for 4 weeks. Two distal adjacent segments of the regenerating nerve were then processed for either conventional histomorphometry using toluidine blue or immunolabeling of neurofilament protein. Myelinated axon and total axon counts were obtained, respectively, to generate estimates of unmyelinated axon numbers, which were then compared to unmyelinated axon counts using EM from the same specimens. For comparison, unmyelinated axons were counted in an uninjured rat laryngeal nerve. ResultsAfter 4 weeks of regeneration, the estimated number of regenerating unmyelinated axons was 4044 ± 232 using this technique, representing 81.3% of the total axonal population. By comparison, the proportion of unmyelinated axons in the uninjured laryngeal nerve was 55% of the total axonal population. Comparison with existing methodThese estimates correlate with electron microscopy measurements, both in terms of the proportion of unmyelinated axons and also by linear regression analysis. ConclusionsThe neurofilament staining method correlates with electron microscopy estimates of the same nerve sections. It is useful for the efficient counting of unmyelinated axons in the regenerating peripheral nerve and can be used by laboratories that do not have access to EM facilities.

Seasonal comparison of the neuromuscular junction morphology of Bufo marinus.

At mammalian neuromuscular junctions (NMJs), prolonged inactivity leads to muscle denervation and atrophy. By contrast, amphibian NMJs do not show such degeneration even though they can remain in a state of drought-imposed dormancy (hibernation) for many years. We have previously reported that during the dry season, toad (Bufo marinus) NMJs display decreased sensitivity to extracellular calcium-dependent neurotransmitter release, which leads to minimal neuromuscular transmission. In the present study, we examined and compared NMJ morphology of toads obtained from the wild during the wet season (February-March) when these toads are active, to toads obtained from dry season (October-November) when toads are inactive. Iliofibularis muscles were isolated and prepared for immunostaining with anti-SV2, a monoclonal antibody that labels synaptic vesicle glycoprotein SV2. The corresponding postsynaptic acetylcholine receptors were stained using Alexa Fluro-555 conjugated α-bungarotoxin. Confocal microscopy and three-dimensional reconstructions were then used to examine the pre-and postsynaptic morphology of toads NMJs from the dry (inactive) and wet (active) seasons. Total axon branch number, the percentage of axon branches with discontinuous distributions of synaptic vesicles, and further the Pearson value of colocalization of pre and postsynaptic elements in each NMJs from both the dry and wet season were compared. While our previous studies on dry toads revealed a significant reduction in evoked neurotransmission, our present findings show that the structure of the NMJs suffered limited level of remodeling, suggesting a mechanism utilized by NMJs in dry season toads to support quick recover from their dormant state after the heavy rain in wet season.

Optic Nerve Head Astrocytes Display Axon-Dependent and -Independent Reactivity in Response to Acutely Elevated Intraocular Pressure.

PurposeOptic nerve head (ONH) astrocytes provide support for axons, but exhibit structural and functional changes (termed reactivity) in a number of glaucoma models. The purpose of this study was to determine if ONH astrocyte structural reactivity is axon-dependent.MethodsUsing rats, we combine retrobulbar optic nerve transection (ONT) with acute controlled elevation of intraocular pressure (CEI), to induce total optic nerve axon loss and ONH astrocyte reactivity, respectively. Animals were euthanized immediately or 1 day post CEI, in the presence or absence of ONT. ONH sections were labeled with fluorescent-tagged phalloidin and antibodies against β3 tubulin, phosphorylated cortactin, phosphorylated paxillin, or complement C3. ONH label intensities were quantified after confocal microscopy. Retrobulbar nerves were assessed for axon injury by light microscopy.ResultsWhile ONT alone had no effect on ONH astrocyte structural orientation, astrocytes demonstrated significant reorganization of cellular extensions within hours after CEI, even when combined with ONT. However, ONH astrocytes displayed differential intensities of actin (phosphorylated cortactin) and focal adhesion (phosphorylated paxillin) mediators in response to CEI alone, ONT alone, or the combination of CEI and ONT. Lastly, label intensities of complement C3 within the ONH were unchanged in eyes subjected to CEI alone, ONT alone, or the combination of CEI and ONT, relative to controls.ConclusionsEarly ONH astrocyte structural reactivity to elevated IOP is multifaceted, displaying both axon dependent and independent responses. These findings have important implications for pursuing astrocytes as diagnostic and therapeutic targets in neurodegenerative disorders with fluctuating levels of axon injury.

Diffusion tensor tractography to visualize axonal outgrowth and regeneration in a 4-cm reverse autograft sciatic nerve rabbit injury model

ABSTRACTBackground: Diffusion tensor tractography (DTT) has recently been shown to accurately detect nerve injury and regeneration. This study assesses whether 7-tesla (7T) DTT imaging is a viable modality to observe axonal outgrowth in a 4 cm rabbit sciatic nerve injury model fixed by a reverse autograft (RA) surgical technique.Methods: Transection injury of unilateral sciatic nerve (4 cm long) was performed in 25 rabbits and repaired using a RA surgical technique. Analysis of the nerve autograft was performed at 3, 6, and 11 weeks postoperatively and compared to normal contralateral sciatic nerve, used as control group. High-resolution DTT from ex vivo sciatic nerves were obtained using 3D diffusion-weighted spin-echo acquisitions at 7-T. Total axons and motor and sensory axons were counted at defined lengths along the graft.Results: At 11 weeks, histologically, the total axon count of the RA group was equivalent to the contralateral uninjured nerve control group. Similarly, by qualitative DTT visualization, the 11-week RA group showed increased fiber tracts compared to the 3 and 6 weeks counterparts. Upon immunohistochemical evaluation, 11-week motor axon counts did not significantly differ between RA and control; but significantly decreased sensory axon counts remained. Nerves explanted at 3 weeks and 6 weeks showed decreased motor and sensory axon counts.Discussion: 7-T DTT is an effective imaging modality that may be used qualitatively to visualize axonal outgrowth and regeneration. This has implications for the development of technology that non-invasively monitors peripheral nerve regeneration in a variety of clinical settings.

Combination of an Electrospun Nanofiber Sheet Incorporating Methylcobalamin and a PGA-Collagen Tube Promotes Nerve Regeneration and Functional Recovery in a Rat Sciatic Nerve Defect Model

Speaker's Bureau: Teijin Nakashima Co., Ltd. (Murase) We previously reported that a novel electrospun nanofiber sheet incorporating methylcobalamin (MeCbl sheet) to deliver it enough locally to the peripheral nerve injury site promotes peripheral nerve regeneration in a rat sciatic nerve crush injury model. The purpose of this study is to investigate the effect of combination of an MeCbl sheet and a PGA-collagen tube (Nerbridge) in a rat sciatic nerve defect model. We used a rat left sciatic nerve 10-mm defect models. Male Wistar rats weighing 180–220 g (n = 37) were used in this study. We divided them into 4 groups: (1) sham operation group (n = 10), (2) Nerbridge group, in which the gap was bridged with Nerbridge (n = 9), (3) Nerbridge + MeCbl sheet group, in which the gap was bridged with Nerbridge wrapped around in an MeCbl sheet (n = 8), and (4) autograft group, in which the sciatic nerve received a reversed autograft (n = 10). At 12 weeks after the operation, motor and sensory function were evaluated by the sciatic functional index, isometric tetanic force of the anterior tibial muscle, wet weight of the anterior tibial muscle, and the von Frey filament test. The electrophysiological analysis was performed by the compound muscle action potentials, the terminal latency, and nerve conduction velocity. We also demonstrated histological analysis by evaluating the number of total axons, the myelinated ratio, and the g-ratio. There was significant improvement in the Nerbridge + MeCbl sheet group for the von Frey filament test (Figure 48-1A), the terminal latency (Figure 48-1B), the ratio of myelinated axon (Figure 48-2A), and the g-ratio (Figure 48-2B) compared with Nerbridge group. The Nerbridge + MeCbl sheet group displayed the same recovery as the autograft group in the von Frey filament test (Figure 48-1A) and the ratio of myelinated axon (Figure 48-2A), although the recovery of the terminal latency (Figure 48-1B) and the g-ratio (Figure 48-2B) in the Nerbridge + MeCbl sheet group did not reach those in the autograft group. •An MeCbl sheet contributed to the recovery of sensory function and the promotion of myelinated ratio and the g-ratio after sciatic nerve injury.•An MeCbl sheet may be a new treatment option to promote nerve regeneration for an injured nerve bridged with an artificial nerve conduit.Figure 48-1BThe values of terminal latency in the Nerbridge + MeCbl sheet group was significantly shorter than that of the Nerbridge group. n = 8-10. *P < .05, **P < .01, ***P < .001; one-way analysis of variance followed by a post hoc, Tukey-Kramer HSD test.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure 48-2AFluorescence micrographs showing cross-sectional slices of sciatic nerves labeled for MBP (red) and NF200 (green). The Nerbridge + MeCbl sheet group displayed a higher ratio of myelinated axon than that in the Nerbridge group and the same as that in the autograft group. Scale bar, 20 μm. n = 5 per group.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure 48-2BPhotomicrographs of cross-sectional slices of sciatic nerves under a transmission electron microscope. The Nerbridge + MeCbl sheet group displayed a lower g-ratio than that in the Nerbridge group. Scale bar, 2 μm. n = 3–5. *P < .05, **P < .01, ***P < .001; one-way analysis of variance followed by a post hoc, Tukey-Kramer HSD test.View Large Image Figure ViewerDownload Hi-res image Download (PPT)

Donor nerve axotomy and axonal regeneration after end-to-side neurorrhaphy in a rodent model.

OBJECTIVE In this study, the authors used a surgical model of end-to-side neurorrhaphy between a nerve graft and a donor tibial nerve in adult rats to investigate the optimal conditions for axonal regeneration induced by the donor nerve. They also assessed the importance of a more favorable pathway using a predegenerated nerve graft to attract regenerating axons to regrow into the graft and then directing and improving their growth toward the target in comparison with results obtained with a fresh nerve graft. METHODS End-to-side neurorrhaphy was performed between a nerve graft and a donor tibial nerve. The nerve graft was obtained from the left tibial nerve, which was either freshly removed or predegenerated 1 week prior to neurorrhaphy. The donor right tibial nerve was injured by epineurium removal alone, injured by epineurium removal with cross section of 20% or 50% of the total axons at the coaptation site, or left intact. The animals were followed postoperatively for a 6-week period, and outcomes were evaluated by optical microscopy and retrograde labeling to detect the regenerated primary sensory neurons located in the lumbar dorsal root ganglia and spinal motor neurons located in the lumbar spinal ventral horn. RESULTS At the end of the follow-up period, no regenerating axons were observed in the nerve grafts when the donor nerve was left intact, and very few axons were detected when the donor nerve was injured by epineurium removal alone. However, numerous regenerating axons appeared in the grafts when the donor nerve was axotomized, and the greatest number was achieved with a 50% cross section axotomized nerve. In the rats with a 50% cross section of the donor nerve, better nerve-like morphology of the grafts was observed, without connective adhesions. When a predegenerated nerve graft was used, more regenerating axons were attracted and elongated with a more regular shape and improved myelination. CONCLUSIONS Axonal regrowth into a nerve graft depends on axotomy of the donor nerve after end-to-side neurorrhaphy. More efficient attraction and an improved structure of the regenerating axons were achieved when a predegenerated nerve graft was used. Furthermore, a nerve graft may require a certain number of regenerating axons to maintain a nerve-like morphology.

Functional characterisation of filamentous actin probe expression in neuronal cells.

Genetically encoded filamentous actin probes, Lifeact, Utrophin and F-tractin, are used as tools to label the actin cytoskeleton. Recent evidence in several different cell types indicates that these probes can cause changes in filamentous actin dynamics, altering cell morphology and function. Although these probes are commonly used to visualise actin dynamics in neurons, their effects on axonal and dendritic morphology has not been systematically characterised. In this study, we quantitatively analysed the effect of Lifeact, Utrophin and F-tractin on neuronal morphogenesis in primary hippocampal neurons. Our data show that the expression of actin-tracking probes significantly impacts on axonal and dendrite growth these neurons. Lifeact-GFP expression, under the control of a pBABE promoter, caused a significant decrease in total axon length, while another Lifeact-GFP expression, under the control of a CAG promoter, decreased the length and complexity of dendritic trees. Utr261-EGFP resulted in increased dendritic branching but Utr230-EGFP only accumulated in cell soma, without labelling any neurites. Lifeact-7-mEGFP and F-tractin-EGFP in a pEGFP-C1 vector, under the control of a CMV promoter, caused only minor changes in neuronal morphology as detected by Sholl analysis. The results of this study demonstrate the effects that filamentous actin tracking probes can have on the axonal and dendritic compartments of neuronal cells and emphasise the care that must be taken when interpreting data from experiments using these probes.

Regional Retinal Ganglion Cell Axon Loss in a Murine Glaucoma Model.

PurposeTo determine if retinal ganglion cell (RGC) axon loss in experimental mouse glaucoma is uniform in the optic nerve.MethodsExperimental glaucoma was induced for 6 weeks with a microbead injection model in CD1 (n = 78) and C57BL/6 (B6, n = 68) mice. From epoxy-embedded sections of optic nerve 1 to 2 mm posterior to the globe, total nerve area and regional axon density (axons/1600 μm2) were measured in superior, inferior, nasal, and temporal zones.ResultsControl eyes of CD1 mice have higher axon density and more total RGCs than control B6 mice eyes. There were no significant differences in control regional axon density in all mice or by strain (all P > 0.2, mixed model). Exposure to elevated IOP caused loss of RGC in both strains. In CD1 mice, axon density declined without significant loss of nerve area, while B6 mice had less density loss, but greater decrease in nerve area. Axon density loss in glaucoma eyes was not significantly greater in any region in either mouse strain (both P > 0.2, mixed model). In moderately damaged CD1 glaucoma eyes, and CD1 eyes with the greatest IOP elevation exposure, density loss differed by region (P = 0.05, P = 0.03, mixed model) with the greatest loss in the temporal and superior regions, while in severely injured B6 nerves superior loss was greater than inferior loss (P = 0.01, mixed model, Bonferroni corrected).ConclusionsThere was selectively greater loss of superior and temporal optic nerve axons of RGCs in mouse glaucoma at certain stages of damage. Differences in nerve area change suggest non-RGC responses differ between mouse strains.

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.

Rational Roots of Sympathetic Overactivity by Neurogenic Pulmonary Edema Modeling Arising from Sympathyco-Vagal Imbalance in Subarachnoid Hemorrhage: An Experimental Study

Autonomous innervations of the lungs are maintained by cervical sympathetic and vagal nerves. Sympathetic overactivity-induced neurogenic pulmonary edema (NPE) is known as a serious complication of subarachnoid hemorrhage, but the rational neuronal mechanism of that overactivity has not yet been clarified fully. The aim of this study was to examine whether there is a relationship between vagal nerve ischemia related sympathetic overactivity and neurogenic pulmonary edema in subarachnoid hemorrhage. This study was conducted on 27 rabbits. A control group was formed of 5 animals, a sham group of 7 to which saline was administered, and a study group of 15 animals that were injected with homologous arterial blood into the cisterna magna. Electrocardiography and respiratory rhythm parameters were monitored for 3 weeks and the animals were then decapitated. Statistical analysis was made of the numbers of degenerated axons in the pulmonary branches of the vagal nerves, the neuron density of stellate ganglions and the vasospasm index of the pulmonary arteries. In the control group, the normal respiration rate was 34 ± 6 bpm, total axon number was 1600 ± 270/mm(2), degenerated axon number was 10 ± 3/mm(2), and vasospasm index was 1.34 ± 0.25. The sham group values were 30 ± 3 bpm, 163 ± 47/mm(2), and 1.95 ± 0.45 and the study group values were 45 ± 8 bpm, 530 ± 92/mm(2), and 2.76 ± 0.83. The mean stellate ganglion neuron density was evaluated as 8.112 ± 1.230/mm(3) in all animals, as 7.420 ± 4.10/mm(3) in animals with slight NPE, and as 12.512 ± 1.236/mm(3) in animals that developed severe NPE. High neuron density of stellate ganglion may have important roots in sympathetic overactivity-related NPE development in subarachnoid hemorrhage.

Semi-automated counting of axon regeneration in poly(lactide co-glycolide) spinal cord bridges

BackgroundSpinal cord injury (SCI) is a debilitating event with multiple mechanisms of degeneration leading to life-long paralysis. Biomaterial strategies, including bridges that span the injury and provide a pathway to reconnect severed regions of the spinal cord, can promote partial restoration of motor function following SCI. Axon growth through the bridge is essential to characterizing regeneration, as recovery can occur via other mechanisms such as plasticity. Quantitative analysis of axons by manual counting of histological sections can be slow, which can limit the number of bridge designs evaluated. In this study, we report a semi-automated process to resolve axon numbers in histological sections, which allows for efficient analysis of large data sets. New methodAxon numbers were estimated in SCI cross-sections from animals implanted with poly(lactide co-glycolide) (PLG) bridges with multiple channels for guiding axons. Immunofluorescence images of histological sections were filtered using a Hessian-based approach prior to threshold detection to improve the signal-to-noise ratio and filter out background staining associated with PLG polymer. ResultsSemi-automated counting successfully recapitulated average axon densities and myelination in a blinded PLG bridge implantation study. Comparison with existing methodsAxon counts obtained with the semi-automated technique correlated well with manual axon counts from blinded independent observers across sections with a wide range of total axons. ConclusionsThis semi-automated detection of Hessian-filtered axons provides an accurate and significantly faster alternative to manual counting of axons for quantitative analysis of regeneration following SCI.

Efficient bridging of 20 mm rat sciatic nerve lesions with a longitudinally micro-structured collagen scaffold

An increasing number of biomaterial nerve guides has been developed that await direct comparative testing with the ‘gold-standard’ autologous nerve graft in functional repair of peripheral nerve defects. In the present study, 20 mm rat sciatic nerve defects were bridged with either a collagen-based micro-structured nerve guide (Perimaix) or an autologous nerve graft. Axons regenerated well into the Perimaix scaffold and, the majority of these axons grew across the 20 mm defect into the distal nerve segment. In fact, both the total axon number and the number of retrogradely traced somatosensory and motor neurons extending their axons across the implant was similar between Perimaix and autologous nerve graft groups. Implantation of Schwann cell-seeded Perimaix scaffolds provided only a beneficial effect on myelination within the scaffold. Functional recovery supported by the implanted, non-seeded Perimaix scaffold was as good as that observed after the autologous nerve graft, despite the presence of thinner myelin sheaths in the Perimaix implanted nerves. These findings support the potential of the Perimaix collagen scaffold as a future off-the-shelf device for clinical applications in selected cases of traumatic peripheral nerve injury.

Beneficial Effect of (‐)‐epigallocatechin‐3‐gallate (EGCG) on Sciatic Nerve Crush Injury: Stereological analysis

The neuroprotective effect of EGCG on sciatic nerve crush injury is associated with significant neurobehavioral recovery (Renno et al. 2014). In this study, we examined the histological changes in the sciatic nerve after crush injury following EGCG treatment. The mean cross sectional area of the axon showed a significant increase in the EGCG treated group starting at week 4 and continued up to week 8 compared to CRUSH animals. However, axon area remained much smaller in CRUSH+EGCG and CRUSH groups compared to SHAM and NAIVE animals. The number of axons per unit area was significantly decreased in CRUSH+EGCG and CRUSH groups compared to control. The CRUSH group displayed a significant increase in total small and thinly myelinated axon number at week 6 and 8 post‐injury. ECGC treated animals showed a significant decrease in the total myelinated axon numbers. CRUSH+EGCG group showed a significant increase in myelin thickness in the distal sciatic nerve segment at week 4 till week 8 compared to CRUSH animals. EGCG treatment significantly increased the fiber diameter to axon diameter (F/A) ratio and the myelin thickness to axon diameter (M/A) ratio starting at week 2 post sciatic nerve injury compared to CRUSH animals. EGCG has profound positive effect on the myelinated axons; increase in the axon area, myelin thickness, F/A ratio and M/A ratio. In summary, quantitative stereological evaluation revealed significant histomorphological evidence of neuroregeneration in the sciatic nerve of CRUSH+EGCG rats compared to control groups between 2 and 4 weeks postoperatively. The morphometric data analysis supports our hypothesis that EGCG treatment stimulates regeneration of myelinated axons and minimizes histomorphological alterations caused by crush injury of sciatic nerve.This work was supported by Kuwait University Research Administration (grant No. MA01/11).

Comparison of acellular nerve allograft modification with Schwann cells or VEGF.

Individual contributions of exogenous Schwann cells (SCs) and vascular endothelial growth factor (VEGF) were evaluated in acellular nerve allografts (ANAs). ANA processing removes SCs and vasculature, likely contributing to reduced regeneration compared to autografts. Exogenous SCs may improve the regenerative microenvironment, and VEGF has been shown to stimulate angiogenesis. Replacing these components in ANAs may improve regeneration. A rat sciatic nerve transection model was used to study 20-mm grafts. Four graft types were studied: (1) isograft, (2) ANA, (3) ANA-SCs, and (4) ANA-VEGF. After 10weeks in vivo, the midgraft and distal nerve to the grafts were analyzed for axonal regeneration using histomorphometry to assess total myelinated axon counts, density, width, and percent neural tissue. The most axons in the distal nerve were regenerated in the isograft followed by the ANA- SC group, with 9171 ± 1822 and 7103 ± 1576 regenerated axons respectively. Both the ANA and ANA-VEGF groups had significantly fewer regenerated axons compared to the isograft (p < 0.05) with 5225 ± 2994 and 5709 ± 2657 regenerated axons, respectively. The ANA and ANA-VEGF groups also had significantly reduced fiber density and percent nerve compared to the isograft; the isograft and ANA-SC groups were not significantly different (p < 0.05). These results show that SCs improve axonal regeneration in a 20mm ANA to a greater extent than VEGF. VEGF treatment showed a trend toward increased axonal regeneration but was not significantly different compared to the untreated ANA. The role of VEGF may be clearer in longer grafts where ischemia is a greater factor.

Adjuvant neurotrophic factors in peripheral nerve repair with chondroitin sulfate proteoglycan-reduced acellular nerve allografts

BackgroundAcellular nerve allografts are now standard tools in peripheral nerve repair because of decreased donor site morbidity and operative time savings. Preparation of nerve allografts involves several steps of decellularization and modification of extracellular matrix to remove chondroitin sulfate proteoglycans (CSPGs), which have been shown to inhibit neurite outgrowth through a poorly understood mechanism involving RhoA and extracellular matrix-integrin interactions. Chondroitinase ABC (ChABC) is an enzyme that degrades CSPG molecules and has been shown to promote neurite outgrowth after injury of the central and peripheral nervous systems. Variable results after ChABC treatment make it difficult to predict the effects of this drug in human nerve allografts, especially in the presence of native extracellular signaling molecules. Several studies have shown cross-talk between neurotrophic factor and CSPG signaling pathways, but their interaction remains poorly understood. In this study, we examined the adjuvant effects of nerve growth factor (NGF) and glial cell line-derived neurotrophic factor (GDNF) on neurite outgrowth postinjury in CSPG-reduced substrates and acellular nerve allografts. Materials and methodsE12 chicken DRG explants were cultured in medium containing ChABC, ChABC + NGF, ChABC + GDNF, or control media. Explants were imaged at 3 d and neurite outgrowths measured. The rat sciatic nerve injury model involved a 1-cm sciatic nerve gap that was microsurgically repaired with ChABC-pretreated acellular nerve allografts. Before implantation, nerve allografts were incubated in NGF, GDNF, or sterile water. Nerve histology was evaluated at 5 d and 8 wk postinjury. ResultsThe addition of GDNF in vitro produced significant increase in sensory neurite length at 3 d compared with ChABC alone (P < 0.01), whereas NGF was not significantly different from control. In vivo adjuvant NGF produced increases in total myelinated axon count (P < 0.005) and motor axon count (P < 0.01), whereas significantly reducing IB4+ nociceptor axon count (P < 0.01). There were no significant differences produced by in vivo adjuvant GDNF. ConclusionsThis study provides initial evidence that CSPG-reduced nerve grafts may disinhibit the prosurvival effects of NGF in vivo, promoting motor axon outgrowth and reducing regeneration of specific nociceptive neurons. Our results support further investigation of adjuvant NGF therapy in CSPG-reduced acellular nerve grafts.

Storage and allogeneic transplantation of peripheral nerve using a green tea polyphenol solution in a canine model

BackgroundIn our previous study, allogeneic-transplanted peripheral nerve segments preserved for one month in a polyphenol solution at 4°C could regenerate nerves in rodents demonstrated the same extent of nerve regeneration as isogeneic fresh nerve grafts. The present study investigated whether the same results could be obtained in a canine model.MethodsA sciatic nerve was harvested from a male beagle dog, divided into fascicules of < 1.5 mm diameter, and stored in a polyphenol solution (1 mg/ml) for one month at 4°C. The nerve fascicles were transplanted into 10 female beagle dogs to bridge 3-cm right ulnar nerve gaps. In the left ulnar nerve in each dog, a 3-cm nerve segment was harvested, turned in the opposite direction, and sutured in situ. Starting one day before transplantation, the immunosuppressant FK506 was administered subcutaneously at doses of 0.1 mg/kg daily in four dogs (PA0.1 group), 0.05 mg/kg daily in four dogs (PA0.05 group), or 0.05 mg/kg every other day in two dogs (PA0.025 group). Twelve weeks after surgery, electrophysiological and morphological studies were performed to assess the regeneration of the right and left ulnar nerves. The data for the right ulnar nerve were expressed as percentages relative to the left ulnar nerve. Polymerase chain reaction (PCR) was used to identify the sex-determining region of the Y-chromosome (Sry) and β-actin to investigate whether cells of donor origin remained in the allogeneic nerve segments. FK506 concentration was measured in blood samples taken before the animals were killed.ResultsThe total myelinated axon numbers and amplitudes of the muscle action potentials correlated significantly with the blood FK506 concentration. Few axons were observed in the allogeneic-transplanted nerve segments in the PA0.025 group. PCR showed clear Sry-specific bands in specimens from the PA0.1 and PA0.05 groups but not from the PA0.025 group.ConclusionsSuccessful nerve regeneration was observed in the polyphenol-treated nerve allografts when transplanted in association with a therapeutic dose of FK506. The data indicate that polyphenols can protect nerve tissue from ischemic damage for one month; however, the effects of immune suppression seem insufficient to permit allogeneic transplantation of peripheral nerves in a canine model.

New Experimental Model of Facial Nerve Function and Integrity to Evaluate Regeneration

Objectives: The ideal animal model for nerve regeneration studies is an object of controversy because all models described by the literature have advantages and disadvantages. Small animals such as rats have been repeatedly used in experimental procedures because of their easy caretaking and handling, resistance to manipulation and infections, and low cost. In this case, rats have the additional benefit of tolerating bilateral facial paralysis. This study aimed to describe the histologic and functional (electroneuromyographic) patterns of the mandibular branch of the facial nerve of Wistar rats in order to create a new experimental model of facial nerve regeneration. Methods: Forty-two male Wistar rats were submitted to neurophysiologic evaluation by a nerve conduction test of the mandibular branch of the facial nerve to obtain the compound muscle action potential. Twelve of these rats had the mandibular branch of the facial nerve surgically removed and submitted to histologic analysis (total axon number, partial axonal density, and axonal diameter) of the proximal and distal nerve segments. Results: There was no statistical difference in the functional and histologic variables studied. Conclusion: These new histologic and functional standards of the mandibular branch of the facial nerve of rats establish an objective, easy, and greatly reproducible model for future facial nerve regeneration studies.

Microfluidic culture platform for studying neuronal response to mild to very mild axonal stretch injury.

A new model for studying localised axonal stretch injury is presented, using a microfluidic device to selectively culture axons on a thin, flexible poly (dimethylsiloxane) membrane which can be deflected upward to stretch the axons. A very mild (0.5% strain) or mild stretch injury (5% strain) was applied to primary cortical neurons after 7 days growth in vitro. The extent of distal degeneration was quantified using the degenerative index (DI, the ratio of fragmented axon area to total axon area) of axons fixed at 24 h and 72 h post injury (PI), and immunolabelled for the axon specific, microtubule associated protein-tau. At 24 h PI following very mild injuries (0.5%), the majority of the axons remained intact and healthy with no significant difference in DI when compared to the control, but at 72 h PI, the DI increased significantly (DI = 0.11 ± 0.03). Remarkably, dendritic beading in the somal compartment was observed at 24 h PI, indicative of dying back degeneration. When the injury level was increased (5% stretch, mild injury), microtubule fragmentation along the injured axons was observed, with a significant increase in DI at 24 h PI (DI = 0.17 ± 0.02) and 72 h PI (DI = 0.18 ± 0.01), relative to uninjured axons. The responses observed for both mild and very mild injuries are similar to those observed in the in vivo models of traumatic brain injury, suggesting that this model can be used to study neuronal trauma and will provide new insights into the cellular and molecular alterations characterizing the neuronal response to discrete axonal injury.

Complete morphologies of basal forebrain cholinergic neurons in the mouse.

The basal forebrain cholinergic system modulates neuronal excitability and vascular tone throughout the cerebral cortex and hippocampus. This system is severely affected in Alzheimer's disease (AD), and drug treatment to enhance cholinergic signaling is widely used as symptomatic therapy in AD. Defining the full morphologies of individual basal forebrain cholinergic neurons has, until now, been technically beyond reach due to their large axon arbor sizes. Using genetically-directed sparse labeling, we have characterized the complete morphologies of basal forebrain cholinergic neurons in the mouse. Individual arbors were observed to span multiple cortical columns, and to have >1000 branch points and total axon lengths up to 50 cm. In an AD model, cholinergic axons were slowly lost and there was an accumulation of axon-derived material in discrete puncta. Calculations based on published morphometric data indicate that basal forebrain cholinergic neurons in humans have a mean axon length of ∼100 meters.DOI: http://dx.doi.org/10.7554/eLife.02444.001.