Axonal Sprouts Research Articles

Lentivirus-siNgR199 Promotes Axonal Regeneration and Functional Recovery in Rats

ABSTRACTBackground: The limited regenerative capability of central neurons and inhibitory factors are the main causes of axonal regeneration failure after spinal cord injury (SCI). Nogo receptors (NgR) are a family of receptors shared by three factors that inhibit axon outgrowth. In a previous study, siNgR199, an effective lentiviral siRNA vector of NgR1, was constructed and transfected into cortical neurons in vitro, and it effectively promoted axon outgrowth. The present study focused on the therapeutic effect of delivery of a recombinant lentivirus containing siNgR199 in vivo in rats. Methods: Rat models of traumatic SCI were constructed according to the method developed by Allen. The animals were randomly divided into three groups: group 1, injected with physiological saline; group 2, injected with empty lentivirus vehicle; and group 3, injected with lentivirus carrying siNgR199. The Basso, Beattie, and Bresnahan (BBB) scale was used for assessing hindlimb locomotor function after SCI. The neural tracer biotinylated dextran amine (BDA) and immunohistochemical methods were employed to study axon outgrowth. Results: After injection for 8 weeks, BBB locomotion scores showed that the motor function of the hindlimb recovered better in animals in group 3 (injected with lentivirus carrying siNgR199) than those in groups 1 and 2, which were injected with saline and empty lentivirus vehicle, respectively. In group 3, regenerative nerve fibers were observed at and across the injury site, while very few axonal sprouts were observed in groups 1 and 2. Conclusions: Injection with lentivirus carrying siNgR199 into the sensorimotor cortex improved axonal regeneration and functional recovery of hindlimb after SCI in rats.

The Capacity of the Distal Stump of Peripheral Nerve to Receive Growing Axons after Two and Six Months Denervation; An Experimental Study with Rats

Peripheral nerve injury may lead to poor recovery outcome in spite of treatment with advanced microsurgical repair techniques. Delayed cross-anastomosis paradigm was used to study the axon grow to the distal nerve stump after denervation separately from the influence of prolonged axotomy in the proximal stump. Left common peroneal nerve of 48 rats was transected and denervated over two or six months. There were two research groups in the study. In the regeneration group (REG) the proximal stump of acutely transected tibial nerve was sutured to denervated distal stump of common peroneal nerve. To our knowledge, this is the first study in which this group was compared to degeneration group (DEG) with both nerve ends denervated over two or six months. This comparison enabled us to study the capacity of denervated distal nerve stump to receive sprouting axons. Axon density in distal nerve stump was calculated after three or six week's follow-up periods. There were no differences in the number of axon sprouts in the distal nerve stump between the denervation periods of two and six months. When compared REG and DEG groups, there was trend to higher axon densities in the REG group, although the differences were not statistically significant. We conclude that the capacity of distal nerve stump to receive the growing axons from the proximal nerve stump does not decrease significantly between two and six months denervation. Cross-anastomosis paradigm provides a useful tool for detailed study of the nerve transfer procedure.

Secretion of bacterial chondroitinase ABC from bone marrow stromal cells by glycosylation site mutation: A promising approach for axon regeneration

Growth-inhibitory chondroitin sulfate proteoglycans (CSPGs) contribute a lot to failure of axon regeneration. Chondroitinase ABC (ChABC) digests glycosaminoglycan chains attached in CSPGs and can thereby promote axonal regeneration beyond a lesion site. However, CSPGs expression are up-regulated for almost 7 weeks after spinal cord injury (SCI) in vivo, so single dose of exogenous ChABC is insufficient for long distance of axon sprout and functional recovery. It is considered an ideal strategy to transfect neurons and/or glia at the injury site with a vector containing the gene encoding chondroitinase, so they can secrete ChABC themselves. Mammalian cells in the current studies, however, can not secret ChABC efficiently. It is well established that glycosylation is a common obstacle for eukaryotic cells to secret bacterial protein. ChABC is a protein heavily glycosylated structurally, and it was reported that inhibiting the glycosylation of xylosyltransferase-1 with a DNA enzyme could reduce GAG chains in the lesion of spinal cord. So presence of glycosylation sites in the bacterial sequence is supposed the barrier that preventing ChABC secretion from mammalian cells. We intend to mutate the key N-glycosylation sites of the bacterial ChABC sequence and transduce it into BMSCs by lentivirus vector. The modified BMSCs are expected to promote axon regeneration through multiple mechanisms, providing sustained ChABC and neurotrophic factors, as well as filling in the cavities formed post-trauma. The transduced BMSCs with gene mutated in key glycosylation sites in the present hypothesis provide a promising strategy to promote axon regeneration.

Spatiotemporal profile of N-cadherin expression in the mossy fiber sprouting and synaptic plasticity following seizures

Recurrent seizures can induce mossy fiber sprouting (MFS), of the hippocampal dentate gyrus, and synaptic reorganization in mature brain. This changes local circuits and provides a structural basis for epileptogenesis in the hippocampus. However, the mechanisms of MFS and synaptic reorganization still remain unclear. Neural-cadherin (N-cadherin), a calcium adhesion molecule, plays an important role in neurite outgrowth, pathfinding, and synaptic specificity of early central nervous system development. It is unknown whether N-cadherin is involved in MFS after seizures in mature brain. To further examine the correlation between MFS and N-cadherin expression, we separately labeled MFS and N-cadherin with Timm staining and antibody in adult rats after status epilepticus (SE). Timm staining revealed that MFS is observed in the inner molecular layer of dentate gyrus of rats 2 and 4 weeks after SE. The observed MFS migrated from the hilus to the granule cell layer, gradually extending axons into the inner molecular layer to form an intense band. Immunohistochemical staining of N-cadherin revealed that the upregulated expression of N-cadherin was concentrated in the position of mossy fiber axonal sprouts of rats 1-4 weeks after SE, and that it was earlier than MFS. The spatial and temporal distribution consistence of N-cadherin and Timm staining supported the correlation that exists between N-cadherin expression and the process of aberrant MFS. This result suggests that N-cadherin may be involved in the pathfinding and synaptic specificity of MFS in mature brain after seizures, and can play an important role in the targeted growth of mossy fibers.

Impaired neurovascular repair in subjects with diabetes following experimental intracutaneous axotomy

Diabetic complications and vascular disease are closely intertwined. Diabetes mellitus is a well-established risk factor for both large and small vessel vascular changes, and conversely other vascular risk factors confer increased risk for diabetic complications such as peripheral neuropathy, nephropathy and retinopathy. Furthermore, axons and blood vessels share molecular signals for purposes of navigation, regeneration and terminal arborizations. We examined blood vessel, Schwann cell and axonal regeneration using validated axotomy models to study and compare patterns and the relationship of regeneration among these different structures. Ten subjects with diabetes mellitus complicated by neuropathy and 10 healthy controls underwent 3 mm distal thigh punch skin biopsies to create an intracutaneous excision axotomy followed by a concentric 4-mm overlapping biopsy at different time points. Serial sections were immunostained against a pan-axonal marker (PGP9.5), an axonal regenerative marker (GAP43), Schwann cells (p75) and blood vessels (CD31) to visualize regenerating structures in the dermis and epidermis. The regenerative and collateral axonal sprouting rates, blood vessel growth rate and Schwann cell density were quantified using established stereology techniques. Subjects also underwent a chemical 'axotomy' through the topical application of capsaicin, and regenerative sprouting was assessed by the return of intraepidermal nerve fibre density through regenerative regrowth. In the healed 3 mm biopsy sites, collateral and dermal regenerative axonal sprouts grew into the central denervated area in a stereotypic pattern with collateral sprouts growing along the dermal-epidermal junction while regenerative dermal axons, blood vessels and Schwann cells grew from their transected proximal stumps into the deep dermis. Vessel growth preceded axon and Schwann cell migration into the denervated region, perhaps acting as scaffolding for axon and Schwann cell growth. In control subjects, Schwann cell growth was more robust and extended into the superficial dermis, while among subjects with diabetes mellitus, Schwann tubes appeared atrophic and were limited to the mid-dermis. Rates of collateral (P=0.0001), dermal axonal regenerative sprouting (P=0.02), Schwann cell migration (P<0.05) and blood vessel growth (P=0.002) were slower among subjects with diabetes mellitus compared with control subjects. Regenerative deficits are a common theme in diabetes mellitus and may underlie the development of neuropathy. We observed that blood vessel growth recapitulated the pattern seen in ontogeny and preceded regenerating nerve fibres, suggesting that enhancement of blood vessel growth might facilitate axonal regeneration. These models are useful tools for the efficient investigation of neurotrophic and regenerative drugs, and also to explore factors that may differentially affect axonal regeneration.

Poor functional recovery and muscle polyinnervation after facial nerve injury in fibroblast growth factor-2 −/− mice can be improved by manual stimulation of denervated vibrissal muscles

Functional recovery following facial nerve injury is poor. Adjacent neuromuscular junctions (NMJs) are “bridged” by terminal Schwann cells and numerous regenerating axonal sprouts. We have recently shown that manual stimulation (MS) restores whisking function and reduces polyinnervation of NMJs. Furthermore, MS requires both insulin-like growth factor-1 (IGF-1) and brain-derived neurotrophic factor (BDNF). Here, we investigated whether fibroblast growth factor-2 (FGF-2) was also required for the beneficial effects of MS. Following transection and suture of the facial nerve (facial-facial anastomisis, FFA) in homozygous mice lacking FGF-2 (FGF-2 −/−), vibrissal motor performance and the percentage of poly-innervated NMJ were quantified. In intact FGF-2 −/− mice and their wildtype (WT) counterparts, there were no differences in amplitude of vibrissal whisking (about 50°) or in the percentage of polyinnervated NMJ (0%). After 2 months FFA and handling alone (i.e. no MS), the amplitude of vibrissal whisking in WT-mice decreased to 22±3°. In the FGF-2 −/− mice, the amplitude was reduced further to 15±4°, that is, function was significantly poorer. Functional deficits were mirrored by increased polyinnervation of NMJ in WT mice (40.33±2.16%) with polyinnervation being increased further in FGF-2 −/− mice (50.33±4.33%). However, regardless of the genotype, MS increased vibrissal whisking amplitude (WT: 33.9°±7.7; FGF-2 −/−: 33.4°±8.1) and concomitantly reduced polyinnervation (WT: 33.9%±7.7; FGF-2 −/−: 33.4%±8.1) to a similar extent. We conclude that, whereas lack of FGF-2 leads to poor functional recovery and target reinnervation, MS can nevertheless confer some functional benefit in its absence.

The effects of low-dose radiation in the treatment of sciatic nerve injury in rats

Peripheral nerve regeneration is often blocked by scar formation and misdirection of axon sprouts. The aim of this study is to evaluate electrophysiological and histopathological effects of low-dose radiation therapy on the prevention of intraneural scar formation in peripheral nerve injury. In this experimental study, twenty rats were randomly divided into two groups. Left sciatic nerves were exposed and clipped by temporary aneurysm clip for 5 minutes in both groups. In all animals, electrophysiological recordings were performed between 22-24 hours after sciatic nerve injury. The control group was not given any treatment. In the experimental group, 700 cGy low-dose radiation was administered on the left sciatic nerves 24 hours after clipping. Six weeks after injury, electrophysiological recordings were performed in both groups and animals were sacrificed to evaluate the injured nerves histopathologically. We observed that low-dose radiotherapy increased the amplitude and improved latency measurements in electrophysiological examinations. Histopathologically, more axonal degeneration and vacuolization was observed in the control group comparing with the experimental group. Endoneural space increased slightly more in the control group than the experimental group. It was observed that low-dose radiotherapy may prevent intraneural scar formation and may improve electrophysiological recovery in sciatic nerve injury performed in rats.

Grafted Human Embryonic Progenitors Expressing Neurogenin-2 Stimulate Axonal Sprouting and Improve Motor Recovery after Severe Spinal Cord Injury

BackgroundSpinal cord injury (SCI) is a widely spread pathology with currently no effective treatment for any symptom. Regenerative medicine through cell transplantation is a very attractive strategy and may be used in different non-exclusive ways to promote functional recovery. We investigated functional and structural outcomes after grafting human embryonic neural progenitors (hENPs) in spinal cord-lesioned rats.Methods and Principal FindingsWith the objective of translation to clinics we have chosen a paradigm of delayed grafting, i.e., one week after lesion, in a severe model of spinal cord compression in adult rats. hENPs were either naïve or engineered to express Neurogenin 2 (Ngn2). Moreover, we have compared integrating and non-integrating lentiviral vectors, since the latter present reduced risks of insertional mutagenesis. We show that transplantation of hENPs transduced to express Ngn2 fully restore weight support and improve functional motor recovery after severe spinal cord compression at thoracic level. This was correlated with partial restoration of serotonin innervations at lumbar level, and translocation of 5HT1A receptors to the plasma membrane of motoneurons. Since hENPs were not detectable 4 weeks after grafting, transitory expression of Ngn2 appears sufficient to achieve motor recovery and to permit axonal regeneration. Importantly, we also demonstrate that transplantation of naïve hENPs is detrimental to functional recovery.Conclusions and Significance Transplantation and short-term survival of Ngn2-expressing hENPs restore weight support after SCI and partially restore serotonin fibers density and 5HT1A receptor pattern caudal to the lesion. Moreover, grafting of naïve-hENPs was found to worsen the outcome versus injured only animals, thus pointing to the possible detrimental effect of stem cell-based therapy per se in SCI. This is of major importance given the increasing number of clinical trials involving cell grafting developed for SCI patients.

Selective Loss and Axonal Sprouting of GABAergic Interneurons in the Sclerotic Hippocampus Induced by LiCl–Pilocarpine

ABSTRACTIn this study, we performed immunohistochemistry for somatostatin (SS), neuropeptide Y (NPY), and parvalbumin (PV) in LiCl–pilocarpine-treated rats to observe quantitative changes and axonal sprouting of GABAergic interneurons in the hippocampus, especially in the sclerotic hippocampus. Fluoro-Jade B (FJB) was performed to detect the specific degeneration of GABAergic interneurons. Compared with age-matched control rats, there were fewer SS/NPY/PV-immunoreactive (IR) interneurons in the hilus of the sclerotic hippocampus in pilocarpine-treated rats; hilar dentritic inhibitory interneurons were most vulnerable. FJB stain revealed degeneration was evident at 2 months after status epilepticus. Some SS-IR and NPY-IR interneurons were also stained for FJB, but there was no evidence of degeneration of PV-IR interneurons. Axonal sprouting of GABAergic interneurons was present in the hippocampus of epileptic rats, and a dramatic increase of SS-IR fibers was observed throughout all layers of CA1 region in the sclerotic hippocampus. These results confirm selective loss and degeneration of a specific subset of GABAergic interneurons in specific subfields of the hippocampus. Axonal sprouting of inhibitory GABAergic interneurons, especially numerous increase of SS-IR neutrophils within CA1 region of the sclerotic hippocampus, may constitute the aberrant inhibitory circum and play a significant role in the generation and compensation of temporal lobe epilepsy.

Axon Sprouting and Synaptic Reorganization of GABAergic Interneurons: A Focused Look at a General Question

Surviving Hilar Somatostatin Interneurons Enlarge, Sprout Axons, and Form New Synapses with Granule Cells in a Mouse Model of Temporal Lobe Epilepsy. Zhang W, Yamawaki R, Wen X, Uhl J, Diaz J, Prince DA, Buckmaster PS. J Neurosci 2009;29(45):14247–14256. In temporal lobe epilepsy, seizures initiate in or near the hippocampus, which frequently displays loss of neurons, including inhibitory interneurons. It is unclear whether surviving interneurons function normally, are impaired, or develop compensatory mechanisms. We evaluated GABAergic interneurons in the hilus of the dentate gyrus of epileptic pilocarpine-treated GIN mice, specifically a subpopulation of somatostatin interneurons that expresses enhanced green fluorescence protein (GFP). GFP-immunocytochemistry and stereological analyses revealed substantial loss of GFP-positive hilar neurons (GPHNs) but increased GFP-positive axon length per dentate gyrus in epileptic mice. Individual biocytin-labeled GPHNs in hippocampal slices from epileptic mice also had larger somata, more axon in the molecular layer, and longer dendrites than controls. Dual whole-cell patch recording was used to test for monosynaptic connections from hilar GPHNs to granule cells. Unitary inhibitory postsynaptic currents (uIPSCs) recorded in control and epileptic mice had similar average rise times, amplitudes, charge transfers, and decay times. However, the probability of finding monosynaptically connected pairs and evoking uIPSCs was 2.6 times higher in epileptic mice compared to controls. Together, these findings suggest that surviving hilar somatostatin interneurons enlarge, extend dendrites, sprout axon collaterals in the molecular layer, and form new synapses with granule cells. These epilepsy-related changes in cellular morphology and connectivity may be mechanisms for surviving hilar interneurons to inhibit more granule cells and compensate for the loss of vulnerable interneurons.

Sprouting capacity of lumbar motoneurons in normal and hemisected spinal cords of the rat

Nerve sprouting to reinnervate partially denervated muscles is important in several disease and injury states. To examine the effectiveness of sprouting of active and inactive motor units (MUs) and the basis for a limit to sprouting, one of three rat lumbar spinal roots was cut under normal conditions and when the spinal cord was hemisected at T12. Muscle and MU isometric contractile forces were recorded and muscle fibres in glycogen-depleted single muscle units enumerated 23 to 380 days after surgery. Enlargement of intact MUs by sprouting was effective in compensating for up to 80% loss of innervation. For injuries that removed >70-80% of the intact MUs, muscle contractile force and weight dropped sharply. For partial denervation of <70%, all MUs increased contractile force by the same factor in both normally active muscles and muscles whose activity was reduced by T12 hemisection. Direct measurements of MU size by counting glycogen-depleted muscle fibres in physiologically and histochemically defined muscle units, provided direct evidence for a limit in MU size, whether or not the activity of the muscles was reduced by spinal cord hemisection. Analysis of spatial distribution of muscle fibres within the outer boundaries of the muscle unit demonstrated a progressive increase in fibres within the territory to the limit of sprouting when most of the muscle unit fibres were adjacent to each other. We conclude that the upper limit of MU enlargement may be explained by the reinnervation of denervated muscle fibres by axon sprouts within the spatial territory of the muscle unit, formerly distributed in a mosaic pattern.

Sprouting of corticospinal tract axons from the contralateral hemisphere into the denervated side of the spinal cord is associated with functional recovery in adult rat after traumatic brain injury and erythropoietin treatment

Erythropoietin (EPO) promotes functional recovery after traumatic brain injury (TBI). This study was designed to investigate whether EPO treatment promotes contralateral corticospinal tract (CST) plasticity in the spinal cord in rats after TBI. Biotinylated dextran amine (BDA) was injected into the right sensorimotor cortex to anterogradely label the CST. TBI was induced by controlled cortical impact over the left parietal cortex immediately after BDA injections. EPO (5000 U/kg) or saline was administered intraperitoneally at Days 1, 2, and 3 post-injury. Neurological function was assessed using a modified neurological severity score (mNSS) and footfault tests. Animals were sacrificed 35 days after injury and brain sections stained for histological analysis. Compared to the saline treatment, EPO treatment significantly improved sensorimotor functional outcome (lower mNSS and reduced footfaults) from Days 7 to 35 post-injury. TBI alone significantly stimulated contralateral CST axon sprouting toward the denervated gray matter of the cervical and lumbar spinal cord; however, EPO treatment further significantly increased the axon sprouting in TBI rats although EPO treatment did not significantly affect axon sprouting in sham animals. The contralesional CST sprouting was highly and positively correlated with sensorimotor recovery after TBI. These data demonstrate that CST fibers originating from the contralesional intact cerebral hemisphere are capable of sprouting into the denervated spinal cord after TBI and EPO treatment, which may at least partially contribute to functional recovery.

Recovery of whisking function after manual stimulation of denervated vibrissal muscles requires brain-derived neurotrophic factor and its receptor tyrosine kinase B

Functional recovery following facial nerve injury is poor. Neuromuscular junctions (NMJs) are “bridged” by terminal Schwann cells and numerous regenerating axonal sprouts. We have shown that this poly-innervation of NMJs can be reduced by manual stimulation (MS) with restoration of whisking function. In addition, we have recently reported that insulin-like growth factor-1 (IGF-1) is required to mediate the beneficial effects of MS. Here we extend our findings to brain derived neurotrophic factor (BDNF). We then examined the effect of MS after facial-facial anastomosis (FFA) in heterozygous mice deficient in BDNF (BDNF+/−) or in its receptor TrkB (TrkB+/−). We quantified vibrissal motor performance and the percentage of NMJ bridged by S100-positive terminal Schwann cells. In intact BDNF+/− or TrkB+/− mice and their wild type (WT) littermates, there were no differences in vibrissal whisking nor in the percentage of bridged NMJ (0% in each genotype). After FFA and handling alone (i.e. no MS) in WT animals, vibrissal whisking amplitude was reduced (60% lower than intact) and the percentage of bridged NMJ increased (27% more than intact). MS improved both the amplitude of vibrissal whisking (not significantly different from intact) and the percentage of bridged NMJ (11% more than intact). After FFA and handling in BDNF+/− or TrkB+/− mice, whisking amplitude was again reduced (53% and 60% lower than intact) and proportion of bridged NMJ increased (24% and 29% more than intact). However, MS failed to improve outcome in both heterozygous strains (whisking amplitude 55% and 58% lower than intact; proportion of bridged NMJ 27% and 18% more than intact). We conclude that BDNF and TRkB are required to mediate the effects of MS on target muscle reinnervation and recovery of whisking function.

Lysophosphatidic acid influences initial neuronal polarity establishment

Neuronal polarity is specified by neurite determination into axons and dendrites. Its establishment requires both extrinsic signals, which regulate axon and dendrite development through repulsive or attractive actions, and intrinsic cellular mechanisms, which include rearrangement and selective transport of the cytoskeleton and localization of intracellular organelles. However, it remains unclear how extrinsic signals activate intrinsic cellular mechanisms to establish neuronal polarity. Here, we examine the effects of lysophosphatidic acid (LPA), a signaling lipid that induces cytoskeletal rearrangement in neuronal cells, on neuronal polarity establishment. In hippocampal neuronal cultures where a concentration gradient of LPA was formed, the bases of axons were located predominantly at the side distal to the LPA source. Furthermore, Golgi apparatus were also positioned distally as early as 1 h after exposure to the LPA source, suggesting that LPA signaling is involved in the initial determination of the area where an axon sprouts, and thereby the establishment of neuronal polarity.

Focal Damage to the Adult Rat Neocortex Induces Wound Healing Accompanied by Axonal Sprouting and Dendritic Structural Plasticity

Accumulating evidence indicates that damage to the adult mammalian brain evokes an array of adaptive cellular responses and may retain a capacity for structural plasticity. We have investigated the cellular and architectural alterations following focal experimental brain injury, as well as the specific capacity for structural remodeling of neuronal processes in a subset of cortical interneurons. Focal acute injury was induced by transient insertion of a needle into the neocortex of anesthetized adult male Hooded-Wistar rats and thy1 green fluorescent protein (GFP) mice. Immunohistochemical, electron microscopy, and bromodeoxyuridine cell proliferation studies demonstrated an active and evolving response of the brain to injury, indicating astrocytic but not neuronal proliferation. Immunolabeling for the neuron-specific markers phosphorylated neurofilaments, α-internexin and calretinin at 7 days post injury (DPI) indicated phosphorylated neurofilaments and α-internexin but not calretinin immunopositive axonal sprouts within the injury site. However, quantitative studies indicated a significant realignment of horizontally projecting dendrites of calretinin-labeled interneurons at 14 DPI. This remodeling was specific to calretinin immunopositive interneurons and did not occur in a subpopulation of pyramidal neurons expressing GFP in the injured mouse cortex. These data show that subclasses of cortical interneurons are capable of adaptive structural remodeling.

Motor Axonal Sprouting and Neuromuscular Junction Loss in an Animal Model of Charcot-Marie-Tooth Disease

Muscle weakness in Charcot-Marie-Tooth Type 1A disease (CMT1A) caused by mutations in peripheral myelin protein 22 (PMP22) has been attributed to an axonopathy that results in denervation and muscle atrophy. The underlying pathophysiological mechanisms involved are not understood. We investigated motor performance, neuromuscular junctions (NMJs), physiological parameters, and muscle morphometry of PMP22 transgenic mice. Neuromuscular junctions were progressively lost in hindlimb muscles of PMP22 transgenic mice, but their motor performance did not completely deteriorate during the observation period. There was considerable variability, including in laterality, in deficits among the animals. Cross-sectional areas and mean fiber size measurements indicated variable myofiber atrophy in hindlimb muscles. There was substantial concomitant axonal sprouting, and loss of neuromuscular junctions was inversely correlated with the accumulated length of axonal branches. Synaptic transmission studied in isolated nerve/muscle preparations indicated variable partial muscle denervation. Acetylcholine sensitivity was higher in the mutant muscles, and maximum tetanic force evoked by direct or indirect stimulation, specific force, and wet weights were markedly reduced in some mutant muscles. In summary, there is partial muscle denervation, and axons may retain some regenerative capacity but fail to reinnervate muscles in PMP22 transgenic mice.

Human Neural Stem Cell Grafts Modify Microglial Response and Enhance Axonal Sprouting in Neonatal Hypoxic–Ischemic Brain Injury

Hypoxic-ischemic (HI) brain injury in newborn infants represents a major cause of cerebral palsy, development delay, and epilepsy. Stem cell-based therapy has the potential to rescue and replace the ischemic tissue caused by HI and to restore function. However, the mechanisms by which stem cell transplants induce functional recovery are yet to be elucidated. In the present study, we sought to investigate the efficacy of human neural stem cells derived from human embryonic stem cells in a rat model of neonatal HI and the mechanisms enhancing brain repair. The human neural stem cells were genetically engineered for in vivo molecular imaging and for postmortem histological tracking. Twenty-four hours after the induction of HI, animals were grafted with human neural stem cells into the forebrain. Motor behavioral tests were performed the fourth week after transplantation. We used immunocytochemistry and neuroanatomical tracing to analyze neural differentiation, axonal sprouting, and microglia response. Treatment-induced changes in gene expression were investigated by microarray and quantitative polymerase chain reaction. Bioluminescence imaging permitted real time longitudinal tracking of grafted human neural stem cells. HI transplanted animals significantly improved in their use of the contralateral impeded forelimb and in the Rotorod test. The grafts showed good survival, dispersion, and differentiation. We observed an increase of uniformly distributed microglia cells in the grafted side. Anterograde neuroanatomical tracing demonstrated significant contralesional sprouting. Microarray analysis revealed upregulation of genes involved in neurogenesis, gliogenesis, and neurotrophic support. These results suggest that human neural stem cell transplants enhance endogenous brain repair through multiple modalities in response to HI.

Recovery of whisking function promoted by manual stimulation of the vibrissal muscles after facial nerve injury requires insulin-like growth factor 1 (IGF-1)

Recently, we showed that manual stimulation (MS) of denervated vibrissal muscles enhanced functional recovery following facial nerve cut and suture (FFA) by reducing poly-innervation at the neuro-muscular junctions (NMJ). Although the cellular correlates of poly-innervation are established, with terminal Schwann cells (TSC) processes attracting axon sprouts to “bridge” adjacent NMJ, molecular correlates are poorly understood. Since quantitative RT-PCR revealed a rapid increase of IGF-1 mRNA in denervated muscles, we examined the effect of daily MS for 2 months after FFA in IGF-1 +/− heterozygous mice; controls were wild-type (WT) littermates including intact animals. We quantified vibrissal motor performance and the percentage of NMJ bridged by S100-positive TSC. There were no differences between intact WT and IGF-1 +/− mice for vibrissal whisking amplitude (48° and 49°) or the percentage of bridged NMJ (0%). After FFA and handling alone (i.e. no MS) in WT animals, vibrissal whisking amplitude was reduced (60% lower than intact) and the percentage of bridged NMJ increased (42% more than intact). MS improved both the amplitude of vibrissal whisking (not significantly different from intact) and the percentage of bridged NMJ (12% more than intact). After FFA and handling in IGF-1 +/− mice, the pattern was similar (whisking amplitude 57% lower than intact; proportion of bridged NMJ 42% more than intact). However, MS did not improve outcome (whisking amplitude 47% lower than intact; proportion of bridged NMJ 40% more than intact). We conclude that IGF-I is required to mediate the effects of MS on target muscle reinnervation and recovery of whisking function.

Earthworm Extracts Facilitate PC12 Cell Differentiation and Promote Axonal Sprouting in Peripheral Nerve Injury

The present study provides in vitro and in vivo evaluations of earthworm (Pheretima aspergilum) on peripheral nerve regeneration. In the in vitro study, we found the earthworm (EW) water extracts caused a marked enhancement of the nerve growth factor-mediated neurite outgrowth from PC12 cells as well as the expressions of growth associated protein 43 and synapsin I. In the in vivo study, silicone rubber chambers filled with EW extracts were used to bridge a 10 mm sciatic nerve defect in rats. Eight weeks after implantation, the group receiving EW extracts had a much higher success percentage of regeneration (90%) compared to the control (60%) receiving the saline. In addition, quantitative histology of the successfully regenerated nerves revealed that myelinated axons in EW group at 31.25 microg/ml was significantly more than those in the controls (p < 0.05). These results showed that EW extracts can be a potential growth-promoting factor on regenerating peripheral nerves.

Peripheral facial nerve axotomy in mice causes sprouting of motor axons into perineuronal central white matter: Time course and molecular characterization

Generation of new axonal sprouts plays an important role in neural repair. In the current study, we examined the appearance, composition and effects of gene deletions on intrabrainstem sprouts following peripheral facial nerve axotomy. Axotomy was followed by the appearance of galanin+ and calcitonin gene-related peptide (CGRP)+ sprouts peaking at day 14, matching both large, neuropeptide+ subpopulations of axotomized facial motoneurons, but with CGRP+ sprouts considerably rarer. Strong immunoreactivity for vesicular acetylcholine transporter (VAChT) and retrogradely transported MiniRuby following its application on freshly cut proximal facial nerve stump confirmed their axotomized motoneuron origin; the sprouts expressed CD44 and alpha7beta1 integrin adhesion molecules and grew apparently unhindered along neighboring central white matter tracts. Quantification of the galanin+ sprouts revealed a stronger response following cut compared with crush (day 7–14) as well as enhanced sprouting after recut (day 8 + 6 vs. 14; 14 + 8 vs. 22), arguing against delayed appearance of sprouting being the result of the initial phase of reinnervation. Sprouting was strongly diminished in brain Jun-deficient mice but enhanced in alpha7 null animals that showed apparently compensatory up-regulation in beta1, suggesting important regulatory roles for transcription factors and the sprout-associated adhesion molecules. Analysis of inflammatory stimuli revealed a 50% reduction 12–48 hours following systemic endotoxin associated with neural inflammation and a tendency toward more sprouts in TNFR1/2 null mutants (P = 10%) with a reduced inflammatory response, indicating detrimental effects of excessive inflammation. Moreover, the study points to the usefulness of the facial axotomy model in exploring physiological and molecular stimuli regulating central sprouting. J. Comp. Neurol. 518:699–721, 2010. © 2009 Wiley-Liss, Inc.