Transplantation of bone-marrow-derived cells into a nerve guide resulted in transdifferentiation into Schwann cells and effective regeneration of transected mouse sciatic nerve.

Peripheral nerves possess the capacity of self-regeneration after traumatic injury. Nevertheless, the functional outcome after peripheral-nerve regeneration is often poor, especially if the nerve injuries occur far from their targets. Aiming to optimize axon regeneration, we grafted bone-marrow-derived cells (BMDCs) into a collagen-tube nerve guide after transection of the mouse sciatic nerve. The control group received only the culture medium. Motor function was tested at 2, 4, and 6 weeks after surgery, using the sciatic functional index (SFI), and showed that functional recovery was significantly improved in animals that received the cell grafts. After 6 weeks, the mice were anesthetized, perfused transcardially, and the sciatic nerves were dissected and processed for transmission electron microscopy and light microscopy. The proximal and distal segments of the nerves were compared, to address the question of improvement in growth rate; the results revealed a maintenance and increase of nerve regeneration for both myelinated and non-myelinated fibers in distal segments of the experimental group. Also, quantitative analysis of the distal region of the regenerating nerves showed that the numbers of myelinated fibers, Schwann cells (SCs) and g-ratio were significantly increased in the experimental group compared to the control group. The transdifferentiation of BMDCs into Schwann cells was confirmed by double labeling with S100/and Hoechst staining. Our data suggest that BMDCs transplanted into a nerve guide can differentiate into SCs, and improve the growth rate of nerve fibers and motor function in a transected sciatic-nerve model.

Distribution of inducible nitric oxide synthase and tumor necrosis factor-alpha in the peripheral nervous system of Lewis rats during ascending paresis and spontaneous recovery from experimental autoimmune neuritis.

BACKGROUND: Inducible nitric oxide synthase (iNOS) and tumor necrosis factor-alpha (TNF-alpha) are pleiotropic molecules with widespread action in autoimmune diseases. OBJECTIVE: This study characterizes the distribution of iNOS and TNF-alpha in the spinal nerve roots, dorsal root ganglia and sciatic nerve of Lewis rats during experimental autoimmune neuritis (EAN). METHODS: Macrophages and neutrophils were identified by immunofluorescence as cellular sources of iNOS and TNF-alpha at various stages of EAN induced by synthetic peptide 26. RESULTS: As the disease progressed, iNOS- and TNF-alpha-bearing cells gradually infiltrated the cauda equina, dorsal root ganglia, Th12-L3 spinal roots, and the sciatic nerve. A severer EAN profile developed when more iNOS- and TNF-alpha-bearing cells were present, and the recovery from EAN was related to the disappearance of these cells and the regeneration of nerve fibers. CONCLUSIONS: This is the first report to show iNOS- and TNF-alpha-immunoreactive cells in dorsal root ganglia during EAN, suggesting an underlying pathology for the neuropathic pain behavior in EAN. Our results suggest that the cells bearing iNOS and TNF-alpha in the different parts of the peripheral nervous system are involved in the development of the clinical signs observed at each stage of EAN.

Ciliary neurotrophic factor fused to a protein transduction domain retains full neuroprotective activity in the absence of cytokine-like side effects.

Ciliary neurotrophic factor (CNTF) is a multifunctional cytokine that can regulate the survival and differentiation of many types of developing and adult neurons. CNTF prevents the degeneration of motor neurons after axotomy and in mouse mutant progressive motor neuronopathy, which has encouraged trials of CNTF for human motor neuron disease. Given systemically, however, CNTF causes severe side effects, including cachexia and a marked immune response, which has limited its clinical application. The present work describes a novel approach for administering recombinant human CNTF (rhCNTF) while conserving neurotrophic activity and avoiding deleterious side effects. rhCNTF was fused to a protein transduction domain derived from the human immunodeficiency virus-1 TAT (transactivator) protein. The resulting fusion protein (TAT-CNTF) crosses the plasma membrane within minutes and displays a nuclear localization. TAT-CNTF was equipotent to rhCNTF in supporting the survival of cultured chicken embryo dorsal root ganglion neurons. Local or subcutaneous administration of TAT-CNTF, like rhCNTF rescued motor neurons from death in neonatal rats subjected to sciatic nerve transection. In contrast to subcutaneous rhCNTF, which caused a 20-30% decrease in body weight in neonatal rats between postnatal days 2 and 7 together with a considerable fat mobilization in brown adipose tissue, TAT-CNTF lacked such side effects. Together, these results indicate that rhCNTF fused with the protein transduction domain/TAT retains neurotrophic activity in the absence of CNTFs cytokine-like side effects and may be a promising candidate for the treatment of motor neuron and other neurodegenerative diseases.

Ciliary neurotrophic factor promotes survival of neonatal rat islets via the BCL-2 anti-apoptotic pathway.

Ciliary neurotrophic factor (CNTF) belongs to the cytokine family and increases neuron differentiation and/or survival. Pancreatic islets are richly innervated and express receptors for nerve growth factors (NGFs) and may undergo neurotypic responses. CNTF is found in pancreatic islets and exerts paracrine effects in neighboring cells. The aim of this study was to investigate possible effects of CNTF on neonatal rat pancreatic islet differentiation and/or survival. For this purpose, we isolated pancreatic islets from neonatal rats (1-2 days old) by the collagenase method and cultured for 3 days in RPMI medium with (CNTF) or without (CTL) 1 nM CNTF. Thereafter, glucose-stimulated insulin secretion (RIA), general metabolism by (NAD(P)H production; MTS), glucose metabolism ((14)CO(2) production), gene (RT-PCR), protein expression (western blotting), caspase-3 activity (Asp-Glu-Val-Asp (DEVD)), and apoptosis (DNA fragmentation) were analyzed. Our results showed that CNTF-treated islets demonstrated reduced glucose-induced insulin secretion. CNTF treatment did not affect glucose metabolism, as well as the expression of mRNAs and proteins that are crucial for the secretory process. Conversely, CNTF significantly increased mRNA and protein levels related to cell survival, such as Cx36, PAX4, and BCL-2, reduced caspase-3 activity, and islet cells apoptosis, suggesting that CNTF does not affect islet cell differentiation and, instead, acts as a survival factor reducing apoptosis by increasing the expression of the anti-apoptotic BCL-2 protein and decreasing caspase-3 activity.

Nitric oxide and TNFalpha effects in experimental autoimmune encephalomyelitis demyelination.

The involvement of inducible nitric oxide synthase (iNOS), which plays various roles in the progression of autoimmune diseases, was studied in iNOS knockout (KO) mice and wild-type (WT) controls with respect to experimental autoimmune encephalomyelitis (EAE). The iNOS (KO) mice presented a less severe form of the disease than the WT control mice. Although the levels of TNFalpha decreased in the periphery in both groups, an increase in the number of TNFalpha-positive cells was detected in the central nervous system during the acute phase of EAE in the WT mice, but not in the KO mice. These findings suggest that NO and TNFalpha contribute to the pathogenesis of acute EAE.

Viability of nerve grafts preseved in different storage medium

We compared the structural features of nerve segments stored in two different solutions previous and after autologous transplantation. Male Wistar rats were divided into groups to obtain normal tibial nerves, freshly transplanted nerves, and nerves stored in Wisconsin/Belzer or Collins solution for 24 or 72 h at 4ºC and transplanted. Stored and transplanted segments were processed for morphologic and morphometric analysis. The cross-sections of segments stored in Wisconsin/Belzer and Collins solution presented aspects similar to that of normal nerves. The density of large-caliber myelinated axons was higher in grafts stored in Wisconsin/Belzer solution than in those preserved in Collins solution. But the density of myelinated axons regenerated through these grafts was around 80% to that registered in the fresh and Wisconsin/Belzer preserved grafts. Moreover, no significant differences in the morphometric parameters were observed between groups. Our data confirm the efficacy of Wisconsin/Belzer to nerve graft preservation and stimulate more detailed physiological, biochemical and molecular studies to rationalize the employment of less expensive and handful storage solutions for short term preservation of peripheral nerve grafts.

Bax and Bcl-2 expression and TUNEL labeling in lumbar enlargement of neonatal rats after sciatic axotomy and melatonin treatment.

Peripheral axotomy in neonatal rats induces neuronal death. We studied the anti-apoptotic protein Bcl-2 and cell death promoter Bax in spinal cord of neonatal rats after sciatic transection and treatment with melatonin, a neuroprotective substance. Pups were unilaterally axotomized at P2 and received melatonin (1 mg/kg; sc) or vehicle 1 h prior to lesion, immediately after, at 1 h, 2 h and then once daily. Rats were sacrificed at 3 h, 6 h, 24 h, 72 h and 5 days postaxotomy. Intact animals were used as controls. Lumbar enlargement was processed for Nissl staining, immunohistochemistry and RT-PCR for Bax or Bcl-2 and TUNEL reaction. Motoneurons (MN) of lesioned (L) and normal (N) sides were counted, and MN survival ratio (MSR=L/N) was calculated. Bax and Bcl-2 showed cytoplasmic immunoreactivity (IR). Bax IR was noticeable in small cells but less evident in MN. In unlesioned pups, some Bax-positive small cells (B+) and TUNEL-positive nuclei (T+) were mainly seen in the dorsal horn. In lesioned animals given vehicle, Bax mRNA levels and numbers of B+ and T+ were increased in comparison with intact controls at 24 h postaxotomy. The basal IR for Bax in MN was not changed by axotomy. Bcl-2 IR was noted in all cells and, like Bcl-2 mRNA, was unaltered after lesion. Melatonin reduced MN loss at 24 h, 72 h and 5 days and T+ at 24 h after lesion but did not interfere with Bax or Bcl-2 expression. These results suggest that (1) sciatic transection at P2 increases Bax mRNA and the amount of B+ and T+ in the lumbar enlargement, (2) Bax IR in immature MN is not altered by axotomy and (3) melatonin protects MN and dorsal horn cells through a mechanism independent of Bax and Bcl-2.

Bone marrow stromal cells and resorbable collagen guidance tubes enhance sciatic nerve regeneration in mice.

We evaluated peripheral nerve regeneration using a tubular nerve guide of resorbable collagen filled with either bone marrow-derived cells (BMDCs) in Dulbecco's cell culture medium (DMEM) or with DMEM alone (control). The control group received just the culture medium (vehicle). The left sciatic nerves of ten isogenic mice were transected and the tubular nerve guides were sutured to the end of the proximal and distal nerve stumps. Motor function was tested at 2, 4 and 6 weeks after surgery using the walking track test. The pawprints were analyzed and the print lengths (PL) were measured to evaluate functional recovery. After 6 weeks, mice were anesthetized, perfused transcardially with fixative containing aldehydes, and the sciatic nerves and tubes were dissected and processed for scanning and transmission electron microscopy. Scanning electron microscopy of the collagen tube revealed that the tube wall became progressively thinner after surgery, proving that the tube can be resorbed in vivo. Quantitative analysis of the regenerating nerves showed that the number of myelinated fibers and the myelin area were significantly increased in the experimental group. Also, motor function recovery was faster in animals that received the cell grafts. These results indicate that the collagen tube filled with BMDCs provided an adequate and favorable environment for the growth and myelination of regenerating axons compared to the collagen tube alone.

Naturally supraorganized collagen increases axonal regeneration after tubulization repair

After axotomy, regeneration can be enhanced by bridging the transected nerve with a biocompatible tube, and the effect of trophic substances or molecules from the extracellular matrix can be investigated by filling the prosthesis. In this study, we assessed the importance of the molecular organization and aggregational state of collagen type I in axonal regeneration and guidance. Two types of collagen were used, namely, a collagen gel derived from bovine tendon that displays supraorganization after extrusion, and collagen from rat tail which does not self-organize under such conditions. Adult male Wistar rats were divided into four groups. In the first group (n=3), the polyethylene tube was filled with bovine collagen, while in the second (n=3), the prosthesis was filled with rat-derived collagen. In the third group (n=3), the tube was left empty, and the fourth group (n=3), consisted of unoperated rats. Six weeks after tubulization, the number of axons was significantly higher with bovine collagen than with rat collagen (7,661 ± 1,018 versus 4,110 ± 1,027, p<0.05), as was the degree of implant absorption. These results support the hypothesis that the use of extracellular matrix substances that self-assembly in an organized pattern can enhance nerve regeneration.

Wallerian degeneration in C57BL/6J and A/J mice: differences in time course of neurofilament and myelin breakdown, macrophage recruitment and iNOS expression.

The lower regeneration potential reported for C57BL/6J mice strain after peripheral nerve lesion may result from alterations in crucial events during Wallerian degeneration. We analysed neurofilament and myelin breakdown, macrophage recruitment, NADPH-diaphorase reaction and inducible nitric oxide synthase (iNOS) expression in transected sciatic nerves of C57BL/6J and A/J mice. The neurofilament volume density was lower in C57BL/6J strain mice at 1 and 3 days after lesion, and later equalled the density observed in A/J. C57BL/6J mice presented a high number of cells containing myelin debris, 3 and 5 days after the lesion. In both strains iNOS immunoreactivity was intense in macrophages and less evident in Schwann cells. However, a delay in macrophage recruitment and a lower percentage of iNOS-expressing macrophages on the third day were observed in C57BL/6J mice. NADPH-diaphorase reaction disclosed a similar pattern for both strains until the seventh day. However, at 5 days, cells with slender processes involving ellipsoid segments showed a well-defined cytoplasmic labelling in C57BL/6J whereas in A/J most of these cells exhibited a more granular and disperse labelling. We propose that these differences between A/J and C57BL/6J strains during Wallerian degeneration may be implicated in the lower regeneration potential observed in the latter.

The Martin-Gruber anastomosis in Brazilians: an anatomical study

Comunications between nerves are relatively common, and individual variations in their anatomical organization have been described. Knowledge of the prevalence of such variations is necessary when establishing the diagnosis of both sexes, were dissected to examine the communication between the median and ulnar nerves (Martin-Gruber anastomosis). This anastomosis was found in five cases (7.8 por cento) one of which was bilateral. There were no significant gender or racial differences in the incidence of this connection. The anastomosis showed secondary branches in two cases, and ran posteriorly to the ulnar artery in three cases, and advanced anteriorly to the flexor digitorum profundus muscles in all cases. Despite the low incidence of Martin-Gruber anastomosis in Brazilians observed here, the importance of an adequate investigation of these connections needs to be underscored. Understanding the existence of this variation, its location and its possible presentation is important for correct patient assistance.

The Martin-Gruber anastomosis in brazilians: an anatomical study

Communications between nerves are relatively common, and individual variations in their anatomical organization have been described. Knowledge of the prevalence of such variations is necessary when estabilishing the diagnosis of neuropathies and surgical landmarks. In this study, 64 anterior forearm regions of cadavers of blacks and whites of both sexes, were dissected to examine the communications between the median and ulnar nerves (Martin-Gruber anastomosis). This anastomosis was found in five cases (7,8%), one of which was bilateral. There were no significant gender or racial differences in the incidence of this connection. The anastomosis in Brazilians observed here, the impoprtance of an adequate investigation of these connections needs to be underscored. Understanding the existence of this variations, its location and its possible presentation is important for correct patient assistance.

Neuroprotective action of melatonin on neonatal rat motoneurons after sciatic nerve transection.

The neuronal isoform of nitric oxide synthase (nNOS), a NADPH-dependent diaphorase, is considered to play a role in motoneuron death induced by sciatic nerve transection in neonatal rats. Neuronal loss in these circumstances has been correlated with nitric oxide (NO) production and NADPH-diaphorase positivity in motoneurons after axotomy. In the present study we looked for a possible protective effect of melatonin, an antioxidant agent and inhibitor of nNOS, on spinal motoneurons after axonal injury. Neonatal Wistar rats (P2) were submitted to sciatic nerve transection and allowed to survive to P7. Melatonin at doses of 1, 5, 10, 50 and 100 mg/kg was given subcutaneously before and at intervals after the surgery. Controls operated in the same way received dilution vehicle or no treatment. The animals were killed by perfusion of fixative and the spinal cord was examined in serial paraffin sections. The motoneurons of the sciatic pool were counted in the axotomized and contralateral sides. Immunohistochemistry for nNOS and glial fibrillary acidic protein was used to evaluate nNOS expression in the axotomized cells and the astrocytic response. We found that melatonin at doses of 1-50 mg/kg decreased neuronal death. Astrocytic hypertrophy in melatonin treated animals was less intense. There were no differences in nNOS expression between treated and control rats, and surviving motoneurons of the sciatic pool did not express the enzyme, suggesting that nNOS may not be involved in neuronal death or survival in these experimental conditions. Possible mechanisms of melatonin neuroprotection, which was equally effective at doses of 1-50 mg/kg, are discussed. Doses of 50 and 100 mg/kg caused failure to thrive, seizures or death. The fact that neuroprotective doses were far smaller than toxic ones should encourage testing of melatonin in neurologic diseases.

Estudo morfológico de ducto deferente em ratos submetidos à vasectomia convencional e a laser de argônio / Morphological study of the vas deferens in rats submitted to conventional and argonium laser vasectomy

Dois grupos de 5 ratos foram submetidos a vasectomia convencional (A) e a raio laser de Argônia (B). No trigésimo dia de pós-operatório os animais foram sacrificados e os ductos deferentes observados sob visäo direta e ao microscópio óptico. Macroscopicamente observou-se em todos os casos a formaçäo de uma tumoraçäo no ápice do coto proximal do ducto deferente. O restante do aparelho permaneceu íntegro. Microscopicamente em ambos os grupos notou-se uma dilataçäo da luz do ducto, o epitélio tornou-se plano com perda de estereocílios e esgarçamento da camada muscular. Além disso, observou-se uma formaçäo tumoral com conteúdo espermático e colóide, circundada por macrófagos e células gigantes, envoltos por uma camada fibrosa. Esta formaçäo constitui-se no granuloma espermático. Näo foram observados significativas entre os métodos aplicados