Number Of Myelinated Fibers Research Articles

A quantitative evaluation of gross versus histologic neuroma formation in a rabbit forelimb amputation model: potential implications for the operative treatment and study of neuromas

BackgroundSurgical treatment of neuromas involves excision of neuromas proximally to the level of grossly "normal" fascicles; however, proximal changes at the axonal level may have both functional and therapeutic implications with regard to amputated nerves. In order to better understand the retrograde "zone of injury" that occurs after nerve transection, we investigated the gross and histologic changes in transected nerves using a rabbit forelimb amputation model.MethodsFour New Zealand White rabbits underwent a forelimb amputation with transection and preservation of the median, radial, and ulnar nerves. After 8 weeks, serial sections of the amputated nerves were then obtained in a distal-to-proximal direction toward the brachial plexus. Quantitative histomorphometric analysis was performed on all nerve specimens.ResultsAll nerves demonstrated statistically significant increases in nerve cross-sectional area between treatment and control limbs at the distal nerve end, but these differences were not observed 10 mm more proximal to the neuroma bulb. At the axonal level, an increased number of myelinated fibers were seen at the distal end of all amputated nerves. The number of myelinated fibers progressively decreased in proximal sections, normalizing at 15 mm proximally, or the level of the brachial plexus. The cross-sectional area of myelinated fibers was significantly decreased in all sections of the treatment nerves, indicating that atrophic axonal changes proceed proximally at least to the level of the brachial plexus.ConclusionsMorphologic changes at the axonal level extend beyond the region of gross neuroma formation in a distal-to-proximal fashion after nerve transection. This discrepancy between gross and histologic neuromas signifies the need for improved standardization among neuroma models, while also providing a fresh perspective on how we should view neuromas during peripheral nerve surgery.

Changes in the connective tissue sheath of Wistar rat nerve with aging

The alterations due to aging in the peripheral nerves can affect the physiology of these structures. Thus, the purpose of the present study was to describe the activity of the MMP-2 and MMP-9, as well as the structure and composition of the extracellular matrix of the rat sciatic nerve during maturation and aging. Our results have shown that the extracellular matrix of the sciatic nerve of 30-, 180- and 730-day-old Wistar rats present ultrastructural, morphometrical and biochemical changes during aging. The perineurium was the structure most affected by age, as evidenced by a decrease in thickness and in collagen fibril content. Cytochemical analysis detected proteoglycans in the basal membrane of Schwann cells and around perineural cells, as well as on the collagen fibrils of the perineurium and endoneurium at all ages. Biochemical analyses showed that the quantity of non-collagenous proteins was higher in 730-day-old animals compared to other ages, while the uronic acid content was higher in 30-day-old animals. Morphometrical analysis detected greater numbers of myelinated fibers and increased myelin thickness in 180-day-old animals. Zymography analysis detected greater amounts and activity of MMP-2 and MMP-9 in 180- and 730-day-old animals compared to younger rats. In conclusion, our results showed changes in the structural organization and composition of extracellular matrix of the sciatic nerve during aging, such as increase in the non-collagenous protein content and higher MMP-2 and MMP-9 activity, decrease in uronic acid concentration and in collagen fibril content in the perineurium, as well as degeneration of nerve fibers.

Novel model for end-neuroma formation in the amputated rabbit forelimb

BackgroundThe forelimb amputee poses many reconstructive challenges in the clinical setting, and there is a paucity of established surgical models for study. To further elucidate the pathogenic process in amputation neuroma formation, we created a reproducible, well-tolerated rabbit forelimb amputation model.MethodsUpon approval from the Institutional Animal Care and Use Committee, 5 New Zealand White rabbits underwent left forelimb amputation. During this initial surgery, the median, radial and ulnar nerves were transected 1.6-2.5 (mean 2.0) cm distal to the brachial plexus, transposed onto the anterior chest wall and preserved at length. Six weeks subsequent to the amputation, the distal 5 mm of each neuroma was excised, and the remaining stump underwent histomorphometric analysis.ResultsThe nerve cross sectional areas increased by factors of 1.99, 3.17, and 2.59 in the median (p = 0.077), radial (p < 0.0001) and the ulnar (p = 0.0026) nerves, respectively. At the axonal level, the number and cross-sectional area of myelinated fibers demonstrated an inverse relationship whereby the number of myelinated fibers in the median, radial and ulnar nerves increased by factors of 5.13 (p = 0.0043), 5.25 (p = 0.0056) and 5.59 (p = 0.0027), and the cross-sectional areas of these myelinated fibers decreased by factors of 4.62 (p < 0.001), 3.51 (p < 0.01), and 4.29 (p = 0.0259), respectively.ConclusionGiven that the surgical model appears well-tolerated by the rabbits and that patterns of morphologic change are consistent and reproducible, we are encouraged to further investigate the utility of this model in the pathogenesis of neuroma formation.

Are we sure to count myelinated fibers entirely? Calibration of a stereological study

Peripheral nerves are subject to many different types of injuries frequently and these injuries may result in partial or total loss of motor and sensory functions. In contrast to the central nervous system, peripheral nervous system are able to undergo spontaneous regeneration. But clinical and experimental studies show that this regeneration is mostly not satisfactory. One of the most important neuropathological indicators of nerve regeneration is quantitative estimation of myelinated axons. Quantitative estimation of the total number of myelinated fibers can be obtained both by light microscopy (LM) and electron microscopy (EM) and depends on using appropriate sampling strategy. In this study, we made a direct comparison between LM and EM estimation of myelinated nerve fibers. Ten female Wistar albino rats were divided into two experimental groups; control group (n=5) and gap group (n=5). In the gap group, 5 mm right sciatic nerve segment of rats were resected to form a gap. Proximal and distal nerve stumps were surrounded by collagen membrane following resection. In the control group, rats received neither any operation nor any treatment. Rats were sacrificed at 12 week after surgery. Nerve samples were embedded in resin. Semi and ultra thin sections (of 1 μm and 100 nm thickness, respectively) were cut and stained with 1% toluidine blue (for LM) and uranyl acetate-lead citrate (for EM). Histological assessment of myelinated fibers was performed with both LM and EM using unbiased stereological counting techniques. There was a significant difference in the total number of myelinated nerve fibers between LM and EM examinations in terms of the both pure and regenerating axons. This difference is probably related to different magnification capacities in LM and EM. LM causes to underestimates as a result of possessing very small myelinated fibers, which are only detectable with EM. According to our results it may be concluded that the combination of both light and electron microscopical results is essential especially in revealing regenerating nerve fibers since very small myelinated fibers can not be distinguishable with LM.

Discrepancies in quantitative assessment of normal and regenerated peripheral nerve fibers between light and electron microscopy.

Quantitative estimation of myelinated nerve fiber number, together with fiber size parameters, is one of the most important tools for nerve regeneration research. In this study we used a design-based stereological method to evaluate the regenerative process in two experimental paradigms: crush injury and autograft repair. Samples were embedded in resin and morphometric counting and measurements were performed using both light and electron microscopes. Results show a significant difference in myelinated fiber number estimation between light and electron microscopes, especially after autograft repair; light microscope significantly underestimates the number of fibers because of the large number of very small axons that can be detected only in electron microscope. The analysis of the size parameters also shows a higher number of small fibers in electron microscopic analysis, especially in regenerated nerves. This comparative study shows that the integration of data obtained in light microscope with those obtained in electron microscope is necessary in revealing very small myelinated fibers that cannot be detected otherwise. Moreover, the difference in the estimation of total number of myelinated fibers between light and electron microscopes must be considered in data analysis to ensure accurate interpretation of the results.

Transplantation of undifferentiated bone-marrow stromal cells into a vein graft accelerates sciatic nerve regeneration in streptozotocin induced diabetic rats.

Influence of undifferentiated bone marrow stromal cells (BMSCs) combined with vein graft on transected sciatic nerve repair was studied in diabetic rats. A nerve segment, 10 mm, was excised and a vein graft (VG) was used to bridge the gap. 10 microliter undifferentiated BMSCs (2 x 10(7) cells /mL) were administered into the graft in treatment group (VG/BMSC). Phosphate buffered saline was only administered into the graft in control group. The regenerated nerve fibers were studied in three time points of 4, 8 and 12 weeks after surgery. Evaluation of the repair process was based on behavioral, functional (Walking Track Analysis), electrophysiological, histomorphometrical and immunohistochemical criteria. The behavioral, functional and electrophysiological studies indicated that there was significant recovery in regeneration of axons in VG/BMSC group (P < 0.05). Morphometric evaluations showed that the number and diameter of myelinated fibers in VG /BMSC group were significantly higher than those in the control group (P < 0.05). This indicates the potential of using undifferentiated BMSCs combined with vein graft in peripheral nerve repair in diabetic rats with no restrictions of donor-site morbidity associated with isolation of Schwann cells. This technique is also cost saving because of decrease in interval from tissue harvest until administration of the cells and simplicity of laboratory techniques in comparison with undifferentiated BMSCs. It may have clinical implications for the surgical management of diabetic patients after nerve transection.

Enhanced axon outgrowth and improved long-distance axon regeneration in sprouty2 deficient mice.

Sprouty (Spry) proteins are negative feedback inhibitors of receptor tyrosine kinase signaling. Downregulation of Spry2 has been demonstrated to promote elongative axon growth of cultured peripheral and central neurons. Here, we analyzed Spry2 global knockout mice with respect to axon outgrowth in vitro and peripheral axon regeneration in vivo. Neurons dissociated from adult Spry2 deficient sensory ganglia revealed stronger extracellular signal-regulated kinase activation and enhanced axon outgrowth. Prominent axon elongation was observed in heterozygous Spry2(+/-) neuron cultures, whereas homozygous Spry2(-/-) neurons predominantly exhibited a branching phenotype. Following sciatic nerve crush, Spry2(+/-) mice recovered faster in motor but not sensory testing paradigms (Spry2(-/-) mice did not tolerate anesthesia required for nerve surgery). We attribute the improvement in the rotarod test to higher numbers of myelinated fibers in the regenerating sciatic nerve, higher densities of motor endplates in hind limb muscles and increased levels of GAP-43 mRNA, a downstream target of extracellular regulated kinase signaling. Conversely, homozygous Spry2(-/-) mice revealed enhanced mechanosensory function (von Frey's test) that was accompanied by an increased innervation of the epidermis, elevated numbers of nonmyelinated axons and more IB4-positive neurons in dorsal root ganglia. The present results corroborate the functional significance of receptor tyrosine kinase signaling inhibitors for axon outgrowth during development and nerve regeneration and propose Spry2 as a novel potential target for pharmacological inhibition to accelerate long-distance axon regeneration in injured peripheral nerves.

Efficacy of the end-to-side neurorrhaphies with epineural window and partial donor neurectomy in peripheral nerve repair: an experimental study in rats.

End-to-side (ETS) neurorrhaphy was a useful tool in peripheral nerve repair and "baby-sitter" procedure. The study was designed to evaluate the long-term efficacy of ETS with epineurial window and 40% partial donor neurectomy in rats. A total of 60 Lewis rats were divided into three groups (n = 20, each group): following peroneal nerve transection, rats in Group A underwent end-to-end neurorrhaphy; rats in Group B underwent ETS neurorrhaphy of the distal peroneal nerve stump to an epineurial window on the tibial nerve; and rats in Group C underwent ETS neurorrhaphy of the distal peroneal nerve stump to the tibial nerve with 40% partial neurectomy. At 6, 12, 18, and 24 weeks after surgery, electrophysiology, muscle tension, myelinated fiber regeneration, cross-sectional area of muscular fiber, and muscle weight were analyzed. Histology exhibited apparently increased number and size of myelinated fibers in peroneal nerves in Group C, compared with those in Group B. More superior recovery was demonstrated in the electrophysiology and axon regeneration of the peroneal nerves, as well as the maintenance of muscle force, wet weight, and fiber size of the anterior tibial muscles in Group C than those in Group B. ETS neurorrhaphy with partial donor neurectomy can achieve higher efficacy in functional and structural recovery of the recipient system. This study provides the evidence of long-term follow-up for the further investigation of ETS neurorrhaphies with different modalities in peripheral nerve repair and in "baby-sitter" procedure.

Two-dimensional sample entropy analysis of rat sural nerve aging.

Entropy analysis of images are usually performed using Shannon entropy, which calculates the probability of occurrency of each gray level on the image. However, not only the pixel gray level but also the spatial distribution of pixels might be important for image analysis. On the other hand, sample entropy (SampEn) is an important tool for estimation of irregularity in time series, which calculates the probability of pattern occurrence within the series. Therefore, we propose here an extension of SampEn to a two-dimensional case, namely SampEn2D, as an entropy method for extracting features from images that accounts for the spatial distribution of pixels. SampEn2D was applied to histological segments of sural nerve obtained from young (30 days) and elderly (720 days) rats. Morphometric indexes, such as the total number of myelinated fibers and the average myelinated fibers area and perimeter were also calculated. Results show that SampEn2D can extract useful information from histological nerve images, classifying elderly rat image as more regular than young rat. As SampEn2D is related to irregularity/unpredictability, we can conclude that the proposed method is complementary to morphometric indexes. Further studies are being built to validate SampEn2D.

Peripheral nerve reconstruction with epsilon-caprolactone conduits seeded with vasoactive intestinal peptide gene-transfected mesenchymal stem cells in a rat model

Objective. Attempts have been made to improve nerve conduits in peripheral nerve reconstruction. We investigated the potential therapeutic effect of a vasoactive intestinal peptide (VIP), a neuropeptide with neuroprotective, trophic and developmental regulatory actions, in peripheral nerve regeneration in a severe model of nerve injury that was repaired with nerve conduits. Approach. The sciatic nerve of each male Wistar rat was transected unilaterally at 10 mm and then repaired with Dl-lactic-ε-caprolactone conduits. The rats were treated locally with saline, with the VIP, with adipose-derived mesenchymal stem cells (ASCs) or with ASCs that were transduced with the VIP-expressing lentivirus. The rats with the transected nerve, with no repairs, were used as untreated controls. At 12 weeks post-surgery, we assessed their limb function by measuring the ankle stance angle and the percentage of their muscle mass reduction, and we evaluated the histopathology, immunohistochemistry and morphometry of the myelinated fibers. Main results. The rats that received a single injection of VIP-expressing ASCs showed a significant functional recovery in the ankle stance angle (p = 0.049) and a higher number of myelinated fibers in the middle and distal segments of the operated nerve versus the other groups (p = 0.046). Significance. These results suggest that utilization of a cellular substrate, plus a VIP source, is a promising method for enhancing nerve regeneration using Dl-lactic-ε-caprolactone conduits and that this method represents a potential useful clinical approach to repairing peripheral nerve damage.

Uncultured undifferentiated adipose-derived nucleated cell fractions combined with inside-out artery graft accelerate sciatic nerve regeneration and functional recovery

Effects of transplantation of adipose-derived nucleated cell fractions (ADNCs) on sciatic nerve regeneration were studied. A 10-mm sciatic nerve defect was bridged using artery graft filled with ADNCs. In control group, artery graft was filled with saline alone. Regenerated nerve fibres were studied for 12 weeks. In sham-operated group, sciatic nerve was only exposed and manipulated. Behavioural and functional studies confirmed faster recovery of regenerated axons in ADNCs transplanted animals than in control group (P<0.05). At the end of study period, animals in ADNCs transplanted group achieved a sciatic functional index (SFI) value of −31.6±−3.14, whereas in control group a value of −42.5±−3.7 was found. Gastrocnemius muscle mass in ADNCs transplanted animals was found to be significantly higher than that in control group (P=0.001). Morphometric indices of regenerated fibres showed the number and diameter of myelinated fibres to be significantly higher in ADNCs transplanted animals than in control group (P=0.001). On immunohistochemistry, there was more positive staining of S100 in the ADNCs transplanted animals than in control group. ADNCs transplantation into an artery graft could be considered a readily accessible technique that improves functional recovery of sciatic nerve.

FK506-loaded chitosan conduit promotes the regeneration of injured sciatic nerves in the rat through the upregulation of brain-derived neurotrophic factor and TrkB

FK506 has been shown to exert neurotrophic and neuroprotective effects, but its long-term application for nerve regeneration is limited. This study evaluated the potential application of a novel FK506-loaded chitosan conduit for peripheral nerve repair, and explored the underlying mechanism. A sciatic nerve injury model was created in male Wistar rats, which were then randomly divided into three treatment groups (n=40, each): chitosan-only, chitosan+FK506 injection, and FK506-loaded chitosan. We found significant recovery of normal morphology of sciatic nerves and higher density of myelinated nerve fibers in rats treated with FK506-loaded chitosan. Similarly, the total number of myelinated nerve fibers, myelin sheath thickness, and axon diameters were significantly higher in this group compared with the others, and the compound muscle action potentials and motor nerve conduction velocity values of sciatic nerves were significantly higher. BDNF and TrkB levels in motor neurons were highest in rats treated with FK506-loaded chitosan. In conclusion, FK506-loaded chitosan promoted peripheral nerve repair and regeneration in a rat model of sciatic nerve injury. These effects are correlated with increased BDNF and TrkB expression in motor neurons.

Experimental diabetes in neonatal mice induces early peripheral sensorimotor neuropathy

Animal models of diabetes do not reach the severity of human diabetic neuropathy but relatively mild neurophysiological deficits and minor morphometric changes. The lack of degenerative neuropathy in diabetic rodent models seems to be a consequence of the shorter length of the axons or the shorter animal life span. Diabetes-induced demyelination needs many weeks or even months before it can be evident by morphometrical analysis. In mice myelination of the peripheral nervous system starts at the prenatal period and it is complete several days after birth. Here we induced experimental diabetes to neonatal mice and we evaluated its effect on the peripheral nerve 4 and 8weeks after diabetes induction. Neurophysiological values showed a decline in sensory nerve conduction velocity at both time-points. Morphometrical analysis of the tibial nerve demonstrated a decrease in the number of myelinated fibers, fiber size and myelin thickness at both time-points studied. Moreover, aldose reductase and poly(ADP-ribose) polymerase activities were increased even if the amount of the enzyme was not affected. Thus, type 1 diabetes in newborn mice induces early peripheral neuropathy and may be a good model to assay pharmacological or gene therapy strategies to treat diabetic neuropathy.

Poly(ε-caprolactone) nerve conduit and local delivery of vegf and fgf2 genes stimulate neuroregeneration.

We studied regeneration of rat sciatic nerve while overcoming of a 5-mm diastasis with the aid of nanostructured conduit made of biocompatible and biodegradable poly(ε-caprolactone) and filled with fibrin hydrogel matrix. Implantation of the conduit into the nerve in combination with local delivery of the expression plasmid carrying genes encoding vascular endothelial growth factor (vegf) and fibroblast growth factor 2 (fgf2) leads to an increase in number of myelinated fibers and S-100(+) cells in the peripheral nerve stump and improved recovery of the nerve function. Under conditions of direct gene therapy, an advantage of electrospun poly(ε-caprolactone) conduit with high-porosity was revealed on the basis of these criteria in comparison with biocompatible silicon conduit.

Sciatic nerve regeneration induced by transplantation of in vitro bone marrow stromal cells into an inside-out artery graft in rat

Traumatic injury to peripheral nerves results in considerable motor and sensory disability. Several research groups have tried to improve the regeneration of traumatized nerves by invention of favorable microsurgery. Effect of undifferentiated bone marrow stromal cells (BMSCs) combined with artery graft on peripheral nerve regeneration was studied using a rat sciatic nerve regeneration model. A 10-mm sciatic nerve defect was bridged using an artery graft (IOAG) filled with undifferentiated BMSCs (2 × 107 cells/mL). In control group, the graft was filled with phosphated buffer saline alone. The regenerated fibers were studied 4, 8 and 12 weeks after surgery. Assessment of nerve regeneration was based on behavioral, functional (Walking Track Analysis), electrophysiological, histomorphometric and immuohistochemical (Schwann cell detection by S-100 expression) criteria. The behavioral, functional and electrophysiological studies confirmed significant recovery of regenerated axons in IOAG/BMSC group (P < 0.05). Quantitative morphometric analyses of regenerated fibers showed the number and diameter of myelinated fibers in IOAG/BMSC group were significantly higher than in the control group (P < 0.05). This demonstrates the potential of using undifferentiated BMSCs combined with artery graft in peripheral nerve regeneration without limitations of donor-site morbidity associated with isolation of Schwann cells. It is also cost saving due to reduction in interval from tissue collection until cell injection, simplicity of laboratory procedures compared to differentiated BMSCs and may have clinical implications for the surgical management of patients after facial nerve transection.

Structural, morphologic and morphometric study of the phrenic nerve: association between experimental diabetes and arterial hypertension (728.24)

Although the association between hypertension and diabetes is common in clinical practice, it is not well known the contribution of each of these diseases in the development of diabetic neuropathy in patients with hypertension. We investigated the presence of peripheral neuropathy, through morphology and morphometry analysis of the phrenic nerve in normotensive rats (Wistar) and spontaneously hypertensive rats (SHR) with experimentally induced chronic diabetes. Diabetic animals received a single injection of streptozotocin (STZ) intravenously while controls received vehicle on the same date. Twelve weeks after injection, arterial pressure and heart rate were recorded and the right and left phrenic nerves were removed and prepared for embedding in epoxy resin and computer morphometry. The phrenic nerves of SHR and diabetic SHR (SHR + STZ) showed reduced number and density of myelinated fibers and Schwann cell nuclei, compared to Wistar. The SHR and SHR+STZ rats showed myelinated fiber size distributions skewed to the right, with an important reduction of the small myelinated fibers. This finding was more pronounced on diabetic SHR when compared to normotensive diabetic animals. We suggest that hypertension causes a reduction of small myelinated fibers and the association of the hypertension and diabetes did not interfere with the small fiber neuropathy present in the hypertensive animals.Grant Funding Source: CAPES, FAPESP, CNPq and FAEPA

The human trochlear and abducens nerves at different ages - a morphometric study.

The trochlear and abducens nerves (TN and AN) control the movement of the superior oblique and lateral rectus muscles of the eyeball, respectively. Despite their immense clinical and radiological importance no morphometric data was available from a wide spectrum of age groups for comparison with either pathological or other conditions involving these nerves. In the present study, morphometry of the TN and AN was performed on twenty post-mortem samples ranging from 12-90 years of age. The nerve samples were processed for resin embedding and toluidine blue stained thin (1µm) sections were used for estimating the total number of myelinated axons by fractionator and the cross sectional area of the nerve and the axons by point counting methods. We observed that the TN was covered by a well-defined epineurium and had ill-defined fascicles, whereas the AN had multiple fascicles with scanty epineurium. Both nerves contained myelinated and unmyelinated fibers of various sizes intermingled with each other. Out of the four age groups (12-20y, 21-40y, 41-60y and >61y) the younger groups revealed isolated bundles of small thinly myelinated axons. The total number of myelinated fibers in the TN and AN at various ages ranged from 1100-3000 and 1600-7000, respectively. There was no significant change in the cross-sectional area of the nerves or the axonal area of the myelinated nerves across the age groups. However, myelin thickness increased significantly in the AN with aging (one way ANOVA). The present study provides baseline morphometric data on the human TN and AN at various ages.

Transplantation of Olfactory Ensheathing Cells to Evaluate Functional Recovery after Peripheral Nerve Injury

Olfactory ensheathing cells (OECs) are neural crest cells which allow growth and regrowth of the primary olfactory neurons. Indeed, the primary olfactory system is characterized by its ability to give rise to new neurons even in adult animals. This particular ability is partly due to the presence of OECs which create a favorable microenvironment for neurogenesis. This property of OECs has been used for cellular transplantation such as in spinal cord injury models. Although the peripheral nervous system has a greater capacity to regenerate after nerve injury than the central nervous system, complete sections induce misrouting during axonal regrowth in particular after facial of laryngeal nerve transection. Specifically, full sectioning of the recurrent laryngeal nerve (RLN) induces aberrant axonal regrowth resulting in synkinesis of the vocal cords. In this specific model, we showed that OECs transplantation efficiently increases axonal regrowth. OECs are constituted of several subpopulations present in both the olfactory mucosa (OM-OECs) and the olfactory bulbs (OB-OECs). We present here a model of cellular transplantation based on the use of these different subpopulations of OECs in a RLN injury model. Using this paradigm, primary cultures of OB-OECs and OM-OECs were transplanted in Matrigel after section and anastomosis of the RLN. Two months after surgery, we evaluated transplanted animals by complementary analyses based on videolaryngoscopy, electromyography (EMG), and histological studies. First, videolaryngoscopy allowed us to evaluate laryngeal functions, in particular muscular cocontractions phenomena. Then, EMG analyses demonstrated richness and synchronization of muscular activities. Finally, histological studies based on toluidine blue staining allowed the quantification of the number and profile of myelinated fibers. All together, we describe here how to isolate, culture, identify and transplant OECs from OM and OB after RLN section-anastomosis and how to evaluate and analyze the efficiency of these transplanted cells on axonal regrowth and laryngeal functions.

Deletion of GABA‐B Receptor in Schwann Cells Regulates Remak Bundles and Small Nociceptive C‐fibers

The mechanisms regulating the differentiation into non-myelinating Schwann cells is not completely understood. Recent evidence indicates that GABA-B receptors may regulate myelination and nociception in the peripheral nervous system. GABA-B receptor total knock-out mice exhibit morphological and molecular changes in peripheral myelin. The number of small myelinated fibers is higher and associated with altered pain sensitivity. Herein, we analyzed whether these changes may be produced by a specific deletion of GABA-B receptors in Schwann cells. The conditional mice (P0-GABA-B1(fl/fl)) show a morphological phenotype characterized by a peculiar increase in the number of small unmyelinated fibers and Remak bundles, including nociceptive C-fibers. The P0-GABA-B1(fl/fl) mice are hyperalgesic and allodynic. In these mice, the morphological and behavioral changes are associated with a downregulation of neuregulin 1 expression in nerves. Our findings suggest that the altered pain sensitivity derives from a Schwann cell-specific loss of GABA-B receptor functions, pointing to a role for GABA-B receptors in the regulation of Schwann cell maturation towards the non-myelinating phenotype.

Remyelination action of olfactory ensheathing cells in contused spinal cord

Objective To detect the myelinating role of olfactory ensheathing cells (OECs in the contused spinal cord and their impact on remyelination.Methods The rats were subjected to spinal cord injury at T10(10 g ×25 mm) using a NYU-Ⅱ impactor.One week later,the rats were transplanted with green fluorescence protein (GFP)-OECs (OECs group) or an equal volume of Dulbecco' s modification of Eagle's medium (DMEM) (control group) at epicenter of the injury as well as its rostral and caudal sites.Six weeks after transplantation,the spinal cords were removed for frozen section.Myelin basic protein (MBP),protein zero (P0),and S100 protein (S100) were determined with qualitative and semi-quantitative immunocytochemical assay.Moreover,plastic embedded semithin and ultrathin sections were prepared for qualitative and semi-quantitative examination under light microscopy and electroscopic study of myelin sheath ultrastructure.Results In OECs group,the nerve fibers labeled with S100,MBP,and PO were extended from the normal tissues to the injured region and even grew through the region with space consuming of 12.3％,11.6％,and 9.3％ respectively.Moreover,there were no statistical differences regarding the number of fibers labeled by the three proteins,but all were significantly larger than that in control group (2.89％,P ＜ 0.01).Number of myelinated nerve fibers in injured regions on hemithin sections was increased significantly to 354.67 ± 59.00 in OECs group,with significant difference compared with 167.33 ± 42.16 in control group (P ＜ 0.01).The regenerated myelin sheaths in OECs group were smaller and thicker than those in control group.Conclusions OECs can accelerate regeneration of myelinated nerve fibers.Additionally,some OECs form myelin sheaths themselves,but the sheath structures are relatively thinner. Key words: Spinal cord injuries; Myelin sheath; Olfactory ensheathing cells