Extradural nerve anastomosis technique for bladder reinnervation in spinal cord injury: anatomical feasibility study in human cadavers.

STUDY DESIGN: An anatomic study of extradural spinal root in 9 embalmed cadavers. OBJECTIVE: To ascertain the anatomical parameters of the extradural spinal root and to demonstrate the feasibility of spinal root anastomoses without opening the spinal dura mater. SUMMARY OF BACKGROUND DATA: Intradural anastomosis of the spinal root has made breakthrough progress in treating neurogenic bladder in spinal cord injury. However, because of the complex surgical procedures and extensive bony destruction, its clinical use is not widely promoted. METHODS: Nine formalin-fixed cadavers were used. The distance between the nerve root outlet and ganglion center, the neighboring nerve root-outlet distance, and the gross anatomy of the extradural spinal root were measured with a surgical microscope. The number of nerve fibers from the T7 to S4 ventral roots (VRs) was calculated by immunohistochemical staining. RESULTS: The longest and shortest lengths of the extradural spinal root were observed at the S4 and T7 levels, with average values of 33.29 and 6.06 mm, respectively. The longest distance between the adjacent nerve root outlets was observed at L1-L2 (mean, 29.16 mm), and shortest at S3-S4 (mean, 11.79 mm). After leaving the dural sac, the spinal root descends in the spinal canal until reaching the corresponding intervertebral foramina, and the motor nerve roots still lie ventrally to the sensory nerve roots. The largest and smallest numbers of nerve fibers were observed at the L3 and S4 levels (mean, 9169 and 1356, respectively). CONCLUSION: The dorsal roots and VRs can both be successfully harvested and identified outside the dural sac. The S1 VR can be anastomosed to the S2 VR extradurally without nerve grafts. For extradural neuroanastomosis of the thoracic VRs to the S2 VR, a nerve graft is required. In addition, there are a sufficient number of nerve fibers for functional bladder recovery at the T7-T12 and S1 levels. This study supports the feasibility of extradural spinal root anastomosis as a modified surgical method for treating neurogenic bladder. LEVEL OF EVIDENCE: N/A.

Retrograde loading of nerves, tracts, and spinal roots with fluorescent dyes.

Retrograde labeling of neurons is a standard anatomical method(1,2) that has also been used to load calcium and voltage-sensitive dyes into neurons(3-6). Generally, the dyes are applied as solid crystals or by local pressure injection using glass pipettes. However, this can result in dilution of the dye and reduced labeling intensity, particularly when several hours are required for dye diffusion. Here we demonstrate a simple and low-cost technique for introducing fluorescent and ion-sensitive dyes into neurons using a polyethylene suction pipette filled with the dye solution. This method offers a reliable way for maintaining a high concentration of the dye in contact with axons throughout the loading procedure.

Identification of motor and sensory fascicles in peripheral nerve trunk using immunohistochemistry and micro-Raman spectroscopy.

OBJECTIVE: To explore a time-efficient method of identifying motor and sensory fascicles in peripheral nerve trunk. METHODS: Thirty Wistar rats were selected to obtain whole spine. The spinal dorsal roots and ventral roots, and sciatic nerve were harvested as sensor, motor, and mixed samples, annexin V and agrin specificities were observed with Western blot and immunohistochemistry. A total of 32 New Zealand rabbits were selected and killed. The roots of spinal nerves were exposed under an operating microscope, and the ventral and dorsal roots, ∼3 mm to 5 mm, were dissociated, and frozen as transverse sections of 30-µm thickness. The sections were examined by micro-Raman spectroscopy. RESULTS: The annexin V and agrin were special substances of sensory and motor nerves, respectively, and can act as specific antigens for identifying different nerve fascicles. Sections of the same type of nerve fascicles showed reproducibility with similar spectral features. Significant differences in the spectral properties, such as the intensity and breadth of the peak, were found between motor and sensory fascicles in the frequency regions of 1,088 cm(-1), 1,276 cm(-1), 1,439 cm(-1), 1,579 cm(-1), and 1,659 cm(-1). With the peak intensity ratio of 1.06 (I(1276)/I(1439)) as a standard, we could identify motor fascicles with a sensitivity of 88%, specificity of 94%, positive predictive value of 93%, and negative predictive value of 88%. In the range of 2,700 cm(-1) to 3,500 cm(-1), the half-peak width of the motor fascicles was narrow and sharp, whereas that of the sensory fascicles was relatively wider. A total of 91% of the peak features were in accordance with the identification standard. CONCLUSION: Motor and sensory fascicles exhibit different characteristics in Raman spectra, which are constant and reliable. Therefore, it is more effective than immunohistochemistry method in identifying different nerve fascicles according to the specific spectrum, and it possesses feasibility for clinical application.

Melanotic schwannoma: a case with strong CD34 expression, with histogenetic implications.

Melanotic schwannomas (MS) are rare tumors composed of cells with both schwannian and melanocytic features, which usually occur in the setting of Carney's Complex. We describe a case of a 36-year-old male who presented with a mass that was attached to the vertebral body as well as the nerve roots of L2 and L3. Immunohistochemical positivity for S-100, HMB-45, and Pan-melanoma markers, as well as characteristic morphologic and ultrastructural findings, suggested that the lesion was a MS. The interest in this case lies in the fact that this case of MS showed strong CD34 expression, a marker that is generally negative in melanocytic tumors. We discuss the biologic significance of the high CD34 expression by the tumor cells and attempt to shed light on the histogenesis of this rare entity.

[Identification of property of nerve fascicles by physical and histochemistry methods].

OBJECTIVE: To explore a method to identify the sensory and motor fascicles in peripheral nerve trunk. METHODS: Thirty Wistar rats were selected to obtain whole spine. The spinal ganglion, its dorsal root and ventral root, and sciatic nerve were harvested, Annexin V and Agrin specificities were observed with Western blot. In the experimental group, anterior branch and posterior branch of spinal nerve, sciatic nerve, and its muscular branch and cutaneous branch were harvested from 15 rats to make the observation of immunohistochemistry. In the other 15 rats, first antibody was replaced by PBS as control group. Different nerve fascicles were studied with Micro Raman scattering technique in 16 12-month-old New Zealand rabbits. RESULTS: The Annexin V and Agrin were special substances of sensory and motor nerves respectively and can act as specific antigens for identifying different nerve fascicles. There were significant differences in the intensity and breadth of the peak of the spectral properties between motor and sensory fascicles at frequencies of 1,088, 1,276, 1,439, 1,579 and 1,659 cm(-1). The peak intensity ratios of 1,276 to 1,439 cm(-1) were 0.95+/-0.06 in motor nerve fascicles and 1.17+/-0.08 in sensory fascicles, showing significant differences (P<0.05). CONCLUSION: The Micro Raman spectra is more effective than immunohistochemistry in identifying different nerve fascicles, and it possesses as feasibility for clinical application.

Evaluation of C5 nerve root repairability in traumatic brachial plexus injuries: proposal of an evaluation scoring system.

We reviewed 36 traumatic brachial plexus injury patients with C5 nerve root injury. We used the choline acetyltransferase (CAT) activity measurement as a gold standard indicator of repairability to assess the reliability of the conventional techniques (preoperative clinical, electrophysiologic, and radiologic examination and intraoperative clinical and electrophysiologic examinations). We proposed a repairability scoring system composed of the correlated conventional techniques and tested the validity of this scoring system using the same reference technique (CAT activity measurement). We found no significant correlation between sensation at C5 dermatome and electromyography study with C5 repairability. On the other hand, we found a significant correlation of Tinel sign, myelography, macroscopic and microscopic examination, and electrical stimulation with C5 repairability. Based on these results, we propose a simple 21-point scoring system to evaluate the repairability of C5 nerve root that is composed of Tinel sign, myelography, macroscopic and microscopic appearance, and electrical stimulation. The scoring system was found to be highly significantly correlated with C5 repairability. We concluded that our proposed evaluation scoring system is a reliable method for C5 repairability evaluation, and it can replace sophisticated techniques.

Immunohistochemical labelling of components of the endoneurial extracellular matrix of intact and rhizotomized dorsal and ventral spinal roots of the rat--a quantitative evaluation using image analysis.

The endoneurial extracellular matrix (ECM) molecules are involved in cell signalling during nervous system development and regeneration. Quantitative differences of immunofluorescence labelling for chondroitin sulfate proteoglycan (CSPG), fibronectin (FN), tenascin-C (TN-C), and thrombospondin (TSP) were evaluated in intact rat dorsal and ventral roots and dorsal and ventral roots 2 and 4 weeks after rhizotomy using image analysis. The distal stumps of spinal roots displayed increased immunolabelling for the molecules with higher immunofluorescence in dorsal than in ventral roots up to 2 weeks from transection. Four weeks after rhizotomy, the immunoreactivity for CSPG, TN-C and TSP decreased in dorsal and increased in ventral root stumps, although a higher level of immunofluorescence for FN remained in both dorsal and ventral root stumps 4 weeks after injury in comparison to 2 weeks after injury. We suggest that the amount of some ECM molecules changed differentially 2 and 4 weeks after rhizotomy to create an appropriate environment in the endoneurium for early and later regrowth of sensory and motor axons. The results presented here are the first report of differences between the endoneurial ECM content of damaged afferent and motor nerve fibers. In addition, the immunohistochemical detection of individual ECM molecules indicated that final extrinsic conditions stimulating the regrowth of regenerating axons probably arise from a balance of both growth-promoting and -inhibiting molecules in the endoneurium.

Is clear cell sarcoma a malignant form of psammomatous melanotic schwannoma? Case report.

The authors present a case of clear cell sarcoma (CCS) in which the tumor originated in the S-1 nerve root and had been previously diagnosed as psammomatous melanotic schwannoma (PMS). This is the third case of a spinal nerve root origin for CCS reported in the English-language literature. The similar histogenesis of CCS and malignant melanoma supports the hypothesis that biological agents or immunotherapy are potentially important areas of investigation. The patient underwent S1-3 laminectomy and gross-total resection of the mass lesion. The border of the resection was extended 1 cm distal to the tumor margin. The postoperative period was uneventful. The new histopathological diagnosis was CCS (malignant melanoma of soft tissue). Despite total resection, the patient returned with disseminated disease at the 18-month follow-up visit. His follow-up magnetic resonance image of the lumbar spine revealed sacral L5-S3 involvement of the vertebral bodies along with disseminated cauda equina seeding. A CCS originating from peripheral nerves is quite rare. The histopathological and immunohistochemical appearance of CCSs resembles those of PMSs. Surgery should be the first choice of treatment.

Effect of mechanical compression on the lumbar nerve root: localization and changes of intraradicular inflammatory cytokines, nitric oxide, and cyclooxygenase.

STUDY DESIGN: Investigation of intraradicular inflammation induced by mechanical compression. OBJECTIVE: To investigate the mechanism of nerve root pain, this study used a lumbar nerve root compression model. SUMMARY OF BACKGROUND DATA: The manifestation of pain at sites of inflammation has a close relationship with the release of mediators from macrophages. However, the mediators involved in inflammation of nerve roots as a result of mechanical compression remain almost unknown. METHODS: In this study, the seventh lumbar nerve root of dogs was compressed with a clip for 3 weeks to observe the changes caused by compression. Immunohistochemistry was performed using the avidin-biotin-peroxidase complex method to observe the changes of T cells (CD45) and macrophages (Mac-1) after compression. Antibodies against as interleukin-1 (IL-1), tumor necrosis factor-alpha (TNF-alpha), inducible nitric oxide synthase (i-NOS), and cyclooxygenase (COX)-1 and 2 were used to examine the localization and changes of these mediators caused by nerve root compression. RESULTS: In control animals, resident T cells were detected, but there were no macrophages. IL-1beta and COX-2 were positive in the Schwann cells and vascular endothelial cells, while COX-1 was detected in the vascular endothelial cells. However, no cells showed TNF-alpha or i-NOS positively. After nerve root compression, numerous T cells and macrophages appeared among the demyelinized nerve fibers. The macrophages were positive for IL-1beta, TNF-alpha, i-NOS, and COX-2. CONCLUSION: Inflammatory cytokines, NO, and COX-2 may be deeply involved in radiculitis caused by mechanical compression, and these mediators seem to be important in the manifestation of root pain.

GalNAc-GD1a in human peripheral nerve: target sites of anti-ganglioside antibody.

BACKGROUND: The authors previously reported that immunoglobulin G (IgG) antibody to the ganglioside N-acetylgalactosaminyl GD1a (GalNAc-GD1a) is associated with the pure motor variant of Guillain-Barré syndrome (GBS). Elucidation of the localization of GalNAc-GD1a in human peripheral nerve tissue may lead to understanding of the pathogenetic role of anti-GalNAc-GD1a antibody in GBS. METHODS: IgG anti-GalNAc-GD1a-monospecific antibody was purified from anti-GalNAc-GD1a antibody-positive rabbit sera through an affinity column. Anti-neurofilament-200 monoclonal and anti-HNK-1 monoclonal antibodies were used as the markers for axon and myelin. Immunohistochemical study using double fluorescence labeling technique was conducted in human ventral roots (VR), dorsal roots (DR), intramuscular nerves, and sural nerves. Human teased ventral fibers also were studied. RESULTS: Anti-GalNAc-GD1a antibody immunostained an inner part of compact myelin and additionally a periaxonal-axolemma-related portion in the VR, small-diameter DR fibers, and IM nerves. In sural nerves, small fibers were selectively stained. In VR, the staining was localized in the paranodal region. CONCLUSION: Anti-GalNAc-GD1a antibodies in patients' sera may bind to those regions in the VR and IM nerves where GalNAc-GD1a is localized, and may function in the pathogenesis of pure motor type GBS. Further investigation is needed to explain the discrepancy between the immunolocalization of GalNAc-GD1a in sensory nerves and the absence of sensory disturbance in patients with GBS with IgG anti-GalNAc-GD1a antibodies.

Extrapituitary GH in the chicken: underestimation of immunohistochemical staining by Carnoy's fixation.

GH has previously been shown to be present in peripheral extrapituitary tIssues of chick embryos, but the cellular distribution of GH immunoreactivity is still uncertain because of differing immunohistochemical findings. The possibility that this uncertainty reflects differences in fixation of the embryonic tIssues was assessed by comparing GH immunoreactivity in tIssues fixed in 4% (w/v) paraformaldehyde or Carnoy's fluid (60% ethanol (v/v); 30% chloroform (v/v); 10% acetic acid (v/v)). A widespread distribution of GH immunoreactivity was seen in paraformaldehyde-fixed tIssues, although it was particularly intense in the spinal cord, dorsal and ventral root ganglia, notochord, myotome, epidermis, crop, heart, lung and humerus. In marked contrast, GH immunoreactivity in embryonic tIssues fixed with Carnoy's was more discrete and mainly restricted to marginal and mantle layers of the spinal cord, spinal nerves, the ventral root ganglia and the extensor nerve of the anterior limb bud. Since these are neural derivatives, Carnoy's fixation appears to preferentially result in neural GH staining, whereas GH staining in neural and non-neural tIssues is seen after paraformaldehyde fixation. Carnoy's, because it is a precipitive fixative, may only fix large GH moieties, whereas GH in peripheral tIssues includes numerous molecular variants, many of which are of relatively small size. Paraformaldehyde, because it is a cross-linking fixative, preferentially fixes peptides and small proteins, and it may therefore fix more GH moieties than Carnoy's fluid. Carnoy's fixation appears to underestimate GH immunoreactivity in immunohistochemical studies on the cellular distribution of GH-like proteins in embryonic chicks.

Crystals of P2 myelin protein in lipid-bound form.

The P2 protein of peripheral nervous system myelin induces experimental allergic neuritis in rats, a model of Guillain-Barré syndrome in humans. Previous purification procedures have used acid extraction to obtain the protein in lipid-free form (LF-P2). Here, we have purified the P2 protein in lipid-bound form (LB-P2) by extracting myelin with the detergent CHAPS, followed by Cu(2+)-affinity column chromatography. All myelin lipids were present in the preparation as shown by high-performance thin-layer chromatography and mass spectrometry. The LB-P2 preparation, which differs from LF-P2 in solubility and in the secondary-structure composition, was dialyzed to remove unbound lipids and excess detergent and crystallized using the hanging-drop vapor diffusion technique. Crystals of lipid-bound P2 appeared usually very reproducibly within 2 weeks at pH 5.7 in polyethylene glycol 6000 (PEG6000) at concentrations of 20-30% (w/v), and larger crystals were obtained by additional sitting-drop crystallization. X-ray diffraction showed reflections up to 2.7A. The crystallization conditions (25-30% PEG6000, pH 5.0) and the unit cell dimensions (a = 94.5A, b = 94.5A, c=74.2A, alpha = beta = 90 degrees, gamma = 120 degrees ) of LB-P2 were different from those earlier described for LF-P2 (10% PEG4000, pH 3, and unit cell dimensions a = 91.8A, b = 99.5A, c = 56.5A, alpha = beta = gamma = 90.0 degrees ). It is important that P2 has been crystallized with specifically bound lipids; therefore, solving this new crystal structure will reveal details of this protein's behavior and role in the myelin sheath.

Repair of ventral root avulsion using autologous nerve grafts in cats.

This study focuses on the capacity of motor axons to elongate from the spinal cord through an autologous nerve graft into a spinal nerve. Applying a ventral surgical approach, C7 ventral roots were avulsed from the cord in 12 cats. Autologous saphenous nerve grafts were implanted into the cord at the ventral root outlet site and coaptated to the spinal nerve. Outgrowth of axons was studied at survival times 7, 14, 30, 60 and 120 days, respectively. The results showed horseradish peroxidase positive motoneurons in the C7 ventral horn after retrograde labeling, as well as neurofilament and acetylcholinesterase positive axons in the entire trajectory from spinal cord to spinal nerve. Neurotization of the C7 spinal nerve started between 14 and 30 days after graft implantation. In addition electrophysiology provided evidence that outgrowing axons had re-established functional contact with the spinodeltoid muscle at 120 days after implantation.

A quantitative immunohistochemical study of the endoneurium in the rat dorsal and ventral spinal roots.

The dorsal and ventral spinal roots contain different types of axons. The endoneurial extracellular matrix (ECM) among them is produced by Schwann cells and fibroblasts under the control of the axons. Chondroitin sulfate proteoglycan, fibronectin, tenascin-C, and thrombospondin are common components of the endoneurial ECM involved in the normal function as well as regeneration of the peripheral nerve. The present paper demonstrates a comparison of immunofluorescence staining for chondroitin sulfate proteoglycan, fibronectin, tenascin-C, and thrombospondin in the endoneurium of the rat dorsal and ventral spinal roots. Sections through the dorsal and ventral roots were cut simultaneously and adhered to the same microscopic slide. They were incubated simultaneously and the intensity of immunofluorescence staining was assessed by computer-assisted image analysis using interactive segmentation of digitized pictures to select the areas of measurement. The measurement of the immunofluorescence brightness revealed that the endoneurium of the dorsal roots was immunostained for the studied molecules at a higher intensity than in the ventral roots. The results suggest quantitative differences of the endoneurial content of the spinal dorsal and ventral roots probably corresponding to the presence of various types of axons. On the other hand, the different concentration of ECM molecules in the endoneurium of dorsal and ventral roots might be related to the formation of extrinsic conditions differently supporting regeneration of afferent and motor axons after their injury.

Expression and localization of insulin receptor in rat dorsal root ganglion and spinal cord.

The expression and localization of the insulin receptor (IR) was examined in rat dorsal root ganglia (DRG) and spinal cord using Western blotting, in situ hybridization and immunocytochemistry. Western blotting showed that the molecular weight of the IR beta subunit was higher in PNS than that found in CNS. Both IR mRNA and protein expressions were highest in small-sized sensory DRG neurons and myelinated sensory root fibers expressed higher levels of IR protein than myelinated anterior root fibers. In the spinal cord, IR immunoreactive neurons were present in lateral lamina V and in lamina X, suggesting the presence of IR in nociceptive pathways. Electronmicroscopy of DRGs revealed a polarized localization of the IR in abaxonal Schwann cell membranes, outer mesaxons in close vicinity to tight junctions of both myelinating and non-myelinating Schwann cells and to plasma membranes of sensory neurons. From these findings, we speculate that insulin may play a role in sensory fibers involved in nociceptive function often perturbed in diabetic neuropathy. The high expression of IR localizing to tight junctions of dorsal root mesaxons of DRGs may suggest a regulatory role on barrier functions compensating for the lack of a blood-nerve barrier in dorsal root ganglia. This is consistent with the colocalization of IR with tight junctions of the paranodal barrier and endoneurial endothelial cells in peripheral nerve.

3-O-acetyl-sphingosine-series myelin glycolipids: characterization of novel 3-O-acetyl-sphingosine galactosylceramide.

Several glycosphingolipids, less polar than galactosylceramide (GalCer), have been purified from rat brain and designated as fast migrating cerebrosides (FMCs). They co-appear with GalCer during myelinogenesis, reach a peak concentration at postnatal day 25-30 and are derivatives of GalCer. Extensive structural analysis of the partially methylated alditol acetates, mass-spectrometry, and (1)H- and (13)C-nuclear magnetic resonance (NMR) spectroscopy unequivocally established the structure of two of these FMCs as 3-O-acetyl-sphingosine GalCer with non-hydroxy and hydroxy fatty N-acylation respectively. That is, an acetyl group is linked at the C3-OH of the sphingosine base of GalCer. In addition, NMR spectroscopy of all of the purified FMCs indicates that they contain a 3-O-acetyl group linked with sphingosine and thus delineates a novel series. Several lines of evidence indicate that FMCs are myelin constituents. FMCs, enriched in both central nervous system (CNS) and peripheral nervous system (PNS) myelin, are concentrated in spinal cord and white matter that are composed of myelinated nerve fibers. There is N-acylation with alpha-hydroxy and C18 and C24 fatty acids, all characteristic of myelin components. They disappear along with GalCer in the murine genetic dysmyelinating disorders, jimpy and quaking, and in a knockout mutant which is devoid of GalCer. In addition, a decrease in FMC and GalCer concentration has been found in Krabbé's disease, a human genetic dysmyelinating disorder.

Nerve fibers in lumbar spine structures and injured spinal roots express the sensory neuron-specific sodium channels SNS/PN3 and NaN/SNS2.

STUDY DESIGN: This prospective study examined the innervation of lumbar spine in tissues from patients with lower back pain and spine nerve roots from patients with traumatic brachial plexus injuries. OBJECTIVES: To demonstrate the presence of nerve fibers in lumbar spine structures and spine nerve roots, and to determine whether they express the sensory neuron-specific sodium channels SNS/PN3 and NaN/SNS2. SUMMARY OF BACKGROUND DATA: The anatomic and molecular basis of low back pain and sciatica is poorly understood. Previous studies have demonstrated sensory nerves in the facet joint capsule and prolapsed intervertebral disc, but not in the ligamentum flavum. The voltage-gated sodium channels SNS/PN3 and NaN/SNS2 are expressed by sensory neurone that mediate pain, but their presence in the lumbar spine is unknown. METHODS: Tissue samples of ligamentum flavum (n = 32), facet joint capsule (n = 20), intervertebral disc (n = 15), and spine roots (n = 8) were immunostained with specific antibodies to protein gene product 9.5 (a panneuronal marker), SNS/PN3, and NaN/SNS2. RESULTS: Protein gene product 9.5 immunoreactive nerve fibers were detected in 72% of the ligamentum flavum specimens and 70% of the facet joint capsule specimens, but in only 20% of the intervertebral disc specimens. The study detected SNS/PN3- and NaN/SNS2-positive fibers, respectively, in 28% and 3% of the ligamentum flavum specimens and 25% and 15% of the facet joint capsule specimens. Numerous SNS/PN3- and NaN/SNS2-positive fibers were found in the acutely injured spine roots, and some were still present in the dorsal roots in the chronic state. CONCLUSIONS: As the findings showed, SNS/PN3- and NaN/SNS2-immunoreactivity is present in a subset of nerve fibers in lumbar spine structures, including ligamentum flavum, and in injured spine roots. Selective SNS/PN3- and NaN/SNS2-blocking agents may provide new therapy for back pain and sciatica.

Computer-assisted quantitative analysis of immunofluorescence staining of the extracellular matrix in rat dorsal and ventral spinal roots.

The endoneurial extracellular matrix (ECM) is produced by Schwann cells and fibroblasts under the control of axons. Dorsal and ventral spinal roots contain different types of axons, but information is not available on differences in the composition of their ECM. A comparison was made of the intensity of immunofluorescence staining of chondroitin sulfate proteoglycan, fibronectin, tenascin and thrombospondin in the endoneurial ECM of rat dorsal and ventral spinal roots. Sections of dorsal and ventral roots were incubated simultaneously for indirect immunofluorescence detection of the epitopes studied. Brightness of immunofluorescence staining was assessed by computer-assisted image analysis using interactive segmentation of digitized images to select areas to be analyzed. Our results revealed quantitative differences in the composition of endoneurial ECM of spinal dorsal and ventral roots, probably due to the presence of different types of axons. The ECM composition of the endoneurium in dorsal and ventral roots may be related with the creation of extrinsic conditions that support differential regeneration of afferent and motor axons after injury.

Intermediate filament proteins in developing and adult human dorsal root and sympathetic ganglia / Las proteínas de los filamentos intermedios en los ganglios simpáticos y de la raíz dorsal humana adulta en desarrollo

Neuronal maturation in the central nervous system, as well as in some cells deriving from neural crest, is accompanied by a swich in the expression of cytoskeletal intermediate filament proteins. Whether this occurs in humans and the exact timing of this change in human dorsal root and sympathetic ganglia are matters still open to debate. The present study was designed to analyze these issues in human embryos (estimated gestational age -e.g.a.- ranging between 6 and 12 weeks), as well as the possible co-expression of more than one intermediate filament protein in both embryos and adults. A panel of commercially available antibodies against vimentin, glial fibrillary acidic protein and neurofilament proteins was used. Glial fibrillary acidic protein was consistently absent in both developing and adult dorsal root or sympathetic ganglia. Conversely, embryonic neurons, satellite glial cells and Schwann cells displayed vimentin immunoreactivity. The number of vimentin immunoreactive neurons decreased progressively, and it was absent from neurons by 12 weeks e.g.a., while it persisted in satellite glial and Schwann cells. By adulthood, the pattern of distribution was identical. The occurrence of neurofilament proteins in peripheral neurons was a regular feature from early developmental stages to adulthood, and a time-dependent increase in the percentage of neurons containing phosphorylated neurofilaments was observed. The present results demonstrate that developing human dorsal root and sympathetic ganglion neurons co-express vimentin and neurofilaments for a short time, but that the intermediate filaments for mature neurons are neurofilaments. Our findings also show that co-expression or a switch in the expression of intermediate filament proteins do not occur in satellite glial cells or Schwann cells, which normally contain vimentin and not glial fibrillary acidic protein (AU)

La maduración neuronal en poblaciones neuronales discretas del sistema central nervioso, así como en algunas células que derivan de la cresta neural es acompañada de un cambio en la expresión de las proteínas citoesqueléticas de filamentos intermedios. El que esto ocurre en los seres humanos y la cronología exacta de este cambio en los ganglios de la raíz dorsal y simpáticos no se ha dilucidado todavía. El presente estudio fue diseñado para analizar estos problemas en embriones humanos (edad gestacional estimada –e.g.e- en el intervalo entre 6 y 12 semanas), así como la posible co-expresión de más de una proteína de filamentos intermedios, tanto en embriones como en adultos. Se utilizó un panel de anticuerpos comerciales frente a la vimentina, a la proteína fibrilar glial ácida, y a las proteínas de los neurofilamentos. La proteína fibrilar glial ácida estaba consistentemente ausente de los ganglios de la raíz dorsal y simpáticos, tanto en el adulto como en embriones en desarrollo. Por el contrario, las neuronas embrionarias, las células gliales satélites y las células de Schwann exhibían inmunoreactividad frente a la vimentina. El número de neuronas con inmunoreactividad para la vimentina descendió progresivamente y este compuesto no se detectaba en las neuronas a una e.g.e. de 12 semanas, mientras que persistía en las células Schwann. Llegada la edad adulta, el patrón de distribución era idéntico. La presencia de proteínas de neurofilamentos en las neuronas periféricas se apreció como una característica consistente a partir de las fases tempranas del desarrollo hasta la edad adulta, observándose un descenso dependiente del tiempo en el porcentaje de neuronas que contenían neurofilamentos fosforilados. Los resultados aquí obtenidos demuestran que las neuronas de los ganglios de la raíz dorsal y simpáticos humanos en desarrollo co-expresan la vimentina y los neurofilamentos durante un periodo corto de tiempo, pero los filamentos intermedios de las neuronas maduras son neurofilamentos. Nuestros hallazgos también muestran que la co-expresión o un cambio en la expresión de las proteínas de los filamentos intermedios no ocurren en las células gliales ni en las de Schwann, las cuales normalmente contienen vimentina y no la proteína fibrilar glial ácida (AU)

Microglial activation, emergence of ED1-expressing cells and clusterin upregulation in the aging rat CNS, with special reference to the spinal cord.

With advancing age, the incidence of neuronal atrophy and dystrophy increases and, in parallel, behavioural sensorimotor impairment becomes overt. Activated microglia has been implicated in cytotoxic and inflammatory processes in neurodegenerative diseases as well as during aging. Here we have used immunohistochemistry and in situ hybridization to examine the expression of OX42, ED1, ED2, GFAP and clusterin in CNS of young adult and behaviourally tested aged rats (30-month-old), to study the occurrence of activated microglia/ED1 positive macrophages in senescence and to what extent this correlates with astrogliosis and signs of sensorimotor impairment among the individuals. The results show a massive region-specific increase in activated microglia and ED1 expressing cell profiles in aged rats. The infiltration was most prominent in the spinal cord dorsal columns, including their sensory relay nuclei, and the outer portions of the lateral and ventral columns. At such sites the occurrence of macrophages coincided with increased levels of GFAP and positive correlations were evident between the labeling for, on the one hand, OX42 and, on the other, GFAP and ED1. Also, the ventral and dorsal roots were heavily infiltrated by ED1 positive cells. The signs of gliosis were most pronounced among aged rats with advanced sensorimotor impairment. In contrast, the grey matter of aged rats showed very few activated microglia/ED1 labeled cells despite signs of focal astrogliosis. ED2 expression was confined to perivascular cells and leptominges with a similar labeling pattern in young and aged rats. In aged rats increased expression of clusterin was observed in GFAP-immunoreactive profiles of the white matter only. It is suggested that this increase may reflect a response to degenerative/inflammatory processes.