Rescuing axons from degeneration does not affect retinal ganglion cell death.

After a traumatic injury to the central nervous system, the distal stumps of axons undergo Wallerian degeneration (WD), an event that comprises cytoskeleton and myelin breakdown, astrocytic gliosis, and overexpression of proteins that inhibit axonal regrowth. By contrast, injured neuronal cell bodies show features characteristic of attempts to initiate the regenerative process of elongating their axons. The main molecular event that leads to WD is an increase in the intracellular calcium concentration, which activates calpains, calcium-dependent proteases that degrade cytoskeleton proteins. The aim of our study was to investigate whether preventing axonal degeneration would impact the survival of retinal ganglion cells (RGCs) after crushing the optic nerve. We observed that male Wistar rats (weighing 200-400 g; n=18) treated with an exogenous calpain inhibitor (20 mM) administered via direct application of the inhibitor embedded within the copolymer resin Evlax immediately following optic nerve crush showed a delay in the onset of WD. This delayed onset was characterized by a decrease in the number of degenerated fibers (P<0.05) and an increase in the number of preserved fibers (P<0.05) 4 days after injury. Additionally, most preserved fibers showed a normal G-ratio. These results indicated that calpain inhibition prevented the degeneration of optic nerve fibers, rescuing axons from the process of axonal degeneration. However, analysis of retinal ganglion cell survival demonstrated no difference between the calpain inhibitor- and vehicle-treated groups, suggesting that although the calpain inhibitor prevented axonal degeneration, it had no effect on RGC survival after optic nerve damage.

Rescuing axons from degeneration does not affect retinal ganglion cell death

After a traumatic injury to the central nervous system, the distal stumps of axons undergo Wallerian degeneration (WD), an event that comprises cytoskeleton and myelin breakdown, astrocytic gliosis, and overexpression of proteins that inhibit axonal regrowth. By contrast, injured neuronal cell bodies show features characteristic of attempts to initiate the regenerative process of elongating their axons. The main molecular event that leads to WD is an increase in the intracellular calcium concentration, which activates calpains, calcium-dependent proteases that degrade cytoskeleton proteins. The aim of our study was to investigate whether preventing axonal degeneration would impact the survival of retinal ganglion cells (RGCs) after crushing the optic nerve. We observed that male Wistar rats (weighing 200-400 g; n=18) treated with an exogenous calpain inhibitor (20 mM) administered via direct application of the inhibitor embedded within the copolymer resin Evlax immediately following optic nerve crush showed a delay in the onset of WD. This delayed onset was characterized by a decrease in the number of degenerated fibers (P<0.05) and an increase in the number of preserved fibers (P<0.05) 4 days after injury. Additionally, most preserved fibers showed a normal G-ratio. These results indicated that calpain inhibition prevented the degeneration of optic nerve fibers, rescuing axons from the process of axonal degeneration. However, analysis of retinal ganglion cell survival demonstrated no difference between the calpain inhibitor- and vehicle-treated groups, suggesting that although the calpain inhibitor prevented axonal degeneration, it had no effect on RGC survival after optic nerve damage.

Double gene therapy with granulocyte colony-stimulating factor and vascular endothelial growth factor acts synergistically to improve nerve regeneration and functional outcome after sciatic nerve injury in mice.

Peripheral-nerve injuries are a common clinical problem and often result in long-term functional deficits. Reconstruction of peripheral-nerve defects is currently undertaken with nerve autografts. However, there is a limited availability of nerves that can be sacrificed and the functional recovery is never 100% satisfactory. We have previously shown that gene therapy with vascular endothelial growth factor (VEGF) significantly improved nerve regeneration, neuronal survival, and muscle activity. Our hypothesis is that granulocyte colony-stimulating factor (G-CSF) synergizes with VEGF to improve the functional outcome after sciatic nerve transection. The left sciatic nerves and the adjacent muscle groups of adult mice were exposed, and 50 or 100 µg (in 50 µl PBS) of VEGF and/or G-CSF genes was injected locally, just below the sciatic nerve, and transferred by electroporation. The sciatic nerves were transected and placed in an empty polycaprolactone (PCL) nerve guide, leaving a 3-mm gap to challenge nerve regeneration. After 6 weeks, the mice were perfused and the sciatic nerve, the dorsal root ganglion (DRG), the spinal cord and the gastrocnemius muscle were processed for light and transmission electron microscopy. Treated animals showed significant improvement in functional and histological analyses compared with the control group. However, the best results were obtained with the G-CSF+VEGF-treated animals: quantitative analysis of regenerated nerves showed a significant increase in the number of myelinated fibers and blood vessels, and the number of neurons in the DRG and motoneurons in the spinal cord was significantly higher. Motor function also showed that functional recovery occurred earlier in animals receiving G-CSF+VEGF-treatment. The gastrocnemius muscle showed an increase in weight and in the levels of creatine phosphokinase, suggesting an improvement of reinnervation and muscle activity. These results suggest that these two factors acted synergistically and optimized the nerve repair potential, improving regeneration after a transection lesion.

Neuroproteomics: an insight into ALS.

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease of unknown aetiology. Diagnosis is made through physical examination, electrophysiological findings, and by excluding other conditions. There is not a single biomarker that concludes the diagnosis. The aim of this study was to investigate differentially expressed proteins in cerebrospinal fluid (CSF) of ALS patients compared to control subjects, with the purpose to identify a panel of possible biomarkers for the disease. The differentially expressed spots/proteins were submitted to two-dimensional (2D) electrophoresis and recognized with matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) mass spectrometry. Parkin-like and many iron and zinc binding were some of the proteins found in ALS CSF. Parkin is a ligase involved in ubiquitin-proteasome pathway and mutations in the parkin gene are the most common cause of recessive familial Parkinson's disease. Iron and zinc are involved with many important metabolic processes and are related to neurodegenerative disease. Common features of ALS comprise failure of the ubiquitin-proteasome system and increased levels of metal ions in the brain. Therefore, the identification of these proteins can be a significant step in ALS research. These and other identified proteins are discussed in this study.

Pulsed ultrasound therapy accelerates the recovery of skeletal muscle damage induced by Bothrops jararacussu venom

We studied the effect of pulsed ultrasound therapy (UST) and antibothropic polyvalent antivenom (PAV) on the regeneration of mouse extensor digitorum longus muscle following damage by Bothrops jararacussu venom. Animals (Swiss male and female mice weighing 25.0 ± 5.0 g; 5 animals per group) received a perimuscular injection of venom (1 mg/kg) and treatment with UST was started 1 h later (1 min/day, 3 MHz, 0.3 W/cm², pulsed mode). Three and 28 days after injection, muscles were dissected and processed for light microscopy. The venom caused complete degeneration of muscle fibers. UST alone and combined with PAV (1.0 mL/kg) partially protected these fibers, whereas muscles receiving no treatment showed disorganized fascicules and fibers with reduced diameter. Treatment with UST and PAV decreased the effects of the venom on creatine kinase content and motor activity (approximately 75 and 48%, respectively). Sonication of the venom solution immediately before application decreased the in vivo and ex vivo myotoxic activities (approximately 60 and 50%, respectively). The present data show that UST counteracts some effects of B. jararacussu venom, causing structural and functional improvement of the regenerated muscle after venom injury.

Pulsed ultrasound therapy accelerates the recovery of skeletal muscle damage induced by Bothrops jararacussu venom.

We studied the effect of pulsed ultrasound therapy (UST) and antibothropic polyvalent antivenom (PAV) on the regeneration of mouse extensor digitorum longus muscle following damage by Bothrops jararacussu venom. Animals (Swiss male and female mice weighing 25.0 ± 5.0 g; 5 animals per group) received a perimuscular injection of venom (1 mg/kg) and treatment with UST was started 1 h later (1 min/day, 3 MHz, 0.3 W/cm(2), pulsed mode). Three and 28 days after injection, muscles were dissected and processed for light microscopy. The venom caused complete degeneration of muscle fibers. UST alone and combined with PAV (1.0 mL/kg) partially protected these fibers, whereas muscles receiving no treatment showed disorganized fascicules and fibers with reduced diameter. Treatment with UST and PAV decreased the effects of the venom on creatine kinase content and motor activity (approximately 75 and 48%, respectively). Sonication of the venom solution immediately before application decreased the in vivo and ex vivo myotoxic activities (approximately 60 and 50%, respectively). The present data show that UST counteracts some effects of B. jararacussu venom, causing structural and functional improvement of the regenerated muscle after venom injury.

Estradiol modulates TGF-ß1 expression and its signaling pathway in thyroid stromal cells.

The higher prevalence of thyroid disease in women suggests that estrogen (E2) might be involved in the pathophysiology of thyroid dysfunction. To approach the question of the effect of stromal cells in the modulation of thyroid epithelial cells activity, we established and characterized a homogeneous stromal cell population (TS7 cells) of rat thyroid gland. These fibroblastic cells synthesize the cytoskeleton proteins α-smooth muscle actin and vimentin, produce basement membrane components and express the cytokine transforming growth factor beta 1 (TGF-ß1). Here, we hypothesized that the effects of E2 on follicular thyroid cells are mediated by TGF-ß1 synthesis and secretion by stromal cells (paracrine action). Thus we investigated the effect of E2 on TGF-ß1 synthesis and its signaling pathway in TS7 cells. In addition, we analyzed the role of TGF-ß1 signaling pathway as mediator of TS7-PC CL3 thyroid epithelial cells interactions. We report that TS7 stromal cells expressed α and ß estrogen receptors (ERα and ERß). Further, both isoforms of TGF-ß1 receptors, TGFRI and TGFRII, were also identified in TS7 cells, suggesting that these cells might be a target for this cytokine in vitro. Treatment of TS7 cells with E2 induced both synthesis and secretion of TGF-ß1. This event was followed by phosphorylation of the transcription factor Smad2, a hallmark of TGF-ß1 pathway activation. Co-culture of PC CL3 cells onto TS7 cells monolayers yielded round aggregates of PC CL3 cells surrounded by TS7 cells. TS7 cells induced a decrease in iodide uptake by PC CL3 cells, probably by a mechanism involving TGF-ß1. Moreover, E2 affected synthesis and organization of the extracellular matrix (ECM) components, tenascin C and chondroitin sulfate, in these co-culture cells. Our results point to the TGF-ß1/Smad-2 signaling pathway as a putative target of estrogen actions on thyroid stromal cells and contribute to understanding the interplay between stromal and follicular cells in thyroid physiology.

Enhancement of sciatic nerve regeneration after vascular endothelial growth factor (VEGF) gene therapy.

AIMS: Recent studies have emphasized the beneficial effects of the vascular endothelial growth factor (VEGF) on neurone survival and Schwann cell proliferation. VEGF is a potent angiogenic factor, and angiogenesis has long been recognized as an important and necessary step during tissue repair. Here, we investigated the effects of VEGF on sciatic nerve regeneration. METHODS: Using light and electron microscopy, we evaluated sciatic nerve regeneration after transection and VEGF gene therapy. We examined the survival of the neurones in the dorsal root ganglia and in lumbar 4 segment of spinal cord. We also evaluated the functional recovery using the sciatic functional index and gastrocnemius muscle weight. In addition, we evaluated the VEGF expression by immunohistochemistry. RESULTS: Fluorescein isothiocyanate-dextran (FITC-dextran) fluorescence of nerves and muscles revealed intense staining in the VEGF-treated group. Quantitative analysis showed that the numbers of myelinated fibres and blood vessels were significantly higher in VEGF-treated animals. VEGF also increased the survival of neurone cell bodies in dorsal root ganglia and in spinal cord. The sciatic functional index and gastrocnemius muscle weight reached significantly higher values in VEGF-treated animals. CONCLUSION: We demonstrate a positive relationship between increased vascularization and enhanced nerve regeneration, indicating that VEGF administration can support and enhance the growth of regenerating nerve fibres, probably through a combination of angiogenic, neurotrophic and neuroprotective effects.

Prevalence of hepatitis C virus among users attending a voluntary testing centre in Rio Grande, southern Brazil: predictive factors and hepatitis C virus genotypes.

We estimated the prevalence of hepatitis C (HCV) infection and associated risk factors in 750 individuals attending the Voluntary Counseling and Testing Center of Rio Grande (VCT/RG), in Southern Brazil, and identified viral genotypes. Demographic data and risk factors for HCV transmission were also collected and analysed. Anti-HCV antibody-positive individuals were tested for HCV-RNA and genotyped by sequencing the 5' untranslated region of the viral genome. Prevalence estimates of anti-HCV and HCV-RNA were 6% and 5.5%, respectively. We identified genotypes 1 (67%), 2 (2%) and 3 (31%); the latter was more prevalent than in other regions of Brazil. Anti-HCV prevalence in VCT/RG users was similar to previous reports. Age, previous blood transfusion, sexual orientation and injecting drug use were independent predictors of HCV infection. The presence of multiple risk factors was also associated with a higher risk for HCV infection. HCV genotype was not associated with any variable analysed in this study.

Mesenchymal stem cells in a polycaprolactone conduit enhance median-nerve regeneration, prevent decrease of creatine phosphokinase levels in muscle, and improve functional recovery in mice.

Although the majority of peripheral-nerve regeneration studies are carried out on the sciatic nerve, lesions of the upper extremities are more common in humans and usually lead to significant physical disabilities. The present study was driven by the hypothesis that a combination of strategies, namely grafts of mesenchymal stem cells (MSC) and resorbable polycaprolactone (PCL) conduits would improve median-nerve regeneration after transection. Mouse median nerves were transected and sutured to PCL tubes that were filled with either green fluorescent protein (GFP(+)) MSC in DMEM or with DMEM alone. During the post-operative period, animals were tested weekly for flexor digitorum muscle function by means of the grasping test. After 8 weeks, the proximal and middle portions of the PCL tube and the regenerating nerves were harvested and processed for light and electron microscopy. The flexor digitorum muscle was weighed and subjected to biochemical analysis for creatine phosphokinase (CK) levels. Scanning electron microscopy of the PCL tube 8 weeks after implantation showed clear signs of wall disintegration. MSC-treated animals showed significantly larger numbers of myelinated and unmyelinated nerve fibers and blood vessels compared with DMEM-treated animals. The flexor digitorum muscle CK levels were significantly higher in the MSC-treated animals, but muscle weight values did not differ between the groups. Compared with the DMEM-treated group, MSC-treated animals showed, by the grasping test, improved functional performance throughout the period analyzed. Immunofluorescence for S-100 and GFP showed, in a few cases, double-labeled cells, suggesting that transplanted cells may occasionally transdifferentiate into Schwann cells. Our data demonstrate that the polycaprolactone conduit filled with MSC is capable of significantly improving the median-nerve regeneration after a traumatic lesion.

HIV-1 vertical transmission in Rio Grande, Southern Brazil.

The aim of this study was to determine the rate and risk factors of HIV-1 mother-to-child transmission (MTCT), the timing of transmission and the transmitted subtype in a population where subtypes B and C co-circulate. One hundred and forty-four babies born to HIV-1-infected mothers were studied. Subtype and timing of transmission were determined by a nested polymerase chain reaction of the gp41 gene. Seven children were infected (4.9%): four were infected intrautero and one intrapartum. The higher frequency of intrautero transmission was statistically significant (P = 0.001). Use of antiretrovirals (ARVs) in the three stages of gestation was a protective risk factor for MTCT (PR = 0.42; CI: 0.21-0.83; P = 0.013). A higher HIV viral load at delivery was the only independent risk factor for MTCT. Early and universal access to ARVs during pregnancy are the most important measures to decrease vertical HIV-1 transmission even in areas where HIV clade distribution differs.

Diversity among satellite glial cells in dorsal root ganglia of the rat.

Peripheral glial cells consist of satellite, enteric glial, and Schwann cells. In dorsal root ganglia, besides pseudo-unipolar neurons, myelinated and nonmyelinated fibers, macrophages, and fibroblasts, satellite cells also constitute the resident components. Information on satellite cells is not abundant; however, they appear to provide mechanical and metabolic support for neurons by forming an envelope surrounding their cell bodies. Although there is a heterogeneous population of neurons in the dorsal root ganglia, satellite cells have been described to be a homogeneous group of perineuronal cells. Our objective was to characterize the ultrastructure, immunohistochemistry, and histochemistry of the satellite cells of the dorsal root ganglia of 17 adult 3-4-month-old Wistar rats of both genders. Ultrastructurally, the nuclei of some satellite cells are heterochromatic, whereas others are euchromatic, which may result from different amounts of nuclear activity. We observed positive immunoreactivity for S-100 and vimentin in the cytoplasm of satellite cells. The intensity of S-100 protein varied according to the size of the enveloped neuron. We also noted that vimentin expression assumed a ring-like pattern and was preferentially located in the cytoplasm around the areas stained for S-100. In addition, we observed nitric oxide synthase-positive small-sized neurons and negative large-sized neurons equal to that described in the literature. Satellite cells were also positive for NADPH-diaphorase, particularly those associated with small-sized neurons. We conclude that all satellite cells are not identical as previously thought because they have different patterns of glial marker expression and these differences may be correlated with the size and function of the neuron they envelope.

Diversity among satellite glial cells in dorsal root ganglia of the rat

Peripheral glial cells consist of satellite, enteric glial, and Schwann cells. In dorsal root ganglia, besides pseudo-unipolar neurons, myelinated and nonmyelinated fibers, macrophages, and fibroblasts, satellite cells also constitute the resident components. Information on satellite cells is not abundant; however, they appear to provide mechanical and metabolic support for neurons by forming an envelope surrounding their cell bodies. Although there is a heterogeneous population of neurons in the dorsal root ganglia, satellite cells have been described to be a homogeneous group of perineuronal cells. Our objective was to characterize the ultrastructure, immunohistochemistry, and histochemistry of the satellite cells of the dorsal root ganglia of 17 adult 3-4-month-old Wistar rats of both genders. Ultrastructurally, the nuclei of some satellite cells are heterochromatic, whereas others are euchromatic, which may result from different amounts of nuclear activity. We observed positive immunoreactivity for S-100 and vimentin in the cytoplasm of satellite cells. The intensity of S-100 protein varied according to the size of the enveloped neuron. We also noted that vimentin expression assumed a ring-like pattern and was preferentially located in the cytoplasm around the areas stained for S-100. In addition, we observed nitric oxide synthase-positive small-sized neurons and negative large-sized neurons equal to that described in the literature. Satellite cells were also positive for NADPH-diaphorase, particularly those associated with small-sized neurons. We conclude that all satellite cells are not identical as previously thought because they have different patterns of glial marker expression and these differences may be correlated with the size and function of the neuron they envelope.

Topographic trace-elemental analysis in the brain of Wistar rats by X-ray microfluorescence with synchrotron radiation.

Knowledge about the spatial distribution and the local concentration of trace elements in tissues is of great importance, since trace elements are involved in many biological functions of living organisms. However, there are few methods available to measure the spatial (two (three)-dimensional) elemental distribution in animal brain. X-ray microfluorescence with synchrotron radiation is a multielemental mapping technique, which was used in this work to determine the topographic of iron, zinc and copper in coronal sections of female Wistar rats of different ages. Young (14 days old) and middle-aged (20 months old) rats (n = 8) were analyzed. The measurements were carried out at the XRF beam line at the Synchrotron Light National Laboratory (Campinas, Brazil). Two-dimensional scanning was performed in order to study the tendency of elemental concentration variation. The acquisition time for each pixel was 10 s/step and the step size was 300 microm/step in both directions. It was observed that the iron distribution was more conspicuous in the cortical area, thalamus and bellow the thalamus. On the other hand, the zinc distribution was more pronounced in the hippocampus. The iron, copper and zinc levels increased with advancing age. Therefore, this study reinforces the idea that these elements are involved in the chemical mechanisms of the brain that induce some neurological diseases, since they are also present in high levels in specific areas of the brain, such as the hippocampus and the substantia nigra of patients with these disorders.

Modulation of extracellular matrix components by metalloproteinases and their tissue inhibitors during degeneration and regeneration of rat sural nerve.

The success of peripheral nervous system regeneration has been associated with changes on the microenvironment, particularly on the extracellular matrix (ECM) components. In the present study we analyzed by indirect immunohistochemistry, electron microscopy and Western blotting, the distribution of ECM components, metalloproteinases (MMPs) and their tissue inhibitors (TIMPs), during Wallerian degeneration (WD) and nerve regeneration (2nd, 7th and 21st days after injury) on crushed rat sural nerves. Our results showed that laminin alpha3-chain and alpha2-chain are over expressed during the early stages of degeneration and regeneration respectively, whereas type IV collagen expression increased progressively after crush. On the other hand, the expression of chondroitin sulfate was down regulated during the early stages of degeneration, returning progressively to normal values during nerve regeneration. The expression of MMP-3 was almost normal immediately after lesion, and then reduced progressively achieving the smallest expression at 21 days after crush; on the contrary, the expression of MMP-7 increased significantly immediately after crush (2nd day) returning to normal values afterwards. TIMP-1 and TIMP-2 were over expressed at the beginning of WD, returning progressively to normal values after that. These results indicate that the modifications of ECM components, which are favorable for nerve regeneration, are correlated with changes on the balance between MMPs and TIMPs.

Expression of ubiquitin and proteasome in motorneurons and astrocytes of spinal cords from patients with amyotrophic lateral sclerosis.

Proteasome, ubiquitin, GFAP and neurofilament were evaluated in motorneurons and astrocytes of spinal cords of ALS and control cases. ALS neurons exhibited ubiquitin positive inclusions and areas of strong immunoreaction for proteasome. Areas of proteasome stain were observed close to neurofilament positive proximal process enlargement. The percentage of neurons strongly immunoreacted, for proteasome was higher in ALS cases than in controls. Many astrocytes were positive for ubiquitin and proteasome. These results suggest that the ubiquitin-proteasome pathway is involved in the ALS pathogenesis and agree with the view that ALS is a disorder of protein aggregation that affects neurons and nonneuronal cells.

Quantitative ultrastructural evidence of myelin malformation in optic nerves of rats submitted to a multideficient diet.

Pups were subjected to malnutrition by feeding the lactating mothers a multi-deficient (8% protein content) diet, known as regional basic diet (RBD), from birth up to weaning. The weanings were fed the same diet until 60 days of age. Ultrastructure of the optic nerve was analyzed at postnatal (P) day P8, P13, P21, P30 and P60. Electron microscopy revealed that at P8 the process of myelination has not started yet in neither groups. At P 13 different stages of myelination were observed and, in the experimental group, the optic nerve showed non-organized axon bundles and empty spaces. Control optic nerve at P21 exhibited axons with fully developed myelin sheath; whereas malnourished group had many axons being enveloped by myelin with anomalous alteration. These alterations were present in malnourished group at P30 and P60. Quantitative analysis showed statistically significant difference between control and malnourished groups when compared to the percentage of myelinated axons, axons with myelin anomalous alterations (MAA) and non-myelinated axons. Also, the myelin area was significantly smaller in malnourished groups when compared to control group. Finally, a high percentage of large non-myelinated fibers were found in the malnourished group. In conclusion, our results show that early malnutrition by a multideficient diet (RBD) affects permanently the optic nerve organization and myelination, indicating an impairment of nerve transmission and a probable dysfunction in the visual ability.

Quantitative evidence for neurofilament heavy subunit aggregation in motor neurons of spinal cords of patients with amyotrophic lateral sclerosis.

Amyotrophic lateral sclerosis (ALS), a neurodegenerative disease of unknown etiology, affects motor neurons leading to atrophy of skeletal muscles, paralysis and death. There is evidence for the accumulation of neurofilaments (NF) in motor neurons of the spinal cord in ALS cases. NF are major structural elements of the neuronal cytoskeleton. They play an important role in cell architecture and differentiation and in the determination and maintenance of fiber caliber. They are composed of three different polypeptides: light (NF-L), medium (NF-M) and heavy (NF-H) subunits. In the present study, we performed a morphological and quantitative immunohistochemical analysis to evaluate the accumulation of NF and the presence of each subunit in control and ALS cases. Spinal cords from patients without neurological disease and from ALS patients were obtained at autopsy. In all ALS cases there was a marked loss of motor neurons, besides atrophic neurons and preserved neurons with cytoplasmic inclusions, and extensive gliosis. In control cases, the immunoreaction in the cytoplasm of neurons was weak for phosphorylated NF-H, strong for NF-M and weak for NF-L. In ALS cases, anterior horn neurons showed intense immunoreactivity in focal regions of neuronal perikarya for all subunits, although the difference in the integrated optical density was statistically significant only for NF-H. Furthermore, we also observed dilated axons (spheroids), which were immunopositive for NF-H but negative for NF-M and NF-L. In conclusion, we present qualitative and quantitative evidence of NF-H subunit accumulation in neuronal perikarya and spheroids, which suggests a possible role of this subunit in the pathogenesis of ALS.

Quantitative evidence for neurofilament heavy subunit aggregation in motor neurons of spinal cords of patients with amyotrophic lateral sclerosis

Amyotrophic lateral sclerosis (ALS), a neurodegenerative disease of unknown etiology, affects motor neurons leading to atrophy of skeletal muscles, paralysis and death. There is evidence for the accumulation of neurofilaments (NF) in motor neurons of the spinal cord in ALS cases. NF are major structural elements of the neuronal cytoskeleton. They play an important role in cell architecture and differentiation and in the determination and maintenance of fiber caliber. They are composed of three different polypeptides: light (NF-L), medium (NF-M) and heavy (NF-H) subunits. In the present study, we performed a morphological and quantitative immunohistochemical analysis to evaluate the accumulation of NF and the presence of each subunit in control and ALS cases. Spinal cords from patients without neurological disease and from ALS patients were obtained at autopsy. In all ALS cases there was a marked loss of motor neurons, besides atrophic neurons and preserved neurons with cytoplasmic inclusions, and extensive gliosis. In control cases, the immunoreaction in the cytoplasm of neurons was weak for phosphorylated NF-H, strong for NF-M and weak for NF-L. In ALS cases, anterior horn neurons showed intense immunoreactivity in focal regions of neuronal perikarya for all subunits, although the difference in the integrated optical density was statistically significant only for NF-H. Furthermore, we also observed dilated axons (spheroids), which were immunopositive for NF-H but negative for NF-M and NF-L. In conclusion, we present qualitative and quantitative evidence of NF-H subunit accumulation in neuronal perikarya and spheroids, which suggests a possible role of this subunit in the pathogenesis of ALS.

Experimental chronic entrapment of the sciatic nerve in adult hamsters: an ultrastructural and morphometric study

Entrapment neuropathy is a group of clinical disorders involving compression of a peripheral nerve and interference with nerve function mostly through traction injury. We have investigated the chronic compression of peripheral nerves as an experimental procedure for detecting changes in ultrastructural nerve morphology. Adult hamsters (Mesocricetus auratus, N = 30) were anesthetized with a 25 percent pentobarbital solution and received a cuff around the right sciatic nerve. Left sciatic nerves were not operated (control group). Animals survived for varying times (up to 15 weeks), after which they were sacrificed and both sciatic nerves were immediately fixed with a paraformaldehyde solution. Experimental nerves were divided into segments based upon their distance from the site of compression (proximal, entrapment and distal). Semithin and ultrathin sections were obtained and examined by light and electron microscopy. Ultrastructural changes were qualitatively described and data from semithin sections were morphometrically analyzed both in control and in compressed nerves. We observed endoneurial edema along with both perineurial and endoneurial thickening and also the existence of whorled cell-sparse structures (Renaut bodies) in the subperineurial space of compressed sciatic nerves. Morphometric analyses of myelinated axons at the compression sites displayed a remarkable increase in the number of small axons (up to 60 percent) in comparison with the control axonal number. The distal segment of compressed nerves presented a distinct decrease in axon number (up to 40 percent) comparatively to the control group. The present experimental model of nerve entrapment in adult hamsters was shown to promote consistent histopathologic alterations analogous to those found in chronic compressive neuropathies