Neuroprotective Effect of Sterculia setigera Leaves Hydroethanolic Extract.

Plants are a valuable source of information for pharmacological research and new drug discovery. The present study aimed to evaluate the neuroprotective potential of the leaves of the medicinal plant Sterculia setigera. In vitro, the effect of Sterculia setigera leaves dry hydroethanolic extract (SSE) was tested on cultured cerebellar granule neurons (CGN) survival when exposed to hydrogen peroxide (H2O2) or 6-hydroxydopamine (6-OHDA), using the viability probe fluorescein diacetate (FDA), a lactate dehydrogenase (LDH) activity assay, an immunocytochemical staining against Gap 43, and the quantification of the expression of genes involved in apoptosis, necrosis, or oxidative stress. In vivo, the effect of intraperitoneal (ip) injection of SSE was assessed on the developing brain of 8-day-old Wistar rats exposed to ethanol neurotoxicity by measuring caspase-3 activity on cerebellum homogenates, the expression of some genes in tissue extracts, the thickness of cerebellar cortical layers and motor coordination. In vitro, SSE protected CGN against H2O2 and 6-OHDA-induced cell death at a dose of 10 µg/mL, inhibited the expression of genes Casp3 and Bad, and upregulated the expression of Cat and Gpx7. In vivo, SSE significantly blocked the deleterious effect of ethanol by reducing the activity of caspase-3, inhibiting the expression of Bax and Tp53, preventing the reduction of the thickness of the internal granule cell layer of the cerebellar cortex, and restoring motor functions. Sterculia setigera exerts neuroactive functions as claimed by traditional medicine and should be a good candidate for the development of a neuroprotective treatment against neurodegenerative diseases.

The effects of exercise training on Kinesin and GAP-43 expression in skeletal muscle fibers of STZ-induced diabetic rats.

Kinesin-1 and Growth Associated Protein 43 (GAP-43) localization in muscle fiber are crucial for proper skeletal muscle hypertrophy. To evaluate this assumption, we investigated the beneficial effects of endurance training on GAP-43 and Kinesin Family Member 5B (KIF5B) expression in gastrocnemius muscle of streptozotocin (STZ)-induced diabetic rats. Fifty-two male rats were randomly divided into four groups: healthy control (C), healthy trained (T), diabetic control (DC) and diabetic trained (DT). Diabetes was induced by a single intraperitoneal injection of STZ (45 mg/kg). The rats in DT and T groups were subjected to treadmill running for 5 days a week over 6 weeks. The results indicated that the GAP-43 and KIF5B protein levels in the DC group were significantly lower than those in the C group. Additionally, chronic treadmill running in diabetic rats was accompanied by significant increase of GAP-43 and KIF5B protein expression, compared to DC group. Furthermore, the endurance training in healthy rats was associated with a significant increase of GAP-43 and KIF5B protein levels. In addition, we found positive correlation between GAP-43 and KIF5B protein levels and myonuclear number per fiber and average gastrocnemius cross-sectional area (CSA). GAP43 and KIF5B protein levels were decreased in skeletal muscles of diabetic rats, and exercise training had beneficial effects and could restore their abnormal expression. Moreover, there is a strong relationship between muscle hypertrophy and GAP-43 and KIF5B protein levels.

Urokinase-type plasminogen activator (uPA) regulates the expression and function of growth-associated protein 43 (GAP-43) in the synapse.

Growth-associated protein 43 (GAP-43) plays a central role in the formation of presynaptic terminals, synaptic plasticity, and axonal growth and regeneration. During development, GAP-43 is found in axonal extensions of most neurons. In contrast, in the mature brain, its expression is restricted to a few presynaptic terminals and scattered axonal growth cones. Urokinase-type plasminogen activator (uPA) is a serine proteinase that, upon binding to its receptor (uPAR), catalyzes the conversion of plasminogen into plasmin and activates signaling pathways that promote cell migration, proliferation, and survival. In the developing brain, uPA induces neuritogenesis and neuronal migration. In contrast, the expression and function of uPA in the mature brain are poorly understood. However, recent evidence reveals that different forms of injury induce release of uPA and expression of uPAR in neurons and that uPA/uPAR binding triggers axonal growth and synapse formation. Here we show that binding of uPA to uPAR induces not only the mobilization of GAP-43 from the axonal shaft to the presynaptic terminal but also its activation in the axonal bouton by PKC-induced calcium-dependent phosphorylation at Ser-41 (pGAP-43). We found that this effect requires open presynaptic N-methyl-d-aspartate receptors but not plasmin generation. Furthermore, our work reveals that, following its activation by uPA/uPAR binding, pGAP-43 colocalizes with presynaptic vesicles and triggers their mobilization to the synaptic release site. Together, these data reveal a novel role of uPA as an activator of the synaptic vesicle cycle in cerebral cortical neurons via its ability to induce presynaptic recruitment and activation of GAP-43.

Geniposide ameliorated fluoxetine-suppressed neurite outgrowth in Neuro2a neuroblastoma cells.

AIM: Fluoxetine (FXT), a selective serotonin reuptake inhibitor (SSRI), is one of the most common psychiatric medications clinically prescribed; while over-produced serotonin may suppress neurite development. The role of major iridoids like geniposide (GPS) and genipin (GNP) from Gardenia jasminoides Ellis fruit (family Rubiaceae) in ameliorating the anti-neurite outgrowth effect of FXT is poorly understood. In this study, the effects of these iridoids on FXT-suppressed neurite outgrowth in Neuro2a neuroblastoma cells were investigated. MAIN METHODS: Neuro2a cells were treated with FXT and GPS. The effect of GPS-FXT co-treatment on neurite outgrowth was observed using inverted phase-contrast microscope imaging system, while neurite outgrowth markers - microtubule-associated protein-2 (MAP2) and growth-associated protein 43 (GAP43) were analyzed using RT-PCR, Western blot and immunofluorescence staining. The transcription factor-cAMP response element binding (CREB), and signaling pathways - mitogen-activated protein kinase (MAPK) and protein kinase B/mammalian target of rapamycin (AKT/mTOR) were also analyzed with the help of Western blot. KEY FINDINGS: The results showed that FXT decreased the neurite outgrowth in Neuro2a cells and also downregulated gene and protein expression of MAP2 and GAP43. It also downregulated the protein expression of phosphorylated-CREB, MAPK, and AKT/mTOR signaling pathways. In contrast, GPS counteracted the effects of FXT. GPS-FXT co-treatment increased the percentage of neurite-bearing cells by 3.6-fold at 200 µM as compared to FXT treatment only. SIGNIFICANCE: This study has provided the possible molecular mechanism showing how FXT exerted its detrimental side-effects on the neurite differentiation, and via the same mechanism how GPS attenuated these side effects.

[The histopathology and immunohistochemistry of granular cell tumour. A study of 12 cases with a brief historical note]. / Análisis histopatológico e inmunohistoquímico del tumor de células granulares. Estudios de 12 casos con una breve nota histórica.

INTRODUCTION AND OBJECTIVE: Granular cell tumour (GCT) is a benign neoplasm of neural/schwannian origin, usually presenting as a single asymptomatic lesion, mainly located in the dermis and subcutaneous tissue or submucosa, although multiple tumours may occur. Microscopically, GCTs are composed of large cells with abundant eosinophilic, granular cytoplasm arranged in sheets, nests, cords or trabeculae. Based on the cytological characteristics and the presence of necrosis, three types are recognized: benign, atypical and malignant. We aim to present the cytological and immunohistochemical characteristics of 12 granular cell tumours. MATERIALS AND METHODS: 12 cases of GCT were selected from the consultation files of one of the authors (COH) The paraffin embedded tissue was processed for immunostaining with S-100 protein, calretinin, CD68, α-inhibin, PGP9.5, CD57 (Leu7), CD63 (NKI / C3), Gap43 (growth-associated protein-43), SOX10, TFE-3 and Ki-67. RESULTS AND CONCLUSIONS: 6 male and 6 female patients, with an average age of 40, made up the study group. The most frequent location for the tumours was in the subcutaneous soft tissues of the arms. There were no malignant cases. All tumours were positive for S-100, CD57, SOX10, calretinin, CD68, PGP9.5, α-inhibin and TFE-3, with a low Ki-67 (1-5%). Additionally, we reported, for the first time, the positive immunoreaction to Gap43 (growth-associated protein-43) in GCT.

Neuroregenerative Effects of Electromagnetic Field and Magnetic Nanoparticles on Spinal Cord Injury in Rats.

The present study aimed to evaluate the effect of iron oxide nanoparticles (IONPs) along with electromagnetic fields (MF) exposure on spontaneous and induced axonal sprouting after spinal cord injury (SCI). Adult male Wistar rats were subjected to spinal cord transection at the T13 segment. The IONP (25 µg/mL) embedded in 3% agarose gel was implanted at the injury site and subsequently exposed to MF (50 Hz, 17.96 µT, 2 hours/day for 5 weeks). Histological analysis of spinal cord tissue showed a significant increase in the expression of the growth-associated protein GAP-43 and it was found to be co-localized with neuronal nuclei marker and neurofilaments. The results show sprouting from mature neurons and axons, significantly less demyelination and more myelinated fibers were evident at the lesion site. However, no motor or somatosensory evoked potential response was observed, suggesting lack of long-distance functional connectivity. These findings highlight the therapeutic potential of IONPs along with MF exposure in promoting neuroregeneration after SCI.

VEGF expression correlates with neuronal differentiation and predicts a favorable prognosis in patients with neuroblastoma.

Neuroblastoma (NB) is a childhood cancer with a low survival rate and great metastatic potential. Vascular endothelial growth factor (VEGF), an angiogenesis factor, has been found to be involved in CRT-related neuronal differentiation of NB cells. In this study, we further confirmed the role VEGF in NB through mouse xenograft model and clinical analysis from NB patients. In xenograft experiments, CRT overexpression effectively inhibited the tumor growth. In addition, the mRNA and protein levels of VEGF and differentiation marker GAP-43 were upregulated by induced CRT expression. However, no significant correlation between the expression level of VEGF and microvessel density was observed in human NB tumors, suggesting a novel mechanism of VEGF participating in NB tumorigenesis through an angiogenesis-independent pathway. In NB patients' samples, mRNA expression levels of CRT and VEGF were positively correlated. Furthermore, positive VEGF expression by immunostaining of NB tumors was found to correlate well with histological grade of differentiation and predicted a favorable prognosis. In conclusion, our findings suggest that VEGF is a favorable prognostic factor of NB and might affect NB tumor behavior through CRT-driven neuronal differentiation rather than angiogenesis that might shed light on a novel therapeutic strategy to improve the outcome of NB.

Liver regeneration: immunohistochemical study of intrinsic hepatic innervation after partial hepatectomy in rats.

BACKGROUND: We examined the intrinsic hepatic innervation after partial hepatectomy (PH) in rats and the presence and pattern of neural sprouting in regenerating liver. METHODS: Male Wistar rats (age 9-13 weeks-w, weight 204-356 g), were submitted to two-thirds PH. Rats were sacrificed at postoperative days (d) 1, 3, 5, 7, at 2 and 4 w, and at 3 and 6 months (m) (6-7 animals/group, control group n = 4). Immunohistochemistry for the pan-neural marker protein gene product 9.5 (PGP9.5) and growth-associated protein 43 (GAP-43), a marker of regenerating nerve axons, was performed on tissue sections from the R1 lobe of the regenerating liver. Portal tracts (PTs) with immunoreactive fibers were counted in each section and computer-assisted morphometric analysis (Image Pro Plus) was used to measure nerve fiber density (number of immuno-positive nerve fibers/mm2 (40x)). RESULTS: Immunoreactivity for PGP9.5 was positive in all groups. The number of PGP9.5 (+) nerve fibers decreased from 0.32 +/- 0.12 (control group) to 0.18 +/- 0.09 (1d post-PH group), and gradually increased reaching pre-PH levels at 6 m (0.3 +/- 0.01). In contrast, immunoreactivity for GAP-43 was observed at 5d post-PH, and GAP-43 (+) PTs percentage increased thereafter with a peak at 3 m post-PH. GAP-43 (+) nerve fiber density increased gradually from 5d (0.05 +/- 0.06) with a peak at 3 m post-PH (0.21 +/- 0.027). At 6 m post-PH, immunoreactivity for GAP-43 was not detectable. CONCLUSIONS: Following PH in rats: 1) nerve fiber density in portal tracts decreases temporarily, and 2) neural sprouting in the regenerating liver lobes starts at 5d, reaches peak levels at 3 m and disappears at 6 m post-PH, indicating that the increase in hepatic mass after PH provides an adequate stimulus for the sprouting process.

Neonatal exposure to a moderate dose of ionizing radiation causes behavioural defects and altered levels of tau protein in mice.

Medical use of ionizing radiation (IR) has great benefits for treatment and diagnostic imaging, but procedures as computerized tomography (CT) may deliver a significant radiation dose to the patient. Recently, awareness has been raised about possible non-cancer consequences from low dose exposure to IR during critical phases of perinatal and/or neonatal brain development. In the present study neonatal NMRI mice were whole body irradiated with a single dose of gamma radiation (0; 350 and 500 mGy) on postnatal day 10 (PND 10). At 2 and 4 months of age, mice of both sexes were observed for spontaneous behaviour in a novel home environment. The neuroproteins CaMKII, GAP-43, synaptophysin and total tau in male mouse cerebral cortex and hippocampus were analysed 24h post-irradiation and in adults at 6 months of age exposed to 0 or 500 mGy on PND 10. A significantly dose-response related deranged spontaneous behaviour in 2- and 4-month-old mice was observed, where both males and females displayed a modified habituation, indicating reduced cognitive function. The dose of 350 mGy seems to be a tentative threshold. Six-month-old male mice showed a significantly increased level of total tau in cerebral cortex after irradiation to 500 mGy compared to controls. This demonstrates that a single moderate dose of IR, given during a defined critical period of brain development, is sufficient to cause persistently reduced cognitive function. Moreover, an elevation of tau protein was observed in male mice displaying reduced cognitive function.

Reactionary Dentinogenesis and Neuroimmune Response in Dental Caries.

Reactionary dentin formation is an adaptive secretory response mediated by odontoblasts to moderate dentin injury. The implications of this process for neuroimmune interactions operating to contain pathogens have not been fully appreciated. The purpose of the present study was to describe the relationship between reactionary dentinogenesis, the neurogenic changes of dental pulp innervation, and dendritic cell recruitment to caries progression, using a comparative immunohistochemical approach in human teeth from young adult individuals. Reactionary dentin formation during dentin caries progression is associated with changes in the integrity of junctional complexes within the odontoblast layer. Diminished coexpression of Cx43 and zonula occludens 1 implies a reduced level of intercellular connectivity between odontoblasts. Dentin caries also causes overexpression of growth-associated protein 43, a modulator of neural plasticity that promotes extensive sprouting of nerve endings into the reactionary dentin matrix. At the same time, an elevated number of HLA-DR-positive dendritic cells infiltrate the odontoblast layer and subsequently invade reactionary dentin formed underneath the early caries-affected regions. Simultaneous odontoblast layer remodeling, nerve fiber sprouting, and activation of dendritic cells during caries progression suggest a coordinated neuroimmune response to fight caries pathogen invasion and to promote dentin-pulp healing. We propose that reactionary dentin formation hinders pathogen invasion and supports defensive neuroimmune interactions against infection. The eventual understanding of this complex scenario may contribute to the development of novel approaches to dental caries treatment.

Growth-associated protein 43 in differentiating peripheral nerve sheath tumors from other non-neural spindle cell neoplasms.

The malignant peripheral nerve sheath tumor is a relatively uncommon type of soft tissue sarcoma arising from a peripheral nerve or extraneural soft tissues and showing nerve sheath differentiation. The diagnosis of malignant peripheral nerve sheath tumor is one of the most challenging tasks in surgical pathology because of its uncommon type (5-10% soft tissue sarcomas), morphologic resemblance to other spindle cell neoplasms and lack of sensitive and specific immunohistochemical markers. The pathologic diagnosis is more straightforward in the clinical setting of neurofibromatosis-1, but problems are mainly centered on the non-neurofibromatosis-1 malignant peripheral nerve sheath tumors. To date, S100 protein is the most widely applied marker in the case of a suspected malignant peripheral nerve sheath tumor, yet its suboptimal sensitivity and its expression in other spindle cell neoplasms, including spindle cell melanoma, clear-cell sarcoma, leiomyosarcoma and monophasic synovial sarcoma, add to the diagnostic conundrum. Growth-associated protein 43 (GAP43), a membrane-associated phosphoprotein expressed in neuronal growth cones and Schwann cell precursors during neural development and axonal regeneration, was applied to a set of nerve sheath and non-nerve sheath spindle cell neoplasms. The findings in this study indicate that GAP43 is expressed in malignant peripheral nerve sheath tumors (n=18/21; 86%) and demonstrates a sensitivity superior to S100 protein (n=13/21; 62%). GAP43 is also positive in neurofibromas (n=17/18; 94%), schwannomas (n=11/12; 92%) and desmoplastic melanomas (n=7/10; 70%). In contrast, it is negative in the non-desmoplastic spindle cell melanomas (n=20/22; 91%). Of the other non-neural soft tissue sarcomas, GAP43 is non-reactive in most leiomyosarcomas (n=14/16; 88%) and clear-cell sarcomas (n=8/8), and only focally positive in monophasic synovial sarcomas (n=3/7; 43%). GAP43 is seemingly a highly sensitive marker for peripheral nerve sheath tumors and may serve as a useful diagnostic adjunct in the diagnosis of malignant peripheral nerve sheath tumor from other spindle cell neoplasms, including spindle cell melanoma.

Growth associated protein 43 is expressed in skeletal muscle fibers and is localized in proximity of mitochondria and calcium release units.

The neuronal Growth Associated Protein 43 (GAP43), also known as B-50 or neuromodulin, is involved in mechanisms controlling pathfinding and branching of neurons during development and regeneration. For many years this protein was classified as neuron-specific, but recent evidences suggest that a) GAP43 is expressed in the nervous system not only in neurons, but also in glial cells, and b) probably it is present also in other tissues. In particular, its expression was revealed in muscles from patients affected by various myopathies, indicating that GAP43 can no-longer considered only as a neuron-specific molecule. We have investigated the expression and subcellular localization of GAP43 in mouse satellite cells, myotubes, and adult muscle (extensor digitorum longus or EDL) using Western blotting, immuno-fluorescence combined to confocal microscopy and electron microscopy. Our in vitro results indicated that GAP43 is indeed expressed in both myoblasts and differentiating myotubes, and its cellular localization changes dramatically during maturation: in myoblasts the localization appeared to be mostly nuclear, whereas with differentiation the protein started to display a sarcomeric-like pattern. In adult fibers, GAP43 expression was evident with the protein labeling forming (in longitudinal views) a double cross striation reminiscent of the staining pattern of other organelles, such as calcium release units (CRUs) and mitochondria. Double immuno-staining and experiments done in EDL muscles fixed at different sarcomere lengths, allowed us to determine the localization, from the sarcomere Z-line, of GAP43 positive foci, falling between that of CRUs and of mitochondria. Staining of cross sections added a detail to the puzzle: GAP43 labeling formed a reticular pattern surrounding individual myofibrils, but excluding contractile elements. This work leads the way to further investigation about the possible physiological and structural role of GAP43 protein in adult fiber function and disease.

Use of poly(DL-lactide-ε-caprolactone) membranes and mesenchymal stem cells from the Wharton's jelly of the umbilical cord for promoting nerve regeneration in axonotmesis: in vitro and in vivo analysis.

Cellular systems implanted into an injured nerve may produce growth factors or extracellular matrix molecules, modulate the inflammatory process and eventually improve nerve regeneration. In the present study, we evaluated the therapeutic value of human umbilical cord matrix MSCs (HMSCs) on rat sciatic nerve after axonotmesis injury associated to Vivosorb® membrane. During HMSCs expansion and differentiation in neuroglial-like cells, the culture medium was collected at 48, 72 and 96 h for nuclear magnetic resonance (NMR) analysis in order to evaluate the metabolic profile. To correlate the HMSCs ability to differentiate and survival capacity in the presence of the Vivosorb® membrane, the [Ca(2+)]i of undifferentiated HMSCs or neuroglial-differentiated HMSCs was determined by the epifluorescence technique using the Fura-2AM probe. The Vivosorb® membrane proved to be adequate and used as scaffold associated with undifferentiated HMSCs or neuroglial-differentiated HMSCs. In vivo testing was carried out in adult rats where a sciatic nerve axonotmesis injury was treated with undifferentiated HMSCs or neuroglial differentiated HMSCs with or without the Vivosorb® membrane. Motor and sensory functional recovery was evaluated throughout a healing period of 12 weeks using sciatic functional index (SFI), extensor postural thrust (EPT), and withdrawal reflex latency (WRL). Stereological analysis was carried out on regenerated nerve fibers. In vitro investigation showed the formation of typical neuroglial cells after differentiation, which were positively stained for the typical specific neuroglial markers such as the GFAP, the GAP-43 and NeuN. NMR showed clear evidence that HMSCs expansion is glycolysis-dependent but their differentiation requires the switch of the metabolic profile to oxidative metabolism. In vivo studies showed enhanced recovery of motor and sensory function in animals treated with transplanted undifferentiated and differentiated HMSCs that was accompanied by an increase in myelin sheath. Taken together, HMSC from the umbilical cord Wharton jelly might be useful for improving the clinical outcome after peripheral nerve lesion.

Angiotensin-(1-7) via the mas receptor alleviates the diabetes-induced decrease in GFAP and GAP-43 immunoreactivity with concomitant reduction in the COX-2 in hippocampal formation: an immunohistochemical study.

We have previously shown that chronic treatment with angiotensin-(1-7) [Ang-(1-7)] can prevent diabetes-induced cardiovascular dysfunction. However, effect of Ang-(1-7) treatment on diabetes-induced alterations in the CNS is unknown. The aim of this study was to test the hypothesis that treatment with Ang-(1-7) can produce protection against diabetes-induced CNS changes. We examined the effect of Ang-(1-7) on the number of cyclooxygenase-2 (COX-2) immunoreactive neurons and the glial fibrillary acidic protein (GFAP)-immunoreactive astrocytes and assessed the changes in the neuronal growth-associated protein-43 (GAP-43) of the hippocampal formation in streptozotocin-induced diabetes in rats. Animals were sacrificed 30 days after induction of diabetes and/or treatment with Ang-(1-7). Ang-(1-7) treatment significantly prevented diabetes-induced decrease in the number of GFAP immunoreactive astrocytes and GAP-43 positive neurons in all hippocampal regions. Co-administration of A779, a selective Ang-(1-7) receptor antagonist, inhibited Ang-(1-7)-mediated protective effects indicating that Ang-(1-7) produces its effects through activation of receptor Mas. Further, Ang-(1-7) treatment through activation of Mas significantly prevented diabetes-induced increase in the number of the COX-2 immunolabeled neurons in all sub-regions of the hippocampus examined. These results show that Ang-(1-7) has a protective role against diabetes-induced changes in the CNS.

Neoinnervation in rotator cuff tendinopathy.

The aim of this study was to determine whether there are more nerves in tendinopathic human tendon, and if so, where are they located. Tendon biopsies were collected from normal, tendinopathic, and torn human rotator cuff tendons and then analyzed using immunohistochemistry and antibodies against a general nerve marker (protein gene product 9.5, PGP9.5), a nerve regeneration marker (growth-associated protein 43, GAP43), and an endothelial cell marker (CD34). Nerve fibers exhibiting PGP9.5 or GAP43 immunoreactivity were often observed intimately in association with tiny blood vessels in the endotendineum of tendinopathic tendons. The expression of PGP9.5 and GAP43 were significantly higher in tendinopathic tendon compared with control tendon and torn tendon. These data support the hypothesis that early tendinopathy is associated with increases of newly grown nerve fibers and blood vessels inside and around tendinopathic tendon, and these may be the source of pain in tendinopathy.

Comparative immunohistochemistry of synaptic markers in the rodent hippocampus in pilocarpine epilepsy.

Pilocarpine-induced epileptic state (Status epilepticus) generates an aberrant sprouting of hippocampal mossy fibers, which alter the intrahippocampal circuits. The mechanisms of the synaptic plasticity remain to be determined. In our studies in mice and rats, pilocarpine-induced seizures were done in order to gain information on the process of synaptogenesis. After a 2-month survival period, changes in the levels of synaptic markers (GAP-43 and Syn-I) were examined in the hippocampus by means of semi-quantitative immunohistochemistry. Mossy fiber sprouting (MFS) was examined in each brain using Timm's sulphide-silver method. Despite the marked behavioral manifestations caused by pilocarpine treatment, only 40% of the rats and 56% of the mice showed MFS. Pilocarpine treatment significantly reduced the GAP-43 immunoreactivity in the inner molecular layer in both species, with some minor differences in the staining pattern. Syn-I immunohistochemistry revealed species differences in the sprouting process. The strong immunoreactive band of the inner molecular layer in rats corresponded to the Timm-positive ectopic mossy fibers. The staining intensity in this layer, representing the ectopic mossy fibers, was weak in the mouse. The Syn-I immunoreactivity decreased significantly in the hilum, where Timm's method also demonstrated enhanced sprouting. This proved that, while sprouted axons displayed strong Syn-I staining in rats, ectopic mossy fibers in mice did not express this synaptic marker. The species variability in the expression of synaptic markers in sprouted axons following pilocarpine treatment indicated different synaptic mechanisms of epileptogenesis.

Decreased innervation of eczematous skin in NC/Nga atopic mice during chronic mild stress.

BACKGROUND AND AIM: A connection between chronic mild stress and altered innervation in the skin of an atopic mouse strain, NC/Nga, was studied. MATERIAL AND METHODS: We used three groups of mice, stressed control (SC, stressed but not immunized with a mite antigen), non-stressed eczematous (NSE, not stressed but immunized) and stressed eczematous (SE, stressed and immunized). RESULTS: There was a decrease of protein gene product (PGP) 9.5 positive nerve fibers in SE compared to SC mice, in both epidermis and dermis, also in SE compared to NSE mice. Also a decrease in growth associated protein (GAP)-43 positive nerve fibers was seen in SE compared to SC, in both epidermis and dermis. CONCLUSION: Chronic mild stress in atopic mice leads to decreased innervation in eczematous lesions.

Effect of intranasal administration of basic fibroblast growth factor on olfactory epithelium.

To further study the effects of basic fibroblast growth factor (bFGF) on the olfactory epithelium, bFGF was intranasally administered twice a day for 6 weeks to 2.5-month-old and 7-month-old mice. The effects were immunohistochemically examined by using antibodies against proliferating cell nuclear antigen, olfactory marker protein, and GAP43. The number of cells positive for proliferating cell nuclear antigen in the supporting cell layer increased dramatically, and that of GAP43-positive cells, or globose basal cells, increased significantly, especially in aging mice. However, no significant changes were observed in the number of olfactory marker protein-positive cells or mature olfactory receptor neurons. These results suggest that topical application of bFGF promotes proliferation of globose basal cells and supporting cells.

Herpes simplex virus utilizes the large secretory vesicle pathway for anterograde transport of tegument and envelope proteins and for viral exocytosis from growth cones of human fetal axons.

Axonal transport of herpes simplex virus (HSV-1) is essential for viral infection and spread in the peripheral nervous system of the host. Therefore, the virus probably utilizes existing active transport and targeting mechanisms in neurons for virus assembly and spread from neurons to skin. In the present study, we used transmission immunoelectron microscopy to investigate the nature and origin of vesicles involved in the anterograde axonal transport of HSV-1 tegument and envelope proteins and of vesicles surrounding partially and fully enveloped capsids in growth cones. This study aimed to elucidate the mechanism of virus assembly and exit from axons of human fetal dorsal root ganglia neurons. We demonstrated that viral tegument and envelope proteins can travel in axons independently of viral capsids and were transported to the axon terminus in two types of transport vesicles, tubulovesicular membrane structures and large dense-cored vesicles. These vesicles and membrane carriers were derived from the trans-Golgi network (TGN) and contained key proteins, such as Rab3A, SNAP-25, GAP-43, and kinesin-1, involved in the secretory and exocytic pathways in axons. These proteins were also observed on fully and partially enveloped capsids in growth cones and on extracellular virions. Our findings provide further evidence to the subassembly model of separate transport in axons of unenveloped capsids from envelope and tegument proteins with final virus assembly occurring at the axon terminus. We postulate that HSV-1 capsids invaginate tegument- and envelope-bearing TGN-derived vesicles and utilize the large secretory vesicle pathway of exocytosis for exit from axons.