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.

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.

Diffuse axonal injury in periventricular leukomalacia as determined by apoptotic marker fractin.

Periventricular leukomalacia (PVL), the major substrate of neurologic deficits in premature infants, is associated with reduced white matter volume. Using immunomarkers of axonal pathology [beta-amyloid precursor protein (beta-APP) and apoptotic marker fractin], we tested the hypothesis that widespread (diffuse) axonal injury occurs in the gliotic white matter beyond the foci of necrosis in PVL, thus contributing to the white matter volume reduction. In a cohort of 17 control cases and 13 PVL cases with lesions of different chronological ages, diffuse axonal damage in PVL was detected by fractin in white matter sites surrounding and distant from acute and organizing foci of necrosis. Using beta-APP, axonal spheroids were detected within necrotic foci in the acute and organizing (subacute) stages, a finding consistent with others. Interestingly, GAP-43 expression was also detected in spheroids in the necrotic foci, suggesting attempts at axonal regeneration. Thirty-one percent of the PVL cases had thalamic damage and 15% neuronal injury in the cerebral cortex overlying PVL. We conclude that diffuse axonal injury, as determined by apoptotic marker fractin, occurs in PVL and that its cause likely includes primary ischemia and trophic degeneration secondary to corticothalamic neuronal damage.

Generating neuron-like cells from BM-derived mesenchymal stromal cells in vitro.

BACKGROUND: The multipotency of stromal cells has been studied extensively. It has been reported that mesenchymal stromal cells (MSC) are capable of differentiating into cells of multilineage. Different methods and reagents have been used to induce the differentiation of MSC. We investigated the efficacy of different growth factors in inducing MSC differentiation into neurons. METHODS: MSC from human BM were isolated and cultured in media supplemented with 10% FBS. These cells were identified and later induced to differentiate into neuron-like cells using different neurotrophic factors. Three different growth factors were used, either alone or in combination: brain-derived neurotrophic factor, epidermal growth factor and neural growth factor. RESULTS: After 10 days of culture, MSC showed neuron-like morphologic changes. Immunostaining showed that these cells expressed markers for neurons (growth-associated protein-43, neuron-specific nuclear protein and neurofilament 200 kDa) and expression of these markers suggested the transition of immature stages to more mature stages of neuron-like cells. DISCUSSION: Our results show that BM-derived MSC can differentiate not only into target cells of mesodermal origin but also neuron-like cells of ectodermal origin. The findings show that a combination of growth factors is more effective in inducing MSC into neuron-like cells.

[The functional change in the 5-HT1A receptor induced by stress and the role of the 5-HT1A receptor in neuroprotection].

Mice exposed to various stresses, especially restrained-stress, revealed the anxiogenic effect detected by the light-dark test. Under this condition, a remarkable decrease in [35S]GTPgammaS binding to membranes from the prefrontal cortex, amygdala and hypothalamus of restrained-stress mice stimulated by the selective 5-HT1A receptor agonist 5-carboxamidotriptamine (5-CT) was clearly observed, whereas a significant increase in [35S]GTPgammaS binding stimulated by the 5-HT1A receptor agonist was clearly observed in the dorsal raphe nuclei (DRN) of restrained-stress mice. The immunohistochemical study showed a drastic reduction in phosphorylated-CREB-like immunoreactivity in the DRN of restrained-stress mice. Furthermore, we found a drastic reduction in myelin-associated glycoprotein (MAG)-like immunoreactivity (MAG-IR) in the DRN, amygdala and hypothalamus, indicating the direct suppression of synaptic transmission in these regions. It has been accepted that GSK3beta in the Wnt signal pathway plays an important role in various neuronal functions including apoptosis, clustering of synapsin I and early growth and axonal remodeling. In the present study, the increase in protein levels of GSK3beta and phosphorylated-GSK3beta to cytosol fractions of the amygdala was noted in restrained-stress mice. Taken together, these results suggest that restrained stress may directly affect the 5-HT1A receptor-regulated synaptic transmission in the brain, leading to the expression of the anxiogenic effect in mice. It is well known that various stresses induce intracellular oxidative stress. The present study was then undertaken to investigate the effect of the stimulation of 5-HT1A receptors on oxidative stress. Treatment with H2O2 caused the activation of caspase-3-positive cells and the reduction in levels of MAG-IR in the limbic neuron/glia cocultures as compared to medium alone. The stimulation of 5-HT1A receptor by 5-CT produced a dramatic protection against H2O2-triggered activation of caspase-3 and reduction in levels of MAG-IR. These results suggest that 5-HT1A receptors were involved in the modulation of anxiety and the understanding of molecular mechanisms of 5-HT1A receptor-related cascades may pave the way for new therapeutic strategies for affective disorders.

Corticothalamic cells in layers 5 and 6 of primary and secondary sensory cortex express GAP-43 mRNA in the adult rat.

The expression of a presynaptic phosphoprotein, growth-associated protein (GAP)-43, is associated with synaptogenesis during development and synaptic remodeling in the adult. This study examined GAP-43 mRNA expression and distribution in primary and secondary areas of visual, auditory, and somatosensory cortex of the adult rat, by in situ hybridization with a digoxigenin-coupled mRNA probe, focusing particularly on the corticothalamic cells in layers 5 and 6. In the six cortical areas studied, GAP-43 mRNA was expressed predominantly in layers 5 and 6 and was greater in secondary than primary areas. There were densely labeled cells in layers 5 and 6 of all areas, which showed a restricted sublaminar distribution in primary areas and more even distribution in secondary areas. Combining retrograde transport of rhodamine beads with in situ hybridization in visual and auditory cortex showed that corticothalamic cells in layers 5 and 6 express GAP-43 mRNA. There are more of these GAP-43 mRNA positive corticothalamic cells in layer 5 of secondary areas than in primary areas. The evidence suggests that in the adult rat, plasticity related to GAP-43 is present in primary and secondary sensory cortex and more so in secondary areas.

Abeta1-42 promotes cholinergic sprouting in patients with AD and Lewy body variant of AD.

BACKGROUND: The neurodegenerative process in Alzheimer's disease (AD) and in the Lewy body variant of AD (LBV) patients is characterized by cholinergic dysfunction and deposition of amyloid beta-peptide (Abeta) 1-40 and 1-42; however, the differential effects of Abeta species on the cholinergic system are not completely clear. OBJECTIVE: To better understand the relationship between levels of Abeta1-40 and 1-42 on cholinergic deficits in AD and LBV patients. METHODS: Levels of choline acetyltransferase (ChAT) activity and ChAT immunoreactivity in the plaques in the frontal cortex of patients with AD and LBV were correlated with Abeta1-42 and 1-40 levels determined by ELISA and with neuropathologic and neurologic markers. RESULTS: Although the overall levels of ChAT activity were reduced in AD and LBV cases, there was a direct correlation with Abeta1-42 levels. Furthermore, patients with high Abeta1-42 levels had more abundant cholinergic dystrophic neurites in the plaques than cases with lower Abeta1-42. CONCLUSION: Abeta1-42 may also trigger cholinergic dysfunction by promoting aberrant neuritic sprouting.

Early development of the hypothalamus of a wallaby (Macropus eugenii).

We have studied the development of the hypothalamus of an Australian marsupial, the tammar wallaby (Macropus eugenii), to provide an initial anatomic framework for future research on the developing hypothalamus of diprotodontid metatheria. Cytoarchitectural (hematoxylin and eosin), immunohistochemical (CD 15 and growth associated protein, GAP-43), tritiated thymidine autoradiography, and carbocyanine dye tracing techniques were applied. Until 12 days after birth (P12), the developing hypothalamus consisted of mainly a ventricular germinal zone with a thin marginal layer, but by P25, most hypothalamic nuclei were well differentiated, indicating that the bulk of hypothalamic cytoarchitectural development occurs between P12 and P25. Strong CD 15 immunoreactivity was found in radial glial fibers in the rostral hypothalamus during early developmental ages, separating individual hypothalamic compartments. Immunoreactivity for GAP-43 was used to reveal developing fiber bundles. The medial forebrain bundle was apparent by P0, and the fornix appeared at P12. Tritiated thymidine autoradiography revealed lateral-to-medial and dorsal-to-ventral neurogenetic gradients similar to those seen in rodents. Dye tracing showed that projections to the posterior pituitary arose from the supraoptic nucleus at P5 and from the paraventricular nucleus at P10. Projections to the medulla were first found from the lateral hypothalamic area at P0 and paraventricular nucleus at P10. In conclusion, the pattern of development of the wallaby hypothalamus is broadly similar to that found in eutheria, with comparable neurogenetic compartments to those identified in rodents. Because most hypothalamic maturation takes place after birth, wallabies provide a useful model for experimentally manipulating the developing mammalian hypothalamus.

Abnormal corticospinal function but normal axonal guidance in human L1CAM mutations.

L1 cell adhesion molecule (L1CAM) gene mutations are associated with X-linked 'recessive' neurological syndromes characterized by spasticity of the legs. L1CAM knock-out mice show hypoplasia of the corticospinal tract and failure of corticospinal axonal decussation and projection beyond the cervical spinal cord. The aim of this study was to determine if similar neuropathology underlies the spastic diplegia of males hemizygous for L1CAM mutations. Studies were performed on eight carrier females and 10 hemizygous males. Transcranial magnetic stimulation excited the corticospinal tract and responses were recorded in biceps brachii and quadriceps femoris. In contralateral biceps and quadriceps the responses had high thresholds and delayed onset compared with normal subjects. Ipsilateral responses in biceps were smaller, with higher thresholds and delayed onsets relative to contralateral responses. Subthreshold corticospinal conditioning of the stretch reflex of biceps and quadriceps was abnormal in both hemizygous males and carrier females suggesting there may also be a reduced projection to inhibitory interneurones. Histological examination of post-mortem material from a 2-week-old male with an L1CAM mutation revealed normal corticospinal decussation and axonal projections to lumbar spinal segments. These data support a role for L1CAM in corticospinal tract development in hemizygous males and 'carrier' females, but do not support a critical role for L1CAM in corticospinal axonal guidance.

Altered expression of synaptic proteins occurs early during progression of Alzheimer's disease.

The expression levels of three synaptic proteins (synaptophysin, synaptotagmin, and growth-associated protein 43 [GAP43]) in AD cases clinically classified by Clinical Dementia Rating (CDR) score were analyzed. Compared with control subjects (CDR = 0), mild (early) AD (CDR = 0.5 to 1) cases had a 25% loss of synaptophysin immunoreactivity. Levels of synaptotagmin and GAP43 were unchanged in mild AD, but cases with CDR of >1 had a progressive decrement in these synaptic proteins. Thus, synaptic injury in frontal cortex is an early event in AD.

Neuron degeneration induced by verapamil and attenuated by EGb761.

Calcium channel blockers are used as neuroprotective agents, as glutamate antagonists. However, it has been found that calcium channel blockers may compromise neuronal survival after long-term exposure. To explore the mechanisms of the toxicity of calcium channel blockers on neurons, we studied the morphological characteristics and biochemical changes of cultured cortical neurons treated with verapamil, a calcium channel blocker. We now report that cerebral cortical cultures exposed to verapamil for 48 h undergo neuronal degeneration in both concentration-dependent and time-dependent fashion, possibly partially through the activation of apoptosis. On the other hand, it was found that Ginkgo biloba extract (EGb761) attenuated verapamil-induced neuronal injury, suggesting the possibility of using verapamil combined with EGb761 clinically. Furthermore, both B-50 immunoactivity (BIA) and the concentration of intracellular calcium in single neurons ([Ca2+]i) decreased after a 48-h exposure to verapamil, suggesting that the mechanisms of verapamil-induced degeneration may be associated with the disruption of intracellular calcium homeostasis and the inhibition of normal axonal elongation.