Evaluation of prenatal calabash chalk geophagy on the developing brain of Wistar rats.

Calabash chalk (CaC) is an aluminium silicate hydroxide compound with heavy metal constituents, making it a potential neurotoxicant. Pregnant women often consume CaC as an antiemetic, which may interfere with the normal development of the foetal brain. Here, we evaluated the effects of CaC administration in pregnant rats on the brain of the offspring. Wistar rat dams were assigned to one of three groups: control, 200 mg/kg and 800 mg/kg of a CaC suspension. Administrations lasted 14 days (gestation days 7-20). On day 14, 5-bromo-2'-deoxyuridine (BrdU) was administered and dams were allowed to term. Behavioural tests were performed on different days as the pups matured, and they were sacrificed on post-natal days 30 and 60. Brains were processed for histology and Western blotting. Results showed no significant differences in surface righting reflex, cliff avoidance, negative geotaxis and open-field activity. No hippocampal and somatosensory cortical cytoarchitectonic alterations and no significant signs of glial fibrillary acidic protein (GFAP) activation were observed. Neuronal nuclei counts showed variability in the somatosensory cortex and hippocampus of the CaC group. BrdU-positive cells were significantly lower in the 200 mg/kg group and higher in the 800 mg/kg group. Doublecortin-X-positive cells were not different in all the CaC groups. Astrocytes and microglia Western blotting quantification confirmed no significant increase in pup glial cells in adulthood. Prenatal consumption of CaC at indicated dosages may not be deleterious to the developing brain, especially after cessation of exposure and during maturation of the animal. However, the differences in neuronal and glial populations may be due to their ability to cope with CaC.

Neuroecotoxicology: Effects of environmental heavy metal exposure on the brain of African giant rats and the contribution of vanadium to the neuropathology.

Increased exploitation of minerals has led to pollution of confined environments as documented in Nigeria Niger Delta. Information on the effects on brain of such exposure is limited. Due to its exploratory activities, the African giant rat (Cricetomys gambianus) (AGR) provides a unique model for neuroecotoxicological research to determine levels of animal and human exposure to different pollutants. This study aims to unravel neuropathological features of AGR sampled from three agro-ecological zones of Nigeria. Fifteen AGR were sampled according to previously determined data on heavy metal exposure: high vanadium, high lead, and low metals. Eighteen AGR were collected from low metal zone and divided into two groups. Control group received vehicle while SMV exposed group received 3 mg/kg sodium metavanadate (SMV) intraperitoneally for 14days. Brain immunohistochemical analyses were conducted, and ultrastructural changes were studied in experimentally exposed group. Results showed significant loss of tyrosin hydroxylase, parvalbumin, orexin-A and melanin concentration hormone containing neuronal populations in brains obtained from high vanadium and high lead zones and in experimentally intoxicated SMV groups. Similarly, significant decrease numbers of dendritic arborations; extracellular matrix density, perineuronal nets; astrocytes and microglia activations are documented in same groups. Ultrastructural studies revealed mass denudation, cilia loss, disintegration of ependymal layer and intense destructions of myelin sheaths in SMV exposed group. These are the first "neuroecotoxicological" findings in distinct neuronal cells. The implications of these findings are highly relevant for human population living in these areas, not only in Nigeria but also in similarly polluted areas elsewhere in the world.

Downregulation of the Astroglial Connexin Expression and Neurodegeneration after Pilocarpine-Induced Status Epilepticus.

Astrocytic networks and gap junctional communication mediated by connexins (Cxs) have been repeatedly implicated in seizures, epileptogenesis, and epilepsy. However, the effect of seizures on Cx expression is controversial. The present study focused on the response of Cxs to status epilepticus (SE), which is in turn an epileptogenic insult. The expression of neuronal Cx36 and astrocytic Cx30 and Cx43 mRNAs was investigated in the brain of rats in the first day after pilocarpine-induced SE. In situ hybridization revealed a progressive decrease in Cx43 and Cx30 mRNA levels, significantly marked 24 h after SE onset in neocortical areas and the hippocampus, and in most thalamic domains, whereas Cx36 mRNA did not exhibit obvious changes. Regional evaluation with quantitative real-time-RT-PCR confirmed Cx43 and Cx30 mRNA downregulation 24 h after SE, when ongoing neuronal cell death was found in the same brain regions. Immunolabeling showed at the same time point marked a decrease in Cx43, microglia activation, and interleukin-1ß induction in some microglial cells. The data showed a transient downregulation of astroglial Cxs in the cortical and thalamic areas in which SE triggers neurodegenerative events in concomitance with microglia activation and cytokine expression. This could potentially represent a protective response of neuroglial networks to SE-induced acute damage.

The ameliorative effects of a phenolic derivative of Moringa oleifera leave against vanadium-induced neurotoxicity in mice.

Vanadium, a transition series metal released during some industrial activities, induces oxidative stress and lipid peroxidation. Ameliorative effect of a pure compound from the methanolic extract of Moringa oleifera leaves, code-named MIMO2, in 14-day old mice administered with vanadium (as sodium metavanadate 3 mg/kg) for 2 weeks was assessed. Results from body weight monitoring, muscular strength, and open field showed slight reduction in body weight and locomotion deficit in vanadium-exposed mice, ameliorated with MIMO2 co-administration. Degeneration of the Purkinje cell layer and neuronal death in the hippocampal CA1 region were observed in vanadium-exposed mice and both appeared significantly reduced with MIMO2 co-administration. Demyelination involving the midline of the corpus callosum, somatosensory and retrosplenial cortices was also reduced with MIMO2. Microglia activation and astrogliosis observed through immunohistochemistry were also alleviated. Immunohistochemistry for myelin, axons and oligodendrocyte lineage cells were also carried out and showed that in vanadium-treated mice brains, oligodendrocyte progenitor cells increased NG2 immunolabelling with hypertrophy and bushy, ramified appearance of their processes. MIMO2 displayed ameliorative and antioxidative effects in vanadium-induced neurotoxicity in experimental murine species. This is likely the first time MIMO2 is being used in vivo in an animal model.

Neuroinflammatory Response in Chronic Hydrocephalus in Juvenile Rats.

Hydrocephalus is especially prevalent in countries with limited resources, where its treatment is still a challenge. However, long-term neuropathological changes in untreated hydrocephalus remain largely unexplored. The present study looks at cortical parenchyma and neuroinflammation in acquired, chronic hydrocephalus. Intracisternal kaolin injections were performed in 3-week-old rats, followed by -1, 4- and 8-week survival; matched control rats received saline injections. Ventriculomegaly has been previously reported to stabilize by the third week in this model. Single and triple immunocytochemical approaches were used to highlight neurones, astrocytes, microglia, and the pro-inflammatory cytokine interleukin (IL)-1ß in the parietal cortex, utilizing cell counts and densitometry. Microglial protein ionized calcium binding adaptor molecule 1 (Iba1) and IL-1ß expressions were monitored with Western blotting in the parietal cortex and hippocampus. In the parietal cortex, which showed progressive disruption of cytoarchitecture, neuronal density was significantly increased at 8weeks post-induction but not at earlier time points, indicating on-going cortical damage in chronic hydrocephalus. Astrocyte and microglia hypertrophy, and Iba1 expression indicated glial cell activation which peaked at 4weeks. IL-1ß expression also peaked at 4weeks and was then down-regulated. Overall the findings indicate that neuroinflammatory features build up in the first month after hydrocephalus induction implicating marked IL-1ß upregulation. The data also show that astrocytes are the main source of IL-1ß in this disorder.

Regional Myelin and Axon Damage and Neuroinflammation in the Adult Mouse Brain After Long-Term Postnatal Vanadium Exposure.

Environmental exposure to vanadium occurs in areas of persistent burning of fossil fuels; this metal is known to induce oxidative stress and oligodendrocyte damage. Here, we determined whether vanadium exposure (3 mg/kg) in mice during the first 3 postnatal months leads to a sustained neuroinflammatory response. Body weight monitoring, and muscle strength and open field tests showed reduction of body weight gain and locomotor impairment in vanadium-exposed mice. Myelin histochemistry and immunohistochemistry for astrocytes, microglia, and nonphosphorylated neurofilaments revealed striking regional heterogeneity. Myelin damage involved the midline corpus callosum and fibers in cortical gray matter, hippocampus, and diencephalon that were associated with axonal damage. Astrocyte and microglial activation was identified in the same regions and in the internal capsule; however, no overt myelin and axon damage was observed in the latter. Double immunofluorescence revealed induction of high tumor necrosis factor (TNF) immunoreactivity in reactive astrocytes. Western blotting analysis showed significant induction of TNF and interleukin-1ß expression. Together these findings show that chronic postnatal vanadium exposure leads to functional deficit and region-dependent myelin damage that does not spare axons. This injury is associated with glial cell activation and proinflammatory cytokine induction, which may reflect both neurotoxic and neuroprotective responses.

Two-photon microscopy imaging of thy1GFP-M transgenic mice: a novel animal model to investigate brain dendritic cell subsets in vivo.

Transgenic mice expressing fluorescent proteins in specific cell populations are widely used for in vivo brain studies with two-photon fluorescence (TPF) microscopy. Mice of the thy1GFP-M line have been engineered for selective expression of green fluorescent protein (GFP) in neuronal populations. Here, we report that TPF microscopy reveals, at the brain surface of these mice, also motile non-neuronal GFP+ cells. We have analyzed the behavior of these cells in vivo and characterized in brain sections their immunophenotype.With TPF imaging, motile GFP+ cells were found in the meninges, subarachnoid space and upper cortical layers. The striking feature of these cells was their ability to move across the brain parenchyma, exhibiting evident shape changes during their scanning-like motion. In brain sections, GFP+ cells were immunonegative to antigens recognizing motile cells such as migratory neuroblasts, neuronal and glial precursors, mast cells, and fibroblasts. GFP+ non-neuronal cells exhibited instead the characteristic features and immunophenotype (CD11c and major histocompatibility complex molecule class II immunopositivity) of dendritic cells (DCs), and were immunonegative to the microglial marker Iba-1. GFP+ cells were also identified in lymph nodes and blood of thy1GFP-M mice, supporting their identity as DCs. Thus, TPF microscopy has here allowed the visualization for the first time of the motile behavior of brain DCs in situ. The results indicate that the thy1GFP-M mouse line provides a novel animal model for the study of subsets of these professional antigen-presenting cells in the brain. Information on brain DCs is still very limited and imaging in thy1GFP-M mice has a great potential for analyses of DC-neuron interaction in normal and pathological conditions.

Excitotoxic lesion of the perirhinal cortex impairs spatial working memory in a delayed-alternation task.

The perirhinal cortex (PRh) is strategically located between the neocortex and memory-related structures such as the entorhinal cortex and the hippocampal formation. The pattern of strong reciprocal connections between these areas, together with experimental evidence that PRh damage induces specific memory deficits, has placed this cortical region at the center of a growing interest for its role in learning and memory mechanisms. The aim of the present study is to clarify the involvement of PRh in learning and retention in a novel experimental model of spatial working memory, the water T-maze. The data show that pre-acquisition neurotoxic PRh lesions caused task-learning deficits. This impairment was observed during the acquisition phase as well as the retrieval phase. On the other hand, a post-acquisition PRh neurotoxic lesion failed to impair the acquisition and the retrieval of the water T-maze task performed 32 day after lesion. These results suggest a possible key role of PRh in the acquisition but not in the retention of a working memory task. Furthermore, these results show that the water T-maze may be a suitable learning paradigm to study different components of learning and memory.

Different patterns of neuronal activation and neurodegeneration in the thalamus and cortex of epilepsy-resistant Proechimys rats versus Wistar rats after pilocarpine-induced protracted seizures.

PURPOSE: To analyze cellular mechanisms of limbic-seizure suppression, the response to pilocarpine-induced seizures was investigated in cortex and thalamus, comparing epilepsy-resistant rats Proechimys guyannensis with Wistar rats. METHODS: Fos immunoreactivity revealing neuronal activation, and degenerating neurons labeled by Fluoro-Jade B (FJB) histochemistry were analyzed on the first day after onset of seizures lasting 3 h. Subpopulations of gamma-aminobutyric acid (GABA)ergic cells were characterized with double Fos-parvalbumin immunohistochemistry. RESULTS: In both cortex and thalamus, degenerating neurons were much fewer in Proechimys than Wistar rats. Fos persisted at high levels at 24 h only in the Proechimys thalamus and cortex, especially in layer VI where corticothalamic neurons reside. In the parietal cortex, about 50% of parvalbumin-containing interneurons at 8 h, and 10-20% at 24 h, were Fos-positive in Wistar rats, but in Proechimys, Fos was expressed in almost all parvalbumin-containing interneurons at 8 h and dropped at 24 h. Fos positivity in cingulate cortex interneurons was similar in both species. In the Wistar rat thalamus, Fos was induced in medial and midline nuclei up to 8 h, when <30% of reticular nucleus cells were Fos-positive, and then decreased, with no relationship with cell loss, evaluated in Nissl-stained sections. In Proechimys, almost all reticular nucleus neurons were Fos-positive at 24 h. DISCUSSION: At variance with laboratory rats, pilocarpine-induced protracted seizures elicit in Proechimys limited neuronal death, and marked and long-lasting Fos induction in excitatory and inhibitory cortical and thalamic cell subsets. The findings implicate intrathalamic and intracortical regulation, and circuits linking thalamus and cortex in limbic seizure suppression leading to epilepsy resistance.

Fos induction and persistence, neurodegeneration, and interneuron activation in the hippocampus of epilepsy-resistant versus epilepsy-prone rats after pilocarpine-induced seizures.

Previous studies demonstrated that the spiny rat Proechimys guyannensis exhibits resistance to experimental epilepsy. Neural activation was studied in the Proechimys hippocampus, using Fos induction, within 24 h after pilocarpine-induced seizures; neurodegenerative events were investigated in parallel, using FluoroJade B histochemistry. These parameters were selected since pilocarpine-induced limbic epilepsy is known to elicit immediate early gene expression and cell loss in the hippocampus of seizure-prone laboratory rodents. At variance with matched experiments in Wistar rats, pilocarpine injection resulted in Proechimys in seizure episodes that, as previously reported, did not develop into status epilepticus. At 3 h and 8 h after seizure onset, Fos immunoreactivity filled the dentate gyrus of both rat species, and was quite marked in pyramidal cells of the Proechimys Ammon's horn. At 24 h, Fos immunoreactivity dropped in the Wistar hippocampus and persisted in Proechimys. At 8 h and 24 h, FluoroJade-stained neurons were very few in the Proechimys hippocampus, whereas they were abundant in that of Wistar rats. Double immunohistochemistry for Fos and parvalbumin, the protein expressed by fast-spiking hippocampal interneurons, indicated that Fos was induced up to 24 h in the vast majority of parvalbumin-containing cells of the Proechimys hippocampus, and in a minority of these cells in the Wistar hippocampus. The findings demonstrate that early postepileptic neurodegeneration is very limited in the Proechimys hippocampus, in which sustained Fos induction persists for several hours. The findings also indicate that Fos induction and persistence may not correlate with seizure intensity and may not be associated with neuronal death. Finally, the data implicate differential mechanisms of interneuron activity in anti-convulsant and pro-convulsant phenomena.