Nisha Patro

S100β upregulation: a possible mechanism of deltamethrin toxicity and motor coordination deficits.

Deltamethrin (DLT) is a type II synthetic pyrethroid with insecticidal properties. It has been considered safe to humans. Excessive exposure of DLT is being variously reported, recently, to cause potential neurotoxicity in adults, as characterized by ataxia, loss of coordination, hyperexcitability, convulsions and paralysis. However, limited information is available on its impact at lower/safe to human doses during development. The present study was designed to assess the postnatal (P) exposure of DLT (as low as 0.7 mg/kg, i.p.) on S-100beta expression in developing rat cerebellum and its impact on Purkinje cell morphogenesis and dendritogenesis, and subsequent spontaneous motor activity (SMA) deficits. Wistar rat pups born to healthy mothers were injected with DLT (Sigma) at a dosage of 0.7 mg/kg body wt., i.p. dissolved in DMSO (Sigma) during P0-7th (DLT-I) and P9-13th day (DLT-II). The control pups were injected with equivalent volumes of DMSO. The pups of both the groups were used to assess the spontaneous motor activity P21 onwards. The cryocut sections (30 microm) of the cerebella were used for anti-S-100beta antibody labeling using streptavidin biotin HRP method. An upregulation of S-100beta expression in Bergmann glial fibers was recorded at P12 and P15 day preparations in both DLT-I and DLT-II treated groups. However, such upregulation of S-100beta was more prominent in DLT-II treated group animals with a large number of strongly S-100beta immunopositive astrocytes flanking around the Purkinje neurons. In Golgi preparation the Purkinje neurons in DLT treated groups had reduced dendritic arbor with short primary dendrites and much reduced dendritic branches which appeared stumpy and hypertrophied. The granule cell proliferation and migration as well as Purkinje cell morphogenesis and dendritogenesis are affected following DLT exposure in the present investigation. This may also affect the mossy fiber-granule cell-parallel pathway formation which in turn may decrease the firing of Purkinje cells (GABAergic inhibitory projections) and thus an increase in the output of the neurons in the deep cerebellar nuclei neurons and disturbed motor coordination.

Experimentally induced diabetes causes glial activation, glutamate toxicity and cellular damage leading to changes in motor function

Behavioral impairments are the most empirical consequence of diabetes mellitus documented in both humans and animal models, but the underlying causes are still poorly understood. As the cerebellum plays a major role in coordination and execution of the motor functions, we investigated the possible involvement of glial activation, cellular degeneration and glutamate transportation in the cerebellum of rats, rendered diabetic by a single injection of streptozotocin (STZ; 45 mg/kg body weight; intraperitoneally). Motor function alterations were studied using Rotarod test (motor coordination) and grip strength (muscle activity) at 2nd, 4th, 6th, 8th, 10th, and 12th week post-diabetic confirmation. Scenario of glial (astroglia and microglia) activation, cell death and glutamate transportation was gaged using immunohistochemistry, histological study and image analysis. Cellular degeneration was clearly demarcated in the diabetic cerebellum. Glial cells were showing sequential and marked activation following diabetes in terms of both morphology and cell number. Bergmann glial cells were hypertrophied and distorted. Active caspase-3 positive apoptotic cells were profoundly present in all three cerebellar layers. Reduced co-labeling of GLT-1 and GFAP revealed the altered glutamate transportation in cerebellum following diabetes. These results, exclusively derived from histology, immunohistochemistry and cellular quantification, provide first insight over the associative reciprocity between the glial activation, cellular degeneration and reduced glutamate transportation, which presumably lead to the behavioral alterations following STZ-induced diabetes.

Neurotoxicological effects of deltamethrin on the postnatal development of cerebellum of rat

Deitamethrin (DLT) has been accepted to be 10,000 times less toxic to man than to insects. While toxicity of DLT in adult animals has been studied using biochemical and electrophysiological tools, reports on its developmental neurotoxicity are rather scanty. Wistar rat pups were exposed to DLT (0·7 mg/kg body wt/day, i.p., dissolved in propylene glycol from postnatal day 9–13. Equal number of age matched pups were used as vehicle controls. The animals were weighed and perfused intracardially on postnatal days 12,15,21, and 30 and their brains dissected out. Cerebellum along with the brainstem was separated by a transverse section at the tectal level and processed for morphometric and toxicological studies. The micro-and inter-neurons in the cerebellum are known to differentiate and mature, both morphologically and biochemically, during the postnatal life of rats. Postnatal exposure to DLT has been observed to delay the cytogenesis and morphogenesis of these neurons. In addition to this, damage to the developing vasculature has also been recorded in the form of thrombus and haemorrhage. Focal degeneration and spongy appearance of the tissue in the vicinity of the damaged blood vessels have also been recorded. The study has opened up several questions on the safety of this substance to the pregnant mothers and infants in the habitats where this substance is in use for vector control.

Flunarizine enhances functional recovery following sciatic nerve crush lesion in rats

We have used the rat sciatic nerve crush (SNC) injury model to assess the neuroprotective effects of flunarizine (FNZ), a calcium channel antagonist and a vasodilator. The animals were treated with FNZ for various durations following SNC (0.33 mg/kg per day, i.p). Employing the physical disector method, we quantitated the rates of neuron loss in the dorsal root ganglion and spinal cord and protective effects of FNZ. FNZ treatment following SNC reduced neuron loss up to 86.6 and 82.5% in DRG sensory and spinal cord motor neurons, respectively. Functional recovery following SNC with or without FNZ treatment was assessed using the measurements of the total, 1-5 and 2-4-toe spread to quantitate percentage relative toe spread in relation to the respective controls. FNZ provided a superior return of function, i.e. near absolute recovery of both sensory and motor functions in 4 weeks, which is consistent with its neuroprotective effects.

Slow Physical Growth, Delayed Reflex Ontogeny, and Permanent Behavioral as Well as Cognitive Impairments in Rats Following Intra-generational Protein Malnutrition.

Environmental stressors including protein malnutrition (PMN) during pre-, neo- and post-natal age have been documented to affect cognitive development and cause increased susceptibility to neuropsychiatric disorders. Most studies have addressed either of the three windows and that does not emulate the clinical conditions of intra-uterine growth restriction (IUGR). Such data fail to provide a complete picture of the behavioral alterations in the F1 generation. The present study thus addresses the larger window from gestation to F1 generation, a new model of intra-generational PMN. Naive Sprague Dawley (SD) dams pre-gestationally switched to LP (8% protein) or HP (20% protein) diets for 45 days were bred and maintained throughout gestation on same diets. Pups born (HP/LP dams) were maintained on the respective diets post-weaningly. The present study aimed to show the sex specific differences in the neurobehavioral evolution and behavioral phenotype of the HP/LP F1 generation pups. A battery of neurodevelopmental reflex tests, behavioral (Open field and forelimb gripstrength test), and cognitive [Elevated plus maze (EPM) and Morris water maze (MWM)] assays were performed. A decelerated growth curve with significantly restricted body and brain weight, delays in apparition of neuro-reflexes and poor performance in the LP group rats was recorded. Intra-generational PMN induced poor habituation-with-time in novel environment exploration, low anxiety and hyperactive like profile in open field test in young and adult rats. The study revealed poor forelimb neuromuscular strength in LP F1 pups till adulthood. Group occupancy plots in MWM test revealed hyperactivity with poor learning, impaired memory retention and integration, thus modeling the signs of early onset Alzehemier phenotype. In addition, a gender specific effect of LP diet with severity in males and favoring female sex was also noticed.

Differential temporal expression of S100β in developing rat brain

Radial glial cells (RGs) originally considered to provide scaffold to the radially migrating neurons constitute a heterogeneous population of the regionally variable precursor cells that generate both neurons as well as glia depending upon the location and the timing of development. Hence specific immunohistochemical markers are required to specify their spatiotemporal location and fate in the neurogenic and gliogenic zones. We hypothesize S100β as a potential and unified marker for both primary and secondary progenitors. To achieve this, cryocut sections from rat brains of varied embryonic and postnatal ages were immunolabeled with a combination of antibodies, i.e., S100β + Nestin, Nestin + GFAP and S100β + GFAP. A large population of the primary and secondary progenitors, lining the VZ and SVZ, simultaneously co-expressed S100β and nestin establishing their progenitor nature. A downregulation of both S100β and nestin noticed by the end of the 1st postnatal week marks their differentiation towards neuronal or glial lineage. In view of the absence of co-expression of GFAP (glial fibrillary acidic protein) either with S100β or nestin, the suitability of accepting GFAP as an early marker of RG’s was eliminated. Thus the dynamic expression of S100β in both the neural stem cells (NSCs) and RGs during embryonic and early neonatal life is associated with its proliferative potential and migration of undifferentiated neuroblasts and astrocytes. Once they lose their potential for proliferation, the S100β expression is repressed with its reemergence in mature astrocytes. This study provides the first clear evidence of S100β expression throughout the period of neurogenesis and early gliogenesis, suggesting its suitability as a radial progenitor cell marker.

Glial alterations in tuberculous and cryptococcal meningitis and their relation to HIV co-infection - A study on human brains

INTRODUCTION Tuberculosis and cryptococcal infection of the central nervous system are common AIDS-associated opportunistic infections in tropical underdeveloped and developing countries. To date, research on these infections has focused on clinical, imaging, laboratory diagnosis, and animal models to elucidate the pathogenesis. There is paucity of information on astroglial and microglial alterations in the human nervous system following these infections. METHODOLOGY The pathomorphologic and morphometric alterations of astroglia and microglia in the prefrontal cortex and hippocampus in cases of tuberculous meningitis (TBM) and cryptococcal meningitis (CM) with and without associated HIV were described and compared with cases of HIV encephalitis without opportunistic infections (OI) and HIV-negative human brain tissue. RESULTS In TBM, the microglia and astrocytes were activated with hypertrophy and hyperplasia, aggregating in the subpial zone and around granulomas in meningeal exudate. In cases of cryptococcal meningitis, reactive changes were less prominent, though activation of both cellular elements was found. Association of HIV with these OIs resulted in muted glial and microglial response. In HIV encephalitis without OI, the level of activation of was low. Both astroglial and microglial cells expressed caspase-3, a pro-apoptotic marker, following HIV and opportunistic infections. Neuronal apoptosis, a mechanism to ensure neuronal survival, was less evident. The reactive astrocytes and microglia following opportunistic infection developed dystrophic changes heralding senescence. CONCLUSIONS Further studies on neuronal-astroglial-microglial interaction will offer deeper insight into the pathogenetic and immune mechanisms in the cellular and pathomorphological evolution of tuberculous and cryptococcal infections.

Intra-generational protein malnutrition impairs temporal astrogenesis in rat brain

ABSTRACT The lack of information on astrogenesis following stressor effect, notwithstanding the imperative roles of astroglia in normal physiology and pathophysiology, incited us to assess temporal astrogenesis and astrocyte density in an intra-generational protein malnutrition (PMN) rat model. Standard immunohistochemical procedures for glial lineage markers and their intensity measurements, and qRT-PCR studies, were performed to reveal the spatio-temporal origin and density of astrocytes. Reduced A2B5+ glia restricted precursor population in ventricles and caused poor dissemination to cortex at embryonic days (E)11-14, and low BLBP+ secondary radial glia in the subventricular zone (SVZ) of E16 low protein (LP) brains reflect compromised progenitor pooling. Contrary to large-sized BLBP+ gliospheres in high protein (HP) brains at E16, small gliospheres and discrete BLBP+ cells in LP brains evidence loss of colonization and low proliferative potential. Delayed emergence of GFAP expression, precocious astrocyte maturation and significantly reduced astrocyte number suggest impaired temporal and compromised astrogenesis within LP-F1 brains. Our findings of protein deprivation induced impairments in temporal astrogenesis, compromised density and astrocytic dysfunction, strengthen the hypothesis of astrocytes as possible drivers of neurodevelopmental disorders. This study may increase our understanding of stressor-associated brain development, opening up windows for effective therapeutic interventions against debilitating neurodevelopmental disorders. Summary: Maternal protein deprivation results in low progenitor pooling, and delayed and compromised astrogenesis, suggesting astrocyte impairment as a driver of neurological diseases owing to their imperative roles in normal and pathological situations.

Microglia in the Physiology and Pathology of Brain

Microglia are the immune cells of the brain involved in regulation and maintenance of brain micro-environment. These cells not only play imperative role in shaping the brain neural networks during development but also protect the brain from any disparaging condition throughout the life as first line of combatants. Interestingly, these cells exhibit dual nature in various neuropathological conditions. Depending upon the quantum of brain insult, they acquire different morphology which in favour either suppress the severity or aggravate the situation by releasing several immune-mediated molecules. This review summarizes the properties of microglia in healthy and diseased brain and explains their dual nature in various neuropathological disorders.

Postnatal exposure to poly (I:C) impairs learning and memory through changes in synaptic plasticity gene expression in developing rat brain

&NA; Viral infection during early stage of life influences brain development and results in several neurodevelopmental disorders such as schizophrenia, autism and behavioral abnormalities. However, the mechanism through which infection causes long‐term behavioral defects is not well known. To elucidate this, we have used synthetic polyinosinic‐polycytidylic acid [poly (I:C)] which acts as a dsRNA molecule and interacts with toll‐like receptor‐3 (TLR‐3) of microglia cells to evoke the immune system, thus mimicking the viral infection. Rat pups of postnatal day (PND) 7 were infused with a single dose of poly (I:C) (5 mg/kg BW) and vehicle alone to controls. When these pups grew to 3, 6 and 12 weeks, their spatial and fear conditioning memory were impaired as assessed by Morris water maze and passive avoidance test, respectively. We checked the immune activation by staining of TNF‐&agr; in the hippocampus and observed that poly (I:C) exposure elevated the number of TNF‐&agr; positive cells immediately after 12 h of infusion in one week rat and it persisted up to postnatal age of 3 and 12 weeks. Moreover, poly (I:C) significantly decreased the binding of 3H‐QNB to the cholinergic receptors in the frontal cortex and hippocampus of 3 and 6 weeks rats as compared to control but did not change significantly in 12 weeks rats. RT‐PCR and immunoblotting results showed that poly (I:C) exposure upregulated the expression of memory associated genes (BDNF, Arc, EGR1) at mRNA and protein level in frontal cortex and hippocampus of 3 weeks rats as compared to control. However, long‐time persistence of poly (I:C) effects significantly decreased the expression of these genes in both brain regions of 12 weeks rats. Taken together, it is evident that early life exposure to poly (I:C) has a long‐term effect and impairs learning and memory, probably through TNF‐&agr; mediated neuroinflammation and alteration in the expression of memory associated genes in frontal cortex and hippocampus of rats.