Microglial Cell Density Research Articles

Traumatic brain injury alterations in the functional connectome are associated with neuroinflammation but not tau in a P30IL tauopathy mouse model

IntroductionTraumatic Brain Injury (TBI) is often associated with long-term cognitive deficits and altered brain networks which have been linked with accumulation of neurofibrillary tau tangles and neuroinflammation. In this work, we investigated the changes in the brain post-TBI in an Alzheimer’s disease pR5 tauopathy model and evaluated the contribution of tauopathy and neuroinflammation to connectivity alterations using resting-state functional Magnetic Resonance Imaging (rs-fMRI). Method26 P301L tau transgenic mice of 8–9 months of age (21–35 g) expressing the human tau isoform carrying the pathogenic P301L mutation were used for the study. Animals were assessed at day 1 and 7 post-injury/craniotomy and were randomly divided into four groups. All animals underwent an MRI scan on a 9.4T Bruker system where rsfMRI was acquired. Following imaging, brains were stained with pSer (396 + 404), glial fibrillary acidic protein (GFAP), and ionised calcium-binding adaptor molecule-1 (Iba-1). Group-information-guided Independent Component Analysis (GIG-ICA) and region-of-interest (ROI)-based network connectivity approaches were applied. Principal Component Regression was applied to predict connectivity network strength from the corresponding ROIs. ResultsTBI mice showed decreased functional connectivity in the dentate gyrus, thalamus, and other areas compared to sham animals at day 1 post-injury with the majority of changes resolving at day 7. Principal Component Regression showed only the contralateral CA1 network strength was correlated with the CA1′s astrocyte and microglia cell density and the ipsilateral thalamus network strength was correlated with the ipsilateral thalamus’ astrocyte and microglia cell density. ConclusionWe present the first report on the temporal alterations in functional connectivity in a P30IL mouse model following TBI. Connectivity between key regions known to be affected in Alzheimer’s disease were short-term and reversible following injury. Connectivity strength in CA1 and thalamus showed significant correlation with astrocyte and microglial cell density but not tau density.

Early Application of Ipsilateral Cathodal-tDCS in a Mouse Model of Brain Ischemia Results in Functional Improvement and Perilesional Microglia Modulation.

Early stroke therapeutic approaches rely on limited options, further characterized by a narrow therapeutic time window. In this context, the application of transcranial direct current stimulation (tDCS) in the acute phases after brain ischemia is emerging as a promising non-invasive tool. Despite the wide clinical application of tDCS, the cellular mechanisms underlying its positive effects are still poorly understood. Here, we explored the effects of cathodal tDCS (C-tDCS) 6 h after focal forelimb M1 ischemia in Cx3CR1GFP/+ mice. C-tDCS improved motor functionality of the affected forelimb, as assessed by the cylinder and foot-fault tests at 48 h, though not changing the ischemic volume. In parallel, histological analysis showed that motor recovery is associated with decreased microglial cell density in the area surrounding the ischemic core, while astrocytes were not affected. Deeper analysis of microglia morphology within the perilesional area revealed a shift toward a more ramified healthier state, with increased processes’ complexity and a less phagocytic anti-inflammatory activity. Taken together, our findings suggest a positive role for early C-tDCS after ischemia, which is able to modulate microglia phenotype and morphology in parallel to motor recovery.

C3a Receptor Signaling Inhibits Neurodegeneration Induced by Neonatal Hypoxic-Ischemic Brain Injury.

Hypoxic-ischemic neonatal encephalopathy due to perinatal asphyxia is the leading cause of brain injury in newborns. Clinical data suggest that brain inflammation induced by perinatal insults can persist for years. We previously showed that signaling through the receptor for complement peptide C3a (C3aR) protects against cognitive impairment induced by experimental perinatal asphyxia. To investigate the long-term neuropathological effects of hypoxic-ischemic injury to the developing brain and the role of C3aR signaling therein, we subjected wildtype mice, C3aR deficient mice, and mice expressing biologically active C3a in the CNS to mild hypoxic-ischemic brain injury on postnatal day 9. We found that such injury triggers neurodegeneration and pronounced reactive gliosis in the ipsilesional hippocampus both of which persist long into adulthood. Transgenic expression of C3a in reactive astrocytes reduced hippocampal neurodegeneration and reactive gliosis. In contrast, neurodegeneration and microglial cell density increased in mice lacking C3aR. Intranasal administration of C3a for 3 days starting 1 h after induction of hypoxia-ischemia reduced neurodegeneration and reactive gliosis in the hippocampus of wildtype mice. We conclude that neonatal hypoxic-ischemic brain injury leads to long-lasting neurodegeneration. This neurodegeneration is substantially reduced by treatment with C3aR agonists, conceivably through modulation of reactive gliosis.

Mice lacking 5-lipoxygenase display motor deficits associated with cortical and hippocampal synapse abnormalities

Neural-immune interactions are related to the synapse plasticity and other dynamic processes in the nervous system. The absence or dysfunction of cellular/molecular elements from the immune system lead to impairments in the central and peripheral nervous system with behavior consequences such as cognitive, sensory, and locomotor deficits as well as social disabilities and anxiety disturbances. Cellular interactions between immune cells such as macrophages, microglia, and neutrophils with glial or neuronal cells have been of increasing interest over the last years. However, little is known about the role of immune-derived soluble factors in the context of homeostasis of the nervous system. Leukotrienes (LTs) are lipid mediators derived from the oxidation of arachidonic acid by 5-lipoxygenase (5-LO), and are classically involved in inflammation, allergies, and asthma. Here, we demonstrated that adult mice lacking 5-LO (5-LO−/−) showed motor deficits in rotarod test and increased repetitive behavior (marble burying test). These behavioral changes are accompanied by increased levels of synapse proteins (PSD95 and synaptophysin) at the motor cortex and hippocampus, but not with BDNF alterations. No changes in microglial cell density or morphology were seen in the brains of 5-LO−/− mice. Furthermore, expression of fractalkine receptor CX3CR1 was increased and of its ligand CX3CL1 was decreased in the cortex of 5-LO−/− mice. Here we provide evidence for the involvement of 5-LO products structuring synapses network with motor behavior consequences. We suggest that the absence of 5-LO products lead to modified microglial/neuron interaction, reducing microglial pruning.

The Effect of Valproic Acid Exposure throughout Development on Microglia Number in the Prefrontal Cortex, Hippocampus and Cerebellum

Microglia serve as resident immune cells in the brain, responding to insults and pathological developments. They have also been implicated in shaping synaptic development and regulation. The present study examined microglial cell density in a number of brain regions across select postnatal (P) ages along with the effects of valproic acid (VPA) on microglia density. Specifically, C57BL/6JCx3CR1+/GFP mice were examined for microglial cell number changes on P7, P14, P30, and P60 under baseline conditions and following 400 mg/kg VPA or saline. The prefrontal cortex (PFC), hippocampus and cerebellum were observed. Under control conditions, the results showed a shift in the number of microglia in these brain areas throughout development with a peak density in the hippocampus at P14 and an increase in PFC microglial numbers from P15 to P30. Interestingly, VPA treatment enhanced microglial numbers in a region-specific manner. VPA at P7 increased microglial cell number in the hippocampus and cerebellum whereas P14 VPA treatment altered microglial density in the cerebellum only. Cerebellar increases also occurred after VPA at P30, and were attended by an effect of increased numbers in the PFC. Finally, animals treated with VPA at P60 exhibited decreased microglia density in the hippocampus only. These results suggest rapid VPA-induced increases in microglial cell density in a developmentally-regulated fashion which differs across distinct brain areas. Furthermore, in the context of prior reports that early VPA causes excitotoxic damage, the present findings suggest early VPA exposure may provide a model for studying altered microglial responses to early toxicant challenge.

The N-Formyl Peptide Receptor 2 (FPR2) Agonist MR-39 Improves Ex Vivo and In Vivo Amyloid Beta (1\u201342)-Induced Neuroinflammation in Mouse Models of Alzheimer\u2019s Disease

The major histopathological hallmarks of Alzheimer’s disease (AD) include β-amyloid (Aβ) plaques, neurofibrillary tangles, and neuronal loss. Aβ 1–42 (Aβ1-42) has been shown to induce neurotoxicity and secretion of proinflammatory mediators that potentiate neurotoxicity. Proinflammatory and neurotoxic activities of Aβ1-42 were shown to be mediated by interactions with several cell surface receptors, including the chemotactic G protein-coupled N-formyl peptide receptor 2 (FPR2). The present study investigated the impact of a new FPR2 agonist, MR-39, on the neuroinflammatory response in ex vivo and in vivo models of AD. To address this question, organotypic hippocampal cultures from wild-type (WT) and FPR2-deficient mice (knockout, KO, FPR2−/−) were treated with fibrillary Aβ1-42, and the effect of the new FPR2 agonist MR-39 on the release of pro- and anti-inflammatory cytokines was assessed. Similarly, APP/PS1 double-transgenic AD mice were treated for 20 weeks with MR-39, and immunohistological staining was performed to assess neuronal loss, gliosis, and Aβ load in the hippocampus and cortex. The data indicated that MR-39 was able to reduce the Aβ1-42-induced release of proinflammatory cytokines and to improve the release of anti-inflammatory cytokines in mouse hippocampal organotypic cultures. The observed effect was apparently related to the inhibition of the MyD88/TRAF6/NFкB signaling pathway and a decrease in NLRP3 inflammasome activation. Administration of MR-39 to APP/PS1 mice improved neuronal survival and decreased microglial cell density and plaque load.These results suggest that FPR2 may be a promising target for alleviating the inflammatory process associated with AD and that MR-39 may be a useful therapeutic agent for AD.

Lipopolysaccharide-induced maternal immune activation modulates microglial CX3CR1 protein expression and morphological phenotype in the hippocampus and dentate gyrus, resulting in cognitive inflexibility during late adolescence

Inflammation during pregnancy can disturb brain development and lead to disorders in the progeny, including autism spectrum disorder and schizophrenia. However, the mechanism by which a prenatal, short-lived increase of cytokines results in adverse neurodevelopmental outcomes remains largely unknown. Microglia—the brain’s resident immune-cells—stand as fundamental cellular mediators, being highly sensitive and responsive to immune signals, which also play key roles during normal development.The fractalkine signaling axis is a neuron-microglia communication mechanism used to regulate neurogenesis and network formation. Previously, we showed hippocampal reduction of fractalkine receptor (Cx3cr1) mRNA at postnatal day (P) 15 in male offspring exposed to maternal immune activation induced with lipopolysaccharide (LPS) during late gestation, which was concomitant to an increased dendritic spine density in the dentate gyrus, a neurogenic niche. The current study sought to evaluate the origin and impact of this reduced hippocampal Cx3cr1 mRNA expression on microglia and cognition. We found that microglial total cell number and density are not affected in the dorsal hippocampus and dentate gyrus, respectively, but that the microglial CX3CR1 protein is decreased in the hippocampus of LPS-male offspring at P15. Further characterization of microglial morphology in the dentate gyrus identified a more ameboid phenotype in LPS-exposed offspring, predominantly in males, at P15. We thus explored maternal plasma and fetal brain cytokines to understand the mechanism behind microglial priming, showing a robust immune activation in the mother at 2 and 4 hrs after LPS administration, while only IL-10 tended towards upregulation at 2 hrs after LPS in fetal brains. To evaluate the functional long-term consequences, we assessed learning and cognitive flexibility behavior during late adolescence, finding that LPS affects only the latter with a male predominance on perseveration. A CX3CR1 gene variant in humans that results in disrupted fractalkine signaling has been recently associated with an increased risk for neurodevelopmental disorders. We show that an acute immune insult during late gestation can alter fractalkine signaling by reducing the microglial CX3CR1 protein expression, highlighting neuron-microglial fractalkine signaling as a relevant target underlying the outcomes of environmental risk factors on neurodevelopmental disorders.

Anesthetic Effects on the Progression of Parkinson Disease in the Rat DJ-1 Model.

Parkinson disease is a chronic and progressive movement disorder characterized by the loss of dopaminergic neurons in the substantia nigra pars compacta (SNpc). The causes of Parkinson disease are not clear but may involve genetic susceptibilities and environmental factors. As in other neurodegenerative disorders, individuals predisposed to Parkinson disease may have an accelerated onset of symptoms following perioperative stress such as anesthesia, surgery, pain, and inflammation. We hypothesized that anesthesia alone accelerates the onset of Parkinson disease-like pathology and symptoms. A presymptomatic Parkinson rat model (the protein, DJ-1, encoded by the Park7 gene [DJ-1], PARK7 knockout) was exposed to a surgical plane of isoflurane or 20% oxygen balanced with nitrogen for 2 hours on 3 occasions between 6 and 7 months of age. Acute and long-term motor and neuropathological effects were examined from 7 to 12 months of age in male DJ-1 rats, using the ladder rung, rotarod, and novel object recognition assays, as well as the immunohistochemical localization of tyrosine hydroxylase in dopaminergic neurons in the substantia nigra and ionized calcium-binding adaptor protein-1 (Iba-1) microglial activation in the substantia nigra and hippocampus. In the acute group, after the third anesthetic exposure at 7 months of age, the isoflurane group had a significant reduction in the density of dopaminergic neurons in the SNpc compared to controls. However, this reduction was not associated with increased microglial activation in the hippocampus or substantia nigra. With the ladder rung motor skills test, there was no effect of anesthetic exposure on the total number of foot faults or the ladder rung pattern in the acute group. The rotarod test also detected no differences before and after the third exposure in controls. For the long-term group, immunohistochemical analyses detected no differences in the density of dopaminergic neurons or microglial cells compared to unexposed DJ-1 rats from 8 to 12 months of age. The ladder rung test in the long-term group showed no differences in the total number of foot faults with time and exposure or between ladder rung patterns. The rotarod test detected no significant effect of exposure with time or between groups at any time point. The novel object recognition task in the long-term group revealed no differences in short- or long-term memory or in the number of rearings as a function of exposure. Multiple isoflurane exposures in this rat model of Parkinson disease transiently enhanced dopaminergic neurodegeneration in the SNpc that resolved over time and had no effects on progression in this Parkinson disease-like phenotype.

A regional and cellular analysis of the early intracellular and extracellular accumulation of Aβ in the brain of 5XFAD mice

Intracellular Aβ (iAβ) expression, extracellular Aβ (eAβ) plaque formation and microglial reactivity are characteristic neuropathological events of Alzheimer’s disease (AD) and have been detected in several transgenic mouse models of this disease. In this work we decided to investigate the early (2–7 months of age) development of these phenomena at both regional and cellular levels in 5XFAD mice, a severe transgenic mouse model of AD. We demonstrated that 1) Aβ pathology develops in many but not all brain regions, 2) iAβ is transient and almost always followed by eAβ in grey matter regions, and the respective levels are roughly proportional, and 3) in about 1/3 of the grey matter regions with Aβ pathology and in several white matter regions, eAβ plaques can appear where no iAβ-positive structures were detected. We also showed that male and female mice share a similar regional and cellular pattern of Aβ pathology development that is more prominent in females. Early iAβ is associated to the activation of microglia, while subsequent formation of eAβ plaques is associated with markedly increased density of microglial cells that acquire a characteristic clustered phenotype. Present analysis is relevant to set a reference for pathophysiological studies and to define specific targets for the test of therapeutic interventions in this widely used AD transgenic model.

Microglial Heterogeneity and Its Potential Role in Driving Phenotypic Diversity of Alzheimer's Disease.

Alzheimer’s disease (AD) is increasingly recognized as a highly heterogeneous disorder occurring under distinct clinical and neuropathological phenotypes. Despite the molecular determinants of such variability not being well defined yet, microglial cells may play a key role in this process by releasing distinct pro- and/or anti-inflammatory cytokines, potentially affecting the expression of the disease. We carried out a neuropathological and biochemical analysis on a series of AD brain samples, gathering evidence about the heterogeneous involvement of microglia in AD. The neuropathological studies showed differences concerning morphology, density and distribution of microglial cells among AD brains. Biochemical investigations showed increased brain levels of IL-4, IL-6, IL-13, CCL17, MMP-7 and CXCL13 in AD in comparison with control subjects. The molecular profiling achieved by measuring the brain levels of 25 inflammatory factors known to be involved in neuroinflammation allowed a stratification of the AD patients in three distinct “neuroinflammatory clusters”. These findings strengthen the relevance of neuroinflammation in AD pathogenesis suggesting, in particular, that the differential involvement of neuroinflammatory molecules released by microglial cells during the development of the disease may contribute to modulate the characteristics and the severity of the neuropathological changes, driving—at least in part—the AD phenotypic diversity.

A loss of mature microglial markers without immune activation in schizophrenia.

Microglia, the immune cells of the brain, are important for neurodevelopment and have been hypothesized to play a role in the pathogenesis of schizophrenia (SCZ). Although previous postmortem studies pointed toward presence of microglial activation, this view has been challenged by more recent hypothesis‐driven and hypothesis‐free analyses. The aim of the present study is to further understand the observed microglial changes in SCZ. We first performed a detailed meta‐analysis on studies that analyzed microglial cell density, microglial morphology, and expression of microglial‐specific markers. We then further explored findings from the temporal cortex by performing immunostainings and qPCRs on an additional dataset. A random effect meta‐analysis showed that the density of microglial cells was unaltered in SCZ (ES: 0.144 95% CI: 0.102 to 0.390, p = .250), and clear changes in microglial morphology were also absent. The expression of several microglial specific genes, such as CX3CR1, CSF1R, IRF8, OLR1, and TMEM119 was decreased in SCZ (ES: −0.417 95% CI: −0.417 to −0.546, p < .0001), consistent with genome‐wide transcriptome meta‐analysis results. These results indicate a change in microglial phenotype rather than density, which was validated with the use of TMEM119/Iba1 immunostainings on temporal cortex of a separate cohort. Changes in microglial gene expression were overlapping between SCZ and other psychiatric disorders, but largely opposite from changes reported in Alzheimer's disease. This distinct microglial phenotype provides a crucial molecular hallmark for future research into the role of microglia in SCZ and other psychiatric disorders.

Fetal Rhesus Monkey First Trimester Zika Virus Infection Impacts Cortical Development in the Second and Third Trimesters

AbstractZika virus is a teratogen similar to other neurotropic viruses, notably cytomegalovirus and rubella. The goal of these studies was to address the direct impact of Zika virus on fetal development by inoculating early gestation fetal rhesus monkeys using an ultrasound-guided approach (intraperitoneal vs. intraventricular). Growth and development were monitored across gestation, maternal samples collected, and fetal tissues obtained in the second trimester or near term. Although normal growth and anatomical development were observed, significant morphologic changes were noted in the cerebral cortex at 3-weeks post-Zika virus inoculation including massive alterations in the distribution, density, number, and morphology of microglial cells in proliferative regions of the fetal cerebral cortex; an altered distribution of Tbr2+ neural precursor cells; increased diameter and volume of blood vessels in the cortical proliferative zones; and a thinner cortical plate. At 3-months postinoculation, alterations in morphology, distribution, and density of microglial cells were also observed with an increase in blood vessel volume; and a thinner cortical plate. Only transient maternal viremia was observed but sustained maternal immune activation was detected. Overall, these studies suggest persistent changes in cortical structure result from early gestation Zika virus exposure with durable effects on microglial cells.

DIPG-56. EXPLORATION OF TUMOR/STROMA INTERACTIONS IN DIPG XENOGRAFT BY SPECIES-SPECIFIC RNA-SEQ DECONVOLUTION INDICATES A ROLE OF MICROGLIA CELL IN DIPG DEVELOPMENT

Diffuse Intrinsic Pontine Glioma (DIPG) and more largely Diffuse Midline Gliomas H3 K27M-mutant (DMG) harbor a unique property of infiltration. Our objective is to elucidate/describe the cellular and molecular determinants of micro-environmental modifications resulting from the tumour/stroma dialogue as it might provide pro-invasive conditions that favour the development of the disease. To this end, we performed RNA-seq analyses to characterize exhaustively the bidirectional molecular modifications of the stroma/tumour in DIPG xenograft models. Gene expression changes in murine microenvironment compartment were investigated as continuous or semi-continuous traits of tumor load by measuring transcriptome in zone with high vs. low infiltration. We observed substantial modulations in gene expression in the microenvironment associated with increasing tumor cell content, pointing to a modification of the macrophage/microglial infiltrate. The expression or overexpression of several modulated genes was validated by IHC in the stroma of DMG primary tumors. Among them, overexpression of the cytokine CCL3 was confirmed, reflecting the activation status of microglial cells. Moreover, we observed in patients that the density of IBA-1 positive microglial cells increases according to the extent of tumor infiltration and that a significant part of them harbor a mitotic status, supporting their interaction with DMG cells. The involvement of this interaction in DMG development needs further evaluation and might represent opportunity to slow down DIPG extension.

The type of stress matters: repeated injection and permanent social isolation stress in male mice have a differential effect on anxiety- and depressive-like behaviours, and associated biological alterations

Chronic stress can alter the immune system, adult hippocampal neurogenesis and induce anxiety- and depressive-like behaviour in rodents. However, previous studies have not discriminated between the effect(s) of different types of stress on these behavioural and biological outcomes. We investigated the effect(s) of repeated injection vs. permanent social isolation on behaviour, stress responsivity, immune system functioning and hippocampal neurogenesis, in young adult male mice, and found that the type of stress exposure does indeed matter. Exposure to 6 weeks of repeated injection resulted in an anxiety-like phenotype, decreased systemic inflammation (i.e., reduced plasma levels of TNFα and IL4), increased corticosterone reactivity, increased microglial activation and decreased neuronal differentiation in the dentate gyrus (DG). In contrast, exposure to 6 weeks of permanent social isolation resulted in a depressive-like phenotype, increased plasma levels of TNFα, decreased plasma levels of IL10 and VEGF, decreased corticosterone reactivity, decreased microglial cell density and increased cell density for radial glia, s100β-positive cells and mature neuroblasts—all in the DG. Interestingly, combining the two distinct stress paradigms did not have an additive effect on behavioural and biological outcomes, but resulted in yet a different phenotype, characterized by increased anxiety-like behaviour, decreased plasma levels of IL1β, IL4 and VEGF, and decreased hippocampal neuronal differentiation, without altered neuroinflammation or corticosterone reactivity. These findings demonstrate that different forms of chronic stress can differentially alter both behavioural and biological outcomes in young adult male mice, and that combining multiple stressors may not necessarily cause more severe pathological outcomes.

A maternal Western diet during gestation and lactation modifies offspring’s microglial cell density and morphology in the hippocampus and prefrontal cortex in Yucatan minipigs

Changes in microglial development and morphology can be induced by inflammatory conditions and associated with eating or mood disorders, such as hyperphagia or depression. In a previous paper in the minipig model, we showed that maternal Western diet during gestation and lactation decreased hippocampus neurogenesis and food-rewarded cognitive abilities in the progeny. Whether these alterations are concomitant with a central inflammatory process in brain structures involved in learning and memory (hippocampus, HPC), cognitive (prefrontal cortex, PFC), or hedonic (orbitofrontal cortex, OFC) control of food intake is still unknown. In the present study, Yucatan minipigs (Sus scrofa) sows were exposed to two different diets during gestation and lactation (standard, SD N = 7 vs. Western diet, WD N = 9). Iba1 is a calcium-binding protein specifically expressed in microglia in the brain, which plays an important role in the regulation of the microglia function. Iba1 expression was examined by immunohistochemical analyses in the PFC, OFC and HPC of piglets. The density of microglial cells, as well as their morphology, were assessed in order to have an indirect insight of microglial cell activation state possibly in relationship with neuroinflammation. The density of Iba1-positive cells was higher in the PFC but not in the HPC of WD compared to SD piglets (p < 0.001). In the HPC, anterior and dorsolateral PFC, WD piglets had more unipolar cells, contrary to SD that had more multipolar cells (P < 0.0001). Opposite effects were observed in the OFC, with SD presenting more unipolar (P < 0.001) microglial cells compared to WD. We showed here that maternal diet during pregnancy and lactation had significant effects on morphological changes of microglial cells in the offspring, and that these effects differed between the HPC and PFC, suggesting different response mechanisms to the early nutritional environment.

Inflammatory cells proliferate in the choroid and retina without choroidal thickness change in early Type 1 diabetes

Increasing evidence points to inflammation as a key factor in the pathogenesis of diabetic retinopathy (DR). Choroidal inflammatory changes in diabetes have been reported and in vivo choroidal thickness (CT) has been searched as a marker of retinopathy with contradictory results. We aimed to investigate the early stages in the retina and choroid in an animal model of Type 1 diabetes. Type 1 diabetes was induced in male Wistar rats via a single i.p. streptozotocin injection. At 8 weeks after disease onset, CT, choroidal vascular density, VEGF and VEGFR2 expression, microglial cell and pericyte distribution were evaluated. Diabetic rats showed no significant change in CT and choroidal vascular density. A widened pericyte-free gap between the retinal pigment epithelium and the choroid was observed in diabetic rats. The immunoreactivity of VEGFR2 was decreased in the retina of diabetic rats, despite no statistically significant difference in the immunoreactivity of VEGF. The density of microglial cells significantly increased in the choroid and retina of diabetic rats. Reactive microglial cells were found to be more abundant in the choroid of diabetic rats. Evidences of the interconnection between the superficial, intermediate, and deep plexuses of the retina were also observed. At early stages, Type 1 diabetes does not affect choroidal thickness and choroidal vascular density. Proliferation and reactivity of microglial cells occurs in the choroidal stroma and the retina. The expression of VEGFR2 decreases in the retina.

A Translational Study on Acute Traumatic Brain Injury: High Incidence of Epileptiform Activity on Human and Rat Electrocorticograms and Histological Correlates in Rats.

Background: In humans, early pathological activity on invasive electrocorticograms (ECoGs) and its putative association with pathomorphology in the early period of traumatic brain injury (TBI) remains obscure. Methods: We assessed pathological activity on scalp electroencephalograms (EEGs) and ECoGs in patients with acute TBI, early electrophysiological changes after lateral fluid percussion brain injury (FPI), and electrophysiological correlates of hippocampal damage (microgliosis and neuronal loss), a week after TBI in rats. Results: Epileptiform activity on ECoGs was evident in 86% of patients during the acute period of TBI, ECoGs being more sensitive to epileptiform and periodic discharges. A “brush-like” ECoG pattern superimposed over rhythmic delta activity and periodic discharge was described for the first time in acute TBI. In rats, FPI increased high-amplitude spike incidence in the neocortex and, most expressed, in the ipsilateral hippocampus, induced hippocampal microgliosis and neuronal loss, ipsilateral dentate gyrus being most vulnerable, a week after TBI. Epileptiform spike incidence correlated with microglial cell density and neuronal loss in the ipsilateral hippocampus. Conclusion: Epileptiform activity is frequent in the acute period of TBI period and is associated with distant hippocampal damage on a microscopic level. This damage is probably involved in late consequences of TBI. The FPI model is suitable for exploring pathogenetic mechanisms of post-traumatic disorders.

Spinal Cord Injury Leads to Hippocampal Glial Alterations and Neural Stem Cell Inactivation.

The hippocampus encodes spatial and contextual information involved in memory and learning. The incorporation of new neurons into hippocampal networks increases neuroplasticity and enhances hippocampal-dependent learning performances. Only few studies have described hippocampal abnormalities after spinal cord injury (SCI) although cognitive deficits related to hippocampal function have been reported in rodents and even humans. The aim of this study was to characterize in further detail hippocampal changes in the acute and chronic SCI. Our data suggested that neurogenesis reduction in the acute phase after SCI could be due to enhanced death of amplifying neural progenitors (ANPs). In addition, astrocytes became reactive and microglial cells increased their number in almost all hippocampal regions studied. Glial changes resulted in a non-inflammatory response as the mRNAs of the major pro-inflammatory cytokines (IL-1β, TNFα, IL-18) remained unaltered, but CD200R mRNA levels were downregulated. Long-term after SCI, astrocytes remained reactive but on the other hand, microglial cell density decreased. Also, glial cells induced a neuroinflammatory environment with the upregulation of IL-1β, TNFα and IL-18 mRNA expression and the decrease of CD200R mRNA. Neurogenesis reduction may be ascribed at later time points to inactivation of neural stem cells (NSCs) and inhibition of ANP proliferation. The number of granular cells and CA1 pyramidal neurons decreased only in the chronic phase. The release of pro-inflammatory cytokines at the chronic phase might involve neurogenesis reduction and neurodegeneration of hippocampal neurons. Therefore, SCI led to hippocampal changes that could be implicated in cognitive deficits observed in rodents and humans.

Lipopolysaccharide-Induced Systemic Inflammation in the Neonatal Period Increases Microglial Density and Oxidative Stress in the Cerebellum of Adult Rats.

Inflammatory processes occurring in the perinatal period may affect different brain regions, resulting in neurologic sequelae. Injection of lipopolysaccharide (LPS) at different neurodevelopmental stages produces long-term consequences in several brain structures, but there is scarce evidence regarding alterations in the cerebellum. The aim of this study was to evaluate the long-term consequences on the cerebellum of a systemic inflammatory process induced by neonatal LPS injection. For this, neonatal rats were randomly assigned to three different groups: naïve, sham, and LPS. Saline (sham group) or LPS solution (1 mg/kg) was intraperitoneally injected on alternate postnatal days (PN) PN1, PN3, PN5, and PN7. Spontaneous activity was evaluated with the open field test in adulthood. The cerebellum was evaluated for different parameters: microglial and Purkinje cell densities, oxidative stress levels, and tumor necrosis factor alpha (TNF-α) mRNA expression. Our results show that administration of LPS did not result in altered spontaneous activity in adult animals. Our data also indicate increased oxidative stress in the cerebellum, as evidenced by an increase in superoxide fluorescence by dihydroethidium (DHE) indicator. Stereological analyses indicated increased microglial density in the cerebellum that was not accompanied by Purkinje cell loss or altered TNF-α expression in adult animals. Interestingly, Purkinje cells ectopically positioned in the granular and molecular layers of the cerebellum were observed in animals of the LPS group. Our data suggest that neonatal LPS exposure causes persistent cellular and molecular changes to the cerebellum, indicating the susceptibility of this region to systemic inflammatory insults in infancy. Further investigation of the consequences of these changes and the development of strategies to avoid those should be subject of future studies.

The Delayed Neuroprotective Effect of Methylene Blue in Experimental Rat Brain Trauma.

After traumatic brain injury (TBI), an increase in dysfunction of the limbs contralateral to injury focus was observed. Using different behavioral tests, we found that a single intravenous injection of methylene blue (MB, 1 mg/kg) 30 min after the injury reduced the impairment of the motor functions of the limbs from 7 to 120 days after TBI. Administration of methylene blue 30 min after the injury and then monthly (six injections in total) was the most effective both in terms of preservation of limb function and duration of therapeutic action. This therapeutic effect was clearly manifested from the seventh day and continued until the end of the experiment—by the 180th day after TBI. MB is known to possess antioxidant properties; it has a protective effect against TBI by promoting autophagy and minimizing lesion volume in the first two weeks after TBI. Studies of the brains on the 180th day after TBI demonstrated that the monthly treatment of animals with MB statistically significantly prevented an increase in the density of microglial cells in the ipsilateral hemisphere and a decrease in the thickness of the corpus callosum in the contralateral hemisphere in comparison with untreated animals. However, on the 180th day after TBI, the magnetic resonance imaging scan of the animal brains did not show a significant reduction in the volume of the lesion in MB-treated animals. These findings are important for understanding the development of the long-term effects of TBI and expand the required therapeutic window for targeted neuroprotective interventions.