Glial Morphology Research Articles

The effect of Brucella abortus on glial activation and cell death in adult male rat's hippocampus

A zoonotic disease called brucellosis can cause flu-like symptoms and heart inflammation. The bacteria responsible for this disease can also enter the brain, causing a condition called neurobrucellosis that can result in long-term neurological problems. In this study, researchers aimed to determine the changes in the hippocampal cells of rats infected with Brucella. For the study, 24 adult male albino rats were inoculated with 1 × 106 CFU Brucella abortus 544. The rats were then deeply anesthetized, and their hippocampus samples were taken for stereological, histological, and molecular studies. The results showed that the infected rats had increased microgliosis and astrogliosis. Furthermore, a high level of caspase-3 in their hippocampal tissue indicated their susceptibility to apoptosis. Additionally, there was a decrease in expression of Ki67, which further supported this. Sholl's analysis confirmed a significant failure in glial morphology. The study demonstrated that the pathogen has the ability to destroy the hippocampus and potentially affect its normal physiology. However, more research is needed to clarify various aspects of neurobrucellosis.

TRPV4 Antagonist Treatment of Hydrocephalus Causes Cell and Molecular Changes in Multiple Brain Cell Types

Hydrocephalus is defined by cerebrospinal fluid (CSF) accumulation in the brain’s ventricles causing ventriculomegaly, an imbalance in CSF secretion, flow, and/or absorption. Treatments for hydrocephalus include invasive surgical procedures, which commonly fail throughout a patient’s life. Transient receptor potential vanilloid member 4 (TRPV4) is a mechanosensitive cation channel implicated in osmotic regulation. Our laboratory found that a TRPV4 antagonist, RN1734, ameliorates hydrocephalus in a rat model of congenital hydrocephalus. It was hypothesized that treatment slowed production of CSF by targeting choroid plexus (CP), a contiguous layer of tightly regulated epithelial cells, that is responsible for CSF production. However, hydrocephalus pathology can have various effects on the brain. This study focuses on changes occurring in CP epithelial cells (CPe) and glia, which have roles in brain-barrier maintenance and brain fluid/electrolyte regulation. Interestingly, CPe and glia contain many of the same channels and transporters, including TRPV4, that are important in regulating both CSF and brain interstitial fluid. These cells contain aquaporins (AQP), AQP1 in CPe, and AQP4 in glia. AQPs have been implicated in diseases associated with fluid regulation and may interact with TRPV4. We hypothesize that TRPV4 and AQPs are important in hydrocephalus pathology, and that changes may be attenuated with RN1734 treatment. A point mutation in TMEM67 results in hydrocephalus (hydro). Changes in cell morphology, gene transcription, protein expression, localization of TRPV4, AQP1, and AQP4 were observed. Postnatal day 15 (P15) untreated (unt) and RN1734-treated (RN;2mg/ml DMSO; 4mg/kg intraperitoneal injection daily P7-P14) wildtype and mutant ( Tmem67−/−) rats were utilized in this study. CPe and periventricular (PV) glia were visualized using fluorescent immunohistochemistry (IHC) of GFAP and a plasma membrane marker. Cell volume of CPe’s and process morphology of glia were altered in hydro rats. RN returned these changes to relatively normal in CPe, but not glia. Next, we used IHC to examine localization of TRPV4 and the AQPs. Increased immunoreactivity of TRPV4 and AQP1 was observed in CPe of unt and RN Tmem67−/− rats. AQP4 and TRPV4 immunoreactivity increased in the subventricular zone and PV white matter of hydro rats. TRPV4 immunoreactivity, but not AQP4, was normalized with RN. We then utilized RTqPCR to inspect transcriptional changes. The data showed increased AQP1 and decreased TRPV4 mRNA in PV cortex and CP of unt and RN Tmem67−/− rats. Previous data from our laboratory found no change in TRPV4 protein in unt Tmem67−/− CPe, but we were curious about AQP expression. The amount of AQP1 protein was increased in the CP of unt and RN Tmem67−/− rats. In PV cortex, AQP4, but not TRPV4, protein was increased in hydro rats. With RN, AQP4 protein was not normalized. In summary, TRPV4 changes in the Tmem67−/− rats returned to normal with RN, glial morphology and AQP changes did not. This may indicate residual inflammation in the Tmem67−/− rats, which we aim to address in future studies. To further elucidate TRPV4’s role in hydrocephalus pathology, future studies will explore post-translational and membrane localization changes. Overall, investigating cell and molecular changes that occur in hydrocephalus will help find better pharmacological targets for this disease. Funding: United States Department of Defense Congressionally Directed Medical Research Program Award; Hydrocephalus Association. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Neurodegeneration and glial morphological changes are both prevented by TRPM2 inhibition during the progression of a Parkinson's disease mouse model

Parkinson's disease (PD) is a neurodegenerative disease characterized by dopaminergic neuron death and neuroinflammation. Emerging evidence points to the involvement of the transient receptor potential melastatin 2 (TRPM2) channel in neuron death and glial activation in several neurodegenerative diseases. However, the involvement of TRPM2 in PD and specifically its relation to the neuroinflammation aspect of the disease remains poorly understood. Here, we hypothesized that AG490, a TRPM2 inhibitor, can be used as a treatment in a mouse model of PD. Mice underwent stereotaxic surgery for 6-hydroxydopamine (6-OHDA) administration in the right striatum. Motor behavioral tests (apomorphine, cylinder, and rotarod) were performed on day 3 post-injection to confirm the PD model induction. AG490 was then daily injected i.p. between days 3 to 6 after surgery. On day 6, motor behavior was assessed again. Substantia nigra (SNc) and striatum (CPu) were collected for immunohistochemistry, immunoblotting, and RT-qPCR analysis on day 7. Our results revealed that AG490 post-treatment reduced motor behavior impairment and nigrostriatal neurodegeneration. In addition, the compound prevented TRPM2 upregulation and changes of the Akt/GSK-3β/caspase-3 signaling pathway. The TRPM2 inhibition also avoids the glial morphology changes observed in the PD group. Remarkably, the morphometrical analysis revealed that the ameboid-shaped microglia, found in 6-OHDA-injected animals, were no longer present in the AG490-treated group. These results indicate that AG490 treatment can reduce dopaminergic neuronal death and suppress neuroinflammation in a PD mouse model. Inhibition of TRPM2 by AG490 could then represent a potential therapeutical strategy to be evaluated for PD treatment.

'Unpredictable chronic mild stress does not exacerbate memory impairment or altered neuronal and glial plasticity in the hippocampus of middle-aged vitamin D deficient mice'.

Vitamin D deficiency is a worldwide health concern, especially in the elderly population. Much remains unknown about the relationship between vitamin D deficiency (VDD), stress-induced cognitive dysfunctions and depressive-like behaviour. In this study, 4-month-old male C57Bl/6J mice were fed with control or vitamin D free diet for 6 months, followed by unpredictable chronic stress (UCMS) for 8 weeks. VDD induced cognitive impairment and reduced grooming behaviour, but did not induce depressive-like behaviour. While UCMS in vitamin D sufficient mice induced expected depressive-like phenotype and impairments in the contextual fear memory, chronic stress did not manifest as an additional risk factor for memory impairments and depressive-like behaviour in VDD mice. In fact, UCMS restored self-care behaviour in VDD mice. At the histopathological level, VDD mice exhibited cell loss in the granule cell layer, reduced survival of newly generated cells, accompanied with an increased number of apoptotic cells and alterations in glial morphology in the hippocampus; however, these effects were not exacerbated by UCMS. Interestingly, UCMS reversed VDD induced loss of microglial cells. Moreover, tyrosine hydroxylase levels decreased in the striatum of VDD mice, but not in stressed VDD mice. These findings indicate that long-term VDD in adulthood impairs cognition but does not augment behavioural response to UCMS in middle-aged mice. While VDD caused cell loss and altered glial response in the DG of the hippocampus, these effects were not exacerbated by UCMS and could contribute to mechanisms regulating altered stress response.

Extracellular matrix substrates differentially influence enteric glial cell homeostasis and immune reactivity.

Enteric glial cells are important players in the control of motility, intestinal barrier integrity and inflammation. During inflammation, they switch into a reactive phenotype enabling them to release inflammatory mediators, thereby shaping the inflammatory environment. While a plethora of well-established in vivo models exist, cell culture models necessary to decipher the mechanistic pathways of enteric glial reactivity are less well standardized. In particular, the composition of extracellular matrices (ECM) can massively affect the experimental outcome. Considering the growing number of studies involving primary enteric glial cells, a better understanding of their homeostatic and inflammatory in vitro culture conditions is needed. We examined the impact of different ECMs on enteric glial culture purity, network morphology and immune responsiveness. Therefore, we used immunofluorescence and brightfield microscopy, as well as 3' bulk mRNA sequencing. Additionally, we compared cultured cells with in vivo enteric glial transcriptomes isolated from Sox10iCreERT2Rpl22HA/+ mice. We identified Matrigel and laminin as superior over other coatings, including poly-L-ornithine, different lysines, collagens, and fibronectin, gaining the highest enteric glial purity and most extended glial networks expressing connexin-43 hemichannels allowing intercellular communication. Transcriptional analysis revealed strong similarities between enteric glia on Matrigel and laminin with enrichment of gene sets supporting neuronal differentiation, while cells on poly-L-ornithine showed enrichment related to cell proliferation. Comparing cultured and in vivo enteric glial transcriptomes revealed a 50% overlap independent of the used coating substrates. Inflammatory activation of enteric glia by IL-1β treatment showed distinct coating-dependent gene expression signatures, with an enrichment of genes related to myeloid and epithelial cell differentiation on Matrigel and laminin coatings, while poly-L-ornithine induced more gene sets related to lymphocyte differentiation. Together, changes in morphology, differentiation and immune activation of primary enteric glial cells proved a strong effect of the ECM. We identified Matrigel and laminin as pre-eminent substrates for murine enteric glial cultures. These new insights will help to standardize and improve enteric glial culture quality and reproducibility between in vitro studies in the future, allowing a better comparison of their functional role in enteric neuroinflammation.

S100B actions on glial and neuronal cells in the developing brain: an overview.

The S100B is a member of the S100 family of "E" helix-loop- "F" helix structure (EF) hand calcium-binding proteins expressed in diverse glial, selected neuronal, and various peripheral cells, exerting differential effects. In particular, this review compiles descriptions of the detection of S100B in different brain cells localized in specific regions during the development of humans, mice, and rats. Then, it summarizes S100B's actions on the differentiation, growth, and maturation of glial and neuronal cells in humans and rodents. Particular emphasis is placed on S100B regulation of the differentiation and maturation of astrocytes, oligodendrocytes (OL), and the stimulation of dendritic development in serotoninergic and cerebellar neurons during embryogenesis. We also summarized reports that associate morphological alterations (impaired neurite outgrowth, neuronal migration, altered radial glial cell morphology) of specific neural cell groups during neurodevelopment and functional disturbances (slower rate of weight gain, impaired spatial learning) with changes in the expression of S100B caused by different conditions and stimuli as exposure to stress, ethanol, cocaine and congenital conditions such as Down's Syndrome. Taken together, this evidence highlights the impact of the expression and early actions of S100B in astrocytes, OL, and neurons during brain development, which is reflected in the alterations in differentiation, growth, and maturation of these cells. This allows the integration of a spatiotemporal panorama of S100B actions in glial and neuronal cells in the developing brain.

Reduced Bergmann glial process terminations and lateral appendages in essential tremor.

Postmortem examination of the essential tremor cerebellum has revealed a variety of pathological changes centered in and around Purkinje cells. Studies have predominantly focused on cerebellar neuronal connections. Bergmann glial morphology has not yet been studied in essential tremor. Among their many roles, Bergmann glia in the cerebellar cortex ensheath Purkinje cell synapses and provide neuroprotection. Specifically, the complex radial processes and lateral appendages of Bergmann glia are structural domains that modulate Purkinje cell synaptic transmission. In this study, we investigate whether Bergmann glia morphology is altered in the essential tremor cerebellum. We applied the Golgi-Kopsch method and used computerized three-dimensional cell reconstruction to visualize Bergmann glia in the postmortem cerebellum of 34 cases and 17 controls. We quantified morphology of terminal structures (number of terminations and lateral appendage density) and morphology of radial processes (total process length, branch length, branch order, and branch volume) in each glial cell. We quantified number of branches and volume as well. Essential tremor cases had a 31.9% decrease in process terminations and a 35.7% decrease in lateral appendage density in Bergmann glia. Total process length and branch length did not differ between essential tremor cases and controls. We found also a reduction in number of secondary and tertiary branches and tertiary branches volume. These findings suggest that Bergmann glia in essential tremor cases have more alterations in their terminal structures, with a relative preservation of radial processes, and highlight a potential role for these astrocytes in the disease pathophysiology.

Method for cryopreservation of trigeminal ganglion for establishing primary cultures of neurons and glia

BackgroundPrimary neuronal cultures are used to elucidate cellular and molecular mechanisms involved in disease pathology and modulation by pharmaceuticals and nutraceuticals, and to identify novel therapeutic targets. However, preparation of primary neuronal cultures from rodent embryos is labor-intensive, and it can be difficult to produce high-quality consistent cultures. To overcome these issues, cryopreservation can be used to obtain standardized, high-quality stocks of neuronal cultures. New methodIn this study, we present a simplified cryopreservation method for rodent primary trigeminal ganglion neurons and glia from Sprague-Dawley neonates, using a 90:10 (v/v) fetal bovine serum/dimethyl sulfoxide cell freezing medium. ResultsCryopreserved trigeminal ganglion cells stored for up to one year in liquid nitrogen exhibited similar neuronal and glial cell morphology to fresh cultures and retained high cell viability. Proteins implicated in inflammation and pain signaling were expressed in agreement with the reported subcellular localization. Additionally, both neurons and glial cells exhibited an increase in intracellular calcium levels in response to a depolarizing stimulus. Cryopreserved cells were also transiently transfected with reporter genes. Comparison with existing methodsOur method is simple, does not require special reagents or equipment, will save time and money, increase flexibility in study design, and produce consistent cultures. ConclusionsThis method for the preparation and cryopreservation of trigeminal ganglia results in primary cultures of neurons and glia similar in viability and morphology to fresh preparations that could be utilized for biochemical, cellular, and molecular studies, increase reproducibility, and save laboratory resources.

Targeting ceramides in 5XFAD mice reduces amyloid plaque deposition and gliosis

AbstractBackgroundAlzheimer’s Disease and other neurodegenerative disorders have been linked to metabolic disorders associated with aberrant lipid deposition. Sphingolipids, such as ceramides, are among the most deleterious lipids that accumulate in the AD brain. Increased ceramides have been measured in post‐mortem AD brain samples and are abundant in Aβ‐plaques. As biomarkers, ceramides in the plasma and cerebrospinal fluid correlate with AD risk. The potential of ceramide‐lowering strategies to treat or prevent AD has been underexplored. We hypothesize that ceramides are causal contributors to AD pathologies and amenable to therapeutic targeting to improve AD outcomes.MethodTo address ceramides in AD, we have targeted serine palmitoyl‐transferase (SPT), the first enzyme and rate‐limiting step in ceramide de novo synthesis, with the drug myriocin in 5XFAD mice on a regular chow diet and an obesogenic high‐fat diet. We have also generated inducible genetic models to study loss‐of‐ceramides (overexpression of the ceramide‐degrading enzyme Asah1) or gain‐of‐ceramides (overexpression of the SPT complex), specifically in neurons in the 5XFAD model. We used immunocytochemistry to assess Aβ plaque numbers and size and both astrocyte and microglial activation and numbers. Cognitive behavior was measured using a Barnes maze.Result5XFAD mice treated with myriocin for 17 weeks had a reduction of hippocampal ceramides. As expected, control vehicle‐treated 5XFAD mice showed a significant memory deficit during behavioral testing. Myriocin‐treated mice had significantly improved memory of the Barnes maze. Treated mice had fewer and smaller Aβ plaques and had a reduction in both astrocyte numbers and arborization in the cortex and hippocampus. Microglia were reduced in the hippocampus of treated mice. In 5XFAD mice with neuron‐specific Asah1 overexpression, Aβ plaque size and number remained unchanged compared to a 5XFAD control. However, Gfap+ astrocytes and Iba1+ microglia/monocyte activation and migration were eliminated, resulting in plaques with no associated glia and glial morphology like non‐5XFAD mice.ConclusionDecreasing ceramide production by myriocin treatment in 5XFAD mice reduced Aβ plaque number and size and reduced gliosis in the cortex and hippocampus. Reducing ceramides in neurons results in the abrogation of gliosis throughout the brain of a 5XFAD mouse.

Extracts from Dendropanax morbifera leaves ameliorates cerebral ischemia-induced hippocampal damage by reducing oxidative damage in gerbil

AimIn this study, we investigated the effects of Dendropanax morbifera extract (DME) on neuroprotection against ischemic damage in gerbils. MethodsDME (100 or 300 mg/kg) was orally administered to gerbils for three weeks, and 2 h after the last DME treatment, transient forebrain ischemia in the common carotid arteries was induced for 5 min. The forebrain ischemia-related cognitive impairments were assessed by spontaneous motor activity and passive avoidance test one and four days after ischemia, respectively. In addition, surviving and degenerating neurons were morphologically confirmed by neuronal nuclei immunohistochemical staining and Fluoro-Jade C staining, respectively, four days after ischemia. Changes of glial morphology were visualized by immunohistochemical staining for each marker such as glial fibrillary acidic protein and ionized calcium-binding protein. Oxidative stress was determined by measurements of dihydroethidium, O2· (formation of formazan) and malondialdehyde two days after ischemia. In addition, glutathione redox system such as reduced glutathione, oxidized glutathione levels, glutathione peroxidase, and glutathione reductase activities were measured two days after ischemia. ResultsSpontaneous motor activity monitoring and passive avoidance tests showed that treatment with 300 mg/kg DME, but not 100 mg/kg, significantly alleviated ischemia-induced memory impairments. In addition, approximately 67 % of mature neurons survived and 29.3 % neurons were degenerated in hippocampal CA1 region four days after ischemia, and ischemia-induced morphological changes in astrocytes and microglia were decreased in the CA1 region after 300 mg/kg DME treatment. Furthermore, treatment with 300 mg/kg DME significantly ameliorated ischemia-induced oxidative stress, such as superoxide formation and lipid peroxidation, two days after ischemia. In addition, ischemia-induced reduction of the glutathione redox system in the hippocampus, assessed two days after the ischemia, was ameliorated by treatment with 300 mg/kg DME. These suggest that DME can potentially reduce ischemia-induced neuronal damage through its antioxidant properties.

PATH-23. CLINICOPATHOLOGIC ANALYSIS OF NOVEL METHYLATION CLUSTERS OF IDH-WILDTYPE DIFFUSE GLIOMAS

Abstract Methylation profiling of brain tumors has revealed novel clusters whose biological and clinical significance is unknown. These include three entities termed by the 12.5 version of the Heidelberg methylation classifier as adult-type diffuse high grade glioma, IDH-wildtype, subtype B (HGG_B), subtype E (HGG_E), and subtype F (HGG_F). We have identified 75 cases belonging to these groups: HGG_B (25 cases, 32% female), HGG_E (40 cases, 42% female), and HGG_F (18 cases, 33% female). Although originally characterized as “adult-type,” we find that 70% of HGG_E cases are pediatric, with a median age at diagnosis of 11 years. HGG_B cases are predominantly seen in young adults, and HGG_F in older adults. The histology varies among methylation classes, with HGG_B cases demonstrating high-grade, predominantly astrocytic, morphology, while HGG_E cases demonstrate high-grade glial histology with distinct nuclear features, and HGG_F cases show highly infiltrative but lower grade glial morphology. Patient outcome data obtained to date shows a collective median survival exceeding 5 years, suggesting re-consideration of the proposed grading for these tumors. MGMT promoter methylation differs across the HGG_B (0%), HGG_E (12%), and HGG_F (0%) subtypes. Mutational analyses show enrichment of TERT promoter mutations in the HGG_F group, while HGG_E samples are enriched for tumors with POLE mutations and elevated tumor mutation burden. HGG_E tumors show epigenetic similarity to pediatric high-grade diffuse gliomas (pedHGG_RTK1A); we therefore propose that HGG_E be considered a pediatric-type HGG. HGG_B tumors show demographic and genomic similarities to high-grade astrocytomas with piloid features, including a subset of cases with CDKN2A/2B loss and ATRX mutation. HGG_F separate into two groups dictated by brain location, and despite the presence of TERT promoter mutations, appear distinct from GBMs. Overall, our work represents an initial effort to understand the morphologic, genomic and clinical characteristics of these previously uncharacterized subtypes of gliomas.

A Drosophila glial cell atlas reveals a mismatch between transcriptional and morphological diversity.

Morphology is a defining feature of neuronal identity. Like neurons, glia display diverse morphologies, both across and within glial classes, but are also known to be morphologically plastic. Here, we explored the relationship between glial morphology and transcriptional signature using the Drosophila central nervous system (CNS), where glia are categorised into 5 main classes (outer and inner surface glia, cortex glia, ensheathing glia, and astrocytes), which show within-class morphological diversity. We analysed and validated single-cell RNA sequencing data of Drosophila glia in 2 well-characterised tissues from distinct developmental stages, containing distinct circuit types: the embryonic ventral nerve cord (VNC) (motor) and the adult optic lobes (sensory). Our analysis identified a new morphologically and transcriptionally distinct surface glial population in the VNC. However, many glial morphological categories could not be distinguished transcriptionally, and indeed, embryonic and adult astrocytes were transcriptionally analogous despite differences in developmental stage and circuit type. While we did detect extensive within-class transcriptomic diversity for optic lobe glia, this could be explained entirely by glial residence in the most superficial neuropil (lamina) and an associated enrichment for immune-related gene expression. In summary, we generated a single-cell transcriptomic atlas of glia in Drosophila, and our extensive in vivo validation revealed that glia exhibit more diversity at the morphological level than was detectable at the transcriptional level. This atlas will serve as a resource for the community to probe glial diversity and function.

Exposure to Δ9-tetrahydrocannabinol leads to a rise in caspase-3, morphological changes in microglial, and astrocyte reactivity in the cerebellum of rats.

The present study aimed to elucidate the effect of 10mg/kg Δ9-tetrahydrocannabinol (THC) on cerebellar neuronal and glial morphology, apoptosis and inflammatory gene expression using a series of histological assays including stereology, Sholl analysis, immunofluorescence and real-time qPCR in male Wistar rats. A decrease in the number of Purkinje neurons and the thickness of the granular layer in the cerebellum was reported in THC-treated rats. Increased expression of Iba-1 and arborization of microglial processes were evidence of microgliosis and morphological changes in microglia. In addition, astrogliosis and changes in astrocyte morphology were other findings associated with THC administration. THC also led to an increase in caspase-3 positive cells and a decrease in autophagy and inflammatory gene expression such as mTOR, BECN1 and LAMP2. However, there were no significant changes in the volume of molecular layers and white matter, the spatial arrangement of granular layers and white matter, or the spatial arrangement of granular layers and white matter in the cerebellum. Taken together, our data showed both neuroprotective and neurodegenerative properties of THC in the cerebellum, which require further study in the future.

The effects of a highly bioavailable curcumin PhytosomeTM preparation on the retinal architecture and glial reactivity in the GFAP-IL6 mice.

Uncontrolled, chronic inflammation in the retina can disturb retinal structure and function leading to impaired visual function. For the first time, in a mouse model of chronic neuroinflammation (GFAP-IL6), we investigated the impact of chronic glial activation on the retinal microglia population and structure. In addition, we tested a curcumin PhytosomeTM preparation with enhanced bioavailability to investigate the effects of a cytokine-suppressing anti-inflammatory drug on retinal architecture. Curcumin PhytosomeTM was fed to 3-month old GFAP-IL6 mice for 4 weeks and compared to their untreated GFAP-IL6 counterparts as well as wild type mice on control diet. Microglial numbers and morphology together with neuronal numbers were characterized using immunohistochemistry and cell reconstruction in the retina, using retinal wholemount and slices. GFAP-IL6 mice showed a significant increase in Iba1-labelled mononuclear phagocytes, including microglia, and displayed altered glial morphology. This resulted in a reduction in cone density and a thinning of the retinal layers compared to wild type mice. Curcumin PhytosomeTM treatment contributed to decreased microglial density, significantly decreasing both soma and cell size compared to control diet, as well as preventing the thinning of the retinal layers. This study is the first to characterize the impact of chronic retinal inflammation in the GFAP-IL6 mouse and the therapeutic benefit of enhanced bioavailable curcumin PhytosomeTM to significantly reduce microglia density and prevent neuronal loss. These data suggest that curcumin could be used as a complementary therapy alongside traditional treatments to reduce associated retinal inflammation in a variety of retinal diseases.

Alterations of Purinergic Receptors Levels and Their Involvement in the Glial Cell Morphology in a Pre-Clinical Model of Autism Spectrum Disorders.

Recent data suggest that defects in purinergic signalling are a common denominator of autism spectrum disorders (ASDs), though nothing is known about whether the disorder-related imbalance occurs at the receptor level. In this study, we investigated whether prenatal exposure to valproic acid (VPA) induces changes in purinergic receptor expression in adolescence and whether it corresponds to glial cell activation. Pregnant dams were subjected to an intraperitoneal injection of VPA at embryonic day 12.5. In the hippocampi of adolescent male VPA offspring, we observed an increase in the level of P2X1, with concomitant decreases in P2X7 and P2Y1 receptors. In contrast, in the cortex, the level of P2X1 was significantly reduced. Also, significant increases in cortical P2Y1 and P2Y12 receptors were detected. Additionally, we observed profound alterations in microglial cell numbers and morphology in the cortex of VPA animals, leading to the elevation of pro-inflammatory cytokine expression. The changes in glial cells were partially reduced via a single administration of a non-selective P2 receptor antagonist. These studies show the involvement of purinergic signalling imbalance in the modulation of brain inflammatory response induced via prenatal VPA exposure and may indicate that purinergic receptors are a novel target for pharmacological intervention in ASDs.

CHANGES IN THE NEUROGENIC NICHE OF THE RAT HIPPOCAMPUS UNDER HYPOXIC EXPOSURE

One of the most vulnerable brain structures to hypoxia is the hippocampus. Maintenance of the neurogenic niche cell pool in the subgranular zone of the hippocampus (SGZ) is provided by adaptive mechanisms. Among them are changes in the functional activity of mitochondrial respiratory chain complexes and the reaction of astroglia, which provides metabolic support for neurons. In order to study the dynamics of adaptive changes in neurons and glia in the dentate gyrus of the hippocampus under hypoxic conditions on a model of intermitten hypobaric hypoxia (5000 m, equivalent to 10.5% O2), with a single (60 min) and multiple (8 and 20 episodes) exposure in low-resistant rats, immunomorphological methods revealed the features of localization and content in the neurons of complex IV mitochondrial respiratory chain (MTCO1), astrocyte marker proteins glutamine synthetase (GS) and GFAP, and doublecortin (DCX) in immature neurons. With a single hypoxia, the content of MTCO1 in neurons significantly increased, and after eight exposures, the amount of glutamine synthetase (GS) in astrocytes of the dentate gyrus of the hippocampus increased. Changes in the content of GS were most pronounced in the processes of astrocytes, which indicates a redistribution of GS. The number of DCX+ neurons in the SGZ significantly decreased after 20 episodes of hypoxia. At the same time, DCX+ cells of glial morphology were found in the polymorphic layer, and staining for GFAP showed an increase in the number of astrocytes. This may be due to a shift in the direction of cell differentiation in the neurogenic niche. Thus, in hypoxia, at the initial stage, a functional restructuring of the respiratory chain of neurons of the granular layer occurs. Subsequently, it is noted by the activation of astrocytes that modulate glutamate metabolism. The presence of a relationship between the dynamics of adaptive reactions of energy exchange in neurons and glia and changes in neurogenesis during 20 episodes of hypoxia suggests that during long-term hypoxia, the differentiation of neural precursors of SGZ in the direction of astroglia occurs, however, this issue requires further study in order to more accurately determine the nature of DCX+ cells.

Modulation of Glial Cell Functions by the Gut–Brain Axis: A Role in Neurodegenerative Disorders and Pain Transmission

Studies on host microbiota and their interactions with the central nervous system (CNS) have grown considerably in the last decade. Indeed, it has been widely demonstrated that dysregulations of the bidirectional gut–brain crosstalk are involved in the development of several pathological conditions, including chronic pain. In addition, the activation of central and peripheral glial cells is also implicated in the pathogenesis and progression of pain and other neurodegenerative disorders. Recent preclinical findings suggest that the gut microbiota plays a pivotal role in regulating glial maturation, morphology and function, possibly through the action of different microbial metabolites, including the most studied short-chain fatty acids (SCFAs). Moreover, altered microbiota composition has been reported in CNS disorders characterized by glial cell activation. In this review, we discuss recent studies showing the role of the gut microbiota and the effects of its depletion in modulating the morphology and function of glial cells (microglia and astrocytes), and we hypothesize a possible role for glia–microbiota interactions in the development and maintenance of chronic pain.

Glial cell reactivity drives cognitive decline in LDLr‐/‐ mice: experimental evidences

AbstractBackgroundFamilial hypercholesterolemia (FH) is a clinical condition characterized by increased plasma cholesterol levels, due to a mutation in the gene encoding the low‐density lipoprotein receptor (LDLr) in cells [Brown and Goldstein, 1984]. Importantly, clinical evidence indicates that FH negatively affects cognition and favors the development of neuropathologies [Ariza et al., 2016; Zambón et al, 2010], however, the mechanisms behind this relationship are still not fully understood. In this study we aimed to investigate the role of glial cell reactivity on the cognitive deficits related to the FH, as well as the possible neuroprotective effect of a pharmacological inhibitor of microglial cell activation.MethodFirstly, male 3‐months‐old and 14‐months‐old C57BL/6 wild‐type and LDLr knockout mice (LDLr‐/‐) mice brains were prepared for the evaluation of glial cell morphology by immunofluorescence. In addition, the mice’s hippocampus was removed to measure the immune content of synaptic and tight‐junctions proteins. Moreover, male and female 6‐months‐old C57BL/6 wild‐type and LDLr‐/‐ mice were treated with minocycline (50 mg/kg) or vehicle (saline) for four weeks. Afterwards, mice were submitted to behavioral tests.ResultMiddle‐aged LDLr‐/‐ mice (14‐months‐old) exhibited increased microgliosis in hippocampus, which was correlated with morphological change of microglial cells and astrocytes in this same brain region. Also, middle‐aged LDLr‐/‐ mice presented a reduction in the immune content of synaptophysin‐1 in the hippocampus. Moreover, young adult (3‐months‐old) LDLr‐/‐ mice exhibited reduced immune content of PSD95 in the hippocampus. Finally, the administration of minocycline to 6‐months‐old LDLr‐/‐ mice ameliorated the performance of hypercholesterolemic mice in hippocampal‐dependent memory tasks, without modifying their locomotor activity and metabolic parameters.ConclusionTaken together, our results demonstrated that FH alters the glial cell reactivity, which was associated with changes in synaptic proteins in the hippocampus. In addition, the pharmacological modulation of microglial cell reactivity ameliorated the hippocampal‐dependent memory in LDLr‐/‐ mice, suggesting that increased glial cell reactivity is a key event for HF‐related cognitive deficits.

Early evolution of glial morphology and inflammatory cytokines following hypoxic-ischemic injury in the newborn piglet brain

Neuroinflammation is a hallmark of hypoxic-ischemic injury and can be characterized by the activation of glial cells and the expression of inflammatory cytokines and chemokines. Interleukin (IL)-1β and tumor necrosis factor (TNF)α are among the best-characterized early response cytokines and are often expressed concurrently. Several types of central nervous system cells secrete IL-1β and TNFα, including microglia, astrocytes, and neurons, and these cytokines convey potent pro-inflammatory actions. Chemokines also play a central role in neuroinflammation by controlling inflammatory cell trafficking. Our aim was to characterise the evolution of early neuroinflammation in the neonatal piglet model of hypoxic-ischemic encephalopathy (HIE). Piglets (< 24 h old) were exposed to HI insult, and recovered to 2, 4, 8, 12 or 24H post-insult. Brain tissue from the frontal cortex and basal ganglia was harvested for assessment of glial cell activation profiles and transcription levels of inflammatory markers in HI piglets with comparison to a control group of newborn piglets. Fluorescence microscopy was used to observe microglia, astrocytes, neurons, degenerating neurons and possibly apoptotic cells, and quantitative polymerase chain reaction was used to measure gene expression of several cytokines and chemokines. HI injury was associated with microglial activation and morphological changes to astrocytes at all time points examined. Gene expression analyses of inflammation-related markers revealed significantly higher expression of pro-inflammatory cytokines tumor necrosis factor-α (TNFα) and interleukin 1 beta (IL-1β), chemokines cxc-chemokine motif ligand (CXCL)8 and CXCL10, and anti-inflammatory cytokine transforming growth factor (TGF)β in every HI group, with some region-specific differences noted. No significant difference was observed in the level of C-X-C chemokine receptor (CCR)5 over time. This high degree of neuroinflammation was associated with a reduction in the number of neurons in piglets at 12H and 24H in the frontal cortex, and the putamen at 12H. This reduction of neurons was not associated with increased numbers of degenerating neurons or potentially apoptotic cells. HI injury triggered a robust early neuroinflammatory response associated with a reduction in neurons in cortical and subcortical regions in our piglet model of HIE. This neuroinflammatory response may be targeted using novel therapeutics to reduce neuropathology in our piglet model of neonatal HIE.

Glia Cell Morphology Analysis Using the Fiji GliaMorph Toolkit.

Glial cells are the support cells of the nervous system. Glial cells typically have elaborate morphologies that facilitate close contacts with neighboring neurons, synapses, and the vasculature. In the retina, Müller glia (MG) are the principal glial cell type that supports neuronal function by providing a myriad of supportive functions via intricate cell morphologies and precise contacts. Thus, complex glial morphology is critical for glial function, but remains challenging to resolve at a sub-cellular level or reproducibly quantify in complex tissues. To address this issue, we developed GliaMorph as a Fiji-based macro toolkit that allows 3D glial cell morphology analysis in the developing and mature retina. As GliaMorph is implemented in a modular fashion, here we present guides to (a) setup of GliaMorph, (b) data understanding in 3D, including z-axis intensity decay and signal-to-noise ratio, (c) pre-processing data to enhance image quality, (d) performing and examining image segmentation, and (e) 3D quantification of MG features, including apicobasal texture analysis. To allow easier application, GliaMorph tools are supported with graphical user interfaces where appropriate, and example data are publicly available to facilitate adoption. Further, GliaMorph can be modified to meet users' morphological analysis needs for other glial or neuronal shapes. Finally, this article provides users with an in-depth understanding of data requirements and the workflow of GliaMorph. © 2023 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Download and installation of GliaMorph components including example data Basic Protocol 2: Understanding data properties and quality 3D-essential for subsequent analysis and capturing data property issues early Basic Protocol 3: Pre-processing AiryScan microscopy data for analysis Alternate Protocol: Pre-processing confocal microscopy data for analysis Basic Protocol 4: Segmentation of glial cells Basic Protocol 5: 3D quantification of glial cell morphology.