Glial Fibrillary Acidic Protein-positive Cells Research Articles

Vagus nerve stimulation rescues persistent pain following orthopedic surgery in adult mice.

Postoperative pain is a major clinical problem imposing a significant burden on patients and society. In a survey 2 years after orthopedic surgery, 57% of patients reported persisting postoperative pain. However, only limited progress has been made in the development of safe and effective therapies to prevent the onset and chronification of pain after orthopedic surgery. We established a tibial fracture mouse model that recapitulates clinically relevant orthopedic trauma surgery, which causes changes in neuropeptide levels in dorsal root ganglia and sustained neuroinflammation in the spinal cord. Here, we monitored extended pain behavior in this model, observing chronic bilateral hindpaw mechanical allodynia in both male and female C57BL/6J mice that persisted for >3 months after surgery. We also tested the analgesic effects of a novel, minimally invasive, bioelectronic approach to percutaneously stimulate the vagus nerve (termed percutaneous vagus nerve stimulation [pVNS]). Weekly pVNS treatment for 30 minutes at 10 Hz for 3 weeks after the surgery strongly reduced pain behaviors compared with untreated controls. Percutaneous vagus nerve stimulation also improved locomotor coordination and accelerated bone healing. In the dorsal root ganglia, vagal stimulation inhibited the activation of glial fibrillary acidic protein-positive satellite cells but without affecting microglial activation. Overall, these data provide novel evidence supportive of the use of pVNS to prevent postoperative pain and inform translational studies to test antinociceptive effects of bioelectronic medicine in the clinic.

Investigation of Naoluoxintong on the neural stem cells by facilitating proliferation and differentiation in vitro and on protecting neurons by up-regulating the expression of nestin in MCAO rats

Ethnopharmacological relevanceThe classic traditional Chinese compound Naoluoxintong (NLXT) has been proven an effective remedy for ischemic stroke (IS). The protective effect of NLXT on neural stem cells (NSCs), however, remains unclear. Aim of the studyTo investigate the protective effect of NLXT on NSCs in rats with middle cerebral artery occlusion (MCAO) and the effect of Nestin expression in vivo. Materials and methodsSprague-Dawley (SD) rats were randomly divided into three groups: the sham-operated group, the MCAO model group and the NLXT group. The MCAO model in rats was established by modified Longa wire embolization method. The sham-operated group, the model group and the NLXT groups were divided into three subgroups according to the sampling time points of 1 d, 3 d and 7 d after successful model-making. Immunofluorescence staining, including bromodeoxyuridine (BrdU)/glial fibrillary acidic protein (GFAP), β-tubulinIII/GFAP, BrdU/doublecortin (DCX) and BrdU/neuronal nuclei (NeuN), was used to detect the proliferation and survival of NSCs in the hippocampal after drug administration. Protein expression of Nestin, DCX, GFAP and NeuN in the hippocampal was detected by Western blot (WB). ResultsImmunofluorescence experiment of Nestin labeled: on the first day, a few Nestin-positive cells were found in the hippocampal DG area. Afterwards, the number of Nestin-labeled positive cells in the model group increased, while the number of cells in the sham group did not fluctuate significantly. The number of positive cells in each administration group increased more than that in the model and normal group. β-tubulin III/GFAP double-labeled: a small amount of double labeled cells was expressed in the normal group, and the number subsequently fluctuated little. In the model group, β-tubulin III/GFAP positive cells increased initially after acute ischemia, and gradually decreased afterwards. In the NLXT-treated group, β-Tubulin III positive cells were significantly increased on day 1, 3 and 7, while GFAP positive cells had little change. BrdU/DCX double-labeled: initially, a small number of BrdU/DCX-labeled positive cells were observed in the normal group and the model group, but there was no increasing trend over time. The positive cells in the NLXT group increased over time, and those in the seven-day group were significantly higher than those in the one-day and three-day groups. BrdU/NEUN double-labeled: in the normal group, BrdU/NEUN positive cells were enriched and distributed regularly. The number of positive cells in the model group was small and decreased gradually with time, and the decrease was most obvious on the third day. The number of positive cells in the NLXT group was significantly higher than that in the model group, and the number of positive cells in the seven-day group was significantly higher than that in the one-day and three-day groups. WB results reflected those three proteins, Nestin, NeuN and DCX, showed an increase in expression, except GFAP, which showed a decreasing trend. ConclusionsPreliminarily, NLXT can promote the migration and differentiation of NSCs. It may have a protective effect on the brain by promoting repair of brain tissue damage through upregulation of Nestin after IS.

Positive effects of running exercise on astrocytes in the medial prefrontal cortex in an animal model of depression.

Depression is one of the most common mental illnesses and seriously affects all aspects of life. Running exercise has been suggested to prevent or alleviate the occurrence and development of depression; however, the underlying mechanisms of these effects remain unclear. Independent studies have indicated that astrocytes play essential roles and that the medial prefrontal cortex (mPFC) is an important brain region involved in the pathology underlying depression. However, it is unknown whether running exercise achieves antidepressant effects by affecting the number of astrocytes and glutamate transport function in the mPFC. Here, animal models of depression were established using chronic unpredictable stress (CUS), and depression-like behavior was assessed by the sucrose preference test. After successfully establishing the depression model, experimental animals performed running exercise. Glial fibrillary acidic protein-positive (GFAP+ ) cell number in the mPFC was precisely quantified using immunohistochemical and stereological methods, and the densities of bromodeoxyuridine-positive (BrdU+ ) and BrdU+ /GFAP+ cells in the mPFC were measured using a semiquantitative immunofluorescence assay. Changes in glutamate transporter gene expression in mPFC astrocytes were detected by mRNA sequencing and qRT-PCR. We found that running exercise reversed CUS-induced decreases in sucrose preference, increased astrocyte number and the density of newborn astrocytes, and reversed decreases in gene expression levels of GFAP, S100b, and the glutamate transporters GLT-1 and GLAST in the mPFC of CUS animals. These results suggested that changes in astrocyte number and glutamate transporter function may be potential meditators of the effects of running exercise in the treatment of depression.

Astrocytic d-amino acid oxidase degrades d-serine in the hindbrain.

d-Serine modulates excitatory neurotransmission by binding to N-methyl-d-aspartate glutamate receptors. d-Amino acid oxidase (DAO) degrades d-amino acids, such as d-serine, in the central nervous system, and is associated with neurological and psychiatric disorders. However, cell types that express brain DAO remain controversial, and whether brain DAO influences systemic d-amino acids in addition to brain d-serine remains unclear. Here, we created astrocyte-specific DAO-conditional knockout mice. Knockout in glial fibrillary acidic protein-positive cells eliminated DAO expression in the hindbrain and increased d-serine levels significantly in the cerebellum. Brain DAO did not influence levels of d-amino acids in the forebrain or periphery. These results show that astrocytic DAO regulates d-serine specifically in the hindbrain.

Comparative Histological Study of Therapeutic Effect of Mesenchymal Stem Cells versus Mesenchymal Stem Cells Co-Cultured with Liver Tissue on Carbon Tetrachloride-Induced Hepatotoxicity in Adult Male Albino Rats.

Liver diseases are major causes of morbidity and mortality. Mesenchymal stem cells (MSCs) have immunomodulatory, anti-inflammatory, and antifibrotic effects, so they can be used in the treatment of liver diseases. MSCs co-cultured with diseased liver tissue improve the homing capacity, survival rate, and paracrine effects of the MSCs, as well as the ability to enhance liver function. This work aimed to study the therapeutic effect of MSCs versus MSCs co-cultured with liver tissue on carbon tetrachloride (CCl4)-induced hepatotoxicity in adult male albino rats. Twenty adult male albino rats were divided into four equal groups; Group I (control group), Group II received CCl4 intraperitoneally (i.p.), Group III received CCl4 i.p. and then injected with MSCs intravenously (i.v.), and Group IV received CCl4 i.p. and then injected with co-cultured MSCs i.v. Finally, liver specimens were processed for light microscopy (LM) and electron microscopy (EM). Statistical analysis was carried out to assess histological scoring, area percentage of collagen fibers, number of glial fibrillary acidic protein-positive cells, and biochemical analysis of alanine aminotransferase and aspartate aminotransferase. Statistical analysis of (histological scoring, area % of collagen fibers, and biochemical analysis) was done by using one-way analysis of variance (ANOVA) test using graphpad software (SanDiego, CA, USA). The means ± standard deviations were used for statistical analysis. LM of Group II revealed loss of hepatic architecture and diffuse fibrosis with dilated congested blood vessels, bile ductular proliferation, and cellular infiltrations. Vacuolated cytoplasm with or without pyknotic nuclei was observed in addition to micro- and macro-steatosis. EM demonstrated disfigured hepatocytes with abnormal organelles surrounding atypical nucleus. Group III showed restoration of the normal liver architecture with greater extent in Group IV. Statistical analysis confirmed the microscopic findings. Co-cultured MSCs with diseased liver tissue augmented the therapeutic effects of MSCs in treating hepatotoxicity induced by CCl4 in adult male albino rats.

Candesartan protects against d-galactose induced – Neurotoxicity and memory deficit via modulation of autophagy and oxidative stress

Purposed-galactose induces neuroinflammation and memory deficit via oxidative stress. Candesartan is an angiotensin II-receptor blocker and has proved neuroprotective properties. This study aimed to investigate the neuroprotective effect of candesartan against d-galactose induced neuroinflammation and memory deficit via autophagy. MethodsTwenty-eight male Wistar rats aged 3 months were divided into four equal groups: control (vehicle), d-gal (100 mg/kg d-galactose), cand (1 mg/kg candesartan), and cand+d-gal (100 mg/kg d-galactose & 1 mg/kg candesartan). All treatments were given orally and daily for 4 weeks. Assessment of memory was done using Morris water maze (MWM) test. Brain tissue was assessed for malondialdehyde (MDA), total thiol, catalase activity, glial fibrillary acidic protein (GFAP) and gene expression of TNF-α, GDNF-1 as well as autophagy genes (Beclin 1 and ATG 5). ResultsProphylactic treatment of candesartan in d-galactose-treated rats significantly (p < 0.001) reduced oxidative stress via reduction of MDA as well as elevation of catalase activity and total thiol levels. Additionally, candesartan prophylactic treatment significantly increased gene expression of GDNF-1 and decreased gene expression of TNF-α. Furthermore, candesartan significantly increased the expression of autophagy related gene (Beclin 1 and ATG 5) in cand+d-gal treated rats. These results were supported by the histopathological findings which showed that candesartan prevented the neuronal injury in the cerebral cortex and hippocampus and decreased GFAP positive cells of the d-galactose-treated rats. Moreover, MWM test showed that candesartan significantly improved memory deficit in cand+d-gal treated rats. ConclusionCandesartan prevents d-galactose-induced neurotoxicity and memory deficit via activating autophagy and decreasing oxidative stress. Therefore, candesartan was a good candidate for age-related neurodegenerative disorders and memory deficit.

A stereological study of glial activation in GFAP-IL6 mice: A model of chronic neuroimmune conditions.

Understanding the role of chronic neuroinflammation, described by increased astro- and microglia activation, in the pathogenesis of Alzheimer's disease (AD) is becoming crucial due to its role throughout the disease progression (Deczkowska et al., 2018). In search of a suitable mouse model of chronic glial activation, we characterized the GFAP-IL6 transgenic mouse model in which the pro-inflammatory cytokine interleukine-6 is overexpressed under the control of the glial fibrillary acidic protein (GFAP) promoter (Campbell et al., 1993). The GFAP-IL6 mice display chronic inflammatory conditions in the brain, with the homozygous mice developing an aggressive pathology characterized by tremor, ataxia, and seizures by 6 months of age (Campbell et al., 1993). While the heterozygous phenotype is milder and characterized by astrocytosis, microgliosis, angiogenesis, and neurodegeneration. Moreover, evidences showed that the progressive motor and cognitive impairment is correlated with the level of microgliosis (Heyser et al., 1997). Using immunohistochemically stained brain sections of 3 months old wild-type (WT) (n=4), GFAP-IL6 heterozygous (n=5), and homozygous (n=5) mice, we estimated the absolute number and morphology of Iba1 and GFAP positive cells, microglia and astrocytes respectively. We focused the analysis on cortex, hippocampus, cerebellum, and medial septum (MS) to find a possible connection with the loss of cholinergic neurons during AD. Homozygous GFAP-IL6 mice showed an increase in the number of microglia by 50-80% in all the brain areas compared to the WT, while the increase in the heterozygous was 35-60%. Moreover, homozygous mice increased the number of microglia by 40% in the MS and nearly 50% in the cerebellum compared to the heterozygous. In the MS astrocytes of the homozygous mice are 70% more than in the heterozygous, which are similar to the WT. The glial morphological analysis showed a different ramification and microglial cell body size in the homozygous compared to the heterozygous and WT. The stronger impact of neuroinflammation in the homozygous GFAP-IL6 mice at 3 months old suggests a possible application in evaluating chronic conditions at early stages. Moreover, the results highlight the high vulnerability to inflammation of the MS. Increased cerebellar glial mediated inflammation emphasized the chance to develop motor deficits.

Effects of cannabinoid (CBD) on blood brain barrier permeability after brain injury in rats

Cannabidiol is a natural herbal medicine known to protect the brain from traumatic brain injury (TBI). Here, a TBI rat model was established, with cannabidiol administered intraperitoneally at doses of 5, 10, or 20 mg/kg, 30 min before surgery and 6 h after surgery until sacrifice. Brain water content, body weight, and modified neurological severity scores were determined, and enzyme-linked immunosorbent assay, immunofluorescence staining, hematoxylin and eosin staining, Nissl staining, Evans-blue dye extravasation, and western blotting were performed. Results showed that cannabidiol decreased the number of aquaporin-4-positive and glial fibrillary acidic protein-positive cells. Cannabidiol also significantly reduced the protein levels of proinflammatory cytokines (TNF-α and IL-1β) and significantly increased the expression of tight junction proteins (claudin-5 and occludin). Moreover, cannabidiol administration significantly mitigated water content in the brain after TBI and blood–brain barrier disruption and ameliorated the neurological deficit score after TBI. Cannabidiol administration improved the integrity and permeability of the blood–brain barrier and reduced edema in the brain after TBI.

Human umbilical cord mesenchymal stem cell-derived extracellular vesicles attenuate experimental autoimmune encephalomyelitis via regulating pro and anti-inflammatory cytokines

The therapeutic effects of mesenchymal stem cells-extracellular vesicles have been proved in many inflammatory animal models. In the current study, we aimed to investigate the effect of extracellular vesicles (EVs) derived from human umbilical cord-MSC (hUCSC-EV) on the clinical score and inflammatory/anti-inflammatory cytokines on the EAE mouse model. After induction of EAE in C57Bl/6 mice, they were treated intravenously with hUCSC-EV or vehicle. The clinical score and body weight of all mice was registered every day. On day 30, mice were sacrificed and splenocytes were isolated for cytokine assay by ELISA. Cytokine expression of pro-/anti-inflammatory cytokine by real-time PCR, leukocyte infiltration by hematoxylin and eosin (H&E) staining, and the percent of glial fibrillary acidic protein (GFAP) and myelin basic protein (MBP) positive cells by immunohistochemistry were assessed in the spinal cord. Our results showed that hUCSC-EV-treated mice have lower maximum mean clinical score (MMCS), pro-inflammatory cytokines, and inflammatory score in comparison to the control mice. We also showed that hUCSC-EV administration significantly improved body weight and increased the anti-inflammatory cytokines and the frequency of Treg cells in the spleen. There was no significant difference in the percent of GFAP and MBP positive cells in the spinal cord of experimental groups. Finally, we suggest that intravenous administration of hUCSC-EV alleviate induce-EAE by reducing the pro-inflammatory cytokines, such as IL-17a, TNF-α, and IFN-γ, and increasing the anti-inflammatory cytokines, IL-4 and IL-10, and also decrease the leukocyte infiltration in a model of MS. It seems that EVs from hUC-MSCs have the same therapeutic effects similar to EVs from other sources of MSCs, such as adipose or bone marrow MSCs.

Shock wave and mesenchymal stem cells as treatment in the acute phase of spinal cord injury: A pilot study

IntroductionSpinal cord injury (SCI) is a complex pathology with thousands of patients worldwide. During the acute early phase, neural tissue shows some regenerative properties that disappear at the chronic phase. Shock Waves and Stem Cells have been proposed as a possible therapy. MethodsHere, we analyse Shock Waves’ immediate effect over spinal cord genetic response in the injured and healthy spinal cord and the effect of Shock Waves and combined Shock Waves plus Stem Cells distally grafted to treat the first month after spinal cord injury. ResultsThe immediate application of shock waves increases VEGF (Vascular Endothelial Growth Factor) but reduces the BDNF (Brain-Derived Growth Factor) RNA (Ribonucleic acid) response. Shock wave therapy increases GFAP (Glial fibrillary acidic protein) positive cells and vascularity during the treatment's acute phase. ConclusionShock wave treatment seems to be enough to produce benefits in the acute phase of spinal cord injury, with no accumulative positive effects when mesenchymal stem cell graft is applied together.

Histological study of the possible protective action of platelet rich plasma on the injurious effect induced by aluminum chloride on albino male rat cerebellar cortex

Background: Aluminum compounds are commonly used in many human activities. They are used in pesticides, detergents, cosmetics, pharmaceuticals, and food additives. Many studies revealed a link between human exposure to aluminum and many neurodegenerative conditions in which there is platelet dysfunction.Aim of the study: The aim of this research was to study the potential protective effect of platelet rich plasma on the deleterious histological effect of aluminum chloride on male rat cerebellar cortex.Materials and methods: In this study, three equal groups of adult male albino rats were used; each consisted of 10 rats: group I a and I b (control group), group II (daily intraperitoneal injection of aluminum chloride dissolved in saline for 60 consecutive days in a dose of 10mg/kg) and group III were given PRP (subcutaneous injection in a dose of 0.5 ml/kg twice weekly) in addition to aluminum chloride in the same previous dose and duration. Cerebellum sections were processed for hematoxylin and eosin staining, GFAB staining and electron microscopic examination.Results: Aluminum chloride administration resulted in many histological alterations. Glial fibrillary acidic protein-positive cells were significantly present in the cerebellum of aluminum chloride-treated animals relative to control animals. Purkinje cells with darkly stained ill-defined nuclei with dark vacuolated cytoplasm with dilated RER cisterns, swollen mitochondria with destroyed crista were observed in an ultrastructural study for the cerebellar cortex of the aluminum chloride-treated group. It also showed degenerated granule cells with the disappearance of the nuclear membrane and vacuolation of the cytoplasm. Some of the myelinated nerve fibers showed degenerative changes. Concomitant administration of PRP decreased these effects.Conclusion: PRP partially minimized the severity of aluminum chloride-induced cerebellar cortex injurious histological effects in male albino rats. Further research is needed to extend these findings to clinical practice.

Dietary fat exacerbates postprandial hypothalamic inflammation involving glial fibrillary acidic protein-positive cells and microglia in male mice.

In humans, obesity is associated with brain inflammation, glial reactivity, and immune cells infiltration. Studies in rodents have shown that glial reactivity occurs within 24 hr of high-fat diet (HFD) consumption, long before obesity development, and takes place mainly in the hypothalamus (HT), a crucial brain structure for controlling body weight. Here, we sought to characterize the postprandial HT inflammatory response to 1, 3, and 6 hr of exposure to either a standard diet (SD) or HFD. HFD exposure increased gene expression of astrocyte and microglial markers (glial fibrillary acidic protein [GFAP] and Iba1, respectively) compared to SD-treated mice and induced morphological modifications of microglial cells in HT. This remodeling was associated with higher expression of inflammatory genes and differential regulation of hypothalamic neuropeptides involved in energy balance regulation. DREADD and PLX5622 technologies, used to modulate GFAP-positive or microglial cells activity, respectively, showed that both glial cell types are involved in hypothalamic postprandial inflammation, with their own specific kinetics and reactiveness to ingested foods. Thus, recurrent exacerbated postprandial inflammation in the brain might promote obesity and needs to be characterized to address this worldwide crisis.

Paraoxon-induced damage in rat hippocampus is associated with alterations in the expression of apoptosis-related proteins

To determine the possible role of apoptosis in the development of paraoxon-induced brain damage, we evaluated expression of apoptosis-related proteins, the extent of neuronal damage, and activation of astrocytes in rat hippocampus. Adult male Wistar rats were intraperitoneally injected with one of three doses of paraoxon (0.3, 0.7, or 1 mg/kg) or corn oil (vehicle). After 14 or 28 days, expression of apoptosis-related proteins, including B-cell leukemia/lymphoma-2 (Bcl-2), Bcl-2 associated X protein (Bax), and caspase-3, as well as the number of neurons and glial fibrillary acidic protein (GFAP) positive cells in hippocampus were examined by western blot, cresyl blue staining, and immunohistochemistry, respectively. After 14 and 28 days, Bax and caspase-3 proteins were significantly increased in rats receiving 0.7 and 1 mg/kg of paraoxon. A significant decrease in Bcl-2 protein levels was also observed in 0.7 and 1 mg/kg groups after 14 days and in 1 mg/kg group after 28 days. Animals treated with 1 mg/kg of paraoxon showed a significant decrease in the number of neurons in the CA1 area. Also, those treated with 0.7 and 1 mg/kg of paraoxon showed an increase in the number of GFAP positive cells in both CA1 and CA3 areas as well as a significant decrease in survived neurons in the CA3 area. Our results indicated that neuronal damage induced by convulsive doses of paraoxon in rat hippocampus is mediated in part through apoptosis mechanism. Activation of astrocytes might lead to reduced extent of damage and damage and consequently increased neuronal survival.

Insulin‐like Growth Factor‐1 Treatment of Experimental Traumatic Brain Injury Increases Newborn Neuron Migration Through the Granular Cell Layer

Traumatic brain injuries (TBI) are a tragic yet surprisingly common problem throughout the world. In the United States alone, 2.8 million people a year are diagnosed with a TBI. TBI causes cellular death and dysfunction within the brain that is connected with cognitive and behavioral dysfunction both clinically and experimentally.The controlled cortical impact (CCI) model is a well characterized contusion model of TBI that emulates both motor and cognitive deficits in addition to cell loss within the dentate gyrus of the hippocampus. Following CCI there is an early loss of the immature neuron population in the subgranular zone of the dentate gyrus followed by a compensatory increase in newborn neurons. Additionally, there is increased glial reactivity in response to injury. Of these newborn neurons, only a small portion survive and incorporate into the granular cell layer (GCL) of the dentate gyrus. However, the surviving trauma‐born neurons display stunted growth and reduced arborization, and not many make it to a fully functional form.Previous research shows that treatment with insulin‐like growth factor‐1 (IGF‐1) conveys behavioral improvements and increases the survival of doublecortin (DCX) positive trauma‐born neurons with improved dendritic arborization. In the current study, C57BL/6J mice underwent a CCI or a sham injury and were administered IGF‐1 (3mg/mg) or vehicle by intracerebroventricular injection (nCCI&amp;IGF‐1 = 7 | nCCI&amp;Veh = 8 | nSham&amp;IGF‐1 = 4 | nSham&amp;Veh = 4) for 7 days following injury. Animals were euthanized 7 days after injury, and the brain was collected, processed, and sectioned at a thickness of 40mm for immunohistochemistry. Tissue was then immuno‐labeled for DCX, a marker of immature neurons, and glial fibrillary acidic protein (GFAP), a marker of astrocytes in the brain, and imaged.We observed that the trauma‐born neurons of CCI injured mice treated with IGF‐1 migrate from the subgranular zone and into the outer 2/3rds of the GCL more frequently than do those observed in animals treated with vehicle. During development, GFAP positive cells send processes out that serve as a scaffolding by which immature neurons can migrate. As such, we hypothesized that after TBI, immature neurons migrate through the GCL by utilizing GFAP positive processes, and that IGF‐1 would increase the number of processes projecting through the GCL compared to vehicle treated animals.We assessed the number of GFAP positive processes projecting into the GCL by counting the number of processes per millimeter in three brain sections collected from the epicenter of injury. Our findings suggest that there is an increase in GFAP processes following CCI, but treatment with IGF‐1 did not increase the number of these processes. Our findings suggest that increased migration seen with IGF‐1 treatment does not occur along glial processes, or that this is not the only means by which DCX positive cells are able to migrate after injury.Support or Funding InformationFunded through American Physiological Society 2019 UGSRF Fellowship

Long Term Cognitive Impairment and Increased Inflammation following Mild Traumatic Brain Injury using the CHIMERA Model

IntroductionTraumatic brain injury (TBI) affects a vast number of people worldwide. Mild TBI (mTBI), including concussions, accounts for most of the TBI cases. mTBI patients experience enduring symptoms, including headaches, mild cognitive dysfunction, emotional instability, psychiatric symptoms, and increased risk of neurodegenerative disease. Repetitive closed head impact model of engineered rotational acceleration (rCHIMERA) is a recently developed nonsurgical animal model of TBI that primarily produces diffuse axonal injury characterized by white matter inflammation and axonal damage, which mimic the mTBI in humans. However, the majority of studies on rCHIMERA were conducted in mice, which are not suitable for assessment of complex behavior.MethodsThis study was designed to validate our rCHIMERA‐induced rat mTBI model using advanced and cutting‐edge behavioral tests and immunofluorescence staining of key biomarkers of TBI. Adult male Sprague‐Dawley rats (~350 g) were divided into sham and injured groups. All rats were anesthetized with dexmedetomidine (IV, 0.2 mg/kg/hr). Following anesthesia, the rats designated for injury received 6 closed‐head impacts of 2 Joules (J) energy over 3 days with 1‐day interval (3 impacts on days 1 &amp; 3, with 30‐seconds between impacts, and rested on day 2). The sham rats were handled the same way as the injured without the impacts. The rats were subsequently subjected to a battery of behavioral tests at specified time points post‐injury: neurological severity score (NSS), automated home‐cage (CleverSys), behavioral spectrometer (Biobserve), rotarod test, and fear conditioning. Histological assessment of the injury was performed in targeted rat brain areas from sham and injured rats. Immunohistochemistry staining for amyloid precursor protein (APP), ionized calcium‐binding adapter molecule 1 (Iba‐1) and glial fibrillary acidic protein (GFAP) were used to determine axonal damage, inflammatory response and astrocytic activation, respectively, in sham and injured rats. The weights of the rats were also monitored for the duration of the studies.Results3 months after mTBI, there was no significant difference in body weight and NSS between sham and injured rats. Injury did not affect motor activity and motor learning with the rotarod test, but did impair associative fear learning and memory in fear conditioning tests 4 weeks and 3 months after mTBI (n=9 shams and 9 mTBI). APP immunohistochemistry in the injured brains revealed induced diffusive axonal injury in the the cortex. The number of GFAP positive cells was increased in the prefrontal cortex and hippocampus after injury and the astrocytes showed distinct hypertrophic morphology. The number of Iba‐1 positive cells was also increased after injury, indicating enhanced inflammation.ConclusionThe results suggest that our mTBI model in rats induces short‐term changes in affect and activity, long‐term cognitive impairments, and histological changes. These observations validate the CHIMERA, using the rat, as a model that replicates clinical mTBI.Support or Funding InformationThis work is supported by the Advancing a Healthier Wisconsin Endowment (#5520444) to AC.

Existence of FGFR1-5-HT1AR heteroreceptor complexes in hippocampal astrocytes. Putative link to 5-HT and FGF2 modulation of hippocampal gamma oscillations

The majority of the fibroblast growth factor receptor 1-serotonin 1 A receptor (FGFR1-5-HT1AR) heterocomplexes in the hippocampus appeared to be located mainly in the neuronal networks and a relevant target for antidepressant drugs. Through a neurochemical and electrophysiological analysis it was therefore tested in the current study if astrocytic FGFR1-5-HT1AR heterocomplexes also exist in hippocampus. They may modulate the structure and function of astroglia in the hippocampus leading to possible changes in the gamma oscillations. Localization of hippocampal FGFR1-5-HT1AR heterocomplexes in astrocytes was found using in situ proximity ligation assay combined with immunohistochemistry using glial fibrillary acidic protein (GFAP) immunoreactivity as a marker for astroglia. Acute i.c.v. treatment with 8-OH-DPAT alone or together with basic fibroblast growth factor (FGF2) significantly increased FGFR1-5-HT1AR heterocomplexes in the GFAP positive cells, especially in the polymorphic layer of the dentate gyrus (PoDG) but also in the CA3 area upon combined treatment. No other hippocampal regions were studied. Also, structural plasticity changes were observed in the astrocytes, especially in the PoDG region, upon these pharmacological treatments. They may also be of relevance for enhancing the astroglial volume transmission with increased modulation of the neuronal networks in the regions studied. The effects of combined FGF2 and 5-HT agonist treatments on gamma oscillations point to a significant antagonistic interaction in astroglial FGFR1-5-HT1AR heterocomplexes that may contribute to counteraction of the 5-HT1AR-mediated decrease of gamma oscillations.This article is part of the special issue entitled ‘Serotonin Research: Crossing Scales and Boundaries’.

Astroglial biotin deprivation under endoplasmic reticulum stress uncouples BCAA-mTORC1 role in lipid synthesis to prolong autophagy inhibition in the aging brain.

Autophagy delays the onset of endoplasmic reticulum (ER) stress by recycling cellular debris. However, the cues that elicit autophagy under the emergence of ER stress and their dysregulation during aging remains obscure. Amino acids, notably branched-chain amino acids (BCAA), get accumulated in the cells once protein synthesis is inhibited by ER stress. The BCAA mimic satiety to inhibit autophagy via mechanistic targets of rapamycin complex 1 (mTORC1) activation and, in contrast, their catabolism supplements de novo lipogenesis for the formation of autophagosome membranes. Thus promoting BCAA utilization is hypothesized to induce autophagy to alleviate ER stress. Nevertheless, except protein synthesis, the rest of BCAA utilization and lipogenesis depends on the co-enzyme biotin. Hence, the levels of biotinylated carboxylases and lipids were assessed in the aging brain of Wistar rats. Despite the increased levels of biotinylated carboxylases and lipids, the aging brain accumulates BCAA. Since astrocytes are the primary site of BCAA and lipid metabolism and the increased expression of glial fibrillary acidic protein (GFAP) denotes astroglial ER stress, co-localization studies were performed to determine the extent of biotinylation in GFAP positive cells. Although total biotin intensity was higher in aged brain slices, the astrocytes specific decrease in biotinylation is attributed to BCAA accumulation, mTORC1 overactivation, autophagy inhibition, and ER stress in the aging brain. The ER stress in primary astrocytes using tunicamycin also mimic the in vivo phenotype. Biotin supplementation ameliorated the changes observed in vitro, corroborating the significance of astrocytes biotin availability to promote autophagy under ER stress in aging.

Selective Regulation of Neurons, Glial Cells, and Neural Stem/Precursor Cells by Poly(allylguanidine)-Coated Surfaces.

Poly(allylguanidine) (PAG) was synthesized and characterized as a polycationic coating material for culturing neurons, glial cells, and neural stem/precursor cells (NSPCs) to apply PAG for neural tissue engineering. For comparison, poly-d-lysine (PDL), the golden benchmark of the neuron cell culture system, was also used in this study. When PAG was subjected to a mixed culture of neurons and glial cells, cell adhesion and neurite extension of neuronal cells were clearly observed but only few glial cells could be found alongside the neurons. Compared to PDL, the significantly lower density of the glial fibrillary acidic protein-positive cells implied that PAG suppressed the glial cell development. Likewise, PAG was demonstrated to dominate the differentiation of NSPCs principally into neurons. To investigate whether the different effects of PAG and PDL on neuron and glial cell behaviors resulted from the difference of guanidinium cations and ammonium cations, poly-l-arginine (PLA) was included and compared in this study. Similar to PDL, PLA supported high neuron and glial cell viability simultaneously. Consequently, glial cell growth and viability restrained on PAG was not only affected by the side-chain guanidino groups but also by the backbone structure property. The absence of the peptide structure in the backbone of PAG and the conformation of coated PAG on tissue culture polystyrene possibly determined the polycationic biomaterial to limit the growth of glial cells.

Effect of Cisplatin on Cerebellar Cortex of Albino Rat and Possible Protective Role of Granulocyte Colony Stimulating Factor versus Citrullus Lanatus Juice: A Histological Study

Background: Cisplatin has a major antitumor effect against a broad range of solid tumors. Yet, its clinical use is limited by its harmful effects especially on the nervous system.Aim of the work: Assessment of the effect of cisplatin on histological structure of cerebellar cortex and the possible protective role of granulocyte colony stimulating factor (G-CSF) versus citrullus lanatus juice. Materials and methods: Forty adult male albino rats were assigned to a group of four: Control group, group I: received cisplatin (10 mg/kg) on the 6th and 13th days of the study via intraperitoneal (IP) injection, group II: received G-CSF (50 µg/kg) for five consecutive days via subcutaneous injection then received as group I, group III: received cisplatin as group I as well as citrullus lanatus juice (4 ml/kg) daily for 16 days via oral gavage. Blood samples were taken to measure plasma malondialdehyde (MDA) and reduced glutathione (GSH) levels. Cerebellar biopsies were processed for light microscopy using H&E and immunohistochemical stains (glial fibrillary acidic protein (GFAP) & caspase-3) as well as for electron microscopy. Morphometric and statistical studies were done for number of Purkinje cells, thickness of granular layer, area percent of GFAP positive cells and optical density of caspase-3 positive cells.Results: Group I showed significantly high MDA and significantly low GSH levels, shrinkage, distortion, deep staining of disarranged Purkinje cells with nuclear pyknosis, swollen mitochondria with destroyed cristae and dilated Golgi apparatus. Separated granule cells were shrunken with dark irregular nuclei. Results were confirmed by morphometric and statistical studies. These changes were ameliorated in both group II and III with great extent in group II than in group III. Conclusion: G-CSF could improve cisplatin induced cerebellar cortical changes better than citrullus lanatus juice.

Juvenile mild traumatic brain injury elicits distinct spatiotemporal astrocyte responses.

Mild-traumatic brain injury (mTBI) represents ~80% of all emergency room visits and increases the probability of developing long-term cognitive disorders in children. To date, molecular and cellular mechanisms underlying post-mTBI cognitive dysfunction are unknown. Astrogliosis has been shown to significantly alter astrocytes' properties following brain injury, potentially leading to significant brain dysfunction. However, such alterations have never been investigated in the context of juvenile mTBI (jmTBI). A closed-head injury model was used to study jmTBI on postnatal-day 17 mice. Astrogliosis was evaluated using glial fibrillary acidic protein (GFAP), vimentin, and nestin immunolabeling in somatosensory cortex (SSC), dentate gyrus (DG), amygdala (AMY), and infralimbic area (ILA) of prefrontal cortex in both hemispheres from 1 to 30 days postinjury (dpi). In vivo T2-weighted-imaging (T2WI) and diffusion tensor imaging (DTI) were performed at 7 and 30 dpi to examine tissue level structural alterations. Increased GFAP-labeling was observed up to 30 dpi in the ipsilateral SSC, the initial site of the impact. However, vimentin and nestin expression was not perturbed by jmTBI. The morphology of GFAP positive cells was significantly altered in the SSC, DG, AMY, and ILA up to 7 dpi that some correlated with magnetic resonance imaging changes. T2WI and DTI values were significantly altered at 30 dpi within these brain regions most prominently in regions distant from the impact site. Our data show that jmTBI triggers changes in astrocytic phenotype with a distinct spatiotemporal pattern. We speculate that the presence and time course of astrogliosis may contribute to pathophysiological processes and long-term structural alterations following jmTBI.