Microglial Activation In Rats Research Articles

The Notch1/Hes1 pathway regulates Neuregulin 1/ErbB4 and participates in microglial activation in rats with VPA-induced autism

The core clinical characteristics of autism, which is a neurodevelopmental disease, involve repetitive behavior and impaired social interactions. Studies have shown that the Notch and Neuregulin1 (NRG1) signaling pathways are abnormally activated in autism, but the mechanism by which these two signaling pathways interact to contribute to the progression of autism has not been determined. Our results suggest that the levels of Notch1, Hes1, NRG1, and phosphorylated ErbB4 in the cerebellum (CB), hippocampus (HC), and prefrontal cortex (PFC) were increased in rats with valproic acid (VPA)-induced autism compared to those in the Con group. However, 3, 5-difluorophenyl-L-alanyl-L-2-phenylglycine tert-butyl (DAPT), which is a Notch pathway inhibitor, ameliorated autism-like behavioral abnormalities and decreased the protein levels of NRG1 and phosphorylated ErbB4 in rats with VPA-induced autism; these results demonstrated that the Notch1/Hes1 pathway could participate in the pathogenesis of autism by regulating the NRG1/ErbB4 signaling pathway. Studies have shown that the Notch pathway regulates microglial differentiation and activation during the onset of neurological disorders and that microglia affect autism-like behavior via synaptic pruning. Therefore, we hypothesized that the Notch1/Hes1 pathway could regulate the NRG1/ErbB4 pathway and thus participate in the development of autism by regulating microglial functions. The present study showed that AG1478, which is an ErbB4 inhibitor, ameliorated the autism-like behaviors in a VPA-induced autism rat model, reduced abnormal microglial activation, and decreased NRG1 and Iba-1 colocalization; however, AG1478 did not alter Notch1/Hes1 activity. These results demonstrated that Notch1/Hes1 may participate in the microglial activation in autism by regulating NRG1/ErbB4, revealing a new mechanism underlying the pathogenesis of autism.

Sciatic nerve block downregulates the BDNF pathway to alleviate the neonatal incision-induced exaggeration of incisional pain via decreasing microglial activation

Sciatic nerve block is under investigation as a possible therapeutic strategy for neonatal injury-induced exaggeration of pain responses to reinjury. Spinal microglial priming, brain-derived neurotrophic factor (BDNF) and Src homology-2 domain-containing protein tyrosine phosphatase-2 (SHP2) participate in exaggerated incisional pain induced by neonatal incision. However, effects of sciatic nerve block on exacerbated incisional pain and underlying mechanisms remain unclear. Here, we demonstrated that sciatic nerve block alleviates pain hypersensitivity and microglial activation in rats subjected to neonatal incision and adult incision (nIN-IN). Chemogenetic activation or inhibition of spinal microglia attenuates or mimics effects of sciatic nerve block on pain hypersensitivity, respectively. Moreover, α-amino-3-hydroxy- 5-methy- 4-isoxazole propionate (AMPA) receptor subunit GluA1 contributes to the exaggeration of incisional pain. The inhibition of BDNF or SHP2 blocks upregulations of downstream molecules in nIN-IN rats. Knockdown of SHP2 attenuates the increase of GluA1 induced by injection of BDNF in adult rats with only neonatal incision. The inhibition of microglia or ablation of microglial BDNF attenuates upregulations of SHP2 and GluA1. Additionally, sciatic nerve block downregulates the expression of these three molecules. Upregulation of BDNF, SHP2 or AMPA receptor attenuates sciatic nerve block-induced reductions of downstream molecules and pain hypersensitivity. Microglial activation abrogates reductions of these three molecules induced by sciatic nerve block. These results suggest that decreased activation of spinal microglia contributes to beneficial effects of sciatic nerve block on the neonatal incision-induced exaggeration of incisional pain via downregulating BDNF/SHP2/GluA1-containing AMPA receptor signaling. Thus, sciatic nerve block may be a promising therapy.

Beneficial effects of running exercise on hippocampal microglia and neuroinflammation in chronic unpredictable stress-induced depression model rats

Running exercise has been shown to relieve symptoms of depression, but the mechanisms underlying the antidepressant effects are unclear. Microglia and concomitant dysregulated neuroinflammation play a pivotal role in the pathogenesis of depression. However, the effects of running exercise on hippocampal neuroinflammation and the number and activation of microglia in depression have not been studied. In this study, rats were subjected to chronic unpredictable stress (CUS) for 5 weeks followed by treadmill running for 6 weeks. The depressive-like symptoms of the rats were assessed with a sucrose preference test (SPT). Immunohistochemistry and stereology were performed to quantify the total number of ionized calcium-binding adapter molecule 1 (Iba1)+ microglia, and immunofluorescence was used to quantify the density of Iba1+/cluster of differentiation 68 (CD68)+ in subregions of the hippocampus. The levels of proinflammatory cytokines in the hippocampus were measured by qRT-PCR and ELISA. The results showed that running exercise reversed the decreased sucrose preference of rats with CUS-induced depression. In addition, CUS increased the number of hippocampal microglia and microglial activation in rats, but running exercise attenuated the CUS-induced increases in the number of microglia in the hippocampus and microglial activation in the dentate gyrus (DG) of the hippocampus. Furthermore, CUS significantly increased the hippocampal levels of inflammatory factors, and the increases in inflammatory factors in the hippocampus were suppressed by running exercise. These results suggest that the antidepressant effects of exercise may be mediated by reducing the number of microglia and inhibiting microglial activation and neuroinflammation in the hippocampus.

Inhibition of microglial activation in rats attenuates paraventricular nucleus inflammation in Gαi2 protein-dependent, salt-sensitive hypertension.

New Findings • What is the central question of this study? We hypothesized that central inflammatory processes that involve activation of microglia and astrocytes contribute to the development of Gαi2 protein‐dependent, salt‐sensitive hypertension.• What is the main finding and its importance? The main finding is that PVN‐specific inflammatory processes, driven by microglial activation, appear to be linked to the development of Gαi2 protein‐dependent, salt‐sensitive hypertension in Sprague–Dawley rats. This finding might reveal new mechanistic targets in the treatment of hypertension. The central mechanisms underlying salt‐sensitive hypertension, a significant public health issue, remain to be established. Researchers in our laboratory have reported that hypothalamic paraventricular nucleus (PVN) Gαi2 proteins mediate the sympathoinhibitory and normotensive responses to high sodium intake in salt‐resistant rats. Given the recent evidence of central inflammation in animal models of hypertension, we hypothesized that PVN inflammation contributes to Gαi2 protein‐dependent, salt‐sensitive hypertension. Male Sprague–Dawley rats received chronic intracerebroventricular infusions of a targeted Gαi2 or control scrambled oligodeoxynucleotide (ODN) and were maintained for 7 days on a normal‐salt (NS; 0.6% NaCl) or high‐salt (HS; 4% NaCl) diet; in subgroups on HS, intracerebroventricular minocycline (microglial inhibitor) was co‐infused with ODNs. Radiotelemetry was used in subgroups of rats to measure mean arterial pressure (MAP) chronically. In a separate group of rats, plasma noradrenaline, plasma renin activity, urinary angiotensinogen and mRNA levels of the PVN pro‐inflammatory cytokines TNFα, IL‐1β and IL‐6 and the anti‐inflammatory cytokine IL‐10 were assessed. In additional groups, immunohistochemistry was performed for markers of PVN and subfornical organ microglial activation and cytokine levels and PVN astrocyte activation. High salt intake evoked salt‐sensitive hypertension, increased plasma noradrenaline, PVN pro‐inflammatory cytokine mRNA upregulation, anti‐inflammatory cytokine mRNA downregulation and PVN‐specific microglial activation in rats receiving a targeted Gαi2 but not scrambled ODN. Minocycline co‐infusion significantly attenuated the increase in MAP and abolished the increase in plasma noradrenaline and inflammation in Gαi2 ODN‐infused animals on HS. Our data suggest that central Gαi2 protein prevents microglial‐mediated PVN inflammation and the development of salt‐sensitive hypertension.

Activation of spinal macrophage-inducible C-type lectin induces mechanical allodynia and microglial activation in rats

Macrophage-inducible C-type lectin (Mincle), a pattern recognition receptor, is a critical component of the innate immune system that is involved in the pathogenesis of chronic pain. Previous studies have reported the expression of Mincle in neuronal and glial cells of the brain, but its expression and role in pain processing at the spinal level remain to be determined. The current study was performed to identify Mincle in the spinal cord and to investigate the effect of Mincle activation on spinal sensitization.Most Mincle immunoreactivity was localized within the grey matter and the dorsal and ventral horns of the lumbar spinal cord in naïve rats. A single intrathecal (i.t.) injection of trehalose-6,6-dibehenate (TDB), a Mincle ligand, induced mechanical allodynia. Immunoreactivity to Mincle and Iba-1 in the spinal cord significantly increased after i.t. injection of TDB. Mechanical allodynia was attenuated by daily i.t. injection of minocycline. However, double immunofluorescence revealed that Mincle co-localizes with NeuN (neurons), but not with Iba-1 (microglia) or GFAP (astrocytes).In conclusion, we found that Mincle was present in spinal cord neurons, but not microglia or astrocytes, and may play a role in microglia-induced spinal sensitization.

Chronic oral methylphenidate treatment increases microglial activation in rats.

Methylphenidate (MP) is a widely prescribed psychostimulant used to treat attention deficit hyperactivity disorder. Previously, we established a drinking paradigm to deliver MP to rats at doses that result in pharmacokinetic profiles similar to treated patients. In the present study, adolescent male rats were assigned to one of three groups: control (water), low-dose MP (LD; 4/10mg/kg), and high dose MP (HD; 30/60mg/kg). Following 3months of treatment, half of the rats in each group were euthanized, and the remaining rats received only water throughout a 1-month-long abstinence phase. In vitro autoradiography using [3H] PK 11195 was performed to measure microglial activation. HD MP rats showed increased [3H] PK 11195 binding compared to control rats in several cerebral cortical areas: primary somatosensory cortex including jaw (68.6%), upper lip (80.1%), barrel field (88.9%), and trunk (78%) regions, forelimb sensorimotor area (87.3%), secondary somatosensory cortex (72.5%), motor cortices 1 (73.2%) and 2 (69.3%), insular cortex (59.9%); as well as subcortical regions including the thalamus (62.9%), globus pallidus (79.4%) and substantia nigra (22.7%). Additionally, HD MP rats showed greater binding compared to LD MP rats in the hippocampus (60.6%), thalamus (59.6%), substantia nigra (38.5%), and motor 2 cortex (55.3%). Following abstinence, HD MP rats showed no significant differences compared to water controls; however, LD MP rats showed increased binding in pre-limbic cortex (78.1%) and ventromedial caudate putamen (113.8%). These findings indicate that chronic MP results in widespread microglial activation immediately after treatment and following the cessation of treatment in some brain regions.

Abstract 105: PVN Inflammation Contributes to Brain GαI2 Protein Dependent Sympathetically Mediated Salt Sensitive Hypertension

Hypothesis: PVN inflammation contributes to salt sensitive hypertension (HTN) that develops following central Gαi2 protein down regulation. Methods: Male Sprague-Dawley (SD) rats receiving a continuous I.C.V. scrambled (S) or a targeted Gαi2 oligodeoxynucleotide (ODN) infusion (25μg/5μl/day) were fed a normal (NS; 0.6% NaCl) or high salt (HS; 4% NaCl) diet for 7-days (N=4-6/group). Additional groups received a S or Gαi2 ODN and minocycline (120 μg/day) co-infusion and a 7-day HS diet. On day 7 24h sodium balance, MAP, plasma norepinephrine content (via ELISA) and PVN specific mRNA levels of IL1β, IL6, IL10 and TNFα (via RT-PCR) were assessed. Separate groups underwent IHC analysis of PVN astrocytes and PVN microglia activation. Results: Central Gαi2 protein down regulation, but not control S ODN treatment, results in HS evoked HTN, sympathoexcitation, sodium retention, increased PVN mRNA levels of pro-inflammatory cytokines IL1β, IL6 and TNFα, decreased PVN anti-inflammatory 1L10 mRNA levels and no change in astrocyte PVN levels. Significantly, Gαi2 protein down regulation increased PVN microglial activation in rats on a NS diet. I.C.V. minocycline (anti-inflammatory antibiotic) co-infusion abolished the HS-evoked pro-inflammatory mRNA cytokine response and attenuated HTN, sympathoexcitation and sodium retention following central Gαi2 protein down regulation. Conclusion: PVN inflammation contribute to central Gαi2 protein dependent salt sensitive HTN by modulating sympathetic outflow and sodium homeostasis. We speculate central Gαi2 protein downregulation evokes microglial activation to prime a pro-inflammatory PVN response to HS intake.

Scutellarin Ameliorates Learning and Memory Deficit via Suppressing β-Amyloid Formation and Microglial Activation in Rats with Chronic Cerebral Hypoperfusion.

Chronic cerebral hypoperfusion is considered as a pivotal factor of cognitive impairment that occurs in cerebrovascular diseases. This study investigated the ameliorating effect of scutellarin (SCT) on spatial cognitive impairment and β-amyloid (Aβ) formation in rats with chronic cerebral hypoperfusion induced by permanent bilateral common carotid artery occlusion (pBCAO). SCT is a flavonoid in medicinal herb of Erigeron breviscapus (vant.) Hand. Mazz. known to have neuroprotective, antioxidative and anti-inflammatory effects. However, the beneficial effect and pivotal mechanism of SCT on cognitive impairment are still unclear. SCT was treated orally with two doses (10 or 30 mg/kg) for 4 weeks. Results of Morris water maze test performed on the ninth week after pBCAO revealed that SCT (30 mg/kg)-treated rats had significantly shortened escape latencies in acquisition training trials, significantly prolonged swimming time at the platform and its surrounding zone, significant increase in memory score, significant reduction in the number of target heading, and significant reduction in the time required for the first target heading during the retention trial compared to rats in the sham-control group. SCT significantly inhibited the production of Aβ(1-40) and Aβ(1–42) in brain tissues. However, SCT significantly upregulated the expression levels of amyloid precursor protein and β-site APP-converting enzyme-1 in the hippocampus. In addition, SCT significantly inhibited the activation of Iba1-expressing microglia in brain tissues. The results suggest that SCT can exert ameliorating effect on spatial cognitive impairment caused by chronic cerebral hypoperfusion through suppressing Aβ formation and microglial activation in brain tissues. Therefore, SCT can be used as a beneficial drug for vascular dementia and Alzheimer's disease.

Neonatal intrahippocampal injection of lipopolysaccharide induces deficits in social behavior and prepulse inhibition and microglial activation in rats: Implication for a new schizophrenia animal model

Several lines of evidence have suggested that the dysregulation of immune system is involved in the pathogenesis of schizophrenia. Microglia are the resident macrophage of the brain and the major player in innate immunity in the brain. We hypothesized that microglia activation may be closely associated with the neuropathology of schizophrenia. Neonatal intrahippocampal injection of lipopolysaccharide (LPS), an activator of microglia, was performed in rats at postnatal day 7 (PD7), and they were separately treated with saline or minocycline for consecutive 3days. Behavioral changes (locomotor activity, social interaction and prepulse inhibition) were examined in adulthood, and the number of microglia was assessed using immunohistochemistry at PD9, PD21 and PD67. The adult rats in LPS-injected group showed obvious behavioral alterations (deficits in social behavior and prepulse inhibition) and a persistently dramatic increase of number of activated microglial cells in the hippocampus, cerebral cortex and thalamus compared to those in saline-injected group. Interestingly, pretreatment with minocycline could significantly rescue the behavioral deficits and prevent microglia activation. Our results suggest that neonatal intrahippocampal LPS injection may serve as a potential schizophrenia animal model, and inhibition of microglia activation may be a potential treatment strategy for schizophrenia.

Abstract WP256: Pretreatment of Rosuvastatin Ameliorates Early Brain Injury after Subarachnoid Hemorrhage through the Inhibition of Microglial Activation in Rats.

BACKGROUND AND PURPOSE: We investigated the protective effects of pretreatment of oral-administered rosuvastatin, a water soluble HMG-CoA reductase inhibitor, on early brain injury (EBI) after subarachnoid hemorrhage (SAH). METHOD: Male Sprague-Dawley rats were operated on with endovascular perforation model and randomly divided into 4 groups: sham-operated, SAH+vehicle, SAH+1mg/kg, or 10mg/kg rosuvastatin. The animals in treatment groups were administered with daily oral rosuvastatin from 7 days before to 1 day after operation. All rats were evaluated regarding neurofunction, brain edema, and blood-brain barrier (BBB) disruption at 24 hours after SAH. Anti-inflammatory responses were also conducted using Western blot and immunohistochemistry at 24 hours after SAH. RESULTS: Compared with the vehicle group, rosuvastatin significantly improved the neurofunction score (modified Garcia score/ 22: 13.0±1.6 vs 17.0±1.0, P <0.05) , brain edema (brain water content: 79.11±0.15% vs 78.68±0.09%, P <0.05) , and BBB disruption (IgG extravasation); data are showed as a mean±SEM. Rosuvastatin reduced number of activated microglial cells , activation of nuclear factor-kappa B(NF-kB), and expression of tumor necrosis factor-alpha (TNF-α) , which were considered as markers of inflammatory response from microglia, and downregulated the expression of neuronal cyclooxygenase-2 (COX-2) and endothelial matrix metalloproteinase-9 (MMP-9). CONCLUSION: The current study showed that pretreatment of rosuvastatin induced anti-inflammatory processes through inhibition of microglial activation and thereby attenuated early brain injury. Rosuvastatin seems to be a promising agent for treatment of SAH.

Adolescent Morphine Exposure Affects Long-Term Microglial Function and Later-Life Relapse Liability in a Model of Addiction

Adolescence in humans represents a unique developmental time point associated with increased risk-taking behavior and experimentation with drugs of abuse. We hypothesized that exposure to drugs of abuse during adolescence may increase the risk of addiction in adulthood. To test this, rats were treated with a subchronic regimen of morphine or saline in adolescence, and their preference for morphine was examined using conditioned place preference (CPP) and drug-induced reinstatement in adulthood. The initial preference for morphine did not differ between groups; however, rats treated with morphine during adolescence showed robust reinstatement of morphine CPP after drug re-exposure in adulthood. This effect was not seen in rats pretreated with a subchronic regimen of morphine as adults, suggesting that exposure to morphine specifically during adolescence increases the risk of relapse to drug-seeking behavior in adulthood. We have previously established a role for microglia, the immune cells of the brain, and immune molecules in the risk of drug-induced reinstatement of morphine CPP. Thus, we examined the role of microglia within the nucleus accumbens of these rats and determined that rats exposed to morphine during adolescence had a significant increase in Toll-like receptor 4 (TLR4) mRNA and protein expression specifically on microglia. Morphine binds to TLR4 directly, and this increase in TLR4 was associated with exaggerated morphine-induced TLR4 signaling and microglial activation in rats previously exposed to morphine during adolescence. These data suggest that long-term changes in microglial function, caused by adolescent morphine exposure, alter the risk of drug-induced reinstatement in adulthood.

Re-induction of hyponatremia after rapid overcorrection of hyponatremia reduces mortality in rats

Osmotic demyelination syndrome is a devastating neurologic disorder often seen after the rapid correction of chronic hyponatremia. The permeability of the blood-brain barrier is increased in experimental osmotic demyelination, and some have suggested that corticosteroids protect against this disorder by keeping the permeability of the blood-brain barrier low. We previously reported that re-lowering of the serum sodium after rapid correction of chronic hyponatremia was beneficial if performed early in the course (12 to 24 h). Here we compared mortality, blood-brain barrier permeability, and microglial activation in rats after the rapid correction of chronic hyponatremia. We studied three groups of rats after correction of chronic hyponatremia: and treated them with sodium chloride, with or without dexamethasone; or with sodium chloride followed by re-induction of hyponatremia. We found that treatment with dexamethasone or re-induction of hyponatremia effectively prevented the opening of the blood-brain barrier, reduced neurological manifestations, and decreased microglial activation; however, only re-induction of hyponatremia resulted in a significant decrease in mortality 5 days after the correction of chronic hyponatremia. Restoring the permeability of the blood-brain barrier to normal levels did not decrease mortality. Our results suggest that after inadvertent rapid correction of hyponatremia, treatment options should favor re-lowering serum sodium. The increased permeability of blood-brain barrier seen in osmotic demyelination syndrome may not be a primary pathophysiologic insult of this syndrome.

Association of Immune Response and Neuronal Cell Death in the Rat Brain After Kainic Acid or Electrical Kindling Treatment

Purpose: In this study, we tried to clarify the possible involvement of immune responses in epilepsy. This attempt was based on our previous reports on the occurrence of immune responses in the rat hippocampus undergoing kainic acid (KA)‐induced neuronal death and in the sclerotic hippocampus of patients with temporal lobe epilepsy. However, it remains unclear whether these immune responses elicited neuronal degeneration in the sclerotic hippocampus. We investigated the relation between neuronal death and microglial activation in rats either pretreated with KA or submitted to electrical kindling. Methods: KA treatment: Adult male Sprague‐Dawley (SD) rats received a single intraperitoneal injection of KA (12 mg/kg). Only rats showing generalized limbic seizures were used in this study. Rats injected with physiological saline were used as controls. At 6, 12, and 18 h; 1, 2, 3, and 7 days; and 2, 4, 6, 8, 10, 12, and 16 weeks after the treatment, the animals were killed. Electrical kindling: Adult male SD rats were stereotaxically implanted with a bipolar electrode into the left perforant pathway near the angular bundle. Two weeks after electrode implantation, rats were submitted to daily kindling. The unrestrained awake rats received kindling stimulation with a 2‐s train of 60‐Hz biphasic constant‐current 1.0‐ms square‐wave pulses twice a day (6 days per week). The evoked behavioral seizures were classified according to standard criteria. Twenty‐four hours after each behavioral seizure stage, the rats were hlled. For rapid kindling, the unrestrained awake animals were subjected to electrical kindling with hourly and repeated electrical stimulation until generalized seizures were elicited 5 times. Sham‐operated rats served as controls. At 1, 3, 6, 9, and 15 days after the final stimulation, the animals were killed. The activated microglial cells and T lymphocytes were detected immunohistochemically with the avidin‐biotinylated peroxidase complex method. The primary antibodies were directed to complement receptors CR3 (OX‐42). MHC 1 (OX‐18), MHC II (OX‐6). and T‐cell surface antigens. Neuronal death was evaluated by determining DNA fragmentation. For this purpose, coronal cryostat sections of the brain were processed according to the TUNEL technique (in situ end‐labeling method). Results: In control rats, numerous resting microglial cells were distributed throughout the brain. However, neither MHC I‐ nor MHC II‐positive staining was observed in the brain. In KA‐treated rats, reactive microglial cells with hypertrophic bodies and thick processes appeared in the CA1 and CA3 fields of the hippocampus, hilus of the dentate gyms, thalamus, amygdala, and piriform cortex as early as 6 h after injection. The number of these reactive microglial cells quickly increased and reached the maximum at 7 days after the KA injection. The expression of MHC I on reactive microglia, DNA fragmentation, and appearance of cytotoxic/suppressor T lymphocytes were noted at 18 h after injection. The helper/inducer T lymphocytes amd MHC II–positive microglial cells were detected in the same brain regions at 48 h after injection. The fragmentation of DNA and T lymphocytes was detected until 4 and 8 weeks, respectively. After 16 weeks, MHC I‐ or MHC II–positive reactive microglial cells or both were reduced in number. In the daily‐kindling model, only a small number of reactive microglia was observed. In contrast, DNA fragmentation, MHC I‐ or MHC II–positive cells, and T lymphocytes were not detected. In the rapid‐kindling model, there were no activated microglial cells, DNA fragmentation, MHC I‐ amd MHC II–positive cells, or T lymphocytes. Conclusions: In the kindling model of epilepsy, neither DNA fragmentation nor immune responses were detected. The result indicates that epileptic seizures do not depend on the immune responses. In the KA‐treated model of epilepsy, immune responses were closely related to DNA fragmentation, suggesting an association of immune responses with neuronal death. We therefore suggest that immune responses play an important role in the neuronal death process induces by KA.