Proteins In Brain Tissue Research Articles

Optimization of protocols for pre-embedding immunogold electron microscopy of neurons in cell cultures and brains

Immunogold labeling allows localization of proteins at the electron microscopy (EM) level of resolution, and quantification of signals. The present paper summarizes methodological issues and experiences gained from studies on the distribution of synaptic and other neuron-specific proteins in cell cultures and brain tissues via a pre-embedding method. An optimal protocol includes careful determination of a fixation condition for any particular antibody, a well-planned tissue processing procedure, and a strict evaluation of the credibility of the labeling. Here, tips and caveats on different steps of the sample preparation protocol are illustrated with examples. A good starting condition for EM-compatible fixation and permeabilization is 4% paraformaldehyde in PBS for 30 min at room temperature, followed by 30 min incubation with 0.1% saponin. An optimal condition can then be readjusted for each particular antibody. Each lot of the secondary antibody (conjugated with a 1.4 nm small gold particle) needs to be evaluated against known standards for labeling efficiency. Silver enhancement is required to make the small gold visible, and quality of the silver-enhanced signals can be affected by subsequent steps of osmium tetroxide treatment, uranyl acetate en bloc staining, and by detergent or ethanol used to clean the diamond knife for cutting thin sections. Most importantly, verification of signals requires understanding of the protein of interest in order to validate for correct localization of antibodies at expected epitopes on particular organelles, and quantification of signals needs to take into consideration the penetration gradient of reagents and clumping of secondary antibodies.

MiR-30c-5p/ATG5 Axis Regulates the Progression of Parkinson's Disease.

Serum miR-30c-5p correlates with Parkinson’s disease (PD), yet its role has not been illustrated. This research analyzed the function of miR-30c-5p in PD. The behavioral evaluation was performed on MPTP-treated PD mice transfected with miR-30c-5p agomiR, antagomiR, siATG5, or 3-MA (an autophagy inhibitor). Oxidative stress-related factors, miR-30c-5p, and apoptosis- and autophagy-associated proteins in brain tissues or cells were determined by molecular experiments. Tyrosine hydroxylase (TH) and dopamine metabolic markers were detected using immunofluorescence and Diode Array Detector (DAD), respectively. Effects of miR-30c-5p and its target gene Autophagy-related gene (ATG) 5 protein (ATG5) on MPP+-treated SH-SY5Y cells were determined through a series of molecular experiments. MiR-30c-5p was upregulated but ATG5 was downregulated in PD mice. MiR-30c-5p antagomiR attenuated the decrease of ATG5 in PD mice. MiR-30c-5p antagomiR partly alleviated the behavioral symptoms and inhibited the increases of malondialdehyde (MDA), catalase (CAT), and SOD in PD mice. The levels of Bcl-2, dopamine, dihydroxyphenylacetic acid (DOPAC), homovanillic acid (HVA), TH, and LC3 II were downregulated in PD mice, while Bax, cleaved caspase-3, P62, and LC3 I were upregulated. However, miR-30c-5p antagomiR partly reversed the levels of these factors in PD mice. 3-MA could block the effects of miR-30c-5p antagomiR on PD mice. MiR-30c-5p antagomiR attenuated apoptosis and induced autophagy in brain tissues of MPTP-treated mice by targeting ATG5. In vitro assay results also showed that silence of ATG5 reduced the protective effect of miR-30c-5p downregulation on the cells. MiR-30c-5p regulates the progression of Parkinson’s disease through attenuating ATG5-inhibited apoptosis and -induced autophagy.

Expression of Acute Phase Protein, Glial Fibrillary Acidic Protein, Epithelial Cadherin, and S100 Protein in Brain Tissues from Natural Cases of Ovine Encephalitic Listeriosis.

Listeriosis is a disease that is induced by infection with the Gram-positive bacterium Listeria monocytogenes. Much is still unknown about the pathogenesis of encephalitic listeriosis. We aimed to identify the contribution of glial fibrillary acidic protein (GFAP), epithelial cadherin (E-cadherin), S100, and acute-phase proteins (APPs) in pathogenesis, clinical and preclinical diagnosis of natural cases of encephalitic listeriosis. Of 1,325 Ossimi sheep, 64 were suspected of having listeriosis from 2018 to 2020. Prospective cohort evaluation of clinical and postmortem findings was performed, in addition to bacterial isolation, the measurement of APPs in serum and cerebrospinal fluid (CSF), and the histopathological and immunohistochemical evaluation of GFAP, S100, and E-cadherin. Infected sheep showed nervous symptoms ranging from neck stretching to complete paralysis. APPs were significantly increased in the CSF of both clinically and preclinically diseased animals; however, serum APPs were only significantly increased in clinically diseased animals. Histopathological evaluation revealed microabscesses, meningoencephalitis, and perivascular cuffing of the brainstem of infected sheep. Immunohistochemical investigations revealed strong expression of GFAP and S100 in necrotic areas and negative expression of E-cadherin. The measurement of CSF APPs could be useful in the preclinical diagnosis of sheep listeriosis. GFAP and S100 proteins could be involved in the pathogenesis of listeriosis; however, E-cadherin does not appear to be involved.

Mechanism of Huoxue Tongluo Decoction in treatment of erectile dysfunction caused by ischemic stroke based on network pharmacology

ObjectiveTo study the therapeutic effect of Huoxue Tongluo Decoction (HXTLD) on erectile dysfunction caused by ischemic stroke and identify the mechanisms involved. MethodsNetwork pharmacology was used to predict the key active ingredients and targets of HXTLD. Surgical methods were used to create a rat model of ischemic stroke. The rats were then given a suspension of HXTLD by ig administration. Erectile function was evaluated by Apomorphine (APO) induction. Real-time fluorescence quantitative reverse transcription-polymerase chain reaction (Real-time PCR) and Western blotting were used to detect the expression of related mRNAs and proteins in rat penile corpus cavernous tissue and brain tissue. Hematoxylin & Eosin (HE) staining was used to investigate structural changes in the penile cavernous tissue. ResultsNetwork pharmacology showed that tumor necrosis factor (TNF), nitric oxide synthase 3 (eNOS), and vascular endothelial growth factor (VEGF) were the key targets of HXTLD in the treatment of erectile dysfunction caused by ischemic stroke. Experimental studies showed that HXTLD improved erectile dysfunction caused by ischemic stroke. HE results showed that HXTLD improved the structure of the corpus cavernosa. HXTLD also inhibited the expression of TNF and VEGF proteins in penile tissue (P < 0.05) and enhanced the expression of eNOS protein in penile tissue (P < 0.05). ConclusionHXTLD improved the erectile function of rats with erectile dysfunction caused by ischemic stroke by regulating the mRNA and protein levels of TNF, eNOS and VEGF.

Connexin43 promotes angiogenesis through activating the HIF-1α/VEGF signaling pathway under chronic cerebral hypoperfusion

Chronic cerebral hypoperfusion, a major vascular contributor to vascular cognitive impairment and dementia, can exacerbate small vessel pathology. Connexin43, the most abundant gap junction protein in brain tissue, has been found to be critically involved in the pathological changes of vascular cognitive impairment and dementia caused by chronic cerebral hypoperfusion. However, the precise mechanisms underpinning its role are unclear. We established a mouse model via bilateral common carotid arteries stenosis on connexin43 heterozygous male mice and demonstrated that connexin43 improves brain blood flow recovery by mediating reparative angiogenesis under chronic cerebral hypoperfusion, which subsequently reduces the characteristic pathologies of vascular cognitive impairment and dementia including white matter lesions and irreversible neuronal injury. We additionally found that connexin43 mediates hypoxia inducible factor-1α expression and then activates the PKA signaling pathway to regulate vascular endothelial growth factor-induced angiogenesis. All the above findings were replicated in bEnd.3 cells treated with 375 µM CoCl2 in vitro. These results suggest that connexin 43 could be instrumental in developing potential therapies for vascular cognitive impairment and dementia caused by chronic cerebral hypoperfusion.

Low-Intensity Pulsed Ultrasound Stimulation Inhibits Cortical Spreading Depression.

Cortical spreading depression (CSD), which is closely correlated with migraine aura, cerebral ischemia, seizure, and brain injury, is a spreading wave of neuronal and glial depolarization. The purpose of this study is to investigate whether low-intensity pulsed ultrasound stimulation (PUS) inhibits CSD by modulating neural activity and hemodynamics. Behavioral test, intrinsic signal optical imaging and western blot analysis were used for evaluating the inhibition effect of PUS on CSD in rat. We found that: 1) 30min of PUS can significantly improve motor activity of rat with CSD. 2) Both 30s and 30min of PUS can significantly reduce count and propagation speed of CSD in rat and the inhibitory effect was enhanced with increase of ultrasound intensity. 3) 30min of PUS significantly enhanced levels of brain-derived neurotrophic factor protein in brain tissue with CSD. These results suggest that PUS has the potential to treat brain disorders associated with CSD.

Neuroprotective effect of aspirin combined with ginkgolide injection on cerebral ischemic stroke rats and its effect on ERK12 signal pathway.

The main aim of this study was to evaluate the neuroprotective effect of aspirin combined with ginkgolide injection on cerebral ischemic stroke model rats and its effect on extracellular regulated protein kinase 1/2 (REK1/2) signaling pathway, and to clarify the possible mechanism of aspirin combined with ginkgolide injection on neuroprotective mechanism. Experimental rats were randomly divided into sham group, model group, aspirin group, ginkgolide group and combination group (aspirin + ginkgolide injection) (n = 20). The results revealed scores of neurological dysfunction and infarct volume in aspirin group, ginkgolide group and combination group rats were lower than those in model group (P < 0.05). Score of neurological dysfunction and the volume of cerebral infarction in combination group rats were lower than those in aspirin group and ginkgolide group (P < 0.05). Combination of aspirin and ginkgolide injection could better reduce brain water content, reduce apoptosis rate of cortical cells P < 0.05, reduce expression levels of caspase-3, Bax and p-REK1/2 proteins in ischemic brain tissue P < 0.05, and increase expression level of Bcl-2 protein than aspirin and ginkgolide injection alone P < 0.05). In conclusion, the synergistic neuroprotective effect of aspirin and ginkgolide injection on cerebral ischemic stroke rats is better than that of aspirin and ginkgolide injection alone. The mechanism of action may be that the two compounds can play a synergistic role and inhibit the activation of REK1/2 signaling pathway, thus inhibiting apoptosis of nerve cells and exerting neuroprotective effect.

Fatty Acid-Binding Protein 3 Expression in the Brain and Skin in Human Synucleinopathies.

Parkinson’s disease (PD) and multiple system atrophy are types of adult-onset neurodegenerative disorders named synucleinopathies, which are characterized by prominent intracellular α-synuclein (αSyn) aggregates. We have previously found that αSyn aggregates and the vulnerability of dopaminergic neurons in the mouse brain are partly associated with the expression of fatty acid-binding protein 3 (FABP3, heart FABP). However, it remains to be elucidated whether FABP3 accumulation is associated with αSyn aggregates in human tissues. Here, we histologically studied FABP3 expression in human tissues obtained from patients with synucleinopathies, patients with Alzheimer disease (AD) and controls. We found that (1) a variety of neurons expressed the FABP3 protein in human brain tissues, (2) FABP3 was colocalized with αSyn aggregates in the brains of individuals with synucleinopathies but not with amyloid β or p-tau aggregates in the brains of individuals with AD, and (3) FABP3 was not present in p-αSyn deposits in biopsied skin tissues from individuals with PD. These findings suggest that FABP3 expression is associated with αSyn aggregation in synucleinopathies and provide new insights into the involvement of FABP3 in synucleinopathies.

Contrast enhanced X-ray computed tomography imaging of amyloid plaques in Alzheimer disease rat model on lab based micro CT system

Amyloid plaques are small (~ 50 μm), highly-dense aggregates of amyloid beta (Aβ) protein in brain tissue, supposed to play a key role in pathogenesis of Alzheimer’s disease (AD). Plaques´ in vivo detection, spatial distribution and quantitative characterization could be an essential marker in diagnostics and evaluation of AD progress. However, current imaging methods in clinics possess substantial limits in sensitivity towards Aβ plaques to play a considerable role in AD screening. Contrast enhanced X-ray micro computed tomography (micro CT) is an emerging highly sensitive imaging technique capable of high resolution visualization of rodent brain. In this study we show the absorption based contrast enhanced X-ray micro CT imaging is viable method for detection and 3D analysis of Aβ plaques in transgenic rodent models of Alzheimer’s disease. Using iodine contrasted brain tissue isolated from the Tg-F344-AD rat model we show the micro CT imaging is capable of precise imaging of Aβ plaques, making possible to further analyze various aspects of their 3D spatial distribution and other properties.

Quantitative Analysis of Mobile Proteins in Normal Brain Tissue by Amide Proton Transfer Imaging: Age Dependence and Sex Differences.

The aims of this study were to evaluate the relationship between age change and amide proton transfer (APT) signal in each region of the whole brain and to derive the standard value of APT signal in each brain region of normal adults. Using the mDIXON 3-dimensional-APT sequence of the fast spin echo method, an APT image was obtained. In total, 60 patients (mean age, 49.8 ± 16.9 years) with no abnormal findings on magnetic resonance imaging data were included. For image analysis, registration parameters were created using the FMRIB Software Library 5.0.11, and then a region of interest was set in the Montreal Neurological Institute structural atlas for analysis. Statistical analyses were performed using the age-dependent and sex differences in APT signals from each brain region. No significant correlation was seen between APT signal and age and sex in all brain regions. Under the APT imaging parameter conditions used in this study, local brain APT signals in healthy adults are independent of age and sex.

Near-Infrared Monoamine Oxidase Inhibitor Biodistribution in a Glioma Mouse Model.

The biodistribution imaging kinetics of near-infrared monoamine oxidase inhibitor (NMI) are reported here. NMI was administered intravenously or orally to mice and detected by NIR fluorescence optical imaging within minutes and the longitudinal signal distribution was measured for up to 1week after a single dose. NMI rapidly reached 3.7-fold higher ventral and 3.2-fold higher brain region fluorescent signal intensity compared to oral route at 24h. Similar patterns of NMI biodistribution were found in mice with or without intracranial implanted GL26 brain tumors. NMI was highly associated with tumors in contrast to adjacent non-tumor brain, confirming diagnostic utility. NMI 5mg/kg imaging signal in brain at 48h was optimal (tumor/non-tumor ratio > 3.5) with minimum off-target distribution. Intravenous NMI imaging signal peaked between 24h and 48h for lung, liver, kidney, blood, brain, and most other tissues. Clearance (signal weaker, but still present) from most tissues occurred by day 7. Intravenous low dose (0.5mg/kg) minimally labeled tumor and other tissues, 5mg/kg showed optimal imaging signal in glioma at a dose we previously reported as efficacious, and 50mg/kg was tolerable but saturated the tissue signals beyond tumor specificity. Gel electrophoresis showed two major bands present in brain tumor and tissue protein lysates. Intravenous 5mg/kg was optimal dose to target brain tumor and identified off-target organs of concern: lungs, liver, and kidneys. These results demonstrate the biodistribution and optimal dose range of NMI for treatment and diagnostic monitoring of glioma.

Genetic background influences LRRK2-mediated Rab phosphorylation in the rat brain

Pathogenic missense mutations in the leucine-rich repeat kinase 2 gene, encoding LRRK2, results in the upregulation of Rab10 and Rab12 phosphorylation in different cells and tissues. Here, we evaluate levels of the LRRK2 kinase substrates pT73-Rab10 and pS106-Rab12 proteins in rat brain tissues from different genetic backgrounds. Whereas lines of Sprague Dawley rats have equivalent levels of pT73-Rab10 and pS106-Rab12 similar to Lrrk2 knockout rats, Long-Evans rats have levels of pT73-Rab10 and pS106-Rab12 comparable to G2019S-LRRK2 BAC transgenic rats. Strong LRRK2 kinase inhibitors are ineffective at reducing pT73-Rab10 and pS106-Rab12 levels in the Sprague Dawley rats, but potently reduce pT73-Rab10 and pS106-Rab12 levels in Long-Evans rats. Oral administration of the PFE-360 LRRK2 kinase inhibitor fails to provide neuroprotection from dopaminergic neurodegeneration caused by rAAV2/1-mediated overexpression of A53T-αsynuclein in Sprague Dawley rats. These results highlight substantial differences in LRRK2-mediated Rab10 and Rab12 phosphorylation in commonly utilized rat genetic backgrounds and suggest LRRK2 may not play a central role in Rab phosphorylation or mutant αsynuclein toxicity in Sprague Dawley rats.

Effects of near-far acupuncture on neuronal function and expression of apoptosis-related protein Bax/Bcl-2/Cleaved caspase-3 in rats with ischemic stroke

Objectives To explore the effects of electroacupuncture on nerve function in rats with ischemic stroke and its mechanism of anti-apoptosis. Methods A total of 80 SPF male SD rats were randomly divided into sham operation group, model group, sham electroacupuncture group, acupuncture group, electroacupuncture group and 16 rats in each group. The rat model of left ischemic stroke was prepared by suture embolization. In the sham group, the left common carotid artery was isolated only and no other treatment was given. In the electroacupuncture group, "Baihui" and "Mingmen" were selected for acupuncture, followed by dilatation wave, frequency 2Hz/100Hz, intensity 1mA, and electroacupuncture for 30min. The sham electroacupuncture group was the same as the electroacupuncture group in acupoint electroacupuncture group was the same as the electroacupuncture group in acupoint selection. The electroacupuncture group was only inserted subcutaneously and then connected with the electroacupuncture group without power supply, and fixed for 30 minutes. The electroacupuncture group and the acupuncture group received electroacupuncture treatment once, for a total of 14 days, 1d after modeling. The Improved Neurological Impairment scale (mNSS) was used to evaluate the degree of neurological impairment in each group after anesthesia and wakefulness. The percentage of cerebral infarction area was determined by TTC staining. HE staining and Nissl staining were used to observe the pathological changes of ischemic brain tissue. The level of apoptosis in ischemic brain tissue was detected by TUNEL assay. Western blot was used to detect protein expression of Bax, Bcl-2 and Cleaved caspase-3 in ischemic brain tissue. Results Compared with the sham operation group, neurological function score, percentage of cerebral infarction area and apoptosis level in the model group were significantly increased (all P &lt; 0.01). Compared with the model group, neurological function score, percentage of cerebral infarction area and apoptosis index of acupuncture group and electroacupuncture group were decreased (all P &lt;0.05). Compared with the model group, the expression levels of Bcl-2 protein in ischemic brain tissue of rats with ischemic stroke were up-regulated in the acupuncture group and electroacupuncture group to different degrees, while the expression levels of Bax and Cleaved caspase-3 protein were down-regulated in the electroacupuncture group. Conclusion Electroacupuncture may inhibit Bax, Cleaved caspase-3 and up-regulate the expression of Bcl-2 against neuronal apoptosis, thereby improving the neurological function injury of ischemic stroke rats.

Mitochondrial dysfunction, mitophagy, and role of dynamin-related protein 1 in Alzheimer's disease.

Alzheimer's disease (AD) is the most common type of dementia and progressive neurodegenerative disease. The presence of β-amyloid (Aβ) plaques and phosphorylated Tau tangles are considered to be the two main hallmarks of AD. Recent findings have shown that different changes in the structure and dynamics of mitochondria play an important role in AD pathology progression. Mitochondrial changes in AD are expressed as enhanced mitochondrial fragmentation, altered mitochondrial dynamics, and changes in the expression of mitochondrial biogenesis genes in vitro and in vivo models. Therefore, targeting mitochondria and associated mitochondrial proteins seems to be a promising alternative instead of targeting Aβ and Tau in the prevention of Alzheimer's disease. The dynamin-related protein (Drp1) is one such protein that plays an important role in the regulation of mitochondrial division and maintenance of mitochondrial structures. Few researchers have shown that inhibition of Drp1 GTPase activity in neuronal cells rescues excessive mitochondrial fragmentation. In addition, the growing evidence revealed that Drp1 can interact with both Aβ and Tau protein in human brain tissues and mouse models. In this review, we would like to update existing knowledge about various changes in and around mitochondria related to the pathogenesis of Alzheimer's disease, with particular emphasis on mitophagy and autophagy.

RETRACTED: MicroRNA-376b-5p targets SOX7 to alleviate ischemic brain injury in a mouse model through activating Wnt/β-catenin signaling pathway

Accumulating evidence has reported the role of microRNA (miR) on ischemic brain injury. We aim to investigate the mechanism of miR-376b-5p/Sex-determining region Y-box 7 (SOX7)/Wnt/β-catenin axis in mice with ischemic brain injury. Transient middle cerebral artery occlusion (tMCAO) model was established by suture method. Expression levels of miR-376b-5p, SOX7, and Wnt/β-catenin pathway-related proteins (Wnt3a and β-catenin) in brain tissues of tMCAO mice were determined by RT-qPCR and western blot analysis. The target relationship between miR-376b-5p and SOX7 was tested by bioinformatics analysis and luciferase activity assay. The neurological scores of mice were recorded and their behaviors were observed. Moreover, the brain damage, oxidative stress indices, hemoglobin (Hb) content, content of brain water, infarct area, TUNEL positive cells, blood-brain barrier permeability and the number of intact neurons in the ischemic-side brain tissues of tMCAO mice were detected via upregulated miR-376b-5p or downregulated SOX7. In mice with ischemic brain injury, miR-376b-5p targeted and downregulated SOX7 in brain tissues, thus activating the Wnt/β-catenin pathway. The tMCAO mice showed higher neurological scores, severe brain damage, decreased number of neurons, as well as elevated blood-brain barrier permeability, Hb content, cerebral edema and infarct area. These symptoms could be alleviated by miR-376b-5p elevation or SOX7 inhibition. SOX7 overexpression reversed the effects of miR-376b-5p elevation on tMCAO mice. Our study suggests that miR-376b-5p could improve the blood-brain barrier permeability, relieve brain edema and decrease infarct area, thus improve ischemic brain injury via the inhibition of SOX7 and activation of Wnt/β-catenin pathway.

Pelargonidin ameliorates MCAO-induced cerebral ischemia/reperfusion injury in rats by the action on the Nrf2/HO-1 pathway.

BackgroundMorbidity and mortality remain high for ischemic stroke victims, and at present these patients lack effective neuroprotective agents, which improve the cure rate. In recent years, studies have shown that pelargonidin has many biological actions. However, few studies are available regarding the pelargonidin treatment of cerebral ischemia.MethodsThe rat middle cerebral artery occlusion (MCAO) model was established to investigate the neuroprotective effect of pelargonidin on cerebral ischemia/reperfusion injury. Reperfusion was performed 2 h after ischemia; magnetic resonance imaging (MRI) and 2, 3, 5-triphenyltetrazolium chloride (TTC) staining were used to measure the volume of cerebral ischemia. Both modified neurological severity scores (mNSSs) and Morris water maze test were used to assess the neurological functions. ELISA was applied to determine the levels of TNF-α, TGF-β, IL-6, IL-10, MDA, and SOD. The expression of Nuclear factor-E2-related factor 2 (Nrf2) and heme oxygenase 1 (HO-1) protein in brain tissue was measured by immunofluorescence and Western blot assays.ResultsThe results showed that pelargonidin could effectively reduce the volume of cerebral ischemia and improve the neurological function in MCAO rats, thereby improving memory and learning ability. With the corresponding decreases in the expression of TNF-α, TGF-β, IL-6, and MDA, the level of IL-10 and SOD increased and also promoted the nuclear metastasis of Nrf2 and the expression of HO-1 in ischemic brain tissues.ConclusionsOur data demonstrated that pelargonidin ameliorated neurological function deficits in MCAO rats, and its potential mechanism of action was associated with overexpression of the Nrf2/HO-1-signaling pathway. This study will provide a new approach to treat cerebral ischemia/reperfusion injury.

Analysis of regulatory effect of miR-149-5p on Sphingosine-1-phosphate receptor 2 of pericytes and its neuroprotective molecular mechanism after acute cerebral ischemia reperfusion in rats

ABSTRACT To investigate the effect of miR-149-5p on sphingosine-1-phosphate receptor 2 (S1PR2) expression level and contents of matrix metalloproteinase (MMP-9) and superoxide dismutase (SOD) in the pericytes after acute cerebral ischemia reperfusion in rats, so as to clarify the neuroprotective molecular mechanism induced by miR-149-5p and provide references for the treatment of neurological diseases, 60 male SD rats aged 7–8 weeks were selected and divided randomly into test group (establishing middle cerebral artery occlusion (MCAO) model) and control group (no modeling). Rat pericytes and peripheral cerebral infarction tissues were collected 12 h, 1 d, 3 d, 5 d, and 7 d after MCAO modeling, respectively. The pericytes were identified by immunofluorescence assay (IFA) and transfected with miR-149-5p. Fluorescence quantitative PCR (FQPCR) and Western blot were adopted to detect S1PR2 expression level. The expression of S1PR2 in MCAO model rats was detected by IFA. Immunohistochemistry (IHC) and quantitative real-time PCR (qRT-PCR) were used to detect the changes of MMP9 protein and mRNA levels of SOD1, SOD2, and SOD3 in brain tissue. The results showed that mRNA level and protein expression level of S1PR2 in the test group were higher than those in the control group three days after MCAO modeling (P < 0.05); the expression of S1PR2 increased 12 h after MCAO modeling and returned to the normal level on the 5th day, and the content of MMP9 protein in brain tissue of the test group was significantly lower than that of the control group (P < 0.05); the mRNA levels and SODs activity of SOD1, SOD2, and SOD3 in the test group were higher than those in the control group (P < 0.05). Therefore, miR-149-5p played a neuroprotective role by regulating S1PR2 to change the expression levels of SODS and MMP9.

Effects of Recombinant Human Erythropoietin on Inflammatory Factors in Rats with Traumatic Brain Injury

To study the effects of recombinant human erythropoietin on inflammatory factors in rats with traumatic brain injury is the main objective. A total of 45 specific-pathogen-free grade male Sprague-Dawley rats were randomly assigned into sham operation group (sham group), model group and recombinant human erythropoietin intervention group (treatment group) (n=15). Model and treatment groups were prepared into traumatic brain injury model by hitting the head through the modified Feeney’s free-fall impact method, while the head of sham group was not hit. After modeling, treatment group was intraperitoneally injected with recombinant human erythropoietin at 5000 IU/kg daily and sham and model groups were intraperitoneally injected with the same dose of normal saline. The rats were killed after 7 d of continuous administration. The changes of brain mitochondrial membrane potential were detected through rhodamine 123 staining and immunocytochemistry and Western blotting were separately employed to measure the expressions of interleukin-1β, interleukin-6 and tumor necrosis factor-α in brain tissues and the expression levels of dynamin-related protein 1, fission 1, mitofusin 2 and optic atrophy 1, mitochondrial dynamics related proteins in brain tissues. Compared with sham group, model group exhibited significantly weakened rhodamine 123 fluorescence intensity, increased expressions of interleukin-1β, interleukin-6 and tumor necrosis factor-α, dynamin-related protein 1 and fission 1 and reduced expressions of mitofusin 2 and optic atrophy 1 in brain tissues (p<0.05). In comparison with model group, treatment group had significantly enhanced rhodamine 123 fluorescence intensity, reduced expressions of interleukin-1β, interleukin-6 and tumor necrosis factor-α, dynamin-related protein 1 and fission 1 and elevated expressions of mitofusin 2 and optic atrophy 1 in brain tissues (p<0.05). Recombinant human erythropoietin can protect the brain after traumatic brain injury by relieving the inflammatory response and mitochondrial injury after traumatic brain injury.

Methylmercury chloride exposure aggravates proinflammatory mediators and Notch-1 signaling in CD14+ and CD40+ cells and is associated with imbalance of neuroimmune function in BTBR T+ Itpr3tf/J mice

Autism spectrum disorder (ASD) is a severe neurodevelopmental disorder characterized by deficits in social interaction, communication, and repetitive behaviors. A key role for immune dysfunction has been suggested in ASD. Recent studies have indicated that inflammatory mediators and Notch-1 signaling may contribute to the development of ASD. Methylmercury chloride (MeHgCl) is an environmental pollutant that primarily affects the central nervous system, causing neurological alterations. Its effects on immunological responses have not been fully investigated in ASD. In this study, we examined the influence of MeHgCl exposure on inflammatory mediators and Notch-1 signaling in BTBR T+ Itpr3tf/J (BTBR) mice, a model of ASD. We examined the effects of MeHgCl on the IL-6-, GM-CSF-, NF-κB p65-, Notch-1-, and IL-27-producing CD14+ and CD40+ cells in the spleen. We assessed the effect of MeHgCl on IL-6, GM-CSF, NF-κB p65, Notch-1, and IL-27 mRNA levels in brain tissue. We also measured IL-6, GM-CSF, and NF-κB p65 protein expression levels in brain tissue. MeHgCl exposure of BTBR mice significantly increased IL-6-, GM-CSF-, NF-κB p65-, and Notch-1-, and decreased IL-27-producing CD14+, and CD40+ cells in the spleen. MeHgCl exposure of BTBR mice upregulated IL-6, GM-CSF, NF-κB p65, and Notch-1, and decreased IL-27 mRNA expression levels in brain tissue. Moreover, MeHgCl resulted in elevated expression of the IL-6, GM-CSF, and NF-κB p65 proteins in brain tissue. Taken together, these results indicate that MeHgCl exposure aggravates proinflammatory mediators and Notch-1 signaling which are associated with imbalance of neuroimmune function in BTBR mice.

Neuroprotective effect of chrysin on isoniazid-induced neurotoxicity via suppression of oxidative stress, inflammation and apoptosis in rats

Isoniazid (INH) is among the most important anti-tuberculosis agents widely prescribed. However, its clinical use is restricted due to its severe side effects associated with neurotoxicity. The aim of the present study was to investigate the neuroprotective effects of chrysin (CR), a natural antioxidant, against INH-induced neurotoxicity in rats. The rats were treated orally with INH (400 mg/kg body weight) alone or with CR (25 and 50 mg/kg body weight) for 7 consecutive days. INH administration significantly increased brain lipid peroxidation and resulted in a significant decrease in antioxidant biomarkers including superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx) and glutathione (GSH). INH treatment also increased levels of nuclear factor kappa B (NF-κB), tumor necrosis factor-α (TNF-α), glial fibrillary acidic protein (GFAP) and activities of p38α mitogen-activated protein kinase (p38α MAPK) while decreasing levels of neural cell adhesion molecule (NCAM). Double immunofluorescence expressions of c-Jun N-terminal kinase (JNK) and Bcl-2 associated X protein (Bax) in brain tissues were increased after INH administration. Furthermore, INH increased mRNA expression levels of nuclear factor erythroid 2-related factor 2 (Nrf-2), heme oxygenase-1 (HO-1), NAD(P)H: quinone oxidoreductase 1 (NQO1), glutamate-cysteine ligase modifier subunit (Gclm), glutamate cysteine ligase catalytic subunit (Gclc), NF-κB, TNF-α, interleukin-1β (IL-1β), interleukin-6 (IL-6) and GFAP in rat brain tissues. Co-treatment with CR increased anti-oxidant capacity as well as regulated inflammation and apoptosis in brain. Additionally, molecular docking results showed that CR had the potential to interact with the active sites of TNF-α and NFκ-B. In conclusion, CR improved INH-induced brain oxidative damage, inflammation and apoptosis, possibly through their antioxidant properties.