Brain Of Wistar Rats Research Articles

Еxpression of cerebral endothelial NLRP3 inflammasome in viral inflammation in vitro

The goal is to study the effects of pro-inflammatory agents on the expression of NLRP3 and the structural integrity of cerebral microvessel endothelial cells in vitro. Materials and methods. The study was conducted on a culture of cerebral endothelial cells. The cells were isolated from Wistar rat brains. Polyinosinic-polycytidylic acid (RolyI: C) – 20 µg / ml was added to cerebral microvessel endothelial cells. In another series of experiments, cells incubated with cerebrospinal fluid (CSF) obtained from patients with enteroviral meningitis (100 µl) were used as a comparison group. CSF was standardized for protein concentration by the Lowry method. The protein concentration was 1 µg / ml. As a control group, cerebral endothelial cells have been cultured in a standard medium. We used the culture insert for 12-well plates. After 24 and 72 h of culturing, we measured transendothelial electrical resistance (TEER) in the endothelial layer. Expression of NLRP3 inflammasomes was assessed with immunocytochemistry 24 hrs from the beginning of cell culture. We used double indirect immunoenzymatic staining method according to the manufacturer’s protocol. Primary antibodies to NLRP3 (Abcam, USA, ab51952) in 1: 100 dilution, secondary antibodies labeled with Alexa Fluor 488 (Abcam, USA, ab150117) in dilution 1: 200 have been applied. Visualization was performed by confocal laser microscopy microscope Olympus FV10i (Olympus, Japan). Results. TEER decreased in PolyI:C and CSF-treated cells after 24 hr from the beginning of incubation. After 72 hours, TEER was significantly lower in these groups compare to the control one. NLRP3 expression was maximal in the cells treated with CSF. After incubation of the cells with PolyI:C, NLRP3 expression in cerebral endothelial cells was elevated compare to the control group, but did not reached the level seen in CSF-treated cells. Conclusion. PolyI:C and CSF obtained from the patients with viral meningitis induce disruption of cerebral microvessels endothelial layer integrity with the corresponding rise in NLRP3 expression in the cells, thereby suggesting mechanism of blood-brain barrier impairment in neuroinfection.

EFEK ASAM ALFA LIPOAT PADA KADAR MDA DAN HISTOLOGI OTAK DIABETES MELLITUS TIPE 1

Background. Diabetic neuropaty is a condition that can affect pyramidal cells and neuronal cells in the hippocampus. Alpha lipoic acid is effective in pathological conditions where ROS (Reactive Oxygen Species) have been implicated, include in brain. Objective. To investigate effects of ALA on oxidative stress in diabetic brain of male Wistar rats. Methods. True experimental design and Posttest Only Control Group are used in this study. Thirty male rats were randomly divided into 5 groups: normal rats without ALA (NTA), diabetic rats without ALA (DTA), diabetic rats with ALA 80 mg, ALA dose 200 mg, and ALA dose 500 mg/kg/day. ALA therapy in mice conducted orally once a day. Diabetes was induced in rats by single intraperitonial injection of streptozotocin (STZ) at 60 mg/kg body weight. The content of malondialdehyde (MDA) in the brain was measured by spectrophotometeric assays. Brain structure (pyramidal cell in hippocampus) was assessed by staining with hematoxylin and eosin. Results. MDA levels in the DTA, DA80 and DA200 is greater than the levels of MDA in the NTA group, but not statistically significant at the MDA values (p = 0,260). Test-Product Moment Pearson correlation showed a weak positive relationship and not significant (r = 0,327) between the groups of NTA, DA80 and DA200 with MDA. No differences pyramidal cell structure between NTA and DTA. Conclusion. The treatment for 4 weeks with ALA had not reduced oxidative stress in diabetic brain.

Detection of drought tolerance gene(s) from some Egyptian wheat cultivars

Acetaminophen is a safe analgesic and an antipyretic agent that is available without a prescription. However, high doses of acetaminophen are a direct cause of hepatic toxicity, neurotoxicity, and even death through induction of oxidative stress. The study aimed to evaluate the possible prophylactic and therapeutic effects of nicotinamide (500 mg/kg B.W.) on the behavioral changes examined by forced swim test and open field test, as well as on the antioxidants status in the brain of male Wistar rats that were intoxicated with a single overdose of acetaminophen (5 g/kg B.W.). Acetaminophen showed depressant like behavior, impaired locomotor activity, induced oxidative damage and neurotoxicity through elevation of lipid peroxidation level, reduction of glutathione level, glutathione peroxidase and catalase activities. On the other hand, nicotinamide showed an obvious antidepressant-like effect in the forced swim test in the acetaminophen-intoxicated rats and treated with nicotinamide either in prophylactic or therapeutic ways. However, nicotinamide increased motor deficits and impaired antioxidants status. Neurohistology of the cerebellum showed neuropil vacuolation, and degenerating Purkinje cells with pyknotic nuclei in groups treated with acetaminophen, nicotinamide or both together. In conclusion, caution must be taken upon administration of overdoses of nicotinamide due to its potential neurotoxicity, particularly, upon combination with acetaminophen therapy.

Determination of Silver Nanoparticle Concentration Ratio in the Blood and Brain of Rats for Different Administration Routes

A study to assess the concentrations ratio of silver nanoparticles (Ag-NPs) in the blood and brain of the male Wistar rats using nuclear physical methods has been carried out. The Ag-NPs suspension including quasi-spherical Ag-NPs was administrated intravenously (in the tail vein for Ag-NPs of 9+2 nm and 94+10 nm), and for Ag-NPs of 94 nm diameter it was also administrated orally and intratracheally. The organ recovery was made in 24 h following the administration (for three types of administration) and one more time in 120 h in case of intravenous administration. Radioactive label in the nuclei of silver was created by irradiation of Ag-NPs suspensions in a flow of reactor thermal neutrons, and its share was 5.6 ∙ 10-7 of the total number of silver atoms. This fact could not affect the overall physical and chemical properties of radiolabeled Ag-NPs. We measured the activity of the 110mAg isotope-label in samples of blood and brain of rats, while the activity of 59Fe isotope was measured after the exposure to samples of these organs.Given the fact that iron is contained mainly in the hemoglobin of blood, on the basis of the measurement of 59Fe activity being the induced by neutron flux, we derived an evaluation of the residual mass of blood in brain capillaries using a standard procedure to prepare the samples. This determination is 0.058±0.010 g, and on average this is about 0 37±0.09% of the total mass of blood in rats. The estimated ratio of the Ag-NPs concentration in brain samples of rats (minus the Ag-NPs number in residual blood capillaries) to their peripheral blood concentration for 9 nm Ag-NPs is 0.16±0.04 and 0.31±0.07, and for 94 nm Ag-NPs - 0.20±0.05 and 0.29±0.07 for times in 24 and 120 hours after intravenous administration, respectively. For Ag-NPs of 9 nm and 94 nm diameter we revealed no significant effect of the Ag-NPs size on the value of this ratio. The same ratio for Ag-NPs of 94 nm diameter in 24 h after the oral and intratracheal administration is 0.29+0.09 and 0.41+0.12, respectively.

RAPD Profiling, DNA Fragmentation, and Histomorphometric Examination in Brains of Wistar Rats Exposed to Indoor 2.5 Ghz Wi-Fi Devices Radiation.

The advent of Wi-Fi connected high technology devices in executing day-to-day activities is fast evolving especially in developing countries of the world and hence the need to assess its safety among others. The present study was conducted to investigate the injurious effect of radiofrequency emissions from installed Wi-Fi devices in brains of young male rats. Animals were divided into four equal groups; group 1 served as control while groups 2, 3, and 4 were exposed to 2.5 Ghz at intervals of 30, 45, and 60 consecutive days with free access to food and water ad libitum. Alterations in harvested brain tissues were confirmed by histopathological analyses which showed vascular congestion and DNA damage in the brain was assayed using agarose gel electrophoresis. Histomorphometry analyses of their brain tissues showed perivascular congestion and tissue damage as well.

Reduction of intracerebral hemorrhage by rivaroxaban after tPA thrombolysis is associated with downregulation of PAR-1 and PAR-2.

This study aimed to assess the risk of intracerebral hemorrhage (ICH) after tissue-type plasminogen activator (tPA) treatment in rivaroxaban compared with warfarin-pretreated male Wistar rat brain after ischemia in relation to activation profiles of protease-activated receptor-1, -2, -3, and -4 (PAR-1, -2, -3, and -4). After pretreatment with warfarin (0.2 mg/kg/day), low-dose rivaroxaban (60 mg/kg/day), high-dose rivaroxaban (120 mg/kg/day), or vehicle for 14 days, transient middle cerebral artery occlusion was induced for 90 min, followed by reperfusion with tPA (10 mg/kg/10 ml). Infarct volume, hemorrhagic volume, immunoglobulin G leakage, and blood parameters were examined. Twenty-four hours after reperfusion, immunohistochemistry for PARs was performed in brain sections. ICH volume was increased in the warfarin-pretreated group compared with the rivaroxaban-treated group. PAR-1, -2, -3, and -4 were widely expressed in the normal brain, and their levels were increased in the ischemic brain, especially in the peri-ischemic lesion. Warfarin pretreatment enhanced the expression of PAR-1 and PAR-2 in the peri-ischemic lesion, whereas rivaroxaban pretreatment did not. The present study shows a lower risk of brain hemorrhage in rivaroxaban-pretreated compared with warfarin-pretreated rats following tPA administration to the ischemic brain. It is suggested that the relative downregulation of PAR-1 and PAR-2 by rivaroxaban compared with warfarin pretreatment might be partly involved in the mechanism of reduced hemorrhagic complications in patients receiving rivaroxaban in clinical trials. © 2016 Wiley Periodicals, Inc.

CUR-CA-THIONE: A NOVEL CURCUMIN CONCOCTION WITH ENHANCED WATER SOLUBILITY AND BRAIN BIO-AVAILABILITY

<p><strong>Objective: </strong>Curcumin, is widely studied as a potential drug in treating various disorders but lacks applicability due to poor water solubility and tissue bioavailability. The main objective of the study was to develop a formulation of curcumin that has enhanced water solubility and brain bioavailability.</p><p><strong>Methods: </strong>A curcumin concoction was prepared using solvent evaporation technique taking casein and glutathione as vectors. Various process parameters were identified namely time, temperature, pH and vector while formulation parameters included drug entrapment, anti-oxidant activity, and water solubility. The concoctions were evaluated for <em>in vitro</em> release kinetics at three pH i.e. 1.2, 4.5 and 6.2 at six-time intervals i.e. 10, 20, 30, 40, 60, 120 min using dialysis bag membrane. The same kinetics was further validated using same time points with wistar rats and giving concoction at a single dose of 2 g/kg via the oral route.</p><p><strong>Results: </strong>A concoction i.e. CUR-CA-THIONE having significant entrapment efficiency (77.83%, 97.75%, 90.19%), water solubility (40, 350 and 45 times than normal curcumin) and DPPH activity (IC<sub>50</sub>: 28.91, 25.07 and 27.89) was evaluated in concoctions CUR-CA-THIONE-T.1, CUR-CA-THIONE-T.2 and CUR-CA-THIONE-T.3 respectively. These formulations were then carried out for <em>in vitro</em> release profile at different pH with average release obtained between 20-30 min. <em>In vivo</em> kinetics was studied by isolating tissues like brain, liver, lung, kidney and spleen in male wistar rats and maximum brain bioavailability was observed for CUR-CA-THIONE-T.3 at 30 min with 75 ng/g of brain tissue.</p><p><strong>Conclusion: </strong>The experiment helps in concluding that CUR-CA-THIONE has improved its water solubility and is able to by-pass systemic circulation to targeted activity.</p>

Redox changes in the brains of reproductive female rats during aging

Reproduction is a critical and demanding phase of an animal's life. In mammals, females usually invest much more in parental care than males, and lactation is the most energetically demanding period of a female's life. Here, we tested whether oxidative stress is a consequence of reproduction in the brains of female Wistar rats. We evaluated the activities of glutathione peroxidase, glutathione S-transferase, and superoxide dismutase; H2O2 consumption; protein carbonylation; NO2 & NO3 levels; and total glutathione, as well as sex hormone levels in brain tissue of animals at 3, 6, 12, and 24months of age. Animals were grouped according to reproductive experience: breeders or non-breeders. Most of the studied parameters showed a difference between non-breeders and breeders at 12 and 24months. At 24months of age, breeders showed higher superoxide dismutase activity, H2O2 consumption, glutathione peroxidase activity, and carbonyl levels than non-breeders. In 12-month-old non-breeders, we observed a higher level of H2O2 consumption and higher superoxide dismutase and glutathione peroxidase activities than breeders. By evaluating the correlation network, we found that there were a larger number of influential nodes and positive links in breeder animals than in non-breeders, indicating a greater number of redox changes in breeder animals. Here, we also demonstrated that the aging process caused higher oxidative damage and higher antioxidant defenses in the brains of breeder female rats at 24months, suggesting that the reproduction process is costly, at least for the female brain. This study shows that there is a strong potential for a link between the cost of reproduction and oxidative stress.

HDAC Inhibitor Sodium Butyrate-Mediated Epigenetic Regulation Enhances Neuroprotective Function of Microglia During Ischemic Stroke

Cerebral ischemia leads to neuroinflammation and activation of microglia which further contribute to stroke pathology. Understanding regulation of microglial activation will aid in the development of therapeutic strategies that mitigate microglia-mediated neurotoxicity in neuropathologies, including ischemia. In this study, we investigated the epigenetic regulation of microglial activation by studying histone modification histone 3-lysine 9-acetylation (H3K9ac) and its regulation by histone deacetylase (HDAC) inhibitors. In vitro analysis of activated microglia showed that HDAC inhibitor, sodium butyrate (SB), alters H3K9ac enrichment and transcription at the promoters of pro-inflammatory (Tnf-α, Nos2, Stat1, Il6) and anti-inflammatory (Il10) genes while inducing the expression of genes downstream of the IL10/STAT3 anti-inflammatory pathway. In an experimental mouse (C57BL/6NTac) model of middle cerebral artery occlusion (MCAO), we observed that SB mediates neuroprotection by epigenetically regulating the microglial inflammatory response, via downregulating the expression of pro-inflammatory mediators, TNF-α and NOS2, and upregulating the expression of anti-inflammatory mediator IL10, in activated microglia. Interestingly, H3K9ac levels were found to be upregulated in activated microglia distributed in the cortex, striatum, and hippocampus of MCAO mice. A similar upregulation of H3K9ac was detected in lipopolysaccharide (LPS)-activated microglia in the Wistar rat brain, indicating that H3K9ac upregulation is consistently associated with microglial activation in vivo. Altogether, these results show evidence of HDAC inhibition being a promising molecular switch to epigenetically modify microglial behavior from pro-inflammatory to anti-inflammatory which could mitigate microglia-mediated neuroinflammation.

Oxidative stress in rat brain but not in liver following oral administration of a low dose of nanoparticulate silver

While it is known that silver nanoparticles (AgNPs) can enter the brain, our knowledge of AgNP-induced neurotoxicity remains incomplete. We investigated the ability of 10 nm citrate-stabilized AgNPs to generate oxidative stress in brain and liver of adult male Wistar rats after repeated oral exposure for 14 days, using a low dose of 0.2 mg/kg b.w. as compared with the same dose of ionic silver (silver citrate). In AgNP-exposed animals, the level of reactive oxygen species (ROS), lipid peroxidation (MDA) and glutathione peroxidase (GPx) activity were found to be significantly higher in brain relative to the control group receiving saline. Administration of ionic silver (silver citrate) increased ROS and MDA levels in both tissues. Activities of GPx in brain so as superoxide dismutase (SOD) and catalase (CAT) in liver of exposed animals were also elevated. Besides, AgNPs and silver ions were both found to cause statistically significant decrease in the reduced-to-oxidized glutathione ratio (GSH/GSSG) in brain. The results show that exposure to a very low dose of particulate silver generates mild oxidative stress in the brain but not in the liver of rats, indicating a role of oxidative stress in AgNP-induced neurotoxicity.

The effect of titanium dioxide nanoparticles on neuroinflammation response in rat brain

Titanium dioxide nanoparticles (TiO2 NPs) are widely used for their whiteness and opacity in several applications such as food colorants, drug additives, biomedical ceramic, and implanted biomaterials. Research on the neurobiological response to orally administered TiO2 NPs is still limited. In our study, we investigate the effects of anatase TiO2 NPs on the brain of Wistar rats after oral intake. After daily intragastric administration of anatase TiO2 NPs (5-10nm) at 0, 50, 100, and 200mg/kg body weight (BW) for 60days, the coefficient of the brain, acethylcholinesterase (AChE) activities, the level of interleukin 6 (IL-6), and the expression of glial fibrillary acidic protein (GFAP) were assessed to quantify the brain damage. The results showed that high-dose anatase TiO2 NPs could induce a downregulated level of AChE activities and showed an increase in plasmatic IL-6 level as compared to the control group accompanied by a dose-dependent decrease inter-doses, associated to an increase in the cerebral IL-6 level as a response to a local inflammation in brain. Furthermore, we observed elevated levels of immunoreactivity to GFAP in rat cerebral cortex. We concluded that oral intake of anatase TiO2 NPs can induce neuroinflammation and could be neurotoxic and hazardous to health.

EFFECTS OF AGING AND 17B-ESTRADIOL ON GLUCOSE TRANSPORTER (GLUT3) AND MEMBRANE FUNCTIONS IN FEMALE RAT BRAIN: A THERAPEUTIC POTENTIAL DRUG FOR ALZHEIMER’S DISEASE

Recently, there has been a growing interest in the action and functions of the ovarian steroid hormone estradiol, particularly on whether they are neuroprotective for such age related disease and neurodegenerative conditions like stroke, Parkinson's disease and Alzheimer's disease. The objective of this study was to observe the changes in activities of mitochondrial enzymes (monoamine oxidase (MAO), Na+K+ATPase and Ca2+ATPase), membrane fluidity, DNA degradation, and glucose transporter 4 (GLUT3) expression in brains of female Wistar rats of 3 months (young), 12 months (adult) and 24 months (old) age groups, and to see whether these changes are restored to normal levels after exogenous administration of 17β-estradiol (E2) (0.1 μg/gm body weight for one month). Controls animals received an equal volume of vehicle. After 30 days of hormone treatment, experimental animals of all the groups were sacrificed and brains were isolated for further study. Whole brains were rapidly excised, and washed with chilled normal saline. Tissue homogenates (1:10) were prepared in homogenizing buffer containing 0.25 M sucrose 0.02 M triethanolamine (pH 7.4) and 0.12 mM dithiothreitol. The pellet obtained after centrifugation at 12,000 rpm using SM 22 rotor on a high speed cooling centrifuge (SORVALL 5CA) containing crude synaptosomes (mitochondria and synaptosomes). The results obtained in the present work revealed that normal aging was associated with significant decrease in the activity of Na+K+ATPase, Ca2+ATPase and membrane fluidity, GLUT3 levels in the brains of aging female rats, and an increase in DNA degradation and MAO activity. The present study showed that estradiol treatment significantly decreased DNA degradation, and MAO activity in brain of aging rats, and a reversal of Na+K+ATPase, Ca2+ATPase and GLUT3 levels was achieved. It can therefore be concluded that estradiol's beneficial effects seemed to arise from its antilipofuscin, antioxidant, antilipidperoxidative effects, implying an overall anti-aging action. The results of this study will be useful for pharmacological modification of the aging process and applying new strategies for control of age related disorders.

Resistance to DNA Damaging Agents Produced Invasive Phenotype of Rat Glioma Cells-Characterization of a New in Vivo Model.

Chemoresistance and invasion properties are severe limitations to efficient glioma therapy. Therefore, development of glioma in vivo models that more accurately resemble the situation observed in patients emerges. Previously, we established RC6 rat glioma cell line resistant to DNA damaging agents including antiglioma approved therapies such as 3-bis(2-chloroethyl)-1-nitrosourea (BCNU) and temozolomide (TMZ). Herein, we evaluated the invasiveness of RC6 cells in vitro and in a new orthotopic animal model. For comparison, we used C6 cells from which RC6 cells originated. Differences in cell growth properties were assessed by real-time cell analyzer. Cells’ invasive potential in vitro was studied in fluorescently labeled gelatin and by formation of multicellular spheroids in hydrogel. For animal studies, fluorescently labeled cells were inoculated into adult male Wistar rat brains. Consecutive coronal and sagittal brain sections were analyzed 10 and 25 days post-inoculation, while rats’ behavior was recorded during three days in the open field test starting from 25th day post-inoculation. We demonstrated that development of chemoresistance induced invasive phenotype of RC6 cells with significant behavioral impediments implying usefulness of orthotopic RC6 glioma allograft in preclinical studies for the examination of new approaches to counteract both chemoresistance and invasion of glioma cells.

Memory deficit, toxic effects and activity of Na(+), K(+)-ATPase and NTPDase in brain of Wistar rats submitted to orally treatment with alpha-terpinene.

The neurotoxic effects and activity of Na(+), K(+)-ATPase and NTPDase in Wistar rats after treatment with α-terpinene (daily oral administration of 0.5, 0.75 and 1.0mLkg(-1) for 10days) were examined. Results of the inhibitory avoidance task showed a memory deficit (p<0.05) in rats treated with all doses of α-terpinene. The evaluation of DNA damage in brain tissue revealed an increase (p<0.05) on frequency of damage and damage index in all concentrations. According to the cytotoxicity assay, doses of 0.5, 0.75 and 1.0mLkg(-1) increase the lactate dehydrogenase levels, and doses of 1.0mLkg(-1) also decrease (p<0.05) cell viability in brain cells. A decrease (p<0.05) on Na(+), K(+)-ATPase activity in brain tissue and on NTPDase activity in serum were observed in all concentrations of α-terpinene. These results suggest that the α-terpinene was cytotoxic and genotoxic to the brain cells by inducing loss of cell viability and DNA damage, as well as causing alterations in Na(+), K(+)-ATPase and NTPDase activity, what may contribute to the memory deficit of treated animals. Thus, α-terpinene cannot be consumed by the population at the doses studied.

Relationships between Mechanical Nociceptive Threshold and Activity of Antioxidant Enzymes in Male Rats with Experimental Type I Diabetes Mellitus.

We analyzed the dynamics of neuropathic pain development and changes in catalase and superoxide dismutase (SOD) activities in the brain, liver, and skeletal muscles of male Wistar rats with 1-month streptozotocin-induced diabetes mellitus. A decrease in mechanical nociceptive threshold was revealed that progressed during the disease progress. Insulin treatment restored nociceptive threshold in diabetic animals to the control values. Catalase activity in the liver and skeletal muscles of diabetic rats increased by 1.5 and 2 times, respectively, in comparison with the control, while insulin treatment reduced enzyme activity to the control level. In the brain, catalase activity was reduced by 1.5 times and insulin therapy did affect this parameter. SOD activity in the studied tissues remained unchanged during diabetes and was not affected by insulin therapy. A strong negative correlation between nociceptive threshold in rats and catalase activity in their liver and skeletal muscles was found.

Concomitants of alcoholism: differential effects of thiamine deficiency, liver damage, and food deprivation on the rat brain in vivo.

Serious neurological concomitants of alcoholism include Wernicke's encephalopathy (WE), Korsakoff's syndrome (KS), and hepatic encephalopathy (HE). This study was conducted in animal models to determine neuroradiological signatures associated with liver damage caused by carbon tetrachloride (CCl4), thiamine deficiency caused by pyrithiamine treatment, and nonspecific nutritional deficiency caused by food deprivation. Magnetic resonance imaging (MRI) and magnetic resonance spectroscopy (MRS) were used to evaluate brains of wild-type Wistar rats at baseline and following treatment. Similar to observations in ethanol (EtOH) exposure models, thiamine deficiency caused enlargement of the lateral ventricles. Liver damage was not associated with effects on cerebrospinal fluid volumes, whereas food deprivation caused modest enlargement of the cisterns. In contrast to what has repeatedly been shown in EtOH exposure models, in which levels of choline-containing compounds (Cho) measured by MRS are elevated, Cho levels in treated animals in all three experiments (i.e., liver damage, thiamine deficiency, and food deprivation) were lower than those in baseline or controls. These results add to the growing body of literature suggesting that MRS-detectable Cho is labile and can depend on a number of variables that are not often considered in human experiments. These results also suggest that reductions in Cho observed in humans with alcohol use disorder (AUD) may well be due to mild manifestations of concomitants of AUD such as liver damage or nutritional deficiencies and not necessarily to alcohol consumption per se.

Analysis of protein phosphorylation in nerve terminal reveals extensive changes in active zone proteins upon exocytosis.

Neurotransmitter release is mediated by the fast, calcium-triggered fusion of synaptic vesicles with the presynaptic plasma membrane, followed by endocytosis and recycling of the membrane of synaptic vesicles. While many of the proteins governing these processes are known, their regulation is only beginning to be understood. Here we have applied quantitative phosphoproteomics to identify changes in phosphorylation status of presynaptic proteins in resting and stimulated nerve terminals isolated from the brains of Wistar rats. Using rigorous quantification, we identified 252 phosphosites that are either up- or downregulated upon triggering calcium-dependent exocytosis. Particularly pronounced were regulated changes of phosphosites within protein constituents of the presynaptic active zone, including bassoon, piccolo, and RIM1. Additionally, we have mapped kinases and phosphatases that are activated upon stimulation. Overall, our study provides a snapshot of phosphorylation changes associated with presynaptic activity and provides a foundation for further functional analysis of key phosphosites involved in presynaptic plasticity.

Abnormal Injury Response in Spontaneous Mild Ventriculomegaly Wistar Rat Brains: A Pathological Correlation Study of Diffusion Tensor and Magnetization Transfer Imaging in Mild Traumatic Brain Injury.

Spontaneous mild ventriculomegaly (MVM) was previously reported in ∼43% of Wistar rats in association with vascular anomalies without phenotypic manifestation. This mild traumatic brain injury (TBI) weight drop model study investigates whether MVM rats (n = 15) have different injury responses that could inadvertently complicate the interpretation of imaging studies compared with normal rats (n = 15). Quantitative MRI, including diffusion tensor imaging (DTI) and magnetization transfer imaging (MTI), and immunohistochemistry (IHC) analysis were used to examine the injury pattern up to 8 days post-injury in MVM and normal rats. Prior to injury, the MVM brain showed significant higher mean diffusivity, axial diffusivity, and radial diffusivity, and lower fractional anisotropy (FA) and magnetization transfer ratio (MTR) in the corpus callosum than normal brain (p < 0.05). Following TBI, normal brains exhibited significant decreases of FA in the corpus callosum, whereas MVM brains demonstrated insignificant changes in FA, suggesting less axonal injury. At day 8 after mild TBI, MTR of the normal brains significantly decreased whereas the MTR of the MVM brains significantly increased. IHC staining substantiated the MRI findings, demonstrating limited axonal injury with significant increase of microgliosis and astrogliosis in MVM brain compared with normal animals. The radiological-pathological correlation data showed that both DTI and MTI were sensitive in detecting mild diffuse brain injury, although DTI metrics were more specific in correlating with histologically identified pathologies. Compared with the higher correlation levels reflecting axonal injury pathology in the normal rat mild TBI, the DTI and MTR metrics were more affected by the increased inflammation in the MVM rat mild TBI. Because MVM Wistar rats appear normal, there was a need to screen rats prior to TBI research to rule out the presence of ventriculomegaly, which may complicate the interpretation of imaging and IHC observations.

The application of Raman microspectroscopy for the study of healthy rat brain tissue

The Raman spectra of three areas of Wistar rat brain tissue: the somatosensory cortex (Sc), the dorsal lateral geniculate nucleus of the thalamus (DLG) and the cerebellar cortex (Cc), excited with 785nm and 442nm laser line, were obtained. The no fixation method to arrange the samples was adopted, and fresh brain scraps were kept in artificial cerebrospinal fluid during the measurement. The CH stretching vibrations region was used to probe the state of the lipids and their arrangement in the studied brain areas. The obtained spectra were discussed by analyzing differences in particular spectral regions by evaluating changes in band intensity ratios as well as using principal component analysis (PCA). The I2880/I2850 intensity ratio of the model unsaturated fatty acids, linoleic (LA) and the docosahexaenoic acid (DHA) were treated as a model of lipid composition and used for a comparison because of their similarity. The I2880/I2850 intensity ratio allowed us to investigate the differences in the microenvironment of the studied areas of the brain. The I2930/I2880 intensity ratio showed the specificity of the lipids disorder because they are a mixture of several hydrocarbon chains of different acyl region compositions. These differences were consistent with the lipid molecular architecture in the studied regions as shown in the PCA loadings plot. Some protein also seemed to differentiate the explored areas of the brain. The obtained results demonstrate that Raman spectroscopy can provide insight into healthy brain tissue composition and structure.

A Search for Mitochondrial Damage in Alzheimer's Disease Using Isolated Rat Brain Mitochondria.

Alzheimer's disease (AD) is a progressive neurodegenerative disorder that affects regions of the brain that control cognition, memory, language, speech and awareness to one's physical surroundings. The pathological initiation and progression of AD is highly complex and its prevalence is on the rise. In his study, Alzheimer's disease was induced with single injection of amyloid-β (Aβ) peptides (30ng, by stereotaxy) in each hemisphere of the Wistar rat brain. Then memory dysfunction, oxidative stress and apoptosis induced by Aβ peptide were investigated on isolated brain mitochondria obtained from infected rat. Our results showed memory impairment in rats after receiving an Aβ peptide. We also found significant rise (P<0.05) at ROS formation, mitochondrial membrane depolarization, mitochondria swelling, cytochrome c release and significant decrease in ATP/ADP ratio on mitochondria isolated from brain of these memory impaired rats compared with those of untreated control rat group. Activation of caspase-3 the final mediator of apoptosis in the brain homogenate of the memory impaired rats was another justification for occurrence of neuron loss in the experimental model of AD. Our results suggest that oxidative stress and mitochondria mediated apoptosis in brain neurons play very important role in initiation of AD.