Brains Of Sprague-Dawley Rats Research Articles

Induction of Neuroinflammation and Brain Oxidative Stress by Brain-Derived Extracellular Vesicles from Hypertensive Rats.

Neuroinflammation and brain oxidative stress are recognized as significant contributors to hypertension including salt sensitive hypertension. Extracellular vesicles (EVs) play an essential role in intercellular communication in various situations, including physiological and pathological ones. Based on this evidence, we hypothesized that EVs derived from the brains of hypertensive rats with salt sensitivity could trigger neuroinflammation and oxidative stress during hypertension development. To test this hypothesis, we compared the impact of EVs isolated from the brains of hypertensive Dahl Salt-Sensitive rats (DSS) and normotensive Sprague Dawley (SD) rats on inflammatory factors and mitochondrial reactive oxygen species (mtROS) production in primary neuronal cultures and brain cardiovascular relevant regions, including the hypothalamic paraventricular nucleus (PVN) and lamina terminalis (LT). We found that brain-derived DSS-EVs significantly increased the mRNA levels of proinflammatory cytokines (PICs) and chemokines, including TNFα, IL1β, CCL2, CCL5, and CCL12, as well as the transcriptional factor NF-κB in neuronal cultures. DSS-EVs also induced oxidative stress in neuronal cultures, as evidenced by elevated NADPH oxidase subunit CYBA coding gene mRNA levels and persistent mtROS elevation. When DSS-EVs were injected into the brains of normal SD rats, the mRNA levels of PICs, chemokines, and the chronic neuronal activity marker FOSL1 were significantly increased in the PVN and LT. Furthermore, DSS-EVs caused mtROS elevation in brain PVN and LT, particularly in neurons. Our study reveals a novel role for brain-derived EVs from hypertensive rats in triggering neuroinflammation, upregulating chemokine expression, and inducing excessive ROS production. These findings provide insight into the complex interactions between EVs and hypertension-associated processes, offering potential therapeutic targets for hypertension-linked neurological complications.

Abstract TP295: Temporal Characterization of Immune Responses in the Rat Brain Following Ischemic Stroke Using In-Depth Flow Cytometry Panels

Introduction: Rat models are widely used in stroke research as they are well suited for behavior studies and brain imaging techniques. However, methods for studying immune responses and identifying distinct leukocyte subsets in rats lag behind mice in part due to lack of specific antibodies for flow cytometry. Several flow cytometry antibody panels have been used to study the immune response of the rat brain after stroke, but they did not distinguish between pro- and anti-inflammatory macrophages, which is critical for stroke-induced neuroinflammation research. Here, we developed flow cytometry panels to measure myeloid and lymphoid leukocyte subsets in rat brain, blood, and spleen, with an emphasis on macrophage subtypes, and validated them in an ischemic stroke model. Method: Adult male (8-12 wk old) Nude and Sprague Dawley (SD) rats were subjected to distal MCAO. Ipsi- and contralateral hemispheres, blood, and spleen were collected and analyzed by flow cytometry at different time points post-stroke. Cell populations were sorted using FACS and characterized using qPCR or morphological analysis to confirm our FACS gating strategy. Results: We discriminated microglia, 3 monocyte/macrophage subsets, granulocytes, dendritic cells, NK cells, B cells, and T cells in the brain and periphery. The temporal profile of leukocyte subsets after stroke resembled those observed in mice, validating the reliability of our antibody panels: granulocytes and monocytes peaked in the first 2-3 days post-stroke, B and T cells peaked around 4-5 days, followed by a gradual increase of ‘microglia’ over 14 days. Using a combination of CD43 and His48 to differentiate monocyte/macrophage subpopulations, we observed a shift in these subsets over time, akin to the shift from pro- to anti-inflammatory macrophages reported in stroke-injured mouse brains. Notably, Nude rats differed from the SD rats in terms of His48 expression; SD rat brain monocyte/macrophage population was predominantly His48lo, while Nude rats had both His48lo and His48hi subsets. Conclusion: Our flow cytometry panels for rats distinguished leukocyte subsets, including monocyte/macrophage subpopulations, providing a valuable tool to study the complex immune responses in rat models of neurological disorders.

In vitro and in vivo evaluation of nanoliposomes loading quercetin and 3-bromopyruvate against glioma

BackgroundDiffuse astrocytoma (a type of glioma) and its prevalence are matters of concern worldwide. Patients with this type of tumour have a poor prognosis because after surgical treatment, radiotherapy and/or chemotherapy, these tumours eventually regrow or progress. To date, there is no effective treatment that can cure affected patients. Quercetin and 3-bromopyruvate are chemical compounds that have been proven to have antitumour effects alone or in combination with other compounds. Nevertheless, combination treatments including these agents are not used for treating diffuse astrocytoma.MethodsThe use of nanoliposomes loaded with quercetin and 3-bromopyruvate as combination therapy was evaluated by treating C6 cells in vitro and in vivo (in Sprague–Dawley rat brain).ResultsThe 0.5 mg/mL quercetin + 0.75 mg/mL 3-bromopyruvate combination treatment decreased the expression of the biomarkers Annexin V and Caspase-3 and inhibited tumour growth; this was consistent with the in vivo results that revealed the administration of this treatment resulted in improved animal survival.ConclusionsThe observations in the present study support the further exploration of this combination of active agents in the treatment of high-grade diffuse astrocytoma, especially in cases for which wide resection is possible.

The effect of Centella asiatica, cinnamon, and spirulina as neuroprotective based on histopathological findings in ratus Sprague Dawley with traumatic brain injury.

Traumatic brain injury (TBI) is a global health problem with the potential to cause dangerous neurological problems. Based on histopathological findings in Sprague Dawley (SD) rats with TBI in the acute phase, the study seeks to discover the effect of Centella asiatica, cinnamon, and spirulina as neuroprotective. We conducted an experimental study with 30 SD rats randomly divided into three groups. The intervention was the administration of C. asiatica, cinnamon, and spirulina to the control and the experimental groups. Histological features were assessed using hematoxylin and eosin (H&E) staining and immunohistochemical examination. The data were analyzed using statistical analysis through correlation tests. The test samples' average body weights had P > 0.05, indicating no significant difference in the test sample body weights. Therefore, the variations in the expression level of the dependent variable were expected to be caused by the induction of brain injury and the administration of C. asiatica, cinnamon, and spirulina. In addition, the variables were not normally distributed. Thus, the Spearman test was carried out and showed the correlation was very strong, with a value of r = 0.818 and P < 0.05. Based on histopathological findings from the brains of SD rats with TBI, pegagan, cinnamon, and spirulina will protect the brain (neuroprotective) in the acute phase.

Thymol abates the detrimental impacts of imidacloprid on rat brains by lessening oxidative damage and apoptotic and inflammatory reactions

Imidacloprid (IMID) is one of the most widely used neonicotinoid insecticides globally and, consequently, a probable widespread environmental contaminant. The potential neurotoxic effects of IMID have been previously reported. This study aimed to investigate the possible beneficial effect of thymol (TML) in relieving IMID-induced harmful effects on the brain of male Sprague-Dawley rats. For this aim, four groups (10 rats/group) were orally administered corn oil, TML (30 mg/kg b.wt), IMID (22.5 mg/kg b.wt), or TML + IMID for 56 days. The brain tissues were biochemically, histopathologically, and immunohistochemically evaluated. The results displayed that TML significantly restored the IMID-induced depletion of the total antioxidant capacity of the brain tissues. At the same time, the IMID-associated increased levels of lipid peroxidation in terms of malondialdehyde content were markedly suppressed in the TML + IMID group. Also, TML oral dosing markedly reduced the release of inflammatory elements, including nitric oxide and myeloperoxidase, resulting from IMID exposure. Furthermore, the IMID-induced decrease in gamma-aminobutyric acid but the increase in acetylcholinesterase was considerably reversed by TML oral dosing. Additionally, TML oral administration significantly counteracted the IMID-induced brainepatic DNA damage, as revealed by the comet assay. Besides, a significant downregulatibrainepatic Caspase-3 was evident in the TML + IMID group compared to the IMID group. However, TML oral dosing has not significantly altered the IMID-induced nuclear factor (NF-κB p65) increase. Therefore, TML could be a protective agent against IMID-induced detrimental impacts on brain tissue, possibly through its antioxidant, antiapoptotic, and anti-inflammatory activities.

Cannabigerol modulates α2-adrenoceptor and 5-HT1A receptor-mediated electrophysiological effects on dorsal raphe nucleus and locus coeruleus neurons and anxiety behavior in rat.

The pharmacological profile of cannabigerol (CBG), which acid form constitutes the main precursor of the most abundant cannabinoids, has been scarcely studied. It has been reported to target α2-adrenoceptor and 5-HT1A receptor. The locus coeruleus (LC) and the dorsal raphe nucleus (DRN) are the main serotonergic (5-HT) and noradrenergic (NA) areas in the rat brain, respectively. We aimed to study the effect of CBG on the firing rate of LC NA cells and DRN 5-HT cells and on α2-adrenergic and 5-HT1A autoreceptors by electrophysiological techniques in male Sprague-Dawley rat brain slices. The effect of CBG on the novelty-suppressed feeding test (NSFT) and the elevated plus maze test (EPMT) and the involvement of the 5-HT1A receptor was also studied. CBG (30μM, 10min) slightly changed the firing rate of NA cells but failed to alter the inhibitory effect of NA (1-100µM). However, in the presence of CBG the inhibitory effect of the selective α2-adrenoceptor agonist UK14304 (10nM) was decreased. Perfusion with CBG (30μM, 10min) did not change the firing rate of DRN 5-HT cells or the inhibitory effect of 5-HT (100μM, 1min) but it reduced the inhibitory effect of ipsapirone (100nM). CBG failed to reverse ipsapirone-induced inhibition whereas perfusion with the 5-HT1A receptor antagonist WAY100635 (30nM) completely restored the firing rate of DRN 5-HT cells. In the EPMT, CBG (10mg/kg, i.p.) significantly increased the percentage of time the rats spent on the open arms and the number of head-dipping but it reduced the anxiety index. In the NSFT, CBG decreased the time latency to eat in the novel environment but it did not alter home-cage consumption. The effect of CBG on the reduction of latency to feed was prevented by pretreatment with WAY100635 (1mg/kg, i.p.). In conclusion, CBG hinders the inhibitory effect produced by selective α2-adrenoceptor and 5-HT1A receptor agonists on the firing rate of NA-LC and 5-HT-DRN neurons by a yet unknown indirect mechanism in rat brain slices and produces anxiolytic-like effects through 5-HT1A receptor.

Classification of Activated Microglia by Convolutional Neural Networks.

Microglia are the resident macrophages in the central nervous system. Brain injuries, such as traumatic brain injury, hypoxia, and stroke, can induce inflammatory responses accompanying microglial activation. The morphology of microglia is notably diverse and is one of the prominent manifestations during activation. In this study, we proposed to detect the activated microglia in immunohistochemistry images by convolutional neural networks (CNN). 2D Iba1 images (40μm) were acquired from a control and a cardiac arrest treated Sprague-Dawley rat brain by a scanning microscope using a 20X objective. The training data were a collection of 54,333 single-cell images obtained from the cortex and midbrain areas, and curated by experienced neuroscientists. Results were compared between CNNs with different architectures, including Resnet18, Resnet50, Resnet101, and support vector machine (SVM) classifiers. The highest model performance was found by Resnet18, trained after 120 epochs with a classification accuracy of 95.5%. The findings indicate a potential application for using CNN in quantitative analysis of microglial morphology over regional difference in a large brain section.

Terminal field volume of the glossopharyngeal nerve in adult rats reverts to prepruning size following microglia depletion with PLX5622.

Programmed reduction of synapses is a hallmark of the developing brain, with sensory systems emerging as useful models with which to study this pruning. The central projections (terminal field) of the gustatory glossopharyngeal nerve (GL) of the rat are a prime example of developmental pruning, undergoing an approximate 66% reduction in volume from postnatal day 15 (P15) to P25. Later in adulthood, developmental GL pruning can be experimentally reversed, expanding to preweaning volumes, suggesting mature volumes may be actively maintained throughout the life span. Microglia are central nervous system glia cells that perform pruning and maintenance functions in other sensory systems, including other gustatory nerves. To determine their role in GL pruning, we depleted microglia from Sprague-Dawley rat brains from P1 to P40 using daily intraperitoneal injections of the colony-stimulating factor 1 receptor inhibitor PLX5622. This prevented GL developmental pruning, resulting in preweaning terminal field volumes and innervation patterns persisting through P40, 2weeks after pruning is normally completed. These findings show microglia are necessary for developmental GL pruning. Ceasing PLX5622 treatments at P40 allowed microglia repopulation, and within 4weeks the GL terminal field had reduced to control volumes, indicating that pruning can occur outside of the typical developmental period. Conversely, when microglia were depleted in adult rats, GL terminal fields expanded, reverting to sizes comparable to the neonatal rat. These data indicate that microglia are required for GL pruning and may continue to maintain the GL terminal field at a reduced size into adulthood.

Biochemical regulatory processes in the control of oxidants and antioxidants production in the brain of rats with iron and copper chronic overloads.

Iron [Fe(II)] and copper [Cu(II)] overloads in rat brain are associated with oxidative stress and damage. The purpose of this research is to study whether brain antioxidant enzymes are involved in the control of intracellular redox homeostasis in the brain of rats male Sprague-Dawley rats (80-90g) that received drinking water supplemented with either 1.0g/L of ferrous chloride (n = 24) or 0.5g/L cupric sulfate (n = 24) for 42days. Nicotinamide adenine dinucleotide phosphate(NADPH) oxidase, superoxide dismutase (SOD), catalase, glutathione peroxidase (GPx) and glutathione transferase (GT) activities in brain were determined by spectrophotometric methods and NO production by the content of nitrite concentration in the organ. Chronic treatment with Fe(II) and Cu(II) led to a significant decrease of nitrite content and SOD activity in brain. Activity of NADPH oxidase increased with Cu(II) treatment. Concerning Fe(II), catalase and GT activities increased in brain after 28 and 4days of treatment, respectively. In the case of Cu(II), catalase activity decreased whereas GT activity increased after 2 and 14days, respectively. The regulation of redox homeostasis in brain involves changes of the activity of these enzymes to control the steady state of oxidant species related to redox signaling pathways upon Cu and Fe overload. NO may serve to detoxify cells from superoxide anion and hydrogen peroxide with the concomitant formation of peroxynitrite. However, the latest is a powerful oxidant which leads to oxidative modifications of biomolecules. These results suggest a common pathway to oxidative stress and damage in brain for Cu(II) and Fe(II).

Short communication: Tissue distribution of major cannabinoids following intraperitoneal injection in male rats.

Currently, peripheral tissue distribution of cannabinoids after treatment is poorly understood. This pilot study sought to examine the early tissue distribution of major cannabinoids 30 minutes following an intraperitoneal injection of vehicle (1:9 Tween 80/SAL), and doses of THC (1 mg/kg) and CBD (5 mg/kg) that are feasible for human consumption in serum, adipose, brain, lung, liver, jejunum, and muscle of male Sprague-Dawley rats. The jejunum and adipose were most enriched in THC. Similarly, CBD was enriched in the jejunum and adipose but also the liver. In contrast, the brain had the lowest concentration of cannabinoids relative to other tissues. The liver had the greatest concentration of the THC metabolites, 11-OH-THC and COOH-THC, compared to all other tissues. Overall, these findings highlight broad tissue distribution and marked differences in tissue concentration not previously appreciated. Thus, as cannabinoid research continues to rapidly grow, consideration of the potential bioactive effects of these molecules in peripheral tissues is warranted in future studies.

Pathophysiological features in the brains of female Spontaneously Diabetic Torii (SDT) fatty rats.

Diabetes mellitus (DM) and obesity are associated with neurodegenerative diseases such as Alzheimer’s disease and psychiatric disorders such as major depression. In this study, we investigated pathophysiological changes in the brains of female Spontaneously Diabetic Torii (SDT) fatty rats with diabetes and obesity. Brains of Sprague-Dawley (SD), SDT and SDT fatty rats were collected at 58 weeks of age. The parietal cortical thickness was measured and the number of pyramidal cells in the hippocampal cornu ammonis 1 and 3 (CA1 and CA3) and the number of granule cells in the dentate gyrus (DG) regions were counted. The area of glial fibrillary acidic protein (GFAP) positivity in CA1, CA3 and DG regions were measured. The parietal cortical thickness and the number of cells in CA3 and DG regions of SDT and SDT fatty rats did not show obvious changes. On the other hand, in the CA1 region, the number of cells in SDT rats and SDT fatty rats was significantly lower than that in SD rats, and that in SDT fatty rats was significantly lower than that in SDT rats. The GFAP-positive area in SDT fatty rats was significantly reduced compared to that in SD rats only in the DG region. Preliminarily result showed that the expression of S100a9, an inflammation-related gene, was increased in the brains of SDT fatty rats. These results suggest that female SDT fatty rat may exhibit central nervous system diseases due to obesity and DM.

Capping Protein Regulator and Myosin 1 Linker 3 (CARMIL3) as a Molecular Signature of Ischemic Neurons in the DWI-T2 Mismatch Areas After Stroke.

Ischemic stroke with a mismatch between diffusion-weighted imaging (DWI) and fluid-attenuated inversion recovery (FLAIR) or T2-weighted images indicates onset within 4.5 h, but the pathological substrates in the DWI-T2 mismatch and T2(+) areas remain elusive. In this study, proteomics was used to explore (1) the protein expression profiles in the T2(+), mismatch, and contralateral areas, and (2) the protein with the highest expression in the T2(+) area in the brains of male Sprague-Dawley rats within 4.5 h after middle cerebral artery occlusion (MCAO). The expression of the candidate protein was further validated in (1) rat brain subjected to MCAO, (2) rat primary cortical neuronal culture with oxygen-glucose deprivation (OGD), and (3) infarcted human brain tissues. This study showed that apoptosis was observed in the T2(+) and mismatch regions and necroptosis in the T2(+) region of rat brains after MCAO. We identified capping protein regulator and myosin 1 linker 3 (CARMIL3) as the candidate molecule in the T2(+) and mismatch areas, exclusively in neurons, predominantly in the cytoplasm, and most abundant in the mismatch area. The CARMIL3(+) neurons and neurites in the mismatch and T2(+) areas were larger than those in the control area, and associated with (1) increased expression of sulfonylurea receptor 1 (SUR1), indicating edema, (2) accumulation of p62, indicating impaired autophagy, and (3) increase in 8-hydroxy-2′-deoxyguanosine (8-OHdG), indicating oxidative stress. The increased expression of CARMIL3 was validated in a cell model of cortical neurons after OGD and in infarcted human brain tissues. In conclusion, this study shows that the mismatch and T2(+) areas within 4.5 h after ischemia are characterized by upregulated expression of CARMIL3 in neurons, particularly the mismatch area, which is associated with neuronal edema, impaired autophagy, and oxidative stress, indicating that CARMIL3 serves as a molecular signature of brain ischemia.

Nicotine Exposure Along with Oral Contraceptive Treatment in Female Rats Exacerbates Post-cerebral Ischemic Hypoperfusion Potentially via Altered Histamine Metabolism.

Smoking-derived nicotine (N) and oral contraceptives (OCs) synergistically exacerbate ischemic brain damage in the female, and the underlying mechanisms remain elusive. Our published study showed that N toxicity is exacerbated by OC via altered mitochondrial electron transport chain function. Because mitochondria play an important role in cellular metabolism, we investigated the global metabolomic profile of brains of adolescent and adult female Sprague-Dawley rats exposed to N with or without OC (N+/-OC). Rats were randomly exposed to saline or N+/-OC for 16-21days followed by random allocation into two cohorts. The first cohort was used to characterize the cortical metabolome. Pathway enrichment analysis showed a significant increase in several histamine metabolites including 1-methylhistamine, 1-methyl-4-imidazoleacetate, and 1-ribosyl-imidazleacetate, along with carnosine and homocarnosine in adolescent and adult animals treated with N and N+OC in relation to respective saline controls, which may be reflective of altered histamine metabolism with nicotine treatment. We also observed reduced levels of the neurotransmitters N-acetyl-aspartyl-glutamate (NAAG), gamma-aminobutyrate (GABA), and N-methyl-GABA in N+OC treatment in adolescent animals. The second cohort underwent bilateral carotid artery occlusion and hypotension followed by cerebral blood flow (CBF) assessment a day later. Autoradiographic images of the brain 24h after ischemic episodes showed severe reduction in cortical and hippocampal local CBF in N+/-OC-exposed rats compared with saline treated. Because GABA and histamine are critical for CBF maintenance, altered metabolism of these neurotransmitters may be responsible for observed severe post-ischemic hypoperfusion, which in turn exacerbates ischemic brain damage.

Central Cholinergic Synapse Formation in Optimized Primary Septal-Hippocampal Co-cultures.

Septal innervation of basal forebrain cholinergic neurons to the hippocampus is critical for normal learning and memory and is severely degenerated in Alzheimer's disease. To understand the molecular events underlying physiological cholinergic synaptogenesis and remodeling, as well as pathological loss, we developed an optimized primary septal-hippocampal co-culture system. Hippocampal and septal tissue were harvested from embryonic Sprague-Dawley rat brain and cultured together at varying densities, cell ratios, and in the presence of different growth factors. We identified conditions that produced robust septal-hippocampal synapse formation. We used confocal microscopy with primary antibodies and fluorescent ligands to validate that this system was capable of generating developmentally mature cholinergic synapses. Such synapses were comprised of physiological synaptic partners and mimicked the molecular composition of in vivo counterparts. This co-culture system will facilitate the study of the formation, plasticity, and dysfunction of central mammalian cholinergic synapses.

Simultaneous nicotine and oral contraceptive exposure alters brain energy metabolism and exacerbates ischemic stroke injury in female rats

Smoking-derived nicotine (N) and oral contraceptives (OC) synergistically exacerbate ischemic brain damage in the females and underlying mechanisms remain elusive. Our published study showed that N toxicity is exacerbated by OC via altered mitochondrial function owing to a defect in the activity of cytochrome c oxidase. Here, we investigated the global metabolomic profile of brains of adolescent female Sprague-Dawley rats exposed to N ± OC. Rats were randomly exposed to saline or N + /−OC for 16–21 days followed by random allocation into two cohorts. One cohort underwent transient middle cerebral artery occlusion and histopathology was performed 30 days later. From the second cohort, cortical tissues were collected for an unbiased global metabolomic profile. Pathway enrichment analysis showed significant decrease in glucose, glucose 6-phosphate and fructose-6-phosphate, along with a significant increase in pyruvate in the N + /−OC exposed groups when compared to saline (p < 0.05), suggesting alterations in the glycolytic pathway which were confirmed by Western blot analyses of glycolytic enzymes. Infarct volume quantification showed a significant increase following N alone or N + OC as compared to saline control. Because glucose metabolism is critical for brain physiology, altered glycolysis deteriorates neural function, thus exacerbating ischemic brain damage.

A Method for Precision Surgical Implantation and Probe Insertion in Sprague-Dawley Rat Brain

Motorized stereotaxic is an advanced tool for surgery and implantation of cannula and electrodes in neuroscience. Stereotaxic surgery and implantation has become increasingly important tool, applied in many experiments. The goal in this present study to determine the surgical implantation and probe insertion at the target location accurately and precisely using motorized stereotaxic. In this study, the method allowed to evaluate the DHEAS fluorescence level through in vivo imaging approach at the target region in the hippocampus rat brain. This present study also described the surgical implantation and probe insertion by motorized stereotaxic as the precise and accurate techniques than conventional stereotaxic procedures. With the rat brain atlas by Paxinos and Watson (2004), the imaging approach can be evaluated precisely at the target location corresponding surgical implantation and probe insertion techniques.

Resveratrol prevents brain injury and neurogranin expression in rats exposed to gaseous formaldehyde

Objective To explore the effects of gaseous formaldehyde exposure on male Sprague-Dawley rat brain function and synaptic protein expression, as well as the neuroprotective effects of resveratrol. Methods Eighty-four male Sprague-Dawley rats were randomly divided into seven group with 12 in each group: control group (N), gaseous formaldehyde accumulation low concentration group (FL group), gaseous formaldehyde accumulation medium concentration group (FM group), gaseous formaldehyde accumulation high concentration group (FH group), gaseous formaldehyde accumulation low concentration + resveratrol group (FLR group), gaseous formaldehyde accumulation concentration + resveratrol group (FMR group) and gaseous formaldehyde accumulation high concentration + resveratrol group (FHR group). The animal model of gaseous formaldehyde accumulation was established by gas formaldehyde exposure method.Learning and memory function was tested by Morris water maze. Cortical and hippocampal neurogranin (Ng) protein expression in brain cortex and hippocampus was measured by Western blot. Results (1) Compared with the escape latency and original platform retention time of rats in group N ((19.75±4.08) s, (43.47±2.43) s), the escape latency of rats in FM and FH groups was prolonged ((25.52±2.98) s, (27.45±2.39) s, t=-4.97, -5.82; both P<0.01), the retention time of the original platform was shortened ((30.99±4.21) s, (18.70±2.58) s, t=10.24, 25.71; both P<0.01). Compared with the corresponding formaldehyde accumulation groups such as the FM and FH groups, the escape latency of the rats in the FMR and FHR groups was significantly longer ((21.37±3.80) s, (24.26±3.56) s, t=1.86, 3.93; both P<0.05), the retention time of the original platform was significantly prolonged ((40.33±3.63)s, (23.46±4.73)s, t=-7.50, -3.95; both P<0.01). (2) Compared with the expression of Ng in cortex and hippocampus of rats in group N ((0.99±0.03), (1.07±0.03)), the expression of Ng protein in cortex and hippocampus of rats in FM and FH groups was significantly reduced((0.62±0.06), (0.39±0.03), (0.77±0.09), (0.46±0.10), t=11.55, 14.08, 11.51, 13.17; all P<0.01). Compared with the corresponding formaldehyde accumulation groups such as the FM and FH groups, the expressions of Ng in the cortex and hippocampus of the rats in the FMR and FHR groups were significantly increased ((0.94±0.11), (0.48±0.04), (0.95±0.05), (0.60±0.09), t=-5.26, -2.09, -5.32, -2.21; all P<0.05). Conclusion Resveratrol can improve the learning and memory function of rats caused by the accumulation of gaseous formaldehyde.The mechanism may be related with the upregulation of Ng. Key words: Gaseous formaldehyde accumulation; Neurogranin; Learning and memory; Resveratrol; Rat

The subcronic effects of acetamipride on the global DNA methylation levels in Sprague-Dawley rat brain and liver

Acetamiprid, which is a neonicotinoid insecticide, is used to control leafy vegetables, fruiting vegetables, fir seeds, citrus fruits, pome fruits, grapes, cotton and ornamental plants and absorbent insects on flowers. The present study aim to evaluate global DNA methylation and gene expression of DNA methylation related enzymes in liver and brain tissues of male Sprague-Dawley rats after a 90-day subchronic exposure to acetamiprid at low doses of 12.5, 25 and 35 mg/kg body weight (b.w.). Global DNA methylation resulted in a significant decrease in the levels of 5-methylcytosine (5-mC%) at the doses of 25 and 35 mg/kg b.w. in the liver and 35 mg/kg b.w. in the brain compared to the vehicle control group. Consistently, expression of DNA methyltransferase enzymes decreased at doses of 12.5, 25 and 35 mg/kg b.w. in liver and 35 mg/kg b.w. in brain. It has been suggested that non-genotoxic (epigenetic) mechanisms may be involved in the toxicity of acetamiprid and further investigations are needed to elucidate the epigenetic effects of neonicotinoid insecticides.

Abstract 355: Effect of Poloxamer 188 on Rat Brain Isolated Mitochondria After Exposure to Hydrogen Peroxide

Introduction: In a pig model of myocardial infarction (MI), intracoronary delivered Poloxamer (P) 188 significantly reduces ischemia/reperfusion (IR) injury when given immediately upon reperfusion, with improved mitochondrial function as a predominant effect. As mitochondria are heavily damaged during IR, a direct effect of P188 on mitochondria may lead to better therapy options during reperfusion. To show not only a similar reduction of IR injury by P188 in the brain, but also a direct P188 effect on mitochondria, we established an in-vitro model of IR that consists of damaging isolated rat brain mitochondria with hydrogen peroxide (H 2 O 2 ), one component of ischemia, then applying P188, and analyzing mitochondrial function. Methods: Male Sprague-Dawley rat brains were removed, and the mitochondria isolated by differential centrifugation and Percoll gradients, then kept on ice to slow their bioenergetics prior to any experimental treatments. Mitochondria were exposed to 200 μM H 2 O 2 for 10 min at room temperature with slight agitation; controls received no H 2 O 2 . Samples were then diluted ½ with buffer ± P188 (250 μM after dilution) to simulate reperfusion and treatment, and kept at room temperature for 10 further minutes. ATP synthesis was measured in a luminometer using a luciferase enzymatic assay. Oxygen consumption was measured by closed cell respirometry with an oxygen meter. In both assays, Complex I and Complex II were examined; Complex I substrates glutamate and malate, Complex II substrate succinate plus the Complex I inhibitor rotenone. Statistics: Data are expressed as mean ± SEM. One-Way ANOVA, SNK-Test; Kruskal-Wallis-Test; α=0.05, * vs control. Results: In both Complex I and II, mitochondrial function was significantly impaired by H 2 O 2 , with ATP synthesis affected more at Complex I and oxygen consumption affected more at Complex II. Addition of P188 did not provide any significant improvement in mitochondrial function. Conclusions: Although P188 significantly reduced IR injury when given during reperfusion in a pig model of MI, it does not appear to provide direct protection to mitochondria in this in-vitro model. Whether the exposure to H 2 O 2 causes the appropriate injury for P188 to become effective remains to be elucidated.

Distribution of neuronal nitric oxide synthase immunoreactivity in adult male Sprague-Dawley rat brain

Neuronal NOS (nNOS) accounts for most of the NO production in the nervous system that modulates synaptic transmission and neuroplasticity. Although previous studies have selectively described the localisation of nNOS in specific brain regions, a comprehensive distribution profile of nNOS in the brain is lacking. Here we provided a detailed morphological characterization on the rostro-caudal distribution of neurons and fibres exhibiting positive nNOS-immunoreactivity in adult Sprague-Dawley rat brain. Our results demonstrated that neurons and fibres in the brain regions that exhibited high nNOS immunoreactivity include the olfactory-related areas, intermediate endopiriform nucleus, Islands of Calleja, subfornical organ, ventral lateral geniculate nucleus, parafascicular thalamic nucleus, superior colliculus, lateral terminal nucleus, pedunculopontine tegmental nucleus, periaqueductal gray, dorsal raphe nucleus, supragenual nucleus, nucleus of the trapezoid body, and the cerebellum. Moderate nNOS immunoreactivity was detected in the cerebral cortex, caudate putamen, hippocampus, thalamus, hypothalamus, amygdala, and the spinal cord. Finally, low NOS immunoreactivity were found in the corpus callosum, fornix, globus pallidus, anterior commissure, and the dorsal hippocampal commissure. In conclusion, this study provides a comprehensive view of the morphology and localisation of nNOS immunoreactivity in the brain that would contribute to a better understanding of the role played by nNOS in the brain.