Different Regions Of Rat Brain Research Articles

Light at night: effect on the daily clock, learning, memory, cognition, and expression of transcripts in different brain regions of rat.

The rapid increase in urbanization is altering the natural composition of the day-night light ratio. The light/dark cycle regulates animal learning, memory, and mood swings. A study was conducted to examine the effect of different quantity and quality of light at night on the daily clock, learning, memory, cognition, and expression of transcripts in key learning centers. Treatment was similar for experiments one to three. Rats were exposed for 30days to 12h light and 12h dark with a night light of 2lx (dLAN group), 250lx (LL), or without night light (LD). In experiment one, after 28days, blood samples were collected and 2days later, animals were exposed to constant darkness. In experiment two, after 30days of treatment, animals were subjected to various tests involving learning, memory, and cognition. In experiment three, after 30days of treatment, animals were sampled, and transcript levels of brain-derived neurotrophic factor, tyrosine kinase, Growth-Associated Protein 43, Neurogranin, microRNA-132, cAMP Response Element-Binding Protein, Glycogen synthase kinase-3β, and Tumor necrosis factor α were measured in hippocampus, thalamus, and cortex tissues. In experiment four, animals were exposed to night light of 0.019W/m2 but of either red (640nm), green (540nm), or blue (450nm) wavelength for 30days, and similar tests were performed as mentioned in experiment 2. While in experiment five, after 30days of respective wavelength treatments, all animals were sampled for gene expression studies. Our results show that exposure to dLAN and LL affects the daily clock as reflected by altered melatonin secretion and locomotor activity, compromises the learning, memory, and cognitive ability, and alterations in the expression levels of transcripts in the hypothalamus, cortex, and thalamus. The effect is night light intensity dependent. Further, blue light at night has less drastic effects than green and red light. These results could be of the potential use of framing the policies for the use of light at night.

N-acetylcysteine elevates Nrf-2/ARE-regulated antioxidant response and mitochondrial biogenesis in different brain regions of monocrotophos exposed rats

Monocrotophos (MCP) is reported to induce the reactive oxygen species-mediated oxidative stress, which is supposed to be one of main mechanisms of MCP-induced neurotoxicity. The present study depicts the positive role of N-acetylcysteine (NAC) against oxidative damage via nuclear factor-erythroid factor 2-related factor 2 (Nrf-2) pathway and mitochondrial biogenesis in different brain regions of rat. Male Albino Wistar rats were divided into control, NAC-treated, MCP and NAC+MCP-treated groups. An intragastric dose of MCP (0.9 mg/kg b.wt) and NAC (200 mg/kg b.wt) was administered for 28 days. Results showed that MCP significantly decreased the activities of mitochondrial complexes, altered the gene expression of Nrf-2/ARE-mediated phase-II detoxifying enzymes and mitochondrial biogenesis factors (peroxiredoxin 1, glutamate-cysteine ligase catalytic subunit, heme oxygenase, NAD(P)H: quinine oxidoreductase 1, peroxisome proliferator-activated receptor gamma coactivator 1-alpha, mitochondrial transcription factor A, nuclear respiratory factors-1 and -2 and manganese superoxide dismutase). The co-administration of NAC modulated these altered mitochondrial biochemicals, restored the gene expressions of phase-II enzymes and mitochondrial biogenesis factors in different regions of rats’ brain. The finding of the present study suggest that NAC administration might protect against the MCP-induced neurotoxicity in rodents.

Neuroprotective Effect of N-acetylcysteine Against Monocrotophos-Induced Oxidative Stress in Different Brain Regions of Rats.

Monocrotophos (MCP) is systemic organophosphate insecticide used against crop pests. It is reported to cause mammalian toxicity through both acute and chronic exposure. In the present study, we have shown the protective role of N-acetylcysteine (NAC) against MCP-induced oxidative stress in frontal cortex, corpus striatum and hippocampus brain regions of rats. Male Albino Wistar rats were divided into control, NAC-treated, MCP and NAC + MCP-treated groups. An oral dose of MCP (0.9mg/kg b.wt) and NAC (200mg/kg b.wt) was administered for 28days. Results showed an increase in lipid peroxidation (LPO) and protein oxidation followed by decreased antioxidant enzymes after 28days of MCP exposure. Histopathological analysis showed that monocrotophos exposure caused neurodegenerative changes as evident by neurons with dystrophic changes in the form of shrunken hyperchromatic nuclei in all the regions of the rat brain. N-acetylcysteine supplementation to MCP-treated rats showed a reduction in oxidative stress and ameliorated cellular alterations in all of the three regions. The results of the study indicate that N-acetylcysteine offers neuroprotection by improving antioxidant response and decreasing oxidative stress in different regions of the rat brain.

Immunohistochemical study of the brain glutamine synthetase expression in the rat septic model

Severe sepsis is accompanied by multiple organ dysfunction where acute liver failure (ALF) plays one of the most critical roles. The principal sign of acute hepatic encephalopathy accompanying ALF is ammonia induced edema of astrocytes. In case of ALF in sepsis, one can suppose increase in systemic and brain ammonia. Astroglia are target cells for ammonia metabolism as they are principal source of glutamine-synthetase (GS). Previous studies have reported that ALF stimulates increase in astroglial GS which correlates with deterioration of the animal state. The aim of the study was to determine the level of glutamine synthetase expression in different brain regions of rats in the conditions of experimental sepsis. Materials and methods. The study was conducted in Wistar rats: 5 sham-operated (control) animals and 20 rats with cecum ligation and puncture (CLP) septic model. Immunohistochemical study of GS expression was performed in the cortex, white matter, hippocampus, thalamus, caudate nucleus/putamen in the period of 20–48 h after CLP. Results. Starting at 12th h after CLP, operated animals displayed progressive impairment finished by profound lethargy and respiratory failure. Between 20–38 h, 9 animals expressed final mentioned symptoms and were euthanized (CLP-B, non-survived), 11 rats displayed less expressed suffering up to 48 h (CLP-A, survived). At 23 h in CLP-B and 48 h in CLP-A rats, liver tissue displayed morphological signs of the focal irreversible damage which was aggravated with time after CLP which could be observed dynamically in non-survived group. Both CLP-A and CLP-B rats showed gradual elevation of GS in all studied brain regions. From 24 to 38 h after CLP, non-survived animals showed significant region-specific dynamic increase in GS expression: in the cortex – by 69.35 %, hippocampus – by 53.6 %, thalamus – by 50.0 %; with the most substantive elevation in the cortex – 1.69-fold increase compared to control. Conclusions. In CLP model, 24 h after operation there is significant dynamic increase in GS level in the cortical, hippocampal and thalamic regions of the rat brain with the most prominent in the cortex. Heterogeneous increase in GS indicates regions more or less vulnerable for incoming systemic agents as well as region-specific reactiveness of the brain tissue, including local astroglia, to these factors in septic conditions. Morphological signs of sepsis-associated liver damage preceding significant increase in the brain GS by 1 h, might suppose addition of the liver failure to the course of sepsis which is accompanied by increased level of hepatogenic toxins (presumably including ammonia) in the blood and in the brain parenchyma by 23 h after CLP. The latter might propose an active implication of hepatogenic detrimental agents in sepsis pathophysiology and involvement of reactively increased brain GS levels in the complex mechanisms of sepsis-associated encephalopathy.

Isoformononetin, a dietary isoflavone protects against streptozotocin induced rat model of neuroinflammation through inhibition of NLRP3/ASC/IL-1 axis activation

AimsIsoformononetin (IFN), a methoxyl isoflavone present in most of human dietary supplements. However, being a highly potent antioxidant and anti-inflammatory molecule, its activity against neuronal oxidative stress and neuroinflammation has not been explored till now. The present study was inquested to assess the antioxidant, anti-apoptotic and anti-inflammatory activity of IFN against streptozotocin induced neuroinflammation in different brain regions of rat. Main methodsFour groups of animals were subjected to treatment as control, toxic control (STZ; single intracerebrovascular injection), third group (STZ + IFN; 20 mg/kg p.o.), fourth group (IFN) for 14 days. The different brain regions of rats were evaluated for inflammatory, apoptotic and biochemical antioxidant markers. The brain tissues were further assessed for gene expression, immunohistochemical and western blotting examination for localization of inflammasome cascade expression that plays a pivotal role in neuroinflammation. Key findingsThe modulation in oxidant/antioxidant status after exposure of STZ was significantly balanced after administration of IFN to rats. Further, IFN was also found to be an apoptotic agent as it modulates the apoptotic gene (Bax) and anti-apoptotic gene (BcL2) expression. IFN significantly curtailed the augmented protein expression of NLRP3, NLRP2, ASC, NFκBP65, IL-1β and caspase-1 due to STZ administration in cortex and hippocampus rat brain regions. SignificanceThe aforementioned results proclaim the neuroprotective functioning of IFN against STZ induced inflammation. IFN significantly prevents the neuroinflammation by decreasing the generation of ROS that reduces the activation of NLRP3/ASC/IL-1 axis thereby exerting neuroprotection as evidenced in rat model of STZ induced neuroninflammation.

GABABR1 in DRN mediated GABA to regulate 5-HT expression in multiple brain regions in male rats with high and low aggressive behavior

The identity of the mechanism that controls aggressive behavior in rodents is unclear. Serotonin (5-HT) and GABA are associated with aggressive behavior in rodents. However, the regulatory relationship between these chemicals in the different brain regions of rats has not been fully defined. This study aimed to clarify the role of GABABR1 in DRN-mediated GABA to regulate 5-HT expression in multiple brain regions in male rats with high and low aggressive behavior. Rat models of highly and less aggressive behavior were established through social isolation plus resident intruder. On this basis, GABA content in the DRN and 5-HT contents in the PFC, hypothalamus, hippocampus and DRN were detected using ELISA. Co-expression of 5-HT and GB1 in the DRN was detected by immunofluorescence and immunoelectron microscopy at the tissue and subcellular levels, respectively. GB1-specific agonist baclofen and GB1-specific inhibitor CGP35348 were injected into the DRN by stereotaxic injection. Changes in 5-HT levels in the PFC, hypothalamus and hippocampus were detected afterward. After modeling, rats with highly aggressive behavior exhibited higher aggressive behavior scores, shorter latencies of aggression, and higher total distances in the open field test than rats with less aggressive behavior. The contents of 5-HT in the PFC, hypothalamus and hippocampus of rats with high and low aggressive behavior (no difference between the two groups) were significantly decreased, but the change in GABA content in the DRN was the opposite. GB1 granules could be found on synaptic membranes containing 5-HT granules, which indicated that 5-HT neurons in the DRN co-expressed with GB1, which also occurred in double immunofluorescence results. At the same time, we found that the expression of GB1 in the DRN of rats with high and low aggressive behavior was significantly increased, and the expression of GB1 in the DRN of rats with low aggressive behavior was significantly higher than that in rats with high aggressive behavior. Nevertheless, the expression of 5-HT in DRN was opposite in these two groups. After microinjection of baclofen into the DRN, the 5-HT contents in the PFC, hypothalamus and hippocampus of rats in each group decreased significantly. In contrast, the 5-HT contents in the PFC, hypothalamus and hippocampus of rats in each group increased significantly after injection with CGP35348. The significant increase in GABA in the DRN combined with the significant increase in GB1 in the DRN further mediated the synaptic inhibition effect, which reduced the 5-HT level of 5-HT neurons in the DRN, resulting in a significant decrease in 5-HT levels in the PFC, hypothalamus and hippocampus. Therefore, GB1-mediated GABA regulation of 5-HT levels in the PFC, hypothalamus and hippocampus is one of the mechanisms of highly and less aggressive behavior originating in the DRN. The increased GB1 level in the DRN of LA-behavior rats exhibited a greater degree of change than in the HA-group rats, which indicated that differently decreased 5-HT levels in the DRN may be the internal mechanisms of high and low aggression behaviors.

The Effect of Ubiquinol on Cerebral Endothelial Cells in Different Regions of Rat Brain

The Effect of Ubiquinol on Cerebral Endothelial Cells in Different Regions of Rat Brain

Gelsolin Encountering Ag Nanorods/Triangles: An Aggregation-Based Colorimetric Sensor Array for in Vivo Monitoring the Cerebrospinal Aβ42% as an Indicator of Cd2+ Exposure-Related Alzheimer's Disease Pathogenesis.

Nowadays, the environmental risk factors for Alzheimer's disease (AD) have received widespread attention. Two major amyloid-β peptide (Aβ) variants, Aβ42 and Aβ40, play a pivotal role in the etiology of AD and the concentration ratio of which (i.e., Aβ42%) has been suggested to be the superior biomarker for AD. In this study, an "aggregation-based colorimetric sensor array" for the simultaneous identification and detection of Aβ40 and Aβ42 with structural similarity was established based on gelsolin-modified silver nanotriangles (Ag NTs) and silver nanorods (Ag NRs). Different aggregation behaviors of gelsolin-modified Ag NTs and Ag NRs in the presence of Aβ42 and Aβ40 resulted in different color and spectral changes, which could be quantitatively analyzed in terms of unique spectral patterns by principal component analysis. With the colorimetric sensor array employed here, the fluctuation of Aβ42% in different brain regions of rats exposed to Cd2+ could be directly monitored. The downward trend of Aβ42% accompanied by variations of other biochemical indicators suggested that subchronic Cd2+ exposure possibly triggered the onset of AD through the intervention of lipid peroxidation pathway. Furthermore, in vivo monitoring the downtrend of Aβ42% in cerebrospinal fluid (CSF) could also be realized, which offers a great opportunity for early diagnosis and treatment of AD that may be induced by environmental factors with CSF Aβ42% as a reliable indicator.

Study of aqueous extract of Terminalia chebula and Allophylus serratus aluminium-induced neurotoxicity: Relevance to sporadic amyotrophic lateral sclerosis

Background: The goal of the current work is to study the possible neuroprotective effect of Terminalia chebula (TC) and Allophylus serratus (AS) leaves extract on ions homeostasis, memory functions, motor learning ability, cytokine production and the oxidative status in different brain regions of rats in aluminium chloride (AlCl3)-induced neurodegeneration rat model.

Effects of Fuhe decoction on behaviors and monoamine neurotransmitters in different brain regions of CUMS combined with social isolation depression model rats

Abstract Objective To investigate the effect of Fuhe decoction on the behavior and levels of monoamine neurotransmitters in different brain regions in a depression rat model induced by chronic unpredictable mild stimulation (CUMS) combined with social isolation. Methods Fifty male SD rats were randomly divided into a blank group, model group, fluoxetine group, Chaiqinwendan decoction group, and Fuhe decoction group. Chronic unpredictable mild stimulation combined with a social isolation method was used to replicate the depression rat model. After 42 days of administration, a tail suspension test and high-performance liquid electrochemical detection (HPLC-ECD) were used to detect the behavioral changes and changes in the content of monoamine neurotransmitters norepinephrine (NE), dopamine (DA), 5-hydroxytrytamine (5-HT), and metabolites in different brain regions of rats in each group before and after treatment. Results Compared with the model group, the epinephrine (E) content in the Fuhe decoction group was highly significantly increased (P Conclusion Fuhe decoction can improve the depression-like behaviors of model rats, and its antidepressant effect may be related to the increase in 5-HT content in the prefrontal cortex and hippocampus of rats.

Metabolite Profile Changes in Different Regions of Rat Brain Affected by Ephedra sinica.

Ephedra sinica Stapf (EP) has a long medication history dating back centuries in the world. There were some reports of adverse effects in the central nervous system (CNS) resulting from administration of a drug containing EP or ephedrine. Compared with alkaloid monomer compounds, the effects of EP on the CNS are usually neglected. It is necessary to explore CNS affection which is helpful to use EP rationally. However, the affection and the changes of substances by EP in the brain are still unknown because the effects of drug on the brain also exhibit different tendency and distribution and usually lead to diversity of metabolite alteration in different regions. In this study, metabolomics based on different brain regions was used to investigate the affection mechanism of EP in the CNS. The metabolites in 6 brain regions from a rat that underwent oral administration with EP for 14 days were determined by UPLC/Q-TOF-MS. Brain histological examinations showed that there were no obvious lesions in EP administration groups. Partial least square-discriminant analysis (PLS-DA) displayed that there were significant separations between control and EP administration groups. 7 CNS biomarkers were found and identified in different regions. 3 metabolic pathways were disturbed by EP, including amino acid metabolism, phospholipid metabolism, and amino sugar metabolism. Furthermore, all biomarkers were significantly changed in the cortex after administration. This study may be helpful to understand the affection mechanism of EP in the CNS and improve cognition of brain regional characteristics.

Ballistic Labeling of Pyramidal Neurons in Brain Slices and in Primary Cell Culture.

It has been reported that the size and shape of dendritic spines is related to their structural plasticity. To identify the morphological structure of pyramidal neurons and dendritic spines, a ballistic labeling technique can be utilized. In the present protocol, pyramidal neurons are labeled with DilC18(3) dye and analyzed using neuronal reconstruction software to assess neuronal morphology and dendritic spines. To investigate neuronal structure, dendritic branching analysis and Sholl analysis are performed, allowing researchers to draw inferences about dendritic branching complexity and neuronal arbor complexity, respectively. The evaluation of dendritic spines is conducted using an automatic assisted classification algorithm integral to the reconstruction software, which classifies spines into four categories (i.e., thin, mushroom, stubby, filopodia). Furthermore, an additional three parameters (i.e., length, head diameter, and volume) are also chosen to assess alterations in dendritic spine morphology. To validate the potential of wide application of the ballistic labeling technique, pyramidal neurons from in vitro cell culture were successfully labeled. Overall, the ballistic labeling method is unique and useful for visualizing neurons in different brain regions in rats, which in combination with sophisticated reconstruction software, allows researchers to elucidate the possible mechanisms underlying neurocognitive dysfunction.

Antiepileptic potential of Bacopa monnieri in the rat brain during PTZ-induced epilepsy with reference to cholinergic system and ATPases

Epilepsy is a chronic central nervous system disorder that occurs not only with the imbalance of glutamatergic neurons and inhibitory gamma-aminobutyric acid (γ-GABA) neurons, but also with abnormal Central cholinergic neuronal regulation. Since long term usage of antiepileptic drugs cause high incidence of pharmacoresistance and untoward side effects, attention has been paid in recent years to screen bioactive compounds from natural medicinal plants for the treatment of several neurological disorders including Epilepsy. Keeping in view of relative importance of natural medicinal plants, the present study is mainly focused to characterize the anti-convulsant effect of Bacopa monnieri (BM), an Indian herb which is being extensively used in Ayurvedic treatments related to neurological complications. The present study is designed to assess the neurotoxicity of Pentylene tetrazole (PTZ), an epileptic compound with particular reference to Cholinergic system and ATPases in different brain regions of rat to explore the possible antiepileptic effect of different extracts of BM in comparison with Diazepam (DZ) (Reference control). The activity levels of Acetyl cholinesterase (AChE) and ATPases were decreased in different regions of brain during PTZ induced epilepsy which were increased in epileptic rats pretreated with different extracts of Bacopa monnieri except EAE and AE. In addition Acetylcholine (ACh), levels were increased during PTZ induced epilepsy when compared with normal control and levels were reversed on pretreatment with different extracts of BM. Recoveries of these parameters suggest that the bioactive factors present in the extracts offer neuroprotection by interrupting the pathological cascade that occurs during epileptogenesis.

Effect of electroacupuncture on expression of GABAA receptor mRNA in different brain regions in rats with chronic inflammatory pain

To observe the expression of GABAA receptor mRNA in different brain regions of the central nervous system in chronic inflammatory pain rats and the intervention effect of electroacupuncture (EA). A total of 48 SPF male SD rats were randomly divided into a blank control group, a model control group, an EA group and a sham EA group, 12 rats in each group. The model of chronic inflammatory pain was established by injecting Freund's complete adjuvant into the foot. The EA group was treated with EA 28 days after the model establishment. The "Housanli" (ST 36) and "Kunlun" (BL 60) were selected and treated with dilatational wave, 2 Hz/100 Hz in frequency, 0.5-1.5 mA for 30 min; EA was given only once. In the sham EA group, the same acupoints were selected but the needles were only inserted into subcutaneous area; EA was connected for 30 min without electrical stimulation. The behavior changes of mechanical pain threshold and thermal pain threshold before model establishment, 1 day, 3 days, 7 days, 14 days, 21 days and 28 days after the model establishment as well as emotional behavior 29 days after the model establishment were observed; the relative expressions of GABAA receptor mRNA in anterior cingulate cortex, amygdala and hypothalamus were observed. Compared with the blank control group, the change rates of mechanical pain threshold and thermal pain threshold in the model control group were decreased significantly 1 day, 3 days, 7 days, 14 days, 21 days, 28 days after model establishment (P<0.01); 29 days after model establishment, the movement distance and staying time in the central area of open field test in the model control group were decreased significantly (P<0.05). After EA intervention, compared with the model control group and the sham EA group, the change rates of mechanical pain threshold and thermal pain threshold, as well as the movement distance and the staying time of central area were significantly increased in the EA group (P<0.01, P<0.05). Twenty-nine days after model establishment, the expression of GABAA receptor mRNA in anterior cingulate cortex and hypothalamus was not significantly different among all groups (P>0.05). Compared with the blank control group, the expression of GABAA receptor mRNA in the amygdala was decreased significantly in the model control group (P<0.01); compared with the model control group and the sham EA group, the expression of GABAA receptor mRNA in amygdala was increased after intervention in the EA group (P<0.01). Single treatment of EA could significantly increase the mechanical pain threshold and thermal pain threshold, improve abnormal emotional behavior in rats with chronic inflammatory pain, which may be related to the increasing of expression of GABAA receptor mRNA in the amygdala.

Assessment of Nuclear and Mitochondrial DNA, Expression of Mitochondria-Related Genes in Different Brain Regions in Rats after Whole-Body X-ray Irradiation.

Studies of molecular changes occurred in various brain regions after whole-body irradiation showed a significant increase in terms of the importance in gaining insight into how to slow down or prevent the development of long-term side effects such as carcinogenesis, cognitive impairment and other pathologies. We have analyzed nDNA damage and repair, changes in mitochondrial DNA (mtDNA) copy number and in the level of mtDNA heteroplasmy, and also examined changes in the expression of genes involved in the regulation of mitochondrial biogenesis and dynamics in three areas of the rat brain (hippocampus, cortex and cerebellum) after whole-body X-ray irradiation. Long amplicon quantitative polymerase chain reaction (LA-QPCR) was used to detect nDNA and mtDNA damage. The level of mtDNA heteroplasmy was estimated using Surveyor nuclease technology. The mtDNA copy numbers and expression levels of a number of genes were determined by real-time PCR. The results showed that the repair of nDNA damage in the rat brain regions occurs slowly within 24 h; in the hippocampus, this process runs much slower. The number of mtDNA copies in three regions of the rat brain increases with a simultaneous increase in mtDNA heteroplasmy. However, in the hippocampus, the copy number of mutant mtDNAs increases significantly by the time point of 24 h after radiation exposure. Our analysis shows that in the brain regions of irradiated rats, there is a decrease in the expression of genes (ND2, CytB, ATP5O) involved in ATP synthesis, although by the same time point after irradiation, an increase in transcripts of genes regulating mitochondrial biogenesis is observed. On the other hand, analysis of genes that control the dynamics of mitochondria (Mfn1, Fis1) revealed that sharp decrease in gene expression level occurred, only in the hippocampus. Consequently, the structural and functional characteristics of the hippocampus of rats exposed to whole-body radiation can be different, most significantly from those of the other brain regions.

The specific absorption rate in different brain regions of rats exposed to electromagnetic plane waves

Accurate dosimetry of a specific brain region in rats exposed to an electromagnetic field (EMF) is essential for studies focusing on dose-effect relationship of the region. However, only dosimetry of whole brain or whole body were evaluated in most of previous studies. In this study, a numerical voxel rat model with 10 segmented brain regions was constructed. Then, the effects of frequency, incidence direction, and E-polarization direction of plane wave EMF on brain region averaged specific absorption rate (BRSAR) of rats were investigated. At last, the reliability of using whole-body averaged SAR (WBDSAR) and whole-brain averaged SAR (WBRSAR) as estimations of BRSAR were also evaluated. Our results demonstrated that the BRSAR depended on the frequency, incidence direction, and E-polarization direction of the EMF. Besides, the largest deviation could be up to 13.1 dB between BRSAR and WBDSAR and 9.59 dB between BRSAR and WBRSAR. The results suggested that to establish an accurate dose-effect relationship, the variance of the BRSAR induced by alteration of frequency, incidence direction, and E-polarization direction of EMF should be avoided or carefully evaluated. Furthermore, the use of WBDSAR and WBRSAR as estimations of BRSAR should be restricted to certain conditions such that the deviations are not too large.

Spatially Resolved Proteome Mapping of Laser Capture Microdissected Tissue with Automated Sample Transfer to Nanodroplets

Current mass spectrometry (MS)-based proteomics approaches are ineffective for mapping protein expression in tissue sections with high spatial resolution because of the limited overall sensitivity of conventional workflows. Here we report an integrated and automated method to advance spatially resolved proteomics by seamlessly coupling laser capture microdissection (LCM) with a recently developed nanoliter-scale sample preparation system termed nanoPOTS (Nanodroplet Processing in One pot for Trace Samples). The workflow is enabled by prepopulating nanowells with DMSO, which serves as a sacrificial capture liquid for microdissected tissues. The DMSO droplets efficiently collect laser-pressure catapulted LCM tissues as small as 20 μm in diameter with success rates >87%. We also demonstrate that tissue treatment with DMSO can significantly improve proteome coverage, likely due to its ability to dissolve lipids from tissue and enhance protein extraction efficiency. The LCM-nanoPOTS platform was able to identify 180, 695, and 1827 protein groups on average from 12-μm-thick rat brain cortex tissue sections having diameters of 50, 100, and 200 μm, respectively. We also analyzed 100-μm-diameter sections corresponding to 10-18 cells from three different regions of rat brain and comparatively quantified ∼1000 proteins, demonstrating the potential utility for high-resolution spatially resolved mapping of protein expression in tissues.

A deep convolutional neural network approach for astrocyte detection

Astrocytes are involved in various brain pathologies including trauma, stroke, neurodegenerative disorders such as Alzheimer’s and Parkinson’s diseases, or chronic pain. Determining cell density in a complex tissue environment in microscopy images and elucidating the temporal characteristics of morphological and biochemical changes is essential to understand the role of astrocytes in physiological and pathological conditions. Nowadays, manual stereological cell counting or semi-automatic segmentation techniques are widely used for the quantitative analysis of microscopy images. Detecting astrocytes automatically is a highly challenging computational task, for which we currently lack efficient image analysis tools. We have developed a fast and fully automated software that assesses the number of astrocytes using Deep Convolutional Neural Networks (DCNN). The method highly outperforms state-of-the-art image analysis and machine learning methods and provides precision comparable to those of human experts. Additionally, the runtime of cell detection is significantly less than that of other three computational methods analysed, and it is faster than human observers by orders of magnitude. We applied our DCNN-based method to examine the number of astrocytes in different brain regions of rats with opioid-induced hyperalgesia/tolerance (OIH/OIT), as morphine tolerance is believed to activate glia. We have demonstrated a strong positive correlation between manual and DCNN-based quantification of astrocytes in rat brain.

Developmental Neurotoxicity of Arsenic: Involvement of Oxidative Stress and Mitochondrial Functions

Over the last decade, there has been an increased concern about the health risks from exposure to arsenic at low doses, because of their neurotoxic effects on the developing brain. The exact mechanism underlying arsenic-induced neurotoxicity during sensitive periods of brain development remains unclear, although enhanced oxidative stresses, leading to mitochondrial dysfunctions might be involved. Here, we highlight the generation of reactive oxygen species (ROS) and oxidative stress which leads to mitochondrial dysfunctions and apoptosis in arsenic-induced developmental neurotoxicity. Here, the administration of sodium arsenite at doses of 2 or 4mg/kg body weight in female rats from gestational to lactational (GD6-PD21) resulted to increased ROS, led to oxidative stress, and increased the apoptosis in the frontal cortex, hippocampus, and corpus striatum of developing rats on PD22, compared to controls. Enhanced levels of ROS were associated with decreased mitochondrial membrane potential and the activity of mitochondrial complexes, and hampered antioxidant levels. Further, neuronal apoptosis, as measured by changes in the expression of pro-apoptotic (Bax, Caspase-3), anti-apoptotic (Bcl2), and stress marker proteins (p-p38, pJNK) in arsenic-exposed rats, was discussed. The severities of changes were found to more persist in the corpus striatum than in other brain regions of arsenic-exposed rats even after the withdrawal of exposure on PD45 as compared to controls. Therefore, our results indicate that perinatal arsenic exposure leads to abrupt changes in ROS, oxidative stress, and mitochondrial functions and that apoptotic factor in different brain regions of rats might contribute to this arsenic-induced developmental neurotoxicity.

An easy-to-use liquid chromatography method with fluorescence detection for the simultaneous determination of five neuroactive amino acids in different regions of rat brain

Introduction: To comprehend the normal function and pathological characteristics of certain neurological disorders it is important to evaluate the neuroactive amino acids levels in animal models.Methods: This work describes a simple liquid chromatography-fluorescence detection (LC-FLD) method for the simultaneous determination of aspartic acid (Asp), glutamic acid (Glu), glutamine (Gln), taurine (Tau) and γ-aminobutyric acid (GABA), using methyl-L-arginine as internal standard, in samples of rat brain tissue. The five target analytes (Asp, Glu, Gln, Tau and GABA) were determined in a single chromatographic run of less 11 min after a derivatization step with o-phthalaldehyde. The derivatives were separated on a reversed-phase C18 column and detected by fluorescence at excitation and emission wavelengths of 340 and 448 nm, respectively.Results: The method was validated in accordance with international guidelines on bioanalytical methods validation and it presented limits of quantification in the range of 25–50 ng mL−1 and calibration curves with determination coefficients (r2) equal to or higher than 0.9920. In addition, the precision (coefficient variation, %) and accuracy (bias, %) of the method meet the established criteria, and the stability of the analytes at the sample handling and storage conditions was demonstrated.Discussion: Unlike other similar bioanalytical assays, the current method was validated using diluted biological matrix, which is advantageous in order to ensure the derivatization process integrity. Moreover, this LC-FLD method was successfully applied for the determination of the compounds of interest in different rat brain tissue regions (frontal cortex, amygdala, hippocampus, cerebellum and striatum). Thus, this bioanalytical assay represents a useful tool to support multiple nonclinical studies in the field of neurosciences, requiring the quantitative profiling and pattern analysis of neuroactive amino acids.