Brain Tissue Homogenates Research Articles

Comparison of toxic effects of lead and copper and protective power of glutathione on oxidative stress parameters

IntroductionLead as an industrial pollutant can be detected at all stages of the working and living environment. Lead, based on its properties, solubility and mobility, accumulates in the soil, so that the average concentrations of oil in the soil are between 15 and 25 mg/kg (Radojević at all., 1999). Due to increased human activity, the amount of copper in the air, soil and water has increased. Glutathione (GSH) is an essential cofactor of many enzymes, such as: formaldehyde dehydrogenase, glyoxalase, prostaglandin endoperoxide isomerase, dehydrochlorinase and others. GSH is a biological redox in the metabolism of erythrocytes, it also plays a role in the transport of amino acids. Reactive forms of oxygen cause oxidative biomolecules (lipids, proteins, DNA) (Freidovich, 1999; Massaad i Klann, 2010).ObjectivesThe aim of this research was to examine the protective role of supplements GSH in conditions of chronic intoxication with sublethal doses of lead acetate and copper II sulfate.MethodsThe preparation of biomaterials for testing and making homogenates of brain tissue of albino rats of Wistar strain was performed and the activity of acid and alkaline DNase was measured spectrophotometrically (Kocić i sar., 1998).ResultsLead otherwise “as soft Lewis acid” has a pronounced affinity for interaction with “soft bases” such as S-atoms of the thiol group in antioxidants, natural biomolecules and supplements in this case in glutathione.ConclusionsIt can be said that GSH is a desirable supplement and antioxidant in the detoxification of reactive oxygen species in rats exposed to lead poisoning.DisclosureNo significant relationships.

Evaluating blood-brain barrier disruption and infarction volume concurrently in rats subjected to ischemic stroke using an optical imaging system

BackgroundBlood-brain barrier (BBB) disruption is pivotal in the pathophysiological process of ischemic stroke and is often measured in rodent stroke studies. Traditionally, rodent BBB permeability increase is determined by measuring cerebral leakage of certain dyes such as Evans Blue or sodium fluorescein (NaFL). However, due to the special processing of samples for BBB permeability measurement, they cannot be used afterward for determining other essential parameters such as cerebral infarction volume. Therefore, using different batches of animals for assessing BBB permeability and infarction volume is typical. However, this would limit the stroke study’s statistical power and scientific value while hindering the implementation of procedures for high standard animal welfare. New methodThe rats subjected to middle cerebral artery occlusion (MCAO) were intraperitoneally injected with NaFL during the reperfusion phase. The brains were sliced and measured for BBB permeability using the small animal optical imaging system (IVIS® Lumia series III). Afterward, the same brain samples were either sliced or homogenized for tests that assessed infarction volume or other molecular changes. ResultsThe sum fluorescence intensity of the ischemic brain slices under the IVIS® Lumia series Ⅲ showed a strong correlation with the infarction volume determined by 2,3,5-triphenyl tetrazolium chloride (TTC) staining (r = 0.7440, P = 0.0087). The fluorescence intensity of the whole ischemic brain was correlated with the NaFL concentration of brain tissue homogenates (r = 0.8653, P = 0.0026) and cerebral infarction volume (r = 0.7282, P = 0.0072). Comparison with existing methodsThe new method enables concurrent measurement of BBB permeability and infarction volume on the same batch of brain tissue samples without affecting most downstream biochemical assays. ConclusionsBy applying the new method, we could use the same batch of ischemic rodent brain tissue for multiple assays, including BBB permeability and infarction volume. Through this, we would reduce the animal numbers in each study and help to maximize the scientific and statistical potential of future rodent ischemic studies.

Changes in Antioxidant/Pro-Oxidant Ratio of Brain Tissue Homogenates in Response to Cocos nucifera Treatment

Objective: This study revealed the effect of Cocos nucifera juice on antioxidant/pro-oxidant ratio in brain tissue homogenates of wistar rats.
 Materials and Methods: 40 male wistar rats were collected and randomly selected into 4 groups. Treatment protocols were; i-normal saline, ii-3ml C. nucifera juice, iii-6ml C.nucifera juice, iv-9ml C. nucifera juice. The study period lasted for 42 days. Brain tissue homogenate was prepared and biochemical analysis for antioxidant enzymes superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPx); pro-oxidant biomarkers hydrogen peroxide (H2O2), thiobarbituric acid reactive substances (TBARS) and nitric oxide (NO) were assayed for.
 Results: The result showed that there was an increase in antioxidants concentration and a simultaneous decrease in pro-oxidant species generation as the dose of treatment was increased. The ratio of antioxidants to pro-oxidants showed a direct proportionality to the treatment dose.
 Conclusion: C. nucifera juice has the ability to improve the level of production or synthesis of the assayed antioxidant enzymes and also has the efficacy to suppress pro-oxidant specie generation.

Efavirenz Biotransformation in Brain Microsomes and Neural Cells

Efavirenz (EFV) is a non‐nucleoside reverse transcriptase inhibitor that is metabolized by both the cytochromes P450 and UDP‐glucuronosyltransferases (UGTs). EFV is one of several anti‐HIV medications that can cross the blood‐brain barrier and is essential to combatting the viral reservoir in the brain that can eventually lead to HIV‐associated neurocognitive disorder (HAND) in nearly 50% of patients. EFV and 8‐hydroxyefavirenz (8‐OHEFV), the primary P450‐dependent metabolite of EFV, have both been linked to neurological adverse events and neurotoxicity. However, very little is known about the fate of EFV in the brain, particularly how it’s metabolized or the precise mechanism of neurotoxicity. To investigate EFV biotransformation in the brain, we first performed microsomal metabolism assays with brain microsomes obtained from mice, cynomolgus macaques, and humans. EFV was incubated with the brain microsomes and NADPH to characterize P450‐dependent metabolism, while EFV or one of its P450‐dependent metabolites, 8‐OHEFV or 8‐,14‐dihydroxyefavirenz (8,14‐diOHEFV), was incubated with brain microsomes and UDPGA to investigate UGT‐catalyzed metabolism. Ultra‐high‐performance liquid chromatography tandem high resolution mass spectrometry (UHPLC‐HRMS) was employed to detect metabolites. Formation of 8‐OHEFV was observed in all three species, as was glucuronidation of 8‐OHEFV and 8,14‐diOHEFV. Interestingly, direct glucuronidation of EFV was only found in the cynomolgus macaque brain microsomes. When comparing brain microsome metabolism to liver microsome metabolism, we see that the liver has a greater capacity to transform EFV and its P450‐dependent metabolites. We tested cell‐type specific metabolism using primary cortical neurons, striatal neurons, astrocytes, and microglia from mice. Cells were cultured and treated with EFV, 8‐OHEFV, or 8,14‐diOHEFV for approximately 24 hours, with DMSO as a vehicle control. In the microglia and astrocytes, we observed P450‐mediated formation of 8‐OHEFV in the EFV‐treated cells. In the cortical neurons, striatal neurons, and astrocytes, both 8‐OHEFV and 8,14‐diOHEFV were glucuronidated. These results shed additional light on the microsome data, indicating which cell‐types may be contributing to EFV metabolism. In male mice dosed with EFV for 5 days in drinking water, UHPLC‐HRMS analysis of brain tissue homogenate and serum revealed 8‐OHEFV in brain tissue in conjunction with serum concentrations in the range of 15‐20 ng/mL. EFV and other metabolites were below the limit of detection in both brain tissue and serum. These data indicate that 8‐OHEFV has the potential to be formed and/or further metabolized in mouse brain. Additionally, inter‐species differences in EFV glucuronidation were observed in brain microsomes. The characterization of EFV metabolism in the brain improves our understanding of tissue‐specific EFV disposition as well as brain glucuronidation more broadly.

Effects of dopants in the imaging of mouse brain by desorption electrospray ionization/post-photoionization mass spectrometry.

Desorption electrospray ionization/post-photoionization (DESI/PI) is a newly developed ionization method by the combination of DESI and post-photoionization for the simultaneous imaging of polar and nonpolar compounds in biological tissues. Dopants are of great importance in DESI/PI for the enhancement of signal intensities through ion-molecule reactions. In this work, to evaluate the performance of dopants in DESI/PI, an efficient homogenate model was developed, and four kinds of dopants (toluene, chlorobenzene, bromobenzene, and anisole) were tested using homogenate of mouse brain tissue as target sample. The influences of the dopants on the signal enhancements of different compounds were explained reasonably by the ionization mechanism. Then, the dopants with their optimum volume contents were applied to the mass spectrometry imaging (MSI). For a comprehensive imaging of various compounds with different polarities, methanol/toluene/formic acid (7:3:0.1) was chosen as the best choice. Finally, the stronger quantitative ability of DESI/PI with toluene as dopant for a few compounds in mouse brain tissue was demonstrated.

Effect of Honey on Oxidative Stress in the Brain Tissue of Sleep Deprived Rats

This study aimed to demonstrate the effect of honey on the oxidative stress in the brain of sleep-deprived rats. Twenty five 6 weeks old male wistar rats were randomly divided into five groups and subjected to paradoxical sleep deprivation and recovery for 5 days using the modified multiple platform (MMP) method. Group I: Normal control; Group II: sleep deprivation (SD); Group III: sleep deprivation and sleep recovery (SD+SR) – received 10 ml/kg distilled water orally each; while Group IV: sleep deprivation and honey (SD+Honey) and Group V: sleep deprivation, recovery with honey (SD+SR+Honey) received honey (1g/kg body weight) orally once daily. Brain tissue of the humanely sacrificed rats were excised and homogenized for assessment of oxidative stress markers. Results indicate a significant decrease (p<0.05) in the concentrations of malondialdehyde (MDA) in brain tissues of rats in Groups IV and V when compared with that from sleep deprived group (Group II). The Increase in reduced glutathione (GSH) concentration and catalase enzyme activity observed in homogenized brain tissue of rats in Groups III, IV, and V differ significantly (p<0.05) when compared with Group II. The results suggest that treatment with honey probably has ameliorative effects on oxidative stress in brain tissue of sleep deprived experimental rats.

Non-invasive imaging of tau-targeted probe uptake by whole brain multi-spectral optoacoustic tomography

PurposeAbnormal tau accumulation within the brain plays an important role in tauopathies such as Alzheimer’s disease and frontotemporal dementia. High-resolution imaging of tau deposits at the whole-brain scale in animal disease models is highly desired.MethodsWe approached this challenge by non-invasively imaging the brains of P301L mice of 4-repeat tau with concurrent volumetric multi-spectral optoacoustic tomography (vMSOT) at ~ 115 μm spatial resolution using the tau-targeted pyridinyl-butadienyl-benzothiazole derivative PBB5 (i.v.). In vitro probe characterization, concurrent vMSOT and epi-fluorescence imaging of in vivo PBB5 targeting (i.v.) was performed in P301L and wild-type mice, followed by ex vivo validation using AT-8 antibody for phosphorylated tau.ResultsPBB5 showed specific binding to recombinant K18 tau fibrils by fluorescence assay, to post-mortem Alzheimer’s disease brain tissue homogenate by competitive binding against [11C]PBB3 and to tau deposits (AT-8 positive) in post-mortem corticobasal degeneration and progressive supranuclear palsy brains. Dose-dependent optoacoustic and fluorescence signal intensities were observed in the mouse brains following i.v. administration of different concentrations of PBB5. In vivo vMSOT brain imaging of P301L mice showed higher retention of PBB5 in the tau-laden cortex and hippocampus compared to wild-type mice, as confirmed by ex vivo vMSOT, epi-fluorescence, multiphoton microscopy, and immunofluorescence staining.ConclusionsWe demonstrated non-invasive whole-brain imaging of tau in P301L mice with vMSOT system using PBB5 at a previously unachieved ~ 115 μm spatial resolution. This platform provides a new tool to study tau spreading and clearance in a tauopathy mouse model, foreseeable in monitoring tau targeting putative therapeutics.

Brain Antigens Stimulate Proliferation of T Lymphocytes With a Pathogenic Phenotype in Multiple Sclerosis Patients.

A method to stimulate T lymphocytes with a broad range of brain antigens would facilitate identification of the autoantigens for multiple sclerosis and enable definition of the pathogenic mechanisms important for multiple sclerosis. In a previous work, we found that the obvious approach of culturing leukocytes with homogenized brain tissue does not work because the brain homogenate suppresses antigen-specific lymphocyte proliferation. We now report a method that substantially reduces the suppressive activity. We used this non-suppressive brain homogenate to stimulate leukocytes from multiple sclerosis patients and controls. We also stimulated with common viruses for comparison. We measured proliferation, selected the responding CD3+ cells with flow cytometry, and sequenced their transcriptomes for mRNA and T-cell receptor sequences. The mRNA expression suggested that the brain-responding cells from MS patients are potentially pathogenic. The T-cell receptor repertoire of the brain-responding cells was clonal with minimal overlap with virus antigens.

Evaluating the effects of anticoagulant rodenticide bromadiolone in Wistar rats co-exposed to vitamin K: impact on blood-liver axis and brain oxidative status.

The aim of this study was to investigate the beneficial effects of vitamin K relate to protection against detrimental effects of bromadiolone. Wistar rats (n = 30) were divided in three groups (n = 10): control group and two groups treated with bromadiolone (0.12mg/kg) and bromadiolone + vitamin K (0.12mg/kg + 100mg/kg) over the period of four days. The main findings in the bromadiolone-exposed rats, such as damaged hepatocytes, high levels of globulin, total proteins and lymphocytes, and altered albumin/globulin ratio, collectively indicate an acute inflammatory process. Morphological changes in erythrocytes include microcytosis, hypochromia, hyperchromia, hemolysis, stomatocytosis, and spherocytosis. Significantly low values of RBC, Hct, and hemoglobin concentrations indicate impairments of the hematopoietic pathway causing combined anemia. The selected dose of bromadiolone caused a non-significant increase of catalase activity and a significant increase of the total protein content in brain tissue homogenates. Vitamin K supplementation reduced many of the harmful effects of bromadiolone. The cytoprotective role of vitamin K was proved to be of great importance for the preservation of structural changes on the membranes of hepatocytes and erythrocytes, in addition to the known role in the treatment of coagulopathies. The results of the study suggest valuable properties of vitamin K in the prevention and treatment of various types of anemia caused by bromadiolone toxicity. Future research is necessary to determine the adequate dose and treatment duration with vitamin K in disorders caused by the cumulative action of bromadiolone and possibly other pesticides.

EXTH-64. COMPARISON OF PANOBINOSTAT CSF PENETRATION WITH CNS PENETRATION FOLLOWING SYSTEMIC ADMINISTRATION IN A PRE-CLINICAL NON-HUMAN PRIMATE MODEL

Abstract Targeted therapies developed for diffuse midline gliomas (DMG) expressing H3K27M have focused on histone deacetylase inhibitors (HDACi). High-throughput drug screening with patient derived DMG cell lines identified the HDACi panobinostat as a prominent clinical agent as well as pre-clinical studies with orthotopic mouse tumors models proving efficacious. Diametrically there is a pronounced lack of measurable panobinostat CSF concentrations in a non-human primate (NHP) non-tumor bearing pre-clinical model and in pediatric brain tumor patients. Notwithstanding, adult and pediatric glioma clinical trials and clinical observation with panobinostat alone or in combination have demonstrated minor responses. Pharmacokinetic models utilize the premise that CSF drug penetration is a surrogate of CNS drug penetration. However, the direct correlation between CSF and CNS drug levels is undefined especially in lieu of geographic CNS extracellular fluid drug variability previously demonstrated in the same NHP pre-clinical model. Utilizing the same NHP model, this study sought to compare panobinostat CSF penetration to CNS penetration via analysis of homogenized normal cerebrum, cerebellum, and brainstem tissue utilizing LC-MS/MS. METHODS: Panobinostat was administered orally as a single dose to three non-human primates. Pre panobinostat plasma and CSF were collected. Following panobinostat administration (1-hr Tmax) CSF, cerebrum, cerebellum, and brain stem tissue were collected as well as plasma to confirm the presence of panobinostat. Tissue slices were individually homogenized and panobinostat extracted via protein precipitation. Plasma, CSF, and tissue panobinostat concentrations were quantified using a LC-MS/MS assay. The lower limit of quantitation (LLOQ) for plasma-0.1 ng/ml, CSF-0.5 ng/ml, and tissue-10.0 pg/mg. RESULTS: Panobinostat was quantifiable in plasma (n=2) at the 1 hour (20.033 ng/mL and 0.153 ng/mL). CSF and CNS tissue samples were below the LLOQ for panobinostat in all samples. CONCLUSIONS: Panobinostat was not measurable from CSF and homogenized brain tissue in a non-tumor bearing NHP model at 1-hour post-administration using LC-MS/MS.

A highly sensitive and rapid LC-MS/MS method for quantification of bexarotene in mouse plasma and brain tissue: Application to mice pharmacokinetic study

Emerging evidence has suggested that bexarotene, a nearly 20-year-old skin cancer drug, may be a potential drug candidate to treat Alzheimer's disease (AD) and other neurodegenerative disorders. As described in this study, a highly sensitive and rapid method, using liquid chromatography–tandem mass spectrometry (LC–MS/MS) to determine bexarotene in mouse plasma and brain tissue, was established and validated for the first time. Single-step protein precipitation utilizing methanol solution (containing 0.05 % acetic acid) as precipitation agent was employed to prepare the samples of plasma and brain tissue. Chromatographic separation in gradient elution mode was conducted via an Agilent ZORBAX SB-C18 column (50 mm × 4.6 mm, 5 µm) employing methanol–ammonium acetate buffer (5 mM, pH adjusted to 4.6 with acetic acid) as mobile phase which flowed at 0.45 mL/min. The total run time was 6 min for each sample. Detection through mass spectrometric technique was operated by selected reaction monitoring (SRM) in negative electrospray ionization mode. The method was linear within the range of 10.0–15000 ng/mL for plasma and 10.0–600 ng/mL for brain tissue homogenate with the lower limit of quantification of 10.0 ng/ml. The plasma or tissue homogenate was only required 20 μL. The intra- and inter-day precision were less than 13.8 %, and the RE was between –7.4 % and 3.4 %. The method was applied to investigate the plasma pharmacokinetics and brain distribution of bexarotene in mice after being intragastrically administered with bexarotene at the dosage of 100 mg/kg. The results showed that both brain and plasma concentrations of bexarotene peaked at 1.0 h. Bexarotene was rapidly eliminated with a half-life of 2.0 h.

Anticonvulsive Effects of Chondroitin Sulfate on Pilocarpine and Pentylenetetrazole Induced Epileptogenesis in Mice.

Chondroitin sulfate is a proteoglycan component of the extracellular matrix (ECM) that supports neuronal and non-neuronal cell activity, provides a negative domain to the extracellular matrix, regulates the intracellular positive ion concentration, and maintains the hypersynchronous epileptiform activity. Therefore, the present study hypothesized an antiepileptic potential of chondroitin sulfate (CS) in pentylenetetrazole-induced kindled epilepsy and pilocarpine-induced status epilepticus in mice. Levels of various oxidative stress markers and inflammatory mediators were estimated in the brain tissue homogenate of mice, and histopathological changes were evaluated. Treatment with valproate (110 mg/kg; i.p.) as a standard drug and chondroitin sulfate (100 & 200 mg/kg, p.o.) significantly (p < 0.01) and dose-dependently prevented the severity of kindled and spontaneous recurrent seizures in mice. Additionally, chondroitin sulfate showed its antioxidant potential by restoring the various biochemical levels and anti-inflammatory properties by reducing NF-kB levels and pro-inflammatory mediators like TNF-alpha, IL-1β, and IL-6, indicating the neuroprotective effect as well as the suppressed levels of caspase-3, which indicated a neuroprotective treatment strategy in epilepsy. The proteoglycan chondroitin sulfate restores the normal physiology and configuration of the neuronal tissue. Further, the molecular docking of chondroitin sulfate at the active pockets of TNF-alpha, IL-1β, and IL-6 showed excellent interactions with critical amino acid residues. In conclusion, the present work provides preclinical evidence of chondroitin sulfate as a new therapeutic approach in attenuating and preventing seizures with a better understanding of the mechanism of alteration in ECM changes influencing abnormal neuronal activities.

Histopathological and ultrastructural assessment of atovaquone-proguanil hydrochloride combination in chronic murine toxoplasmosis

ABSTRACT Over one billion people worldwide are expected to have Toxoplasma gondii infection with anonymous health problems. Available therapies are ineffective for persistent chronic toxoplasmosis. So, there is an imperative need for effective therapies to eliminate chronic tissue stage. In this study, we aimed to assess the effect of a drug combination of atovaquone and proguanil hydrochloride in the treatment of experimental chronic toxoplasmosis. Fifty Swiss Webster mice were used in the study. Forty mice were infected with Me49 type II cystogenic Toxoplasma gondii strain and allocated into four groups: infected untreated (vehicle-administered), infected and treated with cotrimoxazole (CTX) 370 mg/kg/day, infected and treated with atovaquone (ATV) 100 mg/kg/day, and infected and treated with atovaquone/proguanil (ATV/PROG) 50 mg/kg/day. An additional group of uninfected mice was used as an uninfected control group. Drug treatment was initiated 8 weeks post-infection and continued for two weeks. All mice were sacrificed 12 weeks post-infection. Parasitological and histopathological parameters were assessed. Toxoplasma gondii cysts recovered from brain tissue homogenates of both infected untreated and ATV/PROG-treated groups were examined by scanning electron microscopy. Combined ATV/PROG treatment demonstrated a significant reduction of Toxoplasma gondii cyst count in brain tissue (a reduction rate of 84.87%) compared to untreated group (P < .001). Brain tissues obtained from ATV/PROG treated group showed reduction of inflammatory infiltrate and marked attenuation and deformation of recovered Toxoplasma gondii cysts. We conclude that ATV/PROG drug combination could offer a potential drug therapy for Toxoplasma gondii chronic cystic stage.

Using Receptor Autoradiography to Visualize and Quantify Oxytocin and Vasopressin 1a Receptors in the Human and Nonhuman Primate Brain.

Despite its development almost 40years ago, receptor autoradiography remains a regular and reliable practice for the localization of oxytocin and vasopressin receptors in brain tissue sections. It is used across many laboratories, institutions, and animal species to characterize and quantify the distribution and density of these receptors at baseline and/or in response to experimental manipulations or lived experience. This powerful tool and the neuroanatomical receptor maps that it generates have allowed researchers to more accurately investigate and understand the neural substrates upon which oxytocin and vasopressin act to affect behavior. Researchers have used these maps to design site-specific pharmacological manipulations and electrophysiological recordings in animal studies to directly probe the underlying neural mechanisms in this system. This methods chapter describes the specific procedures by which a pharmacologically optimized, competitive binding modification to receptor autoradiography can be used to reliably localize oxytocin and vasopressin receptors in the human brain and in the brains of nonhuman primates. The ability to reliably perform receptor autoradiography for these targets in human brain tissue can finally inform our interpretation of past intranasal oxytocin neuroimaging studies and allows us to move past the reliance on transcriptomic studies using brain tissue homogenates so that we can directly investigate the involvement of oxytocin and vasopressin receptors in human behavior, physiology, and neuropsychiatric disease.

Investigating the Central Nervous System Disposition of Actinomycin D: Implementation and Evaluation of Cerebral Microdialysis and Brain Tissue Measurements Supported by UPLC-MS/MS Quantification.

Actinomycin D is a potent cytotoxic drug against pediatric (and other) tumors that is thought to barely cross the blood–brain barrier. To evaluate its potential applicability for the treatment of patients with central nervous system (CNS) tumors, we established a cerebral microdialysis model in freely moving mice and investigated its CNS disposition by quantifying actinomycin D in cerebral microdialysate, brain tissue homogenate, and plasma. For this purpose, we developed and validated an ultraperformance liquid chromatography–tandem mass spectrometry assay suitable for ultra-sensitive quantification of actinomycin D in the pertinent biological matrices in micro-samples of only 20 µL, with a lower limit of quantification of 0.05 ng/mL. In parallel, we confirmed actinomycin D as a substrate of P-glycoprotein (P-gp) in in vitro experiments. Two hours after intravenous administration of 0.5 mg/kg, actinomycin D reached total brain tissue concentrations of 4.1 ± 0.7 ng/g corresponding to a brain-to-plasma ratio of 0.18 ± 0.03, while it was not detectable in intracerebral microdialysate. This tissue concentration exceeds the concentrations of actinomycin D that have been shown to be effective in in vitro experiments. Elimination of the drug from brain tissue was substantially slower than from plasma, as shown in a brain-to-plasma ratio of approximately 0.53 after 22 h. Because actinomycin D reached potentially effective concentrations in brain tissue in our experiments, the drug should be further investigated as a therapeutic agent in potentially susceptible CNS malignancies, such as ependymoma.

Ameliorating Effect of Combined Cinnamon and Ginger Oils against the Neurotoxicity of Nicotine Administration on the Prefrontal Cortex of Adult Albino Rats: Immunohistochemical and Ultrastructural Study

Background: Nicotine is the active alkaloid in cigarettes. It was reported that tobacco smoking has many hazards; one of these hazards is the effect on the cognitive function of the prefrontal cortex. The aim of our study is to investigate the antioxidant effects of ginger, cinnamon oils, and their combination on morphological changes in the prefrontal cortex that were induced by nicotine. Materials and methods: Fifty adult male albino rats were divided into five groups: group I (control group), group II (nicotine), group III (nicotine + cinnamon), group IV (nicotine + ginger), and group V (nicotine + cinnamon + ginger). The coronal sections from the anterior part of the rat brain at the site of prefrontal cortex were examined by light microscope for (H&amp;E and immunohistochemical staining with TNF-α and GFAP), while the ultrastructure morphology was examined by transmission electron microscopy. Levels of the oxidative stress markers (MDA, GSH) in the rats’ brain tissue homogenate were biochemically assessed. Results: Compared to the control group, the rats that were treated with nicotine (group II) showed a significant oxidative stress in the form of marked elevation of MDA and decrease in GSH, apoptotic changes especially in the pyramidal cells in the form of neuronal cell degeneration and pyknosis, and an elevation in the inflammatory marker TNF-α and GFAP expressions. These changes were observed to a lesser degree in rat group (III) and group (IV), while there was a marked improvement achieved by the combined usage of cinnamon and ginger oils, together compared to the nicotine group. Conclusions: Ginger and cinnamon are powerful antioxidants which ameliorate the degenerative and oxidative effects produced by nicotine on a rat’s prefrontal cortex.

Aptamer-based enrichment of TDP-43 from human cells and tissues with quantification by HPLC-MS/MS

BackgroundThere is great interest in detecting, characterizing and quantifying transactive response DNA binding protein of 43 kDa (TDP-43), and its post-translational modifications, due to its association with frontotemporal dementia (FTD) and amyotrophic lateral sclerosis. Unfortunately, detailed analysis of TDP-43 in human biological matrices by immunometric methods has been hindered by the relatively low abundance of TDP-43 and poor antibody reagent specificity. New methodWith the goal of developing a selective and multiplex method for characterizing TDP-43, we previously developed a high-performance liquid chromatography tandem mass spectrometry (HPLC-MS/MS) assay for relative quantification of TDP-43 in human brain tissue and cells. To improve analytical sensitivity and to perform absolute quantification, we coupled a novel RNA-based aptamer enrichment workflow (and inclusion of a stable isotope-labeled standard) to HPLC-MS/MS. ResultsThe TDP-43 aptamer-enrichment—HPLC-MS/MS assay was linear from 0.37 to 2.55nmol/L, a range suitable for analysis of both human cells and brain tissue homogenates, and had a total CV of 14.8%. Quantitative TDP-43 peptide profiles were developed for cases of FTD with TDP-43 pathology and cases with no neurodegenerative pathology. Comparison with existing methodsCompared to immunoenrichment, aptamer-enrichment yielded cleaner recoveries of TDP-43. The aptamer-enrichment—HPLC-MS/MS method, compared to our previous method without enrichment, increased analytical sensitivity by 8.7-fold and 11.8-fold for endogenous TDP-43 in human cells and brain tissue, respectively. Critically, inclusion of the aptamer enrichment step improved sequence resolution and enabled identification of TDP‐43 C‐terminal fragments. ConclusionsThe aptamer-enrichment—HPLC-MS/MS method enabled highly selective quantification, enhanced sequence coverage and structural characterization of endogenous TDP-43.

Effects of Vitamin C and Dimethylsulfoxide (Dmso) on Neuro-Behaviour, Apoptosis and oxidative stress in ischaemic stroke induced rats

Background: Researches over the years have identified excitotoxicity, oxidative stress, inflammation and cell death as key contributors underlying progression of lesion in ischaemic stroke (IS). This study reports the effect of vitamin C and dimethylsulfoxide (DMSO) in the management of ischaemic stroke (IS) in Wistar rats. Method: Forty rats were divided into four groups of ten rats each. Groups 1, 2 and 3 were induced with IS by middle cerebral artery occlusion (MCAO). Anxiety like behavior and locomotor activity assessments were carried out. Group 1was treated with vitamin C (67.5mg/kg) and group 2 with DMSO (67.5mg/kg) orally for two weeks. Group 4 was neither induced nor treated. Brain tissue homogenates (cerebral cortex) were assessed for the level of calcium binding protein (S100B), antioxidant enzymes [Superoxide dismutase (SOD), Catalae (CAT), Glutathion peroxidase (GPx)] gene expressions, BCL2, Bax and Caspase3. Result: The results of this study showed that in the IS rats, there was significant (P&lt;0.05) increase in anxiety and a significant (P&lt;0.05) decrease in the locomotor activity. There was also significant (P&lt;0.05) increase in S100B, caspase 3 and Bax expressions, while there was significant (P&lt;0.05) decrease in BCL2. Treatment with vitamin C and DMSO resulted in significant (P&lt;0.05) increase in the locomotor activity and significant (P&lt;0.05) decrease in anxiety. There was significant (P&lt;0.05) increase in the expression of SOD, CAT and GPx genes. Also, there was significant (P&lt;0.05) decrease in S100B level, caspase-3 and Bax expressions, while there was significant (P&lt;0.05) increase in BCL2. Conclusion: Conclusively, the results of this research highlighted the potentials for the use of vitamin C and DMSO in the management of IS. Keywords: Stroke; Antioxidant; Locomotor; Neuro-Behavioural; Dimethylsulfoxide Apoptosis; Oxidative Stress

Neuroprotective Outcome of Thymol against Global Cerebral Ischemia Reperfusion injury in Albino Rats

Aims: The purpose of the present study was targeted to explore the possible role of thymol against global cerebral ischemia-reperfusion injury in albino rats.&#x0D; Study Design: Healthy Albino Wistar rats (200–250 gm) were divided randomly into 5 groups (n=6). Group I and II were considered as normal control and sham control, received 2% tween 80 orally, group III was ischemic- reperfusion (disease control) and received 2% tween 80 orally and Group IV and V received thymol at doses of 50 mg/kg, per oral and 100 mg/kg, per oral. respectively.&#x0D; Place and Duration of Study: Department of Pharmacology, Sri Padmavathi School of pharmacy, Tiruchanur, Tirupati in between Sept 2019 to March 2020.&#x0D; Methodology: Group I and II were considered as normal control and sham control, received 2% tween 80 orally, group III was ischemic- reperfusion (disease control) and received 2% tween 80 orally and Group IV and V received thymol at doses of 50 mg/kg, per oral (p.o) and 100 mg/kg, per oral (p.o). respectively. After pretreatment with thymol for 2 weeks, rats were subjected to bilateral common carotid artery occlusion for 1 hour accompanied by 22 hours reperfusion (I/R). After 22 hrs of reperfusion, motor coordination, hanging wire test, despair swim tests were studied. Antioxidant levels of reduced glutathione (GSH), superoxide dismutase (SOD), catalase (CAT) and pro-oxidant level of Malondialdehyde (MDA) were analyzed in brain tissue homogenate. Changes in cerebral infarct size and histopathology were studied.&#x0D; Results: Pretreated groups of thymol (50 and 100 mg/kg p.o) showed significant improvement in neurobehavioral changes and attenuated oxidative damage as indicated by reduced LPO, restored GSH, SOD and CAT levels and decreased infract size when compared to ischemic reperfusion group.&#x0D; Conclusion: This study suggests that thymol may have a beneficial role against global ischemia reperfusion induced damage caused by excessive free radicals and behavioral alterations in rats.

Nanoparticles in the Treatment of Alzheimer’s Disease with Magnetic Resonance

ABSTRACTThis study was to explore the effect of nanoparticles on the cognitive function, learning and memory ability (LMA) of Alzheimer’s disease (AD) rats, and to analyze the changes on magnetic resonance (MR) image. Specifically, the TGN nanoparticles loading H102 (TGN-NP-H102) were prepared, and characterized first. The sprague-dawley (SD) rats were selected as the research subjects, and the AD model was constructed. They were divided into a Sham group (normal SD rats, group A), an AD model group (group B), an H102 group (treated with H102 drugs based on AD model, group C), and a TGN-NP-H102 group (treated with TGN-NP-H102 nanoparticles based on AD model, group D). The changes in T2 value in hippocampal CA1 area (T2-CA1) were analyzed, and the changes in cognitive function and LMA were tested with the Morris water maze experiment (Morris experiment). The results revealed that, the average PS (APS) of TGN-NP-H102 nanoparticles was 122.9±2.8 nm, and its average Zeta potential (AZP) was -28.8±0.2 mV. In group A, the TGN-NP-H102 nanoparticles still remained in the brain tissue homogenate by 74.3 ±4.8% after 10 hours, and the drug-release rate was 53.2 ± 3.2%. After 30 days of treatment, the T2-CA1 value of group D was lower (P &lt;0.05), and the average escape latency (AEL) and swimming distance in the Morris experiment were shorter versus group B (P &lt; 0.05). It indicated that, the brain-targeted TGN-NP-H102 nanoparticles prepared could act on the hippocampus of AD rats, and improve their LMA.