Rat Brain Tissue Research Articles

A bioanalytical assay for estimation of thymoquinone in rats cerebrospinal fluid and brain tissues of nasally administrated thymoquinone loaded lipo-polymeric nanoshells and its pharmacokinetic profiling

Thymoquinone (TH) has been one of the major phytochemical used in the treatment of cancers since long time, especially in the management of glioblastoma multiforme (GBM). The formulation of lipo-polymeric nanoshells (LPNs) and their nasal delivery are fascinating approaches for overcoming the drawbacks of low solubility and poor bioavailability of TH. Hence targeting LPNs to the brain requires a validated bioanalytical method for the assessment of TH concentration in Cerebrospinal fluid (CSF) and brain tissue homogenates (BTH). Therefore, the current work focuses on the development and validation of high-performance liquid chromatography (HPLC) method in CSF by employing nasal simulated fluid (NSF) as one of the major components of the mobile phase. The developed method was checked for linearity in the range of 0.05 to 1.6 μg/mL, having an r2 value of 0.999 with mean % recovery >95% and % RSD values below <2.0%. The developed method gave a clear separation of TH at 6.021 ± 0.17 min with an internal standard at 4.102 ± 0.09 min and a CSF spike at 2.170 ± 0.12 min. The developed method assisted in determining the in-vitro and in-vivo drug release study of LPNs, pharmacokinetic profiling, qualitative in-vivo brain uptake study, in-vitro cellular uptake, and generating stability data of formulated LPNs proposed for intranasal administration in rats.

Effect of Lupeol on the Expression of Neuro Inflammatory Signalling Molecules in Brain Tissues of High Fat Diet and Sucrose Fed Type-2 Diabetic Rats

Lupeol has anti- inflammatory, antimicrobial, anti-protozoal, anti-proliferative, anti-invasion, anti-angiogenic and cholesterol-lowering properties. However, the mechanisms underlying the effect of lupeol on neuroinflammatory signalling molecules have not yet been identified. The study was designed to study the effect of lupeol on the expression of inflammatory signalling molecules in brain tissue of high-fat diet and sucrose-fed type-2 diabetic rats. Adult male albino rats of Wistar 150–180 days old with 180–200 g body weight (b.wt) were divided into four groups of six rats each. Group I: Control (vehicle-treated): Group II: High fat diet-induced type-2 diabetic rats; Group III: Type-2 diabetic rats treated with lupeol (25 mg/kg b.wt/day) orally for 30 days and Group IV: Type-2 diabetic rats treated with metformin (50 mg/kg, b.wt/day orally for 30 days. After 30 days of treatment, the animals were anaesthetized, and brain tissue was dissected and used for the assessment of mRNA expression analysis. Type-2 diabetic animals showed a significant increase (p&lt;0.05) in TNF-α and IL-6 mRNA levels in brain tissue in high-fat diet-induced type-2 diabetic animals. However, lupeol treatment, effectively reduced (p&lt;0.05) the neuroinflammatory signaling molecules (TNF- α and IL-6 mRNA) showing that lupeol has significant role over the control of neuroinflammatory signaling. Our present findings clearly show that lupeol has a significant role in reducing neuroinflammation via the downregulation of TNF-α and IL-6 in brain tissues and hence, lupeol can be a potential natural drug for the treatment of diabetic neuropathy.

Fluorescent α-Conotoxin [Q1G, ΔR14]LvIB Identifies the Distribution of α7 Nicotinic Acetylcholine Receptor in the Rat Brain.

α7 nicotinic acetylcholine receptors (nAChRs) are mainly distributed in the central nervous system (CNS), including the hippocampus, striatum, and cortex of the brain. The α7 nAChR has high Ca2+ permeability and can be quickly activated and desensitized, and is closely related to Alzheimer's disease (AD), epilepsy, schizophrenia, lung cancer, Parkinson's disease (PD), inflammation, and other diseases. α-conotoxins from marine cone snail venom are typically short, disulfide-rich neuropeptides targeting nAChRs and can distinguish various subtypes, providing vital pharmacological tools for the functional research of nAChRs. [Q1G, ΔR14]LvΙB is a rat α7 nAChRs selective antagonist, modified from α-conotoxin LvΙB. In this study, we utilized three types of fluorescein after N-Hydroxy succinimide (NHS) activation treatment: 6-TAMRA-SE, Cy3 NHS, and BODIPY-FL NHS, labeling the N-Terminal of [Q1G, ΔR14]LvΙB under weak alkaline conditions, obtaining three fluorescent analogs: LvIB-R, LvIB-C, and LvIB-B, respectively. The potency of [Q1G, ΔR14]LvΙB fluorescent analogs was evaluated at rat α7 nAChRs expressed in Xenopus laevis oocytes. Using a two-electrode voltage clamp (TEVC), the half-maximal inhibitory concentration (IC50) values of LvIB-R, LvIB-C, and LvIB-B were 643.3 nM, 298.0 nM, and 186.9 nM, respectively. The stability of cerebrospinal fluid analysis showed that after incubation for 12 h, the retention rates of the three fluorescent analogs were 52.2%, 22.1%, and 0%, respectively. [Q1G, ΔR14]LvΙB fluorescent analogs were applied to explore the distribution of α7 nAChRs in the hippocampus and striatum of rat brain tissue and it was found that Cy3- and BODIPY FL-labeled [Q1G, ΔR14]LvΙB exhibited better imaging characteristics than 6-TAMARA-. It was also found that α7 nAChRs are widely distributed in the cerebral cortex and cerebellar lobules. Taking into account potency, imaging, and stability, [Q1G, ΔR14]LvΙB -BODIPY FL is an ideal pharmacological tool to investigate the tissue distribution and function of α7 nAChRs. Our findings not only provide a foundation for the development of conotoxins as visual pharmacological probes, but also demonstrate the distribution of α7 nAChRs in the rat brain.

Radioprotective effect of Cerebrolysin in the Rat's Brain Tissues.

The aim of this study is to evaluate radioprotective effects of Cerebrolysin (CBL) in rats' brain tissues after local irradiation. CBL has demonstrated antioxidant, anti-inflammatory, and tissue repair properties. In this study, the radioprotective effects of CBL in the brain tissues of rats after Irradiation (IR) (50 mg/ kg) were evaluated. The levels of different oxidative stress markers, including malondialdehyde (MDA), nitric oxide (NO), glutathione peroxidase (GPx), catalase (CAT), and superoxide dismutase (SOD) were examined after treatment with radiation and CBL. First, 20 male adult Wistar rats weighing 180-200 g were used. The animals were exposed to a single fraction of 15Gy using a linear accelerator unit at a dose rate of 200 cGy/mine. In this study, to check the amount of oxidative stress following the IR, the level of four markers MDA, NO, GPx, CAT, and SOD were examined and measured using the spectrophotometric method and purchased kits. The results showed that compared to the IR group, the administration of CBL increases the levels of GPX and SOD significantly (p < 0.05). Our finding suggests that CBL has radioprotective effects on the brain by enhancing antioxidant defense mechanisms.

Neutrophil Extracellular Traps Regulate Surgical Brain Injury by Activating the cGAS-STING Pathway

Surgical brain injury (SBI), induced by neurosurgical procedures or instruments, has not attracted adequate attention. The pathophysiological process of SBI remains sparse compared to that of other central nervous system diseases thus far. Therefore, novel and effective therapies for SBI are urgently needed. In this study, we found that neutrophil extracellular traps (NETs) were present in the circulation and brain tissues of rats after SBI, which promoted neuroinflammation, cerebral edema, neuronal cell death, and aggravated neurological dysfunction. Inhibition of NETs formation by peptidylarginine deiminase (PAD) inhibitor or disruption of NETs with deoxyribonuclease I (DNase I) attenuated SBI-induced damages and improved the recovery of neurological function. We show that SBI triggered the activation of cyclic guanosine monophosphate–adenosine monophosphate synthase stimulator of interferon genes (cGAS-STING), and that inhibition of the cGAS-STING pathway could be beneficial. It is worth noting that DNase I markedly suppressed the activation of cGAS-STING, which was reversed by the cGAS product cyclic guanosine monophosphate–adenosine monophosphate (cGMP-AMP, cGAMP). Furthermore, the neuroprotective effect of DNase I in SBI was also abolished by cGAMP. NETs may participate in the pathophysiological regulation of SBI by acting through the cGAS-STING pathway. We also found that high-dose vitamin C administration could effectively inhibit the formation of NETs post-SBI. Thus, targeting NETs may provide a novel therapeutic strategy for SBI treatment, and high-dose vitamin C intervention may be a promising translational therapy with an excellent safety profile and low cost.Graphical The schematic diagram shows the formation of NETs activated cGAS-STING pathway after SBI, leading to increased microglia activation, accompanied with elevation of inflammatory factors, which in turn aggravated brain injury.

Structural, Functional, and Genetic Changes Surrounding Electrodes Implanted in the Brain.

Implantable neurotechnology enables monitoring and stimulating of the brain signals responsible for performing cognitive, motor, and sensory tasks. Electrode arrays implanted in the brain are increasingly used in the clinic to treat a variety of sources of neurological diseases and injuries. However, the implantation of a foreign body typically initiates a tissue response characterized by physical disruption of vasculature and the neuropil as well as the initiation of inflammation and the induction of reactive glial states. Likewise, electrical stimulation can induce damage to the surrounding tissue depending on the intensity and waveform parameters of the applied stimulus. These phenomena, in turn, are likely influenced by the surface chemistry and characteristics of the materials employed, but further information is needed to effectively link the biological responses observed to specific aspects of device design. In order to inform improved design of implantable neurotechnology, we are investigating the basic science principles governing device-tissue integration. We are employing multiple techniques to characterize the structural, functional, and genetic changes that occur in the cells surrounding implanted electrodes. First, we have developed a new "device-in-slice" technique to capture chronically implanted electrodes within thick slices of live rat brain tissue for interrogation with single-cell electrophysiology and two-photon imaging techniques. Our data revealed several new observations of tissue remodeling surrounding devices: (a) there was significant disruption of dendritic arbors in neurons near implants, where losses were driven asymmetrically on the implant-facing side. (b) There was a significant loss of dendritic spine densities in neurons near implants, with a shift toward more immature (nonfunctional) morphologies. (c) There was a reduction in excitatory neurotransmission surrounding implants, as evidenced by a reduction in the frequency of excitatory postsynaptic currents (EPSCs). Lastly, (d) there were changes in the electrophysiological underpinnings of neuronal spiking regularity. In parallel, we initiated new studies to explore changes in gene expression surrounding devices through spatial transcriptomics, which we applied to both recording and stimulating arrays. We found that (a) device implantation is associated with the induction of hundreds of genes associated with neuroinflammation, glial reactivity, oligodendrocyte function, and cellular metabolism and (b) electrical stimulation induces gene expression associated with damage or plasticity in a manner dependent upon the intensity of the applied stimulus. We are currently developing computational analysis tools to distill biomarkers of device-tissue interactions from large transcriptomics data sets. These results improve the current understanding of the biological response to electrodes implanted in the brain while producing new biomarkers for benchmarking the effects of novel electrode designs on responses. As the next generation of neurotechnology is developed, it will be increasingly important to understand the influence of novel materials, surface chemistries, and implant architectures on device performance as well as the relationship with the induction of specific cellular signaling pathways.

The effect of aerobic exercise and food restriction on the expression of alpha-synuclein and sirtuin-1 proteins in the brain tissue of parkinson rats model

Introduction: Alpha-synuclein and sirtuin-1 proteins are genetically and neuropathologically related to the Parkinson's disease. This study aimed to investigate the effect of 12 weeks of aerobic exercise and food restriction on the expression of alpha-synuclein and sirtuin-1 proteins in the brain tissue of Parkinsonian rats. Method: In the current experimental study, 40 white male Wistar rats were randomly divided into five groups of 8 series, including healthy control, Parkinson's control, exercise parkinsonism, food restriction parkinsonism, and exercise+food restriction parkinsonism. The exercise program consisted of 3 months and five weekly sessions with an intensity of 75-80% oxygen consumption. According to the food restriction, the daily intake of each rat was determined to be 11 grams. The western blot method used to determine the expression levels of alpha-synuclein and sirtuin-1 proteins. Data were analyzed using a two-way analysis of variance and Tukey's test with SPSS25 software. Results: There was a significant difference in alpha-synuclein and sirtuin-1 in healthy and Parkinson's rats. The expression of alpha-synuclein in the food restriction+aerobic exercise group significantly decreased compared to the Parkinson's control group (P=0.001). Also, a significant increase in sirtuin-1 was observed in the aerobic training+food restriction group compared to the Parkinson's control group (P=0.001). Conclusion: Three months of aerobic activity have a positive effect on increasing sirtuin-1 and decreasing alpha-synuclein in the hippocampus of rat model of Parkinson, while the effect of exercise with food restriction is greater. Therefore, aerobic exercise and moderate food restriction are recommended as a method of treatment for patients with Parkinson's, with drug interventions.

Effect of ethanol extract of nigella sativa L seeds and propofol on BDNF protein level as neuroplasticity and neuroprotection of traumatic brain injury in rats

Background Traumatic brain injury (TBI) is a change in brain function or evidence of brain pathology caused by external mechanical forces. Brain Derived Neurotrophic Factor (BDNF) is a neurotropin that functions as a neuron protective. Nigella sativa L is reported to have an antioxidant effect, administration of Nigella Sativa L to rats treated with ischemia-reperfusion brain injury. Propofol is an anesthetic agent frequently used intravenously in the management of TBI. The effect of propofol on brain tissue after TBI may be neuroprotective. We aimed to compare the potential of Nigella sativa L and propofol as neuroplasticity and neuroprotection in rats with TBI. Methods This was a laboratory experimental animal model with the post-test only control group design, namely measuring the effect of treatment by comparing the five groups of rats consisting of 30 rats. BDNF levels in rat brain tissue were collected at day 7 of treatment and measured by ELISA. Results The average BDNF protein levels per group, namely G1 (221,243 pg/mL), G2 (172,139 pg/mL), G3 (255,483 pg/mL), G4 (227,089 pg/mL), and G5 (272,603 pg/mL) respectively. Based on the ANOVA statistic, p-value = 0.032 (there was a significant difference between groups), with the Levene Test (0.077) or having variance between the same groups, sequentially the difference in average BDNF protein levels of the five groups is G5&gt;G3&gt;G4&gt;G1&gt;G2, meaning that the combination of Nigella sativa and propofol has more potential to increase BDNF protein levels than Nigella sativa, and Nigella sativa has more potential than propofol. Conclusion We concluded that both nigella sativa and propofol have the potential to increase BDNF protein levels. Nigella Sativa L had a better effect than propofol in repairing damaged neuron cells (neuroplasticity) and increasing BDNF protein levels (neuroprotection) for 7 days of administration in rat traumatic brain injury.

Secondary reagents for immunohistochemical research of rat brain

BACKGROUND: The critical factor when working with immunohistochemistry in laboratory animals is to select appropriate secondary antibodies, that allow clear and specific visualization of tissue antigens. Many reliable secondary reagents are currently not available for purchase, which determines the high relevance of replacing them with other detection systems.AIM: To verify the effectiveness of available secondary reagents for immunohistochemical research of the rat brain.METHODS: Brain samples from Wistar and SHR rats (n=4) were used for the study. Iba-1, GFAP and vimentin immunohistochemistry was carried out using various polymer-based detection systems, namely UltraVision Quanto, N-Histofine Simple stain MAX PO ® and UnoVue Rabbit HRP.RESULTS: All three studied polymer systems demonstrated visualisation of target proteins in brain tissues and cells corresponding to the general understanding of structures containing Iba-1, GFAP and vimentin. The UnoVue Rabbit HRP and Quanto Detection System HRP kits showed good and similarly specific immunohistochemical reaction. However, the UnoVue Rabbit HRP kit was less sensitive compared to the Quanto Detection System HRP. The reaction with N-Histofine® Simple Stain MAX PO was not optimal due to the presence of non-specific background staining that was not present with other reagents.CONCLUSION: Two secondary antibody kits from the three studied showed optimal efficiency of immunohistochemical reaction and minimal background staining. N-Histofine® Simple Stain MAX PO is not suitable for immunohistochemical research of rat brain tissue.

In vitro characterization of [125I]HY-3-24, a selective ligand for the dopamine D3 receptor.

Dopamine D3 receptor (D3R) ligands have been studied for the possible treatment of neurological and neuropsychiatric disorders. However, selective D3R radioligands for in vitro binding studies have been challenging to identify due to the high structural similarity between the D2R and D3R. In a prior study, we reported a new conformationally-flexible benzamide scaffold having a high affinity for D3R and excellent selectivity vs. D2R. In the current study, we characterized the in vitro binding properties of a new radioiodinated ligand, [125I]HY-3-24. In vitro binding studies were conducted in cell lines expressing D3 receptors, rat striatal homogenates, and rat and non-human primate (NHP) brain tissues to measure regional brain distribution of this radioligand. HY-3-24 showed high potency at D3R (Ki = 0.67 ± 0.11 nM, IC50 = 1.5 ± 0.58 nM) compared to other D2-like dopamine receptor subtypes (D2R Ki = 86.7 ± 11.9 nM and D4R Ki > 1,000). The Kd (0.34 ± 0.22 nM) and Bmax (38.91 ± 2.39 fmol/mg) values of [125I]HY-3-24 were determined. In vitro binding studies in rat striatal homogenates using selective D2R and D3R antagonists confirmed the D3R selectivity of [125I]HY-3-24. Autoradiography results demonstrated that [125I]HY-3-24 specifically binds to D3Rs in the nucleus accumbens, islands of Calleja, and caudate putamen in rat and NHP brain sections. These results suggest that [125I]HY-3-24 appears to be a novel radioligand that exhibits high affinity binding at D3R, with low binding to other D2-like dopamine receptors. It is anticipated that [125I]HY-3-24 can be used as the specific D3R radioligand.

Effect of Dl-3-n-Butylphthalide on olfaction in rotenone-induced Parkinson's rats.

Parkinson's disease (PD) is the second most common neurodegenerative disease after Alzheimer's disease. Olfactory dysfunction (OD) is an important nonmotor feature of PD. Dl-3-n-Butylphthalide (NBP) is a synthetic compound isolated from Apium graveolens seeds. The present study was conducted to investigate the effect of NBP on olfaction in rotenone-induced Parkinson's rats to explore the mechanism and pathway of OD in PD. The PD model was established using rotenone-induced SD rats, divided into blank control, model, and treatment groups. A sham group was also established, with 10 rats in each group. The treatment group was given NBP (1 mg/kg, 10 mg/kg, and 100 mg/kg, dissolved in soybean oil) intragastrically for 28 days. Meanwhile, the control group rats were given intra-gastrically soybean oil. After behavioral testing, all rats were executed, and brain tissue was obtained. Proteomics and Proteomic quantification techniques (prm) quantification were used to detect proteomic changes in rat brain tissues. Compared with the control group, the model group showed significant differences in behavioral tests, and this difference was reduced after treatment. Proteomics results showed that after treatment with high-dose NBP, there were 42 differentially expressed proteins compared with the model group. Additionally, the olfactory marker (P08523) showed a significant upregulation difference. We then selected 22 target proteins for PRM quantification and quantified 17 of them. Among them, the olfactory marker protein was at least twofold upregulated in the RTH group compared to the model group.

Antioxidant Properties of Resveratrol in the Brain Tissues of Rats after Radiotherapy

Background:: In recent years, there has been growing interest in the use of herbal products as complementary or alternative therapies for cancer patients. RVT has been extensively studied for its potential role in managing oxidative stress in cancer patients undergoing treatment. Method:: In this study, the radioprotective effects of Resveratrol (RVT) (200 mg/kg) were evaluated in the brain tissues of rats after Radiation Therapy (RT). 20 male adult Wistar rats weighing 180–200 g were used. The animals were exposed to single-fraction Irradiation (IR) (15 Gy) using a linear accelerator unit (Siemens Primus) at a dosage rate of 200 cGy/min at a distance of 100 cm from the source to the axis. Results:: The results of the study showed that compared to the IR group, administration of RVT decreased the levels of MDA and NOx, and increased the levels of CAT and SOD significantly (p&lt;0.05). Conclusion:: Finally, our finding suggests that RVT could have radioprotective effects on the brain by enhancing antioxidant defense mechanisms.

Restoring brain health: Electroacupuncture at GB20 and LR3 for migraine mitigation through mitochondrial restoration.

Electroacupuncture (EA) is a promising alternative therapy for migraine, with mitochondrial dysfunction hypothesized as a pivotal mechanism in migraine pathophysiology. This research endeavors to investigate the therapeutic potential of EA in addressing migraines and shed light on the associated mechanisms linked to mitochondrial anomalies. Migraine in rats was induced by 10 mg/kg nitroglycerin, followed by 2/15 Hz EA treatment at GB20 and LR3. Nociceptive behavior was recorded via a camera and analyzed using EthoVision XT 12.0 software. The hind-paw withdrawal threshold was assessed using the von Frey test. We assessed the levels of calcitonin gene-related peptide (CGRP), nitric oxide (NO), and endothelin (ET) - key parameters in migraine pathophysiology using immunohistochemistry and enzyme-linked immunosorbent assay. Mitochondrial morphology in brain tissues was observed through transmission electron microscopy. Reactive oxygen species (ROS) level in mitochondria was measured by flow cytometry. The levels of PINK1 and Parkin were assessed using Western blot analysis. EA at GB20 and LR3 decreased nociceptive behaviors (resting and grooming) and increased exploratory and locomotor behaviors in migraine rats. The hind-paw withdrawal threshold in migraine rats was significantly elevated following EA treatment. Post-EA treatment, levels of CGRP and NO decreased, while ET level increased, suggesting an alteration in pain and vascular physiology. Notably, EA treatment mitigated the mitochondrial damage and reduced ROS level in the brain tissues of migraine rats. EA treatment upregulated the expression of PINK1 and Parkin in migraine rats. EA at GB20 and LR3 may treat migraine by alleviating PINK1/Parkin-mediated mitochondrial dysfunction.

Biological and physical studies on the protective and therapeutic roles of ashwagandha seed extract against the potential toxic effect of amoxicillin in rats

Background Ashwagandha plant enhances the body’s defense against toxicants through improving the cell-mediated immunity. Objective The present work was performed to assess the protective and therapeutic efficiencies of ashwagandha seed extract (ASE) against the harmful effects of amoxicillin (AM) treatment on biological and physical parameters in the brain, liver, and testes tissues of rats. Materials and methods Total RNA was isolated from brain, liver, and testes tissues to assess the gene expression of steroid 5 alpha-reductase 1 (5α-R1), multidrug resistant 1b (mdr1b), and luteinizing hormone receptor (LHR) genes, respectively. The cDNA was synthesized and real-time polymerase chain reaction (Real-time PCR) was performed, total antioxidant capacity (TAC) was measured. Histological examination and physiological tests for the cerebral cortex of brain tissue were recorded. Results and conclusion Our findings revealed that AM treatment (90 mg/kg. b.wt.) caused detrimental effects in all biological parameters, including up-regulation of gene expressions and reduction of TAC values in brain, liver, and testes tissues, in addition to severe damage to histological architectures in the cerebral cortex, including hemorrhage and neurodegeneration. ASE treatment at different doses (100, 200, and 300 mg/kg) significantly enhanced the biological and physical parameters. As the ASE dose level increased, it was observed that these improvements in gene expressions and TAC also increased. Gene expression enhancements were more pronounced in the therapeutic efficacy of ASE, whereas TAC enhancements were more pronounced in the protective efficacy of this medicinal plant extract, particularly in brain and liver tissues. In terms of histopathological parameters, the damage caused by AM was reduced by using 300 mg/kg of protective ASE than 200 mg/kg of therapeutic ASE. Biophysical investigation indicated that relaxation time and enthalpy were restored and improved, while DC conductivity was not recovered by ASE use against AM damages. The present investigation provided biological and physical evidence for protective and therapeutic efficiencies of ASE against lesion effects of AM in the previously mentioned tissues of rats.

Effect of Xuming Decoction in Records of Proved Prescriptions, Ancient a nd Modern on cerebral ischemic injury and angiogenesis in rat model of acute cerebral infarction

This paper aims to explore the effect of Xuming Decoction in the Records of Proved Prescriptions, Ancient and Modern on cerebral ischemic injury and angiogenesis in the rat model of acute cerebral infarction. SD rats were randomized into 6 groups: sham group, model group, low-, medium-, and high-dose(5.13, 10.26, and 20.52 g·kg~(-1), respectively) Xuming Decoction groups, and butylphthalide(0.06 g·kg~(-1)) group. After the successful establishment of the rat model by middle cerebral artery occlusion(MCAO), rats in the sham and model groups were administrated with distilled water and those in other groups with corresponding drugs for 7 consecutive days. After the neurological function was scored, all the rats were sacrificed, and the brain tissue samples were collected. The degree of cerebral ischemic injury was assessed by the neurological deficit score and staining with 2,3,5-triphenyltetrazolium chloride. Hematoxylin-eosin staining was performed to observe the pathological changes in the brain. Transmission electron microscopy was employed to observe the ultrastructures of neurons and microvascular endothelial cells(ECs) on the ischemic side of the brain tissue. Immunofluorescence assay was employed to detect the expression of von Willebrand factor(vWF) and hematopoietic progenitor cell antigen CD34(CD34) in the ischemic brain tissue. Real-time PCR and Western blot were employed to determine the mRNA and protein levels, respectively, of Runt-related transcription factor 1(RUNX1), vascular endothelial growth factor(VEGF), angiopoietin-1(Ang-1), angiopoietin-2(Ang-2), and VEGF receptor 2(VEGFR2) in the ischemic brain tissue. The results showed that compared with the sham group, the model group showed increased neurological deficit score and cerebral infarction area(P&lt;0.01), pathological changes, and damaged ultrastructure of neurons and microvascular ECs in the ischemic brain tissue. Furthermore, the modeling up-regulated the mRNA levels of RUNX1, VEGF, Ang-1, Ang-2, and VEGFR2(P&lt;0.01) and the protein levels of vWF, CD34, RUNX1, VEGF, Ang-1, Ang-2, and VEGFR2(P&lt;0.05 or P&lt;0.01). Compared with the model group, high-dose Xuming Decoction and butylphthalide decreased the neurological deficit score and cerebral infarction area(P&lt;0.01) and alleviated the pathological changes and damage of the ultrastructure of neurons and microvascular ECs in the ischemic brain tissue. Moreover, they up-regulated the mRNA levels of RUNX1, VEGF, Ang-1, Ang-2, and VEGFR2(P&lt;0.01) and the protein levels of vWF, CD34, RUNX1, VEGF, Ang-1, Ang-2, and VEGFR2(P&lt;0.01). The results suggest that Xuming Decoction in the Records of Proved Prescriptions, Ancient and Modern can promote the angiogenesis and collateral circulation establishment to alleviate neurological dysfunction of the ischemic brain tissue in MCAO rats by regulating the RUNX1/VEGF pathway.

Development and validation of a method for the quantitative determination of monoamine neurotransmitters and their metabolites in rat brain tissue using HPLC-MS/MS

Background. Determining changes in the content of monoamine neurotransmitters and their metabolites in brain structures is a necessary part of studying the pharmacodynamics of antiparkinsonian drugs. A method for the joint determination of norepinephrine, adrenaline, dopamine, serotonin, 5-hydroxyindole-3-acetic acid, 3,4-dihydroxyphenylacetic acid, homovanillic acid, vanillylmandelic acid in rat brain tissue has not previously been developed.The aim of the study. To develop and to validate a method for the quantitative determination of norepinephrine, adrenaline, dopamine, serotonin, 5-hydroxyindole3-acetic acid, 3,4-dihydroxyphenylacetic acid, homovanillic acid, vanillylmandelic acid in rat brain tissue using high-performance liquid chromatography in combination with tandem mass spectrometry (HPLC-MS/MS).Materials and methods. A method for determining monoamine mediators and their metabolites was developed using the HPLC-MS/MS method. Brain tissue homogenates were prepared using a mechanical hand-operated homogenizer. The effect of various antioxidants on the stability of norepinephrine, adrenaline, dopamine and 3,4-dihydroxyphenylacetic acid in the test samples was studied.Results. Chromatographic separation of sample components was carried out using two Synergi Max RP (20 × 2.0 mm, 2.5 µm) and Synergi Fusion RP 80Å (250 × 4.6 mm, 4 µm) chromatographic columns. Elution was carried out in a gradient mode using a mobile phase based on methanol and a 0.1% solution of formic acid in water. To prepare homogenate batches, the samples were diluted with a solution of internal standards in methanol. A 5% aqueous solution of ascorbic acid was chosen as an antioxidant stabilizer.Conclusion. The developed methodology has been fully validated and meets the requirements of Russian and international guidelines. The chosen stabilization method allows samples of brain homogenates to be stored for 30 days after collection.

Imaging Mass Spectrometry of Isotopically Resolved Intact Proteins on a Trapped Ion-Mobility Quadrupole Time-of-Flight Mass Spectrometer.

In this work, we demonstrate rapid, high spatial, and high spectral resolution imaging of intact proteins by matrix-assisted laser desorption/ionization (MALDI) imaging mass spectrometry (IMS) on a hybrid quadrupole-reflectron time-of-flight (qTOF) mass spectrometer equipped with trapped ion mobility spectrometry (TIMS). Historically, untargeted MALDI IMS of proteins has been performed on TOF mass spectrometers. While advances in TOF instrumentation have enabled rapid, high spatial resolution IMS of intact proteins, TOF mass spectrometers generate relatively low-resolution mass spectra with limited mass accuracy. Conversely, the implementation of MALDI sources on high-resolving power Fourier transform (FT) mass spectrometers has allowed IMS experiments to be conducted with high spectral resolution with the caveat of increasingly long data acquisition times. As illustrated here, qTOF mass spectrometers enable protein imaging with the combined advantages of TOF and FT mass spectrometers. Protein isotope distributions were resolved for both a protein standard mixture and proteins detected from a whole-body mouse pup tissue section. Rapid (∼10 pixels/s) 10 μm lateral spatial resolution IMS was performed on a rat brain tissue section while maintaining isotopic spectral resolution. Lastly, proof-of-concept MALDI-TIMS data was acquired from a protein mixture to demonstrate the ability to differentiate charge states by ion mobility. These experiments highlight the advantages of qTOF and timsTOF platforms for resolving and interpreting complex protein spectra generated from tissue by IMS.

Formation of multiple ion types during MALDI imaging mass spectrometry analysis of Mitragyna speciosa alkaloids in dosed rat brain tissue

Mitragyna speciosa, more commonly known as kratom, has emerged as an alternative to treat chronic pain and addiction. However, the alkaloid components of kratom, which are the major contributors to kratom's pharmaceutical properties, have not yet been fully investigated. In this study, matrix-assisted laser desorption/ionization (MALDI) imaging mass spectrometry was used to map the biodistribution of three alkaloids (corynantheidine, mitragynine, and speciogynine) in rat brain tissues. The alkaloids produced three main ion types during MALDI analysis: [M + H]+, [M − H]+, and [M − 3H]+. Contrary to previous reports suggesting that the [M − H]+ and [M − 3H]+ ion types form during laser ablation, these ion types can also be produced during the MALDI matrix application process. Several strategies are proposed to accurately map the biodistribution of the alkaloids. Due to differences in the relative abundances of the ions in different biological regions of the tissue, differences in ionization efficiencies of the ions, and potential overlap of the [M − H]+ and [M − 3H]+ ion types with endogenous metabolites of the same empirical formula, a matrix that mainly produces the [M + H]+ ion type is optimal for accurate mapping of the alkaloids. Alternatively, the most abundant ion type can be mapped or the intensities of all ion types can be summed together to generate a composite image. The accuracy of each of these approaches is explored and validated.

2-Hydroxy-5-nitro-3-(trifluoromethyl)pyridine as a Novel Matrix for Enhanced MALDI Imaging of Tissue Metabolites.

Matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI), which is a label-free imaging technique, determines the spatial distribution and relative abundance of versatile endogenous metabolites in tissues. Meanwhile, matrix selection is generally regarded as a pivotal step in MALDI tissue imaging. This study presents the first report of a novel MALDI matrix, 2-hydroxy-5-nitro-3-(trifluoromethyl)pyridine (HNTP), for the in situ detection and imaging of endogenous metabolites in rat liver and brain tissues by MALDI-MS in positive-ion mode. The HNTP matrix exhibits excellent characteristics, including strong ultraviolet absorption, μm-scale matrix crystals, high chemical stability, low background ion interference, and high metabolite ionization efficiency. Notably, the HNTP matrix also shows superior detection capabilities, successfully showing 185 detectable metabolites in rat liver tissue sections. This outperforms the commonly used matrices of 2,5-dihydroxybenzoic acid and 2-mercaptobenzothiazole, which detect 145 and 120 metabolites from the rat liver, respectively. Furthermore, a total of 152 metabolites are effectively detected and imaged in rat brain tissue using the HNTP matrix, and the spatial distribution of these compounds clearly shows the heterogeneity of the rat brain. The results demonstrate that HNTP is a new and powerful positive-ion mode matrix to enhance the analysis of metabolites in biological tissues by MALDI-MSI.

M6A reader YTHDC2 mediates NCOA4 mRNA stability affecting ferritinophagy to alleviate secondary injury after intracerebral haemorrhage

ABSTRACT Oxidative stress and neuronal dysfunction caused by intracerebral haemorrhage (ICH) can lead to secondary injury. The m6A modification has been implicated in the progression of ICH. This study aimed to investigate the role of the m6A reader YTHDC2 in ICH-induced secondary injury. ICH models were established in rats using autologous blood injection, and neuronal cell models were induced with Hemin. Experiments were conducted to overexpress YTH domain containing 2 (YTHDC2) and examine its effects on neuronal dysfunction, brain injury, and neuronal ferritinophagy. RIP-qPCR and METTL3 silencing were performed to investigate the regulation of YTHDC2 on nuclear receptor coactivator 4 (NCOA4). Finally, NCOA4 overexpression was used to validate the regulatory mechanism of YTHDC2 in ICH. The study found that YTHDC2 expression was significantly downregulated in the brain tissues of ICH rats. However, YTHDC2 overexpression improved neuronal dysfunction and reduced brain water content and neuronal death after ICH. Additionally, it reduced levels of ROS, NCOA4, PTGS2, and ATG5 in the brain tissues of ICH rats, while increasing levels of FTH and FTL. YTHDC2 overexpression also decreased levels of MDA and Fe2+ in the serum, while promoting GSH synthesis. In neuronal cells, YTHDC2 overexpression alleviated Hemin-induced injury, which was reversed by Erastin. Mechanistically, YTHDC2-mediated m6A modification destabilized NCOA4 mRNA, thereby reducing ferritinophagy and alleviating secondary injury after ICH. However, the effects of YTHDC2 were counteracted by NCOA4 overexpression. Overall, YTHDC2 plays a protective role in ICH-induced secondary injury by regulating NCOA4-mediated ferritinophagy.