Browsing by Author "Liu, Ran"
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Item Administration of 5-methoxyindole-2-carboxylic acid that potentially targets mitochondrial dihydrolipoamide dehydrogenase confers cerebral preconditioning against ischemic stroke injury(2018-03-14) Li, Rongrong; Li, Wenjun; Ren, Ming; Thangthaeng, Nopporn; Sumien, Nathalie; Liu, Ran; Yang, Shaohua; Simpkins, James; Forster, Michael J.; Yan, Liang-Jun; Wu, JinziPurpose: The purpose of this study was to investigate a possible role of mitochondrial dihydrolipoamide dehydrogenase (DLDH) as a chemical preconditioning target for neuroprotection against ischemic injury. Methods: We used 5-methoxyindole-2-carboxylic acid (MICA), a reportedly reversible DLDH inhibitor, as the preconditioning agent and administered MICA to rats mainly via dietary intake. Upon completion of 4 week's MICA treatment, rats underwent 1 h transient ischemia and 24 h reperfusion followed by tissue collection. Results: Our results show that MICA protected the brain against ischemic stroke injury as the infarction volume of the brain from the MICA-treated group was significantly smaller than that from the control group. Data were then collected without or with stroke surgery following MICA feeding. It was found that in the absence of stroke following MICA feeding, DLDH activity was lower in the MICA treated group than in the control group, and this decreased activity could be partly due to DLDH protein sulfenation. Moreover, DLDH inhibition by MICA was also found to upregulate the expression of NAD(P)H-ubiquinone oxidoreductase 1(NQO1) via the Nrf2 signaling pathway. In the presence of stroke following MICA feeding, decreased DLDH activity and increased Nrf2 signaling were also observed along with increased NQO1 activity, decreased oxidative stress, decreased cell death, and increased mitochondrial ATP output. We also found that MICA had a delayed preconditioning effect four weeks post MICA treatment. Conclusion: Our study indicates that administration of MICA confers chemical preconditioning and neuroprotection against ischemic stroke injury.Item Artemisinin Prevents Glutamate-Induced Neuronal Cell Death Via Akt Pathway Activation(Frontiers Media S.A., 2018-04-20) Lin, Shao-Peng; Li, Wenjun; Winters, Ali; Liu, Ran; Yang, ShaohuaArtemisinin is an anti-malarial drug that has been in use for almost half century. Recently, novel biological effects of artemisinin on cancer, inflammation-related disorders and cardiovascular disease were reported. However, neuroprotective actions of artemisinin against glutamate-induced oxidative stress have not been investigated. In the current study, we determined the effect of artemisinin against oxidative insult in HT-22 mouse hippocampal cell line. We found that pretreatment of artemisinin declined reactive oxygen species (ROS) production, attenuated the collapse of mitochondrial membrane potential induced by glutamate and rescued HT-22 cells from glutamate-induced cell death. Furthermore, our study demonstrated that artemisinin activated Akt/Bcl-2 signaling and that neuroprotective effect of artemisinin was blocked by Akt-specific inhibitor, MK2206. Taken together, our study indicated that artemisinin prevented neuronal HT-22 cell from glutamate-induced oxidative injury by activation of Akt signaling pathway.Item Artemisinin Protects Oxidative Stress-induced Neuronal Apoptosis Via Up-Regulation of Akt/Bcl-2 Signaling(2017-03-14) Liu, Ran; Xie, Luokun; Li, Wenjun; Winters, Ali; Chaudhari, Kiran; Prah, Jude; Yang, Shao-Hua; Lin, Shao-PengPurpose: Artemisinin is a powerful anti-malarial drug that has been in use for decades. Recently, the novel biological effects of artemisinin on cancer, inflammation-related disorders, and cardiovascular disease were reported. The aim of this study was to explore the neuroprotective actions of artemisinin. Methods: The model of glutamate-induced oxidative injury in HT22 hippocampal cells was established to simulate cellular ischemic model. We investigated the effect of artemisinin on oxidative stress-induced cell apoptosis death and the activity of Akt/Bcl-2 pathway in HT22 cells. Results: Pretreatment with artemisinin attenuated reactive oxygen species (ROS) generations, preventing the decline of mitochondrial membrane potential and rescued the HT22 cells form glutamate-induced apoptosis death. The Akt/Bcl-2 pathway was activated by artemisinin in time dependent manner. Furthermore, the artemisinin inhibitor MK2206 blocked the neuroprotective effect of artemisinin. Conclusions: Artemisinin protects neuronal HT22 cell from glutamate-induced oxidative injury and apoptosis via Akt/Bcl-signaling, thereby might be applicated for clinical neurological therapy.Item Characterizing Region-Specific Glucose Metabolic Profile of the Rodent Brain Using Seahorse XFe96 Analyzer(2022) Wang, Linshu; Chaudhari, Kiran; Winters, Ali; Sun, Yuanhong; Liu, Ran; Yang, ShaohuaPurpose: The brain is highly complex with diverse structural characteristics in accordance with specific functions. Accordingly, differences in regional function, cellular compositions, and active metabolic pathways may link to differences in glucose metabolism at different brain regions. A recent study using imaging mass spectrometry demonstrated that some of the glucose metabolism enzymes and ATP level vary dramatically cross the brain. Disruption of glucose metabolism forms the pathophysiological basis for many brain disorders. Therefore, the brain spatial metabolic signatures are of high relevance in our understanding of the normal brain physiology and neuropathology of neurological diseases. Method: We optimized an acute biopsy punching method and characterized region-specific glucose metabolism of rat and mouse brain by a Seahorse XFe96 analyzer. Results: In the current study, we demonstrated that 0.5 mm diameter tissue punches from 180-µm thick brain sections allow metabolic measurements of anatomically defined brain structures using Seahorse XFe96 analyzer. We found that the cerebellum displays a more quiescent phenotype of glucose metabolism than cerebral cortex, basal ganglia, and hippocampus. In addition, the cerebellum has higher AMPK activation than other brain regions evidenced by the expression of pAMPK, upstream pLKB1, and downstream pACC. Furthermore, rodent brain has relatively low mitochondrial oxidative phosphorylation efficiency with up to 30% of respiration linked to proton leak. Conclusions: The present study determined the region-specific glucose metabolic profile of rodent brain using acute biopsy punches and Seahorse XFe96 analyzer. The metabolic flux analysis indicated that the cerebellum has a more quiescent phenotype of glucose metabolism as compared with the cerebrum. In addition, glucose metabolism might be less efficient in the brain than we expected, with relatively large component of proton leak-linked respiration.Item Determination of metformin bio-distribution by LC-MS/MS in mice treated with a clinically relevant paradigm(PLOS, 2020-06-11) Chaudhari, Kiran; Wang, Jianmei; Xu, Yong; Winters, Ali; Wang, Linshu; Dong, Xiaowei; Cheng, Eric Y.; Liu, Ran; Yang, ShaohuaMetformin, an anti-diabetes drug, has been recently emerging as a potential "anti-aging" intervention based on its reported beneficial actions against aging in preclinical studies. Nonetheless, very few metformin studies using mice have determined metformin concentrations and many effects of metformin have been observed in preclinical studies using doses/concentrations that were not relevant to therapeutic levels in human. We developed a liquid chromatography-tandem mass spectrometry protocol for metformin measurement in plasma, liver, brain, kidney, and muscle of mice. Young adult male and female C57BL/6 mice were voluntarily treated with metformin of 4 mg/ml in drinking water which translated to the maximum dose of 2.5 g/day in humans. A clinically relevant steady-state plasma metformin concentrations were achieved at 7 and 30 days after treatment in male and female mice. Metformin concentrations were slightly higher in muscle than in plasma, while, ~3 and 6-fold higher in the liver and kidney than in plasma, respectively. Low metformin concentration was found in the brain at ~20% of the plasma level. Furthermore, gender difference in steady-state metformin bio-distribution was observed. Our study established steady-state metformin levels in plasma, liver, muscle, kidney, and brain of normoglycemic mice treated with a clinically relevant dose, providing insight into future metformin preclinical studies for potential clinical translation.Item Development of a serum free astrocyte culture method that mimic resting in vivo astrocyte phenotype(2019-03-08) Winters, Ali; Chaudari, Kiran; Hersh, Jessica; Liu, Ran; Yang, Shaohua; Prah, JudePurpose Primary astrocyte cultures have been extensively used for characterization of astrocytes functions in physiological and pathological conditions. The current primary astrocytes are mostly maintained in fetal bovine serum (FBS) containing medium. Although FBS contains growth elements that fulfills many metabolic needs of cultured astrocytes, it alters the genotypic and morphological profiles of primary astrocytes as well as induces astrocyte activation. The aim of this study was to establish a serum-free astrocyte culture medium that maintains primary astrocytes in a quiescent state with phenotypes that mimic in vivo astrocytes. Methods Primary astrocytes were isolated from the cerebral cortex of postnatal day 1 C57BL/6 mice and cultured in serum-free astrocyte basal medium containing FGF2 and EGF (ABM-FGF2-EGF). The phenotype of primary astrocytes cultured in ABM-FGF2-EGF were compared with astrocytes cultured in FBS supplemented DMEM medium (MD-10% FBS). Growth assays, immunostaining, Western blot, quantitative polymerase chain reaction, and metabolic assays were used to access the growth rates, metabolic phenotype, mRNA expression profiles and quiescent or reactive states of astrocytes. Results and Conclusions We demonstrated that the novel serum free ABM-FGF2-EGF medium supports astrocytes growth and enhanced glycolytic metabolism with higher glycogen content, lower GFAP and vimentin expression, and increased glutamate transporter mRNA levels as compared to astrocytes cultured in the MD-10% FBS medium. Our study suggests that our serum free culture method produces astrocytes with a biosynthetic phenotype and morphology similar to in vivo resting astrocytes. Additionally ABM- FGF2-EGF cultured primary astrocytes could be activated by various pathological conditions. The developed serum-free and EGF/FGF2-containing astrocyte basal medium will provide a critical tool for defining the precise function of astrocytes under physiological and pathological conditions.Item Expansion Microscopy for Super-Resolution Imaging of the Rodent Brain(2024-03-21) Ampofo, Hannah; Berry, Raymond; Spann, Claire; Liu, Ran; Yang, ShaohuaExpansion Microscopy for Super-Resolution Imaging of the Rodent Brain Hannah Ampofo, Raymond Berry, Claire Spann, Ran Liu, Shaohua Yang Pharmacology and Neuroscience Department, School of Biomedical Sciences, University of North Texas Health Science Center, Texas, United States. Purpose-To establish Expansion microscopy (ExM) for super-resolution imaging of the rodent brain. Background- ExM is a remarkable imaging technology that enables nanoscale resolution in three-dimensional (3-D) imaging of preserved cells and tissues. ExM, which was invented in 2015, physically expands specimens using a hydrogel, allowing high-resolution imaging to be done with conventional diffraction-limited microscopes. The basic idea is to attach anchors to biomolecules or labels chemically and link them to a hydrogel that is uniformly distributed throughout the material. This polymerization technique separates biomolecules while maintaining their spatial organization by enabling isotropic expansion. The procedure is similar to sketching an outline on an inflating object and blowing it up: the ink particles will move apart, but their relative organization remains the same. Traditional optical imaging is unable to resolve nanoscale structures with dimensions smaller than 200–300 nm due to the fundamental physical limitations imposed by diffraction. ExM offers faster imaging speeds as compared to super-resolution methods, and enhanced antibody efficiency due to the decrowding effect generated by expanding biomolecules. The original ExM could resolve the specimen at 70 nm, however, new variants such as iterative ExM, 10X ExM microscopy, and nine-fold microscopy can resolve down to 15 to 30 nm, comparable to super-resolution microscopes. Method- The Paper-MAP version of expansion microscopy, a modified MAP method that allows immunostaining and expansion within two days was employed. The procedure involved staining floating mouse brain sections and incubating with a Paper-MAP cocktail (consisting of TEMED and sodium acrylate) and ammonium persulfate solution. The hydrogel matrix was created in situ through the crosslinking of sodium acrylate and bisacrylamide, forming a dense polyelectrolyte hydrogel. A denaturing solution was used to mechanically homogenize the sample and then expanded using deionized water. The pre and post-expanded sections were imaged using a Zeiss LSM 510 confocal microscope. Results- Following the addition of the monomer solution, the expansion procedure produced a 2 fold increase in size. This was followed by an evident 4 to 5 fold increase after the expansion was completed. We compared the pre-expansion image to the post-expansion image and observed intricate and detailed structures with significantly enhanced resolution that were previously indistinguishable in the pre-expansion section using confocal microscope. Conclusion- Expansion microscopy is a versatile and accessible imaging technique that resulted in significant improvements in imaging the microscopic configuration of the mouse brain. Its broad application offers a powerful tool for biological research in diverse organisms.Item Experimental ischemic stroke induces secondary white matter degeneration and long-term cognitive impairment(2024-03-21) Berry, Raymond; Liu, Ran; Winters, Ali; Spann, Clair; Ampofo, Hannah; Colon-Perez, Luis; Sumien, Nathalie; Yang, Shao-HuaClinical investigations have detected extensive white matter degeneration in individuals affected by ischemic stroke. Nonetheless, current stroke research has primarily concentrated on the infarct and periinfarct penumbra regions. The exploration of white matter degeneration's role after ischemic stroke and its contribution to post-stroke cognitive impairment and dementia (PSCID) has been limited in experimental models. Understanding the impact of white matter degeneration on PSCID in these models could offer valuable insights into potential therapeutic targets and interventions for alleviating cognitive decline following ischemic stroke. In this study, we analyzed the progression of locomotor and cognitive function up to 4 months after inducing ischemic stroke by middle cerebral artery occlusion in young adult rats. Despite evident ongoing locomotor recovery, long-term cognitive and affective impairment persisted after ischemic stroke, as indicated by Morris water maze, elevated plus maze, and open field performance. At 4-month after stroke, multimodal MRI was conducted to assess white matter degeneration. T2-weighted MRI (T2WI) unveiled bilateral cerebroventricular enlargement after ischemic stroke. Fluid Attenuated Inversion Recovery MRI (FLAIR) revealed white matter hyperintensities in the corpus callosum and fornix across bilateral hemispheres. A positive association between the volume of white matter hyperintensities and total cerebroventricular volume was noted in stroke rats. Further evidence of bilateral white matter degeneration was indicated by the reduction of fractional anisotropy (FA) and quantitative anisotropy (QA) in diffusion-weighted MRI (DWI) analysis. FA measures water diffusion directionality; reduced FA implies decreased white matter tract coherence. QA, linked to diffusion directionality, indicates microstructural white matter changes with decreased QA. Reduced FA and QA in DWI MRI suggest brain microstructural integrity changes, involving myelin sheath disruption, axonal damage, or overall white matter deterioration. Additionally, microglia and astrocyte activation were identified in the bilateral corpus callosum after stroke. This inflammatory response indicates the involvement of glial cells in the post-stroke environment, suggesting a complex interplay between structural alterations and neuroinflammatory processes that may contribute to the observed changes in white matter integrity. Understanding these multifaceted mechanisms is crucial for developing targeted interventions aimed at promoting recovery and minimizing long-term neurological consequences following ischemic stroke. The importance of these results is underscored by their potential connection to neurological or neurodegenerative conditions, given that white matter degeneration is commonly noted in diverse neurological disorders, including Alzheimer's disease, multiple sclerosis, and other related conditions. Our study suggests that experimental ischemic stroke induced by MCAO in young rats replicates long-term cognitive impairment and pervasive white matter degeneration observed in ischemic stroke patients. This model provides an invaluable tool for unraveling the mechanisms underlying post-stroke secondary white matter degeneration and its contribution to PSCID. Researchers and clinicians use these metrics to understand and monitor the progression of neurological diseases, potentially aiding in early diagnosis and treatment planning. This research may pave the way for a more comprehensive understanding of the mechanisms underlying post-stroke cognitive impairment and dementia, ultimately leading to improved strategies for patient care and rehabilitation.Item For the pursuit of oxygen and carbon dioxide channels in mitochondria(Wolters Kluwer - Medknow, 2016-12-30) Yang, Shaohua; Liu, RanItem Hormone treatments reverse stroke-associated declines in cognitive function in a rat model of menopause(2018-03-14) Davis, Delaney; Li, Wenjun; Liu, Ran; Winters, Ali; Forster, Michael; Yang, Shaohua; Sumien, Nathalie; Vann, PhillipPurpose This study addresses the critical questions important to the future of hormone therapy. The purpose of this study was to provide information on how different durations of hormone deprivation can alter the responsiveness of the brain to ischemic injuries and hormonal therapies. Ultimately, these studies will identify a window of opportunity for treatment with hormones preventing brain dysfunction associated with menopause. Methods Eighty-two Sprague-Dawley retired breeder females rats were ovariectomized (ovx). Twelve or two weeks post-surgery, the rats were implanted with hormone pellets containing cholesterol (vehicle), estrogen (E2) or progesterone (P4), which were replaced every 2 weeks. Two weeks post implantation, the rats received either a sham or ischemic stroke (transient Middle Cerebral Artery Occlusion) surgery. After a one week recovery period, the rats were subjected to a behavioral battery of tests measuring affective (plus maze), motor (rotorod) and cognitive (Morris water maze) function. The rats were then euthanized and brain regions were collected for further biochemical analyses. Data were analyzed using 2- or 3-way ANOVAs followed by pairwise comparisons. Results Treatment with E2 or P4 decreased the time spent in the open arms in both 2 and 12 weeks post-ovx groups. There was no effect of stroke or hormone treatment on the rotorod. For spatial learning and memory, stroke impaired the rats in their ability to learn and retain the location of the platform and impairments were worst in the 12-weeks post-ovx group. E2 and P4 treatment improved performance of the stroke rats in both 2 and 12-weeks post-ovx groups. Conclusions These data suggest that the outcome of stroke is worst as a function of time post-ovx, especially on spatial learning and memory. Hormonal treatment with E2 and P4 were successful in reversing the deleterious effects of stroke on cognitive function. Further studies to identify the mechanisms underlying these observations are underway.Item Hyperglycemia Alters Astrocyte Metabolism and Inhibits Astrocyte Proliferation(JKL International, 2018-08-01) Li, Wenjun; Roy Choudhury, Gourav; Winters, Ali; Prah, Jude; Lin, Wenping; Liu, Ran; Yang, ShaohuaDiabetes milieu is a complex metabolic disease that has been known to associate with high risk of various neurological disorders. Hyperglycemia in diabetes could dramatically increase neuronal glucose levels which leads to neuronal damage, a phenomenon referred to as glucose neurotoxicity. On the other hand, the impact of hyperglycemia on astrocytes has been less explored. Astrocytes play important roles in brain energy metabolism through neuron-astrocyte coupling. As the component of blood brain barrier, glucose might be primarily transported into astrocytes, hence, impose direct impact on astrocyte metabolism and function. In the present study, we determined the effect of high glucose on the energy metabolism and function of primary astrocytes. Hyperglycemia level glucose (25 mM) induced cell cycle arrest and inhibited proliferation and migration of primary astrocytes. Consistently, high glucose decreased cyclin D1 and D3 expression. High glucose enhanced glycolytic metabolism, increased ATP and glycogen content in primary astrocytes. In addition, high glucose activated AMP-activated protein kinase (AMPK) signaling pathway in astrocytes. In summary, our in vitro study indicated that hyperglycemia might impact astrocyte energy metabolism and function phenotype. Our study provides a potential mechanism which may underlie the diabetic cerebral neuropathy and warrant further in vivo study to determine the effect of hyperglycemia on astrocyte metabolism and function.Item IMPACT OF T-CELLS ON ASTROCYTES IN VIVO & IN VITRO: IMPLICATIONS POST-ISCHEMIC STROKE(2019-03-05) Xie, Luokun; Li, Wenjun; Liu, Ran; Yang, Shaohua; Hersh, JessicaPurpose: Post-ischemic stroke, T-lymphocytes enter the brain. The role of T-cells in the progression of cerebral infarction or repair mechanisms is unclear. We hypothesized that T-cells interact with astrocytes directly leading to an anti-inflammatory response. Methods: In vivo, ischemic stroke was induced by middle cerebral artery occlusion in young adult C57/B6 male mice. Mice were sacrificed at 3 days or 1-month post-ischemic stroke. Paraffin-embedded brain sections demonstrated co-localization of astrocytes and CD4+ and CD8+ T-cells in the ischemic region 1 month after stroke. T-cells were harvested from the brain by digestion; percoll enriched, and incubated with anti-CD3 and CD25 antibodies. T-cells were sorted via flow cytometry. The cytokine profile of brain infiltrated CD4+ and CD8+ T-cells were compared to spleen T-cells using QT-PCR. In vitro, C8-S murine astrocyte type II clone cell line (ATCC® CRL-2535™), and T-cells extracted from the spleens of 3-month-old C57/B6 female mice were placed in co-culture at a 1:1 for 48 and 72 hours and compared to individual cell cultures. Anti-CTLA-4 antibodies were added to each culture condition as another experimental group. Astrocytes and T-cells were collected separately for QT-PCR analysis. Results: In vivo, the following cytokine gene expressions poststroke, were found to be elevated: IFNγ, IL-10, IL-17, TNFα, and perforin. In vitro, IL-10 gene expression was elevated in astrocytes and T-cells individually harvested from 1:1 co-cultures compared to astrocytes and T-cells alone at 48 and 72 hours respectively. IL-10 was produced primarily by T-cells stimulated by direct contact with astrocytes. Anti-CTLA-4 antibodies blocked the direct cell-to-cell interaction by reducing IL-10 gene expression in both astrocytes and T-cells. Conclusions: Our data suggests that T-cells release pro- and anti- inflammatory cytokines while in close proximity to astrocytes after ischemic stroke. In co-cultures, astrocytes directly interact with T-cells increasing their IL-10 gene expression by 72 h., implying a neuroprotective mechanism exists via astrocyte stimulation of T-cell IL-10 production.Item Metabolic Heterogeneity of Cerebral Cortical and Cerebellar Astrocytes(MDPI, 2023-01-22) Sun, Yuanhong; Winters, Ali; Wang, Linshu; Chaudhari, Kiran; Berry, Raymond; Tang, Christina; Liu, Ran; Yang, ShaohuaAstrocytes play critical roles in regulating neuronal synaptogenesis, maintaining blood-brain barrier integrity, and recycling neurotransmitters. Increasing numbers of studies have suggested astrocyte heterogeneity in morphology, gene profile, and function. However, metabolic phenotype of astrocytes in different brain regions have not been explored. In this paper, we investigated the metabolic signature of cortical and cerebellar astrocytes using primary astrocyte cultures. We observed that cortical astrocytes were larger than cerebellar astrocytes, whereas cerebellar astrocytes had more and longer processes than cortical astrocytes. Using a Seahorse extracellular flux analyzer, we demonstrated that cortical astrocytes had higher mitochondrial respiration and glycolysis than cerebellar astrocytes. Cerebellar astrocytes have lower spare capacity of mitochondrial respiration and glycolysis as compared with cortical astrocytes. Consistently, cortical astrocytes have higher mitochondrial oxidation and glycolysis-derived ATP content than cerebellar astrocytes. In addition, cerebellar astrocytes have a fuel preference for glutamine and fatty acid, whereas cortical astrocytes were more dependent on glucose to meet energy demands. Our study indicated that cortical and cerebellar astrocytes display distinct metabolic phenotypes. Future studies on astrocyte metabolic heterogeneity and brain function in aging and neurodegeneration may lead to better understanding of the role of astrocyte in brain aging and neurodegenerative disorders.Item METHYLENE BLUE INHIBITS PROLIFERATION AND MAINTAINS SELF-RENEWAL OF RAT NEURAL STEM/PROGENITOR CELLS(2014-03) Xie, Luokun; Roy Choudhury, Gourav; Park, Yong; Liu, Ran; Zhang, Chun-Li; Yorio, Thomas; Jin, Kunlin; Yang, ShaohuaNeural progenitor cells (NPCs)are important for neurogenesis and brain damage repair. To elucidate the effect of methylene blue on NPCs, we studied the self-renewal and proliferation of NPCs both in vitro and in vivo. We found that methylene blue restrains NPC proliferation and enahnces NPC self-renewal. Purpose (a): Neural stem cell-based treatment holds a new therapeutic opportunity for treating neurodegenerative disorders. While methylene blue has been shown to be neuroprotective in multiple experimental neurodegenerative disease models, its potential effects on neural stem/progenitor cells (NSPCs) has not been addressed. Methylene blue can easily penetrate the blood brain barrier to access the brain parenchyma. Thus, its effects on NSPCs, whether positive or negative, need to be elucidated. Methods (b): We used in both in vitro culture model and in vivo study to test the effects of methylene blue on the proliferation, self-renewal and differentiation of NSPCs. Neurospheres were generated in vitro and were treated with methylene blue. NSPC proliferation was evaluated by Ki67 staining and propidium iodide staining. NSPC self-renewal was determined by serial passage assay. Real-time PCR was applied to test the expression of neural differentiation markers in NSPCs. To address the signal pathway responsible for the methylene blue-induced changes on NSPCs, expression of cyclins and mTORC1 activation were determined by real-time PCR and Western blot, respectively. The effects of methylene blue on NSPC proliferation was also confirmed by intracerebroventricular infusion of methylene blue in rats followed by BrdU and Nestin staining. Results (c): Methylene blue inhibits porliferation of rat NSPCs in the in vitro culture. Methylene blue treatment decreased most cyclin expression. In addition, methylene blue enhanced the self-renewal capacity of NSPCs, demonstrated by more neurosphere growth and inhibited differentiation marker expression in NSPCs. However, methylene blue did not impair committed neuronal differentiation. The change of cyclin expression is associated with the change of mTOR expression in methylene blue-treated NSPCs. Methylene blue repressed transcription of mTOR rather than enhance mTOR degradation. Consistent with in vitro data, methylene blue inhibited neural stem cell division in the subventricular zone, but did not influenced neuronal development in a short term. Conclusions (d): Our findings indicate that methylene blue could delay NSPCs senescence by enhancing NSPCs self-renewal capacity. However, the long-term effects of methylene blue on the in vivo NSPC pool needs further investigation. The impact of methylene blue on NSPCs should be taken into account in future therapy with methylene blue, either for the peripheral diseases or for the CNS disorders.Item Neural Cytoskeleton Modulation after Transient Ischemic Attack and Region-Specific Brain Metabolism Insights(2022-08) Wang, Linshu; Yang, Shaohua; Sumien, Nathalie; Schreihofer, Derek A.; Liu, RanTransient ischemic attack (TIA) is a symptomatic diagnosis disease characterized as reversible ischemic stroke-like neurological deficit. One-third of the TIA patients have recurrent episodes, and TIA presents as a high vascular risk factor for severe stroke, mild cognitive impairment, and dementia. However, the neuropathophysiology of TIA has been less studied. Here, we established recurrent TIA model in rats with no neurological deficits and no/minimal apoptosis cells detected. Our study demonstrated that recurrent TIA induces neuronal cytoskeleton modification, astrogliosis and microgliosis in the TIAaffected cortical and basal ganglia regions, as well as in the white matter in terms of corpus callosum in the acute and subacute stage. Our data indicate recurrent TIA-induced neuronal cytoskeletal modification and neuroinflammation, may be potentially involved in the vascular contribution to cognitive impairment and dementia. Even though neurological deficits are transient in TIA patients, the brain presents morphologic and metabolic change in response to transient ischemic insult. This can be reflected on the remodeling of cytoskeleton, which plays a critical role in the mitochondria shape and motility maintenance. In addition, the interaction between cytoskeletal components and mitochondria is highly involved in the oxidative phosphorylation and mitochondrial respiration regulation. To investigate the brain metabolic signatures in the normal and pathological conditions, our study optimized a method that enables metabolic function assessment of anatomically defined brain structures by the Seahorse XFe96 analyzer in rodents. Our data demonstrated that the rodent brain has region-specific glucose metabolic profile, the cerebellum displays a more quiescent phenotype than cerebral cortex, basal ganglia, and hippocampus. Additionally, the rodent brain has relatively low mitochondrial oxidative phosphorylation efficiency with high proton leaklinked respiration. Through our proof-of-principle study, we expect to acquire critical insights that will enable future research in pursuit of spatial mapping of the brain glucose metabolism in physiological and pathological conditions (e.g., TIA condition), and further explore the mechanisms and significance of mitochondrial uncoupling of the brain.Item Sex Dependent Alteration in Psychomotor and Cognitive Functions After Chronic Metformin Treatment(2017-03-14) Winters, Ali; Shetty, Ritu; Li, Wenjun; Xie, Luokun; Prah, Jude; Liu, Ran; Sumien, Nathalie; Yang, Shaohua; Chaudhari, KiranPurpose: Metformin, the most commonly used anti-hyperglycemic medication has been proposed to have delayed aging and longevity benefits. Without due consideration to gender/sex influence, metformin administration is being tested for non-diabetic benefits. Amid mixed reports on cognition, the purpose of the current study was to identify the influence of sex variation in the psychomotor and cognitive outcomes after long term metformin treatment. Materials and Methods: Young normo-glycemic male and female C57BL/6J mice (aged 4 mo, n=10 each; total n=40 mice) were randomly assigned to either a control group or metformin group (administered 2 mg/ml in drinking water). After 1 month of treatment, a battery of behavioral tests was initiated to assess the psychomotor and cognitive functions. Metformin treatment was continued during behavior assessment. Results: Overall female mice weighed lesser than male mice. Over the experiment time span, metformin neither altered the body weight nor decreased the blood glucose level significantly. There was no variation in muscle strength or reflexes between male and female mice on either treatment. Male mice were more anxious than female mice and metformin treatment decreased anxiety in male mice only. Female mice had better motor learning and maximum coordinated running performance than male mice. Metformin treatment improved motor learning only in male mice. Metformin treatment improved balance function irrespective of sex. Overall male mice had better retention of long term memory which was deteriorated after metformin treatment. Further, metformin impaired the short term memory and cognitive flexibility only in male mice. Conclusions: This study demonstrated that metformin affects psychomotor or cognitive function differently influenced by sex. Our results suggested that chronic metformin was beneficial for psychomotor function and detrimental for short term and long-term memory in male sex. While, in female sex, metformin had beneficial or no effects on brain functions.Item Tractography as a method for mapping brain connectivity(2024-03-21) Spann, Claire; Yang, Shaohua; Liu, Ran; Berry, Raymond; Ampofo, Hannah; Colon-Perez, LuisPurpose. Mapping the brain and its complex connectivity has proved a challenging feat for neuroscience, though with the development of diffusion tensor imaging and tractography, we are one step closer to understanding brain anatomical connections. This method utilizes diffusion-weighted magnetic resonance imaging, which takes advantage of the Brownian motion of water molecules, to produce a diffusion tensor. In the white matter of the brain, diffusion varies in direction due to cellular membranes and myelin, and the diffusion tensor measures this anisotropic diffusivity to indicate possible tissue orientation. The generalized q-sampling imaging tractography method, developed by Frank Yeh in 2010, uses the diffusion tensor to approximate the course of white matter tracts and can be used to determine the exact location and termination of white matter bundles to assess connectivity between and within different brain regions. Despite limitations that decrease the accuracy of white matter tracking, tractography remains the only method to visualize white matter trajectories in vivo and non-invasively. Though commonly used for human diffusion-weighted images, here we verify tractography as a method to visualize and measure white matter trajectories in the rat brain. Methods. A male 3-month Sprague Dawley rat was used to acquire DWI images that were analyzed using DSI Studio. The DWI was superimposed with the corresponding T2W image and regions of interest (ROIs) were drawn in the corpus callosum and were applied via the built-in Waxholm Space rat atlas. Fiber tracking was seeded from the ROI, and fractional anisotropy, quantitative anisotropy, isotropy, mean diffusivity, axial diffusivity, and radial diffusivity was calculated at each ROI by DSI software. Results. Tractography of the corpus callosum was easily visualized using both drawn and atlas-applied ROIs. Fiber tractography from both ROIs included fibers from the internal and external capsules to ensure the integrity of all corpus callosum fibers. Diffusion metrics were not drastically different between the two seeding methods. Conclusion. This study presents tractography as a tool for visualizing white matter tracts and quantifying different diffusion metrics. Using both hand-drawn regions and regions from the rat atlas, white matter tracts in diseased brains can be compared to controls to measure several aspects of pathology, such as edema, axonal integrity, and axonal density. One application includes the imaging and quantification of both acute and chronic stroke, which exhibit different pathologies that can be visualized and measured with diffusion metrics, allowing for more precise targets of therapy. The use of tractography in adjunct with other established methods can improve the understanding of disease and assist in the development of better treatment.