Browsing by Author "Yang, Shaohua"
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Item 17Beta-Estradiol Suppresses Hydrogen Peroxide-Induced Nuclear Factor Kappa B Activation in HT22 Cells(2008-05-01) Kim, Pil J.; Simpkins; Singh; Yang, ShaohuaKim, Pil J., 17beta-estradiol suppresses hydrogen peroxide-induced nuclear factor κappa B activation in HT22 cells. Master of Science (Biomedical Sciences), May, 2008, 78pp., 20 illustrations, 66 titles. Reactive oxygen species (ROS) are natural byproducts of normal cellular reactions. They are oxygen ions, free (non)radicals, and peroxides that are highly reactive with normal macromolecules, such as lipids, DNA, and proteins. Cells are normally able to defend against the damages of ROS via enzymes that neutralize them into water. However, when cells are not able to cope with the accumulation of ROS, distributions in signaling pathways and gene transcription will occur, which will ultimately lead to cell death. It is now widely accepted that increased oxidative stress-induced damage in the brain is a major cause of neurodegenerative diseases, such as Alzheimer’s disease (AD). Nuclear factor κappa-B (NFκB) is not only a ubiquitously expressed transcription factor but also a signaling protein that is activated by ROS-induced oxidative stress. Our laboratory has demonstrated the neuroprotective effects of 17β-estradiol (E2) are elicited via an anti-oxidant effect. The purpose of this project was to determine the role of NFκB activation in E2-mediated neuroprotection against hydrogen peroxide (H2O2)-induced oxidative stress. HT-22, a murine immortalized hippocampal neuronal cell line, was utilized to determine whether NFκB is activated by hydrogen peroxide-induced oxidative stress and whether E2 suppresses H2O2-induced NFκB activation. We observed that H2O2 activated NFκB by phosphorylation of IκBα (pIκBα), one of the NFκB inhibitor proteins, reduction of total IκBα, and induction of NFκB (p65) nuclear translocation. In contrast, E2 suppressed H2O2-induced NFκB activation by dramatic reducing pIκBα, increasing total IκBα, and inhibiting p65 nuclear translocation. Our results show that one of the mechanisms by which estrogens are neuroprotective against oxidative stress is through the attenuation of H2O2-induced NFκB activation.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 Aging impairs regulatory T cells to affect the mouse model of late-onset multiple sclerosis(2022-08) Wang, Weikan; Su, Dong-Ming; Berg, Rance E.; Bunnell, Bruce A.; Yang, Shaohua; Jones, Harlan P.; Zode, Gulab S.Although multiple sclerosis (MS) primarily onsets in young adults, it can also develop in the elderly, which is termed late-onset (aged) MS. CD4+ Foxp3 + regulatory T (Treg) cells play an ameliorative role in severity of MS or its animal model experimental autoimmune encephalomyelitis (EAE), and the aged immune system accumulates peripheral Treg (pTreg) cells. However, late-onset MS in the aged patients presents a more progressive disease course. We investigated why the accumulated pTreg cells fail to ameliorate the MS severity in the aged individuals by using an aged EAE mouse model to recapitulate late-onset MS in patients. We observed that the onset of EAE is delayed in aged mice, but disease severity is increased compared to young EAE mice. We found that the distribution of Treg cells in aged EAE mice exhibited an increased proportion of polyclonal (pan-) pTreg cells and a decreased proportion of antigen specific-pTreg cells in the periphery, but decreased proportions of both pan- and antigen specificTreg cells in the central nervous system (CNS). Transiently inhibiting Foxp3 or depleting pTreg cells partially corrected Treg distribution and restored the balance of effector T cells (Teff) and Treg cells in the aged inflamed CNS, thereby ameliorating the disease in the aged EAE mice. Furthermore, in the aged inflamed CNS, CNS-Treg cells exhibited a high plasticity and T effector (CNS-Teff) cells presented a great clonal expansion, disrupting the Treg/Teff balance. These results provide evidence and mechanism that accumulated aged pTreg cells play a detrimental role in neuronal inflammation of aged MS.Item AMP-Activated Protein Kinase (AMPK) signaling regulates the age-related decline of hippocampal neurogenesis(2018-05) Wang, Brian S.; Jin, Kunlin; Hodge, Lisa M.; Singh, Meharvan; Sumien, Nathalie; Yang, ShaohuaAging is the progressive decline of physiological function and increased vulnerability to disease and death. By the year 2050, 2 billion people will be over the age of 60. Accompanying this, the incidence of age-associated neurological diseases is expected to rise. Thus, there is an urgent need to find therapies to promote healthy brain aging. The finding that neurogenesis continues into adulthood allows us to target endogenous neurogenesis as a potential therapeutic. However, the number of stem cells can decrease by about 80% in the aged brain and is a main cause for the decrease in brain function. The reasons for the age-related decline in neurogenesis can be due to intrinsic factors such as cell metabolism, which have been studied but its role in neurogenesis remains largely unexplored. Interestingly, neural stem cells (NSCs) possess metabolically different characteristics from their differentiated progeny, suggesting the need for a shift in cellular metabolism to accommodate the requirements for neurogenesis. In the process of the metabolic shift, the AMP-activated protein kinase (AMPK) plays a pivotal role for controlling stem cell proliferation and differentiation as a cell's master metabolic regulator. Additionally, AMPK has been reported to control the functions of signaling pathways that regulate the aging process, which suggests its potential involvement in the age-related decline of neurogenesis. Therefore, we hypothesize that inhibition of AMPK signaling activation (phosphorylation) in the old brain will cause a concomitant increase in hippocampal neurogenesis. Our specific aim is to establish whether AMPK signaling plays a critical role in the age-related decline of hippocampal neurogenesis. Our objectives for this aim are to (i) determine the expression pattern of AMPK in the subgranular and subventricular zones of young-adult and old mice using immunohistochemistry and Western blotting; and (ii) examine the impact of loss or gain of AMPK activation on hippocampal neurogenesis in young-adult and old mice using pharmacological agents Compound C (AMPK inhibitor) and 5-Aminoimidazole-4-carboxamide ribonucleotide (AICAR, AMPK activator). Our results show that (i) AMPK subunit isoforms are differentially expressed in the neurogenic regions – most are localized to the cytoplasm in the subgranular zone (SGZ) with the exception of α2 and β1, while most isoforms are found in the nucleus in the subventricular zone (SVZ) except α1; (ii) AMPK signaling activation was significantly increased in the SGZ and SVZ; and (iii) short-term but not long-term pharmacological inhibition of AMPK signaling could partially rescue hippocampal neurogenesis in the old brain. Taken together, these results indicate that AMPK is a critical mediator in the regulation of downstream processes for the age-related decline in hippocampal neurogenesis.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 Astrocytes & Ischemic stroke(2014-08-01) Roy Choudhury, Gourav; Yang, Shaohua; Singh, Meharvan; Schreihofer, DerekAlthough less appreciated, recent findings introduced critical contributions of astrocytes to numerous CNS functions, like neurogenesis, synaptogenesis, ion homeostasis, neurotransmission, and blood brain barrier formation. Their active participation in the progression of specific CNS pathologies has garnered major attention and culminated in thorough investigation of astrocyte function in brain. Reactive astrogliosis, characterized by increases in glial fibrillary acidic protein (GFAP) and cellular hypertrophy, describes the extensive structural and functional changes that astrocytes undergo in response to tissue injury. Despite of extensive investigation, the molecular mechanism of reactive astrogliosis in ischemic stroke still remains elusive. p38 MAPK is a well studied signal transducing pathway known to be involved in modulating cell type specific responses to ischemic injury. The first study presented in the dissertation delineates the involvement p38 MAPK signaling pathway in reactive astrogliosis after ischemic stroke. Results showed that astrocyte specific deletion of p38 MAPK attenuated oxygen-glucose deprivation (OGD)-induced increase in GFAP expression in primary astrocytes in vitro. Additionally, inhibition of p38 MAPK (SB239063/genetic deletion) slowed astrocyte migration without affecting astrocyte proliferation. In vivo deletion of p38 MAPK from astrocytes attenuated reactive astrogliosis after permanent middle cerebral artery occlusion in mice. These findings strongly indicated that p38 MAPK plays a critical role in reactive astrogliosis after ischemic stroke. During ischemic stroke, astrocyte dysfunction causes extensive cell death through excitotoxicity, disruption of ion and water homeostasis. Restoration of astrocyte function thus may be beneficial to ischemic tissue in the long term. Methylene blue (MB), a metabolic enhancer, has been well studied and known to improve cellular respiration, glucose metabolism and attenuate superoxide production by efficient electron transport in mitochondria. In the second part of this dissertation we determined the effect of MB in astrocytes under oxygen glucose deprivation (OGD) and reoxygenation stress and the underlying protective mechanisms. Our studies demonstrated that MB improved astrocyte bioenergetics and promoted astrocyte survival following OGD and reoxygenation. In conclusion both the studies presented, provide a unique perspective of the importance of astroglial response in ischemic injury and how its modulation can benefit the healing and recovery of the brain following ischemic injury.Item Blood Inflammatory Exosomes with Age Prime Microglia through Complement Signaling for Negative Stroke Outcomes(2020-05) Zhang, Hongxia; Jin, Kunlin; Forster, Michael J.; Yang, Shaohua; Shi, Xiangrong; Cunningham, J. ThomasThe systemic inflammatory milieu plays an important role in the age-related decline of functional integrity, but its contribution to age-related disease (e.g., stroke) remains largely unknown. Here, we found that activated complement molecules (C1q, C3a, C3b) in serum exosomes increased with age, whereas CD46, a C3b/C4b-inactivating factor, was higher in serum exosomes from young rats. These serum inflammatory exosomes passed the blood-brain barrier and primed the microglial response that led to exacerbation of synaptic loss and motor deficits after ischemic stroke via microglial C3a receptor (C3aR). When aged rats were exposed to serum exosomes from young rats, microglia-mediated synaptic loss was reduced and motor deficits after stroke were improved. Administration of C3aR inhibitor or microglial depletion attenuated synaptic loss associated with the treatment of serum exosome from aged rats, in parallel with improved post-stroke outcome. Our data suggest that peripheral circulating old exosomes act as inflammatory mediators and influence ischemic stroke outcome through a complement-microglia axis.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 Chronic testosterone deprivation sensitizes the middle-aged rat brain to damaging effects of testosterone(2020-05) Smith, Charity; Schreihofer, Derek A.; Cunningham, Rebecca L.; Singh, Meharvan; Yang, Shaohua; Jones, Harlan P.Levels of the testosterone (T) fall in aging men. Recently, the number of men obtaining testosterone replacement therapy (TRT) has increased dramatically. However, other consequences of aging, such as increased oxidative stress, may result in detrimental effects when combined with TRT, including an increased stroke risk. Whether such a delay would alter the effects of TRT on stroke is not known. We hypothesized that a delay TRT following castration in middle-aged male rats would result in increased oxidative stress and a reduction in the neuroprotective effects of testosterone following stroke. We evaluated the effects of testosterone treatment after short (2 week) and long-term testosterone deprivation (10 weeks) in middle-aged male rats on cerebral ischemia, oxidative stress and cognitive function. Our data suggest testosterone treatment after long-term hypogonadism can exacerbate functional recovery after focal cerebral ischemia, however in the absence of injury improves cognition. Both effects are regulated by oxidative stress.Item Cocaine-induced stroke susceptibility: motor and cognitive outcomes(2018-03-14) Taylor, Cynthia; Li, Wenjun; Forster, Michael; Yang, Shaohua; Sumien, Nathalie; Vann, PhilipTitle: Cocaine-induced stroke susceptibility: motor and cognitive outcomes Presenters: Philip Vann, Cynthia Taylor, Wenjun Li, Michael J. Forster, Shaohua Yang, Nathalie Sumien Purpose: Epidemiological findings suggest that the number of young individuals suffering from stroke seems to be increasing, and one of the most common cause for such an increase is the use of illicit drugs. Prior work in our laboratory suggested that life-long cocaine intake impaired cognitive function and that short-term intake induces brain changes conferring vulnerability. In this study, we tested the hypothesis that repeated cocaine use will induce brain vulnerability to ischemic stroke. Methods: Fifty seven young male Sprague-Dawley rats (3 months) were injected i.p. with cocaine (10mg/kg) or saline (3 times/wk) for 4 weeks. From each treatment group, half of the rats received an ischemic stroke (transient Middle Cerebral Artery Occlusion) and the other half a sham surgery. After a one month recovery period, the rats were subjected to a behavioral battery of tests measuring balance, motor function, spatial learning and long term memory (locomotor activity, bridge walking, rotorod, and Morris water maze). Once behavioral testing was finalized the rats were euthanized and brain regions were collected for further biochemical analyses. Data were analyzed using 2- or 3-way ANOVAs followed by pairwise comparisons. Results: The stroke surgery resulted in decreased body weights and increased overall activity (total distance travelled and horizontal activity). Maximum performance on the rotorod was lower for the stroked rats than for the shams, and treatment with cocaine did not affect the outcome. However, during training the cocaine-treated rats had higher latencies than the controls. On the bridge walking test, the stroke surgery did not seem to affect performance, however the cocaine-treated stroke rats performed the worst. The stroked rats took longer path length and latencies to reach the platform, and cocaine seem to exacerbate the impairment, more specifically at the end of training and during retention. Conclusions: While preliminary, these results suggest that cocaine-treated rats were more vulnerable to stroke than the saline-treated ones but exhibiting exacerbated impairments on balance and spatial learning and memory. Studies to identify the underlying mechanisms of this vulnerability are underway. IACUC 2016-0022 Intramural grant UNTHSC RI10014Item Combining Injectable Plasma Scaffold with Mesenchymal Stem/Stromal Cells for Repairing Infarct Cavity after Ischemic Stroke(JKL International, 2017-04-01) Zhang, Hongxia; Sun, Fen; Wang, Jixian; Xie, Luokun; Yang, Chenqi; Pan, Mengxiong; Shao, Bei; Yang, Guo-Yuan; Yang, Shaohua; Zhuge, Qichuan; Jin, KunlinStroke survivors are typically left with structural brain damage and associated functional impairment in the chronic phase of injury, for which few therapeutic options exist. We reported previously that transplantation of human embryonic stem cell (hESC)-derived neural stem cells together with Matrigel scaffolding into the brains of rats after focal ischemia reduced infarct volume and improved neurobehavioral performance. Matrigel is a gelatinous protein mixture extracted from mouse sarcoma cells, thus would not be approved for use as a scaffold clinically. In this study, we generated a gel-like scaffold from plasma that was controlled by changing the concentration of CaCl2. In vitro study confirmed that 10-20 mM CaCl2 and 10-40% plasma did not affect the viability and proliferation of human and rat bone marrow mesenchymal stem/stromal cells (BMSCs) and neural stem cells (NSCs). We transplanted plasma scaffold in combination of BMSCs into the cystic cavity after focal cerebral ischemia, and found that the atrophy volume was dramatically reduced and motor function was significantly improved in the group transplanted with scaffold/BMSCs compared with the groups treated with vehicle, scaffold or BMSCs only. Our data suggest that plasma-derived scaffold in combination of BMSCs is feasible for tissue engineering approach for the stroke treatment.Item Connexin 43 Contributes to Estrogen Protection against Oxidative Stress in Cortical Astrocytes(2019-05) Kubelka, Nicholas K.; Singh, Meharvan; Uht, Rosalie M.; Schreihofer, Derek A.; Yang, Shaohua; Planz, John V.Age-related brain disorders are associated with the decline in the ability of brain cells to cope with homeostatic challenge. Although all major brain cell types have the capacity to respond to homeostatic challenges, astrocytes are particularly well-equipped to counteract these challenges. Here, we focused on Connexin 43 (Cx43) as a protein that is not only highly expressed in astrocytes, but whose expression is critical to inter-cellular communication that in turn, can influence cell viability. Most studies to date have focused on the expression (i.e., abundance) of Cx43. However, a critical limitation of these studies is that they did not thoroughly examine functionality of the Cx43 channels. In particular, there is a paucity of data describing the differential contributions of Cx43-containing hemichannels versus Cx43-containing gap junctions to cellular functions. We hypothesized the astrocyte Cx43 hemichannel as a yet unreported target of androgens and estrogens based on three notions. First, our laboratory has determined that astrocytes are a relevant and important target of such gonadal steroid hormones as estrogens (e.g., 17[beta]-estradiol (abbreviated herein as estradiol or E2)) and androgens (such as DHT), through which these hormones promote healthy brain cell function. Second, oxidative stress is associated with an increase in Cx43 opening. Finally, the Cx43 gene promoter contains functional estrogen response element (ERE) half sites, and estradiol, as well as other estrogenic compounds, decrease Cx43 channel opening in peripheral (non-CNS) tissue. Based on these notions, we hypothesized that gonadal androgens and estrogens will inhibit Cx43 hemichannel opening in cortical astrocytes as well. My data revealed that while E2, dihydrotestosterone (DHT), and the estrogenic metabolite of DHT (3[beta]diol) all protect primary cortical astrocytes from the mixed metabolic/oxidative insult, iodoacetic acid (IAA), only DHT decreased astrocyte Cx43 mRNA expression. Consistent with their cytoprotective effects, however, all three steroids decrease astrocyte Cx43 hemichannel opening, and antagonized the increased opening of Cx43 hemichannels induced by IAA. In an effort to pursue the mechanism by which these steroids reduced Cx43 hemichannel opening, we evaluated the phosphorylation of Cx43 at two key residues, Ser 368 and Tyr 265. Phosphorylation at these residues is associated with channel closing, and as such, we predicted that the three hormones would increase the phosphorylation of Cx43 at one or both of these residues. Whereas Tyr265 phosphorylation was unaffected any of the three hormones, DHT significantly reduced the phosphorylation of Cx43 at Ser368. These observations may indicate that while all three steroids contribute to astrocyte protection through a mechanism that involves blocking astrocyte Cx43 hemichannel opening, DHT may induce molecular changes in the astrocytes that are distinct from those induced by estradiol or 3[beta]diol. The knowledge gained through the experiments conducted not only enhance our understanding of how Cx43 hemichannels and Cx43 gap junctions influence astrocyte function and viability but also define Cx43 hemichannels as relevant targets of gonadal steroid hormone induced regulation of cell viability. Such knowledge may facilitate the development of more precise therapeutics (i.e., selectively targeting Cx43 hemichannels without activity at Cx43 gap junctions in the same cells or tissue), the benefit of which would be to better treat age-associated neurodegenerative disorders as well as disorders of peripheral tissueItem 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 Development of methylene blue-loaded nanoparticles for glioblastoma treatment(2015-03) Castañeda-Gill, Jessica M.; Ranjan, Amalendu P.; Yang, Shaohua; Vishwanatha, Jamboor K.Glioblastoma (GBM) is the most common and aggressive primary brain tumor in adults over 45, resulting in an average survival of 15 months post-diagnosis and treatment. While recent research has provided essential information to aid diagnosis and treatment, GBM is known to cause relapse following traditional combinatorial regimens, necessitating the development of more effective and less toxic therapies. Methylene blue (MB), a dye with noted medicinal applications, has received recent consideration as a potential neurotherapeutic due to its ability to infiltrate the blood-brain barrier (BBB) and improve cellular processes within distinct brain cell compartments and types; however, one drawback is an increased administration to produce desired therapeutic effects, leading to excessive brain deposition and potential neurotoxicity. A method commonly used to enhance drug delivery while reducing unwanted side effects is via encapsulation in nanoparticles (NPs) composed of the biodegradable/biocompatible co-polymer, poly(lactic-co-glycolic) acid (PLGA). Based on our previous studies, we are developing a MB-loaded PLGA NP capable of permeating the BBB to treat GBM, to test our hypothesis that MB encapsulation into PLGA NPs will enhance accumulation in cancerous brain regions, resulting in reduced tumor size and prolonged survival. In this study, we formulated and characterized MB-loaded PLGA NPs, with a 3:1 molar ratio of sodium oleate to methylene blue at 5mg, based on particle size, drug loading, encapsulation efficiency, and release kinetics. Currently, we are establishing their in vitro effects in two different commercially-available GBM cell lines, according to their responses to commonly-used chemotherapeutics. Following loading of 5mg MB and their comparison to blank NPs, we obtained NP preparations in the range of 120-145nm, with encapsulation efficiencies from 25-40% and drug loading between 1-2%. Additionally, we have found that 50% of the MB initially added is released at 24 hours, and stays constant up to two weeks, demonstrating sustained drug release. In conclusion, based on studies that have demonstrated in vitro effects with MB at a minimum of 1μM (~0.3mg) and 150nm particles, our formulation should elicit comparable, if not better, results when treating GBM.Item Effect of Progesterone on Calcium Signaling of Hippocampal Neurons(2006-05-01) Hwang, Ji-Yeon; Koulen; Singh, Meharvan; Yang, ShaohuaJi-yeon Hwang, Effects of Progesterone on Calcium Signaling of Hippocampal Neurons. Master of Science (Pharmacology and Neuroscience), May 2006, 74 pp., 18 Figures. Progesterone (P4) is one of the steroid hormones responsible for female sexual behavior. It has been recently show that P4 plays also multiple roles in the central nervous system (CNS) including neuroprotection. Calcium (Ca2+) is involved in numerous cellular processes in nerve cells such as neurotransmitter release and cell death. In the present studies, we present evidence that P4 increases the activity of IP3R-mediated Ca2+ release within nerve cells leading to cell survival and neuroprotection. The purpose of the present study is to identify the subcellular distribution of all IP3Rs and other signaling proteins including Akt and phosphor-Akt, in the primary hippocampal neuron and to test the hypothesis that P4 controls the gain of IP3R-mediated intracellular Ca2+ signaling in neurons. We observed that primary hippocampal neurons express predominantly IP3R type 1, 2, and 3. The cellular distribution of all IP3R isoforms as well as Akt and phospho-Akt was increased in primary hippocampal neurons by P4 treatment. In addition, phospho-Akt was translocated to nucleus in response to P4. P4-pretreated neurons showed potentiated IP3R-mediated intracellular Ca2+ responses. Acute application of P4 resulted in transient elevations of intracellular Ca2+ concentrations. Our results will contribute to establishing potential pharmacological approaches for the treatment of pathological conditions characterized by a dysregulation of cellular Ca2+ concentrations such as Alzheimer’s disease.Item Effects of Sex Steroids on Stroke(2004-02-01) Yang, Shaohua; Simpkins, James W.Yang, Shaohua, Effects of Sex Steroids on Stroke. Doctor of Philosophy (Biomedical Science), February 2004, pp210, 5 tables, 27 illustrations, 64 titles. Estrogens and androgens are recognized as major sex steroids for females and males, respectively. However, it is clear that estrogens as well as androgens are more than gender hormones. Our data indicated that female steroids, such as 17β-estradiol (E2), exert neuroprotective effects on stroke, while male steroids, like testosterone, exert deleterious effects on stroke. The neuroprotective effects of estrogens have been very well demonstrated both in vitro as well as in vivo. Our studies indicated that neuroprotective effects of E2 are exerted both ischemic and hemorrhagic stroke. In our subarachnoid hemorrhage (SAH) model, E2 reduced secondary ischemic damage and mortality consequent to SAH. These effects were not associated with the change of the clot volume in SAH. The neuroprotective effects of estrogens were not only seen in the pre-treatment paradigms. E2 exerted neuroprotective effects even when administered after ischemia, with a therapeutic window of about 3 hours in a permanent focal cerebral ischemia model. This effect of estradiol was associated with no immediate change on blood flow, but with a delayed increasing in cerebral blood flow (CBF). Further our studies indicated that a non-estrogen receptor (ER)-binding analogue possessed both neuroprotective and vasoactive effects, which suggests that both the neuroprotective and vasoactive effects of estrogens are receptor-independent. This molecule also offers the possibility of clinical application for stroke without the side effects of estrogens. We used immunochemistry, immunoblot and mass spectrometry to demonstrate that ERβ is localized to mitochondria. Our data established this ERβ localization in a variety of cell types, suggesting that ERβ is not a nuclear receptor, which was thought to mediate the genomic function of estrogens. In contrast to estrogens, testosterone increased neuronal toxicity and exacerbated cerebral ischemia-reperfusion injury. These results suggest that sex differences in outcome after stroke may result from both the protective effects of estrogens and the damaging effects of testosterone. Further, our study indicated that stress induced testosterone reduction contributes to cerebral ischemia tolerance against ischemia reperfusion injury, providing the first in vivo evidences for a neuroendocrine mechanism for the cerebral preconditioning in males.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 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.
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