Neuroscience

Permanent URI for this collectionhttps://hdl.handle.net/20.500.12503/29935

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Urine sample-derived cerebral organoids suitable for studying neurodevelopment and neuroregeneration
(2020) Wang, Yu-chieh; Lin, Victor; Hu, Jiangnan
Purpose: Human cerebral organoids (COs) have yielded significant discoveries regarding developmental biology, disease mechanisms and pharmacological responses in the central nervous system. Here, we intend to establish cerebral organoids suitable for modeling brain development and neuroregeneration based on somatic cells that are isolated noninvasively. Methods: The development of such organoids began with the collection of urinary epithelial cells (UECs) from human urine samples, followed by reprograming them into induced pluripotent stem cells (hUEC-iPSCs), which were further used for the generation of COs. To comprehensively characterize the cellular and molecular features of our COs, we examined samples at different developmental time points in vitro. To check their survival ability and the neuroregenerative potential, the COs were implanted into immunodeficient mouse brains and further examined. Results: The hUEC-iPSC-developed COs exhibit normal development with neurogenesis and maturation of neuronal cells forming brain layers. These COs produce neurotropic and anti-inflammatory factors that are presumably critical for neurogenesis and neural repair. Several metalloproteases that may facilitate cell migration and microenvironment rearrangement are also present. After transplantation into the mouse cerebrum, vascularization quickly develops in the implanted COs, suggesting their viability and ability to interact with the environment. Conclusions: Our work begins to reveal the promise of generating personalized COs from cells that are isolated from urine samples. With further adaptation, this human CO platform could form a unique and personalized model for the investigation of neural development and facilitate the innovation of novel therapies for treating neurological diseases.
Chronic testosterone deprivation sensitizes the middle-aged rat brain to damaging effects of testosterone replacement
(2020) Vann, Philip; Wong, Jessica; Sumien, Nathalie; Cunningham, Rebecca; Metzger, Daniel; Schreihofer, Derek; Oppong-Gyebi, Anthony; Contreras Garza, Jo; Kasanga, Ella; Smith, Charity
Hypothesis: We hypothesized that a delay in testosterone replacement therapy (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. Background: Levels of the hormone testosterone (T) fall in aging men. Recently, the number of men obtaining TRT has increased dramatically. However, other consequences of aging, such as oxidative stress, may result in detrimental effects when combined with TRT, including stroke risk. Methods: Twelve-month old male Fischer 344 rats were divided into 5 groups (n=9-14): 1) gonad Intact sham stroke (SH), 2) Intact stroke (IN), 3) short term castrate + T (ST), 4) long term castrate (LC), and 5) long term castrate + T (LCT). Rats were castrated 2 weeks (ST) or 10 weeks (LT, LCT) prior to T. Middle cerebral artery occlusion (Stroke) done via stereotaxic injection of endothelin 1 (ET1). Three, 7, and 14 days after stroke several behavior tests were done. Rats were humanely euthanized, and blood/brains were collected. Results: Plasma oxidative stress measured by Advanced Oxidative Protein Products (AOPP) was significantly negatively correlated with T levels. Long-term hypogonadism in middle-aged male Fischer 344 rats TRT exacerbated the detrimental behavioral effects of experimental focal cerebral ischemia. Conclusion: Data suggest that TRT after long-term hypogonadism can exacerbate functional recovery after focal cerebral ischemia.
AT1R sniffer cells detects spontaneous and evoke release of AngII in the AP-NTS pathway
(2020) Paundralingga, Obed; Farmer, George; Cunningham, J. Thomas; Gusson Shimoura Almeida Lima, Caroline
Although angiontesin II (AngII) has multiple actions in the brain, the existence of a brain RAS is still controversial. Our previous studies have used angiotensin sensitive sniffer cells to test whether angiotensin peptides are released from subfornical organ projections to the median preoptic nucleus. In these studies, we examined another pathway involving the area postrema (AP) and nucleus of tractus solitarius (NTS). The AP is angiotensin sensitive and projects to the NTS, so the purpose of this study was to test for the release of angiotensin peptides in the NTS after stimulation of AP. Sniffer cells were produced by transfecting Chinese Hamster Ovary cells with commercially available plasmids for the angiotensin 1a receptor (Origene Tech.) and R-GECO (Addgene #32462). These sniffer cells are sensitive to AngII and III but not angiotensin 1-7, bradykinin, or neurotransmitters such as glutamate or acetylcholine. Sniffer cells were placed on coronal brainstem slices containing both AP and NTS from adult male Sprague and Dawley rats. Changes in fluorescent intensity of sniffer cells in the NTS was determined following electric stimulation of the AP (100Hz, 10ms, 1mA). Electrical stimulation increased fluorescence intensity 134 ± 11%, n=13 of sniffer cells on the NTS with a mean response latency of 4 ± 0.7sec, n=13. Some cells demonstrated spontaneous changes in fluorescence intensity 2±0.1, n=28 that were not observed in cells located outside of the NTS. The results indicate that sniffer cells placed on the NTS demonstrated evidence of spontaneous and evoked release of angiotensin peptides.
PVN-projecting NTS Neurons is Involved in Sympathetic Long-Term Facilitation After Acute Intermittent Optogenetic Stimulation
(2020) Cunningham, J. Thomas; Shimoura, Caroline; Paundralingga, Obed
Acute intermittent optogenetic (AIO) stimulation to the caudal NTS, a visceral sensory nucleus with projections to, among others, the paraventricular nucleus (PVN), induces sympathetic long-term facilitation (LTF), which is a progressive increase of sympathetic nerve activity. The contribution of the NTS to PVN pathway in sympathetic LTF is unknown. We investigated the effects of AIO stimulation of PVN-projecting NTS neurons in rats that were previously injected with rAAV2/Ef1a-DIO-hchR2(H134R)-mCherry in the NTS and retrograde AAV9.hSyn.HI.eGFP-cre.WPRE.SV40 in the PVN. We measured splanchnic sympathetic nerve activity (SSNA), mean arterial pressure (MAP), and heart rate (HR) in anesthetized (urethane and cloralose) and mechanically ventilated adult male Sprague-Dawley rats. Twenty minutes after AIO, defined as train of 10 stimuli each consisting of pulse of light of 10-ms duration delivered at 20 Hz for 1 min every 6 min, SSNA amplitude increased by 26.8 ± 7.09% (n=7), MAP increased by 16.9 ± 11.72% (n=7). This increase in SSNA and MAP occurred whether singular optogenetic train of stimulation increased (n=3) or decreased (n=4) of SSNA. These results suggest that the splanchnic sympathetic LTF induced by acute intermittent stimulation of NTS neurons might be mediated by the PVN-projecting neurons, independently of baroreflex or chemoreflex circuits. Further histological study is needed to clarify whether the differential responses to single optogenetic stimulation is due to activation of collaterals from PVN-projecting NTS neurons that are connected to different circuits or due to activation of different downstream projections from the PVN.
Effect of dietary genistein on functional recovery following cerebral ischemia in ovariectomized middle-aged rats
(2020) Sumien, Nathalie; Vann, Philip; Metzger, Daniel; Schreihofer, Derek; Sun, Fen; Oppong-Gyebi, Anthony
PURPOSE: Advancing age increases women's susceptibility to stroke compared to men, after the menopausal transition, which has been attributed mainly to a drop in estrogen concentrations postmenopause. However, time-dependent mixed benefits and detriments of estrogen therapy for prevention of stroke and cardiovascular diseases after menopause have contributed to widespread mistrust of estrogen use. This has led to the use of other agents like soy isoflavones as alternatives to estrogen therapy. In this study, we hypothesized that the neuroprotective effects of genistein, a soy isoflavone following cerebral ischemia are less sensitive to length of hypogonadism than estrogen. METHOD: Proven retired breeder Sprague-Dawley rats (aged ~9 months old) were ovariectomized, grouped into two hypogonadal time points (2weeks= short-term and 12 weeks= long-term) followed by treatment with isoflavone-free diet or genistein diet (GEN). A group that received 17-β estradiol (E2) was introduced as a control for hormone supplementation. All animals underwent middle cerebral artery occlusion or sham surgery followed by behavioral tests including neurological function, motor function with cylinder and rotarod, cognition with Morris Water Maze (MWM) and gait performance with Catwalk test. RESULTS: We observed effect of stroke on motor and gait performances. Effect of treatment and length of hypogonadism were observed on motor, cognitive and gait performances. GEN but not E2 improved spatial learning on the reversal phase of MWM test. CONCLUSION: Dietary GEN may improve forelimb asymmetry in the acute phase of stroke following long-term hypogonadism and aspects of cognitive functions post-stroke.
Chronic Intermittent Hypoxia Alters the Chloride Gradient in Median Preoptic Nucleus (MnPO) Neurons of Rats
(2020) Cunningham, J. Thomas; Little, Joel; Bachelor, Martha; Rybalchenko, Nataliya; Yuan, Joseph; Farmer, George
Rats exposed to chronic intermittent hypoxia (CIH), an animal model simulating hypoxemia associated with obstructive sleep apnea, exhibit persistent elevations in blood pressure during normoxic periods. In MnPO neurons, angiotensin II type 1 receptor function mediates reduced GABAa inhibition that becomes excitatory following CIH. Here, we use the ratiometric Cl- sensor, ClopHensorN, to monitor the chloride flux of MnPO neurons in normoxic (Norm) and CIH treated rats following GABAa activation. Using isoflurane anesthesia, male Sprague-Dawley rats (250-350g) received microinfusions of AAV9-Cre in the PVN and DIO-ClopHensorN in the MnPO. After recovery, rats underwent 7 consecutive days of CIH (6 min cycles of 3 min 21% O2, 3 min 10% O2 repeated 10x/h for 8 hours) or Normoxia. For ClopHensorN imaging, rats were anesthetized with isoflurane and coronal slices containing the MnPO were cut using standard in vitro slice procedures. Images were captured every 3s. Cl- flux was determined from the ratiometric response to 10s focal application of muscimol (100 uM). Twelve rats (6 Norm, 6 CIH) were used for ClopHensorN studies. In MnPO CIH neurons, 20.1% showed decreased fluorescent ratios while 0.3% showed increased ratios indicative of Cl- efflux. In MnPO Norm neurons, 41.9% showed a muscimol dependent decrease in fluorescent ratio with 0 cells showing an increase. The magnitude of muscimol dependent decreases in fluorescent ratios were reduced in CIH treated rats suggesting reduced GABAa inhibition. Results demonstrate CIH alters Cl- flux in PVN projecting MnPO. These changes may contribute to hypertension associated with CIH.
Circulating exosomes amplify microglial responses and exacerbate synapse loss in aged ischemic stroke brain via a Complement Signaling
(2020) Jin, Kunlin; Zhang, Hongxia
Background: Aging is associated with striking increases in the incidences of stroke, which remains leading cause of disability in the US. Despite progress in understanding molecular mechanisms of neuronal cell death after stroke, effective treatment remains elusive. Recent studies showed systemic factors in the blood can profoundly reverse aging-related impairments, and our previous study show that aging systemic milieu could worse outcome after ischemic stroke. However, the underlying mechanism remain unclear. Method: The exosomes isolated from serum of young and old rats were intravenously injected into aged ischemic rats for 3 days, respectively. Infarct volume was determined by histology and motor deficits were assessed with neurobehavioral tests including running ladder and cylinder. Neuroplasticity was examined after treatment using Golgi-Cox staining. Complement activation and microglial phagocytosis in ischemic brain were measured by Western blot and immunostaining. Results: We found that injection of old serum exosomes into aged animals worsened age-related synaptic loss after stroke, along with increased infarct volume and motor deficits. By proteomic analysis, we found complement activity was increased in old serum exosomes, which could amplify proinflammatory microglial activation via C3a receptor, leading excessive phagocytosis of synapses after stroke. Inhibiting complement cascade or depletion of microglia reduced excessive phagocytosis and attenuated old exosomes-mediated ischemia outcome. Conclusion: Our data suggest that old serum exosomes may drive synapse loss via complement-microglia axis, leading to worsen functional deficits in aged ischemic rats. Targeting complement-microglia axis could potentially be translated into novel therapeutic intervention for ischemic stroke.
Don't Eat that Guacamole: Rare onset of Myasthenia Gravis
(2020) Mederos, Gerardo
Myasthenia Gravis (MG) is an autoimmune disorder that targets the terminal neuro-junctional synapses in muscles leading to weakness and fatigue. Antibodies against the acetylcholine receptors result in a pure motor disorder. Here we present a patient that was diagnosed with MG after eating guacamole. Case Report Patient is a 76 year old caucasian female that presented for ptosis, right facial droop, dysarthria, and dysphagia. Patient initially developed periorbital numbness after eating guacamole at dinner. Patient was diagnosed with a food allergy and treated with prednisone and benadryl. The patient later developed right ptosis and facial droop with dysarthria requiring hospitalization. Imaging was negative for an acute cerebrovascular event, and CT chest was negative for a thymoma. Despite IV steroids her symptoms persisted. Antibodies resulted positive for MG. Patient was started on Pyridostigmine and plasma exchange. After several days of therapy she was back to her baseline function. Discussion The presentation of MG includes muscle weakness with fatigability. MG can include symptoms from mild ptosis to respiratory failure. Approximately 85% of patient's present with ocular muscle weakness. Although most patients present with mild symptoms, up to 39% of patients can have severe muscle weakness associated with dysphagia. Weakness involving the respiratory muscles can develop within 2 years of symptoms onset. In our case, 2 features are unique. First, the patient's initial symptom were periorbital swelling and numbness. MG is a pure motor disease. And second, the patient developed respiratory distress with reduced vital capacity within 72 hours of presentation.
Completion of the Myasthenia Gravis Puzzle by Fitting Together the Seemingly Unrelated Symptom Pieces: A case report
(2020) Sapkota, Bishnu; Muqtadeer, Javeria; Meiling, James
Background: "Common" does not necessarily equate to being "easily recognized." Because of fluctuating skeletal muscle symptoms that may not present equivocally or even at the same time, Myasthenia gravis (MG) often goes undiagnosed, leading to a delay in diagnosis for months to years. This study describes a case of MG which evaded diagnosis multiple times, likely due to fluctuating symptoms and misread presenting symptoms at different individual encounters. Case Information: An 84-year-old female presented with shortness of breath, neck muscle pulling, and worsening generalized weakness. She was unable to stand up from her porch upon arriving home from a cardiology appointment. She exhibited abnormal gait, difficulty swallowing food, and episodes of choking. She also recently received glasses with prism lens by her optometrist due to double vision. Her examination was notable for binocular diplopia, decreased proximal muscle strength in bilateral upper and lower extremities, pressured speech, ptosis, and ataxia. Suspecting a neuromuscular diagnosis, acetylcholine receptor antibodies were ordered and returned positive, indicative of MG. Conclusions: Although MG is a common neuromuscular disease, its diagnosis can be difficult, due to vague fluctuating symptoms and potential for pathologic mimicry that lead physicians down unrelated rabbit holes that delay diagnosis. MG benefits from a broadened approach, taking into account all previous hospital visits, appointments, and symptomatology - as opposed to a limited view on any one symptom present at that encounter. With MG, it is essential to obtain a complete history, combining the seemingly unrelated pieces into the completed puzzle.
FDA-approved antibiotic improves motor function in a rat hemi-parkinsonian model: Potential to extend timeline in early-stage Parkinson's disease
(2020) Salvatore, Michael; McElroy, Christopher; Shifflet, Marla; Kasanga, Ella
Purpose: The motor deficits of Parkinson's disease (PD): bradykinesia, resting tremors, postural instability and/or rigidity are predominantly unilateral early at diagnosis, progressing bilaterally into moderate stages of the disease. In rats, unilateral impairment can be generated using the neurotoxin 6-hydroxydopamine (6-OHDA). Progression of these deficits can be evaluated to provide insight into gradual bilateral impairment and resulting neuropathological changes. This study employed two locomotor function assays: the forepaw adjusting steps (FAS), which is an 'imposed-movement' test and the open-field locomotor (OFL) test, which relies on "at-will' movement, to longitudinally evaluate bradykinesia and impact of an FDA-approved antibiotic, initiated after lesion induction, as a potential repurposed drug in a 6-OHDA model. Method: 3-month old male Sprague-Dawley rats were evaluated at four time-points after lesion induction (days 7, 14, 21 and 28). Results: The OFL tests did not reveal any significant change in activity although there was a trend towards a decrease in ambulatory counts in the lesioned group over the 4-week period. However, FAS revealed a sustained deficit in right forepaw use (~50 %) while a compensatory increase in the use of the unaffected (left) forepaw was observed. The antibiotic further increased left forepaw use although no significant improvement was seen with the OFL test. Conclusion: Lesioning of the nigrostriatal pathway led to a transient compensatory increase in left forepaw use, an effect which was sustained by antibiotic administration suggesting a benefit that may curtail the onset of bilateral impairment.
Sex differences in cognitive function following methamphetamine exposure in young mice.
(2020) Vann, Philip; Wong, Jessica; Shetty, Ritu; Forster, Michael; Sumien, Nathalie; Davis, Delaney
Prescription stimulant misuse among youth has dramatically increased in the United States, with amphetamine compounds gaining popularity in college students. Overall, 20% of college students have reported stimulant misuse to improve their academic performance. There are reported sex differences in psychostimulant use: males use it more frequently but females use it at earlier ages and are more vulnerable to reinforcing effects and dependence. The purpose of this pilot study was to determine if exposure to a prototypical psychostimulant, methamphetamine (meth), would lead to cognitive impairments in young male and female mice. Groups of 4 month old C57BL/6J mice received either saline or methamphetamine (1 mg/kg; i.p.) twice a day for 4 weeks. Two weeks following the last injection, animals were tested for cognitive function using 3 different tests: Morris water maze, active avoidance and fear conditioning. Spatial learning seemed impaired in males but not females, after meth exposure. In active avoidance, female performance was impaired by meth in the learning phase but their cognitive flexibility was not affected, while it was the opposite for males. Fear conditioning response was reduced by meth exposure only in the females. These preliminary results suggest sex-dependent methamphetamine-induced impairments in cognitive function of young mice. Meth exposure impaired spatial memory and cognitive flexibility in males and impaired associative learning in females. While our Ns are too low to obtain significance, these outcomes suggest that further studies are needed and support the use of both sexes in rodent preclinical research of substance abuse.

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