Browsing by Subject "Hypoxia"
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Item A Celebration of the Extraordinary Life of Late Professor Tatiana V. Serebrovskaya (Kyiv, Ukraine) in Advancing Hypoxia Science and Medicine(Mary Ann Liebert, Inc., 2022-08-03) Swenson, Erik R.; Mallet, Robert T.; Xi, Lei; Manukhina, Eugenia B.; Downey, Fred; Burtscher, Johannes; Ehrenreich, Hannelore; Burtscher, MartinItem ANGIOTENSIN CONVERTING ENZYME 1 (ACE1) KNOCKDOWN IN THE MEDIAN PREOPTIC NUCLEUS (MNPO) ATTENUATES SUSTAINED DIURNAL HYPERTENSION FOLLOWING CHRONIC INTERMITTENT HYPOXIA(2014-03) Faulk, Katelynn; Cunningham, J. Thomas; Nedungadi, Thekkethil P.In order to study hypertension associated with Sleep Apnea, our lab uses a hypoxia model on rats. Rats are routinely used to model cardiovascular diseases of humans. We try to discover how the brain controls blood pressure in certain cardiovascular risk groups such as sleep apnea patients. This will help the scientific community better understand how sustained hypertension develops and progresses in sleep apnea patients and may lead to other discoveries about cardiovascular diseases. Purpose (a): Chronic Intermittent Hypoxia (CIH) is a model for the arterial hypoxemia seen in sleep apnea and is associated with a sustained increase in blood pressure throughout the diurnal cycle. Studies indicate that the MnPO contributes to this sustained component of CIH hypertension that persists during normoxia. MnPO neurons from rats show increased expression of the transcription factor FosB following CIH. Dominant-negative inhibition of a FosB splice variant in MnPO attenuates the sustained hypertension in CIH. We identified the pro-hypertensive ACE1 as a possible FosB target gene that may contribute to the sustained hypertension seen in CIH. Methods (b): We tested this hypothesis using a viral vector to knockdown ACE1 in the MnPO. Isoflurane anesthetized adult male rats were microinjected in the MnPO with 500nl of an adeno-associated virus containing GFP and either shRNA against ACE1 (shACE1) or a scrambled shRNA (shSCM). Changes in Mean arterial blood pressure (MAP) were recorded using radio telemetry. Rats were then exposed to CIH for 7 days through 3 minute periods of hypoxia (10% oxygen) and 3 minute periods of normoxia (21% oxygen) for 8 hours per day (0800-1600 h). Normoxic controls were exposed to room air. Laser capture microdissection followed by qRT-PCR showed that shACE1 significantly decreased ACE1 message in MnPO. Results (c): During CIH exposure, MAP significantly increased in both shACE1 and shSCM treated rats. During the normoxic dark phase, knockdown of ACE1 in the MnPO statistically decreased the sustained MAP component of CIH as compared to shSCM controls (P<0.001). Conclusions (d): These results show that ACE1 in the MnPO contributes to the sustained hypertension seen in our CIH model.Item CONTRIBUTION OF THE MNPO ANGIOTENSIN RECEPTORS TO BRAIN STEM ACTIVITY AND HYPERTENSION(2014-03) Shell, Brent; Cunningham, TomSleep apnea can increase blood pressure. It is not understood what changes in the brain occur while experiences lack of oxygen during sleep apnea that results in high blood pressure both during the day and during the sleeping hours. Our lab uses a model of sleep apnea that exposes rodents to periods of reduced oxygen. We have found that removing a receptor for a specific chemical in the front of the brain prevents the increase of blood pressure during the normal oxygen waking hours. This current study shows that the knockdown of this receptor decreases activity in the rear portions of the brain that directly control blood pressure. Understanding the mechanisms how sleep apnea leads to hypertension is essential for effective treatment of the disease. Purpose (a): The repeated bouts of hypoxia experienced by sufferers of sleep apnea results in persistent blood pressure elevation. This pathophysiological increase in pressure exists in both the hypoxic night phase and the normoxic period. Neurological mechanisms that drive this maladaptive blood pressure increase are not well understood. Our lab has shown that knockdown of the Angiotensin type 1a (At1a) receptor in a forebrain nucleus, the median preoptic nucleus (MnPO), prevents the normoxic blood pressure increase. How the MnPO At1a receptors affect downstream nuclei to maintain normal pressure is not known. In the current study, rats were exposed to chronic intermittent hypoxia (CIH) to simulate the hypoxic effects of sleep apnea. We then examined the activity of downstream nuclei by performing immunohistochemistry for ∆FosB, a marker for neuronal activity. We hypothesize that knockdown of AT1a in the MnPO results in decreased ∆FosB expression in downstream hypertensive nuclei such as the caudal ventrolateral medulla (CVLM), the rostral ventrolateral medulla (RVLM), and the nucleus tractus solitaries (NTS). Methods (b): After exposure to chronic intermittent hypoxia (CIH), rats are sacrificed, perfused with 4% paraformaldehyde, dehydrated with sucrose, and serial sectioned at 40 microns on a cryostat. Sections are split into three groups; one group is used for immunohistochemistry. Sections are processed with primary goat antibody for ∆FosB, a secondary biotinilated anti-goat, and finally visualized using diaminobenzidine. Localization of the NTS, CLVM, and RVLM was performed by double labeling for dopamine-β-hydroxylase (DβH), an enzyme used in the production of catecholamines. DβH was visualized using a CY3 fluorophore. Cell counts utilized at least 3 brain sections per nucleus. Results (c): A significant difference was found between the AT1a knockdown rats and the scramble rats in the subpostremal region of the NTS. This region has neurons that are responsible for processing both baroreceptor and chemoreceptor information. Conclusions (d): The MnPO is connected to this region through the paraventricular nucleus. Decreased MnPO activity could cause a decrease in the quantity of inputs to the hindbrain. These results, coupled with the prevention of the normoxic blood pressure increase, indicate that angiotensin acting through the MnPO is affecting the activity of neurons in the brainstem that are directly controlling blood pressure regulation.Item INTERMITTENT HYPOXIA RELATED GENERATION OF REACTIVE OXYGEN SPECIES AND BAROREFLEX REGULATION OF BLOOD PRESSURE(2013-04-12) Lane, AphtonPurpose: The intermittent hypoxia related generation of reactive oxygen species (ROS) associated with obstructive sleep apnea (OSA) increases sympathetic activity. However, the specific role of ROS in establishing the hypertension associated with chronic OSA has not been identified. We hypothesize that the intermittent hypoxia (IH) induced production of ROS will reset the operating point of the Carotid Baroreflex to a higher mean arterial pressure (MAP). Methods: Human subjects (n = 11, 5 female) underwent neck pressure/suction (NP/NS) to assess carotid baroreflex (CBR) function before and after intermittent hypoxia training with or without the antioxidant, N-acetyl cysteine (NAC). During NP/NS, mean arterial pressure was recorded non-invasively using a finometer. Following baseline measurements for hemodynamic variables and CBR function, subjects were asked to ingest a drink containing either NAC or a placebo. One hour after ingestion, hemodynamic variables and CBR function were again measured, following which the subjects were intermittent hypoxia trained (IHT). Immediately after IHT, hemodynamic and CBR function measurements were repeated three times 30 minutes apart. A two-way analysis of variance (ANOVA) was used to analyze differences between treatment groups across time. Results: There were no changes in CBR function with NAC versus placebo across time (p=0.791). In addition, the assessment of the operating point of the MAP with NAC versus placebo across time was not different (p=0.62). Conclusions: These data indicate that the known scavenging effect of ROS on central Nitric Oxide enabling a greater central sympathetic outflow was counteracted by an unidentified local vasodilation resulting in an unchanged MAP. The lack of increase in resting MAP following IHT is in agreement with previous studies that report no change in MAP after IHT.Item Intermittent Hypoxia Training to Foster Brain Recovery after Ischemic Stroke in rats(2018-05) Ruelas, Steven S.; Mallet, Robert T.; Jung, Marianna E.; Schreihofer, Ann M.; Das, Hriday K.Purpose: Ischemic stroke is the leading cause of disability and #5 cause of death in the US. Annually, nearly 800,000 Americans suffer an ischemic stroke, and 130,000 die. The only FDA approved treatment for stroke is recombinant tissue plasminogen activator, but this thrombolytic agent neither protects the affected tissue, nor mitigates the motor or cognitive impairments resulting from stroke. Intermittent hypoxia training (IHT) has been shown to increase cerebral blood flow, reduce oxidative stress, mobilize cerebroprotective signaling cascades and minimize behavioral deficits in a rat model of Alzheimer's Disease. Moreover, a 20 d IHT program attenuated behavioral deficits and protected neurons in ethanol-withdrawn (EW) rats, even when EW began 35 d after IHT. Therefore, we hypothesize that IHT, initiated in rats after stroke, preserves motor and cognitive function, relative to non-IHT rats. Methods: Ischemic stroke will be produced in rats by 90 min occlusion and abrupt reperfusion of the middle cerebral artery (MCA). Motor function and coordination will be evaluated by the rotarod test before and at 1 week intervals after MCA occlusion (MCAO). Rats must balance on a rotating cylinder that accelerates at a constant speed. High fall latency represents intact motor function. The Morris Water Maze (MWM) assesses spatial learning and memory. Rats are placed in an open, circular pool and must find a sunken platform within 90 s. 24 h after stroke, rats undergoing IHT will breathe moderately hypoxic gas (10% O2) for 5-8 cycles, each lasting 5-10 min, with intervening 4 min room air breathing, for 20 consecutive days. These rats will be compared to an MCAO group continuously exposed to 21% O2. At 21 d post-stroke, the brain will be harvested for analyses of infarct and neuroprotective proteins. Results: In pre-stroke testing, the time taken to solve the MWM fell progressively over 10 days, indicating spatial learning and memory, and fall latency on the rotarod lengthened over 5 days, reflecting improved coordination and possibly a training effect. These studies have established the pre-stroke baselines for assessment of IHT's impact on post-stroke recovery. Conclusions: We expect that IHT given after stroke will minimize motor and cognitive impairment by activating neuroprotective signaling cascades culminating in expression of anti-oxidant and anti-inflammatory proteins.Item INTERMITTENT INDUCED CHANGES IN GENE EXPRESSION IN THE MEDIAN PREOPTIC NUCLEUS (MNPO) OF RATS(2013-04-12) Saini, NiharikaPurpose: Our previous work indicated that changes in MnPO gene expression are necessary for the sustained increase in blood pressure produced by chronic intermittent hypoxia (CIH), an animal model of the hypoxemia associated with sleep apnea. This study tested the effects of a 7d CIH exposure on the expression of additional genes in the MnPO that could contribute to changes in the excitability of neurons in this region: Angiotensin receptor 1a (AT1a), Angiotensin receptor 1b (At1b), delta FBJ murine osteosarcoma viral oncogene homolog B (ΔFosB), transient receptor potential channel 4 (TRPC4), potassium-chloride cotransporter 2 (KCC2), and sodium-potassium-chloride cotransporter 1 (NKCC1). Methods: The brain was harvested from adult male rats anesthetized with inactin (100 mg/kg ip) after 7 d of CIH or 7 d of normoxia. Laser Capture Microdissection of MnPO was carried out from 10µ sections. MnPO samples were run through RNA purification, cDNA synthesis and PCR using S18 as the housekeeping gene. Results: PCR results revealed significant increases in the expression of TRPC4, ΔFosB, NKCC1 and AT1a in the MnPO of CIH treated rats relative to normoxic controls. KCC2 and AT1b gene expression in MnPO were not significantly affected by CIH. Conclusions: The results suggest that increased expression of TRPC4, ΔFosB, NKCC1 and especially AT1a could participate in the contribution of MnPO to the sustained hypertensive effects of CIH.Item Interplay between metabolic and myogenic mechanisms in coronary pressure-flow autoregulation(2022-05) Warne, Cooper M.; Tune, Johnathan D.; Dick, Gregory M.; Mallet, Robert T.The local metabolic hypothesis proposes that myocardial oxygen tension, indexed by coronary venous PO2 (CvPO2), determines the degree of coronary pressure-flow autoregulation. Conversely, the myogenic hypothesis proposes that pressure-induced vascular tone, indexed by the pressure at which coronary flow is zero (Pzf), determines autoregulation. My working hypothesis posits that if metabolism predominates, then autoregulation will be directly related to CvPO2, irrespective of reductions in coronary vasomotor tone. Conversely, if a myogenic mechanism predominates, then autoregulation will be directly related to Pzf, regardless of underlying CvPO2. I tested these hypotheses by examining the extent to which exaggeration of the metabolic error signal and attenuation of myogenic tone influences coronary autoregulation. Experiments were performed in anesthetized, open-chest swine in which a coronary artery was cannulated and connected to a servo-controlled roller pump system. This allowed coronary perfusion pressure (CPP) to be incrementally reduced from 140 to 40 mmHg before and during hypoxemia (SO2 ~50%). CvPO2 decreased 13 mmHg and coronary blood flow fell 57% as CPP was reduced. Hypoxemia augmented myocardial oxygen consumption (P < 0.01), increased coronary blood flow (P < 0.0001), and reduced CvPO2 (P < 0.0001) over the same CPP range. Coronary blood flow during hypoxemia maintained myocardial oxygen delivery (P = 0.20). Hypoxemia increased closed-loop autoregulatory gain (Gc) over a CPP range of 120 to 60 mmHg (P = 0.02). Gc was inversely correlated to CvPO2 and Pzf, but the correlation was stronger for CvPO2. These findings support that coronary pressure-flow autoregulation is augmented by hypoxemia-induced increases in the local metabolic error signal, regardless of the myogenic tone.Item Role of catecholaminergic A2 neurons of nucleus of the solitary tract(NTS) in cardiovascular and respiratory adaptations to chronic intermittent hypoxia (CIH) in rats(2014-05-01) Bathina, Chandra Sekhar; Steve MifflinThis study examined the role played by the catecholaminergic A2 neurons of the nucleus of the solitary tract (NTS) of adult male Sprague- Dawley rats in the increased mean arterial pressure (MAP) noticed following exposure to chronic intermittent hypoxia (CIH), a rodent model to simulate arterial hypoxemic conditions occurring in humans suffering from sleep apnea. In one study, we tested the hypothesis that tyrosine hydroxylase (TH) knockdown in NTS reduces the sustained elevation in MAP noticed in the rats exposed to CIH. Adult male Sprague-Dawley rats were implanted with radiotelemetry transmitters and adeno-associated viral constructs with a GFP reporter having either short hairpin RNA for TH (shRNA) or scrambled virus (scrambled) were injected into caudal NTS. shRNA through formation of RNA-induced silencing complex reduced the amount of TH levels in the NTS. Virus injected rats were exposed to 7 days CIH (alternating 6 min periods of 10% O2 and 4 min of 21% O2 from 8am to 4pm; from 4pm to 8am rats were exposed to 21% O2). CIH increased MAP and HR during the day in both the scrambled (n= 14, p Experiments were also conducted to understand the molecular level changes occurring in the A2 neurons, following CIH exposure. mRNA expression changes occurring in the A2 neurons were analyzed by novel technique of laser capture microdissection (LCM) by labeling the A2 neurons using adeno-associated virus with TH promoter attached to green fluorescent protein (GFP). A2 neurons are found to express mRNA of angiotensin receptor subtypes AT1a and AT1b. Moreover, excitatory amino acids (EAAs) like glutamate released from chemoreceptor afferents during chronic intermittent hypoxia (CIH) are found to modulate the activity of the neurons in the region of NTS. The aim of this study was to assess the effect of CIH on the mRNA expression levels of AT1a, AT1b and EAAs receptor subunits in the A2 neurons. We utilized commercially available adeno associated virus (AAV) vector mediated delivery of green fluorescent protein (GFP) labeled tyrosine hydroxylase promoter (AAV-GFP-TH), which will incorporate into the TH genome and express GFP with the TH expression to label the A2 neurons. 7 virus injected rats were exposed to 7 days CIH (alternating 6 min periods of 10% O2 and 4 min of 21% O2 from 8am to 4pm; from 4pm to 8am rats were exposed to 21% O2). Laser capture microdissection was performed to capture the A2 neurons from caudal NTS. Total RNA from these neurons was extracted and the gene expression for different genes were assessed by quantitative real time reverse transcription polymerase chain reaction and compared between the control and CIH rats using 2-ΔΔct method. CIH is found to decrease AT1a (p=0.002; control - 1.08 ± 0.13, n=7; CIH – 0.48 ± 0.07, n= 6) and α-amino-3-hydroxy-5-methyl-4-isoxazolepropionate receptor (AMPA) receptor subunit GluR2 (p=0.03; control - 1.11 ± 0.24, n=7; CIH- 0.52 ± 0.12, n= 6) and increase transcription factor FosB (p=0.03; control - 1.14 ± 0.25, n=7; CIH- 1.97 ± 0.25, n= 5) mRNA expression levels in the A2 neurons. These results suggests that there is increase in activity of these neurons following CIH and a possibility of these neurons becoming more calcium permeable as GluR2 is found to resist calcium permeability. Western blot studies were also conducted from the whole NTS punches, to study the changes in protein levels of the genes studied using LCM. The changes in TH protein levels were not significant in both caudal and sub-postremal NTS (P [greater than] 0.05). GluR1 and GluR2 protein level changes were not significant in the caudal NTS, however, there was a significant decrease (P As the mRNA analysis of A2 neurons suggested, there might be changes occurring in the calcium permeability of A2 neurons following CIH, attempts were made to do calcium imaging studies on the A2 neurons. There was difficulty in the colocalization of GFP with the fura-2AM, the calcium imaging dye. So, calcium imaging was conducted on the NTS neurons of sham Sprague-Dawley rats and CIH exposed rats. 30 μM AMPA application caused a 340/380 ratio change of 0.17 ± 0.01 (n=5) in control rats and this change was significantly higher 0.55 ± 0.13 in CIH rats. The probability of neurons responding to AMPA application was considerably higher in CIH rats. CNQX treatment of the slices abolished the changes in intracellular calcium in neurons from both control and CIH rats, demonstrating that the responses noticed after AMPA application were AMPA receptor mediated. Increases in intracellular calcium levels following 500 μM potassium chloride applications validate the fact that the neurons were viable. Further studies on quantifying the phosphorylated GluR1 and GluR2, subunits of AMPA receptors are required to explain the driving force behind this uniform increase in intracellular calcium levels of NTS neurons after CIH. We conclude that the sustained hypertension observed during CIH can be prevented by TH knockdown and this mechanism might involve paraventricular nucleus (PVN) of forebrain, hypothalamo-pituitary adrenal axis (HPA axis) or intermediolateral cell column (IML) of spinal cord. A2 neurons also undergo molecular alterations that might increase their calcium influx in to the neuron and vise-versa.Item Synergy 2011: Annual Research Report(2011-01-01)