Browsing by Subject "CIH"
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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(2013-04-12) Bathina, Chandra SekharPurpose: To understand the effect of CIH on the mRNA expression levels of AT1a, AT1b and excitatory amino acid (EAAs) receptor subunits in the A2 neurons of NTS and to assess the effect of tyrosine hydroxylase (TH) knockdown in A2 neurons on the cardiovascular responses to CIH Methods: Adult male Sprague-Dawley rats (250-350g) were injected with adeno-associated viruses, either (AAV-GFP-TH) to label the A2 neurons or (AAV-TH-shRNA) to knockdown TH in A2 neurons, into NTS. The control rats for (AAV-TH-shRNA) were injected with virus having scrambled RNA (AAV-sc). All the (AAV-TH-shRNA) and (AAV-sc) injected rats were implanted with radiotelemetry transmitters, 7 days prior to receiving virus injections, to monitor the changes in mean arterial pressure (MAP), heart rate (HR) and respiratory frequency (RF). 14 days after virus injections 7 of AAV-GFP-TH injected rats, all AAV-TH-shRNA and AAV-sc injected rats were exposed to 7 days of 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). 7 of AAV-GFP-TH injected rats were used as controls and were exposed to room air. Laser capture microdissection (LCM) was performed to capture 7-10 A2 neurons for RNA extraction. Gene expression for different genes were assessed by quantitative real time reverse transcription polymerase chain reaction (qRT-PCR) and compared between the control and CIH rats using 2-ΔΔct method. Hindbrains collected from AAV-TH-shRNA and AAV-sc injected rats were utilized for immunohistochemistry and western blot analysis Results: qRT-PCR study showed that CIH decreased AT1a (p=0.002; control - 1.08 ± 0.13, n=7; CIH - 0.48 ± 0.07, n= 6) and 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. CIH increased MAP and HR during the day in both the scRNA (n= 14, p<.001 MAP; p<.001HR) and shRNA (n=13, p<.001 MAP; p<.001 HR) groups. During the night, MAP and HR remained elevated in the scrambled rats (p<0.001 MAP; p<0.001 HR) but not in the shRNA group which can be attributed to the significant reduction in TH-immunoreactivity in NTS (shRNA: 28±lcells/section; scRNA: 35±lcells/section) Conclusions: These results suggest that A2 neurons play a role in the cardiovascular responses to CIH and following CIH, NTS neurons may become more calcium permeable, as GluR2 is found to resist calcium permeabilityItem The role of Angiotensin II in central autonomic and endocrine regulation(2014-08-01) Saxena, Ashwini; Cunningham, J. Thomas; Mifflin, Steve W.; Schreihofer, Ann M.Renin-Angiotensin system (RAS) is a peptidergic hormonal system that is known to regulate hemodynamic and fluid balance. Clinical success of RAS inhibitors in several cardiovascular and renal diseases such as hypertension (HTN), chronic heart failure, and diabetic nephropathy underscore its involvement in their pathophysiology. Recent research efforts are not only helping us understand the mechanisms through which RAS orchestrate the pathophysiology of cardiovascular diseases but are also identifying its involvement in other pathological conditions such as mood disorders and cancer. Although, initially identified as an endocrine system in peripheral circulation, the discovery of renin in the brain ushered in the concept of ‘local’ or ‘tissue’ RAS. Now, local RAS components are increasingly identified to be present in nearly all organ systems. The In the first project we investigated the role played by the communication between circulating Ang II and subfornical organ (SFO), a circumventricular organ lacking blood brain barrier, in chronic intermittent hypoxia (CIH) associated sustained increase in MAP even during period of normoxic breathing. Using viral mediated delivery of shRNA against Ang II type 1a receptors (AT1aR), it was found that the rats that received AT1aRshRNA in their SFO, exhibited increased MAP responses during CIH but their MAP recovered to levels of normoxic control during room air breathing. Also, disruption of this communication led to decreased FosB/ΔFosB staining in the autonomic regions of forebrain. FosB/ΔFosB staining identifies the expression of transcription factor FosB and its splice variant ΔFosB. These transcription factors are known to orchestrate transcriptional adaptations that lead to lasting neuroplastic adaptations. These data suggest that Ang II-SFO communication is essential in neuroplastic adaptations of forebrain autonomic nuclei, which may sustain the CIH associated increase in MAP even during periods of room air breathing. Second project was initiated to study the mechanisms through which synaptically released Ang II could affect post-synaptic neuronal sensitivity and function. It is known that bile duct ligated rats, an experimental model of cirrhosis, exhibit impaired osmoregulation. We previously reported that bile duct ligated rats show increased presence of a non-specific cation channel (TRPV4) in the hypothalamic membrane extracts. In addition, an activated RAS is also associated with fluid-electrolyte imbalance, a hallmark feature of cirrhosis. In this project it was investigated if Ang II could translocate TRPV4 to neuronal surface in vitro, in a hypothalamic neuronal cell line, 4B. In 4B cells, Ang II incubation was associated with increased TRPV4 localization to the cell membrane and was also associated with increased calcium influx in response to specific TRPV4 agonist, GSK 1016790A. In addition, these effects were completely blocked in the presence of AT1R receptor antagonist (Losartan) and Src kinase inhibitor, PP2. Taken together, these data suggest that Ang II could translocate TRPV4 to neuronal membrane via Src kinase pathway. These observations could explain one of the mechanisms through which Ang II could contribute to the pathophysiological adaptations that lead to increased water retention and dilutional hyponatremia associated with chronic liver failure.Item TIME COURSE OF CHANGES IN GLUTAMATERGIC TRANSMISSION WITHIN NTS DURING CIH EXPOSURE AND THE ROLE OF ΔFOSB(2014-03) Wu, Qiong; Mifflin, SteveΔFosB is a transcription factor induced by chronic intermittent hypoxia (CIH), a model of the arterial hypoxemia seen in sleep apnea patients. We reported that 7 days of CIH increases the amplitude of mEPSCs recorded in 2nd order arterial chemoreceptor NTS neurons. We hypothesize that NTS injection of a dominant-negative construct of ΔFosB (provided by Dr. E. Nestler) to block the function of ΔFosB will block the CIH increase in mEPSC. A brain slice preparation was used to record mEPSCs by whole cell patch clamp in a normoxia group and in rats exposed to CIH of differing durations. mEPSC amplitude in normoxia group and after 1, 3, 5, and 7 days CIH exposure averaged 12.3±0.8 pA (n=12), 19.6±1.3 pA (n=11), 17.5±1.6 pA (n=11), 16.7±1.2 pA (n=21), 18.2±0.8 pA (n=7), respectively (all p<.05 vs. normoxia). 1 day after a 7 day exposure, mEPSC amplitude remained increased (17.5±1.6 pA, n=4), after 3 day recovery mEPSC amplitude was similar to normoxia (12.9±1.0 pA, n=7). AAV-GFP-ΔJunD construct was microinjected into NTS to block the function of ΔFosB, then GFP labeled second order NTS neurons were recorded after 1 day and 7 days CIH exposure. ΔFosB inhibition decreased the amplitudes of mEPSCs to normoxia levels in both groups, 13.9±0.6 pA (n=10), 13.0±0.6 pA (n=19), respectively. CIH rapidly enhances the post-synaptic response to glutamatergic synaptic transmission within the NTS and ΔFosB plays a role in mediating this enhancement. ΔFosB is a transcription factor induced by chronic intermittent hypoxia (CIH), a model of the arterial hypoxemia seen in sleep apnea patients. We reported that 7 days of CIH increases the amplitude of mEPSCs recorded in 2nd order arterial chemoreceptor NTS neurons. We hypothesize that NTS injection of a dominant-negative construct of ΔFosB (provided by Dr. E. Nestler) to block the function of ΔFosB will block the CIH increase in mEPSC. A brain slice preparation was used to record mEPSCs from second order NTS neurons by whole cell patch clamp in a normoxia group and in rats exposed to CIH of differing durations. AAV-GFP-ΔJunD construct was microinjected into NTS to block the function of ΔFosB, then GFP labeled second order NTS neurons were recorded after 1 day and 7 days CIH exposure. mEPSC amplitude in normoxia group and after 1, 3, 5, and 7 days CIH exposure averaged 12.3±0.8 pA (n=12), 19.6±1.3 pA (n=11), 17.5±1.6 pA (n=11), 16.7±1.2 pA (n=21), 18.2±0.8 pA (n=7), respectively (all p<.05 vs. normoxia). 1 day after a 7 day exposure, mEPSC amplitude remained increased (17.5±1.6 pA, n=4), after 3 day recovery mEPSC amplitude was similar to normoxia (12.9±1.0 pA, n=7). AAV-GFP-ΔJunD construct was microinjected into NTS to block the function of ΔFosB, then GFP labeled second order NTS neurons were recorded after 1 day and 7 days CIH exposure. ΔFosB inhibition decreased the amplitudes of mEPSCs to normoxia levels in both 1 day and 7 days CIH groups, 13.9±0.6 pA (n=10), 13.0±0.6 pA (n=19), respectively. CIH rapidly enhances the post-synaptic response to glutamatergic synaptic transmission within the NTS and ΔFosB plays a role in mediating this enhancement.