Date of Award

5-1-2014

Degree Type

Dissertation

Degree Name

Doctor of Philosophy

Field of Study

Biomedical Sciences

First Advisor

Steve Mifflin

Abstract

This 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 > 0.05). GluR1 and GluR2 protein level changes were not significant in the caudal NTS, however, there was a significant decrease (P < 0.05) in GluR1 protein levels and significant increase (P < 0.05) in GluR2 protein levels in sub-postremal NTS

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.

Comments

Bathina, Chandra Sekhar, Role of catecholaminergic A2 neurons of nucleus of the solitary tract (NTS) in cardiovascular and respiratory adaptations to chronic intermittent hypoxia (CIH) in rats Doctor of Philosophy (Biomedical Sciences), April 2014, 171 pp. , 3 tables, 28 figures, 211 references. Available May 2015