Browsing by Author "Cunningham, J. Thomas"
Now showing 1 - 20 of 55
- Results Per Page
- Sort Options
Item A Vital role for Median Preoptic AT1a Receptors in the Sustained Hypertension of Chronic Intermittent Hypoxia(2016-03-23) Cunningham, J. Thomas; Shell, BrentPurpose The hypoxemia from Sleep Apnea (SA) results in hypertension during both the hypoxic sleeping period and the normoxic waking period. This pathophysiological sustained hypertension persists during waking hours and is a source of numerous cardiovascular sequlae. In order to better understand the neurological changes that underlie this disease state, our lab utilizes Chronic Intermittent Hypoxia (CIH) to model the hypoxemia and generate the hypertension of SA sufferers. Previously, our lab has shown that the Median Preoptic Nucleus increases in both neuronal activity and Angiotensin Type 1a Receptor (AT1aR) RNA expression in response to CIH. The MnPO is situated in a critical location that allows it to receive inputs from nuclei outside of the blood brain barrier and provide inputs to regions that control sympathetic outflow, and therefore blood pressure. The ability to modulate sympathetics based upon peripheral inputs coupled with increased activity and AT1aR expression leads us to hypothesize that increased Angiotensin signaling to the MnPO is essential for the sustained component of hypertension from CIH. Methods Male Sprague-Dawley rats were microinjected in the MnPO with either a virus to knockdown AT1aR expression (shAT1a) or a scramble virus (SCR) and instrumented with radio telemetry a week later. Radio telemetry provides continuous recording of cardiovascular variables. After a week of surgery recovery the animals were monitored for a 5 day baseline period before experiencing 7 days of CIH. The morning of the final day, the animals were either perfused with formaldehyde for immunohistochemistry (IHC) or their brains were snap frozen for quantitative PCR. Results Rodents injected in the MnPO with the shAT1a did not exhibit the sustained component of hypertension compared to SCR animals (P Conclusions Overall, this data indicates that the increase in AT1aR expression in the MnPO is essential for the development of the sustained component of CIH hypertension. The shAT1a virus successfully prevents the increase in AT1aR expression and this leads to normal waking blood pressure in the CIH exposed rodents. IHC results indicate that there is less activity in the MnPO and RVLM of the rodents most likely related to reduced sympathetic outflow. This data lends support to optimizing our current treatment regiment through blood brain barrier AT1aR blockers and ACE inhibitors. Future treatment methods could focus solely on preventing Angiotensin as a peptide neurotransmitter in the MnPO to ameliorate neurogenic hypertension.Item Androgen Modulation of CNS During Chronic Intermittent Hypoxia(2018-05) Snyder, Brina D.; Cunningham, Rebecca L.; Barber, Robert C.; Cunningham, J. Thomas; Schreihofer, Derek A.; Planz, John V.The underlying causes of age-related neurodegenerative diseases, such as Alzheimer's disease or Parkinson's disease, are unknown. It is likely conditions which contribute to an abundance of oxidative stress throughout life renders an individual more susceptible to late-life neurodegenerative processes. Sex differences are observed in the onset and prevalence of these diseases, suggesting estrogens and androgens influence these processes. This study investigates the early role of androgens under a known oxidative stressor, sleep apnea, which frequently goes untreated in the clinical population but is but is associated with an increased risk of late-life neurodegeneration. The hypoxic events of sleep apnea can be modeled in rats by the use of chronic intermittent hypoxia (CIH). Male rats are more susceptible to hypertensive effects of CIH, a key characteristic of sleep apnea. After one week of CIH treatment, they also exhibit oxidative stress and inflammation in circulation and in brain nuclei associated with early stages of Parkinson's disease or Alzheimer's disease. This led to the hypothesis that oxidative stress and inflammation would be associated with behavior deficits and these effects are mediated by androgens. Results show that oxidative stress and inflammatory dysregulation can be prevented by testosterone, but are highly exacerbated by testosterone's non-aromatizable metabolite, dihydrotestosterone (DHT). Administration of DHT also resulted in significant memory impairments under CIH. In the central nervous system, DHT significantly altered oxidative stress and pro-inflammatory signals, which may underlie its detrimental actions in an oxidative stress environment. There was also evidence of hypothalamic-pituitary-adrenal axis dysregulation, which can influence testosterone and circadian rhythms. These findings have broad implications for clinical populations with conditions which chronically increase oxidative stress and inflammation, while at the same time alter endocrine function. Conditions, such as untreated sleep apnea, may pose a latent risk for neurodegeneration and should be addressed early to prevent later detrimental effects.Item 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 ANGIOTENSIN II RECEPTOR TYPE-1A KNOCKDOWN IN SUBFORNICAL ORGAN PREVENTS SUSTAINED INCREASE IN MEAN ARTERIAL PRESSURE ASSOCIATED WITH CHRONIC INTERMITTENT HYPOXIA(2014-03) Saxena, Ashwini; Little, Joel T.; Nedungadi, Thekkethil P.; Cunningham, J. ThomasPurpose (a): Sleep apnea (SA) is associated with a sustained increase in mean arterial pressure (MAP) even during waking hours. Chronic intermittent hypoxia (CIH) models the hypoxemia associated with SA and produces elevated MAP during CIH and normoxia. Angiotensin II (Ang II) is implicated in the CIH associated increase in MAP. Subfornical organ (SFO), a forebrain circumventricular organ, lacks blood brain barrier and is a major site for the central effects of circulating Ang II. We investigated the effects of Ang II type 1a receptor knockdown (AT1aRKD) in the SFO on CIH hypertension in adult male rats. Methods (b): Adeno-associated viral vectors carrying GFP and either AT1aR shRNA or scrambled shRNA (SCM) were injected in SFO. Continuous measurements of mean arterial pressure, heart rate, respiratory rate, and activity were measured using radio-telemetry device implanted in abdominal aorta. Rats were exposed to cyclic hypoxia (3 min 21% O2 - 3 min 10% O2) for 8 hours/day for 7 days. Rats were sacrificed on Day 8. Results (c): Using laser-capture microdissection and qRT-PCR of amino-allyl RNA, AT1aRKD rats showed decreased SFO AT1aRmRNA in comparison with SCM rats. During intermittently-hypoxic light phase, the AT1aRKD rats exposed to CIH (3 min 10% O2 and 3 min room air cycles for 8 h during light phase for 7 d) exhibited significant increases in MAP vs. AT1aRKD-Normoxia group (p(p<0.05). During the normoxic dark phase, there was no difference in MAP between CIH and normoxic AT1aRKD rats (p=0.69). SCM-CIH group showed significant increase in MAP from SCM-Normoxia group during light phase CIH (p<0.001) and the normoxic dark phase (p<0.001). Conclusions (d): Our data indicate that AT1aRs in SFO may play a role in the sustained increases in MAP during normoxia associated with CIH.Item 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, CarolineAlthough 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.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 Brainstem mechanisms that impair autonomic regulation of blood pressure with obesity(2018-12) Chaudhary, Parul; Schreihofer, Ann M.; Mifflin, Steve W.; Cunningham, J. Thomas; Schreihofer, Derek A.Metabolic syndrome (MetS) is emerging as a global health threat due to its strong association with increased risk for cardiovascular disease and diabetes. Currently, 20-25% of the world's population exhibits some traits of MetS, namely obesity, dyslipidemia, hyperinsulinemia, hypertension, and hyperglycemia. In addition, MetS also promotes the development of impaired short-term regulation of mean arterial pressure (AP) by baroreflexes, which normally act to stabilize AP. The resulting increased AP variability, which is an independent risk factor for poor outcomes, is overlooked as a trait of MetS and goes without evaluation or treatment. People who have controlled hypertension without minimizing elevated AP variability are still at significant risk for detrimental cardiovascular events such as stroke and cognitive decline. Therefore, understanding mechanisms impairing baroreflexes with MetS will help determine appropriate therapeutic management to restore baroreflexes and promote stability of AP. Furthermore, because sex differences in the development of impaired baroreflexes with obesity have been reported, an understanding of how females are protected would provide valuable insights for underlying causes for early onset of impaired baroreflexes in obese males and eventual development of impaired baroreflexes in obese females. In this project, I utilized a rodent model of MetS, obese Zucker rats (OZR), to examine contributions of hypertension and hyperglycemia in the development of impaired baroreflexes in male OZR, and whether hypertensive female OZR have delayed onset of impaired baroreflexes because they have the ability to maintain glycemic control. Male and female OZR have excess weight gain from an early age because the mutation of a leptin receptor renders them insensitive to leptin's actions to regulate appetite and metabolism, promoting excess intake of standard chow and storage of ingested calories. Like obese humans, OZR develop dyslipidemia, hypertension, and insulin resistance that eventually progresses to type 2 diabetes, making them a suitable model for the consequences of MetS. Young adult male OZR (12-15 weeks) develop sympathetically driven hypertension with pronounced attenuation of baroreflex control of heart rate (HR) and sympathetic nerve activity (SNA) compared to juvenile OZR and lean Zucker rats (LZR). In male OZR, the development of impaired baroreflexes coincides with blunted activation of the NTS, the brain stem region that receives baroreceptor afferent inputs to promote baroreflex-mediated changes in HR and SNA, and this deficit likely yields diminished baroreflexes observed in young adult male OZR. In the first project I examined whether improvement of impaired glycemic control in young adult male OZR restores baroreflex-mediated bradycardia and activation of the NTS. Both type 1 and type 2 diabetic rats have impaired vagally-mediated activation of the NTS, in agreement with the reported loss of glucose's ability to enhance glutamatergic neurotransmission within the NTS of hyperglycemic, diabetic rodents. Male OZR develop insulin resistance at an early age, characterized by elevated insulin and triglycerides with impaired glucose tolerance but normal fasting hyperglycemia. We examined glucose homeostasis using chronic measures of blood glucose by telemetry in undisturbed rats because of previous reports of exaggerated stress responses. We observed that although young adult (12-14 weeks old) male OZR have normal fasting blood glucose, they are chronically hyperglycemic with access to food. Treatment of OZR with metformin or pioglitazone restored fed blood glucose levels with access to food and enhanced baroreflex-mediated bradycardia and activation of the NTS, as suggested by phenylehphrine-induced c-Fos expression. In contrast, treatment of LZR did not alter glucose or affect baroreflex-mediated bradycardia and activation of the NTS. Neither treatment reduced elevated AP and insulin in OZR, suggesting the lowering of blood glucose was effective for restoring baroreflexes in young adult male OZR, even in the face of hypertension. In the second project I examined whether the delayed onset of impaired baroreflexes in hypertensive female OZR could be due to their ability to maintain a normal blood glucose and baroreflex-mediated activation of the NTS. Premenopausal obese women protected from diabetes, suggesting they would be protected from deficits produced by hyperglycemia. I observed that intact baroreflex-mediated bradycardia in young adult female OZR extended to preserved sympathetic baroreflexes and baroreflex-mediated activation of the NTS in 12-15-week-old female OZR. Furthermore, although these OZR were hypertensive and hyperinsulinemic, fed glucose levels and glucose tolerance are comparable to LZR. In contrast, by 6 months of age, baroreflex-mediated bradycardia was blunted in female OZR. However, fed glucose was only mildly elevated and baroreflex-mediated activation of the NTS was comparable in OZR and LZR. These data suggest the ability to maintain glucose homeostasis in young adult female OZR coincides with a preservation of baroreflex-mediated bradycardia and activation of the NTS. However, the later development of impaired baroreflex-mediated bradycardia in female OZR occurs through mechanisms distinct from those observed in male OZR. The third project examined whether preventing hypertension in male OZR protected against the development of impaired baroreflexes and activation of the NTS. Treatment with losartan or hydralazine normalized baseline AP in male OZR without affecting hyperinsulinemia, dyslipidemia, or hyperglycemia. Furthermore, these treatments enhanced baroreflex-mediated bradycardia and activation of the NTS in male OZR. However, even when AP was normalized in male OZR, baroreflex-mediated bradycardia was still smaller in treated OZR compared to like-treated LZR, suggesting other mechanisms also contribute to the blunted baroreflexes. Together these studies suggest that the development of hyperglycemia and hypertension in male OZR contribute to impaired baroreflex-mediated bradycardia and activation of the NTS in male OZR. However, the ability of female OZR to maintain glucose homeostasis preserves baroreflexes despite the presence of hypertension and hyperinsulinemia. Furthermore, when female OZR later develop impaired baroreflex-mediated bradycardia, this deficit occurs by mechanisms that differ from male OZR, highlighting the need to examine both sexes for the development of cardiovascular and metabolic disorders.Item Cardiovascular Metrics Associated With Prevention of Aging-Related Parkinsonian Signs Following Exercise Intervention in Sedentary Older Rats(Frontiers Media S.A., 2021-12-15) Kasanga, Ella A.; Little, Joel; McInnis, Tamara R.; Bugnariu, Nicoleta; Cunningham, J. Thomas; Salvatore, Michael F.Preservation of motor capabilities is vital to maintaining independent daily living throughout a person's lifespan and may mitigate aging-related parkinsonism, a progressive and prevalent motor impairment. Physically active lifestyles can mitigate aging-related motor impairment. However, the metrics of physical activity necessary for mitigating parkinsonian signs are not established. Consistent moderate intensity (~10 m/min) treadmill exercise can reverse aging-related parkinsonian signs by 20 weeks in a 2-week on, 2-week off, regimen in previously sedentary advanced middle-aged rats. In this study, we initiated treadmill exercise in sedentary 18-month-old male rats to address two questions: (1) if a rest period not longer than 1-week off exercise, with 15 exercise sessions per month, could attenuate parkinsonian signs within 2 months after exercise initiation, and the associated impact on heart rate (HR) and mean arterial pressure (MAP) and (2) if continuation of this regimen, up to 20 weeks, will be associated with continual prevention of parkinsonian signs. The intensity and frequency of treadmill exercise attenuated aging-related parkinsonian signs by 8 weeks and were maintained till 23 months old. The exercise regimen increased HR by 25% above baseline and gradually reduced pre-intervention MAP. Together, these studies indicate that a practicable frequency and intensity of exercise reduces parkinsonian sign severity commensurate with a modest increase in HR after exercise. These cardiovascular changes provide a baseline of metrics, easily measured in humans, for predictive validity that practicable exercise intensity and schedule can be initiated in previously sedentary older adults to delay the onset of aging-related parkinsonian signs.Item Caspase Lesions of PVN-Projecting MnPO Neurons Blocks the Sustained Component of CIH-Induced Hypertension in Adult Male Rats(2019-03-05) Wang, Lei; Little, Joel; Cunningham, J. Thomas; Marciante, Alexandria B.Purpose: Obstructive Sleep Apnea (OSA) is characterized by cessations in respiration that leads to development of hypertension and persists into the waking period even during normal respiratory patterns. Previous studies show that experimental models of chronic intermittent hypoxia (CIH) produces sustained hypertension similar to that with OSA. It has been proposed that peripheral and CNS renin-angiotensin systems contribute to hypertension associated with CIH. Our working hypothesis is that increased circulating angiotensin II feeds into the forebrain, increasing excitatory signaling through the hypothalamus and hindbrain, creating a vicious cycle. The median preoptic nucleus (MnPO) is an integrative forebrain region that contributes to blood pressure regulation. The MnPO has projections to the paraventricular nucleus (PVN) of the hypothalamus, which contains pre-autonomic centers that project to regions in the hindbrain and regulate sympathetic outflow. We hypothesize that lesioning pathway specific projections from the MnPO to the PVN could attenuate CIH hypertension. Methods: Adult male Sprague-Dawley rats (250-300g) were anesthetized with isoflurane and stereotaxically injected bilaterally in the PVN with a retrograde AAV containing Cre (AAV9.CMV.HI.eGFP-Cre.WPRE.SV40) and with the caspase-3 (AAV5-flex-taCasp3-TEVp) or control virus (AAV5-hSyn-DIO-mCherry) in the MnPO. After 1-week recovery, rats were instrumented with aortic radio telemetry and allowed an additional week recovery. Rats were then moved to new homecages and underwent baseline recording before undergoing our 7-day CIH protocol. Results: The control group exposed to CIH developed chronic hypertension, however, caspase lesions blunted the sustained hypertension. Brain tissue processed for FosB immunohistochemistry showed decreased expression with caspase-induced inhibition in the MnPO and downstream autonomic regulating nuclei. CIH significantly increased plasma advanced oxidative protein products (AOPP) levels in controls. This increase in AOPP levels was blocked in caspase-lesioned rats comparable to normoxic control concentrations. In situ hybridization experiments indicate a reduction in angiotensin type 1a receptors (AT1aR) expression in the caspase-lesioned group exposed to CIH compared to CIH controls. Conclusion: The results indicate that MnPO neurons that project to the PVN play a significant role in blood pressure regulation and in the development of persistent CIH hypertension.Item Characterization of Arterial Pressure and Cerebral Blood Flow Responses To Repeated Thigh Cuff Inflation In Three Experimental Models (Humans, Pigs, Rats)(2022-05) Bhuiyan, Nasrul A.; Rickards, Caroline A.; Tune, Johnathan D.; Cunningham, J. ThomasIn a human model of simulated blood loss, oscillatory patterns of arterial pressure and blood flow, or "pulsatile perfusion", can protect cerebral and peripheral tissue oxygenation, and prolong tolerance to this stress. In this pilot study, we characterize the hemodynamic responses to pulsatile perfusion therapy induced via repeated thigh cuff inflations in humans at rest, and in pig and rat models of actual blood loss. In 2 human participants, 0.1 Hz (10-second cycle) thigh cuff oscillations induced robust 0.1 Hz oscillations in arterial pressure and cerebral blood flow. In the two animal models, all subjects underwent a baseline period, hemorrhage of 55% of total blood volume, then a 30-min period with or without thigh cuff oscillations (0.1 Hz for pigs, and 0.5 Hz for rats). Decreases in mean arterial pressure (MAP) and carotid artery blood flow were observed in response to hemorrhage (P≤0.002) in both pigs and rats. At the end of the PPT period, however, no differences were observed between the oscillation or no oscillation groups for absolute MAP (rats, P=0.44; pigs, P=0.90) or common carotid artery (CCA) peak blood flow (rats, P=0.92; pigs, P=0.93). When examining the frequency power spectrums, there was not a robust increase in 0.5 Hz oscillations for MAP (P=0.23) or CCA flow (P=0.82), but 0.1 Hz oscillations were detected in CCA flow for pigs (P=0.09). While in the human model, large increases in oscillatory power were observed for both arterial pressure and cerebral blood flow, the responses in the two animal models were inconclusive due to high inter-individual variability. These findings indicate the need for further studies and refinement of the thigh cuff approach in the animal models to reliably induce hemodynamic oscillations.Item 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, GeorgeRats 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.Item Chronic Intermittent Hypoxia increases oxidative stress and inflammation(2015-03) Snyder, Brina; Shell, Brent; Cunningham, J. Thomas; Cunningham, RebeccaBackground: Inflammation has been linked with sleep apnea. Sleep apnea is a common comorbidity associated with neurodegenerative disorders, such as Parkinson’s disease and Alzheimer’s disease. Furthermore, neurodegenerative diseases have also been linked with inflammation. A possible mechanism underlying increased inflammation in these disorders is oxidative stress, a hallmark of neurodegeneration. To examine the role of oxidative stress on inflammation, we used chronic intermittent hypoxia (CIH), an established model for the hypoxemia associated with sleep apnea. CIH consists of recurring events of low oxygen followed by reoxygenation. Statement of Hypothesis: We hypothesize that CIH causes oxidative stress, which induces inflammation. Materials and methods: To test this hypothesis, plasma from adult male rats subjected to 7 days of CIH (3 minute periods of hypoxia (10% oxygen) and 3 minute periods of normoxia (21% oxygen) for 8 hours per day) or normoxia (room air) were tested for AOPP, an indicator of oxidative stress, and circulating inflammatory markers (such as IL-1b, IL-10, IL-4, IL-6). Additionally, a group of rats was administered a neurotropic AAV with shRNA for AT1a receptors in their forebrains and instrumented with telemetry for blood pressure recording prior to CIH treatment to determine the effects of angiotensin on CIH hypertension and oxidative stress. Significant results: Our results showed that CIH significantly increased circulating oxidative stress and inflammation. Interestingly, IL-1b, IL-2, and TNF-a inflammatory markers were associated with oxidative stress, unlike IL-10, IL-4, and IL-6 inflammatory markers. These markers were positively associated with IL-1b. Knockdown of angiotensin 1 receptors in the forebrain blocked the diurnal hypertension and CIH induced oxidative stress, indicating the involvement of CIH hypertension and central angiotensin receptors in CIH induced oxidative stress. Conclusions: These results indicate that both neurons and macrophages contribute to CIH induced oxidative stress and inflammation and that CIH oxidative stress and inflammation is dependent on central angiotensin receptors and CIH hypertension.Item Chronic intermittent hypoxia induces oxidative stress and inflammation in brain regions associated with neurodegeneration(2016-03-23) Shell, Brent; Cunningham, J. Thomas; Cunningham, Rebecca; Snyder, Brina D.Age is the highest risk factor for the development of neurodegenerative diseases (ND), such as Alzheimer’s disease (AD) and Parkinson’s disease (PD). As life expectancy increases, the incidence of ND is projected to rise accordingly. Increased ND incidence will be associated with high healthcare costs. Currently, no cure exists for ND and diagnosis occurs at advanced stages, which foreshadows a financial healthcare crisis. Therefore, early identification of patients at risk for ND may provide opportunities for more effective therapies. Since ND is associated with increased oxidative stress (OS) and inflammation, these markers could be used to identify patients at risk for ND. Multiple environmental factors can be an oxidative stressor, and thus exacerbate inflammation induced ND risk. One such environmental factor that increases OS is sleep apnea (SA), a common ND comorbidity. However, it is unknown if SA induced oxidative stress activates neuroinflammation in areas associated with ND. To model SA in rats, chronic intermittent hypoxia was used. Male rats were exposed to six minute chronic intermittent hypoxia (CIH) cycles, during which oxygen levels were rapidly decreased from 21% to 10% then returned to normal room air levels, eight hours a day during the light phase for seven days. Plasma and tissue from hippocampus (HIPP), entorhinal cortex (ETC), substantia nigra (SN), rostral ventrolateral medulla (RVLM), and solitary tract nucleus (NTS) were collected and tested for levels of OS and inflammation, using Advanced Oxidative Protein Products (AOPP) and multiplex immunoassays, respectively. OS markers and inflammation were elevated in the plasma of rats exposed to CIH compared to control rats. Differences in neuroinflammatory markers within tissues were observed. Specifically, inflammatory markers in the RVLM were significantly decreased in animals exposed to CIH while TNF-a and IL-6 were elevated in the SN. TNF-a was positively associated with plasma OS and the cytokine associated with inflammatory cell recruitment, KC-Gro, exhibited the same pattern in the ETC. The ETC and SN are areas associated with initial neurodegenerative processes in AD and PD, respectively. CIH may contribute to processes involved in early ND pathology by elevating OS and inflammation in critical brain regions. These results indicate that SA can exacerbate ND by increasing OS-induced neuroinflammation. Therefore, treatment of SA could be one consideration in preventing ND.Item Contribution of K+/Cl- Cotransporters in AT1aR Dependent GABAa Inhibition in the MnPO Following Chronic Intermittent Hypoxia(2019-03-05) Little, Joel; Marciante, Alexandria B.; Cunningham, J. Thomas; Farmer, George Jr.Purpose: Chronic intermittent hypoxia (CIH) is an animal model that simulates the hypoxemia seen in obstructive sleep apnea (OSA). Rats exposed to CIH exhibit an increase in blood pressure during periods of normoxia, similar to that observed in OSA. The median preoptic nucleus (MnPO) exhibits increases in Angiotensin type 1a receptor (AT1aR) mRNA following CIH and blocking this increase in AT1aR mRNA prevents the sustained increase in blood pressure. Here we investigate the role of AT1aR in the MnPO and the contribution of the K+/Cl- cotransporters KCC2 and NKCC1 on excitatory/inhibitory balance in rats subjected to CIH. Methods: Under isoflurane (2-3%) anesthesia, male Sprague-Dawley rats (250-350g) received microinfusions (0.4 µL) of recombinant AAV construct containing GFP reporter and shRNA against AT1aR (AT1aKD) or an AAV containing the GFP reporter and a shRNA scramble (Scr) targeted to the MnPO. After recovery, rats were subjected to 7 days of CIH (0800-1600 hrs). The CIH protocol consisted of 6 min cycles (3 min 21% O2, 3 min 10% O2) repeated 10x/hr for 8 consecutive hrs (during the normal inactive/sleep phase) on 7 consecutive days. After 7 days CIH, the rats were anesthetized with isoflurane (2-3%) and coronal slices (300 µm) containing the MnPO were cut using standard in vitro slice procedures. Loose patch recordings were obtained from GFP labeled neurons using glass micropipettes containing aCSF as the internal solution (1-3 MΩ). Spontaneous action potentials (APs) were recorded in response to muscimol (100uM, 30s). Results: The GABAa agonist muscimol decreased AP activity of neurons from normoxic/Scr rats. GABAa inhibition was blunted in neurons from CIH/Scr and normoxic/AT1aKD rats. However, GABAa activation from neurons in the CIH/AT1aKD group produced an increase in spontaneous activity. KCC2 block reduced GABAa mediated excitation in CIH/AT1aKD but had no effect GABAa mediated inhibition in CIH/Scr. NKCC1 block reduced GABAa mediated excitation in CIH/AT1aKD and facilitated GABAa mediated inhibition in CIH/Scr. Conclusion: The current study shows AT1aKD mediated reduction in GABAa inhibition is exacerbated such that GABAa activation is excitatory following CIH. KCC2 and NKCC1 contribute to GABAa mediated excitability in CIH/AT1aKD but only NKCC1 contributes to attenuated GABAa function in CIH/Scr. Future studies will address the influence of reduced AT1a signaling and reduced GABAa mediated inhibition on downstream targets of the MnPO.Item DREADD-Induced cFos Expression in the Basal Forebrain of Male Rats(2017-03-14) Cunningham, J. Thomas; Marciante, Alexandria B.Purpose: Designer Receptors Exclusively Activated by Designer Drugs, or DREADDs, are genetically modified G-protein coupled receptors (GPCR) that are sensitive to an exogenous pharmacological agent, clozapine-N-oxide (CNO). DREADDs can be packaged in viral vectors with specific promoters or combined with CRE dependent platforms to express these receptors in specific neuronal phenotypes. This chemogenetic approach can be used to activate (Gq), inhibit (Gi), or stimulate cAMP (Gs) in neurons expressing DREADD receptors. In the present study, we tested the effects of a CRE independent Gq DREADD, using a CaM Kinase (CaMKIIa) promoter, and a mCherry reporter (rAAV5-CaMKIIa-hM3D(Gq)-mCherry) on Fos staining in the basal forebrain. Methods: Adult male Sprague-Dawely rats (250-300 g bw, Charles River) were anesthetized with isoflurane and stereotaxically injected with an AAV containing the hM3D(Gq) or a control virus (rAAV5-CaMKIIa-mCherry) in either the diagonal band of bregma (DBB) or the median preoptic nucleus (MnPO). Animals were monitored for recovery for two weeks and then administered CNO or vehicle. CNO was dissolved into dimethylsulfoxide (DMSO) and saline (ratio 20% to 80%) and given via intraperitoneal injection (IP) at a concentration of 10mg/kg. Rats were food and water deprived for 90 minutes following administration of CNO or vehicle and then perfused transcardially using 4% paraformaldehyde. Brains were harvested and placed into 30% sucrose until proper dehydration of the brain. Forebrains were then sliced into 40 micron segments using cryostat. Immunohistological techniques were performed as previously described using peroxidase staining for Fos to determine activation of the DREADD virus and fluorescent staining for mCherry to verify cells transfected with either the DREADD or control virus. Results: Overall, rats transfected with the Gq DREADD virus and treated with CNO showed significantly elevated Fos staining in the DBB or MnPO than groups transfected with either Gq DREADD and treated with vehicle or the control virus and treated with CNO or vehicle. Those transfected with the Gq DREADD virus in the DBB and treated with CNO, showed significantly more Fos staining than those transfected with either Gq DREADD and treated with vehicle (P Conclusions: These results indicate that Gq DREADD can be used to differentially activate neurons in either the DBB or the MnPO to influence activity in downstream regions that control autonomic and neuroendocrine function.Item DREADD-induced inhibition of the MnPO affects drinking behavior and neuroendocrine function in adult male rats(2018-03-14) Farmer, George; Wang, Lei; Cunningham, J. Thomas; Marciante, Alexandria B.Purpose: Angiotensin II (Ang II) is a peptide hormone that contributes to body fluid balance and hypertension. Forebrain circumventricular organs (CVOs) are sensitive to circulating Ang II and project to the median preoptic nucleus (MnPO). The MnPO projects to the paraventricular nucleus (PVN) and contributes to elevated sympathetic tone and thirst. Methods: We used Designer Receptors Exclusively Activated by Designer Drugs (DREADDs) to test the role of the MnPO in thirst and neuroendocrine responses to Ang II in adult male Sprague-Dawley rats (250-300g). Rats were anesthetized with isoflurane and stereotaxically injected with an inhibitory (Gi) DREADD (rAAV5-CaMKIIa-hM4D(Gi)-mCherry) or control (rAAV5-CaMKIIa-mCherry) virus in the MnPO. After 2 weeks of recovery, each rat was administered 10 mg/kg of exogenous Clozapine-N-Oxide (CNO) ip to inhibit DREADD expressing cells or vehicle ip followed by 2 mg/kg Ang II sc twice per week for 4 weeks. Rats were anesthetized with inactin (10 mg/kg ip) and transcardially perfused 90 minutes after CNO and Ang II treatments. Brains were processed for cFos and mCherry immunohistochemistry. Results: DREADD-injected rats treated with CNO during Ang II exposure had a significantly attenuated drinking response compared to vehicle treatments or to control virus injected rats treated with CNO and Ang II (pIn vitro loose-cell voltage clamp recordings from DREADD-transfected MnPO slices indicated focal CNO (10 uM) application significantly reduces firing rates of these neurons. In situ hybridization experiments of DREADD-transfected MnPO neurons and vesicular glutamate transporter 2 indicated neurons transfected with the DREADD virus containing the CaMKIIa promotor are largely glutamatergic (89.17+1.32%). Conclusion: The results indicate CNO-induced inhibition of excitatory, CaMKIIa-expressing MnPO neurons influences drinking behavior and neuroendocrine function.Item Dysfunctional neuroimmune pathways promote the development and maintenance of lupus hypertension(2020-05) Pham, Grace S.; Mathis, Keisa W.; Rickards, Caroline A.; Goulopoulou, Styliani; Cunningham, J. Thomas; Ma, Rong; Mathew, Stephen O.Hypertension afflicts nearly half of the adults in the United States and the majority of cases have no known cause. Chronic inflammation has been implicated in the development and maintenance of hypertension, and autoimmunity may comprise one of its sources. Hypertension is highly prevalent in the autoimmune disease systemic lupus erythematosus (SLE), in which chronic aberrant inflammation may be a causative factor. Endogenous neuroimmune pathways, such as the hypothalamic-pituitary-adrenal (HPA) axis and the cholinergic anti-inflammatory pathway, likely contribute to this phenomenon. The HPA axis is a classical neuroimmune mechanism that senses peripheral inflammation via afferent vagal fibers, culminating in the release of the anti-inflammatory hormone cortisol. Previous studies have characterized HPA axis dysfunction in SLE, but less is known about how this dysregulation specifically impacts the hypertension that occurs in the setting of SLE. A second neuroimmune interaction, the cholinergic anti-inflammatory pathway, is an efferent vagus nerve-to-spleen mechanism that relies on T cell-produced acetylcholine to quell inflammation in acute settings and may be hypoactive in chronic inflammatory diseases like SLE. Notably, both of these neuroimmune mechanisms depend on vagus nerve function, identifying the vagus as a potential target for neuromodulation. Furthermore, the relationship between chronic inflammation and hypertension validates the investigation of neuroimmune pathway dysfunction towards novel mechanisms of hypertension. Herewithin, the HPA axis and cholinergic anti-inflammatory pathway are investigated using the well-established NZBWF1 mouse model of lupus hypertension. Our findings are that (1) administration of an inflammatory stimulus that activates vagal afferents elicits comparable neuronal activation in the paraventricular nucleus of the hypothalamus, compared to control mice, despite heightened peripheral inflammation; (2) amplification of efferent vagus nerve activity reduces blood pressure and renal inflammation; and (3) chronic unilateral vagotomy paradoxically results in decreased blood pressure and renal inflammation. Taken together, these findings identify dysfunction in two neuroimmune pathways while demonstrating that interventions targeting these pathways may have therapeutic benefits in lupus hypertension. In terms of future impact, these results may promote continuing inquiry in a more recently discovered neuroimmune pathway (i.e., cholinergic anti-inflammatory pathway), as well as reinstate curiosity in an older, abandoned area of research (i.e., HPA).Item Effects of bile duct ligation on the inhibitory control of supraoptic vasopressin neurons(John Wiley & Sons, Inc., 2023-06-20) Aikins, Ato O.; Farmer, George E.; Little, Joel T.; Cunningham, J. ThomasDilutional hyponatremia due to increased plasma arginine vasopressin (AVP) is associated with liver cirrhosis. However, plasma AVP remains elevated despite progressive hypoosmolality. This study investigated changes to inhibitory control of supraoptic nucleus (SON) AVP neurons during liver cirrhosis. Experiments were conducted with adult male Sprague-Dawley rats. Bile duct ligation was used as a model of chronic liver cirrhosis. An adeno-associated virus containing a construct with an AVP promoter and either green fluorescent protein (GFP) or a ratiometric chloride indicator, ClopHensorN, was bilaterally injected into the SON of rats. After 2 weeks, rats received either BDL or sham surgery, and liver cirrhosis was allowed to develop for 4 weeks. In vitro, loose patch recordings of action potentials were obtained from GFP-labeled and unlabeled SON neurons in response to a brief focal application of the GABA(A) agonist muscimol (100 muM). Changes to intracellular chloride ([Cl]i) following muscimol application were determined by changes to the fluorescence ratio of ClopHensorN. The contribution of cation chloride cotransporters NKCC1 and KCC2 to changes in intracellular chloride was investigated using their respective antagonists, bumetanide (BU, 10 muM) and VU0240551 (10 muM). Plasma osmolality and hematocrit were measured as a marker of dilutional hyponatremia. The results showed reduced or absent GABA(A) -mediated inhibition in a greater proportion of AVP neurons from BDL rats as compared to sham rats (100% inhibition in sham vs. 47% in BDL, p = .001). Muscimol application was associated with increased [Cl]i in most cells from BDL as compared to cells from sham rats (chi(2) = 30.24, p < .001). NKCC1 contributed to the impaired inhibition observed in BDL rats (p < .001 BDL - BU vs. BDL + BU). The results show that impaired inhibition of SON AVP neurons and increased intracellular chloride contribute to the sustained dilutional hyponatremia in liver cirrhosis.Item Establishing an STR allele frequency database for the Paraguayan population(2018-05) Giuffrida, Stephanie M.; Planz, John V.; Warren, Joseph E.; Phillips, Nicole R.; Cunningham, J. ThomasThis project determined whether the loci included in the Athos PCR Directa para Identificacao Humana Amplification and AmpFlSTR[R] Identifiler[R] PCR Amplification Kits are suitable for establishment of an STR allele frequency database representative of the Paraguayan population. Allele frequencies for the 21 STR loci included in the Athos PCR Directa para Identificacao Humana Amplification Kit were calculated for 200 individuals in the Paraguayan population. Allele frequencies for the 15 STR loci included in the AmpFlSTR[R] Identifiler[R] PCR Amplification Kit were also calculated for 300 individuals in the Paraguayan population. Performance of these loci was determined by testing the population for Hardy-Weinberg Equilibrium, testing for Linkage Disequilibrium between the loci in the kits, and calculating Power of Discrimination, Power of Exclusion, and Mean Power of Exclusion. Comparability between the common loci included in the two PCR kits was also tested. Results of these tests determined that the Athos PCR Directa para Identificacao Humana Amplification and AmpFlSTR[R] Identifiler[R] PCR Amplification Kits are both suitable for use in an STR allele frequency database. Additionally, the common loci contained in the two PCR kits are comparable, and the populations can be combined to create one database.Item Establishing Equivalent Aerobic Exercise Parameters Between Early-Stage Parkinson's Disease and Pink1 Knockout Rats(IOS Press, 2022-06-28) Salvatore, Michael F.; Soto, Isabel; Kasanga, Ella A.; James, Rachael; Shifflet, Marla K.; Doshier, Kirby; Little, Joel T.; John, Joshia; Alphonso, Helene M.; Cunningham, J. Thomas; Nejtek, Vicki A.BACKGROUND: Rodent Parkinson's disease (PD) models are valuable to interrogate neurobiological mechanisms of exercise that mitigate motor impairment. Translating these mechanisms to human PD must account for physical capabilities of the patient. OBJECTIVE: To establish cardiovascular parameters as a common metric for cross-species translation of aerobic exercise impact. METHOD: We evaluated aerobic exercise impact on heart rate (HR) in 21 early-stage PD subjects (Hoehn Yahr /=3 months, >/=3x/week. In 4-month-old Pink1 knockout (KO) rats exercising in a progressively-increased treadmill speed regimen, we determined a specific treadmill speed that increased HR to an extent similar in human subjects. RESULTS: After completing aerobic exercise for approximately 30 min, PD subjects had increased HR approximately 35% above baseline ( approximately 63% maximum HR). Motor and cognitive test results indicated the exercising subjects completed the timed up and go (TUG) and trail-making test (TMT-A) in significantly less time versus exercise-naive PD subjects. In KO and age-matched wild-type (WT) rats, treadmill speeds of 8-10 m/min increased HR up to 25% above baseline ( approximately 67% maximum HR), with no further increases up to 16 m/min. Exercised KO, but not WT, rats showed increased locomotor activity compared to an age-matched exercise-naive cohort at 5 months old. CONCLUSION: These proof-of-concept results indicate HR is a cross-species translation parameter to evaluate aerobic exercise impact on specific motor or cognitive functions in human subjects and rat PD models. Moreover, a moderate intensity exercise regimen is within the physical abilities of early-stage PD patients and is therefore applicable for interrogating neurobiological mechanisms in rat PD models.
- «
- 1 (current)
- 2
- 3
- »