Browsing by Author "Mabry, Steve"
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Item Chronic Intermittent Hypoxia Increases Oxidative Stress and Impairs Spatial Memory in Male and Female Rats(2023) Gardner, Jennifer J.; Mabry, Steve; Bradshaw, Jessica L.; Wilson, E. Nicole; Little, Joel; Goulopoulou, Styliani; Cunningham, Rebecca L.Obstructive sleep apnea (OSA) is characterized by complex phenotypes and increased long-term risk of neurodegenerative disease. The impact of OSA in women is unknown due to sex differences in clinical presentation contributing to underdiagnosis. Using chronic intermittent hypoxia (CIH) to model OSA in rodents, our previous studies have shown CIH exposure increases oxidative stress and inflammation in male rats. However, the impact of CIH in female rats remains unclear. The objective of this study was to assess sex differences in CIH-mediated oxidative stress and rodent behaviors associated with neurodegenerative disease. Young adult male and female Long Evans and Sprague Dawley rats were exposed to CIH or normoxia for 14-15 days. Spatial memory and fine and gross motor skills were assessed. Plasma oxidative stress was measured and neuronal expression in the dorsal hippocampus was quantified. Female rats exhibited better spatial memory than males with increased neuronal expression in the CA1 region of the hippocampus. In both males and females, CIH impaired spatial memory and increased circulating oxidative stress. Yet, CIH increased CA1 neuronal expression in female rats only. CIH did not impact gross or fine motor skills, regardless of sex. Our preliminary findings indicate CIH increases oxidative stress and impairs spatial memory in males and females, but the impact of CIH on hippocampal neurons and region-specific contributions to spatial memory may be sexually dimorphic.Item Effects of Amyloid β on Recollective Memory: Sex and Hormone Differences(2023) Vera, Edward; Mabry, Steve; Wilson, Nicole; Bradshaw, Jessica L.; Gardner, Jennifer J.; Little, Joel; Rybalchenko, Nataliya; Cunningham, Rebecca L.PURPOSE: Alzheimer’s disease (AD) is linked with increased memory loss and inability to learn new topics. One of the defining neuropathological features of AD is amyloid beta (Aβ) plaques in brain regions, such as the hippocampus. The hippocampus brain region is important for memory and learning. AD risk is elevated in individuals older than 65 years old, especially menopausal women. Menopause is an aging associated endocrine event in which the ovaries stop producing estradiol but continue producing testosterone. Testosterone can be aromatized to estradiol, but aromatase is not functional in women with AD. Therefore, post-menopausal women with AD have more androgens than estrogens than pre-menopausal women and aged men. Androgens can be neuroprotective or neurotoxic depending on the cellular environment. It is unknown what the impact of androgens and sex are on amyloid beta’s effects on the brain, (e.g., hippocampus) and behavior (e.g., memory). We hypothesize that females with the hormonal condition of androgens in the absence of estrogens will exhibit increased recollective memory in response to hippocampal injection of Aβ. METHODS: To investigate the role of androgens and sex on Aβ associated memory impairments, adult male and female Sprague-Dawley rats were gonadectomized to remove circulating sex hormones. A subset of these rats was given either cholesterol or dihydrotestosterone (DHT), which cannot be converted into estrogen. To model AD, rats were injected with 5ug/ul of Aβ oligomer fibrils 1-40 or vehicle shams in the CA1 region of the hippocampus. One week after Aβ hippocampal injections, the rats were assayed for short term and long-term recollective memory via a 1-hour and 24-hour Novel Object behavioral test. The Novel Objective behavioral tests examines recollective memory by quantifying the time spent with a novel object versus the time spent with a known object. Data was quantified with a three-way ANOVA with sex, hormone, and Aβ as independent variables. Tukey’s was used as a post-hoc test. RESULTS: Sex differences were observed between hormone-deficient rats exposed to Aβ. Specifically, males exhibited worse short term recollective memory (1 hour novel object) compared to females. DHT had no effect on recollective memory, regardless of Aβ exposure. No effects were observed in the long-term recollective memory (24-hour novelty test). CONCLUSIONS: Our results indicate that Aβ 's effects on short term recollective memory is influenced by sex chromosomes, as we observed sex differences in the hormone deficient (cholesterol) treated animals. However, DHT did not impact these recollective memory. These results indicate that recollective memory in AD is impacted by the sex chromosomes and not androgens.Item Hypoxia and oxidative stress reduce placental efficiency and impair the balance between autophagy and cell death mechanisms in trophoblasts(2024-03-21) Gardner, Jennifer; Bradshaw, Jessica L.; de Nazare Oliveria da, Renee; Hula, Nataliia; Mabry, Steve; Wilson, E. Nicole; Cunningham, Rebecca L.; Goulopoulou, StylianiIntroduction: Hypoxia and oxidative stress can activate autophagy, a lysosomal degradation pathway that maintains cellular homeostasis. Impairments in autophagy mechanisms have been observed in placentas from obstetric complications associated with placental hypoxia and oxidative stress, such as preeclampsia and intrauterine growth restriction. Purpose: The objective of this study was to investigate the effects of hypoxia and oxidative stress on placental autophagy. We hypothesized that exposure to oxidative stress and hypoxia would alter the balance between cytotoxic and cytoprotective mechanisms in human trophoblast cells and rat placentas and would adversely affect placental efficiency. Methods: We used an in vitro model incorporating human trophoblast cells (BeWo cells) exposed to an oxidative stressor, antimycin A (10, 100, 320 μM) or vehicle for 4 hours. Trophoblast cell death and autophagy mechanisms were assessed via flow cytometry and western blotting. Additionally, we used a rodent model of gestational sleep apnea, a pregnancy complication associated with placental hypoxia. Long Evans timed-pregnant dams were exposed to chronic intermittent hypoxia (CIH; n=6-8) or normoxia (NX; n=8-9) during their sleep cycle from gestational day (GD) 15 to 20 (late pregnancy, term=21-23 days). Results: In trophoblast cells (n=5-9 independent experiments), antimycin A increased necrosis and LC3 A/B II/I ratio (autophagy marker) at 100 μM compared to vehicle (p<0.015). Necrosis remained elevated at 320 μM, while BAX (pro-apoptotic marker) and p62 (autophagosomal flux marker) were reduced compared to vehicle (p<0.0001). LC3 A/B II/I ratio returned to vehicle levels at 320 μM (p>0.05 vs. vehicle). Placental weights from CIH exposed dams were greater (NX: 0.51±0.02 g vs. CIH: 0.60±0.03 g, p=0.015) and fetal to placental weight ratios (marker of placental efficiency) were reduced compared to control pregnancies (NX: 5.25±0.13 vs. CIH: 4.43±0.14, p=0.0006) on GD20. Gestational CIH did not affect (p>0.05) fetal weights (NX: 2.76±0.06 g vs. CIH: 2.61±0.06 g), crown to rump length (NX: 3.32±0.03 cm vs. CIH :3.18±0.12 cm), abdominal girth (NX: 3.22±0.06 cm vs. CIH: 3.32±0.12 cm), or litter size (NX: 11.9±0.90 vs. CIH: 10.5±0.82). Conclusion: Oxidative stress alters the balance between cytotoxic and cytoprotective mechanisms in trophoblast cells, promoting cell necrosis. Although assessment of autophagy machinery and cell death in placentas from hypoxic pregnancies is ongoing, our results indicate that maternal CIH during pregnancy adversely affects placental efficiency.Item Impact of sex and hypoxia on brain region-specific expression of androgen receptor AR45 and G protein Gαq in young adult rats(2024-03-21) Wilson, Elizabeth; Bradshaw, Jessica; Mabry, Steve; Shrestha, Pawan; Gardner, Jennifer; Cunningham, RebeccaPurpose: Sex differences in oxidative stress-associated cognitive decline are influenced by sex hormone levels. However, little is known regarding the expression of hormone receptors in brain regions associated with cognitive function. Notably, oxidative stress-associated neuronal cell death is exacerbated through testosterone signaling via membrane-associated androgen receptor AR45 and G protein Gαq. The objective of this study was to elucidate the expression of AR45 and Gαq in brain regions associated with cognitive function. Additionally, we investigated whether chronic intermittent hypoxia (CIH), an oxidative stressor with sex-specific effects, would modulate AR45 and Gαq expression. Methods: Adult male and female Sprague-Dawley rats were exposed to CIH or normoxia for 14 days. We quantified AR45 and Gαq protein expression in various cognition-associated brain regions [dorsal hippocampal CA1, CA3, DG, and entorhinal cortex (ETC)] via western blotting. For comparisons, AR45 and Gαq protein expression were also assessed in brain regions outside the hippocampal-ETC circuit [thalamus (TH) and striatum (STR)]. Results: The highest AR45 levels were expressed in the CA1 while the lowest expression was observed in the STR. The highest Gαq levels were expressed in the DG and ETC while the lowest expression was observed in the TH. We observed no effect of sex on AR45 or Gαq expression regardless of brain region assessed. Similarly, there was no effect of CIH on AR45 expression in any of the brain regions examined. However, CIH exposure increased Gαq expression only in the CA3 regardless of sex. Conclusions: Our findings reveal enrichment of AR45 and Gαq protein expression within the hippocampal-ETC circuit, which is vulnerable to oxidative stress and neurodegeneration during cognitive decline. Moreover, our data suggest the CA3 is the most vulnerable region to CIH-mediated oxidative stress. Overall, these findings were observed in both sexes, indicating that there are no observed sex differences in AR45 and Gαq expression or their modulation by CIH.Item Long-term effects of late gestational maternal hypoxic stress on mood disorders: Sex and age differences(2021) Mabry, Steve; Wilson, Elizabeth; Rybalchenko, Nataliya; Engelland, Rachel; Fadeyibi, Oluwadarasimi; Osikoya, Oluwatobiloba; Cushen, Spencer; Goulopoulou, Styliani; Cunningham, RebeccaPURPOSE: In utero insults have been linked with increased fear and anxiety in progeny. In utero hypoxic stress is also associated with multiple gestational complications. We hypothesized that exposure to maternal hypoxia during late gestation will have a long-term impact on anxiety in progeny. METHODS: Pregnant female Long-Evans rats were exposed to five days (gestational days: 15-20) of chronic intermittent hypoxia (CIH) or room air (normoxia: 21% O2) for 8 hours during their sleep phase. Each CIH cycle was 6 min of 3 min hypoxia (10% O2) and 3 min normoxia for a total of 10 CIH cycles/hour. At weaning (PND 28), progeny was pair-housed with a conspecific of same sex and similar weight. To examine anxiety disorders, we quantified anxiety-related behaviors (time spent in center of open field arena, marble burying test, social and anti-social behaviors with conspecifics) along with quantifying food intake and circulating sex hormone levels during puberty (postnatal day, PND 40-45) and young adulthood (PND 60-65) in male and female progeny. RESULTS: Maternal CIH did not impact circulating sex hormones or food intake, regardless of sex or age of progeny. However, maternal CIH increased anxiety related behaviors in pubertal females but were not observed in young adulthood. Maternal CIH did not impact male progeny, regardless of age. CONCLUSIONS: Maternal CIH during gestation resulted in increased anxiety related behaviors in pubertal female progeny. Maternal hypoxia during late gestation may temporarily increase the risk for anxiety disorders in pubertal females.Item Long-term effects of late gestational maternal hypoxic stress on neurodegeneration: Sex and age differences(2021) Wilson, Elizabeth; Mabry, Steve; Rybalchenko, Nataliya; Engelland, Rachel; Fadeyibi, Oluwadarasimi; Osikoya, Oluwatobiloba; Cushen, Spencer; Goulopoulou, Styliani; Cunningham, RebeccaIntroduction: In utero insults can lead to onset of neurodegenerative diseases, such as Parkinson's disease (PD). In utero hypoxic insults are associated with maternal sleep apnea or preeclampsia. It is unknown whether late gestational maternal hypoxic insults have long-term effects on brain regions associated with PD, such as the nigrostriatal pathway. We hypothesized that late gestational maternal hypoxia will result in sustained nigrostriatal impairment in male and female progeny. Methods: Timed pregnant Long-Evans rats were exposed to five days (gestational days: 15-20) of chronic intermittent hypoxia (CIH) or room air (normoxia 21% O2) for 8 hours during their sleep phase. Each CIH cycle was 6 min of alternating 3 min hypoxia (10% O2) and normoxia (21% O2) totaling 10 CIH cycles/hour. Gestational age and biometrics were recorded 12-16 hours after birth. At postnatal day, PND 28, progeny were pair-housed with a conspecific of the same sex and similar weight. We focused on PD associated oxidative stress and behavioral impairments in the nigrostriatal pathway. Gross motor (open field), fine motor (ultrasonic vocalizations), and cognition (spatial memory) were examined during puberty and young adulthood. Results: Maternal CIH had no effect on gestational age, progeny biometrics, or progeny circulating oxidative stress. Gross motor and cognitive functions were unaffected by maternal CIH. However, a sustained fine motor impairment was observed in both male and female progeny. Conclusion: Maternal hypoxia during late gestation induced sustained nigrostriatal pathway impairment, which may increase the risk for neurodegeneration.Item Long-term effects of prenatal chronic intermittent hypoxia insult on the substantia nigra(2021) Engelland, Rachel; Fadeyibi, Oluwadarasimi; Rybalchenko, Nataliya; Wilson, Elizabeth; Mabry, Steve; Osikoya, Oluwatobiloba; Cushen, Spencer; Goulopoulou, Styliani; Cunningham, RebeccaPurpose: Prenatal chronic intermittent hypoxia (CIH) was employed to evaluate the effects of hypoxic insults on the substantia nigra (SN), which is impacted by Parkinson's disease (PD). SN loss during PD is linked with oxidative stress (OS) and apoptosis. We hypothesized that exposure to late gestational maternal hypoxia would result in an increase in increased OS, but not apoptosis, in the SN of adult male and female progeny. Methods: During gestational days 15-20, pregnant Long-Evans rats were exposed to CIH or room air (normoxia) for 8 hours. CIH consisted of 3 min hypoxia (10% O2) and 3 min normoxia (21% O2). Animals were sacrificed at puberty (PND 44) or adulthood (PND 66). SN micropunches were obtained. OS was quantified by measuring calpain cleavage of spectrin. Results: OS (calpain cleavage of spectrin) was increased in the SN of adult male and female rats exposed to prenatal CIH compared to control (F1,17 = 3.606; p = 0.075). No effects on OS were observed in pubertal rats. Apoptosis (caspase-3 cleavage of spectrin) was not observed in any of the groups. Conclusions: These data suggest that prenatal CIH programming has a long-lasting impact on the SN of adult progeny, which may increase the susceptibility of SN to damage and PD risk. Although no sex differences were observed in this pilot study, we may see a sex effect upon increasing animal number, especially in male rats. This is consistent with the higher incidence of PD in men than in women.Item Postpartum Maternal Vascular Function in a Rat Model of Gestational Obstructive Sleep Apnea(2020) Osikoya, Oluwatobiloba; Wilson, Elizabeth; Mabry, Steve; Cushen, Spencer; Cunningham, Rebecca; Goulopoulou, Styliani; Singhal, JuhiIntroduction: De novo obstructive sleep apnea (OSA) in pregnancy is associated with adverse gestational outcomes. In pregnant mice, exposure to chronic intermittent hypoxia (CIH), a model of OSA, induces endothelial dysfunction in maternal uterine arteries. It is currently unknown whether gestational OSA has a long-term effect on maternal vascular function. We hypothesized that exposure to gestational CIH during pregnancy will result in postpartum maternal vascular dysfunction. Methods: Pregnant rats were assigned to Normoxia and CIH groups. The CIH group was exposed to five days of intermittent hypoxia [6 min cycles of 3 min hypoxia (10% O2) and 3 min normoxia (21% O2)]. Endothelial function was assessed in isolated maternal uterine arteries at weaning (postnatal day 28). Vascular reactivity to acetylcholine (ACh) was examined in the presence and absence of uterine perivascular adipose tissue (PVAT) using wire myography. Results: In the absence of PVAT, uterine arteries from dams exposed to gestational CIH had exaggerated responses to ACh compared to dams exposed to normoxia [(-)PVAT/pEC50, Normoxia: 6.77±0.08 vs. CIH: 7.13±0.09, p< 0.01], while PVAT normalized this difference [(+)PVAT/pEC50, Normoxia: 6.67±0.08 vs. CIH: 6.70±0.07, p=0.99]. The effects of PVAT were due to its anti-dilatory influences on uterine arteries from CIH-treated rats ([CIH (-PVAT) vs. CIH (+PVAT), p = 0.003), whereas it had no effect on arteries from normoxic rats (p = 0.77). Conclusion: Exposure to gestational CIH exaggerated postpartum uterine vascular smooth muscle relaxation responses. Gestational OSA may impair maternal vascular recovery after birth.Item Sex and age differences in social and cognitive function in offspring exposed to late gestational hypoxia(BioMed Central Ltd., 2023-11-12) Mabry, Steve; Wilson, E. Nicole; Bradshaw, Jessica L.; Gardner, Jennifer J.; Fadeyibi, Oluwadarasimi; Vera, Edward, Jr.; Osikoya, Oluwatobiloba; Cushen, Spencer C.; Karamichos, Dimitrios; Goulopoulou, Styliani; Cunningham, Rebecca L.BACKGROUND: Gestational sleep apnea is a hypoxic sleep disorder that affects 8-26% of pregnancies and increases the risk for central nervous system dysfunction in offspring. Specifically, there are sex differences in the sensitivity of the fetal hippocampus to hypoxic insults, and hippocampal impairments are associated with social dysfunction, repetitive behaviors, anxiety, and cognitive impairment. Yet, it is unclear whether gestational sleep apnea impacts these hippocampal-associated functions and if sex and age modify these effects. To examine the relationship between gestational sleep apnea and hippocampal-associated behaviors, we used chronic intermittent hypoxia (CIH) to model late gestational sleep apnea in pregnant rats. We hypothesized that late gestational CIH would produce sex- and age-specific social, anxiety-like, repetitive, and cognitive impairments in offspring. METHODS: Timed pregnant Long-Evans rats were exposed to CIH or room air normoxia from GD 15-19. Behavioral testing of offspring occurred during either puberty or young adulthood. To examine gestational hypoxia-induced behavioral phenotypes, we quantified hippocampal-associated behaviors (social function, repetitive behaviors, anxiety-like behaviors, and spatial memory and learning), hippocampal neuronal activity (glutamatergic NMDA receptors, dopamine transporter, monoamine oxidase-A, early growth response protein 1, and doublecortin), and circulating hormones in offspring. RESULTS: Late gestational CIH induced sex- and age-specific differences in social, repetitive, and memory functions in offspring. In female pubertal offspring, CIH impaired social function, increased repetitive behaviors, and elevated circulating corticosterone levels but did not impact memory. In contrast, CIH transiently induced spatial memory dysfunction in pubertal male offspring but did not impact social or repetitive functions. Long-term effects of gestational CIH on social behaviors were only observed in female offspring, wherein CIH induced social disengagement and suppression of circulating corticosterone levels in young adulthood. No effects of gestational CIH were observed in anxiety-like behaviors, hippocampal neuronal activity, or circulating testosterone and estradiol levels, regardless of sex or age of offspring. CONCLUSIONS: Our results indicate that hypoxia-associated pregnancy complications during late gestation can increase the risk for behavioral and physiological outcomes in offspring, such as social dysfunction, repetitive behaviors, and cognitive impairment, that are dependent on sex and age. Sleep apnea during late pregnancy is a common pregnancy complication that can impact the brain development of children born to mothers with sleep apnea. Children with impaired brain development may present with decreased social skills, memory issues, anxiety, and compulsivity. It is unclear if there is a cause and effect relationship between sleep apnea during late pregnancy and behavioral changes in offspring. Additionally, it is unknown whether male or female sex or age of the offspring affects these relationships. In this study, we exposed pregnant rats to a model of sleep apnea called chronic intermittent hypoxia (CIH) within late gestation and examined the behavior of the offspring and brain activity during puberty and young adulthood. We found that CIH during late pregnancy had long-term effects in the offspring that were different in males and females. Notably, female offspring displayed social impairments in response to late gestation CIH, whereas male offspring displayed cognitive dysfunction.Item Sex and age differences in social and cognitive function in offspring exposed to late gestational hypoxia(2023) Mabry, Steve; Wilson, E. Nicole; Bradshaw, Jessica L.; Gardner, Jennifer J.; Fadeyibi, Oluwadarasimi; Vera Jr., Edward; Karamichos, Dimitrios; Goulopoulou, Styliani; Cunningham, Rebecca L.Background: Gestational sleep apnea affects 8-26% of pregnancies and can increase the risk for autism spectrum disorder (ASD) in offspring. ASD is a neurodevelopmental disorder associated with social dysfunction, repetitive behaviors, anxiety, and cognitive impairment. To examine the relationship between gestational sleep apnea and ASD, we used a chronic intermittent hypoxia (CIH) protocol between gestational days (GD) 15-19 in pregnant rats to model gestational sleep apnea during the third trimester of pregnancy. We hypothesized that late gestational CIH would produce sex- and age-specific social, mood, and cognitive impairments in offspring. Methods: Timed pregnant Long-Evans rats were exposed to CIH or room air normoxia from GD 15-19. Behavioral testing of offspring occurred during either puberty or young adulthood. To examine ASD phenotype, we quantified ASD-associated behaviors (social function, repetitive behaviors, anxiety-like behaviors, and spatial memory and learning), hippocampal activity (glutamatergic NMDA receptors, dopamine transporter, monoamine oxidase-A, neuronal activation, and neurogenesis), and circulating hormones in offspring. Results: Late gestational CIH induced sex- and age-specific differences in social, repetitive and memory functions in offspring. These effects were mostly transient and present during puberty. In female pubertal offspring, CIH impaired social function, increased repetitive behaviors, suppressed circulating estradiol but did not impact memory. In contrast, CIH impaired spatial memory and suppressed circulating estradiol in pubertal male offspring but did not impact social or repetitive functions. Long term effects of gestational CIH were only observed in female offspring, wherein CIH induced social disengagement and suppression of circulating estradiol during puberty was maintained in young adulthood. No effects of gestational CIH were observed on anxiety-like behaviors, hippocampal activity, circulating testosterone, or circulating corticosterone, regardless of sex or age of offspring. Conclusions: Our results indicate that hypoxia-associated pregnancy complications during the third trimester can increase the risk for ASD, such as pubertal social dysfunction, neuroendocrine suppression, and memory impairments. Current clinical recommendations support ASD screening for all children up to their 24-month checkup. Based on our findings, children from hypoxia-associated pregnancies should be screened for ASD throughout puberty.Item Sex Differences in Oxidative Stress Model of Prodromal Parkinson's Disease(2022) Mabry, Steve; Wilson, Elizabeth; Little, Joel; Romero, Steven; Cunningham, RebeccaINTRODUCTION: Parkinson's disease (PD) is an incurable neurodegenerative disorder that causes deterioration in motor and cognitive function and occurs more commonly in men. Gross motor impairment occurs after 60% dopaminergic neurons are lost in the substantia nigra brain region. However, it is unknown if sex differences are present in the prodromal stage of PD in which no dopaminergic neuronal loss or gross motor dysfunction are observed. Prodromal PD is associated with increased oxidative stress (OS) and OS damage in the substantia nigra, along with cognitive and fine motor skill dysfunction. To examine if sex differences are present in prodromal PD, we will use an animal model, chronic intermittent hypoxia (CIH), that recapitulates many prodromal PD characteristics. Our prior studies using male rats found that CIH induced global OS, OS damage in the substantia nigra, and cognitive dysfunction, which are all consistent with prodromal PD. METHODS: Adult male and female Sprague Dawley rats were exposed CIH to induce a prodromal PD phenotype. The CIH protocol consisted of 10 episodes of hypoxia (12% O2)/hour for a total of 8 hours/day over a 2-week period. Control rats were exposed to room air (normoxia). Rats were behaviorally tested for the following indexes during the last week of CIH exposure: 1) cognitive function (novel object recognition, Morris Water Maze), 2) fine motor behavior (modified open field with an elevated wire mesh), and 3) anxiety (marble test). At the conclusion of behavior testing, rats were sacrificed. Plasma and brain tissue was collected to examine oxidative stress. RESULTS: CIH increased circulating oxidized proteins in both male and female rats compared to control rats. No sex difference was observed in CIH induced circulating oxidative stress. Preliminary analysis indicate that sex differences are present in behavioral tests, especially cognitive function. CONCLUSIONS: These studies indicate sex differences in response to OS. CIH induced OS was consistent across both sexes, as evidenced by similar circulating OS levels. However, each sex responded (behaviorally) differently in response to CIH induced OS. These studies indicate that sex differences may be involved in prodromal PD. Knowledge of these sex differences could lead to earlier detection of PD and possibly the ability to slow conversion of prodromal PD to later stage PD that is exemplified by gross motor loss.Item Sex differences in the activation of central autonomic control regions and neuroinflammation in chronic intermittent hypoxia(2023) Appiah, Cephas; Little, Joel; Mabry, Steve; Cunningham, Rebecca L.; Cunningham, J. ThomasPurpose: Obstructive sleep apnea (OSA) is an independent risk factor for hypertension. Chronic interment hypoxia (CIH), which models episodic hypoxemia of OSA, produces daytime hypertension, oxidative stress, and activation of central autonomic regions that regulate mean arterial pressure (MAP) in male Sprague Dawley (SD) rats. Unlike gonadally intact females, gonadectomized females and males develop CIH hypertension. Lesioning of median preoptic nucleus (MnPO) in males prevents CIH hypertension. We hypothesize that the sex difference observed in CIH hypertension is due sex differences in neuroinflammation and activation of central autonomic regions that support MAP in CIH. Methods: Gonadally intact adult male and female SD rats (250-300g) were continuously exposed to normoxia (CON) or CIH (10% O2 every 3mins cycling 21% O2 every 3mins, 8h/day) for 7 days. Radiotelemetry transmitters were implanted in rats to record MAP and heart rate (HR). After one week of baseline recording, the rats were exposed to either continuous normoxia or CIH and were euthanized (inactin 100 mg/kg ip) on the 8th day for immunohistochemistry and blood analysis. All forebrain sections were stained for FosB/ΔFosB and either neuronal nitric oxide synthase (NOS1) or IBA1 to identify active microglia. Results: CIH males exhibited significantly increased hematocrit indicating erythropoiesis compared to control males (CON 42.1% ± 0.6, n=10; CIH 44.6% ± 0.7, n = 10. P = 0.0173). CIH males exhibited an increase in the average number of FosB positive neurons (CON male 20 ± 2 cells/section, CIH male 35 ± 3; CON female 11 ± 1, CIH female 12 ± 2,) and colocalization of FosB and NOS1 (CON male 10 ± 1 cells/section, CIH male 18 ± 4; CON female 5 ± 1, CIH female 6 ± 1) in the MnPO. CIH females showed a trend for an increase in the average number of IBA1 immunoreactive microglial cells in MnPO (CON 187 ± 16, n = 2; CIH 218 ± 18, n = 4). Conclusion: CIH is associated with increased FosB staining in the MnPO of male rats as opposed to female rats which is consistent with our working hypothesis. In addition, FosB positive MnPO neurons also contained NOS1. In female rats, CIH is associated with a trend for an increase in the numbers of IBA1 positive microglia, indicating increased neuroinflammation in females that is independent of hypertension. CIH was associated with increased FosB staining in NOS1 positive MnPO neurons suggesting that they may be contributing to the sustained hypertension reported in male rats. The research is funded by NIH grant RO1 HL155977.Item Sex-dependent effects of chronic intermittent hypoxia: Implication for obstructive sleep apnea(2024-03-21) Mabry, Steve; Bradshaw, Jessica; Gardner, Jennifer; Wilson, Elizabeth; Cunningham, RebeccaBackground: Obstructive sleep apnea (OSA) is a highly prevalent sleeping disorder in the USA with known sex differences in prevalence and severity. Men have a higher incidence and experience greater severity of OSA than women. However, recent reports indicate the incidence of OSA in women, particularly mild cases of OSA, may be under-reported and left untreated. OSA is characterized by elevated oxidative stress and inflammation, mechanisms that involve mitochondrial function. This study addressed the role of 1) sex and 2) mitochondrial oxidative stress in OSA induced circulatory oxidative stress and inflammatory cytokines. Methods: Adult Sprague-Dawley male and female rats were implanted (s.c.) with an osmotic pump containing either MitoTEMPOL (mitochondrial oxidative stress inhibitor; MT) or saline vehicle and then exposed to a model of OSA, chronic intermittent hypoxia (CIH), or normoxic room-air for 14 days. The CIH protocol consisted of 10 CIH cycles/hour/8 hrs/day, in which each CIH cycle was composed of 3 minutes of normoxia at 21% O2 and 3 minutes of hypoxia at 10% O2. This protocol replicates an apnea-hypopnea index (AHI) of 10, which is consistent with mild OSA in humans. At the conclusion of the CIH protocol, rats were sacrificed and plasma was collected to quantify markers of oxidative stress (Advanced Oxidized Protein Products, AOPP) and inflammation (pro-inflammatory IL-6, anti-inflammatory IL-10, IL-6/IL-10 ratio). To determine statistical significance, ANOVA followed by Tukey’s post-hoc test was used. Significance level was set a p<0.05. Results: We found circulating oxidative stress was dependent on CIH and sex. Sex differences were observed in control normoxic rats, in which females had higher oxidative stress than males. Interestingly, the impact of CIH on oxidative stress was dependent on sex, wherein CIH decreased oxidative stress in females but increased oxidative stress in males. Inhibiting mitochondria-associated oxidative stress reduced oxidative stress in vehicle females, but only blocked the effect of CIH-induced oxidative stress in males. In contrast to oxidative stress, CIH increased the level of IL-6 only in females. Further, CIH overall induced a pro-inflammatory state as measured by an elevated IL6/IL10 ratio in females. The inflammatory effects of CIH in females were blocked by inhibiting mitochondrial-associated oxidative stress, despite no effect on circulating oxidative stress in CIH. Neither CIH nor MT impacted inflammatory markers in males. Discussion: These results indicate CIH-induced mechanisms underlying oxidative stress and inflammation are dependent on sex. Specifically, males experience a mitochondria-associated oxidative stress phenotype and females experience a mitochondria-associated inflammatory phenotype. These findings indicate that the OSA phenotype is sex-dependent, which may be related to the under-reported OSA incidence in women compared to men. Further, these data indicate that women may be at unique risk from OSA, particularly when AHIs are mild. Interestingly, inhibition of mitochondrial oxidative stress may be a potential drug target for both men and women with OSA.Item Sex-dependent effects of chronic intermittent hypoxia: implication for obstructive sleep apnea(BioMed Central Ltd., 2024-04-26) Mabry, Steve; Bradshaw, Jessica L.; Gardner, Jennifer J.; Wilson, E. Nicole; Cunningham, Rebecca L.BACKGROUND: Obstructive sleep apnea (OSA) affects 10-26% of adults in the United States with known sex differences in prevalence and severity. OSA is characterized by elevated inflammation, oxidative stress (OS), and cognitive dysfunction. However, there is a paucity of data regarding the role of sex in the OSA phenotype. Prior findings suggest women exhibit different OSA phenotypes than men, which could result in under-reported OSA prevalence in women. To examine the relationship between OSA and sex, we used chronic intermittent hypoxia (CIH) to model OSA in rats. We hypothesized that CIH would produce sex-dependent phenotypes of inflammation, OS, and cognitive dysfunction, and these sex differences would be dependent on mitochondrial oxidative stress (mtOS). METHODS: Adult male and female Sprague Dawley rats were exposed to CIH or normoxia for 14 days to examine the impact of sex on CIH-associated circulating inflammation (IL-1beta, IL-6, IL-10, TNF-alpha), circulating steroid hormones, circulating OS, and behavior (recollective and spatial memory; gross and fine motor function; anxiety-like behaviors; and compulsive behaviors). Rats were implanted with osmotic minipumps containing either a mitochondria-targeting antioxidant (MitoTEMPOL) or saline vehicle 1 week prior to CIH initiation to examine how inhibiting mtOS would affect the CIH phenotype. RESULTS: Sex-specific differences in CIH-induced inflammation, OS, motor function, and compulsive behavior were observed. In female rats, CIH increased inflammation (plasma IL-6 and IL-6/IL-10 ratio) and impaired fine motor function. Conversely, CIH elevated circulating OS and compulsivity in males. These sex-dependent effects of CIH were blocked by inhibiting mtOS. Interestingly, CIH impaired recollective memory in both sexes but these effects were not mediated by mtOS. No effects of CIH were observed on spatial memory, gross motor function, or anxiety-like behavior, regardless of sex. CONCLUSIONS: Our results indicate that the impact of CIH is dependent on sex, such as an inflammatory response and OS response in females and males, respectively, that are mediated by mtOS. Interestingly, there was no effect of sex or mtOS in CIH-induced impairment of recollective memory. These results indicate that mtOS is involved in the sex differences observed in CIH, but a different mechanism underlies CIH-induced memory impairments. Sleep apnea is a common sleeping condition in adults with a wide range of symptoms that include inflammation, oxidative stress, memory problems, anxiety, and compulsivity. Men are diagnosed with sleep apnea more often than women. Although there is limited information on how sleep apnea affects men and women differently, previous studies suggest that women may exhibit different sleep apnea symptoms than men. To examine the impact of male and female sex on common sleep apnea symptoms, we exposed adult male and female rats to a model of sleep apnea called chronic intermittent hypoxia (CIH). We found that many effects of CIH were different in males and females. CIH females had increased inflammation and motor problems, whereas CIH males had increased oxidative stress and compulsivity. To investigate the reason for these CIH sex differences, we blocked mitochondrial oxidative stress. Blocking mitochondrial oxidative stress decreased CIH associated sex differences. However, blocking mitochondrial oxidative stress had no impact on CIH-induced memory impairment that was observed in male and female rats. Our findings support previous reports that suggest that women exhibit different sleep apnea symptoms than men. Further, we extend these findings by showing that mitochondrial oxidative stress is involved in these sex differences. Clinically, patients diagnosed with sleep apnea are typically treated with continuous positive airway pressure (CPAP) machines, which have high rates of non-compliance (15-40%). Therefore, understanding why sleep apnea is causing these symptoms will be important in developing therapeutics.Item The Role of Lipid Rafts and Membrane Androgen Receptors in Androgen's Neurotoxic Effects(Oxford University Press, 2022-02-21) Fadeyibi, Oluwadarasimi; Rybalchenko, Nataliya; Mabry, Steve; Nguyen, Dianna H.; Cunningham, Rebecca L.Sex differences have been observed in multiple oxidative stress-associated neurodegenerative diseases. Androgens, such as testosterone, can exacerbate oxidative stress through a membrane androgen receptor (mAR), AR45, localized to lipid rafts in the plasma membrane. The goal of this study is to determine if interfering with mAR localization to cholesterol-rich lipid rafts decreases androgen induced neurotoxicity under oxidative stress environments. We hypothesize that cholesterol-rich caveolar lipid rafts are necessary for androgens to induce oxidative stress generation in neurons via the mAR localized within the plasma membrane. Nystatin was used to sequester cholesterol and thus decrease cholesterol-rich caveolar lipid rafts in a neuronal cell line (N27 cells). Nystatin was applied prior to testosterone exposure in oxidatively stressed N27 cells. Cell viability, endocytosis, and protein analysis of oxidative stress, apoptosis, and mAR localization were conducted. Our results show that the loss of lipid rafts via cholesterol sequestering blocked androgen-induced oxidative stress in cells by decreasing the localization of mAR to caveolar lipid rafts.Item Unraveling the Molecular Nexus: Obstructive Sleep Apnea and Glaucoma in a Rat Model(2024-03-21) Donkor, Nina; Mabry, Steve; Wilson, E. Nicole; Gardner, Jennifer J.; Bradshaw, Jessica; Cunningham, Rebecca; Inman, DenisePurpose: Obstructive sleep apnea is a chronic sleep disorder characterized by recurring complete or partial upper airway occlusion. Over the past decade, meta-analyses have established a correlation between this disorder and glaucoma, an ocular neurodegenerative disease, and a leading cause of blindness. However, the link between these pathologies remains elusive. Understanding the mechanisms involved could influence treatment options and reduce the rate of vision loss associated with glaucoma. Using a rat model of sleep apnea, chronic intermittent hypoxia (CIH), we tested the hypothesis that mild sleep apnea initiates morphologic and metabolic changes in the retina that resemble glaucoma. Methods: Rats were randomly assigned to normoxic or CIH groups. The CIH group was exposed to periodic hypoxia during their sleep phase, simulating mild sleep apnea, with oxygen reduction from 21% to 10% and reoxygenation in 6-minute cycles over 8 hours/day for 14 days. The normoxic group experienced similar conditions without changes in oxygen concentration. Subsequently, the eyes were enucleated, and the retina was evaluated for oxidative stress, inflammatory markers, metabolic changes, and hypoxic response modulation using immunohistochemistry and capillary electrophoresis. Results: Immunofluorescence revealed increased expression of 8-OHdG, indicating oxidative stress (nucleic acid damage), as well as the cytokine TNF-α in the CIH group retina compared to controls. No statistically significant differences were observed in HIF-1α protein levels. SIRTUIN-1, a regulator of HIF-1α expression, and the levels of pyruvate dehydrogenase kinase-1 and lactate dehydrogenase-A showed no significant differences between normoxic and CIH groups. Conclusion: The increased oxidative stress and inflammation observed suggest that CIH induces a response in the retina with features shared by early-stage glaucoma. However, the anticipated upregulation of HIF-1α and its targets did not occur, suggesting a greater reduction in oxygen concentration or a longer-term CIH interval may be necessary to observe canonical hypoxic response. Keywords: glaucoma, sleep apnea, chronic intermittent hypoxia, inflammation, oxidative stress