Stress-induced conditions are associated with impaired cerebral blood flow (CBF) and increased risk of dementia and stroke. However, these conditions do not develop in resilient humans and animals. Here the effects of predator stress (PS, cat urine scent, ten days) on CBF and mechanisms of CBF regulation were compared in PS-susceptible (PSs) and PS-resilient (PSr) rats. Fourteen days post-stress, the rats were segregated into PSs and PSr groups based on a behavior-related anxiety index (AI). CBF and its endothelium-dependent changes were measured in the parietal cortex by laser Doppler flowmetry. The major findings are: (1) PS susceptibility was associated with reduced basal CBF and endothelial dysfunction. In PSr rats, the basal CBF was higher, and endothelial dysfunction was attenuated. (2) CBF was inversely correlated with the AI of PS-exposed rats. (3) Endothelial dysfunction was associated with a decrease in eNOS mRNA in PSs rats compared to the PSr and control rats. (4) Brain dopamine was reduced in PSs rats and increased in PSr rats. (5) Plasma corticosterone of PSs was reduced compared to PSr and control rats. (6) A hypercoagulation state was present in PSs rats but not in PSr rats. Thus, potential stress resilience mechanisms that are protective for CBF were identified.

Introduction. Patients with Alzheimer’s disease (AD) have reduced cerebral vascular density (VD), which impairs blood flow to neurons and may contribute to progression of AD. Earlier we showed that prior adaptation to intermittent hypobaric hypoxia (IHH) prevented memory loss and degeneration of cortical neurons in rats with experimental AD (EAD). The aim of this study was to test if IHH might prevent EAD-induced vascular rarefaction in rats. Materials and methods. EAD was induced with bilateral injection of neurotoxic beta-amyloid peptide fragment (A) (25–35) into n. basalis magnocellularis. IHH was simulated at a 4,000 m altitude, for 4 hours a day, for 14 days. Brain blood vessels were stained by transcardiac infusion of Indian ink; brain sections were stained with 0.3 % cresyl violet by Nissle method. Vascular density was assessed in the cortex and hippocampus using the Infinity Analysis Software. Results. In the EAD rats, VD was significantly decreased in the hippocampus (13.3±0.9 vs 17.8±1.0 in field of view, FOV, p<0.03) and in the cortex (17.3±1.5 vs 22.3±1.3 in FOV, p<0.03). AIH increased VD in the hippocampus to 27.0±3.5 in FOV (p=0.01) and in cortex to 26.0±1.1 in FOV (p<0.03). In EAD+AIH rats, VD did not differ significantly from the control rats neither in the hippocampus, nor in the cortex. AIH may stimulate angiogenesis through hypoxia inducible factor-1α-mediated expression of vascular endothelial growth factor and/or by increasing expression and activity of antioxidant enzymes. Conclusion. One of the mechanisms of AIH beneficial effect in AD-related neurodegeneration is preserving the capability for compensatory angiogenesis in brain.

Traumatic stress causes posttraumatic stress disorder (PTSD). PTSD is associated with cardiovascular diseases and risk of sudden cardiac death in some subjects. We compared effects of predator stress (PS, cat urine scent, 10 days) on mechanisms of cardiac injury and protection in experimental PTSD-vulnerable (PTSD) and -resistant (PTSDr) rats. Fourteen days post-stress, rats were evaluated with an elevated plus-maze test, and assigned to PTSD and PTSDr groups according to an anxiety index calculated from the test results. Cardiac injury was evaluated by: 1) exercise tolerance; 2) ECG; 3) myocardial histomorphology; 4) oxidative stress; 5) pro- and anti-inflammatory cytokines. Myocardial heat shock protein 70 (HSP70) was also measured. Experimental PTSD developed in 40% of rats exposed to PS. Exercise tolerance of PTSD rats was 25% less than control rats and 21% less than PTSDr rats. ECG QRS, QT, and OTc intervals were significantly longer in PTSD rats than in control and PTSDr rats. Only cardiomyocytes of PTSD rats had histomorphological signs of metabolic and hypoxic injury and impaired contractility. Oxidative stress markers were higher in PTSD than in PTSDr rats. Pro-inflammatory IL-6 was higher in PTSD rats than in control and PTSDr rats, and anti-inflammatory IL-4 was lower in PTSD than in control and PTSDr rats. Myocardial HSP70 was lower in PTSD rats than in PTSDr and control rats. Our conclusion was that rats with PTSD developed multiple signs of cardiac injury. PTSDr rats were resistant also to cardiac injury. Factors that limit cardiac damage in PS rats include reduced inflammation and oxidative stress and increased protective HSP70.NEW & NOTEWORTHY For the first time, rats exposed to stress were segregated into experimental PTSD (ePTSD)-susceptible and ePTSD-resistant rats. Cardiac injury, ECG changes, and impaired exercise tolerance were more pronounced in ePTSD-susceptible rats. Resistance to ePTSD was associated with decreased inflammation and oxidative stress and with increased protective heat shock protein 70. Results may help identify individuals at high risk of PTSD and also provide a foundation for developing preventive and therapeutic means to restrict PTSD-associated cardiac morbidity.

BACKGROUND:Rats exposed to chronic predator scent stress mimic the phenotype of complex post-traumatic stress disorder (PTSD) in humans, including altered adrenal morphology and function. High- and low-anxiety phenotypes have been described in rats exposed to predator scent stress (PSS). This study aimed to determine whether these high- and low-anxiety phenotypes correlate with changes in adrenal histomorphology and corticosteroid production.METHODS:Rats were exposed to PSS for ten days. Thirty days later, the rats' anxiety index (AI) was assessed with an elevated plus-maze test. Based on differences in AI, the rats were segregated into low- (AI ≤ 0.8, n = 9) and high- (AI > 0.8, n = 10) anxiety phenotypes. Plasma corticosterone (CORT) concentrations were measured by ELISA. Adrenal CORT, desoxyCORT, and 11-dehydroCORT were measured by high-performance liquid chromatography. After staining with hematoxylin and eosin, adrenal histomorphometric changes were evaluated by measuring the thickness of the functional zones of the adrenal cortex.RESULTS:Decreased plasma CORT concentrations, as well as decreased adrenal CORT, desoxyCORT and 11-dehydroCORT concentrations, were observed in high- but not in low-anxiety phenotypes. These decreases were associated with increases in AI. PSS led to a significant decrease in the thickness of the zona fasciculata and an increase in the thickness of the zona intermedia. The increase in the thickness of the zona intermedia was more pronounced in low-anxiety than in high-anxiety rats. A decrease in the adrenal capsule thickness was observed only in low-anxiety rats. The nucleus diameter of cells in the zona fasciculata of high-anxiety rats was significantly smaller than that of control or low-anxiety rats.CONCLUSION:Phenotype-associated changes in adrenal function and histomorphology were observed in a rat model of complex post-traumatic stress disorder.

Posttraumatic stress disorder (PTSD) causes mental and somatic diseases. Intermittent hypoxic conditioning (IHC) has cardio-, vaso-, and neuroprotective effects and alleviates experimental PTSD. IHC’s ability to alleviate harmful PTSD effects on rat heart, liver, and brain was examined. PTSD was induced by 10-day exposure to cat urine scent (PTSD rats). Some rats were then adapted to 14-day IHC (PTSD+IHC rats), while PTSD and untreated control rats were cage rested. PTSD rats had a higher anxiety index (AI, X-maze test), than control or PTSD+IHC rats. This higher AI was associated with reduced glycogen content and histological signs of metabolic and hypoxic damage and of impaired contractility. The livers of PTSD rats had reduced glycogen content. Liver and blood alanine and aspartate aminotransferase activities of PTSD rats were significantly increased. PTSD rats had increased norepinephrine concentration and decreased monoamine oxidase A activity in cerebral cortex. The PTSD-induced elevation of carbonylated proteins and lipid peroxidation products in these organs reflects oxidative stress, a known cause of organ pathology. IHC alleviated PTSD-induced metabolic and structural injury and reduced oxidative stress. Therefore, IHC is a promising preventive treatment for PTSD-related morphological and functional damage to organs, due, in part, to IHC’s reduction of oxidative stress.

Nonpharmacological treatments of stress-induced disorders are promising, since they enhance endogenous stress defense systems, are free of side effects, and have few contraindications. The present study tested the hypothesis that intermittent hypoxia conditioning (IHC) ameliorates behavioral, biochemical, and morphological signs of experimental posttraumatic stress disorder (PTSD) induced in rats with a model of predator stress (10-day exposure to cat urine scent, 15 min daily followed by 14 days of stress-free rest). After the last day of stress exposure, rats were conditioned in an altitude chamber for 14 days at a 1,000-m simulated altitude for 30 min on day 1 with altitude and duration progressively increasing to 4,000 m for 4 h on day 5. PTSD was associated with decreased time spent in open arms and increased time spent in closed arms of the elevated X-maze, increased anxiety index, and increased rate of freezing responses. Functional and structural signs of adrenal cortex degeneration were also observed, including decreased plasma concentration of corticosterone, decreased weight of adrenal glands, reduced thickness of the fasciculate zone, and hydropic degeneration of adrenal gland cells. The thickness of the adrenal fasciculate zone negatively correlated with the anxiety index. IHC alleviated both behavioral signs of PTSD and morphological evidence of adrenal cortex dystrophy. Also, IHC alone exerted an antistress effect, which was evident from the increased time spent in open arms of the elevated X-maze and a lower number of rats displaying freezing responses. Therefore, IHC of rats with experimental PTSD reduced behavioral signs of the condition and damage to the adrenal glands. NEW & NOTEWORTHY Intermittent hypoxia conditioning (IHC) has been shown to be cardio-, vaso-, and neuroprotective. For the first time, in a model of posttraumatic stress disorder (PTSD), this study showed that IHC alleviated both PTSD-induced behavioral disorders and functional and morphological damage to the adrenal glands. Also, IHC alone exerted an antistress effect. These results suggest that IHC may be a promising complementary treatment for PTSD-associated disorders.

Disorders of cerebral circulation are a leading cause of neurological morbidity, disability and death. However, there is currently no promising drug therapy or prevention for these disorders. Non-pharmaceutical therapies might provide new opportunities for protection of brain from hypoxia and ischemia induced by tissue hypoperfusion. Intermittent hypoxic conditioning (IHC) has been studied as a potential therapy. Experimental studies showed that IHC was neuroprotective in hemorrhagic stroke induced by epileptiform seizure and reduced the size of infarct, inflammation, and increased blood-brain barrier permeability after transient middle cerebral artery occlusion. In experimental Alzheimer’s disease (AD), which is considered “a cerebrovascular disorder with neurodegenerative consequences”, IHC exerted pronounced beneficial effects on cerebral circulation. In rats with experimental AD, IHC almost completely prevented endothelial dysfunction of both cerebral and extracerebral blood vessels, rarefaction of brain vascular net and the loss of neurons in the brain cortex. Due to these protective effects, IHC eventually prevented the impairment of memory and development of experimental AD. In spontaneously hypertensive rats, IHC also prevented rarefaction of arterioles and capillaries in brain, which is, interestingly, considered a possible mechanism for the antihypertensive effect of IHC. Despite promising preclinical data, the translation of preclinical hypoxic/ischemic conditioning protocols to a clinical application has been difficult, and clinical studies using this non-pharmacological approach are still scarce. In healthy human subjects, repetitive normobaric IHC significantly diminished variations of ce- rebral perfusion in response to hypercapnia and hypocapnia without compromising cerebral tissue oxygena- tion. This mechanism may be protective in sleep apnea. Paradoxically, elderly people with moderate sleep ap- nea have a survival advantage. Perhaps apneas during sleep may activate adaptive pathways in the elderly. IHC was successfully used for treatment of encephalopathy due to improvement of oxidative homeostasis. Several clinical studies have used repetitive bilateral arm ischemic preconditioning, which reduced stroke recurrence in patients with intracranial arterial stenosis. The ability of the brain to produce an endogenous response to a conditioning stimulus that leads to subsequent protection against future injury has been observed in clinical settings. Patients with previous spontaneous, transient ischemic attacks before cerebral infarction had a more favorable outcome than those without such previous attacks. Deleterious effects of hypoxia are strikingly pronounced during prenatal development and asphyxia at birth resulting in anomalous brain development, schi zophrenia, cerebral palsy, and mental retardation. On the other hand, it appears that moderate IHC in early life accelerates brain development, leading to greater learning and memory capacity. IHC-increased learning capacity is associated with increased brain DNA concentrations, increased neurogenesis, and expression of proteins involved in synaptic plasticity. IHC treatment of mothers at risk of eclampsia resulted in more su- ccessful delivery, less frequent occurrence of nephropathy, fetal hypoxia, premature labor, and better physical condition of newborns. More translational research is needed to more completely utilize beneficial effects of IHC on cerebral circulation.

Previously we have shown that adaptation to hypoxia (AH) is cardio- and vasoprotective in myocardial ischemic and reperfusion injury and this protection is associated with restriction of nitrosative stress. The present study was focused on further elucidation of NO-dependent mechanisms of AH by identifying specific NO synthases (NOS) that could play the major role in AH protection. AH was performed in a normobaric hypoxic chamber by breathing hypoxic gas mixture (9.5-10% O2) for 5-10 min with intervening 4 min normoxia (5-8 cycles daily for 21 days). Expression of neuronal (nNOS), inducible (iNOS), and endothelial (eNOS) protein was measured in the left ventricular myocardium using Western blot analysis with respective antibodies. AH educed iNOS protein expression by 71% (p < 0.05) whereas eNOS protein expression tended to be reduced by 41% compared to control (p < 0.05). nNOS protein expression remained unchanged after AH. Selective iNOS inhibition can mimic the AH-induced protection. Therefore protective effects of AH could be at least partially due to restriction of iNOS and, probably, eNOS expression.

Нарушения мозгового кровообращения являются ведущей причиной неврологической заболеваемости, инвалидизации и смертности. Однако в настоящее время эффективной лекарственной терапии и профилактики этих нарушений не существует. Новые возможности защиты мозга от гипоксии и ишемии, обусловленной гипоперфузией мозговой ткани, могут открыть нефармакологические методы лечения. В качестве одной из таких возможностей изучается адаптация к периодической гипоксии (АПГ). Экспериментальные исследования показали, что АПГ обладает нейропротекторным действием при геморрагическом инсульте, вызванном эпилептиформными судорогами, а также уменьшает размер инфаркта мозга, ограничивает воспаление и повышает проницаемость гематоэнцефалического барьера после транзиторной окклюзии средней мозговой артерии. АПГ оказывает выраженное благоприятное действие на мозговое кровообращение при экспериментальной болезни Альцгеймера (БА), которая рассматривается как «цереброваскулярное расстройство с нейродегенеративными последствиями». У крыс с экспериментальной БА АПГ почти полностью предупреждала дисфункцию эндотелия церебральных и внецеребральных сосудов, изреживание сосудистой сети и утрату нейронов в коре мозга. Благодаря этим защитным эффектам АПГ в конечном счете предупреждала нарушения памяти и развитие экспериментальной БА у животных. У крыс со спонтанной гипертензией АПГ также предупреждала изреживание артериол и капилляров в мозге, что рассматривается как один из возможных механизмов антигипертензивного эффекта АПГ. Несмотря на многообещающие доклинические данные, переход от экспериментальных протоколов адаптации к гипоксии/ишемии к клиническому применению труден, клинические исследования с использованием этого нефармакологического подхода все еще немногочисленны. Тем не менее на здоровых добровольцах показано, что повторная нормобарическая гипоксия значительно уменьшает изменения перфузии мозга, возникающие в ответ на гипер - и гипокапнию, не нарушая при этом оксигенации мозговой ткани. Этот механизм может оказаться протективным при апноэ сна. Парадоксальным образом обнаружилось, что пожилые люди, страдающие умеренным апноэ сна, обладают преимуществом по продолжительности жизни. Возможно, апноэ сна способно активировать адаптивные механизмы в пожилом возрасте. АПГ успешно применялась для лечения энцефалопатии благодаря улучшению окислительного гомеостаза. В нескольких клинических исследованиях использовалось повторное билатеральное ишемическое преконционирование рук, что снижало частоту рецидивов инсульта у пациентов со стенозом внутричерепных артерий. В клинических условиях отмечалась способность мозга формировать реакцию на адаптирующий стимул, которая обеспечивает защиту от последующих повреждений. Исходы инфаркта мозга были более благоприятными у пациентов, ранее перенесших спонтанные транзиторные ишемические атаки, чем у пациентов без предшествующих атак. Повреждающие эффекты гипоксии особенно ярко выражены при эмбриональном развитии и асфиксии у новорожденных, приводя к аномалиям развития мозга, шизофрении, корковому параличу и задержке умственного развития. С другой стороны, оказалось, что умеренная АПГ в ранний период жизни ускоряет развитие мозга и улучшает функции обучения и запоминания, что связывают с увеличением концентраций ДНК в мозге, стимулированием нейрогенеза и экспрессии белков, вовлеченных в синаптическую пластичность. АПГ беременных женщин в группе риска эклампсии приводит к более успешным родам, снижению частоты нефропатии, гипоксии плода, преждевременных родов и улучшает физическое состояние новорожденных. Для более эффективного использования благоприятного действия АПГ на мозговое кровообращение требуется проведение новых трансляционных исследований.

Oligodendrocyte fusion with neurons in the brain cortex is a part of normal ontogenesis and is a possible means of neuroregeneration. Following such fusion, the oligodendrocyte nucleus undergoes neuron-specific reprogramming, resulting in the formation of binuclear neurons, which doubles the functional capability of the neuron. In this study, we tested the hypothesis that the formation of binuclear neurons is involved in long-term adaptation of the brain to intermittent hypobaric hypoxia, which is known to be neuroprotective. Rats were adapted to hypoxia in an altitude chamber at a simulated altitude of 4000 m above sea level for 14 days (30 min increasing to 4 h, daily). One micrometer sections of the left motor cortex were analyzed by light microscopy. Phases of the fusion and reprogramming process were recorded, and the number of binuclear neurons was counted for all section areas containing pyramidal neurons of layers III–V. For the control group subjected to sham hypoxia, the density of binuclear neurons was 4.49 ± 0.32 mm2. In the hypoxia-adapted group, this density increased to 5.71 ± 0.39 mm2 ( P < 0.04). In a subgroup of rats exposed to only one hypoxia session, the number of binuclear neurons did not differ from the number observed in the control group. We suggest that the increased content of binuclear neurons may serve as a structural basis for the neuroprotective effects of the adaptation to hypoxia.

Recently we demonstrated that intermittent, normobaric hypoxia conditioning (IHC) prevented injuries of myocardium and coronary blood vessels induced by myocardial ischemia and reperfusion (IR). This cardio‐ and vasoprotection of was associated with alleviation of nitric oxide overproduction. The aim of this study was to identify specific NO synthase(s) responsible for the IR‐induced NO overproduction and to determine the effect of IHC on these NO synthases. IHC of rats was performed in a normobaric chamber (5‐8 cycles/d for 20 d, FIO2 9.5 ‐ 10% for 5 ‐ 10 min/cycle, with intervening 4‐min normoxia). IR was produced by ligation of the left anterior descending coronary artery for 30 min followed by 60‐min reperfusion. The protein nitration marker, nitrotyrosine (3‐NT) and neuronal (nNOS), inducible (iNOS), and endothelial (eNOS) nitric oxide synthases were measured by immunoblot. IR induced appreciable 3‐NT accumulation in the left ventriclular free wall, increasing the 3‐NT content by 42% (p<0.01), but not in septum. In IHC rats, 3‐NT after IR was similar to that of control rats without IR. IHC decreased iNOS by 71% (p<0.05) and eNOS by 41% (p<0.05) in the left ventricular myocardium; the myocardial content of nNOS remained unchanged. Therefore, IHC prevents IR‐induced NO overproduction in myocardium by restricting myocardial expression of iNOS and eNOS.Grant Funding Source: Supported by the Russian Foundation for Basic Research grant 10‐04‐00980

Patients with Alzheimer's disease (AD) have reduced cerebral vascular density (VD), which impairs blood flow to neurons and may contribute to progression of AD. Earlier we showed that prior adaptation to intermittent hypobaric hypoxia (AH, simulated altitude 4,000 m; 4 h daily, 14 days) prevented memory loss and degeneration of cortical neurons in rats with experimental AD (EAD). Now we tested if AH might prevent EAD‐induced vascular rarefaction in rats with bilateral injection of beta‐amyloid peptide fragment (25–35) into n. basalis magnocellularis. Brain blood vessels were stained by transcardiac injection of Indian ink. 10‐μm brain sections were stained with cresyl violet, and blood vessels were counted. In EAD rats, VD was significantly decreased in hippocampus (13.3±0.9 vs 17.8±1.0 in field of view, FOV, p<0.03) and in cortex (17.3±1.5 vs 22.3±1.3 in FOV, p<0.03). AH increased VD in hippocampus to 27.0±3.5 in FOV (p=0.01) and in cortex to 26.0±1.1 in FOV (p<0.03). In EAD+AD rats, VD did not differ significantly from control rats in the hippocampus (18.0±1.3 vs 17.8±1.0 in FOV, p>0.60) and the cortex (24.2±1.6 vs 22.3±1.3 in FOV, p>0.20). AH may stimulate angiogenesis through hypoxia inducible factor‐1α‐mediated expression of vascular endothelial growth factor.

The role of nitric oxide (NO) in prevention of ischemia and reperfusion (IR) injury is controversial. Earlier we showed that IHC protects the heart and endothelium from IR injury. Now, the role of NO‐dependent mechanisms in this protection was studied. Rats were subjected to normobaric IHC (5–8 cycles/d for 20 d, FIO2 9.5–10% for 5–10 min/cycle, with intervening 4 min normoxia), and control rats were sham‐conditioned. IR was produced by ligation of the left coronary artery for 30 min with 60‐min reperfusion. NO production was evaluated from plasma nitrite+nitrate (NOx). 3‐Nitrotyrosine (3‐NT) and HIF‐1α were measured by immunoblot. IHC tended to increase basal NOx (6.8±0.5 vs 8.1±0.4 μmol/L NOx, p=0.23) and prevented IR‐induced NO overproduction (14±1 vs 9.1±1.1 μmol/L NOx, p<0.005). Without IHC, IR‐induced NO overproduction was associated with significant 3‐NT accumulation in the left ventricle (142±8% vs 100±12% in control, p<0.01) but not in septum or aorta. In IHC rats, 3‐NT after IR was similar to that of control rats without IR. IHC induced marked accumulation of HIF‐1α in the left ventricle (220±38% vs 100±7% in control, p<0.005). HIF‐1α‐induced increase in NO production may prevent subsequent NO overproduction by negative feedback. Thus, IHC cardioprotection is associated with prevention of toxic effects of NO overproduction in the myocardium during IR. Support: RFBR grant 10‐04‐00980.

Nitrosative stress induced by NO overproduction in neurons and glia contributes to neuronal death. However, NO underproduction impaires blood supply to brain cells and aggravates neurodegeneration. In this study, we tested whether NO is detrimental or protective in experimental AD (EAD) modeled in rats by bilateral injection of a toxic β‐amyloid (Aβ) fragment (25–35) into magnocellular nucleus. Memory was evaluated using a conditioned passive avoidance test. NO production was assessed by the plasma level of nitrite and nitrate. During development of EAD rats were injected with a NO synthase inhibitor L‐NNA or a NO donor, dinitrosyl iron complex, DNIC. NO production was significantly decreased in rats with EAD. L‐NNA potentiated the detrimental effect of Aβ and induced memory retention disorders. In contrast, DNIC prevented memory impairment. Resistance to Aβ toxicity was compared in Wistar and August rats, which have lower and higher rates of NO synthesis, respectively. Memory disorders were virtually absent in August rats, and the number of dead neurons in brain cortex was significantly less in August than in Wistar rats. Therefore, reduced NO production in the body plays an important role in development of cognitive disorders induced by Aβ treatment while higher levels of NO may be protective in EAD. Support: Russian Foundation for Basic Research, grant 07‐04‐00650.

This study tested the hypothesis that adaptation to intermittent hypoxia (AIH) can prevent overproduction of nitric oxide (NO) in brain and neurodegeneration induced by beta-amyloid (Aβ) toxicity. Rats were injected with a Aβ protein fragment (25–35) into the nucleus basalis magnocellularis. AIH (simulated altitude of 4000 m, 14 days, 4 h daily) was produced prior to the Aβ injection. A passive, shock-avoidance, conditioned response test was used to evaluate memory function. Degenerating neurons were visualized in stained cortical sections. NO production was evaluated in brain tissue by the content of nitrite and nitrate. Expression of nNOS, iNOS, and eNOS was measured in the cortex and the hippocampus using Western blot analysis. 3-Nitrotyrosine formation, a marker of protein nitration, was quantified by slot blot analysis. Aβ injection impaired memory of rats; AIH significantly alleviated this disorder. Histological examination confirmed the protective effect of AIH. Degenerating neurons, which were numerous in the cortex of Aβ-injected, unadapted rats, were essentially absent in the brain of hypoxia-adapted rats. Injections of Aβ resulted in significant increases in NOx and in expression of all NOS isoforms in brain; AIH blunted these increases. NO overproduction was associated with increased amounts of 3-nitrotyrosine in the cortex and hippocampus. AIH alone did not significantly influence tissue 3-nitrotyrosine, but significantly restricted its increase after the Aβ injection. Therefore, AIH affords significant protection against experimental Alzheimer's disease, and this protection correlates with restricted NO overproduction.

We report here studies addressing the possibility of preventing neurodegenerative changes in the brain using adaptation to periodic hypoxia in rats with experimental Alzheimer’s disease induced by administration of the neurotoxic peptide fragment of β-amyloid (Ab) into the basal magnocellular nucleus. Adaptation to periodic hypoxia was performed in a barochamber (4000 m, 4 h per day, 14 days). The following results were obtained 15 days after administration of Ab. 1. Adaptation to periodic hypoxia significantly blocked Ab-induced memory degradation in rats, as assessed by testing a conditioned passive avoidance reflex. 2. Adaptation to periodic hypoxia significantly restricted increases in oxidative stress, measured spectrophotometrically in the hippocampus in terms of the content of thiobarbituric acid-reactive secondary lipid peroxidation products. 3. Adaptation to periodic hypoxia completely prevented the overproduction of NO in the brains of rats with experimental Alzheimer’s disease, as measured in terms of increases in tissue levels of stable NO metabolites, i.e., nitrites and nitrates. 4. The cerebral cortex of rats given Ab injections after adaptation to periodic hypoxia did not contain neurons with pathomorphological changes or dead neurons (Nissl staining), which were typical in animals with experimental Alzheimer’s disease. Thus, adaptation to periodic hypoxia effectively prevented oxidative and nitrosative stress, protecting against neurodegenerative changes and protecting cognitive functions in experimental Alzheimer’s disease.

Injury of cerebral blood vessels (CBV) and neurons in Alzheimer's disease (AD) partially results from nitric oxide (NO) overproduction in microglia and astrocytes. Formation of NO stores protects CBV against NO toxicity by binding excessive NO. We have shown that prior adaptation to intermittent hypoxia (AH; simulated altitude 4,000 m; 4 h daily, 14 days) reduced memory loss and neurodegeneration in rats with experimental AD (EAD), and we proposed that enhancing protective mechanisms against NO overproduction might contribute to the beneficial effect of AH. Here EAD was modeled in rats by a bilateral injection of beta‐amyloid peptide (Ab) fragment (25‐35) into n. basalis magnocellularis. NO production was assessed in brain tissue by measuring NO2+NO3. NO stores were detected by increases in Doppler‐measured cerebral blood flow (CBF) in response to N‐acetylcysteine (NAC) which releases NO from NO stores. Ab significantly increased NO production in rat brain (25±2.0 vs 40±3.3 μmol/g tissue, respectively). AH prevented NO overproduction in Ab‐treated rats (19±1.6 μmol/g tissue). In rats with EAD, NAC increased CBF by 18.4±6.2%, which reflected formation of NO stores; NO stores were absent in CBV from control rats. Size of NO stores in adapted rats with EAD was significantly larger (31±3.5%) than in nonadapted rats with EAD. Thus, AH protection against Ab‐induced NO overproduction involves two mechanisms: restricting excessive NO synthesis and expanding NO‐storing capacity of CBV. (Supported by RFBR grant 07‐04‐00650)

NO synthesis disturbances play an important role in the development of neurodegenerative damage in Alzheimer disease. We previously showed that adaptation to intermittent hypobaric hypoxia prevents cognitive disturbances in rats with experimental Alzheimer disease [6]. Here we evaluated the role of NO in cognitive disorders and development of adaptive protection during experimental Alzheimer disease. Adaptation to hypoxia in rats was performed in a hypobaric pressure chamber at a simulated altitude of 4000 m (4 h per day for 14 days). Alzheimer disease was simulated by bilateral injections of a toxic fragment of β-amyloid (25–35) into n. basalis magnocellularis. For evaluation of the role of NO in the development and prevention of memory disorders, the rats received intraperitoneally either NO-synthase inhibitor Nω-nitro-L-arginin (L-NNA, 20 mg/kg, every other day for 14 days) or NO-donor dinitrosyl iron complex (200 μg/kg daily for 14 days). NO-synthase inhibitor potentiated the damaging effect of β-amyloid, abolished the protective effect of adaptation to hypoxia, and produced memory disorders in rats similar to those observed during experimental Alzheimer disease. In contrast, the increase in NO level in the body provided by injections of the NO-donor produced a protective effect against memory disorders caused by β-amyloid similar to that induced by adaptation to hypoxia. We concluded that reduced NO production in the organism plays an important role in the development of cognitive disorders produced by injections of β-amyloid, while prevention of NO deficit by administration of NO-donors or nonpharmacological stimulation of NO synthesis can provide a protective effect in experimental Alzheimer disease.

Impaired synthesis of nitric oxide (NO) in cerebral blood vessels (CBV) results in endothelial dysfunction and brain hypoperfusion and thereby contributes to progression of Alzheimer’s disease (AD). Previously we have shown that adaptation to intermittent hypoxia (AH) can stimulate endothelial NO synthesis. We proposed that prior AH (simulated altitude 4,000 m; 4 h daily, 14 days) may restrict NO-dependent disorders in experimental AD. AD was modeled in rats by a bilateral injection of Ab peptide fragment (25–35) into n. basalis magnocellularis. Dead neurons were revealed by Niessle staining. Memory retention was evaluated using the conditioned passive avoidance test. NO production was assessed by plasma level of NO2 and NO3. Endothelium-dependent vasodilation was reflected by changes of local cerebral blood flow in response to acetylcholine using a laser Doppler probe. AH restricted cortical neuron death, memory retention loss, decrease in NO production and endothelial dysfunction. Inhibition of NO synthesis by L-NNA aggravated the retention loss, whereas the NO donor dinitrosyl iron complex improved memory in Ab-treated rats. Therefore stimulation of NO synthesis by AH can prevent neurodegeneration, cognitive disorders and endothelial dysfunction of CBV in experimental AD. (Supported by NWO (47.011.2001.010) and RFBR (03-04-49065)