Potentiation of the protective effect of hypercapnic hypoxia in combination with pharmacological neuroprotective agents

Цель исследования - изучение возможности потенцирования нейропротекторного эффекта гиперкапнической гипоксии при помощи комбинации с фармакологическими активаторами основных механизмов, увеличивающих толерантность головного мозга к ишемии/гипоксии. Методика. Исследования проводились на 140 крысах-самцах Wistar, которые подвергались курсам респираторных воздействий гиперкапнической гипоксии (PO2 - 90 мм рт. ст.; PCO2 - 50 мм рт. ст.) в течении 5 сут по 30 мин ежедневно. После завершения курса тренировок крысам вводили фармакологические препараты (активатор аденозиновых рецепторов, блокатор карбоангидразы, активатор опиоидных рецепторов, блокатор ангиотензин-превращающего фермента) и через 24 ч производилась билатеральная окклюзия общих сонных артерий, а через 72 ч производился подсчет поврежденных/уцелевших клеток в СА1 регионе гиппокампа. Результаты. Морфологическая оценка выживаемости нейронов показала, что применение эналаприла усиливало нейропротекторный эффект гиперкапнической гипоксии, применение АТФ и даларгина не показало значимого прироста эффективности, а блокатор карбоангидразы ацетазоламид полностью устранял нейропротекторный эффект респираторных тренировок. Заключение. Выраженным потенцирующим эффектом на нейропротекцию, обусловленную респираторными тренировками с гиперкапнической гипоксией, обладает ее комбинация с ингибитором АПФ (эналаприлом). Aim. We studied the possibility of potentiating the neuroprotective effect of hypercapnic hypoxia using a combination with pharmacological activators of major mechanisms that increase the brain tolerance to ischemia/hypoxia. Methods. Studies were carried out on 140 male Wistar rats conditioned with respiratory hypercapnic hypoxia (PO2 - 90 mm Hg; PCO2 - 50 mm Hg) for 5 days, 30 min daily. After this exposure, the rats were injected with a drug (adenosine receptor activator (ATP), carbonic anhydrase blocker (acetazolamide), opioid receptor activator (dalargin), angiotensin converting enzyme (ACE) blocker (enalapril). 24 hrs later, the common carotid arteries were occluded bilaterally. 72 hrs after drug injection, the damaged/surviving cells in the CA1 region of the hippocampus were counted. Results. Morphological evaluation of neuronal survival showed that enalapril enhanced the neuroprotective effect of hypercapnic hypoxia. ATP and dalargin did not significantly increase this effect, and acetazolamide completely eliminated this neuroprotective effect. Conclusion. Thus, the neuroprotective effect of hypercapnic hypoxia conditioning was potentiated by its combination with an ACE inhibitor.

Hypercapnia and its combination with hypoxia decrease the permeability of the blood-brain barrier in rats

Цель исследования - оценка проницаемости ГЭБ после сочетанного воздействия гиперкапнии и гипоксии. Методика. Исследования проведены на 40 крысах-самцах Wistar рандомизированых на 4 равные группы (n=10): нормобарическая гипоксия (PO2 - 90 мм рт. ст./13%; PCO2 - 1 мм рт. ст./0,1%); пермиссивная гиперкапния (PO2 - 150 мм рт. ст./21%; PСO2 - 50 мм рт. ст./7%); гиперкапническая гипоксия (PO2 - 90 мм рт. ст./13%; PCO2 - 50 мм рт. ст./7%); контрольная группа (PO2 - 150 мм рт. ст./21%; PCO2 - 1 мм рт. ст./0,1%). Респираторные воздействия проводили в специальной камере в течение 15 сут по 30 мин ежедневно. Газовую смесь подавали в камеру компрессором со скоростью 15 л/мин. Контроль газового состава в камере проводили газоанализатором Microlux (Микролюкс, Россия). Через 24 ч после завершения курсов респираторных воздействий животным внутрибрюшинно вводили 2%-й раствор красителя Evans blue в объеме 4 мл/кг массы животного. Оценку проницаемости ГЭБ осуществляли через 24 ч после введения красителя путем фотометрического измерения его содержания в плазме крови и флуоресцентной микроскопии ткани головного мозга. Анализ интенсивности флуоресценции Evans blue в ткани головного мозга проводился на конфокальном микроскопе FV10i-W (Olympus, Япония). Определяли флуоресцентную интегративную оптическую плотность Evans blue с последующим расчетом индекса проницаемости по концентрации красителя в крови. Определение содержания красителя в крови осуществляли фотометрически (при 610 нм) с использованием планшетного фотометра Multiscan FS (Thermo scientific, США) по калибровочным кривым. Результаты. Индекс проницаемости ГЭБ (содержание в мозге/концентрация в крови) был существенно ниже в группах, которые подвергались респираторным воздействиям с наличием гиперкапнического компонента. Заключение. Интермиттирующая гиперкапническая гипоксия формирует наименьший уровень проницаемости ГЭБ по сравнению с изолированным воздействием гипоксии или гиперкапнии. The aim of study was to evaluate the blood-brain barrier (BBB) permeability after a combined treatment with hypercapnia and hypoxia. Methods. Experiments were performed on 40 Wistar male rats randomized to 4 equal groups (n=10): normobaric hypoxia (PO2 = 90 mm Hg/13%/PCO2 = 1 mm Hg/0.1% CO2,), permissive hypercapnia (PO2 = 150 mm Hg/21%; PCO2 = 50 mm Hg/7%); hypercapnic hypoxia (PO2 = 90 mm Hg/13%, PCO2 = 50 mm Hg/7%); and a control group (PO2 - 150 mm Hg/21%; PCO2 - 1 mm Hg/0.1%) in a special chamber for 15 days, 30 min daily. The gas mixture was delivered to the chamber with a compressor at 15 l/min. Gas composition was monitored with a gas analyzer (Microlux, Russia). At 24 hrs after completion of respiratory exposures, Evans blue dye was administered i.p. (4 ml/kg body weight). BBB permeability was assessed from the content of Evans blue dye in blood plasma photometrically and in brain tissue by fluorescent microscopy. Evans blue fluorescence intensity in brain tissue was analyzed with a confocal microscope FV10i-W (Olympus, Japan). Fluorescent integrative optical density of Evans blue was determined and used for calculation of the permeability index from blood concentration of the dye. Blood concentration of the dye was measured photometrically (at 610 nm) with a plate photometer Multiscan FS (Thermo Scientific, USA) using calibration curves. Results. The BBB permeability index (content of Evans blue dye in the brain / concentration of Evans blue dye in blood) was significantly lower in the groups that underwent respiratory exposures with the presence of a hypercapnic component. Conclusion. Intermittent hypercapnic hypoxia yields the lowest BBB permeability compared to the isolated effect of either hypoxia or hypercapnia.

Evaluation of the activity of antihypoxants with an isothiourea structure in a model of hypercapnic hypoxia with a shutdown of the cerebral hemispheres

PURPOSE: To study the effect of amtizol, 2-aminobenzthiazole (2-ABT) and 2-amino-4-acetylthiazolo[5,4-b]indole (BM-606) on the resistance of male outbred mice to acute hypoxia with hypercapnia under conditions of isolated functioning of one from the hemispheres, as well as both hemispheres of the brain. METHODS: A model of acute hypoxia with hypercapnia (canned hypoxia) was used in mice of the same mass, the lifespan of all animals was determined. Temporary shutdown of the cortex of one of the hemispheres or both hemispheres was achieved by epidural application of filter paper moistened with 25% potassium chloride solution, creating a spreading depression according to Leao. Amtizol, 2-aminobenzthiazole (2-ABT) and 2-amino-4-acetylthiazolo[5,4-b]indole (BM-606) at equimolar doses of 25, 32.5, and 50 mg/kg, respectively were used as pharmacological analyzers, the compounds were injected intraperitoneally 30 min before the hypoxic episode. RESULTS: It was shown that, in contrast to amtizol, 2-ABT and VM-606 increase the life time of experimental animals when any hemisphere is turned off. The use of drugs when both hemispheres were turned off revealed that amtizol has approximately equal effect on the brain and the rest of the body, in 2-ABT antihypoxic activity is 1/3 associated with the brain, in VM-606 exclusively with the brain. CONCLUSION: The experimental model used in this work makes it possible to quite easily evaluate the effect of either one drug or compare several drugs, their role in the functioning of the cerebral hemispheres, on which part of the sample highly resistant or low resistant to hypoxia they have the greatest effect.

Animal Welfare and Meat Quality Assessment in Gas Stunning during Commercial Slaughter of Pigs Using Hypercapnic-Hypoxia (20% CO2 2% O2) Compared to Acute Hypercapnia (90% CO2 in Air)

This study assessed aversion, stunning effectiveness, and product quality of nitrogen and carbon dioxide (CO2) mixtures used for stunning pigs. A total of 1852 slaughter pigs divided into two similar batches was assessed during routine slaughter in a Swedish commercial abattoir using either hypercapnic-hypoxia (20% CO2 and less than 2% O2; 20C2O) or hypercapnia (90% CO2; 90C) gas mixtures. Behavioral indicators of aversion and discomfort were recorded. After exposure, the stunning quality was assessed through brainstem reflexes. After slaughter, the pH and electric conductivity of carcasses were assessed to estimate the incidence of pale, soft, and exudative (PSE) pork, and the presence of ecchymosis were inspected. Compared to 90C, pigs exposed to 20C2O showed a later (p < 0.05) onset of behaviors indicative of aversion, and a lower (p < 0.01) incidence of breathlessness. However, unconsciousness (i.e., losing posture) appeared earlier (p < 0.01) in 90C compared to 20C2O. In 90C, all (100%) pigs were adequately stunned, whereas in 20C2O a 7.4% of pigs showed signs of poor stunning, especially when oxygen concentrations were >2% (p < 0.001). The percentage of PSE carcasses was higher (p < 0.01) in 20C2O than 90C. In conclusion, compared to 90C, 20C2O reduced aversion and discomfort but showed lower stun effectiveness, especially when O2 was above 2%, and a slightly poorer pork quality.

Sex differences in integrated neurocardiovascular control of blood pressure following acute intermittent hypercapnic hypoxia

Repetitive hypoxic apneas, similar to those observed in sleep apnea, result in resetting of the sympathetic baroreflex to higher blood pressures (BP). This baroreflex resetting is associated with hypertension in preclinical models of sleep apnea (intermittent hypoxia, IH); however, the majority of understanding comes from males. There are data to suggest that female rats exposed to IH do not develop high BP. Clinical data further support sex differences in the development of hypertension in sleep apnea, but mechanistic data are lacking. Here we examined sex-related differences in the effect of IH on sympathetic control of BP in humans. We hypothesized that after acute IH we would observe a rise in muscle sympathetic nerve activity (MSNA) and arterial BP in young men ( n = 30) that would be absent in young women ( n = 19). BP and MSNA were measured during normoxic rest before and after 30 min of IH. Baroreflex sensitivity (modified Oxford) was evaluated before and after IH. A rise in mean BP following IH was observed in men (+2.0 ± 0.7 mmHg, P = 0.03), whereas no change was observed in women (−2.7 ± 1.2 mmHg, P = 0.11). The elevation in MSNA following IH was not different between groups (4.7 ± 1.1 vs. 3.8 ± 1.2 bursts/min, P = 0.65). Sympathetic baroreflex sensitivity did not change after IH in either group ( P > 0.05). Our results support sex-related differences in the effect of IH on neurovascular control of BP and show that any BP-raising effects of IH are absent in young women. These data enhance our understanding of sex-specific mechanisms that may contribute to BP changes in sleep apnea.