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L-type Ca2+ channels, found in vascular smooth muscle cells, function to control Ca2+ influx, which directly regulates the degree of tension in the vasculature. An influx of Ca2+ causes these cells to contract while inhibition of this channel causes muscle relaxation, a major goal in treating hypertension. Acute hypoxia inhibits, and chronic hypoxia enhances, Ca2+ channel currents. The mechanisms underlying these hypoxic responses were examined in HEK 293 cells by altering cellular levels of proposed mediators of 0 2 sensing which have previously been shown to be involved in the redox model of 02 sensing in various cell types. In these studies I investigated the roles of mitochondrial complexes and NADPH oxidase function, and changes in cellular ROS levels, on the acute and chronically hypoxic regulation of recombinant L-type Ca2+ channels. An increase in H202, a form of ROS, by exogenous application was found to enhance Ca2+ currents. However neither catalase nor H202 affected the acute hypoxic response. In contrast superoxide dismutase (SOD) abolished hypoxic inhibition of recombinant L-type Ca2+ channels, suggestive of a role of 02- production in 02 sensing. Altered production of this ROS during hypoxia may occur within the mitochondria since acute 02 sensing was abolished in mitochondria-depleted p0 cells. Alterations in NADPH oxidase activity via application of NADPH oxidase inhibitors such as DPl and P AO did not mediate the acute hypoxic response. Hypoxic regulation of mitochondrial complex I may also mediate the response to chronic hypoxia since current enhancement by this stimulus was abolished by rotenone. These findings support the involvement of altered mitochondrial function in the 0 2 sensing pathway which mediates the hypoxic responses of recombinant L-type Ca2+ channel a1c subunits. / Thesis / Master of Science (MSc)

<p>The tumor vasculature is poor and heterogeneous which may result in inadequate oxygenation and changed energy status. In addition the balance between cell proliferation and the rate of cell death is disturbed, which results in tumor growth. </p><p>The aims of this study were 1) to gain more insight into the relation between tumor vascularity, hypoxia, and proliferation in solid tumors, and 2) to study the changes and dynamics of tumor oxygenation in relation to the vascular architecture within individual tumors. For this purpose a double hypoxic marker method was developed, which was subsequently used to 3) determine the turnover rate of hypoxic cells in three different tumor models and 4) to study the effect of cytotoxic drugs on tumor hypoxia and cell death.</p><p>Solid tumor models grown in mice were used. The tumor microenvironment was investigated with exogenous cell markers for hypoxia (pimonidazole and CCI-103F), cell proliferation (BrdUrd) and blood perfusion (Hoechst 33342). The vasculature and the exogenous cell markers were visualized with immunohistochemical techniques. The tumor sections were scanned and quantified with an image analysis systemconsisting of a fluorescence microscope, CCD camera and image analysis software.</p><p>The spatial organization of hypoxia, proliferation, and tumor vasculature was analyzed in several xenograft lines. The study revealed two main hypoxic patterns that seemed to be the consequence of complex relations between vasculature, oxygen delivery, proliferation, and cell loss. The novel double hypoxic cell marker method, with sequential injection of two hypoxic markers, was developed to study dynamic changes of the tumor oxygenation. Based on varying injection intervals between the markers the hypoxic cell half-life was determined in three tumor lines, and ranged from 17 to 49 hours. Intra-tumoral changes in oxygenation status upon oxygen modifying treatments were measured with the double hypoxic marker method. Both decreased levels of tumor hypoxia after carbogen breathing (95%O2 and 5% CO2) and increased levels of tumor hypoxia, as a result of reduced tumor perfusion after hydralazine treatment was detected. Finally the double hypoxic marker assay was used to analyze the effects of the hypoxic cytotoxin tirapazamine in relation to the hypoxic cell population, which caused a reversible decrease of the hypoxic fraction.</p><p>The results presented in this thesis now form the basis for further studies to identify subpopulations of cells that represent specific targets for therapy, and to investigate the effects of different treatment modalities.</p>

The tumor vasculature is poor and heterogeneous which may result in inadequate oxygenation and changed energy status. In addition the balance between cell proliferation and the rate of cell death is disturbed, which results in tumor growth. The aims of this study were 1) to gain more insight into the relation between tumor vascularity, hypoxia, and proliferation in solid tumors, and 2) to study the changes and dynamics of tumor oxygenation in relation to the vascular architecture within individual tumors. For this purpose a double hypoxic marker method was developed, which was subsequently used to 3) determine the turnover rate of hypoxic cells in three different tumor models and 4) to study the effect of cytotoxic drugs on tumor hypoxia and cell death. Solid tumor models grown in mice were used. The tumor microenvironment was investigated with exogenous cell markers for hypoxia (pimonidazole and CCI-103F), cell proliferation (BrdUrd) and blood perfusion (Hoechst 33342). The vasculature and the exogenous cell markers were visualized with immunohistochemical techniques. The tumor sections were scanned and quantified with an image analysis systemconsisting of a fluorescence microscope, CCD camera and image analysis software. The spatial organization of hypoxia, proliferation, and tumor vasculature was analyzed in several xenograft lines. The study revealed two main hypoxic patterns that seemed to be the consequence of complex relations between vasculature, oxygen delivery, proliferation, and cell loss. The novel double hypoxic cell marker method, with sequential injection of two hypoxic markers, was developed to study dynamic changes of the tumor oxygenation. Based on varying injection intervals between the markers the hypoxic cell half-life was determined in three tumor lines, and ranged from 17 to 49 hours. Intra-tumoral changes in oxygenation status upon oxygen modifying treatments were measured with the double hypoxic marker method. Both decreased levels of tumor hypoxia after carbogen breathing (95%O2 and 5% CO2) and increased levels of tumor hypoxia, as a result of reduced tumor perfusion after hydralazine treatment was detected. Finally the double hypoxic marker assay was used to analyze the effects of the hypoxic cytotoxin tirapazamine in relation to the hypoxic cell population, which caused a reversible decrease of the hypoxic fraction. The results presented in this thesis now form the basis for further studies to identify subpopulations of cells that represent specific targets for therapy, and to investigate the effects of different treatment modalities.

This study investigated possible effects of hypoxic training upon speed of high school sprint freestyle. Thirty-eight subjects, grouped as their two schools, performed identical loads during the ten-week program. The Experimental group used hypoxic techniques for about one-half of each workout. Pretests and posttests conducted for the 50-yard and 100-yard distances yielded highly correlated mean scores, with marked differences between the two groups. Analysis of covariance was used, selecting the .05 level for rejection. The comparison of adjusted group means indicated neither group superior at 50 yards, while the 100-yard F-ratio was significant at the .0047 level favoring hypoxic training. It is recommended that hypoxic techniques be incorporated into existing programs, possibly benefitting other strokes.

Author: Bc. Alice Pernicová Title: Motor and cognitive consequences of hypoxic-ischemic damage Objectives: The aim of this diploma work was asses the impact of Enriched Environment on development of rats after hypoxic-ischemic damage. Methods: Experiment was performed in 30 Long Evans rats from breeding in Institute of Physiology, Academy of Sciences of the Czech Republic. All rats were operated for dissection of carotid. Than randomly were half of rats chosen for hypoxic-ischemic damage. Group with hypoxic-ischemic damage were divided into control and experimental group, same procedure was with rats without HIE. For one week were rats from experimental group in special Enriched Environment cage, than they were split into smaller cages with Enriched Environment. Rats from control groups were in normal cages without special equipment. After defined time were all rats tested by chosen test: Reaching test, Ladder rung walking test, Bar holding test, Rotarod test, Morris water maze test, Open filed test. For data analysis were used Microsoft Excel 365, Sigma plot. Results: Results of test showed, that Enriched Environment can positively enhance motoric and cognitive deficit in rat development with hypoxic-ischemic damage. But it is obvious, that Enriched Environment can positively enhance development...

O presente trabalho objetivou explorar a eventual variação da expressão do mRNA e da proteína S100b no hipocampo, cerebelo e córtex cerebral de ratos neonatos em condições de anóxia, comparativamente à condições controle. Este estudo foi desenvolvido em ratos albinos, divididos em dois grupos: o grupo Experimental Anóxia (EA) e o grupo Experimental Controle (EC), que por sua vez foram subdivididos em tempos de 2, 4, 6, 12 e 24 horas no que se refere à coleta de amostras após a aplicação dos estímulos pré-estabelecidos para cada grupo. Dos períodos avaliados, nossos resultados indicaram que a anóxia proporcionou um pico na expressão gênica de S100b após duas horas e proteica após 4 horas nas áreas do hipocampo e cerebelo. O córtex cerebral do grupo EA quando comparado ao grupo EC, não apresentou nenhum aumento significante de S100b nos períodos avaliados. Os resultados obtidos contribuem de forma crucial para elucidação do papel da proteína S100b como biomarcadora na EHI, bem como no esclarecimento parcial da função deste gene com relação à fisiopatologia da doença. / The aim of the present study was to investigate the temporal variation in the expression of S100b mRNA and protein in the hippocampus, cerebellum, and cerebral cortex of newborn rats under conditions of anoxia compared with control rats. The study was performed using two groups albino rats: Experimental Anoxia (EA) and Experimental Control (EC). The animals in both EA and EC were distributed in the following subgroups relative to the time elapsed since the application of the stimuli predefined for each group: two, four, six, 12, and 24 hours. Anoxia induced a peak in the S100b gene expression after two hours and protein expression after 4 hours in the hippocampus and cerebellum. With respect to the cerebral cortex, S100b never exhibited a significant increase in the EA group compared with the EC group. The results of the present study represent a crucial contribution to the elucidation of the role protein S100b plays as a biomarker in HIE, as well as a contribution to the elucidation of the role the corresponding gene plays in the physiopathology of the disease.

A encefalopatia hipóxico-isquêmica neonatal, ou simplesmente hipóxia-isquemia (HI) neonatal, é uma das principais causas de morbidade e mortalidade em neonatos humanos. De 20% a 50% dos recém-nascidos com HI severa morrem no período perinatal. Quando sobrevivem, 25% apresentam deficiências neuropsicológicas, como dificuldade de aprendizado, epilepsia e paralisia cerebral. Devido a isso, a eficácia de possíveis agentes neuroprotetores tem sido testada em modelos animais. Há razão para se pensar que a progesterona tem um forte potencial para o tratamento da HI neonatal, já que a sua utilização tem se mostrado benéfica em pesquisas relacionadas com lesão cerebral traumática, lesão cerebral isquêmica e outros modelos de lesão do sistema nervoso central (SNC) em adultos. Inúmeros estudos têm mostrado que o modelo animal de HI de Rice e Vannucci (1981) em animais neonatos, utilizado no presente trabalho, pode produzir lesões no sistema nervoso central relativamente previsíveis, e que estas lesões encefálicas parecem semelhantes às observadas clinicamente em humanos (SALMASO et al., 2014). Para a realização do modelo de HI foram utilizados ratos Wistar com idade de 7 dias (P7). Após a oclusão da carótida esquerda, os animais foram colocados em câmaras para exposição à atmosfera hipóxica com 8% O2/92% N2 por 90 minutos. Os animais foram divididos em cinco grupos experimentais: SHAM, HI, HI+PROG-PRÉ (PRÉ), HI+PROG-PÓS (PÓS), HI+PROG-PRÉ/PÓS (PP). Os termos PRÉ e PÓS referem-se à administração de progesterona (na dose de 5 mg/kg) antes ou após o procedimento de HI neonatal . Dependendo do grupo experimental, os animais foram tratados com progesterona imediatamente antes da isquemia e/ou 6 e 24 horas após o início da hipóxia. Foram analisados o peso corporal dos animais (imediatamente antes da isquemia e 6, 24 e 48 horas após o início da hipóxia), o volume de lesão cerebral, além da expressão das proteínas p-Akt e caspase-3 pela técnica de Western blotting. / Neonatal hypoxic-ischemic encephalopathy or simply neonatal hypoxia-ischemia (HI) is a main cause of morbidity and mortality in human neonates. Moreover, 25% of survivors show neuropsychological dysfunctions such as learning difficulties, epilepsy and cerebral palsy. Because of this, the effectiveness of potential neuroprotective agents has been tested in animal models. There is a reason to suppose that progesterone has a strong potential for the treatment of neonatal HI since its use has been shown to be beneficial in researches related to traumatic brain injury, ischemic brain injury and other central nervous system injury models (CNS) in adults. Several studies have shown that the newborn animal model of HI developed by Rice and Vannucci (1981), and used in the present study, can produce lesions in the central nervous system which are predictable and similar to those observed clinically in humans. In order to perform the HI model we used 7 days old (P7) Wistar rats. After occlusion of the left carotid, the animals were placed in hypoxic chambers and exposed to the hypoxic atmosphere (8% O2/92% N2 for 90 minutes). The animals were divided into five groups: SHAM, HI, HI+PROG-PRÉ (PRÉ), HI+PROG-PÓS (PÓS), HI+PROG-PRÉ/PÓS (PP).The PRÉ and PÓS terms refer to the administration of progesterone (5 mg/kg) before and/or after the HI procedure. Progesterone was administered immediately before ischemia, 6 and 24 hours after the beginning of hypoxia, depending on the experimental group. Body weight was evaluated immediately before ischemia and/or 6 and 24 hours after the start of hypoxia. The volume of brain damage, in addition to the expression of p-Akt and caspase-3 were also evaluated.