Obstructive sleep apnea (OSA) is a common breathing and sleeping disorder, characterized by repeated episodes of airway obstruction during sleep resulting in intermittent hypoxia (IH). From clinical reports, patients with OSA are associated with behavioral and neuropsychological deficits, including impaired spatial learning memory and cognitive deficiencies. Previous studies proposed that reactive oxygen species (ROS) and apoptosis caused by intermittent hypoxia (IH) contributed to this cognitive deficits. However, the exact mechanism is still poorly understood and not settled. / The endoplasmic reticulum (ER) is a cellular organelle in which all secretory and integral membrane proteins are folded and is also the site where proteins are post-translationally modified in ATP-dependent chaperone-mediated processes. In this study, we hypothesized that ER stress in the hippocampus is initiated in the OSA via elevated levels of ROS. Four groups of adult male mice were used, with two of them exposed to normoxia as control, and the other two exposed to IH treatment, each receiving either vehicle or tauroursodeoxycholic acid (TUDCA), an ER stress inhibitor. Eight-armed radial maze was used to investigate the performance of reference memory during the whole IH/normoxia treatment. After behavior test, long-term potentiation (LTP) was measured to investigate synaptic plasticity in hippocampus. Furthermore, ER stress-associated pro-apoptotic effectors were detected by Western blotting, and ultra-structure of rough ER and the morphology of hippocampal dendritic spines and synapses in the hippocampal CA1 area were observed. / LTP was impaired in the hippocampus after IH treatment, which was rescued by TUDCA. Furthermore, ER stress-associated pro-apoptotic effectors, CHOP and caspase-12, were up-regulated after chronic IH treatment and was abolished by co-infusion of TUDCA. Meanwhile, increased cleaved-caspase-3 after chronic IH treatment was reduced by TUDCA via increased expression of Bcl-2. On the other hand, ultrastructural analysis of rough ER in the hippocampal CA1 revealed IH-induced ER luminal swelling, and was attenuated by TUDCA. In addition, the length of synaptic active zone was significantly reduced after chronic IH treatment and was partially rescued by the application of TUDCA. Golgi staining also showed a decrease in mature dendritic spines in IH group, and reversed by TUDCA. In behavioral analysis, the number of reference memory errors significantly increased after IH treatment and rescued by TUDCA injection. Overall, the data suggest a critical role of ER stress underlying the impairment of long-term synaptic plasticity and neurocognitive deficits in chronic IH. Targeting ER stress could be a potential therapeutic strategy for neural dysfunction in OSA. / On the other hand, neuronal firing, especially robust persistent activity of neuron in hippocampus, is critical role in memory formation. Increased ROS induced by IH has been implicated in long-term potentiation of neural activity. IH could be involved in a variety of K⁺ channels which eventually leads to excitotoxicity by increased Ca2⁺-dependent glutamate release. Although the results were just shown in acute IH treatment, the chronic effect of IH on the firing frequency of hippocampus is still unknown. / Therefore, to investigate the effect of chronic IH treatment on firing activities and local field potentials of hippocampal neurons, implantation of multi-channel micro-wires electrode array into hippocampus of OSA model rat was performed to monitor spontaneous discharge. The results were shown the firing frequency of pyramidal neurons (PNs) was significantly elevated after 8 hours IH in second and third days, on the other hand, interneurons (INs) seem to be more sensitive to intermittent hypoxia since the higher firing frequency was sustained from third day to seventh day after 8 hours IH, however, at the end of 14 days IH treatment, the firing frequencies of PNs and INs are all both dramatically reduced. Meanwhile, the results in this part will enable us to understand the exact change of firing pattern and local field potential during intermittent hypoxia. The percentage of complex burst spikes was decreased after 14 days IH in PNs and the power of theta rhythms was also impaired. It suggests that the disorder of neuronal pattern and the change of local field potential are associated with cognitive impairment in OSA model. After 1 week recovery, the firing frequency of PNs was rescued again, but not for that of INs. We also found that the power of theta rhythms which had an important role in memory formation was weaker after 2 weeks IH treatment, however, the precise mechanism was still unknown. From the effect of intermittent hypoxia on spontaneous discharges and LFP of hippocampal neurons in free moving rat, it may reveal some roles of IH in cognitive impairment via disorder neuronal function in CA1 region. / 阻塞性睡眠呼吸暫停(OSA) 是一種常見的睡眠障礙疾病，這種疾病的主要特徵是在睡眠過程中反復發作的氣道阻塞，從而導致间歇性缺氧(IH)。從臨床報導中發現，OSA患者表現出行為和神經心理缺陷，包括空間學習記憶的受損和認知缺陷。通過之前的研究表明，活性氧(ROS)的增多和細胞凋亡是間歇性缺氧所引起認知功能障礙的主要機制之一，然而，其具體的機制仍不清楚。 / 作為細胞重要的細胞器，內質網是分泌蛋白和膜蛋白折疊組裝的主要場所，同時，由ATP依賴的分子伴侶所介導的蛋白質翻譯後修飾這一過程也主要在內置網中完成。在本課題中，我們假設在OSA模型的海馬組織中，內質網應激的啟動是由於缺氧引起的與活性氧(ROS)的升高。在本課題中，我們使用了四組成年雄性小鼠，其中兩組作為正常對照組，分別接受生理鹽水和牛磺去氧膽酸（一種常用的內質網抑制劑）的腹腔注射，另外兩組接受缺氧處理，同時也分別接受照生理鹽水和牛磺去氧膽酸注射。八臂放射迷宮被用來研究參考記憶的表現。行為學結束之後，長時程增強(LTP)用來測定海馬的突觸可塑性。用免疫印跡的方法檢測內質網應激的相關凋亡蛋白的表達情況，並且觀察海馬CA1區域中，內質網超微結構和海馬樹突棘數目及突觸形態的變化。 / 從實驗結果中，LTP在缺氧後減弱，而TUDCA能夠部分恢復由於缺氧所導致的LTP的降低。除此之外，內質網應激相關的促凋亡蛋白(CHOP和caspase-12)在缺氧組中表達升高，但是在TUDCA組中有所減低，同時，我們還發現，TUDCA也能夠減低缺氧組中cleaved-caspase-3的表達，而這一作用，可能與提高Bcl-2蛋白的表達（一個可標記的抗凋亡蛋白）有關。在間歇性缺氧組的海馬CA1區域中，粗面內質網出現管腔的腫脹，這一超微結構的變化表明在內質網出現官腔中有的許多未折疊蛋白聚集，並通過TUDCA的注射能夠降解未折疊蛋白來緩解這一現象的發生。同時，在IH處理後，突觸超微結構也發生了形態上的變化。突觸活性區的長度在IH處理組中顯著減少，但是在TUDCA組中有一定程度的恢復。高爾基染色顯示，成熟樹突棘（海馬突觸可塑性的結構基礎）的數目在間歇性缺氧組中有所下降，而在TUDCA治療後，成熟樹突棘的數目有所上升。我們發現參考記憶錯誤次數在缺氧後都有明顯的升高，而在注射TUDCA後，參考記憶錯誤次數都有所降低。總之，這些結果證明，內質網應激在間歇性缺氧的所引起的長時程突觸可塑性減弱和神經認知功能的損傷起到關鍵的作用，而抑制內質網應激對OSA中的出現神經功能紊亂起到一定的預防和治療效果。 / 而另一方面，神經元的放電，特別是海馬中神經元穩定持久的放電形式，對記憶的形成起到關鍵的作用。間歇性缺氧所引起的ROS的升高對於長時程增強的神經活動存在一定的關係，因為，通過以往的研究發現，間歇性缺氧可以通過多種鉀離子通道的啟動，最終由於鈣離子依賴的谷氨酸釋放的增多从而導致興奮性毒性的神經遞質的釋放。而這些結果只在急性缺氧模型中發現，慢性的間歇性缺氧對海馬的放電頻率的影響仍是未知之數。 / 因此，為了探討長時程的間歇性缺氧對海馬神經元的放電頻率和局部場電位的影響，多管道微絲電極陣列植入OSA大鼠的海馬中來監控自發放電的影響。結果表明，錐體細胞的放電頻率在第二天和第三天的8小時的間歇性低氧後明顯的升高了。另一方面，我們觀察到中間神經元似乎對間歇性缺氧更敏感，因為，從第三天到第七天缺氧8小時後，神經元的放電頻率都明顯的增高。但是在間歇性缺氧14天后，錐體細胞和中間神經元的放電頻率都所有顯著性的減少。同時，這一部分結果準確表明了海馬神經元的放電模式和局部場電位在間歇性缺氧的模型的是如何變化的。我們發現錐體細胞所具有的複合簇狀放電的比例減少，同時，theta波(與記憶的形成有關)的能量也有所減低。而這種神經元活動和局部的場電位的異常變化可能與OSA模型中出現的總認知功能障礙有關。在恢復一周後，錐體細胞的放電頻率有所增加，基本上可以恢復到缺氧前的狀態，但是中間神經元的頻率卻沒有多大的改變, 但是，其具體機制仍不清楚。從間歇性缺氧對大鼠海馬神經元自發放電和場電位影響的結果，它揭示了間歇性缺氧通過擾亂海馬CA1區域神經元的功能從而導致認知功能損傷。 / Xu, Linhao. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2014. / Includes bibliographical references (leaves 167-199). / Abstracts also in Chinese. / Title from PDF title page (viewed on 03, October, 2016). / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only.

Little is known about the physiology or biochemistry of hypoxia (reduced levels of oxygen) tolerance during development in vertebrate embryos. In most species, relatively brief bouts of severe hypoxia are lethal or teratogenic. An exception to such hypoxia intolerance is the annual killifish Austrofundulus limnaeus, in which populations persist in hypoxic environments. This species inhabits seasonal ponds in Venezuela, surviving through the dry season in the form of diapausing embryos. Embedded in the pond sediment, embryos of A. limnaeus are routinely exposed to hypoxia and anoxia (lack of oxygen) as part of their normal development. Here, we exposed embryos to various levels of PO2 (21.2, 15.6, 10.8, 8.4, 6.1, 3.6, and 2.2 kPa) at two different temperatures (25°C and 30°C) to study the effects on developmental rate and heart rate. We also measured enzyme activity and quantified DNA content of individual embryos to compare differences among the varying levels of hypoxia and temperature. Hypoxia caused a significant decline in developmental rate and caused a stage-specific decline in heart rate. Higher temperature caused an increase in the developmental rate for those embryos incubated at PO2 of 6.1 kPa and greater. Temperature had a negative effect by hindering development below a PO2 of 6.1 kPa. Total embryonic DNA content was reduced at low partial pressures (15.6, 10.8, 8.4, 6.1, 3.6, and 2.2 kPa) of oxygen. Citrate synthase, lactate dehydrogenase, and phosphoenolpyruvate carboxykinase were all down-regulated indicating a complete lack of enzymatic metabolic compensation to combat reduced oxygen levels.

Embryos of the annual killifish Austrofundulus limnaeus display a remarkable tolerance to anoxia during development, most notably during embryonic diapause. Little is known about the metabolic or enzymatic changes that accompany this state of anoxia tolerance. This study examined the metabolic changes associated with exposure to anoxia by measuring the activity of the enzyme phosphoenolpyruvate carboxykinase (PEPCK), and by profiling the concentration of 31 metabolites ranging from amino acids to citric cycle intermediates at 4 different developmental stages, diapause 2 (DII), 4 days post diapause (dpd), 12 and 22 dpd. Embryos of A. limnaeus showed stage specific changes in concentrations of several metabolites. The most notable changes in metabolite concentration in response to anoxia were the increases of lactate, alanine, GABA and succinate as well as a pronounced decrease in aspartate concentrations. However, a complete understanding of the mechanisms by which anoxia tolerance is achieved remains elusive. Further studies into the tissue specific responses of anoxia would enable greater resolution when attempting to explain changes in concentrations of metabolites both during development and in response to anoxic insult.

Indiana University-Purdue University Indianapolis (IUPUI) / Rationale: CD55 down-regulation on airway epithelium correlates with local complement activation observed in hypoxia-associated pulmonary diseases. Therefore, we hypothesized that induction of hypoxia inducible factor 1 alpha (HIF-1α) in hypoxic airway epithelium, mediates CD55 down-regulation. Methods: Chetomin and HIF-1α siRNA inhibited HIF-1α in hypoxic SAECs (1% O2), and mice lungs (10% O2). DMOG mediated HIF-1α stabilization in normoxic SAECs and mice lungs (21% O2). Transduction of SAECs with AdCA5 also stabilized HIF-1α. CD55 and CA9 transcripts were measured by RT-PCR. CD55 and HIF-1α protein expression was assessed by western blots. In vivo, immunohistochemistry (IHC) confirmed CD55 and HIF-1α expression. C3a and C5a levels in bronchoalveolar lavage fluid (BALF) were measured by ELISA. Results: HIF-1α was induced in 6 hour hypoxic SAECs (p<0.05), but CD55 transcripts were repressed (p&lt;0.05). CD55 protein was down-regulated by 72 hours (p<0.05). CA9 transcripts were elevated by 48 -72 hours (p<0.05 and p<0.01, respectively). In vivo, CD55 transcripts and protein were down- regulated by 24 hours post-hypoxia (p<0.01) which corresponded to complement activation (p<0.05) in BALF. However, CA9 was increased (p<0.01). Chetomin (100nM) treatment in 6 hour hypoxic SAECs, recovered CD55 transcripts (p<0.01) and protein (p<0.05), but down-regulated CA9 (p<0.05). Similarly, in vivo chetomin (1mg/ml) treatment recovered CD55 protein (p<0.01) and down-regulated CA9 (p<0.01). Silencing HIF-1α (50nM) in hypoxic SAECs restored CD55 transcripts by 6 hours (p<0.05), and protein expression by 24 hours (p<0.05). However, CA9 was repressed (p<0.01). In vivo silencing of HIF-1α (50µg) restored CD55 protein expression (p<0.05) but down-regulated CA9 (p<0.05). Stabilizing HIF-1α in normoxic SAECs via DMOG (1µM), down-regulated CD55 transcripts and protein (p<0.01), but increased CA9 within 6-24 hours (p<0.05 and p<0.01, respectively). HIF-1α induction by DMOG (1mg/ml) in normoxic mice lungs down-regulated CD55 transcripts (p<0.01) and protein (p<0.01), but increased CA9 (p<0.05). Induction of HIF-1α in AdCA5 (50 PFUs/cell) transduced normoxic SAECs, resulted in CD55 protein down-regulation (p<0.05), but increased CA9 (p<0.001). Conclusions: HIF-1α down-regulates CD55 on airway epithelium. Targeting this mechanism may be a potential therapeutic intervention for attenuating complement activation in hypoxic pulmonary diseases.

Indiana University-Purdue University Indianapolis (IUPUI) / Photoacoustic Imaging is a non-invasive optical imaging modality used to image biological tissues. In this method, a pulsating laser illuminates a region of tissues to be imaged, which then generates an acoustic wave due to thermal volume expansion. This wave is then sensed using an acoustic sensor such as a piezoelectric transducer and the resultant signal is converted into an imaging using the back projection algorithm. Since different types of tissues have different photo-acoustic properties, this imaging modality can be used for imaging different types of tissues and bodily organ systems. This study aims at quantifying the process of light conversion into the acoustic signal. Light travels through tissues and gets attenuated (scattered or absorbed) or reflected depending on the optical properties of the tissues. The process of light propagation through tissues is studied using Monte Carlo simulation software which predicts the propagation of light through tissues of various shapes and with different optical properties. This simulation gives the resultant energy distribution due to light absorption and scattering on a voxel by voxel basis. The Monte Carlo code alone is not sufficient to validate the photon propagation. The success of the Monte Carlo code depends on accurate prediction of the optical properties of the tissues. It also depends on accurately depicting tissue boundaries and thus the resolution of the imaging space. Hence, a validation algorithm has been designed so as to recover the optical properties of the tissues which are imaged and to successfully validate the simulation results. The accuracy of the validation code is studied for various optical properties and boundary conditions. The results are then compared and validated with real time images obtained from the photoacoustic scanner. The various parameters for the successful validation of Monte Carlo method are studied and presented. This study is then validated using the algorithm to study the conversion of light to sound. Thus it is a significant step in the quantification of the photoacoustic effect so as to accurately predict tissue properties.