Effect Of Hyperbaric Oxygen On Expression Of Endothelial Nitric Oxide Synthase In Cultured Rat Cerebral Microvascular Endothelial Cells In Vitro

Hyperbaric oxygen (HBO) has been applied widely in diving operation and clinical treatment. When used correctly, it could exhibit particular effect in many diseases prevention and therapy, including some diving illnesses, and also could markedly improve the efficiency of diving operation. In contrast, if inhaled oxygen partial pressure and duration are beyond a certain threshold, it can damage the body greatly. Especially when the oxygen partial pressure is about 300 kPa and above, oxygen toxicity may occur in a very short time, with the most typical and severe manifestation generalized convulsion, named as central nervous system oxygen toxicity (CNS oxygen toxicity), acute oxygen toxicity, or oxygen convulsion. Obviously, it can greatly restrict diving operation efficiency and further application of HBO in clinical treatment.By now, many hypotheses have been proposed to elucidate the mechanism of oxygen toxicity, including exceptional generation of reactive oxygen species, some important enzymes inhibited, neurotransmitter unbalance, change of cerebral vascular modulation, etc. In recent years, the relativity of cerebral blood flow (CBF) change to hyperoxia-induced seizures has been a focus. There were enough data implying that during the development of oxygen convulsion, CBF decreased in the initial exposure stage, then followed by an abrupt and sustained increase, and subsequently electrical discharges representing seizures appeared in EEG There was a very close relationship between CBF change and the onset of seizures. Further studies indicated that nitric oxide might play a vital role in the change of CBF during exposure, andmodulation of endothelial nitric oxide synthase (eNOS)-derived NO by HBO was responsible for early vasoconstriction, whereas late HBO-induced vasodilation depended upon a large amount of NO from both eNOS and nNOS. However studies in vivo couldn't definitely elucidate the correlation between CBF change and NO from eNOS during HBO exposure.Cerebral microvascular endothelial cells (CMEC) are one of the main components of blood brain barrier. They can regulate cerebral vascular tone and blood flow through the secretion of vasoactive substances especially nitric oxide and endothelin-1. In addition, nitric oxide produced by CMEC may play a key role in the regulation of CBF under basal conditions. In CMEC, NO is produced mainly by eNOS. Endothelial NO is physiologically important for keeping the vessels dilated and maintaining vascular homeostasis.In our studies, we investigated the effect of HBO at 5 ATA mostly used in the studies of hyperoxia-induced seizures on the expression of eNOS in CMEC in vitro. We hoped our studies could provide some direct evidences for explaining the correlation between CBF change and endothelial NO during HBO exposure. Our investigations include:1. Establish a model of primary culture of rat cerebral microvascular endothelial cells (RCMEC)It is difficult and laborious for most labs to perform highly pure primary culture of RCMEC. After a hard exploration, we established a relatively pure primary culture of RCMEC by means of twice enzyme digestions and gradient centrifugation. RCMEC were identified according to morphology, immunochemistry of factor VHI-associated antigen and transmission electron microscopy. Furthermore we optimized the method of primary culture of RCMEC and made it applicable to many studies.2. The effect of HBO on cell morphology and viability rate of cultured RCMECThrough morphology observation and MTT detection, we found that short-term HBO exposure didn't affect cell morphology and viability rate. In contrast, it could promote cell growth lightly.3. The effect of HBO on eNOS expression of cultured RCMECUsing immunocytochemistry and immunoblot, eNOS protein content was detected. In addition eNOS mRNA level was also measured using RT-PCR. The results indicated: (1) compared with hyperbaric air and normobaric air exposure (oxygen partial pressure were all normal in both conditions), 30, 60, 90, 120, and 180 min HBO exposure didn't influence eNOS protein and mRNA levels when detected immediately or 6 hours after exposure; (2) immunoblot analysis indicated that compared with normobaric air exposure, when measured 24 h after HBO exposure, 10 and 20 min exposure had no effects on eNOS protein content, while 30, 60 and 90 min exposure could increase it by 39%, 60%, and 40% respectively. Correspondingly RT-PCR analysis showed that compared with normobaric air exposure, when measured 24 h after HBO exposure, 10 and 20 min exposure could increase eNOS mRNA level by about 15%, and 30, 60 and 90 min exposure could increase it by about 20-30%. These results showed that eNOS expression didn't change immediately after HBO exposure and would be increased significantly 24 h after exposure.In conclusion, through our investigations, a stable and practical method of primary culture of RCMEC had been established in vitro. Furthermore we found that short-term HBO exposure exerted no prominent inhibitory effect on RCMEC growth and reversely could promote growth lightly. eNOS expression didn't change immediately after HBO exposure and would be increased significantly 24 h after exposure. These data indicated 5 ATA HBO exposure could exert definite effect on eNOS expression of cultured RCMEC depending upon exposure duration and the interval of post-exposure. Meanwhile the results showed...