| BackgroundChronic stress refers to the long-term continuous or intermittent exposure to stress source, for instance, the working pressure of daily life. To cope with stress in long period will cause decompensation of the human body and lead to a disfunction of the body, particular the disorder of central nervous, neuroendocrine, immune system, etc. It has been proved that chronic stress is not only as the main reason of various mental illnesses such as depression or post-traumatic stress disorder, but also as a moderate risk factor of cardiovascular disease.According to the report, in America the incident of chronic stress is about 50%, including 25% in high intensive stress. In China, the morbidity of chronic stress is around 42% to 46% in urban. The hyperbaric oxygen treatment center of the navy general hospital receives thousands of patients per year. Most of them suffer from physical disorder cause by mental issue,like family burden or work pressure, such as sudden deafness, cerebral circulation insufficiency, cerebral white matter osteoporosis, etc, who also suffer from abnormal in heart rate, blood pressure, blood sugar, fatty, etc. Finally most of these patients will end up to suffer from various of organic disease, even the serious cardiovascular or cardio-cerebral disease.So far, the study of chronic stress mostly focus on its pathogenic mechanism at home and abroad, and the main means of intervention is about drug or psychological intervention, but their promotion obstruct by the side effects or the high costs.HBO therapy has a significant effect on chronic organic ischemia. According to Yj Bai,etc introduction, the mechanism of HBO therapy including:Improve human body oxygen partial pressure, oxygen diffusion distance, stimulate the formation of collateral circulation, inhibit inflammatory reaction, etc. From clinic observation we find that after receive a course of hyperbaric oxygen therapy, the patient, especially the state of mild depression and anxiety state come from pressure, have reduced more or less. Basis of these observations, we wonder hyperbaric oxygen therapy has the ability to relieve damage cause by chronic stress on the human body?So we designed this experiment, by observing various kinds of physiological signs of rat, to figure out the effect of hyperbaric oxygen therapy in chronic stress rat and explore the feasibility of hyperbaric oxygen therapy intervention in chronic stress.ProposeThrough the observation of chronic stress and hyperbaric oxygen treatment on Wistar rat’s behavioral test, heart rate variability, HPA axis hormone levels, serum nitric oxide system, TNF alpha and glucocorticoid receptor expression in the hippocampus, we aim to discuss the following question:(1) the effect of hyperbaric oxygen therapy to chronic stress in stress relative indication; (2) whether chronic restrict can simulate the physiological state of the chronic stress; (3) whether hyperbaric oxygen therapy can reduce the damage from chronic restrict stress to the body. Therefore we have designed this experiment, aims at providing an experimental basis of hyperbaric oxygen treatment for chronic stress intervention.Materials and methods1. Experimental animals, main equipment and reagents1.1 Experimental animals:60 healthy male Wistar rats in 8 months age weight 200-200 g,5 rats per cage, indoor temperature constant at 25 to 28 "C,50-60% humidity,12 h/d light, free water and food, replace bedding every other day. Adaptive feeding 1 week before the trial.1.2 HBOT equipment:Single medical high pressure pure oxygen therapeutic tank, oxygen is provided by the center air supply of the navy general hospital.1.3 Elevated plus-maze materials:homemade elevated plus-maze:35cm high, two open arms and closed arm of 10cm long 50cm wide,25cm tall in closed arm wall, the bottom and the medial are blacken.1 metre above elevated plus-maze with lighting 500 w lights and cameras.1.4 Open field test materials:an open field environment is 100cm long,100cm wide,35cm high, blacken the bottom and the around. The bottom is separated from white line into 20cm x 20cm smaller, a total of 25 girds.1 m above the field is installed with 500 w lighting light source and the camera.1.5 Immunohistochemical fluorescence materials:FITC (fluorescein isothiocyanate) tag sheep anti rabbit IgG antibody, GR monoclonal antibodies to rat.1.6 Heart rate variability measurement and calculation:multiple conduct physiological measurement instrument1.7 Serological detection:CRH (corticotropin releasing hormone), ATCH (adrenocor ticotropic hormre), GCS (glucocorticoids NOS (nitric oxide synthase NO (nitric oxide) and TNF-α (tumor necrosis factor alpha). Using MULTISKAN EX enzyme standard to test.2. Grouping and experimental, method2.1 effects of hyperbaric oxygen to rat’s elevated plus-maze test20 male Wistar rats were randomly divided into four groups:HBOT group and control group, each group of 10 rats. HBOT group was given 2.01/d ATA hyperbaric oxygen treatment; control group is pure breeding, a total of 21 d.2.2 effects of chronic restricted and hyperbaric oxygen treatment on rat’s heart rate variability and open field test40 male Wistar rats were randomly divided into four groups:restricted group, HBOT group, HBOT combined restricted group and control group, each group of 10 restricted group was received 3 h/d immobility; HBOT group was received 2.0 ATA hyperbaric oxygen treatment 1/d;H HBOT combined restricted group was received hyperbaric oxygen treatment and restricted every day; control group pure breeding, a total of 21 d.The grouping of "Effect of chronic restricted and hyperbaric oxygen treatment on rat’s GR expression in hippocampus and the serum levels of HPA axis hormone" and " Effect of chronic restricted and hyperbaric oxygen treatment on rat’s nitric oxide, nitric oxide eneyme and proinflammatory factor " is simply with 2.2.3. obvervational index3.1 The effect of hyperbaric oxygen of rat’s elevated plus-maze testThe elevated plus maze test was carried out in 1,7,14 and 21 days after one week adaptive phase. The Open arms (OA) time is the main observation. Statistics using two factor analysis of variance of repeated measurements.3.2 The effect of chronic restricted and hyperbaric oxygen treatment on rat’s heart rate variability and open field testObserve the squares(motor lever) and rears(exploratory behavior) of rats in d,11 d,1 d 21 open field test after one week adaptive phase, ECG detection in 21 days to test heart rate variability.3.3 The effect of chronic restricted and hyperbaric oxygen treatment on rat’s GR expression in hippocampus and the serum levels of HPA axis hormoneAfter 21 days treatment, observed the hippocampus of the GR expression level of rat by immunofluorescence staining.Using ELISA to detect the levels of CRH, ACTH and GCS in serum.3.4 The effect of chronic restricted and hyperbaric oxygen treatment on rat’s nitric oxide, nitric oxide eneyme and proinflammatory factorusing ELISA to detect the levels of NOS,NO and TNF-a in serum.4. Statistical processingSPSS 13.0 software was used for statistical processing, and accumulating data is defined as average value plus or minus standard deviation(x±s),enumeration data was defined as rank, using repeated measures analysis of variance to decover the defferenct of behavioral scores in sequential points between groups, factorial experiment design was used for the comparison among groups and correlation between HBO and restrict. LSD pairwise comparison was used for the comparison between each two groups. Kruskal-Wallis test was used for the comparison among group in enumeration datas. taking P<0.05 as a significant difference.Result1. Elevated plus-maze test showed:it is not statistically significant (P= 0.450) between groups, there is no different between HBOT and control group in anxiety degree.2. Open field test shows that:compared with control group, the motor level (115.5±43.4) and exploratory behavior (23.9±10.1) of restricted group were significantly increased (P< 0.001) in 11 d; Compared with restricted group, the growth of HBOT combined restricted group’s scores in 1d to 11 d was decreased (P< 0.01).3. Heart rate variability showed:there was no statistically significant difference between four groups in LF/HF, sympathetic and parasympathetic activity.But it can still condudede to the following trends:restricted and hyperbaric oxygen treatment can lead to decreased sympathetic nerve tension, and hyperbaric oxygen therapy can cause the parasympathetic nervous tension increased.4. Fluorescent immunohistochemical staining showed that the hippocampus GR expression was 0.1546±0.0154 in restrict group; 0.2061±0.0214 in HBOT group; 0.2118±0.0147 in HBOT combind restricted group; 0.2174±0.0123 in control group, the hippocampus GR expression in restricted group was (0.2118+0.0147), lesser than the control group (0.2174+0.0123) (P< 0.001), there is no significant difference (P> 0.05) between HBOT combined restricted group and control group.5. HPA axis hormone showed that there was no statistically significant difference of four groups in serum CRH, ATCH, and GCS.But it can be concluded to the following trends:compared whit control group HBOT group, restricted group, and HBOT combined restricted group was increased in ACTH and GCS levels in serum, but the CRH level in HBOT combined restricted group was decreased among the others.6. There was NO statistically significant difference in 4 groups of serum NO, NOS and of TNF alpha levels. But it can be concluded to the following trends:the level of NOS in restricted group was decreased compared with the others groups, TNF alpha level of HBOT combined restricted group was lesser than restricted group’s.Conclusion1. After hyperbaric oxygen treatment, A. The anxiety of rats has no obvious change; B. the sympathetic nerve tension mild decline of rats and parasympathetic excited; C. the hippocampus GR expression has no obvious change; D. The HPA axis hormone levels of rat in serum were increased, and the levels of NO, NOS and TNF alpha had no obvious change. Indicate that hyperbaric oxygen treatment has the characteristics of stress, but the intensity is not realy strong.2. After restricted treatment, A. Behavior test in rats showed obvious anxiety state; B. sympathetic nerve tension in rat was declined; C. The GR expression of hippocampus was decreased; D. The GCS level of rat was rised, but the CRH and ACTH showed no significant changes; E. The NOS level was dropped in rats. Indicated that the sustain 21 days restricted treatment can simulated the physiological state of chronic stress within a certain scope.3. After hyperbaric oxygen combined restricted treatment, A. The state of anxiety in rats were reduced compared with restricted group; B. The sympathetic nerve tension of rats were declined, but the drops were lesser than restricted group; C. The GR expression of hippocampus has no obviously decreased; D. The levels of ACTH and GCS in serum were increased compared with restrictedl group, but the CRH level of HBOT combined restricted group was decreased compared with the other three groups. E. The level of TNF alpha after restricted combined HBO treatment was decreased compared with control group. Indicated that hyperbaric oxygen can slow the influence of restricted to rat in relative physilogical sign. |
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