Hyperbaric Oxygen Preconditioning Research Articles

Osteopontin Mediates Hyperbaric Oxygen Preconditioning-Induced Neuroprotection Against Ischemic Stroke.

Neurosurgical operations may result in surgical injury which would lead to postoperative neurological deficits. Hyperbaric oxygen preconditioning (HBO-PC) may be beneficial for such people. However, the exact mechanism underlying HBO-PC is not well known yet. The aim of this study is to explore the role of osteopontin (OPN) in HBO-PC-induced neuroprotection. The study consisted of two experiments. In experiment 1, Sprague Dawley (SD) rats were divided into four groups: sham group, HBO-PC sham group, stroke group, and HBO-PC group (HBO-PC + stroke). The animals in the second experiment were randomly assigned to one of two groups: OPN small interfering (siRNA) group (HBO-PC + stroke + OPN siRNA) and control siRNA group (HBO-PC + stroke + negative control siRNA). Neurological outcome in HBO-PC group was better than that of stroke group. After OPN siRNA was administered, neurological function aggravated compared with control siRNA group. Brain morphology and structure seen by light microscopy was diminished in stroke group and OPN siRNA group, while fewer pathological injuries occurred in HBO-PC and control siRNA group. The infarct volume in HBO-PC group was the lowest, followed by OPN siRNA group and stroke group, respectively. Preconditioning with HBO promoted expression of OPN, which reduced the expression of interleukin (IL)-1β/nuclear factor-κ-gene binding (NFκB) and augmented protein kinase B (Akt). OPN siRNA reversed these changes. OPN plays an important role in the neuroprotection elicited by HBO-PC. Pretreatment with HBO may be beneficial for people going to undertake brain surgery.

Nrf2 Activation in Astrocytes Contributes to Spinal Cord Ischemic Tolerance Induced by Hyperbaric Oxygen Preconditioning

In this study, we investigated whether nuclear factor erythroid 2-related factor 2 (Nrf2) activation in astrocytes contributes to the neuroprotection induced by a single hyperbaric oxygen preconditioning (HBO-PC) against spinal cord ischemia/reperfusion (SCIR) injury. In vivo: At 24 h after a single HBO-PC at 2.5 atmospheres absolute for 90 min, the male ICR mice underwent SCIR injury by aortic cross-clamping surgery and observed for 48 h. HBO-PC significantly improved hindlimb motor function, reduced secondary spinal cord edema, ameliorated the reactivity of spinal motor-evoked potentials, and slowed down the process of apoptosis to exert neuroprotective effects against SCIR injury. At 12 h or 24 h after HBO-PC without aortic cross-clamping surgery, Western blot, enzyme-linked immunosorbent assay, realtime-polymerase chain reaction and double-immunofluorescence staining were used to detect the Nrf2 activity of spinal cord tissue, such as mRNA level, protein content, DNA binding activity, and the expression of downstream gene, such as glutamate-cysteine ligase, γ-glutamyltransferase, multidrug resistance protein 1, which are key proteins for intracellular glutathione synthesis and transit. The Nrf2 activity and downstream genes expression were all enhanced in normal spinal cord with HBO-PC. Glutathione content of spinal cord tissue with HBO-PC significantly increased at all time points after SCIR injury. Moreover, Nrf2 overexpression mainly occurs in astrocytes. In vitro: At 24 h after HBO-PC, the primary spinal astrocyte-neuron co-cultures from ICR mouse pups were subjected to oxygen-glucose deprivation (OGD) for 90 min to simulate the ischemia-reperfusion injury. HBO-PC significantly increased the survival rate of neurons and the glutathione content in culture medium, which was mainly released from asctrocytes. Moreover, the Nrf2 activity and downstream genes expression induced by HBO-PC were mainly enhanced in astrocytes, but not in neurons. In conclusion, our findings demonstrated that spinal cord ischemic tolerance induced by HBO-PC may be mainly related to Nrf2 activation in astrocytes.

Hyperbaric oxygen preconditioning attenuates postoperative cognitive impairment in aged rats.

Cognitive decline after surgery in the elderly population is a major clinical problem with high morbidity. Hyperbaric oxygen (HBO) preconditioning can induce significant neuroprotection against acute neurological injury. We hypothesized that HBO preconditioning would prevent the development of postoperative cognitive impairment. Elderly male rats (20 months old) underwent stabilized tibial fracture operation under general anesthesia after HBO preconditioning (once a day for 5 days). Separate cohorts of animals were tested for cognitive function with fear conditioning and Y-maze tests, or euthanized at different times to assess the blood-brain barrier integrity, systemic and hippocampal proinflammatory cytokines, and caspase-3 activity. Animals exhibited significant cognitive impairment evidenced by a decreased percentage of freezing time and an increased number of learning trials on days 1, 3, and 7 after surgery, which were significantly prevented by HBO preconditioning. Furthermore, HBO preconditioning significantly ameliorated the increase in serum and hippocampal proinflammatory cytokines tumor necrosis factor-α, interleukin-1 β (IL-1β), IL-6, and high-mobility group protein 1 in surgery-challenged animals. Moreover, HBO preconditioning markedly improved blood-brain barrier integrity and caspase-3 activity in the hippocampus of surgery-challenged animals. These findings suggest that HBO preconditioning could significantly mitigate surgery-induced cognitive impairment, which is strongly associated with the reduction of systemic and hippocampal proinflammatory cytokines and caspase-3 activity.

Preconditioned hyperbaric oxygenation protects skin flap grafts in rats against ischemia/reperfusion injury

Hyperbaric oxygen (HBO) therapy is an effective therapy for ischemia/reperfusion (I/R) injury of the brain, small intestine, testes and liver. However, the detailed molecular mechanisms underlying the effect of HBO therapy remain undetermined. In the current study, the hypothesis that preconditioning rats with HBO protects grafted skin flaps against subsequent I/R injury was investigated. In addition, the molecular mechanisms underlying HBO therapy were characterized by analyzing the roles of the following important inflammatory factors: High mobility group protein 1 (HMGB1) and nuclear factor-κ B (NF-κB). A total of 40 rats were randomly divided into the following five groups: (i) Sham surgery (SH); (ii) ischemia followed by reperfusion 3 days following surgery (I/R3d); (iii) ischemia followed by reperfusion 5 days following surgery (I/R5d); (iv) HBO preconditioning (HBO-PC) and ischemia followed by reperfusion 3 days following surgery (HBO-PC+3d); and (v) HBO-PC and ischemia followed by reperfusion 5 days following surgery (HBO-PC+5d). For the surgical procedure, all pedicled skin flaps were first measured and elevated (9×6 cm). The feeding vessels of the skin flaps were subsequently clamped for 3 h and released to restore blood flow. The rats in the HBO-PC+3d and HBO-PC+5d groups received 1 h HBO for 3 and 5 consecutive days, respectively, prior to surgery. Following surgery, the rats were euthanized, and grafted tissues were collected for western blotting and immunohistochemistry. HBO-PC increased blood perfusion in epigastric skin flaps and attenuated I/R injury following skin flap graft. Additionally, the elevated expression of HMGB1 and NF-κB proteins during I/R injury was attenuated by HBO-PC treatment. HBO-PC may therefore be applied to reduce I/R injury and improve the survival rate of grafted skin flaps. The molecular mechanisms underlying the effect of HBO therapy are associated with the attenuation of inflammatory responses.

Hyperbaric Oxygen Preconditioning Induces Tolerance against Oxidative Injury and Oxygen-Glucose Deprivation by Up-Regulating Heat Shock Protein 32 in Rat Spinal Neurons

ObjectiveHyperbaric oxygen (HBO) preconditioning (HBO-PC) has been testified to have protective effects on spinal cord injury (SCI). However, the mechanisms remain enigmatic. The present study aimed to explore the effects of HBO-PC on primary rat spinal neurons against oxidative injury and oxygen-glucose deprivation (OGD) and the relationship with heat shock proteins (HSPs).MethodsPrimary rat spinal neurons after 7 days of culture were used in this study. HSPs were detected in rat spinal neurons following a single exposure to HBO at different time points by Western blot. Using lactate dehydrogenase release assay and cell counting kit-8 assay, the injuries induced by hydrogen peroxide (H2O2) insult or OGD were determined and compared among neurons treated with HBO-PC with or without HSP inhibitors.ResultsThe results of Western blot showed that HSP27, HSP70 and HSP90 have a slight but not significant increase in primary neurons following HBO exposure. However, HSP32 expression significantly increased and reached highest at 12 h following HBO exposure. HBO-PC significantly increased the cell viability and decreased the medium lactate dehydrogenase content in cultures treated with H2O2 or OGD. Pretreatment with zinc protoporphyrin IX, a specific inhibitor of HSP32, significantly blocked the protective effects of HBO-PC.ConclusionsThese results suggest that HBO-PC could protect rat spinal neurons in vitro against oxidative injury and OGD mostly by up-regulating of HSP32 expression.

Hemeoxgenase-1 mediates the protective effect of hyperbaric oxygen preconditioning against the brain edema after experimental hemorrhage in rats

Objective To investigate the role of Heme oxygenase-1 in the effect of hyperbaric oxygen preconditioning (HBOP) against the brain edema formation after experimental intracerebral hemorrhage in rats.Methods The study was carried out by animal experiment in two steps by using 54 Spradgue-Dawley rats weighting from 300-350 g.In the first step,rats were treated with HBOP (HBOP group,n =3) or with sham pre-conditioning (Sham pre-conditioning group,n =3).All the rats were sacrificed 24 h after the preconditioning,and basal ganglion of brain tissue was taken for detect HO-1 level by using western blot analysis.In the second step,rats were divided into 4 groups (n =12 in each group):HBOP +ZnPP group,in which rats had a micro-pump intra-peritoneally implanted containing a specific HO-1 inhibitor ZnPPⅨ (Zinc protoporphyrin IX,0.01 mg/kg),Sham pre-conditioning + Znpp group,HBOP + DMSO group,in which rats with a intra-peritoneal micro-pump containing 2 mL Dimethyl sulfoxide (DMSO,a solvent vehicle) and Sham pre-conditioning + DMSO group before HBOP.At 24 hours after the pre-conditioning,rats received an infusion of 100 μL autologous blood into the caudate nucleus to form a simulated intracerebrum hemorrhage (ICH),and were sacrificed 72 h later for brain water content measurements.All data were analyzed by using Stata 7.0 software and statistical analyses were carried out by two-tailed Student t test.Results Compared with the Sham pre-conditioning group,the HBOP group had significant higher level of HO-1.Compared with the Sham pre-conditioning + DMSO group,the HBOP + DMSO group had a significant lower level of water content in the ipsilateral basal ganglion [(81.4 ± 0.9) ％ vs.(82.6 ± 0.8) ％ (P ＜ 0.05)],however,peritoneal infusion of ZnPP Ⅸ before HBOP abolished HBOP-induced protection against brain edema formation after experimental ICH [(82.8 ± 0.9) ％ vs.(82.6 ± 0.7) ％ (P ＞ 0.05)].Conclusions Hyperbaric oxygen preconditioning attenuate brain edema formation after experimental ICH in rats,and this protection is attributed to the activation of HO-1. Key words: Hyperbaric oxygen ; Preconditioning; Rat; Intracerebral hemorrhage ; Brain edema; Heme oxygenase-1 ; Zinc protoporphyrin

Effects of hyperbaric oxygen preconditioning on cardiac stress markers after simulated diving.

Hyperbaric oxygen preconditioning (HBO-PC) can protect the heart from injury during subsequent ischemia. The presence of high loads of venous gas emboli (VGE) induced by a rapid ambient pressure reduction on ascent from diving may cause ischemia and acute heart failure. The aim of this study was to investigate the effect of diving-induced VGE formation on cardiac stress marker levels and the cardioprotective effect of HBO-PC. To induce high loads of VGE, 63 female Sprague–Dawley rats were subjected to a rapid ambient pressure reduction from a simulated saturation dive (50 min at 709 kPa) in a pressure chamber. VGE loads were measured for 60 min in anesthetized animals by the use of ultrasonography. The animals were divided into five groups. Three groups were exposed to either diving or to HBO-PC (100% oxygen, 38 min at 303 kPa) with a 45 or 180 min interval between HBO-PC and diving. Two additional groups were used as baseline controls for the measurements; one group was exposed to equal handling except for HBO-PC and diving, and the other group was completely unexposed. Diving caused high loads of VGE, as well as elevated levels of the cardiac stress markers, cardiac troponin T (cTnT), natriuretic peptide precursor B (Nppb), and αB-crystallin, in blood and cardiac tissue. There were strong positive correlations between VGE loads and stress marker levels after diving, and HBO-PC appeared to have a cardioprotective effect, as indicated by the lower levels of stress marker expression after diving-induced VGE formation.

Effects of hyperbaric oxygen preconditioning on ischemia-reperfusion inflammation and skin flap survival.

Hyperbaric oxygen preconditioning (HBO) is a new method of ischemia preconditioning. In this study, we examined its effects on skin flap survival and the mechanisms involved. Thirty-six rats were divided into three groups: HBO preconditioning, control, and sham groups. An extended epigastric adipocutaneous flap based on the right superficial epigastric artery and vein was raised. A 3-hour period of flap ischemia was induced by clamping the pedicle vessels with a microvascular clamp. At the end of ischemia induction, the clamp was removed and the flap was resutured. Rats in the HBO preconditioning group were treated with HBO four times before surgery. Microcirculation in the skin flap was measured on postoperative days 1, 3 and 5. The size of the flap was measured on postoperative day 5, before the animals were sacrificed. Samples of the skin flap were prepared and stained with hematoxylin and eosin. The levels of tumor necrosis factor (TNF)-α, interleukin (IL)-1β, and IL-6 in the flap samples were measured. Surviving flap size was significantly higher in the HBO preconditioning group compared with controls, with a reduced inflammatory response and increased perfusion. IL-1, TNF-α, and IL-6 levels in the HBO preconditioning group were lower than in controls. HBO preconditioning improved flap survival in this ischemia-reperfusion rat model. The mechanisms responsible for this effect may relate to attenuation of the inflammatory response and increased flap perfusion following HBO preconditioning.

Hyperbaric oxygen preconditioning attenuates hyperglycemia-enhanced hemorrhagic transformation by inhibiting matrix metalloproteinases in focal cerebral ischemia in rats

Hyperglycemia dramatically aggravates brain infarct and hemorrhagic transformation (HT) after ischemic stroke. Oxidative stress and matrix metalloproteinases (MMPs) play an important role in the pathophysiology of HT. Hyperbaric oxygen preconditioning (HBO-PC) has been proved to decrease oxidative stress and has been demonstrated to be neuroprotective in experimental stroke models. The present study determined whether HBO-PC would ameliorate HT by a pre-ischemic increase of reactive oxygen species (ROS) generation, and a suppression of MMP-2 and MMP-9 in hyperglycemic middle cerebral artery occlusion (MCAO) rats. Rats were pretreated with HBO (100% O2, 2.5atmosphere absolutes) 1h daily for 5days before MCAO. Acute hyperglycemia was induced by an injection of 50% dextrose. Neurological deficits, infarction volume and hemorrhagic volume were assessed 24h and 7days after ischemia. ROS scavenger n-acetyl cysteine (NAC), hypoxia-inducible factor-1α (HIF-1α), inhibitor 2-methoxyestradiol (2ME2) and activator cobalt chloride (CoCl2), and MMP inhibitor SB-3CT were administrated for mechanism study. The activity of MMP-2 and MMP-9, and the expression HIF-1α were measured. HBO-PC improved neurological deficits, and reduced hemorrhagic volume; the expression of HIF-1α was significantly decreased, and the activity of MMP-2 and MMP-9 was reduced by HBO-PC compared with vehicle group. Our results suggested that HBO-PC attenuated HT via decreasing HIF-1α and its downstream MMP-2 and MMP-9 in hyperglycemic MCAO rats.

Sirt1 Mediates Hyperbaric Oxygen Preconditioning-Induced Ischemic Tolerance in Rat Brain

Our previous studies have shown that hyperbaric oxygen preconditioning (HBO-PC) induces tolerance to cerebral ischemia/reperfusion (I/R). This study aimed to investigate whether SirT1, a class III histone deacetylase, is involved in neuroprotection elicited by HBO-PC in animal and cell culture models of ischemia. Rats were subjected to middle cerebral artery occlusion for 120 minutes after HBO-PC (once a day for 5 days). Primary cultured cortical neurons were exposed to 2 hours of HBO-PC after 2 hours of oxygen–glucose deprivation (OGD). We showed that HBO-PC increased SirT1 protein and mRNA expression, promoted neurobehavioral score, reduced infarct volume, and improved morphology at 24 hours and 7 days after cerebral I/R. Neuroprotection of HBO-PC was attenuated by SirT1 inhibitor EX527 and SirT1 knockdown by short interfering RNA (siRNA), whereas it was mimicked by SirT1 activator resveratrol. Furthermore, HBO-PC enhanced SirT1 expression and cell viability and reduced lactate dehydrogenase release 24 hours after OGD/re-oxygenation. The neuroprotective effect of HBO-PC was emulated through upregulating SirT1 and, reversely, attenuated through downregulating SirT1. The modulation of SirT1 was made by adenovirus infection carrying SirT1 or SirT1 siRNA. Besides, SirT1 increased B-cell lymphoma 2 (Bcl-2) expression and decrease cleaved caspase 3. These results indicate that SirT1 mediates HBO-PC-induced tolerance to cerebral I/R through inhibition of apoptosis.

Functional recovery in rat spinal cord injury induced by hyperbaric oxygen preconditioning

Background: It is a common belief that neurosurgical interventions can cause inevitable damage resulting from the procedure itself in surgery especially for intramedullary spinal cord tumors. The present study was designed to examine if hyperbaric oxygen preconditioning (HBO-PC) was neuroprotective against surgical injuries using a rat model of spinal cord injury (SCI).Methods: Sprague–Dawley rats were randomly divided into three groups: HBO-PC group, hypobaric hypoxic preconditioning (HH-PC) control group, and normobaric control group. All groups were subjected to SCI by weight drop device. Rats from each group were examined for neurological behavior and electrophysiological function. Tissue sections were analyzed by using immunohistochemistry, TdT-mediated dUTP-biotin nick end labeling, and axonal tract tracing.Results: Significant neurological deficits were observed after SCI and HBO-PC and HH-PC improved neurological deficits 1 week post-injury. The latencies of motor-evoked potential and somatosensory-evoked potential were significantly delayed after SCI, which was attenuated by HBO-PC and HH-PC. Compared with normobaric control group, pretreatment with HBO and hypobaric hypoxia significantly reduced the number of TdT-mediated dUTP-biotin nick end labeling-positive cells, and increased nestin-positive cells. HBO-PC and HH-PC enhanced axonal growth after SCI.Conclusions: In conclusion, preconditioning with HBO and hypobaric hypoxia can facilitate functional recovery and suppress cell apoptosis after SCI and may prove to be a useful preventive strategy to neurosurgical SCI.

Attenuating brain edema, hippocampal oxidative stress, and cognitive dysfunction in rats using hyperbaric oxygen preconditioning during simulated high-altitude exposure

We assessed whether hyperbaric oxygen preconditioning (HBO2P) in rats induced heat shock protein (HSP)-70 and whether HSP-70 antibody (Ab) preconditioning attenuates high altitude exposure (HAE)-induced brain edema, hippocampal oxidative stress, and cognitive dysfunction. Rats were randomly divided into five groups: the non-HBO2P + non-HAE group, the HBO2P + non-HAE group, the non-HBO2P + HAE group, the HBO2P + HAE group, and the HBO2P + HSP-70 Abs + HAE group. The HBO2P groups were given 100% O2 at 2.0 absolute atmospheres for 1 hour per day for 5 consecutive days. The HAE groups were exposed to simulated HAE (9.7% O2 at 0.47 absolute atmospheres of 6,000 m) in a hypobaric chamber for 3 days. Polyclonal rabbit anti-mouse HSP-70-neutralizing Abs were intravenously injected 24 hours before the HAE experiments. Immediately after returning to normal atmosphere, the rats were given cognitive performance tests, overdosed with a general anesthetic, and then their brains were excised en bloc for water content measurements and biochemical evaluation and analysis. Non-HBO2P group rats displayed cognitive deficits, brain edema, and hippocampal oxidative stress (evidenced by increased toxic oxidizing radicals [e.g., nitric oxide metabolites and hydroxyl radicals], increased pro-oxidant enzymes [e.g., malondialdehyde and oxidized glutathione] but decreased antioxidant enzymes [e.g., reduced glutathione, glutathione peroxide, glutathione reductase, and superoxide dismutase]) in HAE. HBO2P induced HSP-70 overexpression in the hippocampus and significantly attenuated HAE-induced brain edema, cognitive deficits, and hippocampal oxidative stress. The beneficial effects of HBO2P were significantly reduced by HSP-70 Ab preconditioning. Our results suggest that high-altitude cerebral edema, cognitive deficit, and hippocampal oxidative stress can be prevented by HSP-70-mediated HBO2P in rats.

Hyperbaric oxygen preconditioning protects cortical neurons against oxygen-glucose deprivation injury: Role of peroxisome proliferator-activated receptor-gamma

Ischemic stroke is one of the leading causes of mortality and disability worldwide. Our previous studies have shown that hyperbaric oxygen (HBO) preconditioning can afford significant neuroprotection against cerebral ischemia–reperfusion (I/R) injury in rats. However, it is still unknown whether HBO preconditioning can directly protect primary cultured cortical neurons against oxygen-glucose deprivation (OGD). Peroxisome proliferator-activated receptor-gamma (PPAR γ) plays a central role in the regulation of apoptosis, oxidative stress and inflammation as well as affords significant neuroprotection against cerebral I/R injury. 15-deoxy-∆12,14-prostaglandin J2 (15d-PGJ2) is an endogenous ligand with a high affinity for PPAR γ. Recently, some studies demonstrate that activation of PPAR γ mediates lipopolysaccharide and anesthetic preconditioning. In the present study, we firstly found that OGD exposure caused the significant damage of cultured cortical neurons evaluated by cell viability, lactate dehydrogenase (LDH) release and caspase-3 activity, which were significantly ameliorated by HBO preconditioning. Furthermore, HBO preconditioning significantly increased the levels of PPAR γ mRNA and protein, PPAR γ DNA binding activity, 15d-PGJ2 and antioxidant enzymatic activities in primary cultured cortical neurons with OGD exposure. Moreover, PPAR γ antagonist GW9662 dose-dependently abolished the protection of HBO preconditioning in OGD-exposed neurons. GW9662 blocked the increase of PPAR γ DNA binding activity and antioxidant enzymatic activities, but did not influence the 15d-PGJ2 level in OGD-exposed neurons with HBO preconditioning. However, the cyclooxygenase (COX)-2 inhibitor NS-398 blocked the production of 15d-PGJ2 in OGD-exposed neurons with HBO preconditioning. In addition, 15d-PGJ2 preconditioning could also protect cultured neurons against OGD injury. These results demonstrate that HBO preconditioning has directly beneficial effects on ODG-exposed cortical neurons by the activation of PPAR γ subsequent to the production of 15d-PGJ2, which in turn increases the downstream antioxidant enzymatic activities.

Hyperbaric oxygen preconditioning attenuates hyperglycemia enhanced hemorrhagic transformation after transient MCAO in rats

BackgroundHemorrhagic transformation (HT) can be a devastating complication of ischemic stroke. Hyperbaric oxygen preconditioning (HBO-PC) has been shown to improve blood-brain barrier (BBB) permeability in stroke models. The purpose of this study is to examine whether HBO-PC attenuates HT after focal cerebral ischemia, and to investigate whether the mechanism of HBO-PC against HT includes up-regulation of antioxidants in hyperglycemic rats.MethodsMale Sprague-Dawley rats (280-320 g) were divided into the following groups: sham, middle cerebral artery occlusion (MCAO) for 2 h, and MCAO treated with HBO-PC. HBO-PC was conducted giving 100% oxygen at 2.5 atm absolute (ATA), for 1 h at every 24 h interval for 5 days. At 24 h after the last session of HBO-PC, rats received an injection of 50% glucose (6 ml/kg intraperitoneally) and were subjected to MCAO 15 min later. At 24 h after MCAO, neurological behavior tests, infarct volume, blood-brain barrier permeability, and hemoglobin content were measured to evaluate the effect of HBO-PC. Western blot analysis of nuclear factor erythroid 2-related factor 2 (Nrf2) and heme oxygenase-1 (HO-1) was evaluated at multiple time-points before and after MCAO.ResultsHBO-PC improved neurological behavior test, and reduced infarction volume, HT and Evans blue extravasation in the ipsilateral hemisphere at 24 h after MCAO. Western blot analysis failed to demonstrate up-regulation of Nrf2 in HBO-PC group before and after MCAO. Paradoxically, HBO-PC decreased HO-1 expression at 24 h after MCAO, as compared with htMCAO group.ConclusionsHBO-PC improved neurological deficits, infarction volume, BBB disruption, and HT after focal cerebral ischemia. However, its mechanism against focal cerebral ischemia and HT may not include activation of Nrf2 and subsequent HO-1 expression.

Heme oxygenase-1 could mediate the protective effects of hyperbaric oxygen preconditioning against hepatic ischemia-reperfusion injury in rats

1. Heme oxygenase 1 (HO-1) has been shown to play a pivotal role in the maintenance of cellular homeostasis when the liver undergoes sublethal stress, such as ischaemia-reperfusion (I/R) injury. In the present study, we investigated the protective role of HO-1 in hyperbaric oxygen (HBO) preconditioning against liver injury after I/R. 2. A total hepatic ischaemia (30 min) and reperfusion (60 min) injury model in rats was used in the present study. Preconditioned groups were exposed to HBO 24 h prior to the induction of I/R injury. Other groups were injected with zinc protoporphyrin IX (ZnPP) intraperitoneally 1 h before I/R to inhibit HO-1 activity. At the end of the reperfusion period, blood and liver samples were collected for the analysis of liver injury markers, morphological changes, and HO-1 expression and activity in the liver. 3. In untreated rats, I/R induced an increase in hepatic injury markers, such as plasma transaminases, inflammatory cytokines (tumour necrosis factor-α and interleukin-1β), and tissue malondialdehyde. However, HBO preconditioning attenuated the I/R-induced increases in these hepatic injury markers, and prevented both the necrosis and apoptosis of hepatocytes induced by I/R injury. Furthermore, HBO preconditioning significantly increased HO-1 mRNA and protein levels in the liver. In rats in which HO-1 activity had been inhibited with ZnPP pretreatment, the protective effects of HBO preconditioning against I/R injury were abolished. 4. In conclusion, HBO preconditioning can protect the liver against I/R injury and it appears that this effect might be mediated by the induction of HO-1.

Reducing Pulmonary Injury by Hyperbaric Oxygen Preconditioning During Simulated High Altitude Exposure in Rats

Hyperbaric oxygen preconditioning (HBO₂P + HAE) has been found to be beneficial in preventing the occurrence of ischemic damage to brain, spinal cord, heart, and liver in several disease models. In addition, pulmonary inflammation and edema are associated with a marked reduction in the expression levels of both aquaporin (AQP) 1 and AQP5 in the lung. Here, the aims of this study are first to ascertain whether acute lung injury can be induced by simulated high altitude in rats and second to assess whether HBO2P + HAE is able to prevent the occurrence of the proposed high altitude-induced ALI. Rats were randomly divided into the following three groups: the normobaric air (NBA; 21% O₂ at 1 ATA) group, the HBO₂P + high altitude exposure (HAE) group, and the NBA + HAE group. In HBO₂P + HAE group, animals received 100% O₂ at 2.0 ATA for 1 hour per day, for five consecutive days. In HAE groups, animals were exposed to a simulated HAE of 6,000 m in a hypobaric chamber for 24 hours. Right after being taken out to the ambient, animals were anesthetized generally and killed and thoroughly exsanguinated before their lungs were excised en bloc. The lungs were used for both histologic and molecular evaluation and analysis. In NBA + HAE group, the animals displayed higher scores of alveolar edema, neutrophil infiltration, and hemorrhage compared with those of NBA controls. In contrast, the levels of both AQP1 and AQP5 proteins and mRNA expression in the lung in the NBA + HAE group were significantly lower than those of NBA controls. However, the increased lung injury scores and the decreased levels of both AQP1 and AQP5 proteins and mRNA expression in the lung caused by HAE was significantly reduced by HBO₂P + HAE. Our results suggest that high altitude pulmonary injury may be prevented by HBO2P + HAE in rats.

Preconditioning with Hyperbaric Oxygen Induces Tolerance Against Renal Ischemia-Reperfusion Injury Via Increased Expression of Heme Oxygenase-1

Renal ischemia/reperfusion (I/R) injury occurs in both native and transplanted kidneys. Hyperbaric oxygen (HBO) has been shown to prevent I/R injury in different tissues. The aim of this study was to evaluate the effect of HBO on renal I/R injury in rats. Male Sprague-Dawley rats were randomly assigned to three groups. The sham group (n = 8) received right nephrectomy. The I/R (n= 8) and HBO + I/R groups (n = 8) received 45 min left renal ischemia followed by 24 h of reperfusion after right nephrectomy. The HBO + I/R group (n = 8) received 100% oxygen at 2.5 atmosphere absolute (ATA), for 1 h at every 12 h interval for 2 d. Reperfusion was performed 24 h later after the last HBO exposure. In HBO + I/R group, blood urea nitrogen (BUN) and creatinine levels decreased significantly compared with the sham and I/R groups (P < 0.01). Activities of superoxide dismutase (SOD) were increased in renal tissue in the HBO + I/R groups. The content of malondialdehyde (MDA) were decreased in the HBO + I/R groups. Kidney samples from HBO + I/R group rats revealed markedly reduced histological damage under histopathological examination. The animals treated with HBO showed significantly elevated heme oxygenase-1 (HO-1) protein and mRNA levels expression compared with I/R group (P < 0.05). Hyperbaric oxygen preconditioning (HBO-PC) can protect renal I/R injury against oxidative stress, and the up-regulation of HO-1 expression plays an essential role in HBO induced preconditioning effect.

Effect of repeated hyperbaric oxygen preconditioning on expression of hypoxia-inducible factor-1 alpha and erythropoietin following spinal ischemia-reperfusion in rats

Objective To investigate the effect of repeated hyperbaric oxygen preconditioning (HOP) on the expression of hypoxia-inducible factor-1 alpha (HIF-1a) and erythropoietin (EPO) following spinal ischemiareperfusion (I/R) in rats. Methods Forty-eight male SD rats weighing 250-300 g were randomly divided into 3 groups (n = 16 each): sham operation group (S group); I/R group and repeated HOP group (HOP group). The animals in HOP group were pretreated with O2 ( ＞ 98 % ) at 2.5 ATA I h/d for 5 consecutive days at 24 h before spinal cord I/R. The animals were anesthetized with intraperitoneal pentobarbital sodium 35 mg/kg. Spinal cord ischemia was produced by cross-clamping of abdominal aorta distal to renal artery for 20 min in I/R and HOP groups. Hind-limb motor function was assessed and scored st 48 h of reperfusion. The animals were then sacrificed and the L5-7 segment of the spinal cord was removed for determination of the expression of HIF-1 α and EPO mRNA and protein and microscopic examination. Results Compared with group S, the hind-limb motor function scores and the number of normal motor neurons in the anterior horn of the spinal cord were significantly decreased, and the expression of HIF-1α and EPO mRNA and protein was up-regulated in I/R and HOP groups ( P ＜ 0.05). Compared with group I/R, the hind-limb motor function scores and the number of normal motor neurons in the anterior horn of the spinal cord were significantly increased, and the expression of HIF-1α and EPO mRNA and protein was up-regulated in group HOP ( P ＜ 0.05). The spinal cord injury was attenuated in group HOP compared with group I/R. Conclusion Repeated HOP can reduce the spinal cord I/R injury though up-regulating the expression of HIF1α and EPO in rats. Key words: Hyperbaric oxygenation; Ischemic preconditioning; Hypoxia-inducible factor 1, alpha subunit; Erythropoietin; Reperfusion injury; Spinal cord

Autophagy activation is involved in neuroprotection induced by hyperbaric oxygen preconditioning against focal cerebral ischemia in rats

Our previous studies have demonstrated that hyperbaric oxygen (HBO) preconditioning induces tolerance to focal cerebral ischemia. The present study aimed to investigate whether autophagy is involved in the neuroprotection elicited by HBO preconditioning in a rat model of transient focal cerebral ischemia. Twenty-four hours after the completion of HBO preconditioning (2.5 atm absolute in 100% oxygen for 60 min per day for 5 consecutive days), male Sprague–Dawley rats were subjected to focal cerebral ischemia by middle cerebral artery occlusion (MCAO) for 120 min. The neurobehavioral score and infarct volume were used to evaluate cerebral ischemic injury. An intracerebroventricular injection of the autophagy inhibitor 3-methyladenine (3-MA) or the autophagy inducer rapamycin was administered before HBO preconditioning or MCAO. We found that after reperfusion the protein expression of LC3-II and Beclin 1 and the formation of autophagosomes were increased by HBO preconditioning or ischemia, but the increase following HBO preconditioning was higher than the increase following ischemia. 3-MA suppressed the increases in LC3-II and Beclin 1 induced by HBO preconditioning and attenuated the neuroprotection of HBO preconditioning against cerebral ischemia. Furthermore, 3-MA treatment before MCAO aggravated subsequent cerebral ischemic injury. In contrast, pretreatment with rapamycin up-regulated LC3-II and Beclin 1 after reperfusion and mimicked the neuroprotective effect of HBO preconditioning. These results indicate that HBO preconditioning elevates autophagic activity, which elicits a neuroprotective effect against ischemic injury in the brain, and suggest a novel mechanism of HBO preconditioning-induced tolerance against transient focal cerebral ischemia.

Cyclooxygenase-2 Mediates Hyperbaric Oxygen Preconditioning in the Rat Model of Transient Global Cerebral Ischemia

Hyperbaric oxygen (HBO) preconditioning (PC) allows brain protection against transient global ischemia. In the present study, we hypothesize that the mechanism of HBO-PC involves the induction of cyclooxygenase-2 (COX-2) in cerebral tissues before ischemia, which leads to a suppression of COX-2 and its downstream targets after global ischemic insult. One hundred twenty-nine male Sprague Dawley rats (body weight 280-300 grams) were allocated to the naive control group and the sham operation group, and 3 groups of animals were subjected to 15-minute 4-vessel occlusion: untreated, preconditioned with HBO 2.5 atmospheres absolutes for 1 hour daily for 5 days, preconditioned as mentioned and administered with COX-2 inhibitor NS-398 (1 mg/kg body weight intraperitoneal) before each preconditioning session, and normal rats preconditioned with HBO without ischemia. The mortality, the incidence of seizures, and T-maze scores were recorded. The quantitative cell count in Nissl stain and TUNEL was conducted on day 7 after ischemia. The brain expression of COX-2 was analyzed with Western blotting and immunofluorescence staining. HBO-PC increased the number of surviving neurons in the Cornu Ammonis area 1, which was associated with the reduced COX-2 expression in the hippocampus and in the cerebral cortex at 1 and 3 days after ischemia. HBO-PC improved functional performance and tended to decrease mortality and the frequency of seizures. These beneficial effects of HBO-PC were abolished by the COX-2 selective inhibitor NS-398. HBO-PC reduced COX-2 expression and provided brain protection after global ischemia. Administration of COX-2 inhibitor with HBO before ischemia abolished preconditioning effect, thereby implicating COX-2 as a mediator of HBO-PC in the ischemic brain.