Isoflurane Preconditioning Protects Gerbil Brain Against Ischemia-reperfusion By A Mechanism Involving Mitochondria

IntroductionPreconditioning (PC) as endogenous protective phenomenon has been more and more paid attention to. Because ischemic preconditioning (IPC) is a stress and injury to the body, so its clinical use has been restrict, and pharmacological PC in place of IPC may represent a safer way of eliciting protection and possess important clinical role.Since two anaesthesia research groups (Cason and Kersten) independently described the preconditioning-mimicking effects of isoflurane for the first time in 1997, subsequent extensive experimental work aimed at elucidating the complex signaling cascade and mechanisms involved in anaesthetic-induced preconditioning. However the exact mechanism has not been clear. Recently studies have showed that the mechanisms of anaesthetic-induced preconditioning ( APC) is same as IPC, namely mitochondria adenosine triphosphate sensitive potassium channel ( mitoK_ATP) has been thought as a target of APC. Recent attentions have showed that cardiac protection by volatile anesthetic-induced precondition is related to the activation of the mitoK_ATP.There are more mitoK_ATP (3-7 times of cardiac cell) in neuronal cell. Recent attentions have showed that volatile anesthetic preconditioning (VAPC) can inhibit platelet aggregate and neutrophil adherence. However the physiologic signals that activate mitoK_ATP and the mechanisms whereby activation of mitoK_ATP protects neurons against ischemic injury are unknown, so the mechanisms of VAPC on brain ischemia will be still studied.Mitochondria is an important organelle which possesses complicated construction with impressionable function. Recent reports have strongly suggested that mitochondrial Ca + overload is the major reason of ischemia-mediated irreversible cellular damage. Mitochondria permeability transition pore (MPTP) is known as involvement of neuronal injury and may be a key step in brain cell damage after ischemic-reperfusion (I/R). Mitochondria plays a critical role in many forms of programmed cell death and triggers apoptosis. Recent studies have clearly established that apoptosis may enhance brain damage after I/R. Antiapoptotic genes such as Bcl-2 can prevent excess expression of apoptotic proteins and reduce cerebral ischemic injury. In turn, decreasing the amount of Bcl-2 may increase brain damage. When proapoptotic gene Bax associates to MPTP, MPTP opens and releases cytochrome c and apotosis inducing factor. Bcl-2 can prevent Bax translocation to MPTP and block apoptosic pathway.In order to determine that the protect effect of isoflurane precondition on gerbil brain against I/R is via apoptosic pathway involved in mitochondrial, this research consists of the following three parts: first, the effect of isoflurane precondition on the mitochondrial ultrastructure and MPTP in gerbil brain against 1/ R;second, the effect of isoflurane preconditioning on the expression of Bcl-2 and Bax mRNA in gerbil brain afterl/R;third, to determine signaling pathway of nitric oxide involved in isoflurane precondition in gerbil brain against I/R.Materials and MethodsMaterials1. Animals;99 healthy male Mongolian gerbils weighting 55-85g were used in this study.2. Main Reagents;Isoflurane (ABBOTT), 5-HD, AG, Fura-2/AM, EGTA ( Sigma) , Bovine serum albumin ( Beijing Hongxing Biochemical) , Takala kit (Dalian Baosheng) , DNA RT-PCR marker ( Dalian Bonder) , Primer, (3-actin (Shanghai Boya).3. Main Apparatus;Datex multiple functional gas monitor (Capnomac Ultima, Finland) , 850-fluorescence spectrophotometer (Japan) , UV-1601 Shimad-zu (Japan) , DF15D high speed cool centrifuge (Japan) , JEM-1200EX transmission electron microscope ( U. S. A) , LKB-V ultramicrotome, KODAK ID gel imaging analytical system ( U. S. A) , PTR-100 PCR amplication device ( U. S. A).Methods1. Isoflurane preconditioning:The gerbils were put in the experiment cabin of 100cm x 100cm X 100cm in which put sodium and isoflurane. The concentration of isoflurane in the cabin was maintained at 1. 2% 1.5% ( end-tidal concentration).2. Brain I/R model:After gerbils were anesthetized with chloral-hydrate 0. 35g kg"1 intraperito-neally ( i. p. ) , the bilateral common carotid arteries were carefully exposed through a ventral midline incision in the neck, and occluded with a aneurismal clip for 5 min, and 24 h after recirculation the forebrain tissues were rapidly removed and cooled in iced saline.3. Experimental designs;SHAM group;namely control group, the bilateral common carotid arteries were exposed, but were not occluded.IR group: subjected to transient forebrain ischemia, the bilateral common carotid arteries were occluded for 5 min and 24h after recirculation.ISO group: 30 min of isoflurane inhalation 1.2% -1.5% followed a 30 min washout period in air before I/R.5-HD + ISO group: received 5-HD ( specific bloker of mitoKATP) 10 mg kg'i. p. and isoflurane inhalation before I/R.5-HD group: 5-HD was given (i. p. ) 30 min before I/R.AG + ISO group: AG (bloker of iNOS) 200mg kg"1 was given (i. p. ) and 30 min later same as ISO group.AG group: AG was given (i. p. ) 30 min before I/R.4. Observation of parameters and method:4. 1 Preparation of isolated mitochondria;as previously described procedure , the mitochondrial pellet was suspended in isolation buffer.4.2 The changes of mitochondrial ultrastructure were observed by electronmicroscopy.4.3 Measurement of mitochondria Ca2+ : According to the Mccormack improvement method, Fura-2/Am as the fluorescence indicator of Ca +, the concentration of Ca + was obtained by using fluorescence photometer at 510 nm, and scan stirring up the spectrum at 430 nm.4.4 Measurement of MPTP;MPTP was detected as an absorbance change of the mitochondria suspension at 540 nm. The protein concentration of mitochondria was 0. 5 mg ml"1. The changes of light absorbance were recorded at 540 nm.4.5 Bel-2 and Bax mRNA involving apoptosis were expressed in brain with RT-PCR.Results1. The effect of isoflurane precondition on the mitochondrial ultrastructure and MPTP in gerbil brain against I/R.1.1 The changes of mitochondrial ultrastructure: The mitochondria massive swelling with disrupted cristae and damaged matrix in IR, 5-HD and 5-HD + ISO groups, in contrast, compact mitochondria in SHAM and ISO groups were displayed.1. 2 The changes of mitochondrial calcium content: Ca + concentration were significantly decreased in SHAM and ISO groups than that in IR group (P <0.01). The mitochondrial Ca + concentration increased in 5-HD + ISO group than that in ISO group ( P < 0. 01). These results showed that isoflurane-in-duced preconditioning can inhibit increasing of Ca2 + concentration in IR group, and 5-HD abolished the effect of isoflurane preconditioning.1. 3 The changes of MPTP opening;MPTP were less opened in SHAM and ISO groups than that in IR group (P <0. Ol). MPTP were more opened in 5-HD+ ISO group than that in ISO group ( P < 0. 01). Statistical significance of the changes above between ISO and SHAM groups was not observed, and there was no statistical significance between IR and 5-HD groups ( P >0.05).2. The effect of isoflurane preconditioning on the expression of Bcl-2 andBax mRNA in gerbil brain after I/R2. 1 Electrophoresis imaging of Bel-2 and Bax: The special expression zone of Bcl-2 were independently observed at 367bp in ISO, IR and 5-HD + ISO groups, but markedly showed in ISO group, and not observed in SHAM and 5-HD groups. The special expression zone of Bax were independently observed at 194bp in ISO, IR, 5-HD + ISO and 5-HD groups, but weakly showed in ISO group, and not observed in SHAM group.2.2 The expression of Bcl-2 and Bax: The mRNA expression of Bcl-2 in ISO group was markedly increased compared with those in SHAM, IR and 5-HD + ISO"groups ( P < 0. 05). The mRNA expressions of Bax in IR, 5-HD + ISO and 5-HD were markedly increased compared with that in SHAM (P <0. 05). There was no statistical significance between IR and 5-HD groups ( P >0.05).2. 3 Bcl-2/Bax: The values of Bcl-2/Bax were markedly lower in IR, 5-HD + ISO and 5-HD groups compared with that in ISO group ( P <0. 01).3. To determine the signaling pathway of NO involved in isoflurane preconditioning in gerbil brain against I/R3. 1 The changes of mitochondrial ultrastructure: We observed large swollen mitochondria with outer membranes appearing ruptured and damaged matrix in IR and AG groups, in contrast, compact mitochondria were displayed in SHAM and ISO groups, and both swollen and compact mitochondria were appeared in AG + ISO group.3. 2 The changes of mitochondrial calcium content: Ca2 + concentration were significantly increased in IR and AG groups compared with those in SHAM, ISO and ISO + AG groups ( P <0. 01) , but there were no significant difference between ISO and ISO + AG groups ( P > 0. 05 ). This result showed that use of AG did not completely abolish the effect of isoflurane preconditioning.3. 3 The changes of AS (MPTP opening) : AS were markedly decreased in IR and AG groups compared with those in SHAM and ISO groups (P <0.01) , and AS were markedly decreased in ISO + AG group compared with that in ISO group (P<0.05).3.4 Electrophoresis imaging of Bcl-2 and Bax: The special expression zoneof Bcl-2 were independently observed in SHAM and ISO groups, but markedly showed in ISO group, and not observed in IR, AG + ISO and AG groups. The special expression zone of Bax were independently observed in ISO, IR, AG + ISO and AG groups, but weakly showed in ISO and AG + ISO groups, and not observed in SHAM group.3.5 The expression of Bcl-2 and Bax: The expression of Bcl-2 mRNA in ISO group was markedly increased compared with those in SHAM, IR and AG + ISO groups ( P < 0. 05). The expression of Bax mRN A in ISO showed decreasing tendence compared with other groups, but there was no significant difference (P > 0. 05 ). The expression of Bax mRN A in IR and AG groups were markedly increased compared with that in SHAM ( P <0.05).Conclusion1. Isoflurane preconditioning can decrease mitochondria calcium overload in the gerbil brain after ischemia-reperfusion.2. Isoflurane preconditioning is mediated by the inhibilation of MPTP opening in the gerbil brain after ischemia-reperfusion.3. Isoflurane preconditioning may be relation to upregulation of Bcl-2 and downregulation of Bax in gerbil brain after ischemia-reperfusion.4. The protective effect of isoflurane preconditioning on gerbil brain after ischemia-reperfusion and upregulation of Bcl-2 and downregulation of Bax may be because of inhibilation of MPTP opening by activation of mitoKATP.5. The signaling pathway of NO was involved in the protective effect of isoflurane preconditioning on gerbil brain against ischemia-repeifusion.