Ischaemia-induced damage to mitochondrial complexes in the neonatal brain--role of NO.

Abstract

Ischaemia can cause irrevekble damage to brain tissue but exactly how this occurs is unclear at the present time. There is however increasing evidence which suggests that disruption of mitochondrial function may take place during recovery (1). Previously we have shown that there is incomplete recovery of the energy state of superfused neonatal cortical brain slices after a 30 minute period of glucoseand oxygen-free conditions (aglycaemic hypoxia)(AH) which is accompanied by elevated intracellular lactate levels indicating damage to the cerebral energy producing systems in the short term (2). Attempts to prevent ischaemic damage in neonatal brain have included the use of nitric oxide synthase inhibitors. It has been reported that N nitro-L-arginine (L-NNA) protected against ischaemia-induced brain damage in the 7 day old rat (3). Whilst N nitro-L-arginine methyl ester (L-NAME) showed a significant reduction in infarct volume after ischaemia in a rat pup model (4). We therefore decided to investigate the effect of AH on the mitochondrial complex activities in cortical slices prepared from 10 day old rat pups and monitor any changes during the recovery period. Furthermore the effect of conditions shown by nuclear magnetic resonance to be beneficial for the recovery of the energy state after AH (2) were measured together with the effect of LNNA. The neonatal cortical brain slices were prepared and rapidly placed in oxygenated glucose-containing medium at 37 OC as previously described (2). The tissues were then superfused in a custom-built apparatus described by Bachelard et al. (5 ) . A third of the slices were removed 1) after 30 minutes under control conditions 2) after 30 minutes AH and 3) after 30 minutes recovery from AH. They were then washed in ice cold isolation medium consisting of 320 mM sucrose, 1 mM EDTA and 10 mM TRIS-HCI, pH 7.4 and then homogenised in 1 ml of this medium. The homogenates were placed in an Eppendorf tube, frozen in liquid nitrogen and stored at -70 OC. In some experiments a) no calcium was added (external calcium = 10 pM) (low calcium), b) external magnesium increased from 1.2 to 10 mM (high magnesium), c) 25 pM diltiazem or d) 25 pM LNNA added, both prior to and during AH. The activities of complex I, 11-III, IV and citrate synthase and the protein concentration were measured in the homogenates after thawing and freeze-thawing a further two times. There was no significant effect on complex I activity by AH or upon recovery. This was the same for low calcium, high magnesium, diltiazem and L-NNA conditions. In the case of complex 11-111 there was no significant change in activity during AH but after the recovery period there was a significant decrease under control conditions to approximately 80% of the control level (Fig. I) . The same results were found with low calcium, high magnesium, and diltiazem. However in the presence of L-NNA there was no significant decrease in complex 11-III activity after the recovery period (Fig.1). When the activity of complex IV was measured during AH there was no significant difference from control values however after the recovery period there was a significant decrease in activity to approximately 80% of the control level (Fig.2). The same results were found with low calcium, high magnesium, and diltiazem. However in the presence of L-NNA there was no decrease in complex IV activity during the recovery period (Fig.2). There was no significant change in the activity of citrate synthase indicating that the mitochondria remained intact following the ischaemic episode. These results show that when glucose and oxygen are drastically reduced in the external medium of neonatal cortical brain slices, for 30 minutes, a significant decrease in complex 11-111 and IV activity of the mitochondria can be detected 30 minutes after restor Nomud Low High DilliaTem L-NNA . = Control CPlaYm magrwium