Adenosine receptors in the immature brain : with special reference to their role in hypoxic ischemia

Abstract

Although the newborn brain tolerates a much longer period of oxygen deprivation and ischemia than does the adult brain, perinatal hypoxic ischemia probably is an important cause of neurological dysfunction, cerebral palsy and epilepsy later in life. Hence it is important to investigate the mechanisms that modulate the extent of perinatal ischemic brain damage.

There is good evidence that endogenous adenosine acts as a neuroprotective agent in models of ischemia in the mature brain. Adenosine activates receptors of four subtypes - A1 A2A, A2B and A3 - each having a distinct pattern of distribution in the brain. Adenosine is present at low levels in the extracellular fluid in the brain under physiological conditions, but increases dramatically during metabolic stress such as ischemia. Adenosine A, receptors play an important role in protecting the mature brain from ischemic injury, but the role of this and other adenosine receptor subtypes in the immature brain remains to be elucidated.

Adenosine A1, and A2A receptors were present in the immature rat brain already before birth. Furthermore, A1, and A2A receptors in the brain (as well as their respective mRNAs) were down-regulated within 1-2 h after combination of unilateral common carotid artery occlusion and timed exposure to 8% oxygen, possibly secondarily to an increase in adenosine levels.

Newborn human babies are sometimes exposed to caffeine and theophylline, which are well-known adenosine receptor antagonists. Since adenosine is thought to be neuroprotective, we hypothesized that these substances then could aggravate an ischemic brain damage.

Surprisingly, we found that brain damage was clearly reduced in rat pups whose mothers received a low dose of caffeine in the drinking water during pregnancy and two weeks after delivery. This was not due to a compensatory upregulation of A1, A2A or GABAA receptors.

Furthermore, a high dose of theophylline (given directly before hypoxic ischemia resulted in a strikingly reduced brain damage. Also in this study, there were no changes in A1, and A2A receptors or mRNA.

To examine the specific role of A1 receptors in the immature brain, we studied the effects of the selective A, antagonist DPCPX, and the A, agonist ADAC given to 7-day-old rats before or after hypoxic ischemia. None of these treatments affected the size of the brain damage, indicating that A, receptors are not critically important during hypoxic ischemia in the immature rat brain. A possible explanation for the difference in this respect between the adult and immature brain is that the A, receptors are not functionally coupled in brains of 7-day-old rats, a theory supported by our study using GTPgammaS-binding. By contrast, A1 agonist had a profound effect on heart rate in these animals, thus confirming that the A, receptors in the heart are functionally coupled.

The role of A2A receptors was examined using the specific antagonist SCH 58261 and A2A knockout mice. The A2A antagonist had a weak protective effect when given 30 min after hypoxic ischemia in a low dose to 7-day-old rats, but not when given in a higher dose or before hypoxia. Conversely, A2A knockout mice of the same age clearly displayed a more severe brain damage than wild type mice after hypoxic ischemia. Thus, it may be that a partial A2A receptor inactivation provides some protection, whereas complete inactivation decreases neuronal survival. We also showed that the increase in size of the ischemic brain injury had long term functional consequences in adult life, such as altered locomotor activity and impaired performance in sensorimotor tests.

Using magnetic resonance imaging (MRI), we found a distinct lesion, as judged by MRI changes, at 3 hours after hypoxic ischemia a timepoint well within the therapeutic interval for neuroprotective intervention. In contrast to previous studies in the mature mouse brain, the maximal area of lesion was attained already at 3-6 h after hypoxic ischemia and then declined over the first 5 days. In some animals, transient MRI changes were also seen on the contralateral side and c-fos mRNA was also induced contralaterally within an hour after hypoxic ischemia in rats. The animals that were found to have early changes using MRI also showed long term behavioral changes.

We conclude that adenosine has important effects during ischemia in the immature brain and that A2A receptors are more important than A, receptors in mediating these effects. Our MRI studies suggest that early morphological changes may be related to long term functional consequences.