Ornithine decarboxylase activity in vitro in response to acute hypoxia: a novel use of newborn rat brain slices.

Abstract

In fetal as well as newborn rats, acute hypoxic exposure results in significantly elevated brain ornithine decarboxylase (ODC) activity, polyamine concentrations, and ODC mRNA. The interpretations of these in vivo hypoxic-induced changes, however, are complicated by maternal confounding effects. To test the hypothesis that acute hypoxia will also increase ODC activity in vitro, we developed a brain slice preparation which eliminates such maternal effects. Sections of whole cerebrum, approximately 300-500 microns thick, were made from 3- to 4-day old Sprague-Dawley rat pups. The slices were equilibrated for 1 h in artificial cerebrospinal fluid (ACSF) continuously bubbled with 95% O2/5% CO2, prior to induction of hypoxia. We induced hypoxia by changing the oxygen concentration to 40%, 30%, 21%, 15%, 10%, or 0% O2, all with 5% CO2 and balance N2. In the normoxic control brain slices, low but stable basal ODC activity persisted for up to 5 h post-sacrifice. Slices in ACSF treated with bovine serum albumin (BSA), or both BSA and fetal bovine serum (FBS), however, showed stable ODC activity values 2- to 3-fold higher than slices in ACSF alone, for up to 5 h. In response to acute hypoxia (i.e., 15, 21, and 30% O2), ODC activity was elevated 1.5- to 2-fold above control values between 1 and 2 h after initiation of hypoxia. Qualitative light and electron microscopic examination of the neonatal brain slices following 2 h hypoxic exposure suggested that the great majority of cells did not show severe hypoxic damage or necrosis. It was concluded that: (1) in neonatal rat brain slices in vitro, stable ODC activity values approximating the whole brain ODC activity seen at sacrifice, can be maintained for several hours; (2) the in vivo hypoxic-induced increase in ODC activity can be approximated in vitro; (3) the neonatal rat brain slice preparation may be an alternative to other methods for studying hypoxic-induced ODC enzyme kinetics, or other brain enzymes, without maternal confounding effects; and (4) ODC activity may be an indicator of active metabolism within the newborn brain slice both in normoxia and hypoxia.