ELSEVIER Brain Research 725 (1996) 184-191 BRAIN RESEARCH Research report The effects in vivo of hypoxia on brain injury Paul Pearigen, Ryder Gwinn, Roger P. Simon * Department of Neurology, Unit'ersity c)[ Cali~brnia, San Francisco, USA Accepted 7 February 1996 Abstract To separately analyze the hypoxic component of hypoxic-ischemic encephalopathy, rats were prepared such that their paO2 was maintained at 20 mmHg while maintaining systemic arterial pressures. During the 20-rain experiment, brain oxygen concentration and extracellular amino acid concentrations were monitored. At sacrifice, the brains were studied for morphologic evidence of injury by immunocytochemical staining for the non-constituitive stress protein HSP-72 or neuronal death by acid fuchsin staining. Oxygenated rats subjected to global ischemia were prepared for comparison. In these experiments, hypoxia resulted in no increase in extracellular glutamate concentration, and no morphologic injury was detected. Thus, hypoxia without ischemia is well tolerated by brain. Keywords: Hypoxia; Heat shock protein; Hypoxia-ischemia; Glutamate 1. Introduction It is a clinical axiom that removal of oxygen from the brain will result in the death of that organ. However, the mechanism by which such brain injury occurs is not quite as clear in the experimental literature. In vivo, hypoxic injury to the brain almost always occurs in the setting of ischemia; what portion of the injury results from oxygen deprivation as opposed to loss of blood flow may be difficult to establish in the intact organism. Clinically, this distinction is usually ignored and the terms hypoxic en- cephalopathy and ischemic encephalopathy are generally used interchangeably. To best order interventions during critical care situations, the relative roles of hypoxia and ischemia in 'hypoxic-ischemic encephalopathy' should be defined. The pathogenesis of hypoxic-ischemic injury to the brain has been clarified over the last decade [6]. Injury begins with elevation of glutamate concentrations in the extracellular compartment, which opens voltage-gated and receptor-operated calcium channels. The resultant intra- cellular calcium toxicity induces catabolic processes within the cell, ultimately resulting in cell death. Early and subtle injury can be detected immunocytochemically by the use of antibodies to nonconstitutive heatshock proteins (HSP) * Corresponding author. Present address: Department of Neurology, University of Pittsburgh Medical School 325 Scaife Hall, Pittsburgh, PA 15213, USA. Fax: (1) (412) 648-1239. induced in nervous system cells by a wide range of stress, including seizures and ischemia [12,16,17]. HSP induction represents a response to the presence of denatured protein in the cell [1]. The presence of denatured proteins activate heatshock factors [18] which bind to heat shock elements, resulting in the transcription of HSP RNA [19,20]. Neu- rons which imunocytochemically stain with HSP antibody therefore contain denatured protein as evidence of injury. We used the techniques of in vivo microdialysis [10] and heat shock protein immunocytochemistry to examine the role of pure hypoxia, in the absence of ischemia, in brain injury. Acid fuchsin staining was used to identify dead cells. 2. Materials and methods 2.1. Animal preparation 2.1.1. Ventilation and physiologic monitoring Adult male Sprague-Dawley rats (325-375 g) were intubated, placed on a homeothermic blanket to maintain rectal temperature at 36.5-37.5°C, and mechanically venti- lated using a Harvard small animal ventilator with 2% halothane in 24% oxygen with a balance of nitrogen gas. A femoral artery catheter was placed for continuous blood pressure recording and for arterial blood gas sampling. A femoral vein was canalized for administration of neuro- muscular paralyzing agents and normal saline. Cutaneous 0006-8993/96/$15.00 Copyright © 1996 Elsevier Science B.V. All rights reserved. Pll S0006-8993(96)00215-6