A multistep validation process of biomarkers for preclinical drug development.

Biomarkers that can be measured in preclinical models in a high-throughput, reproducible manner offer the potential to increase the speed and efficacy of drug development. Development of therapeutic agents for many conditions is hampered by the limited number of validated preclinical biomarkers available to gauge pharmacoefficacy and disease progression, but the validation process for preclinical biomarkers has received limited attention. This report defines a five-step preclinical biomarker validation process and applies the process to a case study of diabetic retinopathy. By showing that a gene expression panel is highly reproducible, coincides with disease manifestation, accurately classifies individual animals and identifies animals treated with a known therapeutic agent, a biomarker panel can be considered validated. This particular biomarker panel consisting of 14 genes (C1inh, C1s, Carhsp1, Chi3l1, Gat3, Gbp2, Hspb1, Icam1, Jak3, Kcne2, Lama5, Lgals3, Nppa, Timp1) can be used in diabetic retinopathy pharmacotherapeutic research, and the biomarker development process outlined here is applicable to drug development efforts for other diseases.

Effect of extreme hypercapnia on hypoxic-ischemic brain damage in the immature rat.

To ascertain the effect of extreme hypercapnia on perinatal hypoxic-ischemic brain damage, 7-d-postnatal rats were exposed to unilateral common carotid artery occlusion followed by hypoxia with 8% oxygen combined with 3, 12, or 15% carbon dioxide (CO2) for 2 h at 37 degrees C. Survivors underwent neuropathologic examination at 30 d of postnatal age, and their brains were characterized as follows: 0 = normal; 1 = mild atrophy; 2 = moderate atrophy; 3 = cystic infarct with external dimensions <3 mm; and 4 = cystic infarct with external dimensions >3 mm. The width of the cerebral hemisphere ipsilateral to the carotid artery occlusion also was determined on a posterior coronal section and compared with that of the contralateral hemisphere to ascertain the severity of cerebral atrophy/cavitation. CO2 tensions averaged 5.08, 11.1, and 13.2 kPa in the 3, 12, and 15% CO2-exposed animals, respectively, during hypoxia-ischemia (HI). Neuropathologic results showed that immature rats exposed to 3 and 12% CO2 had similar severities of brain damage. In contrast, rat pups exposed to HI combined with 15% CO2 were significantly more brain damaged than littermates exposed to 3% CO2. Specifically, eight of 14 animals exposed to 15% CO2 showed cystic infarcts (grades 3 and 4), whereas none of 14 littermates exposed to 3% CO2 developed cystic infarcts (p < 0.01). Analyses of coronal width ratios at each CO2 exposure provided results comparable with those of the gross neuropathology scores. Cerebral blood flow (CBF), measured at 90 min of HI, was lowest in those immature rats exposed to 15% CO2 compared with control (p = 0.04), with higher values in those rat pups exposed to 3 and 12% CO2. The findings indicate that 7-d-postnatal rats exposed to HI with superimposed 12% CO2 are neither less nor more brain damaged than littermates exposed to 3% CO(2) (normocapnia). In contrast, animals exposed to 15% CO2 are the most brain damaged of the three groups. Presumably, extreme hypercapnia produces more severe cardiovascular depression than is seen in animals subjected to lesser degrees of hypercapnia; the cardiovascular depression, in turn, leads to greater cerebral ischemia and ultimate brain damage.

Intracellular calcium accumulation during the evolution of hypoxic-ischemic brain damage in the immature rat.

An excessive intracellular accumulation of calcium (Ca2+) in neurons and glia has been proposed to represent a major 'final common pathway' for cell death arising from hypoxia-ischemia. To clarify the role of altered calcium flux into the perinatal brain undergoing hypoxic-ischemic damage, 7-day postnatal rats underwent unilateral common carotid artery ligation followed by systemic hypoxia with 8% oxygen. This insult is known to produce brain damage in the form of selective neuronal death or infarction largely limited to the cerebral hemisphere ipsilateral to the arterial occlusion. Either prior to or following hypoxia-ischemia, the rat pups received a s.c. injection of 45CaCl2, and specimens of blood, cerebrospinal fluid (CSF), and brain were obtained for isotopic measurements and the calculation of the extent of brain intracellular radioactivity. During hypoxia-ischemia, there was a modest increase in intracellular Ca2+ radioactivity (+28-47%) in both cerebral hemispheres only after 2 h of hypoxia-ischemia. During recovery from 2 h of hypoxia-ischemia, intracellular Ca2+ accumulated progressively only in the ipsilateral cerebral hemisphere for up to 24 h, during which interval intracellular Ca2+ decreased in the contralateral hemisphere. No such progressive accumulation was noted during recovery in animals previously exposed to only 1 h of hypoxia-ischemia. The results suggest that a disruption of intracellular Ca2+ homeostasis is a major contributing factor in the evolution of perinatal hypoxic-ischemic brain damage. Ca2+ accumulation is a relatively modest and late event during the hypoxic-ischemic phase, and a progressive overload occurs during the recovery phase only if infarction occurs. The question remains as to whether or not the intracellular Ca2+ overload occurring during recovery is a contributor to or a consequence of the ultimate brain damage.

Experimental stroke in the female diabetic, db/db, mouse.

Diabetic hyperglycemia increases brain damage after cerebral ischemia in animals and humans, although the underlying mechanisms remain unclear. Gender-linked differences in ischemic tolerance have been described but have not been studied in the context of diabetes. In the current study, we used a model of unilateral common carotid artery ligation, combined with systemic hypoxia, to study the effects of diabetes and gender on hypoxic-ischemic (HI) brain damage in the genetic model of Type II diabetes, the db/db, mouse. Male and female, control and db/db, mice were subjected to right common carotid artery ligation followed by varying periods of hypoxia (8% oxygen/92% nitrogen) to assess mortality, infarct volume, and tissue damage by light microscopic techniques. End-ischemic regional cerebral blood flow (CBF) was determined using [14C] iodoantipyrine autoradiography. Glycolytic and high energy phosphate compounds were measured in blood and brain by enzymatic and fluorometric techniques. Gender and diabetes had significant effects on mortality from HI and extent of brain damage in the survivors. Female mice were more resistant than their male counterparts, such that the severity (mortality and infarction size) in the male diabetics > female diabetics - male controls > female controls. Endischemic CBF and depletion of cerebral high energy reserves were comparable among all groups. Surprisingly, female diabetic mice were more hyperglycemic and demonstrated a greater prolonged lactacidosis than the males; however, they were more resistant to damage. The results suggest a unique pathophysiology of hypoxia-ischemia in the female diabetic brain.

CSF glutamate during hypoxia-ischemia in the immature rat.

Cerebrospinal fluid (CSF) glutamate was measured prior to and during the course of cerebral hypoxia-ischemia in the immature rat to estimate its concentration in the extracellular fluid (ECF). A preliminary experiment was conducted using [14C]glutamate injections into immature rat brain, which showed that equilibration between ECF and CSF occurred within 10 min. Seven-day postnatal rats underwent unilateral common carotid artery ligation followed by hypoxia with 8% oxygen for up to 2 h. Brain damage, in the form of selective neuronal necrosis or apoptosis, commences after 60 min, while infarction commences after 90 min of hypoxia-ischemia. During the course of hypoxia-ischemia, CSF was obtained from the cisterna magna and analyzed for glutamate. No statistically significant increases in CSF glutamate occurred until 105 min, at which time the concentration was 240% of control (20 micromol/l). By 120 min, CSF glutamate had increased over twofold above the control value. In rat pups exposed to 1 h of hypoxia-ischemia, no increases in CSF glutamate occurred for up to 6 h of recovery. In animals exposed to 2 h of hypoxia-ischemia, CSF glutamate decreased to the control value by 1 h of recovery, with a secondary rise at 6 h. Accordingly, the increase in CSF, and presumably ECF, glutamate is a late event, which better corresponds temporally to cerebral infarction than to selective neuronal death. The results suggest that glutamate excitotoxicity, although involved in the occurrence of infarction, neither causes or contributes to selected neuronal death. The secondary elevation in CSF glutamate at 6 h of recovery from 2 h of hypoxia-ischemia occurs coincident with the onset of tissue necrosis, seen histologically.

Effect of carbon dioxide on cerebral metabolism during hypoxia-ischemia in the immature rat.

We previously have demonstrated that hypocapnia aggravates and hypercapnia protects the immature rat from hypoxic-ischemic brain damage. To ascertain cerebral blood flow (CBF) and metabolic correlates, 7-d postnatal rats were subjected to hypoxia-ischemia during which they were rendered either hypo-(3.5 kPa), normo- (5.1 kPa), or hypercapnic (7.3 kPa) by the inhalation of either 0, 3, or 6% CO2, 8% O2, balance N2. CBF during hypoxia-ischemia was better preserved in the normo- and hypercapnic rat pups; these animals also exhibited a stimulation of cerebral glucose utilization. Brain glucose concentrations were higher and lactate lower in the normo- and hypercapnic animals, indicating that glucose was consumed oxidatively in these groups rather than by anaerobic glycolysis, as apparently occurred in the hypocapnic animals. ATP and phosphocreatine were better preserved in the normo- and hypercapnic rats compared with the hypocapnic animals. Cerebrospinal fluid glutamate, as a reflection of the brain extracellular fluid concentration, was lowest in the hypercapnic rats at 2 h of hypoxia-ischemia. The data indicate that during hypoxia-ischemia in the immature rat, CBF is better preserved during normo- and hypercapnia; the greater oxygen delivery promotes cerebral glucose utilization and oxidative metabolism for optimal maintenance of tissue high energy phosphate reserves. An inhibition of glutamate secretion into the synaptic cleft and its attenuation of N-methyl-D-aspartate receptor activation would further protect the hypercapnic animal from hypoxic-ischemic brain damage.

The effect of hyperglycemia on cerebral metabolism during hypoxia-ischemia in the immature rat.

Unlike adults, hyperglycemia with circulating glucose concentrations of 25-35 mM/L protects the immature brain from hypoxic-ischemic damage. To ascertain the effect of hyperglycemia on cerebral oxidative metabolism during the course of hypoxia-ischemia, 7-day postnatal rats underwent unilateral common carotid artery ligation followed by exposure to 8% O2 for 2 h at 37 degrees C. Experimental animals received 0.2 cc s.c. 50% glucose at the onset of hypoxia-ischemia, and 0.15 cc 25% glucose 1 h later to maintain blood glucose concentrations at 20-25 mM/L for 2 h. Control rat pups received equivalent concentrations or volumes of either mannitol or 1 N saline at the same intervals. The cerebral metabolic rate for glucose (CMRglc) increased from 7.1 (control) to 20.2 mumol 100 g-1 min-1 in hyperglycemic rats during the first hour of hypoxia-ischemia, 79 and 35% greater than the rates for saline-and mannitol-injected animals at the same interval, respectively (p < 0.01). Brain intracellular glucose concentrations were 5.2 and 3.0 mM/kg in the hyperglycemic rat pups at 1 and 2 h of hypoxia-ischemia, respectively; glucose levels were near negligible in mannitol- and saline-treated animals at the same intervals. Brain intracellular lactate concentrations averaged 13.4 and 23.3 mM/kg in hyperglycemic animals at 1 and 2 h of hypoxia-ischemia, respectively, more than twice the concentrations estimated for the saline- and mannitol-treated littermates. Phosphocreatine (PCr) and ATP decreased in all three experimental groups, but were preserved to the greatest extent in hyperglycemic animals. Results indicate that anaerobic glycolytic flux is increased to a greater extent in hyperglycemic immature rats than in normoglycemic littermates subjected to cerebral hypoxia-ischemia, and that the enhanced glycolysis leads to greater intracellular lactate accumulation. Despite cerebral lactosis, energy reserves were better preserved in hyperglycemic animals than in saline-treated controls, thus accounting for the greater resistance of hyperglycemic animals to hypoxic-ischemic brain damage.

Paradoxical mitochondrial oxidation in perinatal hypoxic-ischemic brain damage.

Measurements of cytoplasmic and mitochondrial markers of the oxidation-reduction (redox) state of brain tissue were conducted in a perinatal animal model of cerebral hypoxia-ischemia to ascertain underlying biochemical mechanisms whereby ischemia (reduced oxygen and substrate supply) causes brain damage. Seven-day postnatal rats underwent unilateral common carotid artery ligation followed by exposure to 8% oxygen at 37 degrees C for 3 h. During the course of hypoxia-ischemia, the rat pups were quick frozen in liquid nitrogen and their brains processed for the enzymatic, fluorometric measurement of cerebral metabolites necessary for the calculation of intracellular pH and cytoplasmic and mitochondrial redox states. The results showed an early mitochondrial reduction followed by re-oxidation during the course of hypoxia-ischemia. The oxidation reflected a partial depletion in accumulated reducing equivalents and coincides temporally with the duration of hypoxia-ischemia required to convert selective neuronal necrosis into cerebral infarction. The findings suggest that perinatal cerebral hypoxia-ischemia is characterized more by a limitation of substrate than of oxygen supply to the brain, which may explain why glucose supplementation of the immature animal improves neuropathologic outcome, in contrast to adults.

Carbon dioxide protects the perinatal brain from hypoxic-ischemic damage: an experimental study in the immature rat.

BACKGROUND AND OBJECTIVE: Clinical investigations suggest that premature infants who require mechanical ventilation from respiratory distress syndrome are at increased risk for periventricular leukomalacia if hypocapnia occurs during respiratory management. The question remains as to the contribution of hypocapnia to hypoxic-ischemic brain damage and whether or not hypercapnia is neuroprotective. METHODS: Seven-day postnatal rats underwent unilateral common carotid artery ligation followed thereafter by exposure to systemic hypoxia with 8% oxygen (O2) combined with either 0, 3, 6, or 9% carbon dioxide (CO2) for 2.5 hours at 37 degrees C. Survivors underwent neuropathologic examination at 30 days of postnatal age, and their brains were categorized as follows: 0 = normal; 1 = mild atrophy; 2 = moderate atrophy; 3 = atrophy with cystic cavitation < 3 mm; 4 = cystic cavitation > 3 mm of the cerebral hemisphere ipsilateral to the carotid artery ligation. The width of the ipsilateral hemisphere also was determined on a posterior coronal section and compared with that of the contralateral hemisphere to ascertain the severity of cerebral atrophy/cavitation. Data were analyzed by linear models. RESULTS: CO2 tensions averaged 26, 42, 54, and 71 mm Hg in the 0, 3, 6, and 9% CO2 exposed animals, respectively, during systemic hypoxia. Blood O2 tensions during hypoxia were not different among the four groups and averaged 34.7 mm Hg. Neuropathologic results showed that 30/38 (79%) rats exposed to 3% CO2 showed either no or mild brain damage compared with 13/33 (39%) controls (0% CO2). Cystic cavitation occurred in only four CO2 exposed rat pups compared with 14 controls (P = .001). At 6% CO2 exposure, all of 20 rat pups showed either no damage or mild atrophy compared with controls (P < .001); and at 9% CO2 exposure, 19/23 (83%) rat pups showed no or mild damage compared with controls (P < .001). The data also showed that the greatest reduction in brain damage occurred in immature rats exposed to 6% CO2 with slightly less protection at 9% CO2 (P = .012), the latter comparable with the severity of brain damage sustained by animals inhaling 3% CO2. Analyses of coronal width ratios at each CO2 exposure provided results comparable with those of the gross neuropathology scores. CONCLUSIONS: The results indicate that in an immature rat model normocapnic cerebral hypoxia-ischemia is associated with less severe brain damage than in hypocapnic hypoxia-ischemia and that mild hypercapnia is more protective than normocapnia. The findings in an experimental model merit further animal investigations as well as a clinical reappraisal of the ventilatory management of sick newborn human infants.

Glucose transport in developing rat brain: glucose transporter proteins, rate constants and cerebral glucose utilization.

Developing rat brain undergoes a series of functional and anatomic changes which affect its rate of cerebral glucose utilization (CGU). These changes include increases in the levels of the glucose transporter proteins, GLUT1 and GLUT3, in the blood-brain barrier as well as in the neurons and glia. 55 kDa GLUT1 is concentrated in endothelial cells of the blood-brain barrier, whereas GLUT3 is the predominant neuronal transporter. 45 kDa GLUT1 is in non-vascular brain, probably glia. Studies of glucose utilization with the 2-14C-deoxyglucose method of Sokoloff et al., (1977), rely on glucose transport rate constants, k1 and k2, which have been determined in the adult rat brain. The determination of these constants directly in immature brain, in association with the measurement of GLUT1, GLUT3 and cerebral glucose utilization suggests that the observed increases in the rate constants for the transport of glucose into (ki) and out of (k2) brain correspond to the increases in 55 kDa GLUT1 in the blood-brain barrier. The maturational increases in cerebral glucose utilization, however, more closely relate to the pattern of expression of non-vascular GLUT1 (45 kDa), and more specifically GLUT3, suggesting that the cellular expression of the glucose transporter proteins is rate limiting for cerebral glucose utilization during early postnatal development in the rat.

Cerebral mitochondrial redox states during metabolic stress in the immature rat.

The brain mitochondrial NAD+/NADH ratio, as a reflection of the oxidation-reduction (redox) state of cellular compartment, was determined under conditions of hypoxia, anoxia, hypoxia-ischemia, complete ischemia and hypoglycemia in immature rats. NAD+/NADH ratios were calculated from changes in the concentrations of specific oxidative substrates and calculated intracellular pH during cerebral metabolic stress. The results suggest that the use of the acetoacetate/beta-hydroxybutyrate substrate couple provides a more accurate prediction of the mitochondrial redox state under adverse conditions than use of the alpha-ketoglutarate/glutamate couple. It is possible that the mitochondrial oxidation seen with the latter substrate couple during cerebral metabolic stress might reflect a population of cells (neurons or glia) which are substrate-deprived relative to the rest of the brain in the setting of metabolic stress produced by oxygen deficiency.

Cerebral oxidative metabolism during hypothermia and circulatory arrest in newborn dogs.

To ascertain the alterations in cerebral oxidative and energy metabolism that occur during hypothermic circulatory arrest, nitrous oxide-anesthetized, paralyzed, and artificially ventilated newborn dogs were surface cooled to 18-20 degrees C, after which their hearts were arrested with KCl. At 10, 30, 60, and 105 min of circulatory arrest, their brains were prepared by in situ freezing for the regional analysis of glycolytic intermediates and high-energy phosphate reserves. Hypothermia alone was associated with optimal preservation of labile metabolites in brain, even in caudal brainstem and cerebellum, compared with barbiturate-anesthetized littermates. After onset of hypothermic circulatory arrest, glucose decreased progressively in cerebral cortex, caudate nucleus, hippocampus, and subcortical white matter to negligible levels by 30 min. Pyruvate increased transiently (+50%) at 10 min, whereas lactate increased and plateaued (10-11 mmol/kg) at 30 min. The disproportionate increases in pyruvate and lactate resulted in a progressive rise in the lactate/pyruvate ratio. Phosphocreatine fell precipitously to < 0.5 mmol/kg in all structures, with a preservation of ATP for the first 10 min of cerebral ischemia. Thereafter, ATP decreased to < 0.1 mmol/kg in cerebral cortex and between 0.1 and 0.2 mmol/kg in caudate nucleus, hippocampus, and white matter. Total adenine nucleotides (ATP+ADP+AMP) were partially depleted by 30 min in the gray matter structures but were unchanged from control for 60 min in white matter. The findings showed a direct correlation between preservation of cerebral energy stores during hypothermic circulatory arrest and the selective resistance of subcortical white matter to ischemic damage.(ABSTRACT TRUNCATED AT 250 WORDS)

Cerebral energy metabolism during hypoxia-ischemia and early recovery in immature rats.

Persistent alterations in cellular energy homeostasis may contribute to the brain damage that evolves from perinatal cerebral hypoxia-ischemia. Accordingly, the presence and extent of perturbations in high-energy phosphate reserves were analyzed during hypoxia-ischemia and the early recovery period in the immature rat. Seven-day postnatal rats were subjected to unilateral common carotid artery ligation and hypoxia with 8% oxygen at 37 degrees C for 3 h, an insult that produces damage (selective neuronal necrosis or infarction) of the cerebral hemisphere ipsilateral to the common carotid artery ligation in 92% of animals. Rat pups were quick frozen in liquid nitrogen during hypoxia-ischemia and at 10, 30, and 60 min and 4 and 24 h of recovery for enzymatic, fluorometric analysis of phosphocreatine (PCr), creatine, ATP, ADP, and AMP. During hypoxia-ischemia, PCr, ATP, and total adenine nucleotides were decreased by 87, 72, and 50% of control, respectively. During recovery, PCr, ATP, and total adenine nucleotides exhibited a rapid (within 10 min) although incomplete and heterogeneous recovery that persisted for at least 24 h. Mean values for PCr remained between 55 and 85% of control, whereas ATP values remained between 57 and 67% of control. Individual ATP values were inversely related to tissue water content at 10 min of recovery, indicating a close correlation between failure of energy restoration and the extent of cerebral edema as a reflection of brain damage. Thus high-energy phosphate reserves display lingering alterations during recovery from hypoxia-ischemia. The interanimal variability in energy restoration presumably reflects the spectrum of brain damage seen in this model of perinatal cerebral hypoxia-ischemia.

Cerebral oxidative metabolism and redox state during hypoxia-ischemia and early recovery in immature rats.

Intracellular pH (pHi) and cytoplasmic and mitochondrial oxidation-reduction (redox) states of cerebral tissue were examined in relation to perturbations of glycolytic and tricarboxylic acid cycle intermediates and of high-energy phosphate reserves during hypoxia-ischemia and the early recovery period in the immature rat. Seven-day postnatal rats underwent unilateral common carotid artery ligation and exposure to 8% O2 for 3 h, after which they were quick frozen in liquid N2 at the terminus of hypoxia-ischemia and at 10, 30, 60, and 240 min of recovery for enzymatic fluorometric analysis of cerebral metabolites. During hypoxia-ischemia, concentrations of glucose and alpha-ketoglutarate in the cerebral hemisphere ipsilateral to the carotid artery occlusion were depleted to 10 and 70% of control, respectively; pyruvate was unchanged. During recovery, glucose, pyruvate, and alpha-ketoglutarate increased above their respective control values. Calculated pHi decreased from 7.0 (control) to 6.6 during hypoxia-ischemia and normalized by 10 min of recovery. The cytoplasmic NAD+/NADH ratio decreased (increased reduction) to 50% of control during hypoxia-ischemia and remained in the reduced state throughout 4 h of recovery. Paradoxically, mitochondrial NAD+/NADH was oxidized at the terminus of hypoxia-ischemia. The mitochondrial oxidation which developed during hypoxia-ischemia presumably results from a limitation of cellular substrate (glucose) supply, which in turn leads to a depletion of high-energy phosphate reserves, culminating in brain damage.

Carbohydrate and energy metabolism during the evolution of hypoxic-ischemic brain damage in the immature rat.

The brain damage that evolves from perinatal cerebral hypoxia-ischemia may involve lingering disturbances in metabolic activity that proceed into the recovery period. To clarify this issue, we determined the carbohydrate and energy status of cerebral tissue using enzymatic, fluorometric techniques in an experimental model of perinatal hypoxic-ischemic brain damage. Seven-day postnatal rats were subjected to unilateral common carotid artery ligation followed by 3 h of hypoxia with 8% oxygen at 37 degrees C. This insult is known to produce tissue injury (selective neuronal necrosis or infarction) predominantly in the cerebral hemisphere ipsilateral to the carotid artery occlusion in 92% of the animals. Rat pups were quick-frozen in liquid nitrogen at 0, 1, 4, 12, 24, or 72 h of recovery; littermate controls underwent neither ligation nor hypoxia. Glucose in both cerebral hemispheres was nearly completely exhausted during hypoxia-ischemia, with concurrent increases in lactate to 10 mmol/kg. During recovery, glucose promptly increased above control values, suggesting an inhibition of glycolytic flux, as documented in the ipsilateral cerebral hemisphere by measurement of glucose utilization (CMRglc) at 24 h. Tissue lactate declined rapidly during recovery but remained slightly elevated in the ipsilateral hemisphere for 12 h. Phosphocreatine (P approximately Cr) and ATP in the ipsilateral cerebral hemisphere were 14 and 26% of control (p less than 0.001) at the end of hypoxia-ischemia; total adenine nucleotides (ATP + ADP + AMP) also were partially depleted (-46%).(ABSTRACT TRUNCATED AT 250 WORDS)

Regional cerebral blood flow and glucose utilization during hypothermia in newborn dogs.

To ascertain the effect of profound hypothermia on brain function and metabolism, newborn dogs were subjected to surface cooling during which regional cerebral blood flow (rCBF) and glucose utilization (rCGU) were measured with iodo-[14C]-antipyrine and 2-deoxy-[14C]-glucose, respectively. Puppies were anesthetized with nitrous oxide, paralyzed, and their lungs artificially ventilated to maintain arterial normoxia (PaO2 greater than 60 mmHg) and normal acid-base balance (PaCO2 = 35-41 mmHg; pHa = 7.34-7.42). When rectal temperature was decreased from 37 to 20 degrees C, mean arterial blood pressure (MABP) decreased from 75 to 47 mmHg (P less than 0.001) and heart rate from 238 to 64 beats/min (P less than 0.001). Arterial PCO2 was reduced from 38 to 31 mmHg (P less than 0.001) (corrected to 37 degrees C), whereas pHa was unchanged from control (7.40). The electroencephalogram slowed progressively and became isoelectric at 22-25 degrees C. During normothermia (n = 6) blood flow to 16 component structures of brain varied from 17 (occipital white matter) to 65 (medulla) ml.100 g-1.min-1, whereas during hypothermia (n = 6) blood flow was lower in all regions (P less than 0.001) at remarkably uniform levels 8.3-10.3 ml.100 g-1.min-1). Thus, the greatest reductions (range, 16-48% of control) in CBF occurred in those structures with the highest intrinsic flows during normothermia and were proportionately less in low flow structures. Regional CGU also decreased in all brain regions analyzed (P less than 0.001).(ABSTRACT TRUNCATED AT 250 WORDS)