Robert C. Vannucci

Rat model of perinatal hypoxic‐ischemic brain damage

To gain insights into the pathogenesis and management of perinatal hypoxic‐ischemic brain damage, the authors have used an immature rat model which they developed many years ago. The model entails ligation of one common carotid artery followed thereafter by systemic hypoxia. The insult produces permanent hypoxic‐ischemic brain damage limited to the cerebral hemisphere ipsilateral to the carotid artery occlusion. The mini‐review describes recently accomplished research pertaining to the use of the immature rat model, specifically, investigations involving energy metabolism, glucose transporter proteins, free radical injury, and seizures superimposed upon cerebral hypoxia‐ischemia. Future research will focus on molecular mechanisms of neuronal injury with a continuing focus on therapeutic strategies to prevent or minimize hypoxic‐ischemic brain damage. J. Neurosci. Res. 55:158–163, 1999. 

Perinatal hypoxic-ischemic brain damage: evolution of an animal model.

Early research in the Vannucci laboratory prior to 1981 focused largely on brain energy metabolism in the developing rat. At that time, there was no experimental model to study the effects of perinatal hypoxia-ischemia in the rodent, despite the tremendous need to investigate the pathophysiology of perinatal asphyxial brain damage in infants. Accordingly, we developed such a model in the postnatal day 7 rat, using a modification of the Levine preparation in the adult rat. Rat pups underwent unilateral common carotid artery ligation followed by exposure to systemic hypoxia (8% oxygen) at a constant temperature of 37°C. Brain damage, seen histologically, was generally confined to the cerebral hemisphere ipsilateral to the arterial occlusion, and consisted of selective neuronal death or infarction, depending on the duration of the systemic hypoxia. Tissue injury was observed in the cerebral cortex, hippocampus, striatum, and thalamus. Subcortical and periventricular white matter injury was also observed. This model was originally described in the Annals of Neurology in 1981, and during the more than 20 years since that publication numerous investigations utilizing the model have been conducted in our laboratories as well as laboratories around the world. Cerebral blood flow and metabolic correlates have been fully characterized. Physiologic and pharmacologic manipulations have been applied to the model in search of neuroprotective strategies. More recently, molecular biologic alterations during and following the hypoxic-ischemic stress have been ascertained and the model has been adapted to the immature mouse for specific use in genetically altered animals. As predicted in the original article, the model has proven useful for the study of the short- and long-term effects of hypoxic-ischemic brain damage on motor activity, behavior, seizure incidence, and the process of maturation in the brain and other organ systems.

Reduction of Perinatal Hypoxic-Ischemic Brain Damage with Allopurinol

ABSTRACT: Cytotoxic free radicals are generated during cerebral hypoxia-ischemia and reperfusion. We studied the efficacy of allopurinol, a xanthine oxidase inhibitor and free radical scavenger, in reducing posthypoxic-ischemic damage in the developing brain of 7-d-old rat pups. Hypoxic- ischemic injury to the right cerebral hemisphere was produced by ligation of the right common carotid artery followed by 3 h of hypoxia with 8% oxygen. Thirty to 45 min before the hypoxia, the rats received either allopurinol (dose = 130-138 mg/kg) or an equal vol of saline (0.2 mL). Some pups were killed at 42 h of recovery for measurement of cerebral hemispheric water content, whereas others were killed at 30 or more d for neuropathologic examination. A total of 18 allopurinol treated rats had significantly less water content in the right hemisphere (89.07 ± 0.32%) than 23 saline-treated animals (91.64 ± 0.25%, mean ± SEM, p < 0.0001). Rank scoring of neuropathologic alterations revealed that the allopurinol treated rats were less damaged (p = 0.001). Only two of 13 brains from the allopurinol group suffered infarction compared to 10 of the 14 saline-treated animals. The results indicate that allopurinol reduces both cerebral edema and the extent of perinatal hypoxic-ischemic brain damage.

A model of perinatal hypoxic-ischemic brain damage

In conclusion, our immature rat model has gained wide acceptance as the animal model of choice to study basic physiologic, biochemical, and molecular mechanisms of perinatal hypoxic-ischemic brain damage. In addition, the model has been used extensively to study those physiologic and therapeutic variables which either are deleterious or beneficial to the perinatal brain undergoing hypoxia-ischemia. As therapeutic interventions are tested in the animal setting, the results will provide important information regarding the effect of these agents in the human setting.

Neuropathology of remote hypoxic-ischemic damage in the immature rat

SummaryThis study was undertaken to determine: (a) the duration of hypoxia required to produce brain damage in immature rats with unilateral carotid artery ligation (Levine technique); (b) the regions of immature brain most vulnerable to hypoxia-ischemia (HI); and (c) the neuropathology of the remote HI insult. To this end, 7-day postnatal rats, subjected to unilateral carotid artery ligation combined with hypoxia of varying durations (45, 60, 75 or 90 min), were killed at 30 days of postnatal age and their brains examined by light microscopy. The results indicated that a longer duration of HI was more likely to produce brain lesions and that the extent and severity of the lesions closely correlated with the length of HI. Shorter intervals of HI primarily damaged the cerebral cortex and hippocampus, while longer periods resulted in more extensive damage and were often associated with cavitary lesions of the cerebral hemisphere. Comparison of HI brain damage produced by the Levine technique in immature and adult rats suggested that in immature rats: (a) the cavitary lesions were common; (b) the non-cavitary cortical lesions had a tendency to show a vertical band-like distribution — a pattern never seen in adults; and (c) the lesions often showed mineralization. The similarities between these experimentally produced HI cerebral lesions and those observed in the developing human brain, such as ulegyria and porencephaly, are discussed.

Influence of Mild Hypothermia on Hypoxic- Ischemic Brain Damage in the Immature Rat

ABSTRACT: Recent studies in adult animals have shown that even small decreases in brain or core temperature ameliorate the damage resulting from hypoxic-ischemic insults. To determine the effect of minor reductions in ambient temperature either during or after an hypoxic-ischemic insult on the brain of the immature rat, 7-d-postnatal rat pups underwent unilateral common carotid artery ligation followed by exposure to hypoxia in 8% oxygen for 3 h. Control animals were maintained at 37°C during hypoxia-ischemia. Intraischemic hypothermia was induced during the insult at temperatures of 34°C and 31°C. Postischemic hypothermia was induced by exposing rat pups that underwent hypoxia at 37°C to recovering environments of 34°C and 31°C. Temperatures were recorded every 15 min from thermistor probes placed in the ipsilateral hemisphere and rectally. Neuropathologic alterations were assessed at 30 postnatal d. During hypoxia, animals became poikilothermic. Brain damage occurred in 90% of rat pups exposed to hypoxia-ischemia at 37°C. Cerebral injury significantly decreased with decreasing temperatures during hypoxia-ischemia (p < 0.01). Only 30% of rats had brain damage when exposed to hypoxia-ischemia at 34°C, and none of the rats exposed at 31°C had brain damage. In contrast, there was no difference in the extent of cerebral injury between rat pups recovered under hypothermic conditions of either 34°C or 31°C compared with those recovered at 37°C. The results indicate that reductions in temperature of 3 to 6°C have a protective effect during but not immediately after hypoxia-ischemia. These findings have important implications about the thermoregulatory control of the sick newborn infant.

Temporal evolution of neuropathologic changes in an immature rat model of cerebral hypoxia: a light microscopic study

The sequential evolution of neuropathologic changes was studied in an immature model of cerebral hypoxia-ischemia. Accordingly, 7-day postnatal rats were subjected to unilateral common carotid artery ligation combined with 2 h of hypoxia (breathing in 8% oxygen) and their brains were examined by light microscopy at recovery intervals ranging from 0 to 3 weeks. Immediately following hypoxia, a large area with a pale staining border was noted occupying most of the cerebral hemisphere ipstlateral (IL) to the occluded common carotid artery; in approximately half of the brains the dorsomedial cortex of the contralateral (CL) hemisphere was also involved. Most neurons in the pale area had nuclei containing a coarse granular condensation of chromatin. Within a few hours, the majority of neurons in the IL hemisphere had developed pyknotic nuclei and clear or eosinophilic perikarya. After 24 h these changes had evolved in the majority of brains into coagulation necrosis (infarction) in the IL hemisphere sphere and foci of selective neuronal necrosis in the CL ortex. Within a few days infarcts became partially cavirated, and by 3 weeks a smooth-walled cystic infarct had developed. Activated microglia/macrophages and reactive astrocytes were first seen at 4 and 24 h, respectively. No parenchymal neutrophilic infiltrate was seen at any time point.

Regional cerebral blood flow during hypoxia-ischemia in immature rats.

Immature rats subjected to a combination of unilateral common carotid artery ligation and hypoxia sustain brain damage confined largely to the ipsilateral cerebral hemisphere. To ascertain the extent and distribution of ischemic alterations in the brains of these small animals, we modified the Sakurada technique to measure regional cerebral blood flow using carbon-14 autoradiography. Seven-day-old rats underwent right common carotid artery ligation following which they were rendered hypoxic with 8% O2 at 37 degrees C. Before and during hypoxia, the rat pups received an injection of iodo[14C]antipyrine for determination of regional cerebral blood flow. Blood flows to individual structures of the ipsilateral cerebral hemisphere were not influenced by arterial occlusion alone; flows to the contralateral hemisphere and to the brainstem and cerebellum actually increased by 25-50%. Hypoxia-ischemia was associated with decreases in regional cerebral blood flow of the ipsilateral hemisphere such that by 2 hours, flows to subcortical white matter, neocortex, striatum, and thalamus were 15, 17, 34, and 41% of control, respectively. The hierarchy of the blood flow reductions correlated closely with the distribution and extent of ischemic neuronal necrosis. However, unlike the pathologic pattern of this model, the degree of ischemia appeared homogeneous within each brain region. Blood flows to contralateral cerebral hemispheric structures were relatively unchanged from prehypoxic values, whereas flows to the brainstem and cerebellum nearly doubled and tripled, respectively. Thus, ischemia is the predominant factor that determines the topography of tissue injury to major regions of immature rat brain, whereas metabolic factors (intrinsic vulnerability) may influence the heterogeneous pattern of damage seen within individual structures.

Effects of Hypoxia-Ischemia on GLUT1 and GLUT3 Glucose Transporters in Immature Rat Brain:

Cerebral hypoxia-ischemia produces major alterations in energy metabolism and glucose utilization in brain. The facilitative glucose transporter proteins mediate the transport of glucose across the blood–brain barrier (BBB) (55 kDa GLUT1) and into the neurons and glia (GLUT3 and 45 kDa GLUT1). Glucose uptake and utilization are low in the immature rat brain, as are the levels of the glucose transporter proteins. This study investigated the effect of cerebral hypoxia-ischemia in a model of unilateral brain damage on the expression of GLUT 1 and GLUT3 in the ipsilateral (damaged, hypoxic-ischemic) and contralateral (undamaged, hypoxic) hemispheres of perinatal rat brain. Early in the recovery period, both hemispheres exhibited increased expression of BBB GLUT1 and GLUT3, consistent with increased glucose transport and utilization. Further into recovery, BBB GLUT1 increased and neuronal GLUT3 decreased in the damaged hemisphere only, commensurate with neuronal loss.

Hypoxic Preconditioning and Hypoxic-Ischemic Brain Damage in the Immature Rat: Pathologic and Metabolic Correlates

Abstract: It has been reported that immature rats subjected to cerebral hypoxia‐ischemia sustain less brain damage if they are previously exposed to systemic hypoxia compared with animals not exposed to prior hypoxia. Accordingly, neuropathologic and metabolic experiments were conducted to confirm and extend the observation that hypoxic preconditioning protects the perinatal brain from subsequent hypoxic‐ischemic brain damage. Six‐day postnatal rats were subjected to systemic hypoxia with 8% oxygen at 37°C for 2.5 h. Twenty‐four hours later, they were exposed to unilateral cerebral hypoxia‐ischemia for 2.5 h, produced by unilateral common carotid artery ligation and systemic hypoxia with 8% oxygen. Neuropathologic analysis, conducted at 30 days of postnatal age, indicated a substantial reduction in the severity of brain damage in the preconditioned rats, such that only 6 of 14 such animals exhibited cystic infarction, but all 13 animals without prior preconditioning exhibited infarction (p < 0.001). Measurement of cerebral glycolytic and tricarboxylic acid intermediates and high‐energy phosphate reserves at the terminus of and at 4 and 24 h following hypoxia‐ischemia showed no differences in the extent of alterations in the preconditioned and nonpreconditioned immature rats. A difference was seen in the restitution of high‐energy stores during the first 24 h of recovery from hypoxia‐ischemia, with a more optimal preservation of these metabolites in the preconditioned animals, reflecting the less severe ultimate brain damage. Accordingly, the neuroprotection afforded to the preconditioned animals was not the result of any differences in the extent of anaerobic glycolysis, tissue acidosis, or depletion in high‐energy reserves during hypoxia‐ischemia but rather the result of other mechanisms that improved the metabolic status of the immature brain during the early hours of reperfusion following hypoxia‐ischemia.