Hypothermia may be an ideal neuroprotective intervention in hypoxic-ischemic encephalopathy after perinatal asphyxia. The present study describes the long-term effects of prolonged resuscitative whole-body hypothermia initiated 2 h after hypoxicischemic injury on brain morphology and neuropsychological behavior in 7-d-old rats. After right common carotid artery ligation and exposure to hypoxia of 8% O 2 for 105 min, 10 animals were kept normothermic at 37°C and 10 animals were cooled to 30°C rectal temperature for 26 h, starting 2 h after the hypoxic-ischemic insult. All hypoxic-ischemic animals were gavage fed to guarantee long-term survival. Neuroprotection was evaluated by magnetic resonance imaging and behavioral testing. Hypothermia significantly reduced the final size of cerebral infarction by 23% at 6 wk after the insult. The most extended tissue rescue was found in the hippocampus (21%, p ϭ 0.031), followed by the striatum (13%, p ϭ 0.143) and the cortex (11%, p ϭ 0.160). Cooling salvaged spatial memory deficits verified at 5 wk of recovery with Morris Water Maze test; whereas circling abnormalities after apomorphine injection and sensory motor dysfunctions on rotating treadmill improved, yet did not reach statistical significance. When compared with controls, hypoxicischemic animals performed worse in all behavioral tests. Hypothermia did not influence functional outcome in controls. Significant correlations between behavioral performance and corresponding regional brain volumes were found. We conclude that 26 h of mild to moderate resuscitative hypothermia leads not only to brain tissue rescue, but most important to long-lasting behavioral improvement throughout brain maturation despite severity of injury and delayed onset of cooling. (1), a profound, hypoxic-ischemic insult in the human fetus and newborn with a high rate of permanent neuropsychological damage and mortality. A severe acute insult may predominantly affect the thalami, the basal ganglia, the hippocampus, and the brain stem with relative preservation of the cerebral cortex and the subcortical white matter (2). After birth, the clinical signs of perinatal asphyxia include neurologic abnormalities such as coma, seizures, or hypotonia, combined with cardiopulmonary compromise. The newly born infant often requires resuscitation and rapid transfer to the tertiary center for injury severity evaluation and neurologic intensive care. Such support could include specific interventions for hypoxic-ischemic encephalopathy 1 or 2 h into the reperfusion phase from cerebral HI. Despite the fact that perinatal asphyxia closely corresponds to experimental models of cerebral HI, where successful neuroprotective interventions were introduced, presently no agent has been proven useful to ameliorate the chronic handicapping conditions of perinatal asphyxia in the clinical setting (3).In the present clinical situation, neuroprotection can at best be initiated hours after perinatal asphyxia. Such a postischemic or resuscitative neuroprotective measure is hypot...

Magnetic resonance imaging with diffusion- and T2-weighted imaging and 31P magnetic resonance spectroscopy was used to investigate the relationship between development of brain edema and alterations of the brain energy metabolism after hypoxia-ischemia (HI) brain injury in 7-d-old rats. The results were correlated with histologic examinations at various times during recovery up to 5 d. Moderate HI, induced by right common carotid artery ligation and subsequent exposure to 8% O2 for 90 min, produced a cytotoxic edema of 52+/-9% brain volume and depressed the ratio of phosphocreatine to inorganic phosphate from 1.43+/-0.21 to 0.11+/-0.09. Within 1 h of reoxygenation, the edema decreased to 4+/-2% of brain volume, demarcating the core of the lesion. At 5 h of recovery, a secondary cytotoxic edema together with a newly developing vasogenic edema expanded again, reaching its maximal extent of 45+/-10% brain volume at around 24 h. The ratio of phosphocreatine to inorganic phosphate recovered slowly, reaching 1.12+/-0.27 around 13 h. Thereafter it declined again in a manner analogous to the observations made in human newborns after severe perinatal asphyxia, reaching trough values of 0.48+/-0.22 around 24 h after HI. At the cellular level, the vast majority of neuronal death occurred before 15 h. Subsequently, strong glial activation lasted 2-3 d after HI. At 5 d, a cystic infarction of 35+/-12% brain volume was found. We conclude that the biphasic evolution of brain edema and energy metabolism reflects early neuronal and late glial damage in response to moderate HI injury. Therefore, the secondary energy breakdown reflects glial activation and subsequent glial death.

We hypothesized that creatine (Cr) supplementation would preserve energy metabolism and thus ameliorate the energy failure and the extent of brain edema seen after severe but transient cerebral hypoxia-ischemia (HI) in the neonatal rat model. Six-day-old (P6) rats received subcutaneous Cr monohydrate injections for 3 consecutive days (3 g/kg body weight/day), followed by ³¹P-magnetic resonance spectroscopy (MRS) at P9. In a second group, P4 rats received the same Cr dose as above for 3 days prior to unilateral common carotid artery ligation followed 1 h later by 100 min of hypoxia (8% O₂) at P7. Rats were maintained at 37°C rectal temperature until magnetic resonance imaging was performed 24 h after HI. Cr supplementation for 3 days significantly increased the energy potential, i.e. the ratio of phosphocreatine to β-nucleotide triphosphate (PCr/βNTP) and PCr/inorganic phosphate (PCr/Pi) as measured by ³¹P-MRS. Rats with hemispheric cerebral hypoxic-ischemic insult that had received Cr showed a significant reduction (25%) of the volume of edemic brain tissue compared with controls as calculated from diffusion-weighted images (DWI). Thus, prophylactic Cr supplementation demonstrated a significant neuroprotective effect 24 h after transient cerebral HI. We hypothesize that neuroprotection is probably due to the availability of a larger metabolic substrate pool leading to a reduction of the secondary energy failure because DWI has been reported to correlate with the PCr/Pi ratio in the acute phase of injury. Additional protection by Cr may be related to prevention of calcium overload, prevention of mitochondrial permeability transition pore opening and direct antioxidant effects.

Secondary brain damage after transient cerebral hypoxia-ischemia (HI) is caused by a cascade of cellular events. In this study, complementary methods of magnetic resonance imaging and histochemistry were used to investigate the formation of cytotoxic and vasogenic edema during secondary brain damage induced by transient HI in 7-d-old rats. To elicit injury, 21 rats underwent right common carotid artery ligation followed by 1.5 h of 8% O2 exposure. Sequential apparent diffusion coefficient (ADC) and transversal relaxation time (T2) weighted magnetic resonance imaging were recorded for up to 3 d in 13 7-d-old rats. Eight animals were killed at various intervals between the end of HI and 21 h of recovery to perform histochemical assays using neuronal and astrocytic markers. Changes of the ADC revealed a biphasic function for the evolution of cytotoxic edema during the recovery period. At the end of HI, the ADC in the ipsilateral cortex was significantly decreased. Upon reoxygenation, it returned transiently to normal followed by a secondary, although less pronounced, decline after 8-48 h. After this, the ADC rose steadily. From 8 h of recovery, the proportion of vasogenic edema steadily increased as indicated by the T2 prolongation. At 21 h, the majority of glial cells showed immunoreactivity for glial fibrillary acidic protein and were of larger size, whereas the neurons were apoptotic. These results indicate that the delayed cerebral injury is accompanied by late glial swelling in conjunction with an enlarged interstitial space due to cell damage.

The effect of 24h of hypothermic recovery on moderate hypoxic-ischemic brain damage in P7-rats was investigated for 42d after the insult, using magnetic resonance and histopathology. Occlusion of right common carotid artery and 90min exposure to 8% O2 at 37°C body temperature produced cytotoxic edema of 51(±11)% brain volume (BV) and depression of brain energy metabolism (PCr/Pi) from 1.43(±0.21) to 0.14(±0.11). During recovery, the body temperature was reduced to 30°C for 24h in 36 animals, but was kept at 37°C in 34 animals. The edema waned upon reoxygenation leaving only the core lesion at 2h, but reappeared reaching a maximal extent of 11±8% BV under hypothermia compared to 45(±10)% under normothermia at around 24h. PCr/Pi recovered transiently within 13h and declined again to 1.07(±0.19) under hypothermia and to 0.48(±0.22) under normothermia at around 24h.Hypothermia led to significant long term brain protection, leaving permanent tissue damage of 12(±6)% BV compared to 35(±12)% BV under normothermia. However, animals with severe initial injury developed large infarctions, despite hypothermic treatment. Even then, the time to develop infarction was significantly prolonged, leaving the opportunity for additional therapeutic intervention.