Pertubation of intracellular Ca2+(Ca2+)i-regulation during hypoxia-ischemia (HI) is believed to be a critical event in the pathophysiology of brain injury. There is, however, no data available on in vivo changes of (Ca2+)i during or after HI in immature animals. Young rats (n=11) at postnatal day (PND)7-21 were anesthetized with isofluran/urethane. The skull was opened and the parietal cortex of the right hemisphere was exposed (6 × 6 mm window) and the Ca2+-sensitive fluorescence dye Rhod 2-AM (40 μM in 1% Agar gel in Ringer solution) was applied for 30 min and then replaced by Ringer solution in 1% gelatine for 1 h. Using the modified Levine model, the rat pups were subjected to 1h of HI and 3 h of recovery under urethane (1 mg/g ip) anesthesia. Point-analysis demonstrated small increments of(Ca2+)i during HI followed by an increase during reperfusion being more marked at PND 18-21 than in the younger (PND 7-13) rats. Color imaging showed that the cortical area with increase of (Ca2+)i was larger at PND 18-21 than at PND 7-13. Furthermore, differential analysis of images revealed a unidirectional or spreading movement of(Ca2+)i across the cortical surface during the early phase of HI in some animals.

In conclusion, it appears to be technically feasible to apply the Rhod 2-AM macroimaging technique to study changes of (Ca2+)i in response to HI in young rats.