Anderson, Robert E.

Corticotropin releasing factor is an endogenous neuropeptide released by the hypothalamus that activates the pituitary-adrenocortical system in response to stressful stimuli. It has been demonstrated that corticotropin releasing factor increases the excitability of hippocampal neurons in both in vitro and in vivo studies, which may contribute to neurological injury during hypoxia. The purpose of this study was to determine the effects of corticotropin releasing factor and its synthetic competitive antagonist, alpha-CRF, on neuronal synaptic recovery after a hypoxic insult using the hippocampal slice. Wistar rat hippocampal brain slices (n = 120) were treated with various concentrations (10(-6) to 10(-11)) of corticotropin releasing factor or its synthetic antagonist during a 10-minute hypoxic episode. Extracellular recording of population spikes was used during and after the hypoxic insult to assess neuronal recovery. Corticotropin releasing factor provided dose-dependent neuronal protection with maximum recovery (37.95 +/- 8.71%) occurring at 10(-9) concentrations. The competitive antagonist alpha-CRF provided a similar degree of recovery at 10(-6) concentration, whereas 10(-9) molar concentration of competitive antagonist resulted in 16.84 +/- 7.68% recovery. Corticotropin releasing factor provides moderate protection to hypoxic hippocampal neurons in the brain slice preparation. The mechanism of action is unknown but appears to be a direct neuronal effect. These results support the hypothesis that corticotropin releasing factor may act as an endogenous neuroprotective hormone during hypoxia.

The effects of the novel dihydronaphthyridine Ca 2+ antagonist CI-951 on focal cerebral ischemia were assessed during MCA occlusion in 30 white New Zealand rabbits under 1.0% halothane anesthesia. In vivo brain pH i and focal CBF were measured with umbelliferone fluorescense. Baseline normocapnic brain pH i and CBF were 7.02 ± 0.02 and 48.4 ± 2.9 ml/100 g/min, respectively. In the severe ischemic regions, 15 min postocclusion brain pH i , and CBF were 6.62 ± 0.04 and 14.4 ± 0.7 ml/100 g/min in controls vs. 6.60 ± 0.02 and 12.9 ± 2.3 ml/100 g/min, respectively, in animals destined to receive CI-951. Twenty minutes after MCA occlusion, CI-951 was administered at 0.5 μg/kg/min and brain pH i and CBF were determined in both regions of severe and moderate ischemia for 4 h postocclusion. Control severe ischemic sites demonstrated no significant improvement in brain pH i and only mild increases in CBF over the next 4 h. CI-951 caused significant improvement in both of these parameters. Postocclusion 4 h brain pH i and CBF measured 6.69 ± 0.04 and 18.5 ± 3.2 ml/100 g/min in controls vs. 7.01 ± 0.04 and 41.7 ± 5.3 ml/100 g/min, respectively, in CI-951 animals ( p < 0.001). Similar improvements were observed in moderate ischemic sites. In animals that demonstrated postocclusion EEG attenuation, 75% of CI-951 animals had EEG recovery as compared to 18% in controls. CI-951 may be a useful therapeutic agent for focal cerebral ischemia if histological and outcome studies verify these data.

The time course of changes in cortical tissue pH (pHi) and blood flow during cortical seizures in halothane-anesthetized cats was examined. The clearance of the molecular form of umbelliferone (Um) was used to estimate focal cortical blood flow (CBFu), whereas the ratio of the molecular to the ionic form of the molecule was used to concurrently calculate the local pHi. Resting pHi and flow in normocarbic animals was 7.116 ± 0.008 and 46 ± 8 ml/100 g/min, respectively. Respiratory induced alterations of Paco 2 over a range of 20–60 torr revealed a correlated change in pHi from 7.39 ± 0.05 to 7.01 ± 0.03 and a monotonic increase in the rate of Um clearance (slope 0.89 ± 0.13 ml/100 g/min/torr). Focal electrical stimulation of the cortex resulted in a rapid vasodilation (50% dilation = 1–3 s) of pial arterioles and venules and an increase in Um clearance. pHi showed no significant change until around 10 s. The maximum fall in pHi occurred by 30–60 s (6.85 ± 0.054). Longer intervals of stimulation (10 min) resulted in no further decline in pHi, but upon cessation of stimulation, pHi remained acidotic for poststimulation periods up to 10 min, with a mild but statistically significant acidosis being observed at 20 min. The absolute decline in pH observed following stimulation appeared to be closely regulated, as comparable levels following stimulation were observed during hypocarbia and hypercarbia. These observations thus suggest that pHi regulation during intense cortical activation may be considered in three phases: (1) following the onset of activity, an initial acute regulation of pHi at control levels; (2) an intracellular acidosis of around 6.8, which is closely regulated and which can be readily reversed upon termination of stimulation; and (3) during continued stimulation, a change in state where in spite of no further change in pHi, the ability of the cortex to return to control pHi appears to be significantly impaired.

Intracellular brain pH and indicator tissue perfusion were measured with a lipid-soluble, pH-sensitive fluorescent indicator in 10 rabbits who had either severe or moderate focal ischemia depending on whether the middle cerebral artery was occluded at its main trunk or bifurcation. Preocclusion tissue indicator perfusion was 50.1 ml/100 g/min and intracellular brain pH was 7.03. In severe focal ischemia, immediate postocclusion tissue perfusion was 12.7 ml/100 g/min and intracellular brain pH was 6.64. Four hours after occlusion, the perfusion was 5.2 ml/100 g/min and intracellular brain pH was 6.08. There was EEG and histological confirmation of infarction. In the moderate focal ischemia group, immediate postocclusion flow was 20.0 ml/100 g/min and intracellular brain pH was 6.92. At 3 h, postocclusion tissue perfusion was 22.6 ml/100 g/min and intracellular brain pH was 6.86. Therefore, for the first 3 h, this ischemic penumbra was stable. At the fourth hour, both cerebral tissue perfusion and intracellular brain pH worsened. This suggests that the ischemic penumbra is a dynamic state. The rabbit is a good experimental model for the production of both severe and moderate focal ischemia.

Abstract The alterations in metabolism and cerebral blood flow that occur following transient focal ischemia were studied in rabbits anesthetized with halothane and subjected to transient occlusion of an M2 segment of the middle cerebral artery (MCA). The parameters measured included intracellular brain pH and focal cortical blood flow (fCBF)—assessed by the umbelliferone technique—electrocorticograms, and cortical microcirculatory changes. A gradient of ischemia developed in the cortex between the patent and occluded vessels. Cortical sites with moderate and severely diminished flow were examined as a function of time before and after occlusion. Mean preocclusion fCBF was 50.8 ± 2.1 ml/100 g/min, and brain pH was 6.99 ± 0.04. Following occlusion, fCBF fell to 14.6 ± 2.3 ml/100 g/min, with an intracellular pH of 6.53 ± 0.03 in sites of severe ischemia in the territory of the occluded vessel. Sites between the patent and occluded branches revealed moderate changes in fCBF and intracellular pH of 26.7 ± 3.6 ml/100 g/min and 6.74 ± 0.03 ml/100 g/min, respectively. Sites adjacent to the patent M2 branch remained similar to baseline. Pretreatment intravenously with nimodipine, a dihydropyridine class of Ca2+ channel antagonist, improved flow in the territory of the occluded segment of the middle cerebral artery to 30.6 ± 2.2 ml/100 g/min, while maintaining the brain pH at 6.83 ± 0.03. Similarly, at sites intermediate between the patent and occluded M2 segments of the MCA, cortical blood flow remained at 53.5 ± 4.0 ml/100 g/min, and the pH at 6.95 ± 0.04, in 10 animals pretreated with nimodopine. This effect was apparently achieved by blocking the secondary, ischemia-induced vasoconstriction known to occur in areas of focal incomplete ischemia. The diphenylalkylamine class Ca2+ channel blockers was examined using verapamil. This was found to be without significant effect. The vehicle carrying nimodopine was also without effect. To authenticate further the reliability of MCA occlusion, a separate group of animals was examined with cautery occlusion of the M2 segment of the MCA. Nimodopine had an effect in this group of animals identical to that caused by occlusion of the vessel with a clip. Visual inspection of the cortex in control and treated animals revealed a marked difference in the development of cortical pallor and microcirculatory changes. We conclude that nimodopine protects the conducting vessels against the secondary vasoconstriction known to occur in areas of focal incomplete ischemia, and thus increases collateral flow. This agent may have the potential for brain protection in areas of incomplete focal ischemia from occlusion of a single major vessel.