Myron Rosenthal

Rapid preconditioning protects rats against ischemic neuronal damage after 3 but not 7 days of reperfusion following global cerebral ischemia

Earlier studies indicated that sublethal ischemic insults separated by many hours may “precondition” and, thereby, protect tissues from subsequent insults. In Wistar rats, we examined the hypothesis that ischemic preconditioning (IPC) can improve histopathological outcome even if the “conditioning” and “test” ischemic insults are separated by only 30 min. Normothermic (36.5–37°C) global cerebral ischemia was produced by bilateral carotid artery ligation after lowering mean systemic blood pressure. The conditioning ischemic insult lasted 2 min and was associated with a time sufficient to provoke “anoxic depolarization” (AD) (i.e., the abrupt maximal increase in extracellular potassium ion activity). After 30 min of reperfusion, 10-min test ischemia was produced, and histopathology was assessed 3 and 7 days later. After 3 days of reperfusion, neuroprotection was most robust in the left lateral, middle and medial subsections of the hippocampal CA1 subfield and in the cortex, where protection was 91, 76, 70 and 86%, respectively. IPC also protected the right lateral, middle and medial subsections of the hippocampal CA1 region. These data demonstrate that neuroprotection against acute neuronal injury can be achieved by conditioning insults followed by only short (30 min) periods of reperfusion. However, neuroprotection almost disappeared when reperfusion was continued for 7 days. When test ischemia was decreased to 7 min, a clear trend of neuroprotection by IPC was observed. These data suggest that subsequent rescue of neuronal populations could be achieved with better understanding of the neuroprotective mechanisms involved in this rapid IPC model.

Responses of electrical potential, potassium levels, and oxidative metabolic activity of the cerebral neocortex of cats

We measured simultaneously the oxidative metabolic activity, monitored as the tissue fluorescence attribute to intramitochondrial NADH, the extracellular potassium level with ion-selective microelectrodes, and the focal extracellular electrical potential, of one site in intact cerebral cortex of cats. When the cerebral was stimulated by trains of repeated electric pulses applied either directly to its surface or to an afferent pathway, the corrected cortical fluorescence (F-R) declined indicating oxidation of NADH, the activity of extracellular potassium [K+]o increased, and the extracellular potential (Vec) shifted in the negative direction. When mild to moderate stimuli not exceeding 10-15 sec in duration were used, a 3-fold correlation was found between these three variables. The regression of F-R over either Vec, or over log [K+]o had a positive ordinal intercept. The results are in agreement with earlier suggestions 4,24,25,43,45,46 that (a) much but not all the oxidative metabolic response of cortex to electrical stimulation is expended in restoring disturbed ion balance; and (b) that sustained shifts of potential (SP) in response to repetitive electrical stimulation are generated by glia cells depolarized by excess potassium. The magnitude of SP shifts associated with a given elevation of [k+]o are smaller in cerebral cortex than in spinal cord48,49. The correlation of F-R with [K+]o breaks down when pathologic processes of either seizure activity or spreading depression set in. During paroxysmal activity [K+]o tends to remain confined below 10-12 mM, a level observed in non-convulsing cortex as well, but oxidation of NADH progresses beyond that seen in non-convulsing cortex as well, but oxidation of NADH progresses beyond that seen in non-convulsing tissue. This observation is hard to reconcile with the suggestion that excess potassium is a factor in the generation of seizures, at least of the type observed in this study. When [K+]o levels exceeded 10-12 mM, spreading depression invariably followed at least under the unanesthetized condition in these experiments. During spreading depression [K+]o levels rose to exceed 30 mM, sometimes 80 mM. NADH was oxidized during spreading depression to a level comparable to that seen in seizures. The observations are compatible with the suggestion13 that spreading depression occurs whenever the release of potassium into extracellular fluid is overloading its clearance therefrom.

Cytochrome C Is Released From Mitochondria Into the Cytosol After Cerebral Anoxia or Ischemia

Mitochondrial dysfunction may underlie both acute and delayed neuronal cell death resulting from cerebral ischemia. Specifically, postischemic release of mitochondrial constituents such as the pro-apoptotic respiratory chain component cytochrome c could contribute acutely to further mitochondrial dysfunction and to promote delayed neuronal death. Experiments reported here tested the hypothesis that ischemia or severe hypoxia results in release of cytochrome c from mitochondria. Cytochrome c was measured spectrophotometrically from either the cytosolic fraction of cortical brain homogenates after global ischemia plus reperfusion, or from brain slices subjected to severe hypoxia plus reoxygenation. Cytochrome c content in cytosol derived from cerebral cortex was increased after ischemia and reperfusion. In intact hippocampal slices, there was a loss of reducible cytochrome c after hypoxia/reoxygenation, which is consistent with a decrease of this redox carrier in the mitochondrial pool. These results suggest that cytochrome c is lost to the cytosol after cerebral ischemia in a manner that may contribute to postischemic mitochondrial dysfunction and to delayed neuronal death.

Effects of respiratory gases on cytochrome a in intact cerebral cortex: is there a critical PO2?

Changes in the redox level of cytochrome a and in the amount of oxygenated hemoglobin were measured by dual wavelength reflectance spectrophotometry in the intact cerebral cortex of cats (cerveau isolé preparation) and in unanesthetized rabbits with chronically implanted cranial windows. Increases in inspired oxygen were accompanied by an increase in the oxidation level of cytochrome a and an increase in the amount of oxygenated hemoglobin in the optical field. These changes were larger in the presence of 5% CO2. Reduction of the inspired oxygen concentration produced a decrease in the oxidation/reduction ratio of cytochrome a and a disoxygenation of hemoglobin. The presence of CO2 at these lower oxygen levels diminished the reduction of cytochrome a and the disoxygenation of hemoglobin. These data indicate that, in the resting subject, the reduction levels of cytochrome a are well above the low values seen in isolated mitochondria. They also indicate that the blood supply to the cerebral cortex is regulated at a level of slight hypoxia.

Photothrombotic Infarction Triggers Multiple Episodes of Cortical Spreading Depression in Distant Brain Regions

The purposes of this study were to determine whether cortical spreading depression occurs outside of the infarct produced by photothrombotic vascular occlusion, and also the direction of spreading. Focal cerebral thrombotic infarction was produced by irradiating the exposed skull of anesthetized rats with green light (560 nm) following systemic injection of rose bengal dye. At proximal sites (∼2 mm anterior to the infarct border), transient, severe hyperemic episodes (THEs) lasting 1–2 min were intermittently recorded. THE frequency was greatest in the first hour and declined over a 3-h period. THEs were accompanied (and usually preceded) by a precipitous rise in [K+]0 (from ∼3 to >40 mM) and were associated with increases in local tissue oxygen tension (tPO2). Following the rise in [K+]0, clearance of [K+]0 to its pre-THE baseline preceded baseline recovery of CBF. These data indicate that THEs were reactive to physiologic events resembling cortical spreading depression (CSD), which provoked increased demand for oxygen and blood flow, and which spread from proximal sites to areas more distal (∼4 mm) from the rim of the evolving infarct. MK-801 (1 mg/kg, i.v.) inhibited subsequent CSD-like episodes. We conclude that photothrombosis-induced ischemia provoked CSD which was triggered either within the infarct core or in the infarct rim and spread to more distal sites. Whether multiple episodes of CSD during infarct generation are responsible for the remote consequences of focal brain injury remains to be determined.

Effects of age on brain oxidative metabolism in vivo

Non-invasive optical techniques were used to monitor the effects of increasing cerebral energy demand on metabolic capabilities and vascular reactivity in young and aged brain. Low level of electrical stimulation of the cortex, in both young (4--7 months) and aged (24--28 months) rat brain, were accompanied by transient oxidations of NADH and cytochrome oxidase (a,a3) as measured by microfluorometry and reflection spectrophotometry respectively. Stimulation sufficient to produce spreading cortical depression was accompanied by an oxidation of both NADH and cytochrome a,a3 in young brain together with an increase in local blood volume. There was either no change or a slight disoxygenation of hemoglobin. In aged brain, however, spreading depression was associated with an oxidation of NADH and a reduction of cytochrome a,a3 together with an increase in local blood volume and an oxygenation of hemoglobin. The present results indicate that the relationship between microcirculation and the terminal oxidase step of the respiratory chain is altered in aged brain when energy demand is high.

Depth profile of local oxygen tension and blood flow in rat cerebral cortex, white matter and hippocampus

Microregional oxygenation and blood flow were measured in rat cerebral cortical lamina, subcortical white matter and hippocampus. This was done to examine relationships between oxygenation and blood flow at a local level, to determine effects of craniotomy, and to consider whether flow/oxygenation relationships might be predictive of selective vulnerability known to accompany anoxia or ischemia. Blood flow and oxygen tension were measured with closely apposed polarographic microelectrodes. Oxygen tension was highest in white matter, lower in the region of cortical laminae IV-V and lowest in the hippocampus. Blood flow in the hippocampus was higher than that in white matter or laminae IV-V of cerebral cortex. Ratios of blood flow to oxygenation were similar throughout the cortex, higher in white matter but oxygenation in hippocampus was significantly less than expected from measurement of hippocampal blood flow reflecting increased oxygen consumption or relative hypoxia due to increased diffusion distances for oxygen in hippocampus. Comparison of data from closed vs opened skull animals indicated that diffusion of oxygen and hydrogen influenced data to approximately 1000 micron below the cerebral surface.

Antioxidants, mitochondrial hyperoxidation and electrical recovery after anoxia in hippocampal slices

Cerebral injury may occur not only during brain ischemia but also during reperfusion afterward. A characteristic event during reperfusion after cerebral ischemia, or reoxygenation after anoxia in hippocampal slices, is hyperoxidation of the electron carriers of the mitochondrial respiratory chain. Earlier studies suggested that mitochondrial hyperoxidation was produced by an oxyradical mechanism and was linked to neuronal damage. Present studies sought to test this hypothesis by determining whether antioxidants could suppress mitochondrial hyperoxidation and improve electrical recovery after anoxia in hippocampal slices. Both 500 microM ascorbate and 50 microM glutathione decreased post-anoxic hyperoxidation of NADH and improved electrical recovery in hippocampal slices. These data support a role of oxygen free radicals in promoting post-anoxic mitochondrial hyperoxidation and electrical failure, and suggest that these effects of anoxia or ischemia may be linked.

Mitochondrial Hyperoxidation Signals Residual Intracellular Dysfunction After Global Ischemia in Rat Neocortex

Reperfusion after global ischemia (10–60 min in duration) in rat neocortex most commonly provoked transient hyperoxidation of mitochondrial electron carriers, tissue hyperoxygenation, and CBF hyperemia. These responses were normally accompanied by recovery of K+ homeostasis and EEG spike activity. Goals of this research were to understand putative relationships among these postreperfusion events with special emphasis on determining whether mitochondrial hyperoxidation results from intracellular changes that may modulate residual damage. The amplitude of postischemic mitochondrial hyperoxidation (PIMHo) did not increase when CBF increased above an apparent threshold during reperfusion, and tissue hyperoxygenation was not required for PIMHo to occur or to continue. These findings suggest that PIMHo is not merely a response to increased CBF and tissue hyperoxygenation; rather, PIMHo is modulated, at least in part, by residual intracellular derangements that limit mitochondrial electron transport. This suggestion was supported by observations that NAD became hyper-oxidized after reoxygenation in anoxic hippocampal slices. Also, PIMHo occurred and subsequently resolved in many animals, but K+o never was cleared fully to baseline and/or EEG spike activity never was evident. One suggestion is that PIMHo signals or initiates residual intracellular derangements that in turn impair electrical and metabolic recovery of cerebral neurons after ischemia; an alternative suggestion is that PIMHo and tissue hyperoxygenation are not the sole factors modulating the immediate restoration of electrical activity after ischemia. Present data also support the following: Decreased oxygen consumption, despite adequate oxygen delivery, likely contributes to tissue hyperoxygenation after ischemia; and mitochondrial hyperoxidation is modulated by a limitation in the supply of electrons to the mitochondrial respiratory chain.

In situ studies of oxidative energy metabolism during transient cortical ischemia in cats

Changes in oxidative energy metabolism were monitored noninvasively during periods of transient cortical ischemia in cats by means of fluorometric observation of the redox level of intramitochondrial NADH and by dual wavelength reflectance spectrophotometry of cytochrome a. The latter technique also allowed measurements of the oxygenation state of hemoglobin and of blood volume in the optical field. We report here that incomplete ischemia is accompanied by increased levels of NADH and reduced cytochrome a, an increase in the unloading of O2 from hemoglobin and a decrease in the blood volume, but these all turn back toward baseline with the establishment of “collateral” circulation. Complete ischemia is accompanied by sustained increased levels of reduced NAD and cytochrome a, the extent of which equaled the reduction levels produced by terminal anoxia. When short ischemic episodes were repeated, the rates of return of NAD and cytochrome a to baseline redox levels were faster after each successive ischemia, while blood volume returned at the same rate and hemoglobin saturation returned more slowly to its original state of oxygenation. We interpret that the primary lesion of short ischemic episodes is the uncoupling of oxidative phosphorylation.