Effects of Hyperglycemia on the Time Course of Changes in Energy Metabolism and pH during Global Cerebral Ischemia and Reperfusion in Rats: Correlation of 1H and 31P NMR Spectroscopy with Fatty Acid and Excitatory Amino Acid Levels

The effects of hyperglycemia on the time course of changes in cerebral energy metabolite concentrations and intracellular pH were measured by nuclear magnetic resonance (NMR) spectroscopy in rats subjected to temporary complete brain ischemia. Interleaved 31P and 1H NMR spectra were obtained every 5 min before, during, and for 2 h after a 30-min bilateral carotid occlusion preceded by permanent occlusion of the basilar artery. The findings were compared with free fatty acid and excitatory amino acid levels as well as with cations and water content in funnel-frozen brain specimens. One hour before occlusion, nine rats received 50% glucose (12 ml/kg i.p.) and five received 7% saline (12 ml/kg i.p.). Before ischemia, there were no differences in cerebral metabolite levels or pH between hyperglycemic rats and controls. During the carotid occlusion, the lactate/ N-acetylaspartate (Lac/NAA) peak ratio was higher (0.73–1.48 vs. 0.56–0.82; p < 0.05) and pH was lower (<6.0 vs. 6.45 ± 0.05; p < 0.05) in the hyperglycemic rats than in the controls. Phosphocreatine and adenosine triphosphate were totally depleted in both groups. Within 5–15 min after the onset of reperfusion, the Lac/NAA peak ratio increased further in all rats; however, only in extremely hyperglycemic rats (serum glucose > 960 mg/dl) did the lactic acidosis progress rather than recover later during reperfusion. Total free fatty acid and excitatory amino acid levels, but not cation concentration or water content, in brain correlated with serum glucose levels during and after ischemia and with NMR findings after 2 h of reperfusion. Although profound hyperglycemia (serum glucose of 970–1,650 mg/dl) appears to be associated with progression of anaerobic glycolysis and failure of cerebral energy metabolism to recover after temporary complete brain ischemia and with postischemic excitotoxic and lipolytic reactions thought to participate in delayed cellular injury, severe hyperglycemia (490–720 mg/dl) was associated with recovery of energy metabolism.

Astrocytic Acidosis in Hyperglycemic and Complete Ischemia

Nearly complete brain ischemia under normoglycemic conditions results in death of only selectively vulnerable neurons. With prior elevation of brain glucose, such injury is enhanced to include pancellular necrosis (i.e., infarction), perhaps because an associated, severe lactic acidosis preferentially injures astrocytes. However, no direct physiologic measurements exist to support this hypothesis. Therefore, we used microelectrodes to measure intracellular pH and passive electrical properties of cortical astrocytes as a first approach to characterizing the physiologic behavior of these cells during hyperglycemic and complete ischemia, conditions that produce infarction in reperfused brain. Anesthesized rats (n = 26) were made extremely hyperglycemic (blood glucose, 51.4 ± 2.8 m M) so as to create potentially the most extreme acidic conditions possible; then ischemia was induced by cardiac arrest. Two loci more acidic than the interstitial space (6.17–6.20 pH) were found. The more acidic locus [4.30 ± 0.19 (n = 5); range: 3.82–4.89] was occasionally seen at the onset of anoxic depolarization, 3–7 min after cardiac arrest. The less acidic locus [5.30 ± 0.07 (n = 53); range 4.46–5.93)] was seen 5–46 min after cardiac arrest. A small negative change in DC potential [8 ± 1 mV (n = 5); range –3 to –12 mV and 7 ± 2 mV (n = 53); range + 3 to –31 mV, respectively] was always seen upon impalement of acidic loci, suggesting cellular penetration. In a separate group of five animals, electrical characteristics of these cells were specifically measured (n = 17): membrane potential was –12 ± 0.2 mV (range –3 to –24 mV), input resistance was 114 ± 16 MΩ (range 25–250 MΩ), and time constant was 4.4 ± 0.4 ms (range 3.0–7.9 ms). Injection of horseradish peroxidase into cells from either animal group uniformly stained degenerating astrocytes. These findings establish previously unrecognized properties of ischemic astrocytes that may be prerequisites for infarction from nearly complete ischemia: the capacity to develop profound cellular acidosis and a concomitant reduction in cell membrane ion permeability.

Carbonic acid buffer changes during complete brain ischemia

Simultaneous measurements of tissue PCO2 (PtCO2), interstitial H+ concentration ([H+]o), and tissue lactate content were used to examine changes in interstitial HCO3- concentration ([HCO3-]o) during complete ischemia. In normoglycemic rats (blood glucose of 6-8 mM; neocortical ischemic-induced lactate content 8-12 mmol/kg) [H+]o increased from 7.22 +/- 0.02 to 6.79 +/- 0.02 pH (n = 3). By contrast, in hyperglycemic rats (blood glucose 18-75 mM; ischemic-induced lactate content 19-31 mmol/kg) [H+]o rose by a significantly larger amount to 6.19 +/- 0.02 pH (n = 7). Given that HCO3- is the predominant interstitial H+ buffer, changes in peak PtCO2 show why peak [H+]o were bimodally distributed compared with lactate content. Between 8 and 12 mmol/kg lactate, when peak PtCO2 rose from 99 to 186 Torr but [H+]o was constant at 6.79 pH, calculated [HCO3-]o increased from 11.9 to 21.9 mM. Then after transitional changes, peak PtCO2 and [H+]o remained constant at 389 +/- 9 Torr (n = 7) and 6.19 pH despite the fact that tissue lactate ranged from 19 to 31 mmol/kg lactate, respectively; [HCO3-]o must have remained constant at 12.3 +/- 0.7 mM (n = 7). Since ischemic brain continued to produce another 12 more mmol/kg of lactic acid above 19 mmol/kg lactate without further changes in PtCO2 or [H+]o, H+ and HCO3- must have been heterogeneously compartmented. The continued lactic acid production occurred in a compartment that occupied 36% of neocortical space. This compartment is likely to represent glial cells.