Glutamate excitoxicity is the key molecular mechanism which is influenced by body temperature during the acute phase of brain stroke

Abstract

Glutamate excitotoxicity, metabolic rate and inflammatory response have been associated to the deleterious effects of temperature during the acute phase of stroke. So far, the association of temperature with these mechanisms has been studied individually. However, the simultaneous study of the influence of temperature on these mechanisms is necessary to clarify their contributions to temperature-mediated ischemic damage. We used non-invasive Magnetic Resonance Spectroscopy to simultaneously measure temperature, glutamate excitotoxicity and metabolic rate in the brain in animal models of ischemia. The immune response to ischemia was measured through molecular serum markers in peripheral blood. We submitted groups of animals to different experimental conditions (hypothermia at 33°C, normothermia at 37°C and hyperthermia at 39°C), and combined these conditions with pharmacological modulation of glutamate levels in the brain through systemic injections of glutamate and oxaloacetate. We show that pharmacological modulation of glutamate levels can neutralize the deleterious effects of hyperthermia and the beneficial effects of hypothermia, however the analysis of the inflammatory response and metabolic rate, demonstrated that their effects on ischemic damage are less critical than glutamate excitotoxity. We conclude that glutamate excitotoxicity is the key molecular mechanism which is influenced by body temperature during the acute phase of brain stroke.

Figure 1. Brain temperature in the ischemic and the contralateral (healthy) brain hemispheres, as determined from Magnetic Resonance measurements on ischemic animals with body temperatures of 39°C (top), 37°C (middle) and 33°C (bottom).

Statistical significances: *P<0.05.

Figure 2. MRI images and quantification of infarct volumes at 90 min (DWI MR images) and 7 days (density maps from T2-MRI images) after the induction of cerebral ischemia in animals submitted to hypothermia (33°C, n = 8), normothermia (37°C, n = 7), hyperthermia (39°C, n = 7), hypothermia and 7,27 g/kg of glutamate (Glu) (n = 8) and hyperthermia (n = 8) and 35 mg/Kg oxaloacetate (OAA). ).

Color-coded density maps were generated from T2 MR images from one brain slice for each animal (Located at Bregma +0.7 mm). The color code indicates the percentage of animals within a group that shows a lesion in each particular pixel. The grey image used as background is the result of the mean value of all co-registered images per group. Infarct volumes (% of hemispheric volume affected by lesion) are shown as mean ± S.D. Statistical significances: ***P<0.001, **P<0.01 compared to normothermic animals. ##P<0.01 compared to non-treated hypothermic animals, §§P<0.01 compared to non-treated hyperthermic animals.

Figure 3. Pseudocolor T2 weighted MR images of a representative brain of each group.

Images include 7 consecutive 1-mm thick coronal slices, showing the extent of the ischemic damage throughout the whole brain (position of each slice compared to Bregma, to Paxinos and Watson , is presented at the bottom).

Figure 4. Blood glutamate levels (µM) in healthy and in ischemic animals submitted to hypothermia (33°C, n = 8), normothermia (37°C, n = 7), hyperthermia (39°C, n = 7), hypothermia plus 7.27 g/Kg glutamate (Glu) (n = 8) and hyperthermia plus 35 mg/Kg oxaloacetate (OAA) (n = 8) (A).

Basal glutamate levels were determined in all groups under normothermic conditions. Ischemic levels were determined 90 min after occlusion. Levels are shown as mean±S.D. Statistical significances: **P<0.01, *P<0.05 compared to normothermic animals. Brain glutamate levels (corrected as the ratio between glutamate and creatine MRS peak) for the same groups of animals (B). Glutamate levels were determined during the occlusion (90 min) and after reperfusion (180 min). Levels are shown as mean±S.D. ***P<0.001, *P<0.05 compared to normothermic animals.

Figure 5. Blood Interleukin-6 levels (in pg/mL) in animals submitted to hypothermia (33°C, n = 8), normothermia (37°C, n = 7), hyperthermia (39°C, n = 7), hypothermia plus 7.27 g/Kg glutamate (Glu) (n = 8) and hyperthermia plus 35 mg/Kg oxaloacetate (OAA) (n = 8) (A).

Lactate levels during occlusion period (expressed as the ration glutamate/creatine-phosphocreatine peak areas on MRS spectra) on the ischemic brain of the same group of animals (B).