Permeability of the blood-brain barrier depends on brain temperature

Abstract

Increased permeability of the blood-brain barrier (BBB) has been reported in different conditions accompanied by hyperthermia, but the role of brain temperature per se in modulating brain barrier functions has not been directly examined. To delineate the contribution of this factor, we examined albumin immunoreactivity in several brain structures (cortex, hippocampus, thalamus and hypothalamus) of pentobarbital-anesthetized rats (50 mg/kg i.p.), which were passively warmed to different levels of brain temperature (32-42 degrees C). Similar brain structures were also examined for the expression of glial fibrillary acidic protein (GFAP), an index of astrocytic activation, water and ion content, and morphological cell abnormalities. Data were compared with those obtained from drug-free awake rats with normal brain temperatures (36-37 degrees C). The numbers of albumin- and GFAP-positive cells strongly correlate with brain temperature, gradually increasing from approximately 38.5 degrees C and plateauing at 41-42 degrees C. Brains maintained at hyperthermia also showed larger content of brain water and Na(+), K(+) and Cl(-) as well as structural abnormalities of brain cells, all suggesting acute brain edema. The latter alterations were seen at approximately 39 degrees C, gradually progressed with temperature increase, and peaked at maximum hyperthermia. Temperature-dependent changes in albumin immunoreactivity tightly correlated with GFAP immunoreactivity, brain water, and numbers of abnormal cells; they were found in each tested area, but showed some structural specificity. Notably, a mild BBB leakage, selective glial activation, and specific cellular abnormalities were also found in the hypothalamus and piriform cortex during extreme hypothermia (32-33 degrees C); in contrast to hyperthermia these changes were associated with decreased levels of brain water, Na(+) and K(+), suggesting acute brain dehydration. Therefore, brain temperature per se is an important factor in regulating BBB permeability, alterations in brain water homeostasis, and subsequent structural abnormalities of brain cells.

Fig. 1

Brain sections showing regions and cortical areas used for quantitative analyses of albumin and GFAP immunoreactivities and structural cell abnormalities. 1, cingulate cortex; 2, parietal cortex; 3, temporal cortex; 4, piriform cortex; 5, hippocampus; 6, thalamus; and 7, hypothalamus.

Fig. 2

Changes in temperature during pentobarbital anesthesia and the effects of body warming. A, B, and D show, respectively, changes in absolute temperature (NAcc, temporal muscle and skin), relative temperature, and locomotion before and for 15 min after the injection of sodium pentobarbital (50 mg/kg, ip). C shows brain-muscle and skin-muscle temperature differentials. Filled symbols show values significantly different from the pre-injection baseline. The effect of the drug was evaluated with one-way ANOVA with repeated measures (F_16,271=107.75, 47.81 and 16.76 for NAcc, muscle and skin, respectively; F_14,239=27.49 for locomotion; each p<0.001). E shows changes in NAcc temperature during pentobarbital anesthesia in all animals, divided into three groups (hyperthermia, normothermia and hypothermia) according to their final temperatures. Values from −10 to +15 min are means (±SD) for all 16 rats and subsequent values (mean±SD) are shown separately for rats divided into three groups. Circles after 90 min are individual values of NAcc temperature in each of 16 experimental rats.

Fig. 3

Temperature dependence of albumin and GFAP immunoreactivity and cellular brain abnormalities. Data are shown for the sum of all tested structures (A; open circles show original data and closed circles show mean averages), individual brain structures (B), and specific cortical areas (C).

Fig. 4

Original examples of brain slices of the piriform cortex showing albumin immunoreactivity (a–c), GFAP immunoreactivity (d–f) and Nissl staining (g–i) in pentobarbital-anesthetized rats maintained at different brain temperatures (a, d, g: normothermia; b, e, h: hypothermia; c, f, i: hyperthermia). While in normothermic conditions, albumin-labeled cells were largely absent, with only occasional immunoreactivity in the neuropil (a), massive leakage of albumin was seen in the brain taken at extreme hyperthermia (c). There were multiple albumin-positive cells (arrows), which were often distorted and located in areas showing sponginess, edema, and strong immunoreactivity of the neuropil. Upregulation of albumin-positive neurons with mild immunostaining in the surrounding neuropil was seen during extreme hypothermia (b, arrows). In contrast to mild GFAP immunoreactivity in normothermic (d) and hypothermic (e) conditions, many, strongly GFAP-positive astrocytes (arrows) were found during hyperthermia; these cells were preferentially located around the small and large microvessels in the regions showing edema and sponginess. The Nissl staining at normothermic conditions showed a clear nucleus and triangular shaped neurons with clear cell cytoplasm (g, arrows). Pronounced degeneration of cell nuclei and pyknosis and edematous neuropil were observed in the brain taken at hyperthermia (i, arrowheads); most degenerated neurons were seen in areas showing marked sponginess. Although most cortical cells in the brain taken at extreme hypothermia appear to be normal (h, arrow), few showed signs of degeneration, cell body shrinkage and more compact neuropil than in normothermic brains. Bars in each raw are 100 μm.

Fig. 5

Nissl-stained sections from similar areas of the parietal cortex (deep) obtained from rats maintained at different levels of brain temperature (S15=36.05°C; S29=32.30°C; S12=41.80°C). Brain cells during hyperthermia have larger somata and wider axons (arrows) compared to those in normothermia. In contrast, during hypothermia cells are slightly smaller in size, staining is more condensed, and axons are smaller in diameter compared to normothermic conditions (arrows). Bar for each graph is 100 μm.

Fig. 6

Profound changes in structural integrity of the choroids plexus during extreme hyperthermia. All slices were made over lateral ventricles, stained with Hematoxylin-Eosin, and images are shown with equal magnification (bar=200 μm). In contrast to the healthy structure in normothermic conditions, robust desintegration of epithelial cells and profound vacuolization were typical of hyperthermic conditions.

Fig. 7

Temperature dependence of tissue water, Na+, K+, and Cl− shown separately for cortex and thalamus. Horizontal hatched lines show “normal” values evaluated in brains of awake, drug-free rats at normothermia (see Table for quantitative values). Some graphs also show regression lines, regression equations, and coefficients of correlation for the cortex. Each correlation coefficient value is highly significant (p<0.001).

Fig. 8

Correlative relationships between individual brain parameters: the counts of albumin- and GFAP-positive cells evaluated in the brain as a whole (A), the counts of albumin (GFAP-)-positive cells and morphologically abnormal cells (B), the counts of albumin-positive cells and tissue water content (C; evaluated separately in the cortex and thalamus), and tissue water content and amounts of morphologically abnormal cells (D, evaluated separately in the cortex and thalamus). Each graph contains coefficients of correlation and regression lines. Hatched lines in (A) and (B) are lines of equality. Each correlation coefficient value is highly significant (p<0.001).