Heat stress-induced memory impairment is associated with neuroinflammation in mice

Abstract

Background: Heat stress induces many pathophysiological responses and has a profound impact on brain structure. It has been demonstrated that exposure to high temperature induces cognitive impairment in experimental animals and humans. Although the effects of heat stress have long been studied, the mechanisms by which heat stress affects brain structure and cognition not well understood.

Methods: In our longitudinal study of mice exposed to heat over 7, 14, or 42 days, we found that heat stress time dependently impaired cognitive function as determined by Y-maze, passive avoidance, and novel object recognition tests. To elucidate the histological mechanism by which thermal stress inhibited cognitive abilities, we examined heat stress-induced inflammation in the hippocampus.

Results: In mice subjected to heat exposure, we found: 1) an increased number of glial fibrillary acid protein (GFAP)- and macrophage-1 antigen (Mac-1)-positive cells, 2) up-regulated nuclear factor (NF)-κB, a master regulator of inflammation, and 3) marked increases in cyclooxygenase-2 (COX-2), inducible nitric oxide synthase (iNOS), and cytokine interleukin (IL)-1β and tumor necrosis factor (TNF)-α in the mouse hippocampus. We also observed that neuronal and synaptic densities were degenerated significantly in hippocampal regions after heat exposure, as determined by histological analysis of neuronal nuclei (NeuN), postsynaptic density protein 95 (PSD-95), and synaptophysin expression. Moreover, in heat-exposed mice, we found that the number of cells positive for doublecortin (DCX), a marker of neurogenesis, was significantly decreased compared with control mice. Finally, anti-inflammatory agent minocycline inhibited the heat stress-induced cognitive deficits and astogliosis in mice.

Conclusions: Together, these findings suggest that heat stress can lead to activation of glial cells and induction of inflammatory molecules in the hippocampus, which may act as causative factors for memory loss, neuronal death, and impaired adult neurogenesis.

Fig. 1

Effects of heat stress on learning and memory in mice. (a) After mice (n = 12) were exposed to heat for 7, 14, or 42 days, we determined the percentage of spontaneous alternations and the number of arm entries in the Y-maze. (b) Cognitive function was also assessed using a passive avoidance task at different days after heat exposure. Columns represent the latency period for entering the dark box in the acquisition test and in the retention test, 24 h later. (c) Memory function after heat stress was determined by a novel object recognition test. Columns indicate the exploration time during the familiarization session and the percentage of time spent near the novel object among the total time spent exploring the objects during the test session. Values are expressed as means ± standard error of the mean (S.E.M.). *p < 0.05, **p < 0.01, and ***p < 0.001 as compared with the control group

Fig. 2

Time-course analysis of heat shock protein 70 (HSP70) and c-fos protein expression in the hippocampus of adult mice following high temperature exposure for 7, 14, or 42 days. Hippocampal lysates were assayed by Western blotting using specific anti-HSP70 and anti-c-fos antibodies. Values are presented as means ± S.E.M. and compared with the control. *p < 0.05 indicates that the mean value is significantly different from the control group

Fig. 3

Time-course analysis of heat stress-induced activation of astrocytes and microglia in mouse hippocampus. The presence of astrogliosis (a) and microgliosis (b) was determined using glial fibrillary acidic protein (GFAP) and macrophage-1 antigen (Mac-1) staining, respectively. Quantification of GFAP- and Mac-1-stained cells was performed by measuring the area fraction of GFAP and Mac-1-immunoreactive cells/areas in the CA3 of the hippocampus. Scale bar = 50 μm. Values are expressed as means ± S.E.M. *** p < 0.001 indicates that the mean value was significantly different from the control group

Fig. 4

Effects of heat exposure on the nuclear factor (NF)-κB expression in hippocampal whole-tissue lysates and nuclear extracts as measured by Western blot analysis. (a) Representative Western blot illustrating the expression of NF-κB in the hippocampus. The graphs display densitometric analyses of the expression ratios of NF-κB/β-actin in whole protein extracts (b) and of NF-κB/ proliferating cell nuclear antigen (PCNA) in nuclear extracts (c) from the hippocampus. Values are expressed as means ± S.E.M. *p < 0.05 and **p < 0.01 as compared with the control group

Fig. 5

Effects of heat stress on cyclooxygenase 2 (COX-2) and inducible nitric oxide synthase (iNOS) induction in the mouse hippocampus. (a) Representative immunohistochemical photomicrographs of COX-2 expression. Scale bar = 50 μm. (b) The graphs display the optical density of COX-2 immunoreactivity in the CA3 region. Values are expressed as means ± S.E.M. *p < 0.05 and *** p < 0.001 as compared with the control group. (c) Representative Western blot showing the expression of COX-2 and iNOS in the hippocampus. The graphs display densitometric analyses of the expression ratios of COX-2/β-actin (d) and iNOS/β-actin (e) in the mouse hippocampus. Values are expressed as means ± S.E.M. *** p < 0.001 as compared with the control group

Fig. 6

Effects of heat stress on IL-1β and TNF-α production in the mouse hippocampus. Male imprinting control region (ICR) mice were exposed to high temperature for 7, 14, or 42 days. Expression of IL-1β (a) and TNF-α (b) was assessed using a sandwich enzyme-linked immunosorbent assay (ELISA). Values are expressed as means ± S.E.M. *p < 0.05, **p < 0.01, and ***p < 0.001 as compared with the control group

Fig. 7

Effects of heat stress on cell death in the hippocampus. (a) Representative pictures of Cresyl violet staining in the granule cell layer and the pyramidal cell layer of the hippocampus. Cresyl violet-stained cells were markedly lower in the heat-treated groups than the control group. The number of cells was reduced significantly in both the CA1 (b) and CA3 (c) regions. Values are expressed as means ± S.E.M. *p < 0.05, **p < 0.01, and ***p < 0.001 as compared with the control group

Fig. 8

Effects of heat stress on neuronal loss in the hippocampus. (a) Representative Western blot showing the expression of neuronal nuclei (NeuN), a marker for neuronal cells, in the hippocampus. (b) Representative pictures of NeuN staining in the granule cell layer and the pyramidal cell layer of the hippocampus. NeuN-stained cells were markedly lower in the heat-treated groups than the control group. The number of cells was reduced significantly in both the CA3 (c) and CA1 (d) regions. Values are expressed as means ± S.E.M. *p < 0.05, **p < 0.01, and ***p < 0.001 as compared with the control group

Fig. 9

Effects of heat stress on synaptic density in the hippocampus. Representative photomicrographs of postsynaptic density protein (PSD)-95 and synaptophysin immunohistochemistry in CA3. Note that compared with control mice, high temperature-exposed mice exhibit decreased PSD-95 and synaptophysin immunoreactivity. Quantification of the average intensity of the immunostaining of (a) PSD-95 and (b) synaptophysin in the CA3. *p < 0.05 versus the control group. Scale bar = 50 μm

Fig. 10

The effects of heat stress on adult neurogenesis in the hippocampus. (a) Representative images of doublecortin (DCX)-stained cells in the dentate gyrus. Scale bar = 50 μm. (b) Quantification of the number of DCX-expressing cells in the subgranular zone (SGZ). The cell number was decreased significantly in heat-exposed mice compared with control mice. *p < 0.05 and ***p < 0.001 as compared with the control group

Fig. 11

Effects of anti-inflammatory agent on heat-stress-induced memory loss and astrogliosis. (a) Cognitive function was assessed using a passive avoidance task after heat exposure and minocycline treatment. Columns represent the latency period for entering the dark box in the acquisition test and in the retention test, 24 h later. (b) The astrogliosis was analyzed and determined using glial fibrillary acidic protein (GFAP) staining. Quantification of GFAP-stained cells was performed by measuring the area fraction of GFAP-immunoreactive cells/areas in the CA3 of the hippocampus. Scale bar = 50 μm. Values are expressed as means ± S.E.M. *** p < 0.001 indicates that the mean value was significantly different from the control group