It is estimated that recent global warming will cause an increase in patients with heatstroke in the near future. However, there is less understanding of potential long-term adverse outcomes, although patients often exhibit neurological deficits8,20.
In this study, we evaluated the neurological deficits using the rotarod test and brain histological evaluations (in the cerebellum) for 9 weeks using our heatstroke model, because disappearing and swelling of the Purkinje cells have been reported in autopsied brain tissues of patients with heatstroke21. Observation of brain diffusion-weight MRI revealed hyperintensity in the bilateral cerebellar medullas in patients with heatstroke22. These reports indicate that Purkinje cell protection is a key therapeutic target in patients with heatstroke.
In our study, the running time was significantly shorter at 3 weeks post heatstroke and gradually returned to baseline at 9 weeks post heatstroke. These results suggest motor function/coordination impairment in our heatstroke model. In patients with multiple sclerosis, cerebellar ataxia commonly occurs; cerebellar neuronal degeneration has been reported23. Therefore, we examined the morphology of the brain by KB staining, but we could not recognize apparent cerebral infarction or hemorrhage. However, the cerebellum showed decreased intensity with KB stain and was recognized as sponge-like in the HS group. Semi-quantification of the KB-stained region revealed that the cerebellar white matter was significantly demyelinated between 1- and 3-weeks post heatstroke in the HS group. However, the intensity did not significantly differ from that of the Con group at 9 weeks post heatstroke, suggesting remyelination. In contrast, demyelination could not be recognized in the corpus callosum during the experiments; the demyelination was cerebellar-specific and might not have recovered until 9 weeks.
The calbindin-positive Purkinje cells in the Con mice were aligned clearly on the Purkinje cell layer equidistantly. The cell numbers had significantly decreased at 1 week post heatstroke and did not recover during the experimental periods, suggesting that the Purkinje cells degenerated. These results, including the rotarod test, suggested that demyelination after heatstroke-induced cerebellar ataxia resulted in impaired motor coordination. These results resemble the outcome seen in human patients with heatstroke8,20−22.
However, myelin recovered 9 weeks post heatstroke along with the motor coordination, although the number of Purkinje cells did not recover during the experimental periods. To explain this discrepancy, we immunostained for PSD95 and synaptophysin to examine synaptic patency and observed the Purkinje layer at a higher magnification. The PSD95 was highly concentrated and tightly bound to the postsynaptic density of type 1 glutamatergic synapses, suggesting its critical role in protein assembly, synaptic development, and neural plasticity23–25. Synaptophysin is localized to the presynaptic vesicle membrane and contributes to membrane transport26. Its reduction has been considered to correlate with synaptic and neural impairments in Alzheimer’s disease and traumatic brain injury27–30. The PSD95 immunoreactions were recognized in the same direction of the axon plexus surrounding the axon hillock of each Purkinje cell as previously reported31 and distributed widely like plates or sheets along with the Purkinje cells. The degenerated Purkinje cells could not recover, and the cerebellar neuronal connections were temporally disrupted after heatstroke. However, the synaptic patency and myelin staining results, including motor coordination, suggest that the surviving and residual Purkinje cells and the other cerebellar neurons were remyelinated and reconstructed new synaptic connections, probably inducing the recovery of partial motor coordination. It was suggested that these phenomena were associated with late-onset neurological deficits and recovery after heatstroke.
Cerebellum has diversity and dynamism32 and cerebellar dysfunction improved to some extent with time in our rodent model of heatstroke. Thought, there is a case that showed permanent neurological deficit in human heatstroke. Synaptic impairment might not be improved if the Purkinje cell damages were severe after heatstroke. Our results indicated that there is a time before demyelination or synaptic impairment. Therefore, some therapeutic intervention during this period may contribute to improve neurological deficit after heatstroke.
In this study, we neither analyze skeletal muscle impairment nor determine the spinal cord impairment and the peripheral nerve fiber innervation because of a circulatory failure due to massive hemorrhagic shock and ischemia-induced motor neuron necrosis of the spinal cord’s ventral horn33. Moreover, we have not systematically determined diffuse cerebral ischemia. Further analysis is needed to clarify the heat exposure (HE) pathogeny and progression.