Clinically, heat stroke is characterized by CNS dysfunction, multi-organ failure and sharp elevation of body temperature (usually > 40.5 °C) (1–4). At present, there is no uniform and objective diagnostic criterion for diagnosis of heat stroke. Moreover, there is no unified standard for the establishment of heat emission disease animal models. In most experimental studies, the occurrence of animal heat emission diseases is defined as core temperature > 40 °C and low mean arterial pressure (< 50 mmHg) (17, 21). Therefore, based on previous studies, we adopted the experimental condition of exposing rats to 40 °C and 80% humidity until the anal temperature reached 42 °C, as the standard condition for construction of the heart stroke rat model. In the modeling, the anal temperature of the rats increased rapidly at first, which was characterized by restlessness, irritability and more salivation, then the temperature rose slowly and increased again in the later stage. Meanwhile, the activity of the rats decreased significantly. In this experiment, the heat stroke model was established when the rats were awake, and therefore, it failed to monitor the change of mean arterial pressure in rats. Reportedly, after late thermal exposure of critical point (40 °C), the changes in temperature and mean arterial pressure were very similar (22). Hence, the heat stroke rat model was established successfully, which laid a foundation for subsequent heat stroke-induced damage experiments, especially for further research on the effects of the CNS damage.
Heat stroke is one of the most dangerous stages in the course of thermal injury. As the core body temperature continues to rise, the cytotoxic effect and inflammatory response are intensified, leading to a vicious circle as well as extensive tissue and organ damages and eventually resulting in multiorgan failure. If no effective treatment is taken in the early stage of heat stroke, it will raise the mortality (3, 4). At present, there is still no effective and targeted method for treatment of heat stroke. Our study confirmed that the mortality rate was higher after the modeling of heat stroke rats, and was mainly distributed in the early stage (day 3). MSCs therapy, whether in the early stage (day 3) or later stage (day 28), can significantly reduce the mortality rate of heat stroke rats. As reported, the earlier stem cell treatment given led to a lower mortality rate of heat stroke rats (23). MSCs have a good therapeutic effect on various disease models, and the underlying mechanism mainly includes: the secretion of various growth factors and cytokines to regulate the overactivated inflammatory response (24, 25); to promote damaged tissues to regenerate and secret microRNA and mitochondrial microvesicles with regulatory functions (26). The pathogenesis of heat stroke is similar to that of systemic sepsis, a systemic inflammatory response syndrome. Factors such as acute inflammatory response, increased vascular permeability, abnormal coagulation mechanism, and the outbreak of oxygen free radicals all directly affect the survival rate and severity of heat stroke rats (3, 4). MSCs from a variety of sources play an important role in anti-inflammation, immune regulation and organ function improvement, thereby reducing the mortality rate in sepsis animal models (27–29). These studies all suggest that exogenous MSCs therapy can significantly reduce the mortality of heat stroke rats and play a significant role in the early stage.
CNS injury is a common complication of heat stroke. Our previous analysis of clinical data found that almost all patients will suffer central damage to different degrees, which is also the main cause of death or serious sequelae (5). The mechanism of central nerve injury in thermal radiation disease is relatively complex and may be related to two factors. Firstly, the high temperature in the occurrence of thermal radiation diseases directly damages the CNS, causing a series of pathological reactions in brain cells and leading to cerebral edema and metabolic disorders, and further development results in degeneration and necrosis of brain cells. Secondly, cerebral ischemia and hypoxia are the main pathophysiological basis of brain injury during heat stroke. Heat stress caused by high temperature can lead to decreased cerebral blood flow and rapid death of central nerve cells under the stimulation of high temperature and hypoxia (7). In our study, HE staining, neuron Nissl staining, and cerebellar silver staining showed that after the heat stroke injury, the hippocampal vulnerable area (CA1 area), cerebellum, neurons, cones, cerebellar Purkinje cells were found with edema, cavitation, nucleus pycnosis and cell loose arrangement, significant reduction in Nissl bodies, and other damages. Especially, the damages were severe at early stage, but at late stage (day 28), although the above areas still suffered damages, the damages were significantly restored. MSCs therapy significantly reduced the damage of nerve cells in the vulnerable areas of hippocampus and cerebellum in heat stroke rats compared with the HS group at the same time point. It is suggested that MSCs infusion can protect nerve cells (neurons and Purkinje cells) in vulnerable hippocampal and cerebellar areas of heat stroke rats, and the protective effect can happen at the early stage of injury. Extensive studies confirm that MSCs have significant protective effects various CNS damages. MSCs have significant protective effects on cerebral ischemic injuries, external injury caused by brain damage, Alzheimer's disease, dementia, Parkinson's disease and other neurodegenerative diseases. The specific mechanisms include: MSCs can secrete various neurotrophic factors, regulate immune response, improve cerebral vascular permeability, and adjust nerve cell communication (12–14). Therefore, the treatment of MSCs has an important protective effect on the common central system damage of heat stroke, and is of great significance for the survival rate and prognosis of heat stroke rats.
In addition to high fever, ischemia, hypoxia and other factors, inflammatory response also plays an important role in the brain injury caused by heat stroke. A clinical case report shows that in thermal radiation patients, abnormal high signals in the bilateral cortex and sub hippocampal white matter can be observed by craniocerebral magnetic resonance, indicating obvious inflammatory response in brain tissues (30). As reported, the increase of inflammatory cytokines IL-1 and IL-6 in brain tissues after heat stroke is significantly correlated with the nervous system damage, vascular edema and neuron death, while the cortex and hippocampus are the most common damage sites (31). However, the main mechanisms about the occurrence, development and outcome of CNS inflammation during heat stroke are still not fully understood, which leads to the insufficient understanding about the importance of CNS inflammation in clinical treatment of heat stroke. The occurrence and development of inflammation in CNS are closely related to microglia. Microglia cells are widely distributed in the CNS and play roles of nutrition, support, antigen presentation and tissue repair in CNS. They are also the main mediators of CNS immune response and play a crucial role in the pathological and physiological processes of the CNS (8–11). We found that microglial cell activation in the hippocampal vulnerable areas of heat stroke rats was significant in early stage (day 1) and middle stage (day 7 and 14), and significantly decreased in late stage (day 28). Although the microglia cells in the vulnerable hippocampal area of MSCs-treated rats were activated in early stage (day 1), the activation level was low, and the cells maintained a similar stable state at all subsequent time points. It is suggested that the overactivation of microglia cells is involved in the hippocampal injury of heat stroke rats. MSCs therapy can inhibit the overactivation of microglia at the above time points and stabilize the activation state in the whole process. However, the activation of microglia in the cerebellar region of the rats was not obvious during the whole observation period. However, the microglia cells in the cerebellar region of MSCs- treated rats showed a slightly stronger activation state in early period (day 1), and the activation state was not obvious in the subsequent time points. However, microglia cells in the cerebellum did not obviously respond to heat damage, and MSCs may promote the activation of cerebellar microglia cells in heat stroke rats only in early stage (day 1), which is a completely different phenomenon from the hippocampus and needs further study. The mechanism of central inflammatory response involves the activation of microglia, infiltration of inflammatory cells, and release of cytokines and inflammatory chemokines (32). On the one hand, heat stroke can cause central neuron apoptosis or necrosis, such as secondary injury, release of various chemical factors by damaged nerve cells. Moreover, the dead cell fragments can activate the brain neural immune cells-microglia, which then are activated to produce numerous inflammatory factors, resulting in severe inflammation of CNS. On the other hand, a variety of inflammatory factors in the inflammatory response can directly stimulate vascular endothelial cells, and destroy the interconnections between endothelial cells, causing edema in perivascular tissues and aggravating brain damage (33). Studies have confirmed that MSCs have an important regulatory effect on microglia cells after CNS injury, and the main mechanism is that MSCs can regulate the inflammatory response mediated by microglia cells and reduce the release of pro-inflammatory factors (34–36). Our study found that the levels of inflammatory cytokines (IL-1β, IL-6 and TNF-α) and chemokines (GRO-α, MCP-1 and Rantes) in the hippocampal brain tissues of thermophilic rats fluctuated. On the whole, MSCs therapy can significantly inhibit the increase of these cytokines. However, MSCs had no overall significant regulatory effect on the levels of IL-1β, IL-6 and TNF-α in cerebellar tissues of thermally injected rats, but can inhibit the levels of GRO-α, MCP-1 and Rantes at early stage. It is suggested that CNS inflammation plays an important role in the pathogenesis of heat stroke, and the regulation of inflammation may become an important target of MSCs in the treatment of heat stroke.