INTRODUCTION
Heat stroke, the most serious type of heat illness, refers to the presence of hyperthermia (core temperature [Tc] >40°C), accompanied by central nervous system (CNS) dysfunction, such as combativeness, delirium, seizures, or coma (1) (2) (3)
The cerebellum and hippocampus of the brain are particularly vulnerable during the early stage of heat stroke. Heat stroke is associated with not only a high mortality rate but also CNS impairment. Individuals fortunate enough to survive classical heat stroke have high rate of permanent neurological sequelae (4) (5–7) (8)
Iron is a transition metal that plays an essential role in many physiological functions in living organisms. In the CNS, iron participates in myelin synthesis, development of dendritic spines in the hippocampus, and synthesis of neurotransmitters, including monoamine transmitters and gamma-aminobutyric acid. Increasing evidence suggests that dysfunction of brain iron metabolism is involved in many neurodegenerative diseases, such as Alzheimer disease, Parkinson disease, Huntington disease, and Hallervorden-Spatz syndrome (9) in vitro studies have shown that iron acutely promotes platelet reactivity and that iron chelation inhibits activation of tissue factor (10) (11) (12)
Interaction between the iron-regulatory hormone hepcidin and its receptor, the cellular iron exporter ferroportin (Fpn), is central to mammalian iron homeostasis. Fpn1 is an iron exporter expressed on the surface of cells and the sole transporter moving iron from inside cells to the plasma (13) (14)
The liver is the central organ for whole body iron metabolism. Iron and hepcidin, produced mainly by hepatocytes, were significantly increased after repeated exposure to heat stress in older rats, suggesting that iron may play a role in hepatic injury after hyperthermia (15)
In the present study, an in vivo rat model was used to determine whether heat stroke reduces spatial learning and memory ability. We further hypothesized that dysregulation of brain iron metabolism may be involved in the cognitive dysfunction caused by heat stroke and thereby examined changes in iron, as well as expression of hepcidin and Fpn1, in the hippocampus after heat stroke. The goal of the study was to provide preliminary insight into the mechanism of cognitive dysfunction following heat stroke.
MATERIALS AND METHODS
Animals
A total of 144 healthy adult male Sprague-Dawley rats, aged 8 weeks and weighing 260 to 300 g, were purchased from the Beijing Wei Tong Li Hua Laboratory, Beijing, China (license: SCXK(J)2012-0001). The animals were housed in a temperature-controlled (25 ± 0.5°C) and humidity-controlled (25%) environment, with a 12-h light/dark cycle. All rats were allowed to adapt to their living conditions for at least 5 days before the experiment and had access to water and food ad libitum . The handling of animals was in strict accordance with the recommendations of the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health (4th edition, 2008). The Institutional Animal Care and Use Committee of the General Hospital of Chinese Armed Police Forces approved this study.
The rats were randomly divided into a Sham group and Heat Stroke group. The experimental group was further divided into subgroups, investigated at three time points after the heat stroke stimulus: HS0 , representing the acute heat stroke phase (day 0); HS7 , representing the early recovery phase (day 7); and HS21 , representing the late recovery phase (day 21).
Establishment of a rat model of heat stroke
Rats were anesthetized with 1 mL/kg of 3% pentobarbital sodium (Sigma, St. Louis, Mo). Rats in the Sham group underwent unilateral ligation of the femoral artery. They were then housed in a temperature- and humidity-controlled room (25 ± 0.5°C, 25% humidity) with a 12-h light/dark cycle and given free access to food and water.
Rats in the Heat Stroke group underwent transfemoral catheterization for continuous monitoring of arterial blood pressure and transrectal insertion of a thermistor probe (inserted 5 cm into the rectum) to continuously monitor Tc. The animals were then placed in a temperature- and humidity-controlled box with these heat exposure conditions: 39.5 ± 0.2°C and 13.5% humidity. The rats were removed from the box when they met the diagnostic criteria for heat stroke (Tc >42°C or mean arterial pressure <50 mm Hg); this occurred approximately 90 to 120 min after onset of the heat exposure. After the removal, the rats were cooled by exposure to cold water at 4°C for 10 min. The animals were then housed in the same environment as the Sham group animals.
After heat stroke, randomly selected living rats from each group underwent testing in the Morris water maze and other experiments. On days 0, 7, and 21, the remaining rats were euthanized and their hippocampus was excised quickly on ice, immediately transferred to liquid nitrogen, and stored at −80°C for subsequent experiments.
Morris water maze testThe Morris water maze (MWM) consisted of a circular tank (120-cm diameter, 50-cm height), which was filled to a depth of 29 cm with water maintained at 25 ± 1°C and rendered opaque with blue-black ink. A removable circular platform (12-cm diameter, 30-cm height) was added to this pool, with its top surface located 3 cm below the surface of the water. Thus, the platform was hidden from view. The area of the pool was conceptually divided into four quadrants of equal size.
In this behavioral test, rats are placed in the pool just below the surface of the water. They escape from the pool when they reach the platform. Distal visual cues are arrayed around the room and, in general, rats are able to learn the location of the hidden platform based on these cues. Data were collected by a video camera (TOTA-450d, Japan) fixed to the ceiling of the room and connected to a video recorder and an automated tracking system (Beijing zhongshidichuang, Beijing, China).
Spatial learning ability (platform location) test
This portion of the test assessed the spatial learning ability of the rats to find the platform under conditions when they could not directly see the platform but must either remember its location relative to external cues or perform a search for it. Spatial learning was assessed across trials repeated over 5 days. Rats were released into the water facing the wall of the pool from one of four separate quadrants. In all trials, rats were allowed to swim until they landed on the platform. If a rat failed to reach the platform within 120 s, the animal was picked up and placed on the platform for 30 s. This process was repeated for subsequent trials until four trials were completed each day for 5 consecutive days. After the daily sessions, each rat was dried under a heater and returned to the home cage. The time required for the rat to find the platform (escape latency time) was recorded. If the rat failed to locate the platform in the allowed time, a latency time of 120 s was recorded. We also recorded the rat's swimming speed and distance around the platform.
Spatial memory test (probe trial)
On the sixth day, the platform was removed from the pool, and each rat was placed in the pool one time for 120 s, starting from the same starting location as during the initial hidden platform testing. During this probe trial, performance was expressed as the percentage of time spent in each quadrant of the MWM and the number of crossings through the position where the platform was initially located.
Measurement of iron content
The hippocampal tissues were weighed, dried at 65°C for 24 h, and then mixed with nitric acid and 30% hydrogen peroxide. They were subsequently subjected to microwave digestion at optimum heating conditions, and the remaining acid was volatilized by electrothermal heating. The samples were then cooled to room temperature and deionized water was added to generate an identical volume in each sample. Absorbance of a standard iron solution and the hippocampal sample solutions were measured by flame atomic absorption spectrophotometer (contrAA300, Jena, Germany). Total iron content of the samples was calculated based on a standard curve.
Real-time reverse transcription-polymerase chain reaction
Total RNA was purified using TRIzol reagent (Beijing UBIO Biotechnology Co, Ltd, Beijing, China), and reverse transcription-polymerase chain reaction was performed using TaKaRa PrimeScript RT reagent Kit (Takara Biotechnology, Code No. RR047A, Dalian, China), following the manufacturer's instructions. cDNA (1 μL) was then used as a template for PCR. PCR amplification was performed with the following cycling parameters: initial denaturation at 95°C for 2 min; 40 cycles of 95°C for 5 s, 60°C for 30 s, and 72°C for 20 s; and a single final extension period at 72°C for 7 min. Expression of Fpn1 mRNA was determined by normalizing to the Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) mRNA expression level. Each amplification was repeated three times from different reverse transcription reactions, and the data were averaged. The following primers were used for Fpn1 (FWD 5’-AGGCTTAGGGTCTACTGCGG-3’; REV 5’-CCGAAAGACCCCAAAGGACA-3’) and GAPDH (FWD 5’-ACAGCAACAGGGTGGTGGAC-3’; REV 5’-TTTGAGGGTGCAGCGAACTT-3’).
Western blot
Western blot analysis was performed following the manufacturer's instructions. Equal amounts (30 μg) of protein were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis and analyzed by western blots using these primary antibodies: anti-hepcidin-25 antibody (Hepcidin, 1:100; Abcam; ab30760) and anti-SLC40A1 antibody (Fpn1, 1:1000; Abcam; ab85370). Expression of tubulin and GAPDH housekeeping proteins was measured using an anti-tubulin antibody (1:8,000; Beijing UBIO Biotechnology Co, Ltd, Beijing, China) and anti-GAPDH antibody (1:8,000; Beijing UBIO Biotechnology Co, Ltd, Beijing, China). Relative expression levels of hepcidin and Fpn1 proteins were normalized to tubulin and GAPDH expression.
Statistical analysis
The data are expressed as mean ± SEM, and statistical significance was assessed using one-way analysis of variance. A P value < 0.05 was considered statistically significant. Calculations were performed with the SPSS program version 21.0.
RESULTS
Effect of heat stroke on spatial learning ability of Sprague-Dawley rats in the MWM test
To evaluate whether heat stroke causes learning and memory alterations in rats, MWM test were conducted. Rats initially received 5 days of acquisition training with the platform in a fixed location. During this time, there were no significant differences in swimming speed between groups in the MWM (Table 1 ), suggesting that heat stroke did not reduce the rats’ motor ability.
Table 1: Swimming speed during the Morris water maze
The effect of heat stroke on spatial learning ability of rats in the MWM test is shown in Table 2 and Figure 1 . Rats in the HS7 and HS21 groups traveled long distances to reach the platform. As the training days increased, the escape latency time of all groups showed a downward trend. However, a prolonged escape latency time was observed in the HS7 group compared with the Sham group from day 1 to day 5 of training (P < 0.05), likely due to memory deficits resulting from heat stroke impairment of learning and memory. Rats in the HS21 group exhibited a significantly shorter escape latency time than the HS7 group on day 3, day 4, and day 5 (P < 0.05). No significant differences in escape latency times were found between the HS21 and Sham groups. To illustrate the differences described above, Figure 1 depicts representative swimming paths of rats from the Sham, HS7 , and HS21 groups.
Table 2: Escape latency time during the spatial learning ability test
Fig. 1: Representative swimming tracks of rats finding the hidden platform on the fifth day of training in the spatial learning ability test.
Effect of heat stroke on spatial memory of Sprague-Dawley rats in the Morris water maze test
To investigate the effect of heat stroke on spatial memory, performance in the probe trial on day 6 was examined by analyzing the percentage of time spent swimming to the expected position of the platform and the number of times the rats passed through the original location of the hidden platform. A higher percentage of time spent in the platform quadrant was interpreted as a higher level of memory retention. We found that compared with the Sham and HS21 groups, the HS7 group spent less time in the original platform quadrant (Table 3 , P < 0.01). Additionally, the percentage time spent in the original platform quadrant was similar for the HS21 and Sham groups. However, the number of times the rats passed through the original platform location within 120 s was decreased in both the HS7 and HS21 groups compared with the Sham group (P < 0.01). As shown in Figure 2 , Heat Stroke group rats appeared to be disoriented, exhibiting a simple thigmotaxic navigation path, with no apparent sign of remembering the initial platform location and spending more time in the other quadrants. These findings, therefore, indicate that Heat Stroke rats showed clear evidence of impaired spatial memory.
Table 3: Probe trial results showing the percentage of time in the original platform quadrant and frequency of crossing the original platform location
Fig. 2: Representative swimming tracks of rats in the 120-s probe trial test on the sixth day.
Iron content changes in the rat hippocampus after heat stroke
As shown in Figure 3 , the iron concentration in the hippocampus clearly increased in the HS0 , HS7 , and HS21 groups, compared with the Sham group (P < 0.05). There were no significant differences among the HS0 , HS7 , and HS21 groups. These results indicate that heat stroke is associated with iron overload in the rat hippocampus, which persists for at least 3 weeks in this rat model.
Fig. 3: Iron concentration changes in the rat hippocampus after heat stroke.
Expression of Fpn1 mRNA in the rat hippocampus after heat stroke
As shown in Figure 4 , expression of Fpn1 mRNA in the hippocampus of HS7 and HS21 rats was clearly higher than in the Sham and HS0 group rats (P < 0.05). However, there was no difference between the Sham and HS0 groups. These results indicate that heat stroke may upregulate Fpn1 expression in the rat hippocampus.
Fig. 4: Fpn1 mRNA expression levels in rat hippocampus samples after heat stroke.
Expression of hepcidin and Fpn1 proteins in the rat hippocampus after heat stroke
Fpn1 is a unique protein involved in iron efflux. As shown in Figure 5 , expression of Fpn1 protein in the hippocampus was clearly lower in the HS0 (P < 0.001), HS7 (P < 0.01), and HS21 (P < 0.01) groups compared with the Sham group (Figs. 5 , B and D). Fpn1 protein levels were slightly higher in the HS7 and HS21 groups than in the HS0 group, but the difference was only statistically significant between the HS0 and HS7 groups (P < 0.05).
Fig. 5: Expression of hepcidin and Fpn1 at the protein level in the rat hippocampus after heat stroke.
Hepcidin regulates iron efflux by binding to FPN1 and inducing its internalization. To investigate the mechanism of Fpn1 downregulation, we examined protein expression of hepcidin. Expression of hepcidin protein in the rat hippocampus was clearly increased in the HS0 , HS7 , and HS21 groups compared with the Sham group (P < 0.05 for all comparisons, Fig. 5 , A and C). There were no significant differences in hepcidin expression among the HS0 , HS7 , and HS21 groups. The decreased levels of hippocampal Fpn1 are consistent with the increased levels of hippocampal hepcidin.
DISCUSSION
Heat stroke is a life-threatening illness characterized by profound CNS dysfunction and severely elevated Tc, as well as organ and tissue damage resulting from environmental heat exposure. MWM is one of the most common tasks used to assess spatial learning ability and memory in rodents (16, 17) (18) 7 group. The HS21 group also exhibited a decreased number of times the rats crossed through the original platform location. These findings suggest that heat stroke impairs spatial memory in rats, potentially by inducing hippocampal damage.
Iron is the most important trace element in our body. It is involved in many fundamental biological processes, including oxygen transport, DNA synthesis, and mitochondrial respiration. In the CNS, iron is also involved in myelin synthesis, neurotransmitter synthesis, and metabolism. Under physiological conditions, intracellular iron exists mainly in the form of ferritin, with relatively little in the form of free iron. However, under pathological conditions, excessive iron—especially free iron—is toxic to cells, since it can catalyze the generation of hydroxyl radicals through Fenton or Haber-Weiss reactions, leading to lipid, DNA, and protein damage and further neuronal injury (19–22) (23)
But why is the iron content elevated after heat stroke? To investigate the mechanism of iron accumulation, we assessed hippocampal Fpn1 levels. Fpn1 is a recently discovered protein that plays a vital role in iron metabolism as the only known transmembrane iron export protein (24) (25) (26)
Post-translational regulation of Fpn1 is usually controlled by hepcidin. Hepcidin is a small peptide hormone secreted mainly by the liver. It is also expressed in the murine brain, where it is mainly secreted by glial cells (27) (28) (27, 29, 30) 7 and HS21 ), compared with the early phase of heat stroke (HS0 ), but remained lower than in the Sham group.
Interestingly, we found that expression of Fpn1 mRNA was increased after heat stroke. Cellular Fpn levels are regulated by iron through transcriptional and post-transcriptional events. Iron loading, for example, increases levels of Fpn mRNA and heterogeneous nuclear RNA, consistent with increased transcription (31) (32) (33, 34)
Although the mechanisms mediating brain damage in heat stroke remain poorly understood, our research indicates that iron overload may be involved in hippocampal impairment after heat stroke, which in turn results in cognitive dysfunction. Furthermore, hepcidin is upregulated in the hippocampus after heat stroke, which may rapidly decrease cellular Fpn1 protein levels, even under conditions of iron loading, thereby indicating that hepcidin is a more dominant regulator of Fpn than is iron.
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