According to its possible pathophysiology, heatstroke (HS) could be defined as a form of hyperthermia, associated with a systemic inflammatory response, leading to a syndrome of multiorgan dysfunction in which encephalopathy dominates (1). HS occurs due to a complex interplay among the inflammatory response, coagulation disorder, and vascular endothelium injury, which leads to multiorgan dysfunction (2–4). Ischemic gut-derived bioactive toxic factors are associated with organs injury in HS (5). Gut-derived toxic factors via mesenteric lymph while bypassing the portal circulation and liver are a more recent concept (6). Mesenteric lymph has been implicated as the source from which ischemic gut-derived bioactive exudates are delivered into the circulation and result in organ damages in severe acute illnesses (6). In our previous study, we had also found that mesenteric lymph in HS rat activated and injured vascular endothelium, which can be attenuated by mesenteric lymph duct ligation (LDL) by reducing gut-derived factors entering into the systemic circulation via lymphatic vessels (7). However, whether mesenteric lymph could induce organ injuries in HS is to be elucidated.
AS the first vascular bed exposed to mesenteric lymph, HS-induced lung injury may be seemingly secondary to the toxic properties of mesenteric lymph inevitably. Clinically, according to a survey, respiratory failure is the most common dysfunction in the studied HS patients (8). HS animals have also been demonstrated that lung injury presented as overt intralung vascular endothelium injury, inflammation, and hemorrhage or thrombus (4). In the present study, then, we hypothesized and tested whether HS mesenteric lymph could aggravate vascular endothelial cell injury, inflammation, and coagulation, which contribute to lung injury. This hypothesis was tested by determining whether interruption of mesenteric lymph flow by LDL could reduce HS-induced lung injury.
MATERIALS AND METHODS
Because of the fact that estrogen has certain effects on HS pathogenesis and organ injury (9), only adult male pathogen-free Wistar rats (Experimental Animal Center of our hospital), weighing of 220 to 250 g, were used. In the conduct of the experiment involving the rats, we adhered to the guidelines for animal care of our hospital. All procedures were approved by the Institutional Animal Care and Use Committee before the experiments.
LDL was used to prevent gut-derived components leaking into the systemic circulation from mesenteric lymph duct. A midline laparotomy was performed to expose the common mesenteric lymphatic duct, which lies adjacent to the superior mesenteric artery. The mesenteric lymphatic duct was then isolated and double ligated with a 5-0 silk tie. Mesenteric lymphatic ducts of rats in the heatstroke-sham (HSS) and HS groups were threaded by 5-0 silk ties without ligations. The abdomen was then closed with 4-0 monofilament nylons. Immediately following LDL, the animals were then subjected to HS or HSS.
Preparation of HS mode
Eighty rats housed for 6 h at ambient temperature (25 ± 0.5)°C with humidity of (35 ± 5)% initially, in which 40 rats were evenly divided into five groups randomly: control, HSS, HSS-LDL, HS, and HS-LDL group for plasma and tissue indexes measurements, and other 40 rats were divided into control, HSS, HSS-LDL, HS, and HS-LDL groups evenly to observe the HS onset and survival time. Rat models were prepared according to the methods established in our previous study (7). Briefly, an intraperitoneal injection of sodium pentobarbital (50 mg/kg) was applied to abolish the corneal reflex and pain reflex. The right femoral artery of the rat was cannulated with a trocar (24-gauge) for monitoring mean arterial pressure (MAP). Rats in the HS and HS-LDL groups were placed in a prewarmed incubator maintained at (40 ± 0.5)°C and a relative humidity of (60 ± 5)%. The animals in the HSS and HSS-LDL groups were sham-heated at a temperature of (25 ± 0.5)°C and humidity of (35 ± 5)%. MAP and rectal temperature were continuously monitored at 10-min intervals using a Multi-parameter Physiological Monitor (Dräger, Germany). When MAP dropped from the peak to a value of 25 mmHg and core temperature was more than 42°C, HS model was prepared successfully (7, 10). Our pilot study showed that the onset time of HS in rats was about (76.88 ± 1.46) min. Therefore, all heat-stressed animals were exposed to a prewarmed incubator (40 ± 0.5)°C and a relative humidity of (60 ± 0.5)% for exactly 77 min in the following experiments (7, 11).
Blood and tissue sampling
After HS onset, the animals were recovered at room temperature of (25 ± 0.5)°C. At the time points of HS rats’ death, all the rats in other groups were euthanized, blood was withdrawn from the femoral veins, and bronchoalveolar lavage fluid (BALF) was extracted from the lungs of all rats. Blood samples were centrifuged at 300g for 15 min and 5,000g for 5 min to remove the cellular components and acquire acellular plasma. The lung was lavaged, and 5 mL room temperature phosphate-buffered saline (PBS) was injected into the trachea through a 2.0 mm polyethylene tube. The PBS, installed into the lung, was withdrawn and centrifugated (500g, 15 min, 4°C), and supernatant was centrifuged again (16,500g, 10 min, 4°C) to obtain pure BALF. Aliquots of the plasma and BALF were stored at −80°C in polypropylene microcentrifuge tubes. Right lung tissues were obtained at immediate autopsy in all rats for histopathology.
Complete platelet count and coagulation indexes including prothrombin time (PT), activated partial thromboplastin time (APPT), and D-dimer were performed on automated devices.
Plasma and BALF proinflammatory cytokines measurements
Plasma high mobility group box 1 (HMGB1) levels were measured using a commercially available HMGB1 enzyme-linked immunosorbent assay (ELISA) kit II according to the manufacturer's instructions (Shino-Test Corp., Sagamihara, Japan). The plasma and BALF concentrations of tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), and IL-6 were determined by double-antibody sandwich ELISA (R&D, Minneapolis, Minn) according to the manufacturer's instructions. All the samples were run in duplicate.
Endothelial cell injury marker assay
Plasma von Willebrand factor (vWF) and thrombomodulin (TM) were assayed using the ELISA kits (Diagnostica Stago, Asnières, France and Cusabio Biotech Co., Ltd, WuHan, China respectively) according to the manufacturers’ instructions. All the samples were run in duplicate.
Circulating endothelial cell counting
At the time points of HS rats’ death, all rats in other groups were killed. Blood (2 mL) was collected and split into the silicone-coated tubes containing citrate solution (3.8 %). As previously described, after centrifuging the blood samples at 4°C for 20 min at 395g, the supernatants were collected, and ADP (Sigma, St. Louis, Mo; 1 mg/mL solution) was added at a ratio of 5:1 and was agitated for 10 min (7, 12). The mixture was centrifuged at 4°C for 20 min at 395g again to remove platelet aggregates. The supernatants were centrifuged at 4°C for 20 min at 2,100g to obtain circulating vascular endothelial cell precipitates. Then, 0.1 mL 0.9 % NaCl solution was added to prepare cell suspension. A fraction suspension was placed in a hemocytometer to count the numbers of cell under a microscope (Olympus). Each sample was counted four times by two observers, and the mean value was obtained.
Paraffin-embedded right lung tissues were sectioned at 3-μm thickness and stained by hematoxylin & eosin (H&E). Histology analysis was performed by a pathologist blindly and scored according to previously described methods under light microcopy (13). Ten random fields per rat lung tissue were read under ×200 magnification by a pathologist blindly. Histological variables of acute lung injury, each grading from 0 to 3, were as follows: intra-alveolar infiltration of neutrophils, interstitial infiltration of neutrophils, perivenous infiltration of neutrophils, pulmonary congestion, and alveolar hemorrhage. The histologic scores were reported as the sum of the individual values.
Arterial blood gas analysis
Femoral arterial blood gas parameters including pH, PaO2, PaCO2, and lactic acid (LAC) were measured in vitro at 37°C via a blood gas analyzer (Nova Biochemical, Waltham, Mass). The samples were drawn from rats with core temperature 42°C. The results presented were corrected for temperature.
Evans Blue dye lung permeability assay
At the time point of 42°C, 10 mg of Evans Blue dye (EBD) was injected into the rat via the femoral vein catheter. After 5 min (allowing the dye completely circulating), 0.3 mL blood was withdrawn from the femoral vein catheter and centrifuged at 1,500 rpm at 4°C for 20 min. The resultant plasma was serially diluted to form a standard curve. BALF was collected and centrifuged, and then supernatant fluid was assayed spectrophotometrically for dye concentration (14). The percentage of EBD in BALF was determined relative to the standard curve of EBD in plasma.
Bronchoalveolar fluid and plasma protein assay
Both BALF and plasma total protein concentrations were detected using hand Bradford assay (Bio-Rad, Hercules, Calif). The BALF to plasma protein ratio was determined.
Myeloperoxidase (MPO) activity, an index of polymorphonuclear neutrophils (PMNs) accumulation, was determined as previously described (15). The frozen lung tissue was homogenized in a potassium phosphate buffer (pH 7.0, 10 mmol/L) containing 0.5% hexa-decyl-trimethylammonium bromide and centrifuged for 30 min at 20,000g at 4°C. An aliquot of the supernatant was then reacted with a solution of 1.6 mmol/L tetra-methyl-benzedrine and 0.1 mmol/L H2O2. The rate of change in absorbance was measured by spectrophotometry at 650 nm. One unit of MPO activity represents the amount of enzyme that reduced 1 mol/min of peroxide.
vWF immune staining was performed on fixed lung tissue sections using 3,3-Diaminobenzidine tetrahydrochloride (DAB) staining kit (Abcam, Shanghai, China) based on primary antibody to vWF (Santa Cruz, Calif) according to the manufacturer's instructions. The optical densities (ODs) (vWF positive) and the OD0 (vWF negative background) were analyzed under six random fields (×200 magnification) from each lung tissue using the Leica Qwin Image Processing and Analysis System. Corrected ODs (CODs) were gained as following equation: COD = CD1 + CD2 + CD3 + CD4 + CD5 + CD6/(6 × CD0).
All data were expressed as the mean ± SD. Data were analyzed using SPSS Statistical Software 12.0, and comparisons were evaluated using the one-way ANOVA and Student t tests. Values with P < 0.05 were considered significant.
LDL prolonging HS onset time
Rats in control, HSS, and HSS-LDL groups, with the artificial HS time 480 min, did not develop HS. HS onset time in HS-LDL group (86.2 ± 2.6 min) was significantly longer than those in HS group (77.8 ± 2.3 min) (P < 0.01). Rats in control, HSS, and HSS-LDL groups were euthanized at 480 min after experiment initiation; otherwise, they should survive more than 480 min. Although the survival time in HS-LDL rats showed prolonged tendency compared with HS rats, it was not significantly different (20.1 ± 3.4 min vs. 18.4 ± 2.5 min, P = 0.086).
LDL downregulated systemic inflammation
There were no differences between plasma TNF-α, IL-1β, IL-6, and HMGB1 levels in control, HSS, and HSS + LDL groups. However, when compared with control, HSS, and HSS + LDL groups, TNF-α, IL-1β, IL-6, and HMGB1 levels in HS and HS + LDL groups were significantly increased (P < 0.001) (Fig. 1). Compared with HS rats, plasma TNF-α, IL-1β, IL-6, and HMGB1 levels in HS + LDL rats were significantly decreased but not to the control levels (P < 0.05) (Fig. 1).
LDL attenuated endothelial cell injury
There were no differences between circulating endothelial cell (CEC) counts in control, HSS, and HSS + LDL groups (Fig. 2). In contrast, CEC counts in HS and HS-LDL groups were significantly higher than those in control, HSS, and HSS-LDL groups (P < 0.001) (Fig. 2). CEC counts in HS-LDL group were significantly decreased when compared with those in HS group (P < 0.001) (Fig. 2). There were no differences between the plasma vWF and TM concentrations in control, HSS, and HSS-LDL groups. However, when compared with control, HSS, and HSS-LDL groups, both vWF and TM concentrations were significantly increased in HS and HS-LDL groups (P < 0.001) (Fig. 3). Both vWF and TM concentrations in HS-LDL group, though not to the control levels, were significantly decreased when compared with those in HS group (P < 0.001) (Fig. 2).
LDL ameliorated coagulant disorders
APTT, PT, D-Dimer, and platelet count in control, HSS, and HSS + LDL groups were normal. In contrast, obvious coagulant disorders were presented in HS and HS +LDL groups, which showed significantly increased APTT, PT, and D-Dimer levels and decreased platelet counts (P < 0.001) (Fig. 3). All of the above indexes in HS-LDL group were significantly improved when compared with HS group (P < 0.001), though not to the normal levels, which indicated LDL could in part ameliorate HS coagulant disorders.
LDL alleviated lung injury
H&E staining showed that there were almost no lung histopathological injury in control, HSS, and HSS-LDL animals. In contrast, intra-alveolar and interstitial infiltration of neutrophils, perivenous infiltration of neutrophils, pulmonary congestion, and hemorrhage or thrombus were displayed in HS rats (Fig. 4). Although the same histopathologic damages were also seen in HS-LDL rats, they were significantly alleviated (Fig. 4). The lung injury scores for HS rats were significantly higher than those in control, HSS, and HSS-LDL rats (P < 0.001). LDL significantly decreased lung injury scores of HS rats (P < 0.001) (Fig. 4). The histologic changes of lung were further confirmed by lung function indexes analysis. All lung function indexes in control, HSS, and HSS-LDL rats were normal. Compared with control, HSS, and HSS-LDL groups, significantly decreased arterial blood PaO2 and pH levels as well as significantly increased arterial blood PaCO2 and LAC levels were displayed in HS group (P < 0.001). LDL significantly alleviated these lung injury indexes (P < 0.001), though not to normal levels (P > 0.05) (Fig. 5).
LDL downregulated intralung inflammation
To present intralung inflammation quantitatively, cytokines, including TNFα, IL-1β, and IL-6, as well as MPO activity were determinated. BALF TNFα, IL-1β, and IL-6 levels in control, HSS, and HSS-LDL groups were very low and not different (P > 0.05). In HS rats, BALF TNFα, IL-1β, and IL-6 levels were elevated significantly (P < 0.001), which could be downregulated by LDL significantly (P < 0.001), though not to normal levels (P > 0.05) (Fig. 6). Lung MPO levels were also significantly higher in HS rats than those in control, HSS, and HSS-LDL rats (P < 0.001) (Fig. 6). Although LDL reduced pulmonary MPO levels to greater extents (P < 0.001), it could not completely prevent the MPO increase.
LDL alleviated lung endothelial cell injury
There were no differences between lung permeabilities to EBD and protein in control, HSS, and HSS+LDL groups (Fig. 7). In contrast, lung permeabilities to EBD and protein in HS group were significantly higher than those in control, HSS, and HSS + LDL groups (P < 0.001) (Fig. 7). However, LDL could decrease changes in pulmonary permeabilities caused by HS significantly (P < 0.001), though there were not to normal levels (P > 0.05) (Fig. 7). Figure 8 showed an increased vWF immune staining on lung capillary endothelium in HS rats compared with control, HSS, and HSS-LDL animals (P < 0.001). Although LDL reduced pulmonary endothelial vWF staining to greater extents (P < 0.001), it could not completely prevent the increased vWF staining.
Owing to its unclear pathophysiology, HS has a high mortality and morbidity rate with no specific target for treatment. It has been shown in numerous studies that HS is possibly driven by gut-derived septicemia under heat stress (16, 17), which triggers the development of severe inflammation, endothelial injury, and coagulant disorder resulting in multiorgan dysfunctions (1, 4, 18). The traditional view of the gut-derived septicemia in HS is via portal circulation (16, 18). However, recent studies have demonstrated that gut-derived biotoxic factors can enter into the systemic circulation via intestinal lymphatics, rather than portal circulation in burn and trauma hemorrhagic shock animals (19, 20). Subsequent studies confirmed that the lymphatic route was the primary route of damaged gut-derived toxic factors (6, 21). Gathiram et al. also suggested that severe heat stress might cause LPS entering into the systemic circulation via lymphatic vessels (22). Our previous study has also found that HS mesenteric lymph was associated with vascular endothelial injury and inflammation response which can be significantly alleviated by LDL before heat stress (7). Owing to the fact that endothelial injury and inflammation response were the key points associated with organ damages in HS, we presumed that gut-derived toxic components in mesenteric lymph may possibly induce organ injuries. In the present study, we observed whether LDL before heat stress, blocking mesenteric lymph flow, could alleviate lung injury, whereas lung is the first vascular bed exposed to mesenteric lymph and the most common damage organ in HS (4, 8).
First, our results, mostly consistent with previous investigators, showed that HS resembles sepsis in many aspects indeed, presenting as endothelial injury, severe systemic inflammation, and coagulant disorder, all of which jointly initiated and promoted HS development (16–18). HS rats showed significantly increased CEC counts as well as plasma vWF and TM levels compared with HSS rats, indicating vascular endothelial injury in HS. As we know, CEC has been reported to be a specific vascular endothelial injury marker, whose number seems to be associated with the severity of the endothelial lesions, such as vasculitis, acute coronary syndromes, and septic shock (23–25). Many plasma-derived factors have also been regarded as the endothelial injury markers, such as vWF and TM (26, 27). Growing evidences have also shown that systemic inflammation induced by proinflammatory cytokines including TNF-α, IL-1β, IL-6, and HMGB1 may be implicated in HS (28–30). We found that plasma TNF-α, IL-1β, IL-6, and HMGB1 levels were elevated significantly after rats subjected to heat stress, which further verified the previous results (28–30). HS-induced coagulant disorders were assessed by plasma markers, including significantly prolonged PT, APTT, elevated D-dimer, and decreased platelet counts. These findings established a mechanistic link between coagulant host responses to heat stress and HS pathogenesis.
In addition, our results verified the previous results that acute lung injury can be induced in HS rat (15, 31). Lung injuries in HS rats were displayed as following: sever pathological damages showing neutrophils infiltration, pulmonary congestion, hemorrhage or thrombus, as well as deteriorated lung function indexes including arterial blood PaO2, pH, PaCO2, and LAC; increased BALF proinfammatory cytokine levels including TNF-α, IL-1β, and IL-6; increased lung MPO activity (an indicator for PMN cell infiltrating); increased BALF EBD and protein levels (vascular endothelium permeability markers); and increased lung vascular endothelium vWF immune staining, which is consequentially to promote thrombosis in HS possibly, as vWF could facilitate platelets to adhere to subendothelium in injured endothelium (4, 32).
LDL has been indicated its preventive effects on numerous severe illnesses including HS, in which we had showed LDL could significantly alleviate HS-induced vascular endothelial injury and inflammatory response (7, 21). This study provided the further data showing the beneficial effects of LDL on heat stress responses, systemic inflammation, endothelial injury and coagulant disorders, as well as acute lung injury in HS rats. First, LDL significantly protected HS rats systemically as following: prolonging HS onset time; attenuating endothelial cell injury for decreased CEC counts as well as lower plasma vWF and TM concentrations; downregulating systemic inflammation for decreased plasma TNF-α, IL-1β, IL-6, and HMGB1 levels; ameliorating coagulant disorders for decreased APTT, PT, and D-Dimer levels and increased platelet counts. Our results indicated that LDL had protective roles on the key pathogenesis in HS. If these experimental results were true, then, whether LDL could alleviate lung injury in HS, as lung is the first vascular bed exposed to mesenteric lymph. As expected, our results clearly indicated that LDL treatment significantly reduced acute lung inflammtory and hemorrhage/thrombus pathological injury and improved lung function indexes including arterial blood PaO2, pH, PaCO2, and LAC; downregulated intralung inflammtion showing decreased BALF proinfammatory cytokines levels (TNF-α, IL-1β, and IL-6) and decreased lung MPO activity; and alleviated lung endothelial cell injury showing reduced vascular endothelium permeability (increased BALF EBD and protein levels) and lung endothelial vWF expression. Take together, LDL developed protective roles systemically and alleviated lung injury in HS rats. However, our results also showed that the systemic and lung local parameters in the HS + LDL group were still different to those in HSS and HSS + LDL groups significantly. Most of all, LDL did not prolong HS survival time significantly, though there was an extended tendency. Combined with our previous report (7), it was collectively suggested that the pathogenesis of HS was more complex than just mesenteric lymph-derived injury only.
Key questions spontaneously arising from our results include the following: What toxic factors might be present in HS mesenteric lymph, and were there new ways to modulate HS mesenteric lymph composition and flow other than LDL for which would not be a likely therapeutic approach in HS patients? By far now, although a broad range of interventions targeted toward mesenteric lymph have been investigated for its complex bioactive factor composition, it is still hard to know what is exactly responsible for the toxicity of mesenteric lymph (21). Proteomics is possibly beneficial to identify the toxic factors in HS mesenteric lymph (33). Many medical therapies have been explored to modulate mesenteric lymph flow in animal severe illness models (21). It has been demonstrated that hypothermia, particularly below freezing temperatures, may rapidly decrease and virtually stop lymph flow from an intestinal segment (34), which was most exciting to us as hypothermia is the current accepted therapy of choice for HS (35). Whether hypothermia could attenuate HS-induced organ injuries through influencing mesenteric lymph flow is to be elucidated.
There are some limitations in this study. First, similar to other experiments, the HS animal model did not ideally simulate the clinical course of HS patient. Although extensive findings in small animal models are helpful to understand the pathogenesis of HS (7, 9–11, 16), extrapolation of data from small laboratory animals cannot predict reliably the human responses because of interspecies differences. A nonhuman primate model possibly mimics the full spectrum of human HS better (3, 4, 18). Second, we did not determine and isolate the targeted biological factors in mesenteric lymph leading to lung injury for the fact that mesenteric lymph is a complex biological fluid comprising much active protein, lipid factors, endotoxin/bacteria, and cytokines, which makes the identification of the biological factors much more difficult. Third, because of unpredictability, pretreatment in HS is less practical clinically, which results in the need to explore other therapeutic methods targeted to mesenteric lymph in HS.
In summary, LDL partly protected systemic damage and alleviated lung injury in HS rats for its complex pathogenesis. The toxic factors in mesenteric lymph still need further studies to be elucidated. Most of all, there is a need to explore ways to modulate mesenteric lymph flow or composition to prevent further mesenteric lymph-derived injury in HS.
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Keywords:© 2016 by the Shock Society
Coagulant disorders; heatstroke; inflammation; lung injury; mesenteric lymph; vascular endothelial injury