Proteomic analysis of hypothalamic injury in heatstroke rats

Ischemic and oxidative damage to the hypothalamus may be associated with decreased heat tolerance as well as heatstroke formation. The present study explores the hypothalamic proteome mechanisms associated with heatstroke‐mediated hypothalamic ischemia, and oxidative damage. Heatstroke rats had hypotension, hypothalamic ischemia, and lethality. In addition, they had hyperthermia and hypothalamic blood–brain–barrier disruption, oxidative stress, activated inflammation, and neuronal apoptosis and degeneration. 2DE combined LC‐MS/MS revealed that heatstroke‐induced ischemic injury and apoptosis were associated with upregulation of L‐lactate dehydrogenase but downregulation of both dihydropyriminase‐related protein and 14‐3‐3 Zeta isoform protein. Heat‐induced blood–brain–barrier disruption might be related to upregulation of glial fibrillary acidic protein. Oxidative stress caused by heatstroke might be related to upregulation of cytosolic dehydrogenase‐1. Also, heat‐induced overproduction of proinflammatory cytokines might be associated with downregulation of stathmin 1. Heat‐induced hypothalamic ischemia, apoptosis, injury (or upregulation of L‐lactate dehydrogenase), blood–brain–barrier disruption (or upregulation of glial fibrillary acidic protein), oxidative stress (or upregulation of cytosolic dehydrogenase‐1), and activated inflammation (or downregulation of stathmin 1) were all significantly reversed by whole body cooling. Our data indicate that cooling therapy improves outcomes of heatstroke by modulating hypothalamic proteome mechanisms.     

bFGF对热射病大鼠模型ET21, VWF, VEGF水平的影响

目的:研究碱性成纤维细胞生长因子(basic Fibroblast growth factor,bFGF)对热射病大鼠模型ET21、VWF、VEGF水平的影响。方法:选择SD大鼠36只随机分为6组,每组个6只,建立热射病大鼠模型后分为模型组、常温组、常温bFGF组、降温组、降温bFGF组,建模后0 h、2 h,比较不同级别生命征、血管功能因子。结果:建模后0 h,模型组Tr、HR、RR、MAP明显高于空白组(P0.05);建模后2 h,常温bFGF组、降温bFGF组明显低于常温组、降温组,降温组、降温bFGF组明显低于常温组、常温bFGF组(P0.05)。建模后0 h,模型组ET21、VWF的含量均明显高于空白组,VEGF含量明显低于热空白组(P0.05);建模后2 h,常温组ET21、VWF明显升高,VEGF明显降低,常温bFGF组、降温组、降温bFGF组ET21、VWF明显降低,VEGF明显升高(P0.05);降温组、降温bFGF组ET21、VWF明显低于常温组、常温bFGF组,VEGF明显高于常温组、常温bFGF组(P0.05);常温bFGF组、降温bFGF组ET21、VWF明显低于常温组与降温组,VEGF明显高于常温组与降温组(P0.05)。结论:碱性成纤维细胞生长因子有助于改善热射病大鼠生命体征,调节血管功能因子含量,提高热射病大鼠预后。     

Skeletal and cardiac myopathy in HIV-1 transgenic rats

The mechanism by which human immunodeficiency virus (HIV)-1 infection in humans leads to the erosion of lean body mass is poorly defined. Therefore, the purpose of the present study was to determine whether transgenic (Tg) rats that constitutively overexpress HIV-1 viral proteins exhibit muscle wasting and to elucidate putative mechanisms. Over 7 mo, Tg rats gained less body weight than pair-fed controls exclusively as a result of a proportional reduction in lean, not fat, mass. Fast- and slow-twitch muscle atrophy in Tg rats did not result from a reduction in the in vivo-determined rate of protein synthesis. In contrast, urinary excretion of 3-methylhistidine, as well as the content of atrogin-1 and the 14-kDa actin fragment, was elevated in gastrocnemius of Tg rats, suggesting increased muscle proteolysis. Similarly, Tg rats had reduced cardiac mass, which was independent of a change in protein synthesis. This decreased cardiac mass was associated with a reduction in stroke volume, but cardiac output was maintained by a compensatory increase in heart rate. The HIV-induced muscle atrophy was associated with increased whole body energy expenditure, which was not due to an elevated body temperature or secondary bacterial infection. Furthermore, the atrophic response could not be attributed to the development of insulin resistance, decreased levels of circulating amino acids, or increased tissue cytokines. However, skeletal muscle and, to a lesser extent, circulating insulin-like growth factor I was reduced in Tg rats. Although hepatic injury was implicated by increased plasma levels of aspartate and alanine aminotransferases, hepatic protein synthesis was not different between control and Tg rats. Hence, HIV-1 Tg rats develop atrophy of cardiac and skeletal muscle, the latter of which results primarily from an increased protein degradation and may be related to the marked reduction in muscle insulin-like growth factor I.     

Heat shock protein 72 overexpression protects against hyperthermia, circulatory shock, and cerebral ischemia during heatstroke.

This study extends our earlier studies in rats by applying our heatstroke model to a new species. Additionally, transgenic mice are used to examine the role of heat shock protein (HSP) 72 in experimental heatstroke. Transgenic mice that were heterozygous for a porcine HSP70i gene ([+]HSP72), transgene-negative littermate controls ([-]HSP72), and normal Institute of Cancer Research strain mice (ICR) under pentobarbital sodium anesthesia were subjected to heat stress (40 degrees C) to induce heatstroke. In [-]HSP72 or ICR, the values for mean arterial pressure, the striatal blood flow, and the striatal PO2 after the onset of heatstroke were significantly lower than those in preheat controls. The core and brain temperatures, the extracellular concentrations of ischemic and injury markers in the striatum, and the striatal neuronal damage scores were significantly greater than those in the preheat controls. In [-]HSP72 or ICR, the body temperatures, cell ischemia content, and injury marker in the striatum were significantly higher, and the mean arterial pressure, striatal blood flow, and striatal PO2 concentration were significantly lower during heatstroke than in [+]HSP72. Accordingly, the latency and the survival times for [+]HSP72 significantly exceeded those of [-]HSP72 or ICR. These results demonstrate that the overexpression of HSP72 in multiple organs improves survival during heatstroke by reducing hyperthermia, circulatory shock, and cerebral ischemia and damage in mice.     

Cytokine changes in patients with heatstroke during pilgrimage to Makkah

Circulating levels and role of IL-6, IL-1ra, TNFsr-II and CRP in patients with heatstroke is not fully known. This study correlated levels of these mediators with outcome in 26 patients. In survivors (n=20), IL-6 concentration declined on cooling, whereas in non-survivors levels continued to increase at 6 h following admission before declining. Admission TNFsr-II concentrations in survivors were significantly lower than non-survivors and levels continued to rise in both groups. IL-1ra levels were markedly elevated in both groups. Changes in cytokine levels were not influenced by renal function. Elevated C-reactive protein levels were observed for both groups and remained so despite cooling, furthermore, there was no correlation with alanine aminotransferase levels. The study demonstrated the elevation of the above mediators and suggested a role in the pathogenesis of heatstroke. Markedly elevated levels or those that remained elevated despite cooling were associated with mortality.     

Quantitative Significance of Plasma Albumin Metabolism in the Whole Body Protein Turnover in Rats

The amount of extra- and intravascular albumin was estimated in two groups of rats, i.e., those fed a 20% casein (20% protein) diet and a 3% casein (low protein or 3% protein) diet.

The fractional turnover rate of whole body plasma albumin was also measured in the two groups of rats, employing the constant infusion method of Waterlow et al. At the same time, the fractional turnover rate of the whole body protein was measured.

When the diet was changed from the 20% protein to the 3% protein diet, the amount of albumin mass in both extra- and intravascular spaces decreased significantly. During 7 days on the diet, the extra- and intravascular albumin mass per 100 g of body weight did not change significantly in the rats fed the 20% protein diet. On the other hand, rats fed the 3% protein diet lost almost 30% of the extra- and intravascular albumin per lOOg body weight.

The fractional turnover rates of whole body albumin were estimated to be 31.7 and 19.8%/day in the 20% protein and the 3% protein diet-fed rats, respectively. The fractional turnover rates of whole body protein were 16.1 and 10.6%/day in the 20% protein and the 3% protein diet-fed rats, respectively.

The leucine fluxes to albumin synthesis and whole body protein synthesis were calculated to be 5.9 and 83 μmol/hr, respectively, in the 20% protein diet-fed rats. The leucine fluxes in the 3% protein diet-fed rats were 2.5 and 54μmol/hr for the albumin synthesis and for the whole body protein synthesis, respectively.

These results demonstrate the quantitative significance of albumin metabolism in the whole body protein turnover in rats fed on two levels of protein intake.     

阿司匹林对中暑休克大鼠的保护及抗疲劳作用

本研究旨在探讨阿刮匹林是否可以通过降低中暑休克大鼠的白介素-β（interleukin-1β,IL-1β）水平从而发挥抗中暑休克作用。研究包括：（1）预先给产阿刊匹林对人鼠中暑休克的影响;（2）特异性一氧化氮合酶（inducible nitric oxide synthase,iNOS）抑制剂氨基胍（aminoguanidine,AG）对人鼠中暑休克的影响;（3）预先给予阿司匹林对清醒大鼠抗高温疲劳的影响。通过将大鼠置于仿真模拟高温气候舱接受环境离温（环境温度41℃,相对湿度65％）热暴露以诱导中暑休克,建立中暑休克动物模型。实验（1）和（2）分别将大鼠随机分为对照组和阿司匹林处理组,或对照组和AG组,记录热暴露过程中平均动脉压（mean arterial blood pressure,MAP）,结肠温度（colonic temperature,Tco）,心电图（electrocardiograph,ECG）,检测血浆IL-1β或NO浓度。实验（3）将对照组和阿司匹林处理组清醒大鼠置于水温41℃的水箱中,自由游泳,记录生存时间。结果显示,预先给予阿司匹林对大鼠中暑休克形成后血压下降有显著的抑制作用并延长生存时间,抑制血浆IL-1β升高的程度,但对体温变化没有显著影响。预先给予阿司匹林显著延长清醒大鼠在高温疲劳条件下生存时间。AG可以抑制中暑休克形成后大鼠MAP下降并显著延长大鼠生存时间,而且可以显著抑制热暴露后大鼠血浆NO浓度上升,但对大鼠热暴露后体温变化没有显著影响。结果提示,IL-1β可能通过诱导iNOS降低外剧血管张力从而参与中暑休克形成,预防性给予抗炎剂量阿司匹林可能对中暑休克出现的血压降低有一定的保护,同时增强对高温及疲劳耐受性,这种影响可能是通过对IL-1β以及局部iNOS的抑制而实现的。     

Topochemical aspects of nucleic-acid and protein metabolism within the neuron-neuroglia unit of the hypothalamic supraoptic nucleus

The RNA and both the total and basic protein content of individual cells were determined by cytospectrophotometry in neurons and perineuronal oligodendroglia of the hypothalamic supraoptic nucleus in rats subjected to various stresses, as well as in ground squirrels during natural hibernation. Barbiturate narcosis and deep cooling, which induced a decrease in body temperature in rats and hibernation in squirrels, caused a marked decrease of all macromolecular constituents in neurons. A similar decrease was found in the perineuronal oligodendroglia in rats, but an increase was observed in ground squirrels. After cessation of cooling, while the body temperature of the animals returned to normal, the neurons, but not the oligodendroglia, of rats showed a significant accumulation of RNA, while RNA accumulated in both neurons and perineuronal oligodendroglia in ground squirrels. Milder cooling of rats, which did not lower their body temperature, induced reciprocal changes in basic-protein content in neuronal and glial cell nuclei, with the accumulation of protein occurring initially in neurons, and subsequently in glia. When cold adaptation was accomplished, the basic protein content of neurons and glial cells returned to the control level. Four days after adrenalectomy in rats, the RNA content decreased in oligodendroglia but not in neurons of the supraoptic nucleus. This effect was completely abolished by daily injections of cortisol in the adrenalectomized animals. The data obtained indicate the existence of differences in metabolic responses to stress between neurons and glial cells of the supraoptic nucleus of the hypothalamus.     

Heatstroke induces a reduction of natural killer cell activity in rats

Experiments were carried out to determine the changes of natural killer (NK) cell activity that occurred during heatstroke in rats pretreated with or without interleukin-1 (IL-1) receptor antagonist (IL-1ra). After the onset of heatstroke, all the splenic NK cell activity, the effector-target cell conjugation, and the NK cell numbers were decreased in rats. Additionally, an increase in the plasma IL-1 level was associated with arterial hypotension, cerebral ischemia and hyperthermia during rat heatstroke. Pretreatment with an IL-1ra reversed in part the heatstroke-induced inhibition of NK cell activity. Thus it appears that the inhibition of NK cell activity produced by activation of IL-1 receptor mechanism is associated with the increased susceptibility to infection that is well described in heatstroke.     

Increase in rat intestinal permeability to endotoxin during hyperthermia

Victims of heat stroke exhibit several clinical features which are also encountered in endotoxaemia. In order to investigate these similarities hyperthermic rats were used to explore the possibility that high body temperature results in increased permeability of intestinal wall to endotoxin. 125I endotoxin was introduced into intestinal segments taken from non-heat exposed rats. The segments were then incubated at 37 degrees C or 45 degrees C. Intestinal segments from heat stressed rats were similarly prepared and incubated at 37 degrees C. Leakage of endotoxin from segments taken from heat stressed rats was three times greater than from those from non-heat stressed rats, as were the segments from non-heat stressed rats which were incubated at 45 degrees C. These results indicate that the intestinal membrane is damaged by heat and that an increase in outward leakage of microbial endotoxins from the gut then occurs. This might contribute to the pathophysiological picture of heatstroke.     

Biokinetics of 65Zn in liver and whole body and its bio-distribution in nickel treated protein deficient rats

This study was designed to determine the effect of nickel treatment on biological half-lives of 65Zn in whole body and liver as well as on distribution of 65Zn in different organs of protein deficient rats. Nickel sulfate at a dose level of 800mg/l in drinking water was administrated to normal control as well as to protein deficient rats for 8 weeks. A significant increase was found in fast and slow components of biological half lives of 65Zn in whole body and only fast component in liver of protein deficient rats. Interestingly, slow component in whole body and fast component in liver of nickel treated protein deficient rats were not different from normal controls though they were significantly elevated in protein deficient rats. On the other hand, slow component of 65Zn was also not altered in nickel treated protein deficient rats, which however, was significantly decreased in nickel treated rats. Protein deficiency led to a marked elevation in per cent uptake of 65Zn in brain and caused significant depression in liver, kidney and intestine. However, uptake of 65Zn in brain showed a significant depression in nickel treated rats, whereas the uptake was elevated in brain in nickel treated protein deficient rats. In conclusion, protein deficient conditions seem to be playing a dominant role in context with the distribution of 65Zn in different organs when nickel is administered to protein deficient rats. However nickel alone is seen to cause adverse effect on the distribution of 65Zn.     

Plasma-cortisol levels in experimental heatstroke in dogs

The effect of external heat-load, exercise and dehydration on dynamic changes in plasma cortisol during the development of heatstroke was investigated. Thirty-three unanesthetized dogs were tested under two sets of climatic conditions: comfort conditions and hot-dry climatic conditions, half of them while exercising. Half of the dogs in each group were rehydrated. None of the dogs that were investigated at room temperature suffered heatstroke. Of the dogs exposed to high ambient temperature, all of the exercising, as well as five out of six non-hydrated dogs and one rehydrated non-exercising dog suffered heatstroke. Significant dehydration (6%–7% of body weight), occurred only under hgh ambient temperature. Plasma cortisol levels of all dogs that suffered heatstroke rose conspicuously for at least 5 h and returned to normal levels 24 h later. Cortisol levels of dogs who did not experience heatstroke remained within the normal range. Cortisol levels correlated with the severity of the stress leading to heatstroke. High and rising levels of cortisol, several hours after body temperature returns to normal, may support the diagnosis of heatstroke.     

The precision and accuracy of noninvasive measurements of total body calcium in the rat

It has been proposed that the adult castrated male rat may be used as a model of human postmenopausal osteoporosis. If such a model is valid then the total body calcium of castrated rats should progressively decrease compared with normal rats. To search for this age-related difference, it will be necessary to use a measurement technique with a precision which is small compared with the magnitude of the change to be detected. From measurements of total body calcium by neutron activation and by whole body ashing of eight dead rats, it has been shown that total body calcium can be measured with an accuracy and precision which should allow distinction between the expected lifetime changes in normal and castrated rats.     

Physical training under the influence of beta blockade in rats: effect on adrenergic responses.

Rats were trained by daily swimming or running exercises with and without daily propranolol injections. Both training methods resulted in cardiac enlargement, but only swimming exercise caused hypertrophy of the brown adipose tissue. These changes were antagonized by beta blockade. The size of the adrenals reflected the stress of the treatments, but other known stress parameters, such as the size of the thymus or sexual organs dit not. Only swimming training without beta blockade sensitized the rats to the calorigenic action of noradrenaline. The cooling rate of the rats in water, when taking into account the insulative capacity of the body, was decreased in swimming-trained as well as in propranolol-treated rats but increased in running-trained rats. The latter two changes may be due to circulatory alterations, while the delayed body cooling in swimming-trained rats probably results from increased heat production capacity. Training-induced resting bradycardia and enhanced tachycardic response to isoprenaline were observable only in the animal groups trained without beta blockade. The pressor response to noradrenaline tended to be higher in the trained groups and the propranolol-treated group than in the controls and was smaller in the animal groups trained under the influence of beta blockade. On the other hand, the hypotonic response to isoprenaline was smaller in the propranolol-treated and running-trained animals. The results emphasize the importance of the sympathetic nervous system in the adaptation of an organism to physical training.     

Protein synthesis in the organs and tissues of frogs with local exposure to low temperatures]

Intensity of synthesis of total proteins and spectrum of synthesized proteins has been studied in various organs and tissues of sharp-faced frogs during local profound cooling of one of its limbs. It was shown that the effect of low temperatures on a certain part of a body of a poikilothermic animal has induced both a response of the whole organism and a much more acute response of the cooled tissues. The organism responded by a fall of the whole body temperature and a decrease in the activity of synthesis of total proteins of the viscera. Local cooling of a part of the frog body has induced a more significant decrease in the protein synthesis intensity in the cooled tissues alteration in the spectrum of synthesized proteins and appearance of specific proteins.     

Alterations in cardiac contractile performance and sarcoplasmic reticulum function in sucrose-fed rats is associated with insulin resistance

Diabetes mellitus (DM) causes the development of a specific cardiomyopathy that results from the metabolic derangements present in DM and manifests as cardiac contractile dysfunction. Although myocardial dysfunction in Type 1 DM has been associated with defects in the function and regulation of the sarcoplasmic reticulum (SR), very little is known about SR function in Type 2 DM. Accordingly, this study examined whether abnormalities in cardiac contractile performance and SR function occur in the prestage of Type 2 DM (i.e., during insulin resistance). Sucrose feeding was used to induce whole body insulin resistance, whereas cardiac contractile performance was assessed by echocardiography and SR function was measured by SR calcium (Ca²⁺) uptake. Sucrose-fed rats exhibited hyperinsulinemia, hyperglycemia, and hyperlipidemia relative to control rats. Serial echocardiographic assessments in the sucrose-fed rats revealed early abnormalities in diastolic function followed by late systolic dysfunction and concurrent alterations in myocardial structure. The hearts of the 10-wk sucrose-fed rats showed depressed SR function demonstrated by a significant reduction in SR Ca²⁺ uptake. The decline in SR Ca²⁺ uptake was associated with a significant decrease in the cAMP-dependent protein kinase and Ca²⁺/calmodulin-dependent protein kinase II-mediated phosphorylation of phospholamban. The results show that abnormalities in cardiac contractile performance and SR function occur at an insulin-resistant stage before the manifestation of overt Type 2 DM. cardiomyopathy; diabetes mellitus; echocardiography     

Calpain-2 protects against heat stress-induced cardiomyocyte apoptosis and heart dysfunction by blocking p38 mitogen-activated protein kinase activation

Cardiovascular dysfunction is a common complication among heatstroke patients, but its underlying mechanism is unclear. This study was designed to investigate the role of calpain-2 and its downstream signal pathway in heat stress-induced cardiomyocyte apoptosis and heart dysfunction. In cultured primary mouse neonatal cardiomyocytes (MNCs), heat stress (43°C for 2 hr) induced a heat-shock response, as indicated by upregulated heat-shock protein 27 (HSP27) expression and cellular apoptosis, as indicated by increased caspase-3 activity, DNA fragmentation and decreased cell viability. Meanwhile, heat stress decreased calpain activity, which was accompanied by downregulated calpain-2 expression and increased phosphorylation of p38, extraceIIuIar signaI-reguIated protein kinase (ERK1/2) and c-Jun N-terminaI kinase (JNK). Calpain-2 overexpression abrogated heat stress-induced apoptosis and phosphorylation of p38 and JNK, but not of ERK1/2. Blocking only p38 prevented heat stress-induced apoptosis in MNCs. In cardiac-specific calpain-2 overexpressing transgenic mice, p38 phosphorylation and cardiomyocyte apoptosis were decreased in the heart tissue of heatstroke mice, as revealed by western blot and terminal deoxynucleotidyl transferase dUTP nick end labelling assays, respectively. M-mode echocardiography also demonstrated that calpain-2 overexpression significantly improved heatstroke-induced decreases in ventricular end-diastolic volume and cardiac output. In conclusion, our study suggests that heat stress reduces calpain-2 expression, which then activates p38, leading to cardiomyocyte apoptosis and heart dysfunction.     

Differential heating of trunk and extremities. Effects on thermoregulation mechanisms

Experiments in which the whole human body was heated or cooled are compared with others in which one extremity (arm or leg) was simultaneously cooled or heated. With a warm load on the rest of the body resulting in general sweating, a cold load on one extremity did not evoke local shivering; with general body cooling, heating one limb did not stop the shivering. Skin temperatures of the other parts of the body were not influenced by warming or cooling one extremity. Evaporative heat loss was influenced by local, mean skin and core temperature, whereas shivering did not depend on local temperature, and vasomotor control seemed to be controlled predominantly by central temperatures. A cold load on an extremity during whole body heating in most cases induced an oscillatory behaviour of core temperature and of the evaporative heat loss from the body and the extremity. It is assumed that local, mean skin and core temperatures influence the three autonomous effector systems to very different degree.     

A relationship between heat load and plasma enzyme concentration

1. 1. The sensitivity of serum enzyme levels as indicators of tissue damage is less well established in the prodromal period of heatstroke, especially for sub-lethal stress conditions.

2. 2. Anaesthetized rats were exposed to two different sets of thermal conditions.

3. 3. Plasma levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), creatine kinase (CK) and lactate dehydrogenase (LD) were assayed in each group upon termination of stress, 6 h post-stress and 24 h post-stress.

4. 4. The tissue “damage” sustained was mild to moderate and completely reversible.

5. 5. The rate of rise in body temperature may constitute an important factor in the ultimate pathology.

6. 6. CK proved to be the most sensitive parameter of tissue “damage”.