Hyperthermic pretreatment decreases microvascular protein leakage and attenuates hypotension in anaphylactic shock in rats.

Systemic anaphylaxis is a life-threatening allergic reaction and its pathologic conditions, such as edema, bronchospasm, and hypotension, have been attributed to release of vasoactive mediators. Heat shock protein (HSP) is known to play a protective role in living cells under various stresses. In these studies, we investigated the protective role of heat shock response in anaphylactic shock, focusing on changes of blood pressure (BP) and vascular permeability. Adult sensitized rats were injected intravenously with Evans blue (EB) and challenged with bovine serum albumin (BSA). The rats were treated with whole-body hyperthermia at 41.5 +/- 0.5 degrees for 15 min 24 h before BSA challenge. Vascular protein leakage in tissues was analyzed with the EB technique. The results showed that BSA challenge induced EB extravasation in all sensitized rats. EB values (EB/tissue; microg/g) in heart and lung (112.3 +/- 41 and 244.4 +/- 90.6; mean +/- SD; n = 6) in the nonheated rats were significantly higher than those (33.4 +/- 23.3 and 103.4 +/- 63.9; n = 9) in the heated rats (P < 0.05). The results showed that BSA challenge caused BP to fall drastically in the sensitized rats. BP in the heated rats was significantly higher than BP in the nonheated rats from 4 to 15 min during anaphylactic shock (P < 0.001). Inducible HSP72 appeared overexpressed in heart, lung, and liver tissue in the heated rats tested by Western immunoblotting. The results indicate that reduction of increased protein leakage and attenuation of hypotension may result from induction of HSP by whole-body hyperthermia.

Attenuation of sepsis-induced apoptosis by heat shock pretreatment in rats.

Apoptosis is a process by which cells undergo a form of non-necrotic cellular suicide. Although it is a programmed process, apoptosis can be induced by various stressors. During sepsis, apoptosis has been regarded as an important cause of cell death in the immune system, leading to unresponsiveness to treatment. This study was designed to investigate how prior heat shock induction can influence the rate of apoptosis in animals that have experienced sepsis. Sprague-Dawley rats were used, and experimental sepsis was induced by cecal ligation and puncture (CLP). Animals in the heated group were anesthetized and received heat shock by whole-body hyperthermia. They were sacrificed 9 h and 18 h after CLP as early and late sepsis, respectively. Apoptosis was evaluated by "DNA ladder" detection in agarose electrophoresis and Tdt-mediated dUTP nick end-labeling (TUNEL) assay. Hsp72 was detected by Western blot analysis. The results showed that the DNA ladder was detected most clearly in the thymus at the late phase of sepsis with time course dependence, while it showed less clearly in heat shock treated animals. Histopathological study by TUNEL assay obtained similar results in the thymus, where the cortex was more susceptible to apoptosis than the medulla. The Western blot analysis showed that the heat shock induced Hsp72 concomitant with an increase in Bcl-2:Bax ratio. In conclusion, heat shock pretreatment prevents rats from sepsis-induced apoptosis that may account for the better outcome of experimental sepsis. An increase in the Bcl-2:Bax ratio may in part explain the molecular mechanism of the effect of heat shock pretreatment.

Evidence of multi-step regulation of HSP72 expression in experimental sepsis.

Heat shock proteins (Hsps) are a family of highly conserved proteins induced in response to various stresses. Hsps protect cells against subsequent lethal circumstances. Previous work from our laboratory has indicated that Hsp72 is not induced during experimental sepsis in rats, but the regulation of the induction of Hsp72 synthesis in this disease cascade has not been investigated. In the present study, we evaluated the expression of the hsp72gene, focusing on the activation and DNA-binding ability of heat shock factor 1 (HSF1), hsp mRNA accumulation, and Hsp72 synthesis in animal sepsis models induced by cecal ligation and puncture procedure. The results were compared with those of sham-treated and heat-shocked rats. It was shown that the expression of the hsp72 gene in sepsis was a multi-step process, as previously documented in in vitro studies. Hsp72 synthesis was not induced during sepsis, whereas DNA binding of HSF was detectable, suggesting that the induction of Hsp72 is blocked downstream to HSF-DNA complex formation by the metabolic alteration occurring during sepsis. The dissociation failure of the constitutive heat shock element binding factor (CHBF) from the heat shock element may play an important role in this negative regulation.

Sex-specific expression of N-methyl D-aspartate receptor (NMDAR) in the preoptic area of neonatal rats.

The relationship between the N-methyl-D-aspartate receptor (NMDAR) and the sex-specific neurotoxicity of L-glutamate on the preoptic area (POA) of neonatal rats was studied. The NMDAR were semiquantified by western blot analysis. The kinetic change of intracellular calcium and lactate dehydrogenase (LDH) efflux were monitored as rapid and delayed toxic signals, respectively. The results showed that: (1) the NMDAR expression in POA of male rat is higher than that of females; (2) the L-glutamate (500 microM) induced a more significant elevation of intracellular calcium in neuron derived from male rat than that from female; (3) after glutamate-treatment, the LDH efflux in neuronal culture of male rat is higher than that of females. These results suggest that the quantitative difference in NMDAR between male and female rats may contribute to the sex-specific neurotoxicity of L-glutamate on the POA of neonatal rats.

Heat shock treatment decreases the mortality of sepsis in rats.

Sepsis is an increasingly common and lethal diagnosis in hospitalized patients. In spite of the advances in antibiotics and medical equipment, the mortality rate has not been improved in the last decade. Recently, heat shock proteins (Hsps) have been well documented to play a self-protective role in almost all living cells under various pathological and physiological stresses through a mechanism known as thermotolerance or cross tolerance. The present study was designed to investigate the expression of Hsp72 and the protective role of pre-induction of Hsps in the mortality during different phases of sepsis. Adult male Sprague-Dawley rats were employed in the study. Sepsis was induced by cecal ligation and puncture (CLP). Heat shock treatment was performed 16 hrs before sepsis induction by heating the rats whole-bodily with an electric heating pad under general anesthesia. The mortality rates with time in both control and preheated groups were monitored. Hsp72 was detected by SDS-PAGE, Western blotting and immunostaining in the brain, heart, liver, kidney, lung, adrenal gland, muscle and lymphocytes. The results show that both early (9 hrs post-CLP) and late (18 hrs post-CLP) sepsis failed to increase the synthesis of Hsp72. Previous induction of Hsps by heat shock treatment significantly decreased the mortality rate of late sepsis. Applying a sufficient inducer to lymphocytes of late sepsis reversed the synthesis of Hsp72 from inactive state into an over-expressive situation in vitro. These results suggest that Hsps are involved in the pathogenesis of sepsis and the involvement of Hsps during the progression of sepsis could add to a first line of host defense against invasive pathogens. Searching for an acceptable agent or less invasive method that leads to increased Hsps expression may provide a means for better treatment of severe infection.

Effects of Chinese herbal prescriptions on copulatory activity in aged male rats: a preliminary study.

Four herbal prescription medicines, Chi-Pao-Mei-Jan-Tan, Gui-Fu-Ba-Wei-Wan, Huan-Shao-Tan; and San Tsai-Feng-Sui-Tan, were tested for their effects on sexual behavior in aged rats. Crude liquid extracts of these herbs were administered to the rats daily through oral tubing for 14 days. All four herbal prescriptions showed some effects in restoration of mount and intromission behaviors, but there was no effect on restoration of ejaculation in 26 month old rats that had exhibited no copulatory activity (no mount, intromission and ejaculation) previously. The effects of Chi-Pao-Mei-Jan-Tan were further tested in 26 month old rats with low mount and intromission activities but without ejaculation behavior, and in 15 month old rats (middle-age group) that showed normal mount and intromission behavior but no ejaculation activity. Chi-Pao-Mei-Jan-Tan was effective in improving the frequency of both mount and intromission, but failed to restore the ejaculation activity of the old rats with low mount and intromission behaviors. It was, however, very effective in restoration of ejaculation activity in middle-aged rats that exhibited normal mount and intromission behaviors. Serum testosterone (T) levels of Chi-Pao-Mei-Jan-Tan in tested old and middle-aged rats were determined by radioimmunoassay, and showed no difference before and after treatment. Our findings demonstrated that the four herbal prescriptions had some effects in restoration of mount and intromission behaviors, but not ejaculation activity in old rats, and that Chi-Pao-Mei-Jan-Tan was very effective in restoration of ejaculation activity in middle-aged rats. The promotional effect of Chi-Pao-Mei-Jan-Tan on copulatory behavior was not correlated with serum T levels.

Enlarging effects of estradiol on the nuclear volume of neurons in the hypothalamus during aging.

Neuronal nuclear volumes (NNVs) were measured in the medial preoptic nucleus (MPN), anterior hypothalamic area (AHA) and arcuate nucleus (ARN) of young adult, middle-aged, and old rats of both sexes. The NNVs in the darkly stained sexual-dimorphic nucleus of the preoptic area (SDN-POA) and the lighter staining surrounding area (non-SDN-POA) within the MPN were measured separately. Intact young and middle-aged female rats had larger NNVs than those of the males in SDN-POA, non-SDN-POA and AHA but not in ARN. During aging, only intact old female rats manifested significant NNV shrinkage in all the measured areas. Long-term treatment with estradiol benzoate (EB) caused a significant enlargement of the NNVs in non-SDN-POA and ARN of middle-aged and old male rats as well as the NNVs in SDN-POA, non-SDN-POA and ARN of old female rats. The enlarging effect of EB on NNVs in both SDN-POA and non-SDN-POA of female rats could be prevented by ovariectomy. Furthermore, NNVs in SDN-POA and non-SDN-POA of ovariectomized female rats were even smaller than those of the age-matched intact female rats. These results indicate that: (1) the NNVs of MPN and ARN in male and female rats were enlarged after long-term exposure of physiological dose of estradiol; (2) the enlarging effects of EB on NNV in MPN can explain why the NNV of intact female rats is larger than that of males, and (3) during aging, the sex-specific shrinkage of NNVs in MPN, AHA and ARN of female rats may be due to an intrinsic aging process rather than long-term effects of EB.

Protein kinase C activity is increased in rat heart during the early hyperdynamic phase of sepsis.

Changes in protein kinase C (PKC) (calcium- and phospholipid-dependent protein kinase) activity in rat heart during different cardiodynamic phases of sepsis were studied in an attempt to understand the pathophysiology of altered myocardial function during sepsis. Sepsis was induced by cecal ligation and puncture. Experiments were divided into three groups: control, early sepsis, and late sepsis. Early and late sepsis refers to those animals sacrificed at 9 and 18 h, respectively, after cecal ligation and puncture. Cardiac PKC was extracted and partially purified by ammonium sulfate fractionation and diethylaminoethyl-cellulose chromatography. PKC activity was assayed on the basis of the rate of incorporation of 32P from [gamma-32P]adenosine triphosphate into histone. The results show that during early sepsis, cytosolic PKC activity was increased by 42-73%, whereas membrane associated PKC activity was unchanged. During late sepsis, both cytosolic and membrane associated PKC activities remained unchanged. Kinetic analysis of the data on cytosolic PKC during the early phase of sepsis reveals that the Vmax (maximal velocity) values for Ca2+, phosphatidylserine, and diacylglycerol were increased by 58, 42, and 50%, respectively, with no changes in their Km (substrate concentration required for half-maximal enzyme activity) values. These data indicate that cytosolic PKC activity was activated in rat heart during the early hyperdynamic phase of sepsis. Because PKC mediated phosphorylation plays an important role in regulating myocardial contractility, an activation in cytosolic PKC may contribute to the development of a hypercardiodynamic state during the early phase of sepsis.

Protein kinase a activity is increased in rat heart during late hypodynamic phase of sepsis.

Changes in the activities of protein kinase A (PKA, or cAMP-dependent protein kinase) in rat heart during different cardiodynamic phases of sepsis were investigated. Sepsis was induced by cecal ligation and puncture. Experiments were divided into three groups: control, early sepsis, and late sepsis. Early and late sepsis refers to those animals killed at 9 and 18 h, respectively, after cecal ligation and puncture. Cardiac PKA was extracted and partially purified by acid precipitation, ammonium sulfate fractionation, and DEAE-cellulose chromatography. PKA was eluted from DEAE-cellulose column with a linear NaCl gradient. Two peaks of PKA, type I (eluted at low ionic strength) and type II (eluted at high ionic strength), were collected and their activities were determined based on the rate of incorporation of [gamma-32P]ATP into histone. Results obtained show that during early sepsis, both type I and type II PKA activities were unaffected. During late sepsis, type I PKA activities were stimulated by 66.7-97.7%, while type II PKA activities remained constant. Kinetic analysis of the data on type I PKA during late sepsis reveals that the Vmax values for ATP, cAMP, and histone were increased by 84.7, 66.7, and 97.7%, respectively; while the Km values for ATP, cAMP, and histone were unaltered. These data indicate that type I PKA is activated in rat heart during late hypodynamic phase of sepsis. Since kinase-mediated phosphorylation plays an important role in regulating myocardial function and metabolism, an activation of type I PKA during late sepsis may contribute to the development of altered myocardial function during hypodynamic phase of sepsis.

The neonatal neurotoxicity of monosodium L-glutamate on the sexually dimorphic nucleus of the preoptic area in rats.

The neurotoxic effect of monosodium L-glutamate (MSG) on the morphologies in the darkly stained sexually dimorphic nucleus of the preoptic area (SDN-POA) and the lighter-staining surrounding area (non-SDN-POA) within the medial preoptic nucleus (MPN) was evaluated. Male and female Long-Evans rats were used. MSG (4 mg/g of body weight) was administered subcutaneously to pups on days 1 and 3 postnatally. Normal saline was used as the vehicle. At the age of 6 months, the rats were sacrificed and the brain tissues were fixed for histological examination. The morphological changes, i.e., total volume, density, total neuron number, neuronal nuclear volume (NNV) and ratio of pyknosis, of the SDN-POA and non-SDN-POA within the MPN, were estimated using the AMS VIDS III semiautomatic image-analytic system. The results indicate that neonatal MSG treatment caused significant neuronal loss and decreases in total volume of the SDN-POA and non-SDN-POA of male and female rats. However, only the SDN-POA of MSG-treated male rats showed a significant increase of pyknosis and decrease of neuronal density. A significant enlargement of NNV in the SDN-POA and non-SDN-POA was observed in the MSG-treated male rats. These results indicate that the MPN shows sex-specific and area-specific changes after neonatal neurotoxicity due to MSG.

[Effect of pinealectomy on N-methyl-D-aspartate-facilitated receptivity in female rats].

The purpose of this study was to examine the effect of pinealectomy and the possible mechanism of the pineal gland on N-methyl-D-aspartate (NMDA)-associated receptivity in female rats. Monosodium L-glutamate (MSG) was used as a neurotoxin to induce hypogonadal status. Long-Evans rats were divided into four groups: (1) control (C), (2) pinealectomized (Px), (3) MSG-treated (MSG) and (4) pinealectomized MSG-treated (Px-MSG). Two injections of MSG were administered on the first and the third days postnatally with a dose of 4 mg/g body weight. Pinealectomy was performed at six weeks of age. In the first part of the experiment, all four groups of rats were ovariectomized at 3.5 months and implanted subcutaneously with a 2mm silastic capsule filled with 20% estradiol benzoate (EB). One week later, the sexual receptivity was estimated by lordosis quotient (LQ) before and ten minutes after 20 mg/kg B.W. NMDA administration. The result shows that NMDA caused a remarkable increase of LQ in control rats, but no significant effect on MSG-treated rats. There was no significant difference between control and Px rats before NMDA administration, but Px rats exhibited higher LQ than control rats after NMDA treatment. In the second part of the experiment, the effect of pinealectomy on releasability of LHRH neurons was examined indirectly by NMDA-evoked LH secretion. The dosage and sampling schedule were chosen by the dose-response and time course of LH response to NMDA, respectively. Serum samples were collected before and ten minutes after NMDA administration. Serum concentration of LH was measured by radioimmunoassay.(ABSTRACT TRUNCATED AT 250 WORDS)

Electrophysiologic properties of isolated adult cardiomyocytes from septic rats.

In this study, we examined the effect of sepsis on the electrical properties of isolated ventricular myocytes. Sepsis was induced by cecal ligation and puncture (CLP). The control rats were sham-operated. Membrane potentials and ionic currents in isolated cardiac myocytes were measured by the tight-seal, whole-cell patch-clamp technique. The results show that the resting membrane potentials of heart cells were significantly lower in the septic group (18 hr post-CLP) than those in the control group. However, there was no significant difference in action potential duration of 50% and 90% repolarization between the two groups of cells. In voltage-clamp experiments, isoproterenol (10 nM), a beta-adrenergic agonist, caused an increase in L-type calcium current (ICa,L) in a similar magnitude in myocytes isolated from the control and septic rats. Furthermore, isoproterenol failed to modify the time constants for ICa,L inactivation and the overall shape of current-voltage relationship for both groups of cells. These results indicate that formation of a G omega seal and subsequent tight-seal whole-cell recording with patch-clamp technique can be performed in heart cells derived from CLP-induced septic rats, and that septic rat heart is capable of responding effectively to beta-adrenergic stimulation.

[The mechanism of the pineal gland in inhibiting sexual receptivity of female rats treated with monosodium-L-glutamate: (III). Does it concern with the function of the pituitary gland?].

To elucidate whether pituitary function participates in the effect of the pineal gland on sexual receptivity, monosodium L-glutamate (MSG) was used as a neurotoxin to induce hypogonadal status. Long-Evans rats were divided into four groups: (1) normal control (C), (2) pinealectomized (Px), (3) MSG-treated (MSG) and (4) pinealectomized MSG-treated (Px-MSG). Pinealectomy was performed at six weeks of age. In the first part of the experiment, the sexual receptivity was estimated at the age of 2.5 months by lordosis quotient (LQ). The result indicates that the decline of receptivity by neonatal MSG treatment can be significantly improved by pinealectomy. In the second part of the experiment, the effect of pinealectomy on pituitary function was examined by two tests including (1) post-castrational LH rise and (2) pituitary response to LHRH. Ovariectomy was performed at the age of 2.5 months. Four weeks later three consecutive blood samples were collected at 10 minute intervals for LH radioimmunoassay. Then, three doses of LHRH (100 ng, 250 ng and 500 ng/100 g of body weight) were administered separately at two-week intervals, serum samples were taken before as well as 15 and 60 minutes after LHRH administration. The results showed that there was no significant difference in serum LH levels between Px and control rats after ovariectomy. The LHRH-evoked LH elevation in Px-MSG rats was just the same as that of the MSG rats, although the LH level in MSG rats was lower than in the control rats.(ABSTRACT TRUNCATED AT 250 WORDS)

[The mechanism of the pineal gland in inhibiting sexual receptivity of female rats: (II) Does it concern progesterone?].

The present study was designed to find the role of progesterone in modulating the effect of pineal gland upon reproductive behavior in the female rat. Monosodium L-glutamate (MSG) was used as a neurotoxin to induce hypogonadal function. In the first part of the experiment, rats were divided into four groups: (1) normal, (2) pinealectomized (Px), (3) MSG-treated (MSG), and (4) pinealectomized MSG-treated (Px-MSG), as immature rats became mature, the IEC (Incidence of Estrous Cycle) in each group was determined by vaginal smear for three weeks. Serum samples for estimating the levels of progesterone were collected at estrous. Then, ovariectomy was performed. Two weeks later, estradiol was primed for three days following progesterone administration and sexual receptivity as well as solicitation were observed. The results showed that, the serum progesterone levels and IEC of Px-MSG rats were significantly higher than that of the MSG group. In addition, Px and Px-MSG rats showed a higher sexual receptivity than normal and MSG rats, respectively. In the second part of the experiment, rats were divided into two groups: (1) normal and (2) Px. All rats were ovariectomized at 2.5 months of age and implanted with a 2-mm 20% estradiol benzoate-filled silastic capsule. Then, sexual receptivity to various doses of progesterone (0, 0.02, 0.1, 0.5 mg/rat) were observed. The results showed that receptivity of Px rats was significantly higher than that of the normal ones in a dose-dependent manner. It is concluded that pineal gland plays an important role in the regulation of ovary function as well as sexual receptivity in female rats. Also, progesterone may be one of the factors modulating the effect of pineal gland on sexual receptivity.

Sex-specific impairment in sexual and ingestive behaviors of monosodium glutamate-treated rats.

L-monosodium glutamate (MSG) (4 mg/g b.wt.) was injected subcutaneously to pups on days 1 and 3. At age 3.5 months, sexual and ingestive behaviors were observed. Neonatal MSG treatment resulted in severe and widespread neuron destruction in the basomedial hypothalamus of both sexes, but only super-chiasmatic nucleus in male rats. A decline in sexual behavior was also observed. Serum levels of testosterone and dihydrotestosterone in male rats as well as serum level of progesterone in female rats were decreased. Ovariectomized MSG-treated female rats injected with estradiol benzoate followed by progesterone showed a dramatic improvement in sexual behavior, whereas castrated MSG-treated male rats injected with testosterone propionate did not meliorate the decline of sexual behavior. Neonatal MSG treatment also induced reductions of body weight in male rats, higher diurnal percentages of food and water intake in male rats than in female rats, and a decline of water-to-food ratio. These observations suggest that neonatal male rats show higher susceptibility to glutamate-induced dysfunction of sexual and ingestive behaviors than females. Our findings also suggest a sex difference in the mechanism of dysfunction of sexual behavior, i.e., decreased copulatory activity in male rats is mainly due to CNS damage, in contrast to decreased blood progesterone level in female rats.

[The action mechanism of the pineal gland in inhibiting sexual receptivity of female rats: (I). Is it concerned with the extrahypothalamic inhibitory pathway?].

Monosodium L-glutamate (MSG), as a neurotoxin, was administered subcutaneously at a dose of 4 mg/g body weight to rat pups on days 1 and 3 postnatally. The sexual receptivity expressed as lordosis quotient (LQ) in MSG-treated female rats (2.5 months old) was significantly lower than that of the normal female ones, and this poor sexual receptivity of MSG-treated females could be improved by pinealectomy at the age of six weeks. In order to investigate the relationship between the inhibitory role of the pineal gland and the extrahypothalamic inhibitory pathway, normal control and MSG-treated rats were ovariectomized and implanted subcutaneously with estradiol capsule (20% estradiol benzoate, in 2mm silastic tube) at the age of 2.5 months. Three days after implantation, sexual receptivity was observed. Then, the anterior roof deafferentation (ARD) was preformed one week later and the sexual receptivity in both groups was compared again. The results showed that there was no significant difference of the lordosis response between the MSG-treated and the control groups before ARD, and the level of restoration by ARD in LQ of MSG-treated rats was similar to that of the control group. It seems that the extrahypothalamic inhibitory pathway of sexual receptivity may not be influenced by neonatal MSG-treatment. Our previous results indicated that the behavioral sensitivity to estradiol was altered by ARD. However, in the present study, the lordosis responses of MSG-treated and pinealectomized female rats were not significantly different from that of the control group after ovariectomy and exogenous estradiol implantation. This means that the behavioral sensitivities to estradiol were not changed by MSG-treatment or pinealectomy.(ABSTRACT TRUNCATED AT 250 WORDS)