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Human umbilical cord blood cells (HUCBCs) have been shown to be beneficial in reducing neurological deficits in rats with brain fluid percussion injury (FPI). This study aimed to assess the basic mechanisms underlying the neuroprotective effects of HUCBC-derived cluster of differentiation 34-positive (CD34+) cells. Rats were divided into three major groups: (i) sham-operated controls; (ii) FPI rats treated with phosphate-buffered saline (PBS); (iii) FPI rats treated with 0.2%, 50%, or 95% CD34+cells (in 5 × 105cord blood lymphocytes and monocytes). Intravenous (IV) administration of 0.3 ml of PBS, 0.2% CD34+cells, 50% CD34+cells, or 95% CD34+cells was conducted immediately after FPI. It was found that 4 days post-FPI, CD34+cells could be detected in the ischemic brain tissues for 50% CD34+cell- or 95% CD34+cell-treated FPI rats, but not for the PBS-treated FPI rats or the 0.2% CD34+cell-treated FPI rats. CD34+cell (0.2%)-treated FPI rats or PBS-treated FPI rats displayed neurological and motor deficits, cerebral contusion and apoptosis [e.g., increased numbers of both TUNEL (terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling)-positive cells and caspase-3-positive cells], and activated inflammation (e.g., increased serum levels of tumor necrosis factor-α). FPI-induced neurological motor dysfunction, cerebral contusion and apoptosis, and activated inflammation could be attenuated by 50% CD34+or 95% CD34+cell therapy. In addition 50% or 95% CD34+cell therapy but not PBS or 0.2% CD34+cell therapy significantly promoted angiogenesis (e.g., increased numbers of both vasculoendothelial growth factor-positive cells and 5-bromodeoxyuridine (BrdU)-endothelial double-positive cells), neurogenesis (e.g., increased numbers of both glial cell line-derived neurotrophic factor-positive cells and BrdU/neuronal nuclei double-positive cells) in the ischemic brain after FPI, and migration of endothelial progenitor cells from the bone marrow. Our data suggest that IV administration of HUCBC-derived CD34+cells may improve outcomes of FPI in rats by stimulating both angiogenesis and neurogenesis.

The hypothalamus may be involved in regulating homeostasis, motivation, and emotional behavior by controlling autonomic and endocrine activity. The hypothalamus communicates input from the thalamus to the pituitary gland, reticular activating substance, limbic system, and neocortex. This allows the output of pituitary hormones to respond to changes in autonomic nervous system activity. Environmental heat stress increases cutaneous blood flow and metabolism, and progressively decreases splanchnic blood flow. Severe heat exposure also decreases mean arterial pressure (MAP), increases intracranial pressure (ICP), and decreases cerebral perfusion pressure (CPP MAP - ICP), all of which lead to cerebral ischemia and hypoxia. Compared with normothermic controls, rodents with heatstroke have higher hypothalamic values of cellular ischemia (e.g., glutamate and lactate-to-pyruvate ratio) and damage (e.g., glycerol) markers, pro-oxidant enzymes (e.g., lipid peroxidation and glutathione oxidation), proinflammatory cytokines (e.g., interleukin-1 and tumor necrosis factor-), inducible nitric oxide synthase-dependent nitric oxide, and an indicator for the accumulation of polymorphonuclear leukocytes (e.g., myeloperoxidase activity), as well as neuronal damage (e.g., apoptosis, necrosis, and autophagy) after heatstroke. Hypothalamic values of antioxidant defenses (e.g., glutathione peroxidase and glutathione reductase), however, are lower. The ischemic, hypoxic, and oxidative damage to the hypothalamus during heatstroke may cause multiple organ dysfunction or failure through hypothalamic-pituitary-adrenal axis mechanisms. Finding the link between the signaling and heatstroke-induced hypothalamic oxidative and ischemic damage might allow us to clinically attenuate heatstroke. In particular, free radical scavengers, heat shock protein-70 inducers, hypervolemic hemodilution, inducible nitric oxide synthase inhibitors, progenitor stem cells, flutamide, estrogen, interleukin-1 receptor antagonists, glucocorticoid, activated protein C, and baicalin mitigate preclinical heatstroke levels.

We attempted to assess the prophylactic effect of human umbilical cord blood–derived CD34+cells in experimental heatstroke. Anesthetized rats, 1 day before heat stress, were divided into 2 major groups and given CD34−cells (defined by 1 × 106human cord blood lymphocytes and monocytes that contained <0.2% CD34+cells) or CD34+cells (defined by 1 × 106human cord blood lymphocytes and monocytes that contained >95% CD34+cells). They were exposed to ambient temperature of 43°C for 70 min to induce heatstroke. When the CD34−cells–treated or untreated rats underwent heat stress, their survival time values were found to be 20 –24 min. Pretreatment with CD34+cells significantly increased survival time (123 –351 min). As compared with normothermic controls, all CD34−cells–treated heatstroke animals displayed hypotension, hepatic and renal failure, hypercoagulable state, activated inflammation, and cerebral ischemia and injury. However, these heatstroke reactions all were significantly suppressed by CD34+cells pretreatment. In addition, the levels of interlukin-10 in plasma and glial cell line–derived neurotrophic factors in brain were all significantly increased after CD34+cell administration during heatstroke. Our data indicate that human umbilical cord–derived CD34+cells can be used as a prophylactic agent for experimental heatstroke.

Human umbilical cord blood-derived CD34+cells were used to elucidate the mechanisms underlying the beneficial effects exerted by cord blood cells in spinal cord injury (SCI). Rats were divided into four groups (1) sham operation (laminectomy only); (2) laminectomy + SCI + CD34−cells (5 × 105human cord blood lymphocytes and monocytes that contained <0.2% CD34+cells); (3) laminectomy + SCI + CD34+cells (5 × 105human cord blood lymphocytes and monocytes that contained 95% CD34+cells); and (4) laminectomy + SCI + saline (0.3 mL). Spinal cord injury was induced by compressing the spinal cord for 1 min with an aneurysm clip calibrated to a closing pressure of 55 g. CD34 cells or saline was administered immediately after SCI via the tail vein. Behavioral tests of motor function measured by maximal angle an animal could hold to the inclined plane were conducted at days 1 to 7 after SCI. The triphenyltetrazolium chloride staining and terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate-biotin nick end labeling assay were also conducted after SCI to evaluate spinal cord infarction and apoptosis, respectively. To elucidate whether glial cell line-derived neurotrophic factor (GDNF) or vascular endothelial growth factor (VEGF) can be secreted in spinal cord-injured area by the i.v. transplanted CD34+cells, analysis of spinal cord homogenate supernatants by specific enzyme-linked immunosorbent assay for GDNF or immunofluorescence for VEGF was conducted. It was found that systemic administration of CD34+, but not CD34−, cells significantly attenuated the SCI-induced hind limb dysfunction and spinal cord infarction and apoptosis. Both GDNF and VEGF could be detected in the injured spinal cord after transplantation of CD34+, but not CD34−, cells. The results indicate that CD34+cell therapy may be beneficial in reversing the SCI-induced spinal cord infarction and apoptosis and hindlimb dysfunction by stimulating the production of both VEGF and GDNF in a spinal cord compression model.

Multiorgan dysfunction ensuing from severe heatstroke includes hypotension, hepatic and renal failure, hypercoagulable state, activated inflammation, and cerebral ischemia and injury. We attempted to assess whether human umbilical cord blood-derived CD34+cell therapy improves survival during experimental heatstroke by attenuating multiorgan dysfunction. Anesthetized rats, immediately after the onset of heatstroke, were divided into 2 major groups and given CD34−or CD34+cells (1 × 105- 5 × 105/mL/kg body weight) i.v. They were exposed to ambient temperature of 43°C to induce heatstroke. Another group of rats were exposed to room temperature (26°C) and used as normothermic controls. Hypotension, hepatic and renal failure (evidenced by increased serum urea nitrogen, creatinine, aspartate aminotransferase, alanine aminotransferase, and alkaline phosphatase levels in plasma), hypercoagulable state (evidenced by increased prothrombin time, activated partial thromboplastin time, and D-dimer, and decreased platelet count and protein C in plasma), activated inflammation (evidence by increased TNF- levels in serum), and cerebral dysfunction (evidenced by intracranial hypertension, cerebral hypoperfusion and hypoxia, and cerebral ischemia and injury) were monitored. When the CD34−cell-treated or untreated rats underwent heat stress, their survival time values were found to be 19 to 23 min. Resuscitation with CD34+cells significantly improved survival time (duration, 63 - 291 min). As compared with normothermic controls, all CD34−cell-treated heatstroke animals displayed hypotension, hepatic and renal failure, hypercoagulable state, activated inflammation, and cerebral ischemia and injury. However, CD34+cell therapy significantly caused attenuation of all the above-mentioned heatstroke reactions. In addition, the levels of IL-10 in plasma and glial cell line-derived neurotrophic factors in brain were all significantly increased after CD34+cell therapy during heatstroke. Our data indicate that CD34+cell therapy may resuscitate persons who had a heatstroke by reducing multiorgan dysfunction or failure.

The purpose of the current study was to explore the effects of N-methyl-D-aspartate (NMDA)-receptor antagonists (MK-801 and LY235959) administered intracerebroventricularly on the changes of both core temperature and hypothalamic levels of 2,3-dihydroxybenzoic acid (2,3-DHBA) induced by intracerebroventricular injection of glutamate (100 – 400 µg at 10 µl/rabbit) or intravenous administration of lipopolysaccharide (LPS) (2 µg/kg) in rabbits. The measurements of 2,3-DHBA were used as an index of the intrahypothalamic levels of hydroxyl radicals. The rise in both the core temperature and hypothalamic 2,3-DHBA could be induced by intracerebroventricular injection of glutamate or intravenous administration of LPS. The glutamate- or LPS-induced fever and increased hypothalamic levels of 2,3-DHBA were significantly antagonized by pretreatment with injection of MK-801 or LY235959 1 h before glutamate or LPS injection. The increased levels of prostaglandin E2in the hypothalamus induced by glutamate or LPS could be suppressed by MK-801 or LY235959. The data demonstrate that prior antagonism of NMDA receptors in the brain, in addition to reducing prostaglandin E2production in the hypothalamus, suppresses both the glutamate- and LPS-induced fever and increased hypothalamic hydroxyl radicals.

Recent findings have suggested that the N-methyl-D-aspartate (NMDA) receptor-dependent hydroxyl radical pathway in the hypothalamus of rabbit brain may mediate the fever induced by lipopolysaccharide (LPS). The aim of this study was to investigate whether aspirin exerts its antipyresis by suppressing hypothalamic glutamate and hydroxyl radicals in rabbits. The microdialysis probes were stereotaxically and chronically implanted into the preoptic anterior hypothalamus of rabbit brain for determination of both glutamate and hydroxyl radicals in situ. It was found that intravenous (i.v.) injection of LPS, in addition to inducing fever, caused increased levels of both glutamate and hydroxyl radicals in the hypothalamus. Pretreatment with aspirin (10 – 60 mg/kg, i.v.) one hour before an i.v. dose of LPS significantly reduced the febrile response and attenuated the LPS-induced increased levels of both glutamate and hydroxyl radicals in the hypothalamus. The increased levels of prostaglandin E2(PGE2) in the hypothalamus induced by LPS could be suppressed by aspirin pretreatment. The data indicate that systemic administration of aspirin, in addition to suppressing PGE2production, may exert its antipyresis by inhibiting the NMDA receptor-dependent hydroxyl radical pathways in the hypothalamus during LPS fever.

The aim of the present study was to determine whether the possible occurrence of renal ischemia and damage during heatstroke can be suppressed by prior administration of L-N6-(1-iminoethyl) lysine (L-NIL), a selective inducible nitric oxide synthase (iNOS) inhibitor. Urethane-anesthetized rats were exposed to heat stress (43°C) to induce heatstroke. Control rats were exposed to 24°C. Mean arterial pressure and renal blood flow after the onset of heatstroke both were significantly lower in vehicle-treated heatstroke rats than in normothermic controls. However, both the body temperature and renal damage scores were greater in vehicle-treated heatstroke rats compared with normothermic controls. Plasma nitric oxide (NO), creatinine, and blood urea nitrogen (BUN), as well as the renal immunoreactivity of iNOS and peroxynitrite all were significantly higher in vehicle-treated heatstroke rats compared with their normothermic controls. Pretreatment with L-NIL (3 mg/kg, administered intravenously and immediately at the onset of heat stress) significantly attenuated heatstroke-induced hyperthermia, arterial hypotension, renal ischemia and damage, increased renal levels of immunoreactivity of iNOS and peroxynitrite, and increased plasma levels of NO, creatinine, and BUN. Accordingly, pretreatment with L-NIL significantly improved survival during heatstroke. The results suggest that selective inhibition of iNOS-dependent NO and peroxynitrite formation protects against renal ischemia and damage during heatstroke by reducing hyperthermia and arterial hypotension.

The aim of the present study was to ascertain whether the possible occurrence of overproduction of inducible nitric oxide synthase (iNOS)-dependent nitric oxide (NO) in the brain and inflammatory cytokines in the peripheral blood exhibited during heat stroke can be reduced by prior administration of Shengmai San, a Chinese herbal medicine. Aminoguanidine, an iNOS inhibitor, was evaluated at the same time as a reference (positive control). Urethane-anesthetized rats were exposed to heat stress (ambient temperature of 43°C) to induce heat stroke. Control rats were exposed to 24°C. Mean arterial pressure and cerebral blood flow after the onset of heat stroke were all significantly lower than in control rats. However, cerebral iNOS immunoreactivity and NO levels were all greater after the onset of heat stroke. The serum levels of interleukin-1β, interleukin-6, and tumor necrosis factor-αwere all increased after the onset of heat stroke. Shengmai San(1.2 g/ml per rat) or aminoguanidine (30 µmol/ml per rat) was administered orally, daily, and consecutively for 7 days before the initiation of heat stress; and this significantly attenuated the heat stress-induced arterial hypotension, cerebral ischemia, and increased levels of brain iNOS-dependent NO production and serum cytokines formation. Shengmai Sanshared with the aminoguanidine almost the same efficacy in reducing iNOS-dependent NO and cytokines overproduction during heat stroke. These results suggest that Shengmai Sanor aminoguanidine protects against heat stroke-induced arterial hypotension and cerebral ischemia by inhibition of iNOS-dependent NO overproduction in the brain and excessive accumulation of several inflammatory cytokines in the peripheral blood stream.

It has been shown that staphylococcal enterotoxin A (SEA) acts through human peripheral blood mononuclear cells (PBMC) to stimulate synthesis or release of pyrogenic cytokines. Nuclear factor-kappa B (NF-κB) is thought to play an important role in inflammatory responses through the regulation of genes encoding pro-inflammatory cytokines. The purpose of the present study was to determine whether the NF-κB mechanisms in human PBMC are involved in SEA-induced fever. Western blot evaluation revealed SEA was able to induce nuclear translocation of NF-κB from cytosol to nucleus in PBMC, which could be abolished by a NF-κB inhibitor such as pyrrolidine dithiocarbamate (PDTC), sodium pyrithione (Pyri), N-acetyl-L-cysteine (NAC), or curcumin (Cur). Electrophoretic mobility shift assay also showed that the NF-κB DNA-binding activity was increased in the SEA-treated PBMC. Again, the SEA-induced increased NF-κB binding activity was significantly attenuated by either PDTC, Pyri, NAC or Cur. The pyrogenic responses to supernatant fluids obtained from human PBMC stimulated with SEA were associated with increased levels of interleukin 1-β (IL-1β), IL-6, and tumor necrosis factor-α (TNF-α) in the supernatant fluids. Both the fever and the increased levels of IL-1β, IL-6, and TNF-α in supernatant fluids obtained from the SEA-stimulated PBMC were decreased by incubating SEA-PBMC with either PDTC, Pyri, NAC, or Cur. Furthermore, the fever induced by systemic or central administration of SEA in rabbits were attenuated by pre-treatment with an systemic or central dose of either PDTC, Pyri, NAC, or Cur. The data indicate that inhibition of NF-κB prevents SEA-induced fever. (Mol Cell Biochem 262:177–185, 2004)

The present study was attempted to investigate the effect of puerarin, an isoflavone compound isolated from Pueraria lobata, on both the basal body temperature and pyrogenic fever in unanesthetized, restrained rats. Intraperitoneal administration of puerarin or crude extracts of Pueraria lobataelicited hypothermia. Direct administration of a small amount of puerarin into the lateral cerebral ventricle produced the same extent of hypothermia. Systemic or central administration of puerarin causes a decrease in both colonic temperature and hypothalamic 5-HT efflux in rats. The puerarin-induced hypothermia and decreased 5-HT efflux in the hypothalamus were attenuated by selective depletion of hypothalamic 5-HT produced by intracerebroventricular injection of 5,7-dihydroxytryptamine. Furthermore, the puerarin-induced hypothermia was almost completely abolished by treatment with a 5-HT2A-receptor agonist (DOI or quipazine) or a 5-HT1A-receptor antagonist [(−)-pindolol]. A 5-HT2A-receptor antagonist (ketanserin) or a 5-HT1A-receptor agonist (8-OH-DPAT) had additive effects with puerarin. Intracerebroventricular administration of interleukin-1 caused an increase in both colonic temperature and hypothalamic 5-HT efflux. The interleukin-1-induced hyperthermia and increased 5-HT efflux in the hypothalamus were attenuated by treatment with systemic administration of puerarin. The data indicate that puerarin exerts its hypothermic and antipyretic effects by activating 5-HT1Areceptor and/or antagonizing 5-HT2Areceptors in the hypothalamus.

In this study, we investigated the acute hemodynamic effects of an infusion of the endothelin-1 (ET-1)-A-selective receptor antagonists BQ-610 and BQ-123 in heatstroke rats with circulatory shock and cerebral ischemia. Heatstroke was induced by putting the anesthetized adult Sprague-Dawley rats into an ambient temperature of 42°C. The moment in which the mean arterial pressure dropped irreversibly from the peak for an extent of 25 mmHg was taken as the onset of heatstroke. The interval between initiation of heat exposure and heatstroke onset was found to be about 80 min for rats treated with vehicle solution. When the animals were exposed to 42°C for 80 min, hyperthermia, arterial hypotension, decrement of cardiac output (due to decreased stroke volume and decreased total peripheral resistance), increment of plasma ET-1 and tumor necrosis factor-α, and increment of cerebral ischemia and injury markers were manifested. Prior antagonism of ET-1 A receptors with BQ-610 (0.5 mg/kg, i.v.) or BQ-123 (1 mg/kg, i.v.), but not ET-1B receptors with BQ-788 (0.5 mg/kg, i.v.), 60 min before the initiation of heat exposure, appreciably alleviated hyperthermia, arterial hypotension, decreased cardiac output, increment of tumor necrosis factor-α, and increment of cerebral ischemia (e.g., glutamate and lactate/pyruvate ratio) and injury (e.g., glycerol) markers exhibited during heatstroke. The data indicates that ET-1A receptor antagonism may maintain appropriate levels of mean arterial pressure and cerebral circulation during heatstroke by reducing production of tumor necrosis factor-α.

The aim of the present study was to ascertain whether aminoguanidine attenuated intracranial hypertension and cerebral ischemic injury in experimental heatstroke. Urethane-anesthetized rats were exposed to heat stress (ambient temperature of 43°C) to induce heatstroke. Control rats were exposed to 24°C. Mean arterial pressure, cerebral perfusion pressure, and cerebral blood flow after the onset of heatstroke were all significantly lower than in control rats. However, colonic temperature, intracranial pressure, heart rate, cerebral inducible nitric oxide synthase (iNOS)-dependent NO, and neuronal damage score were greater after the onset of heatstroke. Aminoguanidine (30 μmol/kg, i.v.; 30 min before the start of heat exposure) pretreatment significantly attenuated the heatstroke-induced hyperthermia, arterial hypotension, intracranial hypertension, cerebral ischemia and neuronal damage, and increased iNOS-dependent NO formation in the brain. The extracellular concentrations of ischemic (e.g., glutamate and lactate/pyruvate ratio) and damage (e.g., glycerol) markers in the hypothalamus were also increased after the onset of heatstroke. Aminoguanidine pretreatment significantly attenuated the increase in hypothalamic ischemia and damage markers associated with heatstroke. Delaying onset of aminoguanidine administration (i.e., 0 or 30 min after the start of heat exposure) reduced the preventive efficiency on heatstroke-induced hyperthermia, arterial hypotension, intracranial hypertension, cerebral ischemia, and increased iNOS-dependent NO formation in brain. These results suggest that aminoguanidine protects against heatstroke-induced intracranial hypertension and cerebral ischemic injury by inhibition of cerebral iNOS-dependent NO production.

Both the hyperthermia and augmented glutamate release in the organum vasculosum laminae terminalis (OVLT) after an intravenous dose (30 ng/kg) of staphylococcal enterotoxin A (SEA) were significantly reduced by pretreatment with intravenous administration of cyclooxygenase inhibitors such as aspirin (1 – 10 mg/kg), sodium salicylate (1 – 10 mg/kg), or diclofenac (10 mg/kg). Intra-OVLT administration of 50 – 200 μg in 1.0 μl of either aspirin or sodium salicylate 60 min before or 120 min after an intra-OVLT dose (50 μg in 1.0 μl) of glutamate also significantly suppressed the glutamate-induced hyperthermia. These findings suggest that inhibition of cyclooxygenase receptor mechanisms suppresses SEA fever by inhibition of glutamate release in the OVLT of rabbit brain.

Intravenous injection of the supernatant fluids from human peripheral blood mononuclear cells (PBMC) incubated with lipopolysaccharide (LPS) caused fever in rabbits. The fever was in parallel with the levels of either interleukin-1β (IL-1β), IL-6, or tumor necrosis factor-α (TNF-α) in supernatant fluids. When incubating the platonin with the LPS-human PBMC, both the levels of IL-1β, IL-6, or TNF-α in supernatant fluids and the pyrogenicity of supernatant fluids were significantly suppressed. The febrile response to supernatant fluids from the LPS-stimulated PBMC was attenuated almost completely by adding anti-IL-1β, but not anti-IL-6 or anti-TNF-α, monoclonal antibody to supernatant fluids. In addition, both the fever and the increased levels of either IL-1β, IL-6, or TNF-α in rabbit serum following an intravenous administration of LPS were significantly attenuated by pretreatment with an intravenous dose of platonin. Furthermore, the fever induced by intravenous injection of IL-1β was reduced by pretreatment of rabbits with intravenous injection of platonin. The data indicate that platonin inhibits production of pyrogenic cytokines (in particular, IL-1β) from PBMC and results in antipyresis.

At first, we investigated whether both β-endorphin release level in the hypothalamus and body temperature can be altered after intracerebroventricular (i.c.v.) injection of either lipopolysaccharide (LPS), interleukin-1β (IL-1β), or prostaglandin E2(PGE2) in rats. It was found that in the rat, i.c.v. administration of either LPS (0.5 μg in 10 μl), IL-1β (10 ng in 10 μl), or PGE2(200 ng in 10 μl), in addition to producing fever, upregulated the immunoreactivity of β-endorphin in the preoptic anterior hypothalamus of rat brain. Secondarily, we assessed whether the fever induced by either LPS, IL-1β, or PGE2can be altered by pretreatment with buprenorphine (an opioid receptor antagonist). The results revealed that i.c.v. administration of buprenorphine (1 – 10 μg in 10 μl) alone had an insignificant effect on the body temperature. However, the fever induced by i.c.v. injection of either LPS, IL-1β, or PGE2was significantly attenuated by pretreatment with i.c.v. injection of buprenorphine 1 h before the pyrogen injection in rats. The results suggest that pyrogens enhance β-endorphin release in the hypothalamus and trigger fever which can be attenuated by buprenorphine, an opioid receptor antagonist.