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We aimed to elucidate whether and how BRCA1 mislocalization [including membranous nuclear (MN) forms and negative patterns] is associated with the occurrence and the prognosis of breast cancer in young Taiwanese women. A case-control study was carried out to enroll 84 patients with breast cancer and 81 patients with benign breast disease. The subcellular localization of BRCA1 was examined using immunofluorescent assays on fresh tissue touch-imprinting slides to classify staining results into diffuse nuclear (DN), MN, and negative staining. Genetic variations of BRCA1 nuclear localization/transportation-related sequences were analyzed by cDNA sequencing of both nuclear localization signals (NLS) and nuclear export signals (NES). The BRCA1 antibody was verified by two other published ones. Comparisons of immunofluorescent assay with immunohistochemical and H&E staining methods were also performed. BRCA1 mislocalization conferred a 3.13-fold [95% confidence interval (CI): 1.31-7.50] risk of developing breast cancer for the MN form and a 5.79-fold (95% CI:1.58-21.23) risk for BRCA1-negative cases compared with DN staining. However, no genetic variant was found in the NES or the NLS region of the BRCA1 gene. In terms of prognosis, BRCA1 mislocalization showed a 3.5-fold (95% CI: 1.20-10.09) increased risk of breast cancer death compared with DN staining after adjusting for tumor node metastasis stage. BRCA1 MN forms and BRCA1-negative patterns led to a higher risk of carcinogenesis and a poor prognosis of breast cancer. Such BRCA1 mislocalization may not be directly caused by the genetic effects of the NLS and NES domains, but stem from nongenetic modifications (such as epigenetic silencing).

Pregnant women are vulnerable to heat stroke reactions caused by high environmental temperatures. Heat intolerance is associated with hypothalamic impairment. Here, we aim to ascertain whether pregnancy causes heat intolerance by inducing hypothalamic impairment in mice. In the heated groups, mice were exposed to whole body heating (WBH; 41.2 °C for 1 h) in an environment-controlled chamber. Then, they were returned to normal room temperature (26 °C) immediately after WBH. In the hyperbaric oxygen therapy (HBO2T) groups, mice were exposed to 100% O2 at 2.0 atm absolute (ATA) for 4 h immediately post-WBH. Mice that survived after 4 h of WBH were considered survivors. Here, we show that when pregnant mice underwent non-HBO2T (21% O2 at 1.0 ATA for 4 h) after WBH, the survival rate was 4/20, and the core temperature at 4 h post-WBH was 31.2 ± 0.2 °C. Both the survival rate and core temperature of HBO2T pregnant mice (10/10 and 35.2 ± 0.3 °C, respectively) were significantly greater than those in non-HBO2T pregnant mice. Compared to non-HBO2T heated mice, the HBO2T heated mice exhibited lower neurological severity scores, reduced hypothalamic neuronal damage, fewer apoptotic cells, reduced multiorgan damage scores, and lower hypothalamic levels of proinflammatory cytokines and nitrogen and oxygen radical species. Compared to non-HBO2T heated mice, the HBO2T-treated heated mice had significantly higher hypothalamic-pituitary-adrenal axis activity (evidenced by higher serum levels of both adrenocorticotrophic hormone and corticosterone). In conclusion, pregnancy induces heat intolerance by inducing hypothalamic impairment in mice. Additionally, HBO2T protects against heat intolerance in pregnant mice by preserving hypothalamic integrity.

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-1β and tumor necrosis factor-α), 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.

Heatstroke is a form of excessive hyperthermia associated with a systemic inflammatory response that leads to multi-organ dysfunction in which central nervous system disorders predominate. Herein we determined to ascertain whether heat-induced multi-organ dysfunction in rats could be attenuated by granulocyte-colony stimulating factor (G-CSF) preconditioning. Anesthetized rats were divided into 2 major groups and given vehicle solution (isotonic saline, 0.3 ml, subcutaneously) or G-CSF (50–200 μg/kg body weight in 0.3 ml normal saline, subcutaneously) daily and consecutively for 5 days before the start of thermal experiments. They were exposed to an ambient temperature of 43 °C for 68 min to induce heatstroke. G-CSF preconditioning significantly prolonged the survival time in heatstroke rats in a dose-related way (82–98 min vs 127–243 min). The non-preconditioning heatstroke animals showed hyperthermia, arterial hypotension, increased serum levels of systemic inflammatory response molecules, increased hypothalamic apoptotic cell numbers as well as neuronal damage scores, and increased serum levels of renal and hepatic dysfunction indicators. These heatstroke syndromes could be significantly reduced by G-CSF preconditioning. Thus our results revealed a potential for G-CSF used as a prophylactic agent for heatstroke in rats.

Our previous studies showed that electrical stimulation of the nuclei ambiguous (NA) or dorsomotor nuclei of the vagus (DMV) complex in the brain stem of spontaneously breathing pond turtles (Cyclemys fiavomarginata), anesthetized with chloralose (4 mg/100 g) and urethane (40 mg/100 g), produced a marked slowing or even cessation of the heart rate, and resulted in an immediate fall of blood pressure. Results of the present study further demonstrated that the cardioinhibitory responses could also be elicited by microinjection of monosodium glutamate (0.2-20 nl, 50 mM) into the NA/DMV complex in turtles. A two-barrel glass micropipette held in a manipulator was connected to a pneumatic pressure pump for microinjection. The glutamate-induced cardioinhibitory responses could be significantly reduced in a dose-dependent manner by pretreatment with AP-5 (a NMDA receptor antagonist, at 1-8 nmole) or CNQX (a non-NMDA receptor antagonist; at 0.1-0.8 nmole) 20 min before glutamate administration. Histochemical verification by injecting horseradish peroxidase into the cervical vagus nerves revealed that retrogradely labeled glutamatergic neurons in the NA/DMV complex were observed. These results suggest that glutamatergic receptors in the caudal medulla may mediate vagal cardioinhibitory responses in the turtle.

OBJECTIVES To establish mechanisms of neuroprotective actions induced by Premarin (an estrogen sulfate) during traumatic brain injury. DESIGN Chi Mei Medical Center research laboratory. SUBJECTS Male Sprague-Dawley rats 244 to 268 g. INTERVENTIONS Anesthetized rats, immediately after the onset of fluid percussion injury, were divided into three major groups and given the vehicle solution (1 mL/kg of body weight), Premarin (1 mg/kg of body weight), or Premarin (1 mg/kg of body weight) plus the nonselective estrogen receptor-alpha antagonist ICI 182, 780 (0.25 mg/kg of body weight) intravenously and immediately after fluid percussion injury. MEASUREMENTS AND MAIN RESULTS Premarin, in addition to inducing pharmacologic levels of estradiol, causes attenuation of fluid percussion injury-induced cerebral infarction and motor and cognitive function deficits. Fluid percussion injury-induced apoptosis (e.g., increased numbers of both terminal deoxynucleotidyl transferase dUTP nick-end labeling-positive and caspase-3-positive cells) as well as activated inflammation (e.g., increased levels of tumor necrosis factor-alpha) was also significantly Premarin-reduced. In peri-ischemic areas of hippocampus, both angiogenesis (e.g., increased numbers of both 5-bromodeoxyuridine-positive endothelial and vascular endothelial growth factor-positive cells) and neurogenesis (e.g., increased numbers of both 5-bromodeoxyuridine/neuronal-specific nuclear protein double-positive and glial cell line-derived neurotrophic factor-positive cells) were Premarin therapy-promoted. In estrogen receptor-alpha blockade rats, Premarin therapy had less or no effect on fluid percussion injury-induced behavioral deficits, cerebral infarction and apoptosis, and activated inflammation. Furthermore, Premarin-induced angiogenesis and neurogenesis were estrogen receptor-alpha blockade-reduced. CONCLUSIONS Our results indicate that pharmacologic levels of Premarin therapy-induced estradiol protect against cortical and hippocampal programmed cell death after fluid percussion injury through mechanisms stimulating estrogen receptor-alpha in the male rats.

The present study was conducted to assess whether Premarin, a water-soluble estrogen sulfate, can act via estrogen receptors (ERs) to rescue mice from heat-induced lethality. Unanesthetized, unrestrained mice were exposed to ambient temperature of 42.4 degrees C to induce heatstroke (HS). Another group of mice was exposed to room temperature (24 degrees C) and used as normothermic controls. They were given isotonic sodium chloride solution, Premarin (0.1 - 1.0 mg/kg of body weight, i.p.), or Premarin (1 mg/kg of body weight, i.p.) plus the nonselective ER antagonist ICI 182, 780 (0.25 mg/kg of body weight, i.p.) 1 h after the termination of heat stress. Their physiologic and biochemical parameters were continuously monitored. Mice that survived on day 4 of heat treatment were considered survivors. When the vehicle-treated mice underwent heat, the fraction survival and core temperature at +4 h of body heating were found to be 0 of 12 and 34.4 degrees C +/- 3 degrees C, respectively. Administration of Premarin (1 mg/kg) 1 h after the cessation of heat stress rescued the mice from heat-induced death (fraction survival, 12/12) and reduced the hypothermia (core temperature, 37.3 degrees C). The beneficial effects of Premarin in ameliorating lethality and hypothermia can be abolished by simultaneous administration of ICI 182, 780. Both IL-10 (an anti-inflammatory cytokine) and estradiol in the serum were increased significantly in heat-stressed mice administered Premarin compared with vehicle-treated HS group. Heat-induced apoptosis, as indicated by terminal deoxynucleotidyl-transferase-mediated alpha UDP-biotin nick end-labeling staining, in the spleen, liver, and kidney were significantly reduced by Premarin. The increased levels of cellular ischemia (e.g., glutamate, lactate-to-pyruvate ratio, and nitrite) and damage (e.g., glycerol) markers and iNOS expression in the hypothalamus during HS were decreased significantly by Premarin therapy. The levels of proinflammatory cytokines (e.g., IL-1 beta and TNF-alpha) and renal and hepatic dysfunction markers in plasma that are up-regulated in heat stressed mice were significantly lower in Premarin-administered mice. The data indicate that Premarin may act via ERs to rescue mice form HS-induced lethality.

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 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. Keywords:: heatstroke, CD34+cell, cytokine, multiorgan dysfunction, coagulation

1. The aim of the present review is to summarize clinical observations and results of animal models that advance the knowledge of the attenuation of cerebrovascular dysfunction in the setting of heat stroke. It is a narrative review of selected published literature from Medline over the period 1959-2005. 2. All heat-stressed rodents, even under general anaesthesia, have hyperthermia, systemic inflammation, hypercoagulable state, arterial hypotension and tissue ischaemia and injury in multiple organs. These findings demonstrate that rodent heat stroke models can nearly mirror the full spectrum of human heat stroke. Experimental heat stroke fulfills the empirical triad used for the diagnosis of classical human heat stroke, namely hyperthermia, central nervous system alterations and a history of heat stress. 3. These physiological dysfunctions and survival during heat stroke can be improved by whole-body or brain cooling therapy adopted immediately after the onset of heat stroke. 4. However, in the absence of body or brain cooling, these heat stroke reactions can still be reduced by the following measures: (i) fluid replacement with 3% NaCl solution, 10% human albumin or hydroxyethyl starch; (ii) intravenous delivery of anti-inflammatory drugs, free radical scavengers or interleukin-1 receptor antagonists; (iii) hyperbaric oxygen therapy; or (iv) transplantation of human umbilical cord blood cells. 5. In addition, before initiation of heat stress, prior manipulations with one of the following measures was found to be able to protect against heat stroke reactions: (i) systemic delivery of alpha-tocopherol, mannitol, inducible nitric oxide synthase inhibitors, mu-opioid receptor antagonists, endothelin ETA receptor antagonists, serotoninergic nerve depletors or receptor antagonists, or glutamate receptor antagonists; or (ii) heat shock protein 72 preconditioning. 6. There is compelling evidence that cerebrovascular dysfunction is an attractive target for therapy in heat stroke.

Objective: We investigated the effect of estrogen therapy on inflammatory responses, cardiovascular functions, and survival in a rat model of heatstroke. Design: Controlled, prospective study. Setting: Hospital medical research laboratory. Subjects: Sprague-Dawley rats (280 –312 g of body weight, males and females). Interventions: Four major groups of anesthetized rats were designated for experiments: a) vehicle-treated male rats; b) vehicle- or premarin-treated estrus female rats; c) vehicle- or premarin-treated ovariectomized rats; and d) vehicle- or premarintreated leuprolide-treated rats. All animals were exposed to heat stress (ambient temperature 43°C for 70 mins) and then allowed to recover at room temperature (24°C). Their survival time (interval between the onset of heatstroke and animal death) and physiologic and biochemical variables were monitored. Vehicle (normal saline 1 mL/kg of body weight, intravenously) or premarin (1 mg/mL/kg of body weight, intravenously) was administered 70 mins after initiation of heat stress. Ovariectomy or leuprolide (100 g/kg/day, subcutaneously) injection was conducted 4 wks before the start of heat stress experiments. Another group of rats were exposed to 24°C and used as normothermic controls. Measurements and Main Results: Compared with the estrus female rats, the ovariectomized rats, the leuprolide-treated rats, and male rats all had lower levels of plasma estradiol and lower survival time values. However, after an intravenous dose of premarin, both the plasma estradiol and survival time values were significantly increased. Compared with the normothermic controls, the vehicle-treated male and ovariectomized rats all displayed higher levels of serum tumor necrosis factor-, which could be suppressed by premarin therapy. In contrast, the serum levels of IL-10 in these groups were significantly elevated by premarin during heatstroke. Furthermore, the heatstroke-induced hyperthermia, arterial hypotension, intracranial hypertension, and cerebral hypoperfusion, hypoxia, and ischemia were significantly attenuated by premarin therapy in ovariectomized rats. Conclusions: We successfully demonstrated that estrogen replacement may improve survival during heatstroke by ameliorating inflammatory responses and cardiovascular dysfunction. (Crit Care Med 2006; 34:1113–1118)

We assess the effects of ipsapirone (a 5-HT1A receptor agonist), ketanserin (a 5-HT2A receptor antagonist), (-)-pindolol (a 5-HT1A receptor antagonist), and DOI (a 5-HT2A receptor agonist) on heatstroke in a rat model. Animals, under urethane anesthesia, were exposed to high ambient temperature of 42 degrees C until mean arterial pressure and local cerebral blood flow in the striatum began to decrease, which was arbitrarily defined as the onset of heatstroke. Normothermic controls were exposed to room temperature of 24 degrees C. In rats treated with normal saline immediately before the initiation of heat stress, the values for survival time were found to be 21 to 25 min. Systemic administration of ipsapirone (10 mg/kg) or ketanserin (2 mg/kg) immediately before the initiation of heat stress significantly increased the survival time to new values of 92 to 104 min. Combined treatment with ipsapirone and ketanserin had additive effects (survival time of 156-194 min). In contrast, systemic administration of (-)-pindolol (2 mg/kg) or DOI (2 mg/kg) significantly decreased the survival time to new values of 2 to 3 min. In vehicle-treated heatstroke rats, the values for core temperature, intracranial pressure, and the extracellular levels of cellular ischemia (e.g., glutamate and lactate/pyruvate ratio) or damage (e.g., glycerol) markers and neuronal damage scores in striatum were significantly higher than those of normothermic controls. On the other hand, the values for mean arterial pressure, cerebral perfusion pressure, cerebral blood flow, and brain partial pressure of O2 were significantly lower than those of normothermic controls. The heatstroke-induced hyperthermia, arterial hypotension, intracranial hypertension, cerebral hypoperfusion and hypoxia, and increased levels of cellular ischemia and damage markers in striatum were all significantly attenuated by prior administration of ipsapirone or ketanserin. The present results strongly suggest that previous activation of 5-HT1A receptors or antagonism of 5-HT2A receptors protects against heatstroke by reducing circulatory shock and cerebral ischemia, whereas prior antagonism of 5-HT1A receptors or activation of 5-HT2A receptors exacerbates heatstroke.

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. Keywords:: aminoguanidine, heatstroke, nitric oxide, cerebral ischemia, hypothalamus

The efficacy of hepatitis B immunoglobulin (HBIG) in infants of hepatitis B e antigen (HBeAg)-negative hepatitis B surface antigen (HBsAg) carrier mothers in Taiwan is not clear.To describe the responses of infants born to HBeAg-negative carrier mothers receiving HBIG combined with hepatitis B vaccine.Term babies born to HBeAg-negative carrier mothers were assigned based on chart number to 1 of the 2 treatment groups. Group A infants (n = 94) received 0.5 ml (145 IU) of HBIG within 24 h of birth and 3 subsequent doses of recombinant hepatitis B virus (HBV) vaccine at 3 to 5 days, 1 month and 6 months of age. Group B infants (n = 122) received 3 doses of vaccines only. Infants (n = 19) born to HBeAg-positive carrier mothers were treated like those in Group A and are referred to as Group C. Sera obtained from infants at 2 and 7 months of age were tested for hepatitis B virus (HBV) markers.There were 2 (1%; one in Group A and one in Group B) subclinical breakthrough hepatitis B infections among studied infants. One (5%) child of Group C had asymptomatic HBV infection at the age of 7 months and became a chronic carrier. The rate of protective anti-hepatitis B surface antibody (anti-HBs) titers achieved (10 mIU/ml) by 2 months of age was significantly higher in Group A than that in Group B (98% vs. 57%, P0.001). However, it was not different by 7 months of age. Infants (Group A) immunized with HBIG and vaccine had a significantly higher geometric mean titer (GMT, milli-International Units/ml) of anti-HBs than those (Group B) with vaccines only at 2 months of age (P0.001). Conversely at 7 months of age, the GMT of anti-HBs was significantly higher in infants who received vaccine only (P = 0.001).A protective level of antibodies was achieved earlier in those infants receiving both passive and active immunizations. However, infants receiving active immunizations alone achieved a higher GMT at 7 months of age. There was no clear benefit of passive-active vs.active immunization alone for chronic HBV infection in infants of HBsAg-positive, HBeAg-negative mothers.

Intolerance to heat exposure is believed to be associated with hypothalamo-pituitary-adrenocortical (HPA) axis impairment [reflected by decreases in blood concentrations of both adrenocorticotrophic-hormone (ACTH) and corticosterone]. The purpose of this study was to determine the effect of human recombinant factor VIIa (rfVIIa) on heat intolerance, HPA axis impairment, and hypothalamic inflammation, ischemic and oxidative damage, and apoptosis in mice under heat stress. Immediately after heat stress (41.2 °C for 1 h), mice were treated with vehicle (1 mL/kg of body weight) or rfVIIa (65-270 µg/kg of body weight) and then returned to room temperature (26 °C). Mice still alive on day 4 of heat exposure were considered survivors. Cellular ischemia markers (e.g., glutamate, lactate-to-pyruvate ratio), oxidative damage markers (e.g., nitric oxide metabolite, hydroxyl radials), and pro-inflammatory cytokines (e.g., interleukin-6, interleukin-1β, tumor necrosis factor-α) in hypothalamus were determined. In addition, blood concentrations of both ACTH and corticosterone were measured. Hypothalamic cell damage was assessed by determing the neuronal damage scores, whereas the hypothalamic cell apoptosis was determined by assessing the numbers of cells stained with terminal deoxynucleotidyl transferase-mediated αUTP nick-end labeling, caspase-3-positive cells, and platelet endothelial cell adhesion molecula-1-positive cells in hypothalamus. Compared with vehicle-treated heated mice, rfVIIa-treated heated mice had significantly higher fractional survival (8/10 vs 1/10), lesser thermoregulatory deficit (34.1 vs 24.8 °C), lesser extents of ischemic, oxidative, and inflammatory markers in hypothalamus, lesser neuronal damage scores and apoptosis in hypothalamus, and lesser HPA axis impairment. Human recombinant factor VIIa appears to exert a protective effect against heatstroke by attenuating hypothalamic cell apoptosis (due to ischemic, inflammatory, and oxidative damage) in mice.

Objective Intravenous or intraspinal transplantation of human umbilical cord blood cells-derived CD34 + cells (human CD34 + cells) or mesenchymal stem cells after spinal cord injury (SCI) improved hind limb functional recovery in adult rats. The objective of this study is to ascertain whether SCI in rats can be attenuated by conditioned medium (CM) or secretome obtained from cultured human CD34 + stem cells. Materials and methods Sprague-Dawley rats were assigned to one of the following five groups: the sham group, the SCI group treated with vehicle solution (SCI + V), the SCI group treated with CM (SCI + CM), the SCI group treated with 17β-estradiol E 2 (10 μg; SCI + E 2 ), and the SCI group treated with CM plus E 2 (SCI + CM + E 2 ). A 0.5-mL volume of CM or vehicle solution was administered intravenously immediately after SCI. Results Compared with the sham group, the (SCI + V) group had significantly higher scores of neurological motor dysfunction as well as inflammation apoptosis, oxidative stress, and astrogliosis in the injured spinal cord. The neurological deficits, numbers of apoptotic cell, extent of inflammation, oxidative stress, and astrogliosis in the injured spinal cord were significantly attenuated by CM, E 2 , or CM plus E 2 , but not by the vehicle solution. In addition, the neuroprotective effect exerted by a combination of CM and E 2 is superior to that exerted by CM- or E 2 -alone therapy. Conclusion The neuroprotective effects of CM from cultured human CD34 + cells are similar to those of human CD34 + cells and the CM was found to enhance the neuroprotective effects of E 2 in rat SCI.

Changes in the peroxisome proliferator-activated receptors-δ(PPARδ) expression in rats after spinal cord injury (SCI) have been previously reported. Diabetic animals show a higher mortality after SCI. However, the relationship between the progress of diabetes and PPARδin SCI remains unknown. In the present study, we used compressive SCI in streptozotocin-(STZ-) induced diabetic rats. GW0742, a PPARδagonist, was used to evaluate its merit in STZ rats after SCI. Changes in PPARδexpression were detected by Western blot. Survival rates were also estimated. A lower expression of PPARδin spinal cords of STZ-diabetic rats was observed. In addition, the survival times in two-week induction diabetes were longer than those in eight-week induction group, which is consistent with the expression of PPARδin the spinal cord. Moreover, GW0742 significantly increased the survival time of STZ rats. Furthermore, their motor function and pain response were attenuated by GSK0660, a selective PPARδantagonist, but were enhanced by GW0742. In conclusion, the data suggest that higher mortality rate in STZ-diabetic rats with SCI is associated with the decrease of PPARδexpression. Thus, change of PPARδexpression with the progress of diabetes seems responsible for the higher mortality rate after SCI.

Heatstroke is characterized by excessive hyperthermia associated with systemic inflammatory responses, which leads to multiple organ failure, in which brain disorders predominate. This definition can be almost fulfilled by a mouse model of heatstroke used in the present study. Unanesthetized mice were exposed to whole body heating (41.2°C for 1 hour) and then returned to room temperature (26°C) for recovery. Immediately after termination of whole body heating, heated mice displayed excessive hyperthermia (body core temperature ~42.5°C). Four hours after termination of heat stress, heated mice displayed (i) systemic inflammation; (ii) ischemic, hypoxic, and oxidative damage to the hypothalamus; (iii) hypothalamo-pituitary-adrenocortical axis impairment (reflected by plasma levels of both adrenocorticotrophic-hormone and corticosterone); (iv) decreased fractional survival; and (v) thermoregulatory deficits (e.g., they became hypothermia when they were exposed to room temperature). These heatstroke reactions can be significantly attenuated by human umbilical cord blood-derived CD34+cells therapy. Our data suggest that human umbilical cord blood-derived stem cells therapy may improve outcomes of heatstroke in mice by reducing systemic inflammation as well as hypothalamo-pituitary-adrenocortical axis impairment.

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 × 105 cord 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.

Human umbilical cord blood derived CD34+ stem cells are reported to mediate therapeutic effects in stroke animal models. Estrogen was known to protect against ischemic injury. The present study wished to investigate whether the protective effect of CD34+ cells against ischemic injury can be reinforced with complemental estradiol treatment in female ovariectomized rat and its possible mechanism. Experiment 1 was to determine the best optimal timing of CD34+ cell treatment for the neuroprotective effect after 60-min middle cerebral artery occlusion (MCAO). Experiment 2 was to evaluate the adjuvant effect of 17β-estradiol on CD34+ cell neuroprotection after MCAO. Experiment 1 showed intravenous infusion with CD34+ cells before MCAO (pre-treatment) caused less infarction size than those infused after MCAO (post-treatment) on 7T magnetic resonance T2-weighted images. Experiment 2 revealed infarction size was most significantly reduced after CD34+ + estradiol pre-treatment. When compared with no treatment group, CD34+ + estradiol pre-treatment showed significantly less ADC reduction at 2 h and 2 d, less CBF reduction at 2 h and less hyperperfusion at 2 d. The immunoreactivity of c-Fos, c-Jun and GFAP was attenuated, and BDNF showed significant recovery from 2 h to 2 d after MCAO, especially after CD34+ + estradiol pre-treatment. The present study suggests pre-treatment with CD34+ cells with complemental estradiol can be most protective against ischemic injury, which may act through stabilization of cerebral hemodynamics and normalization of the expressions of immediate early genes and BDNF.

Flutamide has been used as an adjunct for decreasing the mortality from subsequent sepsis. Heatstroke resembles septic shock in many aspects. We hypothesized that heat-induced multiple organ dysfunction syndromes and lethality could be reduced by flutamide therapy. In heatstroke groups, mice were exposed to whole body heating (41.2°C, for 1h) in a controlled-environment chamber. The heat-stressed mice were returned to normal room temperature (24°C) after whole body heating. Mice still alive on day 4 of WBH treatment were considered survivors. Physiological and biochemical parameters were monitored for 2.5h post-WBH. Heatstroke mice were subcutaneously treated with flutamide (12.5-50mg/kg body weight in 0.05 ml) or vehicle solution (0.05 ml/kg body weight) once daily for 3 consecutive days post-WBH. We evaluated the effect of flutamide in heatstroke mice and showed that flutamide significantly (i) attenuated hypothermia, (ii) reduced the number of apoptotic cells in the hypothalamus, the spleen, the liver, and the kidney, (iii) attenuated the plasma index of toxic oxidizing radicals (e.g., nitric oxide metabolites and hydroxyl radicals), (iv) diminished the plasma index of the organ injury index (e.g., lactate dehydrogenase), (v) attenuated plasma systemic inflammation response molecules (e.g., tumor necrosis factor-α and interleukin-6), (vi) reduced the index of infiltration of polymorphonuclear neutrophils in the lung (e.g., myeloperoxidase activity), and (vii) allowed three times the fractional survival compared with vehicle. Thus, flutamide appears to be a novel agent for the treatment of mice with heatstroke or patients in the early stage of heatstroke.

To assess whether systemic delivery of kynurenic acid improves the outcomes of heatstroke in rats. Anesthetized rats were divided into 2 major groups and given vehicle solution (isotonic saline 0.3 mL/kg rat weight) or kynurenic acid (30–100 mg in 0.3 mL saline/kg) 4 h before the start of thermal experiments. They were exposed to an ambient temperature of 43 °C for 68 min to induce heatstroke. Another group of rats were exposed to room temperature (26 °C) and used as normothermic controls. Their core temperatures, mean arterial pressures, serum levels of systemic inflammatory response molecules, hypothalamic values of apoptotic cells and neuronal damage scores, and spleen, liver, kidney and lung values of apoptotic cells were determined. The survival time values during heatstroke for vehicle-treated rats were decreased from the control values of 475–485 min to new values of 83–95 min. Treatment with KYNA (30–100 mg/kg, iv) 4 h before the start of heat stress significantly and dose-dependently decreased the survival time to new values of 152–356 min (P

When the vehicle-treated, sham-operated mice underwent heat stress, the fraction survival and core temperature at +4 h of body heating were found to be 5 of 15 and34.4∘C±0.3∘C, respectively. Castration 2 weeks before the start of heat stress decreased the plasma levels of testosterone almost to zero, protected the mice from heat-induced death (fraction survival, 13/15) and reduced the hypothermia (core temperature,37.3∘C). The beneficial effects of castration in ameliorating lethality and hypothermia can be significantly reduced by testosterone replacement. Heat-induced apoptosis, as indicated by terminal deoxynucleotidyl- transferase- mediatedαUDP-biotin nick end-labeling staining, were significantly prevented by castration. In addition, heat-induced neuronal damage, as indicated by cell shrinkage and pyknosis of nucleus, to the hypothalamus was also castration-prevented. Again, the beneficial effects of castration in reducing neuronal damage to the hypothalamus as well as apoptosis in multiple organs during heatstroke, were significantly reversed by testosterone replacement. The data indicate that testosterone depletion by castration may protect mice from heatstroke-induced multiple organ damage and lethality.

Prior administration of naltrexone or cyclic d-phe-cys-try-arg-thr-pen-thr-NH,2 (CTAP) (a μ-opioid receptor antagonist), but not nor-binaltorphine (a K-opioid receptor antagonist) or ICI-174864 (a δ-opioid receptor antagonist), improve heat tolerance in the rat. As compared to those of normothermic controls, all vehicle-pretreated heatstroke rats displayed higher levels of creatinine, blood urea nitrogen, alkaline phosphatase, glutamic oxaloacetic transaminase, glutamic pyruvic transaminase, tumor necrosis factor-alpha, prothrombin time, activated partial thromboplastin time, and d-dimer in the plasma, cellular ischemia and injury markers in brain, and intracranial pressure. In contrast, all vehicle pretreated heatstroke animals had lower levels of mean arterial pressure, cerebral perfusion pressure, cerebral blood flow, brain PO2, and platelet count and protein C in the plasma. Twenty minutes before the start of heat exposure, prior administration of CTAP or naltrexone improves heat tolerance by reducing the systemic inflammation, hyper-coagulable state, and tissue ischemia and injury that occurred during heatstroke. The results show that μ-opioid receptor antagonism is effective for attenuation of heatstroke reactions.

Background Human umbilical cord blood cells (HUCBCs) were used to investigate the mechanisms underlying the beneficial effects of cord blood cells in spinal cord injury (SCI). Methods Rats were divided into three groups: (1) sham operation (laminectomy only); (2) laminectomy + SCI + human adult peripheral blood mononucleocytes (PBMCs) (5 × 10 6 /0.3 mL); and (3) laminectomy + SCI + HUCBCs (5 × 10 6 /0.3 mL). SCI was induced by compressing the spinal cord for 1 minute with an aneurysm clip calibrated to 55 g closing pressure. HUCBCs were infused immediately after SCI via the tail vein. Behavioral function tests measuring the maximal angle at which an animal could hold onto the inclined plane were conducted on days 1, 4 and 7 after SCI. Serum levels of tumor necrosis factor (TNF)-α and interleukin (IL)-10, were assayed. Further more, to determine if glial cell line-derived neurotrophic factor (GDNF) or vascular endothelial growth factor (VEGF) could be detected in the spinal cord injured area after systemic HUCBC infusion, analysis of these two molecules was conducted by immunofluorescence. Results Systemic HUCBC infusion significantly attenuated SCI-induced hind limb dysfunction. The serum IL-10 levels were increased, but TNF-α levels were decreased after HUCBC infusion. Both VEGF and GDNF could be detected in the injured spinal cord after transplantation of HUCBC, but not PBMC, cells. Conclusion Our results demonstrate that HUCBC therapy may be beneficial for the recovery of SCI-induced hind limb dysfunction by increasing serum levels of IL-10, VEGF and GDNF in SCI rats.

The aim of present study was to examine whether systemically delivered glial cell-derived neurotrophic factor (GDNF) was beneficial in reversing the spinal cord injury (SCI) in a spinal cord compression model. Rats were divided into three major groups: (1) sham operation (laminectomy only); (2) laminectomy+SCI+normal saline (1 ml/kg, i.v.); (3) laminectomy+SCI+GDNF (50 ng/kg, i.v.). 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. GDNF 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 1-7 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. Both GDNF and vascular endothelial growth factor (VEGF) in the injured spinal cord were assayed by immunofluorescence. It was found that systemically delivered GDNF, but not vehicle solution, significantly attenuated the SCI-induced hind limb dysfunction and spinal cord infarction and apoptosis. Both GDNF and VEGF could be detected in the injury spinal cord after GDNF, but not vehicle solution, therapy. The results indicate that GDNF treatment may be beneficial in reversing hind limb dysfunction by reducing spinal cord infarction and apoptosis in a spinal cord compression model.

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 x 10(5) human cord blood lymphocytes and monocytes that contained

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 bloodYderived 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 j or CD34 + cells (1 � 10 5 Y 5 � 10 5 /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 j cellYtreated 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 Y 291 min). As compared with normothermic controls, all CD34 j cellYtreated 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 lineYderived 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. KEYWORDS—Heatstroke, blood pressure, cytokines, coagulation, umbilical cord blood cells

Intravenously delivered human umbilical cord blood cells (HUCBC) have been previously shown to improve both morphologic and functional recovery of heat-stroked rats. To extend these findings, we examined both the morphologic and functional alterations in the presence of HUCBC or human peripheral mononuclear cells (PBMC) 24 h before initiation of heatstroke. Anesthetized rats, 1 day before the initiation of heatstroke, were divided into three major groups and given the following: (a) serum-free lymphocyte medium (0.3 ml) intravenously; (b) PBMC (5 x 10(6) in 0.3 ml serum-free lymphocyte medium); or (c) HUCBC (5 x 10(6) in 0.3 ml serum-free lymphocyte medium). Another group of rats were exposed to room temperature (26 degrees C) and used as normothermic controls. In vehicle-treated heatstroke rats, their mean arterial pressure, cerebral blood flow, and brain PO(2) were all lower than in normothermic controls after the onset of heatstroke. However, their body temperatures and striatal levels of inducible nitric oxide synthase (iNOS)-dependent NO, ischemia and damage markers (e.g., glycerol, glutamate, and lactate/pyruvate ratio), and neuronal damage in the striatum were all greater. The heatstroke-induced arterial hypotension, cerebral ischemia and hypoxia, and increased levels of iNOS-dependent NO in the striatum were all significantly reduced by pretreatment with HUCBC, but not with PBMC. Moreover, HUCBC were localized by immunohistochemistry and PCR analysis in the injured brain structures and spleen. These findings indicate that HUCBC transplantation, in addition to having therapeutic values, can be a good choice for preventing heatstroke occurrence.