Sheng-Hsien Chen

Human umbilical cord blood-derived CD34+ cells may attenuate spinal cord injury by stimulating vascular endothelial and neurotrophic factors.

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 × 105 human cord blood lymphocytes and monocytes that contained <0.2% CD34+ cells); (3) laminectomy + SCI + CD34+ cells (5 × 105 human 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.

Human Umbilical Cord Blood Cells Protect Against Hypothalamic Apoptosis and Systemic Inflammation Response During Heatstroke in Rats

BACKGROUND Intravenous administration of human umbilical cord blood cells (HUCBC) has been shown to improve heatstroke by reducing arterial hypotension as well as cerebral ischemia and damage in a rat model. To extend these findings, we assessed both hypothalamic neuronal apoptosis and systemic inflammatory responses in the presence of HUCBCs or vehicle medium immediately after initiation of heatstroke. METHODS Anesthetized rats, immediately after the initiation of heat stress, were divided into two groups and given either serum-free lymphocyte medium (0.3mL per rat, intravenously) or HUCBCs (5 x 10(6) in 0.3 mL serum-free lymphocyte medium, intravenously). Another group of rats were exposed to room temperature (26 degrees C) and used as normothermic controls. Heatstroke was induced by exposing the anesthetized rats to a high ambient temperature of 43 degrees C for 68 minutes. RESULTS After the onset of heatstroke, animals treated with serum-free lymphocyte medium displayed hyperthermia, hypotension, bradycardia, hypothalamic neuronal apoptosis and degeneration, and up-regulation of systemic inflammatory response molecules including serum tumor necrosis factor-alpha, soluble intercellular adhesion molecule-1 and E-selectin. Heatstroke-induced hypotension, bradycardia, hypothalamic neuronal apoptosis and degeneration, and increased systemic inflammatory response molecules were significantly inhibited by HUCBC treatment. Although heatstroke-induced hyperthermia was not affected by HUCBC treatment, the serum levels of the anti-inflammatory cytokine interleukin-10 were significantly increased by HUCBC therapy during hyperthermia. CONCLUSIONS These findings suggest that HUCBC transplantation may prevent the occurrence of heatstroke by reducing hypothalamic neuronal damage and the systemic inflammatory responses.

Human umbilical cord blood-derived CD34+ cells cause attenuation of multiorgan dysfunction during experimental heatstroke.

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-&agr; 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.

Premarin can act via estrogen receptors to rescue mice from heatstroke-induced lethality.

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°C to induce heatstroke (HS). Another group of mice was exposed to room temperature (24°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°C ± 3°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°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 &agr;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&bgr; and TNF-&agr;) 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.

Premarin stimulates estrogen receptor-alpha to protect against traumatic brain injury in male rats.

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-&agr; 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-&agr;) 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-&agr; 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-&agr; 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-&agr; in the male rats.

Umbilical Cord Blood-Derived CD34+ Cells Improve Outcomes of Traumatic Brain Injury in Rats by Stimulating Angiogenesis and Neurogenesis

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.

Premarin improves outcomes of spinal cord injury in male rats through stimulating both angiogenesis and neurogenesis.

Objective:To ascertain whether Premarin improves spinal cord injury outcomes in male rats by stimulating both angiogenesis and neurogenesis. Design:Chi Mei Medical Center research laboratory. Subjects:Male Sprague-Dawley rats 240–258 g. Interventions:Anesthetized rats, after the onset of spinal cord injury, were divided into two groups and given the vehicle solution (1 mL/kg of body weight) or Premarin (1 mg/kg of body weight). Saline or Premarin solutions were administered intravenously and immediately after spinal cord injury. Measurements and Main Results:Premarin (an estrogen sulfate) causes attenuation of spinal cord injury-induced spinal cord infarction and hind limb locomotor dysfunction. Spinal cord injury-induced apoptosis as well as activated inflammation was also significantly Premarin-reduced. In injured spinal cord, angiogenesis, neurogenesis, and production of an antiinflammatory cytokine were all Premarin therapy-promoted. Conclusions:Our results indicate that Premarin therapy may protect against spinal cord apoptosis after spinal cord injury through mechanisms stimulating both angiogenesis and neurogenesis in male rats.

Transplantation of human dental pulp-derived stem cells protects against heatstroke in mice.

Stem cells from human exfoliated deciduous tooth pulp (SHED) is a promising approach for the treatment of stroke and spinal cord injury. In this study, we investigated the therapeutic effects of SHED for the treatment of multiple organ (including brain, particularly hypothalamus) injury in heatstroke mice. ICR male mice were exposed to whole body heating (WBH; 41.2°C, relative humidity 50-55%, for 1 h) and then returned to normal room temperature (26°C). We observed that intravenous administration of SHED immediately post-WBH exhibited the following therapeutic benefits for recovery after heatstroke: (a) inhibition of WBH-induced neurologic and thermoregulatory deficits; (b) reduction of WBH-induced ischemia, hypoxia, and oxidative damage to the brain (particularly the hypothalamus); (c) attenuation of WBH-induced increased plasma levels of systemic inflammatory response molecules, such as tumor necrosis factor-a and intercellular adhesion molecule-1; (d) improvement of WBH-induced hypothalamo-pituitary-adrenocortical (HPA) axis activity (as reflected by enhanced plasma levels of both adrenocorticotrophic hormone and corticosterone); and (e) attenuation of WBH-induced multiple organ apoptosis as well as lethality. In conclusion, post-WBH treatment with SHED reduced induction of proinflammatory cytokines and oxidative radicals, enhanced plasma induction of both adrenocorticotrophic hormone and corticosterone, and improved lethality in mouse heatstroke. The protective effect of SHED may be related to a decreased inflammatory response, decreased oxidative stress, and an increased HPA axis activity following the WBH injury.

Therapeutic treatment with ascorbate rescues mice from heat stroke-induced death by attenuating systemic inflammatory response and hypothalamic neuronal damage

The impact of ascorbate on oxidative stress-related diseases is moderate because of its limited oral bioavailability and rapid clearance. However, recent evidence of the clinical benefit of parenteral vitamin C administration has emerged, especially in critical care. Heatstroke is defined as a form of excessive hyperthermia associated with a systemic inflammatory response that results in multiple organ dysfunctions in which central nervous system disorders such as delirium, convulsions, and coma are predominant. The thermoregulatory, immune, coagulation and tissue injury responses of heatstroke closely resemble those observed during sepsis and are likely mediated by similar cellular mechanisms. This study was performed by using the characteristic high lethality rate and sepsis-mimic systemic inflammatory response of a murine model of heat stroke to test our hypothesis that supra-physiological doses of ascorbate may have therapeutic use in critical care. We demonstrated that parenteral administration of ascorbate abrogated the lethality and thermoregulatory dysfunction in murine model of heat stroke by attenuating heat stroke-induced accelerated systemic inflammatory, coagulation responses and the resultant multiple organ injury, especially in hypothalamus. Overall, our findings support the hypothesis and notion that supra-physiological doses of ascorbate may have therapeutic use in critical care.

Dengue Virus Infection in Early Gestation With Delivery of an Unaffected Fetus and No Vertical Transmission

112 Dengue fever is an infectious, tropical viral disease. There have been a number of epidemics in Taiwan during the last century [1]. In addition, dengue hemorrhagic fever (DHF) and even dengue shock fever have been reported in recent decades. Dengue infection may cause disease in neonates born to infected mothers, but the teratogenic and abortion effects of dengue infection on pregnant women and their fetuses, especially during early gestation, are unclear. Prenatal genetic counseling is, therefore, crucial. Here, we present a pregnant woman of 11+ weeks’ gestation who suffered from DHF, but finally gave birth to an unaffected full-term baby. A 30-year-old, gravida 2, para 1, woman at 11+ weeks’ gestation was sent to our emergency department with a 3-day history of intermittent high fever (> 39°C) and headache. Her previous obstetric history and prenatal care were uneventful. Her body temperature was 38.4°C, and the results of urine analysis were within normal limits. Her white blood cell count was 7,100/μL, and her platelet count was 245,000/μL (Figure). The cause of her fever remained undetermined after the initial workup. After triage, the patient was admitted to the infection ward. She was also closely monitored for serial full blood counts and urea levels, and by liver function tests and fetal echographic screening. Three days later, she developed nausea, petechiae, severe thrombocytopenia, and leucopenia (Figure). Her body temperature rose to a maximum of 39.7°C, and her platelet count dropped to 25,000/μL on the sixth day after admission (Figure). Acute dengue virus infection was highly suspected based on the patient’s reported recent mosquito bite history, and this was subsequently confirmed by serologic tests. She was provided with supportive care, and the fever gradually subsided. Her blood platelet count had increased to normal by the ninth day. Regular prenatal care was arranged after discharge at our obstetrics outpatient department. An uncomplicated pregnancy led to the delivery of a normal healthy male infant at 39 weeks’ gestation. Delivery was by cesarean section owing to a breech presentation. Serologic testing for immunoglobulins, IgG and IgM, against dengue virus was negative in the neonate, although IgG was still detected in the mother after delivery. Dengue fever is a major public health concern in Taiwan and in other countries in Southeast Asia. DENGUE VIRUS INFECTION IN EARLY GESTATION WITH DELIVERY OF AN UNAFFECTED FETUS AND NO VERTICAL TRANSMISSION