Infusion of human umbilical cord blood cells protect against cerebral ischemia and damage during heatstroke in the rat

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Abstract

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 × 106 in 0.3 ml serum-free lymphocyte medium); or (c) HUCBC (5 × 106 in 0.3 ml serum-free lymphocyte medium). Another group of rats were exposed to room temperature (26°C) and used as normothermic controls. In vehicle-treated heatstroke rats, their mean arterial pressure, cerebral blood flow, and brain PO2 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.

Introduction

Heatstroke afflicts many humans engaged in severe exercising, firefight, or some military or mining activities (Halle and Repasy, 1987, Knochel, 1989, Simon, 1993). Many predisposing diseases (such as cardiovascular disease, diabetes mellitus, alcoholism, and impaired sweat production) or factors (such as salt and water depletion and fever after immunization) may increase susceptibility to heatstroke (Dahmash et al., 1993, Knochel, 1989) Unless promptly recognized and treated, hyperthermia, central nervous system dysfunctions (such as restlessness, delirium and coma), and multiple organ failure may occur and result in high rate of mortality (Bouchama and Knochel, 2002).

It is well known that the lactate/pyruvate ratio is a marker of cell ischemia, whereas glycerol is a marker of how severely cells are affected by ongoing pathology (Hillered and Persson, 1999, Hillered et al., 1990, Hillered et al., 1998). Excessive accumulation of glutamate has been shown in ischemic brain tissue (Nilsson et al., 1996, Persson and Hillered, 1992, Ungerstedt, 1997). Indeed, when the animals are exposed to hot environment, cerebral ischemia and hypoxia which occurred during heatstroke were associated with an increased production of glycerol, lactate/pyruvate ratio, glutamate, and inducible nitric oxide synthase (iNOS)-dependent NO in the brain (Chang et al., 2004, Chen et al., 2005, Kuo et al., 2003). In addition, the PO2 in rat brain was decreased after the onset of heatstroke onset. Thus, it appears that excessive accumulation of glycerol, glutamate, lactate/pyruvate ratio, and iNOS-dependent NO in the brain may be secondary to cerebral ischemia, hypoxia, and/or injury in the rat.

It is estimated that approximately 2% of the human umbilical blood cells (HUCBC), similar to the percentage observed in bone marrow, are stem cells capable of reconstituting blood lineages (Bender et al., 1991, Ho et al., 1996, Nieda et al., 1997, Wu et al., 1999). Intravenously delivered HUCBC found in the brain 4 weeks after middle cerebral artery occlusion in the rat were excessively localized to the ischemic hemispheres (Wang et al., 2004). HUCBC have emerged as an alternative to bone marrow since they possess greater availability, lower risk of mediating viral transmission, and weaker immunogenicity (Lewis, 2002). Accumulating evidence has demonstrated that HUCBC transplantation is a promising new therapeutic method against neurodegenerative diseases, such as stroke, traumatic brain injury, and spinal cord injury as well as blood diseases (Chen et al., 2001b, Lu et al., 2002, Saporta et al., 2003, Wang et al., 2004, Willing et al., 2003). More recently, we have also demonstrated that HUCBC therapy may resuscitate rats with heatstroke by reducing circulatory shock, and cerebral NO overload and ischemic injury; central delivery of HUCBC seems superior to systemic delivery of HUCBC in resuscitating rats with heatstroke (Chen et al., 2005). However, to our knowledge, evidence is not available about the preventive effects of HUCBC administered before the initiation of cerebral ischemia and injury.

To deal with the question, the present study was designed to investigate whether an early in exposure to HUCBC treatment could still promote attenuation of both the morphologic and functional alterations which occurred during heatstroke in the rat. We attempted to elucidate whether HUCBC pretreatment could be a good choice for preventing heatstroke occurrence.

Section snippets

Animals

Adult Sprague–Dawley rats (weight, 287 ± 16 g) were obtained from the Animal Resource Center of the National Science Council of the Republic of China (Taipei, Taiwan). The animals were housed 4 in a group at an ambient temperature (Ta) of 22 ± 1°C, with a 12-h light/dark cycle. Pellet rat chow and tap water were available ad libitum. All protocols were approved by the Animal Ethics Committee of the Chi-Mei Medical Center (Tainan, Taiwan) in accordance with the Guide for the Care and Use of

HUCBC pretreatment extends latency and improves survival during heatstroke

Table 1 summarizes the latency and survival time for vehicle-pretreated, PBMC-pretreated, and HUCBC-pretreated rats during heatstroke. It can be seen from the table that the latency and the survival time values were found to be 68–70 min (n = 8) and 18–22 min (n = 8) respectively for vehicle-treated heatstroke rats. The values of latency and survival time for PBMC-pretreated heatstroke rats were not distinguishable from those of the vehicle-pretreated heatstroke rats. However, as compared with

Discussion

Pretreatment (present results) or posttreatment (Chen et al., 2005) with HUCBC significantly attenuates the arterial hypotension, cerebral ischemia and hypoxia, and increased levels of ischemia and damage markers in the brain during heatstroke. These findings demonstrate that HUCBC is effective for prevention and repair of circulatory shock and ischemic damage in the brain during heatstroke by reducing iNOS-dependent NO formation in the brain. However, treatment with PBMC fails to produce any

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