Granulocyte Macrophage Colony Stimulating Factor (GM-CSF) Prevents Apoptosis and Improves Functional Outcome in Experimental Spinal Cord Contusion Injury

Objective: Granulocyte macrophage colony stimulating factor (GM-CSF) is a potent hematopoietic cytokine, which stimulates stem cell proliferation in the bone marrow and inhibits apoptotic cell death in leukocytes. However, the effects of GM-CSF in the central nervous system are still unclear. The present study was undertaken to determine if GM-CSF can rescue neuronal cells from apoptosis and improve neurologic function in a spinal cord injury (SCI) model. Methods: To study the effect of GM-CSF on apoptotic neuronal death, we used a staurosporine-induced neuronal death model in a Neuro 2A (N2A) cell line (in vitro) and in a rat SCI model (in vivo). N2A cells were preincubated with GM-CSF for 60 minutes before being exposed to staurosporine for 24 hours. To inhibit GM-CSF, we pretreated N2A cells with antibodies of the GM-CSF receptor for 60 minutes. SCI was made by clip compression. Rats were treated with daily GM-CSF (20 ƒÝg/d) for 5 days. The number of apoptotic cells in the spinal cord and neurologic improvements were checked. Results: GM-CSF pretreatment was found to significantly protect N2A cells from apoptosis, and neutralizing antibodies for the GM-CSF receptors inhibited the rescuing effect of GM-CSF on apoptosis. In the rat SCI model, neurologic functions improved significantly in the GM-CSF¡Vadministered group versus the phosphate buffered saline (PBS)-treated control. TUNEL (terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeled) staining showed that GM-CSF administration reduced apoptosis in the injured spinal cord. Conclusion: Treatment of SCI with GM-CSF showed some beneficial effects. Neuronal protection against apoptosis is viewed as a likely mechanism underlying the therapeutic effect of GM-CSF in SCI. Traumatic SCI affects many people and can result in severe neurological damage. After SCI, the central region of the spinal cord may be critically damaged by the primary mechanical injury and show hemorrhagic necrosis, which expands with time because of the activation of secondary injury processes (23). Moreover, apoptosis may play an important role in these secondary injury processes after SCI (25). Apoptosis was noted around the injury site in a histological study of the spinal cords of patients that died after traumatic SCI (5). These findings are the basis of the therapeutic principle of neuronal apoptosis inhibition after SCI. Hematopoietic and neural stem cells have many similarities at the transcriptional level during differentiation (20). Furthermore, some investigators suggest that stem cells in adult tissue have the ability to transdifferentiate into other cells (10). Vescovi et al.(24) reported that nestin-positive cells from adult subventricular zone neural stem cells can proliferate extensively in vitro to form clonal progeny that are capable of differentiating into neurons or blood cells (24). Hematopoietic differentiation was achieved by grafting nestin-positive cells into the tail veins of partially irradiated mice. However, hematopoietic cells can adopt a neuronal fate (17, 18). These studies suggest that neuronal and hematopoietic systems use associated differentiation mechanisms. In view of these similarities, it is perhaps not surprising that similar cytokines regulate the developments of both neuronal and hematopoietic systems. In addition, hematopoietic cytokines have been reported to control the activation, proliferation, differentiation, and survival of neural stem cells. The proliferation of early rat oligodendroglial progenitors in vitro is promoted by interleukin (IL)2 and IL4 and negatively regulated by transforming growth factor-b superfamily factors (3, 21). In terms of the neuronal lineage, IL5, IL7, IL9, and IL11 have been shown to enhance the elaboration of neuroblasts from a conditionally immortalized cell line derived from embryonic murine hippocampal progenitor cells (16). Thus, hematopoietic cytokines may participate in the differentiation of all three major cell types (neurons, oligodendrocytes, and astrocytes) in the developing brain. GM-CSF is a well-known hematopoietic cytokine. GM-CSF was originally identified because of its ability to stimulate the differentiation and function of hematopoietic cells (1, 9). GM-CSF stimulates bone marrow stem cell proliferation and reduces leukocyte apoptosis, increasing the white blood cell number in the peripheral blood. Because of these hematopoietic stimulating effects, GM-CSF has been used as a therapeutic cytokine in patients suffering from diseases related to bone marrow suppression. However, it remains unclear whether GM-CSF stimulates differentiation and prevents apoptosis in the neuronal system. To study the role of GM-CSF in neuronal protection, we hypothesized that GM-CSF would be able to rescue N2A cells from staurosporine-induced apoptosis and that it would improve neurologic functioning after SCI in a rat model. MATERIALS AND METHODS