The Effects of Mitotic Inhibition on the Spinal Cord Response to the Superimposed Injuries of Spinal Cord Hemisection and Peripheral Axotomy

The present study was carried out to test the hypothesis that dividing microglia are responsible for the depression of crossed phrenic nerve activity documented at 2 weeks postphrenicotomy in an injury model which superimposes the effects of spinal cord injury on peripheral axotomy. Crossed phenic nerve activity is defined as the respiratory activity recorded from the phrenic nerve during the crossed phrenic phenomenon (CPP) which is a respiratory reflex induced by respiratory stress following an ispsilateral spinal cord hemisection. Young adult female Sprague-Dawley rats were subjected to left intrathoracic phrenicotomies. Cytarabine (Cyt-A, a powerful antimitotic drug) or saline-filled miniosmotic pumps were then implanted into the cisterna magna and 2 weeks were allowed to pass at which time the CPP was induced by a left C2 spinal cord hemisection and transection of the contralateral phrenic nerve. Control studies including bromodeoxyuridine labeling of mitotic cells and a triple immunofluorescent protocol were carried out to verify that microglial cells were the primary cell type undergoing mitosis in the current injury model and that Cyt-A completely inhibited cellular proliferation. Quantitative electrophysiological analysis of crossed phrenic nerve activity showed that there is a statistically significant depression of activity at 2 weeks postphrenicotomy when animals were infused with saline compared to controls. Crossed phrenic nerve activity levels were not significantly different, however, from control levels when 2-week postphrenicotomized rats were infused with Cyt-A. Immunofluorescent studies showed that the majority of cells dividing in response to phrenicotomy were microglia. Furthermore, there were no astrocytes seen dividing at any time. From the results, we conclude that activated microglial cells may be responsible for the depression in crossed phrenic activity normally seen 2 weeks postphrenicotomy. Further, the activation of microglia may be related to the astrocytic response to injury. The activated microglial cell may be acting as a coordinator of various aspects of the injury response. Alternatively, the activation of microglia may be a necessary step in the cascade of multiple events that take place in the spinal cord after injury.