The Early Protective Role Of Neuregulin-1 In Rat Spinal Cord Ischemia Injury

It has been reported that nearly 70% of Spinal cord injury were caused by hypoxia and ischemia. The survival of neurons after ischemia was closely related to prognosis and recovery of the patients. Neuregulin-1 (NRG-1) is a signaling protein among cells produced by glial cells and neurons. It plays a critical role via ErbB receptors not only in maturation and development of the nervous system, but also in neural protection in ischemia nerve system damage. Astrocytes are the major components of neuroglia and compose of 50% of the brain volume. As we know, the number of astrocytes is about 10-50 times greater than that of neurons and so astrocytes play an important role in the brain functions. However, questions remain unknown: what would happen to astrocytes in number and morphology and in express of the NRG-1 under Spinal cord ischemia and Hypoxia? And what would be the significance if changes happened in astrocytes? In order to answer these questions, we have done as follows:1. In order to clarify the role of NRG-1 and astrocytes under Spinal cord ischemia and Hypoxia condition in vivo, we constructed Spinal cord ischemia and hypoxia animal model. The fluorescent immunohistochemical method was used to detect both the expressions of NRG-1 and NRG-1R (ErbB4) . The Western blot method was used to detect the expressions of NRG-1 protein. We observed that the numbers of the reactive astrocytes were increased in hypoxia region. The NRG-1 was observed in both astrocytes and neurons, and the expression of NRG-1 obviously increased in ischemia spinal cord (P<0.05). The ErbB4 was observed in neurons, and the expression of ErbB4 increased in ischemia group in comparison with normal group. These results suggested that in the early duration of ischemia, the protective roles to neurons were carried out by both increased numbers of the astrocytes and by their increased expression quantity of the NRG-1.2. To investigate advanced protection of NRG-1 to the apoptosis neurons, we injected NRG-1 to the subarachnoid space in the ischemic rats. The caspase3 mRNA was detected by semi-quantitative RT-PCR method. The results showedthat the expression of caspase3 mRNA increased after ischemia compared with the normal group and sham-operated group (P<0.05) . And the expression of caspase3 mRNA decreased in the NRG-1 injection group (P<0.05) while the vesicle group is same with the ischemia group (P>0.05). The results suggested that injection of NRG-1 may protect the neurons from apoptosis.3. To investigate the changes of the expression of NRG-1 in the cultured astrocytes after conditioned hypoxia, we cultured the rat cortical astrocytes and developed the hypoxia model. The hypoxia model was developed in 95% Niand 5%CO₂ lower O₂ chamber for 20min. After that, the culture was returned to normoxia 5%CO₂ incubator. The expression of NRG-1 was examined in both normal and hypoxia cultures by means of fluorescent immunocytochemical labeling and semi-quantitative Western blot. Results showed that normal cultured astrocytes expressed a relative lower level of NRG-1. The expression of NRG-1 slowly increased after hypoxia/reoxygenation, and dramatically increased to reach the highest level at 8 h after reoxygenation, then decreased again. The results suggest that the astrocytes could not express enough NRG-1 immediately after hypoxia and ischemia; therefore, astrocytes secreted NRG-1 might play a protective role in hypoxia injury.In summary, astrocytes, which are comprised of 50% the total volume of the Spinal cord ischemia and Hypoxia, increase the expression level of NRG-1 in hypoxia condition, due to the special anatomical relationship between astrocytes and neurons. The increasing of NRG-1 would alleviate the death of neurons by different ways. On one hand, NRG-1 acts on astrocytes to produce more nerve protective factors by autocrine. On the other hand, NRG-1 binds to the receptors of neurons to against apoptosis by paracrine. These results clearly indicated that NRG-1 plays a critical protective role in Spinal cord ischemia and hypoxia condition.