Correlation between Activity of Nitric Oxide Synthases and Apoptotic Change in the Closed Head Injury Animal Model

Kim, Jeong Hoon; Park, Chong Oon; Hyun, Dong Keun; Ha, Young Soo

Abstract

OBJECTIVE
The authors present an investigation of alterations in nitric oxide synthase(NOS) activity and histopathological response after moderate diffuse axonal injury(mDAI).
METHODS
A total of 40 anesthetized Sprague-Dawley adult rats were injured utilizing the Marmarou's weight-drop device through a Plexiglas guide tube. These rats were divided into 8 groups(control, 1 hr, 2 hr, 3 hr, 6 hr, 12 hr, 24 hr, 48 hr after trauma). The temporal pattern of apoptosis after mDAI in the adult rat brain was characterized using TUNEL stain and cDNA for NOS activity was amplified using RT-PCR. The PCR products were electrophoresed on a 2% agarose gel and then observed under a UV-transilluminator. The enhanced chemiluminescence method(Pierce, Rockford, IL) was used to visualize the protein bands, and the quantification was performed by using the image analysis soft-ware Bio-1D.
RESULTS
eNOS activity was not detected at all groups, but nNOS activity was expressed at 3 hr and continuously by 48 hr after impact , which was especially showed about 2 times larger than control group at 12 and 24 hr(mean value=28314+/-35.07 in injury group, 13386+/-26.14 in control group, p<0.05), followed by being on the decrease at 48 hr. iNOS activity was showed fivetmes larger than control group at 12 hr and 24 hr(mean value=15264+/-38.37 in injury group, 3002+/-31.62 in control group, p<0.05) followed by dramatically decrease below the level of control group(2299+/-14.06). Histologically, significant apoptotic changes after brain injury were occurred from 12 to 24 hr post-injury(AI=24.8%, 27.5%, p<0.001).
CONCLUSION
These data indicate that nNOS and iNOS activity are affected after mDAI in a time-dependent manner and there is a close relation between apoptotic changes and NOS activity. nNOS activity is not stronger than iNOS, which lately expressed. However, iNOS activity is markedly reduced by 48 hr compared to nNOS.