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

Original Article

Journal of Korean Neurosurgical Society 2002;32(2): 142-148.

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

Department of Neurosurgery, Inha Hospital, College of Medicine, Inha University, Sungnam, Korea.

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.

Key Words: Nitric oxide synthase; Apoptosis; TUNEL stain; RT-PCR