Study on the Relationship between the Expression Ratio of Induced Nitric Oxide Synthase over Glial Fibrillary Acidic Protein and the Time of Injury after Brain Concussion in Rats_West China Medical Journal

Authors：

 Dong HewenHewen , DaiHao , XiaYongjun , ZengDa , JinWei ,  LiuMin

West China School of Basic and Forensic Medicine, Sichuan University, Chengdu, Sichuan 610041, P. R. China;

Corresponding?author：

LiuMin, Email: min8liu@hotmail.com

Keywords：

Induced nitric oxide synthase; Glial fibrillary acidic protein; Brain concussion; Injury time determination; Ratio of the percentage of positive cells; Rats

DOI：

10.7507/1002-0179.201600195

Video：

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Abstract Full text Figures/Tables Video References Cited by

Objective To study the relationship between the expression ratio of induced nitric oxide synthase (iNOS) over glial fibrillary acidic protein (GFAP) and the time of injury after brain concussion in rat, in order to acquire a new visual angle for determining injury time of cerebral concussion. Methods Eighty-five healthy Sprague-Dawley rats were divided into three groups randomly: model group (n=25), experimental group (n=55), and control group (n=5). The rats in the model group were used to confirm the attack hight to make the model of brain concussion; according to the time of execution, rats in the experimental group were then subdivided into 11 groups with 5 rats in each subgroup, and their execution time was respectively hour 0.5, 1, 3, 6, 12, 24, 48, 96, 168, 240, and 336; the rats in the control group were executed after fed for 24 hours. After the model of cerebral concussion was established through freefalling dart method, hematoxylin-eosin staining and immunohistochemistry staining of iNOS and GFAP were conducted for the brain of the rats. All related experimental results were studied by using microscope with image analytical system and homologous statistics. Results The ratio of positive expression of iNOS over that of GFAP increased gradually during hour 0.5- 3 after injury in brain (from 5.03 to 10.47). At the same time, the positive expression of iNOS increased significantly (from 14.61% to 37.45%). However, the increase of the positive expression of GFAP was not obvious. Between hour 3 and 12, the ratio began to decline to 4.98, which was still at a high level, and during the same time period, the positive expressions of iNOS and GFAP also experienced the same change pattern. Later, the ratio began to decline between hour 12 and 336 after injury (from 4.98 to 0.95). All ratios at this time were lower than those between hour 0.5 and 12. The positive expression of iNOS and GFAP both increased to a climax before declining. Conclusions The ratio of positive expression of iNOS over GFAP and the respective change pattern of iNOS and GFAP can be used as the evidence of estimating the injury time of cerebral concussion. We can use the ratio of two or more markers to provide a new visual angle for concluding the concussion injury time.

Citation： Dong HewenHewen, DaiHao, XiaYongjun, ZengDa, JinWei, LiuMin. Study on the Relationship between the Expression Ratio of Induced Nitric Oxide Synthase over Glial Fibrillary Acidic Protein and the Time of Injury after Brain Concussion in Rats. West China Medical Journal, 2016, 31(4): 718-723. doi: 10.7507/1002-0179.201600195 Copy

1.	?Nylén K, Ost M, Csajbok LZ, et al. Increased serum-GFAP in patients with severe traumatic brain injury is related to outcome[J]. J Neurol Sci, 2006, 240(1/2): 85-91.
2.	Deng P, Xu HH, Zhu JZ. Preliminary study on using changes in astrocytes and neurons in the brain-stem of the rat after stab wounds for determining the time of injury[J]. Forensic Sci Int, 1999, 103(3): 181-191.
3.	王煒煜, 秦啟生, 易繼林. 大鼠腦挫傷后GFAP和iNOS表達與損傷時間關系的實驗研究[J]. 中國法醫學雜志, 2004, 19(5): 271-274.
4.	Nieto-Sampedro M, Lewis ER, Cotman CW, et al. Brain injury causes a time-dependent increase in neuronotrophic activity at the lesion site[J]. Science, 1982, 217(4562): 860-861.
5.	楊靜, 王曄, 陳曉剛, 等. 大鼠腦液壓沖擊傷caspase-8的表達[J]. 法醫學雜志, 2006, 22(1): 1-3.
6.	陳銳, 余彬華, 胡玲, 等. 大鼠腦震蕩傷后腦組織HSP70、bFGF及TGF-β1表達變化[J]. 法醫學雜志, 2009, 25(4): 249-253.
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8.	Marmarou A, Foda MA, van den Brink W, et al. A new model of diffuse brain injury in rats. Part I: Pathophysiology and biomechanics[J]. J Neurosurg, 1994, 80(2): 291-300.
9.	陳銳, 嚴治, 胡玲, 等. 大鼠腦震蕩模型建立法的改良和評價[J]. 廣東醫學院學報, 2010, 28(4): 362-363.
10.	Kamijo R, Harada H, Matsuyama T, et al. Requirement for transcription factor IRF-1 in NO synthase induction in macrophages[J]. Science, 1994, 263(5153): 1612-1615.
11.	Saura M, Zaragoza C, Bao G, et al. Interaction of interferon regulatory factor-1 and nuclear factor kappa B during activation of inducible nitric oxide synthase transcription[J]. J Mol Biol, 1999, 289(3): 459-471.
12.	Martin E, Nathan C, Xie Q. Role of interferon regulatory factor 1 in induction of nitric oxide synthase[J]. J Exp Med, 1994, 180(3): 977-984.
13.	Xie K, Huang S, Dong Z, et al. Destruction of bystander cells by tumor cells transfected with inducible nitric oxide (NO) synthase gene[J]. J Natl Cancer Inst, 1997, 89(6): 421-427.
14.	Robertson FM, Long BW, Tober KL, et al. Gene expression and cellular sources of inducible nitric oxide synthase during tumor promotion[J]. Carcinogenesis, 1996, 17(9): 2053-2059.
15.	Palmieri D, Capponi S, Geroldi A, et al. TNF alpha induces the expression of genes associated with endothelial dysfunction through p38MAPK-mediated down-regulation of miR-149[J]. Biochem Biophys Res Commun, 2014, 443(1): 246-251.
16.	Ono K, Suzuki H, Sawada M. Delayed neural damage is induced by iNOS-expressing microglia in a brain injury model[J]. Neurosci Lett, 2010, 473(2): 146-150.
17.	Togashi H, Sasaki M, Frohman E, et al. Neuronal (type Ⅰ) nitric oxide synthase regulates nuclear factor kappaB activity and immunologic (type Ⅱ) nitric oxide synthase expression[J]. Proc Natl Acad Sci USA, 1997, 94(6): 2676-2680.
18.	Huang D, Shenoy A, Cui J, et al. In situ detection of AP sites and DNA Strand breaks bearing 3’-phosphate termini in ischemic mouse brain[J]. FASEB J, 2000, 14(2): 407-417.
19.	Middeldorp J, Hol EM. GFAP in health and disease[J]. Prog Neurobiol, 2011, 93(3): 421-443.
20.	Hozumi I, Chiu FC, Norton WT. Biochemical and immunocytochemical changes in glial fibrillary acidic protein after stab wounds[J]. Brain Res, 1990, 524(1): 64-71.
21.	李玉紅, 閆平, 孟曉萍, 等. 實驗性大鼠顱腦損傷后GFAP的表達研究[J]. 傷殘醫學雜志, 2004, 12(2): 1-3.

1. ?Nylén K, Ost M, Csajbok LZ, et al. Increased serum-GFAP in patients with severe traumatic brain injury is related to outcome[J]. J Neurol Sci, 2006, 240(1/2): 85-91.
2. Deng P, Xu HH, Zhu JZ. Preliminary study on using changes in astrocytes and neurons in the brain-stem of the rat after stab wounds for determining the time of injury[J]. Forensic Sci Int, 1999, 103(3): 181-191.
3. 王煒煜, 秦啟生, 易繼林. 大鼠腦挫傷后GFAP和iNOS表達與損傷時間關系的實驗研究[J]. 中國法醫學雜志, 2004, 19(5): 271-274.
4. Nieto-Sampedro M, Lewis ER, Cotman CW, et al. Brain injury causes a time-dependent increase in neuronotrophic activity at the lesion site[J]. Science, 1982, 217(4562): 860-861.
5. 楊靜, 王曄, 陳曉剛, 等. 大鼠腦液壓沖擊傷caspase-8的表達[J]. 法醫學雜志, 2006, 22(1): 1-3.
6. 陳銳, 余彬華, 胡玲, 等. 大鼠腦震蕩傷后腦組織HSP70、bFGF及TGF-β1表達變化[J]. 法醫學雜志, 2009, 25(4): 249-253.
7. 董陽. 一重和三重腦震蕩鼠BB、BW和FS行為學變化及腦組織GFAP和MBP變化研究[D]. 昆明: 昆明醫科大學, 2013.
8. Marmarou A, Foda MA, van den Brink W, et al. A new model of diffuse brain injury in rats. Part I: Pathophysiology and biomechanics[J]. J Neurosurg, 1994, 80(2): 291-300.
9. 陳銳, 嚴治, 胡玲, 等. 大鼠腦震蕩模型建立法的改良和評價[J]. 廣東醫學院學報, 2010, 28(4): 362-363.
10. Kamijo R, Harada H, Matsuyama T, et al. Requirement for transcription factor IRF-1 in NO synthase induction in macrophages[J]. Science, 1994, 263(5153): 1612-1615.
11. Saura M, Zaragoza C, Bao G, et al. Interaction of interferon regulatory factor-1 and nuclear factor kappa B during activation of inducible nitric oxide synthase transcription[J]. J Mol Biol, 1999, 289(3): 459-471.
12. Martin E, Nathan C, Xie Q. Role of interferon regulatory factor 1 in induction of nitric oxide synthase[J]. J Exp Med, 1994, 180(3): 977-984.
13. Xie K, Huang S, Dong Z, et al. Destruction of bystander cells by tumor cells transfected with inducible nitric oxide (NO) synthase gene[J]. J Natl Cancer Inst, 1997, 89(6): 421-427.
14. Robertson FM, Long BW, Tober KL, et al. Gene expression and cellular sources of inducible nitric oxide synthase during tumor promotion[J]. Carcinogenesis, 1996, 17(9): 2053-2059.
15. Palmieri D, Capponi S, Geroldi A, et al. TNF alpha induces the expression of genes associated with endothelial dysfunction through p38MAPK-mediated down-regulation of miR-149[J]. Biochem Biophys Res Commun, 2014, 443(1): 246-251.
16. Ono K, Suzuki H, Sawada M. Delayed neural damage is induced by iNOS-expressing microglia in a brain injury model[J]. Neurosci Lett, 2010, 473(2): 146-150.
17. Togashi H, Sasaki M, Frohman E, et al. Neuronal (type Ⅰ) nitric oxide synthase regulates nuclear factor kappaB activity and immunologic (type Ⅱ) nitric oxide synthase expression[J]. Proc Natl Acad Sci USA, 1997, 94(6): 2676-2680.
18. Huang D, Shenoy A, Cui J, et al. In situ detection of AP sites and DNA Strand breaks bearing 3’-phosphate termini in ischemic mouse brain[J]. FASEB J, 2000, 14(2): 407-417.
19. Middeldorp J, Hol EM. GFAP in health and disease[J]. Prog Neurobiol, 2011, 93(3): 421-443.
20. Hozumi I, Chiu FC, Norton WT. Biochemical and immunocytochemical changes in glial fibrillary acidic protein after stab wounds[J]. Brain Res, 1990, 524(1): 64-71.
21. 李玉紅, 閆平, 孟曉萍, 等. 實驗性大鼠顱腦損傷后GFAP的表達研究[J]. 傷殘醫學雜志, 2004, 12(2): 1-3.

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West China Medical Journal

Study on the Relationship between the Expression Ratio of Induced Nitric Oxide Synthase over Glial Fibrillary Acidic Protein and the Time of Injury after Brain Concussion in Rats

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