Establishment and assessment of experimental periventricular leukomalacia models in newborn rats / 中国组织工程研究

ABSTRACT

BACKGROUND: The researches on the onset mechanism and intervention of periventricular leukomalacia (PVL) are affected due to few cases of generally-acknowledged animal model, so it is necessary to establish a reliable animal model for the study of PVL.0BJECTIVE To establish PVL animal model of the 2-day-old SD rats.DESIGN: Randomized control animal trial.SETTING: Department of Pediatrics, West China Second University Hospital, Sichuan University.MATERIALS A total of 36 2-day-old SD rats of cleaning grade and either gender, weighing 6-8 g, were provided by the West China Experimental Animal Center of Sichuan University; Mice anti-O4 was purchased by Chemicon Company,rabbit anti-glial fibrillary acidic protein (GFAP), rabbit anti-β-amyloid precursor protein (β-APP), rabbit anti-myeline basic protein (MBP), SABC immunohistochemical kit and DAB color reagent were all offered by Wuhan Boster Biological Technology Co., Ltd. Rabbit anti-mice IgG-FITC was obtained from Zhongshan Golden Bridge BioTechnology Co., Ltd.METHODS: The experiment was accomplished in the Laboratory of Women and Children, West China Second Hospital of Sichuan University between May and December in 2005. Totally 36 rats Were randomly divided into experimental PVL group and control group, 18 in each. The experimental PVL group was subjected to unilateral carotid ligation (UCL), and then they were put into a box filled with 6% oxygen and 94% nitrogen for 4 hours. Six rats were executed at ischemic 72hours, 14 days and 28 days respectively. Meanwhile sham surgeries were performed on the control group without ligation or exposure to hypoxia. And the time segment was identical with that of experimental group. ①Histopathological examination Rat hearts were fixed by perfusion and stained with hematoxylin-eosin (HE). Light and electronic microscopy were used to observe the brain pathological and ultrastructure changes, ②Immunohistochemistry method was used to detect the distribution and expression of GFAP, β-APP, MBP and O4 in the white matter of both experimental and the control groups 72 hours post-operation. ③Neuroethology examination Hanging test (rats were forced to hold the horizontal glass rod with forelegs, and the time of dropping was recorded in the distance of 45 cm.Scoring 1 point ＜ 10 s; 2 points 10-30 s; 3 points 30 s-2 minutes; 4 points 2-5 minutes; 5 points ＞ 5 minutes),inclined plane test (rats were laid on the inclined plate at the angle of 45° while rat heads turning upwards at the angle of more than 135°), open field test (square box without summit was divided into 9 equal grills at the bottom, rats were placed in the central grill to observe the activity within 30 seconds. Scoring 1 point as rats entered the neighbor cage above half the body; 1 point as standing by hind limbs; total scores were the addition of the two), and cylinder test (rats were put in the cylinder of 20 cm×30 cm×5 cm to record the time of initial forepaw of each weight-bearing contact with the wall during a full rear, right (ipsilateral) or left (contralateral) percentage of total forepaw contacts at initiation was calculated.) were tested on the SD rats at 28 days post-operation. Then statistical management was conducted.MATN OUTCOME MEASURES: ①HE stain and electronic microscope were used to detect the histopathology changes after ischemia and hypoxia.②lmmunohistochemistry method was used to detect the expressions of GFAP, β-APP, MBP and O4 in the corresponding cell and tissue after ischemia and hypoxia. ③Neuroethology examination was used to evaluate the rats after ischemia and hypoxia by scores of each test.RESULTS: All 36 rats were involved in the result analysis. ①White matter damage was observed in the periventricular white matter in the PVL group by light and electronic microscopy at the early stage of post-operation; Ventricular dilatation and the loss of medullary sheath were detected in the white matter at the latter stage. ②The integrated optical density (IOD) of GFAP in PVL group was stronger than that of the control group (6 566.93±455.56, 1 069.32±791.71,P ＜ 0.05), and the mean diameter of GFAP-immunoreactive cells was increased in the brain tissue of PVL group compared with those of the control group [(11.69±0.97), (8.24±0.22), P ＜ 0.05]; β-APP immunohistochemistry demonstrated the IOD of PVL group was stronger than that of the control group [(59 304.07±6 864.03), (15 132.29±2 455.52),P＜ 0.05]; MBP IOD of the PVL group was decreased compared with the control group [(21 764.29±1 981.63), (69 174.72±3 199.90), P ＜ 0.05]; The density of O4-immunoreactive pyknotic cells was dramatically increased in the PVL group compared with the control group [(54.08±11.99), (1.25±0.51), P＜ 0.05].③In PVL group, the hanging time was shorter in the hanging test than that of the control group [(1.27±0.14), (4.24±0.59) minutes, P ＜ 0.05]; The turning-around time was longer in the inclined plane test than that of the control group [(7.17±2.32), (3.27±0.82) s, P ＜ 0.05]; The score in the open field test was decreased than that of the control group [(3.68±0.82), (12.67±1.00) s, P ＜ 0.05]; In the cylinder test the activity of the left limb was less than that of the right limb [(19.25±2.77), (64.55±0.36)%, P ＜ 0.05].CONCLUSION: PVL animal model can be successfully established by the method of UCL-hypoxia using the 2-day-old SD rat, and appears the obvious white atter damage, abnormal neurobehavior, reasonable pathological and behavior change.