| Objective1.To construct an animal model of intermittent hypoxia and verify it.2.To observe the effects of chronic intermittent hypoxia on the ocular surface of mice,and to investigate the changes of tight junction protein in corneal epithelium.3.To investigate the effect of chronic intermittent hypoxia on lacrimal gland in mice.Method1.Constructing of an animal model of intermittent hypoxia and verifying it1.1 Constructing of an animal model of intermittent hypoxiaThe oxygen concentration detector is placed in the customized hypoxia box to record the oxygen concentration in real time.The time and flow rate of nitrogen,oxygen and air are set by the time controller and gas flowmeter,so that the oxygen concentration in the hypoxia box changes periodically.1.2 Verifying the effectiveness of the intermittent hypoxia animal modelTo further verify that periodic changes in oxygen concentration in the hypoxia box cause intermittent hypoxia in mice.After the mice were anesthetized with Ulatan,which had little effect on respiratory circulation,the tail of the mice was clamped with a blood oxygen saturation monitor and sent into the hypoxia box to record the blood oxygen saturation of the mice in real time.2.To investigate the effects of chronic intermittent hypoxia on the tight junction of corneal epithelium and lacrimal gland in mice2.1 Constructing the animal model of chronic intermittent hypoxiaThirty male 6-week-old BALB/c mice were randomly divided into chronic intermittent hypoxia group(CIH group)and normoxic control group(Normoxia group).The mice in the CIH group were placed in the hypoxia box under hypoxia / reoxygenation cycle circumstance every day,and the living condition of the mice in the Normoxia goup was the same as that in the CIH group except that the air was ventilated.The experiment lasted 8 hours a day for 4 weeks.2.2 At the end of the experiment,the ocular surface indexes of the two groups were checked,including corneal fluorescein sodium staining and tear secretion test.2.3 All mice were executed at the same day.The morphological changes of corneal epithelium and lacrimal gland in mice were observed by hematoxylin eosin staining.The apoptosis of corneal epithelium and lacrimal gland were detected by TUNEL kit.Western Blot and Real-time PCR were used to detect the protein and m RNA expression of NOX4,Occludin,Claudin-1 and ZO-1 in corneal epithelium.Immunohistochemistry was used to observe the expression of NOX4 in lacrimal gland.Result1.In each hypoxia / reoxygenation cycle,the oxygen concentration in the box and the blood oxygen saturation of mice showed periodic changes with time,suggesting that the intermittent hypoxia mouse model was successfully constructed.2.Compared with the Normoxia group,the corneal fluorescein score of CIH group increased and the level of tear secretion decreased.The number of TUNEL positive cells in corneal epithelium increased.Western Blot and Real-time PCR showed that the expression of NOX4 protein and m RNA increased,while the protein and m RNA expression of tight junction proteins Occludin,Claudin-1 and ZO-1 decreased.3.Compared with the Normoxia group,the acinar cells in the lacrimal gland of the CIH group decreased,the number of TUNEL positive cells and the expression of NOX4 in the lacrimal gland increased significantly.ConclusionChronic intermittent hypoxia cause corneal epithelial damage and decrease tear secretion in mice.The injury of corneal epithelium may be related to the increase of oxidative stress,apoptosis of epithelial cells and decrease of tight junction protein in corneal epithelium.The possible mechanism of the decrease of tear secretion is related to the atrophy of acinar cells,the apoptosis of lacrimal gland and the increase of oxidative stress. |
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