Source: Hou, A. et al., A Chronic Autoimmune Dry Eye Rat Model with Increase in Effector Memory T Cells in Eyeball Tissue. J. Vis. Exp. (2017)
This video demonstrates a method for assessing corneal smoothness and tear film stability in an autoimmune rat model. It highlights the impact of gland dysfunction on tear film stability, crucial for understanding dry eye disease.
All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
1. Preparation of Lacrimal Gland Extract
NOTE: Rats were anesthetized with the intraperitoneal injection of ketamine (75 mg/kg) and xylazine (10 mg/kg). Proper anesthetization was confirmed by toe pinching and tail pinching. Ophthalmic gel was applied to the rat's eyes to prevent dryness after each procedure. The anesthetized rats were placed under far infrared lights to keep the animals warm until they fully recovered. During the procedures and recovery time, animals were closely monitored by researchers. All the materials and surgery tools were sterile before use. At the end of the experiment, rats were euthanized by the intraperitoneal injection of pentobarbital (80 mg/kg). Complete euthanasia was verified by lack of cardiac pulse and no blink reflex by touching the eyeball. Rats were housed in standard conditions: room temperature, 21-23 °C; relative humidity, 30-70%; light-dark cycle, alternating 12 h (7 AM to 7 PM).
2. Preparation of Emulsion and Pertussis Toxin
3. Immunization of the Lewis Rats
4. Injection of the Antigen Mixture into the Forniceal Subconjunctiva and Lacrimal Gland to Recruit Antigen-specific Immune Cells and Cause Local Inflammation
5. Assessment of Dry Eye Features
NOTE: The anesthetized rats should be held gently by a gloved hand in an upright position on a flat surface to avoid movement.
The authors have nothing to disclose.
Reagents | |||
Protease inhibitor cocktail | Sigma-Aldrich | P2714-1BTL | |
Pierce BCA Protein Assay Kit | Thermal Scientific | 23227 | |
Mycobacterium tuberculosis H37Ra | Becton, Dickinson and company | 231141 | |
Complete Freund's adjuvant | Becton, Dickinson and company | 231131 | |
Ovalbumin | Sigma-Aldrich | A5503-10G | |
Incomplete Freund's adjuvant | Sigma-Aldrich | F5506-6X10ML | |
Pertussis toxin | Sigma-Aldrich | P7208-50UG | |
Equipment | |||
Sonicator | Sonics | Vibra-Cell | |
Stereo microscope with ring light illuminator and camera | Carl Zeiss | NA |