difficult or impossible. Therefore, if animals are going to be asleep for more than just a few minutes, it is necessary to place an ointment (e.g., any sterile opthalmic ointment or petroleum jelly) on the eyes to prevent drying of the cornea. Also, it is important not to expose the eyes of anesthetized animals that have had their pupils dilated to light for any longer than necessary, as strong light can damage the photore-ceptor cells.
The severity and clinical course of EAU in mice vary depending on the strain and the immunization regimen. In the B10.A strain, it is possible to control the course of disease by adjusting the respective doses of antigen and PTX, so that the whole spectrum of disease, from chronic to hyperacute, can be obtained (Caspi et al., 1990). High-intensity immunization results in an acute form of disease with an early onset (day 12 to 14) and in diffuse photoreceptor damage, whereas lower antigen and PTX doses will result in milder disease with progressively later onset. In the milder form of the disease, pathology is typically focal and a large portion of the photoreceptor cell layer may be spared. This type of disease tends to take on a chronic form, or may remit and be followed by a relapse after a brief disease-free period. The number of relapses that can occur has not been determined.
Enucleation should be performed carefully, without squeezing the eyeball, to avoid maceration of delicate ocular tissues that become even more fragile when inflamed. Eyes should be collected within 15 min of euthanasia, because autolysis sets in very rapidly and will preclude correct evaluation of the results. It is important to prefix the eyes in 4% buffered glutaraldehyde for 1 hr, after which the eyes should be transferred to 10% buffered formalin at least overnight. This brief fixation in 4% glutaraldehyde prevents artifactual detachment of the retina from the choroid. However, leaving the eyes in glutaraldehyde for too long will cause excessive hardening of the lens, which will make sectioning difficult. Eyes should not be harvested too early after disease onset, as retinal damage takes time to develop: a good time to collect them is 1 week after clinical onset. Whenever possible, both eyes should be evaluated clinically as well as histopathologi-cally, as disease is not always equally severe in both eyes and may even be unilateral. If the experimental setup permits taking only one eye for histopathology, always collect the right (or always the left) eyes to average out this random variation across the group.
Lewis rats sometimes develop very severe anterior chamber inflammation, which can lead to corneal perforation. This is more likely to occur with retinal soluble antigen (S-Ag), or its peptides, and pertussis than with IRBP peptide R16. Complications can also result from use of complete Freund's adjuvant (CFA). Typical problems are development of arthritis and/or severe granulomatous inflammation of the injection site that can sometimes lead to tissue necrosis. In such cases the animal(s) should be euthanized promptly. Hunters adjuvant, which causes fewer undesirable side effects, can in some cases be successfully substituted for CFA (Roberge et al., 1992).
If no disease is obtained and the animals have been immunized with the correct doses of antigen (and, in mice, Bordetella pertussis toxin), there may be problems with the anti-genic material. Incorrectly synthesized peptide may contain an altered uveitogenic epitope. There may not be enough mycobacteria in the complete Freund's adjuvant (CFA) because the suspension had settled or was improperly mixed before taking the aliquot. A poorly prepared antigen/adjuvant emulsion is less effective; a well-prepared emulsion has the consistency of thick cream and a drop will not disperse when added to water.
If injected animals are harboring an infection or if the wrong strain of animal (genetically resistant) is used, the response can also be affected.
In Lewis rats, onset of experimental autoimmune uveoretinitis (EAU) occurs 9 to 10 days after immunization with interphotoreceptor retinoid-binding protein (IRBP) peptide R16 in complete Freund's adjuvant (CFA), or 4 to 6 days after adoptive transfer of R16-specific lymphocytes. Disease grades, evaluated by his-topathology in eyes collected 7 days after clinical onset, range from 1 to 3 in most rats. The R16 epitope rarely if ever causes disease having a grade higher than 3; however, higher EAU grades are frequently obtained with other uvei-togenic epitopes, e.g., peptide 35 of retinal soluble antigen (S-Ag).
In B10.RIII mice, EAU onset can occur as early as day 9 after immunization with IRBP peptide 161-180. EAU grades, evaluated by histopathology on day 21 after immunization with 50 |g peptide, range between 2 and 4 for most mice.
Adoptively transferred disease usually appears 4 to 6 days after intraperitoneal transfer of cells. It can be as severe or more severe than after active immunization, depending on the number of cells transferred.
Carrying out a uveitis protocol from beginning to end, including histological evaluation of the tissue, takes several weeks, although only a minority of that time is spent in performing the experimental procedures. Preparing the enriched complete Freund's adjuvant (CFA) takes 15 to 20 min. Preparing the antigen/adjuvant emulsion and immunizing 10 to 15 animals takes ~1 hr. Whereas follow-up of clinical disease in rats (observation with a flashlight) may take only 15 to 20 min, fundoscopic examination of that number of mice, especially if it includes anesthesia, takes 1 to 1.5 hr. Euthanizing the animals, followed by collecting and fixing the eyes, takes slightly over 1 hr, including the fixative change. If lymph nodes are collected and processed for culture, 3 to 4 additional hr are required. Harvesting the lymph node cells after culture and performing the adoptive transfer procedure takes 2 to 3 hr.
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Animal Models for Autoimmune and
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