This model can be used to study cellular mechanisms involving the pathogenesis of immune-mediated human posterior uveitis. The implication of this technique extend toward the translational study of human uveitis in monitoring novel drug efficacy and for providing mechanistic insight into chemical disease progression. The main advantage of using this model is the reliability of disease induction and use of non-invasive endoscopic techniques that allow us to monitor disease progression.
Here we describe how to induce the disease and assess the pathology using several readouts To prepare IRBP 1-20 complete Freund's Adjuvant or CFA, resuspend a calculated amount of the peptide in a minimum volume of 100%DMSO. When the powder has completely dissolved, add small volumes of PBS to the tube until reaching to the final calculated volume of PBS according to the number of mice to be injected using gentle agitation to mix the solution. After preparing the complete Freund's Adjuvant, gently and frequently pipette the solution to form a viscus and evenly distributed emulsion while adding the DMSO PBS peptide solution dropwise at a one-to-one ratio to the suspension.
To aerate the solutions, use a 1000 microliter pipette set to 700 microliters to repeatedly pipette the solution until a thick, creamy emulsion has been obtained. Before injecting the peptide suspension, place each mouse to be injected into a separate cage and use a one milliliter syringe equipped with a 23 gauge needle to intraperitoneal deliver 1.5 micrograms of Bordetella pertussis toxin in 100 microliters of RPMI 1640 medium supplemented with 1%mouse serum to each animal. When pertussis toxin solution has been injected, mouse should be held in a scruff like position and the skin pinched to form a tent like structure on the back of the neck.
Thread the needle to the space between the finger and thumb, and inject 200 microliters of the IRBP emulsion to the tented skin, maintaining pressure after the injection and rotating the needle head to close the skin before retraction. To score the clinical disease, on day 21 after injection, confirm a lack of response to petal reflex in the anesthetized peptide injected mouse and restrain the animal in a scruff. Topically apply 1%tropicamide and 2%phenylephrine to the cornea of each eye immediately after injection.
When the pupils have fully dilated, place the mouse onto a purpose-built microscope stage. Position the microscope for full access to the retina. Adjust the eye piece throughout the imaging process as necessary to see the optic disc and retina and acquire images of the entire retinal area, adjusting and covering all of the corners of the periphery.
To measure vessel leakage, administer 100 microliters of 2%fluorescein subcutaneously at the back of the neck and reposition the mouse such that the retina is center on the middle of the live image. Set the fundoscope to a blue light excitation filter at 465 to 490 nanometers before acquiring an image of each at 1.5 and seven minutes after fluorescent injection. When all of the images have been acquired, intraperitoneally deliver an anti sedation injectable for recovery and place the mouse in a cage on a preheated mat with access to wet soaked diet with monitoring until the mouse regains consciousness.
For clinical disease scoring of the images, base the clinical assessment on the severity of the optic disc inflammation, retinal vessel cuffing, retinal tissue infiltrate, and structural damage. Score each of these parameters on a scale from one to five. The collective total is representative of the clinical disease for the whole eye with a maximum score of 20 obtainable per eye.
After fundus imaging for the final clinical endpoint of the study, euthanize the mouse. For histological analysis, prize apart the eyelids for access to the entire eye, then place curved forceps behind the globe to grasp the orbital connective tissue and optic nerve and enucleate the eye. Place the enucleated eye in one to five milliliters of 4%glutaraldehyde for a minimum of 15 minutes before transferring the organ to one to five milliliters of 10%formaldehyde for at least 24 hours.
After histological tissue staining, according to standard protocols, assigned scores on a scale of zero to three based on the level of immune cell infiltration and the degree of retinal damage. Fundoscopic changes are classified during disease progression as inflammatory changes that include retinal tissue and vascular and optic disc inflammation and retinal structural damage in addition to histological changes based on infiltrating immune cells and structural impairments. These clinical and histopathological changes can be graded and scored to assess disease progression and to evaluate therapeutic intervention efficacy.
Vascular leakage is also a pathological feature of the model and of human uveitis. As illustrated in this analysis, fluorescein can be used to visualize vascular leakage as another readout of this model. It is essential for researchers to ensure the emulsion is prepared correctly without signs of separation between the solutions.
They should be mixed thoroughly and the final product should be smooth in texture. This procedure can be combined with other methods of drug administration and with for cytometry for phenotyping infiltrating retinal immune cell populations.
