A Murine Model of Ischemic Retinal Injury Induced by Transient Bilateral Common Carotid Artery Occlusion

The mouse model established with this protocol can be utilized to investigate the pathophysiology of ischemic retinopathies and for in vivo drug screening. The current method could reduce reliance on various surgical instruments and shorten operation time to induce retinal ischemia. Pathogenesis of vascular diseases, such as diabetic retinopathy, occlusion of retinal veins or arteries and ocular ischemic syndrome can be understood.

Demonstrating the procedure will be Deokho Lee, our PhD student from my laboratory. Yukihiro Miwa, DVM, PhD, and assistant professor from my laboratory will help to demonstrate the procedure. Autoclave all the surgical instruments and keep them in 70%ethyl alcohol prior to the surgical procedure.

Prepare male BALB/c mice in a specific pathogen-free room to maintain sterile conditions before, during and after the surgery. Apply one drop of 0.1%purified sodium hyaluronate eyedrop solution to the eyes of the animal to prevent dryness under anesthesia. Place the mouse on its back and fix its paws using adhesive tape.

Disinfect the neck area using 70%ethyl alcohol before the surgery. Using a blade, make a sagittal incision on the neck. Carefully separate the salivary glands using two forceps and set them aside to visualize the underlying CCA.

Isolate the right CCA from the respective vagal nerves and accompanying veins and place two 6-0 silk sutures under the CCA. Tie the two ends tightly to block the blood flow. Wipe the blood oozing out from the damaged small veins during the procedure for clear visibility of the CCA.

Find the left CCA without harming the accompanying veins and place a 6-O suture needle under it to mark a site for clamping, occlude it for two seconds. Softly remove the clamp to avoid damaging the arterial wall and reopen the left CCA. After reopening, suture the wounds on the neck with a silk suture.

When finished, apply a dab of antibiotic to the neck to inhibit bacterial infection. Inject the mouse with 0.75 milligrams per kilogram of atipamezole hydrochloride intraperitoneally to help it recover from deep anesthesia quickly, then returned the mouse to a cage with preheated pads. When the mouse wakes up, inject it with 0.4 milligrams per kilogram butorphanol tartrate for pain management.

Eyelid drooping was examined after tBCCAO. The right eye showed mild and severe eyelid drooping, while the left eyes had no drooping except for one mouse. Immunoblots and quantitative analyses for HIF-1 alpha and beta-actin showed that HIF-1 alpha was stabilized in the right retina three and six hours after tBCCAO.

Retinal reactive gliosis was examined three days after tBCCAO. The average of morphology scores for GFAP labeling in the right retina was the highest among the both retinas in the sham operated mice and the left retina in tBCCAO operated mice. The amplitudes of b-wave in the right eye decreased three and seven days after tBCCAO as shown in dark adapted ERG.

Whereas quantitative analyses showed a decrease in the amplitudes of b-wave in the right eye without any change of a-wave amplitude. Representative OCT images in the sham and tBCCAO operated retinas and quantitative analyses showed that there was an increase in retinal thickness in the right retina and no change in the left retina. When attempting this protocol, it is important to remember that the right CCA has to be occluded clearly.

And then the left CCA has to be stringently occluded. With this method, hypertension or hypoglycemia can be applied to mimic conditions of human ocular diseases. This model shows the results of common retinal ischemia and the mechanisms of retinal ischemia.