Induction of Retinal Ischemia

1. The next step involves administration of an endogenous growth factor and vessel damage to promote blood vessel growth in the retina. Fully and robustly engrafted animals are selected and anesthetized.

2. VEGF is administered directly into the vitreous using a 36-gage needle and Hamilton syringe (see Note 8). Either purified (40 ^g/kg) VEGF protein or AAV-VEGF virus (2 x 108 viral particles), where the CMV promotor drives expression of VEGF in an adeno-associated virus (AAV) vector, can be used. VEGF is an endothelial cell-specific mito-gen that is transcriptionally regulated by the cytomegalovirus promoter/enhancer when packaged in AAV. AAV mediates the long-term expression in nondividing cells, which allows for stable expression and constant amounts of VEGF to reach the area of ischemia to promote neovascularization (Fig. 3; ref. 12).

3. The study of clinical diseases, such as diabetic retinopathy and retinopathy of prematurity, has led to an understanding of the pathology that occurs in these diseases. In these conditions, the eye "detects" a lack of oxygen, either as a result of the diabetic condition or the removal from the incubator's oxygen-rich environment of a prematurely born baby. As a result, the cells signal new blood vessel growth in the region in an attempt to relieve the perceived ischemia. We take advantage of this neovascularization by creating a local region of ischemia in the eye through cauterizing of large blood vessels with a laser. This induces new blood vessel growth into the region in an attempt to relieve the ischemic pressure.

4. Peak expression of VEGF by AAV has been determined to be at 3 to 6 wk; therefore, the physical disruption of the blood vessels is done during this time (unpublished data). Begin by anesthetizing the mice normally with a general anesthetic, and concurrently administer a 10% sodium fluorescein solution intraperitoneally, which labels blood vessels for facilitating visualization during procoagulation.

5. Dilate the eyes with 1% atropine for 5 min, wash with PBS, and subsequently dilate with 2.5% phenylephrine for 5 min (see Note 9).

6. Immediately after the two 5-min treatments, the mice undergo laser treatment. An Argon Green laser system (HGM Corporation, Salt Lake City, UT) can be used for retinal vessel photocoagulation with the aid of a 78-diopter lens. The blue-green argon laser (wavelength 488-514 nm) is applied to various venous sites around the optic nerve.

7. Venous occlusions are accomplished with >60 burns of 1-s duration, 50 mM spot size, and 50 to 100 mW intensity.

Fig. 3. Illustration and sectional representation of the eye for growth factor administration. Arrows indicate angle and placement of needle for growth factor injection (see Note 8).

Oblique section /1

Fig. 3. Illustration and sectional representation of the eye for growth factor administration. Arrows indicate angle and placement of needle for growth factor injection (see Note 8).

8. Again, allow the animals to recover for 30 d while the transplanted HSC, directed by the ischemia and induced by the VEGF, contribute to the neovascularization to relieve the hypoxia produced by the cauterizing of the existing vessels.

Was this article helpful?

0 0
10 Ways To Fight Off Cancer

10 Ways To Fight Off Cancer

Learning About 10 Ways Fight Off Cancer Can Have Amazing Benefits For Your Life The Best Tips On How To Keep This Killer At Bay Discovering that you or a loved one has cancer can be utterly terrifying. All the same, once you comprehend the causes of cancer and learn how to reverse those causes, you or your loved one may have more than a fighting chance of beating out cancer.

Get My Free Ebook


Post a comment