Ординатура / Офтальмология / Английские материалы / Ocular Neuroprotection_Levin, Polo _2003

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3.Schwartz M, Yoles E. Optic nerve degeneration and potential neuroprotection: implications for glaucoma (abst). Eur J Ophthalmol 1999; 9 (suppl 1): S9–11.

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M.Autoimmune T cells retard the loss of function in injured rat optic nerves. J Neuroimmunol 2000; 106:189–197.

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E.RGC death in mice after optic nerve crush injury: oxidative stress and neuroprotection. Invest Ophthalmol Vis Sci 2000; 41:4169–4174.

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plexus via the collector channels. The venous plexus drains into the episcleral veins, which are radially oriented and are called radial veins. They run posteriorly and merge with the large vessels near the extraocular muscle insertion. The large vessels join posteriorly with the inferior and superior ophthalmic veins and enter the cavernous sinus. Any interruption of the episcleral drainage will cause a rise in IOP and, possibly, secondary glaucoma. It is therefore post-trabecular glaucoma. Because the outflow is affected, this procedure may lead to open-angle glaucoma [1,2]. If one increases the post-trabecular meshwork resistance by cauterizing the deep episcleral veins, one would expect an increase in IOP.

Described below are the essential steps in creating elevation of IOP in adult rat eyes. This method is reliable and reproducible. The range of elevated IOP pressure correlates with the number of cauterized veins. This model does not involve introduction of exogenous material in the eye.

III.PROCEDURE

1.Adult Wistar rats (250–300 g) are anesthetized with intraperitoneal injection of 0.1 to 0.15 mL mixture of acepromazine maleate (1.2 mg/kg), xylazine (8 mg/kg), and ketamine (40 mg/kg).

Anesthetic injection can be given intramuscularly; however, it takes between 15 and 20 min to induce deep anesthetic condition to perform surgery. Certain hyperactive rats may require a second or third injection of the anesthetic mixture administered in much smaller doses than the initial dose.

2.To maintain the animals’ body temperature during the procedure, they should be placed on a heated pad.

3.For each animal, the contralateral eye should serve as a comparative control and should either have a sham operation or be left undisturbed. Because of individual variations in IOP, preand postoperative IOP should be compared between normal and experimental eyes for each animal.

4.In order to keep the eye open, place the sutures on the eyelids.

5.Four to five aqueous-containing radial veins emerge from the circumferential venous plexus in the rat eye. The supranasal vein should be located first. In order to stabilize the globe and expose the veins, make an incision 2–3 mm long within the limbal periphery using sterile microscissors. Then make two radial relaxing incisions at the edges of the initial incision and recess the tissue posteriorly to expose the underlying extraocular muscle.

6.Isolate the muscle (superior rectus) and anchor with a suture to expose the underlying episcleral-radial vein. Special attention should be paid

Figure 1 The photograph shows the location of the episcleral radial (arrow) merging caudally with the ciliary vein. Following the incision to conjunctiva in the rat eye, superior rectus muscle is anchored by a suture (black) and pulled forward to expose the circumferential veins.

to minimize the blood loss and damage to the conjunctiva and, especially, the sclera. Aqueous-containing radial veins lie slightly deeper to the ocular muscles. The radial veins can be distinguished by their darker color. The radial veins travel on the surface of the sclera (Fig. 1).

7.The radial veins merge with the ciliary veins at about one-third the distance between the episcleral venous plexus and the optic nerve head. Near the junction, isolate the radial vein with the least damage to the sclera and put an open number-five forcep under it to create a bridge. Apply the cautery at the center of the isolated vein. The cautery tip should be about the same size as the outer diameter of the vein. The purpose of this procedure is to minimize thermal damage to the sclera during cauterization. Different material, such as wood or metal, can be placed under the isolated vein to protect from heat. As the vein is cauterized, a global mass appears on both ends of the severed vein. At the proximal portion of the cauterized vein, there is a distention of the radial vein, as the drainage is blocked. Even a

minor leak of the fluid at the cauterized end will encourage neovascularization. Incidentally, this happens in 10 to 15% of the surgeries. In each of these cases, we have observed formation of microvessels within a week. Measure IOP 2 to 3 days after surgery and check the sites of cauterization; if any are leaky, recauterize them. A successful operation requires that vessels not be reconnected. Another method is to isolate the vein, ligate it, and then cauterize.

8.In Wistar rats there are four to five major venous trunks that run posteriorly in each eye. Usually they are equidistant around the circumference of the globe. On the dorsal aspect of the globe there are two major vessels equidistant from the superior oblique and the superior rectus muscles. Near the superior rectus muscle, the radial vein is located closer to the temporal border. Similarly, there are two episcleral venous trunks located on the ventral aspect of the eye. One trunk is located between inferior rectus and inferior oblique and the other located at the inferior border of the lateral rectus muscle. Occasionally, some rats have a fifth venous trunk that is located deeper than the medial rectus. In order to elevate IOP to approximately double the value of normal IOP, it is advisable to cauterize a minimum of two and a maximum of three vessels. If all venous trunks are cauterized, the eye becomes necrotic within a week.

9.Expose the second and third venous trunks by manipulating the anchored superior rectus muscle or superior oblique muscle. The isolation procedure is similar to that described above. After the venous occlusion, flush the eyes with saline and apply antibiotic ophthalmic ointment.

10.The veins can be cauterized at the junction of the episcleral vein and ciliary vein (Fig. 2, incision 2) which leads to elevated IOP. This procedure does not lead to any change in constriction and or dilation of the pupil. No inflammation was noticed in the anterior aspect of the eye.

11.Some practitioners of this model have utilized neovascularizaton inhibitors such as 5-fluorouracil applied subconjunctively for the first few days after venous occlusion. It is highly advisable not to measure the IOP on the first day after surgery, as chances of dislodging the cauterized plug are very high. Monitoring IOP can begin within 2– 3 days, when conjuctival incisions are healed.

12.Following the occlusion of three veins, the IOP is usually in the range of 28 to 30 mmHg (normal average being 13.0 mmHg). Within 2 to 3 days following surgery, there is a small drop in the IOP. Subsequently, the elevated IOP remains high (22 to 25 mmHg) for the longest period of study (120 days).

Figure 2 Diagramatic representation of the rat eye showing circumferential venous plexus (ven. plex.) and the Schlemm canal (Schl. can.). Notice the anchoring of superior rectus (sup. rec.) muscle. Collector channels (coll. cha.) connect the Schlemm canal with the venous plexus, which subsequently exit via radial veins on the scleral surface, and carry aqueous (rad. vein. aqu.). Interruptions 1 and 2 on the scleral vessel mark the point where cauterization leading to the elevation of IOP can be made.

IV. METHODS FOR MEASURING IOP

For rats in the awake state, animals are held on a flat surface gently with minimal pressure applied to the shoulder. Press gently on the head. Apply topical anesthesia (a drop or two of proparacaine), and measure the IOP. Using Tonopen X-L (Mentor Ophthalmics) applanation is made and a reading is noted. The Tonopen probe usually requires three to four applications to the cornea before its processor is activated. Routinely, four readings are obtained and averaged and the mean values recorded. The IOP of each experimental eye is compared with the contralateral unoperated eye. For the sake of consistency and to exclude variations in IOP due to diurnal cycle, measurements should be made at one set time. We measure IOP between 9 and 10 a.m. Measurements are made twice a week in

the first 2 weeks following the surgery and once a week thereafter for the duration of the experiments.

When using Mentor 1 pneumotonometer, we mildly anesthetize the rat, with the same anesthetic used for the initial surgery. Three consecutive IOP measurements are recorded. Mentor 1 pneumotonometer provides consistent IOP preferences, and experience of each investigator will play a greater role in choosing the method of IOP measurement. In anesthetized Wistar rats the mean IOP of the normal eye is 12.5 mmHg, whereas in restrained but awake rats it is 13.5 mmHg. The range of normal IOP in Wistar rats is between 10 and 16 mmHg.

V.ADVANTAGES AND DISADVANTAGES

1.This procedure requires careful surgical manipulations and experience. In initial trials, our success rate was 25%. After realizing the formation of new vessels at the occluded end and being exceedingly careful in all surgical procedures, we reached a success rate of 80 to 90%.

2.If more than one rat is transferred to a cage, this usually leads to some aggressive behavior, which amplifies the chance of reopening the wound. It is preferable to keep rats isolated, one per cage.

3.In less than 5% of cases, corneal lesions or hemorrhaging of the globe occurred. Affected animals should be excluded from any studies.

The elevated IOP can be maintained consistently in a large number of animals for a considerable period of time. Therefore, this procedure leads to the development of an excellent experimental system for studying the mechanisms of cell death of retinal ganglion cells and the effects of various neuroprotective agents. Using this model system, we have studied the effects of neurotrophins and gene transfer to the retinal ganglion cells in glaucomatous animals and animals with optic nerve transections [3–8]. Prelabeling of all retinal ganglion cells exclusively in the retina was achieved by use of Fluorogold (Fluorochrome Inc., Englewood, CO) following protocols described in the above references.

VI. CONCLUSIONS

The procedure described above serves as a simplified means of inducing chronic elevation of intraocular pressure and experimental glaucoma in rodents. This procedure clearly allows viewing of the optic disc [9] and does not induce a strong inflammatory response within the eye [10]. This procedure has furthered our understanding of the relationship between IOP and optic neuropathy on one hand

and the evaluation of various neuroprotective agents on the other—and will continue to do so.

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