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

ods to protect or preserve the optic nerve in glaucoma. The optic nerve head is a highly complicated structure, consisting of axons surrounded by glial cells passing through a pressure gradient, with a unique vascular supply. Because of this, such models must be created in the intact, functioning eye. Once such a model is in place, it provides a reproducible system for testing the effectiveness of potential neuroprotective compounds.

II.OVERVIEW OF METHODS IN THE CONTEXT OF NEUROPROTECTION

The primate model of trabecular meshwork sclerosis from argon laser treatment has proven highly useful over the past 3 decades [4–6]. The advantages of this approach are that the primate optic nerve anatomy is very similar to that of the human, and the pathology of chronically elevated IOP in monkeys bears many similarities to that of human glaucoma. However, the cost and difficulty of working with this model precludes its use for studies assessing the cellular response to IOP and other experimental manipulations, where individual variability necessitates the use of large numbers of animals. This is also true for studies designed to test the effect of potential neuroprotective agents.

The development of chronic IOP elevation in laboratory rats provides an alternative model, which has many advantages [7–9]. First, these animals are easy to handle, allowing frequent measurement of IOP with the animal awake. Second, their relatively low cost makes it possible to use them in studies requiring large numbers of animals. Third, a large body of knowledge based primarily on the rat already exists with regard to the cellular and molecular biology of central nervous system and optic nerve damage. This provides opportunities for understanding the cellular mechanisms of pressure-induced optic nerve damage. Finally, the anatomy of the rat optic nerve head bears several features in common with that of the human [10,11] and the pathology of elevated pressure in the model described here has many similarities to that of human glaucoma [12].

A reliable model of pressure-induced optic nerve damage must have three components: a method of creating elevated IOP; a reliable, unbiased method of measuring IOP that will provide objective understanding of the pressure experienced by the eye and the optic nerve head; and a rapid, reproducible system for assessing the resulting optic nerve damage. By using all of these features such a model can be used to develop a reliable understanding of the mechanism of pressureinduced optic nerve damage and to assess potential neuroprotective therapies.

Three basic methods of creating chronically elevated IOP in rats have been described. These include cauterization of large episcleral veins [13], laser photocoagulation of the angle vessels [14], and episcleral vein injection of hypertonic saline [7]. The former two methods are described and discussed in other chapters of this book. This chapter will describe our method of producing scarring of the aqueous humor outflow pathways using hypertonic saline injection of the episcleral veins.

In addition to the method itself, we will present our experience with measuring IOP and determining the extent of optic nerve damage produced by this technique.

III.CREATING AQUEOUS OUTFLOW OBSTRUCTION BY HYPERTONIC SALINE INJECTION OF EPISCLERAL VEINS

A. Procedure Objectives

The major objective in creating a chronic model of elevated IOP is to selectively obstruct aqueous outflow. In the rat, this is complicated by the close proximity of the aqueous outflow pathways within the angle of the eye and the blood supply to the ciliary body that comes from the major arterial circle of the iris. A too vigorous destruction of the outflow system can easily compromise the blood supply to the ciliary body and produce unwanted hypotony. Our method produces selective injury to this outflow system, based on the anatomy of aqueous humor outflow.

In the rat, the primary route of aqueous humor outflow is through the trabecular meshwork and into Schlemm’s canal (Fig. 1). Following this, it escapes through numerous aqueous collector channels and into the circular venous plexus

Figure 1 Schematic of the anatomy of aqueous humor outflow in the rat eye. Aqueous humor (dots) moves into Schlemm’s canal (SC), through trans-scleral collector channels and into the limbal venous plexus. From here, flow occurs posteriorly within radial aqueous-containing veins (which also contain blood) (AV). The arterial supply to the anterior segment is also illustrated, showing arterial supply from anterior ciliary arteries (ACA) and long posterior ciliary arteries (LPCA). These arterioles interconnect via a circular limbal artery. The iris (I) and ciliary process (CP) arterial blood supply arise from the major arterial circle of the iris. (From Ref. 15.)

Table 1 Injection Equipment

Operating microscope (at least 16 3 with 10 3 oculars) with foot-drive focus

Dumont #5 forceps (0.05 3 0.01 mm) Curved Vannas scissors (Storz)

Curved Mosquito hemostatic forceps Heavy tissue (iris) scissors

Weck cell sponges

Dumont #7, reverse action curved forceps (blunted tip) Pump (settings to microliters per minute)

Plastic (delrin) ring

Hypertonic saline (0.22 Fm millipore filter) Microneedle

that encircles the limbus. Numerous radial veins drain aqueous (and blood) away from this plexus, posteriorly within the episclera [15].

We create isolated scarring of the trabecular meshwork by injecting a sclerosing agent (hypertonic saline) into the episcleral vein in retrograde fashion. By applying a resilient plastic ring around the equator of the eye, with a gap straddling the vein to be injected, the other episcleral veins are temporarily blocked off. The injection, via a microneedle into episcleral veins, is confined to the limbal plexus, and the saline is forced into Schlemm’s canal and the trabecular meshwork. We have found that the needle insertion is best done by hand, as a micromanipulator is too cumbersome and not easily adaptable to the anatomical variations in limbal vasculature.

B.Equipment

The necessary equipment for the entire procedure is listed in Table 1. Methods of manufacturing the microneedle used for injecting hypertonic saline and a description of the plastic ring are presented in detail below.

C.Microneedle Construction

The equipment needed to construct the microneedle is listed in Table 2. The steps in constructing the microneedle consist of heating the polyethylene tubing over a bunsen burner and stretching it to an even taper. This produces a segment leading from normal diameter to a thinnest dimension that is approximately 15 to 20 cm long. A 5–6 mm section of glass microneedle (pulled out on a needle puller) is then inserted into the tip of the tubing and a drop of the glue is applied

Table 2 Equipment for Manufacturing the Microneedle

P50 tubing

10 Fl borosilicate micropipettes Bunsen burner

Epoxy glue

Needle puller (to pull micropipette to approx 50 m outer diameter and 30 m inner diameter

23-G needle (tip broken off )

1 cc syringe

Abrasive wheel (Dremel tool)

to the junction. This microneedle must have little or no taper, so that the needle will provide an effective seal of the vessel wall where it is inserted. A tapered needle, as seen with a standard micropipette, will leak if the needle is not held perfectly still within the vessel, and it is allowed to slide back and forth. A small amount of the glue will naturally migrate between the tubing and the needle, so an equal length segment of glass must be inside the tubing to prevent the glue from covering and plugging up the internal opening of the glass needle. The large end of the tubing is then swedged onto the 23-G needle shaft (with the tip broken off) and secured with a drop of glue (Fig. 2). Once this is entirely dry (24 hs), the needle is beveled using the fine abrasive wheel on a dremel tool. Water applied to the stone provides good lubrication and produces a smooth, sharp bevel. When a needle is no longer usable, it can be cut off the end of the tubing and a new one glued in its place. This allows the tubing, which can be difficult to pull to a taper, to be reused several times.

D. Plastic Ring

The plastic ring is essential for isolating the injection to the limbus. It compresses all of the veins draining aqueous away from the limbus, except the one being injected, and confines the saline to the limbal vessels and Schlemm’s canal. This ring is manufactured from Delrin stock using a mini lathe. Dimensions and appearance of the current ring are shown in Figure 3. There is a 1 mm wide groove machined in the inner aspect of the ring. This gives the ring a U shape when cut in cross section and improves its stability when applied to the equator of the eye. A small gap is cut in this ring, allowing the ring to straddle the vein to be injected. We have found that this gap is most easily cut with a razor blade, with the sides beveled away from each other. This allows better access to the vessel with the microinstruments, both for dissection and for the needle placement.

Figure 2 Microneedle and syringe construction (above), with detail of needle attachment to tubing (below). (From Ref. 7.)

E.Steps of Episcleral Vein Injection

1.A lateral canthotomy is made to improve access to the eye following a 5 s compression with a hemostat. This heals quickly and does not require suturing.

2.The best position for the ring is at the equator of the eye, and it should be placed under the dissecting or surgical microscope. The ring is spread, with a fine mosquito forceps, just large enough to slip over the equator, then slid off the forceps with a finger. This should immediately blanch out the limbal vessels (except possibly the artery) for a few minutes, due to aqueous being forced out of the anterior chamber. The plexus and episcleral veins will refill with blood once IOP falls enough to allow blood to refill the limbal plexus, either from filling of the long posterior ciliary artery, the anterior ciliary artery, or both. At this point, any possible routes of saline escape from the limbus should be identified and the ring shifted to occlude them, still leaving the injection vein unobstructed.

3.Generally, the most accessible and largest veins are located superiorly.

(a) (b)

Figure 3 Delrin ring used to isolate saline injection to the limbal vasculature and aqueous outflow pathways. (a) Appearance of the ring, both whole and when cut in cross section; (b) overall dimensions of the ring, as well as the gap cut in the ring, with beveled sides to improve access to the vessel.

Thus, the animal is best positioned on his chest, with the superior aspect of the globe pointed up. Excellent illumination is essential, particularly when working with the higher magnifications. An assistant, who is operating and timing the injection pump, can rotate the eye down to improve access to the vein, approximately at the equator.

4.The vessel wall is first exposed through a conjunctival incision. This is performed using the Dumont forceps to put traction on the conjunctiva or Tenons just over the vessel. Complete removal of connective tissue improves the chances of inserting the needle into the vessel lumen, without creating a false passage between the outside of the vessel wall and perivascular adventitia.

5.Once the vessel is exposed, the surgeon grasps the needle, bevel up, with curved, reverse action forceps. The best place to grasp the needle is at the bead of glue that forms at the junction of the needle and the tubing. The glue protects the tubing and the glass from being crushed by the forceps. It is also helpful to place a groove in the inner edges

of the forceps blades. This provides a more positive grasp of the needle and keeps the needle from pivoting, since the sides of the glue joint are usually curved.

6.Needle insertion is performed by grasping the vein with Dumont forceps posterior to the intended insertion site. This stabilizes the vein and increases its size by temporarily obstructing the venous blood as it flows away from the limbal plexus. It is best to choose a straight section of the vein to decrease the chance that the needle will be passed out of the vein accidentally.

The technique for needle insertion involves placing the tip of the needle (which is held nearly parallel to the vein) against the vein wall. By advancing the needle toward the limbus slightly, the tip will engage the vessel wall. This is usually aided by slightly loosening tension on the vein, thus allowing it to gain its maximum size. Once engaged, the needle is passed forward into the lumen of the vein. Generally, this requires increasing tension on the vein slightly, so that it will not move. The needle can then be advanced into the vessel lumen, approximately 2 to 3 mm. Often, this is accompanied by a slight reflux of blood into the needle. Tipping the needle up slightly after entering the vessel lumen will also decrease the chance of accidentally passing the tip out of the opposite wall of the vein.

7.The injection is begun once the needle is safely in the vessel. Accidental extravasation usually prevents successful cannulation and injection, although it can still be attempted. A failed injection can sometimes be remedied by finding and injecting an alternative vessel, or going to the fellow eye. Usually, the eye has to be abandoned, but injection can be reattempted in 1 to 2 weeks.

The injection is timed by noting when the pressure in the needle builds enough to fill the limbal vessels. This is the point at which timing starts. At the end of the injection time, the needle is removed and the vessel clamped with the Dumont forceps for several seconds to prevent reflux of the saline.

8.Typically, good signs of a successful injection are immediate blanching of the limbal vasculature and deepening of the anterior chamber. A temporary lens opacity usually develops due to osmotic effects of the hyertonic saline in the anterior chamber. All of these are consistent with the saline being forced into Schlemm’s canal and across the trabecular meshwork. Following the injection, the limbal vessels will refill slowly, usually beginning with the limbal artery.

9.Antibiotic ointment is instilled without suturing and the animal allowed to recover from the anesthesia. We do not typically use post-operative steroids or repeated dosing with antibiotics.

The typical postoperative course involves a mild amount of inflammation, usually manifested by slight corneal haze for a day or two. This almost always is self-limited. Following the injection, the IOP may initially show a brief elevation, but generally is normal or low for a few days. This is then followed by a gradual increase. Most eyes will demonstrate a rise in IOP by 10 days to 2 weeks after the injection. This is most likely due to gradual scarring of the trabecular meshwork and angle, producing a variable degree of angle closure. Once aqueous humor formation returns to normal, the closed angle is not able to accommodate the increased flow, resulting in elevated IOP.

We have found that, when performed on animals housed in a 12-h light: dark cycle, the scarring of the aqueous ouflow pathways produces elevated pressures that can vary markedly with the circadian cycle [16]. This can range from the low 20s to the mid 40s from the light to the dark phase of the cycle, representing a doubling of the normal circadian rhythm [17]. We have also found that placing the animals in a constant, low level light environment will increase the mean IOP to approximately 27 mm Hg and minimize the extent of circadian fluctuation. In this situation, the typical range of mean IOP following the procedure varies from the low 30s to low 40s, as determined by the Tonopen in awake animals (see below).

The success rate of producing an elevated pressure with this method is quite high. Review of a recent group of 40 eyes shows that pressures were significantly elevated in 33 eyes following a single injection. A second injection is always possible in eyes that fail to respond, and it can increase the success rate even further [7].

F.Factors That Influence the Success of a Given Injection in Producing Elevated IOP

Three main factors influence the success of saline injection. These factors appear to affect each other, and experience, correlated with success in producing elevated IOP, is required to find the proper combination of saline concentration, injection force, and duration of injection.

1.Molarity of the saline solution. We have found that a 1.75–2.5 M solution produces a reliable increase in IOP. Concentrations higher than this will result in excessive inflammation and, often, long-lasting hypotony.

2.Force of injection. This influences the ability of the saline to gain access to the trabecular meshwork. Too low a force will fail to perfuse the angle structures sufficiently. An excessive force may produce too much inflammation, with a result similar to that of using too high a concentration of the saline. This may be due to subtle interconnections that exist between the episcleral vasculature and emissary veins that