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

drain venous blood from the ciliary body to the episclera. A high injection force may also cause perfusion of the ciliary processes with the saline. We have found that using a pump for this injection, aiming for a force sufficient to inject at a rate of 50 L over 30 s, helps achieve more reproducible injections.

3.Duration of injection. We generally try to inject for 15 to 30 s. Longer times will produce excessive inflammation.

G.Reasons for Failure of Injection to Produce Elevated IOP

These generally correspond to inadequate delivery of the saline into the outflow system. They often correlate with a lack of anterior chamber deepening and lens opacity, and with poor blanching of the limbal vasculature. The most common cause of this is if the ring fails to adequately occlude all vessels leading away from the limbus. This may be due to improper placement of the ring, or anatomic peculiarities of vessels that make it impossible to adequately occlude all of these vessels. Occasionally there are large, deep veins that appear to drain directly from the ciliary body, emptying into the limbal plexus. We generally avoid these if it is not possible to occlude the vessel with the ring, as these vessels are large enough to allow much of the saline to drain away from the limbal plexus. Accidental injection of these vessels may also produce inadvertent injection of the ciliary body vasculature, which could produce ciliary body shutdown and hypotony.

IV. IOP MEASUREMENT

IOP measurement must be done in an unbiased fashion, using a method that is objective and calibrated to actual IOP [18,19]. We use the Tonopen XL tonometer for measuring IOP. This is done using topical anesthesia, taking 10 consecutive readings and then using the average as the measured IOP. Acceptable readings are those that register immediately upon contact of the tonometer tip with the cornea, using firm but not excessive force. The eye itself should be moved posterior slightly with each applanation of the tonometer.

The operator must learn to recognize valid readings and objectively ignore all those that do not meet these criteria. A very light touch with the corneal tear film may produce a low single digit reading that is not accurate. In addition, readings that occur when the tip breaks contact with the cornea (“off ” readings) and the instrument generated averages are also not accurate. The key challenge with this tonometry is that the high curvature of the rat cornea requires that the

tonometer be held exactly perpendicular to the corneal surface. This positioning is acquired with practice.

Measuring IOP in the rat eye requires considerable experience and this skill is best acquired by first calibrating the instrument on a cannulated rat eye connected to a low pressure transducer and an extra syringe to adjust the IOP, described below. With this setup, the individual learns, through immediate feedback, how to judge an acceptable reading. With time and practice, the ability to measure IOP in an unbiased fashion improves, and this can be seen by a steadily diminishing standard deviation in the measurements obtained.

Finally, we have found that general anesthetics can produce a rapid and substantial decrease in measured IOP [20]. Because of this, we now measure all IOPs with animals in the awake state, using only topical anesthesia. This allows the best understanding of the pressure to which the eye and optic nerve are actually exposed. This technique also requires considerable practice. However, Brown Norway rats are very docile and rapidly become accustomed to these measurements. Once this technique is mastered, measuring awake IOP is actually much faster, and obviously more accurate, than under any general anesthetic. Periodic “refresher” calibration sessions are useful, even after the practitioner becomes skillful with this technique. These help the practitioner maintain the ability to sense acceptable readings and help uncover any possible systematic errors that may develop. Such sessions should also be done whenever the Tonopen is serviced or a new one is purchased.

V.TONOMETER CALIBRATION

The tonometer is best calibrated in the living rat eye. The objective is to manipulate the IOP in known amounts while allowing simultaneous measurements with the tonometer. To do this, the eye of an anesthetized rat is cannulated and connected to a manifold equipped with 3-way stopcocks. The manifold is also connected to a low pressure transducer and to a Hamilton syringe for altering IOP. A third port is connected to a 60 cc syringe to allow periodic refilling of the system, as needed, while another is connected to a sphygmomanometer for calibrating the transducer and chart recorder. By keeping the system between the eye, the transducer and the chart recorder open at the same time, actual IOP can be rapidly manipulated by one operator, who also records tonometer readings made by another directly on the chart strip as they are made. With this arrangement, IOP measurements can be obtained with the tonopen and compared immediately to actual IOP, obtained through the transducer. This provides immediate feedback, helping the Tonopen operator improve technique and learn to recognize valid IOP readings in an unbiased fashion.

with all tonometers in all eyes, we have found that the Tonopen readings are less than actual pressure at the higher end of this range.

VI. ASSESSING NERVE DAMAGE AND ITS

RELATIONSHIP TO IOP LEVEL

Optic nerve damage can be assessed using many techniques. We use a qualitative grading scale of damage determined by histological examination of a crosssection of the myelinated portion of the optic nerve. This is rapid, reproducible, easily taught to others, and correlates well with actual numbers of axons lost, as determined by careful ultrastructure analyses. This grading system is described in Table 3.

When using this system to assess injury, four or five masked graders record their assessment of the grade, and the average grade is calculated as the score for that nerve. When using this system to compare the nerve injury grade to IOP level between two groups of animals, we determine the best fit line for one group (e.g., the control group) to generate a formula to predict each data point for both groups. The difference between observed and expected values for each data point are then calculated. The mean difference values for each group can then be compared by t-test to determine the significance of the differences. Alternatively, data can be linearized before statistical analysis. Sample size analyses using data like this indicate that group sizes of 20 will provide a test with sufficient power to detect a 10% difference in the number of optic nerve axons between groups.

Table 3 Grading System for Assessing Extent of Optic Nerve Cross-Section Injury

1Normal optic nerve morphology with, at most, only a few randomly scattered degenerating axons

2Densely staining, degenerating axons appear focally, with a few axonal swellings

3Numerous degenerating axons and axonal swellings appear to spread away

from the focal area. Central damage tends to exceed that in the periphery

4Numerous degenerating and axonal swellings appear throughout the nerve, in-

terspersed with apparently normal axons. Numbers of degenerating and normal axons appear to be approximately equivalent

5Degenerating axons and axonal swellings make up nearly the entire mass of the nerve, with scattered, apparently normal axons. Gliosis may appear in severe cases

VII. ADVANTAGES AND DISADVANTAGES

The major advantages of the method discussed here is the low cost of purchasing and maintaining laboratory rats. This allows relatively large numbers of animals to be used for studies, both of injury mechanism as well as for testing neuroprotection effectiveness of new agents. In this manner, it is possible to minimize difficulties imposed by individual variation in response to elevated IOP that exists among animals. The equipment needed for the injections and for making the microneedle is standard and readily available.

A second advantage of this method is that it clearly relies on obstructing aqueous outflow, which is also the primary mechanism by which elevated IOP occurs in glaucoma. This is supported by our studies of the aqueous outflow system of the rat eye, histologic documentation of angle closure in these specimens, and the increased fluctuation of IOP that we see in these animals [17]. In the latter respect, this model clearly mimics human glaucoma, in which increased IOP fluctuation is a characteristic finding in many patients.

A third advantage of this model is that these animals are easy to work with and allow us to measure IOP while they are awake. This provides a very accurate assessment of the IOP experienced by the eye throughout a given experiment. This presents a distinct advantage over obtaining IOP from animals that are under the influence of general anesthetics, commonly employed for studies of primate glaucoma models.

Fourth, experience to date strongly suggests that the optic nerve damage experienced by these animals results solely from the pressure elevation. This is supported by our observation of no optic nerve damage in eyes that received an injection but failed to develop elevated IOP. In addition, we have found a very strong correlation between the extent of IOP elevation and optic nerve damage. This validates the accuracy of our pressure measurements, as well as our method of determining optic nerve damage. In addition, we have found that specific cellular responses, such as message production for Thy-1 [21] and neurofilament protein are closely linked to the extent of pressure elevation.

Finally, this model provides a unique opportunity to understand the sequence of cellular events that follows elevation of IOP. The entire knowledge of neuropathology and cell biology of CNS injury developed in other rat CNS models is now available to the vision researcher to understand the cell biology of pressure-induced optic nerve damage. This provides a powerful opportunity to develop directed, rational strategies of neuroprotection for patients with extensive glaucomatous optic nerve damage who have progressive loss of visual function despite maximally controlled IOP.

As stated above, the primary difficulty with creating elevated IOP in these small eyes lies in the challenge of scarring the aqueous outflow pathways without damaging the immediately adjacent structures that are necessary for maintaining aqueous humor formation. Damage to the latter will often produce hypotony, rather than ocular hypertension.

A potential disadvantage, common to all methods of modeling glaucoma in rats, lies in the difficulty of obtaining accurate measurements of IOP. Due to fluctuations of IOP, a characteristic of all eyes with aqueous outflow obstruction, frequent daily measurements are essential. Measuring IOP in awake rats is an important skill that requires considerable experience. However, once this skill is acquired, we have found that measuring IOP in awake animals is faster and less traumatic for the animals than using general anesthetics.

Disadvantages of our method in particular primarily lie in the surgical skill needed to successfully cannulate these small vessels for injection and the time and patience needed to learn to measure IOP in rats. However, with practice and careful attention to detail, most researchers should be able to acquire this skill. Several laboratories have already successfully reproduced this model. However, this success requires a strong commitment, and nearly all of the individuals learning this technique have included in a personal visit, often more than once, to our laboratory to gain first-hand knowledge of these skills.

VIII. CONCLUSIONS

Our method of producing chronic elevation of IOP by episcleral vein injection of hypertonic saline is both reproducible and reliable. It combines the advantages of using laboratory rats with a simple, elegant method of scarring the aqueous humor outflow pathways, while minimizing the effects on other anterior segment structures. Our experience with this model demonstrates that the resulting pressure elevation has many similarities to that of human glaucoma, as does the pathology of the optic nerve damage. With careful assessment of IOP and the extent of optic nerve damage, it is possible to understand the relationship between pressure and optic nerve damage. This understanding will allow us to use this model to test the effectiveness of potential neuroprotective agents and improve our understanding of the mechanism of pressure-induced optic nerve damage.

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keys (Macaca mulatta), and squirrel monkeys (Saimiri sciurea), but the most work has been done in cynomolgus and rhesus.

II. MONKEY MODELS

A.Trabecular Laser

1. Historical

Most current studies are done with laser-induced glaucoma model in the rhesus and cynomolgus monkey, which was first described by Gaasterland and Kupfer in 1974 [4]. Ba´ra´ny, studying large numbers of vervet monkeys in Uganda, had noted elevated intraocular pressure (IOP) and glaucomatous cupping in an occasional monkey following apparent ocular trauma and postulated its cause as trauma-induced damage and malfunction of the trabecular meshwork (TM) (personal communication). Gaasterland and Kupfer reasoned correctly that controlled trauma and scarification confined to the TM could be induced by the application of high energy laser burns delivered by standard clinical methods, and could elevate IOP similar to human traumatic glaucoma, but without inducing other anterior segment abnormalities that could themselves impair vision and preclude evaluation of the posterior segment.

2. Method

Laser photocoagulation of the TM is conducted in the anesthetized monkey. Typically, ketamine (10 mg/kg, I.M.) or ketamine plus diazepam (1 mg/kg, I.M.) or acepromazine (0.2–1 mg/kg, I.M.) are sufficient to minimize eye movements during the procedure. If additional short duration sedation is necessary, methohexital sodium (10 mg/kg, I.M.) can be added and will usually last about 1 h. Monkeys are placed prone on a wooden, plastic, or metal board with a post on which a custom-fabricated head holder is mounted. The head holder has a metal bar that fits in the monkey’s mouth, can be adjusted to hold the head securely, and can be further adjusted to position the eye for lasering. Alternatively, an assistant can simply hold the head in the proper position. Topical anesthetic is administered and a custom-fabricated [5] mirrored Goniolens (Ocular Instruments) filled with hydroxypropyl methylcellulose (Gonak , Akorn, Buffalo Grove, IL), is placed on the eye. The custom lenses have a smaller diameter lens to easily fit through the small palpebral fissure. Also, the curvature of the lens and angle of the mirrors are different due to the smaller eye. In some larger animals, a standard adult or pediatric Goldmann-type lens can be used or a canthotomy can be performed to facilitate lens placement. However, in smaller monkeys, the procedure is greatly aided by the smaller and properly constructed monkey-specific lenses.

Table 1 Laser Parameters for Producing Experimental Glaucoma in Monkeys

A standard clinical argon laser (e.g., Coherent, model 900), or portable red diode (Iris Medical Oculight SLX) or green diode (Iris Medical Oculight GL) may be used. Typical settings are as follows: argon—1500 mW power, 500– 1000 ms duration, 50 m spot size, 50–250 burns; red diode—1250 mW, 500– 1000 ms, 75 m, 50–250 burns [6]; green diode—1000 mW, 500–1000 ms, 75 m, 50–250 burns (Table 1). Contiguous burns are placed in the mid-TM over 180—270° of the circumference of the TM in each session. Care should be taken to avoid burn spread posteriorly over the ciliary muscle, as this will increase the post-treatment inflammatory reaction and prolong the post-treatment hypotony (see below). Scarification of the anterior ciliary muscle may obstruct uveoscleral outflow and result in greater IOP elevation. These factors are very variable and hard to control, and placing the center of the burn at the junction of the pigmented and nonpigmented portion of the TM seems like the best compromise. At least one quadrant is left untreated to avoid very high IOP rises. Immediately after laser treatment, moderate iridocyclitis occurs with resultant ocular hypotony, which usually resolves within 3–4 weeks. IOP will then usually return to normal, or rise above normal if the session was effective (Fig. 1). However, additional treatment sessions are usually necessary, and the number of sessions required and their effectiveness can vary greatly between monkeys. The intensity and location of subsequent sessions can nonetheless be titrated to the target IOP. Unless one is striving for a very high IOP, it is generally advisable never to treat the entire 360° circumference with contiguous burns at any one sitting. We always leave at least 90° untreated at each sitting, although the “untreated” quadrant may have been treated at a prior session. In our experience, it is usually necessary to treat the entire circumference at least once, however the sessions are split. If no sustained pressure rise is achieved after the first session and resolution of the posttreatment inflammation, we will again treat 270°, encompassing the previously untreated quadrant. Third and subsequent sessions are titrated according to the response and the desired target IOP. A final IOP of within 10 mmHg of target can usually be obtained by varying the treatment strategy. Only rarely does one