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

could increase uveoscleral outflow [101]. Cyclodialysis clefts can also increase uveoscleral facility in monkeys [101]. The increased uveoscleral outflow in experimental glaucoma was not sufficient to prevent the IOP rise [9].

Apparent aqueous humor flow in lasered eyes measured by fluorophotometry significantly decreased by 46% at 36–75 days after laser at a time when IOP was increased by 17.0 9.3 mmHg [9]. If real, the aqueous flow decrease may have been caused by inflammation and damage to the ciliary processes [102]. Aqueous flow returned to normal when measured at least one year after laser treatment [9].

Accumulated protein in and around the ciliary body would likely not hinder egress of aqueous humor. If anything, it would slightly increase osmotic pressure, which would draw more water, although this effect would likely be small. A breakdown of the blood-aqueous barrier should also not affect aqueous humor production [103].

However, both increased protein concentration and blood–aqueous barrier breakdown could affect fluorophotometric measurement of flow. If fluorescein binds to albumin and is quenched, the fluorophotometer would not “see” as much as is actually present. Also, the increased scatter from macromolecules could increase the fluorescence signal (by multiple scatter of excitation light and fluorescence into the measurement window, and by some scattered excitation light passing the barrier filter, which is not 100% efficient) leading to an overestimate of fluorescein concentration. It is difficult to predict how both of these processes would affect the fluorophotometric estimate of flow rate. The flow estimate is proportional to the change in mass of fluorescein in the cornea and the anterior chamber divided by the mean concentration in the anterior chamber on an interval. If fluorescein concentration in the anterior chamber is overor underestimated, flow rate would be underor overestimated respectively. Thus, quenching could increase the estimate of flow, whereas scatter might decrease it.

Breakdown of the blood–aqueous barrier would also allow more fluorescein to diffuse directly into the blood. One assumes that this loss normally accounts for 8% or less of fluorescein loss [104]. Diffusional loss in an eye with a compromised blood–aqueous barrier has not been measured, but it would likely be higher. This increased loss would lead to an overestimate of flow rate rather than an underestimate and lower flow, as Toris and Pederson observed.

One might also expect that aqueous humor flow would decrease during inflammation. A diminished flow would increase concentration of macromolecules that respond to the insult and help repair the damage, and would slow the release of any toxins into the venous blood. Reduced flow would give the TM more time to break down and remove debris and invading substances. The increased protein concentration in the aqueous humor may reflect this lower flow rate as well as breakdown of the blood–aqueous barrier, although it would be difficult to measure either independently.

Flow rate could be measured in an inflamed eye by using a large radioactive tracer, large enough not to cross the corneal endothelium or to leave the anterior chamber by diffusional pathways. Any fluorescence methods to measure flow would likely be difficult because of the increased scatter.

7. Blood Flow

Blood flow in the retina, optic nerve head, and retrobulbar optic nerve measured with tritiated iodoantipyrine did not differ between control and glaucomatous eyes [105].

8. Vitreous Sampling/Injections/Glutamate

Visualization of the area of the vitreous for injections and for sampling can be accomplished by placing the monkey on its back, dilating the pupil, and placing a well made of tubing on the cornea and filling it with gonioscopic gel. Under microscopic visualization, a 23-G needle attached to a tuberculin syringe can be inserted and 0.1–0.2 mL of vitreous withdrawn from the desired location without producing hemorrhage. The pressure in the eye can be restored by then injecting viscoelastic material into the vitreous. Drugs of interest can be delivered to the vitreous with a similar set up but using a smaller (30-G) needle and injecting, usually, up to 50 L of material.

Vitreous glutamate was shown to be elevated in experimental glaucoma of 18–32 weeks duration to concentrations potentially toxic to RGCs [106]. Anterior and posterior vitreous levels were, respectively, 59.7 7.3 and 80.3 7.8 mol/ L in experimental glaucoma and 12.3 1.5 and 12.3 2.3 mol/L in control eyes. However, in a group of 20 cynomolgus and rhesus monkeys with laserinduced glaucoma from 3–51.7 weeks, glutamate levels in the posterior vitreous sampled from near the posterior pole were no different between the experimental glaucoma and contraleral normal control eyes (experimental glaucoma 32.9 6.8 M; control 36.0 7.6 M) (Kaufman, unpublished data).

9. Neuroprotection

Memantine, a noncompetitive NMDA receptor antagonist (NMDA-type glutamatergic open-channel blocker), was administered orally to monkeys with experimental glaucoma for 15 months. Significantly less loss of visually evoked cortical potential amplitude was demonstrated in memantine-treated compared with untreated animals with experimental glaucoma [107].

Ganglion cell survival was enhanced in regions of the retina where the photoreceptors had been focally destroyed by retinal laser photocoagulation prior to the induction of experimental glaucoma in the monkey [108]. These findings demonstrate that RGCs can be protected from pressure-induced damage in the

monkey and that the outer retina may be involved in the pathophysiology of glaucomatous optic neuropathy. The monkey is a good model for such studies. Morphological changes in the photoreceptors have been observed in experimental glaucoma in the monkey [37]. These changes are remarkably similar to what has been found in chronic human glaucoma [37]. In both cases, there is selective, ischemic-like swelling of the L/M-cones (redand green-sensitive cones), perhaps due to decreased choroidal blood flow [109,110]. (The L/M-cones are numerically the dominant cone population since the S- or blue-sensitive cones make up only about 9% of all cones.) At the molecular level, preliminary studies have shown decreased production of mRNA that encodes for the L/M-cone opsins in both human glaucoma and experimental glaucoma in monkeys [111]. Rhodopsin mRNA levels, by contrast, remain relatively unaffected. Interestingly, the L/M- cones that have the greatest reduction in mRNA in monkeys may be those in the arcuate region. Independent evidence for outer retinal injury in human as well as experimental glaucoma has now been obtained using the mERG [75,112]. Delays in an early feature (N1) of the mERG seem to be most prominent in the arcuate region, as well.

10.IOP-Lowering Strategies

The experimental glaucoma monkey has been used to investigate the efficacy and mechanism of clinical and potential glaucoma therapeutic agents. Timolol (0.5%), epinephrine (2%), pilocarpine (4%), vanadate (1%), PGF2α (500 µg), and forskolin (1%) were all found to significantly decrease IOP after single or multiple treatments [113,114]. Other prostaglandin derivatives have been tested extensively in this model [115,116], including some of the newest antiglaucoma therapies (e.g., latanoprost) [116,117]. The combined effects of brimonidine, dorzolamide, or latanoprost with timolol were all found to decrease IOP more than timolol alone [118].

Trabeculectomy surgery to construct a guarded fistula between the anterior chamber of the eye and the subconjunctival space is often complicated by the ability of the surrounding tissue to scar over the fistula, thus blocking the route of exit for the aqueous and reducing the ability of the treatment to lower IOP. Strategies to prevent wound healing and the scarring process can be studied in experimental glaucoma in monkeys. Typically, mitomycin C has been used in humans for this purpose, but recently studies in monkeys using a recombinant adenovirus carrying the coding region for human p21 has shown this to be just as effective without the other adverse effects often encountered with mitomycin C.

11. Histological Findings

Light and electron microscopic examinations of the filtration angle treated by laser several months previously indicate “blunting” of the trabecular beams and

Figure 4 (A) Anterior chamber angle after laser photocoagulation on two occasions. IOP, 40 mmHg. Noteworthy are scarring of TM and adjacent tissue (arrow), obliteration of canal of Schlemm, and pigment dispersion with melanin phagocytosis within iris root and TM. (B) Anterior chamber angle of control monkey eye. Normal trabecular area (small arrow) separates anterior chamber from canal of Schlemm (large arrow). (From Ref. 4.)

scattered peripheral anterior synechiae [119]. Scarring of the TM and obliteration of the canal of Schlemm is observed in treated eyes (Fig. 4) [4]. (Kaufman, Carassa, Albert, Tamm, unpublished data).

In the retina, there is a selective loss of ganglion cells and thinning of the nerve fiber layer (Fig. 5) [4]. The large RGCs located in the midperiphery and fovea appear to be preferentially damaged in laser-induced experimental glaucoma [120,121].

In the optic nerve, optic nerve fibers larger than the mean axon diameter atrophy more rapidly in the glaucomatous eye [122]. RGCs in glaucomatous eyes undergo a pattern of degeneration that, morphologically, originates with the dendritic arbor of the cell. Both midget and parasol cells show signs of atrophy, although parasol cells appeared to be slightly more sensitive to the degenerative effects of prolonged IOP elevation [123]. Parafoveal cone loss was not found in experimental glaucoma in which there was extensive damage to RGCs [124].

Small RGCs that project to the parvocellular layers of the lateral geniculate nucleus belong to the P pathway or the color system. Large RGCs project to the magnocellular layers and belong to the M pathway or the luminance system. Rapid-phase axonal transport, measured by radioactive labeling, to the dorsal lateral geniculate body in monkeys with chronic experimental glaucoma is de-

Figure 5 (A) Macular retina and optic nerve head of treated monkey eye from Figure 4A. There is selective loss of ganglion cells with thinning of the nerve fiber layer (small arrow) and cupping of the nervehead with posterior bowing of the lamina cribrosa (large arrow). Splitting of the thin retina at the juncture with optic nerve is an artifact. (B) Macular retina and optic nervehead of control monkey eye. A thick layer of ganglion cells is present next to the fovea (small arrow). There is no cupping of the nervehead; the lamina cribrosa goes straight across the anterior optic nerve (large arrow). (A and B at same low-power magnification.) (From Ref. 4.)

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Figure 6 Part 1. Primate retinogeniculate pathway showing the regions of the LGN examined and the approximate locations of their retinal inputs. Layers 1 and 2 are the M-layers, and layers 3 through 6 are the p-layers. Ganglion cells in nasal retina project to the contralateral LGN, whereas those in the temporal retina project ipsilaterally. The nasal region of the LGN receives its afferent input from ganglion cells in the superior retina (B, C), and the temporal region of the LGN is innervated by ganglion cells located in inferior retina (A, D). (From Ref. 126.)

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Figure 6 (cont.) Part 2. Photomicrographs of cresyl violet-stained coronal sections from the right LGN of a normal rhesus monkey (A) and animals that had the pressure in one eye elevated for 2.5 (B), 8 (C), and 24 (D) weeks. In all cases, the nasal region of the nucleus is to the left, and the temporal region is to the right. Layers 1, 4, and 6 receive retinal input from the normal contralateral eye, whereas layers 2, 3, and 5 are innervated by the glaucomatous ipsilateral eye. Prolonged elevation of IOP resulted in a decrease in the size and Nissl substance within neurons receiving input from the glaucomatous eye, resulting in their pale appearance. Scale bar, 500 m. (From Ref. 126.)

creased more in the magnocellular layers than in the parvocellular layers [125]. Unilateral elevation of IOP severely affects the size, density, and number of neurons in those LGN layers receiving input from the affected eye. One study of increased IOP for 2.5–27 weeks shows a greater degenerative effect on magnocellular than parvocellular regions [126]; while another study showed equal loss of neurons in the magnocellular and parvocellular layers with experimentally increased IOP for 14 months (Fig. 6) [127]. Significant loss of CaMKII-a immunoreactivity (indicating blue-on neurons) between and within the principal layers of the LGN occurs in all ocular hypertensive monkeys with or without significant

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Figure 6 (cont.) Part 3. Low-power microphotographs of coronal sections of the left LGN from control (left) and glaucomatous (14 months) (right) cynomolgus monkeys, immunostained for parvalbumin. All six layers in the control are strongly immunoreactive for parvalbumin as indicated by numbers. There is overall shrinkage of the LGN and a decrease in immunoreactivity in parvocellular layers 4 and 6 in the glaucomatous LGN compared with control. The bar indicates 0.5 mm. (From Ref. 127.)

optic nerve fiber loss [128]. Changes in the distribution of certain neurochemicals occurs in the visual cortex of glaucomatous primates, suggesting cortical plasticity in recovery from glaucomatous visual damage [129]. Cytochrome oxidase reactivity in the neurons in the LGN was reduced to the same degree in both the P- and M-cellular layers with increasing severity of experimental glaucoma [130].

In eyes with IOP increased for 14–60 months, ganglion cells and nitrergic nerve fibers in the choroid are significantly reduced. Whole mount preparations of the retina stained for NADPH diaphorase revealed a significant reduction in positively stained amacrine cells, reduction in diameter of arterioles and changes in the staining pattern of the retinal vasculature, particularly in the perimacular region [131]. Vascular and glial changes of the retrolaminar optic nerve was studied in monkeys with elevated pressures for 1 to 4 years. Independently of axon loss, the number of capillaries remained constant or decreased only slightly. Some vessels, especially in the most severely damaged regions, were occluded.

The density of glial cells increased. In glaucomatous optic nerves, the density of alphaB-crystallin and glial fibrillary acidic protein-positive cells significantly increased [132].

Optic nerve head changes have been evaluated in the laser-induced chronic glaucoma monkey model by several methods. Optic disc cupping occurs symmetrically in early stages of experimental glaucoma but occurs predominantly in the vertical axis in later stages. Spontaneous reduction of IOP led to a reversal of cupping, which was significantly less in later stages of glaucoma [133]. Cupping in the monkey may have both a compliance component and a true tissue loss component, analogous to the situation in human infants with congenital glaucoma [134]. Lowering the IOP and re-examination may be the only way to tell. When IOP is acutely lowered, there is significantly less anterior movement of optic nerve heads with larger deeper cups than those with smaller cups [135].

Glaucomatous nerve heads showed increased labeling for collagen type IV along the margins of beams in the lamina cribrosa, due to accumulation of basement membrane–like materials. Material in the pores of the laminar beams labeled with antibodies to collagen types I, II and IV. These changes were not seen in optic nerve transected eyes [136]. In glaucomatous nerve heads, there is a major disruption of the lamina cribrosa beam structure, including a decrease in collagen density [137,138]. The destruction of collagen beams accompanies an accumulation and enlargement of collagen-associated proteoglycan filaments. Accumulation of chondroitin sulfate proteoglycans was most evident. Prominent filamentous heparan sulfate/heparin proteoglycans were also noted in thickened astrocytic and vascular basal laminae [139].

Elastin is an important component of the ECM of the lamina cribrosa that provides resiliency and deformability to the tissue. The elastic properties of the lamina cribrosa are important for buffering the constant fluctuations in IOP [140]. Abnormal elastin synthesis by astrocytes of the lamina cribrosa in experimental glaucoma optic neuropathy was shown to be specific to elevated IOP and not secondary to axonal loss, as demonstrated by comparison with optic nerve transected eyes [10].

B. Other Monkey Models

1. Optic Nerve Transection

Optic nerve transection (ONT) can be used to distinguish between elevated IOP– induced changes versus those due to the loss of axons that occurs in transection. Six animals underwent transection of the optic nerve in one eye, preserving the central vessels as verified by the absence of hemorrhage by indirect ophthalmoscopy at the completion of the transection and again several days later. Briefly, an oculoplastic surgeon performs a lateral orbitotomy [141] using pentobarbital

(15 mg/kg I.V. or 35 mg/kg I.M.) or isofluorane (1.5–2% inhalation) anesthesia. The intraconal space is entered by gentle dissection between the lateral and superior recti muscles, under 2.5 -loupe magnification. A malleable retractor is used to gently retract the globe medially. At all times, pressure on the globe is kept as light as possible and pressure is released for a few seconds every 2 to 3 mins. Under visualization with an operating microscope, the optic nerve is exposed and a sickle knife is used to make a 3 mm linear incision in the dura parallel to the nerve, as far posteriorly as practical (at least 15 mm posterior to the globe) in order to avoid damage to the central retinal artery. Dural vessels and use of cautery are avoided. Neurosurgical angled fine scissors are then used to extend the incision posteriorly several millimeters. The scissors are then inserted within the dural sheath, and the nerve transected (two cuts each two-thirds through the nerve or complete transection) under direct visualization [10]. The nerve may be transected within the sheath, or the sheath and the nerve can be transected as one unit. The ciliary ganglion is used as a marker to verify that we are sufficiently far posterior. The transection is performed approximately 12 mm posterior to the globe in rhesus monkeys. The retina is then observed by direct and indirect ophthalmoscopy, to ensure that no central retinal artery occlusion occurred. The wounds are closed and the animals treated with systemic benzathine and procaine penicillin (30,000 U/kg, Phoenix Pharmaceutical, Inc., St. Joseph, MO) daily for 5 days and systemic methylprednisolone acetate (Depo-Medrol, 1 mg/kg I.M. Pharmacia Corp, Pepack, NJ) for 3 weeks, tapering to 0.1 mg/kg over the course of 7–10 days. An alternative to the prolonged depomedrol treatment is dexamethasone (1 mg/kg, I.M., Phoenix Pharmaceutical, Inc., St. Joseph, MO) administered for 3–5 days. Analgesia is provided with buprenorphine HCl (0.1 mg/kg, I.M., Abbott Labs, Chicago, IL) every 12 h for 3 days, if necessary.

Disc pallor and loss/attenuation of the nerve fiber layer may be evident within one month (Fig. 2) although, in most cases, indirect ophthalmoscopy, slit lamp biomicroscopic funduscopy, and stereoscopic fundus photography detect no clear-cut changes in the fundus of eyes approximately 1 month after ONT compared with their presurgical baseline or with their contralateral control. Changes in the clinical appearance of the optic disc begin to be visible 5 weeks after experimental optic nerve trauma [142]. Consistent with ONT, an afferent pupillary defect develops in the ONT eye. The pupil in the transected eye was generally larger than in the control eye, and the consensual response to light was weak in some ONT eyes throughout the 4 weeks. The dilated pupil and absent or weak consensual response to light may have resulted from damage to the ciliary ganglion or the parasympathetic efferent fibers traveling with the ciliary nerves during the dissection, so that the iris sphincter muscle received reduced innervation. No systematic alteration in IOP is measured in ONT eyes, although transient elevations may occur, presumably related to orbital swelling and pressure on the globe, as can occur after orbitotomy in humans.

As mentioned previously, ONT does not elicit responses in astrocytes of the optic nerve head nor alter the structure of the tissues as seen after experimental glaucoma [10].

2. Endothelin

Endothelin-1 delivered to the perineural region of the anterior optic nerve via osmotically driven minipumps has been used to study the effect of chronic vasoconstriction and reduced blood flow to the optic nerve. Optic nerve blood flow determined by colored microspheres after 7 days for endothelin-1 administration was significantly decreased [143]. Chronic ischemia for up to 2 to 6 months resulted in diffuse loss of axons without a change in the IOP [144]. Confocal scanning laser ophthalmoscopic topographic analysis 3–5 months after the onset of ischemia showed increased cup area, cup volume, and mean cup depth. mERG after 5 months showed functional changes consistent with inner-retinal damage [145].

3. Steroids

Early attempts to induce IOP elevation and outflow facility reduction by chronic administration of glucocorticosteroids in a nonhuman primate, analogous to ste- roid-induced ocular hypertension in the human, were not successful [146]. More recently, steroid-induced ocular hypertension has been induced for the first time in cynomolgus monkeys (Fig. 7) [147]. In this model, monkeys received topical ocular drops (10 L) of 0.1% dexamethasone 3 times a day for 28 days; 45% (5 of 11) of monkeys tested developed and maintained ocular hypertension of greater than 5 mmHg, similar to the frequency in humans [148,149]. Discontinuation of dexamethasone treatment resulted in IOP returning to normal in 2 weeks. The response in responder monkeys was reproducible but nonresponder monkeys remained nonresponders. This study identified no statistically significant evidence for a link between myocilin mutations and steroid-induced ocular hypertension in both monkeys and humans.

The use of sustained-release intraocular steroid implants is awaiting testing in monkeys as another possible mode for inducing ocular hypertension in this species.

4. Particles/Gels

Intracameral injection of ghost red blood cells causes abrupt IOP elevations of 70–90 mmHg lasting 2 to 42 days [150,151]. IOP elevation of 45 mmHg to 70 mmHg for only 2 to 4 days could cause significant retinal ganglion and axonal degeneration. Cross-linked polyacrylamide (microgels), an altered form of the synthetic viscoelastic Orcolon, elevated IOP by 5–10 mmHg and decreased out-