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

Figure 1 IOP and its control following argon laser treatment of the TM in one eye (experimental glaucoma) of rhesus monkey 1035. This monkey was unusual in that only one laser session was required to elevate the IOP; usually two or three sessions are required. Topical Timoptic-XE 0.5% was given once daily or every other day where indicated to maintain IOP at the desired level. Lowering IOP pharmacologically (with Timoptic, Alphagan, Trusopt and PGF2α-1-isopropylester) for 1 week prior to sacrifice resulted in no change in the surprisingly modest disc cupping (experimental glaucoma C/D 0.4; Control C/D 0.2), thus suggesting that there was actual loss of tissue, rather than simply a pressure-induced mechanical backward bowing of the elastic lamina cribrosa. The axonal loss for experimental glaucoma was mild (0.28). Reproduced with permission. (From Ref. 10.)

encounter an animal in which the IOP never rises with multiple treatments. Typically, 2–3 treatment sessions are required to achieve a sustained IOP rise. Some animals may need to be re-treated after a sustained period of IOP elevation that is then followed by a gradual decrease in IOP.

If IOP exceeds the desired level or if the monkey displays any signs of discomfort (usually when IOP exceeds 60 mmHg by Goldmann applanation tonometry), standard antiglaucoma medications can be administered once or twice a day. Monkeys can be trained to enter an “iron maiden” (a modified squeeze cage) that has been adapted to immobilize the conscious monkey while tilting it to a vertical position so that the eyelid may be retracted and the medications dropped onto the cornea. Alternatively, for uncooperative monkeys, 5–10 mg/kg ketamine I.M. can be given for sedation and the medications then administered.

However, some monkeys may show a decrease in appetite with frequent anesthesia so this must be assessed on a case-by-case basis. The medications that have been used successfully alone or in combination include Timoptic-XE (0.5% timolol maleate in gel-forming vehicle, Merck & Co, West Point, PA); Alphagan (0.2% brimonidine tartrate, Allergan, Irvine, CA); Trusopt (2% dorzolamide hydrochloride, Merck); and PGF2α-1-isopropylester (2 µg in 5 µl saline, Caymen Chemical Co, Ann Arbor, MI) or Xalatan (0.005% latanoprost, Pharmacia Corp, Peapack, NJ). If necessary, acetazolamide (5 mg/kg, Ben Venue Laboratories, Bedford, OH) I.M. has been given once or twice daily.

3. Clinical

IOP under ketamine anesthesia is monitored weekly or more frequently if medications are being implemented to target a specific pressure range. IOP is measured with a minified Goldmann (Haag-Streit, Ko¨niz, Switzerland) applanation tonometer [7]. Occasionally, these are backed up by measurements with a Tonopen XL (Mentor O&O, Norwell, MA) if corneal edema or neovascularization, or head and eye movements under ketamine anesthesia preclude readings with the Goldmann [8]. Others have also used pneumotonometry for IOP determinations [9]. In some cases, IOP can fluctuate greatly. Slit lamp biomicroscopy of the anterior and posterior segments, including stereoscopic optic disc evaluation with a fundus lens are performed once a month when IOP has stabilized. The size, shape, and pallor of the optic disc, the cup-to-disc ratio, and the retinal nerve fiber layer are evaluated. The pupil in the lasered eye usually becomes dilated, probably due to damaging the parasympathetic motor nerves to the iris by the laser treatments (energy spread to the anterior ciliary muscle through which the nerves travel to reach the iris). Iridolenticular adhesions may develop independently of corneal changes or duration of IOP elevation. Anterior and posterior synechiae are often observed. However, IOP may be elevated even if most or all of the angle remains open and there is no papillary block. The TM is invariably heavily pigmented. As the duration of IOP elevation becomes longer, some animals may develop corneal edema followed, in some cases, by neovascularization of the cornea.

Cupping of the optic nerve head, with posterior bowing of the lamina cribrosa is typical [4]. Optic disc cupping occurs more rapidly in the monkey than in the human. For a given monkey, this is dependent on the IOP elevation and duration. However, results can fluctuate between monkeys. In order to accurately assess cupping due to loss of neural/glial tissue versus posterior bowing of the elastic monkey lamina, the IOP should be lowered. This can be done with the combinations of pharmacological agents mentioned above to control IOP. Alternatively, I.V. mannitol (1.5 g/kg) can be administered over a 30 min period. If

IOP is still too high, give I.V. acetazolamide (5 mg/kg) and wait 30 min more. If IOP is still greater than 20 mmHg, give I.V. methohexital (5 mg/kg) or pentobarbital (10 mg/kg). If IOP after mannitol is still greater than 40 mmHg, give both acetazolamide and methohexital or pentobarbital. Fundus stereo photography (Topcon TRC 50IA fundus camera (Topcon America Corporation, Paramus, NJ) can be used to document the time course of the changes. An example of glaucomatous damage is shown (Fig. 2) after 4 months of elevated pressure where the entire optic disc surface is excavated; the disc margin is undermined; there is substantial peripapillary atrophy; and the retinal nerve fiber layer is substantially attenuated [10]. Other instrumentation used to image the optic nerve in humans produce results that are more difficult to interpret in the monkeys due to a lack of corrections for the smaller eye, steeper corneal curvature, uncompensated corneal birefringence, and poor ocular fixation. Confocal scanning laser ophthalmoscopy (TopSS Topographic Scanning System, Laser Diagnostic Technologies, Inc., San Diego, CA) [11,12] and Heidelberg retinal tomography (HRT; Heidelberg Engineering, Heidelberg, Germany) [13,14] can be used for optic disc and peripapillary retinal contour analysis. Scanning laser polarimeter (GDx, Laser Diagnostic Technologies, Inc., San Diego, CA) to assess retinal nerve fiber layer thickness may be used for generalized qualitative evaluation of differences between the eyes of a given monkey. Adaptations of the HRT and GDx are being made for more accurate and quantitative use in the monkey and even for rodents [15] (R. Weinreb, personal communication). Optical coherence tomography (OCT) is under study for evaluation of retinal nerve fiber layer in glaucoma in humans [16– 18] and may be applicable to monkeys. For all these photographic/imaging procedures, pupils are dilated with 2.5% phenylephrine HCl (Mydfrin, Alcon, Ft. Worth, TX) and 1% tropicamide (Mydriacyl, Alcon). Anesthesia for these procedures is ketamine (10 mg/kg, I.M.) acepromazine (0.2–1 mg/kg I.M.), methohexital sodium (15 mg/kg, I.M.) if needed to eliminate eye movements.

4. Perimetry

Behavioral perimetry in monkeys shows the same intersubject variability in the effects of elevated IOP on visual field sensitivities that are common with hightension glaucoma or ocular hypertension patients [19]. Perimetry regimens with either white or monochromatic stimuli are not useful predictors of ganglion cell loss until a substantial proportion of cells have died. The variance in ganglion cell loss is large for mild defects, which would be diagnostic of early glaucoma, and for visual field locations near the fovea, where sensitivity losses occur relatively late in the disease process [20]. Monkeys with laser-induced glaucoma exhibit the same type of Humphrey visual field defects as do glaucomatous humans.

Figure 2 (A) Fundus photographs of experimental glaucoma and control (B) eyes of a rhesus monkey at 4.5 months after unilateral laser-induced IOP elevation (experimental glaucoma 40 mmHg; control 20 mmHg). The entire experimental glaucoma disc surface is excavated, the disc margins are undermined (white arrowhead), there is substantial peripapillary atrophy (white arrow), and the retinal nerve fiber layer (asterisks in control) is substantially attenuated (white star). (C) Fundus photographs of ONT and of control (D) eyes of a rhesus monkey, 3.5 wk after transection, 1 week prior to sacrifice. Note normal retinal vasculature, absence of retinal or vitreous hemorrhage, and presence of pallor but absence of disc cupping and very early, mild attenuation of nerve fiber layer (absence of striations emanating from the temporal disc margin) as a result of ONT. (Reproduced with permission from Ref. 10.)

Spectral sensitivity defects occur in experimental glaucoma similar to those found in patients with glaucoma. Elevated IOP resulted in short wavelength sensitivity losses. The optimum condition identifying the greatest short wavelength sensitivity reduction is a yellow background of moderate intensity. In the early stages of experimental glaucoma, the cone mechanisms and the rod mechanism typically showed decreased test and field sensitivities. In advanced stages of experimental glaucoma, the largest sensitivity losses are in the longer wavelength, red-green opponent mechanisms [21].

5. Electrophysiology

Objective, noninvasive electrophysiological measures of retinal ganglion cell (RGC) function may be of value for monitoring glaucomatous damage in humans and for providing comparable functional measures in experimental animal models.

Ganzfeld ERG. The a- and b-waves of the full-field ERG reflect a massed electrical response from the entire retina that is dominated by photoreceptor and bipolar activity with little apparent contribution from the ganglion cell layer [22]. Most ERG studies of human patients with glaucoma found no correlation between visual loss and scotopic or photopic full-field ERG [23–26] perhaps due to variability in severity, disease progression, and treatment regimens. This is of less concern in laser-induced experimental glaucoma in nonhuman primates. In agreement with human investigations, several studies of experimental glaucoma have found no effect on the scotopic a- and b-waves of the traditional full-field ERG [27–31].

However, some recent investigations in human glaucoma patients do find alterations of certain features of the a- and b-waves [32–36] suggesting that layers of the retina distal to the ganglion cells may be involved in glaucoma. Support for outer retinal changes are recent studies showing swelling of cell bodies in the outer nuclear and outer plexiform layers in both human and experimental glaucoma [37].

Oscillatory Potentials. The dark-adapted full-field ERG elicited by a bright flash can be filtered to reveal time-locked high frequency wavelets riding on the a- and b-waves known as oscillatory potentials. Oscillatory potentials are thought to be generated within the inner plexiform layer [38]. Several studies have found that oscillatory potentials are reduced in human glaucoma [34,39,40]. Investigations of oscillatory potentials in nonhuman primate models of glaucoma are mixed [28,41].

Photopic Negative Response. Another feature of the full-field ERG that appears to be affected in glaucoma is a negative wave that follows the b-wave recorded under photopic conditions. In some studies the photopic negative re-

sponse was found to be greatly reduced in nonhuman primate experimental glaucoma [31,42], and in human patients [26,43,44] whereas other investigators found no consistent changes in patients with advanced glaucoma [45].

Scotopic Threshold Response. The scotopic threshold response is the dark-adapted ERG to a very dim flash of light that first appears as a cornea negative wave and peaks approximately 200 ms after stimulation [46]. On the basis of pharmacologic manipulations, the scotopic threshold response is thought to reflect proximal, or inner retinal activity [46]. However, the scotopic threshold response is not abolished in humans with long-standing optic atrophy or in cats with optic nerve transection (ONT) [47], suggesting that a substantial part of the scotopic threshold response may be due to amacrine cell activity. The scotopic threshold response in human glaucoma patients is highly variable and generally unchanged in humans with glaucomatous field loss [48]. In contrast, the scotopic threshold response is greatly altered or abolished in nonhuman primate experimental glaucoma [28]. Frishman [28] suggests the scotopic threshold response may receive contributions from both rod amacrine (a short-latency component) and ganglion cells (a long-latency component). There appears to be marked species differences in the relative contribution of the two cell types.

Pattern ERG. The pattern pattern ERG is a small amplitude ERG response to pattern “reversal” (e.g., exchange of the black and white checks of a chessboard pattern) or onset of a pattern in which there is no global change in luminance. It is not a full-field stimulus; rather the pattern ERG is the summed response from a large area of the central field. Many studies have shown that the pattern ERG reflects ganglion cell activation [22,49,50].

The pattern ERG is reduced in human diseases of the optic nerve [51], including glaucoma [52–63]. Changes in the pattern ERG also have been reported in ocular hypertension [57,58,64–70]; see Korth [71] for a review of the pattern ERG in glaucoma. The pattern ERG has also been examined in animal models of glaucoma. Reduction of pattern ERG amplitude is correlated with the degree of disc cupping in monkeys with chronic ocular hypertension [27,41]. The pattern ERG is not used extensively in the diagnosis and monitoring of disease progression, perhaps due to the technical difficulties with recording small amplitude signals and the finding that substantial peripheral loss must occur before the pattern ERG is affected [71].

In addition, some of the inconsistencies among these studies have been attributed to differences in the relative insensitivity of the full-field ERG to ganglion cell loss and the inability of the mass ERG response to reflect localized or patchy loss of function that is characteristic in glaucoma. Several novel electrophysiological techniques have been introduced that appear promising for detecting and monitoring glaucoma because they are thought to reflect inner retinal activity.

Multifocal ERG (mERG). The mERG technique derives the local electrical response of many small patches of the retina (typically 103) using a sparse binary m-sequence cross correlation technique introduced by Sutter [72,73]. The technique has attracted considerable attention in the study of glaucoma for two reasons. First, it can image the patchy, localized areas of retinal dysfunction that characterize the glaucomatous visual field loss. Second, the binary m-sequence method permits a single recording to be analyzed in first-order (linear) and higherorder components, or “kernels.” Higher-order kernels reflect complex nonlinear dynamic responses that are presumed to originate in layers of the retina proximal to photoreceptors, possibly including ganglion cells.

Chan [74] found reductions in the amplitudes of both the firstand secondorder kernels in humans with glaucoma. Delays in mERG waveforms also have been found in primary open angle glaucoma [75–77]. Amplitude differences were not evident in these studies.

The relationship between the mERG and glaucomatous field losses as measured by standard automated perimetry has not been established [78–80].

The mERG has also been examined in experimental glaucoma. In normal eyes of rhesus monkeys, the first-order mERG response kernel contains more prominent oscillatory potentials ( 60 Hz) than in comparable recordings in humans [81,82]. The rhesus macaque mERG oscillatory potentials are larger in central than in peripheral locations. The second-order kernel also contains larger oscillatory potentials than seen in human recordings. In addition, there are prominent naso-temporal variations in the oscillatory potentials of both kernels; responses near the optic nerve head have larger amplitude oscillatory potentials than temporal locations [81,82]. One approach to reversibly simulating glaucoma in nonhuman primates is to pharmacologically suppress sodium-based spiking activity of the inner retinal with tetrodotoxin and NMDA. Four effects on the mERG have been noted following intravitreal administration of tetrodotoxin and NMDA: (1) a marked increase in the amplitude of the mERG first and second order responses; (2) retinotopic changes, with the largest increased amplitudes occurring in the foveal region; and (3) a reduction of the prominent oscillatory potentials; and (4) removal of the naso-temporal variations. These findings differ from human glaucoma studies that found no effect or reduced amplitude foveal responses.

In nonhuman primates with advanced experimental glaucoma, Frishman found the effects were similar to those produced by suppressing inner retinal activity with tetrodotoxin and NMDA [82]. In addition, the later portion of the first order kernel waveform was altered, lacking a dip after the large positive wave, similar to the changes seen in the photopic negative response. mERG changes increased over the time course of glaucoma and were more diffusely distributed across the visual field [82]. Another study of nonhuman primates with

IOPs elevated for over 16 months, and histologically documented ganglion cell loss, found the amplitude of both first and second order mERG responses were attenuated and were highly correlated with RGC density (Fig. 3) [29,30]. A similar effect was found in nonhuman primates in the early stages of experimental glaucoma [83]. mERG after 4 weeks of elevated IOP ( 25 mmHg) showed an attenuation of the negative waveform complex at 40–70 ms following the prominent positive (P1) wave. A similar alteration of these waves following ONT suggests this waveform feature of the mERG, to some extent, reflects ganglion cell activity [83].

Visual Evoked Potential (VEP). Amplitude reductions and delays in peak latency of the VEP elicited by patterned stimuli have been reported in human glaucoma [52,60,84–94]. In addition, distortion of waveforms, rendering them unscoreable using traditional metrics, were found in a high proportion of glaucoma patients [52,89]. The correlation with standard automated perimetry defects has been attempted but has yielded poor results [71,84]. Pattern VEP has been considered less sensitive than standard automated perimetry for the early detection of glaucoma.

Recently, a multifocal VEP (mVEP) method has been introduced that appears to correlate well with field loss [80,95]. When all factors are taken into account, the mVEP appears to capture standard automated perimetry field losses with exceptional accuracy. Its role in early detection remains to be established. The mVEP has not been reported in studies of experimental glaucoma.

6. Aqueous Humor Dynamics

Outflow facility can be measured by constant pressure perfusion [96], Schiotz tonography [97,98], pneumotonography [99] and by a fluorophotometric technique [9,100]. In vivo constant pressure outflow facility measurement decreases from prelaser baseline of 0.33 L/min/mmHg–0.75 L/min/mmHg, to 0.02 L/ min/mmHg–0.11 L/min/mmHg post-laser [4]. Toris also showed tonographic outflow facility was decreased by 71% at 36–75 days and fluorophotometric outflow facility was decreased by 63% at least 1.7 years later [9]. Outflow facility by constant pressure perfusion in a group of 16 cynomolgus monkeys at 1–5 months post laser, when IOP averaged 32.9 3.5 mmHg in lasered versus 17.0 1.2 mmHg in control eyes, was 0.049 0.012 L/min/mmHg versus 0.424 0.038 L/min/mmHg, respectively (Kiland J, Kaufman PL, unpublished data). Uveoscleral outflow measured with tracers or calculated was increased at least one year after laser treatment [9]. This could be partially due to the persistent low-grade inflammation that may be associated with chronic endogenous prostaglandin release. Also, artificial openings (small cyclodialysis clefts) between the scleral spur and the ciliary body may have resulted from the laser burns, which

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Figure 3 Part 1. First order mERG responses from both eyes of a monkey with severe ocular hypertension. (A) Normotensive (OS) eye. Trace array of 61 responses, each of which represents the local retinal response corresponding to the stimulus element at that location in the stimulus field. The central seven traces (shaded) represent responses from macular retina extending to approximately 8° retinal eccentricity. The surrounding 54 traces (unshaded) represent responses from perimacular retina extending from approximately 8 to 25° retinal eccentricity. Calibration bars 200 nV, 100 ms. Note that amplitude of individual traces is expressed in units of volts because each trace represents the response from retinal areas of the same size. (B) Macular and perimacular responses obtained by summation of either the central seven or surrounding 54 response traces, respectively, from (A). Amplitude measures for the five peaks of the macular response were made as indicated. Calibration bars, 5 nV/degree squared (deg2) and 25 ms. Note that macular and perimacular response amplitude is expressed as response density (volts/unit retinal area), because these responses reflect retinal stimulus areas of different size. (C) Sixty-one response array obtained from the hypertensive (OD) eye of the same animal whose responses are shown in (A) and (B). Calibration as for (A). (D) Macular (top trace) and perimacular (bottom trace) responses obtained from (C); calibration as for (B).

Part 2. Second-order mERG responses from the same recordings that produced the first-order responses shown in Part 1. (A) As for Part 1A. (B) As for Part 1B. A single peak-to-peak measure of second-order macular response amplitude was made as indicated. Calibration bars, 2.5 nV/deg2 and 25 ms. (C) As for Part 1C. (D) As for Part 1D.

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Part 3. Comparison of macular and perimacular responses from the hypertensive (OD, light traces) and normotensive (OS, heavy traces) eyes shown in Parts 1 and 2. Histological analysis showed that normalized (OD/OS) perifoveal RGC density in the hypertensive eye was 0.11. First order response calibration bars, 5 nV/deg2 and 25 ms; second-order calibration bars, 2.5 nV/deg2 and 25 ms. (From Ref. 30.)