Influence of Experimental Chronic High-Pressure Glaucoma on Age-Related Macular Degeneration in Rhesus Monkeys

Abstract

purpose. To assess prospectively whether development of age-related macular degeneration is influenced by experimentally induced chronic high-pressure glaucoma, and whether age-related macular degeneration influences the appearance of the optic nerve head in experimental chronic high-pressure glaucoma in older rhesus monkeys.

methods. The longitudinal study included 102 eyes of 52 rhesus monkeys. The total study group was divided into a group with experimentally induced unilateral chronic high-pressure glaucoma (n = 40 eyes) and a normal control group (n = 62 eyes). Additionally, arterial hypertension and atherosclerosis were experimentally induced in both study groups in a similar percentage of monkeys. Mean monkey age at the end of the study was 19.6 ± 3.1 years (range, 13–24 years). The macular region, optic disc, and retinal nerve fiber layer were morphometrically evaluated by color wide-angle fundus photographs taken at baseline and at the end of the study.

results. The degree of age-related macular degeneration, measured as number and area of drusen in the foveal and extrafoveal region of the macula, did not differ significantly between the two study groups. In the glaucomatous group, the degree of macular degeneration was statistically independent of the development of parapapillary atrophy, loss of neuroretinal rim, and decrease in the visibility of the retinal nerve fiber layer.

conclusions. Development of age-related macular degeneration in rhesus monkeys is independent of concomitant chronic high-pressure glaucoma, including the development of glaucomatous parapapillary chorioretinal atrophy. Conversely, age-related macular degeneration does not markedly influence the course of experimental chronic high-pressure glaucoma or the development of parapapillary atrophy in monkeys.

Age-related macular degeneration and glaucomatous optic neuropathy are among the leading causes of marked visual impairment in the elderly population in Western countries. ^{1 2 3 4} The purpose of the present longitudinal experimental study in rhesus monkeys was to evaluate whether experimentally induced chronic high-pressure glaucoma influences the development of age-related macular degeneration, and conversely, whether age-related macular degeneration influences the degree of glaucomatous optic neuropathy. 

Materials and Methods

The study comprised 102 eyes of 52 rhesus monkeys (Macaca mulatta; Table 1 . Selection criterion of inclusion of the eyes in the study was the availability of wide-angle color fundus photographs with the posterior pole of the fundus fully illuminated. The total study group was divided into an arterial hypertensive–atherosclerotic subgroup consisting of 33 monkeys in which systemic arterial hypertension and/or diet-induced atherosclerosis had been induced experimentally, and into a second subgroup including 19 monkeys without chronic arterial hypertension or atherosclerosis (Table 1) . The reason for inducing arterial hypertension and atherosclerosis in a subgroup of monkeys was that in humans, age-related macular degeneration and glaucoma are diseases of the elderly population, who often have systemic arterial hypertension and atherosclerosis. Atherosclerosis was produced experimentally by feeding the animals a special atherogenic diet (consisting of 1 mg cholesterol per calorie [0.8% by weight]) and 43% of total calories as fat) ⁵ continuously for many years. Chronic arterial hypertension was produced by modified Goldblatt’s procedure ⁶ and maintained for a period of years, as confirmed by serial blood pressure measurements. 

To examine the relationship between age-related macular degeneration and glaucoma, experimental high-pressure glaucoma was unilaterally produced in 40 eyes of 40 monkeys by multiple applications of argon laser to the trabecular meshwork. ⁷ Mean age (±SD) was 19.6 ± 3.3 years, mean duration of elevation of intraocular pressure was 24.0 ± 13.0 months (median, 21 months; range, 4–55 months). Before the application of laser photocoagulation, intraocular pressure was measured three times on 3 days to establish a baseline for each eye. During the follow-up period, the frequency of intraocular pressure measurements depended on the level of intraocular pressure in each eye. The higher the intraocular pressure, the more frequently it was measured: two to three times a week when the intraocular pressure was more than 60 mm Hg, weekly for intraocular pressure of 40 to 50 mm Hg, and monthly for intraocular pressures of less than 40 mm Hg. Our objective was to maintain an intraocular pressure between 30 and 40 mm Hg to mimic the clinical situation of moderate ocular hypertension. Because, with the argon laser trabecular application, it is impossible to achieve a desired level of intraocular pressure on a long-term basis, the intraocular pressure often tended to go higher than the desired levels if left alone. Therefore, to maintain our desired level of intraocular pressure (i.e., 30–40 mm Hg), we had to use ocular hypotensive drops, such as topical β-blockers and miotics, in 90% of the glaucomatous eyes. Because age was significantly different between the glaucomatous group and the nonglaucomatous control group, a second nonglaucomatous control group was formed, consisting of 43 eyes with a mean age of 19.53 ± 2.31 years (Table 2) . The percentage of monkeys with arterial hypertension, atherosclerosis, and combined arterial hypertension with atherosclerosis, respectively, in each study group did not differ significantly (P > 0.50; χ² test) between the nonglaucomatous control group and the group with experimental glaucoma. 

The study design complied with the National Institutes of Health’s and the University of Iowa’s Institutional Guidelines for the Care and Use of Laboratory Animals, and the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. All animals were examined at baseline of the study and serially thereafter under ketamine anesthesia (8–10 mg/kg body weight) and before and during follow-up of elevation of intraocular pressure in the glaucomatous group. These examinations included intraocular pressure measurement by Goldmann applanation tonometry, ophthalmoscopic examination, and stereoscopic color fundus photography. All experimental procedures were performed, and the fundus photographs were taken at the University of Iowa, Iowa City, and morphometrically evaluated at Friedrich-Alexander University, Erlangen, Germany, as has previously been described in detail. ⁸ We evaluated the areas of the neuroretinal rim and of the alpha zone and beta zone of the parapapillary atrophy (Fig. 1) and the visibility of the retinal nerve fiber layer. The degree of age-related macular degeneration was evaluated by counting the number of drusen, separated in the foveal region and in the extrafoveal region within the temporal vascular arcade. The mean size of the drusen was classified according to three grades scaled from 1 for very small to 3 for very large. The possibility of grading the severity of age-related macular degeneration on fundus photographs has been described in detail. ^{9 10} To determine the reproducibility of the assessment of age-related macular degeneration, photographs of 10 randomly selected eyes were re-evaluated five times. 

For evaluation of the statistical significance of differences in measurements between the study groups, the Mann–Whitney test was applied. For the comparison of frequencies, theχ ² test was used. For assessment of differences in the follow-up examinations, the Wilcoxon test was applied. The coefficient of variation was calculated as the ratio of the mean of the SD divided by the mean of the mean. 

Results

All measurements considered, the number of drusen increased significantly (P = 0.007) with increasing age. Comparing the glaucomatous group (40 eyes) with the age-matched nonglaucomatous control group (43 eyes), no significant (P ≥ 0.30) differences were detected in total drusen count, drusen count in the foveal and extrafoveal regions, total drusen area, drusen area in the foveal region, or drusen area in the extrafoveal region (Table 2) . In the follow-up examination, when the photographs taken at baseline and the end of the study were compared, the increase in total drusen count, foveal drusen count, and extrafoveal drusen count in the total drusen, foveal drusen, and extrafoveal drusen areas did not vary statistically (P > 0.40) between the glaucomatous group and the nonglaucomatous age-matched control group (Table 3) . 

In the whole study group, at baseline and the end of the study, respectively, total drusen count (P = 0.75 and P = 0.28, respectively), foveal drusen count (P = 0.26 and P = 0.95, respectively), extrafoveal drusen count (P = 0.26 and P = 0.08, respectively), total drusen area (P = 0.28 and P = 0.35, respectively), foveal drusen area (P = 0.42 and P = 0.77, respectively), and extrafoveal drusen area (P = 0.22 and P = 0.28, respectively) were statistically independent of neuroretinal rim area. The same held true if only the eyes of the glaucomatous group were taken into account (P > 0.25). Neuroretinal rim area decreased significantly (P < 0.001) during the period of elevation of intraocular pressure within the glaucomatous group. Within the glaucomatous group, number and area of drusen at end of the study, and the change in number and area of drusen during the period of elevation of intraocular pressure were statistically (P > 0.30) independent of the decrease in neuroretinal rim area. When the glaucomatous group was compared with the nonglaucomatous age-matched control group, neuroretinal rim area was significantly (P < 0.001) smaller in the glaucomatous group. In the normal eyes, neuroretinal rim area was independent (P = 0.96) of age. 

In the entire study group, size of beta and alpha zones of parapapillary atrophy were statistically independent of total drusen count (P = 0.28 and P = 0.94, respectively), foveal drusen count (P = 0.98 and P = 0.33, respectively), extrafoveal drusen count (P = 0.76 and P = 0.33, respectively), total drusen area (P = 0.70 and P = 0.63, respectively), foveal drusen area (P = 0.95 and P = 0.23, respectively), and extrafoveal drusen area (P = 0.61 and P = 0.95, respectively). The same result was found when the glaucomatous group (P > 0.25) and the nonglaucomatous age-matched control group (P > 0.20) were analyzed separately. Within the glaucomatous group, number and area of drusen at the end of the study, and the change in number and area of drusen during the period of elevation of intraocular pressure were statistically (P > 0.30) independent of the development and enlargement of the beta zone and alpha zones of parapapillary atrophy. 

In the entire study group and in each of the subgroups, total drusen count (P = 0.40), foveal drusen count (P = 0.65), extrafoveal drusen count (P = 0.35), total drusen area (P = 0.13), foveal drusen area (P = 0.15), and extrafoveal drusen area (P = 0.19) were statistically (P > 0.30) independent of the visibility of the retinal nerve fiber layer, with squared correlation coefficients lower than 0.04. Visibility of the retinal nerve fiber layer decreased significantly (P < 0.001) during the period of elevation of intraocular pressure within the glaucomatous group. When the glaucomatous group and the nonglaucomatous age-matched control group were compared, visibility of the retinal nerve fiber layer was significantly (P < 0.001) lower in the glaucomatous group. 

If in the statistical analysis the total study group was divided into monkeys with and without arterial hypertension–atherosclerosis, count and size of macular drusen were not significantly (P > 0.10) correlated with any of the parameters: neuroretinal rim area, size of alpha and beta zones of parapapillary atrophy, and retinal nerve fiber layer visibility. Correspondingly, the differences in size and count of macular drusen between the glaucomatous eyes and the nonglaucomatous eyes were not statistically significant (P > 0.20). 

The coefficient of variation for the reassessment of the number of drusen in the foveal region was 0.189, and for the count of the drusen in the extrafoveal region, it was 0.174. 

Discussion

The results of the present study suggest that the courses of both diseases, age-related macular degeneration and experimental chronic high-pressure glaucoma, are independent of each other. Monkey eyes with pronounced age-related macular degeneration and those without did not vary significantly in degree of neuroretinal rim loss and size and increase of beta zone of parapapillary atrophy. As a corollary, monkeys with glaucoma and monkeys without glaucoma did not vary significantly in the number and size of macular drusen. In the glaucomatous group, the severity of age-related macular degeneration was statistically independent of neuroretinal rim area and visibility of the retinal nerve fiber layer, which decreased significantly during the time of elevated intraocular pressure. However, the median and mean number and size of macular drusen tended to be smaller, although not statistically lower, in the glaucomatous eyes compared with the nonglaucomatous age-matched control eyes (Tables 2 and 3) . There were monkeys in which the number of drusen decreased after elevation of intraocular pressure (Figs. 1A 1B) , whereas in the contralateral nonglaucomatous eye the number of drusen increased. 

Although these differences can be explained by the physiologic fluctuation in the appearance of macular drusen, ¹¹ they at least suggest that the induction of glaucoma in the present study did not favor the development of age-related degeneration. This may be astonishing, because vascular insufficiency has been thought responsible for the development of both diseases. ^{12 13 14 15 16 17 18 19 20 21 22} Studies have suggested that patients with age-related macular degeneration have a reduced choroidal blood flow that may lead to the development of the disease. ^{12 13 14 15 16 17 18} Other studies have suggested that, besides elevated intraocular pressure, vascular insufficiency, including vasospasm with vascular dysregulation, is among the main risk factors of glaucoma. ^{19 20 21} Correspondingly, calcium channel blockers as vasospasmolytics have been reported to be helpful in the treatment of patients with glaucoma. ²²  

Interestingly, age-related macular degeneration and parapapillary chorioretinal atrophy in monkey eyes with glaucoma were statistically independent of each other, although both are associated with degenerative changes of the retinal pigment epithelium, and although for both of them, again a vascular pathogenesis has been discussed. ^{8 12 13 14 15 16 17 18 19 20} Previous histomorphometric and perimetric studies have shown that parapapillary atrophy in glaucomatous eyes is the clinical–histopathologic equivalent of structural and pigmentary irregularities and of loss of retinal pigment epithelium in the parapapillary region. ^{8 23} It corresponds psychophysically to relative and absolute scotomata in the visual field. Vascular insufficiency in the choroid has been thought pathogenetically responsible for the development of parapapillary atrophy in glaucomatous eyes. ^{8 20 21} In a parallel manner, age-related macular degeneration shows morphologic changes in the macular retinal pigment epithelial layer, leading to structural and pigmentary irregularities and finally to a loss of retinal pigment epithelial cells. ²⁴ Microperimetric studies have revealed that the lesions of age-related macular degeneration represent relative and absolute visual field defects. ²⁵ Pathogenetically, an impairment of the choroidal circulation has been reported. ^{12 13 14 15 16 17 18}  

Despite these similarities between age-related macular degeneration and parapapillary atrophy, no connection between them has been discovered, either in the present experimental study or in clinical investigations in patients. ²⁶ It can be concluded that parapapillary atrophy is neither a risk factor nor a protective factor for age-related macular degeneration and that they have a different pathogenesis. 

In conclusion, development of age-related macular degeneration in rhesus monkeys may be independent of concomitant chronic high-pressure glaucoma, including the development of glaucomatous parapapillary chorioretinal atrophy. Conversely, age-related macular degeneration may not markedly influence the course of experimental chronic high-pressure glaucoma or the development of parapapillary atrophy in monkeys. Although the two diseases may not influence each other in their development, the coexistence of both diseases in patients has a cumulative negative effect on visual function from a clinical point of view. The central visual field, which may be relatively spared by the glaucomatous process, is markedly affected by age-related macular degeneration. 

 Supported by Grant EY-1576 from the National Institutes of Health; in part by unrestricted grants from Research to Prevent Blindness; and by Grant SFB 539 Deutsche Forschungsgemeinschaft. SSH is a Research to Prevent Blindness Senior Scientific Investigator.

 Submitted for publication December 13, 1999; revised February 23, 2000; accepted March 15, 2000.

 Commercial relationships policy: N.

 

Table 1.

 

Composition of the Study Groups

	Total Study Group	Atherosclerotic Group	Arterial Hypertension Group	Atherosclerotic + Arterial Hypertension Group	Control Group
Monkeys/eyes (n)	52/102	6/11	12/24	15/30	19/37
At the start of the study
Age (y)	15.0 ± 4.4	14.0 ± 6.2	14.2 ± 4.4	13.7 ± 4.5	16.9 ± 2.8
Median	15	11	14	12	16
Range	7–23	7–23	7–22	7–22	12–23
At the end of the study
Age (y)	19.6 ± 3.1	18.0 ± 3.9	21.8 ± 1.6	19.0 ± 3.2	19.4 ± 2.8
Median	20	17	22	19	20
Range	13–24	13–23	19–24	13–23	13–24

 Mean age was not significantly (P > 0.35) different between the three groups with arterial hypertension or atherosclerosis. For two monkeys, one eye had to be excluded from the study due to poor quality of the fundus photograph.

Table 2.

 

Number and Relative Size of Macular Drusen

	Glaucoma Group	Age-Matched Control Group	P
Eyes (n)	40	43
Age (y)
Mean± SD	19.73 ± 3.25	19.53 ± 2.31	0.40 (NS)
Median	20	20
Range	16–24	13–24
Total drusen (n)
Mean± SD	19.10 ± 29.96	30.49 ± 41.30	0.43 (NS)
Median	4	7
Range	0–108	0–58
Foveal drusen (n)
Mean± SD	12.88 ± 22.53	15.09 ± 17.70	0.43 (NS)
Median	3	7
Range	0–96	0–58
Extrafoveal drusen (n)
Mean± SD	6.23 ± 12.37	15.40 ± 29.20	0.30 (NS)
Median	0	0
Range	0–50	0–135
Total drusen area
Mean± SD	20.51 ± 33.56	64.44 ± 115.25	0.47 (NS)
Median	5	9
Range	0–147	0–525
Foveal drusen area
Mean± SD	11.76 ± 19.51	25.98 ± 39.22	0.37 (NS)
Median	4	7
Range	0–97.50	0–144
Extrafoveal drusen area
Mean± SD	8.75 ± 18.02	38.47 ± 83.03	0.35 (NS)
Median	0	0
Range	0–72	0–405

 Mean ± SD. NS, not statistically significant.

Figure 1.

 

Fundus photographs of two rhesus monkeys before (A, C) and after (B, D) experimental elevation of intraocular pressure. Note: alpha zone (small arrows) and beta zone (large arrows) of parapapillary atrophy enlarged at the follow-up examination (B, C).

Table 3.

 

Change in Count and Area of Macular Drusen during the Study Period

	Glaucoma Group	Control Group	P
Eyes (n)	40	43
Total drusen (n)
Mean± SD	5.22 ± 20.44	8.03 ± 18.48	0.41 (NS)
Median	0	0
Range	−37–86	−3–76
Total drusen area
Mean± SD	8.55 ± 29.35	20.69 ± 59.29	0.41 (NS)
Median	0	0
Range	−74–96	−3–232
Foveal drusen (n)
Mean± SD	1.45 ± 14.77	2.81 ± 10.19	0.63 (NS)
Median	0	0
Range	−27–76	−14–40
Foveal drusen area
Mean± SD	2.35 ± 15.02	4.93 ± 10.92	0.44 (NS)
Median	0	0
Range	−50–40.50	−3–46
Extrafoveal drusen (n)
Mean± SD	3.78 ± 9.81	5.22 ± 16.02	0.45 (NS)
Median	0	0
Range	−12–36	0–62
Extrafoveal drusen area
Mean± SD	6.20 ± 17.20	15.76 ± 52.60	0.61 (NS)
Median	0	0
Range	−24–72	0–209

 Mean ± SD. NS, not statistically significant.