Aqueous Humor Dynamics and Trabecular Meshwork and Anterior Ciliary Muscle Morphologic Changes with Age in Rhesus Monkeys

B’Ann True Gabelt,1 Johannes Gottanka,2 Elke Lu¨tjen-Drecoll,2 and Paul L. Kaufman1,3

PURPOSE. To determine in rhesus monkeys the age-dependence ntraocular pressure (IOP) in normal healthy humans remains of uveoscleral outflow (Fu) and morphology of the trabecular Irelatively stable or slightly increases with age in many West- meshwork (TM) and anterior ciliary muscle (CM). ern populations,1–4 while decreasing with age in the Japanese population.5 Studies of aqueous dynamics in humans have METHODS. Intraocular pressure (IOP) was measured by Gold- shown that, with age, aqueous flow,2,6–8 mann applanation tonometry in monkeys under ketamine an- tonographic outflow facility,2,6,8,9 and anterior chamber volume all decrease,3,8 esthesia. After anterior chamber cannulation under pentobar- whereas episcleral venous pressure is unchanged.2,8 Until re- bital anesthesia, aqueous humor formation (AHF), anterior cently, the only measurements of uveoscleral outflow (Fu) in chamber volume, trabecular outflow, and Fu were determined humans were obtained in a few eyes with ocular melanomas.10 isotopically. The CM and TM were examined by light and Fluorophotometric estimates of outflow facility in humans electron microscopy. have shown a decrease with age.8 RESULTS. IOP increased significantly with age in monkeys Similarly, IOP in normal, healthy, free-ranging rhesus mon- aged 3 to 29 years. AHF and anterior chamber volume were keys remains relatively constant with age, after an initial juve- unchanged. Fu was decreased, and trabecular outflow in- nile hypertensive phase.11–13 Outflow facility, as well as its creased in monkeys aged 25 to 29 years compared with the response to intracameral pilocarpine, declines with age.14 Mor- remaining monkeys. Morphologically, there was a significant phologic changes in the ciliary muscle (CM) in aging rhesus increase in the thickness of the elastic fibers of the trabecu- monkeys, including increases in the number of pigmented cells lum ciliare covering the anterior tips of the CM, and an between the CM bundles with the anterior longitudinal region increase in extracellular material between the muscle tips. being affected last, suggests that uveoscleral outflow (Fu) may 15,16 The number of TM cells decreased with age, whereas the also be affected. amount of fibrillar material and sheath-derived plaques in- In the present study we determined in rhesus monkeys the creased. This increase was less pronounced in the middle correlation with age of Fu, rate of aqueous humor formation filtering portion of the cribriform region than in the anterior (AHF), rate of trabecular outflow, and anterior segment vol- and posterior portions. ume. We also examined the possible role of previously unstud- ied age-related morphologic changes in the anterior portion of CONCLUSIONS. The decline in Fu in very old rhesus monkeys the CM and the trabecular meshwork (TM) that could alter with normal IOP parallels that seen in normotensive aging uveoscleral and trabecular outflow. humans. This may be correlated with thickening of the elastic fiber sheath in the CM tips in addition to other morphologic changes. The TM findings are analogous to those in the aging METHODS human eye and are consistent with the age-related decrease in outflow facility reported in both humans and monkeys. (Invest Animals and Anesthesia Ophthalmol Vis Sci. 2003;44:2118–2125) DOI:10.1167/ On the day before or on the same day as Fu studies, IOP (Goldmann iovs.02-0569 applanation)17 and slit lamp biomicroscopy were performed in monkeys that were under intramuscular ketamine anesthesia (10 mg/kg). For Fu studies, 24 rhesus monkeys (Macaca mulatta), ages 3 to 29 ϳ 1 years (human equivalent, 8–73 years) were anesthetized with intra- From the Department of Ophthalmology and Visual Sciences and muscular injection of ketamine followed by intravenous administration the 3Wisconsin Regional Primate Research Center, University Of Wis- of pentobarbital (10–15 mg/kg; maintenance dose of 5–10 mg/kg as consin, Madison, Wisconsin; and the 2Department of Anatomy, Univer- sity Erlangen-Nu¨rnberg, Erlangen, Germany. needed, usually every 1 to 1.5 hours). A femoral artery was then This is Wisconsin Regional Primate Research Center publication cannulated for subsequent blood sampling. Eight additional animals number 42-012. were used for determinations of blood-equivalent albumin space (de- Supported by National Eye Institute Grant EY02698 (PLK); an scribed later). unrestricted departmental grant from Research to Prevent Blindness Quantitative morphologic studies were conducted on CM and TM and physician-scientist awards (PLK); Grant RR00167 from the Wiscon- from 12 other rhesus monkeys, aged 3.5 to 34 years, killed at the sin Regional Primate Research Center; and Grant SFB319 from the University of Wisconsin-Madison and subsequently sent to the Univer- Academie of Science, Mainz, Germany (EL-D). sity of Erlangen. Different structural parameters from monkeys in the Submitted for publication June 11, 2002; revised November 12, current study have been reported earlier15 (Table 1). 2002; accepted November 17, 2002. Disclosure: B.T. Gabelt, None; J. Gottanka, None; E. Lu¨tjen- Drecoll, None; P.L. Kaufman, None AHF, Flow to Blood, Fu, Anterior Chamber The publication costs of this article were defrayed in part by page Volume, Blood-Equivalent Albumin Space

charge payment. This article must therefore be marked “advertise- 20 ment” in accordance with 18 U.S.C. §1734 solely to indicate this fact. AHF was measured by dilution of radioiodinated monkey albumin 19 Corresponding author: Paul L. Kaufman, Department Ophthalmol- dissolved in Ba´rańy perfusate according to the method of Sperber ogy and Visual Sciences, F4/328 CSC, 600 Highland Avenue; Madison, and Bill.21 Each anterior chamber was cannulated temporally with WI 53792-3220; [email protected]. three 23-gauge needles: outflow on top, pressure in the center, inflow

Downloaded from iovs.arvojournals.org on 09/30/2021 IOVS, May 2003, Vol. 44, No. 5 Aqueous and Trabecular Morphologic Changes with Age 2119

TABLE 1. Monkeys Used for Morphology Studies

ID Germany Age (y) Sex Health Fixative Pretreatments

149/83 3.5 M Healthy Ito’s* ϩ 3% Pilo (1/8) or 3% Atr (1/8) None 101/86 5 F Healthy Ito’s ϩ 1% Atr 1% Atr 106/84 8 Ito’s None 74/92 9 F Healthy 10% Para ϩ 10% Pilo or 1% Atr 10% Pilo OD, 1% Atr OS 56/85 9 F Progressive paresis Perf ﬁx with Ito’s; Ito’s ϩ 10% Pilo 1% Atr OD, 10% Pilo OS or 1% Atr 137/89 10 F Chronic wasting PLP None amyloid liver 39/86 20 F Endometriosis Ito’s ϩ 10% Pilo or 1% Atr 10% Pilo OD, 1% Atr OS 176/82 25 Declined with age Ito’s None 79/85 26 M Decline; shigella Ito’s None 106/83 30 F Decline with age; Ito’s Carbachol iontophoresis OD iridx OD 43/85 33–36 F Near death Perf ﬁx with Ito’s; Ito’s ϩ 10% Pilo 10% Pilo OD; 1% Atr OS or 1% Atr 95/83 34 M Decline with age Ito’s None

Atr, atropine sulfate; Pilo, pilocarpine HCl; PLP, periodate/lysine/paraformaldehyde. * Ito’s ﬁxative.18

on the bottom. The inflow and outflow needles were connected to a All experiments were in accordance with the ARVO Statement for closed circuit containing a peristalic pump and a loop that passed the Use of Animals in Ophthalmic and Vision Research and the Uni- through a well detector (Canberra, Meriden, CT). The circuit for the versity of Wisconsin institutional animal care and use committee. right eye contained I-125 albumin solution, and the circuit for the left The fixed specimens were cut into 1- to 2-mm wedges encompass- eye contained I-131 albumin solution, each at a concentration of ing cornea, TM, and iris and ciliary body posterior to the ora serrata and approximately 5 ϫ 106 counts per minute (cpm)/mL. Cold albumin embedded in Epon. Semithin sections (1 ␮m) and ultrathin sections was added to a final concentration of 0.1%. The circuit volume was (60 nm) were cut with an ultramicrotome (Reichert, Vienna, Austria). 1.231 mL, and the circulation rate was 150 to 200 ␮L/min. There was Semithin sections were stained with toluidine blue, and ultrathin sec- no additional infusion of isotope solution during the experiment. The tions were counterstained with lead citrate and uranyl acetate. Semi- entire experiment was run at spontaneous IOP, which was approxi- and ultrathin sections of the TM and CM tips were investigated in four mately 10 mm Hg for monkeys under pentobarbital anesthesia. different sectors of the circumference of the eye. After cannulation of the eyes and a 5- to 10-minute equilibration Quantitative evaluation was performed in semithin sagittal sections period at spontaneous IOP, the mixing pump was started. Radioactivity of the four different portions of the circumference. The number of in the well detector loop was measured for 1 minute with a multichan- trabecular cells (defined as cells with nucleus in the section) were nel analyzer (series 30; Canberra) every 5 minutes for the next 120 counted in the entire meshwork in that section. In ultrathin sections of minutes. Blood samples of 1 mL were taken from the femoral artery the chamber angle, the area occupied by sheath-derived plaques (the every 10 minutes beginning at 40 minutes and the radioactivity mea- sheaths of the elastic fibers) and fibrillar material under the inner wall sured with a gamma counter (Packard Instrument Co., Meriden, CT). of Schlemm’s canal was measured. Measurements were performed Anterior chamber volume was calculated from the initial decrease in circuit isotope concentration. AHF was determined from the slope of the disappearance of radioactivity from the circuit. Flow into the bloodstream (flow to blood) was calculated from the radioactivity recovered in the blood samples (corrected for the decreasing concentration in the anterior chamber) and the blood-equivalent albumin space. Fu was calculated as the difference between AHF and flow to blood. Blood-equivalent albumin space was determined on a separate occasion in six of these same animals, in five additional animals aged 4 to 22 years, and in three of these same animals plus three additional animals aged 24 to 30 years, by constantly infusing I-125 monkey albumin into the saphenous vein and sampling blood from a femoral artery at 10- or 15-minute intervals for 2 hours. The resultant regression equation for the 4- to 22-year age group was applied to flow to blood calculations for animals 3 to 23 years of age, and the equation from the 24- to 30-year group was applied to flow to blood calculations for animals 25 to 29 years of age, unless the monkey’s own data were available (n ϭ 9), in which case those data were used. Analysis

AHF, Fu, and ﬂow to blood data were analyzed with the two-tailed, FIGURE 1. IOP increased in the monkeys with age. The slope of the unpaired t-test for differences with unequal variances compared with linear regression line (not shown) of IOP with age for all animals (3–29 0.0. Probabilities for slopes were calculated by regression analysis years, n ϭ 22) was signiﬁcantly different from 0.0 (P ϭ 0.039; r2 ϭ using Minitab software (Minitab, Inc., State College, PA). 0.197).

Downloaded from iovs.arvojournals.org on 09/30/2021 2120 Gabelt et al. IOVS, May 2003, Vol. 44, No. 5

TABLE 2. Effects of Aging on Aqueous Formation and Drainage in Rhesus Monkeys

3–10 y 19–23 y 25–29 y 3–23 y (19 ؍ n) (5 ؍ n) (8 ؍ n) (11 ؍ n)

IOP (mm Hg) 16.1 Ϯ 0.9* 19.1 Ϯ 1.1† 18.3 Ϯ 1.6 17.3 Ϯ 0.8‡ AC Volume (␮L) 148 Ϯ 9 142 Ϯ 8 141 Ϯ 7 146 Ϯ 6 AHF (␮L/min) 1.68 Ϯ 0.07 1.71 Ϯ 0.09 1.65 Ϯ 0.05 1.69 Ϯ 0.06 Fu (␮L/min) 0.64 Ϯ 0.11 0.62 Ϯ 0.07 0.33 Ϯ 0.08§ 0.63 Ϯ 0.07 Fu/AHF (%) 38.3 Ϯ 6.5 36.0 Ϯ 3.9 19.8 Ϯ 5.4§ 37.3 Ϯ 4.0 FTB (␮L/min) 1.04 Ϯ 0.13 1.09 Ϯ 0.08 1.36 Ϯ 0.10 1.06 Ϯ 0.08 FTB/AHF (%) 61.5 Ϯ 6.4 64.0 Ϯ 3.9 80.2 Ϯ 5.4§ 62.6 Ϯ 4.0

Data are the mean Ϯ SEM. Results in the 25- to 29-year age group were signiﬁcantly different from those in the 3- to 10-, 19- to 23-, and 3- to 23-year age groups, according to the unpaired t-test for differences with unequal variances compared with 0.0. * n ϭ 10. † n ϭ 7. ‡ n ϭ 17. § P Ͻ 0.05.

with a computer-based morphometric system (Quantimed 500; Leica, However, there was no significant difference in the mean IOP Cambridge, UK) at ϫ3000 on a video screen. The area of the cribriform between the various groups. region analyzed was 315 ␮m2 and was located in the middle filtering Total AHF remained constant throughout life (Fig. 2, Table part of the meshwork, avoiding both the anterior and posterior ends of 2). The increase in flow to blood and decline in Fu were Schlemm’s canal. The length of the area was 45 ␮m along the inner precipitous in the 25- to 29-year group, rather than changing wall of the canal and extended 7 ␮m from the inner wall into the uniformly throughout the life span. The regression of flow to cribriform region. In addition, the diameter of cross sections through blood and Fu versus age in the 3- to 23-year-old animals was not elastic fibers was measured. In sagittal ultrathin sections through the CM tips of the longitudinal and reticular part of the muscle the diameter of 10 elastic fibers per section in the trabeculum ciliare was measured. As the morphology of the elastic fibers was in general similar in the different quadrants, quantitative evaluations were performed only in one randomly chosen section per eye.

RESULTS Physiology Slit lamp biomicroscopy showed that the anterior segments of all animals were free of cells, ﬂare, and ocular abnormalities before experimentation. IOP increased signiﬁcantly as age increased from 3 to 29 years (n ϭ 22, P ϭ 0.039, Fig. 1, Table 2).

FIGURE 3. Flow to blood (FTB) increased very little with age in rhesus monkeys until rather late in life. The slopes of the linear regression lines (not shown) for rate of (A) FTB or (B) FTB/AHF (aqueous humor FIGURE 2. Aqueous humor formation (AHF) rate did not change with formulation) versus age in all animals (3–29 years, n ϭ 24) or for age in rhesus monkeys (n ϭ 24). animals 3 to 23 years (n ϭ 19) were not signiﬁcantly different from 0.0.

Downloaded from iovs.arvojournals.org on 09/30/2021 IOVS, May 2003, Vol. 44, No. 5 Aqueous and Trabecular Morphologic Changes with Age 2121

FIGURE 5. Anterior chamber (AC) volume remained essentially constant with age (n ϭ 24).

significant (Figs. 3, 4). However, mean Fu, Fu/AHF, and flow to blood/AHF were significantly different between the 25- to 29-year group and the others groups (Table 2). Anterior chamber volume estimated by isotope dilution did not change with age in this study (Fig. 5 and Table 2). The regression equation in pooled data from animals aged 4 to 22 years for blood-equivalent albumin space as a percentage of body weight versus time (n ϭ 11) was 8.157 ϩ (0.011 ϫ minutes from the start of I-125 albumin infusion). A slightly FIGURE 4. Uveoscleral outflow (Fu) decreased very little with age in different regression equation was obtained for blood-equiva- rhesus monkeys until rather late in life. The slopes of the linear lent albumin space measured in pooled data from monkeys 24 regression lines (not shown) for rates of (A)Fuor(B) Fu/AHF (aqueous to 30 years of age (n ϭ 6): 7.623 ϩ (0.011 ϫ minutes from the humor formulation) versus age in all animals (3–29 years, n ϭ 24) or in start of I-125 albumin infusion). animals 3 to 23 years (n ϭ 19) were not significantly different from 0.0.

FIGURE 6. Electron micrographs of the ciliary muscle tips of a 3.5-year- old monkey (A, B) and a 34-year-old monkey (C, D). The extracellular material within the trabeculum ciliare surrounding the muscle tips increased with increasing age. Thick bundles of collagen ﬁbers appeared in the old monkey’s eye (C, arrows). The sheath of the elastic ﬁbers (arrows) in old monkeys (D, arrows)is also much thicker than in younger animals (B, arrows) MC, ciliary muscle cell. Scale bar, 1 ␮m.

Downloaded from iovs.arvojournals.org on 09/30/2021 2122 Gabelt et al. IOVS, May 2003, Vol. 44, No. 5

Schlemm’s canal in the middle filtration area was highly variable, but there was a tendency toward an increase with age (Fig. 9A). The area of optically empty spaces within this region remained constant with age (Fig. 9B), as did the area occupied by fibrillar material and by cells (Figs. 9C, 9D). This was true, however, only in the middle filtering portion of Schlemm’s canal. In the adjacent posterior portion of the inner wall, the increase in fibrillar material and the increase in thickness of the sheaths of the elastic fibers were much more pronounced in old monkeys than in young animals (Figs. 10, 11).

DISCUSSION In this study, Fu appeared to decline significantly in rhesus monkeys older than 25 years compared with younger age groups, even as close in age as 19 to 23 years. Based on existing human data,8 Fu also decreases in healthy humans older than 60 years (equivalent to our 25- to 29-year old monkeys) compared with 20- to 30-year-olds. Whether there is a gradual or a dramatic decline in Fu in humans has not been determined. Townsend and Brubaker22 calculated Fu to be 34% Ϯ 12% of AHF in control eyes of 24-year-old humans, similar to our finding in our 3- to 23-year-old monkeys (37% Ϯ 4%). Purported monkey versus human differences in Fu in earlier comparisons are probably due largely to age and/or disease state differences, rather than to species differences. A study by Bill and Philips10 of Fu in humans was undertaken in 48- to 73-year-old eyes, most of which harbored malignant melanomas. They suggested that the increase in polysaccharide material that often occurs in choroidal melanomas probably impedes flow through the TM and CM. The large scatter and the weak correlation in the plots of Fu or flow to blood or Fu versus age in our monkeys (r2 Յ 0.1; Figs. 3, 4, respectively) indicates that experimental noise or other factors are also at play. FIGURE 7. Elastic fiber thickness changed with age. (A) In the ciliary Previous studies showed an age-related decrease in total muscle (CM) tips, elastic fiber thickness including the sheath material outflow facility in both monkeys14 and humans.2,6,9 In humans, increased significantly with age (slope of linear regression, P Ͻ 0.001, reduced formation of giant vacuoles in the inner wall endothe- not shown). (B) Elastic fiber thickness changes in the trabecular mesh- lium of Schlemm’s canal has been proposed to account for the work (TM) in the subendothelial region of Schlemm’s canal were more age-related increase in outflow resistance (Roy S, Boldea R, variable but also increased significantly with age (slope of linear re- Leuba S, Mermoud A, ARVO Abstract 733, 2001) but this is only gression, P ϭ 0.024, not shown). r2 ϭ 0.764 (A) and ϭ 0.413 (B). one of many possibilities. The amount and induction of mRNA for various matrix metalloproteinases decreased with age of Morphology porcine trabecular cell cultures exposed to 15 and 50 mm Hg In rhesus monkeys the trabeculum ciliare connecting the uveal portion of the TM with the iris root and thereby covering the CM tips consisted of a network of collagen and elastic fibers. On the aspect facing the anterior chamber, this network was covered by TM cells. On the aspect facing the CM bundles, the elastic and collagen fibers of the trabeculum ciliare were continuous with the connective tissue between the CM bundles and the basement membrane of the CM cells. With increasing age, the fibrillar material within the trabeculum ciliare and the intermuscular spaces at the muscle tips increased. The elastic fibers gained a homogeneous-appearing electron-light sheath that also increased with age. Quantitative evaluation of the diameter of the elastic fibers including the sheath material revealed that the increase in thickness was linear (Figs. 6, 7A). In the TM the number of TM cells decreased significantly with increasing age (Fig. 8). In the cribriform region there was also an increase in the amount of extracellular material and increased thickness of the elastic fiber sheaths. Quantitative evaluation of the diameter of the elastic fibers in the middle filtration area of the cribriform region showed that the thickening in that area was less prominent and the interindividual FIGURE 8. Trabecular meshwork (TM) cell counts significantly de- variability greater than at the muscle tips (Fig. 7B). The area of crease with age (slope of linear regression, P ϭ 0.002, not shown). sheath-derived plaques directly underneath the inner wall of r2 ϭ 0.723.

Downloaded from iovs.arvojournals.org on 09/30/2021 IOVS, May 2003, Vol. 44, No. 5 Aqueous and Trabecular Morphologic Changes with Age 2123

FIGURE 9. In the middle, filtering portion of Schlemm’s canal, the area occupied by sheath-derived plaques (A), fibrillar material (B), empty space (C), or cells (D), became more variable with age, but the correlation was not significant.

of pressure for 72 hours (Ehrich D, Tripathi BJ, Tripathi RC, tion in matrix metalloproteinase activity (Williams GC, Borra´s Duncker GIW, Gotsis S, ARVO Abstract 748, 2001). In human T, Pizzo SV, ARVO Abstract 764, 2001). Findings in both of the TM cells, there may also be a passage-number–related reduc- latter studies suggest a reduced capacity to break down extra-

FIGURE 10. Electron micrographs of the inner wall region of Schlemm’s canal (SC) in a 2-year-old (A, B) and a 34-year-old (C, D) monkey. In both the young and the old animals, there was more extracellular material in the posterior part of SC (B, D) than in the middle, presumably main ﬁl- tering, portion (A, C). In the old monkey, there was more extracellular material directly adjacent to the inner wall than in the young one. Part of this material was formed by thickened sheaths of elastic ﬁbers (sheath-derived plaques; arrows). Scale bar, 2 ␮m.

Downloaded from iovs.arvojournals.org on 09/30/2021 2124 Gabelt et al. IOVS, May 2003, Vol. 44, No. 5

in monkeys aged more than 25 years would be expected to be greater than in those aged less than 19 years, which was not the case. In adult humans, leakiness remained largely unchanged from 18 to 77 years.27 Scleral hydraulic conductivity does not appear to change with age in humans (Noury AM, Jackson TL, Hodgetts A, Marshall J, ARVO Abstract 3580, 2001). If we assume the same blood-equivalent albumin space in all animals, then the difference in Fu in the two age groups is even more striking (Gabelt BT, Kaufman PL, ARVO Abstract 2749, 2000). There has been a debate about whether Fu actually passes through the sclera versus flowing into the choroidal blood.28 However, these arguments in no way negate the use of protein tracers as a marker. Any protein appearing in the blood shortly after its introduction into the anterior chamber can be assumed to have been carried to the blood by transtrabecular flow. Therefore the calculation of Fu as the difference of AHF and flow to blood would not depend on the exact route of Fu. Prior studies of the aging CM have revealed some cellular changes that seem unlikely to have consequences for bulk fluid FIGURE 11. Higher magnification of the sheath-derived plaques (arrows) underneath the inner wall of Schlemm’s canal (SC) in a 34-year- flow through the tissue, which is more likely to depend on old monkey. EL, elastic fiber. Scale bar, 1 ␮m. events in the intermuscular spaces and perhaps especially at the anterior entrance to those spaces at the trabeculum ciliare. The trabeculum ciliare is a network of elastic and collagen cellular material, which could contribute to a reduction of fibers between the iris root, the uveal portion of the TM, and outflow either through the TM or through the Fu pathway. 23 the CM tips. It is covered by cells anteriorly and connected to Also, a reduction in CM movement reflected by the onset of the basement membrane of the CM cells and the connective presbyopia in humans and monkeys which is complete by tissue between the CM bundles at their tips posteriorly. Aque- approximately 55 years in humans and 25 years in rhesus 24 ous humor must pass through the spaces of the trabeculum monkeys, could contribute to an environment in which ex- ciliare to gain entrance to the spaces between the CM tips, tracellular material could accumulate in the CM and TM out- which constitute the entrance to the Fu pathway. flow pathways.25 In the present study, there was a significant increase in In our study and inferred from Toris et al.,8 the flow of fluid thickness of the elastic fibers of the trabeculum ciliare with age through the trabecular outflow pathway actually appears to in rhesus monkeys. Cross sections through the elastic fibers increase with age when the starting IOP is normal. Trabecular and their sheaths form the so called sheath-derived plaques.29 outflow facility was not measured as part of the present study. Sheath-derived plaques at the muscle tips increase with age in An increase in trabecular flow would not by itself rule out a 29 decrease in trabecular outflow facility. human eyes and especially so in glaucomatous eyes. The plaques at the muscle tips in old rhesus monkeys presumably However, if trabecular outflow facility (Ctrab) and Fu decrease while AHF remains stable, IOP must increase or epi- are not large enough to reduce Fu themselves but may indicate scleral venous pressure (Pe) must decline, based on the Gold- a general increase in extracellular material in the CM tips and ϭ Ϫ ϩ a consequent reduction in the size of the “gateway” openings mann equation: AHF Ctrab (IOP Pe) Fu. Because IOP appears to increase with age in the monkeys we studied (Fig. for entrance of aqueous humor into the Fu pathways. The number of pigmented cells in the inner and posterior 1), whereas AHF remained unchanged and Fu decreased, a 15 decrease in trabecular outflow facility with age is possible. Our parts of the CM in rhesus monkeys increases with age, but morphologic and physiological data suggest that the age-re- the most anterior portions are spared until after age 20 years, lated increases in IOP in this sample of monkeys may reflect at which point the spaces between the muscle fiber bundles changes in the uveoscleral as well as the trabecular outflow contain a greatly increased number of pigmented cells com- pathway (discussed later). However the physiologic changes in pared with younger animals. This is especially prominent after aqueous humor drainage in our studies appear not to occur age 25 years when pigmented cells are present even between uniformly throughout life but change rather precipitously at 25 the tips of the anterior longitudinal portion of the muscle. The years of age. Morphologic changes seem to occur more uni- correlation with the dramatic decrease of Fu over age 25 is formly with age. It may be that a threshold in morphologic noteworthy but could be either a contributing factor to or the changes must occur before there are any changes in physio- result of decreased Fu. logic response. With increasing age in rhesus monkeys, we found an in- The increased appearance of isotope in the blood in older crease in sheath-derived plaques and fibrillar material in the animals can be explained in two ways: Either almost all the juxtacanalicular region and a decrease in overall cellularity of fluid goes through the TM, or Fu channels empty into the blood the TM. All these findings also occur in human eyes with more promptly. In the first case, the normal elevation in IOP increasing age. After filtration surgery in young normal mon- with age may produce a widening of the paracellular channels keys increased plaque formation also occurred in the TM, between the inner-wall endothelium26 and a loosening of the presumably consequent to underperfusion.30 If underperfu- cell–cell attachments that form them, allowing more fluid to sion causes plaque formation in our old monkeys, this could flow through this route in a nondiseased eye. The second explain why changes in the TM show more interindividual possibility suggests that Fu channels become more “leaky” or variability (between animals of the same age) than do the CM are otherwise altered with age. However the change in Fu was entrance changes. Because AHF does not decline noticeably not continuous throughout life, but rather occurred precipi- with age, the flow may wash out and degrade sheath material tously in very old age. If the vasculature in general becomes in the TM. This would be less true in the muscle tips with more leaky with age, then the blood-equivalent albumin space severely reduced Fu.

Downloaded from iovs.arvojournals.org on 09/30/2021 IOVS, May 2003, Vol. 44, No. 5 Aqueous and Trabecular Morphologic Changes with Age 2125

In conclusion, our results in monkeys and those of Toris et 15. Lu¨tjen-Drecoll E, Tamm E, Kaufman PL. Age changes in rhesus al.8 in humans, using independent techniques with different monkey ciliary muscle: light and electron microscopy. Exp Eye assumptions and potential flaws, both indicate that Fu de- Res. 1988;47:885–899. creases at older ages in eyes with normal IOP and normal 16. Lu¨tjen-Drecoll E, Tamm E, Kaufman PL. Age-related loss of mor- findings in biomicroscopy. This decrease in Fu with age could phologic responses to pilocarpine in rhesus monkey ciliary mus- be especially important in elderly patients with glaucoma, in cle. Arch Ophthalmol. 1988;106:1591–1598. whom outflow facility through the TM is also compromised.31 17. Kaufman PL, Davis GE. “Minified” Goldmann applanating prism for tonometry in monkeys and humans. Arch Ophthalmol. 1980;98: 542–546. Acknowledgments 18. Ito S, Karnovsky MJ. Formaldehyde-glutaraldehyde fixatives con- The authors thank the Wisconsin Regional Primate Research Center for taining trinitro compounds (Abstract). J Cell Biol. 1968;39:168a. making available to us rhesus monkeys of various age groups for the in 19. Ba´rańy EH. Simultaneous measurements of changing intraocular vivo studies and for providing eyes from animals euthanatized for other pressure and outflow facility in the vervet monkey by constant protocols, and Jennifer Seeman, DVM, for providing expertise in per- pressure infusion. Invest Ophthalmol. 1964;3:135–143. forming femoral artery cannulations for blood collection. 20. Gabelt BT, Kaufman PL. Prostaglandin F2␣ increases uveoscleral outflow in the cynomolgus monkey. Exp Eye Res. 1989;49:389– References 402. 21. Sperber GO, Bill A. A method for near-continuous determination of 1. Armaly MF. The genetic determination of ocular pressure in the aqueous humor flow: effects of anaesthetics, temperature and normal eye. Arch Ophthalmol. 1967;78:187–192. indomethacin. Exp Eye Res. 1984;39:435–453. 2. Gaasterland D, Kupfer C, Ross K. Studies of aqueous humour 22. Townsend DJ, Brubaker RF. Immediate effect of epinephrine on dynamics in man. III. Measurements in young normal subjects aqueous formation in the normal human eye as measured by using norepinephrine and isoproterenol. Invest Ophthalmol Vis fluorophotometry. Invest Ophthalmol Vis Sci. 1980;19:256–266. Sci. 1973;12:267–279. 23. Tamm E, Lu¨tjen-Drecoll E, Jungkunz W, Rohen JW. Posterior at- 3. Brubaker RF, Nagataki S, Townsend DJ, Burns RR, Higgins RG, tachment of ciliary muscle in young, accommodating old, presby- Wentworth W. The effect of age on aqueous humor formation in opic monkeys. Invest Ophthalmol Vis Sci. 1991;32:1678–1692. man. Ophthalmology. 1981;88:283–287. 24. Bito LZ, DeRousseau CJ, Kaufman PL, Bito JW. Age-dependent loss 4. Kaufman PL. Aging and aqueous humor dynamics (review). Atti of accommodative amplitude in rhesus monkeys: an animal model Fondazione Giorgio Ronchi. 1985;40:465–470. for presbyopia. Invest Ophthalmol Vis Sci. 1982;23:23–31. 5. Shiose Y. Intraocular pressure: new perspectives. Surv Ophthal- 25. Kaufman PL, Gabelt BT. Presbyopia, prostaglandins and primary mol. 1990;34:413–435. open angle glaucoma. In: Krieglstein GK, ed. Glaucoma Update V. 6. Becker B. The decline in aqueous secretion and outflow facility Proceedings of the Symposium of the Glaucoma Society of the with age. Am J Ophthalmol. 1958;46:731–736. International Congress of Ophthalmology in Quebec City, June 7. Brubaker RF. The flow of aqueous humor in the human eye. Am 1994. New York: Springer-Verlag; 1995:224–241. Ophthalmol Soc. 1982;80:391–414. 26. Epstein DL, Rohen JW. Morphology of the trabecular meshwork 8. Toris CB, Yablonski ME, Wang YL, Camras CB. Aqueous humor and inner wall endothelium after cationized ferritin perfusion in dynamics in the aging human eye. Am J Ophthalmol. 1999;127: the monkey eye. Invest Ophthalmol Vis Sci. 1991;32:160–171. 407–412. 27. Gamble J, Bethell D, Day NP, et al. Age-related changes in micro- 9. Croft MA, Oyen MJ, Gange SJ, Fisher MR, Kaufman PL. Aging vascular permeability: a significant factor in the susceptibility of effects on accommodation and outflow facility responses to pilo- children to shock? Clin Sci. 2000;98:211–216. carpine in humans. Arch Ophthalmol. 1996;114:586–592. 28. Johnson M, Erickson K. Mechanisms and routes of aqueous humor 10. Bill A, Phillips I. Uveoscleral drainage of aqueous humor in human drainage. In: Albert DM, Jakobiec FA, eds. Principles and Practice eyes. Exp Eye Res. 1971;21:275–281. of Ophthalmology. Philadelphia: WB Saunders; 2000 2577–2595. 11. Bito LZ, Merritt SQ, DeRousseau CJ. Intraocular pressure of rhesus 29. Lu¨tjen-Drecoll E, Shimuzu R, Rohrbach M, Rohen JW. Quantitative monkeys (Macaca mulatta). I. An initial survey of two free-breeding analysis of “plaque material” in the inner and outer wall of colonies. Invest Ophthalmol Vis Sci. 1979;18:785–793. Schlemm’s canal in normal and glaucomatous eyes. Exp Eye Res. 12. DeRousseau CJ, Bito LZ. Intraocular pressure of rhesus monkeys 1986;42:443–455. (Macaca mulatta). II. Juvenile ocular hypertension and its apparent 30. Lu¨tjen-Drecoll E, Ba´rańy EH. Functional and electron microscopic relationship to ocular growth. Exp Eye Res. 1981;32:407–417. changes in the trabecular meshwork remaining after trabeculec- 13. Kaufman PL, Bito LZ. The occurrence of senile cataracts, ocular tomy in cynomolgus monkeys. Invest Ophthalmol Vis Sci. 1974; hypertension and glaucoma in rhesus monkeys. Exp Eye Res. 13:511–524. 1982;34:287–291. 31. Allingham RR, de Kater AW, Ethier CR. Schlemm’s canal and 14. Gabelt BT, Crawford K, Kaufman PL. Outflow facility and its primary open angle glaucoma: correlation between Schlemm’s response to pilocarpine decline in aging rhesus monkeys. Arch canal dimensions and outflow facility. Exp Eye Res. 1996;62:101– Ophthalmol. 1991;109:879–882. 109.

Downloaded from iovs.arvojournals.org on 09/30/2021