Morphology and Accommodative Function of the Vitreous Zonule in Human and Monkey Eyes

Abstract

Purpose.: To explore the attachments of the posterior zonule and vitreous in relation to accommodation and presbyopia in monkeys and humans.

Methods.: Novel scanning electron microscopy (SEM) and ultrasound biomicroscopy (UBM) techniques were used to visualize the anterior, intermediate, and posterior vitreous zonule and their connections to the ciliary body, vitreous membrane, lens capsule, and ora serrata, and to characterize their age-related changes and correlate them with loss of accommodative forward movement of the ciliary body. α-Chymotrypsin was used focally to lyse the vitreous zonule and determine the effect on movement of the accommodative apparatus in monkeys.

Results.: The vitreous attached to the peripheral lens capsule and the ora serrata directly. The pars plana zonule and the posterior tines of the anterior zonule were separated from the vitreous membrane except for strategically placed attachments, collectively termed the vitreous zonule, that may modulate and smooth the forward and backward movements of the entire system. Age-dependent changes in these relationships correlated significantly with loss of accommodative amplitude. Lysis of the intermediate vitreous zonule partially restored accommodative movement.

Conclusions.: The vitreous zonule system may help to smoothly translate to the lens the driving forces of accommodation and disaccommodation generated by the ciliary muscle, while maintaining visual focus and protecting the lens capsule and ora serrata from acute tractional forces. Stiffening of the vitreous zonular system may contribute to age-related loss of accommodation and offer a therapeutic target for presbyopia.

The age-related loss of accommodation (presbyopia) in human and nonhuman primates clearly involves loss of deformability of the crystalline lens. In addition, ciliary muscle mobility, as measured by both histologic ^1,2 and ultrasonographic (monkey) ³ and magnetic resonance (human) ⁴ imaging techniques, also diminishes with age. However, the isolated ciliary muscle in nonhuman primates maintains its full ability to contract in response to appropriate pharmacologic stimulation throughout life, ^1,2,5 and its contractile machinery and receptor biology also remain normal. ^6,7 Therefore, posterior restriction of muscle movement in the aging eye has been hypothesized to play a role in the pathophysiology of presbyopia as well, at least in monkeys. ⁸  

In addition, there are zonular attachments to the vitreous membrane in both species that have not yet been fully investigated. Earlier studies were focused mainly on the anterior hyaloid membrane. Wieger ⁹ was among the first to describe attachments of the zonules to the anterior vitreous membrane. They were later termed Wieger's ligament. ^10–12 Streeten and Pulaski ¹³ doubted the strength of Wieger's ligament, because, after lens extraction, it is not uncommon for a superficial flap of either the capsule or anterior hyaloid to be torn from Wieger's area. ¹³ Bernal et al., ¹⁴ using a combination of environmental scanning electron microscopy (ESEM) combined with a custom-made manual lens stretcher to facilitate inside microscopy, were able to further analyze the complex connection between the anterior vitreous membrane and zonule. ¹⁴ Attachments between the posterior pars plana zonule and the vitreous membrane have been observed in histologic sections and by SEM. ^15–17  

Improved in vivo imaging by ultrasound biomicroscopy (UBM) allows visualization of zonular fibers in the living eye. ¹⁸ Using UBM, we observed a straight line parallel to but separated from the pars plana by a cleft approximately 2 mm wide, extending from the region of the ciliary processes to the region of the ora serrata (see Fig. 1). There has been debate as to whether the structure observed by UBM was (1) the pars plana zonule, which in the living eye is normally separated from the pars plana ciliary epithelium, but after fixation for histology or air drying for scanning electron microscopy (SEM) becomes apposed to the pars plana ciliary epithelium (CE) ¹⁹ ; (2) the vitreous membrane ²⁰ ; or (3) another component of the vitreozonular system that has not been well described or characterized. 

We investigated the zonule, the posterior vitreous, and their attachments by novel scanning SEM and UBM techniques, and we now can hypothesize about how this system may affect ciliary muscle movement. In addition, we provide preliminary physiological evidence that ciliary muscle movement may be increased by perturbing the system. 

Materials and Methods

Morphology

Monkey Eyes.

Twenty-seven normal eyes of 22 rhesus monkeys (Macaca mulatta) of either sex and eyes of 1 cynomolgus monkey (Macaca fascicularis), ranging in age from 3.5 to 26 years, were obtained from caged colonies of the Wisconsin National Primate Research Center, the Harlow Primate Laboratory, and Covance, Inc. (all in Madison, WI) and analyzed by SEM. The animals were euthanatized in conjunction with various nonocular protocols. All procedures conformed to the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research and were in accordance with institutionally approved animal protocols. 

Before death, the animals were deeply anesthetized after ketamine induction (3–30 mg/kg IM) with pentobarbital Na (10–15 mg/kg IV, supplemented with 0.5–10 mg/kg IV, as needed) and then perfused transcardially with 1 L of 0.1 M PBS (phosphate-buffered saline) followed by paraformaldehyde (PFA) 4% for 10 to 15 minutes. After systemic perfusion fixation, the eyes were enucleated, slits were cut in the posterior sclera, and a window was cut in the anterior cornea to enhance the penetration of the fixative while preserving the architecture of the ciliary muscle and its posterior attachment to the elastic lamina of Bruch's membrane. ^1,21 The entire eyes were then placed in lto's fixative ²² and sent to Erlangen. 

Before preparation for SEM, the eyes were placed in cacodylate buffer overnight. Small pie-shaped sectors were cut from all four quadrants (of the anterior globe). These sectors contained the ciliary body; the adjacent cornea, sclera, and neighboring part of the lens; and the vitreous membrane. 

Visualization of the vitreous membrane and the zonule is difficult and requires several approaches, as the consistency of the vitreous does not allow good preparation in non–critical-point-dried specimens. However, after critical-point drying the delicate membrane is extremely rigid and friable. Without customized preparation methods, the vitreous membrane completely covers the ciliary body and zonule, as the spaces between the structures collapse during the drying process. 

Ultimately, three different approaches were used to visualize the different portions of the zonular connections to the vitreous membrane. 

Approach 1.

To analyze the zonular connections to the anterior vitreous membrane and the posterior lens capsule, we kept the lens in place. The anterior vitreous membrane was carefully reflected posteriorly, trying not to disrupt its connections to the adjacent structures. In some specimens, the ciliary processes were separated from one another to visualize the connection of the zonule to the ciliary epithelium. In other specimens, the tips of the processes were excised for the same purpose. After critical-point drying, the specimens were viewed from a sagittal, an interior chamber, and a posterior chamber approach. 

Approach 2.

For visualization of the zonular connections in the area between the pars plana zonule and the vitreous membrane over its entire anterior– posterior sagittal length, the specimens were viewed from their sagittal aspect, and the vitreous membrane was carefully lifted from the pars plana region. With this approach, it became obvious that overlying most of the pars plana region there was a cleft between the pars plana zonule and the neighboring vitreous that disappeared in the most posterior pars plana in the region of the ora serrata. 

Approach 3.

To analyze this area of connection in more detail, either the anterior or the posterior part of the vitreous membrane was carefully scrolled. The critical-point–dried specimens were then investigated, either from a sagittal or an intraocular en face perspective. 

For critical-point drying, after the various preparatory protocols, the specimens were carefully dehydrated in graded alcohol and acetone and were dried in a critical-point dryer (see Rohen ¹⁹ ). The specimens were then put on a disc, coated with gold palladium, and examined with a scanning electron microscope (Stereoscan; Cambridge Instrument Co., Ltd., Cambridge, UK). 

Human Eyes.

Specimens of each quadrant of eight human eyes aged 55 to 100 years (mean ± SD, 67 ± 15.3) with postmortem times between 7 and 18 hours (mean ± SD 11.2 ± 3.5) were evaluated in the same manner as the monkey eyes. The eyes were obtained by the Department of Anatomy of the University of Erlangen-Nürnberg, after appropriate consent, in accordance with the Declaration of Helsinki for research involving human tissue. Immediately after enucleation, the anterior portion of the cornea was removed, slits were cut into the sclera, and the eyes were immersion fixed in Ito's solution for 3 to 5 hours. The anterior eye segment was then dissected and postfixed in 1% osmic acid for 3 hours. After that, small pie-shaped sectors were cut from the anterior eye segment and prepared in the same manner. 

Accommodation in Live Monkeys

A Hartinger coincidence refractometer (Carl Zeiss Meditec, Jena, Germany) was used to measure resting refractive error and accommodation in response to electrical stimulation of the Edinger-Westphal (E-W) nucleus via an implanted electrode. ³ Supramaximal stimulus settings were chosen that induced maximum forward ciliary body movement and maximum accommodation, allowing comparisons to be made of accommodative responses between young and older eyes. Maximal stimulus is defined as the level of E-W stimulus current necessary to induce maximum accommodative change, measured by refractometry. Supramaximal stimulus is a level of E-W stimulus current ∼25% (or ∼0.10–0.20 mA) above the maximal stimulus that ensures maximum forward ciliary body movement. 

UBM in Live Monkeys

A 50-MHz UBM instrument (model 840; Humphrey Instruments; Carl Zeiss Meditec) was used to image the posterior vitreous zonule and ciliary body, and the images were then used to measure the width of the cleft between the pars plana and the vitreous membrane/vitreous zonule in the resting eye (Fig. 1). In addition, a 35-MHz UBM instrument (model MHF-1; Ultraview System P60; E-Technologies, Bettendorf, IA) was used to collect images encompassing the entire sagittal extent of the ciliary body to the region of the ora serrata. Dynamic UBM images were obtained during central stimulation of accommodation and then recorded to videotape. ³ From these images, measurements were taken of the distance between the scleral spur and the posterior insertion point of the posterior vitreous zonule at rest and during supramaximal central stimulation. 

Figure 1.

 

UBM overview image in a live rhesus monkey shows a prominent straight line (arrow) extending from the pars plicata region of the ciliary body to the ora serrata region and separated from the pars plana epithelium by a cleft. CP, ciliary processes, CB, ciliary body.

The eye was stabilized with extraocular muscle sutures during UBM imaging, to dampen or eliminate convergence eye movement. The transducer was held in place by a stabilization arm to eliminate any change in angle of the transducer to the eye during accommodation, and the anterior and posterior ends of the ciliary body were oriented in a sagittal direction within all images, to ensure stability and reproducibility. The temporal region was selected as providing the best ultrasound imagery, with clear and distinct edges of the ciliary body and posterior vitreous zonule. Comparisons were made of the angle between the inner aspect of the cornea and the anterior aspect of the muscle apex (clinically called the ciliary face and not including the ciliary processes) in the unaccommodated versus the accommodated state. The narrowing of this angle was used as a surrogate indicator of forward ciliary body movement. ³ With respect to comparing the angle measurement in the unaccommodated and accommodated states, the location of the anterior aspect of the muscle apex was far enough inward to reflect the translocation of the innermost aspect of the muscle. 

Lysis and Disruption of the Posterior Vitreous Zonule

Surgical Procedure.

In one eye each of seven rhesus monkeys (four aged 6–9 years and the remaining three aged 15, 25, and 26 years) and one eye of one cynomolgus monkey (aged 3.5 years), lysis of the posterior zonule was attempted by insertion of 8 to 16 μL of 83 U/mL α-chymotrypsin in heavy sucrose medium via a ∼1-mm incision through the sclera and the choroid in the midregion of the pars plana. The eye was oriented so that the solution fell by gravity onto the strands of the zonule. The contralateral eyes served as the control. One of the seven rhesus monkeys, aged 25 years, had undergone bilateral extracapsular lens extraction (ECLE) ²³ 19 months before unilateral α-chymotrypsin injection and transcorneal lens capsulectomy and vitrectomy. Three of the seven monkeys, aged 6 to 9 years, were pseudophakic after bilateral ECLE 8 months before unilateral α-chymotrypsin injection. 

Ultrasound Biomicroscopy.

Maximum accommodative responses were induced by corneal iontophoresis of 40% carbachol in agar (a supramaximal dose) ²⁴ in three monkeys, before and after α-chymotrypsin injection. In three other rhesus monkeys, maximum accommodative responses were induced by supramaximal E-W stimulation before and after α-chymotrypsin injection. UBM imaging was undertaken in the resting and accommodated state before and after α-chymotrypsin injection in these six monkeys. Three of these monkeys were randomly selected to undergo morphologic examination. 

Morphology.

Three of the pseudophakic rhesus monkeys (aged 6–9 years), the one aphakic rhesus monkey, and the one phakic 3.5-year-old cynomolgus monkey were studied. Specimens from the region of injection of α-chymotrypsin were prepared for SEM as for UBM. In addition, 1- to 2-mm wide specimens of the anterior eye segment that included the entire anterior–posterior extent of the ciliary body and the adjacent cornea and sclera were embedded in Epon, and 1-μm semithin sections were cut and stained with toluidine blue. In these semithin sections, the apical position and width of the ciliary muscle were determined as described previously. ²¹ The opposite eye served as the control. 

Statistical Analysis

A two-tailed paired t-test was used to detect significant differences. A multiple regression analysis was performed to evaluate the relationships between accommodative amplitude and age or the forward movement of the posterior insertion zone. Stepwise regression analysis based on the Akaike information criterion (AIC) was used for model selection. ²⁵ P < 0.05 was considered significant; 0.05 < P < 0.10 was considered to indicate a trend, given the small number of monkeys in some groups. Throughout this article, the mean ± s.e.m. is reported. However, with respect to the age range and postmortem time range of the eight human eyes that were studied, the mean ± SD are reported. 

Results

Morphology in Monkey Eyes

Anterior Vitreous Zonule.

An overview of the insertion of the vitreous membrane and posterior tines of the anterior zonule indicated that both systems inserted into the posterior lens capsule at the lens periphery (Figs. 2A, 2B). Careful lifting of the vitreous membrane from the zonule showed that both the zonule and the vitreous inserted at nearly the same place, but the vitreous zonule covered the insertion of the zonular plate and inserted somewhat more centrally. Most of the posterior zonular tines were not attached to the vitreous membrane but formed a zonular plate running from the zonular plexus to the posterior lens capsule (Fig. 2A). As the vitreous could easily be lifted from this zonular plate, we assume that, in the live monkey, the zonular plate and vitreous are separated by an aqueous-filled small cleft in this region. There were, however, strands of zonular fibers, deriving from the zonular plexus that inserted into the vitreous membrane at fairly regular intervals of approximately 50 to 75 μm (Fig. 2C). These connections were termed anterior vitreous zonules. 

Figure 2.

 

A 75-year-old human eye. (A) Scanning electron micrograph of a sagittal aspect of the anterior vitreous membrane (VM), the posterior tine of the zonular fork (*), and the posterior lens capsule (LC). (B) Scanning electron micrograph of the anterior VM and its attachment to the posterior LC. (C) Careful lifting of the VM reveals that the majority of the zonule (Z) inserts directly into the posterior LC without attachment to the vitreous membrane that itself forms a separate layer also directly inserting into the posterior LC. However, some bundles of zonular fibers (anterior vitreous zonule, aVZ) do insert into the anterior VM at spatial intervals of approximately 75 μm.

Intermediate and Posterior Vitreous Zonules.

After careful elevation of the vitreous membrane overlying the middle part of the pars plana, taking care not to remove the vitreous membrane itself, we could clearly identify the presence of a cleft between the vitreous membrane and the pars plana zonules. This cleft was bridged by zonular fiber bundles, called intermediate vitreous zonules. These structures were spaced uniformly around the entire circumference of the pars plana region, separated by approximately the width of a ciliary process (Figs. 3A, 3B). Indeed, each intermediate vitreous zonular fiber bundle split anteriorly and formed a fork with tines that inserted into the zonular plexus on either side of an individual ciliary process (Fig. 3B). The intermediate vitreous zonular bundles had no direct contact with the pars plana zonular apparatus. 

Figure 3.

 

(A) Scanning electron micrographs of a sagittal and oblique internal view of the ciliary body (CB), the vitreous membrane (VM), the zonular plexus (ZP), and the zonular connections bridging the cleft between the pars plana zonules (ppZ) and VM (rhesus monkey, aged 8 years). The bridging bundles of zonular fibers run from the region of the ZP in the valleys of the posterior pars plicata toward the VM in the region of the ora serrata (*). These zonular bundles were termed intermediate vitreous zonule (iVZ). (B) Scanning electron micrograph showing the inner aspect of the posterior pars plicata (ppl), pars plana (pp), VM, and iVZ of a 10-year-old rhesus monkey. Anteriorly, the intermediate vitreous zonular bundles split into a fork with tines that insert on both sides of the processes in the valleys of the ppl (*). Posteriorly, each main bundle splits into several smaller bundles that merge with the VM (arrows).

In contrast, posteriorly in the ora serrata region, each intermediate vitreous zonular fiber bundle split into several fine fibrils that became part of the vitreous membrane (Fig. 4A). The vitreous membrane, with its brushlike posterior extensions of the intermediate vitreous zonule, was attached to the pars plana zonule by numerous oblique-running fine fibrils (Fig. 4B). Thus, at the site of attachment of the vitreous to the posterior pars plana and ora region, a multilayered, interconnected spongelike ring, termed the posterior vitreous zonule, was formed. 

Figure 4.

 

(A) Scanning electron micrographs of sagittal sections of the ora serrata region (rhesus monkey, aged 6 years). The vitreous membrane (VM), the posterior extensions of the intermediate vitreous zonule (iVZ), and the pars plana zonule (ppZ) form an interconnected spongelike structure (*). (B) After careful elevation of the VM, the connections of the ppZ to the posterior VM become visible (arrows).

Morphology in Human Eyes

In general, the SEM appearance and anatomic relationships of the vitreous zonule and vitreous membrane were similar in humans and monkeys. The most striking difference involved the posterior vitreous zonule and its attachment to the pars plana zonule in the region of the ora serrata. In the monkey, the spongelike insertion encompassed a sagittal length approximately one sixth (∼0.5 mm) the distance between the scleral spur and ora serrata. In the human, the insertion was disproportionately thicker and encompassed between one third and one half (∼3.4 mm) the sagittal distance between the scleral spur and the ora serrata (Fig. 5A). SEM of the posterior vitreous zonule showed that this system is more complex in the human eye than in the monkey eye. In the entire area of attachment, vitreous zonular fibers ran from the posterior zonular insertion anteriorly to the vitreous membrane and also from the more anterior pars plana zonule into the posterior vitreous zonule, thereby forming a latticelike structure (Fig. 5B). 

Figure 5.

 

(A) Histologic sagittal section through a 100-year-old human eye showing the posterior adherence of the vitreous membrane to the posterior pars plana and ora region (*). Note that this region in humans encompasses nearly one half the sagittal length between the scleral spur and ora serrata. (B) Scanning electron micrograph of the posterior vitreous zonule (sagittal aspect, 85-year-old human eye). Note the oblique-running fibrils between the pars plana zonules (ppZ) and the vitreous membrane (VM); in contrast to the monkey (Fig. 4B), these fibrils form a latticelike structure (arrows).

UBM in Live Monkeys

UBM imaging, involving slightly oblique rather than absolutely perpendicular cuts, revealed that the pars plana zonule lay immediately adjacent to the pars plana ciliary epithelium (Fig. 6). 

Figure 6.

 

UBM images of unaccommodated (A) and accommodated (B) ciliary muscle in a live rhesus monkey, aged 25 years. In these somewhat oblique sections, one can see the pars plana zonules immediately adjacent to the pars plana epithelium (arrowheads). This structure is more apparent in the accommodated than in the unaccommodated state. Straight line: the ciliary processes and the ora serrata represent the vitreous zonule. The numbers represent the angle between the anterior face of the ciliary body and the inner surface of the peripheral cornea, as defined by the white lines. Narrowing of the ciliary body (CB)–corneal angle in the accommodated versus the unaccommodated state was used as a surrogate indicator of forward ciliary body movement.

Nearly serial cuts revealed two distinct linear structures separated from the pars plana by a wide cleft—the vitreous zonule extending from the ora serrata region to the valleys of the pars plicata and the vitreous membrane branching from and lying internal to the vitreous zonule, as both extended anteriorly from the ora region (Fig. 7). The relative prominence of each linear structure varied from cut to cut. 

Figure 7.

 

UBM images were obtained in a 15-year-old rhesus monkey. Care was taken to assure that the vitreous zonule appeared as a continuous prominent straight line parallel to the focus line (*) imprinted by the instrument within all images, indicating that these were true sagittal sections. (A) Anteriorly, the vitreous membrane is separated from the vitreous zonule. The vitreous zonule fork (seen by SEM, Fig. 3B) occurs just posterior to its insertion to the zonular plexus and, as the fibers split at the fork, they course out of the UBM image plane (arrowhead). Thus, the white line that represents the vitreous zonule appears to discontinue (arrowhead) immediately posterior to reaching the ciliary process region in this image. This drop out of the vitreous zonule strand is not always apparent in UBM images, due to the orientation of the UBM probe and the close proximity of the vitreous zonule fork to the ciliary processes (CP), as in (B).

The apparent discontinuities in the vitreous zonule in some of the perpendicular sections (Fig. 7A) most likely represented the forking of the vitreous zonule fibers as they approached the posterior aspects of the ciliary processes as seen by SEM (Fig. 3B). The similarity of the SEM and UBM images is easily seen in Figure 8. 

Figure 8.

 

UBM image (A) analogous to SEM sections (B) and (A) of Figure 3 reproduced for ease of comparison, from the same 8-year-old rhesus monkey. CB, ciliary body, pp, pars plana zonule, VZ, vitreous zonule, VM, vitreous membrane.

Age-Related Changes in Live Monkeys

In the resting eye, the width of the cleft between the pars plana region of the ciliary body and the intermediate vitreous zonular bundles, at the cleft's widest point, increased with age by 0.012 ± 0.0022 mm/y (Figs. 9A–D; P = 0.001, r = 0.79, n = 19). In addition, the thickness of the ciliary muscle at the point of the maximum cleft width decreased with age, and the curvature of the pars plana and the posterior pars plicata increased with age (Figs. 9A–C). Further, with increased cleft width, accommodative amplitude declined significantly (−36.1 ± 11.1 D/mm; Fig. 9E); P = 0.007, r = 0.63, n = 17). 

Figure 9.

 

UBM obtained in three rhesus monkeys aged 6, 16, and 25 years, showing the age-related change in cleft width (A–C). Cleft width plotted versus age (D) and accommodative amplitude (E). Note also the decreasing width of the ciliary muscle overlying the widest part of the cleft and the increased curvature of the arc formed by the inner portion of the ciliary body.

In the resting eye, the distance between the scleral spur and the posterior insertion zone of the posterior vitreous zonule (Figs. 10A, 10B) did not significantly change with age (Fig. 10C). During accommodation, it could clearly be seen that the posterior insertion zone of the posterior vitreous zonule moved forward (Figs. 10A, 10B; Movie S1). Thus, the distance between the scleral spur and the posterior insertion zone of the posterior zonule diminished with accommodation (P < 0.001) and did so more markedly in the young eyes (1.052 ± 0.067 mm, n = 4) than in the older eyes (0.403 ± 0.118 mm, n = 4; Table 1, Fig. 10D). 

Figure 10.

 

UBM images in a 21-year-old rhesus monkey eye before (A) and during (B) central electrical supramaximal stimulation of the E-W nucleus. The distance between the scleral spur and the posterior insertion zone of the vitreous zonule was measured in the unaccommodated and accommodated states. CB, ciliary body; c, cornea. (C) Distance between the scleral spur and vitreous zonule insertion zone in the resting eye, and (D) accommodative forward movement of the vitreous zonule insertion zone plotted versus age. (E) Accommodative amplitude plotted versus the forward movement of the vitreous zonule insertion zone during accommodation.

Table 1.

 

Accommodative Forward Movement of the Vitreous Zonule Posterior Insertion Zone in Monkey Eyes

		n	P
Young (6–9 y)	1.052 ± 0.067	4	0.001
Older (19–21 y)	0.403 ± 0.118	4

 Data are expressed as the mean ± SEM (in millimeters) and show the accommodative forward movement of the intermediate posterior zonule's posterior insertion zone during supramaximal central stimulation, to induce accommodation in monkeys. The accommodative forward movement of the posterior insertion zone declined by −60% in the older versus the young monkey eyes.

Results of the regression analysis showed that accommodative amplitude (P = 0.0003; not shown) and accommodative forward movement of the posterior insertion zone (P = 0.017) declined significantly with age (Fig. 10D). The accommodative forward movement of the posterior insertion zone declined by ∼60% in the old versus the young eyes (P = 0.001; Table 1). Accommodative amplitude increased significantly with increased accommodative forward movement of the vitreous zonule's posterior insertion zone (Fig. 10E). 

The results of the stepwise regression analysis showed that age was a better predictor of accommodative amplitude than was either the forward movement of the posterior insertion zone or the cleft width. 

Disruption of the Intermediate Vitreous Zonule in Live Monkeys

Morphology.

SEM showed that the intermediate vitreous zonular bundles in the region of the α-chymotrypsin injection were absent (Fig. 11). In all four rhesus monkeys (aged 6–9 and 25 years) independent of phakic status, at rest, the muscle in the enzyme-injected temporal quadrant was positioned more anteriorly than the temporal quadrant in the contralateral controls (Fig. 12). This positioning was true to a lesser degree in the single young cynomolgus monkeys. In all five monkeys considered together, the anterior translation was 0.256 ± 0.079 mm (n = 5; P = 0.032; Table 2). The difference in apical position between treated versus control eyes became far less pronounced as the distance from the injection site increased (Table 2). The muscle tended to be thicker in the α-chymotrypsin–injected eyes than in the contralateral control eyes, but the difference was not significant. 

Figure 11.

 

Scanning electron micrograph of the temporal region of a 9-year-old rhesus monkey eye after a pars plana injection of α-chymotrypsin shows the absence of intermediate vitreous zonular fibers in this region. VM, vitreous membrane; ppZ, pars plana zonule; CB, ciliary body.

Figure 12.

 

Semithin sections of the temporal regions of the right and left eye of a 9-year-old rhesus monkey. In the resting eye (A), the inner edge of the ciliary muscle was located much more anteriorly after α-chymotrypsin injection with disruption of the intermediate vitreous zonule than in the contralateral noninjected eye (B). Sp, scleral spur. Arrow: inner apex.

Table 2.

 

Morphologic Findings with and without α-Chymotrypsin Treatment to Lyse the Vitreous Zonule in Monkey Eyes

A. Apical Position and Muscle Width
Monkey	Age (y)		Apical Position (mm)				Muscle Width (mm)
			Temporal	Inferior	Superior	Nasal	Temporal	Inferior	Superior	Nasal
1	3.50	Postlysis	0.51	0.49	0.53	0.61	0.67	0.54	0.60	0.76
		Control	0.55	0.51	0.53	0.64	0.60	0.53	0.53	0.62
2	6–8	Postlysis	0.32	0.55	0.57	0.76	0.60	0.60	0.70	0.59
		Control	0.59	0.72	0.55	0.47	0.68	0.51	0.59	0.60
3	9.00	Postlysis	0.31	0.27	0.45	0.64	1.01	0.98	1.04	1.04
		Control	0.53	0.64	0.63	0.39	0.86	0.80	0.85	0.80
4	9.50	Postlysis	0.23	0.31	0.37		0.94	0.91	0.88	0.88
		Control	0.76	0.85	0.85	0.55	0.67	0.72	0.73	0.79
5†	25.00	Postlysis	0.42	0.45	0.53	0.59	0.71	0.72	0.67	0.72
		Control	0.64	0.56	0.58	0.47	0.63	0.73	0.71	0.64

B. Differences in Apical Position and Muscle Width
Monkey	Apical Position (mm) α-Chymotrypsin–Treated minus Control Eye				Muscle Width (mm) α-Chymotrypsin–Treated minus Control Eye
	Temporal	Inferior	Superior	Nasal	Temporal	Inferior	Superior	Nasal
1	−0.04	−0.02	0.0	−0.03	0.07	0.01	0.07	0.14
2*	−0.27	−0.17	0.02	0.29	−0.08	0.09	0.11	−0.01
3*	−0.22	−0.37	−0.18	0.25	0.15	0.18	0.19	0.24
4*	−0.53	−0.54	−0.48		0.27	0.19	0.15	0.09
5†	−0.22	−0.11	−0.05	0.12	0.08	−0.01	−0.04	0.08
Mean	−0.256*	−0.242	−0.138	0.158	0.098	0.092	0.096	0.108
SEM	0.079	0.094	0.092	0.072	0.057	0.042	0.039	0.041
P	0.044	0.062	0.209	NS	0.161	0.091	0.072	0.058

 Data show the apical position and muscle width (A) and the differences in muscle apical position and muscle width (B) between α-chymotrypsin-treated and control monkey eyes in the respective quadrants. The eyes received α-chymotrypsin injection in the temporal quadrant to lyse the vitreous zonule (Postlysis). The apical position of the muscle was farther anterior in the α-chymotrypsin–injected eyes than in the contralateral control eyes. The difference was most pronounced near the injection site (temporal quadrant; P = 0.044, by two-tailed paired t-test). Overall the muscle width tended to be thicker in the α-chymotrypsin–injected eyes than in the contralateral control eyes, but the change in width was not dependent on proximity to the injection site. NS, not significant. Monkey 1 was cynomolgus and monkeys 2–5 were rhesus.

*  Pseudophakic eyes.

†  Aphakic eye.

UBM in Live Monkeys.

UBM imaging of the ciliary body in the temporal quadrants of six rhesus monkey eyes (aged 6–26 years) before and after α-chymotrypsin lysis of the vitreous zonule showed increased accommodative forward ciliary body movement after versus before lysis (P = 0.044; Table 3, Fig. 13; Movie S2). This movement was observed in phakic eyes, pseudophakic eyes with intact posterior capsule, and the one truly aphakic eye without capsule. The latter eyes typically exhibited markedly decreased accommodative ciliary body movement when the intermediate vitreous zonule was intact. ²¹  

Table 3.

 

Forward Ciliary Body Movement in Monkey Eyes as Measured by UBM before and after α-Chymotrypsin

Monkey	Age (y)	Pre α-Chymotrypsin	Post α-Chymotrypsin	Post minus Pre α-Chymotrypsin	FCB Movement Increase (%)
1	25*	37.4	66.9	29.5	78.9
2	15	30.4	44.9	14.5	47.7
3	26	13.2	31.3	18.1	137.1
4	8†	91.0	80.8	−10.2	−11.2
5	8.5†	61.7	100.0	38.3	62.1
6	8†	49.8	103.6	53.8	108.0
Mean	—	47.3	71.3	24.0	70.4
SEM		11.1	11.9	9.0	21.0
P				0.044	0.02

 Data show the forward ciliary body (FCB) movement in six rhesus monkey eyes before and after α-chymotrypsin injection. FCB was determined as the narrowing of the angle that is formed between the inner aspect of the cornea and the anterior aspect of the ciliary body in the accommodated versus the unaccommodated state measured in degrees (Fig. 13). The eyes received an α-chymotrypsin injection in the temporal quadrant. Measurements were taken in the region of the injection site. FCB movement was significantly higher after α-chymotrypsin (Post) versus before (Pre) by two-tailed paired t-test. Monkeys 1–3 were stimulated to accommodate by central electrical stimulation, and monkeys 4–6 were stimulated to accommodate pharmacologically by carbachol iontophoresis.

*  Aphakic eye.

†  Pseudophakic eyes.

Figure 13.

 

Ultrasound biomicroscopic images of the 25-year-old rhesus monkey eye at rest and during supramaximal central stimulation to induce accommodation. Left: the aphakic eye after ECLE; right: after subsequent α-chymotrypsin lysis of the intermediate vitreous zonule and surgical removal of the lens capsule. The change in the ciliary body to cornea angle during maximal accommodation (bottom) is increased after lysis of the intermediate vitreous zonule fibers (arrows), although the difference at rest (top) is minimal.

Discussion

This study describes the complexity of a vitreous zonular system that helps to smoothly translate to the lens the driving forces for accommodation and disaccommodation generated by ciliary muscle contraction and relaxation. Our study also suggests how that structure allows and assists the very fine and rapid refocusing and maintenance of focus at multiple distances required by primate visual needs. ²⁶  

In relation to the Helmholtz theory, ²⁷ accommodation requires both centripetal and forward ciliary body movement, and the age-related loss of accommodation may be related to the loss of forward ciliary body movement. In relation to the Coleman (catenary) theory of accommodation, which suggests that accommodative shape changes in the lens are induced by vitreous pressure, ^28–30 age-related loss of ciliary body movement combined with increased lens size ^4,31,32 may alter the central catenary shape and thereby reduce accommodative amplitude. We prefer to not go beyond what we have just stated, to avoid overspeculation with respect to the various theories of accommodation. We have opted simply to publish the findings and provide some discussion of their meaning. 

This study unveils the potential for novel mechanisms and suggests the need to establish methods to include the vitreous membrane and vitreous zonule, along with the anterior and posterior zonular complexes, in future finite-element models of the primate accommodation system. This article also raises several interesting questions about the future of antipresbyopia surgery and treatment. 

The presence of vitreous strands in living humans was observed earlier by Coleman ³³ and in monkeys by Glasser et al. ¹⁸ using UBM. However, neither the strands themselves nor their anatomic relationships to other structures were definitively identified and characterized. Indeed, some researchers have labeled the vitreous strands simply as anterior hyaloid membrane. ²⁰  

With respect to forward ciliary body movement, the angle measured includes the inner aspect of the cornea and the anterior aspect of the muscle apex (not including the ciliary processes) which is quite distinct and easily measured in the iridectomized eye. Forward ciliary body movement as measured by UBM does not discern the individual contributions of the different areas of the muscle (i.e., longitudinal, circular, and reticular muscle portions) and what occurs in the different internal anatomic regions of the muscle cannot be precisely quantified. Nonetheless, the technique is a reliable indicator of overall forward ciliary body/muscle movement (i.e., muscle apex) ^3,21 based on the following: (1) The angle change reflects the translocation of the innermost aspect of the muscle (apex); (2) the change in forward ciliary body movement as measured by UBM after removal of the posterior restriction is corroborated by the morphologic findings in this article and historically ^1,2,5 ; (3) a qualitative examination of the dynamic video images (Movies S1 and S2) shows that the overall muscle movement is forward and inward without isolated pockets remaining static (with the exception of the outermost anterior longitudinal portion of the muscle which is anchored/fixed anteriorly at the scleral spur). Therefore, the overall forward movement of the muscle (including the muscle apex) could be inferred from the forward ciliary body movement measured by UBM, and the inward movement of the circular portion of the muscle could be inferred from gonioscopically measured inward ciliary process movement. Comparisons of forward versus centripetal/inward ciliary body movement (measured by UBM and goniovideography, respectively) in living monkey eyes are made elsewhere and are beyond the scope of this article. ³⁴  

There are only two regions in which the vitreous membrane is firmly attached to structures of the accommodative system over the entire circumference: anteriorly at the peripheral posterior lens capsule and posteriorly in the region of the ora serrata. The intervening separation allows for the movement of the zonular system needed for accommodation and disaccommodation. However, there are three other less-fixed fibrillar attachments, comprising the vitreous zonule that may stabilize and smooth the movement of the entire system and allow for finer accommodative control and reduced stress on the ora serrata and the lens capsule. This vitreous zonule, visualized by our improved SEM and UBM techniques, is surprisingly robust and is intimately related to the anterior, intermediate, and posterior vitreous membrane. 

Our findings concerning the anterior vitreous zonule add new information to that described previously in humans ^9–17 and to goniovideography observations previously reported for monkeys. ²³ The earlier goniovideography study included a video that showed the zonular attachments between the valleys of the ciliary processes and Wieger's ligament in vivo in the resting eye and during accommodation. ²³ The current SEM investigations indicate that, at most places, the anterior vitreous membrane and posterior zonular tines of the anterior zonular fork attaching to the posterior lens capsule are separated from each other, presumably allowing unrestricted movement of the system. There are, however, strands of anterior vitreous zonule that connect the zonular plexus with the anterior vitreous membrane. This anterior vitreous zonule may help to stabilize the anterior vitreous and thereby the position of the lens during accommodation. 

The cleft between the pars plana zonule of the ciliary body and the vitreous membrane is bridged by bands of intermediate vitreous zonule fibers. The cleft could provide a low-friction interface between the ciliary epithelium/pars plana zonule, and vitreous membrane when the ciliary muscle moves anteriorly and posteriorly during accommodation and disaccommodation, increasing the efficiency of the system. The intermediate vitreous zonule may also stabilize the vitreous membrane in its optimal contour, while dampening and smoothing the muscle's forward and inward movement. 

The posterior vitreous zonule with its adjacent vitreous membrane, the pars plana zonule, and the inner limiting membrane of the ciliary epithelium are joined in an attachment zone of approximately 0.5 mm in monkeys and an astonishing 3 to 4 mm in humans in anterior–posterior length, extending circumferentially around the eye in the region of the ora serrata. This loose fibrillar attachment of the several structures may also permit the forward–inward movement of the ciliary muscle/zonular vitreous system in a dampened manner so as not to acutely overstretch the peripheral retina and its attachment at the ora. This arrangement would minimize the risk of tearing the peripheral retina by accommodative effort. The greater length and thickness of this zone in humans than in monkeys may reflect the need for protecting the retina over the much longer human lifespan. 

With age, there is widening of the space between the ciliary body and vitreous membrane and reduction of forward movement of the posterior insertion zone. This change could restrict forward movement of the zonular plexus on accommodative effort. However, our regression analysis showed that age was more important in predicting accommodative amplitude than was either the cleft width or the forward movement of the posterior insertion zone. This finding is not surprising, given that other age-related changes that also affect accommodation are likely to occur in the eye concurrently. 

Nonetheless, consistent with the hypothesis of age-related posterior restriction of ciliary muscle/zonular movement, lysis of the posterior vitreous zonular system in a few eyes increased the forward movement of the ciliary muscle on central electrical or peripheral pharmacologic stimulation. Although the technique is not clinically applicable, the findings and interpretations further support age-related posterior restriction of ciliary muscle and zonular movement as both a pathophysiologic factor and a potential therapeutic target in presbyopia. 

Supplementary Materials

Movie S1 - 4.7 MB (.wmv) 

Movie S2 - 2.7 MB (.wmv) 

Footnotes

 Supported in part by NEI Grants R01EY10213 and R21EY018370 (PLK); the Ocular Physiology Research and Education Foundation; and DFG DR 124/7 (EL-D). The Wisconsin National Primate Research Center, University of Wisconsin-Madison, is funded by Base Grant 5P51 RR 000167 and Core Grant for Vision Research P30EY016665.

Footnotes

 Disclosure: E. Lütjen-Drecoll, None; P.L. Kaufman, None; R. Wasielewski, None; L. Ting-Li, None; M.A. Croft, None

The authors thank Johannes Rohen for invaluable help in preparing the specimens for SEM investigations and Elke Kretzschmar and Marco Gösswein for technical assistance.