Superior cervical ganglionectomy in monkeys: light and electron microscopy of the anterior eye segment.

Morphological changes in the anterior eye segment of eight cynomolgus monkeys were investigated 2 days to 2.2 years after unilateral surgical superior cervical ganglionectomy (SCGx). SCGx was confirmed by histologic examination of the excised surgical specimen and persistent ipsilateral miosis. In four short-term monkeys (2, 4, 7 and 11 days), iris, ciliary muscle and trabecular meshwork were studied by electron microscopy. In the other four longer-term monkeys (3 week, 4 week, 5 week, 2.2 year) the anterior eye segment was investigated with tyrosine hydroxylase immunohistochemistry (TH-IR) and catecholamine fluorescence (CF). Electron microscopy of experimental eyes showed characteristic signs of Wallerian degeneration in numerous nerve fibers and terminals in the iris, but to a lesser extent in the ciliary muscle and the trabecular meshwork. TH-IR and CF showed marked interindividual differences. In all experimental eyes, there was a marked reduction, but never a complete absence of adrenergic nerves in the iris. In two animals (4 week and 2.2 years), the adrenergic innervation of the ciliary body and the chamber angle was similarly reduced. In contrast, in the experimental eyes of the other two animals (3 and 5 weeks), changes in adrenergic innervation to the ciliary body and chamber angle were minimal or absent. The results indicate that following apparently complete SCGx in the cynomolgus monkey, reduction of adrenergic innervation to the iris as evidenced by pupillary physiology, electron microscopy, TH-IR and CF does not guarantee reduction in adrenergic innervation to the ciliary body and trabecular meshwork. SCGx may not extirpate all third order sympathetic neurons in the distal stump, or there may be a significant contribution of accessory ganglion cells to the adrenergic innervation of the anterior eye segment.

Scanning electron microscopic study of episcleral arteriovenous anastomoses in the owl and cynomolgus monkey.

In the present study we have analyzed the architecture of the episcleral microvasculature in the owl and cynomolgus monkey using scanning electron microscopy of resin casts. Due to the topical pretreatment with nitroprusside the complete vasculature including segments of the aqueous humor outflow channels could be visualized. We found that 1-3 mm posterior to the limbus corneae the episcleral vessels form numerous arteriovenous anastomoses (AVA) in the size of small arterioles and venules. These AVA are also located at the collector channels near Schlemm's canal and at the episcleral venous plexus which drains the collector channels. It is assumed that the AVA might influence not only the circulation in the episcleral venous plexus but also in the aqueous humor outflow routes.

Short-term hemodynamic changes in episcleral arteriovenous anastomoses correlate with venous pressure and IOP changes in the albino rabbit.

In the present study we have determined the effects of acute diameter changes in the recently discovered episcleral arteriovenous anastomoses (AVA) on episcleral venous pressure and intraocular pressure (IOP) in the rabbit eye. The pressure was measured in episcleral arterioles, AVA and veins (vascular pressure) with a pressure chamber mounted on the tip of a microendoscope. After constriction of the AVA following topical administration (100 micrograms) of epinephrine we observed a decrease in the episcleral vascular pressure as well as in the IOP. Acute widening of the AVA after topical administration of 5mg nitroprusside led to a significant increase in the pressure of arterioles, AVA, veins and IOP which lasted several min. After 0.5mg topical nitroprusside the blood flow in the AVA increased. No significant effects were seen in the intravascular pressures; the IOP had a tendency to decrease. Experimental lowering of the IOP to 10mmHg caused an increase of vascular diameters and of blood flow in the AVA. It is assumed that-at least in drastic hemodynamic disorders-the degree of the AVA-perfusion might influence aqueous humor outflow and IOP due to changes of the episcleral venous pressure.

In vivo observations of the episcleral vasculature in the albino rabbit.

SUMMARY: The recently described arteriovenous anastomoses of the episcleral vasculature were studied in the albino rabbit by in vivo microscopy and microendoscopy. The arteriovenous anastomoses lead directly into the episcleral venous plexus which drains aqueous humor via well-visible aqueous veins. In the arteriolar segment of the arteriovenous anastomoses the blood flow velocity, measured by intra-arterial bolus injections of Evans blue, decreases drastically toward the venular segment of the arteriovenous anastomoses. After topical application of epinephrine, the arteriolar segment contracts faster and to a greater degree than all other parts of the episcleral vasculature. In the aqueous veins this stoppage of the blood inflow from the arteriovenous anastomoses may allow an increase in the amount of aqueous humor drained from the anterior chamber. The simultaneously measured intraocular pressure decreases significantly. Experimental elevation of the intraocular pressure from 18 to 30 mm Hg causes a decrease in diameter and blood flow in the arteriovenous anastomoses. These effects are reversed by returning to normal intraocular pressure levels. It is assumed that the arteriovenous anastomoses play a role in aqueous humor drainage by the episcleral venous plexus.

Functional morphology of the episcleral vasculature in rabbits and dogs: presence of arteriovenous anastomoses.

SUMMARY: Scanning electron microscopic analysis of corrosion casts of the intra- and episcleral vasculature in the limbic region of two species (rabbit and dog) revealed typical arteriovenous anastomoses representing shunts between the long radial arterioles, branches of the anterior ciliary arteries, and the episcleral venous plexus. The arteriovenous anastomoses consist of a small arteriolar segment and a wide, funnel-like venous segment. Using immunohistochemical techniques it was shown that the wall of the arteriolar segment consists of smooth muscle cells containing alpha-smooth muscle actin. In the dog, the vessel wall of the arteriovenous anastomoses also contain typical epithelioid cells, the structure of which was analyzed by transmission electron microscopy. In the episcleral vasculature, a capillary network is lacking, an elaborate wide venous plexus is developed instead. It is assumed that this plexus with the help of the newly described arteriovenous anastomoses may influence aqueous humor drainage and thereby also intraocular pressure.

Ultrastructure of the trabecular meshwork in untreated cases of primary open-angle glaucoma (POAG).

Thirty-one trabeculectomy specimens of patients suffering from primary open-angle glaucoma (POAG) who had received no, or only minor, medication prior to surgery were ultrastructurally and quantitatively analysed. Most of the specimens revealed thickened trabeculae, increased amounts of plaque-material deposited within the cribriform layer and an abundance of long spacing (lattice) collagen. The uveal meshwork was partly deprived of cells whereas the cribriform layer often contained numerous enlarged, light cells with many small mitochondria and lysosomes but no prominent endoplasmic reticulum or Golgi complexes. The quantitation of sheath-deprived plaque material (SD plaques) in a defined area of inner and outer wall of Schlemm's canal showed no significant difference between the untreated cases studied here and the treated ones studied previously. However, both groups had significantly higher amounts of SD plaque material in the inner wall than normal controls of a similar age range.

[Immunocytochemical and ultrastructural studies of human scleral spur. Characterization of a contractile cell population]. / Immunzytochemische und ultrastrukturelle Untersuchungen am menschlichen Skleralsporn. Charakterisierung einer kontraktilen Zellpopulation.

Contraction of the ciliary muscle induced by cholinergic drugs causes movement of the scleral spur and separation of the trabecular meshwork lamellae. The effect of epinephrine, however, does not seem to be mediated by ciliary muscle tone. We investigated the scleral spur in 37 human eyes (age 17-87 years). Serial tangential sections were studied with ultrastructural and immunocytochemical methods. The ciliary muscle cells do not enter the scleral spur, but their tendons, which consist of elastic fibers, are continuous with the elastic fibers in the scleral spur. Within the scleral spur is found a population of circularly oriented and spindle-shaped cells. The scleral spur cells form no bundles, but are loosely aggregated. They have long cytoplasmic processes and are connected to each other by adherence type and gap junctions. The scleral spur cells show intense staining for smooth-muscle-specific alpha-actin and myosin. Ultrastructurally, the scleral spur cells contain abundant actin filaments, but otherwise do not show the typical ultrastructure of ciliary muscle cells. The scleral spur cells do not express a complete basal lamina. They form individual tendinous connections with the elastic fibers in the scleral spur, which are continuous with the elastic fibers of the trabecular meshwork. The scleral spur cells are in close contact with nerve terminals containing small granular vesicles that are typical for adrenergic terminals. We conclude that the scleral spur cells are contractile myofibroblasts. Their contraction might be involved in the effects of epinephrine on the aqueous outflow.

Postnatal development of skull base, neuro- and viscerocranium in man and monkey: morphometric evaluation of CT scans and radiograms.

Postnatal development of the neuro- and viscerocranium with special reference to the maxillodental structures was studied morphometrically by analyzing computer tomograms and radiograms of human and monkey heads of different age groups. The following parameters were used: the prognathic angle, the clivus angle, the palate-incisivus angle, the interincisival angle and the viscerocranial quotient. In the newborn primates including man, all parameters measured were relatively similar; postnatally, however, characteristic differences in the growth pattern between man and monkey were developing. In monkey, a marked prognathic growth of the viscerocranium was found associated with characteristic positional changes of the frontal teeth, whereas the growth of the neurocranium was retarded. Here, unlike the human, a flattening of the skull base was observed. In contrast, the human skull showed no major proportional changes during its postnatal development compared with the original spherical skull form of the newborn.

Contractile cells in the human scleral spur.

The scleral spur in 37 human (age 17-87 years) and six cynomolgus monkey eyes (2-4 years) was investigated. Serial meridional and tangential sections were studied with ultrastructural and immunocytochemical methods. The bundles of the ciliary muscle do not enter the scleral spur, but their tendons, which consist of elastic fibres join the elastic fibres in the scleral spur. Within the scleral spur a population of circularly oriented and spindle-shaped cells is found. In contrast to the ciliary muscle cells, the scleral spur cells form no bundles, but are loosely aggregated. They have long cytoplasmic processes and are connected to each other by adherens-type and gap junctions. They stain intensely for alpha-smooth muscle actin, myosin and vimentin. In contrast to the ciliary muscle cells, they do not stain for desmin. Ultrastructurally, the scleral spur cells contain abundant thin (actin) filaments, but do not otherwise show the typical ultrastructural features of ciliary muscle cells. The scleral spur cells do not express a complete basal lamina. They form individual tendinous connections with the elastic fibres in the scleral spur, which are continuous with the elastic fibres of the trabecular meshwork. The scleral spur cells are in close contact with nerve terminals containing small agranular (30-60 nm) and large granular (65-110 nm) vesicles but also with terminals containing small granular (30-60 nm) vesicles which are regarded as typical for adrenergic terminals. We conclude that the scleral spur cells are contractile myofibroblasts. Their contraction might influence the rate of the aqueous outflow.

Age-related changes of the human ciliary muscle. A quantitative morphometric study.

The age-related changes of the ciliary muscle of human eyes (33-87 years) were studied on histological meridional sections. Eighty-five melanoma eyes and 10 eyes of normal donors were investigated. The total area and the length of the muscle, the area of the three main portions and the distance of the inner apex of the muscle to the scleral spur were determined and correlated with age. Total area and length of the muscle show a continuous and significant decrease with age. The area of the longitudinal and reticular portion continuously decreases, whereas the area of the circular portion significantly increases with age. The decrease in area is more pronounced in the longitudinal portion than in the reticular portion of the muscle, which shows an age-related increase in connective tissue. In addition, the distance of the inner apex of the muscle to the scleral spur shortens continuously. Thus, with increasing age the ciliary muscle adopts an anterior-inward position. A similar form is seen in young eyes after ciliary muscle contraction only. There might be a functional relationship between the observed age-changes in the ciliary muscle system and the phenomenon of the so-called 'lens paradox' (steepening of the anterior and posterior curvatures of the disaccommodated lens with age).

The effect of epinephrine on ciliary process vasculature and IOP studied by intraocular microendoscopy in the albino rabbit.

The effect of epinephrine on intraocular pressure (IOP) and the hemodynamics of the ciliary process vasculature in albino rabbits was studied by intraocular microendoscopy. Intraarterial application of epinephrine (15, 50, 250 ng/kg bw) lead to an immediate vasoconstriction and a reduction in blood flow velocity (BFV) in the iridial and major ciliary processes lasting from 30 to 120 sec. This anemic phase was followed by a hyperemic phase of about 60 to 240 sec. showing a vasodilation up to 150% of the initial diameters and an increase in BFV. The hyperemic phase can be prevented by pretreatment with indomethacin. Simultaneously measured IOP decreases in the anemic and increases in the hyperemic phase parallel with the changes in vascular diameter. After topical administration of epinephrine (25-50 micrograms/kg bw) a marked vasoconstriction followed by a vasodilatory phase was similarly found. However, the reactive changes of the ciliary process vasculature lasted considerably longer. The anemic phase lasted 15 minutes, the hyperemic phase 40 to 60 min. Again, this hyperemia can be prevented by indomethacin-pretreatment. In the iridial processes the anemic phase persisted till 70 minutes. No hyperemia and no substantial influence of indomethacin-pretreatment was found in this territory. In the anemic phase the IOP decreased in average from 20 mmHg to 15 mmHg. However, in contrast to the reactive changes of the IOP after intraarterial epinephrine application, the IOP did not increase again in the hyperemic phase, but decreased further to about 12 mmHg. After pretreatment with indomethacin the IOP remained at the level of 15 mmHg. The short-term IOP-changes after i.a. application of epinephrine, mirror the vasoconstrictory and vasodilatory reactions in the ciliary processes and might be due to volume changes in the eye (plethysmographic effect). However, the long lasting IOP reduction after topical epinephrine in the hyperemic phase can not be due to vascular reactions in the ciliary processes. There must be other factors responsible for the long lasting pressure reducing effect of epinephrine.

The craniofacial proportions and laryngeal position in monkeys and man of different ages (a morphometric study based on CT-scans and radiographs).

Using CT-scans and radiographs, sagittal planes through the head and neck of men and monkeys at different ages were analyzed morphometrically for their craniofacial proportions and laryngeal position. In monkeys, a continuous prognathic growth of the splanchnocranium was found within the first 3 years. The neurocranial growth, however, was markedly reduced. The larynx of monkeys showed only a slight descensus with age. In contrast to this, the growth of the splanchnocranium in man did not change the craniofacial proportions significantly. The larynx, however, descended markedly within the first two years of life. In adults, the final position of the larynx was nearly 3 vertebral bodies further caudally than in the newborn. The differences in the postnatal position of the larynx, which is essential for the development of speech, are explained by differences in the growth pattern of human and monkey skulls.

Outflow facility studies in the perfused human ocular anterior segment.

We have recently developed a tissue model of the human aqueous outflow pathway involving placement of the eviscerated anterior corneoscleral shell, [with lens and uveal tissue removed but trabecular meshwork (TM) attached] onto a specialized perfusion apparatus. The TM and associated outflow tissues are perfused with culture medium at a physiologically-relevant perfusion pressure in a 5% CO2 environment at 37 degrees C. Under these conditions, the perfused outflow tissues are similar for several days, to the human and/or subhuman primate outflow system in vivo with regard to morphology as well as several functional parameters. Measured facility of outflow (0.271 +/- 0.018 microliters min-1 mmHg-1, n = 79) is similar to facility values obtained by tonography in living human beings. Moreover, outflow facility decreases in a linear fashion with increased perfusion pressure by 1.4% mmHg-1. Finally the removal of the TM results in a 41% decrease in measured outflow resistance. The ability to study viable human outflow tissue for at least several days and the opportunity to establish a model which serves as an alternative to animal testing, point to the potential importance of this technique in investigating the biology of the aqueous outflow system.

Parasympathetic denervation of the ciliary muscle following panretinal photocoagulation.

Cynomolgus monkeys underwent unilateral panretinal scatter photocoagulation (PRP) and/or nasal and temporal horizontal retinal meridional photocoagulation (HRMP) with xenon arc or argon or krypton laser light. Shortly thereafter, in the PRP-treated eyes, accommodative responsiveness to topical eserine and electrical stimulation of the Edinger-Westphal nucleus (EWN) was diminished, accommodative responsiveness to intramuscular (i.m.) pilocarpine was enhanced, and the number of muscarinic receptors in the ciliary muscle was reduced compared to the contralateral controls. In most instances, these parameters returned to normal over 6-12 wks and the abnormalities could be induced again by another round of PRP. However, in some PRP-treated eyes, accommodative responsiveness to EWN stimulation and topical eserine remained subnormal permanently (greater than 1 yr). Shortly after HRMP alone, accommodative responses to i.m. pilocarpine, topical eserine, and central stimulation did not differ markedly in the treated and control eyes. Morphologic studies 1 to 78 wk following PRP revealed that myelinated and unmyelinated nerves within the entire circumference of the choroid and ciliary muscle were severely damaged early on. The number of unmyelinated nerves between the individual ciliary muscle fibers was drastically reduced, those which remained were swollen or deteriorated, and agranular synaptic vesicles were rarely seen. Thereafter, the nerves in the choroid and ciliary muscle gradually regenerated. Following HRMP, only the choroidal nerves which passed through the photocoagulated areas and the ciliary muscle nerves in the corresponding meridians showed signs of deterioration, and there was minimal effect on the physiologic responses examined. These findings collectively indicate that intraocular parasympathetic denervation of the ciliary muscle is produced by PRP, although all nerve types are likely damaged.

Posterior attachment of ciliary muscle in young, accommodating old, presbyopic monkeys.

The authors studied the posterior attachment of the ciliary muscle in seven young (3-10 yr) and five old (26-34 yr) rhesus monkeys by light microscopy and electron microscopy. Posterior attachment of the muscle bundles consisted of elastic tendons, exclusively. The elastic tendons were continuous with the elastic lamina of Bruch's membrane and were also connected by smaller elastic fibers to an elastic meshwork that surrounds the pars plana vessels. In some areas, the tendons formed focal contacts with the endothelial cells. The authors found that in old eyes, the tendons and the elastic fibers of the posterior ciliary body showed pronounced structural changes. The tendons appeared thickened, showed increased amounts of associated microfibrils, and were surrounded by dense layers of thick collagen fibrils. An increased amount of collagen fibrils was also seen between the elastic layer of Bruch's membrane and the pigmented epithelium. A mechanical link between those collagen fibrils and the elastic fibers is suggested by the presence of osmiophilic points of contact. The age-related increase in elastic fibrillar material could cause decreased compliance of the posterior insertion of ciliary muscle and could be an essential factor for presbyopia in rhesus monkeys.

Spectrometric measurements in the anterior eye vasculature of the albino rabbit--a study with the EMPHO I.

Intracapillary haemoglobin oxygenation (oxygen saturation) and haemoglobin concentration (corresponding with blood volume) were measured in different regions of the albino rabbit anterior eye segment using the Erlangen micro-lightguide spectrophotometer (EMPHO I). The tip of the microlightguide (active diameter 210 microns, cover diameter 0.5 mm) was placed in the vitreous by a goniometric mounting and controlled by a micromanipulator. After stepwise elevation of intraocular pressure (IOP) the haemoglobin oxygenation decreased slightly in the iris at an IOP-level of 60 mmHg, whereas in the three regions of the ciliary processes and in the peripheral choroid haemoglobin oxygenation did not decrease until values of 80 mmHg were applied. In contrast, haemoglobin concentration decreases when the IOP increases in all regions except in the pars plana where the haemoglobin concentration increased at 40 and 60 mmHg. The most pronounced reactive hyperaemia was found in the major ciliary processes. Quite passive changes of haemoglobin concentration were seen in the peripheral choroid. Effective regulatory mechanisms must exist which result in IOP-independent (20-60 mmHg) haemoglobin oxygenation, whereas pronounced changes in intracapillary haemoglobin concentration were observed. The decay of both parameters at IOP 80 mmHg is indicative of an exhaustion of their regulatory capacity. Epinephrine topically applied onto the conjunctival sac or injected into the common carotid artery led to a short-term decrease of haemoglobin concentration and, later, a hyperaemic response. A short-term haemoglobin deoxygenation was found especially in the major ciliary processes.

Morphology of the trabecular meshwork and inner-wall endothelium after cationized ferritin perfusion in the monkey eye.

Nine eyes of five cynomolgus monkeys were perfused through the anterior chamber with cationized ferritin (CF) at normal and increased intraocular pressure. After perfusion with glutaraldehyde, the morphologic appearance of the endothelial lining of Schlemm's canal (SC) and of the adjacent regions was analyzed using tangential and sagittal serial sections. The CF, which binds to negatively charged surfaces, was observed to be adherent to the free surfaces of trabecular cell membranes and to accumulate in the cribriform layer underlining the endothelial lining of SC. Tangential sections of the inner-wall endothelium demonstrated that separations of the adjacent cell membranes occur between the tight junctions forming openings or lacunae and bent, tunnel-like channels that represent continuous paracellular pathways. Complete staining of these inner-wall paracellular pathways with CF were observed indicating that the adjoining membranes are negatively charged and that the perfused fluid had passed through these intercellular channels. These paracellular pathways appeared enlarged and were more easily identified at elevated perfusion pressure. In general, intracytoplasmic vacuoles demonstrated heavy staining with CF on their luminal surface but only faint staining on the adluminal (cribriform-facing) surface. Apparent giant vacuoles were observed to be often not real intracellular vacuoles but rather dilatations of the paracellular spaces. This study demonstrates that there are paracellular routes through the inner-wall endothelium by which high molecular-weight substances such as ferritin and macrophages can leave the anterior chamber. Probably there are both transcytoplasmic and paracellular mechanisms of aqueous outflow that may vary under different conditions of pressure or flow.

Scanning electron microscopic study on the vasculature of the human anterior eye segment, especially with respect to the ciliary processes.

The architecture of the vasculature of the human anterior eye segment was studied by scanning electron microscopy of vascular resin casts. Regarding the major vessels it was found that the perforating branches of the anterior ciliary arteries (ACA) form an anastomozing circle which lies in the posterior portion of the ciliary muscle (intramuscular circle). The ACAs supply the outer and posterior parts of the ciliary muscle, partly the iris, and the peripheral choroid by recurrent ACA branches. The major arterial circle of iris (MACI) which lies more anteriorly is formed mainly by the long posterior ciliary arteries and supplies the inner and anterior portion of the ciliary muscle, the iris and the ciliary processes. The ciliary process vasculature consists of three different vascular territories with discrete arterioles and venules. The first vascular territory which is located at the anterior end of the major processes, is drained posteriorly by venules which pass the ciliary body without greater connections to the venules of the major ciliary processes. The second and third territories comprise the vasculature of the major and minor ciliary processes drained posteriorly by venules which are located at the margin of the ciliary processes. In supravital experiments with human autopsy eyes, a characteristic segment of arterioles supplying the first and second territory was found to be constricted after immersion with epinephrine in a similar way as in cynomolgus monkeys in vivo. Although the general arrangement of the ciliary process vessels is similar to that of the cynomolgus monkey, there are characteristic differences in the size of the territories and in the pattern of the capillary networks. The division of the ciliary process vasculature into three vascular territories may reflect a functional differentiation in the process of aqueous humour production.

Age-related changes of the ciliary muscle in comparison with changes induced by treatment with prostaglandin F2 alpha. An ultrastructural study in rhesus and cynomolgus monkeys.

The relationship between individual ciliary muscle cells and the surrounding connective tissue was studied in the eyes of three normal, young (3-4 years) cynomolgus monkeys (Macaca fascicularis), three aged (34-36 years) rhesus monkeys (Macaca mulatta) and seven young (3-7 years) cynomolgus monkeys topically treated with prostaglandin F2 alpha (PGF2 alpha) for 4-8 days. In normal eyes, collagen fibrils and microfibrils are in places in continuity with the muscle cells' basal lamina, which is connected to the cell membrane by fine fibrillous material. In old eyes, the basal lamina is markedly thickened, masking the connection of fibrils with the muscle cells' membrane. A distinctive finding in several muscle cells of old eyes are electronlucent clefts, 60-80 nm wide, between basal lamina and cell membrane, which are not transversed by fibrils or fibrillous material. The cell membrane of these muscle cells shows large folds filled with disarranged myofilaments. Additionally, these cells contain inclusion bodies consisting of concentrically arranged double membranes. Following treatment with PGF2 alpha, similar changes are seen in young animals, too. Here, the muscle cells have lost their connection to the extracellular fibrils due to a PGF2 alpha-induced lysis of extracellular material. Lack of attachment between basal lamina and altered muscle cells in aged eyes might indicate an involvement of the extracellular matrix in age-related changes of the individual ciliary muscle cells.