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.

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).

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.

Age-related changes in the composition of proteins in the trabecular meshwork of the human eye.

The composition of the trabecular meshwork proteins of human eyes ranging in age from 36 days to 84 years was examined by polyacrylamide gel electrophoresis and amino acid analysis. Proteins of different molecular weights could be extracted from the tissue with acetic acid. Although their electrophoretic patterns became less distinct with increasing age, proteins of molecular weights ranging from 50 000 to 69 000 always prevailed. The amino acid compositions of the acetic acid-insoluble trabecular meshwork residues revealed the prevalence of collagenous proteins. The peptide maps produced by treatment with cyanogen bromide indicate that most of the fragments solubilized from the trabecular meshwork of younger eyes are derived from type I collagen. Beyond 40 years of age, the trabecular meshwork was resistant to cyanogen bromide and pepsin digestion. A rough estimate of the distribution of collagen types in the trabecular meshwork was based on 3-hydroxyproline/4-hydroxyproline ratios, indicating an age-related increase of type I collagen from about 55 to 70 per cent, and of type IV collagen from about 2 to 5 per cent of the total protein present. During ageing, some of the protein-bound methionine is oxidized to methionine sulfoxide, reaching about 35 per cent of the total methionine content at the age of 20 years and, with a slower rate of oxidation, a mean value of 40 per cent at 80 years of age. Electron-microscopic analysis of specimens remaining undissolved after cyanogen bromide cleavage and pepsin treatment no longer revealed regular collagenous fibrils but rather elastic-like fibers surrounded by wide sheaths consisting of fine fibrils with a regular cross-banding periodicity of 40-50 nm. In addition, clusters of so-called curly (lattice) collagen were found. The amino acid composition of this insoluble material suggests that altered collagen-like molecules prevail among the proteins of the residues.

Scanning electron microscopic studies of the zonular apparatus in human and monkey eyes.

Scanning electron microscopic studies of the zonular apparatus in 10 human and 17 monkey eyes revealed two functionally different sets of zonular fibers: the "main fiber" and the "tension fiber" system. The two systems are connected, forming a broad, sagittally oriented "zonular plexus" within the pars plicata of the ciliary body. The zonular plexus is attached to the ciliary epithelium by the tension fibers, which leave the main system and run obliquely forward to the epithelium deep in the valleys of the ciliary processes. Anteriorly, the zonular plexus splits into the two branches of the "zonular fork," which run respectively to the anterior and posterior aspect of the lens. Thus the zonular plexus can act as a fulcrum. The three-dimensional architecture of the zonular apparatus is consistent with a new concept of accommodation.

Regional differences in the fine structure of the ciliary epithelium related to accommodation.

The ciliary bodies of five monkey eyes and one human eye were subdivided into five zones. The ciliary epithelium with its bordering stroma was investigated electron microscopically. The number of cell organelles of the nonpigmented (NPE) and pigmented (PE) epithelium (mitochondria, rough endoplasmic reticulum, Golgi complexes); intercellular junctions between NPE and NPE, PE and PE, and NPE and PE (desmosomes, puncta adhaerentia, gap junctions, tight junctions); and fenestrations of the capillary endothelium were quantitatively evaluated. All these types of cell organelles, fenestrations of the capillary endothelium, and gap junctions in the NPE were found in greater numbers at the crests of the ciliary processes than in the valleys between processes. On the other hand, the number of puncta adhaerentia is significantly higher in the valleys than at the crests. In the valleys, the internal limiting membrane performs an elaborate network of electron-dense strands in which many fine zonular fibers terminate. These fibers are believed to belong to the "tension fiber system." Their firm attachment to the ciliary epithelium and the great number of intercellular junctions known as mechanical structures lend further support to our concept that these structures function as a fulcrum in the process of accommodation.

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.

Ultrahistochemical studies on tangential sections of the trabecular meshwork in normal and glaucomatous eyes.

The composition of the extracellular material of the cribriform meshwork was compared in five normal and 13 glaucomatous eyes of the same age group (58 to 70 years). Enzymatic digestion and histochemical methods applicable to electron microscopy were used. In both groups of eyes the ground substance was sensitive to chondroitinase ABC, and also the other methods showed no qualitative differences between the groups. In contrast, the fibrillar components of the extracellular material, which partly could be studied only after enzymatic treatment, showed qualitative differences between normal and glaucomatous eyes. In addition to the normal fibrous components, the glaucoma tissue contained large amounts of very fine fibrils. In seven eyes with end-stage glaucoma these fibrils filled the whole cribriform region. In these cases collagen fibers also appeared. Whether these were formed from the fine fibrils is not clear.

The fine structure of the cribriform meshwork in normal and glaucomatous eyes as seen in tangential sections.

Electron microscopic serial sections in a tangential plane through the inner wall of Schlemm's canal and the trabecular meshwork in normal and glaucomatous eyes revealed a characteristic network of elastic-like fibers (cribriform plexus), which is directly connected to the inner wall endothelium by a special fiber system (connecting fibrils). This cribriform plexus is also connected to the ciliary muscle system. Ciliary muscle tendons were found that not only show the same fine structure as the cribriform plexus but also join it. The ciliary muscle tone can therefore directly influence the fiber system of the cribriform plexus and its connections to the inner wall of Schlemm's canal. in eyes with chronic simple glaucoma, three types of plaques in the cribriform meshwork have been described after studies of sagittal sections. A comparison with tangential sections of the same piece of tissue shows that plaques of type II and III are in fact sections through the cribriform plexus and that only type I plaques are a separate entity.

Hyperopia and loss of accommodation following ciliary muscle disinsertion in the cynomolgus monkey: physiologic and scanning electron microscopic studies.

Twenty-three cynomolgus monkeys underwent 360-degree disinsertion and retrodisplacement of the ciliary muscle in one eye. Ten to 12 weeks after unilateral disinsertion, resting refraction in the "disinserted" eyes was more hyperopic than in the opposite eyes by 1.12 +/- 0.21 (mean +/- S.E.M.) diopters (p less than 0.001). Accomodative responses to intramuscular pilocarpine (2 or 3 mg/kg) were 0.90 +/- 0.14 (mean +/- S.E.M.) diopters in the disinserted eyes and 13.88 +/- 0.79 diopters in the opposite eyes. The induced hyperopia and loss of accommodation in the disinserted eyes seemed permanent, persisting for at least 14 months in one monkey and 29 months in three monkeys tested periodically after disinsertion. By light microscopy, the ciliary muscle in the disinserted eyes appeared normal and was contracted by pilocarpine. Scanning electron microscopy of the accommodative apparatus revealed retrodisplacement of the ciliary muscle, ciliary processes, and zonular plexus in the disinserted eyes. Structural alterations in the zonular apparatus seemed insufficient to account for the physiological findings. Hyperopia and loss of accommodation following ciliary muscle retrodisplacement are consistent with a new theory of zonular action during accommodation.

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.

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.

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.

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.

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.

Outflow facility studies in the perfused bovine aqueous outflow pathways.

We have recently developed a technique for constant pressure perfusion of the aqueous outflow pathway of the eye. Our preliminary studies, conducted in the calf eye, show surprisingly that the manipulations necessary for preparing the outflow pathways and attached corneoscleral shell for perfusion do not greatly disrupt normal aqueous outflow physiology and anatomy according to the following criteria: 1. facility of outflow is similar before and during outflow pathway perfusion 2. as in the intact eye, facility of outflow decreases with increased IOP 3. removal of outflow resistance tissue greatly increases facility of outflow 4. morphology of outflow tissues remains normal Use of the perfused outflow pathway model may enable the creation of valuable in vitro preparations which may provide much needed information about the pathogenesis of primary open-angle glaucoma.

Oxygen delivery to the anterior chamber of the eye--a novel function of the anterior iris surface.

By means of pO2-needle electrodes the oxygen tension in the anterior chamber of rabbit and cynomolgus monkey eyes was measured and correlated with the topography. It can be clearly shown that the pO2 in front of the pupil is substantially lower than in front of the anterior iris surface. It is evident that the O2-supply of the anterior chamber is provided by the iris vasculature. This result was confirmed by measurements after partial or total iridectomy. A brief recapitulation of the morphology of the iris showed that the abundance of iris vasculature is well understandable in view of these pO2 measurements.

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.

In vitro cultures of trabecular meshwork cells of the human eye as a model system for the study of cellular aging.

Cells from the human trabecular meshwork providing a drainage system for the outflow of aqueous humour in the eye were isolated and propagated in monolayer culture. Following serial subcultivation of the primary cultures, there was a gradual decline in the fraction of dividing cells with increasing population doubling level (PDL) resulting finally in growth cessation and disintegration of these 'senescent' cultures. The number of population doublings was at most 20. Senescent cultures revealed reduced glycosaminoglycan synthesis rates (as measured by [14C]glucosamine incorporation) with a relative decrease of hyaluronic acid and increase of heparan sulfate. Medium-supplied (exogenous) hyaluronic acid enhanced hyaluronic acid synthesis of trabecular meshwork cells cultured in a defined, serum-free medium. Ascorbic acid (25-200 micrograms/ml), which is found in very high concentration in the ocular aqueous humour, stimulated hyaluronic acid synthesis of confluent cultures, also. The functional significance of decreased hyaluronic acid (and elevated heparan sulfate) synthesis in the process of cellular aging in vitro (and in vivo?), as well as the importance of hyaluronic acid for the structural integrity and functional activity of the trabecular meshwork were discussed.