Cassandra Flügel

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.

Different cell populations in bovine trabecular meshwork: An ultrastructural and immunocytochemical study

Bovine outflow tissue differs markedly from that of humans. Tissue culture studies on the cells of this region are often compared with those of primate trabecular meshwork cells. A thorough cytological and immunocytochemical characterization of the cells of the bovine chamber angle is lacking. We have therefore investigated the cells of the pectinate ligament, the reticular meshwork, the region adjacent to the aqueous plexus, the connective tissue region between reticular meshwork and ciliary muscle and the ciliary muscle itself, ultrastructurally and immunocytochemically with staining for the cytoskeletal proteins vimentin and desmin, for alpha-smooth muscle-actin and rough endoplasmic reticulum (rER). In the pectinate ligament and in the region adjacent to the aqueous plexus, the cells were found to have especially abundant rER and glycogen in their cytoplasm. Vimentin was abundant in the reticular meshwork as positive staining was seen both in frozen and paraffin sections. Alpha-smooth muscle-actin could be found in the region connecting ciliary muscle and reticular meshwork as well as in a small area adjacent to the posterior capillary loops of the aqueous plexus. Ultrastructurally, these cells resembled myofibroblasts. The ciliary muscle cells stained both for vimentin and for alpha-smooth muscle actin.

Age does not affect contractile responses of the isolated rhesus monkey ciliary muscle to muscarinic agonists

In primates, ciliary muscle contraction causes accommodation and facilitates aqueous outflow. In living rhesus monkeys, accommodative, outflow facility, and ciliary muscle movement responses to cholinergic agonists all decline with age. We developed an apparatus to determine in vitro whether the latter is related to intra- or extra-ciliary muscle factors, and whether ciliary muscle contraction in the coronal (putatively more accommodation-relevant) and longitudinal (putatively more facility-relevant) vectors can be dissociated pharmacologically. In fresh ciliary muscle strips, carbachol and aceclidine each induced dose-dependent contraction in the longitudinal and coronal vectors. With neither drug was there any apparent dissociation of the responses in the two vectors. Atropine pretreatment completely prevented a supramaximal dose of carbachol from inducing ciliary muscle contraction in either vector. Ciliary muscle strips responded to several cholinergic agonists as well on day 2 (24-32 hours post-enucleation) as on day 1 (1-9 hours post-enucleation) when kept in a cell culture medium at 4 degrees C. By light microscopy, the general architecture of the ciliary muscle, the muscle bundles, and the single muscle cells appeared normal; however, cellular and nuclear swelling were apparent following the 32-hour culturing period. Contractile responses to near-maximal doses of carbachol and aceclidine did not vary markedly with age in either vector, suggesting that the age-related decrease in ciliary muscle mobility in vivo is due to extra-muscular restrictive factors rather than diminished muscular contractility.

Cell cultures of human ciliary muscle: growth, ultrastructural and immunocytochemical characteristics

Primary ciliary muscle cell cultures derived from human donors (16-91 years) were established and characterized by comparing them with ciliary muscle in tissue sections using immunocytochemical and ultrastructural methods. Monoclonal antibodies against desmin, vimentin, alpha-actinin, smooth muscle (sm) specific alpha-actin and von Willebrand factor were used. In tissue sections of the ciliary body, ciliary muscle cells, vascular muscle cells, pericytes, endothelial cells and fibroblasts stain for vimentin. Both types of muscle cells and the pericytes stain for alpha-sm-actin, but only ciliary muscle cells stain for desmin. For tissue cultures, explants of the meridional and partly the reticular portion of the ciliary muscle were dissected and grown directly or after digestion of the explant with collagenase. Ten primary cell cultures with a typical hill-and-valley growth pattern similar to smooth muscle cells and two with a growth pattern similar to fibroblasts were established. All cultures could be subcultured up to the fifth passage. In fibroblast-like cultures 5-10% of the cells stained for alpha-sm-actin. Staining for desmin was not observed. In smooth muscle-like cultures, all cells stained positive for alpha-sm-actin. Desmin staining was not seen in growing non-confluent smooth muscle-like cultures. In confluent cultures, about 10% of the cells stained positive for desmin, preferentially in areas where the cells had formed hills. No culture stained for von Willebrand factor. Staining for alpha-actinin in smooth muscle-like cultures showed that the dense bands of the myofilaments were arranged in register, similar to the typical ciliary muscle cell morphology seen in tissue sections. Ultrastructurally, the smooth muscle-like cultures showed the typical morphology of cultured smooth muscle cells. We conclude that the smooth muscle-like cultures consist of ciliary muscle cells.

Age-Related Loss of α-Smooth Muscle Actin in Normal and Glaucomatous Human Trabecular Mesh work of Different Age Groups

The trabecular meshwork of 45 normal human eyes ranging in age from 19 to 81 years and of six glaucomatous human eyes (33–78 years) was studied for the presence of contractile cells. For this purpose, the chamber angle was immunocytochemically stained with antibodies against α-smooth muscle (sm) actin. Within the various ages, the presence of α-sm actin labelled cells showed differences. In younger eyes, α-sm actin filaments were seen in nearly all cells of the trabecular meshwork. With increasing age, fewer cells stained for α-sm actin and were mainly seen in the posterior part of the trabecular meshwork and in the scleral spur. The main filtering portion of the meshwork contained nearly no stained cells. No differences were found between glaucomatous eyes and normal eyes of the same age group.

Histochemical differences within the ciliary muscle and its function in accommodation.

During accommodation, the ciliary muscle is known to move forward-inward. This movement depends on the stiffness of the ciliary muscle connections with the scleral spur. These connections are mediated by the tips of the meridional muscle. If the tips are weakened by pharmacological or surgical means, accommodation suffers. For normal accommodation, it is therefore necessary that the tips stiffen before the contraction of the main part of the muscle. We have therefore looked at the primate eye for enzymatic and ultrastructural differences between the tips and the bulk of the muscle viz, the reticular and circular portion. Myosin ATPase was stained after either alkaline or acid preincubation. Lactate dehydrogenase (LDH), succinic dehydrogenase (SDH), NADH-tetrazoliumreductase (TR) and lipids were stained using conventional methods. The results of the enzyme staining were a modest difference between the meridional tips and the bulk. The tips stained stronger for ATPase following both preincubation methods, and for LDH, whereas the bulk cells stained stronger for SDH, NADH-TR and lipids. The tips contained fewer mitochondria and more myofibrils. In all these respects, the tips of the meridional muscle resemble the fast fibers of striated muscle.

Hyaluronan synthase immunoreactivity in the anterior segment of the primate eye

The anterior segment of human and cynomologus monkey eyes was investigated for the presence of hyaluronan (HA) synthesizing cells using a polyclonal antibody against the enzyme HA synthase (HAS). In the chamber angle region the most intense staining was seen in the cell membranes of the corneal endothelium and in monkey eyes in the cells covering the posterior extension of the cornea (the operculum). The trabecular meshwork cells of the uveal and inner corneoscleral lamellae were also intensely stained. On the other hand, no staining was observed in the trabecular cells of the outer corneoscleral and the cribriform meshwork. The cell membranes of the inner wall endothelium of Schlemms canal were labelled only at their luminal surface.In the iris stroma and the trabeculum ciliare (the ciliary body band), labelled cells were also found, whereas the connective tissue of the ciliary muscle and the muscle itself did not contain HAS-positive cells. In the ciliary processes immunoreactivity was seen in the non-pigmented epithelial cells (NPE) covering the anterior tips of the processes, suggesting that HA found in the aqueous humor is produced by these cells. The pars plana NPE showed the most intense staining in the cells directly adjacent to the ora serrata region. The hyalocytes found in the neighborhood of the pars plana also showed intense HAS immunoreactivity. It is likely that both hyalocytes and NPE cells of the posterior pars plana release HA into the vitreous.

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.

Distribution of αB-crystallin in the anterior segment of primate and bovine eyes

The presence and distribution of αB-crystallin in the anterior segment of human, monkey and bovine eyes was investigated immunocytochemically. In all three species the most intense staining was seen in the lens and in the nonpigmented and pigmented epithelial cells covering the tips of the pars plicata of the ciliary body. The staining intensity of the ciliary epithelial cells was comparable to that seen in the lens fibers. Strong labeling was also found in the corneal endothelium.In bovine eyes the presence of αB-crystallin in lens, ciliary epithelium of the pars plicata and corneal endothelium was also shown by biochemical analysis using SDS-PAGE and immunoblotting. Cum. Eye Res. 12: 871-876, 1993.

Histochemical demonstration of carbonic anhydrase in gills and opercular epithelium of seawater- and freshwater-adapted killyfish (Fundulus heteroclitus)

Gills and operculum of seawater- and freshwater-adapted killyfish (Fundulus heteroclitus) were stained histochemically for carbonic anhydrase (CA). In the seawater-acclimatized specimens, CA was found predominantly in the chloride cells which were considerably larger than in the freshwater-adapted ones. Within these cells, the reaction products were concentrated in the apical parts of the cytoplasm. In contrast, chloride cells of freshwater-adapted fish were not, or only faintly, stained both in gills and opercular epithelium. Reaction products for CA were seen additionally in the cytoplasm of the outer respiratory cells lining the lamellae of gills both in seawater- and freshwater-adapted fish.