Characterization of an in vivo model for the study of eyelash biology and trichomegaly: mouse eyelash morphology, development, growth cycle, and anagen prolongation by bimatoprost.

BACKGROUND: Hypertrichosis or alopecia of the eyelashes is associated with various diseases or may be drug induced. Although neither increase nor loss of eyelashes is life threatening, eyelash disorders can be psychologically disturbing. However, as control of eyelash growth and the underlying mechanisms of eyelash hypo- or hypertrichosis are largely obscure, available therapy is limited. OBJECTIVES: To improve this situation, we sought to establish a pragmatic, well-defined mouse model for the study and pharmacological investigation of eyelash follicle biology. METHODS: We took a morphometric approach to establish an eyelash model using female C57BL/6J mice by comparing with pelage hairs and highlighting the differences. We next applied a hypertrichosis-triggering agent and investigated its effect using the model. RESULTS: In eyelashes, a synchronized growth cycle was observed after morphogenesis but was completed earlier than pelage hairs. Exogen was strictly regulated and occurred in every cycle in the eyelash. Otherwise, general morphological features of mouse eyelashes (shafts, follicles, morphogenesis and growth cycle) were comparable with those of pelage hairs. The eyelash growth-stimulatory agent in humans, bimatoprost, significantly extended the duration of anagen, resulting in more and longer eyelashes, but there was no evidence of follicle neogenesis. CONCLUSIONS: This study shows that mouse eyelashes offer an excellent in vivo model for the quantitative and qualitative analysis of eyelash morphology, development, growth cycle, exogen and pharmacological modulation. This model will help to elucidate the unknown molecular controls of eyelash growth, and to develop novel drugs to treat eyelash disorders.

Antiglaucoma EP2 Agonists: A Long Road That Led Somewhere.

For >2 decades, EP2 agonists have been the subject of antiglaucoma research and development by scientists in industry and academia around the world. The road has led to the recent approval of the first drug of this class. This article reviews the development of EP2 agonists from conception to clinical approval, discussing pharmacology, structure, biodistribution, therapeutics, and drug delivery. An extensive list of source references is provided for the reader's benefit.

[Primary open angle glaucoma. Morphological bases for the understanding of the pathogenesis and effects of antiglaucomatic substances]. / Primäres Offenwinkelglaukom. Morphologische Grundlagen zum Verständnis der Pathogenese und der Wirkung antiglaukomatöser Substanzen.

The pathogenesis of glaucomatic illnesses is poorly understood. An increase in ocular pressure can be caused by an increase in the secretion of aqueous humour or a reduction in its outflow. In the elderly, outflow is reduced while at the same time less aqueous humour is produced. This balance is easily disturbed, so that age represents a risk factor for glaucoma in addition to increased ocular pressure. Therapeutic possibilities involve, on the one hand, reducing the secretion of aqueous humour, for example using, beta blockers, carbonic anhydrase inhibitors and clonidine. On the other hand, aqueous humour outflow can also be influenced by drugs. Conventional outflow is increased by the administration of miotics. The uveoscleral outflow can be increased by prostaglandin derivates. Drugs which only influence trabecular outflow are not yet available. Future therapeutic possibilities involve new aspects of the pathophysiology, e.c. the use of growth factors, free radical scavenging enzymes and choroidal blood flow.

Functional morphology of the trabecular meshwork in primate eyes.

The trabecular meshwork forms most of the resistance to aqueous humor outflow needed for maintenance of a pressure gradient between intraocular pressure of approximately 17 mmHg and venous pressure of approximately 10 mmHg. The composition of the extracellular material in the subendothelial or cribriform layer seems to be mainly responsible for outflow resistance. The aqueous humor pathways through the subendothelial layer can be influenced by ciliary muscle contraction and presumably also by contractile elements recently found both in trabecular meshwork and scleral spur. Pharmacologically induced disconnection of inner wall and cribriform cells leads to wash out of extracellular material through breaks of the endothelial lining of Schlemm's canal and to increase of outflow facility. In glaucomatous eyes the resistance to aqueous humor outflow is increased due to an increase in different forms of extracellular material deposited within the cribriform layer. The amount of this newly developed extracellular material is correlated with loss of axons in the optic nerve, indicating that a common factor is responsible for both changes. To investigate the effect of various factors on the biology of trabecular cells monolayer cultures derived from cribriform and corneoscleral trabecular meshwork have been established. The two cell lines can be differentiated because cribriform cells in vivo as in vitro stain for alphabeta-crystallin whereas the corneoscleral cells remain unstained. The effect of TGFbeta, a growth factor increased in aqueous humor of glaucomatous eyes and glycocorticoids on trabecular meshwork cells show typical changes in formation of extracellular matrix components and of stress proteins. Dexamethasone and oxidative damage also lead to increase of trabecular meshwork inducible glucocorticoid response (TIGR) protein. A mutation of the TIGR-gene family has recently been found in families with juvenile and chronic simple glaucoma. Future research has to clarify the significance of these genetic factors for the pathophysiology of glaucoma and the role of trabecular cell activity in this respect.

Classification of human scleral spur cells in monolayer culture.

Aqueous humor outflow in primate eyes can be facilitated by ciliary muscle contraction, thereby widening fluid pathways through the trabecular meshwork. Recently in the scleral spur smooth muscle (sm) alpha-actin positive myofibroblast-like cells have been described which are in contact with the elastic fiber system of both the spur and trabecular meshwork. In the vicinity of these cells nerve terminals have been described. It is speculated that contraction of scleral spur cells can facilitate aqueous humor outflow, too. To provide a tool for further physiological and pharmacological studies monolayer cell cultures of human scleral spur have been established and characterized. For this purpose, cells derived from scleral spur, outer and inner trabecular meshwork and ciliary muscle tips from 7 donor eyes (43-87 years-old respectively, obtained 3-7 h post mortem) were grown in tissue culture medium and the different monolayer cells classified by their growth characteristics, and by immunohistochemical staining for vimentin, alpha-sm-actin, desmin, and alpha B-crystallin, respectively. In addition, the presence of alpha B-crystallin mRNA and desmin mRNA was verified using the polymerase chain reaction (PCR)-method. We were able to characterize and distinguish human scleral spur cells from adjacent ciliary muscle and trabecular meshwork cells. Scleral spur cells (SPC) grew slower than ciliary muscle cells (CMC) but much faster than trabecular meshwork cells (TMC). All cells showed the same staining characteristics in vitro as they did in vivo. Scleral spur cells stained for vimentin and alpha-sm-actin, but not for desmin and alpha B-crystallin. The corresponding mRNAs of the latter two proteins could not be detected by PCR in the spur cells. Cells grew out from all donor eyes so that they actually provide a tool for further experimental studies.

alpha B-crystallin in the primate ciliary muscle and trabecular meshwork.

The presence of alpha B-crystallin, a protein with heat-shock protein-like properties, has been demonstrated in ciliary muscle and trabecular meshwork derived from human and monkey eyes using immunohistochemical and polymerase chain reaction methods. Both frozen sections and cultured cells have been analyzed. In the ciliary muscle, alpha B-crystallin staining is localized in the region of the dense bands, in the cytoplasm of the muscle cells and in the Schwann cells of the nerves supplying the muscle. In the trabecular meshwork, two cell types could be distinguished on the basis of alpha B-crystallin occurrence. Whereas the trabecular cells covering the lamellae were virtually devoid of the protein, the subendothelial or cribriform cells contained relatively large amounts in parallel with a higher alpha B-crystallin mRNA level.

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.

Immunohistochemical study of extracellular material in the aged human synovial membrane.

Types III, IV, VI collagen and laminin distribution in synovial tissue of seven autopsy knee joints from old human donors (69-94 years of age) were investigated with immunocytochemical and immunohistochemical methods. The synovial intima is separated from the subintimal tissue by an intermediate fibrillar zone rich in staining for type III collagen. In the intima basement membrane-like material associated with synovial lining cells stains for type IV collagen and laminin. Fine fibrils surrounding the lining cells stain for type VI collagen. In two of the cases type VI collagen occurs mainly as long-spacing collagen, the distinct aggregated form of type VI collagen. This staining pattern was qualitatively the same in all different regions and cases investigated. However, considerable quantitative differences were seen.

Echothiophate-induced structural alterations in the anterior chamber angle of the cynomolgus monkey.

Four cynomolgus monkeys were treated topically twice daily in one eye with echothiophate iodide (PI) doses of 63, 75, or 250 micrograms per treatment for 7.7 weeks to 7 months. The opposite eyes of two monkeys received a control solution (diluent). The anterior ocular segments of all six eyes were studied by light and transmission electron microscopy during treatment. In the PI-treated eyes, the cribriform and outer corneoscleral meshwork were unusually dense. The trabecular meshwork was collapsed and the lamellae showed thickened basement membranes and thickened sheaths of elastic-like material. Most endothelial cells were enlarged and activated. Some contained many glycogen particles; others showed evidence of degeneration. The cribriform meshwork contained much more extracellular fine fibrillar material than normal, and the endothelium of the inner wall of Schlemm's canal was damaged. The PI-contracted ciliary muscle had a more rectangular shape than normally contracted muscles, and the inner edge extended so far anteriorly that in some areas it overlapped and occluded the trabecular meshwork. The muscle cells appeared damaged, and their basement membranes were thickened. The nonpigmented epithelial cells of the ciliary processes showed signs of degeneration, and within the pars plana some contained large, weakly osmiophilic inclusions. The basement membrane of the ciliary epithelium was thickened everywhere. The stromal vessels of the pars plana were dilated, and signs of inflammation were present. The sphincter iridis was damaged and there were iridocorneal adhesions. The 5 month diluent-treated eye demonstrated mild structural abnormalities in the meshwork.

Carbonic anhydrase distribution in the rabbit eye by light and electron microscopy.

The distribution of carbonic anhydrase (CA) in the rabbit eye was studied by light and electron microscopy according to the histochemical method of Hansson. In the cornea, CA staining was found in the cytoplasm of the endothelium. The filtering tissue in the chamber angle did not stain. The pigmented epithelium of the iris and the non-pigmented epithelium of the ciliary body showed intense staining distinctly located at lateral and apical cell membranes, without clearcut regional differences. In the Müller cells of the retina cytoplasmic staining was found. The dilatator muscle of the iris, the pigmented epithelium of the ciliary body, and the pigment epithelium of the retina showed similar and intense staining: cytoplasmic, mitochondrial, and at the cell membranes. The similarity of CA staining in these areas suggests similarity in function, possibly as transport processes important for the nutrition of the overlying cells. However, the role of CA in the various locations remains speculative, except in the corneal endothelium and the prelenticular ciliary processes, where the enzyme is evidently concerned with transport of salt and water.

Effects of intracameral Na2EDTA and EGTA on aqueous outflow routes in the monkey eye.

Intracameral perfusion with 4 to 6 mM Na2EDTA or 4 mM EGTA for 40 to 80 min caused a very large increase in gross outflow facility. This effect was partly reversible when followed by perfusion with mock aqueous humor. Eyes perfused with Na2EDTA were studied morphologically. In the trabecular meshwork the cells separated due to a splitting of the cell junctions. A distention of the cribriform meshwork, a wash-out of extracellular material , and a disintegration of the denuded trabecular cores were also noticed. The inner wall of Schlemm's canal protruded in a "balloonlike" manner into the lumen of the canal and showed frank ruptures, especially after prolonged perfusion times. The conventional outflow pathways beyond Schlemm's canal showed no abnormalities. In the uveoscleral outflow routes the anterior and middle part of the ciliary muscle demonstrated very wide intermuscular clefts and many degenerated muscle fibers. The posterior third of the muscle was normal. So were the ciliary epithelium, the choroid, and the retina. The pupillary sphincter also showed degeneration. The corneal endothelial cells separated, starting at the apical junctional complex.

Association of a choroidal ganglion cell plexus with the fovea centralis.

PURPOSE: After recently demonstrating an NADPH-diaphorase-, nitric oxide synthase (NOS)-positive ganglion cell plexus in the human choroid that was absent in rabbit and rat eyes, the authors extended their comparative studies to nonhuman primates and to subprimate mammals. METHODS: The authors investigated the choroids of diurnal cynomolgus monkeys with well-developed fovea centralis and accommodative systems; diurnal tree shrews without a fovea centralis or accommodative capacity; nocturnal owl monkeys with substantial accommodative capacity but without a fovea centralis; cats with an area centralis but no fovea centralis; and pigs without an area centralis or a fovea centralis. The latter two species have moderately developed ciliary muscles. Wholemounts of the choroid of eight cynomolgus monkey, two owl monkey, four tree shrew, four cat, and four pig eyes were stained for NADPH-diaphorase. In addition, frozen sections through the cynomolgus monkey choroid were stained for NOS and vasoactive intestinal polypeptide (VIP). RESULTS: In all species, the choroidal vessels were surrounded by NADPH-diaphorase-positive nerve fibers. A ganglion cell plexus, however, was seen only in cynomolgus monkey eyes. The ganglion cells stained for NOS and VIP. CONCLUSIONS: The presence of intrachoroidal nitrergic nerve cells restricted to species with a fully developed fovea centralis may indicate a functional correlation of these structures.

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.

Efferent and afferent innervation of primate trabecular meshwork and scleral spur.

PURPOSE: To determine the correlation between nerve terminals and cells or extracellular matrix (ECM) components in different portions of the primate trabecular meshwork (TM) and scleral spur (SS). METHODS: Serial sagittal and tangential sections through the anterior segments of 10 cynomolgus monkey eyes and 12 human eyes were investigated immunohistochemically with antibodies against the vesicular acetylcholine transporter (VACHT), vasoactive intestinal polypeptide (VIP), tyrosine-hydroxylase (TH), neuropeptide Y (NPY), substance P (SP), calcitonin gene-related peptide (CGRP), and galanin (GAL) and with a reduced nicotinamide adenine dinucleotide phosphate-diaphorase (NADPHd) reaction. The distribution of the terminals was compared with that of alpha-smooth-muscle actin (SMA) staining in TM and SS. The relationship between terminals and adjacent cells or ECM components was also studied in ultrathin sections through the TM and SS of 11 monkey eyes cut in sagittal, tangential, and frontal planes. RESULTS: NADPHd-positive nerve terminals were present, especially in the outer portion of both human and monkey TM and in the SS. VACHT-immunoreactive (IR) fibers were found in human but not in monkey SS and TM. The fibers were most numerous in the elongated SS and posterior TM where most cells also stained for SMA. SP- and CGRP-IR nerve endings were also more numerous in the outer TM and SS than in the inner TM. Ultrastructurally, staining for SP was seen in nerve endings containing mitochondria and dense core vesicles and was in contact with the cribriform elastic network. In the posterior SS of monkey eyes were large terminals similar to those previously described in human eyes. CONCLUSIONS: The results show for the first time that in the primate TM and SS, there are cholinergic and nitrergic nerve terminals that could induce contraction and relaxation of TM and SS cells. Terminals in contact with the elastic-like network of the TM and containing SP-IR resemble afferent mechanoreceptor-like terminals in other parts of the body. These findings raise the possibility that the TM may have some ability to self-regulate aqueous humor outflow.

Induction of tissue transglutaminase in the trabecular meshwork by TGF-beta1 and TGF-beta2.

PURPOSE: To study whether human trabecular meshwork (HTM) cells are capable of expressing and secreting tissue transglutaminase (tTgase), an enzyme cross-linking extracellular matrix (ECM) proteins, and whether tTgase and synthesis of cross-linked fibronectin are increased after treatment of HTM cells with transforming growth factor (TGF)-beta1 or -beta2. METHODS: Anterior segments of six normal human eyes were stained with antibodies to tTgase. Tissues from three eyes were analyzed for tTgase using Western blot analysis. Monolayer cultures of HTM cells from eyes of five human donors were treated with 1.0 ng/ml TGF-beta1, -beta2, or 5 X 10(-7) M dexamethasone (DEX) for 12 to 96 hours. Induction of tTgase was investigated by Western and Northern blot analysis. External tTgase activity was measured by the ability to form polymerized fibronectin and the incorporation of biotinylated cadaverine into fibronectin. RESULTS: Labeling for tTgase was observed throughout the entire HTM. Cultured HTM cells expressed tTgase intra- and extracellularly. Treatment of cultured HTM cells with TGF-beta1 and -beta2 increased the tTgase mRNA and protein levels, whereas DEX had no effect. TGF-beta-treated HTM cells showed a significant increase in polymerized and unpolymerized fibronectin. Incorporation of biotinylated cadaverine was markedly increased when HTM cells were treated with TGF-beta for 24 hours before seeding. CONCLUSIONS: The enzyme tTgase is expressed in the HTM and is inducible by TGF-beta1 or -beta2 in cultured HTM cells. Extracellular tTgase is able to polymerize fibronectin. Increased levels of TGF-beta2 in the aqueous humor may lead to an increase of tTgase expression and activity in the HTM, causing an increase of irreversibly cross-linked ECM proteins. This mechanism might play a role for the increased outflow resistance seen in glaucomatous eyes.

Nerve endings with structural characteristics of mechanoreceptors in the human scleral spur.

PURPOSE: The innervation of the scleral spur region was investigated to learn whether mechano-receptors are present in this region. METHODS: Serial tangential sections and whole-mount preparations of the scleral spur region of 18 human eyes of different ages were investigated with electronmicroscopic and immunohistochemical methods. For immunohistochemistry antibodies against neurofilament-proteins, synaptophysin, substance P (SP), calcitonin gene-related peptide (CGRP), vasoactive intestinal polypeptide (VIP), neuropeptide Y (NPY), tyrosine-hydroxylase, dopamine-beta-hydroxylase, and acetylcholinesterase were used. RESULTS: Club- or bulb-shaped nerve endings with a diameter of 5 microns to 25 microns were identified in the scleral spur region throughout the whole circumference of the eyes. The terminals derive from myelinated axons with a diameter of approximately 3 microns and stain with antibodies against neurofilament-proteins and synaptophysin but do not stain for tyrosine-hydroxylase, dopamine-beta-hydroxylase, acetylcholinesterase, NPY, VIP, SP, or CGRP. Electronmicroscopically, the endings contain abundant neurofilaments, granular and agranular vesicles of different sizes, numerous mitochondria, and lysosome-like lamellated structures. The endings are incompletely ensheathed by Schwann cells. Those areas of the cell membrane of the endings that are not covered by Schwann cells are in intimate contact with the fibrillar connective tissue elements of the scleral spur. CONCLUSION: These structural features are highly characteristic for mechanoreceptive nerve endings in other tissues of the human body. The authors therefore hypothesize that the club-or bulb-shaped nerve endings in the human scleral spur are afferent mechanoreceptors that measure stress or strain in the connective tissue elements of the scleral spur. Such changes might be induced by ciliary muscle contraction and/or by changes in intraocular pressure.

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.

Anterior chamber-associated immune deviation elicited via primate eyes.

PURPOSE: To determine whether injection of a soluble antigen, ovalbumin (OVA), into the anterior chamber of cynomolgus monkey eyes would impair the ability of these animals to subsequently develop delayed hypersensitivity when confronted by this antigen in immunogenic form. METHODS: OVA or phosphate-buffered saline was injected into the anterior chamber of adult cynomolgus monkeys that were subsequently immunized with OVA in adjuvant and then skin challenged for delayed hypersensitivity with OVA. RESULTS: Recipients of intracameral OVA proved unable to acquire antigen-specific delayed hypersensitivity when they received an immunogenic regimen of OVA in adjuvant. Since the flow of aqueous humor through the uveoscleral pathway of primate eyes can be promoted by topical treatment with PGF2 alpha isopropylester, a preliminary experiment is described in which induction of anterior chamber-associated immune deviation by OVA was prevented when the antigen was first introduced into monkey eyes treated with PGF2 alpha isopropylester. CONCLUSIONS: Monkeys resemble rodents in displaying anterior chamber associated immune deviation (impaired ability to acquire antigen-specific delayed hypersensitivity) when they first encounter an antigen via the anterior chamber. The findings suggest that the cellular and molecular mechanisms of immune privilege, recently described in rodents, may apply to immune responses to intraocular antigens and pathogens in primates, including humans. Primate eyes offer an opportunity to explore the mechanisms of anterior chamber-associated immune deviation using pharmacologic agents that modify the aqueous outflow tracts.

Nerve cells in the human ciliary muscle: ultrastructural and immunocytochemical characterization.

PURPOSE: Intrinsic nerve cells in the human ciliary muscle were identified and characterized by immunohistochemical and ultrastructural methods. METHODS: Serial sections through the ciliary muscle of 10 human donors (age range, 53 to 91 years) were investigated by electron microscopy, NADPH-diaphorase (NADPH-d) staining, and immunohistochemistry. Antibodies against nitric oxide synthase (NOS), protein gene product 9.5 (PGP 9.5), neurofilament proteins, tyrosine hydroxylase (TH), neuropeptide Y (NPY), vasoactive intestinal peptide (VIP), substance P (SP) and calcitonin gene-related peptide (CGRP) were used. Nerve cell density per millimeter of circumferential width was analyzed in three eyes, and in one eye the total number of neurons in the entire ciliary muscle was evaluated. RESULTS: Small (70% of the total; longitudinal diameter 10 to 14 microns) and large (longitudinal diameter 20 to 30 microns) ganglion cells were identified in the inner parts of the muscles' reticular and circular portions. No nerve cells were observed in the anterior longitudinal portion. The cells were in contact with unmyelinated axons and synaptic boutons containing small agranular and large granular vesicles. Axo-somatic and axo-dendritic synapses were observed. Histochemically and ultrahistochemically, the neurons stained intensely for NADPH-d. Both cell types were multipolar and expressed long filamentous processes. Axonal processes with periodic swellings suggesting varicosities ran close and parallel to neighboring muscle bundles. Some nerve cells were connected with each other by axonal processes. No perivascular NADPH-d-positive nerves were seen around ciliary muscle vessels, but they were present in the wall of the major arterial circle of the iris. A small number of ganglion cells contributed to this perivascular network. NADPH-d-positive neurons stained for PGP 9.5 and NOS. No TH, NPY, or VIP-positive ciliary muscle neurons were observed. In double labeling experiments, 70% of the nerve cells were in contact with nerve endings expressing SP-like and CGRP-like immunoreactivity. Seventeen to 32 NADPH-d-positive neurons were counted per millimeter of ciliary muscle circumferential width, with 923 in the entire ciliary muscle of one donor eye. CONCLUSIONS: The presence of intrinsic NOS-positive nerve cells concentrated in the inner parts of the ciliary muscle might indicate a physiological role of nitric oxide for disaccommodation or fluctuations during accommodation.