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Nerve Cells in the Human Ciliary Muscle: Ultrastructural and Immunocytochemical Characterization Ernst R. Tamm,* Cassandra Fliigel-Koch,* Bernd Mayer,-\ and Elke Lutjen-Drecolt* 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 fim) and large (longitudinal diameter 20 to 30 fim) 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-posilive 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. Invest Ophthalmol Vis Sci. 1995; 36:414-426. i n m a t e ciliary muscle has classically been subdivided into three parts: the outer longitudinal portion, the intermediate reticular portion, and the inner circular portion.1 Contraction of the inner portions causes an anterior-inward movement of the muscle and induces From the *Depattment of Anatomy II, University of Erlangen-Numberg, Erlangen, Germans, and the +Institute for Phamuicology and Toxicology, University of'Gnu, Gnu Austria Presented in pan at the annual meeting of the Association for Research in Vision l p ^ l ^ nd by grant PSS36 from the Fond xur Fordemng der wiss. Forschung in Ostetreich c B ^ ' - i r i . ,,• • ,.. - i s , „ „ , • ; c « i J < : ; o o ^ ^ , , Submtttedfor publication May 23, 1994; revised September 16, 1994; accepted So/Member 20,1994. Proprietary Merest category, N. liepnnt requests: bmsl R. 1 amm, Department of Anatomy 11, University of Erlangen-Nilmberg, Universilatsslrasse 19, 91054 Erlangen, Germany. 414 accommodation,1 whereas contraction of the outer longitudinal portion, which is attached to scleral spur and trabecular meshwork,2'3 facilitates aqueous outflow.4 Morphologically, the different portions are not separate entities but are connected with each other. Nonetheless, histochemical and pharmacologic studies indicate that a functional separation might be possible.5'6 Ciliary muscle as a whole is a fast, multi-unit SmOOth mUSCle' innervated by paraSympathetiC aXOnS of the oculomotor nerve (N. III). The fibers take their Origin in the Edinger-WeStphal nucleus and Synapse in the ciliary ganglion.8'9 Degeneration studies in monloo o keys indicate that 97% of the ciliary ganglion neurons supply the ciliary muscle, whereas only 3% innervate the iris Sphincter. Accordingly, the individual Ciliary Investigative Ophthalmology & Visual Science, February 1995, Vol. 36, No. 2 Copyright © Association for Research in Vision and Ophthalmology Downloaded From: http://iovs.arvojournals.org/pdfaccess.ashx?url=/data/journals/iovs/933410/ on 06/17/2017 Ciliary Muscle Nerve Cells muscle cells in monkeys 10 " and humans' 2 express an extremely dense innervation. The density of muscarinic cholinergic receptors is much higher in ciliary muscle than in other cholinergically innervated tissues.1314 In contrast, both morphologic1""17 and physiological studies18 indicate that the sympathetic innervation of the primate ciliary muscle is of minor physiological significance. More recently, some varicose nerve terminals have been identified in the human ciliary muscle that are immunoreactive with antibodies against various neuropeptides, such as neuropeptide Y (NPY), substance P (SP), calcitonin generelated peptide (CGRP), and vasoactive intestinal polypeptide (VIP).19 The physiological role of these peptides for the human ciliary muscle is unclear. In vitro studies indicate that SP is a potent agent to induce contraction of human ciliary muscle strips.20 In addition to the extrinsic innervation deriving from nerve cells outside the eye, intrinsic nerve cells are present in human ciliary muscle. Although these ciliary muscle nerve cells (Pkxus gangliosus ciliaris) were discovered more than one hundred years ago21"23 and were mentioned in several studies during the last few decades,24"28 nothing is known about their location, axonal projection, and specific function. In the present study, we characterized the intrinsic neurons of human ciliary muscle by means of immunohistochemistry and electron microscopy. MATERIALS AND METHODS Ten pairs of human autopsy eyes (age range, 53 to 91 years) were investigated. No donor had a history of chamber angle abnormality. Methods for securing human tissue were humane, included proper consent and approval, and complied with the Declaration of Helsinki. The eyes were cut equatorially behind the ora serrata, and the anterior segment was dissected in quadrants. From each quadrant, wedge-shaped pieces containing the ciliary muscle and trabecular meshwork were cut. The specimens were immersed in Zamboni's2a or Ito's solution30 for 24 hours at 4°C, or in neutral buffered formalin (4%) for 4 hours at 4°C. All specimens from autopsy eyes were placed in fixative within 4 hours of death; specimens from two pairs of eyes (ages 63 and 91 years) were fixed within 20 minutes of death. Electron Microscopy Specimens fixed in Ito's solution were processed for electron microscopy. After postfixation with 1% osmium tetroxide, the specimens were dehydrated with graded alcohols and embedded in Epon (Roth, Karlsruhe, Germany). Serial meridional, frontal, and tan- 415 gential semithin sections were cut on a microtome, stained with Richardson's stain,31 and investigated for nerve cells. Specimens that showed nerve cells in semithin sections were further processed for electron microscopy. Serial ultrathin sections were treated with lead citrate and uranyl acetate and were viewed using a Zeiss EM 902 microscope (Zeiss, Oberkochen, Germany) . Immunohistochemistry Specimens fixed in Zamboni's solution were washed for 24 hours in phosphate-buffered saline (PBS) and quick frozen in isopentane precooled with liquid nitrogen. Meridonal and serial tangential cyrostat sections were cut at a thickness of 15 to 20 //m. The sections were placed on slides covered with 0.1 % polyL-lysine and preincubated for 45 minutes in Blotto's dry milk solution.32 After preincubation, the sections were incubated overnight at room temperature with the primary antibodies listed in Table 1. The presence of nitric oxide synthase (NOS) was shown with polyclonal antibodies raised against NOS purified from porcine cerebellum. This antibody has been successfully used for Western blot analysis33 and immunohistochemistry.34 After overnight incubation, the sections were washed in PBS, reacted for 1 hour with biotinylated secondary antibodies (Amersham Buchler, Braunschweig, Germany), washed again, and covered with streptavidin-FITC (Dakopatts, Hamburg, Germany) . Double-staining experiments were performed in the two pairs of eyes that were fixed within 20 minutes of death. Sections were incubated with rabbit NOS antibodies in combination with the nonrabbit antibodies listed in Table 1. Binding of mouse, rat, sheep, and goat antibodies was visualized using biotinylated secondary antibodies specific for each species and streptavidin-FITC. The rabbit antiserum was stained with Texas red (Amersham) or Cy 3 (Dianova, Hamburg, Germany) conjugated anti-rabbit IgG. After washing in PBS, the sections were mounted in Entellan (Merck, Darmstadt, Germany) containing 1,4-Diazabicyclo [2,2,2] octan (DABCO, Merck)39 and viewed with a Leitz Aristoplan microscope (Ernst Leitz GmbH, Wetzlar, Germany). Kodak T-max 400 film (Eastman Kodak, Rochester, NY) was used for photography. Staining with antibodies against the different neuropeptides visualized varicose terminals with the typical spatial distribution, as described for each of these peptides in the anterior segment of the human eye.19 Sections of the small intestine, containing parts of the enteric nervous system, served as additional positive control. Negative control experiments were performed using either PBS or preimmune serum from Downloaded From: http://iovs.arvojournals.org/pdfaccess.ashx?url=/data/journals/iovs/933410/ on 06/17/2017 416 TABLE l. Investigative Ophthalmology & Visual Science, February 1995, Vol. 36, No. 2 Detailed Description of Antisera Used for Immunocytochemistry Primary Antisera Nitric oxide synthase (NOS) Tyrosine hydroxylase (TH) Substance P (SP) Calcitonin gene-related peptide (CGRP) Neuropeptide Y (NPY) Vasoactive intestinal peptide (VIP) Pan-neuronal marker (NA 1298)* Protein gene product 9.5 (PGP 9.5)f Host Species Source 3334 Bernd Mayer (Graz, Austria) Eugene Tech (Ridgefield Park, NJ) Eugene Tech Chemicon63 (Temecula, CA) Rabbit Rabbit Rabbit 611 Euro Diagnostica AB (Malmo, Sweden) Affiniti Res. Prod. Ltd. (Nottingham, UK) Amersham Buchler (Braunschweig, Germany) Affihiti65 Euro-Diagnostica AB Affiniti UltraClone Ltd66 (Isle of Wight, UK) Dilution Rat 1:500 1:200 1:200 1:200 Rabbit Sheep Rabbit Goat Rabbit Mouse Rabbit 1:1000 1:800 1:80 1:200 1:1000 1:1000 1:200 * NA 1298 is composed of a number of mouse IgGi and IgM antibodies that react with nonposphorylated neurofilamenl H (5!00 kcU epitopes. t PGP 9.5 is a neuron-specific, cytoplasmic, ubiquitin carboxyl-terminal hydrolase.6' the same host species, substituted for the primary antibody. NADPH-Diaphorase Staining Serial tangential and frontal sections from specimens fixed in neutral buffered formalin were incubated in a moist chamber at 37°C using the following medium: /5-nicotinamide adenine dinucleotide phosphate-tetrasodium salt (reduced NADPH; Biomol, Hamburg, Germany), 1 mg/ml; nitroblue tetrazolium chloride (Serva, Heidelberg, Germany), 0.1 mg/ml; 0.3% Triton X-100 in 0.1 M PBS, pH 7.4. After incubation for 1 hour, the sections were rinsed in PBS and mounted in Kaiser's glycerin gelatine (Merck). For combination with immunohistochemistry, specimens fixed in formalin and Zamboni's solution were used. Sections were first stained with antibodies listed in Table 1, stained nerve cells were photographed, and the sections were thoroughly rinsed in PBS and stained for NADPH-d. For ultrahistochemistry, sections were mounted with a small drop of 2% agar solution on Thermanox plates (Vogel GmbH, Giessen, Germany) and stained for NADPH-d. After staining, the sections were dehydrated and embedded in Epon without previous osmium fixation. Ultrathin sections were not or only slightly counterstained and viewed with the electron microscope. The number of positively stained neurons was quantified in four eyes from different donors. The anterior segment of these eyes was dissected in specimens that contained the total anterior-posterior length of the ciliary muscle and 1 mm of its circumferential width. In three eyes, one of these specimen from each quadrant was randomly selected and quantitatively analyzed. Serial 20-/xm tangential sections were cut through the entire specimen, all sections (20 to 35 per specimen) were placed on slides and stained for NADPH-diaphorase, and the total number of NADPH-d-positive neurons visualized in these sections was counted. Only cells in which the nucleus was cut were considered. Care was taken not to count the same cell in adjacent sections; for example, if a cell was cut twice, it was only counted once. In one eye, similar specimens from the entire circumference of the ciliary body (containing the entire ciliary muscle of this eye) were analyzed. RESULTS Light Microscopy The ganglion cells in the human ciliary muscle were characteristically localized between the muscle bundles of the reticular and circular portion (Fig. 1). Most of them were situated in the connective tissue spaces between the ciliary muscle bundles or in close association with the fibroblast sheaths of the bundles. No nerve cells were observed within an individual muscle bundle. Several ganglion cells were located along the inner border of the ciliary muscle. None were found in the anterior longitudinal portion of the muscle. The vast majority of the cells appeared to be solitary; only occasionally were two ganglion cells found close to each other. Nerve cells were characterized by their large euchromatic nucleus, prominent nucleoli, and clear cytoplasm. They were surrounded by a thin ring of peripheral glia cells. The cytoplasm and nucleus of glia cells were densely stained. Frequently, myelinated axons were seen close to the nerve cells. Ciliary muscle nerve cells had a common shape, oval, but they varied Downloaded From: http://iovs.arvojournals.org/pdfaccess.ashx?url=/data/journals/iovs/933410/ on 06/17/2017 417 Ciliary Muscle Nerve Cells x . ••'«• FIGURE 1. Nerve cells in the human ciliary muscle (semithin sections, Richardson's stain). (A) An oval-shaped neuron (arrow) with a longitudinal diameter of 30 urn. is situated between the muscle bundles of the circular portion. M = ciliary muscle; S = scleral spur; SC = Schlemm's canal; TW = trabecular meshwork; MA = major arterial circle of the iris. Donor age, 56 years. Original magnification, X330. (B) Higher magnification of A (original magnification, X1000). The nerve cell (arrow) is characterized by a large euchromatic nucleus, lipofuscin particles, and a clear cytoplasm. Myelinated axons (arrowheads) are seen close to the nerve cell. (C) Small (longitudinal diameter 11 ym) ciliary muscle neuron (arrow) between the muscle bundles of the reticular portion. Similar to large neurons, small neurons are commonly associated with myelinated axons (arrowheads). Donor age, 71 years. Original magnification, X1000. considerably in size. Small ganglion cells (approximately 70% of the total) had a longitudinal diameter of 10 to 14 fim, whereas large ganglion cells measured 20 to 30 fxm. Both types of ganglion cells appeared to be evenly distributed throughout the inner regions of the ciliary muscle. Electron Microscopy The perikaryon of ciliary muscle nerve cells contained numerous mitochondria and profiles of short, rough endoplasmic reticulum cisterns and free polysomes (Fig. 2). Scattered throughout the cytoplasm were large granular vesicles, lipid droplets, and irregularly shaped electron-opaque lipofuscin granules. Each ganglion cell was surrounded by electron-dense, flat processes of glia cells, which also surrounded numerous preterminal, unmyelinated axons and several terminal boutons. The boutons, which formed axo-somatic synaptic contacts with the perikaryon, contained mitochondria, numerous small agranular vesicles (40 Downloaded From: http://iovs.arvojournals.org/pdfaccess.ashx?url=/data/journals/iovs/933410/ on 06/17/2017 Investigative Ophthalmology & Visual Science, February 1995, Vol. 36, No. 2 FIGURE 2. Electron micrograph of a large ganglion cell (same neuron as in Figs. 1A and IB; original magnification, X7600). The perikaryon of the neuron contains numerous mitochondria and profiles of short, rough endoplasmic reticulum cisterns and free polysomes {black asterisk). Scattered throughout the cytoplasm are large granular vesicles and irregularly shaped lipofuscin granules (white asterisk). The neuron is surrounded by flat processes of glia cells (G), which also surround numerous preterminal, unmyelinated axons (arrozvs). NU = nucleus. Downloaded From: http://iovs.arvojournals.org/pdfaccess.ashx?url=/data/journals/iovs/933410/ on 06/17/2017 Ciliary Muscle Nerve Cells 419 NADPH-Diaphorase Staining Large and small ciliary muscle ganglion cells showed a prominent NADPH-d reaction (Fig. 5). Both soma and dendrites of the nerve cells were darkly stained. The large ganglion cells were multipolar and sometimes expressed numerous lamellar dendrites (Fig. 5A) or several long and short filamentous processes (Figs. 5B, 5C). Small neurons had smoother surfaces, some short filamentous processes, and one to two long filamentous processes (Figs. 5D, 5E). In favorable sections, long, presumably axonal, processes of both cell types could be traced for 80 to 100 //m. The majority of these processes ran close and parallel to the neighboring muscle bundles (Figs. 5C, 5E). During their course, the processes sometimes expressed periodic swellings, suggesting varicosities (Fig. 5E). Occasionally, neighboring nerve cells were connected with each other by axonal processes (Fig. 5F). Faint NADPH-d reaction was seen in ciliary muscle cells (Figs. 5C, 5D). In addition, ciliary muscle capillaries showed faint endothelial staining for NADPH-d but no positive perivascular nervous networks. In contrast, numerous NADPH-d positive varicose nerve fibers were observed F1GURE 3. Electron microscopy of ciliary muscle neurons (donor age, 56 years). (A) Ciliary muscle nerve cells are in contact with terminal boutons that form axo-somatic synaptic contacts (arrows). The boutons contain numerous agranular vesicles (40 to 60 nm, asterisk) and some large granular vesicles (60 to 120 nm, arrowheads). Original magnification, X50000. (B, C) Ciliary muscle neurons (N) express spinelike (B, arrowheads) and lamellar (C) dendritic processes where axo-dendritic synapses (arrows) are formed. In addition to agranular vesicles (asterisk), some of the axo-dendritic synaptic boutons contain larger amounts of granular vesicles than axo-somatic contacts (C, arrowheads). (D) Ultrahistochemical investigation of NADPH-d-stained ganglion cells (N) shows the electron-dense NADPH-d reaction product in the cytoplasm (arrowheads). The neurons show similar structural features already observed in conventional electron microscopy. Original magnification, X6000. NU = nucleus. to 60 n m ) , and some large (60 to 120 nm) granular vesicles (Fig. 3A). In places, the ganglion cells expressed spine-like (Fig. 3B) or lamellar (Fig. 3C) dendritic processes. In these regions, axo-dendritic synapses were formed. In some of the boutons contacting the dendrites, the n u m b e r of large granular vesicles seemed to be greater than in axo-somatic contacts (Fig. 3C). In general, ultrastructural features of large and small ciliary muscle neurons were similar, but synaptic contacts were less frequent with small neurons (Fig. 4). FIGURE 4. Electron micrograph of a small ganglion cell in the ciliary muscle (consecutive section of Fig. 1C; original magnification, X12700). The neuron is surrounded by processes of glia cells (arrowheads). Contacts with nerve terminals (arrow) and preterminal axons are less frequent than in large neurons. NU = nucleus. Downloaded From: http://iovs.arvojournals.org/pdfaccess.ashx?url=/data/journals/iovs/933410/ on 06/17/2017 420 Investigative Ophthalmology 8c Visual Science, February 1995, Vol. 36, No. 2 in the media of the major arterial circle of the iris, which runs close or within the circular portion of the human ciliary muscle (Fig. 5G). Axonal processes of neighboring ganglion cells seemed to contribute to this perivascular nervous network (Fig. 5H). Ultrahistochemical investigation of NADPH-dstained ganglion cells showed the same structural features observed by conventional electron microscopy (Fig. 3D). In double-labeling experiments, the NADPH-d positive neurons and the nerve fibers that contacted the perikaryon of the neurons stained for the neuronal marker PGP 9.5. Quantitative measurements in all quadrants of three eyes showed a density of 17 to 32 NADPH-dstained neurons per millimeter of circumferential ciliary muscle width (Table 2). In one donor eye, 923 such neurons were counted throughout the entire ciliary muscle. Immunohistochemistry The combination of NOS immunostaining and NADPH-d-staining revealed complete colocalization in neuronal perikarya and fibers (Figs. 6A, 6B). No NOS-immunoreactivity was observed in ciliary muscle cells or vascular endothelium. No ciliary muscle nerve cells that showed immunoreactivity for tyrosine hydroxylase, NPY, or VIP were observed. Double-labeling experiments showed that ~70% of small and large NOS-positive neurons were in contact with varicose nerve endings expressing SP-like and CGRP-like immunoreactivity (Figs. 6C to 6F). These nerve terminals sometimes encircled the NOS-immunoreactive neurons (Figs. 6C, 6D) or formed distinct arborizing boutons along the perikarya of the nerve cells (Figs. 6E to 6G). Perikarya of ciliary muscle nerve cells did not stain for CGRP or SP. Double staining for the pan- neuronal marker NA 1298 and NOS showed positive staining for NA 1298 in the axonal processes of the ganglion cells and in the thin nerve fibers that contacted their perikaryon, but not in the cytoplasm of the neurons. DISCUSSION Our study confirms earlier reports of a distinct population of solitary ganglion cells in the human ciliary muscle.21"28 Compared to human choroidal ganglion cells,34 or to most mammalian peripheral ganglion cells in the enteric nervous system or in other visceral organs,36 ciliary muscle nerve cells appear to be relatively small. This may explain why some authors investigating ciliary muscle innervation either did not observe or denied the presence of such intrinsic ganglion cells.l0'37'38 In the guinea pig small intestine, Furness et al39 identified a population of neurons with comparable size by means of an intracellular dye injection. Although in enteric nerve cells an association between cell shape, chemistry, and function seems to exist,40 functional properties of these small neurons have not been identified. In addition to the neuronal marker PGP 9.5, ciliary muscle nerve cells stain for NADPH-d and NOS and most probably use nitric oxide (NO) as a neurotransmitter. The colocalization of NOS-immunoreactivity and NADPH-d stain, which is amply documented in brain and peripheral nervous system,41'42 was also demonstrated in our study. Quantitatively, 17 to 32 NADPH-d-NOS-positive cells were counted per millimeter of circumferential ciliary muscle width in three of the investigated eyes. Given a total circumferential ciliary muscle width of approximately 36 mm,43 these counts are in good agreement with 923 ganglion cells FIGURE 5. NADPH-diaphorase (NADPH-d)-positive nerve cells in the ciliary muscle (tangential sections). (A, B) Large ciliary muscle ganglion cells that show a prominent NADPH-d reaction. Both soma and dendrites of the nerve cells are darkly stained. The cells are multipolar and express numerous lamellar dendrites (A, arrows) or several long and short filamentous processes (B, arrows; original magnification, X1000). (C) Long filamentous processes of large neurons (arrows) run close and parallel to adjacent ciliary muscle bundles, that show a faint NADPH-d-reaction (asterisk; original magnification, X1000). (D) A small ciliary muscle neuron expresses two long, filamentous processes (arrows) that encircle an adjacent muscle bundle (asterisk; original magnification, XI000). (E) During their course along the muscle bundles, filamentous processes of ciliary muscle neurons express periodic swellings, suggesting varicosities (arrows; original magnification, X1000). (F) Two neighboring nerve cells are in contact with each other by axonal processes (arrows; original magnification, X250). (G) Numerous NADPH-d-positive nerve fibers (arrows) are stained in the wall of the major arterial circle of the iris (asterisk; original magnification, X300). (H) The axon of an adjacent nerve cell runs close to and seems to contribute to the perivascular nervous network of the major arterial circle (asterisk; original magnification, X150). Downloaded From: http://iovs.arvojournals.org/pdfaccess.ashx?url=/data/journals/iovs/933410/ on 06/17/2017 Ciliary Muscle Nerve Cells 421 \ .-JIT Downloaded From: http://iovs.arvojournals.org/pdfaccess.ashx?url=/data/journals/iovs/933410/ on 06/17/2017 422 Investigative Ophthalmology & Visual Science, February 1995, Vol. 36, No. 2 TABLE 2. NADPH-Diaphorase Positive Cells Counted in the Different Quadrants of Human Ciliary Muscle Age (years) Temporal Nasal Superior Inferior Mean ± SD Number of Positive Cells in 1 mm Circumferential Length 91 56 21 29 32 26 17 24 19 24 28 31 21 29 24.25 ± 5.4 22.75 ± 5.05 28.25 ± 3.3 Number of Positive Cells Per Quadrant (Total: 923) 53 255 187 found in the one eye in which the total number of NADPH-d-NOS-positive nerve cells in the entire ciliary muscle was evaluated. The number is smaller than that reported for NADPH-d-NOS-positive nerve cells in the entire human choroid (1555 to 2579)34 or the total number of nerve cells in the ciliary ganglion (1088 to 6835, mean 2394 ± 1036),44 but, in our opinion, it is still large enough to indicate physiological significance. Similar intrinsic NOS-positive ganglion cells have been described in numerous visceral organs, such as intestine,45 gallbladder,46 urinary bladder,47 and trachea.48 In the enteric nervous system, NO mediates nonadrenergic-noncholinergic relaxation of intestinal smooth muscle.49 NO released by perivascular nerves relaxes vascular smooth muscle and mediates vasodilation.50 In the posterior part of the human eye, NOS-positive neurons contribute to the nitrergic vasodilative innervation of the choroidal vasculature.34 In the ciliary muscle, neurons along the major arterial circle of the iris may similarly contribute to the nitrergic innervation of this vessel. Most of the NOSpositive ciliary muscle neurons, however, seem not to innervate the ciliary muscle vasculature but the ciliary muscle cells proper. It seems reasonable to assume that this nitrergic innervation induces relaxation of the ciliary muscle cells. Recent in vitro studies that demonstrate a NO-induced relaxation of isolated bovine ciliary muscle support this assumption.51 In hu- 242 239 man ciliary muscle, the NOS-positive neurons are situated in the inner regions of the muscle, which predominandy serve accommodation. During accommodation, the contracting ciliary muscle moves in an anterior-inward direction and stretches its posterior elastic tendons.1'52 The stretched posterior elastic tendons pull the ciliary muscle backward during relaxation and disaccommodation. A relaxing nitrergic innervation of the ciliary muscle might facilitate the backward movement during disaccommodation. Another possibility is that nitrergic ciliary muscle neurons contribute to the small fluctuations of accommodation under steady viewing conditions. These fluctuations may help to indicate the direction (contraction or relaxation) in which change should occur to obtain perfect focus.53'34 Ciliary muscle neurons are innervated by synaptic boutons. Although some of the contacts may derive from neighboring neurons, it seems likely that the neurons have an extrinsic preganglionic innervation from outside the eye. The nature and origin of such an innervation remains to be clarified. Some of the nerve fibers contacting ciliary muscle neurons show SP-like and CGRP-like immunoreactivity. SP-immunoreactive nerve cells have been demonstrated in the Edinger-Westphal nucleus of cats55 and CGRP- and/ or SP-immunoreactive nerve cells in the ciliary ganglion of rats, cats, and monkeys.5''"58 Similar cells may also be present in humans. On the other hand, in FIGURE 6. Immunocytochemistry of ciliary muscle neurons. (A, B) Combinations of NOS immunostaining (B) and NADPH-d (A) reveals complete colocalization in neuronal perikarya and fibers (original magnification, X1800). (C, D) Double-labeling of ciliary muscle neuron for NOS (C) and substance P (D). Substance P-like immunoreactivity is seen in varicose axons (arrows) that encircle the NOS-immunoreactive neuron (original magnification, X2000). (E-H) Large (E and F, original magnification, X1800) and small (G and H, original magnification, X2000) ciliary muscle neurons that stain for NOS (E and G) are in contact with arborizing boutons that express CGRP-like immunoreactivity (F and H, arroius). Downloaded From: http://iovs.arvojournals.org/pdfaccess.ashx?url=/data/journals/iovs/933410/ on 06/17/2017 Ciliary Muscle Nerve Cells Downloaded From: http://iovs.arvojournals.org/pdfaccess.ashx?url=/data/journals/iovs/933410/ on 06/17/2017 423 424 Investigative Ophthalmology & Visual Science, February 1995, Vol. 36, No. 2 various species, ocular SP- and CGRP-immunoreactive axons have been shown to derive from sensory trigeminal ganglion cells.19 It is assumed that these peptides are locally released by means of an axon reflex in the course of an ocular irritative response.5960 Collaterals of SP- or CGRP-containing trigeminal axons that innervate nitrergic ciliary muscle neurons may promote relaxation of the ciliary muscle in such events. Uveoscleral outflow is increased in cynomolgus monkeys after experimental iridocyclitis.61 Release of NO might contribute to this phenomenon because relaxation of the ciliary muscle and widening of the intermuscular spaces is known to cause a significant increase in uveoscleral outflow.62 9. Ruskell GL, Griffiths T. Peripheral nerve pathway to the ciliary muscle. Exp Eye Res. 1979;28:277-284. 10. van der Zypen E. Licht—und elektronenmikroskopische Untersuchungen uber den Bau und die Innervation des Ziliarmuskels bei Mensch und Affe {Cercopithecus ethiops). Albrecht v Graefes Arch klin exp Opthalmol. 1967; 174:143-168. In German. 11. Tamm E, Lutjen-Drecoll E, Rohen JW. Age-related changes of the ciliary muscle in comparison with changes induced by treatment with prostaglandin Fia: An ultrastructural study in rhesus and cynomolgus monkeys. Mech Aging Develop. 1990;51:101-120. 12. Ishikawa T. Fine structure of the human ciliary muscle. Invest Opthalmol Vis Sci. 1962; 1:587-608. 13. 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