Download Nerve cells in the human ciliary muscle: ultrastructural and

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
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nd by grant PSS36 from the Fond xur Fordemng der wiss. Forschung in Ostetreich
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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
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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
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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
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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
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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.
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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.
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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).
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Ciliary Muscle Nerve Cells
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\ .-JIT
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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).
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Ciliary Muscle Nerve Cells
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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.
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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
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13. Barany EH, Berrie CP, Birdsall NJM, Burgen ASV,
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Key words
the response to the induction of agonist subsensitivity.
Br J Pharmacol. 1982; 77:731-739.
ciliary muscle, neurotransmitter, nerve cell, human eye, ni14. Erickson-Lamy KA, PolanskyJR, Kaufman PL, Zlock
tric oxide
DM. Cholinergic drugs alter ciliary muscle response
and receptor content. Invest Opthalmol Vis Sci.
Acknowledgments
1987; 28:375-383.
15. Ehinger B. Adrenergic nerves to the eye and to related
The authors thank Anke Fischer, Angelika Hauser, Sandra
structures in man and in the cynomojgus monkey
Herrmann, Elke Kretzschmar and Gertrud Link for their
(Macaco, irus). Invest Opthalmol Vis Sci. 1966;5:42-54.
expert technical help and Marco GofJwein for his excellent
16. Laties AM, Jacobowitz D. A comparative study of the
preparation of the photographs.
autonomic innervation of the eye in monkey, cat and
rabbit. Anat Rec. 1966; 156:383-396.
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