March 2002
Volume 43, Issue 3
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Anatomy and Pathology/Oncology  |   March 2002
Characterization of Nitrergic Neurons in the Porcine and Human Ciliary Nerves
Author Affiliations
  • C. Albrecht May
    From the Department of Anatomy II, University of Erlangen-Nürnberg, Erlangen, Germany.
  • Anne V. Fuchs
    From the Department of Anatomy II, University of Erlangen-Nürnberg, Erlangen, Germany.
  • Michael Scheib
    From the Department of Anatomy II, University of Erlangen-Nürnberg, Erlangen, Germany.
  • Elke Lütjen-Drecoll
    From the Department of Anatomy II, University of Erlangen-Nürnberg, Erlangen, Germany.
Investigative Ophthalmology & Visual Science March 2002, Vol.43, 581-586. doi:
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      C. Albrecht May, Anne V. Fuchs, Michael Scheib, Elke Lütjen-Drecoll; Characterization of Nitrergic Neurons in the Porcine and Human Ciliary Nerves. Invest. Ophthalmol. Vis. Sci. 2002;43(3):581-586.

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Abstract

purpose. To further characterize a subpopulation of choroidal ganglion cells associated with the ciliary nerves.

methods. Isolated long ciliary nerves of porcine and human eyes containing ciliary nerve–associated ganglion cells (CNGCs) were embedded in Epon for ultrastructural investigation, or wholemounts were stained with antibodies against nitric oxide synthase (NOS), vasoactive intestinal polypeptide (VIP), vesicular acetylcholine transporter, neuropeptide Y (NPY), tyrosine hydroxylase (TH), calcitonin gene-related peptide (CGRP), substance P (SP), and synaptophysin. In addition, wholemount preparations of the choroid and of the anterior segment were stained for reduced nicotinamide adenine dinucleotide phosphate-diaphorase (NADPH-D). Serial sections through choroid and anterior segment were stained with the prior antibodies listed.

results. In the porcine choroid only CNGCs were present. They stained for brain (b)NOS and VIP and were surrounded by SP and VIP-immunoreactive (IR) nerve terminals. The axonal processes of the CNGCs followed the ciliary nerves to the anterior eye segment, where they formed a nerve fiber plexus that terminated in the trabecular meshwork. None of the axons passed into the sparse NOS-IR nerve fiber plexus surrounding the choroidal vasculature. The CNGCs in the human choroid morphologically resembled those seen in the pig.

conclusions. The CNGC proportion of choroidal ganglion cells is presumably involved in the intrinsic (peripheral) innervation of the aqueous outflow tissues and of the choroid.

The human and primate choroids contain intrinsic nerve cells that stain for reduced nicotinamide adenine dinucleotide phosphate-diaphorase (NADPH-D) and with antibodies against brain nitric oxide synthase (bNOS). 1 2 3 These nerve cells probably use nitric oxide (NO) as a neurotransmitter. The axons of NADPH-D-bNOS–positive choroidal nerve cells contact choroidal vessels and contribute to the vasodilative action of NO on choroidal blood flow. 4 5 6  
It is not clear, however, whether all choroidal nerve cells serve a vasodilative function or whether at least some of these cells may be involved in other functional processes in the eye that also require regulation mediated by the autonomic nervous system. In support of this, human choroidal ganglion cells differ in immunoreactivity for various neurotransmitters, in size, and in location. Within sections of the human choroid, nerve cells stain with antibodies against vasoactive intestinal polypeptide (VIP) 2 7 and single cells against neuropeptide Y (NPY). 8 In addition, not all cells are widely distributed throughout the suprachoroidal tissue. Some nerve cells seem to be directly associated with ciliary nerves. These ciliary nerve–associated ganglion cells (CNGCs) are localized between the axons of individual ciliary nerves. Further characterization of CNGCs and determination of their functional significance are difficult, because most of the axons join a nerve fiber plexus. Therefore, they cannot be observed in wholemount preparations or serial sections. For cell injections the number of appropriate human eyes is limited. In the present study, we report that in the choroid of porcine eyes CNGCs were the only type of ganglion cell present. The porcine eye could therefore be used as a model to further characterize this type of choroidal ganglion cell and to follow the course of the postganglionic nerve fibers. 
Methods
Porcine Tissue Samples
The eyes of 62 domestic pigs were obtained from the local abattoir within 30 minutes after death, bisected equatorially, and immersion fixed in 4% neutral buffered formalin for 4 hours. After rinsing in phosphate-buffered saline (PBS; pH 7.4) wholemounts of the posterior (24 eyes) and the anterior (7 eyes) uvea were isolated. Ten additional eyes were immersed in 20% saccharose PBS and deep frozen. Fifteen-micrometer-thick frozen sections were cut in sagittal or tangential orientation. The sections were mounted on poly-l-lysine–coated glass slides. In 28 eyes, the ciliary nerves of the inferotemporal quadrant were isolated and examined under a microscope (magnification, ×100). Nerves that contained CNGCs were identified and mounted on glass slides. 
Enzyme Histochemistry
For NADPH-D staining, wholemounts of the posterior and anterior uvea and serial sections through four anterior eye segments were used. The samples were incubated for 2 hours (37°C) with 1 mg/mL NADPH (Biomol, Hamburg, Germany), 0.2 mg/mL nitroblue tetrazolium chloride (Serva, Heidelberg, Germany), and 0.3% Triton X-100 in 0.1 M PBS. After incubation, the wholemounts were depigmented using 3% H2O2 and 1% KOH. This depigmentation method was the same as described in primate eyes. 2 3 (The observed species differences indicate that depigmentation had no influence on the differences in NOS innervation between primate and porcine choroids.) In addition, the RPE was partly removed mechanically using a blunt plastic stick. Wholemounts and sections were mounted with Kaiser’s glycerin gelatin (Merck, Darmstadt, Germany) and examined by microscope (Dialux 20; Leitz, Wetzlar, Germany). 
Immunohistochemistry
Frozen sections through the choroid and anterior segment and isolated ciliary nerves containing CNGCs were stained. After blocking of tissue-specific peroxidase, using 0.3% H2O2 in Tris-buffered saline (TBS; pH 7.4), the samples were incubated in 2% dry milk solution for 20 minutes followed by the primary antibodies (Table 1) . After overnight incubation at 4°C, the sections were rinsed in TBS. Sections were incubated with a host-specific Cy3-fluorescein–conjugated secondary antibody (1:500–1:800; Dianova, Hamburg, Germany) for 1 hour at room temperature, the tissue again was rinsed in TBS and mounted with Kaiser’s glycerin gelatin. The slides were examined with a fluorescence microscope (Aristoplan; Leitz). 
Electron Microscopy
Isolated ciliary nerves containing CNGCs were either fixed in Ito solution 9 or in 4% paraformaldehyde containing 0.1% glutaraldehyde. In the latter group, enzyme histochemical staining for NADPH-D was performed as described earlier. 
All specimens were postfixed in osmium tetroxide, dehydrated in an ascending series of alcohol, and embedded in Epon. Ultrathin sections through the CNGCs were stained with uranyl acetate and/or leaded citrate and examined with an electron microscope (EM 902; Zeiss, Oberkochen, Germany). 
Human Tissue Samples
Both eyes of nine human donors (aged 42–89 years) were obtained 4 to 12 hours after death from the Anatomy Institute in Erlangen, from donors who had given permission to use their bodily tissues for research. Tissue observation was in accordance with the Declaration of Helsinki. The eyes were enucleated, the anterior segments dissected, and the posterior segments fixed as described in the prior section. 
The posterior eye segments of 10 eyes of six donors were divided into four segments, and wholemounts of the choroid and sclera were stained enzyme histochemically for NADPH-D. Three complete sets of wholemounts were used for quantitative evaluation of the CNGCs. 
Immunohistochemical staining was performed on ciliary nerve preparations of three eyes (three donors) and on tangential sections of the choroid that contain ciliary nerves. The primary antibodies included NOS, VIP, substance P (SP), and synaptophysin (see Table 1 ). 
Single NADPH-D–stained ciliary nerves containing CNGCs were postfixed in Ito solution and embedded in Epon. Semithin sections of the CNGCs were stained with toluidine blue. 
Results
Porcine Eyes
Choroid.
In all 24 wholemounts of the porcine choroid that were stained for NADPH-D, positive nerve cells were found adjacent to only one or two ciliary nerves. The number of these CNGCs varied between the eyes studied. In most of the eyes, clusters of 2 to 5 cells with diameters of 30 to 45 μm were found (Fig. 1a) . In others, single neurons were seen that were relatively large in diameter (∼100 μm). The CNGCs were located in the inferotemporal quadrant. Independent of size and number, all CNGCs stained with antibodies against protein gene product (PGP) 9.5, bNOS, and VIP. The CNGCs were surrounded by nerve terminals that stained for synaptophysin, SP, and VIP, but not for bNOS, NPY, tyrosine hydroxylase (TH), vesicular acetylcholin transporter (VAChT), or calcitonin gene-related peptide (CGRP). The terminals were mainly located at the poles of the CNGCs around their processes, and only single synaptic endings were located at the cell body (Fig. 1b) . The CNGCs were surrounded by glial cells positive for glial fibrillary acidic protein (GFAP; Fig. 1c ). 
In semithin and ultrastructural sections, the ganglion cells were characterized by large rounded nuclei with a prominent nucleolus and light, fine, granular nucleoplasm (Fig. 2a) . The less electron-dense perikarya contained organelles including mitochondria, rough endoplasmic reticulum, Golgi apparatus, and free ribosomes. Only a few medium-sized vesicles were seen, sometimes near sacs of the Golgi apparatus. Nerve fibers leaving the ganglion were always nonmyelinated. 
The ganglion cells were ensheathed by glial cells, which formed a basal membrane. They were generally distinguished from the ganglion cells by smaller size, densely stained nuclei, and numerous cytoplasmic processes (Fig. 2b) . At the poles of the CNGCs several axons were enlarged, were not surrounded by glial cells, and showed membrane specializations and dense core or empty vesicles (Figs. 3a 3b)
Wholemount preparations and serial sections revealed that none of the axons of the CNGCs passed into the choroid. The arteries and arterioles in the choroid proper were accompanied by only single perivascular nerve fibers (Fig. 4a) that stained positive for bNOS, VIP, TH, and NPY. A dense NADPH-D–positive suprachoroidal nerve plexus comparable to that in other species (human, monkey, dog, cat, cow, rat) did not exist. However, the endothelial staining for NADPH-D and endothelial (e)NOS was intense in all choroidal vessels, but was most pronounced in the arteries and arterioles (Fig. 4b)
Anterior Eye Segment.
Wholemount preparations of the chamber angle region and serial tangential sections through the tissues of the outflow regions that were stained for NADPH-D or bNOS showed that nerve fibers deriving from the CNGC joined a delicate NADPH-D-bNOS–positive nerve fiber plexus in the trabecular meshwork (TM) and scleral spur. The nerve fibers entered the outflow region in a posterior-anterior course. At the level of the scleral spur, they bent nearly perpendicularly into the TM. Within the TM the thin circularly running nerve fibers formed distinct varicosities (Fig. 5) . In addition to the TM–scleral spur nerve fiber plexus, single NADPH-D-bNOS–positive nerve fibers were located adjacent to smaller arteries and arterioles located in the iris root. In the porcine ciliary body, iris stroma, and cornea, no NOS-positive nerve fibers were found. 
Human Eyes
Within the short ciliary nerves of the human eyes, various numbers of NADPH-D– and bNOS-positive ganglion cells were found, with no preference in quadrant localization (Table 2) . These CNGCs appeared either solitarily or in smaller groups and had a spindle-shaped appearance (Fig. 6a) . Their size varied between 15 to 25 μm in length. The processes of these cells followed the ciliary nerves and did not branch into the suprachoroidal nerve plexus containing the choroidal ganglion cells. Staining of ciliary nerve wholemounts with antibodies against synaptophysin revealed only a few synaptic contacts of nerve fibers with the CNGCs (Fig. 6b)
Discussion
The presence of intrinsic uveal ganglion cells has been described by several investigators. 10 11 12 13 14 15 Further classification of the cells as a first step toward understanding their functional significance, however, was performed only recently. 1 2 3 In choroid and ciliary muscle the ganglion cells stain for NOS and VIP. They are most numerous in primate and avian eyes, which posses a well-developed fovea centralis and accommodation system. 3 16 In the human choroid, nearly 2000 CNGCs were counted. Most of the postganglionic nerve fibers appeared unmyelinated and passed into a perivascular nerve fiber plexus. They presumably contribute to the nitrergic vasodilative innervation of the choroidal vasculature. In the human ciliary muscle, in addition, approximately 900 smaller ganglion cells were found. 16 Most of the postganglionic nerve fibers at this location seem not to innervate the vasculature, but the ciliary muscle cells of the inner circular portion of the muscle. It was hypothesized that this nitrergic innervation might lead to relaxation of the inner ciliary muscle cells especially during the small fluctuations that may help to change shape of the muscle to obtain perfect focus during accommodation. 16  
In this study we describe for the first time that a certain proportion of the intrinsic uveal NOS-immunoreactive (IR) ganglion cells, the CNGCs, do not innervate the choroidal vasculature. We were able to follow the course of the axons of these ganglion cells in porcine eyes, which contain only this type of ganglion cell in the choroid. The postganglionic nerve fibers followed the ciliary nerves toward the outflow tissues, where they entered a nerve fiber plexus innervating, for example, the TM. The CNGCs were surrounded by synaptic boutons that stained for SP and CGRP. The origin of these nerve fibers is not known, because there are no studies describing a detailed immunohistochemistry of the cranial and cervical ganglia in pigs. In various other species, SP- and CGRP-IR nerve fibers derive from the trigeminal ganglion, but nerve cells staining for these neurotransmitters have also been described in the ciliary ganglion. 
In human eyes, because of the dense choroidal innervation, it is difficult to follow the course of the postsynaptic nerve fibers. The localization of the ganglion cells in the ciliary nerves, their spindle shape and smaller size, and the fact that the axons follow the nerves toward the anterior segment led to the assumption that these ganglion cells in human eyes are comparable to the porcine CNGCs. 
NOS-IR nerve fibers are abundant in the primate TM, especially in the cribriform region adjacent to the inner wall of the Schlemm canal. 17 Physiological studies performed in enucleated human and bovine anterior eye segments have shown that NO induces relaxation of TM cells and an increase in outflow facility, whereas contraction of TM cells has an opposite effect. 18 19 Contraction and relaxation of TM cells may be induced by neurotransmitters released from nerve terminals or varicosities. Presence of these structures in the TM have been described by several investigators. 17 20 21 22 23 24 NO released from nerve terminals could cause relaxation of TM cells and allow an increase in outflow facility independent from ciliary muscle contraction. 
In porcine eyes we found a similar network of NOS-IR fibers in the TM. In contrast to other nonprimate mammals, such as rat, rabbit, and cat, which do not regularly contain NOS-IR ganglion cells in the choroid, the porcine eyes, similar to the primates, show a well-developed scleral spur and nearly lamellated TM expanded between spur and cornea. 25 In the inferotemporal quadrant, the location of the CNGCs, there is also a well-developed ciliary muscle inserting into the scleral spur region. Although the innervation described in the TM may be related to some metabolic requirement or function of these cells, we assume that in porcine and primate eyes, the CNGCs may be involved in relaxation of trabecular cells and increase in outflow facility. 
 
Table 1.
 
Specification of the Primary Antibodies Used in the Study
Table 1.
 
Specification of the Primary Antibodies Used in the Study
Antigen Host Dilution Selling Agency
VIP Rabbit 1:400 Euro Diagnostica AB, Malmö, Sweden
NPY Goat 1:100 Amersham Buchler, Braunschweig, Germany
TH Rabbit 1:400 Biotrend, Köln, Germany
SP Rabbit 1:500 Biotrend
CGRP Rabbit 1:500 Euro Diagnostica AB
bNOS Rabbit 1:250 B. Mayer, Graz, Austria
eNOS Mouse 1:400 Transduction Laboratories, Lexington, KY
Synaptophysin Mouse 1:100 Dako A/S, Glostrup, Denmark
VAChT Rabbit 1:1000 Biomar, Marburg, Germany
PGP 9.5 Mouse 1:200 Biotrend
GFAP Rabbit 1:500 Dako A/S
Figure 1.
 
(a) NADPH-D staining of a porcine ciliary nerve preparation. Both elongated spindle-shaped ganglion cells and their processes stained intensely. (b) Synaptophysin staining of porcine CNGCs revealed numerous synaptic contacts at the origin of the processes and only single positive staining at the remaining cell surface. (c) Immunohistochemical staining for GFAP showed positive staining surrounding the ganglion cells. CN, ciliary nerve; ( Image not available ) soma of ganglion cells. Magnification, (a) ×400; (b) ×380; (c) ×360.
Figure 1.
 
(a) NADPH-D staining of a porcine ciliary nerve preparation. Both elongated spindle-shaped ganglion cells and their processes stained intensely. (b) Synaptophysin staining of porcine CNGCs revealed numerous synaptic contacts at the origin of the processes and only single positive staining at the remaining cell surface. (c) Immunohistochemical staining for GFAP showed positive staining surrounding the ganglion cells. CN, ciliary nerve; ( Image not available ) soma of ganglion cells. Magnification, (a) ×400; (b) ×380; (c) ×360.
Figure 2.
 
(a) Semithin section of two CNGCs in the porcine eye. The cells were completely surrounded by glial cells (arrowheads). (b) Electron micrograph of the glial cells showed numerous cytoplasmic processes adjacent to the ganglion cells. N, nucleus of the glial cell. Magnification, (a) ×450; (b) ×14,000.
Figure 2.
 
(a) Semithin section of two CNGCs in the porcine eye. The cells were completely surrounded by glial cells (arrowheads). (b) Electron micrograph of the glial cells showed numerous cytoplasmic processes adjacent to the ganglion cells. N, nucleus of the glial cell. Magnification, (a) ×450; (b) ×14,000.
Figure 3.
 
(a) Electron micrograph of synapses around the porcine CNGCs showed different kinds of vesicles (arrowheads). (b) Higher magnification of one of the synapses. Magnification, (a) 6,500; (b) 15,200.
Figure 3.
 
(a) Electron micrograph of synapses around the porcine CNGCs showed different kinds of vesicles (arrowheads). (b) Higher magnification of one of the synapses. Magnification, (a) 6,500; (b) 15,200.
Figure 4.
 
NADPH-D–stained porcine choroid wholemounts. (a) Only few perivascular nerve fibers were seen surrounding the arteries (arrowheads). The endothelium of the arteries was intensely stained, whereas that of the veins was only faintly stained. (b) The endothelial cells of the capillaries (arrows) also stained for NADPH-D. Magnification, ×60.
Figure 4.
 
NADPH-D–stained porcine choroid wholemounts. (a) Only few perivascular nerve fibers were seen surrounding the arteries (arrowheads). The endothelium of the arteries was intensely stained, whereas that of the veins was only faintly stained. (b) The endothelial cells of the capillaries (arrows) also stained for NADPH-D. Magnification, ×60.
Figure 5.
 
NADPH-D–stained wholemount preparation of the porcine trabecular meshwork (TM) and scleral spur (SS) regions. Several positive nerve fibers ran circumferentially within the TM. Processes deriving from the CNGCs pass into these fibers. Magnification, ×120.
Figure 5.
 
NADPH-D–stained wholemount preparation of the porcine trabecular meshwork (TM) and scleral spur (SS) regions. Several positive nerve fibers ran circumferentially within the TM. Processes deriving from the CNGCs pass into these fibers. Magnification, ×120.
Table 2.
 
NADPH-D–Positive CNGCs Counted in the Various Quadrants of Human Eyes
Table 2.
 
NADPH-D–Positive CNGCs Counted in the Various Quadrants of Human Eyes
Quadrant Male, 42 y Male, 73 y Female, 89 y
Temporal 21 8 43
Nasal 18 17 15
Superior 22 10 21
Inferior 15 4 27
Total 76 39 106
Figure 6.
 
(a) Immunohistochemical staining for NOS of a human ciliary nerve (CN). In contrast to the other choroidal ganglion cells, the NOS-positive CNGCs are spindle shaped. (b) Staining for synaptophysin showed numerous synaptic endings around the CNGCs. Magnification, (a) ×200; (b) ×360.
Figure 6.
 
(a) Immunohistochemical staining for NOS of a human ciliary nerve (CN). In contrast to the other choroidal ganglion cells, the NOS-positive CNGCs are spindle shaped. (b) Staining for synaptophysin showed numerous synaptic endings around the CNGCs. Magnification, (a) ×200; (b) ×360.
The authors thank Marco Gösswein for excellent preparation of the photographs. 
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Figure 1.
 
(a) NADPH-D staining of a porcine ciliary nerve preparation. Both elongated spindle-shaped ganglion cells and their processes stained intensely. (b) Synaptophysin staining of porcine CNGCs revealed numerous synaptic contacts at the origin of the processes and only single positive staining at the remaining cell surface. (c) Immunohistochemical staining for GFAP showed positive staining surrounding the ganglion cells. CN, ciliary nerve; ( Image not available ) soma of ganglion cells. Magnification, (a) ×400; (b) ×380; (c) ×360.
Figure 1.
 
(a) NADPH-D staining of a porcine ciliary nerve preparation. Both elongated spindle-shaped ganglion cells and their processes stained intensely. (b) Synaptophysin staining of porcine CNGCs revealed numerous synaptic contacts at the origin of the processes and only single positive staining at the remaining cell surface. (c) Immunohistochemical staining for GFAP showed positive staining surrounding the ganglion cells. CN, ciliary nerve; ( Image not available ) soma of ganglion cells. Magnification, (a) ×400; (b) ×380; (c) ×360.
Figure 2.
 
(a) Semithin section of two CNGCs in the porcine eye. The cells were completely surrounded by glial cells (arrowheads). (b) Electron micrograph of the glial cells showed numerous cytoplasmic processes adjacent to the ganglion cells. N, nucleus of the glial cell. Magnification, (a) ×450; (b) ×14,000.
Figure 2.
 
(a) Semithin section of two CNGCs in the porcine eye. The cells were completely surrounded by glial cells (arrowheads). (b) Electron micrograph of the glial cells showed numerous cytoplasmic processes adjacent to the ganglion cells. N, nucleus of the glial cell. Magnification, (a) ×450; (b) ×14,000.
Figure 3.
 
(a) Electron micrograph of synapses around the porcine CNGCs showed different kinds of vesicles (arrowheads). (b) Higher magnification of one of the synapses. Magnification, (a) 6,500; (b) 15,200.
Figure 3.
 
(a) Electron micrograph of synapses around the porcine CNGCs showed different kinds of vesicles (arrowheads). (b) Higher magnification of one of the synapses. Magnification, (a) 6,500; (b) 15,200.
Figure 4.
 
NADPH-D–stained porcine choroid wholemounts. (a) Only few perivascular nerve fibers were seen surrounding the arteries (arrowheads). The endothelium of the arteries was intensely stained, whereas that of the veins was only faintly stained. (b) The endothelial cells of the capillaries (arrows) also stained for NADPH-D. Magnification, ×60.
Figure 4.
 
NADPH-D–stained porcine choroid wholemounts. (a) Only few perivascular nerve fibers were seen surrounding the arteries (arrowheads). The endothelium of the arteries was intensely stained, whereas that of the veins was only faintly stained. (b) The endothelial cells of the capillaries (arrows) also stained for NADPH-D. Magnification, ×60.
Figure 5.
 
NADPH-D–stained wholemount preparation of the porcine trabecular meshwork (TM) and scleral spur (SS) regions. Several positive nerve fibers ran circumferentially within the TM. Processes deriving from the CNGCs pass into these fibers. Magnification, ×120.
Figure 5.
 
NADPH-D–stained wholemount preparation of the porcine trabecular meshwork (TM) and scleral spur (SS) regions. Several positive nerve fibers ran circumferentially within the TM. Processes deriving from the CNGCs pass into these fibers. Magnification, ×120.
Figure 6.
 
(a) Immunohistochemical staining for NOS of a human ciliary nerve (CN). In contrast to the other choroidal ganglion cells, the NOS-positive CNGCs are spindle shaped. (b) Staining for synaptophysin showed numerous synaptic endings around the CNGCs. Magnification, (a) ×200; (b) ×360.
Figure 6.
 
(a) Immunohistochemical staining for NOS of a human ciliary nerve (CN). In contrast to the other choroidal ganglion cells, the NOS-positive CNGCs are spindle shaped. (b) Staining for synaptophysin showed numerous synaptic endings around the CNGCs. Magnification, (a) ×200; (b) ×360.
Table 1.
 
Specification of the Primary Antibodies Used in the Study
Table 1.
 
Specification of the Primary Antibodies Used in the Study
Antigen Host Dilution Selling Agency
VIP Rabbit 1:400 Euro Diagnostica AB, Malmö, Sweden
NPY Goat 1:100 Amersham Buchler, Braunschweig, Germany
TH Rabbit 1:400 Biotrend, Köln, Germany
SP Rabbit 1:500 Biotrend
CGRP Rabbit 1:500 Euro Diagnostica AB
bNOS Rabbit 1:250 B. Mayer, Graz, Austria
eNOS Mouse 1:400 Transduction Laboratories, Lexington, KY
Synaptophysin Mouse 1:100 Dako A/S, Glostrup, Denmark
VAChT Rabbit 1:1000 Biomar, Marburg, Germany
PGP 9.5 Mouse 1:200 Biotrend
GFAP Rabbit 1:500 Dako A/S
Table 2.
 
NADPH-D–Positive CNGCs Counted in the Various Quadrants of Human Eyes
Table 2.
 
NADPH-D–Positive CNGCs Counted in the Various Quadrants of Human Eyes
Quadrant Male, 42 y Male, 73 y Female, 89 y
Temporal 21 8 43
Nasal 18 17 15
Superior 22 10 21
Inferior 15 4 27
Total 76 39 106
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