Secretoneurin in the Peripheral Ocular Innervation

Abstract

purpose. To evaluate whether secretoneurin represents a sensory neuropeptide innervating the anterior segment of the eye.

methods. The presence and distribution of secretoneurin was investigated in human eyes by radioimmunoassay and immunofluorescence and compared with that of the rat eye. The source of secretoneurin-positive nerves in the eye was established by measuring the concentration in eye tissues, the trigeminal and superior cervical ganglia both in control rats and in rats treated with capsaicin, and by performing immunofluorescence in one rat subjected to sympathectomy. In the rat trigeminal ganglion, the corresponding mRNA was verified by in situ hybridization and the processing of secretogranin II into secretoneurin by gel filtration chromatography.

results. In human eyes, the highest levels of the peptide were found in the choroid. Nerve fibers were visualized in both species in the upper corneal and limbal stroma; in the trabecular meshwork; in the ciliary muscle, the ciliary body stroma, and processes; and in clear association with the dilator muscle, which disappeared after sympathetic denervation in rats; and also innervating the sphincter muscle in the iris and the choroidal stroma and surrounding blood vessels. Significant amounts of secretoneurin were present in the rat trigeminal ganglion and rat eye tissues. Capsaicin pretreatment led to a 57.0% ± 4.3% and 59.1% ± 11.9% decrease of the concentration in the trigeminal ganglion and the iris/ciliary body complex, respectively. Despite high levels in the rat superior cervical ganglion, sympathetic denervation failed to lower the concentration in eye tissues. The secretogranin II probe labeled numerous small-sized ganglion cells within the rat trigeminal ganglion, and the precursor of the peptide was found to become completely processed into secretoneurin.

conclusions. Apart from the sympathetically innervated dilator muscle, there is unequivocal evidence that secretoneurin represents a constituent of capsaicin-sensitive sensory neurons in the rat trigeminal ganglion and of unmyelinated C-fibers in the rat iris/ciliary body complex, which indicates a participation of this peptide in the ocular irritative response, a model for neurogenic inflammation in lower mammals. Because of the association of nerves with blood vessels and potent angiogenic properties, secretoneurin may be involved in neovascularization processes.

Secretoneurin (SN), a 33-amino acid polypeptide, is generated by proteolytic processing of secretogranin II (SgII), formerly also called chromogranin C. ¹ SgII belongs to the family of chromogranins that comprise chromogranins A and B (for a recent review, see Ref. ² ), SgII (for a recent review, see Ref. ³ ), 7B2, ⁴ and the novel chromogranin-like protein NESP55. ⁵ The chromogranins were originally characterized as the acidic proteins of chromaffin granules. Later on, their widespread expression in large dense core secretory granules of various nerves and neuroendocrine tissues was recognized. In contrast to chromogranin A and B, SgII is expressed in higher amounts in the brain and peripheral neurons relative to the adrenal medulla, ^{6 7} and it is proteolytically processed in the brain to high degrees to smaller peptides. ¹ One of these peptides represents SN. ^{1 8}  

SN is highly conserved during evolution and found in mammals, birds, amphibians, and fish. ⁹ It is distinctly distributed throughout the central nervous system, both in humans ^{10 11 12 13 14} and rats ^{15 16 17 18 19 20} and is released from brain tissue in a Ca²⁺-dependent manner. ²¹ To date, several biological activities have been established for SN. SN induces dopamine release in the rat striatum in vivo and in vitro, ^{22 23 24} and it exerts a very strong chemotactic effect on monocytes and eosinophils but not on granulocytes. ^{25 26 27 28 29} Very recently, a potent angiogenic effect comparable to that of VEGF was reported. ³⁰ Specific binding sites for SN with an affinity in the nanomolar range were found in human monocytes and two monocytic cell lines. ^{31 32}  

SN has been detected in various endocrine organs, including the adrenal medulla; thyroid C cells; the anterior pituitary; the A and B cells of the pancreas; and the endocrine cells of the gastrointestinal tract or bronchial mucosa. ³³ In the peripheral nervous system, it has been found in preganglionic neurons projecting to ³⁴ as well as in postganglionic neurons derived from the superior cervical ganglion, ³⁵ indicating a participation in sympathetic transmission. Furthermore, SN is present within the human enteric neuroendocrine system, ³⁶ and in various sympathetic, enteric, and sensory ganglia, ³⁷ and in rat pelvic neurons and vas deferens. ³⁸ It also innervates the rat uterus. ³⁹ Thus, a widespread distribution of this neuropeptide within the neuroendocrine vegetative system is evident. 

With respect to the eye, there is little knowledge available. SN is expressed in amacrine cells in the proximal inner nuclear layer and in displaced amacrine cells in the ganglion cell layer in the human retina, ⁴⁰ which is reminiscent of the classic localization of typical neuropeptides in the eye. No studies on the distribution of SN in the anterior segment of the eye are available. The anterior segment is innervated by sympathetic and parasympathetic neurons, as well as sensory neurons. There are clear indications that SN is part of the sensory innervation of the eye. First, topical application of formaldehyde elevates the concentration of SN in the rabbit aqueous humor. ⁴¹ This kind of irritation is well known to provoke neurogenic inflammation exclusively by releasing sensory peptides that derive from the trigeminal ganglion, in particular substance P (SP) and calcitonin gene-related peptide (CGRP) (for a review, see Ref. ⁴² ). Second, SN immunoreactivity has been detected in cells of the rat trigeminal ganglion by immunohistochemistry. ³⁷ Third, this peptide is present in capsaicin-sensitive sensory neurons in the peripheral nervous system—in particular in rat dorsal root ganglion cells ⁴³ and in nerves innervating the rat uterus. ³⁹  

In this study, we investigated whether SN is present in capsaicin-sensitive sensory neurons innervating the eye. First, the levels and distribution of SN-immunoreactivity were studied in the human and rat eye, and then the source of nerve fibers was explored in detail in animal experiments. Capsaicin represents an ideal tool to investigate sensory neurons, as it destroys more than a half of these neurons when injected into newborn rats—in particular, small neurons giving rise to unmyelinated C-fibers. ^{44 45 46} By such experiments, SN was found to be present in sensory neurons. These findings were further corroborated by an analysis of the corresponding mRNA by in situ hybridization in the rat trigeminal ganglion. To establish whether SN also contributes to the sympathetic innervation of the eye, the levels of the peptide were measured in the rat superior cervical ganglion and in various eye tissues subsequent to sympathetic denervation of the eye by axotomy. Axotomy was also used to evaluate an effect on nerve staining by immunofluorescence. 

Materials and Methods

Capsaicin Treatment of Rats

Newborn Sprague-Dawley rats were injected subcutaneously under the neck fold with a single dosage of 50 mg/kg capsaicin (obtained from Sigma-Aldrich, Vienna, Austria) on the first day after birth. Capsaicin was dissolved in saline containing 10% ethanol and 10% Tween 80. Untreated animals served as control subjects. The animals were housed in cages with a dark–light cycle of 12 hours each (lights on at 7 AM and off at 7 PM, in 23 ± 1°C) and fed a commercial chow and water ad libitum. The animals were allowed to grow for 3 months and were then killed by an overdose of CO₂ followed by dissection of tissues. 

Sympathetic Denervation of the Rat Eye by Axotomy

Eight young adult male Sprague-Dawley rats (6–8 weeks old) were used. The rats were anesthetized with intramuscular injections of fentanyl (0.05 mg/kg body weight; Janssen, Neuss, Germany) and dehydroxybenzperidol (5 mg/kg; Janssen) and the left superior cervical ganglion was exposed by a midline incision. Axotomy was performed by transecting the internal as well as the external carotid nerve close to the ganglion with the aid of a stereoscopic dissecting microscope while carefully sparing the blood supply of the ganglion. The contralateral nonsurgical side served as the control. Each surgically altered animal exhibited the signs of an ipsilateral Horner syndrome with miosis and ptosis of the left eyelid. After a survival period of 7 days, the animals were killed by an overdose of CO₂,_. and the eyes were removed. Subsequently, the cornea, the iris/ciliary body complex, the retina, and the choroid/sclera were dissected, weighed, and stored at −80°C until further processing by radioimmunoassay. 

All experimental and animal care procedures were performed in compliance with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research, and the animal experiments were approved by the Ministry of Science in Austria. 

Preparation of Human Eyes

Human donor eyes obtained postmortem for corneal transplantation were provided by the local cornea bank. Ten eyes of five donors, including three men (mean age 77.4 years) and two women (mean age 67.8 years), were processed for determination of SN. None of the donor eyes had signs of ocular diseases, infections, or pseudophakia. At the beginning, an incision was made at the limbus, and the cornea inclusive of the limbus was circumferentially excised. Next, the sclera was detached, and a small piece cut off. After excision of the cornea and sclera, the eye cup was turned around and the free-lying choroid was gently removed. Then the retina was dissected and finally the iris/ciliary body complex was removed. The study protocol adhered to the provisions of the Declaration of Helsinki for research involving human tissue. 

Radioimmunoassay for Determination of the Concentration of SN in Rat and Human Eye Tissues, in the Rat Trigeminal and Superior Cervical Ganglion

SN was heat extracted from the various eye specimens and ganglia. For this purpose, the tissues were sonicated in distilled water, boiled for 10 minutes, and centrifuged at 14,000g for 20 minutes at 4°C, and the clear supernatant was then analyzed for the presence of SN by radioimmunoassay, as described previously. ¹ The cornea and sclera were chopped up with a razor blade before sonication. 

In brief, iodination of SN (Neosystems, Strasbourg, France) was performed with the chloramine T method to a specific activity of 7.7 × 10⁴ cpm/ng. SN and ¹²⁵I-labeled peptides were used as a standard and a tracer, respectively. Samples and standards were incubated with the antiserum (dilution 1:18,000) and tracer (10⁴ cpm) for 48 hours at 4°C. All dilutions were made with radioimmunoassay buffer (150 mM NaH₂PO₄ [pH 7.4], 15 mM NaCl, 0.02% NaN₃, 0,0006% phenol red, 0.1% bovine serum albumin, and 0.06% gelatin). Bound and free activities were separated by adding 1 mL of dextran-coated charcoal. After a 15-minute incubation at 4°C, samples were centrifuged for 15 minutes at 3200g followed by counting of the supernatant in a γ-spectrophotometer. The antiserum against synthetic rat SN (SgII 154-186) coupled to keyhole limpet hemocyanin was raised in Chinchilla bastard rabbits with a standard immunization protocol. ¹ It reacts with the free peptide and all larger proteins containing the SN sequence including SgII equally well. The antiserum fully cross-reacts with human SN, which differs in only one amino acid with the rat sequence. 

In Situ Hybridization in the Rat Trigeminal Ganglion

For in situ hybridization, the ganglia of three untreated rats were dissected, mounted in optimal cutting temperature compound (Tissue-Tec; Sakura Finetec, Loeterwoude, The Netherlands), and frozen in 2-methylbutane (−30°C). Serial sections (20 μm) were cut on a cryostat (Microm, Heidelberg, Germany), mounted onto polylysine-coated slides (Menzel-Glazer, Braunschweig, Germany) and stored at −20°C. The sections then were fixed for 10 minutes in 2% paraformaldehyde and rinsed twice in phosphate-buffered saline (PBS) followed by 0.25% acetic anhydride in 0.1 M triethanolamine and 0.95% sodium chloride (pH 8) for 10 minutes. Tissues were dehydrated through a series of ethanol dilutions, delipidated in chloroform for 5 minutes, rehydrated, and air dried. For SgII, a 48-mer nucleotide corresponding to amino acids 25-40 of rat SgII was used as described previously. ⁴⁷ The oligonucleotides were 3′-labeled with terminal deoxynucleotidyl transferase (Roche, Mannheim, Germany) and ³⁵S-α-thio-dATP (NEG 034H; NEN, Boston, MA) at 37°C for 30 minutes. Labeled probe of known radioactivity (1.5 × 10⁶ cpm) was applied to individual sections in 50 μL of hybridization buffer containing 50% formamide. ⁴⁷ The sections were placed in humid chambers and incubated for 18 hours at 42°C. Posthybridization washes included four changes at 15-minute intervals of 300 mM sodium chloride and 30 mM sodium citrate with 50% formamide at 42°C preceded and followed by 150 mM sodium chloride and 15 mM sodium citrate at room temperature. Finally, slides were rinsed in water and 70% ethanol and left to dry. Dried sections were exposed to ³H-sensitive film (Biomax MR; Eastman Kodak, Rochester, NY), dipped in photographic emulsion (NTB2, diluted 1:1 with water; Eastman Kodak), and exposed at 4°C for 14 to 28 days, counterstained with cresyl violet, and coverslipped. 

Control sections incubated with a scrambled oligonucleotide probe gave no signal. 

High-Performance Gel Filtration Chromatography in the Rat Trigeminal Ganglion

SN was heat-extracted from two trigeminal ganglia, as described earlier; the supernatant was lyophilized; and the lyophilized tissue extract was redissolved in column buffer. An aliquot of the sample was loaded on a gel filtration column (Superose 12HR 10/30; Amersham Pharmacia, Uppsala, Sweden) at a flow rate of 0.4 mL/min. Filtered (Millipore, Bedford, MA) 75 mM sodium phosphate buffer (pH 7.4; containing 75 mM NaCl and 0.02% NaN₃) was used as column buffer. One-minute fractions were collected and analyzed by radioimmunoassay. 

Immunofluorescence for the Visualization of SN-Immunoreactive Nerves in Human and Rat Eye Tissues

The eyes of a 75-year-old donor and a 67-year-old donor obtained from the local cornea bank were placed in an ice-cold solution of 4% paraformaldehyde in PBS for 4 hours. The eyes were separated into four parts and, after removal of the lens and vitreous, the tissues again were immersed in 4% paraformaldehyde in PBS for another 4 hours. The blocks were placed in a solution containing 20% sucrose in PBS for at least 24 hours and then frozen in cold (−60°C) isopentane and stored at −70°C. Six- to 15-μm-thick sections were cut from the specimens on a cryostat (Reichert Jung; Leica-Reichert, Vienna, Austria) at −20°C and mounted on poly-l-lysine–coated slides. Fixed human eye sections ⁴⁸ were washed for 1 hour at room temperature in Tris-buffered saline (TBS; 50 mM Tris [pH 7.5] and 0.9% NaCl) with 0.3% Triton X-100. The sections were then preincubated for 1 hour with 20% normal goat serum (NGS) in TBS with 0.3% Triton X-100 in disposable immunostaining chambers (Shandon Coverplate, Cat. No. 7211013; Thermo Electron Corp., Woburn, MA) and subsequently incubated for 72 hours at 4°C with polyclonal rabbit SN antiserum ¹ at a dilution of 1:3000 in TBS containing 2% normal goat serum (NGS) and 0.3% Triton X-100. After three washes with TBS, sections were incubated with the secondary antibody (Cy3-conjugated AffiniPure goat anti-rabbit IgG; catalog no. 111-165-006; Jackson ImmunoResearch Laboratories, West Grove, PA) diluted 1:400 for 24 hours at 4°C. Stained sections were washed three times with TBS, mounted with 0.4% gelatin containing 0.04% chrom(III)sulfate, and coverslipped. Sections were visualized with an optical microscope (Axioplan; Carl Zeiss Meditec, Jena, Germany) and micrographs obtained (AxioCam HR; Carl Zeiss Meditec). In control experiments, no immunoreactivity was detected with antibodies adsorbed with an excess of SN (1 μM) or when the primary antibody was omitted. Furthermore, sections were processed for double immunofluorescence with the SN- and a SP-antibody (Research Diagnostics Inc., Flanders, NJ) at equal dilutions. 

Rat eyes were also examined for the presence of SN-immunoreactive nerves by immunofluorescence. For this purpose, one male rat (200g) was deeply anesthetized with an intraperitoneal injection of pentobarbital sodium (100 mg/kg) and perfused transcardially with saline followed by 4% paraformaldehyde. The eyes were removed, and the tissues dissected and immersion fixed in 4% paraformaldehyde for 2 hours followed by rinses in 20% sucrose (pH 7.4). Sections were cut and processed by immunofluorescence as described earlier. 

Furthermore, one male rat (200 g) was sympathetically denervated on the left side and, after 7 days, the animal was transcardially perfused and the eye tissues conveyed for immunofluorescence. 

Results

Concentration of SN in Human Eye Tissues

The amounts of SN in various tissues of the human eye are given in Table 1 . The highest levels were found in the choroid, but significant amounts were detected in the retina and in the iris/ciliary body complex. Lower levels were present in the sclera and in the cornea. 

Visualization of SN-Immunoreactive Nerves in the Human and Rat Eye by Immunofluorescence

SN immunoreactivity was found to be distinctly distributed throughout the anterior segment of the human eye (Fig. 1) . 

In the cornea, nerve fibers were visualized in the stroma (Fig. 1A) , whereas the epithelium and endothelium featured autofluorescence but were devoid of SN. At the corneoscleral limbus, a dense network of immunoreactive nerves was present in the upper stroma surrounding blood vessels (Fig. 1B) . In the chamber angle, individual nerve fibers were seen in the trabecular meshwork adjacent to Schlemm’s canal (Fig. 1C) . In the iris, a moderate number of SN-positive nerves was observed within the stroma (Fig. 1D) , and sometimes immunoreactive nerves surrounded blood vessels (not shown). Intense staining was present in clear association with the dilator muscle (Fig. 1D) , but nerve fibers were also abundantly found within the sphincter muscle (Fig. 1E) . The ciliary body featured rich innervation. Dense immunoreactive nerves were observed within the ciliary muscle (Fig. 1F) . There was a dense network of SN-immunoreactive nerves present in the stroma at the base of the ciliary processes (Fig. 1G) , and dispersed nerve fibers were found within the stroma of the ciliary processes (Fig. 1G) . Finally, abundant SN-positive innervation occurred in the choroid, particularly in the stroma and surrounding blood vessels (Fig. 1H) , which was also seen with SP (Fig. 1I) , indicating colocalization. 

In rat eyes, the distribution was similar to that in human eyes. As an example, an intense presence of immunoreactive nerves associated with blood vessels is demonstrated in Figure 1Jin the choroid, where also an intrinsic ganglion cell is shown. The main difference between human and rat eyes was a more dense presence of nerve fibers in the iris stroma (Fig. 1K) , and in each eye tissue nerves remained despite sympathetic denervation, whereas the association with the dilator muscle disappeared after axotomy (Fig. 1L) . 

Concentration of SN in Various Rat Tissues after Capsaicin Pretreatment and Sympathetic Denervation

The concentrations of SN in the rat trigeminal ganglion and in various rat eye tissues including the cornea, the iris/ciliary body complex, the choroid/sclera, and the retina in untreated control animals and after capsaicin pretreatment and sympathetic denervation of eyes by axotomy are given in the Tables 2 and 3 . 

SN immunoreactivity was found to be present in each of the tissues studied in various amounts. In the trigeminal ganglion, SN averaged 21.1 ± 4.3 fmol/mg wet weight in control subjects, and it was significantly decreased in capsaicin-pretreated rats (43.0% ± 4.3% of the control). In the rat eye, the highest concentrations were found in the retina followed by the iris/ciliary body complex and the choroid/sclera. Lower amounts of SN were detected in the cornea. Capsaicin pretreatment significantly lowered the levels of SN in the iris/ciliary body complex by 59.1% (±11.9%). In the retina, cornea, and choroid/sclera, no significant changes were found. 

In the rat superior cervical ganglion, the levels of SN averaged 232.2 ± 21.4 fmol/total ganglion and were found to be higher than those of SP, CGRP, neurokinin A (NKA) and vasoactive intestinal polypeptide (VIP) (Troger J, unpublished observation, 2004). Capsaicin pretreatment had no influence on the concentration of SN in the superior cervical ganglion (data not shown). 

The concentration of SN in the cornea, the iris/ciliary body complex, the retina and choroid/sclera in the nonsurgical right eyes and axotomized left eyes is given in Table 3 . There was a moderate decrease of the levels of SN found in each of the sympathetically denervated tissues, but statistical calculation revealed no difference in SN levels between control and axotomized tissues. 

The Molecular Form of SN in the Rat Trigeminal Ganglion

Gel filtration chromatography of tissue extracts of the rat trigeminal ganglion followed by radioimmunoassay for SN in each of the collected fractions revealed the presence of a major peak in position 37-43 (Fig. 2) . This position corresponds to the peptide SN standard. In the position of the precursor SgII, no immunoreactivity was found, indicating complete proteolytic processing of the precursor molecule to the neuropeptide SN already present at the site of synthesis. 

Expression of SgII mRNA in the Rat Trigeminal Ganglion

The SgII probe distinctly labeled nerve cell bodies within the trigeminal ganglion, indicating the presence of SgII mRNA (Fig. 3) . The labeled cells were mainly of small size and as a rule, the cells labeled with the SgII probe were observed numerously (Fig. 3C)and occurred in scattered locations (Figs. 3B 3C) , although occasionally small clusters or rows of cells were also seen (Fig. 3A) . The probe did not label nerve fibers or non-neuronal cells in the ganglia. Incubation of sections with a scrambled probe failed to produce a signal (Fig. 3D) . Counting cells expressing SgII mRNA (with a signal of at least five times or more over background) revealed that 31.1% ± 1.8% of principal ganglion cells contained the signal. 

Discussion

Our results obtained from animal experiments unequivocally establish SN as a novel constituent of sensory neurons innervating the anterior segment of the eye for the following reasons: First, the peptide was present within the rat trigeminal ganglion in significant amounts. The levels of SN were lower than those of CGRP, which is the most abundant sensory peptide in the ganglion—in particular they are approximately one half of those of CGRP (Troger J, unpublished observation, 2004). This finding is in agreement with the lower amount of SgII mRNA-expressing cells (30%) versus those expressing CGRP mRNA (40%–50%). ⁴⁹ Secondly, neonatal capsaicin treatment led to a decrease of SN-immunoreactivity in the rat trigeminal ganglion in the range of 50%. Capsaicin destroys predominantly small neurons, giving rise to unmyelinated C-fibers, ^{44 45 46} indicating that SN is present in these small neurons. This is in accordance with in situ hybridization experiments where mainly small-sized cell bodies of the ganglion expressed the signal. Finally, capsaicin treatment also decreased the levels of SN in the rat iris/ciliary body complex, which indicates the presence of the peptide in unmyelinated C-fibers there. 

The minor effect of capsaicin on SN levels in the rat cornea and choroid/sclera argues for the presence of either capsaicin-insensitive unmyelinated C-fibers (as shown in the rat enteric nervous system ⁴⁶ ) and/or the presence of SN in myelinated fibers, most obviously in Aδ-fibers, which also arise from small-sized cells and are less sensitive to capsaicin. ^{44 45 46} Furthermore, sprouting of surviving sensory neurons in the cornea after systemic capsaicin treatment ⁵⁰ may have masked a more pronounced drop of corneal SN levels. Alternatively, SN-immunoreactive nerves of other origin than the sensory ones may exist in the cornea and choroid/sclera. High concentrations of SN were indeed measured in the rat superior cervical ganglion. The levels are in the range of those of neuropeptide Y (NPY; Troger J, unpublished observation, 2004), which is a well-known constituent of the postganglionic sympathetic innervation of the eye. ^{51 52 53} However, although the levels of SN tended to decrease moderately, sympathetic denervation of the rat eye by axotomy failed to lower them significantly which is in sharp contrast to NPY. In accordance, most of the SN immunoreactivity within the superior cervical ganglion was present in nerve endings and varicosities of preganglionic origin, whereas the ganglion cells exhibited weaker staining. ^{35 37} Thus, apart from the dilator muscle (discussed later), SN does not contribute to the postganglionic sympathetic innervation of the eye. An involvement of SN in parasympathetic transmission cannot be excluded but is difficult to explore, because the postganglionic parasympathetic neurons supplying the eye derive both from the ciliary and the pterygopalatine ganglion, ⁵⁴ making denervation studies difficult. The role of SN in parasympathetic transmission must be examined in a further study. 

In human eyes, the following interesting conclusions can be drawn: First, the levels of SN in the cornea were higher than those of VIP but lower than those of CGRP or NKA (Troger J, unpublished data). CGRP- and also SP-immunoreactive nerves were found to be abundant around limbal blood vessels, and sparse fibers are also present in the stroma reaching the epithelium in various species. ^{54 55 56} In contrast, VIP could be made visible by Jones and Marfurt ⁵⁷ in the rat cornea for the first time, whereas Stone et al. ^{54 58} found nerves certainly around blood vessels at the limbus but failed to find nerves within the cornea. The relative low levels of SN in the human cornea are in contrast to the abundant presence of fluorescent immunoreactivity, particularly within the upper limbal stroma. Second, there was a prominent innervation in the ciliary body including the ciliary muscle which is similar to SP, CGRP, or NPY. ^{54 55} This may indicate a participation of this peptide in the regulation of the muscle tonus and consequently in accommodation. Third, there was a clear association of SN immunoreactivity with the dilator muscle, which is similar to NPY. ^{54 59} This association was found in both species and disappeared after sympathetic denervation in the rat, indicating a sympathetic origin. Thus, there is a dichotomy—that is, the sympathetically innervated dilator muscle and the sensory origin of stromal nerves in the iris. In contrast to NPY SN-positive nerve fibers were also prominent within the sphincter muscle, indicating a dual innervation for SN similar to SP ^{54 55} and VIP. ^{54 58} Whether SN induces a contractile response on these muscles remains to be elucidated. The minor presence of nerves in the human iris stroma can be explained by age-related atrophy in humans rather than by species differences. Finally, the abundance of SN-positive nerve fibers in the human choroid reflects the high concentration of the peptide as measured by radioimmunoassay. Furthermore, there is a clear association with the innervation of blood vessels that is similar to most of the classic neuropeptides in the eye. ⁵⁴ The higher levels of SN in the human choroid when compared with the rat can be explained by the way that the choroid was not separated from the sclera in the rat, and the sclera with low amounts of SN is the tissue that makes up the major part of the wet weight. 

The demonstration of nerve fibers in the iris/ciliary body complex together with the expression of SgII mRNA in nonpigmented ciliary epithelial cells ^{60 61} completes the picture of the innervation pattern in this tissue. SN has been detected in significant amounts in the rabbit ⁴¹ and human aqueous humor (manuscript submitted) indicating either secretion from the nonpigmented ciliary epithelium and/or release from nerve fibers of the iris/ciliary body complex. Sensory neuropeptides in the iris/ciliary body complex are well known to provoke the irritative response in the eye, which represents a classic model for neurogenic inflammation in lower mammals (for a review, see Ref. ⁴² ). SP is known to be mainly responsible for the miosis and CGRP for the vascular effects, at least in the rabbit, whereas prostaglandins act in a modulatory way. ⁴² Further mediators of neurogenic inflammation in the eye are pituitary adenylate cyclase-activating polypeptide ⁶² and nitric oxide, which activates C-fibers and mediates the vascular responses of the peptides, at least in the inflammation induced by electroconvulsive treatment ⁶³ and by intravitreally injected endotoxin. ⁶⁴ Whether SN is involved in this irritative response of the eye remains to be elucidated. It must be emphasized that this peptide features biological effects that contribute to neurogenic inflammation in general (for a review, see Ref. ⁶⁵ ). It attracts human monocytes, ^{25 26 27 28} stimulates migration of fibroblasts, ⁶⁶ stimulates migration and inhibits proliferation of endothelial cells, ⁶⁷ stimulates migration and proliferation of smooth muscle cells, ⁶⁸ and is a potent attractant for human eosinophils. ²⁹ SN may be involved in the neurogenic inflammation of the eye, as this represents also a process with the function of neurons to maintain homeostasis in the face of irritation to the tissue, as SN is a constituent of C-fibers in the rat iris/ciliary body complex, which are known to mediate the response and as the peptide is released by formaldehyde irritation into the rabbit aqueous humor. ⁴¹  

Another main finding that may be clinically relevant concerns the clear association of SN-immunoreactive nerves with blood vessels. Very recently a potent angiogenic effect of SN comparable to that of VEGF was reported in the eye. ³⁰ Clinically, rubeosis iridis and neovascularizations on the retinal surface result frequently from ischemia due to diabetes mellitus and central retinal vein occlusion. In the avascular cornea, neovascularizations originating from limbal blood vessels may also occur in a variety of diseases. The most accepted explanation for the appearance of new vessel formation is the ischemia-induced synthesis of proangiogenic growth factors and cytokines—in particular, VEGF and bFGF. ⁶⁹ The chemotactic properties of SN on endothelial cells in vitro, ⁶⁷ the angiogenic effect of the peptide, ³⁰ the close interaction of SN-containing nerve fibers with blood vessels in the eye, and the increased synthesis of SN in the setting of ischemia ⁷⁰ may indicate that this peptide contributes to neovascularization processes in the eye. This is strengthened by the observation that other neuropeptides closely associated with blood vessels in the eye have also been reported to induce angiogenesis—in particular, SP ⁷¹ and NPY. ⁷²  

In conclusion, SN was found to be present in capsaicin-sensitive sensory neurons innervating the rat eye. SN innervates particular eye tissues both in the rat and in humans, as evidenced by the presence of immunoreactivities and of immunoreactive nerves, respectively. In the corresponding trigeminal ganglion, the precursor SgII is completely processed to SN and numerous small-sized ganglion cells contain the signal there. The functional significance of this peptide in the eye must be evaluated in further studies, and the present study provides the basis for these experiments. 

 Supported by Grant P14022-Med from the Austrian Science Foundation (JT).

 Submitted for publication April 16, 2004; revised July 21 and October 1, 2004; accepted October 12, 2004.

 Disclosure: J. Troger, None; A. Doblinger, None; J. Leierer, None; A. Laslop, None; E. Schmid, None; B. Teuchner, None; M. Opatril, None; W. Philipp, None; L. Klimaschewski, None; K. Pfaller, None; W. Göttinger, None; R. Fischer-Colbrie, None

 The publication costs of this article were defrayed in part by page charge payment. This article must therefore be marked “advertisement” in accordance with 18 U.S.C. §1734 solely to indicate this fact.

 

Table 1.

 

Concentration of Secretoneurin in Various Human Eye Tissues

Tissue	SN
Cornea	0.2 ± 0.02
Iris/ciliary body	1.5 ± 0.1
Retina	2.3 ± 0.1
Choroid	4.6 ± 0.4
Sclera	0.3 ± 0.04

 Data are expressed as mean femtomoles per milligram of wet tissue weight ± SEM. n = 10.

Figure 1.

 

SN-immunoreactive nerves in human (A–I) and rat (J–L) eyes. SN-immunoreactive nerves were visualized by immunofluorescence. In the cornea, nerve fibers are shown in the anterior two thirds of the stroma (A, arrows). Arrowheads: autofluorescence in the epithelium. At the limbus, a dense network of immunoreactive nerves was present in association with vessels (B, arrows). An immunoreactive nerve was present in the trabecular meshwork adjacent to the inner wall of Schlemm’s canal (C, arrow). In the iris, a moderate number of nerves were observed within the stroma (D, arrowheads) but there was a clear association evident with the dilator muscle (arrows) and sometimes blood vessels (data not shown), and nerves were also visualized in the region of the sphincter muscle (E, arrows). A dense innervation of the ciliary muscle was observed (F). A dense network was present in the ciliary body stroma at the base of the ciliary processes (G, arrowheads) with nerves sometimes passing to the stroma of the processes (arrows). Finally, in the choroid, blood vessels and the stroma were richly supplied both by (H, arrows) SN- and (I, arrows) SP-immunoreactive nerves, indicating colocalization. In rat eyes, the distribution was similar to that of human eyes. As an example, a dense network of fine-scattered varicosities was observed in the choroid both in the stroma and around blood vessels (J, arrows). Arrowhead: a highly fluorescent structure, most obviously an intrinsic ganglion cell. By contrast with humans, a more dense presence of nerves was found in the iris stroma (K, arrows), whereas the association of nerves with the dilator muscle was also evident in the rat (arrowheads). In sympathetically denervated eyes, the association with the dilator muscle disappeared (L, arrowheads) but nerves in the stroma (arrows) and in other eye tissues did not (data not shown). CM, ciliary muscle; Ep, epithelium; TM, trabecular meshwork; Sc, Schlemm’s canal; Sph, sphincter; St, stroma; V, vessel.

Table 2.

 

Concentration of Secretoneurin in the Rat Trigeminal Ganglion and Various Rat Eye Tissues in Control and Capsaicin-Pretreated Specimens

	SN
		Control Subjects	Capsaicin
TG	21.1 ± 4.3	9.1 ± 0.4*
Cornea	1.6 ± 0.2	1.1 ± 0.2
Iris/ciliary body	23.3 ± 6.0	9.5 ± 2.8, †
Retina	44.1 ± 6.5	43.5 ± 5.4
Choroid/sclera	8.0 ± 1.4	6.2 ± 0.7

 Data represent mean femtomoles per milligram wet weight ± SEM (n = 4–6). Calculation of differences between control subjects and capsaicin data was done with the Mann-Whitney test.

*   P < 0.01.

†   P < 0.05.

Table 3.

 

Concentration of Secretoneurin in Various Rat Eye Tissues after Axotomy of the Left Eye

	SN
		Control Eyes	Axotomized Eyes
Cornea	1.9 ± 0.1	1.8 ± 0.2
Iris/ciliary body complex	21.2 ± 2.8	17.8 ± 2.3
Retina	30.3 ± 4.4	26.7 ± 3.3
Choroid/sclera	9.0 ± 0.8	7.7 ± 0.6

 The right eye served as a control. Data represent mean femtomoles per milligram wet weight ± SEM of eight eyes, respectively. There were no significant differences observed between control and sympathetically denervated tissues.

Figure 2.

 

Gel filtration chromatography of tissue extracts of two trigeminal ganglia. Note the presence of a single peak corresponding to the position of the peptide SN and the absence of a peak in the position of SgII.

Figure 3.

 

Expression of SgII mRNA in the rat trigeminal ganglion by in situ hybridization. (A, B) Cells are shown in the bright-field micrograph. The SgII-expressing cells were predominantly of small size (cells in A–C) and appeared mainly in scattered locations (B, C), although occasionally clusters were also observed (A). The SgII probe labeled numerous ganglion cells, as illustrated in the dark-field micrograph (C). In control sections, no signal was obtained with a scrambled probe (D).

The authors thank Hildegunde Knaus for excellent technical assistence.