Distinct Substance P and Calcitonin Gene-Related Peptide Immunoreactive Nerves in the Guinea Pig Eye

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1 Distinct Substance P and Calcitonin Gene-Related Peptide Immunoreactive Nerves in the Guinea Pig Eye Yasuaki Kuwayama* ond Richard A. Srone Using a double labeling indirect immunofluorescent technique, we studied the guinea pig trigeminal ganglion and eye for co-localization of substance P and calcitonin gene-related peptide. In the trigeminal ganglion, the number of neurons immunoreactive for calcitonin gene-related peptide significantly outnumber those immunoreactive for substance P, but virtually all substance P positive neurons are immunoreactive for calcitonin gene-related peptide. In the eye, a complex pattern of co-localization is present; both peptides co-localize in most immunoreactive nerve fibers. Nerve fibers immunoreactive only for calcitonin gene-related peptide tend to be concentrated in the cornea and posterior ciliary body. Nerve fibers immunoreactive only for substance P are present in relation to both iris muscles. Sensory denervation by intracranial transection of the ophthalmic and maxillary nerves fails to eliminate these substance P positive but CGRP negative iris nerve fibers. These findings indicate an alternative origin for substance P immunoreactive nerves supplying the iris muscles in this species. Invest Ophthalmol Vis Sci 28: ,1987 Three neuropeptides have been localized to date in sensory nerves of the guinea pig eye: substance P (SP), cholecystokinin (CCK), and calcitonin gene-related peptide (CGRP). 1 SP and CCK partially co-localize both in nerve fibers of the uvea and in their cells of origin in the guinea pig trigeminal ganglion. 2 SP also co-localizes with CGRP in rat 3 " 7 and guinea pig 8-9 trigeminal ganglia, and the coexistence of SP- and CGRP-like immunoreactivities is found in nerve fibers of the rat iris 8 and the guinea pig cornea and iris. 3 While not directly studied in the eye, the latter two peptides now are known to coexist even in the same neurosecretory vesicle. 10 In the present study, we have investigated further the co-localization of SP and CGRP in the nerve fibers of the guinea pig eye. Materials and Methods All procedures in this study conformed to the ARVO Resolution on the Use of Animals in Research. From the Department of Ophthalmology, University of Pennsylvania School of Medicine, Scheie Eye Institute, Philadelphia, Pennsylvania. * Current address: Department of Ophthalmology, Osaka University Medical School, Osaka, Japan. Supported by a fellowship from Research to Prevent Blindness, Inc. (Y.K.), NEI Grant EY-05454, an unrestricted grant from Allergan Pharmaceuticals, and the Gretel and Eugene Ormandy Teaching and Research Fund of the Scheie Eye Institute. Submitted for publication: February 11, Reprint requests: Richard A. Stone, 418 Johnson Pavilion, University of Pennsylvania School of Medicine, Philadelphia, PA Surgical Procedures Eight ( g) albino male guinea pigs were anesthetized with a mixture of intramuscular ketamine hydrochloride and acepromazine. Under direct visualization, unilateral intracranial neurotomy of the ophthalmic and maxillary nerves as far distal as possible from the trigeminal ganglion was performed on five guinea pigs; on the other three, unilateral superior cervical ganglionectomy and intracranial neurotomy of the ophthalmic and maxillary nerves were performed on the same side. The ipsilateral eyelid of each operated animal was sutured shut to protect the cornea. Two to three weeks after surgery, the animals were processed for immunohistochemistry. In all of these animals, histologic sections confirmed the complete interruption of the ophthalmic and maxillary nerves and the absence of identifiable ganglion cells distal to the site of nerve section. Tissue Preparation Normal and operated guinea pigs were anesthetized with intraperitoneal sodium pentobarbital and perfused through the left ventricle, first with saline and then with Zamboni's fixative." The eyes and trigeminal, superior cervical, pterygopalatine and ciliary ganglia were removed, post-fixed for 24 hr at 4 C, and then washed overnight at 4 C in 0.1 M phosphate buffer, ph 7.4, with 30% sucrose. Sixteen to twenty micron thick cryostat tissue sections were thaw-mounted on gelatin-coated slides, dried at room temperature, and stored at 20 C until stained by an indirect immunofluorescence technique. 1947

2 1948 INVESTIGATIVE OPHTHALMOLOGY & VISUAL SCIENCE / December 1987 Vol. 28 Antibodies Two primary antisera against CGRP were generated in rabbits: one against the synthetic C-terminal sequence of CGRP 12 ; the other, against a conjugate of rat CGRP. 4 A monoclonal antibody to SP (Pel-Freeze Biologicals, Rogers, AR; Lot No. B3K35) was raised in rats against synthetic SP conjugated with carbodiimide to bovine serum albumin; it recognizes the carboxy terminal part of SP. 13 Immunohistochemical Processing The immunohistochemical technique was similar to that previously employed. 2 All washes and antibody dilutions used 0.05 M phosphate buffered saline, ph 7.4. Final dilutions of all primary and secondary antibody solutions contained 0.3% Triton X-100. To stain for one antigen at a time, washed tissue sections were incubated overnight at room temperature either with rabbit anti-cgrp antiserum or with rat anti-sp antibody, each diluted 1:400. Tissue sections then were incubated for 30 min at room temperature with 1:300 dilution either of goat anti-rabbit IgG conjugated to fluorescein isothiocyanate (FITC) or of goat anti-rat IgG conjugated to FITC (Cappel Laboratories, Malvern, PA). The tissue sections were mounted in a tris-glycerin mixture. To demonstrate simultaneously CGRP-like and SP-like immunoreactivities, the tissue sections were incubated overnight at room temperature with a mixture of anti-cgrp serum and anti-sp monoclonal antibody, each diluted 1:400. They then were incubated for 30 min at room temperature with a mixture of goat anti-rabbit IgG conjugated to rhodamine isothiocyanate (RITC) (Cappel) diluted 1:400 and goat anti-rat IgG conjugated to FITC diluted 1:300. Mounted, the sections were examined with a Zeiss epi-illumination system that allowed for rapid alternation of filter combinations for optimum fluorescence visualization of FITC ( nm exciter filter; 510 nm dichromatic beam splitter; 520 nm barrierfilter)or RITC ( nm exciterfilter;580 nm dichromatic beam splitter; 590 nm barrier filter) fluorescence. Controls To assess immunohistochemical specificity, the diluted primary antisera were pre-incubated overnight at 4 C with 10" 6 M of synthetic SP and/or CGRP (Peninsula Laboratories, Inc., Belmont, CA); and the preabsorbed antisera were substituted for nonabsorbed antisera in the histochemical procedure. Additional controls, analogous to those previously described, 2 were undertaken to demonstrate the lack of inappropriate cross-reactivity between the primary and secondary antibodies. As previously discussed in relationship to ocular sensory innervation, 14 potential cross-reactivity among related antigens must be considered in applying immunohistochemical techniques. SP, a member of the tachykinin family of peptides, has been characterized biochemically in the rabbit uvea. 15 Two related tachykinins, neurokinin A and neurokinin B, also have been isolated biochemically from the rabbit sphincter muscle 16 ; these more recently discovered tachykinins have not been studied histochemically in the eye. For CGRP, two highly homologous peptides expressed by different genes have been identified by molecular biology techniques in the trigeminal ganglion; they are called a-cgrp and j8-cgrp. 17 Biochemical studies are needed to learn whether both or just one of these peptides occur in ocular nerves. Thus, while the present results satisfy currently accepted immunohistochemical criteria of specificity, the term "-like immunoreactive" (-LI) appropriately is applied to the findings described here. Results Co-Localization in CGRP and SP in the Trigeminal Ganglion As previously reported in guinea pig, 914 the number of CGRP-LI nerve cells was two to three times greater than that of SP-LI cells. When studied for the simultaneous demonstration of CGRP and SP, almost all the SP-LI nerve cells also were immunoreactive for CGRP (Fig. 1). Consistent with the disparity in total number, nerve cells immunoreactive only for CGRP were found commonly, while those immunoreactive only for SP were rare. Many individual nerve fibers within the ganglion also appeared to be immunoreactive to both CGRP and SP; other nerve fibers were immunoreactive only for CGRP; a few, only for SP. Co-Localization of SP and CGRP in the Eye In sections from eyes stained simultaneously for SP and CGRP, we observed fine varicose nerve fibers immunoreactive for both peptides. In cornea (Figs. 2A, B), the immunoreactive nervefiberswere present in the anterior two thirds of the cornea. Most showed co-localization of CGRP and SP, but nervefibersimmunoreactive only for CGRP were common in the limbal area. No corneal nervefiberswere observed to be immunoreactive only to SP. In the ciliary body (Figs. 2C, D), many nerve fibers were immunoreactive for both peptides, but some were immunoreactive only for one. Nerve fibers immunoreactive only

3 No. 12 SUBSTANCE P AND CGRP OCULAR NERVES / Kuwoyomo ond Srone Fig. 1. Simultaneous immunohistochemistry for SP (A, C) and CGRP (B,D) in the guinea pig tngeminal ganglion. A, C: the distribution of SP is demonstrated using FITC-conjugated antibody; B, D: the distribution of CGRP, using RITC-conjugated second antibody. A:B or CD are identical fields. A and B: Arrows indicate SP immunoreactive nerve cells that also are immunoreactive for CGRP. Note that more nerve cells are immunoreactive only for CGRP. C and D: SP and CGRP co-localize in this cell body and in the axon originating from it. Fluorescence micrographs; magnification bars, 50 p. for CGRP tended to be localized posteriorly; those immunoreactive only for SP were more prominent anteriorly near the iris root. In the ciliary processes (Figs. 2E, F) and choroid (Figs. 2G, H), almost all the immunoreactive nerve fibers showed co-localization of both peptides. The co-localization pattern in iris nerves was distinctive. In the anterior stroma (Figs. 3A, B), most of the immunoreactive nerve fibers related to blood vessels contained both CGRP and SP. However, the innervation along the anterior surface of dilator muscle (Figs. 3A, B) and within the sphincter muscle (Figs. 3C, D) contained many SP-LI nerve fibers; only rarely were any of these nerve fibers simultaneously immunoreactive to CGRP. Immunostaining of Other Cranial Ganglia In the superior cervical ganglion, a moderate number of SP-LI and CGRP-LI nerve fibers, mostly varicose but sometimes non-varicose in appearance, were present (Fig. 4A); very few SP-LI nerve cells (Fig. 4B) and no CGRP-LI nerve cells were visualized. In the ciliary ganglion, no nerve cells immunostained for either peptide, but nerve fibers immunoreactive for either SP (Fig. 4C) or CGRP were seen. In the pterygopalatine ganglion, a small number of SP-LI nerve cells were localized but with no apparent somatotopical organization (Fig. 4D); no CGRP-LI cells were found. Many fine varicose SP-LI and a moderate number of fine varicose CGRP-LI nerve fibers were seen; occasionally, immunoreactive nerve fibers surrounded unstained nerve cells. Sensory Denervation Eyes that had undergone intracranial ophthalmic and maxillary neurotomy were examined for CGRPand SP-LI nerve fibers by the double labeling immunofluorescence technique. Under these conditions, CGRP-LI nerve fibers were totally eliminated from the ipsilateral eye. In contrast, many SP-LI nerve

4 1950 INVESTIGATIVE OPHTHALMOLOGY G VISUAL SCIENCE / December 1987 Vol. 28 J w. Fig. 2. Simultaneous localization of SP-like (A, C, E, G) and CGRP-like (B, D, F, H) immunoreactivities in nervefibersof the guinea pig eye. A:B, C:D, E:F and G:H are identical fields. A and B: This field of cornea illustrates a nerve fiber just below the epithelium that is immunoreactive only for CGRP (arrowhead). A nerve trunk deeper in the stroma has too many nervefibersto resolve individually but shows immunoreactivity to both CGRP and SP (arrow). C and D: Within the ciliary body, many of SP-LI nervefiberssimilarly appear to be positive for CGRP. E and F: Nervefibers (arrows) running toward the ciliary process show immunoreactivity both for SP and CGRP. G and H: SP-LI nervefibersin the choroid also are immunoreactive for CGRP (arrow). Fluorescence micrographs; magnification bars, 50 fi.

5 No. 12 5UDSTANCE P AND CGRP OCULAR NERVES / Kuwoyomo ond Srone 1951 Fig. 3. Simultaneous immunohistochemistry for SP (A, C) and CGRP (B, D) in the iris of guinea pig. A:B or C:D are identicalfields.a and B: Many SP-LI nervefibersin the iris stroma are also immunoreactive for CGRP, but nervefibersalong the anterior surface of dilator muscle (arrowhead) show only SP-like immunoreactivity. C and D: The iris sphincter muscle contains numerous SP-LI nervefibers,but comparatively few of these show immunoreactivity to CGRP. Fluorescence micrographs; magnification bars, 50 fi. fibers persisted in the iris, especially along the anterior surface of the dilator and within the sphincter. A few SP-LI nerve fibers also remained in the anterior ciliary body. No SP-LI nerve fibers were seen elsewhere in the eye. In the contralateral eyes, there were no differences in the number or in the distribution of CGRP-LI and SP-LI nerve fibers compared to the normal. Combination of Sensory and Sympathetic Denervation Combined sensory and sympathetic denervation produced the same result as sensory denervation alone (Fig. 5). No CGRP-LI nerve fibers were visualized in any intraocular tissues of these denervated eyes. However, SP-LI nerve fibers persisted in the iris and anterior ciliary body of the ipsilateral eye. All other SP-LI nerve fibers were eliminated from these eyes. The contralateral eyes showed no differences in immunoreactive nerve fibers compared to normal eyes. Discussion In parallel with previous findings, 914 more of CGRP-LI neurons occur in the guinea pig trigeminal ganglion than SP-LI neurons. Almost all SP-LI trigeminal neurons also are immunoreactive to CGRP; but consistent with their greater number, many CGRP-LI nerve cells are not immunoreactive to SP. We also confirm co-localization of SP- and CGRPlike immunoreactivities in many peripheral nerve fibers of the eye. 3-8 Like the trigeminal ganglion, most nervefibersobserved here are immunoreactive either to both peptides or just to CGRP. For the iris, however, wefinda notable discrepancy in the pattern of peptide co-localization. Only a minority of iris nerves appear immunoreactive to both CGRP and SP; and contrary to findings in the trigeminal ganglion, almost all of the many SP-LI nerve fibers in the sphincter and on the anterior surface of the dilator fail to react for CGRP. Alternative explanations for this discrepancy in iris co-localization include: some trigeminal neurons may express both

6 Fig. 4. SP-like immunorcactivity in cranial ganglia of guinea pig. (A) Superior cervical ganglion. A moderate number of both varicose and non-varicose SP-LI nerve fibers are illustrated. (B) Superior cervical ganglion. A single SP-LI nerve cell (arrow) is shown in this section. (C) Ciliary ganglion. SP-LI nerve fibers but no immunoreactive cells are visualized. (D) Pterygopalatine ganglion. SP-LI nerve cells (arrows) and fibers are present. Fluorescence micrographs; magnification bars, 50 fi. Fig. 5. The ipsilateral iris of the guinea pig 14 days after combined sensory and sympathetic denervation, simultaneously immunostained for SP (A, C) and CGRP (B, D). A: Many SP-LI nerve fibers persist, particularly along the anterior surface of the dilator muscle. B: CGRP-LI nerve fibers are completely depleted in the same field as Fig. 5A. C: Many residual SP-LI nerve fibers also are visualized in the iris sphincter muscle. D: In the same field as Fig. 5C, CGRP immunoreactivity is totally eliminated. Fluorescence micrographs; magnification bars, 50 fi.

7 No. 12 SUBSTANCE P AND CGRP OCULAR NERVES / Kuwayama and Srone 1953 peptides in their soma but selectively transport only SP peripherally; the concentration of CGRP in iris SP-LI nerve fibers is below the sensitivity of the immunohistochemical technique; and some iris SP-LI nerve fibers derive from an origin different from iris CGRP-LI nerve fibers. After intracranial neurotomy of the ophthalmic and maxillary nerves, the residual SP-LIfibersalways and exclusively are located in the same areas of the eye that contain SP positive but CGRP negative nerve fibers in the double immunolabeling studies on normal eyes. Because a large number of CGRP-LI neurons project to the eye, because the CGRP-LI trigeminal neurons projecting to the eye lack somatotopical organization within the trigeminal ganglion 14 and because sensory denervation eliminates all CGRP-LI nervefibersfrom the eye, we conclude that the sensory denervations in these experiments interrupted the direct pathway from the trigeminal ganglion to the eye. Taken together, these observations indicate the existence of an origin outside the trigeminal ganglion for the SP-LI positive but CGRP negative nerve fibers in the guinea pig iris. This conclusion contradicts previous work suggesting a sensory source for all ocular SP-LI nerves. Prior immunohistochemical studies have reported that virtually all SP-LI axons in the iris disappear following damage to the trigeminal system of rat, 1819 rabbit 20 and guinea pig. 21 Radioimmunoassay assay data have indicated a 71% and 88% loss of SP from the rabbit iris-ciliary body 22 ' 23 and a 42% loss from the guinea pig iris 21 following sensory denervation. These radioimmunoassay data have been interpreted to support a sensory origin for ocular SP-LI nerves, but they also are consistent with a nonsensory origin for a proportion of SP-LI nerves. No ready explanation reconciles the differences between reports, but species variations or differences in technique may contribute. The ganglion of origin for the SP positive but CGRP negative iris nerves in guinea pig is not known. Among the three autonomic ganglia serving the guinea pig eye, SP-LI nerve cells are present in the superior cervical ganglion and in the pterygopalatine ganglion but not in the ciliary ganglion. Because the SP-LI iris nerves persist after combined sensory and sympathetic denervation, the superior cervical ganglion is not their source. Suggesting a possible pterygopalatine origin, radioimmunoassay levels of SP in the iris sphincter region fall 70% and those in the iris dilator-ciliary body region fall 71% after pterygopalatine ganglion damage in rabbits. 24 Intraorbital accessory ganglia or a novel sensory supply to the eye are other potential sources for intraocular SP-LI nerve fibers, but their true origin remains speculative. Despite the rich intraocular distribution of SP-LI nerve fibers' the only generally accepted functional effect of SP in the eye is miosis. 25 This effect is consistent with the occurrence of SP-LI nerves in the sphincter muscle CGRP, a potent vasodilator, has no pupillary effect. 26 Based on physiological responses, it has been proposed that these two neuropeptides participate jointly in the neurogenic ocular injury response, SP inducing miosis and CGRP mediating disruption of the blood-ocular barrier and elevation of intraocular pressure. 26 The miotic response to SP, however, shows marked species variation; SP is a potent miotic in rabbit, pig and cow but is inactive in man, baboon, cat and dog. 27 The reactivity of the guinea pig sphincter muscle to SP has not been tested to our knowledge, and receptor studies on the sphincter muscle have been reported only in rabbit. 28 In rhesus monkey, cat and pig, we find that the SP-LI innervation to the iris muscles is much less than in guinea pig and that essentially all SP-LI nerve fibers in the rhesus monkey, cat and pig iris are simultaneously immunoreactive for CGRP (Kuwayama and Stone, unpublished); these obseivations preclude clear relationships among the SP-LI innervation density, CGRP co-localization and sphincter muscle contractility to SP. The iris physiology studies assume a sensory origin for its SP-LI innervation. The occurrence of SP-LI nervefibersof non-trigeminal origin in the guinea pig indicates that the nature of the SP-LI ocular innervation is more complex than previously believed. It is important to identify the origin and species differences of these presumed non-sensory SP-LI ocular nerves to elucidate further the role of this peptide in the eye and to clarify its interactions with CGRP. Key words: substance P, calcitonin gene-related peptide, eye, trigeminal ganglion, innervation, immunohistochemistry, guinea pig Acknowledgments The authors thank Drs. Julia M. Polak, Giorgio Terenghi, and Wylie W. Vale for providing antisera to CGRP. Ms. Alice McGlinn provided excellent technical assistance. References 1. Stone RA, Kuwayama Y, and Laties AM: Regulatory peptides in the eye. Experientia 43:791, Kuwayama Y and Stone R: Cholecystokinin-like immunoreactivity occurs in ocular sensory neurons and partially co-localizes with substance P. Brain Res 381:266, Gibbins IL, Furness JB, Costa M, Maclntyre I, Hillyard CJ, and Girgis S: Co-localization of calcitonin gene-related peptide-like immunoreactivity with substance P in cutaneous, vascular and visceral sensory neurons of guinea pigs. Neurosci Lett 57:125, Terenghi G, Polak JM, Ghatei MA, Mulderry PK, Butler JM,

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