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The distribution and origin of substance P immunoreactive nerve fibres in the rat conjunctiva
Exp. Eye Res. (1991) 53, 641-646
The
Distribution
and Origin of Substance P lmmunoreactive Nerve Fibres in the Rat Conjunctiva J. LUHTALA”
Eye Research
Laboratory,
(Received
Department
25 October
AND
H. UUSITALO
of Anatomy,
1990 and accepted
University in revised
of Helsinki,
Helsinki,
form 30 January
Finland
1997)
Indirect immunohistochemistry was used to examine the presence and origin of substance P immunoreactive nerve fibres in the rat conjunctiva. Fluorescent substance P immunoreactive nerve fibres were visualized both in the epithelium and stroma, their number being higher in the stroma where they were associated with blood vessels, the smooth muscle of Miiller and the Meibomian glands. Ten to twenty percent of the ganglion cells in the trigeminal ganglion were immunoreactive to substance P. most of them being small in size. Sensory denervation by electrocoagulating the ophthalmic and maxillary branches of the trigeminal nerve caused a complete disappearance of the epithelial fibres and greatly reduced the number of the stromal fibres. These results indicate that the majority of the demonstrated fibres are sensory nerves originating from the trigeminal ganglion. Since sympathectomy had no detectable effect on the number or distribution of substance P immunoreactive nerve fibres, and since some of the fibres remained after sensory denervation it is suggested that at least some of the remaining fibres could be of parasympathetic origin. Key words: substance P ; immunohistochemistry ; conjunctiva ; trigeminal ganglion : superior cervical ganglion.
1. Introduction
2. Material
Substance P (SP) is an 11 amino acid peptide located in some central and peripheral neurones (Hokfelt
Male and female Wistar Hannover albino rats weighing 180-300 g were kept in a constant dark/ light cycle and allowed water and food pellets ad libitum.
et al., 1975). To date, convincing evidence has been gathered to indicate that it functions within the mammalian nervous system as a neurotransmitter or modulator (Otsuka and Konishi, 1983; Salt and Hill, 1983; Sweeney and Sawynok, 1986). In earlier studies, SP has been demonstrated in sensory ganglions as well as in primary sensory nerves in various tissues, including the eye (Cuello, Del Fiaggo and Paxinos, 1978; Miller et al., 1981). Conjunctiva is known to receive sensory, sympathetic and parasympathetic innervation from the trigeminal, superior cervical and pterygopalatine ganglia, respectively (Macintosh, 1974). Neurotransmitters-not to mention their physiological role-in the conjunctival nerves are largely undefined due to the very limited number of studies in this tissue. Previous investigations have revealed catecholaminecontaining, acetylcholinesterase-positive and calcitonin gene-related peptide (CGRP) immunoreactive nerve fibres in the conjunctiva (Karjalainen, Tervo and Palkama, 1978; Luhtala, Palkama and Uusitalo, 1991). In this paper conjunctival SP-immunoreactive nerve fibres were examined by indirect immunohistochemistry and their origin was evaluated by performing sensory and sympathetic denervations.
and Methods
Denervation Experiments
Altogether 20 rats were used in this study. Nine animals underwent unilateral sensory denervation, seven uni- and two bilateral sympathectomy ; two rats were kept intact as controls. The animals were anaesthetized with intraperitoneal sodium pentobarbital (60 mg kg-’ body weight: Mebunat, Orion, Finland). Sensory denervations were performed by electrocoagulating the ophthalmic and maxillary branches of the trigeminal nerve through craniotomy 6-12 days before further processing. The site of the coagulation was checked at autopsy and additionally the loss of the cornea1 blink reflex in the denervated eye was used as verification of the success of the operation. Sympathectomies were done by surgically extirpating the right (and in the bilaterally sympathectomized animals also the left) superior cervical ganglion 12-2 5 days prior to fixation. Sympathectomies were confirmed by checking the disappearance of tyrosine hydroxylase and dopamine ,+hydroxylase immunoreactivities in the anterior segment of the eye. Immunohistochemistry
* For correspondence at: Department of Helsinki,
Siltavuorenpenger
0014-4835/91/110641+06
20A.
00170
Anatomy, University Helsinki, Finland.
$03.00
of
Anaesthetized rats were perfusion-fixed through the left heart ventricle in sodium pentobarbital anaes0 199 1 Academic Press Limited
642
J. LUHTALA
AND
H. UUSITALO
FIG. 1. SP-immunoreactive nerve fibres (arrows) in palpebral epithelium (EP). MG, Meibomian gland: STR, stroma. x 375. FIG. 2. Immunofluorescent nerve flbre (arrow) right beneath the epithelium (EP) in palpebral conjunctiva. MG, Meibomian gland. x 375. FIG. 3. Nerve fibres (arrows) running between Meibomian glands (MG). x 375. FIG. 4. A single free nerve ending (arrow) in fornical epithelium. GC. Goblet cell: STR. stroma. x 450. FIG. 5. Fornical stroma displaying a branching SP-immunoreactive nerve bundle (arrow). x 300. FIG. 6. The smooth muscle of Miiller (MM) surrounded by immunofluorescent libres. El?, Epithelium. x 250.
thesia. After the blood was washed out with saline the animals were perfused with 4 y0 formaldehyde in 0.1 M phosphate buffer, pH 7.4. The eyes were dissected out together with both lids and immersion fixed in the same fixative for 2 hr at + 4°C. In order to prevent the formation of ice-crystals, the fixated tissues were
soaked for at least 24 hr at +4”C in 0.1 M phosphate buffer containing 30 % sucrose. Cryostat sections (S-20 /tm) were mounted on chrome-alum-gelatin-coated glass slides. air-dried for 60 min at + 20°C and incubated with O.l(%, Triton-X saline, pH 7.2 (Tritonin 0.05 M phosphate-buffered
SP IN THE
RAT
CONJUNCTIVA
643
FIG. 7. Three stromal blood vessels surrounded by SP-immunoreactive fibres (arrows) in fornix. x 250. FK 8. Nerve fibres (arrows) immunoreactive to SP in bulbar epithelium (EP). SCL, Sclera. x 375. FIG. 9. Immunofluorescent nerve fibres around episcleral veins (EV). SCL. Sclera: BEP, Bulbar epithelium; PEP, Palpebral epithelium. x 300. FIG. 10. No immunofluorescent fibres detectable in the palpebral conjunctiva of a sensorily denervated rat. EP. Epithelium; MG. Meibomian gland. x 3 75. FIG. 1 1. Two SP-immunoreactive nerve fibres (arrows) in palpebral stroma after sensory denervation. MM. Mtiller’s muscle. x 450. FIG. 12. SP-immunoreactive nerve fibres (arrows) following a stromal blood vessel (v) in an ipsilaterally sympathectomized specimen. x 375. 5% normal swine serum for 2 hr at + 4°C. This and all other incubations were performed in humid chambers. Primary polyclonal rabbit antisera to SP (RPN 1842 Amersham, U.K.) diluted
PBS) containing
1: 10004000 in Triton-PBS was incubated at +4”C for 2448 hr. The sections were thoroughly rinsed for 60 min with Triton-PBS and incubated at + 20°C for 60 min with biotinylated antisera to rabbit IgG (RPN
J. LUtiTALA
AND
H. UUSITALO
13. Ganglion cell bodies and nerve fibres immunoreactive to SP in trigeminal ganglion. x 300. 14. An immunofluorescent fibre (arrow) visible between the ganglion cells in superior cervical ganglion while the ganglion cell bodies remain non-immunoreactive to SP. x 450. FIG. FIG.
1004 Amersham, U.K.) diluted 1: 100 in PBS. After a 60-min washing with PBS, 1 :lOO fluorescein-conjugated streptavidin (RPN 1232 Amersham) in PBS was incubated at +2O”C for 15 min. The sections were rinsed again with PBS, glycerin-PBS (3 : 1) mixture was applied on the glasses and they were then coated with cover slides. The specimens were viewed using a Leitz Orthoplan or Aristoplan fluorescence microscope equipped with an epi-illuminator and a specific filter block 12. Photographs were taken on Kodak T-max film with an automatic Leitz OrthoVariomat microscope camera.
followed the course of the smooth muscle of Miiller and occasional fibres were observed near or between the acini of the Meibomian glands (Fig. 3). Fornical Conjunctiva Epithelial fibres were rare in the fornices (Fig. 4). The SP-immunoreactive nerve fibres were more numerous in the stroma, where they were also seen as thicker bundles (Fig. 5). The MiilIer’s muscle received a moderate number of surrounding fibres (Fig. 6) and the stromal blood vessels were often associated with immunofluorescent fibres (Fig. 7).
Controls Omission of the primary antiserum, replacement of the primary antiserum with normal rabbit serum and absorption of the primary antiserum with 0.1-1.0 ,uM SP (Peninsula, U.S.A.) were utilized as control experiments. All these controls were negative, thus confirming the results to be immunohistochemically specific.
Bulbar Conjunctiva The bulbar epithelium showed few free nerve endings (Fig. 8) and the number of stromal fibres was smaller than elsewhere in the conjunctiva. Nevertheless, fibres in association with blood vessels were discovered and, in particular. the episcleral veins were variably surrounded by SP-immunoreactive fibres (Fig. 9).
3. Results SP-immunoreactive nerves were found as thin varicose fibres throughout the conjunctiva being most numerous in the substantia propria. No clear difference could be observed in the number nor localization of fibres between upper and lower lids. Pulpehul
Conjunctiva
Immunofluorescent free nerve endings and fibres were irregularly observed in the palpebral epithelium (Fig. 1). Despite the relatively small number of SPimmunoreactive fibres in the epithelium itself, many fibres were seen just beneath the epithelium (Fig. 2). A modest amount of SP-immunoreactive nerve fibres
Denervafion Experiments After the unilateral coagulation of the ophthalmic and maxillary nerves all the epithelial and most of the stromal SP-immunoreactive nerve tibres disappeared in the ipsilateral conjunctiva (Fig. 10). A few libres. however, were detectable in fornical and bulbar stroma (Fig. 11). Sympathetic denervations on their part had no apparent effect on the SP-immunoreactive fibres (Fig. 12). Ganglions By visual examination about IO-LO% of the ganglion cells in the trigeminal ganglion were im-
SP IN THE
RAT
645
CONJUNCTIVA
munoreactive to SP this immunoreactivity being primarily localized in the small-size ganglion cells, and furthermore a substantial amount of immunofluorescent fibres were detected (Fig. 13). There were no SP-immunoreactive ganglion cells in the superior cervical ganglion but some SP-immunoreactive fibres were observed between the cells (Fig. 14). 4. Discussion
The conjunctival epithelium showed a modest number of immunofluorescent fibres through its entire length. SP-immunoreactive nerve fibres were observed in greatest amounts in the substantia propria, especially in the fornices. While most of the detected fibres seemed to have no specific destination, the stromal blood vessels were often, and the Miiller’s muscle regularly though to a lesser extent, associated with SP-immunoreactive fibres. Infrequent fibres were seen near or within the Meibomian glands. Sensory denervation resulted in a major reduction in the number of the SP-immunoreactive nerve fibres. As in earlier studies, the trigeminal ganglion was found to contain nerve cells immunoreactive to SP (Hokfelt et al., 1975; Cue110 et al.. 1978) and it is conceivable that a majority of the demonstrated fibres originate from the trigeminal ganglion. These nerves are likely to be sensory due to the general sensory nature of the trigeminal neurons, the previous evidence of sensory nerves in the conjunctiva (Macintosh, 1’374) and the current description of SP as a sensory neurotransmitter (Hiikfelt et al., 1975 : Takahashi and Otsuka, 1975; Olgarth et al., 1977). A minor portion of the SP-immunoreactive fibres remained after sensory denervation in the bulbar and palpebral stroma. One explanation of this phenomenon may be that the electrocoagulation of the sensory nerve was not complete. Although no definite conclusion of their origin can be made based on our results, it is possible that these fibres could be parasympathetic nerves from the pterygopalatine ganglion since the pterygopalatine ganglion contains SP-immunoreactive ganglion cells (Kuwayama and Stone. 198 7) and the conjunctiva is known to receive its parasympathetic innervation from the pterygopalatine ganglion (Macintosh, 1974). When compared to the number and distribution of CGKP-immunoreactive fibres in the conjunctiva (Luhtala et al.. 1991). the most prominent difference is that the overall number of the SP-immunoreactive fibres was clearly smaller, particularly in the epithelium, which is consistent with the observation that the trigeminal ganglion contains about twice as many CGRP- as SP-immunoreactive neurons (Lee et al., 198 5). Nonetheless, the stromal blood vesselsseemed
to be at least as frequently innervated by nerve fibres immunoreactive to SP. In the present study SP-immunoreactive nerve fibres were observed in association with the episcleral veins
where CGRP immunoreactivity has been previously demonstrated (Luhtala et al., 1991). Considering the vasomotor properties of these peptides (Lembeck and Holzer, 1979 ; Brain et al., 198 5), it may be suggested that SP and CGRP might regulate the venous flow in the episcleral veins and thus have a part in the regulation of the aqueous outflow. Physiological data indicate that SP increases vascular permeability and that this is based on a release of histamine and other substances from mast cells which induce an inflammatory reaction (Johnson and Erdos, 1973 : Lembeck and Holzer, 1979 ; Fewtrell et al.. 1982). SP can also exert its effect directly via specific vascular receptors (Foreman et al., 1983). In the rat and human skin, SP has been shown to increase the permeability of the vascular bed, causing short-lasting wheal and flare (Brain and Williams, 1985; Wallengren and Hakanson, 1987). Furthermore, SP and CGRP, which are often co-localized in the same primary afferent fibres (Wiesenfeld-Hallin et al., 1984: Lee et al., 1985), seem to potentiate and modify the effects of each other as do also histamine, serotonin and other inflammatory mediators (Gamse and Saria, 1985: Brain and Williams, 1985, 1988, 1989). Although there is no experimental data on the physiological effects of SP and CGRP in the conjunctiva, these neuropeptides could be of importance, e.g. in inflammation of the conjunctiva. Acknowledgements Financial support was received and A. Gyllenberg Foundations, all authors are grateful to Mrs Paula technical assistance and to Mr Reijo the photographs.
from TEKES. S. Juselius of Helsinki, Finland. The Hasenson for her skillful Karppinen for producing
References Brain, S. D. and Williams, T. J. (1985). Inflammatory oedema induced by synergism between calcitonin generelated peptide (CGRP) and mediators of increased vascular permeability. Br. J. Pharmacol. 86, 855-60. Brain, S. D. and Williams, T. J. (1988). Substance P regulates the vasodilator activity of calcitonin gene-related peptide. Nature 335, 73-5.
Brain. S. D. and Williams, T. J. (1989). Interactions between the tachykinins and calcitonin gene-related peptide lead to the modulation of oedema formation and blood flow in rat skin. Br. I, Pharnracol. 97. 77.-82. Brain, S. D.. Williams, T. J.. Tippins, J. R., Morris, H. R. and Maclntyre. I. (1985). Calcitonin gene-related peptide is a potent vasodilator. Nature 313. 54-6. Cuello, A. C., Del Fiacco, M. and Paxinos. G. ( 1978). The central and peripheral ends of substance P-containing sensory neurones in the rat trigeminal system. Brain Res. 152. 499-509. Fewtrell, C. M. S., Foreman, J. C., Jordan, C. C.. Oehme, P.. Renner, H. and Stewart, H. M. (1982). The effects of substance P on histamine and 5-hydroxytryptamine release in rat. 1. PhysioI. 330. 393-411. Foreman. J. C.. Jordan, C. C., Oehme, P. and Renner. H. ( 1983). Structure-activity relationships for some substance P-related peptides that cause wheal and flare reactions in human skin. 1. Physiol. 335, 449-65.
646 Gamse. R. and Saria, A. (198 5). Potentiation of tachykinin induced plasma protein extravasation by calcitonin gene-related peptide. Eur. J. Pharmarol. 114. 61-6. Hakfelt. T., Kellerth, J.-O., Nilsson. G. and Pernow. B. (19 75). Experimental immunohistochemical studies on the localization and distribution of substance P in cat primary sensory neurons. Brain Res. 100, 235-52. Johnson. A. R. and Erdb;s, E. G. (1973). Release of histamine from mast cells by vasoactive peptides. Proc. Sot. Exp. Biol. Med. 142. 1253-6. Karjalainen K., Tervo. T. and Palkama, A. (1978). Catecholamine-containing and acetylcholinesterase-positive nerve fibres in the rabbit conjunctiva. Actn Ophthdmol. 56, 91 l-20. Kuwayama. Y. and Stone, R. A. (1987). Distinct substance P and calcitonin gene-related peptide immunoreactive nerves in the guinea pig eye. Invest. Ophthalmol. Vis. Sci. 28, 1947-54. Lee, Y., Kawai. Y.. Shiosaka, S.. Takami. K.. Kiyama. K., Hillyard. C. J., Girgis, S., MacIntyre, I., Emson. P. C. and Tohyama, M. (1985). Coexistence of calcitonin generelated peptide and substance P-like peptide in single cells in the trigeminal ganglion of the rat: immunochemical analysis. Bruin Res. 330, 194-6. Lembeck, F. and Holzer, P. (1979). Substance P as neurogenic mediator of antidromic vasodilatation and neurogenic plasma extravasation. Arch. Pharmacol. 310, 175-83. Luhtala, J.. Palkama. A. and IJusitalo, H. (I 991). Calcitonin gene-related peptide immunoreactive nerve fibres in the rate conjunctiva. invest. Ophthalmol. Vis. Sci. 32, 640-5. Macintosh, S. R. (1974). The innervation of the conjunctiva
J. LUHTALA
AND
H. UUSITALO
in monkeys: an electron microscopic and nerve degeneration study. Albrecht v. Graefes Arch. Klin. Erp. Ophthalmol. 192, 105-16. Miller, A., Costa, M.. Furness, J. B. and Chubb. I. W. ( 198 1). Substance P immunoreactive sensory nerves supply the rat iris and cornea. Neurosci. Lett. 23. 24 3-9. Olgarth. I,.. Gazelius, B.. Brodin. E. and Nilsson. C. I 19771. Release of substance P-like immunoreactivity from the cat dental pulp. Arttr Physiol. Scud. 101. 510-L. Otsuka. M. and Konishi, S. (I 983). Substance P-the first peptide neurotransmitter ? Trends .!kwosci. 6. 3 17-20. Salt. T. E. and Hill. R. G. (1983). Neurotransmitter candidates of somatosensory primary afferent fibres. Neuroscience 10. 1083-103. Sweeney. M. I. and Sawynok, J. (1986). Evidence that substance P may be a modulator rather than a transmitter of noxious mechanical stimulation. Corn. I. Physiol. 64. 1 324-7. Takahashi, T. and Otsuka. M. ( 1975). Regional distribution of substance P in the spinal cord and nerve roots of the cat and the effect of dorsal root section. Brrrin Res. 87. l-l 1. Wallengren. J. and HBkanson, R. ( 1987). Effects of substance P, neurokinin A and calcitonin gene-related peptide in human skin and their involvement in sensory nerve mediated responses. Eur. 1. Pharmnrol. 143. 267-Y 3. Wiesenfeld-Hallin, Z., HGkfelt, T.. Lundberg, J. .%I.. Forssmann. W. G.. Reinecke. M.. Tschopp, F. A. and Fischer. J. A. (1984). Immunoreactive calcitonin gene-related peptide and substance P coexists in sensory neurons to the spinal cord and interact in spinal behavioral responses of the rat. i\jeurosci. Lett, 52. 199-20~.
The
Distribution
and Origin of Substance P lmmunoreactive Nerve Fibres in the Rat Conjunctiva J. LUHTALA”
Eye Research
Laboratory,
(Received
Department
25 October
AND
H. UUSITALO
of Anatomy,
1990 and accepted
University in revised
of Helsinki,
Helsinki,
form 30 January
Finland
1997)
Indirect immunohistochemistry was used to examine the presence and origin of substance P immunoreactive nerve fibres in the rat conjunctiva. Fluorescent substance P immunoreactive nerve fibres were visualized both in the epithelium and stroma, their number being higher in the stroma where they were associated with blood vessels, the smooth muscle of Miiller and the Meibomian glands. Ten to twenty percent of the ganglion cells in the trigeminal ganglion were immunoreactive to substance P. most of them being small in size. Sensory denervation by electrocoagulating the ophthalmic and maxillary branches of the trigeminal nerve caused a complete disappearance of the epithelial fibres and greatly reduced the number of the stromal fibres. These results indicate that the majority of the demonstrated fibres are sensory nerves originating from the trigeminal ganglion. Since sympathectomy had no detectable effect on the number or distribution of substance P immunoreactive nerve fibres, and since some of the fibres remained after sensory denervation it is suggested that at least some of the remaining fibres could be of parasympathetic origin. Key words: substance P ; immunohistochemistry ; conjunctiva ; trigeminal ganglion : superior cervical ganglion.
1. Introduction
2. Material
Substance P (SP) is an 11 amino acid peptide located in some central and peripheral neurones (Hokfelt
Male and female Wistar Hannover albino rats weighing 180-300 g were kept in a constant dark/ light cycle and allowed water and food pellets ad libitum.
et al., 1975). To date, convincing evidence has been gathered to indicate that it functions within the mammalian nervous system as a neurotransmitter or modulator (Otsuka and Konishi, 1983; Salt and Hill, 1983; Sweeney and Sawynok, 1986). In earlier studies, SP has been demonstrated in sensory ganglions as well as in primary sensory nerves in various tissues, including the eye (Cuello, Del Fiaggo and Paxinos, 1978; Miller et al., 1981). Conjunctiva is known to receive sensory, sympathetic and parasympathetic innervation from the trigeminal, superior cervical and pterygopalatine ganglia, respectively (Macintosh, 1974). Neurotransmitters-not to mention their physiological role-in the conjunctival nerves are largely undefined due to the very limited number of studies in this tissue. Previous investigations have revealed catecholaminecontaining, acetylcholinesterase-positive and calcitonin gene-related peptide (CGRP) immunoreactive nerve fibres in the conjunctiva (Karjalainen, Tervo and Palkama, 1978; Luhtala, Palkama and Uusitalo, 1991). In this paper conjunctival SP-immunoreactive nerve fibres were examined by indirect immunohistochemistry and their origin was evaluated by performing sensory and sympathetic denervations.
and Methods
Denervation Experiments
Altogether 20 rats were used in this study. Nine animals underwent unilateral sensory denervation, seven uni- and two bilateral sympathectomy ; two rats were kept intact as controls. The animals were anaesthetized with intraperitoneal sodium pentobarbital (60 mg kg-’ body weight: Mebunat, Orion, Finland). Sensory denervations were performed by electrocoagulating the ophthalmic and maxillary branches of the trigeminal nerve through craniotomy 6-12 days before further processing. The site of the coagulation was checked at autopsy and additionally the loss of the cornea1 blink reflex in the denervated eye was used as verification of the success of the operation. Sympathectomies were done by surgically extirpating the right (and in the bilaterally sympathectomized animals also the left) superior cervical ganglion 12-2 5 days prior to fixation. Sympathectomies were confirmed by checking the disappearance of tyrosine hydroxylase and dopamine ,+hydroxylase immunoreactivities in the anterior segment of the eye. Immunohistochemistry
* For correspondence at: Department of Helsinki,
Siltavuorenpenger
0014-4835/91/110641+06
20A.
00170
Anatomy, University Helsinki, Finland.
$03.00
of
Anaesthetized rats were perfusion-fixed through the left heart ventricle in sodium pentobarbital anaes0 199 1 Academic Press Limited
642
J. LUHTALA
AND
H. UUSITALO
FIG. 1. SP-immunoreactive nerve fibres (arrows) in palpebral epithelium (EP). MG, Meibomian gland: STR, stroma. x 375. FIG. 2. Immunofluorescent nerve flbre (arrow) right beneath the epithelium (EP) in palpebral conjunctiva. MG, Meibomian gland. x 375. FIG. 3. Nerve fibres (arrows) running between Meibomian glands (MG). x 375. FIG. 4. A single free nerve ending (arrow) in fornical epithelium. GC. Goblet cell: STR. stroma. x 450. FIG. 5. Fornical stroma displaying a branching SP-immunoreactive nerve bundle (arrow). x 300. FIG. 6. The smooth muscle of Miiller (MM) surrounded by immunofluorescent libres. El?, Epithelium. x 250.
thesia. After the blood was washed out with saline the animals were perfused with 4 y0 formaldehyde in 0.1 M phosphate buffer, pH 7.4. The eyes were dissected out together with both lids and immersion fixed in the same fixative for 2 hr at + 4°C. In order to prevent the formation of ice-crystals, the fixated tissues were
soaked for at least 24 hr at +4”C in 0.1 M phosphate buffer containing 30 % sucrose. Cryostat sections (S-20 /tm) were mounted on chrome-alum-gelatin-coated glass slides. air-dried for 60 min at + 20°C and incubated with O.l(%, Triton-X saline, pH 7.2 (Tritonin 0.05 M phosphate-buffered
SP IN THE
RAT
CONJUNCTIVA
643
FIG. 7. Three stromal blood vessels surrounded by SP-immunoreactive fibres (arrows) in fornix. x 250. FK 8. Nerve fibres (arrows) immunoreactive to SP in bulbar epithelium (EP). SCL, Sclera. x 375. FIG. 9. Immunofluorescent nerve fibres around episcleral veins (EV). SCL. Sclera: BEP, Bulbar epithelium; PEP, Palpebral epithelium. x 300. FIG. 10. No immunofluorescent fibres detectable in the palpebral conjunctiva of a sensorily denervated rat. EP. Epithelium; MG. Meibomian gland. x 3 75. FIG. 1 1. Two SP-immunoreactive nerve fibres (arrows) in palpebral stroma after sensory denervation. MM. Mtiller’s muscle. x 450. FIG. 12. SP-immunoreactive nerve fibres (arrows) following a stromal blood vessel (v) in an ipsilaterally sympathectomized specimen. x 375. 5% normal swine serum for 2 hr at + 4°C. This and all other incubations were performed in humid chambers. Primary polyclonal rabbit antisera to SP (RPN 1842 Amersham, U.K.) diluted
PBS) containing
1: 10004000 in Triton-PBS was incubated at +4”C for 2448 hr. The sections were thoroughly rinsed for 60 min with Triton-PBS and incubated at + 20°C for 60 min with biotinylated antisera to rabbit IgG (RPN
J. LUtiTALA
AND
H. UUSITALO
13. Ganglion cell bodies and nerve fibres immunoreactive to SP in trigeminal ganglion. x 300. 14. An immunofluorescent fibre (arrow) visible between the ganglion cells in superior cervical ganglion while the ganglion cell bodies remain non-immunoreactive to SP. x 450. FIG. FIG.
1004 Amersham, U.K.) diluted 1: 100 in PBS. After a 60-min washing with PBS, 1 :lOO fluorescein-conjugated streptavidin (RPN 1232 Amersham) in PBS was incubated at +2O”C for 15 min. The sections were rinsed again with PBS, glycerin-PBS (3 : 1) mixture was applied on the glasses and they were then coated with cover slides. The specimens were viewed using a Leitz Orthoplan or Aristoplan fluorescence microscope equipped with an epi-illuminator and a specific filter block 12. Photographs were taken on Kodak T-max film with an automatic Leitz OrthoVariomat microscope camera.
followed the course of the smooth muscle of Miiller and occasional fibres were observed near or between the acini of the Meibomian glands (Fig. 3). Fornical Conjunctiva Epithelial fibres were rare in the fornices (Fig. 4). The SP-immunoreactive nerve fibres were more numerous in the stroma, where they were also seen as thicker bundles (Fig. 5). The MiilIer’s muscle received a moderate number of surrounding fibres (Fig. 6) and the stromal blood vessels were often associated with immunofluorescent fibres (Fig. 7).
Controls Omission of the primary antiserum, replacement of the primary antiserum with normal rabbit serum and absorption of the primary antiserum with 0.1-1.0 ,uM SP (Peninsula, U.S.A.) were utilized as control experiments. All these controls were negative, thus confirming the results to be immunohistochemically specific.
Bulbar Conjunctiva The bulbar epithelium showed few free nerve endings (Fig. 8) and the number of stromal fibres was smaller than elsewhere in the conjunctiva. Nevertheless, fibres in association with blood vessels were discovered and, in particular. the episcleral veins were variably surrounded by SP-immunoreactive fibres (Fig. 9).
3. Results SP-immunoreactive nerves were found as thin varicose fibres throughout the conjunctiva being most numerous in the substantia propria. No clear difference could be observed in the number nor localization of fibres between upper and lower lids. Pulpehul
Conjunctiva
Immunofluorescent free nerve endings and fibres were irregularly observed in the palpebral epithelium (Fig. 1). Despite the relatively small number of SPimmunoreactive fibres in the epithelium itself, many fibres were seen just beneath the epithelium (Fig. 2). A modest amount of SP-immunoreactive nerve fibres
Denervafion Experiments After the unilateral coagulation of the ophthalmic and maxillary nerves all the epithelial and most of the stromal SP-immunoreactive nerve tibres disappeared in the ipsilateral conjunctiva (Fig. 10). A few libres. however, were detectable in fornical and bulbar stroma (Fig. 11). Sympathetic denervations on their part had no apparent effect on the SP-immunoreactive fibres (Fig. 12). Ganglions By visual examination about IO-LO% of the ganglion cells in the trigeminal ganglion were im-
SP IN THE
RAT
645
CONJUNCTIVA
munoreactive to SP this immunoreactivity being primarily localized in the small-size ganglion cells, and furthermore a substantial amount of immunofluorescent fibres were detected (Fig. 13). There were no SP-immunoreactive ganglion cells in the superior cervical ganglion but some SP-immunoreactive fibres were observed between the cells (Fig. 14). 4. Discussion
The conjunctival epithelium showed a modest number of immunofluorescent fibres through its entire length. SP-immunoreactive nerve fibres were observed in greatest amounts in the substantia propria, especially in the fornices. While most of the detected fibres seemed to have no specific destination, the stromal blood vessels were often, and the Miiller’s muscle regularly though to a lesser extent, associated with SP-immunoreactive fibres. Infrequent fibres were seen near or within the Meibomian glands. Sensory denervation resulted in a major reduction in the number of the SP-immunoreactive nerve fibres. As in earlier studies, the trigeminal ganglion was found to contain nerve cells immunoreactive to SP (Hokfelt et al., 1975; Cue110 et al.. 1978) and it is conceivable that a majority of the demonstrated fibres originate from the trigeminal ganglion. These nerves are likely to be sensory due to the general sensory nature of the trigeminal neurons, the previous evidence of sensory nerves in the conjunctiva (Macintosh, 1’374) and the current description of SP as a sensory neurotransmitter (Hiikfelt et al., 1975 : Takahashi and Otsuka, 1975; Olgarth et al., 1977). A minor portion of the SP-immunoreactive fibres remained after sensory denervation in the bulbar and palpebral stroma. One explanation of this phenomenon may be that the electrocoagulation of the sensory nerve was not complete. Although no definite conclusion of their origin can be made based on our results, it is possible that these fibres could be parasympathetic nerves from the pterygopalatine ganglion since the pterygopalatine ganglion contains SP-immunoreactive ganglion cells (Kuwayama and Stone. 198 7) and the conjunctiva is known to receive its parasympathetic innervation from the pterygopalatine ganglion (Macintosh, 1974). When compared to the number and distribution of CGKP-immunoreactive fibres in the conjunctiva (Luhtala et al.. 1991). the most prominent difference is that the overall number of the SP-immunoreactive fibres was clearly smaller, particularly in the epithelium, which is consistent with the observation that the trigeminal ganglion contains about twice as many CGRP- as SP-immunoreactive neurons (Lee et al., 198 5). Nonetheless, the stromal blood vesselsseemed
to be at least as frequently innervated by nerve fibres immunoreactive to SP. In the present study SP-immunoreactive nerve fibres were observed in association with the episcleral veins
where CGRP immunoreactivity has been previously demonstrated (Luhtala et al., 1991). Considering the vasomotor properties of these peptides (Lembeck and Holzer, 1979 ; Brain et al., 198 5), it may be suggested that SP and CGRP might regulate the venous flow in the episcleral veins and thus have a part in the regulation of the aqueous outflow. Physiological data indicate that SP increases vascular permeability and that this is based on a release of histamine and other substances from mast cells which induce an inflammatory reaction (Johnson and Erdos, 1973 : Lembeck and Holzer, 1979 ; Fewtrell et al.. 1982). SP can also exert its effect directly via specific vascular receptors (Foreman et al., 1983). In the rat and human skin, SP has been shown to increase the permeability of the vascular bed, causing short-lasting wheal and flare (Brain and Williams, 1985; Wallengren and Hakanson, 1987). Furthermore, SP and CGRP, which are often co-localized in the same primary afferent fibres (Wiesenfeld-Hallin et al., 1984: Lee et al., 1985), seem to potentiate and modify the effects of each other as do also histamine, serotonin and other inflammatory mediators (Gamse and Saria, 1985: Brain and Williams, 1985, 1988, 1989). Although there is no experimental data on the physiological effects of SP and CGRP in the conjunctiva, these neuropeptides could be of importance, e.g. in inflammation of the conjunctiva. Acknowledgements Financial support was received and A. Gyllenberg Foundations, all authors are grateful to Mrs Paula technical assistance and to Mr Reijo the photographs.
from TEKES. S. Juselius of Helsinki, Finland. The Hasenson for her skillful Karppinen for producing
References Brain, S. D. and Williams, T. J. (1985). Inflammatory oedema induced by synergism between calcitonin generelated peptide (CGRP) and mediators of increased vascular permeability. Br. J. Pharmacol. 86, 855-60. Brain, S. D. and Williams, T. J. (1988). Substance P regulates the vasodilator activity of calcitonin gene-related peptide. Nature 335, 73-5.
Brain. S. D. and Williams, T. J. (1989). Interactions between the tachykinins and calcitonin gene-related peptide lead to the modulation of oedema formation and blood flow in rat skin. Br. I, Pharnracol. 97. 77.-82. Brain, S. D.. Williams, T. J.. Tippins, J. R., Morris, H. R. and Maclntyre. I. (1985). Calcitonin gene-related peptide is a potent vasodilator. Nature 313. 54-6. Cuello, A. C., Del Fiacco, M. and Paxinos. G. ( 1978). The central and peripheral ends of substance P-containing sensory neurones in the rat trigeminal system. Brain Res. 152. 499-509. Fewtrell, C. M. S., Foreman, J. C., Jordan, C. C.. Oehme, P.. Renner, H. and Stewart, H. M. (1982). The effects of substance P on histamine and 5-hydroxytryptamine release in rat. 1. PhysioI. 330. 393-411. Foreman. J. C.. Jordan, C. C., Oehme, P. and Renner. H. ( 1983). Structure-activity relationships for some substance P-related peptides that cause wheal and flare reactions in human skin. 1. Physiol. 335, 449-65.
646 Gamse. R. and Saria, A. (198 5). Potentiation of tachykinin induced plasma protein extravasation by calcitonin gene-related peptide. Eur. J. Pharmarol. 114. 61-6. Hakfelt. T., Kellerth, J.-O., Nilsson. G. and Pernow. B. (19 75). Experimental immunohistochemical studies on the localization and distribution of substance P in cat primary sensory neurons. Brain Res. 100, 235-52. Johnson. A. R. and Erdb;s, E. G. (1973). Release of histamine from mast cells by vasoactive peptides. Proc. Sot. Exp. Biol. Med. 142. 1253-6. Karjalainen K., Tervo. T. and Palkama, A. (1978). Catecholamine-containing and acetylcholinesterase-positive nerve fibres in the rabbit conjunctiva. Actn Ophthdmol. 56, 91 l-20. Kuwayama. Y. and Stone, R. A. (1987). Distinct substance P and calcitonin gene-related peptide immunoreactive nerves in the guinea pig eye. Invest. Ophthalmol. Vis. Sci. 28, 1947-54. Lee, Y., Kawai. Y.. Shiosaka, S.. Takami. K.. Kiyama. K., Hillyard. C. J., Girgis, S., MacIntyre, I., Emson. P. C. and Tohyama, M. (1985). Coexistence of calcitonin generelated peptide and substance P-like peptide in single cells in the trigeminal ganglion of the rat: immunochemical analysis. Bruin Res. 330, 194-6. Lembeck, F. and Holzer, P. (1979). Substance P as neurogenic mediator of antidromic vasodilatation and neurogenic plasma extravasation. Arch. Pharmacol. 310, 175-83. Luhtala, J.. Palkama. A. and IJusitalo, H. (I 991). Calcitonin gene-related peptide immunoreactive nerve fibres in the rate conjunctiva. invest. Ophthalmol. Vis. Sci. 32, 640-5. Macintosh, S. R. (1974). The innervation of the conjunctiva
J. LUHTALA
AND
H. UUSITALO
in monkeys: an electron microscopic and nerve degeneration study. Albrecht v. Graefes Arch. Klin. Erp. Ophthalmol. 192, 105-16. Miller, A., Costa, M.. Furness, J. B. and Chubb. I. W. ( 198 1). Substance P immunoreactive sensory nerves supply the rat iris and cornea. Neurosci. Lett. 23. 24 3-9. Olgarth. I,.. Gazelius, B.. Brodin. E. and Nilsson. C. I 19771. Release of substance P-like immunoreactivity from the cat dental pulp. Arttr Physiol. Scud. 101. 510-L. Otsuka. M. and Konishi, S. (I 983). Substance P-the first peptide neurotransmitter ? Trends .!kwosci. 6. 3 17-20. Salt. T. E. and Hill. R. G. (1983). Neurotransmitter candidates of somatosensory primary afferent fibres. Neuroscience 10. 1083-103. Sweeney. M. I. and Sawynok, J. (1986). Evidence that substance P may be a modulator rather than a transmitter of noxious mechanical stimulation. Corn. I. Physiol. 64. 1 324-7. Takahashi, T. and Otsuka. M. ( 1975). Regional distribution of substance P in the spinal cord and nerve roots of the cat and the effect of dorsal root section. Brrrin Res. 87. l-l 1. Wallengren. J. and HBkanson, R. ( 1987). Effects of substance P, neurokinin A and calcitonin gene-related peptide in human skin and their involvement in sensory nerve mediated responses. Eur. 1. Pharmnrol. 143. 267-Y 3. Wiesenfeld-Hallin, Z., HGkfelt, T.. Lundberg, J. .%I.. Forssmann. W. G.. Reinecke. M.. Tschopp, F. A. and Fischer. J. A. (1984). Immunoreactive calcitonin gene-related peptide and substance P coexists in sensory neurons to the spinal cord and interact in spinal behavioral responses of the rat. i\jeurosci. Lett, 52. 199-20~.