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Pharmacological Characterization of Receptors on Blood Vessels in the Tympanic Membrane Involved in Otitis Media
Auris·Nasus·Larynx (Tokyo) 12 (Supp!. I) S 135-S 137,1985
PHARMACOLOGICAL CHARACTERIZATION OF RECEPTORS ON BLOOD VESSELS IN THE TYMPANIC MEMBRANE INVOLVED IN OTITIS MEDIA Sten HELLSTROM, Nils ALBIIN, Pamela GOLDIE, Bengt SALEN, and Lars-Eric STENFORS Departments of Anatomy and Otolaryngology, University of Umed, S-901 87 Umed, Sweden
Acetylcholine dropped onto the meatal surface of the tympanic membrane evoked vasodilatation and a vascular leakage to the middle ear cavity. The vasoreactions were atropine-resistent. Substance P and vasoactive intestinal polypeptide (VIP) injected intravenously caused a marked vascular leakage and VIP also vasodilatation. These blood vessel changes seem to be regulated by the parasympathetic nerves as they were inhibited by vagotomy. Constriction of the tympanic membrane vessels was mediated through a-receptors. Under normal conditions the tympanic membrane (TM) looks transparent and exhibits only minor blood vessels. The vascular supply of the TM has been described in the literature (LUSCHER, 1929; HAMBERGER and LINDGREN, 1941; SAINI, 1964; PAU, 1983; ALBIIN et al., 1985). The blood supply of both the human and the rat TM in a healthy ear seems to be quite minimal, as myringotomy can be performed without causing any macroscopically visible bleeding. In an animal model, the rat, it was recently shown that the earliest changes in experimentally provoked otitis media with effusion are noted in the flaccid portion of the tympanic membrane. Upon histological examination the pars flaccida appears edematous and exhibits dilated vessels. By vital staining, e.g. use of intravenously administered Evans Blue, leakage from these dilated vessels is indicated. These vascular events, the vasodilatation and increased permeability, should be regulated by neural and humoral substances. The innervation of the tympanic membrane
is intriguing. Catecholaminergic nerve fibers have been identified throughout both portions of the tympanic membrane, while neuropeptide-containing (substance P and vasoactive intestinal polypeptide (VIP)) nerves are located exclusively in the pars flaccida (WIDEMAR et al., 1985). These neuropeptides are potent edema-provoking substances. The pars flaccida is also unique in that it contains abundant mast cells rich in histamine-containing granules (ALM et al., 1983). Against this background the present study was aimed to pharmacologically characterize the receptors regulating the tympanic membrane vessels. Material and Methods
Healthy adult male Sprague-Dawley rats were used. The animals were anesthetized by intraperitoneally administered pentobarbital and subjected to the following procedures: a) Cholinergic (acetylcholine), a-adrenergic (phenylephrine), and /J-adrenergic (isoproterenol) agonists, at concentrations 10- 1 , 10- 8, 10- 5, 10- 7 M, were dropped onto the meatal surface of the tympanic membrane. Some animals were injected with atropine (50 nM/kg b.w.) i.p. to block cholinergic receptors prior to the application of acetylcholine. b) The neuropeptides, substance P (75 nM/kg) and VIP (100 nM/kg) were injected i.v. c) Mechanical stimulation of the external auditory canal through a stream of chilled air (14°C, 2.5 liters/min for 20 min). One week prior to the stimulation one group of rats had been subjected to surgically per-
S. HELLSTROM et al.
S 136
Table 1. Effects of vasoactive drugs on tympanic membrane vessels and vascular leakage to the attic space. Substance
Concentration
Vasodilatation
Acetylcholine
10-7 M 10-5 M 10-3 M 10-1 M 10-3 M
o o
Atropine prior to acetylcholine Phenylephine (a-agonist)
+
+
+t +t o o o
10-7 M 10-5 M 10-3 M
o
1O- 1 M
Substance P VIP
+t,
pronounced;
75 nM/kg 100 nM/kg
+, moderate;
o
+ + +
+t
10-7 M 10-5 M 10-3 M
o +
Vascular leakage
o
+
1O-1 M
Isoproterenol CO-agonist)
Vasoconstriction
o o o
o o
o o
+t +t
0, no effect.
formed unilateral sympathectomy. Another group of rats had been extracranially vagotomized. After administering the drugs or after mechanically stimulated, the tympanic membrane was continuously observed for 20 min through an ear speculum under an otomicroscope. Any change in the diameter of the tympanic membrane vessels was registered by the eye and the degree of vascular leakage was determined by administration of the intravital stain Evans Blue. Results
The results of the different drugs applied are shown in Table 1. Acetylcholine caused both a marked vasodilatation and vascular leakage. Pretreatment with atropine did not block the effects evoked by acetylcholine. The most pronounced vascular leakage was evoked with the neuropeptides substance P and VIP. Of these only VIP appeared to have a vasodilatatory capacity. Of the adrenergic agonists tested, the areceptor stimulatory drug phenylephrine caused vasoconstriction. A stream of chilled air at 14°C into the
external auditory canal caused only moderate vasodilatation but pronounced vascular leakage. In vagotomized animals the leakage ceased while sympathectomy appeared to enhance this vascular phenomenon. Discussion
In summary, acetylcholine and the neuropeptide VIP, evoked vasodilation and an increased permeability. The atropine resistance of the cholinergic effect and the abolished vascular leakage after vagotomy support the suggestion that like e.g. in some cerebral vessels (BEVAN et al., 1984) the acetylcholine and VIP coexist in the same perivascular neurons belonging to the parasympathetic system. Whether or not the vascular leakage of substance P is mediated through a release of histamine (FOREMAN and PIOTROWSKI, 1984) from the abundant pars flaccida mast cells cannot be answered at present. Upon inflammation of the middle ear cavity the appearance of the TM is dramatically altered. Thus, a characteristic finding in acute otitis media in the early stage is a diffuse redness of the pars flaccid a and the adjacent skin. In secretory otitis media,
Lecture
however, the appearance of the TM is characterized by dilated radiating vessels in the periphery of the pars tensa. One may speculate that these blood vessels take an active part in the pathogenesis of otitis media, rather than merely reflect an underlying inflammatory condition. If so, knowledge of the receptor identity may help us to design pharmacological drugs which could hinder the disorder from becoming manifest. This study was supported by grants from the Swedish Medical Research Council No. B85-17X06578-03B.
References ALBIIN, N., STENFORS, L.-E., and HELLSTROM, S.: The vascular supply of the rat tympanic membrane. Anat. Rec. 212: 17-22, 1985. ALM, P.-E., BLOOM, G. D., HELLSTROM, S., STENFORS, L.-E., and WIDEMAR, L.: Mast cells in the pars flaccida of the tympanic membrane. A quantitative morphological and biochemical study in the rat. Experientia 39: 287-289, 1983. BEVAN, J. A., MOSCOWITZ, M., SAID, S. I., and BUGA, G.: Evidence that vasoactive intestinal poly-
Physiology
S 137
peptide is a dilator transmitter to some cerebral and extracerebral cranial arteries. Peptides 5: 385-388, 1984. FOREMAN, J. C. and PIOTROWSKI, W.: Peptides and histamine release. J. Allergy Clin. Immunol. 74: 127-131, 1984. HAMBERGER, c.-A. and LINDGREN, A. G. H.: Uber die Gefiissversorgung des Trommelfells. Acta Otolaryngol. 29: 99-112, 1941. HELLSTROM, S., SALEN, B., and STENFORS, L.-E.: The site of initial production and transport of effusion materials in otitis media serosa. Acta Otolaryngol. 93: 435-440, 1982. LUSCHER, E.: Zur topographie und Physiologie der cutanen oberflachlichen Trommelfellgefiisse. Z. Hals. Nasen. Ohrenheilkd. 22: 13-52,1929. PAU, H. W.: Fluoreszenzangiographie des Trommelfelles. Entwickling der Methode und Normalbefunde. Laryngol. Rhinol. Otol. 62: 8696, 1983. SAINI, V. K.: Vascular pattern of human tympanic membrane. Arch. Otolaryngol. 79: 193-196, 1964. WIDEMAR, L., HELLSTROM, S., SCHULTZBERG, M., and STENFORS, L.-E.: Autonomic innervation of the tympanic membrane. An immunohistochemical and histofluorescence study. Acta Otolaryngol. 100: 58-65, 1985.
PHARMACOLOGICAL CHARACTERIZATION OF RECEPTORS ON BLOOD VESSELS IN THE TYMPANIC MEMBRANE INVOLVED IN OTITIS MEDIA Sten HELLSTROM, Nils ALBIIN, Pamela GOLDIE, Bengt SALEN, and Lars-Eric STENFORS Departments of Anatomy and Otolaryngology, University of Umed, S-901 87 Umed, Sweden
Acetylcholine dropped onto the meatal surface of the tympanic membrane evoked vasodilatation and a vascular leakage to the middle ear cavity. The vasoreactions were atropine-resistent. Substance P and vasoactive intestinal polypeptide (VIP) injected intravenously caused a marked vascular leakage and VIP also vasodilatation. These blood vessel changes seem to be regulated by the parasympathetic nerves as they were inhibited by vagotomy. Constriction of the tympanic membrane vessels was mediated through a-receptors. Under normal conditions the tympanic membrane (TM) looks transparent and exhibits only minor blood vessels. The vascular supply of the TM has been described in the literature (LUSCHER, 1929; HAMBERGER and LINDGREN, 1941; SAINI, 1964; PAU, 1983; ALBIIN et al., 1985). The blood supply of both the human and the rat TM in a healthy ear seems to be quite minimal, as myringotomy can be performed without causing any macroscopically visible bleeding. In an animal model, the rat, it was recently shown that the earliest changes in experimentally provoked otitis media with effusion are noted in the flaccid portion of the tympanic membrane. Upon histological examination the pars flaccida appears edematous and exhibits dilated vessels. By vital staining, e.g. use of intravenously administered Evans Blue, leakage from these dilated vessels is indicated. These vascular events, the vasodilatation and increased permeability, should be regulated by neural and humoral substances. The innervation of the tympanic membrane
is intriguing. Catecholaminergic nerve fibers have been identified throughout both portions of the tympanic membrane, while neuropeptide-containing (substance P and vasoactive intestinal polypeptide (VIP)) nerves are located exclusively in the pars flaccida (WIDEMAR et al., 1985). These neuropeptides are potent edema-provoking substances. The pars flaccida is also unique in that it contains abundant mast cells rich in histamine-containing granules (ALM et al., 1983). Against this background the present study was aimed to pharmacologically characterize the receptors regulating the tympanic membrane vessels. Material and Methods
Healthy adult male Sprague-Dawley rats were used. The animals were anesthetized by intraperitoneally administered pentobarbital and subjected to the following procedures: a) Cholinergic (acetylcholine), a-adrenergic (phenylephrine), and /J-adrenergic (isoproterenol) agonists, at concentrations 10- 1 , 10- 8, 10- 5, 10- 7 M, were dropped onto the meatal surface of the tympanic membrane. Some animals were injected with atropine (50 nM/kg b.w.) i.p. to block cholinergic receptors prior to the application of acetylcholine. b) The neuropeptides, substance P (75 nM/kg) and VIP (100 nM/kg) were injected i.v. c) Mechanical stimulation of the external auditory canal through a stream of chilled air (14°C, 2.5 liters/min for 20 min). One week prior to the stimulation one group of rats had been subjected to surgically per-
S. HELLSTROM et al.
S 136
Table 1. Effects of vasoactive drugs on tympanic membrane vessels and vascular leakage to the attic space. Substance
Concentration
Vasodilatation
Acetylcholine
10-7 M 10-5 M 10-3 M 10-1 M 10-3 M
o o
Atropine prior to acetylcholine Phenylephine (a-agonist)
+
+
+t +t o o o
10-7 M 10-5 M 10-3 M
o
1O- 1 M
Substance P VIP
+t,
pronounced;
75 nM/kg 100 nM/kg
+, moderate;
o
+ + +
+t
10-7 M 10-5 M 10-3 M
o +
Vascular leakage
o
+
1O-1 M
Isoproterenol CO-agonist)
Vasoconstriction
o o o
o o
o o
+t +t
0, no effect.
formed unilateral sympathectomy. Another group of rats had been extracranially vagotomized. After administering the drugs or after mechanically stimulated, the tympanic membrane was continuously observed for 20 min through an ear speculum under an otomicroscope. Any change in the diameter of the tympanic membrane vessels was registered by the eye and the degree of vascular leakage was determined by administration of the intravital stain Evans Blue. Results
The results of the different drugs applied are shown in Table 1. Acetylcholine caused both a marked vasodilatation and vascular leakage. Pretreatment with atropine did not block the effects evoked by acetylcholine. The most pronounced vascular leakage was evoked with the neuropeptides substance P and VIP. Of these only VIP appeared to have a vasodilatatory capacity. Of the adrenergic agonists tested, the areceptor stimulatory drug phenylephrine caused vasoconstriction. A stream of chilled air at 14°C into the
external auditory canal caused only moderate vasodilatation but pronounced vascular leakage. In vagotomized animals the leakage ceased while sympathectomy appeared to enhance this vascular phenomenon. Discussion
In summary, acetylcholine and the neuropeptide VIP, evoked vasodilation and an increased permeability. The atropine resistance of the cholinergic effect and the abolished vascular leakage after vagotomy support the suggestion that like e.g. in some cerebral vessels (BEVAN et al., 1984) the acetylcholine and VIP coexist in the same perivascular neurons belonging to the parasympathetic system. Whether or not the vascular leakage of substance P is mediated through a release of histamine (FOREMAN and PIOTROWSKI, 1984) from the abundant pars flaccida mast cells cannot be answered at present. Upon inflammation of the middle ear cavity the appearance of the TM is dramatically altered. Thus, a characteristic finding in acute otitis media in the early stage is a diffuse redness of the pars flaccid a and the adjacent skin. In secretory otitis media,
Lecture
however, the appearance of the TM is characterized by dilated radiating vessels in the periphery of the pars tensa. One may speculate that these blood vessels take an active part in the pathogenesis of otitis media, rather than merely reflect an underlying inflammatory condition. If so, knowledge of the receptor identity may help us to design pharmacological drugs which could hinder the disorder from becoming manifest. This study was supported by grants from the Swedish Medical Research Council No. B85-17X06578-03B.
References ALBIIN, N., STENFORS, L.-E., and HELLSTROM, S.: The vascular supply of the rat tympanic membrane. Anat. Rec. 212: 17-22, 1985. ALM, P.-E., BLOOM, G. D., HELLSTROM, S., STENFORS, L.-E., and WIDEMAR, L.: Mast cells in the pars flaccida of the tympanic membrane. A quantitative morphological and biochemical study in the rat. Experientia 39: 287-289, 1983. BEVAN, J. A., MOSCOWITZ, M., SAID, S. I., and BUGA, G.: Evidence that vasoactive intestinal poly-
Physiology
S 137
peptide is a dilator transmitter to some cerebral and extracerebral cranial arteries. Peptides 5: 385-388, 1984. FOREMAN, J. C. and PIOTROWSKI, W.: Peptides and histamine release. J. Allergy Clin. Immunol. 74: 127-131, 1984. HAMBERGER, c.-A. and LINDGREN, A. G. H.: Uber die Gefiissversorgung des Trommelfells. Acta Otolaryngol. 29: 99-112, 1941. HELLSTROM, S., SALEN, B., and STENFORS, L.-E.: The site of initial production and transport of effusion materials in otitis media serosa. Acta Otolaryngol. 93: 435-440, 1982. LUSCHER, E.: Zur topographie und Physiologie der cutanen oberflachlichen Trommelfellgefiisse. Z. Hals. Nasen. Ohrenheilkd. 22: 13-52,1929. PAU, H. W.: Fluoreszenzangiographie des Trommelfelles. Entwickling der Methode und Normalbefunde. Laryngol. Rhinol. Otol. 62: 8696, 1983. SAINI, V. K.: Vascular pattern of human tympanic membrane. Arch. Otolaryngol. 79: 193-196, 1964. WIDEMAR, L., HELLSTROM, S., SCHULTZBERG, M., and STENFORS, L.-E.: Autonomic innervation of the tympanic membrane. An immunohistochemical and histofluorescence study. Acta Otolaryngol. 100: 58-65, 1985.