Archivesof Oto-Rhino-Laryngology 9 Springer-Verlag 1982
Arch Otorhinolaryngol (1982) 236:7-14
Adrenergic and Peptidergic Innervation of Cochlear Blood Vessels* R, Uddman 1, O. Ninoyu 2, and F. Sundler 3 1Lgkargruppen Gustav Adolf, Gustav Adolfs Torg 43, S-211 53 Malmt, Sweden 2Dept. of Otolaryngology, Kansai Medical University, Moriguchi, Osaka 570, Japan 3Dept. of Histology, University of Lund, S-223 62 Lund, Sweden
Summary. Guinea pig cochlear blood vessels were investigated with regard to their supply of adrenergic and peptidergic nerve fibers. Using the glyoxylic acid histofluorescence technique, numerous adrenergic fibers were seen around the labyrinthine artery, whereas the spiral modiolar artery contained only few such fibers. Immunocytochemistry revealed nerve fibers containing immunoreactive avian pancreatic polypeptide, vasoactive intestinal peptide, substance P, or gastrin-releasing peptide around the labyrinthine and spiral modiolar arteries. Adrenergic or peptidergic nerve fibers were not seen around the blood vessels of the stria vascularis. Upon removal of the superior cervical ganglion, adrenergic fibers disappeared and fibers displaying avian pancreatic polypeptide immunoreactivity were reduced in number. These data suggest co-occurrence of catecholamines and immunoreactive avian pancreatic polypeptide in a population of adrenergic nerves. Key words: Cochlear blood vessels - Neuropeptides - Immunocytochemistry Cochlear blood vessels are richly supplied with nerve fibers. Although early studies [14, 23] did not disclose the identity of these fibers modern histochemical techniques have revealed that the cochlear vessels harbour adrenergic [21, 24, 25, 29, 30] and acetylcholinesterase (AChE)-positive [8] nerve fibers. Catecholamines and acetylcholine are known to have a variety of vasomotor actions and there are several reports on their effects on cochlear blood vessels [7, 18, 27,
311. Immunocytochemical studies have revealed that the cerebrovascular bed receives an abundant supply of nerve fibers containing vasoactive intestinal * Supported by grants from the Swedish Medical Research Council (14X-4499) Offprints requests to: R. Uddman, MD, Dept. of Otolaryngology, University of Lurid, Malm6
General Hospital, S-21401 Malmt, Sweden
0302-9530/82/0236/0007/$ 01.60
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peptide (VIP) [4], substance P (SP) [5], or immunoreactive avian pancreatic polypeptide (APP) [32], and a number of investigations have indicated a role of neuropeptides in the control of blood vessel tone. So far nothing is known about the existence of peptide-containing fibers around the blood vessels in the inner ear. In the present study, we have analyzed the occurrence and distribution of nerve fibers around cochlear blood vessels of the guinea pig using the glyoxylic acid technique for the demonstration of adrenergic and immunocytochemical techniques for the demonstration of peptide-containing nerve fibers.
Material and Methods Twenty guinea pigs of both sexes weighing 200- 500 g were used. They were killed by exsanguination under diethyl ether anesthesia. In four guinea pigs the left cervical sympathetic ganglion was removed under Ketalar (Ketamin chloride, Parke-Davis, Gwent, UK) anesthesia. These animals were killed 1 - 6 weeks later. The cochleae were rapidly removed and further dissection was performed in either a glyoxylic acid solution or in a formaldehyde-picric acid fixative.
Demonstration of Adrenergic Nerve Fibers Segments of the stria vascularis, the labyrinthine, and spiral modiolar arteries were dissected free in a freshly made solution of glyoxylic acid in phosphate buffer [6, 12]. They were immersed in the glyoxylic acid solution for at least 30 rain, stretched as whole mounts on microscope slides, dried under a hair drier (70~ C) for 1 h, and further dehydrated in a desiccator for 1 h. The specimens were then mounted in Entellan (Merck Darmstadt, FRG) and examined with a fluorescence microscope.
Demonstration of Peptide-containing Nerve Fibers Segments of the stria vascularis, the labyrinthine, and spiral modiolar arteries were stretched on microscope slides as whole mounts and fixed in a buffered solution of formaldehyde and picric acid overnight. They were then dehydrated, cleared in xylene, and hydrated in a series of ethanol solutions [2]. The specimens were processed for the immunocytochemical demonstration of APP, VIP, SP, or gastrin-releasing peptide (GRP). For the demonstration of APP-immunoreactive nerve fibers, an antiserum (generous gift from Dr J. R. Kimmel, University of Kansas, Kansas City, USA) raised against pure avian pancreatic polypeptide (PP) and known to demonstrate neuronal APP was used. The antiserum was applied at a dilution of 1:80. It has been used in several immunocytochemical studies [13, 15, 32]. Neuronal APP can not be demonstrated using antibodies directed against mammalian PP nor can it be demonstrated by all antibodies against APP [13]. Neuronal APP is, therefore, probably distinct from hormonal APP although sharing an amino acid sequence [13, 32]. For the demonstration of VIP immunoreactivity, an antiserum against pure porcine VIP was used. The antiserum (code No. 7852, Milab, Malm6, Sweden) has been used in several immunocytochemical studies [26, 33]. It was used at a dilution of 1 : 160. For the demonstration of SP immunoreactivity, an antiserum against synthetic bovine SP was used [the antiserum (code No. SP-8) was a kind gift from Dr P. Emson, Cambridge, UK]. It has been applied and characterized in previous immunocytochemical studies [1, 5]. It was used at a dilution of 1 : 20. The antiserum is directed against the C-terminal portion of the SP molecule. It does not cross-react with bombesin or GRP, which share the COOH-terminal two amino acids with SP. It does, however, cross-react with physalaemin [1], which shares five amino acid residues with SP.
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Physalaemin was recently claimed to occur in nerve fibers in the mammalian gastrointestinal tract [111. The GRP antiserum [(code No. 6902) kindly provided by Dr N. Yanaihara, Laboratory of Biorganic Chemistry, Shizuoka College of Pharmacy, Shizuoka, Japan] was raised in rabbits against (synthetic) GRP [35], a peptide originally isolated from the porcine antrum [17]. The COOH-terminal portion of this peptide closely resembles bombesin. The antiserum has been found to cross-react with authentic bombesin but not with SP (unpublished observations). The antiserum was used at a dilution of 1 : 320-640. The site of antigen-antibody reaction was revealed by fluorescein isothiocyanate labeled goat anti-rabbit IgG (Dako Immunoglobulin AB, Stockholm, Sweden) diluted at 1 : 20. Some of the whole mounts were subsequently washed, incubated with a peroxidase-antiperoxidase complex, and stained for peroxidase. Specimens incubated with antiserum inactivated by the addition of excess antigen (10-100 ~tg of pure avian PP, pure porcine VIP, synthetic SP, and synthetic bombesin, respectively) served as controls. Pure avian PP was a gift from Dr J. R. Kimmel. Pure porcine VIP was a gift from Dr V. Mutt, Karolinska Institutet, Stockholm, Sweden. SP and bombesin were obtained from Beckman S. A., Geneva, Switzerland. Cross-reactivity with unknown peptides containing immunoreactive amino acid sequences recognized by the different antisera cannot be excluded. It would, therefore, be appropriate to name the immunoreactive material APP-like, VIP-like, SP-like, or GRP-like. For brevity, the immunoreactive nerve fibers are referred to as APP-, VIP-, SP-, or GRP-nerve fibers in the text.
Results
Adrenergic Nerve Fibers Glyoxylic acid induced fluorescence in nerve fibers in the wall of both the labyrinthine and the spiral modiolar arteries (Fig. 1). The labyrinthine artery contained a very dense plexus of fine-beaded nerve fibers. The spiral modiolar artery on the other hand contained only few single fibers. Adrenergic fibers disappeared upon removal of the superior cervical ganglion. The stria vascularis was devoid of adrenergic fibers.
Peptide-containing Nerve Fibers The APP-, VIP-, SP-, or GRP-immunoreactive nerve fibers were found both around the labyrinthine and spiral modiolar arteries. The labyrinthine artery contained plexuses of each of these types of fibers. The APP fibers were fairly numerous, while those containing VIP, SP, or GRP were few (Figs. 2 - 5 ) . The spiral modiolar artery contained only single, scattered, fine, varicose fibers of each type. Generally, the peptide-containing nerve fibers were less numerous than adrenergic nerve fibers in the corresponding locations. After removal of the superior cervical ganglion, there was a diminished supply of APP fibers. The number of VIP-, SP-, or GRP-containing nerve fibers was not overtly reduced. Peptide-containing fibers were not seen around the vessels of the stria vascularis.
Fig. 1. Glyoxylic acid treatment. Adrenergic nerve fibers form a dense plexus around the labyrinthine artery (x 240)
Fig. 2 Fig. 3 Fig. 2. Nerve fibers containing immunoreactive avian pancreatic polypeptide forming a meshwork around a the labyrinthine artery and b the spiral modiolar artery. Immunofiuorescence technique. (• 240) Fig. 3. Vasoactive intestinal polypeptide containing nerve fibers around the labyrinthine artery. Immunoperoxidase Staining (• 240)
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Fig. 4. A fine network of substance P containing nerve fibers around a the labyrinthine artery (x 240) and b the spiral rnodiolar artery (x 330). Immunofluorescence technique
Fig. 5. A fine plexus of nerve fibers around the labyrinthine artery displaying gastrin releasing peptide immunofluorescence (x 240)
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Discussion
A dense supply of adrenergic nerve fibers has been demonstrated around cochlear blood vessels using the Falck-Hillarp histofluorescence method [21, 24, 25, 29, 30]. The glyoxylic acid technique is claimed to be superior to the standard Falck-Hillarp technique for the demonstration of noradrenaline stores in whole-mount preparations [6]. So far, the glyoxylic acid technique has not been used in inner-ear studies. In the present study, a dense plexus of varicose adrenergic nerve fibers was seen around the labyrinthine artery, while the spiral modiolar artery contained only single, beaded fibers. Following superior cervical ganglionectomy the adrenergic fibers to the cochlear vessels disappeared. On the whole, these results confirm previous studies [21, 24, 25, 29, 30]. The action of noradrenaline and sympathetic stimulation on cochlear blood flow has been described with somewhat conflicting results [27, 31]. According to Hultcrantz et al. [7] sympathetic stimulation causes a reduction of the cochlear blood flOW.
Pancreatic polypeptide (PP) is a peptide hormone candidate [9], produced by a population of cells in the pancreas and in the gastrointestinal tract [10, 28]. Immunocytochemical studies have revealed that certain antibodies raised against avian pancreatic polypeptide (APP) also demonstrate neuronal elements [13, 15, 32]. Since these neurons are not demonstrable with antibodies against mammalian PP nor with all antibodies against APP, neuronal PP may represent an APP fragment. This possibility may gain support from the recent finding of Vaillant and Taylor [34] that an antiserum raised against the COOH-terminal hexapeptide of mammalian PP was capable of demonstrating nerve fibers in several peripheral organs. In the present study, a fine network of APP-immunoreactive fibers was noted both around the labyrinthine and the spiral modiolar arteries. Removal of the superior cervical ganglion caused a reduction of the number of APP-immunoreactive fibers. APP-immunoreactive nerve cell bodies are numerous in sympathetic ganglia and it has recently been suggested that APP-immunoreactive nerve fibers may constitute a subpopulation of adrenergic fibers [15, 32]. With regard to its distribution and pharmacologic effects VIP has been studied in fairly great detail [22]. There is a rich supply of VIP fibers around pial blood vessels [4]; VIP elicits dilatation of pial vessels and systemic administration of VIP results in an augmented cerebral blood flow [16]. In this study, a moderate supply of VIP fibers was noted around cochlear vessels. Like VIP, SP is a neuropeptide with a widespread distribution in the central and peripheral nervous systems. In particular, SP fibers are numerous in tissues known to receive a heavy sensory innervation [19]; SP fibers are numerous around cerebral blood vessels and SP dilates pial vessels both in vitro and in vivo [5]. In the present study, a moderate supply of SP fibers was found around the labyrinthine and spiral modiolar arteries. Bombesin is a tetradecapeptide isolated from the skin of amphibians. Recently, a 27-amino-acid peptide, gastrin-releasing peptide (GRP) was isolated from the porcine stomach [17]. It is thought that GRP represents the mammalian counterpart of bombesin since most of the amino acid sequence of bombesin is
Innervation of Cochlear Blood Vessels
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contained within the COOH-terminal part of the GRP molecule and since the two peptides seem to have a very similar spectrum of biologic activities [17]. Bombesin-immunoreactive nerve fibers have previously been demonstrated in the gastrointestinal tract and in the central nervous system [3, 20]. In the present study, delicate GRP-immunoreactive nerve fibers were noted around the cochlear vessels. In conclusion, the occurrence of adrenergic nerve fibers and fibers containing immunoreactive APP, VIP, SP, or GRP around cochlear blood vessels suggests multiple and complex neurogenic mechanisms in the regulation of cochlear blood flow.
References 1. Brodin E, Alumets J, HSkanson R, Leander S, Sundler F (1981) Immunoreactive substance P in chicken gut, distribution, development and possible functional. Cell Tissue Res 216 : 455-469 2. Costa M, Buffa R, Furness JB, SoMa E (1980) Immunohistochemical localization of potypeptides in peripheral autonomic nerves using whole mount preparations. Histochemistry 65 : 157-165 3. Dockray G J, Vaillant C, Walsh JH (1979) The neuronal origin of bombesin-like immunoreactivity in the rat gastrointestinal tract. Neuroscience 4:1561-1568 4. Edvinsson L, Fahrenkrug J, Hanko J, Owman C, Sundler F, Uddman R (1980) VIP (vasoaetive intestinal polypeptide)-containing nerves of intracranial arteries in mammals. CelI Tissue Res 208 : 135-142 5. Edvinsson L, McCulloch J, Uddman R (1981) Substance P: immunohistochemical localization and effect upon cat pial arteries in vitro and in situ. J Physiol 318:251-258 6. Furness JB, Costa M (1975) The use of glyoxylic acid for the fluorescence histochemical demonstration of peripheral stores of noradrenaline and 5-hydroxytryptamine in whole mounts. Histochemistry 41:335-352 7. Hultcrantz E, Linder J, Angelborg C (1977) Sympathetic effects on cochlear blood flow at different blood pressure levels. INSERM 68:271-278 8. Ishii T (1971) Acetylcholinesterase activity in the perivascular nerve plexus of the basilar and labyrinthine arteries. Acta Otolaryngol (Stockh) 72:281-287 9. Kimmel JR, Pollock HG, Hazelwood RL (1971) A new pancreatic polypeptide hormone. Fed Proc 30:I318 10. Larsson L-I, Sundler F, Hgtkanson R, Pollock HG, Kimmel JR (1974) Localization of APP, a postulated new hormone to a pancreatic endocrine cell type. Histochemistry 42:377-382 11. Lazarus LH, Linnoila RI, Hernandez O, DiAugustine RP (1980) A neuropeptide in mammalian tissue with physalaemin-Iike immunoreactivity. Nature 287:555-558 12. Lindvalt O, Bj6rklund A (1974) The glyoxylic acid fluorescence histochemical method: a detailed account of the methodology for the visualization of central eatecholamine neurons. Histochemistry 39: 97-127 13. Lorgn I, Alumets J, Hfikanson R, Sundler F (1979) Immunoreactive pancreatic polypeptide (PP) occurs in the central and peripheral nervous system: preliminary immunocytochemical observations. Cell Tissue Res 200:179-186 14. Lorente de N6 R (1937) The neural mechanism of hearing. Laryngoscope 47:373-377 15. Lundberg JM, Hrkfelt T, Anggfird A, Kimmel J, Goldstein M, Markey K (1980) Coexistence of an avian pancreatic polypeptide (APP) immunoreactive substance and catecholamines in some peripheral and central neurons. Acta Physiol Scand 110:107-109 16. McCutloch J, Edvinsson L (1980) The effects of vasoaetive intestinal polypeptide upon piat arteriolar calibre, cerebral blood flow, cerebral oxygen consumption and the electroencephalogram. Am J Physiol 238:H449-456
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17. McDonald TJ, J6rnvall H, Nilsson G, Vagne M, Ghatei M, Bloom SR, Mutt V (1979) Characterization of a gastrin releasing peptide from porcine non-antral gastric tissue. Biochem Bi0phys Res Commun 90: 227-233 18. Nomura Y (1961) Observations on the microcirculation of the cochlea. Ann Otol Rhinol Laryngol 70: 1037-1054 19. Olgart L, H61delt T, Nilsson G, Pernow B (1977) Localization of substance P-like immunoreactivity in nerves in the tooth pulp. Pain 4:153-159 20. Polak JM, Ghatei MA, Wharton J, Bishop AE, Bloom SR, SoMa E, Brown MR, Pearse AGE (1978) Bombesin-like immunoreactivity in the gastrointestinal tract, lung and central nervous system. Scand J Gastroenterol [Suppl] 49:148 21. Ross MD (1971) Fluorescence and electron microscopic observations of the general visceral, efferent innervation of the inner ear. Acta Otolaryngol [Suppl] (Stockh) 286:1-18 22. Said SI (1982) Vasoactive intestinal peptide. Raven Press, New York 23. Smith CA (1951) Capillary areas of the cochlea in the guinea pig. Laryngoscope 61 : 1073-1095 24. Spoendlin H, Lichtensteiger W (1966) The adrenergic innervation of the labyrinth. Acta Otolaryngol (Stockh) 61 : 423-434 25. Spoendlin H, Lichtensteiger W (1967) The sympathetic nerve supply to the inner ear. Arch Klin Exp Ohren-Nasen-Kehlkopfheilkd 189:346-359 26. Sporrong B, Falkmer S, Robboy SJ, Alumets J, Hf~kansonR, Ljungberg O, Sundler F (1982) Neurohormonal peptides in ovarian carcinoids. An immunohistochemical study of eighty-one primary carcinoids and of intra-ovarian metastases from six mid-gut carcinoids. Cancer 48 : 68-74 27. Suga F (1976) Neural control of cochlear blood flow. Acta Otolaryngol (Stockh) 81 : 26-35 28. Sundler F, HSkanson R, Larsson L-I (1977) Ontogeny of rat pancreatic polypeptide (PP) cells. Cell Tissue Res 178:303-306 29. Terayama Y, Holz E, Beck C (1966) Adrenergic innervation of the cochlea. Ann Otol Rhinol Laryngol 75: 69-86 30. Terayama Y, Shige E, Sakamoto T (1973) Distribution and origin of adrenergic nerve fibers in the vestibular apparatus and their arterial supply in the guinea pig. Acta Otolaryngol (Stockh) 76: 244-253 31. Todd NW, Dennard JE, Clairmont AA, Jackson RT (1974) Sympathetic stimulation and otic blood flow. Ann Otol Rhinol Laryngol 83:1-8 32. Uddman R, Edvinsson L, H~kanson R, Owman C, 8undler F (1982) Immunohistochemical demonstration of APP (avian pancreatic polypeptide)-immunoreactive nerve fibres around cerebral blood vessels. Brain Res Bull (in press) 33. Uddman R, Kitajiri M, Sundler F (1982) The autonomic innervation of the middle ear. Ann Otol Rhinol Laryngol (in press) 34. Vaillant C, Taylor IL (1981) Demonstration of carboxyl-terminal PP-like peptides in endocrine cells and nerves. Peptides [Suppl2] 2:31-36 35. Yanaihara N, Yanaihara C, Mochizuki T, Iwahara K, Fujita T, Iwanaga T (1981) Immunoreactive GRP. Peptides [Suppl2] 2:185-192 Received March 18, 1982/Accepted March 22, 1982