Hannu Uusitalo

Calcitonin gene-related peptide (CGRP) immunoreactive sensory nerves in the human and guinea pig uvea and cornea

The presence of calcitonin gene-related peptide (CGRP) immunoreactive nerves in the uvea and cornea of human and guinea pig eyes was evaluated using immunohistochemical techniques. CGRP immunoreactivity was found in thin, varicose nerve fibers in both species. Most of the fibres were localized in the ciliary body, and were mainly associated with blood vessels. In the human ciliary body, a moderate number of CGRP immunoreactive nerves were also seen in the ciliary muscle. In the iris and cornea, CGRP immunoreactive fibres were relatively uncommon. In the iris, they were mostly found associated with blood vessels, while in the cornea they were seen sub-epithelially or as free nerve endings in the epithelium. In the trigeminal ganglion, small sized ganglion cells displayed CGRP immunoreactivity. About 40% of all ganglion cells were immunoreactive nerves in the guinea pig, while sympathetic denervation did not change the staining pattern of CGRP immunoreactivity. The present findings, together with previous physiological data, suggest that CGRP might play a role in the regulation of the blood flow, aqueous humour dynamics, and neurogenic inflammation, not only in experimental animals but also in man.

CGRP-immunoreactive sensory nerve fibers in the submandibular gland of the rat.

SummaryIndirect immunofluorescence technique was used to study the occurrence and distribution of CGRP immunoreactivity in the submandibular gland of normal rats and after unilateral sensory and sympathetic denervations. In normal rats, CGRP-immunoreactive nerve fibers and nerve trunks were seen around or in close contact with interlobular salivary ducts as well as around small blood vessels of the gland. Occasionally, CGRP-immunoreactive nerve fibers were also detected between or around the acini of the gland.The submandibular ganglia contained CGRP-immunoreactive nerve fibers, but the ganglion cells were not immunoreactive for CGRP. The trigeminal ganglion contained a population of CGRP-immunoreactive, mainly small sized ganglion cells and nerve fibers distributed throughout the ganglion. Unilateral electrocoagulation of the trigeminal nerve caused a significant reduction in the number of immunoreactive nerve fibers in the gland, although some fibers still were present in the ipsilateral glandular tissue. Unilateral superior cervical ganglionectomy caused no detectable effect on the number of CGRP-immunoreactive nerve fibers in the gland.The present results suggest that the rat submandibular gland contains CGRP-immunoreactive nerve fibers both around blood vessels and in glandular secretory elements. Denervation experiments support the view that the majority, but perhaps not all of them originate from the trigeminal ganglion.

Effect of neurogenic irritation and calcitonin gene-related peptide (CGRP) on ocular blood flow in the rabbit

The effects of sensory nerve stimulation (topical neutral formaldehyde, 1%) and intracameral injection of calcitonin gene-related peptide (CGRP) on regional ocular blood flow, intraocular pressure (IOP), the blood-aqueous barrier, pupil size, and blood pressure were studied in the rabbit. Sensory nerve stimulation elicited a typical irritative response in the rabbit eye, with vasodilation in the ciliary body (from 128 +/- 31 to 363 +/- 105 mg/min, p less than 0.05) accompanied with a breakdown of the blood-aqueous barrier, rise in the IOP, and miosis. CGRP caused similar, but not identical, changes in the eye: vasodilation in the ciliary body (from 60 +/- 14 to 258 +/- 75 mg/min, p less than 0.05), breakdown of the blood-aqueous barrier and rise in the IOP, accompanied with systemic hypotension. Miosis was not observed after CGRP. In the present study, the vasodilatory action of CGRP on the rabbit eye has been shown. This makes our understanding of the mechanism of the ocular irritative response after sensory nerve stimulation more complete. Thus, CGRP through vasodilation disrupts the blood-aqueous barrier and raises the IOP. The more intense increase in the IOP after sensory nerve stimulation than after CGRP is probably caused by a CGRP-induced vasodilation and breakdown of the blood-aqueous barrier, enhanced by a miosis-induced pupillary block.

THE EFFECT OF FIXATION ON CORNEAL ENDOTHELIAL CELL DIMENSIONS AND MORPHOLOGY IN SCANNING ELECTRON MICROSCOPY

Soft tissue specimens shrink during fixation, dehydration and critical point drying when prepared for scanning electron microscopy (SEM). This can cause serious artifacts not only in ‘compact’ tissues but especially in hollow structure, like the eye, where the chambers are lined by delicate layers such as the corneal endothelium. In this study various glutaraldehyde and formaldehyde fixations at different concentrations with or without 5% sucrose were tested. Dimensional as well as morphological changes of rabbit corneal endothelial cells were evaluated. The mean surface diameter and area of rabbits central corneal endothelial cells were measured first in vivo by specular microscope. Thereafter the same corneas were fixed in 9 different solutions and processed for scanning electron microscopy. The surface structure of the same endothelium was then photographed with SEM. The cell dimensions were remeasured. According to our results 1.25% or 2.50% glutaraldehydes without sucrose gave the best surface preservation and caused the least shrinkage.

Distribution of adrenergic nerves in the lacrimal glands of guinea-pig and rat

The distribution of adrenergic nerves in the ex- and intraorbital lacrimal glands of guinea-pig and rat was studied using the sucrose-potassium phosphate-glyoxylic acid (SPG) technique. Blood vessels and secretory acini of guinea-pig lacrimal glands were demonstrated as having a rich adrenergic innervation. Adrenergic fibers in the rat were, however, much more sparse, and most of them were seen in association with glandular blood vessels, with only a few being found between secretory acini. Pretreatment of animal with a monoamine oxidase inhibitor and L-dopa did not change the morphological distribution of catecholamine fluorescent fibers, although the treatment improved the fluorescence, especially in the rat. Extirpation of the ipsilateral superior cervical ganglion eliminated all the fluorescent fibers in both normal and pretreated animals. The presence of adrenergic innervation of the lacrimal glands, especially in close connection with secretory acini, supports the theory that catecholamine-containing nerves play a role in the regulation of lacrimal secretion.

Biochemical and immunohistochemical determination of 5-hydroxytryptamine located in mast cells in the trigeminal ganglion of the rat and guinea pig

SummaryThe presence of 5-hydroxytryptamine (5-HT), as well as its precursor (5-HTP) and metabolite (5-HIAA), were biochemically determinated in the trigeminal ganglion of the guinea pig and rat. The distribution of 5-HT in the ganglion and in its posterior root was studied using both indirect immunofluorescence and the peroxidase-antiperoxidase method. In order to increase the possible 5-HT content of primary sensory neurons for subsequent immunohistochemical visualization, animals were first treated with nialamide, an inhibitor of monoamine oxidase, and then loaded with l-tryptophan. Another group of animals received colchicine to inhibit intra-axonal transport of transmitter substances. However, even combined use of loading and colchicine treatment did not reveal 5-HT immunoreactivity in ganglion cells.The only source of 5-HT immunoreactivity in the trigeminal ganglion and its posterior root was mast cells. These cells were located around the ganglion in adjacent leptomeningeal and connective tissues, as well as between the ganglion cells and nerve fibers. Only occasionally were mast cells found in the posterior root of the ganglion.

The distribution and origin of substance P immunoreactive nerve fibres in the rat conjunctiva

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 Müller 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.

Nerve fibers showing immunoreactivities for proenkephalin A-derived peptides in the lacrimal glands of the guinea pig

The extra- and intraorbital lacrimal glands of guinea pigs were studied for the presence and distribution of enkephalin-like immunoractive nerve fibers. Four specific antisera against the four different enkephalin sequences contained in pre-proenkephalin A (Met-Enk, Met-Enk-Arg-Phe, Met-Enk-Arg-Gly-Leu, and Leu-Enk) were used. All of these immunoreactivities were identically distributed in varicose nerve fibers in both the extra- and intraorbital lacrimal glands. These fibers densely surrounded the glandular acini and also innervated the secretory ducts. Sympathetic denervation had no effect on these nerve fibers. The results suggest that enkephalins derived from proenkephalin A may play a role in the nervous control of the lacrimal secretion.

Production and characterization of a monoclonal antibody against human calcitonin gene-related peptide (CGRP) and its immunohistochemical application to salivary glands.

SummaryA monoclonal antibody (mAb), 129CD8 was raised against a C-terminal fragment (aa28–37) of α-human calcitonin gene-related peptide (CGRP) coupled to bovine serum albumin. The specificity of the monoclonal antibody 129CD8 was corroborated by dot immunobinding experiments, enzyme-linked immunoassay and immunostaining of tissue sections. In vitro studies showed that the mAb 129CD8 readily recognized the fragment 28–37 of α-human CGRP and to a slightly lesser degree whole α-human CGRP and the fragments containing the C-terminal part of the molecule. The mAb 129CD8 also recognized the β-human CGRP but not the α-rat CGRP. The mAb 129CD8 did not react with substance P, katacalcin, calcitonin, amylin or fragments of α-human CGRP lacking the C-terminal part of the molecule.Immunocytochemical staining was performed on human skin, guinea-pig thyroid and salivary glands and the trigeminal ganglion, and rat thyroid gland. Our findings demonstrate, in keeping with previous studies, that in human skin, nerve fibres containing CGRP immunoreactivity are found in both epidermis and dermis. In accordance with previous investigators, the Merkel cells were immunoreactive for CGRP. In the guinea-pig and rat thyroid gland CGRP immunoreactivity was localized in the C-cells. The distribution of CGRP immunoreactivity in the guinea-pig salivary glands is different from that previously reported for rat salivary glands. In the guinea-pig trigeminal ganglion, CGRP immunoreactivity was localized mainly in small-sized neurons and fibres traversing the ganglion. Double staining with substance P performed on guinea-pig trigeminal ganglion revealed four types of sensory neurons, those containing both peptides, those containing only substance P or CGRP and those lacking both peptides. Guinea-pig parotid gland, but not the submandibular or sublingual glands, contained periacinar fibres exhibiting both immunoreactivities. Substance P-positive, CGRP-positive fibres were also seen around parotid and submandibular, but not around sublingual, gland ducts. All glands received perivascular innervation showing immunoreactivities for both peptides. The present results support the idea that in the peripheral nervous system only a subpopulation of sensory neurons contains both substance P and CGRP. Consequently, colocalization of substance P and CGRP indicates a sensory nerve, while those containing either substance P or CGRP may be sensory or parasympathetic.

Substance P and neurokinin A immunoreactive nerve fibres in the developing salivary glands of the rat

The time of appearance and distribution of substance P (SP) and neurokinin A (NKA) immunoreactive nerve fibres in developing salivary glands of the rat were studied by the use of indirect immunohistochemical methods. The glands were examined at daily intervals from the 15th day in utero (i.u.) until birth, and subsequently on the 2nd, 5th, 7th, 12th, 16th and 30th postnatal day. The findings were compared to samples from adult. The first SP- and NKA-immunoreactive (IR) nerve fibres appeared on the 19th day i.u. in the parotid and submandibular glands and were abundantly distributed around developing ductal branches. In the mesenchyme around the developing ductal branches of the parotid gland the fibres appeared on the 20th day i.u. In the submandibular gland NKA-IR fibres appeared in the mesenchyme surrounding the developing ductal branches on the 19th day i.u. and SP-IR fibres on the 21st day i.u. Around blood vessels of both glands, SP- and NKA-IR fibres made their appearance only much later, on the second postnatal day. The number of SP- and NKA-IR nerve fibres in the developing salivary glands was already high on the 19th day i.u. when they were first detected. From this point up to the 16th postnatal day the glands were richly innervated by the fibres, but later the numbers slowly decreased to adult levels. The abundance of SP- and NKA-IR nerve fibres especially around the ductal branches and secretory structures in the developing salivary glands suggests a role in the functional maturation of salivary glands.