Intraocular Neurografts as a Model for Studying of Organization of Synaptic Connections in a Denervated Brain Area.

Intraocular neurografts of the septal region of rats were used as the model of deafferentiated brain area where the lack of adequate innervation is compensated for own interneuronal connections. Septum anlage from the brain of a 17-day fetus served as the donor material. The grafts developing in the anterior eye chamber over 3 months represented well-differentiated samples of the nervous tissue. A comparative morphometric study of the tripartite organization of synapses in the grafts and in the septum in situ was conducted. In the grafts, the mean volume and perimeter of synaptic terminals were below the normal. At the same time, postsynaptic densities did not differ from the control. A significant difference was found in the degree of surrounding of presynaptic terminals by astrocytic processes: in the grafts this parameter was higher by 1.8 times. Our results attest to an important role of perisynaptic glia in the formation of functionally active synaptic contacts with unusual neuronal targets.

Changes of the vasculature and innervation in the anterior segment of the RCS rat eye.

Investigating the anterior eye segment vasculature and innervation of dystrophic RCS rats, two major unique findings were observed: in the iris, young adult animals with retinal dystrophy showed an increase in substance P nerve fibres and a dilation of arterioles and capillaries. This finding continued during ageing. In the pars plana region, the surface covered by venules decreased continuously with age. In older animals, this decrease was parallelled by a local decrease of sympathetic TH-positive nerve fibres supplying these venules. For both conditions, no comparable data exists so far in the literature. They might point to a unique situation in the anterior eye segment of the dystrophic RCS rat.

Anterior segment growth and peripheral neural pathways in chick.

PURPOSE: To learn if peripheral nerve pathways are necessary for corneal expansion and anterior segment growth under a 12-hr light:dark cycle or for the inhibition of corneal expansion under constant light rearing. METHODS: Recently hatched White Leghorn chicks under anesthesia received unilateral ciliary ganglionectomy (CGx), cranial cervical ganglionectomy (Sx), or section of the ophthalmic nerve (TGx), along with sham-operated and/or never-operated control cohorts. Chicks were reared postoperatively under either a 12-hr light:dark cycle or under constant light. After 2 weeks and with the chicks under anesthesia, corneal radii of curvature and diameters were obtained with a photokeratoscope, refractometry and A-scan ultrasonography were performed, and the axial and equatorial dimensions of enucleated eyes were measured with digital calipers. Corneal areas were calculated from corneal curvatures and diameters. RESULTS: Despite the rich peripheral innervation to the eye, the selective denervations performed here exerted remarkably limited effects on corneal expansion and anterior segment development in chicks reared under either lighting condition. Ophthalmic nerve section did reverse in large part the inhibition of equatorial expansion of the vitreous chamber occurring under constant light rearing. CONCLUSIONS: The ciliary, sympathetic, or ophthalmic peripheral nerve pathways to the eye are not required either for corneal expansion and anterior segment development under a 12-hr light:dark cycle or for the inhibition of corneal expansion under constant light rearing. The ocular sensory innervation may be a means for regulating vitreous cavity shape.

Neurokinin a is a main constituent of sensory neurons innervating the anterior segment of the eye.

PURPOSE: To study the innervation pattern of the anterior segment of the eye by neurokinin (NK)-A-immunoreactive nerves and to determine their sensory origin. METHODS: The presence and distribution of NKA was examined in human eyes by radioimmunoassay and immunofluorescence. The source of nerves was determined by measuring the concentration of NKA in the trigeminal ganglion (TG) in comparison with that of the classic sensory peptides substance P (SP) and calcitonin gene-related peptide (CGRP) and in eye tissues in capsaicin-pretreated rats versus control subjects. The NKA-like immunoreactivities were further characterized by reversed phase HPLC in the rat TG and the human iris-ciliary body complex. The presence of gamma-PPT-A mRNA was studied in the rat TG by in situ hybridization. RESULTS: The levels of NKA in human eye tissues were approximately 10 times higher than those of SP but lower than those of CGRP. Nerve fibers were visualized in the cornea, the trabecular meshwork, the iridial stroma, and, prominently, in the sphincter muscle, the ciliary body stroma and muscle and processes, and the choroidal stroma and surrounding blood vessels. In the rat TG, the concentration of NKA was approximately five times higher than that of SP. Capsaicin led to a >60% decrease of the concentration of the peptide in the rat TG and rat eye tissues except for the retina. NKA-like immunoreactivities were present in a single peak corresponding to synthetic NKA, both in the rat TG and in the human iris-ciliary body complex, and numerous ganglion cells of small size were labeled by a gamma-PPT-A probe in the rat TG. CONCLUSIONS: The present results clearly demonstrate that NKA is a main constituent of sensory neurons innervating the anterior segment of the eye. The presence of the peptide in C fibers in ocular tissues indicates a participation in sensory transmission and an involvement in the irritative response in the eye, a model for neurogenic inflammation in lower mammals.

Secretoneurin in the peripheral ocular innervation.

PURPOSE: To evaluate whether secretoneurin represents a sensory neuropeptide innervating the anterior segment of the eye. METHODS: The presence and distribution of secretoneurin was investigated in human eyes by radioimmunoassay and immunofluorescence and compared with that of the rat eye. The source of secretoneurin-positive nerves in the eye was established by measuring the concentration in eye tissues, the trigeminal and superior cervical ganglia both in control rats and in rats treated with capsaicin, and by performing immunofluorescence in one rat subjected to sympathectomy. In the rat trigeminal ganglion, the corresponding mRNA was verified by in situ hybridization and the processing of secretogranin II into secretoneurin by gel filtration chromatography. RESULTS: In human eyes, the highest levels of the peptide were found in the choroid. Nerve fibers were visualized in both species in the upper corneal and limbal stroma; in the trabecular meshwork; in the ciliary muscle, the ciliary body stroma, and processes; and in clear association with the dilator muscle, which disappeared after sympathetic denervation in rats; and also innervating the sphincter muscle in the iris and the choroidal stroma and surrounding blood vessels. Significant amounts of secretoneurin were present in the rat trigeminal ganglion and rat eye tissues. Capsaicin pretreatment led to a 57.0% +/- 4.3% and 59.1% +/- 11.9% decrease of the concentration in the trigeminal ganglion and the iris/ciliary body complex, respectively. Despite high levels in the rat superior cervical ganglion, sympathetic denervation failed to lower the concentration in eye tissues. The secretogranin II probe labeled numerous small-sized ganglion cells within the rat trigeminal ganglion, and the precursor of the peptide was found to become completely processed into secretoneurin. CONCLUSIONS: Apart from the sympathetically innervated dilator muscle, there is unequivocal evidence that secretoneurin represents a constituent of capsaicin-sensitive sensory neurons in the rat trigeminal ganglion and of unmyelinated C-fibers in the rat iris/ciliary body complex, which indicates a participation of this peptide in the ocular irritative response, a model for neurogenic inflammation in lower mammals. Because of the association of nerves with blood vessels and potent angiogenic properties, secretoneurin may be involved in neovascularization processes.

Angioarchitecture and innervation of the primate anterior episclera.

PURPOSE: To investigate the primate episcleral vasculature and its innervation with respect to morphological specializations. METHODS: Serial sections of the anterior episclera of 8 monkey eyes and 20 human eyes were investigated enzyme- and immunohistochemically using antibodies against smooth-muscle alpha-actin (SMA), neurofilament, synaptophysin, substance P (SP), calcitonin gene-related peptide (CGRP), vesicular acetylcholine transporter (VACHT), vasoactive intestinal polypeptide (VIP), neuropeptide Y (NPY), tyrosine hydroxylase (TH), vesicular monoamine transporter II (VMAT II), as well as the NADPH-diaphorase reaction. Arteriovenous anastomoses (AVA) were quantified. RESULTS: All episcleral vessels including veins showed intense staining for SMA. Capillary loops were only seen in the limbal arcades, not in the episclera itself. Instead, AVA connected the episcleral arteries with the veins, which formed an interlacing vascular network. In the monkey episclera, 4-6/mm2 AVA were found; in the human episclera, 0.5-1/mm2. Numerous nerve endings staining for NADPHd (NADPHdiaphorase) and TH surrounded all episcleral vessels including anastomoses and veins. NPY, VIP, and VACHT-immunoreactive (IR) nerve terminals were less numerous. CGRP and SP-IR terminals were seen both at the vessels and in the intervascular connective tissue. CONCLUSIONS: The episcleral vasculature shows a specialized morphology with absence of capillaries, numerous arteriovenous anastomoses, a muscle-rich venous network, and intense innervation by vasodilative and vasoconstrictive nerves. This might allow regulation of blood flow and volume in the episcleral vessels and Voigt's capillaries for thermoregulation and modulation of episcleral venous pressure and thereby outflow facility.

Innervation of myofibroblast-like scleral spur cells in human monkey eyes.

PURPOSE: To study the innervation of the presumably contractile, myofibroblast-like scleral spur cells in human and cynomolgus monkey eyes. METHODS: Serial tangential sections of the scleral spur region of the eyes of 16 human donors and 6 cynomolgus monkeys were investigated with immunocytochemical methods. Antibodies against acetylcholinesterase, synaptophysin, alpha-smooth muscle actin, calcitonin gene-related peptide (CGRP), neuropeptide Y (NPY), nitric oxide synthase (NOS), substance P (SP), tyrosine hydroxylase (TH), and vasoactive intestinal peptide (VIP) were used. In addition, sections were processed for glyoxylic acid-induced catecholamine fluorescence (CF) and for NADPH-diaphorase (NADPH-d). RESULTS: In the eyes of both species, circumferentially oriented varicose axons were observed in the scleral spur region of all quadrants. Double labeling showed that most of these scleral spur axons were in close contact with the alpha-smooth muscle actin-positive, myofibroblast-like scleral spur cells. In human eyes, the axons showed like-immunoreactivity (LI) for SP, CGRP, NPY, VIP, and NOS. In addition, numerous scleral spur axons stained for NADPH-d. Most SP-LI scleral spur axons were double-labeled for CGRP-LI, and none for VIP-LI. All NPY-LI scleral spur axons were double labeled for VIP-LI but lacked immunoreactivity to TH. Some VIP-LI axons were not labeled for NPY-LI. Nerve fibers immunoreactive (IR) for TH or positively stained for CF were not observed in association with scleral spur cells. In contrast, in cynomolgus monkey eyes, circumferentially oriented TH-IR and CF-positive varicose axons were observed frequently in the scleral spur region. In addition, SP-LI, CGRP-LI, and NPY-LI/TH-IR axons were present in the chamber angle of monkey eyes, whereas VIP-LI, VIP-LI/NPY-LI, NOS-positive, or NADPH-d-positive nerve fibers were absent. In both species, positive staining for acetylcholinesterase was seen only in the ciliary muscle, not in the scleral spur region. CONCLUSIONS: The close association of varicose axons with the myofibroblast-like scleral spur cells indicates that nervous signals modulate scleral spur cell tone. A sympathetic scleral spur cell innervation is present only in cynomolgus monkeys but seems to be absent in humans. Conversely, scleral spur axons of presumably parasympathetic origin (NOS-IR or NADPH-d-positive, VIP-LI, and VIP-LI/NPY-LI) are absent in the cynomolgus monkeys but present in humans. In both species, a cholinergic innervation of the scleral spur cells seems to be rare or absent.

Ocular amyloid deposition in familial amyloidosis, Finnish: an analysis of native and variant gelsolin in Meretoja's syndrome.

PURPOSE: To analyze the deposition of amyloid and its precursors in eyes of patients with familial amyloidosis, Finnish (FAF; Meretoja's syndrome), a hereditary systemic amyloidosis. METHODS: Autopsy eyes from three patients with FAF and ten control eyes were studied by Congo red staining and with antibodies to the nonmutated part of gelsolin (GS-2C4), the mutated gelsolin Asn-187 fragment (AGel), and amyloid-P component (AP). RESULTS: Congo red and antisera to AP and AGel bound to amyloid deposits in the cornea and conjunctiva, the sclera, the perineurium of ciliary nerves, the walls of ciliary vessels, the optic nerve sheaths, the stroma of the ciliary body, and along the choriocapillaris. mAb GS-2C4 bound weakly and focally to most deposits and strongly around the choriocapillaris. It labeled the corneal epithelium and endothelium, keratocytes, scleral fibroblasts, trabecular and lens epithelial cells, the ciliary muscle and epithelium, the iris sphincter and dilator, and stromal cells of the conjunctiva and uveal tract. CONCLUSIONS: Local production, especially in the cornea, conjunctiva, sclera, and ciliary muscle, and systemic deposition, particularly in blood vessles and in the sclera, may contribute to amyloid deposits in FAF. To explain the complex pattern of deposition, microenvironmental factors such as lamellar architecture of the cornea and sclera, altered processing of gelsolin, or blood-tissue barriers must be invoked. In addition to corneal lattice dystrophy type II, the observed deposits help to explain glaucoma in patients with FAF.

The allocation of nerve fibres to the anterior eye segment and peripheral ganglia of rats. I. The sensory innervation.

The distribution of sensory trigeminal nerve fibres in the anterior eye segment and the autonomic eye related ganglia, i.e. the parasympathetic ciliary and pterygopalatine ganglia and the sympathetic superior cervical ganglion, was studied in rats. For this the trigeminal ganglion was injected with tritiated leucine and wheat germ agglutinin coupled to horseradish peroxidase (WGA-HRP). After injection of WGA-HRP into the trigeminal ganglion, ganglion cell somata in the superior cervical and the pterygopalatine ganglion were labelled. As labelling of these cell bodies with WGA-HRP is the result of retrograde transport it must be assumed that cell bodies in these ganglia project to the trigeminal ganglion. [3H]Leucine injection into the trigeminal ganglion revealed the presence of labelled nerve fibres in the pterygopalatine ganglion and the nodose ganglion i.e. the sensory ganglion of the vagus nerve. Labelled nerve fibres were absent in the ciliary and superior cervical ganglion. As [3H]leucine labelling of nerve fibres is the result of anterograde transport exclusively, it can be concluded that trigeminal nerve fibres project to the nodose ganglion and the pterygopalatine ganglion, but not to the ciliary and superior cervical ganglion. In the retrobulbar structures, sensory nerve fibres occurred between the inferior oblique and the lateral rectus muscle and were present medial to the medial rectus muscle. Within the anterior eye segment, sensory nerve fibres were found in the cornea epithelium, stroma and adjacent to the endothelium. In addition, labelled fibres were found in the anterior stroma of the ciliary body, throughout the iris up to the pupillary border and in the conjunctiva. Most sensory nerve fibres which innervate the cornea, the iris and the ciliary body traverse the ciliary cleft.

Immunohistochemical localization of dopamine-beta-hydroxylase in human and monkey eyes.

PURPOSE: To investigate the pattern of dopamine-beta-hydroxylase (DBH)-containing fibers in human and monkey eyes. METHODS: DBH-containing fibers were detected by immunohistochemistry. The primary antibody used recognized DBH, the key enzyme in the conversion of dopamine to noradrenaline. RESULTS: In the anterior segment, DBH immunoreaction product was found in the peripheral corneal endothelium layer, in both the dilator and sphincter muscles of the iris, as well as in the anterior border layer of the iris. The ciliary muscle and the stroma of the ciliary processes were also zones of concentration. In the posterior segment, staining was seen around blood vessels in the choroid, in the vascular walls of the short posterior ciliary arteries and in the ciliary nerves. The retina was also immunopositive, with specific labeling in cones and rods of photoreceptors, inner and outer plexiform layers and ganglion cell layer. There was no significant difference in the distribution of DBH-related immunoreactivity in human and monkey eyes. CONCLUSIONS: The localization of DBH-related immunoreactivity is generally consistent with the known physiological roles of noradrenaline. However, an apparently high concentration of the enzyme in the anterior border layer of the iris and in retinal photoreceptors raises questions about the possible role of DBH-containing fibers in these structures.

Irritation of the anterior segment of the eye by ultraviolet radiation: influence of nerve blockade and calcium antagonists.

The aim of this investigation was to determine the influence on anterior segment inflammation elicited by UV radiation, of ocular denervation and pharmacological blockade of sensory nerve fibers with capsaicin, tetrodotoxin and calcium antagonists. Both eyes of pigmented rabbits were exposed for 5 min to UV radiation (254 nm); 24 h later, inflammatory signs were evaluated by biomicroscopy of the corneal epithelium, the stroma and the endothelium and scored from 0 to 4. Conjunctival vasodilation and miosis were also assessed. Two weeks before UV exposure, a group of rabbits received a retrobulbar injection of ethanol or of 1% capsaicin. Intact, capsaicin-treated and alcohol-denervated animals were treated topically, prior to UV exposure, with tetrodotoxin (0.78 mM) and the calcium antagonists diltiazem (1-28 mM) and nifedipine (10 mM). UV radiation produced at 24 h signs of corneal irritation, conjunctival hyperemia, miosis and elevated protein content of the aqueous humor. Retrobulbar injection of 99% alcohol or 1% capsaicin did not diminish significantly the inflammation of tissues directly exposed to UV radiation, although extension of inflammatory signs to unaffected areas was prevented. Pre-treatment of normal and denervated eyes with diltiazem attenuated UV-induced eye irritation signs at concentrations of 10 mM or over. The effect was less pronounced with tetrodotoxin and was not obtained with nifedipine. These findings suggest that the contribution of a neurogenic mechanism to anterior segment inflammation induced by UV exposure is modest. They also show that high concentrations of diltiazem, but not of nifedipine, effectively reduced inflammation of the anterior segment of the eye evoked by UV radiation.(ABSTRACT TRUNCATED AT 250 WORDS)

Autonomic nerves containing substance P in the aqueous outflow channels and scleral spur of the guinea pig.

PURPOSE: To study the innervation of the aqueous outflow channels and scleral spur by autonomic nerves containing substance P. METHODS: The experiments were conducted on guinea pigs. Immunohistochemical techniques and capsaicin-ablation of the sensory nerves were used to investigate nerves containing substance P at the light and electron microscopic level. RESULTS: Nerves containing substance P were observed in the aqueous outflow channels and scleral spur regions. The fine structures of these nerves had a similar pattern in those regions, and the labeled elements had abundant small vesicles, a few large vesicles, and numerous neurotubuli. Following capsaicin treatment, these nerves remained intact and no degenerated substance P-like immunoreactive nerves were found. CONCLUSIONS: Nerves containing substance P are most likely of autonomic origin in view of their ultrastructural features. These nerves innervate the aqueous outflow channels and scleral spur, and are probably important for neurogenic influences on the intraocular pressure by the autonomic nervous system.

Substance P and opioid peptidergic innervation of the anterior eye segment of the rat: an immunohistochemical study.

Recently discovered endogenous opioid peptides such as nociceptin are known to modulate neurotransmitter release of primary afferent neurons (especially substance P, SP) and they have also been demonstrated in peripheral nerve fibres. The aim of this study was to investigate the opioid peptidergic innervation of the anterior eye segment and to compare it with the innervation pattern of SP in order to shed light on the functional relationship between these peptides. Anterior eye segments of 20 rat eyes were cut in a tangential plane and the sections stained with antibodies against SP, nociceptin, nocistatin, endomorphin 1 and 2, leu-enkephalin and met-enkephalin. Sections of the spinal cord or brain were used as positive controls. Numerous SP-immunoreactive nerve fibres were found in the conjunctiva, cornea, episclera, trabecular meshwork, iris and ciliary body. A weak staining for met-enkephalin and leu-enkephalin could only be found in the iris and anteriormost ciliary body. Nerve fibres immunoreactive for nociceptin, nocistatin, and endomorphin 1 or 2 could not be detected in any part of the anterior eye segment. It is tempting to speculate that the opioid peptidergic innervation of the anterior ciliary body may play a role in the modulation of intraocular inflammation.

Peripheral neural circuits regulating IOP? A review of its anatomical backbone.

The peripheral nervous system is classically separated into a somatic division containing both afferent and efferent pathways and an autonomic division composed of efferents only. The somatic afferent division is divided in A- and B-neurons. The B-neurons are supposed to be autonomic afferents as part of a reflex system involved in homeostasis. Recent data obtained by neuronal tracing and immunohistochemical experiments concerning the eye related peripheral nervous system endorse the existence of these peripheral reflex systems. Somatic afferents of trigeminal origin synaptically innervate parasympathetic neurons in the pterygopalatine ganglion. This probably represents a pathway mediating autonomically regulated ocular activity in response to sensory stimulation. In addition, it has been hypothesized that trigeminal sensory nerve fibres have an efferent function in response to noxious stimuli e.g. the ocular injury response. Sympathetic nerve fibres originating in the superior cervical ganglion course through the trigeminal and pterygopalatine ganglion without forming direct synaptic contacts. These fibres, however, contain clusters of vesicles suggesting some kind of interneural communication. Parasympathetic nerve fibres of pterygopalatine origin course through the ciliary ganglion. These nerve fibre terminals also contain clusters of vesicles without direct synaptic contacts. Experimental data concerning the distribution of neuropeptides revealed a more detailed knowledge of the anterior eye segment innervation. These experimental data are subject to some debate. The pros and cons of different techniques are discussed. Neural circuits regulating IOP have long been postulated. The possible role of peripheral reflex systems in the regulation of IOP is discussed.

Review: mediation of the ocular response to injury.

The structure of the anterior segment of the eye provide aqueous humour for metabolic traffic, regulation of intraocular pressure and the maintenance of a functional permeability barrier to separate internal compartments from general systemic influences. Irritative and injurous insults to the eye elicit an acute defensive miotic and vascular response which upsets the aqueous dynamics and provokes the influx of blood plasma proteins into the aqueous chambers. These events are initiated by antidromic activation of sensory elements within the anterior segment, releasing substance P and calcitonin gene-related peptide (CGRP) which, in lower mammals at least, stimulate respectively the miotic and vascular reactions. Considerable species differences can be found in the responsiveness of the eye to injury and in the effects of exogenous CGRP and substance P.

Sympathetic innervation of the rat's eye and peripheral ganglia: an electron microscopic autoradiographic tracing study.

The sympathetic innervation of the rat anterior eye segment and related peripheral ganglia was studied using light and electron microscopic autoradiography after anterograde tracing with 3H-leucine from the superior cervical ganglion. In the trigeminal and pterygopalatine ganglia, unmyelinated nerve fibers were labeled. Some fibers contained accumulations of small vesicles. Close apposition of these labeled sympathetic fibers with other unmyelinated fibers was common, and was also observed at sites where accumulations of vesicles were found. In the iris, ciliary body and trabeculum, numerous fibers and vesicle-containing varicosities were labeled, which all had a similar morphology. No labeling was found in the cornea. Sympathetic fibers traversing the trigeminal and pterygopalatine ganglion closely appose other unmyelinated fibers and contain clusters of vesicles without forming classical synaptic contacts. However, non-synaptic information transfer needs further investigation. The anterior eye segment, except for the cornea, is densely innervated by sympathetic nerve terminals.

Anterior segment autonomic dysfunction in ocular hypertension.

Autonomic nerve function in the anterior segment of the eye was assessed by measurement of the pupil cycle time in 189 patients with ocular hypertension and 70 age- and sex-matched control subjects. Parasympathetic dysfunction was significantly present in ocular hypertensive patients with narrow irido-corneal angles, though not in wide-angle subjects. The results are discussed, with particular reference to the accumulating evidence implicating autonomic dysfunction as a significant factor in the pathogenesis of the primary glaucomas.

Autonomic denervation hypersensitivity in the primary glaucomas.

Autonomic denervation hypersensitivity of the iris was assessed in 44 patients with primary closed-angle glaucoma, 20 patients with primary open-angle glaucoma, and 40 age- and sex-matched control subjects. Significantly increased pupillary constriction in response to 2.5% methacholine chloride, indicating parasympathetic denervation hypersensitivity, was present in both closed-angle (p less than 0.02) and open-angle glaucoma patients (p less than 0.001), compared with the control group. Significantly increased pupillary dilatation in response to 0.5 per cent phenylephrine hydrochloride, indicating sympathetic denervation hypersensitivity of the iris, was also present in both closed-angle glaucoma (p less than 0.01) and open-angle glaucoma patients (p less than 0.05). The association between autonomic neuropathy and the primary glaucomas is discussed, with particular reference to the potential effects of autonomic dysfunction on aqueous dynamics.