PurposeThis study was designed to compare and contrast quantitative data of the human corneal sub-basal nerve plexus (SBP) evaluated by two different methods: in vivo confocal microscopy (IVCM), and ...immunohistochemical staining of ex vivo donor corneas.MethodsSeven parameters of the SBP in large-scale IVCM mosaicking images from healthy subjects were compared with the identical parameters in ex vivo donor corneas stained by β-III-tubulin immunohistochemistry. Corneal nerve fiber length (CNFL), corneal nerve fiber density (CNFD), corneal nerve branch density (CNBD), average weighted corneal nerve fiber tortuosity (CNFTo), corneal nerve connection points (CNCP), average corneal nerve single-fiber length (CNSFL), and average weighted corneal nerve fiber thickness (CNFTh) were calculated using a dedicated, published algorithm and compared.ResultsOur experiments showed significantly higher values for CNFL (50.2 vs 21.4 mm/mm
), CNFD (1358.8 vs 277.3 nerve fibers/mm
), CNBD (847.6 vs 163.5 branches/mm
), CNFTo (0.095 vs 0.081 μm
), and CNCP (49.4 vs 21.6 connections/mm
) in histologically staining specimens compared with IVCM images. In contrast, CNSFL values were higher in IVCM images than in histological specimens (32.1 vs 74.1 μm). No significant difference was observed in CNFTh (2.22 vs 2.20 μm) between the two groups.ConclusionsThe results of this study have shown that IVCM has an inherently lower resolution compared with ex vivo immunohistochemical staining of the corneal SBP and that this limitation leads to a systematic underestimation of several SBP parameters. Despite this shortcoming, IVCM is a vital clinical tool for in vivo characterization, quantitative clinical imaging, and evaluation of the human corneal SBP.
In the 1990s the Swiss agricultural policy was reformed and new environmental objectives were formulated. The aims of the reform were to halt the loss of agro-biodiversity and to enable the spread of ...endangered species. As a result, the utilised agricultural area (UAA) is now interspersed with low input ecological compensation areas (ECA), making up 13% of the UAA (extensified grassland 90,000
ha, traditional orchards 25,000
ha, hedgerows 3000
ha, other elements 23,000
ha). To assess whether ECA contribute to the enhancement of biodiversity, plant composition was recorded on 1914 ECA of the Swiss plateau and 1966 territories of 27 bird species, which typically breed in open and semi-open farmland, were mapped and related to ECA. Eighty-six percent of ECA litter meadows and 50% of ECA hedgerows were of good ecological quality and attracted wetland and hedgerow birds. Most ECA hay meadows and traditional orchards, on the other hand, still reflected their former intensive management with only 20 and 12%, respectively, being of good ecological quality. Hardly any benefits for grassland and orchard birds were observed. Ecological quality of ECA was generally higher in the bio-geographical region ‘Basin of Lake Geneva and Upper Rhine Valley’ than in the other two regions of the Swiss plateau and it was higher in the agricultural production zone ‘Pre-alpine Hills’ than in the ‘Lowland Zone’.
Summary
Assessment of various morphological parameters of the corneal subbasal nerve plexus is a valuable method of documenting the structural and presumably functional integrity of the corneal ...innervation in health and disease. The aim of this work is to establish a rapid, reliable and reproducible method for visualization of the human corneal SBP using femtosecond laser cut corneal tissue sections. Trephined healthy corneal buttons were fixed and processed using TissueSurgeon—a femtosecond laser based microtome, to obtain thick tissue sections of the corneal epithelium and anterior stroma cut parallel to the ocular surface within approximately 15 min. A near infrared femtosecond laser was focused on to the cornea approximately 70–90 μm from the anterior surface to induce material separation using TissueSurgeon. The obtained corneal sections were stained following standard immunohistochemical procedures with anti‐neuronal β‐III tubulin antibody for visualization of the corneal nerves. Sections that contained the epithelium and approximately 20–30 μm of anterior stroma yielded excellent visualisation of the SBP with minimal optical interference from underlying stromal nerves. In conclusion, the results of this study have demonstrated that femtosecond laser cutting of the human cornea offers greater speed, ease and reliability than standard tissue preparation methods for obtaining high quality thick sections of the anterior cornea cut parallel to the ocular surface.
Lay description
Human cornea is one of the most highly innervated tissues in the body. Sectioning of the cornea for the quantitative analysis of its innervation by immunohistochemistry is necessary during health and disease. Attempts to obtain frozen or paraffin‐sections of whole corneas parallel to the plane of its innervation are technically challenging and also time consuming due to the curvature of the tissue. In an effort to overcome this difficulty, here we report a contact‐free and rapid femtosecond laser based cutting method to obtain high quality surface parallel sections of the human cornea in less than 15 min. Furthermore, femtosecond based laser microtomy can be adopted easily for sectioning of all kind of biological samples including hard tissues (teeth and bone) and offers a more standardized way of tissue sectioning by avoiding the laborious processes of sample preparation and embedding thereby preventing the loss of valuable tissue sample.
Corneal nerves: structure, contents and function Müller, Linda J.; Marfurt, Carl F.; Kruse, Friedrich ...
Experimental Eye Research,
05/2003, Letnik:
76, Številka:
5
Book Review, Journal Article
Recenzirano
This review provides a comprehensive analysis of the structure, neurochemical content, and functions of corneal nerves, with special emphasis on human corneal nerves. A revised interpretation of ...human corneal nerve architecture is presented based on recent observations obtained by in vivo confocal microscopy (IVCM), immunohistochemistry, and ultrastructural analyses of serial-sectioned human corneas. Current data on the neurotransmitter and neuropeptide contents of corneal nerves are discussed, as are the mechanisms by which corneal neurochemicals and associated neurotrophins modulate corneal physiology, homeostasis and wound healing. The results of recent clinical studies of topically applied neuropeptides and neurotrophins to treat neurotrophic keratitis are reviewed. Recommendations for using IVCM to evaluate corneal nerves in health and disease are presented.
To determine the architectural pattern and neuropeptide content of canine corneal innervation.
Corneal nerve fibers in normal dog eyes were labeled immunohistochemically with antibodies against ...protein gene product (PGP)-9.5, calcitonin gene-related peptide (CGRP), substance P (SP), vasoactive intestinal polypeptide (VIP), and tyrosine hydroxylase (TH). Relative innervation densities and distribution patterns for each fiber population were assessed qualitatively by serial line-drawing reconstructions and quantitatively by computer-assisted analyses.
More than 99% of all corneal PGP-9.5-immunoreactive (IR) nerves contained both CGRP and SP, approximately 30% contained TH, and none contained VIP. Distribution patterns of corneal PGP-9.5-, CGRP-, SP-, and TH-IR nerves were indistinguishable, except that TH-IR fibers were absent from the corneal epithelium. Morphologically, canine corneal innervation consisted of a rich anterior stromal plexus, divided on the basis of morphologic criteria into anterior and posterior levels, and a rich epithelial innervation, characterized by large numbers of horizontally oriented, basal epithelial "leash" formations. Leash axons in all quadrants of the corneal epithelium oriented preferentially toward a common locus in the perilimbal cornea.
The results of this study demonstrate for the first time the detailed architectural features, distinctive basal epithelial leash orientations, and peptidergic content of canine corneal innervation. The normal innervation pattern described in this study will provide other investigators with essential baseline data for assessing corneal nerve alterations in canine patients with spontaneous chronic corneal epithelial defects (SCCED) and other ocular diseases or injuries.
To delineate the clinical features and alterations in innervation and substance P (SP) content in spontaneous chronic corneal epithelial defects (SCCED) in dogs and to conduct a preliminary ...investigation evaluating the efficacy of topical SP, with or without insulin-like growth factor (IGF)-1, in the treatment of this disorder.
Complete ophthalmic examinations, including Cochet-Bonnet aesthesiometry, were performed in 45 canine patients that had spontaneous corneal epithelial defects of at least 3 weeks' duration and with no identifiable cause. Eighteen patients had superficial keratectomies performed, and the corneal nerves were labeled immunohistochemically with antibodies against protein gene product (PGP)-9.5, SP, vasoactive intestinal peptide (VIP), and tyrosine hydroxylase (TH). Relative fiber densities were assessed qualitatively and quantitatively. Corneal epithelial cell and tear SP contents were determined in affected and normal dogs by an enzyme immunoassay. A preliminary open-label treatment trial of topical SP, with and without IGF-1, was conducted in 21 dogs.
The duration of the erosion before admittance into the study was a mean of 9.22 weeks (range, 3-52). The average patient was middle aged (mean, 9.25 +/- 1.85 years SD); no sex predisposition of the disease was identified. Boxers, golden retrievers, and keeshonds were overrepresented when compared with the normal hospital population. Corneal sensation was normal. Marked alterations in corneal innervation were identified in affected dogs with abnormal increased SP and calcitonin gene-related peptide (CGRP)-immunoreactive nerve plexuses identified surrounding the periphery of the epithelial defect. The SP content of epithelial cells surrounding the defect increased, whereas the tear SP content remained unchanged. Of the canine patients treated with SP, with or without IGF-1, 70% to 75% had complete healing of the defect.
This idiopathic spontaneous corneal disease in dogs shares clinical features with chronic epithelial defects in humans. The presence of marked alterations in peptidergic innervation and positive response to topical therapy with SP suggest that SP plays a critical role in corneal wound-healing processes.
Corneal nerves regulate corneal epithelial integrity, proliferation, and wound healing. The mechanisms by which the nerves mediate their effects remain poorly understood; however, the release of ...biologically active neuropeptides has been hypothesized. The purpose of the current investigation was to determine the relative densities, distribution patterns, and origins of rat corneal nerves containing each of eight different neuropeptides, calcitonin gene-related peptide (CGRP), substance P (SP), galanin (GAL), neuropeptide Y (NPY), methionine-enkephalin (M-ENK), vasoactive intestinal polypeptide (VIP), somatostatin (SOM), and cholecystokinin (CCK). In the first set of experiments, immunohistochemical demonstrations of the above neuropeptides were performed on free-floating corneal sections cut tangential to the corneal surface. The results showed that six of the peptides, CGRP, SP, GAL, NPY, M-ENK, and VIP were present in rat corneal nerves. The innervation patterns of corneal nerves containing each of these six peptides were then documented by mapping all fibers in serial sections from select corneal quadrants onto a series of line drawings by using a drawing tube. In the second set of experiments, the origins of the corneal peptidergic nerve fibers were determined by selective ocular denervations. Unilateral combined sensory and sympathetic ocular denervations or unilateral sympathetic ocular denervations were performed in adult rats by transecting the ophthalmomaxillary nerve and/or removing the superior cervical ganglion. After 5–7 days, each of the ipsilateral corneas was sectioned and processed immunohistochemically for the presence of one of the six peptides found in experiment one, and the fibers that survived the ocular denervations were plotted onto line drawings. Ocular denervations revealed that corneal peptidergic nerves have sensory (CGRP, SP, and GAL), sympathetic (NPY), and parasympathetic (GAL, NPY, M-ENK, and VIP) origins. The results of this investigation have shown that the peptidergic innervation of the rat cornea is more extensive and complex than previously reported. This is the first investigation to show the presence of GAL in the rat cornea, and the first to demonstrate the presence of NPY-, VIP-, and M-ENK-IR nerve fibers in the cornea of any species.
Abstract
Background
In 2022, a free-of-charge screening to eliminate the HCV virus circulation was offered countrywide to the citizens born from 1969 to 1989. Recent studies reported low adherence ...rates to HCV screening campaigns, highlighting the need for policy makers to know the determinants of adherence, aimed to set up more effective communication campaigns.
Methods
A target population random sample answered by phone a questionnaire based on the Health Belief Model (HBF), containing 4 sections on perceived HCV susceptibility and severity, screening benefits and barriers. Each interviewed was finally invited to adhere to the screening. Prevalence was estimated using a 95% C.I., and association between sociodemographic characteristics, HBF attitudes, and screening adherence following the interview were assessed.
Results
Survey involved 641 participants. HCV susceptibility was perceived by 4.8% (C.I.95% 3,23-6,57) and HCV severity by 69,4% (I.C.95% 65,83-72,97), 95% (C.I.95% 93,3-96,6) acknowledged screening benefits and 88% (85,4-90,5) perceived no obstacles. It was found that 49.0% (C.I.95% 45,1-52,8) received no information. Perception of screening barriers was associated with low education level (OR 1.83, P = 0.02). In the 2 months following the interview, 17.6% (C.I.95% 14,6-20,5) carried out the HCV screening. Severity (OR 1.74, P = 0.03) and no barriers perception (OR 2.63, P = 0.01) were associated factors to post phone call adherence to UCV screening.
Conclusions
Insufficient communication campaign toward target population results in poor knowledge of HCV susceptibility and gravity and low adherence to HCV screening. Target population needs to acquire better awareness about HCV and HCV screening, through targeted and effective messages especially focusing on the susceptibility and gravity of HCV infection.
Key messages
• Target population does not feel susceptible to hepatitis c and has poor perception of severity.
• It is necessary to improve communication through effective messages.
The peptidergic and serotoninergic innervation of the rat dura mater was investigated by reacting dural wholemounts immunohistochemically with antibodies to calcitonin gene-related peptide (CGRP), ...substance P (SP), neuropeptide Y (NPY), vasoactive intestinal polypeptide (VIP), and serotonin (5-HT). CGRP and SP innervations of the dura were robust and the patterns of distribution of these neuropeptides were essentially the same. The majority of the fibers were perivascular and distributed to branches of the anterior and middle meningeal arteries and to the superior sagittal and transverse sinuses. Other CGRP/SP fibers appeared to end "free" within the dural connective tissue. NPY-immunoreactive fibers were extremely numerous and also distributed heavily to the branches of the meningeal arteries, the venous sinuses, and to the dural connective tissue. The pattern of NPY innervation resembled in many ways that of CGRP/SP; however, NPY innervation of the sinuses was greater and NPY perivascular fibers supplying the meningeal arteries formed more intimate contacts with the walls of the vessels. The pattern of VIP innervation was, in general, similar to that observed for the three previous neuropeptides; however, the overall density was considerably less. Small to moderate numbers of serotoninergic nerve fibers were observed in some, but not all, of the duras processed for 5-HT. The latter fibers were almost exclusively perivascular in distribution. Dural mast cells were prominently stained in the 5-HT preparations because of their serotonin content. Mast cells were also labeled in a nonspecific fashion in some of the tissues reacted immunohistochemically for neuropeptides; some of them were located in close apposition to passing nerve fibers. This study represents, to our knowledge, the first comprehensive work on the peptidergic and serotoninergic innervation of the mammalian dura mater. The results should increase our understanding of the roles that these fibers play in normal dural physiology and of their potential interactions in the pathogenesis of vascular headache.
To determine the effect of ocular sympathetic nerves on corneal epithelial proliferation in the rat.
Osmotic pumps filled with bromodeoxyuridine (BrdU) were implanted subcutaneously in adult rats to ...label corneal epithelial cells entering the S-phase of the cell cycle during a 24-hour period. Corneas in some animals were wounded with n-heptanol. Several days or weeks before pump implantation, animals were subjected to either unilateral superior cervical ganglionectomy (SCGectomy), unilateral transection of the cervical sympathetic trunk (sympathetic decentralization), bilateral SCGectomy plus unilateral topical norepinephrine administration, or unilateral SCGectomy plus systemic capsaicin administration. Differences in BrdU-labeling indices between experimental and control eyes in each group were determined from cell counts on paraffin sections.
Superior cervical ganglionectomy and sympathetic decentralization significantly decreased epithelial proliferation in nonwounded and wounded corneas. Topical applications of norepinephrine to eyes that had been deprived of their sympathetic innervation completely reversed the antiproliferative effect of ocular sympathectomy. Systemic administration of the neurotoxin capsaicin, in conjunction with unilateral SCGectomy, did not alter the proliferative rate; comparison was made to animals that received unilateral SCGectomy only.
Ocular sympathetic nerves stimulate rat corneal epithelial proliferation under normal physiological conditions and during corneal wound healing. The promotion of DNA synthesis by these nerves occurs independently of functional interactions with capsaicin-sensitive, ocular sensory nerves and appears to be related, at least in part, to the release of norepinephrine.
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