Neuromediatorii in Ochiul Uscat

8/11/2019 Neuromediatorii in Ochiul Uscat

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CLINICAL SCIENCES

Alterations of Tear Neuromediatorsin Dry Eye Disease

Alessandro Lambiase, MD, PhD; Alessandra Micera, PhD; Marta Sacchetti, MD, PhD;Magdalena Cortes, MD; Flavio Mantelli, MD; Stefano Bonini, MD

Objectives:To evaluate tear levels of neuromediatorsin patients with dry eye disease and to identify statisti-cal correlations with the clinical findings.

Methods: Nineteen patientswith dry eyedisease(Sjogrensyndrome, n=5 patients; nonSjogren syndrome, n=10;and ocular cicatricial pemphigoid, n=4) and 12 healthyvolunteers were enrolled.The eyes of all participantswere

evaluatedby slitlamp examination, Schirmer testing, fluo-rescein staining, and tear film break-up time. Grading ofdry eye severity was recorded. Tear samples were col-lected, and substance P, calcitonin generelated peptide(CGRP), neuropeptide Y (NPY), vasoactive intestinal pep-tide, and nerve growth factor (NGF) concentrations wereevaluated by enzyme-linked immunoassay and corre-lated with the clinical findings.

Results: Nerve growth factor tear levels were signifi-cantly increased in participants with dry eyedisease; CGRPand NPY concentrations weresignificantly decreased when

compared with those in healthy participants. Dry eye se-verity showed a direct correlation with NGF and an in-verse correlation with CGRP and NPY tear levels. Nervegrowth factor tear levels showed a direct correlation withconjunctival hyperemia and fluorescein staining results,CGRP directlycorrelated with Schirmer testvalues, andNPYinversely correlated with tear film break-up time. Sub-group analysis showed that CGRP and NPY but not NGF

were changed in autoimmune (ie, Sjogren syndrome andocular cicatricial pemphigoid) dry eye disease.

Conclusions: Thedecreased tear levels of NPYand CGRPin dry eye disease are related to impaired lacrimal func-tion, and tear levels of NGF are more closely related tocorneal epithelial damage. Our findings suggest that NPY,CGRP, and NGF could become useful markers of dry eyeseverity.

Arch Ophthalmol. 2011;129(8):981-986

THE OCULAR SURFACE IS EX-tensively supplied by sen-sory and autonomic nervefibers that play a crucial rolein maintaining healthy epi-

thelia and represent the main sources ofneurogenic inflammation.1- 3 Cross-communication between the nervous andimmune systems is shown by the releaseand binding of common neuromediatorsand cytokines.4-6 Specifically, substance P(SP), calcitonin generelated peptide(CGRP), vasoactive intestinal peptide(VIP), and neuropeptide Y (NPY) are re-

leased from ocular surface epithelial cells,lacrimal gland tissues, and nerve endingsat inflammatory sites, modulating the in-filtration and activation of the immunecells andtriggering thereflex of tearing andocular discomfort.7,8

Evidence suggests that neuromedia-tors are involved in chronic ocular sur-face diseases such as dry eye. Dry eye is acomplex disease characterized by changesin the ocular surface related to reduced

quality and/or quantity of tears, inflam-matory reaction, and impairment of ocu-lar surface sensitivity.9,10 Lacrimal glandfunction is regulated by the nervous sys-tem, with autonomic and sensory nervesbeing capable of influencing acinar glandsecretion and inflammatory reactionsthrough the release of neuromedia-tors.8,11,12 Substance P and CGRP pro-mote local inflammationby inducing bloodvessel dilatation, leukocyte extravasa-tion, immune cell activation, and synthe-sis and release of several cytokines.8,13-15

Vasoactive intestinal peptide and NPY ex-

ert anti-inflammatory actions by inhibit-ing T-cell proliferation and helper T-celltype 1 response and modulating the re-lease of cytokines, chemokines, and ni-tric oxide.16-20 This neuroimmune cross-communication is capable of modulatinglocal inflammation by enhancing sensorynerve excitability and triggering the acti-vation of different immune cell types.

Changes in circulating levels of neuro-peptides and neurotrophins, as well as the

Author Affiliations:Department of Ophthalmology,Campus Bio-Medico, Universityof Rome (Drs Lambiase, Micera,Sacchetti, Cortes, Mantelli, andBonini), and Istituto diRecovero e Cura a CarattereScientifico G. B. Bietti EyeFoundation (Dr Micera),Rome, Italy.

ARCH OPHTHALMOL/ VOL 129 (NO. 8), AUG 2011 WWW.ARCHOPHTHALMOL.COM981

2011 American Medical Association. All rights reserved.

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impairment of salivary gland innervation in patients withSjogren syndrome, have been demonstrated.21-24 Thesechanges arehypothesizedto be a cause of salivary fluid flowdecrease during Sjogren syndrome. In fact, a remarkablediscrepancy can be observed between the inflammatory in-volvementof theglands andthe decrease in fluid flow. Onepossibleexplanation is that thisautoimmune diseaseand/orlocal inflammation may cause vasoneural dysregulation ofand injury to the peripheral nerves, leading to decreasedfluid flow andatrophy of the acinar cells.25,26 Patients with

autoimmune diseases are known to have a decrease in cir-culating levels of NPY, and the salivary glands of patientswith Sjogren syndromehave reducedNPY-containing nerveterminals.25 Of interest, an increase in nerve growth factor(NGF) tear levels also has been reported in patients withdry eye disease.27 Nerve growth factor is a neurotrophinexerting a pleiotropic action on the ocular surface, modu-lating immune reaction, sensitivity, corneal and conjunc-tival epithelia proliferation, and differentiation and stimu-lation of mucin production by goblet cells.28

The aim of this study was to measure the tear con-tent of VIP, NPY, CGRP, SP, and NGF in patients af-fected by Sjogren syndrome and nonSjogren syndromedry eye disease compared with that of healthy volun-

teers. Potential correlations with the clinical character-istics of the patients also were evaluated.

METHODS

PARTICIPANTS ANDTEAR SAMPLE COLLECTION

The study was performed in accordance with the Declarationof Helsinki for research involving humans, and the Intramural

Ethics Committee approved the project.Informed consentwaiv-ers were signed by each participant.

Participants included 12 healthy individuals (8 men and 4women; mean [SD] age, 41 [21] years) and 19 patients (4 menand 15 women; 68 [13] years) with dry eye disease due toSjogren syndrome (n = 5), nonSjogren syndrome (n = 10), orocular cicatricial pemphigoid (n= 4), with no history of otherocular diseases.

The diagnosis of dry eye disease was based on clinical his-tory and the results of slitlamp examination, Schirmer testing,ocular surface fluorescein staining, and tear film break-up time.Conjunctivalhyperemia wasgraded as follows: 0, absent; 1, mild;2, moderate; and 3, severe. Corneal staining was recorded andgraded according to the Oxford scheme. Dry eye severity grad-ing from 1 to 4 was recorded according to the Dry Eye Work-Shop classification.29,30 Evaluation by an immunologist and bio-chemical investigations were performed to identify systemicautoimmune diseases.

Tear samples were collected without anesthetic from allparticipants using dry microsponges (Sharp-tip Micro-sponges; Alcon Laboratory Inc, Fort Worth, Texas) insertedsimultaneously in the inferior conjunctival fornix of botheyes, removed after 60 seconds, and then immediately im-

mersed in a 1.5-mL Eppendorf vial containing 50 L of tissueprotein extractor solution (T-PER; Thermo Scientific PierceProtein Research Products, Rockford, Illinois) with 10 g/mLof aprotinin and 1 mM of phenyl methyl sulfonyl fluoride. Mi-crosponges were then centrifuged to dryness at 13 000 rpmfor 3 minutes to recover tears. The amount of fluid recoveredwas determined, as previously described.31 Briefly, it was ob-tained by weighing the sponge and the tube after the absorp-tion and centrifugation steps. The protein profile of the tearswas then recorded (A280 program of a NanoDrop ND-1000UV-Vis Spectrophotometer; NanoDrop Technologies Inc,

Wilmington, Delaware).

Table 1. Characteristics of ParticipantsWith Dry Eye Disease

Characteristic Value

Patients, No. 19

Sex, No.

Male 4Female 15

Age, y

Range 45-82Mean (SD) 68 (13)

Dry eye type, No.

NonSjogren syndrome 10

Sjogren syndrome 5Ocular ci catri cial pemphigoi d 4

Conjunctival hyperemia score

Median (range) 1 (0-2)

Mean (SD) 0.9 (0.7)

Schirmer test, mm/5 min


Mean (SD) 4.4 (3.1)Tear film break-up time, s

Median (range) 4.0 (3.0-5.0)

Mean (SD) 3.7 (1.3)

Oxford score


Mean (SD) 1.8 (1.6)

9.0

7.0

8.0

6.0

3.0

2.0

1.0

5.0

4.0

0SP

NP

YTearLevel,ng/mL

A HealthyDry eye

HealthyDry eye

CGRP NPY VIP

250.0

150.0

100.0

200.0

50.0

0

TearLevel,pg/mL

B

NGF

Figure 1.Changes in tear levels in patients with dry eye disease comparedwith healthy participants. A, Significant decrease in tear levels of calcitoningenerelated peptide (CGRP) (P=.001) and neuropeptide Y (NPY) (P=.01),but not of substance P (SP) and vasoactive intestinal peptide (VIP) inpatients with dry eye. B, Nerve growth factor (NGF) tear concentrationsignificantly increased in patients with dry eye (P=.02).



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Schirmer test and break-up time values) were not sig-nificantly different between subgroups (Table 4).

COMMENT

Dry eye is a multifactorial disease characterized by de-creased tear flow, conjunctival inflammation, impairmentof corneal sensitivity, decrease in goblet cell density, epi-thelial metaplasia, and corneal damage, frequently lead-ing to visual impairment.9 It is well known33 that normaltear flow is regulated by a fine balance between the para-sympathetic, sympathetic, and sensory nerves. In fact, theautonomic and sensory nerves locally release several me-

diators that are able to influence acinar gland secretion aswell as theinflammatory reaction at thelacrimal glandandthe conjunctiva.1 The main causes of this neurogenic in-flammation are considered to be SP and CGRP, both re-leased by the sensory nerves. In this study, we demon-strated a significantdecreaseinCGRP inthetears ofpatientsaffected by dry eye; although not significant, a trend of de-

creasein SPalso was observedin patientswith dryeye. Also,in those patients,we observed a significant decrease in NPYand a significant increase in NGF; changes in VIP levelswere not significant. These results suggest that at leastCGRP,NPY, andNGF areinvolvedin dryeye disease. How-ever, because the source of these factors was not estab-lished in this study, it is unclear whether these alterationsin the tears of patients with dry eye represent a patho-genic mechanism of the disease or whether they may re-sultfromchanges inthe ocular surface ofdry eyes. Wehopethat these aspects will be investigated in studies aimed at

6.0

4.0

3.0

5.0

1.0

2.0

0

TearLevel,ng/mL

A

=

5 mm/5 min

Schirmer Test Values

BUT Values

NPY

CGRP


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Medico, Via Alvaro del Portillo 200, 00128 Rome, Italy([email protected]).Financial Disclosure:None reported.

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Neuromediatorii in Ochiul Uscat