meibomian glands are large sebaceous glands of the eyelids whose lipid secretions are essential for the maintenance , health and integrity of ocular surface .
several studies have reported on the neurotransmitters of the meibomian glands in different species ; in guinea pigs : neuropeptide y , substance p and vasoactive intestinal polypeptide ( vip ) ; in humans : calcitonin gene - related peptide ( cgrp ) and substance p ; in cynomolgus monkeys : substance p , crgp , neuropeptide y and vip ; in rat : nitric oxide , enkephalin , tyrosine hydrolase and dopamine - beta - hydroxylase .
meibomian gland dysfunction is common , but yet often overlooked lid margin disease , as a result of altered or reduced lipid excretion that lead to disruption of the tear film with resultant irritation to the cornea , conjunctiva , and lids .
it may thus be acknowledged that its importance has conceivably been underestimated , and possibly the most frequently cause of dry eye syndrome could be due to increased evaporation of the aqueous tears .
dry eye syndrome affects about 75% of population that may not blink so often ( work with pc , watching tv or reading ) or wear contact lens .
this can also result from chemical burns , different autoimmune diseases , bacteria that may breakdown the oil , or taking oral isotretinoin medications .
toxicity and subtoxicity of vitamins has been associated with overdosing of vitamin supplements , which are added to a variety of food in industrialize countries , or with extremely high doses of multivitamins taken by pregnant women .
vitamin a and its biologically active metabolite , retinoic acid ( ra ) are essential for embryonic development , regulate vital functions of the embryo , modulate neurogenesis , neuronal survival and synaptic plasticity .
it is very important to have the right balance of vitamin a in the maternal diet for a successful developmental outcome , because it can act as a teratogen in critical developmental periods .
excess intake of ra shows remarkable teratogenic similarities to those generated by a lack of ra during prenatal periods . as retinoids play a key role in fin formation , differentiation and maturation of many types of cells , they help reverse the pathogenesis of malignancies .
ra promotes cellular differentiation and can induce regeneration in a number of different organ systems therefore it is used for treatment of skin diseases , cancers , immunotherapy and vaccine strategies .
unfortunately , malformations and set of behavioural anomalies were seen in the human embryo exposed to 13-cis ra during treatment of the mother with the acne drug isotretinoin ( accutane ) .
isotretinoin also alters the meibomian gland function and structure , causing acinar atrophy and hyposecretion of the meibomian oil .
many nerve fibers occur around and within the wall of the small vessels that build up a dense meshwork around the acini .
nitric oxide ( no ) is an unconventional neurotransmitter , free radical , involved in many physiological and pathological processes .
the production of no is indicated immunohistochemically by the demonstration of nitric oxide synthase ( nos ) , the enzyme responsible for no generation .
the presence of constitutive nos ( neuronal and endothelial nos ) may be inferred by positive nadph - d staining method , which is widely used in the investigation of the nervous system .
staining of other structures is non - specific nadph - d stain that is not equivalent to the presence of nos .
previous studies have determined the localization and morphology of the nadph - d positive neural elements of the meibomian glands in adult rats and also during postnatal development , thereby implying participation of no in meibomian glands innervation . in this work
the presence of nadph - d activity in and around the meibomian glands was investigated in order to discover the prenatal effect of excess of ra to its development .
the experimental animals , wistar pups of both genders ( n=18 ) weighing 3035 g and aged 14 days post partum were housed in cages together with their mothers under standard conditions of temperature and lighting .
the animals were handled in accordance with the regulations of the association for research in vision and ophthalmology .
all experiments were conducted in accordance with the committee for ethics on animal experiments at the faculty of medicine , pavol jozef safarik university in kosice , slovakia , and the state veterinary and food administration of the slovak republic no .
all the experiments were made at the same hour daily and season of the year .
experimental animals were divided in two groups : control group and ra group . in the control group 16 eyelids of 14 days old rats with no experimental intervention were studied . in ra group
20 eyelids of rat pups with pre - natal administration of the excess of ra were used .
1 mg / kg of all - trans ra ( sigma - aldrich , r2625 ) , was applied to pregnant wistar rats of ra group intraperitoneally during the gestational period in each of the 1214 embryonic days , totally 3 mg / kg . the upper eyelids were carefully excised after killing animals with an overdose of diethyl ether and stored in the 4% paraformaldehyde + 0,1% glutaraldehyde buffered with 0,1 m sodium phosphate , ph -7,4 for 34 h. the fixatives were freshly made up immediately prior to perfusion .
samples were stored in the above mentioned solution for 24 h , then 1 h in increasing concentrations of sucrose ( 1530% ) and stored overnight at 4c .
both upper eyelids were sampled at their centre and sectioned parasagittaly in a freezing microtome at a thickness of 45 m .
ten sections / eyelid were processed with nadph - d histochemistry using the technique according to scherer - singler et al . , which was modified to suit our laboratory conditions , as was reported in our previous studies .
the sections were incubated for an hour at 37c in a solution containing 1.5 mm nitroblue tetrazolium ( nbt , sigma - aldrich , n6876 ) , 1.0 mm  - nadph ( nadph , sigma - aldrich n1630 ) , 10.0 mm monosodium malate ( malic acid , sigma - aldrich m1125 ) and
0.5% triton x-100 dissolved in 0,1 m phosphate buffer ( ph 7.4 ) . as a control for nadph - d staining we carried out the processing with the omission of the substrate  - nadph in the reaction medium .
sections were rinsed in 0,1 m phosphate buffer ( ph 7,4 ) , mounted on glass slides , air - dried overnight and finally cover slipped with mounting medium entellan ( merck 100869 ) .
the morphologic appearance of the nadph - d positive structures in meibomian glands were evaluated histologically , on slides using light microscope olympus ch20 .
images were obtained with a moticam 2300 camera mounted on the microscope optika b600 ti coupled to a capture board in a computer .
the experimental animals , wistar pups of both genders ( n=18 ) weighing 3035 g and aged 14 days post partum were housed in cages together with their mothers under standard conditions of temperature and lighting .
the animals were handled in accordance with the regulations of the association for research in vision and ophthalmology .
all experiments were conducted in accordance with the committee for ethics on animal experiments at the faculty of medicine , pavol jozef safarik university in kosice , slovakia , and the state veterinary and food administration of the slovak republic no .
all the experiments were made at the same hour daily and season of the year .
experimental animals were divided in two groups : control group and ra group . in the control group 16 eyelids of 14 days old rats with no experimental intervention were studied . in ra group
20 eyelids of rat pups with pre - natal administration of the excess of ra were used .
1 mg / kg of all - trans ra ( sigma - aldrich , r2625 ) , was applied to pregnant wistar rats of ra group intraperitoneally during the gestational period in each of the 1214 embryonic days , totally 3 mg / kg . the upper eyelids were carefully excised after killing animals with an overdose of diethyl ether and stored in the 4% paraformaldehyde + 0,1% glutaraldehyde buffered with 0,1 m sodium phosphate , ph -7,4 for 34 h. the fixatives were freshly made up immediately prior to perfusion .
samples were stored in the above mentioned solution for 24 h , then 1 h in increasing concentrations of sucrose ( 1530% ) and stored overnight at 4c .
both upper eyelids were sampled at their centre and sectioned parasagittaly in a freezing microtome at a thickness of 45 m .
ten sections / eyelid were processed with nadph - d histochemistry using the technique according to scherer - singler et al . , which was modified to suit our laboratory conditions , as was reported in our previous studies .
the sections were incubated for an hour at 37c in a solution containing 1.5 mm nitroblue tetrazolium ( nbt , sigma - aldrich , n6876 ) , 1.0 mm  - nadph ( nadph , sigma - aldrich n1630 ) , 10.0 mm monosodium malate ( malic acid , sigma - aldrich m1125 ) and
0.5% triton x-100 dissolved in 0,1 m phosphate buffer ( ph 7.4 ) . as a control for nadph - d staining we carried out the processing with the omission of the substrate  - nadph in the reaction medium .
following the reaction , sections were rinsed in 0,1 m phosphate buffer ( ph 7,4 ) , mounted on glass slides , air - dried overnight and finally cover slipped with mounting medium entellan ( merck 100869 ) .
the morphologic appearance of the nadph - d positive structures in meibomian glands were evaluated histologically , on slides using light microscope olympus ch20 .
images were obtained with a moticam 2300 camera mounted on the microscope optika b600 ti coupled to a capture board in a computer .
nadph - diaphorase histochemical procedure results in the deposition of a dark blue water insoluble formazan reaction product .
we observed nadph - d positive staining in nerve fibers and endothelial cells of blood vessels coursing around the meibomian glands , between acini and in the submucosa .
histochemical analysis of control eyelids revealed more numerous nadph - d well - stained acini of meibomian glands arranged tightly into groups .
darker acini were in preponderance over lightly stained acini , the majority of acini showed a uniform dark blue staining ( figure 1a ) .
intensively stained vessels and nadph - d positive nerve fibers bordered acini of meibomian glands .
these structures were present in the submucosal layer as well ( figure 1b ) . 
 figure 1parasagittal section through the rat upper eyelid on 14 postnatal day , control group , nadph - diaphorase staining .
a ) round shaped acini of meibomian glands ( a ) lie near the conjunctival surface ( c ) of the eyelid ; skin ( s ) ; hairs ( h ) ; scale bar : 500 m .
b ) inset from a : blood vessels accompanied by nerve fibers ( arrows ) are running mostly between acini of the meibomian glands ( a ) and rarely in submucosa ( sm ) ; scale bar : 100 m . 
 
parasagittal section through the rat upper eyelid on 14 postnatal day , control group , nadph - diaphorase staining .
a ) round shaped acini of meibomian glands ( a ) lie near the conjunctival surface ( c ) of the eyelid ; skin ( s ) ; hairs ( h ) ; scale bar : 500 m .
b ) inset from a : blood vessels accompanied by nerve fibers ( arrows ) are running mostly between acini of the meibomian glands ( a ) and rarely in submucosa ( sm ) ; scale bar : 100 m . 

figure 2parasagittal section through the rat upper eyelid on 14 postnatal day , ra group , nadph - diaphorase staining .
a ) shrunken acini of meibomian glands ( a ) lie near the conjunctival surface ( c ) of the eyelid ; skin ( s ) ; hairs ( h ) ; junction between upper and lower eyelid ( j ) ; blood vessel ( v ) ; scale bar : 500 m .
b ) inset from a : blood vessels accompanied by nerve fibers ( arrows ) are running mostly in submucosa ( sm ) ; and rarely between acini of the meibomian glands ( a ) ; scale bar : 100 m . 
 
parasagittal section through the rat upper eyelid on 14 postnatal day , ra group , nadph - diaphorase staining .
a ) shrunken acini of meibomian glands ( a ) lie near the conjunctival surface ( c ) of the eyelid ; skin ( s ) ; hairs ( h ) ; junction between upper and lower eyelid ( j ) ; blood vessel ( v ) ; scale bar : 500 m .
b ) inset from a : blood vessels accompanied by nerve fibers ( arrows ) are running mostly in submucosa ( sm ) ; and rarely between acini of the meibomian glands ( a ) ; scale bar : 100 m .
the analysis of ra group showed less numerous , shrunken acini of meibomian glands , which were seen not only smaller in size , but also the density of their staining was considerably lowered ( figure 2a ) . in the ra group in the submucosa
marked differences were noticed compared to eyelids of control group , there were more numerous nadph - d stained vessels accompanied by nadph - d positive nerve fibers . although the nerve fibers and blood vessels were less numerous between the acini , only a few fine nerve fibers and blood vessels were present running between the acini of meibomian glands ( figure 2b ) .
generally accepted fact is that the excess of vitamin a during pregnancy has teratogenic effects towards offspring .
hence the composition of human and rat meibomian glands are very similar , the development of nitrergic innervation in the rat 's meibomian gland was investigated in our previous experiment .
nadph - d histochemical activity in nerve fibers and vessels suggests utilization of no . in this work ,
histochemical analysis revealed changes in morphology and nitrergic innervations of meibomian glands in 14-day - old rats prenatally treated with excess of ra .
acini were smaller , shrunken and less intensively nadph - d stained , more nerve fibers and vessels were present in submucosal layer of eyelid , although they were less between acini . these findings corresponded with results in rabbits by lambert and smith .
mathers et al . found , that during usage of accutane by human , meibomian glands appeared significantly less dense and were atrophied , with consequent increased tear 's evaporation and osmolarity . during systemic ra treatment , eye dryness
it is very important to select such dose of ra that will have the less effect to meibomian glands while maintaining therapeutic effects . after a lot of pilot experiments we finally considered to use all - trans ra in doses of 1 mg / kg on pregnant rat in 1416 embryonic day to investigate the influence of prenatal administered subtoxic doses of ra to offspring 's postnatal development . with isotretinoin usage , all glands
are approximately equally affected , while by influence of infectious diseases some glands are normal and others are severely affected .
this was confirmed as well in our experiment , as all meibomian glands were equally affected by excess of ra .
changes of meibomian glands are reversible on discontinuing the drug , whereas meibomian glands after prenatal treatment are altered permanently .
the nadph - d histochemistry was used as a method for visualisation of enzyme nos in neurons , their fibres and blood vessel endothelia . in other tissues ,
positive staining for nadph - d is non - specific . in many regions of the central and peripheral nervous system ,
the presence of nos is taken to indicate a functional role for no in the involved tissues .
this innervation appears in early development and is fully established after third postnatal week in rat nervous system .
the picture of meibomian glands of prenatally influenced rats showed developmental stagnation compared to control animals .
ra may similarly cause an alteration in the composition of meibomian gland excretions , resulting in increased viscosity . in the early phases of development ,
up to 1 postnatal week it was observed that nerve fibers and blood vessels were running in the submucosa , not in the septa between the acini . in samples of adult rats ,
typically observed intensely stained nerve fibers running as a network around blood vessels were seen .
they were mostly seen between acini and around the acinar ducts , less in the submucosal layer .
other useful investigations were also performed during rat embryonic development , and age - related changes of nadph - diaphorase positivity in the rat were reported .
the excess of retinoic acid during prenatal period influences the development and morphology of nitrergic structures of the meibomian glands
. it may be that innervation is more related to regulating the periacinar vascular network and meeting nutritional demands .
future functional studies of this model may yield important insights into the relationships between ra treatment and meibomian gland morphology and innervation .
 the purpose of this study was to investigate the effect of prenatal administration of retinoic acid ( ra ) on the development of nicotinamide adenine dinucleotide diaphorase ( nadph - d ) positive structures in the rat meibomian glands . 
 one mg / kg of ra was applied to pregnant wistar rats intraperitonaelly during the gestational period in each of the 12th14th embryonic days ( totally 3 mg / kg ) . 
 sections of the central upper eyelids were investigated in rat pups on the 14th postnatal day . 
 they were processed histochemically for nadph - d , to study the presence and distribution of nitric oxide synthase ( nos ) positive nerve structures . 
 nadph - d staining of meibomian glands was compared in two groups of rat pups . in the control group , 
 eyelids of 14 day - old rats were studied with no experimental intervention . 
 the second group consisted of rat pups which were prenatally administered the excess of ra . 
 histochemical analysis of control eyelids revealed numerous nadph - d well - stained acini of meibomian glands arranged tightly into groups . intensively stained vessels and nadph - d / nos - positive nerve fibers bordered acini of meibomian glands . 
 these structures were present in the submucosal layer as well . 
 the analysis of ra group showed less numerous , shrunken acini of meibomian glands that were seen not only smaller in size , but also in density of their staining and the amount of nitrergic nerve fibers around acini were considerably lowered . in the submucosa differences 
 were noticed compared to the control group , there were numerous nadph - d stained vessels accompanied by nadph - d / nos - positive nerve fibers . 
 the excess of ra during the prenatal period may influence on the development and morphology of nadph - d positive structures of rat 's meibomian glands . 
Introduction
Materials and Methods
Animal treatment and tissue preparation
Histochemical procedure
Assessment
Results
Discussion