Age-related Changes And Sex Differences Of Human Palpebral Conjunctiva And Meibomian Gland On In Vivo Laser Scanning Confocal Microscopy

Dry eye-related research is the hot spot in ophthalmic research in recent years since its prevalence increased gradually, being a rather common eye disease. Evaporative dry eye caused by meibomian gland (MG) dysfunction accounted for a large proportion. Meibomian gland secretion of a variety of lipid components to form the lipid layer of tear film and determine tear evaporation and tear film stability. Meibomian glands with normal numbers and morphology play an important role in maintaining a stable ocular surface. Previous studies mainly involved pathological and animal studies. Heretofore, no reports on the changes of normal meibomian glands and tarsal conjunctiva under in vivo condition have been published. It remained unclear that the morphology and density of meibomian glands in the normal subjects ere affected by various internal and external impact factors such as gender, age, hormone levels and living conditions..Laser scanning confocal microscopy (LSCM) provided a new way to observe the ocular surface tissue under in vivo condition. With the application of LSCM, we observed in vivo structure of tarsal conjunctiva and meibomian glands in normal subjects, and discussed the changes of density and shape in conjunctiva epithelial cells, goblet cells (GCs), dendritic cells (DCs) and MG acinar units. Moreover, correlations of these parameters and age as well as gender were explored.PartⅠAge-Related changes of human palpebral conjunctiva and meibomian gland on in vivo laser scanning confocal microscopyPurpose:To analyzed age-related changes of human conjunctiva and tissue under the palpebral conjunctiva by in vivo laser scanning confocal microscopy (LSCM).Methods:86 eyes of 86 healthy subjects were enrolled in this study after a routine slit-lamp examination. They were divided into four groups according to the age:A (0～20 years), B (21～40 years), C (41～60 years) and D (over 60 years). There are about 20 cases in each group. Flipping upper eyelid, in vivo LSCM was performed on the subjects' palpebral conjunctivas and the images were recorded. The morphology of palpebral conjunctiva epithelial cells, GCs, DCs, and MG acinar units were analyzed and the density of epithelial basal cells, GCs, DCs, MG acinar units and the diameters of MG acinar units were calculated. One-way analysis of variance (ANOVA) was used to compare the means of the density of palpebral epithelial basal cells, GCs, DCs, MG acinar units and the diameters of MG acinar units in different groups. Subsequently the data between four groups were analyzed by Least Significant Difference(LSD). The palpebral conjunctiva and the MG structures with aging changes were evaluated.Result:(1) By LSCM, superficial epithelial cell bodies of palpebral conjunctiva were characterized as large, cytoplasm of low reflective, body around of high reflective, loose-arranged cells around with hyperreflective and fuzzy. The cells' shape are like round or oval. Epithelial morphology is closely related to its normal physiological process. The more flat shape, larger, the closer is to the surface epithelium. Conjunctiva under the basal layer of the confocal microscope showed bright and clear around the border nearly round, oval cells. The cytoplasm was low reflection. As the basal layer are closer to MGs and boundary are clearer, so we statistics epithelial basal cell density of palpebral conjunctiva cells. The average densities in group A, B, C, D were (5357±304), (4917±413), (4925±521), (4943±484) cells/mm2 respectively, using analysis of variance showing a significant differences between groups (p=0.002, F=5.217). But the density did not decrease with age. (2) GC is the main source of mucus layer of tear and plays an important role of maintenance of tear film stability. GC bodies were large, loose-arranged cells full of bright intracellular particles. The shape of GCs were oval or round cells. GCs were larger by 3 to 4 times than the surrounding epithelial cells. The mean densities of the GC were (1422±590), (1409±711), (791±457), (779±546) cells/mm2, respectively to the four age groups (A, B, C, D), using analysis of variance and indicating a significant reduction (p=0.000, F=14.168). (3) At present, DCs were the most powerful antigen presenting cells in the body. The images in vivo showed that the typical DCs appeared to be hyperreflective corpuscular particles with dendritic processes, scattered among the palpebral conjunctival epithelial cells. There were several kinds of atypical morphologies could be seen as well, such as DC bodies lacking dendrites, having short or long dendrites, or the wire netting pattern of DCs with long entangled dendrites. The branching patterns were decreasing with increasing age. The mean densities of the DC bodies were (29±15), (30±12), (41±23), (36±15) cells/mm2. The DC bodies densities using analysis of variance and indicated a no significantly decreasing tendency with increasing age (p=0.06, F=2.573). (4) MG acinar cells showed different forms were due to different optical section. The outer fo MG acinar cells were tire-like, accompanied by bright reflective, and gray cell cavities with high reflective dot secretion. MGs were distribution of group-like aggregation and arranged irregularly. The mean densities of the MG acinar uints were (92±26), (86±27), (62±20), (56±16) cells/mm2, respectively to the four age groups (A, B, C, D), using analysis of variance and indicating a significant reduction (p=0.000,13.133). The mean diameters of MG acinar units were (99.06±16.64), (98.22±18.77), (85.91±15.55), (88.26±16.71)μm respectively to each age groups (A-D) using analysis of variance and showing significantly (p=0.022,F=3.385), but not significantly reduced with age.Conclusions:Using LSCM can clearly observe the normal palpebral conjunctiva and meibomian glands of the microstructure. The structure of palpebral conjunctiva cells and density of DCs did not change with age significantly. The branches morphology of DC, and the densities of GCs and MG acinar units were the trend of degradation with age. The tips was that MG function was reduced.PartⅡSex differences in human palpebral conjunctiva and meibomian gland by in vivo laser scanning confocal microscopyPurpose:To analyzed sex difference in human conjunctiva and tissue under the palpebral conjunctiva by in vivo laser scanning confocal microscopy (LSCM).Methods:86 eyes of 86 healthy subjects were enrolled in this study after a routine slit-lamp examination. They were divided into four groups according to the sex, below 40 years old and above 41 years old:A (male,0～40 years), B (female.0-40 years), C (male, above 41 years) and D (female, above 41 years). There are about 20 cases in each group. Flipping upper eyelid, in vivo LSCM was performed on the subjects' palpebral conjunctivas and the images were recorded. The morphology of palpebral conjunctiva epithelial cells, GCs, DCs, and MG acinar units were analyzed and the density of epithelial basal cells, GCs, DCs, MG acinar units and the diameters of MG acinar units were calculated. T test was used to compare the means of the density of palpebral epithelial basal cells, GCs, DCs, MG acinar units and the diameters of MG acinar units in different groups. The palpebral conjunctiva and the MG structures with sex difference were evaluated.Result:(1) By LSCM, the average densities in group A, B, C, D were (5162±324), (5042±608), (4902±374), (5047±345) cells/mm2 respectively. Both using t test and regardless of the age below 40 years (p=0.469,t=0.827), nor the age above 41 years (p=0.607,t=-1.295) were neither a significant difference. (2) The mean densities of the GC were (1406±496), (1426±367), (840±375), (744±307) cells/mm2, respectively to the four age groups (A, B, C, D), both using t test indicating a no significant relation with sex in groups of age below 40 years (p=0.288, t=-0.135) and above 41 years (p=0.406,t=0.801). (3) The mean densities of the DC bodies were (28±12), (30±13), (46±20), (34±18) cells/mm2. The DC bodies densities indicated a no significantly decreasing tendency with sex, no matter the age below 40 years (p=0.551,t=-0.310) and the age above 41 years(p=0.149, t=2.010) both using t test. (4) The mean densities of the MG acinar uints were (86±26), (95±24), (59±17), (58±19) cells/mm2, respectively to the four age groups (A, B, C, D). Both using t test there were no significant differences with sex in the age below 40 years (p=0.566,t=-1.130) and the age above 41 years (p=0.550,t=0.285). The mean diameters of MG acinar units were (96.30±14.98), (99.54±19.45), (92.72±16.79), (84.88±17.03)μm respectively to each age groups (A-D) both using t test showing no significant difference with sex in the age below 40 years (p=0.270,t=-0.622) and the age above 41 years (p=0.950,t=1.473). There were not significantly changes with sex.Conclusions:Using LSCM can clearly observe the normal palpebral conjunctiva and meibomian glands of the microstructure. The structure of palpebral conjunctiva cells and MGs did not change with age significantly. The densities of palpebral conjunctiva epithelium basal cells, DC bodies, GCs and MG acinar units were no relation with sex. Normal population palpebral conjunctiva and MGs of microstructure can offer a diagnosis of histological basis in meibomian gland-related diseases (MGD) and evaporative dry eye.