Dipeptidyl peptidase IV (DPPIV) activity in the tear fluid as an indicator of the severity of corneal injury: a histochemical and biochemical study

Comparative histochemical and biochemical studies on the catalytically active protease Dipeptidyl peptidase IV (DPPIV), have been performed in the rabbit cornea and the tear fluid using a sensitive fluorogenic substrate, Gly-Pro-7-amino-4-Trifluoromethyl Coumarine (AFC). In both normal and experimentally injured corneas, DPPIV activity was detected histochemically and in the tear fluid biochemically. In contrast to the normal cornea where DPPIV activity was absent and in the tear fluid where it was low, during continuous wearing of contact lenses or repeated irradiation of the cornea with UVB rays, slight DPPIV activity appeared first in the superficial layers of the corneal epithelium, while later increased activity was present in the whole epithelium. This paralleled elevated DPPIV activity in the tear fluid. Moreover, during continuous contact lens wear, the increased DPPIV activity in the tear fluid was, in many cases, coincidental with the presence of capillaries in the limbal part of the corneal stroma. After severe alkali burns when corneal ulcers appeared, collagen fragments were active for DPPIV, which was associated with high DPPIV activity in the tear fluid. In conclusion, Gly-Pro-AFC was found to be useful for comparative histochemical and biochemical studies on DPPIV activity in the experimentally injured rabbit eye. Using the method of the tear film collection by a short touch of substrate punches to the respective site of the cornea or conjunctiva we can show that in experimental injuries (wearing of contact lenses, irradiation of the cornea with UVB rays), the damaged corneal cells were the main source for DPPIV activity in the tear fluid. It is suggested that the activity of DPPIV measured in the tear fluid might serve as an indicator of early corneal disorders, e.g. corneal vascularization related to contact lens wear.     

Corneal epithelium as a platform for secretion of transgene products after transfection with liposomal gene eyedrops

BACKGROUND: The first objective of the study was to evaluate the transfection of corneal epithelium with non-viral vectors to secrete transgene products into the tear fluid and aqueous humor. The second goal was to evaluate the differentiated corneal epithelial cell culture for transfection studies. METHODS: The human corneal epithelial (HCE) cell line was cultured to different stages of differentiation and transfected with complexes of pCMV-SEAP2 with DOTAP/DOPE, DOTAP/DOPE/protamine sulfate (PS) and polyethylenimine (PEI). The complexes of DOTAP/DOPE with plasmid (CMV-SEAP2 or pCMV-Luc4) were subsequently applied topically to the rabbit eyes. Secreted alkaline phosphatase (SEAP) was analyzed using chemiluminescent assay. Luciferase (Luc) was detected at the mRNA level in cornea and conjunctiva using a qRT-PCR. RESULTS: The transfection levels decreased with differentiation of HCE cells. PEI was effective in transfecting both the dividing and partly differentiated cells, but ineffective in differentiated cells. DOTAP/DOPE showed high activity in differentiated cell cultures, while added PS did not improve transfection. Significant SEAP expression was observed for three days after in vivo transfection in the tear fluid and aqueous humor. The luciferase mRNA was found both in the cornea and conjunctiva. The rates of SEAP secretion from both the basolateral side of differentiated HCE cells and cornea in vivo were within the same range. CONCLUSIONS: Corneal epithelium can be transfected topically to secrete gene products to the tear fluid and aqueous humor. The differentiated HCE model is a useful tool in the evaluation of non-viral carriers for corneal transfection.     

The Expression of Tenascin-X in Developing and Adult Rat and Human Eye

Tenascin-X has been studied in developing and adult rat eye and in foetal and adult human eyes, using immunohistochemistry and frozen sections. The data were compared with the distribution of tenascin-C. The immunoreactivity for tenascin-X was seen in a basement membrane-like feature in different structures of embryonic (E) day 16–17 rat eyes. Postnatal (P) day 2 and older rat eyes showed immunoreactivity for tenascin-X in different connective tissues. In the epithelial basement membrane zone of the cornea, immunostaining was positive in P5 eyes, negative in P10 and P15 eyes and again positive in P30 and adult eyes. In the 20-week-old human foetus, immunoreactivity for the tenascin was seen in the posterior parts of the conjunctival stroma adjacent to the sclera and in a basement membrane-like fashion in anterior conjunctiva. In the adult human eye, immunoreactivity for tenascin-X was seen in the anterior one-third stroma of cornea as thin fibrils, in the stroma of the limbus and conjunctiva, and in blood vessels. Immunostaining for tenascin-C was seen in the posterior aspect of the further cornea, and in mesenchyme adjacent to cornea in E16–17 rat eyes. Corneal keratocytes and Descemet's membrane showed immunoreactivity for tenascin-C in P2–P15 rat eyes. Sclera and the junction of the cornea, and sclera expressed tenascin-C in P2 and older rat eyes. In human foetal eyes, immunostaining for tenascin-C was seen in the anterior parts of the corneal stroma, in the basement membrane zone and Bowman's membrane of the corneal epithelium, in the posterior one-fifth of the corneal stroma and the sclera starting from the junction of the cornea and sclera. In normal human adult eyes, immunostaining for tenascin-X was seen in the anterior one-third stroma of cornea, in the stroma of limbus and conjunctiva, and in blood vessels. The association of tenascin-X and basement membranes in early development evokes a question of its potential function in the development of the basement membrane. The results also suggest the association of tenascin-X with connective tissue development as well as the association of tenascin-C with the migration of keratocytes during the development of the corneal stroma.     

Corneal alternations induced by topical application of benzalkonium chloride in rabbit

Benzalkonium chloride (BAC) is the most common preservative in ophthalmic preparations. Here, we investigated the corneal alternations in rabbits following exposure to BAC. Twenty-four adult male New Zealand albino rabbits were randomly divided into three groups. BAC at 0.01%, 0.05%, or 0.1% was applied twice daily to one eye each of rabbits for 4 days. The contralateral untreated eyes were used as control. Aqueous tear production and fluorescein staining scores of BAC-treated eyes were compared with those of controls. The structure of the central cornea was examined by in vivo confocal microscopy. Expression of mucin-5 subtype AC (MUC5AC) in conjunctiva was detected by immunostainig on cryosections. Corneal barrier function was assessed in terms of permeability to carboxy fluorescein (CF). The distribution and expression of ZO-1, a known marker of tight junction, and reorganization of the perijunctional actomyosin ring (PAMR) were examined by immunofluorescence analysis. Although there were no significant differences between control and BAC-treated eyes in Schirmer scores, corneal fluorescein scores and the number of conjunctival MUC5AC staining cells, in vivo confocal microscopy revealed significant epithelial and stromal defects in all BAC-treated corneas. Moreover, BAC at 0.1% resulted in significant increases in central corneal thickness and endothelial CF permeability, compared with those in control eyes, and endothelial cell damage with dislocation of ZO-1 and disruption of PAMR. Topical application of BAC can quickly impair the whole cornea without occurrence of dry eye. A high concentration of BAC breaks down the barrier integrity of corneal endothelium, concomitant with the disruption of PAMR and remodeling of apical junctional complex in vivo.     

Limbal stem cells, a review of their identification and culture for clinical use

The surface of the eye is covered by two distinct epithelial populations, the conjunctival and corneal epithelia. The stem cell population for the corneal epithelia has been found to be located at the area known as the limbus. This is a narrow ring of tissue at the transitional zone between the cornea and conjunctiva. This stem cell population is responsible for generating transient amplifying cells which are responsible for renewing the cornea epithelia. There are currently no definitive markers for the stem cell population in the limbus. Instead using morphological features, such as small cells with a high nucleus-to-cytoplasm ratio, in conjunction with the presence of certain markers e.g. ΔNP63α and the absence of others, e.g. the cytokeratin pair 3 & 12, are taken as being indicative of the stem cell population. Damage can occur to the corneal epithelium due to a number of causes including, Steven-Johnson syndrome, and chemical or thermal burns. This results in invasion of the cornea by the conjunctival epithelium resulting in impaired vision. In 1997 Pellegrini et al. (Lancet 349, 990) successfully used cells sheets from cultured limbal cells to successfully treat patients with corneal damage. Since then several other groups, have successfully treated patients, using similar methods.     

The effects of PEP-1-FK506BP on dry eye disease in a rat model

As FK506 binding proteins (FK506BPs) are known to play an important role in the regulation of a variety of biological processes related to cell survival, this study was designed to examined the protective effects of FK506 binding protein 12 (FK506BP) on low humidity air flow induced dry eye in a rat model using transduced PEP-1-FK506BP. After the topical application of PEP-1-FK506BP, tear volumes were markedly increased and significant prevention of cornea damage was observed compared with dry eye rats. Further, immunohistochemical analysis demonstrated that PEP-1-FK506BP markedly prevented damage to the cornea, the bulbar conjunctiva, and the palpebral conjunctiva epithelial lining compared with dry eye rats. In addition, caspase-3 and PARP expression levels were found to be decreased. These results demonstrated that topical application of PEP-1-FK506BP significantly ameliorates dry eye injury in an animal model. Thus, we suggest that PEP-1-FK506BP can be developed as a new ophthalmic drop to treat dry eye diseases. [BMB Reports 2015; 48(3): 153-158]     

Astragalus IV ameliorates the dry eye injury in rabbit model via MUC1-ErbB1 pathway

The therapeutic effects and potential mechanisms of astragaloside IV on a rabbits dry eye model induced by benzalkonium chloride (BAC) was examined. In our study, a BAC-induced dry eye rabbit model was treated with eye drops containing astragaloside IV (5, 10 μM) or solvent four times a day. The clinical evaluations, such as tear break-up time (BUT) and Schirmer tear test (STT), were performed on days 0, 7, 14, 21, and 28. On day 28, the cornea and bulbar conjunctiva tissues (left eye and right eye) were collected with histology, and immunofluorescent staining conducted. The levels of MUC1 and ErbB1in the corneas were determined by realtime quantitative PCR (qRT-PCR) and the proteins levels of MUC1 and ErbB1 were detected by Western blot. It was demonstrated that both astragaloside IV (5, 10 μM) treatments resulted in an increased STT and BUT on days 7, 14, 21 and 28. Additionally, the astragaloside IV (5, 10 μM)-treated group showed increasing PAS-positive goblet cells than model group (0 μM). Moreover, the MUC1 in model group (0 μM) was decreased, while the expression of MUC1 in astragaloside IV (5, 10 μM) group was increased. Furthermore, astragaloside IV had a protective effect on BAC-induced rabbits’ dry eye and demonstrated clinical improvements, which indicated that astragaloside IV served as a potential protective agent in the clinical treatment of dry eye.Key words: Astragaloside IV, dry eye model, MUC1, ErbB1     

Contributions of Ocular Surface Components to Matrix-Metalloproteinases (MMP)-2 and MMP-9 in Feline Tears following Corneal Epithelial Wounding

Purpose

This study investigated ocular surface components that contribute to matrix-metalloproteinase (MMP)-2 and MMP-9 found in tears following corneal epithelial wounding.

Methods

Laboratory short-haired cats underwent corneal epithelial debridement in one randomly chosen eye (n = 18). Eye-flush tears were collected at baseline and during various healing stages. Procedural control eyes (identical experimental protocol as wounded eyes except for wounding, n = 5) served as controls for tear analysis. MMP activity was analyzed in tears using gelatin zymography. MMP staining patterns were evaluated in ocular tissues using immunohistochemistry and used to determine MMP expression sites responsible for tear-derived MMPs.

Results

The proMMP-2 and proMMP-9 activity in tears was highest in wounded and procedural control eyes during epithelial migration (8 to 36 hours post-wounding). Wounded eyes showed significantly higher proMMP-9 in tears only during and after epithelial restratification (day 3 to 4 and day 7 to 28 post-wounding, respectively) as compared to procedural controls (p<0.05). Tears from wounded and procedural control eyes showed no statistical differences for pro-MMP-2 and MMP-9 (p>0.05). Immunohistochemistry showed increased MMP-2 and MMP-9 expression in the cornea during epithelial migration and wound closure. The conjunctival epithelium exhibited highest levels of both MMPs during wound closure, while MMP-9 expression was reduced in conjunctival goblet cells during corneal epithelial migration followed by complete absence of the cells during wound closure. The immunostaining for both MMPs was elevated in the lacrimal gland during corneal healing, with little/no change in the meibomian glands. Conjunctival-associated lymphoid tissue (CALT) showed weak MMP-2 and intense MMP-9 staining.

Conclusions

Following wounding, migrating corneal epithelium contributed little to the observed MMP levels in tears. The major sources assessed in the present study for tear-derived MMP-2 and MMP-9 following corneal wounding are the lacrimal gland and CALT. Other sources included stromal keratocytes and conjunctiva with goblet cells.     

A Biodegradable,Sustained-Released,Prednisolone Acetate Microfilm Drug Delivery System Effectively Prolongs Corneal Allograft Survival in the Rat Keratoplasty Model

Frequent and long-term use of topical corticosteroids after corneal transplantation is necessary to prevent graft rejection. However, it relies heavily on patient compliance, and sustained therapeutic drug levels are often not achieved with administration of topical eye drops. A biodegradable drug delivery system with a controlled and sustained drug release may circumvent these limitations. In this study, we investigated the efficacy of a prednisolone acetate (PA)-loaded poly (d,l-lactide-co-ε-caprolactone) (PLC) microfilm drug delivery system on promoting the survival of allogeneic grafts after penetrating keratoplasty (PK) using a rat model. The drug release profiles of the microfilms were characterized (group 1). Subsequently, forty-eight PK were performed in four experimental groups: syngeneic control grafts (group 2), allogeneic control grafts (group 3), allogeneic grafts with subconjunctivally-implanted PA microfilm (group 4), and allogeneic grafts with PA eye drops (group 5; n = 12 in each). PA-loaded microfilm achieved a sustained and steady release at a rate of 0.006–0.009 mg/day, with a consistent aqueous drug concentration of 207–209 ng/ml. The mean survival days was >28 days in group 2, 9.9±0.8 days in group 3, 26.8±2.7 days in group 4, and 26.4±3.4 days in group 5 (P = 0.023 and P = 0.027 compared with group 3). Statistically significant decrease in CD4+, CD163+, CD 25+, and CD54+ cell infiltration was observed in group 4 and group 5 compared with group 3 (P<0.001). There was no significant difference in the mean survival and immunohistochemical analysis between group 4 and group 5. These results showed that sustained PA-loaded microfilm effectively prolongs corneal allograft survival. It is as effective as conventional PA eye drops, providing a promising clinically applicable alternative for patients undergoing corneal transplantation.     

Proteomic analysis of rabbit tear fluid: Defensin levels after an experimental corneal wound are correlated to wound closure

The cornea is the major refracting optical element of the eye and therefore critical for forming a retinal image. The exposed surface of the eye is protected from pathogens by the innate immune system whose components include defensins, naturally occurring peptides with antimicrobial properties, and the physical barrier formed by the outer epithelial layer of the cornea. The proteomic approach has revealed that tear levels of defensins are correlated with the course of healing of an experimental corneal wound. Tears were collected from New Zealand White rabbits prior to (day 0) and daily for 5 days (days 1-5) following a standard unilateral 6 mm diameter corneal epithelial abrasion. Tear protein profiles obtained from wounded and contra-lateral control eyes were compared using SELDI ProteinChip technology. Peptides and proteins of interest were purified by RP-HPLC and characterized by nanoESI-MS/MS. Mass spectra of tears on post-wound day 1, revealed 13 peaks whose level decreased and five that increased. During wound healing the tear protein profile correlated with wound closure. An important finding was that the levels of rabbit defensins (NP-1 and NP-2), which were elevated after wounding returned to normal levels by the time the corneal abrasion healed. Relative quantification of NP-2 in tear fluid prior to (day 0) and after corneal wounding (days 1- 3) was determined using iTRAQ technology. A corneal wound eliminates the barrier function of innate immunity and puts the cornea at risk from microbial attack until the epithelial cells restore the surface barrier. The increased availability of defensins in the tears during healing suggests that these peptides could protect the cornea from microbial attack during a period of increased vulnerability.     

Histochemical changes in the rabbit cornea and plasmin activity in the tear fluid during contact lens wear. Favourable influence of protease inhibitors (aprotinin,PC5, elastatinal)

Summary Plasmin activity in the tear fluid of the rabbit eye was examined during the wearing of soft contact lenses (SCL) and compared with the occurrence of corneal disturbances assessed in cryostat sections. Plasmin activity was determined with a semiquantitative method using dry punches of filter paper previously soaked in 0.1 M Tris-HCl buffer solution containing mmol/l d-Val-Leu-Lys-FCA (trifluoromethylaminocoumarine), pH 7.2. Punches were applied to the corneal surface for 5 s (tear collection) and incubated in wet chamber. The time of appearance of the bright yellow fluorescence in UV light was recorded and taken as a measure of plasmin activity. For calibration punches soaked in solutions containing plasmin in various concentrations, and processed in the same manner were used. Changes in the cornea were examined histochemically using methods of choice for acid glycosidases, proteases, dehydrogenases, and Na⁺-K⁺-ATPase. SCL with high and low water content were worn in rabbits in 1, 2, 4, 7, 14, 21 and 28 days.Decreased activity of Na⁺-K⁺-ATPase, GGT, and SDH in the corneal endothelium and epithelium were not accompanied by detectable plasmin activity in the tear fluid. Pronounced damage of the corneal epithelium (increased activities of acid glycosidases, acid proteases, LDH, markedly decreased activity of SDH) was accompanied by low concentration of plasmin (0.4–1.0 g/ml) in the tear fluid. Middle activity of plasmin (1.0–2.0 g/ml) was detectable when PMNs were present in the corneal stroma. High plasmin activity (2.0–3.0 g/ml) correlated with corneal ulceration and vascularization. The occurrence of both — plasmin activity and corneal disturbances was highly dependent on the water content of SCL (which goes parallel with oxygen permeability), duration of SCL wear, mechanical stress, and bacterial contamination. Mechanical irritation is considered to be the main factor leading to the appearance of plasmin activity in the tear fluid. The local application of aprotinin which inhibits plasmin and some other serine proteases, enables us to prolong the harmless wear of SCLH (approximately one week). The combination of aprotin-in with leukocyte elastase inhibitors (elastatinal and particularly PC5), prevents ulceration of the cornea and inhibits corneal vascularization after SCLL wear. Vascularization of the cornea does not occur if protease inhibitors are combined with flurbiprofen, an anti-inflammatory drug of cyclooxygenase pathway of arachidonic acid. Protease inhibitors also improved the course of bacterial keratitis.     

The Effect of Tear Supplementation on Ocular Surface Sensations during the Interblink Interval in Patients with Dry Eye

Purpose

To investigate the characteristics of ocular surface sensations and corneal sensitivity during the interblink interval before and after tear supplementation in dry eye patients.

Methods

Twenty subjects (41.88±14.37 years) with dry eye symptoms were included in the dry eye group. Fourteen subjects (39.13±11.27 years) without any clinical signs and/or symptoms of dry eye were included in the control group. Tear film dynamics was assessed by non-invasive tear film breakup time (NI-BUT) in parallel with continuous recordings of ocular sensations during forced blinking. Corneal sensitivity to selective stimulation of corneal mechano-, cold and chemical receptors was assessed using a gas esthesiometer. All the measurements were made before and 5 min after saline and hydroxypropyl-guar (HP-guar) drops.

Results

In dry eye patients the intensity of irritation increased rapidly after the last blink during forced blinking, while in controls there was no alteration in the intensity during the first 10 sec followed by an exponential increase. Irritation scores were significantly higher in dry eye patients throughout the entire interblink interval compared to controls (p<0.004). NI-BUT significantly increased after HP-guar (p = 0.003) but not after saline drops (p = 0.14). In both groups, either after saline or HP-guar the shape of symptom intensity curves remained the same with significantly lower irritation scores (p<0.004), however after HP-guar the decrease was significantly more pronounced (p<0.004). Corneal sensitivity to selective mechanical, cold and chemical stimulation decreased significantly in both groups after HP-guar (p<0.05), but not after saline drops (p>0.05).

Conclusion

Ocular surface irritation responses due to tear film drying are considerably increased in dry eye patients compared to normal subjects. Although tear supplementation improves the protective tear film layer, and thus reduce unpleasant sensory responses, the rapid rise in discomfort is still maintained and might be responsible for the remaining complaints of dry eye patients despite the treatment.     

The histochemical pattern of mechanically or chemically injured rabbit cornea after aprotinin treatment: relationships with the plasmin concentration of the tear fluid

Summary Plasmin, a serine protease, was recently found to be involved in corneal ulcerative processes in humans and rabbits. In our experiments, plasmin activity was found in the tear fluid after mechanical and chemical damage of the rabbit cornea, such as de-epithelization and burning with alkali. The plasmin concentrations in the tear fluid were dependent on the severity of injury. The highest plasmin activity (2.0–3.0 g ml^–1) occurred after severe alkali damage to large areas of the cornea, and the lowest activity (0.4–1.0 g ml^–1) after mechanical injury (de-epithelization).Plasmin concentrations up to 1.0 ml^–1 were associated with increased activities of lysosomal hydrolases in epithelial cells and keratocytes beneath the epithelium. Plasmin activities increased as the inflammatory reaction developed. When plasmin activity in the tear fluid was higher than 1.0 g ml^–1, inflammatory cells were found in the corneal stroma. Levels of 1.5–2.0 g ml^–1 were connected with higher numbers of inflammatory cells (particularly polymorphonuclear leukocytes) with increased activities of lysosomal hydrolases. Very high plasmin activities (2.5–3.0 g ml^–1) accompanied corneal ulcerative processes.The local application of aprotinin (Trasylol, Bayer), an inhibitor of plasmin, and also of some other proteases, was found to be necessary for the healing of severe corneal injuries in which highly elevated plasmin activity in the tear fluid and inflammatory cellulization of the cornea occurred (severe damage). It was beneficial in cases in which medium plasmin activity occurred in the tear fluid and inflammatory changes in the cornea were not too extensive. If used very early after injury, aprotinin prevents the appearance of high plasmin activity in the tear fluid, reduces the invasion of inflammatory cells into the corneal stroma, and accelerates the healing. Even the corneal transparency is restored in many cases.     

An X-Ray Scattering Study into the Structural Basis of Corneal Refractive Function in an Avian Model

Avian vision diseases in which eye growth is compromised are helping to define what governs corneal shape and ultrastructural organization. The highly specific collagen architecture of the main corneal layer, the stroma, is believed to be important for the maintenance of corneal curvature and hence visual quality. Blindness enlarged globe (beg) is a recessively inherited condition of chickens characterized by retinal dystrophy and blindness at hatch, with secondary globe enlargement and loss of corneal curvature by 3–4 months. Here we define corneal ultrastructural changes as the beg eye develops posthatch, using wide-angle x-ray scattering to map collagen fibril orientation across affected corneas at three posthatch time points. The results disclosed alterations in the bulk alignment of corneal collagen in beg chicks compared with age-matched controls. These changes accompanied the eye globe enlargement and corneal flattening observed in affected birds, and were manifested as a progressive loss of circumferential collagen alignment in the peripheral cornea and limbus in birds older than 1 month. Progressive remodeling of peripheral stromal collagen in beg birds posthatch may relate to the morphometric changes exhibited by the disease, likely as an extension of myopia-like scleral remodeling triggered by deprivation of a retinal image.     

Transduced PEP-1-FK506BP ameliorates corneal injury in Botulinum toxin A-induced dry eye mouse model

FK506 binding protein 12 (FK506BP) belongs to a family of immunophilins, and is involved in multiple biological processes. However, the function of FK506BP in corneal disease remains unclear. In this study, we examined the protective effects on dry eye disease in a Botulinum toxin A (BTX-A) induced mouse model, using a cell-permeable PEP-1-FK506BP protein. PEP-1-FK506BP efficiently transduced into human corneal epithelial cells in a time- and dose-dependent manner, and remained stable in the cells for 48 h. In addition, we demonstrated that topical application of PEP-1-FK506BP was transduced into mouse cornea and conjunctiva by immunohistochemistry. Furthermore, topical application of PEP-1-FK506BP to BTX-A-induced mouse model markedly inhibited expression levels of pro-inflammatory cytokines such as interleukin-1β (IL-1β), tumor necrosis factor-α (TNF-α) and macrophage inhibitory factor (MIF) in corneal and conjunctival epithelium. These results suggest PEP-1-FK506BP as a potential therapeutic agent for dry eye diseases. [BMB Reports 2013; 46(2): 124-129]     

Central Adaptation to Repeated Galvanic Vestibular Stimulation: Implications for Pre-Flight Astronaut Training

Healthy subjects (N = 10) were exposed to 10-min cumulative pseudorandom bilateral bipolar Galvanic vestibular stimulation (GVS) on a weekly basis for 12 weeks (120 min total exposure). During each trial subjects performed computerized dynamic posturography and eye movements were measured using digital video-oculography. Follow up tests were conducted 6 weeks and 6 months after the 12-week adaptation period. Postural performance was significantly impaired during GVS at first exposure, but recovered to baseline over a period of 7–8 weeks (70–80 min GVS exposure). This postural recovery was maintained 6 months after adaptation. In contrast, the roll vestibulo-ocular reflex response to GVS was not attenuated by repeated exposure. This suggests that GVS adaptation did not occur at the vestibular end-organs or involve changes in low-level (brainstem-mediated) vestibulo-ocular or vestibulo-spinal reflexes. Faced with unreliable vestibular input, the cerebellum reweighted sensory input to emphasize veridical extra-vestibular information, such as somatosensation, vision and visceral stretch receptors, to regain postural function. After a period of recovery subjects exhibited dual adaption and the ability to rapidly switch between the perturbed (GVS) and natural vestibular state for up to 6 months.     

Effect of lacrimal plugs combined with deproteinized calf blood extract eye gel for filamentary keratitis

This study aims to investigate the efficacy of lacrimal plugs combined with deproteinized calf blood extract eye gel on the treatment of dry-eye-associated filamentary keratitis. Lacrimal plugs were inserted into both the upper and lower puncta of 15 patients (28 eyes). Deproteinized calf blood cxtract eye gel was applied in two patients who were not cured after this operation. All patients were asked to complete three questionnaires 1 day before the surgery and at 1 week and 1 month after the surgery. Ophthalmologic examinations were carried out and repeated at 1 day and at 1 week and 1 month after plug insertion, which include fluorescein staining, tear break-up time (BUT), Schirmer test I (STI), corneal confocal microscope (HRT-III), and impression cytology (IC). Symptoms were relieved in all patients 1 month after the application of lacrimal plugs. Deproteinized calf blood extract eye gel was applied to two patients whose symptoms remained after lacrimal plug implantation for 1 week. At 3 weeks later, the symptoms disappeared and cornea fluorescein stain was hardly identified. The lacrimal river in all patients became broader after the surgery, 11 of whom reached 0.3 mm. Cornea filamentary disappeared in all patients. The average of BUT and ST were increased to 7.50 ± 1.897 s (p < 0.001) and 8.12 ± 1.996 mm at 1 week after the operation and 7.94 s and 9.00 ± 1.897 mm at 1 month later, respectively. Photography from HRT-III suggested that the state of corneal epithelium was markedly improved, including the squamous metaplasia of the epithelial layer of the cornea and the bend of nerve fibers under the corneal epithelium. IC also suggested that the squamous metaplasia of epithelial layer of conjunctiva in these patients was improved. The symptoms of patients suffering from severe filamentary keratitis were remarkably relieved by using lacrimal plugs, which could increase the tear volume of the ocular surface, improve the condition of tear film, and promote the recovery of corneal diseases. For patients with more severe symptoms, additional usage of deproteinized calf blood extract eye gel could assist the treatment. Therefore, lacrimal plugs combined with deproteinized calf blood extract eye gel are suggested to be an effective method to treat severe filamentary keratitis.     

不同角膜表面处理方式在全飞秒SMILE手术中的应用效果分析

摘要 目的：探讨不同角膜表面处理方式在全飞秒SMILE手术中的应用效果。方法：选择2021年10月至2022年9月来我院择期行全飞秒SMILE手术的患者50例,根据先右后左手术原则,观察眼与对照眼没有固定左右眼,观察眼25例选择右眼,25例选择左眼,对照眼反之选择另一眼,观察眼使用开睑器开睑后用三角海绵擦拭清洁角膜表。对照眼眼自净（眨眼）后使用开睑器开眼睑。对比观察眼与对照眼的角膜光密度、角膜表面颗粒物质计数、角膜表面湿度、术中负压吸引完成时间、失吸比例、OBL发生率,分析50例患者的舒适度。结果：观察眼的角膜光密度为16.33±2.12,对照组为16.85±2.58,组间对比无统计学意义（t=-1.101,P=0.274）。对照眼的表面颗粒物质计数明显较观察眼低,角膜表面湿度明显较观察眼高（P<0.05）。对照眼与观察组术中负压吸引完成时间、失吸比例对比无统计学意义（P>0.05）。观察眼中OBL发生率为6.00 %,对照眼中OBL发生率为2.00 %,组间对比无统计学意义（P=0.617>0.05）。50例患者中对三角海绵擦拭角膜感觉恐惧者占比32.00%（16/50）,三角海绵擦拭角膜后对注视绿点存在影响者占比20.00 %（10/50）,手术中选择三角海绵擦拭角膜者20例,占比40.00 %（20/50）,选择眼自净（眨眼）者30例,占比60.00 %（30/50）。结论：眼自净（眨眼）可以代替三角海绵擦拭角膜,用于全飞秒SMILE手术,提高患者舒适度。     

Analysis of the Viscoelastic Properties of the Human Cornea Using Scheimpflug Imaging in Inflation Experiment of Eye Globes

Purpose

To demonstrate a Scheimpflug-based imaging procedure for investigating the depth- and time-dependent strain response of the human cornea to inflation testing of whole eye globes.

Methods

Six specimens, three of which with intact corneal epithelium, were mounted in a customized apparatus within a humidity and temperature-monitored wet chamber. Each specimen was subjected to two mechanical tests in order to measure corneal strain resulting from application of cyclic (cyclic regimen) and constant (creep regimen) stress by changing the intra-ocular pressure (IOP) within physiological ranges (18–42 mmHg). Corneal shape changes were analyzed as a function of IOP and both corneal stress-strain curves and creep curves were generated.

Results

The procedure was highly accurate and repeatable. Upon cyclic stress application, a biomechanical corneal elasticity gradient was found in the front-back direction. The average Young''s modulus of the anterior cornea ranged between 2.28±0.87 MPa and 3.30±0.90 MPa in specimens with and without intact epithelium (P = 0.05) respectively. The Young''s modulus of the posterior cornea was on average 0.21±0.09 MPa and 0.17±0.06 MPa (P>0.05) respectively. The time-dependent strain response of the cornea to creep testing was quantified by fitting data to a modified Zener model for extracting both the relaxation time and compliance function.

Conclusion

Cyclic and creep mechanical tests are valuable for investigating the strain response of the intact human cornea within physiological IOP ranges, providing meaningful results that can be translated to clinic. The presence of epithelium influences the results of anterior corneal shape changes when monitoring deformation via Scheimpflug imaging in inflation experiments of whole eye globes.