Minocycline Inhibits Inflammation and Squamous Metaplasia of Conjunctival Tissue Culture in Airlift Conditions.

PURPOSE: Inflammation and squamous metaplasia is a common pathological process that occurs in many ocular surface diseases such as dry eye, Stevens-Johnson syndrome, mucous membrane pemphigoid, and chemical/thermal burns. At present, there is no ideal medicinal treatment for this abnormality. We report herein on an ex vivo conjunctival inflammation and squamous metaplasia model by culturing human conjunctival tissues at an air-liquid interface for up to 8 days to study the effects of minocycline on inflammation and squamous metaplasia. METHODS: The levels of inflammatory mediators including interleukin-1ß, tumor necrosis factor-α, and metalloproteinase-9 in the cultured human conjunctival tissues were determined by enzyme-linked immuno-sorbent assay and real-time polymerase chain reaction. The total and phosphorylated nuclear factor-κB were detected by western blot. Differentiation markers K10, MUC5AC, and Pax6 and proliferation markers Ki67, p63, and K14 were determined by immunofluorescence or immunohistochemical staining. RESULTS: The results indicated that minocycline inhibited inflammation, decreased the expression of interleukin-1ß, tumor necrosis factor-α, and metalloproteinase -9, and squamous metaplasia features such as hyperproliferation and abnormal epidermal differentiation of conjunctival epithelium. CONCLUSIONS: These findings highlight the possibility that minocycline could be used to treat dry eye and other ocular surface diseases exhibiting epithelial cell inflammation and squamous metaplasia.

Postoperative Shifts in Adult Strabismus Patients with Visual Deficits.

OBJECTIVE: To evaluate the effects of corrective surgery in adult strabismic patients with visual deficits and the stability of postoperative alignments. METHODS: Retrospective study. Alignment and binocular visual function were evaluated in adult patients with visual deficits (best corrective visual acuity (BCVA) <0.3 in one or both eyes) with at least 6 months postoperative follow-up. The results in these patients were compared to those in patients with normal or nearly normal vision (BCVA ≥ 0.6 in the worse eye) matched by the type of strabismus, magnitude of deviation, age at the time of corrective surgery, and length of time before final follow-up. RESULTS: Sixty-four patients aged 28.3 ± 12.0 (17~65) years were included in the visual deficit group, and 64 subjects aged 26.8 ± 9.4 (17~58) years were included in the matched control group. No significant difference was found in the age or deviation angle between the two groups. The surgery was considered a success in 42 patients (65.6%) in the visual deficit group and 55 patients (86%) in the control group at the last visit (Χ2 = 7.19, p = 0.008). Normal or partially normal stereo acuity was achieved by three patients in the visual deficit group and 29 patients in the control group. An average shift toward exotropia of 7.3 prism diopter (PD) in the visual deficit group and 4.9 PD in the control group was observed between postoperative day 1 and the final visit (t = 2.08, p = 0.04). Of the 22 patients in the visual deficit group that were defined as "not a success," 20 were under-corrected, and two were over-corrected. CONCLUSION: Adults with poor vision experienced positive results from corrective surgery via a postoperative shift toward exotropia. Full correction of deviation and postoperative shift should be considered before surgery.

[Effects of amniotic extraction on epithelial wound healing and stromal remodelling after excimer laser keratectomy in rabbit cornea].

OBJECTIVE: To investigate the effects and mechanism of amniotic extraction on corneal healing after photorefractive keratectomy (PRK). METHODS: Experimental Study. Thirty-six New Zealand rabbit corneas were performed with PRK models (-10 diopters, 6.5 mm diameter). According to random number table, all eyes were divided into three groups, including treated with amniotic extraction, 0.1% dexamethasone and excipient respectively after operation. Clinical and histopathologic examinations were taken by slit-lamp microscope and light microscope. Corneal epithelium reparation was observed by fluorescent staining. Corneal stroma cell apoptosis was evaluated by terminal deoxyribonucleotidyl transferase-mediated deoxyuridine triphosphate nick end labeling (TUNEL) assay. Myofibroblast generation was evaluated by immunofluorescence checking the expression of alpha-smooth muscle actin (α-SMA). The number of TUNEL and α-SMA positive cells was analyzed to explore the effects on corneal haze. The haze grading was compared between groups using Kruskal-Wallis H test. Mean values for each experiment were compared between groups using a one-way analysis of variance and LSD-t test.Spearman rank analysis was used to evaluate the correlation between the haze grading and the expression of TUNEL positive cells and α-SMA. RESULTS: The corneas of seventy-two eyes reepithelialized in 6 days after operation. The average epithelium repair time of the AE group was (4.12 ± 0.62) d, the dexamethasone group was (5.25 ± 0.78) d, and the excipient group was (4.96 ± 0.73) d. The progression of reepithelialization was significantly faster in the AE group than the other two groups (F = 14.144, P < 0.01). The haze appeared in the first week after the PRK in all three groups, increased after 3-4 weeks, and relieved after 8 weeks. The degree of haze was significantly lower in the AE group than the other two groups in the first week (Vs. dexamethasone group, H = 3.995, P < 0.05; vs. excipient group, H = 12.77, P < 0.01), in the 4th week (Vs. dexamethasone group, H = 4.468, P < 0.05;vs. excipient group, H = 9.003, P < 0.01), and 8th week (Vs dexamethasone group, H = 4.397, P < 0.05;vs. excipient group, H = 5.744, P < 0.05) after PRK. The TUNEL-positive cells appeared in the central anterior stroma at the first week after surgery. And the number of TUNEL-positive cells in the AE group was (2.2500 ± 0.3750) cells/HP, the dexamethasone group was (4.5000 ± 0.7500) cells/HP, and the excipient group was (7.1250 ± 0.9063) cells/HP. The number of TUNEL-positive cells in AE group was less than those in the other two groups (Vs. dexamethasone group, t = 4.26, P < 0.01; vs. excipient group, t = 8.13, P < 0.01). The TUNEL-positive cells were only found in the excipient group (2.8750 ± 0.6563)/HP in 4th week after operation.It was significantly different between the dexamethasone group and the excipient group (t = 9.01, P < 0.01). There were no significant TUNEL-positive cells in 8th weeks in all three groups.α-SMA-positive cells started to appear apparently at the first week after surgery in the dexamethasone and excipient groups, and the peaks appeared at the 4th week after treatments, and there were still a lot of α-SMA-positive cells in corneal stroma at the 8th week after operation in both groups.On the contrary, there were no significant α-SMA-positive cells in the AE group all the time after surgery. The statistical significant difference can be found between the AE group vs. the dexamethasone and excipient groups in the first week (t = 28.62, 36.55;P < 0.01), in the 4th week (t = 30.40, 35.96; P < 0.01), and in the 8th week (t = 34.02, 38.32; P < 0.01).Spearman rank analysis demonstrated that the formation of haze was proportional to the expression of TUNEL positive cells (r = 0.881, P < 0.01) and α-SMA (r = 0.710, P < 0.01). CONCLUSION: Amniotic extraction can reduce the formation of haze, which was more effective than 0.1% dexamethasone.It might release certain factors which were transported into corneal matrix, then affected the healing of epithelial cell by interacting with the corneal cell factors, reducing the cell apoptosis, corneal wound healing response and rebuilding the corneal matrix with less myofibroblast, collagen and scar and finally reduce the formation of haze.

[Therapeutic effects of Pyranoprofen on the mouse dry eye induced by topical medication of Benzalkonium Chloride].

OBJECTIVE: Randomized controlled experimental study to investigate the therapeutic effect and the possible mechanism of Pranoprofen on the recovery of dry eye induced by topical medication of Benzalkonium Chloride (BAC) in mouse. METHODS: It was an experimental study. Seventy BALB/c mice were treated with topical administration of 0.25% BAC to establish the dry eye condition. Based on the consistency of break-up time of tear-film (BUT), corneal fluorescein staining scores and inflammation index, the eyes were re-selected and randomly divided into four groups on day (D) 21 after the BAC treatment. Group A was set up as blank control, while group B, C and D were treated respectively with 0.1% sodium hyaluronate eye drops, 0.1% fluorometholone eye drops plus 0.1% sodium hyaluronate eye drops, 0.1% pyranoprofen eye drops plus 0.1% sodium hyaluronate eye drops. BUTs, tear volumes, corneal fluorescein staining scores and inflammation index were evaluated in each group on D0, 1, 3 and 5 after the therapeutic treatment. Global specimens were collected on D6. Sections were stained with hematoxylin and eosin (HE) or by periodic acid-schiff (PAS) assay, and labeled with cytokeratin 10 (K10) antibody. The expression of tumor necrosis factor-α (TNF-α) in the cornea and conjunctiva was quantified by western blot. RESULTS: 72 eyes were included in the sequential experiment, 18 eyes for each group. On D0, 1 and 3, no clinical differences were observed among the groups. On D5, the BUT was (2.933 ± 0.320), (2.900 ± 0.280), (3.464 ± 0.498) and (3.643 ± 0.413) s in group A, B, C and D respectively; the BUTs in group C and D were significant longer than those of group A and B (F = 13.774, P = 0.000). The corneal fluorescein staining score was (11.640 ± 1.008), (11.790 ± 1.188), (10.330 ± 1.371) and (10.270 ± 1.104)s in group A, B, C and D respectively; the scores in group C and D were significant lower than those of group A and B (F = 6.145, P = 0.001). The corneal inflammatory index was (0.232 ± 0.059), (0.229 ± 0.078), (0.151 ± 0.055) and (0.154 ± 0.056) in group A, B, C and D respectively; the index in group C and D were significant lower than those of group A and B (F = 6.703, P = 0.001). No significant difference was found in tear volume among groups. No significant difference was found between Pyranoprofen and Fluorometholone treatment in BUT, corneal fluorescein score or inflammatory index. Corneal morphology showed the feature of thicker corneal epithelial layer in group A and uniformity in group C and D. PAS assay revealed similar goblet cell numbers in group C and D, but less goblet cells in group A and B. Cytokeratin 10 was almost negatively expressed in Pranoprofen or Fluorometholone treated groups, and remained positive in the corneal epithelium with other treatments. The level of TNF-α in the cornea was down-regulated in Pranoprofen or Fluorometholone treated groups. CONCLUSIONS: Pranoprofen or Fluorometholone combined with sodium hyaluronate treatment presented similar therapeutic effects on BAC-induced mouse dry eye, with the more stable tear film, the better regularity of epithelium recovery, the down-regulation of inflammatory TNF-α, the increased number of goblet cells, and the elimination of squamous metaplasia, when compared with the treatment of sodium hyaluronate eye drops only. Our results showed the great potentialities of Pranoprofen in the clinical treatment of ocular surface inflammation in the mild and severity dry eye.

Amniotic membrane inhibits squamous metaplasia of human conjunctival epithelium.

Squamous metaplasia is a common pathological process that occurs in the ocular surface epithelium. At present, there is no effective treatment for this abnormality. In the current study, we established an ex vivo conjunctival squamous metaplasia model by culturing human conjunctival tissues at an air-liquid interface for durations of up to 12 days. We then investigated the effects of amniotic membrane (AM) on squamous metaplasia through coculture of conjunctival tissues with AM or AM extract. We found that metaplasia features such as hyperproliferation and abnormal epidermal differentiation of conjunctival epithelium could be inhibited by AM or its extract. In addition, existing squamous metaplasia of conjunctival epithelium could be reversed to a nearly normal phenotype by AM. The mechanism by which AM prevents squamous metaplasia may involve downregulation of p38 mitogen-activated protein kinase and Wnt signaling pathways, which were activated in conjunctival explants cultured with an airlift technique. In conclusion, AM can inhibit and reverse squamous metaplasia of conjunctival epithelium. This finding may shed new light on prevention and treatment of diseases that involve epithelial squamous metaplasia.

[Effect of doxycycline on inflammation-related cytokines and apoptosis in human conjunctival epithelial cells].

OBJECTIVE: To evaluate the effects of doxycycline on the regulation of intercellular adhesion molecule-1 (ICAM-1, CD54), interleukin-1beta (IL-1beta), human leukocyte antigen DR (HLA-DR) and apoptosis in human conjunctival epithelial cells. METHODS: Human primary conjunctival epithelial cells were isolated and cultured from donors and identified by immunohistochemistry. Cultured epithelial cells were treated with either 0 U/ml IFN-gamma, 300 U/ml IFN-gamma, 300 U/ml IFN-gamma with 10 microg/ml doxycycline, 300 U/ml IFN-gamma with 20 microg/ml doxycycline, 300 U/ml IFN-gamma with 40 microg/ml doxycycline or 300 U/ml IFN-gamma with 100 microg/ml dexamethasone for 24 hours. The amount of CD54, HLA-DR and IL-1beta was measured by flow cytometry and western blot analysis. Apoptosis was evaluated by flow cytometry after the cultured epithelial cells were treated for 72 hours. RESULTS: Cultured conjunctival epithelial cells can express CD54 and IL-1beta. IFN-gamma increased the amount of CD54 and IL-1beta (P < 0.01). Doxycycline and dexamethasone inhibited the IFN-gamma induced increase of express of CD54 and IL-1beta of cultured conjunctival epithelial cells, and the inhibiting effect was dependent on the concentration of doxycycline (P < 0.01). Very little HLA-DR and apoptosis were detected before and after treatment with IFN-gamma. CONCLUSION: Doxycycline can suppress the expression of inflammatory cytokine such as CD54 and IL-1beta, which suggests that doxycycline may be a potent drug for the treatment of ocular surface inflammatory disease.

An experimental study of anti-angiogenesis with recombinant human kringle 5.

OBJECTIVE: To investigate the anti-angiogenesis effects of recombinant human kringle 5 (rhk5) eye drops on corneal neovascularization (CNV) in rabbits induced by alkali burn. METHODS: Forty New Zealand albino rabbits were burned on the central corneas of their right eyes by 1 mol/L NaOH for 60s. Animals were randomly divided into 4 groups, 10 rabbits each group. Then rhk5 eye drops with different concentrations were applied four times daily for four weeks on each group: 5 mg/L in group 1, 10 mg/L in group 2, 20 mg/L in group 3 and carrier solution in group 4 (controls). The occurrence and development of CNV was observed every other day by slit-lamp microscope, and the area of CNV was calculated. Then the rabbits were killed on the 28th day (d), and the corneas were taken for histopathological examinations. Pearson's analysis was used to analyze the correlation between the number of inflammatory cells and the area of CNV. RESULTS: The occurrence of CNV in group 1 was (3.4 +/- 0.5) d, group 2 (6.8 +/- 0.4) d, group 3 (6.7 +/- 0.7) d and group 4 (3.7 +/- 0.5) d. Significant difference (P < 0.05) in the occurrence time of CNV was found between group 2 and the control. Compared with the control group, the CNV occurrence was significantly delayed in the group treated by 10 mg/L rhk5. No significant difference was found between control group and group 1, and between group 2 and group 3. The CNV areas of group 2 and group 3 were also smaller than the control group (P < 0.05). The area of CNV had close relationship with the retrocorneal membrane and with the number of inflammatory cells (P < 0.05). CONCLUSION: Topical application of recombinant human rhk5 has prominent effectiveness on the inhibition of angiogenesis induced by alkali burn.

[Tear film changes after phacoemulsification].

OBJECTIVE: To evaluate tear film changes after phacoemulsification. METHODS: This prospective randomized study involved 68 consecutive patients with age-related cataract (79 eyes) who underwent phacoemulsification. Tear break-up time, Schirmer I test (S I t) value, corneal fluorescein staining, tear film pattern by DR-1 tear interfermetry and the height of tear meniscus were measured preoperatively and 1 day and 2, 7, 14, 30, 180 days postoperatively. RESULTS: At 1 day and 2 days postoperatively, the mean tear break-up time reduced greatly, and the number of patients who showed III to V tear film pattern, the mean S I t value, the height of tear meniscus and fluorescein staining scores increased significantly (P < 0.001 for day 1, P < 0.005 for day 2). S I t value at 7 days postoperatively (P = 0.831) and the height of tear meniscus at 14 days returned to their preoperative values (P = 1.000). The tear break-up time, corneal fluorescein staining scores and the grades of tear film pattern all recovered at 30 days postoperatively (P > 0.05). At 30 days postoperatively, 19.3% of patients showed shorter tear break-up time and less S I t value than that of preoperative day. In addition, 1/9 of the patients with normal preoperative tear film experienced dry eye at 30 days postoperatively. The cases with tear break-up time < 10 seconds compared to patients with tear break-up time >/= 10 seconds were more easily to show tear instability postoperatively (relative risk 13.5, 8.25, 20.0, 39.0, 8.6, 3.9 at 1 day and 2, 7, 14, 30, 180 postoperative days, respectively). CONCLUSION: Phacoemulsification significantly alters the tear break-up time, S I t value, corneal fluorescein staining, tear film pattern and the height of tear meniscus. Some patients with normal tear film may experience dry eye after surgery. Patients with preexisting tear break-up time < 10 seconds are easily at risk of experiencing the tear film instability postoperatively.