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AIM: To analyze the effect of human amniotic homogenate extract on corneal neovascularization after corneal alkali burn in the process of pigment epithelium derived factor (PEDF) and vascular endothelial growth factor (VEGF) expression and the effect of corneal neovascularization.METHODS: Totally 32 patients with corneal alkali burn were selected from June 2015 to June 2016 in Foshan,and were randomly divided into Group A and Group B,with a total of 37 eyes.Group A of 17 cases,with a total of 19 eyes,were treated with 40mg/L human amniotic homogenate extract;Group B (n=15),and 18 eyes,treated with 3g/L prednisolone eye drops.In the treatment of 1,4,7,14,21 and 28d at different time points,we observed the growth of corneal neovascularization,and detected the expression of PEDF and VEGF during angiogenesis.RESULTS: Group A of patients in the use of human amniotic homogenate extract after the treatment,the expression level of PEDF was significantly higher than that in Group B(P=0.001),after 28d treatment,the expression level of PEDF reached 0.721±0.314.While patients in Group B the expression level of PEDF was only 0.538±0.253.Two groups had significant difference between the expression level of PEDF (P<0.05).The expression level of VEGF in Group A was lower than in Group B at different time points in the test.After the treatment of 28d patients in the Group A,the expression level of VEGF was 0.152±0.020,in Group B the expression level of VEGF was0.302±0.031.Two groups of patients with VEGF expression level between the differences were statistically significant (P<0.05).The patients number in Group A with corneal neovascularization was significantly lower than that in Group B,the difference was statistically significant (P<0.05).CONCLUSION: Human amniotic homogenate extract can increase the expression of PEDF in corneal neovascularization after corneal alkali burn,inhibit the expression of VEGF and the proliferation of corneal neovascularization.
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AIM:To investigate the effects of amnion epithelial cell ( AEC) suspension liquid on the biological behavior of the rabbit's corneal epithelium, combined with the in vitro and in vivo experiments. · METHODS: The rabbit's corneal epithelium were cultured in the lower chamber of transwell, and AEC suspension liquid was dropwised in the upper chamber. There was only culture medium in the upper chamber of the control group. The proliferation of rabbit's corneal epithelium was observed with CCK-8 automated colorimetry and the expression of PCNA was detected by immunocytochemistry. We used the scratch wound assay to detect the migration of corneal epithelial cell ( CEC ) . The in vivo models were established by placing a 10mm diameter corneal trephine in the center of the cornea, within 1mol/L NaOH for 1min. We divided those into three groups: treatment group of AEC suspension liquid eye drop, AEC suspension liquid subconjunctival injection and the control group without any treatment. Using the slit- lamp biomicroscope and fluorescence staining to observe the cornea per week. After 28d we took the eyeballs with the HE staining. The expression of VEGF was detected by immunohistochemistry. ·RESULTS: The activity of CEC with AEC treatment was much higher than the control group ( P< 0. 05 ). The expression of PCNA increased in AEC group (P<0. 05). And the migration of CEC in the AEC group was faster than the control one. In vivo, the inflammation of the corneal and the CNV of the AEC group were all significantly reduced compared with the control group (P<0. 05 ). There were less invasive cells and more ordered organization arrangement in ACE group observed by the HE staining. The expression of VEGF and mcp-1 in these two AEC treatment groups all significantly decreased compared with the control group (P<0. 05). ·CONCLUSION: AEC suspension liquid can promote the proliferation and migration of the rabbit's corneal epithelium. The potential of AEC suspension liquid as a therapy for acute corneal alkali burn.
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Background To maintain corneal transparency is important for good visual function.A new treatment concept and the selection of surgical techniques and timing of surgery are critical for stopping the infringement of cornea tissue after alkali burning and other chemical warfare agents.Objective This study was to investigative the ultrastructure and histopathological status following the femtosecond laser-assisted deep lamellar keratoplasty (DLK) for acute alkali burn of cornea.Methods Acute corneal alkali burn models were established in 12 New Zealand rabbits by putting the 6 mm filter paper with 1 mol/L NaOH at the central cornea for 30 seconds.The rabbits were randomly allocated to femtosecond laser-assisted DLK group and model control group according to the randomized number table method.Femtosecond laser-assisted DLK was performed to transplant the corneal grafts of domestic rabbits to the model rabbits 24 hours after burning.The rabbits were sacrificed 1 week,2 weeks and 4 weeks after modeling,and the corneas were extracted for the preparation of corneal section.The cornea were performed with hematoxylin and eosin staining to assess the histopathological status under the optical microscope,and the ultrastructure of grafts and corneas was examined under the transmission electron microscope (TEM).Results Acute corneal alkali bourn models were successfully eatablished.In the fourth week after surgery,corneal graft was clear in the femtosecond laser-assisted DLK group.However,corneal swelling,conjunctival congestion and neovascularization were found in the model control group.Histopathological examination revealed the defect of corneal epithelium,edema of stroma,loose arrangement of collagen fibers,much vacuoles,few neovascularization and infiltration of a large number of inflammatory cells in the model control group,but in the femtosecond laser-assisted DLK group,the inflammatory response was slight.More desmosomes among the endothelial cells were seen,and the nuclei were intact in the grafts.In the fourth week after surgery,the transplanted corneas were transparent with the regular arrangement of collagen fibers and entire fibroblasts in the femtosecond laser-assisted DLK group under the TEM.However,flat surface corneal epithelial cells and shedding of some epithelial cells were exhibited in the modelcontrol group.Conclusions Femtosecond laser-assisted DLK can effectively alleviate the inflammatory response,promote epithelial healing and enhance intercellular tight junction in the cornea with acute alkali burn.
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BackgroundIt has been proved that as a chemokine,interferon-inducible protein-10(IP-10)can regulate the immuno-inflammatory reaction.Some new researches showed that IP-10 also played role in regulating the neovascular vessel formation.Corneal neovascularization (CNV) is associated with multiple cellular factors,but its mechanism is below clear.Objective The present study was to address the roles of exogenous mouse IP-10 in alkali burn-induced CNV.Methods Eighty-two SPF BALB/c mice were used in this experiment and grouped according to random number table.The corneal alkali burn models were established by putting the filter paper with 1 mol/L NaOH at the central corneas of the left eyes for 40 seconds.10 mg/L IP-10 was topically administered from the first day or 14 days after modeling in the early intervene group( 10 eyes)or middle-late intervene group(5 eyes).CNV area was measured as a percentage of whole cornea.0.2％ sodium hyaluronate(HA) as vehicle was utilized in the model control group.Angiogenic factor expression in corneal tissue in the early intervene group was quantified by reverse transcriptase polymerase chain reaction (RT-PCR)and compared with model control group.All animal experiments were performed in accordance with the Association for Research in Vision and Ophthalmology Statement for the Use of Animals in Ophthalmic and Vision Research and complied with the standards of Guidelines for the Care and Use of Laboratory Animals of Soochow University. ResultsThe CNV percentage was(88.67±10.22) ％ in the model control mice,showing a significant increase in comparison with that of IP-10 early intervene group (70.06±12.21)％ (t=3.77,P=0.00).In 21 days after corneal alkali burn,the CNV percentage was(87.33±13.47)％ in the model control mice,and that of the IP-10 middle-late intervene group was ( 86.56± 12.47 ) ％ without significant difference between them ( t =1.26,P＞0.05 ).Two days or four days after IP-10 early intervene,the expressions of chemokine receptor type 3 ( CXCR3 ) in corneal tissue were significant higher than model control group( t =3.13,3.07,P＜0.05 ),but the expressions of vascular endothelial growth factor (VEGF) in cornea were lowed ( t =5.99,6.27,P＜0.01),and so were transforming growth factor-β1 (TGF-β1) (t =8.50,P＜0.01;t =4.53,P＜0.05).Conclusions The early topical administration of the exogenous mouse IP-10 can inhibit CNV by up-regulating CXCR3 expression and down-regulating VEGF and TGF-β1 expression in cornea.However,middle-later usage of the IP-10 is uneffective.
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Background Corneal chemical burns,especially hazards of alkali burn become increasingly prominent.Clinically,anti-inflammatory,immuno-suppression,corneal transplantation are the common treating method for corneal alkali burn.But the research of tissue repair under the microenvironment of corneal alkali burn is necessary.Objective The aim of this study was to investigate the therapeutic effect of deproteinised calf serum eye gel on the corneal alkali burn.Methods Alkali burn model of cornea was established on the right eyes by putting the filter paper with 0.5 mol/L NaOH on the center cornea for 1 minute in 24 white rabbits.The model rabbits were divided randomly into 4 groups.Normal saline solution,deproteinised calf serum eye gel,blank matrix gel or recombinant bovine basic fibroblast growth factor(rb-bFGF)eye-gel was topically administered 4 times per day for 14days in the 4 groups,respectively.The inflammatory reaction was examined under the slit lamp and scored based on Ando' s criteria.Corneal fluorescine staining was performed to calculate the corneal ulcer area and scored based on Trousdale' s criteria.Histopathological examination of corneas was performed on the fourtcenth day after experiment.The use of the experiment animals complied with ARVO Statement.Results Corneal edema and opacification were seen in the model eyes with the modeling successful rate 100％.On the seventh day after experiment,the severe ulcer of cornea and hypopyon appeared in the normal saline solution group.Corneal epithelium was intact but the intrarocular structure was invisible in the blank matrix gel group.In th(c) rb-bFCF group,corneal new vessels were seen,however,the corneal ulcer completely regrow in the deproteinised calf serum eye gel group.In 3,5,7,10 and 14days after examination,the corneal inflammatory scores were significantly lower in the deproteinised calf serum eye gel group and rb-bFGF group than those of the normal saline solution(P＜0.01).No significant difference was found in the inflammatory score between the deproteinised calf serum eye gel group and rb-bFGF group (P＞0.05) but was significantly lower than the blank gel matrix group (P ＜ 0.05).With respect to the corneal ulcer,the score was decreased in the deproteinised calf serum eye gel group compared with the normal saline solution group and blank gel matrix group (P ＜ 0.05).Howcver,no significant difference was found in the corneal ulcer score between the deproteinised calf serum eye gel group and rb-bFGF group in various time points (P＞ 0.05).Conclusions Deproteinised calf serum eye gel can promote the healing of corneal ulcer and remit the inflammatory response afler corneal alkali burns with a better effectiveness than rb-bFGF.
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Background Corneal neovascularization ( CNV ) is a complication of many ocular surface diseases.It often worsen the pathological course.Effective therapy for CNV is still researching. Objective This study was to investigate the inhibitory effect of irradiation on CNV. Methods CNV models were established in 70 right eyes of 70 clean Wistar rats by corneal alkali burning.The models were randomized into β ray 10 Gy once irradiation group( 2 eyes),β ray 7 Gy multiple irradiation group( 17 eyes),β ray 10 Gy multiple irradiation group( 17 eyes),1％ cyclosporin A ( CsA ) eye drops group ( 17 eyes) and model group ( 17 eyes),and 6 matched normal rats were used as normal controls.All treatments started from the first day of the corneal alkali burning.CNV length and area were measured under the slit lamp every day.Corneal samples and homogenate were prepared 3,5,7 days after corneal alkali burning.The expressions of bcl-2,bax,vascular endothelial growth factor(VEGF) in rat corneas were detected by immunochemistry,VEGF proteins and VEGF mRNA were detected by Western blot and reverse transcription polymerase chain reaction (RT-PCR),respectively. Results Corneal ulceration was found in the βray 10 Gy once irradiation group and β ray 10 Gy multiple irradiation group.CNV length and area were much less in the β ray 7 Gy multiple irradiation group and 1％ CsA eye drops group compared with the model group on the seventh day after experiment( length:q=14.40,24.20,P＜0.01 ;area:q=17.80,14.00,P＜0.01 ).Immunochemistry revealed that compared with the model group,expressions of bcl-2 and VEGF proteins were weaker,but the expression of bax protein was stronger in the β ray 7 Gy multiple irradiation group and 1％ CsA eye drops group.RT-PCR showed that the expression of VEGF mRNA in cornea was lower in the β ray 10 Gy multiple irradiation group,β ray 7 Gy multiple irradiation group and 1％ CsA eye drops group in comparison with that in model group,and the results from Western blot showed the same pattern as RT-PCR. Conclusions Low dose irradiation of 90Sr-90Y ophthalmic applicator inhibits CNV formation after alkali burn.The study provide a new understanding of the irradiation for the treatment of CNV.
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Background Microvessels are composed of two interacting cell types: endothelial cells and pericytes. Over the past decades, studies of corneal angiogenesis have concentrated mainly on endothelial cells, while interest in pericytes has lagged behind. Objective The present study aimed to investigate the recruitment of vascular endothelial cells and pericytes in rat corneas after alkali burn. Methods Corneal alkali burn models were established in the right eyes of 36 adult SPF SD rats by putting 4 mm medicators containing a 1% 1 mol/L NaOH solution at the central corneas for 30 seconds, and 3 matched normal rats were used as controls. Corneas were excised 1,2,3,5,7 and 10 days after surgery. Frozen sections that parallel with the corneoscleral limbus were constructed. Double immunofluorescence staining was used to observe the dynamic expression of CD31 and α-smooth muscle actin (α-SMA) in corneal tissue for the evaluation of the number of endothelial cells and pericytes. The pericyte coverage index (PCI) was calculated to quantify the recruitment of pericytes to neovascular sites. The use of experimental animals followed the Statement of Association for Research in Vision and Ophthalmology. Results CD31 was expressed in the superficial stromal layer of the cornea on the 1 st day, showing the presence of red fluoresence. The positive cell number for CD31 was gradually increased with the passage of time and proceeded into the deep stromal layer from days 2 through 5 but decreased after that. However,α-SMA was positively expressed on the 2nd day in the cornea after alkali burn with the presence of green fluorescence. The positive cell number for α-SMA was less than those of CD31 throughout the experimental period. The PCI was 0, 16.07%, 11.95%, 43.84%, 73.97% and 86. 21% , respectively, 1,2,3,5,7 and 10 days after surgery. Conclusion Pericytes recruitment to corneal new vessels may play a key role in the stabilization and maturation of angiogeneis.
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PURPOSE: The therapeutic effects of amniotic membrane transplantation (AMT) contain anti-fibrosis, anti-inflammation and facilitation of epithelialization. Thus we investigated whether applying of amniotic membrane (AM) ointment could get the same effect as AMT. METHODS: Alkali burn was induced by applying 6 mm round filter paper which was soaked with 1 N NaOH, onto the central cornea for 60 seconds on both eyes of 10 white rabbits. Then we applied AM ointment on one eye and base ointment on the other eye, four times a day for 1 week. The corneas were harvested for histopathologic studies at 1 day and 3 weeks. Corneal opacity and thickness were measured in 3 days and 1, 2, 3 weeks. RESULTS: In the AM ointment applicated group, the number of the infiltrated PMNs (polymorphonuclear cells) and apoptotic keratocytes were significantly less than that of the control group (p< 0.05). The degree of lipid peroxidation and myofibroblast differentiation were less than those of the control group. Corneal opacity and corneal edema were less in AM ointment group than control group. CONCLUSIONS: AM ointment application after alkali burn is beneficial to reduce inflammation, keratocyte apoptosis and lipid peroxidation, and is considered to suppress corneal haze by these effects. Therefore, this report may be a basic study for the AM ointment research to treat recalcitrant keratitis.
Subject(s)
Rabbits , Alkalies , Amnion , Apoptosis , Burns , Cornea , Corneal Edema , Corneal Opacity , Inflammation , Keratitis , Lipid Peroxidation , MyofibroblastsABSTRACT
PURPOSE: Amniotic membrane (AM) contains various proteinase inhibitors and when used as a graft, it could enhance healing process by blocking insult of inflammatory cells and inhibiting proteolytic damage. Thus we evaluated whether applying of amniotic membrane extract as eyedrops could get the same effect as amniotic membrane patching. METHODS: Alkali wounds were inflicted on the central corneas of rabbits by applying a round filter paper, 6.0 mm in diameter, soaked in 1N NaOH for 30 seconds. A total of 16 rabbits were divided into four groups: (1) applied with amniotic membrane extract; (2) applied with amniotic membrane extract and Healon(R); (3) applied with methylcellulose; and (4) control. Each material was applied for 1 week. During follow-ups, epithelial defects, corneal thickness and its opacity were measured. RESULTS: The epithelial healing was faster and the corneal thickness was thinner in amniotic membrane extract applied groups than in non-applied. Corneal opacity was much less in AM extract applied groups. CONCLUSIONS: AM extract as eyedrops promotes wound healing and it could be an effective method for treating various keratitis due to its convenience and good effect.
Subject(s)
Rabbits , Alkalies , Amnion , Burns , Cornea , Corneal Opacity , Follow-Up Studies , Keratitis , Methylcellulose , Ophthalmic Solutions , Transplants , Wound Healing , Wounds and InjuriesABSTRACT
A retrospective study was made for 12 penetrating keratoplasties (9 patients) for corneal opacity due to severe corneal alkali burn from January 1987 to December 1991 at St. Mary's Hospital, Catholic University Medical College. 5 eyes which received penetrating keratoplasty within a year after alkali burn have never improved in visual acuity, but 3 out of 7 eyes which received penetrating keratoplasty after a year obtained improved visual acuity during the follow up period of 6 months After keratoplasty, the corrected visual acuity of 11 out of the 12 transplants (91.7%) were temporary improved in comparision with the preoperative levels. But 3 eyes (27.2%) have kept improved visual acuity during the follow up period of 6 months. The relationship between preoperative corneal scarring type according to Kinoshita and Manabe's classification and postoperative corneal transparency was reviewed; 3 among 6 eyes with scarring type A have kept clear cornea, but all of 6 eyes with scarring type B were noted to have .opaque cornea during the follow up period of 6 months.