Effects of salicylate and steroid on neutrophil migration and corneal blood vessel growth

Cauterization of the cornea results in emigration of neutrophils from the limbal blood vessels into the corneal tissue. Blood vessel proliferation follows, the stimulus for which is unknown. In this study, 0.1 M sodium salicylate drops administered topically to cauterized rat corneas over a 48-h period had an inhibitory effect on the migration of neutrophils from the limbal vessels 6 h after injury, but this was not maintained at 48 h. After 6 days of treatment, the salicylate had no effect on vessel growth into the cauterized rat cornea. Application of prednisolone disodium phosphate ointment to cauterized corneas also inhibited neutrophil migration at 6 h, but increased the extravascular neutrophils at 48 h. After 6 days of treatment, corneal blood vessel growth was significantly reduced. It was concluded that there is no consistent relation between the number of extravascular neutrophils at the corneal limbus and the extent of corneal blood vessel growth.     

Limbal autografting: comparison of results in the acute and chronic phases of ocular surface burns

PURPOSE: To compare outcomes of limbal autograft transplantation (LAT) in the acute and chronic phases of ocular surface burns. METHODS: Retrospective analysis of case records of 16 consecutive patients who underwent LAT for ocular surface burns, at our institute, between April 1994 and March 1997. RESULTS: Limbal autograft transplantation was successful in reconstructing the corneal surface and restoring ocular comfort in 15 (93.8%) eyes. Limbal autografting failed to reconstruct the ocular surface in one patient undergoing surgery 2 weeks after grade IV alkali burns. In 13 eyes with counting fingers or worse vision, functional success (visual acuity >20/400) was attained after LAT in nine (69.2%) eyes. Visual acuity > or = 20/80 was achieved in two (25%) of eight eyes undergoing surgery for a persistent epithelial defect (PED) and five of six (83.3%) eyes undergoing surgery after the epithelial defect had healed (p = 0.03). Nine patients underwent simultaneous superior and inferior limbal autografting. Mean epithelial healing time in six of these patients undergoing surgery in the acute phase of injury (<4 months) was 15+/-6.1 days. In three patients undergoing a similar procedure in the chronic phase of injury, the healing time was 8.3+/-6.7 days. CONCLUSIONS: Limbal autograft transplantation is successful in reconstructing the corneal surface and restoring ocular comfort after ocular surface burns. Surgery in the acute phase of injury (<4 months), in the presence of a PED, could result in delayed corneal reepithelialization and poorer visual prognosis. If performed in the acute phase of injury, LAT should be performed after adequate limbal vascularization and resolution of surface inflammation in the recipient eye, avoiding graft placement over ischemic limbus.     

活体异体结膜缘和羊膜移植治疗化学性眼外伤造成的角膜缘干细胞缺失(英文)

目的:评价利用活体异体结膜缘和羊膜移植治疗化学性眼外伤造成的角膜缘干细胞缺失的临床效果。方法:从2005-07/2007-12,本研究包括了9名男性化学性眼外伤患者(10眼)。所有患者接受了亲属活体异体结膜缘和羊膜移植,2例眼接受了睑缝术。用环孢菌素和泼尼松龙进行全身性免疫抑制。结果:在3例眼中观察到完全角膜上皮化(30%),其中1例在术后1.5mo出现免疫排斥,角膜溶解引起穿孔,加大全身性免疫抑制剂量来控制病情。3例眼中植片无法在角膜表面重新形成上皮,被定为原发性失败。其余4眼有部分上皮形成,但上皮细胞无法完全覆盖角膜表面。术前最佳矫正视力从手动到1m处数指,术后最佳矫正视力从光感到20/80。有5眼视力得到改进,不需其他治疗。手术失败的主要原因为干眼症和持续性炎症。结论:对于能控制泪量和眼部炎症的病例,亲属活体异体角膜缘和羊膜移植是治疗化学性眼外伤造成的角膜缘干细胞缺失最佳方法之一。     

The inflammatory milieu associated with conjunctivalized cornea and its alteration with IL-1 RA gene therapy

PURPOSE: This study was designed to gain an insight into the inflammatory milieu into which a donor limbal graft is routinely introduced. The objective of this study was to modulate this environment by gene therapy with the anti-inflammatory cytokine interleukin-1 receptor antagonist (IL-1 RA). METHODS: In a mouse model, the ocular surface cytokine environment associated with a conjunctivalized cornea was assessed 4 weeks after injury. Total corneal epithelial and limbal debridement was performed with a combination of alkali and scrape injury. The cytokines and adhesion molecules measured included IL-1alpha, IL-1beta, IL-6, VEGF, intercellular adhesion molecule (ICAM)-1, and vascular adhesion molecule (VCAM)-1, by real-time PCR or ELISA. Injured corneas were transfected with IL-1 RA by injection of naked plasmid vector pIRES-EGFP-IL-1 RA immediately after injury. Corneas transfected with pIRES-EGFP served as the control. Expression of corneal IL-1 RA after transfection with pIRES-EGFP-IL1-RA was assessed over a 2-week period by real-time PCR and Western blot analysis. In addition, limbal stem cell grafts transfected with IL-1 RA were assessed for leukocyte influx. RESULTS: Conjunctivalized corneas showed increased expression of IL-1alpha, IL-1beta, IL-1 RA, IL-6, VEGF, ICAM-1, and VCAM-1, compared with normal cornea. Transfection-efficiency experiments indicated that corneal expression of IL-1 RA peaked between 12 and 24 hours and lasted up to 2 weeks after the initial transfection. IL-1 RA corneal gene therapy resulted in a downregulation of IL-1beta and VCAM-1 expression at 4 weeks after injury, whereas downregulation of IL-6 was evident only at 1 week after injury. Corneal neovascularization was also reduced. In addition, corneal limbal stem cell grafts transfected with IL-1 RA showed a decreased leukocyte influx compared with control grafts. CONCLUSIONS: Transfection of a cornea with IL-1 RA immediately after epithelial injury selectively altered the cytokine profile of the resultant conjunctivalized cornea and suppressed corneal neovascularization. Transfection of corneal limbal donor tissue with IL-1 RA before engraftment can reduce leukocyte influx into the graft. The findings demonstrate the feasibility of using transient cytokine gene expression, either in donor or recipient corneal tissue, to alter the ocular surface environment beneficially.     

大鼠角膜缘上皮细胞培养与同体移植的实验研究

目的：观察以明胶为载体培养的角膜缘上皮细胞移植治疗角膜缘干细胞缺乏症的疗效。 方法：大鼠角膜缘上皮细胞在铺有明胶载体的细胞培养板上进行培养5d后,角膜上皮细胞移植术前24h用3H胸腺嘧啶核苷标记培养的角膜缘上皮细胞,培养标记的角膜缘上皮细胞对角膜缘干细胞缺乏的大鼠动物模型行角膜缘上皮细胞同体移植术,对移植术后角膜进行观察、病理学检查及同位素检测。 结果：大鼠角膜缘上皮细胞可以在明胶载体上培养、增殖、分化为密集角膜上皮细胞层;角膜缘上皮细胞移植术后角膜上皮完整、基质细胞浸润减轻、新生血管减少。病理学检查角膜缘及周边部上皮细胞为多层结构,角膜新生血管消失及基质中炎性细胞浸润减轻。角膜缘上皮细胞移植术后4wk受眼角膜仍可测到3H胸腺嘧啶核苷。 结论：角膜缘上皮细胞移植治疗角膜缘干细胞缺乏症可恢复角膜缘干细胞缺乏病变角膜上皮结构的完整性,减少角膜新生血管的形成,维持角膜缘干细胞的屏障功能,为角膜移值提供更好的条件。     

Expression of HGF, KGF, EGF and receptor messenger RNAs following corneal epithelial wounding

Hepatocyte growth factor (HGF), keratinocyte growth factor (KGF), epidermal growth factor (EGF), and their receptors have been associated with homeostasis and wound healing in the cornea. The purpose of this study was to examine the expression of the messenger RNAs for these growth factors and receptors in a wounded series of mouse corneas using in situ hybridization. In situ hybridization was performed with 3H-labeled riboprobes on unwounded corneas and corneas at 30 minutes, 4, 12, 24, 48 and 72 hr, and 7 days after epithelial scrape wounds in Balb/C mice. Qualitative and semi-quantitative analyses were performed. Expression of HGF, KGF and EGF mRNAs in keratocytes in the unwounded cornea was low. EGF mRNA was also expressed in unwounded corneal epithelium. Following wounding, however, these growth factor mRNAs were markedly upregulated in keratocytes. EGF mRNA expression in the epithelium appeared unaffected by wounding. At seven days after wounding and several days following closure of the epithelial defect, HGF mRNA and KGF mRNA were still expressed at higher levels in keratocytes compared with unwounded corneas. No difference in expression of HGF or KGF mRNAs between limbal, peripheral corneal, or central corneal keratocytes was noted in the unwounded cornea, KGF receptor mRNA was prominently expressed throughout the unwounded corneal epithelium. HGF receptor mRNA and EGF receptor mRNAs were expressed at low levels in unwounded cornea epithelium. Following scrape injury, expression of HGF receptor mRNA and KGF receptor mRNA were markedly upregulated in the corneal epithelium, while no significant increase in EGF receptor mRNA expression was noted. These studies suggest a prominent role for HGF and KGF in modulating corneal epithelial wound healing following injury. Less prominent changes in EGF mRNA and EGF receptor mRNA in the corneal epithelium following wounding may suggest that EGF has more of a role in homeostasis in the mouse corneal epithelium.     

羊膜移植对碱烧伤大鼠角膜缘干细胞的影响

目的观察羊膜移植对碱烧伤大鼠角膜缘干细胞的影响。方法SD大鼠40只制作眼碱烧伤模型,单眼行眼碱烧伤;随机选取20只大鼠（20眼）为实验组,20只大鼠（20眼）为对照组;实验组行新鲜羊膜移植,对照组不处理,观察大鼠眼表情况。并于术后1周、2周、3周、4周取角膜缘组织,采用免疫组化技术观察p63蛋白在角膜缘干细胞的表达情况。结果p63在实验组和对照组的角膜缘干细胞中均有表达,位于角膜缘上皮细胞基底层的细胞核内。但实验组明显高于对照组,两者比较有统计学意义（P〈0．05）。且随着烧伤时间的延长,p63的表达呈线性下降趋势。结论眼碱烧伤后行羊膜移植,能促进角膜缘干细胞的增生、分化,利于角膜上皮的修复。     

Neurotrophic corneal ulcer development following cataract surgery with a limbal relaxing incision

A 60-year-old man with bilateral corneal opacity underwent cataract extraction surgery involving the use of a limbal relaxing incision in his left eye. He had lower lid ectropion and lagophthalmos in both eyes. Eleven days after the surgery, a slit-lamp examination revealed a neurotrophic corneal ulcer with a punch-out epithelial defect and rolled edges at the center of the pre-existing corneal opacity. The patient was treated with sodium hyaluronate, autologous serum, and oral doxycycline. Six weeks after the surgery an improvement in corneal sensation was observed and the neurotrophic corneal ulcer subsequently healed over the course of one year. In this report, we present a case of neurotrophic keratitis that occurred after performing cataract surgery concurrent with a limbal relaxing incision. As such, we suggest that limbal relaxing incisions should be performed cautiously in patients with causative risk factors for corneal hypesthesia.     

原发性翼状胬肉两种治疗方法的疗效观察

目的:探讨原发性翼状胬肉两种治疗方法的疗效观察。方法:A组25例采用翼状胬肉切除联合丝裂霉素术治疗,B组10例采用自体球结膜+角膜缘干细胞移植术治疗。根据角膜创面恢复、结膜伤口愈合、胬肉有无复发进行观察。结果:A组患者术后角膜上皮第3～7d恢复,结膜充血10～15d消退,3例复发;B组患者术后角膜上皮第2d恢复,结膜充血7～10d消退,1例复发。结论:翼状胬肉切除术联合丝裂霉素和自体球结膜+角膜缘干细胞移植术都能降低胬肉复发率,二者相比较,自体球结膜+角膜缘干细胞移植术优于翼状胬肉切除联合丝裂霉素。     

Phacoemulsification after penetrating keratoplasty with autologous limbal transplant and amniotic membrane transplant in chemical burns

We report a patient who had earlier penetrating keratoplasty with amniotic membrane transplant and autologous limbal cell transplant for chemical injury who underwent cataract surgery by phacoaspiration. A posterior limbal incision with corneal valve was made superotemporally with extreme caution to avoid damage to the limbal graft. Aspiration flow rates and vacuum were kept low to avoid any turbulence during surgery. A 6.0 mm optic diameter acrylic foldable intraocular lens was inserted in the bag. The patient achieved a best-corrected visual acuity of 6/12 at 10 months' follow-up with a clear corneal graft. We conclude that caution during wound construction and phacoaspiration can help preserve corneal and limbal graft integrity in patients undergoing cataract surgery after corneal graft and limbal transplantation.     

骨髓间充质干细胞在角膜损伤修复中的应用和研究进展

骨髓间充质干细胞(bone marrow mesenchymal stem cell,BMSC)可在体内外培养诱导分化为角膜上皮细胞、角膜缘上皮细胞和角膜基质细胞等。BMSC移植后可分化为角膜上皮细胞,有效修复损伤的角膜上皮细胞组织,能表达多种细胞因子,减少炎症细胞浸润,减轻细胞损伤,并抑制细胞凋亡,降低角膜移植后的排斥反应; BMSC还能分化促进角膜缘干细胞增殖。也可在载体上利用BMSC构建生物角膜,进行眼表修复,此为基础研究提供了新的研究方向,为临床治疗角膜损伤类疾病提出了新的临床治疗思路,在基础研究和临床运用方面都有非常巨大的潜力和广阔的前景。本文就BMSC在角膜损伤修复中的研究现状和进展进行综述。     

The potential for eye bank limbal rings to generate cultured corneal epithelial allografts.

PURPOSE: Patients with severe limbal deficiencies are unable to maintain a stable corneal surface. If sheets of cultured allogeneic corneal epithelium could be prepared from eye banked corneal limbal rings, which are normally discarded after keratoplasty, the sheets may be beneficial for grafting onto patients with limbal stem cell deficiencies. METHOD: Biopsies of limbal tissue (2-3 mm2) removed from organ-cultured corneal limbal rings or from fresh whole globes were either trypsinized or set up as explants to assess their potential for corneal epithelial cell production. RESULTS: Several biopsies were taken from each of 21 organ-cultured limbal rings and 10 fresh cadaveric globes. Cultures were generated from every cadaveric eye (10/10), although not all biopsies from the same eye gave rise to cultures. Confluent sheets of cultured cells were also produced successfully from limbal rings that had been in organ culture for up to 25 days, but the success rate from limbal ring material was variable (14/21). An analysis of parameters associated with each limbal ring was carried out in an attempt to identify the reasons for the different efficiencies of epithelial production. No obvious single parameter correlation was detected, although there was a trend to poorer efficiency with increased donor age. CONCLUSIONS: Confluent sheets of cultured corneal epithelial cells, suitable for grafting, can be produced from limbal tissue taken from eye bank organ-cultured corneas, although it takes longer, on average, to reach confluence (17-21 days) than an equivalent sample from a fresh eye (9-12 days).     

兔同种异体角膜缘移植角膜印迹细胞学检测

目的 研究兔角膜缘干细胞缺乏和同种异体角膜缘移植后临床和角膜上皮表型的改变。方法 建立兔角膜缘干细胞缺乏模型,1个月后对治疗组进行同种异体角膜缘移植,术后联合使用免疫抑制剂。比较治疗组与非治疗组的临床表现和角膜表型的改变。结果 兔角膜缘干细胞缺乏后,角膜混浊,新生血管化,持续性上皮缺损;角膜上皮为结膜细胞表型。移植术后角膜上皮完整,新生血管减少,角膜透明度增加;上皮恢复角膜表型。结论 兔同种异体角膜缘移植联合术后使用免疫抑制剂是治疗角膜缘干细胞缺乏症的有效方法。印迹细胞学检查是角膜缘干细胞缺乏症诊断和角膜缘移植术后的评价手段。     

Early limbal autograft after alkali burn of the ocular surface

Limbal autograft transplantation is recommended for treatment of limbal stem cell destruction and its complications. It is appropriate to restore corneal reepithelialization and arrest corneal vascularization and scarring. In unilateral alkali injury, limbal transplantation was first recommended for late management of corneal conjunctivalization. Our patient had severe ocular unilateral alkali injury. Early autologous limbal transplant was effective in restoring an excellent corneal surface and a good visual function.     

Amniotic membrane as a substrate for cultivating limbal corneal epithelial cells for autologous transplantation in rabbits

PURPOSE: To examine the viability of using human amniotic membrane as substrate for culturing corneal epithelial cells and transplanting them onto severely injured rabbit eyes. METHODS: An ocular-surface injury was created in the right eye of eight rabbits by a lamellar keratectomy extending 5 mm outside the limbus. Next, from the limbal region of the uninjured left eyes of five of these animals, a small biopsy of corneal epithelial cells was taken and cultured on acellular human amniotic membrane. One month later, the invading conjunctiva that covered the corneal surface of all eight injured eyes was surgically removed. Five of the eyes then received grafts of amniotic membrane containing autologous cultured epithelial cells, whereas the other three received grafts of acellular amniotic membrane alone. RESULTS: A confluent primary culture of limbal corneal epithelial cells was established on acellular human amniotic membrane after 14 days. Cells were partially stratified and fairly well attached to the underlying amniotic membrane, although a fully formed basement membrane was not evident. The three rabbits that received amniotic membrane transplantation alone all had total epithelial defects on the graft in the early postoperative period. Eyes that were grafted with amniotic membrane that contained cultivated epithelial cells, however, were all successfully epithelialized up to 5 days after surgery. CONCLUSION: Autologous transplantation of cultivated corneal epithelium is feasible by using acellular amniotic membrane as a carrier.     

Limbal autograft and allograft transplantations in patients with corneal burns

AIM: To investigate and compare the surgical outcomes of limbal autograft and limbal allograft transplantations in patients with corneal burns. METHODS: In total, 20 patients (n=22 eyes) with chemical burn and two patients (n=2 eyes) with thermal burn were included in this study. Limbal autograft or limbal allograft transplantation surgery was performed in all patients. HLA-typing was tested before allograft surgeries. Limbal allografting was performed in all eyes using donor tissue from live relatives. Systemic cyclosporine A was administered for immunosuppression. RESULTS: The corneal surface was successfully reconstructed in all eyes (100%) after limbal autografting, two eyes required additional amniotic membrane transplantation and one eye required allografting. The mean follow-up period for limbal autografts was 13.9 +/- 7.0 months. Limbal allografting failed to reduce corneal vascularity and opacification in five (55.6%) eyes and was successful only in four (44.4%) eyes (mean follow-up 16.2 +/- 11.2 months) (P=0.002). In all, 15 eyes undergoing limbal autografting completed re-epithelialization of the cornea at a mean of 35.6 +/- 60.2 days. The mean epithelial healing time in nine eyes undergoing limbal allografting was 13.0 +/- 7.3 days (P=0.525). After limbal autografting, functional vision (> or =1/10) was attained in 12 (80%) eyes. Only one eye (11.1%) achieved functional vision after limbal allografting (P=0.036). Penetrating keratoplasty was performed in three patients following limbal allografting. No cyclosporine-associated side effects were observed. CONCLUSIONS: Limbal autograft transplantation is an effective and safe procedure for unilateral corneal burns. It seems that limbal allograft transplantation is better combined with penetrating keratoplasty for a better visual outcome and higher graft survival rate. Systemic immunosuppression seems to be necessary for limbal allografts even in the presence of HLA-matched donor tissues.     

Stepwise surgical approach for in vivo expansion of epithelial stem cells to treating severe acute chemical burns with total limbal deficiency

To describe the clinical outcome of a new surgical treatment for the acute stages of severe corneal burn injury and its complications, a prospective study of five acute corneal burn patients with severe limbal damage was performed. Amniotic membrane transplantation (AMT) and conjunctival limbal autograft (CLAU) was performed at the acute stage of corneal burn injury to reconstruct the damaged ocular surface (step I). Three to six months later, the opaque central part of the amniotic membrane containing in vivo grown corneal stem cells were removed and retransplanted to the defect created after the removal of pseudopterygium (step II). All injured eyes were successfully treated, but in one eye with marked stromal lysis, three-layered AMT and penetrating keratoplasty with retransplantation of in vivo grown corneal stem cells was performed. In the former cases, visual acuity was greatly improved more than three lines (ranging from 3 to 12 lines). In short, retransplantation of in vivo grown corneal stem cells after AMT and CLAU is a recommendable modality for restoring a stable corneal epithelium of a severely burned ocular surface in the acute stage and can be considered a preventative measure for avoiding late onset complications.     

Corneal vascularization in hydrogel contact lens wearers

Limbal injection, peripheral corneal edema, and adjacent leukocyte infiltration may all be involved in the neovascularization of corneal tissue. Hydrogel lenses should be made with a minimum average thickness an maximum water content to increase oxygen transmission to the cornea. Lenses should not compress the limbal area in order to decrease the risk of limbal injection and, therefore, possible leukocyte invasion. Following these guidelines will decrease the risk of corneal vascularization in the hydrogel contact lens wearer. Keeping these principles in mind will also help the practitioner evaluate corneal health in all patients.     

Protein kinase C subspecies in rabbit corneal epithelium: increased activity of alpha subspecies during wound healing.

Protein kinase C (PKC) has been implicated in cell proliferation and differentiation. Multiple forms of PKC have been isolated, principally from the brain where PKC is most abundant. In rabbit corneal epithelium, two distinct major peaks of PKC activity were resolved by hydroxyapatite column chromatography. Peak 2, with 65% of the total PKC activity, corresponds to alpha-PKC, based on its mobility in the column and Western blot analysis using specific monoclonal antibodies. Peak 1 did not react with either polyclonal or monoclonal antibodies to PKC alpha-, beta-, and gamma-isoforms suggesting the presence of isoforms specific to the corneal epithelium, or of another member of the PKC family. To investigate possible changes in the amounts of the various PKC subspecies during wound healing, the enzyme activities of the isolated subspecies were assayed 2, 5, and 7 days after corneal de-epithelialization. Two days after wounding, by which time the migratory limbal epithelium had covered the denuded area, total PKC activity was unchanged but alpha-PKC activity had increased to 77% of the total activity, compared with 65% in non-wounded epithelium. An increased proportion of alpha-PKC activity was also observed 5 and 7 days after wounding, during which time proliferation of epithelium continued. We hypothesize that alpha-PKC plays a role in long-term responses after injury such as gene expression and corneal epithelial proliferation. Moreover, these studies indicate that the cornea provides a good model of in vivo wound healing for PKC studies.     

Lipopolysaccharide from Escherichia coli induces the expression of vascular endothelial growth factor via toll-like receptor 4 in human limbal fibroblasts

Corneal neovascularization can be induced by a severe ocular infection, injury or immunological diseases. The vascular endothelial growth factor (VEGF) is the main cytokine involved in this phenomenon, inducing angiogenesis from the vascularized ocular tissues. As the limbal tissue is located between conjunctival and corneal tissues, we suggest that the limbal cells are participating in the production of VEGF induced by bacterial components as LPS. In this work, RT-PCRs and immunoblots were used to investigate the expression of VEGF and other pro-angiogenic genes in primary cultures of human limbal fibroblasts (PCHLF) treated with lipopolysaccharide (LPS) from Escherichia coli. We found that the expression of VEGF was initiated at 6 h and reaches its highest expression at 72 h after stimulation with LPS. Up-regulation of toll-like receptor 4 (TLR4) after 3 h of treatment was also observed. LPS-induced the expression of VEGF in a dose-dependent manner, and the blocking of TLR4 with an anti-TLR4 antibody prevented VEGF expression. We also analyzed the molecules that modulate VEGF expression. LPS did not induce the up-regulation of LL-37 nor the hypoxia induced factor 1 alpha (HIF-1alpha) mRNA expression, however, an up-regulation of interleukin 13 receptor alpha 1 (IL-13Ralpha1) and interleukin 4 receptor alpha (IL-4Ralpha) were observed after 3 and 12 h of stimulation, respectively. The expression of interleukin 13 did not change throughout the treatment. These results suggest that TLR4, IL-13Ralpha1 and IL-4Ralpha induced by LPS in PCHLF could be playing an important role in the corneal neovascularization.