Volume 42 ·  Issue 08 ·  2006

Keratopathy is one of the main blinding eye diseases in China, and the improvement of its clinical diagnosis and treatment level depends on the depth and breadth of basic and applied basic research. Some fields of basic and applied basic research on keratopathy in China have been at the international advanced level. However, generally speaking, there is still a big gap between basic research on keratopathy in China and developed countries, which is embodied in the lack of systematic research, limited research funds, and insufficient quality and quantity of researchers. In the future, basic research on keratopathy in China should mainly focus on the pathogenesis of infectious keratopathy, the pathogenic genes of corneal dystrophy, corneal tissue engineering, the pathogenesis of dry eye, tissue reconstruction of severe ocular surface chemical injury, the pathogenesis and prevention of ocular surface neovascularization, and the development of therapeutic drugs for transplant rejection.

The basic and clinical research of limbal stem cells promotes the development of ocular surface diseases.This paper analyzes the anatomical characteristics, proliferative ability, the role and existing problems of surface marker proteins in the identification of limbal stem cells, puts forward the clinical methods for diagnosing limbal stem cell deficiency, and summarizes the therapeutic effects and cell outcomes of various limbal epithelial transplantation, and puts forward the key problems to be solved for further research on limbal stem cells.

Objective to evaluate the clinical effect of autologous limbal stem cell transplantation using fibrogel membrane as carrier in vitro for the treatment of limbal stem cell deficiency. Methods A rabbit model of complete limbal stem cell deficiency was made. Stem cells were cultured from limbal tissue of contralateral eye, and tissue engineered corneal epithelium was made using fibrogel membrane as carrier. The experimental animals were randomly divided into 4 groups, among which groups Ⅰ ~ Ⅲ were the experimental groups, and tissue engineered corneal epithelium transplantation was performed. Group Ⅰ was observed for 3 months after transplantation, group Ⅱ was observed for 1 month after transplantation, group Ⅲ was observed for 2 weeks after transplantation. Group Ⅳ was the control group, and the gel membrane without stem cells was transplanted for 3 months after transplantation. The clinical efficacy was evaluated by corneal epithelial staining, corneal opacity and neovascularization. The corneal epithelial repair at different postoperative stages was evaluated by pathological examination, and the cell phenotype of corneal epithelium before and after transplantation was examined by blotting cytology. Immunohistochemical staining was used to observe the expression of keratin K3, MUC5AC and transcription factor p63 in transplanted corneal epithelium. Results the corneal epithelium healed gradually, the transparency increased, and the neovascularization decreased or disappeared in the experimental group; In the control group, the cornea showed persistent opacity, the epithelial defect persisted and did not heal, and finally a large number of new blood vessels grew into the epithelium, and the epithelium became conjunctival. The difference was statistically significant (P =0.021). The corneal epithelial imprinting cytology showed that the conjunctival cell phenotype was PAS (+) in the control group, while the epithelial cell phenotype was PAS (-) in the experimental group. Histopathology showed that the experimental group showed normal corneal epithelial structure, while the control group showed conjunctival metaplasia epithelium, with blood vessels and goblet cells. Immunohistochemistry showed that the corneal epithelium of the experimental group expressed corneal epithelium-specific keratin K3 (AE5), but did not express conjunctival-specific MUC5AC, and expressed transcription factor p63 in the epithelial base. The epithelium of the control group continued to express MUC5AC. Conclusion Tissue engineering corneal epithelial transplantation is an effective method for the treatment of limbal stem cell deficiency. It can reconstruct ocular surface, repair corneal epithelial damage, inhibit corneal epithelial conjunctivization and neovascularization. Fibrogel membrane is a new tissue engineering material with fast absorption, high transparency and good histocompatibility, and is an ideal carrier material for transplantation.

Objective to investigate the efficacy and safety of PEGFP-IL-1ra plasmid injected into rabbit corneal stroma mediated by linear polyethyleneimine (PEI). Methods the recombinant plasmid PEGFP-hIL-1ra was constructed by polymerase chain reaction (PCR) using human cDNA library as template. Corneal endothelial cells were transfected with cationic polymer, and the expression of IL-1ra gene and protein after transfection was detected by green fluorescent protein (GFP) tracer and Western blot technique.30 Wistar rats in the experimental group were injected with 20 μ l of mixed solution of pEGFP-hIL-1RA plasmid and PEI-in-vivo (containing 10 μ g plasmid) in the corneal stroma, and 15 Wistar rats in the control group were injected with 20 μ l of PEI-in-vivo solution. At 1, 3, 6, 14 and 21 days after injection, the corneas were collected by HE staining, transmission electron microscopy, trypanosome blue-alizarin red staining and immunohistochemistry. The expression site and intensity of IL-1ra-GFP fusion protein in cornea were observed under fluorescence microscope. Results the recombinant plasmid PEGFP-hIL-1ra was amplified by cDNA library as a template, and PEGFP-hIL-1ra was constructed. The direction and size of the inserts were confirmed by PstI and BamHI digestion and DNA sequencing. After 12 hours of PEGFP-hIL-1ra transfection in corneal endothelial cells mediated by PEI-in-vitro, GFP fluorescence expression was observed in 10% ~15% of the cells. Western-blotting detection showed the expression of hIL-1ra-GFP protein with relative molecular mass of 44 000. Fluorescence bands were observed in corneal epithelial basal cells 1 day after PEGFP-hIL-1ra plasmid and PEI-in-vivo corneal matrix injection, and the fluorescence intensity reached the peak at 6 days The combined staining of trypanosome blue and alizarin red showed no damage to corneal endothelial cells; Conclusion cationic polymer-mediated injection of PEGFP-hIL-1ra plasmid can quickly and effectively transfer IL-1ra gene into the cornea and express it, which provides a new technical platform for gene therapy of corneal immuno-inflammatory response-related diseases using anti-inflammatory cytokine IL-1ra.

Objective to investigate the histopathological characteristics of corneal wound healing after small incision deep lamellar corneal endothelial transplantation. Methods Forty-five guinea pigs were divided into recipient group (30 eyes) and donor group (15 eyes), and allogeneic deep lamellar corneal endothelial transplantation was performed. The right eye of the recipient group was the surgical eye, and the normal cornea of the left eye was the histopathological control. Two guinea pigs in the recipient group were sacrificed at 1, 2 weeks, 1, 2, 3 and 4 months after operation, respectively. The corneal tissues of the left and right eyes were stained with alizarin red, hematoxylin-eosin and/or periodate Schiff staining, and then light microscopy was performed to count the intrastromal cells of the cornea. Results The cornea of 22 eyes in the recipient group was transparent after operation. After the corneal endothelial cells stained with alizarin red, the morphology and arrangement of the endothelial cells on the graft were normal, and there was a circular endothelial cell abnormal area between the graft bed and the graft. The collagen fibers of the posterior lamella and the posterior elastic layer were interrupted and the stump of the latter was rolled up at the junction of the implant bed and the graft; The gap at the junction is filled by tissues with disordered structure and more cellular components; Conclusion the healing characteristics of stromal wound in small incision deep lamellar corneal endothelial transplantation are related to the form of injury, collagen is arranged neatly at the lamellar interface, and the healing mode of fibrosis can be seen at the junction between the graft and the bed.

Objective to investigate the expression of nuclear transcription factor kappa B (NF-κ B) and its induced cytokines in rat keratitis and the effect of pyrrolidine dithiocarbamate (PDTC) on its expression.Methods 112 rats were selected to establish a keratitis model, and divided into 56 rats in inflammatory group and 56 rats in PDTC pretreatment group according to random number table.30 minutes before the model was made, the rats were injected with normal saline (inflammatory group) and PDTC (PDTC pretreatment group) under the bulbar conjunctiva, respectively. Each group was divided into 0.5, 1.0, 3.0, 6.0, 12.0, 24.0 and 72.0 h subgroups according to different times after lipopolysaccharide (LPS) stimulation, with 8 rats in each subgroup. Morphological changes of cornea were observed by pathological tissue section; The expression of NF-κ B p65 in cornea was detected by immunohistochemical staining; The expression of corneal tumor necrosis factor α (TNF-α) mRNA was detected by reverse transcription polymerase chain reaction (RT-PCR). Results the corneal tissue in the inflammatory group was obviously edema after LPS stimulation, a large number of inflammatory cells infiltrated, and collagen fibers were disordered. Immunohistochemical staining showed that NF-κ B positive cells were observed at 0.5 h after LPS stimulation, and the expression of NF-κ B positive cells increased gradually, the strongest at 3.0-12.0 h, and the expression of NF-κ B positive cells increased significantly between 0.5-24.0 h compared with PDTC pretreatment group (P<0.01). The expression of TNF-α mRNA began to increase at 0.5 h after LPS stimulation, and was strongest at 3.0-12.0 h. The expression of TNF-α mRNA increased significantly between 0.5-24.0 h compared with PDTC pretreatment group (P<0.01). Conclusion NF-κ B and its induced TNF-α play an important role in keratitis, and PDTC may reduce corneal damage by inhibiting the activity of NF-κ B.

Objective to investigate the causes and changes of penetrating keratoplasty (PKP) in Shandong Ophthalmology Institute during 6 years from 1997 to 2002. Methods All PKP cases in Shandong Ophthalmology Institute from January 1997 to December 2002 were retrospectively investigated. The causes of PKP were divided into 10 categories: suppurative keratitis, herpes simplex keratitis, keratoconus, bullous keratopathy, leukoplakia, corneal rupture, corneal burn, re-PKP, genetically related corneal dystrophy and corneal degeneration and others. Results A total of 1702 patients with PKP were investigated. The causes of PKP were 542 (31.9%), 299 (17.6%), 219 (12.9%), 164 (9.6%), 118 (6.9%), 105 (6.2%), 77 (4.5%), 70 (4.1%), 67 (3.9%) and 41 (2.4%). The proportions of keratoconus (x2=26.399, P<0.001) and bullous keratopathy (x2=11.4229, P<0.001) showed an obvious upward trend, while the other causes showed no obvious change. Fungal infection ranked first in purulent keratitis (65.9%), and the proportion of each pathogen did not change significantly in the past 6 years. Most patients with bullous keratopathy had a history of intraocular surgery, The main reasons for reoperation were immune rejection (61.0%), graft infection (14.3%) and recurrence of herpes simplex keratitis (10.4%). Conclusion Infectious keratopathy is still the main indication of PKP, among which fungal infection is the first cause. Keratoconus and bullous keratopathy are both on the rise.

Objective to investigate the predictability, safety and effectiveness of implantation of angle-supported rigid intraocular lens in the anterior chamber in the treatment of high myopia. Methods The preoperative diopter was-9.50~ -26.25 D, mean (-15.89±3.78) D, and the preoperative best corrected visual acuity was 0.5~1.0. The mean follow-up time of implantation of angle-supported rigid intraocular lens (Phakic 6H) in the treatment of high myopia was 1 year. The changes of naked eye visual acuity, corrected visual acuity, diopter, intraocular pressure, corneal endothelium and anterior segment were observed before and after operation. Results One year after operation, the naked eye visual acuity was 0.3~1.5, the best corrected visual acuity was 0.5~1.5, the residual diopter was-2.00~ +0.50 D, mean (-0.40±0.64) D, and the diopter was within ≤ ±1.00D, accounting for 96.1%. The corneal endothelial cell counts were (3174±248) /mm2 and (3067±320) /mm2 before and after operation, respectively One patient had macular hemorrhage in both eyes at 8 months postoperative.Conclusion Phakic rigid intraocular lens with atrial angle support is safe and effective in the treatment of ultra-high myopia, and it is worthy of further clinical study, and the long-term efficacy and safety need to be further observed.

Objective to investigate the feasibility of non-contact method for measuring corneal thickness and the consistency between the results and the results of contact method.Methods 107 patients (211 eyes) were randomly sampled in our hospital before excimer laser subkeratomileusis (LASEK) and micro keratome excimer laser subkeratomileusis (Epi-LASIK). According to the degree of myopia, they were divided into 4 groups and measured by non-contact three-dimensional anterior chamber analyzer Pentacam and conventional contact A-ultrasound keratometer AL-3000 respectively.The results of central corneal thickness were compared in groups.Results The mean value of central corneal thickness measured by Pentacam system was (535.58±36.34) μ m, and the mean value of A-ultrasound was (536.73±37.18) μ m. The 95% confidence interval of the mean value difference was (-1.88, -0.42). The difference of central corneal thickness between the groups was not statistically significant (P>0.05). Conclusion The results of central corneal thickness measured by Pentacam system were slightly different from those of A-ultrasound, but there was no contact and no trauma.

Objective to investigate the clinical characteristics, diagnosis and differential diagnosis of acute zonal occult outer retinopathy (AZOOR). Methods Six patients diagnosed with AZOOR in our hospital and other hospitals in the past two years were examined by routine ocular examination, fluorescein fundus angiography, visual electrophysiology, visual field and blood routine, immune and nervous system examination, and all the medical history and examination data of the patients were collected and comprehensively analyzed. Results Among the 6 patients (12 eyes) with AZOOR, 5 were female and 1 was male; The age ranged from 26 to 42 years old, with an average of 35 years old, were both eyes. The follow-up time was 4 to 18 months, with an average of (7.5±3.2) months. Six eyes had myopia before the onset of the disease, and 3 eyes had flash in front of the eye at the onset of the disease, and the visual acuity decreased slightly. Only one eye had an exponential visual acuity/40 cm. Visual field defects or decreased local sensitivity were found in at least one eye of all patients, one eye showed enlarged physiological blind spots in both eyes, four eyes showed active inflammatory lesions in the anterior segment of the eye, 10 eyes showed mild inflammatory changes in the vitreous body, 4 eyes showed yellow-white spotty lesions in the posterior pole of the fundus, and 6 eyes showed gray-white spotty lesions in the posterior pole to the middle periphery of the retina, which were located in the outer layer of the retina or the layer of pigment epithelium-vitreous-choroidal capillary complex. Fluorescein fundus angiography showed fundus depigmentation in one eye, and the other patients showed fundus hyperfluorescence spots. One eye showed macular-choroidal neovascularization. All patients showed abnormalities in electroretinogram detection to varying degrees. During the follow-up, except for 1 eye with decreased visual acuity, the symptoms of other patients were similar to those at first diagnosis. 5 eyes showed no obvious visual field changes, 1 eye showed increased visual field defects, 1 case showed increased retinal spots in both eyes, 1 case showed decreased retinal lesions in both eyes, 1 case showed disappearance of retinal spots in both eyes, Conclusion AZOOR is a rare ocular disease, which is more common in young women, with flash sensation in front of the eye, visual field defect, electroretinogram change, and slight fundus lesion, which are common characteristics of AZOOR. Its differential diagnosis is complicated, and it is easy to misdiagnose and miss diagnosis.

Objective to investigate the diagnostic value of Heidelberg retinal tomography (HRT-Ⅱ) and coherence optical tomography (OCT) in the diagnosis of macular edema. Methods 212 eyes diagnosed by fluorescein fundus angiography (FFA) were measured by HRT-Ⅱ and foveal retinal thickness by OCT. Then, the quantitative diagnosis of macular edema was performed by HRT-Ⅱ and OCT, respectively, and the consistency between the results of FFA and FFA was compared. Results The results of FFA were used as the gold standard and compared with the other two methods, the Kappa values of macular edema diagnosis were 0.553 for OCT and 0.403 for HRT-Ⅱ. The odds ratio (OR) of macular edema diagnosis was 8.519 for OCT and 3.210 for HRT-Ⅱ. The difference between the macular edema index of patients diagnosed by FFA and those without macular edema was statistically significant (P<0.01). The difference of macular retinal thickness between the two groups was also statistically significant (P<0.01). The optimal critical value of HRT-Ⅱ in the diagnosis of macular edema was E =2.00, and the ROC curve area was 0.673. The optimal critical value of retinal thickness in the diagnosis of macular edema by OCT was 170 μ m, and the ROC curve area was 0.774. Conclusion For the diagnosis of macular edema, HRT-Ⅱ has higher sensitivity than OCT, lower missed diagnosis rate, but poor specificity, and higher misdiagnosis rate. For the localization observation of macular edema, HRT-Ⅱ has obvious advantages. Compared with FFA, the diagnostic results of OCT for macular edema are better than that of HRT-Ⅱ. The clinical diagnostic value of OCT for macular edema is higher than that of HRT-Ⅱ. The combination of the two examination methods can accurately perform the quantitative analysis of macular edema.

Objective to establish a rapid, accurate and simple method for screening the mutation of mtDNA G11778A in Leber hereditary optic neuropathy (LHON), and to preliminarily judge the proportion of wild and mutant in heterogeneous individuals.Methods Fluorescent MGB probe real-time polymerase chain reaction (PCR) technique was used to introduce wild and mutant probes into the same reaction system, and the blood samples of 20 patients and 17 normal people in LHON family were detected by multi-fluorescence channel PCR, respectively, and the results were compared with the results of nucleic acid sequencing. Results The results of normal blood samples were as follows: the VIC channel and the FAM channel were positive by fluorescence PCR, and the LHON mtDNA was judged to be wild type, which was in perfect agreement with the sequencing results; Sequencing results of clinical LHON patients and family members: 12 patients with LHON mtDNA variant were positive for LHON mtDNA variant by fluorescence PCR, among which 5 cases were mixed with LHON mtDNA variant and wild strain, and the Ct values of the mixed variant and wild strain were both greater than 25%; The sequencing results showed that there were 8 cases of LHON mtDNA wild type, 5 cases of LHON mtDNA wild type, and 3 cases of LHON mtDNA mutant and mixed type by fluorescence PCR. The Ct values of mutant and wild strains were both less than 25%; Fluorescence quantitative PCR can detect LHON mtDNA G11778A gene mutation in 80 min, which is much shorter than sequencing time. Conclusion Fluorescence MGB probe real-time PCR can detect LHON mtDNA G11778A gene mutation simply, quickly and accurately, and judge the proportion of wild and mutation in individual heterogeneity, which is of great value to further investigate whether there is a threshold of mtDNA gene mutation in LHON patients.

Limbal damage caused by various factors is the most common cause of ocular surface injury, and the main feature is corneal conjunctivization in the damaged area. Different degrees and extents of limbal damage lead to different degrees of ocular surface injury, and their treatment methods are also different. This experiment intends to classify the limbal damage according to the degree of stem cell loss, and explore the reasonable treatment scheme for each grade of injury.

The phacoemulsification combined with intraocular lens implantation is the mainstream of cataract surgery with small incision and mild postoperative reaction. However, the influence of surgery on blood aqueous barrier function has not been completely solved at present, and the disorder of blood aqueous barrier function will lead to prolonged postoperative recovery time and medication time, and also increase the incidence of postoperative complications, such as glaucoma, cystoid macular edema, pupil adhesion, etc. To reduce the disturbance of blood aqueous barrier caused by surgery, the work required at present mainly focuses on two aspects: first, further improve the surgical technique and reduce the irritation of anterior uvea caused by surgery itself; The second is to improve the material and design of intraocular lens, reduce its interference to the function of blood aqueous humor barrier, and speed up the recovery of blood aqueous humor barrier function.

Timolol is an effective intraocular pressure lowering drug and has been used in the treatment of chronic open-angle glaucoma for nearly 30 years [1]. Even now, timolol is still one of the most important anti-glaucoma drugs, but the bioavailability of ordinary eye drops in the eye is very low, and most of them are excreted by the nasolacrimal duct [2].

The patient was a 52-year-old female with oblique eyes since childhood, aggravated with visual impairment for 7 years. The patient complained of oblique eyes since childhood, poor visual acuity, but could do a few housework. In 1997, the patient gradually felt oblique eyes aggravated, and could not see objects, more obviously in the right eye. It was necessary to lift the upper lid of the left eye with fingers to see objects, but still could not see objects when lifting the upper lid of the right eye.

The patient, a 22-year-old male, came to the ophthalmology department of our hospital on May 7, 2004 due to uninduced blurred vision with black spots fluttering in front of his eyes for half a year, aggravated for 2 months, and red, swollen, swollen and painful in both eyes in the past 2~3 days, with the right eye as the focus.

the appearance of hyaline calcified substances in the optic head is called drusen of the optic disc. Most patients have binocular onset and have different clinical manifestations due to different locations.

Ophthalmologists always pay more attention to the updating of equipment, the advancement of technology, the improvement of methods, the adjustment of parameters and the application of new intraocular lens in cataract surgery, and think that as long as viscoelastic can maintain the anterior chamber space, capsulorhexis and implantation of intraocular lens is enough.