Kan Koizumi

A central role for inflammation in the pathogenesis of diabetic retinopathy

Diabetic retinopathy is a leading cause of adult vision loss and blindness. Much of the retinal damage that characterizes the disease results from retinal vascular leakage and nonperfusion. Diabetic retinal vascular leakage, capillary nonperfusion, and endothelial cell damage are temporary and spatially associated with retinal leukocyte stasis in early experimental diabetes. Retinal leukostasis increases within days of developing diabetes and correlates with the increased expression of retinal intercellular adhesion molecule‐1 (ICAM‐1) and CD18. Mice deficient in the genes encoding for the leukocyte adhesion molecules CD18 and ICAM‐1 were studied in two models of diabetic retinopathy with respect to the long‐term development of retinal vascular lesions. CD18−/− and ICAM‐1−/− mice demonstrate significantly fewer adherent leukocytes in the retinal vasculature at 11 and 15 months after induction of diabetes with STZ. This condition is associated with fewer damaged endothelial cells and lesser vascular leakage. Galactosemia of up to 24 months causes pericyte and endothelial cell loss and formation of acellular capillaries. These changes are significantly reduced in CD18‐ and ICAM‐1‐deficient mice. Basement membrane thickening of the retinal vessels is increased in long‐term galactosemic animals independent of the genetic strain. Here we show that chronic, low‐grade subclinical inflammation is responsible for many of the signature vascular lesions of diabetic retinopathy. These data highlight the central and causal role of adherent leukocytes in the pathogenesis of diabetic retinopathy. They also underscore the potential utility of anti‐inflammatory treatment in diabetic retinopathy.

Nonsteroidal anti-inflammatory drugs prevent early diabetic retinopathy via TNF-alpha suppression.

Leukocyte adhesion to the diabetic retinal vasculature results in blood‐retinal barrier breakdown, capillary nonperfusion, and endothelial cell injury and death. Intercellular adhesion molecule‐1 (ICAM‐1) and the leukocyte integrin CD18 are required for these processes. Diabetes was induced in Long Evans rats, resulting in a two‐ to threefold increase in retinal leukocyte adhesion. Following one week of diabetes, neutrophil CD11a, CD11b, and CD18 expression was increased significantly, as were retinal ICAM‐1 levels. Animals were treated with aspirin, a cyclooxygenase 2 (COX‐2) inhibitor (meloxicam), or a soluble tumor necrosis factor α (TNF‐α) receptor/Fc construct (TNFR‐Fc, etanercept). High‐dose aspirin, etanercept, and high‐dose meloxicam each reduced leukocyte adhesion and suppressed blood‐retinal barrier breakdown. High‐dose aspirin also reduced the expression of CD11a, CD11b, and CD18, whereas meloxicam and etanercept did not. High‐dose aspirin, etanercept, and high‐dose meloxicam each reduced retinal ICAM‐1 expression. Aspirin and meloxicam both lowered retinal TNF‐α levels. Notably, aspirin, meloxicam, and etanercept did not change retinal vascular endothelial growth factor levels. High‐dose aspirin, etanercept and high‐dose meloxicam, each suppressed the retinal expression of eNOS and the DNA‐binding capacity of retinal nuclear factor‐κΒ. High‐dose aspirin also suppressed Erk kinase activity, which is involved in CD18 up‐regulation. Taken together, these data identify COX‐2 and TNF‐α as operative in the early signature pathologies of diabetic retinopathy, a newly recognized inflammatory disease.

Comparison of intact and denuded amniotic membrane as a substrate for cell-suspension culture of human limbal epithelial cells

BackgroundWe have previously developed a limbal epithelial culture system using a cell-suspension method on denuded amniotic membrane (AM). However, other workers reported that intact AM is advantageous for limbal epithelial culture in that it preserves stem cell characteristics. In this study, we cultivated human limbal epithelial cell-suspensions on both intact and denuded AM and compared the morphology and adhesion of the limbal epithelial cells on these two substrates.MethodsHuman limbal epithelial cells were dissociated from donor eyes using dispase and gentle pipetting and then seeded onto intact and denuded AM as cell suspension. Limbal epithelial cells on AM were co-cultured with a MMC-treated 3T3 fibroblast feeder layer and epithelial differentiation was promoted by air lifting. Cultures were examined by light, scanning and transmission electron microscopy and differences in cellular attachments and intercellular spacing were quantified. Basement membrane complexes were examined by indirect immunofluorescence.ResultsLimbal cells grown on denuded AM were well stratified and differentiated. Cells were well attached to each other and to the basement membrane. In contrast, limbal cells cultured on intact AM failed to stratify and in places formed a monolayer.The culture on denuded AM had significantly (P<0.001) more desmosomal junctions as well as significantly (P<0.001) more junctional attachments to the carrier than the intact culture. In addition, the intercellular spaces between cells cultivated on denuded AM were significantly (P<0.001) smaller than those between cells grown on the intact substrate. In cultures on both denuded and intact AM, the basement membrane zone displayed a positive staining for collagen VII, integrins alpha-6 and beta-4 and laminin 5.ConclusionsWe successfully cultivated well-stratified and -differentiated limbal cells on denuded AM, while on the intact AM limbal cells failed to stratify and in places formed only a monolayer of cells. The limbal cells cultivated on denuded AM were well attached to the AM stroma and were morphologically superior to the limbal epithelium cultivated on intact AM. We conclude that for purposes of transplantation of differentiated epithelial sheets, denuded AM is probably the more practical carrier for human limbal epithelial cell cultures when using our cell-suspension culture system.

Paraoxonase gene polymorphisms and plasma oxidized low-density lipoprotein level as possible risk factors for exudative age-related macular degeneration

PURPOSE Paraoxonase (E.C.3.1.1.2) is a polymorphic protein shown to prevent low-density lipoprotein oxidation. Our purpose is to evaluate the hypothesis that paraoxonase gene polymorphisms and plasma oxidized low-density lipoprotein level play a role in the occurrence of exudative age-related macular degeneration. METHODS We analyzed paraoxonase genotypes (A/B, Gln-Arg192 and L/M, Leu-Met54) and plasma oxidized low-density lipoprotein levels in 72 unrelated Japanese patients with exudative age-related macular degeneration and compared the results with those of 140 age-matched and sex-matched control subjects. RESULTS The distribution of paraoxonase 192 and paraoxonase 54 polymorphisms was significantly different between the patients with age-related macular degeneration and control subjects (chi-square = 6.226, P =.0445, and chi-square = 6.863, P =.0323, respectively). The high frequency of the BB genotype at position 192 was observed in the exudative age-related macular degeneration group compared with control subjects (52.8% vs 35.0%, respectively; P =.0127). The high frequency of the LL genotype at position 54 was observed in the patients more than the controls (91.7% vs 77.1%, respectively; P =.0090). The mean (+/- SE) oxidized low-density lipoprotein levels in the patients was significantly higher than in the controls (19.1 +/- 1.0 vs 16.2 +/- 0.6 U/ml, P <.01). CONCLUSION These results indicate that the paraoxonase gene polymorphisms may represent a possible genetic risk factor for age-related macular degeneration and that increased plasma oxidized low-density lipoprotein may be involved in the pathogenesis of age-related macular degeneration.

Visual outcome of patients with macular edema after pars plana vitrectomy and indocyanine green-assisted peeling of the internal limiting membrane

PurposeTo evaluate the efficacy of inner limiting membrane (ILM) peeling in persistent macular edema.MethodsThis retrospective review analyzed a series of 23 eyes from 23 patients with persistent macular edema treated by pars plana vitrectomy (PPV) with indocyanine green (ICG)-assisted peeling of the ILM. Thirteen female and 10 male patients with a mean age of 57.2±15.6 (24–77) years underwent operation between May 2000 and October 2001. The main diagnoses were uveitis (anterior, intermediate, posterior and panuveitis) (n=9), central retinal vein occlusion (CRVO) (n=4), diabetic retinopathy (DR) (n=5), vitreoretinal traction syndrome (n=2), and Irvine–Gass syndrome (n=3). Nine eyes had undergone phacoemulsification (PE) previously and two eyes had been subjected to combined PE and ILM peeling. The eyes were tamponaded with gas (3), silicone oil (5) or air (11). In four cases no endotamponade was used. Improvement in visual acuity of 2 lines or more was regarded as significant.ResultsVisual acuity improved after 3 months in 9 of the 23 patients. After 6 months and at the follow-up, a significant improvement was found in 6/21 and 7/21 patients. This improvement was predominantly seen in patients with uveitis (5/9), or diabetic maculopathy (3/5); One patient with Irvine–Gass syndrome showed a significant reduction, one with vitreoretinal traction an improvement in visual acuity. The group of patients with CRVO showed no significant change during the follow-up. The choice of endotamponade did not alter the visual acuity outcome.ConclusionsDifferent patient groups respond differently to ILM peeling. Although overall significant visual acuity improvement was observed in only one third of all cases 12 months after ILM peeling for persistent macular edema, patients with uveitis and nonproliferative diabetic maculopathy demonstrated a benefit. The lack of long-term improvement in the majority of cases is in accordance with the hypothesis that ILM peeling may reduce the intraretinal edema, but does not affect the underlying mechanism causing macular edema. So far, only diabetics have shown improvement (still unproven) from ILM peeling, and this study provides no justification for extending the treatment to macular edema of other causes. Large-scale investigations are needed to evaluate the efficacy in certain diagnosis groups.

Pharmacokinetics and safety of intravitreally delivered etanercept

BackgroundThe anti-inflammatory drug etanercept may be an effective therapeutic agent in diabetic retinopathy. In order to further evaluate its potential, the pharmacokinetics and safety of this drug after intravitreal delivery were investigated.MethodsAfter intravitreal administration of etanercept in rabbits, clinical examination, electroretinography (ERG), visually evoked potentials (VEP) and histology were evaluated. The pharmacokinetics and distribution of etanercept were analyzed using fluorescence-coupled protein at 0, 2, 4, and 8 weeks after injection in vitreous, retina, and choroid.ResultsNo adverse effects and signs of toxicity were found. Etanercept showed peak concentrations after 4 weeks in the retina and choroid.ConclusionsIntravitreally delivered etanercept is safe and results in high concentrations in the retina and choroid over a long period of time.

Retinectomy for treatment of intractable glaucoma: long term results

Aim: To report long term efficacy and complications of retinectomy as an intraocular pressure lowering procedure for intractable glaucoma. Methods: This was a consecutive interventional case series. In 44 consecutive eyes (39 patients, 22 men and 17 women) retinectomy was performed to lower the intraocular pressure (IOP) in patients with uncontrolled IOP (>35 mm Hg for more than 4 months) despite conventional filtering surgery and drug treatment. Pars plana vitrectomy was performed and the peripheral retina was surgically excised to various degrees. The procedure was concluded by an intraocular gas tamponade of 20% C3F8. Included were patients with neovascular glaucoma (12 eyes), infantile and juvenile glaucoma (three eyes), secondary glaucoma due to aphakia (13 eyes), severe ocular trauma (seven eyes), uveitis (seven eyes), and glaucoma in Ehlers-Danlos syndrome (two). Results: All patients underwent successful surgical retinectomy. All patients were followed for 5 years. Mean postoperative IOP after 4 years was 15.7 (SD 9.4) mm Hg, representing a decrease of IOP by 61% compared to the preoperative level (41.2 (9.4) mm Hg). In 52.3% of eyes long term regulation of IOP could be achieved without complications. Retinectomy was least effective in neovascular glaucoma because of central retinal vein occlusion (CRVO). Eyes with glaucoma secondary to uveitis showed a tendency towards low IOP levels with subsequent phthisis bulbi. The initial visual acuity of all patients was lower than 20/50 (mean 1.8 (0.8) logMAR) in the treated eye. Final visual acuity was 2.3 (0.6) logMAR. 21 out of 44 cases developed retinal complications (retinal detachment or proliferative vitreoretinopathy (PVR)) after surgery, requiring silicone tamponade in 11 eyes (52%) either for persistent low IOP or for PVR. Nine eyes developed phthisis, seven of which were enucleated during the follow up. Conclusions: Long term results after retinectomy demonstrate its efficacy in otherwise intractable glaucoma. Efficacy and safety of retinectomy are dependent on the underlying disease.

Vitrectomy for rhegmatogenous or tractional retinal detachment with familial exudative vitreoretinopathy

OBJECTIVE To examine the anatomic features and surgical indications of familial exudative vitreoretinopathy (FEVR) complicated with rhegmatogenous or tractional retinal detachment. DESIGN Retrospective noncomparative case series. PARTICIPANTS Twenty-eight eyes of 25 patients who had either clinically suspected or fully diagnosed FEVR. Of these, 25 had rhegmatogenous retinal detachment, 2 had tractional retinal detachment, and 1 had tractional retinal detachment plus vitreous hemorrhage. INTERVENTIONS The authors carefully observed the vitreoretinal interface during surgery, studied the clinical and anatomic features of FEVR, and then evaluated the surgical results. RESULTS The vitreoretinal adhesions were so strong in the peripheral avascular area that iatrogenic retinal breaks easily occurred in 22 of 28 eyes. In all cases, the bimanual technique with vitreous scissors and forceps was required to dissect the posterior vitreous membrane from the retinal surface. The retina was reattached in 24 of 28 cases (85.7%), and visual acuity improved in 20 eyes (71.4%). CONCLUSION Dissection of the vitreous in the peripheral avascular area is very difficult in FEVR, and those patients for whom this procedure was not successfully performed may have a poorer prognosis.

Intentional complete interruption of a retinal vein after vitrectomy might improve the rate of successful chorioretinal venous anastomosis formation in central retinal vein occlusion.

Abstract Background. Laser or surgical chorioretinal venous anastomosis has not become widespread as suitable treatment for central retinal vein occlusion (CRVO). We report here the effectiveness of cutting off a retinal vein with vitrectomy in making a chorioretinal venous anastomosis in CRVO. Methods. We performed a vitrectomy accompanied by a chorioretinal venous anastomosis procedure for seven consecutive patients with CRVO. These patients had shown no improvement in their visual acuity in the month following their first visit to our hospital and had a visual acuity of less than 20/200. Their preoperative visual acuity ranged from counting fingers to 20/300. The procedure included the complete cutting of the affected retinal vein, and the making of a small incision at both sides of the vein interruption through the full thickness of the retina, the retinal pigment epithelium and Bruchs membrane. Results. In five of the seven patients, successful chorioretinal venous anastomosis was observed. All five patients showed an improvement of two or more lines in visual acuity 6 months postoperatively. In three of the five, visual acuity was 20/100 or better. A postoperative complication of recurrent vitreous hemorrhage and fibrous proliferation was observed in one patient, and a second operation was necessary. Conclusions. Surgical interruption of an affected vein was a feasible procedure and could raise the rate of successful chorioretinal venous anastomosis in CRVO.

Multicenter genetic study of retinitis pigmentosa in Japan: I. Genetic heterogeneity in typical retinitis pigmentosa☆

A nationwide, multicenter study of typical retinitis pigmentosa (RP) was carried out in collaboration with 18 hospitals throughout Japan to obtain current information for genetic counseling. We analyzed the genetic heterogeneity of RP based on the parental consanguinity of 434 probands registered during a 6-month period in 1990. A gradual decline in the frequency of consanguineous marriage was recognized among the normal parents of RP patients. The relative frequencies of inheritance patterns were estimated as: autosomal recessive, 25.2%; autosomal dominant, 16.9%; X-linked, 1.6%; and simplex, 56.3%. A comparison of these results with previous reports in Japan revealed a decline in the relative frequency of autosomal recessive cases and an increase in simplex cases. This suggests a decrease in the incidence of autosomal recessive retinitis pigmentosa in Japan, as well as the necessity for exhaustive investigations aimed at identifying inheritance patterns for RP patients seeking genetic counseling.