Shunji Kusaka

Serum concentrations of bevacizumab (avastin) and vascular endothelial growth factor in infants with retinopathy of prematurity.

PURPOSEnTo determine the serum concentrations of bevacizumab and vascular endothelial growth factor (VEGF) in infants with retinopathy of prematurity (ROP) who received intravitreal bevacizumab; and to determine whether the changes in the serum concentration of bevacizumab were significantly correlated with the serum concentration of VEGF after intravitreal bevacizumab.nnnDESIGNnCase series.nnnMETHODSnEleven infants (4 girls and 7 boys) with ROP were studied. They received 0.25 mg or 0.5 mg of intravitreal bevacizumab to either 1 eye (unilateral cases) or both eyes (bilateral cases) with vascularly active ROP. Serum samples were collected before and 1 day, 1 week, and 2 weeks after the intravitreal bevacizumab. The serum concentrations of bevacizumab and VEGF were measured by enzyme-linked immunosorbent assay, and the correlation in the serum levels between the 2 was determined.nnnRESULTSnThe serum concentration of bevacizumab before and 1 day, 1week, and 2 weeks after a total of 0.5 mg of intravitreal bevacizumab was 0 ng/mL, 195 ± 324 ng/mL, 946 ± 680 ng/mL, and 1214 ± 351 ng/mL, respectively. The serum bevacizumab level before and 1 day and 1 week after a total 1.0 mg of intravitreal bevacizumab was 0 ng/mL, 248 ± 174 ng/mL, and 548 ± 89 ng/mL, respectively. The serum concentration of VEGF before and 1 day, 1 week, and 2 weeks after a total of 0.5 mg intravitreal bevacizumab was 1628 ± 929 pg/mL, 427 ± 140 pg/mL, 246 ± 110 pg/mL, and 269 ± 157 pg/mL, respectively. There was a significant negative correlation (r = -0.575, P = .0125) between the serum concentration of bevacizumab and VEGF when a total of 0.25 mg or 0.5 mg of bevacizumab was injected.nnnCONCLUSIONSnThese results indicate that bevacizumab can escape from the eye into the systemic circulation and reduce the serum level of VEGF in infants with ROP. Continued extensive evaluations of infants are warranted for possible effects after intravitreal bevacizumab in ROP patients.

Fluorescein angiographic observations of peripheral retinal vessel growth in infants after intravitreal injection of bevacizumab as sole therapy for zone I and posterior zone II retinopathy of prematurity.

Aim To evaluate vascularisation of the peripheral retina using fluorescein angiography (FA) digital recordings of infants who had been treated with intravitreal bevacizumab (IVB) as sole therapy for zone I and posterior zone II retinopathy of prematurity (ROP). Methods A retrospective evaluation was performed of medical records, RetCam fundus images and RetCam fluorescein angiogram videos of 10 neonates (20 eyes) who received intravitreal bevacizumab injections as the only treatment for zone I and posterior zone II ROP between August 2007 and November 2012. Results All eyes had initial resolution of posterior disease after IVB injection as documented by RetCam colour fundus photographs. Using a distance of 2u2005disc diameters from the ora serrata to vascular termini as the upper limit of allowable avascular retina in children, the FA of these infants demonstrated that 11 of 20 eyes had not achieved normal retinal vascularisation. Conclusions Although bevacizumab appears effective in bringing resolution of zone I and posterior zone II ROP and allowing growth of peripheral retinal vessels, in our series of 20 eyes, complete normal peripheral retinal vascularisation was not achieved in half of the patients.

Intravitreal injection of bevacizumab for retinopathy of prematurity

AbstractPurposenTo evaluate the outcomes of intravitreal injection of bevacizumab (IVB) for retinopathy of prematurity (ROP).MethodsIVB was selected to be the first treatment for type 1 ROP in 8 eyes (4 patients). Bevacizumab (0.25xa0mg/eye) was injected into the vitreous cavity under either general anesthesia or sedation. Fundus photography and fluorescein angiography were performed before the IVB. One infant was observed to the age of 1xa0year 6xa0months, the second to 1xa0year 9xa0months, the third to 1xa0year 10xa0months, and the fourth to 2xa0years 0xa0month.ResultsBefore the IVB, 6 eyes (3 patients) had ROP in zone II and 2 eyes (one patient) had ROP in zone I. The 3 infants with ROP in zone II weighed 652, 476, and 579xa0g with gestational ages of 24, 27, and 24xa0weeks at birth, respectively. The infant with ROP in zone I weighed 972xa0g with a gestational age of 26xa0weeks at birth. IVB was performed at postmenstrual ages of 33–37xa0weeks. The IVB was effective in all eyes with ROP in zone II and additional treatment was not required, whereas vitreous hemorrhage and cataract were found at 19xa0weeks and 5xa0months after the initial IVB in the two eyes with ROP in zone I. These two eyes required additional IVB, laser photocoagulation, and surgery.ConclusionsOur findings suggest that eyes with type 1 ROP in zone II can be treated with IVB. Further studies are needed with a larger number of eyes.

Mutations in the TSPAN12 Gene in Japanese Patients with Familial Exudative Vitreoretinopathy

PURPOSEnTo search for mutations in the TSPAN12 gene in 90 Japanese probands with familial exudative vitreoretinopathy (FEVR) and their family members and to determine the types and frequencies of the mutations.nnnDESIGNnLaboratory investigation and clinical case analyses.nnnMETHODSnDirect sequencing after polymerase chain reaction of the coding exons of TSPAN12 was performed for 90 probands with FEVR and some of their family members. The clinical signs and symptoms that were characteristic of individuals with TSPAN12 mutations were determined.nnnRESULTSnThree families were found to carry 2 mutations in TSPAN12. One of these mutations was a new missense change, L245P, and the other was an already reported nonsense mutation, L140X, in 2 families. Mutations in TSPAN12 accounted for 3% of Japanese FEVR patients and 8% of the FEVR families who did not have mutations in any of the known FEVR genes, FZD4, LRP5, and NDP. The clinical signs and symptoms varied among the patients, but the retinal findings with TSPAN12 mutations were not different from those with mutations in the known FEVR-causing genes.nnnCONCLUSIONSnMutant TSPAN12 is responsible for approximately 3% of FEVR patients in Japan. The results provide further evidence that mutations in TSPAN12 are FEVR causing and that the gene products most likely play a role in the development of retinal vessels.

TGF-β2 promotes RPE cell invasion into a collagen gel by mediating urokinase-type plasminogen activator (uPA) expression.

Transforming growth factor-beta (TGF-β) is one of the main epithelial-mesenchymal transition (EMT)-inducing factors. In general, TGF-β-induced EMT promotes cell migration and invasion. TGF-β also acts as a potent regulator of pericellular proteolysis by regulating the expression and secretion of plasminogen activators. Urokinase-type plasminogen activator (uPA) is a serine protease that binds to its cell surface receptor (uPAR) with high affinity. uPA binding to uPAR stimulates uPARs interaction with transmembrane proteins, such as integrins, to regulate cytoskeletal reorganization and cell migration, differentiation and proliferation. However, the influence of TGF-β and the uPA/uPAR system on EMT in retinal pigment epithelial (RPE) cells is still unclear. The purpose of this study was to determine the effect of TGF-β2, which is the predominant isoform in the retina, and the uPA/uPAR system on RPE cells. In this study, we first examined the effect of TGF-β2 and/or the inhibitor of uPA (u-PA-STOP(®)) on the proliferation of a human retinal pigment epithelial cell line (ARPE-19 cells). Treatment with TGF-β2 or u-PA-STOP(®) suppressed cell proliferation. Combination treatment of TGF-β2 and u-PA-STOP(®) enhanced cell growth suppression. Furthermore, western blot analysis, fibrin zymography and real-time reverse transcription PCR showed that that TGF-β2 induced EMT in ARPE-19 cells and that the expression of uPA and uPAR expression was up-regulated during EMT. The TGF-β inhibitor SB431542 suppressed TGF-β2-stimulated uPA expression and secretion but did not suppress uPAR expression. Furthermore, we seeded ARPE-19 cells onto Transwell chambers and allowed them to invade the collagen matrix in the presence of TGF-β2 alone or with TGF-β2 and u-PA-STOP(®). TGF-β2 treatment induced ARPE-19 cell invasion into the collagen gel. Treatment with a combination of TGF-β2 and the uPA inhibitor strongly inhibited ARPE-19 cell invasion compared with treatment with TGF-β2 alone. Furthermore, the interaction between uPA and ARPE-19 cells was analyzed using a surface plasmon biosensor system. The binding of uPA to ARPE-19 cells was observed. In addition, TGF-β2 significantly promoted the binding activity of uPA to ARPE-19 cells in a time-dependent or cell-number-dependent fashion. These results indicate that TGF-β-induced EMT-associated phenotype changes in ARPE-19 cells and the invasiveness of ARPE-19 cells into a collagen gel matrix are mediated, at least in part, by uPA.

Longitudinal clinical course of three Japanese patients with Leber congenital amaurosis/early-onset retinal dystrophy with RDH12 mutation

AbstractPurposennTo report the longitudinal clinical course of three Japanese patients from two families with Leber congenital amaurosis/early-onset retinal dystrophy (LCA/EORD), and the results of next-generation DNA sequences on them.Patients and methodsThe patients were three Japanese children: a 4-year-old girl, a 6-year-old boy, and a 3-year-old girl. Patients 1 and 2 were siblings, and patient 3 was from an unrelated family. Standard ophthalmic examinations including perimetry, electroretinography, optical coherence tomography, and ultrasonography were performed on each patient. The patients were observed for 28, 16, and 10xa0years. Whole exomes of the patients and their non-symptomatic parents were analyzed using a next-generation sequence technique.ResultsThe decimal visual acuity varied between 0.07 and 0.6 at the initial visit and decreased to counting finger to hand motion in their teens. Funduscopy showed diffuse retinal and macular degeneration. During the follow-up period, a posterior staphyloma developed and the macular area became atrophic. Patient 1 developed cataracts in her early twenties. Genetic analysis revealed a homozygous A126V substitution in the RDH12 gene in all patients.ConclusionsThe three patients with LCA/EORD had a progressive decrease of their vision with the formation of a posterior staphyloma. This is the first report of Japanese patients with LCA/EORD with a RDH12 mutation.

Combined treatment for Coats' disease: retinal laser photocoagulation combined with intravitreal bevacizumab injection was effective in two cases

BackgroundThe exact pathogenetic mechanisms of Coats’ disease remain unknown. In this report, we show two cases of Coats’ disease that achieved a favorable prognosis with the combined treatment of intravitreal bevacizumab (IVB) injection prior to photocoagulation, although both initially resisted photocoagulation therapy.Case presentationsCase 1 was a 15-year-old boy with initial visual acuity of 0.4 OD. At the temporal retina, aneurysms and abnormal telangiectatic vessels were observed. Hard exudates and an exudative retinal detachment extended to the fovea. He was diagnosed as having Coats’ disease at stage 3A and we performed laser photocoagulation as an initial approach to treat peripheral aneurysms and telangiectatic vessels. After the treatment, the exudative retinal detachment was eased and visual acuity improved to 1.0; however, recurrence occurred after 5 months. The exudative change was resistant against laser photocoagulation therapy and we therefore added IVB as an adjuvant before photocoagulation. Fourteen days after IVB injection phased laser photocoagulation was given to cover the abnormal capillaries, aneurysms and the leakage area spotted in FA. A good prognosis was obtained with decreased exudation and improved visual acuity.Case 2 was an 11-year-old boy with decreased visual acuity of 0.15 OS at the initial visit. Hard exudates, retinal edema and serous retinal detachment were seen at the macula and peripheral retina. Fluorescein angiography revealed telangiectatic capillaries at the temporal retina. Our diagnosis was Coats’ disease at stage 3A. Extensive photocoagulation was performed as an initial treatment to the lesion. However, the exudative change was severe and resistant against the photocoagulation treatment. Therefore, we added IVB as an adjuvant before photocoagulation. Exudative change in the retina seemed to be eased 7 days after IVB injection, therefore, phased laser phototherapy was added to cover the abnormal capillaries. After the combination therapy, exudative change was remarkably ameliorated and better visual acuity was achieved.ConclusionBevacizumab is considered an effective adjuvant for Coats’ disease with exudative change resistant to retinal photocoagulation therapy.

Efficacy of Short-term Postoperative Perfluoro-n-octane Tamponade for Pediatric Complex Retinal Detachment

PURPOSEnTo evaluate the efficacy of perfluoro-n-octane as a postoperative short-term tamponade after vitrectomy in pediatric cases with complex retinal detachment (RD) and proliferative vitreoretinopathy (PVR).nnnDESIGNnProspective, noncomparative, interventional case series.nnnMETHODSnThe medical records of 10 eyes of 9 children (6 boys and 3 girls), whose age ranged from 3 months to 11 years, with a median of 7.5 months, were reviewed. The cause of the PVR was retinopathy of prematurity (7 eyes of 6 patients); familial exudative vitreoretinopathy; or tractional RD associated with congenital optic nerve anomalies,(1) and persistent fetal vasculature. Perfluoro-n-octane was injected into the eyes at the primary surgery in 2 eyes and at the repeat surgeries in 8 eyes. The perfluoro-n-octane was removed after 1 to 4 postoperative weeks. The patients were followed for 5 to 43 months.nnnRESULTSnAt the last examination, the retinas were reattached in 8 eyes (80%). In the other two eyes, a retinal attachment was not obtained. Postoperatively, the best-corrected visual acuity improved from hand motion to 0.1 in 1 eye and could not be measured in the other 9 patients because of their ages. No apparent adverse events related to the use of perfluoro-n-octane were noted.nnnCONCLUSIONSnAlthough cautions should be exercised regarding potential mechanical retinal injuries by heavy liquids in the eye, short-term perfluoro-n-octane tamponade was effective in pediatric cases with severe PVR in which retinal reattachment is considered to be difficult with conventional gas or silicone oil tamponade.

Intraocular pressure elevation is a delayed-onset complication after successful vitrectomy for stages 4 and 5 retinopathy of prematurity.

Purpose: To determine the incidence and risk factors for delayed-onset intraocular pressure (IOP) elevations after vitrectomy for Stages 4 and 5 retinopathy of prematurity and, in addition, to determine the results of treating the IOP elevations. Methods: Fifty-five consecutive eyes with successful retinal reattachment and at least 24 months of follow-up after vitrectomy were studied. The ophthalmic examinations included slit-lamp biomicroscopy, wide-field digital retinal imaging, and IOP measurements. Eyes were classified into 2 groups: eyes with a postoperative IOP elevation to ≥21 mmHg and eyes whose IOP was always <21 mmHg. Results: Eight eyes (14.5%) developed an IOP elevation to ≥21 mmHg from 2 months to 4 months (median: 2.5 months) after the vitrectomy. In two of these eyes, the IOP was controlled with antiglaucoma medications. In the other six eyes, trabeculotomy for eyes with open angles and trabeculectomy or goniosynechialysis for eyes with closed angles were performed. The IOPs were successfully controlled after an average of 1.5 glaucoma surgeries. Multivariate logistic regression analyses identified that a young gestational age (odds ratio = 1.147, 95% confidence interval: 1.024–1.346) and lensectomy (odds ratio = 8.795, 95% confidence interval: 1.262–183.3) were significantly associated with the IOP elevation. Conclusion: Delayed-onset IOP elevation after vitrectomy for retinopathy of prematurity occurs in 14.5% of the eyes, and it is significantly associated with a young gestational age and presence of lensectomy.

Novel nonsense and splice site mutations in CRB1 gene in two Japanese patients with early-onset retinal dystrophy

AbstractPurposenTo report novel mutations in the CRB1 gene in two patients with early-onset retinal dystrophy (EORD) and the longitudinal clinical course of EORD.nPatients and methodsThe patients were two unrelated Japanese children. Standard ophthalmic examinations including perimetry, electroretinography, and optical coherence tomography were performed on both patients. Whole exomes of the patients and their nonsymptomatic parents were analyzed using a next-generation sequence (NGS) technique.ResultsPatient 1 was noted to have esotropia and hyperopia at age 3. His decimal best-corrected visual acuity (BCVA) was 0.6 OD and 0.3 OS at age 6 with de-pigmentation of the retinal pigment epithelium (RPE). At age 19, his central vision was still preserved; however, numerous pigment granules were present in the retina. NGS analysis revealed a p.R632X nonsense and c.652xa0+xa01_652xa0+xa04delGTAA splice site mutations in the CRB1 gene. Patient 2 was noted to have hyperopia at age 3. His decimal BCVA at age 6 was 0.3 OD and 0.4 OS with de-pigmented RPE. The degree of retinal pigmentation was increased but his BCVA was good until the age of 14xa0years. NGS analysis revealed c.652xa0+xa01_652xa0+xa04delGTAA and c.652xa0+xa01_652xa0+xa02insT splice site mutations in the CRB1 gene.ConclusionsThe phenotypes of these novel mutations for EORD are typical of CRB1-associated EORD (LCA8). They were slowly progressive until the second decade of life.