Retro-mode imaging of fibrovascular membrane in proliferative diabetic retinopathy after intravitreal bevacizumab injection

The F10 is a new commercially available scanning laser confocal ophthalmoscope (SLO) that can perform multiple functions. We determined the usefulness of noninvasive evaluation of proliferative diabetic retinopathy (PDR) pathologies before and after intravitreal injection of bevacizumab (IVB) using the new indirect viewing system of the retro-mode function of the F10 SLO, and compared the images histologically with surgically excised fibrovascular membrane from two cases. In PDR, neovascular vessels in fibrovascular membrane were clearly seen with the retro-mode, even after IVB and without blood flow. The F10 SLO may be useful in evaluating neovascular vessels in fibrovascular membrane in PDR and for determining the precise retinal changes in diabetic retinopathy

The novel functional nucleic acid iRed effectively regulates target genes following cytoplasmic delivery by faint electric treatment

An intelligent shRNA expression device (iRed) contains the minimum essential components needed for shRNA production in cells, and could be a novel tool to regulate target genes. However, general delivery carriers consisting of cationic polymers/lipids could impede function of a newly generated shRNA via electrostatic interaction in the cytoplasm. Recently, we found that faint electric treatment (fET) of cells enhanced delivery of siRNA and functional nucleic acids into the cytoplasm in the absence of delivery carriers. Here, we examined fET of cells stably expressing luciferase in the presence of iRed encoding anti-luciferase shRNA. Transfection of lipofectamine 2000 (LFN)/iRed lipoplexes showed an RNAi effect, but fET-mediated iRed transfection did not, likely because of the endosomal localization of iRed after delivery. However, fET in the presence of lysosomotropic agent chloroquine significantly improved the RNAi effect of iRed/fET to levels that were higher than those for the LFN/iRed lipoplexes. Furthermore, the amount of lipid droplets in adipocytes significantly decreased following fET with iRed against resistin in the presence of chloroquine. Thus, iRed could be a useful tool to regulate target genes following fET-mediated cytoplasmic delivery with endosomal escape devices

Microarray-based global mapping of integration sites for the retrotransposon, intracisternal A-particle, in the mouse genome

Mammalian genomes contain numerous evolutionary harbored mobile elements, a part of which are still active and may cause genomic instability. Their movement and positional diversity occasionally result in phenotypic changes and variation by causing altered expression or disruption of neighboring host genes. Here, we describe a novel microarray-based method by which dispersed genomic locations of a type of retrotransposon in a mammalian genome can be identified. Using this method, we mapped the DNA elements for a mouse retrotransposon, intracisternal A-particle (IAP), within genomes of C3H/He and C57BL/6J inbred mouse strains; consequently we detected hundreds of probable IAP cDNA–integrated genomic regions, in which a considerable number of strain-specific putative insertions were included. In addition, by comparing genomic DNAs from radiation-induced myeloid leukemia cells and its reference normal tissue, we detected three genomic regions around which an IAP element was integrated. These results demonstrate the first successful genome-wide mapping of a retrotransposon type in a mammalian genome

Generation of four induced pluripotent stem cell lines (FHUi003-A, FHUi003-B, FHUi004-A and FHUi004-B) from two affected individuals of a familial neurohypophyseal diabetes insipidus family

Four disease-specific induced pluripotent stem cell (iPSC) lines were respectively derived from peripheral blood mononuclear cells of two affected individuals in a family affected by familial neurohypophyseal diabetes insipidus carrying the c.314G>C mutation. The expression of pluripotency markers (NANOG, OCT4, and SOX2), maintenance of a normal karyotype, absence of episomal vectors used for iPSC generation, and presence of the original pathogenic mutation were confirmed for each iPSC line. The ability to differentiate into three germ layers was confirmed by a teratoma formation assay. These iPSC lines can help in disease recapitulation in vitro using organoids and elucidation of disease mechanisms

Succinate Increases in the Vitreous Fluid of Patients With Active Proliferative Diabetic Retinopathy

Purpose: To examine vitreous succinate levels from proliferative diabetic retinopathy (PDR) patients and ascertain their association with PDR activity. Design: Comparative case series. Methods: A total of 81 eyes of 72 PDR patients were divided into active PDR (22 eyes), quiescent PDR (21 eyes), and active PDR with intravitreal bevacizumab injection (38 eyes). Twenty epiretinal membrane (ERM) patients (21 eyes) served as controls. Results: Mean vitreous succinate levels were 1.27 μM in ERM and 2.20 μM in PDR, with the differences statistically significant (P = .03). When comparing mean vitreous succinate levels (active PDR: 3.32 μM; quiescent PDR: 1.02 μM; active PDR with intravitreal bevacizumab injection: 1.20 μM), significant differences were found between active and quiescent PDR (P < .01) and between active PDR and active PDR with intravitreal bevacizumab injection (P < .01). Even though succinate levels were low, retinopathy activities were very high in patients with active PDR with intravitreal bevacizumab injection. Mean vitreous vascular endothelial growth factor (VEGF) levels (active PDR: 1696 pg/mL; quiescent PDR: 110 pg/mL; active PDR with intravitreal bevacizumab injection: n.d.) were similar to previous reports. Mean vitreous erythropoietin levels (active PDR: 703 mIU/mL; quiescent PDR: 305 mIU/mL; active PDR with intravitreal bevacizumab injection: 1562 mIU/mL) suggested very high retinopathy activities in patients with active PDR with intravitreal bevacizumab injection. Conclusions: Succinate, like VEGF, may be an angiogenic factor that is induced by ischemia in PDR. Although succinate is reported to promote VEGF expression, VEGF inhibition decreases succinate. Thus, VEGF, via a positive feedback mechanism, may regulate succinate

RETINAL BLOOD FLOW LEVELS MEASURED BY LASER SPECKLE FLOWGRAPHY IN PATIENTS WHO RECEIVED INTRAVITREAL BEVACIZUMAB INJECTION FOR MACULAR EDEMA SECONDARY TO CENTRAL RETINAL VEIN OCCLUSION

Purpose: To report retinal blood flow levels measured by Laser speckle flowgraphy in three patients after they received an intravitreal bevacizumab injection (IVB) for macular edema secondary to central retinal vein occlusion (CRVO). Methods: Three patients (3 eyes) being treated with IVB (1.25 mg/0.05 mL) for secondary macular edema of CRVO were examined. Laser speckle flowgraphy analyses of the blood flow were based on the examinations of mean blur rate (MBR) at the major vessels of the optic disk. Central retinal thickness (CRT) was measured by optical coherence tomography using Macular Cube 512 128 scanning protocol. Results: After the first IVB, Case 1 exhibited an increase in MBR and decrease in CRT. After 4months, an additional injectionwas required because of a subsequentMBR decrease and CRT increase, which led to an increase inMBR and decrease in CRT similar to that observed after the first treatment. Subsequently, blood flow has continued to improve without additional IVB. Macular edema recurrence in Case 2 led to 3 further IVBs over a 6-month period. Although increases inMBRanddecreases inCRTwerenoted,MBRvalues tendedtodeclineafter eachIVB. In Case 3, macular edema recurrence led to 5 additional IVBs being carried out within a 1-year period. Continuous MBR increases and CRT decreases were observed in the patient after each IVB. By measuringMBR using laser speckle flowgraphy,wemay predict the prognosis of CRVO. Conclusion: Mean blur rate increases after IVB were confirmed by laser speckle flowgraphy in three patients. Even though CRVO pathology backgrounds can vary, laser speckle flowgraphy may be useful in both determining the CRVO prognosis and in evaluating treatment efficacy

Predicting the outcome of chronic kidney disease by the estimated nephron number: The rationale and design of PRONEP, a prospective, multicenter, observational cohort study

<p>Abstract</p> <p>Background</p> <p>The nephron number is thought to be associated with the outcome of chronic kidney disease (CKD). If the nephron number can be estimated in the clinical setting, it could become a strong tool to predict renal outcome. This study was designed to estimate the nephron number in CKD patients and to establish a method to predict the outcome by using the estimated nephron number.</p> <p>Methods/Design</p> <p>The hypothesis of this study is that the estimated nephron number can predict the outcome of a CKD patient. This will be a multicenter, prospective (minimum 3 and maximum 5 years follow-up) study. The subjects will comprise CKD patients aged over 14 years who have undergone a kidney biopsy. From January 2011 to March 2013, we will recruit 600 CKD patients from 10 hospitals belonging to the National Hospital Organization of Japan. The primary parameter for assessment is the composite of total mortality, renal death, cerebro-cardiovascular events, and a 50% reduction in the eGFR. The secondary parameter is the rate of eGFR decline per year. The nephron number will be estimated by the glomerular density in biopsy specimens and the renal cortex volume. This study includes one sub-cohort study to establish the equation to calculate the renal cortex volume. Enrollment will be performed at the time of the kidney biopsy, and the data will consist of a medical interview, ultrasound for measurement of the kidney size, blood or urine test, and the pathological findings of the kidney biopsy. Patients will continue to have medical consultations and receive examinations and/or treatment as usual. The data from the patients will be collected once a year after the kidney biopsy until March 2016. All data using this study are easily obtained in routine clinical practice.</p> <p>Discussion</p> <p>This study includes the first trials to estimate the renal cortex volume and nephron number in the general clinical setting. Furthermore, this is the first prospective study to examine whether the nephron number predicts the outcome of CKD patients. The results from this study should provide powerful new tools for nephrologists in routine clinical practice.</p> <p>Trial registration</p> <p>UMIN-Clinical Trial Registration, UMIN000004784.</p

A novel in vivo corneal trans-epithelial electrical resistance measurement device

Purpose: To develop a device that is capable of easily measuring corneal transepithelial electrical resistance (TER) and changes in the corneal barrier function. Methods: We had previously developed an in vivo method for measuring corneal TER using intraocular electrode. This method can be used to precisely measure the decline of the corneal barrier function after instillation of benzalkonium chloride (BAC). In order to lessen the invasiveness of that procedure, we further refined the method for measuring the corneal TER by developing electrodes that could be placed on the cornea and in the conjunctival sac instead of inserting them into the anterior chamber. TER was then calculated by subtracting the electrical resistance, which lacked the corneal epithelial input, from the whole electrical resistance that was measured between the electrodes. Slit lamp examination and scanning electron microscopy (SEM) were used to determine safety of the new device. Corneal TER changes after exposure to 0.02% BAC were determined using the new device as well as SEM and transmission electron microscopy (TEM). Results: Slit lamp examination before and after exposure of rabbits\u27 corneas to the sensor confirmed safety of the device. SEM examination revealed no difference of the corneal epithelium which exposed to the new device with normal corneas. SEM and TEM pictures revealed damaged microvilli and tight junctions after instillation of 0.02% BAC. TER change after treatment with 0.02%BAC was similar to those determined by the established anterior chamber method. Conclusion: We succeeded to develop a less invasive device for corneal TER measurement in vivo in animals. This new device may be applicable in the future for clinical use in humans