[show abstract][hide abstract] ABSTRACT: To evaluate and compare early corneal wound healing and inflammatory responses after small incision lenticule extraction (SMILE) versus femtosecond laser laser in situ keratomileusis (LASIK).
Thirty-six eyes of 36 rabbits underwent SMILE, while another 36 eyes of 36 rabbits were treated with femtosecond laser LASIK. All the eyes were subjected to the same refractive correction of -6.00 DS/-1.00 DC. Twelve eyes that had no surgery were included for control. After euthanisation, corneal tissue sections were evaluated with terminal deoxyribonucleotidyl transferase-mediated dUTP-digoxigenin nick-end labelling (TUNEL) assay to detect apoptosis at postoperative 4 and 24 h, immunocytochemistry for Ki67 to detect keratocyte proliferation at postoperative day 3, week 1 and month 1, and immunocytochemistry for CD11b to detect inflammation at postoperative day 1, day 3 and week 1, respectively.
No adverse effects were noted after SMILE or LASIK. Corneal healing postoperatively was uneventful in all cases. There were significantly fewer TUNEL-positive corneal stromal cells after the SMILE procedure at 4 and 24 h postoperatively (p<0.01) compared with the LASIK procedure. In addition, immunocytochemistry showed significantly fewer Ki67-positive cells in the SMILE group than those in the femtosecond laser LASIK group at day 3 and week 1 postoperatively (p<0.05), but there was little expression of Ki67 at month 1 postoperatively in both groups. The CD11b-positive cells were significantly fewer in the SMILE group at day 1, day 3 and week 1 postoperatively (p<0.01).
SMILE induces less keratocyte apoptosis, proliferation and inflammation compared with femtosecond laser LASIK.
The British journal of ophthalmology 11/2013; · 2.92 Impact Factor
[show abstract][hide abstract] ABSTRACT: PURPOSE: To determine the effect of travoprost 0.004% on Schlemm's canal (SC) in healthy human eyes using Fourier-domain optical coherence tomography (FD-OCT). METHODS: Twelve healthy volunteers were recruited for a double-blind, placebo-controlled, randomized, and paired comparison study. Right eyes of subjects were randomly assigned to receive either travoprost 0.004% or placebo; the contralateral eye received the other treatment. FD-OCT imaging of SC and measurements of intraocular pressure (IOP) were carried out before and at 1, 2, 4, 6, 8, 12, 24, 36, 48, 60, 72 and 84 hours after eye drop instillation. Results: After instillation of travoprost eye drops, IOP gradually reduced, and the SC lumens expanded, while those values remained unchanged in placebo treated eyes. At 8 hours after the travoprost administration, the mean SC area increased 90.30% and 90.20%, respectively, in nasal and temporal quadrant of the treated eyes as compared to the placebo group. The SC area and IOP showed a similar pattern of changes at most time points examined. In travoprost-treated eyes, a statistically significant correlation between SC area and IOP is observed (r=-0.2817; p=0.0004). Measurements of the SC area showed sufficient repeatability and reproducibility. Conclusions: SC can be noninvasively imaged and quantitatively assessed in the living healthy human eye by FD-OCT. Travoprost treatment leads to SC lumen expansion accompanied by a drop of IOP in the healthy eye, likely as a result of the enhancement of pressure sensitive trabecular meshwork outflow induced by travoprost.
[show abstract][hide abstract] ABSTRACT: Recombinant adeno-associated viral vector serotype 2 (rAAV2) has been used with success to deliver retina-targeted gene therapeutics in retinal degeneration. However, one of the major limitations of this approach is the vector's low transduction efficiency. This study is designed to increase AAV2 transduction efficiency in vitro and in vivo.
Green fluorescence protein (GFP) or luciferase reporter gene-carried rAAV2 vectors were applied to cultured human RPE cells (ARPE-19) or animal eyes with or without chemotherapeutic agents. GFP transduction efficiency was evaluated by image, flow cytometry analysis, and Western blot. The ciliary neurotrophic factor (rAAV2-CNTF)-carried AAV2 vector was coinjected to subretinal space with or without chemotherapeutic agent. The therapeutic efficacy was evaluated by counting numbers of remaining photoreceptors in retina sections of treated or untreated eyes.
Coadministration of 0.1 μg/mL doxorubicin (DXR), 0.14 μg/mL cytarabine (Ara-C), 1 μg/mL etoposide (VP-16), or 20 μg/mL cisplatin (DDP) significantly increased rAAV2-mediated GFP and/or luciferase expression in cultured hRPE cells without any detectable toxicity. Pretreatment with DXR for 24 h prior to infection was most effective in enhancing rAAV2 transgene expression in hRPE cells. In addition, subretinal coinjection of rAAV2-CMV-ciliary neurotrophic factor (rAAV2-CNTF) and DXR into the eyes of rats with inherited retinal degeneration resulted in an approximately 2-fold increase in photoreceptor layer thickness and cellular density of the outer nuclear layer (ONL) compared to rAAV2-CNTF alone, reflecting a pronounced protection effect mediated by the enhanced expression of CNTF.
The method described here to improve rAAV2-based gene delivery is simple and feasible without any detectable toxicity. This strategy might be therapeutically exploited in the gene therapy of degenerative retinal diseases.
[show abstract][hide abstract] ABSTRACT: Images from cultured lens cells do not convey enough spatial information, and imaging of fixed lens specimens cannot reveal dynamic changes in the cells. As such, a real-time, convenient approach for monitoring label-free imaging of dynamic processes of living cells within the whole lens is urgently needed.
Female Wistar rat lenses were kept in organ culture. Insulin-like growth factor-I was added to the culture medium to induce cell mitosis. A novel method of ultraviolet (UV) irradiation was used to induce cell apoptosis and fiber damage. The cellular morphological dynamics within the whole lens were monitored by inverted phase contrast microscopy. Apoptosis was assessed using a commercial kit with Hoechst 33342/YO-PRO®-1/propidium iodide (PI).
The intrinsic transparency and low-light scattering property of the rat lens permitted direct imaging of the lens epithelial cells (LECs) and the superficial fiber cells. We visualized the processes of mitosis and apoptosis of the LECs, and we obtained dynamic images of posterior fiber cells following UVA irradiation.
This method opens a new window for observing lens cells in their physiologic location, and it can be readily applied in studies on lens physiology and pathology.
[show abstract][hide abstract] ABSTRACT: To determine sonic hedgehog (Shh) expression and whether it exerts neuroprotective effects on retinal ganglion cells (RGCs) in a rat chronic ocular hypertension model.
Intraocular pressure (IOP) elevation in adult rat was induced by episcleral vein cautery. Retinal expression of Shh protein and mRNA was determined by immunohistochemistry, Western blot analysis, and real-time PCR. Exogenous Shh and its inhibitor cyclopamine were intravitreally injected to examine their effects on RGC survival after ocular hypertension by the counting of retrograde DiI-labeled RGCs. Shh pathway components mediating neuroprotective effects were characterized using Western blot analysis and real-time PCR.
Shh was mainly detected in the RGCs in normal adult rat. Retinas from the elevated IOP group had 2.1- to 4.4-fold greater Shh expression than control retinas (P < 0.05). Shh promoted RGC survival at 2 and 4 weeks after IOP elevation in a dose-dependent manner, resulting in a loss of only 4.54% +/- 0.36% RGCs at 2 weeks (P < 0.01; vs. PBS-treated groups). In contrast, cyclopamine increased RGC loss. Protein and mRNA levels of the Shh signal transducer Smo and the downstream transcription factor Gli1 were significantly upregulated in RGCs after chronic ocular hypertension or intravitreal injection of Shh.
Shh and Smo are upregulated in a time-dependent manner in retinas exposed to ocular hypertension, and Shh has neuroprotective effects on damaged RGCs in a rat chronic hypertension model. Shh may exert neuroprotective effects by relieving the inhibition of Smo and subsequently activating Gli1.
[show abstract][hide abstract] ABSTRACT: To investigate the feasibility of introducing brain-derived neurotrophic factor (BDNF) gene into retinal pigment epithelial cells in vivo by electroporation and whether this method can rescue photoreceptors of retinitis pigmentosa in Royal College Surgeons (RCS) rats.
The BDNF-GFP fusion eukaryotic-expressing plasmid was constructed and subretinally or intravitreously injected into the eyes of RCS rats followed by in vivo electroporation. The expression of BDNF mRNA and protein was detected by RT-PCR and Western immunoblot analysis. The number of surviving photoreceptors was counted, and the TdT-dUTP terminal nick-end labeling (TUNEL) method was used to detect the apoptotic retinal cells at different timepoints after introduction of BDNF plasmid.
Treated eyes showed a significantly higher rescue ratio and a lower number of TUNEL-positive photoreceptors than did the control eyes at various timepoints.
These findings provide evidence that electroporation is an effective method for gene transfer into retinal pigment epithelial cells, and the rescue of photoreceptors can be achieved by BDNF gene transfection with electroporation.
Current eye research 09/2009; 34(9):791-9. · 1.51 Impact Factor
[show abstract][hide abstract] ABSTRACT: Recombinant adeno-associated virus (rAAV) is widely used in retinal gene therapy. Enhanced rAAV transduction may be important for better therapeutic effects in some retinal gene therapies. In this study, we examined the effects of adenovirus 5 (Ad5) on retina transduction mediated by rAAV2. Our results provide the first evidence that low levels of either replication-incompetent or conditional replication-competent Ad5 significantly enhance and accelerate transgene expression in human and rat retinal cells. This effect occurs principally at the transcriptional level, rather than through enhanced viral entry or DNA replication. In in vivo analyses with the SD rat, the Balb/c mouse, and the RCS rat, strong enhancement and acceleration of transgene expression, as well as therapeutic effects, were confirmed. Low levels of Ad5 may enhance the utility of rAAV2-mediated transduction strategies in future clinical investigations.
Vision Research 08/2008; 48(15):1648-54. · 2.14 Impact Factor
[show abstract][hide abstract] ABSTRACT: Engraftment of marrow stromal cells (MSCs) has been proposed as a therapeutic approach for degenerative diseases. In this study we investigated the fate and dynamic progress of grafted MSCs in living retina with the aim of evaluating the use of transplanted MSCs to treat retinal degeneration. Approximately 1x10(5) gfp-MSCs in 2 microl phosphate-buffered saline were injected into the subretinal space of adult Sprague-Dawley rats. Two weeks later, approximately 0.174%+/-0.082% of the transplanted cells had survived and diffused into the subretinal space. Nine weeks after transplantation the surviving gfp-MSCs accounted for 0.049%+/-0.023% of the number of cells injected and were mainly located at the injection site. The same number of MSCs were transplanted into the left eye subretinal space of 3-week-old hereditary retinal degenerative Royal College of Surgeons rats, and phosphate-buffered saline was injected into their right eyes as a control. Five weeks after transplantation, the amount of rudimentary photoreceptors was more significantly increased in grafted eyes than in control eyes. The results indicated that grafted MSCs could survive and rescue retinal degeneration.