[Experimental study on eyelid reconstruction with acellular xenogenic dermal matrix].

OBJECTIVE: To investigate the histocompatibility of acellular xenogenic pig dermal matrix (Xeno-ADM) after transplanted into the rabbit eyelids. METHODS: Thirty-eight New Zealand rabbits were divided into two groups randomly. After excising a 5 mm x 4 mm tarso-conjunctiva flap from one lower eyelid of each rabbit, Xeno-ADM and allogenic sclera were implanted in these two groups separately. Samples of implanted material were collected for histological examination in 1, 2, 4, 6, 8, 12 and 16 weeks after the operation. RIA method was employed to determine the contents of IL-2 and IL-6 in the homogenate fluid from local tissues and in peripheral blood. RESULTS: Compared to the control group, after the operation, the conjunctival congestion, effusion, inflammatory reaction and red swelling of the eyelid subsided faster in the treated group. No rejection or necrosis occurred in transplanted rabbits. At 16 weeks after operation, SNK-q test showed there was no obvious difference in the average breadth and height of the eyelids (P > 0.05) between these two groups and normal rabbits. Histological examination of sections of eyelid tissues stained by HE staining and observed under light microscope showed that at 1, 2, 4, 6, 8, 12 and 16 weeks postoperatively, eyelids implanted with Xeno-ADM had less inflammatory reaction, fewer lymphocytes infiltrating and higher vascularization with faster ingrowths of new collagen fibers, as compared with the sclera-implanted group. This also indicated that Xeno-ADM had a good compatibility. Statistical analysis of immunogenic indexes indicated that the level of IL-2 and IL-6 in the homogenate fluid at 6-8 weeks postoperatively was greater than those in 1, 2, 4 weeks after implantation. The level of IL-2 and IL-6 rose significantly after the operation and reached a peak value at the 6 th week (experimental group, IL-2, 0.292 81 ng/ml) and 8 th week (experimental group, IL-6, 118.258 pg/ml; control group, IL-2, 0.277 99 ng/ml and IL-6, 255.871 pg/ml). The level of IL-2 and IL-6 dropped significantly at the 12th week and raised again at the 16th week, therefore, the concentration achieved another peak value in the control group (IL-2, 1.363 41 ng/ml; IL-6, 622.863 pg/ml), which was much higher than those in the first peak. The level of IL-2 and IL-6 in peripheral blood 1 week after the transplantation was higher than the preoperative level. These two indexes increased gradually with marked fluctuation, particularly in 4 - 8 weeks after implantation, both of them reached the peak values at that period, which was similar to the inflammation reaction changes in the implanted groups. With replicated testing and analysis of variance (ANOVA), neither the type of treatment nor the time factor had any effect on the levels of IL-2 and IL-6 in the serum, and there was no significant difference between experimental group and control group (P > 0.05). CONCLUSIONS: Xeno-ADM shows good histocompatibility to New Zealand rabbits, which can induce neovascular and collagen fibers grow into implanted tissues. Therefore, this can be used to support the eyelids as a substitution for the tarsus. High levels of IL-2 and IL-6 can be detected in local tissues and in the serum after Xeno-ADM transplantation, but no statistical differences present between the dynamic changes of the level of both ILs in Xeno-ADM group and allogenic sclera group. Xeno-ADM could be an ideal substitute for allogenic sclera in the reconstruction of eye lids and could be used broadly in ophthalmology. However, as a hetero-transplantation material used clinically, further ethical and immunological studies are required.

Pigment epithelium-derived factor protects retinal ganglion cells from hypoxia-induced apoptosis by preventing mitochondrial dysfunction.

AIM: To investigate the potential of pigment epithelium-derived factor (PEDF) to protect the immortalized rat retinal ganglion cells-5 (RGC-5) exposed to CoCl2-induced chemical hypoxia. METHODS: After being differentiated with staurosporine (SS), RGC-5 cells were cultured in four conditions: control group cells cultured in Dulbecco's modified eagle medium (DMEM) supplemented with 10% fetal bovine serum, 100 µmol/mL streptomycin and penicillin (named as normal conditions); hypoxia group cells cultured in DMEM containing 300 µmol/mL CoCl2; cells in the group protected by PEDF were first pretreated with 100 ng/mL PEDF for 2h and then cultured in the same condition as hypoxia group cells; and PEDF group cells that were cultured in the presence of 100 ng/mL PEDF under normal conditions. The cell viability was assessed by MTT assay, the percentage of apoptotic cells was quantified using Annexin V-FITC apoptosis kit, and intra-cellar reactive oxygen species (ROS) was measured by dichloro-dihydro-fluorescein diacetate (DCFH-DA) probe. The mitochondria-mediated apoptosis was also examined to further study the underlying mechanism of the protective effect of PEDF. The opening of mitochondrial permeability transition pores (mPTPs) and membrane potential (Δψm) were tested as cellular adenosine triphosphate (ATP) level and glutathione (GSH). Also, the expression and distribution of Cyt C and apoptosis inducing factor (AIF) were observed. RESULTS: SS induced differentiation of RGC-5 cells resulting in elongation of their neurites and establishing contacts between outgrowths. Exposure to 300 µmol/mL CoCl2 triggered death of 30% of the total cells in cultures within 24h. At the same time, pretreatment with 100 ng/mL PEDF significantly suppressed the cell death induced by hypoxia (P<0.05). The apoptosis induced by treatment of CoCl2 was that induced cell death accompanied with increasing intra-cellar ROS and decreasing GSH and ATP level. PEDF pre-treatment suppressed these effects (P<0.05). Additionally, PEDF treatment inhibited the opening of mPTPs and suppressed decreasing of Δψm in RGC-5 cells, resulting in blocking of the mitochondrial apoptotic pathway. CONCLUSION: Pretreatment of RGC-5 cells with 100 ng/mL PEDF significantly decreases the extent of apoptosis. PEDF inhibits the opening of mPTPs and suppresses decreasing of Δψm. Moreover, PEDF also reduces ROS production and inhibits cellular ATP level's reduction. Cyt C and AIF activation in PEDF-pretreated cultures are also reduced. These results demonstrate the potential for PEDF to protect RGCs against hypoxic damage in vitro by preventing mitochondrial dysfunction.

Panretinal photocoagulation versus panretinal photocoagulation plus intravitreal bevacizumab for high-risk proliferative diabetic retinopathy.

AIM: To evaluate the effects of panretinal photocoagulation (PRP) compared with PRP plus intravitreal bevacizumab (IVB) in patients with high-risk proliferative diabetic retinopathy (PDR) according to the Early Treatment Diabetic Retinopathy Study criteria. METHODS: The data were collected retrospectively from the eyes of high-risk PDR patients, which were divided into two groups. After treated with standard PRP, the eyes were randomly assigned to receive only PRP (PRP group) or PRP plus intravitreal injection of 1.25 mg of bevacizumab (PRP-Plus group). Patients underwent complete ophthalmic evaluation, including best corrected visual acuity (BCVA), intraocular pressure (IOP), and new vessel size in fluorescein angiography (FA) and optical coherence tomography for the assessment of central subfield macular thickness (CSMT) at baseline and at weeks 12 (±2), 16 (±2), 24 (±2) and 48 (±2). Main outcome measures also included vitreous clear-up time and neovascularization on the disc (NVD) regression time. Adverse events associated with intravitreal injection were investigated. RESULTS: Thirty consecutive patients (n=36 eyes) completed the 48-week follow-up. There was no significant difference between the PRP and PRP-Plus groups with respect to age, gender, type or duration of diabetes, area of fluorescein leakage from active neovascularizations (NVs), BCVA or CSMT at baseline. The mean vitreous clear-up time was 12.1±3.4wk after PRP and 8.4±3.5wk after PRP combined with IVB. The mean time interval from treatment to complete NVD regression on FA examination was 15.2±3.5wk in PRP group and 12.5±3.1wk in PRP-Plus group. No significant difference in CSMT was observed between the groups throughout the study period. However, the total area of actively leaking NVs was significantly reduced in the PRP-Plus group compared with the PRP group (P<0.05). Patients received an average of 1.3 injections (range: 1-2). Ten eyes (27.8%) underwent 2 injections. Two eyes had ocular complication of PDR progression to dense vitreous hemorrhage (VH). No major adverse events were identified. CONCLUSION: The adjunctive use of IVB with PRP is associated with a greater reduction in the area of active leaking NVs than PRP alone in patients with high-risk PDR. Short-term results suggest combined IVB and PRP achieved rapid clearance of VH and regression of retinal NV in the treatment of high-risk PDR. Further studies are needed to determine the effect of repeated intravitreal bevacizumab injections and the proper number of bevacizumab injections as an adjuvant.

Diffusion tensor imaging of optic nerve and optic radiation in primary chronic angle-closure glaucoma using 3T magnetic resonance imaging.

AIM: To evaluate the value of quantitative diffusion tensor imaging (DTI) in assessing the axonal and myelin damage of the optic nerves and optic radiations in patients with chronic primary angle-closure glaucoma (PACG) by using high-field magnetic resonance (MR) imaging (3T). METHODS: Twenty patients with bilateral chronic PACG and twenty age- and sex matched disease-free control subjects were enrolled. Conventional MRI and DTI were performed on all subjects using 3T MR scanner. Mean diffusivity (MD), fractional anisotropy (FA), axial diffusivities (AD) and radial diffusivities (RD) of each optic nerve and each optic radiation were measured by using post-processing software of DTI studio 2.3, and then compared between left eyes and right eyes and between patients group and control group. The paired-sample t- test were used. RESULTS: There was no abnormality in the shape and signal intensity of the optic nerves and optic radiations in patients group and control group on the conventional MRI. No significant differences were observed in the FA, MD, AD and RD between the right and left optic nerves and optic radiations within patients group and control group (P>0.05). The optic nerves and optic radiations of patients with chronic PACG, as compared with control subjects, had significantly higher MD, AD, RD and significantly lower FA (P<0.05). CONCLUSION: The diffusivity of optic nerves and optic radiations in chronic PACG group showed abnormal and diffusivity parameters could be used markers of axonal and myelin injury in glaucoma.

Alteration of UCP2 and ZO-1 expression in trabecular meshwork of neovascular glaucoma patients.

PURPOSE: The aim of this study was to investigate the expression of uncoupling protein 2 (UCP2) and zonula occludens-1 (ZO-1) in the trabecular meshwork (TM) of neovascular glaucoma (NVG) patients treated with trabeculectomy. MATERIALS AND METHODS: Six eyes with NVG underwent trabeculectomy for therapeutic purposes. The data consisted of patient demographics, presurgical and postsurgical visual acuity, intraocular pressure, gonioscopy, and neovascularization of iris and/or the anterior chamber angle. TM samples were obtained from the NVG eyes that had undergone surgery. Immunofluorescence and confocal laser scanning microscopy were carried out to determine the expression of UCP2 and ZO-1 in the TM cells. RESULTS: The baseline median visual acuity was light perception, and the mean intraocular pressure (standard error) was 52.5 (8.3) mm Hg. All eyes displayed neovascularization of the iris and the anterior chamber angle. The expression of UCP2 was significantly decreased in TM cells of NVG compared with the control (P=0.000), whereas increase in ZO-1 expression was detected in staining cells with NVG in comparison with the control (P=0.000). The necrotic cells in the TM were increased (P=0.000), whereas the viable cells were reduced (P=0.000) in comparison with the control. CONCLUSIONS: The decreased UCP2 expression and increased ZO-1 expression suggest that the oxidative stress-induced mitochondrial dysfunction and tight junction formation may play pivotal roles in the progress of NVG.

Surgical treatment for 42 patients with traumatic annular ciliochoroidal detachment.

AIM: To analyze the clinical features of traumatic annular ciliochoroidal detachment (CCD) with ultrasound biomicroscopy (UBM) images, to investigate the surgical outcomes of ciliary body suturing and the prognostic factors. METHODS: Forty-two patients with traumatic annular CCD who had undergone ciliary body suturing were enrolled for complete ocular examinations, including visual acuity (VA), slitlamp microscopy, tonometer, indirect ophthalscopy and UBM. Comparisons of clinical features were performed among baseline and follow-ups, and the morphologic alterations on UBM images were analyzed between pre- and post-surgery. RESULTS: The mean intraocular pressure (IOP) was 5.54mmHg, and the median VA was 0.1 in traumatic eyes at baseline. The pre-surgical morphological features on UBM images consisted of supraciliochoroidal effusion (33.33%), multilayer splits (40.48%) and CCD with cyclodialysis cleft (26.19%). After surgery, the median VA was 0.4 at the final follow-up. IOPs were significantly increased, which the mean final IOP was to 10.36mmHg (P<0.01). UBM images displayed complete reattachment in 40.48% of patients, partial reattachment in 50.00% of patients and 360-degree detachment in 9.52% of patients. Analyzing the prognostic factors, the significant factors were duration, VA at baseline, ocular laterality (P<0.01), gender, age and the presence of hypotonous maculopathy (P<0.05). CONCLUSION: Ciliary body suturing is the optimized procedure for traumatic annular CCD. UBM is a useful equipment for diagnosis and monitoring post-surgical morphological changes. The periodical detection of IOP and UBM is necessary for the observation of surgical outcomes.

[Experimental study of the eyelid reconstruction in situ with the acellular xenogeneic dermal matrix].

OBJECTIVE: To investigate the histocompatibility of acellular xenogeneic dermal matrix implanted in the rabbit eyelid reconstruction in situ and to compare the histological change of acellular xenogeneic dermal matrix and sclera replacing tarsus. METHODS: Thirty-six New Zealand rabbits were divided into two groups randomly. Establishment of the rabbits unilateral eyelid defect model, the eyelid reconstruction in situ were performed with either acellular xenogeneic dermal matrix or allogeneic sclera at random. The rabbits were clinically examined for inflammation and implant exposure and sacrificed 1, 2, 4, 6, 8 and 12 weeks after implantation. The eyelid with implant (Xeno-ADM or allogeneic sclera) were dislodged and the specimens were assessed histopathologically and ultrastructurally with light microscopies respectively for evaluation of change of juncture between implant and autoallergic tarsal plates including inflammation, vascularization and confluence. The 4, 8 and 12 weeks specimens were assessed with transmission electron microscope micro structural changes of the above organizations. RESULTS: Light microscopy and electron microscopy showed no statistical difference between two groups. But histological examination showed that eyelid implanted with acellular xenogeneic dermal matrix had less immunological and inflammatory reaction than sclera-implanted group. Acellular xenogeneic dermal matrix could induce neovascular and collagenous fibers into implanted tissue. CONCLUSION: Acellular xenogeneic dermal matrix is histocompatible to New Zealand rabbit, it can be used to support the eyelid as a substitution for tarsus.