Long-term Outcomes of Baerveldt Glaucoma Implant Shunts as a Primary Versus Secondary Procedure.

PURPOSE: To determine the 5-year success rate for Baerveldt glaucoma implant (BGI) in patients who received the implant as a primary (primary BGI, used as the initial surgical procedure) or secondary (secondary BGI, used after trabeculectomy) by a single surgeon between year 1994 and 2010. PATIENTS AND METHODS: A total of 117 eyes from patients who underwent BGI placement as a primary or secondary procedure were included in this study. Demographics, previous history, type of glaucoma, intraocular pressure (IOP), visual acuity (VA), glaucoma medication use, and early and late postoperative complications were collected. Postoperative data were collected at day 1, 30, 60, and every year until the last visit (minimum of 2 y). IOP was the main outcome measure. Secondary outcome measures included VA, glaucoma medication use, and early and late postoperative complications. Overall success rates were calculated for primary and secondary BGI using Kaplan-Meier survival analysis. Differences between survival curves were determined using log-rank test. Risk factors for success were defined by Cox proportional-hazards regression model. RESULTS: At the 5-year follow-up, the overall success rates (determined as IOP between 6 and 21 mm Hg) in the primary and secondary BGI were 58% (49/79) and 53% (20/38), respectively (P=0.56). The overall success rate dropped by an average 10% and 13% per year for the primary and secondary BGI groups, respectively (P=0.05). The complete success rates at the 5-year follow-up for primary and secondary BGI were 24% (19/79) and 13% (5/38), respectively, whereas the qualified success rates were 34% (27/79) and 39% (15/38), respectively. There was a significant decrease in IOP [from 31.23 (±9.51) to 12.86 (±4.84) mm Hg (P<0.001) for the primary BGI group, and from 26.35 (±7.22) to 13.92 (±5.90) mm Hg (P<0.001) for the secondary BGI group]. There was also a significant drop in medication use but a significant worsening in VA in both groups (P<0.05) likely from cataract. The difference in the incidence of postoperative complications was not statistically significant between the groups. CONCLUSIONS: Although, the success rates were similar for the primary and secondary BGI at the 5-year follow-up, the drop in the success rate per year was significantly higher in the secondary BGI group. In contrast, both procedures had similar incidence of postoperative complications.

Non-invasive Detection of Unique Molecular Signatures in Laser-Induced Retinal Injuries.

Unintentional laser exposure is an increasing concern in many operational environments. Determining whether a laser exposure event caused a retinal injury currently requires medical expertise and specialized equipment that are not always readily available. The purpose of this study is to test the feasibility of using dynamic light scattering (DLS) to non-invasively detect laser retinal injuries through interrogation of the vitreous humor (VH). Three grades of retinal laser lesions were studied: mild (minimally visible lesions), moderate (Grade II), and severe (Grade III). A pre-post-treatment design was used to collect DLS measurements in vivo at various time points, using a customized instrument. VH samples were analyzed by liquid chromatography/tandem mass spectrometry (LC-MS/MS) and relative protein abundances were determined by spectral counting. DLS signal analysis revealed significant changes in particle diameter and intensity in laser-treated groups as compared with control. Differences in protein profile in the VH of the laser-treated eyes were noted when compared with control. These results suggest that laser injury to the retina induces upregulation of proteins that diffuse into the VH from the damaged tissue, which can be detected non-invasively using DLS.

Lymphatic and Blood Vessel Density in Human Conjunctiva After Glaucoma Filtration Surgery.

PURPOSE: To investigate the lymphatic vascular microvessel density (LVD) and the blood vascular microvessel density (MVD) and their distribution in excised leaking blebs after mitomycin C trabeculectomy and normal conjunctiva specimens. MATERIALS AND METHODS: LVD and MVD in normal human conjunctiva (n=8) and excised blebs in the hypocellular stroma and peribleb tissue (conjunctiva adjacent to hypocellular bleb tissue) (n=8) were evaluated by immunohistochemistry using antibodies raised against Lymphatic Vessel Endothelial Receptor 1 (D2-40, lymphatic endothelium) and CD34 (vascular endothelium). LVD and MVD counts were performed by light microscopy in 5 fields at ×20 magnification by 3 observers. Differences were determined using Mann-Whitney U test (P<0.05 was considered significant). RESULTS: The leaking blebs showed typical epithelial-stromal domes with areas of acellular stroma covered by attenuated epithelium and surrounded by normal conjunctival epithelium and a dense scar-like matrix replacing the substantia propria. The LVD and MVD were significantly reduced to nil in the hypocellular conjunctival stroma of the excised blebs compared with normal conjunctiva (21.42 vs. 1.16, P<0.002 and 24.28 vs. 1, P<0.008, respectively). The LVD and MVD was also reduced (2- to 2.5-fold) in the peribleb stroma when compared with normal conjunctiva specimens. CONCLUSIONS: In this study we show reduced LCD and MVD in the hypocellular and peribleb stroma. These results may suggest a role of these vessels in an altered immune response in leaking blebs leading to an increased risk for blebitis.

Proteomics in the Study of Bacterial Keratitis.

Bacterial keratitis is a serious ocular infection that can cause severe visual loss if treatment is not initiated at an early stage. It is most commonly caused by Staphylococcus aureus, Pseudomonas aeruginosa, Streptococcus pneumoniae, or Serratia species. Depending on the invading organism, bacterial keratitis can progress rapidly, leading to corneal destruction and potential blindness. Common risk factors for bacterial keratitis include contact lens wear, ocular trauma, ocular surface disease, ocular surgery, lid deformity, chronic use of topical steroids, contaminated ocular medications or solutions, and systemic immunosuppression. The pathogenesis of bacterial keratitis, which depends on the bacterium-host interaction and the virulence of the invading bacterium, is complicated and not completely understood. This review highlights some of the proteomic technologies that have been used to identify virulence factors and the host response to infections of bacterial keratitis in order to understand the disease process and develop improved methods of diagnosis and treatment. Although work in this field is not abundant, proteomic technologies have provided valuable information toward our current knowledge of bacterial keratitis. More studies using global proteomic approaches are warranted because it is an important tool to identify novel targets for intervention and prevention of corneal damage caused by these virulent microorganisms.

Proteomic analysis of keratitis-associated Pseudomonas aeruginosa.

PURPOSE: To compare the proteomic profile of a clinical isolate of Pseudomonas aeruginosa (P. aeruginosa) obtained from an infected cornea of a contact lens wearer and the laboratory strain P. aeruginosa ATCC 10145. METHODS: Antibiotic sensitivity, motility, biofilm formation, and virulence tests were performed using standard methods. Whole protein lysates were analyzed with liquid chromatography/ tandem mass spectrometry (LC-MS/MS) in triplicate, and relative protein abundances were determined with spectral counting. The G test followed by a post hoc Holm-Sidak adjustment was used for the statistical analyses to determine significance in the differential expression of proteins between the two strains. RESULTS: A total of 687 proteins were detected. One-hundred thirty-three (133) proteins were significantly different between the two strains. Among these, 13 were upregulated, and 16 were downregulated in the clinical strain compared to ATCC 10145, whereas 57 were detected only in the clinical strain. The upregulated proteins are associated with virulence and pathogenicity. CONCLUSIONS: Proteins detected at higher levels in the clinical strain of P. aeruginosa were proteins known to be virulence factors. These results confirm that the keratitis-associated P. aeruginosa strain is pathogenic and expresses a higher number of virulence factors compared to the laboratory strain ATCC 10145. Identification of the protein profile of the corneal strain of P. aeruginosa in this study will aid in elucidating novel intervention strategies for reducing the burden of P. aeruginosa infection in keratitis.

MicroRNA in aqueous humor from patients with cataract.

MicroRNAs (miRNAs) are small non-coding RNA molecules with regulatory function and marked tissue specificity that can modulate multiple gene targets. They have been detected in body fluids and are associated with various physiologic and pathologic processes. We analyzed aqueous humor (AH) from human subjects undergoing cataract surgery to establish the presence and relative quantities of known miRNAs. AH was collected from patients without known ocular diseases other than cataract and a normal systemic history. Quantitative real-time PCR in an array platform was used to detect known miRNAs present in the AH. Among the 264 miRNAs tested, 110 were present in the AH. The top 5 abundant miRNAs identified were miR-202, miR-193b, miR-135a, miR-365, and miR-376a. The presence of miRNAs in AH suggests that they may have functional roles in regulating target genes in tissues lining the anterior chamber. Further analysis of the AH miRNA population may identify potential gene targets and provide insights regarding their roles in AH regulation, glaucoma and anterior segment disease processes.

Immunohistochemical evidence of specific iris involvement in Blau syndrome.

PURPOSE: To evaluate iris involvement in Blau syndrome using histology and immunohistochemistry. METHODS: Iridectomy specimen of a patient with treated Blau syndrome and a normal control were evaluated by light microscopy and immunohistochemistry using antibodies against CD4(+), CD8(+), HLA-DR, CD68(+), NF-κB and IL-17. RESULTS: Blau iris tissue demonstrated increased numbers of CD4(+) lymphocytes and CD68 negative, HLA-DR positive spindle shaped cells compared to normal iris tissue. Blau iris tissue also demonstrated elevated CD4(+)/CD8(+) ratio and IL-17 and NF-κB immunolabeling. No macrophages, epithelioid cells, or granulomas were noted in the Blau specimen. CONCLUSIONS: The persistent immunolocalization of inflammatory markers in an iris specimen from an aggresively treated patient with proven Blau syndrome suggests that further pathologic and immunohistochemical investigation of Blau ocular tissue is necessary to better understand the complexities of NOD2 activating mutations in the eye.

Animal models of glaucoma.

Glaucoma is a heterogeneous group of disorders that progressively lead to blindness due to loss of retinal ganglion cells and damage to the optic nerve. It is a leading cause of blindness and visual impairment worldwide. Although research in the field of glaucoma is substantial, the pathophysiologic mechanisms causing the disease are not completely understood. A wide variety of animal models have been used to study glaucoma. These include monkeys, dogs, cats, rodents, and several other species. Although these models have provided valuable information about the disease, there is still no ideal model for studying glaucoma due to its complexity. In this paper we present a summary of most of the animal models that have been developed and used for the study of the different types of glaucoma, the strengths and limitations associated with each species use, and some potential criteria to develop a suitable model.

Anti-retinal antibodies in serum of laser-treated rabbits.

PURPOSE: Retinal injuries that affect the photoreceptors and/or the retinal pigment epithelium (RPE) may result in the leakage of retinal proteins into the systemic circulation. This study was designed to determine whether an immune response is elicited after an acute retinal injury resulting in circulating anti-retinal antibodies in the serum. METHODS: Fifty laser burns of different grades (minimally visible lesion [MVL], grade II [GII], or grade III [GIII] lesions) were created in the retinas of Dutch Belted rabbits. The degree of laser burns was confirmed by fundus imaging and histology. Serum samples were collected from the animals 3 months after the retinal injury. Candidate autoantigens were identified by two-dimensional (2-D) Western blots of rabbit retinal lysate probed with sera from either control or laser-treated animals. Candidate autoantigens were further characterized by immunostaining to confirm their retinal localization. RESULTS: Seven and 11 protein spots were selected from the MVL and GII laser-treated samples, respectively, for autoantigen identification. No protein spots were detected in the GIII laser-treated samples. Four candidate autoantigens were common to both MVL and GII lesions: dihydropyrimidinase-related protein 2, fructose-bisphosphate aldolase C, chaperonin-containing T-complex polypeptide 1 subunit zeta, and pyruvate kinase isozyme. CONCLUSIONS: Laser-induced retinal injuries resulted in circulating anti-retinal antibodies that were detectable 3 months after the injury. The response appeared to vary with the severity of the laser retinal damage. The identification of the candidate antigens in this study suggest that this approach may permit future development of new diagnostic methods for retinal injuries.

Novel serum proteomic signatures in a non-human primate model of retinal injury.

PURPOSE: To identify candidate protein biomarkers in sera indicative of acute retinal injury. METHODS: We used laser photocoagulation as a model of acute retinal injury in Rhesus macaques. In a paired-control study design, we collected serum from each animal (n=6) at 4 h, 1 day, and 3 days following a mock procedure and then again following retinal laser treatment that produced mild lesions. Samples were fractionated by isoelectric focusing, digested with trypsin, and analyzed by liquid chromatography/tandem mass spectrometry (LC-MS/MS). Spectral counting was used to determine relative protein abundances and identify proteins with statistically significant differences between control and treated sera. RESULTS: Mild retinal injury was confirmed by fundus photography and histological examination. The average number of total proteins detected by LC-MS/MS was 908±82 among samples from all three time points. Following statistical analysis and employing stringent filtering criteria, a total of 19 proteins were identified as being significantly more abundant in sera following laser-induced retinal injury, relative to control sera. Many of the proteins detected were unique to one time point. However, four proteins (phosphoglycerate kinase 1, keratin 18, Lewis alpha-3-fucosyltransferase, and ephrin receptor A2) showed differences that were significant at both 4 h and 1 day after laser treatment, followed by a decrease to baseline levels by day 3. CONCLUSIONS: A serum biomarker response to mild retinal laser injury was demonstrated in a primate model. Among the proteins detected with highest significant differences, most are upregulated within 24 h, and their appearance in the serum is transient. It is conceivable that a panel of these proteins could provide a means for detecting the acute-phase response to retinal injury. Further investigation of these candidate biomarkers and their correlation to retinal damage is warranted.

Suppression of transforming growth factor-ß effects in rabbit subconjunctival fibroblasts by activin receptor-like kinase 5 inhibitor.

PURPOSE: Transforming growth factor-ß (TGF-ß) activity has been implicated in subconjunctival scarring in eyes following glaucoma filtration surgery (GFS). The purpose of this study is to determine whether an inhibitor for activin receptor-like kinase (ALK) 5 (also known as TGF-ß receptor type I) could suppress TGF-ß activity and thereby promote filtering bleb survival after GFS in a rabbit model. METHODS: An ALK-5 inhibitor, SB-505124, was used. A docking study was performed to investigate the interaction between the inhibitor and the receptor. Immunofluorescence for connective tissue growth factor (CTGF) and α-smooth muscle actin (α-SMA) was performed in cultured rabbit subconjunctival fibroblasts. Immunoblotting for phosphorylated Smad2 (pSmad2), CTGF, and α-SMA was also performed. In an in vivo rabbit GFS model, SB-505124 was delivered in a lactose tablet during surgery. Eyes were examined by slit-lamp and intraocular pressure (IOP) was measured until the time of bleb failure or up to 28 days after surgery. Tissue sections on day 5 after surgery were histologically evaluated after staining with hematoxylin and eosin. The sections were also immunostained for CTGF and α-SMA. In addition, cell outgrowth from dissected subconjunctival tissues placed in a cell culture flask with media was investigated. RESULTS: The docking study indicated hydrogen bond interactions between SB-505124 and amino acids His-283 and Ser-280 of ALK-5. Suppression of pSmad2, CTGF, and α-SMA by SB-505124 was observed in cultured fibroblasts. Filtering blebs in the GFS with SB-505124 group were maintained for more than 10 days, and the period of bleb survival was significantly longer than that in controls. IOP levels after surgery seemed to be related to bleb survival. Histologically, subconjunctival cell infiltration and scarring at the surgical site in the GFS with SB-505124 and mitomycin C (MMC) groups were much subsided compared to controls. Suppression of CTGF and α-SMA by SB-505124 was also observed by immunofluorescence. Cell outgrowth from explants dissected from eyes to which SB-505124 was applied during GFS was robust while outgrowth was poor from those treated with MMC. CONCLUSIONS: The ALK-5 inhibitor SB-505124 was efficacious both in vitro and in vivo in suppressing the TGF-ß action. The inhibitor may provide a novel therapy for preventing ocular inflammation and scarring.

Genomic and expression analysis of multiple Sry loci from a single Rattus norvegicus Y chromosome.

BACKGROUND: Sry is a gene known to be essential for testis determination but is also transcribed in adult male tissues. The laboratory rat, Rattus norvegicus, has multiple Y chromosome copies of Sry while most mammals have only a single copy. DNA sequence comparisons with other rodents with multiple Sry copies are inconsistent in divergence patterns and functionality of the multiple copies. To address hypotheses of divergence, gene conversion and functional constraints, we sequenced Sry loci from a single R. norvegicus Y chromosome from the Spontaneously Hypertensive Rat strain (SHR) and analyzed DNA sequences for homology among copies. Next, to determine whether all copies of Sry are expressed, we developed a modification of the fluorescent marked capillary electrophoresis method to generate three different sized amplification products to identify Sry copies. We applied this fragment analysis method to both genomic DNA and cDNA prepared from mRNA from testis and adrenal gland of adult male rats. RESULTS: Y chromosome fragments were amplified and sequenced using primers that included the entire Sry coding region and flanking sequences. The analysis of these sequences identified six Sry loci on the Y chromosome. These are paralogous copies consistent with a single phylogeny and the divergence between any two copies is less than 2%. All copies have a conserved reading frame and amino acid sequence consistent with function. Fragment analysis of genomic DNA showed close approximations of experimental with predicted values, validating the use of this method to identify proportions of each copy. Using the fragment analysis procedure with cDNA samples showed the Sry copies expressed were significantly different from the genomic distribution (testis p < 0.001, adrenal gland p < 0.001), and the testis and adrenal copy distribution in the transcripts were also significantly different from each other (p < 0.001). Total Sry transcript expression, analyzed by real-time PCR, showed significantly higher levels of Sry in testis than adrenal gland (p, 0.001). CONCLUSION: The SHR Y chromosome contains at least 6 full length copies of the Sry gene. These copies have a conserved coding region and conserved amino acid sequence. The pattern of divergence is not consistent with gene conversion as the mechanism for this conservation. Expression studies show multiple copies expressed in the adult male testis and adrenal glands, with tissue specific differences in expression patterns. Both the DNA sequence analysis and RNA transcript expression analysis are consistent with more than one copy having function and selection preventing divergence although we have no functional evidence.