Purpose To visualize and quantify conventional outflow directly in its anatomic location. Methods We obtained fluorescein canalograms in six porcine whole eyes and six porcine anterior segment cultures. Eyes were perfused with a constant pressure of 15 mmHg using media containing 0.017 mg/ml fluorescein. Flow patterns were visualized using a stereo dissecting microscope equipped for fluorescent imaging. Images were captured every 30 seconds for 20 minutes for time lapse analysis. Anterior chamber cultures were imaged again on day three of culture. Canalograms were first analyzed for filling time per quadrant. We then wrote a program to automatically compute focal flow fits for each macropixel and to detect convergent perilimbal flow patterns with macropixels grouped into 3 equal-radial width rings around the cornea. A generalized additive model was used to determine fluorescence changes of individual macropixels. Results The resulting imaging algorithm deployed 1024 macropixels that were fit to determine maximum intensity and time to fill. These individual fits highlighted the focal flow function. In whole eyes, significantly faster flow was seen in the inferonasal (IN) and superonasal (SN) quadrants compared to the superotemporal (ST) and inferotemporal (IT) ones (p<0.05). In anterior chamber cultures, reduced flow on day 1 increased in all quadrants on day 3 except in IT (p<0.05). Perilimbal ring analysis uncovered convergent perilimbal flow. Conclusions An algorithm was developed that analyzes regional and circumferential outflow patterns. This algorithm found flow patterns that changed over time and differ in whole eyes and anterior segment cultures.

Regionally Discrete Aqueous Humor Outflow Quantification using Fluorescein Canalograms

Bacterial keratitis is a devastating condition that can rapidly progress to serious complications if not treated promptly. Certain causative microorganisms such as Staphylococcus aureus and Pseudomonas aeruginosa are notorious for their resistance to antibiotics. Resistant bacterial keratitis results in poorer outcomes such as scarring and the need for surgical intervention. Thorough understanding of the causative pathogen and its virulence factors is vital for the discovery of novel treatments to avoid further antibiotic resistance. While much has been previously reported on P. aeruginosa, S. aureus has been less extensively studied. This review aims to give a brief overview of S. aureus epidemiology, pathophysiology and clinical characteristics as well as summarise the current evidence for potential novel therapies.

https://www.mdpi.com/2077-0383/10/4/758/pdf

Staphylococcus aureus Keratitis: Incidence, Pathophysiology, Risk Factors and Novel Strategies for Treatment

OBJECTIVES To report the spectrum and trends of isolated microorganisms and their antibiotic susceptibility profile in patients with infectious keratitis in a 6-year period at a referral centre in Tehran. METHODS The microbiology records of all corneal scrapings with a diagnosis of infectious keratitis were reviewed. RESULTS A total of 6282 corneal scrapings were performed during the study period, of which 2479 (39.5%) samples were culture positive. Pseudomonas aeruginosa was found to be the most common causative agent in patients with keratitis, although Streptococcus pneumonia was the most prevalently isolated microorganism in patients older than 50 years. Fusarium sp. was the most common responsible pathogen in patients with fungal keratitis. The prevalence of bacterial keratitis due to gram positive microorganisms increased over time, however the number of Pseudomonas keratitis decreased in the second half of the study. Gram negative organisms showed a good sensitivity to levofloxacin, however, 34.1% of S. aureus isolates and 29.7% of coagulase negative staphylococci were resistant to this antibiotic. The odds of ciprofloxacin and levofloxacin resistance increased 1.25 and 1.15 for each 1-year increase in culture date, respectively (P < 0.001, P = 0.004). CONCLUSIONS We documented an increasing trend in the percentage of gram positive bacteria. Levofloxacin monotherapy might still be a good option in patients with gram negative bacterial keratitis, however owing to increasing resistance of staphylococci to fluoroquinolones, a regimen consisting of a combination of fortified antibiotics may be more effective in staphylococcal keratitis.

Infectious keratitis: trends in microbiological and antibiotic sensitivity patterns.

Coagulase-negative staphylococci (CoNS) are frequently occurring ocular opportunistic pathogens that are not easily identifiable to the species level. The goal of this study was to speciate CoNS and document antibiotic susceptibilities from cases of endophthalmitis (n = 50), keratitis (n = 50), and conjunctivitis/blepharitis (n = 50) for empiric therapy. All 150 isolates of CoNS were speciated using (1) API Staph (biochemical system), (2) Biolog GEN III Microplates (phenotypic substrate system), and (3) DNA sequencing of the sodA gene. Disk diffusion antibiotic susceptibilities for topical and intravitreal treatment were determined based on serum standards. CoNS identification to the species level by all three methods indicated that S. epidermidis was the predominant species of CoNS isolated from cases of endophthalmitis (84–90%), keratitis (80–86%), and conjunctivitis/blepharitis (62–68%). Identifications indicated different distributions of CoNS species among endophthalmitis (6), keratitis (10), and conjunctivitis/blepharitis (13). Antibiotic susceptibility profiles support empiric treatment of endophthalmitis with vancomycin, and keratitis treatment with cefazolin or vancomycin. There was no clear antibiotic choice for conjunctivitis/blepharitis. S. epidermidis was the most frequently found CoNS ocular pathogen, and infection by other CoNS appears to be less specific and random. Antibiotic resistance does not appear to be a serious problem associated with CoNS.

https://www.mdpi.com/2079-6382/10/6/721/pdf

Speciation and Antibiotic Susceptibilities of Coagulase Negative Staphylococci Isolated from Ocular Infections.

Recent advances in the medical and surgical management of open-angle glaucoma have increased the number of treatment options available. Several new intraocular pressure (IOP)-lowering treatments target the conventional aqueous outflow (AO) system. However, success rates are variable and outcomes in individual patients are often difficult to predict. Variable treatment responses remain unexplained and highlight deficiencies in our current understanding of AO regulation and IOP homeostasis. Imaging is often relied upon to confirm diagnoses and monitor treatment responses in other ocular and systemic pathologies. As yet no suitable AO imaging tool has been developed to fulfil this role in glaucoma. A variety of imaging techniques have been used to study the AO tracts of humans and animals in ex vivo and in vivo eyes. In this review, results from novel imaging techniques that assess aqueous drainage through the episcleral venous system are considered and we argue these provide new insights into AO regulation. We suggest that the ability to objectively measure AO responses to interventions would be a significant clinical advance, and we have demonstrated that this can be achieved with direct visualisation of aqueous drainage. We predict that the evolution of AO imaging technology will continue to reveal critical components of AO and IOP regulation, and that personalised IOP-lowering treatment in glaucoma care may well become a reality in the near future.

/pdf/aqueous-outflow-imaging-techniques-and-what-they-tell-us-2b3no1l2bk.pdf

Aqueous outflow imaging techniques and what they tell us about intraocular pressure regulation.

Methicillin-Resistant Staphylococcus aureus Keratitis: Initial Treatment, Risk Factors, Clinical Features, and Treatment Outcomes.

PURPOSE To investigate whether microsurgical excision of trabecular meshwork (TM) in an ex vivo pigmentary glaucoma model can normalize the hypertensive phenotype. METHODS Eight eyes of a porcine pigmentary glaucoma model underwent 90° of microsurgical TM excision with an aspirating dual-blade (Goniotome (G)). 24 hours later, additional 90° of TM were removed. Anterior segments with sham surgeries served as the control (C). Outflow facility and intraocular pressure (IOP) were analyzed. Histology with hematoxylin and eosin (H&E) was obtained. RESULTS After the first 90° TM excision, IOP was significantly lower in G (10.2±2.4 mmHg, n = 7) than C (20.0±2.0mmHg, n = 8, P<0.01). Outflow facility in G (0.38±0.07 μl/min/mmHg) was higher than C (0.16±0.02 μl/min/mmHg, P<0.01). After the second 90° TM excision, IOP in G (6.46±0.81 mmHg, n = 7) was significantly lower than C (20.3±1.7 mmHg, n = 8, P<0.001), while the outflow facility in G (0.50±0.05 μl/min/mmHg, n = 7) was higher than C (0.16±0.01 μl/min/mmHg, n = 8, P<0.001). Compared to the first excision, excision of an additional 90° did not change of IOP (P = 0.20) or outflow facility (P = 0.17) further. CONCLUSIONS Excision of 90° of TM in a pigmentary glaucoma model using an aspirating dual-blade decreased IOP and increased outflow facility.

Intraocular pressure reduction in a pigmentary glaucoma model by Goniotome Ab interno trabeculectomy.

To characterize the effects of netarsudil on the aqueous humor outflow tract distal to the trabecular meshwork (TM). We hypothesized that netarsudil increases outflow facility in eyes with and without circumferential ab interno trabeculectomy (AIT) that removes the TM. Sixty-four porcine anterior segment cultures were randomly assigned to groups with (n = 32) and without circumferential AIT (n = 32). Cultures were exposed to 0.1, 1, and 10 μM netarsudil (N = 8 eyes per concentration). For each concentration, IOP and vessel diameters were compared with their respective pretreatment baselines. Outflow tract vessel diameters were assessed by spectral-domain optical coherence tomography (SDOCT) and rendered in 4D (XYZ time series). Netarsudil at 1 μM reduced IOP both in eyes with TM (− 0.60 ± 0.24 mmHg, p = 0.01) and in eyes without TM (− 1.79 ± 0.42 mmHg, p < 0.01). At this concentration, vessels of the distal outflow tract dilated by 72%. However, at 0.1 μM netarsudil elevated IOP in eyes with TM (1.59 ± 0.36 mmHg, p < 0.001) as well as in eyes without TM (0.23 ± 0.32 mmHg, p < 0.001). Vessels of the distal outflow tract constricted by 31%. Similarly, netarsudil at a concentration of 10 μM elevated IOP both in eyes with TM (1.91 ± 0.193, p < 0.001) and in eyes without TM (3.65 ± 0.86 mmHg, p < 0.001). At this concentration, outflow tract vessels constricted by 27%. In the porcine anterior segment culture, the dose-dependent IOP changes caused by netarsudil matched the diameter changes of distal outflow tract vessels. Hyper- and hypotensive properties of netarsudil persisted after TM removal.

/pdf/dose-dependent-effects-of-netarsudil-a-rho-kinase-inhibitor-je16p6eggh.pdf

Dose-dependent effects of netarsudil, a Rho-kinase inhibitor, on the distal outflow tract.

Purpose: To characterize the effects of netarsudil on the aqueous humor outflow tract distal to the trabecular meshwork (TM). We hypothesized that netarsudil increases outflow facility in eyes with and without circumferential ab interno trabeculectomy (AIT) that removes the TM.
Methods: 64 porcine anterior segment cultures were randomly assigned to groups with (n=32) and without circumferential AIT (n=32). Cultures were exposed to 0.1, 1, and 10 microM netarsudil (N= 8 eyes per concentration). For each concentration, IOP and vessel diameters were compared to their respective pretreatment baselines. Outflow tract vessel diameters were assessed by spectral-domain optical coherence tomography (SDOCT) and rendered in 4D (XYZ time-series). 
Results: Netarsudil at 1 microM reduced IOP in both eyes with TM (-0.60+/-0.24 mmHg, p = 0.01) and in eyes without TM (-1.79+/-0.42 mmHg, p<0.01). At this concentration, vessels of the distal outflow tract dilated by 72%. However, at 0.1 microM netarsudil elevated IOP in eyes with TM (1.59+/-0.36 mmHg, p<0.001) as well as in eyes without TM (0.23+/-0.32 mmHg, p<0.001). Vessels of the distal outflow tract constricted by 31%. Similarly, netarsudil at a concentration of 10 microM elevated IOP both in eyes with TM (1.91+/-0.193, p<0.001) and in eyes without TM (3.65+/-0.86 mmHg, p<0.001). At this concentration, outflow tract vessels constricted by 27%. 
Conclusion: In the porcine anterior segment culture, the dose-dependent IOP changes caused by netarsudil matched the diameter changes of distal outflow tract vessels. Hyper- and hypotensive properties of netarsudil persisted after TM removal. 
Keywords: Rho-kinase inhibitor; netarsudil; distal outflow tract; anterior chamber perfusion model; porcine eyes

/pdf/dose-dependent-effects-of-netarsudil-a-rho-kinase-inhibitor-fuq1zazhrc.pdf

Dose-dependent effects of netarsudil, a Rho-kinase inhibitor, on the distal outflow tract

Purpose The risk for glaucoma is driven by the microanatomy and function of the anterior segment. We performed a computation-intense, high-resolution, full-thickness ribbon-scanning confocal microscopy (RSCM) of the outflow tract of two human eyes. We hypothesized this would reveal important species differences when compared to existing data of porcine eyes, an animal that does not spontaneously develop glaucoma. Methods After perfusing two human octogenarian eyes with lectin-fluorophore conjugate and optical clearance with benzyl alcohol benzyl benzoate (BABB), anterior segments were scanned by RSCM and reconstructed in 3D for whole-specimen rendering. Morphometric analyses of the outflow tract were performed for the trabecular meshwork (TM), limbal, and perilimbal outflow structures and compared to existing porcine data. Results RSCM provided high-resolution data for IMARIS-based surface reconstruction of outflow tract structures in 3D. Different from porcine eyes with an abundance of highly interconnected, narrow, and short collector channels (CCs), human eyes demonstrated fewer CCs which had a 1.5x greater cross-sectional area (CSA) and 2.6x greater length. Proximal CC openings at the level of Schlemm's canal (SC) had a 1.3x larger CSA than distal openings into the scleral vascular plexus (SVP). CCs were 10.2x smaller in volume than the receiving SVP vessels. Axenfeld loops, projections of the long ciliary nerve, were also visualized. Conclusion In this high-resolution, volumetric RSCM analysis, human eyes had far fewer outflow tract vessels than porcine eyes. Human CCs spanned several clock-hours and were larger than in porcine eyes. These species differences may point to factors downstream of the TM that increase our vulnerability to glaucoma.

/pdf/3d-reconstruction-of-the-human-conventional-outflow-system-3r3cjbgc4k.pdf

Sarah Atta

Papers

Methicillin-Resistant Staphylococcus aureus Keratitis: Initial Treatment, Risk Factors, Clinical Features, and Treatment Outcomes.

Intraocular pressure reduction in a pigmentary glaucoma model by Goniotome Ab interno trabeculectomy.

Dose-dependent effects of netarsudil, a Rho-kinase inhibitor, on the distal outflow tract.

Dose-dependent effects of netarsudil, a Rho-kinase inhibitor, on the distal outflow tract

3D-Reconstruction of the human conventional outflow system by ribbon scanning confocal microscopy.