Functional, structural, and molecular identification of lymphatic outflow from subconjunctival blebs.

The purpose of this study is to evaluate outflow pathways from subconjunctival blebs and to identify their identity. Post-mortem porcine (n = 20), human (n = 1), and bovine (n = 1) eyes were acquired, and tracers (fluorescein, indocyanine green, or fixable/fluorescent dextrans) were injected into the subconjunctival space to create raised blebs where outflow pathways were visualized qualitatively and quantitatively. Rodents with fluorescent reporter transgenes were imaged for structural comparison. Concurrent optical coherence tomography (OCT) was obtained to study the structural nature of these pathways. Using fixable/fluorescent dextrans, tracers were trapped to the bleb outflow pathway lumen walls for histological visualization and molecular identification using immunofluorescence against lymphatic and blood vessel markers. Bleb outflow pathways could be observed using all tracers in all species. Quantitative analysis showed that the nasal quadrant had more bleb-related outflow pathways compared to the temporal quadrant (nasal: 1.9±0.3 pathways vs. temporal: 0.7±0.2 pathways; p = 0.003). However, not all blebs resulted in an outflow pathway (0-pathways = 18.2%; 1-pathway = 36.4%; 2-pathways = 38.6%; and 3-pathways = 6.8%). Outflow signal was validated as true luminal pathways using optical coherence tomography and histology. Bicuspid valves were identified in the direction of flow in porcine eyes. Immunofluorescence of labeled pathways demonstrated a lymphatic (Prox-1 and podoplanin) but not a blood vessel (CD31) identity. Therefore, subconjunctival bleb outflow occurs in discrete luminal pathways. They are lymphatic as assessed by structural identification of valves and molecular identification of lymphatic markers. Better understanding of lymphatic outflow may lead to improved eye care for glaucoma surgery and ocular drug delivery.

Cellular and cytoskeletal alterations of scleral fibroblasts in response to glucocorticoid steroids.

Steroid-induced ocular hypertension can be seen even after trabecular meshwork (TM) bypass/ablation. Thus, the purpose was to investigate steroid-response in cells distal to the TM by using primary scleral fibroblasts. Primary scleral cell cultures were generated using mid-depth scleral wedges from human donor corneo-scleral rims (n = 5) after corneal transplantation. Cells were treated with dexamethasone (DEX; 100 nM) and compared to media (MED)/vehicle (DMSO) controls. Cell size, shape, and migration were studied using the IncuCyte Live-Cell Analysis System. Cytoskeleton was compared using Alexa Fluor-568 Phalloidin and senescence tested by evaluating beta-galactosidase. Western blot comparison was performed for α-SMA, FKBP-51, fibronectin, phospho-myosin light chain, and myocilin. Scleral fibroblasts upregulated FKBP-51 in response to DEX indicating the existence of steroid-responsive pathways. Compared to controls, DEX-treated cells proliferated slower (~50%; p < 0.01-0.02), grew larger (~1.3-fold; p < 0.001), and migrated less (p = 0.01-0.006). Alexa Fluor 568 Phalloidin actin stress fiber labeling was more diffuse in DEX-treated cells (p = 0.001-0.004). DEX-treated cells showed more senescence compared to controls (~1.7-fold; p = 0.01-0.02). However, DEX-treated cells did not show increased cross-linked actin network formation or elevated myocilin/fibronectin/α-SMA/phospho-myosin light chain protein expression. For all parameters, MED- and DMSO-treated control cells were not significantly different. Primary scleral fibroblasts, grown from tissue collected immediately distal to the TM, demonstrated scleral-response behaviors that were similar to, but not identical with, classic TM steroid-response. Further study is needed to understand how these scleral cellular alterations may contribute to steroid-response IOP elevation after TM bypass/ablation surgery.

Photoreceptor rescue of pigment epithelium-derived factor-impregnated nanoparticles in Royal College of Surgeons rats.

PURPOSE: To investigate the protective effect of intravitreal injection of pigment epithelium-derived factor-impregnated nanoparticles (PEDF-NPs) against photoreceptor degeneration in Royal College of Surgeons (RCS) rats. METHODS: Three-week-old RCS rats received an intravitreal injection of PBS, blank NPs, PEDF (2.5 µg), or PEDF-NPs (2.5 µg). Eyes were assessed with morphological, immunohistochemical, and physiologic analysis over the following 8 weeks. Cell death was examined using the terminal deoxynucleotidyl transferase-mediated uridine 5'-triphosphate-biotin nick end labeling (TUNEL) assay. RESULTS: In RCS rats, the a- and b-wave amplitudes on electroretinograms in eyes treated with PEDF-NPs were greater than those in retinas receiving other treatment. Immunocytochemistry showed consistently greater opsin preservation in eyes treated with PEDF-NPs. A significantly higher number of photoreceptors and significantly fewer TUNEL-positive cells were present after treatment with PEDF-NPs, compared to PEDF-treated eyes. CONCLUSIONS: The results suggest that intravitreally injected PEDF-NPs delayed photoreceptor degeneration by inhibiting apoptosis in the RCS rat retina due to targeting and sustained release of PEDF.

Bleb-related Porcine Lymphatic Outflow is Greater from Subconjunctival compared to Subtenon Blebs.

Aim: Understanding the mechanism of fluid outflow by comparing the subconjunctival and subtenon spaces can lead to improved ocular therapeutics. The purpose of the current study is to evaluate subconjunctival vs subtenon lymphatic outflow by creating tracer-filled blebs in each location. Methods: Porcine (n = 20) eyes received subconjunctival or subtenon injection(s) of fixable and fluorescent dextrans. Blebs were angiographically imaged using a Heidelberg Spectralis ([Heidelberg Retina Angiograph] HRA + OCT; Heidelberg Engineering) and bleb-related lymphatic outflow pathways were counted. Optical coherence tomography (OCT) imaging of these pathways was used to assess structural lumens and the presence of valve-like structures. Furthermore, a comparison between tracer injection locations (superior/inferior/temporal/nasal) was made. Histologic analyses for subconjunctival and subtenon outflow pathways were performed, to confirm tracer co-localization with molecular lymphatic markers. Results: Subconjunctival blebs demonstrated a greater number of lymphatic outflow pathways compared to subtenon blebs in every quadrant [superior: 6.10 ± 1.18 (subconjunctival) vs 0.50 ± 0.27 (subtenon); temporal: 2.30 ± 0.40 vs 0.10 ± 0.10; nasal: 5.30 ± 0.60 vs 0.30 ± 0.21; inferior: 6.00 ±1.29 vs 0.1 ± 0.1; all comparisons p < 0.001]. For subconjunctival blebs, the temporal quadrant showed fewer lymphatic outflow pathways compared to the nasal side (p = 0.005). Discussion: Subconjunctival blebs accessed greater lymphatic outflow compared to subtenon blebs. Furthermore, regional differences existed, with fewer lymphatic vessels temporal than at the other locations. Clinical significance: Aqueous humor drainage after glaucoma surgery is incompletely understood. The present manuscript adds to our understanding of how lymphatics might influence filtration bleb function. How to cite this article: Lee JY, Strohmaier CA, Akiyama G, et al. Bleb-related Porcine Lymphatic Outflow Is Greater from Subconjunctival compared to Subtenon Blebs. J Curr Glaucoma Pract 2022;16(3):144-151.

Aqueous humour outflow imaging: seeing is believing.

Elevated intraocular pressure (IOP) is the primary risk factor for blindness in glaucoma. IOP is determined by many factors including aqueous humour production and aqueous humour outflow (AHO), where AHO disturbance represents the primary cause of increased IOP. With the recent development of new IOP lowering drugs and Minimally Invasive Glaucoma Surgeries (MIGS), renewed interest has arisen in shedding light on not only how but where AHO is occurring for the trabecular/conventional, uveoscleral/unconventional, and subconjunctival outflow pathways. Historical studies critical to understanding outflow anatomy will be presented, leading to the development of modern imaging methods. New biological behaviours uncovered by modern imaging methods will be discussed with relevance to glaucoma therapies emphasized.

Visual Assessment of Aqueous Humor Outflow.

In the past decade, many new pharmacological and surgical treatments have become available to lower intraocular pressure (IOP) for glaucoma. The majority of these options have targeted improving aqueous humor outflow (AHO). At the same time, in addition to new treatments, research advances in AHO assessment have led to the development of new tools to structurally assess AHO pathways and to visualize where aqueous is flowing in the eye. These new imaging modalities have uncovered novel AHO observations that challenge traditional AHO concepts. New behaviors including segmental, pulsatile, and dynamic AHO may have relevance to the disease and the level of therapeutic response for IOP-lowering treatments. By better understanding the regulation of segmental, pulsatile, and dynamic AHO, it may be possible to find new and innovative treatments for glaucoma aiming at these new AHO behaviors.

Micropulse Transscleral Diode Laser Cyclophotocoagulation in Refractory Glaucoma: Short-Term Efficacy, Safety, and Impact of Surgical History on Outcomes.

PURPOSE: To assess the short-term efficacy and safety of micropulse transscleral diode laser cyclophotocoagulation (MP-TSCPC) in the management of refractory glaucoma and to compare outcomes based on prior glaucoma surgeries. DESIGN: Retrospective analysis. PARTICIPANTS: Patients with refractory glaucoma who underwent MP-TSCPC at a single institution by 1 of 4 surgeons. METHODS: Chart review of cases of MP-TSCPC using the Iridex Cyclo G6 (Mountain View, CA) laser with standard parameters and laser duration at the discretion of each treating physician. MAIN OUTCOME MEASURES: Probability of postoperative success was estimated by the Kaplan-Meier method. Success parameters included intraocular pressure (IOP) 6 to 21 mmHg with or without topical antihypertensive therapy, 20% or more IOP reduction from baseline for any 2 consecutive visits after 3 postoperative months, and no subsequent glaucoma surgery. RESULTS: One hundred sixteen eyes of 116 patients (mean age, 65.8±16.9 years) were included. Baseline IOP was 22.2±7.9 mmHg, and mean postoperative follow-up time was 6.3±3.4 months (range, 3-12 months.) Postoperative IOP at the final follow up was 15.3±6.6 mmHg (P < 0.01), corresponding to a reduction of approximately 6.9 mmHg (31.1%). Most eyes (66.4%) underwent at least 6 months of follow-up. Short-term probability of success was 93.1% at 3 months and 74.3% at 6 months. Eyes that had undergone prior traditional glaucoma surgery (trabeculectomy, tube shunt, excessive pressure-regulating shunt system miniature glaucoma shunt [Alcon, Fort Worth, TX], or a combination thereof) demonstrated a higher probability of success (67.6%) compared with eyes that had not (41.4%; P = 0.014). The most common complications were decline in best-corrected visual acuity (7.8%) and hypotony (1.7%). CONCLUSIONS: Micropulse transscleral diode laser cyclophotocoagulation has a significant short-term ocular hypotensive effect and favorable safety profile in eyes with refractory glaucoma. The probability of successful outcome was greater in eyes that had undergone prior traditional glaucoma surgery.

Edaravone Prevents Retinal Degeneration in Adult Mice Following Optic Nerve Injury.

Purpose: To assess the therapeutic potential of edaravone, a free radical scavenger that is used for the treatment of acute brain infarction and amyotrophic lateral sclerosis, in a mouse model of optic nerve injury (ONI). Methods: Two microliters of edaravone (7.2 mM) or vehicle were injected intraocularly 3 minutes after ONI. Optical coherence tomography, retrograde labeling of retinal ganglion cells (RGCs), histopathology, and immunohistochemical analyses of phosphorylated apoptosis signal-regulating kinase-1 (ASK1) and p38 mitogen-activated protein kinase (MAPK) in the retina were performed after ONI. Reactive oxygen species (ROS) levels were assessed with a CellROX Green Reagent. Results: Edaravone ameliorated ONI-induced ROS production, RGC death, and inner retinal degeneration. Also, activation of the ASK1-p38 MAPK pathway that induces RGC death following ONI was suppressed with edaravone treatment. Conclusions: The results of this study suggest that intraocular administration of edaravone may be a useful treatment for posttraumatic complications.

Valproic acid and ASK1 deficiency ameliorate optic neuritis and neurodegeneration in an animal model of multiple sclerosis.

Optic neuritis, which is an acute inflammatory demyelinating syndrome of the central nervous system, is one of the major complications in multiple sclerosis (MS). Herein, we investigated the therapeutic potential of valproic acid (VPA) on optic neuritis in experimental autoimmune encephalomyelitis (EAE), a mouse model of MS. EAE was induced in C57BL/6 mice by immunization with MOG35-55 and VPA (300mg/kg) was administered via intraperitoneal injection once daily from day 3 postimmunization until the end of the experimental period (day 28). VPA treatment suppressed neuroinflammation and decreased the clinical score of EAE at an early phase (from day 12-14 after immunization). We also examined the effects of apoptosis signal-regulating kinase 1 (ASK1), an evolutionarily conserved signaling intermediate for innate immunity, in EAE mice. ASK1 deficiency strongly suppressed microglial activation and decreased the clinical score of EAE at a late phase (day 25, 27 and 28 after immunization). When VPA was administered to ASK1-deficient EAE mice, the clinical score was suppressed in both early and late phases (from day 12-28 after immunization) and showed synergistic effects on protection of retinal neurons. Our findings raise intriguing possibilities that the widely prescribed drug VPA and ASK1 inhibition may be useful for neuroinflammatory disorders including optic neuritis and MS.

Caloric restriction promotes cell survival in a mouse model of normal tension glaucoma.

Glaucoma is characterized by progressive degeneration of retinal ganglion cells (RGCs) and their axons. We previously reported that loss of glutamate transporters (EAAC1 or GLAST) in mice leads to RGC degeneration that is similar to normal tension glaucoma and these animal models are useful in examining potential therapeutic strategies. Caloric restriction has been reported to increase longevity and has potential benefits in injury and disease. Here we investigated the effects of every-other-day fasting (EODF), a form of caloric restriction, on glaucomatous pathology in EAAC1-/- mice. EODF suppressed RGC death and retinal degeneration without altering intraocular pressure. Moreover, visual impairment was ameliorated with EODF, indicating the functional significance of the neuroprotective effect of EODF. Several mechanisms associated with this neuroprotection were explored. We found that EODF upregulated blood ß-hydroxybutyrate levels and increased histone acetylation in the retina. Furthermore, it elevated retinal mRNA expression levels of neurotrophic factors and catalase, whereas it decreased oxidative stress levels in the retina. Our findings suggest that EODF, a safe, non-invasive, and low-cost treatment, may be available for glaucoma therapy.

Intraoperative electrophysiological evaluations of macular function during peripheral scleral indentation.

Scleral indentation is widely used to examine the peripheral fundus, however it can increase the intraocular pressure (IOP) to high levels which can then affect retinal function. We evaluated the effects of scleral indentation on the macular function electrophysiologically. Intraoperative focal macular electroretinograms (iFMERGs) were recorded with and without controlling the IOP in 7 eyes. Without IOP control, the IOP increased from 21.7 ± 4.9 to 92.7 ± 20.2 mmHg significantly (P = 0.020) and the amplitudes of the b-wave (from 6.29 ± 1.160 to 3.71 ± 1.98 uV, P = 0.007), on-photopic negative response (from 2.29 ± 0.99 to 0.72 ± 0.47 uV, on-PhNR, P = 0.005), and d-wave (from 2.57 ± 0.41 to 1.64 ± 0.69 uV, P = 0.007) decreased significantly soon after beginning the indentation. All values returned to the baseline levels after releasing the indentation. In the eyes with IOP controlled, the IOP and the amplitude of all components did not change significantly during and after the indentation except the on-PhNR amplitude which was significantly reduced during the indentation. The changes in the iFMERGs and macular function caused by scleral indentation were transient and reversible. The changes can be minimized by controlling the IOP.

Intraoperative Electroretinograms before and after Core Vitrectomy.

PURPOSE: To evaluate retinal function by intraoperative electroretinograms (ERGs) before and after core vitrectomy. DESIGN: Retrospective consecutive case series. METHOD: Full-field photopic ERGs were recorded prior to the beginning and just after core vitrectomy using a sterilized contact lens electrode in 20 eyes that underwent non-complicated vitreous surgery. A light-emitted diode was embedded into the contact lens, and a stimulus of 150 ms on and 350 ms off at 2 Hz was delivered. The amplitudes and latencies of the a-, b-, and d-waves, photopic negative response (PhNR), and oscillatory potentials (OPs) were analyzed. The intraocular temperature at the mid-vitreous was measured at the beginning and just after the surgery with a thermoprobe. RESULTS: The intraocular temperature was 33.2 ± 1.3°C before and 29.4 ± 1.7°C after the vitrectomy. The amplitudes of the PhNR and OPs were significantly smaller after surgery, and the latencies of all components were prolonged after the surgery. These changes were not significantly correlated with the changes of the temperature. CONCLUSION: Retinal function is reduced just after core vitrectomy in conjunction with significant temperature reduction. The differences in the degree of alterations of each ERG component suggests different sensitivity of each type of retinal neuron.

Association between the major histocompatibility complex and clinical response to infliximab therapy in patients with Behçet uveitis.

PURPOSE: Our aim was to investigate whether major histocompatibility complex (MHC) polymorphisms are associated with response to infliximab therapy in Japanese patients with Behçet uveitis (BU). METHODS: We retrospectively reviewed 24 patients (17 men and seven women) treated with infliximab for BU. Of them, ten patients were genotyped as HLA A*2601, and nine as HLA B*5101. Therapeutic response levels in the two groups were compared based on ocular attacks and the Behçet disease ocular attack score 24 (BOS24) over 24 months of treatment. RESULTS: Mean frequencies of ocular attacks at 13-18 and 19-24 months after the start of treatment were significantly higher in the HLA A*2601 group (P = 0.0392 and 0.0177, respectively). Mean BOS24-6 M values for months 1-6, 7-12, 13-18, and 19-24 were also significantly higher in the HLA A*2601 group (P = 0.0459, 0.0150, 0.0394, and 0.0178, respectively). Shortening of the infusion interval was required in eight patients in the HLA A*2601 group but in one only in the HLA B*5101 group. Behçet-disease-related adverse events occurred in eight patients in the HLA A*2601 group and two in the HLA B*5101 group. Nonocular adverse events occurred in four patients in the HLA A*2601 group and none in the HLA B*5101 group. CONCLUSIONS: Although mean change from baseline in the number of ocular attack scores in the HLA A26 and HLA B51 groups seemed to be similar, the HLA-A26 group had a more severe disease course under infliximab therapy for ocular/extraocular involvement. These data suggest that response to infliximab therapy in Japanese patients with BU is partly due to genetic determinants in the HLA complex.

Health- and vision-related quality of life in patients receiving infliximab therapy for Behcet uveitis.

OBJECTIVE: To evaluate the changes in health-related and vision-related quality of life (HR-QoL and VR-QoL) in patients with Behçet uveitis (BU) receiving infliximab therapy. METHODS: Twenty patients with recurrent BU attacks were enrolled. All patients were treated with infliximab. We evaluated the mean number of uveitis attacks and the mean score of extraocular manifestations by Behçet disease current activity form (BDCAF) during the 6 months before and the 6 and 12 months after initiation of infliximab. The EuroQol-5D questionnaire (EQ-5D) and the 25-item National Eye Institute Visual Function Questionnaire (NEI VFQ-25) were self-administered in all patients before treatment and, 6 and 12 months after treatment. Patients' pre- and post-treatment EQ-5D and NEI VFQ-25 scores were compared. RESULTS: By the 6- and 12-month follow-up, the frequency of uveitis attacks and the BDCAF scores was significantly decreased compared with the 6 months before starting infliximab (p<0.0001). Fully completed questionnaires were received from all patients. Infliximab therapy was associated with a significant improvement of the EQ-5D (p<0.0001), NEI VFQ-25 composite score (p=0.0001), general health score (p=0.0001) and mental health score (p=0.0001). This result shows a significant decrease in inflammatory activity of the disease and consequently improvement in HR-QoL and VR-QoL scores with a rising response pattern in all dimensions. CONCLUSIONS: Relief of uveitis attacks and extraocular manifestations by infliximab therapy significantly improved the HR-QoL and VR-QoL in patients with BU.

A Case of Retinitis Pigmentosa Requiring Vitrectomy because of Repeated Vitreous Hemorrhage.

A male patient with retinitis pigmentosa (RP) accompanied by repeated vitreous hemorrhage in both eyes underwent unilateral vitrectomy. Visual acuity recovered to 0.8 in the operated right eye, and no hemorrhage, complications or progression of RP were observed. Fluorescein angiography performed 2 months after surgery detected neovascularization at the optic disc in the operated right eye, but not in the non-operated left eye, and no avascular areas were found in either eye. Vitrectomy may be effective for the treatment of RP accompanied by vitreous hemorrhage.

Therapeutic effect of stealth-type polymeric nanoparticles with encapsulated betamethasone phosphate on experimental autoimmune uveoretinitis.

PURPOSE: The therapeutic effects of betamethasone phosphate (BP) encapsulated in biocompatible and biodegradable blended nanoparticles of poly(lactic acid) (PLA) homopolymers and PEG-block-PLA copolymers (stealth nanosteroids) were examined in an experimental autoimmune uveoretinitis (EAU) model in Lewis rats. METHODS: EAU was induced by S-antigen peptide in Lewis rats. Accumulation of systemically administered Cy7-labeled stealth nanoparticles in inflamed eyes of rats with EAU was assessed using in vivo fluorescence imaging, and the therapeutic effect of stealth nanosteroids, nonstealth nanosteroids, or saline on EAU was examined. The eyes were obtained 7 days after the treatment, and the histologic score was determined using pathologic findings. The expression of inflammatory cytokines including IL-6, IL-17, and VEGF was determined immunohistochemically. RESULTS: Cy7-stealth nanoparticles accumulated in inflamed eyes of rats with EAU and remained in situ for a 3-day period. Systemically administered stealth nanosteroids (100 µg of BP) reduced the clinical scores of rats with EAU within 1 day and maintained the effect for 2 weeks. This treatment also decreased the histologic scores and the expression of inflammatory cytokines in the retina of EAU. CONCLUSIONS: The strong therapeutic benefit on EAU obtained with the stealth nanosteroids may have been due to prolonged blood circulation and targeting to the inflamed uvea and retina, in addition to sustained release in situ.