Increased Post-Hypoxic Oxidative Stress and Activation of the PERK Branch of the UPR in Trap1-Deficient Drosophila melanogaster Is Abrogated by Metformin.

Hypoxia is known to impair mitochondrial and endoplasmic reticulum (ER) homeostasis. Post-hypoxic perturbations of the ER proteostasis result in the accumulation of misfolded/unfolded proteins leading to the activation of the Unfolded Protein Response (UPR). Mitochondrial chaperone TNF receptor-associated protein 1 (TRAP1) is reported to preserve mitochondrial membrane potential and to impede reactive oxygen species (ROS) production thereby protecting cells from ER stress as well as oxidative stress. The first-line antidiabetic drug Metformin has been attributed a neuroprotective role after hypoxia. Interestingly, Metformin has been reported to rescue mitochondrial deficits in fibroblasts derived from a patient carrying a homozygous TRAP1 loss-of-function mutation. We sought to investigate a putative link between Metformin, TRAP1, and the UPR after hypoxia. We assessed post-hypoxic/reperfusion longevity, mortality, negative geotaxis, ROS production, metabolic activity, gene expression of antioxidant proteins, and activation of the UPR in Trap1-deficient flies. Following hypoxia, Trap1 deficiency caused higher mortality and greater impairments in negative geotaxis compared to controls. Similarly, post-hypoxic production of ROS and UPR activation was significantly higher in Trap1-deficient compared to control flies. Metformin counteracted the deleterious effects of hypoxia in Trap1-deficient flies but had no protective effect in wild-type flies. We provide evidence that TRAP1 is crucially involved in the post-hypoxic regulation of mitochondrial/ER stress and the activation of the UPR. Metformin appears to rescue Trap1-deficiency after hypoxia mitigating ROS production and downregulating the pro-apoptotic PERK (protein kinase R-like ER kinase) arm of the UPR.

Microglial-specific depletion of TAK1 is neuroprotective in the acute phase after ischemic stroke.

Transforming growth factor-ß-activated kinase 1 (TAK1) is upregulated after cerebral ischemia and contributes to an aggravation of brain injury. TAK1 acts as a key regulator of NF-ΚB and the MAP kinases JNK and p38 and modulates post-ischemic neuroinflammation and apoptosis. Microglia are the main TAK1-expressing immunocompetent cells of the brain. However, little is known about the function and regulation of microglial TAK1 after cerebral ischemia. Tamoxifen-dependent conditional depletion of TAK1 in microglial cells was induced in Cx3cr1creER-Tak1fl/fl mice. The creER-negative Tak1fl/fl mice and vehicle-treated (corn oil) mice served as control groups. A transient intraluminal middle cerebral artery occlusion of 30 min followed by 6 h and 72 h of reperfusion was performed in male mice. Oxygen-glucose-deprivation (OGD) was performed with primary cortical glial cell cultures to examine the effect of microglial-specific and general (5Z-7-Oxozeaenol) TAK1 inhibition after different reperfusion times (1 h, 6 h, and 72 h). Cx3cr1creER-Tak1fl/fl mice showed reduced infarct sizes and improved neurological outcomes compared to the control group. The mRNA and protein levels of pro-inflammatory Il1b/IL-1ß and Tnf/TNF-α in the peri-infarct zones of microglial-specific TAK1-depleted mice were significantly reduced. Furthermore, TAK1 depletion in vitro led to reduced cell death rates after OGD. Moreover, hypoxia-mediated activation of TAK1 and its downstream signalling proteins, JNK and p38, were dampened by microglial TAK1 depletion. In contrast, 5Z-7-Oxozeaenol-induced pharmacological inhibition of TAK1 completely diminished MAPK-signalling including the kinases JNK and p38 in all cells. Microglial TAK1 depletion abrogates post-ischemic neuroinflammation and apoptosis in the acute phase, hence might be considered as a potential target in the treatment of cerebral hypoxia. KEY MESSAGES: TAK1 is activated after cerebral ischemia and induces MAP kinases p38 and JNK. Activated TAK1 increases apoptosis rate and the level pro-inflammatory cytokines IL-1ß and TNF-α. Microglial cells seem to be the main source of TAK1-mediated post-ischemic neuroinflammation. Microglial-specific TAK1-depletion mediates sustainable neuroprotective effects, which might be superior to global TAK1 inhibition.

EPO regulates neuroprotective Transmembrane BAX Inhibitor-1 Motif-containing (TMBIM) family members GRINA and FAIM2 after cerebral ischemia-reperfusion injury.

BACKGROUND AND PURPOSE: Transmembrane BAX Inhibitor-1 Motif-containing (TMBIM) family members exert inhibitory activities in apoptosis and necroptosis. FAIM2 (TMBIM-2) is neuroprotective against murine focal ischemia and is regulated by erythropoietin (EPO). Similar to FAIM2, GRINA (TMBIM-3) is predominantly expressed in the brain. The role of GRINA in transient brain ischemia, its potential synergistic effects with FAIM2 and its regulation by EPO treatment were assessed. METHODS: We performed transient (30 min) middle cerebral artery occlusion (tMCAo) followed by 72 h of reperfusion in GRINA-deficient (GRINA-/-), FAIM2-deficient (FAIM2-/-), double-deficient (GRINA-/-FAIM2-/-) and wildtype littermates (WT) mice. We administered EPO or saline 0, 24 and 48 h after tMCAo. We subjected primary murine cortical neurons (pMCN) of all mouse strains to oxygen-glucose deprivation (OGD) after GRINA and/or FAIM2 gene transfection. RESULTS: Compared to wildtype controls GRINA deficiency led to a similar increase in infarct volumes as FAIM2 deficiency (p < .01). We observed the highest neurological deficits and largest infarct sizes in double-deficient mice. EPO administration upregulated GRINA and FAIM2 mRNA levels in wildtype littermates. EPO decreased infarct sizes and abrogated neurological impairments in wildtype controls. GRINA and/or FAIM2 deficient mice showed increased expression levels of cleaved-caspase 3 and of pro-apoptotic BAX mRNA. Further, caspase 8 was upregulated in FAIM2-/- and caspase 9 in GRINA-/- mice. Overexpression of GRINA and FAIM2 in wildtype and in double deficient pMCN decreased cell death rate after OGD. CONCLUSIONS: GRINA and FAIM2 are highly expressed in the brain and convey EPO-mediated neuroprotection after ischemic stroke involving different caspases.

Greater vertical spot spacing to improve femtosecond laser capsulotomy quality.

PURPOSE: To evaluate the effect of adapted capsulotomy laser settings on the cutting quality in femtosecond laser-assisted cataract surgery. SETTING: Ruhr-University Eye Clinic, Bochum, Germany. DESIGN: Prospective randomized case series. METHODS: Eyes were treated with 1 of 2 laser settings. In Group 1, the regular standard settings were used (incisional depth 600 µm, pulse energy 4 µJ, horizontal spot spacing 5 µm, vertical spot spacing 10 µm, treatment time 1.2 seconds). In Group 2, vertical spot spacing was increased to 15 µm and the treatment time was 1.0 seconds. Light microscopy was used to evaluate the cut quality of the capsule edge. The size and number of tags (misplaced laser spots, which form a second cut of the capsule with high tear risk) were evaluated in a blinded manner. Groups were compared using the Mann-Whitney U test. RESULTS: The study comprised 100 eyes (50 eyes in each group). Cataract surgery was successfully completed in all eyes, and no anterior capsule tear occurred during the treatment. Histologically, significant fewer tags were observed with the new capsulotomy laser setting. The mean score for the number and size of free tags was significantly lower in this group than with the standard settings (P < .001). CONCLUSIONS: The new laser settings improved cut quality and reduced the number of tags. The modification has the potential to reduce the risk for radial capsule tears in femtosecond laser-assisted cataract surgery. With the new settings, no tags and no capsule tears were observed under the operating microscope in any eye.

Cataract formation after YAG laser vitreolysis: importance of femtosecond laser anterior capsulotomies in perforated posterior capsules.

PURPOSE: To report a patient who developed a white cataract after Nd:YAG laser vitreolysis with a posterior capsule defect. METHODS: Femtosecond laser-assisted capsulotomy was performed for optic capture fixation in a patient with a cataract due to a posterior capsule defect after Nd:YAG laser-vitreolysis. RESULTS: A 55-year-old, highly myopic woman presented with visual impairment 4 days after Nd:YAG laser vitreolysis due to preexisting floaters. The slit-lamp examination showed a mature white cataract; therefore, the intactness of the posterior capsule could not be judged. The patient underwent cataract surgery with femtosecond laser-assisted capsulotomy. Intraoperatively, a highly disrupted posterior capsule was observed. Intraocular lens (IOL) was implanted into the ciliary sulcus and the anterior circular and centered capsulotomy was used for posterior optic capture fixation. Then vitrectomy was performed. No intraoperative or postoperative complications occurred. CONCLUSIONS: YAG laser vitreolysis presents a new and promising therapeutic approach for floaters. However, the complications are unknown. We describe the induction of cataract as a major complication. Furthermore, the femtosecond laser can ensure a circular and complete anterior capsulotomy, which is essential for optic capture fixation in these cases.

Femtosecond Laser-Assisted Cataract Surgery After Radial Keratotomy.

PURPOSE: To determine the efficacy of femtosecond laser-assisted cataract surgery in eyes with radial keratometry. METHODS: Femtosecond laser-assisted cataract surgery was performed in 3 patients (6 eyes) who had six to eight radial keratotomy incisions. RESULTS: In all cases, the anterior segment of the eye was visualized with integrated three-dimensional optical coherence tomography and it was possible to position the laser corneal incisions between the radial keratotomy incisions. No intraoperative complications occurred. In particular, no corneal perforation, anterior capsular tears, or discontinuities could be detected. No corneal leakage or other postoperative complications were noted on follow-up after 6 weeks and 6 months. CONCLUSIONS: Femtosecond laser-assisted cataract surgery presents a new, feasible, and safe surgical technique for patients with cataract who had previous radial keratotomy. [J Refract Surg. 2016;32(6):426-428.].

Simultaneous Complement Response via Lectin Pathway in Retina and Optic Nerve in an Experimental Autoimmune Glaucoma Model.

Glaucoma is a multifactorial disease and especially mechanisms occurring independently from an elevated intraocular pressure (IOP) are still unknown. Likely, the immune system contributes to the glaucoma pathogenesis. Previously, IgG antibody depositions and retinal ganglion cell (RGC) loss were found in an IOP-independent autoimmune glaucoma model. Therefore, we investigated the possible participation of the complement system in this model. Here, rats were immunized with bovine optic nerve homogenate antigen (ONA), while controls (Co) received sodium chloride (n = 5-6/group). After 14 days, RGC density was quantified on flatmounts. No changes in the number of RGCs could be observed at this point in time. Longitudinal optic nerve sections were stained against the myelin basic protein (MBP). We could note few signs of degeneration processes. In order to detect distinct complement components, retinas and optic nerves were labeled with complement markers at 3, 7, 14, and 28 days and analyzed. Significantly more C3 and MAC depositions were found in retinas and optic nerves of the ONA group. These were already present at day 7, before RGC loss and demyelination occurred. Additionally, an upregulation of C3 protein was noted via Western Blot at this time. After 14 days, quantitative real-time PCR revealed significantly more C3 mRNA in the ONA retinas. An upregulation of the lectin pathway-associated mannose-serine-protease-2 (MASP2) was observed in the retinas as well as in the optic nerves of the ONA group after 7 days. Significantly more MASP2 in retinas could also be observed via Western Blot analyses at this point in time. No effect was noted in regard to C1q. Therefore, we assume that the immunization led to an activation of the complement system via the lectin pathway in retinas and optic nerves at an early stage in this glaucoma model. This activation seems to be an early response, which then triggers degeneration. These findings can help to develop novel therapy strategies for glaucoma patients.

Systemic ocular antigen immunization leads only to a minor secondary immune response.

The immunization with optic nerve homogenate antigen (ONA) or S100 induced retinal degeneration. Since many neurological diseases are reinforced or initiated by immune cells, leucocytes were analyzed. CD3(+) T-cells in the retina increased slightly in ONA rats, but not in S100 treated retinas. No CD45R(+) B-cells and granulocytes could be detected in the retinas. At early stages, CD3(+) cells, Iba1(+) macrophages and granulocytes of the secondary lymphoid organs were not affected. Yet, the sole injection of pertussis toxin led to a shift to fewer CD45R(+) cells and more granulocytes in spleens. Later, splenic Iba1(+) macrophages were increased in both groups. We conclude that the retinal infiltration of lymphocytes is not crucial for the degeneration process and rather an epiphenomenon.

Retinal and Optic Nerve Damage is Associated with Early Glial Responses in an Experimental Autoimmune Glaucoma Model.

It is well established that the immunization with ocular antigens causes a retinal ganglion cell (RGC) decline, which is accompanied by glia alterations. In this study, the degenerative effects of the immunization with an optic nerve homogenate (ONA) and its purified compound S100 were analyzed on retinas and optic nerves. Since a participation of glia cells in cell death mechanisms is currently discussed, rats were immunized with S100 or ONA. At 14 and 28 days, immune-histological and Western blot analyses were performed to investigate the optic nerve structure (SMI-32), retinal ganglion cells (Brn-3a), apoptosis (cleaved caspase 3, FasL), and glial profile (Iba1, ED1, GFAP, vimentin). Neurofilament dissolution in S100 animals was evident at 14 days (p = 0.047) and increased at 28 days (p = 0.01). ONA optic nerves remained intact at early stages and degenerated later on (p = 0.002). In both groups, RGC loss was detected via immune-histology and Western blot at 28 days (ONA: p = 0.02; S100: p = 0.005). Additionally, more Iba1(+) retinal microglia could be detected at early stages (ONA: p = 0.006; S100: p = 0.028). A slight astrocyte response was detected on Western blots only on ONA retinas (p = 0.01). Hence, the RGC and optic nerve decline was partly antigen dependent, while neuronal loss is paralleled by an early microglial response.

Evaluation of uveal and capsule biocompatibility of a single-piece hydrophobic acrylic intraocular lens with ultraviolet-ozone treatment on the posterior surface.

PURPOSE: To evaluate a single-piece hydrophobic acrylic intraocular lens (IOL) with ultraviolet-ozone (UV-O3) treatment on the posterior surface and compare it with an identical untreated IOL in a rabbit model. SETTING: John A. Moran Eye Center, University of Utah, Salt Lake City, Utah, USA. DESIGN: Experimental study. METHODS: Study IOLs were implanted in the right eyes and control IOLs in the left eyes of 10 New Zealand rabbits. Slitlamp examinations were performed 1 to 6 weeks postoperatively. Neodymium:YAG (Nd:YAG) posterior capsulotomy was performed in both eyes of 5 rabbits after the 4-week slitlamp examination. At 6 weeks, the rabbits were killed humanely and their globes were enucleated. Capsular bag opacification was scored from the posterior aspect (Miyake-Apple view), and the eyes were processed for histopathology. RESULTS: At 4 weeks, the mean posterior capsule opacification (PCO) scores were 0.88 ± 0.33 (SD) in the study eyes and 2.55 ± 1.13 in the control eyes (P=.003, 2-tailed paired t test). Performance of Nd:YAG posterior capsulotomy was similar in both groups. Gross postmortem examination also showed statistically less peripheral PCO in eyes with the study IOLs than in control eyes. There was no difference in histopathologic findings between study eyes and control eyes and no signs of untoward inflammation or toxicity in any eye evaluated. CONCLUSIONS: Treatment of the posterior surface of a single-piece hydrophobic acrylic IOL with UV-O3 appears to prevent PCO, likely by increasing adhesion between the posterior capsule and the IOL while retaining uveal biocompatibility. Performance of Nd:YAG posterior capsulotomy was similar between treated IOLs and untreated IOLs.

Long-term uveal and capsular biocompatibility of a new accommodating intraocular lens.

PURPOSE: To evaluate long-term uveal and capsular biocompatibility of a new accommodating intraocular lens (IOL). SETTING: John A. Moran Eye Center, University of Utah, Salt Lake City, Utah, USA. DESIGN: Experimental study. METHODS: Bilateral phacoemulsification was performed in 14 rabbits; 1 eye received the accommodating IOL (Fluidvision) and the other received a hydrophobic acrylic control IOL. Slitlamp examinations were performed at postoperative weeks 1 to 4 and months 2, 3, 4, and 6. Six rabbits were humanely killed at 2 months and 8 rabbits at 6 months. After gross examination with the Miyake-Apple view, selected IOLs were removed for implant cytology. All globes were then sectioned and processed for histopathologic examination. RESULTS: Uveal biocompatibility of study and control IOLs was similar in clinical and pathologic examinations up to 6 months postoperatively. In the study group, anterior capsule opacification appeared absent and posterior capsule opacification (PCO) was significantly less than in the control group. At the gross examination at 6 months, central PCO was 0.8 ± 0.5 (SD) in the study IOLs and 3.7 ± 0.4 in the control IOLs (P < .0001, 2-tailed paired t test). Histopathologic examination confirmed the relative lack of capsule opacification in study eyes compared with controls and the absence of untoward inflammatory reaction or toxicity in all eyes. CONCLUSIONS: The accommodating IOL maintained an expanded capsular bag secondary to the large size of the haptic elements without significant contact with the anterior capsule. This appeared to prevent overall capsular bag opacification and to retain uveal and capsular biocompatibility.