Silencing of ocular transthyretin, a gene responsible for hereditary transthyretin amyloidosis, by intravitreal injection of an siRNA conjugate into rabbit eyes.

The first small interfering RNA (siRNA) therapeutic received approval for hereditary transthyretin (ATTRv) amyloidosis, and the patients' lifespan extension by specific inhibition of hepatic synthesis of transthyretin (TTR) is expected. However, ocular amyloidosis in these patients has been a crucial issue. This study aims to evaluate the efficacy and safety of intravitreal TTR siRNA conjugate injection into rabbit eyes. Rabbit (r) TTR siRNA is a screened TTR siRNA conjugate from 53 candidates. The intraocular pressure (IOP) immediately after injection was high despite the 65.9 % decrease of aqueous humor TTR protein levels in the rTTR siRNA group compared with those in the Control siRNA group 2 weeks after the 50 µL siRNA injection. The IOP spike was milder after the 30 µL siRNA injection, and aqueous humor TTR levels decreased by ∼50 % in the rTTR siRNA group, which is consistent with the mRNA levels in the retina. The parameters of dark-adapted, light-adapted, and light-adapted 30 Hz electroretinogram and the thickness of each retinal layer in histological analysis demonstrated no significant differences between the groups. In conclusion, we developed TTR siRNA conjugates for rabbit eyes, and the results indicate that intravitreal TTR siRNA conjugate injection could be a therapeutic option for ocular amyloidosis caused by ATTRv amyloidosis.

Mitochondria transfer from early stages of erythroblasts to their macrophage niche via tunnelling nanotubes.

Adult erythropoiesis entails a series of well-coordinated events that produce mature red blood cells. One of such events is the mitochondria clearance that occurs cell-autonomously via autophagy-dependent mechanisms. Interestingly, recent studies have shown mitochondria transfer activities between various cell types. In the context of erythropoiesis, macrophages are known to interact closely with the early stages of erythroblasts to provide a specialized niche, termed erythroblastic islands (EBI). However, whether mitochondria transfer can occur in the EBI niche has not been explored. Here, we report that mitochondria transfer in the EBI niche occurs in vivo. We observed mitochondria transfer activities from the early stages of erythroblasts to macrophages in the reconstituted in vitro murine EBI via different modes, including tunnelling nanotubes (TNT). Moreover, we demonstrated that Wiskott-Aldrich syndrome protein (WASp) in macrophages mediates TNT formation and mitochondria transfer via the modulation of F-actin filamentation, thus promoting mitochondria clearance from erythroid cells, to potentially enhance their differentiation. Taken together, our findings provide novel insight into the mitochondria clearance machineries that mediate erythroid maturation.

In vivo imaging of axonal transport of mitochondria in the diseased and aged mammalian CNS.

The lack of intravital imaging of axonal transport of mitochondria in the mammalian CNS precludes characterization of the dynamics of axonal transport of mitochondria in the diseased and aged mammalian CNS. Glaucoma, the most common neurodegenerative eye disease, is characterized by axon degeneration and the death of retinal ganglion cells (RGCs) and by an age-related increase in incidence. RGC death is hypothesized to result from disturbances in axonal transport and in mitochondrial function. Here we report minimally invasive intravital multiphoton imaging of anesthetized mouse RGCs through the sclera that provides sequential time-lapse images of mitochondria transported in a single axon with submicrometer resolution. Unlike findings from explants, we show that the axonal transport of mitochondria is highly dynamic in the mammalian CNS in vivo under physiological conditions. Furthermore, in the early stage of glaucoma modeled in adult (4-mo-old) mice, the number of transported mitochondria decreases before RGC death, although transport does not shorten. However, with increasing age up to 23-25 mo, mitochondrial transport (duration, distance, and duty cycle) shortens. In axons, mitochondria-free regions increase and lengths of transported mitochondria decrease with aging, although totally organized transport patterns are preserved in old (23- to 25-mo-old) mice. Moreover, axonal transport of mitochondria is more vulnerable to glaucomatous insults in old mice than in adult mice. These mitochondrial changes with aging may underlie the age-related increase in glaucoma incidence. Our method is useful for characterizing the dynamics of axonal transport of mitochondria and may be applied to other submicrometer structures in the diseased and aged mammalian CNS in vivo.

Circumpapillary retinal nerve fiber layer thickness, anterior lamina cribrosa depth, and lamina cribrosa thickness in neovascular glaucoma secondary to proliferative diabetic retinopathy: a cross-sectional study.

BACKGROUND: To compare the lamina cribrosa between eyes with and without neovascular glaucoma (NVG) using enhanced depth imaging spectral-domain optical coherence tomography. METHODS: Forty-six patients with proliferative diabetic retinopathy were enrolled in this cross-sectional study. The patients were divided into two groups based on the absence or presence of NVG (the non-NVG group and the NVG group, respectively). The intraocular pressure (IOP), circumpapillary retinal nerve fiber layer (cpRNFL) thickness, anterior lamina cribrosa depth (ALD), and laminar thickness (LT) were compared between the groups. RESULTS: In the non-NVG group, the mean age was 66.2 ± 2.4 (mean ± standard error) years, mean maximum IOP was 18.8 ± 1.8 mmHg, mean cpRNFL thickness was 91.2 ± 3.9 µm, mean ALD was 407.0 ± 22.9 µm, and mean LT was 155.0 ± 4.7 µm. In the NVG group, the mean age was 61.4 ± 2.1 years, mean maximum IOP was 33.1 ± 1.6 mmHg, mean cpRNFL thickness was 73.6 ± 3.4 µm, mean ALD was 403.9 ± 20.1 µm, and mean LT was 156.9 ± 4.2 µm. The IOP was significantly higher and the cpRNFL was significantly thinner in the NVG group (P < 0.001 and P = 0.002, respectively). However, the age, ALD, and LT were not statistically different between the groups (P = 0.151, 0.919, and 0.757, respectively). CONCLUSIONS: Although the cpRNFL was thinner, the structure of the lamina cribrosa was unchanged in the NVG eyes. Axonal loss of the retinal ganglion cells in NVG patients was prior to lamina cribrosa deformation.

The relationship between self-reported habitual exercise and visual field defect progression: a retrospective cohort study.

BACKGROUND: Exercise reduces intraocular pressure (IOP) in the short term. However, it is not known whether exercise contributes to slower glaucomatous visual field defect progression. METHODS: Twenty-four primary open-angle glaucoma or exfoliation glaucoma patients who were evaluated by the Humphrey Field Analyzer (HFA) 24-2 program ≥ four times in 3 years were enrolled. Patients with a history of intraocular surgery in past 3 years or other eye diseases threatening visual fields were excluded. Patients were classified into two groups whether they had exercise habits or not. RESULTS: Eleven patients had exercise habits. The mean ± standard error of IOP and MD slope were 14.8 ± 0.9 mmHg and +0.20 ± 0.20 dB/year in the exercise group and 13.3 ± 0.8 mmHg and -0.53 ± 0.18 dB/year in the non-exercise group (P = 0.24 and P = 0.01, respectively). Higher IOP [odds ratio (OR) = 0.44/1 mmHg increase; P = 0.02] and habitual exercise (OR = 0.04; P = 0.02) reduced the visual field defect progression risk in logistic regression analyses. CONCLUSIONS: Patients with self-reported exercise habits had slower glaucoma progression.

Determinants of anterior chamber angle narrowing after mydriasis in the patients with cataract.

PURPOSE: To identify the determinants of anterior chamber angle narrowing after mydriasis by quantitative assessment of the anterior chamber components using anterior segment optical coherence tomography (AS-OCT). METHODS: In this intervention study, anterior chamber angle analysis was performed using AS-OCT in patients with cataract before pharmacological pupillary dilation and 30 min afterwards. Angle narrowing was quantified by the change of angle opening distance 500 after mydriasis (ΔAOD500). Multivariate linear regression and stepwise selection regression were used to identify which parameters in AS-OCT significantly determined ΔAOD500. RESULTS: One hundred and two Japanese patients (age, mean ± standard deviation, 71.9 ± 11.2 years) were enrolled. Multivariate analysis indicated that smaller ΔAOD500 were significantly associated with larger lens vault (LV; P < 0.001), larger iris area (IA; P = 0.003), and posterior corneal arch distance (PCAL; P = 0.01). Stepwise linear regression analysis revealed that smaller ΔAOD500 was independently associated with larger LV (partial R (2) = 0.232, P < 0.001), larger IA (partial R (2) = 0.080, P = 0.001), and smaller PCAL (partial R (2) = 0.066, P = 0.002) before mydriasis. These three factors explained 37.7 % of the decrease in ΔAOD500. CONCLUSION: Larger LV, larger IA, and smaller PCAL contribute considerably to anterior chamber angle narrowing after mydriasis in patients with cataract.

Trabeculotomy Using the Kahook Dual Blade for Exfoliation Glaucoma and Primary Open Angle Glaucoma: Comparison of Outcomes According to Incision Range.

PRCIS: Large amount of trabeculotomy with Kahook Dual Blade (KDB) confers better intraocular pressure (IOP) lowering. PURPOSE: We compared the postoperative outcomes of trabeculotomy using the KDB in patients with exfoliation glaucoma (EXG) and primary open angle glaucoma between the 120-degree incision and the 210- to 240-degree incision groups. PATIENTS AND METHODS: We retrospectively analyzed the postoperative outcomes of trabeculotomy performed using the KDB in 101 eyes with EXG and primary open angle glaucoma, who received 120- and 210- to 240-degree incisions at Kumamoto University Hospital between April 25, 2018, and August 11, 2021. Kaplan-Meyer survival curves were used to assess the outcomes. Surgical failure was defined as IOP ≥21 mm Hg (criterion A) and ≥19 mm Hg (criterion B), or an IOP ≤4 mm Hg in either criterion and the need for additional glaucoma surgery. RESULTS: In total, 64 and 37 eyes were included in the 120 and 210- to 240-degree groups, respectively. The 210- to 240-degree group had a higher 1-year success rate compared with the 120-degree group, both when considering all eyes and when considering only those with EXG (P<0.05). CONCLUSIONS: In trabeculotomy with KDB, a 210- to 240-degree incision was more effective than a 120-degree incision in lowering IOP in EXG cases.

Aldose reductase inhibitor counteracts the enhanced expression of matrix metalloproteinase-10 and improves corneal wound healing in galactose-fed rats.

PURPOSE: We investigated the effect of an aldose reductase inhibitor (ARI) and the role of matrix metalloproteinase (MMP)-10 on recovery after corneal epithelium removal in a rat diabetic keratopathy model. METHODS: Three-week-old Sprague-Dawley rats were fed the following diets for 6 weeks: normal MF chow (MF), 50% galactose (Gal), and 50% Gal containing 0.01% ARI (Gal +ARI). The corneal epithelium was removed using n-heptanol, and the area of epithelial defects was photographed and measured every 24 h. Real-time reverse transcriptase PCR, western blotting, and immunohistochemistry were used to determine the expression profile of MMP-10 and integrin α3. RESULTS: Compared to the MF control group, the amount of galactitol in the Gal group increased approximately 200-fold, which was reduced to sevenfold by ARI treatment. The area of corneal erosion in the Gal group was significantly larger than in the MF group at 72 h and thereafter (p<0.01, unpaired t test). The expression level of MMP-10 was enhanced at both the protein and mRNA levels by exposure to a high concentration of Gal, while integrin α3 expression decreased at the protein level but remained unchanged at the mRNA level. Delayed epithelial wound healing and alterations in the expression levels of MMP-10 and integrin α3 were normalized by ARI. The corneal erosion closure rate was significantly decreased with topical recombinant MMP-10. CONCLUSIONS: These studies confirm that the increased expression of MMP-10 induced by Gal feeding is counteracted by ARI treatment and suggest a role of MMP-10 in modulating corneal epithelial wound healing.

[A prospective study of postoperative aqueous flare in trabeculectomy alone versus phacotrabeculectomy].

PURPOSE: To compare trabeculectomy alone with phacotrabeculectomy in postoperative aqueous flare. SUBJECTS AND METHODS: Aqueous flare was prospectively measured using a laser flare-cell meter in open-angle glaucoma patients with cataract undergoing trabeculectomy with either mitomycin C or phacotrabeculectomy with mitomycin C. Flare was measured before surgery and at 2 and 4 weeks after surgery. RESULTS: Twenty-four patients who underwent trabeculectomy and 26 patients who underwent phacotrabeculectomy completed the study. There were no significant differences in flare values between the two groups at any point. Logistic regression analysis demonstrated that exfoliation glaucoma (odds ratio = 8.978; p = 0.0028) and postoperatively shallow anterior chamber (odds ratio = 15.539 ; p = 0.0074) were risk factors for high values of aqueous flare at 2 weeks after surgery. CONCLUSIONS: Combined phacoemulsification does not enhance postoperative aqueous flare in trabeculectomy. Exfoliation glaucoma and postoperative shallow anterior chamber are factors in the elevation of aqueous flare.

Bone marrow imaging reveals the migration dynamics of neonatal hematopoietic stem cells.

Hematopoietic stem cells (HSCs) are produced from the blood vessel walls and circulate in the blood during the perinatal period. However, the migration dynamics of how HSCs enter the bone marrow remain elusive. To observe the dynamics of HSCs over time, the present study develops an intravital imaging method to visualize bone marrow in neonatal long bones formed by endochondral ossification which is essential for HSC niche formation. Endogenous HSCs are labeled with tdTomato under the control of an HSC marker gene Hlf, and a customized imaging system with a bone penetrating laser is developed for intravital imaging of tdTomato-labeled neonatal HSCs in undrilled tibia, which is essential to avoid bleeding from fragile neonatal tibia by bone drilling. The migration speed of neonatal HSCs is higher than that of adult HSCs. Neonatal HSCs migrate from outside to inside the tibia via the blood vessels that penetrate the bone, which is a transient structure during the neonatal period, and settle on the blood vessel wall in the bone marrow. The results obtained from direct observations in vivo reveal the motile dynamics and colonization process of neonatal HSCs during bone marrow formation.

Factors associated with the surgical outcomes of Baerveldt glaucoma implant for open-angle glaucoma, an age-related eye disease.

To identify the factors associated with the surgical outcomes of Baerveldt glaucoma implant (BGI) for open-angle glaucoma (OAG), the medical records of 51 consecutive OAG patients (age, 43-91 years) who underwent BGI were retrospectively reviewed (median follow-up, 21.7 months). Surgical success was defined as the following postoperative intraocular pressures (IOPs, mmHg): (A) 6 ≤ IOP ≤ 21; (B) 6 ≤ IOP ≤ 18; and (C) 6 ≤ IOP ≤ 15 without loss of light perception or additional glaucoma surgery. Univariate analysis showed that age (all criteria), glaucoma type (criterion C), and preoperative IOP (criteria A and B) were the candidate factors (P < 0.20). When the patients were divided into two groups according to median age (72 years), the success probability was higher in the older group for criteria B (P = 0.047) and C (P = 0.02), and the postoperative IOP was lower in the older group 1-year post-surgery (P = 0.002). Furthermore, the multivariate Cox proportional hazards model revealed that older age was independently associated with surgical success for criteria B (relative risk [RR], 0.94; P = 0.02) and C (RR, 0.94; P = 0.01). In conclusion, older age is a factor associated with the surgical success of BGI for OAG.

Correction: TGF-ß-induced activation of conjunctival fibroblasts is modulated by FGF-2 and substratum stiffness.

[This corrects the article DOI: 10.1371/journal.pone.0242626.].

TGF-ß-induced activation of conjunctival fibroblasts is modulated by FGF-2 and substratum stiffness.

PURPOSE: This study aimed to investigate the effects of substratum stiffness on the sensitivity of human conjunctival fibroblasts to transforming growth factor (TGF)-ß, and to explore the molecular mechanism of action. METHODS: Human conjunctival fibroblasts were cultured on collagen-coated plastic or silicone plates. The stiffness of the silicone plates was 0.2 or 64 kPa. Cells were treated by 2.5 ng/mL TGF-ß2 with or without fibroblast growth factor (FGF)-2 (0-100 ng/mL) for 24 h or 48 h. The protein expression levels were determined by Western blot analysis. Cell proliferation was assessed using the WST-8 assay. RESULTS: FGF-2 suppressed the TGF-ß-induced expression of α-smooth muscle actin (SMA) and collagen type I (Col I), but not fibronectin (FN). Both FGF-2 and TGF-ß2 increased cell proliferation without an additive effect. The induction of α-SMA by TGF-ß2 was decreased on the soft substratum, without any change in the expression level or subcellular location of Yes-associated protein/transcriptional coactivator with PDZ-binding motif (YAP/TAZ). FGF-2 suppressed TGF-ß-induced α-SMA expression even on the soft substratum. CONCLUSIONS: FGF-2 treatment and a soft substratum suppressed TGF-ß-induced transdifferentiation of conjunctival fibroblasts into myofibroblasts. FGF-2 attenuated the TGF-ß-induced expression of α-SMA, even on a soft substratum.

Restricted post-trabeculectomy bleb formation by conjunctival scarring.

PURPOSE: To evaluate the effects of radial conjunctival incision during fornix-based trabeculectomy, and conjunctival scarring associated with previous ocular surgeries, for the restriction of bleb formation. METHODS: Digital photo-slit lamp images of filtering blebs, which had undergone fornix-based trabeculectomy with mitomycin C previously, were analysed. The area and the height of the bleb in the conjunctivae with radial incision (Ain and Hin, respectively) were compared with the area and the height of the bleb in the conjunctivae without radial incision (Ano and Hno, respectively). We compared the parameters of bleb area (total bleb area, Ain and Ano) and bleb height (the bleb height at the centre of the scleral flap; Hc, Hin and Hno) in eyes without previous ocular surgeries, with those in eyes that underwent previous ocular surgeries. RESULTS: The study population consisted of 51 eyes. The Ano and the Hno were significantly larger than the Ain (p < 0.001) and the Hin (p < 0.001) respectively. Subgroup analyses in eyes without previous ocular surgeries and eyes with phacoemulsification cataract surgery also showed that the Ano was significantly larger than the Ain. However, there was no significant difference between the Ano and the Ain in eyes with previous trabeculectomy. A comparison of bleb formation between eyes without previous ocular surgeries and eyes with cataract surgery showed no significant differences in each of the bleb area and bleb height parameters. By contrast, the total bleb area (p = 0.021), the Ano (p = 0.017) and the Hc (p = 0.045) were significantly smaller in eyes with previous trabeculectomy than in eyes without previous ocular surgeries. CONCLUSIONS: Our data demonstrate that bleb formation depends on radial conjunctival incision during fornix-based trabeculectomy and surgical conjunctival scarring from previous trabeculectomy.

Prospective cohort study of corneal endothelial cell loss after Baerveldt glaucoma implantation.

PURPOSE: To evaluate corneal endothelial cell loss after Baerveldt glaucoma implantation. METHODS: We prospectively evaluated changes in endothelial cell density (ECD) in the cornea of 59 patients (59 eyes) who underwent Baerveldt glaucoma implantation. Tubes were inserted into the anterior chamber in 45 eyes and pars plana in 14 eyes. The primary outcome measure was the change in corneal ECD after Baerveldt glaucoma implantation. Secondary outcome measures included relationships between corneal ECD and optical coherence tomography images of tube parameters (tube-cornea distance and tube-cornea angle) and prognostic factors for corneal ECD loss. RESULTS: Corneal ECD decreased significantly by 9.2% at 12 months after surgery (P = 0.001). In the anterior chamber Baerveldt glaucoma implantation group, corneal ECD at the tube insertion quadrant decreased significantly by 9.6% at 3 months (P = <0.001), 10.7% at 6 months (P = <0.001), and 13.1% at 12 months (P = <0.001). Corneal ECD at the center decreased significantly at 6 months after surgery (7.2% loss; P = 0.012) and at 12 months after surgery (12.1% loss; P = 0.001). Corneal ECD at the contralateral quadrant decreased significantly at 12 months after surgery only (10.3% loss; P = 0.004). In the pars plana Baerveldt glaucoma implantation group, no significant loss of corneal ECD was found in any corneal areas at any post-surgery follow-up visits. Tube-cornea angle was negatively correlated with the rate of corneal ECD loss at the tube insertion quadrant; r = -0.55 (P = 0.0013). In multivariable analyses, exfoliation glaucoma and narrower tube-cornea angle were significant prognostic factors for severe corneal ECD loss (P = 0.0068 and P = 0.046, respectively). CONCLUSIONS: Anterior chamber Baerveldt glaucoma implantation causes corneal endothelial cell loss. Corneal endothelial cell loss starts at the tube insertion quadrant. Exfoliation glaucoma and narrower tube-corneal angle are associated with severe loss of corneal endothelial cells.

Effect of Microtubule Disruption on Dynamics of Acidic Organelles in the Axons of Primary Cultured Retinal Ganglion Cells.

PURPOSE: Axonal transport is fundamental to autophagy in neuronal cells. To understand its biological significance in various conditions, it is necessary to monitor the process of autophagy. However, monitoring methods are often limited to static analyses, such as protein expression and histological observations. Autophagy has multistep process and is highly dynamic; therefore, additional techniques are necessary to study autophagy. In this study, we quantified the dynamics of autophagy-related organelle transport under conditions of dynamic instability and catastrophic disruption of microtubules using in vitro live imaging. MATERIALS AND METHODS: Retinal ganglion cells (RGCs) were isolated from postnatal day 3 Sprague-Dawley rats by immunopanning. After 7 days of culture, acidic organelles were stained by LysoTracker. Dynamics of acidic organelles was quantified using kymographs. Colchicine was used to induce microtubule disruption. Movement of acidic organelles was observed at five time points: before, and at 6, 24, 72, and 120 h after colchicine stimulation. Ethidium homodimer-1 (EthD-1) was used to determine cell viability. RESULTS: The status of axonal transport of acidic organelles (n = 363) from 27 RGCs was classified into four categories: anterograde (1.4%), retrograde (90%), stationary (8.0%), and fluttering (0.28%). Six hours after the induction of microtubule disruption in 14 of 27 RGCs, almost all acidic organelles (n = 236) were stationary. All acidic components had completely stopped moving 24 h later. At 72 h after stimulation, axonal fragmentation, and shrinking and disappearance of soma were observed in 71% of RGCs. Finally, the remaining RGCs became positive for EthD-1. In the control (13 of 27 RGCs), axonal transport was maintained for 120 h and EthD-1-positive RGCs were not observed. CONCLUSION: Almost all acidic organelles were transported retrogradely along the axon, which was inhibited by colchicine. Understanding the dynamics of acidic organelles may provide useful parameters for characterizing autophagy of neuronal cells in pathophysiological conditions.

Culture Systems of Dissociated Mouse and Human Pluripotent Stem Cell-Derived Retinal Ganglion Cells Purified by Two-Step Immunopanning.

Purpose: We aimed to establish purification and culture systems for retinal ganglion cells (RGCs) differentiated from mouse and human pluripotent stem cells (PSC) for in vitro and regenerative medicine studies. Methods: We used a two-step immunopanning method to purify RGCs from mouse and human PSC-derived three-dimensional (3D) retinal organoids. To assess the method, we purified RGCs from 3D retinal organoids derived from embryonic stem cells (ESCs) generated from Thy1-EGFP transgenic (TG) mice. In addition, 3D retinal organoids differentiated from human induced PSCs (iPSCs) were cultured for up to differentiation day (DD) 120, and RGCs were purified by immunopanning. RGC marker expressions were confirmed by immunostaining and reverse transcription-quantitative PCR. The purified RGCs were cultured, and neurite outgrowth was measured and analyzed using an IncuCyte Zoom system. Results: Mouse RGCs purified from Thy1-EGFP TG mouse retinas and the ESC-derived 3D retinas could be maintained for approximately 2 to 3 weeks, expressing the markers BRN3B and SMI-312. Purified RGCs from human iPSC-derived retinal organoids expressed RGC markers and could be maintained for up to 4 weeks. The RGCs collected at DD 90 to 110 extended longer neurites than those collected at younger stages. Conclusions: We successfully purified RGCs from mouse and human PSC-derived 3D retinal organoids cultured for approximately 120 days. RGCs from older retinal organoids would be useful for neurite tracking. This method would be effective not only for studying the pathology of human RGC diseases but also for therapeutic drug studies and RGC transplantation.

Thrombopoietin Metabolically Primes Hematopoietic Stem Cells to Megakaryocyte-Lineage Differentiation.

During acute myelosuppression or thrombocytopenia, bone marrow (BM) hematopoietic cells respond rapidly to replenish peripheral blood platelets. While the cytokine thrombopoietin (Thpo) both regulates platelet production and maintains HSC potential, whether Thpo controls megakaryocyte (Mk)-lineage differentiation of HSCs is unclear. Here, we show that Thpo rapidly upregulates mitochondrial activity in HSCs, an activity accompanied by differentiation to an Mk lineage. Moreover, in unperturbed hematopoiesis, HSCs with high mitochondrial activity exhibit Mk-lineage differentiation in vitro and myeloid lineage-biased reconstitution in vivo. Furthermore, Thpo skewed HSCs to express the tetraspanin CD9, a pattern correlated with mitochondrial activity. Mitochondria-active HSCs are resistant to apoptosis and oxidative stress upon Thpo stimulation. Thpo-regulated mitochondrial activity associated with mitochondrial translocation of STAT3 phosphorylated at serine 727. Overall, we report an important role for Thpo in regulating rapid Mk-lineage commitment. Thpo-dependent changes in mitochondrial metabolism prime HSCs to undergo direct differentiation to an Mk lineage.

Comparison of subconjunctival scarring after microincision vitrectomy surgery using 20-, 23-, 25- and 27-gauge systems in rabbits.

PURPOSE: To compare subconjunctival scarring after vitrectomy in rabbit eyes using different gauge systems by analysing anterior segment optical coherence tomography (AS-OCT) images and histological sections. METHODS: Vitrectomy using 20-, 23-, 25- and 27-gauge systems was performed for rabbits. Anterior segment optical coherence tomography (AS-OCT) images of the incision sites were obtained before and at day 1, 7 and 1 month after surgery. We measured the thickness of conjunctival epithelium, stroma, Tenon's capsule and total conjunctiva of these three layers, then determined the preservation rates of the borderlines between each layer. Surgical invasion was estimated by histological observation. RESULTS: The thickness of total conjunctiva, or the thickness of both conjunctival stroma and Tenon's capsule in the 20-gauge group was significantly thicker than that in the 27-gauge group at day 1 and day 7 after surgery. Preservation rates of the conjunctival stroma/Tenon's capsule borderline were significantly lower in the 20-gauge group than in the 25- and 27-gauge groups at day 1, day 7 and 1 month. Preservation rates of the Tenon's capsule/sclera borderline were significantly lower in the 20-gauge group than in the 25- and 27-gauge groups at 1 month. In the 27-gauge group, the number of α-smooth muscle actin-positive fibroblasts was significantly smaller than in the 20-gauge group at day 7. CONCLUSION: Based on the finding of AS-OCT and histology, micro incision vitreous surgery, especially using 27-gauge, contributed to less subconjunctival scarring postoperatively. Therefore, the 27-gauge pars plana vitrectomy (PPV) may be a more effective technique for preserving the structure of conjunctiva.

Evaluation of Bleb Fluid After Baerveldt Glaucoma Implantation Using Magnetic Resonance Imaging.

We evaluated bleb fluid images taken after Baerveldt glaucoma implantation. T2-weighted images of bleb fluid were scanned with 3 Tesla magnetic resonance imaging in 52 patients who had undergone tube-shunt surgery using the 350-mm2 endplate Baerveldt glaucoma implant; three-dimensional images were constructed from these images. Bleb fluid images were classified into either a layer of bleb fluid on either side of the endplate (double bleb layer group; n = 24) or one layer outside the endplate (single bleb layer group; n = 28). Despite there being no correlation between the bleb volume and the postoperative IOP (r = -0.080; P = 0.57), the double bleb layer group had significantly lower postoperative IOPs than the single bleb layer group (12.3 ± 3.8 mmHg vs. 14.7 ± 4.1 mmHg, respectively; P = 0.033). The single bleb layer was significantly related to higher numbers of prior intraocular surgeries (relative risk = 2.85; P = 0.0014). Formation of a layer of bleb fluid on either side of the endplate may have resulted in the lower postoperative IOPs after Baerveldt glaucoma implantation. Repeated intraocular surgery adversely affects formation of the double bleb layer.