Investigating retinal explant models cultured in static and perfused systems to test the performance of exosomes secreted from retinal organoids.

BACKGROUND: Ex vivo cultures of retinal explants are appropriate models for translational research. However, one of the difficult problems of retinal explants ex vivo culture is that their nutrient supply needs cannot be constantly met. NEW METHOD: This study evaluated the effect of perfused culture on the survival of retinal explants, addressing the challenge of insufficient nutrition in static culture. Furthermore, exosomes secreted from retinal organoids (RO-Exos) were stained with PKH26 to track their uptake in retinal explants to mimic the efficacy of exosomal drugs in vivo. RESULTS: We found that the retinal explants cultured with perfusion exhibited significantly higher viability, increased NeuN+ cells, and reduced apoptosis compared to the static culture group at Days Ex Vivo (DEV) 4, 7, and 14. The perfusion-cultured retinal explants exhibited reduced mRNA markers for gliosis and microglial activation, along with lower expression of GFAP and Iba1, as revealed by immunostaining. Additionally, RNA-sequencing analysis showed that perfusion culture mainly upregulated genes associated with visual perception and photoreceptor cell maintenance while downregulating the immune system process and immune response. RO-Exos promoted the uptake of PKH26-labelled exosomes and the growth of retinal explants in perfusion culture. COMPARISON WITH EXISTING METHODS: Our perfusion culture system can provide a continuous supply of culture medium to achieve steady-state equilibrium in retinal explant culture. Compared to traditional static culture, it better preserves the vitality, provides better neuroprotection, and reduces glial activation. CONCLUSIONS: This study provides a promising ex vivo model for further studies on degenerative retinal diseases and drug screening.

Tissue engineering RPE sheet derived from hiPSC-RPE cell spheroids supplemented with Y-27632 and RepSox.

BACKGROUND: Retinal pigment epithelium (RPE) cell therapy is a promising way to treat many retinal diseases. However, obtaining transplantable RPE cells is time-consuming and less effective. This study aimed to develop novel strategies for generating engineered RPE patches with physiological characteristics. RESULTS: Our findings revealed that RPE cells derived from human induced pluripotent stem cells (hiPSCs) successfully self-assembled into spheroids. The RPE spheroids treated with Y27632 and Repsox had increased expression of epithelial markers and RPE-specific genes, along with improved cell viability and barrier function. Transcriptome analysis indicated enhanced cell adhesion and extracellular matrix (ECM) organization in RPE spheroids. These RPE spheroids could be seeded and bioprinted on collagen vitrigel (CV) membranes to construct engineered RPE sheets. Circular RPE patches, obtained by trephining a specific section of the RPE sheet, exhibited abundant microvilli and pigment particles, as well as reduced proliferative capacity and enhanced maturation. CONCLUSIONS: Our study suggests that the supplementation of small molecules and 3D spheroid culture, as well as the bioprinting technique, can be effective methods to promote RPE cultivation and construct engineered RPE sheets, which may support future clinical RPE cell therapy and the development of RPE models for research applications.

Light-sheet laser speckle imaging for cilia motility assessment.

Mucociliary clearance is an important innate defense mechanism predominantly mediated by ciliated cells in the upper respiratory tract. Ciliary motility on the respiratory epithelium surface and mucus pathogen trapping assist in maintaining healthy airways. Optical imaging methods have been used to obtain several indicators for assessing ciliary movement. Light-sheet laser speckle imaging (LSH-LSI) is a label-free and non-invasive optical technique for three-dimensional and quantitative mapping of velocities of microscopic scatterers. Here, we propose to use an inverted LSH-LSI platform to study cilia motility. We have experimentally confirmed that LSH-LSI can reliably measure the ciliary beating frequency and has the potential to provide many additional quantitative indicators for characterizing the ciliary beating pattern without labeling. For example, the asymmetry between the power stroke and the recovery stroke is apparent in the local velocity waveform. PIV (particle imaging velocimetry) analysis of laser speckle data could determine the cilia motion directions in different phases.

Effects of extended pharmacological disruption of zebrafish embryonic heart biomechanical environment on cardiac function, morphology, and gene expression.

BACKGROUND: Biomechanical stimuli are known to be important to cardiac development, but the mechanisms are not fully understood. Here, we pharmacologically disrupted the biomechanical environment of wild-type zebrafish embryonic hearts for an extended duration and investigated the consequent effects on cardiac function, morphological development, and gene expression. RESULTS: Myocardial contractility was significantly diminished or abolished in zebrafish embryonic hearts treated for 72 hours from 2 dpf with 2,3-butanedione monoxime (BDM). Image-based flow simulations showed that flow wall shear stresses were abolished or significantly reduced with high oscillatory shear indices. At 5 dpf, after removal of BDM, treated embryonic hearts were maldeveloped, having disrupted cardiac looping, smaller ventricles, and poor cardiac function (lower ejected flow, bulboventricular regurgitation, lower contractility, and slower heart rate). RNA sequencing of cardiomyocytes of treated hearts revealed 922 significantly up-regulated genes and 1,698 significantly down-regulated genes. RNA analysis and subsequent qPCR and histology validation suggested that biomechanical disruption led to an up-regulation of inflammatory and apoptotic genes and down-regulation of ECM remodeling and ECM-receptor interaction genes. Biomechanics disruption also prevented the formation of ventricular trabeculation along with notch1 and erbb4a down-regulation. CONCLUSIONS: Extended disruption of biomechanical stimuli caused maldevelopment, and potential genes responsible for this are identified.

Multifunctional laser speckle imaging.

We have developed a multi-functional laser speckle imaging system, which can be operated in both the surface illumination laser speckle contrast imaging (SI-LSCI) mode and the line scan laser speckle contrast imaging (LS-LSCI) mode. The system has been applied to imaging the chicken embryos to visualize both the blood flow and morphological details of the vasculature. The experimental results demonstrated that LS-LSCI is capable of detecting and quantifying blood flow in blood vessels smaller and deeper than those detectable by conventional SI-LSCI. Furthermore, the line scan mode is also capable of producing depth-resolved absorption-based morphological images of tissue, augmenting flow-based functional images.

Quadrature demodulation in line-scan focal modulation microscopy for imaging three-dimensional zebrafish neural structure.

Visualizing biological processes in neuroscience requires in vivo functional imaging at single-neuron resolution, high image acquisition speed and strong optical sectioning ability. However, due to light scattering of in tissue, very often conventional wide-field fluorescence microscopes are unable to resolve cells in the presence of a strong out-of-focus background. Line-scan focal modulation microscopy enables high temporal resolution and good optical sectioning ability at the same time. Here we demonstrate a quadrature demodulation method to extract the focal information with an extended frequency bandwidth and therefore higher spatial resolution. The performance of the demodulation scheme in line-scan focal modulation microscope has been evaluated by performing imaging experiments with fluorescence beads and zebrafish neural structure. Reduced background, reduced artifacts and more detailed morphological information are evident in the obtained images.

A cerebral arteriovenous malformation mistakenly diagnosed as dry eye and glaucoma: a case report.

BACKGROUND: To report the first case of a cerebral arteriovenous malformation (AVM) with ocular symptoms and review the characteristics of this case and the main point of confusion for the diagnosis of such a case. CASE PRESENTATION: A 58-year-old woman presented to the ophthalmology clinic with 1 and a half years of right eye redness, ocular hypertension and recurrent headache. One and a half years ago she was diagnosed with right eye dry eye and glaucoma and had received treatment according to this diagnosis. However, none of the treatments led to any improvement in redness and headache. Physical examination revealed dry eye and severe corkscrew hyperaemia with dilated vessels in the right eye. The results of fundoscopic examination of both eyes were normal. After we considered that the symptoms may be related to abnormal intracranial vessels, computed tomography angiography and venography (CTA + CTV) were performed, and the results showed an arteriovenous malformation in the right parietal-occipital area in the brain. The AVM was definitively located by further examination with digital subtraction angiography (DSA). After AVM endovascular embolism treatment, the conjunctival congestion of the right eye was significantly relieved, and the intraocular pressure decreased to normal. CONCLUSION: In clinical practice, when corkscrew hyperaemia accompanied by neurological symptoms is found, cerebral vascular diseases might be considered. In this case, the ophthalmologist's diagnosis should combine disease history and imaging examination.

Numerical modeling of two-photon focal modulation microscopy with a sinusoidal phase filter.

A spatiotemporal phase modulator (STPM) is theoretically investigated using the vectorial diffraction theory. The STPM is equivalent to a time-dependent phase-only pupil filter that alternates between a homogeneous filter and a stripe-shaped filter with a sinusoidal phase distribution. It is found that two-photon focal modulation microscopy (TPFMM) using this STPM can significantly suppress the background contribution from out-of-focus ballistic excitation and achieve almost the same resolution as two-photon microscopy. The modulation depth is also evaluated and a compromise exists between the signal-to-background ratio and signal-to-noise ratio. The theoretical investigations provide important insights into future implementations of TPFMM and its potential to further extend the penetration depth of nonlinear microscopy in imaging multiple-scattering biological tissues.

Effects of pupil filter patterns in line-scan focal modulation microscopy.

Line-scan focal modulation microscopy (LSFMM) is an emerging imaging technique that affords high imaging speed and good optical sectioning at the same time. We present a systematic investigation into optimal design of the pupil filter for LSFMM in an attempt to achieve the best performance in terms of spatial resolutions, optical sectioning, and modulation depth. Scalar diffraction theory was used to compute light propagation and distribution in the system and theoretical predictions on system performance, which were then compared with experimental results.

Corticosteroids effects on LPS-induced rat inflammatory keratocyte cell model.

PURPOSE: Corticosteroids are efficient anti-inflammation treatments. However, there are still arguments on whether it should be used in keratitis. This study was to observe the effect of corticosteroids on keratocytes both in normal condition and inflammation status in vitro. METHODS: Rat keratocytes were cultured and used for examination. 10 µg/ml lipopolysaccharide (LPS) was used to establish the inflammatory keratocyte cell model, and prednisolone acetate (PA), dexamethasone (Dex) and fluorometholone (Flu) were used as corticosteroids treatments. 5 d-growth curve and cell viabilities were assayed by CCK8, and cell morphologies and migration rate were studied. TNF-α, IL-6 and IL-1ß levels were examined by ELISA. Western blotting was used to quantified type VI collagen (Col VI) and matrix metalloproteinase 9 (MMP9) expressions, and immunofluorescence staining assays of Col I and Col VI were carried out. RESULTS: In normal condition, proliferation and migration of keratocytes were slightly influenced in PA, Dex and Flu groups. The secretion of Col I and Col VI was suppressed and MMP9 expression increased in corticosteroids groups. But no significant difference was seen in TNF-α, IL-6 and IL-1ß expression levels. In inflammatory status, TNF-α, IL-6 and MMP9 levels increased in LPS group, while they significantly decreased in corticosteroids groups. Although keratocytes viabilities and migration were slightly affected in 24 h, no significant differences were seen between LPS group and corticosteroids groups in 5-d proliferation. Col I and Col VI secretion in LPS-keratocytes was maintained with corticosteroids treatments. CONCLUSIONS: Corticosteroids showed lightly effects on keratocytes proliferation and migration, but it successfully decreased TNF-α, IL-6 level and maintained the secretion of and Col I and Col VI, while suppressed the expression of MMP9 in LPS-induced keratocytes. PA was suggested to use in early stage of keratitis clinical treatment.

Stripe-shaped apertures in confocal microscopy.

We have theoretically verified that, compared with the aperture shapes of previous research, combining two stripe-shaped apertures in a confocal microscope with a finite-sized pinhole improves the axial resolution to a certain extent. Because different stripe shapes cause different effects, we also investigated the relationships among resolution, shapes, pinhole size, and the signal-to-background ratio.

Numerical studies of focal modulation microscopy in high-NA system.

High spatial resolution with deep imaging penetration depth is the main advantage of focal modulation microscopy (FMM). This paper investigates effects of polarization on FMM in a high-NA system based on vectorial diffraction theory. Compared with confocal microscopy, FMM shows a 20.1% improvement in axial resolution. The performance of different polarization patterns is also discussed numerically. The study on polarization modulation may provide a new way to obtain a tighter focal spot.

A novel fibrinogenase from Agkistrodon acutus venom protects against LPS-induced endotoxemia via regulating NF-κB pathway.

CONTEXT: Endotoxins including lipopolysaccharide (LPS) could cause endotoxemia which often results in excessive inflammation, organ dysfunction, sepsis, disseminated intravascular coagulation (DIC) or even death. Previously, a novel fibrinogenase (FII) showed protective effects on LPS-induced DIC via activating protein C and suppressing inflammatory cytokines. OBJECTIVE: To evaluate whether FII has protective effect on LPS-induced endotoxemia in mice and learn about the role of NF-κB pathway in TNF-α producing process. METHODS: BALB/C mice were intraperitoneally injected (i.p.) with (a) 30 mg/kg LPS, (b) LPS + 0.3 mg/kg FII, (c) LPS + 1.0 mg/kg FII, (d) LPS + 3.0 mg/kg FII or (e) saline. Both survival rate and organ function were tested, including alanine aminotransferase (ALT), blood urine nitrogen (BUN) and tissue section, and TNF-α was examined by ELISA. RAW 264.7 macrophage was administered with (a) LPS, (b) LPS + FII, (c) FII alone or (d) saline, and TNF-α and phosphorylation (P)-NF-κB (P65) were determined by Western blot. RESULTS: The administration of LPS led to 65% mortality rate, a rise of serum TNF-α, BUN and ALT levels, and both liver and renal tissue damage were observed. While FII treatment significantly increased the survival rate of LPS-induced endotoxemia mice model, histopathology and protein analysis results also revealed that FII remarkably protected liver and renal from LPS damage as well as decreasing TNF-α level. In vitro, FII significantly decreased LPS-induced TNF-α production and the expression of P-NF-κB (P65). CONCLUSIONS: Our findings suggested that FII had protective effect on LPS-induced endotoxemia and organ injuries by suppressing the activation of NF-κB which decreased TNF-α level.

Purification, Partial Characterizations, and N-Terminal Amino Acid Sequence of a Procoagulant Protein FV-2 from Daboia Russelli Siamensis (Myanmar) Venom.

In this study, we purified and characterized the procoagulant protein FV-2 from Daboia russelli siamensis (Myanmar) venom using ion-exchange chromatography on CM-Sephadex C-50 and gel filtration on SuperdexTM G-75 column. The activation of factor X and prothrombin was determined, respectively, by specific chromogenic substrates. The fibrinogen-clotting activity, thermal stability, and pH stability were also determined. The N-treminal sequence was determined by the National Center of Biomedical Analysis of China. In the end, FV-2 was achieved with a molecular weight of 13,608.0 Da. It could activate factor X, but did not affect prothrombin or fibrinogen. The suitable pH was 6.5-7.5, and the suitable temperature ranged from 25 to 60°C. The N-terminal sequence was Asn-Phe-Phe-Gln-Phe-Ala-Glu-Met-Ile-Val-Lys-Met-Thr-Gly-Lys. Taken together, our studies suggest that FV-2 is a factor X-activating enzyme, which can activate factor X to factor Xa, but it has no effect on prothrombin and fibrinogen.