Bongsu Kim

Retinal MMP-12, MMP-13, TIMP-1, and TIMP-2 Expression in Murine Experimental Retinal Detachment

PURPOSE Matrix metalloproteinases (MMPs) and their inhibitors play a role in the pathobiology of retinal detachment (RD) and proliferative vitreoretinopathy (PVR). Proliferative vitreoretinopathy is facilitated by chronic retinal detachment and involves excessive deposition of extracellular matrix (ECM) proteins. Matrix metalloproteinase-2 and -13 are important modulators of the ECM which have not been evaluated in RD. The purpose of this study was to investigate the retinal expression of select MMPs, including MMP-12, MMP-13, and associated inhibitors in a murine model of retinal detachment. METHODS Transient or chronic retinal detachments (RDs) were induced by subretinal injection of either saline (SA) or hyaluronic acid (HA) in C57BL/6 mice. To confirm that the HA-RD model has features consistent with PVR-like changes, glial activation and subretinal fibrosis were evaluated with immunofluorescence, dilated fundus examination, and spectral-domain optical coherence tomography (SD-OCT). Gene expression was quantified by qRT-PCR. Proteins were assayed by immunoblot and immunohistochemistry. RESULTS Hyaluronic acid RD eyes developed gliosis and subretinal fibrosis on dilated exam, SD-OCT, and immunofluorescence analysis. Gene expression of Mmp-12 and Mmp-13, and Timp-1 was strongly upregulated at all time points in RD compared with controls. Timp-2, Mmp-2, and Mmp-9 expression was modest. Hyaluronic acid RDs exhibited more MMP and TIMP expression than SA-RDs. MMP-12, -13, and TIMP-1 proteins were elevated in RDs compared with controls. Immunohistochemistry revealed moderate to strong MMP-13 levels in subretinal space macrophages. CONCLUSIONS Fibrosis can develop in the HA-RD model. There is an upregulation of select MMPs that may modulate the wound healing process following RD.

Fabrication of porous microtent structures toward an in vitro endothelium model

This paper reports the fabrication of compliant and permeable thin films with controlled curvature preferable to serve as engineering scaffolds for the production of in vivo like vascular endothelial constructs. A simple fabrication process was developed to fabricate three-dimensional ‘tent’ like microstructures by combining electrospinning and microfabrication. In particular, the ‘microtents’ were created by electrospinning mechanically flexible poly(etherurethane)urea(PEUU) polymer on a microstructured collecting substrate. The shape of the ‘microtents’ can be tuned by adjusting the geometries of microstructures on the collecting substrate and the operational parameters of electrospinning. Mechanical characterization showed the nonlinear mechanical behavior of porous polymeric thin films is similar to those of soft tissues, indicating that these thin films may serve as scaffolds for mimicking local mechanical environment of vascular tissues. Human endothelial cells were successfully cultured on the concave side of the porous thin film, constituting an endothelium model in vitro. This work addresses the need for engineered tissue scaffolds that can mimic both morphological and mechanical environments of natural vascular endothelium. The coupled effects of mechanical, structural and biochemical factors on vascular endothelium can thus be investigated.

MIF Inhibitor ISO-1 Protects Photoreceptors and Reduces Gliosis in Experimental Retinal Detachment

Photoreceptor death and retinal gliosis underlie the majority of vision threatening retinal diseases including retinal detachment (RD). Although the underlying pathobiology of vision limiting processes in RD is not fully understood, inflammation is known to play a critical role. We conducted an iTRAQ proteomic screen of up- and down-regulated proteins in a murine model of RD to identify potential targetable candidates. Macrophage migration inhibitory factor (MIF) was identified and evaluated for neurotoxic and pro-gliotic effects during RD. Systemic administration of the MIF inhibitor ISO-1 significantly blocked photoreceptor apoptosis, outer nuclear layer (ONL) thinning, and retinal gliosis. ISO-1 and MIF knockout (MIFKO) had greater accumulation of Müller glia pERK expression in the detached retina, suggesting that Müller survival pathways might underlie the neuroprotective response. Our data show the feasibility of the MIF-inhibitor ISO-1 to block pathological damage responses in retinal detachment and provide a rationale to explore MIF inhibition as a potential therapeutic option for RD.

Porous microfluidics: A unique platform for transvascular study

This work reports the development of a porous microfluidic system which can serve as an in vitro model of natural vessels in circulatory and respiratory systems. The model possesses both structural and mechanical characteristics of the natural counterparts. To mimic the semi-permeable wall of natural vessels, microfluidic channels with porous walls are fabricated by spinning polymeric nanofibers on the collecting substrate with three-dimensional microelectrodes. The mechanical properties of the porous substrate are evaluated using conventional tensile testing. The permeability of the fibrous membrane is characterized by perfusion experiments. The results collectively show the utility of the membrane in transvascular study. Human umbilical vein endothelial cells (HUVECs) are cultured on the fibrous membrane and form an endothelial monolayer. The resulting structure is elastic and distensible, similar as natural blood vessels. The curved endothelium surface also helps to mimic the complex mechanical loading states of natural vascular endothelium under pulsatile flow. This work is expected to add a new dimension to the widely used microfluidic systems by allowing both in-vessel transport and trans-vessel transport. The immediate impacts of the porous microfluidic systems can be found in in vitro study of circulatory and respiratory systems.

A new multichannel method quantitating TUNEL in detached photoreceptor nuclei

&NA; Nuclear co‐localization labels are critical to ocular research. Among these, the TUNEL assay has been established as a gold standard of cell death and apoptosis. While several validated computer‐based methods exist to quantitate these markers, including ImageJ Retina Analysis (RA) Toolkit and ImagePro, none verify the count with the nuclear counter stain to confirm nuclear co‐localization. We established a new ImageJ‐based automated multichannel thresholding (MCT) method to quantitate nuclear co‐localized labeling. The MCT method was validated by comparing it with the two published TUNEL analysis in TUNEL‐positive photoreceptors in an experimental retinal detachment (RD) model. RDs were induced in murine eyes and cross‐sectional images of TUNEL and DAPI counter stain were obtained. Images were classified as “typical” or high density ”hotspot” TUNEL regions (n = 10/group). Images were analyzed and compared between the MCT method and the published techniques including both ”standard” and ”high” settings of the RA Toolkit for detecting lower or higher TUNEL densities, respectively. Additional testing of the MCT method with built‐in ImageJ thresholding algorithms was performed to produce fully automated measurements. All images were compared with Bland‐Altman mean difference plots to assess the difference in counts and linear regression plots to assess correlation. Comparison between the MCT method and the ImagePro method were found to be well correlated (typical: R2 = 0.8972, hotspot: R2 = 0.9000) with minor to non‐significant differences. The RA Toolkit settings were found to be mostly well correlated as well (standard/typical: R2 = 0.8036, standard/hotspot: R2 = 0.4309, high/typical: R2 = 0.7895, high/hotspot: R2 = 0.8738) but were often found to have significantly higher counts than the MCT. In conclusion, the MCT method compared favorably with validated computer‐based methods of nuclear marker immunofluorescence quantitation and avoids staining artifacts through the incorporation of the nuclear counter stain to confirm positive cells. HighlightsCurrent cell death analysis methods do not verify cells with nuclear counterstain.This can lead to artificial or false‐positive results.Multichannel thresholding allows counting of nuclear verified cells.Compared favorably to multiple verified and published methods.Automated thresholding algorithms allows rapid and efficient batch analysis.

Programmable micropatterning of nanofibers for functional tissue engineering

This paper reports programmable micropatterning of electrospun nanofibrous materials using micropatterned collecting chips that consist of independently programmable microelectrodes. By regulating the local electrical field using activated and floating electrodes, the collecting chip allows creating the pattern of electrospun fibrous microstructures with controllable local orientation. This work provides a simple yet highly reliable way to produce micro/nanostructures of polymer nanofibers in a cost effective manner. It has a great potential in creating functional tissue engineered scaffolds with morphological and functional similarities with natural tissues, and can also be used as filtering substrates in microdevices with designed performance for on-chip biochemical analysis.

Fabrication and Characterization of a Microscale Cellular Loading Device for Cellular Biomechanical Study

Mechanical stimuli interfere with cellular behaviors under many physiological conditions. To understand the role of mechanical stimuli, engineered devices are developed to apply mechanical loads to cells in vitro. Despite of their usefulness, these devices are limited since they often lack the capacity of spatial load control, which is essential for intercellular study. Moreover, application of both compressive and tensile loads using a single loading device is challenging. Here, we fabricate and characterize a microdevice for applying programmable compressive/tensile loads to live cells. The device consists of two PDMS substrates. The top substrate consists of nine circular membranes with patterned microdots array on the top surfaces. Each membrane is connected with a microfluidic channel built in the bottom substrate. Upon actuation, the fluid in the channels deforms the membranes and applies controllable strain to cells cultured on the membranes. In this design, each membrane can be individually controlled to apply desired strain levels. The surface strain of the PDMS membranes is characterized by mapping the displacement of the dot array. The result of strain analysis shows that, the radial strain at the center of a circular membrane upon deformation ranges from about 5% compressive strain to about 20% tensile strain, validating the capacity of the device in applying both tensile and compressive stresses. Cell testing is performed using trabecular meshwork endothelial cells. Cells on different membranes are subjected to 0.5Hz of compressive or tensile stresses. The result shows that compressive and tensile stresses have different effects on the cells, indicating the device a promising solution for cellular biomechanical study.Copyright

Exploring Electrical Impedance Spectroscopy as an Innovative Tool for On-Chip Analysis of Human Trabecular Meshwork

This work reports the investigation of in vitro cultured human trabecular meshwork (TM) using electric impedance spectroscopy. The outflow facility of aqueous humor contains a three-dimensional network composed of TM endothelial cells and the extracellular matrix (ECM). The network is a critical determinant of intraocular pressure (IOP), the increase of which is the main cause of primary open angle glaucoma (POAG). Electric impedance spectroscopy was chosen over other technologies for on-chip TM cell study because of its non-invasive and label-free natures as well as the ease of on-chip integration. An array of interdigitated microelectrodes (IMEs) was fabricated for impedance sensing. Polydimethylsiloxane (PDMS) was used as the passivation layer and to form the cell culture wells. TM cell growth and proliferation under external stimuli can thus be in situ monitored. In this work, the utility of the impedance spectroscopy was demonstrated by treating TM cells with dexamethasone (DEX), a synthetic glucocorticoid which is found to increase the IOP. Electric impedance was monitored for 3 days during DEX treatment. The impedance measurement at 40 kHz showed that a smaller increase of impedance magnitude occurred in the DEX treated group than in the control group, which suggested that DEX inhibits the proliferation of TM cells. The higher the DEX concentration, the greater inhibition was observed. The measurement was validated using AlamarBlue proliferation assay and microscopic observation. The measurement provides a reasonable explanation of glucocorticoid induced glaucoma, where the glucocorticoid alters the metabolic and physiological functions of TM cells by inhibiting normal cell growth and proliferation.Copyright

Programmable Micropatterning of Polymer Nanofibers

This paper reports programmable micropatterning of electrospun nanofibrous materials using a collector chip that consists of an array of independently controllable microelectrodes. The microelectrodes on the collecting chip are prepared by standard photolithography. By programming the local electrical field using excited and floating electrodes, the collector chip allows patterning of microstructures with controllable characteristics. The difference of electrostatic force between the excited and the floating electrodes increases the patterning contrast of electrospun nanofibers. The arbitrary geometries are successfully patterned on the array of 6 × 6 electrodes by independently programmable control of the voltage of each electrode. The experimental result also shows that it is possible to control the porosity and alignment of fibers. This method provides a simple yet highly reliable approach for creating combined micro/nanostructures of polymer nanofibers in a cost effective manner, which has great potential in functional tissue engineering, filtration, and chemical sensing. The work is also expected to foster the use of nanofibers in microdevices for on-chip biochemical analysis, and controlled infiltration and proliferation. The resulting nanofibers with controllable porosity are especially useful for the construction of tissue engineering scaffolds with morphological and functional similarity with natural tissues.Copyright