Whitney Greene

Pathophysiology of blast‐induced ocular trauma in rats after repeated exposure to low‐level blast overpressure

The incidence of blast‐induced ocular injury has dramatically increased due to advances in weaponry and military tactics. A single exposure to blast overpressure (BOP) has been shown to cause damage to the eye in animal models; however, on the battlefield, military personnel are exposed to BOP multiple times. The effects of repeated exposures to BOP on ocular tissues have not been investigated. The purpose of this study is to characterize the effects of single or repeated exposure on ocular tissues.

Pathophysiology of blast-induced ocular trauma with apoptosis in the retina and optic nerve

BACKGROUND Blast-induced ocular trauma is a frequent cause of morbidity for survivors of improvised explosive devices. Blast overpressure (BOP) of 120 ± 7 KPa has been shown to cause damage to lungs, brain, and gut in a rat model; however, the effects of BOP on ocular tissues have not been characterized. To elucidate the pathophysiology of blast-induced ocular trauma, ocular tissues from rats subjected to blast were examined for evidence of apoptosis by the detection of activated caspase 3 and TUNEL assay in their ocular tissues. METHODS A compressed air shock tube was used to deliver 120 ± 7 KPa of BOP for duration of 2 msec to the right side of the rats. Rats were then euthanized at specific time points after blast exposure (3 hours, 24 hours, 48 hours). Ocular tissues were processed for immunohistochemistry to detect activated caspase 3 and TUNEL assay. Tissues were evaluated for relative levels of positive signal as compared to nonblast exposed controls. RESULTS Activated caspase 3 was detected in the optic nerve, ganglion layer, and inner nuclear layer post blast exposure. At 24 and 48 hours, the inner nuclear layer from the right side had more cells with activated caspase 3. In the optic nerve, the highest levels of activated caspase 3 were detected on the right side at 24 hours post blast. CONCLUSION BOP of 120 ± 7 KPa induces optic neuropathy and retinal damage. In both the optic nerve and retina, caspase 3 was activated in the right and left sides following blast exposure. The results of this study reveal that blast exposure induces apoptosis in both the optic nerve and retinal tissues.

Secretion Profile of Induced Pluripotent Stem Cell-Derived Retinal Pigment Epithelium During Wound Healing

Purpose The purpose of this study was to characterize the secretion profile of induced pluripotent stem cell-derived retinal pigment epithelium (iPS-RPE) during wound healing. iPS-RPE was used to develop an in vitro wound healing model. We hypothesized that iPS-RPE secretes cytokines and growth factors which act in an autocrine manner to promote migration and proliferation of cells during wound healing. Methods iPS-RPE was grown in transwells until fully confluent and pigmented. The monolayers were scratched to induce a wound. Levels of Ki-67, β-catenin, e-cadherin, n-cadherin, and S100A4 expression were analyzed by immunofluorescent labeling. Cell culture medium samples were collected from both the apical and basolateral sides of the transwells every 72 hours for 21 days. The medium samples were analyzed using multiplex ELISA to detect secreted growth factors and cytokines. The effects of conditioned medium on collagen gel contraction, cell proliferation, and migration were measured. Results iPS-RPE underwent epithelial-mesenchymal transition (EMT) during wound healing as indicated by the translocation of β-catenin to the nucleus, cadherin switch, and expression of S100A4. GRO, GM-CSF, MCP-1, IL-6, and IL-8 were secreted by both the control and the wounded cell cultures. VEGF, FGF-2, and TGFβ expression were detected at higher levels after wounding than those in control. The proteins were found to be secreted in a polarized manner. The conditioned medium from wounded monolayers promoted collagen gel contraction, as well as proliferation and migration of ARPE 19 cells. Conclusions These results indicate that after the monolayer is wounded, iPS-RPE secretes proteins into the culture medium that promote increased proliferation, contraction, and migration.

MicroRNA expression profiles of human iPS cells, retinal pigment epithelium derived from iPS, and fetal retinal pigment epithelium.

The objective of this report is to describe the protocols for comparing the microRNA (miRNA) profiles of human induced-pluripotent stem (iPS) cells, retinal pigment epithelium (RPE) derived from human iPS cells (iPS-RPE), and fetal RPE. The protocols include collection of RNA for analysis by microarray, and the analysis of microarray data to identify miRNAs that are differentially expressed among three cell types. The methods for culture of iPS cells and fetal RPE are explained. The protocol used for differentiation of RPE from human iPS is also described. The RNA extraction technique we describe was selected to allow maximal recovery of very small RNA for use in a miRNA microarray. Finally, cellular pathway and network analysis of microarray data is explained. These techniques will facilitate the comparison of the miRNA profiles of three different cell types.

Deriving retinal pigment epithelium (RPE) from induced pluripotent stem (iPS) cells by different sizes of embryoid bodies.

Pluripotent stem cells possess the ability to proliferate indefinitely and to differentiate into almost any cell type. Additionally, the development of techniques to reprogram somatic cells into induced pluripotent stem (iPS) cells has generated interest and excitement towards the possibility of customized personal regenerative medicine. However, the efficiency of stem cell differentiation towards a desired lineage remains low. The purpose of this study is to describe a protocol to derive retinal pigment epithelium (RPE) from iPS cells (iPS-RPE) by applying a tissue engineering approach to generate homogenous populations of embryoid bodies (EBs), a common intermediate during in vitro differentiation. The protocol applies the formation of specific size of EBs using microwell plate technology. The methods for identifying protein and gene markers of RPE by immunocytochemistry and reverse-transcription polymerase chain reaction (RT-PCR) are also explained. Finally, the efficiency of differentiation in different sizes of EBs monitored by fluorescence-activated cell sorting (FACS) analysis of RPE markers is described. These techniques will facilitate the differentiation of iPS cells into RPE for future applications.