Poster BB5 - Efficacy of Poloxamer P188 for Wound Healing in a Brain Endothelium Model

Introduction: Initially approved by the FDA for its role in reducing blood viscosity during transfusion, Poloxamer 188 (P188) has since emerged as a promising candidate for therapeutic purpose, including wound healing. The P188's ability to restore compromised cell membranes and re-establish their structural integrity has been extensively documented. P188 has also demonstrated its efficacy in traumatic brain injury (TBI) models [1], where the feasibility of restoring the function of brain endothelium and promoting recovery was established. We hypothesize that P188 may enhance wound healing in response to a mechanical injury in an in vitro brain endothelium model. In addition to restoring the membrane integrity, evidence from our laboratory suggests that P188 is transported across the cell membrane and interact with intracellular organelles. Elucidation of reparative mechanisms using the injury model may provide valuable insights into optimizing therapeutic effects of P188. In the current study, we detected the wound area, monitored the wound closure as a function of time, and characterized the accelerated wound closure by P188.

Materials and

Methods: BALB/c Mouse Primary Brain Microvascular Endothelial Cells (MPBMECs) were cultured until confluent and then seeded onto glass-bottom imaging dishes. Cell confluency and viability were first assessed. A monolayer of brain endothelial cells that mimics the brain endothelium was confirmed by labeling ZO-1 protein immunostaining, indicating the formation of tight junctions and a functional monolayer. To create the wound model, scratches were made across the monolayer using a sterile pipette tip, and the cells were incubated in either complete media or P188 solution. For P188 treatment, a stock solution of 2 mM concentration was prepared and further diluted into 10 or 100 uM concentrations. NucBlue was utilized to stain the nuclei. Image analysis was performed by outlining of Regions of Interest (ROIs) using ImageJ FIJI to quantify the wound area.

Results, Conclusions, and Discussions: Wounds were imaged, and the wound area was identified and monitored over 24 hours (Fig. 1). While untreated cells appear to close the wound area without P188, the wound was not completely closed in 24 hours. In contrast, treatment of either 10 or 100 uM P188 showed a complete wound closure. As shown in Figure 2, the wound closure is well described by a linear regression, and that the rate of wound closure, e.g., the normalized wound area/hour, increased from 3.4%/hr in untreated control wound to 5.2%/hr in response to the P188 treatment. Interestingly, no significant difference was observed between the two treatment groups (10 vs. 100 uM P188), suggesting that the lower concentration of P188 may exert a comparable effect on wound closure rates. It should be noted that, during the first 6 hours, the P188 treatment does not appear to affect wound healing but noticeably improved the wound closure after 12-hour treatment. While these findings highlight the potential of P188 in promoting wound healing of a brain endothelium injury, the mechanisms that mediate such reparative effects are yet to be fully elucidated.

In summary, the results of this study demonstrated the efficacy of P188 in enhancing wound closure rates in a brain endothelium model. While these findings hold promise for wound healing, the exact mechanisms underlying P188's effects appear complex. Our recent results indicate that P188 is transported across the cell membrane and found to co-localize with intracellular organelles. In addition to the already established mechanism of membrane resealing by P188, it may facilitate the intrinsic reparative cellular machinery. Detailed experiments are underway to identify the intracellular organelles that do interact with P188 and formulate a more comprehensive integrated wound healing mechanism from the cell membrane to subcellular level that might represent an important next step in advancing the therapeutic applications of P188.

Acknowledgements (Optional): Authors acknowledge generous support from Dr. Walter Cabe for this work.