(F-223) Reparative Role of Copolymer P188 in an Injured Brain Endothelium Model

Introduction:: Introduction: Blast-induced traumatic brain injury (bTBI) has been studied extensively. Using tissue engineering approaches, our laboratory has previously shown that FDA-approved Poloxamer 188 (P188) is a potential candidate to repair traumatically injured brain tissues. Treatment with P188 has shown reparative effects by restoring the integrity of the injured cell membrane. In order to mimic mechanical trauma to the blood brain barrier (BBB), a shockwave chamber was custom-designed and used to create reproducible injuries to confluent monolayer of mouse brain endothelial cells. Such a biosystem was proven useful to determine the reparative potential of P188. Successful conjugation of P188 with fluorophores allowed the investigator to monitor and examine the reparative mechanisms at the cell surface as well as probable coupling to the intrinsic cellular repair machinery. Our preliminary results indicate that P188 is not only rapidly transported into the injured cells but co-localizes with the cell membrane. Such findings should provide a deeper insight into how FDA-approved P188 can be repurposed to treat bTBI.

Materials and Methods:: Methods: Mouse brain endothelial cells (BECs) were commercially purchased and cultured to produce confluent monolayers. A shockwave chamber has been utilized to produce shockwaves with a peak pressure of 12 MPa. The chamber created a lesion on the monolayer. The size of the lesion has been controlled by the input parameters of the shockwave generation [1]. Fluorescently conjugated P188 with Rhodamine 110 (10 mM) was added to the cell culture immediately following a blast. To visualize the cell membrane, it was stained with a fluorescent Membrite (Biotium, 488/515 nm) for 5 min. Co-localization of P188 (red) and the cell membrane (green) was identified by yellow color. After collecting fluorescent images, concentric rings in an increment of 50 mm radius were drawn from the center of traumatically produced lesion (Fig. 1). Total fluorescence intensity was calculated in each ring and normalized by its cell density. Fluorescent images were further analyzed to differentiate P188 that accumulated in the cytoplasm vs. P188 that was inserted into the cell membrane to reseal the membrane defects and pores induced by the simulated blast.

Results, Conclusions, and Discussions:: Results and Discussion: Using the shockwave chamber, a nearly circular central damage lesion of ~ 200 mm in size was created. Cells were completely detached from the lesion and presumed to be non-viable. While P188 uptake was observed both in control and shockwave-damaged cells, the uptake was greater in the cells that were located peripherally around the lesion. These cells were shown to be partially damaged and up-regulated apoptotic biomarkers. However, the cells located sufficiently away from the damage site still indicated some P188 uptake, but its accumulation was significantly lower, suggesting the transport mechanisms in undamaged viable cells are different. In fact, the P188 accumulation in the cells in the 6^th ring (i.e., 300 mm away from the lesion edge) begin to resemble that observed in the control cells (Fig. 1). Also, evidence of P188 resealing the damaged cell membrane of the cells around the lesion was shown by co-localization of P188 and Membrite (i.e., visualized in yellow color) and indicates the copolymer was likely inserted into the membrane defects and restored the membrane integrity.

Conclusion: FDA-approved P188 has demonstrated beneficial effects to repair injured brain endothelium. Both control cells and damaged cells were observed to internalize P188. However, the amount of P188 uptake in the injured cells was significantly elevated. Evidence is growing that P188 is inserted into the cell membrane to reseal the membrane defects and co-localizes with organelles (e.g., lysosomes) that are involved in the intrinsic cellular repair machinery. Based on these findings, a postulate is formulated that the reparative effects of P188 are not only confined to the cell membrane but also intracellular signaling pathways to promote restoration of the cell’s structure and function. Further studies are underway to determine multiple reparative mechanisms of P188 in response to bTBI. Finally, development of machine learning algorithms [2] is expected to accelerate the efforts to identify and validate crosstalk between intracellular signaling pathways that are involved regenerative tissue repair.






Acknowledgements (Optional): : Acknowledgement: Authors gratefully acknowledge financial support from the Office of Naval Research (N00014 21-1-2039) and from the Alfred R. and Janet H. Potvin Endowment.


References (Optional): : References:
1. Inyang, E., Abhyankar, V., Chen, B., and Cho, M. Sci. Reports. 2020
2. Alsup A, Fowlds K, Cho M, Luber J. BetaBuddy: An end-to-end computer vision pipeilne for the automated analysis of insulin secreting b-cells. Published online April 6, 2023. doi:10.1101/2023.04.06.535890.