(J-367) Ultra-thin poly-dimethyl siloxane (PDMS) cranial windows for 2-photon imaging, drug delivery, and electrophysiology in awake mice.

Introduction: Soft, transparent poly-dimethyl-siloxane (PDMS) cranial windows allow for 2-photon imaging, intracortical injections, and implantation of microelectrode arrays (MEAs) [1]. Because thin and soft sheets of PDMS are difficult to handle during implantation, PDMS cranial windows are typically 300μm thick and have a Young’s Modulus of >2.4MPa [2]. These properties make it challenging to penetrate the window with fragile materials like pulled glass pipettes and MEAs. Furthermore, physiological motion artifacts, heterogeneous bubble formation under the PDMS window, and skull regrowth limit the lifetime of chronic imaging. Here, we present a methodology that allows for easy implantation of ultra-thin (< 100μm) and soft (~0.7MPa [2]) PDMS cranial windows using 3D-printed tools. This modified design reduces physiological motion artifacts, prevents bubble formation, and delays skull regrowth which will be beneficial to greatly improve studies involving 2p imaging combined with fluid delivery and/or MEA implantation. Specifically in the study of neural device interfaces, improving chronic tissue integration and performance of MEAs requires multi-modal strategies. This setup allows us to administer drugs to the tissue-device interface to explore pharmacological approaches for reducing the foreign body response to implanted neural interfaces.

Materials and Methods: Ultra-thin PDMS windows are fabricated by spin-coating wafers of Sylgard184 mixed at a 20:1 polymer-to-curing agent ratio. The resulting PDMS has a Young’s Modulus that is less than 1/3^rd that of more commonly used ratios like 10:1. A 3D-printed frame is designed to have an inner border that sits into the skull and an outer border that sits on top of the skull. A small sheet is cut from the wafer and adhered to the 3D-printed frame to assemble the window. The frame interferes with skull regrowth and makes the PDMS lay closer to the brain like an artificial dura. For implantation in mice, a 3mm-by-3mm square of the skull is removed over the right hemisphere. The frame + PDMS ensemble is positioned and depressed into the craniotomy using a stereotaxic arm. The edges are sealed with silicone and covered in dental cement. This provides sufficient room to install a metal frame for head-fixing awake mice. An additional 3D-printed insert acts as an “artificial skull flap” and prevents bubble formation under the window between imaging sessions. A removable, protective hardcover is installed over the window via screws embedded in the dental cement of the head cap. Flexible, MEAs are implanted using a tungsten shuttle through a pre-punctured hole in the window. The exiting interface is sealed with KwikSil for chronic implantation. Intracortical microinjections are delivered using pulled glass pipettes (pore size < 6μm) using a microsyringe infusion pump.


Results, Conclusions, and Discussions:: We have demonstrated that this cranial window design can maintain chronic imaging clarity to image neuronal calcium activity (Thy1-jRGECO1a) up to 450μm deep and microglia (CX3CR1-GFP) up to 250μm deep for at least 21 weeks. We also demonstrate that the soft and thin PDMS sheet enables intracortical virus/drug delivery after MEA implantation. The well created by the frame allows us to prevent and even reverse bubble formation under PDMS windows. Lastly, the frame design allows agarose to be used during imaging to reduce motion artifacts. With this setup, we can explore the effects of local drug delivery to the tissue-device interface with the ability to readily manipulate the concentration and timing of dosing. This will allow us to rapidly develop pharmacological strategies for reducing FBR that can later be administered through drug coatings or microfluidic devices.


Acknowledgements (Optional): This material is based upon work supported by the National Science Foundation Graduate Research Fellowship Program and the NIH BRAIN Initiative (Grant No. R01NS110564-01). Any opinions, findings, and conclusions or recommendations expressed in this material are those of the author(s) and do not necessarily reflect the views of the National Science Foundation or the NIH BRAIN Initiative.


References (Optional): [1] C. Heo et al., ‘A soft, transparent, freely accessible cranial window for chronic imaging and electrophysiology’, Sci Rep, vol. 6, Jun. 2016, doi: 10.1038/srep27818.

[2] J. Vaicekauskaite, P. Mazurek, S. Vudayagiri, and A. L. Skov, ‘Mapping the mechanical and electrical properties of commercial silicone elastomer formulations for stretchable transducers’, J Mater Chem C Mater, vol. 8, no. 4, pp. 1273–1279, 2020, doi: 10.1039/c9tc05072h.