Cranial window implantation in rats and mice

The animals were anaesthetized in an induction chamber with 3.0% isoflurane (Hana Pharm., South Korea). They were supplied with pure O₂ during anaesthesia (VetEquip) for 1–2 min, and then maintained with 2.0% of isoflurane during surgery for the cranial window implantation and 1.3% isoflurane during imaging. The level of anaesthesia was assessed through toe pinching during surgery and experimentation. The animals’ body temperatures were maintained at 36.5–37.5 °C using a heating pad connected to a controller (DC temperature controller, FHC, USA), and their eyes were covered with eye protecting gel (Solcorin, Solco Basie Ltd). During experimentation, heart rate (mouse = 300–450 beat/min, rat = 300–400 beat/min) and O₂ saturation (SpO₂; mouse >95%, rat >99%) were monitored using a pulse oximeter (Rat: Nonin Medical, USA and mouse: Kent Scientific, USA). Their heads were firmly fixed with ear bars in a stereotaxic frame (David Kopf Instruments, USA). An incision was carefully made over the skin of the right hemisphere (Fig. 2A for rats). The remaining epidermis and debris were removed with isopropyl alcohol and betadine liquid swabs. For the rat surgeries, a craniotomy (3 mm above bregma and 6 mm below bregma with a width of 4 mm) was performed through careful drilling using a dental drill (MICROTORQUE II, NJ, USA). After craniotomy, the dura mater was quickly removed with fine forceps. The exposed brain tissue was subsequently covered with PDMS (size = 6 mm × 11 mm, thickness = approximately 0.4 mm), and the edge of the PDMS was immediately glued onto the skull using cyanoacrylate glue (Loctite, USA). When air was trapped between the cortical tissue and PDMS, the air bubbles were removed by pushing ACSF solution into the space between the cortical tissue and the PDMS using a syringe. For the mouse surgeries, we implanted a PDMS cranial window and used a head-fixing stereotaxic frame for two-photon microscopy. To secure the head-fixing frame onto the skull, one microscrew (1.6 mm, Plastics one, USA) was embedded in the olfactory bulb site and another microscrew was embedded in the contralateral hemisphere. We then made an outline of a circle with a 4–5 mm inner diameter over the somatosensory area, between the bregma and lambda, and the head-fixing square frame (size = 12 mm ×19 mm ×1 mm, Narishige, Japan) was secured to the sketched circle using cyanoacrylate glue. Immediately, we carefully cut along the circle line to remove the skull using a dental drill. The sterilized PDMS was prepared and soaked in saline solution. Subsequently, a sterilized PDMS film (circle with a diameter of 6 mm and thickness of 0.25 mm–0.35 mm) was applied to fully cover the exposed circle-shaped brain tissue, and the boundary of the PDMS was glued onto the skull. We waited approximately 10 min to allow for drying. After tight sealing of the PDMS onto the skull using cyanoacrylate glue in both rats and mice, a dental resin (OA2, Dentist Inc., South Korea) was applied along the edges of the PDMS and exposed to UV light for 10 s to ensure permanent implantation within the skull (steps showed in Fig. 2). The remaining incision area was sutured and disinfected with antiseptic liquid. The animals were administered Meloxicam (1 mg/kg, Boehringer Ingelheim, Germany) and Baytril (5 mg/kg, Bayer, Germany) via subcutaneous injection and returned to their cages. Subsequently, Tylenol (1 ml/10 ml, Janssen, USA) was administered to ameliorate any potential pain, and Septrin (1 mg/ml, Samil Pharmaceutical Company, South Korea) was provided in the drinking water to relieve inflammation. A video showing a freely moving mouse with a PDMS cranial window is shown in the supplementary Video S3 (25 weeks). Before each imaging session, 70% Ethanol or distilled water was used for PDMS cleaning. Transparent PDMS surface becomes opaque by Acetone. In some cases, air bubbles can be found inside PDMS window (between the PDMS and cortex). These bubbles usually disappear naturally in 2–5 weeks post-surgery. In very rare cases, it takes longer than 5 weeks for the bubbles to disappear. So far, we have conducted PDMS cranial window surgeries on over 130 animals including rats (n = ~30) and mice (n = ~100). Out of over 130 animals with the implantation, only one male mouse broke the whole window frame with dental resin 15 months post-implantation. Furthermore, less than 10% of the rats and 5% of the mice showed inflammation inside the window. The percentage of successful animal preparations for imaging is at least as high, if not higher, than the glass window preparation method.