The experiments were approved by our Institutional Animal Care and Use Committee. As a preclinical trial, surgery was performed in order to perform CED into the striatum of five purposely bred sheep (mean weight 26.5 kg). The animals were anesthetized by intramuscular injection of acepromazine (0.05 mg/kg), buprenorphine (0.01 mg/kg), glycopyrrolate (0.01 mg/kg) and thiopental (15.0 mg/kg) and intravenous injection of ketamine (3–6 mg/kg) and diazepam (0.1–0.3 mg/kg). Anesthesia was maintained during the entire procedure. Ventilation was performed mechanically with 2% isoflurane in oxygen. Physiological monitoring including heart rate, blood pressure, arterial oxygen saturation, temperature, end tidal CO2 and blood gases was performed and recorded every 15 min during the procedure.
Prior to surgery, MRI was performed on a 3 Tesla whole body MRI scanner (Achieva, Philips Healthcare) using an eight-channel knee coil to obtain subject-specific data. Multislice T1- and T2-weighted turbo spin echo (TSE) and 3D magnetization prepared rapid gradient echo (MPRAGE) images were acquired with a field of view of 128×128×80 mm3
, an in-plane resolution of 1×1 mm and slice thickness of 2 mm. The total imaging time was approximately 25 min. Upon completion, the animals were transferred to the adjacent angiography suite and prepared for sterile surgery. They were secured on the non-radio-opaque table with a surgical beanbag. A non-invasive frame designed to hold and manipulate the access sheath was mounted onto the animal's skull. A single incision and burr hole (0.5 cm diameter) were created approximately 1 cm anterior of the coronal suture and 1 cm mediolateral to the sagittal suture. A metal sheath was inserted into the manipulator with the distal end touching the dura mater. CBCT data were acquired for surgery planning and navigation using an angiographic c-arm system (Allura FD20, Philips Healthcare). Preoperative MRI data were imported into the corresponding work station and registered with CBCT data. The path of the CED microcannula was planned on merged datasets using XperGuide. This dedicated navigation software provides real-time guidance of radio-dense medical devices by fluoroscopic imaging shown relative to 3D multimodal image data and the planned path in a single view. Fluoroscopic data were updated in conjunction with every adjustment of the manipulator in order to see the effects in relation to 3D image data and the planned path. By alternating between the entry view (ie, parallel to the planned path) and progression view (ie, perpendicular to the planned path), both automatically calculated and provided by navigation software, the sheaths were readily aligned with the planned paths. The cannula with a distal tip length of 3.0 mm and outer diameter of 300 μm was introduced and progressed towards the end position without fluoroscopic guidance because the cannula tip was not visible with x-ray imaging. Instead, progression depth measured from the dura mater during the planning phase was applied. The end position of the cannula tip was validated by acquisition of high resolution CBCT data (XperCT) using a 22 cm detector size and an unbinned reconstruction algorithm.19
High resolution CBCT was registered to T2w-TSE data to confirm the position of the cannula tip and, if necessary, the cannula was progressed by distance to the target measured on the fused image data.
In order to visualize the approximate spread of the injected substance with MRI, the therapeutic compound was mixed with 2.0 mmol/l gadolinium (Gd). The mixture was infused at a rate of 0.3 μl/min for a total of 100 μl using a syringe pump (PHD 2000 Infusion, Harvard Apparatus, Holliston, Massachusetts, USA). After infusion, the skull and wound were closed and the animal was transferred to the MRI system where the same image data were acquired as before surgery. Upon completion, the animals were recovered.