Refinement of a chronic cranial window implant in the rat for longitudinal in vivo two–photon fluorescence microscopy of neurovascular function

Longitudinal studies using two–photon fluorescence microscopy (TPFM) are critical for facilitating cellular scale imaging of brain morphology and function. Studies have been conducted in the mouse due to their relatively higher transparency and long term patency of a chronic cranial window. Increasing availability of transgenic rat models, and the range of established behavioural paradigms, necessitates development of a chronic preparation for the rat. However, surgical craniotomies in the rat present challenges due to craniotomy closure by wound healing and diminished image quality due to inflammation, restricting most rat TPFM experiments to acute preparations. Long-term patency is enabled by employing sterile surgical technique, minimization of trauma with precise tissue handling during surgery, judicious selection of the size and placement of the craniotomy, diligent monitoring of animal physiology and support throughout the surgery, and modification of the home cage for long-term preservation of cranial implants. Immunohistochemical analysis employing the glial fibrillary acidic protein (GFAP) and ionized calcium-binding adaptor molecule-1 (Iba-1) showed activation and recruitment of astrocytes and microglia/macrophages directly inferior to the cranial window at one week after surgery, with more diffuse response in deeper cortical layers at two weeks, and amelioration around four weeks post craniotomy. TPFM was conducted up to 14 weeks post craniotomy, reaching cortical depths of 400 µm to 600 µm at most time-points. The rate of signal decay with increasing depth and maximum cortical depth attained had greater variation between individual rats at a single time-point than within a rat across time.