Abstract
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.
Highlights
Longitudinal in vivo brain studies have become widely recognized as critical for progress in neuroscience
All experimental procedures in this study were approved by, and performed in accordance with the regulations established by the Animal Care Committee of the Sunnybrook Research Institute, which adheres to the Policies and Guidelines of the Canadian Council on Animal Care (CCAC) and meets all the requirements of the Provincial statute of Ontario, Animals for Research Act as well as those of the Federal Health of Animals Act, and the International Council for Laboratory Animal Science (ICLAS)
We found that using a scalpel blade to scrape the bone created micro-abrasions and chronic bleeding into or around the craniotomy
Summary
Extra vigilance ensured the drill bit never contacted the dura or pial surface At this point, room temperature saline was used to flush the cranial region. Our preference was not to cover the entire dorsal skull surface with dental cement, as some protocols suggest This requires a greater amount of skin to be removed and may produce greater tissue irritation from the chronic implant. Cyanoacrylate glue or surgical skin glue (Vetbond, 3M Animal Care Products, USA) was applied sparingly between the skin and cement well to anchor the skin onto the skull and prevent growth over the craniotomy (Fig. 1A–D). The cranial window was examined for glass coverslip integrity, purulent infection, cortical edema or deformation, translucent or opaque fibrotic tissue growth (i.e. wound healing), and general appearance of the pial vessels. Averaged values were normalized to the signal maximum in the pial surface to allow comparison between animals
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