Abstract
BackgroundImaging of the brain surface vasculature following inflammatory insults is critical to study structural and functional changes in the living brain under normal and pathological conditions. Although there have been published reports relating to the changes that occur in the blood brain barrier (BBB) during the inflammatory process, the ability to visualize and track such changes in vivo and over time has proven to be problematic. Different techniques have been used to achieve visualization of pial vessels, but the approach has limits, which can jeopardize the well-being of the animals. Development of the cranial window technique provided a major advance in the acquisition of live images of the brain vasculature and its response to different insults and treatments.MethodsWe describe in detail a protocol for delivery of a localized inflammatory insult to the mouse brain via a craniula (cranial window and adjacent cannula) and subsequent imaging of the mouse brain vasculature by intravital microscopy and two-photon laser scanning microscopy. The surgical implantation of the craniula can be completed in 30-45 min and images can be acquired immediately and for several months thereafter. The technique is minimally invasive and permits serial injections directly to the brain, thereby allowing longitudinal imaging studies. The craniula technique permits the study of structural and functional changes of the BBB following inflammatory insult and as such has wide application to neuroscience research.
Highlights
MethodsWe describe in detail a protocol for delivery of a localized inflammatory insult to the mouse brain via a craniula (cranial window and adjacent cannula) and subsequent imaging of the mouse brain vasculature by intravital microscopy and two-photon laser scanning microscopy
Imaging of the brain surface vasculature following inflammatory insults is critical to study structural and functional changes in the living brain under normal and pathological conditions
Applications of the procedure To demonstrate the utility of the craniula technique for delivery of an inflammatory insult and tracking subsequent changes in the brain vasculature without affecting other organs, we studied the effects of three poly(ADPribose) type 1 (PARP-1) inhibitors in response to release of a localized inflammatory stimulus (TNFα)
Summary
Animals We have followed the approach of the well-known cranial window technique for mice that has been described in detail by Zhang and colleagues [9, 18]. The first major variation from a regular cranial window technique is to create a 0.5 mm circular foramen for the IC cannula with a high-speed drill over the parietal bone, 0.1 mm posterior to bregma and 0.1 mm to the right of the sagittal suture (Fig. 2b). This is one of the most delicate steps in the procedure; in case of failure (drilling though the skull bone), the experiment should be discontinued.
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