Combining in vivo two-photon microscopy with ex vivo pressure myography as a novel approach to assess neurovascular coupling
Authors: Evans Lowri , Gray Anna , Schiessl Ingo , Pritchard Harry , Allan Stuart , Greenstein Adam 
Published: May 2023

The brain’s vascular network maintains cerebral blood flow and crucially, responds neuronal demands by rapidly altering local perfusion. In vivo techniques using cranial windows can visualise this neurovascular communication and measure real-time blood flow changes. In turn, the ex vivo capillary-to-parenchymal-arteriole preparation (CaPA Prep) can examine the mechanisms of signalling between capillaries and arterioles in response to physiological or pharmacological stimuli, and study these in health and disease models. In order to promote the 3R’s of animal research, and reduce the number of animals required in studies of neurovascular function, we wished to examine whether vessels from mice which have undergone cranial window surgery are comparable to vessels from mice without cranial window surgery by using the ex vivo CaPA Prep.C57 mice underwent craniotomies to implant chronic cranial windows and were left to recover. In order to examine capillary-to-arteriole signalling, we utilised the CaPA Prep, where parenchymal arterioles (<45μms) with attached capillary branches were isolated and pressurised. The capillary branch and arteriole are independently stimulated via a micropipette and the arteriole diameter is recorded. This approach enables study of multiple ion channels integral to neurovascular coupling to be assessed.Cranial window implantation did not significantly affect the function of cerebral vessels, compared to control C57 mice. Mice underwent craniotomies and were left to recover for up to 8 weeks before euthanization. Parenchymal arterioles with attached capillary branches were pressurised to 40mmHg. No significant difference in tone was observed between control and implanted animals (32.7 ± 4.7 vs 29.7 ± 2.57, N = 4-6, P= .56). We next examined the capillary-to-arteriolar KIR2.1 signalling mechanism via application of 10mM K+ to the capillary branch. Responses were not significantly different between control and implanted animals (68.1 ± 13.2 vs 75.2 ± 8.4, N = 4, P= .66) and KIR2.1 function via directly stimulating the arteriole was also not significantly different (74.7 ± 9.4 vs 60.7 ± 5.3, N = 4, P= .24). Arteriole responses to NS309 (3mM) and 60mM K+ were also not significantly different between groups (73.5 ± 11.51 vs 104.8 ± 19.6, N = 4-5, P= .23, 60.6 ± 5.5 vs 64.9 ± 5.5, N = 4-5, P= .59, respectively).Moreover, our preliminary data suggests that in vivo imaging in mice with cranial windows also has no effect on vascular function in relation to pressure induced tone, KIR2.1 capillary-to-arteriolar signalling and responses to NS309 and 60mMK+. Overall, our data suggests cranial window implantation and in vivo imaging preserves vascular function and these mice can be utilised for ex vivo analysis of vessel physiology. Minimising the number of animals avoids the need for additional animals for different techniques, and maximises the data obtained per animal. British Heart Foundation This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.