2-Photon imaging of fluorescent proteins in living swine
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Abstract
A common point of failure in translation of preclinical neurological research to successful clinical trials comes in the giant leap from rodent models to humans. Non-human primates are phylogenetically close to humans, but cost and ethical considerations prohibit their widespread usage in preclinical trials. Swine have large, gyrencencephalic brains, which are biofidelic to human brains. Their classification as livestock makes them a readily accessible model organism. However, their size has precluded experiments involving intravital imaging with cellular resolution. Here, we present a suite of techniques and tools for in vivo imaging of porcine brains with subcellular resolution. Specifically, we describe surgical techniques for implanting a synthetic, flexible, transparent dural window for chronic optical access to the neocortex. We detail optimized parameters and methods for injecting adeno-associated virus vectors through the cranial imaging window to express fluorescent proteins. We introduce a large-animal 2-photon microscope that was constructed with off-the shelf components, has a gantry design capable of accommodating animals > 80 kg, and is equipped with a high-speed digitizer for digital fluorescence lifetime imaging. Finally, we delineate strategies developed to mitigate the substantial motion artifact that complicates high resolution imaging in large animals, including heartbeat-triggered high-speed image stack acquisition. The effectiveness of this approach is demonstrated in sample images acquired from pigs transduced with the chloride-sensitive fluorescent protein SuperClomeleon.
Article activity feed
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Two burr holes displaying the PDMS overlaying the cortex immediately after AAV injection and the pristine window 1 month later
These images are an impressive proof of principle! I'm wondering, why is the color of the 1 month post-op tissue different than that at the time of surgery? Is this a difference in lighting, discoloration of the tissue, from fluorescent protein expression, or something else? It also looks to me like the large vasculature observed during surgery does not appear the same post-operatively, why is this the case?
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Cranial imaging window
I'm very impressed by the technical challenges surmounted in order to develop and implement this challenging preparation and imaging. I think this demonstration would be even stronger if there was a quantification of success rate and post-operative tissue health.
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Rapid and shallow (1mm depth) injection of SClm-AAV and other AAV vectors successfully transduced of the cortex with wide-spread SClm expression without damage to the cortex
This is fantastic! A quantitative comparison of the efficacy of transduction vectors would be very useful. In addition, histology with some marker(s) of cortical damage could help demonstrate the (lack of) tissue damage using this methodology.
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Rapid and shallow (1mm depth) injection of SClm-AAV and other AAV vectors successfully transduced of the cortex with wide-spread SClm expression without damage to the cortex
This is fantastic! A quantitative comparison of the efficacy of transduction vectors would be very useful. In addition, histology with some marker(s) of cortical damage could help demonstrate the (lack of) tissue damage using this methodology.
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Cranial imaging window
I'm very impressed by the technical challenges surmounted in order to develop and implement this challenging preparation and imaging. I think this demonstration would be even stronger if there was a quantification of success rate and post-operative tissue health.
-
Two burr holes displaying the PDMS overlaying the cortex immediately after AAV injection and the pristine window 1 month later
These images are an impressive proof of principle! I'm wondering, why is the color of the 1 month post-op tissue different than that at the time of surgery? Is this a difference in lighting, discoloration of the tissue, from fluorescent protein expression, or something else? It also looks to me like the large vasculature observed during surgery does not appear the same post-operatively, why is this the case?
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