2-Photon imaging of fluorescent proteins in living swine.

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.

2-Photon imaging of fluorescent proteins in living swine.

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.

Robust, long-term video EEG monitoring in a porcine model of post-traumatic epilepsy.

To date, post-traumatic epilepsy (PTE) research in large animal models has been limited. Recent advances in neocortical microscopy have made possible new insights into neocortical PTE. However, it is very difficult to engender convincing neocortical PTE in rodents. Thus, large animal models that develop neocortical PTE may provide useful insights that also can be more comparable to human patients. Because gyrencephalic species have prolonged latent periods, long-term video EEG recording is required. Here, we report a fully subcutaneous EEG implant with synchronized video in freely ambulatory swine for up to 13 months during epileptogenesis following bilateral cortical impact injuries or sham surgery The advantages of this system include the availability of a commercially available system that is simple to install, a low failure rate after surgery for EEG implantation, radiotelemetry that enables continuous monitoring of freely ambulating animals, excellent synchronization to video to EEG, and a robust signal to noise ratio. The disadvantages of this system in this species and age are the accretion of skull bone which entirely embedded a subset of skull screws and EEG electrodes, and the inability to rearrange the EEG electrode array. These disadvantages may be overcome by splicing a subdural electrode strip to the electrode leads so that skull growth is less likely to interfere with long-term signal capture and by placing two implants for a more extensive montage. This commercially available system in this bilateral cortical impact swine model may be useful to a wide range of investigators studying epileptogenesis in PTE.SignificancePost-traumatic epilepsy (PTE) is a cause of significant morbidity after traumatic brain injury (TBI) and is often drug-resistant. Robust, informative animal models would greatly facilitate PTE research. Ideally, this biofidelic model of PTE would utilize a species that approximates human brain anatomy, brain size, glial populations, and inflammatory pathways. An ideal model would also incorporate feasible methods for long-term video EEG recording required to quantify seizure activity. Here, we describe the first model of PTE in swine and describe a method for robust long-term video EEG monitoring for up to 13 months post-TBI. The relatively easy "out-of-the-box" radiotelemetry system and surgical techniques described here will be adaptable by a wide array of investigators studying the pathogenesis and treatment of PTE.

Toxic expanded GGGGCC repeat transcription is mediated by the PAF1 complex in C9orf72-associated FTD.

An expanded GGGGCC hexanucleotide of more than 30 repeats (termed (G4C2)30+) within C9orf72 is the most prominent mutation in familial frontotemporal degeneration (FTD) and amyotrophic lateral sclerosis (ALS) (termed C9+). Through an unbiased large-scale screen of (G4C2)49-expressing Drosophila we identify the CDC73/PAF1 complex (PAF1C), a transcriptional regulator of RNA polymerase II, as a suppressor of G4C2-associated toxicity when knocked-down. Depletion of PAF1C reduces RNA and GR dipeptide production from (G4C2)30+ transgenes. Notably, in Drosophila, the PAF1C components Paf1 and Leo1 appear to be selective for the transcription of long, toxic repeat expansions, but not shorter, nontoxic expansions. In yeast, PAF1C components regulate the expression of both sense and antisense repeats. PAF1C is upregulated following (G4C2)30+ expression in flies and mice. In humans, PAF1 is also upregulated in C9+-derived cells, and its heterodimer partner, LEO1, binds C9+ repeat chromatin. In C9+ FTD, PAF1 and LEO1 are upregulated and their expression positively correlates with the expression of repeat-containing C9orf72 transcripts. These data indicate that PAF1C activity is an important factor for transcription of the long, toxic repeat in C9+ FTD.