The Major and Minor Users for this application request funds to purchase a High Pressure Freezing (HPF) machine to carry-out ultrastructural studies for NIH-funded projects. The broad, long-term objective of this shared instrument is to improve the quality of tissue processing for electron microscopy (EM), in particular to improve the ultrastructure of mitochondria, ER, and other cellular components in brown adipose tissue (BAT), skeletal muscle, intestine, liver, and stomach. The requested instrument is the Wohlwend HPF, which was chosen for its flexibility, ease of use, excellent repair and service record, and outstanding performance. The machine will be placed into an existing EM facility, which is partially supported by the Beth Israel Deaconess Medical Center and partially supported by an active NIH P30 Center Grant for digestive diseases. The Major User group consists of 5 investigators with NIH-funded grants, one Minor User, who has an active biomedical research pro- gram, and dedicated time for outside users. The individual research goal for each user is different, but each requires improved tissue ultrastructure. Dr. Arany studies changes in skeletal muscle mitochondria in different models of metabolic pathophysiology. Drs. Cohen, Goodyear, and Tseng study the pathological significance of mitochondrial changes in BAT and/or liver, which relate to energy metabolism in obesity, exercise, or diabetes. Dr. Blumberg studies the role of intestinal Paneth cells in the pathogenesis of Inflammatory Bowel Disease, in particular the inflammation-induced activation of ER stress and the unfolded protein response. Lastly, Dr. Ha- gen studies the pathogenic role of tight junction abnormalities in stomach cancer pathogenesis. Preliminary data, collected with the assistance of an expert in the field who was also recruited as a consultant for this application, clearly demonstrate that HPF followed by super-quick freeze substitution methods resulted in significantly improved tissue morphology compared to conventional processing techniques. The overall benefit to the health-related goals of each project will be that HPF/SQFS limited cell shrinkage, mitochondrial swelling and disruption, and the extraction of important proteins in the mitochondrial matrix, lumen of the ER, nuclei, and cytoplasm. With the capability of the requested HPF machine to freeze thicker and larger tissue pieces, the user groups were able to prepare numerous thick plastic sections for analysis followed by numerous thin sections for EM analysis that were in the proper orientation and free of freezing damage. The differences in tissue ultrastructure using HPF/SQFS compared to conventional processing were so striking that the User group is reluctant to continue their studies with significant artifacts that cannot be fixed without the benefit of this machine. Administratively, the requested HPF will be managed by the EM Core Director and Research Associate, who will do the training, upkeep, and oversight of the instrument. There will be a local advisory committee to handle conflicts and new user applications. Lastly, there is strong institutional support for this instrumentation, with an appropriate budget, space, and financial commitment to ensure success.
| Palmer, Colin J; Bruckner, Raphael J; Paulo, Joao A et al. (2017) Cdkal1, a type 2 diabetes susceptibility gene, regulates mitochondrial function in adipose tissue. Mol Metab 6:1212-1225 |
