COBRE Core Facilities

ANIMAL MODELS AND HISTOLOGY CORE

Dr. Karen Stokes serves as the Overall Core Director and is responsible for the administrative and budgetary needs of the Animal Models Core. Dr. Hugh Price serves as the Leader of the Histology and Genotyping Subcore. This subcore genotypes the mutant mice for COBRE project leaders, and offers a centralized histology service with cardiovascular disease-specific stains. Furthermore, Subcore A serves as an information hub for sharing mouse strains and tissues. The Cardiovascular Phenotyping Subcore is led by Dr. Shenu Bhuiyan. This Subcore provides a centrally located, state-of-the-art, highly specialized laboratory capable of non-invasive monitoring of cardiac and vascular function, and telemetry in normal and disease rodent models. Although priority will be given to project leaders, Core services are available to all faculty at our institute.

Specific aims of the Animal Models Core are to:

1. To develop a core facility that complements the Redox Molecular Signaling Core by allowing project PIs to test the importance of redox modifications in relevant in vivo models of cardiovascular disease
2. To provide the investigators with a new genotyping service that will help streamline and improve the generation of animal models for their studies
3. To provide centralized histological serves to ensure high quality morphometric analysis of tissue in various pathological models.
4. To offer the technical capabilities and expertise necessary for investigators to design and perform studies using cutting edge technologies in vascular and cardiovascular phenotypic measurements, such as telemetry, ultrasound and laser speckle imaging.
5. To facilitate access to expertise for troubleshooting genotyping, histological and phenotyping difficulties.
6. To ensure the trainees of junior faculty are exposed to the latest methodologies in cardiovascular research, animal models, and cardiovascular histology and phenotyping.

Histology and Genotyping Subcore A:
The genotyping component of this subcore is located in animal resources (9th floor of the medical school building), and will genotype more than 2000 mice for the 3 COBRE projects, as well as mice for other investigators. This subcore also includes a centralized histology service with access to several cardiovascular-relevant histological stains. It will be incorporated into the current Morphometry Core Facility associated with the Department of Cell Biology and Anatomy, located on the 8th floor of the Medical School Building in Room 8-303 and occupying approximately 1,200 sq. ft. of laboratory space.

Cardiovascular Phenotyping Subcore B:
This subcore is in a dedicated suite (825 sq. ft.) of 4 rooms located on the 4th floor of the medical school building. The current modalities (telemetry, laser speckle imaging and ultra high frequency ultrasound) are housed in separate rooms of the suite. Ongoing renovation will allow for the inclusion of analysis workstations and a desk for the subcore research associate in close proximity to the technologies. The telemetry room also contains two cage racks to house animals being monitored by telemetry. Our animal facility is AAALAC accredited and is staffed by two veterinarians with formal training in Laboratory Animal Medicine. Animal resources will supply biosafety cage change stations, cages, autoclaves, and cage washer facilities as well as husbandry staff and veterinarian support. The freight elevator outside the suite of the phenotyping subcore provides easy access for animal transport and movement of husbandry supplies from Animal Resources Facility to the subcore and back to housing rooms/cage wash facility on the 9th floor.

REDOX MOLECULAR SIGNALING CORE

The Redox Molecular Signaling Core occupies 3,375 sq. ft. of laboratory space on the 3rd floor of the Medical School building and on the 6th floor of the adjacent Biomedical Research Institute. As part of the COBRE Center for Redox Biology and Cardiovascular Disease, this core is able to offer services at a significantly reduced rate to facilitate the study of redox regulation of cardiovascular disease at LSU Health – Shreveport. In addition, the COBRE grant provides money to expand this core facility over time, as discussed below. Currently, this core is divided functionally and spatially into two distinct sub-cores: the Analytic Redox Biology Sub-Core and the Molecular Signaling Sub-Core.

Analytical Redox Biology Sub-Core: The Analytic Redox Biology Sub-Core occupies space in Room F6-12 (690 sq. ft.) of the Biomedical Research Institute and Room 3-449 (1,074 sq. ft) in the adjacent Medical School Building. This core facility provides high quality, accurate measurements of reactive oxygen species, reactive nitrogen species, and reactive sulfide species in cell culture and tissue samples. High performance liquid chromatography (HPLC) systems coupled with UV-vis and fluorescence detectors are used to specifically quantify cellular and mitochondrial superoxide production, hydrogen sulfide pools (free sulfide, sulfide bound to transitional metals, and sulfane sulfur), and thiols (glutathione, GSH/GSSG, cysteine, cystine, homocysteine, persulfides, and glutathionylation). In addition, this core facility employs a versatile and highly sensitive Sievers NO Analyzer (NOA 280i, GE) capable of measuring nitric oxide, nitrite, nitrate, nitrosoheme, and nitrosothiols in a variety of biological samples. Lastly, this core utilizes a collection of mass spectrometery equipment, including a SYNAPT HDMS (Waters Corp.), a LCQ-DECA XP (Thermo), and a Q-TOF micro (Waters Corp.), to facilitate protein identification, identify post-translational modifications, and quantify fatty acid oxidation products.

Molecular Signaling Sub-Core: The Molecular Signaling Sub-Core is located on the 3rd floor of the Medical School Building in a bank of adjacent laboratories (3-236, 3-324, 3-326, 3-328, 3-330, 3-332, 3-334) occupying approximately 1,612 sq. ft. of laboratory space. The facility offers services for molecular cloning and site-directed mutagenesis, as well as design and production of vectors for CRISPR/Cas9 modification of vascular cells. Through the use of three dedicated cell culture rooms, this core provides services for endothelial, smooth muscle, and cardiac myocyte cell isolation, for generation of vascular cell lines, and for adenovirus and lentivirus production for transient or stable modification of cardiovascular cells. In addition, this core provides access to equipment and expertise for exposing vascular cells to hemodynamic forces using parallel plate flow chambers and access to equipment for the study of cellular effects of hypoxia/reoxygenation injury (Coy Hypoxic Chamber). As part of the ongoing COBRE grant, this core will purchase a hypoxia-capable CLARIOstar spectrofluorometer in 2020 to allow investigators to assess the cellular redox state over an extended, uninterrupted time course, including assessment of redox signaling during hypoxia and reoxygenation. In addition, this facility will purchase a ProteinSimple Wes Simple Western system in 2021 to provide investigators with an automated, high throughput analysis of changes in protein expression and function with reduced variability and improved quantitation compared to typical methods of manual Western blotting.