Animals. VLCAD−/− mice were created by Exil et al. and purchased from Mutant Mouse Regional Resource Centers (MMRC, University of Missouri, Columbia, MO). VLCAD−/− was created by breeding VLCAD–/+ animals creating VLCAD−/− and VLCAD+/+ littermate controls. Mice were maintained in a pathogen-free facility with 12-hour light and dark schedule. Mice were fed PicoLab Mouse Diet 20 5058 (LabDiet, Richmond IN) until 2 weeks postinjection and then were placed on a standard chow. Mice received both diets and water ad libitum. Animals were fasted for 18–24 hours before bleeds, magnetic resonance spectrometry or cold fast challenge. For cold fast challenge, after the 18-hour fast, mice were singly housed in minimal bedding cages and placed in a 4 °C cold room for 150 minutes or until rectal temperatures were below 20 °C. Blood glucose levels were measured by Nova Max Glucose Monitor and Strips (Nova Biomedical, Waltham, MA).
All animal procedures were approved by University of Massachusetts Medical School Institutional Animal Care and Use Committee as well as University of Florida Institutional Animal Care and Use Committee in accordance with the Association for Assessment and Accreditation of Laboratory Animal Care International specifications.
. rAAV9/2 pseudotyped vectors were generated to express human VLCAD under the transcriptional control of the cytomegalovirus enhancer/chicken β-actin promoter.19
rAAV vectors were produced, purified and tittered as previously described (UMMS Gene Therapy Center, Worcester, MA).31
Genomic DNA extraction and quantitative-PCR
. DNA was extracted and quantified as previously described.32
Animals were sacrificed at indicated time points and tissues were harvested in a manner to avoid cross-contamination, snap frozen in liquid nitrogen and stored at −80 °C. gDNA was extracted using a DNeasy blood and tissue kit (Qiagen, Valencia, CA) according to the manufacturer's instructions. gDNA concentrations were determined using an Eppendorf Biophotometer (Eppendorf, Hamburg, Germany).
rAAV genome copies in the gDNA from heart, liver, tibialis anterior, and brown fat were quantified by quantitative-PCR with an ABI 7900 HT sequence detection system (Applied Biosystems, Carlsbad, CA) according to the manufacturer's instructions and results were analyzed using SDS 2.3 software. Briefly, primers and probe were designed to amplify SV40 poly-A tail of the rAAV9-CBA-VLCAD vector. A standard curve was generated using plasmid DNA containing the same SV40 poly-A target sequence. PCR contained a total volume of 100 µl and were run at the following conditions: 50 °C for 2 minutes, 95 °C for 10 minutes, and 45 cycles of 95 °C for 15 seconds and 60 °C for 1 minute.
DNA samples were assayed in triplicate. In order to assay PCR inhibition, the third replicate was spiked with plasmid DNA at a ratio of 100-copies/µg gDNA. If this replicate was greater than 40-copies/µg gDNA then the results were considered acceptable. If a sample contained greater than or equal to 100-copies/µg gDNA it was considered positive for vector genomes. If a sample contained less than 100 copies/µg gDNA it was considered negative for vector genomes. If less than 1 µg of gDNA was analyzed to avoid PCR inhibition, the vector copy number reported was normalized per µg gDNA and the plasmid spike in was reduced to maintain the ratio of 100-copies/µg gDNA.
Immunohistochemistry. Tissues were fixed in 10% neutral buffered formalin, and embedded in paraffin. Immunohistochemistry was carried out by the Molecular Pathology and Immunology Core at University of Florida using the DAKO Autostainer plus protocol. Briefly, 4 µm serial sections were removed of paraffin and incubated with 3% H2O2 in methanol to block endogenous peroxidase activity. Sections were treated with Trilogy (Cell Marque, Rocklin, CA) at 95 °C for 25 minutes and blocked with Sniper (Biocare Medical, Walnut Creek, CA) to reduce nonspecific background staining. Sections were incubated with rabbit anti-mouse VLCAD at room temperature for 1 hour. Then sections were incubated with Mach2 Gt × Rb HRP polymer (Biocare Medical) for 30 minutes. Staining was visualized with DAB chromagen (Biocare Medical).
. Blood acyl carnitines were extracted, derivatized, and analyzed as described previously.33
The following labeled carnitine standards (Cambridge Isotope Laboratories, Andover, MA) were used for quantification: l
) and l
). Blood acyl carnitine samples were analyzed as butyl esters on a Micromass Quattro II (Manchester, UK) with an electrospray ionization source operating in the positive ion mode by direct infusion. Tissue samples were processed as previously described.34
In brief, frozen tissue samples were ground into powder and resuspended in 80% acetonitrile containing carnitine standards described above at a concentration of 60 mg/ml. Samples were homogenized and centrifuged, supernatants were dried under nitrogen. Samples were then resuspended in 10% acetyl chloride in 1-butanol and heated at 65 °C for 15 minutes, dried under nitrogen and brought up in 80% acetonitrile for analysis. Samples were run in triplicate on a Waters (Milford, MA) Premier XE triple quadrupole mass spectrometer similarly to the blood samples.
. Single voxel 1
H-MRS was obtained from the tibialis anterior muscle and liver at 11.1T and 4.7T, respectively. 11.1T localized proton muscle spectra were obtained using a Bruker spectrometer (PV3) as described previously.9
A custom-made loop gap coil with 1.4-cm inner diameter tuned to 470.5 MHz was used. Localizer anatomical proton magnetic resonance images were acquired using FLASH (matrix, 256 × 256; echo time = 4.3 msec; repetition time = 207 msec, FOV = 20 × 20 mm2
) were acquired in three orthogonal directions for the precise localization of a volume of interest from which proton MRS would be obtained. Water suppression was optimized by chemical shift-selective water suppression. Localized spectra were acquired by point-resolved spatially localized spectroscopy (echo time = 18 msec; repetition time = 3,000 msec; number of excitations = 512, bandwidth = 8,013 Hz, number of complex points = 2,048). Data were processed using jMRUI (http://www.mrui.uab.es/mrui/mrui_Overview.shtml
). Similarly localized single voxel proton liver spectra were obtained at 4.7T using point-resolved spatially localized spectroscopy (echo time = 14 msec; repetition time = 2,500 msec; number of excitations = 16, bandwidth = 3,005 Hz, number of complex points = 8,196) on a Varian/Agilent spectrometer. Liver MRS was processed using in house software (IDL; ITT) in order to determine the integral of water and lipid resonances.
Statistics. All statistics were carried out using Prism 5 Software (GraphPad Software, San Diego, CA). All data are presented as means ± SEM. The n value designates number of animals used. Significant differences were determined by unpaired Student's t-test. When looking at the effect of two variables, such as reduction of acyl carnitines and time, or temperature and time, a two-way analysis of variance were used to determine significance. Differences were considered significant if P < 0.05.
Inflammation of liver postinjection.
Reduction of acyl carnitines in rAAV9-VLCAD with GFP control.
Acyl carnitine profiles over time in uninjected mice.
Representative video of VLCAD−/−
PBS control mouse after 18-hour fast and 120 minutes at 4 °C.
Representative video of VLCAD−/−
AAV9-treated mouse after 18-hour fast and 150 minutes at 4 °C.
Representative video of VLCAD+/+
PBS control mouse after 18-hour fast and 150 minutes at 4°C.