Principal Investigator: ZHEN YAN · 2016-12-15 · Principal Investigator: ZHEN YAN PI Department...

Principal Investigator: ZHEN YANPI Department (LDAP, UVa display dept.): Department of Medicine, Cardiovascular MedicineProtocol Title: Skeletal muscle plasticity and its impact on catabolic muscle wasting and type 2 diabetesProtocol Number: 3762-02-15Protocol Submittal Type: Modification only

Cover Letter provided by person who submitted protocol

Date letter (protocol) was submitted: 06/23/2016Letter was submitted by: YAN, ZHEN (zy3m)Dear Committee,

I am writing to request a modification without adding mice. I request to perform introvital imaging of some of the mice wehave in our protocol to allow for imaging in FDB muscle that has been transfected with reporter gene. This will enhancethe significance of the finding. Mice will be euthanized immediately after the imaging acquisition. Please let me know ifyou have any questions and concerns. I look forward to your feedback.

Warm regards,

Zhen Yan

Date letter (protocol) was submitted: 07/22/2016Letter was submitted by: YAN, ZHEN (zy3m)Dear Committee,

Thank you very much for your careful review of our request for modification of the protocol. I have made the changesaccording to your suggestions. Please let me know if you have any questions.

Thank you,

Zhen

SUMMARY OF SPECIES PROCEDURES

Species Procedure # 1: Exercise in pregnancy in prevention of diabetes Species: Mice Animal Handler(s): FISHER, CARLEIGH (ccf2vj) - Health Status: OK FOR WORK , Must return by: 03/09/17 (982-4477) WILSON, REBECCA (rjw7jc) - Health Status: OK FOR WORK , Must return by: 01/12/17 (982-4477) ZHANG, MEI (mz3a) - Health Status: OK FOR WORK , Must return by: 01/14/17 (434-982-4473)

Species Procedure # 2: Exercise on diet-induced insulin resistance Species: Mice Animal Handler(s): CUI, DI (dc5hh) - Health Status: OK FOR WORK , Must return by: 09/29/17 (982-4473) HARRISON, LOGAN (lwh9dp) - Health Status: OK FOR WORK , Must return by: 06/08/17 (982-4473) OSINSKI, VICTORIA (vo3sc) - Health Status: OK FOR WORK , Must return by: 01/23/17 (982-0879) WILSON, REBECCA (rjw7jc) - Health Status: OK FOR WORK , Must return by: 01/12/17 (982-4477) ZHANG, MEI (mz3a) - Health Status: OK FOR WORK , Must return by: 01/14/17 (434-982-4473) ZHAO, HENAN (hz4p) - Health Status: OK FOR WORK , Must return by: 09/29/17 (982-4473)

Species Procedure # 3: Exercise-induced mitochondrial biogenesis Species: Mice Animal Handler(s): CUI, DI (dc5hh) - Health Status: OK FOR WORK , Must return by: 09/29/17 (982-4473) DRAKE, JOSHUA (jcd6g) - Health Status: OK FOR WORK , Must return by: 07/05/17 (982-4473) FISHER, CARLEIGH (ccf2vj) - Health Status: OK FOR WORK , Must return by: 03/09/17 (982-4477) HARRISON, LOGAN (lwh9dp) - Health Status: OK FOR WORK , Must return by: 06/08/17 (982-4473) SVINDRYCH, ZDENEK (zs4d) - Health Status: OK FOR WORK , Must return by: 06/06/17 (982-4869) WILSON, REBECCA (rjw7jc) - Health Status: OK FOR WORK , Must return by: 01/12/17 (982-4477)

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mailto:[email protected]















WILSON, REBECCA (rjw7jc) - Health Status: OK FOR WORK , Must return by: 01/12/17 (982-4477) ZHANG, MEI (mz3a) - Health Status: OK FOR WORK , Must return by: 01/14/17 (434-982-4473) ZHAO, HENAN (hz4p) - Health Status: OK FOR WORK , Must return by: 09/29/17 (982-4473)

Species Procedure # 4: NO protection against catabolic muscle wasting Species: Mice Animal Handler(s): LEITNER, LUCIA (lml8j) - Health Status: OK FOR WORK , Must return by: 04/15/17 (982-4473) WILSON, REBECCA (rjw7jc) - Health Status: OK FOR WORK , Must return by: 01/12/17 (982-4477) ZHANG, MEI (mz3a) - Health Status: OK FOR WORK , Must return by: 01/14/17 (434-982-4473) ZHAO, HENAN (hz4p) - Health Status: OK FOR WORK , Must return by: 09/29/17 (982-4473)

Principal Investigator: YAN, ZHENDept (ACUC for billing):MEDICINE-CARDIOVASCULAR RESEARCH CTPO Box: 801394 E-Mail: [email protected] Phone: 434-982-4477Fax: 434-982-3139Home Phone: 919-968-1938

Contact Person: ZHANG, MEIDept (ACUC for billing): UNKNOWNPO Box: UNKNOWNE-Mail: [email protected] Phone: 434-982-4473Fax: 434-982-3139Home Phone: 919-242-5328

Protocol Identification: RESEARCH

Current Funding Source Status Dates DescriptionDHHS/NIH ACTIVE 7/1/2011-6/30/2016; 4/1/2015-1/31/2019 N/AFOUNDATION ACTIVE 12/15/14 – 12/14/16 Friedreich's Ataxia Research AllianceUVA DEPARTMENTAL ACTIVE 1/1/2009-Continuous N/A

Agency Grant Numbers & Titles: NIH 2R01AR050429-07 NIH R01GM114840

FARA 183

Protocol Abstract: Mammalian skeletal muscle is remarkably plastic such that alteration in its usage, hormonal changes, or systemicdiseases can induce profound functional and structural changes. On the other hand, skeletal muscle function hassignificant impact on disease conditions. Research in this laboratory focuses on the mechanisms by which skeletalmuscle undergoes beneficial changes (adaptation) in response to exercise (repeated contractile activity and undergoesabnormal changes (maladaptation) under pathological conditions and the impacts on other tissues/organs and viceversa. We will also test whether a physiological model of resistance training would improve muscle mass, strength,mitochondria, capillary density and fiber type composition. Specifically, we are interested in three interrelated questions:

1) Exercise-induced mitochondrial biogenesis. In the last period of three years, we have found that exercise trainingpromotes mitochondrial biogenesis through activation of the p38 MAPK- PGC-1alpha regulatory axis and promotesmitophagy. We plan to study the importance of p38 MAPK isoforms in skeletal muscle adaptation and their roles indiet-induced insulin resistance and catabolic muscle wasting induced by sepsis. We plan to address the regulation andimportance of autophagy genes in exercise training-induced muscle adaptation and their impact in diet- induced insulinresistance. In addition, we have secured new funding to study the impact of exercise on cardiac and skeletal musclesin a mouse model of Friedreich’s Ataxia, a human genetic disorder due to loss of frataxin expression and impairedmitochondrial function as a result of abnormal insertion GAA repeats in the intron of the frataxin gene. Based on aprototype cage the PI has developed during his previous employments at UT Southwestern and Duke, we will buildcustom-made cages with a lever plate for mice to push against weight to get food as a voluntary weight lifting model totest if this will induce adaptations that are normally seen in humans in response to resistance exercise training.

2) NO protection against catabolic muscle wasting. We have in the last three years shown that NO-dependentantioxidant defenses in skeletal muscle can provide profound protection against cardiac cachexia (loss of lean bodymass due to catabolic muscle wasting) and diabetic cardiomyopathy, a heart failure condition induced by diabetes. Weplan to use cecal ligation puncture (CLP) and parabiosis in combination with genetic models in mice to dissect the

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mechanism by which EcSOD, which is induced by nitric oxide in skeletal muscle, protects against multiple organdysfunction syndrome (MODS).

3) Exercise in pregnancy in prevention of diabetes. In the last three years, we have shown that maternal exerciseprevents hypermethylation of the Pgc-1alpha gene in offspring skeletal muscle, preventing age-dependent metabolicdysfunction. We plan to use powerful molecular genetics combined with diet- induced paternal and maternal obesityand exercise training to further dissect the mechanisms.

In summary, we will employ models of physiology and pathology in genetically engineering in mice in combination withthe state- of-the-art technologies, such as in vivo imaging to improve the understanding of the molecular and signalingmechanisms underlying physiological adaptation and pathological maladaptation in skeletal muscle and their impact onhealth and diseases.

Animal Use Justification: Muscle plasticity and its impact on health are not simple muscle cell phenomena. It involves complicated cell-to-celland organ-to- organ interactions. I have studied muscle plasticity for more than 25 years. An in vitro model systemsuitable for long-term treatment mimicking the effects of exercise training and impact on chronic diseases does notexist. Neither does a model exist for studying the impact of a chronic disease on muscle function in an integrativemanner similar to humans. The major reason is that the complex architecture and diversity of physiological inputsacting upon intact skeletal muscles can not be simply replicated in a model system like cell culture, tissue culture orcomputer modeling at this point. Mice are excellent models for this type of work because (1) they are mammals that areeasily housed and handled; (2) various exercise and disease models have been well established; (3) many of theexperimental procedures are well developed ones in the mice; for example, Parabiosis is the surgical act of artificiallycreating conjoined twins of two animals. This experiment has been performed on mice for the physiological study ofhow internal bodily secretions (such as hormones) affect organisms precisely as the blood is shared; (4) thephysiological and histological data describing muscle adaptation and maladaptation are well- established andreproducible (in our lab), making the studies very feasible; and (5) a variety of well-defined and well-characterizedinbred genetic strains of mice exist for genetic mapping experiments. There is currently one muscle hypertrophy model that is near physiological: ladder climbing. This model requiresresearchers to attaching weights to mice by the tails and train mice individually to climb a 2-meter ladder to get food. Itis extremely lab intensive. Human manipulation makes this model not physiological completely. Other models ofhypertrophy using surgical ablation of synergistic muscles are prone of injury and inflammation. The PI has developedthe prototype weight lifting cage during my time UT Southwestern Medical Center and Duke University. This is aprotocol that does not need surgery or human presence. Mice will push against a lever plate to get food. There havebeen no adverse effects observed. Mice will be provided with water all the time. Food will be put in the cage after eachnight to ensure plenty food consumption. This may well be the first true physiological model of resistant exercise.

Alternatives to the Use of Animals REPLACEMENT: REPLACEMENT: We have always tried to use non-animal techniques whenever possible, such as C2C12 cell culture,to address some questions and test the feasibility before we use animal models. We are conscious that theseexperimental approaches not only over-simplify the system, but also fail to accurately reflect the complicatedinteractions that occur during skeletal muscles phenotypic changes. In addition, we have used and will continue to useDrosphilla (fly) model to address some of the questions related to autophagy in indirect flight muscle and the heart.Unfortunately, the processes that exercise involve complex interactions among various cells, tissues and organs. Theuse of animal models is essential to appropriately reflect the complexity of events that occur during muscle adaptationand maladaptation.

REDUCTION: REDUCTION. We strike to perfect our assays and analyses for the in vivo physiological and histological data toimprove reproducibility and to minimize the number of animals used. We have used and will continue to use statisticalanalysis diligently to minimize the number of animals needed to reach meaningful conclusions for the studies.

REFINEMENT: REFINEMENT) Pain and distress will be minimized by adhering to the IACUC and Federal Guidelines relating tosurgical procedures, anesthesia and analgesic use. Mice will be monitored for pain and distress following surgery, andappropriate medication will be administered.

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PI Assurance for NON-Duplication:As Principal Investigator, I have reviewed the literature relevant to my area of investigation, and am providing myassurance that the animal studies proposed in this protocol do not unnecessarily duplicate previous published orunpublished studies.

DEA Controlled Substances:Will any DEA Controlled Substances be given to LIVE ANIMALS in this protocol? YES

NOTE: DEA Controlled Substances must be securely locked in a manner that cannot be easily removed, and up-to dateusage logs (including inventory, amount used and balance remaining) must be maintained.

Location(s) of DEA Controlled Substances: MR-4: 6031 Molecular Imaging Core (MIC):Will you utilize the MIC in this Protocol? YES

Radioactive Materials: YES, Authorized User Number Selected in this Protocol: 0416.Name registered to Authorized User Number (0416): HOEHN, KYLE.

Hazardous Chemicals:Will any chemicals be administered to LIVE ANIMALS in this protocol that are carcinogenic, mutagenic,teratogenic or otherwise toxic to humans or other animals? YES

Animals with Induced or Spontaneous Genetic Mutations (Transgenic):Does this research involve the creation (de novo generation by gene manipulation) or generation (creation ofnovel transgenics by breeding two transgenic lines) of transgenic animals (animals in which the germ line isaltered by genomic DNA insertion or removal)? YES

Animals with Induced or Spontaneous Genetic Mutations (Mutations):Does the animal have a genetic mutation (either induced or spontaneous) that will lead to a phenotype whichadversely impacts its health or well-being? YES

Social Housing:Does the nature of your research require an exemption from Social Housing? YES

Environmental Enrichment (other than social housing):Does the nature of the research require an exemption from Environmental Enrichment? YES

SPECIES PROCEDURE # 1

Species: MiceProcedure ID: Exercise in pregnancy in prevention of diabetes

USDA Pain and Distress Category:B: 54 Animals/YearC: 400 Animals/YearD: 0 Animals/YearE: 0 Animals/Year

Anticipated maximum number of animals per year: 454 Justify the Number of Animals Needed: We have obtained data that exercise in pregnancy render protection in offspring against diet-induced type 2 diabetes.We have also obtained data to show that this protection may be related to hypermethylation of the PGC-1alpha gene inthe liver. To further the study, we plan to 1) test if paternal and maternal high-fat diet can induce hypermethylation ofPgc-1alpha equally in the offspring and lead to increased susceptibility to insulin resistance and whether maternalexercise during pregnancy will mitigate these abnormalities; 2) test if overexpressed Pgc-1alpha gene and deletion ofthe Pgc-1alpha in skeletal muscle lead to increased and decreased susceptibility to develop insulin resistance,respectively; 3) conduct a studies to have the offsping with either wild type or deleted DMNT3b gene that isresponsible for demethylating the PGC-1alpha gene. Breeding colonies Three (3) genetic mouse lines: Myogenin-Cre (Dr. Olson), MCK- Pgc-1alpha (Dr. Spiegelman) andfloxed DMNT3b (We have rederived the mice from UNC) Nine (9) mice per line: 3 male and 6 female per line (1 maleand 2 female per breeding cage) Two (2) generations per year Total: 3 lines x 9 breeding mice x 2 generations = 54mice

Study 1. Maternal exercise on offspring's susceptibility to develop insulin resistance from male or female dams onhigh-fat diet

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We have previously obtained comprehensive data indicating that maternal exercise during completely blocks obesepregnancy- induced hypermethylation of the Pgc-1alpha gene and age- dependent insulin resistance in the offspring.However, we do not know if paternal high-fat diet can induce hypermethylation of Pgc-1alpha equally in the offspringand lead to increased susceptibility to insulin resistance and whether maternal exercise during pregnancy will mitigatethese abnormalities. We now propose to study the impact of maternal exercise during pregnancy with male dam onhigh-fat diet.

Paternal diet and maternal exercise Five (3) groups of mice: Normal pregnancy control (NC), PaternalHigh-fat-Sedentary (PHF-Sed), Paternal High-fat-Exercise (PHF- Ex). Note maternal high-fat diet-exercise studies havebeen done. Breeding mice: Four (4) female mice and four (4) male mice per group. Total: 3 groups x 8 mice = 24 mice

Insulin resistance in the offspring Three (3) groups: Offspring from above mentioned groups. Two (2) genders: Maleand Female Based on the glucose tolerance test data from our previous experiment (MHF-Sed 31,097 mg/dl/min vs.MHF-Ex 22,333 mg/dl/min) and the variation (SD = 6,368), we need n = 13 to achieve statistical significance with analpha=0.05 and power = 0.9 (www.biomath,info/power/ttest.htm). Total: 3 groups x 2 genders x 13 per group = 78 mice

Study 2. Muscle Pgc-1alpha in maternal exercise-mediated prevention the transmission of susceptibility to developinsulin resistance from female dams on high-fat diet

We have previously obtained comprehensive data indicating that maternal exercise during completely blocks obesepregnancy- induced hypermethylation of the Pgc-1alpha gene and age- dependent insulin resistance in the offspring.However, we do not know altered Pgc-1alpha expression is causal to the to changes in susceptibility to insulinresistance. We now propose to use molecular genetics to dissect the mechanism.

Maternal exercise set up Three (3) groups of dams: Normal pregnancy control (NC), Maternal High-fat-Sedentary(MHF-Sed), Maternal High-fat- Exercise (MHF-Ex). Two (2) genetic genetic breedings: Transgenic Pgc-1alphoverexpression (TG) and muscle-specific Pgc-1alpha knockout (MKO) with wild mice from the same litters in the sameuterus. Breeding mice: Four (4) female mice and four (4) male mice per group. Total: 2 genetic breedings x 3 groups x8 mice = 48 mice

Insulin resistance in the offspring Six (6) groups: Offspring from above mentioned groups with either TG, KO or WTbackground. Two (2) genders: Male and Female Based on the glucose tolerance test data from our previousexperiment (MHF-Sed 31,097 mg/dl/min vs. MHF-Ex 22,333 mg/dl/min) and the variation (SD = 6,368), we need n =13 to achieve statistical significance with an alpha=0.05 and power = 0.9 (www.biomath,info/power/ttest.htm). Total: 6groups x 2 genders x 13 per group = 156 mice

Study 3. The role of DNA methyltransferase 3b (DMNT3b) in exercise-mediated epigenetic regulation by maternalexercise during pregnancy. Recent studies have shown that diabetic patients or cells treated with saturated fatty acidleads to hypermethylation of the PGC- 1alpha promoter at -260, which reduces PGC-1alpha expression by DMNT3benzyme. We have confirmed that maternal exercise prevents hypermethylation of this CpG site in female offspring'sliver. We therefore propose to determine if DMNT3b is responsible for the effects of maternal exercise on the offspring.

Maternal diet and crossbreeding set up One (1) breeding scheme: Male myogenin-cre positive and floxed DMNT3bmice (n = 4) will be bred with female floxed DMNT3b mice (n = 4). Two (2) diet interventions: normal chow (NC) andhigh-fat diet (HF) Total: 1 breeding x 2 diets x 8 mice per breeding = 16 mice

Pgc-1alpha methylation in the offspring Two (2) genotypes: Wild type (WT, floxed DNMT3b without cre) andtissue-specific DMNT3b knockout mice (KO) Two (2) genders: male and female Two (2) maternal diet conditions: NCvs HF Based on the glucose tolerance test data from our previous experiment (Sed-HF 31,097 mg/dl/min vs. Ex-HF22,333 mg/dl/min) and the variation (SD = 6,368), we need n = 13 to achieve statistical significance with analpha=0.05 and power = 0.9 (www.biomath,info/power/ttest.htm). Total: 2 genotypes x 2 genders x 2 maternal dietconditions x 13 mice per group = 78 mice

Summary Total number of mice used for maintenance: 54 Total number of mice used for studies in the offspring: 400

Adverse Events:

What anticipated adverse events could occur in a research animal within this species procedure? The only adverse event that might occur is that change in diet and addition of exercise at the same time lead tounsuccessful pregnancy. We will never change the diet and put mice in the running protocol at the same time.

Provide the percentage of animals that will have an adverse event that prevents the data from being used in the

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experiment. If we avoid simultaneous changing of diet and exercise, the percentage of animals that will have adverse event ofunsuccessful pregnancy is estimated to be zero.

Molecular Imaging Core:Will you utilize the Molecular Imaging Core (MIC) in this Species Procedure? YES

Imaging modalities to be used: Positron Emission Tomography (PET) - involves positron emitting radioisotopes

NOTE: The Molecular Imaging Core imaging modalities will be conducted in accordance with Dr. Stuart Berr's ACUCprotocol #3539. All ordering and use of radioactive material (RAM) will be conducted under the Molecular Imaging CoreUser Authorization Number.

If the PI intends to return imaged animals that possess either RAM or biohazardous material to the vivarium, the PI mustnotify the vivarium supervisor at least one day in advance of the imaging session in order to have suitable returnhousing available.

Radioactive Materials: YES

I (YAN, ZHEN) accept responsibility for the following: Notifying the vivarium supervisor prior to introducing radioactive materials into the vivarium. 1.Ensuring that each cage that houses animal(s) harboring experimentally introduced radioactive materials isproperly labeled.

2.

Contacting the vivarium supervisor for assistance with obtaining radioactive material labels, if needed. 3.

Isotopes selected to be used in LIVE ANIMALS in this Species Procedure: F-18

Describe the route, volume and frequency of Isotope administration AND how contaminated bedding andcarcasses will be disposed. The Bijoy Kundu Lab will assist with radioactivity work. 200 µCi (200 ul) of 18F-FDG will be injected intraperitoneally(i.p.) ONCE. Contaminated bench coat, bedding, and carcasses will be disposed of according to environmental healthand safety and radiation safety guidelines. For example, the radioactive carcasses will be placed in green or blackplastic bags and labeled on the outside with radioactive warning tape. We will organize with the Radiation SafetyOffice (982-4917) for pickup.

Describe the duration of hazardous shedding after Isotope administration AND any precautionary measurestaken to protect personnel. 18F-FDG (200 µCi in 200 ul) will be diluted in normal saline injected via i.p. No sample collections will be done within24 hours. Only PET imaging will be done. Twenty-four hours after the injection, mice will be euthanized by CO2. Toprotect persons from possible shedding at these points they will wear full personal protective gear including full-lengthlab coats, safety glasses, and gloves. The entire PET imaging experiment will be conducted at Snyder 068. The areaand personnel clothing will be swipe tested after every experiment to ensure there is no contamination.

Does this research involve the creation (de novo generation by gene manipulation) or generation (creation ofnovel transgenics by breeding two transgenic lines) of transgenic animals (animals in which the germ line isaltered by genomic DNA insertion or removal)? YES

Describe the genetic alteration and means by which the alteration will be achieved. To study the role of epigenetic regulation of the Pgc-1alpha gene in the transmission of the negative impact of parentalobesity, we will breed wild type mice with MCK-Pgc-1alpha (Dr. Spiegelman) to have both wild type andMCK-Pgc-1alpha mice in the same uterus. To study the role of DNMT3b in methylating Pgc-1alpha gene in thetransmission of the negative impact of parental obesity, we will crossbreed Myogenin-Cre mice (Dr. Eric Olson) withfloxed DMNT3b (We have rederived the mice from UNC) to have both wild type and muscle-specific DMNT3b knockoutmice in the same uterus. All the breeding mice are already in our colonies. None of the mice have any knownanatomical and function deficits.

The NIH Guidelines for Research Involving Recombinant or Synthetic Nucleic Acid Molecules requires thefollowing:

Transgenic experiments involving the generation of transgenic animals require notification of the InstitutionalBiosafety Committee (IBC) simultaneous with initiation.Transgenic experiments involving human or animal pathogens require IBC approval prior to initiation!

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Transgenic experiments using animals which have been genetically modified, that when infected, poseenhanced risks to personnel (humans) require IBC approval prior to initiation!

Permanent HousingLocation(s) of Permanent Housing:

CCM-Managed:Aurbach Vivarium (Fontaine Vivarium, 450 Ray C. Hunt Drive)MR4 VivariumSnyder Vivarium

Animal Husbandry (Mice): NON-STANDARD HUSBANDRY

NON-STANDARD Housing and Care: YES

NON-STANDARD Housing and Care description (Mice)Only study 1 and study 2 require non-standard housing and care. 1. We will place 1 male mouse with 1 female mouseper cage for breeding. Every morning, we will check plug for signs of pregnancy.

2. Once confirmed having plug, the female mouse will be randomly assigned to either a cage (15 x 32 cm) with alocked running wheel (sedentary) or a unlocked running wheel (exercise).

3. The mice in the cages with unlocked running wheel are allow to run voluntarily. Our preliminary data showed thatpregnant mice run about 10 kilometers per night for the first 10 days of pregnancy and then gradually decrease therunning distance to 0 before giving birth. There were no signs of distress or any other side effects.

4. The running wheels of the exercise group will be locked on day 20 of pregnancy allowing for normal delivery of thepups. The number of pups will be recorded daily and normal lactation period is maintained. Normal weaning will takeplace at 21 days of age for the offspring.

RESTRICTED or SCHEDULED ACCESS to Food and/or Water: NO

NON-STANDARD Provision of Food and/or Water: YES

NON-STANDARD Provision of Food and/or Water description (Mice)All 3 studies require non-standard provision of food. 1. We will feed female or male mice in the high-fat diet group(HFD) with high-fat diet (60% fat, Research Diets) for 6 weeks before mating. For the female HFD group, they will bekept on high-fat diet during pregnancy whether they are in the exercise or sedentary group.

2. The dams and offspring in Study 1 and 2 will be on normal chow diet after the lab.

Social Housing:Does the nature of the research require an exemption for Social Housing? YES

A scientific justification must be provided. Be specific - Are you requesting an exemption for a specific group ofanimals or all animals? How many animals will be involved? What is the duration of the exemption? Voluntary running studies requires precise recording of running distance for each individual mouse throughout theexperiments, which prohibits social housing.

Environmental Enrichment (other than social housing):Does the nature of the research require an exemption for Environmental Enrichment? YES

A scientific justification must be provided. Be specific! Are you requesting an exemption to a specific form ofenrichment or to all enrichment? Include which group(s) of animals, the duration, and the scientific rationale. It is well-known that cages with voluntary wheel running even when locked is an excellent housing condition for micepromoting exploratory activities.

Live Animal Work Outside of Vivarium: YES

Location(s) of Live Animal Work Outside of Vivarium: Aurbach (Fontaine, 450 Ray C. Hunt Dr.): G136MR-4: 6031Sheridan G. Snyder Translational Research Building: 033, 034 Pharmaceutical(s) Used: (Mice)

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Pre-Anesthetic:NONE SELECTED Inductional Anesthetic:NONE SELECTED Maintenance Anesthetic:NONE SELECTED PRIMARY Post-procedural analgesic:NONE SELECTED SECONDARY Post-procedural analgesic:NONE SELECTED Euthanasia Agent:NONE SELECTED Confirmation of Death

Special Considerations for RodentsRodents, especially neonates, are particularly resistant to euthanasia by overdose of inhaled agents such as CO2 oreven injectable agents; for this reason, IACUC policy requires a secondary physical method of euthanasia FOR ALLRODENTS after the animal is profoundly anesthetized, prior to carcass disposal.

Acceptable physical methods for adult and neonatal rodents include:DecapitationCardiac perfusionRemoval of vital organs (e.g. heart, lungs, brain)Opening of the chest cavity to induce bilateral pneumothoraxCutting the major blood vessels to induce exsanguination (e.g. aorta, vena cava)Cervical dislocation - formal training is recommended

One of these procedures must be performed in all rodents euthanized via CO2 inhalation once they have lostconsciousness. These procedures may not be performed in conscious animals without specific IACUC approval.

Main Procedure

Procedures, which require Humane Endpoints and Criteria for Euthanasia, to be performed on animals used inthis procedure section:

NONE

Main Procedure Description: Diet and exercise interventions 1. We will subject male or female breeders of wild type or different geneticallyengineered backgrounds (as described in Justification of Number of Animals) to normal chow or high-fat diet (D12492,Research Diet, 60% fat) for 6 weeks (as described in NON- STANDARD Provision of Food) before placing 1 malemouse with 1 female mouse per cage for breeding. Every morning, we will check plug for signs of pregnancy.

2. Once confirmed having plug, the female mouse will be randomly assigned to either a cage (15 x 32 cm) with alocked running wheel (sedentary) or an unlocked running wheel (exercise).

3. The mice in the cages with unlocked running wheel are allowed to run voluntarily. Our preliminary data showed thatpregnant mice run about 10 kilometers per night for the first 10 days of pregnancy and then gradually decrease therunning distance to 0 before giving birth. There were no signs of distress or any other side effects.

4. The running wheels of the exercise group will be locked on day 20 of pregnancy allowing for normal delivery of thepups.

5. The number of pups will be recorded daily and normal lactation period is maintained. Normal weaning will take placeat 21 days of age for the offspring.

Determination of obesity and insulin resistance During the lactation period, all mice are maintained on normal chowdiet. Body weight of the offspring will be monitored every week and glucose tolerance test and insulin tolerance testwill be performed at 6 and 9 months of age. At the end of the experiment (9 months), metabolic cage and micorPETwill be performed (see below).

Glucose tolerance test 1. This is a well-established protocol by Joslin Diabetes Center and will be performed in MR4vivarium. Mice are maintained in a normal light/dark cycle (7:00/19:00). Remove food by changing to a new cage

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https://researchcompliance.web.virginia.edu/acuc/pi/policy/Policy_Euthanasia_Confirmation_of_Death_Carcass_Disposal.pdf

without food and with water at 5:00 pm the day before the experiment (place DO NOT FEED card is placed in thecardholder).

2. Nick the tail with a pair of scissors at the very end (clean the scissor after each mouse with 70% EtOH).

3. Baseline blood glucose is measured using a glucose meter from Bayer. Mice are transferred to individually labeledcages.

4. Inject filter sterilized D-glucose in normal saline (200 mg/ml) at 2 mg/g (Keep glucose on slidewarmer beforeinjection). Set up the timer.

5. At 15, 30, 60, and 120 minutes blood glucose is sampled from the tail of each mouse by gently massaging a smalldrop of blood onto the glucometer strip.

6. Return the mice to the housing quarter.

Insulin tolerance test protocol 1. Mice are maintained in a normal light/dark cycle (7:00/19:00).

2. Remove food by changing to a new cage without food and with water at 8:00 am 6 hours before the experiment(place DO NOT FEED card is placed in the cardholder).

3. Prepare a 0.1U/mL insulin solution with filter-sterilized saline immediately before the test.

4. Assess basal glucose level (time point 0) by clipping the tip of the tail.

5. Transfer animal to new cage without food.

6. Prepare syringes with insulin (1U/kg of body weight).

7. Inject animals IP with 1 min-intervals, and if possible, include longer intervals between cages.

8. Pick up animals from cages ~40 sec before each time point measurement.

9. Assess glucose at 15, 30 and 60 minutes post-injection.

Metabolic cage and Dual Energy X-Ray Absorptiometry/Densitometry (Dexa) studies 1. This will be done in theAinimal Characterization Core under the protocol #3351.

2. Transfer mice to Fontaine Research Park the Animal Characterization Core in covered cages by Animal Care.

3. Mice are accustomed to the cages (Comprehensive Laboratory Animal Monitoring System (CLAMS) from ColumbusInstruments) during a 72-hour period

4. After the mice are accustomed to the cages, a second and third 72-hour period in the cages will then serve theactual measurements.

5. Four mice (from each experiment groups) are run simultaneously.

6. During the entire 72-hour period measurements are collected from each mouse every 15 minutes in an automatednon-invasive fashion. Metabolic parameters (O2 consumption and CO2 production, food intake, water intake and cageactivity) will be measured in two 72-hour periods. The data from the last 48 hours of the second and third period areevaluated.

7. Dual Energy X-Ray Absorptiometry/Densitometry (Dexa) scan will then be done after the metabolic cage studies.Percent body fat and lean body mass will be measured using a LUNAR PIXImus mouse densitometer (software v.2.00; GE Medical Systems). Mouse head will be excluded from the DEXA analysis.

MicroPET scanning 1. Mice are fasted for 16 hours overnight.

2. Transfer mice to Fontaine Research Park in vivo imaging facility in covered cages by Animal Care.

3. Anesthetize the mice under isofluorene anesthesia system.

4. Inject 150-200 µCi of FDG i.p. and place the mouse in a prone position.

5. Acquire image at 25’ using microPET scanner with the long axis of the mouse parallel to the long axis of thescanner (10 minutes)

6. Inject insulin (1 Unit/kg) i.p. and perform the same scanning at 25’ and 50’ after insulin injection.

7. Between each scans, mice will be allowed to recover from anesthesia.

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8. Keep the mice in the imaging facility for 24 hours to allow for sufficient decay the isotope

6. After 30 min, mice were euthanized and tissues removed and frozen in liquid nitrogen for further analyses.

Post-Procedural Details: NA

Procedural Training: All the personnel involved in this project have excellent experience in animal handling, including diet intervention,exercise, crossbreeding, glucose and insulin tolerance tests. The Metabolic cage, Dual Energy X-Ray as well as PETimaging will be conducted by the core facilities with faculty who have expertise in each of the techniques.

Animal Handler Responsibilities

FISHER, CARLEIGH (ccf2vj) ACUC Defined General Requirements UVa - Orientation Seminar: 04/01/2015 UVa - Animal Handler Refresher Training: Not required, UVa - Orientation Seminar is less than 3-years old UVa - Working Safely with Animals Training: 01/27/2015 LATA - Mouse Training: 03/24/2015 Biomethodology (restraint, blood collection, gavage, and/or injection) Critical Clinical Monitoring - Non-surgical (Humane Endpoints) Euthanasia

WILSON, REBECCA (rjw7jc) ACUC Defined General Requirements UVa - Orientation Seminar: 03/12/2013 UVa - Animal Handler Refresher Training: 04/13/2016 UVa - Working Safely with Animals Training: 01/26/2014 LATA - Mouse Training: 01/26/2014 Biomethodology (restraint, blood collection, gavage, and/or injection) Critical Clinical Monitoring - Non-surgical (Humane Endpoints) Euthanasia Supervise and/or Train Others

ZHANG, MEI (mz3a) ACUC Defined General Requirements UVa - Orientation Seminar: 02/18/2009 UVa - Animal Handler Refresher Training: 08/02/2013 Out-of-date, Must Retake UVa - Working Safely with Animals Training: 02/18/2009 LATA - Mouse Training: 08/02/2013 Biomethodology (restraint, blood collection, gavage, and/or injection) Critical Clinical Monitoring - Non-surgical (Humane Endpoints) Euthanasia Supervise and/or Train Others


Species: MiceProcedure ID: Exercise on diet-induced insulin resistanceDoes this Species Procedure (in part or in whole) contain a Breeding Colony? Yes

Will your breeding of rodents require an exemption to the breeding scheme or weaning age beyond thosespecified in the ACUC Policy on Breeding and Weaning of Rats and Mice? NO


Anticipated maximum number of animals per year: 1080

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Justify the Number of Animals Needed: We have recently obtained data in this study indicating the importance of p38 MAPK, mitochondrial autophagy inmitochondrial degeneration, steatosis and insulin resistance. In addition, mitochondrial permeability transition (MPT, aprocess of forming pores on the mitochondiral membranes for small molecules) appear to be the trigger of theautophagy process. We propose to use both pharmacological and genetic approaches to determine if inhibitingautophagy would prevent high-fat diet-induced mitochondrial dysfunction and insulin resistance. Mice (C57BL/6) withdisrupted Cyclophilin D (CyD), ATG1 (Ulk1), ATG5 or ATG6 gene will be used. Mice with floxed p38gamma, p38beta,p38gamma, ATG1 (Ulk1), ATG7, or CyD alleles will be crossbred with myogenin-CRE mice (C57BL/6) to generateskeletal muscle- specific p38gamma, p38beta, p38gamma, ATG1 (Ulk1), ATG7, or CyD knockout of mice,respectively. We would like to generate liver-specific CypD knockout mice as well by crossing floxed CypD mice withalbumin-Cre mice.Wild type littermates will be used as control. These mice will be used to determine if disruption ofautophagy gene function and/or the gene required for MPT will lead to resistance to high-fat diet-induced mitochondrialdysfunction, steatosis and insulin resistance. To monitor mitochondrial damage, we have generated a novelrecombinant DNA pMito-Timer, which allow for detection of freshly synthesized mitochondria and damaged/oxidizedmitochondria. We plan to generate tissue-specific inducible trangenic mice to achieve the measurement ofmitochondrial quality in vivo. Transgenic mice breeding colonies for maintenance Floxed p38alpha (from boehringer-ingelheim) Floxed p38beta(from boehringer-ingelheim) Floxed p38gamma (from boehringer-ingelheim) ATG5 knockout mice (from Dr. NoboruMizushima) ATG6 knockout mice (from Dr. Beth Levine) Floxed ATG6 mice (generated at UVa) Floxed ATG7 mice(from Masaaki Komatsu) CyD knockout mice (from Jeffrey Molkentin) Floxed CyD mice (Jackson Laboratory) ATG1(Ulk1) knockout mice (from Mondira Kundo) Floxed ATG1 (Ulk1) mice (from Mondira Kundo) GFP-LC3 mice (from Dr.Noboru Mizushima) CAG-CAT-MitTimer mice (to be generated at UVa) Myogenin-Cre (from Dr. Olson)-For generationof muscle- specific knockout mice Albumin-Cre (from the Jackson laboratories)--For generation of liver-specificknockout mice MCK-Cre (from the Jackson Laboratories)--For generation of muscle-specific knockout miceHSA-ERTM (from Dr. Kyle Hoehn)--For generation of inducible muscle-specific knockout mice 4 breeding pairs foreach line (1 male and 2 female) will be replaced every 6 months = 12 mice x 17 lines x 2 sets/year = 408 mice

High-fat diet-induced change in mitophagy and mitochondrial quality in skeletal muscle Two (2) conditions: normalchow (NC) and high-fat diet (HF) Three (3) lines: Wild type mice, Skeletal muscle-MitoTimer mice, and GFP-LC3 miceWe need n = 12 for each group (based on statistical analysis (http://www.biomath.info/power/ttest.htm) with alpha =0.05 and beta = 0.10 (power = 0.9) and take account for standard deviation of 40% and change of 50%. Total mice: 2conditions x 3 line x 12 mice per time point = 72 mice.

Genetic approach to prevent high-fat diet-induced mitochondrial dysfunction and insulin resistance in skeletal muscleTwo (2) conditions: normal chow (NC) and high-fat diet (HF) Seven (7) lines: ATG5 KO, ATG6 KO, muscle-specificp38alpha KO (p38alpha MKO), p38beta MKO, p38gamma MKO, ATG7 MKO or ATG1 MKO mice Two genotypes:Wild type (WT) and knockout (KO) We need n = 8 for each group (based on statistical analysis(http://www.biomath.info/power/ttest.htm) with alpha = 0.05 and beta = 0.10 (power = 0.9) and take account forstandard deviation of 33% and change of 50%). Considering the possibility of losing samples due non-responders tohigh-fat diet, we prefer n = 10 for each condition. Total mice: 2 conditions x 7 lines x 2 genotypes x 10 mice per timepoint = 280 mice.

Genetic approach to block MPT in autophagy in high-fat diet- induced mitochondrial dysfunction and insulin resistancein skeletal muscle Two (2) conditions: normal chow (NC) and high-fat diet (HF) Three (3) lines: CyD knockout,Liver-specific CyD KO mice (CypD LKO), CypD MKO mice Two genotypes: Wild type (WT) and knockout (KO) Weneed n = 8 for each group (based on statistical analysis (http://www.biomath.info/power/ttest.htm) with alpha = 0.05and beta = 0.10 (power = 0.9) and take account for standard deviation of 33% and change of 50%). Considering thepossibility of losing samples due non-responders to high-fat diet, we prefer n = 10 for each condition. Total mice: 2conditions x 3 lines x 2 genotypes x 10 mice per time point = 120 mice.

Pharmacological approach to prevent high-fat diet-induced mitochondrial dysfunction and insulin resistance Two (2)conditions: normal chow (NC) and high-fat diet (HF) Two (2) treatments: saline (SL) and chloroquine (CQ) injectionsWe need n = 8 for each group (based on statistical analysis (http://www.biomath.info/power/ttest.htm) with alpha = 0.05and beta = 0.10 (power = 0.9) and take account for standard deviation of 33% and change of 50%). Considering thepossibility of losing samples due non-responders to high-fat diet, we prefer n = 10 for each condition. Total mice: 2conditions x 2 treatments x 10 mice per time point = 40 mice.

Monitor of mitochondrial quality control by autophagy genes under the condition of high-fat diet Two (2) conditions:normal chow (NC) and high-fat diet (HF) Eight (8) lines: ATG5 KO, ATG6 KO, p38alpha MKO, p38beta MKO,p38gamma MKO, ATG7 MKO, ATG1 MKO, CypD MKO mice One (1) transgene transfected mice: electricpulse-mediated gene transfer of pMitoTimer We need n = 8 for each group (based on statistical analysis(http://www.biomath.info/power/ttest.htm) with alpha = 0.05 and beta = 0.10 (power = 0.9) and take account for

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(http://www.biomath.info/power/ttest.htm) with alpha = 0.05 and beta = 0.10 (power = 0.9) and take account forstandard deviation of 33% and change of 50%). Considering the possibility of losing samples due non-responders tohigh-fat diet, we prefer n = 10 for each condition. Total mice: 2 conditions x 8 lines x 1 genotypes x 10 mice per timepoint = 160 mice.

Adverse Events:

What anticipated adverse events could occur in a research animal within this species procedure? Somatic gene transfer may lead to infection at the transfection site in the foot.

Provide the percentage of animals that will have an adverse event that prevents the data from being used in theexperiment. Due to our stringent practice of sterilizing DNA solution right before the procedure with filter sterilization, thepercentage of animals that will have the adverse event is estimated to be 1%.







2.


Isotopes selected to be used in LIVE ANIMALS in this Species Procedure: C-14,H-3

Describe the route, volume and frequency of Isotope administration AND how contaminated bedding andcarcasses will be disposed. The Bijoy Kundu Lab will assist with radioactivity work. 0.05uCi 3H- glucose will be administered to the mouse perminute over a 3 hour period = 9uCi. Tracer stock solution will be 50uCi/mL so 1uL will be infused per minute.Contaminated bench coat, bedding, and carcasses will be disposed of according to environmental health and safetyand radiation safety guidelines. For example, the radioactive carcasses will be placed in green or black plastic bagsand labeled on the outside with radioactive warning tape. We will organize with the Radiation Safety Office (982-4917)for pickup.

Describe the duration of hazardous shedding after Isotope administration AND any precautionary measurestaken to protect personnel. 3H-glucose will be mixed with unlabeled glucose and continuously infused. Blood will be sampled from the tail tip at 15minute intervals and there is a chance that 3H may be excreted in urine of mice. 90 minutes after glucose injection themice will be euthanized by cervical dislocation after being made unconscious by isofluorane anesthesia or CO2. Toprotect persons from possible shedding at these points they will wear full personal protective gear including full lengthlab coats, safety glasses, and gloves. The area and personnel clothing will be swipe tested after every experiment toensure there is no contamination.

Does this research involve the creation (de novo generation by gene manipulation) or generation (creation ofnovel transgenics by breeding two transgenic lines) of transgenic animals (animals in which the germ line is

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altered by genomic DNA insertion or removal)? YES

Describe the genetic alteration and means by which the alteration will be achieved. We plan to generate an inducible transgenic mice expressing the fluorescent protein MitoTimer that we generated.This will be generated by pro-nuclear injection (at UVa Transgenic Core) of Chicken beta-actin promoter driving floxedchloramphenicol acetyltransferase followed by MitoTimer. The transgene is inserted into the genome in a randomfasion.


Transgenic experiments involving the generation of transgenic animals require notification of the InstitutionalBiosafety Committee (IBC) simultaneous with initiation.Transgenic experiments involving human or animal pathogens require IBC approval prior to initiation!Transgenic experiments using animals which have been genetically modified, that when infected, poseenhanced risks to personnel (humans) require IBC approval prior to initiation!


CCM-Managed:MR4 Vivarium

Animal Husbandry (Mice): NON-STANDARD HUSBANDRY

NON-STANDARD Housing and Care: YES

NON-STANDARD Housing and Care description (Mice)Mice in the high-fat diet group (described above) will be fed with Western diet, 45% high-fat diet (Research Diets,D12451) for a duration of 8-12 weeks starting at the age of 10-12 weeks. We (the post-doctoral fellows and the labmanager) are responsible for feeding.

RESTRICTED or SCHEDULED ACCESS to Food and/or Water: YES

RESTRICTED or SCHEDULED ACCESS to Food and/or Water description (Mice)An overnight fasting (no more than 16 hours) is necessary prior to glucose tolerance testing and/or PET scanning.Water is provided ad libitum, only food is removed.

NON-STANDARD Provision of Food and/or Water: YES

NON-STANDARD Provision of Food and/or Water description (Mice)Mice in the high-fat diet group (described above) will be fed with Western diet, 45% high-fat diet (Research Diets,D12451) for a duration of 8-12 weeks starting at the age of 10-12 weeks. We (the post-doctoral fellows and the labmanager) are responsible for feeding.


Location(s) of Live Animal Work Outside of Vivarium: Aurbach (Fontaine, 450 Ray C. Hunt Dr.): G136MR-4: 6041Sheridan G. Snyder Translational Research Building: 033, 034 Pharmaceutical(s) Used: (Mice)

Pre-Anesthetic:NOT REQUIRED Inductional Anesthetic:NOT REQUIRED Maintenance Anesthetic:Isoflurane to Effect PRIMARY Post-procedural analgesic:NOT REQUIRED SECONDARY Post-procedural analgesic:

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NOT REQUIRED Euthanasia Agent:Cervical Dislocation Under Anesthesia Confirmation of Death




Main Procedure


NONE

Main Procedure Description: Breeding colony management 1. Standard breeding colony husbandry will be followed including IACUC policy onbreeding and weaning. We will maintain the colony breeding by put one adult male mouse with two adult female mice(at least 6 weeks of age). 2. Once the female mice are confirmed of being preganent, they will be separated from eachother to ensure correct tracing of the lineage. 3. Pups will be weaned at 21 days of age, ear-tagged and genotyped byPCR of the tail DNA. 4. Mice will be house with a density no more than 5 mice/cage. 5. Pups that will not be used forexperiments or breeding will be euthanized in a carbon dioxide chamber upon confirmation of the genotype.

Isolation of mouse embryonic fibroblast (MEF) cells from pregnant mice 1. Regular breeding and check for plug torecord the date of pregnancy. 2. Uthanize the pregnant mice at E13.5 under isofluorene-induced anesthesia followedby cervical dislocation in MR4 6041. 3. Harvest mouse embryonic fibroblast (MEF) cells from each individual embryoand genomic DNA from placenta for genotyping.

High-fat diet feeding and genetic intervention studies 1. Feed half of the mice with normal chaw or high-fat diet(D12492, Research Diet; 60% fat or D12451, Research Diet; 45% fat), with the other half on normal chow diet asdescribed in the justification for the number of mice. 2. Perform glucose tolerance test overnight fasting after 12 weeksof intervention (see below).

Perform the metabolic cage and Dual Energy X-Ray 1. Absorptiometry/Densitometry (DEXA) studies in the AnimalCharacterization Core under the protocol #3351 (see below). 2. Perform microPET scanning (see below). 3. Euthanizemice at under anesthesia by isofluorene or i.p. injection of pentobarbital (50 mg/kg) followed by cervical dislocation. 4.Harvest skeletal muscles and other tissues for further analyses.

Anti-autophagy drug treatment 1. Feed wild type mice with normal chow or high-fat diet (D12492, Research Diet; 60%fat). 2. For mice under each condition, half will be injected with chloroquine (60 mg/kg/day, i.p.). 3. Perform glucosetolerance test overnight fasting after 12 weeks of intervention (see below). 4. Perform the metabolic cage and DualEnergy X-Ray Absorptiometry/Densitometry (DEXA) studies in the Animal Characterization Core under the protocol#3351 (see below). 5. Perform microPET scanning (see below). 6. Euthanize mice at under anesthesia by isofluoreneor i.p. injection of pentobarbital (50 mg/kg) followed by cervical dislocation. 7. Harvest skeletal muscles and othertissues for further analyses.

Glucose tolerance test (GTT) protocol 1. This is a well-established protocol by Joslin Diabetes Center and will beperformed in MR4 vivarium. 2. Mice are maintained in a normal light/dark cycle (7:00/19:00). 3. Remove food bychanging to a new cage without food and with water at 5:00 pm the day before the experiment (place DO NOT FEEDcard is placed in the cardholder). 4. Nick the tail with a pair of scissors at the very end (clean the scissor after eachmouse with 70% EtOH). 5. Baseline blood glucose is measured using a glucose meter from Bayer. 6. Mice aretransferred to individually labeled cages. 7. Inject filter sterilized D-glucose in normal saline (200 mg/ml) at 2 mg/g

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(Keep glucose on slidewarmer before injection). Set up the timer. 8. At 30, 60, and 120 minutes blood glucose issampled from the tail of each mouse by gently massaging a small drop of blood onto the glucometer strip. 9. Returnthe mice to the housing quarter. 10. The GTT tests will be done at the end of 8-12 weeks of diet intervention.

Insulin tolerance test (ITT) protocol 1. Mice are maintained in a normal light/dark cycle (7:00/19:00). 2. Remove food bychanging to a new cage without food and with water at 8:00 am 6 hours before the experiment (place DO NOT FEEDcard is placed in the cardholder). 3. Prepare a 0.1U/mL insulin solution with filter-sterilized saline immediately beforethe test. 4. Assess basal glucose level (time point 0) by clipping the tip of the tail. 5. Transfer animal to new cagewithout food. 6. Prepare syringes with insulin (1U/kg of body weight). 7. Inject animals IP with 1 min-intervals, and ifpossible, include longer intervals between cages. Please note the interval is between mice to make sure that we couldhandle the injections with accuracy. 8. Pick up animals from cages ~40 sec before each time point measurement. 9.Assess glucose at 15, 30 and 60 minutes post-injection. 10. The ITT test will be done 2 days after recovery from GTTtest.

Metabolic cage and Dual Energy X-Ray Absorptiometry/Densitometry (Dexa) studies 1. This will be done in theAinimal Characterization Core under the protocol #3351. 2. Transfer mice to Fontaine Research Park the AnimalCharacterization Core in covered cages by Animal Care. 3. Mice are accustomed to the cages (ComprehensiveLaboratory Animal Monitoring System (CLAMS) from Columbus Instruments) during a 72-hour period 4. After the miceare accustomed to the cages, a second and third 72-hour period in the cages will then serve the actual measurements.5. Four mice (from each experiment groups) are run simultaneously. 6. During the entire 72-hour period measurementsare collected from each mouse every 15 minutes in an automated non-invasive fashion. Metabolic parameters (O2consumption and CO2 production, food intake, water intake and cage activity) will be measured in two 72- hour periods.The data from the last 48 hours of the second and third period are evaluated. 7. Dual Energy X-RayAbsorptiometry/Densitometry (Dexa) scan will then be done after the metabolic cage studies. Percent body fat andlean body mass will be measured using a LUNAR PIXImus mouse densitometer (software v. 2.00; GE MedicalSystems). Mouse head will be excluded from the DEXA analysis.

MicroPET scanning 1. Mice are fasted for 16 hours overnight. 2. Transfer mice to Fontaine Research Park in vivoimaging facility in covered cages by Animal Care. 3. Anesthetize the mice under isofluorene anesthesia system. 4.Inject 150-200 µCi of FDG i.p. and place the mouse in a prone position. 5. Acquire image using microPET scannerwith the long axis of the mouse parallel to the long axis of the scanner (10 minutes) 6. Inject insulin (1 Unit/kg) i.p. andperform the same scanning at 30’, 60’ and 120’ after insulin injection. 7. Between each scans, mice will be allowed torecover from anesthesia. 8. Keep the mice in the imaging facility for 24 hours to allow for sufficient decay the isotope

In vivo insulin stimulated muscle glucose clearance (this assay will be performed at Jordan 5224 by Dr. Hoehn with aradiation user number 0416) 1. 5h fasted mice will be anesthetized with propofol (250 mg/kg i.p) 2. Inject IV with0.05U/animal insulin for the fat-feeding studies and 10µCi [3H]-2-DOG and [14C]-D- Glucose. 3. Samples of blood willbe taken from the tail at 1, 2, 5, 10, and 30 min, mixed with 100µl each of 0.3N BaOH and 0.3N ZnSO4 4. Bloodglucose will be simultaneously measured using an Accu- Check Advantage meter. 5. Blood radioactivity willdetermined at each time point. 6. After 30 min, mice were euthanized and tissues removed and frozen in liquidnitrogen for further analyses. 7. This procedure is done at the very end of the studies.

Whole body tension test 1. Since high-fat diet impairs mitochondrial function, this established non-invasivephysiological test of muscle function (Siegel, 2009; Krediet, 2005; Carlson, 2010; Carlson, 1990) may reveal defects inmuscle contractile function in vivo. A whole-body tension test device has been customed made according to theseprevious studies. 2. A plastic adhesive tape is gently wrapped around the base of the tail attaching a flexible steelthread with a hook at the other end and the mouse is placed at the entry of a metal mesh tube. 3. Once the mouse hasfully entered the tube, the hooked end of the steel thread is attached to an isometric tension transducer (Grass FT103) to measure tensions ranging up to 500 g. 4. The position of the mouse within the tube was examined in order toensure that all four paws were inside the enclosure and gripping the wire mesh. 5. Forward pulling movements areelicited by a standardized stroke of the tail with serrated forceps and the corresponding forward pulling tensions (FPTs)are recorded using a computer recording system. 6. Ten FPTs are recorded during each session with at least 5seconds of rest between each. 7. The average of the top 5 FPTs is normalized by the body weight. 8. The mouse willbe returned to the cage immediately after the test.

Protocol for gene transfer in skeletal muscle 1. The mouse will be anesthetized with isofluorene anesthesia and placedon a slidewarmer set at 37 degree to avoid hypothermia. 2. For the injection of FDB muscles, 10 ul of hyaluaronidase(2 mg/ml) will be injected with a 0.3 cc insulin syringe with a 30 gauge needle subcutaneously above the FDB muscleat the bottom of the feet and wait for 30 min. 3. Endotoxin free plasmid DNA (1 µg/µl in normal saline) will be injectedinto the tibialis anterior muscles (65-100 ug each) and FDB muscles (10 ug each). 4. Fifteen minutes after the DNAinjection, electrical field is delivered to the injected muscle by a S88K square pulse stimulator through a Model 5332-needle array (BTX) (Eight pulses at 100 ms, 1 Hz and 100 volt were applied to the muscle). 5. Allow mice to recover

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for 10 days before imaging analysis. We will follow the ACUC Policy on Rodent Surgery and Post-Operative Care. 6.Euthanize mice under anesthesia by isofluorene or i.p. injection of pentobarbital (50 mg/kg) followed by cervicaldislocation 7. Harvest the tibialis anterior muscles for mRNA and protein analyses

Post-Procedural Details: NA

Procedural Training: Mei Zhang and Josh Drake have extensive experience in using these techniques. Dr. Kyle Hoehn has very extensiveexperience in using isotope-base assessment of insulin sensitivity in various tissues and this assay will be performedat Hoehn Lab at Jordan Hall. Rhianna Laker and Mitsuharu Okutsu have excellent skills in eletric pulse-mediated genetransfer. The metabolic cage and Dexa scan will be done by the Ainimal Characterization Core. The microPET imagingwill be done by the Molecular Imaging Core.


CUI, DI (dc5hh) ACUC Defined General Requirements UVa - Orientation Seminar: 10/01/2015 UVa - Animal Handler Refresher Training: Not required, UVa - Orientation Seminar is less than 3-years old UVa - Working Safely with Animals Training: 09/14/2015 LATA - Mouse Training: 09/14/2015 Biomethodology (restraint, blood collection, gavage, and/or injection) Breeding Colony Management Critical Clinical Monitoring - Non-surgical (Humane Endpoints) Euthanasia Supervise and/or Train Others

HARRISON, LOGAN (lwh9dp) ACUC Defined General Requirements UVa - Orientation Seminar: 06/02/2016 UVa - Animal Handler Refresher Training: Not required, UVa - Orientation Seminar is less than 3-years old UVa - Working Safely with Animals Training: 06/06/2016 LATA - Mouse Training: 06/06/2016 Biomethodology (restraint, blood collection, gavage, and/or injection) Breeding Colony Management Critical Clinical Monitoring - Non-surgical (Humane Endpoints) Euthanasia

OSINSKI, VICTORIA (vo3sc) ACUC Defined General Requirements UVa - Orientation Seminar: 12/03/2014 UVa - Animal Handler Refresher Training: Not required, UVa - Orientation Seminar is less than 3-years old UVa - Working Safely with Animals Training: 02/09/2015 LATA - Mouse Training: 02/09/2015 Biomethodology (restraint, blood collection, gavage, and/or injection) Critical Clinical Monitoring - Non-surgical (Humane Endpoints) Euthanasia

WILSON, REBECCA (rjw7jc) ACUC Defined General Requirements UVa - Orientation Seminar: 03/12/2013 UVa - Animal Handler Refresher Training: 04/13/2016 UVa - Working Safely with Animals Training: 01/26/2014 LATA - Mouse Training: 01/26/2014 Biomethodology (restraint, blood collection, gavage, and/or injection) Breeding Colony Management Critical Clinical Monitoring - Non-surgical (Humane Endpoints) Euthanasia Supervise and/or Train Others

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ZHANG, MEI (mz3a) ACUC Defined General Requirements UVa - Orientation Seminar: 02/18/2009 UVa - Animal Handler Refresher Training: 08/02/2013 Out-of-date, Must Retake UVa - Working Safely with Animals Training: 02/18/2009 LATA - Mouse Training: 08/02/2013 Biomethodology (restraint, blood collection, gavage, and/or injection) Breeding Colony Management Critical Clinical Monitoring - Non-surgical (Humane Endpoints) Euthanasia Supervise and/or Train Others

ZHAO, HENAN (hz4p) ACUC Defined General Requirements UVa - Orientation Seminar: 11/04/2015 UVa - Animal Handler Refresher Training: Not required, UVa - Orientation Seminar is less than 3-years old UVa - Working Safely with Animals Training: 10/16/2015 LATA - Mouse Training: 10/15/2015 Biomethodology (restraint, blood collection, gavage, and/or injection) Breeding Colony Management Critical Clinical Monitoring - Non-surgical (Humane Endpoints) Euthanasia


Species: MiceProcedure ID: Exercise-induced mitochondrial biogenesisDoes this Species Procedure (in part or in whole) contain a Breeding Colony? Yes



Anticipated maximum number of animals per year: 1365 Justify the Number of Animals Needed: Our recent studies suggest that 1) p38 MAPK isoforms and PGC- 1alpha genes have different functions in skeletalmuscle in response to exercise training; 2) Exercise training induces autopahgy/mitophagy that may be dependent onAMPK function; 2) Exercise trainning induces ketone body converting enzyme BDH1 in skeletal muscle. We thereforepropose to study the functional role of these signaling and metabolic molecules. Transgenic mice breeding colonies for maintenance Floxed p38alpha mice (from boehringer-ingelheim)---Described inprotocol #2 formaintenance. Floxed p38beta mice (from boehringer-ingelheim)---Described in protocol #2 formaintenance. Floxed p38gamma mice (from boehringer-ingelheim)--- Described in protocol #2 for maintenance. ATG6knockout mice (from Dr. Beth Levine)---Described in protocol #2 for maintenance. Floxed ATG7 mice (from MasaakiKomatsu)---Described in protocol #2 for maintenance. ATG1 (Ulk1) knockout mice (from Mondira Kundo)---Describedin protocol #2 for maintenance. Floxed ATG1 (Ulk1) mice (from Mondira Kundo)---Described in protocol #2 formaintenance. CAG-CAT-MitoTimer mice (generated at UVa)---Described in protocol #2 for maintenance.Myogenin-CRE mice (from Eric Olson Lab)---Described in protocol #2 for maintenance. MCK-Cre (JacksonLaboratory)---Described in protocol #2 for maintenance. HSA-Cre-ERTE (from Kryn Esser Lab)---Described in protocol#2 for maintenance. MCK-PGC-1alpha transgenic (from Bruce Speigelman at Harvard). Floxe-PGC-1alpha mice (fromBruce Speigelman at Harvard) MCK-BDH1 transgenic mice (from Yan Lab generated at Duke) MCK-constitutive activeAMPKalpha (CaAMPKalpha) (from Laurie Goodyear at Harvard) MCK-dominant negative AMPKalpha(DnAMPKalpha) (from Laurie Goodyear at Harvard) Fxntm1MknFxntm1Pand (Jackson Laboratory, Stock# 014162),also known as KIKO mice Fxntm1Pand mice (Jackson Laboratory, Stock# 008470), also known as KI mice Total 18lines 2 breeding pairs trios for each line (1 male and 2 female) will be replaced every 6 months = 6 mice x 18 lines x 2sets/year = 216 mice

Study 1. Exercise training-induced basal autophagy and improvement of mitochondrial quality Two (2) geneticpage 17/35



Study 1. Exercise training-induced basal autophagy and improvement of mitochondrial quality Two (2) geneticbackgrounds: Wild type mice and muscle- specific MitoTimer TG mice (offspring of crossbreeding between MCK-Creand CAG-CAT-MitoTimer mice) Six (6) time points: 0, 1, 3, 7, 14, and 28 days of voluntary running exercise We need n= 11 for each group (based on statistical analysis http://www.biomath.info/power/ttest.htm with alpha = 0.05 and beta =0.10 (power = 0.9) and take account for standard deviation of 30% and change of 50% with a potential loss (20%) inanimals due to failure in running protocol). Total mice: 2 genotypes x 6 time points x 11 mice per time point = 132 mice

Study 2. The importance of PGC-1alpha, p38 MAPK and AMPK in exercise training-induced autophagy in skeletalmuscle Six (6) genetic mouse lines: MCK-PGC-1a, PGC-1a MKO, p38alpha/beta MKO, p38gamma MKO,CaAMPKalpha, DnAMPKalpha Two (2) genotypes: Wild-type mice (WT) and knockout mice (KO) Two (2) conditions:sedentary control (Sed) and exercise (Ex, 4 weeks of voluntary running) We need n = 11 for each group (based onstatistical analysis http://www.biomath.info/power/ttest.htm with alpha = 0.05 and beta = 0.10 (power = 0.9) and takeaccount for standard deviation of 30% and change of 50% with a potential loss (20%) in animals due to failure inrunning protocol). Total mice: 6 lines x 2 genotypes x 2 conditions x 11 mice per time point = 264 mice

Study 3. The importance of autophagy in exercise training- induced improvement of mitochondrial quality in skeletalmuscle One (1) time point: 4 weeks Eight (8) genetic mouse lines: ATG6 KO, ATG7 MKO, ATG1 MKO, PGC-1a MKO,p38alpha/beta MKO, p38gamma MKO, CaAMPKalpha, DnAMPKalpha in muscel-specific MitoTimer TG mice. Two (2)genotypes: Wild-type mice (WT) and transgenic (TG) or knockout mice (KO) Two (2) conditions: sedentary control(Sed) and exercise (Ex, 4 weeks of voluntary running) We need n = 11 for each group (based on statistical analysishttp://www.biomath.info/power/ttest.htm with alpha = 0.05 and beta = 0.10 (power = 0.9) and take account for standarddeviation of 30% and change of 50% with a potential loss (20%) in animals due to failure in running protocol). Totalmice: 1 time pint x 8 lines x 2 genotypes x 2 conditions x 11 mice per time point = 352 mice

Study 4. The importance of autophagy/mitophagy in exercise training-induced attenuation of insulin resistance inskeletal muscle in mice on high-fat diet Three (3) genetic mouse lines: ATG6 KO, ATG7 MKO, ATG1 MKO. Two (2)genotypes: Wild-type mice (WT) and knockout mice (KO) Two (2) activity conditions: sedentary control (Sed) andexercise (Ex, 4 weeks of voluntary running) Two (2) feeding conditions: normal chow (NC) and high-fat diet (HF) Weneed n = 11 for each group (based on statistical analysis http://www.biomath.info/power/ttest.htm with alpha = 0.05 andbeta = 0.10 (power = 0.9) and take account for standard deviation of 30% and change of 50% with a potential loss(20%) in animals due to failure in running protocol). Total mice: 3 lines x 2 genotypes x 2 activity conditions x 2 feedingconditions x 11 mice per time point = 263 mice

Study 5. Contractile activity-induced mitophagy in skeletal muscle We will determine if treadmill running or motor nervestimulation will acutely induce mitochondrial stress and activate mitophagy. One (1) genotypes: muscle-specificMitoTimer TG mice Three (3) activity conditions: Sedentary mice (Sed), Exercised mice (Ex, treadmill running test) andnerve-stimulated mice (Stim, 2 h) with the contralateral TA muscle as control We need n = 8 for each group (based onstatistical analysis http://www.biomath.info/power/ttest.htm with alpha = 0.05 and beta = 0.10 (power = 0.9) and takeaccount for standard deviation of 30% and change of 50% with a potential loss (20%) in animals due to surgicalprocedure and motor nerve stimulation). Total mice: 1 genotypes x 3 activity conditions x 8 mice per time point = 24mice

Study 6. The impact of acute and exercise training on mitochondrial and muscle function in FA mice It is not knownwhether exercise has positive or negative impacts on mitochondrial function and muscle function. We need to breedcrossbreed Fxntm1MknFxntm1Pand (Jackson Laboratory, Stock# 014162), also known as KIKO mice, withhomozygous Fxntm1Pand mice (Jackson Laboratory, Stock# 008470), also known as KI mice, to obtainFxntm1MknFxntm1Pand mice for experiments. The KIKO mice may display very mild gait abnormality late in life (>6months of age). This will not affect their ability of mating and pregnancy and delivery for this study (< 3 months of age).Two (2) genotypes: Wild type, KIKO mice Two (2) conditions: Sedentary and Exercise Two (2) durations: Acuteexercise (treadmill running test) and 4 weeks of training. We need n = 11 for each group (based on statistical analysishttp://www.biomath.info/power/ttest.htm with alpha = 0.05 and beta = 0.10 (power = 0.9) and take account for otherreasons for not being able to run on treadmill. Total mice: 2 genotypes x 2 conditions x 2 durations x 11 mice condition= 88 mice

Study 7. Test EMG activity of hindlimb muscles of mice on orbital shaker. We are interested in different types ofexercise in protection against muscle wasting. Literature exists suggesting that Tai Chi, a slow movement, low impactexercise derived from traditional Chinese martial art, is beneficial in improving muscle function and quality of life invarious chronic diseases associated with frailty and in aged population. There is no animal model of Tai Chi exercise.We are planning to set a mouse model of Tai Chi exercise by putting mice on an orbital shaker. We need EMGevidence to support and instruct us about the model establishment. Two (2) groups of mice: Sedentary control (Sed)and Orbital shaking (OS) We need n = 5 for each group (based on statistical analysishttp://www.biomath.info/power/ttest.htm with alpha = 0.05 and beta = 0.10 (power = 0.9) in c-fos mRNA expression

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from a pilot study we did years ago in a different exercise model in mice, weight lifting). Total mice: 2 conditions x 5mice in each group = 10 mice.

Study 8. Test if a voluntary resistance exercise induces hypertrophy adaptation. We are interested in different types ofexercise in protection against chronic diseases. Literature exists suggesting that ladder climbing in mice can inducemuscle hypertrophy, which is an adaptation you normally see in response to resistance exercise in human. However,this model is not totally physiological due to the requirement human manipulation. It is also very tedious, whichprevents from have enough number of animals in studies in a reasonable period of time. Based on our previousprototype development, we would like to test if our new resistance exercise in mice induces physiological adaptation.Two (2) groups of mice: Sedentary control (Sed) and resistance exercise (RE). We need n = 8 for each group (basedon statistical analysis http://www.biomath.info/power/ttest.htm with alpha = 0.05 and beta = 0.10 (power = 0.9) inmuscle weight from other models that mimic resistance exercise. Total mice: 2 conditions x 8 mice in each group = 16mice.

Total number mice needed for maintenance breeding 216 Total number of mice needed for experiments withoutinvasive surgical procedures: 1149

Adverse Events:

What anticipated adverse events could occur in a research animal within this species procedure? Motor nerve stimulation involves insertion of electrodes near the nerve, which might lead to infection.

Provide the percentage of animals that will have an adverse event that prevents the data from being used in theexperiment. We always use betadine and 70% ethanol to sterilize the electrodes to avoid infection. The rate of infection is expectedto be less than 1% (has never happened to us before)







2.


Isotopes selected to be used in LIVE ANIMALS in this Species Procedure: F-18

Describe the route, volume and frequency of Isotope administration AND how contaminated bedding andcarcasses will be disposed. The Bijoy Kundu Lab will assist with radioactivity work. 200 µCi (200 ul) of 18F-FDG will be injected intraperitoneally(i.p.) ONCE. Contaminated bench coat, bedding, and carcasses will be disposed of according to environmental healthand safety and radiation safety guidelines. For example, the radioactive carcasses will be placed in green or blackplastic bags and labeled on the outside with radioactive warning tape. We will organize with the Radiation SafetyOffice (982-4917) for pickup.

Describe the duration of hazardous shedding after Isotope administration AND any precautionary measurestaken to protect personnel. 18F-FDG (200 µCi in 200 ul) will be diluted in normal saline injected via i.p. No sample collections will be done within

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24 hours. Only PET imaging will be done. Twenty-four hours after the injection, mice will be euthanized by CO2. Toprotect persons from possible shedding at these points they will wear full personal protective gear including full-lengthlab coats, safety glasses, and gloves. The entire PET imaging experiment will be conducted at Snyder 068. The areaand personnel clothing will be swipe tested after every experiment to ensure there is no contamination.


Describe the genetic alteration and means by which the alteration will be achieved. To investigate the role of p38 MAPK in exercise training in diet- induced insulin resistance, floxed p38alpha, beta orgamma mice (from boehringer-ingelheim) with be crossbred with Myogenin-Cre mice. To study the importance ofautophagy/mitophagy, ATG6 knockout mice (from Dr. Beth Levine) and ATG1 (Ulk1) knockout mice (from MondiraKundo) will be used in exercise studies. In addition, floxed ATG7 mice (from Masaaki Komatsu) and floxed ATG1(Ulk1) mice (from Mondira Kundo) will be crossbred with Myogenin-Cre mice to generate muscle-specific KO mice,and floxed PGC-1alpha mice (from Bruce Spiegelman) will be crossbred with Myogenin-Cre mice to generatemuscle-specific KO mice. To obtain mice with FA, Fxntm1MknFxntm1Pand (Jackson Laboratory, Stock# 014162), alsoknown as KIKO mice will be crossbred with Fxntm1Pand mice (Jackson Laboratory, Stock# 008470), also known as KImice or Wild type mice to obtain KIKO mice or Wild type mice with similar genetic background. KIKO mice do notshow any abnormal function at young age (

Does the animal have a genetic mutation (either induced or spontaneous) that will lead to a phenotype whichadversely impacts its health or well-being? YES

Describe the known or anticipated phenotypic result and any adverse impact on the well-being of the animal. Only potential phenotypic alterations that could potentially have negative impact on the well-being of the animals arethe age- dependent neuromuscular dysfunction (abnormal gait and loss of muscle strength) in KIKO mice. The findingsare parts of the studies. No reports have shown severe adverse impact on KIKO mice in terms of survival and fertility.All the other genetically engineered mice do not have any detectable abnormalities.


Transgenic experiments involving the generation of transgenic animals require notification of the InstitutionalBiosafety Committee (IBC) simultaneous with initiation.Transgenic experiments involving human or animal pathogens require IBC approval prior to initiation!Transgenic experiments using animals which have been genetically modified, that when infected, poseenhanced risks to personnel (humans) require IBC approval prior to initiation!


CCM-Managed:Aurbach Vivarium (Fontaine Vivarium, 450 Ray C. Hunt Drive)MR4 VivariumSnyder Vivarium

Animal Husbandry (Mice): STANDARD HUSBANDRY

Social Housing:Does the nature of the research require an exemption for Social Housing? YES

A scientific justification must be provided. Be specific - Are you requesting an exemption for a specific group ofanimals or all animals? How many animals will be involved? What is the duration of the exemption? Voluntary running studies requires precise recording of running distance for each individual mouse throughout theexperiments, which prohibits social housing.

Voluntary weight lifting studies requires precise recording of weight lifting activity for each individual mouse throughoutthe experiments, which prohibits social housing.

Environmental Enrichment (other than social housing):Does the nature of the research require an exemption for Environmental Enrichment? YES

A scientific justification must be provided. Be specific! Are you requesting an exemption to a specific form ofenrichment or to all enrichment? Include which group(s) of animals, the duration, and the scientific rationale.

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It is well-known that cages with voluntary wheel running even when locked is an excellent housing condition for micepromoting exploratory activities.

Other than group housing, which is not possible due to the need of accurate recording, we will provide Innodome™ toenrich the living environment.


Location(s) of Live Animal Work Outside of Vivarium: Aurbach (Fontaine, 450 Ray C. Hunt Dr.): G136Gilmer (Biology, Psychology, Life Sciences, Gilmer Plaza): PLSB 047, PLSB 053MR-4: 6031, G147GSheridan G. Snyder Translational Research Building: 034 Pharmaceutical(s) Used: (Mice)

Pre-Anesthetic:NOT REQUIRED Inductional Anesthetic:Isoflurane to Effect Maintenance Anesthetic:Isoflurane to Effect PRIMARY Post-procedural analgesic:NOT REQUIRED SECONDARY Post-procedural analgesic:NOT REQUIRED Euthanasia Agent:Cervical Dislocation Under Anesthesia Confirmation of Death




Main Procedure


NONE

Main Procedure Description: Breeding colony management 1. Standard breeding colony husbandry will be followed including IACUC policy onbreeding and weaning. We will maintain the colony breeding by put one adult male mouse with two adult female mice(at least 6 weeks of age).

2. Once the female mice are confirmed pregnant, they will be separated from each other to ensure correct tracing ofthe lineage.

3. Pups will be weaned at 21 days of age, ear-tagged and genotyped by PCR of the tail DNA.

4. Mice will be house with a density no more than 4 mice/cage.

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5. Pups that will not be used for experiments or breeding will be euthanized in a carbon dioxide chamber uponconfirmation of the genotype

Protocol for voluntary wheel running. 1. Obtain mice from breeding colony (at least 8 weeks old).

2. House mice individually in cage (15 x 32 cm) equipped with running wheel.

3. Accustom the mice in the running cages for at least 3 days with the running wheel locked.

4. Remove the lock allowing the mice to run for different durations (Study 1) or 4 weeks (all the other studies withvoluntary running as exercise training). The cages will be changed every two weeks. Since we have one mouse percage, this is sufficient to maintain a good sanitation.

5. Euthanize mice under anesthesia by isofluorene or i.p. injection of pentobarbital (50 mg/kg) followed by cervicaldislocation.

6. Harvest muscles and other tissues/organs for additional analyses.

Protocols for motor nerve stimulation 1. Obtain the mice from the breeding colony (at leaset 8 weeks old).

2. Transfer mice to the surgery within the Vivarium in covered cages.

3. Mice are anesthetized with isofluorene.

4. Insert electrodes to flank one of the common peroneal nerves under sterilized condition.

5. Stimulate the peroneal nerve at 10 Hz, 1V, and 0.25 ms duration for 2 hour under aneasthesia.

6. Euthanize mice under anesthesia by isofluorene or i.p. injection of pentobarbital (50 mg/kg) followed by cervicaldislocation

7. Harvest the stimulated tibialis anterior muscle and the contralateral control muscle for analyses.

Protocol for gene transfer in skeletal muscle 1. The mouse will be anesthetized with isofluorene anesthesia and placedon a slide-warmer set at 37 degree to avoid hypothermia.

2. For the injection of FDB muscles, 10 ul of hyaluaronidase (Pharmacological grade, 2 mg/ml) will be injected with a0.3 cc insulin syringe with a 30 gauge needle subcutaneously above the FDB muscle at the bottom of the feet and waitfor 30 min.

3. Endotoxin-free plasmid DNA (1 µg/µl in normal saline) will be injected into the gastrocnemius muscles (65-100 ugeach) and FDB muscles (10 ug each).

4. Fifteen minutes after the DNA injection, electrical field is delivered to the injected muscle by a S88K square pulsestimulator through a Model 533 2-needle array (BTX) (Eight pulses at 100 ms, 1 Hz and 100 volt were applied to themuscle).

5. Allow mice to recover for 10 days before imaging analysis. We will follow the ACUC Policy on Rodent Surgery andPost- Operative Care.

6. Euthanize mice under anesthesia by isofluorene or i.p. injection of pentobarbital (50 mg/kg) followed by cervicaldislocation

7. Harvest the muscles and other tissues/organs if needed for mRNA and protein analyses

Treadmill running test 1. We will subject voluntary exercise trained mice to treadmill running test as following.

2. The mice will be acclimatized to the treadmill test by walking on the treadmill for 10 min each day for 3 days at aspeed of 0.5 mph (at 3 hours into the dark cycle of the 12-h/12-h dark/light cycle) with a 0 inclination.

3. On the day of the test, the mice will be fasted for 5 hours before the test. The test will start with mice running on thetreadmill with 5% inclination and 0.5 mph speed for 30’. The speed will be increased by 0.1 mph every 30’ (not exceed1 mph) until exhaustion, which is defined as that mice refuse to run when encouraged by brushing the tail (a brush onthe back door of the channel in the custom designed mouse treadmill).

4. During the running test, blood glucose and lactate will be measured using Ascensia® CONTOUR™ Blood GlucoseMeter (Bayer) and Lactate Scout (EKF Diagnostic, Magdeburg) from the tail vein every 20 min as well as at the end ofthe test (exhaustion). The duration of the running test will be recorded and the total distance will be calculated.

Whole body tension test Since high-fat diet impairs mitochondrial function, this established non-invasive physiological

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test of muscle function (Siegel, 2009; Krediet, 2005; Carlson, 2010; Carlson, 1990) may reveal defects in musclecontractile function in vivo. A whole-body tension test device has been customed made.

1. A plastic adhesive tape is gently wrapped around the base of the tail attaching a flexible steel thread with a hook atthe other end

2. The mouse is placed at the entry of a metal mesh tube.

3. Once the mouse has fully entered the tube, the hooked end of the steel thread is attached to an isometric tensiontransducer (Grass FT 103) to measure tensions ranging up to 500 g.

4. The position of the mouse within the tube was examined in order to ensure that all four paws were inside theenclosure and gripping the wire mesh.

5. Forward pulling movements are elicited by a standardized stroke of the tail with serrated forceps and thecorresponding forward pulling tensions (FPTs) are recorded using a computer recording system.

6. Ten FPTs are recorded during each session with at least 5 seconds of rest between each.

7. The average of the top 5 FPTs is normalized by the body weight.

8. The mouse will be returned to the cage immediately after the test.

High-fat diet feeding and genetic intervention studies 1. Feed half of the mice with normal chaw or high-fat diet(D12492, Research Diet; 60% fat or D12451, Research Diet; 45% fat), with the other half on normal chow diet asdescribed in the justification for the number of mice.

2. Perform glucose tolerance test overnight fasting after 12 weeks of intervention (see below).

Glucose tolerance test protocol 1. This is a well-established protocol by Joslin Diabetes Center and will be performed inMR4 vivarium.

2. Mice are maintained in a normal light/dark cycle (7:00/19:00).

3. Remove food by changing to a new cage without food and with water at 5:00 pm the day before the experiment(place DO NOT FEED card is placed in the cardholder).

4. Nick the tail with a pair of scissors at the very end (clean the scissor after each mouse with 70% EtOH).

5. Baseline blood glucose is measured using a glucose meter from Bayer.

6. Mice are transferred to individually labeled cages.

7. Inject filter sterilized D-glucose in normal saline (200 mg/ml) at 2 mg/g (Keep glucose on slidewarmer beforeinjection). Set up the timer. 8. At 15, 30, 60, and 120 minutes blood glucose is sampled from the tail of each mouse bygently massaging a small drop of blood onto the glucometer strip.

9. Return the mice to the housing quarter.

Insulin tolerance test protocol 1. Mice are maintained in a normal light/dark cycle (7:00/19:00).

2. Remove food by changing to a new cage without food and with water at 8:00 am 6 hours before the experiment(place DO NOT FEED card is placed in the cardholder).

3. Prepare a 0.1U/mL insulin solution with filter-sterilized saline immediately before the test.

4. Assess basal glucose level (time point 0) by clipping the tip of the tail.

5. Transfer animal to new cage without food.

6. Prepare syringes with insulin (1U/kg of body weight).

7. Inject animals IP with 1 min-intervals, and if possible, include longer intervals between cages.

8. Pick up animals from cages ~40 sec before each time point measurement.

9. Assess glucose at 15, 30 and 60 minutes post-injection.

Metabolic cage and Dual Energy X-Ray Absorptiometry/Densitometry (Dexa) studies only when we see significantdifferences in body weight in mice

1. This will be done in the Ainimal Characterization Core under the protocol #3351.

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2. Transfer mice to Fontaine Research Park the Animal Characterization Core in covered cages by Animal Care.

3. Mice are accustomed to the cages (Comprehensive Laboratory Animal Monitoring System (CLAMS) from ColumbusInstruments) during a 72-hour period

4. After the mice are accustomed to the cages, a second and third 72-hour period in the cages will then serve theactual measurements.

5. Four mice (from each experiment groups) are run simultaneously.

6. During the entire 72-hour period measurements are collected from each mouse every 15 minutes in an automatednon- invasive fashion. Metabolic parameters (O2 consumption and CO2 production, food intake, water intake and cageactivity) will be measured in two 72-hour periods. The data from the last 48 hours of the second and third period areevaluated.

7. Dual Energy X-Ray Absorptiometry/Densitometry (Dexa) scan will then be done after the metabolic cage studies.Percent body fat and lean body mass will be measured using a LUNAR PIXImus mouse densitometer (software v.2.00; GE Medical Systems). Mouse head will be excluded from the DEXA analysis.

MicroPET scanning 1. Mice are fasted for 16 hours overnight.

2. Transfer mice to Fontaine Research Park in vivo imaging facility in covered cages by Animal Care.


4. Inject 200 µCi of FDG i.p. and place the mouse in a prone position.

5. Acquire image using microPET scanner at 25 min with the long axis of the mouse parallel to the long axis of thescanner (10 minutes)

6. Inject insulin (1 Unit/kg) i.p. and perform the same scanning at 25 min and 50 min after insulin injection.

7. Between each scans, mice will be allowed to recover from anesthesia.

8. Keep the mice in the imaging facility for 24 hours to allow for sufficient decay the isotope

Electromyography (EMG) recording in mice on an orbital shaker This procedure will be set up with the help andinstruction of Dr. Joe Hart from orthopedic surgery. 1. Mice are fasted for 16 hours overnight and moved to MR4 6031surgery room.


3. Insert a sterile 20 gauge needle percutaneously into the left gastrocnemius muscle (~2 mm deep) and insert a sterileEMG wire through the needle.

4. Back the needle out and keep the wire in the muscle. This setting allows free movement of the mouse.

5. Connect this wire lead is connected to a biopac data acquisition system - MP150 A-D board with a universalinterface module to digitize amplified EMG signal using EMG100C.

6. Let the mice recover from anesthesia (within minutes). Each mouse will receive a single dose i.p. injection ofanalgesic after EMG wire placement (tramadol hydrochloride 20 mg/kg body weight).

7. Place the mice on an orbital shaker and start orbital shaking at 100 rpm and record EMG activity. We will thenincrementally increase the speed 10 rpm each time until we see clear rhythmic EMG activity. This speed will be usedfor all mice in the exercise group. We do not know the speed yet, but this test will allow us to find a rotation speed thatleads to activation of gastrocnemius muscle.

8. Shake mice at this speed for 1 hour and then euthanize the mice under anesthesia and harvest the GA and otherhindlimb muscles for gene expression and protein analyses.

9. Perform the same procedure without shaking for mice in the sedentary group.

Voluntary weight lifting 1. We will put a 11 mm-diameter collar made of plastic coated metal wire (2.7 mm thick) aroundthe neck for each mouse under isoflurane anesthesia and make sure that the collar can be turned freely. Mice will behoused in the custom-made weight lifting cage for 3 days before the experiment with food and water provided in thecage. 2. On the first training day in the evening, food will be move to the top of the cage and the mouse will get thefood by pushing the lever plate with no weight added and the lifting activity recorded. Water will be provided in thecage with free access. 3. Food will be added back to the cage next morning. 4. On the second day of training, the

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same procedure will be repeated with a weight of 25% of the body weight added to the lever plate and weight liftingactivity recording. 5. Food will be added back to the cage next morning. 6. Step 2 and 3 will be repeated with 50, 75and 100% of body weight on the 3rd, 4th and 5th day. The weight will stay at 100% of body weight for the rest of thetraining day for a total of 10 days. 7. Mice kept in similar cage with food and water provided with free access will serveas sedentary control. 8. We will humanely euthanize the mice under isofluorane anesthesia with cervical dislocationfollowed by muscle harvesting at 24 hours after the last training. 9. To fully assess the benefit of exercise, we will have5 mice in this protocol have simultaneous access to a free running wheel in the cage to have both weightlifting andweight lifting activity. Both these protocols are non-invasive and physiological. No pain will be elicited. No anesthesiainvolved. All activities are voluntary. Food and water will be plentifully provided.

Laser Doppler scaning imaging for blood flow in FA mice 1. We will measure blood flow one time at ~6.5 months ofage for KIKO and WT mice at MR5 G225. 2. Blood flow in both the hindlimbs will be measured using a PeriScan PIM-IIblood perfusion monitor (laser Doppler scanner) (Perimed, Inc. North Royalton, OH). This technique for measuringblood flow is a purely non-invasive procedure that uses a light beam that moves across the skin to measure blood flowa few millimeters below the skin. 3. No surgery is involved in this measurement; however, to prepare the animal for thescan, each animal will receive light sedation with ketamine at half the dose used for normal surgical procedures toprevent undue stress during immobilization required for the measurement. 4. Three sites from the upper, middle andlower portions of the hindlimb will be selected for measurement. The average blood flow of these three sites will becalculated. The estimated duration of the measurement procedure will be 10 - 15 minutes. 5. Mice will be euthanizedafter the measurement.

Introvital imaging of FDB muscle This is a protocol that will allow us examine the response to reporter gene to motornerve stimulation in mice. 1. We need to perform this analysis in 5 mice in our existing protocol. 2. 10 days after genetransfer (an approved procedure in this protocol), we will request transfer of mice from MR4 by ARC to Keck Center(Room PLSB 047, Physical and Life Science Building). Only the Center for Comparative Medicine be used for transferof the mice initially to Gilmer vivarium the day before terminal intravital imaging. 3. On the same day, we willanesthetize the mouse with isoflurane on a heated stage in supine position (PLSB 053). 4. A small incision will bemade to expose the left FDB muscle at the bottom of the foot after preparation with betadine and 70% enthanol. 5.Introvital imaging of FDB muscle will be acquired at the incision. 6. Two electrodes will be inserted at the thigh nearthe sciatic nerve to elicit electric stimulation-induced contraction at 100 Hz, 10 ms pulse per second at 15 volt for 20min. 7. Periodic imaging will last for 2 hrs. 8. Mice will be euthanized at the end of the experiments under anesthesia bycervical dislocation. 9. The DNA construct that will be used for this study is mitochondrial AMPK activity reporter(mitoABKAR).

Post-Procedural Details: There are NO survival surgery procedures involved, but some procedures require anesthesia. Mice are monitoredcarefully while under anesthesia and kept warm throughout the procedure. Isofluorene or i.p. injection of pentobarbital(50 mg/kg) followed by cervical dislocation will be use to euthanize the mice at the end of the study.

The EMG wire insertion is a minimally invasive procedure no more than the procedure involved for in vivo musclecontractility assay in vivo. The experiment is also one time, short term. We will euthanize the mice immediately afterthe experiment for sample harvesting.

Procedural Training: Mei Zhang, Josh Drake and Zdenek Svindrych have extensive animal research experience with the proceduresdescribed above. They will provide the training for other and future lab members.


CUI, DI (dc5hh) ACUC Defined General Requirements UVa - Orientation Seminar: 10/01/2015 UVa - Animal Handler Refresher Training: Not required, UVa - Orientation Seminar is less than 3-years old UVa - Working Safely with Animals Training: 09/14/2015 LATA - Mouse Training: 09/14/2015 LATA - Anesthesia and Analgesia of Rodents: 09/14/2015 Biomethodology (restraint, blood collection, gavage, and/or injection) Breeding Colony Management Critical Clinical Monitoring - Non-surgical (Humane Endpoints) Euthanasia Supervise and/or Train Others

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DRAKE, JOSHUA (jcd6g) ACUC Defined General Requirements UVa - Orientation Seminar: 07/02/2014 UVa - Animal Handler Refresher Training: Not required, UVa - Orientation Seminar is less than 3-years old UVa - Working Safely with Animals Training: 07/03/2014 LATA - Mouse Training: 07/03/2014 LATA - Anesthesia and Analgesia of Rodents: 07/03/2014 Biomethodology (restraint, blood collection, gavage, and/or injection) Breeding Colony Management Critical Clinical Monitoring - Non-surgical (Humane Endpoints) Euthanasia Supervise and/or Train Others

FISHER, CARLEIGH (ccf2vj) ACUC Defined General Requirements UVa - Orientation Seminar: 04/01/2015 UVa - Animal Handler Refresher Training: Not required, UVa - Orientation Seminar is less than 3-years old UVa - Working Safely with Animals Training: 01/27/2015 LATA - Mouse Training: 03/24/2015 LATA - Anesthesia and Analgesia of Rodents: 03/24/2015 Biomethodology (restraint, blood collection, gavage, and/or injection) Breeding Colony Management Critical Clinical Monitoring - Non-surgical (Humane Endpoints) Euthanasia

HARRISON, LOGAN (lwh9dp) ACUC Defined General Requirements UVa - Orientation Seminar: 06/02/2016 UVa - Animal Handler Refresher Training: Not required, UVa - Orientation Seminar is less than 3-years old UVa - Working Safely with Animals Training: 06/06/2016 LATA - Mouse Training: 06/06/2016 LATA - Anesthesia and Analgesia of Rodents: 06/06/2016 Biomethodology (restraint, blood collection, gavage, and/or injection) Breeding Colony Management Critical Clinical Monitoring - Non-surgical (Humane Endpoints) Euthanasia

SVINDRYCH, ZDENEK (zs4d) ACUC Defined General Requirements UVa - Orientation Seminar: 06/02/2016 UVa - Animal Handler Refresher Training: Not required, UVa - Orientation Seminar is less than 3-years old UVa - Working Safely with Animals Training: 05/19/2016 LATA - Mouse Training: 05/27/2016 LATA - Anesthesia and Analgesia of Rodents: 05/27/2016

WILSON, REBECCA (rjw7jc) ACUC Defined General Requirements UVa - Orientation Seminar: 03/12/2013 UVa - Animal Handler Refresher Training: 04/13/2016 UVa - Working Safely with Animals Training: 01/26/2014 LATA - Mouse Training: 01/26/2014 LATA - Anesthesia and Analgesia of Rodents: 01/26/2014 Biomethodology (restraint, blood collection, gavage, and/or injection) Breeding Colony Management Critical Clinical Monitoring - Non-surgical (Humane Endpoints) Euthanasia Supervise and/or Train Others

ZHANG, MEI (mz3a) ACUC Defined General Requirements UVa - Orientation Seminar: 02/18/2009 UVa - Animal Handler Refresher Training: 08/02/2013 Out-of-date, Must Retake UVa - Working Safely with Animals Training: 02/18/2009 LATA - Mouse Training: 08/02/2013 LATA - Anesthesia and Analgesia of Rodents: 02/06/2009 Biomethodology (restraint, blood collection, gavage, and/or injection)

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Breeding Colony Management Critical Clinical Monitoring - Non-surgical (Humane Endpoints) Euthanasia Supervise and/or Train Others

ZHAO, HENAN (hz4p) ACUC Defined General Requirements UVa - Orientation Seminar: 11/04/2015 UVa - Animal Handler Refresher Training: Not required, UVa - Orientation Seminar is less than 3-years old UVa - Working Safely with Animals Training: 10/16/2015 LATA - Mouse Training: 10/15/2015 LATA - Anesthesia and Analgesia of Rodents: 10/15/2015 Biomethodology (restraint, blood collection, gavage, and/or injection) Breeding Colony Management Critical Clinical Monitoring - Non-surgical (Humane Endpoints) Euthanasia


Species: MiceProcedure ID: NO protection against catabolic muscle wastingDoes this Species Procedure (in part or in whole) contain a Breeding Colony? Yes



Anticipated maximum number of animals per year: 1028 Justification for Category E procedures: We have obtained strong evidence that muscle-specific SOD3 transgenic mice have SOD3 redistributed to allperipheral tissues/organs. To ascertain if muscle-derived SOD3 expression provide protection against multiple organdysfunction syndrome (MODS), we choose the gold-standard for human sepsis and MODS in humans, mouse sepsismodel of cecal ligation and puncture (CLP). CLP will be employed in EcSOD transgenic, knockout and wild typelittermate mice followed by analyses for the survival and biochemical and morphological evidence of protection bymuscle-derived EcSOD. This research direction is entirely novel. The findings will be extremely valuable for futuredevelopment of therapies for this detrimental disease that affect millions of human patients each year. After CLP, wewill administer 4 mg/kg mouse Ketoprofen subcutaneously every 12 hours or 24 hours or as required. Death mayoccur for the mice for survival curve, which is an outcome of MODS. We do not intend to terminate the experimentsprematurely to alter the outcome. The protection observed in muscle-specific EcSOD transgenic mice against MODS could be consequences ofsecondary changes the transgenic mouse skeletal muscles and peripheral tissues/organs. To elegantly confirm thatmuscle-derived EcSOD provides direct protective function, we propose to take advantage of parabiosis tounequivocally confirm the findings. Systemic administration of recombinant EcSOD in a clinical trial has proven to beunsuccessful due to lack of continuous supply and sufficient targeting of the molecule to the action site of reactiveoxygen species overproduction. It is therefore critical to use this technology in addressing the question.

Some pilot studies have been done in the last year or so in this laboratory supporting further research in this area. Wehave also received a favorable, fundable score on an NIH R01 grant. However, we need to perform formalinvestigations to substantially firm the conclusions.

Justify the Number of Animals Needed: Several recent works from this lab suggest that oxidative myofibers have a robust intrinsic protective mechanism thatrender oxidative/exercise trained myofibers resistant to oxidative damage and catabolic muscle wasting in a iNos/nitricoxide-dependent manner. Most of the findings were obtained in culture myofibers and some were in mice. We plan tofurther investigate this important phenomenon with a focus on the therapeutic value of antioxidant genes in livinganimals with the wild type transgenic or knockout backgrounds.

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animals with the wild type transgenic or knockout backgrounds. Transgenic mice breeding colonies for maintenance Myogenin-Cre mice (from Eric Olson)---Maintained as described inprotocol #3 MCK-Cre mice (from the Jackson Laboratories)---Maintained as described in protocol #3 Floxed SOD3mice (from David Harrison) GSNOR KO mice (from Jonathan Stamler’s lab) MCK-SOD3 mice (Yan lab at UVa) 2breeding trios for each line (1 male and 2 female) will be replaced every 6 months = 6 mice x 5 lines x 2 sets/year = 60mice

Endothelial injury and multiple organ dysfunction induced by CLP in muscle-specific SOD3 transgenic and KO miceFour (4) genotypes: SOD3 TG and wild type littermates (WT) and SOD3 KO and WT Two (2) treatments: CLP andmock surgery control (CON) Two (3) time points: 6 hours, 24 hours and 48 hours We need n = 17 for each group(based on statistical analysis (http://www.biomath.info/power/ttest.htm) with alpha = 0.05 and beta = 0.10 (power = 0.9)and take account for standard deviation of 30% and change of 40%). Total mice: 4 genotypes x 2 treatments x 3 timepoint x 17 mice per time point = 408 mice

Prevention of cardiac dysfunction induced by STZ injection in muscle-specific SOD3 mice Two (2) genotypes: Wildtype and muscle-specific SOD3 mice Two (2) treatments: Saline and STZ injections One (1) time point: 10 weeks Weneed n = 10 for each group (based on statistical analysis (http://www.biomath.info/power/ttest.htm) with alpha = 0.05and beta = 0.10 (power = 0.9) and take account for standard deviation of 10% and change of 20%). Total mice: 2genotypes x 2 treatments x 1 time point x 10 mice per time point = 40 mice

Parabiosis-mediated protection from muscle-derived EcSOD against endothelial injury and multiple organ dysfunctioninduced by LPS injection Two (2) groups: Wild type (WT)-WT followed by LPS injection and EcSOD-WT followed byLPS injection. Three (3) time points: 6, 24 and 48 hours Considering the potential loss of mice due to surgery failure,we plan to have 10 pairs for each group to begin with. When we obtain preliminary findings, we will adjust the samplesize following statistical analysis Total mice: 2 groups x 2 mice per pair x 3 time points x 10 pairs = 120 mice

Protection by exercise training or EcSOD expression against ischemia/reperfusion induced damage to skeletal muscleFour (4) groups: Sedentary (Sed), Exercise (Ex, 4 weeks of voluntary running), wild type (WT) and EcSOD transgenic(TG) Seven (7) time point: Con, 0 h, 3 h, day 1, day3, day 7 and day 14 Based on power analysis of the publishedfindings, we need n = 7 for each condition. Total mice: 4 groups x 7 time point x 7 mice/group = 196 mice.

Summary Number of mice needed for maintenance breeding: 60 mice Number of mice needed for experiments: 764mice

Adverse Events:

What anticipated adverse events could occur in a research animal within this species procedure? Surgery to have parabiosis may lead to infection and cause death.

Provide the percentage of animals that will have an adverse event that prevents the data from being used in theexperiment. We have a sterile procedures including a complete sterilization of all the instruments and sutures, betadine+70%ethanol preparation of the incisions. The estimated infection rate is 5%.

Alternatives to Painful and Distressful ProceduresSEARCH # 1

Database or source consulted: MEDLINE Date search was performed: 02/25/2016 Years covered by the search: 66 TO 16 Keywords: SOD3 (extracellular superoxide dismutase),multiple organ dysfunction syndrome, parabiosis orcecal ligation and puncture Summarize your findings: Searching Sod3 and multiple organ dysfunctionsyndrome led to 1 hit with a paper on SOD3 variantR213G on aging mice, which is completely consistentwith the them of our project, but on a different issue(SNP of Sod3 gene variant). Adding the other terms ledto no hit.

Alternatives to Painful and Distressful ProceduresSEARCH # 2

Database or source consulted: Google Scholar Date search was performed: 02/25/2016 Years covered by the search: 66 TO 16 Keywords: SOD3 (extracellular superoxide dismutase),multiple organ dysfuction syndrome, parabiosis orcecal ligation and puncture Summarize your findings: Did not find directly any relevant publications.

Did you find a less painful/less distressful alternative thatcould accomplish the goals of your animal use protocol? NO

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Did you find a less painful/less distressful alternative thatcould accomplish the goals of your animal use protocol? NO


Imaging modalities to be used: Magnet Resonance Imaging and Spectroscopy (MRI-Clinscan 7.0T)



Hazardous Chemicals: YES

I (YAN, ZHEN) accept responsibility for the following: Notifying the vivarium supervisor prior to introducing hazardous chemicals into the vivarium. 1.Ensuring that each cage that houses animal(s) harboring experimentally introduced hazardous chemicals isproperly labeled.

2.

Contacting the vivarium supervisor for assistance with obtaining hazardous chemical labels, if needed. 3.

Hazardous Chemical(s) to be administered to live animals in this Species Procedure: Chemical 01: Streptozocine (STZ)

Describe the route, volume and frequency of Hazardous Chemical administration AND how contaminatedbedding and carcasses will be disposed. STZ (50 mg/kg) will be used for intraperitoneal injections daily for 5 days. The solution will be prepared in the fumehood in the lab. The volume of injection will be in the range of 0.1-0.3 ml depending on the body weight of the mice.The STZ bedding during the injection days and first 24 hrs after the last injection be collected and placed in the whitebuckets to be disposed of by EHS. The carcasses can be disposed of as per normal procedure.

Describe the duration of hazardous shedding after Hazardous Chemical administration AND any precautionarymeasures taken to protect personnel. The duration of hazardous shedding will be 5 days plus the first 24 hrs after the last injection. We will take extremeprecautionary measure to ensure safety. We will hang a door sign and the cages will be clearly labelled with the dateand time of administration. Safety glasses, gloves and a protective gown will always be used. The solution will beprepared in the fume hood in the lab. while performing injections. All the personnel involved in this procedure willundergo extensive training by the experienced personnel in the lab before performing the injection independently.


Describe the genetic alteration and means by which the alteration will be achieved. Our research with NIH R21 funding showed that EcSOD overexpression in skeletal muscle significantly protectsskeletal muscle and other peripheral tissues/organs in the model of LPS injection-induced endotoxemia (a model ofsevere sepsis). However, the gold standard (more relevant to human sepsis) in the filed is the model of cecal ligationand pucture (CLP). We expect that the EcSOD expression in skeletal muscle will be sufficient and necessary forprotections against multiple organ dysfunction in this model. We will cross myogenin-Cre mice with floxed EcSODmice to obtain muscle-specific EcSOD KO mice. We also want to test if GSNOR KO mice, which will have elevatedlevels of NO donor and elevated EcSOD expression, have resistance to CLP-induced MODS. The findings will providevaluable insights about human therapy.


Transgenic experiments involving the generation of transgenic animals require notification of the Institutional

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Biosafety Committee (IBC) simultaneous with initiation.Transgenic experiments involving human or animal pathogens require IBC approval prior to initiation!Transgenic experiments using animals which have been genetically modified, that when infected, poseenhanced risks to personnel (humans) require IBC approval prior to initiation!


CCM-Managed:MR4 Vivarium

Animal Husbandry (Mice): STANDARD HUSBANDRY


Location(s) of Live Animal Work Outside of Vivarium: MR-4: 1184, 3143, 6031, G147G Pharmaceutical(s) Used: (Mice)

Pre-Anesthetic:NONE SELECTED Inductional Anesthetic:Isoflurane to Effect Maintenance Anesthetic:NOT REQUIRED PRIMARY Post-procedural analgesic:NOT REQUIRED SECONDARY Post-procedural analgesic:NOT REQUIRED Euthanasia Agent:Cervical Dislocation Under Anesthesia Confirmation of Death




Main Procedure


NONE

Main Procedure Description: Breeding colony management 1. For experiments involving genetically engineered mice, standard breeding colonyhusbandry will be followed including IACUC policy on breeding and weaning. We will maintain the colony breeding byput one adult male mouse with two adult female mice (at least 6 weeks of age).

2. Once the female mice are confirmed pregnancy, they will be separated from each other to ensure correct tracing ofthe lineage.

3. Pups will be weaned at 21 days of age, ear-tagged and genotyped by PCR of the tail DNA.

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4. Mice will be house with a density no more than 4 mice/cage.

5. Pups that will not be used for experiments or breeding will be euthanized in a carbon dioxide chamber uponconfirmation of the genotype.

Protocol for STZ injections 1. Perform daily injection for 5 days for wild type and genetically engineered mice withnormal saline (NS) or STZ (50 mg/kg).

2. On the day of euthanasia, measure blood glucose and ketone bodies using commercial glucose and ketone bodystripes in the tail vein blood.

3. 10 weeks after STZ injection, euthanize mice under anesthesia by isofluorene or i.p. injection of pentobarbitalfollowed by cervical dislocation

4. Harvest skeletal muscles and other tissues for further analyses

Protocol for exercise capacity test 1. To evaluate exercise capacity, we will subject the mice to treadmill running test.This functional test will be performed in Rm 14 of MR4, as a routine in our lab.

2. Mice will be acclimatized to the test by walking on the treadmill at 0.5 mph (0%) for 5 min each day for 3 days.

3. The treadmill test starts at 0.6 mph (5%) and the speed increases by 0.1 mph every 30 min with the highest speedat 1 mph.

4. The test is terminated when mice show signs of exhaustion and refuse to run upon stimulation of the tail by acustom-built brush. Normal mice run about 1.5 miles. Maximum time on the treadmill will be around 1.5 hours. Micewith muscle wasting is expected to have shorter running distance as little as 0.5 mice (30 min).

5. The total distance will be calculated as an index of exercise capacity

Protocol for Whole body tension test Since high-fat diet impairs mitochondrial function, this established non-invasivephysiological test of muscle function (Siegel, 2009; Krediet, 2005; Carlson, 2010; Carlson, 1990) may reveal defects inmuscle contractile function in vivo. A whole-body tension test device has been customed made.

1. A plastic adhesive tape is gently wrapped around the base of the tail attaching a flexible steel thread with a hook atthe other end

2. The mouse is placed at the entry of a metal mesh tube.

3. Once the mouse has fully entered the tube, the hooked end of the steel thread is attached to an isometric tensiontransducer (Grass FT 103) to measure tensions ranging up to 500 g.

4. The position of the mouse within the tube will be examined in order to ensure that all four paws were inside theenclosure and gripping the wire mesh.

5. Forward pulling movements are elicited by a standardized stroke of the tail with serrated forceps and thecorresponding forward pulling tensions (FPTs) are recorded using a computer recording system.

6. Ten FPTs are recorded during each session with at least 5 seconds of rest between each.

7. The average of the top 5 FPTs is normalized by the body weight.


MRI imaging for cardiac function analysis 1. Transfer mice to molecular imaging core at MR4 1184.

2. Perform MRI imaging under anesthesia induced by isofluorene by the core facility (~15 min per mouse).


4. Mice will be transferred to MR4 6031 for euthanasia and sample harvesting.

Protocol for parabiosis surgery This is well established surgical protocol developed by: Wright, D.E., Wagers, A.J.,Gulati, A.P., Johnson, F.L. & Weissman, I.L. Physiological migration of hematopoietic stem and progenitor cells.Science 294, 1933-1936 (2001). After receiving a comprehensive training by Dr. Jony Kipni's lab at UVa who hasperfected this technique, Dr. Mei Zhang with very extensive training of animal surgery is able to do the procedure as aroutine in this laboratory.

1. The surgery will always performed for parabiosis between mice of the same gender and in the same litter tominimize rejection.

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2. Mice are anesthetized and the fur on the corresponding lateral aspects of each mouse is shaved.

3. Matching skin incisions from the olecranon to the knee joint are made on each mouse, bluntly dissecting thesubcutaneous fascia to create 0.5cm of free skin on each side of the incision.

4. Lay the mice side-by-side so that there is contact between the body walls. The olecranon and knee joints areattached by a single 2-0 silk suture and tie (joints are sutured together to reduce direct pulling on the sutures of theskin).

5. Press the dermis of the parabiotic partners together (excluding epidermal layers in the junction of the dermis) andclose the skin incisions with wound clips.

6. Parts of the skin incisions that are difficult to reach with the wound clip applier are sutured.

7. Parabiotic mice will be kept for 7-14 days after the surgery and will be closely monitored.

Protocol for cecal ligation and puncture (CLP) This is a well established protocol widely used by the researchcommunity as a model of sepsis with the closest relevance to human sepsis and MODS. The protocol is available inCurrent Protocols in Immunology 19.13.1-19.13.11, November 2012. Dr. Borna Mehrad here at UVA has personalexperience with this techniuque and has trained Dr. Mei Zhang with the procedure (Steinhauser et. al, J Immunol.1999;162:392-399)

1. CLP will be performed to induce sepsis in adult mice (at least 8 wks of age) as described.

2. After 16 hrs of fasting, mice will be anesthetized with isoflurane, and submitted to a midline laparotomy (2 cmincision).

3. The cecum will be carefully isolated and a 3.0 cotton ligature will be placed below the ileocecal valve to preventbowel obstruction.

4. The cecum will be punctured once with an 18-gauge needle, and the mice will recover from anesthesia.

5. For control mice, a shame operation will be performed with the abdominal cavity opened and the cecum isolatedwithout ligation and puncture.

6. All mice will receive subcutaneous injections of 1 ml of warm (37°C) saline with tramadol hydrochloride (20 mg/kgbody weight) during the postoperative period.

7. After CLP, we will administer 4 mg/kg mouse Ketoprofen subcutaneously every 12 hours or 24 hours or as required.

Protocol for microbubble-enhanced imaging of VCAM-1 signaling This is a non-invasive imaging protocol that iswell-established by Dr. Klibanov here atUVA. We plan to take advantage of this novel imaging approach in micefollowing either LPS or CLP (as described above) without adding more mice to the protocol to maximize the use of themice.

1. At 6h after LPS injection or 12/24 hr after the induction of CLP, the mouse will be placed on a stage forcontrast-enhanced ultrasound imaging.

2. The mouse will be anesthetized by intraperitoneal administration of ketamine/xylazine (100 mg/kg, i.p.).

3. Body temperature will be maintained with an electric heat pad.

4. Animal fur will be first removed by shaving, followed with Nair application.

5. VCAM-1 antibody-conjugated microbubble in saline will be administered via retro-orbital sinus injection or a tailveincatheter using a 0.5-cc syringe and 28 g needle.

6. The needle tip will be carefully introduced to penetrate the retro-orbital sinus, and up to 150 uL of microbubblesolution will be injected.

7. The anterior cervical and abdominal region of the mouse will be depilated using an electric clipper or depilatorycream.

8. Ultrasound imaging will be used to detect microbubble accumulation in several fields around the kidneys 10 minafter the injection.

9. Warmed acoustic coupling gel will be used to couple the ultrasound transducer to the animal skin.

10. All mice will be euthanized after the experiment using cervical dislocation after anesthesia overdose for sample

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harvest.

Ischemia/reperfusion in skeletal muscle induced by tourniquet This is an established protocol by Tran et al. (Eur JPharm 2011, 650:328-34). This approach will allow us to induced ischemia/reperfusion injury that mimic certainaspects of peripheral arterial disease for skeletal muscle

1. Mice will be anesthetized under isoflurane on a heating pad to maintain body temperature at 37 °C.

2. Fur will be completely removed from both hind limbs with an electric shaver.

3. Unilateral hind limb ischemia is induced by placing an orthodontic rubber band (4 oz) at the hip joint using aMcGivney hemorrhoidal ligator (Crawford et al., 2007). Mice are allow to recover from the anesthesia and they walkwith limited use of the occluded limb.

4. After 2 h ischemia, the orthodontic rubber band tourniquet is released and the hindlimb undergoes reperfusion(normal perfusion without occlusion). No other procedures are performed.

5. The contralateral limb will be used as control.

6. During the entire procedure, mice are kept hydrated with intraperitoneal injection of 0.2 ml normal saline every 2 h.

In vivo muscle contractile function This is a well-established protocol for assessment of hindlimb muscle contractility inmice. Aurora Scientific 305C Dual-Mode Muscle Lever System is the state-of-the-art system for non- invasiveassessment of muscle contractile function in vivo (http://www.aurorascientific.com/). With the recent funding fromSchool of Medicine General Equipment Solicitation 2012, we have set up this system as shared equipment in MR46031B with heated stage and isoflurane anesthesia. We will add this assay to the existing protocol for our micefollowing hindlimb ischemia reperfusion (196 mice) before euthanization of sample harvesting to maximized the datacollection capability and elevate the significance of the findings. Here are the simple steps involved. 1. Anesthetize themice using 2% isoflurane and oxygen flow of 0.5 L/min. 2. Place mice in supine position on the heated (38C) stage ofthe Aurora Scientific 305C Dual-Mode Muscle Lever System with the left or right foot (the limb of ischemia reperfusion)placed on the footplate and the hindlimb fixed at the knee. 3. Insert sterile electrodes percutaneous at the thigh of thefixed limb flanking the sciatic nerve. 4. Obtain single twitch, force-frequency curve and fatigability using the AuroraScientific in vivo muscle contractility system. 5. Let the mice recover from anesthesia completely. We need to wait forabout 24 hours to avoid impact of muscle contractions on gene expression and other biochemical changes. 6.Euthanized the mice for sample harvesting within 24 hours.

Echocardiography This is non-invasive protocol for assessment of cardiac function in mice. We add this non-invasiveprotocol to the existing protocol for the STZ study for cardiac function assessment (40 mice) 1. Remove the fur of thechest with a hair clipper and Nair. 2. Anesthetize the mouse under isoflurane 2% isoflurane and oxygen flow of 0.5L/min on a heated stage with temperature set to 38C. 3. Apply pre-warmed (37C) ultrasound gel to the chest. 4.Acquire short-axis echocardiograph using the Philip iE33 Echocardiograph. 5. Let the mice recover from anesthesiacompletely. 6. Euthanized the mice for sample harvesting within 24 hours.

Post-Procedural Details: After injection of STZ, CLP, we will monitor the mice daily. Decreased food intake and loss of body weight are theexpected outcome of the treatments, we do not intend to intervene. We have not had premature death after theseprocedures previously. But we predict that CLP will lead to some death after 96 hours. We need this information todetermine if overexpression of SOD3 in skeletal muscle prevent multiple organ dysfunction. After CLP, we willadminister 4 mg/kg mouse Ketoprofen subcutaneously every 12 hours or 24 hours or as required.

During and after the surgery of parabiosis, we will monitor the mice daily. If we find that mice display significantdiscomfort, such as frequent licking of the injection site with signs of infection, we will terminate the experiment byeuthanasia.

Humane Endpoints and Criteria for Euthanasia

What is your definition of humane endpoint(s) as it applies to these studies?Be specific, e.g. greater than 15% weight loss from baseline or age matched controls, lack of food and/or waterconsumption for more than 48 hours, extreme lethargy, hindlimb paralysis, neurologic signs, etc.Definition of humane endpoints If we observe any of the following signs, we will humanely euthanize the mice:

1. The body weight loss is more than 15% from baseline baseline or age matched controls (weighing the body weight).

2. We observe lack of food and/or water consumption for more than 48 hours (observe the marked water level andpage 33/35

2. We observe lack of food and/or water consumption for more than 48 hours (observe the marked water level andnumber of chow pallets in the cage).

Frequency of observation We will monitor the change in body weight (everyday for short- term experiment withduration 7 days) and record the changes in body weight in our lab animal record book. For long-term experiments, foodand water consumption will be monitored and recorded every 48 hours.

For LPS or CLP-induced endothelial activation and multiple organ dysfunction, our experiments will last less than 48hours and loss of body weight and reduced food and water intake are part of the pathological process. For theseexperiments, these will not be used as criterion for humane endpoints.

How frequently will animals be observed and assessed for these criteria?If the frequency of observations will change over time (e.g. once weekly until tumor size reaches 1 cm2, thenevery other day for duration), this information must be provided.No changes in frequency over time.

You are responsible for training your staff!How will your staff be trained to recognize these specific humane endpoints?How are laboratory personnel trained to recognize and respond to unanticipated outcomes?Training of our personnel Our experienced staff with more than 10 years experience in animal research will train eachof the new personnel coming to the lab about these criteria to ensure that the procedures are followed.

What is the required action to be taken when an endpoint is reached?Actions for humane endpoints We will perform isoflurane anesthesia followed by cervical dislocation followed bynecessary sample harvesting.

Procedural Training: Mei Zhang and Josh Drake in our laboratory have extensive experience in following these procedures. They will teachthe other personnel in the lab listed in this protocol.


LEITNER, LUCIA (lml8j) ACUC Defined General Requirements UVa - Orientation Seminar: 04/05/2016 UVa - Animal Handler Refresher Training: Not required, UVa - Orientation Seminar is less than 3-years old UVa - Working Safely with Animals Training: 04/14/2016 LATA - Mouse Training: 04/14/2016 LATA - Anesthesia and Analgesia of Rodents: 04/14/2016 Biomethodology (restraint, blood collection, gavage, and/or injection) Breeding Colony Management Critical Clinical Monitoring - Non-surgical (Humane Endpoints) Euthanasia

WILSON, REBECCA (rjw7jc) ACUC Defined General Requirements UVa - Orientation Seminar: 03/12/2013 UVa - Animal Handler Refresher Training: 04/13/2016 UVa - Working Safely with Animals Training: 01/26/2014 LATA - Mouse Training: 01/26/2014 LATA - Anesthesia and Analgesia of Rodents: 01/26/2014 Biomethodology (restraint, blood collection, gavage, and/or injection) Breeding Colony Management Critical Clinical Monitoring - Non-surgical (Humane Endpoints) Euthanasia Supervise and/or Train Others

ZHANG, MEI (mz3a) ACUC Defined General Requirements UVa - Orientation Seminar: 02/18/2009 UVa - Animal Handler Refresher Training: 08/02/2013 Out-of-date, Must Retake UVa - Working Safely with Animals Training: 02/18/2009

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LATA - Mouse Training: 08/02/2013 LATA - Anesthesia and Analgesia of Rodents: 02/06/2009 Biomethodology (restraint, blood collection, gavage, and/or injection) Breeding Colony Management Critical Clinical Monitoring - Non-surgical (Humane Endpoints) Euthanasia Supervise and/or Train Others

ZHAO, HENAN (hz4p) ACUC Defined General Requirements UVa - Orientation Seminar: 11/04/2015 UVa - Animal Handler Refresher Training: Not required, UVa - Orientation Seminar is less than 3-years old UVa - Working Safely with Animals Training: 10/16/2015 LATA - Mouse Training: 10/15/2015 LATA - Anesthesia and Analgesia of Rodents: 10/15/2015 Biomethodology (restraint, blood collection, gavage, and/or injection) Breeding Colony Management Critical Clinical Monitoring - Non-surgical (Humane Endpoints) Euthanasia

Principal Investigator's Assurance for the Humane Care and Use of Laboratory Animals

This protocol will comply with the regulations set forth in the Animal Welfare Act (Public Law 91-579) as amended,the Guide for the Care and Use of Laboratory Animals (NRC, 2011), PHS Policy (OLAW/NIH, 2002), and thepolicies and procedures of the University of Virginia Institutional Animal Care and Use Committee (IACUC).

1.

All responsibilities for public health hazards and precautions associated with the performance of this protocol restwith the Principal Investigator and his/her department. All biohazardous agents, radioactive materials, andpotentially hazardous chemicals used in this animal research protocol have been listed in the protocol.

2.

The use of other biological or non-living model systems and alternative methods have been considered in thepreparation of this protocol and have been found not to be usable for scientific reasons at this time.

3.

All procedures involving live animals will be performed under the supervision of a trained and qualified scientist ortheir trained and qualified designee.

4.

All personnel associated with the research in this protocol are enrolled in the University's health surveillanceprogram for exposure to laboratory animals administered by Occupational Health and Student Health.

5.

All personnel associated with the research in this protocol have completed the University's required laboratoryanimal training program. Certain species and research projects may require additional training as determined bythe Institutional Veterinarian or the IACUC.

6.

Any variations from the approved protocol MUST be submitted to the Institutional Animal Care and Use Committee(IACUC) AND BE APPROVED PRIOR to their implementation into the experimental paradigm. Examples of suchchanges include, but are not limited to, changes in:

USDA category for pain and distress a.Species b.Experimental procedure c.Number of animals d.Acute to chronic experiments e.Personnel f.Post-operative care of animals undergoing survival surgery g.Husbandry h.

7.

The IACUC is required by federal law to inspect all animal facilities and animal procedure areas (includingresearch laboratories) at least twice per year. These inspections may be unannounced and conducted at any time.The PI will make all records available for inspection.

8.

FAILURE TO RECEIVE APPROVAL PRIOR TO MODIFICATION OF EXPERIMENTS INVOLVING LABORATORYANIMALS MAY RESULT IN REVOCATION OF THE EXISTING PROTOCOL, LOSS OF ANIMAL USE PRIVILEGES,

AND OTHER DISCIPLINARY ACTIONS AT THE DISCRETION OF THE IACUC.**** END OF PROTOCOL ****

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Principal Investigator: ZHEN YAN · 2016-12-15 · Principal Investigator: ZHEN YAN PI Department...

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