Principal Investigator: ZHEN YAN
PI Department (LDAP, UVa display dept.): Department of Medicine, Cardiovascular Medicine
Protocol Title: Skeletal muscle plasticity and its impact on catabolic muscle wasting and type 2 diabetes
Protocol Number: 3762-02-15
Protocol Submittal Type: 1st or 2nd annual review - NO modifications (other than personnel & literature search
updates)
PI Training: PI has valid Animal Research Refresher for PIs
(Animal Research Refresher for PIs greater than or equal to 04/13/2013, i.e. training is valid for 3 years) Animal Research Refresher for PIs: 02/26/2016
Cover Letter provided by person who submitted protocol
Date letter (protocol) was submitted: 01/25/2016
Letter was submitted by: YAN, ZHEN (zy3m)
Dear Committee, This is a request of the first year renewal of the animal protocol. I have added grant information of a grant that was
funded in April of 2015. No other changes were made. Please let me know if you have any questions regarding this
request. Thank you very much for your generous support and service, Zhen Date letter (protocol) was submitted: 02/25/2016
Letter was submitted by: YAN, ZHEN (zy3m)
Dear Committee, Sorry for being slow in responding due to cancelled flights on my trips in the last two weeks. Please let me know if you
have any questions. Warm regards, 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) LEWELLEN, BEVAN (bml4dv) - Health Status: OK FOR WORK , Must return by: 01/26/17 (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: 09/21/16 (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: 10/20/16 (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: 09/21/16 (982-4473) DRAKE, JOSHUA (jcd6g) - Health Status: OK FOR WORK , Must return by: 06/26/16 (982-4473) page 1/34
FISHER, CARLEIGH (ccf2vj) - Health Status: OK FOR WORK , Must return by: 03/09/17 (982-4477) LEWELLEN, BEVAN (bml4dv) - Health Status: OK FOR WORK , Must return by: 01/26/17 (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) ZHANG, JIUZHI (jz9x) - Health Status: OK FOR WORK , Must return by: 07/16/16 (4349824473) ZHAO, HENAN (hz4p) - Health Status: OK FOR WORK , Must return by: 10/20/16 (982-4473) Species Procedure # 4: NO protection against catabolic muscle wasting
Species: Mice
Animal Handler(s):
LAKER, RHIANNA (rcl6n) - Health Status: UNFIT FOR WORK , Must return by: 03/26/16 (Out-of-Date!)
(982-4473) WILSON, REBECCA (rjw7jc) - Health Status: OK FOR WORK , Must return by: 01/12/17 (982-4477) ZHANG, JIUZHI (jz9x) - Health Status: OK FOR WORK , Must return by: 07/16/16 (4349824473) 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: 10/20/16 (982-4473) Principal Investigator: YAN, ZHEN
Dept (ACUC for billing):
MEDICINE-CARDIOVASCULAR RESEARCH CT
PO Box: 801394
E-Mail: zy3m@virginia.edu
Work Phone: 434-982-4477
Fax: 434-982-3139
Home Phone: 919-968-1938 Contact Person: ZHANG, MEI
Dept (ACUC for billing): UNKNOWN
PO Box: UNKNOWN
E-Mail: mz3a@virginia.edu
Work Phone: 434-982-4473
Fax: 434-982-3139
Home Phone: 919-242-5328
Protocol Identification: RESEARCH Current Funding Source Status
Dates
Description
DHHS/NIH
ACTIVE 7/1/2011-6/30/2016; 4/1/2015-1/31/2019 N/A
FOUNDATION
ACTIVE 12/15/14 – 12/14/16
Friedreich's Ataxia Research Alliance
UVA 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 systemic
diseases can induce profound functional and structural changes. On the other hand, skeletal muscle function has
significant impact on disease conditions. Research in this laboratory focuses on the mechanisms by which skeletal
muscle undergoes beneficial changes (adaptation) in response to exercise (repeated contractile activity and undergoes
abnormal changes (maladaptation) under pathological conditions and the impacts on other tissues/organs and vice
versa. 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 training
promotes mitochondrial biogenesis through activation of the p38 MAPK- PGC-1alpha regulatory axis and promotes
mitophagy. We plan to study the importance of p38 MAPK isoforms in skeletal muscle adaptation and their roles in
diet-induced insulin resistance and catabolic muscle wasting induced by sepsis. We plan to address the regulation and
importance of autophagy genes in exercise training-induced muscle adaptation and their impact in diet- induced insulin
resistance. In addition, we have secured new funding to study the impact of exercise on cardiac and skeletal muscles
in a mouse model of Friedreich’s Ataxia, a human genetic disorder due to loss of frataxin expression and impaired
mitochondrial function as a result of abnormal insertion GAA repeats in the intron of the frataxin gene. Based on a
prototype cage the PI has developed during his previous employments at UT Southwestern and Duke, we will build
custom-made cages with a lever plate for mice to push against weight to get food as a voluntary weight lifting model to
test if this will induce adaptations that are normally seen in humans in response to resistance exercise training. page 2/34
2) NO protection against catabolic muscle wasting. We have in the last three years shown that NO-dependent
antioxidant defenses in skeletal muscle can provide profound protection against cardiac cachexia (loss of lean body
mass due to catabolic muscle wasting) and diabetic cardiomyopathy, a heart failure condition induced by diabetes. We
plan to use cecal ligation puncture (CLP) and parabiosis in combination with genetic models in mice to dissect the
mechanism by which EcSOD, which is induced by nitric oxide in skeletal muscle, protects against multiple organ
dysfunction syndrome (MODS). 3) Exercise in pregnancy in prevention of diabetes. In the last three years, we have shown that maternal exercise
prevents hypermethylation of the Pgc-1alpha gene in offspring skeletal muscle, preventing age-dependent metabolic
dysfunction. We plan to use powerful molecular genetics combined with diet- induced paternal and maternal obesity
and exercise training to further dissect the mechanisms. In summary, we will employ models of physiology and pathology in genetically engineering in mice in combination with
the state- of-the-art technologies, such as in vivo imaging to improve the understanding of the molecular and signaling
mechanisms underlying physiological adaptation and pathological maladaptation in skeletal muscle and their impact on
health and diseases. Animal Use Justification: Muscle plasticity and its impact on health are not simple muscle cell phenomena. It involves complicated cell-to-cell
and organ-to- organ interactions. I have studied muscle plasticity for more than 25 years. An in vitro model system
suitable for long-term treatment mimicking the effects of exercise training and impact on chronic diseases does not
exist. Neither does a model exist for studying the impact of a chronic disease on muscle function in an integrative
manner similar to humans. The major reason is that the complex architecture and diversity of physiological inputs
acting upon intact skeletal muscles can not be simply replicated in a model system like cell culture, tissue culture or
computer modeling at this point. Mice are excellent models for this type of work because (1) they are mammals that are
easily housed and handled; (2) various exercise and disease models have been well established; (3) many of the
experimental procedures are well developed ones in the mice; for example, Parabiosis is the surgical act of artificially
creating conjoined twins of two animals. This experiment has been performed on mice for the physiological study of
how internal bodily secretions (such as hormones) affect organisms precisely as the blood is shared; (4) the
physiological and histological data describing muscle adaptation and maladaptation are well- established and
reproducible (in our lab), making the studies very feasible; and (5) a variety of well-defined and well-characterized
inbred genetic strains of mice exist for genetic mapping experiments. There is currently one muscle hypertrophy model that is near physiological: ladder climbing. This model requires
researchers to attaching weights to mice by the tails and train mice individually to climb a 2-meter ladder to get food. It
is extremely lab intensive. Human manipulation makes this model not physiological completely. Other models of
hypertrophy using surgical ablation of synergistic muscles are prone of injury and inflammation. The PI has developed
the prototype weight lifting cage during my time UT Southwestern Medical Center and Duke University. This is a
protocol that does not need surgery or human presence. Mice will push against a lever plate to get food. There have
been no adverse effects observed. Mice will be provided with water all the time. Food will be put in the cage after each
night 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 these
experimental approaches not only over-simplify the system, but also fail to accurately reflect the complicated
interactions that occur during skeletal muscles phenotypic changes. In addition, we have used and will continue to use
Drosphilla (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. The
use of animal models is essential to appropriately reflect the complexity of events that occur during muscle adaptation
and maladaptation. REDUCTION: REDUCTION. We strike to perfect our assays and analyses for the in vivo physiological and histological data to
improve reproducibility and to minimize the number of animals used. We have used and will continue to use statistical
analysis diligently to minimize the number of animals needed to reach meaningful conclusions for the studies. page 3/34
REFINEMENT: REFINEMENT) Pain and distress will be minimized by adhering to the IACUC and Federal Guidelines relating to
surgical procedures, anesthesia and analgesic use. Mice will be monitored for pain and distress following surgery, and
appropriate medication will be administered. PI Assurance for NON-Duplication:
As Principal Investigator, I have reviewed the literature relevant to my area of investigation, and am providing my
assurance that the animal studies proposed in this protocol do not unnecessarily duplicate previous published or
unpublished 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 date
usage 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 of
novel transgenics by breeding two transgenic lines) of transgenic animals (animals in which the germ line is
altered 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 which
adversely 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: Mice
Procedure ID: Exercise in pregnancy in prevention of diabetes
USDA Pain and Distress Category:
B: 54 Animals/Year
C: 400 Animals/Year
D: 0 Animals/Year
E: 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 in
the liver. To further the study, we plan to 1) test if paternal and maternal high-fat diet can induce hypermethylation of
Pgc-1alpha equally in the offspring and lead to increased susceptibility to insulin resistance and whether maternal
exercise during pregnancy will mitigate these abnormalities; 2) test if overexpressed Pgc-1alpha gene and deletion of
the 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 is
responsible for demethylating the PGC-1alpha gene. Breeding colonies Three (3) genetic mouse lines: Myogenin-Cre (Dr. Olson), MCK- Pgc-1alpha (Dr. Spiegelman) and
floxed DMNT3b (We have rederived the mice from UNC) Nine (9) mice per line: 3 male and 6 female per line (1 male
page 4/34
floxed DMNT3b (We have rederived the mice from UNC) Nine (9) mice per line: 3 male and 6 female per line (1 male
and 2 female per breeding cage) Two (2) generations per year Total: 3 lines x 9 breeding mice x 2 generations = 54
mice Study 1. Maternal exercise on offspring's susceptibility to develop insulin resistance from male or female dams on
high-fat diet We have previously obtained comprehensive data indicating that maternal exercise during completely blocks obese
pregnancy- 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 offspring
and lead to increased susceptibility to insulin resistance and whether maternal exercise during pregnancy will mitigate
these abnormalities. We now propose to study the impact of maternal exercise during pregnancy with male dam on
high-fat diet. Paternal diet and maternal exercise Five (3) groups of mice: Normal pregnancy control (NC), Paternal
High-fat-Sedentary (PHF-Sed), Paternal High-fat-Exercise (PHF- Ex). Note maternal high-fat diet-exercise studies have
been 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: Male
and 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 an
alpha=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 develop
insulin resistance from female dams on high-fat diet We have previously obtained comprehensive data indicating that maternal exercise during completely blocks obese
pregnancy- 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 insulin
resistance. 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-1alph
overexpression (TG) and muscle-specific Pgc-1alpha knockout (MKO) with wild mice from the same litters in the same
uterus. Breeding mice: Four (4) female mice and four (4) male mice per group. Total: 2 genetic breedings x 3 groups x
8 mice = 48 mice Insulin resistance in the offspring Six (6) groups: Offspring from above mentioned groups with either TG, KO or WT
background. Two (2) genders: Male and 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 an alpha=0.05 and power = 0.9 (www.biomath,info/power/ttest.htm). Total: 6
groups x 2 genders x 13 per group = 156 mice Study 3. The role of DNA methyltransferase 3b (DMNT3b) in exercise-mediated epigenetic regulation by maternal
exercise during pregnancy. Recent studies have shown that diabetic patients or cells treated with saturated fatty acid
leads to hypermethylation of the PGC- 1alpha promoter at -260, which reduces PGC-1alpha expression by DMNT3b
enzyme. We have confirmed that maternal exercise prevents hypermethylation of this CpG site in female offspring's
liver. 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 DMNT3b
mice (n = 4) will be bred with female floxed DMNT3b mice (n = 4). Two (2) diet interventions: normal chow (NC) and
high-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) and
tissue-specific DMNT3b knockout mice (KO) Two (2) genders: male and female Two (2) maternal diet conditions: NC
vs HF Based on the glucose tolerance test data from our previous experiment (Sed-HF 31,097 mg/dl/min vs. Ex-HF
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: 2 genotypes x 2 genders x 2 maternal diet
conditions 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 Molecular Imaging Core:
Will you utilize the Molecular Imaging Core (MIC) in this Species Procedure? YES page 5/34
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 ACUC
protocol #3539. All ordering and use of radioactive material (RAM) will be conducted under the Molecular Imaging Core
User Authorization Number.
If the PI intends to return imaged animals that possess either RAM or biohazardous material to the vivarium, the PI must
notify the vivarium supervisor at least one day in advance of the imaging session in order to have suitable return
housing available. Radioactive Materials: YES I (YAN, ZHEN) accept responsibility for the following: 1. Notifying the vivarium supervisor prior to introducing radioactive materials into the vivarium. 2. Ensuring that each cage that houses animal(s) harboring experimentally introduced radioactive materials is
properly labeled. 3. Contacting the vivarium supervisor for assistance with obtaining radioactive material labels, if needed. 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 and
carcasses 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 health
and safety and radiation safety guidelines. For example, the radioactive carcasses will be placed in green or black
plastic bags and 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 measures
taken 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
24 hours. Only PET imaging will be done. Twenty-four hours after the injection, mice will be euthanized by CO2. To
protect persons from possible shedding at these points they will wear full personal protective gear including full-length
lab coats, safety glasses, and gloves. The entire PET imaging experiment will be conducted at Snyder 068. The area
and 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 of
novel transgenics by breeding two transgenic lines) of transgenic animals (animals in which the germ line is
altered 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 parental
obesity, we will breed wild type mice with MCK-Pgc-1alpha (Dr. Spiegelman) to have both wild type and
MCK-Pgc-1alpha mice in the same uterus. To study the role of DNMT3b in methylating Pgc-1alpha gene in the
transmission of the negative impact of parental obesity, we will crossbreed Myogenin-Cre mice (Dr. Eric Olson) with
floxed DMNT3b (We have rederived the mice from UNC) to have both wild type and muscle-specific DMNT3b knockout
mice in the same uterus. All the breeding mice are already in our colonies. None of the mice have any known
anatomical and function deficits. The NIH Guidelines for Research Involving Recombinant or Synthetic Nucleic Acid Molecules requires the
following:
Transgenic experiments involving the generation of transgenic animals require notification of the Institutional
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, pose
enhanced risks to personnel (humans) require IBC approval prior to initiation!
Permanent Housing
Location(s) of Permanent Housing:
Aurbach Vivarium (Fontaine Vivarium, 450 Ray C. Hunt Drive)
MR4 Vivarium
page 6/34
MR4 Vivarium
Snyder 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 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 a
locked 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 that
pregnant mice run about 10 kilometers per night for the first 10 days of pregnancy and then gradually decrease the
running 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 the
pups. The number of pups will be recorded daily and normal lactation period is maintained. Normal weaning will take
place 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 be
kept 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 of
animals 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 the
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 of
enrichment 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 mice
promoting 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.): G136
MR-4: 6031
Sheridan G. Snyder Translational Research Building: 033, 034 Pharmaceutical(s) Used: (Mice) Pre-Anesthetic:
NONE SELECTED Inductional Anesthetic:
NONE SELECTED Maintenance Anesthetic:
NONE SELECTED
page 7/34
NONE SELECTED PRIMARY Post-procedural analgesic:
NONE SELECTED SECONDARY Post-procedural analgesic:
NONE SELECTED Euthanasia Agent:
NONE SELECTED Main Procedure
Procedures, which require Humane Endpoints and Criteria for Euthanasia, to be performed on animals used in
this procedure section: NONE
Main Procedure Description: Diet and exercise interventions 1. We will subject male or female breeders of wild type or different genetically
engineered 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 male
mouse 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 a
locked 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 that
pregnant mice run about 10 kilometers per night for the first 10 days of pregnancy and then gradually decrease the
running 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 the
pups. 5. The number of pups will be recorded daily and normal lactation period is maintained. Normal weaning will take place
at 21 days of age for the offspring. Determination of obesity and insulin resistance During the lactation period, all mice are maintained on normal chow
diet. Body weight of the offspring will be monitored every week and glucose tolerance test and insulin tolerance test
will be performed at 6 and 9 months of age. At the end of the experiment (9 months), metabolic cage and micorPET
will be performed (see below). Glucose tolerance test 1. This is a well-established protocol by Joslin Diabetes Center and will be performed in MR4
vivarium. Mice are maintained in a normal light/dark cycle (7:00/19:00). 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). 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 labeled
cages. 4. Inject filter sterilized D-glucose in normal saline (200 mg/ml) at 2 mg/g (Keep glucose on slidewarmer before
injection). 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 small
drop 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. page 8/34
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 the
Ainimal 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 Columbus
Instruments) 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 the
actual 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 automated
non-invasive fashion. Metabolic parameters (O2 consumption 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-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 the
scanner (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. 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 PET
imaging 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
UVa - Animal Facility Rules and Procedures Training: Required before entering any vivarium at UVa, provided
in-person by the vivarium supervisors, unique for each vivarium at UVa.
LATA - Mouse Training: 03/24/2015
page 9/34
LATA - Mouse Training: 03/24/2015
Biomethodology (restraint, blood collection, gavage, and/or injection)
Critical Clinical Monitoring - Non-surgical (Humane Endpoints)
Euthanasia
LEWELLEN, BEVAN (bml4dv)
ACUC Defined General Requirements
UVa - Orientation Seminar: 02/05/2015
UVa - Animal Handler Refresher Training: Not required, UVa - Orientation Seminar is less than 3-years old UVa - Working Safely with Animals Training: 02/03/2015
UVa - Animal Facility Rules and Procedures Training: 02/26/2015
LATA - Mouse Training: 02/03/2015
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: Required for protocol approval UVa - Working Safely with Animals Training: 01/26/2014
UVa - Animal Facility Rules and Procedures Training: Required before entering any vivarium at UVa, provided
in-person by the vivarium supervisors, unique for each vivarium at UVa.
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 UVa - Working Safely with Animals Training: 02/18/2009
UVa - Animal Facility Rules and Procedures Training: Required before entering any vivarium at UVa, provided
in-person by the vivarium supervisors, unique for each vivarium at UVa.
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 PROCEDURE # 2
Species: Mice
Procedure ID: Exercise on diet-induced insulin resistance
Does 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 those
specified in the ACUC Policy on Breeding and Weaning of Rats and Mice? NO
USDA Pain and Distress Category:
B: 408 Animals/Year
C: 672 Animals/Year
D: 0 Animals/Year
E: 0 Animals/Year Anticipated maximum number of animals per year: 1080 Justify the Number of Animals Needed: We have recently obtained data in this study indicating the importance of p38 MAPK, mitochondrial autophagy in
mitochondrial degeneration, steatosis and insulin resistance. In addition, mitochondrial permeability transition (MPT, a
process of forming pores on the mitochondiral membranes for small molecules) appear to be the trigger of the
autophagy process. We propose to use both pharmacological and genetic approaches to determine if inhibiting
page 10/34
autophagy would prevent high-fat diet-induced mitochondrial dysfunction and insulin resistance. Mice (C57BL/6) with
disrupted 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 generate
skeletal 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 with
albumin-Cre mice.Wild type littermates will be used as control. These mice will be used to determine if disruption of
autophagy gene function and/or the gene required for MPT will lead to resistance to high-fat diet-induced mitochondrial
dysfunction, steatosis and insulin resistance. To monitor mitochondrial damage, we have generated a novel
recombinant DNA pMito-Timer, which allow for detection of freshly synthesized mitochondria and damaged/oxidized
mitochondria. We plan to generate tissue-specific inducible trangenic mice to achieve the measurement of
mitochondrial 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. Noboru
Mizushima) 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 generation
of muscle- specific knockout mice Albumin-Cre (from the Jackson laboratories)--For generation of liver-specific
knockout mice MCK-Cre (from the Jackson Laboratories)--For generation of muscle-specific knockout mice
HSA-ERTM (from Dr. Kyle Hoehn)--For generation of inducible muscle-specific knockout mice 4 breeding pairs for
each 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: normal
chow (NC) and high-fat diet (HF) Three (3) lines: Wild type mice, Skeletal muscle-MitoTimer mice, and GFP-LC3 mice
We 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: 2
conditions 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 muscle
Two (2) conditions: normal chow (NC) and high-fat diet (HF) Seven (7) lines: ATG5 KO, ATG6 KO, muscle-specific
p38alpha 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 for
standard deviation of 33% and change of 50%). Considering the possibility of losing samples due non-responders to
high-fat diet, we prefer n = 10 for each condition. Total mice: 2 conditions x 7 lines x 2 genotypes x 10 mice per time
point = 280 mice. Genetic approach to block MPT in autophagy in high-fat diet- induced mitochondrial dysfunction and insulin resistance
in 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) 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 standard deviation of 33% and change of 50%). Considering the
possibility of losing samples due non-responders to high-fat diet, we prefer n = 10 for each condition. Total mice: 2
conditions 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) injections
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 standard deviation of 33% and change of 50%). Considering the
possibility of losing samples due non-responders to high-fat diet, we prefer n = 10 for each condition. Total mice: 2
conditions 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: electric
pulse-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
standard deviation of 33% and change of 50%). Considering the possibility of losing samples due non-responders to
high-fat diet, we prefer n = 10 for each condition. Total mice: 2 conditions x 8 lines x 1 genotypes x 10 mice per time
point = 160 mice. page 11/34
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 ACUC
protocol #3539. All ordering and use of radioactive material (RAM) will be conducted under the Molecular Imaging Core
User Authorization Number.
If the PI intends to return imaged animals that possess either RAM or biohazardous material to the vivarium, the PI must
notify the vivarium supervisor at least one day in advance of the imaging session in order to have suitable return
housing available. Radioactive Materials: YES I (YAN, ZHEN) accept responsibility for the following: 1. Notifying the vivarium supervisor prior to introducing radioactive materials into the vivarium. 2. Ensuring that each cage that houses animal(s) harboring experimentally introduced radioactive materials is
properly labeled. 3. Contacting the vivarium supervisor for assistance with obtaining radioactive material labels, if needed. 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 and
carcasses will be disposed. The Bijoy Kundu Lab will assist with radioactivity work. 0.05uCi 3H- glucose will be administered to the mouse per
minute 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 safety
and radiation safety guidelines. For example, the radioactive carcasses will be placed in green or black plastic bags
and 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 measures
taken to protect personnel. 3H-glucose will be mixed with unlabeled glucose and continuously infused. Blood will be sampled from the tail tip at 15
minute intervals and there is a chance that 3H may be excreted in urine of mice. 90 minutes after glucose injection the
mice will be euthanized by cervical dislocation after being made unconscious by isofluorane anesthesia or CO2. To
protect persons from possible shedding at these points they will wear full personal protective gear including full length
lab coats, safety glasses, and gloves. The area and 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 of
novel transgenics by breeding two transgenic lines) of transgenic animals (animals in which the germ line is
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 floxed
chloramphenicol acetyltransferase followed by MitoTimer. The transgene is inserted into the genome in a random
fasion. The NIH Guidelines for Research Involving Recombinant or Synthetic Nucleic Acid Molecules requires the
following:
Transgenic experiments involving the generation of transgenic animals require notification of the Institutional
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, pose
enhanced risks to personnel (humans) require IBC approval prior to initiation!
Permanent Housing
page 12/34
Location(s) of Permanent Housing:
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 lab
manager) 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 lab
manager) are responsible for feeding. Live Animal Work Outside of Vivarium: YES Location(s) of Live Animal Work Outside of Vivarium: Aurbach (Fontaine, 450 Ray C. Hunt Dr.): G136
MR-4: 6041
Sheridan 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:
NOT REQUIRED Euthanasia Agent:
Cervical Dislocation Under Anesthesia Main Procedure
Procedures, which require Humane Endpoints and Criteria for Euthanasia, to be performed on animals used in
this procedure section: NONE
Main Procedure Description: Breeding colony management 1. Standard breeding colony husbandry will be followed including IACUC policy on
breeding 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 each
other to ensure correct tracing of the 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 5 mice/cage. 5. Pups that will not be used for
experiments 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 to
page 13/34
Isolation of mouse embryonic fibroblast (MEF) cells from pregnant mice 1. Regular breeding and check for plug to
record the date of pregnancy. 2. Uthanize the pregnant mice at E13.5 under isofluorene-induced anesthesia followed
by cervical dislocation in MR4 6041. 3. Harvest mouse embryonic fibroblast (MEF) cells from each individual embryo
and 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 as
described in the justification for the number of mice. 2. Perform glucose tolerance test overnight fasting after 12 weeks
of intervention (see below). Perform the metabolic cage and Dual Energy X-Ray 1. Absorptiometry/Densitometry (DEXA) studies in the Animal
Characterization Core under the protocol #3351 (see below). 2. Perform microPET scanning (see below). 3. Euthanize
mice 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 glucose
tolerance test overnight fasting after 12 weeks of intervention (see below). 4. Perform the metabolic cage and Dual
Energy 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 isofluorene
or i.p. injection of pentobarbital (50 mg/kg) followed by cervical dislocation. 7. Harvest skeletal muscles and other
tissues for further analyses. Glucose tolerance test (GTT) protocol 1. This is a well-established protocol by Joslin Diabetes Center and will be
performed in MR4 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 before injection). Set up the timer. 8. At 30, 60, and 120 minutes blood glucose is
sampled from the tail of each mouse by gently massaging a small drop of blood onto the glucometer strip. 9. Return
the 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 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. Please note the interval is between mice to make sure that we could
handle 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 GTT
test. Metabolic cage and Dual Energy X-Ray Absorptiometry/Densitometry (Dexa) studies 1. This will be done in the
Ainimal 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 Columbus Instruments) 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 the actual 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 automated non-invasive fashion. Metabolic parameters (O2
consumption 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-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 using microPET scanner
with the long axis of the mouse parallel to the long axis of the scanner (10 minutes) 6. Inject insulin (1 Unit/kg) i.p. and
perform the same scanning at 30’, 60’ and 120’ 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 page 14/34
In vivo insulin stimulated muscle glucose clearance (this assay will be performed at Jordan 5224 by Dr. Hoehn with a
radiation user number 0416) 1. 5h fasted mice will be anesthetized with propofol (250 mg/kg i.p) 2. Inject IV with
0.05U/animal insulin for the fat-feeding studies and 10µCi [3H]-2-DOG and [14C]-D- Glucose. 3. Samples of blood will
be 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. Blood
glucose will be simultaneously measured using an Accu- Check Advantage meter. 5. Blood radioactivity will
determined at each time point. 6. After 30 min, mice were euthanized and tissues removed and frozen in liquid
nitrogen 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-invasive
physiological test of muscle function (Siegel, 2009; Krediet, 2005; Carlson, 2010; Carlson, 1990) may reveal defects in
muscle contractile function in vivo. A whole-body tension test device has been customed made according to these
previous studies. 2. A plastic adhesive tape is gently wrapped around the base of the tail attaching a flexible steel
thread with a hook at the other end and 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 tension transducer (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 the enclosure and gripping the wire mesh. 5. Forward pulling movements are
elicited 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 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. Protocol for gene transfer in skeletal muscle 1. The mouse will be anesthetized with isofluorene anesthesia and placed
on 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 muscle
at the bottom of the feet and wait for 30 min. 3. Endotoxin free plasmid DNA (1 µg/µl in normal saline) will be injected
into the tibialis anterior muscles (65-100 ug each) 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 pulse stimulator through a Model 533
2-needle array (BTX) (Eight pulses at 100 ms, 1 Hz and 100 volt were applied to the muscle). 5. Allow mice to recover
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 cervical
dislocation 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 extensive
experience in using isotope-base assessment of insulin sensitivity in various tissues and this assay will be performed
at Hoehn Lab at Jordan Hall. Rhianna Laker and Mitsuharu Okutsu have excellent skills in eletric pulse-mediated gene
transfer. The metabolic cage and Dexa scan will be done by the Ainimal Characterization Core. The microPET imaging
will be done by the Molecular Imaging Core. Animal Handler Responsibilities
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
UVa - Animal Facility Rules and Procedures Training: 10/29/2015
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
OSINSKI, VICTORIA (vo3sc)
page 15/34
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
UVa - Animal Facility Rules and Procedures Training: 02/12/2015
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: Required for protocol approval UVa - Working Safely with Animals Training: 01/26/2014
UVa - Animal Facility Rules and Procedures Training: Required before entering any vivarium at UVa, provided
in-person by the vivarium supervisors, unique for each vivarium at UVa.
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
ZHANG, MEI (mz3a)
ACUC Defined General Requirements
UVa - Orientation Seminar: 02/18/2009
UVa - Animal Handler Refresher Training: 08/02/2013 UVa - Working Safely with Animals Training: 02/18/2009
UVa - Animal Facility Rules and Procedures Training: Required before entering any vivarium at UVa, provided
in-person by the vivarium supervisors, unique for each vivarium at UVa.
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
UVa - Animal Facility Rules and Procedures Training: 11/12/2015
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 PROCEDURE # 3
Species: Mice
Procedure ID: Exercise-induced mitochondrial biogenesis
Does 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 those
specified in the ACUC Policy on Breeding and Weaning of Rats and Mice? NO
USDA Pain and Distress Category:
B: 216 Animals/Year
C: 1149 Animals/Year
D: 0 Animals/Year
E: 0 Animals/Year page 16/34
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 skeletal
muscle in response to exercise training; 2) Exercise training induces autopahgy/mitophagy that may be dependent on
AMPK function; 2) Exercise trainning induces ketone body converting enzyme BDH1 in skeletal muscle. We therefore
propose to study the functional role of these signaling and metabolic molecules. Transgenic mice breeding colonies for maintenance Floxed p38alpha mice (from boehringer-ingelheim)---Described in
protocol #2 formaintenance. Floxed p38beta mice (from boehringer-ingelheim)---Described in protocol #2 for
maintenance. Floxed p38gamma mice (from boehringer-ingelheim)--- Described in protocol #2 for maintenance. ATG6
knockout mice (from Dr. Beth Levine)---Described in protocol #2 for maintenance. Floxed ATG7 mice (from Masaaki
Komatsu)---Described in protocol #2 for maintenance. ATG1 (Ulk1) knockout mice (from Mondira Kundo)---Described
in protocol #2 for maintenance. Floxed ATG1 (Ulk1) mice (from Mondira Kundo)---Described in protocol #2 for
maintenance. 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 (Jackson
Laboratory)---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 (from
Bruce Speigelman at Harvard) MCK-BDH1 transgenic mice (from Yan Lab generated at Duke) MCK-constitutive active
AMPKalpha (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 18
lines 2 breeding pairs trios for each line (1 male and 2 female) will be replaced every 6 months = 6 mice x 18 lines x 2
sets/year = 216 mice Study 1. Exercise training-induced basal autophagy and improvement of mitochondrial quality Two (2) genetic
backgrounds: Wild type mice and muscle- specific MitoTimer TG mice (offspring of crossbreeding between MCK-Cre
and 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%) in
animals 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 skeletal
muscle 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 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%) in animals due to failure in
running 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 skeletal
muscle 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 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%) in animals due to failure in running protocol). Total
mice: 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 in
skeletal 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) and
exercise (Ex, 4 weeks of voluntary running) Two (2) feeding conditions: normal chow (NC) and high-fat diet (HF) 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%) in animals due to failure in running protocol). Total mice: 3 lines x 2 genotypes x 2 activity conditions x 2 feeding
conditions 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 nerve
stimulation will acutely induce mitochondrial stress and activate mitophagy. One (1) genotypes: muscle-specific
MitoTimer TG mice Three (3) activity conditions: Sedentary mice (Sed), Exercised mice (Ex, treadmill running test) and
nerve-stimulated mice (Stim, 2 h) with the contralateral TA muscle as control 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
page 17/34
account for standard deviation of 30% and change of 50% with a potential loss (20%) in animals due to surgical
procedure and motor nerve stimulation). Total mice: 1 genotypes x 3 activity conditions x 8 mice per time point = 24
mice Study 6. The impact of acute and exercise training on mitochondrial and muscle function in FA mice It is not known
whether exercise has positive or negative impacts on mitochondrial function and muscle function. We need to breed
crossbreed Fxntm1MknFxntm1Pand (Jackson Laboratory, Stock# 014162), also known as KIKO mice, with
homozygous Fxntm1Pand mice (Jackson Laboratory, Stock# 008470), also known as KI mice, to obtain
Fxntm1MknFxntm1Pand mice for experiments. The KIKO mice may display very mild gait abnormality late in life (>6
months 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: Acute
exercise (treadmill running test) and 4 weeks of training. 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 other
reasons 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 of
exercise in protection against muscle wasting. Literature exists suggesting that Tai Chi, a slow movement, low impact
exercise derived from traditional Chinese martial art, is beneficial in improving muscle function and quality of life in
various 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 EMG
evidence 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 analysis
http://www.biomath.info/power/ttest.htm with alpha = 0.05 and beta = 0.10 (power = 0.9) in c-fos mRNA expression
from a pilot study we did years ago in a different exercise model in mice, weight lifting). Total mice: 2 conditions x 5
mice in each group = 10 mice. Study 8. Test if a voluntary resistance exercise induces hypertrophy adaptation. We are interested in different types of
exercise in protection against chronic diseases. Literature exists suggesting that ladder climbing in mice can induce
muscle 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, which
prevents from have enough number of animals in studies in a reasonable period of time. Based on our previous
prototype 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 (based
on statistical analysis http://www.biomath.info/power/ttest.htm with alpha = 0.05 and beta = 0.10 (power = 0.9) in
muscle weight from other models that mimic resistance exercise. Total mice: 2 conditions x 8 mice in each group = 16
mice. Total number mice needed for maintenance breeding 216 Total number of mice needed for experiments without
invasive surgical procedures: 1149 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 ACUC
protocol #3539. All ordering and use of radioactive material (RAM) will be conducted under the Molecular Imaging Core
User Authorization Number.
If the PI intends to return imaged animals that possess either RAM or biohazardous material to the vivarium, the PI must
notify the vivarium supervisor at least one day in advance of the imaging session in order to have suitable return
housing available. Radioactive Materials: YES I (YAN, ZHEN) accept responsibility for the following: 1. Notifying the vivarium supervisor prior to introducing radioactive materials into the vivarium. 2. Ensuring that each cage that houses animal(s) harboring experimentally introduced radioactive materials is
properly labeled. 3. Contacting the vivarium supervisor for assistance with obtaining radioactive material labels, if needed. page 18/34
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 and
carcasses 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 health
and safety and radiation safety guidelines. For example, the radioactive carcasses will be placed in green or black
plastic bags and 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 measures
taken 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
24 hours. Only PET imaging will be done. Twenty-four hours after the injection, mice will be euthanized by CO2. To
protect persons from possible shedding at these points they will wear full personal protective gear including full-length
lab coats, safety glasses, and gloves. The entire PET imaging experiment will be conducted at Snyder 068. The area
and 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 of
novel transgenics by breeding two transgenic lines) of transgenic animals (animals in which the germ line is
altered by genomic DNA insertion or removal)? YES
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 or
gamma mice (from boehringer-ingelheim) with be crossbred with Myogenin-Cre mice. To study the importance of
autophagy/mitophagy, ATG6 knockout mice (from Dr. Beth Levine) and ATG1 (Ulk1) knockout mice (from Mondira
Kundo) 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 generate
muscle-specific KO mice. To obtain mice with FA, Fxntm1MknFxntm1Pand (Jackson Laboratory, Stock# 014162), also
known as KIKO mice will be crossbred with Fxntm1Pand mice (Jackson Laboratory, Stock# 008470), also known as KI
mice or Wild type mice to obtain KIKO mice or Wild type mice with similar genetic background. KIKO mice do not
show any abnormal function at young age (
Does the animal have a genetic mutation (either induced or spontaneous) that will lead to a phenotype which
adversely 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 are
the age- dependent neuromuscular dysfunction (abnormal gait and loss of muscle strength) in KIKO mice. The findings
are 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. The NIH Guidelines for Research Involving Recombinant or Synthetic Nucleic Acid Molecules requires the
following:
Transgenic experiments involving the generation of transgenic animals require notification of the Institutional
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, pose
enhanced risks to personnel (humans) require IBC approval prior to initiation!
Permanent Housing
Location(s) of Permanent Housing:
Aurbach Vivarium (Fontaine Vivarium, 450 Ray C. Hunt Drive)
MR4 Vivarium
Snyder 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 of
animals or all animals? How many animals will be involved? What is the duration of the exemption? page 19/34
Voluntary running studies requires precise recording of running distance for each individual mouse throughout the
experiments, which prohibits social housing. Voluntary weight lifting studies requires precise recording of weight lifting activity for each individual mouse throughout
the 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 of
enrichment 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 mice
promoting exploratory activities. Other than group housing, which is not possible due to the need of accurate recording, we will provide Innodome™ to
enrich the living environment. Live Animal Work Outside of Vivarium: YES Location(s) of Live Animal Work Outside of Vivarium: Aurbach (Fontaine, 450 Ray C. Hunt Dr.): G136
MR-4: 6031, G147G
Sheridan 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 Main Procedure
Procedures, which require Humane Endpoints and Criteria for Euthanasia, to be performed on animals used in
this procedure section: NONE
Main Procedure Description: Breeding colony management 1. Standard breeding colony husbandry will be followed including IACUC policy on
breeding 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 of
the 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. 5. Pups that will not be used for experiments or breeding will be euthanized in a carbon dioxide chamber upon
confirmation 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. page 20/34
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 with
voluntary running as exercise training). The cages will be changed every two weeks. Since we have one mouse per
cage, 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 cervical
dislocation. 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 cervical
dislocation 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 placed
on 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 a
0.3 cc insulin syringe with a 30 gauge needle subcutaneously above the FDB muscle at the bottom of the feet and wait
for 30 min. 3. Endotoxin-free plasmid DNA (1 µg/µl in normal saline) will be injected into the gastrocnemius muscles (65-100 ug
each) 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 pulse
stimulator through a Model 533 2-needle array (BTX) (Eight pulses at 100 ms, 1 Hz and 100 volt were applied to the
muscle). 5. Allow mice to recover 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 cervical
dislocation 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 a
speed 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 the
treadmill with 5% inclination and 0.5 mph speed for 30’. The speed will be increased by 0.1 mph every 30’ (not exceed
1 mph) until exhaustion, which is defined as that mice refuse to run when encouraged by brushing the tail (a brush on
the 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 Glucose
Meter (Bayer) and Lactate Scout (EKF Diagnostic, Magdeburg) from the tail vein every 20 min as well as at the end of
the 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
test of muscle function (Siegel, 2009; Krediet, 2005; Carlson, 2010; Carlson, 1990) may reveal defects in muscle
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 at
the other end 2. The mouse is placed at the entry of a metal mesh tube. page 21/34
3. Once the mouse has fully entered the tube, the hooked end of the steel thread is attached to an isometric tension
transducer (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 the
enclosure and gripping the wire mesh. 5. Forward pulling movements are elicited 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 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 as
described 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 in
MR4 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 before
injection). Set up the timer. 8. At 15, 30, 60, and 120 minutes blood glucose is sampled from the tail of each mouse by
gently 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 significant
differences in body weight in mice 1. This will be done in the Ainimal 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 Columbus
Instruments) 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 the
page 22/34
actual 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 automated
non- invasive fashion. Metabolic parameters (O2 consumption 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-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 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 the
scanner (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 and
instruction of Dr. Joe Hart from orthopedic surgery. 1. Mice are fasted for 16 hours overnight and moved to MR4 6031
surgery room. 2. Anesthetize the mice under isofluorene anesthesia system. 3. Insert a sterile 20 gauge needle percutaneously into the left gastrocnemius muscle (~2 mm deep) and insert a sterile
EMG 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 universal
interface 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 of
analgesic 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 then
incrementally increase the speed 10 rpm each time until we see clear rhythmic EMG activity. This speed will be used
for all mice in the exercise group. We do not know the speed yet, but this test will allow us to find a rotation speed that
leads 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 other
hindlimb 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) around
the neck for each mouse under isoflurane anesthesia and make sure that the collar can be turned freely. Mice will be
housed in the custom-made weight lifting cage for 3 days before the experiment with food and water provided in the
cage. 2. On the first training day in the evening, food will be move to the top of the cage and the mouse will get the
food by pushing the lever plate with no weight added and the lifting activity recorded. Water will be provided in the
cage with free access. 3. Food will be added back to the cage next morning. 4. On the second day of training, the
same procedure will be repeated with a weight of 25% of the body weight added to the lever plate and weight lifting
activity recording. 5. Food will be added back to the cage next morning. 6. Step 2 and 3 will be repeated with 50, 75
and 100% of body weight on the 3rd, 4th and 5th day. The weight will stay at 100% of body weight for the rest of the
training day for a total of 10 days. 7. Mice kept in similar cage with food and water provided with free access will serve
as sedentary control. 8. We will humanely euthanize the mice under isofluorane anesthesia with cervical dislocation
followed by muscle harvesting at 24 hours after the last training. page 23/34
followed by muscle harvesting at 24 hours after the last training. Laser Doppler scaning imaging for blood flow in FA mice 1. We will measure blood flow one time at ~6.5 months of
age for KIKO and WT mice at MR5 G225. 2. Blood flow in both the hindlimbs will be measured using a PeriScan PIM-II
blood perfusion monitor (laser Doppler scanner) (Perimed, Inc. North Royalton, OH). This technique for measuring
blood flow is a purely non-invasive procedure that uses a light beam that moves across the skin to measure blood flow
a few millimeters below the skin. 3. No surgery is involved in this measurement; however, to prepare the animal for the
scan, each animal will receive light sedation with ketamine at half the dose used for normal surgical procedures to
prevent undue stress during immobilization required for the measurement. 4. Three sites from the upper, middle and
lower portions of the hindlimb will be selected for measurement. The average blood flow of these three sites will be
calculated. The estimated duration of the measurement procedure will be 10 - 15 minutes. 5. Mice will be euthanized
after the measurement. Post-Procedural Details: There are NO surgical procedures involved, but some procedures require anesthesia. Mice are monitored carefully
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 muscle
contractility assay in vivo. The experiment is also one time, short term. We will euthanize the mice immediately after
the experiment for sample harvesting. Procedural Training: Mei Zhang and Josh Drake have extensive animal research experience with the procedures described above. They
will provide the training for other and future lab members. Animal Handler Responsibilities
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
UVa - Animal Facility Rules and Procedures Training: 10/29/2015
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
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
UVa - Animal Facility Rules and Procedures Training: Required before entering any vivarium at UVa, provided
in-person by the vivarium supervisors, unique for each vivarium at UVa.
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
UVa - Animal Facility Rules and Procedures Training: Required before entering any vivarium at UVa, provided
page 24/34
UVa - Animal Facility Rules and Procedures Training: Required before entering any vivarium at UVa, provided
in-person by the vivarium supervisors, unique for each vivarium at UVa.
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
LEWELLEN, BEVAN (bml4dv)
ACUC Defined General Requirements
UVa - Orientation Seminar: 02/05/2015
UVa - Animal Handler Refresher Training: Not required, UVa - Orientation Seminar is less than 3-years old UVa - Working Safely with Animals Training: 02/03/2015
UVa - Animal Facility Rules and Procedures Training: 02/26/2015
LATA - Mouse Training: 02/03/2015
LATA - Anesthesia and Analgesia of Rodents: 02/03/2015
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: Required for protocol approval UVa - Working Safely with Animals Training: 01/26/2014
UVa - Animal Facility Rules and Procedures Training: Required before entering any vivarium at UVa, provided
in-person by the vivarium supervisors, unique for each vivarium at UVa.
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, JIUZHI (jz9x)
ACUC Defined General Requirements
UVa - Orientation Seminar: 07/08/2015
UVa - Animal Handler Refresher Training: Not required, UVa - Orientation Seminar is less than 3-years old UVa - Working Safely with Animals Training: 07/10/2015
UVa - Animal Facility Rules and Procedures Training: 07/10/2015
LATA - Mouse Training: 06/09/2015
LATA - Anesthesia and Analgesia of Rodents: 06/09/2015
Biomethodology (restraint, blood collection, gavage, and/or injection)
Breeding Colony Management
Critical Clinical Monitoring - Non-surgical (Humane Endpoints)
Euthanasia
ZHANG, MEI (mz3a)
ACUC Defined General Requirements
UVa - Orientation Seminar: 02/18/2009
UVa - Animal Handler Refresher Training: 08/02/2013 UVa - Working Safely with Animals Training: 02/18/2009
UVa - Animal Facility Rules and Procedures Training: Required before entering any vivarium at UVa, provided
in-person by the vivarium supervisors, unique for each vivarium at UVa.
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
page 25/34
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
UVa - Animal Facility Rules and Procedures Training: 11/12/2015
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 PROCEDURE # 4
Species: Mice
Procedure ID: NO protection against catabolic muscle wasting
Does 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 those
specified in the ACUC Policy on Breeding and Weaning of Rats and Mice? NO
USDA Pain and Distress Category:
B: 60 Animals/Year
C: 196 Animals/Year
D: 40 Animals/Year
E: 732 Animals/Year 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 all
peripheral tissues/organs. To ascertain if muscle-derived SOD3 expression provide protection against multiple organ
dysfunction syndrome (MODS), we choose the gold-standard for human sepsis and MODS in humans, mouse sepsis
model of cecal ligation and puncture (CLP). CLP will be employed in EcSOD transgenic, knockout and wild type
littermate mice followed by analyses for the survival and biochemical and morphological evidence of protection by
muscle-derived EcSOD. This research direction is entirely novel. The findings will be extremely valuable for future
development of therapies for this detrimental disease that affect millions of human patients each year. After CLP, we
will administer 4 mg/kg mouse Ketoprofen subcutaneously every 12 hours or 24 hours or as required. Death may
occur for the mice for survival curve, which is an outcome of MODS. We do not intend to terminate the experiments
prematurely to alter the outcome. The protection observed in muscle-specific EcSOD transgenic mice against MODS could be consequences of
secondary changes the transgenic mouse skeletal muscles and peripheral tissues/organs. To elegantly confirm that
muscle-derived EcSOD provides direct protective function, we propose to take advantage of parabiosis to
unequivocally confirm the findings. Systemic administration of recombinant EcSOD in a clinical trial has proven to be
unsuccessful due to lack of continuous supply and sufficient targeting of the molecule to the action site of reactive
oxygen 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. We
have also received a favorable, fundable score on an NIH R01 grant. However, we need to perform formal
investigations 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 that
render oxidative/exercise trained myofibers resistant to oxidative damage and catabolic muscle wasting in a iNos/nitric
oxide-dependent manner. Most of the findings were obtained in culture myofibers and some were in mice. We plan to
further investigate this important phenomenon with a focus on the therapeutic value of antioxidant genes in living
animals with the wild type transgenic or knockout backgrounds. Transgenic mice breeding colonies for maintenance Myogenin-Cre mice (from Eric Olson)---Maintained as described in
protocol #3 MCK-Cre mice (from the Jackson Laboratories)---Maintained as described in protocol #3 Floxed SOD3
mice (from David Harrison) GSNOR KO mice (from Jonathan Stamler’s lab) MCK-SOD3 mice (Yan lab at UVa) 2
breeding trios for each line (1 male and 2 female) will be replaced every 6 months = 6 mice x 5 lines x 2 sets/year = 60
mice page 26/34
mice Endothelial injury and multiple organ dysfunction induced by CLP in muscle-specific SOD3 transgenic and KO mice
Four (4) genotypes: SOD3 TG and wild type littermates (WT) and SOD3 KO and WT Two (2) treatments: CLP and
mock 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 time
point x 17 mice per time point = 408 mice Prevention of cardiac dysfunction induced by STZ injection in muscle-specific SOD3 mice Two (2) genotypes: Wild
type and muscle-specific SOD3 mice Two (2) treatments: Saline and STZ injections One (1) time point: 10 weeks We
need n = 10 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 10% and change of 20%). Total mice: 2
genotypes 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 dysfunction
induced by LPS injection Two (2) groups: Wild type (WT)-WT followed by LPS injection and EcSOD-WT followed by
LPS 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 sample
size 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 muscle
Four (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 published
findings, 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: 764
mice Alternatives to Painful and Distressful Procedures
SEARCH # 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 or
cecal ligation and puncture Summarize your findings: Searching Sod3 and multiple organ dysfunction
syndrome led to 1 hit with a paper on SOD3 variant
R213G on aging mice, which is completely consistent
with the them of our project, but on a different issue
(SNP of Sod3 gene variant). Adding the other terms led
to no hit. Alternatives to Painful and Distressful Procedures
SEARCH # 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 or
cecal ligation and puncture Summarize your findings: Did not find directly any relevant publications. Did you find a less painful/less distressful alternative that
could accomplish the goals of your animal use protocol? NO Did you find a less painful/less distressful alternative that
could accomplish the goals of your animal use protocol? NO Molecular Imaging Core:
Will you utilize the Molecular Imaging Core (MIC) in this Species Procedure? YES Imaging modalities to be used: Magnet Resonance Imaging and Spectroscopy (MRI-Clinscan 7.0T) NOTE: The Molecular Imaging Core imaging modalities will be conducted in accordance with Dr. Stuart Berr's ACUC
protocol #3539. All ordering and use of radioactive material (RAM) will be conducted under the Molecular Imaging Core
User Authorization Number.
If the PI intends to return imaged animals that possess either RAM or biohazardous material to the vivarium, the PI must
notify the vivarium supervisor at least one day in advance of the imaging session in order to have suitable return
housing available. page 27/34
Hazardous Chemicals: YES I (YAN, ZHEN) accept responsibility for the following: 1. Notifying the vivarium supervisor prior to introducing hazardous chemicals into the vivarium. 2. Ensuring that each cage that houses animal(s) harboring experimentally introduced hazardous chemicals is
properly labeled. 3. Contacting the vivarium supervisor for assistance with obtaining hazardous chemical labels, if needed. 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 contaminated
bedding 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 fume
hood 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 white
buckets 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 precautionary
measures 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 extreme
precautionary measure to ensure safety. We will hang a door sign and the cages will be clearly labelled with the date
and time of administration. Safety glasses, gloves and a protective gown will always be used. The solution will be
prepared in the fume hood in the lab. while performing injections. All the personnel involved in this procedure will
undergo extensive training by the experienced personnel in the lab before performing the injection independently. Does this research involve the creation (de novo generation by gene manipulation) or generation (creation of
novel transgenics by breeding two transgenic lines) of transgenic animals (animals in which the germ line is
altered by genomic DNA insertion or removal)? YES
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 protects
skeletal muscle and other peripheral tissues/organs in the model of LPS injection-induced endotoxemia (a model of
severe sepsis). However, the gold standard (more relevant to human sepsis) in the filed is the model of cecal ligation
and pucture (CLP). We expect that the EcSOD expression in skeletal muscle will be sufficient and necessary for
protections against multiple organ dysfunction in this model. We will cross myogenin-Cre mice with floxed EcSOD
mice to obtain muscle-specific EcSOD KO mice. We also want to test if GSNOR KO mice, which will have elevated
levels of NO donor and elevated EcSOD expression, have resistance to CLP-induced MODS. The findings will provide
valuable insights about human therapy. The NIH Guidelines for Research Involving Recombinant or Synthetic Nucleic Acid Molecules requires the
following:
Transgenic experiments involving the generation of transgenic animals require notification of the Institutional
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, pose
enhanced risks to personnel (humans) require IBC approval prior to initiation!
Permanent Housing
Location(s) of Permanent Housing:
MR4 Vivarium
Animal Husbandry (Mice): STANDARD HUSBANDRY Live Animal Work Outside of Vivarium: YES Location(s) of Live Animal Work Outside of Vivarium: MR-4: 1184, 3143, 6031, G147G Pharmaceutical(s) Used: (Mice) Pre-Anesthetic:
NONE SELECTED page 28/34
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 Main Procedure
Procedures, which require Humane Endpoints and Criteria for Euthanasia, to be performed on animals used in
this procedure section: NONE
Main Procedure Description: Breeding colony management 1. For experiments involving genetically engineered mice, standard breeding colony
husbandry will be followed including IACUC policy on breeding 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 pregnancy, they will be separated from each other to ensure correct tracing of
the 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. 5. Pups that will not be used for experiments or breeding will be euthanized in a carbon dioxide chamber upon
confirmation of the genotype. Protocol for STZ injections 1. Perform daily injection for 5 days for wild type and genetically engineered mice with
normal saline (NS) or STZ (50 mg/kg). 2. On the day of euthanasia, measure blood glucose and ketone bodies using commercial glucose and ketone body
stripes in the tail vein blood. 3. 10 weeks after STZ injection, euthanize mice under anesthesia by isofluorene or i.p. injection of pentobarbital
followed 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 speed
at 1 mph. 4. The test is terminated when mice show signs of exhaustion and refuse to run upon stimulation of the tail by a
custom-built brush. Normal mice run about 1.5 miles. Maximum time on the treadmill will be around 1.5 hours. Mice
with 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-invasive
physiological test of muscle function (Siegel, 2009; Krediet, 2005; Carlson, 2010; Carlson, 1990) may reveal defects in
muscle 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 at
the other end 2. The mouse is placed at the entry of a metal mesh tube. page 29/34
3. Once the mouse has fully entered the tube, the hooked end of the steel thread is attached to an isometric tension
transducer (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 the
enclosure and gripping the wire mesh. 5. Forward pulling movements are elicited 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 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. 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). 3. The mouse will be returned to the cage immediately after the test. 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 has
perfected this technique, Dr. Mei Zhang with very extensive training of animal surgery is able to do the procedure as a
routine in this laboratory. 1. The surgery will always performed for parabiosis between mice of the same gender and in the same litter to
minimize rejection. 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 the
subcutaneous 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 are
attached by a single 2-0 silk suture and tie (joints are sutured together to reduce direct pulling on the sutures of the
skin). 5. Press the dermis of the parabiotic partners together (excluding epidermal layers in the junction of the dermis) and
close 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 research
community as a model of sepsis with the closest relevance to human sepsis and MODS. The protocol is available in
Current Protocols in Immunology 19.13.1-19.13.11, November 2012. Dr. Borna Mehrad here at UVA has personal
experience 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 cm
incision). 3. The cecum will be carefully isolated and a 3.0 cotton ligature will be placed below the ileocecal valve to prevent
bowel 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 isolated
without ligation and puncture. 6. All mice will receive subcutaneous injections of 1 ml of warm (37°C) saline with tramadol hydrochloride (20 mg/kg
body weight) during the postoperative period. page 30/34
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 is
well-established by Dr. Klibanov here atUVA. We plan to take advantage of this novel imaging approach in mice
following either LPS or CLP (as described above) without adding more mice to the protocol to maximize the use of the
mice. 1. At 6h after LPS injection or 12/24 hr after the induction of CLP, the mouse will be placed on a stage for
contrast-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 tailvein
catheter 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 microbubble
solution will be injected. 7. The anterior cervical and abdominal region of the mouse will be depilated using an electric clipper or depilatory
cream. 8. Ultrasound imaging will be used to detect microbubble accumulation in several fields around the kidneys 10 min
after 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
harvest. Ischemia/reperfusion in skeletal muscle induced by tourniquet This is an established protocol by Tran et al. (Eur J
Pharm 2011, 650:328-34). This approach will allow us to induced ischemia/reperfusion injury that mimic certain
aspects 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 a
McGivney hemorrhoidal ligator (Crawford et al., 2007). Mice are allow to recover from the anesthesia and they walk
with 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 in
mice. Aurora Scientific 305C Dual-Mode Muscle Lever System is the state-of-the-art system for non- invasive
assessment of muscle contractile function in vivo (http://www.aurorascientific.com/). With the recent funding from
School of Medicine General Equipment Solicitation 2012, we have set up this system as shared equipment in MR4
6031B with heated stage and isoflurane anesthesia. We will add this assay to the existing protocol for our mice
following hindlimb ischemia reperfusion (196 mice) before euthanization of sample harvesting to maximized the data
collection capability and elevate the significance of the findings. Here are the simple steps involved. 1. Anesthetize the
mice using 2% isoflurane and oxygen flow of 0.5 L/min. 2. Place mice in supine position on the heated (38C) stage of
the 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 the
fixed limb flanking the sciatic nerve. 4. Obtain single twitch, force-frequency curve and fatigability using the Aurora
Scientific in vivo muscle contractility system. 5. Let the mice recover from anesthesia completely. We need to wait for
about 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-invasive
page 31/34
Echocardiography This is non-invasive protocol for assessment of cardiac function in mice. We add this non-invasive
protocol to the existing protocol for the STZ study for cardiac function assessment (40 mice) 1. Remove the fur of the
chest with a hair clipper and Nair. 2. Anesthetize the mouse under isoflurane 2% isoflurane and oxygen flow of 0.5
L/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 anesthesia
completely. 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 the
expected outcome of the treatments, we do not intend to intervene. We have not had premature death after these
procedures previously. But we predict that CLP will lead to some death after 96 hours. We need this information to
determine if overexpression of SOD3 in skeletal muscle prevent multiple organ dysfunction. After CLP, we will
administer 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 significant
discomfort, such as frequent licking of the injection site with signs of infection, we will terminate the experiment by
euthanasia. 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 water
consumption 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 and
number of chow pallets in the cage). Frequency of observation We will monitor the change in body weight (everyday for short- term experiment with
duration 7 days) and record the changes in body weight in our lab animal record book. For long-term experiments, food
and 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 48
hours and loss of body weight and reduced food and water intake are part of the pathological process. For these
experiments, 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 , then
every 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 each
of 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 by
necessary sample harvesting. Procedural Training: Mei Zhang and Josh Drake in our laboratory have extensive experience in following these procedures. They will teach
the other personnel in the lab listed in this protocol. Animal Handler Responsibilities
page 32/34
LAKER, RHIANNA (rcl6n)
ACUC Defined General Requirements
UVa - Orientation Seminar: 06/03/2011
UVa - Animal Handler Refresher Training: 06/04/2014 UVa - Working Safely with Animals Training: 05/10/2011
UVa - Animal Facility Rules and Procedures Training: Required before entering any vivarium at UVa, provided
in-person by the vivarium supervisors, unique for each vivarium at UVa.
LATA - Mouse Training: 05/10/2011
LATA - Anesthesia and Analgesia of Rodents: 05/10/2011
Biomethodology (restraint, blood collection, gavage, and/or injection)
Breeding Colony Management
Critical Clinical Monitoring - Non-surgical (Humane Endpoints)
Euthanasia
Supervise and/or Train Others
WILSON, REBECCA (rjw7jc)
ACUC Defined General Requirements
UVa - Orientation Seminar: 03/12/2013
UVa - Animal Handler Refresher Training: Required for protocol approval UVa - Working Safely with Animals Training: 01/26/2014
UVa - Animal Facility Rules and Procedures Training: Required before entering any vivarium at UVa, provided
in-person by the vivarium supervisors, unique for each vivarium at UVa.
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, JIUZHI (jz9x)
ACUC Defined General Requirements
UVa - Orientation Seminar: 07/08/2015
UVa - Animal Handler Refresher Training: Not required, UVa - Orientation Seminar is less than 3-years old UVa - Working Safely with Animals Training: 07/10/2015
UVa - Animal Facility Rules and Procedures Training: 07/10/2015
LATA - Mouse Training: 06/09/2015
LATA - Anesthesia and Analgesia of Rodents: 06/09/2015
Biomethodology (restraint, blood collection, gavage, and/or injection)
Breeding Colony Management
Critical Clinical Monitoring - Non-surgical (Humane Endpoints)
Euthanasia
ZHANG, MEI (mz3a)
ACUC Defined General Requirements
UVa - Orientation Seminar: 02/18/2009
UVa - Animal Handler Refresher Training: 08/02/2013 UVa - Working Safely with Animals Training: 02/18/2009
UVa - Animal Facility Rules and Procedures Training: Required before entering any vivarium at UVa, provided
in-person by the vivarium supervisors, unique for each vivarium at UVa.
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
UVa - Animal Facility Rules and Procedures Training: 11/12/2015
LATA - Mouse Training: 10/15/2015
LATA - Anesthesia and Analgesia of Rodents: 10/15/2015
Biomethodology (restraint, blood collection, gavage, and/or injection)
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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
1. 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 the
policies and procedures of the University of Virginia Institutional Animal Care and Use Committee (IACUC). 2. All responsibilities for public health hazards and precautions associated with the performance of this protocol rest
with the Principal Investigator and his/her department. All biohazardous agents, radioactive materials, and
potentially hazardous chemicals used in this animal research protocol have been listed in the protocol. 3. The use of other biological or non-living model systems and alternative methods have been considered in the
preparation of this protocol and have been found not to be usable for scientific reasons at this time. 4. All procedures involving live animals will be performed under the supervision of a trained and qualified scientist or
their trained and qualified designee. 5. All personnel associated with the research in this protocol are enrolled in the University's health surveillance
program for exposure to laboratory animals administered by Occupational Health and Student Health. 6. All personnel associated with the research in this protocol have completed the University's required laboratory
animal training program. Certain species and research projects may require additional training as determined by
the Institutional Veterinarian or the IACUC. 7. 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 such
changes include, but are not limited to, changes in: a. USDA category for pain and distress b. Species c. Experimental procedure d. Number of animals e. Acute to chronic experiments f. Personnel g. Post-operative care of animals undergoing survival surgery h. Husbandry 8. The IACUC is required by federal law to inspect all animal facilities and animal procedure areas (including
research 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. FAILURE TO RECEIVE APPROVAL PRIOR TO MODIFICATION OF EXPERIMENTS INVOLVING LABORATORY
ANIMALS 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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