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Institutional Animal Care and Use Committee
Protocol Application
PI Last Name:___________________
PI First Name:___________________
Protocol Title: Vascular Smooth muscle phenotype modulation and extracellular matrix remodeling in coronary collateral growth
Directions: Please answer all questions as completely as possible. Fields will expand to allow for unlimited text to
be answered in all descriptive question fields. You may cut and paste text into fields. Any figures may be appended
in IRBNet as separate documents.
Administrative Information
1. In conducting this project, I agree to have my laboratory and all animals assigned to this protocol available for examination
by IACUC members or Department of Comparative Medicine personnel at all times during the project. I understand that
according to the regulations under which the IACUC is authorized, any time any animal is deemed to be in unacceptable
distress or is receiving inhumane treatment, in the professional opinion of the attending veterinarian, the animal may be
subjected to immediate euthanasia and/or the procedure immediately halted. The IACUC will be informed of any action
taken. The Director of University Biological Resources may suspend a protocol, for cause, until the IACUC has reviewed the
incidents leading to the suspension.
If the animals assigned to this protocol are supported by a grant or contract, I further certify that the procedures described in
this protocol agree with the procedures described in the grant or contract application. I agree to provide the IACUC with one
copy of my grant or contract proposal to allow verification that the description of the animal use portion of the grant or
contract agrees with the protocol. I understand that changes in animal use procedures during the grant or contract period
must be described on the annual monitoring/review form.
I understand that IACUC approval of this protocol does not guarantee availability of resources or services from the
Department of Comparative Medicine.
I certify the information provided herein is correct. I agree to accept responsibility for this project in accordance with all
federal, state and local laws and regulations, NIH/ILAR guidelines, PHS Animal Welfare Assurance, and institutional policies
and procedures.
I certify I have read and agree to the above:
I agree
I disagree
Objectives
Please describe your objectives (255 characters MAX)
1) Determine the importance of vascular smooth muscle (VSM) phenotype modulation and ECM remodeling for collateral growth
2) Restore normal VSM phenotype modulation and ECM remodeling in the metabolic syndrome rat model to restore collateral growth
Rationale
Please describe your rationale for pursing the following protocol procedures (500 character MAX)
Collateral circulation can provide adequate perfusion to ischemic heart tissue distal to the site of coronary artery occlusion. For functional
collaterals to form, vascular smooth muscle cells (VSMCs) must undergo precise phenotypic modulation. This is accompanied by
extracellular matrix (ECM) remodeling. Our preliminary data indicate that both VSMC phenotype switching and ECM remodeling are
altered in the metabolic syndrome rat model, which fails to develop coronary collaterals.
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Description
Please provide a general description of the following procedures.
Coronary collateral growth (CCG), an adaptive response to transient, repetitive coronary artery occlusion/myocardial ischemia (RI) such as
occurs in stable angina, is impaired in human metabolic syndrome and in animal models of the metabolic syndrome. Even though
multiple factors that contribute to impaired collateral development in the metabolic syndrome are known, all interventions to attempt to
induce collateral growth in metabolic syndrome patients and animal models to date have been inadequate.
In order to form functional coronary collaterals with an enlarged lumen, vascualr smooth muscle cells (VSMCs) must switch to the
synthetic, proliferative and migratory phenotype in the early phase of collateral remodeling then switch back to the contractile
phenotype characterized by low proliferation and migration rates in the later phases of collateral remodeling. An integral component of
collateral growth is also extracellualr matrix (ECM) remodeling. We hypothesize that abnormal VSMC phenotype regulation and
abnormal ECM remodeling may provide the common link behind the failure of attempts to restore collateral growth in the metaboic
syndrome.
Russell (JCR) and Zucker obese fatty (ZOF) rats will be used as two rat models of the metabolc syndrome. Both rat phenotypes are obese
with fatty livers, highly insulin resistant, dyslipidemic (high LDL, vLDL and triglycerides, low HDL). The JCR rats are also mildly
hypertensive and slightly hyperglycemic. The Spreague-Dawley (SD) rats will used as healthy controls.
We mimick transient, repetative coronary artery occlusion in the rat model by placing an occluder over the left anterior descending
coronary artery (LAD). Details of the survival surgery are described in question #11. Following surgery, rats are allowed to recover for 2
days (in our experience, this time period allows the animals to recover from post-operative pain and no longer require analgesics for pain
management, resume normal eating, drinking and grooming, as well as allows for complete clearance of the acute inflammatory reaction
at the occluder implantation site). The RI protocol for rat consists of: 8 40 sec occlusions, administered every 20 minutes for a total of 2
hours and 20 minutes. This is repeated every 8 hours over a period of 3, 6, 9 or 10 days. Sham (control) animals will be instrumented but
will not undergo the RI protocol.
The goals of these studies are to: 1) determine whether the regulation of VSMC phenotype switching and ECM remodeling by MMP
activation over the time course of CCG is altered in normal vs. the metabolic sydrome animals, and 2) restore VSMC phenotype regulation
and MMP activation in the metabolic syndrome animals to that observed in normal animals with the goal of restoring CCG in the
metabolic syndrome.
Our preliminary data indicate that VSMC phenotype during collateral growth is critically regulated by microRNAs (miRs)-145 and -21.
Moreover, miR-145 and miR-21 expression is markedly altered (miR-145 is downregulated while miR-21 is upregulated) in the JCR rats,
which correlates with abberrent VSMC phenotype in JCR rats. Therefore, we will use miR-145, delivered in adenoviral vectors (miR-145Adv, 2x10^12 particles/mL), and the miR-21 antagonist (antimiR-21, 5 mg/kg) to manipulate VSMC phenotype. Delivery of both
constructs to rats is approved by the Institutional Biosafety Committee (protocol number B1106). Regarding the experiments involving
Adv-mediated gene delivery, we have extensive experience with this procedure. We have previously used the same Adv constructs,
approved in the PI’s current IACUC protocol (protocol number XXXX), as well as virtually identical Adv constructs (identical Adv
backbone with a different gene insert), which are published in XXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXX. From a
practical stand-point, Adv delivery is identical to delivery of microspheres used to measure coronary blood flow and involves direct
micro-injection (26 GA insulin syringe) of 100 μl saline+Adv construct solution over 20 sec during LAD occlusion (occluder is inflated) at
the time of initial surgery. Treatment with these Adv constructs does not cause any harm to the animals. Adv-treated animals will be
housed in a separate/isolated BSL-2 approved space for the duration of infection, as in the past. The Adv constructs cannot be
aerosolized or shed from the animals, and therefore do not present any significant risk to the DCM staff. AntimiR-21 delivery is technically
identical to miR-145-Adv delivery. Although antimiR constructs have not yet been delivered via intracardiac injection, i.v. antimiR-21
delivery at 2X the dose (10 mg/kg) via tail injection did not cause any harm to the animals (Obad et al, Nat Genet. 2011;43:371-378).
AntimiR-21 presents no biological hazard to DCM staff.
Our recent findings also show that MMP activation is altered in the metabolic syndrome. Pharmacological inhibitors of MMPs 3, 7, 8, 12
and MT-1-MMP (MMP14) will be used to block their activation to asses whether their activation is required for CCG in the normal rats and
whether restoration of their activation to the normal profile can restore normal ECM remodeling and CCG in the metabolic syndrome
(JCR and ZOF rats). These inhibitors will be delivered via a jugular vein catheter implanted at the time of occluder implantation (initial
surgery). Details of catheter implantation are described in question #11. All of these inhibitors have been used chronically in rodents in
vivo and have not caused any adverse effects at the doses given for the time span of <10 days of treatment. We will treat the rats for a
maximum of 4 days (see table in separate file).
We will use these animals for measurements of: 1) coronary blood flow, as an indication of coronary collateral growth (CCG), 2) VSMC
phenotype (Western blot), 3) VSMC proliferation and apoptosis (TUNEL, immunohistochemistry, Western blot), 4) matrix
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metalloproteinase (MMP) activation (Western blot), 5) microRNA expression (qPCR, in-situ hybridization).
Coronary blood flow will be measured by microspheres (color, non-radioactive) at day 0 and day 10 of the RI protocol. Two different
microspheres (5 x 105, approximately 150 μl), 15 μm diameter, which lodge pre-capillary and thus stay in the heart, will be injected into
the left ventricular cavity by direct micro-injection (26 GA insulin syringe) over 20 sec during LAD occlusion (occluder is inflated) at the
time of initial surgery (first type) and at day 10 of RI (second type) under anesthesia (75 mg/kg ketamine and 5 mg/kg xylazine + 1-2%
sevofluorene), immediately prior to euthanasia. This method of microsphere injection results in no hemodynamic change or arrythmia, as
previously confirmed by ECG. The exact number of animals used for these measurements is specified in the table below.
All other measurements will be performed post-mortem on isolated heart tissue.
For these experiments, we will need a total of 1,125 rats (484 SD and 641 JCR or ZOF (we will use JCR and ZOF rats interchangebly
depending on availability of the JCR rats because our past and preliminary results show no difference between the two phenotypes)).
The JCR and ZOF numbers are estimated based on the projected availability of the JCR rats over the next 3 years (6 per month, 8 months
during the year excluding May-September due to temperature-related shipping restrictions). The treatments groups, the number of
animals per treatment group and the measurements to be acquired are described in detail in the table attached as a separate file. Where
obvious groups of animals are missing or proposed to be done in only one rat phenotype, this is because our preliminary data indicate
that doing them in both phenotypes would be illogical and a waste of animals. Minimal numbers of animals are used to obtain all of the
necessary measurements. Additional animals are added due to surgery survival rate based on 8 years of experience with these rat
models.
2. Describe the methods used to determine the number of animals needed for this project. List statistical tests or other methods
used to determine these numbers.
The animal numbers needed for the study were determined from a power analysis to determine the required sample size for each
treatment group to achieve a power of 0.90 to 0.95 with a probability of a type 1 error of 0.05. Based on preliminary and previously
published results, a total of 8 animals is required per treatment group to achieve a power of at least 95% for testing all between and
within group comparisons for in vivo measurements (coronary flow), and 8 animals per treatment group are required for all Western blot,
immunohistochemistry and miR measurements, using p<0.05. An additional 10% of animals are added to the SD, 20% to the JCR and
30% ZOF groups to account for our surgical survival rate of ~90% in the SD, ~80% in the JCR and ~70% in the ZOF phenotype.
Alternative Methodologies
3. Describe what alternatives to the use of animals were considered (e.g., computer modeling, tissue/cell culture, etc.) and
explain why they cannot be used in place of this animal use protocol.
Both computer modeling and cell culture experiments were considered. Because of the complex make-up of animal and human
physiology (the interaction of many hormones, growth factors, multiple cell types, etc), there is no way to model the effect of hormones
and growth factors on coronary vascular growth in a non-animal model. For this reason, and to determine the therapeutic potential of
such treatments, we must perform experiments on animals. Cell culture experiments cannot be used because they cannot mimic the
complex interaction mentioned above. There are no cell culture models that adequately address the complexities on the intact animal
involving multiple cell types, growth factors, and interactions between the heart and the neuroendocrine system. Insufficient knowledge
is available to conduct computer simulation type experiments.
4. State why a lower order would not be used in this study. Ex. mice instead of swine.
The circulatory systems of lower species of animals (non-mammals) are inadequate for the study of the collateral circulation with respect
to mammalian VSM phenotype modulation and ECM remodeling which is relevant to abnormalities observed in the metabolic syndrome.
5. Describe considerations given to the use of alternatives to painful procedures which will be performed on the animal(s) in this
project. If such alternative procedures cannot be used, provide justification for the use of painful procedures.
All surgical procedures will take place under general anesthesia; thus the only pain associated with this protocol is postoperative
recovery, during which the animals will be given appropriate analgesics. There are no alternatives to left thoracotomy for occluder
placement over the LAD. There are alternatives to i.v. drug delivery via the jugular vein catheter; however, these involve daily tail vein
injections. We consider an implanted catheter to be far less stressful to the animal. Alternatives to the metal theters were considered, but
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were found to be inadequate, as they resulted in experimental failure (inability to inflate the occluder when the animals destroyed the
unprotected catheters), and thus higher numbers of animals used.
Non-Duplication of Research
6. Has the desired information been previously published?
No.
7. Regarding this proposed project, indicate which databases were searched, the keywords used for the search, and the date(s) of
those searches.
PubMed, Medline
3.8.2012
collateral growth AND - VSMC phenotype regulation, - contractile VSMC, - VSMC proliferation, - VSMC apoptosis, - miR-145, - miR-21,
miR intracardiac delivery
collateral growth AND - MMP 1, 3, 7, 8, 12, 13, MT-1(14)
metabolic syndrome AND MMP AND endostatin, angiostatin
Education, Training, and Assurances
8. I certify that each person associated with this project has completed the IACUC-mandated training in the appropriate
laboratory animal research techniques and procedures outlined in this submission. I further assure that documentation in
support of this training is on file in the IACUC office (with a copy maintained in the animal use laboratory).
Please indicate Yes training is complete and records on file or No, read 8a.
Yes
8a. If 'No', then the individual(s) cannot be involved in the animal-related portions of this project until
such time as the appropriate training has been completed and documentation forwarded to the IACUC office.
9. Are all personnel associated with the animal-related portions of this project enrolled in the College of Medicine Occupational
Health Program (OHP)?
Yes
9a. If 'No", identify personnel NOT participating in the Occupational Health Program (OHP). NOTE: A waiver must
be signed by each individual not participating in the OHP. The signed waiver must be on file in the Research
Compliance office. Individuals not participating in the OHP may be prohibited from working with certain species.
Please list the names of those with waivers.
Survival Surgery
10. Will any animal be subjected to a survival surgical procedure ( a surgical procedure from which the animal is EXPECTED TO
RECOVER from anesthesia)? Please indicate Yes or No.
Yes
10a. If 'Yes', Survival Surgery will be performed under which of the following conditions?
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Aseptic - REQUIRED FOR cats, dogs, rabbits, non-human primates, and livestock: requires a dedicated surgical environment, sterile
instruments, and sterile garments
Non-Aseptic
- Acceptable for rodent surgery; requires clean surgical environment using sterile surgical instruments.
10b. If 'Yes', At what locations will survival surgery be performed.
Please indicate building and Room numbers for all sites.
MSB XXXX
11. Provide a detailed description of the survival surgical procedure. Please provide the appropriate information under each of the
italicized headers below (boxes will expand to fit content).
Type of survival surgical procedure to be performed:
This is a survival surgical protocol carried out in rats. All surgical procedures will be performed using sterile surgical instruments in a
clean surgical environment and in accordance with DCM protocols. 3-4 month old, 650-700g, male Russell (JCR), 3-4 month old,
600-700g, male Zucker obese fatty (ZOF), and 3-4 month old, 300-350g male Sprague-Dawley (SD) rats will be used for chronic (3-10
days) implantation of a pneumatic occluder over the left anterior descending coronary artery (LAD). Some rats will also have a
jugular vein catheter inserted at the same time for i.v. drug delivery. Rats will be anesthetized, intubated, and prepared for surgery.
Involved areas (chest, neck and area between the scapulae) will be shaved and scrubbed with betadine. Rats will be anesthetized
with 75 mg/kg ketamine and 5 mg/kg xylazine given i.p (27 GA needle). Rats will then be orally intubated using a 16 GA 48 mm BD
Angiocath, ventilated (respiratory rate = 75/min; tidal volume = 8 ml/kg, 95% oxygen), and anesthesia during surgery will be
maintained by sevoflurane inhalation (1.0-2.0%). Physiological body temperature will be maintained with a heating pad throughout
the surgery. Appropriate depth of anesthesia will be monitored by toe pinch reflex.
For i.v. drug delivery, a catheter (PE-50 tubing flushed once with heparinized saline then filled with saline only) will be introduced
into the jugular vein. Following establishment of a plane of anesthesia, the vein will be isolated from the surrounding tissue by
blunt dissection. Sutures will be looped proximal and distal to area of injury to temporarily occlude flow when retracted. The vein
will then be tied off distally and the catheter will be inserted 1.5 cm into the jugular vein and secured in place using 4-0 silk sutures
in two places. The catheter will then be looped and secured again to the muscle over the sternum using 4-0 silk suture before it is
externalized together with the occluder catheter. The neck incision will be sutured closed with 3-0 silk suture or stapled with wound
closure staples. The catheters will be flushed with 3 cc saline every 24 hours. With the saline flush, they remain patent for up to 14
days.
Following placement of the jugular catheter (if needed), midline incision and traction of the skin and overlaying muscle followed by
left thoracotomy at the 3rd-5th intercostal space is performed, and the pericardium is incised to expose the base of the heart
between the pulmonary arterial outflow tract and the left appendage. 5-0 Prolene suture is then threaded around the proximal
portion of the LAD, and a balloon occluder is tied onto the surface of the heart. The correct position of the occluder and the success
of LAD occlusion are monitored by color change, blanching, of the left ventricular (LV) wall upon occluder inflation, and reactive
hyperemia (blushing of the myocardium during reperfusion) upon occluder deflation. The occluder catheter is tunneled through
the chest wall using a 14GA angiocath (which is the exact diameter of the catheter preventing pneumothorax occurance) and the
chest wall is closed using 3.0 silk suture (3-4 stitches on either side of the ribs bordering the inscision). The chest cavity is then
evacuated of air and negative pressure confirmed using a 22 GA angiocath connected to a 3 cc syringe. The occluder catheter is
tunneled subcutaneously and externalized between the scapulae. The skin is sutured, the animal is turned into prone position, and
a metal spring coil (tether) is secured to the back using a metal button as a base, to protect the occluder catheter from damage and
allow easy access for future occluder inflation. The tether is flexible and allows the animal access to all parts of the cage, but
prevents the animal from destroying the occluder. We have found that this tether is well tolerated by the animals and allows us to
make measurements without handling/stressing the animals. After closure, the incision sites are thoroughly cleaned with betadine.
No additional post-operative care is provided we have never had any problems (infection or irritation) with this approach, but the
incision sites are monitored daily. In case of infection or irritation (redness, swelling, evidence of excessive grooming), the incision
sites will be cleaned with betadine and topical antibiotic ointment applied daily and/or the animals will be treated according to
advice given by DCM veterinary staff. Sevoflurane is then withdrawn with continuous oxygen inhalation. Upon confirmation of
spontaneous respiration, the animal is extubated, and observed for a minimum of 2 hours, or until the rat is fully awake. Rats are
then allowed to recover for 2 days before the start of the repetitive ischemia/repetitive occlusion (RI) protocol.
Post-operative pain will be treated by injection of the analgesic Buprenex, 0.02 mg/kg sub-cutaneous, given post-operatively, after
the animals have been taken of the ventilator and have been breathing normally on their own for 10 minutes (at approximately 2-3
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pm on the day of surgery) but before the animals are awake. Subsequent doses of 0.02 mg/kg will be given the next day and on
subsequent days (~8-9 am and ~4-5 pm) if the animals shows signs of pain or distress such as increased respiratory rate, abnormal
resistance to handling, pain upon palpation of the surgery site, guarding of the surgery site, or refusal to eat and/or drink, anorexia
and/or lethargy and/or as determined by the USA veterinary staff. Animals will be euthanized in case of infection not responsive to
treatment, pain/distress or illness not responsive to treatment, damage to instrumentation, failure to eat or drink for longer than 48
hours, weight loss of more than 20% of pre-operative weight and/or in accordance with the opinion of the USA veterinary staff.
Surgical and post-surgical logs will be kept as required. The RI protocol for rat consists of: 8 40 sec occlusions, administered every 20
minutes for a total of 2 hours and 20 minutes. This is repeated every 8 hours over a period of 3, 6, 9 or 10 days. The occlusions do
not appear to produce any discomfort, nor are they associated with any untoward effects on cardiac function-there is no infarction
or arrythmias (as previously established by ECG and echocardiography). Sham (control) animals will be instrumented but will not
undergo the RI protocol.
Name ALL the personnel to be involved in the survival surgery:
XXXX, XXXXX
XXXX, XXXXX
Anesthetic Regimen (anesthetic agent, dose, and route of administration):
Rats are anesthetized with 75 mg/kg ketamine and 5 mg/kg xylazine given i.p (27 GA needle). Rats are then orally intubated using a
16 GA 48 mm BD Angiocath, ventilated (respiratory rate = 75/min; tidal volume = 8 ml/kg, 95% oxygen), and anesthesia during
surgery is maintained by sevoflurane inhalation (1.0-2.0%).
Method of assuring surgical plane of anesthesia is reached and maintained:
Toe pinch reflex.
Post-operative care (include anticipated recovery time and location, medications, analgesics, post-op procedures, and names of personnel
who will provide post-op care):
Upon confirmation of spontaneous respiration, the animal is extubated, and observed for a minimum of 2 hours, or until the rat is
fully awake in the PI's laboratory where surgeries will be performed (MSB 2178). JCR and ZOF rats will receive Yobine (yohimbine)
0.1 mg/kg immediately after they have been extubated. A second dose of 0.1 mg/kg will be given if the animal has not began to
move around the cage within 30 minutes.
Post-operative pain will be treated by injection of the analgesic Buprenex, 0.02 mg/kg sub-cutaneous 1 time/day on the day of
surgery and the following day. Subsequent doses will be given if the animal shows signs of pain or distress such as abnormal
resistance to handling, pain upon palpation of the surgery site, guarding of the surgery site, or anorexia and/or lethargy and/or as
determined by the USA veterinary staff.
Post-op care will be provided by XXXXXXXXX and XXXXXXXX, Research Assistants in the lab. If neither of them are available,
post-op care will be provided by the PI, XXXX, XXX.
12. I understand that I am responsible for post survival care of my animals, unless informed otherwise by DCM.
Please indicate Yes to signify your understanding.
Yes
13. Will major survival surgery be performed more than once to any single animal?
Please indicate Yes to signify your understanding.
No
13a. If 'Yes', please explain/Justify the need for multiple survival surgeries on a single animal.
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Segment Information
Segment Number: (Ex. 1, 2, 3,...) 1
Segment USDA Category D
Species: Rats
IF other 'other', please indicate:
Genetic Designation: Sprague-Dawley (SD)
Allow Transfer:
Strain/Stock/Breed: SD
No
If transfer, to what Protocol#
Number Authorized:
Euthanasia Primary:
Ketamine/xylazine and sevofluorene overdose
Euthanasia Secondary:Tissue harvest (heart removal)
Segment Description (field will expand to accommodate content):
Outbred white rat
S1. Specify the Age and/or weight of the animals to be used in this segment of the project:
10-12 weeks
S2.. Specify the sex of the animals to be used in this segment of the project:
Male
S3. Will any of the following requirements or conditions apply to the animals to be procured for this project: lactating (with or
without litter), Neutered/Spayed, Proven or Retired Breeder, Untimed pregnant, Timed Pregnant, Genetically altered,
Surgically altered, or Other similar characteristics.
Please indicate Yes, the animals have at least one of these characteristics or No, none have these characteristics.
No
S3a. Fully describe the special conditions, alterations, or modifications for this segment of animals.
(The box will expand to fit content).
Animal Care and Husbandry
S4. Will animals be subjected to food or water restriction/deprivation (other than peri-operative fasting) during this project?
No
S4a. Describe the food or water restriction/deprivation regimen. Be sure to include a discussion of how the restriction
will be monitored, what actions will be taken to remedy adverse effects of such restrictions/deprivations, and
what (and where) records will be maintained.
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S5. Are there any special husbandry requirements (food, bedding, caging, room temperature, lighting, or water) for the animals
described in this segment of the project?
Yes
S5a. Fully Describe the special Husbandry requirement(s).
Post-surgery, rats will be in special cages (provided by the PI). These are plastic cages with plastic and metal-reinforced
tops with a hole to accommodate the tether. Rats will be tethered in the cage for 5-12 days; however, the tether allows the
animal complete free movement inside the cage. We have not had to acclimate the animals to the tethers. We have found
that they accept the tether well, and they still have access to food and water, and can readily position themselves for their
normal functions (e.g., position during sleeping). We believe that the tethers are less stressful than repeated handling of
the animals, and also increases the success of our experiments, which means that fewer animals are needed, because the
animals cannot destroy the externalized catheters.
The personnel from the PI’s lab will be fully responsible for changing the cages (2 times per week in accordance with
DCM’s scheduled cage changes) and supplying food and water to the animals in these cages.
S6. Is this a breeding protocol from which offspring will be transferred to a different protocol after weaning?
No
S6a. If ''Yes', please provide a valid protocol number to which offspring from the breeding protocol are to be
transferred. If that experimental protocol is under review concurrently, please indicate.
Transportation of Animals
S7. If animals assigned to this project require specific investigator attention or are found dead, should authorized personnel be
contacted outside of regular working hours? If 'Yes', every attempt will be made to contact you; however, DCM is not responsible if you cannot be reached.
Yes
S8. If animals assigned to this project DIE, should they be saved for your examination?
Yes
S8a. If 'Yes', please select your preferred storage method for the animal prior to your examination:
Other (Contact DCM):
Segment Number: (Ex. 1, 2, 3,...) 2
Segment USDA Category D
Species: Rats
IF other 'other', please indicate:
Genetic Designation: Zucker obese fatty (ZOF)
Allow Transfer:
Strain/Stock/Breed: ZOF
If transfer, to what Protocol#
Number Authorized:
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Freeze
Page 9 of 12
Euthanasia Primary:
ketamine/xylazine and sevofluorene overdose
Euthanasia Secondary:tissue harvest (heart removal)
Segment Description (field will expand to accommodate content):
Zucker obese fatty rat is a leptin receptor knockout rat. It is obese with fatty liver, increased circulating leptin, insulin
resistant and dyslipidemic (high triglycerides, LDL and low HDL). Coat color is brown and white.
S1. Specify the Age and/or weight of the animals to be used in this segment of the project:
8-10 weeks
S2.. Specify the sex of the animals to be used in this segment of the project:
Male
S3. Will any of the following requirements or conditions apply to the animals to be procured for this project: lactating (with or
without litter), Neutered/Spayed, Proven or Retired Breeder, Untimed pregnant, Timed Pregnant, Genetically altered,
Surgically altered, or Other similar characteristics.
Please indicate Yes, the animals have at least one of these characteristics or No, none have these characteristics.
No
S3a. Fully describe the special conditions, alterations, or modifications for this segment of animals.
(The box will expand to fit content).
Animal Care and Husbandry
S4. Will animals be subjected to food or water restriction/deprivation (other than peri-operative fasting) during this project?
No
S4a. Describe the food or water restriction/deprivation regimen. Be sure to include a discussion of how the restriction
will be monitored, what actions will be taken to remedy adverse effects of such restrictions/deprivations, and
what (and where) records will be maintained.
S5. Are there any special husbandry requirements (food, bedding, caging, room temperature, lighting, or water) for the animals
described in this segment of the project?
Yes
S5a. Fully Describe the special Husbandry requirement(s).
Post-surgery, rats will be in special cages (provided by the PI). These are plastic cages with plastic and metal-reinforced
tops with a hole to accommodate the tether. Rats will be tethered in the cage for 5-12 days; however, the tether allows the
animal complete free movement inside the cage. We have not had to acclimate the animals to the tethers. We have found
that they accept the tether well, and they still have access to food and water, and can readily position themselves for their
normal functions (e.g., position during sleeping). We believe that the tethers are less stressful than repeated handling of
the animals, and also increases the success of our experiments, which means that fewer animals are needed, because the
animals cannot destroy the externalized catheters.
The personnel from the PI’s lab will be fully responsible for changing the cages (2 times per week in accordance with
DCM’s scheduled cage changes) and supplying food and water to the animals in these cages.
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S6. Is this a breeding protocol from which offspring will be transferred to a different protocol after weaning?
No
S6a. If ''Yes', please provide a valid protocol number to which offspring from the breeding protocol are to be
transferred. If that experimental protocol is under review concurrently, please indicate.
Transportation of Animals
S7. If animals assigned to this project require specific investigator attention or are found dead, should authorized personnel be
contacted outside of regular working hours? If 'Yes', every attempt will be made to contact you; however, DCM is not responsible if you cannot be reached.
No
S8. If animals assigned to this project DIE, should they be saved for your examination?
Yes
S8a. If 'Yes', please select your preferred storage method for the animal prior to your examination:
Freeze
Other (Contact DCM):
Segment Number: (Ex. 1, 2, 3,...) 3
Species: Rats
Segment USDA Category D
IF other 'other', please indicate:
Genetic Designation: Russell (JCR)
Allow Transfer:
Strain/Stock/Breed: JCR
If transfer, to what Protocol#
Number Authorized:
Euthanasia Primary:
ketamine/xylazine and sevofluorene overdose
Euthanasia Secondary:tissue harvest (heart removal)
Segment Description (field will expand to accommodate content):
The JCR rat a leptin receptor knockout. It is obese with fatty liver and increased plasma leptin, insulin resistant (elevated
insulin with glucose intolerance), dyslipidimic (elevated triglycerides, LDL and vLDL and low HDL), mildly hyperglycemic,
and hypertensive. The JCR rat is unique among the rodent models of obesity, insulin resistance, diabetes and the
metabolic syndrome in that it develops the cardiovascular pathology which mimics that of the human metabolic
syndrome, including impaired endothelial-dependent and -independent vasorelaxation, wide-spread atherosclerosis, and
at 16+ weeks of age, myocardial infarcts and strokes.
S1. Specify the Age and/or weight of the animals to be used in this segment of the project:
8-10 weeks
Confidential- USA Internal Use Only
Page 11 of 12
S2.. Specify the sex of the animals to be used in this segment of the project:
Male
S3. Will any of the following requirements or conditions apply to the animals to be procured for this project: lactating (with or
without litter), Neutered/Spayed, Proven or Retired Breeder, Untimed pregnant, Timed Pregnant, Genetically altered,
Surgically altered, or Other similar characteristics.
Please indicate Yes, the animals have at least one of these characteristics or No, none have these characteristics.
No
S3a. Fully describe the special conditions, alterations, or modifications for this segment of animals.
(The box will expand to fit content).
Animal Care and Husbandry
S4. Will animals be subjected to food or water restriction/deprivation (other than peri-operative fasting) during this project?
No
S4a. Describe the food or water restriction/deprivation regimen. Be sure to include a discussion of how the restriction
will be monitored, what actions will be taken to remedy adverse effects of such restrictions/deprivations, and
what (and where) records will be maintained.
S5. Are there any special husbandry requirements (food, bedding, caging, room temperature, lighting, or water) for the animals
described in this segment of the project?
Yes
S5a. Fully Describe the special Husbandry requirement(s).
Post-surgery, rats will be in special cages (provided by the PI). These are plastic cages with plastic and metal-reinforced
tops with a hole to accommodate the tether. Rats will be tethered in the cage for 5-12 days; however, the tether allows the
animal complete free movement inside the cage. We have not had to acclimate the animals to the tethers. We have found
that they accept the tether well, and they still have access to food and water, and can readily position themselves for their
normal functions (e.g., position during sleeping). We believe that the tethers are less stressful than repeated handling of
the animals, and also increases the success of our experiments, which means that fewer animals are needed, because the
animals cannot destroy the externalized catheters.
The personnel from the PI’s lab will be fully responsible for changing the cages (2 times per week in accordance with
DCM’s scheduled cage changes) and supplying food and water to the animals in these cages.
S6. Is this a breeding protocol from which offspring will be transferred to a different protocol after weaning?
No
S6a. If ''Yes', please provide a valid protocol number to which offspring from the breeding protocol are to be
transferred. If that experimental protocol is under review concurrently, please indicate.
Transportation of Animals
S7. If animals assigned to this project require specific investigator attention or are found dead, should authorized personnel be
contacted outside of regular working hours? If 'Yes', every attempt will be made to contact you; however, DCM is not responsible if you cannot be reached.
No
Confidential- USA Internal Use Only
Page 12 of 12
S8. If animals assigned to this project DIE, should they be saved for your examination?
Yes
S8a. If 'Yes', please select your preferred storage method for the animal prior to your examination:
Other (Contact DCM):
Confidential- USA Internal Use Only
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