WPI IACUC Animal Use Protocol Review Form (continued)
IACUC DATE STAMP:Shaded Areas for IACUC use only. / PROTOCOL NUMBER:
#03-02
APPROVAL DATES: / MODIFICATION DATE(S):
Date Filed:
Approval:
1st Renewal:
2nd Renewal:
Protocol Summary
Protocol Title
Oxygen Tension Imaging in the Rodent EyePrincipal Investigator (PI) Name and Address
First Name: / Ross D. / Department: / Biomedical EngineeringLast Name: / Shonat / Phone Number: / 508-831-6086
WPI ID Number: / [COMMENT1]349-60-5577 / Email Address: /
Campus Address: / Department of Biomedical Engineering (SL 411), WPI
Project Personnel[COMMENT2]
Name(s) of all individuals involved in project / IACUCCertification No. / Role in Project(Indicate who will do euthanasia, anesthesia, surgery, and any other non-surgical procedure).
Ross D. Shonat / PI-98-101 / PI (all procedures)
Adam S. Norige / ST-03-112 / Graduate Student (all procedures)
Student Status (Complete this Section if PI is a Student)
Status (Graduate/Undergraduate):Faculty Sponsor/Supervisor Name: / [COMMENT3]
Sponsor/Supervisor’s Signature: / Date:
Type of Submission (Check One)Provide Related IACUC Numbers
X / New Protocol / (Not applicable for new protocols)Amendment of IACUC Protocol #
Year 3 Renewal of IACUC Protocol #
Salary Support/Fellowship-related IACUC #
Identical to IACUC Protocol #
Funding Source(s):
Biomedical Engineering Research Grant, The Whitaker Foundation, Rosslyn, VA, USA (current)Corporate Funding, Biomedical Research Models, Inc, Worcester, MA, USA (current)
CAREER Award, National Science Foundation, Washington, DC, USA (pending)
Project Start Date:
/ 11/20/03 /Project End Date:
/ 11/19/06Species
/ Est. Proj. / Procedures: Check ALL that apply(See below for corresponding List of Procedures) / USDA Pain
Common Name and Strain
/ #/Yr / Tot. / a / b / c / d / e / f / G / h / i / j / k / l / m / z /Level
1 / Rat (disease control) / 100 / 300 / X / X / 22 / Rat (disease) / 100 / 300 / X / X / 2
3 / Mouse (disease control) / 100 / 300 / X / X / 2
4 / Mouse (disease) / 100 / 300 / X / X / 2
5
List of Procedures: / h. Food / Water Deprivation
a. Survival Surgery
b. Non-survival Surgery / i. Biohazard (i.e. Radioisotopes, Infectious Agents,
Toxin/Mutagen/Carcinogen, Recombinant DNA)
c. Multiple Survival Surgery / j. Burns or Trauma
d. Prolonged Restraint / k. Drugs
e. Collection of Cells, Tissues, or Organs / l. Antibody production
f. Aversive Conditioning / m Diagnostic X-rays
g. Special Diet / z. Other (Specify:)
USDA Pain Levels:1 = negligible, 2 = pain/distress avoided by appropriate drug use, 3 = pain/distress NOT avoided by appropriate drug use. (Level 3 procedures require appropriate documentation and justification)
Facility and Room # Where Procedures Will Take Place (If Multiple, so Indicate)
WPI Salisbury Labs, Rm 414Proposed Facility where Animals will be Housed (Must be an IACUC Approved Facility)
WPI Central Animal Facility (SL 313-319)Special Conditions / Situations
/YES
/NO
Housing Outside of the Central Animal Facility for More than 12 Hours (in a Study Area) / XTeaching / Training Protocol (If YES, complete “Teaching/Training Protocol” section in this packet) / X
Drug USE Summary
Analgesics / Anesthetics
Generic Name / Species / Dose (mg/kg) / Route1 / Avertin (2.5%) – anesthetic / Mice / 0.015 ml/g / IP
2 / Alpha-chloralose – anesthetic / Mice/Rats / 0.05 mg/g / IV
3 / Urethane – anesthetic / Mice/Rats / 0.75 mg/g / IV
4 / Halothane – anesthetic / Mice/Rats / 0.5-5% / Inhaled
5 / Isoflurane – anesthetic / Mice/Rats / 0.5-5% / Inhaled
6 / Pentobarbital sodium (50 mg/ml) / Mice/Rats / 30-50 mg/kg / IP
7 / Xylazine (20 mg/ml) / Mice/Rats / 5-10 mg/kg / IP
8 / Ketamine Hydrochloride (100 mg/ml) / Mice/Rats / 65-100 mg/kg / IP
9 / Mepivacaine (1%) / Mice/Rats / 0.1 ml / SC
Sedatives / Tranquilizers
Generic Name / Species / Dose (mg/kg) / Route1
2
3
Antibiotics
Generic Name / Species / Dose (mg/kg) / Route1
2
3
Miscellaneous and Other Drugs (Including IV Fluids)
Generic Name / Species / Dose (mg/kg) / Route1 / Pancuronium bromide (muscle paralyzer) / Mice/Rats / 0.20 mg/kg/hr / IV
2 / 1% tropicamide (Mydriacyl) – (pupil dilator) / Mice/Rats / Drop / Eye
3 / Lactated Ringers/Sodium Bicarbonate (acid-base) / Mice/Rats / As needed / IV
4 / Pd-porphyrin (10 mg/ml) – (oxygen probe) / Mice/Rats / 10 mg/kg / IV
5 / Fixative solutions (formalin or paraformaldyehyde) / Mice/Rats / As needed / Aortic cannula
Euthanasia Method(s) / Drug(s)
Euthanasia Method / Generic Drug Name / Species / Dose (mg/kg) / Route1 / Cervical dislocation under anesthesia / Mice / N/A / N/A
2 / Euthanasia Solution (200 mg/ml) / Mice/Rats / 100 mg/kg / IV
3
4
5
6
Note: All drugs used on animals before, during, or after an experiment or surgical procedure must be obtained from legal sources. All controlled substances should be kept in a double-locked compartment. Records should be kept documenting each use of a controlled substance. USE ONLY DRUGS THAT ARE WITHIN THEIR EXPIRATION DATE. All drugs should be disposed of properly when out of date or no longer needed. This applies to IV fluids as well.
Principal Investigator’s Assurance
Read Carefully, then Sign and Date Below
I have provided an accurate description of the proposed animal care and use protocol and agree to the following conditions:
All experiments involving live animals will be performed under my supervision or that of other qualified individuals as indicated on this form. The personnel involved have been, or will be, trained prior to any animal work in proper procedures of animal handling, administration of anesthetics and analgesics, and the AVMA recommended methods of euthanasia to be used in this project.I agree to report all animal purchases to the WPI Institutional Animal Care and Use Committee (IACUC) and to purchase only those animals approved for use and only up the maximum allowed by the IACUC. I understand that a failure to report all animal purchases to IACUC or to exceed the maximum number allowed is a violation and may result in the suspension of my approved protocol.
All personnel will be informed that any concerns for inhumane care and treatment of animals or unlawful acts involving animals should be reported to the IACUC and that anyone reporting such concerns cannot be discriminated against or be subject to any reprisal for reporting their concerns.
I agree to abide by governmental regulations and WPI policies concerning the use of animals.
I will ensure that veterinary care is provided to animals showing evidence of pain or illness.
I agree to give consideration to tissue sharing and will do so whenever possible.
I certify that any animal use proposed in a grant or contract proposal to support this research corresponds to the information provided herein.
If the procedures concerning animal use in this research activity are to be revised or changed, I will so notify the IACUC of these changes before the change is implemented. I understand that failure to request an amendment for changes in animal use may place WPI and myself in violation of Federal regulations and the Animal Welfare Act.
As required by Federal regulations, I assure that the activities described do not unnecessarily duplicate previous experiments and I assure the animal models proposed are the most appropriate for achieving the objectives of this project and have provided justification for each model used in the protocol (Animal Research Plan Rationale below).
Principal Investigator Signature: / Date:
CONFIRMATION OF SCIENTIFIC / INSTRUCTIONAL MERIT REVIEW
Before any project utilizing animals can be initiated, it must be reviewed and approved based on scientific or instructional merit. To assure the IACUC that this review is in place, the following information is needed regarding the review process that is applicable for this protocol. (check one of the two boxes below far left).
X / This project will only be initiated after it has been peerreviewed outside of WPI (e.g. NIH, NSF, etc.)or within WPI by a formal interdepartmental review group. If so, identify which group, agency or board has reviewed or will review this project for scientific or instructional merit. (Note: a signature is not required if you checked this box)
Name of Review Agency, Committee, or Board:
The Whitaker Foundation, Rosslyn, VA, USA (current), National Science Foundation (pending), National Institutes of Health (for age-related macular degeneration project at University of Pennsylvania).OR
This project is 1) ONLY being reviewed within a department; or 2) you would like to initiate theproject prior to receipt of an extramural award notice. If so, the chairperson of your department must attest to the scientific or instructional merit of this project. Please have him/her sign below on the signature line and indicate who conducted the review (check one of the two boxes below):
Departmental Committee
Name of Committee
Name of Committee Chairperson or Official Designee
OROther Review Process
Describe Review Process
Name of Department Chairperson
Department Chairperson Signature:
/Date:
Animal Research Plan
Project Overview / Objective
[COMMENT4]The survival of the retina in the eye is dependent on an adequate supply of oxygen and nutrients to fuel the metabolic processes that are vital for tissue homeostasis and primary visual function. Insufficient oxygen delivery and retinal hypoxia have been implicated as causal for the development of many devastating diseases of the eye, including diabetic retinopathy, glaucoma, retinopathy of prematurity, and age-related macular degeneration. While hypoxia is likely present at the end stages, the temporal and spatial relationships between blood flow insufficiency, vessel patency, and tissue hypoxia in the early stages of retinal disease remain largely unknown. We have recently completed construction of a microscope-based imaging system for measuring the oxygen tension (PO2) inside the retinal and choroidal vessels of rats and mice. Two dimensional PO2 maps are generated from the phosphorescence lifetime of an injected palladium-porphyrin compound using a frequency-domain measurement. With the development of this technology for use rats and mice, the potential for studying retinal diseases (diabetic retinopathy, glaucoma, retinopathy of prematurity, and age-related macular degeneration) where oxygen tension is implicated is substantially advanced in this rodent animal model. We will use this new technology to investigate a number of different retinal diseases that are hypothesized as associated with retinal hypoxia.The following retinal diseases are specifically targeted in this animal use protocol:
Diabetic Retinopathy is a devastating disease of the eye resulting from the long-term effects of diabetes mellitus. Abnormalities in retinal tissue oxygen delivery and consumption are thought to be significant in the progression of the disease, yet no study has adequately correlated the development and progression of diabetic retinopathy with changes in the oxygenation state of the retina. We will follow control and diabetic rodents (both rats and mice) at various points in the disease progression.
Age-Related Macular Degeneration (AMD)is the leading cause of irriversible blindness in people over 65. Although the majority of AMD cases are the less severe dry form, the wet or exudative form accounts for majority of the cases with severe vision loss. The exudative AMD is characterized by new blood vessels invasion of subretinal space from the choroidal capillaries (choroidal neovascularization, or CNV), leading to hemorrhage, fibrosis, and loss of central vision. To study the mechanism of CNV development and to search for effective and non-distructive treatments, reliable small animal models are essential. Although the pathogenesis of CNV is poorly understood, we do know that CNV is consistently associated with abnormal accumulation of extracellular matrix in the Bruch’s membrane. Collaborators at the University of Pennsylvania have recently developed a new technique to reliably generate choroidal neovascularization (CNV) in experimental animals by using Matrigel, a bioactive substance derived from the Engelbroth-Holm-Swarm (EHS) mouse sarcoma that contains protein composition comparable to basement membrane. Subretinal injection of 2 ul of Matrigel to a rat reliably induces CNV in 7-10 days. This animal model closely mimics CNV in human and is therefore a good model to the pathology of this disorder and also for testing potential agents that may inhibit the development of CNV. Technologies developed by the PI to generate oxygen tension (PO2) maps will be used to measure, for the first time, the oxygen tension in and around the injected Matrigel site. The critical question is whether hypoxia precedes, is associated with, or follows the choroidal neovascularization. The results of this study should have a substantial impact on our understanding of CNV and lead to more long-term studies using this animal model.
It is expected that this research will: 1) offer a more complete understanding of oxygen delivery to the retina by the two distinct and separate vasculatures that supply it, 2) provide new insights into the role of oxygen metabolism and delivery during the early phases of diabetic retinopathy and age-related macular degeneration, and 3) reveal correlations between the oxygenation and sub-clinical pathological changes in diabetic retinopathy and age-related macular degeneration. It is also expected that the efficacy of certain drug therapies for treating diabetic retinopathy and age-related macular degeneration can be assessed rapidly using rodent animal models and the PO2 imaging technique developed in the lab.
Protocol Synopsis / Experimental Plan
[COMMENT5] The following is the experimental plan for each animal:1) Each animal (mouse or rat) studied will be fully anesthetized during all surgical and experimental procedures and euthanized afterwards. As all procedures require substantial intervention and instrumentation, survival is not an option and no animals will be allowed to survive.
2) Anesthesia will be initiated by one of the following procedures:
a) Mice: 2.5% Avertin (0.015 ml/g, IP) and maintained under anesthesia with urethane (0.75 mg/g, IP) and alpha-chloralose (0.05 mg/g, IP) as needed.
b) Rats: Initial anesthesia with bolus urethane (1.5 mg/g, IP) and alpha-chloralose (0.10 mg/g, IP) and maintained under anesthesia with urethane (0.75 mg/g, IP) and alpha-chloralose (0.05 mg/g, IP).
c) Rats and Mice: Anesthesia initiated via a nose cone with halothane or isoflurane (2-5%) and maintained at a surgical plane of anesthesia with continuous halothane or isoflurane (0.5% - 1.5%) delivery. The minute volume of waste anesthetic gas will be removed with a halogenated-gas filter unit (F/Air canister).
d) Rats and Mice: Pentobarbital sodium (0.038 mg/g, IP) and xylazine (0.007 mg/g, IP) for induction and maintenance, as needed.
e) Rats and Mice: Ketamine (0.075 mg/g, IP) and xylazine (0.007 mg/g, IP) for induction and maintenance as needed.
Note: Method chosen will be dictated by past studies used for comparison. A volatile anesthetic will be chosen by default unless the procedure dictates that it may not be appropriate.
3) Cannulas will be placed in the trachea for anesthetic and regulated gas delivery, in the femoral artery for blood pressure monitoring, and in the femoral vein for drug infusion and fluid maintenance. Lactated Ringer's and sodium bicarbonate will be used for base buffering and fluid balance.
4) Paralysis with pancuronium bromide (0.2 mg/kg IV loading, 0.15-0.2 mg/kg/hr IV maintenance) will be used as needed. Continuous monitoring of blood pressure and heart rate will be used to insure adequate anesthesia with paralysis. A small animal ventilator will support the paralyzed animals. Paralysis is sometimes necessary to prevent unwanted eye movements.
5) Tidal CO2 and body temperature will be continuously monitored. Arterial blood sampling (50-80 ul) may be used to determine and manipulate blood-gases. In mice, only one blood-gas arterial sample is expected, as mice do not tolerate more than one blood withdrawal.
6) Animals will be secured to a stereotaxic head holder after a topical anesthetic (1% mepivacaine, SC) is applied under the scalp. This is necessary to stabilize the head and eye.
7) A lateral canthotomy will be performed on one eye and the eyelids reflected back. Tropicamide (1%) and Goniosol will be applied to the cornea to dilate the iris and coat the surface. A coverslip will be applied to negate the refractive power of the cornea and permit observation of the retinal surface using ordinary microscope objectives.
8) A palladium-porphyrin compound, used as a vascular oxygen tension probe in these studies, will be injected into the femoral vein in small volumes (20-50 ul, mice; 100 – 300 ul, rats). The probe will be dissolved in a physiologic-buffered saline solution. No acute reaction to the injection has ever been observed or is expected here.
9) In some animals, additional procedures will be performed in addition to the PO2 mapping procedures. These procedures include:
a) Perfusion fixation of tissues for subsequent histological procedures: Under anesthesia, fixative solutions, wash solutions, and histological marker solutions will be perfused by gravity feed through the aorta. Eyes and other organs will be harvested for subsequent histological procedures. All fixative and histological procedures will be done under deep anesthesia before euthanasia.
b) Removal of the sciatic nerve for nerve conduction tests: Diabetes is thought to slow the conduction of nerve action potentials. The sciatic nerve will be harvested in some deeply anesthetized rodents for in vitro tests of nerve conduction velocity.
c) Removal of the lower leg for tendon performance studies: Collaborative research groups not associated with this protocol wish to study the tendons in the diabetic animals used in these studies. This constitutes a dual use for an animal.
10) At the end of the experiment, animals will be euthanized with a lethal dose of Euthanasia solution (denatured pentobarbital sodium 200 mg/ml) followed by cervical dislocation (in mice).
Rationale, Appropriateness, Alternatives, and Numbers[COMMENT6]
Very briefly state the objective(s) and potential significance of the activities involving animal use (Explain why animals are required for your studies)
[COMMENT7]This project involves a study of two different retinal vascular diseases (diabetic retinopathy and age-related macular degeneration) in rodentanimal models. To study these diseases using the oxygen imaging system developed in the lab, the eye and retina must be similar to human (mammal). Studies on human beings are not possible because the injectable probes used in the study are not currently appropriate for human use.Additional comments on age-related macular degeneration: While a CNV model has been developed and mimics many of the characteristics of human age-related macular degeneration, the oxygen environment in and around the site of Matrigel injection has never been determined. Since neovascularization is strongly linked to hypoxia, a knowledge of the oxygen environment during CNV development would have high significance. Given a very poor understanding of the human disease and the paucity of animal models, a need to measure oxygen tension in this new animal model is necessary.