AMREP AEC Application Form

Transcription

1 AMREP AEC Application Form PART 2: Project information NOTE: Non-technical language must be used throughout Part 2. Application Number: 5240 Chief Investigator Name: Xiao-Jun Du 1. Title of project Significance of programmed cell necrosis in cardiac injury and inflammation after a heart attack 2. Summary of the project (Use non-technical terms) (Omitting the details that are required in section 5, briefly describe the proposed project in not more than 200 words.) Heart attack (myocardial infarction) induces loss of working heart muscle cells initially and then evokes an inflammatory response that causes further damage to the heart. Thus, protection against cardiac cell death and/or ameliorating inflammation would be expected to better preserve heart function. In addition to known types of cell death due to non-programmed necrosis or programmed apoptosis, recent studies have revealed that cell death in the setting of organ infarction may occur in the form of programmed necrosis called necroptosis. In contrast to apoptosis which is dependent on a group of proteins called caspases and does not evoke inflammation, necroptosis is executed by a distinct group of proteins including MLKL and promotes severe inflammation. With the documentation of the mechanism for necroptosis, it has been proposed that MLKL that induces necroptosis and inflammation is a good therapeutic target. However, up to now there has been no clear demonstration on the role of MLKL in necroptosis of cardiac cells following myocardial infarction. Hypothesis: MLKL-mediated necroptosis is important to cardiac injury following a heart attack through mediating cardiac cell death and inflammation. We will test this hypothesis by conducting experiments on wild-type and MLKL deficient mice subjected to a heart attack. The extent of cell death, inflammation and decline in heart function following a heart attack will be determined. 3. Justification for the use of animals 3.1 Significance and potential benefit of the proposed project (in words, using non-technical terms) (Provide information that will assist the AEC in weighing the predicted scientific or educational outcome of the project against the potential negative impact on the welfare of the animals.)

2 Each year, around 55,000 Australians suffer from heart attack this equates to one heart attack every 10 minutes (from Heart Foundation website). It is a serious and life-threatening condition. Understanding how the heart muscle is damaged following a heart attack and subsequent inflammatory response that further exacerbates cardiac damage, is imperative in developing therapies. The mouse heart attack model mimics in many ways the clinical situation (cell death, inflammatory response, scar formation, decline in heart function). We have extensive experience in inducing a heart attack in mice by surgical means and studying cardiac cell death, inflammation and heart function following the heart attack. We also have access to MLKL deficient mice (MLKL KO), which will enhance our study capabilities and quality. The findings from this study would reveal a novel therapeutic approach for the management of patients after a heart attack leading to improved outcomes. Potential negative impact on animals Mice will be subjected to open heart surgery to induce a heart attack by occluding a coronary artery (a heart blood vessel) causing ischemia (lack of oxygen) to the heart muscle serviced by this artery. Operated animals will experience pain and distress after regaining consciousness from open-chest surgery which will be managed with administration of analgesia. Animals may die suddenly during surgery of complications such as accidental damage to or breaking an artery, or may fail to regain consciousness after surgery (for example, due to respiratory problem resulting from lung or trachea (breathing tube) damage), which accounts for up to 5% loss from surgical risk and complications. Animals may develop acute and/or chronic heart failure after a heart attack, which accounts for up to 10% of animal loss post surgery. Mice will be monitored post surgery and will be euthanised if they develop this condition and do not respond to treatment (detailed below). Potential benefit of the proposed project Results from this project will improve our understanding on the mechanisms responsible for cardiac cell death and inflammation which lead to heart damage after myocardial infarction and the role of MLKL, providing important experimental evidence for developing new therapeutic strategies targeting MLKL to combat post ischemia heart damage. 3.2 Aims of the proposed project and details of why the use of animals is essential The aims are to compare between wild-type and MLKL KO mice subjected to myocardial infarction: (i) the amount of cardiac cell death, (ii) the scale of cardiac inflammation, and (iii) the extent of cardiac functional damage during sub-acute and chronic phases. In this project we need to investigate heart muscle cell death and inflammatory response and damage after a heart attack in a live/whole-animal model and, as such, this study cannot be replaced by a non-animal based system. In addition the need to access heart muscle tissue to investigate inflammatory changes and damage at specific time-points after a heart attack precludes conducting this study in humans. As aforementioned, the inflammatory response and heart muscle damage seen in mice after a heart attack in many ways mimics the clinical situation. Furthermore, our extensive experience in the mouse model of heart attack and the availability of MLKL KO mice, makes the mouse the species of choice for this study. 3.3 Features of the model (Briefly describe the animal model/s and the time of onset and rate of progression of phenotypic changes, focussing on welfare of the animal. Include a table that summarises for each strain of genetically modified animal the phenotype and health status over time in the context of the proposed experiments and how/why the strain will be used in the proposed studies. January

3 Phenotypic changes: Inducing a heart attack in mice is a commonly used model in heart research. Generally, mice tolerate this insult well. After inducing a heart attack, the heart starts to remodel (e.g. becomes larger), and develop dysfunction with time (e.g. over 4 weeks), which we can monitor by noninvasive echocardiography. Some mice may develop heart failure after a heart attack which can be identified by the presence of the following symptoms: oedema, laboured breathing, and reduced activity (more details below). Species, strain: We will use MLKL KO mice and their wild-type littermates (in C57/Bl6 background) in this study. We have considerable experience in cardiovascular research using this species and C57/Bl6 strain, including surgically inducing a heart attack and studying the heart after the heart attack. Also, the inflammatory response and heart muscle damage following a heart attack simulate the characteristics seen in human disease. Use of MLKL KO mice would allow us to identify the role of MLKL in inflammation and cardiac changes after a heart attack. The MLKL KO strain of mice has been generated for several years, with no adverse phenotype reported under baseline conditions, but shows attenuated cell death when exposed to cell death inducing pathogens. Age, sex: Adult male or female mice (~12 weeks of age or ~25-30g in body weight) will be used in order to exclude age as a factor (eg response of juvenile hearts and aged hearts differ). 4. The principles of replacement, reduction and refinement (the 3Rs) 4.1 Replacement: Code s Methods that replace or partially replace the use of animals must be investigated, considered and, where applicable, implemented. This study involves a complex disease condition, i.e. heart disease, inflammation and dysfunction, and can only be simulated and conducted in a live animal, in which all biological systems are able to function normally, interact and respond to disease stimulus. These studies cannot be replicated in non-animal based systems. 4.2 Reduction: Code s The number of animals used in a project must be the minimum necessary to achieve the proposed aim(s) and to satisfy good statistical design. We have designed the proposed studies to use the minimum number of animals required to ensure scientific and statistical validity to achieve the scientific aims. The number of mice required for this project was determined by a power calculation (see Section 5), using values determined from our previous studies and experience on heart disease models in mice over 15 years. 4.3 Refinement: Code s.1.28 Steps must be taken at all times to support and safeguard animal wellbeing. The effectiveness of strategies for supporting and safeguarding animal wellbeing must be kept under review during the lifetime of the project. January

4 Over the years we have incorporated some refinements to our studies which have helped with animal care and welfare: 1. Only experienced staff will be allowed to perform surgical procedures to minimize loss of animals due to surgical complications. 2. Animals will be carefully monitored and cared to reduce peri-operative loss (e.g. due to hypothermia). 3. Animals will always be handled with care and consideration. Intraperitoneal and subcutaneous injections when required will be administered by experienced well-trained staff. 5. Details of each experiment (Copy this section for each different experiment described in the application) (5.1) Diagrammatic (i.e., a figure, not text) timeline or flowchart for each group of animals In addition to the timeline, provide responses under each of the following subheadings for each timeline: 1. Justification of the time points, scientific end points, blood volumes, and dose and frequency of any treatments 2. Statistical or other justification of animal numbers and a summary table of total numbers. If embryos are required, include the number of mothers and all embryos (i.e. not only those of the correct genotype) beyond mid-gestational age of 10 days.) 3. The total number of each procedure to which an individual or group of animals will be subjected. 4. Outline the expected adverse impact of the experiment on the welfare of the animals (e.g. describe the potential impact of disease models in which animals develop diabetes, cancer, heart failure etc). Distinguish between treatment and control groups where appropriate. Note: The potential adverse impact of individual procedures should be described in Section Provide details of how the experiment has been designed to minimise the overall adverse impact on the animals. For example, by minimising the number of procedures per animal, providing intervals that allow the animals to recover fully between procedures, minimising the duration and severity of disease, choosing the earliest endpoints possible to provide the data needed and using bio-markers if possible in the determination of end points. Note: The way in which the impact of each procedure will be minimised should be described in Section 6. For further information and examples: eriment Aim-1: To determine whether MLKL knockout is associated with a reduced infarct size ischemia 0.5h/1h reperfusion 24h blood/heart collection 1. Justification of the time points, scientific end points, blood volumes, and dose and frequency of any treatments Ischemia/Reperfusion surgery: Surgery will be performed by experienced personnel to induce a heart attack (and heart muscle ischemia) for either 30 or 60 minutes, after which the ligation will be removed allowing oxygenated blood to return to the muscle that was affected (reperfusion). We wish to test in two different ischemia durations, i.e. 30 and 60 minute, respectively, representing moderate and severe degree of cardiac ischemia. Our previous studies have shown that the infarct size, expressed as the percentage of total ischemic myocardium is approximately 40% and 65%, respectively January

5 following ischemia for 30 or 60 minutes. Blood/Tissue collection: At 24h after reperfusion, a cardiac puncture will be performed on anaesthetised animals to obtain a blood sample (a 25G needle is used to puncture the heart through the chest wall and up to 1ml of blood is withdrawn). The chest wall is then cut open and the heart removed. The animal will die of exsanguination. Hearts will be used for histological measurement of infarct size. The technique applied for infarct size determination requires as exclusive use of an entire heart. 2. Statistical or other justification of animal numbers Based on our previous studies, to obtain a significance level of 0.05 and power of 0.8, expected inter-group difference is 15%, and intra- group variation is 12%, the group size is 11 by Sigma Plot Power Calculation. Thus, this study needs 44 mice (11/group x 2 genotypes x 2 ischemic durations). We require ~10% extra mice (n=4) to compensate for the loss of animals (see section 3.1). Thus, the total number is 48 (24 wildtype and 24 MLKL KO). 3. The total number of each procedure to which an individual or group of animals will be subjected Procedure Ischemia/Reperfusion surgery Blood/Tissue collection Frequency of procedure 4. Outline the expected adverse impact of the experiment on the welfare of the animals Cardiovascular changes are expected to occur after inducing a heart attack, however, this is not expected to be worse in the MLKL KO mice. Mice may develop acute heart failure after a heart attack (see section 6). The study endpoint is too short for development of long-term cardiovascular consequences. 5. Provide details of how the experiment has been designed to minimise the overall adverse impact on the animals. By using experienced surgeons mice recover quickly after this surgery. Aim-2: To determine influence of MLKL on cardiac inflammation following ischemia-reperfusion ischemia 1h reperfusion 24h blood/heart collection 72h blood/heart collection 1. Justification of the time points, scientific end points, blood volumes, and dose and frequency of any treatments Ischemia/Reperfusion surgery: Surgery will be performed by experienced personnel to induce a heart attack for 60 minutes (to induce severe cardiac ischemia), after which the ligation will be removed allowing reperfusion. Following cardiac ischemia-reperfusion, the inflammatory response becomes evident at hours and reaches the maximum around 72 hours. To compare the scale of inflammatory responses, blood and hearts will be harvested from mice subjected to 60-min ischemia followed by reperfusion for 24 and 72 hours (two time-points). Wild-type and MLKL KO mice will also be operated to induce sham-surgery (i.e. without induction of cardiac ischemia to control for potential influences by the surgical procedure per se). Blood/Tissue collection: At 24h or 72h after reperfusion, a cardiac puncture will be performed on January

6 anaesthetised animals to obtain a blood sample (a 25G needle is used to puncture the heart through the chest wall and up to 1ml of blood is withdrawn). The chest wall is then cut open and the heart removed. The animal will die of exsanguination. Plasma and heart samples will be used for measurement of a number of inflammatory markers at gene (RNA) and protein levels (eg IL-1β, IL-6, TNFα, MCP-1, MMP9, MIF, ICAM1, VCAM1) following the ischemic insult will be examined by biochemical assays using methods including realtime PCR and Western blot. Circulating levels of selected cytokines (eg TNFα, IL-1β, MIF) and MMP9 will be determined by ELISA using plasma. We will also determine inflammatory cell density in the infarct myocardium by using immunohistochemistry (this part requires a separate set of heart tissues). 2. Statistical or other justification of animal numbers Based on power calculation as shown above, the group size is 11. The number of animals required for this Aim is: N = 11 x 2 (ischemia/reperfusion or sham) x 2 (genotypes) x 2 (24h, 72h) x 2 (sets) = 176. We require ~10% extra mice (n=16) to compensate for the loss of animals subjected to surgery. Thus, the total number is 192 (96 C57Bl6 and 96 MLKL KO mice). 3. The total number of each procedure to which an individual or group of animals will be subjected Procedure Ischemia/Reperfusion or sham surgery Blood/Tissue collection Frequency of procedure 4. Outline the expected adverse impact of the experiment on the welfare of the animals Cardiovascular changes are expected to occur after inducing a heart attack, however, this is not expected to be worse in the MLKL KO mice compared to wild-type. Mice may develop acute heart failure after a heart attack (see section 6). The study endpoint is too short for development of long-term cardiovascular consequences. 5. Provide details of how the experiment has been designed to minimise the overall adverse impact on the animals. By using experienced surgeons mice recover quickly after this surgery. Aim-3. To explore the influence of MLKL on cardiac remodeling and function at subacute and chronic phases ischemia reperfusion 1h week 1 week 2 week 4 blood/heart collection echocardiography catheterisation 1. Justification of the time points, scientific end points, blood volumes, and dose and frequency of any treatments Ischemia/Reperfusion surgery: Surgery will be performed by experienced personnel to induce a heart attack for 60 minutes (severe cardiac ischemia), after which the ligation will be removed allowing reperfusion. If January

7 depletion of MLKL could reduce the infarct size and suppress the extent of inflammation, then it is very likely that MLKL KO mice would have less severity of heart chamber dilatation and dysfunction at the late phase following a heart attack. We therefore elect to study the potential role of MLKL over 4 weeks duration after inducing a heart attack. Both wild-type and MLKL KO mice will be studied with and without (sham) cardiac ischemia. Echocardiography: Non-invasive echocardiography will be performed on lightly anaesthetised animals prior to surgery, and 1, 2 and 4 weeks post surgery in order to assess any changes in heart chamber size (remodelling) and function in both the early (week 1 and 2) and later phase (week 4). Catheterisation: This is an invasive and terminal procedure. Four weeks after surgery in anesthetised animals, a pressure catheter is surgically inserted into the vasculature and advanced into the heart (more details below). This allows assessment of heart function (different measures than can be obtained with echocardiography) and blood pressure. Catheter will be performed a day or so after the final echocardiography (this allows time for the animals to fully recover from the light anaesthesia used during echocardiography). Blood/Tissue collection: At the completion of catheter, while the animal is anaesthetised, a cardiac puncture will be performed to obtain a blood sample (a 25G needle is used to puncture the heart through the chest wall and up to 1ml of blood is withdrawn). The chest wall is then cut open and the heart removed. The animal will die of exsanguination. Hearts will be used for histological/biochemical assays. 2. Statistical or other justification of animal numbers As in Aim-1, the number of mice required per group is 11, the number of animals required are as follows: N = 11 x 2 (ischemia/reperfusion surgery or sham) x 2 (genotypes) = 44. We require ~10% extra mice (n=4) to compensate for the loss of animals during surgical procedure or during the study period. Thus, the total number is 48 (24 C57Bl6 and 24 MLKL KO mice). 3. The total number of each procedure to which an individual or group of animals will be subjected Procedure Ischemia/Reperfusion or sham surgery Echocardiography Catheterisation Blood/Tissue collection Frequency of procedure 4 times 4. Outline the expected adverse impact of the experiment on the welfare of the animals Cardiovascular changes are expected to occur after inducing a heart attack, however, this is not expected to be worse in the MLKL KO mice compared to wild-type. Mice may develop acute or chronic heart failure after a heart attack (see section 6). 5. Provide details of how the experiment has been designed to minimise the overall adverse impact on the animals. By using experienced surgeons mice recover quickly after this surgery. January

8 6. Details of all (i.e., with and without anaesthesia) procedures and 6.1 Procedure 1 Inducing myocardial infarction in the mouse Description of Procedure, including the building, level and room number in which it will be conducted and any special equipment that will be used. We will follow SOP #7/2015: Surgical method of inducing myocardial infarction in the mouse. Briefly, to induce a heart attack (myocardial infarction) animals will be anesthetised by a mixture of ketamine (100 mg/kg), xylazine (20 mg/kg) and atropine (1.2 mg/kg) i.p. (refer to section 8). This anaesthetic mixture induces rapid and adequate anaesthesia lasting for about 30 minutes, the period long enough to complete surgery. During the procedure the anaesthetic level will be monitored constantly by respiratory pattern (both rate and depth) and pedal reflex (response to stimuli). Endotracheal intubation will be conducted to insert a 20G cannula for mechanic ventilation during open-chest surgery. The ventilator is set at breaths/minute and ml of tidal volume with room air or room air supplemented with oxygen. The chest will be shaved and cleaned with 70% ethanol and the animal will be placed on a heat pad (~32-35ºC) in a supine position. With the aid of a surgical microscope, a left thoracotomy will be made via the forth intercostal space to expose the heart. After identification of the left coronary artery, a 7-0 silk suture will be placed to surround the artery together with inclusion of two releasing loops (made with 5-0 silk suture). Regional ischemia will be confirmed at the time of occlusion by visualising the presence of a pale colour on the heart. Then the chest cage is closed by 6-0 monofilament suture after expansion of the lung to remove any extra air in the chest cavity. The muscle and skin layers will be closed using 6-0 monofilament suture. After suturing the chest incision, the ends of two releasing rings will be left outside of the chest. At the scheduled time, reperfusion will be induced by pulling the rings to loosen the ligation. This procedure does not interfere with animal recovery from surgery and does not require anesthetization. The skin wound will be spread with the antiseptic ointment, betadine. Mice will be administered antisedan (0.2 mg/kg, s.c.) to help with recovery from anaesthesia. The animal s eyes will be covered with drops of liquid tears immediately before and soon after the completion of surgery to prevent optical damage due to dryness of the eyes. This surgical procedure and tissue collection will be conducted in a PC2-certified surgical room in PAC and at the Small Animal Procedure room at the Third Floor of Baker IDI Institute Potential adverse impact of the procedure on the animals wellbeing Possible sources of pain and distress in mice subjected to cardiac surgery may include some discomfort resulting from surgery itself, such as fluid retention, pain resulting from ischemia (reduced blood flow to heart muscle) or other post-surgical pain, as well as any other discomfort due to handling. Surgical related pain may slow animal recovery immediately after surgical procedure, cause animal inactivity and difficulty to access food and water, thereby increasing risk of poor recovery and post surgical death. Following the heart attack, acute heart failure may develop over the first few days after surgery manifested with signs of drowsier, inactive, oedema/laboured breathing (e.g., puffiness ) and/or low body temperature, and chronic heart failure (manifested with the same symptoms as for acute heart failure) may also develop in some animals in the long-term study groups Steps taken to minimise and manage the potential adverse impact to the animal of the procedure January

9 1. Only experience surgeons will perform open-chest surgery to minimize distress, pain and loss of animals. 2. After regaining consciousness, animals are kept on a heating pad until fully recovered. If a mouse is found to have signs of heart failure (labour-breathing, oedema, inactivity, low body temperature (felt by touch)), the cage will be placed half on and half off a heat pad, and a dose of diuretic will be given (frusemide, 4 mg/kg, s.c.). Sick mice will be placed on a care form and checked at least twice daily. Body weight will be monitored. 3. Use of lignocaine as a supplementary measure to further reduce the extent of surgical related pain and facilitate animal recovery. Lignocaine, a local anaesthetic/pain killer, will be used to minimize pain by subcutaneous injection under skin incision site after skin closure. 4. In addition, the cage will be provided with soaked cotton wool and crushed food on the cage floor (for easy access). 5. We will carefully follow SOP#6/2015: Post-operative, post-anaesthesia and post-experimental care of rats and mice. Animals will be carefully inspected 3 times in the first 24 hours and then twice a day for 72h and then daily until study endpoint. 6. If the animal s condition does not improve and/or body weight drops by more than 15% within the period of 24 hours, the mouse will be humanely euthanized Details of for the procedure Signs to be monitored (include only those relevant to the procedure) Name of person/s responsible for Frequency of Trigger(s) for changes to regimen (List changes in wellbeing, symptoms, and the conditions that will result in intervention, describe the corresponding intervention e.g., increased frequency of, the provision of supportive care, euthanasia etc, and the criteria that will determine the next step in if supportive care is given i.e. what will be done if the wellbeing of the animal deteriorates, remains stable or improves while it is on supportive care) During surgery and immediately post surgery: Anaesthetic depth, heart rate, skin colour, breathing. As the animal regains consciousness, its ability to regain full movement is assessed. XJ Du XM Gao H Kiriazis Continuously during surgery and every ~5-10min after surgery until mouse regains consciousness and is fully recovered Usually, after surgical induced heart attack, animals are able to recover. However, if infarct size is big (> 50% of the left ventricle), animals may develop acute heart failure with a slow regain of consciousness and failure to disconnect from the mechanic ventilation. In this case, mice will be continuously ventilated and diuretic administered to help recovery; if situation is unable to improve, the animal will be euthanized. After surgery ( mouse has regained consciousness): We will follow SOP #6/2015 Post-operative, post-anaesthesia and postexperimental care of rats and mice. We will check the wound, assess general health and XJ Du XM Gao H Kiriazis with the help of PAC staff Three times in the 24h after surgery, then twice daily until 72h after surgery, then daily until the end of study Crushed food and cotton soaked with water will be placed on the cage floor for easy access and the cage put on a heating pad to prevent low body temperature until fully recovered (1-3 days post-surgery). If animals develop signs of heart failure as aforementioned, diuretic will be given to help reducing fluid retention, and SOP#6/2015 will be followed. If there is no improvement or the condition of the animal January

10 wellbeing, including status of alertness, body temperature, body weight, breathing pattern, feeding behaviour, activity level and posture. deteriorates within 24 hour period, it will be euthanized. Provide a customised score sheet template if used: - attach as an appendix and refer to the attachment (title / number) here: 6.2 Procedure 2 Echocardiography Description of Procedure, including the building, level and room number in which it will be conducted and any special equipment that will be used. This non-invasive procedure uses ultrasound to provide valuable information about heart size and function. We will follow SOP #10/2014: Echocardiography in the mouse. Briefly, mice will be anesthetised using isoflurane anaesthesia with (~4.5% for induction, ~2% for maintenance). A small transducer will be used to capture heart images for subsequent analysis, and then the animal is taken off the isoflurane and allowed to recover (usually within minutes). Echocardiography will be conducted in the PC2-certified Echo/Vevo Room located at level 3, in Baker IDI building Potential adverse impact of the procedure on the animals wellbeing Based on our extensive previous experience (have performed echocardiography in 1000 s of mice), we do not expect any adverse impact of this procedure on the animals. Though, anaesthetic overdose (particularly during induction) is a possibility Steps taken to minimise and manage the potential adverse impact to the animal of the procedure Mice are carefully monitored during anaesthesia if they show any signs of gasping they will be removed to room air and/or supplemented with oxygen. We have not loss any mice due to isoflurane overdose Details of for procedure Signs to be monitored (include only those relevant to the procedure) Name of person/s responsible for Frequency of Trigger(s) for changes to regimen (List changes in wellbeing, symptoms, and the conditions that will result in intervention, describe the corresponding intervention e.g., increased frequency of, the provision of supportive care, euthanasia etc, and the criteria that will determine the next step in if supportive care is given i.e. what will be done if the wellbeing of the animal deteriorates, remains stable or improves while it is on supportive care) Anaesthetic depth including conscious status, breathing pattern and heart XJ Du XM Gao During induction and echocardiography If the animal gasps under isoflurane anaesthesia it will be removed to fresh air/supplemented with oxygen January

11 rate H Kiriazis examination Provide a customised score sheet template if used: - attach as an appendix and refer to the attachment (title / number) here: 6.3 Procedure 3 Catheterisation Description of Procedure, including the building, level and room number in which it will be conducted and any special equipment that will be used. Cardiac catheterisation is used for the determination of blood pressure, and heart developed pressure and contractility, and is a way to assess heart function differently from that by echocardiography. We will follow SOP #9/2015: Cardiac catheterisation in the mouse. Briefly, mice will be anaesthetized using ketamine/xylazine/atropine (see section 8) and placed on a heating pad at a supine position. A midline incision at the ventral side of the neck is made to dissect and expose the right carotid artery. A 1.4 Fr Millar catheter with micro-manometer is inserted through the carotid artery to the aorta to record the blood pressure, then the catheter is sent further down to the heart to measure left ventricular pressure and contractility parameters. After recording all desired parameters (usually takes 5-10 min), heart tissues will be collected from exsanguinated mice. This procedure will be conducted in the PC2-certified catheter room at Experimental Cardiology Laboratory (level 3) in the Baker IDI building Potential adverse impact of the procedure on the animals wellbeing Animals may experience pain during the skin incision, if not fully anaesthetised Steps taken to minimise and manage the potential adverse impact to the animal of the procedure Researcher will make sure that a proper anaesthetic level is achieved (eg by checking the pedal reflex) before the skin incision Details of for procedure Signs to be monitored (include only those relevant to the procedure) Name of person/s responsible for Frequency of Trigger(s) for changes to regimen (List changes in wellbeing, symptoms, and the conditions that will result in intervention, describe the corresponding intervention e.g., increased frequency of, the provision of supportive care, euthanasia etc, and the criteria that will determine the next step in if supportive care is given i.e. what will be done if the wellbeing of the animal deteriorates, remains stable or improves while it is on supportive care) Anaesthetic depth (pedal reflex), breathing pattern, heart rate, and blood pressure XJ Du XM Gao H Kiriazis Continuously during procedure If there are early signs of the anaesthesia wearing off (eg heart rate increase), an anaesthetic top-up will be administered (1/3 rd dose). January

12 Provide a customised score sheet template if used: - attach as an appendix and refer to the attachment (title / number) here: 6.4 Procedure 4 Collection of blood via cardiac puncture Description of Procedure, including the building, level and room number in which it will be conducted and any special equipment that will be used. At the end-point a (~1ml) blood sample will be collected via cardiac puncture. Mice will be anaesthetized by a mixture of ketamine, xylazine and atropine i.p. (refer to Section 8). The skin overlying the chest will be cut open, and a syringe with 25 G needle will be inserted on the left side to puncture the heart (through the chest wall) and withdraw blood. Following this, the chest wall will be opened and the heart removed. The anaesthetised mouse will die by exsanguination. This procedure will be conducted in a PC2-certified room at Experimental Cardiology Laboratory (level 3) in the Baker IDI building Potential adverse impact of the procedure on the animals wellbeing Animals may experience pain during the skin incision and cardiac puncture, if not fully anaesthetised Steps taken to minimise and manage the potential adverse impact to the animal of the procedure Researcher will make sure that a proper anaesthetic level is achieved (eg by checking the pedal reflex) before the skin incision/cardiac puncture Details of for procedure Signs to be monitored (include only those relevant to the procedure) Name of person/s responsible for Frequency of Trigger(s) for changes to regimen (List changes in wellbeing, symptoms, and the conditions that will result in intervention, describe the corresponding intervention e.g., increased frequency of, the provision of supportive care, euthanasia etc, and the criteria that will determine the next step in if supportive care is given i.e. what will be done if the wellbeing of the animal deteriorates, remains stable or improves while it is on supportive care) Anaesthetic depth (pedal reflex), breathing pattern XJ Du XM Gao H Kiriazis Continuously during procedure If anaesthesia depth is not sufficient (eg pedal reflex is evident) an anaesthetic top-up will be administered (1/3 rd dose). Blood collection will not commence until the animal is fully anaesthetised. Provide a customised score sheet template if used: - attach as an appendix and refer to the January

13 attachment (title / number) here: 6.5 Procedure 5 injections Description of Procedure, including the building, level and room number in which it will be conducted and any special equipment that will be used. Animals will be injected intraperitoneally with ketamine/xylazine and atropine to induce anesthesia for surgical purpose or at the end for blood and tissue collection. Cardiac puncture will be done for blood collection under full anesthesia as the terminal procedure. Injection will be conducted in a PC2-certified room at PAC or at Experimental Cardiology Laboratory (level 3) in the Baker IDI building Potential adverse impact of the procedure on the animals wellbeing Animals may experience pain during the skin incision and cardiac puncture, if not fully anaesthetised Steps taken to minimise and manage the potential adverse impact to the animal of the procedure Researcher will ensure that a proper anaesthetic level is achieved (eg by checking the pedal reflex) before the skin incision/cardiac puncture Details of for procedure Signs to be monitored (include only those relevant to the procedure) Name of person/s responsible for Frequency of Trigger(s) for changes to regimen (List changes in wellbeing, symptoms, and the conditions that will result in intervention, describe the corresponding intervention e.g., increased frequency of, the provision of supportive care, euthanasia etc, and the criteria that will determine the next step in if supportive care is given i.e. what will be done if the wellbeing of the animal deteriorates, remains stable or improves while it is on supportive care) Animal breathing, speed of induction of anesthesia XJ Du XM Gao H Kiriazis Continuously during procedure Attention will be given to ensure the correct position and depth of injection. Blood collection will not commence until the animal is fully anaesthetised. Provide a customised score sheet template if used: - attach as an appendix and refer to the attachment (title / number) here: 7. Details of general day-to-day Signs to be monitored (relevant to each model) Name of person/s responsible for Frequency of Trigger(s)( i.e. Changes in wellbeing, symptoms, and conditions that will result in intervention, and what will the corresponding interventions be? i.e. January

14 supportive care and euthanasia) The MLKL KO mice do not have any health issues and are not expected to require additional care. They will be monitored for general well being (eg activity, grooming etc) XJ Du XM Gao H Kiriazis with the help of PAC staff Weekly prior to entering the study; daily after surgery If mice are found to be inactive or have an unkempt appearance they will be closely monitored following SOP#6/2015 Post-operative, post-anaesthesia and post-experimental care of rats and mice. Provide a customised score sheet template if used: - attach as an appendix and refer to the attachment (title / number) here: 8. Details of administration of anaesthetic agents, analgesics, experimental agents NOTE: The term Experimental agents includes drugs, infectious agents, vaccines, vehicle (for controls), antibiotic in drinking water after irradiation, cells etc. (Copy the table if more than 4 agents will be used.) Agent Name (active ingredient) of agent/drug Ketamine, Xylazine, Atropine (KXA) Lignocaine Antisedan (atipamezole) Frusemide Isoflurane Purpose Anaesthetic prior to Surgery/catheterisa tion/ tissue collection local anaesthetic Assistance to recover from KXA anaesthesia Diuretic Anaesthetic for echocardiography Route of administration i.p. s.c. s.c. s.c. inhalation Dose(s) (mg/kg) K, 100 mg/kg, X, 20 mg/kg (10mg/kg for catheter) A, 1.2 mg/kg 2-4 mg/kg 0.2 mg/kg 4 mg/kg ~4.5% for induction ~2% for maintenance Needle gauge (if applicable) Volume/dose (if applicable) 30G 30G 30G 30G N/A ul 50 ul ul ul N/A Frequency Once initially at time of surgery and, if top-up required, additional ~1/3 full dose will be given At time of surgery/post surgery as required January

15 Total number of times administered Side effects Suppress heart function (avoid overdose) and dryness of eyes (liquid tears to be used to prevent this) nil nil nil Suppress heart function (if overdosed) Vehicle if applicable saline saline saline saline N/A 9. Fate of the animals What will happen to the animals at the completion of the experiment? All mice will be killed at the completion of the experiment. Animals will be under anaesthesia to enable blood to be sampled, prior to removal of tissues after which the chest will be opened and the heart removed. Mice will die of exsanguination. Feedback: To leave feedback regarding this AEC application form please click here. January