What does this study involve doing to the animals?	What will the animals experience? How much suffering might it cause? What might it make it worse?	How will suffering be reduced to a minimum?
	Adverse effects	Methodology and interventions	End-points
Behavioural tests training for 1 week preceding MCAO: tactile stimulation, hill-and-valley staircase, six-tube choice, adhesive removal	These are behavioural observations, perform voluntarily by the animals and will not cause pain, distress or lasting harm	All tests are performed in the modified home cage	Typically, a maximum time to perform the requested task is set, and a final score is given. Animals not reaching a baseline performance will be excluded from the study
Under general anaesthesia, transient (3 h) occlusion of the MCA using an intraluminal thread advanced via the common carotid artery.	Pain and discomfort associated with surgery Potential for unexpected surgical complications, e.g. subarachnoid haemorrhage, ipsilateral retinal injury, intraluminal thrombus formation, brain oedema, hypothalamus involvement with consequent hyperthermia or temporal muscle necrosis. These can present clinically in a number of different ways, for example: sudden collapse, paralysis, severe head tilts, seizures Potential effects of anaesthesia on physiological variables, e.g. hypothermia, hypotension, hypoxia Poor nutritional intake resulting from reduced consciousness level, impaired mastication and poor motility, generally in the first 48 h post-MCAO	Use of appropriate anaesthetics, with appropriate analgesics (i.e. effective yet with minimal neuroprotective properties) Well-trained surgeon using appropriate aseptic surgical technique (with regular reviews of success rates) Maintenance of homeostasis during anaesthesia Use of standardised monofilaments and surgical technique to reduce variability and complications derived from extensive lesions Intensive post-operative care for first 3–5 days, including external heat sources. Regular body weight checks; daily observation and wound care Providing easy accessible food and water during the recovery period, or additional food (mash, liquid) and assistance with feeding if necessary; rehydrate (e.g. via saline injection) if necessary	Animals will be humanely killed if any of the following occur – • Significant technical problems during surgery. • Failing to fully recover from anaesthesia • Signs of unexpected surgical complications • If animal’s bodyweight loss exceeds 20% pre-surgical weight, despite additional feeding and/or rehydration, or if they remain immobile for over 24 h
Intravenous bolus of saline/test compound, followed by subcutaneous implantation of a mini-pump under general anaesthesia (as part of the MCAO surgical procedure, i.e. no additional anaesthesia requested)	Pain and discomfort associated with surgical procedure (mini-pump implantation) No adverse effect expected at the dose levels administered	Animals will be closely observed for adverse effects of test substances	Animals will be humanely killed if any severe side effects due to the novel therapeutic agents are noted
Behavioural tests performed daily during the first week post-MCAO, and weekly for 6 weeks: tactile stimulation, hill-and-valley staircase, six-tube choice, adhesive removal	Animals may find the tasks stressful if their motor abilities are compromised	Monitor for behavioural indicators of anxiety or distress Animals will be continuously observed by experienced staff	Typically, a maximum time (cut-off) to perform the requested task is set, and a final score is given
Longitudinal imaging (MRI) under anaesthesia at 1 h, 8 and 45 days after MCAO	Repeated anaesthesia Effect of anaesthesia, and physiological variables (hypothermia, arterial blood pressure, arterial blood gases) on stroke outcome	Use of appropriate anaesthetics and analgesics (i.e. effective yet with minimal neuroprotective properties) Maintenance of homeostasis during anaesthesia	Animals failing to fully recover from anaesthesia will be euthanised