L2

Question 1

What are the perspectives on animal research?

Answer 1

1. Animal Rights (any conscious animal has rights)
2. Utilitarian (act to maximize benefit overall and minimize harm; welfare)
3. Contractarian (animals do not have any moral status)

Question 2

What is the Tri-Council Policy statement?

Answer 2

The Tri-Council Policy statement is an ethical conduct for research involving humans.

A harm-benefit analysis, which is difficult to do, but necessary.

“In all clinical research, the REB should carefully evaluate previous laboratory, animal and human research with a drug or other therapy, and/or have an expert evaluation undertaken on its behalf, to ensure that the foreseeable risk from its use is: (a) justified by the potential benefits to be gained; and (b) appropriately minimized.”

Question 3

What are the factors for weighing animal welfare concerns with potential benefits?

Question 4

How do animal studies translate into successful human research?

Answer 4

ACCURACY OF BIOMEDICAL RESEARCH (2006).

• • searched seven leading journals (science, nature, etc.)
• • papers with 500+ citations (treatments/prevention)
• • 37% translated; 18% contradicted; 45% untested
• • ~1/2 were considered “good” quality, but this wasn’t predicative
• • keep in mind these were big papers that had a 37% translation (into clinical trial) rate; the more average papers likely don’t translate as ‘well’.

Question 5

What is face validity?

Answer 5

How well the model mimics the disease (ie. does the method of disease in the animal model match how it occurs in humans?)

Question 6

What is the CCAC?

Answer 6

CCAC = Canadian Council for Animal Care

Community representatives (have no bias since they aren’t paid by the U of A or any of the researchers)

[definition] a national organization responsible for setting and maintaining standards for the care and use of animals in science.

1. the use of animals in research, teaching, and testing is acceptable only if it promises to contribute to understanding of fundamental biological principles, or to the development of knowledge that can reasonably be expected to benefit humans or animals.
2. people involved with the use of animals in science must prepare and review scientific protocols with a view to replace, reduce, and refine animal use whenever possible.

Question 7

What is UAPWC?

Answer 7

University Animal Policy and Welfare Committee

Question 8

What is ACUC?

Answer 8

Animal Care and Use Committe

Question 9

What is research merit?

Answer 9

An indication that the study has potential for benefit enough to outweigh the harm.

Question 10

What are the evaluating protocols at the U of A?

Answer 10

1. Structure
2. Follows CCAC guidelines
3. PI submits application to ACUC
4. Decision (accept, revise, reject)
5. Information requested (people involved, species and number to be used, procedures to be used, research merit)
6. Follow up evaluations

Question 11

What are the three R’s of animal research?

Answer 11

1. Replacement: computer simulations instead of animals, rodents instead of primates, and in vitro instead of in vivo.
2. Reduction: using fewer animals and maximizing information gained.
3. Refinement: changing procedures to minimize pain and distress, reducing the level of invasiveness

Question 12

What are the levels of invasiveness?

Answer 12

(A) use of invertebrates or live isolates (not a lot of review here)
(B) experiments causing little or no harm (pure observation in natural environment, for example)
(C) experiments causing minor pain or stress for a short period without causing significant changes in animal appearance or physiology (behavioural training, for example)
(D) moderate to severe distress or discomfort but without prolonged clinical distress (doing surgery, taking blood samples, for example – a lot of stroke research happens here)
(E) experiments that causes severe stress at or above pain threshold (prolonged, inescapable pain, such as tumor growth in animals until death; these are fiarly rare to receive/accept without revision)

Question 13

What are some type of stroke models?

Answer 13

1. In Vitro
   - - cultured cells
   - - brain slice preparations
   - - insults (oxygen glucose deprivation [OGD], excitotoxicity [glutamate], other ‘toxins’ [blood])
   - - these models are used to study the mechanisms of damage and putative neuroprotectants, but are not very relevant for plasticity/rehab work
2. In Vivo
   - - ischemia (global or focal)
   - - hemorrhage (ICH, SAH)
   - - others (devascularization, suction lesions, knife cuts, excitotoxins [glutamate], trauma)

Question 14

What type of animals are used for stroke research?

Answer 14

Rodents, mostly (cost is fine, they’re simple, ethics are well defined); also used are primates and pigs, justified by their relevance to humans (as a final step prior to clinic work).

Question 15

Why study models?

Answer 15

– to study pathophysiology; allowing for the development and evaluation of neuroprotectants, drugs which target the mechanisms of cell death)

– to study neuroplasticity; development and evaluation of treatments, rehab

– to look for side-effects; ie. toxicity

Question 16

What are some things to consider when looking at animal models?

Answer 16

1. reliability
2. validity (clinical relevance)
3. limitations/problems with models (cost, complexity, surgical side effects and ability to broadly reflect mechanisms of injury/repair occurring in humans)
4. ethics

Question 17

What level is stroke research/why is it this level?

Answer 17

Level D (surgery with recovery; post-op pain).

• • causes neurological impairments
• • various agents/treatments given (ie. experimental treatments with potential side-effects, various physiological manipulations, such as food deprivation, and repeated handling and testing which may involve restraints)
• • possibility of complications (loss of body weight, cerebral edema, risk of infection, seizures, and risk of death)

– NOT a level E because it is risk of death, not death as an endpoint

Question 18

Global Ischemia Models (description)

Answer 18

PURPOSE: to mimic cardiac arrest and related conditions (ie. severe hypotension); also used to predict results in focal ischemia.

[variability] results from method and duration of the ischemia, species and animal characteristics, such as age, and interventions (CPR, temperature, etc.).

Question 19

What are some issues with global ischemia models?

Answer 19

They tend to look at mild cases, ignoring that often with cardiac arrest in humans there are multiple comorbidities (narrowed vessels, diabetes, etc.) and ignoring the possibility of more severe cases not benefiting from neuroprotective effects.

Question 20

What are some tests for rodents to take after global ischemia that test memory?

Answer 20

1. morris water maze
2. t-maze
3. habituation
4. active and passive avoidance
5. novel objects
6. radial arm maze

Question 21

What are some confounds for the tests on rodents for memory following a GIS?

Answer 21

If there is ischemia to the MCA then there may be motor damage that could reflect latency in the morris water maze, or a bias to go in circles due to damage in one of the hemispheres; strokes could affect motivation, etc..

– there are ways to test these; ie. for motivation, make the platform visible in the morris water maze to see if they’ll go for it, can test how fast the animal is swimming to see if it’s a motor deficit, test for depression/apathy (floating instead of swimming, for example).

Question 22

Focal Ischemia Models (descriptions)

Answer 22

PURPOSE: to mimic thrombotic and embolic stroke.

[injury profile] usual a focal infarct with distal damage

[functional impairments] focal neurological signs (such as arm weakness), dementia in patients (can occur depending upon insult severity); usually somatosensory and motor impairments in animal models

[variability] results from the method, the duration, and the location of the ischemia; the species and animal/patient characteristics; interventions (tPA, temperature, etc.)

Question 23

What are some focal ischemia models?

Answer 23

[models]:

1. intraluminal suture occlusion of MCA
   - - ISO
   - - thread a needle up the MCA where there is an eventual stopping of blood flow (a common model)
2. clip occlusion or cauterization of MCA
   - - hole in the side of the head of the rat/mouse, find the MCA, and then clip/cauterize it
3. embolic occlusion of MCA
   - - take blood from the rate and let it clot, then inject the clot and let it move up the arteries on its own (very variable and not often used in research on plasticity/rehab studies)
4. photothrombosis (via. Rose Bengal dye)
   - - inject light sensitive dye into the animal which then circulates; when you shine a light on it, it causes a clot to occur in the region which is illuminated
   - - a good model for studying recovery
5. Vasoconstriction with endothelin 1 (ET1)
   - - ET1 is a basoconstrictive peptide; injected into the brain causes constriction of vessels (collapses enough and causes ischemia; can make small lesion in localized areas of the brain)

*messing with the MCA cause large areas of damage, while the latter two are smaller and more localized

Question 24

What are some global ischemia models?

Answer 24

[models]:

1. true cardiac arrest (mechanical or drug induced)
2. 2-VO (two vessel occlusion of carotid arteries)
   - - 2-VO alone
   - - 2-VO with hypotension
   - - (2-VO alone doesn’t cause ischemia in rats due to the Circle of Willis, but 2-VO + hypotension does)
3. 4-VO
   * models will vary with species (for example, rats have the CoW but other animals may not)

Question 25

Intracerebral Hemorrhage Model (description)

Answer 25

PURPOSE: to model ICH

[injury profile] hematoma with tissue destruction, selective peri-hematoma cell death, distal injury

[functional impairments] deficits similar to focal ischemia (size, location); animal models usually see motor impairments because of damaging the striatum

[variability] results from model, location/amount of bleeding, species and animal characteristics, and interventions

Question 26

What are some ICH models?

Answer 26

1. collagenase (an enzyme that’s natural in the body; it digests the blood vessel so it ruptures, resulting in a spontaneous bleed in the brain)
2. blood injection
3. injection of blood components (ie. thrombin, iron)
4. spontaneous ICH (SHR’s = spontaneous hemorrhagic rats, who are more likely to experience strokes; problem with this model is the variability, which makes it impractical, but with the mimicked physiology)

Question 27

Subarachnoid Hemorrhage Models (description)

Answer 27

PURPOSE: to model SAH in humans

[injury profile] spread of blood within subarachnoid space with hematoma development, selective neuronal damage with infarction sometimes occurring

[functional impairment] mild to severe impairments with high mortality, rodent models tend to cause modest memory impairments with up to 30% mortality

[variability] results from the model and the amount of bleeding, the species and animal characteristics, and any interventions

Question 28

What are some SAH models?

Answer 28

1. suture perforation (intraluminal suture perforation model, where you poke through the suture)
2. blood injections

Question 29

What are some common post-stroke deficits in rodent models?

Answer 29

• • sensory function (usually somatosensory)
• • motor function (walking, reaching, balancing)
• • cognition (learning and memory, attention; neglect)

Question 30

How do you test walking abilities in rodents?

Answer 30

• • beam (walking on a beam)
• • grid/ladder
• • rotarod
• • paw placement/weight bearing

Question 31

How would you test spontaneous limb use?

Answer 31

• • cylinder test (exploratory; rats with stroke may favour ipsilateral limb)
• • corner turn test (may have bias in how they turn)

Question 32

How would you test reaching in rodents?

Answer 32

• • staircase test
• • single pellet test
• • tray task

Question 33

What is the staircase test?

Answer 33

The rat has to reach to get to the pellets, which are laid down; there are higher shelves with easy access, and lower ones which the rat has to reach for.

Researchers typically count the number of pellets the rat gets and at a certain level of performance they’ll give them stroke controlateral to the preferred limb.

Question 34

What is the single pellet test?

Answer 34

A barrier is set up so that the rat must reach across it to get to the food; useful to asses how often they reach and how successful they are.

In stroke research it is also good to look at data besides endpoints – how good their reach is, if they aim correctly, how the grasping is, can they release it, etc.

(how successful versus how good)

Question 35

What is the tray task?

Answer 35

Rats are in a ‘jail’ of sorts (bars which prevent them from simply putting their head down to get at food which is placed on the opposite side); the rats need to reach to get to the food.

Researchers can learn which hand is preferred (most rats use both but have one they use more). After a stroke the rat may rely on their ‘good’ limb, and you can force them to use the impaired one to get to food (assess qualitative and quantitative measures).

Question 36

Why is looking at cell death after stroke important?

Answer 36

The mechanisms and timing of cell death are critically important to developing neuroprotectants.

[recovery]:

• • the location and extend of cell death determines the type and severity of impairments
• • mechanisms and rate of damage can influence and be influence by treatments (rehab)

Question 37

What does maturation of damage mean?

Answer 37

The injury doesn’t occur instantly but rather continues over a matter of days (in terms of cell death)

Question 38

What influences cell damage?

Answer 38

1. insult
2. severity
3. brain location

etc.

Question 39

What sort of damage would you expect to see first in a stroke rat model in the hippocampus?

Answer 39

CA1 is particularly vulnerable and is damaged early on; the CA4/hilus and subiculum are also vulnerable.
– after ischemia the cells in CA1 tend to die by four days

CA3 is more resistant to damage.

Question 40

What is the maturation phenomenon?

Answer 40

That cell death and atrophy take time to mature; the rate of maturation depends upon many factors.

• • so, for example, CA1 neurons take typically 2 - 4 days to die after moderate severity global ischemia, but sometimes they can die later
• • this is affected by insult severity (the longer the occlusion the quicker the cells die)

Question 41

What is a “dark neuron”?

Answer 41

An artifact; the tissue processing problem causes these cells to often be mistaken for cell death or injury.

Question 42

What is cell death like after focal ischemia?

Answer 42

It varies by insult severity and other factors (ie. CBF, which is critical). The visual appearance and mechanisms of injury overlap extensively with those for global ischemia.

• • the core is re most likely to target for therapies (promotes functional recovery)
• • is important for plasticity

Question 43

What is a TTC stain?

Answer 43

A stain for living cells; triphenyltetrazolium chloride.

– pale on a scan is metabolically inactive

Question 44

What is Wallerian degeneration?

Answer 44

Anterograde or orthograde degeneration; forward, up the neuron.

• • in this context there is also: transneuronal, retrograde, and anterograde
• • damage can also occur in white matter tracts and glial cells

Question 45

What are the injury rates after a hemorrhagic stroke?

Answer 45

[primary] trauma, mechanical (cells in the hematoma die quickly)

[secondary] peri-hematoma, due to toxicity of blood, etc. (these cells die more slowly; a maturation effect from days to months)

Question 46

What are some mechanisms of cell death in ischemia?

Answer 46

1. ischemia
2. loss of atp
3. depolarization
   - - it is a matter of seconds between loss of atp and depolarization; if you re-establish blood flow than it’ll be fine, but it depends a great deal on how long the cell is depolarized; without the Na+/K+ pump there’s an influx of ions (osmatic forces, pull water in – cytotoxic edema)
   - - although this can happen most cells actually recover and die days later
4. influx of ions
   - - Na+ and Cl- causes cell swelling
   - - influx of Ca++ leads to delayed injury (can also lead to activation of enzymes which are harmful to the cell as well)
5. release of nt (ie. glutamate)
6. further Ca++ entry
   - - NMDA
   - - non-NMDA (AMPA)
   - - exess activation of NMDA channels is thought to be harmful
   - - difficult to treat though since blocking NMDA blocks potential plasticity (blocks compensation as well as harmful effects)
7. activation of lipases, protease, endonucleases
8. protein synthesis inhibition
9. organelle damage (mitochondria)

• • and so on
• • a lot of what contributes to cell death/damage also contributes to cell recovery

Question 47

What are some mechanisms of cell death in hemorrhage?

Answer 47

1. trauma (mechanical forces)
2. ischmia (?)
3. neurotoxicity (ie. iron)
   - - iron is released from degrading rbc’s
   - - iron stimulates the production of fee radicals leading to cell death and cerebral edema
   - - iron is thought to have a considerably greater role in injury after a ICH than ischemia
4. inflammation (ie. microglia activation)

Question 48

What is the relationship between cell death and recovery?

Answer 48

The amount and location of cell death is key to predicting outcome; the type and rate of cell death critically affects the efficacy of neuroprotectants.

• • cell death stimulates neuroplasticity
• • cell death mechanisms overlap with repair processes
• • rehabilitation can affect cell death

[CELL DEATH AND REPAIR]:

1. share mechanisms
2. overlap in time
3. spatially overlap

Question 49

What are the differences between basic science and clinical science?

Answer 49

[BASIC SCIENCE]

1. generated many possible targets (ie. understanding mechanisms of injury)
2. screen many targets for promising candidate treatments
3. leading compounds

[CLINICAL SCIENCE]

1. evaluate putative targets in clinical studies (confirmation of animal work)
2. evaluate best treatments in phase i - iii trials
   - - phase iii deals with efficacy and is very important in clinical trials
3. widespread useage

Question 50

What are some effective treatments for strokes?

Answer 50

While no neuroprotective drug has been proven to reduce damage (this is after focal ischemia in people), there are treatments:

1. tPA and related treatments (removes clot and improves CBF)
2. hemicraniotomy for massive MCA occlusion
3. hypothermia or prevention of fever (improves outcome and reduces mortality in neonates after HIE and in adults with cardiac arrest; data indicates that it will work after focal ischemia and maybe after SAH/ICH)

Question 51

What are some problems with animal studies?

Answer 51

1. choice of species (prefer rodents to most anything else), model, comorbidities, etc.
2. choice of treatment (possible conflicts of interest, other biases)
3. study design (randomization, group sizes, endpoints)
   - - endpoints: histology (lesion size, not perfect predictor of behavioural effects), behaviour, or duration of assessment
4. physiological controls (temperature, anesthesia)
5. treatment delivery (side-effects, feasibility, use of combination of treatments)
6. dissemination of results (ie. publication bias)

Question 52

What are some consideration needed for designing phase iii acute stroke trials?

Answer 52

1. dose selection based on preclinical and phase 1 and 2 data
   - - the does needs to be scaled to some measure, translated from animal model to human; the dose selection needs to be based on these models
2. time window for initiation of drug (a lot of stroke patients will be given neuroprotective drugs way too late; same with rehab)
3. patient selection based on mechanisms of action
4. outcome measures (one type of primary outcome or global assessment; it is important to outline beforehand the method of measurement and the type of primary outcome)
5. severity of stroke population to be studied
6. length of follow-up period
7. use of surrogate markers to provide support for drug efficiency (ie. protein released due to brain damage/stroke will go into the blood stream, and you can do a blood test to check to see if the drug lowers it)
8. prespecification of covariate analysis
9. fostering of appropriate and effective relationships between sponsors, academicians, and investigators

Question 53

What are some issues with clinical studies?

Answer 53

1. the type and sequence of trials (often studies, esp. rehab ones, do not follow phase i – iii progression)
2. choice and timing of therapy
   - - not always well founded in animal research
   - - rehab therapies are often poorly defined compared to drug ones (less risk of consequences)
   - - treatments may be given too late
3. patient selection, group size, etc.
   - - diversity creates variability while selectivity limits your ability to generalize (rehab. therapies often use compliant patients)
   - - rehab trials are often small (especially compared to neuroprotection; usually due to funding, but creates low statistical power)
4. choice of endpoint(s) and assessment time
   - - function scales; sometimes it is difficult to explain how a therapy actually improves outcome
5. procedural details and protocol violations
   - - blinding
   - - randomization
   - - control groups