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32 Cards in this Set
- Front
- Back
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What is the protocol number?
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10052
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What strains of mice are we using?
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C57Bl/6 and Trpv4 KO
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Anesthesia of mice
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Pentobarbital sodium 50mg/kg i.p.
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What size catheter is inserted into trachea?
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24 gauge angiocath catheter
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What dose of Pseudomonas should be delivered?
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6 x 10^4 CFU
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What is the delivery volume?
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50 µl phosphate buffered saline, p.H. 7.4
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What dose causes pneumonia and severe sepsis in C56Bl/6 mice, associated with little mortality within the first 48 hours after innoculation?
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5 x 10^4 CFU
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What causes lung edema, lung epithelial injury but no mortality within 48 hours in C3H/HeJ mice?
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6 x 10^4 CFU (similar dose to 5 x 10^4 CFU)
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What is the delivery volume?
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50 microliters
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What strain of Pseudomonas are we using?
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Pa103
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What type of secretion system and exotoxins are expressed in the Pa103 strain?
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Type III secretion system
Exotoxin ExoU, ExoT and ExoY |
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How does the type III secretion system work?
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P. aeruginosa literally injects exotoxins into host cells
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How many innoculation groups?
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5 innoculation groups
1. The parent Pa103 strain 2. Pa103 lacking functional type III secretion system 3. Pa103 lacking all exotoxins 4. Pa103 expressing a mutant ExoU and no other exotoxins which has attenuated phospholipase A2 activity 5. Vehicle control |
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Where do animals go after inoculation?
1. 1 hour time point 2. 4 hour time point 3. 16 hour time point |
1. Remain in the room
2. Remain in the room 3. Back to the vivarium |
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What will be maintained in the vivarium for the 16 hour time point animals?
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A post-procedure care log, noting:
innoculation time time projected for terminal studies |
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If animals in vivarium group are in distress, what should be given to them?
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Buprenex
.15mg/kg, s.c. |
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Is fluid replacement allowed in the mice under study?
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No. This study is a measure of edema, and fluid replacement would confound the study.
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At the selected time point for each mouse, what do you do?
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Anesthetize with pentobarbitol (50mg/kg)
Open the Chest Inject 100U heparin into right ventricle |
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After heparin injection into right ventricle, what do you do?
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The pulmonary artery will be cannulated via the right ventricle
Left atrial appendage clipped open Lung vasculature briefly rinsed with sterile phosphate buffered saline |
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Subgroup 1
1. What operation is initially performed? 2. Assessment part 1 3. Assessment part 2 |
1. Trachea cannulation and BAL using 1 ml of PBS
2. Cytospin to assess inflammatory cell influx 3. Protein assay to determine lung injury |
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Subgroup 2
1. What we do to lungs 2. What we do to one lung 3. What we do to the other |
1. Randomly ligate lung at the hilus
2. Wet-dry ratio 3. Inflate, ligate and immerse in fixative for assessment of alveolar fluid volume fraction and lung structure |
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How many animals are to be assigned each sub group?
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8 animals
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What is trpv4 (according to this protocol)
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TRPV4 is a Ca and Mg permeable cation channel within the vanilloid receptor subgroup of the transient receptor potential TRP family.
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What are some trpv4 activating stimuli?
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Osmotic cell swelling
increased vascular and airway pressure |
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TRPV4 activation requires release of what?
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Arachidonic acid from membrane phospholipids via endogenous phospholipase A2 and synthesis of epoxyeicosatrienoic acids (cytochrome P450 epoxygenase metabolites of arachindonic acid)
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Objective of protocol
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Determine whether exogenous phospholipase A2, like that produced by P. aeruginosa exotoxins, can elicit trpv4 activation in mouse lung
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What have we already shown about the action of endogenous phosphlipase A2?
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That it activate TRPV4 Channel
It provides the substrate arachidonic acid |
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Which exotoxin produced by P. aeruginosa is a phospholipase A2?
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ExoU
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What is the breeding protocol for the trpv4 knockout mice?
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10047
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What is the plane of anesthesia monitored by?
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breathing patterns
motor tone limb withdrawal in response to toe pinch palpebral reflex |
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Animals anesthetized to a surgical plane should show
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slow and deep breathing
loss of motor tone lack of limb withdrawal after toe pinch loss of palbebral reflex |
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Immediately following innoculation with pseudomonas, what do we do with the mice
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return them to their cages
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