Upload
berniece-doyle
View
228
Download
0
Tags:
Embed Size (px)
Citation preview
Division of Laboratory Animal Resources (DLAR)
Evaluation of CO2 Displacement Rates During Mouse Euthanasia
in Balb/C Mice
Joseph T. Newsome DVM, DACLAM
Clinical Director, Lab Animal Resources
Associate Professor, Pathology
Univ. of Pittsburgh
Division of Laboratory Animal Resources (DLAR)
• Goal - identify ranges of CO2 displacement rates & CO2 concentration and their impacts on pain and stress during mouse euthanasia
• Today – present early data related to displacement rates & CO2 concentrations using measures of neurophysiology and mouse behavior.
Objectives
2015 ACLAM Forum New Orleans 2
Division of Laboratory Animal Resources (DLAR)
3
• 2013 AVMA Euthanasia Guidelines– a displacement rate from 10% to 30% of the
chamber volume/min is recommended
• NIH Acceptance• Guide 8th ed. – Performance Standards• Literature Review• Employee Concerns
Background
2015 ACLAM Forum
Division of Laboratory Animal Resources (DLAR)
4
AVMA CO2 POE info• ….inhaled agents should be administered under
conditions where animals are most comfortable.• ….inhaled anesthetic vapors associated with some
degree of aversion in laboratory rodents, with increasing aversion noted as concentration increased Leach et al (pp 162–164)
• Inhalation of CO2 causes respiratory acidosis and produces a reversible anesthetic state by rapidly decreasing intracellular pH. (p211)
2015 ACLAM Forum
Division of Laboratory Animal Resources (DLAR)
5
AVMA CO2 POE info• Both basal and evoked neural activity are depressed soon after
inhalation of 100% CO2. (pp 211–214 ) • Inhalation of CO2 at 7.5% increases pain threshold, and >
30% cause deep anesthesia and death with exposure.• Carbon dioxide distress in animals via 3 mechanisms:
1. pain due to formation of carbonic acid on respiratory and ocular membranes,
2. production of so-called air hunger & feeling of breathlessness,
3. direct stimulation of ion channels within the amygdala associated with the fear response.
Division of Laboratory Animal Resources (DLAR)
6
Literature Review• Mice detect very small changes to CO2 levels
(Hu et al, 2007), and rats abandon food rewards in a chamber when CO2 reach only 16-18% (Niel & Weary, 2007).
• Rodents are very sensitive to CO2 changes, escape if able, and prolonged exposure to CO2
without the ability to escape is likely stressful.
2015 ACLAM Forum
Division of Laboratory Animal Resources (DLAR)
7
Key Literature Issues• Slow displacement rate of 20% / min. is more
humane than fast exposure to very high CO2
concentrations, as occurs with pre-filled chambers (Hawkins et al., 2011).
• Mice euthanized with 20% versus 100% CO2
displacement/min, demonstrated no significant difference in plasma corticosterone or cfos levels in the hypothalamus (Valentine et al., 2012).2015 ACLAM Forum
Division of Laboratory Animal Resources (DLAR)
8
Literature review cont.• Valentine evaluated cfos after four min. CO2
exposure• Cfos is not induced until approximately 15 min.
post exposure (Hu et al., 1994), and optimally expressed at > 30 min. post exposure (Imaki et al, 1994; Johnson et al., 2011).
• Choose to measure activation of Extracellular signal Regulated Kinase (ERK), a rapidly expressed indirect measure of cellular activity (Ji et al., 1999). 2015 ACLAM Forum
Division of Laboratory Animal Resources (DLAR)
9
ANATOMY: Stress/Anxiety
2015 ACLAM Forum – New Orleans
Para Ventricular Nucleus of the hypothalamus (PVN)
Central Amygdala (AMYG
Zieman etal. Cell 139 2009
Division of Laboratory Animal Resources (DLAR)
10
ANATOMY: Pain
2015 ACLAM Forum – New Orleans
Nociception: Trigeminal Nucleus Caudalis (TNC) & Nucleus Tractus Solitarius (NTS)
Division of Laboratory Animal Resources (DLAR)
11
Hypothesis
• CO2 exposure at moderate chamber displacement rates (above 30% and below 110% per minute), will reduce time to unconsciousness without significantly increasing nociception, stress, and respiratory pathology, when compared to current AVMA POE displacement rate recommendations of 10%-30% per minute2015 ACLAM Forum
Division of Laboratory Animal Resources (DLAR)
12
Reporting Results
2015 ACLAM Forum
1. Time to unconsciousness,2. Plasma corticosterone levels3. ERK activation in the Trigeminal
Nucleus Caudalis, NTS, PVN, and the Central Amygdala
4. Behavioral measures of fear & anxiety5. Histopathology
Division of Laboratory Animal Resources (DLAR)
13
Methodology -
2015 ACLAM Forum
50 mice
5 mice each group
10%, 35%, 60%, 85%, or 110%displacement
CO2
MedicalGrade Air
Division of Laboratory Animal Resources (DLAR)
14
Methodology – Pilot Two Arms
• 10% and 110%• Controls Decapitation
– Identical mean time
• Capsacin vs 28%, 43%, and 100% CO2
2015 ACLAM Forum
Pilot Arm 1
Pilot Arm 2
Division of Laboratory Animal Resources (DLAR)
15
Methodology – Arm 1
2015 ACLAM Forum
• Balb/cJ mice Male, ages 8-11 weeks – Acclimated 4-7 days full microisolator techniques
• Nesting material (Nestlets®) and cardboard huts (Sheppard Shacks®)
• All studies performed 9-11 am
Division of Laboratory Animal Resources (DLAR)
16
Methodology:
2015 ACLAM Forum
A B
C
B
Dimensions: 42.5cm L x 42.5cm W x 30.5 cm H
Division of Laboratory Animal Resources (DLAR)
172015 ACLAM Forum
Rodent Behavioral Analysis Core
Med Associates Inc
Division of Laboratory Animal Resources (DLAR)
18
Methodology – ERK Brain Regions
2015 ACLAM Forum
ERK Activity during CO2 Euthanasia: Active ERK (p-ERK/t-ERK) was > in the trigeminal nucleus caudalis and lowest in the paraventricular nucleus of the hypothalamus pERK is measurable in brain regions of interest
Division of Laboratory Animal Resources (DLAR)
19
Time to Unconsciousness
2015 ACLAM Forum – New Orleans
Time to unconsciousness • Affected by
displacement• p-value =
4.701e-05
Division of Laboratory Animal Resources (DLAR)
20
Results: Histopathology• Nasal Turbinates – No significant
differences - Mild to moderate multifocal hemorrhage all.
• Lungs – hemorrhage, fibrin, alveolar wall thickening– 10% CO2 >> 110% CO2
– Dyspnea??
2015 ACLAM Forum – New Orleans
Division of Laboratory Animal Resources (DLAR)
21
RESULTS: Corticosterone Elisa Assay (ng/ml)
2015 ACLAM Forum
cortiso
l
●
●
●
150
●
100 ●
●
●
●
●
●
50 ●
●
● ● ● ●
● ●
0
N.10% N.110% Y.10% Y.110% C O 2 .d isp lace
• 10% vs controls
P=.02• 110% vs
controls NS
• 10% vs 110%
P = .001
Division of Laboratory Animal Resources (DLAR)
22
Results ERK Ratio: AMYG PERK/TERK• CO2 vs Med Air
– P< 0.003
• Displacement 10% vs 110%– Not Significant
– P< 0.21
N.10% N.110% Y.10% Y.110%
CO2 Displaced
AM
YG
PE
RK
/TE
RK
Division of Laboratory Animal Resources (DLAR)
23
Results ERK Ratio: Other Regions• PVN stress• TNC and NTS –
Pain perception– No Significant
Differences
N.10% N.110% Y.10% Y.110%
CO2 Displaced
PV
N P
ER
K/T
ER
K
Division of Laboratory Animal Resources (DLAR)
24
Methodology – Behavior Assessment
2015 ACLAM Forum
• Video Scoring - Blinded
• Med Associates Behavioral Data Mean Speed Total Distance Rearings and Jumps Ambulatory Time
Division of Laboratory Animal Resources (DLAR)
25
Behavioral Results - Video scoring • Tail slapping• Face/Whisker rubbing• Stereotypy - fine movements anxiety?
Ongoing pending statistical evaluations– Preliminary no noticeable trends or differences
between controls or exposure groups
2015 ACLAM Forum
Division of Laboratory Animal Resources (DLAR)
26
Mean Speed
2015 ACLAM Forum – New Orleans
• Mean speed is not affected by CO2 or displacement
• Mean distance is not affected by CO2 or displacement.
• Total distance obvious 10%>110%
P<.01
Distance
Division of Laboratory Animal Resources (DLAR)
27
Vertical Rearing
2015 ACLAM Forum – New Orleans
• Adjusted conscious time• CO2 is significant• Displacement rate is
highly significant• Interaction between CO2
and displacement is not significant
p-value CO2 0.02646
displace < 2e-16 CO2:displace 0.36589
Division of Laboratory Animal Resources (DLAR)
28
Ambulatory Time
2015 ACLAM Forum – New Orleans
10
• Higher displacement reduces ambulatory time, but the effect of CO2 is small and NS
• Time of last movement is related to displacement (p<<0.0001)
• Time in Zone 1 is strongly related to CO2(p <0.0007)
Division of Laboratory Animal Resources (DLAR)
29
Interpreting Behavioral Results
2015 ACLAM Forum
• Zone analysis & movement. – Calm vs Ambulatory CO2 or displacement
– CO2 induced Freezing
– Exploratory, Zone crossing
• Zone 1 suggests lower anxiety
• Thigmotactic vs. Central
0
Division of Laboratory Animal Resources (DLAR)
30
Methodology – CO2 Concentration Arm 2• Capsaicin (positive control) 10 mM stock
solution in EtOH.• Chloral Hydrate - 370-400 mg/kg• C02 28% vs 43%Fixed Flow • Decapitation
– Core Blood: pending cortisone
– Brain Harvest
2015 ACLAM Forum
Division of Laboratory Animal Resources (DLAR)
31
Results: ERK Activity during CO2 Euthanasia Compared to Capsaicin: AMYG
2015 ACLAM Forum
• Capsaicin and 100% CO2 detectible & both significant altered vs. 28% and 43% (P<0.01)
• ERK is detectible but not significant differences in PVN, NTS, or TNC (Pain)
Division of Laboratory Animal Resources (DLAR)
32
Conclusions: Whats it all mean?• ERK Ratios - effective measure of brain
activation related to CO2 and or displacement effects (AMYG yes, +/- PVN
• Pain Region Assessments– Not Painful or equivocal
– Detection not timely or sensitive enough
• Behavioral Assessments - Ongoing
2015 ACLAM Forum
Division of Laboratory Animal Resources (DLAR)
33
Future Directions Lessons Learned
2015 ACLAM Forum
• More mice different displacements– 35%,60
• Strain and species variations– Genetics role in CO2 response – Immature animals
• Behavioral Interpretations• The AVMA and POE have made a commitment
to ensure the Guidelines reflect an expectation and paradigm of continuous improvement
Division of Laboratory Animal Resources (DLAR)
34
Acknowledgements• ACLAM Foundation and the Mice• DLAR Kathleen McDonald BS CMAR, RLATG; Lora
Rigatti, DVM DACVP; Chris Janssen DVM, Mark Scales • Pain Specialist: Michael Gold Ph.D. Anesthesia• Neurobiology: Edda Thiels Ph.D. Jocelyn Mauna, Ms.
Laura Miller RBAC - Rodent Behavioral Analysis Core• Stats: Daniel Normolle Ph.D. Biostatistics UPCIThis project was supported in part by award P30CA047904 (AF & Stat)
2015 ACLAM Forum
Division of Laboratory Animal Resources (DLAR)
35Funny Musicians – Funnystack.com