Division of Laboratory Animal Resources (DLAR) Evaluation of CO2 Displacement Rates During Mouse Euthanasia in Balb/C Mice Joseph T. Newsome DVM, DACLAM

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


• 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

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• 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

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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)

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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.


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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.

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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


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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


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ANATOMY: Stress/Anxiety

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Para Ventricular Nucleus of the hypothalamus (PVN)

Central Amygdala (AMYG

Zieman etal. Cell 139 2009


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ANATOMY: Pain


Nociception: Trigeminal Nucleus Caudalis (TNC) & Nucleus Tractus Solitarius (NTS)


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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


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Reporting Results

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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


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Methodology -

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50 mice

5 mice each group

10%, 35%, 60%, 85%, or 110%displacement

CO2

MedicalGrade Air


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Methodology – Pilot Two Arms

• 10% and 110%• Controls Decapitation

– Identical mean time

• Capsacin vs 28%, 43%, and 100% CO2

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Pilot Arm 1

Pilot Arm 2


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Methodology – Arm 1

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• 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


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Methodology:

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A B

C

B

Dimensions: 42.5cm L x 42.5cm W x 30.5 cm H


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Rodent Behavioral Analysis Core

Med Associates Inc


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Methodology – ERK Brain Regions

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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


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Time to Unconsciousness


Time to unconsciousness • Affected by

displacement• p-value =

4.701e-05


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Results: Histopathology• Nasal Turbinates – No significant

differences - Mild to moderate multifocal hemorrhage all.

• Lungs – hemorrhage, fibrin, alveolar wall thickening– 10% CO2 >> 110% CO2

– Dyspnea??



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RESULTS: Corticosterone Elisa Assay (ng/ml)

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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


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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


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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


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Methodology – Behavior Assessment

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• Video Scoring - Blinded

• Med Associates Behavioral Data Mean Speed Total Distance Rearings and Jumps Ambulatory Time


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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

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Mean Speed


• 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


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Vertical Rearing


• 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


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Ambulatory Time


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• 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)


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Interpreting Behavioral Results

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• Zone analysis & movement. – Calm vs Ambulatory CO2 or displacement

– CO2 induced Freezing

– Exploratory, Zone crossing

• Zone 1 suggests lower anxiety

• Thigmotactic vs. Central

0


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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

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Results: ERK Activity during CO2 Euthanasia Compared to Capsaicin: AMYG

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• 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)


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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

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Future Directions Lessons Learned

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• 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


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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)

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Documents

Division of Laboratory Animal Resources (DLAR) Evaluation of CO2 Displacement Rates During Mouse Euthanasia in Balb/C Mice Joseph T. Newsome DVM, DACLAM