Table 1.
Characteristics of the 15 adult mouse studies included in the systematic review.
| References | Strain, Sex, and Age | Intervention treatments of interest (group size) | Euthanasia or anesthesia induction with recovery | Frequency of exposure or re-use | Control intervention (group size) | Outcome measures (identification of effect) |
|---|---|---|---|---|---|---|
| (16) | Quackenbush, females, mature | 1. sublimation of dry ice 2. Chloroform into bedding 3. Ether-soaked cotton (n = 12) |
Euthanasia | None, three replicates | Untreated controls in same environment in two densities, n = 1 and 3 Three replicates for each group |
Chamber escape behaviors: ND LOP: shorter for CO2 Time to death: shorter for CO2 |
| (17) | C57BL/6N, males, 16 week | 1. CO2 15% VDR 2. CO2 30% VDR 3. CO2 50% VDR 4. CO2 100% VDR (n = 12–13) |
Definitive: euthanasia | Re-used control mice in euthanasia studies | Pilot: air flow Controls: noise and air movement-only exposure, n = 28 |
Anxiety: ND Heart rate: ND between groups; blood pressure: ND LOP: faster as [CO2] increased Plasma cort: ND Time to death: decreased as [CO2] increased |
| (18) | C57BL/6NTac, males, 16 week | 1. Isoflurane 5% in O2 1 L/min then 100% CO2 after induction 2. Pentobarbital IP (n = 11–14) |
Euthanasia | None | Used same control and CO2 comparator data as published in (17) | Anxiety: increased as [CO2] increased distance traveled: increased as [CO2] increased Heart rate: ND or increased as [CO2] increased Blood pressure: ND or decreased as [CO2] increased Plasma ACTH: reduced with pentobarb LOP: ND or faster as [CO2] increased Time to death: ND or faster as [CO2] increased |
| (19) | 129 × S1/SVJ, mixed sex, 6–24 week | 1. CO2 100% facemask 2. CO2 70% facemask 3. KCl 4. cervical dislocation 5. decapitation (n = 5–10) |
euthanasia under succinyl choline | None | Each animal used as own control for baseline data | Time to loss of EEG signal: shorter for CO2 100% = KCl = cervical dislocation = decapitation Visual evoked potential: reduced for CO2 100% |
| (20) | NMRI, males, 12–16 week | 1. CO2 0.2 L/min FR 2. 8% sevoflurane in O2 0.2 L/min FR 3. 4% isoflurane in O2 0.2 L/min FR (n = 6–9) |
Gas exposure with recovery | 4 days chamber habituation, no gas | Air only central chamber, with two lateral chambers into which gas pumped, food restriction to maintain 85–95% pre-study weight, food pellets placed in lateral chambers during testing to motivate entry, compared combinations of gas exposures in three trials: sevo vs. CO2, CO2 vs. iso, iso vs. sevo | Approach/avoidance with food reward: time in sevo chamber increased compared with air only, ND between sevo vs. CO2 chambers, ND between time in iso vs. CO2 chambers, ND between time in iso vs. sevo chambers, ND in food reward eaten between three chambers |
| (21) | Mixed strains, cull genotypes, females, mature, and pregnant | 1. CO2 20% VDR 2. Pentobarbital IP 3. Cervical dislocation 4. Halothane in O2 5. KCl under anesthesia 6. Cervical dislocation under anesthesia (n = 5–7) |
Euthanasia or anesthesia induction with euthanasia | None | None, all compared to CO2 | LOP: not consistently measured time to cardiac arrest: fastest KCl, next fastest CO2 = cervical dislocation (with or without anesthesia) |
| (22) | BALB/C, females, 9–15 weekk | 1. CO2 53% FR 2. argon 99% FR 3. CO2 20%/argon 4.5% FR 4. CO2 30%/argon 5.3% FR (n = 30 total) -high concentrations only |
Definitive: gas exposure with recovery | Yes, up to 4 exposures per mouse over 6 week study | Pilot: gas level testing at low, medium, high, n = 30 (not re-used in definitive study) Definitive: 30 min chamber acclimation period, air-only 3 min control (each animal used as own control), measured time dwelling, time to withdrawal, and time to re-entry in chamber |
Rearing: increased for CO2 53% FR from baseline, ND between treatment groups approach/avoidance (time dwelling and time to withdrawal latencies): all [CO2] shorter vs. argon alone |
| (23) | BALB/C, females, 9–15 week | 1. CO2 50.8% FR 2. CO2 34.9% FR 3. CO2 25.5% FR 4. Argon 99.2% FR (n = 30 total) -high concentrations only |
Definitive: gas exposure with recovery | Up to 7 exposures per mouse | Pilot: gas level testing at low, medium, high, n = 30 (not re-used in definitive study) Definitive: 30 min chamber acclimation period, air-only 3 min control (each animal used as own control), measured time dwelling, time to withdrawal, and time to re-entry in chamber |
Anxiety/pain behaviors (urination, defecation, rearing): ND from baseline, ND between groups Approach avoidance latencies: time dwelling: shorter for all compared with air only, ND between groups; time to withdrawal latency: ND |
| (24) | CD1, males, surplus mature | 1. CO2 70% FR 2. AR 160% FR 3. CO 9% FR 4. isoflurane 3%/O2 70% FR (n = 6–7) |
Gas exposure with recovery | >50 exposures per mouse over 4 month study, three replicates per concentration | Control: air only, each animal as its own control -initial training to lower chamber with food reward -multiple CO2 concentrations evaluated | Approach/avoidance: latency to withdrawal decreased with increasing [CO2] (always left when chamber concentrations 13.5–18.2%), latency to withdraw for isoflurane = CO, latency to withdraw longest for argon |
| (25) | C57BL/6J-Tyr, females, 20 week | 1. CO2 50% FR 2. CO2 40% FR 3. CO2 30% FR 4. CO2 20% FR (n = 5–6) |
Euthanasia | None | None | Labored breathing: seen in all groups prior to LOP, ND seen between groups LOP: faster with increased [CO2] LORR: faster with increased [CO2] |
| (26) | C57BL/6J, males, 8 week | 1. CO2 20% VDR 2. isoflurane 5%/O2 4 L/min 3. isoflurane 5% drop (n = 8–9) |
Gas exposure with recovery | Two replicates | Three acclimation trials to test apparatus | Light/dark aversion pairing chamber latency: time to withdrawal: iso/O2 > CO2 = isoflurane drop, re-entry time: iso/O2 < CO2 = isoflurane drop; re-exposure: time to withdrawal reduced for iso/O2 |
| (27) | C57BL/6J, males, 12–16 week | 1. CO2 20% VDR 2. isoflurane 5% in O2 2 L/min (n = 6–7/group) |
Euthanasia | None | None | LOP: ND LORR: faster with CO2 time to death: faster with CO2 |
| (28) | CD1, retired sentinels, sex, and age not specified | 1. CO2 70% FR, 325 lux 2. CO2 30% FR, 325 lux 3. isoflurane 3% then 70% CO2 FR after induction, 325 lux 4. CO2 30% FR, 500 lux 5. CO2 30% FR, 5 lux (n = 6) |
Euthanasia | None, three replicates | Chamber light levels during euthanasia evaluated, euthanized in pairs in home cages, no acclimation period for lighting prior to euthanasia | Anxiety combined: highest for CO2 70% = CO2 70% + iso, increased at 500 vs. 40 lux = CO2 30% 325 lux LOP: CO2 70% < CO2 70% + iso < CO2 30% 325 lux Plasma cort: increased for CO2 70% + iso |
| (29) | CD1, mixed sex, 10–12 week | 1. CO2 20% VDR 2. CO2 20% + 60% N2O VDR 3. 20% CO2 + 60% N2 VDR (n = 3/sex/strain) |
Definitive: euthanasia | Pilot: 3 × with 48 h washout between trial | Pilot: exposure with recovery, (n = 12 F) | Rearing: ND Jumping: increased for CO2 + N2 LORR: fastest for CO2 + N2O |
| (30) | CD1, females, 8–11 week | 1. CO2 100% VDR 2. CO2 20% VDR 3. iso 5% in O2 1.2 L/min then CO2 100% VDR after induction 4. CO2 20% VDR + ACE premed 5. CO2 20% VDR + MDZ premed (n = 10) |
Euthanasia | None | Exposure to CO2 gas for euthanasia with or without sedation | Increased activity: higher for iso Ultrasonic vocalization: increased for iso = CO2 100% VDR Other behaviors: ND LORR: fastest for CO2 100% VDR Plasma corticosterone: increased MDZ plasma ACTH: ND c-fos: decreased with CO2 100% VDR Time to death: fastest for CO2 100% VDR |
ACE, acepromazine; ACTH, adrenocorticotropin hormone; cort, corticosterone; EEG, electroencephalograph; FR, flow rate; IP, intraperitoneal; iso, isoflurane; KCl, potassium chloride; LOP, time to loss of posture; LORR, time to loss of righting reflex; MDZ, midazolam; ND, no difference; sevo, sevoflurane VDR, volume displacement rate.