Evidence for the use of isoflurane as a replacement for chloral hydrate anesthesia in experimental stroke: an ethical issue

Abstract

Since an ethical issue has been raised regarding the use of the well-known anesthetic agent chloral hydrate, owing to its mutagenic and carcinogenic effects in animals, attention of neuroscientists has turned to finding out an alternative agent able to meet not only potency, safety, and analgesic efficacy, but also reduced neuroprotective effect for stroke research. The aim of this study was to compare the potential of chloral hydrate and isoflurane for both modulating the action of the experimental neuroprotectant MK801 and exerting analgesia. After middle cerebral artery occlusion in rats, no difference was observed in 24 h survival rate, success of ischemia, or infarct volume reduction between both anesthetics. However, isoflurane exerted a more pronounced analgesic effect than chloral hydrate as evidenced by formalin test 3 hours after anesthesia onset, thus encouraging the use of isoflurane in experimental stroke models.

Figure 1

Experimental design and classification of animal groups. I/R: ischemia/reperfusion animals; VEH: vehicle group (saline); ISO: isoflurane anesthesia (4% induction, 2% maintenance); CHLO: chloral hydrate anesthesia (300 mg/kg ip). MK801 group corresponded to animals who received MK801 IV injection (0.5 mg/kg) 3 minutes before MCAO.

Figure 2

Effects of chloral hydrate and isoflurane on 24 h survival and ischemic events rates after MCAO. Frequency of infarct patterns, subcortical infarct areas and total infarct areas (ischemic), nonischemic and dead animals related to anesthesia type: (a) chloral hydrate; (b) isoflurane. N = 45 animals per group. Only ischemic animals were kept in the study.

Figure 3

Compared effects of chloral hydrate and isoflurane on infarct volume after MCAO. Volumes are corrected from edema and expressed in mm³ (mean ± SEM); n = 8 animals per group. Differences were not significant on total infarct volume according to t-test analysis (P = 0.9862), cortical infarct (P = 0.7577), and subcortical infarct (P = 0.4079).

Figure 4

Neuroprotective effect of MK801 in rats anesthetized with chloral hydrate (CHLO) or isoflurane (ISO). (a) Representative brain slices after Nissl staining for each group treated with vehicle (VEH) or MK801 (MK801). (b) Infarct volumes (corrected from oedema) were determined after one-hour MCAO followed by 23 hours of reperfusion period. MK801 was injected three minutes before MCAO (IV, 0.5 mg/kg). Volumes are expressed in mm³ (mean ± SEM); n = 8 animals per group. Differences in infarct volume were significant according to one-way ANOVA test (P < 0.05).

Figure 5

Assessment of pain after surgery including anesthesia with either chloral hydrate (CHLO) or isoflurane (ISO). Pain scores (ranging from 0 to 3) were pooled every 5 minutes. Formalin test was performed on healthy animals, sham animals, and I/R animals in each condition of anesthesia; n = 5 animals per group. In the SHAM group (Figure 5(a)), a significant difference was observed between isoflurane treated and healthy animals at 15 min of the formalin injection according to one-way ANOVA test (P = 0.02). At the other time points, no significant difference was observed between the different groups according to one-way ANOVA test. In the group I/R (Figure 5(b)), a significant difference was observed between isoflurane group and healthy animals at 35 min (P = 0.03) and at 50 min (P = 0.005) after formalin injection according to one-way ANOVA test.