Isoflurane does not affect brain cell death, hippocampal neurogenesis, or long-term neurocognitive outcome in aged rats

Abstract

Background: Roughly, 10% of elderly patients develop postoperative cognitive dysfunction. General anesthesia impairs spatial memory in aged rats, but the mechanism is not known. Hippocampal neurogenesis affects spatial learning and memory in rats, and isoflurane affects neurogenesis in neonatal and young adult rats. We tested the hypothesis that isoflurane impairs neurogenesis and hippocampal function in aged rats.

Methods: Isoflurane was administered to 16-month-old rats at one minimum alveolar concentration for 4 h. FluoroJade staining was performed to assess brain cell death 16 h after isoflurane administration. Dentate gyrus progenitor proliferation was assessed by bromodeoxyuridine injection 4 days after anesthesia and quantification of bromodeoxyuridine+ cells 12 h later. Neuronal differentiation was studied by determining colocalization of bromodeoxyuridine with the immature neuronal marker NeuroD 5 days after anesthesia. New neuronal survival was assessed by quantifying cells coexpressing bromodeoxyuridine and the mature neuronal marker NeuN 5 weeks after anesthesia. Four months after anesthesia, associative learning was assessed by fear conditioning. Spatial reference memory acquisition and retention was tested in the Morris Water Maze.

Results: Cell death was sporadic and not different between groups. We did not detect any differences in hippocampal progenitor proliferation, neuronal differentiation, new neuronal survival, or in any of the tests of long-term hippocampal function.

Conclusion: In aged rats, isoflurane does not affect brain cell death, hippocampal neurogenesis, or long-term neurocognitive outcome.

Fig. 1

Minimum alveolar concentration (MAC) of isoflurane (iso) in 16-month-old rats. MAC was determined by tail clamping of simultaneously anesthetized rats (n = 7–10). Data are from five separate experiments.

Fig. 2

Physiologic variables over 4 h of isoflurane anesthesia in 16-month-old rats. Data are pH (A), arterial carbon dioxide tension (PaCO₂) (B), arterial oxygen tension (PaO₂) (C), mean arterial blood pressure (D), blood hemoglobin (Hb) concentration (E), blood glucose concentration (F), and pericranial temperature (G) from five cardiorespiratory control animals that were not used for any other part of the study. The exception is pericranial temperature, which was computer-controlled and recorded in all animals individually (n = 37).

Fig. 3

FluoroJade (FJ) stain of the hippocampus of 16-month-old rats 16 h after isoflurane (n = 6) or no isoflurane (control, n = 6). Extensive cell death in the CA-1 area of a positive control animal injected with kainic acid (10 mg/kg) 16 h before transcardiac perfusion (A). Almost complete absence of cell death in the hippocampus of a representative isoflurane-treated animal (B). Magnified view of one FJ-positive cell (arrow). Sham-anesthetized animals likewise had no significant detectable cell death (data not shown).

Fig. 4

Progenitor proliferation in the dentate gyrus (DG) after 4 h of isoflurane (iso) in 16-month-old rats. Bromodeoxyuridine (BrdU) (50 mg/kg) was injected twice with a 12-h interval 4 days after 4 h of isoflurane (n = 6) or no isoflurane (control, n = 6). Immunocytochemical staining of bromodeoxyuridine+ cells (arrows in A, main image 4×, inset 20× objective) of every twelfth coronal section of the hippocampus and quantification of every bromodeoxyuridine+ cell (B) revealed no difference between groups (P > 0.2, Mann–Whitney U test).

Fig. 5

Neuronal differentiation and new neuronal density are not affected by isoflurane (iso) in aged rats. Stack of microscopic images acquired 1 mm apart in the Z-plane (A) showing co-staining of the S-phase marker bromodeoxyuridine (BrdU) with the immature neuronal marker NeuroD 12 h after the last of two BrdU injections given intraperitoneally to aged rats on the fourth day after 4 h of isoflurane anesthesia. Neither the proportion of BrdU+ cells that coexpress NeuroD on day 5 after anesthesia (B) nor the density of BrdU+ /NeuroD+ cells (C) is affected by isoflurane. New neuronal survival 28 days after a 4-day pulse of bromodeoxyuridine (50 mg/kg × 8 at 12-hourly intervals) starting 4 days after 4 h of isoflurane anesthesia (D, G). Immunofluorescent image of a bromodeoxyuridine+ /NeuN+ cell (D, arrow) and an image stack acquired on a confocal microscope showing colocalization of two bromodeoxyuridine+ /NeuN+ cells (E). The proportion of bromodeoxyuridine+ cells that coexpress the mature neuronal marker NeuN 35 days after isoflurane (n = 5) or no isoflurane (control, n = 6) does not differ between groups (F). The density of bromodeoxyuridine+ /NeuN+ cells in the DG is not different between groups either (G). DAPI = 4′,6-diamidino-2-phenylindole.

Fig. 6

Fear conditioning 4 months after 4 h of isoflurane (iso) anesthesia (n = 13) or sham anesthesia (control, n = 11) administered to 16-month-old rats. Unconditioned fear is measured by freeze scores at baseline before (A) and 24 h after exposure to the conditioned stimulus–unconditioned stimulus pairings (B). The time curve (C) and summary (D) of the tone test reveals no difference between groups in the degree to which the nonhippocampal-dependent association between the tone and the foot shock were formed. During the hippocampal-dependent context portion (E and F) of the test, there was also no difference in freeze scores between groups (P > 0.24, Mann–Whitney U test).

Fig. 7

Morris water maze test 4 months after 4 h isoflurane (iso) anesthesia (n = 13) or sham anesthesia (control, n = 11) in 16-month-old rats. During the cued trials (data not shown), the submerged platform was marked with a flag, which was removed during the place trials (A), in which spatial memory is tested by measuring time-integrated distance to the platform allowing some credit to be assigned for searching in the general vicinity of the platform. The 90-s probe trial (platform removed) tests memory retention (B). The dotted line indicates the likelihood of time spent in quadrants by chance. No difference was found between groups in any of the measured parameters. CW = clockwise.