Isoflurane does not cause neuroapoptosis but reduces astroglial processes in young adult mice

Abstract

Background: Isoflurane, a volatile anesthetic widely used clinically, has been implicated to be both neuroprotective and neurotoxic. The claim about isoflurane causing neural apoptosis remains controversial. In this study, we investigated the effects of isoflurane exposures on apoptotic and anti-apoptotic signals, cell proliferation and neurogenesis, and astroglial processes in young adult mouse brains.

Methods: Sixty 6-week-old mice were randomly assigned to four anesthetic concentration groups (0 as control and 0.6%, 1.3%, and 2%) with four recovery times (2 h and 1, 6, and 14 d) after 2-h isoflurane exposure. Immunohistochemistry measurements of activated caspase-3 and Bcl-xl for apoptotic and anti-apoptotic signals, respectively, glial fibrillary acidic protein (GFAP) and vimentin for reactive astrocytosis, doublecortin (Dcx) for neurogenesis, and BrdU for cell proliferation were performed.

Results: Contrary to the previous conclusion derived from studies with neonatal rodents, we found no evidence of isoflurane-induced apoptosis in the adult mouse brain. Neurogenesis in the subgranule zone of the dentate gyrus was not affected by isoflurane. However, there is a tendency of reduced cell proliferation after 2% isoflurane exposure. VIM and GFAP staining showed that isoflurane exposure caused a delayed reduction of astroglial processes in the hippocampus and dentate gyrus.

Conclusion: Two-hour exposure to isoflurane did not cause neuroapoptosis in adult brains. The delayed reduction in astroglial processes after isoflurane exposure may explain why some volatile anesthetics can confer neuroprotection after experimental stroke because reduced glial scarring facilitates better long-term neuronal recoveries.

Figure 1

Immunohistochemical staining of apoptotic and anti-apoptotic signals in the hippocampal Cornu Ammonis layer 1 (CA1) regions. A-C: Activated caspase-3 staining (green) revealed no apoptosis in isoflurane-exposed animals (A), whereas positive control from a mouse after 20-min of hypoxia exhibited activated caspase-3⁺ cells (B). C is higher magnification of area outlined in B. D-F: Bcl-xl immunostaining in the hippocampal CA1 regions (red) revealed no anti-apoptotic response in isoflurane-exposed animals (D), whereas positive control from a mouse after 20 min of hypoxia exhibited Bcl-xl⁺ cells (E). F is a higher magnification of area outlined in E. Cell nuclei are stained with DAPI (blue).

Figure 2

Counting of the number of BrdU⁺ cells in the subgranular zone of dentate gyrus. All animals that received BrdU injection were grouped by isoflurane (isof.) concentrations (Control = 0%), irrespective of recovery time. Error bars show the median and the 25^th and 75^th percentiles.

Figure 3

Quantification of doublecortin⁺ (Dcx⁺) cell densities (in unit of Dcx⁺ cells/mm²) in the dentate gyrus subgranular zone, showing no statistically significant difference among groups.

Figure 4

Immunohistochemical analysis of GFAP and VIM in representative regions of the dentate gyrus granule layer, hilus, and hippocampal molecular layer. Notice the reduced immunoreactivities at the later time points in the isoflurane-exposed animals.

Figure 5

Quantification of percent GFAP⁺ and percent VIM⁺ areas in the dentate gyrus granule layer, hilus, and hippocampal molecular layer at different anesthetic concentrations over days of observation. Red, blue, green, and purple bars represent control, 0.6%, 1.3%, and 2% isoflurane exposure groups, respectively. Depicted are percentage of GFAP⁺ immunoreactivity in the granule layer (A), hilus (B), and hippocampal molecular layer (C) and percentage of VIM⁺ immunoreactivity in the granule layer (D), hilus (E), and hippocampal molecluar layer (F). Notice the marked reduction in GFAP and VIM at later time points especially at the higher concentration of isoflurane. *, p < 0.05; #, p < 0.01.