Brain regional vulnerability to anaesthesia-induced neuroapoptosis shifts with age at exposure and extends into adulthood for some regions

Abstract

Background: General anaesthesia facilitates surgical operations and painful interventions in millions of patients every year. Recent observations of anaesthetic-induced neuronal cell death in newborn animals have raised substantial concerns for young children undergoing anaesthesia. However, it remains unclear why some brain regions are more affected than others, why certain neurones are eliminated while neighbouring cells are seemingly unaffected, and what renders the developing brain exquisitely vulnerable, while the adult brain apparently remains resistant to the phenomenon.

Methods: Neonatal (P7), juvenile (P21), and young adult mice (P49) were anaesthetized with 1.5% isoflurane. At the conclusion of anaesthesia, activated cleaved caspase 3 (AC3), a marker of apoptotic cell death, was quantified in the neocortex (RSA), caudoputamen (CPu), hippocampal CA1 and dentate gyrus (DG), cerebellum (Cb), and olfactory bulb (GrO) and compared with that found in unanaesthetized littermates.

Results: After anaesthetic exposure, increased AC3 was detected in neonatal mice in RSA (11-fold, compared with controls), CPu (10-fold), CA1 (three-fold), Cb (four-fold), and GrO (four-fold). Surprisingly, AC3 continued to be elevated in the DG and GrO of juvenile (15- and 12-fold, respectively) and young adult mice (two- and four-fold, respectively).

Conclusions: The present study confirms the findings of previous studies showing peak vulnerability to anaesthesia-induced neuronal cell death in the newborn forebrain. It also shows sustained susceptibility into adulthood in areas of continued neurogenesis, substantially expanding the previously observed age of vulnerability. The differential windows of vulnerability among brain regions, which closely follow regional peaks in neurogenesis, may explain the heightened vulnerability of the developing brain because of its increased number of immature neurones.

Keywords: anaesthesia, paediatric; anaesthetics volatile, isoflurane; brain, injury; safety, drug; toxicity.

Fig 1

Prolonged exposure to isoflurane triggers neuronal apoptotic cell death in several brain regions in newborn, juvenile, and young adult mice. Boxplots represent density counts of dying cells, as assessed by expression of cleaved caspase 3, a marker of apoptotic cell death. Thick horizontal lines signify respective group medians, boxes are 25th–75th percentiles, whiskers are 10th–90th percentiles, open circles and triangles depict outliers. Littermates were randomly assigned to 6 h of 1.5% isoflurane (Anaesthesia) or 6 h of room air (No Anaesthesia) at three different ages: as newborns [post-natal day (P)7], as juveniles (P21), or as young adults (P49). Density of cleaved caspase 3-positive cells was assessed in superficial layers II/III of the retrosplenial agranular cortex (RSA; a), caudoputamen (CPu; b), the pyramidal layer of cornu ammonis 1 (CA1; c), cerebellar cortex (Cb; d), and white matter (Cb wm; e), the subgranular zone and granule cell layer of dentate gyrus (DG; f), and the granule layer of the olfactory bulb (GrO; g). P7, Anaesthesia (n=10), No Anaesthesia (n=8); P21, Anaesthesia (n=8), No Anaesthesia (n=7); P49, Anaesthesia (n=16), No Anaesthesia (n=13). *P<0.05, **P<0.01, ***P<0.001, compared with the respective No Anaesthesia group.

Fig 2

Exposure to prolonged anaesthesia induces significant neuroapoptosis in several brain regions in newborn mice. Representative high-power magnification photomicrographs of layers II/III of the retrosplenial agranular cortex (RSA), caudoputamen (CPu), the pyramidal layer of cornu ammonis region 1 (CA1), subgranular zone and granule cell layer of dentate gyrus (DG), the granule layer of the olfactory bulb (GrO) and cerebellar cortex (Cb), and both internal granule layer (arrows) and external granule layer (arrow head) from a newborn mouse (P7) anaesthetized for 6 h with 1.5% isoflurane (Anaesthesia, a–f) and from an unanaesthetized and fasted littermate (No Anaesthesia, g–l). Sections were stained with propidium iodide (PI; red) to label all cells, for neuronal nuclei (NeuN; blue) to label post-mitotic neurones, and for activated, cleaved caspase 3 (AC3; green) to label apoptotic cells (arrows). All brain regions shown demonstrated statistically significantly increased neuroapoptosis after anaesthetic exposure, compared with unanaesthetized littermates, except for the dentate gyrus, which revealed no significantly increased cell death in newborn animals. For quantification, see Figure 1. Scale bars=50 µm.

Fig 3

Exposure to prolonged anaesthesia induces significant neuroapoptosis in the dentate gyrus, olfactory bulb, and cerebellum of juvenile mice. Representative high-power magnification photomicrographs of the subgranular zone and granule cell layer of dentate gyrus (DG), the granule layer of the olfactory bulb (GrO) and cerebellar white matter (Cb wm) from a juvenile mouse (P21) anaesthetized for 6 h with 1.5% isoflurane (Anaesthesia; a–c) and from an unanaesthetized, fasted littermate (No Anaesthesia; d–f). Sections were stained with propidium iodide (PI; red) to label all cells, for neuronal nuclei (NeuN; blue) to label post-mitotic neurones, and for activated, cleaved caspase 3 (AC3; green) to label apoptotic cells (arrows). Of all the brain regions examined, the dentate gyrus, olfactory bulb, and cerebellar white matter were the only regions demonstrating significantly increased neuroapoptosis in juvenile animals, compared with unanaesthetized littermates. For quantification, see Figure 1. Scale bars=50 µm.

Fig 4

Exposure to prolonged anaesthesia induces significant neuroapoptosis in the dentate gyrus and olfactory bulb of young adult mice. Representative high-power magnification photomicrographs of the subgranular zone and granule cell layer of the dentate gyrus (DG) and the granule layer of the olfactory bulb (GrO) from a young adult mouse (P49) anaesthetized for 6 h with 1.5% isoflurane (Anaesthesia; a and b) and from an unanaesthetized and fasted littermate (No Anaesthesia; c and d). Sections were stained with propidium iodide (PI; red) to label all cells, for neuronal nuclei (NeuN; blue) to label post-mitotic neurones, and for activated, cleaved caspase 3 (AC3; green) to label apoptotic cells (arrows). Of all the brain regions examined, the dentate gyrus and olfactory bulb were the only regions in young adult animals demonstrating significantly increased neuroapoptosis, compared with unanaesthetized littermates. For quantification, see Figure 1. Scale bars=50 µm.

Fig 5

Age windows of vulnerability to anaesthesia-induced neuroapoptosis differ by brain region. Graph indicates the severity of anaesthesia-induced neuroapoptosis depicted as the median fold-increase over physiological apoptotic cell death, as observed in the respective littermate controls. Apoptotic neuronal density was quantified for superficial layers II/III of the retrosplenial agranular cortex (RSA), caudoputamen (CPu), the pyramidal layer of cornu ammonis 1 (CA1), cerebellum (Cb at P7, CB wm at P21), the subgranular zone and granule cell layer of dentate gyrus (DG), and the granule layer of the olfactory bulb (GrO) after a 6 h exposure to 1.5% isoflurane in newborn (P7), juvenile (P21), and adult mice (P49), compared with fasted, unanaesthetized littermates. Maximum vulnerability was observed in the neocortex, caudoputamen, and CA1 at P7, whereas the number of vulnerable neurones peaked at P21 for the cerebellum, dentate gyrus, and olfactory bulb.