*Contributed equally
Isoflurane (Iso) is a commonly used inhalational anesthetic and is associated with the incidence of postoperative cognitive dysfunction (POCD). Cannabinoid receptor 2 (CB2R) was previously reported to have a promising neuroprotective function in cases of POCD, but the specific mechanisms have remained to be fully explored. The aim of the present study was to investigate the effect of CB2R deficiency on spatial cognitive performance in adult mice exposed to Iso. A total of 20 adult CB2R knockout (KO) and 20 wild-type (WT) mice were exposed to Iso (1.4% in oxygen for 4 h) or 100% oxygen. The Morris water maze (MWZ) test was performed 10 days after Iso exposure. Immunofluorescence staining and reverse transcription-quantitative PCR were performed to assess the expression of microglial marker ionized calcium-binding adaptor molecule-1 (Iba1) and the mRNA expression levels of microglial phenotype markers (M1: Interleukin-6, tumor necrosis factor-α, inducible nitric oxide synthase; M2: Chitinase-3 like protein) in the hippocampus. Changes in hippocampal neurogenesis and neuroplasticity were assessed by 5-bromodeoxyuridine (BrdU) immunostaining and Golgi staining. Compared with control mice, WT Iso-exposed mice had impaired spatial performance in the MWZ test. Furthermore, hippocampal Iba1 immunoreactivity and the number of microglial branches were notably increased in Iso-exposed WT mice. This was paralleled by significant upregulation of M1-associated markers and downregulation of M2-associated markers in the hippocampus. An obviously reduced number of BrdU+ neurons and decreased spine density were observed in WT Iso-exposed mice compared with control mice. Of note, CB2R deficiency exacerbated the spatial cognition impairment induced by Iso in the MWZ test. The alterations in the activation, morphology and M1 polarization of microglia, the number of BrdU+ neurons and spine density were more pronounced in CB2R-deficient Iso-exposed KO mice than in WT Iso-exposed mice. These results suggested that CB2R has a crucial role in Iso-induced cognitive impairment, which may be related to changes in hippocampal neuroinflammation, neurogenesis and neuroplasticity.
Postoperative cognitive dysfunction (POCD) is one of the most common and devastating complications in patients undergoing major surgery under general anesthesia (
An accumulating body of evidence suggests that POCD shares a significant mechanical connection and commonality with Alzheimer's disease, in which altered microglia and astrocytic cells release extensive proinflammatory cytokines, resulting in persistent neuroinflammation and synaptic impairment (
Cannabinoid receptor 2 (CB2R) is a crucial neuromodulatory target in the central nervous system and has an important role in homeostatic control and numerous neurodegenerative diseases. CB2R is reported to be expressed in both microglia and neurons in healthy brains at low levels and is markedly upregulated by microglia during pathological conditions. Previous studies have demonstrated the vital role of CB2R in microglial activity and polarization in the regulation of neuroinflammatory processes (
Five female CB2R knockout (KO) mice (aged 3 months; weight, 25-30 g) were purchased from the Jackson Laboratory and originally created on the background of C57BL/6J mice. Based on the protocol of a previous study (
All experiments were performed with the approval of the Animal Care Committee of the Fourth Hospital of Hebei Medical University (Shijiazhuang, China).
A total of 20 CB2R KO mice and 20 WT littermates (CB2R WT) (aged 3 months; weight, 25-30 g) were randomly divided into groups (n=10 in each group): CB2R WT+O2, CB2R KO+O2, CB2R WT+Iso and CB2R KO+Iso. The level of Iso exposure was based on previous reports indicating that 1.4% Iso for >4 h resulted in cognitive impairment in young adult C57BL/6 mice (
After a 10 day washing period between Iso treatment and examination based on previous literature (
After the cognitive performance experiments, mice were injected with a dose of sodium pentobarbital (50 mg/kg, intraperitoneally) and sacrificed by cervical dislocation. Their brains were removed from skulls and rapidly dissected into two hemispheres. One hemisphere was post-fixed in 4% paraformaldehyde for 24 h, incubated in 30% sucrose for 12 h and then processed for immunofluorescence staining and BrdU immunostaining. The other hemisphere was frozen immediately on dry ice, stored at -80˚C and processed for RNA extraction.
Immunofluorescence staining of the hippocampal slices was performed as described in a previous study by our group (
Total RNA was extracted from the hippocampal tissue using the RNeasy mini kit (Qiagen GmbH) following the manufacturer's protocol. RT to generate cDNA was performed using the ReverTra Ace qPCR RT Master Mix with gDNA Remover kit (Toyobo Co., Ltd.) according to the manufacturer's protocol. qPCR analysis was performed using the FastStart Essential DNA Green Master kit (Roche) according to the manufacturer's protocol. The primer sequences of the target genes were as follows: Interleukin-6 (IL-6), 5'-TGCAAGAGACTTCCATCCAGTT-3' (forward) and 5'-GAAGTAGGGAAGGCCGTGG-3' (reverse); tumor necrosis factor-α (TNF-α), 5'-GCACCACCATCAAGGACTC-3' (forward) and 5'-TGAGACAGAGGCAACCTGAC-3' (reverse); inducible nitric oxide synthase (iNOS), 5'-GGCAGCCTGTGAGACCTTTG-3' (forward) and 5'-GCATTGGAAGTGAAGCGTTTC-3' (reverse); chitinase-3 like protein (Ym1/2), 5'-CAGGGTAATGAGTGGGTTGG-3' (forward) and 5'-CACGGCACCTCCTAAATTGT-3' (reverse); GAPDH, 5'-ACTCCACTCACGGCAAATTC-3' (forward) and 5'-TCTCCATGGTGGTGAAGACA-3' (reverse). The PCR amplification conditions were adjusted based on the manufacturer's protocol and previous research (
All mice received an intraperitoneal injection of BrdU (Sigma-Aldrich; Merck KGaA) at a dose of 50 mg/kg twice a day for five consecutive days and were sacrificed 24 h after the last BrdU injection (
Golgi staining was performed according to the protocol of the FD Rapid Golgistain™ kit (FD NeuroTechnologies, Inc.), as previously described (
All statistical analyses were performed using GraphPad Prism version 7.0 (GraphPad Software, Inc.). Values are expressed as the mean ± standard error of mean. Results of the escape latencies and swimming speeds from the MWZ tests were analyzed using two-way repeated-measures ANOVA followed by Tukey's post-hoc test. For other data, a two-way ANOVA was performed, if appropriate. P<0.05 was considered to indicate a statistically significant difference.
The timeline of the experimental procedures is presented in
To investigate whether CB2R deficiency affected microglial activation and morphology in the Iso-induced POCD model mice, microglia were subjected to immunofluorescence staining for Iba1, an efficient and specific microglial biomarker (
In general, in numerous neurodegenerative diseases, activated microglia are driven to polarize to two opposite subtypes: M1 phenotype expressing the markers IL-6, TNF-α and iNOS or the M2 phenotype characterized by Ym1/2 expression (
Inhibition of hippocampal neurogenesis has been demonstrated to be a vital hallmark of the POCD model (
Aberrant synaptic plasticity has been reported to be closely related to Iso-induced POCD (
POCD is one of the most urgent concerns for patients and clinicians worldwide, as it significantly increases medical costs, morbidity and mortality. Therefore, extensive research has been performed to investigate its underlying etiology and potential therapeutic targets. A large and growing body of evidence supports the theory that CB2R has a vital neuroprotective role in cognitive dysfunction diseases via modulation of microglia-associated neuroinflammation (
The MWZ test, a classical tool used to monitor hippocampus-related spatial learning and memory ability, has been widely applied in laboratory research (
The neuroinflammatory hypothesis is considered one of the leading mechanisms of POCD, based on which various promising candidates for the prevention and treatment of POCD have been developed (
In addition to neuroinflammation, neurogenesis and neuroplasticity are considered crucial mechanisms for the endogenous cannabinoid system to exert regulatory roles in numerous neurocognitive disorders (
Dendritic spines are post-synaptic structures at a majority of excitatory synapses in the mammalian brain. The number and size of dendritic spines are closely related to cognitive function in different neurological diseases (
There are certain limitations to the present study. RT-qPCR experiments were performed to explore the impact of CB2R deficiency on microglial phenotype changes in Iso-induced POCD mice based on a previous study by our group (
In conclusion, the present study indicated that CB2R deficiency aggravated spatial cognitive impairment in the Iso-induced POCD mouse model. This was partly explained by the aggravated neuroinflammatory reactivity of microglia and enhanced injury to neurogenesis and neuroplasticity in the hippocampus. These results further suggest that CB2R is a promising pharmacological target for Iso-induced POCD; however, further research is required to demonstrate its validity.
Not applicable.
The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.
CL and JPS were responsible for designing the study, performing the experiment, collecting the data and writing the manuscript. JGS and YS were responsible for designing the study, performing the experiment, and collecting the data and reviewing the manuscript. HJ was responsible for providing experimental ideas and reviewing the manuscript. CL and HJ were responsible for the confirming the authenticity of all the raw data. All authors read and approved the final manuscript.
All experiments were performed with the approval of the Animal Care Committee of the Fourth Hospital of Hebei Medical University (Shijiazhuang, China).
Not applicable.
The authors declare that they have no competing interests.
CB2R deficiency enhances Iso-induced spatial memory impairment in the MWZ test. (A) Schematic diagram of the experimental procedures. (B) Swimming speed. (C) Mean escape latency. (D) Crossing the platform. (E) Representative path tracing on the sixth day of the MWZ test. Values are expressed as the mean ± standard error of the mean (n=10). Results of escape latency and swimming speed were assessed by two-way repeated-measures ANOVA and other statistical comparisons were performed by two-way ANOVA. *P<0.05, ***P<0.001 and #P<0.05. CB2R, cannabinoid receptor 2; MWZ, Morris water maze; WT, wild-type; KO, knockout; sec, seconds; Iso, isoflurane.
CB2R deficiency enhances Iso-induced activation and affects the morphology of microglia in the hippocampus of adult mice. (A) Representative images of immunofluorescence staining for Iba1 (green) and nuclei stained in blue (white scale bar, 100 µm). (B) Quantification of Iba1 immunofluorescence staining intensity in the hippocampus. (C) Representative images of Iba1 (green) and nuclei (blue) by immunofluorescence staining (red scale bar, 10 µm). (D) Number of Iba1+ microglial branches in the hippocampus. Values are expressed as the mean ± standard error of the mean (n=6) and analyzed using two-way ANOVA, **P<0.01, #P<0.05 and ##P<0.01. CB2R, cannabinoid receptor 2; Iba1, ionized calcium-binding adaptor molecule-1; WT, wild-type; KO, knockout; Iso, isoflurane.
CB2R deficiency promotes microglial M1 polarization in the hippocampus of adult mice exposed to Iso. Quantification of the hippocampal mRNA levels of M1 microglia-associated biomarkers (A) IL-6; (B) TNF-α; (C) iNOS; and (D) M2 microglia-associated marker Ym1/2. Values are expressed as the mean ± standard error of the mean (n=6) and analyzed using two-way ANOVA. *P<0.05, ***P<0.001, #P<0.05 and ##P<0.01. CB2R, cannabinoid receptor 2; Iba1, ionized calcium-binding adaptor molecule-1; IL-6, interleukin-6; iNOS, inducible nitric oxide synthase; TNF-α, tumor necrosis factor; Ym1/2, chitinase-3 like protein; Iso, isoflurane.
CB2R deficiency enhances neurogenetic damage in the hippocampus of adult mice exposed to Iso. (A) Representative images of immunostaining for BrdU (positive brown staining indicated with blue arrows; black scale bar, 200 µm; red scale bar in magnified window, 20 µm). (B) Quantification of the hippocampal BrdU+ cells. Values are expressed as the mean ± standard error of the mean (n=6) and analyzed using two-way ANOVA. ***P<0.001 and #P<0.05. BrdU, 5-bromodeoxyuridine; CB2R, cannabinoid receptor 2; WT, wild-type; KO, knockout; Iso, isoflurane; DG, dentate gyrus.
CB2R deficiency reduces dendritic complexity in the hippocampus of adult mice exposed to Iso. (A) A representative image of neurons with Golgi staining in the whole brain section (scale bar, 200 µm). (B) Representative images of hippocampal dendrite fragments with visible spines (scale bar, 5 µm). (C) Quantification of spine density in the hippocampal dentate gyrus. Values are expressed as the mean ± standard error of the mean (n=4) and analyzed using two-way ANOVA */#P<0.05. CB2R, cannabinoid receptor 2; WT, wild-type; KO, knockout; Iso, isoflurane.
Schematic representation of the effects of CB2R deficiency on Iso-induced cognitive impairment. Iso exposure leads to spatial memory impairment of adult mice, accompanied by alterations in the activation, morphology and M1 polarization of microglia, BrdU+ neurogenesis and spine density in the hippocampus. CB2R deficiency makes adult mice more vulnerable to Iso neurotoxicity, as indicated by more significant injury in terms of neuroinflammation, neurogenesis and neuroplasticity after Iso exposure. BrdU, 5-bromodeoxyuridine; CB2R, cannabinoid receptor 2; WT, wild-type; KO, knockout; Iso, isoflurane.