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. 2018 Jan 17;24(3):212–221. doi: 10.1111/cns.12794

Isoflurane anesthesia in aged mice and effects of A1 adenosine receptors on cognitive impairment

Chun‐Long Zuo 1,2, Chun‐Man Wang 1,2, Jin Liu 1,2, Ting Shen 1,2, Jiang‐Ping Zhou 1,2, Xin‐Rui Hao 1,2, Yi‐Zhao Pan 1,2, Hua‐Cheng Liu 1,2, Qing‐Quan Lian 1,2,✉, Han Lin 1,2,✉
PMCID: PMC6489773  PMID: 29345054

Summary

Aims

Isoflurane may not only accelerate the process of Alzheimer's disease (AD), but increase the risk of incidence of postoperative cognitive dysfunction (POCD). However, the underlying mechanisms remain unknown. This study was designed to investigate whether isoflurane contributed to the POCD occurrence through A1 adenosine receptor (A1AR) in aged mice.

Methods

We assessed cognitive function of mice with Morris water maze (MWM) and then measured expression level of two AD biomarkers (P‐tau and Aβ) and a subtype of the NMDA receptor (NR2B) in aged wild‐type (WT) and homozygous A1 adenosine receptor (A1AR) knockout (KO) mice at baseline and after they were exposed to isoflurane (1.4% for 2 hours).

Results

For cognitive test, WT mice with isoflurane exposure performed worse than the WT mice without isoflurane exposure. However, A1AR KO mice with isoflurane exposure performed better than WT mice with isoflurane exposure. WT mice exposed to isoflurane had increased levels of Aβ and phosphorylated tau (P‐tau). Levels of Aβ and P‐tau were decreased in A1AR KO mice, whereas no differences were noted between KO mice with and without isoflurane exposure. NR2B expression was inversely related to that of P‐tau, with no differences found between KO mice with and without isoflurane exposure.

Conclusions

We found an association between isoflurane exposure, impairment of spatial memory, decreasing level of NR2B, and increasing levels of A‐beta and P‐tau, presumably via the activation of the A1A receptor.

Keywords: adenosine A1, cognitive dysfunction, hippocampus, isoflurane, receptor

1. INTRODUCTION

Alzheimer's disease (AD) is an insidious and progressive neurodegenerative disorder characterized by extracellular amyloid plaques and intracellular neurofibrillary tangles (NFTs) as well as synaptic loss, neuronal death, and glial activation.1, 2, 3, 4 Currently, the prevalence of AD in those over the age of 65 in the United States is 1 in 9 (11%).5 Isoflurane and sevoflurane can exacerbate AD pathology from prospective evidence of biophysical, animal model, and clinical reports.6 Isoflurane also can increase Aβ generation and aggregation both in vitro and in vivo,7, 8 besides promoting levels of phosphorylated tau.9 Nevertheless, underlying molecular mechanisms for how anesthetics affect AD pathological progress in particular on whole animal subjects remain unclear.

Postoperative cognitive dysfunction (POCD) is an often mild yet persistent neurocognitive disorder. Symptoms typically include difficulties with memory, concentration, and attention after surgery and anesthesia. Severe symptoms may include memory loss, impaired higher‐level cognitive function, and psychomotor dysfunction.10 Approximately up to 40% of patients over age 60 hospitalized for surgery are reported to suffer POCD on discharge, with an additional 10% suffering POCD 3 months later.11 Importantly, POCD has been associated with the risk of leaving labor market prematurely and even increased mortality.10 Previous studies have been suggested a close association between AD and POCD12 regarding risk factors and pathomechanisms of both conditions.13, 14 Furthermore, AD patients are more susceptible to development of cognitive function decline after anesthesia and surgery.10, 15 Repeated exposure to general anesthetics, such as isoflurane, is a potential risk factor for development of POCD and AD.16, 17 Furthermore, high level of Aβ in aging brain following general anesthetic exposure is likely to increase the risk of the POCD development.10 Thus, general anesthetics might lead to POCD through induction of AD neuropathogenesis,13 but the underlying mechanisms remain unclear.

Most functions of adenosine, which plays an important role in many physiological and pathological processes in the mammalian (CNS), are through activating adenosine receptors. Caffeine, the most consumed natural psychoactive stimulant, as a nonselective blocker of adenosine receptors (A1, A2A, A2B, and A3), is associated with a lower incidence of cognitive impairment in consumers.18 Caffeine can even reverse cognitive impairment and decrease the level of Aβ in aged AD mice.19 The A1AR receptors are involved in β‐amyloid precursor protein (APP) processing and tau phosphorylation.20 In necropsies of AD patients, these receptors are redistributed accompanied by increased expression. Thus, we postulated that A1AR might be involved in cognitive function by regulating Aβ production and tau phosphorylation.

The neurophysiological basis for learning and memory involves long‐term potentiation (LTP) and long‐term depression (LTD), both dependent on activation of NMDA receptors (NMDARs).21 NR2B is one of NMDAR subunits and plays an important role in learning and memory.22 We therefore set out to determine whether isoflurane can induce cognitive dysfunction and whether its effects are associated with the changes in AD pathogeneses and NR2B expression in wild and A1AR KO aged mice.

2. MATERIALS AND METHODS

2.1. Animals

All animal experimental protocols were approved by the Animal Care and Use Committee of Wenzhou Medical University (Wenzhou, China), and all procedures were performed in accordance with the National Institutes of Health (NIH) guidelines of animal care. A1AR KO mice (A1 KO, 129/OlaHsd/C57BL) and their littermates, male, 18‐22 months old, 26‐33 g, were housed under a 12/12‐h light‐dark cycle at 22‐24°C with free access to food and water and randomly assigned to WT mice control (WTC), WT mice anesthetized with isoflurane (WTI), and KO mice control (KOC) and KO mice anesthetized with isoflurane (KOI) groups. The initial pairs of heterozygous mice were a gift from Dr. Jiang Fan Chen at the Department of Neurology, Boston University School of Medicine, Boston, Massachusetts, USA.

2.2. Characterization of A1AR KO mice

Heterozygote mice were intercrossed to produce homozygote (A1 KO) (A1R−/−) and wild‐type (WT) littermates (A1R+/+). Genotypes were confirmed by PCR and agarose gel electrophoresis on tail sample DNA. Briefly, genomic DNA was isolated from tail biopsies. PCR was performed at cycling temperatures of 94°C, 3‐min denaturing, 39 cycles of 94°C, 55°C each 30 s annealing, and 72°C, 60 s extension. Primer pairs for genotyping were described as follows: Primer pairs for A1AR were forward 5′ TAC TTC AAC TTC TTC GTC TGG GT 3′ and reverse 5′ GTT GTG GAT TCG GAA GGC ATA GA 3′; primer pairs for A1AR KO were forward 5′ GAA TTC TTG AAG ACG AAA GG 3′ and reverse 5′AAG GCT GAG GAG GAA CAG TG 3′. Products were separated on a 2% agarose gel with 0.5%? ethidium bromide and visualized under ultraviolet light.

2.3. Mice anesthesia

A1 KO and WT mice were anesthetized at 1 minimum alveolar concentration (MAC) as determined by tail clamping. The 1 MAC was determined to be 1.4%. WTI and KOI group were flushed continuously with isoflurane and 100% oxygen for two hours. WTC and KOC group received 100% oxygen for two hours at the identical condition. During anesthesia, mice breathed spontaneously. The temperature of chamber floor was kept at 37°C and was covered with soda lime. The concentration of isoflurane was continuously monitored with a gas analyzer (ARYM‐0054 Vamos, Dräger, Germany). When mice were exposed to chambers, respiratory rate and invasive arterial blood pressure were monitored and blood was sampled for blood gas analysis. Anesthesia was terminated by discontinuing isoflurane; mice were kept in chamber containing 100% oxygen until return of righting reflex. Subsequently, they were returned to their individual home cages.8

2.4. Hemodynamic monitor and blood gas analysis

Blood pressure and blood gases were measured in a separate cohort (n = 8/group) as previously described23 to confirm whether such anesthesia regimen affects cardiorespiratory function. Briefly, arterial blood was sampled from WT and KO mice with isoflurane exposure via 24‐gauge arterial puncture needle (IntroCan®‐W, B/Braun) through abdominal aorta using a dissecting microscope (PS100; Nikon, Tokyo, Japan). Mean arterial blood pressure (MAP) was measured by anesthesia monitor (M3046; Philips, Boeblingen, Germany). Blood sample (0.2‐.03 mL) was immediately analyzed to determine pH, arterial oxygen, and carbon dioxide with blood gas analyzer (GEM Premier 3000, Bedford, MA, USA).

2.5. Morris water maze

MWM was done 24 hours after isoflurane exposure.24 Before trials, all mice were placed into water of the swimming arena with a 6‐cm‐diameter platform submerged 0.5‐1 cm above the surface of water on day 0. Each mouse was allowed to swim for 30 s for them to locate the platform. Mice that had vision problems or did not swim were removed from the arena and excluded for further experiments. Subsequently, spatial acquisition trials were conducted, where platform was submerged 0.5‐1 cm below surface of water. Animals underwent four trials each day in the pool at four different starting positions facing the tank wall. A trail limit of 1 minute per trial allowed mice to find the platform within a 30‐min intertrial interval. Animals not finding the platform within the time allotted were guided onto the platform for 15 s. The swim speed, distance (path length), and time (escape latency) in finding the platform were calculated from recorded videos using MWM software (SLY‐WMS Morris water maze, Shuolinyuan, Beijing, China). On day 5, a probe trial was performed, in which the platform was removed and animals were placed in a novel start position 180° from the original platform position to swim freely for 60 s. The percentage of time spent in target quadrant and time of platform‐site crossovers were recorded.

2.6. Western blot

Eight animals from each group were deeply anesthetized with 5% chloral hydrate and transcardially perfused with normal saline 24 hours after MWM. The brains were quickly removed. Brain tissue was homogenized in a mixture composed of RIPA lysis buffer, phosphatase, and protease inhibitors, and incubated for 30 minutes on ice. The lysate was then sonicated and centrifuged at 13 000 g for 30 minutes, at 4°C. Protein samples were quantitated by BCA protein assay kit (Thermo Scientific, Waltham, MA, USA), and the concentrations were measured using a spectrophotometer (Thermo Scientific, MULTISKAN MK3). Subsequently, samples were admixed with 5 ×  sample buffer, equalized using double distilled H2O, and heated for 5 minutes at 100°C. An equal amount of protein from each sample was separated by sodium dodecyl sulfate‐polyacrylamide gel electrophoresis (SDS‐PAGE) on a 10% gel and electrophoretically transferred to a polyvinylidene fluoride (PVDF) membrane (Bio‐Rad, Hercules, CA, USA). Blots were blocked with 10% skim milk in Tris‐buffered saline and Tween‐20 (0.1%) (TBST) for 2 hours at room temperature (RT) and incubated at 4°C overnight with rabbit anti‐tau antibody (1:1000, ab47579; Abcam, Cambridge, UK), rabbit anti‐P‐tau antibody (1:1000, 44‐750G; Thermo), rabbit anti‐NR2B antibody (1:1000, ab65875; Abcam), or rabbit anti‐actin antibody (1:1000, AP0060; Bioworld Technology, St. Louis Park, MN, USA). After incubation with primary antibody, the blots were incubated for 2 hours at RT with secondary antibodies (anti‐rabbit antibodies, 1:5000, ab6721; Abcam). Between steps, blots were washed with TBST. Blots were visualized with ECL Western blot detection system (ImageQuant LAS 4000mini). Bands were analyzed by Quantity one software version 4.6.2.

2.7. Immunohistochemistry

Five animals from each group were deeply anesthetized with 5% chloral hydrate and transcardially perfused with normal saline with simultaneous exsanguination from right atrium, followed with 4% paraformaldehyde in 0.1 mol/L phosphate buffer with a pH of 7.4 24 hours after MWM. Brains were removed and kept at 4°C overnight in 4% paraformaldehyde. Serial coronal 4‐μm sections were cut into paraffin blocks using a microtome (Lecai RM2235, Wetzlar, Germany), and at least three slides from each animal were used for Aβ staining with DAB. Briefly, sections were deparaffinized and rehydrated with xylene, a series of graded alcohols, and washed in distilled water followed by PBS. Sections were then incubated in 3% hydrogen peroxide for 15 minutes to quench endogenous peroxidase activity and then washed. Antigen retrieval was performed by incubation in 10 mmol/L sodium citrate buffer with pH of 6.0 for 20 minutes in a microwave oven at 100°C followed by PBS. Sections were then incubated in primary antibody to Aβ 1‐42 (1:100, ab10148; Abcam) overnight at 4°C. Subsequently, sections were washed at RT, incubated in biotinylated secondary antibody (K5007; Dako, Glostrup, Denmark) for 30 minutes, washed again, and incubated with avidin‐biotin‐peroxidase complex. Sections were then incubated in DAB working solution (K5007; Dako) followed by washing with distilled water and counter‐staining with hematoxylin. Finally, sections were dehydrated with a gradient of ethanol solutions, cleared in xylene, covered with a cover slip, and viewed and quantitated with a microscope (Leica DM3000, Wetzlar, Germany). We acquired the images on the microscope. Color threshold and planimetry were used for quantitation. All quantitations were determined by calculating the percentage of the Aβ‐positive staining area to the total cross‐sectional hippocampus area in 5 sections per mouse using Image‐Pro Plus software version 6.0. The methods were analyzed as previously described.25

2.8. ELISA

For analyses of Aβ42 and Aβ40 burden, the hippocampi were homogenized with an extraction buffer containing 5 mol/L guanidine HCl/50 mmol/L Tris‐HCl and a protease inhibitor cocktail with AEBSF (a serine protease inhibitor) according to the manufacturer's instructions. The mouse Aβ42 or Aβ40 ELISA kit (Invitrogen, Waltham, MA, USA) was used. In brief, samples were centrifuged at 16 000 g for 20 minutes at 4°C and supernatant diluted 1:2 to 1:10 for Aβ42 or Aβ40 before adding to ELISA plates coated with anti‐mouse Aβ42 or Aβ40 antibody and then incubated 2 hours at RT. Samples were incubated 1 hour at RT with 100 μL of Ms Aβ42 or Aβ40 Detection Antibody after washing and then washed before incubating 100 μL of horseradish peroxidase‐labeled anti‐rabbit antibody for 30 minutes at RT. After washing again, samples were incubated with 100 μL of stabilized chromogen for 30 minutes at RT and the absorbance was measured at a wavelength of 450 nm using spectrophotometer after the addition of a stop solution. Additionally, protein samples were quantitated by BCA protein assay kit and the concentrations were measured using spectrophotometer. Aβ‐concentrations were expressed as pg/mg tissue.

2.9. Statistics

All the data were expressed as mean ± SEM except those data derived from the probe trails of MWM that were expressed as median and interquartile range. The data of spatial acquisition trials were analyzed by a three‐way ANOVA with repeated‐measures (ie, isoflurane exposure and genotype as two factors between subjects, day as a repeated‐measures factor) followed with LSD post hoc test comparison. MANOVA was used to test the main effects for group at each time point. The data of probe trial were analyzed with a two‐way ANOVA (with isoflurane and the genotype as the two variables). The data of level of protein expression and the percentage of Aβ level in four groups were analyzed with two‐way ANOVA followed by a post hoc via Scheffe's test. A P values < 0.05 was considered statistically significant. SPSS software (SPSS for Windows, version 19.0, SPSS, Chicago, IL, USA) was used to analyze the data.

3. RESULTS

3.1. Isoflurane does not induce cardiorespiratory distress

To assess effects of isoflurane on cardiorespiratory effects in aged mice, mice were exposed to 1.4% isoflurane for 2 hours. The respiratory rate did not differ in either WT mice (A1R+/+) or A1AR KO mice (A1R−/−) during isoflurane exposure (data not shown). Blood gas analysis revealed that PaO2, PaCO2, and pH were all in normal range, and MAP did not differ either among groups (Table 1).

Table 1.

The data of MAP and blood gas value of mice at 1 hr and 2 hrs during anesthesia

Parameters WTI KOI
1 hr 2 hrs 1 hr 2 hrs
pH 7.40 ± 0.01 7.38 ± 0.01 7.41 ± 0.01 7.39 ± 0.01
PaCO2 (mm Hg) 39.5 ± 0.8 40.6 ± 0.6 39.9 ± 0.9 41.4 ± 0.6
PaO2 (mm Hg) 443.0 ± 27.7 427.4 ± 25.1 415.0 ± 20.1 398.9 ± 14.7
MAP (mm Hg) 94 ± 2 91 ± 1 94 ± 1 90 ± 1
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Measurement of the MAP and blood gas value of mice at 1 hr and 2 hrs during anesthesia from the WTI group and the KOI group. 1 hr of WTI, n = 8; 2 hrs of WTI, n = 8; 1 hr of KOI, n = 8; 2 hrs of KOI, n = 8. Mean ±SEM. pH, P = 0.288; One‐way ANOVA (F = 1.318; df = 3); PaCO2, P = 0.313; one‐way ANOVA (F = 1.243, df = 3); PaO2, P = 0.563; one‐way ANOVA (F = 0.694, df = 3); MAP, P = 0.123; one‐way ANOVA (F = 2.1; df = 3).

WTI: WT mice anesthetized with isoflurane group.

KOI: KO mice anesthetized with isoflurane group.

3.2. Characterization of A1AR KO mice

Genomic DNA was isolated from tail biopsies of WT and KO mice and used to identify genotypes using PCR and agarose gel electrophoresis. WT alleles yielded a 350‐bp product, while mutant alleles yielded a 200‐bp product.

3.3. Behavior analysis with Morris water maze

On days 3 and 4, escape latency of MWM decreased in KOC group (n = 8) compared to that in WTC group (n = 8) (Figure 1A; P = 0.024; MANOVA (F = 2.396, df = 3) for day 3; P = 0.049; MANOVA (F = 18.579, df = 3) for day 4; interaction, P = 0.03). On day 4, escape latency decreased in KOI group (n = 8) compared to that in WTI group (n = 8) (Figure 1A; P = 0.000; MANOVA (F = 18.579, df = 3); interaction, P = 0.03). However, escape latency increased in WTI group (n = 8) compared to that in WTC group (n = 8) (Figure 1A; P = 0.000; MANOVA (F = 18.579, df = 3); interaction, P = 0.03). Although escape latency in KOI group (n = 8) tended to increase compared to that in KOC group (n = 8), differences were not significant (Figure 1A; P = 0.467; MANOVA (F = 18.579, df = 3); interaction, P = 0.03). Swim speeds were similar in each day among all groups (Figure 1B; P = 0.991; 0.638; 0.541; 0.62; MANOVA (F = 0.036, df = 3; F = 0.572, df = 3; F = 0.733, df = 3; F = 0.601, df = 3); interaction, P = 0.342). During probe trial (on day 5), although KO mice tended to take more time at platform‐site crossovers and spend less time in the target quadrant compared to WT mice, differences were not significant among all groups (Figure 1C,D; one‐way ANOVA (F = 0.41; df = 3), P = 0.747 for the percentage of time in the target quadrant; interaction, P = 0.512; one‐way ANOVA (F = 1.579; df = 3), P = 0.217 for crossovers; interaction, P = 0.339).

Figure 1.

Figure 1

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Spatial learning and memory were assessed 24 hrs after 2 hrs of isoflurane on aged mice. Aged mice received 2 hrs of isoflurane and underwent assessment of spatial learning and memory in the MWM. Mice navigated to the hidden platform in place trials. During a probe trial, memory retention was assessed 24 hrs after the place trials. (A) Time to reach the platform (escape latency) was recorded in the MWM. On days 3 and 4, the mice in the KOC group performed better than the mice in the WTC group. On day 4, the mice in the WTI group performed worse than the mice in the WTC group. However, the mice in the KOI group performed better than the mice in the WTI group. No differences were found between the KOI group and the KOC group. (B) Swim speeds were recorded in the MWM. No differences were found among all groups. (C) The percentage of time spent in the target quadrant was recorded in the MWM. No differences were found among all groups. (D) The time of platform‐site crossovers was recorded in the MWM. No differences were found among all groups. All data points represent the mean ± SEM except those data derived from the probe trails of MWM that were expressed as median and interquartile range

The results suggest cognitive function can be impaired in aged mice after isoflurane exposure and knocking out of A1AR may ameliorate isoflurane‐induced cognitive impairment.

3.4. Western blot

The Western blot tests (for the P‐tau and the NR2B) were done 24 hours after the Morris water maze test. The two‐way ANOVA statistical results indicated that isoflurane (P = 0.001; for NR2B; P = 0.006; for P‐tau; P = 0.735; for T‐tau) or A1AR KO (P = 0.000; for NR2B; P = 0.000; for P‐tau; P = 0.348; for T‐tau) had significant effects on the NR2B and P‐tau, but not on the T‐tau. Also, there seemed no significant interaction between the two factors (two‐way ANOVA; interaction, P = 0.329; for NR2B; P = 0.815; for P‐tau; P = 0.770; for T‐tau). Results revealed level of P‐tau protein was increased, and level of NR2B was decreased in WTI group (n = 8) compared to KOI group (n = 8) (Figure 2A,B). However, level of P‐tau was decreased in KOC group (n = 8) and level of N2RB was decreased in WTI group (n = 8) compared to WTC group (n = 8) (Figure 2A,B). Level of T‐tau did not differ among all groups (Figure 2A,B).

Figure 2.

Figure 2

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Isoflurane increases the level of P‐tau and decreases the level of NR2B, but A1AR knocked out can inhibit the effect of isoflurane. (A) Expression of P‐tau, T‐tau, NR2B in the hippocampus in four groups was detected 24 hrs after the MWM. Actin served as a loading control. (B) Quantification of the levels of P‐tau, T‐tau, NR2B in the hippocampus in four groups was detected 24 hrs after the MWM. P‐tau: WTC, n = 8; KOC, n = 8; WTI, n = 8; KOI, n = 8. Mean ± SEM. *, P = 0.035; #, P = 0.017; one‐way ANOVA (F = 10.2; df = 3), with Scheffe's post hoc comparisons. T‐tau: WTC, n = 8; KOC, n = 8; WTI, n = 8; KOI, n = 8. Mean ±SEM. n.s, not significant; P = 0.774; one‐way ANOVA (F = 0.371; df = 3), with Scheffe's post hoc comparisons. NR2B: WTC, n = 8; KOC, n = 8; WTI, n = 8; KOI, n = 8. Mean ± SEM. *, P = 0.029; #, P = 0.006; one‐way ANOVA (F = 11.61; df = 3), with Scheffe's post hoc comparisons. WTC: WT mice control group. KOC: KO mice control group. WTI: WT mice anesthetized with isoflurane group. KOI: KO mice anesthetized with isoflurane group

3.5. Immunohistochemistry

The immunohistochemistry for the Aβ levels was done 24 hours after the MWM. The two‐way ANOVA statistical results indicated that isoflurane (P = 0.005) or A1AR KO (P = 0.000) had a significant effect on the Aβ (two‐way ANOVA; interaction, P = 0.014). In hippocampus, level of Aβ was increased in WTI group compared to that in WTC group (Figure 3A,C,E). Furthermore, level of Aβ accumulation was decreased in KOI group compared to that in WTI group (Figure 3C,D,E). No differences were found between KOC and KOI groups (Figure 3B,D,E).

Figure 3.

Figure 3

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Isoflurane increases the level of Aβ accumulation, but knocked out A1AR can inhibit the effect of isoflurane. Immunohistology of hippocampal neurons from the WTC (A), KOC (B), WTI (C), and KOI (D) groups stained with anti‐Aβ 1‐42 antibodies. Scale bars, 50 μm. (E) Quantification of the level of Aβ level in the hippocampus from four groups detected 24 hrs after the MWM. WTC, n = 5; KOC, n = 5; WTI, n = 5; KOI, n = 5. Mean ± SEM. **, P = 0.006, #, P = 0.000; one‐way ANOVA (F = 18.625; df = 3), with Scheffe's post hoc comparisons

3.6. ELISA

The two‐way ANOVA statistical results indicated that isoflurane (P = 0.943; for Aβ40; P = 0.071; for Aβ42; P = 0.008; for Aβ42: Aβ40) or A1AR KO (P = 0.115; for Aβ40; P = 0.003; for Aβ42; P = 0.001; for Aβ42:Aβ40) had a significant effect on the Aβ42:Aβ40, but not on the Aβ40 (two‐way ANOVA; interaction, P = 0.431; for Aβ40; P = 0.153; for Aβ42; P = 0.009; for Aβ42:Aβ40). When we examined Aβ levels in aged mice, we found an increase in Aβ42:Aβ40 ratio in WTI group compared to WTC group (Figure 4A,C). However, level of Aβ42 or Aβ42:Aβ40 ratio was decreased in KOI group compared to that in WTI group (Figure 4A,C). No significant differences were found among all groups for level of Aβ40 (Figure 4B).

Figure 4.

Figure 4

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Isoflurane treatment modified Aβ levels in aged mice. (A) It showed an increase in Aβ42 levels in the WTI group compared to the WTC group or the KOI group. WTC, n = 4; KOC, n = 4; WTI, n = 4; KOI, n = 4. Mean ± SEM. *, P = 0.024; one‐way ANOVA (F = 6.634; df = 3), with Scheffe's post hoc comparisons. (B) It showed no significant difference among all groups for Aβ40 levels. WTC, n = 4; KOC, n = 4; WTI, n = 4; KOI, n = 4. Mean ± SEM. n.s, not significant; one‐way ANOVA (F = 1.188; df = 3), with Scheffe’s post hoc comparisons. (C) It showed an increase of Aβ42:Aβ40 ratio in the WTI group compared to the WTC group or the KOI group. WTC, n = 4; KOC, n = 4; WTI, n = 4; KOI, n = 4. Mean ± SEM. **, P = 0.007, #, P = 0.002; one‐way ANOVA (F = 13.000; df = 3), with Scheffe's post hoc comparisons

4. DISCUSSION

4.1. Isoflurane and hemodynamic

We have shown 1.4% isoflurane exposure for 2 hours as previously described8, 9, 26 has no effect on the blood pressure of mice. Furthermore, result of blood gas analysis is also in normal range. If respiratory depression, hypoxia, hypercapnia, and acidosis manifest during anesthesia, neurotoxicity can also occur and mortality can be increased in rodents as we described previously.23 The above risk factors must therefore be excluded. In this study, when mice were exposed to 1.4% isoflurane for 2 hours, mortality was not increased and physiological derangement was not found during anesthesia.

4.2. Isoflurane effect of adenosine release

Clinical relevant concentrations of isoflurane can increase adenosine generation in cultured human umbilical vein or mouse glomerular endothelial cells.27 The adenosine generation was reported to be either enhanced or decreased during isoflurane cardioprotection.28, 29

Isoflurane can induce opening of mitochondrial permeability transition pore (mPTP) and decrease the generation of adenosine‐5′‐triphosphate (ATP).30 However, it was reported that isoflurane initially increases cellular ATP levels through opening of mitochondrial ATP‐sensitive potassium channel (mitoKATP), whereas long exposure of isoflurane resulted in continuous reduction in ATP levels.31

As we know, adenosine is generated intracellularly as a metabolic intermediary for nucleic acids synthesis and the biological energy currency ATP and extracellularly by enzymatical conversion from released ATP into adenosine through coupled ectonucleotidases (CD73 and CD39).32 Furthermore, this intracellular production of adenosine is traditionally considered as the most important source of extracellular adenosine. Therefore, it seemed that isoflurane might decrease adenosine generation from ATP metabolism.

4.3. Neurotoxic effect of isoflurane

It has been reported that exposure to general anesthetics (GAs), such as isoflurane, preponderantly during the early postnatal period or during later life, triggers long‐term morphological and functional alterations in the brain. In the developing brain, numerous rodent studies have demonstrated that early‐life anesthesia induces persistent learning and behavioral deficits. In the aged brain, animal model studies have revealed that brains are more vulnerable to GA‐induced cognitive impairment than young adult brains.33 The neurotoxic effects of isoflurane are attributed to altered calcium homeostasis,34 reactive oxygen species (ROS) accumulation, enhancement of endogenous neurodegenerative mechanisms35 (eg, induced apoptosis and increased production of Aβ), and neuroinflammation.36 However, in contrast to the developing CNS, little is known about the structural changes induced by GAs in the aged brain. However, in line with other in vitro and in vivo work,7, 8 our data may indicate a link of isoflurane‐induced apoptosis to Aβ generation.

4.4. Isoflurane, A1AR, and learning and memory

Our study found that 1.4% isoflurane exposure for 2 hours impairs spatial memory in aged mice, a finding consistent with previous studies in mice and rats.24, 37 On days 3 and 4, escape latency became shorter in A1R−/− mice of KOC group compared to that in A1R+/+ mice of WTC group. On day 4, escape latency became longer in A1R+/+ mice of WTI group compared to that in A1R+/+ mice of WTC group. However, escape latency was shorter in A1R−/− mice of the KOI group compared to that in A1R+/+ mice of WTI group. This suggests isoflurane could impair learning and memory of aged mice. Additionally, A1R−/− mice performed better than A1R+/+ mice in MWM. As escape latency was no difference between KOI and KOC groups, we concluded knocked out A1AR inhibited isoflurane‐induced behavior impairment.

Levels of A1ARs are increased in the hippocampus of AD brain.20 Caffeine or other adenosine receptor antagonists protected against cognitive impairment.19 Our results were consistent with these reports. Thus, the study revealed isoflurane might induce cognitive impairment through A1AR. Furthermore, A1AR knockout could not only improve spatial learning and memory of aged mice, but reverse isoflurane‐induced cognitive impairment.

4.5. Isoflurane, A1AR, Aβ, and P‐tau

Aβ, the key component of senile plaques in AD patients,4 is produced from APP by step‐wise cleavage by β‐secretase and γ‐secretase.13 However, Aβ deposition also takes place during healthy aging.38 In the study, we worked mouse model in physiological conditions, whereas most articles research on transgenic models which overexpress mutated variants of AD‐related genes.9 Actually, equilibrium of different Aβ oligomers in healthy aged animals can be different from pathological models. Additionally, because of three mutations in the amino acid sequence, murine animals do not develop amyloid plaques.39 Nonetheless, soluble endogenous Aβ can be detected in brain from WT mice using appropriate assays.40 Thus, endogenous Aβ can contribute to age‐related physiological and biochemical alteration in brain of WT mice. Tau, a microtubule‐associated protein, stabilizes microtubules and regulates axonal transport. Under pathological conditions, tau is phosphorylated and detaches from microtubules, accumulating in the somatodendritic compartment of neurons, and finally forms NFTs, one of pathological hallmarks in AD patients.41 Our research revealed levels of Aβ were increased in WTI group compared to those in WTC group. However, Aβ and P‐tau were decreased in KOI group compared to those in WTI group, similar as P‐tau in KOC group compared to those in WTC group. Furthermore, levels of Aβ or P‐tau showed no differences between KOC and KOI groups. This was consistent with previous studies.3, 4, 42 This study showed an association between the administration of isoflurane, impairment of spatial memory, and increasing levels of A‐beta and P‐tau, presumably via the activation of the A1AR receptor.

4.6. Isoflurane, A1AR, and NR2B

NR2B is one of NMDAR subtypes. Overexpression of NR2B improves learning and memory in a transgenic mouse.43 NR2B may thus be involved in learning and memory. Our studies showed levels of NR2B were decreased in WTI group compared to those in WTC or KOI group. Furthermore, levels of NR2B showed no differences between KOC and KOI groups. Isoflurane can inhibit NMDAR, possibly main mechanism of this effect in animals.44 Activation of A1ARs attenuates LTP and LTD of synaptic transmission by inhibiting NMDAR, impairing function of working memory.21, 45 Inhalation anesthetics, such as halothane,46 isoflurane,47 sevoflurane, and enflurane,48 can depress spontaneously synchronized calcium oscillations (initiated by activating NMDARs on neuron) via A1AR activation. This anesthetic‐induced inhibition can be partly reversed by knocking out of A1AR. This suggests isoflurane might decrease level of NR2B likely via A1AR. Our results were consistent with the aforementioned studies. Thus, isoflurane could depress NMDAR‐mediated neural excitatory activity by activating A1AR, and anesthetic‐induced neurotoxicity would be reversed by knocking out A1AR.

4.7. Aβ, tau, and NR2B

Levels of NR2B mRNA and protein are decreased in AD brain.49 Hippocampal NMDAR loss correlates with AD progression.50 Therefore, prolonged depression of NMDAR‐evoked currents may contribute to AD pathogenesis.

Aβ is associated with neurite breakage and reductions in the number of dendritic spines in animal models of AD.51 Aβ decreases surface expression of NR2B by promoting endocytosis of receptor proteins and reduces synaptic NMDARs.52 Thus, we considered NMDAR downregulation is a major downstream effect exerted by Aβ.53

Increased phosphorylation of tau decreased level of NR2B in selectively vulnerable brain regions, leading to NMDAR hypofunction.54 Additionally, overexpression of tau might inhibit LTP and disrupt memory.55 LTD‐inducing NMDAR activation triggered an increase in tau phosphorylation with phosphomimetic tau also facilitating LTD induction. Furthermore, tau protein was required for Aβ‐induced impairment of LTP.56 Tau phosphorylation might therefore be part of a regulatory mechanism to control NMDAR activity.57

Aβ may interact with P‐tau and Aβ accumulation may induce hyperphosphorylation of tau; in turn, hyperphosphorylation of tau may also increase Aβ production and induce neurotoxicity.41, 56

Taken together, this study considers isoflurane might induce cognitive impairment resulting from depressing level of NR2B and NMDAR‐evoked currents by increasing levels of Aβ and P‐tau via activation of A1AR in aged mice. Furthermore, isoflurane‐induced neurotoxicity can be reversed by knocking out A1AR. A1AR can therefore be considered a potential target in therapy of AD and POCD in elderly patients. This study provides novel evidence of isoflurane inducing cognitive impairment via A1AR and of knockout of A1AR reversing cognitive impairment in aged mice. Certainly, other mechanisms also play major roles in pathogenesis, such as cellular apoptosis, neuroinflammation, and mitochondrial dysfunction, including aberrant Ca2+ homeostasis, mutated presenilin‐1, microglia‐derived cytokines, and other immune mediators.58, 59 Further studies will be necessary to understand precisely how inhalation anesthetics induce neurotoxicity and whether and how this contributes to AD and/or POCD.

There are a few limitations of our study. First, the time spent in the target quadrant during the probe test was less than 30% in all groups which is surprised but similar to that in a previous study.60 The possible explanation could be that more stress condition as to no platform can be located with initial attempts during probe test may force animal moving away from the target quadrant. On the other hand, declining cognitive function is associated with increasing age. In general, old age is less focusing on doing things per se even in the absence of pathological conditions.60 Second, P‐tau is a very sensitive biomarker which can be changed by many factors including body temperature change which was well documented previously.3 Although animals’ body temperature was maintained at the level closed to 37°C, its fluctuation during the course of study is likely to contribute to the changes of P‐tau. Thirdly, Aβ is also a sensitive biomarker which can be varied in adult mice and humans as a function of natural sleep61 or interrupted sleep.62 As the mice of all groups were housed under a 12/12‐h light‐dark cycle, the sleep/wake conditions for these animals were consistent throughout the experiments. Therefore, Aβ changes found in our study are unlikely due to sleep changes. Lastly, isoflurane‐induced “brain damage” less in A1AR knockout than wild‐type aged mice may be just an association rather the “real” mechanism as knockout procedure could trigger other biological compensative mechanism which may contribute to such effects. Therefore, other study protocol including pharmacological intervention(s) should be considered in future study to confirm the role of A1AR in this context. Additionally, behavior of long‐term effects longer than 24 hours should be evaluated and other behavioral tests except MWM should be applied.

5. CONCLUSIONS

In our study, we showed an association between the administration of isoflurane, impairment of spatial memory, decreasing level of NR2B, and increasing levels of Aβ and P‐tau, presumably via the activation of the A1 AR. Specifically, we confirmed the key role of A1 AR for isoflurane‐induced cognitive impairment in aged mice. However, more studies are needed to establish the cause‐effect relationships among these factors.

CONFLICT OF INTEREST

The authors declare no conflict of interest.

ACKNOWLEDGEMENTS

We would like to thank Dr. Jiang Fan Chen at the Department of Neurology, Boston University School of Medicine, Boston, Massachusetts, USA, for their generous gift of the A1AR KO mice and article revision. This work was supported by grants from National Natural Science Foundation of China (No.81471448), Natural Science Foundation of Zhejiang Province (No. Y14H090071) and Science Technology Department of Zhejiang Province(No.2012R10073).

Zuo C‐L, Wang C‐M, Liu J, et al. Isoflurane anesthesia in aged mice and effects of A1 adenosine receptors on cognitive impairment. CNS Neurosci Ther. 2018;24:212–221. 10.1111/cns.12794

Contributor Information

Qing‐Quan Lian, Email: lianqingquanmz@163.com.

Han Lin, Email: nanlinhannansh@gmail.com.

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