Adiponectin deficiency is a critical factor contributing to cognitive dysfunction in obese mice after sevoflurane exposure

doi:10.21203/rs.3.rs-4875994/v1

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Research Article

Adiponectin deficiency is a critical factor contributing to cognitive dysfunction in obese mice after sevoflurane exposure

https://doi.org/10.21203/rs.3.rs-4875994/v1

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published 16 Oct, 2024

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Background

The growing number of obese individuals is expected to create an increase in the number of major operations to be performed in those patients. Obesity is a risk factor for a range of postoperative complications including perioperative neurocognitive disorders. However, the neurotoxic effects of general anaesthesia in the obese subjects are not yet determined. We hypothesize that general anaesthesia such as sevoflurane induces neurotoxicity in obese animals while no significant impact is induced in lean mice. This vulnerability depends on the reduction of the adiponectin in the obese mice.

Methods

Obese mice were bred by subjecting C57BL/6 mice to a 60% high fat diet. Both obese mice lean mice were exposed to 2 hours of sevoflurane. To confirm the role of adiponectin deficiency in sevoflurane induced neurotoxicity, adiponectin knockout mice were established and exposed to the sevoflurane. Finally, the neuroprotective effects of adiponectin receptor agonist (AdipoRon) were examined. Cognitive functions, neuroinflammatory responses and neuronal degeneration were accessed.

Results

Sevoflurane triggered significant cognitive dysfunction, neuroinflammatory response and neuronal degeneration in obese mice while no significant impact was observed in lean mice. Similar cognitive dysfunction and neuronal degeneration were also observed in the adiponectin knockout mice after sevoflurane exposure. Administration of AdipoRon prevented the deleterious effects of sevoflurane in both obese and adiponectin knockout mice.

Conclusions

Our findings demonstrated that obese mice are more susceptible to sevoflurane triggered neurotoxicity and cognitive impairment compared with lean animals. Adiponectin deficiency in obese subjects is one of the underlying mechanisms and treatment with adiponectin receptor agonist ameliorates this vulnerability. These findings may have therapeutic implications to reduce the incidence of anaesthesia induced neurotoxicity in obese subjects.

Obesity

Sevoflurane induced neurotoxicity

Cognitive dysfunction

Neuroinflammation

Adiponectin

The post exposure effects on the brain from sevoflurane has been subjected to debate. Sevoflurane can be neuroprotective in various circumstances, including cerebral ischaemia/reperfusion cerebral injury [1] and liposaccharide-induced neuroinflammation [2]. However, it also exhibits neurotoxic effects in certain animal groups such as the aged [3] or those with pre-existing cognitive impairment [4]. Previously, sevoflurane was shown to induce neurotoxicity via increasing proinflammatory cytokines released by microglia [5, 6] and activating neurotoxic tau proteins [7]. Interestingly, significant decline in memory function was also observed in diabetic rats following sevoflurane exposure [6]. To date, it remains inconclusive whether brains from obese individuals are more vulnerable to sevoflurane-induced neurotoxicity (SIN). With obesity often coexisting in those with diabetes as part of the metabolic syndrome, it is worthwhile investigating whether this condition puts such patients at increased risk for SIN.

Adiponectin is a neuroprotective adipokine produced by adipose tissues which regulates inflammatory and neurotrophic responses [8, 9]. Adiponectin deficiency is commonly observed in obese patients and animal models [10]. Recent research reported that adiponectin deficiency impaired cognitive function through exacerbating neuroinflammation while intervention with adiponectin suppressed inflammatory cytokine release via AdipoR-AMPK signalling [11]. Based on the neuroprotective and neurotrophic features of adiponectin, we hypothesized that adiponectin deficiency places obese individuals at increased risk of SIN, presenting with cognitive dysfunction following sevoflurane exposure [12]. We first aim to evaluate the differences in susceptibility of obese mice to SIN. Then we aim to determine whether adiponectin deficiency is involved in this susceptibility by utilizing a mouse model with the background of adiponectin knockout (APN-KO). Our findings indicate that sevoflurane triggered neurotoxicity and cognitive impairment in obese and APN-KO but not in lean mice. Supplementation with an adiponectin receptor agonist, AdipoRon, in both obese and APN-KO mice reduced SIN and cognitive dysfunction. Taken together, our findings support the role of adiponectin deficiency which contribute to the SIN in obese subjects.

Animal groups

3-month-old male C57BL/6 mice obtained from the Centre of Comparative Medicine Research, The University of Hong Kong. Animals were kept on a 12/12-hour light-dark cycle and had access to food and water ad libitum. All animal experimental protocols were approved by the Department of Health, HKSAR, China and Committee on the Use of Live Animals in Teaching and Research (CULATR, approval number: 5349-20), The University of Hong Kong. All animal houses and facilities are accredited by the AAALAC International. All animal protocols were carried out in Centre of Comparative Medicine Research and Department of Anaesthesiology, Laboratory Block, Sassoon Road, Hong Kong.

Obese mice were bred from 3 months C57BL/6 mice fed with a diet comprising of 60% fat content for 3 months, whereas lean mice were fed with a standard diet. Body weights were monitored once per week. APN-KO mice with C57 genetic background were kindly provided by Prof. Aimin Xu from Department of Medicine, The University of Hong Kong [13]. Similarly aged 4-month-old male APN-KO mice were used in the experiments. The timeline for sevoflurane exposure, behavioural test and animal sacrifice is shown in Fig. 1a.

Sevoflurane exposure and AdipoRon treatment

Concentration of sevoflurane and the exposure duration were chosen according to previous report [4]. Briefly, mice were first placed in an induction chamber filled with 5% of sevoflurane (SEVOrane, 100% w/w, Abbvie, USA) delivered in medical oxygen for 5 mins (5% sevoflurane, 1L/min airflow). Anaesthesia was then maintained for 2 hours using 2.5% sevoflurane delivered at 1L/min airflow and respiration rate was monitored. Mice were then allowed to recover and transferred to their home cages until further behavioural experiments.

AdipoRon is an orally active selective agonist for the adiponectin receptor [14]. The stock was dissolved with 10% of DMSO and 90% of corn oil (Sigma-Aldrich, USA) for oral administration. Obese and knockout mice were randomly pretreated with either 300µl of vehicle or AdipoRon at a dose of 50µg/kg/day [15] once every second day for 1 week. The animals were then exposed to sevoflurane as described above, with AdipoRon or vehicle continuing for another 7 days until the start of behavioural assessments. The timeline for AdipoRon treatment, sevoflurane exposure and animal sacrifice is illustrated in Fig. 6a. Animals were finally euthanized by CO2 asphyxiation followed by tissue harvesting.

Open field and novel object recognition tests

Locomotor function and anxiety level of the animals were accessed by the open field test [4]. On day 5 post-exposure, mice were placed in the centre of an open arena box measuring 40cm x 40cm x 40cm and were allowed to freely explore the environment for 10 mins. The length of distance travelled and the centre duration time were then analysed using the SMART VIDEO TRACKING software (Harvard Apparatus, USA). On the video display, the base of the box was divided into 25 equal squares and centre duration time was defined as the length of the time that the animal stayed within the central 9 squares. A longer centre duration time represents a less anxious state of the mice.

Object recognition memory was determined by the novel object recognition (NOR) test as described [4]. Briefly, on post exposure day 6, the mice were placed in the same box as in the open field test. Two identical objects were placed in the box and the mice were allowed to interact with the objects for 10 mins. 24 hours after this familiarization, one of the objects was replaced by an novel object. The mice were then allowed to interact with both objects for 10 mins and their behaviour was analysed by the SMART VIDEO TRACKING software. The discrimination index is calculated using the formula of Tn/Tt, where Tn is the duration of time interacting with the novel object and Tt is the total time interacting with both the familiar and the novel object. A higher discrimination index indicates a better recognition memory in the test. Exclusion criteria for this test included inadequate total object exploration time (less than 20s) or recognition bias towards a specific side of object during the familiarization period.

2. Puzzle box test

The memory function and problem solving abilities of the animals were examined by the puzzle box task as previously described [16, 17]. The puzzle box consists of a brightly lit zone and a covered dark zone that are separated by a movable barrier. Mice were trained to move from the light zone into the dark zone through a narrow underpass located under the barrier. They underwent a total of nine trials (T1-T9), with three trials per day. Increasing difficulty of passing through the barrier were introduced day by day. Training began on day 5 post exposure and the underpass was unobstructed during the first trial (T1). In T2 and T3, a channel was introduced through which the mice should enter the dark zone. The burrowing test was introduced on day 6. It began with T4 which was identical to T3. For T5 and T6, the underpass was filled with bedding that the mice needed to dig through to reach the dark zone. On day 7, the plug test was introduced with T7 being a repetition of T6 but for T8 and T9, a plug was placed and blocked the underpass that needed removal using teeth and paws. Problem solving ability (T2, T5 and T8), short term memory (T3 and T6 and T9) and long-term memory (T4 and T7) were examined according to the sequence of the trials. A trial was started by placing the mouse in the light zone, and ended when the mouse entered the dark zone or after a total time of 5 mins (training and burrowing tests) or 10 mins (plug test). After finishing each trial, the mice were allowed to rest for 10 mins before the next trial. The performance of mice was compareed by measuring the escape latency in reaching the dark zone in each trial.

TUNEL assay

DNA fragmentation in the hippocampus were examined using the TUNEL as described [4]. Counterstaining of nuclei was performed by staining with DAPI solution (Sigma-Aldrich, USA). Fluorescent images were obtained using a Carl Zeiss LSM780 laser scanning confocal microscope (ZEISS, Germany). Immunofluorescence intensities was analysed by ZEN software.

Western Blotting for protein analysis

Different phosphorylated and endogenous protein expressions were examined by Western blot. The hippocampal tissues were first homogenized in RIPA buffer supplemented with protease and phosphatase inhibitors (Roche, Switzerland). Protein samples were then extracted and resolved by SDS-PAGE, followed by transferring onto a PVDF membrane. The membrane was then incubated with different primary and HRP conjugated secondary antibodies. The protein signals were visualized with enhanced chemiluminescence reagents (Advansta Inc, USA) and captured by Chemidoc imaging system (Bio-Rad, USA). Quantification of the signals was performed by densitometric analysis of the Image Lab Software (Bio-Rad, USA).

4. Immunofluorescence staining

Microglial and astrocytic activation in the hippocampus were examined by immunofluorescence staining. Briefly, brain tissues were removed after perfusion with 0.9% saline and tissues were fixed with 4% paraformaldehyde solution for 24 hours. 20µm coronal sections containing the hippocampal region were prepared and mounted on the glass slide. Sections were first blocked by PBS with 1% BSA and 0.1% of Triton X reagent, then stained with the microglial marker IBA1 and the astrocyte marker GFAP antibodies overnight. Afterwards sections were incubated with a secondary antibody conjugated with Alexa Fluor dyes for 2 hrs. After incubation, sections were mounted with prolong gold medium containing DAPI (Thermofisher Scientific, USA). Fluorescent images were captured by the confocal microscope and the immunofluorescence intensities were analysed by ZEN software.

Golgi staining for the number of dendritic spines

Dendritic spines in the hippocampus were examined by Hito Golgi-Cox OptimStain™ kit as described [18]. Briefly, brain tissues were removed without perfusion and rinsed with distilled water. Impregnation of the Golgi stain was performed according to the manufacturer’s protocol. 150µm coronal brain sections of the hippocampal region were taken using a cryostat. Dendritic spines in hippocampal neurons were then examined under a light microscope. The number of spines was counted, and the results were expressed as number of spines per µm of dendrite.

ELISA assay and Multiplex cytokine assays

ELISA and multiplex assays were used to examine adiponectin and inflammatory cytokine markers in the serum and hippocampus. Hippocampal tissues were harvested and extracted in lysis buffer supplemented with a protease inhibitor. The adiponectin level in the serum and hippocampus was examined by ELISA according to the manufacturer’s protocol. Inflammatory cytokine expression in the hippocampus was examined by MILLIPLEX MAP mouse cytokine/chemokine magnetic bead panels according to the manufacturer’s protocol. Data were collected using a Bio-Plex 200 system (Bio-Rad, USA).

Statistical analyses

Normality of the data were assessed using the Shapiro–Wilk normality test. The sample size to detect a significance for behavioural experiments and biochemistry studies is based on our previous studies [4, 19]. Data were analysed using an unpaired two-tailed t test or one way ANOVA with post-hoc test of Tukey. Data were presented as the mean ± standard error of the mean (SEM). Statistical significance was considered when p ≤ 0.05.

1. Sevoflurane induced cognitive dysfunction in obese but not in lean mice

We first established the obese model with the significant increase of body weight after 12 weeks of high fat diet exposure (Supplementary figure; Fig. S1). No significant difference in the total distance travelled and centre duration in the open field test was observed in both groups of animals (Fig. 1b), suggesting that sevoflurane did not exert any residual effects on locomotion and anxiety. On the other hand, a significant reduction in the discrimination index measured by the NOR was observed in the obese mice but no in lean mice, indicating that sevoflurane triggered a decline in object recognition memory only in obese mice (Fig. 1b). Results from the puzzle box test further indicated that sevoflurane adversely affected memory functions (T4, T6 and T9) and problem-solving ability (T5 and T9) in the obese mice which was reflected by the significant increase of escape latency in the sevoflurane exposure group (Fig. 1c). On the contrary, no significant changes were found in the lean control groups (Fig. 1c). These results confirm that sevoflurane exposure triggers cognitive deterioration in obese but not in lean mice.

2. Sevoflurane induced the loss of dendritic spines and triggered apoptosis in the hippocampus of obese and not in lean mice

The number of dendritic spines on hippocampal neurons were assessed by Golgi staining and apoptosis was examined by TUNEL staining. In the lean groups, no significant differences in dendritic spines were observed while a significant decrease in the number of dendritic spines was found in obese mice after sevoflurane exposure (Fig. 1d). Similarly, no significant increase of TUNEL positive signals and pro-apoptotic Bax/Bcl-2 ratio was indicated in lean groups (Fig. 1e and f). Conversely, a significant increase in TUNEL positive signals and Bax/Bcl-2 ratio was evident in the obese mice after sevoflurane exposure (Fig. 1e and f). These results demonstrated that sevoflurane initiated synaptic loss and neuronal apoptosis in the hippocampus of obese mice while minimum impact was seen in lean mice.

3. Sevoflurane induced neuroinflammatory changes in the hippocampus of obese mice and not in lean mice

In obese mice, significant increases in IBA1 and GFAP immunofluorescence signals in hippocampus were observed whereas no significant changes were found in lean mice after sevoflurane exposure (Fig. 2a and b). From the multiplex assay, significant increases in inflammatory cytokines IL-1β and MCP-1 were found in the obese groups while no significant differences were observed between the lean groups (Fig. 2c). These data revealed that sevoflurane specifically triggered neuroinflammatory responses in the brains from obese but not from lean mice.

4. Sevoflurane modulated the AMPK/JNK pathway activity and increased neuropathological changes in the hippocampus of obese mice

Adiponectin can bind to the adiponectin receptor and inhibits neuroinflammatory responses through the AMPK pathway [12]. We first demonstrated a significant reduction of adiponectin in the serum and hippocampus of obese mice (Supplementary figure; Fig. S2a) without the modulation of adiponectin receptor 1 among all animal groups (Supplementary figure; Fig. S2b). Next, a significant reduction in phosphorylated AMPK was noted in the obese mice after sevoflurane exposure (Fig. 3a). Besides, a significant up-regulation in phosphorylated JNK and c-jun, the downstream transcriptional factor of JNK, were observed in obese animals (Fig. 3a).

As neuroinflammation is shown to correlate with the presence of different hallmarks of neuropathology such as tauopathy [20], tau phosphorylation was also assessed by Western blot. Consistent with the above findings, a significant increase in phosphorylated tau at the Ser 199/202 and Ser 396 epitopes were observed in the obese mice (Fig. 3b). These findings together imply that sevoflurane exacerbates neuroinflammatory responses and the development of neuropathological changes in obese but not in lean mice.

5. Sevoflurane induced cognitive impairment, neuroinflammatory responses, tau phosphorylation, apoptosis and dendritic spine loss and modulated the AMPK/JNK pathway activities in APN-KO mice

To confirm if adiponectin deficiency renders animals more susceptible to SIN, we repeated the above investigations in APN-KO mice fed with a standard diet. First, no significant weight differences were observed between wild type and APN-KO mice at the same age (Supplementary figure; Fig. S3). From the open field test, no significant changes in locomotor activity or anxiety level were observed in APN-KO mice after sevoflurane exposure (Fig. 4a). On the other hand, a significant reduction in the discrimination index from the NOR test was noted in the APN-KO mice, which was in alignment with the results in the obese animals. Similarly, the puzzle box test also revealed a longer escape latency in both memory (T6 and T9) and problem-solving tasks (T5) (Fig. 4b). In line with the cognitive decline, significant increase in IBA1, GFAP immunofluorescence and IL-1β were also observed in the APN-KO mice (Fig. 4c and d). Consistent with the findings in obese mice, a significant reduction in phosphorylated AMPK, an increase in the expression of phosphorylated JNK, c-jun and phosphorylated tau in Ser 199/202 were seen in APN-KO mice (Fig. 4e and 5a). In terms of apoptosis, an increase in TUNEL immunofluorescence (Fig. 5b), coupled with an increased Bax/Bcl2 ratio (Fig. 5c) in the APN-KO mice post sevoflurane exposure. Finally, reduced dendritic spines was also observed in the APN-KO mice (Fig. 5d). These data from APN-KO mice strongly support the hypothesis that adiponectin deficiency plays a critical part in SIN and cognitive decline.

6. Supplementation of AdipoRon improved sevoflurane induced cognitive decline and neuropatholgy in obese and APN-KO mice

To examine whether administration of adiponectin receptor agonist can confer protection against SIN, AdipoRon was given to both obese and APN-KO mice starting 7 days before and continued for another 7 days after sevoflurane exposure. NOR test performed in both these types of mice indicated a significant increase in the discrimination index after AdipoRon supplementation (Fig. 6b). Moreover, a significant increase of dendritic spines (Fig. 6c) and amelioration of microglial activation (Fig. 6d) were observed in the AdipoRon plus sevoflurane groups. From Western blotting, significant up-regulation of phosphorylated AMPK, together with the down-regulation of phosphorylated tau (Ser/199/202) and Bax/Bcl2 ratio were shown in both AdipoRon treated groups (Fig. 6e). These data suggest that AdipoRon can partially reverse the deleterious effects of sevoflurane in obese and APN-KO mice which may have translational value in the clinical setting.

Consistent with previous findings regarding obesity and cognition, current study demonstrated that obese animals with a lower level of adiponectin have a greater tendency for poorer cognitive performance following sevoflurane exposure. Concurrently our findings demonstrate that sevoflurane exposure triggered neuroinflammation with increased apoptosis, reduction of dendritic spines and increased tau phosphorylation in the obese mice compared with their lean counterparts.

Normally both astrocytes and microglia play essential roles in supporting neuronal development and plasticity within the CNS by controlling metabolic and neurotrophic tasks [5, 21]. However, when glial cells are stimulated and activation occurs, as was the case we shown in our obese and APN-KO mice following sevoflurane exposure, they can also induce an neuroinflammatory state by releasing chemokines and inflammatory cytokines including IL-1β and TNF-α [21]. These neuroinflammatory changes are implicated in cognitive dysfunction and correlate with the observation of increase in astroglial activation and cytokines accompanying neuropathological change in other forms of dementia such as Alzheimer's disease (AD) [22].

In addition to demonstrating astroglial activation and increased inflammatory cytokines, we also investigated the downstream modulation of the intracellular AMPK/JNK pathways following sevoflurane exposure. Activation of JNK leads to phosphorylation of transcription factors, such as c-jun, which stimulates the expression of pro-inflammatory genes [23]. Conversely, AMPK plays a key role inhibiting inflammatory responses by influencing JNK and NF-κB signalling [24, 25]. Neuroinflammatory conditions such as diabetes or AD have been shown to cause AMPK inhibition alongside JNK and NF-κB pathway upregulation. [25, 26] Our data demonstrated a significant reduction of phosphorylated AMPK in obese animals after sevoflurane exposure, associated with increased phosphorylated JNK and c-Jun, while no significant corresponding changes were observed in lean mice.

There was also a significant loss of dendritic spine and increased apoptotic signals in hippocampus of obese following sevoflurane exposure (Fig. 1). The synapse stands as a critical locus of neurodegeneration preceding the onset of cognitive deficits [27, 28] and the degree of synaptic loss and neuronal apoptosis correlate with the extent of cognitive decline.[27, 28]. In apoptotic cells, DNA fragmentation and the activation of the pro-apoptotic Bax/Bcl2 pathway are characteristically seen [29]. Bax is the key component for increasing mitochondrial permeability, which initiating apoptosis [29], while anti-apoptotic Bcl2 controls mitochondrial permeabilization, thus inhibiting the downstream apoptotic pathway and facilitating cell survival [29].

We then assessed for increases in phosphorylated tau after sevoflurane exposure. Phosphorylation of tau and the subsequent tauopathy are believed to be associated with the progression of neuroinflammation [20]. Phosphorylation of tau results in its dissociation from axonal microtubules and form oligomers, which cause neurotoxicity and subsequent synaptic dysfunction [30]. Under neuroinflammatory conditions, JNK acts as a MAP kinase and regulate tau phosphorylation on different amino acid residues, including serine 199, 202, 396, and 404 [31]. Moreover, inflammatory cytokines including IL-1β can promote tau phosphorylation through various signalling pathways [32]. Previous reports described sevoflurane induced tau phosphorylation in postnatal mice resulting in later cognitive dysfunction [33, 34]. Consistent with these findings, our data also revealed a significant increase of tau phosphorylation in obese mice, but not in lean mice.

Whether one develops post operative cognitive decline is dependent on multiple factors. Patients with major risk factors for developing perioperative neurocognitive disorders (PNDs) such as advanced age or pre-existing cognitive impairment, are likely to have a propensity towards a neuroinflammatory state. This pro-inflammatory tendency may result in a greater neuroinflammatory response in the brain when further triggered. Neuroinflammation is an early event involved in the neurotoxicity of sevoflurane in other models [4, 35]. Major surgical trauma triggers an acute systemic inflammation and in a significant proportion of cases can lead to a neuroinflammatory response that could disrupt cognitive processes.

Therefore, the presence of cognitive deficits in the absence of surgery in this study is a rather significant finding. The impact of general anaesthesia alone on the brain remains a topic of debate, as both neuroprotective [1, 36] and detrimental effects [3, 4, 37] have been reported. It has been proposed that the differential effects of sevoflurane are based on the state of neuronal development and pathological background [35]. With regards to the CNS, obesity is associated with the decreased grey matter volume, accumulation of neuropathology and neuroinflammatory response [38–40]. Recently it has been identified as an independent risk factor for post operative cognitive dysfunction [41]. Furthermore, in both clinical observations and animal studies, subjects with metabolic syndrome have a higher risk in developing different postoperative complications, including experiencing cognitive decline [42, 43]. These findings imply that the brain from an obese individual is already under “stressed” condition which may make it more vulnerable to the SIN.

Adiponectin is one of the most abundant adipokine possessing anti-inflammatory properties, as well as regulating energy expenditure via lipid and glucose metabolism [44, 45]. Circulating adiponectin can pass through the blood brain barrier and bind to adiponectin receptors, thereby regulate cerebral energy homeostasis, hippocampal neurogenesis and synaptic plasticity [44]. Apart from obese patients, a reduced circulating adiponectin level is also observed in patients with mild cognitive impairment and AD [46]. Chronic adiponectin deficiency is associated with the accumulation of AD related neuropathological changes and cognitive deficits in aged animals [47]. In contrast, treatment with an adiponectin receptor agonist can enhance insulin sensitizing effects and improve cognitive dysfunction in AD animals [15]. These reports indicate the important role of adiponectin in cognitive dysfunction and adiponectin deficiency may render the brain more vulnerable to exogenous insults. Owing to a deficiency of adiponectin in subjects with excess adiposity, we hypothesized that obese subjects are more susceptible to the neurotoxicity of sevoflurane because of lower levels of this adipokine. The results observed in obese mice in part supports the hypothesis.

To confirm if adiponectin deficiency is indeed a key factor contributing to this vulnerability, APN-KO mice, fed with a standard diet to reduce the chance of developing obesity, were similarly exposed to sevoflurane. Similar neurotoxic effects and cognitive deficits were observed in these mice including cognitive impairment, neuroinflammatory responses, inhibition of AMPK, phosphorylated JNK and c-jun and increased tau phosphorylation at residue 199, apoptosis and dendritic spine loss. These data from APN-KO mice confirm that the pre-existing of deficiency of adiponectin, but not necessarily adiposity, is sufficient to induce cognitive decline, neuroinflammatory response and neuronal degeneration by sevoflurane exposure (Figs. 4 and 5), which lending support to our hypothesis that obese mice are more susceptible to the neurotoxic effects of sevoflurane due to adiponectin deficiency.

To further confirm the key role of adiponectin, we proceed to evaluate whether augmenting the biological actions of adiponectin in both obese and APN-KO mice would negate the adverse effects from sevoflurane. We used the synthetic selective adiponectin receptor agonist AdipoRon to mimic the actions of adiponectin. This orally active agent circumvents some limitations of converting the native protein into a viable pharmacological agent, such as the diverse range of protein structure expressed and the insolubility of the C-terminal domain [48]. This molecule can cross the blood brain barrier and its use have been shown to ameliorate Alzheimer like neuropathological traits in AD animal models [15, 49]. We demonstrated that supplementation of AdipoRon attenuated sevoflurane induced microglial activation, synaptic loss, and cognitive decline in both obese and knockout mice, which are in agreement with previous studies and confirm the critical role of adiponectin deficiency in SIN.

Though our data is compelling for a protective effect of adiponectin against the neurotoxic effects of sevoflurane in the obese, our experimental design has a few limitations which need to be highlighted. First, few patients would clinically be exposed to anaesthesia in the absence of surgery, so we did not use a true representative model of PNDs. We examined the protective effect of only one adiponectin substitute that have been previously studied experimentally while others are also available. We administered AdipoRon for some time across the peri-exposure period but did not assess the cognitive behaviour just prior to the animal being subjected to anaesthesia so we may have already altered the animal’s baseline with the supplement. Finally, the beneficial cognitive effects of AdipoRon may not be limited to obese animals and it would be interesting to test AdipoRon in other models of PNDs.

In conclusion, the brains of obese subjects exposed to sevoflurane maybe more vulnerable to develop adverse cerebral effects in part due to the relative lack of neurotrophic effects from adiponectin. Supplementation with the adiponectin substitute AdipoRon provides mitigation against SIN. Given the scope of the global obesity epidemic and the demand for surgical procedures in this population, perioperative neurocognitive disorders may place an increasing burden to health systems worldwide and erode into affected patients’ post operative quality of life in increasing numbers. As adiponectin substitutes are undergoing active investigation for a range of obesity related metabolic disease, our findings provide an impetus to examine the role of these agents in cerebral protection during the perioperative period for these patients.

AD Alzheimer's disease

APN-KO Adiponectin knockout

CNS Central nervous system

GFAP Glial fibrillary acidic protein

IBA1 Ionized calcium binding adaptor molecule 1

NOR Novel object recognition

SIN Sevoflurane induced neurotoxicity

TUNEL TdT-mediated dUTP nick-end labelling

Ethics approval

All animal experimental protocols were approved by the Department of Health, HKSAR, China and the Committee on the Use of Live Animals in Teaching and Research (CULATR, approval number: 5349-20), The University of Hong Kong.

Author contributions

Design and Conceptualization: JMTC, RCCC and GTCW; Investigation and Formal analysis: JMTC, SPWC and JW; Writing-original draft: JMTC and SPWC; Writing-review and editing: JMTC, RCCC and GTCW. Funding acquisition, Resources, Supervision and Project administration: JMTC, RCCC and GTCW. All authors read and approved the final manuscript.

Competing interests

The authors declare that they have no competing interests.

Funding

The work described in this paper was partially supported by the grants from the Research Grants Council of the Hong Kong Special Administrative Region, China (HKU 17101020, HKU 17100622 and HKU 17100523) and Seed Fund for Basic Research, The University of Hong Kong (2202100768).

Acknowledgements

Authors would like to thank for the support from Prof. Aimin Xu for donating the mouse strain of adiponectin knockout. Imaging data were acquired using equipment maintained by the University of Hong Kong, Li Ka Shing Faculty of Medicine Faculty Core Facility. The authors acknowledge the assistance of the University of Hong Kong Li Ka Shing Faculty of Medicine Faculty Core Facility.

Availability of data and materials

The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.

Supplementary figures and tables

Supplementary data in this manuscript are included in the supplementary figures. Antibodies and reagent used in this study are described in the supplementary table.

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No competing interests reported.

Fig.S1.tif
Supplementary Figure 1 (S1): High fat diet increased body weight of C57 mice Significant weight gain was observed in high fat diet group after 4 weeks of high fat diet exposure.
Fig.S2.tif
Supplementary Figure 2 (S2): High fat diet reduced adiponectin in C57 mice without modulating adiponectin receptor expression (a) Significant reduction of adiponectin was observed in the serum and hippocampus of obese mice. n = 4. *p < 0.05, **p < 0.01 in comparison to the respective control groups. Values were analysed using the unpaired t-test. (b) No significant change in adiponectin receptor expression was observed in the brain after high fat diet exposure. n = 3.
Fig.S3.tif
Supplementary Figure 3 (S3): No significant weight changes seen in wild type and adiponectin knockout mice No significant weight change was observed between wild type and APN-KO mice at the time point of 3-month and 4-month old.
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Adiponectin deficiency is a critical factor contributing to cognitive dysfunction in obese mice after sevoflurane exposure

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Abstract

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Introduction

Methods

Animal groups

Sevoflurane exposure and AdipoRon treatment

Open field and novel object recognition tests

2. Puzzle box test

TUNEL assay

Western Blotting for protein analysis

4. Immunofluorescence staining

Golgi staining for the number of dendritic spines

ELISA assay and Multiplex cytokine assays

Statistical analyses

Results

Discussion

Conclusions

Abbreviations

Declarations

References

Additional Declarations

Supplementary Files

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Journal Publication

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