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. 2015 Jan 14;35(4):585–594. doi: 10.1007/s10571-014-0154-6

Minocycline Alleviates Sevoflurane-Induced Cognitive Impairment in Aged Rats

Yue Tian 1, Shanbin Guo 2, Xiuying Wu 1,✉, Ling Ma 1, Xiaochun Zhao 1
PMCID: PMC11486178  PMID: 25585814

Abstract

Minocycline has been implicated in the treatment for multiple diseases in the nervous system for its neuroprotective properties. However, the mechanism by which minocycline benefits postoperative anesthesia-induced cognitive dysfunction is still unclear. In this study, we introduced minocycline to a rat model of anesthetic-induced learning and memory impairment, to investigate the effects of minocycline on neuroinflammation, beta amyloid (Aβ) deposition, and activation of nuclear factor κB (NF-κB) signaling pathway in the hippocampus. Aged rats were treated with sevoflurane to induce cognitive impairment with and without pre-administration of minocycline. The rats were then subjected to Morris water maze tests to evaluate their learning and memory performance. Subsequently, apoptosis in the hippocampal tissue was assessed with TUNEL assays. Furthermore, the levels of apoptosis-related proteins and pro-inflammatory cytokines, Aβ responses, and activation of the NF-κB signaling pathway in the hippocampus were examined by Western blot analysis. Our results revealed that minocycline effectively alleviated sevoflurane-induced cognitive impairment in aged rats. Minocycline reduced sevoflurane-induced neuronal apoptosis and inflammation, as well as suppressed sevoflurane-induced Aβ accumulation and activation of NF-κB signaling pathway in the hippocampus of aged rats. In conclusion, our findings indicate that minocycline is a potent agent to counteract sevoflurane-induced cognitive impairment and neurotoxicity in the nervous system of aged rats, which is likely to be mediated via NF-κB signaling pathway.

Keywords: Minocycline, Sevoflurane, Anesthesia-induced cognitive impairment, Neurotoxicity, NF-κB signaling

Introduction

Postoperative cognitive dysfunction (POCD) is a clinical phenomenon characterized by cognitive impairment in patients after anesthesia and surgery, especially in the elderly surgical patients. It has been reported that about 25 % of elderly (60 years old and above) patients exhibit POCD one week after surgery, and about 10 % of elderly patients exhibit persistent POCD for three months or longer (Moller et al. 1998). POCD can be self-limiting in most patients, but it may be long-term or even permanent in some patients and associated with postoperative complications and mortality (Bekker and Weeks 2003, Rasmussen et al. 2003). For example, anesthetics-induced POCD has been reported to be a risk factor for Alzheimer’s disease in elderly surgery patients (Steinmetz et al. 2009; Bittner et al. 2011). In the past decade, extensive work has been done to elucidate the mechanisms for anesthetics-induced POCD, and it was found that anesthetics such as sevoflurane can induce neuronal apoptosis in experimental animal models and in elderly Alzheimer’s patients, suggesting that anesthetics neurotoxicity may play an important part in POCD (Silbert et al. 2011; Brosnan and Bickler 2013; Xiong et al. 2013). Hence, it is a practically and clinically important task to seek agents that can counteract anesthetics-induced neurotoxicity and cognitive dysfunction, especially for the well-being of the elderly surgery patients.

Minocycline is a semi-synthetic, second-generation tetracycline derivative capable of penetrating the blood–brain barrier (Yang et al. 2007a). Minocycline is commonly used to treat infectious diseases in the clinic for its anti-inflammation (Desjarlais et al. 2014), anti-apoptosis (Liu et al. 2014), and anti-oxidation (Karachitos et al. 2012) properties. Interestingly, experimental data have indicated that minocycline exhibits neuroprotective functions in many nervous system-associated diseases, such as cerebral ischemia (Yrjanheikki et al. 1999), spinal cord injury (Stirling et al. 2004), Parkinson’s disease (Wu et al. 2002), Huntington’s disease (Palazuelos et al. 2009), and Alzheimer’s disease (Choi et al. 2007). Recently, it was reported that minocycline could alleviate isoflurane anesthesia-induced cognitive impairment in aged rats (Kong et al. 2013), suggesting that minocycline might be a potential agent to antagonize the anesthetics-induced adverse effects in the brain, thereby protecting the nervous system of the elderly surgery patients. However, whether minocycline can mitigate the cognitive dysfunction induced by other anesthetics such as sevoflurane remains unknown, and it is worth addressing for the potential clinical implication in elderly surgery.

Herein, we employed an aged rat model of learning and memory impairment resulting from sevoflurane inhalation. We investigated the effects of minocycline administration on sevoflurane-induced cognitive impairment and the underlying molecular mechanisms. Our results revealed that minocycline effectively alleviated sevoflurane-induced learning and memory impairment in aged rats as evidenced by Morris water maze (MWM) tests. Moreover, minocycline exerted protection for the hippocampal neurons by suppressing sevoflurane-induced apoptosis, inflammation, beta amyloid (Aβ) accumulation, and activation of nuclear factor κB (NF-κB) signaling pathway in the hippocampus of aged rats. In summary, our findings demonstrate a neuroprotective property of minocycline in sevoflurane-induced neurotoxicity and cognitive impairment, which is likely to be mediated through NF-κB signaling pathway.

Materials and Methods

Establishment of a Rat Model of Cognitive Impairment and Minocycline Treatment

Male, 20-month-old SD rats were purchased from the Laboratory Animal Center of China Medical University. Prior to the experiments, the animals were maintained under specific pathogen free (SPF) conditions for a week for adaptation, during which period they were allowed to consume food and water ad libitum. The animal experiment protocol was approved by the Animal Ethics Committee of China Medical University.

Twenty-four rats were randomly divided into four treatment groups with six animals in each group: control group, minocycline group, sevoflurane group, and minocycline + sevoflurane group. The rats in minocycline group and minocycline + sevoflurane group received intraperitoneal injection of 45 mg/kg minocycline 12 h prior to anesthesia, whereas the animals in the other two groups received a same volume of saline. The animals in sevoflurane group and minocycline + sevoflurane group were anesthetized in the environment containing 2 % sevoflurane for 5 h. 24 h later, physiological data of the animals including heart rate (HR), mean arterial pressure (MAP), arterial carbon dioxide tension (PaCO2), and arterial oxygen tension (SPO2) were documented. After MWM tests, the animals were sacrificed. For each rat, half of the brain was fixed in 4 % paraformaldehyde, paraffin-embedded, and the hippocampal tissue in the other half was frozen at −70 °C and kept for subsequent experiments.

Morris Water Maze

MWM test consisting of acquisition test and special probe trial is a well-established method to evaluate learning and memory ability in small animals (Blokland et al. 2004). Acquisition test: this experiment lasted for four days with two training sessions on each day and a 15-min interval between the two sessions. During training, each animal was placed, facing the tank wall, at a designated starting point in different quadrants of the maze every day. All groups shared the same starting point in each training session. If a rat swam for an amount of time before climbing onto the platform and remained there for over 3 s, its swimming time was documented and considered to be the latency for seeking escape. If a rat failed to find the platform within 120 s, or it was ushered to the platform and remained there for 30 s, the latency of these animals was recorded as 120 s. The latency of finding the hidden platform to escape reflects an animal’s performance at acquiring experience, or learning ability.

Spatial probe trial: this experiment was only performed once, whereby the platform was removed. A rat was placed at any starting point not adjacent to the platform while facing the wall. The frequency of the rat swimming across the original platform location within 120 s was documented. This experiment reveals an animal’s performance at recalling experience, or memory ability.

TUNEL Assay

Apoptosis was examined using In Situ Cell Death Detection Kit (Roche Diagnostics, Mannheim, Germany) following the manufacturer’s instructions. Specifically, 5-μm sections of the paraffin-embedded brain tissues were dewaxed, permeabilized, and blocked. The sections were treated with 50 μl TUNEL reaction solution and incubated in a humidified, dark box at 37 °C for 60 min. Subsequently, 50 μl Converter-POD was added to the sections for another incubation for 30 min in a dark box, and 50 μl DAB substrate solution was then added for color development. Hematoxylin was used to stain the nuclei, followed by routine dehydration, decolorization, and mounting. The sections were microscopically examined and images were captured. For each captured image, the numbers of total nuclei and TUNEL-positive nuclei were counted and the apoptosis ratio was calculated as follows: apoptosis ratio = (number of TUNEL-positive nuclei/number of total nuclei) × 100 %.

Western Blot Analysis

Hippocampal tissues were lysed with radio-immunoprecipitation assay (RIPA) lysis buffer (Beyotime Institute of Biotechnology, Haimen, China) to extract total proteins and nucleoproteins as previously described (Maguire et al. 2011). Protein concentration was determined using a bicinchoninic acid (BCA) protein assay kit (Beyotime). 40 μg total proteins from each sample were separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) and transferred to polyvinylidene fluoride (PVDF) membranes (Millipore, Bedford, MA, USA), which were probed with primary antibodies against the proteins of interest at 4 °C overnight. The primary antibodies against cleaved caspase-3, Bax, Bcl-2, tumor necrosis factor (TNF)-α, interleukin (IL)-6, IL-1β, nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor α (IκBα), and NF-κB were from Santa Cruz (Dallas, TX, USA). Anti-BACE, anti-Aβ (1–42), and anti-p-IκBα antibodies were purchased from Bioss (Beijing, China). The membranes were then incubated with corresponding HRP-conjugated secondary antibodies (1:5,000; Beyotime) at room temperature for 1 h. The signals of the proteins of interest were visualized with the ECL system (7SeaPharmTech, Shanghai, China) followed by exposure to films in a darkroom. The films were scanned and analyzed with Gel-Pro-Analyzer to reveal the densitometry values of the target bands. β-actin and lamin A were used as the internal controls for cytoplasmic proteins and nuclear proteins, respectively, in the experiment for detecting NF-κB nuclear translocation, otherwise, β-actin was the internal control for total cellular proteins.

Statistical Analysis

The data are expressed as the mean ± standard deviation. One-way analysis of variance was used for comparisons between groups. Bonferroni post hoc analysis was performed for multiple comparisons. GraphPad Prism 5.0 was employed for data processing and graph plotting. The differences were considered statistically significant when P < 0.05.

Results

Minocycline Alleviated Sevoflurane-Induced Learning and Memory Impairment in Aged Rats

The cognitive impairment model of aged rats was induced by sevoflurane inhalation. At 24 h post-treatment, the physiological conditions of the rats were examined before MWM tests, and the indicators of arterial blood gas were not affected by sevoflurane or minocycline (Table 1). MWM tests were performed to assess the effects of minocycline on learning and memory ability of the sevoflurane-treated rats. Compared with control group, sevoflurane-treated rats displayed an extended latency and a reduced number of swimming passes above the platform in MWM tests, and the differences were statistically significant (Fig. 1a, b, P < 0.001 and P < 0.001, respectively), indicating an impaired learning and memory ability due to sevoflurane anesthesia. Compared with the rats treated with sevoflurane only, those treated with both minocycline and sevoflurane exhibited a remarkably shortened latency and an increased number of passes above the platform (Fig. 1a, b, P < 0.05). The MWM results indicated that minocycline alleviated sevoflurane-induced learning and memory impairment in aged rats.

Table 1.

Arterial blood gas index

Groups (n = 6) HR (beats/min) MAP (mmHg) PaCO2 (mmHg) SPO2 (mmHg)
Control 382.7 ± 13.5 105.78 ± 5.92 37.95 ± 1.31 97.78 ± 1.13
Minocycline 384.3 ± 19.4 107.68 ± 5.58 37.28 ± 1.18 97.57 ± 1.24
Sevoflurane 390.0 ± 17.1 103.05 ± 6.13 38.43 ± 1.29 97.78 ± 1.10
Minocycline + sevoflurane 389.3 ± 17.5 104.43 ± 6.39 37.53 ± 1.32 97.70 ± 1.10
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Fig. 1.

Fig. 1

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Minocycline alleviated sevoflurane-induced cognitive impairment in aged rats. Learning and memory ability of the sevoflurane-treated rats with and without pre-administration of minocycline was examined with Morris water maze (MWM) tests (n = 6 for each group). a Acquisition tests of MWM were performed to examine rats’ learning ability as indicated by the latency to find the platform; b MWM navigation tests were performed to examine rats’ memory ability of recalling the original position of the platform. All experimental data are expressed as the mean ± SD. Compared with the untreated control group, ***P < 0.001; compared with sevoflurane treatment group, # P < 0.05

Minocycline Mitigated Sevoflurane-Induced Neuronal Apoptosis in Rat Hippocampus

After MWM tests, the rats were sacrificed and the brains were harvested. To investigate the effect of minocycline on neuronal apoptosis in sevoflurane-treated rats, TUNEL assay was performed to detect the apoptotic neurons in the hippocampus. The results revealed that sevoflurane induced degeneration of rat hippocampal neurons, as evidenced by the sparse, irregular arrangement of cells that manifested a pronounced tail-like appearance, whereas minocycline ameliorated sevoflurane-induced neuronal degeneration to a significant degree (Fig. 2a). Statistical analysis on the TUNEL results showed that sevoflurane anesthesia resulted in elevated numbers of TUNEL-positive nuclei in the hippocampal region, which was significantly reduced by minocycline co-treatment (Fig. 2b, P < 0.05), implying that minocycline could attenuate sevoflurane-induced cell apoptosis in the hippocampal region of aged rats. Furthermore, the levels of several apoptosis-related proteins including cleaved caspase-3, Bax, and Bcl-2 (Wang et al. 2004) in the rat hippocampus were examined by Western blot analysis. Sevoflurane inhalation resulted in increased levels of cleaved caspase-3 and Bax in rat hippocampus, which was significantly inhibited by minocycline (Fig. 2c, d, P < 0.01). Meanwhile, minocycline administration reversed sevoflurane-induced downregulation of Bcl-2 in rat hippocampus (Fig. 2d, P < 0.01). In summary, these results indicated that minocycline suppressed sevoflurane-induced apoptosis in the hippocampal neurons of aged rats.

Fig. 2.

Fig. 2

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Minocycline inhibited sevoflurane-induced hippocampal neuronal apoptosis in aged rats. After MWM tests, the rats were sacrificed and the rat brains were harvested. a The brains were fixed, paraffin-embedded, and sectioned, followed by TUNEL assay to detect chromosomal fragmentation in the apoptotic cells. Cell nuclei were stained blue with hematoxylin, whereas fragmented DNA was labeled and turned brownish under an optical microscope; b statistical analysis of the TUNEL results (n = 6 for each group). The apoptosis ratio is calculated as (number of TUNEL-positive nuclei/number of total nuclei) × 100 %; c–e the hippocampi of the experimental rats were isolated and lysed with RIPA to extract total proteins. The levels of cleaved caspase-3, Bax, and Bcl-2 in rat hippocampus were determined by Western blots followed by densitometry analysis using β-actin as the internal control. This figure shows the representative images of all the animals examined, and the data are expressed as the mean ± SD (n = 6 for each group). Compared with control group, **P < 0.01, ***P < 0.01; compared with sevoflurane group, # P < 0.05, ## P < 0.01

Minocycline Inhibited Sevoflurane-Induced Aβ Accumulation

Amyloid precursor protein is present in neurons and can be cleaved to generate Aβ peptide by β-site APP-converting enzyme (BACE) (Lammich et al. 2004; Dong et al. 2011). Aβ deposition is frequently observed in the neurons of Alzheimer’s patients and it is assumed to play a critical role in the neuropathogenesis of the diseases (O’Brien and Wong 2011). Western blot analysis was performed to examine the levels of Aβ and BACE in the hippocampus of sevoflurane-treated rats with and without minocycline pre-administration (Fig. 3). The results revealed that the levels of both of Aβ and BACE were significantly elevated in the hippocampus of sevoflurane-treated rats compared with the untreated rats (P < 0.01 for Aβ, P < 0.001 for BACE), indicating an activated Aβ response to sevoflurane anesthesia. Notably, minocycline reduced sevoflurane-induced elevation of Aβ and BACE in rat hippocampus, compared with the rats treated with sevoflurane alone (P < 0.05 and P < 0.01, respectively). Thus, these results indicated that minocycline suppressed sevoflurane-induced upregulation of BACE and resultant Aβ accumulation in rat hippocampus.

Fig. 3.

Fig. 3

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Minocycline inhibited sevoflurane-induced Aβ accumulation in rat hippocampus. The levels of Aβ and BACE in rat hippocampus were revealed by Western blots. Densitometry analysis was performed using β-actin as the internal control. Figure shows the representative images of all the experimental rats, and the values are expressed as the mean ± SD (n = 6 for each group). Compared with control group, **P < 0.01, ***P < 0.001; compared with sevoflurane group, # P < 0.05, ## P < 0.01

Minocycline Suppressed Sevoflurane-Induced Inflammation in the Hippocampus of Aged Rats

To investigate the effects of minocycline on sevoflurane-induced neuroinflammation, Western blot analysis was performed to detect the levels of inflammatory cytokines including TNF-α, IL-1β, and IL-6 in rat hippocampus (Fig. 4). Compared with the untreated rats, the levels of TNF-α, IL-1β, and IL-6 in the hippocampus of sevoflurane-treated rats were all markedly elevated (P < 0.01, P < 0.01 and P < 0.01, respectively) and such elevation was significantly suppressed by minocycline (P < 0.05, P < 0.01 and P < 0.01, respectively). Minocycline inhibited sevoflurane-induced upregulation of inflammatory cytokines in the rat hippocampus, suggesting that sevoflurane-induced neuroinflammation could be attenuated by minocycline.

Fig. 4.

Fig. 4

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Minocycline suppressed sevoflurane-induced elevation of inflammatory cytokines in the hippocampus of aged rats. The levels of TNF-α, IL-1β, and IL-6 in rat hippocampus were examined by Western blot analysis. Densitometry analysis was performed using β-actin as the internal control. This figure shows the representative images of all the experimental rats, and the values are expressed as the mean ± SD (n = 6 for each group). Compared with control group, **P < 0.01; compared with sevoflurane group, # P < 0.05, ## P < 0.01

Minocycline Inhibited Sevoflurane-Induced Activation of the NF-κB Signaling Pathway in the Hippocampus of Aged Rats

NF-κB signaling pathway is one of the key pathways mediating inflammation and stress responses in the cells (Abdullah et al. 2013). Proteolysis and phosphorylation of IκBα as well as NF-κB nuclear translocation are the major events for the activation of the NF-κB signaling (Huang et al. 2013; Nguyen et al. 2014). To elucidate whether minocycline attenuated sevoflurane-induced neuronal apoptosis and inflammation via NF-κB signaling pathway, the levels of full-length IκBα and phosphorylated form p-IκBα, as well as nuclear and cytoplasmic levels of NF-κB in rat hippocampus were determined by Western blot analysis. The levels of the active form p-IκBα and nuclear NF-κB were significantly increased in the hippocampus of sevoflurane-treated rats (Fig. 5a, c, P < 0.01), resulting in reduced levels of full-length IκBα and cytoplasmic NF-κB, compared with the untreated control (Fig. 5b, d, P < 0.01, P < 0.05), indicating that NF-κB signaling pathway was activated by sevoflurane anesthesia. However, sevoflurane-induced IκBα phosphorylation and NF-κB nuclear translocation were inhibited by minocycline to a significant extent. Hence, these results suggest that the minocycline inhibited sevoflurane-induced activation of the NF-κB signaling pathway in the hippocampus of aged rats.

Fig. 5.

Fig. 5

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Minocycline inhibited sevoflurane-induced activation of the NF-κB signaling pathway in the hippocampus of aged rats. The levels of p-IκBα, full-length IκBα, nuclear NF-κB, and cytoplasmic NF-κB in rat hippocampus were examined by Western blot analysis. Densitometry analysis was performed using β-actin as the internal control for total proteins in a and b, whereas β-actin and lamin A were used as the internal controls for cytoplasmic proteins and nuclear proteins, respectively, in c and d. This figure shows the representative images of all the experimental rats, and the values are expressed as the mean ± SD (n = 6 for each group). Compared with control group, *P < 0.05, **P < 0.01; compared with sevoflurane group, # P < 0.05, ## P < 0.01

Discussion

Preclinical studies have shown that sevoflurane, one of the most commonly used inhalational anesthetics, could induce widespread cerebral neuronal apoptosis and cognitive impairment (Jevtovic-Todorovic et al. 2003). Moreover, it has been reported that sevoflurane could induce neuroinflammation and deposition of Aβ in the hippocampus (Liu et al. 2013). In the present study, aged rats that were exposed to 2 % sevoflurane for 5 h displayed cognitive impairment and neurodegenerative symptoms including apoptosis, inflammation, and Aβ accumulation in the hippocampus. Minocycline administration significantly alleviated sevoflurane-induced cognitive impairment, hippocampal neurodegeneration, inflammation, and Aβ deposition, suggesting that minocycline could be a potent agent to counteract cognitive impairment and neurotoxicity induced by sevoflurane anesthesia. It has been reported that long-term anesthesia may cause hypoxia in the brain, therefore damaging the neurons and resulting in cognitive impairment (Kong et al. 2013). However, in our study, the indicators of arterial blood gas were not affected by sevoflurane or minocycline, implying that sevoflurane-induced neurotoxicity was not due to cerebral hypoxia. In fact, we found that sevoflurane anesthesia led to activation of NF-κB signaling, which was suppressed by minocycline, suggesting that NF-κB signaling pathway may be an important mechanism accounting for sevoflurane-induced neurotoxicity as well as the neuroprotective effects of minocycline.

MWM, which is a well-established test to evaluate learning and memory performance in animals (Blokland et al. 2004), was employed to study the effects of minocycline on sevoflurane-induced cognitive impairment in aged rats. The differences in the escape latency and number of passes over the original platform location revealed that sevoflurane compromised the animals’ learning and memory ability, which was effectively improved by the administration of minocycline. We further investigated the molecular mechanism for minocycline-mediated neuroprotection. Western blot analysis showed that minocycline counteracted sevoflurane-induced elevation of the anti-apoptotic protein Bcl-2, and reduction of the pro-apoptotic proteins Bax and cleaved caspase-3 in rat hippocampus, implying that minocycline significantly mitigated sevoflurane-induced apoptosis in the hippocampal neurons, which was consistent with results of TUNEL assays. Minocycline also inhibited sevoflurane-induced upregulation of BACE, resulting in suppression of β-secretory pathway of APP and ultimate generation of Aβ, indicating that minocycline inhibited sevoflurane-induced Aβ accumulation in the hippocampus of aged rats. In addition, minocycline suppressed sevoflurane-induced elevation of pro-inflammatory cytokines including TNF-α, IL-1β, and IL-6 in rat hippocampus. It is generally believed that pro-inflammatory cytokines in the hippocampus are secreted by activated microglials, which are key mediators for neuroinflammation (King et al. 2001; Streit 2004). Thus, these results suggest that minocycline may attenuate sevoflurane-induced microglial activation and the consequent neuroinflammation in the hippocampus of aged rats. Overall, the above results demonstrate the neuroprotective effects of minocycline in sevoflurane-induced neurotoxicity by suppressing sevoflurane-induced neuronal apoptosis, Aβ accumulation, and inflammation in the hippocampus. These data are consistent with the work by Kong et al. on the neuroprotective effects of minocycline on isoflurane-induced neural injury in aged rats (Kong et al. 2013) and also confirms the neuroprotective mechanisms of minocycline including promoting neuronal survival (Huang et al. 2010), inhibiting caspase cascades and microglial-mediated neuroinflammation (Chen et al. 2000), and diminishing Aβ deposition in hippocampal neurons (Seabrook et al. 2006). In addition, inhalational anesthetics have been reported to induce cell injury by disrupting intracellular calcium homeostasis (Yang et al. 2008), and minocycline is able to target mitochondria and maintain intracellular calcium homeostasis (Fernandez-Gomez et al. 2005; Garcia-Martinez et al. 2010), thereby exerting neuroprotective effects. Hence, the involvement of minocycline in intracellular calcium homeostasis of hippocampal neurons may be also involved in the neuroprotective actions of minocycline against sevoflurane anesthesia.

In addition to the aforementioned roles of minocycline in neuroprotection, in the present study, we bring new insights into NF-κB signaling pathway as a critical molecular mechanism in anesthesia-induced cognitive impairment and in minocycline-mediated neuroprotection. NF-κB is a homologous or heterologous dimer consisting of polypeptide chain p50 and p65. The activity of NF-κB is inhibited by IκB, which binds to NF-κB dimer to form a trimer that is stably present in the cytoplasm. In response to an external stimulus, IκB is phosphorylated, resulting in its dissociation from the trimer. Subsequently, NF-κB dimmers translocate from cytoplasm into nucleus to activate transcription of downstream genes that play critical roles in inflammation and apoptosis (Yang et al. 2007b; Gilmore and Wolenski 2012). NF-κB signaling has been implicated to play a part in cognitive deficits in animal models (Mattson and Camandola 2001; Camandola and Mattson 2007), thus we further investigated whether minocycline counteracted sevoflurane-induced cognitive impairment and neurotoxicity via NF-κB signaling pathway. Our results revealed that sevoflurane induced phosphorylation of IκB, resulting in NF-κB nuclear translocation and activation of transcription of downstream genes, whereas minocycline significantly inhibited sevoflurane-induced activation of NF-κB signaling pathway in rat hippocampus. These data suggest that NF-κB signaling pathway may play a critical role in sevoflurane-induced cognitive impairment and neural injury, and it might serve as an important mechanism accounting for the neuroprotective effects of minocycline.

Alzheimer’s disease is characterized by increased Aβ deposition, extracellular senile plaques, intracellular neurofibrillary tangles, and massive neuronal loss in the brain (Blennow et al. 2006). Cognitive impairment is commonly a transition stage between normal aging and pathogenesis of Alzheimer’s disease, and POCD has been reported to be a risk factor for Alzheimer’s disease in elderly surgery patients (Steinmetz et al. 2009; Bittner et al. 2011). In the present study, we found that minocycline could mitigate sevoflurane-induced cognitive impairment accompanying with downregulation of BACE and reduction of Aβ in rat hippocampus, providing new evidence for the potential beneficial effects of minocycline on Alzheimer’s disease (Choi et al. 2007).

Minocycline has long been proposed to treat POCD and neurodegenerative diseases (Blum et al. 2004; Fan et al. 2011), and our study reinforces the clinical potential of minocycline in POCD prevention/treatment. In humans, long-term treatment with minocycline at doses of up to 200 mg/day is generally safe and well tolerated as demonstrated by tolerability tests and clinical trials in rheumatoid arthritis, acne vulgaris, and Huntington’s disease (Blum et al. 2004). In some rare cases, minocycline treatment can cause some minor side effects such as digestive disorders, staining of teeth, or benign intracranial hypertension, but the side effects are mostly reversible upon termination of use (Donnet et al. 1992; Shapiro et al. 1997; Good and Hussey 2003). Hence, minocycline is a safe compound and can be further evaluated as a promising agent to alleviate POCD in clinical studies.

In summary, our study reveals that minocycline displays neuroprotective properties against sevoflurane-induced learning and memory impairment in aged rats and that this effect correlates with minocycline-mediated inhibition of sevoflurane-induced apoptosis, inflammation, Aβ accumulation, and activation of NF-κB signaling pathway in rat hippocampus. Our data suggest that minocycline may counteract sevoflurane-induced cognitive impairment and neurotoxicity through NF-κB signaling pathway, and that minocycline has potential clinical values as a neuroprotective agent to prevent or treat POCD.

Acknowledgments

This study was supported by grants from the National Natural Science Foundation of China (Nos. 81473285, 81101402, and 81171782).

Conflict of interest

The authors declare that they have no conflict of interest.

Ethical approval

All applicable international, national, and institutional guidelines for the care and use of animals were followed. All procedures performed in studies involving animals were approved by the Animal Ethics Committee of China Medical University and in accordance with the ethical standards of the institution.

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