Minocycline mitigates isoflurane-induced cognitive impairment in aged rats

brain research 1496 (2013) 84–93

Available online at www.sciencedirect.com

www.elsevier.com/locate/brainres

Research Report

Minocycline mitigates isoflurane-induced cognitive impairment in aged rats Shi-Yong Lia, Li-Xia Xiab, Yi-Lin Zhaoa, Liu Yanga, Ye-Lin Chena, Jin-Tao Wanga, Ai-Lin Luoa, a Department of Anesthesiology, Tongji Hospital of Tongji Medical College, Huazhong University of Science and Technology, 1095 JieFang Avenue, Wuhan, Hubei 430030, China b Operating Room, Tongji Hospital of Tongji Medical College, Huazhong University of Science and Technology, 1095 JieFang Avenue, Wuhan, Hubei 430030, China

ar t ic l e in f o

abs tra ct

Article history:

Postoperative cognitive dysfunction (POCD) is a severe neurological sequela that occurs in

Accepted 6 December 2012

individuals who have undergone anesthesia and surgery, especially in the geriatric surgical

Available online 25 December 2012

population. Although it is known that isoflurane exposure impairs cognitive function in

Keywords:

aged rodents, there are few clinical interventions for the prophylaxis and treatment of this

Minocycline

disorder. Minocycline, a derivative of tetracycline, produces neuroprotection from several

Isoflurane

neurodegenerative diseases. Therefore, we set out to investigate the effects of minocycline

Postoperative cognitive dysfunction

pretreatment on isoflurane-induced cognitive impairment in aged rats. We found that

TNF-a

pretreatment with minocycline remarkably alleviated isoflurane-induced cognitive dysfunction and inhibited the isoflurane-induced over expression of TNF-a, IL-1b, and IL-6, possibly by inhibiting the degradation of IkBa. In addition, minocycline downregulated the isoflurane-induced increase in the protein levels of cleaved caspase 3 and bax, and upregulated the bcl-2 protein level. These findings highlight the beneficial role of minocycline in preventing isoflurane-induced cognitive impairment and suggested that minocycline can be used as a clinical treatment to mitigate the cognitive impairment induced by isoflurane in elderly patients. & 2013 Elsevier B.V. All rights reserved.

1.

Introduction

Postoperative cognitive dysfunction (POCD) is characterized by a continuous deterioration of cognitive performance after anesthesia and surgery, as evaluated by preoperative and postoperative cognitive testing (Moller et al., 1998; Rasmussen, 1998). It is reported that at the time of discharge from the Abbreviations: AD,

Alzheimer’s disease; BBB,

polypeptide gene enhancer in B-cells; NF-kB,

hospital, 41.4% of elderly patients (60 years or older) were subjected to POCD after non-cardiac surgery (Monk et al., 2008; Steinmetz et al., 2009). Although perioperative morbidity and mortality have been dramatically reduced over the past decades, little progress has been made in alleviating the prevalence of POCD, which imposes a serious burden on quality of life, as well as on healthcare costs.

blood brain barrier; IL,

interleukin; IkB,

inhibitor of nuclear factor of kappa light

nuclear factor kappa-light-chain-enhancer of activated B-cells; POCD,

cognitive dysfunction; TNF, tumor necrosis factor Corresponding author. Fax: þ86 27 83665480. E-mail address: [email protected] (A.-L. Luo). 0006-8993/$ - see front matter & 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.brainres.2012.12.025

postoperative

brain research 1496 (2013) 84–93

85

Although the etiology of POCD remains elusive, factors such as advanced age and duration of anesthesia have been considered as major risk factors (Moller et al., 1998; Monk et al., 2008; Ramaiah and Lam, 2009). Some studies suggest that there is no difference in the incidence of POCD attributed to general anesthesia vs. regional anesthesia (Newman et al., 2007). Nevertheless, exposure to general anesthetics remains a cardinal cause of POCD (Lin et al., 2012; Mawhinney et al., 2012; Su et al., 2011; Wan et al., 2007). The exact role of general anesthetics in POCD is yet to be fully elucidated, but extensive information gained over the past decade indicates that the excessive release of proinflammatory cytokines, including tumor necrosis factor (TNF)-a, interleukin (IL)-1b and IL-6, is involved in cognitive impairment after surgery and anesthesia (Cibelli et al., 2010; Lin et al., 2012; Lucas et al., 2006; Terrando et al., 2010; Wan et al., 2010, 2007; Wu et al., 2012). The degradation of the inhibitory nuclear factor kappa light polypeptide gene enhancer in B-cells (IkB)a is associated with neuroinflammation including nuclear factor of kappa light polypeptide gene enhancer in B-cells (NF-kB) activation and proinflammatory cytokines production (Nikodemova et al., 2006). Furthermore, overproduction of TNF-a, IL-b and IL-6 can impair neuronal cells function including impairment of synaptic plasticity, apoptosis (Lynch and Lynch, 2002; Shaftel et al., 2008; Sheng et al., 2005; Wei et al., 2011). A burgeoning series of studies demonstrate that isofluraneinduced hippocampal neuronal apoptosis is associated with the hippocampus-dependent memory deficit (Lin and Zuo, 2011; Xie et al., 2006; Zhang et al., 2012). Our previous results also demonstrated that isoflurane exposure induces neuronal apoptosis (Zhao et al., 2011a, 2011b) and increases the secretion of proinflammatory cytokines in the hippocampus of neonatal rats (Shi et al., 2010). Theoretically, drugs with anti-neuroinflammatory and anti-neuroapoptotic properties could be effective, at least in part, inpreventing the cognitive dysfunction that occurs after general anesthesia. Minocycline is a tetracycline derivative that easily crosses the blood brain barrier (BBB) (Arvin et al., 2002; Yrjanheikki et al., 1999). Accumulating evidence suggests that the neuroprotective effect of minocycline is mainly caused by the inhibition of inflammation and neuroapoptosis (Choi et al., 2007; Lucas et al., 2006; Tikka et al., 2001). An increasing number of studies have revealed that minocycline improves cognitive function in neurodegenerative diseases such as Alzheimer’s disease, Huntington’s disease and Parkinson’s disease (Choi et al., 2007; Thomas et al., 2003; Tikka et al., 2001). Hence, we hypothesize that pretreatment with minocycline could alleviate the cognitive impairment that is triggered by isoflurane in aged rats.

reduced mean blood pressure (Fig. 1A) and heart rate (Fig. 1B), but the difference was not statistically significant (P ¼ 0.45). To determine if isoflurane anesthesia caused hypoxia, arterial blood was drawn by cardiac puncture and arterial gas analysis was performed immediately after the anesthesia ended. As shown in Table 1, PaCO2, PaO2, pH, blood glucose and arterial oxygen saturation (SaO2) did not change significantly.

2.

Fig. 1 – Isoflurane anesthesia had no significant effect on mean blood pressure and heart rate. (A) Mean blood pressure was relatively stable in both the isoflurane untreated group and the isoflurane treated group. (B) Heart rate did not change significantly in isoflurane untreated rats or isoflurane treated rats. The isoflurane untreated group included the CON and MINO groups, and the isoflurane treated group comprised the ISO and MINOþISO groups. The results are presented as the mean7SEM (n ¼30).

Results

2.1. Isoflurane anesthesia did not induce circulatory or respiratory distress To exclude the possibility that isoflurane depressed the circulatory and respiratory systems, mean blood pressure and heart rate were continuously monitored and were recorded hourly. As shown in Fig. 1, isoflurane anesthesia

2.2. Minocycline pretreatment improved cognitive function after isoflurane exposure To determine the effect of minocycline on cognitive function after isoflurane anesthesia, the Morris water maze (MWM) was used to assess learning and memory. As shown in Fig. 2A, both the repeated factor (training days) and the over-group factor significantly affected the latency of the rats to locate the platform. However, no interactive effect between

86

brain research 1496 (2013) 84–93

Table 1 – Effect of isoflurane exposure on physiological parameters of arterial blood gas analysis. ABG

CON

ISO

MINOþISO

MINO

pH PaCO2 (mm Hg) PaO2 (mm Hg) Glucose (mmol/l) SaO2 (%)

7.3170.05 36.274.3 108714 4.570.4 9971

7.3270.06 36.172.7 106713 4.470.7 9770.6

7.3570.06 39.273.6 10179 3.970.7 9870.9

7.3670.04 40.772.1 10578 4.470.5 9970.8

pH, PaCO2, PaO2, glucose and SaO2 levels did not differ significantly among the four groups. The results are presented as the mean7SEM (n ¼ 5).

group and training days was found (P ¼0.68). On the third and fourth training days, rats in the minocyclineþisofluranegroup (MINOþISO) spent less time locating the platform (Day 3, Po0.001; Day 4, P ¼0.007, MINOþISO vs. ISO). There was no significant difference in the latency between the control (CON) and MINO groups (P¼ 0.72). In the probe trial, the percentage time of rats in the MINOþISO group spent in the target quadrant was much greater than the rats in the ISO group (Po0.001) (Fig. 2B). There was no significant difference between the MINOþISO and CON groups (P¼ 0.55).

2.3. Minocycline reduced the isoflurane-induced upregulation of inflammatory cytokine levels in the hippocampus The levels of TNF-a, IL-1b and IL-6 detected at a series of time points after anesthesia were respectively shown in Fig. 3A, B and C. After isoflurane exposure the expression of TNF-a, IL-1b and IL-6 increased significantly immediately, peaked 3 h after isoflurane exposure; and the level of IL-1b (Fig. 3B) and IL-6 (Fig. 3C) persisted till 6 h after anesthesia, then fell to baseline levels 12 h after anesthesia; but the increase of TNF-a (Fig. 3A) lasted to 12 h and fell to baseline 24 h after anesthesia. When rats were pretreated with minocycline, the elevated levels of TNF-a, IL-1b and IL-6 in the hippocampus were reversed. Sole minocycline treatment did not change the expression of TNF-a, IL-1b and IL-6 at all detected time points. To investigate the mechanism involved in the suppression of proinflammatory cytokines by minocycline, the level of IkBa protein was detected by western blot. As shown in Fig. 3D, isoflurane markedly decreased the expression of IkBa from the end of anesthesia to 6 h after anesthesia (0 h: Po0.001; 3 h: Po0.001; 6 h: P ¼0.002), which was reversed by treatment with MINO. Sole intraperitoneal injection of minocycline did not affect the protein levels of IkBa at any time point after anesthesia.

2.4. Minocycline decreased the levels of TNF-a mRNA and protein in vitro To determine whether minocycline could inhibit the secretion of neuronal TNF-a that was induced by isoflurane, we assayed both the mRNA and protein levels of TNF-a in hippocampal neurons in vitro. In line with previous studies, isoflurane increased TNF-a expression at both the mRNA and protein levels (Po0.05). Pretreatment with minocycline 30 min before isoflurane exposure resulted in the downregulation of TNF-a

Fig. 2 – Minocycline pretreatment mitigated the isofluraneinduced spatial memory impairment. (A) The effect of minocycline pretreatment on average latency to reach the platform in the spatial acquisition trials. (B) The effect of minocycline pretreatment on the percentage of time spent in the target quadrant. The results are presented as the mean7SEM (n ¼10). Po0.05 MINOþISO vs. ISO; #Po0.05 ISO vs. CON.

mRNA (Fig. 4A) and TNF-a protein (Fig. 4B) (Po0.05, MINOþISO vs. ISO). To further explore the mechanisms involved in the inhibition of TNF-a expression, we determined the protein level of IkBa. As shown in Fig. 4C, minocycline increased the level of IkBa protein, which was reduced by isoflurane (Po0.001, MINOþISO vs. ISO).

brain research 1496 (2013) 84–93

87

Fig. 3 – The effect of minocycline on the expression of TNF-a, IL-1b, IL-6 and IjBa in hippocampus. (A)–(C) respectively showed that minocycline decreased the expression of TNF-a, IL-1b and IL-6 after isoflurane exposure in aged rats. (D) Quantification of western blot results showed that minocycline increased the expression of IjBa after isoflurane exposure in aged rats. The results are presented as the mean7SEM (n ¼ 5). Po0.05 MINOþISO vs. ISO; #Po0.05 ISO vs. CON.

2.5. Minocycline decreased isoflurane-mediated neuronal apoptosis in the hippocampi of aged rats To evaluate the effect of minocycline on isoflurane-induced neuroapoptosis in the hippocampus, we set out to examine the activity of caspase 3 and the protein expression of cleaved caspase 3. As shown in Fig. 5, isoflurane activated caspase 3 (Fig. 5A, B) (caspase 3 activity: Po0.001; cleaved caspase 3: Po0.001). To further investigate the mechanisms involved in the anti-neuroapoptotic properties of minocycline, we revealed the expression of bax and bcl-2 by western blot. It was observed that isoflurane upregulated the expression of bax (Fig. 5C) (P¼0.006) and reduced the expression of bcl-2 (Fig. 5D) (P¼0.004). However, pretreatment with minocycline inhibited the activation of caspase 3 (caspase 3 activity: Po0.001; cleaved caspase 3: Po0.001), decreased the expression of bax (Po0.001), and increased the expression of bcl-2 (P¼0.027) as compared to the ISO group.

3.

Discussion

Although the neurobiological basis of POCD remains unclear, it is has been determined that POCD is age-dependent by clinical

trials (Monk et al., 2008) and experimental models (Mawhinney et al., 2012; Stratmann et al., 2009; Su et al., 2011; Zhang et al., 2012). In addition, a recent meta-analysis revealed that general anesthesia is a possible cause of POCD (Mason et al., 2010), despite previous reports which suggest that the incidence of POCD in general anesthesia compared to regional anesthesia is not significantly different (Rasmussen et al., 2003; WilliamsRusso et al., 1995). As the global population ages, the number of elderly patients who are subjected to anesthesia and surgery is increasing. We therefore sought to investigate the effect of minocycline on isoflurane-induced cognitive disorder. In present study, we found that pretreatment with minocycline alleviated cognitive impairment in aged rats exposed to 1.4% isoflurane for 6 h. The protective role of minocycline was associated with significant suppression of the excessive release of proinflammatory cytokines and a marked reduction of neuroapoptosis in the hippocampus. No significant changes of basic physiological parameters were observed in all groups, which implying that the side effect of isoflurane on the circulatory and respiratory systems did not significantly influence the results gained in present study, although heart rate and mean blood pressure were slightly inhibited. It has been demonstrated that hippocampal neuroinflammation triggered by increases in TNF-a and IL-1b underlies

88

brain research 1496 (2013) 84–93

Fig. 4 – The effect of minocycline on TNF-a production in hippocampal neurons. (A) Pretreatment with minocycline led to a decline in TNF-a mRNA levels that were upregulated by isoflurane exposure. (B) Pretreatment with minocycline led to a decrease in the levels of TNF-a protein that were upregulated by isoflurane exposure. (C) Pretreatment with minocycline increased the level of IjBa protein that was reduced by isoflurane exposure. The results are presented as the mean7SEM (n¼ 5). Po0.05 MINOþISO vs. ISO; #Po0.05 ISO vs. CON.

cognitive deficits, while the blockade of TNF-a and IL-1b improves cognitive function (Bluthe et al., 1995; Cibelli et al., 2010; Kent et al., 1992; Terrando et al., 2010). Consistent with previous studies (Vizcaychipi et al., 2011; Wu et al., 2012), we found that isoflurane elevated the levels of TNF-a, IL-1b and IL-6 in vivo and resulted in the induction of neuroinflammatory processes. However, an elaborate research demonstrates isoflurane does not induce increase of IL-1b and IL-6 in the hippocampi of nearly three-month-old mice (Cibelli et al., 2010). Several other studies using different protocols of anesthesia also display that anesthesia does not change the level of proinflammatory cytokines (Rosczyk et al., 2008; Wan et al., 2007). To our knowledge, the different age of used animal might be the principal reason as it is reported that increasing age is an independent risk factor of POCD (Monk et al., 2008). Furthermore, the isoflurane concentration and anesthesia duration should be taken into account. Previously, surgery-induced secretion of proinflammatory cytokines were glial-derived (Rosczyk et al., 2008; Terrando et al., 2010; Wan et al., 2007), but in our results showed isoflurane-modulated TNF-a increase derived from neuron. Two other studies support the result that production of TNF-a induced by neurotoxins is from neurons (Takahashi et al., 2008; Wu et al., 2012). This inconsistency is probably due to that surgery-stimulated cytokines originated from peripheral immune system and then activate the neuroinflammatory response in hippocampus (Terrando et al., 2010), while

isoflurane can readily spread to the brain and directly target the neurons. It seems that the neuroflammatory response is activated by sole anesthesia or surgery in different pathways. Of note, proinflammatory cytokine likely have bidirectional effect on brain functions in different contexts (Liu et al., 2011; Pickering et al., 2005; Wan et al., 2007; Wei et al., 2011). However, it is confirmed that increased IL-1b in hippocampus is contributed to age-related impairment in long-term potentiation and age-related neuronal apoptosis (Lynch and Lynch, 2002). Although the relationship between isoflurane-induced neuroinflammation and cognitive disorder is undetermined, studies reveal that proinflammatory cytokines are involved in the pathogenesis of neurodegenerative diseases such as AD (Akiyama et al., 2000; Eikelenboom et al., 2002; Rozemuller et al., 2012), which is exacerbated after isoflurane anesthesia (Wei and Xie, 2009; Xie et al., 2007). Taken together, the isoflurane-induced upregulation of TNF-a, IL-1b and IL-6 and the resulting inflammatory response may interrupt cognitive function in aged rats. A recent study verifies that the mitochondrial pathway of neuroapoptosis was responsible for isoflurane-induced cognitive dysfunction in aged mice (Zhang et al., 2012). A number of studies revealed that clinically relevant concentration of isoflurane activates mitochondrial apoptotic pathway and finally increases the levels of cleaved caspase 3 (the active form of caspase) in neurons or in neuroglial lines transfected with amyloid precursor protein (Xie et al., 2006, 2007; Zhang

brain research 1496 (2013) 84–93

89

Fig. 5 – Minocycline alleviated neuroapoptosis in the hippocampus after isoflurane anesthesia. (A) The activity of caspase 3 was suppressed by minocycline pretreatment. The relative levels of cleaved caspase 3 (B), bax (C) and bcl-2 (D) in hippocampi of aged rats, normalized to the internal reference b-actin. The upper immunoblots are representative of cleaved caspase 3, bax, and bcl-2. The results are presented as the mean7SEM (n ¼10). Po0.05 MINOþISO vs. ISO; #Po0.05 ISO vs. CON. clvd casp3 is an abbreviation of cleaved caspase 3.

et al., 2012). In mitochondrial apoptotic pathway, the proapoptotic and anti-apoptotic members of Bcl family initiate the dysfunction of mitochondrial membrane (Gross et al., 1999). In present study, we found that isoflurane activated caspase 3, upregulated bax and downregulated bcl-2. However, neuroleptic general anesthesia (fentanyl–droperidol) decreases the ratio of bcl-2: bax while does not activate caspase 3 in adult rats (Wan et al., 2007). This difference may be owing to using different aging rats and different anesthetics. It is noteworthy that proinflammatory cytokines can induce neuronal apoptosis. It is reported TNF-a modulates caspase 3-dependent and caspase 3-independent apoptosis in neural cells (Alvarez et al., 2011; Huang et al., 2005). In the process of normal aging, increased level of IL-1b and IL-1 receptor is accompanied by elevated activity of caspase 3 in hippocampus (Lynch and Lynch, 2002). Coupled with the results that isoflurane can activate caspase 3, the question comes out whether neuroinflammation and neuroapoptosis are two coinstantaneously distinguished mechanisms or the former is initial while the latter is secondary. This is also the caveat of the present study. Further work need to be done to establish the relevance between isoflurane-induced neuroinflammation and neuroapoptosis.

Suppressing the inflammatory response by minocycline inhibits glial activation and therefore improves cognitive function (Fan et al., 2007; Krady et al., 2005; Seabrook et al., 2006; Yrjanheikki et al., 1999). As aforementioned, our study and other study demonstrate that isoflurane-induced upregulation of TNF-a is neuron-derived. Thus, we investigate whether minocycline can inhibit isoflurane-induced increase of proinflammatory cytokines originating from neurons. Interestingly, we found that pretreatment with minocycline inhibited the neuronal secretion of TNF-a in vivo and in vitro. The mechanism of minocycline on the regulation of TNF-a production seems to be associated with inhibiting the degradation of IkBa. The degradation of IkBa is pivotal for the release of NF-kB. Of interest, minocycline exerts its inhibitory role on NF-kB transcriptional activity by attenuating the degradation of IkBa in microglia (Nikodemova et al., 2006). In the present study, we showed that minocycline restored the isoflurane-induced downregulation of IkBa in aged rats. Similar changes were observed in cultured neurons. Minocycline suppresses the mitochondrial apoptotic pathway by inhibiting the loss of the mitochondrial membrane potential, thus reducing the cytoplasmic level of cytochrome C in animal models of Huntington’s disease (Wang et al.,

90

brain research 1496 (2013) 84–93

2003). It is noted that minocycline induces bcl-2 accumulation in neuronal mitochondria, in which bcl-2 antagonizes pro-apoptotic molecules such as bax (Wang et al., 2003; Wang et al., 2004; Zhang et al., 2012). According to these results, it is indicated that the inhibition of hippocampal neuroapoptosis may be another mechanism involved in the neuroprotective properties of minocycline against isoflurane insult in aged rats. In conclusion, minocycline alleviated the impairment in cognitive function that was caused by clinical concentrations of isoflurane in aged rats. Minocycline was involved in the suppression of the neuroinflammatory response and apoptosis in the hippocampus. Considering the inexpensive cost, the good safety record and the high BBB permeability, minocycline is a potential candidate for the clinical prophylaxis and treatment of POCD.

4.

Experimental procedures

4.1.

Animals and drug administration

All animal experiments were approved by the Ethical Committee on Animal Experimentation of Tongji Medical College, Huazhong University of Science and Technology, China. The present study used 20-month-old male Sprague Dawley rats, weighing 350–400 g, which were purchased from the Center of Experimental Animal (Tongji Medical College) and raised under standard laboratory conditions (room temperature: 2272 1C, relative humidity: 6075%,12 h light/dark cycle). Standard rodent food and water were available ad libitum. Rats were randomly assigned into four groups: control (CON), isoflurane (ISO), minocyclineþisoflurane (MINOþISO) and minocycline (MINO) (n¼ 40 rats per group). Rats in the MINO and MINOþISO groups received a dose of 50 mg/kg minocycline (Sigma, St. Louis, MO) by intraperitoneal injection 12 h before exposure to isoflurane anesthesia. Rats in the CON and ISO groups were intraperitoneally injected with an identical volume of saline. Rats in the CON and MINO groups were exposed to vehicle gas (30% oxygen and 70% nitrogen) for 6 h, and rats in the ISO and MINOþISO groups were exposed to 1.4% isoflurane for 6 h. Anesthetic exposure was performed in anesthesiainduction chambers kept in a homoeothermic incubator to maintain the environmental temperature at 37 1C Rats were exposed to 1.4% isoflurane for 6 h through a calibrated isoflurane vaporizer, using 30% oxygen and 70% nitrogen as the vehicle gas, or just to the vehicle gas at the same flow rate (Su et al., 2011). Two liters of total gas flow were used to ascertain a steady state of anesthetic gas and prevent the accumulation of expired carbon dioxide in the chamber. An infrared probe (OhmedaS/5 Compact, Datex-Ohmeda, Louisville, CO) was adopted to continuously monitor the concentrations of oxygen, carbon dioxide and isoflurane in the exhalant gas. Rats were visually inspected for respiratory effort and skin color. At the end of anesthesia, five rats in each group were randomized to dynamically assess the expression of TNF-a, IL-6 and IL-1b (0 h, 3 h, 6 h, 12 h and 24 h after isoflurane exposure). Arterial gas analysis was performed in the rats

used to detect proinflammtory cytokines 0 h after anesthesia. Half of the hippocampus was used for the detection of TNF-a, IL-6 and IL-1b by ELISA and the other half was used for extraction of total protein to detect the expression of IkBa. Two weeks after isoflurane exposure, rats underwent MWM testing to examine their spatial memory abilities. After MWM testing, the rats were decapitated and the hippocampi were dissociated for evaluation of caspase 3 activity and the protein levels of cleaved caspase 3, bax, and bcl-2.

4.2.

Primary neuron culture and drug administration

Pregnant rats were purchased from the Center of Experimental Animal of Tongji Medical College. Rats with a gestation stage of Day 18 were killed with carbon dioxide and a Cesarean section was performed. The hippocampi were dissected from the embryonic brains for neuron culture. Primary culture of the hippocampal neurons was performed as described in our previous studies (Xiang et al., 2009; Zhao et al., 2011a, 2011b). Ten days after the harvest, the neurons were divided into four groups: CON, ISO, MINOþISO and MINO. Each group contained ten dishes, five for mRNA extraction and five for the detection of IkBa protein. The minocycline pretreatment protocol was described in previous studies (Choi et al., 2007; Gonzalez et al., 2007). Briefly, minocycline (final concentration of 10 mM) was added to neurons in the MINOþISO and MINO groups 30 min prior to isoflurane or vehicle gas exposure. The TNF-a protein level in the media was assessed by a TNF-a ELISA kit (BioSource International Inc., Camarillo, CA).

4.3.

Arterial gas analysis and hemodynamic monitoring

Mean blood pressure and heart rate were dynamically monitored during anesthesia using a noninvasive blood pressure meter (BP-98 A, Softron, Beijing, China). Five rats in each group were used for arterial gas analysis with an ABL-800FLEX analyzer (Radiometer, Denmark). At the end of anesthesia, arterial blood was drawn by cardiac puncture for arterial blood gas analysis (Jevtovic-Todorovic et al., 2003).

4.4.

Morris water maze test

The MWM trials were performed as previously described (D’Hooge and De Deyn, 2001; Vorhees and Williams, 2006). The apparatus, consisting of a circular pool (120 cm diameter and 50 cm high) containing a hidden platform in a dimly lit room, was employed to train and test the learning and memory of rats. The rats were trained to identify the location of the hidden platform using only distal extra-maze cues attached to the walls of the room. The pool was divided into four equal quadrants and was filled with a water and carbonic ink mixture to a height of 1.5 cm above the top of the black 15-cm-diameter platform. The pool was kept at 20 1C. A video camera was mounted above the pool to track the rats. The experiment was recorded and analyzed using a camera connected to a video recorder and the EthoVision tracking system (Noldus Information Technology, Wageningen, the Netherlands). On the 15th day following isoflurane exposure, the MWM training and testing began. Testing lasted for five days.

brain research 1496 (2013) 84–93

The spatial acquisition trial was performed on the first four days, beginning at 9:00 A.M. every day. At the beginning of each trial, the rat was placed into the water, facing the wall of the pool, into one of the three quadrants that did not contain the platform. Each rat was given 60 s to search and mount the platform, and could remain on the platform for 15 s after it was located. Then, the rat was sent back to its home cages, wiped dry with a towel, and warmed with a heating lamp. Rats that failed to find the platform in 60 s were manually guided to the platform and remained on the platform for 15 s before being returned to cages. The time spent on searching and mounting the platform (latency) was calculated. A probe trial, in which the platform was removed, was given to assess reference memory on the fifth day. Rats were randomly placed into a quadrant that did not contain the platform and were allowed to swim freely for 60 s. The percentage of time spent in the target quadrant was considered an indicator of memory performance.

4.5.

Western blot analysis

The dissected rat hippocampi were homogenized in RIPA buffer (150 mM sodium chloride, Triton X-100, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulfate,50 mM Tris, pH 8.0) containing protease inhibitors (1 mg/ml antipine, 5 mg/ ml pepstatin, 1 mg/ml leupeptin, 1 mg/ml aprotinin) (Beyotime Institute Biotechnology, Haimen, China) and phosphatase inhibitors (1 mM NaF, 0.4 mM Na3VO4, 0.5 mM okadaic acid). After centrifugation at 12,000 g/min for 10 min, the supernatant was harvested. The protein samples were denatured with 5  loading buffer in boiling water for 5 min. The samples (75 mg) were separated on 12% sodium dodecyl sulfate polyacrylamide gels using electrophoresis and were transferred to 0.22 mm nitrocellulose membranes (Millipore, Billerica, MA). The membranes were blocked with 5% milk in Tris-buffered saline with 0.1% Tween 20 (TBST) for 1 h at room temperature and then incubated at 4 1C with the appropriate primary antibody. The primary antibodies were: rabbit anti-cleaved caspase 3 (1:1000, Cell Signaling Technology, Beverly, MA), rabbit anti-IkBa (1:1000, Cell Signaling Technology), mouse anti-bcl-2, anti-bax (1:1000, BD bioscience, MA) or mouse anti-b actin (1:5000, Sigma, St. Louis, MO). After incubation in the primary antibodies, the membranes were rinsed and then incubated for 2 h at room temperature with horseradish peroxidase (HRP)-conjugated secondary antibody (anti-mouse or anti-rabbit (1:7500, Abcam, Cambridge, MA)). Following the species-appropriate HRP-conjugated secondary incubation, detection was performed using SuperSignals West Pico (ThermoScientific, Rockford, IL) and the blots were photographed using Image LabTM Software Systems (BIO-RAD, Hercules, CA). Software Image Lab 3.0 (BIO-RAD) was used to analyze the relative intensity of the bands.

4.6.

Assay of caspase 3 activity

Caspase 3 activity was assayed by the Caspase 3 Colorimetric Assay Kit (R&D Systemss, Minneapolis, MN). The procedure was performed following the supplier’s instructions.

91

4.7. Quantification of TNF-a, IL-6 and IL-1b with enzymelinked immunosorbent assay (ELISA) The protein levels of TNF-a, IL-6 and IL-1b in hippocampal tissues and the protein level of TNF-a in the media of cultured hippocampal neurons were determined by commercially available ELISA kits (BioSource International Inc, Camarillo, CA) following the protocols provided by manufacturer. All samples were assayed in duplicate. The readings were normalized to the amount of standard protein.

4.8. RNA isolation and quantitative real-time polymerase chain reaction Total RNA extraction of hippocampal neurons and the reverse transcription procedure were performed as previously described with minor modifications (Zhao et al., 2011a, 2011b). In brief, the total RNA of neurons was extracted with TRIzols (Invitrogen, Carlsbad, CA) according to the manufacturer’s instructions. One microgram of total RNA was reverse transcribed using random primers and Superscript II Reverse Transcriptase (Takara, Shiga, Japan) in a 20 ml reaction system. Specific primers for TNF-a (forward:50 -AACTGGCAGAGGAGGCG-30 ; and reverse: 50 -CAGAAGAGCGT GGTGGC-30 ), and for the endogenous control GAPDH (forward: 50 -GGCACAGTCAAGGCTGAGAATG-30 ; and reverse: 50 -ATGGTGGTGAAGACGCCAGTA-30 ) were designed and synthesized by Takara (Takara BioTechnology (Dalian), China). ABI Stepone version 2.0 (Applied Biosystems, Foster City, CA) was used to conduct the qRT-PCR using the Power SYBRTM Green PCRMaster Mix (Takara). Each sample was run in triplicate. Five independent polymerase chain reaction amplification experiments were performed for each sample. Data were analyzed using Sequence Detection Software version 3.0 (Applied Biosystems). Relative quantification was performed by means of the 2DDCt method (Livak and Schmittgen, 2001; Schmittgen and Livak, 2008). Data are expressed as fold changes normalized to control groups.

4.9.

Statistical analysis

All data were presented and graphed as the mean7SEM. The Statistical Package for the Social Sciences 16.0 software was used for the statistical analyses. Data acquired from the detection of protein and mRNA were analyzed with an analysis of variance (ANOVA), followed by a least square difference (LSD) multiple comparison test. Data collected from the spatial acquisition trials were analyzed using a repeated measures ANOVA (the different treatments were the between groups factors and time was the repeated measures factor), followed by a post-hoc test to compare four groups. Differences were deemed statistically significant if Po0.05.

Acknowledgments The present work was supported by a grant from the National Natural Science Foundation of China (Nos. 30772086, 30901390, 81271233, and 81200880).

92

brain research 1496 (2013) 84–93

references

Alvarez, S., Blanco, A., Fresno, M., Munoz-Fernandez, M.A., 2011. TNF-alpha contributes to caspase-3 independent apoptosis in neuroblastoma cells: role of NFAT. PLoS One 6, e16100. Akiyama, H., Barger, S., Barnum, S., Bradt, B., Bauer, J., Cole, G.M., Cooper, N.R., Eikelenboom, P., Emmerling, M., Fiebich, B.L., Finch, C.E., Frautschy, S., Griffin, W.S.T., Hampel, H., Hull, M., Landreth, G., Lue, L.F., Mrak, R., Mackenzie, I.R., McGeer, P.L., Banion, O., Pachter, M.K., Pasinetti, J., Plata Salaman, G., Rogers, C., Rydel, J., Shen, R., Streit, Y., Strohmeyer, W., Tooyoma, R., Van Muiswinkel, I., Veerhuis, F.L., Walker, R., Webster, D., Wegrzyniak, S., Wenk, B., Wyss Coray, T., G., 2000. Inflammation and Alzheimer’s disease. Neurobiol. Aging 21, 383–421. Arvin, K.L., Han, B.H., Du, Y., Lin, S.Z., Paul, S.M., Holtzman, D.M., 2002. Minocycline markedly protects the neonatal brain against hypoxic–ischemic injury. Ann. Neurol. 52, 54–61. Bluthe, R.M., Beaudu, C., Kelley, K.W., Dantzer, R., 1995. Differential effects of IL-1ra on sickness behavior and weight loss induced by IL-1 in rats. Brain Res. 677, 171–176. Choi, Y., Kim, H.S., Shin, K.Y., Kim, E.M., Kim, M., Kim, H.S., Park, C.H., Jeong, Y.H., Yoo, J., Lee, J.P., Chang, K.A., Kim, S., Suh, Y.H., 2007. Minocycline attenuates neuronal cell death and improves cognitive impairment in Alzheimer’s disease models. Neuropsychopharmacology 32, 2393–2404. Cibelli, M., Fidalgo, A.R., Terrando, N., Ma, D., Monaco, C., Feldmann, M., Takata, M., Lever, I.J., Nanchahal, J., Fanselow, M.S., Maze, M., 2010. Role of interleukin-1beta in postoperative cognitive dysfunction. Ann. Neurol. 68, 360–368. D’Hooge, R., De Deyn, P.P., 2001. Applications of the Morris water maze in the study of learning and memory. Brain Res. Brain Res. Rev. 36, 60–90. Eikelenboom, P., Bate, C., Van Gool, W.A., Hoozemans, J.J., Rozemuller, J.M., Veerhuis, R., Williams, A., 2002. Neuroinflammation in Alzheimer’s disease and prion disease. Glia 40, 232–239. Fan, R., Xu, F., Previti, M.L., Davis, J., Grande, A.M., Robinson, J.K., Van Nostrand, W.E., 2007. Minocycline reduces microglial activation and improves behavioral deficits in a transgenic model of cerebral microvascular amyloid. J. Neurosci. 27, 3057–3063. Gonzalez, J.C., Egea, J., Del, C.G.M., Fernandez-Gomez, F.J., Sanchez-Prieto, J., Gandia, L., Garcia, A.G., Jordan, J., Hernandez-Guijo, J.M., 2007. Neuroprotectant minocycline depresses glutamatergic neurotransmission and Ca(2þ) signalling in hippocampal neurons. Eur. J. Neurosci. 26, 2481–2495. Gross, A., McDonnell, J.M., Korsmeyer, S.J., 1999. BCL-2 family members and the mitochondria in apoptosis. Genes Dev. 13, 1899–1911. Huang, Y., Erdmann, N., Peng, H., Zhao, Y., Zheng, J., 2005. The role of TNF related apoptosis-inducing ligand in neurodegenerative diseases. Cell Mol. Immunol. 2, 113–122. Jevtovic-Todorovic, V., Hartman, R.E., Izumi, Y., Benshoff, N.D., Dikranian, K., Zorumski, C.F., Olney, J.W., Wozniak, D.F., 2003. Early exposure to common anesthetic agents causes widespread neurodegeneration in the developing rat brain and persistent learning deficits. J. Neurosci. 23, 876–882. Kent, S., Bluthe, R.M., Dantzer, R., Hardwick, A.J., Kelley, K.W., Rothwell, N.J., Vannice, J.L., 1992. Different receptor mechanisms mediate the pyrogenic and behavioral effects of interleukin 1. Proc. Natl. Acad. Sci. USA 89, 9117–9120. Krady, J.K., Basu, A., Allen, C.M., Xu, Y., LaNoue, K.F., Gardner, T.W., Levison, S.W., 2005. Minocycline reduces proinflammatory cytokine expression, microglial activation, and caspase-3

activation in a rodent model of diabetic retinopathy. Diabetes 54, 1559–1565. Lin, D., Cao, L., Wang, Z., Li, J., Washington, J.M., Zuo, Z., 2012. Lidocaine attenuates cognitive impairment after isoflurane anesthesia in old rats. Behav. Brain Res. 228 (2), 319–327. Liu, Z., Qiu, Y.H., Li, B., Ma, S.H., Peng, Y.P., 2011. Neuroprotection of interleukin-6 against NMDA-induced apoptosis and its signal-transduction mechanisms. Neurotoxic. Res. 19, 484–495. Lin, D., Zuo, Z., 2011. Isoflurane induces hippocampal cell injury and cognitive impairments in adult rats. Neuropharmacology 61, 1354–1359. Livak, K.J., Schmittgen, T.D., 2001. Analysis of relative gene expression data using real-time quantitative PCR and the 2(Delta DeltaC(T)) method. Methods 25, 402–408. Lucas, S.M., Rothwell, N.J., Gibson, R.M., 2006. The role of inflammation in CNS injury and disease. Br. J. Pharmacol. 147 (1), S232–S240. Lynch, A.M., Lynch, M.A., 2002. The age-related increase in IL-1 type I receptor in rat hippocampus is coupled with an increase in caspase-3 activation. Eur. J. Neurosci. 15, 1779–1788. Mason, S.E., Noel-Storr, A., Ritchie, C.W., 2010. The impact of general and regional anesthesia on the incidence of postoperative cognitive dysfunction and post-operative delirium: a systematic review with meta-analysis. J. Alzheimers Dis. 22, 67–79. Mawhinney, L.J., de Rivero, V.J., Alonso, O.F., Jimenez, C.A., Furones, C., Moreno, W.J., Lewis, M.C., Dietrich, W.D., Bramlett, H.M., 2012. Isoflurane/nitrous oxide anesthesia induces increases in NMDA receptor subunit NR2B protein expression in the aged rat brain. Brain Res. 1431, 23–34. Moller, J.T., Cluitmans, P., Rasmussen, L.S., Houx, P., Rasmussen, H., Canet, J., Rabbitt, P., Jolles, J., Larsen, K., Hanning, C.D., Langeron, O., Johnson, T., Lauven, P.M., Kristensen, P.A., Biedler, A., van Beem, H., Fraidakis, O., Silverstein, J.H., Beneken, J.E., Gravenstein, J.S., 1998. Long-term postoperative cognitive dysfunction in the elderly ISPOCD1 study. ISPOCD investigators. International study of post-operative cognitive dysfunction. Lancet 351, 857–861. Monk, T.G., Weldon, B.C., Garvan, C.W., Dede, D.E., van der Aa, M.T., Heilman, K.M., Gravenstein, J.S., 2008. Predictors of cognitive dysfunction after major noncardiac surgery. Anesthesiology 108, 18–30. Newman, S., Stygall, J., Hirani, S., Shaefi, S., Maze, M., 2007. Postoperative cognitive dysfunction after noncardiac surgery: a systematic review. Anesthesiology 106, 572–590. Nikodemova, M., Duncan, I.D., Watters, J.J., 2006. Minocycline exerts inhibitory effects on multiple mitogen-activated protein kinases and IkappaBalpha degradation in a stimulusspecific manner in microglia. J. Neurochem. 96, 314–323. Pickering, M., Cumiskey, D., O’Connor, J.J., 2005. Actions of TNFalpha on glutamatergic synaptic transmission in the central nervous system. Exp. Physiol. 90, 663–670. Ramaiah, R., Lam, A.M., 2009. Postoperative cognitive dysfunction in the elderly. Anesthesiol. Clin. 27, 485–496. Rasmussen, L.S., 1998. Defining postoperative cognitive dysfunction. Eur. J. Anaesthesiol. 15, 761–764. Rasmussen, L.S., Johnson, T., Kuipers, H.M., Kristensen, D., Siersma, V.D., Vila, P., Jolles, J., Papaioannou, A., Abildstrom, H., Silverstein, J.H., Bonal, J.A., Raeder, J., Nielsen, I.K., Korttila, K., Munoz, L., Dodds, C., Hanning, C.D., Moller, J.T., 2003. Does anaesthesia cause postoperative cognitive dysfunction? A randomised study of regional versus general anaesthesia in 438 elderly patients. Acta Anaesthesiol. Scand. 47, 260–266. Rosczyk, H.A., Sparkman, N.L., Johnson, R.W., 2008. Neuroinflammation and cognitive function in aged mice following minor surgery. Exp. Gerontol. 43, 840–846.

brain research 1496 (2013) 84–93

Rozemuller, A.J., Jansen, C., Carrano, A., van Haastert, E.S., Hondius, D., van der Vies, S.M., Hoozemans, J.J., 2012. Neuroinflammation and common mechanism in Alzheimer’s disease and prion amyloidosis: amyloid-associated proteins, neuroinflammation and neurofibrillary degeneration. Neurodegener Dis. 10, 301–304. Schmittgen, T.D., Livak, K.J., 2008. Analyzing real-time PCR data by the comparative C(T) method. Nat. Protoc. 3, 1101–1108. Seabrook, T.J., Jiang, L., Maier, M., Lemere, C.A., 2006. Minocycline affects microglia activation, Abeta deposition, and behavior in APP-tg mice. Glia 53, 776–782. Shaftel, S.S., Griffin, W.S., O’Banion, M.K., 2008. The role of interleukin-1 in neuroinflammation and Alzheimer disease: an evolving perspective. J. Neuroinflammation 5, 7. Sheng, W.S., Hu, S., Ni, H.T., Rowen, T.N., Lokensgard, J.R., Peterson, P.K., 2005. TNF-alpha-induced chemokine production and apoptosis in human neural precursor cells. J. Leukocyte Biol. 78, 1233–1241. Shi, Q.Y., Luo, A.L., Li, S.Y., 2010. Effect of Isoflurane on mRNA expression of proinflammatorial cytokines in the hippocampus of immature rats. Chin. J. Anesthesiol. 30 (3), 324–326. Steinmetz, J., Christensen, K.B., Lund, T., Lohse, N., Rasmussen, L.S., 2009. Long-term consequences of postoperative cognitive dysfunction. Anesthesiology 110, 548–555. Stratmann, G., Sall, J.W., May, L.D., Bell, J.S., Magnusson, K.R., Rau, V., Visrodia, K.H., Alvi, R.S., Ku, B., Lee, M.T., Dai, R., 2009. Isoflurane differentially affects neurogenesis and long-term neurocognitive function in 60-day-old and 7-day-old rats. Anesthesiology 110, 834–848. Su, D., Zhao, Y., Wang, B., Xu, H., Li, W., Chen, J., Wang, X., 2011. Isoflurane-induced spatial memory impairment in mice is prevented by the acetylcholinesterase inhibitor donepezil. PLoS One 6, e27632. Takahashi, K., Funata, N., Ikuta, F., Sato, S., 2008. Neuronal apoptosis and inflammatory responses in the central nervous system of a rabbit treated with Shiga toxin-2. J. Neuroinflammation 5, 11. Terrando, N., Monaco, C., Ma, D., Foxwell, B.M., Feldmann, M., Maze, M., 2010. Tumor necrosis factor-alpha triggers a cytokine cascade yielding postoperative cognitive decline. Proc. Natl. Acad. Sci. USA 107, 20518–20522. Thomas, M., Le, W.D., Jankovic, J., 2003. Minocycline and other tetracycline derivatives: a neuroprotective strategy in Parkinson’s disease and Huntington’s disease. Clin. Neuropharmacol. 26, 18–23. Tikka, T., Fiebich, B.L., Goldsteins, G., Keinanen, R., Koistinaho, J., 2001. Minocycline, a tetracycline derivative, is neuroprotective against excitotoxicity by inhibiting activation and proliferation of microglia. J. Neurosci. 21, 2580–2588. Vizcaychipi, M.P., Lloyd, D.G., Wan, Y., Palazzo, M.G., Maze, M., Ma, D., 2011. Xenon pretreatment may prevent early memory decline after isoflurane anesthesia and surgery in mice. PLoS One 6, e26394. Vorhees, C.V., Williams, M.T., 2006. Morris water maze: procedures for assessing spatial and related forms of learning and memory. Nat. Protoc. 1, 848–858. Wan, Y., Xu, J., Meng, F., Bao, Y., Ge, Y., Lobo, N., Vizcaychipi, M.P., Zhang, D., Gentleman, S.M., Maze, M., Ma, D., 2010. Cognitive

93

decline following major surgery is associated with gliosis, beta-amyloid accumulation, and tau phosphorylation in old mice. Crit. Care Med. 38, 2190–2198. Wan, Y., Xu, J., Ma, D., Zeng, Y., Cibelli, M., Maze, M., 2007. Postoperative impairment of cognitive function in rats: a possible role for cytokine-mediated inflammation in the hippocampus. Anesthesiology 106, 436–443. Wang, J., Wei, Q., Wang, C.Y., Hill, W.D., Hess, D.C., Dong, Z., 2004. Minocycline up-regulates Bcl-2 and protects against cell death in mitochondria. J. Biol. Chem. 279, 19948–19954. Wang, X., Zhu, S., Drozda, M., Zhang, W., Stavrovskaya, I.G., Cattaneo, E., Ferrante, R.J., Kristal, B.S., Friedlander, R.M., 2003. Minocycline inhibits caspase-independent and -dependent mitochondrial cell death pathways in models of Huntington’s disease. Proc. Natl. Acad. Sci. USA 100, 10483–10487. Wei, H., Xie, Z., 2009. Anesthesia, calcium homeostasis and Alzheimer’s disease. Curr. Alzheimer Res. 6, 30–35. Wei, H., Zou, H., Sheikh, A.M., Malik, M., Dobkin, C., Brown, W.T., Li, X., 2011. IL-6 is increased in the cerebellum of autistic brain and alters neural cell adhesion, migration and synaptic formation. J. Neuroinflammation 8, 52. Williams-Russo, P., Sharrock, N.E., Mattis, S., Szatrowski, T.P., Charlson, M.E., 1995. Cognitive effects after epidural vs general anesthesia in older adults. A randomized trial. JAMA 274, 44–50. Wu, X., Lu, Y., Dong, Y., Zhang, G., Zhang, Y., Xu, Z., Culley, D.J., Crosby, G., Marcantonio, E.R., Tanzi, R.E., Xie, Z., 2012. The inhalation anesthetic isoflurane increases levels of proinflammatory TNF-alpha, IL-6, and IL-1beta. Neurobiol. Aging 33 (7), 1364–1378. Xiang, Q., Tan, L., Zhao, Y.L., Wang, J.T., Jin, X.G., Luo, A.L., 2009. Isoflurane enhances spontaneous Ca(2þ) oscillations in developing rat hippocampal neurons in vitro. Acta Anaesthesiol. Scand. 53, 765–773. Xie, Z., Dong, Y., Maeda, U., Alfille, P., Culley, D.J., Crosby, G., Tanzi, R.E., 2006. The common inhalation anesthetic isoflurane induces apoptosis and increases amyloid beta protein levels. Anesthesiology 104, 988–994. Xie, Z., Dong, Y., Maeda, U., Moir, R.D., Xia, W., Culley, D.J., Crosby, G., Tanzi, R.E., 2007. The inhalation anesthetic isoflurane induces a vicious cycle of apoptosis and amyloid beta-protein accumulation. J. Neurosci. 27, 1247–1254. Yrjanheikki, J., Tikka, T., Keinanen, R., Goldsteins, G., Chan, P.H., Koistinaho, J., 1999. A tetracycline derivative, minocycline, reduces inflammation and protects against focal cerebral ischemia with a wide therapeutic window. Proc. Natl. Acad. Sci. USA 96, 13496–13500. Zhang, Y., Xu, Z., Wang, H., Dong, Y., Shi, H.N., Culley, D.J., Crosby, G., Marcantonio, E.R., Tanzi, R.E., Xie, Z., 2012. Anesthetics isoflurane and desflurane differently affect mitochondrial function, learning and memory. Ann. Neurol. 71 (5), 687–698. Zhao, Y.L., Xiang, Q., Shi, Q.Y., Li, S.Y., Tan, L., Wang, J.T., Jin, X.G., Luo, A.L., 2011a. GABAergic excitotoxicity injury of the immature hippocampal pyramidal neurons’ exposure to isoflurane. Anesth. Analg. 113, 1152–1160. Zhao, Y., Jin, X., Wang, J., Tan, L., Li, S., Luo, A., 2011b. Isoflurane enhances the expression of cytochrome C by facilitation of NMDA receptor in developing rat hippocampal neurons in vitro. J. Huazhong Univ. Sci. Technol. Med. Sci. 31, 779–783.