- Email: [email protected]
Sevoflurane aggregates cognitive dysfunction and hippocampal oxidative stress induced by β-amyloid in rats
Sevoflurane aggregates cognitive dysfunction and hippocampal oxidative stress induced by β-amyloid in rats Tian Yue, Guo Shanbin, Ma Ling, Wang Yuan, Xu Ying, Zhao Ping PII: DOI: Reference:
S0024-3205(15)30066-7 doi: 10.1016/j.lfs.2015.11.002 LFS 14549
To appear in:
Life Sciences
Received date: Revised date: Accepted date:
12 March 2015 9 October 2015 3 November 2015
Please cite this article as: Yue Tian, Shanbin Guo, Ling Ma, Yuan Wang, Ying Xu, Ping Zhao, Sevoflurane aggregates cognitive dysfunction and hippocampal oxidative stress induced by β-amyloid in rats, Life Sciences (2015), doi: 10.1016/j.lfs.2015.11.002
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT Sevoflurane Aggregates Cognitive Dysfunction and Hippocampal Oxidative Stress
T
Induced by β-amyloid in Rats
IP
Running title: Sevoflurane affects hippocampal function
SC R
Tian Yue1, Guo Shanbin2, Ma Ling1, Wang Yuan1, Xu Ying1, Zhao Ping1*
1Department of Anesthesiology, Shengjing Hospital of China Medical University, Shenyang 110004,
NU
China.
TE
*Corresponding author: Zhao Ping
D
MA
2Department of Pharmacy, Shengjing Hospital of China Medical University, Shenyang 110004, China.
CE P
Department of Anesthesiology, Shengjing Hospital of China Medical University, 36 Sanhao Street,
Tel: None
AC
Shenyang 110004, China.
Fax: None
Email: [email protected]
ACCEPTED MANUSCRIPT Abstract
T
Aims: To investigate the effects of sevoflurane inhalation on β-amyloid (Aβ)-induced cognitive disorders
IP
and hippocampal oxidative stress in rat models.
SC R
Materials and Methods: Cognitive dysfunction is induced by hippocampal injection of Aβ1-40 (10 μg in 2 μl) for 22 days. To explore the effect of sevoflurane inhalation on Aβ1-40 induced cognitive disorder, two
NU
doses of sevoflurane inhalation are used: 1.3% (Aβ+S1) and 2.6% (Aβ+S2). Sham operation (Sham, for
MA
operation control), saline injection (Control, for injection control) and 30% oxygen inhalation after Aβ1-40
D
injection (Aβ+O2, for inhalation control) were used as controls. All rats were further tested in electrical
TE
Y-maze and Morris water maze. Serum S100β levels, hippocampal superoxide dismutase (SOD) activity,
CE P
S100β expression and malonyldialdehyde (MDA) concentrations were further quantified.
Key findings: Rats in Aβ+O2, Aβ+S1 and Aβ+S2 group had lower number of correct actions in the
AC
electrical Y maze task, longer escape latencies, less time exploring the original platform, elevated serum S100β levels, depressed hippocampal SOD activity, S100β expression and higher MDA concentrations
compared to control group (p<0.05). Such difference was not significant between Aβ+S1 and Aβ+O2 rats.
Rats in Aβ+S2 group, however, showed significantly impaired performances compared to Aβ+S1 group
(p<0.05).
Significance: Sevoflurane (2.6%) can aggravate the Aβ-induced cognitive dysfunction, possibly via the
intracerebral oxidative stress response.
Keywords: Sevoflurane inhalation; Amyloid protein; Cognitive dysfunctions; Oxidative stress
ACCEPTED MANUSCRIPT Introduction
T
Alzheimer’s disease (AD) is clinically manifested with advancing hypomnesia and cognitive disorders, and
IP
affecting about 26.6 million people worldwide, making it a major cause of senile dementia [1]. The most
SC R
prominent pathological feature of AD is the occurrence of extracellular senile plaque formed by β-amyloid
proteins deposition [2, 3]. Such accumulation of Aβ oligomers has neurotoxicity and impairs synaptic
NU
functions and memory [4] via stimulating the production of intracellular active oxides and free radicals
MA
such as ROS, thereby increasing the level of lipid peroxidation products malonyldialdehyde (MDA).
D
Meanwhile Aβ can also disrupt cellular innate anti-oxidation system by the inactivation of key enzymes
TE
such as superoxide dismutase (SOD) and glutathione peroxidase GSH-Px [5]. In a word, the oxidative
[6-8].
CE P
stress is the major pathological mechanism underlying AD related neuronal dysfunction and degeneration
AC
Mainly produced in astrocytes of central nervous system (CNS), S100β is a calcium-binding protein that
regulates the growth, proliferation and differentiation of neural glial cells, in addition to intracellular calcium homeostasis. S100β normally cannot penetrate the blood-brain barrier (BBB). When there are acute injuries in CNS, however, S100β may enter the cerebrospinal fluid (CSF) and the general circulation through BBB. Therefore CSF or serum S100β level can be used as an index evaluating the severity of CNS injury [9]. It has been reported that S100β level may elevate in AD patients, suggesting the potential relationship between S100β and AD-related neural injuries [10], although its detailed mechanism remained
unknown.
ACCEPTED MANUSCRIPT As one commonly used inhaled anesthetics, sevoflurane has in vitro neurotoxicity via its facilitation of
T
Aβ oligomerization, but not significant cognitive impairments at moderate concentrations [11-13].
IP
Recently it has been reported that sevoflurane impaired both short-term and long-term cognitive functions
SC R
[14], especially in aged rats [15]. Meanwhile sevoflurane may elevated serum S100β levels [16]. Based on
these evidences, it is possible that sevoflurane may aggravate AD-related memory and learning functions
NU
via the stimulation of S100β release. This study thus aimed to evaluate the effect of sevoflurane on the
MA
cognitive dysfunctions and oxidative stress response in the hippocampus of Aβ-induced AD rats.
D
1. Materials and Methods
TE
1.1. Animals
CE P
A total of 120 male SD rats (body weight between 250 and 300 g) were purchased from the animal
experiment center of China Medical University. All rats were kept in an SPF-grade animal house,
AC
which was kept at 25 °C temperature and 40% relative humidity with food and water provided ad
libitum. Animals were randomly divided into 5 groups including the sham group, control group, amyloid protein group (Aβ+O2), 1.3% sevoflurane group (Aβ+S1) and 2.6% sevoflurane (Aβ+S2). All
experimental protocols have been pre-approved by the ethical committee of using animals in
experiments in China Medical University. 1.2. Aβ1-40 injection and sevoflurane inhalation Following previously established procedures [17], rats were anesthetized by intraperitoneal injection
of 10% chloral hydrate (250 mg/kg) and fixed on the stereotaxic apparatus to locate the bilateral
ACCEPTED MANUSCRIPT hippocampal CA1 regions (coordinates: 3.2 mm posterior, 2.0 mm bilateral and 3.9 mm ventral; all
T
relative to Bregma). The skull was removed to expose the dura, through which a micro-syringe was
IP
inserted vertically into hippocampal CA1 region. 2 µl of Aβ1-40 (Sigma, US), which has been
SC R
re-suspended in 5 µg/µl solutions in sterilized saline, was slowly injected on rats of Aβ+O2, Aβ+S1
and Aβ+S2 groups within 5 min, followed by 5-min retention of the syringe. Gentamicin (20 000 U)
NU
was intraperitoneally injected after the surgery. Rats in the control group received isosmotic saline
MA
injection on bilateral hippocampus. Rats in the sham group received all surgical procedures but no
D
injection.
TE
Sevoflurane was applied using a self-made anesthesia chamber (50 cm X 30 cm X 30 cm), which
CE P
was kept at 37 °C and had two ventilating holes inside: the superior one connected to an anesthesia
pump (flow rate=3 L/min, concentration of O2=30%); the inferior one connected to a gaseous monitor.
AC
22 days after the injection, rats in Aβ+S1 and Aβ+S2 group were received 1.3% and 2.6% of
sevoflurane (Abbott, US) inhalation for 4 h, respectively. These concentrations and time periods were
selected based on our pilot studies showing no respiratory arrest under 2.6% sevoflurane, in addition to
various similar studies. The respiration rhythm and mucosal color of animals were continuously monitored to avoid hypoxia. Rats in C group and Aβ+O2 group received 30% O2 for 4 h. Rats in the
sham group received 4 h of air inhalation in the chamber.
1.3. Electrical Y-maze test
The test apparatus consists of three arms, each of which has a light with electrical wire mesh at the end
ACCEPTED MANUSCRIPT side. In each trial only the light in one arm (safe area) was bright, with no electrical currents.
T
Meantime lights in the other two arms (unsafe areas) were dimmed with 50~70 V electrical currents.
IP
Before training, rats were allowed to freely explore the apparatus for 5 min.
SC R
In the training and test session, the rat was firstly put in the safe area. Once the rat stayed in the safe
area for more than 30 sec, the location of safe area was changed to force the rat to find the new safe
NU
area via the electrical shock. A correct trial was recorded when the rat successfully entered the safe
MA
area within 10 sec, otherwise an incorrect trial was recorded. The whole session lasted for 5 min and
D
total number of correct trials were collected and counted. Twenty and twenty-one days after the
TE
intra-hippocampal injection, all rats were trained in the electrical Y-maze test. Before Morris
CE P
water-maze task, electrical Y-maze test was tested on the first day (D1) after anesthesia.
1.4. Morris water-maze task
AC
17 days after the intra-hippocampal injection, rats were tested in Morris water-maze, which had a
circular pool (160 cm diameter, 50 cm height, 35 cm depth of water) divided into four equal quadrants.
A hidden platform (15 cm diameter) was placed 2 cm under the water surface in quadrant IV. The
navigation experiment began by placing the rat at each of the four quadrants, and recording its time
latency to find the hidden platform. The Any-Maze video system (RD1101, Yishu Info, China)
analyzed the swimming speed and escape latency. A cut-off limit was set as 90 sec for rats that cannot
locate the platform. Four test sessions (for each quadrant) were performed in one day for five
consecutive days.
ACCEPTED MANUSCRIPT One day after navigation test, spatial probe test was conducted by placing the rat into quadrant II.
T
The time spent in exploring the original quadrant of the hidden platform (quadrant IV) was recorded.
IP
Performances of navigation test on the fifth day (21 days after the injection) and spatial probe test (22
SC R
days after the injection) were recorded as the pre-treatment score. The post-treatment performance was evaluated at D1, D3 and D7 after sevoflurane inhalation (for Aβ+S1 and Aβ+S2 groups) or O2
NU
treatment (for C and Aβ+O2 groups). Eight randomly selected rats from each group were tested for
MA
their swimming speed, escape latency and time spent in exploring the original platform. An overview
D
of animal injection, inhalation and behavioral studies and time schedules is illustrated in Figure 1.
TE
1.5. Serum S100β assay
CE P
Immediately after each behavioral task, 1 ml venous blood were collected, centrifuged at 3000 rpm for
10 min and separated for the serum. S100β concentration was quantified by ELISA using S100β test
AC
kit (Xitang Biotech, China) following the manual instruction. The absorption value at 540 nm was
obtained by a microplate reader, and was estimated for serum S100β levels based on the standard
curve. All measurements were performed in triplicates.
1.6. SOD and MDA expression in hippocampus
Rats after the behavioral test were immediately sacrificed and extracted for hippocampus. Tissue
samples were homogenized in iced saline and centrifuged at 4 °C under 2500 rpm for 10 min. The
supernatant was saved and quantified for SOD activity using the hydroxylamine method, or MDA
content using the thiobarbituric acid method.
ACCEPTED MANUSCRIPT 1.7. Immunohistochemical (IHC) staining for hippocampal expression of S100β
T
Hippocampal tissues were extracted, embedded in paraffin and sectioned into 4 µm tissue slices,
IP
which were dewaxed, rehydrated, immersed in 3% H2O2 and processed by antigen retrieval in boiled
SC R
citric acid buffer. Nonspecific binding sites were blocked in fetal bovine serum, followed by primary
and secondary antibody incubation. After development in SABC, slices were counter-stained in
NU
hematoxylin and dehydrated. Images were captured using light field microscope at 400X
MA
magnification, and were analyzed for the area of positive cells and integral optical density (IOD)
D
values using IPP image analysis software.
TE
1.8. Statistics
CE P
GraphPad Prism 5 software processed all collected data, which were presented as mean ± standard
deviation (SD). One-way analysis of variance (ANOVA) and post-hoc Tukey test was used to
AC
determine significant difference and for between-group-comparisons. For analysis involving two
independent factors, 2-way ANOVA and post-hoc Bonferroni test was employed. A statistical
significance was defined when p<0.05.
2. Results
2.1. Impaired learning after high sevoflurane inhalation in Y-maze
No significant motor dysfunctions have been discovered in any group. Test results of electrical Y-maze (Figure 2) showed significantly lowered number of correct trials in Aβ+O2, Aβ+S1 and Aβ+S2 rats
compared to control (F=3.768, p<0.05). Further comparisons showed no significant difference
ACCEPTED MANUSCRIPT between Aβ+S1 and Aβ+O2 animals (p>0.05) but lower scores in Aβ+S2 rats compared to Aβ+O2 or
T
Aβ+S1 animals (p<0.05). These results suggest that the high concentration treatment of sevoflurane
IP
impairs learning and memory function.
SC R
2.2. High dose sevoflurane leads to longer escape latency in water-maze
Pre-treatment water-maze test results (Figure 3) showed longer escape latencies (F(4, 115) = 12.782,
NU
p<0.001, post-hoc Tukey test, p<0.05) and shorter time spent (p<0.05) exploring the original platform
MA
in Aβ+O2, Aβ+S1 and Aβ+S2 rats, compared to control ones. No significant difference has been found
D
among Aβ+O2, Aβ+S1 and Aβ+S2 rats (p>0.05). All rats had similar swimming speeds (F=0.09,
TE
p>0.05).
CE P
Post-treatment water-maze performance (Figure 4 and 5) showed elongated escape latencies and shorter time spent exploring the original platform in Aβ+O2, Aβ+S1 and Aβ+S2 rats, compared to
AC
control ones (F=17.9, p<0.001 with respect to interaction effects between group and time; p<0.05 in
post-hoc Bonferroni test). Further comparisons showed no significant difference of performance between Aβ+S1 and Aβ+O2 animals (p>0.05 in post-hoc Bonferroni test). Aβ+S2 group, however, had longer escape latencies and shorter exploration time compared to Aβ+O2 or Aβ+S1 rats (p<0.05 in post-hoc Bonferroni test). All rats displayed similar swimming speeds (F=0.15, p>0.05 with respect to
the interaction effect or either of main effect). Therefore, sevoflurane at high dose further aggravates
spatial learning in AD rats. 2.3. Serum and hippocampal levels of S100β were affected by sevoflurane
ACCEPTED MANUSCRIPT Post-treatment serum S100β levels were elevated in Aβ+O2, Aβ+S1 and Aβ+S2 rats, compared to
T
control ones (Table 1, F=5.67, p<0.05). Further comparisons showed no significant difference between
IP
Aβ+S1 and Aβ+O2 animals at each time point (p>0.05 using post-hoc Tukey test). Compared to
SC R
Aβ+S1 rats, Aβ+S2 rats showed significantly elevated S100β concentrations (p<0.05). In contrast with serum assays, IHC staining results (Figure 6, Table 2) showed depressed S100β
NU
expression in Aβ+O2, Aβ+S1 and Aβ+S2 rats compared to control ones (p<0.05 by post-hoc Tukey
MA
test). It can be found that S100β was abundantly expressed in the cytoplasm of neurons and glial cells
D
in control and sham rats. The application of high dosage of sevoflurane, however, depleted the
TE
cytoplasmic expression of S100β in neurons but not in glial cells. Aβ+S2 rats, showed significantly
CE P
suppressed hippocampal S100β expressions compared to Aβ+S1 littermates (p<0.05). All these results clearly suggest the release of S100β from hippocampus into the general circulation after CNS injury.
AC
2.4. SOD and MDA expressions were alternated in hippocampal tissues by sevoflurane
Compared to control animals, significantly depressed SOD activities and elevated MDA amounts were observed in hippocampus of Aβ+O2, Aβ+S1 and Aβ+S2 rats (Table 3, F=6.78, p<0.05). Further comparisons showed no significant difference in either index between Aβ+S1 and Aβ+O2 animals (p>0.05 in post-hoc Tukey test). Compared to Aβ+S1 rats, Aβ+S2 rats, however, showed significantly
suppressed SOD activities and elevated MDA contents (p<0.05). Therefore high dose of sevoflurane
disrupts the anti-oxidation balance inside hippocampus.
3. Discussion
ACCEPTED MANUSCRIPT Aβ is the critical factor in the pathogenesis and development of AD [1, 2]. It is a by-product of normal
T
metabolism and has important physiological roles for neural functions [18]. The over-deposition of Aβ,
IP
however, exerts neurotoxicity in a dose-dependent manner [19]. The intra-hippocampal injection of Aβ
SC R
thus can mimic the formation of senile plaque in AD patient’s brain and cause CNS damages, as
shown by the lower N-acetyl, higher creatine and choline peaks in MRI, impaired structures of glial
NU
cells, morphological changes such as neuron atrophy and mitochondria fractures, in addition to lower
MA
density and abnormal morphology of synaptic vesicles of hippocampus [20]. This study generated an
D
AD rat model by injecting Aβ1-40 into bilateral hippocampal CA1 regions and found significantly
TE
longer escape latencies and shorter time spent exploring the original platform in Aβ treated rats
CE P
compared to control ones (Figure 3), suggesting the cognitive impairments induced by Aβ1-40.
In clinical practice, sevoflurane is commonly used as an anesthesia medicine at 1.3%~2.6%
AC
concentration, which is recognized as low to moderate dose of anesthesia in animals [13]. Thus we applied 1.3% and 2.6% sevoflurane for 4 h in Aβ+S1 and Aβ+S2 rats, respectively, to induce cognitive
dysfunctions including impairments of memory, focus, learning and motor abilities [21, 22]. The
Y-maze task is employed to evaluate learning and flexible memory. As it is generally believed that the
decrease of cognitive function is the primary manifestation of AD and is related to hippocampal
functions [23, 24], we performed Y-maze test after anesthesia exposure as it is sensitive for cognitive
flexibility. Morris water-maze task, on the other hand, is usually used to measure the spatial learning
and memory [24, 25]. During both Y-maze and water-maze tasks, rats after high-dose sevoflurane
ACCEPTED MANUSCRIPT treatment (Aβ+S2) had worse performance as shown by lower correct times in Y-maze, longer escape
T
latencies and shorter time exploring the original platform in water-maze task. Rats with lower
IP
sevoflurane (Aβ+S1), however, had no significant difference in terms of all these indexes. These
SC R
results collectively suggest that the Aβ-induced neurotoxicity can be aggravated by the inhalation of
2.6% sevoflurane, which can further impair the cognitive function of rats. Lower sevoflurane (1.3%)
NU
had no effects on neural functions.
MA
Caused by the endogenous free radicals, oxidative stress plays an important role in the pathogenesis
D
of various neural degenerative diseases due to neuronal aging and apoptosis [26]. Aβ proteins exert its
TE
neurotoxicity via the induction of oxidative stress response by: (1) the production of free oxygen
CE P
radicals; (2) the interruption of membrane lipids, acid esters and lipoproteins, leading to cell damage;
(3) the release of inflammatory cytokines, which produce large amounts of free radicals, further
AC
aggravating the oxidative stress [27, 28]. MDA level evaluates the peroxidation of membrane lipids, while SOD reflects the body’s anti-oxidation ability, making them key indexes reflecting oxidative
stress [29]. Results in this study showed significantly depressed SOD activities and elevated MDA contents after sevoflurane application compared to control animals (Table 3), suggesting that Aβ1-40 can increase brain free oxygen radical level, decrease SOD activity, thereby inducing oxidative stress response. Rats in Aβ+S1 group had no significant changes in SOD levels or MDA activity, while
higher sevoflurane significantly depressed SOD activities and elevated MDA levels, indicating the potentiating effects on Aβ-induced oxidative stress response from 2.6% sevoflurane. This can be
ACCEPTED MANUSCRIPT explained by the release of pro-inflammatory cytokine after sevoflurane application [30].
T
As a sensitive index reflecting neural damage, S100β protein level was lower in hippocampus but
IP
higher in serum after Aβ injection (Table 1 and 2, Figure 6), and Aβ+S2 rats having significant
SC R
differences compared to Aβ+O2 and Aβ+S1 animals. This pattern indicates that Aβ deposition may damage glial cells, induce the release of S100β from hippocampus into CSF, and further into the
NU
general circulation through the damaged BBB. This process can be can be further facilitated by the
MA
2.6% sevoflurane.
D
This study further tested the cognitive function of Aβ-induced AD rats after sevoflurane inhalation,
TE
in addition to sevoflurane-related mechanism in aggravating Aβ-induced neurotoxicity in terms of
CE P
oxidative stress. In CNS injury, inflammation is closely related to the oxidative stress response.
Therefore, sevoflurane may exert its neurotoxicity via both inflammatory response and oxidative stress,
AC
both of which are pathological features of AD [31]. Past studies have proved the potential relationship between oxidative stress and inflammation underlying Aβ-induced neurotoxicity. For example, alphaGlycerylphosphorylethanolamine can rescue astrocytes from redox rearrangements induced by Aβ1-40
and protect them against inflammation [32]. All these studies collectively indicate that sevoflurane
aggravates AD severity via both inflammation and oxidative stress in glial cells. Although no direct
study has been performed, it is interesting that sevoflurane can suppress oxidative stress and
inflammation response in cerebral ischemia-reperfusion rats [33, 34]. These results seem contradictory
to our results, but as different disease models were used, the opposite function of sevoflurane is not out
ACCEPTED MANUSCRIPT of expectations. In word, a future study can be performed in the field of inflammatory related
T
pathways underlying the aggravation of Aβ-induced neurotoxicity by sevoflurane.
IP
4. Conclusion
SC R
In summary, this study demonstrated the aggravated cognitive functions on Aβ-induced AD rat after
the inhalation of 2.6% sevoflurane, possibly through the facilitation of brain oxidative stress response.
NU
Our results provide further knowledge about the pathology of AD and indicate potential drug targets in
AC
CE P
TE
D
MA
further studies.
ACCEPTED MANUSCRIPT Acknowledgement This work was supported by NSFC (81302534，81171782) and Shengjing Hospital of China
SC R
IP
T
Medical University, Shenyang (No.: 2014sj08).
Conflict of interest
AC
CE P
TE
D
MA
NU
None.
ACCEPTED MANUSCRIPT Reference: 1.
Huang Y, Mucke L. Alzheimer mechanisms and therapeutic strategies. Cell. 2012;
IP
O'Brien RJ, Wong PC. Amyloid precursor protein processing and Alzheimer's disease. Annu Rev Neurosci. 2011; 34: p. 185-204.
Tang K, Hynan LS, Baskin F, Rosenberg RN. Platelet amyloid precursor protein
NU
3.
SC R
2.
T
148(6): p. 1204-22.
MA
processing: a bio-marker for Alzheimer's disease. J Neurol Sci. 2006. 240(1-2): p. 53-8.
D
Sakono M, Zako T. Amyloid oligomers: formation and toxicity of Abeta oligomers.
TE
4.
5.
CE P
Febs j. 2010; 277(6): p. 1348-58. Wang, X., et al., Oxidative stress and mitochondrial dysfunction in Alzheimer's
6.
AC
disease. Biochim Biophys Acta. 2014. 1842(8): p. 1240-7. Abdulwahid Arif I, Ahmad Khan H. Environmental toxins and Parkinson's disease: putative roles of impaired electron transport chain and oxidative stress. Toxicol Ind Health. 2010; 26(2): p. 121-8. 7.
Eghwrudjakpor PO, Allison AB Oxidative stress following traumatic brain injury: enhancement of endogenous antioxidant defense systems and the promise of improved outcome. Niger J Med. 2010; 19(1): p. 14-21.
8.
Allen CL, Bayraktutan U. Oxidative stress and its role in the pathogenesis of
ACCEPTED MANUSCRIPT ischaemic stroke. Int J Stroke. 2009; 4(6): p. 461-70. Rothermundt M, Ohrmann P, Abel S, Siegmund A, Pedersen A, Ponath G, Suslow T,
T
9.
IP
Peters M, Kaestner F, Heindel W, Arolt V, Pfleiderer B. Glial cell activation in a
SC R
subgroup of patients with schizophrenia indicated by increased S100B serum
Psychiatry. 2007; 31(2): p. 361-4.
Chaves ML, Camozzato AL, Ferreira ED, Piazenski I, Kochhann R, Dall'Igna O,
MA
10.
NU
concentrations and elevated myo-inositol. Prog Neuropsychopharmacol Biol
D
Mazzini GS, Souza DO, Portela LV. Serum levels of S100B and NSE proteins in
Eberspacher E, et al. Effects of sevoflurane on cognitive deficit, motor function, and
CE P
11.
TE
Alzheimer's disease patients. J Neuroinflammation. 2010; 7: p. 6.
histopathology after cerebral ischemia in rats. Acta Anaesthesiol Scand. 2009; 53(6):
12.
AC
p. 774-82.
Schoen J, Husemann L, Tiemeyer C, Lueloh A, Sedemund-Adib B, Berger KU, Hueppe M, Heringlake M.. Cognitive function after sevoflurane- vs propofol-based anaesthesia for on-pump cardiac surgery: a randomized controlled trial. Br J Anaesth. 2011; 106(6): p. 840-50.
13.
Callaway JK, Jones NC, Royse AG, Royse CF. Sevoflurane anesthesia does not impair acquisition learning or memory in the Morris water maze in young adult and aged rats. Anesthesiology. 2012; 117(5): p. 1091-101.
ACCEPTED MANUSCRIPT 14.
Xie H, She GM, Wang C, Zhang LY, Liu CF. The gender difference in effect of
T
sevoflurane exposure on cognitive function and hippocampus neuronal apoptosis in
IP
rats. European review for medical and pharmacological sciences. 2015;19(4): p.
15.
SC R
647-57.
Tian Y, Guo S, Wu X, Ma L, Zhao X. Minocycline alleviates sevoflurane-induced
NU
cognitive impairment in aged rats. Cellular and molecular neurobiology. 2015;35(4): p.
Singh SP, Kapoor PM, Chowdhury U, Kiran U. Comparison of S100beta levels, and
D
16.
MA
585-94.
TE
their correlation with hemodynamic indices in patients undergoing coronary artery
CE P
bypass grafting with three different anesthetic techniques. Annals of cardiac anaesthesia. 2011;14(3): p. 197-202. Miguel-Hidalgo JJ, Cacabelos R. Beta-amyloid(1-40)-induced neurodegeneration in
AC
17.
the rat hippocampal neurons of the CA1 subfield. Acta Neuropathol. 1998; 95(5): p. 455-65. 18.
Muller UC, Zheng H. Physiological functions of APP family proteins. Cold Spring Harb Perspect Med. 2012; 2(2): p. a006288.
19.
Lopez OL, Klunk WE, Mathis C, Coleman RL, Price J, Becker JT, Aizenstein HJ, Snitz B, Cohen A, Ikonomovic M, McDade E, DeKosky ST,Weissfeld L, Kuller LH. Amyloid, neurodegeneration, and small vessel disease as predictors of dementia in the
ACCEPTED MANUSCRIPT oldest-old. Neurology. 2014; 83(20): p. 1804-11. Zhang L, Dong S, Zhao G, Ma Y. 7.0T nuclear magnetic resonance evaluation of the
T
20.
21.
SC R
verification. Neural Regen Res. 2014; 9(4): p. 430-5.
IP
amyloid beta (1-40) animal model of Alzheimer's disease: comparison of cytology
Lin D, Zuo Z. Isoflurane induces hippocampal cell injury and cognitive impairments
Paule MG, Li M, Allen RR, Liu F, Zou X, Hotchkiss C, Hanig JP, Patterson TA,
MA
22.
NU
in adult rats. Neuropharmacology. 2011; 61(8): p. 1354-9.
D
Slikker W Jr, Wang C. Ketamine anesthesia during the first week of life can cause
23.
CE P
p. 220-30.
TE
long-lasting cognitive deficits in rhesus monkeys. Neurotoxicol Teratol. 2011; 33(2):
Havekes R, Abel T, Van der Zee EA. The cholinergic system and neostriatal memory
24.
AC
functions. Behav Brain Res 2011; 221(2): p. 412-23. Mizoguchi K, Shoji H, Tanaka Y, Tabira T. Orbitofrontal dopaminergic dysfunction causes age-related impairment of reversal learning in rats. Neuroscience. 2010; 170(4): p. 1110-9. 25.
Shu SY, Wu YM, Bao XM, Wen ZB, Huang FH, Li SX, Fu QZ, Ning Q. A new area in the human brain associated with learning and memory: immunohistochemical and functional MRI analysis. Mol Psychiatry. 2002; 7(9): p. 1018-22.
26.
Paradies G, Petrosillo G, Paradies V, Ruggiero FM. Mitochondrial dysfunction in brain
ACCEPTED MANUSCRIPT aging: role of oxidative stress and cardiolipin. Neurochem Int. 2011; 58(4): p. 447-57. Abeti R, Duchen M.R. Activation of PARP by oxidative stress induced by
T
27.
IP
beta-amyloid: implications for Alzheimer's disease. Neurochem Res. 2012; 37(11): p.
28.
SC R
2589-96.
Querfurth HW, LaFerla F.M. Alzheimer's disease. N Engl J Med. 2010; 362(4): p.
Mao X, Hao S, Zhu Z, Zhang H, Wu W, Xu F, Liu B. Procyanidins protects against
MA
29.
NU
329-44.
D
oxidative damage and cognitive deficits after traumatic brain injury. Brain Inj
Shen X. Dong Y, Xu Z, Wang H, Miao C, Soriano SG, Sun D, Baxter MG, Zhang Y,
CE P
30.
TE
2015;29(1):86-92
Xie Z. Selective anesthesia-induced neuroinflammation in developing mouse brain
31.
AC
and cognitive impairment. Anesthesiology. 2013; 118(3): p. 502-15. Malm TM, Iivonen H, Goldsteins G, Keksa-Goldsteine V, Ahtoniemi T, Kanninen K, Salminen A, Auriola S, Van Groen T, Tanila H, Koistinaho J. Pyrrolidine dithiocarbamate activates Akt and improves spatial learning in APP/PS1 mice without affecting beta-amyloid burden. J Neurosci. 2007; 27(14): p. 3712-21. 32.
Bisaglia M, Venezia V, Biglieri M, Russo C, Mancini F, Milanese C, Schettini G. alpha-Glycerylphosphorylethanolamine
rescues
astrocytes
from
mitochondrial
impairment and oxidative stress induced by amyloid beta-peptides. Neurochem Int.
ACCEPTED MANUSCRIPT 2004; 44(3): p. 161-70. Zhang Y, Zhang FG, Meng C, Tian SY, Wang YX, Zhao W, Chen J, Zhang XS, Liang
T
33.
IP
Y, Zhang SD, Xing YJ Inhibition of sevoflurane postconditioning against cerebral
SC R
ischemia reperfusion-induced oxidative injury in rats. Molecules. 2012; 17(1): p. 341-54.
NU
Bedirli N, Bagriacik EU, Emmez H, Yilmaz G, Unal Y, Ozkose Z Sevoflurane and
MA
isoflurane preconditioning provides neuroprotection by inhibition of apoptosis-related
CE P
TE
2012; 24(4): p. 336-44.
D
mRNA expression in a rat model of focal cerebral ischemia. J Neurosurg Anesthesiol.
AC
34.
ACCEPTED MANUSCRIPT Figure legends
T
Figure 1 Schematic illustrations of animal injection and behavioral studies. On P22，the water-maze
IP
probe test was performed before sevoflurane inhalation and on D1, Y-maze test was performed before the
SC R
water-maze. “P” stands for the day after intra-hippocampal injection while “D” stands for days after
sevoflurane inhalation.
NU
Figure 2 Scores in electrical Y-maze of rats after anesthesia. The injection of Aβ1-40 impaired the
MA
Y-maze performance, which was further aggravated by addition of sevoflurane. a, p<0.05 compared to the
D
control group; b, p<0.05 compared to Aβ+O2 group; c, p<0.05 compared to Aβ+S1 group. Between-group
CE P
(SD). N=24 per group.
TE
comparisons were performed using post-hoc Tukey test. Data were presented as mean ± standard deviation
Figure 3 Water-maze performances of pre-anesthesia rats. (A) No major difference existed in average
AC
swimming speed across all groups. (B) The escape latency was increased after the application of Aβ1-40. (C)
The time spent in exploration of the original platform was also lowered in all three treatment groups. These
results supported aggravated learning and memory functions. Between-group comparisons were performed using post-hoc Tukey test. a, p<0.05 compared to the control group. Data were presented as mean ±
standard deviation (SD). N=24 per group.
Figure 4 Water-maze performance of post-anesthesia rats. (A) No difference existed in average
swimming speed. (B) Elevated escape latency and (C) time spent in exploration of the original platform
was also increased in sevoflurane-treated animals. Between-group comparisons were performed using
ACCEPTED MANUSCRIPT post-hoc Bonferroni test. Data were presented as mean ± standard deviation (SD). a, p<0.05 compared to
T
the control group; c, p<0.05 compared to Aβ+S1 group. N=8 per group.
IP
Figure 5 Representative movement path in post-anesthesia rats in water-maze. Rats from the sham
SC R
group, control group, Aβ+O2 group, Aβ+S1 and Aβ+S2 group were tested in D1 (left panel), D3 (middle
panel) and D7 (right panel) in Morris water-maze task, with their movement path (red curves) in finding
NU
the hidden platform (green circle). Roman numbers (I to IV) indicate four arbitrary divided quadrants
MA
along with the orientations (NE, northeast; SE, southeast; SW, southwest; NW, northwest). Only one
D
representative path was shown at each group. N=8 per group.
TE
Figure 6 S100β expression profiles in post-anesthesia rat hippocampus. IHC staining technique was
CE P
employed to reveal the expression of S100β proteins in hippocampal CA1 regions. Representative staining
images (X400 under a light-field microscope) were shown on D1 (left panel), D3 (middle panel) and D7
AC
(right panel) after anesthesia. S100β was abundantly expressed in cytoplasm of neurons and glial cells in control and sham rats. The addition of Aβ1-40 decreased expression levels in neurons but not in glial cells. The application of sevoflurane further aggravated the depletion. Scale bar, 20 μm.
ACCEPTED MANUSCRIPT
AC
CE P
TE
D
MA
NU
SC R
IP
T
Figure 1
D
TE
CE P
AC
2
20
10
T
30 a
IP
SC R
NU
0
β+ S
1
β+ S
a
A
MA
A
2
β+ O
on tr ol
A
C
Sh am
Number of correct trials in Y-maze
ACCEPTED MANUSCRIPT
Figure 2
40
a,b,c
a
S2
60
S2
D
a
β+
S1 A β+
A β+
S2
S1
2
O
tr ol
am
A β+
C on
Sh
Swimming speed (cm/s)
T
30
IP
20
10
0
SC R
NU
MA
40
A
β+
TE
(B)
A β+
S1
A
O 2
30
A β+
2
O
β+
A
CE P
ro l
20
A β+
tr ol
0
on t
C
Escpae latency (s) 10
C on
am
Sh
AC
(C)
Sh am
Time spent in exploration of the original platform (%)
ACCEPTED MANUSCRIPT
Figure 3
(A) 40
a a
80
a
a
40
20
0
2
2
S2
β+
A
20
30
T
IP
SC R
NU
S1
0
β+ S
a β+ S
A
1
β+
A
2
O
tr ol
β+
A
C on
am
Sh
Swimming speed (cm/s) 10
A
1
60
β+ S
a
MA
50
β+ S
D
TE
40
A
2
β+ O
A
ol
tr
(B)
A
2
O
β+
l
tr o
30
A
on
CE P
10
C on
Sh
Escpae latency (s) 20
C
am
am
0
AC
(C)
Sh
Time spent in exploration of the original platform (%)
ACCEPTED MANUSCRIPT
Figure 4
(A) 40
D1 D3 D7
a,b,c
a
80
a
a,b,c
40
20
0
D1 D3 D7
D1 D3 D7
ACCEPTED MANUSCRIPT
AC
CE P
TE
D
MA
NU
SC R
IP
T
Figure 5
ACCEPTED MANUSCRIPT
AC
CE P
TE
D
MA
NU
SC R
IP
T
Figure 6
ACCEPTED MANUSCRIPT Table 1 Post-anesthesia serum S100β levels (pg/ml) D3
D7
Sham
10.9±1.4
11.6±2.2
Control
11.2±1.2
12.1±2.0
Aβ+O2
19.0±3.3 a
19.7±3.5a
18.9±2.1a
Aβ+S1
20.9±3.7a
21.3±4.8 a
21.1±4.2a
Aβ+S2
28.8±5.1abc
33.0±5.2abc
33.9±4.6 abc
T
D1
NU
SC R
IP
11.1±2.8 11.9±2.2
MA
Group
TE
D
Note: a , p<0.05 compared to the control group; b, p<0.05 compared to Aβ group; c, p<0.05
AC
CE P
compared to S1 group. N=8 per group.
ACCEPTED MANUSCRIPT Table 2 IOD/area ratios in post-anesthesia rats D3
D7
Sham
0.27±0.02
0.32±0.03
Control
0.25±0.01
0.31±0.03
Aβ+O2
0.20±0.01a
0.21±0.02a
0.21±0.03a
Aβ+S1
0.21±0.03a
0.21±0.01a
0.22±0.01a
Aβ+S2
0.11±0.01abc
0.09±0abc
0.09±0.01 abc
T
D1
NU
SC R
IP
0.26±0.04 0.24±0.05
MA
Group
TE
D
Note: a , p<0.05 compared to the control group; b, p<0.05 compared to Aβ group; c, p<0.05
AC
CE P
compared to S1 group. N=8 per group.
ACCEPTED MANUSCRIPT Table 3 Hippocampal SOD activity and MDA contents in post-anesthesia rats SOD (U/mg)
MDA (nmol/mg)
D3
D7
D1
Sham
30.2±3.1
31.5±3.0
28.7±2.0
1.90±0.28
Control
28.2±1.9
28.9±1.7
30.1±1.5
Aβ+O2
21.2±1.6a
20.3±1.5a
Aβ+S1
22.2±2.0a
21.2±2.1a
1.82±0.21
1.79±0.19
1.78±0.19
21.0±1.8a
4.71±0.39a
4.55±0.45a
4.61±0.51a
22.9±1.8a
5.06±0.69a
5.11±0.76a
5.30±0.81a
8.23±0.54a
9.30±1.05a
9.51±0.92a
bc
bc
bc
MA
NU
SC R
1.81±0.20
16.0±1.1abc
TE
D
16.8±1.4abc
D7
1.82±0.23
Aβ+S2 17.3±1.5abc
D3
IP
D1
T
Group
CE P
Note: a , p<0.05 compared to the control group; b, p<0.05 compared to Aβ group; c, p<0.05
AC
compared to S1 group. N=8 per group.
S0024-3205(15)30066-7 doi: 10.1016/j.lfs.2015.11.002 LFS 14549
To appear in:
Life Sciences
Received date: Revised date: Accepted date:
12 March 2015 9 October 2015 3 November 2015
Please cite this article as: Yue Tian, Shanbin Guo, Ling Ma, Yuan Wang, Ying Xu, Ping Zhao, Sevoflurane aggregates cognitive dysfunction and hippocampal oxidative stress induced by β-amyloid in rats, Life Sciences (2015), doi: 10.1016/j.lfs.2015.11.002
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT Sevoflurane Aggregates Cognitive Dysfunction and Hippocampal Oxidative Stress
T
Induced by β-amyloid in Rats
IP
Running title: Sevoflurane affects hippocampal function
SC R
Tian Yue1, Guo Shanbin2, Ma Ling1, Wang Yuan1, Xu Ying1, Zhao Ping1*
1Department of Anesthesiology, Shengjing Hospital of China Medical University, Shenyang 110004,
NU
China.
TE
*Corresponding author: Zhao Ping
D
MA
2Department of Pharmacy, Shengjing Hospital of China Medical University, Shenyang 110004, China.
CE P
Department of Anesthesiology, Shengjing Hospital of China Medical University, 36 Sanhao Street,
Tel: None
AC
Shenyang 110004, China.
Fax: None
Email: [email protected]
ACCEPTED MANUSCRIPT Abstract
T
Aims: To investigate the effects of sevoflurane inhalation on β-amyloid (Aβ)-induced cognitive disorders
IP
and hippocampal oxidative stress in rat models.
SC R
Materials and Methods: Cognitive dysfunction is induced by hippocampal injection of Aβ1-40 (10 μg in 2 μl) for 22 days. To explore the effect of sevoflurane inhalation on Aβ1-40 induced cognitive disorder, two
NU
doses of sevoflurane inhalation are used: 1.3% (Aβ+S1) and 2.6% (Aβ+S2). Sham operation (Sham, for
MA
operation control), saline injection (Control, for injection control) and 30% oxygen inhalation after Aβ1-40
D
injection (Aβ+O2, for inhalation control) were used as controls. All rats were further tested in electrical
TE
Y-maze and Morris water maze. Serum S100β levels, hippocampal superoxide dismutase (SOD) activity,
CE P
S100β expression and malonyldialdehyde (MDA) concentrations were further quantified.
Key findings: Rats in Aβ+O2, Aβ+S1 and Aβ+S2 group had lower number of correct actions in the
AC
electrical Y maze task, longer escape latencies, less time exploring the original platform, elevated serum S100β levels, depressed hippocampal SOD activity, S100β expression and higher MDA concentrations
compared to control group (p<0.05). Such difference was not significant between Aβ+S1 and Aβ+O2 rats.
Rats in Aβ+S2 group, however, showed significantly impaired performances compared to Aβ+S1 group
(p<0.05).
Significance: Sevoflurane (2.6%) can aggravate the Aβ-induced cognitive dysfunction, possibly via the
intracerebral oxidative stress response.
Keywords: Sevoflurane inhalation; Amyloid protein; Cognitive dysfunctions; Oxidative stress
ACCEPTED MANUSCRIPT Introduction
T
Alzheimer’s disease (AD) is clinically manifested with advancing hypomnesia and cognitive disorders, and
IP
affecting about 26.6 million people worldwide, making it a major cause of senile dementia [1]. The most
SC R
prominent pathological feature of AD is the occurrence of extracellular senile plaque formed by β-amyloid
proteins deposition [2, 3]. Such accumulation of Aβ oligomers has neurotoxicity and impairs synaptic
NU
functions and memory [4] via stimulating the production of intracellular active oxides and free radicals
MA
such as ROS, thereby increasing the level of lipid peroxidation products malonyldialdehyde (MDA).
D
Meanwhile Aβ can also disrupt cellular innate anti-oxidation system by the inactivation of key enzymes
TE
such as superoxide dismutase (SOD) and glutathione peroxidase GSH-Px [5]. In a word, the oxidative
[6-8].
CE P
stress is the major pathological mechanism underlying AD related neuronal dysfunction and degeneration
AC
Mainly produced in astrocytes of central nervous system (CNS), S100β is a calcium-binding protein that
regulates the growth, proliferation and differentiation of neural glial cells, in addition to intracellular calcium homeostasis. S100β normally cannot penetrate the blood-brain barrier (BBB). When there are acute injuries in CNS, however, S100β may enter the cerebrospinal fluid (CSF) and the general circulation through BBB. Therefore CSF or serum S100β level can be used as an index evaluating the severity of CNS injury [9]. It has been reported that S100β level may elevate in AD patients, suggesting the potential relationship between S100β and AD-related neural injuries [10], although its detailed mechanism remained
unknown.
ACCEPTED MANUSCRIPT As one commonly used inhaled anesthetics, sevoflurane has in vitro neurotoxicity via its facilitation of
T
Aβ oligomerization, but not significant cognitive impairments at moderate concentrations [11-13].
IP
Recently it has been reported that sevoflurane impaired both short-term and long-term cognitive functions
SC R
[14], especially in aged rats [15]. Meanwhile sevoflurane may elevated serum S100β levels [16]. Based on
these evidences, it is possible that sevoflurane may aggravate AD-related memory and learning functions
NU
via the stimulation of S100β release. This study thus aimed to evaluate the effect of sevoflurane on the
MA
cognitive dysfunctions and oxidative stress response in the hippocampus of Aβ-induced AD rats.
D
1. Materials and Methods
TE
1.1. Animals
CE P
A total of 120 male SD rats (body weight between 250 and 300 g) were purchased from the animal
experiment center of China Medical University. All rats were kept in an SPF-grade animal house,
AC
which was kept at 25 °C temperature and 40% relative humidity with food and water provided ad
libitum. Animals were randomly divided into 5 groups including the sham group, control group, amyloid protein group (Aβ+O2), 1.3% sevoflurane group (Aβ+S1) and 2.6% sevoflurane (Aβ+S2). All
experimental protocols have been pre-approved by the ethical committee of using animals in
experiments in China Medical University. 1.2. Aβ1-40 injection and sevoflurane inhalation Following previously established procedures [17], rats were anesthetized by intraperitoneal injection
of 10% chloral hydrate (250 mg/kg) and fixed on the stereotaxic apparatus to locate the bilateral
ACCEPTED MANUSCRIPT hippocampal CA1 regions (coordinates: 3.2 mm posterior, 2.0 mm bilateral and 3.9 mm ventral; all
T
relative to Bregma). The skull was removed to expose the dura, through which a micro-syringe was
IP
inserted vertically into hippocampal CA1 region. 2 µl of Aβ1-40 (Sigma, US), which has been
SC R
re-suspended in 5 µg/µl solutions in sterilized saline, was slowly injected on rats of Aβ+O2, Aβ+S1
and Aβ+S2 groups within 5 min, followed by 5-min retention of the syringe. Gentamicin (20 000 U)
NU
was intraperitoneally injected after the surgery. Rats in the control group received isosmotic saline
MA
injection on bilateral hippocampus. Rats in the sham group received all surgical procedures but no
D
injection.
TE
Sevoflurane was applied using a self-made anesthesia chamber (50 cm X 30 cm X 30 cm), which
CE P
was kept at 37 °C and had two ventilating holes inside: the superior one connected to an anesthesia
pump (flow rate=3 L/min, concentration of O2=30%); the inferior one connected to a gaseous monitor.
AC
22 days after the injection, rats in Aβ+S1 and Aβ+S2 group were received 1.3% and 2.6% of
sevoflurane (Abbott, US) inhalation for 4 h, respectively. These concentrations and time periods were
selected based on our pilot studies showing no respiratory arrest under 2.6% sevoflurane, in addition to
various similar studies. The respiration rhythm and mucosal color of animals were continuously monitored to avoid hypoxia. Rats in C group and Aβ+O2 group received 30% O2 for 4 h. Rats in the
sham group received 4 h of air inhalation in the chamber.
1.3. Electrical Y-maze test
The test apparatus consists of three arms, each of which has a light with electrical wire mesh at the end
ACCEPTED MANUSCRIPT side. In each trial only the light in one arm (safe area) was bright, with no electrical currents.
T
Meantime lights in the other two arms (unsafe areas) were dimmed with 50~70 V electrical currents.
IP
Before training, rats were allowed to freely explore the apparatus for 5 min.
SC R
In the training and test session, the rat was firstly put in the safe area. Once the rat stayed in the safe
area for more than 30 sec, the location of safe area was changed to force the rat to find the new safe
NU
area via the electrical shock. A correct trial was recorded when the rat successfully entered the safe
MA
area within 10 sec, otherwise an incorrect trial was recorded. The whole session lasted for 5 min and
D
total number of correct trials were collected and counted. Twenty and twenty-one days after the
TE
intra-hippocampal injection, all rats were trained in the electrical Y-maze test. Before Morris
CE P
water-maze task, electrical Y-maze test was tested on the first day (D1) after anesthesia.
1.4. Morris water-maze task
AC
17 days after the intra-hippocampal injection, rats were tested in Morris water-maze, which had a
circular pool (160 cm diameter, 50 cm height, 35 cm depth of water) divided into four equal quadrants.
A hidden platform (15 cm diameter) was placed 2 cm under the water surface in quadrant IV. The
navigation experiment began by placing the rat at each of the four quadrants, and recording its time
latency to find the hidden platform. The Any-Maze video system (RD1101, Yishu Info, China)
analyzed the swimming speed and escape latency. A cut-off limit was set as 90 sec for rats that cannot
locate the platform. Four test sessions (for each quadrant) were performed in one day for five
consecutive days.
ACCEPTED MANUSCRIPT One day after navigation test, spatial probe test was conducted by placing the rat into quadrant II.
T
The time spent in exploring the original quadrant of the hidden platform (quadrant IV) was recorded.
IP
Performances of navigation test on the fifth day (21 days after the injection) and spatial probe test (22
SC R
days after the injection) were recorded as the pre-treatment score. The post-treatment performance was evaluated at D1, D3 and D7 after sevoflurane inhalation (for Aβ+S1 and Aβ+S2 groups) or O2
NU
treatment (for C and Aβ+O2 groups). Eight randomly selected rats from each group were tested for
MA
their swimming speed, escape latency and time spent in exploring the original platform. An overview
D
of animal injection, inhalation and behavioral studies and time schedules is illustrated in Figure 1.
TE
1.5. Serum S100β assay
CE P
Immediately after each behavioral task, 1 ml venous blood were collected, centrifuged at 3000 rpm for
10 min and separated for the serum. S100β concentration was quantified by ELISA using S100β test
AC
kit (Xitang Biotech, China) following the manual instruction. The absorption value at 540 nm was
obtained by a microplate reader, and was estimated for serum S100β levels based on the standard
curve. All measurements were performed in triplicates.
1.6. SOD and MDA expression in hippocampus
Rats after the behavioral test were immediately sacrificed and extracted for hippocampus. Tissue
samples were homogenized in iced saline and centrifuged at 4 °C under 2500 rpm for 10 min. The
supernatant was saved and quantified for SOD activity using the hydroxylamine method, or MDA
content using the thiobarbituric acid method.
ACCEPTED MANUSCRIPT 1.7. Immunohistochemical (IHC) staining for hippocampal expression of S100β
T
Hippocampal tissues were extracted, embedded in paraffin and sectioned into 4 µm tissue slices,
IP
which were dewaxed, rehydrated, immersed in 3% H2O2 and processed by antigen retrieval in boiled
SC R
citric acid buffer. Nonspecific binding sites were blocked in fetal bovine serum, followed by primary
and secondary antibody incubation. After development in SABC, slices were counter-stained in
NU
hematoxylin and dehydrated. Images were captured using light field microscope at 400X
MA
magnification, and were analyzed for the area of positive cells and integral optical density (IOD)
D
values using IPP image analysis software.
TE
1.8. Statistics
CE P
GraphPad Prism 5 software processed all collected data, which were presented as mean ± standard
deviation (SD). One-way analysis of variance (ANOVA) and post-hoc Tukey test was used to
AC
determine significant difference and for between-group-comparisons. For analysis involving two
independent factors, 2-way ANOVA and post-hoc Bonferroni test was employed. A statistical
significance was defined when p<0.05.
2. Results
2.1. Impaired learning after high sevoflurane inhalation in Y-maze
No significant motor dysfunctions have been discovered in any group. Test results of electrical Y-maze (Figure 2) showed significantly lowered number of correct trials in Aβ+O2, Aβ+S1 and Aβ+S2 rats
compared to control (F=3.768, p<0.05). Further comparisons showed no significant difference
ACCEPTED MANUSCRIPT between Aβ+S1 and Aβ+O2 animals (p>0.05) but lower scores in Aβ+S2 rats compared to Aβ+O2 or
T
Aβ+S1 animals (p<0.05). These results suggest that the high concentration treatment of sevoflurane
IP
impairs learning and memory function.
SC R
2.2. High dose sevoflurane leads to longer escape latency in water-maze
Pre-treatment water-maze test results (Figure 3) showed longer escape latencies (F(4, 115) = 12.782,
NU
p<0.001, post-hoc Tukey test, p<0.05) and shorter time spent (p<0.05) exploring the original platform
MA
in Aβ+O2, Aβ+S1 and Aβ+S2 rats, compared to control ones. No significant difference has been found
D
among Aβ+O2, Aβ+S1 and Aβ+S2 rats (p>0.05). All rats had similar swimming speeds (F=0.09,
TE
p>0.05).
CE P
Post-treatment water-maze performance (Figure 4 and 5) showed elongated escape latencies and shorter time spent exploring the original platform in Aβ+O2, Aβ+S1 and Aβ+S2 rats, compared to
AC
control ones (F=17.9, p<0.001 with respect to interaction effects between group and time; p<0.05 in
post-hoc Bonferroni test). Further comparisons showed no significant difference of performance between Aβ+S1 and Aβ+O2 animals (p>0.05 in post-hoc Bonferroni test). Aβ+S2 group, however, had longer escape latencies and shorter exploration time compared to Aβ+O2 or Aβ+S1 rats (p<0.05 in post-hoc Bonferroni test). All rats displayed similar swimming speeds (F=0.15, p>0.05 with respect to
the interaction effect or either of main effect). Therefore, sevoflurane at high dose further aggravates
spatial learning in AD rats. 2.3. Serum and hippocampal levels of S100β were affected by sevoflurane
ACCEPTED MANUSCRIPT Post-treatment serum S100β levels were elevated in Aβ+O2, Aβ+S1 and Aβ+S2 rats, compared to
T
control ones (Table 1, F=5.67, p<0.05). Further comparisons showed no significant difference between
IP
Aβ+S1 and Aβ+O2 animals at each time point (p>0.05 using post-hoc Tukey test). Compared to
SC R
Aβ+S1 rats, Aβ+S2 rats showed significantly elevated S100β concentrations (p<0.05). In contrast with serum assays, IHC staining results (Figure 6, Table 2) showed depressed S100β
NU
expression in Aβ+O2, Aβ+S1 and Aβ+S2 rats compared to control ones (p<0.05 by post-hoc Tukey
MA
test). It can be found that S100β was abundantly expressed in the cytoplasm of neurons and glial cells
D
in control and sham rats. The application of high dosage of sevoflurane, however, depleted the
TE
cytoplasmic expression of S100β in neurons but not in glial cells. Aβ+S2 rats, showed significantly
CE P
suppressed hippocampal S100β expressions compared to Aβ+S1 littermates (p<0.05). All these results clearly suggest the release of S100β from hippocampus into the general circulation after CNS injury.
AC
2.4. SOD and MDA expressions were alternated in hippocampal tissues by sevoflurane
Compared to control animals, significantly depressed SOD activities and elevated MDA amounts were observed in hippocampus of Aβ+O2, Aβ+S1 and Aβ+S2 rats (Table 3, F=6.78, p<0.05). Further comparisons showed no significant difference in either index between Aβ+S1 and Aβ+O2 animals (p>0.05 in post-hoc Tukey test). Compared to Aβ+S1 rats, Aβ+S2 rats, however, showed significantly
suppressed SOD activities and elevated MDA contents (p<0.05). Therefore high dose of sevoflurane
disrupts the anti-oxidation balance inside hippocampus.
3. Discussion
ACCEPTED MANUSCRIPT Aβ is the critical factor in the pathogenesis and development of AD [1, 2]. It is a by-product of normal
T
metabolism and has important physiological roles for neural functions [18]. The over-deposition of Aβ,
IP
however, exerts neurotoxicity in a dose-dependent manner [19]. The intra-hippocampal injection of Aβ
SC R
thus can mimic the formation of senile plaque in AD patient’s brain and cause CNS damages, as
shown by the lower N-acetyl, higher creatine and choline peaks in MRI, impaired structures of glial
NU
cells, morphological changes such as neuron atrophy and mitochondria fractures, in addition to lower
MA
density and abnormal morphology of synaptic vesicles of hippocampus [20]. This study generated an
D
AD rat model by injecting Aβ1-40 into bilateral hippocampal CA1 regions and found significantly
TE
longer escape latencies and shorter time spent exploring the original platform in Aβ treated rats
CE P
compared to control ones (Figure 3), suggesting the cognitive impairments induced by Aβ1-40.
In clinical practice, sevoflurane is commonly used as an anesthesia medicine at 1.3%~2.6%
AC
concentration, which is recognized as low to moderate dose of anesthesia in animals [13]. Thus we applied 1.3% and 2.6% sevoflurane for 4 h in Aβ+S1 and Aβ+S2 rats, respectively, to induce cognitive
dysfunctions including impairments of memory, focus, learning and motor abilities [21, 22]. The
Y-maze task is employed to evaluate learning and flexible memory. As it is generally believed that the
decrease of cognitive function is the primary manifestation of AD and is related to hippocampal
functions [23, 24], we performed Y-maze test after anesthesia exposure as it is sensitive for cognitive
flexibility. Morris water-maze task, on the other hand, is usually used to measure the spatial learning
and memory [24, 25]. During both Y-maze and water-maze tasks, rats after high-dose sevoflurane
ACCEPTED MANUSCRIPT treatment (Aβ+S2) had worse performance as shown by lower correct times in Y-maze, longer escape
T
latencies and shorter time exploring the original platform in water-maze task. Rats with lower
IP
sevoflurane (Aβ+S1), however, had no significant difference in terms of all these indexes. These
SC R
results collectively suggest that the Aβ-induced neurotoxicity can be aggravated by the inhalation of
2.6% sevoflurane, which can further impair the cognitive function of rats. Lower sevoflurane (1.3%)
NU
had no effects on neural functions.
MA
Caused by the endogenous free radicals, oxidative stress plays an important role in the pathogenesis
D
of various neural degenerative diseases due to neuronal aging and apoptosis [26]. Aβ proteins exert its
TE
neurotoxicity via the induction of oxidative stress response by: (1) the production of free oxygen
CE P
radicals; (2) the interruption of membrane lipids, acid esters and lipoproteins, leading to cell damage;
(3) the release of inflammatory cytokines, which produce large amounts of free radicals, further
AC
aggravating the oxidative stress [27, 28]. MDA level evaluates the peroxidation of membrane lipids, while SOD reflects the body’s anti-oxidation ability, making them key indexes reflecting oxidative
stress [29]. Results in this study showed significantly depressed SOD activities and elevated MDA contents after sevoflurane application compared to control animals (Table 3), suggesting that Aβ1-40 can increase brain free oxygen radical level, decrease SOD activity, thereby inducing oxidative stress response. Rats in Aβ+S1 group had no significant changes in SOD levels or MDA activity, while
higher sevoflurane significantly depressed SOD activities and elevated MDA levels, indicating the potentiating effects on Aβ-induced oxidative stress response from 2.6% sevoflurane. This can be
ACCEPTED MANUSCRIPT explained by the release of pro-inflammatory cytokine after sevoflurane application [30].
T
As a sensitive index reflecting neural damage, S100β protein level was lower in hippocampus but
IP
higher in serum after Aβ injection (Table 1 and 2, Figure 6), and Aβ+S2 rats having significant
SC R
differences compared to Aβ+O2 and Aβ+S1 animals. This pattern indicates that Aβ deposition may damage glial cells, induce the release of S100β from hippocampus into CSF, and further into the
NU
general circulation through the damaged BBB. This process can be can be further facilitated by the
MA
2.6% sevoflurane.
D
This study further tested the cognitive function of Aβ-induced AD rats after sevoflurane inhalation,
TE
in addition to sevoflurane-related mechanism in aggravating Aβ-induced neurotoxicity in terms of
CE P
oxidative stress. In CNS injury, inflammation is closely related to the oxidative stress response.
Therefore, sevoflurane may exert its neurotoxicity via both inflammatory response and oxidative stress,
AC
both of which are pathological features of AD [31]. Past studies have proved the potential relationship between oxidative stress and inflammation underlying Aβ-induced neurotoxicity. For example, alphaGlycerylphosphorylethanolamine can rescue astrocytes from redox rearrangements induced by Aβ1-40
and protect them against inflammation [32]. All these studies collectively indicate that sevoflurane
aggravates AD severity via both inflammation and oxidative stress in glial cells. Although no direct
study has been performed, it is interesting that sevoflurane can suppress oxidative stress and
inflammation response in cerebral ischemia-reperfusion rats [33, 34]. These results seem contradictory
to our results, but as different disease models were used, the opposite function of sevoflurane is not out
ACCEPTED MANUSCRIPT of expectations. In word, a future study can be performed in the field of inflammatory related
T
pathways underlying the aggravation of Aβ-induced neurotoxicity by sevoflurane.
IP
4. Conclusion
SC R
In summary, this study demonstrated the aggravated cognitive functions on Aβ-induced AD rat after
the inhalation of 2.6% sevoflurane, possibly through the facilitation of brain oxidative stress response.
NU
Our results provide further knowledge about the pathology of AD and indicate potential drug targets in
AC
CE P
TE
D
MA
further studies.
ACCEPTED MANUSCRIPT Acknowledgement This work was supported by NSFC (81302534，81171782) and Shengjing Hospital of China
SC R
IP
T
Medical University, Shenyang (No.: 2014sj08).
Conflict of interest
AC
CE P
TE
D
MA
NU
None.
ACCEPTED MANUSCRIPT Reference: 1.
Huang Y, Mucke L. Alzheimer mechanisms and therapeutic strategies. Cell. 2012;
IP
O'Brien RJ, Wong PC. Amyloid precursor protein processing and Alzheimer's disease. Annu Rev Neurosci. 2011; 34: p. 185-204.
Tang K, Hynan LS, Baskin F, Rosenberg RN. Platelet amyloid precursor protein
NU
3.
SC R
2.
T
148(6): p. 1204-22.
MA
processing: a bio-marker for Alzheimer's disease. J Neurol Sci. 2006. 240(1-2): p. 53-8.
D
Sakono M, Zako T. Amyloid oligomers: formation and toxicity of Abeta oligomers.
TE
4.
5.
CE P
Febs j. 2010; 277(6): p. 1348-58. Wang, X., et al., Oxidative stress and mitochondrial dysfunction in Alzheimer's
6.
AC
disease. Biochim Biophys Acta. 2014. 1842(8): p. 1240-7. Abdulwahid Arif I, Ahmad Khan H. Environmental toxins and Parkinson's disease: putative roles of impaired electron transport chain and oxidative stress. Toxicol Ind Health. 2010; 26(2): p. 121-8. 7.
Eghwrudjakpor PO, Allison AB Oxidative stress following traumatic brain injury: enhancement of endogenous antioxidant defense systems and the promise of improved outcome. Niger J Med. 2010; 19(1): p. 14-21.
8.
Allen CL, Bayraktutan U. Oxidative stress and its role in the pathogenesis of
ACCEPTED MANUSCRIPT ischaemic stroke. Int J Stroke. 2009; 4(6): p. 461-70. Rothermundt M, Ohrmann P, Abel S, Siegmund A, Pedersen A, Ponath G, Suslow T,
T
9.
IP
Peters M, Kaestner F, Heindel W, Arolt V, Pfleiderer B. Glial cell activation in a
SC R
subgroup of patients with schizophrenia indicated by increased S100B serum
Psychiatry. 2007; 31(2): p. 361-4.
Chaves ML, Camozzato AL, Ferreira ED, Piazenski I, Kochhann R, Dall'Igna O,
MA
10.
NU
concentrations and elevated myo-inositol. Prog Neuropsychopharmacol Biol
D
Mazzini GS, Souza DO, Portela LV. Serum levels of S100B and NSE proteins in
Eberspacher E, et al. Effects of sevoflurane on cognitive deficit, motor function, and
CE P
11.
TE
Alzheimer's disease patients. J Neuroinflammation. 2010; 7: p. 6.
histopathology after cerebral ischemia in rats. Acta Anaesthesiol Scand. 2009; 53(6):
12.
AC
p. 774-82.
Schoen J, Husemann L, Tiemeyer C, Lueloh A, Sedemund-Adib B, Berger KU, Hueppe M, Heringlake M.. Cognitive function after sevoflurane- vs propofol-based anaesthesia for on-pump cardiac surgery: a randomized controlled trial. Br J Anaesth. 2011; 106(6): p. 840-50.
13.
Callaway JK, Jones NC, Royse AG, Royse CF. Sevoflurane anesthesia does not impair acquisition learning or memory in the Morris water maze in young adult and aged rats. Anesthesiology. 2012; 117(5): p. 1091-101.
ACCEPTED MANUSCRIPT 14.
Xie H, She GM, Wang C, Zhang LY, Liu CF. The gender difference in effect of
T
sevoflurane exposure on cognitive function and hippocampus neuronal apoptosis in
IP
rats. European review for medical and pharmacological sciences. 2015;19(4): p.
15.
SC R
647-57.
Tian Y, Guo S, Wu X, Ma L, Zhao X. Minocycline alleviates sevoflurane-induced
NU
cognitive impairment in aged rats. Cellular and molecular neurobiology. 2015;35(4): p.
Singh SP, Kapoor PM, Chowdhury U, Kiran U. Comparison of S100beta levels, and
D
16.
MA
585-94.
TE
their correlation with hemodynamic indices in patients undergoing coronary artery
CE P
bypass grafting with three different anesthetic techniques. Annals of cardiac anaesthesia. 2011;14(3): p. 197-202. Miguel-Hidalgo JJ, Cacabelos R. Beta-amyloid(1-40)-induced neurodegeneration in
AC
17.
the rat hippocampal neurons of the CA1 subfield. Acta Neuropathol. 1998; 95(5): p. 455-65. 18.
Muller UC, Zheng H. Physiological functions of APP family proteins. Cold Spring Harb Perspect Med. 2012; 2(2): p. a006288.
19.
Lopez OL, Klunk WE, Mathis C, Coleman RL, Price J, Becker JT, Aizenstein HJ, Snitz B, Cohen A, Ikonomovic M, McDade E, DeKosky ST,Weissfeld L, Kuller LH. Amyloid, neurodegeneration, and small vessel disease as predictors of dementia in the
ACCEPTED MANUSCRIPT oldest-old. Neurology. 2014; 83(20): p. 1804-11. Zhang L, Dong S, Zhao G, Ma Y. 7.0T nuclear magnetic resonance evaluation of the
T
20.
21.
SC R
verification. Neural Regen Res. 2014; 9(4): p. 430-5.
IP
amyloid beta (1-40) animal model of Alzheimer's disease: comparison of cytology
Lin D, Zuo Z. Isoflurane induces hippocampal cell injury and cognitive impairments
Paule MG, Li M, Allen RR, Liu F, Zou X, Hotchkiss C, Hanig JP, Patterson TA,
MA
22.
NU
in adult rats. Neuropharmacology. 2011; 61(8): p. 1354-9.
D
Slikker W Jr, Wang C. Ketamine anesthesia during the first week of life can cause
23.
CE P
p. 220-30.
TE
long-lasting cognitive deficits in rhesus monkeys. Neurotoxicol Teratol. 2011; 33(2):
Havekes R, Abel T, Van der Zee EA. The cholinergic system and neostriatal memory
24.
AC
functions. Behav Brain Res 2011; 221(2): p. 412-23. Mizoguchi K, Shoji H, Tanaka Y, Tabira T. Orbitofrontal dopaminergic dysfunction causes age-related impairment of reversal learning in rats. Neuroscience. 2010; 170(4): p. 1110-9. 25.
Shu SY, Wu YM, Bao XM, Wen ZB, Huang FH, Li SX, Fu QZ, Ning Q. A new area in the human brain associated with learning and memory: immunohistochemical and functional MRI analysis. Mol Psychiatry. 2002; 7(9): p. 1018-22.
26.
Paradies G, Petrosillo G, Paradies V, Ruggiero FM. Mitochondrial dysfunction in brain
ACCEPTED MANUSCRIPT aging: role of oxidative stress and cardiolipin. Neurochem Int. 2011; 58(4): p. 447-57. Abeti R, Duchen M.R. Activation of PARP by oxidative stress induced by
T
27.
IP
beta-amyloid: implications for Alzheimer's disease. Neurochem Res. 2012; 37(11): p.
28.
SC R
2589-96.
Querfurth HW, LaFerla F.M. Alzheimer's disease. N Engl J Med. 2010; 362(4): p.
Mao X, Hao S, Zhu Z, Zhang H, Wu W, Xu F, Liu B. Procyanidins protects against
MA
29.
NU
329-44.
D
oxidative damage and cognitive deficits after traumatic brain injury. Brain Inj
Shen X. Dong Y, Xu Z, Wang H, Miao C, Soriano SG, Sun D, Baxter MG, Zhang Y,
CE P
30.
TE
2015;29(1):86-92
Xie Z. Selective anesthesia-induced neuroinflammation in developing mouse brain
31.
AC
and cognitive impairment. Anesthesiology. 2013; 118(3): p. 502-15. Malm TM, Iivonen H, Goldsteins G, Keksa-Goldsteine V, Ahtoniemi T, Kanninen K, Salminen A, Auriola S, Van Groen T, Tanila H, Koistinaho J. Pyrrolidine dithiocarbamate activates Akt and improves spatial learning in APP/PS1 mice without affecting beta-amyloid burden. J Neurosci. 2007; 27(14): p. 3712-21. 32.
Bisaglia M, Venezia V, Biglieri M, Russo C, Mancini F, Milanese C, Schettini G. alpha-Glycerylphosphorylethanolamine
rescues
astrocytes
from
mitochondrial
impairment and oxidative stress induced by amyloid beta-peptides. Neurochem Int.
ACCEPTED MANUSCRIPT 2004; 44(3): p. 161-70. Zhang Y, Zhang FG, Meng C, Tian SY, Wang YX, Zhao W, Chen J, Zhang XS, Liang
T
33.
IP
Y, Zhang SD, Xing YJ Inhibition of sevoflurane postconditioning against cerebral
SC R
ischemia reperfusion-induced oxidative injury in rats. Molecules. 2012; 17(1): p. 341-54.
NU
Bedirli N, Bagriacik EU, Emmez H, Yilmaz G, Unal Y, Ozkose Z Sevoflurane and
MA
isoflurane preconditioning provides neuroprotection by inhibition of apoptosis-related
CE P
TE
2012; 24(4): p. 336-44.
D
mRNA expression in a rat model of focal cerebral ischemia. J Neurosurg Anesthesiol.
AC
34.
ACCEPTED MANUSCRIPT Figure legends
T
Figure 1 Schematic illustrations of animal injection and behavioral studies. On P22，the water-maze
IP
probe test was performed before sevoflurane inhalation and on D1, Y-maze test was performed before the
SC R
water-maze. “P” stands for the day after intra-hippocampal injection while “D” stands for days after
sevoflurane inhalation.
NU
Figure 2 Scores in electrical Y-maze of rats after anesthesia. The injection of Aβ1-40 impaired the
MA
Y-maze performance, which was further aggravated by addition of sevoflurane. a, p<0.05 compared to the
D
control group; b, p<0.05 compared to Aβ+O2 group; c, p<0.05 compared to Aβ+S1 group. Between-group
CE P
(SD). N=24 per group.
TE
comparisons were performed using post-hoc Tukey test. Data were presented as mean ± standard deviation
Figure 3 Water-maze performances of pre-anesthesia rats. (A) No major difference existed in average
AC
swimming speed across all groups. (B) The escape latency was increased after the application of Aβ1-40. (C)
The time spent in exploration of the original platform was also lowered in all three treatment groups. These
results supported aggravated learning and memory functions. Between-group comparisons were performed using post-hoc Tukey test. a, p<0.05 compared to the control group. Data were presented as mean ±
standard deviation (SD). N=24 per group.
Figure 4 Water-maze performance of post-anesthesia rats. (A) No difference existed in average
swimming speed. (B) Elevated escape latency and (C) time spent in exploration of the original platform
was also increased in sevoflurane-treated animals. Between-group comparisons were performed using
ACCEPTED MANUSCRIPT post-hoc Bonferroni test. Data were presented as mean ± standard deviation (SD). a, p<0.05 compared to
T
the control group; c, p<0.05 compared to Aβ+S1 group. N=8 per group.
IP
Figure 5 Representative movement path in post-anesthesia rats in water-maze. Rats from the sham
SC R
group, control group, Aβ+O2 group, Aβ+S1 and Aβ+S2 group were tested in D1 (left panel), D3 (middle
panel) and D7 (right panel) in Morris water-maze task, with their movement path (red curves) in finding
NU
the hidden platform (green circle). Roman numbers (I to IV) indicate four arbitrary divided quadrants
MA
along with the orientations (NE, northeast; SE, southeast; SW, southwest; NW, northwest). Only one
D
representative path was shown at each group. N=8 per group.
TE
Figure 6 S100β expression profiles in post-anesthesia rat hippocampus. IHC staining technique was
CE P
employed to reveal the expression of S100β proteins in hippocampal CA1 regions. Representative staining
images (X400 under a light-field microscope) were shown on D1 (left panel), D3 (middle panel) and D7
AC
(right panel) after anesthesia. S100β was abundantly expressed in cytoplasm of neurons and glial cells in control and sham rats. The addition of Aβ1-40 decreased expression levels in neurons but not in glial cells. The application of sevoflurane further aggravated the depletion. Scale bar, 20 μm.
ACCEPTED MANUSCRIPT
AC
CE P
TE
D
MA
NU
SC R
IP
T
Figure 1
D
TE
CE P
AC
2
20
10
T
30 a
IP
SC R
NU
0
β+ S
1
β+ S
a
A
MA
A
2
β+ O
on tr ol
A
C
Sh am
Number of correct trials in Y-maze
ACCEPTED MANUSCRIPT
Figure 2
40
a,b,c
a
S2
60
S2
D
a
β+
S1 A β+
A β+
S2
S1
2
O
tr ol
am
A β+
C on
Sh
Swimming speed (cm/s)
T
30
IP
20
10
0
SC R
NU
MA
40
A
β+
TE
(B)
A β+
S1
A
O 2
30
A β+
2
O
β+
A
CE P
ro l
20
A β+
tr ol
0
on t
C
Escpae latency (s) 10
C on
am
Sh
AC
(C)
Sh am
Time spent in exploration of the original platform (%)
ACCEPTED MANUSCRIPT
Figure 3
(A) 40
a a
80
a
a
40
20
0
2
2
S2
β+
A
20
30
T
IP
SC R
NU
S1
0
β+ S
a β+ S
A
1
β+
A
2
O
tr ol
β+
A
C on
am
Sh
Swimming speed (cm/s) 10
A
1
60
β+ S
a
MA
50
β+ S
D
TE
40
A
2
β+ O
A
ol
tr
(B)
A
2
O
β+
l
tr o
30
A
on
CE P
10
C on
Sh
Escpae latency (s) 20
C
am
am
0
AC
(C)
Sh
Time spent in exploration of the original platform (%)
ACCEPTED MANUSCRIPT
Figure 4
(A) 40
D1 D3 D7
a,b,c
a
80
a
a,b,c
40
20
0
D1 D3 D7
D1 D3 D7
ACCEPTED MANUSCRIPT
AC
CE P
TE
D
MA
NU
SC R
IP
T
Figure 5
ACCEPTED MANUSCRIPT
AC
CE P
TE
D
MA
NU
SC R
IP
T
Figure 6
ACCEPTED MANUSCRIPT Table 1 Post-anesthesia serum S100β levels (pg/ml) D3
D7
Sham
10.9±1.4
11.6±2.2
Control
11.2±1.2
12.1±2.0
Aβ+O2
19.0±3.3 a
19.7±3.5a
18.9±2.1a
Aβ+S1
20.9±3.7a
21.3±4.8 a
21.1±4.2a
Aβ+S2
28.8±5.1abc
33.0±5.2abc
33.9±4.6 abc
T
D1
NU
SC R
IP
11.1±2.8 11.9±2.2
MA
Group
TE
D
Note: a , p<0.05 compared to the control group; b, p<0.05 compared to Aβ group; c, p<0.05
AC
CE P
compared to S1 group. N=8 per group.
ACCEPTED MANUSCRIPT Table 2 IOD/area ratios in post-anesthesia rats D3
D7
Sham
0.27±0.02
0.32±0.03
Control
0.25±0.01
0.31±0.03
Aβ+O2
0.20±0.01a
0.21±0.02a
0.21±0.03a
Aβ+S1
0.21±0.03a
0.21±0.01a
0.22±0.01a
Aβ+S2
0.11±0.01abc
0.09±0abc
0.09±0.01 abc
T
D1
NU
SC R
IP
0.26±0.04 0.24±0.05
MA
Group
TE
D
Note: a , p<0.05 compared to the control group; b, p<0.05 compared to Aβ group; c, p<0.05
AC
CE P
compared to S1 group. N=8 per group.
ACCEPTED MANUSCRIPT Table 3 Hippocampal SOD activity and MDA contents in post-anesthesia rats SOD (U/mg)
MDA (nmol/mg)
D3
D7
D1
Sham
30.2±3.1
31.5±3.0
28.7±2.0
1.90±0.28
Control
28.2±1.9
28.9±1.7
30.1±1.5
Aβ+O2
21.2±1.6a
20.3±1.5a
Aβ+S1
22.2±2.0a
21.2±2.1a
1.82±0.21
1.79±0.19
1.78±0.19
21.0±1.8a
4.71±0.39a
4.55±0.45a
4.61±0.51a
22.9±1.8a
5.06±0.69a
5.11±0.76a
5.30±0.81a
8.23±0.54a
9.30±1.05a
9.51±0.92a
bc
bc
bc
MA
NU
SC R
1.81±0.20
16.0±1.1abc
TE
D
16.8±1.4abc
D7
1.82±0.23
Aβ+S2 17.3±1.5abc
D3
IP
D1
T
Group
CE P
Note: a , p<0.05 compared to the control group; b, p<0.05 compared to Aβ group; c, p<0.05
AC
compared to S1 group. N=8 per group.