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Preoperative vitamin-rich carbohydrate loading alleviates postoperative cognitive dysfunction in aged rats
Accepted Manuscript Title: Preoperative vitamin-rich carbohydrate loading alleviates postoperative cognitive dysfunction in aged rats Authors: Youbo Zuo, Xingping Hu, Qiuyan Yang, Lei Zhao, Xueli Chen, Jiangtao Lin, Tiande Yang PII: DOI: Article Number:
S0166-4328(19)30218-9 https://doi.org/10.1016/j.bbr.2019.112107 112107
Reference:
BBR 112107
To appear in:
Behavioural Brain Research
Received date: Revised date: Accepted date:
9 February 2019 15 July 2019 20 July 2019
Please cite this article as: Zuo Y, Hu X, Yang Q, Zhao L, Chen X, Lin J, Yang T, Preoperative vitamin-rich carbohydrate loading alleviates postoperative cognitive dysfunction in aged rats, Behavioural Brain Research (2019), https://doi.org/10.1016/j.bbr.2019.112107 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.
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Preoperative vitamin-rich carbohydrate loading alleviates postoperative cognitive dysfunction in aged rats Youbo Zuoa,b, Xingping Huc, Qiuyan Yangc, Lei Zhaob, Xueli Chend, Jiangtao Lind, Tiande Yanga,* Department of Anesthesiology, Xinqiao Hospital, Army Medical University,
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a
Chongqing 400037, China
Department of Anesthesiology, Affiliated Hospital of North Sichuan Medical
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b
College, Nanchong 637000, China. E-mail: [email protected] c
Department of Emergency, Affiliated Hospital of North Sichuan Medical College,
Department of Anesthesiology, North Sichuan Medical College, Nanchong 637007,
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d
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Nanchong 637000, China. E-mail: [email protected]
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China. E-mail: [email protected]
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* Corresponding author: Tian-de Yang
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Department of Anesthesiology, Xinqiao Hospital, Army Medical University, 183, Xinqiao Street, Shapingba District, Chongqing 400037, China
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Tel: +86 17308171218
E-mail address: [email protected]
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Non-standard abbreviations: CHO, carbohydrates; IR, insulin resistance; ER, endoplasmic reticulum; ERAS, enhanced recovery after surgery; GRP-78, glucoseregulated protein 78; eIF2a, eukaryotic translation initiation factor 2 subunit a
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Highlights
Preoperative carbohydrate loading alleviates POCD.
Preoperative carbohydrate loading attenuates endoplasmic reticulum stress.
Preoperative carbohydrate loading suppresses apoptosis.
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Abstract With the acceleration of an aging population, postoperative cognitive dysfunction
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(POCD) has become a large problem. Preoperative carbohydrate (CHO) loading has
been reported to attenuate surgery stress response and insulin resistance. The present study aimed to investigate whether preoperative vitamin-rich CHO loading has an
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effect on POCD, endoplasmic reticulum (ER) stress, and apoptosis. Eighty male
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Sprague-Dawley rats (20-month old) were randomly assigned to four groups (20 per
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group): control group (no anesthesia and surgery), fasting group (fasting 14 h before
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surgery), water group (oral water 3 h before surgery), and CHO group (oral vitamin-
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rich CHO 3 h before surgery). The POCD rat model was established by splenectomy under intraperitoneal injection of pentobarbital sodium. Cognitive function was
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assessed using the Morris water maze (MWM) after surgery. The levels of endoplasmic reticulum (ER) stress markers and apoptosis related proteins in the
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hippocampus were examined by western blot analysis. The vitamin-rich CHO treated animals performed better in the MWM tests than the animals in the fasting and water
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groups. Furthermore, preoperative CHO loading reduced ER stress and neuronal apoptosis in the hippocampus of aged rats, as indicated by the protein biomarkers of GRP78, eIF2a, Beclin1, Bax, and Bcl-2. In conclusion, preoperative vitamin-rich CHO loading could improve POCD by attenuating ER stress and neural apoptosis, providing a basis as a potential treatment against POCD.
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Keywords: Carbohydrate; Postoperative cognitive dysfunction; Rats; Hippocampus; Endoplasmic reticulum stress; Apoptosis.
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1. Introduction
Postoperative cognitive dysfunction (POCD) is recognized as a common nervous
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system complication characterized by impairment of learning and memory and
difficulty in language comprehension after anesthesia and surgery, especially in
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elderly patients. POCD seriously affects patients’ quality of life and leads to a high
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risk of disability and mortality [1,2]. A recent increase in the number of elderly
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patients undergoing surgery has resulted in an increased incidence of POCD [3].
POCD remains unclear.
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However, there is still a lack of effective treatment for POCD, and the pathogenesis of
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Advancing age, the severity of surgery, the occurrence of complications, and coexisting illnesses have been implicated as the main risk factors for POCD [4,5].
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Surgery gives rise to circulating cortisol and inflammatory cytokines, resulting in
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insulin resistance (IR), which contributes to many postoperative complications. Previous reports have shown that inflammation plays a critical role in the development of POCD [6]. POCD is correlated with the concentration of peripheral
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and cerebrospinal fluid (CSF) inflammatory markers, such as CRP and IL-6 [7]. Growing evidence also supports the concept that IR is important in the pathogenesis of cognitive impairment and neurodegeneration [8]. Additionally, nutrient deprivation, inflammatory response, and IR during surgery, can also trigger ER stress and apoptosis [9]. Recent studies have reported that endoplasmic reticulum (ER)
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stress and ER stress-related apoptosis play a pivotal role in certain neurological disorders and neurodegenerative diseases, including Alzheimer's disease (AD) and Parkinson’s disease [10,11]. Preoperative carbohydrate (CHO) loading is an important element of the Enhanced Recovery After Surgery (ERAS) protocol [12]. It has been reported that
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preoperative CHO loading is able to reduce IR [13,14] and decrease circulating IL-6
and CRP concentrations [15,16], suggesting that preoperative CHO loading may have
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a positive impact on cognitive impairment. The present study aimed to investigate
whether preoperative vitamin-rich CHO loading can attenuate splenectomy-induced
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cognitive decline and to determine whether it has an impact on ER stress and
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apoptosis in rats after surgery.
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2. Materials and Methods 2.1. Animals
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All experiments were approved by the Animal Ethics Committee of North Sichuan Medical College and were performed in accordance with the National
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Institutes of Health guide for the care and use of Laboratory animals (NIH
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Publications No. 8023, revised 1978). Male Sprague-Dawley rats (weight, 500-650 g; age, 20 months) were supplied by the Laboratory Animal Center of North Sichuan Medical College (Nanchong, China). All animals were initially housed in groups
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under a normal 12/12 h light/dark cycle in a temperature and humidity-controlled room (22 ± 1°C and 50–60%, respectively). The animals were allowed to acclimate for a week prior to the experiments, with free access to standard rat chow and tap water.
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2.2. Vitamin-rich carbohydrates The vitamin-rich CHO beverage (14.2% carbohydrates, Outfast, YICHANG HUMANWELL PHARMACEUTICAL CO., LTD. China) used in this study contained water, maltodextrin, crystalline fructose, glucose, food additives (sodium citrate, citric acid monopotassium phosphate, potassium sorbate, L-malic acid),
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taurine, zinc gluconate, vitamin B1, vitamin B6, vitamin B12, and flavor (Table 1).
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2.3. Study design
Animals (n = 80) were randomly assigned to four groups (20 per group): control, fasting, water, and CHO. Except for those in the control group, all rats were fasted
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beginning at 7:00 pm on the night prior to surgery. Rats in the fasting group did not
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receive anything, but were fasted for 14 h prior to undergoing a splenectomy. Rats in
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the water and CHO groups voluntarily ingested 12 ml of clear water or vitamin-rich
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CHO beverage 3 h prior to splenectomy (during 6:00 to 7:00 am on the day of surgery). The selected dosage of the CHO is equivalent to the standard morning CHO dose for
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surgical patients [17].
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humans, which is grade A recommended as part of the preoperative care of most
For the splenectomy, rats were anesthetized by an intraperitoneal injection of
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pentobarbital sodium 35 mg/kg. After anesthesia induction, animals were fixed on the operating table. The entire surgical process was performed under sterile conditions. The spleen was exposed through a 2-cm incision in the left upper abdominal quadrant and
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was removed from the abdomen. The blood vessels of the spleen were ligated using 70 silk suture, and the spleen was removed by transecting the blood vessels near to the spleen. The wound was infiltrated with 0.25% bupivacaine and closed using sterile sutures. The body temperature of the animals was maintained at 37℃, and SpO2 was monitored via pulse oximetry during the surgery. Animals were housed individually in
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cages with free access to food and water after surgery. The surgery process lasted about 30 minutes. After splenectomy, half of the rats in each group were anesthetized with sodium pentobarbital via an intraperitoneal injection (60 mg/kg) on postoperative day 1. The brains were quickly removed and placed on ice. The right hemisphere of the brain was
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dissected and stored at -70℃ in a refrigerator and used for western blot analysis.
Biomarkers, such as glucose-regulated protein 78 (GRP-78), eukaryotic initiation factor
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2 (eIF2a), Beclin-1, Bax, and Bcl-2, were measured in our study. The remaining rats in each group underwent the Morris water maze test on postoperative day 2, and were
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sacrificed on postoperative day 7 following the probe test.
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2.4. Morris water maze test
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Spatial learning and memory were assessed using the classic Morris water maze, which consists of an orientation navigation test and a spatial probe test [18]. The maze
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includes a swimming pool with a diameter of 120 cm and a height of 50 cm, and a camera system. The pool was filled with water (22℃) to a depth of 25 cm. The
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addition of black nontoxic paint to the pool provided an opaque surface. Visual cues were positioned around the pool, which was divided into four equal quadrants. A
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hidden platform (10 cm in diameter) was fixed at 2 cm below the water surface in one quadrant of the pool, and this position of the platform remained unchanged for all the
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training sessions. The orientation navigation test was started on postoperative day 2 and lasted 5 days. The rats were randomly placed into the water four times per day in the four different quadrants. The swimming paths of the rats were automatically recorded by the camera system and analyzed using the corresponding software. The maximum time for each rat to search for the hidden platform was limited to 60 s. The
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rats were then allowed to stay on the platform for 15 s to remember the surrounding markers. If the rat failed to find the hidden platform within 60 s, it was guided to the platform and allowed to stay there for 15 s, and the escape latency was recorded as 60 s. Spatial probe tests were conducted on postoperative day 7. For this test, the platform was removed and the rats were allowed to swim for 60 s. The swim time to
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the target quadrant and the times across the platform were recorded. All the trials
2.5. Protein extraction and western blotting analysis
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were performed between 8:00 am and 12:00 am.
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Tissue sampling, protein extraction, and western blot analysis were carried out as
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previously described [19]. Total protein was extracted from the hippocampus, and the
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concentration of the protein was determined using the Bradford method. In brief,
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hippocampal samples were weighed and lysed with cold radio-immunoprecipitation assay lysis buffer (RIPA, Boster, AR0105-100, Wuhan, China) and protease inhibitor
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(Boster, AR1182, Wuhan, China). After being kept on the ice for 30 min, the samples were centrifuged at 12,000 rpm for 15 min. The total quantity of protein in the
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supernatants was measured using the BCA method. The hippocampus samples were run on a precast SDS-PAGE gel with 10 μg
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proteins in each lane for 2 h and transferred to a polyvinylidene difluoride membrane (PVDF). After washing with 0.1% Tris-HCl buffer solution and Tween (TBST) for 5
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min, the membranes were blocked in 5% non-fat milk for 1 h. Afterward, the proteins were incubated with the following primary antibodies overnight (4 ℃): GRP78 (1:2000, Bioss), eIF2a (1:1000, Bioss), Beclin-1 (1:1000, CST), Bax (1:1000, CST), Bcl-2 (1:400, Boster), and GAPDH (1:8000, Boster). The membrane then was washed with 0.1% TBST five times, for 5 min each, and incubated with secondary antibodies,
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horseradish peroxidase (HRP)-conjugated goat anti-rabbit (1:7500, Boster, China), for 1 h. Finally, the membrane was washed as above and the ECL reagents (Boster, China) were used to detect the proteins. The bands were visualized by a Chemi DocXRS+ Imaging System (Bio-Rad) and analyzed using the Quantity-One software. The density ratio represented the relative intensity of each band against GAPDH as
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the loading control.
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2.6. Statistics
All data were analyzed by SPSS 17.0 statistical software (SPSS Inc, Chicago, IL, USA). All of the quantitative data were expressed as mean ± SEM. Data analyzed by
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repeated measures ANOVA were firstly assessed for sphericity using Mauchly’s test.
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The Greenhouse-Geisser correction was used to correct the F statistic and assess
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significance if the data violated the assumption of sphericity. Data analyzed by ANOVA were assessed for homogeneity of variance with Levene’s test. Data with
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equal variances were assessed post hoc using the least significant difference (LSD) test, while data that violated Levene’s test were assessed post hoc using Games-
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Howell. Behavioral data from the orientation navigation test were analyzed by mixeddesign ANOVA. Data from the probe test and western blotting studies were analyzed
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using one-way ANOVA. P<0.05 was considered to be statistically significant. 3. Results
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3.1. MWM test
The MWM test was conducted to investigate the spatial learning and memory of
the experimental rats. In the Water maze tests, mixed two-way ANOVA with the between-subject factor “Treatment” and the within-subject factor “Days” was used to analyze the difference of escape latency. Data were firstly assessed for sphericity
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using Mauchly’s test. The test confirmed that the data violated the assumption of sphericity, and the Greenhouse-Geisser correction was used to correct the F statistic and assess significance. Escape latency decreased over the 5 d training period (Fig. 1A). Repeated-measures ANOVA identified a significant reduction in escape latency with training days (F (3.3, 117.4) =109.884, p<0.001). Significant difference among
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groups was also identified (F (3, 36) =3.143, p = 0.037). Post hoc analysis using LSD test revealed that rats in the fasting group had a greater latency than those in the
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control group (p = 0.016), while rats received vitamin-rich CHO had a shorter latency than the fasting group (p = 0.029).
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To assess spatial memory, a probe trial was administered on postoperative day 7.
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One-way ANOVA of the time spent in the target quadrant (F (3, 35) = 4.781, p =
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0.007) and number of platform crossings (F (3, 35) = 5.227, p = 0.004) showed a
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significant difference among groups. Post hoc multiple comparison (LSD) showed that the time spent in the target quadrant in the fasting group (p=0.006) and water
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group (p = 0.007) was shorter than those in the control group. The time spent in the target quadrant in the CHO group was longer than those in the fasting group (p =
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0.021) and water group (p = 0.025) (Fig. 1B). The number of platform crossings in the fasting group (p = 0.047) and water group (p = 0.012) were lower than those in the
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control group. The number of platform crossings was higher in the CHO group, compared with the fasting group (p = 0.008) and water group (p = 0.002) (Fig. 1C).
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The data suggest that preoperative vitamin-rich CHO loading could improve the
cognitive impairment induced by splenectomy and fasting. 3.2 ER stress To evaluate whether the administration of vitamin-rich CHO could attenuate surgery-induced ER stress, we measured the expression levels of ER stress associated
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proteins in the hippocampus. The western blot results revealed that the expression levels of GRP78 and eIF2a were significantly up-regulated in the hippocampus on postoperative day 1 in the fasting and water groups, when compared with those of the control group (P<0.01; Fig. 2). Preoperative vitamin-rich CHO treatment significantly inhibited the expression of GRP78 and eIF2a in the hippocampus after surgery
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(P<0.01; Fig. 2). There was no significant difference in the GRP78 and eIF2a
expression levels in rats between the control group and CHO group. These results
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indicate that preoperative vitamin-rich CHO loading has a positive impact on ER stress during surgery.
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3.2 Apoptosis
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To evaluate cell apoptosis, apoptosis-associated proteins were detected by
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western blot. On postoperative day 1, the results of the western blot analysis showed
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that the expression levels of Beclin-1 and Bax were increased after splenectomy, and
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the vitamin-rich CHO treatment significantly attenuated the surgery-induced upregulation of Beclin-1 and Bax levels (P<0.01; Fig. 3A,B). On the other hand,
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splenectomy and fasting down-regulated the expression of Bcl-2, and vitamin-rich CHO treatment significantly blocked surgery-induced decreases of Bcl-2 (P<0.01;
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Fig. 3C). There was no significant difference in the Beclin-1, Bax, and Bcl-2 expression levels between the control group and CHO group; the treatment with water
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exhibited the same effects as the fasting group. These findings suggest that preoperative oral vitamin-rich CHO significantly attenuated apoptosis after splenectomy and fasting.
4. Discussion
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In this study, we investigated the neuroprotective effects of vitamin-rich CHO in a splenectomy animal model. The results showed that preoperative vitamin-rich CHO loading significantly improved postoperative cognitive impairment. Furthermore, we studied whether the vitamin-rich CHO affected ER stress and apoptosis induced by surgery. The results revealed that preoperative vitamin-rich CHO loading can relieve
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ER stress and suppress apoptosis in the hippocampus of rats that underwent splenectomy. Our study results indicate that ER stress and apoptosis in the
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hippocampus may be associated with impairment in spatial learning and memory induced by surgery and fasting. Thus, preoperative CHO loading might be an
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effective treatment against POCD in clinical practice.
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The main purpose of this study was to evaluate the effect of vitamin-rich CHO
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loading on POCD. POCD is a frequent complication following major surgery in the
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elderly. The MWM test, which is a common way to evaluate learning and memory ability in animals [18], was employed to study the effects of the vitamin-rich CHO on
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splenectomy-induced cognitive impairment in aged rats. Previous studies have demonstrated that inflammation and IR are correlated with cognitive impairment [6-
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8]. Recently, it has been reported that preoperative CHO loading reduces postoperative IR [13,14,20], decreases circulating IL-6 and CRP concentrations
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[15,16], and even has a positive effect on gut recovery after surgery [21,22], indicating that preoperative CHO loading may have an impact on POCD.
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In the current study, rats undergoing splenectomy and fasting showed
significant decreases in the learning and memory measured by way of the MWM test, suggesting that a cognitive impairment model was successfully established. The addition of a vitamin-rich CHO beverage diminished the cognitive impairment after surgery, resulting in outcomes comparable to those of the control animals. The results
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demonstrate that preoperative vitamin-rich CHO loading has a positive effect on POCD. ER stress is one of the pivotal molecular mechanisms underlying POCD. The ER is a cellular organelle which plays a critical role in protein folding. Various harmful stimuli and pathological conditions, such as nutrient deprivation, inflammation,
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oxidative stress, and hypoxia, may impair ER function and eventually lead to ER
stress. Tian et al. [23] showed that minor abdominal surgery triggered ER stress and
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apoptosis in the hippocampus of mice. To evaluate ER stress after splenectomy,
protein levels of ER stress markers, such as GRP78 and eIF2a, were measured in the
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hippocampus in the present study. GRP 78 is an important biomarker of ER stress,
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which plays a critical role in the regulation of ER functioning [24], and eIF2a is a key
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downstream mediator of PERK. Our study found that preoperative vitamin-rich CHO
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loading alleviated the upregulation of GRP-78 and eIF2a in the hippocampus of splenectomy rats, indicating that the potential neuroprotective effects of preoperative
hippocampus.
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vitamin-rich CHO loading is associated with the alleviation of ER stress in the
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ER stress and ER stress-induced inflammation and insulin resistance form a vicious cycle, which ultimately leads to neuronal cell death through autophagy and
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apoptosis [9]. Previous studies have shown that ER stress-related apoptosis in the hippocampus is a predominant pathological mechanism in several neurological
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disorders [10,11,25]. To further learn the effect of the vitamin-rich CHO on apoptosis, the protein levels of Beclin-1, Bax, and Bcl-2 were measured in the hippocampus of rats in this study. Beclin-1 is a main component of the autophagy machinery, playing a crucial role in the initiation of autophagy. Bax and Bcl-2 are two separate members of a gene family that is important in the regulation of apoptosis [26]. It is commonly
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considered that Bax is a pro-apoptotic protein, while Bcl-2 is anti-apoptotic protein [27]. The interaction between the autophagy protein Beclin-1 and the anti-apoptotic protein Bcl-2 has been shown to regulate the switch between the autophagic and apoptotic machinery. In this study, splenectomy and starvation induced ER stress, resulting in
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autophagy and the production of high levels of Beclin-1. Severe and consistent ER
stress will lead to apoptosis. The binding of Beclin-1 to Bcl-2 disrupts the balance of
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Bax and Bcl-2, which are closely associated to apoptosis. Our study found that the
expression of Bcl-2 was down-regulated, whereas Bax and Beclin-1 levels were up-
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regulated in the fasting and water groups. However, the vitamin-rich CHO loading
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reversed these results and blocked the apoptosis induced by splenectomy.
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Although this study discovered the effects of the vitamin-rich CHO on POCD,
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ER stress, and apoptosis in aged rats, some limitations could not be ignored. Firstly, besides carbohydrates, other components of the vitamin-rich CHO, such as vitamin,
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zinc, could also exert an effect on neurodegenerative diseases [28,29]. Further study should explore the neuroprotective effects of carbohydrates only. Secondly, only a
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morning dose of CHO was administrated in this study, without further investigation of a combined evening dose. Therefore, further studies focusing on suitable doses of
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CHO are needed.
In conclusion, the present study demonstrated that preoperative vitamin-rich
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CHO loading improves POCD induced by splenectomy, which may be associated with the inhibition of ER stress and apoptosis in the hippocampus of rats. Our findings suggest that preoperative vitamin-rich CHO loading may be a potential treatment strategy for preventing POCD.
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Statement of Authorship YBZ and TDY contributed to the conception and design of the study and had primary responsibility for the interpretation of the data; YBZ generated and analyzed the experimental data and drafted the article; XPH, QYY, LZ, XLC, and JTL performed the animal experiments and the laboratory tests; all authors approved the
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final draft of the manuscript be submitted.
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Funding
This work was supported by the Foundation of Sichuan Educational Committee,
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China [grant number 17ZB0179].
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Declarations of interest
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None.
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Acknowledgements
We would like to thank Mei Zeng from the School of Basic Medical Science,
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North Sichuan Medical College for providing helpful advice and invaluable support
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regarding the experiments.
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[23] Tian A, Ma H, Zhang R, Cui Y, Wan C. Edaravone improves spatial memory and modulates endoplasmic reticulum stress-mediated apoptosis after abdominal surgery in mice. Exp Ther Med 2017; 14: 355-360. http://dx.doi.org/10.3892/etm.2017.4489. [24] Xu C, Bailly-Maitre B, Reed JC. Endoplasmic reticulum stress: cell life and death decisions. J Clin Invest 2005; 115: 2656-2664.
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http://dx.doi.org/10.1172/JCI26373.
[25] Sun H, Yang Y, Shao H, Sun W, Gu M, Wang H, et al. Sodium arsenite-induced
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http://dx.doi.org/10.3389/fnmol.2017.00286.
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[26] Hwang L, Choi IY, Kim SE, Ko IG, Shin MS, Kim CJ, et al. Dexmedetomidine
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ameliorates intracerebral hemorrhage-induced memory impairment by inhibiting
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apoptosis and enhancing brain-derived neurotrophic factor expression in the rat hippocampus. Int J Mol Med 2013; 31: 1047-1056.
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http://dx.doi.org/10.3892/ijmm.2013.1301.
[27] Reed CJ. Apoptosis and cancer: Strategies for integrating programmed cell death.
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Semin Hematol 2000; 37: 9-16. http://dx.doi.org/10.1016/S0037-1963(00)90055-6. [28] Moosavirad SA, Rabbani M, Sharifzadeh M, Hosseini-Sharifabad A. Protective
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effect of vitamin C, vitamin B12 and omega-3 on lead-induced memory impairment in rat. Res Pharm Sci. 2016; 11: 390–396. http://dx.doi.org/ 10.4103/1735-
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5362.192490.
[29] Kawahara M, Tanaka KI, Kato-Negishi M. Zinc, carnosine, and neurodegenerative diseases. Nutrients 2018; 10: 147. http://dx.doi.org/10.3390/nu10020147.
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Figure captions
Fig. 1. Effects of vitamin-rich CHO on cognitive function in aged rats tested by MWM. (A) Mean
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latency in the orientation navigation was tested for 5 consecutive days. Data presented as mean ±
SEM. n = 10. *P < 0.05 vs. fasting group. (B) The percent of the time spent in the target quadrant (where the platform was located during hidden platform training) during the probe test. (C)
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Number of crossings over the former location of the platform during the probe test. Data presented
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as mean ± SEM. n = 10. *P < 0.05 vs. control group; **P < 0.01 vs. control group; #P < 0.05 vs.
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CHO group; ##P < 0.01 vs. CHO group. MWM, Morris water maze.
Fig. 2. The expression of GRP78 and eIF2a in the hippocampus on postoperative day 1. (A) Representative western blot for GRP78 in the hippocampus and relative density analysis of the GRP78 protein bands. The relative density is expressed as a ratio (GRP78/GAPDH). (B) Representative western blot for eIF2a in the hippocampus and relative density analysis of the eIF2a protein bands. The relative density is expressed as a ratio (eIF2a/GAPDH). Data were
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expressed as mean ± SEM. n=10. **P < 0.01 vs. control group; #P < 0.05 vs. CHO group; ##P <
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0.01 vs. CHO group.
Fig. 3. The expression of Beclin-1, Bax, and Bcl-2 in the hippocampus on postoperative day 1. (A) Representative western blot for Beclin-1 in the hippocampus and relative density analysis of
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the Beclin-1 protein bands. The relative density is expressed as a ratio (Beclin-1/GAPDH). (B)
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Representative western blot for Bax in the hippocampus and relative density analysis of the Bax
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protein bands. The relative density is expressed as a ratio (Bax/GAPDH). (C) Representative western blot for Bcl-2 in the hippocampus and relative density analysis of the Bcl-2 protein bands.
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The relative density is expressed as a ratio (Bcl-2/GAPDH). Data were expressed as mean ± SEM.
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n = 10. **P < 0.01 vs. control group; #P < 0.05 vs. CHO group; ##P < 0.01 vs. CHO group.
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Table 1. Content of the vitamin-rich carbohydrate beverage per 100 mL Per 100 ml 57.6 kCal 14.2 g 45 mg 0.24 mg 0.13 mg 0.14 mg 1.48 mg
NRV% 3% 5% 2% 17% 9% 6% 10%
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Ingredient Calories Carbohydrates Sodium VitaminB1 VitaminB6 VitaminB12 Zinc
S0166-4328(19)30218-9 https://doi.org/10.1016/j.bbr.2019.112107 112107
Reference:
BBR 112107
To appear in:
Behavioural Brain Research
Received date: Revised date: Accepted date:
9 February 2019 15 July 2019 20 July 2019
Please cite this article as: Zuo Y, Hu X, Yang Q, Zhao L, Chen X, Lin J, Yang T, Preoperative vitamin-rich carbohydrate loading alleviates postoperative cognitive dysfunction in aged rats, Behavioural Brain Research (2019), https://doi.org/10.1016/j.bbr.2019.112107 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.
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Preoperative vitamin-rich carbohydrate loading alleviates postoperative cognitive dysfunction in aged rats Youbo Zuoa,b, Xingping Huc, Qiuyan Yangc, Lei Zhaob, Xueli Chend, Jiangtao Lind, Tiande Yanga,* Department of Anesthesiology, Xinqiao Hospital, Army Medical University,
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a
Chongqing 400037, China
Department of Anesthesiology, Affiliated Hospital of North Sichuan Medical
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b
College, Nanchong 637000, China. E-mail: [email protected] c
Department of Emergency, Affiliated Hospital of North Sichuan Medical College,
Department of Anesthesiology, North Sichuan Medical College, Nanchong 637007,
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d
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Nanchong 637000, China. E-mail: [email protected]
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China. E-mail: [email protected]
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* Corresponding author: Tian-de Yang
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Department of Anesthesiology, Xinqiao Hospital, Army Medical University, 183, Xinqiao Street, Shapingba District, Chongqing 400037, China
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Tel: +86 17308171218
E-mail address: [email protected]
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Non-standard abbreviations: CHO, carbohydrates; IR, insulin resistance; ER, endoplasmic reticulum; ERAS, enhanced recovery after surgery; GRP-78, glucoseregulated protein 78; eIF2a, eukaryotic translation initiation factor 2 subunit a
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Highlights
Preoperative carbohydrate loading alleviates POCD.
Preoperative carbohydrate loading attenuates endoplasmic reticulum stress.
Preoperative carbohydrate loading suppresses apoptosis.
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Abstract With the acceleration of an aging population, postoperative cognitive dysfunction
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(POCD) has become a large problem. Preoperative carbohydrate (CHO) loading has
been reported to attenuate surgery stress response and insulin resistance. The present study aimed to investigate whether preoperative vitamin-rich CHO loading has an
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effect on POCD, endoplasmic reticulum (ER) stress, and apoptosis. Eighty male
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Sprague-Dawley rats (20-month old) were randomly assigned to four groups (20 per
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group): control group (no anesthesia and surgery), fasting group (fasting 14 h before
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surgery), water group (oral water 3 h before surgery), and CHO group (oral vitamin-
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rich CHO 3 h before surgery). The POCD rat model was established by splenectomy under intraperitoneal injection of pentobarbital sodium. Cognitive function was
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assessed using the Morris water maze (MWM) after surgery. The levels of endoplasmic reticulum (ER) stress markers and apoptosis related proteins in the
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hippocampus were examined by western blot analysis. The vitamin-rich CHO treated animals performed better in the MWM tests than the animals in the fasting and water
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groups. Furthermore, preoperative CHO loading reduced ER stress and neuronal apoptosis in the hippocampus of aged rats, as indicated by the protein biomarkers of GRP78, eIF2a, Beclin1, Bax, and Bcl-2. In conclusion, preoperative vitamin-rich CHO loading could improve POCD by attenuating ER stress and neural apoptosis, providing a basis as a potential treatment against POCD.
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Keywords: Carbohydrate; Postoperative cognitive dysfunction; Rats; Hippocampus; Endoplasmic reticulum stress; Apoptosis.
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1. Introduction
Postoperative cognitive dysfunction (POCD) is recognized as a common nervous
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system complication characterized by impairment of learning and memory and
difficulty in language comprehension after anesthesia and surgery, especially in
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elderly patients. POCD seriously affects patients’ quality of life and leads to a high
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risk of disability and mortality [1,2]. A recent increase in the number of elderly
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patients undergoing surgery has resulted in an increased incidence of POCD [3].
POCD remains unclear.
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However, there is still a lack of effective treatment for POCD, and the pathogenesis of
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Advancing age, the severity of surgery, the occurrence of complications, and coexisting illnesses have been implicated as the main risk factors for POCD [4,5].
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Surgery gives rise to circulating cortisol and inflammatory cytokines, resulting in
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insulin resistance (IR), which contributes to many postoperative complications. Previous reports have shown that inflammation plays a critical role in the development of POCD [6]. POCD is correlated with the concentration of peripheral
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and cerebrospinal fluid (CSF) inflammatory markers, such as CRP and IL-6 [7]. Growing evidence also supports the concept that IR is important in the pathogenesis of cognitive impairment and neurodegeneration [8]. Additionally, nutrient deprivation, inflammatory response, and IR during surgery, can also trigger ER stress and apoptosis [9]. Recent studies have reported that endoplasmic reticulum (ER)
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stress and ER stress-related apoptosis play a pivotal role in certain neurological disorders and neurodegenerative diseases, including Alzheimer's disease (AD) and Parkinson’s disease [10,11]. Preoperative carbohydrate (CHO) loading is an important element of the Enhanced Recovery After Surgery (ERAS) protocol [12]. It has been reported that
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preoperative CHO loading is able to reduce IR [13,14] and decrease circulating IL-6
and CRP concentrations [15,16], suggesting that preoperative CHO loading may have
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a positive impact on cognitive impairment. The present study aimed to investigate
whether preoperative vitamin-rich CHO loading can attenuate splenectomy-induced
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cognitive decline and to determine whether it has an impact on ER stress and
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apoptosis in rats after surgery.
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2. Materials and Methods 2.1. Animals
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All experiments were approved by the Animal Ethics Committee of North Sichuan Medical College and were performed in accordance with the National
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Institutes of Health guide for the care and use of Laboratory animals (NIH
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Publications No. 8023, revised 1978). Male Sprague-Dawley rats (weight, 500-650 g; age, 20 months) were supplied by the Laboratory Animal Center of North Sichuan Medical College (Nanchong, China). All animals were initially housed in groups
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under a normal 12/12 h light/dark cycle in a temperature and humidity-controlled room (22 ± 1°C and 50–60%, respectively). The animals were allowed to acclimate for a week prior to the experiments, with free access to standard rat chow and tap water.
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2.2. Vitamin-rich carbohydrates The vitamin-rich CHO beverage (14.2% carbohydrates, Outfast, YICHANG HUMANWELL PHARMACEUTICAL CO., LTD. China) used in this study contained water, maltodextrin, crystalline fructose, glucose, food additives (sodium citrate, citric acid monopotassium phosphate, potassium sorbate, L-malic acid),
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taurine, zinc gluconate, vitamin B1, vitamin B6, vitamin B12, and flavor (Table 1).
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2.3. Study design
Animals (n = 80) were randomly assigned to four groups (20 per group): control, fasting, water, and CHO. Except for those in the control group, all rats were fasted
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beginning at 7:00 pm on the night prior to surgery. Rats in the fasting group did not
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receive anything, but were fasted for 14 h prior to undergoing a splenectomy. Rats in
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the water and CHO groups voluntarily ingested 12 ml of clear water or vitamin-rich
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CHO beverage 3 h prior to splenectomy (during 6:00 to 7:00 am on the day of surgery). The selected dosage of the CHO is equivalent to the standard morning CHO dose for
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surgical patients [17].
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humans, which is grade A recommended as part of the preoperative care of most
For the splenectomy, rats were anesthetized by an intraperitoneal injection of
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pentobarbital sodium 35 mg/kg. After anesthesia induction, animals were fixed on the operating table. The entire surgical process was performed under sterile conditions. The spleen was exposed through a 2-cm incision in the left upper abdominal quadrant and
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was removed from the abdomen. The blood vessels of the spleen were ligated using 70 silk suture, and the spleen was removed by transecting the blood vessels near to the spleen. The wound was infiltrated with 0.25% bupivacaine and closed using sterile sutures. The body temperature of the animals was maintained at 37℃, and SpO2 was monitored via pulse oximetry during the surgery. Animals were housed individually in
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cages with free access to food and water after surgery. The surgery process lasted about 30 minutes. After splenectomy, half of the rats in each group were anesthetized with sodium pentobarbital via an intraperitoneal injection (60 mg/kg) on postoperative day 1. The brains were quickly removed and placed on ice. The right hemisphere of the brain was
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dissected and stored at -70℃ in a refrigerator and used for western blot analysis.
Biomarkers, such as glucose-regulated protein 78 (GRP-78), eukaryotic initiation factor
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2 (eIF2a), Beclin-1, Bax, and Bcl-2, were measured in our study. The remaining rats in each group underwent the Morris water maze test on postoperative day 2, and were
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sacrificed on postoperative day 7 following the probe test.
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2.4. Morris water maze test
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Spatial learning and memory were assessed using the classic Morris water maze, which consists of an orientation navigation test and a spatial probe test [18]. The maze
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includes a swimming pool with a diameter of 120 cm and a height of 50 cm, and a camera system. The pool was filled with water (22℃) to a depth of 25 cm. The
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addition of black nontoxic paint to the pool provided an opaque surface. Visual cues were positioned around the pool, which was divided into four equal quadrants. A
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hidden platform (10 cm in diameter) was fixed at 2 cm below the water surface in one quadrant of the pool, and this position of the platform remained unchanged for all the
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training sessions. The orientation navigation test was started on postoperative day 2 and lasted 5 days. The rats were randomly placed into the water four times per day in the four different quadrants. The swimming paths of the rats were automatically recorded by the camera system and analyzed using the corresponding software. The maximum time for each rat to search for the hidden platform was limited to 60 s. The
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rats were then allowed to stay on the platform for 15 s to remember the surrounding markers. If the rat failed to find the hidden platform within 60 s, it was guided to the platform and allowed to stay there for 15 s, and the escape latency was recorded as 60 s. Spatial probe tests were conducted on postoperative day 7. For this test, the platform was removed and the rats were allowed to swim for 60 s. The swim time to
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the target quadrant and the times across the platform were recorded. All the trials
2.5. Protein extraction and western blotting analysis
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were performed between 8:00 am and 12:00 am.
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Tissue sampling, protein extraction, and western blot analysis were carried out as
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previously described [19]. Total protein was extracted from the hippocampus, and the
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concentration of the protein was determined using the Bradford method. In brief,
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hippocampal samples were weighed and lysed with cold radio-immunoprecipitation assay lysis buffer (RIPA, Boster, AR0105-100, Wuhan, China) and protease inhibitor
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(Boster, AR1182, Wuhan, China). After being kept on the ice for 30 min, the samples were centrifuged at 12,000 rpm for 15 min. The total quantity of protein in the
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supernatants was measured using the BCA method. The hippocampus samples were run on a precast SDS-PAGE gel with 10 μg
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proteins in each lane for 2 h and transferred to a polyvinylidene difluoride membrane (PVDF). After washing with 0.1% Tris-HCl buffer solution and Tween (TBST) for 5
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min, the membranes were blocked in 5% non-fat milk for 1 h. Afterward, the proteins were incubated with the following primary antibodies overnight (4 ℃): GRP78 (1:2000, Bioss), eIF2a (1:1000, Bioss), Beclin-1 (1:1000, CST), Bax (1:1000, CST), Bcl-2 (1:400, Boster), and GAPDH (1:8000, Boster). The membrane then was washed with 0.1% TBST five times, for 5 min each, and incubated with secondary antibodies,
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horseradish peroxidase (HRP)-conjugated goat anti-rabbit (1:7500, Boster, China), for 1 h. Finally, the membrane was washed as above and the ECL reagents (Boster, China) were used to detect the proteins. The bands were visualized by a Chemi DocXRS+ Imaging System (Bio-Rad) and analyzed using the Quantity-One software. The density ratio represented the relative intensity of each band against GAPDH as
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the loading control.
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2.6. Statistics
All data were analyzed by SPSS 17.0 statistical software (SPSS Inc, Chicago, IL, USA). All of the quantitative data were expressed as mean ± SEM. Data analyzed by
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repeated measures ANOVA were firstly assessed for sphericity using Mauchly’s test.
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The Greenhouse-Geisser correction was used to correct the F statistic and assess
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significance if the data violated the assumption of sphericity. Data analyzed by ANOVA were assessed for homogeneity of variance with Levene’s test. Data with
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equal variances were assessed post hoc using the least significant difference (LSD) test, while data that violated Levene’s test were assessed post hoc using Games-
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Howell. Behavioral data from the orientation navigation test were analyzed by mixeddesign ANOVA. Data from the probe test and western blotting studies were analyzed
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using one-way ANOVA. P<0.05 was considered to be statistically significant. 3. Results
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3.1. MWM test
The MWM test was conducted to investigate the spatial learning and memory of
the experimental rats. In the Water maze tests, mixed two-way ANOVA with the between-subject factor “Treatment” and the within-subject factor “Days” was used to analyze the difference of escape latency. Data were firstly assessed for sphericity
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using Mauchly’s test. The test confirmed that the data violated the assumption of sphericity, and the Greenhouse-Geisser correction was used to correct the F statistic and assess significance. Escape latency decreased over the 5 d training period (Fig. 1A). Repeated-measures ANOVA identified a significant reduction in escape latency with training days (F (3.3, 117.4) =109.884, p<0.001). Significant difference among
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groups was also identified (F (3, 36) =3.143, p = 0.037). Post hoc analysis using LSD test revealed that rats in the fasting group had a greater latency than those in the
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control group (p = 0.016), while rats received vitamin-rich CHO had a shorter latency than the fasting group (p = 0.029).
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To assess spatial memory, a probe trial was administered on postoperative day 7.
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One-way ANOVA of the time spent in the target quadrant (F (3, 35) = 4.781, p =
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0.007) and number of platform crossings (F (3, 35) = 5.227, p = 0.004) showed a
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significant difference among groups. Post hoc multiple comparison (LSD) showed that the time spent in the target quadrant in the fasting group (p=0.006) and water
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group (p = 0.007) was shorter than those in the control group. The time spent in the target quadrant in the CHO group was longer than those in the fasting group (p =
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0.021) and water group (p = 0.025) (Fig. 1B). The number of platform crossings in the fasting group (p = 0.047) and water group (p = 0.012) were lower than those in the
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control group. The number of platform crossings was higher in the CHO group, compared with the fasting group (p = 0.008) and water group (p = 0.002) (Fig. 1C).
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The data suggest that preoperative vitamin-rich CHO loading could improve the
cognitive impairment induced by splenectomy and fasting. 3.2 ER stress To evaluate whether the administration of vitamin-rich CHO could attenuate surgery-induced ER stress, we measured the expression levels of ER stress associated
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proteins in the hippocampus. The western blot results revealed that the expression levels of GRP78 and eIF2a were significantly up-regulated in the hippocampus on postoperative day 1 in the fasting and water groups, when compared with those of the control group (P<0.01; Fig. 2). Preoperative vitamin-rich CHO treatment significantly inhibited the expression of GRP78 and eIF2a in the hippocampus after surgery
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(P<0.01; Fig. 2). There was no significant difference in the GRP78 and eIF2a
expression levels in rats between the control group and CHO group. These results
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indicate that preoperative vitamin-rich CHO loading has a positive impact on ER stress during surgery.
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3.2 Apoptosis
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To evaluate cell apoptosis, apoptosis-associated proteins were detected by
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western blot. On postoperative day 1, the results of the western blot analysis showed
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that the expression levels of Beclin-1 and Bax were increased after splenectomy, and
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the vitamin-rich CHO treatment significantly attenuated the surgery-induced upregulation of Beclin-1 and Bax levels (P<0.01; Fig. 3A,B). On the other hand,
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splenectomy and fasting down-regulated the expression of Bcl-2, and vitamin-rich CHO treatment significantly blocked surgery-induced decreases of Bcl-2 (P<0.01;
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Fig. 3C). There was no significant difference in the Beclin-1, Bax, and Bcl-2 expression levels between the control group and CHO group; the treatment with water
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exhibited the same effects as the fasting group. These findings suggest that preoperative oral vitamin-rich CHO significantly attenuated apoptosis after splenectomy and fasting.
4. Discussion
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In this study, we investigated the neuroprotective effects of vitamin-rich CHO in a splenectomy animal model. The results showed that preoperative vitamin-rich CHO loading significantly improved postoperative cognitive impairment. Furthermore, we studied whether the vitamin-rich CHO affected ER stress and apoptosis induced by surgery. The results revealed that preoperative vitamin-rich CHO loading can relieve
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ER stress and suppress apoptosis in the hippocampus of rats that underwent splenectomy. Our study results indicate that ER stress and apoptosis in the
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hippocampus may be associated with impairment in spatial learning and memory induced by surgery and fasting. Thus, preoperative CHO loading might be an
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effective treatment against POCD in clinical practice.
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The main purpose of this study was to evaluate the effect of vitamin-rich CHO
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loading on POCD. POCD is a frequent complication following major surgery in the
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elderly. The MWM test, which is a common way to evaluate learning and memory ability in animals [18], was employed to study the effects of the vitamin-rich CHO on
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splenectomy-induced cognitive impairment in aged rats. Previous studies have demonstrated that inflammation and IR are correlated with cognitive impairment [6-
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8]. Recently, it has been reported that preoperative CHO loading reduces postoperative IR [13,14,20], decreases circulating IL-6 and CRP concentrations
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[15,16], and even has a positive effect on gut recovery after surgery [21,22], indicating that preoperative CHO loading may have an impact on POCD.
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In the current study, rats undergoing splenectomy and fasting showed
significant decreases in the learning and memory measured by way of the MWM test, suggesting that a cognitive impairment model was successfully established. The addition of a vitamin-rich CHO beverage diminished the cognitive impairment after surgery, resulting in outcomes comparable to those of the control animals. The results
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demonstrate that preoperative vitamin-rich CHO loading has a positive effect on POCD. ER stress is one of the pivotal molecular mechanisms underlying POCD. The ER is a cellular organelle which plays a critical role in protein folding. Various harmful stimuli and pathological conditions, such as nutrient deprivation, inflammation,
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oxidative stress, and hypoxia, may impair ER function and eventually lead to ER
stress. Tian et al. [23] showed that minor abdominal surgery triggered ER stress and
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apoptosis in the hippocampus of mice. To evaluate ER stress after splenectomy,
protein levels of ER stress markers, such as GRP78 and eIF2a, were measured in the
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hippocampus in the present study. GRP 78 is an important biomarker of ER stress,
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which plays a critical role in the regulation of ER functioning [24], and eIF2a is a key
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downstream mediator of PERK. Our study found that preoperative vitamin-rich CHO
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loading alleviated the upregulation of GRP-78 and eIF2a in the hippocampus of splenectomy rats, indicating that the potential neuroprotective effects of preoperative
hippocampus.
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vitamin-rich CHO loading is associated with the alleviation of ER stress in the
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ER stress and ER stress-induced inflammation and insulin resistance form a vicious cycle, which ultimately leads to neuronal cell death through autophagy and
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apoptosis [9]. Previous studies have shown that ER stress-related apoptosis in the hippocampus is a predominant pathological mechanism in several neurological
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disorders [10,11,25]. To further learn the effect of the vitamin-rich CHO on apoptosis, the protein levels of Beclin-1, Bax, and Bcl-2 were measured in the hippocampus of rats in this study. Beclin-1 is a main component of the autophagy machinery, playing a crucial role in the initiation of autophagy. Bax and Bcl-2 are two separate members of a gene family that is important in the regulation of apoptosis [26]. It is commonly
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considered that Bax is a pro-apoptotic protein, while Bcl-2 is anti-apoptotic protein [27]. The interaction between the autophagy protein Beclin-1 and the anti-apoptotic protein Bcl-2 has been shown to regulate the switch between the autophagic and apoptotic machinery. In this study, splenectomy and starvation induced ER stress, resulting in
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autophagy and the production of high levels of Beclin-1. Severe and consistent ER
stress will lead to apoptosis. The binding of Beclin-1 to Bcl-2 disrupts the balance of
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Bax and Bcl-2, which are closely associated to apoptosis. Our study found that the
expression of Bcl-2 was down-regulated, whereas Bax and Beclin-1 levels were up-
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regulated in the fasting and water groups. However, the vitamin-rich CHO loading
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reversed these results and blocked the apoptosis induced by splenectomy.
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Although this study discovered the effects of the vitamin-rich CHO on POCD,
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ER stress, and apoptosis in aged rats, some limitations could not be ignored. Firstly, besides carbohydrates, other components of the vitamin-rich CHO, such as vitamin,
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zinc, could also exert an effect on neurodegenerative diseases [28,29]. Further study should explore the neuroprotective effects of carbohydrates only. Secondly, only a
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morning dose of CHO was administrated in this study, without further investigation of a combined evening dose. Therefore, further studies focusing on suitable doses of
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CHO are needed.
In conclusion, the present study demonstrated that preoperative vitamin-rich
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CHO loading improves POCD induced by splenectomy, which may be associated with the inhibition of ER stress and apoptosis in the hippocampus of rats. Our findings suggest that preoperative vitamin-rich CHO loading may be a potential treatment strategy for preventing POCD.
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Statement of Authorship YBZ and TDY contributed to the conception and design of the study and had primary responsibility for the interpretation of the data; YBZ generated and analyzed the experimental data and drafted the article; XPH, QYY, LZ, XLC, and JTL performed the animal experiments and the laboratory tests; all authors approved the
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final draft of the manuscript be submitted.
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Funding
This work was supported by the Foundation of Sichuan Educational Committee,
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China [grant number 17ZB0179].
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Declarations of interest
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None.
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Acknowledgements
We would like to thank Mei Zeng from the School of Basic Medical Science,
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North Sichuan Medical College for providing helpful advice and invaluable support
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regarding the experiments.
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Figure captions
Fig. 1. Effects of vitamin-rich CHO on cognitive function in aged rats tested by MWM. (A) Mean
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latency in the orientation navigation was tested for 5 consecutive days. Data presented as mean ±
SEM. n = 10. *P < 0.05 vs. fasting group. (B) The percent of the time spent in the target quadrant (where the platform was located during hidden platform training) during the probe test. (C)
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Number of crossings over the former location of the platform during the probe test. Data presented
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as mean ± SEM. n = 10. *P < 0.05 vs. control group; **P < 0.01 vs. control group; #P < 0.05 vs.
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CHO group; ##P < 0.01 vs. CHO group. MWM, Morris water maze.
Fig. 2. The expression of GRP78 and eIF2a in the hippocampus on postoperative day 1. (A) Representative western blot for GRP78 in the hippocampus and relative density analysis of the GRP78 protein bands. The relative density is expressed as a ratio (GRP78/GAPDH). (B) Representative western blot for eIF2a in the hippocampus and relative density analysis of the eIF2a protein bands. The relative density is expressed as a ratio (eIF2a/GAPDH). Data were
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expressed as mean ± SEM. n=10. **P < 0.01 vs. control group; #P < 0.05 vs. CHO group; ##P <
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0.01 vs. CHO group.
Fig. 3. The expression of Beclin-1, Bax, and Bcl-2 in the hippocampus on postoperative day 1. (A) Representative western blot for Beclin-1 in the hippocampus and relative density analysis of
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the Beclin-1 protein bands. The relative density is expressed as a ratio (Beclin-1/GAPDH). (B)
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Representative western blot for Bax in the hippocampus and relative density analysis of the Bax
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protein bands. The relative density is expressed as a ratio (Bax/GAPDH). (C) Representative western blot for Bcl-2 in the hippocampus and relative density analysis of the Bcl-2 protein bands.
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The relative density is expressed as a ratio (Bcl-2/GAPDH). Data were expressed as mean ± SEM.
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n = 10. **P < 0.01 vs. control group; #P < 0.05 vs. CHO group; ##P < 0.01 vs. CHO group.
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Table 1. Content of the vitamin-rich carbohydrate beverage per 100 mL Per 100 ml 57.6 kCal 14.2 g 45 mg 0.24 mg 0.13 mg 0.14 mg 1.48 mg
NRV% 3% 5% 2% 17% 9% 6% 10%
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Ingredient Calories Carbohydrates Sodium VitaminB1 VitaminB6 VitaminB12 Zinc