Nicotine-Induced Neuroprotection against Cognitive Dysfunction after Partial Hepatectomy Involves Activation of BDNF/TrkB Signaling Pathway and Inhibition of NF-κB Signaling Pathway in Aged Rats

Nicotine-Induced Neuroprotection against Cognitive Dysfunction after Partial Hepatectomy Involves... Abstract Introduction The main purpose of this study was to investigate the effects and possible mechanisms of nicotine pre-treatment on postoperative cognitive dysfunction (POCD) in aged rats. Methods Nicotine (0.5 mg/kg) was given i.p. immediately after anesthesia induction. After the Morris water maze test was used to evaluate the rats’ spatial learning and memory, serum and hippocampal tissues were harvested 1 and 3 days after intervention. Inflammatory cytokines in the serum were evaluated by Enzyme-linked Immunosorbent Assay (ELISA). Brain-derived neurotrophic factor (BDNF), p-TrkB, neuroinflammation cytokines, NF-κB p65, and cleaved caspase-3 were measured by western blotting; neuronal apoptosis in the hippocampal CA1 region was also evaluated by TUNEL staining. Results We found that nicotine markedly attenuated the POCD and reduced the elevated levels of inflammatory cytokines in the serum, including IL-1β and high mobility group box-1 (HMGB1), on postoperative day 1. Additionally, nicotine suppressed the surgery-induced release of IL-1β, TNF-ɑ, HMGB1, and NF-κB p65 in the hippocampus on postoperative day 1 and day 3. In addition, operated rats displayed lower BDNF and p-TrkB in the hippocampus on postoperative day 1, returning to baseline by postoperative day 3. However, nicotine pre-treatment clearly reversed the surgical stress-induced decrease in both BDNF and p-TrkB expression in the hippocampus. Furthermore, nicotine pre-treatment significantly alleviated the surgery-induced increase in the neuronal apoptosis in the hippocampus on postoperative day 1 and day 3. Conclusions Our results showed that nicotine-induced neuroprotection against POCD may involve activation of the BDNF/TrkB signaling pathway and inhibition of the NF-κB signaling pathway. Implications Nicotine has long been considered a potent therapeutic agent for neuroprotection. This study reported the positive effect of nicotine treatment on cognitive dysfunction after partial hepatectomy in aged rats. Furthermore, the underlying mechanism may involve activation of the BDNF/TrkB signaling pathway and inhibition of the NF-κB signaling pathway in the hippocampus. Introduction Postoperative cognitive dysfunction (POCD) is an important clinical issue in perioperative care following major surgery and general anesthesia in elderly individuals. It refers to an impairment of memory, concentration, information processing, language comprehension, and social integration.1 The International Study of POCD (ISPOCD) has concluded that the incidence of POCD was about 26% at one week postoperatively and 10% at 3 months after surgery in patients aged over 60 year.2 Cognitive impairment after surgery has been associated with prolonged hospital day and increased cost of hospitalization and out-of-hospital care.3 POCD also adversely affects postoperative quality of life, surgical morbidity, and mortality.4 Although many etiologic and pathophysiological mechanisms have been implicated in the development of POCD, the exact cascade remains elusive. In the past decade, neuroinflammation has emerged as a key event in the development of POCD.5 The peripheral inflammatory mediators induced by surgical stress result in the release of inflammatory cytokines in the central nervous system.6 Neuroinflammation will harm the neurons and cause irreversible neuronal apoptosis, contributing to the brain functional changes.7 Our previous studies have also confirmed the critical role of neuroinflammation in the development of POCD.8 We further showed that anti-inflammatory agents effectively attenuated the cognitive impairment induced by surgical trauma.9 Neurotrophins are also critical for the development of POCD. Brain-derived neurotrophic factor (BDNF), an extensively studied neurotrophin that is mediated via tyrosine kinase B (TrkB),10 plays a critical role in neuron survival, plasticity, neurogenesis, and synaptogenesis in the developing brain.11 Mounting evidence has suggested that changes in its level are highly correlated with the development of several human diseases, including depression, neurodegeneration, and psychiatric disorders.12 A recent study has identified a reduction of surgical stress-induced BDNF as an important factor that influences the risk of POCD in aged mice.13 A clinical study conducted by our study team also showed that reduced BDNF levels in the serum are related to cognitive impairment after hip replacement surgery in elderly patients (unpublished data). Therefore, it might be possible to prevent surgical stress-induced cognitive dysfunction by activating BDNF/TrkB signaling pathways and inhibiting neuroinflammation-related signaling pathways. Nicotine, an α7 nicotinic acetylcholine receptor agonist (α7nAChR), has been demonstrated to have neuroprotective effects in experimental models of Alzheimer’s disease14 and Parkinson’s disease.15 Studies also reported that nicotine resolved neuroinflammation and reversed cognitive decline after tibial fracture operation in aged mice.6 However, the specific mechanisms underlying the nicotine pre-treatment-induced neuroprotective effect on POCD remain to be explored. Recently, we observed that nicotine pre-treatment effectively attenuated lipopolysaccharide-induced cognitive impairment by increasing BDNF expression and inhibiting neuroinflammation in the rat hippocampus.8 Therefore, we hypothesized that nicotine might be able to improve spatial learning and memory in aged rats with cognitive impairment induced by partial hepatectomy. We further hypothesized that the mechanism may involve activation of the BDNF/TrkB signaling pathways and inhibition of neuroinflammation-related signaling pathways. Materials and Methods Ethics Statement All animals protocols were approved by the Animal Care and Use Committee of Qilu Hospital (Qingdao) and performed in accordance with the guidelines for experiment animals use established by the Institutional Animal Care and Use Committee of Shandong University. Every possible efforts were made to minimize the number of rats used and their suffering. Animals and Grouping A total of 60 male Sprague Dawley rats (18 months old, weighing 480–600 g) were purchased from Shandong University Experiment Animal Center. The rats were raised in the specific pathogen—free animal laboratory at 23 ± 2°C with a relative humidity of 55 ± 5% on a 12:12 h light:dark cycle in the Experiment Animal Center of Shandong University. All rats were housed in standard cages and were provided with access to food and water ad libitum. Animals were allowed to acclimate for 7 days before beginning the experiments. The rats were randomly divided into four groups (n = 15): (1) Control (C); (2) Anesthesia only (A); (3) Anesthesia plus Surgery (S); (4) Nicotine plus Surgery (Nic + S). Rats were sacrificed at days 1 and 3 after intervention. Surgical Procedures The rats in group C received sterile saline to control for the effects of injection stress. The rats in group A were intraperitoneally anesthetized with a combination of 20 μg/kg fentanyl and 500 μg/kg droperidol. The rats underwent partial hepatectomy under general anesthesia in the S and Nic + S groups. Partial hepatectomy was performed under aseptic conditions as described previously.16 Briefly, the liver was exposed through a 2 cm midline abdominal incision. The left lateral lobes of the liver were visualized, isolated, and excised. The wound was then infiltrated with 0.25% bupivacaine. After checking, the muscles and skin were closed by sterile suture. Nicotine hydrogen tartrate (Sigma, St. Louis, MO, USA) in 0.9% saline was used. Nicotine was were given i.p. immediately at final concentrations of 0.5 mg/kg nicotine-free base in the Nic + S group after anesthesia induction. The dose used was based on our previous study.8 The exploratory laparotomy was performed under anesthesia before the blood and tissue sample collection. The rats suffering from the intra-abdominal infection and hemorrhage were excluded. Morris Water Maze (MWM) The experimental design is shown in Figure 1A. The MWM test was used to evaluate the rats’ hippocampus-dependent spatial learning and memory, as described previously.17 A video tracking system recording the rat’s movement in a water maze was used in conjunction with the Color Video Camera. Rats were tested in a black circular tank (1.2 m diameter, 50 cm deep; water, 22°C) filled to 30 cm. The nonvisible platform (15 cm diameter) was placed in the first quadrant (target quadrant) and positioned 2 cm below the water surface. There were many visual cues around the tank. Every rat was placed on the platform for 30 s before the start of each trial. In each trial, the rats were placed in a fixed starting position in the maze, facing the tank wall. They were given 60 s to locate the platform in the first quadrant. The rats that found the platform within this time limit were allowed to remain there for 15 s. Otherwise, the animals that did not locate the platform were guided to it and were allowed to remain there for 15 s. Throughout the experiment, the location of the platform was fixed and rats were trained for four trials per day for 6 consecutive days. The learning parameters that were recorded included swimming speed and latency (time from the start of the trial until the rat reached the platform). On day 7, rats received partial hepatectomy and/or general anesthesia. Animals were subjected to a memory test on postoperative days 1 and 3. After removing the platform from the first quadrant, the third quadrant was used as the starting position to evaluate the memory. The rats were allowed to swim freely for 60 s. The latency for the first time to reach the target area, the percentage of time spent in the target quadrant and the number of platform crossings were recorded on preoperative day 1 and postoperative days 1 and 3. Figure 1. View largeDownload slide Effects of nicotine pre-treatment on spatial learning and memory parameters in the Morris Water Maze test. (A) Experimental design. (B) Mean escape latencies of the four groups in the acquisition phase. n = 12 per group. (C) Average swimming speed of each group in the acquisition phase. n = 12 per group. (D) Latencies for the first time to the platform for each group at 1 day before surgery and at 1 day (n = 12) and 3 days (n = 6) after surgery in the probe trials. (E) Time spent in the target quadrant for each group at 1 day before surgery and at 1 day (n = 12) and 3 days (n = 6) after surgery in the probe trials. (F) The number of platform crossings with a 60-s limit for each group at 1 day before surgery and at 1 day (n = 12) and 3 days (n = 6) after surgery in the probe trials. All data are expressed as the means ± SD. *p < .05. Figure 1. View largeDownload slide Effects of nicotine pre-treatment on spatial learning and memory parameters in the Morris Water Maze test. (A) Experimental design. (B) Mean escape latencies of the four groups in the acquisition phase. n = 12 per group. (C) Average swimming speed of each group in the acquisition phase. n = 12 per group. (D) Latencies for the first time to the platform for each group at 1 day before surgery and at 1 day (n = 12) and 3 days (n = 6) after surgery in the probe trials. (E) Time spent in the target quadrant for each group at 1 day before surgery and at 1 day (n = 12) and 3 days (n = 6) after surgery in the probe trials. (F) The number of platform crossings with a 60-s limit for each group at 1 day before surgery and at 1 day (n = 12) and 3 days (n = 6) after surgery in the probe trials. All data are expressed as the means ± SD. *p < .05. Enzyme-linked Immunosorbent Assay (ELISA) The rats in each group were anesthetized using Nembutal. Their blood was collected via cardiac puncture. The blood was allowed to clot for 2 h at room temperature and was then centrifuged for 15 min at 2000g at 4°C. The serum fraction was removed and stored at −80°C for further analysis. The hippocampus was rinsed with buffered saline (PBS), homogenized in 1 mL of PBS, and stored overnight at −20°C. The homogenates were subjected to two freeze–thaw cycles to break the cell membrane and then centrifuged for 5 min at 5000g at 2°C–8°C. The supernatants were stored at −80°C. The levels of serum IL-1β, TNF-α, high mobility group box-1 (HMGB-1), and hippocampal BDNF were measured using commercially available ELISA kits (Neobioscience Technology Company: IL-1β, TNF-α, BDNF; R&D Systems: HMGB-1) according to the manufacturer’s protocol. Western Blot Analysis Rats were sacrificed under deep anesthesia and hippocampal tissues were removed. Subsequently, the harvested tissues were homogenized on ice using 1 mL of ice-cold RIPA buffer containing the protease inhibitor phenylmethanesulfonyl fluoride (2 mM) in added protease inhibitor. The homogenates were centrifuged at 13000 rpm at 4°C for 30 min. The protein concentration in the supernatant was determined using the bicinchoninic acid (BCA) method. Equal amounts of protein (40 μg) from each sample were separated via 12% SDS–PAGE and were transferred to PVDF membranes (Millipore, Boston, MA, USA). The membranes were immunoblotted overnight at 4°C using a rabbit anti-IL-1β antibody (Proteintech, China; dilution 1:500), goat anti-TNF-α antibody (Santa Cruz Biotechnology Inc., USA; dilution 1:500), rabbit anti-NF-κB p65 antibody (Proteintech, China; dilution 1:1000), rabbit anti-caspase-3 antibody (Proteintech, China; dilution 1:500), rabbit anti-BDNF antibody (Santa Cruz Biotechnology Inc., USA; dilution 1:1000), rabbit anti-phospho-TrkB antibody (Abcam, USA; dilution 1:1000), rabbit anti-HMGB-1 antibody (Abcam, USA; dilution 1:10000) and rabbit anti-β-actin antibody (Proteintech, China; dilution 1:1000). After three 5 min washes, the membranes were incubated in an HRP-conjugated secondary antibody (Proteintech, China; dilution 1:2000) for 1 h at room temperature, followed by washing and subsequent visualization via enhanced chemiluminescence (ECL; Millipore, Billerica, MA, USA). Finally, the optical densities of the resultant bands were recorded and analyzed using C-Digit software (LI-COR, USA). The relative expression was normalized to β-actin. TUNEL Staining Under deep anesthesia, the thoracic cavity was opened to expose the heart, and its right atrium was severed. Next, 0.01 M PBS (4°C) followed by 200 mL of ice-cold 4% paraformaldehyde were transcardially perfused into the left ventricle. The brains were removed and post-fixed in 4% paraformaldehyde overnight at 4°C, followed by rehydration and embedding in paraffin. The sections were deparaffinized before antigen retrieval by microwaving the sections in sodium citrate buffer. The samples were preincubated in 3% H2O2 at room temperature for 10 min to eliminate nonspecific peroxidase activity and were then blocked in 3% normal goat serum at room temperature for 1 h. The sections were stained using an in situ cell death detection kit (POD; Roche Diagnostics Corp., Indianapolis, IN, USA) per the manufacturer’s instructions. Five sections in each rat were collected from CA1 of the dorsal hippocampus and were examined in each group. In each section, five independent fields (400×) were selected for examination by two independent observers who were blinded to the experimental groups. The percentage of TUNEL-positive nuclei in the dorsal CA1 region was calculated to evaluate apoptosis. Statistical Analysis All data are presented as the means ± SD. Training behavioral parameters were analyzed by two-way repeated measures ANOVA. Memory behavioral parameters, ELISA, Western blot and TUNEL staining were analyzed using one-way ANOVA followed by Tukey–Kramer post hoc tests for multiple comparisons. The differences between the values were considered to be significant at p < .05. All statistical tests and graphs were performed using Statistical Program for Social Sciences 20.0 software (SPSS, Inc., Chicago, IL, USA). Results Nicotine Attenuated the Surgery-Induced Cognitive Dysfunction Sixty rats were enrolled in the study. Twelve rats were excluded because of the intra-abdominal infection and hemorrhage, and there were 48 rats (n = 12) entered the final analysis. To evaluate the effect of nicotine on surgery stress-induced cognitive dysfunction, the Morris water maze was employed to analyze cognition changes. Comparisons by two-way repeated-measures ANOVA revealed no significant difference in latency and swimming speeds among the four groups (Figure 1B and C, p > 0.05). In the following probe trials, the latency for the first time to the platform, the percentage of time spent in the target quadrant (first quadrant) and the number of original platform crossings were analyzed (Figure 1D, E, and F). Significant differences in the latency (1 d aft: F3,44=46.357, p = .000; 3 d aft: F3,20=20.529, p = .000), the time spent in the target quadrant (1 d aft: F3,44=7.634, p = .000; 3 d aft: F3,20=19.225, p = .000) and the number of platform crossings (1 d aft: F3,44=16.310, p = .000; 3 d aft: F3,20=22.101, p = .000) were observed among the four groups at days 1 and 3 after surgery. Compared with group C, the latency for the first time to the platform increased in group S (p < .05), and the time spent in the target quadrant (p < .05) and the number of platform crossings (p < .05) decreased in group S at days 1 and 3 after surgery. However, compared with group S, the latency for the first time to the platform significantly decreased in group Nic+S (p < .05), and the time spent in the target quadrant and the number of platform crossings significantly increased in group Nic+S (p < .05). Nicotine Decreased Inflammatory Cytokine Expression in the Hippocampus To understand whether nicotine inhibited inflammation in the central compartment, the protein expression levels of the pro-inflammatory cytokines IL-1β, TNF-α, HMGB-1, and NF-κB p65 were measured in the hippocampus at 1 day and 3 days after surgery. As shown in Figure 2, western blot analysis indicated that exposure to surgery significantly increased hippocampus IL-1β (F3,8 = 15.534, p = .001), TNF-α (F3,8 = 33.656, p = .000), HMGB-1 (F3,8 = 38.874, p = .000), and NF-κB p65 (F3,8 = 76.238, p = .000) expression versus the control group on postoperative days 1 (p < .05). Levels of these pro-inflammatory cytokines in the hippocampus remained elevated in the surgical group up to postoperative day 3 (IL-1β: F3,8 = 10.133, p = .004; TNF-α: F3,8 = 22.295, p = .000; HMGB-1: F3,8 = 34.908, p = .000; NF-κB p65: F3,8 = 21.961, p = .000). However, the administration of nicotine potently reduced the levels of IL-1β, TNF-α, HMGB-1, and NF-κB p65 on postoperative days 1 and 3 (p < .05). Figure 2. View largeDownload slide Effects of nicotine pre-treatment on surgery-induced IL-1β, TNF-α, HMGB-1, and NF-κB p65 levels in the hippocampus at 1 day and 3 days after surgery. Western blot analyses were employed to measure IL-1β (A), TNF-α (B), HMGB-1 (C), and NF-κB p65 (D) expression. The quantification analysis showed that nicotine treatment significantly decreased the protein levels of TNF-α, IL-1β, HMGB-1 and NF-κB p65 in the hippocampus at 1 day and 3 days after surgery. All data are expressed as the means ± SD. n = 3 per group. *p < .05, **p < .01. Figure 2. View largeDownload slide Effects of nicotine pre-treatment on surgery-induced IL-1β, TNF-α, HMGB-1, and NF-κB p65 levels in the hippocampus at 1 day and 3 days after surgery. Western blot analyses were employed to measure IL-1β (A), TNF-α (B), HMGB-1 (C), and NF-κB p65 (D) expression. The quantification analysis showed that nicotine treatment significantly decreased the protein levels of TNF-α, IL-1β, HMGB-1 and NF-κB p65 in the hippocampus at 1 day and 3 days after surgery. All data are expressed as the means ± SD. n = 3 per group. *p < .05, **p < .01. Nicotine Reduced Inflammatory Cytokine Levels in the Serum Serum samples were collected from all the animals at 1 day and 3 days after surgery to evaluate the systemic levels of IL-1β, TNF-α and HMGB-1 (Figure 3A, B and C). No changes in serum TNF-α were observed on postoperative days 1 and 3 (1 d aft: F3,16 = 0.231, p = .874; 3 d aft: F3,16 = 0.135, p = .938) between the four groups. Surgery significantly upregulated serum levels of IL-1β (F3,16 = 46.838, p = .000) and HMGB-1 (F3,16 = 5.003, p = .012) on postoperative day 1, which returns to baseline by day 3 (IL-1β: F3,16 = 0.446, p = .724; HMGB-1: F3,16 = 0.067, p = .977). However, the elevated levels of these inflammatory cytokines were dramatically attenuated by nicotine administration (p < .05). Nicotine Attenuated the Surgery-Induced Down-Regulation of BDNF and p-TrkB in the Hippocampus To explore the mechanism by which nicotine administration ameliorates cognitive dysfunction, we measured the expression of BDNF and p-TrkB. As shown in Figure 3D, E and F, exposure to surgery significantly decreased BDNF versus the control group on postoperative day 1(ELISA methodologies: F3,8 = 9.649, p = .005; western blot analysis: F3,8 = 9.704, p = .005). Furthermore, the down-regulation of BDNF inhibited the TrkB receptor and the downstream signaling pathway. As expected, the expression of p-TrkB was reduced on postoperative day 1 (F3,8 = 8.478, p = .007). However, nicotine effectively upregulated BDNF and p-TrkB expression in the hippocampus on postoperative day 1 (p < .05). No significant difference in hippocampal BDNF and p-TrkB expression was observed on postoperative day 3 between the four groups (BDNF: western blot analysis: F3,8 = 0.399, p = .757, ELISA methodologies: F3,8 = 0.138, p = .935; TrkB: F3,8 = 0.145, p = .930). Figure 3. View largeDownload slide Effects of nicotine pre-treatment on the levels of IL-1β, TNF-α and HMGB-1 in serum and the levels of BDNF and p-TrkB in the hippocampus at 1 day and 3 days after surgery. (A) No changes in serum TNF-α were observed on at 1 day and 3 days after surgery. Surgery increased the levels of IL-1β (B) and HMGB-1 (C) in serum at these time points. However, nicotine treatment clearly reduced the levels of IL-1β and HMGB-1. (D) ELISA methodologies of BDNF protein levels in hippocampus. (E) Western blot analysis of BDNF and p-TrkB protein levels in the hippocampus. (F) The quantification analysis of BDNF and p-TrkB protein levels in the hippocampus. All data are expressed as the means ± SD. n = 3 per group. *p < .05, **p < .01. Figure 3. View largeDownload slide Effects of nicotine pre-treatment on the levels of IL-1β, TNF-α and HMGB-1 in serum and the levels of BDNF and p-TrkB in the hippocampus at 1 day and 3 days after surgery. (A) No changes in serum TNF-α were observed on at 1 day and 3 days after surgery. Surgery increased the levels of IL-1β (B) and HMGB-1 (C) in serum at these time points. However, nicotine treatment clearly reduced the levels of IL-1β and HMGB-1. (D) ELISA methodologies of BDNF protein levels in hippocampus. (E) Western blot analysis of BDNF and p-TrkB protein levels in the hippocampus. (F) The quantification analysis of BDNF and p-TrkB protein levels in the hippocampus. All data are expressed as the means ± SD. n = 3 per group. *p < .05, **p < .01. Nicotine Suppressed Cleaved Caspase-3 Expression in the Hippocampus and Neuron Apoptosis in the Dorsal Hippocampal CA1 Region To further investigate whether neuronal apoptosis occurred in the hippocampus, the hippocampal cleaved caspase-3 level and the percentage of TUNEL-positive nuclei in the dorsal hippocampal CA1 region were assessed. Surgery exposure markedly increased the expression of cleaved caspase-3 in the hippocampus, which was reversed by treatment with nicotine (Figure 4A, 1 d aft: F3,8 = 39.689, p = .000; 3 d aft: F3,8 = 52.097, p = .000). Compared with group C, neuron apoptosis was also clearly detected at 1 day and 3 days after surgery in group S (Figure 4B, 1 d aft: F3,8 = 143.523, p = .000; 3 d aft: F3,8 = 463.438, p = .000). Nicotine clearly reduced the number of apoptotic cells in the hippocampus at 1 day and 3 days after the treatment (p < .05). Figure 4. View largeDownload slide Effects of nicotine pre-treatment on the levels of cleaved caspase-3 in the hippocampus and neurons apoptosis in the dorsal hippocampal CA1 region. (A) Western blot analyses were employed to measure cleaved caspase-3; the quantification analysis showed that nicotine treatment significantly decreased the protein levels of cleaved caspase-3 in the hippocampus at 1 day and 3 days after surgery. (B) Representative TUNEL staining (400×) of hippocampal sections is shown; the data are quantitatively expressed in the histogram. Scale bars represent 50 μm. All data are expressed as the means ± SD. n = 3 per group. *p < .05, **p < .01. Figure 4. View largeDownload slide Effects of nicotine pre-treatment on the levels of cleaved caspase-3 in the hippocampus and neurons apoptosis in the dorsal hippocampal CA1 region. (A) Western blot analyses were employed to measure cleaved caspase-3; the quantification analysis showed that nicotine treatment significantly decreased the protein levels of cleaved caspase-3 in the hippocampus at 1 day and 3 days after surgery. (B) Representative TUNEL staining (400×) of hippocampal sections is shown; the data are quantitatively expressed in the histogram. Scale bars represent 50 μm. All data are expressed as the means ± SD. n = 3 per group. *p < .05, **p < .01. Discussion In the current study, we first reported the protective effect of nicotine pre-treatment on cognitive dysfunction induced by partial hepatectomy in aged rats. Our results showed the following findings: (i) nicotine effectively attenuated cognitive dysfunction induced by partial hepatectomy, as indicated by the MWM test. (ii) Nicotine suppressed the surgery-induced excessive release of inflammatory cytokines in the serum and hippocampus. (iii) Moreover, nicotine decreased the neuronal apoptosis in the hippocampus. (iv) Importantly, this study indicated that nicotine activated the BDNF-TrkB signaling pathway and inhibited the NF-κB signaling pathway in the hippocampus. Collectively, these findings indicated that nicotine may be an effective neuroprotective agent for the prevention of POCD. Currently, there are few available therapeutic interventions to prevent POCD. Thus, we sought to investigate the effect of nicotine on POCD in aged rats. Several recent rodent models have successfully mimicked POCD in humans, such as splenectomy,18 minor abdominal surgery,7 partial hepatectomy,17 and tibial fracture.6 In this study, we generated the experimental POCD model by performing partial hepatectomy in aged rats. Additionally, studies have indicated that the hippocampus, one of the most vulnerable tissues to stress, played a critical role in spatial memory formation and consolidation; exposure to surgical stress negatively affected functional and structural effects on the hippocampus.19 Hippocampal damage causes brain disorders, resulting in cognitive and behavioral impairment.10 Therefore, we focused on the hippocampus. Recently, we also observed that nicotine pre-treatment (0.5 mg/kg, i.p.) significantly attenuated lipopolysaccharide-induced deficits in learning and memory function in rats.8 Based on our previous results, we hypothesize that nicotine may provide a pharmacological strategy to prevent POCD. To determine the effect of nicotine on cognitive dysfunction after partial hepatectomy, the Morris water maze, one of the most widely accepted models in rodents, was used to evaluate learning and memory in this study. Our results showed that the aged rats had deficits in the hippocampus-dependent spatial memory as manifested by the longer latency to reach the platform for the first time, the lower percentage of time spent in the target quadrant and the lower number of original platform crossings in the MWM test at day 1 and day 3 after surgery, which were in line with other studies.17,18 However, those deficits could be clearly reversed by nicotine pre-treatment. To investigate the potential mechanisms of how nicotine improves learning and memory, expressed as the results of the MWM test, we focused our biochemical analyses on the neuroinflammation-related signaling pathway and the BDNF-TrkB signaling pathway in the hippocampus. Neuroinflammation has been considered the main mechanism involved in the pathogenesis of POCD.20 Surgical stress engages the immune system and initiates an inflammatory cascade involving inflammatory cytokines in the serum. These cytokines readily compromise endothelial function and disrupt the blood-brain barrier (BBB). Peripheral macrophages migrate into the hippocampus through a permeable BBB. Upon macrophage and resident microglia activation in the hippocampus, NF-κB is activated to enhance transcription, promoting neuroinflammation, including IL-1β, IL-6, TNF-a, and HMGB-1, which further augment NF-κB transcription, consequently contributing to cognitive impairment.3,6 In a further study, Terrando et al. found that TNF-α release is an important upstream event that can provoke IL-1β expression and amplify the production of neuroinflammation in the hippocampus.21 Our results suggested that the pre-treatment of nicotine inhibited surgery-induced proinflammatory cytokine release in the serum and hippocampus. Interestingly, the inflammation-enhanced translocation of the phosphorylated form of NF-κB p65 was blocked by nicotine pre-treatment. Taken together, we demonstrated that nicotine may attenuate POCD via its anti-inflammatory effects and suppressed the NF-κB signaling pathway in the hippocampus. BDNF, a growth factor, is widely expressed in the brain.22 Recent studies have found that changes in its level were correlated with the development of several human diseases, such as neurodegeneration, depression, and psychiatric disorders.12 BDNF exerts its neuronal protective functions through binding to the TrkB receptor; BDNF/TrkB signaling is critical for synaptic function and plasticity. Studies have previously reported its role in learning, memory and neurogensis.23 Before our study, the function of BDNF/TrkB signaling on nicotine-induced neuroprotective effects on POCD development was unclear. In our study, we observed that surgery significantly decreased hippocampal BDNF levels in the hippocampus and then inhibited the TrkB receptor, causing down-regulation of p-TrkB in the hippocampus. The observed inhibition in the BDNF/TrkB signaling pathway in aged rats may be linked to the performance of aged rats in spatial memory tasks. However, pre-treatment with nicotine effectively activated the BDNF/TrkB signaling pathway and resulted in a marked increase in hippocampal BDNF and p-TrkB levels in the hippocampus, an event that appears to be linked to improvement in memory and cognitive ability. It has been demonstrated that neuroapoptosis was responsible for surgery-induced cognitive dysfunction.24 Both increased proinflammatory cytokine levels and reduced BDNF levels in the hippocampus can induce neuronal apoptosis.24,25 Combined with the previous results from other studies or ours,8,26 we deduced that the excessive release of pro-inflammatory cytokines caused by the surgery stress impaired BDNF induction in the hippocampus, which, consequently, elicited neuronal apoptosis. Interestingly, we found that pre-treatment with nicotine markedly decreased the levels of cleaved caspase-3 (the active form of caspase-3), a pro-apoptotic protein involved in cell apoptosis in the hippocampus, and the percentage of TUNEL-positive nuclei in the hippocampal CA1 region. Per our results, it has been suggested that the inhibition of hippocampal neuroapoptosis may be another mechanism involved in the neuroprotective properties of nicotine against POCD in aged rats. Conclusion In conclusion, the obtained results confirmed our hypothesis that nicotine exhibited neuroprotective effects in surgery-induced cognitive dysfunction in aged rats, which might be associated with activation of the BDNF/TrkB signaling pathway and inhibition of the NF-κB signaling pathway in the hippocampus. Supplementary Material Supplementary data are available at Nicotine and Tobacco Research online. Funding This work was supported by grants from the Shandong Province Science and Technology Program (Grant No. 2011GSF11801). Declaration of Interests None declared. References 1. Zhang C, Li C, Xu Zet al.   The effect of surgical and psychological stress on learning and memory function in aged C57BL/6 mice. Neuroscience . 2016; 320: 210– 220. Google Scholar CrossRef Search ADS PubMed  2. Moller JT, Cluitmans P, Rasmussen LSet al.   Long-term postoperative cognitive dysfunction in the elderly ISPOCD1 study. ISPOCD investigators. International Study of Post-Operative Cognitive Dysfunction. Lancet . 1998; 351( 9106): 857– 861. Google Scholar CrossRef Search ADS PubMed  3. Wang H-L, Liu H, Xue Z-G, Liao Q-W, Fang H. Minocycline attenuates post-operative cognitive impairment in aged mice by inhibiting microglia activation. J Cell Mol Med . 2016; 20( 9): 1632– 1639. Google Scholar CrossRef Search ADS PubMed  4. Silbert BS, Evered LA, Scott DA. 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Acute nicotine treatment attenuates lipopolysaccharide-induced cognitive dysfunction by increasing BDNF expression and inhibiting neuroinflammation in the rat hippocampus. Neurosci Lett . 2015; 604: 161– 166. Google Scholar CrossRef Search ADS PubMed  9. Chen K, Wei P, Zheng Q, Zhou J, Li J. Neuroprotective effects of intravenous lidocaine on early postoperative cognitive dysfunction in elderly patients following spine surgery. Med Sci Monit . 2015; 21: 1402– 1407. Google Scholar CrossRef Search ADS PubMed  10. Zhong Y, Zhu Y, He T, Li W, Yan H, Miao Y. Rolipram-induced improvement of cognitive function correlates with changes in hippocampal CREB phosphorylation, BDNF and Arc protein levels. Neurosci Lett . 2016; 610: 171– 176. Google Scholar CrossRef Search ADS PubMed  11. Zhu L, Nang C, Luo Fet al.   Esculetin attenuates lipopolysaccharide (LPS)-induced neuroinflammatory processes and depressive-like behavior in mice. Physiol Behav . 2016; 163: 184– 192. Google Scholar CrossRef Search ADS PubMed  12. Fulgenzi G, Tomassoni-Ardori F, Babini Let al.   BDNF modulates heart contraction force and long-term homeostasis through truncated TrkB.T1 receptor activation. J Cell Biol . 2015; 210( 6): 1003– 1012. Google Scholar CrossRef Search ADS PubMed  13. Tian XS, Tong YW, Li ZQet al.   Surgical stress induces brain-derived neurotrophic factor reduction and postoperative cognitive dysfunction via glucocorticoid receptor phosphorylation in aged mice. CNS Neurosci Ther . 2015; 21( 5): 398– 409. Google Scholar CrossRef Search ADS PubMed  14. Shim SB, Lee SH, Chae KRet al.   Nicotine leads to improvements in behavioral impairment and an increase in the nicotine acetylcholine receptor in transgenic mice. Neurochem Res . 2008; 33( 9): 1783– 1788. Google Scholar CrossRef Search ADS PubMed  15. Quik M, Parameswaran N, McCallum SEet al.   Chronic oral nicotine treatment protects against striatal degeneration in MPTP-treated primates. J Neurochem . 2006; 98( 6): 1866– 1875. Google Scholar CrossRef Search ADS PubMed  16. Cao XZ, Ma H, Wang JKet al.   Postoperative cognitive deficits and neuroinflammation in the hippocampus triggered by surgical trauma are exacerbated in aged rats. Prog Neuropsychopharmacol Biol Psychiatry . 2010; 34( 8): 1426– 1432. Google Scholar CrossRef Search ADS PubMed  17. Xie Z, Le Y, Liu Set al.   Aging differentially affects the loss of neuronal dendritic spine, neuroinflammation and memory impairment at rats after surgery. Plos One . 2014; 9: e106837. Google Scholar CrossRef Search ADS PubMed  18. He HJ, Wang Y, Le Yet al.   Surgery upregulates high mobility group box-1 and disrupts the blood-brain barrier causing cognitive dysfunction in aged rats. CNS Neurosci Ther . 2012; 18( 12): 994– 1002. Google Scholar CrossRef Search ADS PubMed  19. Chen J, Buchanan JB, Sparkman NL, Godbout JP, Freund GG, Johnson RW. Neuroinflammation and disruption in working memory in aged mice after acute stimulation of the peripheral innate immune system. Brain Behav Immun . 2008; 22( 3): 301– 311. Google Scholar CrossRef Search ADS PubMed  20. Scavone C, Chen C, Cai Jet al.   Protective effect of RNase on unilateral nephrectomy-induced postoperative cognitive dysfunction in aged mice. Plos One . 2015; 10: e0134307. Google Scholar CrossRef Search ADS PubMed  21. Terrando N, Monaco C, Ma D, Foxwell BM, Feldmann M, Maze M. Tumor necrosis factor-alpha triggers a cytokine cascade yielding postoperative cognitive decline. Proc Natl Acad Sci USA . 2010; 107( 47): 20518– 20522. Google Scholar CrossRef Search ADS PubMed  22. Andresen JH, Løberg EM, Wright M, Goverud IL, Stray-Pedersen B, Saugstad OD. Nicotine affects the expression of brain-derived neurotrophic factor mRNA and protein in the hippocampus of hypoxic newborn piglets. J Perinat Med . 2009; 37( 5): 553– 560. Google Scholar CrossRef Search ADS PubMed  23. Fan D, Li J, Zheng B, Hua L, Zuo Z. Enriched Environment Attenuates Surgery-Induced Impairment of Learning, Memory, and Neurogenesis Possibly by Preserving BDNF Expression. Mol Neurobiol . 2016; 53( 1): 344– 354. Google Scholar CrossRef Search ADS PubMed  24. Li SY, Xia LX, Zhao YLet al.   Minocycline mitigates isoflurane-induced cognitive impairment in aged rats. Brain Res . 2013; 1496: 84– 93. Google Scholar CrossRef Search ADS PubMed  25. Lu B, Nagappan G, Lu Y. BDNF and synaptic plasticity, cognitive function, and dysfunction. Handb Exp Pharmacol . 2014; 220: 223– 250. Google Scholar CrossRef Search ADS PubMed  26. Kirsten TB, Chaves-Kirsten GP, Bernardes Set al.   Lipopolysaccharide Exposure Induces Maternal Hypozincemia, and Prenatal Zinc Treatment Prevents Autistic-Like Behaviors and Disturbances in the Striatal Dopaminergic and mTOR Systems of Offspring. PLoS One . 2015; 10( 7): e0134565. Google Scholar CrossRef Search ADS PubMed  © The Author(s) 2017. Published by Oxford University Press on behalf of the Society for Research on Nicotine and Tobacco. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com. http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Nicotine and Tobacco Research Oxford University Press

Nicotine-Induced Neuroprotection against Cognitive Dysfunction after Partial Hepatectomy Involves Activation of BDNF/TrkB Signaling Pathway and Inhibition of NF-κB Signaling Pathway in Aged Rats

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Abstract

Abstract Introduction The main purpose of this study was to investigate the effects and possible mechanisms of nicotine pre-treatment on postoperative cognitive dysfunction (POCD) in aged rats. Methods Nicotine (0.5 mg/kg) was given i.p. immediately after anesthesia induction. After the Morris water maze test was used to evaluate the rats’ spatial learning and memory, serum and hippocampal tissues were harvested 1 and 3 days after intervention. Inflammatory cytokines in the serum were evaluated by Enzyme-linked Immunosorbent Assay (ELISA). Brain-derived neurotrophic factor (BDNF), p-TrkB, neuroinflammation cytokines, NF-κB p65, and cleaved caspase-3 were measured by western blotting; neuronal apoptosis in the hippocampal CA1 region was also evaluated by TUNEL staining. Results We found that nicotine markedly attenuated the POCD and reduced the elevated levels of inflammatory cytokines in the serum, including IL-1β and high mobility group box-1 (HMGB1), on postoperative day 1. Additionally, nicotine suppressed the surgery-induced release of IL-1β, TNF-ɑ, HMGB1, and NF-κB p65 in the hippocampus on postoperative day 1 and day 3. In addition, operated rats displayed lower BDNF and p-TrkB in the hippocampus on postoperative day 1, returning to baseline by postoperative day 3. However, nicotine pre-treatment clearly reversed the surgical stress-induced decrease in both BDNF and p-TrkB expression in the hippocampus. Furthermore, nicotine pre-treatment significantly alleviated the surgery-induced increase in the neuronal apoptosis in the hippocampus on postoperative day 1 and day 3. Conclusions Our results showed that nicotine-induced neuroprotection against POCD may involve activation of the BDNF/TrkB signaling pathway and inhibition of the NF-κB signaling pathway. Implications Nicotine has long been considered a potent therapeutic agent for neuroprotection. This study reported the positive effect of nicotine treatment on cognitive dysfunction after partial hepatectomy in aged rats. Furthermore, the underlying mechanism may involve activation of the BDNF/TrkB signaling pathway and inhibition of the NF-κB signaling pathway in the hippocampus. Introduction Postoperative cognitive dysfunction (POCD) is an important clinical issue in perioperative care following major surgery and general anesthesia in elderly individuals. It refers to an impairment of memory, concentration, information processing, language comprehension, and social integration.1 The International Study of POCD (ISPOCD) has concluded that the incidence of POCD was about 26% at one week postoperatively and 10% at 3 months after surgery in patients aged over 60 year.2 Cognitive impairment after surgery has been associated with prolonged hospital day and increased cost of hospitalization and out-of-hospital care.3 POCD also adversely affects postoperative quality of life, surgical morbidity, and mortality.4 Although many etiologic and pathophysiological mechanisms have been implicated in the development of POCD, the exact cascade remains elusive. In the past decade, neuroinflammation has emerged as a key event in the development of POCD.5 The peripheral inflammatory mediators induced by surgical stress result in the release of inflammatory cytokines in the central nervous system.6 Neuroinflammation will harm the neurons and cause irreversible neuronal apoptosis, contributing to the brain functional changes.7 Our previous studies have also confirmed the critical role of neuroinflammation in the development of POCD.8 We further showed that anti-inflammatory agents effectively attenuated the cognitive impairment induced by surgical trauma.9 Neurotrophins are also critical for the development of POCD. Brain-derived neurotrophic factor (BDNF), an extensively studied neurotrophin that is mediated via tyrosine kinase B (TrkB),10 plays a critical role in neuron survival, plasticity, neurogenesis, and synaptogenesis in the developing brain.11 Mounting evidence has suggested that changes in its level are highly correlated with the development of several human diseases, including depression, neurodegeneration, and psychiatric disorders.12 A recent study has identified a reduction of surgical stress-induced BDNF as an important factor that influences the risk of POCD in aged mice.13 A clinical study conducted by our study team also showed that reduced BDNF levels in the serum are related to cognitive impairment after hip replacement surgery in elderly patients (unpublished data). Therefore, it might be possible to prevent surgical stress-induced cognitive dysfunction by activating BDNF/TrkB signaling pathways and inhibiting neuroinflammation-related signaling pathways. Nicotine, an α7 nicotinic acetylcholine receptor agonist (α7nAChR), has been demonstrated to have neuroprotective effects in experimental models of Alzheimer’s disease14 and Parkinson’s disease.15 Studies also reported that nicotine resolved neuroinflammation and reversed cognitive decline after tibial fracture operation in aged mice.6 However, the specific mechanisms underlying the nicotine pre-treatment-induced neuroprotective effect on POCD remain to be explored. Recently, we observed that nicotine pre-treatment effectively attenuated lipopolysaccharide-induced cognitive impairment by increasing BDNF expression and inhibiting neuroinflammation in the rat hippocampus.8 Therefore, we hypothesized that nicotine might be able to improve spatial learning and memory in aged rats with cognitive impairment induced by partial hepatectomy. We further hypothesized that the mechanism may involve activation of the BDNF/TrkB signaling pathways and inhibition of neuroinflammation-related signaling pathways. Materials and Methods Ethics Statement All animals protocols were approved by the Animal Care and Use Committee of Qilu Hospital (Qingdao) and performed in accordance with the guidelines for experiment animals use established by the Institutional Animal Care and Use Committee of Shandong University. Every possible efforts were made to minimize the number of rats used and their suffering. Animals and Grouping A total of 60 male Sprague Dawley rats (18 months old, weighing 480–600 g) were purchased from Shandong University Experiment Animal Center. The rats were raised in the specific pathogen—free animal laboratory at 23 ± 2°C with a relative humidity of 55 ± 5% on a 12:12 h light:dark cycle in the Experiment Animal Center of Shandong University. All rats were housed in standard cages and were provided with access to food and water ad libitum. Animals were allowed to acclimate for 7 days before beginning the experiments. The rats were randomly divided into four groups (n = 15): (1) Control (C); (2) Anesthesia only (A); (3) Anesthesia plus Surgery (S); (4) Nicotine plus Surgery (Nic + S). Rats were sacrificed at days 1 and 3 after intervention. Surgical Procedures The rats in group C received sterile saline to control for the effects of injection stress. The rats in group A were intraperitoneally anesthetized with a combination of 20 μg/kg fentanyl and 500 μg/kg droperidol. The rats underwent partial hepatectomy under general anesthesia in the S and Nic + S groups. Partial hepatectomy was performed under aseptic conditions as described previously.16 Briefly, the liver was exposed through a 2 cm midline abdominal incision. The left lateral lobes of the liver were visualized, isolated, and excised. The wound was then infiltrated with 0.25% bupivacaine. After checking, the muscles and skin were closed by sterile suture. Nicotine hydrogen tartrate (Sigma, St. Louis, MO, USA) in 0.9% saline was used. Nicotine was were given i.p. immediately at final concentrations of 0.5 mg/kg nicotine-free base in the Nic + S group after anesthesia induction. The dose used was based on our previous study.8 The exploratory laparotomy was performed under anesthesia before the blood and tissue sample collection. The rats suffering from the intra-abdominal infection and hemorrhage were excluded. Morris Water Maze (MWM) The experimental design is shown in Figure 1A. The MWM test was used to evaluate the rats’ hippocampus-dependent spatial learning and memory, as described previously.17 A video tracking system recording the rat’s movement in a water maze was used in conjunction with the Color Video Camera. Rats were tested in a black circular tank (1.2 m diameter, 50 cm deep; water, 22°C) filled to 30 cm. The nonvisible platform (15 cm diameter) was placed in the first quadrant (target quadrant) and positioned 2 cm below the water surface. There were many visual cues around the tank. Every rat was placed on the platform for 30 s before the start of each trial. In each trial, the rats were placed in a fixed starting position in the maze, facing the tank wall. They were given 60 s to locate the platform in the first quadrant. The rats that found the platform within this time limit were allowed to remain there for 15 s. Otherwise, the animals that did not locate the platform were guided to it and were allowed to remain there for 15 s. Throughout the experiment, the location of the platform was fixed and rats were trained for four trials per day for 6 consecutive days. The learning parameters that were recorded included swimming speed and latency (time from the start of the trial until the rat reached the platform). On day 7, rats received partial hepatectomy and/or general anesthesia. Animals were subjected to a memory test on postoperative days 1 and 3. After removing the platform from the first quadrant, the third quadrant was used as the starting position to evaluate the memory. The rats were allowed to swim freely for 60 s. The latency for the first time to reach the target area, the percentage of time spent in the target quadrant and the number of platform crossings were recorded on preoperative day 1 and postoperative days 1 and 3. Figure 1. View largeDownload slide Effects of nicotine pre-treatment on spatial learning and memory parameters in the Morris Water Maze test. (A) Experimental design. (B) Mean escape latencies of the four groups in the acquisition phase. n = 12 per group. (C) Average swimming speed of each group in the acquisition phase. n = 12 per group. (D) Latencies for the first time to the platform for each group at 1 day before surgery and at 1 day (n = 12) and 3 days (n = 6) after surgery in the probe trials. (E) Time spent in the target quadrant for each group at 1 day before surgery and at 1 day (n = 12) and 3 days (n = 6) after surgery in the probe trials. (F) The number of platform crossings with a 60-s limit for each group at 1 day before surgery and at 1 day (n = 12) and 3 days (n = 6) after surgery in the probe trials. All data are expressed as the means ± SD. *p < .05. Figure 1. View largeDownload slide Effects of nicotine pre-treatment on spatial learning and memory parameters in the Morris Water Maze test. (A) Experimental design. (B) Mean escape latencies of the four groups in the acquisition phase. n = 12 per group. (C) Average swimming speed of each group in the acquisition phase. n = 12 per group. (D) Latencies for the first time to the platform for each group at 1 day before surgery and at 1 day (n = 12) and 3 days (n = 6) after surgery in the probe trials. (E) Time spent in the target quadrant for each group at 1 day before surgery and at 1 day (n = 12) and 3 days (n = 6) after surgery in the probe trials. (F) The number of platform crossings with a 60-s limit for each group at 1 day before surgery and at 1 day (n = 12) and 3 days (n = 6) after surgery in the probe trials. All data are expressed as the means ± SD. *p < .05. Enzyme-linked Immunosorbent Assay (ELISA) The rats in each group were anesthetized using Nembutal. Their blood was collected via cardiac puncture. The blood was allowed to clot for 2 h at room temperature and was then centrifuged for 15 min at 2000g at 4°C. The serum fraction was removed and stored at −80°C for further analysis. The hippocampus was rinsed with buffered saline (PBS), homogenized in 1 mL of PBS, and stored overnight at −20°C. The homogenates were subjected to two freeze–thaw cycles to break the cell membrane and then centrifuged for 5 min at 5000g at 2°C–8°C. The supernatants were stored at −80°C. The levels of serum IL-1β, TNF-α, high mobility group box-1 (HMGB-1), and hippocampal BDNF were measured using commercially available ELISA kits (Neobioscience Technology Company: IL-1β, TNF-α, BDNF; R&D Systems: HMGB-1) according to the manufacturer’s protocol. Western Blot Analysis Rats were sacrificed under deep anesthesia and hippocampal tissues were removed. Subsequently, the harvested tissues were homogenized on ice using 1 mL of ice-cold RIPA buffer containing the protease inhibitor phenylmethanesulfonyl fluoride (2 mM) in added protease inhibitor. The homogenates were centrifuged at 13000 rpm at 4°C for 30 min. The protein concentration in the supernatant was determined using the bicinchoninic acid (BCA) method. Equal amounts of protein (40 μg) from each sample were separated via 12% SDS–PAGE and were transferred to PVDF membranes (Millipore, Boston, MA, USA). The membranes were immunoblotted overnight at 4°C using a rabbit anti-IL-1β antibody (Proteintech, China; dilution 1:500), goat anti-TNF-α antibody (Santa Cruz Biotechnology Inc., USA; dilution 1:500), rabbit anti-NF-κB p65 antibody (Proteintech, China; dilution 1:1000), rabbit anti-caspase-3 antibody (Proteintech, China; dilution 1:500), rabbit anti-BDNF antibody (Santa Cruz Biotechnology Inc., USA; dilution 1:1000), rabbit anti-phospho-TrkB antibody (Abcam, USA; dilution 1:1000), rabbit anti-HMGB-1 antibody (Abcam, USA; dilution 1:10000) and rabbit anti-β-actin antibody (Proteintech, China; dilution 1:1000). After three 5 min washes, the membranes were incubated in an HRP-conjugated secondary antibody (Proteintech, China; dilution 1:2000) for 1 h at room temperature, followed by washing and subsequent visualization via enhanced chemiluminescence (ECL; Millipore, Billerica, MA, USA). Finally, the optical densities of the resultant bands were recorded and analyzed using C-Digit software (LI-COR, USA). The relative expression was normalized to β-actin. TUNEL Staining Under deep anesthesia, the thoracic cavity was opened to expose the heart, and its right atrium was severed. Next, 0.01 M PBS (4°C) followed by 200 mL of ice-cold 4% paraformaldehyde were transcardially perfused into the left ventricle. The brains were removed and post-fixed in 4% paraformaldehyde overnight at 4°C, followed by rehydration and embedding in paraffin. The sections were deparaffinized before antigen retrieval by microwaving the sections in sodium citrate buffer. The samples were preincubated in 3% H2O2 at room temperature for 10 min to eliminate nonspecific peroxidase activity and were then blocked in 3% normal goat serum at room temperature for 1 h. The sections were stained using an in situ cell death detection kit (POD; Roche Diagnostics Corp., Indianapolis, IN, USA) per the manufacturer’s instructions. Five sections in each rat were collected from CA1 of the dorsal hippocampus and were examined in each group. In each section, five independent fields (400×) were selected for examination by two independent observers who were blinded to the experimental groups. The percentage of TUNEL-positive nuclei in the dorsal CA1 region was calculated to evaluate apoptosis. Statistical Analysis All data are presented as the means ± SD. Training behavioral parameters were analyzed by two-way repeated measures ANOVA. Memory behavioral parameters, ELISA, Western blot and TUNEL staining were analyzed using one-way ANOVA followed by Tukey–Kramer post hoc tests for multiple comparisons. The differences between the values were considered to be significant at p < .05. All statistical tests and graphs were performed using Statistical Program for Social Sciences 20.0 software (SPSS, Inc., Chicago, IL, USA). Results Nicotine Attenuated the Surgery-Induced Cognitive Dysfunction Sixty rats were enrolled in the study. Twelve rats were excluded because of the intra-abdominal infection and hemorrhage, and there were 48 rats (n = 12) entered the final analysis. To evaluate the effect of nicotine on surgery stress-induced cognitive dysfunction, the Morris water maze was employed to analyze cognition changes. Comparisons by two-way repeated-measures ANOVA revealed no significant difference in latency and swimming speeds among the four groups (Figure 1B and C, p > 0.05). In the following probe trials, the latency for the first time to the platform, the percentage of time spent in the target quadrant (first quadrant) and the number of original platform crossings were analyzed (Figure 1D, E, and F). Significant differences in the latency (1 d aft: F3,44=46.357, p = .000; 3 d aft: F3,20=20.529, p = .000), the time spent in the target quadrant (1 d aft: F3,44=7.634, p = .000; 3 d aft: F3,20=19.225, p = .000) and the number of platform crossings (1 d aft: F3,44=16.310, p = .000; 3 d aft: F3,20=22.101, p = .000) were observed among the four groups at days 1 and 3 after surgery. Compared with group C, the latency for the first time to the platform increased in group S (p < .05), and the time spent in the target quadrant (p < .05) and the number of platform crossings (p < .05) decreased in group S at days 1 and 3 after surgery. However, compared with group S, the latency for the first time to the platform significantly decreased in group Nic+S (p < .05), and the time spent in the target quadrant and the number of platform crossings significantly increased in group Nic+S (p < .05). Nicotine Decreased Inflammatory Cytokine Expression in the Hippocampus To understand whether nicotine inhibited inflammation in the central compartment, the protein expression levels of the pro-inflammatory cytokines IL-1β, TNF-α, HMGB-1, and NF-κB p65 were measured in the hippocampus at 1 day and 3 days after surgery. As shown in Figure 2, western blot analysis indicated that exposure to surgery significantly increased hippocampus IL-1β (F3,8 = 15.534, p = .001), TNF-α (F3,8 = 33.656, p = .000), HMGB-1 (F3,8 = 38.874, p = .000), and NF-κB p65 (F3,8 = 76.238, p = .000) expression versus the control group on postoperative days 1 (p < .05). Levels of these pro-inflammatory cytokines in the hippocampus remained elevated in the surgical group up to postoperative day 3 (IL-1β: F3,8 = 10.133, p = .004; TNF-α: F3,8 = 22.295, p = .000; HMGB-1: F3,8 = 34.908, p = .000; NF-κB p65: F3,8 = 21.961, p = .000). However, the administration of nicotine potently reduced the levels of IL-1β, TNF-α, HMGB-1, and NF-κB p65 on postoperative days 1 and 3 (p < .05). Figure 2. View largeDownload slide Effects of nicotine pre-treatment on surgery-induced IL-1β, TNF-α, HMGB-1, and NF-κB p65 levels in the hippocampus at 1 day and 3 days after surgery. Western blot analyses were employed to measure IL-1β (A), TNF-α (B), HMGB-1 (C), and NF-κB p65 (D) expression. The quantification analysis showed that nicotine treatment significantly decreased the protein levels of TNF-α, IL-1β, HMGB-1 and NF-κB p65 in the hippocampus at 1 day and 3 days after surgery. All data are expressed as the means ± SD. n = 3 per group. *p < .05, **p < .01. Figure 2. View largeDownload slide Effects of nicotine pre-treatment on surgery-induced IL-1β, TNF-α, HMGB-1, and NF-κB p65 levels in the hippocampus at 1 day and 3 days after surgery. Western blot analyses were employed to measure IL-1β (A), TNF-α (B), HMGB-1 (C), and NF-κB p65 (D) expression. The quantification analysis showed that nicotine treatment significantly decreased the protein levels of TNF-α, IL-1β, HMGB-1 and NF-κB p65 in the hippocampus at 1 day and 3 days after surgery. All data are expressed as the means ± SD. n = 3 per group. *p < .05, **p < .01. Nicotine Reduced Inflammatory Cytokine Levels in the Serum Serum samples were collected from all the animals at 1 day and 3 days after surgery to evaluate the systemic levels of IL-1β, TNF-α and HMGB-1 (Figure 3A, B and C). No changes in serum TNF-α were observed on postoperative days 1 and 3 (1 d aft: F3,16 = 0.231, p = .874; 3 d aft: F3,16 = 0.135, p = .938) between the four groups. Surgery significantly upregulated serum levels of IL-1β (F3,16 = 46.838, p = .000) and HMGB-1 (F3,16 = 5.003, p = .012) on postoperative day 1, which returns to baseline by day 3 (IL-1β: F3,16 = 0.446, p = .724; HMGB-1: F3,16 = 0.067, p = .977). However, the elevated levels of these inflammatory cytokines were dramatically attenuated by nicotine administration (p < .05). Nicotine Attenuated the Surgery-Induced Down-Regulation of BDNF and p-TrkB in the Hippocampus To explore the mechanism by which nicotine administration ameliorates cognitive dysfunction, we measured the expression of BDNF and p-TrkB. As shown in Figure 3D, E and F, exposure to surgery significantly decreased BDNF versus the control group on postoperative day 1(ELISA methodologies: F3,8 = 9.649, p = .005; western blot analysis: F3,8 = 9.704, p = .005). Furthermore, the down-regulation of BDNF inhibited the TrkB receptor and the downstream signaling pathway. As expected, the expression of p-TrkB was reduced on postoperative day 1 (F3,8 = 8.478, p = .007). However, nicotine effectively upregulated BDNF and p-TrkB expression in the hippocampus on postoperative day 1 (p < .05). No significant difference in hippocampal BDNF and p-TrkB expression was observed on postoperative day 3 between the four groups (BDNF: western blot analysis: F3,8 = 0.399, p = .757, ELISA methodologies: F3,8 = 0.138, p = .935; TrkB: F3,8 = 0.145, p = .930). Figure 3. View largeDownload slide Effects of nicotine pre-treatment on the levels of IL-1β, TNF-α and HMGB-1 in serum and the levels of BDNF and p-TrkB in the hippocampus at 1 day and 3 days after surgery. (A) No changes in serum TNF-α were observed on at 1 day and 3 days after surgery. Surgery increased the levels of IL-1β (B) and HMGB-1 (C) in serum at these time points. However, nicotine treatment clearly reduced the levels of IL-1β and HMGB-1. (D) ELISA methodologies of BDNF protein levels in hippocampus. (E) Western blot analysis of BDNF and p-TrkB protein levels in the hippocampus. (F) The quantification analysis of BDNF and p-TrkB protein levels in the hippocampus. All data are expressed as the means ± SD. n = 3 per group. *p < .05, **p < .01. Figure 3. View largeDownload slide Effects of nicotine pre-treatment on the levels of IL-1β, TNF-α and HMGB-1 in serum and the levels of BDNF and p-TrkB in the hippocampus at 1 day and 3 days after surgery. (A) No changes in serum TNF-α were observed on at 1 day and 3 days after surgery. Surgery increased the levels of IL-1β (B) and HMGB-1 (C) in serum at these time points. However, nicotine treatment clearly reduced the levels of IL-1β and HMGB-1. (D) ELISA methodologies of BDNF protein levels in hippocampus. (E) Western blot analysis of BDNF and p-TrkB protein levels in the hippocampus. (F) The quantification analysis of BDNF and p-TrkB protein levels in the hippocampus. All data are expressed as the means ± SD. n = 3 per group. *p < .05, **p < .01. Nicotine Suppressed Cleaved Caspase-3 Expression in the Hippocampus and Neuron Apoptosis in the Dorsal Hippocampal CA1 Region To further investigate whether neuronal apoptosis occurred in the hippocampus, the hippocampal cleaved caspase-3 level and the percentage of TUNEL-positive nuclei in the dorsal hippocampal CA1 region were assessed. Surgery exposure markedly increased the expression of cleaved caspase-3 in the hippocampus, which was reversed by treatment with nicotine (Figure 4A, 1 d aft: F3,8 = 39.689, p = .000; 3 d aft: F3,8 = 52.097, p = .000). Compared with group C, neuron apoptosis was also clearly detected at 1 day and 3 days after surgery in group S (Figure 4B, 1 d aft: F3,8 = 143.523, p = .000; 3 d aft: F3,8 = 463.438, p = .000). Nicotine clearly reduced the number of apoptotic cells in the hippocampus at 1 day and 3 days after the treatment (p < .05). Figure 4. View largeDownload slide Effects of nicotine pre-treatment on the levels of cleaved caspase-3 in the hippocampus and neurons apoptosis in the dorsal hippocampal CA1 region. (A) Western blot analyses were employed to measure cleaved caspase-3; the quantification analysis showed that nicotine treatment significantly decreased the protein levels of cleaved caspase-3 in the hippocampus at 1 day and 3 days after surgery. (B) Representative TUNEL staining (400×) of hippocampal sections is shown; the data are quantitatively expressed in the histogram. Scale bars represent 50 μm. All data are expressed as the means ± SD. n = 3 per group. *p < .05, **p < .01. Figure 4. View largeDownload slide Effects of nicotine pre-treatment on the levels of cleaved caspase-3 in the hippocampus and neurons apoptosis in the dorsal hippocampal CA1 region. (A) Western blot analyses were employed to measure cleaved caspase-3; the quantification analysis showed that nicotine treatment significantly decreased the protein levels of cleaved caspase-3 in the hippocampus at 1 day and 3 days after surgery. (B) Representative TUNEL staining (400×) of hippocampal sections is shown; the data are quantitatively expressed in the histogram. Scale bars represent 50 μm. All data are expressed as the means ± SD. n = 3 per group. *p < .05, **p < .01. Discussion In the current study, we first reported the protective effect of nicotine pre-treatment on cognitive dysfunction induced by partial hepatectomy in aged rats. Our results showed the following findings: (i) nicotine effectively attenuated cognitive dysfunction induced by partial hepatectomy, as indicated by the MWM test. (ii) Nicotine suppressed the surgery-induced excessive release of inflammatory cytokines in the serum and hippocampus. (iii) Moreover, nicotine decreased the neuronal apoptosis in the hippocampus. (iv) Importantly, this study indicated that nicotine activated the BDNF-TrkB signaling pathway and inhibited the NF-κB signaling pathway in the hippocampus. Collectively, these findings indicated that nicotine may be an effective neuroprotective agent for the prevention of POCD. Currently, there are few available therapeutic interventions to prevent POCD. Thus, we sought to investigate the effect of nicotine on POCD in aged rats. Several recent rodent models have successfully mimicked POCD in humans, such as splenectomy,18 minor abdominal surgery,7 partial hepatectomy,17 and tibial fracture.6 In this study, we generated the experimental POCD model by performing partial hepatectomy in aged rats. Additionally, studies have indicated that the hippocampus, one of the most vulnerable tissues to stress, played a critical role in spatial memory formation and consolidation; exposure to surgical stress negatively affected functional and structural effects on the hippocampus.19 Hippocampal damage causes brain disorders, resulting in cognitive and behavioral impairment.10 Therefore, we focused on the hippocampus. Recently, we also observed that nicotine pre-treatment (0.5 mg/kg, i.p.) significantly attenuated lipopolysaccharide-induced deficits in learning and memory function in rats.8 Based on our previous results, we hypothesize that nicotine may provide a pharmacological strategy to prevent POCD. To determine the effect of nicotine on cognitive dysfunction after partial hepatectomy, the Morris water maze, one of the most widely accepted models in rodents, was used to evaluate learning and memory in this study. Our results showed that the aged rats had deficits in the hippocampus-dependent spatial memory as manifested by the longer latency to reach the platform for the first time, the lower percentage of time spent in the target quadrant and the lower number of original platform crossings in the MWM test at day 1 and day 3 after surgery, which were in line with other studies.17,18 However, those deficits could be clearly reversed by nicotine pre-treatment. To investigate the potential mechanisms of how nicotine improves learning and memory, expressed as the results of the MWM test, we focused our biochemical analyses on the neuroinflammation-related signaling pathway and the BDNF-TrkB signaling pathway in the hippocampus. Neuroinflammation has been considered the main mechanism involved in the pathogenesis of POCD.20 Surgical stress engages the immune system and initiates an inflammatory cascade involving inflammatory cytokines in the serum. These cytokines readily compromise endothelial function and disrupt the blood-brain barrier (BBB). Peripheral macrophages migrate into the hippocampus through a permeable BBB. Upon macrophage and resident microglia activation in the hippocampus, NF-κB is activated to enhance transcription, promoting neuroinflammation, including IL-1β, IL-6, TNF-a, and HMGB-1, which further augment NF-κB transcription, consequently contributing to cognitive impairment.3,6 In a further study, Terrando et al. found that TNF-α release is an important upstream event that can provoke IL-1β expression and amplify the production of neuroinflammation in the hippocampus.21 Our results suggested that the pre-treatment of nicotine inhibited surgery-induced proinflammatory cytokine release in the serum and hippocampus. Interestingly, the inflammation-enhanced translocation of the phosphorylated form of NF-κB p65 was blocked by nicotine pre-treatment. Taken together, we demonstrated that nicotine may attenuate POCD via its anti-inflammatory effects and suppressed the NF-κB signaling pathway in the hippocampus. BDNF, a growth factor, is widely expressed in the brain.22 Recent studies have found that changes in its level were correlated with the development of several human diseases, such as neurodegeneration, depression, and psychiatric disorders.12 BDNF exerts its neuronal protective functions through binding to the TrkB receptor; BDNF/TrkB signaling is critical for synaptic function and plasticity. Studies have previously reported its role in learning, memory and neurogensis.23 Before our study, the function of BDNF/TrkB signaling on nicotine-induced neuroprotective effects on POCD development was unclear. In our study, we observed that surgery significantly decreased hippocampal BDNF levels in the hippocampus and then inhibited the TrkB receptor, causing down-regulation of p-TrkB in the hippocampus. The observed inhibition in the BDNF/TrkB signaling pathway in aged rats may be linked to the performance of aged rats in spatial memory tasks. However, pre-treatment with nicotine effectively activated the BDNF/TrkB signaling pathway and resulted in a marked increase in hippocampal BDNF and p-TrkB levels in the hippocampus, an event that appears to be linked to improvement in memory and cognitive ability. It has been demonstrated that neuroapoptosis was responsible for surgery-induced cognitive dysfunction.24 Both increased proinflammatory cytokine levels and reduced BDNF levels in the hippocampus can induce neuronal apoptosis.24,25 Combined with the previous results from other studies or ours,8,26 we deduced that the excessive release of pro-inflammatory cytokines caused by the surgery stress impaired BDNF induction in the hippocampus, which, consequently, elicited neuronal apoptosis. Interestingly, we found that pre-treatment with nicotine markedly decreased the levels of cleaved caspase-3 (the active form of caspase-3), a pro-apoptotic protein involved in cell apoptosis in the hippocampus, and the percentage of TUNEL-positive nuclei in the hippocampal CA1 region. Per our results, it has been suggested that the inhibition of hippocampal neuroapoptosis may be another mechanism involved in the neuroprotective properties of nicotine against POCD in aged rats. Conclusion In conclusion, the obtained results confirmed our hypothesis that nicotine exhibited neuroprotective effects in surgery-induced cognitive dysfunction in aged rats, which might be associated with activation of the BDNF/TrkB signaling pathway and inhibition of the NF-κB signaling pathway in the hippocampus. Supplementary Material Supplementary data are available at Nicotine and Tobacco Research online. Funding This work was supported by grants from the Shandong Province Science and Technology Program (Grant No. 2011GSF11801). Declaration of Interests None declared. References 1. Zhang C, Li C, Xu Zet al.   The effect of surgical and psychological stress on learning and memory function in aged C57BL/6 mice. Neuroscience . 2016; 320: 210– 220. Google Scholar CrossRef Search ADS PubMed  2. Moller JT, Cluitmans P, Rasmussen LSet al.   Long-term postoperative cognitive dysfunction in the elderly ISPOCD1 study. ISPOCD investigators. International Study of Post-Operative Cognitive Dysfunction. Lancet . 1998; 351( 9106): 857– 861. Google Scholar CrossRef Search ADS PubMed  3. Wang H-L, Liu H, Xue Z-G, Liao Q-W, Fang H. Minocycline attenuates post-operative cognitive impairment in aged mice by inhibiting microglia activation. J Cell Mol Med . 2016; 20( 9): 1632– 1639. Google Scholar CrossRef Search ADS PubMed  4. Silbert BS, Evered LA, Scott DA. 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Acute nicotine treatment attenuates lipopolysaccharide-induced cognitive dysfunction by increasing BDNF expression and inhibiting neuroinflammation in the rat hippocampus. Neurosci Lett . 2015; 604: 161– 166. Google Scholar CrossRef Search ADS PubMed  9. Chen K, Wei P, Zheng Q, Zhou J, Li J. Neuroprotective effects of intravenous lidocaine on early postoperative cognitive dysfunction in elderly patients following spine surgery. Med Sci Monit . 2015; 21: 1402– 1407. Google Scholar CrossRef Search ADS PubMed  10. Zhong Y, Zhu Y, He T, Li W, Yan H, Miao Y. Rolipram-induced improvement of cognitive function correlates with changes in hippocampal CREB phosphorylation, BDNF and Arc protein levels. Neurosci Lett . 2016; 610: 171– 176. Google Scholar CrossRef Search ADS PubMed  11. Zhu L, Nang C, Luo Fet al.   Esculetin attenuates lipopolysaccharide (LPS)-induced neuroinflammatory processes and depressive-like behavior in mice. Physiol Behav . 2016; 163: 184– 192. Google Scholar CrossRef Search ADS PubMed  12. Fulgenzi G, Tomassoni-Ardori F, Babini Let al.   BDNF modulates heart contraction force and long-term homeostasis through truncated TrkB.T1 receptor activation. J Cell Biol . 2015; 210( 6): 1003– 1012. Google Scholar CrossRef Search ADS PubMed  13. Tian XS, Tong YW, Li ZQet al.   Surgical stress induces brain-derived neurotrophic factor reduction and postoperative cognitive dysfunction via glucocorticoid receptor phosphorylation in aged mice. CNS Neurosci Ther . 2015; 21( 5): 398– 409. Google Scholar CrossRef Search ADS PubMed  14. Shim SB, Lee SH, Chae KRet al.   Nicotine leads to improvements in behavioral impairment and an increase in the nicotine acetylcholine receptor in transgenic mice. Neurochem Res . 2008; 33( 9): 1783– 1788. Google Scholar CrossRef Search ADS PubMed  15. Quik M, Parameswaran N, McCallum SEet al.   Chronic oral nicotine treatment protects against striatal degeneration in MPTP-treated primates. J Neurochem . 2006; 98( 6): 1866– 1875. Google Scholar CrossRef Search ADS PubMed  16. Cao XZ, Ma H, Wang JKet al.   Postoperative cognitive deficits and neuroinflammation in the hippocampus triggered by surgical trauma are exacerbated in aged rats. Prog Neuropsychopharmacol Biol Psychiatry . 2010; 34( 8): 1426– 1432. Google Scholar CrossRef Search ADS PubMed  17. Xie Z, Le Y, Liu Set al.   Aging differentially affects the loss of neuronal dendritic spine, neuroinflammation and memory impairment at rats after surgery. Plos One . 2014; 9: e106837. Google Scholar CrossRef Search ADS PubMed  18. He HJ, Wang Y, Le Yet al.   Surgery upregulates high mobility group box-1 and disrupts the blood-brain barrier causing cognitive dysfunction in aged rats. CNS Neurosci Ther . 2012; 18( 12): 994– 1002. Google Scholar CrossRef Search ADS PubMed  19. Chen J, Buchanan JB, Sparkman NL, Godbout JP, Freund GG, Johnson RW. Neuroinflammation and disruption in working memory in aged mice after acute stimulation of the peripheral innate immune system. Brain Behav Immun . 2008; 22( 3): 301– 311. Google Scholar CrossRef Search ADS PubMed  20. Scavone C, Chen C, Cai Jet al.   Protective effect of RNase on unilateral nephrectomy-induced postoperative cognitive dysfunction in aged mice. Plos One . 2015; 10: e0134307. Google Scholar CrossRef Search ADS PubMed  21. Terrando N, Monaco C, Ma D, Foxwell BM, Feldmann M, Maze M. Tumor necrosis factor-alpha triggers a cytokine cascade yielding postoperative cognitive decline. Proc Natl Acad Sci USA . 2010; 107( 47): 20518– 20522. Google Scholar CrossRef Search ADS PubMed  22. Andresen JH, Løberg EM, Wright M, Goverud IL, Stray-Pedersen B, Saugstad OD. Nicotine affects the expression of brain-derived neurotrophic factor mRNA and protein in the hippocampus of hypoxic newborn piglets. J Perinat Med . 2009; 37( 5): 553– 560. Google Scholar CrossRef Search ADS PubMed  23. 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Published by Oxford University Press on behalf of the Society for Research on Nicotine and Tobacco. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

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Nicotine and Tobacco ResearchOxford University Press

Published: Apr 1, 2018

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