Postoperative Cognitive Deficits And Neuroinflammation In The Hippocampus Triggered By Surgical Trauma Are Exacerbated In Aged Rats

IntroductionElderly patients undergoing surgical intervention often suffer from postoperative cognitive dysfunction (POCD), a condition characterized by the progressive deterioration of cognitive function, following surgery. These cognitive impairments, either transient or permanent, affect a significant number of patients, and result in prolonged hospitalization and delayed recovery from illness. POCD is more frequently reported following cardiac surgery and is also known to occur in other surgical procedures.Although the neurobiological basis of POCD remains unknown, major risk factors, such as advanced age, poor education, preexisting cognitive impairment, severity of coexisting illness, duration of anesthesia, respiratory complications and second operation, have been identified. Of these, age has been increasingly reported as the most prominent risk factor for the development of POCD. Surprisingly, hypoxaemia or hypotensive episodes do not appear to influence the incidence of POCD,suggesting more involvement of surgical trauma itself in the impairment of cognitive function.Several theories have been advanced to explain the mechanisms of POCD. It remains unclear whether its occurrence is directly related to surgical trauma. Stress activates the peripheral innate immune system, resulting in the release of inflammatory mediators, which impairs cognitive function [13]. Age-related neuroinflammatory changes have been reported, including the increased expression of pro-inflammatory cytokines IL-1β, IL-6 and TNF-α. A substantial body of evidence indicates that significantly elevated expression of pro-inflammatory cytokines, particularly in the hippocampus, results in impairments in long-term potentiation (LTP) and performance deficits in hippocampal-mediated cognitive tests. In the present study, we investigated the effects of partial hepatectomy on spatial learning and memory and hippocampal pro-inflammatory cytokines in adult and aged rats.Materials and methodsAnimalsBoth adult (3-6 months old) and aged (20-24 months old) Sprague-Dawley male rats were randomly divided into a total of six groups:control (n=12 for each group), anesthesia alone (n=36 for each group), and operation group (n=36 for each group). All animals were housed in a temperature controlled room on a 12-h light and 12-h dark cycle with ad libitum access to food and water. All rats were adapted to their environment for a minimum of 7-10 days before experimentation. Control animals received sterile saline to control for effects of injection stress. The anesthesia alone group received 1.5% to 2% isoflurane with intubation and mechanical ventilation. The operation group underwent partial hepatectomy under general anesthesia. In brief, the liver was exposed through a 1-2 cm midline abdominal incision. The left lateral lobes of the liver (approximately corresponding to 30% of the organ) were excised. The wound was then infiltrated with 0.25% bupivacaine, and closed by sterile suture. All experimental procedures were performed in accordance with the Declaration of the National Institutes of Health Guide for Care and Use of Laboratory Animals.Experimental proceduresRats were sacrificed on postoperative days 1,3, and 7 (n=12/time point) after spatial working tests. Hippocampal tissues of half of the rats in each group were quickly dissected and stored at-70℃until they were assayed for central cytokine mRNAs. The remaining animals in each group were killed and perfused transcardially and postfixed for immunohistochemistry.Cognitive testingThe Morris water maze (MWM) is a hippocampal-dependent test of spatial learning for rodents. Rats were placed on the platform for 30 s before the start of each trial, and released into the water facing the wall of the pool from one of four randomly assigned release points (N, W, S and E). In all trials, rats were allowed to swim until they landed on the platform. If a rat failed to find the platform within 60 s, it was usually picked up and placed on the platform for 15 s. The animal remained on the platform for 30 s between trials. Rats were trained with the platform in a fixed location for three trials per day for 6 consecutive days. Animals underwent surgery on day 7. On postoperative days 1,3,7 rats were subjected to a reversal test in which the platform was relocated to the opposite quadrant of the pool. Reversal learning reveals whether animals can extinguish their initial learning of the platform's position and acquire a direct path to the new goal position. Swimming distance, speed, and latency to the platform were recorded by video tracking mounted on the ceiling and digital images were analyzed by water maze software (HVS image, United Kingdom).Brain cytokine mRNA extraction and reverse-transcription PCRTotal RNA was extracted from homogenization of 200 mg hippocampal tissue samples per manufacturer's instructions for TRIZOL Reagent (Invitrogen, US). Reverse-transcription polymerase chain reacrtion (RT-PCR) was performed as previously reported. Briefly, synthesis of the first strand of complementary DNA was conducted using RNA PCR Kit (AMV) Version 3.0 (TaKaRa, Japan). Reverse transcription was performed at 30℃for 10 min, 42℃for 30 min and terminated by heating to 99℃for 5 min. The products were amplified by PCR. Primer sequences and amplification sizes are given in Table 1. The amount of mRNA was expressed as a ratio of densitometric measurements derived from target mRNA andβ-actin. ImmunohistochemistryImmunohistochemistry was performed as previously reported. Sections were deparaffinized and rehydrated, then incubated with 3% H2O2 in methanol at room temperature for 10 min to block endogenous peroxidase and washed three times with PBS, and blocked in 1% normal goat serum at room temperature for 30 min. Primary antibodies were used as follows:mouse rabbit anti-rat glial fibrillary acidic protein (GFAP) antibody(1:80; Sigma) and mouse anti-rat S100βantibody (1:200; Sigma). Incubation with primary antibody was carried out at 4℃for overnight, followed by rinsing several times. Biotinylated goat anti-rabbit or goat anti-mouse IgG secondary antibody was applied at 37℃for 30 min. After thorough washing, the reaction product was visualized using the DAB method. The sections were counterstained with hematoxylin, dehydrated, and mounted. Control samples were run in parallel omitting primary antibody. Every fifth section was selected and generated 5-10 sections per reference space in a systematic-random manner. The positive cells in the hippocampus were estimated using the MetaMorph software system (US).Statistical AnalysisData from immunohistochemistry and RT-PCR were analyzed with two-way ANOVA in which age and operation were dependent variables. Repeated-measures ANOVA as a two-way design was used to analyze training behavioral parameters. A separate two-way ANOVA examined the effects of age and operation on working memory performance during reversal testing. Post hoc Student's t-test was employed when ANOVA revealed significance. A p-value< 0.05 was considered to be statistically significant.ResultsAged rats exhibit impaired performance on spatial learning and memory exacerbated by surgical traumaTo evaluate the effects of surgery on hippocampus-dependent spatial learning and memory, a MWM was constructed. Repeated-measures of ANOVA of swim data revealed significant effects of day (p<0.001) and age (p<0.001) on both latency and distance (Figs. 1A & 1B), but not on speed (p=0.194) (Fig. 1C).These results indicated that while both adult and aged rats showed improvement in spatial learning and memory over time, aged rats swam further and longer in order to reach the target quadrant during the acquisition phase. The lack of effect of speed suggests that the poorer performance of aged rats did not result from lack of motivation or reduced motor ability. Analysis of distance and latency to platform during reversal testing revealed significant effects of age (p<0.001), operation (p<0.001) and age×operation interaction (p=0.047, p=0.054, respectively) (Figs. 2A & 2B), but no main effect of speed (p=0.634) (Fig.2C). Impaired performance on distance and latency parameters was found on postoperative day 1 (p< 0.001) in adult rats, and improved on day 3 (p=0.848, p=0.973, respectively). In aged rats, the same impairments were found on postoperative day 1 (p< 0.001) and continued to day 3 (p=0.002, p=0.001, respectively), only improving on day 7 (p=0.966, p=0.775, respectively) (Figs. 2A & 2B). Anesthesia alone did not significantly impair cognitive function on days 1,3,7 postoperatively (data not shown). These results demonstrate that the surgical procedure impaired spatial learning and memory, and induced a prolonged sickness response in aged rats.Aged rats exhibit elevated pro-inflammatory cytokine expression exacerbated by surgical traumaTo investigate whether anesthesia or surgical trauma alters pro-inflammatory cytokine expression, hippocampal IL-1(3, IL-6 and TNF-a mRNA were measured. Two-way ANOVA of hippocampal IL-1β, IL-6 and TNF-a expression revealed significant effects of age (p< 0.001), operation (p< 0.001) and age×operation interaction (p< 0.001, p< 0.001 and p= 0.003, respectively) (Figs.3A-3C). Significant differences were observed for basal hippocampal IL-1β, IL-6 and TNF-a mRNA levels between adult and aged rats (p=0.041, p=0.012, and p=0.035 respectively). Anesthesia alone did not significantly alter hippocampal pro-inflammatory cytokine levels in adult or aged animals when compared to their age-matched naive controls at any time point (data not shown). IL-1βand IL-6 levels were upregulated by surgical trauma on postoperative day 1 (p<0.001) in adult rats; these cytokines remained upregulated until postoperative day 3 in aged rates (p< 0.001). (Figs.3A & 3B). TNF-a mRNA only increased on day 1 postoperatively in aged subjects (p< 0.001), but did not do so in adult subjects (p= 0.095) (Fig.3C). There was an amplified and prolonged pro-inflammatory cytokine response in aged brains following surgical procedure. These results indicate that the surgical procedure performed resulted in a further amplification of pro-inflammatory cytokine levels in aged hippocampal tissues.Aged rats exhibit upregulated GFAP and S100βexpression in the hippocampus following surgical traumaTo explore why there were age-related differences in cytokine expression following surgery procedure, hippocampal GFAP and S100βwere examined. Analysis of GFAP and S100βrevealed significant effects of age (p<0.001, respectively) and operation (p<0.001, respectively), and agexoperation interaction (p<0.001, respectively) (Figs.4A & 4B). Significant differences were observed for basal hippocampal GFAP and S100βbetween adult and aged rats (p=0.039, p=0.002, respectively). While the GFAP-positive cells of the adult subjects downregulated on day 3 (p=0.09), GFAP expression in aged subjects remained significantly upregulated on day 3 (p<0.001) and improved on day 7 postoperatively (p=0.823) (Fig.4A). A similar pattern change was seen with S100β(Fig. 4B). These results indicate that aging may potentiate hippocampal glial cell activation when challenged by the surgical procedure performed here.CD200 and CD200R mRNA expression following surgical traumaTo investigate potential mechanisms on how surgical trauma sensitizes glial cell activation, CD200 and CD200R mRNA were measured. CD200 expression was downregulated on postoperative day 1 in adult and aged rats (p< 0.001, respectively), and improved on day 3 (p=0.067, p=0.582, respectively) (Fig.5A). Surgical trauma did not significantly alter hippocampal CD200R levels in adult and aged animals at any time point (data not show) (Fig. 5B). These results indicate that trauma-induced sensitization of glial cell activation may be mediated, in part by the downregulation of hippocampal CD200 expression.DiscussionThe findings of this study indicate that moderate surgical trauma induced an exaggerated neuroinflammatory response and exacerbated cognitive function impairments in aged rats when compared to their adult counterparts. Anesthesia alone did not impair cognitive function or alter hippocampal pro-inflammatory cytokine levels in adult and aged rats. Our study revealed an age-related increase in the vulnerability to cognitive impairments triggered by surgical trauma. Postoperative cognitive impairment appears to parallel an increase in central pro-inflammatory cytokines expression.Cytokines have been described as double-edged swords. They protect, repair but also impair neuronal function during excessive or chronic neuroinflammation.It has been postulated that overexpression of central inflammatory cytokines may lead to profound disturbances in sensory-motor coordination and cognition. The hippocampus, a brain region which highly expresses pro-inflammatory cytokine receptors, appears to be more sensitive to excessive or prolonged cytokine exposure.Blockade of pro-inflammatory cytokine receptors in the brain blunts many aspects of the sickness response to peripheral immune challenge. It is known that the central administration of exogenous IL-1βinduces pro-inflammatory immune responses and cognitive deficits which are attenuated by intracerebroventricular administration of IL-1 receptor antagonist. Other studies have indicated that administration of IL-6 neutralizing antibodies prolongs LTP and improves spatial alternation behavior and facilitates recovery from LPS-induced sickness behavior. These studies emphasize the role of pro-inflammatory cytokines in regulating behavioral and cognitive changes.The differential inflammatory responses in the hippocampus following surgical trauma challenge between aged and adult subjects may be explained by age-specific differences in glial cells. Glial cells generally exhibit a quiescent phenotype in the healthy adult brain. In normal aging however, glial cells are primed. In this primed state, the cells exhibit increases in the expression of GFAP, S100βand major histocompatibility complex-Ⅱ. Primed glial cells do not secrete appreciable levels of pro-inflammatory cytokines under basal conditions, but they are hyper-responsive to secondary stimuli and can produce an exaggerated and prolonged neuroinflammatory response when further provoked.Thus, the inflammatory response in aged brain is magnified when challenged with peripheral stimulus such as surgical trauma, resulting in greater cognitive impairments.Peripheral immune challenge is transmitted to the brain via multiple humoral and neural routes. This immune-to-brain signaling results in the de novo production of pro-inflammatory cytokines within the brain, largely by glial cell. Vagal sensory pathways are known to be important in mediating cytokine-induced sickness behavior. In rats, subdiaphragmatic vagotomy attenuates the upregulation of IL-1βin the hippocampus and alleviates behavioral depression induced by peripheral injection of LPS. Moreover, vagal stimulation also increases IL-1βin the brain. These findings explain why activation of the peripheral innate immune system by surgical trauma induces brain glial cell activation to produce pro-inflammatory cytokines responsible for cognitive deficits.To investigate potential mechanisms on how surgical trauma sensitizes glial cell activation, CD200 and CD200R were measured. CD200 expressed on the surface of neurons is thought to constitutively maintain glial cell in a quiescent state through interactions with its receptor CD200R. Recent evidence indicates that CD200 expression is reduced in aged animals, suggesting that reduced neuronal control of glial cell may be an age-related feature which results in glial cell activation. The effects of surgical trauma on glial cell in aged rats suggest that trauma-induced sensitization of glial cell may be mediated, in part, by the attenuation of neuronal control of microglia through downregulation of hippocampal CD200 expression. Therefore, modulating the inhibitory CD200-CD200R interplay to silence glial cell activation may be a promising strategy for improving recovery from sickness and reducing neurobehavioral deficits.In conclusion, the results of this study indicate that pro-inflammatory cytokines may play a significant role in disrupting normal cognitive function in the hippocampus. Surgical trauma resulted in exaggerated and prolonged neuroinflammatory response in the hippocampus and greater cognitive impairments in aged rats when compared to those of their adult counterparts. Inhibition of neuroinflammation triggered by surgical trauma may be a promising strategy for POCD prevention.AcknowledgementsThis research was financed by a grant from the Natural Science Foundation of Liaoning Education Committee (Grant No.2008796).