postoperative cognitive dysfunction ( pocd ) is the deterioration of cognitive function , especially learning and memory , which may last for days , months , or even years [ 13 ] .
pocd occurs after cardiac and noncardiac surgeries and increases first - year morbidity and mortality after surgery [ 46 ] .
there is no doubt that aged individuals are more likely to develop pocd [ 79 ] .
maze 's group demonstrated that learning and memory were impaired after anesthesia and surgery in 3 - 4-month - old mice .
.   demonstrated that there were no signs of neuroinflammation or cognition impairment after surgery in adult mice ( 46-month - old ) .
similarly , wuri et al .   observed no learning or memory changes after partial hepatectomy in adult mice ( 4-month - old ) . according to the work of finlay and darlington , mice after two months
accordingly , 36-month - old mice and 2-month - old mice are biologically equivalent to 3040-year - old human and college freshmen , respectively . while maze 's group investigated the learning and memory after anesthesia and surgery in 3 - 4-month - old mice , no data in younger adult mice , that is , 2-month - old , are available .
unfortunately , this is an age at which particular diseases will arise and may need surgeries , such as appendicitis , osteosarcoma , and leukemia . in the present study , we investigated whether the spatial reference memory of 2-month - old mice will be affected after anesthesia and surgery .
this study was approved by the animal care and use committee of shanghai jiao tong university , school of medicine .
all animal procedures were performed in accordance with the national institutes of health ( nih ) animal care guidelines .
two - month - old male c57bl/6j mice were provided by the animal research center of shanghai jiaotong university , school of medicine .
the animals were housed in standard cages under controlled laboratory conditions ( temperature of 22  2c , 12-hour light/12-hour dark cycle ) with free access to regular rodent pellets and water .
all mice were allowed to adapt to their new environment for 7 days before beginning the experiments .
mice were randomly divided into three groups : nave group , anesthesia group , and surgery group .
splenectomy   was performed with neuroleptic anesthesia ( intraperitoneal injection of 200  g / kg fentanyl and 10  mg / kg droperidol , as reported previously [ 15 , 16 ] ) in the surgery group . for the splenectomy ,
a small incision was made in the left upper abdominal quadrant , and the spleen was mobilized , isolated , and removed .
a single dose of butorphanol ( 0.4  mg / kg , s.c . ) was administered for postoperative analgesia at the end of surgery .
spatial reference memory was evaluated in the mwm using a computerized video tracking system ( days 37 and days 6266 in figure 1 , n = 15 ) .
the reference memory test was performed on four days ( days 36 and days 6265 in figure 1 ) . for the reference memory ,
briefly , a hidden round platform was placed 1  cm below the water surface and located in the center of the northeast quadrant in a circular pool ( 110  cm in diameter and 30  cm in depth ) .
the water was maintained at 2325c , and the pool was situated in a room with visual cues .
in all the trials , each mouse was released into the water facing the pool wall from one of four separate quadrants and allowed to swim until it landed on the platform .
once the mouse found the platform , the trial was terminated , and the mouse was allowed to stay on the platform for 15  s. if the mouse failed to find the platform within 60  s , it was gently guided to the platform and allowed to remain on the platform for 15  s. four trials were conducted per day , separated by a 5-minute intertrial interval , and the platform remained at the same location throughout the test .
the amount of time spent finding and mounting the platform ( escape latency ) and the swimming speed were calculated from the recorded videos using mwm software ( shanghai jiliang software technology co. ltd . , china ) .
the probe test was performed on the day after the reference memory test ( day 7 and day 66 in figure 1 ) . in this test , the platform was absent , and the animals were allowed to swim freely for 60  s , starting from the quadrant opposite the platform .
seven days or 66 days after surgery ( day 8 and day 67 in figure 1 , n = 15 ) , the avoidance learning task was performed as previously described [ 17 , 18 ] in a y - maze equipped with electric grids in the floor .
the grids were controlled by a computer , and a camera on the top of the y - maze recorded and provided the position of the animals to the computer .
the animals had to leave the  start arm  within 5 seconds and escape into the  correct arm  to avoid foot shocks .
active avoidance errors were recorded if the animals did not leave the  start arm  within 5 seconds . if the mouse chose the 
foot shocks were administered for 7 seconds each until the animals chose the  correct arm .
 the foot shock level was changed individually ( maximum : 40  v ) according to the performance of the mouse in the first trial or until the mouse suddenly lifted one or two paws from the grid at the bottom of the y - maze after the shock .
one trial per minute was performed until the mouse reached the final criterion of correctly performing seven out of eight consecutive trials . in order to avoid odor confounding ,
the numbers of total trials , active avoidance errors , and discrimination errors were recorded .
mice in each group were sacrificed 2  h , 6  h , 24  h , and 48  h after surgery .
hippocampus dissections were performed on ice - cold frosted glass , and tissues were quickly frozen in liquid nitrogen .
the mouse hippocampus was homogenized in sterile 0.1  m pbs containing a complete protease inhibitor cocktail ( roche ) .
the homogenates were centrifuged at 10,000  rpm for 15  min at 4c , and the supernatants were analyzed for il-1 and il-6 using elisa kit ( r&d systems , minneapolis , mn ) .
the protein concentrations of all samples were measured using a bca protein assay kit ( pierce ) .
for immunohistochemical analysis of microglia , all mice ( n = 4 ) were deeply anesthetized using equithesin [ 1% pentobarbital/4% ( v / v ) chloral hydrate ; 3.5  ml / kg , i.p . ] and perfused intracardially with saline followed by 4% paraformaldehyde in 0.1  m phosphate buffer ( pb , ph 7.4 ) .
brains were then harvested , postfixed in the same fixative for 4 hours at 4c , and immersed in 1030% gradient sucrose in pb for 2448 hours at 4c for cryoprotection .
brain tissue was freeze - mounted in oct embedding medium , and 16  m thick coronal sections of hippocampus were cut sequentially and mounted on superfrost plus slides .
slices were permeabilized in 0.4% triton x-100 , blocked with 5% bovine serum albumin in 0.1% triton x-100 , and incubated overnight at 4c with mouse anti - cd11b ( abcam , cambridge , uk , 1  :  100 ) . after rinsing in 0.1% triton x-100 in pbs ,
sections were incubated with secondary antibodies conjugated with alexa fluor 488 ( 1  :  500 ; invitrogen ; paisley , uk ) for 1 hour in the dark .
all the procedures for negative controls were processed in the same manner except omitting primary antibody .
the statistical package for the social sciences ( spss ) v.20.0 was used for the statistical analyses .
two - way anova with repeated measures was used to analyze the water maze escape latency and average speed . one - way anova was used for the probe quadrant trial data , avoidance learning task data , and the il-1 and il-6 , followed by post hoc bonferroni correction .
upregulation of these two cytokines was observed at 6 hours after surgery and decreased again by 24 hours postoperatively ( figures 2(a ) and 2(b ) ) .
anesthesia per se did not affect the level of il-1 or il-6 at any time point compared with the nave controls .
cd11b immunostaining showed that very few microglia were activated in the hippocampus from mice of nave or anesthesia groups ( figure 3 ) . on the contrary ,
surgery profoundly induced microglia activation in the hippocampus , which was intensive at 6 and 24 hours after surgery but started to decline by 48 hours ( figure 3 ) .
the results of the morris water maze performed two days and two months after surgery revealed no differences among the three groups in latency , swimming speed , or swimming time in the target quadrant during the probe test ( figures 4 and 5 ) .
similarly , no significant differences were observed in the results of the avoidance learning task in the y - maze , regardless of the number of learning trials , avoidance errors , or discrimination errors or voltage ( figures 4 and 5 ) .
we demonstrated in the present study that ( a ) anesthesia and surgery but not anesthesia only temporally increased hippocampal il-1 and il-6 , as well as microglia activation in the hippocampus in 2-month - old young adult mice and that ( b ) anesthesia and surgery can not hurt the short - term and long - term reference memory of such aged mice .
these findings indicate that central inflammation induced by surgery does not necessarily lead to reference memory impairment in young adult mice .
our present results are consistent with previous studies demonstrating that neuroinflammation can be induced by surgery .
.   demonstrated that the concentration of il-6 in cerebrospinal fluid significantly increased 1 week after cardiac surgery .
[ 11 , 20 , 21 ] demonstrated that serum tnf- induced by surgery disturbed the blood - brain barrier ( bbb ) , which then stimulated macrophage migration into the hippocampus and promoted hippocampal neuroinflammation .
we assume that the increases of il-1 and il-6 in hippocampus after surgery in the present study may arise through the same pathway , but more studies are needed to confirm the assumption in the young adult mice .
other proinflammatory cytokines may also be involved in the development of pocd in aged animals .
ma et al .   demonstrated that tnf- , il-1 , il-4 , and il-6 in the hippocampus increased after surgery and the tnf- receptor antagonist attenuated the elevation of these cytokines .
wang et al .   found that the hippocampal il-1 , tnf- , and ifn- were overexpressed after surgery in aged mice .
.   demonstrated that the hippocampal il-6 , il-12 , and il1 increased after surgery in aged rats .
more studies should be done to investigate whether such proinflammatory cytokines changed after surgery in young adult animals . however , the neuroinflammation detected in the current study is insufficient to cause learning or memory impairment in such young adult mice , as we did not observe any short- or long - term changes in these aspects after surgery .
we postulate that this is because the immune system in young adult mice is very strong so that the anti - inflammatory pathways , such as the vagus nerve pathway , are rapidly activated to reduce the inflammation . in many previous experiments reporting
impaired learning or memory ability after surgery , at least one other contributing factor exists .
for example , fidalgo et al .   observed impaired learning and memory after surgery in adult mice when they were simultaneously infected with lps ( 50  ng / kg , a subclinical dose ) .
the surgery - induced learning and memory impairment demonstrated by he et al . , wan et al . , and cao et al .  
together , results from current and previous findings all indicate that other factors , such as old age or subclinical infection , are required so that the neuroinflammation induced by surgery would be deleterious enough to alter spatial reference memory in mice .
morris water maze and active avoidance test were used in the present study to measure the spatial reference memory changes after anesthesia and surgery .
spatial reference memory is the ability to remember the relevance of spaces , which is a relative long - term memory compared with working memory .
working memory is a limited capacity that is responsible for the transient holding , processing , and manipulation of information , which is a relative short - term memory .
more studies should be done to investigate the changes of working memory after anesthesia and surgery .
age is probably one of the key determinants of the responses to anesthesia and surgery . as most previous studies utilized 46-month - old mice [ 15 , 20 , 21 , 30 ] , the present study used for the first time two - month - old mice , which are biologically equivalent to 18-year - old human .
differences in ages among our study and previous studies are very possibly the reason for the different behavior tests results .
since the spleen is an immunoregulatory organ and splenectomy may deteriorate the immunological system and therefore lead to an exaggerated inflammatory status , we are unable to rule out the possibility that the absence of spleen also contributed to the development of neuroinflammation .
other surgical procedures are needed to further determine the role of surgical trauma in the neuroinflammation and the following learning and memory changes .
anesthesia and surgery lead to neuroinflammation . however , such neuroinflammation is insufficient to impair the spatial reference memory of young adult mice .
 postoperative cognitive dysfunction ( pocd ) increases morbidity and mortality after surgery . 
 but the underlying mechanism is not clear yet . 
 while age is now accepted as the top one risk factor for pocd , results from studies investigating postoperative cognitive functions in adults have been controversial , and data about the very young adult individuals are lacking . 
 the present study investigated the spatial reference memory , il-1 , il-6 , and microglia activation changes in the hippocampus in 2-month - old mice after anesthesia and surgery . 
 we found that hippocampal il-1 and il-6 increased at 6 hours after surgery . 
 microglia were profoundly activated in the hippocampus 6 to 24 hours after surgery . however , no significant behavior changes were found in these mice . 
 these results indicate that although anesthesia and surgery led to neuroinflammation , the latter was insufficient to impair the spatial reference memory of young adult mice . 