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Logo of nihpaAbout Author manuscriptsSubmit a manuscriptHHS Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
 
Anesthesiology. Author manuscript; available in PMC 2012 April 1.
Published in final edited form as:
PMCID: PMC3063324
NIHMSID: NIHMS265910

Hsp72 Overexpression Prevents Early Postoperative Memory Decline after Orthopedic Surgery Under General Anesthesia in Mice

Marcela P. Vizcaychipi, MB.Bs., M.D., E.D.I.C.M., F.R.C.A.,*+ Lijun Xu, M.D.,#^ George Barreto, Ph.D.,#ς Daqing Ma, M.D., Ph.D.,*[reverse epsilon] Mervyn Maze, M.B.Ch.B., F.R.C.A., F.R.C.P.,ψ[nabla] and Rona G. Giffard, Ph.D., M.D.#

Abstract

Background

Problems with learning and memory are common after surgery in the elderly, and are associated with high morbidity. Heat Shock Protein 72 (Hsp72) confers neuroprotection against acute neurological injury. We hypothesized that over expression of Hsp72 would prevent the development of postoperative memory loss.

Methods

C57BL/6 wild type and Hsp72 over-expressing transgenic mice were randomly allocated to: control, isoflurane anesthesia alone, or tibial fracture under isoflurane anesthesia. Animals were trained 24 h prior to surgery using a fear conditioning protocol and assessed in their training environment and in a novel context on post-treatment days 1, 3, and 7. Microglial activation was assessed by immunostaining.

Results

Adult male C57BL/6 wild type mice exhibited reduced memory evidenced by decreased percentfreezing time on days 1 and 3 after anesthesia alone (58.8 +/− 5, 46.5 +/− 5 mean+/−SEM) and following surgery 53.4+/−6, 44.1+/−7 compared to controls 78.8+/−5, 63.4+/−6; P< 0.05 and P< 0.001 respectively). Hsp72 mice showed no difference by treatment on any day. Similarly, non-hippocampal dependent memory was significantly impaired on days 1 and 3 after surgery and day 3 after anesthesia. The genotype effect was significant on day 1 and 7. CD68 immunopositive activated microglia in the hippocampus varied modestly with subregion and time, on day 7 there was a significant treatment effect with no genotype effect with more activated microglia after surgery in all regions.

Conclusion

Hsp72 overexpression is associated with prevention of postoperative hippocampal-dependent and -independent memory deficit induced by anesthesia and/or surgery. Memory deficit is not correlated with numbers of activated hippocampal microglia.

Introduction

Postoperative cognitive dysfunction (POCD) is characterized by long-term cognitive including memory impairment persisting at least three months after a major surgical intervention.12 When POCD manifests soon after surgery it may be referred to as early POCD.34 POCD is defined as a change in performance on neuropsychological tests after surgery compared to before, while postoperative delirium that manifests in the early postoperative period is diagnosed on the basis of the Diagnostic and Statistical Manual of Mental Disorders fourth edition* criteria, which include fluctuating mental status, disturbance in attention and may include memory deficits.5 The relationship between these two is not clear. Early POCD may be linked to anesthesia,68 fluid and analgesic regimen.3,8 Early POCD is seen more frequently after general anesthesia and it is associated with higher mortality.6,910 Steinmetz et al showed that patients with early POCD are at higher risk of leaving the labor market prematurely because of disability or voluntary early retirement.1 This condition has been noted in a diverse group of patients; particularly those undergoing cardiac and orthopedic procedures,1011 though others have suggested that the burden of POCD has been overestimated due to statistical and methodological difficulties.12

Since its initial description, the worsened outcomes associated with POCD have stimulated a broad range of investigations to ascertain the prevalence, long-term consequences and appropriate diagnostic criteria.11,13 There is some evidence suggesting that the level of stress response, which depends on the type of insult and duration, may contribute to the development of POCD.1415 Despite the identification of risk factors for POCD 1618 the molecular mechanisms responsible for its development largely remain elusive although central inflammatory response, specifically increased cytokines in the hippocampus, following surgery has been reported in a rat model of POCD19 and increased hippocampal interleukin (IL)-1β was suggested to play a role in memory deficit following infection.2021

At the level of individual cells, heat shock proteins are induced by many forms of stress, and their expression is associated with resistance to a subsequent stress. Heat shock protein 72 (Hsp72) is a highly stress inducible cytosolic heat shock protein that performs many cell protective functions including acting as a protein chaperone, regulating inflammation, and regulating cell death and survival pathways.22 Reduced induction of heat shock proteins has been reported in aged rodents23 and under some circumstances induced levels of Hsp72 may be insufficient to prevent the injurious effect of stress.2325 We have previously shown in animal and cell models of stroke that injury is reduced in animals or brain cells over-expressing Hsp7226 and that this is associated with reduced activation of the proinflammatory transcription Nuclear Factor κlight chain-enhancer of activated B cells (NFkB).27 In addition to its roles in stress, survival and inflammation, Hsp72 has also been implicated in learning and plasticity, with upregulation of Heat Shock Cognate 70 reported in a learning paradigm28 and changes in Hsp72 noted in the hippocampus of animals trained in a radial maze task.29

Only a few animal models of POCD have been developed to date.19 To better study cellular and molecular mechanisms responsible for POCD we developed a model using a surgery akin to arthroplasty, the commonest major elective surgical procedure in the cohort of patients of an age in which POCD is common, and used it to test the hypothesis that Hsp72 overexpression can prevent or reduce POCD after orthopedic surgery under general anesthesia.

Material and Methods

Animals

Experiments were performed in accordance with a protocol approved by the Stanford Animal Care and Use Committee, Stanford University, Stanford, California, and following the National Institutes of Health guidelines. All animals were supplied with food and water ad libitum. Temperature, humidity and night-day cycle were maintained according to the standards set up by the research animal services at Stanford University. Adult male mice expressing a chimerictransgene of the rat-inducible Hsp72 gene (Hsp72-Tg) under control of the chicken-actin promoter and human cytomegalovirus enhancer were originally produced by Dillmann and coworkers 30 and back bred into the C57BL/6 background and maintained as heterozygous transgenics by always crossing with C57BL/6 wild type (C57BL/6-WT) females, so littermates could be used for experiments after genotyping by polymerase chain reaction analysis of tail DNA. Brain levels of Hsp72 were previously shown to be elevated about 10 fold in this strain.27 C57BL/6-WT mice were purchased from Charles River Laboratories (Wilmington, MA).

Adult male mice were randomly allocated to one of three treatment groups: control (naïve animals without treatment; (C), anesthesia alone (A) and tibial fracture surgery (S). Within each treatment group separate groups were used for each assessment timepoint, days 1, 3, and 7. A total of 4 mice died in the postoperative period, all were from surgery groups, 2 of each genotype, and were excluded from analysis. Animal ages were 3 to 14 months with a median of 6 (range: 3 – 14) for C57BL/6-WT and 7 (3 – 14) for Hsp72-Tg group. Animals weighed from 21 to 51 g with a median of 37.2 (range: 23 – 51) for C57BL/6-WT and 32.0 (21 – 49) for Hsp72-Tg group.

Surgical model

Animals from both genotypes either had anesthesia alone or anesthesia plus tibial fracture with pinning. General anesthesia, which lasted for 20 min, consisted of induction with 5 % isoflurane in 30 % oxygen in air at a flow rate of one L/min (vaporizer from Ohio, Model 100F, Louisville, KY); anesthesia was maintained with 2.0 ± 0.4 % isoflurane, approximately 1.4 Minimal Alveolar Concentration for mice. The concentrations of isoflurane and oxygen were continuously monitored (VetEquip. Inc., Pleasanton, CA). Temperature was monitored throughout the surgical procedure using a rectal probe. Five minutes after induction of anesthesia, supplemental analgesia was provided by buprenorphine, 0.3 mg/kg in saline, intraperitoneally in < 1 ml given over 5 min. Although morphine is known to have effects on the immune response buprenorphine does not alter the immune response at different concentrations up to 7 days after nociceptive stimuli,31 therefore it was chosen for this experiment. Eutectic mixture of local anesthetic was applied to the surgical site on completion of the surgical procedure, followed by daily application until day 3 postoperatively.

The surgical model was based on Harry et al.32 In brief, after skin incision below the knee, soft tissue was reflected to expose bone through which a 0.3 mm stainless steel rod was inserted into the medullary cavity. Once the tibia was internally fixated, the bone was fractured in the mid-diaphysis (tibial, mid-shaft) using surgical pliers. The wound was closed using 2–0 Nylon suture (Ethicon Inc., Somerville, NJ). Post-intervention animals were left to recover on heated pads, and then transferred to their own cages, where food and water were available.

Behavioral tests

Fear Conditioning (FC)

Room temperature was kept between 20 – 22°C and humidity between 35 – 55 % during behavioral assessment. One day prior to surgery or anesthesia animals were trained for FC in order to learn the task and establish long-term memory. Training and testing were performed in chambers with a video camera positioned at the top of the chamber to allow the subjects’ behavior to be observed and recorded by an experimenter both on- and off-line. The floor consisted of 32 stainless steel rods (1 mm diameter) spaced 0.5 cm apart (center-to-center); wired to a shock generator and scrambler to deliver a 0.70 mA foot shock.

The chambers were cleaned with a 5% sodium hydroxide solution scented with 0.2% mint extract, and pans containing a thin film of the same solution were placed underneath the grid floors. Background noise (60 dB) was provided by a fan positioned underneath the video camera.

The FC paradigm consists of a training phase prior to surgery and an evaluation phase after surgery when memory is assessed (fig. 1). A contextual interval of 25 s (Trace), that is, the time between conditional and unconditional stimuli for assessment of hippocampal dependent memory was chosen as it was previously shown that less than 18 s is not enough for establishment of hippocampal memory and above 30 s other regions of the brain may be involved.33 If the conditional stimulus (Tone) is too weak (less than 50 dB), it does not increase connectivity within the learning centers and if too strong, more than 75 dB, it acts as an unconditional stimulus; therefore, we chose a 70 dB tone. For the unconditional stimulus (Shock) used to generate fear, too weak a stimulus may not trigger fear while too strong may activate pain pathways creating a confounding variable; therefore we chose a 0.70 mA shock which triggers maximum freezing behavior.34

Fig. 1
Schematic representation of the fear conditioning protocol. (A) Fear conditioning training. (B) Contextual and conditional assessment after surgery and or anesthesia. Time is not to scale.

Training was carried out 24 h prior to surgery. Mice were allowed to familiarize themselves with the surroundings (Context) for 120 s followed by a 20 s 70 dB tone (Conditional stimulus) and then a delay of 25 sec. This contextual interval was terminated by an unconditional stimulus, a 0.70 mA for 2 ms electrical foot-shock. After six pairs of conditional-unconditional stimuli the mice learned the association and established long-term memory. The pairs of conditional-unconditional stimuli were separated by random intervals from 45 to 60 s, the inter-training interval. The inter-training interval allowed the mice to disengage from the process of association before a new set of stimuli was introduced. After fear was established the mice would freeze and the percentage of time spent not moving was determined (% Freezing time). Freezing time measured during exposure to the known context or after a conditional stimulus in the known context reflects hippocampal dependent memory while assessment during delivery of the conditional stimulus (Tone) assesses hippocampal independent memory.

Following surgery or anesthesia we assessed hippocampal-dependent and -independent memory in the known context and in a novel context/environment 1, 3, and 7 days after surgery, exposure to anesthesia or neither. Analysis was performed with Freeze-Frame / Freeze-View modes. Threshold for freezing was chosen according to the individual animals motion index histogram. An epoch of 0.75 s was used to count an episode of freezing. Percentage of total time spent freezing was used as an indicator of memory formation during training and retrieval of information after treatment.

Pain threshold

Separate groups of C57BL/6-WT and Hsp72-Tgmice that were subjected to neither anesthesia nor surgery were assessed for sensory and motor activity to detect possible behavioral differences between C57BL/6-WT and Hsp72-Tg mice. Pain threshold was assessed using a hot plate at 55°C Integrated Information Technology Center, Life Science Inc., Woodland Hills, CA) to test acute thermal nociception. The latency to the nociceptive response was the time until the mouse started licking the hind paw or jumping. A cutoff time of 30 s was applied to protect animals from burn injury or distress.

Acoustic startle reflex

Acoustic function was tested in a mouse startle reflex chamber made of plexiglass vented with holes that was attached to a spring-loaded platform hinged to a support stand. A transducer assessed the displacement force of the animal chamber. The entire apparatus was housed in an acoustically insulated wooden box containing speakers (Med Associates Inc., St Albans, VT). The startle stimulus consisted of applying 110 dB of white noise lasting 10 s. Latency to startle response was used as a measure of sensorineural hearing (Acoustic reflex). The Stanford Neuroscience Behavior Phenotyping and Pharmacology Behavior Core directed by Dr. MehrdadShamloo, Ph.D., provided all behavioral equipment and training except the use of the Smart-Home Cage.

Assessment of spontaneous activity

Spontaneous locomotion was assessed for a period of 60 min, 72 h prior to surgery using the Smart-Home Cage System (Afasci, Inc., Redwood City, CA; loaned by Dr. Xinmin (Simon) Xie, M.D., Ph.D.) to compare C57BL/6-WT and Hsp72-Tg mice. Active count and travel distance were used as measures of motor activity detected as breaks of infrared sensors crossing the cage.

Immunohistochemistry and morphometric analysis

Animals were sacrificed 2 h following behavioral assessment on day 1 and 7. All animals received terminal anesthesia (isoflurane 5%), followed by transcardial bleeding and perfusion with 20 ml of cold saline followed by 4% paraformaldehyde. Brains were removed, postfixed, then sections 50-μm thick, were obtained using a Vibratome (VT 1000 S, Leica Microsystems, Wetzlar, Germany). Immunohistochemistry was performed on free-floating sections under moderate shaking. All washes and incubations were done in 0.1M phosphate buffer pH 7.4 containing 0.3% triton X-100. Sections were incubated for 1 h in blocking solution (0.1M phosphate buffer, 0.3% Triton X-100 and 5% equine serum). After three washes in phosphate buffer, sections were incubated overnight at 4ºC with a rat anti-mouse antibody for CD68 diluted 1:200 (MCA1957GA, Serotec, Raleigh, NC). Sections were then rinsed in phosphate buffer and incubated for 2 h at room temperature with an Alexa 488-conjugated goat anti-rat (diluted 1:200, Invitrogen, Carlsbad, CA) secondary antibody, washed and mounted on glass slides using Vectashield mounting medium with 4′, 6-diamidino-2-phenylindole (DAPI; Vector Laboratories, Burlingame, CA). Immunostaining was absent when the primary antibody was omitted.

For each animal, the number of CD68 immunoreactive cells in four subregions of the hippocampus, CA1, CA2, CA3 and hilus of dentate gyrus (−1.70 to −2.18 mm relative to Bregma) were estimated from photomicrographs with a counting frame size of 0.4 mm2. Sections immunostained for CD68 were photographed at 20x magnification using a digital camera attached to a ZeissAxiovert 200M inverted epifluorescence microscope (Carl ZeissMicroimaging, Inc., Thornwood, NY). The total numbers of CD68 immunoreactive cells were counted in 3 counting frames per region (total 12 frames per animal) using Image J software.§ The numbers of cells in the 3 frames per regions were then averaged. The age range of animals studied was 6–12 months (C57BL/6-WT: 7.5 months ± 2.3 and Hsp72-Tg: 8.2 months ± 2.6 mean ± SD; P= 0.6), not different between genotypes.

Statistical analysis

The observer was unaware of the treatment received by mice at the time of assessment of behavior and assessment of cell counting for immunohistochemistry. Results are expressed as mean ± SEM. To study the treatment effect on memory and microglial activation repeated two-way ANOVA and a post hocTukey’s multiple comparison test for genotype and condition was performed using a statistical package (GraphPad Prism 5, GraphPad, San Diego, CA, or SAS 9.1 SAS, Cary, NC). A P value of < 0.05 was considered statistically significant.

Results

Development of the FC paradigm

We developed a FC paradigm in which mice learned conditional reflexes to establish both hippocampal-dependent and hippocampal-independent long-term memory, for use to assess retrograde memory after surgery. In developing our paradigm we standardized the time of day for training, the number of conditional stimuli, and the intensity of stimuli for establishment of long-term memory. We investigated the time of exposure to a familiar environment called context or contextual period prior to a subsequent conditional stimulus.

There was no difference in acquisition between training during light and dark times of the cycle; however, animals trained during daytime freeze more than mice trained at night during the assessment phase indicating that recollection was better during daytime. Therefore, animals were trained and assessed during the light cycle, consistent with prior results.35

We trained a cohort of C57BL/6-WT mice with a short (2-pair of conditional-unconditional stimuli), medium length paradigm (6-pair of conditional-unconditional stimuli) and a long paradigm (8-pair of conditional-unconditional stimuli) to ascertain the numbers of conditional-unconditional pairs of stimuli required to develop and consolidate new information (acquisition). We found that 2 or 8 pairs were less effective for establishment of long-term memory than 6 (data not shown). Therefore, 6 pairs were used for the subsequent study. The pairs of conditional-unconditional stimuli were separated by random intervals between 45 and 60 s each to prevent associative conditioning in between trials.

Finally, we assessed the time required to establish long-term memory. We identified 24 h as the minimum interval to establish long-term memory. This was assessed in a different group of C57BL/6-WT mice prior to this experiment. Animals were trained with a set of conditional stimuli and then assessed for memory formation in their training context and also in a novel context at different time points. Once we identified that memory was established at 24 h post-training we decided to perform surgery 24 h post-training (data not shown).

Behavioral Phenotype of naïve mice

There were no differences at baseline between C57BL/6-WT and Hsp72-Tg mice for acoustic reflex, pain threshold or motor spontaneous activity (table 1). Acquisition of memory during training prior to surgery was similar between C57BL/6-WT and Hsp72-Tg. Asymptotic performance was achieved by the end of the 6th training session (fig. 2). There were no differences prior to surgery in acquisition during trace intervals, conditional, unconditional response, or inter-training intervals during training between the strains (data not shown).

Fig. 2
Acquisition of memory during the training phase of the fear conditioning protocol is similar in C57BL/6-WT (n = 55) and Hsp72-Tg mice (n = 45). Both mouse strains achieved maximal performance by the 6th training session. No differences in establishment ...
Table 1
Baseline Behavioral Variables

Physiological variables

No statistical differences were detected among monitored physiological variables between groups. Animal ages were range 3 to 14 m, median of 6 for C57BL/6-WT and 7 m (3 – 14) for Hsp72-Tg group. Animals weighed from 23 to 51 g, median 37.2 g for C57BL/6-WT and 32.0 g (21 – 49 g) for Hsp72-Tg group. Rectal temperature during surgery and anesthesia were 35.9°C ± 1.0°C in C57BL/6-WT mice and 36.1°C ± 0.7°C in Hsp72-Tg. Respiratory rate was between 20 and 30 breaths/min in both groups during maintenance of anesthesia with 1.4 – 1.6 % isoflurane. Major bleeding was observed in three animals (1 C57BL/6-WT, 2 Hsp72-Tg), which were removed from the study and sacrificed immediately.

Effect of Surgery on Behavior

Contextual conditional response is a hippocampal dependent memory task. Treatment effect: C57BL/6-WT mice showed reduced memory based on treatment on day 1 (n=14 for C, A; n=12 for S) and 3 (n=11 all groups) after anesthesia alone; mean ± SEM (95% CI): 58.8 ± 5 (48 to 69.6) and 46.5 ± 4.8 (35.7 to 57.2) respectively and following surgery also on days 1 and 3; 53.4 ± 5.8 (40.6 to 66.2) and 44.1 ± 6.9 (28.7 to 59.5) respectively (P< 0.05 and P< 0.001 respectively) when compared to controls 78.78 ± 4.6 (68.6 to 88.8) and 63.4 ± 6.2 (49.5 to 77.4) respectively, but not on day 7 (n= 11 all groups) (fig. 3). Hsp72-Tg mice showed no difference by treatment on any of the days (fig. 3). On day 3 the genotype had a strong effect on the contextual conditional response (P < 0.0001). While on day 7, no differences between groups were observed (fig. 3C). A sub-analysis to identify apossible age-related difference on memory on day 1 and 3 after surgery and/or anesthesia was performed and no age-related statistical differences were found (fig. 4). It should be noted however, that the study was not designed or powered to assess the effect of age.

Fig. 3
Hippocampal-dependent memory is impaired following anesthesia or surgery. Contextual conditional response (CCR), a form of hippocampal dependent memory, was assessed in separate groups of mice as % freezing time on days 1 (n = 14/group), 3 (n = 11) and ...
Fig. 4
Contextual conditional response (CCR) was reanalyzed as percent freezing time after separating mice by age into two groups (young adult: 3 – 6 m and adult: 6.1 – 14 m) on days 1 and 3 after surgery. Groups are Control (C), Anesthesia (A) ...

Hippocampal-independent memory

Freezing behavior during the conditional stimulus assesses hippocampal independent memory, e.g., prefrontal cortex and amygdala. We assessed the dependence of the non-conditional response (Tone) on either treatment or genotype (fig. 5). Treatment effect: We found that the response of Hsp72-Tg mice was not affected by treatment on any day while C57BL/6-WT mice showed reduced memory following surgery on day1 (n= 12) and 3 (n= 11); mean ± SEM (95% CI): 31.9 ± 5.2 (20.3 to 43.5) and 36.6 ± 6.0 (23.1 to 50.0) respectively and day 3 after anesthesia 37.3 ± 3.5 (29.5 to 45.1) (P < 0.05) when compared to the control group n= 14; mean ± SEM (95% CI): 49.2 ± 5.04 (38.3 to 60.1) or anesthesia alone group n= 14; mean ± SEM (95% CI): 46.1 ± 3.5 (38.5 to 53.8) on day 1, while those exposed to anesthesia alone differed from control only on day 3. Genotype effect: We found that on day 1, the nonconditional response was strongly dependent on genotype after surgery (P < 0.001). On day 3 the genotype effect was significant after anesthesia alone and surgery (P < 0.05). Such dependence was still significant on day 7 for the surgery group by two-way ANOVA. (P < 0.05) while the anesthesia alone C57BL/6-WT mice did not differ from control (fig. 5). No age-related difference on day 1, 3 and 7 after surgery and anesthesia were found (data not shown).

Fig. 5
Hippocampal-independent memory is impaired after anesthesia or surgery. Conditional response (CR), a form of hippocampal independent memory was assessed as percent freezing time following the tone on days 1 (n = 14), 3 (n = 11) and 7 (n = 11) after treatment. ...

Microglial activation

Activated microglia assessed by CD68 immunostaining were more prominent in animals that underwent surgery (fig. 6) and were observed throughout the brain, but there were also some region specific differences between treatment groups. We analyzed numbers of CD68 positive cells in the CA1, CA2, CA3 and hilus of dentate gyrus, subsections of the hippocampal formation on days 1 and 7 after treatment. Two-way ANOVA for genotype, treatment, and their interaction was performed and the genotype effect did not reach significance after Benjamini Hochberg correction for multiple testing in any subsections (raw P values are 0.0638, 0.0405, 0.0694 and 0.0277 in CA1, CA2, CA3 and hilus of dentate gyrus, respectively and adjusted P values are all 0.0694). In contrast, on day 7 there was a significant treatment effect with no genotype effect, with more activated microglia after surgery in all hippocampal regions and both genotypes(tables 2, ,33).

Fig. 6
Microglial activation was assessed by immunoreactivity for CD68 and cell counting. The subregions within the hippocampus assessed in this study are indicated by squares on the brain section at the top. Representative photomicrographs of the CA1 and CA3 ...
Table 2
Number of CD68 Immunopositive Cells in CA1 and CA3 on Day 1 and Day 7
Table 3
Number of CD68 Immunopositive Cells in CA2 and Hilus of Dentate Gyrus on Day 1 and Day 7

Discussion

This study establishes a new paradigm for postoperative cognitive dysfunction following anesthesia and tibial fracture surgery, in which FC training is performed prior to anesthesia and surgery, and memory deficits in C57BL/6-WT mice are detectable through 7 days postexposure. This model is sensitive to changes induced by exposure to anesthesia alone, as well as changes due to the combined effects of anesthesia and surgery. Mice overexpressing Hsp72 showed no cognitive impairment following the same treatments. This is consistent with a prior study showing heat shock pretreatment was associated with improved Morris water maze performance after traumatic brain injury. 36

While both hippocampal dependent and independent memory was impaired by both anesthesia and surgery, the effect of anesthesia alone was most notable on day 3. By day 7 no treatment showed a difference in hippocampal dependent memory, while the WT surgical group still showed significant hippocampal independent memory deficit at 7 days. While we cannot rule out an effect of anesthetic on blood pressure or blood gases, the anesthetic was fairly brief, and held constant for all groups, so any hemodynamic effect was unlikely to be different between genotypes. In addition, neither reduced blood pressure, oxygenation, or depth of anesthesia were associated with POCD.2,37 Further studies are warranted to examine the effect of depth of anesthesia on memory retrieval as the current literature is unclear on this issue.

Isoflurane has been reported to suppress learning in a dose-dependent fashion.38 Furthermore, it has been demonstrated that isoflurane interferes with anterograde but not retrograde memory for Pavlovian fear conditioning.3940 In that study, FC training took place during delivery of anesthesia while in this study we trained animals prior to surgery or anesthesia. This allowed us to remove the influence of the acquisition phase on assessment of memory postoperatively. While previous studies have demonstrated that ketamine, xylazine and acepromazine anesthesia interfere with retrograde memory after FC training,41 to the best of our knowledge this is the first report showing impaired recall of FC after isoflurane-induced anesthesia. Furthermore, this phenomenon is not observed in mice overexpressing the chaperone Hsp72. However a dose response curve and exposure to different anesthetic agents are warranted to identify the effect of concentration and type of anesthesia on induction of Hsp72 in the future.

Work from several laboratories including ours suggests that an inflammatory component could contribute to impaired memory and that inflammatory changes particularly, increased levels of IL-1β in the hippocampus may be implicated in the setting of infection18,39 or as we previously reported, following splenectomy surgery,19 or traumatic brain injury.42 It is likely that properly regulated levels of IL-1β are required for memory as the lack of hippocampal IL-1β interferes with cognition and IL-1β is necessary for memory formation. Due to this prior work suggesting a link between inflammation in the hippocampus and memory impairment we assessed the number of activated microglia in subregions of the hippocampus, however we did not observe a significant correlation. This suggests either that the memory deficit is not correlated with numbers of activated microglia, or that the relationship is complex, possibly different early and later after injury.

When compared to prior reports linking inflammation and impaired memory there are several likely factors that may explain why we did not observe a correlation with numbers of activated microglia. Our previous work19 and that of Maier’s group2021 was performed in rats. There are known differences in immune response between rats and mice. Our previous paper used a model of splenectomy, which directly affects the immune system, and a learning paradigm that was employed only after treatment, not as here a memory that was established prior to treatment and assessed after treatment. Microglial activation was not assessed in that report, only evidence of astrocyte activation. Thus there are many differences between the two studies. Still, it is surprising that we did not find a stronger correlation between numbers of microglia and memory impairment. This could reflect the observation that microglia can play a positive role in plasticity, as well as contribute to memory impairment in some settings. Conceivably the early activation is deleterious, but the late activation may not be, and in contrast may contribute to plasticity and the reestablishment of connectivity. Further studies will be needed to test this hypothesis. In the current study it is also possible that differences in peripheral inflammatory response could influence the response to injury and this could be affected by Hsp72 overexpression. Further studies would be needed to evaluate this possibility.

Hsp72 was previously shown to be lower in DBA mice that had learned a radial maze, but unchanged in C57Bl6 mice after learning the maze,29 while in contrast Heat Shock Cognate 70 was increased in rats learning the Morris water maze.28 The exact relationship between plasticity and expression levels of Hsp72 is thus not yet clear. We found no difference in acquisition of FC between C57BL/6-WT and Hsp72-Tg mice that chronically overexpressed Hsp72 in all cell types, consistent with another report.43 The differences observed after anesthesia/surgery may reflect differences in Hsp72 levels preceding exposure to anesthesia and/or surgery as well as responses to these exposures that varied with genotype and age. Whether the different genotypes had different changes in Hsp72 expression patterns following anesthesia and surgery is unknown. However, the pathways underlying memory acquisition, transition from short-term to long-term memory, and memory retrieval are not fully understood, so it was not possible to evaluate possible differences in Hsp72 levels in the relevant pathways or how they may have changed following exposure to anesthesia and surgery. New investigations to explore the effect of age on memory retrieval, and to measure Hsp72 in different brain regions, may shed light on these factors. We are also not able to determine at this point whether effects of Hsp72 directly on memory processes is most important in the differences observed, or to what extent the differences reflect a downstream effect via modulation of inflammation or other mediators that also affect memory.

Retrieval of information was fully protected in Hsp72-Tg mice up to day 7, though our sensitivity is reduced at this point as all mice show reduction in freezing time with time following training. These findings suggest that Hsp72 is important either to memory retrieval or protection from a process that degrades memory retrieval following anesthesia or surgery. Hsp72 is known to inhibit inducible nitric oxide synthase gene transcription via the inhibition of activation of its transcription factor NFkB.27,44 Hsp72 also stabilizes mitochondrial membrane potential and ameliorates reactive oxygen species production.45 Whether the observed protection of memory involves reduced oxidative stress such as from inhibition of induction of nitric oxide synthase, or inhibition of cytokine production, or both remains to be evaluated.

In summary, Hsp72 overexpression is associated with full maintenance of memory following isoflurane anesthesia and/or surgery. However, whether the mechanism of this improved cognitive performance compared to WT reflects direct effects on processes in the pathways serving memory establishment and retrieval and/or modulation of inflammation which in turn affects memory still remains to be elucidated.

Acknowledgments

Sources of financial support for the work; This work was supported in part by a National Institutes of Health grant, Bethesda, Maryland, R01 GM 49831 to Rona G. Giffard, and the Intensive Care Society Traveling Fellowship Award, London, United Kingdom to Marcela P. Vizcaychipi

Individuals or organizations whose assistance is acknowledged:The authors would like to thank Ming Zheng, Ph.D., Research Associate at Stanford University, Stanford, California, for help with statistical analysis, MehrdadShamloo, Ph.D., Director, Behavioral and Functional Neuroscience Laboratory, Stanford, California, and the Neuroscience Behavior Phenotyping and Pharmacology Core, Palo Alto, California, for help with behavioral assessment and Xinmin (Simon) Xie, M.D., Ph.D., Consulting Associate Professor, Department of Medicine Stanford University and Chief Scientific Officer, AfaSci, Inc., Burlingame, California for the loan of the Smart-Home cage.

Footnotes

Department/institution to which the work is attributed: Department of Anesthesia Stanford University, Stanford, California

Dr. Maze is a consultant for Air Products (Allentown, Pennsylvania), a company that is interested in developing clinical applications for xenon.

Part of this work was presented as a poster at the Intensive Care Society State of The Art Meeting, December 14, 2009, London, United Kingdom

*Available at: http://www.dsmivtr.org. Last accessed April 9, 2010.

§Available at: http://rsb.info.nih.gov/ij/. Last accessed April 15, 2010.

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