Prolonged mechanical ventilation–induced neuroinflammation affects postoperative memory dysfunction in surgical mice

The experiments were performed in accordance with a protocol approved by the animal use and care committee of Wuhan University, Hubei, China, and in accordance with the National Institutes of Health guidelines. This study was approved by the animal ethics committee at the Zhongnan Hospital and Research Centre, Hubei, China.

Normal male C57BL/6 wild-type mice weighing between 20 and 25 g, 6 to 8 weeks of age, were purchased from medical the experimental animal center of Hubei province. The animals were housed in individual cages in a temperature-, humidity- and light-controlled room (12-hour light-dark cycle) and were acclimated to these conditions for at least 7 days prior to use in experiments. Under aseptic conditions, mice were subjected to an open tibial fracture of the left hind paw with an intramedullary fixation [10]. Briefly, mice received general anesthesia with 2% isoflurane, and analgesia was achieved with buprenorphine 0.1 mg/kg administered subcutaneously, immediately after anesthetic induction and before surgical insult. A midline incision was performed on the left hind paw, and a 0.38-mm pin was inserted into the intramedullary canal, the periosteum was stripped and an osteotomy was performed. Temperature-controlled, light-emitting diode shower lights were used to maintain body temperature at 37 ± 0.5°C. The entire procedure, from induction of anesthesia to the end of surgery, lasted 12 ± 5 minutes, and then the mice were suspended at a 45° angle, with a light source in the neck area and a homemade metal laryngoscope adjusted to provide the best visualization of the vocal cords. A 22-gauge venous catheter with a needle core was inserted 3 mm into the trachea of the mice [11]. Then, with the needle core pulled out, the catheter was connected to the ventilator (TOPO; Kent Scientific, Torrington, CT, USA). The respiratory rate was set at 100 breaths/min, and the pressure control mode was set with a peak inspiratory pressure of 12 to 15 cmH₂O. The fraction of inspiration oxygen (FiO₂) was kept at 0.5. Arterial oxygen saturation was measured noninvasively using a MouseOx pulse oximetry system (Starr Life Sciences, Oakmont, PA, USA) during anesthesia. After surgery, the mice randomized to the MV group received 1.2% isoflurane/oxygen (FiO₂ =0.5) for 1 hour, 3 hours or 6 hours, and the S group was placed in an anesthetizing chamber flushed with 1.2% isoflurane/oxygen (FiO₂ =0.5) for 1 hour, 3 hours or 6 hours and did not undergo tracheal intubation. Anesthetic and oxygen concentrations were measured continuously (GE Healthcare, Wauwatosa, WI, USA), and the temperature of the anesthetizing chamber was controlled to maintain rat body temperature at 37 ± 0.5°C [12,13]. After MV, the anesthetics were discontinued, and all animals were allowed to recover for 20 minutes in a box flushed with 100% oxygen and then placed in their home cages.

The animals were tagged and randomly allocated to each group before any treatment or procedure. Researchers were blinded to the group assignment, which was revealed only after the analysis phase.

Freezing behavior is an indicator of aversive memory that is measured when subject mice are reexposed to the conditional stimulus. For this study, we used a previously published paradigm [14]. The FC paradigm consists of a training phase prior to surgery and an evaluation phase after surgery or MV when memory is assessed. Briefly, 1 day prior to surgery or MV, control (n =12), surgery (n =36) and MV (n =36) animals were trained for FC to learn the task and establish long-term memory. Mice were allowed to familiarize themselves with the surroundings (context) for 120 seconds, followed by a 20-second, 70-dB tone (conditional stimulus) and then a delay of 25 seconds. This contextual interval was terminated by an unconditional stimulus, a 0.70-mA electrical foot shock for 2 milliseconds. The pairs of conditional–unconditional stimuli were separated by random intervals from 45 to 60 seconds, which was the intertraining interval. The intertraining interval allowed the mice to disengage from the process of association before a new set of stimuli was introduced. After six pairs of conditional–unconditional stimuli, the mice learned the association and established long-term memory. After surgery or MV, mice were placed back in the original conditioning chamber, where no tone or shock was presented, to assess recall of context and/or environment 6 hours, 1 day and 3 days after surgery, exposure to MV or neither (Figure 1). Our measure of associative learning was the percentage of time spent not moving (percentage freezing time). Behavior was captured with an infrared video camera (Sony Corporation, Tokyo, Japan).The observer was unaware of the treatment received by mice at the time of the behavioral assessment.

Blood was collected into heparin-coated syringes at 6 hours, 1 day and 6 days post-MV after thoracotomy under terminal isoflurane anesthesia. Samples were centrifuged at 3,400 rotations per minute for 10 minutes, and plasma was collected and stored at −80°C until assayed. Hippocampal tissues were homogenized on ice in 20 mM Tris-HCl buffer (pH 7.3) containing protease inhibitors. Homogenates were centrifuged at 10,000 × g for 10 minutes at 4°C. The supernatant was then ultracentrifuged at 150,000 × g for 2 hours. Plasma and hippocampal tissue IL-1β, IL-6 and TNFα were measured using commercially available enzyme-linked immunosorbent assay kits according to the manufacturer’s instructions (Santa Cruz Biotechnology, Santa Cruz, CA, USA). All samples were assayed in duplicates. The readings were normalized to the amount of standard protein.

At 6 hours, 1 day and 3 days after surgery (n =12) and MV (n =12), the animals were anesthetized with isoflurane. The thoracic cavities were opened and perfused intracardially with 40 ml of cold saline, followed by 4% paraformaldehyde. Then, the brain was rapidly taken out, postfixed in 4% paraformaldehyde at 4°C, embedded in optimal cutting temperature tissue-freezing medium and sectioned for immunofluorescence. Tissue sections were blocked in 5% bovine serum albumin. After three washes in phosphate buffer, the sections were incubated with a mouse monoclonal anti-CD11b antibody (1:200; Abcam, Cambridge, UK) at 4°C overnight. After several washes, the sections were incubated with a goat anti-mouse immunoglobulin G secondary antibody (1:100; Jackson ImmunoResearch Laboratories, West Grove, PA, USA) for 1 hour in the dark. After rinsing with phosphate-buffered saline, the sections were mounted on slides with Hoechst 33342 dye for 5 minutes. After washes, the sections were subsequently observed, and images were acquired using an imaging system equipped with a fluorescence microscope (Olympus, Tokyo, Japan). For each animal, the number of CD11b-positive cells in three hippocampal subregions—CA1, CA2 and CA3—were estimated from photomicrographs with a counting frame size of 0.4 mm². The number of CD11b-positive cells per square millimeter were counted in three counting frames per region (total of nine frames per animal) by using Image J software (National Institutes of Health, Bethesda, MD, USA), and the numbers of cells in the three frames per region were then averaged.

The animals were anesthetized with isoflurane. The thoracic cavities were opened and perfused intracardially with ice-cold saline, followed by perfusion with 4% paraformaldehyde fixative for 10 minutes. Coronal sections were cut 150 μm thick on a vibrotome, and the area of the CA1 pyramidal layer and stratum radiatum was dissected out. Briefly, microdissected areas were washed in 0.1 M/L sodium phosphate buffer and postfixed at room temperature for 1 hour in 1% osmium tetroxide. Samples were then rinsed in ultrapure water, and tissue blocks were dehydrated at room temperature through graded ethanols from 30% to 100% for 10 minutes each, including 1% uranyl acetate in 70% ethanol for 40 minutes, and embedded in Epon epoxy medium (Momentive Specialty Chemicals/Hexion, Columbus, OH, USA). Twenty-four hours later, 120-nm sections were cut with an ultramicrotome (DuPont, Wilmington, DE, USA) and stained with 4% uranyl acetate for 20 minutes and 0.5% lead citrate for 5 minutes. Ultrastructural changes of synapses in the CA1 were observed under a Hitachi HT7700 TEM microscope (Hitachi, Tokyo, Japan) and subsequently processed using Adobe PhotoShop software (Adobe Systems, San Jose, CA, USA). Synaptic structure in CA1 was analyzed in at least 20 images per mouse (n =3). Measurement of postsynaptic density (PSD) areas, width of synaptic cleft and number of vesicles were included only if synaptic terminal profiles were clearly visible, and these were performed using ImageJ software. The identities of images were coded and revealed to the observer only after the data analysis was complete.

Cytoplasmic and nuclear proteins were prepared using a nuclear and cytoplasmic protein extraction kit (KeyGen Biotech, Nanjing, China) following the manufacturer’s instructions. Hippocampal tissues were homogenized on ice and resuspended in the cytoplasmic protein extraction reagent, then centrifuged at 12,000 × g at 4°C for 5 minutes. The supernatant was cytoplasmic protein, and the pellet was resuspended in the nuclear protein extraction reagent and centrifuged at 12,000 × g at 4°C for 10 minutes. The supernatant was nuclear protein. The nuclear protein was subjected to Western blot analysis.

Statistical analysis was performed with IBM SPSS software (version 19.0; IBM, Armonk, NY, USA). The results are expressed as mean ± standard error of the mean. Statistical analysis was performed with analysis of variance followed by the Student-Newman-Keuls multiple-comparisons test for numerical data. Differences between two groups were assessed with Student’s t-test for data normally distributed and with the Mann–Whitney rank-sum test for data non-normally distributed, under the supervision of an expert statistician. Significance was set at P <0.05.