Animals and treatments
All animal procedures were approved by the Institutional Animal Care and Use Committee of Nanjing Medical University(IACUC: 2103051), and the Number of Animal Use Permit is SYXK(Su)2020-0022. Totally 60 adult male Sprague Dawley rats (Jiangsu Laboratory Animal Center, China; 8 weeks, 280-320g) were housed under a 12/12 h light-dark cycle at 22-24℃ with 50%–60% humidity. Food and water were available ad libitum.
Our animals experiment included four groups: Sham operation group (n=15), CLP group (n=15), CLP+MFGE8 group (n=15), and CLP+MFGE8+Cilengitide group (n=15). Totally 3.3ug MFGE8 (LMAI Bio, ICA286Hu01) was injected intracerebroventricularly (i.c.v.) on the first day after CLP surgery for rats in CLP+MFGE8 group and CLP+MFGE8+Cilengitide group using a microsyringe with stereotaxic co-ordinates of AP-1.0 mm, ML2.0 mm, DV3.8 mm[14]. Cilengitide (MCE, HY-16141), inhibitor of αVβ3 and αVβ5 integrins receptor [15], was diluted with normal saline (10 mg/kg, 1 ml/animal), and injected intraperitoneal one hour after CLP surgery for rats in CLP+MFGE8+Cilengitide group[16]. The rats were weighed every day during the experiment. Three days after CLP, the neurological behavior tests were measured continuously for five days. The Morris water maze (MWM) and open filed test (OFT) were performed on third day post-surgery. After behavioral observation finished, the animals were observed by fMRI scan to detect the brain changes of structure and function. And then, the rats were sacrificed, and the brain as well as serum were collected for proteins analysis and pathological observation.
Cecal ligation and puncture
Sepsis-associated encephalopathy model were induced by cecal ligation and puncture (CLP) surgery in male Sprague Dawley rats. After fasting for 6 hours, the rats were anesthetized by intraperitoneal injection 35 mg/kg pentobarbital sodium according to Rittirsch D’s research[17]. A median incision (2 cm in length) was made on lower quadrants of abdominal wall, and the mesentery and cecum were dissociated under aseptic conditions. The cecum was exteriorized and ligated distal to the ileocecal valve about 3 cm with 4-0 non-absorbable suture. The cecum was perforated twice using a 22-gauge needle and a small amount of feces extruded through the perforation site. Animals were injected with saline solution (5 ml/100 g) subcutaneously (s.c.) immediately after the CLP surgery for fluid supplement. The rats were allowed free access to food and water after surgery. In the sham group, animals underwent all surgical procedures, but the cecum was neither ligated nor perforated. After the sepsis model successfully established, some rats were attacked by acute respiratory failure or renal failure. The survival rate was recorded every 24 h for seven days and the survival curve was plotted.
Open field test
Motor function and anxiety were assessed by the open field test (OFT). The open field apparatus was a black square (100×100 cm; height of 50 cm), and the arena was divided into the center area (60 cm×60 cm square) and the edge area (20 cm away from the walls). The light was maintained at minimum (~ 300 lx) to avoid anxiety behavior. The test sessions were recorded by a video camera placed 160 cm above the arena and analyzed with ANY maze Video Tracking System (Stoelting Co. Wood Dale, IL, USA). The experiment was performed on the third day after surgery. The total distance the animals travelled and duration in central area were measured in a ten minutes session. After one round of test, the arena was cleaned by 75% alcohol for eliminating odors.
Morris water maze test
Cognitive functions of spatial learning and memory were evaluated by Morris water maze test according to the methods of Wang[18]. The apparatus for the MWM consisted of a circular pool (diameter 180 cm; height 45 cm) filled with water made opaque with black dye (22–24 °C) and a platform (diameter 4 cm, smooth-faced) hidden submerged 1 cm below the surface of the water located at a fixed location in the target quadrant. The maze was geographically divided into four quadrants that were equally spaced around the perimeter of the pool. The tests were recorded by a camera which was hanging above the pool and the results were analyzed by ANY maze Video Tracking System. The experiment, including the navigation and probe tests, was trained on the 3rd day after operation. Before the first day of MWM test, each rat was allowed to swim freely in water for one minute without the platform, in order to adapt the environment and eliminate unqualified rates. The navigation tests were performed on the third to sixth days post-CLP. During the continuous four training days, each rat was released into the pool to find a platform placed under the dark water 2 cm below, and trained four times from every different quadrant each day. On the fifth day of Morris water maze test, the platform was removed for space exploration training. The rats faced to the wall was placed in the water from the opposite side of the original platform quadrant. The time spent in the target quadrant and the times of crossing the platform were recorded.
Resting-State MRI scan and fMRI data processing
MRI scans were performed in a 7 Tesla horizontal bore magnet (Bruker Biospec 7 T/20 USR; Bruker, Karlsruhe, Germany). Using a rat head volume coil, we acquired blood oxygen level dependent (BOLD) fMRI responses. Eight rats in each group were included in imaging examinations. Fast spin-echo T2 weighted MR images were obtained to visualize the structure of the rats’ brains. The rats were anesthetized by 2 % isoflurane, and then, the head was fixed in a capsule with a tooth-bar to limit the movement. Inhaling with 2% isoflurane and 2 L/min oxygen, the rats breathed at a rate of 60 times per minute. Fast spin-echo T2 weighted MR images were obtained with the following parameters: slice=25, repetition time (TR)=3000 ms, echo time (TE) =11 ms, TA=4 min 48 s, SI=1.00/1.00 mm, FOV=3.17/2.50 cm, MTX=256. The scan parameters of BOLD: slice=18, TR=2000ms, TE= 25ms, Slice Thickness=1 mm, TA=5 min, image size =128×128.
After preprocessing of EPI series (discarding the first 10 volumes, slice timing, correction, realignment, motion correction, coregistration with T2 images to rat brain atlas) with SPM8 (https://www.fil.ion.ucl.ac.uk/spm/) and DPABI (http: //rfmri.org/dpabi) [19], the normalized images were linearly detrended. Those on which the head moved by more than 0.2 mm and 2degree were excluded. After the images were smoothed with a Gaussian kernel of 4 mm× 4 mm× 4 mm full width at half-maximum (FWHM) to improve the signal-to-noise ratio (SNR), amplitude of the low-frequency fluctuations (ALFF) was abstracted from the BOLD images. For rs-fMRI data, regions of interest (ROIs) were extracted from SIGMA Anatomical Brain Atlas using dpabi software, four brain regions were chosen as seed ROIs for analyses of brain functional connection. The functional connectivity (FC) score between CA1 of hippocampus and lateral entorhinal cortex (LEnt) on the same side of the cerebral hemisphere were calculated through using Pearson’s correlation coefficients and converted to normally distributed Z-scores using the Fisher transformation.
Enzyme-linked immunosorbent assay
Blood was collected from left ventricle, and then samples were centrifuged at 12000 g for 10 min at 4 ℃. The serum levels of IL-1β, IL-6, CRP were determined by ELISA kits (Solarbio, Beijing, China) according to the manufacturer's instructions, and the absorbance was measured with a micro-plate reader at 450 nm (optical density (OD) value).
Western blot analysis
The hippocampus on both sides were lysed and homogenized in RIPA lysis buffer containing phenylmethylsulfonyl fluoride immediately. After protein concentration assayed with the BCA kit, Total protein samples were separated by 12 % sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene difluoride (PVDF) filters. Membranes were probed with anti-rat monoclonal antibodies raised in rats against MFG-E8 (Abcam, ab9324), Rac1 (Abcam, ab155938) and LC3 (Abcam, 192890), overnight at 4℃. On the second day, the membrane were washed three times using PBS with 0.1% Tween20, reacted with HRP-conjugated affinipure rabbit Anti-rat IgG (H+L) (Abcam, ab6734) for two hours at 22-25℃. The integral optical density (IOD) of each band was measured using ImageJ software.
Hematoxylin-Eosin (HE) Staining for pathology observation
After 48 hours of fixation with 4% paraformaldehyde, brain tissues were cut into slices in the coronal direction. Brain slices were dehydrated with gradient alcohol, cleared in xylene, and embedded in paraffin. Subsequently, brain tissues were cut into 5-μm slides for HE staining (Boster, Wuhan, China) and captured under a microscope.
Statistical Analysis
Data presented as mean ± standard deviation (S.D.) was analyzed with SPSS 22.0 and graph pad prism 8. Multiple groups comparisons were performed by one-way ANOVA followed by Tukey test for multiple comparisons. In behavior test results, western blot results and ELISA results, p-value less than 0.05 (p < 0.05) was considered statistically significant. The difference of ALFF value was analysed by two sample t-tests with REST software (p < 0.001). The ALFF value were compared between CLP group and CLP +MFGE8 group, as well as between CLP group and CLP +MFGE8+cilengitide group. The correction thresholds were limited by false discovery rate (FDR) p = 0.05, cluster connectivity criterion: mm=5, cluster size=21, p=0.001.
The zFC between CA1 and LEnt on the same side of the cerebral hemisphere were analysed by one-way ANOVA followed by Tukey test. The relationships between FC of hippocampal CA1 and LEnt and total time in target quadrant in Morris water maze test were explored by Spearman’s correlation analysis.