Male Sprague‒Dawley (SD) rats weighing 280 to 300 g were provided by the Experimental Animal Center of the Fourth Military Medical University. The animals were housed in a controlled environment (25 $\pm$ 2 °C, 50% humidity, and 12 h light-dark cycle from 8 to 8) and were given free access to food and water. All animal experiments were carried out in compliance with the Guidelines for the Care and Use of Laboratory Animals of the Fourth Military Medical University. A flowchart of the experimental design is shown in Fig. 1. The animals were randomly assigned to the control (Con, MCAO model without HBO preconditioning) group or the HBO group. HBO preconditioning combined with the ischaemia/reperfusion model was employed to screen predictive biomarkers for the protective effect of HBO preconditioning. The infarct volumes between the Con and HBO groups were calculated and analysed by independent sample T test analysis. Based on the mean and standard deviation of the infarct volume of the Con group, the rats in the HBO group in which the infarct volumes were less than the means minus the standard deviation (Mean (Con)-SD) were defined as the protective group (HBOP, hyperbaric oxygen protective), and the other rats in the HBO group were defined as the nonprotective group (HBOU, hyperbaric oxygen nonprotective).

Fig. 1.

Experimental design of the proteomic screening and subsequent validation of biomarkers for the neuroprotective effect of HBO preconditioning against cerebral ischaemia‒reperfusion injury. HBO, hyperbaric oxygen; MCAO, middle cerebral artery occlusion; Con, control; HBOP, hyperbaric oxygen protective; HBOU, hyperbaric oxygen nonprotective.

Rats in the HBO group accepted oxygen (2.5 ATA, 100% O${}_2$, 1 h/d) for five days, while rats in the control group did not receive HBO preconditioning but were placed into a hyperbaric chamber breathing normal baric air under the same conditions. Rat blood was got from the femoral artery at 6 h after the last time of HBO preconditioning. The samples were lef to clot for 2 h at 4 °C followed by 3000 g centrifugation force for 15 min at 4 °C. The serum was then aliquoted and stored at –80 °C before processing.

As was previously described, the rat transient focal cerebral ischaemia was induced by middle cerebral artery occlusion (MCAO) [11, 12]. Briefly, anesthesia was induced by an intraperitoneal injection of sodium pentobarbital (50 mg/kg in normal saline). The right common carotid artery (CCA) and right external carotid artery (ECA) were exposed through a ventral midline incision of the neck and were ligated proximally. The 4/0 single silk nylon suture (Sunbio Biotechnology Co. Ltd., Beijing, China) was inserted into the common carotid artery below the carotid bifurcation through a small incision, followed by inserting into the internal carotid artery (ICA) about 18–20 mm distal to the carotid bifurcation until slight resistance was felt. Two hours after ischemia, the animals were anesthetized again and reperfusion was accomplished by retracting the sutures.

Regional cerebral blood flow (rCBF) of all animals subjected to ischaemia/reperfusion injury was monitored using a PF 5000 Laser Doppler Perfusion Monitoring Unit (PeriFlux 5000, Perimed AB, Stockholm, Sweden). Stroke models were considered successful if rCBF decreased to 30% of the baseline level immediately after MCAO and increased to 70% of the baseline within 10 min after reperfusion.

At 72 h after reperfusion, neurologic behaviours of rats were assessed according to the Garcia scoring system (an 18-point scoring system) by an observer who was blinded to the grouping information [13]. Six aspects of neurological deficit were measured, including (1) spontaneous activity, (2) symmetry of four limb movements, (3) forepaw outward extension, (4) climbing ability, (5) body proprioception, and (6) response to whisker touch. Each aspect was scored from 0 to 3 points, and the total score was calculated. All behavioral tests were performed in a quiet room. The higher the score was lead to the better the neurobehavioral function. The animals were then sacrificed under deep anaesthesia with pentobarbital sodium and decapitated, and their brains were evacuated and sliced into six 2-mm-thick coronal sections with a matrix and stained with 2% TTC (Cat.#17779, Sigma, St. Louis, MO, USA) in normal saline at 37 °C for 10 min to evaluate the infarct volume ratio. The stained slices were then transferred to 4% formalin for postfixation before being photographed with a digital camera (Canon, Tokyo, Japan). White or pale pink areas were defined as infarction and were measured using image analysis software (Adobe Photoshop CS5, Adobe Systems, CA, USA). To exclude the effect of cerebral oedema that is induced by ischaemia on the infarct size, the percentage of the infarct volume was calculated using a corrected algorithm: (total contralateral hemispheric volume – total ipsilateral hemispheric stained volume)/total contralateral hemispheric volume $\times$ 100%.

Immunoaffinity Depletion of High-Abundance Proteins Collected serum was pooled into three groups, each containing serum from 6 rats. Serum pools were depleted of the three most abundant proteins (albumin, IgG, and transferrin) using Multiple Affinity Removal Column (M-3) following the manufacturer’s protocol (Agilent Technologies Co., Ltd., Santa Clara, CA, USA). Briefly, the serum samples were diluted 5-fold with Agilent buffer A. The 100 µL diluted sample was loaded into a 0.22 µm spin cartridge and centrifuged at 16000 g for 2 min. Then, the bound proteins were eluted with buffer B at 1 mL/min for 3.5 min. Desalination was performed in 5K ultrafiltration tubes. The depleted serum was concentrated using the EZQ${}^{\mathrm{\circledR }}$ protein quantification kit (Bio-Rad, Hercules, CA, USA). The collected samples were exchanged with a buffer of sodium bicarbonate and 0.5 M sodium carbonate solution.

A 2-D clean-up kit (GE Healthcare, Bucks, United Kingdom) was used to remove the substances that interfered with plasma two-way electrophoresis. Serums from 6 rats in each group were pooled. The serums of the three groups were labelled respectively with different fluorescent dyes later and then mixed before analysis. The protein concentration in the serum samples was determined with the Ettan TM 2-D Quant Kit (GE Healthcare Life Sciences, Piscataway, NJ, USA). protein quantification kit. Fifty micrograms of six serum samples from each group were randomly selected. Labelling reactions were performed with either Cy3 or Cy5 fluorescent dyes. The internal standard was prepared by mixing 25 µg of each sample protein and performing the labelling reaction with Cy2 dye. Fluorescent dye-labelled plasma proteins top-sample to solid-phase nonlinear dry adhesive strips for hydration. The Ettan${}^{\text{TM}}$ IPGPhor (GE Healthcare) isoelectric focusing apparatus was applied, and the Ettan${}^{\text{TM}}$ DALT (GE Life Sciences) Six vertical electrophoresis system was used to perform first- and second-direction electrophoresis.

Cy2, Cy3 and Cy5 were scanned with excitation light at 488 nm, 532 nm and 633 nm. The glue images were collected and analysed with the Typhoon multifunction scanning imaging system (GE Healthcare). The images of samples labelled with Cy2, Cy3, and Cy5 fluorescent dyes are coloured blue, green, and red, respectively. The DeCyder V6.5 analysis software (GE Healthcare) detected and matched the analysed collagen points. 3D images and manual proofreading were used to select the differential protein points and then match the corresponding protein points on the preparation glue (Fig. 2). The differential protein points were dug using the Ettan${}^{\text{TM}}$ Spot picker automatic spot cutting system (GE Healthcare).

Fig. 2.

Screening process for the differential proteins by 2D-DIGE-MALDI-TOF-MS.

Digestion and picking were done by preparing the gels. Two-dimensional electrophoresis was performed. immobilized pH gradient (IPG) bands were loaded with 500 to 1000 µg of protein and gels were stained with Coomassie blue. Protein points of interest were excised and counterstained with 25 mM ammonium bicarbonate and 50% acetonitrile. The gel was freeze-dried by centrifugation. It was performed with 0.01 µg/µL trypsin in 25 mM ammonium bicarbonate 10 for 15 hours at room temperature. The supernatant was collected and tryptic peptides were extracted with 5% trifluoroacetic acid at 40 °C for 1 h, then with 2.5% trifluoroacetic acid (TFA) and 50% Acetonitrile (ACN) for 1 h at 30 °C for 1 h.

The peptide mixture was dissolved in 0.5% TFA, the 1 µL peptide solution was mixed with 1 µL matrix (4-hydroxy-cyanogenic acid in 30% ACN, 0.1% TFA) and then found on the target plate. Matrix-assisted laser desorption/ionization time of flight (MALDI-TOF) mass spectrometry and tandem TOF/TOF mass spectroscopy were performed on a 4800 Plus MALDI TOF/TOF instrument (Applied Biosystems, Foster City, CA, USA). Peptide mass maps were obtained in positive reflection mode, with an average of 5000 laser per MALDI-TOF spectrum and 5500 laser per TOF/TOF spectrum (20000 resolution). The 4800 calibration mixtures (Applied Biosystems, Foster City, CA, USA) were used to calibrate the spectrum to a mass tolerance of 0.1 Da. The parent mass peak with a minimum signal to noise ratio of 15 was selected for tandem TOF/TOF analysis. The Mascot database search algorithm (version 2.0, https://www.matrixscience.com) was used to interrogate the rat sequences in the International Protein Index Database (IPI_Rat V3.52, https://www.ebi.ac.uk/IPI). All automated data analysis and database searches were performed using GPS Explorer TM software (Biosystems Applied Systems version 3.6, Applied Biosystems, Foster City, CA, USA). Confidence identification was statistically significant (p 0.05) for a protein score (based on the mass/mass spectrometry combination) and for an optimal ion score (based on mass/mass spectrometry). Any redundant proteins appearing with different names and accession numbers in the database were eliminated. If multiple proteins are identified at one point, the protein with the highest protein score (the highest ranking) is removed. The molecular weight and isoelectric points of most proteins agree with the gel region where the spots were excised.

The serum potential biomarkers were verified by western blot technique, which was carried out as previously described [14]. Another set of animals was used in this study and was randomly assigned into the control (n = 8) and HBO groups (n = 16). Blood serum was collected at 6 h after the last session of HBO, and the animals were subjected to 120 min MCAO at 24 h after HBO preconditioning. The brain infarct volume ratio was assessed at 72 h after reperfusion. Serum from the HBO group was regrouped as described above into HBOP (n = 10) and HBOU (n = 6) groups (infarct volume ratio for each group: control group 0.39 $\pm$ 0.032; HBOP group 0.16 $\pm$ 0.036; HBOU group 0.36 $\pm$ 0.014. p 0.05, HBOP vs. Con and HBOP vs. HBOU). The serum samples in each group were randomly picked for a subsequent western blot analysis. An equal amount of protein (5 µL) was loaded onto each lane of 12% polyacrylamide-S ml DS gels. The gels were electrophoresed and transferred onto PVDF membranes. The membrane was then blocked at room temperature for 1 h and incubated with primary antibody at 4 °C overnight. Rabbit polyclonal anti-haemopexin (1:500 dilution, Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA) and anti-rabbit $\beta$-actin antibodies (1:1000 dilution, CWBIO, Peking, China) were used. The membranes were then incubated for 1 h at room temperature with the appropriate horseradish peroxidase-conjugated secondary antibodies (1:1000; Sigma Aldrich, St. Louis, MO, USA). Immunoreactive bands were visualized using enhanced chemiluminescence reagents (Applygen Technologies Inc., Beijing, China). The optical density was determined using Gelpro32 software (Media Cybernetics, Marlow, UK).

Serum haemopexin levels were measured by a quantitative competitive enzyme linked immunosorbent assay (ELISA) kit (Westang, shanghai, China). The same serum samples used in the western blot study above were applied here. All standards and samples were tested in duplicate. Absorbance was measured at 450 nm using a monochromotor microplate reader (Denley Dragon Wellscan MK 3, Thermo, Finland).

SPSS 20.0 (SPSS Inc., Chicago, IL, USA) was used to conduct statistical analysis. Measurement data were obtained, and tests of normality were performed using the Kolmogorov‒Smirnov normality test. All the data except the neurologic score are presented as the Mean $\pm$ SD and were analysed using a two-tailed Student’s t test or one-way ANOVA followed by LSD post-hoc test. The neurologic scores were expressed as the median (range) and were analysed with the Kruskal‒Wallis test followed by the Mann‒Whitney U test using Bonferroni correction. Pearson correlation analysis was used to analyse the correlation between infarct size and hemopexin (HPX) serum concentration. Receiver operating characteristic (ROC) curve analysis for serum HPX concentration at 2 h after HBO preconditioning was used to calculate the area under the ROC curve (AUC), sensitivity, and specificity. p 0.05 was considered significantly different.

During occlusion, the cerebral blood flow (CBF) value decreased to 30% of the baseline in all rats. After reperfusion, CBF increased back to 70% of the baseline immediately and gradually grew back to 100% of the baseline (Fig. 3A).

Fig. 3.

Regional cerebral blood flows, infarction volume, and neurological outcome at 3 d after reperfusion. (A) Reginal cerebral blood flows in the ischaemic hemisphere of rats during the surgery procedure. Con: control group, in which the animals underwent MCAO without HBO preconditioning; HBO: hyperbaric oxygen preconditioning (2.5 ATA, 100% O${}_2$, 1 h/day, 5 d) group. (B) Representative TTC-stained brain slices of rats after regrouping at 3 d after reperfusion. HBOP: subjects that experienced a protective effect of HBO preconditioning with an infarct volume ratio Mean-SD of Con group. HBOU: subjects in the HBO group that showed no benefit from HBO preconditioning with an infarct volume ratio $\ge$Mean-SD of the Con group at 3 d after reperfusion. (C–E) Infarct volume ratio after regrouping at 3 d after reperfusion. (F–H) Neurological functioning scores after regrouping at 3 d after reperfusion. N = 8 for Con, n = 10 for HBOP, and n = 6 for HBOU. * p 0.05 compared to Con group; ** p 0.01 compared to Con group.

To examine the long-term neuprotection of HBO preconditioning, 72 h long-lasting neurological function and neuroprotective effects were assessed by infarction volume evaluation and behavioral assessment. The neurobehavioral evaluation was done through an 18-point scoring system Garcia score method and the neurobehavioral scores (NBS) were got in each group [13]. As shown in Fig. 3F, HBO preconditioning improved neurological function effectively at 3 d after transient cerebral ischaemia and reperfusion (p = 0.024 Con vs. HBO). In addition, TTC staining revealed that HBO preconditioning also reduced the infarct volume ratio at 3 d after MCAO (p = 0.007 Con vs. HBO, Fig. 3B–C). As expected, we found that not every subject in the HBO group exhibited a smaller infarct volume ratio than that of the control group. The outcome in some of the subjects in the HBO preconditioning group was even worse than that in the Con group. Therefore, rats in the HBO group were subgrouped into HBOP (subjects that benefited) and HBOU (subjects that did not benefit) groups based on the infarct volume ratio. Briefly, we regrouped the serum according to different infarct volume ratios at 72 h after reperfusion within the HBO preconditioning group as the HBOP group (n = 10, with an infarct volume ratio (Mean-SD) of the control group) and HBOU (n = 6, with an infarct volume ratio $\ge$(Mean-SD) of the Con group). After regrouping, the HBOP group showed significant improvement in both neurological function (p 0.01 Con vs. HBOP, Fig. 3G) and a reduced brain infarct volume ratio (p 0.01 Con vs. HBOP, Fig. 3D), whereas worse outcomes were observed for the animals in the HBOU group compared to the HBOP group, which were similar to those in the Con group (p $>$ 0.05 Con vs. HBOU, Fig. 3B,E,H).

After regrouping, serum collected before MCAO was analysed by 2D-DIGE-MALDI-TOF-MS/MS. After 2-D DIGE, the Cy2, Cy3, and Cy5 fluorescence intensities of each gel were individually imaged and analysed with DeCyder 6.5 software (GE Healthcare, Little Chalfont, UK). To reveal the specific protein changes in the subjects that benefited (protective effect) from HBO preconditioning, serum proteomic comparisons were designed as follows: we separately investigated the differences in AV. ratios for the control versus HBOP group and HBOU versus HBOP group. Proteins that showed significant differences (p 0.05) in both comparisons were selected as candidates for further verification. Among the 1926 matched protein spots, 15 spots were differentially expressed in the HBOP group compared to the other groups (AV.Ratio $>$1.2 or –1.2, p 0.05; Table 1). Pzp (Alpha-1-macroglobulin), Apoa1 (Apolipoprotein A-I), Alb (serum albumin), Haemopexin, B allele, and Complement C3 expression levels were increased in HBOP samples, whereas LOC500180 Ig kappa chain C region, Gc Vitamin D-binding protein, Apoe (Apolipoprotein E), Orm 1 (Alpha-1-acid lycoprotein), Hp (Isoform 2 of Haptoglobin), Srprb, Cfb complement factor B, RGD 1564318 (Ig lambda-2 chain C region), and Fetub Fetuin-B showed lower expression levels in the HBOP group (AV.Ratio $>$1.2 or –1.2, p 0.05; Table 1) compared to the other groups.

Among the 15 differentially expressed spots in the HBOP group, 3 spots corresponding to 3 different genes (haptoglobin, serum albumin, and haemopexin) products were clearly identified by MALDI-TOF-MS/MS (Fig. 4). Serum albumin and haemopexin were upregulated, and haptoglobin was downregulated in the HBOP group (p 0.05 compared to other groups).

Fig. 4.

Protein identification by matrix assisted laser desorption/ionization time-of-flight mass spectrometry. MW, molecular weight; PI, protein isoelectric point.

We first excluded albumin as a biomarker candidate that can predict the outcome of stroke after preconditioning because it is the most abundant protein in plasma. Haemopexin (HPX) and haptoglobin proteins, which stand out differently in both comparisons with the HBOP group, were selected for further verification using enzyme-linked immunosorbent assay (ELISA). As shown in Fig. 5A, the changes in the HPX protein concentration in the HBOP groups were upregulated compared with that in the HBOU group and Con group, which was consistent with the data obtained from the MALDI-TOF-MS/MS proteomic analysis (p 0.05 HBOP vs. Con; p 0.05 HBOP vs. HBOU). To further verify the results, we validated the expression of HPX in individual samples using western blot analysis (n = 6 for each group). After Bradford assay quantification, equal amounts of protein (5 µg) from each sample were loaded onto each lane of polyacrylamide-SDS gels. After the western blot study, the changes in haemopexin were validated and exhibited similar changes in subjects from the HBOP group in accordance with MALDI-TOF-MS/MS proteomic analysis and ELISA analysis. As shown in Fig. 5B, the overall level of HPX in the HBOP group was higher than that in the control group and HBOU group (p 0.05 HBOP vs. Con; p 0.05 HBOP vs. HBOU). We also verified the expression of the haptoglobin protein among the different groups using the ELISA method and western blot, and there was no significant difference among the three groups (data not shown).

Fig. 5.

Enzyme-linked immunosorbent assay (ELISA) and Western blot (WB) analysis of HPX levels in the serum of rats at 6 h after HBO preconditioning. (A) Enzyme-linked immunosorbent assay; (B) Western blot analysis. (n = 8 in the control group, n = 6 in the HBOU group, n = 10 in the HBOP group). The statistical results of WB after regrouping are shown under the representative bands. * p 0.05 compared to the Con group; # p 0.05 compared to the HBOP group; ## p 0.01 compared to the HBOP group.

To verify the feasibility of HPX as a biomarker for the neuroprotective effect of HBO pretreatment, the correlation between serum HPX content at different time points after HBO pretreatment and the infarct volume at 72 h following the MCAO procedure was evaluated. The serum HPX content was determined by ELISA. The results of the correlation analysis showed that the haemopexin concentration in the serum of rats at 2 h after HBO preconditioning was negatively correlated with the infarct volume ratio (correlation coefficient = –0.6199, 95% confidence interval (CI) (–0.8428, –0.2153), p 0.05, Fig. 6A). The ROC curve that discriminates the HBOP group from the HBOU group indicated that the area under the ROC curve was 0.9 (95% CI: 0.7141–1; p 0.01); Youden’s index = 0.9 at HPX concentration = 0.6265 mg/mL (n = 18, Fig. 6B). We also determined the baseline HPX concentration in the serum of the rats, and the HPX concentration at 24 h after HBO preconditioning was not correlated with the infarct volume ratio at 72 h following MCAO (p $>$ 0.05, Fig. 6C,D).

Fig. 6.

Analysis of HPX serum concentration at different time points after HBO pretreatment and prognosis of the rats in different groups. Quantification of HPX by standard ELISA tests. (A) The HPX level at 2 h after HBO preconditioning was negatively correlated with the infarct volume at 3 d after reperfusion (r = –0.62, p 0.05). (B) The receiver operating characteristic (ROC) curves of prognostic factors for distinguishing HBOP with HBOU. The area under the ROC curve was 0.9; 95% CI: 0.7141–1; p 0.01; Youden’s index = 0.9 at HPX concentration = 0.6265 mg/mL. (C) No correlation was detected between the baseline HPX before HBO preconditioning and the infarct volume at 3 d after reperfusion (r = 0.01, p $>$ 0.05). (D) No correlation was detected between HPX levels at 24 h after HBO preconditioning and the infarct volume at 3 d after reperfusion (r = –0.15, p $>$ 0.05). n = 18. ROC, receiver operating characteristic; ELISA, enzyme linked immunosorbent assay.