The animal study was approved by the Animal Ethics Committee of the Guangzhou Red Cross Hospital, China. All animals were carefully handled in compliance with established guidelines.

Forty male SD rats (weight approximately 100 $ \pm $ 20 g, four-five weeks old) were supplied by the Animal Center of Jinan University (Guangzhou, China). Rats were housed in a temperature and humidity-controlled room on a 12/12 hour light/dark cycle and food and water were freely available. After one week, all rats were randomly divided into four groups (10 rats per group): Control, Day 3, Day 7, and Day 14 after ischemia injury surgery. Focal cortical ischemia was generated by photothrombosis of the cortical microvessels as described in a previous study [24]. Briefly, rats were anesthetized and body temperature maintained at 37 $ \pm $ 0.5$^\circ$C by heat pad. The rat skull was exposed at 0.5 mm anterior and 3.7 mm lateral from the bregma, the skull was laser illuminated at a wavelength of 561 nm, eight mm diameter for 10-15 min, after infusion with Rose Bengal (RB, 50 mg/kg body weight, Sigma-Aldrich, CA, USA) which was dissolved in normal saline into the right femoral vein for two minutes [24]. The scalp was sutured and rats were allowed to wake before being returned to their cages. A sham control group received the same vein injection of Rose Bengal and the same surgery but without laser exposure to avoid any photochemical reaction. A schematic diagram of this surgery is given in Fig.1A. At scheduled time points, rats were sacrificed and their brains removed and immersed overnight in a fixative solution. The hippocampi were dissected out under microscopy, frozen immediately in liquid nitrogen, and stored at $-$80$^\circ$C until required.

Fig. 1.

(A) A schematic diagram showing how photothrombosis induces focal ischemic injury in rats. (B) Lesion areas visualized by TTC staining. TTC staining colors: red-viable tissue, white-ischemic tissue lesions.

Ischemic lesion were measured after surgery at days 3, 7, and 14. Rats were anesthetized with carbon dioxide and sacrificed with a rat guillotine. Brains were immediately obtained, placed into a rat brain matrix (RBM-4000, ASI Instrument, USA), and frozen at $-20^\circ$C for 20 min. The brain was then cut into 2-mm blocks and immersed in PBS at room temperature. Each slice was then incubated in a solution of 2% 2,3,5-triphenyltetrazolium hydrochloride (TTC) (Sigma-Aldrich, St. Louis, MO, USA) and maintained at 37$^\circ$C in a heater in the dark for 30 min, before excess TTC was drained and slices were fixed with 4% poly (tetrafluoroethylene-co-perfluoropropyl vinyl ether) (PFA) for 20 min. Infarcted brain tissue remained unstained (white), whereas normal tissue stained red.

Proteins and three biological replicates were collected from each group using the phenol extraction method [25]. Briefly, about 200 $\mu$g protein samples were mixed into 30 $\mu$L STD buffer, the mixture was incubated at 100$^\circ$C for five minutes and cooled to about 25$^\circ$C. The solution was then diluted with a 200 $\mu$L UA buffer and a 30 kDa ultrafiltration and centrifuged at 14,000g for 15 min at room temperature. These steps were repeated with 100 $\mu$L UA buffer added to the sediment and dark incubated for about 20 min. Samples were then centrifuged at 12,000g for 10 min. Filters were washed with 100 $\mu$L UA buffer and 100 $\mu$L DS buffer, then 2 $\mu$g trypsin (Promega, USA) in 40 $\mu$L DS buffer, which was added to each filter. Samples with other buffers were incubated overnight at 37$^\circ$C and the resulting peptides were processed by centrifugation. Filters were rinsed with 40 $\mu$L 10 $ \times $ DS buffer, recentrifuged, and finally the peptide content was tested for spectral density with UV light at 280 nm. About 100 $\mu$g of peptides from each treatment group were labeled with iTRAQ reagents following the manufacturer's instructions (Applied Biosystems, CA, USA).

Peptides were purified from excess labeling reagent by High Performance Liquid Chromatography (HPLC) (Shimadzu Corporation, Japan) prior to the mass spectrometric analysis. Labeled samples were dried and then diluted with 20-fold cation exchange binding buffer A (containing 10 mM KH$ {}_{2} $ PO$ {}_{4} $, at pH 3.0, and 25% ACN). The labeled samples were then separated into ten fractions by gradient elution in a poly-sulfoethyl 4.6 $ {\times} $ 100 mm A column (5 m, 200 A; Poly LC, Columbia, MD, USA) [26].

The heat map was generated using Heatmap V2.0 (R-package). The biological processes, molecular functions, and cellular components of the identified differentially expressed proteins were examined with KOBAS software to perform gene ontology (GO) annotation (http://kobas.cbi.pku.edu.cn/home.do) [27]. The GO enrichment analysis of the differentially expressed proteins was implemented with WEGO software (http://wego.genomics.org.cn/cgi-bin/wego/index.pl), in which the gene length bias was corrected. A corrected p value $ < $ 0.05 was chosen to include as many items as possible [28]. Kyoto encyclopedia of genes and genomes (KEGG) is a database resource for the analysis of high-level gene functions and biological gene systems (http://www.genome.jp/kegg/) [29]. Cluster Profiler software (R-package) was employed to test the statistical enrichment of differentially expressed proteins in KEGG pathways. A P value $ < $ 0.05 was also chosen so as to include as many items as possible.

A PPI network analysis provides new insights into protein function. A PPI network was constructed to further analyze the function of 25 common differentially expressed proteins. The Search Tool for the Retrieval of Interacting Genes/Proteins (STRING) (https://string-db.org/) provides the information for both predicted interactions. With this tool, differentially expressed protein pairs with significant interactions (combined score $ > $ 0.9) were identified and used for the construction of the PPI network, which in turn was visualized using Cytoscape (http://cytoscape.org/).

The brain infarct size after surgery was evaluated by TTC assay. Infarcted brain tissue was white, whereas the normal tissue was stained red. No infarct damage was found in control rats (Fig.1B), while infarct regions were identified in Day 3 rats. Furthermore, the infarct size in Day 7 rats was slightly bigger than that of the Day 3 group, which was also the case for Day 14 rats (Fig.1B). These results suggest that brain ischemia and ischemic damage was successfully induced in rats following focal cortical ischemia surgery.

A total of 4,081 peptides from the unique trypsin-digested proteins of all four groups were identified. 260 non-redundant proteins were identified with significantly differential expression that was more common in rats from Day 3, 7, and 14 groups than in the control group (Fig.2). In a comparison of Day 3 and control groups, 90 proteins were markedly upregulated and 170 were markedly downregulated. In a comparison of the control and Day 7 group, 83 proteins were markedly upregulated and 167 were markedly downregulated. The comparison of the Day 14 and the control groups was even more dramatic, with 98 markedly upregulated and 162 markedly downregulated proteins. Hierarchical cluster analysis revealed that Day 3, 7, and 14 groups had clearly different expression patterns among the 260 identified proteins, of which 160 were upregulated in the three different groups when compared to the control (Fig.2) and 80 proteins were downregulated. Sample clustering results indicated that protein expression patterns in the Day 3, 7, and 14 groups were more similar to each other than to those of the control group. 34.6%, 31.9%, and 37.7% of proteins were downregulated in the three different groups, while 65.4%, 64.2%, and 62.3% of proteins were upregulated, respectively, when compared to controls.

Fig. 2.

Hierarchical clustering of 260 differentially expressed proteins. Four main protein expression patterns were identified: the control was the protein expression atlas occurring within the first day after surgical simulation of the healthy side of the hippocampus; Day 3, 7, and 14 were the protein expression atlases occurring on the third, seventh, and fourteenth day, respectively, after surgical simulation of the diseased side of the hippocampus. Increasing red color indicates an increasing protein expression level.

GO annotation and enrichment analysis of 260 differentially expressed proteins that were more common in the three groups than the control were implemented with KOBAS and WEGO software. All GO terms with corrected P values less than 0.05 were considered to be significantly enriched. There were 4,944 GO items harvested in this study. Myelin sheath (GO: 0043209) and cell junction (GO: 0030054) were the two most enriched items. The distribution bar charts of the biological processes, cellular components, and molecular functions are shown in Fig.3. From the perspective of biological processes, those of cellular processes, metabolic processes, and pigmentation were the top three significantly enriched terms. In the molecular function category, binding, catalytic, and structural molecular functions were the top three. In the cellular component category, cell and cell parts were the top two significantly over-represented terms.

Fig. 3.

Gene ontology(GO) enrichment analysis of the 260 differentially expressed proteins identified in this study.

KOBAS software (http://www.genome.jp/kegg/) was employed to test the statistical enrichment of differentially expressed proteins using KEGG pathways. In this study, 260 differentially expressed proteins involved 220 pathways. Fig.4 shows the results of the KEGG pathway enrichment, clearly showing ribosome pathways as the top enriched term (Fig.4). In addition, 12 differentially expressed proteins identified in this study were identified as participants in ribosomal pathways. Moreover, it is noteworthy that proximal tubule bicarbonate reclamation, endocrine and other factor-regulated calcium reabsorption, and regulation of actin cytoskeleton and synaptic vesicle cycle were also significantly enriched.

Fig. 4.

Scatter diagram of enriched KEGG pathways of the 260 differentially expressed proteins identified in this study. Degree of enrichment was measured by Rich factor, $Q$ value, and the number of genes enriched in one pathway. The Rich factor is the ratio of the number of differential expression genes enriched in one pathway and the GO annotation number. The greater the Rich factor value, the higher the degree of enrichment. The $Q$ value is a variant of a $P$ value, for which lower values equate to significant enrichment. The $Y$-axis represents the name of pathway and the $X$-axis represents the Rich factor. The point size is the number of differentially expressed genes in one pathway, and the color of the point indicates the range of the $Q$ value.

The numbers of proteins with significantly differential expression and their overlap under different groups, as illustrated by Venn diagram analysis, are shown in Fig.5 and Table 1. A Venn diagram was created to depict the overlap of differentially expressed proteins from the four groups (Control, Day 3, Day 7, and Day 14), showing that 25 were shared by the Control vs. Day 3, Control vs. Day 7, and Control vs. Day 14 (Fig.5). The details of these 25 proteins are shown in Table 1. Among these, vimentin (Vim), albumin (Alb), inositol polyphosphate-4-phosphatase type I A (Inpp4a), Immunoglobulin Superfamily Member 1 (Igsf1), Annexin A2 (Anxa2), adhesion G protein-coupled receptor B2 (Bai2), heat shock protein family B (small) member 1 (Hspb1), Janus kinase and microtubule interacting protein 3 (Jakmip3), Superantigen (Speg), Uridine phosphorylase 1 (Upp1), BSD domain-containing 1 (Bsdc1), protein kinase AMP-activated non-catalytic subunit gamma 1 (Prkag1), potassium calcium activated channel subfamily M regulatory beta subunit 4 (Kcnmb4), Eukaryotic translation initiation factor 4E nuclear import factor 1 (Eif4enif1), ATP-binding cassette sub-family F member 1 (Abcf1), Molecule interacting with casL-like 1 (Micall1), nudix hydrolase 5 (Nudt5), ATPase secretory pathway Ca$ {}^{2+} $ transporting 1 (Atp2c1), GAR1 ribonucleoprotein (Gar1), exportin for tRNA (Xpot), general transcription factor IIF subunit 1 (Gtf2f1), and kinase non-catalytic C-lobe domain containing 1 (Kndc1) were upregulated, but Keratin 42 (Krt42), Keratin 10 (Krt10), and Keratin 1 (Krt1) were downregulated (Table 1).

Fig. 5.

Venn diagrams of differentially expressed proteins detected in the four different groups of this study. The overlap represents 25 differentially expressed proteins common to the four different groups.

The GO annotation and enrichment analysis of these 25 differentially expressed proteins was also implemented using KOBAS and WEGO software, respectively. GO terms with corrected $ {P} $ values less than 0.05 were considered significantly enriched. There were 1,142 GO items harvested in this study. Intermediate filament (GO: 0043209) and intermediate filament cytoskeleton (GO: 0045111) were the two most enriched items. The distribution bar charts of the biological processes, cellular components, and molecular functions are shown in Fig.6. From the perspective of biological processes, cellular processes, metabolic processes, and pigmentation were the top three significantly enriched terms. From the perspective of molecular function, binding, catalytic, and enzyme regulator were the top three significantly enriched terms. From the perspective of cellular component, cell and cell parts were the top two significantly over-represented terms (Fig.6).

Fig. 6.

GO enrichment analysis of 25 differentially expressed proteins identified in this study.

Moreover, KOBAS software was also employed to test the statistical enrichment of these 25 differentially expressed genes in KEGG pathways. It was found that the 25 proteins were involved in 30 pathways. Fig.7 shows the results of pathway enrichment. It clearly displays Epstein-Barr virus infection as the top enriched term (Fig.7). Additionally, two differentially expressed genes identified in this study are known to participate in Epstein-Barr virus infection pathways. Moreover, it is worth noting that Circadian rhythm, Basal transcription factors, and Drug metabolism-other enzymes, Staphylococcus aureus infection, and the vascular endothelial growth factor (VEGF) signaling pathway were also found to be significantly enriched here.

Fig. 7.

Scatter diagram of enriched KEGG pathways of 25 differentially expressed proteins identified in this study. The degree of enrichment was measured by Rich factor, $Q$ value, and the number of genes enriched in one pathway. The Rich factor is the ratio of the number of differentially expressed genes enriched in one pathway and the GO annotation number. The greater the value of the Rich factor, the higher the degree of enrichment. The $Q$ value is a variant of a $P$ value, for which lower values equate to significant enrichment. The $Y$-axis represents the name of pathway and the $X$-axis represents the Rich factor. The point size is the number of differentially expressed genes in one pathway, and the color of the point indicates the range of the $Q$ value.

The interactive protein network for the 25 differentially expressed proteins was predicted by STRING, and a total of 223 interactive proteins were searched and are represented by red arrow nodes. Black lines indicate the predicted protein directly with the differentially expressed proteins, which are represented by circular nodes. PPI network analysis found that Vim and Alb had the top two highest degrees in the constructed network (Fig.8).

Fig. 8.

A protein-protein interaction network of differentially expressed proteins. The circular nodes indicate differentially expressed proteins. Arrowed nodes stand for essential proteins predicted by STRING.