Hemopexin alleviates cognitive dysfunction after focal cerebral ischemia-reperfusion injury in rats

All protocols carried out in this article were approved by the Medical University of Tianjin experimental animal management committee (Aecl2015–0158 [JIN]; October 27, 2015). Male Sprague–Dawley (SD) rats (7 to 8 weeks old, weighting 250 g to 280 g) provided by Experimental Animal Laboratories of the Academy of Military Medical Sciences (license number: SCXK_ (Army) 2009–003, Beijing, China), were housed individually with a 12-h light/dark cycle, relative humidity of 55 to 75% and a constant surrounding temperature (22 ± 2 °C), with water and food available ad libitum. All rats were randomized into treatment groups (Sham n = 36; MCAO n = 36; Vehicle n = 36; HPX n = 36; HPX + ZnPPIX n = 30), and researchers were blinded to treatments in all experiments below. Rats in the sham group received a sham operation; in the other groups, rats all received MCAO with or without intracerebroventricular injection of HPX (HPX group) and HPX with ZnPPIX (HPX + ZnPPIX group). Euthanasia was performed by intraperitoneal injectition of pentobarbital sodium (200 mg/kg) after the experiments.

The middle cerebral artery occlusion (MCAO) method was applied to induce the focal cerebral ischemia/reperfusion (I/R) injury model. Briefly, rats were fasted for 12 h before surgery with water accessible and were then anesthetized with chloral hydrate (10%, 3 ml/kg) by intraperitoneal injection. Then, a ventral midline neck incision was made, followed by the identification and careful free dissection of the right common carotid artery (CCA), external carotid artery (ECA), and internal carotid artery (ICA) from surrounding tissues. A surgical filament (Beijing Cinontech Co. Ltd.) was inserted into the ICA after the proximal ligation of the CCA and ECA until a mild resistance was felt, indicating the occlusion of the middle cerebral artery (MCA), which resulted in a transient cessation of blood flow and subsequent brain infarction in the area supplied by the MCA. The skin was closed with a 4–0 silk suture, followed by 1 h of ischemia. After that, the filament was withdrawn to initiate a reperfusion process. The body temperature of the rats was maintained at 37 ± 0.5 °C during the whole procedure.

Right after reperfusion, rats were anesthetized as before and placed on a stereotaxic frame. The skull was fixed and secured with four stainless-steel screws, and a scalp incision was performed to expose the surface of the skull and bregma. A burr hole (1.5 mm lateral to and 0.8 mm posterior to bregma) was drilled into the right cranial bone. A 25-μl microsyringe (Hamilton) was very slowly inserted 3.5 mm beneath the dural surface to inject saline (10 μl, 0.9% sodium chloride for the MCAO group rats), vehicle (10 μl, 0.1% sodium azide for the vehicle group rats), rat hemopexin reference serum (10 μl, 1.86 g/l HPX dissolved in 0.1% sodium azide, ICL, Portland for the HPX group rats) or solution mix (10 μl, HPX 1.86 g/l + ZnPPIX 20 μM for the HPX + ZnPPIX group rats) once at a rate of approximately 3.3 μl·min^− 1 within 3 min with a semi-automatic control device. The microsyringe was kept in place for 5 min to ensure the effectiveness of injection before withdrawal. After withdrawal, the incision was sutured, and the rats were allowed to recover from anesthesia. Sham rats underwent the same surgical procedure without exposure to MCAO injury and cerebroventricular injection.

The MWM assay consists of two parts: a spatial probe test and a hidden platform test [10]. Briefly, the test system included a large four-quadrant circular tank (210 cm in diameter and 50 cm in height) filled with water (temperature was maintained at 19 to 22 °C, 30 cm in depth) and colored cards on the walls that remained constantly throughout the whole experiment. In the spatial test, a black target platform was fixed 1 cm below the surface of the water in the middle of the southwest (SW) quadrant, which ensured that the platform was invisible to rats. Each rat was tested four times a day, with a 5-min interval between each trial, for 5 consecutive days and randomly released in quadrant positions as described below. Each trial had a maximum duration of 120 s, and rats were allowed to swim freely in the tank to find the hidden platform. Once the hidden platform was found, the rats were allowed to rest on the platform for 30 min, and the time spent to find the platform was recorded as their escape latency. If rats failed to find the platform within 120 s, they were placed on the platform for 30 s by the researcher, and the escape latency was recorded as 120 s. After the 5 days of tests, the hidden platform test was carried out. In this test, the hidden platform described above was removed to observe and record the time spent (time percentage) in the target quadrant (quadrant SW) and the number of platform crossings. The escape latency, time percentage and number of platform crossings in the MWM were recorded by a computerized video tracking system (Ethovision 3.0; Noldus Information Technology, Wageningen, Netherlands).

At 7 days after ischemia-reperfusion, rats were sacrificed to quickly obtain the brain tissue of the ischemic penumbra, which was snap-frozen in liquid nitrogen. The ischemic penumbra brain tissue was then ground in radioimmunoprecipitation (RIPA) assay buffer (Sigma-Aldrich) containing protease inhibitors to obtain the protein extracts. An equal amount (40 μg) of protein sample among the groups was loaded into each lane of a 10% sodium dodecyl sulfate (SDS) polyacrylamide gel and separated by electrophoresis, followed by electrotransference to a polyvinylidene difluoride (PVDF) membrane (Millipore, Billerica, MA). After being blocked with 5% nonfat milk for 2 h at room temperature, membranes were incubated with the following primary antibodies: anti-HO-1 (rabbit polyclonal, 1:1000 in dilution, Santa Cruz Biotechnology Inc., USA), anti-VE-cadherin (rabbit polyclonal,1:3000 in dilution, abcam, USA) and anti-rabbit β-actin (rabbit polyclonal, 1:1000 in dilution, Abcam, USA) with gentle shaking at 4 °C overnight. The membranes were washed in TBST (Tris-buffered saline Tween-20) 3 times (5 min each time), followed by incubation with the corresponding goat anti-rabbit secondary antibodies (11,000 dilution, CWBIO, Peking, China) to recognize the bound antibodies at room temperature for 2 h. The immunoblots were immersed in enhanced chemiluminescent (ECL) reagent to visualize the specific protein bands, followed by exposure to ECL-hyperfilm (Amersham Biosciences, Piscataway, NJ, USA).

Rats were sacrificed after being intraperitoneally anesthetized at 7 days after reperfusion. The rats were anesthetized intraperitoneally as described before and transcardially perfused with heparinized saline, followed by transcardial perfusion with 4% paraformaldehyde. After perfusion, the rats were decapitated to remove the brain tissue from the skull, embed it within OCT medium (Leica 0201 06926, Germany) and store it at − 80 °C. Coronal sections (8 μm) were obtained from the frozen brain tissue for immunohistochemical staining. CD31 and vWF are two endothelial cell markers. After deparaffinization and dehydration, non-specific endogenous peroxidase activity was blocked by treating sections with 3% hydrogen peroxide in methanol for 30 min. Antigen recovery was performed by boiling the sections for 10 min in 10 mM citrate buffer (pH 6.0). Nonspecific binding was blocked with 1% non-immune serum in PBS for 30 min. The sections were then incubated with a goat monoclonal antibody (mAb) against CD31 (1:100, Abcam, England) and vWF (1:100, Abcam, England) overnight at 4 °C. The sections were then washed with PBS, incubated with a biotinylated anti-goat IgG (1:100, Santa Cruz Biotechnology) for 2 h at 37 °C, washed and then incubated with an avidin peroxidase conjugate solution (1:100, Santa Cruz Biotechnology) for 1 h. Finally, the sections were developed with diaminobenzidine for 5 min. Negative controls were similarly processed but without the primary antibody.

The vascular density and the number of CD31-positive cells in the injured brain were measured by vWF and CD31 immunofluorescence staining. Here, five brain tissue sections at 50-mm intervals were analyzed under a light microscope (200×, Leica). The vWF-positive vessels in the boundary zone of the lesion and in the injured hippocampus were counted. The vascular density and the number of CD31-positive cells in both regions were determined by dividing the immuno-reactive vessels by the corresponding area [11]. Data presented as total number of vessels or CD31 per mm.

The BBB permeability was evaluated by Evans blue (EB) dye extravasation. Two days after MCAO, EB (2.5%, 2 ml/kg) was injected through the femoral vein 1 h before euthanasia. The brains were harvested after perfusion with 4% paraformaldehyde, and were then incubated in dimethylformamide at 60 °C for 24 h. Then the brain tissue was homogenized and centrifuged at 1000 rpm for 5 min. Standard curve was drawn by testing the absorbance of gradient-dosed Evans blue. The absorbance of the supernatant was detected at 625 nm to calculate the relative content of EB. Brain water content was measured by the dry-wet method as previously reported and was expressed as a percentage of wet weight [12].

For quantitative real-time polymerase chain reaction (qRT-PCR), Total RNA was extracted from the frozen brain tissue described above with Trizol reagents (Roche, Germany). In addition, the concentration of total RNA was measured by an OD of 260 nm on a Smart Spec™ plus spectrophotometer (Bio-Rad, Hercules, CA). RNA was reversely transcribed to complementary DNA (cDNA) using a Transcriptor First Strand cDNA Synthesis Kit (Roche, Germany). qRT-PCR was performed according to the instructions of the manufacture’s amplification kit (Roche, Germany), using the SYBR Green method on an ABI 7500 instrument (Applied Biosystems, Foster City, CA). The RT-PCR amplification was performed with a volume of 50 μl using a ready-to-use qRT-PCR mix (Roche, Germany). All reactions were performed in triplicate. The messenger RNA (mRNA) levels were normalized to the endogenous reference gene GAPDH. Relative quantification was achieved by the comparative 2^−ΔΔct method as described [13]. The sequences of the primers used for RT-PCR are shown below (Tab. 1).

The baseline escape latency in the spatial probe test among the five groups before sham operation and focal cerebral I/R injury (− 24 h) was not significantly different (P > 0.05, n = 6, Fig. 1a). Compared with the escape latency of the sham group, the escape latency of the MCAO group in the spatial probe test on day 2 to 7 after focal cerebral I/R injury was significantly longer (Sham vs. MCAO: 48.58 ± 5.99 s vs. 86.56 ± 5.24 s, 27.23 ± 5.82 s vs. 62.76 ± 5.53 s, 18.76 ± 5.14 s vs. 42.39 ± 5.91 s, 9.10 ± 4.41 s vs. 34.09 ± 4.89 s, 6.03 ± 2.18 s vs. 29.47 ± 3.05 s, 3.11 ± 1.67 s vs. 18.96 ± 3.55 s; P < 0.05, n = 6, Fig. 1b), and the time spent in the target quadrant and number of platform crossings in the hidden platform test were dramatically lower in the MCAO group than in the sham group (Sham vs. MCAO: 46.29 ± 2.51 s vs. 26.66 ± 2.32 s and 10.17 ± 1.94 vs. 4.67 ± 2.16; P < 0.05, n = 6, Fig. 1c and d). In the HPX group, compared with the measures in the vehicle group, the escape latencies in the spatial probe test on day 2 to 7 after focal cerebral I/R injury were significantly lower (Vehicle vs. HPX: 84.32 ± 5.01 s vs. 64.01 ± 5.98 s, 60.37 ± 5.01 s vs. 40.22 ± 5.62 s, 40.72 ± 5.59 s vs. 28.61 ± 5.55 s, 32.67 ± 4.22 s vs. 22.80 ± 4.12 s, 27.53 ± 3.44 s vs. 13.34 ± 3.78 s, 16.32 ± 3.79 s vs. 6.87 ± 3.03 s; P < 0.05, n = 6, Fig. 1b), and the time spent in the target quadrant and number of platform crossings in the hidden platform test were dramatically increased (Vehicle vs. HPX: 26.96 ± 2.13 s vs. 39.00 ± 2.69 s and 4.50 ± 1.52 vs. 7.17 ± 2.14, respectively; P < 0.05, n = 6, Fig. 1c and d). The escape latencies in the spatial probe test on day 2 to 7 after focal cerebral I/R injury in the HPX + ZnPPIX group were obviously longer than those in the HPX group (HPX vs. HPX + ZnPPIX: 64.01 ± 5.98 s vs. 85.76 ± 4.51 s, 40.22 ± 5.62 s vs. 62.03 ± 5.01 s, 28.61 ± 5.55 s vs. 43.44 ± 4.03 s, 22.80 ± 4.12 s vs. 33.65 ± 3.98 s, 13.34 ± 3.78 s vs. 28.87 ± 3.47 s, 6.87 ± 3.03 s vs. 17.77 ± 2.89 s; P < 0.05, n = 6, Fig. 1b), and the time spent in the target quadrant and number of platform crossings in the hidden platform test were both dramatically decreased in the HPX + ZnPPIX group (HPX vs. HPX + ZnPPIX: 39.00 ± 2.69 s vs. 26.86 ± 2.13 s and 7.17 ± 2.14 vs. 4.83 ± 1.33, respectively; P < 0.05, n = 6, Fig. 1c and d). However, there were no significant differences in the escape latency, time spent in target quadrant and number of platform crossings between the MCAO and vehicle group (MCAO vs. Vehicle: P > 0.05, n = 6, Fig. 1b, c and d).

Compared with the sham group, the MCAO group exhibited an increase in HO-1 protein expression (Sham vs. MCAO: 2.16 ± 0.86 vs. 5.33 ± 1.45, P < 0.05, n = 6, Fig. 2) in the ischemic penumbra on day 7 after focal cerebral I/R injury. In the HPX group, compared with the protein expression of HO-1 in the MCAO group and vehicle group, the protein expression of HO-1 (MCAO vs. HPX: 3.28 ± 1.07 vs. 8.53 ± 2.01, P < 0.05; Vehicle vs. HPX: 3.16 ± 1.15 vs. 8.53 ± 2.01, P < 0.05, n = 6, Fig. 2) were markedly higher. However, there were no significant differences between the MCAO and vehicle group in the protein expression levels of HO-1 (MCAO vs. Vehicle: P > 0.05, n = 6, Fig. 2).

Compared with the sham group, the MCAO group exhibited a higher new vessel density in the ischemic penumbra hippocampus areas at 7 days after focal cerebral I/R injury (Sham vs. MCAO: 1.17 ± 0.75 vs. 3.67 ± 1.37; P < 0.05, n = 6, Fig. 3a and b). The new vessel density of the ischemic penumbra hippocampus area at 7 days after focal cerebral I/R injury witnessed a marked growth in those treated with HPX compared with the new vessel density in those in the MCAO group treated with vehicle (Vehicle vs. HPX: 4.00 ± 1.41 vs. 9.17 ± 2.48; P < 0.05, n = 6, Fig. 3a and b). When the inhibitor of HO-1, ZnPPIX, was given along with HPX to rats subjected to focal cerebral I/R injury, the new vessel density of the ischemic penumbra hippocampus area at 7 days after focal cerebral I/R dropped substantially compared with the respective densities in the HPX group (HPX vs. HPX + ZnPPIX: 9.17 ± 2.48 vs. 3.33 ± 1.97; P < 0.05, n = 6, Fig. 3a and b). However, the new vessel density of the ischemic penumbra hippocampus area at 7 day after focal cerebral I/R injury in the vehicle group was not significantly different from that in the MCAO group (P > 0.05, n = 6, Fig. 3a and b).

We evaluated the effect of HPX given via intracerebroventricular injection on alleviating the structural and functional impairment of the BBB by assessing BBB permeability, brain integrity, and new vessel stability at 7 days after focal cerebral I/R injury. Compared with that observed in the sham group, the extravasation of Evans blue dye, which is indicative of the BBB permeability, in the MCAO group increased significantly (Sham vs. MCAO: 0.23 ± 0.14 vs. 1.77 ± 0.45; P < 0.05, n = 6, Fig. 4a). Furthermore, the extent of brain edema measured by water content, which is indicative of BBB integrity, in rats subjected to focal cerebral I/R injury was more severe than in the sham group (Sham vs. MCAO: 72.33 ± 2.66 vs. 84.50 ± 5.65; P < 0.05, n = 6, Fig. 4b), and the Ang1/Ang2 ratio, which is associated with the vascular stability, was substantially reduced (Sham vs. MCAO: 3.87 ± 0.76 vs. 0.67 ± 0.27; P < 0.05, n = 6, Fig. 4c). VE - cadherin plays an important role in endothelial cell migration and survival, angiogenesis and maintaining the integrity of the blood vessels [14]. In our study, VE - cadherin expression decreased after MCAO in contrast to the sham group (Sham vs. MCAO: 1.13 ± 0.11 vs. 0.54 ± 0.13; P < 0.05, n = 6, Fig. 4d).

When compared with that observed in rats treated with vehicle, the extravasation of Evans blue dye (Vehicle vs. HPX: 1.85 ± 0.48 vs. 0.97 ± 0.29; P < 0.05, n = 6, Fig. 4a), the water content (Vehicle vs. HPX: 84.33 ± 6.98 vs. 78.33 ± 3.27; P < 0.05, n = 6, Fig. 4b) and VE - cadherin levels (Vehicle vs. HPX: 0.59 ± 0.14 vs. 0.96 ± 0.23; P < 0.05, n = 6, Fig. 4d) in HPX group were much higher than vehicle group, and the Ang1/Ang2 ratio was obviously increased (Vehicle vs. HPX: 0.57 ± 0.36 vs. 2.47 ± 0.55; P < 0.05, n = 6, Fig. 4c) by HPX injection treatment at 7 days after focal cerebral I/R injury. Similarly, these changes were consistent when the MCAO group and HPX group were compared. In rats that received both HPX and ZnPPIX, the antagonist of HO-1, the positive effects of HPX on BBB function were blocked. Specifically, the extravasation of Evans blue dye (HPX + ZnPPIX vs. HPX: 0.97 ± 0.29 vs. 1.83 ± 0.43; P < 0.05, n = 6, Fig. 4a), the water content (HPX + ZnPPIX vs. HPX: 78.33 ± 3.27 vs. 85.17 ± 4.02; P < 0.05, n = 6, Fig. 4b) and the VE - cadherin levels (HPX + ZnPPIX vs. HPX: 78.33 ± 3.27 vs. 85.17 ± 4.02; P < 0.05, n = 6, Fig. 4d) were significantly higher, the Ang1/Ang2 ratio was obviously lower (HPX + ZnPPIX vs. HPX: 0.61 ± 0.0.08 vs. 0.96 ± 0.23, P < 0.05, n = 6, Fig. 4c) in the HPX + ZnPPIX group than in the HPX group. However, the extravasation of Evans blue dye, the water content, the Ang1/Ang2 ratio and the VE - cadherin protein levels did not differ significantly between the MCAO and vehicle groups (P > 0.05, n = 6, Fig. 4a-d).