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of hypothermia therapy, there are not effective the-rapies so far to reduce brain damage in infants2.

In recent years, several studies have shown that the erythropoietin (EPO) has neuroprotecti-ve activities on hypoxic-ischemic brain damage (HIBD)3-6. EPO is functioning in decreasing the apoptosis, reactive oxygen species, glutamate excitotoxicity and inflammation, and then stimu-lating the neurogenesis and angiogenesis7.

Mounting evidence has shown that the metal-loprotein kinases (MMPs), especially the MMP-2 are dramatically increased in the adult brain after cerebral ischemia8. Moreover, MMP-2 shows an increased activity during the HIBD in the imma-ture brain and contributes to the protective role in the brain injury9. On the other hand, MMP-2 seems to be correlated with the protective effects of EPO on focal cerebral ischemia10.

The aim of this paper was to determine the role of EPO in HIBD induced MMP-2 expression, and to elucidate the role of EPO/MMP-2 after neona-tal hypoxia-ischemia.

Materials and Methods

Animal Preparation7-day-old (P7) Sprague-Dawley rats of each

gender with weight of 8-15 g were purchased from the Animal Research Center of Taishan Medical College (Taian, Shandong, China). This study was approved by the Animal Ethics Committee of the Animal Center of Central Hospital Taian City (Taian, Shandong, China).

Experimental Neonatal Hypoxia-IschemiaCerebral hypoxia-ischemia was induced by a uni-

lateral permanent ligation of the common carotid ar-tery; then systemic hypoxia was induced3. Besides,

Abstract. – OBJECTIVE: Erythropoietin (EPO), as a type of the tissue-protective cytokines, is a 30.4 kDa hematopoietic glycoprotein. The pur-pose of this study was to explore the neuropro-tective effects of EPO on the neonatal hypox-ic-ischemic-induced hippocampus injury and the MMP-2 expression.

MATERIALS AND METHODS: Neonatal Sprague-Dawley (SD) rats were randomly di-vided into an untreated group (control) and two hypoxia-ischemia (HI) groups treated with sa-line control or EPO. Hippocampi were harvest-ed at various times after return to normoxia (6 h, 24 h, 3 days and 7 days post-HI) for analy-ses of infarct areas and expression using his-tology, Western blot and reverse transcrip-tase-polymerase chain reaction (RT-PCR).

RESULTS: EPO injections reduced the infarc-tion and loss of brain tissue. HI group exhibited an enhanced MMP-2 positive staining compared to controls at 24 h, 3 and 7 days post-HI by immu-nohistochemistry. These results were confirmed by Western blot analysis of MMP-2 expression at 7 days post-HI. Levels of MMP-2 mRNA in the in-jured hippocampi increased significantly at 24 h and 7 days post-HI. In particular, the EPO treat-ment further significantly enhanced this increase.

CONCLUSIONS: EPO protected hypoxic-isch-emic-induced neonatal brain damage by up-reg-ulating the MMP-2 expression. Hence, systemic EPO may have potential utility for the treatment of HI injury in human newborns.

Key Words: Erythropoietin, Hippocampus, Hypoxia-ischemia

brain, Matrix metalloproteinase 2.

Introduction

In all causes of neonatal death and neurologi-cal injury, hypoxic-ischemic (HI) damage still re-mains the major cause1. Due to severe limitations

European Review for Medical and Pharmacological Sciences 2017; 21: 4327-4332

L. ZHANG1, L. WANG2, F.-B. NING1, T. WANG1, Y.-C. LIANG1, Y.-L. LIU1

1Department of Neurology, Central Hospital of Taian City, Shandong Province, China2Department of Intervention Radiology, Central Hospital of Taian City, Shandong Province, China

Corresponding Author: Yunlin Liu, MD; e-mail: fulang3086217@163.com

Erythropoietin reduces hippocampus injury in neonatal rats with hypoxic ischemic brain damage via targeting matrix metalloprotein-2

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rat pups were anesthetized with sodium with 4% pentobarbital (40 mg/kg, i.p.). After the anesthesia, the midline neck was incised in rat pups and then the left common carotid artery was separated and cut off. The Sham operated animals experienced the same surgical procedure without the ligation of the carotid artery. After the surgery, the rat pups were sent to their primary home cage to recover for 1 h. Hypoxia was then caused by subjecting the animals in a special chamber (37 °C) with humidified nitro-gen mixture of 8% oxygen for 2 h. The age-matched sham-operated animals were the controls. Pups which underwent hypoxia-ischemia were random-ly assigned to the control group (vehicle treatment) and the EPO group. EPO group was injected intra-peritoneally with recombinant human erythropoie-tin (rhEPO, Beijing Sihuan Biopharmaceutical Co., Ltd., Beijing, China) at a dose of 5000 U/kg. Control group was injected intraperitoneally with an equal volume of saline. Animals were sacrificed at speci-fic time points (6 h, 24 h, 3 days and 7 days) after the injury (12 rats per group and time point).

Tissue PreparationAt scheduled time points, anesthetized animals

were first transcardially perfused by phosphate buffered saline (PBS) with 4% paraformaldehyde. Then the brains were quickly removed and sub-merged in paraformaldehyde for 12 h at 4°C and embedded in paraffin. For biochemical analysis, animals were sacrificed through decapitation and the hippocampi were stored at -80 °C.

Nissl StainingCoronal sections (including the hippocampus)

were cut (4 μm thick), and then deparaffinized in xylene, dehydrated in gradient ethanol, and final-ly washed with PBS. The sections were stained followed by the manufacturer’s protocols with 0.5% cresyl violet acetate (Sigma-Aldrich, St. Louis, MO, USA).

Immunohistochemical AnalysisThe expression of MMP-2 in hippocampus

was detected by immunohistochemical staining. Coronal sections (including the hippocampus) (4 μm thick) were deparaffinized, dehydrated and washed by phosphate buffered saline (PBS). The sections were incubated in 10 mM EDTA (pH 8.0) and heated 2 min at 100°C. And the sections were pretreated with 3% H2O2 in PBS for 15 min and blocked with 5% BSA at 37°C, then dipped in the mouse anti-MMP-2 (1:100, Abcam, Cambridge, MA, USA) at 4°C overnight. On the next day, the

experiment with the immunohistochemical stai-ning kit (Zhongshan, Beijing, China) was con-ducted. The sections were firstly washed and then stained with 3,3-diaminobenzidine and finally counterstained with hematoxylin. We considered the brown staining was apparent in the cytoplasm as positive MMP-2 expression.

Evaluation of ImmunohistochemistryThe number of positively stained cells was

counted in each of the 5 randomly selected con-secutive fields under 400-fold magnification. Considering the staining diffuseness, the stained sections areas were grouped as the following cri-terias: 0: no staining, 1: < 25% of the area stai-ned, 2: 25% to 50% of the area stained, 3: 50% to 75% of the area stained, and 4: >75% of the area stained. In consideration of the staining intensity, the sections were graded as the following crite-ria: 0: no staining, 1: weak but detectable staining above the control level, 2: distinct staining, and 3: intense staining. Total IHC scores were acquired by addition of staining intensity and diffuseness scores. In the hippocampus samples, scores <1.5 were considered negative staining, whereas sco-res >1.5 were considered positive staining.

Western BlotAfter treatment, the hippocampus was rinsed

with ice-cold phosphate buffered saline (PBS) and subjected to total protein extraction with radioim-munoprecipitation assay (RIPA) lysis buffer. The protein concentrations were quantified by the bi-cinchoninic assay (BCA) method. 30 mg protein samples were run on 10% gels, and then transferred to the PVDF membrane. After 1 hour of blocking with the 5% skim milk, the membranes were in-cubated with the primary mouse anti-MMP-2 an-tibody (1:1000, Abcam, Cambridge, MA, USA), the rabbit anti-GAPDH antibody (1:1000, Ab-cam, Cambridge, MA, USA) overnight at 4 °C. After washing in tris-buffered saline and Tween 20 (TBST) for 3 times, the membranes were then incubated with a peroxidase (HRP)-labeled secon-dary antibody (1:5000, Santa Cruz Biotechnology, Santa Cruz, CA, USA) for 2 h. The bands were wa-shed again, enhanced with chemiluminescence re-agents and visualized with the ChemiDocTM MP Imaging System (Bio-Rad, Hercules, CA, USA).

Quantitative Real-time PCRAfter treatment, the hippocampus was collected

to determine the expression of MMP-2 mRNA by qRT-PCR. Total RNA was extracted by the Tri-

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zol RNA extraction reagent (Invitrogen, Carlsbad, CA, USA) following the protocols and quantified by spectrophotometer method. Purified RNA with equal volume was reverse transcribed (RevertAid Fist Strand cDNA Synthesis Kit, Thermo, K1622, Waltham, MA, USA). The cDNA product was subjected to real-time quantification on an ABI PRISM 7000 Sequence Detection System (TaKa-Ra Biotechnology, Dalian, Liaoning, China), with the following PCR cycling: 95 °C for 20 s, followed by 39 cycles of 95 °C for 10 s and 60 °C for 20 s, then 70 °C for 10 s. Final extension was completed from 70 °C to 95 °C at 0.4 °C/ 5 s. The primers were designed as follows: MMp-2: Sense: 5’-GCC CAG AGA CTG CTA TGT CC-3’; Antisense: 5’-TC-GTAG TTG GTT GTG GTT GC-3’; GAPDH: Sen-se: TGG GTG TGAACC ACG AGAA-3’; Antisen-se: 5’-GGC ATG GAC TGTGGT CAT GA-3’. The relative expression of MMP-2 was measured by the comparative method with the formula 2-ΔΔCt and nor-malized with the corresponding GAPDH Ct values. All data presented three independent experiments.

Statistical AnalysisAll data were expressed as the mean ± standard

error of the mean (SEM), and were analyzed using a one-way analysis of variance followed by LSD

(Least Significant Difference). Statistical analysis was performed using SPSS 19.0 software (IBM, Armonk, NY, USA).

Results

EPO Attenuates Brain Injury in HI Rats Compared to the sham group, the treatment of

hypoxia significantly increased the infarction and loss of brain tissue (Figure 1 A, B). Both morphological and quantitative analysis revealed that the EPO treat-ment dramatically down-regulated the infarction and loss of brain tissue in comparison with those in the HI group (Figure 1 C, D). These results were further con-firmed by Nissl-staining in hippocampus sections. The Hypoxia-ischemia treatment significantly induced a cell loss, deformation and swelling of the neurons in HI rats, while EPO treatment partially reversed this dama-ge (Figure 1E). All the results have demonstrated that EPO attenuates brain injury in HI rats.

EPO Treatment Increased the MMP-2 Expression in HI Rats

As shown in Figure 2A, the MMP-2 positive cells were appeared to be dispersed throughout the hippocampus in sham animals. At 24 h after

Figure 1. Nissl-staining for the brain sections in 3 different groups. Coronal Nissl-stained sections for brains in SHAM (A), HIBD (B) and EPO (C) groups. Quantitative evaluation of infarcted areas (D); Nissl-staining for hippo-campus sections (E). (*Compared with Sham, p<0.05; # Compared with HIBD, p<0.05).

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HI, a marked increase in the number and inten-sity of MMP-2 positive staining cells was obser-ved in hippocampus in treatment with or without EPO animals. Quantitative analysis revealed that the treatment with HI significantly increased the MMP-2 protein levels in a time dependent manner. Furthermore, EPO treatment elevated the MMP-2 expression in HI rats at specific time points (24 h, 3 days and 7 days) after the injury (Figure 2B). These results indicated that EPO enhanced MMP-2 expression in HI rats. Increased level of MMP-2 in hippocampus after treatment with EPO for 7 days (Figure 3) further confirmed the results.

EPO Treatment Increased mRNA Level of MMP-2

As shown in Figure 4, the results of MMP-2 mRNA expression in hippocampus in HI rats

treated with or without EPO were similar to the MMP-2 immunochemical staining.

Discussion

This study showed that EPO and MMP-2 may be involved in the events induced by hypoxia ischemia in the newborn brain of rat. It also de-monstrated that the treatment with EPO enhan-ced the MMP-2 expression to HI challenge. The administration of EPO not only prevented brain injury in immature rat during HI, but also led to the increase of MMP-2 expression after injury.

MMPs contains a multigene family of zinc-de-pendent endopeptidases which participate in the healing, the inflammatory response and the an-giogenesis of many common tissues11. MMP-2, a

Figure 2. EPO treatment promoted MMP-2 expression in hippocampus sections after hypoxia-ischemia. A, Immunohisto-chemical staining of MMP-2 for different groups at different time points (× 400); B, Quantitative analysis of the immunohisto-chemical staining results. (*Compared with Sham, p<0.05; # Compared with HIBD, p<0.05).

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subfamily of MMPs, secreted by neurons, glial cells, vascular endothelial cells and neutrophils in the nervous system played an important role in pathological functions within the development of brain injury12. The enhanced activity of MMP-2 was associated with the breakdown of blo-od-brain barrier (BBB) and neuro-inflammatory response in the early phage of brain injury after focal and global brain ischemia. Furthermore, it was proved that the MMP-2 activity is expressed in wound healing by modulating the glial scar formation, which is associated with the involve-

ment in neural repair and spinal cord injury. It has been shown that MMP-2 was up-regulated during the brain injury after 7 days13. MMP-2 is considered to have functions in promoting the repair and extension of synapse by degrading glial scar, and taking part in neuroprotection14. Another work demonstrated a significant increase in both MMP-2 activity and VEGF levels during ischemia-reperfusion injury after 7 and 14 days in rats. It suggested that the increase of MMP-2 and secretion caused by treadmill training may be involved in the increase of neuroprotection whi-ch correlated with the angiogenesis increase. The timing of MMP-2 expression changed after HIBD in this study, which is similar to the time frame of MMP-2 activation previously observed in glial scar formation and angiogenesis.

Erythropoietin (EPO) is a hematopoietic glyco-protein produced in the fetal liver and adult ki-dney. It has been widely used in clinical practi-ce to treat anemia due to the beneficial effect on hematopoiesis15. Some recent researches16-18 have demonstrated that the EPO receptor (EPOR) was expressed in various tissues and cells, including neurons and glial cells. EPO may exert a neuro-protective effect through the enhancement of the EPOR levels on rats during hypoxic-ischemic brain damage. Intraperitoneal administration of recombinant human erythropoietin (rhEPO) re-sults in an attenuation of brain damage in HIBD19. In addition, the angiogenic, anti-inflammatory and endothelial cell stabilizating effects of EPO, including the improvement of the neurogenesis, cognitive and behavioral performance, have been

Figure 3. mRNA expression of MMP-2 in hippocampus for different groups. (*Compared with Sham, p<0.05; #Com-pared with HIBD, p<0.05).

Figure 4. Enhanced expression of MMP-2 was detected by Western blotting in hippocampus after treatment with EPO for 7 days. (*Compared with Sham, p<0.05; #Compared with HIBD, p<0.05).

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previously described20. EPO affects the angio-genesis and neurogenesis by activating various signaling pathways, including PI3K/Akt and ERK1/2 pathway.

Conclusions

We observed that the EPO treatment of HIBD protected hypoxic-ischemic-induced neonatal brain damage by up-regulating the MMP-2 expres-sion. The close correlation between the MMP-2 and EPO administration provided evidence for the possible effect of the EPO against HIBD.

Conflict of interestThe authors declare no conflicts of interest.

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