Background Erythropoietin (EPO) functions as a tissue-protective cytokine in addition to its crucial hormonal role in red cell production and neuron protection. This study aimed to determine the neuron protective effect of erythropoietin on experimental rats enduring spinal cord injury (SCI) by assessing thrombospondin-1 (TSP-1) level and transforming growth factor-β (TGF-β) in the development of a rat model of SCI.

Methods Sixty Sprague-Dawley rats were randomly assigned to three groups: sham operation control group, SCI group and EPO treatment group. By using a weight-drop contusion SCI model, the rats in the SCI group and EPO treatment group were sacrificed at 24 hours and 7 days subsequently. The Basso, Beattie, and Bresnahan (BBB) scores were examined for locomotor function. Pathological changes were observed after HE staining. The expressions of thrombospondin-2 (TSP-1) and TGF-β were determined by immunohistochemical staining and Western blotting .

Results Slighter locomotor dysfunction was discovered and it was recovered abruptly as higher BBB scores were found in the EPO treatment group than in the SCI group (P <0.01). Pathologically, progressive disruption of the dorsal white matter and regeneration of a few neurons were also observed in SCI rats. TSP-1 and TGF-β expression increased at 24 hours and 7 days after SCI in the injured segment, and it was higher in the SCI group than in the EPO treatment group. Spinal cord samples from the animals demonstrated a TSP-1 optical density of 112.2±6.8 and TSP-1 positive cells of 5.7±1.3 respectively. After injury, the TSP-1 optical density and cell number increased to 287.2±14.3/mm² and 23.2±2.6/mm² at 24 hours and to 232.1±13.2/mm² and 15.2±2.3/mm² at 7 days respectively. When EPO treated rats compared with the SCI rats, the TSP-1 optical density and cell number decreased to 213.1±11.6/mm² and 11.9±1.6/mm² at 24 hours and to 189.9±10.5/mm² and 9.3±1.5/mm² at 7 days, respectively (P <0.01). In the SCI rats, the TGF-β optical density and positive neuron number were 291.4±15.2/mm² and 28.8±4.9/mm² at 24 hours and 259.1±12.3/mm² and 23.9±4.1/mm² at 7 days respectively. They decreased in the EPO treated rats to 222.8±11.9/mm² and 13.7±2.1/mm² at 24 hours and to 196.5±9.7/mm² and 8.7±2.2/mm² at 7 days (P <0.01).

Conclusions Increased expression of TSP-1 and TGF-β can be found in the injured segment of the spinal cord at 24 hours and 7 days after injury. EPO treatment can effectively prevent pathological alterations from severe spinal cord injury by reduced expression of TSP-1 and TGF-β.

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Spinal cord injury (SCI) is a severe complication worldwide and it usually causes life-long disability for the patients.¹ In the state of South Carolina, the rate of SCI mortality was 27.4 per million population between 1981 and 1998.² An ideal treatment would be one that is administered systemically and without significant side-effects.³ Erythropoietin (EPO) has recently been shown to play roles in the nervous system in both normal and pathological conditions.⁴ The evidence for EPO as a potent inhibitor of apoptosis and a promising therapeutic agent in a variety of neurological insults including trauma is mounting. With the recent interest in clinical trials of EPO in stroke, it is both timely and prudent to consider the use of this pharmaceutical avenue in traumatic brain injury in humans.⁵ Traumatic spinal cord injury results in disruption of the neural and vascular structures and then damage to the intact neighboring tissue.⁶

Thrombospondin-1 (TSP-1) has been recognized the best member of the family of five isoforms which are termed as matricellular proteins with multiple biological functions.⁷ It is one of the major physiological activators of transforming growth factor-β (TGF-β) in vivo, and accounts for 60% of the total TGF-β activity released.⁸ The role of TSP-1 and TGF-β in brain injury has been suggested,^9,10 including angiogenesis, scar deposition, inflammation, anti-inflammation, neuroprotection, and influence on astrocyte phenotype and mobility. Erythropoietin is found to decrease renal fibrosis in mice by inhibiting TGF-beta-induced epithelial-to-mesenchy- mal transition. Therefore, identifying the specific molecular pathway mediating the EPO neurons protection against SCI would be of great value to the patients. In the present study, we employed weight-drop SCI rats to assess the expression of TSP-1 and TGF-β at different time points after SCI in EPO treated SCI rats, attempting to investigate their role in protection of EPO after SCI.

Animals
Sprague-Dawley (SD) female rats were purchased from the Experimental Animal Center of Zhejiang University. Monoclone TSP-1 and polyclone TGF-β antibodies were purchased from Oncogene Company, USA. The NIH Principles of Laboratory Animal Care and the China National Regulations for Experimental Animal Care were followed in the study. All experiments and procedures were approved by the Committee on Animal Care and Use of Zhejiang University.

Animal model
Sixty Sprague-Dawley female rats weighing 230–250 g were randomly assigned to three groups: sham-operation control group, SCI group, and EPO treatment group. Under anesthesia of sodium pentobarbital (40 mg/kg, i.p.), the vertebral column of the rats was exposed and a laminectomy was performed at the T10 level. A contusion injury was then made using a weight-drop device in rats of the SCI group and EPO treatment group. A weight of 10 g was dropped from a height of 50 mm on the exposed spinal cord, and the impounder left for 20 seconds before being withdrawn to produce a moderate contusion. In the SCI group, normal saline (via i.p. injection) was given immediately after enclosure of the incision. The rats of the EPO treatment group received single doses of EPO (1000 units/kg, i.p.) immediately after the incision was closed. The control group animals received the same surgical procedure but sustained no impact injury and their spinal cord was left open for 5 minutes. Following the contusion, the incision was closed in layers and penicillin G (40 000 IU for each, i.m.) was injected. In all animals surgery was performed in an aseptic manner on a warming pad set at 37°C. The rats had their bladders expressed at least twice a day until self-empty was apparent. Each rat was scored using the Basso, Beattie, Bresnahan (BBB) Locomotor Rating Scale at 24 hours and 7 days after operation. The rats were sacrificed at 24 hours and 7 days for subsequent histological study, immunohistochemistry assay, and Western blotting respectively.

Immunohistochemistry and HE staining
Immunohistochemistry was used to localize the TSP-1 and TGF-β protein. Five rats in each group were sacrificed by pentobarbital overdose at the 24 hours and 7 days after injury. Subsequently, their thoracic cavities were opened and perfused intracardially with normal saline. Following saline perfusion, the rats were perfused with 300–400 ml of fixative solution containing 4% paraformaldehyde in 0.1 mol/L phosphate buffer (pH 7.4). After perfusion, the injured spinal cord of each rat was taken out. The tissues of the injured spinal cord were fixed in the same fixative solution for 4 hours and then placed in 30% phosphate buffered sucrose until tissue sinking. Eight-micrometer-thick sections were cut on freezing microtome through the coronary planes of the heart. The sections were then rinsed in 0.01 mol/L phosphate-buffered saline (PBS) and mounted onto 0.02% poly-L-lysine-coated slides.

The SABC system was used with 3,3′-diaminobenzidine hydrochloride (DAB) as the chromagen. Briefly, tissue sections were washed in PBS, incubated in 1% bovine serum albumin (BSA) for 30 minutes, and then incubated overnight at 4°C in the primary antibody (monoclone TSP-1 antibody, or polyclone TGF-β antibody) plus 1% BSA in PBS. The primary antibody was diluted at a ratio of 1:150. Control sections were incubated in PBS. The next day, the sections were incubated in a biotinylated goat anti-mouse secondary antibody (diluted to 1:200 in PBS), and subsequently in an avidin-HRP solution. Immunolabeling was visualized with 0.05% DAB plus 0.3% H₂O₂ in PBS. The sections were then dehydrated through ethanol and xylene before coverslips with permount. HE staining was also applied to determine morphological changes.

Western blotting
The 1–2 mm injury point of the spinal cord was used for Western blotting. The rest 5 rats per group at 24 hours after injury were sacrificed and the samples were taken and lysed in lysis buffer containing 50 mmol/L Tris-Cl (pH 8.0), 150 mmol/L NaCl, 0.02% sodium azide, 100 µg/ml phenylmethylsulfonyl fluoride, 1 µg/ml aprotinin, and 1% Triton X-100. After centrifugation at 12 000 r/min, 50 µg of total protein of each sample was loaded into a 12% SDS-PAGE gel and transferred to nitrocellulose membrane (Amersham Biosciences Inc., Piscataway, USA). The blocked membranes were then incubated with the indicated antibody (monoclone TSP-1 antibody, or polyclone TGF-β antibody), and the immunoreactive bands were visualized using the chemiluminescent reagent recommended by the Supersignal West Dura Extended Duration Substrate kit (Pierce Chemical, Rockford, USA). The signals of the bands were quantified using the GS-710 calibrated imaging densitometer (Bio-Rad, USA). The results were expressed as a relative density. Equal protein loading in each lane was confirmed by hybridization with a 1:2000 dilution of β-actin antibody (Santa Cruz Biotechnology, Inc., USA).

Image analysis and statistical analysis
For immunohistochemistry, each slide was examined at a magnification of 400 times and analyzed with UTHSCSA Image Tool 3.0 (University of Texas Medical School at San Antonio, TX, USA). The number and optical density of TSP-1 and TGF-β positive cells were measured. The protein concentration was determined using Bio-Rad Quantity One 4.6.2 (Bio-Rad). All data were analyzed by Student′s t test. The data were expressed as mean ± standard deviation (SD). A P value <0.05 was considered statistically significant.

The rats with SCI demonstrated dramatic and bilateral hind limb paralysis with no movement at all or only slight movement of a joint from one hour after injury while open-field walking. The rats in the sham operation group walked normally after recovery from the anesthesia. The rats were followed up for 24 hours and 7 days respectively after surgery to score their locomotor activity according to the BBB scale. The locomotor dysfunction was reproducible and the BBB score recovered to a plateau below the range of 10 points, indicating that the contusion injury produced was of moderate severity and quickly recovered in the EPO treated rats (P <0.01) (Figure 1). Consistently, HE staining of the lesioned spinal cord revealed the formation of a large cavity involving the dorsal and partially the lateral funiculi and also the dorsal and central gray matter. The central lesion area consisted of spared fibers, variable cyst formations and gliosis. Twenty-four hours after injury, blood cells, some dead neurons and increased number of gliocytes were seen in the spinal cord, but they were comparatively less in the EPO treated rats. There was a progressive disruption of tissue architecture of the dorsal white and central gray matter, a gliocyte filling in the injury area 7 days after injury, and a reduction in the volume of cavitation in the EPO treatment rats. More fiber sprouting and neuron bodies were observed in the gray matter around the lesion, indicating more neuron regeneration in the EPO treated rats than in the SCI rats 7 days after SCI. Moreover, the lesions were severe in the SCI group than in the EPO treated group (Figure 2).

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Figure 1. BBB scores in the three groups at 1 day and 7 days after SCI.

In the spinal cord of the injured rats, TSP-1 and TGF-β immunohistochemistry staining showed positive cells with buffy granules as shown by DAB staining. Predominantly the neurons (distinguished from glial cells by a relatively lager nucleus, prominent cytoplasm, and the pericellular space created by shrinkage) demonstrated increased TSP-1 expression compared to the sham- operated group. The samples of the spinal cord from the animals demonstrated a TSP-1 optical density of 112.2±6.8 and the number of TSP-1 positive cells 5.7±1.3 respectively. After injury, the TSP-1 optical density and the cell number increased to 287.2±14.3 and 23.2±2.6/mm² at 24 hours, 232.1±13.2 and 15.2±2.3/mm² at 7 days. The TSP-1 optical density and the cell number decreased to 213.1±11.6 and 11.9±1.6/mm² at 24 hours, and to 189.9±10.5 and 9.3±1.5/mm² at 7 days in the EPO treated rats, compared with the SCI rats (P <0.01). In the SCI rats, the TGF-β optical density and the number of positive neurons were 291.4±15.2 and 28.8±4.9/mm² respectively at 24 hours, and 259.1±12.3 and 23.9±4.1/mm² respectively at 7 days. They decreased in the EPO treated rats to 222.8±11.9 and 13.7±2.1/mm² respectively at 24 hours, and 196.5±9.7 and 8.7±2.2/mm² respectively at 7 days (P <0.01). The TSP-1 and TGF-β optical density and the number of positive neurons peaked in the injured segment at 24 hours (Figures 3 and 4).

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Figure 2. HE staining of the spinal cord segment 24 hours and 7 days after injury. A: sham-operated rat (24 hours after injury); B: EPO treated rat (24 hours after injury); C: SCI rat (24 hours after injury); D: Sham-operated rat (7 days after injury); E: EPO treated rat (7 days after injury); F: SCI rat (7 days after injury). Twenty-four hours after injury blood cells, some dead neurons and an increased number of gliocytes were observed in the spinal cord, but they were comparatively less in the EPO treated rats (Figure 2B and 2C). There was a progressive disruption of tissue architecture of the dorsal white and central gray matter, a gliocyte filling in the injured area 7 days after injury and a reduction in the volume of cavitation in the erythropoietin treated rats (Figure 2E and 2F). Figure 3. TSP-1 immunohistochemistry staining in the spinal cord segment. A: sham-operated rat (24 hours after injury); B: EPO treated rat (24 hours after injury); C: SCI rat (24 hours after injury); D: sham-operated rat (7 days after injury); E: EPO treated rat (7 days after injury); F: SCI rat (7 days after injury). TSP-1 immunohistochemistry staining showed positive cells, predominantly neurons (distinguished from glial cells by a relatively lager nucleus, prominent cytoplasm, and the pericellular space created by shrinkage). After injury, TSP-1 optical density and cell number increased in the SCI rats (Figure 3C and 3F). In the EPO treated rats, the TSP-1 positive optical density and cell number decreased (Figure 3B and 3E). Figure 4. TGF-β immunohistochemistry staining in the spinal cord segment. A: sham-operated rat (24 hours after injury); B: EPO treated rat (24 hours after injury); C: SCI rat (24 hours after injury); D: sham-operated rat (7 days after injury); E: EPO treated rat (7 days after injury); F: SCI rat (7 days after injury). After injury, the TGF-β optical density and the cell number increased in the SCI rats (Figure 4C and 4F). In the EPO treated rats, the TGF-β positive optical density and the cell number decreased (Figure 4B and 4E).

Concentrations of TSP-1 and TGF-β protein obtained from the injured segment were significantly increased than those of the sham operation group (P <0.01). The relative intensity of TSP-1 was 100.0%, 191.1% and 268.2%, and those of TGF-β was 100.0%, 179.4% and 287.5% in the sham operated group, EPO treatment group and SCI group at 24 hours after SCI (Figure 5).

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Figure 5. Western blotting of TGF-β and TSP-1 protein concentrations in the spinal cord segment.

SCI has a high morbidity and mortality in athletes, drivers, and passengers.^11,12 It is diverse in causes as well as pathological and functional consequences. There are several animal models mimicking human SCI including trauma, ischemia and inflammation. The modeling of such a lesion, however, is necessarily parallel to the pursuit of clinical interventions that will interrupt the cytopathology of chronic spinal destruction. In models where such information is available, the experimental biomechanical descriptors of the injury process are able to predict the subsequent acute and chronic patho- physiology of the spinal cord. A weight-drop contusion SCI model is commonly used in research into acute SCI and can serve as an adequate animal model for the study of functional and morphological changes after SCI and the effects of new treatment strategies.^13,14

Several treatments of SCI have been developed in the last two decades. The present study demonstrated that there is a neurological benefit associated with EPO injection after SCI. A single dose of EPO was found to be associated with a marked clinical course of motor function recovery, characterized by BBB scores in a period of 7 days despite dramatic differences in the histological level after the period. A significant hemorrhage occurred after the severe parenchymal disruption caused by absorption of kinetic energy. In the later stages of response to the injury caused by contusion, cellular debris and grossly disrupted axons elicited pronounced inflammatory and degenerative processes that initiate a secondary phase of injury, which could be reduced by EPO treatment. Immunohistochemistry study and Western blotting revealed that TSP-1 and TGF-β expressions increased after injury, but decreased in the EPO treatment rats.

TSP-1, a member of the matricellular family, can regulate cell proliferation, migration and apoptosis in a variety of physiological and pathological conditions including wound healing, inflammation, angiogenesis and neoplasia.^15,16 It also inhibits angiogenesis,¹⁷ maybe due to local vascular alterations and ischemia in the spinal cord, which are the most important aspects of secondary injury.¹⁸ TGF-β is a potent regulatory cytokines involving in many biological processes.¹⁹ However, it is secreted in a biologically inactive form which is unable to interact with its receptors and elicit biological responses.²⁰ TSP-1 utilizes a two-step mechanism to activate latent TGF-β, which decreases the accumulation of mononuclear phagocytes in and around the injury site, while reducing the possibility of secondary injury.²¹ After SCI, TGF-β signaling may be crucial to inflammatory gene synthesis in healing of the injury by mediating the resolution of inflammatory infiltrate. EPO has been shown to protect neuronal cells in vitro not only from apoptosis induced by hypoxia but also from excitotoxins and glucose deprivation.⁴ Recent studies have also revealed that EPO suppresses the up-regulated expressions of TGF-β and increases the production of collagen induced by angiotensin II in rat cardiac fibroblasts²² or decreases renal fibrosis in mice by inhibiting TGF-β.²³ The present study revealed that EPO effectively prevents pathological alterations and severe spinal cord injury by lowering the expression of TSP-1 and TGF-β. However, further studies are needed to find the underlying mechanism of the treatment.

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