GART expression in rat spinal cord after injury and its role in inflammation

brain research 1564 (2014) 41–51

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Research Report

GART expression in rat spinal cord after injury and its role in inflammation Dongmei Zhanga,b,1, Ying Yuec,1, Shengyang Jiangc, Aihong Lid, Aisong Guod, Xinming Wud, Xiaopeng Xiae, Hongbing Chenge, Jinlong Zhangf, Tao Taoa,g,n, Xingxing Guc,nn a

Department of Pathogen Biology, Medical School, Nantong University, Nantong 226001, People's Republic of China Basic Medical Research Center, Medical School, Nantong University, Nantong 226001, People's Republic of China c The Jiangsu Key Laboratory of Neuroregeneration, Nantong University, Nantong 226001, People's Republic of China d Department of Neurology, Affiliated Hospital of Nantong University, Nantong 226001, People's Republic of China e Department of Orthopaedics, Traditional Chinese Medical Hospital of Nantong City, Nantong 226001, People's Republic of China f Department of Spine Surgery, The Second Affiliated Hospital of Nantong University, Nantong 226001, People's Republic of China g Department of Chemistry and Institutes of Biomedical Sciences, Fudan University, Shanghai 200433, People's Republic of China b

art i cle i nfo

ab st rac t

Article history:

The glycinamide ribonucleotide transformylase (GART) gene, a trifunctional polypeptide,

Accepted 28 March 2014

has phosphoribosylglycinamide formyltransferase, phosphoribosylglycinamide synthe-

Available online 4 April 2014

tase, and phosphoribosylaminoimidazole synthetase activity, and is required for de novo

Keywords: Spinal cord injury GART Rats Inflammatory

purine biosynthesis. GART is highly conserved in vertebrates. Alternative splicing of GART results in two transcript variants encoding different isoforms. However, the expression and function of GART in the central nervous system lesion are still unclear. In this study, we used a traumatic spinal cord injury (SCI) model in adult Sprague-Dawley rats and investigated the dynamic changes of GART protein expression in the spinal cord. Western blot analysis revealed that GART was present in sham-operated spinal cord. It gradually increased, reached a peak at day 3 after SCI, and then declined during the following days. Double immunofluorescence staining revealed a widespread of GART, and the majority of GARTs are detected in astrocytes. After injury, GART expression was increased predominantly in astrocytes, positively correlated with the highly expressed proliferating cell nuclear antigen (PCNA). Knockdown of GART expression in cultured primary astrocytes by siRNA revealed that expression of GART in astrocytes plays a role in the LPS-induced

Abbreviations: GART,

Glycinamide ribonucleotide transformylase; SCI,

Spinal cord injury; PCNA,

Proliferating cell nuclear antigen;

CNS, Central nervous system n Corresponding author at: Department of Pathogen Biology, Medical School, Nantong University, Nantong 226001, People's Republic of China. nn Corresponding author. Fax: þ86 51385051999. E-mail addresses: [email protected] (T. Tao), [email protected] (X. Gu). 1 Both authors contributed equally to this paper. http://dx.doi.org/10.1016/j.brainres.2014.03.044 0006-8993/& 2014 Elsevier B.V. All rights reserved.

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brain research 1564 (2014) 41–51

release of pro-inflammatory factors, such as TNF-α and IL-6. These results showed that GART may participate in the pathophysiology of SCI, and more research is needed to have a good understanding of its function and mechanism. & 2014 Elsevier B.V. All rights reserved.

1.

Introduction

Traumatic spinal cord injury (SCI) is a devastating and common neurological disorder that has profound influences on modern society from psychosocial, physical, and socioeconomic perspectives (McDonald and Sadowsky, 2002; Sekhon and Fehlings, 2001; Wyndaele and Wyndaele, 2006). SCI triggers the inflammatory response (Beattie, 2004) and astrocyte activation. It is widely accepted that activated asctrocytes release several kinds of proinflammatory cytokines, reactive oxygen (MunozFernandez and Fresno, 1998), proteases, and nitrogen radicals, which even lead to and exaggerate the neuronal dysfunction and cell death, if such inflammatory responses are not properly resolved (Hausmann, 2003). Astrocytes, as the major glial cell population in the central nervous system (CNS) (Rivieccio et al., 2005), are mainly responsible for CNS homeostasis (Aschner, 1998). After ischemic or traumatic brain insults, astrocytes undergo phenotypic changes (Ridet et al., 1997), proliferation, and cellular hypertrophy, which is called ‘astrogliosis’. Astrogliosis, a typical neuroinflammatory process, is a shared feature of many chronic and acute neurodegenerative diseases (Barcia et al., 2012; Serrano-Pozo et al., 2011). Astrocytes proliferation and reactive gliosis result in the formation of a dense astrocytic scar (McGraw et al., 2001; Raghupathi et al., 2004). This glial scar provides a physical and biochemical barrier to regeneration and plasticity, and acts as a source of multiple inhibitory factors that affect functional recovery from SCI (Davies et al., 1996; Silver and Miller, 2004). However, it remains to be further elucidated about the molecular mechanisms of post-traumatic pathology of spinal cord. Thus, exploring the molecular and cellular mechanisms of astrocytes activation and identifying the proteins involved will be helpful to understand the regeneration of the spinal cord following injury. The glycinamide ribonucleotide transformylase (GART) gene, located on chromosome 21, encodes the trifunctional enzyme of glycinamide ribonucleotide synthetase (GARS)– aminoimidazole ribonucleotide synthetase (AIRS)–glycinamide ribonucleotide transformylase (GART) (Brodsky et al., 1997). GARS–AIRS–GART catalyzes three independent steps in the de novo purine nucleotide biosynthesis pathway. GART was first discovered and partially characterized from pigeon liver in pioneering investigations by Warren and Buchanan (1957). Investigator reported that human tumor cells respond to de novo purine synthesis blockade by GART inhibitors with p53 stabilization (Bronder and Moran, 2003). Several studies showed that extracellular purines can activate astrocytes and microglial cells in response to injury or neurodegeneration (Rathbone et al., 1999). However, the expression and possible roles of GART in spinal cord injury are still unknown.

In the present study, we reported the distribution and expression of GART in rat spinal cord after injury for the first time. Our result is conducted to gain a better insight into the physiologic functions of GART in spinal cord and its association with the molecular and cellular mechanisms underlying nervous system injury and repair.

2.

Results

2.1. Behavioral changes following traumatic spinal cord injury The spontaneous recovery of locomotor function after spinal cord injury was tested using the BBB rating scale (Basso et al., 1995). Scores were recorded and the averages are shown in Fig. 1. All animals slacked hind limb locomotion after contusion to their spinal cords. Some spontaneous improvement in function occurred with time after SCI.

2.2. Time-dependent expression of GART protein following spinal cord injury We examined the temporal expression patterns of GART and GFAP following spinal cord contusion injury by Western blot. It was proved that GART expression was low but detectable in sham operated spinal cords, increased at 12 h after SCI, and reached a peak at day 3 (nPo0.05); then, it gradually decreased to the control levels. The expression of GFAP with an astrocytes marker was similar to that of GART following

Fig. 1 – Time course and degree of functional recovery in rats after SCI. Rats (n ¼ 3) were killed at each time point (at 6 h and 12 h and at 1, 3, 5, 7, and 14 days post-injury). Data are reported as mean7values of open-field locomotion BBB scores.

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Fig. 2 – Time-dependent expression of GART protein in rat spinal cord after SCI. Spinal cord tissues from rats at various survival times after SCI were homogenized and subjected to immunoblot analysis. Sample immunoblots probed with GART, GFAP and GAPDH are shown above (A). The bar chart below demonstrates the ratio of GART/GFAP to GAPDH for each time point ( B). Data are means7SEM (n ¼3;n, #Po0.05, significantly different from the sham groups).

SCI (Fig. 2A and B ). This result demonstrated that GART might be involved in the process of SCI in a time-dependent manner.

2.3. ‘Dose–Response Effects’ between the SCI and GART expression To further explore the possible correlation between GART expression and SCI progression, we investigated the effects of contusion induced by dropping the rod from different heights (0, 6.25, 12.5, 25, 50, 75, 100, and 125 mm) at day 3 after SCI and observed the expression patterns of GART by Western blot. As the contusion height elevated, GART expression was gradually enhanced, significantly higher than the sham group at 25 mm height, reached a peak at 100 mm height, and declined weakly in the case of 125 mm injury (Fig. 3A and B). To further strengthen the correlation hypothesized between lesion size/ astrocyte presence and GART overexpression, we also examined the expression of GFAP. Changes in GFAP showed activation of astrocytes after SCI under different conditions. These data further demonstrated that the up-regulation of GART is indeed related to the development of spinal cord injury.

2.4.

The staining changes of GART in the spinal cord

To identify the cellular localization and the temporal changes of GART immunoreactivity in spinal cord, we carried out immunohistochemisry experiments with anti-GART mouse

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Fig. 3 – ‘Dose–Response Effects’ between the SCI and GART expression. Spinal cord tissues from rats at various degrees of contusion induced by different heights (0, 6.25, 12.5, 25, 50, 75, 100, and 125 mm) at day 3 after SCI was homogenized and subjected to immunoblot analysis. Sample immunoblots probed with GART, GFAP and GAPDH are shown above (A). The bar chart below demonstrates the ratio of GART/GFAP to GAPDH for each height point (B). Data are means 7SEM (n ¼3; n, #Po0.05, significantly different from the sham group).

polyclonal antibody on transverse cryosections of the spinal cord, 2 mm rostral to epicenter. As shown above, GART protein has the maximal protein expression at day 3. Thus, we chose 3 days after SCI as the time point for our microscopic work. According to the positive cell profiles, GART was widely expressed in both the ventral horn and white matter (Fig. 4a and b) including most of glial cells, regardless of whether sham or injury. Interestingly, at high magnification, injury increased GART significantly in gray matter (Fig. 4d) and white matter (Fig. 4f). No staining was observed in the negative control sections (Fig. 4g). At the same time, we found their quantitative changes to be parallel with the Western blot results (Fig. 4h).

2.5. The colocalization of GART with different phenotype specific markers in spinal cord after SCI To further address the expression of GART in the spinal cord, we performed double immunofluorescence microscopy studies in transverse cryosections of spinal cord tissues within 2 mm distance from the lesion site by colabeling GART with NeuN, GFAP, CD11b and CNPase. We found GART expression (yellow) in a lot of astrocytes (Fig. 5i) and very few neurons (Fig. 5c and f) but not in microglia and oligodendrocytes (Fig. 5l, o, r and u). Significantly, such expression was clearly increased in astrocytes at day 3 after SCI compared with the sham spinal cord (Fig. 5l). No staining was observed in the

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Fig. 4 – Immunohistochemical expression of GART in adult rat spinal cord. Low-power views of cross-sections (2 mm rostral to epicenter) immunostained with antibody specific for GART in sham spinal cord (a) and 3 days after injury (b). Higher-power views in the ventral horn (c and d) and white matter (e and f) of the spinal cord. Quantitative analysis of GART-positive cells/ mm2 between sham and SCI groups at day 3 after operation (g). nPo0.05 compared with sham spinal cord. Error bars represent SEM. Scale bars, 200 μm (a and b) and 20 μm (c–g).

negative control sections (Fig. 5Y). As shown in (Fig. 5z), the numbers of astrocytes and GART-positive astrocytes section increased prominently after injury compared with the

sham-operated spinal cord (nPo0.05), while such change was not detected in neurons. It may also be the reason why we do not see the obvious neuron in Fig. 4.

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Fig. 5 – Double immunofluorescence staining for GART and different phenotype-specific markers in the spinal cord. In the adult rat spinal cord 2 mm to epicenter at day 3 after SCI, horizontal sections were labeled with GART (red) and different phenotype-specific markers (green), such as NeuN, GFAP, CD11b and CNPase. The colocalization of GART with different phenotype specific markers (yellow) are shown in c, f, i, l, o, r, u and x. Negative control is shown in Y. Quantitative analysis of different phenotype-specific markers positive cells expressing GART in sham spinal cord and at 3 days after SCI in n (nPo0.05, the changes of GART expression after SCI were obvious in astrocytes compared with the sham group). Error bars represent SEM. Scale bars, 20 μm (a–m).

2.6. Colocalization of GART and the cellular proliferation marker in the adult rat spinal cord after SCI The following studies were designed to demonstrate the relationship between GART and the cell proliferation in rat spinal cord after SCI. We first examined PCNA expression in the injured spinal cord, which has been used as a general marker of dividing cells (Xu et al., 2012; Zhao et al., 2011b). At day 1 following SCI, the expression of PCNA gradually increased, reached a peak at 3–5 days, and slightly deceased thereafter

(Fig. 6A). We next performed double-labeling immunofluorescence staining of GART together with GFAP (or PCNA) in the injured and sham spinal cords (Fig. 6B). As shown in Fig. 6Bf the majority of activated astrocytes were PCNA-positive. More importantly, there was obvious colocalization between GART and PCNA in those activated astrocytes of the SCI groups (Fig. 6Bi and l). No detectable expression of PCNA was observed in sham groups (Fig. 6Bb and h). We also costained with a microglial marker (CD11b), in order to more clearly show how many of these GFAPþ cells are PCNAþ (Fig. 6Bt).

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Fig. 6 – Association of GART with cell proliferation after SCI. Western blot analysis of PCNA in spinal cord after SCI (A and B). The expression of PCNA was increased after SCI and peaked at day 3. Double immunofluorescence staining of PCNA, GFAP, GART and CD11b in spinal cord after SCI (C). In adult spinal cord at 3 days after injury, sections were labeled with PCNA and GFAP/CD11b and the colocalization of PCNA with GFAP/CD11b (yellow) were shown in the spinal cord. The majority of reactive astrocytes/microglial were PCNA-positive at 3 days after SCI (f/r). Moreover, there was colocalization (yellow) between GART and PCNA (l). However, we observed hardly any expression of PCNA in sham groups (b, h, and n). Scale bars, 20 μm (C).

2.7. The roles of GART in LPS-induced astrocyte activation and proinflammatory cytokine production To further examine the functions of GART during astrocyte activation and neuroinflammation, we stimulated the rat primary astrocytes with LPS, a widely accepted stimulus of neuroinflammation, and detected GART expression during this process. As shown in Fig. 7A and B, iNOS expression was greatly stimulated by LPS treatment, which proved the successfully LPS-induced astrocyte activation (Beurel, 2011). More importantly, LPS significantly up-regulated GART expression in astrocytes at 4 h post-treatment, reached the maximum at 6 h, and then gradually decreased. To evaluate the effects of GART on astrocyte activation and the subsequent neuroinflammation response, we knocked down GART expression by the RNAi technique. The knockdown efficiency was confirmed by Western blot analysis. As demonstrated in Fig. 7C, GART RNAi treatment inhibited astrocyte GART expression to 68.26%, compared with control siRNA. It is

reported that, when activated, astrocytes release several proinflammatory cytokines, such as IL-1β, IL-6 and TNF-α. Consistent with this, ELISA assay showed that LPS (1 μg/ml, 6 h) addition successfully triggered the production of TNF-α (Fig. 7D) and IL-6 (Fig. 7E) in rat primary astrocytes. Interestingly, inhibiting GART expression by RNAi apparently increased the astrocyte secretion of IL-6 and TNF-α. Taken together, our results suggested that GART participates in LPSinduced astrocyte activation and pro-inflammatory cytokine secretion.

3.

Discussion

Human glycinamide ribonucleotide transformylase (GART) gene localizes on chromosome 21q22.1 within the Down syndrome critical region (Hards et al., 1986; Patterson et al., 1981). It is a validated target for cancer chemotherapy (Manieri et al., 2007). Previous researches showed GART

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Fig. 7 – GART was involved in the activation of astrocytes induced by LPS. LPS induced GART protein expression in a timedependent manner in rat primary astrocytes. Data are means7SEM of the maximum response observed; n ¼ 3, nPo0.05 versus the corresponding control (A and B). Effect of silencing GART in astrocytes. Astrocytes were transfected with indicated plasmids for 72 h, and the knockdown efficiency was ensured by immunoblotting for GART (C). After transfected with indicated plasmids, astrocytes were treated with LPS (1 ug/ml) for 6 h. ELISA showed the effects of GART on LPS induction of TNF-α (D) and IL-6 (E) in astrocytes. Data were expressed as mean7SEM of the maximum response observed. Statistical differences compared with the normal astrocytes were given as n ¼3, nPo0.05 and with the LPS-treated normal astrocytes group as n¼ 3, #Po0.05.

subserves a critical biochemical function in terms of de novo purine biosynthesis (Aimi et al., 1990). It is also known that elevated levels of purine nucleotides may promote astrocytic hypertrophy (Banerjee and Nandagopal, 2007), and extracellular purines can activate astrocytes in response to injury or neurodegeneration (Rathbone et al., 1999). But to date, few structural or protein mechanism studies have been reported for human GART in the central nervous system. In this study, we revealed the temporal–spatial expressions of GART in adult rat after spinal cord injury for the first time. Western blot analysis showed that GART was significantly increased at day 3 after injury. Meanwhile, the protein level of PCNA increased markedly from 3 to 5 days after injury. Immunohistochemical staining revealed that the expression of GART was enhanced obviously in the gray matter and white matter. In additional, the colocalization of GART/PCNA was detected in injured spinal cord. In conclusion, SCI might enhance the

expression of GART in the activated astrocytes. These findings might provide an important clue to learn the molecular and cellular mechanisms underlying inflammation. And these data were parallel with the hypothesis that GART was implicated in CNS pathophysiology after SCI. Traumatic insult to the adult mammalian central nervous system (CNS), such as spinal cord injury (SCI), initiates a cascade of events that ultimately lead to regenerative failure that profoundly influences the quality of patients’ lives. According to experimental and clinical studies, acute SCI causes primary and secondary damages to the spinal cord (Tator, 1995; Wu et al., 2011; Zhao et al., 2011a). Primary injury is due to tissue detritions, which are the result of external mechanical forces. Then, direct trauma to the spinal cord induces secondary injury, including spinal cord ischemia, edema, electrolyte imbalance, free radical damage, excitotoxicity, and inflammatory injury, which eventually cause

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neuronal apoptosis and astrocyte proliferation (Choi et al., 2010; Kwon et al., 2004). These events severely impact on the prognosis of spinal cord by aggravating the injury degree and expanding the range of lesion. Damage to the spinal cord leads to neuronal apoptosis, oxidative stress, reactive gliosis, and inflammation (Choi et al., 2010; Liu et al., 2010; Lopez-Vales et al., 2010). These factors cause proliferation, hypertrophy, and migration of astrocytes (Frisen et al., 1995; White et al., 2010). Recent findings revealed that astrocytes are not only supportive cells for neuronal function, but also actively participate in the initiation and modulation of inflammatory reactions in CNS injury, including Parkinson's disease, Alzheimer's disease, prion diseases, and multiple sclerosis (Ridet et al., 1997). These reactive astrocytes are capable of producing a variety of potentially neurotoxic compounds and proinflammatory mediators, such as IL-6 and TNF-α (Kang and Hebert, 2011; Pekny and Nilsson, 2005). IL-6 is a regulator of inflammatory and immunological responses. Under physiological conditions, the expression levels of IL-6 in the CNS are limited. However, both CNS injury and inflammation can significantly elevate the IL-6 levels (Benveniste, 1998). The cytokine TNF-α is an important factor in the regulation of neuronal apoptotic cell death. These cytokines mediate neuroinflammation and contribute to the neuropathology and pathophysiology associated with the inflamed CNS (Benveniste, 1997). To further discuss the roles of GART during SCI progression and its molecular mechanisms, we next established the astrocyte activation model by LPS-treatment, inhibited GART expression by RNAi, and explored the expression and possible function of GART during this process (Sofroniew and Vinters, 2010). We found that the knockdown of GART expression greatly increased the release of TNF-α and IL-6 in LPS activated astrocytes. All of these results indicated that GART was involved in the activation of astrocytes. In addition to the above we are talking about the neuronal apoptosis, astrocyte proliferation, and inflammation response. SCI can also cause an important physiological phenomenon in the formation of glial scar. Astrocytes are broadly distributed throughout the spinal cord (Sofroniew, 2009). After SCI, astrocytes proliferate to form a scar that preserves the integrity of surrounding cells. However, the persistence of a glial scar is detrimental to functional recovery of a damaged spinal cord, largely because this scar not only forms a physical barrier but also secretes inhibitors of axonal growth (Yiu and He, 2006). Found in our experiments GART involved gliosis and inflammation by SCI, but the GART and glial scar formation have any contact is worth further research work in the future. In summary, we investigated the protein expression and cellular localization of GART during SCI and demonstrated that GART might be involved in the activation of astrocytes. These data will be important in addressing molecular and

cellular mechanisms underlying SCI. However, further studies should be performed to confirm the intrinsic mechanisms and functions of GART in the central nervous system injury and repair.

4.

Experimental procedures

4.1. Animals, surgery, and locomotor function experiments Animals, surgeries and locomotor function experiments were performed in accordance with the National Institutes of Health Guidelines for the Care and Use of Laboratory Animals; all animal protocols were approved by the Department of Animal Center, Medical College of Nantong University. Male Sprague-Dawley rats with an average body weight of 250 g (220–275 g) were used in this study. Dorsal laminectomies at the level of the ninth thoracic vertebra (T9) were carried out under anesthesia with ketamine (90 mg/kg)/xylazine (10 mg/kg), and surgery was performed under aseptic conditions. Ketoprofen (5 mg/kg) was administered to minimize postsurgical pain and discomfort. Contusion injury groups (n ¼51) were performed using the NYU impactor (Gruner, 1992); the exposed spinal cord was contused by dropping a rod 2.0 mm in diameter and 10 g in weight from a height of 100 mm for 33 rats (Shen et al., 2008; Wu et al., 2011; Zhang et al., 2013). The rest of the contusion injuries (n ¼21) were divided randomly into seven groups of three animals. The seven groups were contused from different heights (6.25, 12.5, 25, 50, 75, 100, and 125 mm). Sham operated animals (n¼ 12) were anesthetized and surgically prepared but did not receive spinal injury. After SCI, the overlying muscles and skin were closed in layers with 0–4 silk sutures and staples. The animals were allowed to recover on a 30-1C heating pad. Postoperative treatments included saline (2.0 ml, s.c.) for rehydration and Baytril (0.3 ml, 22.7 mg/ml, s. c. once daily) to prevent urinary tract infection. Bladders were manually expressed twice daily until reflex bladder emptying returned. Animals were housed under a 12 h light/dark cycle in a pathogen-free area with free access to water and food. The spontaneous recovery of locomotor function after SCI was examined using the Basso, Beattie and Bresnahan (BBB) rating scale (Basso et al., 1995). Animals were killed 6, 12 h, 1, 3, 5, 7, and 14 days after injury (Table 1). All efforts were made to minimize the number of animals used and their suffering.

4.2.

Western blot analysis

To obtain samples for Western blot, the sham operated or injured spinal cords were excised and snap frozen at 80 1C until use. The portion of spinal cord extending 5 mm rostral

Table 1 – Animals used for each experiment.

Western blot Immunohistochemistry Immunofluorescence

Sham

6h

12 h

1 day

3 days

5 days

7 days

14 days

6 3 3

3 – –

3 – –

3 – –

24 3 6

3 – –

3 – –

3 – –

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and 5 mm caudal to the injury epicenter was immediately removed. To prepare sates, frozen spinal cord samples were minced with eye scissors in ice. The samples were then homogenized in lysis buffer (1% NP-40,50 mmol/l Tris, pH 7. 5, 5 mmol/l EDTA, 1% sodium dodecyl sulfate (SDS), 1% sodium deoxycholate, 1% Triton X-100, 1 mmol/l PMSF, 10 mg/ml aprotinin, and 1 mg/ml leupeptin) and clarified by centrifuging for 20 min in a microcentrifuge at 4 1C. After determination of its protein concentration with the Bradford assay (Bio-Rad), the resulting supernatant (50 μg of protein) was subjected to SDS-polyacrylamide gel electrophoresis. The separated proteins were transferred to a polyvinylidene difluoride membrane (Millipore) by a transfer apparatus at 350 mA for 2.5 h. The membrane was then blocked with 5% nonfat milk and incubated with primary antibody against GART (anti-mouse, 1:1000; Santa Cruz), proliferating cell nuclear antigen (PCNA, anti-rabbit, 1:1000; Santa Cruz), glial fibrillary acidic protein (GFAP, anti-rabbit, 1:1000; Sigma) or glyceraldehyde-3-phosphatedehy drogenase (GAPDH, antirabbit, 1:1000; Sigma). After incubating with the fluorescently labeled secondary antibodies (Odyssey, 1:10,000), the membranes were detected on the Odyssey instrument.

4.3.

Immunohistochemistry

After defined survival times, sham-operated and injured rats were terminally anesthetized and perfused through the ascending aorta with saline, followed by 4% paraformaldehyde. After perfusion, the sham-operated and injured spinal cords were removed and post-fixed in the same fixative for 3 h and then replaced with 20% sucrose for 2–3days, following 30% sucrose for 2–3 days. After treatment with sucrose solutions, the tissues were embedded in O.T.C. compound. Then, 8 μm frozen cross sections at two spinal cord levels (2 mm rostral and caudal to the injury epicenter) were prepared and examined. All of the sections were blocked with 10% goat serum with 0.3% Triton X-100 and 1% (w/v) bovine serum albumin (BSA) for 2 h at room temperature (RT) and incubated overnight at 4 1C with anti-GART antibody (anti-mouse, 1:100; Santa Cruz), followed by incubation in biotinylated secondary antibody (Vector Laboratories, Burlingame, CA). Staining was visualized with diaminobenzidine (Vector Laboratories). The primary antibodies were deleted from the procedure in the negative control. Cells with brown staining were counted as positive; cells with no staining were counted as negative.

4.4.

Double immunofluorescence staining

For double immunofluorescence staining, sections were first blocked with 10% normal serum blocking solution species, the same as the secondary antibody, containing 3% BSA and 0.1% Triton X-100 and 0.05% Tween 20, 2 h at RT in order to avoid unspecific staining. Then, the sections were incubated with both mouse polyclonal primary antibodies for GART (1:100; Santa Cruz), rabbit monoclonal primary antibodies for PCNA (1:100; Santa Cruz), or different phenotype-specific markers as follows: NeuN (neuron marker, 1:100; Chemicon), GFAP (astrocyte marker, 1:200; Sigma), CD11b (microglia marker, 1:50; Serotec), CNPase (oligodendrocyte marker,

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1:100; Chemicon). Sections were incubated with both primary antibodies over-night at 4 1C, followed by a mixture of FITCand TRITC-conjugated secondary antibodies for 2 h at 4 1C. The stained sections were examined with a Leica fluorescent microscope (Germany). The primary antibodies were deleted from the procedure in the negative control.

4.5.

Quantitative analysis

The numbers of GART-positive cells in the spinal cord 2 mm rostral to the epicenter were counted in a 500  500-μm measuring frame. For each animal, a measure was taken in a section through the dorsal horn, the lateral funiculus, and the ventral horn. To avoid counting the same cell in more than one section, we counted every fifth section (50 μm apart). The cell counts were then used to determine the total number of GART-positive cells per square millimeter. Cells double labeled for GART and the other phenotypic specific markers used in the experiment were also quantified. The total numbers of each phenotype specific marker-positive cells and GART-positive cells were counted in each section. Then, the percentage of double-positive cells was recorded. Two or three adjacent sections per animal were sampled 2 mm to the epicenter.

4.6.

siRNA constructions and transfection

Primer pairs for the rat GART siRNA expression vector were target of the sequence, GCUGGAGAAACAAUUGUCAdTdT; for transient transfection, the GART siRNA vector and the control vector (vector with scramble siRNA) were carried out using lipofectamine 2000 (Invitrogen) and plus reagent in OptiM EM (Invitrogen) as suggested by the manufacturer. Cells were used for the subsequent experiments 72 h after transfection.

4.7.

Primary astrocyte cultures and cell treatment

Astrocyte cultures were prepared from spinal cords of adult male Sprague-Dawley rats using a previously described method (Codeluppi et al., 2009; Tawfik et al., 2006) with a few modifications. The spinal cords were ejected from the vertebral column using a saline-filled syringe. The tissue was chemically dissociated with 0.25% trypsin–EDTA for 10 min followed by mechanical trituration in modified essential medium (DMEM). After centrifugation at 1500 rpm for 5 min, the cells were suspended with DMEM/F12 culture medium, containing 10% heat-inactivated FBS and 1% glutamine, and plated in a flask. The cultures were maintained in a humidified atmosphere of 95% O2/5% CO2 at 37 1C for 48–72 h to allow sufficient time for the cells to adhere and begin multiplying. The medium was changed after every 24 h. Approximately on days 10 and 11, flasks were wrapped in plastic, placed on a shaker plat form in a horizontal position with the medium covering the cells, and were shaken at 350 rpm for 6 h at 37 1C to separate the microglia from the astrocytes. The next day, the cells were trypsinized and replanted in six-well plates (40,000 cells per well). Prior to experimental treatments, cultures of astrocytes were passaged twice. Cell culture medium was switched to serum-free DMEM/F12 culture medium. Transient transfection was performed using

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lipofectamine 2000 as suggested by the manufacturer and incubated for 2 days. The astrocytes were synchronized for 24 h in the absence of serum and then incubated in the absence of serum and in the presence or absence of 1 ug/ml lipopolysaccharide (LPS). LPS-induced astrocytes were then harvested for Western blot analysis and ELISA analysis.

4.8.

ELISA IL-6 and TNF-α detection assay

Cells in 100 μl of medium were seeded onto 96-well plates and treated under different conditions. At the appropriate time, 100 μl of supernatants was harvested for ELISA assay according to the instruction of the manufacturer (BD PharMingen).

4.9.

Statistical analysis

All values are expressed as means7SEM. The statistical significance of differences between groups was determined by the Kruskal–Wallis test because of the small numbers in the group and Dunnett's test. Po0.05 or Po0.01 was considered significant. Each experiment consisted of at least three replicates per condition.

Acknowledgments This work was supported by National Basic Research Program of China (973 Program, No. 2012CB822104); National Natural Science Foundation of China (81202368 and 31072188); Key Project Natural Science Foundation of Jiangsu University and College (No. 11KJA310002); Nantong City Social Development Projects funds (HS2012032); a Project funded by the Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD).

references

Aimi, J., Qiu, H., Williams, J., Zalkin, H., Dixon, J.E., 1990. De novo purine nucleotide biosynthesis: cloning of human and avian cDNAs encoding the trifunctional glycinamide ribonucleotide synthetase–aminoimidazole ribonucleotide synthetase– glycinamide ribonucleotide transformylase by functional complementation in E. coli. Nucleic Acids Res. 18, 6665–6672. Aschner, M., 1998. Astrocytes as mediators of immune and inflammatory responses in the CNS. Neurotoxicology 19, 269–281. Banerjee, D., Nandagopal, K., 2007. Potential interaction between the GARS–AIRS–GART gene and CP2/LBP-1c/LSF transcription factor in Down syndrome-related Alzheimer disease. Cell. Mol. Neurobiol. 27, 1117–1126. Barcia, C., Ros, C.M., Annese, V., Gomez, A., Ros-Bernal, F., Aguado-Llera, D., Martinez-Pagan, M.E., de Pablos, V., Fernandez-Villalba, E., Herrero, M.T., 2012. IFN-gamma signaling, with the synergistic contribution of TNF-alpha, mediates cell specific microglial and astroglial activation in experimental models of Parkinson’s disease. Cell Death Dis. 3, e379. Basso, D.M., Beattie, M.S., Bresnahan, J.C., 1995. A sensitive and reliable locomotor rating scale for open field testing in rats. J. Neurotrauma 12, 1–21.

Beattie, M.S., 2004. Inflammation and apoptosis: linked therapeutic targets in spinal cord injury. Trends Mol. Med. 10, 580–583. Benveniste, E.N., 1997. Cytokines: influence on glial cell gene expression and function. Chem. Immunol. 69, 31–75. Benveniste, E.N., 1998. Cytokine actions in the central nervous system. Cytokine Growth Factor Rev. 9, 259–275. Beurel, E., 2011. HDAC6 regulates LPS-tolerance in astrocytes. PLoS One 6, e25804. Brodsky, G., Barnes, T., Bleskan, J., Becker, L., Cox, M., Patterson, D., 1997. The human GARS–AIRS–GART gene encodes two proteins which are differentially expressed during human brain development and temporally overexpressed in cerebellum of individuals with Down syndrome. Hum. Mol. Genet. 6, 2043–2050. Bronder, J.L., Moran, R.G., 2003. A defect in the p53 response pathway induced by de novo purine synthesis inhibition. J. Biol. Chem. 278, 48861–48871. Choi, D.C., Lee, J.Y., Moon, Y.J., Kim, S.W., Oh, T.H., Yune, T.Y., 2010. Acupuncture-mediated inhibition of inflammation facilitates significant functional recovery after spinal cord injury. Neurobiol. Dis. 39, 272–282. Codeluppi, S., Svensson, C.I., Hefferan, M.P., Valencia, F., Silldorff, M.D., Oshiro, M., Marsala, M., Pasquale, E.B., 2009. The RhebmTOR pathway is upregulated in reactive astrocytes of the injured spinal cord. J. Neurosci. 29, 1093–1104. Davies, S.J., Field, P.M., Raisman, G., 1996. Regeneration of cut adult axons fails even in the presence of continuous aligned glial pathways. Exp. Neurol. 142, 203–216. Frisen, J., Haegerstrand, A., Risling, M., Fried, K., Johansson, C.B., Hammarberg, H., Elde, R., Hokfelt, T., Cullheim, S., 1995. Spinal axons in central nervous system scar tissue are closely related to laminin-immunoreactive astrocytes. Neuroscience 65, 293–304. Gruner, J.A., 1992. A monitored contusion model of spinal cord injury in the rat. J. Neurotrauma 9, 123–126. Hards, R.G., Benkovic, S.J., Van Keuren, M.L., Graw, S.L., Drabkin, H.A., Patterson, D., 1986. Assignment of a third purine biosynthetic gene (glycinamide ribonucleotide transformylase) to human chromosome. Am. J. Hum. Genet. 39, 179–185. Hausmann, O.N., 2003. Post-traumatic inflammation following spinal cord injury. Spinal Cord 41, 369–378. Kang, W., Hebert, J.M., 2011. Signaling pathways in reactive astrocytes, a genetic perspective. Mol. Neurobiol. 43, 147–154. Kwon, B.K., Tetzlaff, W., Grauer, J.N., Beiner, J., Vaccaro, A.R., 2004. Pathophysiology and pharmacologic treatment of acute spinal cord injury. Spine J. 4, 451–464. Liu, Y., Figley, S., Spratt, S.K., Lee, G., Ando, D., Surosky, R., Fehlings, M.G., 2010a. Fenretinide promotes functional recovery and tissue protection after spinal cord contusion injury in mice. J Neurosci. 30, 3220–3226. Liu, Y., Figley, S., Spratt, S.K., Lee, G., Ando, D., Surosky, R., Fehlings, M.G., 2010a. An engineered transcription factor which activates VEGF-A enhances recovery after spinal cord injury. Neurobiol Dis. 37, 384–393. Lopez-Vales, R., Redensek, A., Skinner, T.A., Rathore, K.I., Ghasemlou, N., Wojewodka, G., DeSanctis, J., Radzioch, D., David, S., 2010. Fenretinide promotes functional recovery and tissue protection after spinal cord contusion injury in mice. J. Neurosci 30, 3220–3226. Manieri, W., Moore, M.E., Soellner, M.B., Tsang, P., Caperelli, C.A., 2007. Human glycinamide ribonucleotide transformylase: active site mutants as mechanistic probes. Biochemistry 46, 156–163. McDonald, J.W., Sadowsky, C., 2002. Spinal-cord injury. Lancet 359, 417–425. McGraw, J., Hiebert, G.W., Steeves, J.D., 2001. Modulating astrogliosis after neurotrauma. J. Neurosci. Res. 63, 109–115.

brain research 1564 (2014) 41–51

Munoz-Fernandez, M.A., Fresno, M., 1998. The role of tumour necrosis factor, interleukin 6, interferon-gamma and inducible nitric oxide synthase in the development and pathology of the nervous system. Prog. Neurobiol. 56, 307–340. Patterson, D., Graw, S., Jones, C., 1981. Demonstration, by somatic cell genetics, of coordinate regulation of genes for two enzymes of purine synthesis assigned to human chromosome. Proc. Natl. Acad. Sci. USA 78, 405–409. Pekny, M., Nilsson, M., 2005. Astrocyte activation and reactive gliosis. Glia 50, 427–434. Raghupathi, R., Mehr, M.F., Helfaer, M.A., Margulies, S.S., 2004. Traumatic axonal injury is exacerbated following repetitive closed head injury in the neonatal pig. J. Neurotrauma 21, 307–316. Rathbone, M.P., Middlemiss, P.J., Gysbers, J.W., Andrew, C., Herman, M.A., Reed, J.K., Ciccarelli, R., Di Iorio, P., Caciagli, F., 1999. Trophic effects of purines in neurons and glial cells. Prog. Neurobiol. 59, 663–690. Ridet, J.L., Malhotra, S.K., Privat, A., Gage, F.H., 1997. Reactive astrocytes: cellular and molecular cues to biological function. Trends Neurosci. 20, 570–577. Rivieccio, M.A., John, G.R., Song, X., Suh, H.S., Zhao, Y., Lee, S.C., Brosnan, C.F., 2005. The cytokine IL-1beta activates IFN response factor 3 in human fetal astrocytes in culture. J. Immunol. 174, 3719–3726. Sekhon, L.H., Fehlings, M.G., 2001. Epidemiology, demographics, and pathophysiology of acute spinal cord injury. Spine 26, S2–S12 (Phila Pa 1976). Serrano-Pozo, A., Mielke, M.L., Gomez-Isla, T., Betensky, R.A., Growdon, J.H., Frosch, M.P., Hyman, B.T., 2011. Reactive glia not only associates with plaques but also parallels tangles in Alzheimer’s disease. Am. J. Pathol. 179, 1373–1384. Shen, A., Liu, Y., Zhao, J., Qin, J., Shi, S., Chen, M., Gao, S., Xiao, F., Lu, Q., Cheng, C., 2008. Temporal-spatial expressions of p27kip1 and its phosphorylation on serine-10 after acute spinal cord injury in adult rat: implications for post-traumatic glial proliferation. Neurochem. Int. 52, 1266–1275. Silver, J., Miller, J.H., 2004. Regeneration beyond the glial scar. Nat. Rev. Neurosci. 5, 146–156.

51

Sofroniew, M.V., 2009. Molecular dissection of reactive astrogliosis and glial scar formation. Trends Neurosci. 32, 638–647. Sofroniew, M.V., Vinters, H.V., 2010. Astrocytes: biology and pathology. Acta Neuropathol. 119, 7–35. Tator, C.H., 1995. Update on the pathophysiology and pathology of acute spinal cord injury. Brain Pathol. 5, 407–413. Tawfik, V.L., Lacroix-Fralish, M.L., Bercury, K.K., NutileMcMenemy, N., Harris, B.T., Deleo, J.A., 2006. Induction of astrocyte differentiation by propentofylline increases glutamate transporter expression in vitro: heterogeneity of the quiescent phenotype. Glia 54, 193–203. Warren, L., Buchanan, J.M., 1957. Biosynthesis of the purines. XIX. 2-Amino-N-ribosylacetamide 50 -phosphate (glycinamide ribotide) transformylase. J. Biol. Chem. 229, 613–626. White, R.E., McTigue, D.M., Jakeman, L.B., 2010. Regional heterogeneity in astrocyte responses following contusive spinal cord injury in mice. J. Comp. Neurol. 518, 1370–1390. Wu, G., Cao, J., Peng, C., Yang, H., Cui, Z., Zhao, J., Wu, Q., Han, J., Li, H., Gu, X., Zhang, F., 2011. Temporal and spatial expression of cyclin H in rat spinal cord injury. Neuromol. Med. 13, 187–196. Wyndaele, M., Wyndaele, J.J., 2006. Incidence, prevalence and epidemiology of spinal cord injury: what learns a worldwide literature survey?. Spinal Cord 44, 523–529. Xu, T., Wang, X., Cao, M., Wu, X., Yan, Y., Fu, H., Zhao, W., Gong, P., Ke, K., Gu, X., 2012. Increased expression of BAG-1 in rat brain cortex after traumatic brain injury. J. Mol. Histol. 43, 335–342. Yiu, G., He, Z., 2006. Glial inhibition of CNS axon regeneration. Nat. Rev. Neurosci. 7, 617–627. Zhang, S., Huan, W., Wei, H., Shi, J., Fan, J., Zhao, J., Shen, A., Teng, H., 2013. FOXO3a/p27kip1 expression and essential role after acute spinal cord injury in adult rat. J. Cell Biochem. 114, 354–365. Zhao, J., Zhang, S., Wu, X., Huan, W., Liu, Z., Wei, H., Shen, A., Teng, H., 2011a. KPC1 expression and essential role after acute spinal cord injury in adult rat. Neurochem. Res. 36, 549–558. Zhao, W., Yang, J., Shi, W., Wu, X., Shao, B., Wu, Q., Chen, J., Ni, L., 2011b. Upregulation of p21-activated kinase 6 in rat brain cortex after traumatic brain injury. J. Mol. Histol. 42, 195–203.