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. 2018 Mar 2;97(9):e9913. doi: 10.1097/MD.0000000000009913

Therapeutic time window for the effects of erythropoietin on astrogliosis and neurite outgrowth in an in vitro model of spinal cord injury

Hea Nam Hong a, Ju Hee Shim a, You Jin Won a, Jong Yoon Yoo b,∗, Chang Ho Hwang c,∗
Editor: Anser Azim
PMCID: PMC5851719  PMID: 29489692

Abstract

Background:

The objective of this study was to investigate the underlying molecular mechanisms and the therapeutic time window for preventing astrogliosis with erythropoietin (EPO) treatment after in vitro modeled spinal cord injury (SCI).

Methods:

Cultured rat spinal cord astrocytes were treated with kainate and scratching to generate an in vitro model of SCI. EPO (100U/mL or 300U/mL) was added immediately or 2, 4, or 8 hours after injury. Some cultures were also treated with AG490, an inhibitor of the EPO-EPO receptor (EpoR) pathway mediator Janus kinase 2 (JAK2). To evaluate neurite extension, rat embryonic spinal cord neurons were seeded onto astrocyte cultures and treated with EPO immediately after injury in the presence or absence of anti-EpoR antibody.

Results:

EPO treatment at up to 8 hours after injury reduced the expression of axonal growth inhibiting molecules (glial fibrillary acidic protein, vimentin, and chondroitin sulfate proteoglycan), cytoskeletal regulatory proteins (Rho-associated protein kinase and ephephrin A4), and proinflammatory cytokines (tumor necrosis factor-alpha, transforming growth factor-beta, and phosphorylated-Smad3) in a dosedependent manner (P < .001). Most effects peaked with EPO treatment 2–4hours after injury. Additionally, EPO treatment up to 4 hours after injury promoted expression of the EpoR (>2-fold) and JAK2 (>3-fold) in a dose-dependent manner (P < .001), whereas co-treatment with AG490 precluded these effects (P < .001). EPO treatment up to 4hours after injury also enhanced axonal b-III tubulin-immunoreactivity (>12-fold), and this effect was precluded by co-treatment with an anti-EpoR antibody (P < .001).

Conclusions:

EPO treatment within 8 hours after injury reduced astrogliosis, and EPO treatment within 4 hours promoted neurite outgrowth. EPO therapy immediately after spinal cord injury may regulate glia to generate an environment permissive of axonal regeneration.

Keywords: astrocyte, erythropoietin, neurite growth, spinal cord injury

1. Introduction

The limited functional recovery after spinal cord injury (SCI) is partly related to a growth-inhibiting environment created by activated glia. In response to SCI, astrocytes undergo reactive astrogliosis, which is characterized by the upregulation of 2 intermediate filaments, glial fibrillary acidic protein (GFAP) and vimentin. Reactive astrocytes also secrete various neuro-inhibitory molecules such as chondroitin sulfate proteoglycans (CSPGs)[1] and produce pro-inflammatory cytokines such as tumor necrosis factor-alpha (TNF-α), transforming growth factor-beta (TGF-β), and interleukins (IL-1, IL-6).[2] Although these changes lead to transient beneficial effects in the early phase of injury,[2] activated glia ultimately create an environment precluding axonal regeneration and may facilitate secondary neuronal damage after primary insult.[3] In particular, activation of the Rho-associated protein kinase (ROCK) pathway after SCI is associated with neuronal apoptosis[4] and ephrinA4 (EphA4) signaling prevents axonal regeneration after SCI.[5] Accordingly, regulation of astrogliosis is an important potential therapeutic approach to improving functional recovery after SCI.

Erythropoietin (EPO) promotes neural regeneration after the central nervous system (CNS) injury by decreasing glutamate toxicity, increasing the generation of anti-apoptotic factors, reducing inflammation, and decreasing nitric oxide-mediated injury through direct and indirect antioxidant effects.[6] EPO-mediated neuro-protection is partly attributed to Epo receptor (EpoR) activation and subsequent Janus kinase 2 (JAK2) signaling as well as the activation of other downstream signaling pathways such as the phosphoinostitide 3-kinase (PI3K)/protein kinase B (AKT) pathway.[7–9] Although the neuro-protective effects of EPO are well known, few studies to date have evaluated the therapeutic time window for administering EPO to prevent astrogliosis after SCI.

The goal of this study was to investigate the molecular mechanisms and therapeutic time window for the effects of recombinant human EPO (rhEPO) on glial activation after SCI. Specifically, authors used an in vitro model of SCI to examine the effects of rhEPO on the generation of inhibitory molecules, pro-inflammatory cytokine expression, EPO-EpoR signaling, and neurite outgrowth.

2. Materials and methods

2.1. Cell culture

Authors developed an in vitro model of SCI that combines chemical [(kainic acid (KA)] and mechanical (scratch) injury to simulate real-time SCI.[10]

2.2. rhEPO treatments

Ethical approval was not necessary because it was an in-vitro experiment.

After scratching and incubation with 50 μM KA, cells were rinsed twice in Hank's balanced salt solution and the medium was replaced with fresh Dulbecco's modified Eagle's medium. rhEPO (Epokine, 10,000 units/mL; CJ Pharmaceutical Co., Republic of Korea) was added at different concentrations (100 U/mL or 300 U/mL) was added at t = 0 (immediately), 2, 4, or 8 hours after injury. Some cultures were also treated with the EPO-EpoR pathway inhibitor (AG490) at the indicated time points.

2.3. Western blotting

Cells were lysed by the addition of sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) sample buffer (62.5 mM Tris–HCl [pH 6.8], 2% SDS, 7.8% glycerol, 4.5% mercaptoethanol and 0.1% bromphenol blue) and subsequent boiling for 5 minutes at 100°C. Lysates were cleared by centrifugation and then supernatant protein concentrations were determined relative to a bovine serum albumin (BSA) standard. Cell lysates were subjected to SDS-PAGE on a 4% stacking gel and 10% polyacrylamide separating gel for 70 minutes at 130 V. Then, proteins were transferred onto nitrocellulose membranes with a Bio-Rad transfer unit for 120 minutes at 200 mA. Protein blots were incubated in blocking buffer (2% BSA in Tween20/Tris-buffered saline) for 1 hour at room temperature on a rotating platform. Blots were incubated overnight with primary antibodies against GFAP, CSPG, vimentin, ROCK, EphA4, TNF-α, TGF-β, phosphorylated-Smad3 (p-Smad3: a mediator of TGF-β actions), EpoR, or JAK2. Then, blots were incubated with horseradish peroxidase-conjugated secondary antibodies for 1 hour, followed by 3 washes. Immuno-reactive bands were visualized by chemiluminescence reagents (PIERCE, Rockford, IL). Band optical densities were analyzed with an Imaging Densitometer (Bio-rad, GS-670). More than 3 independent experiments were performed for each experiment.

2.4. Neurite outgrowth analysis

Spinal cord neurons were prepared from embryonic day 16 (E16) Sprague–Dawley rats as previously described.[11] After 7 days in vitro, spinal neurons were trypsinized and seeded onto astrocytes immediately after model SCI injury. A concentration of 100 U/mL rhEPO was added at t = 0 (immediately), 2, 4, or 8 hours after injury. Some cultures were also treated with anti-rhEPO receptor antibody (AbEpoR, 4 μg/mL, R&D Systems, Minneapolis) at the indicated times. After treatment, cultures were incubated in neuro-basal medium for 48 hours and subsequently fixed with 4% paraformaldehyde. Three independent experiments were performed (n = 2 wells per group per experiment), and 10 fields per well were randomly selected at a magnification of ×10. Neurite outgrowth was evaluated in each filed by measuring the area fraction occupied by neurites stained with anti-Tuj1 antibody (anti-Tubulin, beta III isoform, Millipore, Darmstadt, Germany) using ImageJ software (version 1.43u, National Institutes of Health, Bethesda, MA).

2.5. Statistical analysis

Adobe Photoshop 7.0 (Adobe Systems, San Jose, CA) was used for image quality optimization and figures preparation. Data were analyzed using GraphPad Instat 3.05 (GraphPad, San Diego). Statistical comparisons were performed using a one-way analysis of variance with Tukey-Kramer post hoc test. A P-value > .05 was the threshold for statistical significance.

3. Results

3.1. EPO treatment prevents astroglial expression of neuroinhibitory molecules after model SCI

Western blotting revealed that EPO treatment reduced the expression of GFAP, vimentin, and CSPG compared to control at 48 hours after treatment. GFAP expression was decreased when EPO was applied immediately after injury (100 U: 81.1 ± 8.3%; 300 U: 49.5 ± 2.7%) and 4 hour after injury (100 U: 41.6 ± 6.4%; 300 U: 38.6 ± 9.7%) compared to the control level. Vimentin expression was most remarkably decreased when EPO was applied 8 hours after injury (300 U: 39.6 ± 2.2%) while CSPG expression was most notably decreased by EPO treatment 4 hours after injury (300 U: 39.1 ± 8.3%) compared to control. Thus, EPO treatment reduced GFAP and CSPG expression compared to control when applied up to 8 hours after injury (Fig. 1A and B).

Figure 1.

Figure 1

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Effects of EPO treatment on astroglial expression of axonal growth inhibiting molecules. (A) Western blots showing the relative expression of target proteins with β-actin as a loading control. (B) Quantification of bands shown in panel A relative to the control condition. Values represent the mean ± standard deviation (n = 3 per time point in each group). CSPG = chondroitin sulfate proteoglycan, EPO = erythropoietin, GFAP = glial fibrillary acidic protein, S/KA = scratch and kainate injury model. ∗ indicates P < .001 vs the S/KA group.

3.2. EPO treatment decreases the expression of cytoskeletal protein regulators after model SCI

EPO treatment reduced the expression of ROCK compared to control when applied up to 8 hours after injury (100 U: 63.7 ± 6.9%; 300 U: 43.0 ± 7.6%). EPO treatment decreased EphA4 expression below that in control cells all time points with a maximum decrease observed at 4 hours after injury (100 U: 38.7 ± 4.1%; 300 U: 29.0 ± 6.6%); this decrease was still evident when EPO was applied 8 hours after injury (100 U: 78.4 ± 3.9%; 300 U: 60.9 ± 2.6%) (Fig. 2). Meanwhile, the effect of 300 U EPO on the expression of EphA4 at both 0 hour and 2 hours were opposed with the time point 4 and 8 hours.

Figure 2.

Figure 2

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Effects of EPO on cytoskeletal protein regulatory molecule expression. Top: Western blots showing the relative expression of target proteins with β-actin as a loading control. Bottom: Quantification of bands shown in the top panel relative to the control condition. Values represent the mean ± standard deviation (n = 6 per time point in each group). EphA4 = ephrinA4, EPO = erythropoietin, ROCK = rho-associated protein kinase, S/KA = scratch and kainate injury model. ∗ indicates P < .001 vs the S/KA group.

3.3. EPO treatment decreases pro-inflammatory cytokine expression after model SCI

EPO treatment reduced the expression of TNF-α, TGF-β, and p-Smad3 compared to control when applied up to 4 to 8 hours after injury, with a peak effect when applied 4 hours after injury. Effects on TGF-β and p-Smad3 expression were not statistically significant when EPO was applied at 8 hours after injury (Fig. 3). Meanwhile, the effect of 300 U EPO on the expression of p-Smad3 at both 0 hour and 2 hours were opposed with the time point 4 and 8 hours and the effect of 300 U EPO on the expression of TNF-α at both 4 and 8 hours were opposed with the time point 0 hour and 2 hours.

Figure 3.

Figure 3

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Effects of EPO on TNF-α, TGF-β, and p-Smad3 expression. Top: Western blots showing the relative expression of target proteins with β-actin as a loading control. Bottom: Quantification of bands shown in the top panel relative to the control condition. Values represent the mean ± standard deviation (n = 6 per time point in each group). EPO = erythropoietin, S/KA = scratch and kainate injury model, TGF-β = transforming growth factor-beta, TNF-α = tumor necrosis factor-alpha. ∗ indicates P < .001 vs the S/KA group.

3.4. Effects of EPO treatment on EpoR signaling after model SCI

EPO treatment increased EpoR expression nearly 2-fold compared to control when applied 2 hours after injury, but showed little-to-no effect when applied 4 hours after injury. In contrast, EPO treatment increased JAK2 expression when applied 4 hours after injury. EPO treatment also reduced the expression of phosphorylated protein kinase B (pAKT) when applied 4 hours after injury. Co-treatment with AG490 prevented EPO-mediated decreases in TGF-β expression after application at 2 hours after injury and reductions in pAKT after application at 4 hours after injury (Fig. 4).

Figure 4.

Figure 4

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Effects of EPO on EpoR and JAK2 expression. Top panels: Western blots showing the relative expression of target proteins with β-actin as a loading control (upper) and AKT as a total protein control (lower). Bottom panels: Quantification of bands shown in the top panel relative to the control condition. Values represent the mean ± standard deviation (n = 3 per time point in each group). EPO = erythropoietin, EpoR = EPO receptor, JAK2 = Janus kinase 2, pAKT = phosphorylated protein kinase B, S/KA = scratch and kainate injury model, TGF-β = transforming growth factor-beta. ∗ indicates P < .001 vs the S/KA group and ∗∗ indicates P < .001 vs the S/KA + EPO100 U group.

3.5. EPO treatment increases neurite outgrowth after model SCI

Spinal cord neurons grown in the absence of EPO were shorter and fewer in number compared to those grown in the presence of EPO. EPO treatment dramatically increased the number of β-III tubulin-immuno-reactive axons when applied up to 4 hours after injury. EPO treatment immediately after injury increased neurite outgrowth by almost 13-fold (12.71 ± 1.76) compared to control, whereas treatment at 8 hours after injury had no significant effect on neurite outgrowth. Co-treatment anti-EpoR antibody prevented EPO-mediated increases in neurite outgrowth at all time points (Fig. 5A–C).

Figure 5.

Figure 5

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Effects of EPO on neurite outgrowth. (A) Immunocytochemical staining with anti-Tuj1 antibody. (B, C) Quantification of immunofluorescence shown in panel A relative to the control condition. Values represent the mean ± standard deviation (n = 6). Scale bar = 100 μm. AbEpoR = anti-rhEPO receptor antibody, EPO = erythropoietin, S/KA = scratch and kainate injury model. ∗ indicates P < .001 vs the S/KA group, ∗∗ indicates P < .001 vs the S/KA + EPO100 U group, and # indicates P < .005 v. the S/KA group.

4. Discussion

In the present study, authors examined the ability of EPO to limit the extent of astrogliosis after modeled SCI in vitro and found that EPO applied up to 8 hours after injury regulated the expression of axonal growth inhibiting molecules, cytoskeletal regulatory proteins, pro-inflammatory cytokines, and EPO-EpoR signaling molecules. Additionally, EPO applied up to 4 hours after injury promoted neurite extension in seeded spinal neurons. Effects were dose dependent and partially prevented by co-application of AG490 or anti-EpoR antibody.

Authors’ finding that EPO treatment mitigated increases of astrocytic GFAP, vimentin, and CSPG expression after injury is compatible with the findings reported in previous literature describing the anti-apoptotic and neuroprotective effects of EPO in SCI [12,13] as well as the reported ability of EPO to prevent gliosis.[14,15] Previous research has focused on the expression of neuron-glial antigen 2 (NG2) and phosphacan in glial scarring and demonstrated important roles for these molecules in the inhibition of neuronal regeneration after human SCI.[16] It was reported that, among other CSPGs, phosphacan was selectively decreased by EPO treatment after SCI.[14,15] Our results challenge this notion and suggest that other glial scar components may be regulated by EPO treatment after SCI.

Authors also found that EPO treatment attenuated the expression of cytoskeletal regulatory proteins, ROCK, and EphA4, after modeled SCI. Astroglial activation is associated with increases in ROCK and ephrin expression, which regulate cytoskeletal proteins via RhoA and Eph/ephrin signaling, respectively.[17,18] The EphA4 receptor is highly expressed on astrocytes following SCI and is a major inhibitor of neurite regrowth.[19] In contrast, ROCK is a member of a family of serine-threonine kinases that inhibits cell migration by promoting actin stabilization and the secondary loss of actin monomers. Accordingly, ROCK inhibitors such as Fasudil and Y-27632 increase neurite regeneration and outgrowth in various CNS disorders.[20] From this perspective, the inhibitory effects of EPO treatment on ROCK and EphA4 expression in astrocytes may be an important component of its neuroregenerative mechanism.

It is generally recognized that EPO exerts antiapoptotic and anti-inflammatory effects in the CNS disorders.[21,22] Here, authors found that EPO treatment reduced the expression of pro-inflammatory cytokines after modeled SCI. TNF-α is a cytokine involved in systemic inflammation and apoptosis. EPO has been previously reported to reduce the expression of IL-6 and TNF-α as well as astroglial reactivity.[12] TGF-β stimulates astrocytes and fibroblasts to form glial scars.[23,24] Buss et al[25] reported that TGF-β1 expression was dramatically increased at 2 days after SCI and followed by the induction of TGF-β2 expression. This finding suggests a role for TGF-β1 in induction of acute inflammatory response and glial scar formation, and a role for TGF-β2 in glial scar maintenance. A recent study by Fang et al[26] revealed that EPO treatment decreased TGF-β expression after SCI and effectively prevented a number of injury-related pathological alterations. However, TGF-β regulates many biological processes through the Smad and DAXX pathways.[27,28] On the basis of these findings and authors’ results, authors hypothesize that the ability of EPO treatment to affect TGF-β expression after model SCI is critical for preventing glial scar formation and facilitating neuroregeneration.

Protective effects of EPO in nonhematopoietic tissues are thought to be mediated by EPO-EpoR signaling and subsequent activation of JAK2 and other downstream mediators including signal transducer and activator of transcription (STAT) and PI3K/AKT.[7–9] In the current study, EpoR expression was increased when EPO was applied 2 hours after injury, whereas JAK2 expression was increased when by EPO was applied 4 hours after injury. Apparent dose-dependency and significant effects of EPO treatment on EpoR and JAK2 expression are consistent with previous studies investigating the neuroprotective effects of EPO. Authors’ findings are also consistent with the observation of Ostroseki et al., who reported that AG490 co-treatment abolished the neuroprotective effects of EPO in cultured brain neuron.[7] Contrary to authors’ expectation and previous reports, AKT expression was reduced when EPO was applied 4 hours after injury, and this effect was sensitive to AG490 co-treatment. Miljus et al[29] reported that AG490, but not PI3K inhibitor co-treatment, abolished the protective effects of EPO in insect brain neurons, and proposed that the PI3K/AKT signaling pathway was not involved in the mechanism of EPO mediated neuroprotection. Considering the heterogeneity of the nonhematopoietic EpoRs[30,31] and the phylogenetic age of these receptors,[7] undiscovered interactions in the EPO-EpoR pathway may account for the effects of EPO on astrocytes.

Finally, authors observed a facilitative effect of EPO on neurite outgrowth. Several previous reports have indicated a positive effect of EPO on axonal and/or dendritic outgrowth,[32–34] in agreement with authors’ finding. However, previous studies utilized cultured brain cell and especially hippocampal cell, such that this is the first study to demonstrate an effect of EPO on the growth of spinal neurons. It is notable that the peak effect of EPO on neurite outgrowth was observed when EPO was applied immediately after injury, in contrast with other effects of EPO that peaked when EPO was applied at 2 or 4 hours after injury. This finding revives controversy about whether EPO inhibits further neural damage or promotes neuronal regeneration after SCI study.[35]

In case of being treated before injuries, EPO showed the dose-dependent, protective effects on hippocampal, cortical neurons and spinal neurons on normal conditions in wide range of concentration (0–30 pM or 0–100 U) in a study by Morishita et al and by Yoo et al.[36,37] However, at higher concentration (300 pM or 200–300 U), it showed no dose-dependency and even revealed decreased effects. It might happen by super-saturation or ceiling effects of EpoRs. As thermal injuries can induce denaturation of EpoRs,[38] chemical or mechanical injuries can also make any changes in structure of EpoRs.[39] In the present study, the threshold of supersaturation or ceiling effects might be shifted so that subsequent dose-dependency could be noticed again at higher concentrations (300 U). However, the effect of 300 U EPO on the expression of p-Smad3, EphA4, and TNF-α at some time points was opposed with the different time points. It might take different time, depending on the different downstream cascades (ROCK vs EphA4 and TNF-α vs p-Smad3), how quickly denatured EpoR readapt to changes in molecular or genetic levels. In addition, heterogeneity of the non-hematopoietic EpoRs with the different phylogenetic age could booster these diversities.[7,30,31]

The present work had some limitations. First, despite authors’ efforts to simulate SCI in vitro, it is notable that various neuronal populations react differently to injury, and even similarly sized neurons within a population can exhibit different responses to astrocytic scar-like co-cultures.[40,41] Thus, authors’ model was not a definitively reliable representation of SCI. Second, it is difficult to compare authors’ findings with those of previous studies, as previous studies have used: different rat strains, sexes, and weights; various methods and devices for SCI induction; and various schedules of therapeutic treatment.[42] No positive control for the EPO and no control group for the EPO on normal conditions are another issue. It is clear that delay between EPO and SCI in part mediates the neuro-protective efficacy of treatment. Therefore, future studies should expand our findings to an in vivo model of SCI.

5. Conclusions

The application of EPO to astrocytic cultures after modeled SCI enhanced EPO-EpoR signaling and specifically JAK signaling, but not PI3K/AKT signaling. These changes were associated with decreased TNF-α, TNF-β, and Smad expression and the reduced expression of cytoskeletal regulatory proteins ROCK and EphA4. Finally, EPO treatment promoted neurite outgrowth when applied within 4 hours after injury. Our findings suggest that it may be effective if provided around 2to 4 hours after injury in the inhibition of further neuronal deterioration point of view, and it may be optimal if administered immediately after injury in the neuro-regenerative point of view.

Footnotes

Abbreviations: AKT = protein kinase B, BSA = bovine serum albumin, CNS = central nervous system, CSPGs = chondroitin sulfate proteoglycans, EphA4 = ephrinA4, EPO = erythropoietin, EpoR = EPO receptor, GFAP = glial fibrillary acidic protein, IL = interleukins, JAK2 = Janus kinase 2, KA = kainic acid, NG2 = neuron-glial antigen 2, pAKT = phosphorylated protein kinase B, PI3K = phosphoinostitide 3-kinase, p-Smad3 = phosphorylated-Smad3, rhEPO = recombinant human EPO, ROCK = Rho-associated protein kinase, SCI = spinal cord injury, SDS-PAGE = sodium dodecyl sulfate polyacrylamide gel electrophoresis, STAT = signal transducer and activator of transcription, TGF-β = transforming growth factor-beta, TNF-α = tumor necrosis factor-alpha.

This work was supported by a grant (2011-412) from the ASAN Institute for Life Science, Seoul, Korea and by the National Research Foundation of Korea (NRF) grant funded by the Korea government (Ministry of Science, ICT & Future Planning) (NRF-2017R1A2B4011478).

The authors have no conflicts of interest to disclose.

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