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. Author manuscript; available in PMC: 2011 Sep 1.
Published in final edited form as: Stroke. 2010 Jul 29;41(9):2071–2076. doi: 10.1161/STROKEAHA.110.586198

Erythropoietin in combination of tissue plasminogen activator exacerbates brain hemorrhage when treatment is initiated 6h after stroke

Longfei Jia 1, Michael Chopp 1,2, Li Zhang 1, Mei Lu 3, Zheng Gang Zhang 1
PMCID: PMC2950698  NIHMSID: NIHMS229501  PMID: 20671252

Abstract

Background and Purpose

Erythropoietin (EPO), a hematopoietic cytokine, exerts neuroprotective effects in experimental stroke. In the present study, we investigated the effect of recombinant human EPO (rhEPO) in combination with tissue plasminogen activator (tPA) on embolic stroke.

Methods

Rats subjected to embolic middle cerebral artery occlusion (MCAo) were treated with rhEPO (5,000 units/kg) in combination with tPA (10 mg/kg) at 2 or 6 h after MCAo. Control groups consisted of ischemic rats treated with rhEPO (5,000 units/kg) alone, tPA (10 mg/kg) alone, or saline at 2 or 6h after MCAo.

Results

The combination therapy of rhEPO and tPA initiated 6h after MCAo did not reduce the ischemic lesion volume and significantly (p<0.05) increased the incidence of brain hemorrhage measured by frequency of gross hemorrhage and a quantitative spectrophotometric hemoglobin assay compared with rats treated with rhEPO alone and tPA alone. However, when the combination therapy was initiated 2h after MCAo, the treatment significantly (p<0.05) reduced the lesion volume and did not substantially increase the incidence of hemorrhagic transformation compared with saline-treated rats. Immunostaining analysis revealed that the combination therapy of rhEPO and tPA at 6h significantly (p<0.05) increased MMP9, NF-κB, and interleukin-1 receptor-associated kinase-1 (IRAK-1) immunoreactive cerebral vessels compared with rats treated with rhEPO alone and saline.

Conclusions

EPO exacerbates tPA-induced brain hemorrhage without reduction of ischemic brain damage when administered 6h after stroke in a rat model of embolic MCAo and that MMP9, NF-κB, and IRAK-1 upregulated by the delayed combination therapy may contribute to augmentation of brain hemorrhage.

Introduction

Recombinant human tissue-type plasminogen activator (tPA) is an effective treatment for acute ischemic stroke only when given within 4.5h of stroke onset 1. However, tPA treatment increases the incidence of hemorrhagic transformation 2, 3. Experimental studies show that neuroprotective or anti-thrombotic agents used in conjunction with tPA increases safety and efficacy of thrombolytic therapy in experimental stroke4–6.

Erythropoietin (EPO) is a naturally occurring cytokine and has been used for treatment of anemia for more than two decades 7. Administration of exogenous recombinant human EPO (rhEPO) after focal or global cerebral ischemia augments the cytoprotective and restorative EPO response pathway leading to a substantial improvement in neurobehavioral outcome 8–10. However, a recent report on the Phase III clinical trial for the treatment of acute ischemic stroke with EPO demonstrated increased mortality and hemorrhage in EPO treated patients compared with control 11. The reasons for the apparent adverse effects of the EPO treatment stand in contrast to the robust preclinical data indicating a therapeutic benefit of EPO treatment 12, 13. Careful review of the trial shows that 63% of patients enrolled in this trial also received tPA, and that many of these patients were treated at or beyond the therapeutic window for tPA 11. Data from the clinical trial also indicate that it was these patients that drove the adverse response to EPO 11. Consistent with the imperative to test preclinically the interaction between tPA and EPO and as a part of our ongoing studies on the neuroprotective effects of EPO treatment, we investigated the effects of combination treatment with EPO and tPA in a model of embolic stroke in the rat. We demonstrate that the combination treatment performed early after stroke (i.e. 2 hours) is neuroprotective; however, outside the therapeutic window (i.e. 6 hours), combination tPA and EPO significantly exacerbates hemorrhagic transformation and negates any benefit of EPO therapy

Methods

All experimental procedures were approved by the Henry Ford Hospital Committee forn the Care of Experimental Animals.

Animal model

Male Wistar rats weighing 350 to 450 g (n=194) were subjected to embolic middle cerebral artery occlusion (MCAo) by placement of an embolus at the origin of the MCA, as described previously14.

Experimental Protocols

Recombinant human EPO (rhEPO, epoietin α, AMGEN) was given (i.p) at a dose of 5,000 units/kg 2 or 6h after embolic MCAo and was followed by a second and third dose of 5,000 units/kg 24 and 48h after the first dose. Recombinant human tPA (Genentech) was infused intravenously at a dose of 10 mg/kg (10% bolus 2 or 6h after MCAo, and the remainder at a continuous infusion over a 30 min interval using a syringe infusion pump, (Harvard Apparatus). After MCAo, animals were randomly assigned to monotherapy with rhEPO at 2h (n=27) or 6h (n=24), tPA at 2h (n=26) or 6h (n=26), combination therapy with rhEPO and tPA at 2h (n=39), 6h (n=36), or saline (n=26). Rats were killed at 1 or 7 days after MCAo.

Measurements of infarct volume and hemorrhage

Seven days after MCAo, infarct volume and gross hemorrhage were measured as previously described 14 (see Supplemental Methods for more details).

Spectrophotometric measurement of intracerebral hemorrhage

The hemorrhage volume wasquantified with a spectrophotometric hemoglobin assay 24h after stroke onset (see Supplemental Methods)15.

Immunohistochemistry and Weestern blots

Immunohistochemistry and Western blots were performed on brain tissue from rats sacrificed 24 h after ischemia 6. The following antibodies were used in the present study: a mouse anti-MMP9 (1:100, Chemicon), a mouse anti-fibrin/fibrinogen antibody (1:1000, Accurate Chemical& Scientific), a mouse anti-endothelial barrier antigen (EBA, 1:1000, SMI 71, Sternberger Monoclonals Inc), a mouse anti-NF-κB (P65, 1:150, Chemicon), which is specific for the detection of activated NF-κB 16, and a mouse anti-interleukin-1 receptor-associated kinase-1 (IRAK-1) (1:50, Santa Cruz). (see Supplemental Methods).

Statistics

Data were evaluated for normality. Ranked data were used in the analysis when they were not normally distributed. The incidence of hemorrhage was compared using Chi-square analysis. The 2X2 factorial design and 2-way ANOVA were considered for each rhEPO and tPA combination at each administration time. The analysis started testing for overall group effect/treatment interactions, followed by the pair-wise group comparisons if the overall group effect/treatment interaction was detected at the 0.05 level; otherwise, the pair-wise group comparisons would be considered as exploratory analysis. P<0.05 was considered a significant difference. All values are presented as mean ± SE.

Results

Mortality

The mortality rates were 15, 20, 17, 30, 33, and 38% for rats treated with saline, EPO at 2h, EPO at 6h, tPA at 6h, the combination of EPO and tPA at 2 and 6h, respectively. No significant differences were detected among the groups. The majority of animals died within 24h of onset of MCAo. Rats that died were excluded from further evaluation.

The effect of rhEPO in combination with tPA on ischemic lesion volume

To examine whether a combination therapy of rhEPO and tPA has a neuroprotective effect, ischemic rats were treated 2 or 6h after MCAo and killed 7 days after MCAo. When the treatment initiated at 2h of onset of stroke, monotherapy of rhEPO or tPA significantly (p<0.05) reduced ischemic lesion volume (Figure 1), which is consistent with published studies 13, 18. The combination therapy with rhEPO and tPA also significantly (p<0.05) reduced the lesion volume compared with rats treated with saline (Figure 1), but no treatment interaction (synergistic effect) or additive effect on the lesion volume was detected compared with the monotherapy. However, when the combination of rhEPO with tPA was administered 6h after stroke, the combination did not reduce the lesion volume compared with the saline group. As expected in this model 13, monotherapy of rhEPO but not tPA given 6h after stroke substantially reduced the lesion volume (Figure 1). These data indicate that the efficacy of 6h treatment of stroke with rhEPO is negated by tPA.

Figure 1. Infarct volume.

Figure 1

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Bar graph shows the infarct volume assessed 7 days after MCAo. Values are mean ± SE. *P < 0.05 as compared with the saline-treated group.

The effect of rhEPO in combination with tPA on hemorrhage

A major concern of thrombolysis is the incidence of hemorrhagic transformation 3. To examine whether the combination of rhEPO with tPA affects the incidence of brain hemorrhage, we measured frequency of the gross hemorrhage and quantified brain hemorrhagic volume with a spectrophotometric hemoglobin assay 24h after stroke 19. Neither the combination therapy of rhEPO with tPA (35%) nor monotherapy of rhEPO (25%) or tPA (28%) initiated 2h after stroke significantly increased the incidence of hemorrhagic transformation compared with saline treated rats (14%). In contrast, the combination of rhEPO and tPA at 6h significantly increased (p<0.05) the incidence of gross hemorrhage from 14% in the saline group to 59% in the combination-treated group. The EPO monotherapy at 6h did not significantly increase the incidence of gross hemorrhage (25%) compared saline treated rats. The monotherapy of tPA at 6h resulted in a trend toward increase in the incidence of gross hemorrhage (43%), which did not reach statistical significance. However, quantitative data analysis of the brain hemorrhage revealed that the tPA monotherapy at 6h significantly (p<0.05) increased the hemorrhagic volume (4.4 μl, Figure 2) compared with the volume in the saline group (1.4 μl, Figure 2). Moreover, the combination of rhEPO and tPA further augmented (p<0.05) the hemorrhage volume (9 μl, Figure 2) compared with the tPA alone group (Figure 2). The monotherapy of rhEPO did not significantly increase the hemorrhage volume compared with the volume in the saline group (Figure 2).

Figure 2. Effects of EPO and tPA on hemorrhage.

Figure 2

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Panel A shows the hemorrhage volume assessed with the spectrophotometric assay 24h after stroke onset. Panels B and C show intracerebral hemorrhage in H&E-stained coronal brain section from representative rats treated saline (B) and the combination of rhEPO and tPA (C) 6h after stroke onset. Values are mean ± SE. *P < 0.05 as compared with the saline-treated group. +P < 0.05 as compared with tPA-treated at 6h group. Bar=100μm.

Brain parenchymal deposition of the large plasma protein fibrin/fibrinogen is an indicator of BBB leakage 20. To further examine vascular leakage, we measured fibrin/fibrinogen deposition in ischemic brain. The combination of rhEPO and tPA at 6h substantially increased the number of cerebral vessels with extravascular fibrin deposition compared with ischemic rats treated with saline or the combination therapy at 2h (Figure 3), suggesting that the delayed combination therapy exacerbates BBB disruption.

Figure 3. Expression of MMP9, NF-κB, IRAK1, fibrin(ogen) and eNOS.

Figure 3

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Panel A shows the MMP9 immunoreactivity measured 24h after MCAo. Panels B and C show MMP9 positive vessels in representative rats treated with saline (B) and the combination of rhEPO and tPA initiated at 6h (C). Panels D to F show the double immunofluorescent staining of NF-κB (green, D and E) with EBA (red, D) and fibrin(ogen) (red, E), and IRAK1 (red, F) with fibrin(ogen) (green, F) from a representative rat treated with rhEPO in combination with tPA at 6h. An arrow in panels indicates NF-κB (E) and IRAK1 positive vessels with appearances of extravascular fibrin deposition 24h after stroke onset. Quantitative data show that the combination treatment with rhEPO and tPA at 6h after stroke onset significantly increases the number of vessels with NF-κB (G), extravascular fibrin (H), and IRAK1 (I) immunoreactivity compared with the combination therapy at 2h or saline treated rats. Western blots show phospho-eNOS (J) and nuclear NF-κB (K) levels. Values are mean ± SE. *P < 0.05 as compared with the saline-treated group. Bars in panels B through F=50μm.

The effect of rhEPO in combination with tPA on expression of MMP9, NF-κB, and IRAK1

Upregulation of MMP9 contributes to hemorrhagic transformation after stroke 21, 22. To examine whether the delayed (6h) combination therapy of rhEPO and tPA affects MMP9 expression, immunohistochemical staining with antibodies against MMP9 was performed on coronal brain sections obtained from rats sacrificed 24h after stroke. Combination treatment with rhEPO and tPA at 6h significantly increased the MMP9 immunoreactive area compared with saline treated rats (Figure 3), suggesting that the increased MMP9 may contribute to exacerbation of hemorrhagic transformation.

In addition to its many functions, activation of NF-κB triggers upregulation of MMP9 and acts as a primary transactivator for tPA-induced BBB breakdown and hemorrhage transformation23, 24. We therefore measured NF-κB protein nuclear translocation at 2 and 24h after the treatments using Western blots. The combination treatment with EPO and tPA at 2 or 6h did not significantly alter the nuclear NF-κB protein levels compared with that in the saline, rhEPO alone and tPA alone groups (Figure 3). However, immunostaining analysis revealed that the delayed combination rhEPO and tPA significantly increased the number of NF-κB immunoreactive vessels compared with the combination therapy at 2h (Figure 3). Concurrently, the delayed combination treatment robustly increased the number of IRAK1 immunoreactive vessels compared with the number in rats treated with the combination therapy at 2h. IRAK1 triggers activation of NF-κB 25. In addition, double immunostaining revealed that some of NF-κB immunoreactive vessels exhibited parenchymal fibrin/fibrinogen deposition (Figure 3). The anti-NF-κB antibody used in the present study is specific for the detection of activated NF-κB 16. Thus, our data suggest that the delayed combination treatment upregulates IRAK1 and activates NF-κB, which may trigger MMP9 expression, leading to brain hemorrhage.

Activation of eNOS enhances thrombolysis and reduces brain hemorrhage in the ischemic brain 26. EPO elevates levels of phosphorylated eNOS protein in cerebral vessels 27. To examine whether the combination of rhEPO and tPA affects eNOS levels, Western blot analysis was performed. The combination treatment with EPO and tPA at 2 or 6h had no effects on eNOS protein levels (Figure 3).

Discussion

The present study demonstrated that the combination of rhEPO and tPA initiated 6h after embolic MCAo failed to reduce ischemic lesion volume and exacerbated hemorrhagic transformation, whereas the combination therapy started 2h after stroke significantly reduced ischemic damage without increasing the hemorrhagic transformation. These data suggest that rhEPO exacerbates tPA-induced brain hemorrhage when the combination treatment starts 6h after stroke in a rat model of embolic MCA occlusion.

Previous experimental studies in the treatment of acute ischemic stroke demonstrated that tPA in combination with neuroprotective agents such as neuroserpin, minocycline, and atorvastatin given 4 to 6h after embolic stroke enhances the neuroprotective effect by attenuating the side effects of thrombolysis 28–30. Since the efficacy of EPO in experimental stroke has been well demonstrated, we hypothesized that the combination treatment of rhEPO with tPA extends the therapeutic window for tPA. However, to our surprise, the present study showed that the combination of rhEPO and tPA initiated 6h after stroke substantially augmented the incidence of brain hemorrhage compared with monotherapy of rhEPO or tPA given at the same time point. The efficacy of the monotherapy of rhEPO at 2 and 6h observed in the present study are consistent with published reports 13. In this model of embolic stroke, we have demonstrated that the therapeutic window for tPA is less than 4h after stroke onset, and delayed tPA treatment increases the incidence of hemorrhagic transformation 28, 31. The present study shows that monotherapy of tPA at 6h augmented the incidence of brain hemorrhage. However, rhEPO in combination with tPA at 6h aggravated the hemorrhage, suggesting that EPO likely exacerbates tPA-induced hemorrhage. These experimental results are consistent with observations made from the recent double-blind, placebo-controlled, randomized German Multicenter EPO Stroke Trial 11, reporting that with a high proportion of tPA treated patients enrollment (63%), there was a significant increase in death rate when stroke patients received EPO within 6h of the onset of stroke compared with the death rate in the placebo group 11. The elevated tPA protocol violation rate (50%) including treatment beyond the 3h time window may have contributed to the high death rate in the EPO treated patients.

Mechanisms underlying the exacerbation and the increased incidence of hemorrhage are currently unknown when the combination therapy of rhEPO and tPA was initiated 6h after stroke. Thrombolysis with tPA upregulates MMP9, a protein-digesting enzyme which degrades the major extracellularmatrix components, and thereby exacerbates BBB disruption and hemorrhagic transformation 32–34. The present study shows that tPA in combination with rhEPO at 6h but not 2h significantly increased the MMP9 levels, which was concurrent with an increase in the number of leaking vessels, suggesting that upregulation of MMP9 contributes to augmentation of brain hemorrhage. NF-κB triggers upregulation of MMP9 expression 35. EPO regulates the NF-κB signaling pathway that mediates EPO-induced neuroprotection 36. Thus, EPO could activate the NF-κB signaling pathway leading to an upregulation of MMP9 expression when EPO is administered in combination of tPA. Indeed, the delayed (6h) combination treatment substantially activated NF-κB signaling pathway in cerebral vessels, although our Western blot analysis did not reveal significant differences of NF-κB levels among experimental groups either at acute (2h after initiated treatments) or delayed (24h after initiated treatments) time point. Moreover, the delayed combination treatment upregulated IRAK1 in cerebral vessels. IRAK1 plays an important role in signal transduction of Toll-like receptors and interleukin-1 receptors 25. IRAK1 along with other molecules activate NF-κB 25. Collectively, we speculate that the delayed combination therapy may activate the NF-κB signaling pathway and IRAK1, leading to upregulation of MMP9 that mediates augmentation of brain hemorrhage observed in the present study. A possible limitation of the present study is that the dose of tPA employed was 10 times higher than the dose used in humans, and this dose may upregulate IRAK1 and NF-κB and thereby contribute to hemorrhagic transformation. Future studies focusing on investigating the cause-effect of the NF-κB signaling pathway and IRAK1 upregulated by the delayed combination of rhEPO and tPA on exacerbating brain hemorrhage are warranted.

In summary, the present study indicates that the combination treatment with rhEPO and tPA carries a higher risk of intracerebral hemorrhage without reduction of ischemic brain damage when administered 6h after embolic stroke, while the combination therapy initiated 2h after stroke does not exacerbate brain hemorrhage and reduces ischemic cell damage.

Supplementary Material

supplement

Acknowledgments

The authors wish to thank Cindi Roberts and Qing-e Lu for technical assistance. This work was supported by NINDS grants PO1 NS23393 and RO1 HL64766.

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