Post-ischemic treatment with erythropoietin or carbamylated erythropoietin reduces infarction and improves neurological outcome in a rat model of focal cerebral ischemia

Abstract

Background and purpose: Recombinant human erythropoietin (rhEPO; Epoetin-alpha; PROCRITtrade mark) has been shown to exert neuroprotective and restorative effects in a variety of CNS injury models. However, limited information is available regarding the dose levels required for these beneficial effects or the neuronal responses that may underlie them. Here we have investigated the dose-response to rhEPO and compared the effects of rhEPO with those of carbamylated rhEPO (CEPO) in a model of cerebral stroke in rats.

Experimental approach: Rats subjected to embolic middle cerebral artery occlusion (MCAo) were treated with rhEPO or CEPO, starting at 6 h and repeated at 24 and 48 h, after MCAo. Cerebral infarct volumes were assessed at 28 days and neurological impairment at 7, 14, 21 and 28 days, post-MCAo.

Key results: rhEPO at dose levels of 500, 1150 or 5000 IU kg(-1) or CEPO at a dose level of 50 microg kg(-1) significantly reduced cortical infarct volume and reduced neurologic impairment. All doses of rhEPO, but not CEPO, produced a transient increase in haematocrit, while rhEPO and CEPO substantially reduced the number of apoptotic cells and activated microglia in the ischemic boundary region.

Conclusions and implications: These data indicate that rhEPO and CEPO have anti-inflammatory and anti-apoptotic effects, even with administration at 6 h following embolic MCAo in rats. Taken together, these actions of rhEPO and CEPO are likely to contribute to their reduction of neurologic impairment following cerebral ischemia.

Figure 1

Changes in haematocrit before, during and after treatment with rhEPO and CEPO. Zero and 1 day time points represent prior to MCA occlusion and CEPO or rhEPO treatment, respectively. ^*P<0.05 and ^**P<0.01 vs the vehicle group. N=10 rats per group. CEPO, carbamylated rhEPO; MCA, middle cerebral artery; rhEPO, recombinant human erythropoietin.

Figure 2

Infarct volumes 28 days after embolic MCA occlusion. Panel a shows infarction on a coronal section stained with H&E of a representative rat from vehicle, rhEPO 5000 IU kg⁻¹ and CEPO 50 μg kg⁻¹ groups. Panel b shows quantitative analysis revealing that delayed (6 h) treatment with CEPO or rhEPO reduced infarct volume. Infarct volumes were measured as a whole hemisphere (Whole), cortex and subcortex. N=12 rats for control, rhEPO 500 IU kg⁻¹, and rhEPO 5000 IU kg⁻¹ groups. N=6 rats for rhEPO 50 IU kg⁻¹, rhEPO 1150 IU kg⁻¹, and CEPO 50 μg kg⁻¹ groups. ^*P<0.05 vs the vehicle group. CEPO, carbamylated rhEPO; H&E, haematoxylin and eosin; MCA, middle cerebral artery; rhEPO, recombinant human erythropoietin.

Figure 3

The effects of CEPO and rhEPO on neurological function. Delayed (6 h) treatment with CEPO or rhEPO improves neurological function measured by foot-fault test (a) and mNSS (b) compared with the vehicle group. ^*P<0.05 vs the vehicle group and ⁺P<0.05 vs rhEPO 500 and 1150 IU kg⁻¹ groups. N=10 rats per group. CEPO, carbamylated rhEPO; mNSS, modified neurological severity score; rhEPO, recombinant human erythropoietin.

Figure 4

The effect of CEPO and EPO on apoptosis and microglial responses. Panels a–f are images of activated microglial cells identified by IB4-positive cells in the cortical boundary region from representative rats treated with vehicle (a and b), rhEPO 5000 IU kg⁻¹ (c and d) or CEPO 50 μg kg⁻¹ (e and f). Panels b, d and f are high magnification images from the box area in panels a, c and e, respectively. (g and h) Quantitative data of TUNEL-positive cells and activated microglial cells, respectively, in the ischaemic boundary region. Core in the panels a, c and e indicates the ischaemic core. Bar=80 μm for panels a, c and e; Bar=20 μm for panels b, d, and f. CEPO, carbamylated rhEPO; EPO, erythropoietin; rhEPO, recombinant human erythropoietin; TUNEL, deoxynucleotidyl transferase-mediated biotinylated UTP nick end labelling model. ^*P<0.05 vs the vehicle group.