rhEPO Administration Research Articles

A re-assessment of erythropoietin as a neuroprotective agent following rat spinal cord compression or contusion injury

This study was initiated due to an NIH “Facilities of Research — Spinal Cord Injury” contract to support independent replication of published studies that appear promising for eventual clinical testing. We repeated a study reporting the beneficial effects of recombinant human erythropoietin (rhEPO) treatment after spinal cord injury (SCI). Moderate thoracic SCI was produced by two methods: 1) compression due to placement of a modified aneurysm clip (20 g, 10 s) at the T3 spinal segment ( n=45) [followed by administration of rhEPO 1000 IU/kg/IP in 1 or 3 doses (treatment groups)] and 2) contusion by means of the MASCIS impactor ( n = 42) at spinal T9 (height 12.5 cm, weight 10 g) [followed by the administration of rhEPO 5000 IU/kg/IP for 7d or single dose (treatment groups)]. The use of rhEPO following moderate compressive or contusive injury of the thoracic spinal cord did not improve the locomotor behavior (BBB rating scale). Also, secondary changes (i.e. necrotic changes followed by cavitation) were not significantly improved with rhEPO therapy. With these results, although we cannot conclude that there will be no beneficial effect in different SCI models, we caution researchers that the use of rhEPO requires further investigation before implementing clinical trials.

Effect of Erythropoietin on Blood Pressure and on the Vascular Endothelial ET-1/ETB Receptor System

Recombinant human erythropoietin (rhEPO) increases blood pressure (BP) and the vascular production of endothelin-1 in renal failure rats. This study was designed to investigate the effect of rhEPO on BP and on the ET-1/ET(B)R system in rats with normal renal function. To further characterize the effect of rhEPO on the ET-1/ET(B)R system, we also studied heterozygous (+/-) ET(B)R knockout (KO) mice. The animals received either the vehicle or rhEPO (100 U/kg subcutaneously three times per week). ET(B)R(+/-) mice were compared with ET(A)R(+/-) and wild-type (WT) mice. In rats, the ET(B)R mRNA expression was assessed in blood vessels as well as the vascular ET(B)R density using immunohistochemistry. In mice, ET-1 concentration was measured in the thoracic aorta. RhEPO administration increased hematocrit levels in all treated animals. This therapy had no effect on BP in normal rats, but it did increase vascular and renal cortex ET(B)R mRNA expression. Immunohistochemistry confirmed that the ET(B)R density was increased in blood vessel endothelium in these normal rats. In contrast, rhEPO increased BP in ET(B)R(+/-) mice and this pressor effect was associated with higher ET-1 concentrations in the thoracic aorta. RhEPO exerts a pleotropic effect on the endothelial ET-1/ET(B)R system. The increase in endothelial ET(B)R expression may contribute to maintaining normal BP during rhEPO administration in normal animals. Conversely, conditions with deficient ET(B)R expression, such as in ET(B)R(+/-) mice, may lead to hypertension while receiving the same therapy.

The efficacy of erythropoietin on acute spinal cord injury. An experimental study on a rat model

The accumulated knowledge of erythropoietin (EPO) interaction in neural injury has led to potentially novel therapeutic strategies. Previous experimental studies of recombinant human EPO (rhEPO) administration have shown favorable results after central and peripheral neural injury. In the present study we used the aneurysmal clip model to evaluate the efficacy of two different regimes of rhEPO administration on the functional outcome after severe acute spinal cord injury (ASCI). Thirty rats were operated on with posterior laminectomy at thoracic 10th vertebra. Spinal cord trauma produced by extradural placement of the aneurysm clip, for 1 min. Animals were divided into three groups; the first group received a low total EPO dose (EPO-L), (2 doses of 1,000 IU each s.c.). The second group was administered the high total EPO dose (EPO-H), (14 doses of 1,000 IU each s.c.), and the third was the Control group, which received normal saline in the same time fashion with EPO-H group. Follow-up was for 6 weeks. Estimation of the functional progress of each rat was calculated using the locomotor rating scale of Basso et al, with a range from 0 to 21. After surgery the animals suffered paraplegia with urinary disturbances. Rats that received EPO demonstrated statistically significant functional improvement compared to the Control group, throughout study interval. On the last follow-up at 6 weeks the EPO-L rats achieved a mean score 17.3 +/- 1.15, the EPO-H 14.7 +/- 1.82, and the control group 8.2 +/- 0.78. Comparison between the two EPO groups reveals superior final outcome of the group treated with lower total dose. Our study supports current knowledge, that EPO administration has a positive effect on functional recovery after experimental ASCI. These data reflect the positive impact of EPO on the pathophysiologic cascade of secondary neural damage. However, we observed a dose-related effect on functional recovery. Interestingly, large doses do not seem to favor the neurological recovery as lower doses do.

Differential Neuroprotective Properties of Endogenous and Exogenous Erythropoietin in a Mouse Model of Traumatic Brain Injury

Both heat acclimation (HA) and post-injury treatment with recombinant human erythropoietin (Epo, rhEpo, exogenous Epo) are neuroprotective against traumatic brain injury (TBI). Our previous data demonstrated that HA-induced neuroprotection includes improved functional recovery and reduced cerebral edema formation. Additionally, in earlier Western-blot analyses, we found that HA mice display increased expression of the specific erythropoietin receptor (EpoR) and of hypoxia-inducible factor-1 alpha (HIF-1 alpha), the inducible subunit of the transcription factor, which regulates Epo gene expression, but not of Epo itself. In light of this, the aim of the current study was threefold: (1) to assess Epo expression in the trauma area and hippocampus following HA, rhEpo administration, or combined HA-rhEpo treatment, using immunohistochemical methods that offer enhanced anatomical resolution; (2) to examine the effects of endogenous and exogenous Epo on edema formation in normothermic (NT) mice; and (3) to evaluate the effects of exogenous Epo administration on neuroprotective outcome measures in HA animals. HA induced enhanced expression of endogenous Epo in the trauma area and the hippocampus. Treatment with anti-Epo antibody given to NT mice increased edema formation, whereas rhEpo induced no beneficial effect. Cognitive performance testing and immunohistochemical findings reinforced HA and rhEpo as separate protective interventions but showed no advantage to combining the two strategies. We therefore suggest that HA-induced neuroprotection is shaped by pre-existing mediators but cannot be modified by post-injury treatment aimed at increasing the levels of neuroprotective agents.

Possible Involvement of Erythropoietin in Remote Renal Preconditioning-Induced Cardioprotection in Rats

Remote preconditioning is a unique phenomenon in which brief episodes of ischemia and reperfusion to remote organs protect the target organ against sustained ischemia/reperfusion (I/R)-induced injury. Protective effects of remote renal preconditioning are well established in the heart, but their mechanisms still remain to be elucidated. Hence, the present study was designed to investigate the possible involvement of erythropoietin in remote renal preconditioning (RRPC)-induced cardioprotection in rats. RRPC was performed by 4 episodes of 5 min renal artery occlusion followed by 5 min reperfusion. Gentamicin (100 mg/kg intraperitoneal) was administered for 6 days for induction of renal failure. Isolated rat hearts were perfused on Langendorff apparatus and were subjected to global ischemia for 30 min ischemia followed by 120 min reperfusion. The levels of lactate dehydrogenase (LDH) and creatine kinase (CK) were measured in coronary effluent to assess the degree of myocardial injury. Extent of myocardial infarct size and coronary flow rate was also measured. RRPC prevented I/R-induced myocardial injury and produced cardioprotective effects. However, cardioprotective effects of RRPC were not observed in renal failure rats, indicating the protective role of humoral factor was released from functional kidneys. In renal failure rats, exogenous administration of rhEPO (5,000 IU/kg intraperitoneal) with RRPC restored the cardioprotective effects of later. These results implicate that RRPC-induced cardioprotective effects may be mediated through release of erythropoietin from kidney.

Recombinant human erythropoietin administration protects cortical neurons from traumatic brain injury in rats

We explored the regulation of erythropoietin and erythropoietin receptor on traumatic brain injury (TBI), as well as the antiapoptotic effects of recombinant human erythropoietin (rhEPO) treatment. Female Wistar rats were randomly divided into three groups: rhEPO-treated TBI, vehicle-treated TBI, and sham-operated. TBI was induced by the Feeney free falling model. Rats were killed 5, 12, 24, 72, 120, or 168 h after TBI. Regulation of EPO, EPOR and Bcl-2 was detected by reverse transcription-polymerase chain reaction (RT-PCR), western blotting and immunofluorescence. Terminal deoxynucleotidyl transferase-mediated biotin-dUTP nick-end labeling (TUNEL) was used to assess DNA fragmentation after TBI. Induction of EPOR expression persisted for 168 h after TBI, whereas EPO was only slightly elevated for 72 h. In the rhEPO-treated TBI, Bcl-2 mRNA and protein levels were greater than in the vehicle-treated TBI. Bcl-2 mRNA peaked at 24 h and remained stable for 72-120 h. The number of TUNEL-positive cells in the rhEPO-treated TBI was far fewer than in the vehicle-treated TBI. EPOR regulation is enhanced for almost a week after TBI. Administration of rhEPO protects neurons by enhancing Bcl-2 expression, thereby inhibiting TBI-induced neuronal apoptosis.

Erythropoietin Pretreatment Protects Against Acute Chemotherapy Toxicity in Isolated Rat Hearts

The use of chemotherapeutic agents, such as anthracycline or trastuzumab, in oncology is limited by their cardiac toxicity. Recent experimental studies suggest that recombinant human erythropoietin (rhEPO) can be considered as a protective agent because its administration protects against cardiac ischemic injury, improving functional recovery, and reducing cell death. The aim of this study was to investigate whether pretreatment by rhEPO protects against acute cardiotoxicity induced by doxorubicin and trastuzumab, using the isolated rat heart model. Rats were treated with rhEPO (5000 IU/kg, intraperitoneally [i.p.]) or vehicle. One hour later, hearts were isolated and retrogradely perfused at constant flow. Following 20 mins of stabilization, hearts were perfused for 60 mins with modified-Krebs solution containing 6 mg/l doxorubicin or 10 mg/l trastuzumab. Hearts receiving doxorubicin were paced; those receiving trastuzumab were unpaced. Control hearts were perfused with modified-Krebs solution only. Doxorubicin exposure decreased left ventricular developed pressure (LVDP; approximately -40% of baseline) and increased end diastolic pressure (EDP; approximately +390% of baseline) and coronary perfusion pressure (CPP; approximately +70% of baseline). Incidence of ventricular tachycardia or fibrillation (VT/VF) was also significantly enhanced (86% vs. 0% in control group). Trastuzumab exposure increased CPP and EDP (approximately +70% of baseline for the both) without affecting LVDP. Prior rhEPO treatment significantly prevented doxorubicin-induced deleterious effects on LVDP, EDP, and VT/VF incidence. rhEPO administration also prevented trastuzumab-induced deleterious effects on CPP and EDP. This study shows that pretreatment by rhEPO protects myocardium against functional damage and electrophysiologic injury induced by acute doxorubicin or trastuzumab exposure. Further investigations are required to elucidate the precise mechanisms involved.

Erythropoietin for neuroprotection in preterm infants: feasiblity and safety study

Background: Long-term disability remains a major problem in very preterm infants. Novel strategies to protect developing organs, in particular the brain, are therefore of greatest interest in neonatal intensive care medicine. Erythropoietin has been shown to be protective against hypoxic-ischemic and inflammatory injuries in cell culture, animal models of brain injury, and in clinical trials in human adults. Objective: The rationale for our study was that early administration of high dose recombinant human erythropoietin (rhEpo) may reduce perinatal brain injury (IVH and PVL) in very preterm infants and improve neuro-developmental outcome. In a first step, we investigated whether high dose rhEpo given shortly after birth and subsequently over the first two days to very preterm infants is safe in terms of short-term outcome. Patients and Methods: Randomized, double masked single-centre trial with a 2 to 1 allocation in favor of rhEpo. Preterm infants (gestational age 24 0/7–31 6/7 weeks) were given rhEpo (n t=30; 3,000 U/kg body weight) or NaCl 0.9% (n c=15) intravenously at 3, 12–18 and 36–42 hours after birth. Results: The percentage of infants surviving without brain injury or retinopathy was 53% in the rhEpo group and 60% in the placebo group. There were no relevant differences regarding short-term outcomes such as IVH, retinopathy, sepsis, necrotizing enterocolitis, and bronchopulmonary dysplasia. In five infants of the rhEpo group with a gestational age <26 0/7 weeks, withdrawal of intensive care was decided (3/5 with severe bilateral IVH, 2/5 with pulmonary insufficiency); no infant of the control group died. rhEpo treatment did not result in significant differences in blood pressure, cerebral oxygenation, hemoglobin, leukocyte and platelet count. Conclusions: No significant adverse effects of early high-dose rhEpo treatment in very preterm infants were identified. These results enable us to embark on a large multicentre trial with the aim to determine if early high dose administration of rhEpo in very preterm infants improves neurodevelopmental outcome at 24 months and 5 years corrected age.

Recombinant human erythropoietin attenuates hepatic injury induced by ischemia/reperfusion in an isolated mouse liver model

Apoptosis is a central mechanism of cell death following reperfusion of the ischemic liver. Recombinant human erythropoietin (rhEPO) have an important role in the treatment of myocardial ischemia/reperfusion (I/R) injury, by preventing apoptosis. The aim of the study was to investigate the effect of different regimens of rhEPO in preventing apoptosis following I/R-induced hepatic injury. Isolated mouse livers were randomly divided into five groups: (1) control group, perfused for the whole study period (105 min); (2) 30-min perfusion followed by 90 min of ischemia and 15 min of reperfusion; (3), (4) and (5) like group 2, but with administration of rhEPO 5,000 units/kg i.p. at 30 min, 24 h, or both 30 min and 24 h respectively, before induction of ischemia. Perfusate liver enzyme levels and intrahepatic caspase-3 activity were measured, and apoptotic cells were identified by morphological criteria, TUNEL assay, and immunohistochemistry for caspase-3. Using immunoblot the expression of the proapoptotic JNK and inhibitor of NFkappaB (IkappaBalpha) were also evaluated. von Willebrand factor (vWF) immunohistochemistry was used as a marker of endothelial cells. Compared to the I/R livers, all 3 rhEPO pretreated groups showed: a significant reduction in liver enzyme levels (P < 0.05) and intrahepatic caspase-3 activity (P < 0.05), fewer apoptotic hepatocytes (P < 0.05) and positive vWF staining in numerous endothelial cells lining the sinusoids. EPO decreased JNK phosphorylation and the degradation of the inhibitor of NFkappaB (IkappaBalpha) during I/R. There was no added benefit of the multiple- over the single-dose rhEPO regimen. Pretreatment with one dose of rhEPO can attenuate post-I/R hepatocyte apoptotic liver damage. NFkappaB and JNK activation is likely to play a pivotal role in the pathophysiology of I/R hepatic injury and might have a key role in EPO-mediated protective effects. This effect is associated with the increase in sinusoidal vWF immunostaining suggests an additional effect of rhEPO in liver angiogenesis recovery. These findings have important implications for the potential use of rhEPO in I/R injury during liver transplantation.

Erythropoietin protects retinal neurons and glial cells in early-stage streptozotocin-induced diabetic rats

The purpose of this study was to investigate the efficacy of recombinant human erythropoietin (rhEPO) in preventing and reversing dysfunction of retinal neurons and glial cells in early-stage streptozotocin (STZ)-induced diabetic rats. Two weeks after STZ [60mg/kg body weight (b.w.), i.p.] injection, diabetic rats had been administered rhEPO (5000IU/kg of b.w., i.p.) injection three times weekly for 2weeks. The changes to the electroretinogram, retina ultrastructure, erythropoietin receptors (EPO-Rs) and the content of glutamate in retinas before and after the experiment in test and control groups were compared. The amplitudes of b-wave and oscillatory potentials (OPs) decreased at the end of the experiment in STZ-induced diabetic rats compared with the age-matched controls, whereas the amplitudes of b-wave and OPs showed no decrease in diabetic rats with rhEPO injection. Mitochondrial metamorphosis in ganglion cells occurred in STZ-induced diabetic rats but was not found in those rats with rhEPO treatment. EPO-Rs in retinas and the retinal glutamate of STZ-induced diabetic rats at the end of the experiment had increased obviously compared with rhEPO-injected diabetic rats. The abnormalities of retinal electrophysiological activity, ganglion cells with swollen mitochondria in the retinas, increased retinal glutamate and EPO-R in the retinas occurred early in the process of the disease course in diabetic rats. These aspects can all be improved by intraperitoneal administered rhEPO. The administration of rhEPO may be useful in the neural treatment of diabetic retinopathy at the early stage.

Recombinant Human Erythropoietin Administration Decreases the Frequency of Excessive Apoptosis in Erythroid Progenitors in the Model Mouse of Anemia Due to Chronic Renal Failure.

Recombinant human erythropoietin (rh-EPO) administration results in a dramatic improvement of anemia due to chronic renal failure (ACRF). We reported that excessive apoptosis in erythroid progenitors was observed in ACRF patients before rh-EPO administration, and that we have established the model mouse of ACRF by administration of adenine-rich diets (ADRD) in ASH meetings 2001 and 2006. In this study, we evaluated the frequency of apoptosis in bone marrow erythroid cells of the model mouse before and after erythropoietin (EPO) administration, to confirm the hypothesis that excessive apoptosis in erythroid progenitors induced anemia via insufficient production of EPO. ADRD was administrated to C57B6 male mice for 8 weeks. Every day subcutaneous administration of EPO started at 6 weeks and continued for 2 weeks. Serum BUN and Crt levels began to elevate from 2 weeks after ADRD administration (Table1). Hb levels began to decrease from 5 weeks after ADRD administration and increased after EPO administration in a dose dependent manner (Table1, Figure1). The frequencies of apoptosis in erythroid progenitors and mature erythroblasts were evaluated by three color flow cytometric analysis using anti-CD34-antibody, anti-EPO-receptor (EPO-R)-antibody and AnnexinV. Erythroid progenitors were identified as CD34(+) and EPO-R(+) cells, and mature erythroblasts identified as CD34(−) and EPO-R(+) cells. Much higher frequency of apoptosis was observed in erythroid progenitors at 6 weeks after ADRD administration (76.4±5.3%, n=4), while, the frequency of apoptosis decreased dramatically after EPO administration in a dose dependent manner (0U/kg/day: 70.1±6.9% at 7 weeks, 73.8±8.7% at 8 weeks, 10U/kg/day: 48.1±5.4%* at 7 weeks, 46.9±4.2%* at 8 weeks, 100U/kg/day: 36.9±2.5%* at 7 weeks, 21.2±6.8%* at 8 weeks, 1000U/kg/day: 18.4±2.9%* at 7 weeks, 15.1±3.5%* at 8 weeks, each n=4, *p<0.01) (Figure2). The frequency of apoptosis was not increased in mature erythroblasts at each week after ADRD administration. Our findings suggested that excessive apoptosis occurred mainly in CD34(+) erythroid progenitors by insufficient production of EPO, and that EPO had the anti-apoptotic effect to erythroid progenitors. Insufficient production of EPO might be one of the important causes of ACRF.Table 1BUN, Crt and Hb levels in C57B6 mice after ADRD administration.0 week2 weeks4 weeks5 weeks6 weeks7 weeks8 weeksBUN (mg/dl)40.1±4.6073.2±14.4150.7±11.6148.8±4.60164.0±8.40161.8±16.4193.0±9.10Crt (mg/dl)0.06±0.040.30±0.030.51±0.070.52±0.090.68±0.090.65±0.040.80±0.17Hb (g/dl)13.6±0.413.3±0.313.2±0.211.1±0.110.2±0.59.6±0.18.3±0.5 [Display omitted] [Display omitted]

Chronic erythropoietin-mediated effects on the expression of astrocyte markers in a rat model of contusive spinal cord injury

Using a standardized rat model of contusive spinal cord injury (SCI; [Gorio A, Gokmen N, Erbayraktar S, Yilmaz O, Madaschi L, Cichetti C, Di Giulio AM, Vardar E, Cerami A, Brines M (2002) Recombinant human erythropoietin counteracts secondary injury and markedly enhances neurological recovery from experimental spinal cord trauma. Proc Natl Acad Sci U S A 99:9450–9455]), we previously showed that the administration of recombinant human erythropoietin (rhEPO) improves both tissue sparing and locomotory outcome. In the present study, to better understand rhEPO-mediated effects on chronic astrocyte response to SCI in rat, we have used immunocytochemical methods combined with confocal and electron microscopy to investigate, 1 month after injury, the effects of a single rhEPO administration on the expression of a) aquaporin 4 (AQP4), the main astrocytic water channel implicated in edema development and resolution, and two molecules (dystrophin and syntrophin) involved in its membrane anchoring; b) glial fibrillary acidic protein (GFAP) and vimentin as markers of astrogliosis; c) chondroitin sulfate proteoglycans of the extracellular matrix which are upregulated after SCI and can inhibit axonal regeneration and influence neuronal and glial properties. Our results show that rhEPO administration after SCI modifies astrocytic response to injury by increasing AQP4 immunoreactivity in the spinal cord, but not in the brain, without apparent modifications of dystrophin and syntrophin distribution. Attenuation of astrogliosis, demonstrated by the semiquantitative analysis of GFAP labeling, was associated with a reduction of phosphacan/RPTPζ/β, whereas the levels of lecticans remained unchanged. Finally, the relative volume of a microvessel fraction was significantly increased, indicating a pro-angiogenetic or a vasodilatory effect of rhEPO. These changes were consistently associated with remarkable reduction of lesion size and with improvement in tissue preservation and locomotor recovery, confirming previous observations and underscoring the potentiality of rhEPO for the therapeutic management of SCI.

Role of gender in outcome after traumatic brain injury and therapeutic effect of erythropoietin in mice

The aim of this study was to investigate the role of gender in histological and functional outcome, angiogenesis, neurogenesis and therapeutic effects of recombinant human erythropoietin (rhEPO) in mice after traumatic brain injury (TBI). TBI caused both tissue loss in the cortex and cell loss in the dentate gyrus (DG) in the injured hemisphere at day 35 post TBI without a significant gender difference. After TBI, sensorimotor deficits were significantly larger in male mice compared to females, while similar spatial learning deficits were present in both genders. TBI alone significantly stimulated angiogenesis and neurogenesis in the cortex and in the DG of injured hemispheres in both genders. rhEPO at a dose of 5000 units/kg body weight administered intraperitoneally at 6 h, and 3 and 7 days after injury significantly reduced lesion volume and DG cell loss examined at day 35 after TBI as well as dramatically improved sensorimotor and spatial learning performance without an obvious gender proclivity. rhEPO significantly enhanced neurogenesis in the cortex and the DG of the ipsilateral hemisphere in male TBI mice. rhEPO did not affect angiogenesis in the ipsilateral cortex and DG in both genders after TBI. The present data demonstrate that posttraumatic administration of rhEPO improves histological and functional outcome in both genders, which may be mediated by reducing cortical tissue damage and DG cell loss in the ipsilateral hemisphere. In addition, the major gender propensity observed in the present study with mice after TBI without treatment is limited to sensorimotor deficits and cell proliferation.

Cellular Protection by Erythropoietin: New Therapeutic Implications?

Erythropoietin (EPO), the principal hematopoietic hormone produced by the kidney and the liver in fetuses, regulates mammalian erythropoiesis and exhibits diverse cellular effects in nonhematopoietic tissues. The introduction of recombinant human EPO (rhEPO) has marked a significant advance in the management of anemia associated with chronic renal failure. At the same time, experimental studies have unveiled its potential neuroprotective and cardioprotective properties, occurring independently of its hematopoietic action. As with other cytoprotective agents, administration of exogenous rhEPO can confer cerebral and myocardial protection against ischemia-reperfusion injury in terms of reduction in cellular apoptosis and necrosis, as well as improvement in functional recovery. Very recent studies even suggest that this drug could have beneficial applications in oncology, protecting against chemotherapy cardiotoxicity. The purpose of this letter is to review current information regarding the various conditions in which rhEPO and its derivates could confer cellular protection. We also address clinical perspectives and novel therapeutic strategies that could be developed based on these studies. Thus, EPO seems to be a very promising agent for protecting cellular survival during both acute and chronic diseases, and its future should be considered with enthusiasm.

Inhibitory effect on cerebral inflammatory agents that accompany traumatic brain injury in a rat model: A potential neuroprotective mechanism of recombinant human erythropoietin (rhEPO)

Erythropoietin (EPO) has recently been shown to have a neuroprotective effect in animal models of traumatic brain injury (TBI). However, the precise mechanisms remain unclear. Cerebral inflammation plays an important role in the pathogenesis of secondary brain injury after TBI. We, therefore, tried to analyze how recombinant human erythropoietin (rhEPO) might effect the inflammation-related factors common to TBI: nuclear factor kappa B (NF-kappaB), interleukin-1beta (IL-1beta), tumor necrosis factor-alpha (TNF-alpha), interleukin-6 (IL-6) and intercellular adhesion molecule-1 (ICAM-1) in a rat TBI model. Male rats were given 0 or 5000 units/kg injections of rhEPO 1h post-injury and on days 1, 2 and 3 after surgery. Brain samples were extracted at 3 days after trauma. We measured NF-kappaB by electrophoretic mobility shift assay (EMSA); IL-1beta, TNF-alpha and IL-6 by enzyme-linked immunosorbent assay (ELISA); ICAM-1 by immunohistochemistry; brain edema by wet/dry method; blood-brain barrier (BBB) permeability by Evans blue extravasation and cortical apoptosis by terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) method. We found that NF-kappaB, pro-inflammatory cytokines and ICAM-1 were increased in all injured animals. In animals given rhEPO post-TBI, NF-kappaB, IL-1beta, TNF-alpha and ICAM-1 were decreased in comparison to vehicle-treated animals. Measures of IL-6 showed no change after rhEPO treatment. Administration of rhEPO reduced brain edema, BBB permeability and apoptotic cells in the injured brain. In conclusion, post-TBI rhEPO administration may attenuate inflammatory response in the injured rat brain, and this may be one mechanism by which rhEPO improves outcome following TBI.

L-Arginine Administration Reverses Anemia Associated with Renal Disease

Recombinant human erythropoietin (rhEpo) has proved to be remarkably safe and effective for the treatment of anemia. Despite the use of rhEpo, concerns about its cost, the need for frequent parenteral administration, and the development of anti-Epo antibodies have prompted the development of improved agents to rescue anemia. Patients with anemia associated with renal disease are usually treated by intravenous or subcutaneous rhEpo administration; however, some patients do not respond well to rhEpo, because of the presence of Epo antibody or other unknown reasons. A new, orally administered drug is needed as an economical and effective method to treat such patients. We administered 1.3 g/day of L-arginine to 8 elderly patients with anemia associated with renal disease. All 8 patients responded to the treatment with increases in hemoglobin levels. Six of the patients showed improved renal function. There were no significant adverse effects. Our data show that oral administration of 1.3 g/day of L-arginine significantly improves Epo production and reverses anemia without adverse effects in elderly patients who have anemia associated with renal disease and are in the predialysis state of chronic renal failure.

Erythropoietin Protects from Post-Traumatic Edema in the Rat Brain

Erythropoietin (Epo) is gaining interest in various neurological insults as a possible neuroprotective agent. We determined the effects of recombinant human Epo (rhEpo, 5000 IU per kg bw) on brain edema induced in rats by traumatic brain injury (TBI; impact-acceleration model; rhEpo administration 30 mins after injury). Magnetic resonance imaging (MRI) and a gravimetric technique were applied. In the MRI experiments, the apparent diffusion coefficient (ADC) and the tissue T(1) relaxation time were measured hourly in the neocortex and caudoputamen, during a 6 h time span after TBI. In the gravimetric experiments, brain water content (BWC) was determined in these two regions, 6 h after TBI. Apparent diffusion coefficient measurements showed that rhEpo decreased brain edema early and durably. Gravimetric measurements showed that rhEpo decreased BWC at H(6) in the neocortex as well as in the caudoputamen. No significant differences in ADC, in T(1), or in BWC were found between rhEpo treated-TBI rats and sham-operated rats. Our findings show that post-traumatic administration of rhEpo can significantly reduce the development of brain edema in a model of diffuse TBI. Further studies should be conducted to identify the biochemical mechanisms involved in these immediate effects and to assess the use of rhEpo as a possible therapy for post-traumatic brain edema.

Intrastriatal administration of erythropoietin protects dopaminergic neurons and improves neurobehavioral outcome in a rat model of Parkinson’s disease

Erythropoietin (EPO), a hematopoietic cytokine, has recently been demonstrated to protect nigral dopaminergic neurons in a mouse model of Parkinson’s disease (PD). In the present study, we tested the hypothesis that recombinant human erythropoietin (rhEPO) could protect dopaminergic neurons and improve neurobehavioral outcome in a rat model of PD. rhEPO (20 units in 2 μl of vehicle) was stereotaxically injected into one side of the striatum. 6-Hydroxydopamine (6-OHDA) was injected into the same side 1 day later. Another group of rats received rhEPO (5000 u/kg, i.p.) daily for 8 days, and unilateral injection of 6-OHDA in the striatum 3 days after systemic administration of rhEPO. We observed that intrastriatal administration, but not systemic administration of rhEPO significantly reduced the degree of rotational asymmetry. The rhEPO-treated rats also showed an improvement in skilled forelimb use when compared with control rats. The number of tyrosine hydroxylase (TH)-immunoreactive (IR) neurons in the ipsilateral substantia nigra (SN) was significantly larger in intrastriatal rhEPO-treated rats than that in control rats. TH-IR fibers in the 6-OHDA-lesioned striatum were also increased in the intrastriatal rhEPO-treated rats when compared with control rats. In addition, there were lower levels of expression of major histocompatibility complex (MHC) class II antigens and a smaller number of activated microglia in the ipsilateral SN in intrastriatal rhEPO-treated rats than that in control rats at 2 weeks, suggesting that intrastriatal injection of rhEPO attenuated 6-OHDA-induced inflammation in the ipsilateral SN. Our results suggest that intrastriatal administration of rhEPO can protect nigral dopaminergic neurons from cell death induced by 6-OHDA and improve neurobehavioral outcome in a rat model of PD. Anti-inflammation may be one of mechanisms responsible for rhEPO neuroprotection.

Deleterious myocardial consequences induced by intermittent hypoxia are reversed by erythropoietin

The aim of this work was to investigate the effects of different recombinant human erythropoietin (rhEPO) doses on the infarction development and if rhEPO could protect against the deleterious consequences induced by a previous intermittent hypoxia (IH, FiO(2) 5%, 4h). First, isolated rat hearts were submitted to an ischemia-reperfusion. rhEPO was infused before or after ischemia, at different doses. Secondly, rats were exposed to of IH. Twenty-four hours later, hearts underwent the ischemia-reperfusion protocol. For some hearts, 5Uml(-1) rhEPO was infused as previously. We observed that rhEPO has a dose-dependant effect on infarct size since it was significantly reduced by rhEPO infusions before or after ischemia. We also showed that 4h of IH induced a higher sensitivity to the infarction which was prevented by rhEPO. In conclusion, rhEPO administration before or after ischemia can protect isolated rat myocardium in a dose dependent manner and efficiently prevents the higher sensitivity to the infarction induced by previous intermittent hypoxia.

Anemia of Chronic Renal Failure Is Associated with Much Higher Frequency of Apoptosis in Erythroid Progenitor in a Mouse Model.

Insufficient production of erythropoietin (EPO) is one of the important causes of anemia of patients with chronic renal failure (CRF). Recombinant human erythropoietin (rh-EPO) administration results in a dramatic improvement of anemia in these patients. In ASH meeting 2001, we reported that excessive apoptosis in erythroid progenitor was observed in anemia of CRF patients before rh-EPO administration. In this study, we established a mouse model of anemia of CRF to confirm the hypothesis that excessive apoptosis in erythroid progenitor induced anemia. C57B6 male mice was treated with adenine-rich diets (ADRD) for 8 weeks. Serum blood urea nitrogen (BUN), creatinine (Crt) and complete blood count (CBC) were measured every week. The frequency of apoptosis in erythroid progenitor and mature erythroblasts were evaluated by three color flow cytometric analysis. Apoptotic cells were analyzed by AnnexinV. The cell populations of erythroid progenitor were identified as CD34(+)erythropoietin-receptor (EPO-R)(+) cells and mature erythroblasts identified as CD34(-)EPO-R(+) cells. Serum BUN and Crt levels began to elevate from 2 weeks after ADRD administration (BUN; 40.1±4.6, 73.2±14.4, 150.7±11.6, 164.0±8.4, 193.0±23.3mg/dl, Crt; 0.06±0.04, 0.30±0.03, 0.51±0.07, 0.68±0.09, 0.80±0.17mg/dl, n=5: mean±SD at 0, 2, 4, 6 and 8 weeks after ADRD administration, respectively). Hb and Ht began to decrease from 6 and 4 weeks after ADRD administration (Hb; 13.6±0.4, 13.3±0.3, 13.2±0.2, 10.2±0.5, 8.3±0.5 g/dl, Ht; 52.4±1.4, 49.4±1.7, 43.6±0.2, 35.4±1.8, 30.8±1.6%, n=5: mean±SD at 0, 2, 4, 6 and 8 weeks after ADRD administration, respectively). Serum EPO levels were not elevated after ADRD administration, regardless of anemia. Much higher frequency of apoptosis was observed in erythroid progenitor from 4 to 8 weeks after ADRD administration, compared to that at 0 week (CD34(+)EPO-R(+); 18.2±7.4, 41.7±7.3, 56.0±4.8, 76.4±5.3, 70.1±6.9, 73.8±8.7%, n=5, mean±SD at 0, 4, 5, 6, 7 and 8 weeks after ADRD administration, p<0.05, respectively). While, the frequency of apoptosis was not high in mature erythroblasts after ADRD administration (CD34(-)EPO-R(+); 1.58±1.4, 0.21±0.1, 0.58±0.1, 0.77±0.7, 0.49±0.2, 0.25±0.2%, n=5, mean±SD at 0, 4, 5, 6, 7 and 8 weeks after ADRD administration, N.S, respectively). Pathological examination of kidney showed that tubular damage was diffusely observed from 2 to 8 weeks after ADRD administration, and tubular necrosis was suspected as the major cause of renal failure.Our findings suggested that excessive apoptosis occurred mainly in CD34(+) erythroid progenitor by insufficient production of EPO, which induced anemia in mice with CRF.