Necati Gökmen

Erythropoietin prevents motor neuron apoptosis and neurologic disability in experimental spinal cord ischemic injury

The cytokine erythropoietin (EPO) possesses potent neuroprotective activity against a variety of potential brain injuries, including transient ischemia and reperfusion. It is currently unknown whether EPO will also ameliorate spinal cord injury. Immunocytochemistry performed using human spinal cord sections showed abundant EPO receptor immunoreactivity of capillaries, especially in white matter, and motor neurons within the ventral horn. We used a transient global spinal ischemia model in rabbits to test whether exogenous EPO can cross the blood–spinal cord barrier and protect these motor neurons. Spinal cord ischemia was produced in rabbits by occlusion of the abdominal aorta for 20 min, followed by saline or recombinant human (rHu)-EPO (350, 800, or 1,000 units/kg of body weight) administered intravenously immediately after the onset of reperfusion. The functional neurological status of animals was better for rHu-EPO-treated animals 1 h after recovery from anesthesia, and improved dramatically over the next 48 h. In contrast, saline-treated animals exhibited a poorer neurological score at 1 h and did not significantly improve. Histopathological examination of the affected spinal cord revealed widespread motor neuron injury associated with positive terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling in control but not in rHu-EPO-treated animals. These observations suggest both an acute as well as a delayed beneficial action of rHu-EPO in ischemic spinal cord injury. Because rHu-EPO is currently used widely with an excellent safety profile, clinical trials evaluating its potential to prevent motor neuron apoptosis and the neurological deficits that occur as a consequence of ischemic injury are warranted.

Asialoerythropoietin is a nonerythropoietic cytokine with broad neuroprotective activity in vivo

Erythropoietin (EPO) is a tissue-protective cytokine preventing vascular spasm, apoptosis, and inflammatory responses. Although best known for its role in hematopoietic lineages, EPO also affects other tissues, including those of the nervous system. Enthusiasm for recombinant human erythropoietin (rhEPO) as a potential neuroprotective therapeutic must be tempered, however, by the knowledge it also enlarges circulating red cell mass and increases platelet aggregability. Here we examined whether erythropoietic and tissue-protective activities of rhEPO might be dissociated by a variation of the molecule. We demonstrate that asialoerythropoietin (asialoEPO), generated by total enzymatic desialylation of rhEPO, possesses a very short plasma half-life and is fully neuroprotective. In marked contrast with rhEPO, this molecule at doses and frequencies at which rhEPO exhibited erythropoiesis, did not increase the hematocrit of mice or rats. AsialoEPO appeared promptly within the cerebrospinal fluid after i.v. administration; intravenously administered radioiodine-labeled asialoEPO bound to neurons within the hippocampus and cortex in a pattern corresponding to the distribution of the EPO receptor. Most importantly, asialoEPO exhibits a broad spectrum of neuroprotective activities, as demonstrated in models of cerebral ischemia, spinal cord compression, and sciatic nerve crush. These data suggest that nonerythropoietic variants of rhEPO can cross the blood–brain barrier and provide neuroprotection.

Recombinant human erythropoietin counteracts secondary injury and markedly enhances neurological recovery from experimental spinal cord trauma

Erythropoietin (EPO) functions as a tissue-protective cytokine in addition to its crucial hormonal role in red cell production. In the brain, for example, EPO and its receptor are locally produced, are modulated by metabolic stressors, and provide neuroprotective and antiinflammatory functions. We have previously shown that recombinant human EPO (rhEPO) administered within the systemic circulation enters the brain and is neuroprotective. At present, it is unknown whether rhEPO can also improve recovery after traumatic injury of the spinal cord. To evaluate whether rhEPO improves functional outcome if administered after cord injury, two rodent models were evaluated. First, a moderate compression of 0.6 N was produced by application of an aneursym clip at level T3 for 1 min. RhEPO (1,000 units per kg of body weight i.p.) administered immediately after release of compression was associated with partial recovery of motor function within 12 h after injury, which was nearly complete by 28 days. In contrast, saline-treated animals exhibited only poor recovery. In the second model used, rhEPO administration (5,000 units per kg of body weight i.p. given once 1 h after injury) also produced a superior recovery of function compared with saline-treated controls after a contusion of 1 N at level T9. In this model of more severe spinal cord injury, secondary inflammation was also markedly attenuated by rhEPO administration and associated with reduced cavitation within the cord. These observations suggest that rhEPO provides early recovery of function, especially after spinal cord compression, as well as longer-latency neuroprotective, antiinflammatory and antiapoptotic functions.

Erythropoietin improves long-term spatial memory deficits and brain injury following neonatal hypoxia-ischemia in rats

It is well known that neonatal hypoxic-ischemic brain injury leads to mental retardation and deficits in cognitive abilities such as learning and memory in human beings. The ameliorative effect of erythropoietin (Epo) on experimental hypoxic-ischemic brain injury in neonatal rats has been recently reported. However, the effect of Epo on cognitive abilities in the hypoxic-ischemic brain injury model is unknown. The aim of this study is to investigate the effects of Epo on learning-memory, behavior and neurodegeneration induced by hypoxia-ischemia. Seven days old Wistar Albino rat pups have been used in the study (n = 28). Experimental groups in the study were: (1) saline-treated hypoxia-ischemia group, (2) Epo-treated (i.p., 1000 U/kg) hypoxia-ischemia group, (3) sham-operated group, (4) control group. In hypoxia-ischemia groups, left common carotid artery was ligated permanently on the seventh postnatal day. Two hours after the procedure, hypoxia (92% nitrogen and 8% oxygen) was induced for 2.5 h. Epo was administered as a single dose immediately after the hypoxia period. When pups were 22 days old, learning experiments were performed using Morris water maze. On the 20th week, when brain development is accepted to be complete, learning experiments were repeated. Rats were then perfused and brains removed for macroscopic and microscopic evaluation. Epo treatment immediately after hypoxic-ischemic insult significantly improved long-term neurobehavioral achievements when tested during the subsequent phase of brain maturation and even into adulthood. Histopathological evaluation demonstrated that Epo also significantly diminished brain injury and spared hippocampal CA1 neurons. In conclusion, Epo administrated as a single dose immediately after neonatal hypoxic-ischemic insult provides benefit over a prolonged period in the still developing rat brain. Since the wide use of Epo in premature newborns, this agent may be potentially beneficial in treating asphyxial brain damage in the perinatal period.

Neuroprotective Effect of Erythropoietin on Hypoxic-Ischemic Brain Injury in Neonatal Rats

Erythropoietin (Epo) prevents ischemia and hypoxia-induced neuronal death in vitro. Recent studies have shown that this cytokine also has in vivo neuroprotective effects in cerebral and spinal ischemia in adult rodents. In this study, we aimed to investigate the effect of systemically administered recombinant human Epo on infarct volume and apoptotic neuronal death in a newborn rat hypoxic-ischemic brain injury model. Our results showed that a single dose of intraperitoneal Epo treatment (1,000 U/kg) significantly decreased the mean infarct volume as compared to the control group. In contrast to the Epo-treated group, histopathological examination by positive terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling of the affected brain in control animals revealed widespread neuronal injury associated with numerous apoptotic cells. Morphometric analysis to determine the extent of damage quantitatively ascertained that the mean infarct volume was significantly lower in the Epo-treated group (p < 0.003). These results suggest the beneficial neuroprotective effect of Epo in this model of neonatal hypoxic-ischemic brain injury. To our knowledge, this is the first study that demonstrates a protective effect of Epo against hypoxia-ischemia in the developing brain.

Erythropoietin Increases Glutathione Peroxidase Enzyme Activity and Decreases Lipid Peroxidation Levels in Hypoxic-Ischemic Brain Injury in Neonatal Rats

Background: We have previously shown that erythropoietin (Epo) exerts neuroprotective effects in the Rice-Vannucci model of neonatal hypoxic-ischemic brain injury. However, the mechanisms of Epo protection in this model are still unclear. Objectives: In the present study, we studied the effects of systemically administered Epo on lipid peroxidation levels and antioxidant enzyme (superoxide dismutase and glutathione peroxidase) activities following hypoxic-ischemic brain injury in neonatal rats. Methods: Seven-day-old Wistar rat pups were subjected to left carotid artery occlusion followed by 2.5 h of hypoxic exposure. Brain lipid peroxidation levels and antioxidant enzyme activities were measured in the injured hemispheres 24 h after the hypoxic-ischemic insult. Results: Hypoxic-ischemic injury significantly increased the thiobarbituric acid-reactive substance levels in the injured hemispheres as compared to the control group. In addition, glutathione peroxidase activity was significantly elevated in Epo-treated animals compared to saline-treated animals and the control group. Conclusions: These results suggest that Epo exerts neuroprotective effects against hypoxic-ischemic brain injury at least partially via the modulation of antioxidant enzyme activity.

Erythropoietin exerts neuroprotective effect in neonatal rat model of hypoxic–ischemic brain injury

Hypoxic-ischemic encephalopathy seen in survivors of perinatal asphyxia is a frequently encountered and a major clinical problem for which there is currently no effective treatment. Hematopoietic neuroprotective agents, such as erythropoietin (EPO) may rescue neurons from cell death in this setting. EPO is a cytokine hormone that has neuroprotective effect in vitro and in vivo. In this study, we evaluated the effect of posthypoxic EPO administration in an animal model of neonatal hypoxic-ischemic injury. Our results show that a single intracerebroventricular injection of EPO immediately after hypoxic-ischemic insult in neonatal rat model of hypoxic-ischemia reduced the extent of hypoxic-ischemic brain damage. The mean infarct volume assessed 7 days after hypoxia was significantly smaller in EPO-treated group than in the control group. These findings suggest that EPO may provide benefit after hypoxic-ischemic events in the developing brain, a major contributor to static encephalopathy and cerebral palsy.

Selective Inhibition of Nitric Oxide in Hypoxic-Ischemic Brain Model in Newborn Rats: Is It an Explanation for the Protective Role of Erythropoietin?

Erythropoietin (Epo) exerts neuroprotection against neuronal death induced by ischemia and hypoxia in vitro and in vivo. Recent studies suggest that the neuroprotective effects of Epo may depend upon different mechanisms, including the inhibition of nitric oxide (NO). We recently demonstrated that Epo exerts neuroprotection in a model of neonatal hypoxic-ischemic brain damage. In the present study, we directly determined whether systemic administration of recombinant Epo modulates cerebral NO production in a neonatal rat model of hypoxic-ischemic brain injury. Seven-day-old Wistar rat pups were subjected to left carotid artery occlusion followed by 2.5 h of hypoxic exposure. Brain nitrite levels were evaluated in both hemispheres (carotid ligated or nonligated) by Griess reagent 72 h after the hypoxic-ischemic insult. Our results show that hypoxic-ischemic insult results a significant increase in NO production as compared with NO levels in hypoxic hemispheres and control animals. A single dose of Epo treatment (1,000 U/kg i.p.) significantly decreased NO overproduction in the hypoxic-ischemic hemisphere, whereas no significant change appeared in hypoxia alone or in controls. These data suggest that the selective inhibitory effect of Epo on NO overproduction could have a neuroprotective effect in neonatal hypoxic-ischemic brain injury.

Erythropoietin Downregulates Bax and DP5 ProApoptotic Gene Expression in Neonatal Hypoxic-Ischemic Brain Injury

Background: Perinatal asphyxia is an important cause of neonatal mortality and subsequent serious sequelae such as motor and cognitive deficits and seizures. The ameliorative effect of erythropoietin (Epo) on experimental hypoxic-ischemic brain injury in neonatal rats has been recently reported. Recent studies also confirm the antiapoptotic effect of Epo in a variety of in vitro and in vivo neuronal injury models including hypoxic-ischemic brain injury. However, molecular mechanisms of Epo protection and antiapoptotic effect in this model are unclear. Epo may exert its antiapoptotic effect via the differential regulation of the expression of genes involved in the apoptotic process. Objectives: Thus, in the present study, we studied the effects of systemically administered Epo on antiapoptotic (bcl-2, bcl-XL), proapoptotic (bax and DP5) gene expression following hypoxic-ischemic brain injury in neonatal rats. Methods: Seven- day-old Wistar rat pups were divided into three groups: control group (n = 15), saline-treated group (n = 17), and Epo-treated group (n = 18). Rat pups were subjected to left carotid artery occlusion followed by 2.5 h of hypoxic exposure. Epo-treated group received an intraperitoneal injection of recombinant human Epo at a dose of 1,000 units/kg, saline-treated group received an intraperitoneal injection of saline at the same volume of Epo. Forty-eight hours after hypoxia, 3 animals in each group were killed for histopathological evaluation. To detect DNA fragmentation in cell nuclei, terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling reaction was applied. Bcl-2 and bax protein expression were also analyzed with immunohistochemistry. For reverse transcriptase-polymerase chain reaction (RT-PCR) analysis, rats were sacrificed 4, 12, and 24 h after hypoxia. Bcl-2, bcl-XL, bax, and DP5 mRNA expression were analyzed by RT-PCR. Results: Epo significantly prevented hypoxia-ischemia-induced bax and DP5 mRNA upregulation in brain tissue. Epo did not show any effect on bcl-XL transcription altered by injury. However, Epo reversed injury-induced downregulation in bcl-2 transcription. Modulating effects of Epo on bcl-2 and bax protein expression were also revealed by immunohistochemistry. Conclusions: These results suggest that Epo exerts a neuroprotective effect against hypoxic-ischemic brain injury, at least partially, via the differential regulation of the expression of genes involved in apoptotic process.

Erythropoietin-induced changes in brain gene expression reveal induction of synaptic plasticity genes in experimental stroke

Erythropoietin (EPO) is a neuroprotective cytokine in models of ischemic and nervous system injury, where it reduces neuronal apoptosis and inflammatory cytokines and increases neurogenesis and angiogenesis. EPO also improves cognition in healthy volunteers and schizophrenic patients. We studied the effect of EPO administration on the gene-expression profile in the ischemic cortex of rats after cerebral ischemia at early time points (2 and 6 h). EPO treatment up-regulated genes already increased by ischemia. Hierarchical clustering and analysis of overrepresented functional categories identified genes implicated in synaptic plasticity—Arc, BDNF, Egr1, and Egr2, of which Egr2 was the most significantly regulated. Up-regulation of Arc, BDNF, Dusp5, Egr1, Egr2, Egr4, and Nr4a3 was confirmed by quantitative PCR. We investigated the up-regulation of Egr2/Krox20 further because of its role in neuronal plasticity. Its elevation by EPO was confirmed in an independent in vivo experiment of cerebral ischemia in rats. Using the rat neuroblastoma B104, we found that wild-type cells that do not express EPO receptor (EPOR) do not respond to EPO by inducing Egr2. However, EPOR-expressing B104 cells induce Egr2 early upon incubation with EPO, indicating that Egr2 induction is a direct effect of EPO and that EPOR mediates this effect. Because these changes occur in vivo before decreased inflammatory cytokines or neuronal apoptosis is evident, these findings provide a molecular mechanism for the neuroreparative effects of cytokines and suggest a mechanism of neuroprotection by which promotion of a plastic phenotype results in decreased inflammation and neuronal death.