SEARCH

SEARCH BY CITATION

Keywords:

  • Erythropoietin;
  • Neonatal hypoxia;
  • Status Epilepticus;
  • Neuronal apoptosis;
  • Seizure susceptibility

Abstract

  1. Top of page
  2. Abstract
  3. METHODS
  4. RESULTS
  5. DISCUSSION
  6. REFERENCES

Summary: Purpose: To determine if posthypoxia treatment with erythropoietin (EPO) has protective effects against subsequent susceptibility to seizure related neuronal injury in rat pups subjected to acute hypoxia at P10.

Methods: Four groups of rats were manipulated at P10, as described below, then all received kainic acid (KA) (10 mg/kg i.p.) at P29: Hypoxia-NS-KA group (n = 11): subjected to acute hypoxia (down to 4% O2), and then immediately received saline i.p. Hypoxia-EPO-KA group (n = 10): subjected to acute hypoxia and then immediately received EPO (1,000 U/Kg i.p.). Normoxia-NS-KA group (n = 11): sham manipulated and injected with saline. Normoxia-EPO-KA group (n = 10): sham manipulated then immediately injected with EPO (1000 U/Kg i.p.). After receiving KA at P29, all rats were monitored using videotape techniques, and were sacrificed at P31. TUNEL and Hoechst stains to assess for apoptosis, and regular histology for hippocampal cell counts were performed.

Results: Administration of the single dose of erythropoietin directly after an acute hypoxic event at P10 resulted at P29 in increased latency to forelimb clonus seizures, reduced duration of these seizures, protection against hippocampal cell loss, and decreased hippocampal apoptosis in the Hypoxia-EPO-KA group as compared to the Hypoxia-NS-KA group.

Conclusion: These data support the presence of favorable protective effects of erythropoietin against the long-term consequences of acute hypoxia in the developing brain and raise the possibility of its investigation as a potential neuroprotective agent after human neonatal hypoxic encephalopathy.

Neonatal seizures are often the result of hypoxic encephalopathy (Volpe, 2000). The development of epilepsy, often associated with cerebral palsy and mental retardation, is seen in many infants who experienced prior EEG-proven neonatal seizures due to birth asphyxia (Legido et al., 1991). In a well-established rat model of hypoxic encephalopathy, it has been demonstrated that hypoxia induces acute seizures only if it occurs during a time span ranging from postnatal day (P) 10 to 12 (Jensen et al., 1991). In this age group it also enhances long term excitability within the hippocampal neuronal network, increases susceptibility to seizure induced neuronal injury later in life, and leads to hippocampal neuronal cell loss and memory impairment in adulthood (Jensen et al., 1992; Jensen et al., 1998; Koh and Jensen, 2001; Mikati et al., 2005). Evidence in the literature shows that angiogenesis and its regulatory factors are affected after neonatal rat hypoxic-ischemic encephalopathy (Huang et al., 2004), and that impaired angiogenesis is associated with neuronal apoptosis in the developing brain (Raab et al., 2004). One of the emerging novel neuroprotective angiogenesis stimulators is erythropoietin (EPO) (Maiese et al., 2004). EPO was originally thought to have only one function, namely erythropoiesis. However, now we know that it has receptors on multiple central nervous system cell types and that it is produced in multiple sites in the brain (Chong et al., 2003a; Maiese et al., 2004; Marti, 2004). It also has a modulatory effect on multiple signal transduction factors in neural tissues (Brunet et al., 1999; Zhou et al., 2000; Kawakami et al., 2001; Shingo et al., 2001; Somervaille et al., 2001; Chong et al., 2002a; Figueroa et al., 2002; Mahmud et al., 2002; Vairano et al., 2002; Chong et al., 2003b, 2003c, 2003d; Kang et al., 2003a, 2003b; Matsushita et al., 2003; Olsen, 2003; Chong et al., 2004; Chong and Maiese, 2004; Dzietko et al., 2004; Wang et al., 2004; Weishaupt et al., 2004; Kumral, 2006), and its production in the brain is a highly regulated process involving several Hypoxia inducible factors (HIF) (Heidbreder et al., 2003).

It is not known if EPO has protective effects in the P10 rat hypoxia model. This model mimics hypoxic encephalopathy in the human newborn at birth, a condition for which no effective postnatal intervention exists to prevent subsequent brain injury and long term neurologic sequalae (Koh et al., 2004). Our objective was to determine if EPO has protective effects against subsequent susceptibility to seizure related neuronal injury in rat pups subjected to acute hypoxia at P10.

METHODS

  1. Top of page
  2. Abstract
  3. METHODS
  4. RESULTS
  5. DISCUSSION
  6. REFERENCES

Forty-two Sprague-Dawley rat pups were divided into three groups. All animals were maintained in restricted-access rooms with a controlled temperature (23°C) and a 12 h light-dark cycle. Standard laboratory chow and drinking water were provided ad libitum. All experimental procedures were approved by the Institutional Review Board, and all attempts were made to reduce pain and discomfort to experimental animals. Four groups were studied. The Normoxia-NS-KA group (n = 11): rats were sham manipulated at P10. The sham-manipulated pups were placed in a normoxic chamber for the same period needed to complete the acute hypoxia experiment. When these rats were taken out of the chamber, they were injected with the appropriate volumes of saline according to their weights. The Normoxia-EPO-KA group (n = 10): rats were sham manipulated at P10 as in the previous group except that they were injected with recombinant human EPO (1000 U/Kg i.p.) (Eprex, Cilag AG, Switzerland) immediately after being taken out of the normoxia chamber. The Hypoxia-NS-KA group (n = 11): rats were subjected to acute hypoxia at P10 according to the following protocol of progressively decreasing oxygen (O2) tensions: 8 min in 7% O2, 2 min 30 s in 7%–6%, 4 min in 6%, 3 min 40 s in 6%–5%, 2 min in 5%, 5 min in 5%–4%, and 1 min in 4% O2. This protocol, which is similar to, but slightly modified from, that of Jensen et al. (1991) was found by us to result in acute hypoxic seizures in all the pups with minimal if any mortality (Jensen et al., 1991; Koh et al., 2004; Mikati et al., 2005). Rats consistently started seizing 2 min after being placed in 7% O2, had similar degree of seizure activity, and stopped seizing 2 min after returning to room air. These rats were subsequently immediately saline injected. The Hypoxia-EPO-KA group (n = 10): rats were similarly subjected to acute hypoxia at P10, and then immediately injected with recombinant human EPO (1000 U/Kg i.p.). This EPO dose was previously shown to improve long-term spatial memory deficits and brain injury resulting from neonatal hypoxia-ischemia in rats (Kumral et al., 2004). All rats were injected with KA (10 mg/kg i.p.) at P29 and seizure latency and duration were documented using videotape techniques, which are in routine use in our laboratory (Mikati et al., 2004a; Mikati et al., 2004b). The animals were observed and videotaped for 3 h after KA injection. The videotapes were reviewed and the seizures scored by an observer blinded to the treatment groups. Latency to forelimb clonus with rearing with or without falling (FLC) and duration of FLC were documented. At P31, all rats were sacrificed exactly 48 h after KA injection. In addition, the right and left hemispheres were dissected for immediate frozen sectioning and subsequent TUNEL staining and Hoechst 33342 nuclear staining for assessment of DNA fragmentation and chromatin condensation respectively. Two contiguous slices/animal, taken at a specific standardized level, were used to generate data. These were coronal slices taken at the level, which is 0.43 mm behind the bregma. This is the level of first appearance of the ventral hippocampus with a full view of the dorsal hippocampus. The first slice was used for the combined Hoechst 33342 (Molecular Probes, Eugene, OR, U.S.A.) and TUNEL (Roche Diagnostics Corporation, IN, U.S.A.) staining and the second for the H&E. The thickness of the sections was 7 μm. For each section, the following hippocampal subfields were analyzed (CA3/CA4, CA2, and CA1 regions) at 20x magnification. The, combined, Hoechst and TUNEL staining procedure we used consists of coupling these two stains on the same slide. The Hoechst stain labels all the nuclei on the slide and in addition shows the ones, which have chromatin condensation. As a result, the Hoechst ordinal scale is based on the numbers of shrunken and chromatin condensed nuclei as a percentage of the total number of nuclei stained by the Hoechst stain. Similarly, the TUNEL ordinal scale is based on the number of fluorescent nuclei as a percentage of the total number of nuclei as shown by the Hoechst stain. This assessment was performed, by an observer blinded to the treatment groups, according to an ordinal severity scale from 0 to 4: 0, no positive fluorescent cells; and 4, 75%–100% of the cells in a specific region (CA1, CA2 or CA3/CA4 in the hippocampus) were positively fluorescent. Scores 1, 2, and 3 indicated 1%–25%, 25%–50%, and 50%–75% positive cells in the area of interest, as described previously (Mikati et al., 2003). A similar scoring system was used to assess the extent of chromatin condensation using the Hoechst stain in the hippocampus.

Hematoxylin and eosin staining was also performed for the study of CA3/CA4 and CA1 cell density (at 20x magnification) in the three groups and that for neuronal loss assessment by using the free UTHSCSA ImageTool program (developed at the University of Texas Health Science Center at San Antonio, Texas and available from the Internet by anonymous FTP from ftp://maxrad6.uthscsa.edu).

Analysis of variance (ANOVA) with post hoc analysis with the least significant difference (LSD) test was used to analyze the seizure latency and duration data, as well as the cell density data. The Kruskal–Wallis median test was used to analyze the TUNEL staining data.

RESULTS

  1. Top of page
  2. Abstract
  3. METHODS
  4. RESULTS
  5. DISCUSSION
  6. REFERENCES

Latency to FLC

Preexisting hypoxia at P10 resulted in shortening of the latency to FLC after KA injection at P29 (Hypoxia-NS-KA group) and EPO administration directly after hypoxia at P10 (Hypoxia-EPO-KA group) prevented this shortening (F = 6.81; p = 0.001, see post hoc paired comparisons that follow). Post hoc paired comparisons showed that the Hypoxia-NS-KA group was different from the Normoxia-NS-KA group (p = 0.001), from the Normoxia-EPO-KA group (p = 0.0001), and from the Hypoxia-EPO-KA group (p = 0.019). The later three groups were not different (p = 0.35) (Fig. 1).

image

Figure 1. Latency to FLC seizures according to group. *Different from the Normoxia-NS-KA, Normoxia-EPO-KA, and the Hypoxia-EPO-KA groups (p < 0.05). Mean ± standard error (SE) of the latency to FLC was the following (in minutes): Normoxia-NS-KA group: 67.87 ± 8.52, Normoxia-EPO-KA group: 75.62 ± 8.63, Hypoxia-NS-KA group: 32.55 ± 4.44, Hypoxia-EPO-KA group 3: 56.7 ± 10.29.

Download figure to PowerPoint

Duration of FLC

Preexisting hypoxia at P10 resulted in longer duration of FLC at P29 (Hypoxia-NS-KA group) and EPO administration directly after hypoxia at P10 prevented this elongation (F = 4.62; p = 0.007, see post hoc paired comparisons that follow). Post hoc paired comparisons showed that the Hypoxia-NS-KA group was different from the Normoxia-NS-KA group (p = 0.005), from the Normoxia-EPO-KA group (p = 0.001), and from the Hypoxia-EPO-KA group (p = 0.023). The later three groups were not different (p = 0.64) (Fig. 2).

image

Figure 2. Duration of FLC seizures according to group. *Different from the Normoxia-NS-KA, Normoxia-EPO-KA, and the Hypoxia-EPO-KA groups (p < 0.05). Duration of FLC seizures was the following (in minutes): Normoxia-NS-KA group: 3.27 ± 0.85, Normoxia-EPO-KA group: 2.62± 0.26, Hypoxia-NS-KA group: 8.27 ± 2.04, Hypoxia-EPO-KA group: 4.2 ± 0.71.

Download figure to PowerPoint

TUNEL scores

The four groups differed in the extent of total TUNEL scores (H = 6.38, p = 0.04), and in CA1 TUNEL score (H = 8.41, p = 0.01). Paired comparisons showed that the total scores of the Hypoxia-NS-KA group were different from the Normoxia-NS-KA group (H = 5.12; p = 0.02), from the Normoxia-EPO-KA group (H = 10.81, p = 0.0008), and from those of the Hypoxia-EPO-KA group (H = 3.85; p = 0.04). In addition, paired comparisons also showed that the CA1 scores of the Hypoxia-NS-KA group were different from the Normoxia-NS-KA group (H = 6.93; p = 0.008), Normoxia-EPO-KA group (H = 6.47, p = 0.007) and from those of the Hypoxia-EPO-KA group (H = 4.17; p = 0.04). The Normoxia-NS-KA and Hypoxia-EPO-KA groups did not show any statistically significant differences (H = 0.25; p = 0.62, and H = 0.83; p = 0.36 for the total and CA1 scores respectively). Similarly, The Normoxia-EPO-KA and Hypoxia-EPO-KA groups did not show any statistically significant differences (H = 0.85, p = 0.37, and H = 0.29, p = 0.46 for the total and CA1 scores, respectively). Other comparisons were not significant (Fig. 3A and C).

image

Figure 3. (A) Illustrations (20x) of the Hoechst, TUNEL and H & E stains of representative CA1 subfields according to treatment group. Note the increased chromatin condensation (Hoechst), DNA fragmentation (TUNEL) and neuronal cell loss in the Hypoxia-NS-KA group. (B) Hoechst scores of hippocampal subfields according to treatment group. The Mean ± SE for Normoxia-NS-KA, Normoxia-EPO-KA, Hypoxia-NS-KA and Hypoxia-EPO-KA groups were the following: CA3/CA4: 1.125 ± 0.12, 1.00 ± 0.00, 1.44 ± 0.24, 1.14 ± 0.14; CA2: 1.00 ± 0.00, 1.00 ± 0.00, 1.00 ± 0.00, 1.00 ± 0.00; CA1: 1.00 ± 0.00, 1.60 ± 0.23, 2.55 ± 0.24, 1.14 ± 0.14; Total combined scores for the CA3/CA4, CA2 and the CA1 subfields: 4.12 ± 0.12, 4.22 ± 0.23, 6.00 ± 0.16, 4.14 ± 0.14 respectively. *Different from the Normoxia-NS-KA, Normoxia-EPO-KA and the Hypoxia-EPO-KA groups (p < 0.05). (C) TUNEL scores of hippocampal subfields according to treatment group. The Mean ± SE for Normoxia-NS-KA, Normoxia-EPO-KA, Hypoxia-NS-KA, and Hypoxia-EPO-KA groups were the following: CA3/CA4: 0.25 ± 0.16, 0.00 ± 0.00, 0.55 ± 0.24, 0.28 ± 0.18; CA2: 0.12 ± 0.12, 0.00 ± 0.00, 0.33 ± 0.16, 0.00 ± 0.00; CA1: 0.37 ± 0.18, 0.43 ± 0.25, 2.11 ± 0.45, 0.71 ± 0.28; Total combined scores for the CA3/CA4, CA2 and the CA1 subfields: 0.75 ± 0.31, 0.43 ± 0.25, 3.00 ± 0.74, 1.00 ± 0.38 respectively. *Different from the Normoxia-NS-KA, Normoxia-EPO-KA, and the Hypoxia-EPO-KA groups (p < 0.05). (D) Cell density in the CA3/CA4 and CA1 hippocampal subfields according to treatment group. The Mean density ± SE for Normoxia-NS-KA, Normoxia-EPO-KA, Hypoxia-NS-KA, and Hypoxia-EPO-KA groups were the following (cell count/pixel2): CA3/CA4: 4.93 × 10−4± 4.29 × 10−5, 5.19 × 10−4± 4.32 × 10−5, 4.05 × 10−4± 2.64 × 10−5, 5.98 × 10−4± 3.61 × 10−5; CA1: 8.7 × 10−4± 5.56 × 10−5, 9.04 × 10−4± 5.53 × 10−5, 6.57 × 10−4± 2.89 × 10−5, 9.52 × 10−4± 1.2 × 10−4. *Different from the Normoxia-NS-KA, Normoxia-EPO-KA, and the Hypoxia-EPO-KA groups (p < 0.05).

Download figure to PowerPoint

Hoechst Scores

The four groups differed in their total Hoechst stain scores (H = 10.61; p = 0.01), and in their CA1 Hoechst score (H = 11.15; p = 0.01). Paired comparisons showed that the total scores of the Hypoxia-NS-KA group were different from the Normoxia-NS-KA group (H = 9.72; p = 0.001), Normoxia-EPO-KA group (H = 9.63, p = 0.006), and from those of the Hypoxia-EPO-KA group (H = 5.85; p = 0.01). Paired comparisons also showed that the CA1 scores of the Hypoxia-NS-KA group were different from the Normoxia-NS-KA group (H = 11.72; p = 0.0006), Normoxia-EPO-KA group (H = 9.84, p = 0.001), and from those of the Hypoxia-EPO-KA group (H = 6.95; p = 0.008). The Normoxia-NS-KA and Hypoxia-EPO-KA groups were not different (H = 0.009; p = 0.92, and H = 1.14; p = 0.28 for the total and CA1 scores, respectively). Similarly, the Normoxia-EPO-KA and Hypoxia-EPO-KA groups did not show any statistically significant differences (H = 1.35, p = 0.64, and H = 0.009, p = 0.91 for the total and CA1 scores, respectively). Other comparisons were not significant (Fig. 3A and B).

Cell counts

The four groups also differed in their CA1 cell density (F = 9.67; p = 0.003). Paired comparisons showed that the CA1 cell density of the Hypoxia-NS-KA group was different from the Normoxia-NS-KA group (p = 0.002), from the Normoxia-EPO-KA group (p = 0.0001), and from that of the Hypoxia-EPO-KA group (p = 0.0004). The Normoxia-NS-KA and Hypoxia-EPO-KA groups were not different (p = 0.29). Similarly, the Normoxia-EPO-KA and Hypoxia-EPO-KA groups did not show any statistically significant differences (p = 0.44). Other comparisons were not significant (Fig. 3A and D).

DISCUSSION

  1. Top of page
  2. Abstract
  3. METHODS
  4. RESULTS
  5. DISCUSSION
  6. REFERENCES

Our study reproduced the effects of acute hypoxia that have been previously reported in the developing brain (Dell'Anna et al., 1991; Jensen et al., 1991, 1992, 1998). In addition it demonstrated that a single dose of EPO given directly after an acute hypoxic event at P10 increased the latency to FLC seizures, reduced the duration of these seizures, and protected against seizure induced cell loss and programmed cell death caused by subsequent status epilepticus. Our data show no difference between the Normoxia-EPO-KA group, Normoxia-NS-KA group and the Hypoxia-EPO-KA group and also show striking differences between each of these groups and the Hypoxia-NS-KA group. This indicates that a single dose of EPO administered directly after hypoxia at P10 was protective against the sequalae of a “second hit” of KA induced Status Epilepticus. This is consistent with several other recent studies showing neuroprotective effects of EPO in in vivo models of neonatal hypoxia-ischemia (Kumral et al., 2004; Maiese et al., 2004; Demers et al., 2005; Diaz et al., 2005; Kerendi et al., 2005). In addition, comparable long-term results involving recovery of sensorimotor deficits, attenuation of brain injury, preservation of the integrity of cerebral cortex, and improvement of neurobehavioral achievements, have been achieved by using a single dose of EPO in the P7 model of hypoxia-ischemia (Kumral et al., 2004; Spandou et al., 2005). Our study, however, differs from those studies in that it investigated the P10 developmental stage using a hypoxia rather than a hypoxia-ischemia model. The P10 developmental stage is of particular interest as the rat brain at this stage is at a developmental stage close to that of the full term human newborn and as this stage is particularly vulnerable to the effects of hypoxia and hypoxia induced seizures (Jensen et al., 1991; Jensen et al., 1992; Jensen et al., 1998; Mikati et al., 2005).

Based on studies of EPO effects in the models of chemical ischemia (Kawakami et al., 2001) and of in vivo hypoxia ischemia (Juul, 2002), we postulate that the mechanisms that underlie the effects we observed may involve the JAK2 and NF-kB signaling pathways. Activation of neuronal EPO receptors triggers a cross-talk between the signaling pathways of Jak2 and NF-kB, which leads to phosphorylation of the inhibitor of NF-kB (IkB), subsequent nuclear translocation of NF-kB, and then to NF-kB-dependent transcription of neuroprotective genes (Digicaylioglu and Lipton, 2001).

EPO also has neuroprotective effects in other models. These include the following: (1) experimental subarachnoid hemorrhage (Grasso et al., 2002; Olsen, 2003); (2) NMDA toxicity (Dzietko et al., 2004); (3) neuronal axotomy (Weishaupt et al., 2004); (4) anoxia in endothelial cells in culture (Chong et al., 2002a), and (5) oxidative stress in cerebral microvascular endothelial cells and in primary neurons and cerebral microglia in culture (Chong et al., 2003b; Chong et al., 2003c; Chong et al., 2003d).

Recombinant Human EPO has been demonstrated to result in neuronal and vascular protection not only through the maintenance of cellular integrity, but also through the prevention of cellular inflammation (Maiese et al., 2004). The neuroprotective effects of EPO are believed to involve the following: (1) the Janus tyrosine kinase 2 (JAK2) and protein kinase B (Akt) (Kawakami et al., 2001); (2) microglial inflammatory activation (Chong et al., 2003a, 2003b, 2003c, 2003d; Marti, 2004); (3) the forkhead transcription factor FOXO3a (also known as FKHRL1) and glycogen synthase kinase 3b (GSK-3b) (Brunet et al., 1999; Chong et al., 2004); (4) the transcriptional coactivator p300 associated with FOXO3a (Mahmud et al., 2002); (5) c-MYC, c-JUN, b-catenin, and cytochrome c (Chong and Maiese, 2004; Maiese et al., 2004); (6) Bax, Bad, Bcl-2, and Bcl-xL (Somervaille et al., 2001; Vairano et al., 2002); (7) apoptotic protease activating factor 1 (APAF-1) (Chong et al., 2003b); (8) nuclear factor kB (NF-kB) (Marti, 2004); (9) HIF-1 (Figueroa et al. 2002); (10) IkB kinase (IKK) (Shingo et al., 2001; Matsushita et al., 2003; Wang et al., 2004), and (11) caspase-mediated pathways (Zhou et al., 2000; Chong et al., 2002a; Kang et al., 2003a, 2003b).

Years of clinical administration in patients with anemia and chronic kidney diseases have shown EPO to be well tolerated and safe (Jelkmann, 1992). More recently, one clinical trial has demonstrated some efficacy and safety of EPO in a limited number of patients with acute ischemic stroke (Ehrenreich et al., 2002). Additional trials are seeking to examine the role of EPO in other disorders (Chong et al., 2003a; Ehrenreich et al., 2004; Genc et al., 2004). In our study we have demonstrated that a single EPO dose given directly after P10 hypoxia results in reduced vulnerability to a second hit (KA induced status epilepticus). Whether the mechanism of these effects involves any of the above-mentioned pathways and/or the reduction of electrographic seizures would require further investigation. In addition, data available from the study of Koh et al. (2004) and from our current study raise several questions concerning the extent of the brain injury contributed by hypoxia and the means of assessment of the neuronal damage induced by hypoxia alone. These questions emphasize the need of further investigations in this area.

In future therapeutic trials of EPO several cautions are warranted. Both acute and long-term administration of this drug can precipitate hypertensive emergencies accompanied by hypertensive encephalopathy and seizures in humans with renal failure (Beccari, 1994; Beccari et al., 1995; Miyashita et al., 2004). EPO can also result in the formation of antierythropoietin antibodies, in red cell aplasia (Casadevall et al., 2002), and in decreased expression of EPO receptors on the cell surface (Verdier et al., 2000). Maintenance treatment with EPO, in humans, has also been associated with myocardial infarction, vascular thrombosis, pyrexia, vomiting, shortness of breath, paresthesias, and upper respiratory tract infection (Henry et al., 2004) and, in experimental animals, in cerebral ischemia in mice (Wiessner et al., 2001). These side effects could limit the use of EPO for the treatment of diseases of the nervous system. Therefore, strategies to develop derivatives of EPO, such as carbamylated EPO and neurotrophic sequences of EPO (Campana et al., 1998; Leist et al., 2004), have been suggested to remove erythropoietic activity and potential toxicity. However, present work on animal models suggests that currently available derivatives of EPO possess only limited utility (Gil et al., 2004). Whether EPO or its derivatives will be clinically useful neuroprotective agents clearly requires further investigations (Bernaudin et al., 1999; Chong et al., 2002a, 2002b).

Our data demonstrate the presence of a favorable protective effect of EPO against the long-term consequences of acute hypoxia in the developing brain, and raise the possibility of its investigation as a potential neuroprotective agent after human neonatal hypoxic encephalopathy.

REFERENCES

  1. Top of page
  2. Abstract
  3. METHODS
  4. RESULTS
  5. DISCUSSION
  6. REFERENCES
  • Beccari M. (1994) Seizures in dialysis patients treated with recombinant erythropoietin. Review of the literature and guidelines for prevention. International Journal of Artificial Organs 17:513.
  • Beccari M, Romagnoni M, Sorgato G. (1995) Seizures in dialysis patients treated with recombinant erythropoietin. Nephrology Dialysis Transplantation 10:423424.
  • Bernaudin M, Marti HH, Roussel S, Divoux D, Nouvelot A, MacKenzie ET, Petit E. (1999) A potential role for erythropoietin in focal permanent cerebral ischemia in mice. Journal of Cerebral Blood Flow and Metabolism 19:643651.
  • Brunet A, Bonni A, Zigmond MJ, Lin MZ, Juo P, Hu LS, Anderson MJ, Arden KC, Blenis J, Greenberg ME. (1999) Akt promotes cell survival by phosphorylating and inhibiting a Forkhead transcription factor. Cell 96:857868.
  • Campana WM, Misasi R, O'Brien JS. (1998) Identification of a neurotrophic sequence in erythropoietin. International Journal of Molecular Medicine 1:235241.
  • Casadevall N, Nataf J, Viron B, Kolta A, Kiladjian JJ, Martin-Dupont P, Michaud P, Papo T, Ugo V, Teyssandier I, Varet B, Mayeux P. (2002) Pure red-cell aplasia and antierythropoietin antibodies in patients treated with recombinant erythropoietin. New England Journal of Medicine 346:469475.
  • Chong ZZ, Kang JQ, Maiese K. (2002a) Erythropoietin is a novel vascular protectant through activation of Akt1 and mitochondrial modulation of cysteine proteases. Circulation 106:29732979.
  • Chong ZZ, Kang JQ, Maiese K. (2002b) Angiogenesis and plasticity: role of erythropoietin in vascular systems. Journal of Hematotherapeutic Stem Cell Research 11:863871.
  • Chong ZZ, Kang JQ, Maiese K. (2003a) Erythropoietin: cytoprotection in vascular and neuronal cells. Current Drug Targets Cardiovascular and Haematological Disorders 3:141154.
  • Chong ZZ, Kang JQ, Maiese K. (2003b) Apaf-1, Bcl-xL, cytochrome c, and caspase-9 form the critical elements for cerebral vascular protection by erythropoietin. Journal of Cerebral Blood Flow and Metabolism 23:320330.
  • Chong ZZ, Kang JQ, Maiese K. (2003c) Erythropoietin fosters both intrinsic and extrinsic neuronal protection through modulation of microglia, Akt1, Bad, and caspase-mediated pathways. British Journal of Pharmacology 138:11071118.
  • Chong ZZ, Lin SH, Kang JQ, Maiese K. (2003d) Erythropoietin prevents early and late neuronal demise through modulation of AKt1 and induction of caspace 1, 3, and 8. Journal of Neuroscience Research 71:659669.
  • Chong ZZ, Lin SH, Maiese K. (2004) The NAD+ precursor nicotinamide governs neuronal survival during oxidative stress through protein kinase B coupled to FOXO3a and mitochondrial membrane potential. Journal of Cerebral Blood Flow and Metabolism 24:728743.
  • Chong ZZ, Maiese K. (2004) Targeting WNT, protein kinase B, and mitochondrial membrane integrity to foster cellular survival in the nervous system. Histology and Histopathology 19:495504.
  • Dell'Anna ME, Calzolari S, Molinari M, Iuvone L, Calimici R. (1991) Neonatal anoxia induces transitory hyperactivity, permanent spatial memory deficits and CA1 cell density reduction in developing rats. Behavioral Brain Research 45:125134.
  • Demers EJ, McPherson RJ, Juul SE. (2005) Erythropoietin protects dopaminergic neurons and improves neurobehavioral outcomes in juvenile rats after neonatal hypoxia-ischemia. Pediatric Research 58:297301.
  • Diaz Z, Assaraf MI, Miller WH Jr., Schipper HM. (2005) Astroglial cytoprotection by erythropoietin pre-conditioning: implications for ischemic and degenerative CNS disorders. Journal of Neurochemistry 93:392402.
  • Digicaylioglu M, Lipton SA. (2001) Erythropoietin-mediated neuroprotection involves cross-talk between Jak2 and NF-kappaB signalling cascades. Nature 412:641647.
  • Dzietko M, Felderhoff-Mueser U, Sifringer M, Krutz B, Bittigau P, Thor F, Heumann R, Buhrer C, Ikonomidou C, Hansen HH. (2004) Erythropoietin protects the developing brain against N-methyl-D-aspartate receptor antagonist neurotoxicity. Neurobiology of Disease 15:177187.
  • Ehrenreich H, Hasselblatt M, Dembowski C, Cepek L, Lewczuk P, Stiefel M, Rustenbeck HH, Breiter N, Jacob S, Knerlich F, Bohn M, Poser W, Ruther E, Kochen M, Gefeller O, Gleiter C, Wessel TC, De Ryck M, Itri L, Prange H, Cerami A, Brines M, Siren AL. (2002) Erythropoietin therapy for acute stroke is both safe and beneficial. Molecular Medicine 8:495505.
  • Ehrenreich H, Degner D, Meller J, Brines M, Behe M, Hasselblatt M, Woldt H, Falkai P, Knerlich F, Jacob S, Von Ahsen N, Maier W, Bruck W, Ruther E, Cerami A, Becker W, Siren AL. (2004) Erythropoietin: a candidate compound for neuroprotection in schizophrenia. Molecular Psychiatry 9:4254.
  • Figueroa YG, Chan AK, Ibrahim R, Tang Y, Burow ME, Alam J, Scandurro AB, Beckman BS. (2002) NF-kappaB plays a key role in hypoxia-inducible factor-1-regulated erythropoietin gene expression. Experimental Hematology 30:14191427.
  • Genc S, Koroglu TF, Genc K. (2004) Erythropoietin as a novel neuroprotectant. Restorative Neurology and Neuroscience 22:105119.
  • Gil JM, Leist M, Popovic N, Brundin P, Petersen A. (2004) Asialoerythropoietin is not effective in the R6/2 line of Huntington's disease mice. BMC Neuroscience 5:17.
  • Grasso G, Buemi M, Alafaci C, Sfacteria A, Passalacqua M, Sturiale A, Calapai G, De Vico G, Piedimonte G, Salpietro FM, Tomasello F. (2002) Beneficial effects of systemic administration of recombinant human erythropoietin in rabbits subjected to subarachnoid hemorrhage. Proceedings of the National Academy of Sciences U.S.A. 99:56275631.
  • Heidbreder M, Frohlich F, Johren O, Dendorfer A, Qadri F, Dominiak P. (2003) Hypoxia rapidly activates HIF-3alpha mRNA expression. Federation of American Societies for Experimental Biology Journal 17:15411543.
  • Henry DH, Bowers P, Romano MT, Provenzano R. (2004) Epoetin alfa. Clinical evolution of a pleiotropic cytokine. Archives of Internal Medicine 164:262276.
  • Huang YF, Zhuang SQ, Chen DP, Liang YJ, Li XY. (2004) [Angiogenesis and its regulatory factors in brain tissue of neonatal rat hypoxic-ischemic encephalopathy]. Zhonghua Er Ke Za Zhi 42:210214.
  • Jelkmann W. (1992) Erythropoietin: structure, control of production, and function. Physiological Reviews 72:449489.
  • Jensen FE, Applegate CD, Holtzman D, Belin TR, Burchfiel JL. (1991) Epileptogenic effect of hypoxia in the immature rodent brain. Annals of Neurology 29:629637.
  • Jensen FE, Holmes GL, Lombroso CT, Blume HK, Firkusny IR. (1992) Age-dependent changes in long-term seizure susceptibility and behavior after hypoxia in rats. Epilepsia 33:971980.
  • Jensen FE, Wang C, Stafstrom CE, Liu Z, Geary C, Stevens MC. (1998) Acute and chronic increases in excitability in rat hippocampal slices after perinatal hypoxia In vivo. Journal of Neurophysiology 79:7381.
  • Juul S. (2002) Erythropoietin in the central nervous system, and its use to prevent hypoxic-ischemic brain damage. Acta Paediatrica Supplement 91:3642.
  • Kang JQ, Chong ZZ, Maiese K. (2003a) Akt1 protects against inflammatory microglial activation through maintenance of membrane asymmetry and modulation of cysteine protease activity. Journal of Neuroscience Research 74:3751.
  • Kang JQ, Chong ZZ, Maiese K. (2003b) Critical role for Akt1 in the modulation of apoptotic phosphatidylserine exposure and microglial activation. Molecular Pharmacology 64:557569.
  • Kawakami M, Sekiguchi M, Sato K, Kozaki S, Takahashi M. (2001) Erythropoietin receptor-mediated inhibition of exocytotic glutamate release confers neuroprotection during chemical ischemia. Journal of Biological Chemistry 276:3946939475.
  • Kerendi F, Halkos ME, Kin H, Corvera JS, Brat DJ, Wagner MB, Vinten-Johansen J, Zhao ZQ, Forbess JM, Kanter KR, Kelley ME, Kirshbom PM. (2005) Upregulation of hypoxia inducible factor is associated with attenuation of neuronal injury in neonatal piglets undergoing deep hypothermic circulatory arrest. Journal of Thoracic and Cardiovascular Surgery 130:1079.
  • Koh S, Jensen FE. (2001) Topiramate blocks perinatal hypoxia-induced seizures in rat pups. Annals of Neurology 50:366372.
  • Koh S, Tibayan FD, Simpson JN, Jensen FE. (2004) NBQX or topiramate treatment after perinatal hypoxia-induced seizures prevents later increases in seizure-induced neuronal injury. Epilepsia 45:569575.
  • Kumral A, Uysal N, Tugyan K, Sonmez A, Yilmaz O, Gokmen N, Kiray M, Genc S, Duman N, Koroglu TF, Ozkan H, Genc K. (2004) Erythropoietin improves long-term spatial memory deficits and brain injury following neonatal hypoxia-ischemia in rats. Behavioral Brain Research 153:7786.
  • Kumral A, Genc S, Ozer E, Yilmaz O, Gokmen N, Koroglu TF, Duman N, Genc K, Ozkan H. (2006) Erythropoietin downregulates bax and DP5 preapoptotic gene expression in neonatal hypoxic-ischemic brain injury. Biology of the Neonate 89:205210.
  • Legido A, Clancy RR, Berman PH. (1991) Neurologic outcome after electroencephalographically proven neonatal seizures. Pediatrics 88:583596.
  • Leist M, Ghezzi P, Grasso G, Bianchi R, Villa P, Fratelli M, Savino C, Bianchi M, Nielsen J, Gerwien J, Kallunki P, Larsen AK, Helboe L, Christensen S, Pedersen LO, Nielsen M, Torup L, Sager T, Sfacteria A, Erbayraktar S, Erbayraktar Z, Gokmen N, Yilmaz O, Cerami-Hand C, Xie QW, Coleman T, Cerami A, Brines M. (2004) Derivatives of erythropoietin that are tissue protective but not erythropoietic. Science 305:239242.
  • Mahmud DLMGA, Deb DK, Platanias LC, Uddin S, Wickrema A. (2002) Phosphorylation of forkhead transcription factors by erythropoietin and stem cell factor prevents acetylation and their interaction with coactivator p300 in erythroid progenitor cells. Oncogene 21:15561562.
  • Maiese K, Li F, Chong ZZ. (2004) Erythropoietin in the brain: can the promise to protect be fulfilled? Trends in Pharmacological Sciences 25:577583.
  • Marti HH. (2004) Erythropoietin and the hypoxic brain. Journal of Experimental Biology 207:32333242.
  • Matsushita H, Johnston MV, Lange MS, Wilson MA. (2003) Protective effect of erythropoietin in neonatal hypoxic ischemia in mice. Neuroreport 14:17571761.
  • Mikati MA, Abi-Habib RJ, El Sabban ME, Dbaibo GS, Kurdi RM, Kobeissi M, Farhat F, Asaad W. (2003) Hippocampal programmed cell death after status epilepticus: evidence for NMDA-receptor and ceramide-mediated mechanisms. Epilepsia 44:282291.
  • Mikati MA, Holmes GL, Werner S, Bakkar N, Carmant L, Liu Z, Stafstrom CE. (2004a) Effects of nimodipine on the behavioral sequalae of experimental status epilepticus in prepubescent rats. Epilepsy and Behavior 5:168174.
  • Mikati MA, Kurdit RM, Rahmeh AA, Farhat F, Abu Rialy S, Lteif L, Francis E, Geha G, Maraashli W. (2004b) Effects of creatine and cyclocreatine supplementation on kainate induced injury in pre-pubescent rats. Brain Injury 18:12291241.
  • Mikati MA, Zeinieh MP, Kurdi RM, Harb SA, El Hokayem JA, Daderian RH, Shamseddine A, Obeid M, Bitar FF, El Sabban M. (2005) Long-term effects of acute and of chronic hypoxia on behavior and on hippocampal histology in the developing brain. Brain Research Developmental Brain Research 157:98102.
  • Miyashita K, Tojo A, Kimura K, Goto A, Omata M, Nishiyama K, Fujita T. (2004) Blood pressure response to erythropoietin injection in hemodialysis and predialysis patients. Hypertension Research 27:7984.
  • Olsen NV. (2003) Central nervous system frontiers for the use of erythropoietin. Clinical Infectious Diseases 37(suppl 4):S323330.
  • Raab S, Beck H, Gaumann A, Yuce A, Gerber HP, Plate K, Hammes HP, Ferrara N, Breier G. (2004) Impaired brain angiogenesis and neuronal apoptosis induced by conditional homozygous inactivation of vascular endothelial growth factor. Thrombosis and Haemostasis 91:595605.
  • Shingo T, Sorokan ST, Shimazaki T, Weiss S. (2001) Erythropoietin regulates the in vitro and in vivo production of neuronal progenitors by mammalian forebrain neural stem cells. J Neuroscience 21:97339743.
  • Somervaille TC, Linch DC, Khwaja A. (2001) Growth factor withdrawal from primary human erythroid progenitors induces apoptosis through a pathway involving glycogen synthase kinase-3 and Bax. Blood 98:13741381.
  • Spandou E, Papadopoulou Z, Soubasi V, Karkavelas G, Simeonidou C, Pazaiti A, Guiba-Tziampiri O. (2005) Erythropoietin prevents long-term sensorimotor deficits and brain injury following neonatal hypoxia-ischemia in rats. Brain Research 1045:2230.
  • Vairano M, Dello Russo C, Pozzoli G, Battaglia A, Scambia G, Tringali G, Aloe-Spiriti MA, Preziosi P, Navarra P. (2002) Erythropoietin exerts anti-apoptotic effects on rat microglial cells in vitro. European Journal of Neuroscience 16:584592.
  • Verdier F, Walrafen P, Hubert N, Chretien S, Gisselbrecht S, Lacombe C, Mayeux P. (2000) Proteasomes regulate the duration of erythropoietin receptor activation by controlling down-regulation of cell surface receptors. Journal of Biological Chemistry 275:1837518381.
  • Volpe J. (2000) Neurology of the newborn. WB Saunders, Philadelphia .
  • Wang L, Zhang Z, Wang Y, Zhang R, Chopp M. (2004) Treatment of stroke with erythropoietin enhances neurogenesis and angiogenesis and improves neurological function in rats. Stroke 35:17321737.
  • Weishaupt JH, Rohde G, Polking E, Siren AL, Ehrenreich H, Bahr M. (2004) Effect of erythropoietin axotomy-induced apoptosis in rat retinal ganglion cells. Investigative Ophthalmology and Visual Science 45:15141522.
  • Wiessner C, Allegrini PR, Ekatodramis D, Jewell UR, Stallmach T, Gassmann M. (2001) Increased cerebral infarct volumes in polyglobulic mice overexpressing erythropoietin. Journal of Cerebral Blood Flow and Metabolism 21:857864.
  • Zhou H, Li XM, Meinkoth J, Pittman RN. (2000) Akt regulates cell survival and apoptosis at a postmitochondrial level. Journal of Cell Biology 151:483494.