Postnatal erythropoietin treatment mitigates neural cell loss after systemic prenatal hypoxic-ischemic injury

Laboratory investigation

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Object

Brain injury from preterm birth predisposes children to cerebral palsy, epilepsy, cognitive delay, and behavioral abnormalities. The CNS injury often begins before the early birth, which hinders diagnosis and concurrent treatment. Safe, effective postnatal interventions are urgently needed to minimize these chronic neurological deficits. Erythropoietin (EPO) is a pleiotropic neuroprotective cytokine, but the biological basis of its efficacy in the damaged developing brain remains unclear. Coordinated expression of EPO ligand and receptor expression occurs during CNS development to promote neural cell survival. The authors propose that prenatal third trimester global hypoxiaischemia disrupts the developmentally regulated expression of neural cell EPO signaling, and predisposes neural cells to death. Furthermore, the authors suggest that neonatal exogenous recombinant human EPO (rhEPO) administration can restore the mismatch of EPO ligand and receptor levels, and enhance neural cell survival.

Methods

Transient systemic hypoxia-ischemia (TSHI) on embryonic Day 18 in rats mimics human early-thirdtrimester placental insufficiency. This model was used to test the authors' hypothesis using a novel clinically relevant paradigm of prenatal injury on embryonic Day 18, neonatal systemic rhEPO administration initiated 4 days after injury on postnatal Day 1, and histological, biochemical, and functional analyses in neonatal, juvenile, and adult rats.

Results

The results showed that prenatal TSHI upregulates brain EPO receptors, but not EPO ligand. Sustained EPO receptor upregulation was pronounced on oligodendroglial lineage cells and neurons, neural cell populations particularly prone to loss from CNS injury due to preterm birth. Postnatal rhEPO administration after prenatal TSHI minimized histological damage and rescued oligodendrocytes and γ-aminobutyric acidergic interneurons. Myelin basic protein expression in adult rats after insult was reduced compared with sham controls, but could be restored to near normal levels by neonatal rhEPO treatment. Erythropoietin-treated TSHI rats performed significantly better than their saline-treated peers as adults in motor skills tests, and showed significant seizure threshold restoration using a pentylenetetrazole increasing-dose paradigm.

Conclusions

These data demonstrate that neonatal rhEPO administration in a novel clinically relevant paradigm initiated 4 days after a global prenatal hypoxic-ischemic insult in rats rescues neural cells, and induces lasting histological and functional improvement in adult rats.

Abbreviations used in this paper: DAPI = 4,6'-diamino-2-phenylindole-dihydrochloride; ELISA = enzyme-linked immunosorbent assay; EPO = erythropoietin; EPOR = EPO receptor; GABA = γ-aminobutyric acid; GAD = glutamic acid decarboxylase; GFAP = glial fibrillary acidic protein; MBP = myelin basic protein; PTZ = pentylenetetrazole; rhEPO = recombinant human EPO; RT-PCR = reverse transcriptase polymerase chain reaction; TSHI = transient systemic hypoxia-ischemia; TUNEL = terminal deoxynucleotidyl transferase–mediated deoxyuridine triphosphate nick-end labeling.

Article Information

Address correspondence to: Shenandoah Robinson, M.D., Pediatric Neurosurgery, B501, Rainbow Babies & Children's Hospital, 11100 Euclid Avenue, Cleveland, Ohio 44106. email: shenandoah.robinson@UHhospitals.org.

© AANS, except where prohibited by US copyright law.

Headings

Figures

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    Summary diagram illustrating the various experiments performed at each time point after TSHI on embryonic Day 18 (E18). Three neonatal rhEPO dosing regimens were used: low (L; 500 IU/kg intraperitoneally on postnatal Day 1 [P1]); moderate (MrEPO; 1000 IU/kg intraperitoneally on postnatal Days 1–3); and high (HighrEPO; 2000 IU/kg intraperitoneally on postnatal Days 1–5). Efforts were made to use as few litters as possible while generating adequate data, including using rats pups from more than 1 litter for every experiment. Two litters of TSHI and 2 sham control litters were used for studies at embryonic Day 19. Three insult and 3 sham control litters were used for PCR, ELISA, and Western blots (WESTERN) at postnatal Days 0, 2, and 5, and cell culture on postnatal Day 1 immunolabeled on postnatal Day 2 (immunocytochemistry [ICC]). For hematocrit (HCT) measurements on postnatal Day 5, 3 control and 3 insult litters were used to generate the pups with one-quarter of each litter receiving either saline or the low, moderate, or high EPO regimen; these brains were collected for other studies (immunohistochemistry [IHC], PCR, and Western blots). For immunohistochemistry at postnatal Days 5 and 9, 4 control and 4 insult litters were used to generate the pups, with one-half of each litter receiving saline or the moderate rhEPO regimen. For motor tests (MOTOR) at postnatal Day 15, 4 control and 4 insult litters were generated with one-quarter of each litter treated with saline or 1 of the 3 EPO regimens. Most saline-treated and high-dose rhEPO-treated rats from postnatal Day 15 were used at postnatal Day 24. For the adult studies, 6 control and 7 insult litters were generated with one-half of each litter treated with saline or the high-dose rhEPO regimen. Adult motor testing was completed first, followed by seizure threshold (SEIZURE). Brains from surviving adults were used for immunohistochemistry and biochemical analyses. The number of samples used for each experiment is listed with the data presentation.

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    Graphs and images showing that prenatal TSHI on embryonic day 18 disrupts the coordinated developmental transcription of EPO ligand and receptor in the cerebrum in vivo. a: In sham controls, RT-PCR using the Ct technique shows stable EPO ligand transcription from embryonic Day 19 (E19) through postnatal Day 2 (P2), and that more cycles are required to reach the threshold at postnatal Day 5, consistent with postnatal downregulation of EPO (*p < 0.01). Transcription levels appear unaffected by prenatal injury. A lower Ct for a target gene indicates fewer cycles are necessary to reach the threshold, and therefore that more mRNA transcripts are present in a sample, with each sample normalized to the endogenous ribosomal 18S mRNA (ΔCt). b: Reverse transcriptase PCR for EPOR demonstrates the TSHI insult induces more EPOR mRNA transcription at embryonic Day 19 compared with sham controls (*p = 0.043), disrupting the developmentally coordinated expression of ligand and receptor. c: During development the threshold cycle ratio (ΔCt EPO/ΔCt EPOR) for EPO ligand to receptor in controls gradually increases from embryonic Day 19 to postnatal Day 5, consistent with a rate-limiting amount of ligand regulating receptor-mediated signaling. Because a higher ΔCt means fewer mRNA transcripts are present, a higher EPO to EPOR ΔCt ratio indicates a greater discrepancy between the amount of EPO and EPOR transcription. After the TSHI insult on embryonic Day 18, the ratio is increased at embryonic Day 19 by about 30% compared with sham controls, and returns to control levels by postnatal Day 5. d: In situ hybridization with EPOR on embryonic Day 19 localized the marked increase in EPOR transcription after the insult compared with sham controls, particularly in the subplate (sp) and overlying cortex (ctx). Upper panels, Bar = 100 μm. Dotted line in higher magnification of boxed regions (lower panels) demarcates the gray matter-white matter junction just below the subplate. Bar = 10 μm. e: Enzyme-linked immunosorbent assay for EPO ligand shows developmental decrease in EPO expression from embryonic Day 19 through postnatal Day 5, with no significant difference induced by embryonic Day 18 injury (p = 0.003). f: Representative Western blot of EPOR and β-actin from postnatal Day 0 brain samples. g: Western blot EPOR levels at postnatal Days 0 and 5 from the frontal lobe brain, standardized to β-actin levels, showed a 2-fold increase in EPOR expression after the insult (n = 5–10; *p = 0.037, **p < 0.001).

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    Images demonstrating that TSHI insult on embryonic Day 18 has minimal effect on postnatal EPO ligand expression, but alters expression of the EPOR. a: Erythropoietin ligand expression visualized with immunoperoxidase labeling gradually decreases postnatally from postnatal Day 2 through Day 9 in the cortex (ctx), subplate (sp), and white matter (wm) in sham controls, with minimal difference induced by the prenatal injury. Bar = 100 μm. b: Erythropoietin receptor immunolabeling also gradually decreases from postnatal Day 2 through Day 9 in sham controls, but remains elevated through postnatal Day 9 after the embryonic Day 18 insult, especially in the subplate. Bar = 100 μm c: Immunolabeling for the EPO ligand at postnatal Day 5 did not demonstrate any difference in the cortex or subplate after the embryonic Day 18 insult, and fewer cells appear immunolabeled with EPO ligand antibodies in the white matter after the embryonic Day 18 TSHI. Insets shown on right side for control (upper) and insult (lower). Bar = 100 μm. d: Erythropoietin receptor immunolabeling in the subplate and periventricular white matter at postnatal Day 5. Although fewer cells are present in the subplate after the insult, a greater proportion of the remaining subplate cells are labeled with EPORs by immunoperoxidase staining. More white matter cells express EPORs after the insult, in contrast to the lack of increased ligand expression observed after the insult. Bar = 10 μm.

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    Images showing EPORs are present on neural cells in the developing brain, including oligodendroglial lineage cells. a: White matter (corpus callosum) at postnatal Day 5 double-labeled in vivo with O4 and EPOR antibodies show EPORs are present on O4-immunopositive oligodendrocytes. Bar = 10 μm. b: Frontal lobe cells from postnatal Day 1 and 1 day in vitro sham control pups double-labeled with O4 and EPOR antibodies in vitro show EPORs are present on oligodendroglial cell bodies and processes. Similar cultures from insult animals showed EPOR-immunopositive expression on β-tubulin–positive neurons, GFAP-positive astrocytes, and ED1-positive microglia. Bar = 10μm. c: A gradual increase in EPOR expression on individual postnatal Day 2 equivalent (postnatal Day 1 and 1 day in vitro) sham control cells in vitro visualized using double-labeling with immunofluorescence correlates with the maturation stage of cells in the oligodendroglial lineage, with more EPOR labeling visible on more mature oligodendroglial lineage cells (Semiquantitative Intensity Scale: 0 = none, 1 = faint, 2 = strong). After the embryonic Day 18 insult the average intensity of EPOR expression per cell is elevated compared with sham control cells (*p < 0.02, **p < 0.0001). d: The average intensity of EPOR expression per cell after the embryonic Day 18 insult was also increased in vitro at postnatal Day 2 on neurons, astrocytes, and microglia (*p < 0.0001, **p = 0.0003).

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    Neonatal intraperitoneal administration of exogenous rhEPO results in systemic absorption, influences the hematocrit, and decreases the number of activated caspase-3–positive cells in the brain. a: Serum human rhEPO levels collected on postnatal Day 2, 6 hours after rhEPO (2000U/kg intraperitoneally on postnatal Days 1–2) and measured by ELISA, were elevated from baseline of zero, with no difference noted between insult and control rats. b: Hematocrit comparison between control and insult rats at postnatal Day 5 after treatment with saline or 1 of the 3 rhEPO dosing regimens. The hematocrit of saline-treated insult pups is significantly lower than saline-treated sham controls (*p = 0.003), reflecting the impact of the systemic insult on embryonic Day 18. The high-dose rhEPO regimen (2000 IU/kg for postnatal Days 1–5) increases the hematocrit in both control and insult rats compared with the other 3 groups (saline, low, or moderate dose rhEPO regimens: **p < 0.0001). The hematocrit elevation induced by high-dose rhEPO in insult pups is counterbalanced by the insult-induced decrease in hematocrit. After high-dose rhEPO treatment, insult pup hematocrit does not differ from normal saline-treated sham control hematocrits. c: Anticleaved caspase-3 antibodies visualized with immunoperoxidase-labeled cells in the periventricular white matter undergoing cell death at postnatal Day 5. The number of cleaved caspase-3–positive cells is increased (arrows) after TSHI on embryonic Day 18 compared with sham controls. Neonatal EPO treatment (1000 U/kg on postnatal Days 1–3) causes a marked decrease in the number of activated caspase-3–positive cells. Bar = 100 μm. d: Quantification of cleaved caspase-3–positive expression in the periventricular white matter at postnatal Days 5 and 9 (*p < 0.0001).

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    Neonatal rhEPO treatment after prenatal TSHI restores oligodendrocyte development and myelin production. a: Neonatal rhEPO treatment restores the number of O4-immunoperoxidase labeled oligodendrocyte lineage cells observed in the corpus callosum in vivo in postinsult rats at postnatal Day 9. Bar = 10 μm. OPCs = oligodendrocyte precursor cells. b: The number of O4-immunopositive oligodendrocytes immunolabeled at postnatal Day 9 is diminished in the periventricular white matter after prenatal TSHI, compared with sham controls, and restored to control density with rhEPO (*p < 0.0001). c: Western blot of MBP in juvenile (postnatal Day 24) periventricular white matter from saline-treated sham control and prenatal TSHI insult animals, and from EPO-treated controls and insults. d: Relative proportion of MBP to β-actin at postnatal Day 24 and in adults shows insult animals had significantly less MBP present (*p = 0.028). Myelin production in rhEPO-treated postinsult adults was restored (**p = 0.002), and supranormal MBP production appeared in adult EPO-treated sham controls (***p = 0.001, 2-way ANOVA).

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    Exogenous neonatal EPO treatment restores GABAergic neuronal counts after prenatal insult. a: Loss of GAD-67 immunoperoxidase-labeled cells in layer IV of the cingulate gyrus in vivo occurs in insult rats at postnatal Day 9, compared with sham controls (*p < 0.0004), and is restored after neonatal rhEPO treatment (**p < 0.0001), comparable to sham controls. b: At postnatal Day 24, loss of GAD-67–immunopositive cells persists in the deep parietal cortex of postinsult rats compared with sham controls (*p < 0.0001), and is restored in rhEPO-treated postinsult rats compared with saline-treated insult rats (**p < 0.01). c: Parvalbumin (PVA) immunoperoxidase-labeling in postnatal Day 24 deep parietal cortex shows the loss of PVA-immunopositive neurons after the insult, and improvement with rhEPO treatment. Bar = 100 μm. d: Graph shows significant loss of PVA-immunopositive neurons at postnatal Day 24 after the embryonic Day 18 insult, and after rhEPO treatment in insult rats, numbers comparable to controls (*p < 0.0001). e: In adult rats, significantly fewer deep parietal PVA-immunopositive neurons are present after the embryonic Day 18 insult (*p < 0.0001). Improvement in PVA-immunopositive neuronal number occurs after neonatal EPO treatment.

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    Graphs showing motor skills and seizure threshold deficits from prenatal TSHI are improved by neonatal rhEPO treatment. a: Insult rats demonstrate dose-responsive improvement in bar holding with neonatal EPO treatment. While a single low nonneuroprotective dose (500 IU/kg) of rEPO on postnatal Day 1, or 3 moderate doses (1000 IU/kg on postnatal Days 1–3) failed to produce significant improvement in individual time trials at postnatal Day 15 on the bar test compared with saline-treated insult rats, a high neuroprotective dose (2000 IU/kg on postnatal Days 1–5) induced significant improvement in insult rats (*p = 0.002, 2-way ANOVA), such that the insult rats were indistinguishable from control rats treated with saline. b: Stride length in adult rats is decreased after the prenatal insult (*p < 0.0001) compared with sham controls, and improves after high-dose neonatal rhEPO (2000 IU/kg for postnatal Days 1–5; **p = 0.014, 2-way ANOVA). c: Adult rats after the prenatal insult made more total errors on the horizontal ladder with irregular rungs than sham controls. Insult rats with high-dose neonatal rhEPO made significantly fewer total errors than saline-treated rats (**p = 0.006, 2-way ANOVA). d–f: The seizure threshold was lowered using PTZ, a GABA antagonist, in an escalating dose paradigm. Seizures were evaluated on a scale with 3 grades. The dose required to induce each seizure grade was significantly lower in postinsult rats, compared with sham controls (*Grade 1: p = 0.012; Grade II: p = 0.02; Grade III: p = 0.03). With high-dose neonatal rhEPO, the PTZ dose necessary to induce each grade of seizure in adult rats after the prenatal insult was significantly higher than in saline-treated postinsult rats (**Grade I: p = 0.0001; Grade II: p = 0.006; Grade III: p = 0.006; 2-way ANOVA), and comparable to doses for control rats.

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