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Complex pattern of interaction between in utero hypoxia-ischemia and intra-amniotic inflammation disrupts brain development and motor function.

Jantzie LL, Corbett CJ, Berglass J, Firl DJ, Flores J, Mannix R, Robinson S - J Neuroinflammation (2014)

Bottom Line: At postnatal day 15, myelin basic protein expression is reduced by 31% in TSHI + LPS pups compared to shams (P < 0.05).Both myelin basic protein expression (P < 0.01) and the phosphoneurofilament/neurofilament ratio, a marker of axonal dysfunction, are reduced in postnatal day 28 TSHI pups (P < 0.001).Both injuries cause significant gait deficits.

View Article: PubMed Central - HTML - PubMed

Affiliation: Departments of Neurology and Neurosurgery, F,M, Kirby Center for Neurobiology, Boston Children's Hospital, Harvard Medical School, 300 Longwood Avenue, Boston, MA 02115, USA. Shenandoah.Robinson@childrens.harvard.edu.

ABSTRACT

Background: Infants born preterm commonly suffer from a combination of hypoxia-ischemia (HI) and infectious perinatal inflammatory insults that lead to cerebral palsy, cognitive delay, behavioral issues and epilepsy. Using a novel rat model of combined late gestation HI and lipopolysaccharide (LPS)-induced inflammation, we tested our hypothesis that inflammation from HI and LPS differentially affects gliosis, white matter development and motor impairment during the first postnatal month.

Methods: Pregnant rats underwent laparotomy on embryonic day 18 and transient systemic HI (TSHI) and/or intra-amniotic LPS injection. Shams received laparotomy and anesthesia only. Pups were born at term. Immunohistochemistry with stereological estimates was performed to assess regional glial loads, and western blots were performed for protein expression. Erythropoietin ligand and receptor levels were quantified using quantitative PCR. Digigait analysis detected gait deficits. Statistical analysis was performed with one-way analysis of variance and post-hoc Bonferonni correction.

Results: Microglial and astroglial immunolabeling are elevated in TSHI + LPS fimbria at postnatal day 2 compared to sham (both P < 0.03). At postnatal day 15, myelin basic protein expression is reduced by 31% in TSHI + LPS pups compared to shams (P < 0.05). By postnatal day 28, white matter injury shifts from the acute injury pattern to a chronic injury pattern in TSHI pups only. Both myelin basic protein expression (P < 0.01) and the phosphoneurofilament/neurofilament ratio, a marker of axonal dysfunction, are reduced in postnatal day 28 TSHI pups (P < 0.001). Erythropoietin ligand to receptor ratios differ between brains exposed to TSHI and LPS. Gait analyses reveal that all groups (TSHI, LPS and TSHI + LPS) are ataxic with deficits in stride, paw placement, gait consistency and coordination (all P < 0.001).

Conclusions: Prenatal TSHI and TSHI + LPS lead to different patterns of injury with respect to myelination, axon integrity and gait deficits. Dual injury leads to acute alterations in glial response and cellular inflammation, while TSHI alone causes more prominent chronic white matter and axonal injury. Both injuries cause significant gait deficits. Further study will contribute to stratification of injury mechanisms in preterm infants, and guide the use of promising therapeutic interventions.

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Injury impacts erythropoietin ligand and receptor mRNA levels. (A) At postnatal day 2 (P2), erythropoietin receptor (EPOR) mRNA levels are significantly higher in transient systemic hypoxia-ischemia (TSHI) pups compared to sham levels, without a concomitant increase in erythropoietin (EPO) ligand mRNA levels (n = 5-14, ***P < 0.001). (B) By P15 no significant differences in EPO or EPOR mRNA exists between injury groups (n = 3-7). (C) The patterns of the ratio of EPO ligand to EPO receptor shift from P2 to P15, with sustained ligand to receptor mismatch in TSHI pups, in contrast to the shift observed in pups exposed to lipopolysaccharide (LPS) or TSHI + LPS. The persistent ligand deficiency correlates with the sustained loss of myelin basic protein and the reduced phosphoneurofilament/neurofilament ratio observed at P28 in TSHI white matter, but not LPS or TSHI + LPS white matter.
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Figure 5: Injury impacts erythropoietin ligand and receptor mRNA levels. (A) At postnatal day 2 (P2), erythropoietin receptor (EPOR) mRNA levels are significantly higher in transient systemic hypoxia-ischemia (TSHI) pups compared to sham levels, without a concomitant increase in erythropoietin (EPO) ligand mRNA levels (n = 5-14, ***P < 0.001). (B) By P15 no significant differences in EPO or EPOR mRNA exists between injury groups (n = 3-7). (C) The patterns of the ratio of EPO ligand to EPO receptor shift from P2 to P15, with sustained ligand to receptor mismatch in TSHI pups, in contrast to the shift observed in pups exposed to lipopolysaccharide (LPS) or TSHI + LPS. The persistent ligand deficiency correlates with the sustained loss of myelin basic protein and the reduced phosphoneurofilament/neurofilament ratio observed at P28 in TSHI white matter, but not LPS or TSHI + LPS white matter.

Mentions: EPO signaling influences the genesis, survival and maturation of developing neural cells after prenatal injury, primarily through the balance of the ligand and receptor levels, which are transcriptionally regulated [37,38]. To determine if the different in utero lesions altered postnatal EPO signaling in the brain, quantitative PCR analyses of EPO and EPO receptor (EPOR) mRNA were performed at P2 and P15. At P2, EPO ligand mRNA was consistent across the injured groups compared to shams (Figure 5A). In contrast, significant increases in EPOR mRNA were observed in TSHI pups (2.0 ± 0.1, n = 7) compared to sham controls (0.9 ± 0.1, n = 7, P = 0.001, Figure 5A). The relative levels of mRNA expression had shifted by P15 (Figure 5B). Levels of white matter EPO lig and mRNA trended down following TSHI, while in the presence of LPS-induced inflammation levels were stable, without or with TSHI. Similarly, EPOR mRNA levels were lower at P15 in the presence of LPS. As shown in Figure 5C, the pattern of the EPO ligand:receptor ratio shifted from P2 to P15. The ligand:receptor mismatch and relative ligand deficiency persisted in TSHI pups from P2 through P15, consistent with the reduced MBP expression and pNF/NF ratio observed in white matter from TSHI rats at P28. By contrast, in the presence of LPS, the EPO ligand:receptor ratio stabilized by P15, consistent with the lack of significant white matter MBP and NF changes observed in LPS or TSHI + LPS rats at P28. These data indicate that the impact of prenatal injury on EPO signaling may vary depending on whether concurrent inflammation is present or not, and that prenatal TSHI may induce a more severe, sustained injury to the developing white matter. Further detailed analyses are underway to clarify these complex interactions.


Complex pattern of interaction between in utero hypoxia-ischemia and intra-amniotic inflammation disrupts brain development and motor function.

Jantzie LL, Corbett CJ, Berglass J, Firl DJ, Flores J, Mannix R, Robinson S - J Neuroinflammation (2014)

Injury impacts erythropoietin ligand and receptor mRNA levels. (A) At postnatal day 2 (P2), erythropoietin receptor (EPOR) mRNA levels are significantly higher in transient systemic hypoxia-ischemia (TSHI) pups compared to sham levels, without a concomitant increase in erythropoietin (EPO) ligand mRNA levels (n = 5-14, ***P < 0.001). (B) By P15 no significant differences in EPO or EPOR mRNA exists between injury groups (n = 3-7). (C) The patterns of the ratio of EPO ligand to EPO receptor shift from P2 to P15, with sustained ligand to receptor mismatch in TSHI pups, in contrast to the shift observed in pups exposed to lipopolysaccharide (LPS) or TSHI + LPS. The persistent ligand deficiency correlates with the sustained loss of myelin basic protein and the reduced phosphoneurofilament/neurofilament ratio observed at P28 in TSHI white matter, but not LPS or TSHI + LPS white matter.
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Figure 5: Injury impacts erythropoietin ligand and receptor mRNA levels. (A) At postnatal day 2 (P2), erythropoietin receptor (EPOR) mRNA levels are significantly higher in transient systemic hypoxia-ischemia (TSHI) pups compared to sham levels, without a concomitant increase in erythropoietin (EPO) ligand mRNA levels (n = 5-14, ***P < 0.001). (B) By P15 no significant differences in EPO or EPOR mRNA exists between injury groups (n = 3-7). (C) The patterns of the ratio of EPO ligand to EPO receptor shift from P2 to P15, with sustained ligand to receptor mismatch in TSHI pups, in contrast to the shift observed in pups exposed to lipopolysaccharide (LPS) or TSHI + LPS. The persistent ligand deficiency correlates with the sustained loss of myelin basic protein and the reduced phosphoneurofilament/neurofilament ratio observed at P28 in TSHI white matter, but not LPS or TSHI + LPS white matter.
Mentions: EPO signaling influences the genesis, survival and maturation of developing neural cells after prenatal injury, primarily through the balance of the ligand and receptor levels, which are transcriptionally regulated [37,38]. To determine if the different in utero lesions altered postnatal EPO signaling in the brain, quantitative PCR analyses of EPO and EPO receptor (EPOR) mRNA were performed at P2 and P15. At P2, EPO ligand mRNA was consistent across the injured groups compared to shams (Figure 5A). In contrast, significant increases in EPOR mRNA were observed in TSHI pups (2.0 ± 0.1, n = 7) compared to sham controls (0.9 ± 0.1, n = 7, P = 0.001, Figure 5A). The relative levels of mRNA expression had shifted by P15 (Figure 5B). Levels of white matter EPO lig and mRNA trended down following TSHI, while in the presence of LPS-induced inflammation levels were stable, without or with TSHI. Similarly, EPOR mRNA levels were lower at P15 in the presence of LPS. As shown in Figure 5C, the pattern of the EPO ligand:receptor ratio shifted from P2 to P15. The ligand:receptor mismatch and relative ligand deficiency persisted in TSHI pups from P2 through P15, consistent with the reduced MBP expression and pNF/NF ratio observed in white matter from TSHI rats at P28. By contrast, in the presence of LPS, the EPO ligand:receptor ratio stabilized by P15, consistent with the lack of significant white matter MBP and NF changes observed in LPS or TSHI + LPS rats at P28. These data indicate that the impact of prenatal injury on EPO signaling may vary depending on whether concurrent inflammation is present or not, and that prenatal TSHI may induce a more severe, sustained injury to the developing white matter. Further detailed analyses are underway to clarify these complex interactions.

Bottom Line: At postnatal day 15, myelin basic protein expression is reduced by 31% in TSHI + LPS pups compared to shams (P < 0.05).Both myelin basic protein expression (P < 0.01) and the phosphoneurofilament/neurofilament ratio, a marker of axonal dysfunction, are reduced in postnatal day 28 TSHI pups (P < 0.001).Both injuries cause significant gait deficits.

View Article: PubMed Central - HTML - PubMed

Affiliation: Departments of Neurology and Neurosurgery, F,M, Kirby Center for Neurobiology, Boston Children's Hospital, Harvard Medical School, 300 Longwood Avenue, Boston, MA 02115, USA. Shenandoah.Robinson@childrens.harvard.edu.

ABSTRACT

Background: Infants born preterm commonly suffer from a combination of hypoxia-ischemia (HI) and infectious perinatal inflammatory insults that lead to cerebral palsy, cognitive delay, behavioral issues and epilepsy. Using a novel rat model of combined late gestation HI and lipopolysaccharide (LPS)-induced inflammation, we tested our hypothesis that inflammation from HI and LPS differentially affects gliosis, white matter development and motor impairment during the first postnatal month.

Methods: Pregnant rats underwent laparotomy on embryonic day 18 and transient systemic HI (TSHI) and/or intra-amniotic LPS injection. Shams received laparotomy and anesthesia only. Pups were born at term. Immunohistochemistry with stereological estimates was performed to assess regional glial loads, and western blots were performed for protein expression. Erythropoietin ligand and receptor levels were quantified using quantitative PCR. Digigait analysis detected gait deficits. Statistical analysis was performed with one-way analysis of variance and post-hoc Bonferonni correction.

Results: Microglial and astroglial immunolabeling are elevated in TSHI + LPS fimbria at postnatal day 2 compared to sham (both P < 0.03). At postnatal day 15, myelin basic protein expression is reduced by 31% in TSHI + LPS pups compared to shams (P < 0.05). By postnatal day 28, white matter injury shifts from the acute injury pattern to a chronic injury pattern in TSHI pups only. Both myelin basic protein expression (P < 0.01) and the phosphoneurofilament/neurofilament ratio, a marker of axonal dysfunction, are reduced in postnatal day 28 TSHI pups (P < 0.001). Erythropoietin ligand to receptor ratios differ between brains exposed to TSHI and LPS. Gait analyses reveal that all groups (TSHI, LPS and TSHI + LPS) are ataxic with deficits in stride, paw placement, gait consistency and coordination (all P < 0.001).

Conclusions: Prenatal TSHI and TSHI + LPS lead to different patterns of injury with respect to myelination, axon integrity and gait deficits. Dual injury leads to acute alterations in glial response and cellular inflammation, while TSHI alone causes more prominent chronic white matter and axonal injury. Both injuries cause significant gait deficits. Further study will contribute to stratification of injury mechanisms in preterm infants, and guide the use of promising therapeutic interventions.

Show MeSH
Related in: MedlinePlus