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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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Myelin basic protein and neurofilament expression is significantly decreased in transient systemic hypoxia-ischemia pups on postnatal day 28. (A) Following transient systemic hypoxia-ischemia (TSHI) on embryonic day 18, western blot analyses at postnatal day 28 reveal protein expression of myelin basic protein (MBP), phosphoneurofilament (pNF), and neurofilament (NF) is significantly altered in microdissected white matter. (B) MBP expression is significantly decreased in microdissected white matter from TSHI pups compared to sham levels (n = 3-10, **P < 0.01). (C) Similarly, the ratio of pNF/NF is significantly decreased in TSHI pups compared to shams, indicative of axonal dysfunction (n = 4-13, ***P < 0.001). LPS, lipopolysaccharide.
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Figure 4: Myelin basic protein and neurofilament expression is significantly decreased in transient systemic hypoxia-ischemia pups on postnatal day 28. (A) Following transient systemic hypoxia-ischemia (TSHI) on embryonic day 18, western blot analyses at postnatal day 28 reveal protein expression of myelin basic protein (MBP), phosphoneurofilament (pNF), and neurofilament (NF) is significantly altered in microdissected white matter. (B) MBP expression is significantly decreased in microdissected white matter from TSHI pups compared to sham levels (n = 3-10, **P < 0.01). (C) Similarly, the ratio of pNF/NF is significantly decreased in TSHI pups compared to shams, indicative of axonal dysfunction (n = 4-13, ***P < 0.001). LPS, lipopolysaccharide.

Mentions: To determine whether the reduced expression of MBP observed at P15 was transient or sustained, white matter was also analysed at P28, a time point equivalent to an older teenager when myelination is complete. These studies revealed an apparent shift from an acute white matter injury pattern with MBP loss in combined TSHI + LPS at P15 to a different chronic injury pattern at P28 juvenile rats. Notably, the chronic white matter injury pattern was present in TSHI pups, but not observed in rats exposed to LPS. At P28, TSHI pups had significantly decreased MBP protein expression compared to shams (Figure 4A). Specifically, P28 TSHI pups had a 28% reduction in MBP (0.72 ± 0.07, n = 10) compared to sham animals (1.04 ± 0.10, n = 7, P = 0.038, Figure 4B). Analysis of the axonal proteins, NF and pNF, corroborated MBP levels and revealed chronic axonal injury was also present in TSHI pups. The ratio of pNF/NF, a measure of axonal health, was markedly reduced at P28 in TSHI pups (0.79 ± 0.03, n = 9) compared to shams (1.00 ± 0.04, n = 6, P = 0.005, Figure 4C), consistent with the chronic loss of MBP observed at P28. Interestingly, these results indicate prenatal TSHI causes a more sustained white matter injury than prenatal LPS-induced inflammation, suggesting that LPS and TSHI have differing mechanisms of injury that cause varying patterns of injury to the immature white matter and developing axons during the first month of postnatal CNS maturation.


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)

Myelin basic protein and neurofilament expression is significantly decreased in transient systemic hypoxia-ischemia pups on postnatal day 28. (A) Following transient systemic hypoxia-ischemia (TSHI) on embryonic day 18, western blot analyses at postnatal day 28 reveal protein expression of myelin basic protein (MBP), phosphoneurofilament (pNF), and neurofilament (NF) is significantly altered in microdissected white matter. (B) MBP expression is significantly decreased in microdissected white matter from TSHI pups compared to sham levels (n = 3-10, **P < 0.01). (C) Similarly, the ratio of pNF/NF is significantly decreased in TSHI pups compared to shams, indicative of axonal dysfunction (n = 4-13, ***P < 0.001). LPS, lipopolysaccharide.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4128546&req=5

Figure 4: Myelin basic protein and neurofilament expression is significantly decreased in transient systemic hypoxia-ischemia pups on postnatal day 28. (A) Following transient systemic hypoxia-ischemia (TSHI) on embryonic day 18, western blot analyses at postnatal day 28 reveal protein expression of myelin basic protein (MBP), phosphoneurofilament (pNF), and neurofilament (NF) is significantly altered in microdissected white matter. (B) MBP expression is significantly decreased in microdissected white matter from TSHI pups compared to sham levels (n = 3-10, **P < 0.01). (C) Similarly, the ratio of pNF/NF is significantly decreased in TSHI pups compared to shams, indicative of axonal dysfunction (n = 4-13, ***P < 0.001). LPS, lipopolysaccharide.
Mentions: To determine whether the reduced expression of MBP observed at P15 was transient or sustained, white matter was also analysed at P28, a time point equivalent to an older teenager when myelination is complete. These studies revealed an apparent shift from an acute white matter injury pattern with MBP loss in combined TSHI + LPS at P15 to a different chronic injury pattern at P28 juvenile rats. Notably, the chronic white matter injury pattern was present in TSHI pups, but not observed in rats exposed to LPS. At P28, TSHI pups had significantly decreased MBP protein expression compared to shams (Figure 4A). Specifically, P28 TSHI pups had a 28% reduction in MBP (0.72 ± 0.07, n = 10) compared to sham animals (1.04 ± 0.10, n = 7, P = 0.038, Figure 4B). Analysis of the axonal proteins, NF and pNF, corroborated MBP levels and revealed chronic axonal injury was also present in TSHI pups. The ratio of pNF/NF, a measure of axonal health, was markedly reduced at P28 in TSHI pups (0.79 ± 0.03, n = 9) compared to shams (1.00 ± 0.04, n = 6, P = 0.005, Figure 4C), consistent with the chronic loss of MBP observed at P28. Interestingly, these results indicate prenatal TSHI causes a more sustained white matter injury than prenatal LPS-induced inflammation, suggesting that LPS and TSHI have differing mechanisms of injury that cause varying patterns of injury to the immature white matter and developing axons during the first month of postnatal CNS maturation.

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