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Systemic inflammation in early neonatal mice induces transient and lasting neurodegenerative effects.

Cardoso FL, Herz J, Fernandes A, Rocha J, Sepodes B, Brito MA, McGavern DB, Brites D - J Neuroinflammation (2015)

Bottom Line: However, the progressive effects on the murine neurodevelopmental program over the week that follows systemic inflammation are not known.The impaired myelination was associated with alterations in the proliferation and differentiation of NG2 progenitor cells early after LPS administration, rather than with excessive phagocytosis by CNS myeloid cells.Quantification of inflammatory biomarkers revealed decreased expression of ATX with concurrent increases in HMGB1, TLR-4, and MMP-9 expression levels.

View Article: PubMed Central - PubMed

Affiliation: Research Institute for Medicines (iMed.ULisboa), Faculdade de Farmácia, Universidade de Lisboa, Avenida Professor Gama Pinto, 1649-003, Lisbon, Portugal. filipacardoso@ff.ulisboa.pt.

ABSTRACT

Background: The inflammatory mediator lipopolysaccharide (LPS) has been shown to induce acute gliosis in neonatal mice. However, the progressive effects on the murine neurodevelopmental program over the week that follows systemic inflammation are not known. Thus, we investigated the effects of repeated LPS administration in the first postnatal week in mice, a condition mimicking sepsis in late preterm infants, on the developing central nervous system (CNS).

Methods: Systemic inflammation was induced by daily intraperitoneal administration (i.p.) of LPS (6 mg/kg) in newborn mice from postnatal day (PND) 4 to PND6. The effects on neurodevelopment were examined by staining the white matter and neurons with Luxol Fast Blue and Cresyl Violet, respectively. The inflammatory response was assessed by quantifying the expression/activity of matrix metalloproteinases (MMP), toll-like receptor (TLR)-4, high mobility group box (HMGB)-1, and autotaxin (ATX). In addition, B6 CX3CR1(gfp/+) mice combined with cryo-immunofluorescence were used to determine the acute, delayed, and lasting effects on myelination, microglia, and astrocytes.

Results: LPS administration led to acute body and brain weight loss as well as overt structural changes in the brain such as cerebellar hypoplasia, neuronal loss/shrinkage, and delayed myelination. The impaired myelination was associated with alterations in the proliferation and differentiation of NG2 progenitor cells early after LPS administration, rather than with excessive phagocytosis by CNS myeloid cells. In addition to disruptions in brain architecture, a robust inflammatory response to LPS was observed. Quantification of inflammatory biomarkers revealed decreased expression of ATX with concurrent increases in HMGB1, TLR-4, and MMP-9 expression levels. Acute astrogliosis (GFAP(+) cells) in the brain parenchyma and at the microvasculature interface together with parenchymal microgliosis (CX3CR1(+) cells) were also observed. These changes preceded the migration/proliferation of CX3CR1(+) cells around the vessels at later time points and the subsequent loss of GFAP(+) astrocytes.

Conclusion: Collectively, our study has uncovered a complex innate inflammatory reaction and associated structural changes in the brains of neonatal mice challenged peripherally with LPS. These findings may explain some of the neurobehavioral abnormalities that develop following neonatal sepsis.

No MeSH data available.


Related in: MedlinePlus

Lipopolysaccharide (LPS) administration triggers transient shrinkage of cerebellar layers, acute neuronal loss, and sustained atrophy. Paraffin sections from CD1 wild-type mice at 1 and 9 days post-LPS administration were stained with Luxol Fast Blue (myelin, blue) followed by Cresyl Violet (Nissl bodies, purple). (A) Representative images are shown for each condition in the pons, hippocampus, and cerebellum. (B) The widths of each cerebellar layer [external germinal layer (EGL), molecular layer (ML), Purkinje layer, internal granular layer (IGL), white matter layer (WML)] were measured. (C,D) Intensity of Cresyl Violet staining in the EGL and of Luxol Fast Blue in WML were quantified per square micrometer at 1 and 9 days after LPS administration, respectively. (E,F) The number of neurons per field and the area of neuronal cell body (soma) was quantified throughout the pons, in the CA3 hippocampal region, and in the cerebellar Purkinje layer (PL). All determinations were done using ImageJ software (NIH, USA). Results are mean ± SEM from at least five animals. *P < 0.05 vs. without (W/O) LPS.
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Fig3: Lipopolysaccharide (LPS) administration triggers transient shrinkage of cerebellar layers, acute neuronal loss, and sustained atrophy. Paraffin sections from CD1 wild-type mice at 1 and 9 days post-LPS administration were stained with Luxol Fast Blue (myelin, blue) followed by Cresyl Violet (Nissl bodies, purple). (A) Representative images are shown for each condition in the pons, hippocampus, and cerebellum. (B) The widths of each cerebellar layer [external germinal layer (EGL), molecular layer (ML), Purkinje layer, internal granular layer (IGL), white matter layer (WML)] were measured. (C,D) Intensity of Cresyl Violet staining in the EGL and of Luxol Fast Blue in WML were quantified per square micrometer at 1 and 9 days after LPS administration, respectively. (E,F) The number of neurons per field and the area of neuronal cell body (soma) was quantified throughout the pons, in the CA3 hippocampal region, and in the cerebellar Purkinje layer (PL). All determinations were done using ImageJ software (NIH, USA). Results are mean ± SEM from at least five animals. *P < 0.05 vs. without (W/O) LPS.

Mentions: Given the observed decrease in the cerebellar area, we next explored the width of each layer: the external germinal layer (with proliferating neuroepithelial cells), the molecular layer (containing the axons of granule cells and dendrites of Purkinje cells), the Purkinje neuronal layer, the granular layer (with small neurons called granule cells), and the white matter layer (with myelin fibers) (representative images of cerebellum are shown in Figure 3A). There was a significant shrinkage of the neuron-containing layers at 24 h after the last LPS injection (P < 0.05 for the Purkinje layer; P < 0.01 for the external germinal layer and granular layer) but not at LPS9 (Figure 3B). In agreement, the density of neurons based on the intensity of staining per square micrometer of cells in the external germinal layer was also markedly reduced at LPS1 (approximately twofold, P < 0.05) (Figure 3C). Lastly, a negative impact on the density of Luxol Fast Blue-labeled myelin fibers in the cerebellum was evident at LPS9 (Figure 3D, P < 0.05).Figure 3


Systemic inflammation in early neonatal mice induces transient and lasting neurodegenerative effects.

Cardoso FL, Herz J, Fernandes A, Rocha J, Sepodes B, Brito MA, McGavern DB, Brites D - J Neuroinflammation (2015)

Lipopolysaccharide (LPS) administration triggers transient shrinkage of cerebellar layers, acute neuronal loss, and sustained atrophy. Paraffin sections from CD1 wild-type mice at 1 and 9 days post-LPS administration were stained with Luxol Fast Blue (myelin, blue) followed by Cresyl Violet (Nissl bodies, purple). (A) Representative images are shown for each condition in the pons, hippocampus, and cerebellum. (B) The widths of each cerebellar layer [external germinal layer (EGL), molecular layer (ML), Purkinje layer, internal granular layer (IGL), white matter layer (WML)] were measured. (C,D) Intensity of Cresyl Violet staining in the EGL and of Luxol Fast Blue in WML were quantified per square micrometer at 1 and 9 days after LPS administration, respectively. (E,F) The number of neurons per field and the area of neuronal cell body (soma) was quantified throughout the pons, in the CA3 hippocampal region, and in the cerebellar Purkinje layer (PL). All determinations were done using ImageJ software (NIH, USA). Results are mean ± SEM from at least five animals. *P < 0.05 vs. without (W/O) LPS.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Fig3: Lipopolysaccharide (LPS) administration triggers transient shrinkage of cerebellar layers, acute neuronal loss, and sustained atrophy. Paraffin sections from CD1 wild-type mice at 1 and 9 days post-LPS administration were stained with Luxol Fast Blue (myelin, blue) followed by Cresyl Violet (Nissl bodies, purple). (A) Representative images are shown for each condition in the pons, hippocampus, and cerebellum. (B) The widths of each cerebellar layer [external germinal layer (EGL), molecular layer (ML), Purkinje layer, internal granular layer (IGL), white matter layer (WML)] were measured. (C,D) Intensity of Cresyl Violet staining in the EGL and of Luxol Fast Blue in WML were quantified per square micrometer at 1 and 9 days after LPS administration, respectively. (E,F) The number of neurons per field and the area of neuronal cell body (soma) was quantified throughout the pons, in the CA3 hippocampal region, and in the cerebellar Purkinje layer (PL). All determinations were done using ImageJ software (NIH, USA). Results are mean ± SEM from at least five animals. *P < 0.05 vs. without (W/O) LPS.
Mentions: Given the observed decrease in the cerebellar area, we next explored the width of each layer: the external germinal layer (with proliferating neuroepithelial cells), the molecular layer (containing the axons of granule cells and dendrites of Purkinje cells), the Purkinje neuronal layer, the granular layer (with small neurons called granule cells), and the white matter layer (with myelin fibers) (representative images of cerebellum are shown in Figure 3A). There was a significant shrinkage of the neuron-containing layers at 24 h after the last LPS injection (P < 0.05 for the Purkinje layer; P < 0.01 for the external germinal layer and granular layer) but not at LPS9 (Figure 3B). In agreement, the density of neurons based on the intensity of staining per square micrometer of cells in the external germinal layer was also markedly reduced at LPS1 (approximately twofold, P < 0.05) (Figure 3C). Lastly, a negative impact on the density of Luxol Fast Blue-labeled myelin fibers in the cerebellum was evident at LPS9 (Figure 3D, P < 0.05).Figure 3

Bottom Line: However, the progressive effects on the murine neurodevelopmental program over the week that follows systemic inflammation are not known.The impaired myelination was associated with alterations in the proliferation and differentiation of NG2 progenitor cells early after LPS administration, rather than with excessive phagocytosis by CNS myeloid cells.Quantification of inflammatory biomarkers revealed decreased expression of ATX with concurrent increases in HMGB1, TLR-4, and MMP-9 expression levels.

View Article: PubMed Central - PubMed

Affiliation: Research Institute for Medicines (iMed.ULisboa), Faculdade de Farmácia, Universidade de Lisboa, Avenida Professor Gama Pinto, 1649-003, Lisbon, Portugal. filipacardoso@ff.ulisboa.pt.

ABSTRACT

Background: The inflammatory mediator lipopolysaccharide (LPS) has been shown to induce acute gliosis in neonatal mice. However, the progressive effects on the murine neurodevelopmental program over the week that follows systemic inflammation are not known. Thus, we investigated the effects of repeated LPS administration in the first postnatal week in mice, a condition mimicking sepsis in late preterm infants, on the developing central nervous system (CNS).

Methods: Systemic inflammation was induced by daily intraperitoneal administration (i.p.) of LPS (6 mg/kg) in newborn mice from postnatal day (PND) 4 to PND6. The effects on neurodevelopment were examined by staining the white matter and neurons with Luxol Fast Blue and Cresyl Violet, respectively. The inflammatory response was assessed by quantifying the expression/activity of matrix metalloproteinases (MMP), toll-like receptor (TLR)-4, high mobility group box (HMGB)-1, and autotaxin (ATX). In addition, B6 CX3CR1(gfp/+) mice combined with cryo-immunofluorescence were used to determine the acute, delayed, and lasting effects on myelination, microglia, and astrocytes.

Results: LPS administration led to acute body and brain weight loss as well as overt structural changes in the brain such as cerebellar hypoplasia, neuronal loss/shrinkage, and delayed myelination. The impaired myelination was associated with alterations in the proliferation and differentiation of NG2 progenitor cells early after LPS administration, rather than with excessive phagocytosis by CNS myeloid cells. In addition to disruptions in brain architecture, a robust inflammatory response to LPS was observed. Quantification of inflammatory biomarkers revealed decreased expression of ATX with concurrent increases in HMGB1, TLR-4, and MMP-9 expression levels. Acute astrogliosis (GFAP(+) cells) in the brain parenchyma and at the microvasculature interface together with parenchymal microgliosis (CX3CR1(+) cells) were also observed. These changes preceded the migration/proliferation of CX3CR1(+) cells around the vessels at later time points and the subsequent loss of GFAP(+) astrocytes.

Conclusion: Collectively, our study has uncovered a complex innate inflammatory reaction and associated structural changes in the brains of neonatal mice challenged peripherally with LPS. These findings may explain some of the neurobehavioral abnormalities that develop following neonatal sepsis.

No MeSH data available.


Related in: MedlinePlus