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Increased expression of the chemokines CXCL1 and MIP-1α by resident brain cells precedes neutrophil infiltration in the brain following prolonged soman-induced status epilepticus in rats.

Johnson EA, Dao TL, Guignet MA, Geddes CE, Koemeter-Cox AI, Kan RK - J Neuroinflammation (2011)

Bottom Line: Chemokines with significantly increased protein levels were localized to resident brain cells (i.e. neurons, astrocytes, microglia and endothelial cells).We observed significant concentration increases for CXCL1 and MIP-1α after seizure onset.This process may play a key role in the progressive secondary brain pathology observed in this model though further study is warranted.

View Article: PubMed Central - HTML - PubMed

Affiliation: Research Division, Pharmacology Branch, US Army Medical Research Institute of Chemical Defense, Aberdeen Proving Ground, MD 21010, USA. erik.a.johnson1@us.army.mil

ABSTRACT

Background: Exposure to the nerve agent soman (GD) causes neuronal cell death and impaired behavioral function dependent on the induction of status epilepticus (SE). Little is known about the maturation of this pathological process, though neuroinflammation and infiltration of neutrophils are prominent features. The purpose of this study is to quantify the regional and temporal progression of early chemotactic signals, describe the cellular expression of these factors and the relationship between expression and neutrophil infiltration in damaged brain using a rat GD seizure model.

Methods: Protein levels of 4 chemokines responsible for neutrophil infiltration and activation were quantified up to 72 hours in multiple brain regions (i.e. piriform cortex, hippocampus and thalamus) following SE onset using multiplex bead immunoassays. Chemokines with significantly increased protein levels were localized to resident brain cells (i.e. neurons, astrocytes, microglia and endothelial cells). Lastly, neutrophil infiltration into these brain regions was quantified and correlated to the expression of these chemokines.

Results: We observed significant concentration increases for CXCL1 and MIP-1α after seizure onset. CXCL1 expression originated from neurons and endothelial cells while MIP-1α was expressed by neurons and microglia. Lastly, the expression of these chemokines directly preceded and positively correlated with significant neutrophil infiltration in the brain. These data suggest that following GD-induced SE, a strong chemotactic response originating from various brain cells, recruits circulating neutrophils to the injured brain.

Conclusions: A strong induction of neutrophil attractant chemokines occurs following GD-induced SE resulting in neutrophil influx into injured brain tissues. This process may play a key role in the progressive secondary brain pathology observed in this model though further study is warranted.

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MIP-1α is primarily expressed by neurons and dystrophic microglia after GD-induced SE. MIP-1α immunolabeling (A-G, green) is present in the piriform cortex, hippocampus and thalamus 12 hours after GD-induced SE (A, B & C; left) but absent in vehicle controls of these same regions (A, B & C; right). In the piriform cortex (A), labeling is observed primarily in layers II and III. In the hippocampus (B), labeling was less robust and found primarily in the CA1 and CA3 pyramidal layers as well as in the polymorphic layer of the dentate gyrus (PoDG) but not the granular layer of the dentate gyrus (GrDG). MIP-1α labeling was also less robust in the thalamus (C) and was found primarily in the laterodorsal and lateral posterior nuclei. Neurons (D, red) and MIP-1α frequently co-localized (D, yellow), while no co-localization with astrocytes (E, red) was observed. MIP-1α was primarily expressed by microglia with a dystrophic morphology (F, red). Limited co-localization was observed in endothelial cells (G, red). Scale bar: 250 μm (A-C), 50 μm and 10 μm (D-F) for regular and confocal fluorescent microscopy respectively; n = 9 for 12-hour and n = 4 for vehicle controls.
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Figure 4: MIP-1α is primarily expressed by neurons and dystrophic microglia after GD-induced SE. MIP-1α immunolabeling (A-G, green) is present in the piriform cortex, hippocampus and thalamus 12 hours after GD-induced SE (A, B & C; left) but absent in vehicle controls of these same regions (A, B & C; right). In the piriform cortex (A), labeling is observed primarily in layers II and III. In the hippocampus (B), labeling was less robust and found primarily in the CA1 and CA3 pyramidal layers as well as in the polymorphic layer of the dentate gyrus (PoDG) but not the granular layer of the dentate gyrus (GrDG). MIP-1α labeling was also less robust in the thalamus (C) and was found primarily in the laterodorsal and lateral posterior nuclei. Neurons (D, red) and MIP-1α frequently co-localized (D, yellow), while no co-localization with astrocytes (E, red) was observed. MIP-1α was primarily expressed by microglia with a dystrophic morphology (F, red). Limited co-localization was observed in endothelial cells (G, red). Scale bar: 250 μm (A-C), 50 μm and 10 μm (D-F) for regular and confocal fluorescent microscopy respectively; n = 9 for 12-hour and n = 4 for vehicle controls.

Mentions: Twelve hours following seizure onset, MIP-1α immunolabeling was present in piriform cortex (Figure 4A, left), hippocampus (dentate gyrus shown; Figure 4B, left) and thalamus (Figure 4C, left) but not in vehicle controls (Figure 4A, B &4C, right). Weak to moderate diffuse co-localization with neurons (Figure 4D) was observed in layers II and III of the piriform cortex, the CA1 and CA3 pyramidal layers of the hippocampus and the polymorphic (PoDG) but not the granular layer (GrDG) of the dentate gyrus. Astrocytes were not found to express MIP-1α in any region observed (Figure 4E). Activated microglia strongly expressed MIP-1α in all regions investigated. These cells had a myriad of morphological features including hypertrophy, spheroid shape, blebbing and dystrophy. Specifically, MIP-1α shows a high degree of cellular localization with the dystrophic microglial morphology (Figure 4F). Lastly, MIP-1α-positive cells were closely associated with large blood vessel endothelial cells in the piriform cortex and thalamus but co-localization was not observed (Figure 4G).


Increased expression of the chemokines CXCL1 and MIP-1α by resident brain cells precedes neutrophil infiltration in the brain following prolonged soman-induced status epilepticus in rats.

Johnson EA, Dao TL, Guignet MA, Geddes CE, Koemeter-Cox AI, Kan RK - J Neuroinflammation (2011)

MIP-1α is primarily expressed by neurons and dystrophic microglia after GD-induced SE. MIP-1α immunolabeling (A-G, green) is present in the piriform cortex, hippocampus and thalamus 12 hours after GD-induced SE (A, B & C; left) but absent in vehicle controls of these same regions (A, B & C; right). In the piriform cortex (A), labeling is observed primarily in layers II and III. In the hippocampus (B), labeling was less robust and found primarily in the CA1 and CA3 pyramidal layers as well as in the polymorphic layer of the dentate gyrus (PoDG) but not the granular layer of the dentate gyrus (GrDG). MIP-1α labeling was also less robust in the thalamus (C) and was found primarily in the laterodorsal and lateral posterior nuclei. Neurons (D, red) and MIP-1α frequently co-localized (D, yellow), while no co-localization with astrocytes (E, red) was observed. MIP-1α was primarily expressed by microglia with a dystrophic morphology (F, red). Limited co-localization was observed in endothelial cells (G, red). Scale bar: 250 μm (A-C), 50 μm and 10 μm (D-F) for regular and confocal fluorescent microscopy respectively; n = 9 for 12-hour and n = 4 for vehicle controls.
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Figure 4: MIP-1α is primarily expressed by neurons and dystrophic microglia after GD-induced SE. MIP-1α immunolabeling (A-G, green) is present in the piriform cortex, hippocampus and thalamus 12 hours after GD-induced SE (A, B & C; left) but absent in vehicle controls of these same regions (A, B & C; right). In the piriform cortex (A), labeling is observed primarily in layers II and III. In the hippocampus (B), labeling was less robust and found primarily in the CA1 and CA3 pyramidal layers as well as in the polymorphic layer of the dentate gyrus (PoDG) but not the granular layer of the dentate gyrus (GrDG). MIP-1α labeling was also less robust in the thalamus (C) and was found primarily in the laterodorsal and lateral posterior nuclei. Neurons (D, red) and MIP-1α frequently co-localized (D, yellow), while no co-localization with astrocytes (E, red) was observed. MIP-1α was primarily expressed by microglia with a dystrophic morphology (F, red). Limited co-localization was observed in endothelial cells (G, red). Scale bar: 250 μm (A-C), 50 μm and 10 μm (D-F) for regular and confocal fluorescent microscopy respectively; n = 9 for 12-hour and n = 4 for vehicle controls.
Mentions: Twelve hours following seizure onset, MIP-1α immunolabeling was present in piriform cortex (Figure 4A, left), hippocampus (dentate gyrus shown; Figure 4B, left) and thalamus (Figure 4C, left) but not in vehicle controls (Figure 4A, B &4C, right). Weak to moderate diffuse co-localization with neurons (Figure 4D) was observed in layers II and III of the piriform cortex, the CA1 and CA3 pyramidal layers of the hippocampus and the polymorphic (PoDG) but not the granular layer (GrDG) of the dentate gyrus. Astrocytes were not found to express MIP-1α in any region observed (Figure 4E). Activated microglia strongly expressed MIP-1α in all regions investigated. These cells had a myriad of morphological features including hypertrophy, spheroid shape, blebbing and dystrophy. Specifically, MIP-1α shows a high degree of cellular localization with the dystrophic microglial morphology (Figure 4F). Lastly, MIP-1α-positive cells were closely associated with large blood vessel endothelial cells in the piriform cortex and thalamus but co-localization was not observed (Figure 4G).

Bottom Line: Chemokines with significantly increased protein levels were localized to resident brain cells (i.e. neurons, astrocytes, microglia and endothelial cells).We observed significant concentration increases for CXCL1 and MIP-1α after seizure onset.This process may play a key role in the progressive secondary brain pathology observed in this model though further study is warranted.

View Article: PubMed Central - HTML - PubMed

Affiliation: Research Division, Pharmacology Branch, US Army Medical Research Institute of Chemical Defense, Aberdeen Proving Ground, MD 21010, USA. erik.a.johnson1@us.army.mil

ABSTRACT

Background: Exposure to the nerve agent soman (GD) causes neuronal cell death and impaired behavioral function dependent on the induction of status epilepticus (SE). Little is known about the maturation of this pathological process, though neuroinflammation and infiltration of neutrophils are prominent features. The purpose of this study is to quantify the regional and temporal progression of early chemotactic signals, describe the cellular expression of these factors and the relationship between expression and neutrophil infiltration in damaged brain using a rat GD seizure model.

Methods: Protein levels of 4 chemokines responsible for neutrophil infiltration and activation were quantified up to 72 hours in multiple brain regions (i.e. piriform cortex, hippocampus and thalamus) following SE onset using multiplex bead immunoassays. Chemokines with significantly increased protein levels were localized to resident brain cells (i.e. neurons, astrocytes, microglia and endothelial cells). Lastly, neutrophil infiltration into these brain regions was quantified and correlated to the expression of these chemokines.

Results: We observed significant concentration increases for CXCL1 and MIP-1α after seizure onset. CXCL1 expression originated from neurons and endothelial cells while MIP-1α was expressed by neurons and microglia. Lastly, the expression of these chemokines directly preceded and positively correlated with significant neutrophil infiltration in the brain. These data suggest that following GD-induced SE, a strong chemotactic response originating from various brain cells, recruits circulating neutrophils to the injured brain.

Conclusions: A strong induction of neutrophil attractant chemokines occurs following GD-induced SE resulting in neutrophil influx into injured brain tissues. This process may play a key role in the progressive secondary brain pathology observed in this model though further study is warranted.

Show MeSH
Related in: MedlinePlus