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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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CXCL1 is expressed in neurons and, to a much lesser extent, in endothelial cells after GD-induced SE. Prominent CXCL1 (A-I, green) immunolabeling is present in the piriform cortex, hippocampus and thalamus 12 hours after GD-induced SE (A, B & C; left). CXCL1 is 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 primarily confined to the CA3 pyramidal layer and granular layer of the dentate gyrus (GrDG) but not evident in the polymorphic layer of the dentate gyrus (PoDG). CXCL1 was located to the laterodorsal and lateral posterior nuclei of the thalamus (C). Labeling was absent in the secondary controls for both 12-hour GD-exposed (D) and vehicle control tissues (E), exemplified by the piriform cortex. Neurons (F, red) and CXCL1 were often found to co-localize (F, yellow). Co-localization was not observed in hypertrophic astrocytes (G, red) or activated microglia (H, red) and was limited in endothelial cells (I, yellow). DAPI (A-I, blue) was used to label the nuclei of each cell. Scale bar: 250 μm (A-E), 50 μm and 20 μm (F-I) for regular and confocal fluorescent microscopy respectively; n = 7 for 12-hour, n = 4 for vehicle controls.
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Figure 2: CXCL1 is expressed in neurons and, to a much lesser extent, in endothelial cells after GD-induced SE. Prominent CXCL1 (A-I, green) immunolabeling is present in the piriform cortex, hippocampus and thalamus 12 hours after GD-induced SE (A, B & C; left). CXCL1 is 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 primarily confined to the CA3 pyramidal layer and granular layer of the dentate gyrus (GrDG) but not evident in the polymorphic layer of the dentate gyrus (PoDG). CXCL1 was located to the laterodorsal and lateral posterior nuclei of the thalamus (C). Labeling was absent in the secondary controls for both 12-hour GD-exposed (D) and vehicle control tissues (E), exemplified by the piriform cortex. Neurons (F, red) and CXCL1 were often found to co-localize (F, yellow). Co-localization was not observed in hypertrophic astrocytes (G, red) or activated microglia (H, red) and was limited in endothelial cells (I, yellow). DAPI (A-I, blue) was used to label the nuclei of each cell. Scale bar: 250 μm (A-E), 50 μm and 20 μm (F-I) for regular and confocal fluorescent microscopy respectively; n = 7 for 12-hour, n = 4 for vehicle controls.

Mentions: Twelve hours following GD-induced SE, CXCL1 immunolabeling was present in the piriform cortex (Figure 2A, left), hippocampus (dentate gyrus shown; Figure 2B, left) and thalamus (Figure 2C, left), while CXCL1 labeling was absent in vehicle controls in the same regions (Figure 2A, B, &2C, right). Specific labeling was also absent in secondary only controls at the 12-hour time point (Figure 2D) and in vehicle controls (Figure 2E) as exemplified by the piriform cortex. In the piriform cortex, CXCL1-positive cells were found predominantly in layer II but also in layer III. In the hippocampus, CXCL1-positive cells were found primarily in the granular layer of the dentate gyrus (GrDG) and the CA3 pyramidal layer closest to the dentate gyrus. CXCL1-positive cells were also found in the laterodorsal and lateral posterior nuclei of the thalamus. To identify these cells, sections were co-labeled with antibodies specific for neurons, astrocytes, microglia and endothelial cells and for CXCL1. CXCL1 immunoreactivity was found in neuronal populations in the regions mentioned above (Figure 2F). CXCL1 diffusely labeled the cytoplasm in these cells with interspersed fine punctate labeling. No co-localization was observed between CXCL1 and astrocytes (Figure 2G) or microglia (Figure 2H) regardless of state of activation. Co-localization was limited in endothelial cells, though a prevalent, but not exclusive, association between CXCL1 and the vasculature was often observed (Figure 2I).


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)

CXCL1 is expressed in neurons and, to a much lesser extent, in endothelial cells after GD-induced SE. Prominent CXCL1 (A-I, green) immunolabeling is present in the piriform cortex, hippocampus and thalamus 12 hours after GD-induced SE (A, B & C; left). CXCL1 is 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 primarily confined to the CA3 pyramidal layer and granular layer of the dentate gyrus (GrDG) but not evident in the polymorphic layer of the dentate gyrus (PoDG). CXCL1 was located to the laterodorsal and lateral posterior nuclei of the thalamus (C). Labeling was absent in the secondary controls for both 12-hour GD-exposed (D) and vehicle control tissues (E), exemplified by the piriform cortex. Neurons (F, red) and CXCL1 were often found to co-localize (F, yellow). Co-localization was not observed in hypertrophic astrocytes (G, red) or activated microglia (H, red) and was limited in endothelial cells (I, yellow). DAPI (A-I, blue) was used to label the nuclei of each cell. Scale bar: 250 μm (A-E), 50 μm and 20 μm (F-I) for regular and confocal fluorescent microscopy respectively; n = 7 for 12-hour, n = 4 for vehicle controls.
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Figure 2: CXCL1 is expressed in neurons and, to a much lesser extent, in endothelial cells after GD-induced SE. Prominent CXCL1 (A-I, green) immunolabeling is present in the piriform cortex, hippocampus and thalamus 12 hours after GD-induced SE (A, B & C; left). CXCL1 is 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 primarily confined to the CA3 pyramidal layer and granular layer of the dentate gyrus (GrDG) but not evident in the polymorphic layer of the dentate gyrus (PoDG). CXCL1 was located to the laterodorsal and lateral posterior nuclei of the thalamus (C). Labeling was absent in the secondary controls for both 12-hour GD-exposed (D) and vehicle control tissues (E), exemplified by the piriform cortex. Neurons (F, red) and CXCL1 were often found to co-localize (F, yellow). Co-localization was not observed in hypertrophic astrocytes (G, red) or activated microglia (H, red) and was limited in endothelial cells (I, yellow). DAPI (A-I, blue) was used to label the nuclei of each cell. Scale bar: 250 μm (A-E), 50 μm and 20 μm (F-I) for regular and confocal fluorescent microscopy respectively; n = 7 for 12-hour, n = 4 for vehicle controls.
Mentions: Twelve hours following GD-induced SE, CXCL1 immunolabeling was present in the piriform cortex (Figure 2A, left), hippocampus (dentate gyrus shown; Figure 2B, left) and thalamus (Figure 2C, left), while CXCL1 labeling was absent in vehicle controls in the same regions (Figure 2A, B, &2C, right). Specific labeling was also absent in secondary only controls at the 12-hour time point (Figure 2D) and in vehicle controls (Figure 2E) as exemplified by the piriform cortex. In the piriform cortex, CXCL1-positive cells were found predominantly in layer II but also in layer III. In the hippocampus, CXCL1-positive cells were found primarily in the granular layer of the dentate gyrus (GrDG) and the CA3 pyramidal layer closest to the dentate gyrus. CXCL1-positive cells were also found in the laterodorsal and lateral posterior nuclei of the thalamus. To identify these cells, sections were co-labeled with antibodies specific for neurons, astrocytes, microglia and endothelial cells and for CXCL1. CXCL1 immunoreactivity was found in neuronal populations in the regions mentioned above (Figure 2F). CXCL1 diffusely labeled the cytoplasm in these cells with interspersed fine punctate labeling. No co-localization was observed between CXCL1 and astrocytes (Figure 2G) or microglia (Figure 2H) regardless of state of activation. Co-localization was limited in endothelial cells, though a prevalent, but not exclusive, association between CXCL1 and the vasculature was often observed (Figure 2I).

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