Limits...
Monocyte chemoattractant protein-1-deficiency impairs the expression of IL-6, IL-1β and G-CSF after transient focal ischemia in mice.

Strecker JK, Minnerup J, Gess B, Ringelstein EB, Schäbitz WR, Schilling M - PLoS ONE (2011)

Bottom Line: Here we report that MCP-1-deficiency leads to a shift towards a less inflammatory state following experimental occlusion of the middle cerebral artery including an impaired induction of interleukin-6, interleukin-1β and granulocyte-colony stimulating factor expression as well as a subsequent diminished influx of hematogenous cells.Investigations revealed no differences in transcription of tumor necrosis factor-α and astrogliosis 12 and 36 hours after onset of ischemia.These novel results help to understand post ischemic, inflammatory mechanisms and might give further arguments towards therapeutical interventions by modulation of MCP-1 expression in post stroke inflammation.

View Article: PubMed Central - PubMed

Affiliation: Department of Neurology, University of Münster, Münster, Germany. strecker.jan@gmx.de

ABSTRACT
Monocyte chemoattractant protein-1 (MCP-1), a chemokine secreted by neurons and astrocytes following stroke is known to aggravate ischemia-related damage. Previous studies revealed that MCP-1-deficient mice develop smaller infarcts and have an improved neurological outcome, whereas mice overexpressing MCP-1 show worsened brain damage and impaired neurological function. The aim of the present study was to elucidate the molecular background of the enhanced recovery in MCP-1-deficient mice after stroke. For this purpose, we (1) performed expression analyses on crucial post-stroke related inflammatory genes in MCP-1-deficient mice compared to wildtype controls, (2) analyzed a possible impact of MCP-1 on astrocyte activation (3) investigated the cellular origin of respective inflammatory cytokines and (4) analyzed the impact of MCP-1 secretion on the migration of both neutrophil granulocytes and T-cells. Here we report that MCP-1-deficiency leads to a shift towards a less inflammatory state following experimental occlusion of the middle cerebral artery including an impaired induction of interleukin-6, interleukin-1β and granulocyte-colony stimulating factor expression as well as a subsequent diminished influx of hematogenous cells. Additionally, MCP-1-deficient mice developed smaller infarcts 36 hours after experimental stroke. Investigations revealed no differences in transcription of tumor necrosis factor-α and astrogliosis 12 and 36 hours after onset of ischemia. These novel results help to understand post ischemic, inflammatory mechanisms and might give further arguments towards therapeutical interventions by modulation of MCP-1 expression in post stroke inflammation.

Show MeSH

Related in: MedlinePlus

Laser capture microdissection was performed on PEN-membrane mounted and toluidine-stained brain sections.A: Three areas, namely cerebral cortex (cor), infarct core (inf) and contralateral striatum (cnl) were cut out of specimen and total RNA for subsequent analyses was extracted. B: Total RNA integrity of each sample was measured with an Agilent 2100 Bioanalyzer (exemplary data shown). Distinct peaks for 18S and 28S rRNA indicate intact RNA. Only total RNA with a RNA integrity number greater than or equal to 6 was used for subsequent reverse transcription and qRT-PCR. C: Real-Time PCR data showed no significant differences of IL-6, IL-1β, TNF-α and G-CSF expression between wildtype and MCP-1-deficient control animals. D: Infarct size assessed 12 and 36 hours after MCAO. Lesion volume is expressed as percentage of the ipsilateral hemisphere. Values represent mean ± SD, *p<0.05.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC3198727&req=5

pone-0025863-g001: Laser capture microdissection was performed on PEN-membrane mounted and toluidine-stained brain sections.A: Three areas, namely cerebral cortex (cor), infarct core (inf) and contralateral striatum (cnl) were cut out of specimen and total RNA for subsequent analyses was extracted. B: Total RNA integrity of each sample was measured with an Agilent 2100 Bioanalyzer (exemplary data shown). Distinct peaks for 18S and 28S rRNA indicate intact RNA. Only total RNA with a RNA integrity number greater than or equal to 6 was used for subsequent reverse transcription and qRT-PCR. C: Real-Time PCR data showed no significant differences of IL-6, IL-1β, TNF-α and G-CSF expression between wildtype and MCP-1-deficient control animals. D: Infarct size assessed 12 and 36 hours after MCAO. Lesion volume is expressed as percentage of the ipsilateral hemisphere. Values represent mean ± SD, *p<0.05.

Mentions: Once air-dried, slides were stored at −80°C until further use. Three different areas of interest were isolated using a Zeiss/PALM LMD microscope (Carl Zeiss MicroImaging GmbH, Germany): the ipsilateral cortex (covering the primary motor- and primary somatosensory-cortex), the ipsilateral ischemic area and the contralateral caudate putamen (Fig.1 A). Regions of interest were isolated and collected in microfuge caps containing 45 µl of RNA extraction buffer RLT (Qiagen, Hilden, Germany). To avoid RNA-degradation, lysis buffer was supplemented with 2-mercaptoethanol (143 mM, Sigma-Aldrich, Munich, Germany). Three areas of 20 brain slices from each animal were cut and collected in separate caps for subsequent analysis of gene expression.


Monocyte chemoattractant protein-1-deficiency impairs the expression of IL-6, IL-1β and G-CSF after transient focal ischemia in mice.

Strecker JK, Minnerup J, Gess B, Ringelstein EB, Schäbitz WR, Schilling M - PLoS ONE (2011)

Laser capture microdissection was performed on PEN-membrane mounted and toluidine-stained brain sections.A: Three areas, namely cerebral cortex (cor), infarct core (inf) and contralateral striatum (cnl) were cut out of specimen and total RNA for subsequent analyses was extracted. B: Total RNA integrity of each sample was measured with an Agilent 2100 Bioanalyzer (exemplary data shown). Distinct peaks for 18S and 28S rRNA indicate intact RNA. Only total RNA with a RNA integrity number greater than or equal to 6 was used for subsequent reverse transcription and qRT-PCR. C: Real-Time PCR data showed no significant differences of IL-6, IL-1β, TNF-α and G-CSF expression between wildtype and MCP-1-deficient control animals. D: Infarct size assessed 12 and 36 hours after MCAO. Lesion volume is expressed as percentage of the ipsilateral hemisphere. Values represent mean ± SD, *p<0.05.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3198727&req=5

pone-0025863-g001: Laser capture microdissection was performed on PEN-membrane mounted and toluidine-stained brain sections.A: Three areas, namely cerebral cortex (cor), infarct core (inf) and contralateral striatum (cnl) were cut out of specimen and total RNA for subsequent analyses was extracted. B: Total RNA integrity of each sample was measured with an Agilent 2100 Bioanalyzer (exemplary data shown). Distinct peaks for 18S and 28S rRNA indicate intact RNA. Only total RNA with a RNA integrity number greater than or equal to 6 was used for subsequent reverse transcription and qRT-PCR. C: Real-Time PCR data showed no significant differences of IL-6, IL-1β, TNF-α and G-CSF expression between wildtype and MCP-1-deficient control animals. D: Infarct size assessed 12 and 36 hours after MCAO. Lesion volume is expressed as percentage of the ipsilateral hemisphere. Values represent mean ± SD, *p<0.05.
Mentions: Once air-dried, slides were stored at −80°C until further use. Three different areas of interest were isolated using a Zeiss/PALM LMD microscope (Carl Zeiss MicroImaging GmbH, Germany): the ipsilateral cortex (covering the primary motor- and primary somatosensory-cortex), the ipsilateral ischemic area and the contralateral caudate putamen (Fig.1 A). Regions of interest were isolated and collected in microfuge caps containing 45 µl of RNA extraction buffer RLT (Qiagen, Hilden, Germany). To avoid RNA-degradation, lysis buffer was supplemented with 2-mercaptoethanol (143 mM, Sigma-Aldrich, Munich, Germany). Three areas of 20 brain slices from each animal were cut and collected in separate caps for subsequent analysis of gene expression.

Bottom Line: Here we report that MCP-1-deficiency leads to a shift towards a less inflammatory state following experimental occlusion of the middle cerebral artery including an impaired induction of interleukin-6, interleukin-1β and granulocyte-colony stimulating factor expression as well as a subsequent diminished influx of hematogenous cells.Investigations revealed no differences in transcription of tumor necrosis factor-α and astrogliosis 12 and 36 hours after onset of ischemia.These novel results help to understand post ischemic, inflammatory mechanisms and might give further arguments towards therapeutical interventions by modulation of MCP-1 expression in post stroke inflammation.

View Article: PubMed Central - PubMed

Affiliation: Department of Neurology, University of Münster, Münster, Germany. strecker.jan@gmx.de

ABSTRACT
Monocyte chemoattractant protein-1 (MCP-1), a chemokine secreted by neurons and astrocytes following stroke is known to aggravate ischemia-related damage. Previous studies revealed that MCP-1-deficient mice develop smaller infarcts and have an improved neurological outcome, whereas mice overexpressing MCP-1 show worsened brain damage and impaired neurological function. The aim of the present study was to elucidate the molecular background of the enhanced recovery in MCP-1-deficient mice after stroke. For this purpose, we (1) performed expression analyses on crucial post-stroke related inflammatory genes in MCP-1-deficient mice compared to wildtype controls, (2) analyzed a possible impact of MCP-1 on astrocyte activation (3) investigated the cellular origin of respective inflammatory cytokines and (4) analyzed the impact of MCP-1 secretion on the migration of both neutrophil granulocytes and T-cells. Here we report that MCP-1-deficiency leads to a shift towards a less inflammatory state following experimental occlusion of the middle cerebral artery including an impaired induction of interleukin-6, interleukin-1β and granulocyte-colony stimulating factor expression as well as a subsequent diminished influx of hematogenous cells. Additionally, MCP-1-deficient mice developed smaller infarcts 36 hours after experimental stroke. Investigations revealed no differences in transcription of tumor necrosis factor-α and astrogliosis 12 and 36 hours after onset of ischemia. These novel results help to understand post ischemic, inflammatory mechanisms and might give further arguments towards therapeutical interventions by modulation of MCP-1 expression in post stroke inflammation.

Show MeSH
Related in: MedlinePlus