Limits...
Purinergic receptor stimulation reduces cytotoxic edema and brain infarcts in mouse induced by photothrombosis by energizing glial mitochondria.

Zheng W, Watts LT, Holstein DM, Prajapati SI, Keller C, Grass EH, Walter CA, Lechleiter JD - PLoS ONE (2010)

Bottom Line: Delayed 2MeSADP treatment, 24 hours after the initial thrombosis, also significantly reduced cytotoxic edema and the continued growth of the brain infarction.Pharmacological and genetic evidence are presented indicating that 2MeSADP protection is mediated by enhanced astrocyte mitochondrial metabolism via increased inositol trisphosphate (IP(3))-dependent Ca(2+) release.Enhancement of this energy source could have similar protective benefits for a wide range of brain injuries.

View Article: PubMed Central - PubMed

Affiliation: Department of Cellular and Structural Biology, University of Texas Health Science Center at San Antonio, San Antonio, Texas, United States of America.

ABSTRACT
Treatments to improve the neurological outcome of edema and cerebral ischemic stroke are severely limited. Here, we present the first in vivo single cell images of cortical mouse astrocytes documenting the impact of single vessel photothrombosis on cytotoxic edema and cerebral infarcts. The volume of astrocytes expressing green fluorescent protein (GFP) increased by over 600% within 3 hours of ischemia. The subsequent growth of cerebral infarcts was easily followed as the loss of GFP fluorescence as astrocytes lysed. Cytotoxic edema and the magnitude of ischemic lesions were significantly reduced by treatment with the purinergic ligand 2-methylthioladenosine 5' diphosphate (2-MeSADP), an agonist with high specificity for the purinergic receptor type 1 isoform (P2Y(1)R). At 24 hours, cytotoxic edema in astrocytes was still apparent at the penumbra and preceded the cell lysis that defined the infarct. Delayed 2MeSADP treatment, 24 hours after the initial thrombosis, also significantly reduced cytotoxic edema and the continued growth of the brain infarction. Pharmacological and genetic evidence are presented indicating that 2MeSADP protection is mediated by enhanced astrocyte mitochondrial metabolism via increased inositol trisphosphate (IP(3))-dependent Ca(2+) release. We suggest that mitochondria play a critical role in astrocyte energy metabolism in the penumbra of ischemic lesions, where low ATP levels are widely accepted to be responsible for cytotoxic edema. Enhancement of this energy source could have similar protective benefits for a wide range of brain injuries.

Show MeSH

Related in: MedlinePlus

2-MeSADP treatment decreases in vivo lesions induced by single vessel photothrombosis.(A) Mosaic reconstruction of approximately 30 imaging fields of the parietal cortex of live transgenic mice expressing GFP in astrocytes (GFAP-GFP). The same cortical region was imaged at day 0 (pre-photothrombosis) and at 24, 48 hours post-photothrombosis. RB was tail vein injected and irradiated (543 nm) at the single blood vessel indicated by the open white arrows, triggering photothrombosis at 0 hours (labeled as clot in 48 hours). Astrocyte cell lysis at 24 and 48 hours is apparent by the absence of GFP fluorescence. White dashed rectangle indicates region that is presented at higher magnification in Fig. S4A. (B) Experimental mouse that was co-injected with RB and the P2Y1-R ligand, 2-MeSADP. Note the dramatic reduction in the stroke lesion at 24 and 48 hours compared to panels in (A). (C) Mosaic image of day 2 panel in (A) overlaid with mosaic image of rhodamine-dextran filled vasculature. (D) Line plots of the average lesion size of 5 control mice (RB only) and 5 experimental mice (RB+ 2-MeSADP). **p<0.01.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC3008710&req=5

pone-0014401-g002: 2-MeSADP treatment decreases in vivo lesions induced by single vessel photothrombosis.(A) Mosaic reconstruction of approximately 30 imaging fields of the parietal cortex of live transgenic mice expressing GFP in astrocytes (GFAP-GFP). The same cortical region was imaged at day 0 (pre-photothrombosis) and at 24, 48 hours post-photothrombosis. RB was tail vein injected and irradiated (543 nm) at the single blood vessel indicated by the open white arrows, triggering photothrombosis at 0 hours (labeled as clot in 48 hours). Astrocyte cell lysis at 24 and 48 hours is apparent by the absence of GFP fluorescence. White dashed rectangle indicates region that is presented at higher magnification in Fig. S4A. (B) Experimental mouse that was co-injected with RB and the P2Y1-R ligand, 2-MeSADP. Note the dramatic reduction in the stroke lesion at 24 and 48 hours compared to panels in (A). (C) Mosaic image of day 2 panel in (A) overlaid with mosaic image of rhodamine-dextran filled vasculature. (D) Line plots of the average lesion size of 5 control mice (RB only) and 5 experimental mice (RB+ 2-MeSADP). **p<0.01.

Mentions: To further investigate the impact of P2Y1R stimulation on cerebral ischemic lesions, we performed a series of in vivo imaging experiments utilizing transgenic mice expressing green fluorescent protein (GFP) in astrocytes (FVB/N-Tg(GFAPGFP)14Mes/J, JAX® Mice). The cell bodies of individual astrocytes were easily imaged with a confocal microscope through a thinned skull window in the parietal cortex of these mice (Fig. S1 and S2). When a single bolus of RB was tail-vein injected, a luminal increase in blood vessel fluorescence was transiently observed in the imaging window. Single vessel photothrombosis could be induced by irradiating a zoomed region of an RB-filled arteriole with green light (543 nm) for ∼5 minutes (Fig. S1D). Large mosaic images of the cortex were then seamlessly reconstructed from ∼30 individual z-stacks to follow the effect of a single vessel clot. Twenty-four hours after photothrombosis, an infarction border could easily be identified by the absence (lysis) of GFP labeled astrocytes near the clotted single vessel. The clot itself was also easy to identify by the red fluorescence of trapped RB dye (Fig. 2A). We found that the average size of an RB-induced lesion at 24 hours was 1.02±0.07 µm2×105 (n = 5). This area increased to 2.14±0.22 µm 2×105 (n = 5) by 48 hours. Pharmacological agents are known to leak into local brain tissue following blood clotting due to the disruption of the blood brain barrier (BBB). An increase in BBB permeability was observed in these preparations as leakage of RB dye into the extravascular space (Fig. S2). Extravasation of re-injected RB dye from a clotted arteriole was also obvious two days after the initial photothrombosis (Fig. S2). We took advantage of the BBB permeability breakdown to introduce P2Y1R agonist into the ischemic region. We determined that a tail-vein injection of 2MeSADP (100 µM) along with RB dye at the initial photothrombosis significantly decreased the stroke lesion size at 24 hours to 0.30±0.02 µm2×105 (n = 5) and to 0.59±0.07 µm2×105 (n = 5) at 48 hours (Fig. 2D). Moreover, tail-vein injections of 2MeSADP could again be delayed 24 hours after the initial photothrombosis and still significantly slow the progression of the lesion. Specifically, RB-induced lesions in both control and test mice were permitted to expand to 0.95±0.17×105 µm2 (n = 3) and 0.89±0.12 µm2×105 (n = 3) for 24 hours, respectively (Fig. 3). The average lesion size of mice subsequently injected with 2MeSADP was 0.88±0.15×105 µm2 (n = 3) at 48 hours compared to 1.97±0.15×105 µm2 (n = 3) for non-treated mice.


Purinergic receptor stimulation reduces cytotoxic edema and brain infarcts in mouse induced by photothrombosis by energizing glial mitochondria.

Zheng W, Watts LT, Holstein DM, Prajapati SI, Keller C, Grass EH, Walter CA, Lechleiter JD - PLoS ONE (2010)

2-MeSADP treatment decreases in vivo lesions induced by single vessel photothrombosis.(A) Mosaic reconstruction of approximately 30 imaging fields of the parietal cortex of live transgenic mice expressing GFP in astrocytes (GFAP-GFP). The same cortical region was imaged at day 0 (pre-photothrombosis) and at 24, 48 hours post-photothrombosis. RB was tail vein injected and irradiated (543 nm) at the single blood vessel indicated by the open white arrows, triggering photothrombosis at 0 hours (labeled as clot in 48 hours). Astrocyte cell lysis at 24 and 48 hours is apparent by the absence of GFP fluorescence. White dashed rectangle indicates region that is presented at higher magnification in Fig. S4A. (B) Experimental mouse that was co-injected with RB and the P2Y1-R ligand, 2-MeSADP. Note the dramatic reduction in the stroke lesion at 24 and 48 hours compared to panels in (A). (C) Mosaic image of day 2 panel in (A) overlaid with mosaic image of rhodamine-dextran filled vasculature. (D) Line plots of the average lesion size of 5 control mice (RB only) and 5 experimental mice (RB+ 2-MeSADP). **p<0.01.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3008710&req=5

pone-0014401-g002: 2-MeSADP treatment decreases in vivo lesions induced by single vessel photothrombosis.(A) Mosaic reconstruction of approximately 30 imaging fields of the parietal cortex of live transgenic mice expressing GFP in astrocytes (GFAP-GFP). The same cortical region was imaged at day 0 (pre-photothrombosis) and at 24, 48 hours post-photothrombosis. RB was tail vein injected and irradiated (543 nm) at the single blood vessel indicated by the open white arrows, triggering photothrombosis at 0 hours (labeled as clot in 48 hours). Astrocyte cell lysis at 24 and 48 hours is apparent by the absence of GFP fluorescence. White dashed rectangle indicates region that is presented at higher magnification in Fig. S4A. (B) Experimental mouse that was co-injected with RB and the P2Y1-R ligand, 2-MeSADP. Note the dramatic reduction in the stroke lesion at 24 and 48 hours compared to panels in (A). (C) Mosaic image of day 2 panel in (A) overlaid with mosaic image of rhodamine-dextran filled vasculature. (D) Line plots of the average lesion size of 5 control mice (RB only) and 5 experimental mice (RB+ 2-MeSADP). **p<0.01.
Mentions: To further investigate the impact of P2Y1R stimulation on cerebral ischemic lesions, we performed a series of in vivo imaging experiments utilizing transgenic mice expressing green fluorescent protein (GFP) in astrocytes (FVB/N-Tg(GFAPGFP)14Mes/J, JAX® Mice). The cell bodies of individual astrocytes were easily imaged with a confocal microscope through a thinned skull window in the parietal cortex of these mice (Fig. S1 and S2). When a single bolus of RB was tail-vein injected, a luminal increase in blood vessel fluorescence was transiently observed in the imaging window. Single vessel photothrombosis could be induced by irradiating a zoomed region of an RB-filled arteriole with green light (543 nm) for ∼5 minutes (Fig. S1D). Large mosaic images of the cortex were then seamlessly reconstructed from ∼30 individual z-stacks to follow the effect of a single vessel clot. Twenty-four hours after photothrombosis, an infarction border could easily be identified by the absence (lysis) of GFP labeled astrocytes near the clotted single vessel. The clot itself was also easy to identify by the red fluorescence of trapped RB dye (Fig. 2A). We found that the average size of an RB-induced lesion at 24 hours was 1.02±0.07 µm2×105 (n = 5). This area increased to 2.14±0.22 µm 2×105 (n = 5) by 48 hours. Pharmacological agents are known to leak into local brain tissue following blood clotting due to the disruption of the blood brain barrier (BBB). An increase in BBB permeability was observed in these preparations as leakage of RB dye into the extravascular space (Fig. S2). Extravasation of re-injected RB dye from a clotted arteriole was also obvious two days after the initial photothrombosis (Fig. S2). We took advantage of the BBB permeability breakdown to introduce P2Y1R agonist into the ischemic region. We determined that a tail-vein injection of 2MeSADP (100 µM) along with RB dye at the initial photothrombosis significantly decreased the stroke lesion size at 24 hours to 0.30±0.02 µm2×105 (n = 5) and to 0.59±0.07 µm2×105 (n = 5) at 48 hours (Fig. 2D). Moreover, tail-vein injections of 2MeSADP could again be delayed 24 hours after the initial photothrombosis and still significantly slow the progression of the lesion. Specifically, RB-induced lesions in both control and test mice were permitted to expand to 0.95±0.17×105 µm2 (n = 3) and 0.89±0.12 µm2×105 (n = 3) for 24 hours, respectively (Fig. 3). The average lesion size of mice subsequently injected with 2MeSADP was 0.88±0.15×105 µm2 (n = 3) at 48 hours compared to 1.97±0.15×105 µm2 (n = 3) for non-treated mice.

Bottom Line: Delayed 2MeSADP treatment, 24 hours after the initial thrombosis, also significantly reduced cytotoxic edema and the continued growth of the brain infarction.Pharmacological and genetic evidence are presented indicating that 2MeSADP protection is mediated by enhanced astrocyte mitochondrial metabolism via increased inositol trisphosphate (IP(3))-dependent Ca(2+) release.Enhancement of this energy source could have similar protective benefits for a wide range of brain injuries.

View Article: PubMed Central - PubMed

Affiliation: Department of Cellular and Structural Biology, University of Texas Health Science Center at San Antonio, San Antonio, Texas, United States of America.

ABSTRACT
Treatments to improve the neurological outcome of edema and cerebral ischemic stroke are severely limited. Here, we present the first in vivo single cell images of cortical mouse astrocytes documenting the impact of single vessel photothrombosis on cytotoxic edema and cerebral infarcts. The volume of astrocytes expressing green fluorescent protein (GFP) increased by over 600% within 3 hours of ischemia. The subsequent growth of cerebral infarcts was easily followed as the loss of GFP fluorescence as astrocytes lysed. Cytotoxic edema and the magnitude of ischemic lesions were significantly reduced by treatment with the purinergic ligand 2-methylthioladenosine 5' diphosphate (2-MeSADP), an agonist with high specificity for the purinergic receptor type 1 isoform (P2Y(1)R). At 24 hours, cytotoxic edema in astrocytes was still apparent at the penumbra and preceded the cell lysis that defined the infarct. Delayed 2MeSADP treatment, 24 hours after the initial thrombosis, also significantly reduced cytotoxic edema and the continued growth of the brain infarction. Pharmacological and genetic evidence are presented indicating that 2MeSADP protection is mediated by enhanced astrocyte mitochondrial metabolism via increased inositol trisphosphate (IP(3))-dependent Ca(2+) release. We suggest that mitochondria play a critical role in astrocyte energy metabolism in the penumbra of ischemic lesions, where low ATP levels are widely accepted to be responsible for cytotoxic edema. Enhancement of this energy source could have similar protective benefits for a wide range of brain injuries.

Show MeSH
Related in: MedlinePlus