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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.

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2-MeSADP treatment significantly reduces RB-induced post-ischemic cytotoxic swelling and necrosis of cortical astrocytes.(A) Confocal image at the boundary of an RB-induced lesion. The white arrow indicates a gradient of astrocyte swelling nearest the infarct 24 hours post-clot in a GFAP-GFP mouse. The insert shows a lower magnification mosaic image of cortex near the infarct, initially presented in Fig. 3A. Dashed rectangle corresponds to higher magnification image. (B) Rapid astrocyte swelling near RB-induced photothrombosis in GFAP-GFP transgenic mouse. The same region of the cortex was periodically imaged just prior to (0 min) and after ischemia at 30, 80, 140 min. The insert highlights a single swollen astrocyte. (C) Lineplot of average size of astrocyte somas near clot as a function of time. The swelling is close to maximum by 3 hours of the initial clot. Means +/− SD (n = 38 cells) pooled from 5 control RB only mice and 5 2-MeSADP treated mice. ** p<0.01 *** p<0.001 (D) High magnification images of cortical region near a blood vessel just prior to (0 hrs) and after photothombosis (3 and 24 hrs). Oncotic cell death (the absence of green fluorescence) is apparent at 24 hours post-thrombosis, although some astrocytes further from the lesion appear to have reversed swelling. (E) Comparable high magnification images near clotted vessel treated with 2-MeSADP. Note that astrocyte swelling proximal to the clotted vessel is absent 3 hours post-thrombosis and that many of these cells have not lysed even at 24 hours.
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pone-0014401-g004: 2-MeSADP treatment significantly reduces RB-induced post-ischemic cytotoxic swelling and necrosis of cortical astrocytes.(A) Confocal image at the boundary of an RB-induced lesion. The white arrow indicates a gradient of astrocyte swelling nearest the infarct 24 hours post-clot in a GFAP-GFP mouse. The insert shows a lower magnification mosaic image of cortex near the infarct, initially presented in Fig. 3A. Dashed rectangle corresponds to higher magnification image. (B) Rapid astrocyte swelling near RB-induced photothrombosis in GFAP-GFP transgenic mouse. The same region of the cortex was periodically imaged just prior to (0 min) and after ischemia at 30, 80, 140 min. The insert highlights a single swollen astrocyte. (C) Lineplot of average size of astrocyte somas near clot as a function of time. The swelling is close to maximum by 3 hours of the initial clot. Means +/− SD (n = 38 cells) pooled from 5 control RB only mice and 5 2-MeSADP treated mice. ** p<0.01 *** p<0.001 (D) High magnification images of cortical region near a blood vessel just prior to (0 hrs) and after photothombosis (3 and 24 hrs). Oncotic cell death (the absence of green fluorescence) is apparent at 24 hours post-thrombosis, although some astrocytes further from the lesion appear to have reversed swelling. (E) Comparable high magnification images near clotted vessel treated with 2-MeSADP. Note that astrocyte swelling proximal to the clotted vessel is absent 3 hours post-thrombosis and that many of these cells have not lysed even at 24 hours.

Mentions: Astrocyte swelling and necrosis are considered major causes of tissue damage after focal brain ischemia [16]. Consistent with this etiology, we observed severely swollen astrocytes near the edge of RB-induced infarcts with a gradient of cell swelling towards thrombosis (Fig. 4A). Temporal imaging of perivascular astrocytes immediately after a single RB-induced photothrombosis revealed rapid swelling with the average size of the cell soma increasing over 6-fold within 3 hours, from 63±15 µm2 at rest to 382±61 µm2 (mean ± SD, n = 38 cells pooled from 5 mice, Fig. 4B, C). Swelling gradually spread outward from the ischemic core to adjacent tissue (Fig. 4A). By 24 hours, many of the swollen astrocytes had lysed (necrosis), although some of the cells distal to the clot exhibited reversible swelling (Fig. 4D, 6F). In contrast, astrocyte swelling in the presence of 2MeSADP post-photothrombosis was significantly reduced. At 3 hours, the average soma size was only 99±22 µm2, and at 24 hours, many of the astrocytes near the ischemic core had still not lysed (Fig. 4C, E). Neither laser illumination nor the RB dye by itself affected astrocyte swelling (data not shown). These data suggest that a key mechanism of action underlying 2MeSADP-mediated protection is its ability to reduce astrocyte swelling and necrosis.


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 significantly reduces RB-induced post-ischemic cytotoxic swelling and necrosis of cortical astrocytes.(A) Confocal image at the boundary of an RB-induced lesion. The white arrow indicates a gradient of astrocyte swelling nearest the infarct 24 hours post-clot in a GFAP-GFP mouse. The insert shows a lower magnification mosaic image of cortex near the infarct, initially presented in Fig. 3A. Dashed rectangle corresponds to higher magnification image. (B) Rapid astrocyte swelling near RB-induced photothrombosis in GFAP-GFP transgenic mouse. The same region of the cortex was periodically imaged just prior to (0 min) and after ischemia at 30, 80, 140 min. The insert highlights a single swollen astrocyte. (C) Lineplot of average size of astrocyte somas near clot as a function of time. The swelling is close to maximum by 3 hours of the initial clot. Means +/− SD (n = 38 cells) pooled from 5 control RB only mice and 5 2-MeSADP treated mice. ** p<0.01 *** p<0.001 (D) High magnification images of cortical region near a blood vessel just prior to (0 hrs) and after photothombosis (3 and 24 hrs). Oncotic cell death (the absence of green fluorescence) is apparent at 24 hours post-thrombosis, although some astrocytes further from the lesion appear to have reversed swelling. (E) Comparable high magnification images near clotted vessel treated with 2-MeSADP. Note that astrocyte swelling proximal to the clotted vessel is absent 3 hours post-thrombosis and that many of these cells have not lysed even at 24 hours.
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pone-0014401-g004: 2-MeSADP treatment significantly reduces RB-induced post-ischemic cytotoxic swelling and necrosis of cortical astrocytes.(A) Confocal image at the boundary of an RB-induced lesion. The white arrow indicates a gradient of astrocyte swelling nearest the infarct 24 hours post-clot in a GFAP-GFP mouse. The insert shows a lower magnification mosaic image of cortex near the infarct, initially presented in Fig. 3A. Dashed rectangle corresponds to higher magnification image. (B) Rapid astrocyte swelling near RB-induced photothrombosis in GFAP-GFP transgenic mouse. The same region of the cortex was periodically imaged just prior to (0 min) and after ischemia at 30, 80, 140 min. The insert highlights a single swollen astrocyte. (C) Lineplot of average size of astrocyte somas near clot as a function of time. The swelling is close to maximum by 3 hours of the initial clot. Means +/− SD (n = 38 cells) pooled from 5 control RB only mice and 5 2-MeSADP treated mice. ** p<0.01 *** p<0.001 (D) High magnification images of cortical region near a blood vessel just prior to (0 hrs) and after photothombosis (3 and 24 hrs). Oncotic cell death (the absence of green fluorescence) is apparent at 24 hours post-thrombosis, although some astrocytes further from the lesion appear to have reversed swelling. (E) Comparable high magnification images near clotted vessel treated with 2-MeSADP. Note that astrocyte swelling proximal to the clotted vessel is absent 3 hours post-thrombosis and that many of these cells have not lysed even at 24 hours.
Mentions: Astrocyte swelling and necrosis are considered major causes of tissue damage after focal brain ischemia [16]. Consistent with this etiology, we observed severely swollen astrocytes near the edge of RB-induced infarcts with a gradient of cell swelling towards thrombosis (Fig. 4A). Temporal imaging of perivascular astrocytes immediately after a single RB-induced photothrombosis revealed rapid swelling with the average size of the cell soma increasing over 6-fold within 3 hours, from 63±15 µm2 at rest to 382±61 µm2 (mean ± SD, n = 38 cells pooled from 5 mice, Fig. 4B, C). Swelling gradually spread outward from the ischemic core to adjacent tissue (Fig. 4A). By 24 hours, many of the swollen astrocytes had lysed (necrosis), although some of the cells distal to the clot exhibited reversible swelling (Fig. 4D, 6F). In contrast, astrocyte swelling in the presence of 2MeSADP post-photothrombosis was significantly reduced. At 3 hours, the average soma size was only 99±22 µm2, and at 24 hours, many of the astrocytes near the ischemic core had still not lysed (Fig. 4C, E). Neither laser illumination nor the RB dye by itself affected astrocyte swelling (data not shown). These data suggest that a key mechanism of action underlying 2MeSADP-mediated protection is its ability to reduce astrocyte swelling and necrosis.

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