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Dehydroascorbic Acid Attenuates Ischemic Brain Edema and Neurotoxicity in Cerebral Ischemia: An in vivo Study.

Song J, Park J, Kim JH, Choi JY, Kim JY, Lee KM, Lee JE - Exp Neurobiol (2015)

Bottom Line: Dehydroascorbic acid (DHA) as the oxidized form of ascorbic acid (AA) acts as a cellular protector against oxidative stress and easily enters into the brain compared to AA.We also examined the expression of claudin 5 for confirming the BBB breakdown, and the expression of bcl 2 associated X protein (Bax), caspase-3, inducible nitric oxide synthase (iNOS) for checking the effect of DHA on the neurotoxicity.Based on our findings, we propose that DHA might alleviate the pathogenesis of ischemic brain injury by attenuating edema, neuronal loss, and by improving synaptic connection.

View Article: PubMed Central - PubMed

Affiliation: Department of Anatomy, Yonsei University College of Medicine, Seoul 120-752, Korea.

ABSTRACT
Ischemic stroke results in the diverse phathophysiologies including blood brain barrier (BBB) disruption, brain edema, neuronal cell death, and synaptic loss in brain. Vitamin C has known as the potent anti-oxidant having multiple functions in various organs, as well as in brain. Dehydroascorbic acid (DHA) as the oxidized form of ascorbic acid (AA) acts as a cellular protector against oxidative stress and easily enters into the brain compared to AA. To determine the role of DHA on edema formation, neuronal cell death, and synaptic dysfunction following cerebral ischemia, we investigated the infarct size of ischemic brain tissue and measured the expression of aquaporin 1 (AQP-1) as the water channel protein. We also examined the expression of claudin 5 for confirming the BBB breakdown, and the expression of bcl 2 associated X protein (Bax), caspase-3, inducible nitric oxide synthase (iNOS) for checking the effect of DHA on the neurotoxicity. Finally, we examined postsynaptic density protein-95 (PSD-95) expression to confirm the effect of DHA on synaptic dysfunction following ischemic stroke. Based on our findings, we propose that DHA might alleviate the pathogenesis of ischemic brain injury by attenuating edema, neuronal loss, and by improving synaptic connection.

No MeSH data available.


Related in: MedlinePlus

Immunochemical image for confirmation of reduced cleaved caspase-3 expression by DHA treatment. (A) Immunochemical images showed that cleaved caspase-3 positive cells (red) were densely expressed in the EC group. In DHA treatment group, cleaved caspase-3 expression was decreased in rat cortex, compared with the EC group. (B) In DHA treatment group, cleaved caspase-3 positive cells were decreased in rat striatum due to DHA treatment. (C) The graph showed the percentage (%) of cleaved caspase-3 positive cells to compare the difference of cleaved caspase-3 fluorescence intensity. Statistical significance with EC group was determined by t-test. (D) The graph of cleaved caspase-3 protein level showed the same pattern with immunochemical images. Differences were considered significant at *p<0.05, (ANOVA followed by Bonferroni post hoc multiple comparison test). Scale bar=100 µm, Cleaved caspase-3: red, 4', 6-diamidino-2-phenylindole (DAPI): blue. NC: normal control group, EC: experimental control; reperfusion 24 hr after MCAO injury, DHA: DHA treatment and reperfusion 24 hr after MCAO injury.
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Figure 5: Immunochemical image for confirmation of reduced cleaved caspase-3 expression by DHA treatment. (A) Immunochemical images showed that cleaved caspase-3 positive cells (red) were densely expressed in the EC group. In DHA treatment group, cleaved caspase-3 expression was decreased in rat cortex, compared with the EC group. (B) In DHA treatment group, cleaved caspase-3 positive cells were decreased in rat striatum due to DHA treatment. (C) The graph showed the percentage (%) of cleaved caspase-3 positive cells to compare the difference of cleaved caspase-3 fluorescence intensity. Statistical significance with EC group was determined by t-test. (D) The graph of cleaved caspase-3 protein level showed the same pattern with immunochemical images. Differences were considered significant at *p<0.05, (ANOVA followed by Bonferroni post hoc multiple comparison test). Scale bar=100 µm, Cleaved caspase-3: red, 4', 6-diamidino-2-phenylindole (DAPI): blue. NC: normal control group, EC: experimental control; reperfusion 24 hr after MCAO injury, DHA: DHA treatment and reperfusion 24 hr after MCAO injury.

Mentions: We performed immunohistochemical staining using cleaved caspase-3 antibody at reperfusion 24 hr after MCAO injury to examine whether DHA influences on the cell death both in cortex (Fig. 5A) and in striatum (Fig. 5B). Cleaved caspase-3 immunopositive cells were not observed in the rat cortex of the normal control (NC) group (Fig. 5A). However, cleaved caspase-3 positive cells were strongly expressed in the cortex in reperfusion 24 hr after MCAO injury group (EC group) (Fig. 5A). In addition, DHA treated rat cortex did not exhibit the expression of cleaved caspase-3 strongly compared to 24 hr MCAO group (EC group) (Fig. 5A). In striatum, cleaved caspase-3 expression showed the same pattern of the cortex (Fig. 5B). Fig. 5C was shown that the fluorescent intensity of cleaved caspase-3 was attenuated significantly over 4 times in the DHA treatment group compared to EC group (Fig. 5C). The western blot data (Fig.5D) also showed the reduction of cleaved caspase-3 expression in the DHA treatment group in spite of the MCAO injury. Following these data, DHA may inhibit the cell death under ischemic injury by attenuating the expression of cleaved caspase-3. Second, we conducted immunohistochemistry using Bax antibody at reperfusion 24 hr after MCAO injury to examine whether DHA influences on the alteration of marker that affects mitochondrial cell death both in cortex (Fig. 6A) and in striatum (Fig. 6B). We did not observe Bax immunoreactivity in the rat cortex of the normal control (NC) group (Fig. 6A). However, the expression of Bax was increased strongly in the cortex at reperfusion 24 hr after MCAO injury group (EC group) (Fig. 6A). In addition, the expression of Bax were not strongly exhibited in the cortex of DHA treated MCAO group compared to 24 hr MCAO group (EC group) (Fig. 6A). In striatum, Bax expression showed the same pattern as the cortex (Fig. 6B). Fig. 6C was shown that the significantly reduced fluorescent intensity of Bax over 2 times in the DHA treatment group compared to EC group (Fig. 6C). The western blot data (Fig. 6D) also showed the reduction of Bax expression in the DHA treatment group compared to the MCAO injury (Fig. 6D). Considering these data, DHA may influence on the cellular protection against the mitochondrial cell death under ischemic injury by attenuating the expression of Bax. Additionally, we conducted immunohistochemistry using iNOS antibody at reperfusion 24 hr after MCAO injury to confirm whether there was change of marker as the inflammatory mediator in both cortex (Fig. 7A) and striatum (Fig. 7B) or not. In the EC group, iNOS was considerably expressed in both in cortex (Fig. 7A) and striatum (Fig. 7B). However, iNOS expression was not found in both cortex (Fig. 7A) and striatum (Fig. 7B) at NC group. In the DHA treatment group, iNOS expression was decreased in both cortex (Fig. 7A) and striatum (Fig. 7B) compared to EC group. Fig. 7C was shown that the significantly decreased fluorescent intensity of iNOS over 9 times in the DHA treatment group compared to EC group (Fig. 7C). Based on these results, we suggest that DHA may inhibit the expression of iNOS in ischemic brain and also block the expression of inflammatory mediators.


Dehydroascorbic Acid Attenuates Ischemic Brain Edema and Neurotoxicity in Cerebral Ischemia: An in vivo Study.

Song J, Park J, Kim JH, Choi JY, Kim JY, Lee KM, Lee JE - Exp Neurobiol (2015)

Immunochemical image for confirmation of reduced cleaved caspase-3 expression by DHA treatment. (A) Immunochemical images showed that cleaved caspase-3 positive cells (red) were densely expressed in the EC group. In DHA treatment group, cleaved caspase-3 expression was decreased in rat cortex, compared with the EC group. (B) In DHA treatment group, cleaved caspase-3 positive cells were decreased in rat striatum due to DHA treatment. (C) The graph showed the percentage (%) of cleaved caspase-3 positive cells to compare the difference of cleaved caspase-3 fluorescence intensity. Statistical significance with EC group was determined by t-test. (D) The graph of cleaved caspase-3 protein level showed the same pattern with immunochemical images. Differences were considered significant at *p<0.05, (ANOVA followed by Bonferroni post hoc multiple comparison test). Scale bar=100 µm, Cleaved caspase-3: red, 4', 6-diamidino-2-phenylindole (DAPI): blue. NC: normal control group, EC: experimental control; reperfusion 24 hr after MCAO injury, DHA: DHA treatment and reperfusion 24 hr after MCAO injury.
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Figure 5: Immunochemical image for confirmation of reduced cleaved caspase-3 expression by DHA treatment. (A) Immunochemical images showed that cleaved caspase-3 positive cells (red) were densely expressed in the EC group. In DHA treatment group, cleaved caspase-3 expression was decreased in rat cortex, compared with the EC group. (B) In DHA treatment group, cleaved caspase-3 positive cells were decreased in rat striatum due to DHA treatment. (C) The graph showed the percentage (%) of cleaved caspase-3 positive cells to compare the difference of cleaved caspase-3 fluorescence intensity. Statistical significance with EC group was determined by t-test. (D) The graph of cleaved caspase-3 protein level showed the same pattern with immunochemical images. Differences were considered significant at *p<0.05, (ANOVA followed by Bonferroni post hoc multiple comparison test). Scale bar=100 µm, Cleaved caspase-3: red, 4', 6-diamidino-2-phenylindole (DAPI): blue. NC: normal control group, EC: experimental control; reperfusion 24 hr after MCAO injury, DHA: DHA treatment and reperfusion 24 hr after MCAO injury.
Mentions: We performed immunohistochemical staining using cleaved caspase-3 antibody at reperfusion 24 hr after MCAO injury to examine whether DHA influences on the cell death both in cortex (Fig. 5A) and in striatum (Fig. 5B). Cleaved caspase-3 immunopositive cells were not observed in the rat cortex of the normal control (NC) group (Fig. 5A). However, cleaved caspase-3 positive cells were strongly expressed in the cortex in reperfusion 24 hr after MCAO injury group (EC group) (Fig. 5A). In addition, DHA treated rat cortex did not exhibit the expression of cleaved caspase-3 strongly compared to 24 hr MCAO group (EC group) (Fig. 5A). In striatum, cleaved caspase-3 expression showed the same pattern of the cortex (Fig. 5B). Fig. 5C was shown that the fluorescent intensity of cleaved caspase-3 was attenuated significantly over 4 times in the DHA treatment group compared to EC group (Fig. 5C). The western blot data (Fig.5D) also showed the reduction of cleaved caspase-3 expression in the DHA treatment group in spite of the MCAO injury. Following these data, DHA may inhibit the cell death under ischemic injury by attenuating the expression of cleaved caspase-3. Second, we conducted immunohistochemistry using Bax antibody at reperfusion 24 hr after MCAO injury to examine whether DHA influences on the alteration of marker that affects mitochondrial cell death both in cortex (Fig. 6A) and in striatum (Fig. 6B). We did not observe Bax immunoreactivity in the rat cortex of the normal control (NC) group (Fig. 6A). However, the expression of Bax was increased strongly in the cortex at reperfusion 24 hr after MCAO injury group (EC group) (Fig. 6A). In addition, the expression of Bax were not strongly exhibited in the cortex of DHA treated MCAO group compared to 24 hr MCAO group (EC group) (Fig. 6A). In striatum, Bax expression showed the same pattern as the cortex (Fig. 6B). Fig. 6C was shown that the significantly reduced fluorescent intensity of Bax over 2 times in the DHA treatment group compared to EC group (Fig. 6C). The western blot data (Fig. 6D) also showed the reduction of Bax expression in the DHA treatment group compared to the MCAO injury (Fig. 6D). Considering these data, DHA may influence on the cellular protection against the mitochondrial cell death under ischemic injury by attenuating the expression of Bax. Additionally, we conducted immunohistochemistry using iNOS antibody at reperfusion 24 hr after MCAO injury to confirm whether there was change of marker as the inflammatory mediator in both cortex (Fig. 7A) and striatum (Fig. 7B) or not. In the EC group, iNOS was considerably expressed in both in cortex (Fig. 7A) and striatum (Fig. 7B). However, iNOS expression was not found in both cortex (Fig. 7A) and striatum (Fig. 7B) at NC group. In the DHA treatment group, iNOS expression was decreased in both cortex (Fig. 7A) and striatum (Fig. 7B) compared to EC group. Fig. 7C was shown that the significantly decreased fluorescent intensity of iNOS over 9 times in the DHA treatment group compared to EC group (Fig. 7C). Based on these results, we suggest that DHA may inhibit the expression of iNOS in ischemic brain and also block the expression of inflammatory mediators.

Bottom Line: Dehydroascorbic acid (DHA) as the oxidized form of ascorbic acid (AA) acts as a cellular protector against oxidative stress and easily enters into the brain compared to AA.We also examined the expression of claudin 5 for confirming the BBB breakdown, and the expression of bcl 2 associated X protein (Bax), caspase-3, inducible nitric oxide synthase (iNOS) for checking the effect of DHA on the neurotoxicity.Based on our findings, we propose that DHA might alleviate the pathogenesis of ischemic brain injury by attenuating edema, neuronal loss, and by improving synaptic connection.

View Article: PubMed Central - PubMed

Affiliation: Department of Anatomy, Yonsei University College of Medicine, Seoul 120-752, Korea.

ABSTRACT
Ischemic stroke results in the diverse phathophysiologies including blood brain barrier (BBB) disruption, brain edema, neuronal cell death, and synaptic loss in brain. Vitamin C has known as the potent anti-oxidant having multiple functions in various organs, as well as in brain. Dehydroascorbic acid (DHA) as the oxidized form of ascorbic acid (AA) acts as a cellular protector against oxidative stress and easily enters into the brain compared to AA. To determine the role of DHA on edema formation, neuronal cell death, and synaptic dysfunction following cerebral ischemia, we investigated the infarct size of ischemic brain tissue and measured the expression of aquaporin 1 (AQP-1) as the water channel protein. We also examined the expression of claudin 5 for confirming the BBB breakdown, and the expression of bcl 2 associated X protein (Bax), caspase-3, inducible nitric oxide synthase (iNOS) for checking the effect of DHA on the neurotoxicity. Finally, we examined postsynaptic density protein-95 (PSD-95) expression to confirm the effect of DHA on synaptic dysfunction following ischemic stroke. Based on our findings, we propose that DHA might alleviate the pathogenesis of ischemic brain injury by attenuating edema, neuronal loss, and by improving synaptic connection.

No MeSH data available.


Related in: MedlinePlus