Whereas almost no IgG immunostaining could be detected in shamoperated animals, intense levels of IgG immunolabeling were BI6727 Volasertib observed in vehicletreated mice 24 hr post pMCAO. In contrast, the intensity of IgG staining was markedly diminished after administration of AA. To evaluate the degree of AAinduced changes in IgG expression, we determined semiquantitatively the average IgG immunostaining intensity in both vehicle and AA treated mice at 24 hr postischemia. Quantitative analysis confirmed the qualitative observations and revealed a statistically significant 33% decrease in the intensity of IgG immunostaining in pMCAO induced ischemic animals treated with AA. Reduction of Cytochrome c Immunostaining by AA Mitochondrial dysfunction has been shown to contribute to ischemia induced brain injury.
Focal cerebral ischemia increases the mitochondrial membrane Krishnamurthy et al. Page 6 J Neurosci AC480 HER2 inhibitor Res. Author manuscript, available in PMC 2010 September 19. NIH PA Author Manuscript NIH PA Author Manuscript NIH PA Author Manuscript permeabilization, facilitating thereby the release of cytochrome c, which in turn activates cell death programs. To gain further insight into the mechanisms of action of AA, we therefore examined the distribution pattern of immunoreactivity for cytochrome c in ischemic mice. As illustrated in Figure 4A, immunoreactivity for cytochrome c was readily detected throughout the cortex in vehicletreated mice at 24 hr following pMCAO. Immunostaining for cytochrome c was detected in the intracellular space but also in cell bodies and their processes.
Cytochrome cpositive cells displayed a medium intensity of staining throughout the cortex. However, cells located at the periphery of the infarct area displayed a robust increase in the intensity of cytochrome c immunostaining. In contrast, such an increase in cytochrome c labeling was not observed in cells at the edges of the infarct region in AA treated ischemic animals. Inhibition of Mitochondrial Cytochrome c Release by AA The observation of an AA related decrease in the intensity of cytochrome c staining at the infarct periphery suggests that AA could play a role in the release of cytochrome c. To put this hypothesis to the test, we examined in isolated mouse brain mitochondria whether treatment with AA could prevent the release of cytochrome c induced by Ca2 and oxidative stress.
Indeed, both Ca2 overloading and oxidative stress to mitochondria have been shown to be involved in stroke related cell death and tissue damage. In a first experiment, we analyzed whether AA could prevent cytochrome c release induced by Ca2 overloading. As shown in Figure 4B, immunoblot analysis of the supernatant of mitochondria revealed a robust release of cytochrome c after exposure to 3 mM Ca2. This release was completely inhibited by addition of 100 M AA. AA itself, however, did not induce any cytochrome c release. In a second set of experiments, we studied the effects of AA on the release of cytochrome c induced by oxidative stresses, such as nitric oxide and H2O2. Results show that 100 M AA was also efficient at preventing cytochrome c release induced by both nitric oxide and H2O2.
AA Protection Against OGD Induced Decline in Cell Viability At the end of 5 hr of OGD, HT 22 neuronal cultures were treated with 1 g/ml or 10 g/ml AA. Cell viability was assessed 24 hr later by the Alamar blue assay, an index of mitochondrial function. Compared with controls, OGD reduced viability by 38%. This decline in viability was significantly reduced by AA in a dose dependent manner. Effect of AA on Mitochondrial Membrane Potential After 5 hr of OGD, AA was added to HT 22 neuronal cultures, and change inΔΨm was assessed 20 min later with TMRE with a fluorescence microscope. OGD induced a 55% decline in