Effects of Citrus Flavonoids Against Microvascular Damage Induced by Hypoperfusion and Reperfusion in Rat Pial Circulation.

OBJECTIVE: The aim of the present study was to investigate the in vivo protective effects of hesperidin or diosmin or apigenin on damage induced by transient BCCAO and reperfusion. METHODS: Rat pial microcirculation was observed through a closed cranial window, using fluorescence microscopy. Pial arterioles were classified in five orders according to the Strahler's method. RESULTS: After 30 BCCAO and 60 minutes reperfusion, rats showed decreased arteriolar diameter, microvascular leakage, leukocyte adhesion, and reduction in capillary perfusion. Hesperidin and diosmin abolished the reduction in arteriolar diameter, while higher dose apigenin induced dilation by 21.7 ± 2.0% in order three arterioles RE. Nitric oxide synthase inhibition attenuated significantly hesperidin or diosmin or apigenin's effects on arteriolar diameter. Moreover, all these substances reduced microvascular leakage as well as leukocyte adhesion in dose-related manner, while capillary perfusion was protected. Furthermore, reduction in infarcted area and decrease in ROS production were observed. CONCLUSIONS: Hesperidin, diosmin, and apigenin showed dose-related protective effects on hypoperfusion-reperfusion injury, causing nitric oxide release and attenuating tissue edema and leukocyte adhesion.

Effects of oleuropein and pinoresinol on microvascular damage induced by hypoperfusion and reperfusion in rat pial circulation.

OBJECTIVE: The present study was aimed to assess the in vivo acute effects of oleuropein or/and pinoresinol, polyphenols widely diffused in natural sources, on rat pial microvascular responses during transient BCCAO and reperfusion. METHODS: Rat pial microcirculation was visualized by fluorescence microscopy through a closed cranial window. Pial arterioles were classified into five orders of branching. Capillaries were assigned order 0, the smallest arterioles order 1 and the largest ones order 5. RESULTS: Rats subjected to BCCAO and reperfusion showed: arteriolar diameter decrease, microvascular leakage, leukocyte adhesion in venules, and reduction in capillary perfusion. Pretreatment with oleuropein or pinoresinol, a higher dose before BCCAO determined dilation in all arteriolar orders RE. Microvascular leakage was reduced as well as leukocyte adhesion and ROS formation, while capillary perfusion was protected. Inhibition of endothelium nitric oxide synthase prior to oleuropein or pinoresinol reduced the effect of these polyphenols on pial arteriolar diameter and leakage. These substances, administered together, prevented microvascular damage to a larger extent. CONCLUSION: Oleuropein and pinoresinol were both able to protect pial microcirculation from I-reperfusion injury, to increase nitric oxide release and to reduce oxidative stress preserving pial blood flow distribution.

Rat Pial Microvascular Changes During Cerebral Blood Flow Decrease and Recovery: Effects of Cyanidin Administration.

The reactive oxygen species (ROS) are known to play a major role in many pathophysiological conditions, such as ischemia and reperfusion injury. The present study was aimed to evaluate the in vivo cyanidin (anthocyanin) effects on damages induced by rat pial microvascular hypoperfusion-reperfusion injury by cerebral blood flow decrease (CBFD) and subsequent cerebral blood flow recovery (CBFR). In particular, the main purpose was to detect changes in ROS production after cyanidin administration. Rat pial microvasculature was investigated using fluorescence microscopy through a cranial window (closed); Strahler's method was utilized to define the geometric features of pial vessels. ROS production was investigated in vivo by 2'-7'-dichlorofluorescein-diacetate assay and neuronal damage was measured on isolated brain sections by 2,3,5-triphenyltetrazolium chloride staining. After 30 min of CBFD, induced by bilateral common carotid artery occlusion, and 60 min of CBFR, rats showed decrease of arteriolar diameter and capillary perfusion; furthermore, increase in microvascular leakage and leukocyte adhesion was observed. Conversely, cyanidin administration induced dose-related arteriolar dilation, reduction in microvascular permeability as well as leukocyte adhesion when compared to animals subjected to restriction of cerebral blood flow; moreover, capillary perfusion was protected. ROS generation increase and marked neuronal damage were detected in animals subjected to CBFD and CBFR. On the other hand, cyanidin was able to reduce ROS generation and neuronal damage. In conclusion, cyanidin treatment showed dose-related protective effects on rat pial microcirculation during CBFD and subsequent CBFR, inducing arteriolar dilation by nitric oxide release and inhibiting ROS formation, consequently preserving the blood brain barrier integrity.

Laser Speckle Imaging of Rat Pial Microvasculature during Hypoperfusion-Reperfusion Damage.

The present study was aimed to in vivo assess the blood flow oscillatory patterns in rat pial microvessels during 30 min bilateral common carotid artery occlusion (BCCAO) and 60 min reperfusion by laser speckle imaging (LSI). Pial microcirculation was visualized by fluorescence microscopy. The blood flow oscillations of single microvessels were recorded by LSI; spectral analysis was performed by Wavelet transform. Under baseline conditions, arterioles and venules were characterized by blood flow oscillations in the frequency ranges 0.005-0.0095 Hz, 0.0095-0.021 Hz, 0.021-0.052 Hz, 0.052-0.150 Hz and 0.150-0.500 Hz. Arterioles showed oscillations with the highest spectral density when compared with venules. Moreover, the frequency components in the ranges 0.052-0.150 Hz and 0.150-0.500 were predominant in the arteriolar total power spectrum; while, the frequency component in the range 0.150-0.500 Hz showed the highest spectral density in venules. After 30 min BCCAO, the arteriolar spectral density decreased compared to baseline; moreover, the arteriolar frequency component in the range 0.052-0.150 Hz significantly decreased in percent spectral density, while the frequency component in the range 0.150-0.500 Hz significantly increased in percent spectral density. However, an increase in arteriolar spectral density was detected at 60 min reperfusion compared to BCCAO values; consequently, an increase in percent spectral density of the frequency component in the range 0.052-0.150 Hz was observed, while the percent spectral density of the frequency component in the range 0.150-0.500 Hz significantly decreased. The remaining frequency components did not significantly change during hypoperfusion and reperfusion. The changes in blood flow during hypoperfusion/reperfusion caused tissue damage in the cortex and striatum of all animals. In conclusion, our data demonstrate that the frequency component in the range 0.052-0.150 Hz, related to myogenic activity, was significantly impaired by hypoperfusion and reperfusion, affecting cerebral blood flow distribution and causing tissue damage.

Malvidin's Effects on Rat Pial Microvascular Permeability Changes Due to Hypoperfusion and Reperfusion Injury.

The present study was aimed to evaluate the malvidin's protective effects on damage induced by 30 min bilateral common carotid artery occlusion (BCCAO) and 60 min reperfusion (RE) in rat pial microcirculation. Rat pial microcirculation was observed using fluorescence microscopy through a closed cranial window. Western blotting analysis was performed to investigate the endothelial nitric oxide synthase (eNOS), phosphorylated eNOS (p-eNOS) and matrix metalloproteinase 9 (MMP-9) expression. Moreover, MMP-9 activity was evaluated by zymography. Finally, neuronal damage and radical oxygen species (ROS) formation were assessed. In all animals, pial arterioles were classified in five orders of branching according to Strahler's method. In hypoperfused rats, 30 min BCCAO and 60 min RE caused a decrease in arteriolar diameter, an increase in microvascular leakage and leukocyte adhesion, accompanied by decreased capillary perfusion and red blood cell velocity (VRBC). Moreover, marked neuronal damage and evident ROS generation were detected. Conversely, malvidin administration induced arteriolar dilation in dose-related manner, reducing microvascular leakage as well as leukocyte adhesion. Capillary perfusion and VRBC were protected. Nitric oxide (NO) synthase inhibition significantly attenuated malvidin's effects on arteriolar diameter. Western blotting analysis revealed an increase in eNOS and p-eNOS expression, while zymography indicated a decrease in MMP-9 activity after malvidin's administration. Furthermore, malvidin was able to prevent neuronal damage and to decrease ROS generation. In conclusion, malvidin protects rat pial microcirculation against BCCAO/RE injury, preventing blood-brain impairment and neuronal loss. Malvidin's effects appear to be mediated by eNOS activation and scavenger activity.