Elevated Aminopeptidase P Attenuates Cerebral Arterial Responses to Bradykinin in Fawn-Hooded Hypertensive Rats

Cerebral arterial myogenic and autoregulatory responses are impaired in Fawn Hooded hypertensive (FHH) rats. Cerebral autoregulatory responses are restored in the congenic rat strain in which a segment of chromosome 1 from the Brown Norway (BN) rat was transferred into the FHH genetic background (FHH.1BN). The impact of this region on cerebral arterial dilator responses remains unknown. Aminopeptidase is a gene that was transferred into the FHH genetic background to generate the FHH.1BN rats and is responsible for degradation of the vasodilator bradykinin. Thus, we hypothesized that FHH rats will have increased aminopeptidase P levels with impaired cerebral arterial responses to bradykinin compared to BN and FHH.1BN rats. We demonstrated higher cerebral arterial expression of aminopeptidase P in FHH compared to BN rats. Accordingly, we demonstrated markedly impaired cerebral arterial dilation to bradykinin in FHH compared to BN rats. Interestingly, aminopeptidase P expression was lower in FHH.1BN compared to FHH rats. Decreased aminopeptidase P levels in FHH.1BN rats were associated with increased cerebral arterial bradykinin-induced dilator responses. Aminopeptidase P inhibition by apstatin improved cerebral arterial bradykinin dilator responses in FHH rats to a level similar to FHH.1BN rats. Unlike bradykinin, cerebral arterial responses to acetylcholine were similar between FHH and FHH.1BN groups. These findings indicate decreased bradykinin bioavailability contributes to impaired cerebral arterial dilation in FHH rats. Overall, these data indicate an important role of aminopeptidase P in the impaired cerebral arterial function in FHH rat.     

Sex-specific differences in cerebral arterial myogenic tone in hypertensive and normotensive rats

Cerebral blood flow (CBF) is maintained constant despite changes in systemic blood pressure (BP) through multiple mechanisms of autoregulation such as vascular myogenic reactivity. Our aim was to determine myogenic characteristics of cannulated middle cerebral arteries (MCA) in male and female stroke-prone spontaneously hypertensive rats (SHRSP) and Wistar-Kyoto rats (WKY) at 12 wk of age under pressurised no-flow conditions. MCA pressure-diameter relationships (20-200 mmHg) were constructed in active (with calcium) and passive (without calcium) conditions, and myogenic and mechanical properties were determined. Myogenic reactivity in WKY (P < 0.05) and SHRSP (P < 0.05) males was impaired compared with their female counterparts. Comparison of SHRSP with WKY in males revealed similar myogenic reactivity, but in females SHRSP exhibited augmented myogenic reactivity (P < 0.05). In both sexes, myogenic tone yielded at lower pressure in SHRSP compared with WKY vessels (120-140 vs. 140-180 mmHg). Stress-strain relationships and elastic moduli in WKY rats showed that vessels were stiffer in females than in males. Conversely, in SHRSP, male vessels were stiffer than female vessels. Comparison of strains in males indicated that stiffness was increased in SHRSP compared with WKY vessels, whereas the converse was observed in females. These findings demonstrate that MCA myogenic and distensibility characteristics exhibit significant sex- and strain-dependent differences. Inappropriate myogenic adaptation and augmented vascular stiffness, particularly in male SHRSP, are potential limiting factors in blood flow autoregulation and may increase the predisposition for stroke-related cerebrovascular events.     

Temporal characterization of the development of renal injury in FHH rats and FHH.1BN congenic strains

The present study examined the effect of transfer of portions of chromosome 1 that includes (FHH.1(BN) AR(+) strain) or excludes (control FHH.1(BN) AR(-) strain) a 4.3-Mb region from the Brown Norway (BN) rat that restores the autoregulation (AR) of renal blood flow (RBF) on the development of hypertension and renal injury in congenic strains of Fawn Hooded Hypertensive (FHH) rats. FHH and control AR(-) rats exhibited poor autoregulation of RBF, and glomerular capillary pressure (Pgc) rose by 19 ± 2 mmHg in FHH rats when renal perfusion pressure (RPP) was increased from 100 to 150 mmHg. In contrast, RBF was well autoregulated in the AR(+) strain, and Pgc only increased by 3 ± 1 mmHg when RPP was increased over this range. Baseline mean arterial pressure (MAP) at 12 wk of age was similar in all strains and averaged 122 mmHg. MAP increased significantly in FHH rats and was significantly higher by 12 mmHg in 21-wk-old FHH rats than in the FHH.1(BN) congenic strains. Protein excretion rose from 5 ± 1 to 397 ± 29 mg/day in 6- vs. 21-wk-old FHH rats. In contrast, protein excretion only increased to 139 ± 21 mg/day in the control AR(-) strain, and it did not increase significantly in the AR(+) strain. Glomerular permeability to albumin was similar in all strains at 6 wk of age. It increased significantly in 9-wk-old FHH and control AR(-) rats, but not in the AR(+) strain. The levels of matrix metalloproteinase (MMP)-2 and transforming growth factor (TGF)-β2 protein were significantly higher in the renal cortex of 9-wk-old FHH rats compared with the levels seen in the AR(+) strain. These data indicate that transfer of a 4.3-Mb region of BN chromosome 1 into the FHH genetic background improves autoregulation of RBF, normalizes Pgc, and slows the progression of renal disease.     

Nicotinamide Attenuates Focal Ischemic Brain Injury in Rats: With Special Reference to Changes in Nicotinamide and NAD+ Levels in Ischemic Core and Penumbra

We investigated the neuroprotective action of nicotinamide in focal ischemia. Male spontaneously hypertensive rats (5–7 months old) were subjected to photothrombotic occlusion of the right distal middle cerebral artery (MCA). Either nicotinamide (125 or 250 mg/kg) or vehicle was injected IV before MCA occlusion. Changes in the cerebral blood flow (CBF) were monitored using laser-Doppler flowmetry, and infarct volumes were determined with TTC staining 3 days after MCA occlusion. In another set of experiments, the brain nicotinamide and nicotinamide adenine dinucleotide (NAD⁺) levels were analyzed by HPLC using the frozen samples dissected from the regions corresponding to the ischemic core and penumbra. In the 250-mg/kg nicotinamide group, the ischemic CBF was significantly increased compared to that the untreated group, and the infarct volumes were substantially attenuated (–36%). On the other hand, the ischemic CBF in the 125 mg/kg nicotinamide group was not significantly different from the untreated CBF, however, the infarct volumes were substantially attenuated (–38%). Cerebral ischemia per se did not affect the concentrations of nicotinamide and NAD⁺ both in the penumbra and ischemic core. In the nicotinamide groups, the brain nicotinamide levels increased significantly in all areas examined, and brain NAD⁺ levels increased in the penumbra but not in the ischemic core. Increased brain levels of nicotinamide are considered to be primarily important for neuroprotection against ischemia, and the protective action may be partly mediated through the increased NAD⁺ in the penumbra.     

Restoration of normal vascular relaxation mechanisms in cerebral arteries by chromosomal substitution in consomic SS.13BN rats

This study sought to identify the mechanisms of vascular relaxation that are rescued in middle cerebral arteries (MCA) of SS.13BN consomic rats by substituting chromosome 13 containing the renin gene from Brown Norway (BN) rats into the Dahl salt-sensitive (SS) genetic background. Isolated MCA from SS rats exhibited an indomethacin-sensitive constriction in response to acetylcholine (ACh) and hypoxia. ACh-induced dilation was NO dependent and hypoxic dilations were cyclooxygenase (COX) dependent in BN and SS.13BN rats. In SS rats, hypoxic dilation was restored by indomethacin and abolished by inhibiting cytochrome P-450 epoxygenases, suggesting a role for epoxyeicosatrienoic acids. MCA from SS and SS.13BN rats constricted and MCA from BN rats dilated in response to the stable prostacyclin analog iloprost. MCA from SS.13BN and BN rats (but not SS rats) dilated in response to the prostaglandin E2 receptor agonist butaprost. Hypoxia increased prostacyclin release in cerebral arteries from all the strains, whereas thromboxane A2 production was reduced in BN rat vessels only. These data suggest that SS rats may be less sensitive to vasodilator prostaglandins and that normalization of renin-angiotensin system regulation causes a switch from production of COX-derived vasoconstrictor metabolites (in SS rats) toward NO-dependent relaxation in response to ACh- and prostaglandin-dependent dilation in response to hypoxia in SS.13(BN) rats.     

Dual-specificity phosphatase 6 deletion protects the colonic epithelium against inflammation and promotes both proliferation and tumorigenesis

The Ras/mitogen-activated protein kinase (MAPK) pathway controls fundamental cellular processes such as proliferation, differentiation, and apoptosis. The dual-specificity phosphatase 6 (DUSP6) regulates cytoplasmic MAPK signaling by dephosphorylating and inactivating extracellular signal-regulated kinase (ERK1/2) MAPK. To determine the role of DUSP6 in the maintenance of intestinal homeostasis, we characterized the intestinal epithelial phenotype of Dusp6 knockout (KO) mice under normal, oncogenic, and proinflammatory conditions. Our results show that loss of Dusp6 increased crypt depth and epithelial cell proliferation without altering colonic architecture. Crypt regeneration capacity was also enhanced, as revealed by ex vivo Dusp6 KO organoid cultures. Additionally, loss of Dusp6 induced goblet cell expansion without affecting enteroendocrine and absorptive cell differentiation. Our data also demonstrate that Dusp6 KO mice were protected from acute dextran sulfate sodium-induced colitis, as opposed to wild-type mice. In addition, Dusp6 gene deletion markedly enhanced tumor load in Apc ^Min/+ mice. Decreased DUSP6 expression by RNA interference in HT29 colorectal cancer cells enhanced ERK1/2 activation levels and promoted both anchorage-independent growth in soft agar as well as invasion through Matrigel. Finally, DUSP6 mRNA expression in human colorectal tumors was decreased in advanced stage tumors compared with paired normal tissues. These results demonstrate that DUSP6 phosphatase, by controlling ERK1/2 activation, regulates colonic inflammatory responses, and protects the intestinal epithelium against oncogenic stress.     

Angiogenesis and capillary maturation phenotypes associated with the Edpm3 locus on rat chromosome 3

The quantitative trait locus (QTL) Edpm3 is one of a group of additively acting QTL \responsible for the difference in estrogen-induced pituitary tumor growth between the tumor-susceptible F344 and tumor-resistant BN rat strains. The F344.BN-Edpm3^BN rat strain was produced by moving the segment of rat Chr 3 between D3Mgh7 and D3Mgh13, which contains the Edpm3 QTL, from the BN strain into the F344 genetic background. In a previous study, we used this congenic line to find that the BN allele of the Edpm3 QTL reduces tissue mass and S-phase fraction in the estrogen-induced rat pituitary tumor. We now report on the use of this congenic line to investigate the linkage of Edpm3 to tumor angiogenesis. Contrary to expectation, the F344.BN-Edpm3^BN strain has significantly greater angiogenic activity than does F344 in both treated and untreated rats. Microvessel count (MVC), perivascular space, and number of nonattached pericytes/pericapillary fibroblasts are all elevated in the pituitary by chronic estrogen treatment and their values are significantly greater in F344.BN-Edpm3^BN than F344. Thus, although there is greater angiogenic activity in the pituitary of estrogen-treated F344.BN-Edpm3^BN rats, there is a deficiency in capillary maturation compared with F344.     

Transient middle cerebral artery occlusion causes different structural, mechanical, and myogenic alterations in normotensive and hypertensive rats

Transient focal cerebral ischemia in the rat alters vessel properties, and spontaneously hypertensive rats (SHR) show a poorer outcome after ischemia. In the present study we examined the role of hypertension on vessel properties after ischemia-reperfusion. The right middle cerebral artery (MCA) was occluded (90 min) and reperfused (24 h) in SHR (n = 12) and Wistar-Kyoto rats (WKY; n = 11). Sham-operated rats (SHR, n = 10; WKY, n = 10) were used as controls. The structural, mechanical, and myogenic properties of the MCA were assessed by pressure myography. Nuclei distribution and elastin content and organization were analyzed by confocal microscopy. Infarct volume was larger in SHR than in WKY rats. Ischemia-reperfusion induced adventitial hypertrophy associated with an increase in the total number of adventitial cells. In addition, fenestrae area and arterial distensibility increased and myogenic tone decreased in the MCA of WKY rats after ischemia-reperfusion. Hypertension per se induced hypertrophic inward remodeling. Ischemia-reperfusion decreased the cross-sectional area of the MCA in SHR, without significant changes in distensibility, despite an increase in fenestrae area. In addition, MCA myogenic properties were not altered after ischemia-reperfusion in SHR. Our results indicate that in normotensive rats, MCA develops a compensatory mechanism (i.e., enhanced distensibility and decreased myogenic tone) that counteracts the effect of ischemia-reperfusion and ensures correct cerebral irrigation. These compensatory mechanisms are lost in hypertension, thereby explaining, at least in part, the greater infarct volume observed in SHR.     

Oxidant stress and constrictor reactivity impair cerebral artery dilation in obese Zucker rats

This study tested the hypothesis that evolution of the metabolic syndrome in obese Zucker rats (OZR) leads to impaired dilator reactivity of cerebral resistance arteries vs. responses determined in lean Zucker rats (LZR). Middle cerebral arteries (MCA) from 17-wk-old male LZR and OZR were isolated and cannulated with glass micropipettes. Vascular reactivity was assessed in response to challenge with ACh, sodium nitroprusside (SNP), reductions and elevations in Po2, 5-HT, and increased intralumenal pressure. Vessels were treated with the free radical scavenger 4-hydroxy-2,2,6,6-tetramethylpiperidine 1-oxyl (tempol) to assess the role of superoxide production in altering reactivity, and passive vascular wall mechanics was assessed in each vessel. Vascular superoxide production was assessed in isolated arteries using fluorescence microscopy. Vessel dilation to ACh and hypoxia was impaired in OZR vs. LZR, although responses to SNP were normal. Vessel constriction to 5-HT, elevated Po2, and elevated intralumenal pressure was enhanced in OZR vs. LZR. Fluorescence microscopy demonstrated an increased superoxide production in arteries of OZR vs. LZR, correctable by incubation with tempol. Although treatment of vessels from OZR with tempol improved dilation to ACh and hypoxia, constrictor responses to 5-HT, elevated Po2, and pressure were not altered by tempol treatment. Indexes of vessel wall mechanics were comparable between groups. These results suggest that vasodilator reactivity of MCA of OZR in response to endothelium-dependent dilator stimuli is impaired vs. LZR and that this may represent a reduced bioavailability of signaling molecules due to oxidant scavenging. However, oxidative stress-independent increases in myogenic tone and constrictor reactivity may contribute to blunted dilator responses of cerebral microvessels.     

Postnatal maturation attenuates pressure-evoked myogenic tone and stretch-induced increases in Ca2+ in rat cerebral arteries

Although postnatal maturation potently modulates agonist-induced cerebrovascular contractility, its effects on the mechanisms mediating cerebrovascular myogenic tone remain poorly understood. Because the regulation of calcium influx and myofilament calcium sensitivity change markedly during early postnatal life, the present study tested the general hypothesis that early postnatal maturation increases the pressure sensitivity of cerebrovascular myogenic tone via age-dependent enhancement of pressure-induced calcium mobilization and myofilament calcium sensitivity. Pressure-induced myogenic tone and changes in artery wall intracellular calcium concentrations ([Ca(2+)](i)) were measured simultaneously in endothelium-denuded, fura-2-loaded middle cerebral arteries (MCA) from pup [postnatal day 14 (P14)] and adult (6-mo-old) Sprague-Dawley rats. Increases in pressure from 20 to 80 mmHg enhanced myogenic tone in MCA from both pups and adults although the normalized magnitudes of these increases were significantly greater in pup than adult MCA. At each pressure step, vascular wall [Ca(2+)](i) was also significantly greater in pup than in adult MCA. Nifedipine significantly attenuated pressure-evoked constrictions in pup MCA and essentially eliminated all responses to pressure in the adult MCA. Both pup and adult MCA exhibited pressure-dependent increases in calcium sensitivity, as estimated by changes in the ratio of pressure-induced myogenic tone to wall [Ca(2+)](i). However, there were no differences in the magnitudes of these increases between pup and adult MCA. The results support the view that regardless of postnatal age, changes in both calcium influx and myofilament calcium sensitivity contribute to the regulation of cerebral artery myogenic tone. The greater cerebral myogenic response in P14 compared with adult MCA appears to be due to greater pressure-induced increases in [Ca(2+)](i), rather than enhanced augmentation of myofilament calcium sensitivity.     

Catechin treatment improves cerebrovascular flow-mediated dilation and learning abilities in atherosclerotic mice

Severe dyslipidemia and the associated oxidative stress could accelerate the age-related decline in cerebrovascular endothelial function and cerebral blood flow (CBF), leading to neuronal loss and impaired learning abilities. We hypothesized that a chronic treatment with the polyphenol catechin would prevent endothelial dysfunction, maintain CBF responses, and protect learning abilities in atherosclerotic (ATX) mice. We treated ATX (C57Bl/6-LDLR(-/-)hApoB(+/+); 3 mo old) mice with catechin (30 mg · kg(-1) · day(-1)) for 3 mo, and C57Bl/6 [wild type (WT), 3 and 6 mo old] mice were used as controls. ACh- and flow-mediated dilations (FMD) were recorded in pressurized cerebral arteries. Basal CBF and increases in CBF induced by whisker stimulation were measured by optical coherence tomography and Doppler, respectively. Learning capacities were evaluated with the Morris water maze test. Compared with 6-mo-old WT mice, cerebral arteries from 6-mo-old ATX mice displayed a higher myogenic tone, lower responses to ACh and FMD, and were insensitive to NOS inhibition (P < 0.05), suggesting endothelial dysfunction. Basal and increases in CBF were lower in 6-mo-old ATX than WT mice (P < 0.05). A decline in the learning capabilities was also observed in ATX mice (P < 0.05). Catechin 1) reduced cerebral superoxide staining (P < 0.05) in ATX mice, 2) restored endothelial function by reducing myogenic tone, improving ACh- and FMD and restoring the sensitivity to nitric oxide synthase inhibition (P < 0.05), 3) increased the changes in CBF during stimulation but not basal CBF, and 4) prevented the decline in learning abilities (P < 0.05). In conclusion, catechin treatment of ATX mice prevents cerebrovascular dysfunctions and the associated decline in learning capacities.     

Efficacy and Dose-Dependent Safety of Intra-Arterial Delivery of Mesenchymal Stem Cells in a Rodent Stroke Model

Intra-arterial (IA) delivery of mesenchymal stem cells (MSCs) for acute ischemic stroke is attractive for clinical translation. However, studies using rat model of stroke have demonstrated that IA MSCs delivery can decrease middle cerebral artery (MCA) flow, which may limit its clinical translation. The goal of this study is to identify a dose of IA MSCs (maximum tolerated dose; MTD) that does not compromise MCA flow and evaluate its efficacy and optimal timing in a rat model of reversible middle cerebral artery occlusion (rMCAo). We sought to determine if there is a difference in efficacy of acute (1 h) versus sub-acute (24 h) IA MSCs treatment after rMCAo. Adult female Sprague-Dawley rats underwent rMCAo (90 min) and an hour later a single dose of MSCs (at de-escalating doses 1×10⁶, 5×10⁵, 2×10⁵, 1×10⁵ and 5×10⁴) was given using IA route. MSCs were suspended in phosphate buffered saline (PBS) and PBS alone was used for control experiments. We measured the percent change in mean laser Doppler flow signal over the ipsilateral MCA in de-escalating doses groups to determine MTD. The results demonstrated that the lowering of IA MSC dose to 1×10⁵ and below did not compromise MCA flow and hence an IA MSC dose of 1×10⁵ considered as MTD. Subsequently, 1 h and 24 h after rMCAo, rats were treated with IA MSCs or PBS. The 24 h delivery of IA MSCs significantly improved neurodeficit score and reduced the mean infarct volume at one month as compared to control, but not the 1 h delivery. Overall, this study suggests that the IA delivery of MSCs can be performed safely and efficaciously at the MTD of 1×10⁵ delivered at 24 hours in rodent model of stroke.     

Middle cerebral artery alterations in a rat chronic hypoperfusion model

Chronic cerebral hypoperfusion (CHP) induces microvascular changes that could contribute to the progression of vascular cognitive impairment and dementia in the aging brain. This study aimed to analyze the effects of CHP on structural, mechanical, and myogenic properties of the middle cerebral artery (MCA) after bilateral common carotid artery occlusion (BCCAO) in adult male Wistar rats. Sham animals underwent a similar surgical procedure without carotid artery (CA) ligation. After 15 days of occlusion, MCA and CA were dissected and MCA structural, mechanical, and myogenic properties were assessed by pressure myography. Collagen I/III expression was determined by immunofluorescence in MCA and CA and by Western blot in CA. mRNA levels for 1A1, 1A2, and 3A1 collagen subunits were quantified by quantitative real-time PCR in CA. Matrix metalloproteinase (MMP-1, MMP-2, MMP-9, and MMP-13) and hypoxia-inducible factor-1α (HIF-1α) protein expression were determined in CA by Western blot. BCCAO diminished cross-sectional area, wall thickness, and wall-to-lumen ratio. Nevertheless, whereas wall stress was increased, stiffness was not modified and myogenic response was diminished. Hypoperfusion triggered HIF-1α expression. Collagen I/III protein expression diminished in MCA and CA after BCCAO, despite increased mRNA levels for 1A1 and 3A1 collagen subunits. Therefore, the reduced collagen expression might be due to proteolytic degradation, since the expression of MMP-1 and MMP-9 increased in the CA. These data suggest that BCCAO induces hypotrophic remodeling by a mechanism that involves a reduction of collagen I/III in association with increased MMP-1 and MMP-9 and that decreases myogenic tone in major arteries supplying the brain.     

Ventilatory phenotypes among four strains of adult rats.

Our purpose in this study was to identify different ventilatory phenotypes among four different strains of rats. We examined 114 rats from three in-house, inbred strains and one outbred strain: Brown Norway (BN; n = 26), Dahl salt-sensitive (n = 24), Fawn-hooded Hypertensive (FHH: n = 27), and outbred Sprague-Dawley rats (SD; n = 37). We measured eupneic (room air) breathing and the ventilatory responses to hypoxia (12% O(2)-88% N(2)), hypercapnia (7% CO(2)), and two levels of submaximal exercise. Primary strain differences were between BN and the other strains. BN rats had a relatively attenuated ventilatory response to CO(2) (P < 0.001), an accentuated ventilatory response to exercise (P < 0.05), and an accentuated ventilatory roll-off during hypoxia (P < 0.05). Ventilation during hypoxia was lower than other strains, but hyperventilation during hypoxia was equal to the other strains (P > 0.05), indicating that the metabolic rate during hypoxia decreased more in BN rats than in other strains. Another strain difference was in the frequency and timing components of augmented breaths, where FHH rats frequently differed from the other strains, and the BN rats had the longest expiratory time of the augmented breaths (probably secondary to the blunted CO(2) sensitivity). These strain differences not only provide insight into physiological mechanisms but also indicate traits (such as CO(2) sensitivity) that are genetically regulated. Finally, the data establish a foundation for physiological genomic studies aimed at elucidating the genetics of these ventilatory control mechanisms.     

15-HETE参与的脑缺血再灌注损伤中p-ERK1/2表达变化的研究

目的:通过应用15-脂氧化酶(15-Lipoxygenase,15-LOX)抑制剂去甲二氢愈创木酸(nordihydroguaiaretic acid,NDGA)抑制15-羟基-二十碳四烯酸(15-hydroxyeicosatetraenoic acid,15-HETE)的生成,观察缺血再灌注损伤中大鼠脑组织磷酸化细胞外信号调节酶(phosphor-extracellular signal-regulated kinase,p-ERK1/2)表达的变化,探讨p-ERK1/2在15-HETE参与的脑缺血再灌注损伤中的作用及其表达变化。方法:应用大脑中动脉线栓栓塞法(MCAO)制作大鼠脑梗死2小时再灌注模型。将大鼠随机分为三组:假手术组(sham组)、DMSO对照组、NDGA处理组。后两组再根据不同的灌注时间分为三个亚组:再灌注1小时组、再灌注6小时组、再灌注24小时组。采用TTC染色法检测再灌注24小时大鼠脑梗死体积;免疫印迹(Western blot)法测定梗死后再灌注不同时间点梗死核心区和梗死周围区的p-ERK1/2的表达。结果:假手术组仅有少量p-ERK1/2的表达。DMSO对照组梗死核心区p-ERK1/2的表达从梗死再灌注后1小时即开始逐渐升高(1.43±0.06),6小时达高峰(2.02±0.14),24小时有所下降(1.16±0.21),与假手术组(0.62±0.08)比较P值均0.01;梗死周围区p-ERK1/2表现出相同的变化趋势。与DMSO对照组比较,NDGA处理组大鼠脑梗死体积显著减小(20.10±0.12%vs 17.24±0.16%,P=0.009,P0.05),各时间点p-ERK1/2的表达均下降。与梗死核心区相比较,梗死周围区24小时仍可检测到较高含量的p-ERK1/2(1.16±0.21 vs 1.86±0.14),但梗死核心区表达相对较少。结论:脑缺血再灌注损伤中,p-ERK1/2的表达增加,说明p-ERK1/2参与其中;应用NDGA后,p-ERK1/2的表达降低,脑梗死体积减小,证实p-ERK1/2参与了15-HETE介导的脑缺血再灌注损伤,并在此过程中可能参与了细胞的凋亡。     

Endothelin-1 Induced Middle Cerebral Artery Occlusion Model for Ischemic Stroke with Laser Doppler Flowmetry Guidance in Rat

Stroke is the number one cause of disability and third leading cause of death in the world, costing an estimated $70 billion in the United States in 2009^{1, 2}. Several models of cerebral ischemia have been developed to mimic the human condition of stroke. It has been suggested that up to 80% of all strokes result from ischemic damage in the middle cerebral artery (MCA) area³. In the early 1990s, endothelin-1 (ET-1) ⁴ was used to induce ischemia by applying it directly adjacent to the surface of the MCA after craniotomy. Later, this model was modified ⁵ by using a stereotaxic injection of ET-1 adjacent to the MCA to produce focal cerebral ischemia. The main advantages of this model include the ability to perform the procedure quickly, the ability to control artery constriction by altering the dose of ET-1 delivered, no need to manipulate the extracranial vessels supplying blood to the brain as well as gradual reperfusion rates that more closely mimics the reperfusion in humans^5-7. On the other hand, the ET-1 model has disadvantages that include the need for a craniotomy, as well as higher variability in stroke volume⁸. This variability can be reduced with the use of laser Doppler flowmetry (LDF) to verify cerebral ischemia during ET-1 infusion. Factors that affect stroke variability include precision of infusion and the batch of the ET-1 used⁶. Another important consideration is that although reperfusion is a common occurrence in human stroke, the duration of occlusion for ET-1 induced MCAO may not closely mimic that of human stroke where many patients have partial reperfusion over a period of hours to days following occlusion^{9, 10}. This protocol will describe in detail the ET-1 induced MCAO model for ischemic stroke in rats. It will also draw attention to special considerations and potential drawbacks throughout the procedure.     

Introgression of chromosome 13 in Dahl salt-sensitive genetic background restores cerebral vascular relaxation

To evaluate the potential role of impaired renin-angiotensin system (RAS) function in contributing to reduced vascular relaxation in Dahl salt-sensitive (S) rats, responses to ACh (10(-6) mol/l) and hypoxia (Po(2) reduction to 40-45 mmHg) were determined in isolated middle cerebral arteries of Dahl S rats, Brown Norway (BN) rats, and consomic rats having chromosome 13 (containing the renin gene) or chromosome 16 of the BN rat substituted into the Dahl S genetic background (SS-13(BN) and SS-16(BN), respectively). Arteries of BN rats on a low-salt (LS) diet (0.4% NaCl) dilated in response to ACh and hypoxia, whereas dilation in response to these stimuli was absent in Dahl S rats on LS diet. Vasodilation to ACh and hypoxia was restored in SS-13(BN) rats on an LS diet but not in SS-16(BN) rats. High-salt diet (4% NaCl), to suppress ANG II, eliminated vasodilation to hypoxia and ACh in BN and in SS-13(BN) rats. Treatment of SS-13(BN) rats with the AT(1) receptor antagonist losartan also eliminated the restored vasodilation in response to ACh and hypoxia. These studies suggest that restoration of normal RAS regulation in SS-13(BN) consomic rats restores vascular relaxation mechanisms that are impaired in Dahl S rats.     

Divergent coronary flow responses to uridine adenosine tetraphosphate in atherosclerotic ApoE knockout mice

Uridine adenosine tetraphosphate (Up₄A) exerts potent relaxation in porcine coronary arteries that is reduced following myocardial infarction, suggesting a crucial role for Up₄A in the regulation of coronary flow (CF) in cardiovascular disorders. We evaluated the vasoactive effects of Up₄A on CF in atherosclerosis using ApoE knockout (KO) mice ex vivo and in vivo. Functional studies were conducted in isolated mouse hearts using the Langendorff technique. Immunofluorescence was performed to assess purinergic P2X₁ receptor (P2X₁R) expression in isolated mouse coronary arteries. In vivo effects of Up₄A on coronary blood flow (CBF) were assessed using ultrasound. Infusion of Up₄A (10^?9–10^?5 M) into isolated mouse hearts resulted in a concentration-dependent reduction in CF in WT and ApoE KO mice to a similar extent; this effect was exacerbated in ApoE KO mice fed a high-fat diet (HFD). The P2X₁R antagonist MRS2159 restored Up₄A-mediated decreases in CF more so in ApoE KO + HFD than ApoE KO mice. The smooth muscle to endothelial cell ratio of coronary P2X₁R expression was greater in ApoE KO + HFD than ApoE KO or WT mice, suggesting a net vasoconstrictor potential of P2X₁R in ApoE KO + HFD mice. In contrast, Up₄A (1.6 mg/kg) increased CBF to a similar extent among the three groups. In conclusion, Up₄A decreases CF more in ApoE KO + HFD mice, likely through a net upregulation of vasoconstrictor P2X₁R. In contrast, Up₄A increases CBF in vivo regardless of the atherosclerotic model.