摘要
脑缺血是一种常见疾病,具有高发病率、高死亡率和高致残率的特点,严重危害人类健康。脑组织维持正常代谢活动,需氧量巨大,但脑本身的无氧代谢能力及能量贮备能力极差,所需要的能量几乎全部来自葡萄糖的有氧代谢。在脑缺血、缺氧状态下,脑动脉床内所含的氧仅够供脑消耗几秒钟,可使有氧代谢很快转化为无氧代谢,ATP生成的效率仅为正常时的1/18,因而脑缺血后常伴有脑细胞的能量降低、自由基和兴奋性氨基酸增多、钙离子超载以及细胞结构破坏等一系列病理生理反应。
Na~+, K~+-ATPase是广泛存在于哺乳动物细胞膜上的一种ATP依赖性离子转运体,主要功能是维持细胞内外的离子平衡。脑组织Na~+, K~+-ATPase主要存在于突触膜(SPM)和微血管的内皮细胞(CMV),在血管紧张度的维持和调节中有着重要作用。脑缺血发生后由于ATP的生成减少,Na~+, K~+-ATPase活性降低,使细胞内外离子平衡被打破。其中,细胞外液中K~+在短时间内的骤然升高,可刺激血管平滑肌细胞去极化,开放钠离子通道使细胞内Na~+进一步升高而逆转Na~+/Ca~(2+)交换,开放电压依赖性钙通道(VDCC)而使Ca~(2+)内流增加,二者均可导致细胞内Ca~(2+)超载,从而增强细胞对缩血管物质的敏感性,引起血管收缩。
但目前对缺氧所致的血管损伤的研究多集中在肺和肾小血管的缺氧性损伤,认为缺氧引起的肺血管收缩主要与体液因素、交感神经作用及对血管平滑肌的直接作用有关。本实验拟在大鼠脑基底动脉,通过观察缺氧前后血管直径的变化,探讨缺氧对脑血管收缩的影响以及Na~+, K~+- ATPase在其中的作用。
目的:采用大鼠离体血管基底动脉,观察缺氧前后血管直径的变化以及Na~+, K~+-ATPase抑制剂对其影响,从而探讨Na~+, K~+-ATPase是否参与缺氧诱发的脑血管收缩。
方法:取大鼠,腹腔注射10%水合氯醛,待动物深度麻醉后快速断头取脑,将脑组织连同基底动脉一并浸泡于预冷的(4℃)PSS液中(浓度mmol/L: NaCl 119, KCl 4.7, CaCl_2 2.5, MgSO_4·7H_2O 1.17, NaHCO_3 25, KH_2PO_4 1.18, ECTA 0.027, Glucose 5.5),去除软脑膜,分离出基底动脉。选取约5~8mm无分枝的一段血管,置于装满4℃PSS液的微血管直径测定仪器浴槽中,将血管一端固定在浴槽中与压力感受器P1相连的玻璃电极上,冲洗血管内腔,再用同样的方法将血管另一端固定在浴槽中与压力感受器P2相连玻璃电极上。该电极连有一个阻力传感器,可以读取血管纵轴方向的阻力。浴槽底部连有成像系统,血管直径和血管两端压力的变化情况通过myoview acquisition软件处理后可显示在微机上(如Fig.1,Fig.2所示)。开启加热系统,将浴槽内温度缓慢升高到37±0.5℃,并持续通以95%O_2~+5%CO_2混合气体,分别记录灌流PSS液、缺氧PSS液、K-PSS液、缺氧K-PSS液、Oua-PSS液、缺氧Oua-PSS液、Oua-K-PSS液、缺氧Oua-K-PSS液、无钾PSS液及缺氧无钾PSS液后5、10、15、30和60min时血管血管直径的变化。
结果:
1血管稳定性研究
在无外界刺激下,本研究所用大鼠基底动脉血管直径在持续灌流5min、10min、15min、30min和60min后无明显改变(P>0.05),且间隔1h先后两次给予高K-PSS液后,血管收缩后的相对直径(RD)分别为:58.09±0.53和58.49±0.74,二者之间也无显著性差别(P>0.05),提示在本实验条件下,该基底动脉的反应活性至少可以稳定60min。
2缺氧对基底动脉直径的影响
缺氧灌流5 min后,基底动脉即出现收缩,可使血管直径由151.33±23.00μm缩小至146.17±23.17μm,其变化幅度虽然仅有3.52%(从100.0%降至96.40%),却已具有统计学显著意义(P<0.05),提示缺氧可引起脑血管收缩。当缺氧灌流15 min时此收缩达到高峰(140.33±21.00μm,92.76),再继续缺氧灌流,无论30 min或60 min,均未见血管进一步收缩(P>0.05)。
3缺氧对高K~+预刺激基底动脉直径的影响
用高K-PSS液灌流大鼠基底动脉,可使其产生明显收缩,血管直径可由132.00±20.80μm缩小至107.41±19.04μm,其变化幅度为18.88%(P<0.01)。在此基础上再给予无氧高K-PSS液灌流,缺氧5分钟即可使基底动脉进一步收缩,且收缩幅度随着缺氧时间的延长而进一步加大。但缺氧对正常或高K~+预刺激基底动脉的直径变化幅度的影响并无显著区别(P>0.05)。
4哇巴因对基底动脉直径的影响
哇巴因为Na~+,K~+-ATP酶的抑制剂,在有氧情况下,哇巴因(10~(-9)～10~(-4) mol/L)可浓度依赖性引起基底动脉收缩,10~(-8) mol/L即可显著缩小血管直径的变化幅度(P<0.01))。但10~(-6)、10~(-5)和10~(-4) mol/L三个剂量收缩血管的幅度与5×10~(-7)mol/L相比并无明显差异(P>0.05),提示哇巴因5×10~(-7)mol/L已使血管达最大收缩(69.98±8.45)。但将此有氧的含哇巴因PSS液改用无氧的含相应浓度哇巴因PSS液灌流时,缺氧仍可使10~(-8)和10~(-7) mol/L哇巴因预刺激的基底动脉进一步收缩(P<0.01),而对5×10~(-7)mol/L哇巴因预刺激已达最大收缩的血管直径则无明显影响(P>0.05),提示缺氧诱发的脑血管收缩可能与Na~+,K~+-ATP酶的抑制程度有关。
5哇巴因对高K~+预刺激基底动脉直径的影响
用有氧的含10~(-8) mol/L和10~(-7) mol/L哇巴因的高钾PSS液灌流血管,可使基底动脉直径由原来的189.00±8.00μm和199.33±12.89μm收缩到108.50±7.00μm和94.6±5.56μm,其变化幅度分别为42.62%和52.35%(P<0.01);但将此有氧的含哇巴因的高钾PSS液改用无氧的含相应浓度哇巴因的高钾PSS液灌流时,则缺氧不再使10~(-8)和10~(-7) mol/L哇巴因和高钾预刺激的基底动脉进一步收缩(P>0.05),提示高K可提高Na~+,K~+-ATP酶对哇巴因的亲和力,使10~(-8)和10~(-7) mol/L的哇巴因即可完全抑制Na~+,K~+-ATP酶而使血管达到最大收缩,进一步提示缺氧诱发的脑血管收缩可能与Na~+,K~+-ATP酶的抑制程度有关。
6无钾PSS液对基底动脉直径的影响
无钾PSS液可取消Na~+,K~+-ATP酶的活性。用含氧无钾PSS液灌流5 min后,基底动脉即达最大收缩,可使血管直径由194.20±19.84μm缩小至113.60±17.92μm,其变化幅度为41.74%±3.23%(P<0.01);改用无氧无钾PSS液灌流后,基底动脉仍在5 min内即达最大收缩,变化幅度为41.53%±3.84%(P<0.01),与前者相似。这些结果进一步提示,Na~+,K~+-ATP酶参与了缺氧诱发的脑血管收缩。
结论:缺氧可引起脑血管收缩,脑血管上的Na~+,K~+-ATP酶可能参与了这种缺氧诱发的脑血管收缩。其机制可能与缺氧导致的Na~+,K~+-ATPase活性降低有关。
Cerebral ischemia, as a common disease, with characteristic of high morbidity and mortality rate, is hazardous to human being’s health. To maintain normal metabolism, the oxygen requirement of brain tissue is very huge, but its capability of anaerobic metabolism and oxygen reserve is small. The energy almost comes from aerobic metabolism of glucose. If cerebral ischemia happened, oxygen in cerebral vascularture can be used for only seconds, the store of glucose in brain can maintenants for only minutes, and aerobic metabolism is converted to anaerobic metabolism and the production of ATP in the ischemic cell is only 1/18 in the non-ischemic cells. So that, cerebral ischemia is always followed by low energy metabolism, high level of free radical and excitatory amino acids, overload of intracellular Ca~(2+), and destruction of cell structure.
So far, Na~+, K~+-ATPase is the only one receptor of cardiac glycosides drugs. Na~+, K~+-ATPase in brain tissue exists in the synaptic membrane (SPM) and microvascular endotheliocyte (CMV). Na~+,K~+-ATPase has an important role in the maintenance of the vascular tone and vascular contraction. When cerebral ischemia happened, the activity of Na~+, K~+-ATPase is reduced, the ion balance inside and outside of cells is broken, and the extracellular K~+ concentration ([K~+]O) increases quikly in very short time. In turn, The high [K~+]O can make cell depolarization, intracellular sodium concentration ([Na~+]i) and intracellular calcium concentration ([Ca~(2+)]i) are increased by activating Na~+-Ca~(2+) exchanger or openning voltage-dependent Ca+ channels. This extra calcium increases the sensitivity of intracellular pool to vasoconstrictors and makes vessel contraction.
Nevertheless previous studies about vascular injury induced by ischemia are focused on small vessels in lung and kidney. The contracted mechanism of lung small vessels concern three factors: direct action of ischemia on the vascular sooth muscle, humoral factor and sympathetic nerve. The present study is to explore the effects of anoxia on vascular contraction and the effects of Na+,K+-ATPase in the anoxic vasoconstriction by observing the changes of the basilar artery diameter before and after anoxia.
Objective: To investigate the effects of anoxia on the vasoconstriction in isolated rat basilar arteries and the effects of Na~+,K~+-ATPase inhibitor ouabain (oua) in the anoxic vasoconstriction by observing the changes of the basilar artery diameter before and after anoxia in order to explore if Na~+,K~+-ATPase involves in the hypoxia response.
Methods: Animals are anesthetized with the overdose of chloral hydrate (10%, 3.5 ml/Kg) and the brain is quickly removed and placed in a chamber containing cold physiologic salt solution (PSS) (mmol/L: NaCl 119, KCl 4.7,CaCl_2 2.5,MgSO_4·7H_2O 1.17,NaHCO_3 25,KH_2PO_4 1.18,ECTA 0.027,Glucose 5.5). Microvessels of the desired size are dissected free under a stereo microscope and transferred to a temperature- controlled chamber with cold PSS located on an inverted microscope. The one end of the vessel was carefully cannulated on the right side glass cannula connected to P1 transducer and the lumen of the mounted vessel was gently flushed with PSS to remove any blood or debris. Then the other end of the vessel was carefully cannulated on the left side glass cannula connected to P2 transducer. And the normal PSS was set the temperature at 37℃and gassed with 95% O_2 and 5% CO2 mixtrue. The isolated basilar artery was perfused with PSS, K-PSS, oua-PSS, oua-K-PSS, or K-free-PSS under normal or anoxia condition and recorded the diameter changes in 5, 10, 15, 30 and 60 minutes after perfusion.
Result:
1 The stability of isolated rat basilar artery
There were no change in the diameters of isolated rat basilar artery at 5, 10, 15, 30 and 60 minutes after perfusion with normal PSS (p>0.05). There were no significant difference in diameter changes stimulated by two perfusions of high K- PSS in the interval of 60 minutes, and the relative diameters after contraction was 58.09±0.53 and 58.49±0.74, respectively. These results indicate that the activity of the vessels is stable under our experiment condition.
2 Effects of anoxia on the diameter of rat basilar artery
Perfused with normal PSS in anoxia condition, the rat basilar artery started to contract in 5 minuters and the diameter was reduced from 151.33±23.00μm to 146.17±23.17μm. Although the change percentage of vessel diameters was only 3.52%, there is a significant differentce between normal (100%) and anoxia (96.40%±1.41%), suggesting that anoxia could induce a cerebral vasoconstriction. And this contraction reached the maximal in 15 minuters (p<0.01), but did not reveal the further contraction at 30 and 60 minute after continuate perfusion in anoxia condition (p>0.05).
3 Effects of anoxia on the diameter of rat basilar artery suffered from high K-PSS pretreatment
Perfused with high K-PSS, basilar arteries were contracted obviously, their diameter was constricted from 132.00±20.80μm to 107.41±19.04μm, and their change percentage was 18.88%. But perfused with K-PSS in anoxia condition, basilar artery was further constricted in 5 minutes (p<0.05), and contracted extent was increased as the anoxia time was elonged. However, there was no significant difference in the change percentage of basilar artery diameter prestimulated by high K-PSS between normal and anoxic condition (p>0.05).
4 Effects of oubain on the diameter of rat basilar artery Ouabain is an inhibitor of the Na~+,K~+-ATPase. In normal condition, ouabain (10~(-9)～10~(-4) mol/L) could induce the contraction of rat basilar artery in a concentration-dependent manner and decrease the change percentage of vessel diameter as lower as 10~(-8) mol/L (p<0.01). But there is no significant difference between 10~(-6),10~(-5),or 10~(-4) and 5×10~(-7) mol/L of ouabain (p>0.05), suggesting that ouabain 5×10~(-7) mol/L has produced a maximal vasocontriction (69.98±8.45). However, in anoxic condition, the rat basilar arteries preconstricted by 10~(-8) and 10~(-7) mol/L ouabain were still further constricted by anoxia, but those preconstricted by 5×10~(-7) mol/L ouabain, which had reached the maximal contraction, did not reveal any change in diameter (p>0.05), indicating that the cerebral vasocontriction induced by anoxia may related to the degree of the Na~+,K~+-ATPase inhibtion.
5 Effects of oubain on the diameter of rat basilar artery suffered from high K-PSS pretreatment
In normal condition, 10~(-8) and 10~(-7) mol/L ouabain could decrease the diameter of rat basilar arteries pretreated by high K-PSS from 189.00±8.00μm and 199.33±12.89μm to 108.50±7.00μm and 94.60±5.56μm, and their change percentage was 42.62% and 52.35%, respectively. However, in anoxic condition, the rat basilar arteries preconstricted by 10-8 and 10~(-7) mol/L ouabain and by high K-PSS were not further constricted by anoxia (p>0.05). These results indicate that the high K~+ could increase the affinity of the Na~+,K~+-ATPase binding to ouabain, which make ouabain reach a maximal inhibition of the Na~+,K~+-ATPase and a maximal vasoconstriction in 10~(-8) and 10~(-7) mol/L, and further suggesting the cerebral vasocontriction induced by anoxia may related to the degree of the Na~+,K~+-ATPase inhibtion.
6 Effects of K~+-free PSS on the diameter of rat basilar artery K~+-free PSS could completely abolish the activity of the Na~+,K~+-ATPase. In normal or anoxic condition, the rat basilar arteries could reach the maximal contraction in 5 minuters and the diameters were reduced from 194.20±19.84μm to 113.60±17.92μm and 114.00±18.40μm, and their change percentages were similar, 41.74%±3.23% and 41.53%±3.84%, respectively (P<0.01). These results further suggest that the Na~+ , K~+-ATPase involves in the cerebral vasoconstriction induced by anoxia.
Conclusion: Anoxia results in cerebral vasocontriction. Na~+,K~+-ATPase situated in cerebral vessels involes in anoxic cerebral vasoconstriction. Its mechanism may relate to the activity of the Na+,K+-ATPase lowered by anoxia.
引文
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