大鼠坐骨神经慢性压迫后脊髓背角GCH1基因表达与疼痛的关系

目的:探讨大鼠坐骨神经慢性压迫(chronic constriction injury,CCI)后,脊髓背角内三磷酸鸟苷环化水解酶1 (GTP cyclohydrolase 1,GCH1)基因的表达变化与神经病理性疼痛产生的关系.方法:wistar大鼠64只被随机分为CCI组和Sham组,每组32只.对CCI组大鼠行右侧坐骨神经结扎;建立CCI模型,Sham组仅暴露坐骨神经不结扎,于术前1天及术后1、3、7、10、14、21、28天测定大鼠机械痛阈值,并分别在术后3、7、14、28天职腰4～6段脊髓,进行免疫组化和Western Blotting检测,观察脊髓背角GCH1表达的变化情况.结果:CCI组大鼠术后各时间点后肢机械痛阈值均显著低于Sham组(P＜0.05),术后第1天开始疼痛阈值便明显降低,直至术后第28天,均低于术前水平(P＜0.05).免疫组化结果显示:CCI组大鼠术后脊髓背角神经元GCH1的表达呈先升高后降低的趋势;与Sham组相比,术后各时间点GCH1的表达均明显增高(P＜0.05).Western Blotting检测与免疫组化结果基本一致,CCI组术后3天、7天、14天GCH1表达均高于Sham组(P＜0.05).结论:周围神经损伤引起的痛觉过敏随脊髓背角内GCH1表达升高而增强,随其表达降低而减轻,可以认为GCH1可能参与了神经病理性疼痛的形成.     

鞘内注射GDNF对神经病理性疼痛大鼠脊髓水平GAD65表达的影响

目的:研究神经病理性疼痛大鼠脊髓背角谷氨酸脱羧酶65(Glutamate decarboxylase65,GAD65)的表达变化及蛛网膜下腔注射胶质细胞源性神经营养因子(glial cell line-derived neurotrophic factor,GDNF)对其表达的影响。方法:实验1:雄性SD大鼠随机分为假手术组(sham组)和坐骨神经结扎组(CCI组)。CCI组又分为术后1 d(D1)、3 d(D3)、7 d(D7)和14 d(D14)组。实验2:雄性SD大鼠随机分为:sham组、D14组、D14+GDNF组和D14+PBS组。两实验分别于实验前及CCI术后第1、3、7、14 d测定热缩足潜伏期(thermal withdrawal latency,TWL)及机械缩足阈值(mechanical withdrawal threshold,MWT),处死后取其L4~L5脊髓背角,采用Western blot方法测定GAD65蛋白表达情况。结果:与sham组相比,CCI组大鼠TWL及MWT均降低,GAD65表达水平于术后3 d开始降低,一直持续到术后14 d;与D14组和D14+PBS组相比,D14+GDNF组大鼠TWL及MWT均升高,GAD65表达水平升高。结论:神经病理性疼痛的发生机制可能和大鼠脊髓背角内GAD 65表达降低相关,而GDNF对其镇痛作用可能部分是通过增加脊髓GAD65的表达实现的。     

神经病理性疼痛大鼠脊髓Ⅱ层突触形态结构的变化

目的:观察神经病理性疼痛大鼠脊髓背角Ⅱ层突触形态结构的可塑性,探讨神经病理性疼痛的发病机制。方法:SD大鼠15只,随机分为正常组、假手术组和模型组各5只,采用坐骨神经慢性压迫性损伤(CCI)建立大鼠神经病理性疼痛模型。应用透射电镜观察和体视学定量分析脊髓Ⅱ层突触形态结构的参数变化。结果:与正常组及假手术组比较,模型组大鼠脊髓背角Ⅱ层的突触后致密物质增厚(P<0.05),突触间隙增宽及突触界面曲率明显增大(P<0.05),凹型棘突触的数密度显著增高(P<0.01),总突触、棘突触和穿孔性突触的数密度增高明显(P<0.05)。结论:脊髓Ⅱ层突触形态结构可塑性变化可能参与神经病理性疼痛的发生。     

脉冲射频对坐骨神经慢性压迫模型大鼠的痛觉过敏及脊髓背角NR2B亚基表达的作用

目的观察脉冲射频(PRF)对坐骨神经慢性压迫(CCI)后神经病理性疼痛模型大鼠疼痛行为学、损伤坐骨神经结组织病理学的影响,检测脊髓背角NR2B亚基的表达。方法 48只Sprague-Dawley大鼠随机等分为Sham-Sham(SS)组、Sham-PRF(SP)组、CCI-Sham(CS)组和CCI-PRF(CP)组。CS、CP组大鼠结扎右侧坐骨神经干,制作CCI模型;SS、SP组只分离坐骨神经而不结扎。术后15 d,CP、SP组于坐骨神经干结扎处行PRF,SS、CS组仅放置电极针不通电。造模前,造模后3 d、7 d、11 d、15 d,治疗后1 d、3 d、7 d、11 d、15 d测量大鼠右后足机械缩足阈值(HWT);治疗后15 d光镜下行右侧坐骨神经干评分;Western blotting测定L4-6脊髓背角中NR2B亚基蛋白表达。结果造模后CS、CP组HWT较SS、SP组降低;治疗后,CP组HWT高于CS组。光镜下CP组较CS组神经轴索直径、髓鞘厚度明显增加(P0.01)。CP组大鼠脊髓背角NR2B表达低于CS组(F=10.769,P0.05)。结论PRF能降低CCI模型脊髓背角NR2B亚基表达,修复受损神经,降低机械痛敏。     

鞘内注射GDNF对神经病理性疼痛大鼠脊髓水平p-Shc表达的影响

目的:探讨神经病理性疼痛大鼠脊髓背角水平p-Shc的表达变化及鞘内注射胶质细胞源性神经营养因子(glial cell line-derived neurotrophic factor,GDNF)对其表达的影响。方法:实验1:雄性SD大鼠随机分为假手术(sham)组和CCI组。CCI组又分为术后1 d组(D1组)、3 d组(D3组)、7 d组(D7组)和14 d组(D14组),分别于术后1、3、7、14 d测定热缩足潜伏期(thermal withdraw latency,TWL)和机械缩足阈值(mechanical withdrawal threshold,MWT)后处死并取其脊髓背角,采用Western blot方法测定Shc/p-Shc蛋白表达情况。实验2:雄性SD大鼠分为sham组、D14组、D14+PBS组、D14+GDNF组。与实验1相同的时间点测定TWL和MWT,并于第14 d处死并取其L4~5脊髓背角,采用Western blot方法检测Shc/p-Shc的表达变化。结果:与sham组相比,CCI组大鼠TWL和MWT明显降低(P<0.01);与D14组和D14+PBS组相比,D14+GDNF组大鼠TWL和MWT明显升高(P<0.01)。CCI组脊髓背角内p-Shc表达水平于术后3 d开始升高(P<0.05),一直持续到术后14 d(P<0.01);D14+GDNF组p-Shc表达水平较D14组和D14+PBS组显著下降(P<0.05)。结论:大鼠脊髓背角内接头蛋白Shc可能参与了神经病理性疼痛的发生,鞘内注射GDNF减轻神经病理性疼痛可能与降低脊髓背角Shc的磷酸化水平有关。     

鞘内注射右美托咪定对坐骨神经慢性缩窄性损伤大鼠脊髓背角肿瘤坏死因子α蛋白表达的影响

目的探讨鞘内注射右美托咪定（ DEX）对坐骨神经慢性缩窄性损伤（ CCI）大鼠脊髓背角肿瘤坏死因子α（ TNF-α）蛋白表达的影响。方法成年Sprague-Dawley雄性大鼠44只随机分为四组（ n＝11）：假手术组（ sham组）,坐骨神经慢性缩窄性损伤组（ CCI组）,生理盐水组（ CCI＋NS组）和右美托咪定组（ CCI＋DEX组）。采用von Frey纤毛分别测定了sham组和CCI组术前及术后1、3、5、7、14 d,以及CCI＋NS组和CCI＋DEX组术后第7天鞘内给药前和给药后1、2、3 h的机械刺激抬腿反应阈值（ PWT）。 sham组和CCI组在术后第7天,CCI＋NS组和CCI＋DEX组在术后第7天鞘内给药后1 h时间点分别余5只大鼠处死取脊髓背角,采用Werstern blot法测定TNF-α蛋白表达。结果与sham组相比,CCI组PWT自术后第3天开始出现下降,并持续至第14天（ P＜0.001）;与CCI＋NS组相比,CCI＋DEX组在给药后1 h即出现明显镇痛作用,且持续至给药后2 h（P＜0.01或P＜0.001）。 Werstern blot结果显示,和sham组比较,CCI组和CCI＋NS组大鼠脊髓背角TNF-α蛋白表达上调（ P＜0.05）;与CCI＋NS组比较,CCI＋DEX组TNF-α蛋白表达下调（ P＜0.05）。结论鞘内注射右美托咪定可通过抑制CCI大鼠脊髓背角TNF-α蛋白表达减轻神经病理性疼痛。     

腹腔注射右美托咪啶对神经病理性疼痛大鼠镇痛的实验研究

目的:评价右美托咪啶对神经病理性疼痛大鼠行为学、痛敏及脊髓背角神经元磷酸化胞外反应激酶(phosphorylated extracellular regulated protein kinases,p ERK)、磷酸化c AMP反应元件结合蛋白(phosphorylated c AMP response element bound protein,p CREB)、c-fos蛋白表达的影响。方法:健康成年雄性Wistar大鼠54只,6~8周龄,体重180~220 g,采用随机数字表法,将其分为3组(n=18):假手术组(S组)、慢性神经病理性疼痛组(C组)和右美托咪啶组(D组)。S组仅分离坐骨神经但不结扎,C组和D组采用结扎坐骨神经的方法制备大鼠坐骨神经慢性压迫性损伤(chronic constriction injury,CCI)的神经病理性疼痛模型,D组于术后即刻开始至处死前1d腹腔注射右美托咪啶50μg/kg,1次/d,S组和C组注射等容量生理盐水。于术前1 d、术后3、7、14 d时以缩足阈值(paw withdrawal threshold,PWT)测定大鼠机械痛阈和辐射热的缩足潜伏期(paw withdrawl latency,PWL)测定大鼠的热痛阈,并于术后测定痛阈后灌注处死大鼠,取L4~6脊髓组织,采用免疫组织化学法检测脊髓背角神经元p ERK、p CREB、c-fos的表达水平。结果:与S组比较,C组和D组术后3、7、14 d时MWT降低,TWL缩短,脊髓背角p ERK、p CREB、c-fos表达上调(P<0.05);与C组比较,D组术后3、7、14 d时MWT升高,TWL延长,脊髓背角p ERK、p CREB、c-fos表达下调(P<0.05)。与术前1 d比较,C组和D组术后3、7、14 d时MWT降低,TWL缩短;与术后3 d时比较,C组和D组7、14 d时MWT降低,TWL缩短,脊髓背角p ERK、p CREB、c-fos表达上调(P<0.05)。结论:右美托咪啶可减轻大鼠慢性神经病理性疼痛,抑制p ERK、p CREB、c-fos的表达可能是其作用机制之一。     

鞘内注射银杏内酯B对神经病理痛大鼠痛阈及脊髓c-fos表达的影响

目的:观察鞘内注射银杏内酯B(BN52021)对SNI神经病理痛大鼠痛阈及脊髓c-fos表达的影响,探讨脊髓血小板活化因子及其受体参与痛觉信号调节的可能机制.方法:鞘内置管后的SD大鼠24只随机等分为4组:假手术组(sham组),SNI组,SNI+DMSO对照组和SNI+BN52021组,建立SNI疼痛模型,手术后1,3,5,7,10和14d鞘内给药并测痛阈,第14d取大鼠腰段脊髓,冰冻切片,免疫组织化学染色检测Fos免疫阳性细胞.结果:SNI神经损伤大鼠机械缩爪阈明显降低(P<0.05),同侧脊髓背角浅层内Fos阳性神经元明显增多(P<0.05);鞘内应用银杏内酯B明显减少脊髓背角神经元c-fos的表达,同时伴有大鼠机械异常痛敏的减轻,各组大鼠辐射热缩爪潜伏期无明显差异.结论:鞘内注射银杏内酯B可减轻SNI大鼠机械异常痛敏,抑制神经损伤后脊髓背角c-fos的表达.     

SNI模型大鼠脊髓背角N-甲基-D-天冬氨酸受体和降钙素基因相关肽的表达

【目的】观察坐骨神经分支选择结扎切断模型(Spared nerve injury model,SNI)痛阈及脊髓背角N-甲基-D-天冬氨受体(NMDAR)、降钙素基因相关肽(CGRP)表达的变化。【方法】健康雄性SD大鼠27只,体重220~280 g。随机分为3组(n=9)。对照组(C组);假手术组(sham组);坐骨神经分支选择结扎切断模型组(SNI组)。在模型制作前1 d和模型制作后14 d内,每组均进行疼痛行为学观察。所有组别均在模型制作后d2、d7、d14天观察行为学改变后分批取材(每批3只),用免疫组化法观察大鼠L5节段水平脊髓背角NMDAR和CGRP的表达。【结果】与C组和sham组比较,SNI组在SNI术后均出现机械性异常疼痛痛阈降低(P<0.01),但热刺激后爪退缩潜伏时间无统计学意义(P>0.05);同时SNI组大鼠脊髓背角NMDAR和CGRP免疫阳性产物数量和表达增加(P<0.01)。【结论】SNI模型大鼠出现典型的疼痛行为学特征时,其脊髓背角NMDAR和CGRP表达也明显增强,并且脊髓背角NMDAR和CGRP的变化趋势大体相同。     

加巴喷丁联合吗啡对神经病理性疼痛大鼠脊髓胶质原纤维酸性蛋白表达的影响

目的:观察加巴喷丁联合吗啡镇痛对坐骨神经慢性压迫(CCI)大鼠脊髓背角胶质原纤维酸性蛋白(GFAP)表达的影响。方法:选择重量在180～220g的成年雄性SD大鼠24只,随机分为4组(n=6):假手术组(S组)、模型组(M组)、预防性镇痛组(P组)和常规镇痛组(N组)。M组、P组和N组采用CCI建立神经病理性疼痛模型,P组在术前1d至手术后9d使用加巴喷丁联合吗啡镇痛,N组在手术当日至手术后第9天加巴喷丁联合吗啡镇痛,S组和M组仅给予生理盐水。分别于手术后第3、5、7、9天测定各组50%机械刺激缩足阈值,手术后第10天采用免疫组化测定脊髓GFAP表达。结果:与M组相比,P组和N组50%缩足阈值升高,同时脊髓背角GFAP表达降低(M组:0.6237±0.04903,P组:0.4616±0.03890,N组:0.5215±0.02691)。结论:加巴喷丁联合吗啡可以减轻CCI神经病理性疼痛和脊髓背角GFAP的表达。     

ON THE PHYSICOCHEMICAL REGULATION OF THE GROWTH OF TISSUES : THE EFFECTS OF THE DILUTION OF THE MEDIUM ON THE GROWTH OF THE SPLEEN.

It may be concluded that the degree of dilution of the culture medium has a marked influence on the rate of growth of splenic tissue. The maximum acceleration was obtained in a medium composed of three volumes of normal plasma and two volumes of distilled water. The growth in this hypotonic plasma was very much larger than in normal plasma. On the contrary, the growth of the spleen in hypertonic plasma was always less than in normal plasma. In other experiments, we found that in diluted plasma there was also an acceleration of the growth of the skin, the heart, and the liver of chickens. The skin of adult frogs also grew more actively in this plasma. The optimum degree of dilution varied according to the nature of the tissues and to the species of the animals. While the plasma containing two fifths distilled water produced the largest growth of splenic tissue, a slightly less diluted medium was more favorable for the liver and the heart, and generally for the skin also. The action of hypertonic plasma varied also in a large measure. While the spleen did not grow at all in the medium containing 0.0124 and 0.0144 sodium chlorid, the skin, on the other hand, could stand a high concentration of the sodium chlorid. Even its growth was activated in media containing 0.0094 and 0.0124 sodium chlorid and was greater than with normal plasma. The spleen of kittens was very easily affected by the changes of the dilution of the plasma, while the skin of the frog presented its best growth in plasma containing one half distilled water. Marked variations in the sensitiveness of tissues to hypertonic and hypotonic media will probably be observed in animals of different species. From these experiments, three conclusions can be drawn: namely, that certain laws of growth, discovered by Loeb, in lower organisms are true also for higher organisms; that normal plasma is not the optimum medium for the growth of tissue; and that each tissue has probably its optimum medium. The growth of the spleen is, without doubt, considerably modified by the variations of the dilution and perhaps of the osmotic tension of the plasma. It is possible then that the influence of osmotic tension, discovered by Loeb, in the growth of certain organisms, is a general law applicable as well to higher forms of life— frogs, cats, and chickens—as to lower organisms—tubularia and sea-urchins. In placing tubularia in different dilutions of sea-water and distilled water, Loeb found that the greatest rate of regeneration was observed when two volumes of distilled water were added to three volumes of sea-water. But fertilized eggs of sea-urchins were more sensitive to the action of hypertonic plasma, and they all died in a dilution of sea-water with two fifths distilled water. If only one fifth distilled water was added to the sea-water they developed normally. We found that the cells of certain tissues of the chicken follow a similar rule, since the maximal growth of the spleen is obtained in plasma containing two fifths distilled water, while other tissues grow better in a less hypotonic medium. Normal plasma is certainly not the ideal medium for the growth of tissues, since slight modifications of the tension, the alkalinity, or the addition of certain inorganic salts to normal plasma, increase the rate of the growth of tissues.     

THE PATHOGENESIS OF THE HEMORRHAGIC DISEASES OF THE NEW-BORN

Those conditions of the new-born characterized by a hemorrhagic tendency, icterus, and fatty changes, are probably all syndromes which may occur as the result of a number of toxic agents. All of them, however, have been produced, in these experiments, by the action of a single experimental agent. Thus, a picture indistinguishable from that called Buhl''s disease has been obtained by the use of chloroform, as have also the pictures known as Winckel''s disease, melæna neonatorum, etc. Chloroform is not held to be the only substance that has this power. It stands rather as one member of a group of agents, the effects of which in general and in individual organs are similar to those caused by lack of oxygen. The essential features of these conditions have also been produced by direct asphyxiation of the fetus. The suggestion is therefore made that underlying all these symptoms and pathological complexes, there is a deficiency of oxidation, general, local, or selective, thus bringing this group of diseases into the general category of acute yellow atrophy of the liver, eclampsia, pernicious vomiting, cyclic vomiting, phosphorus poisoning, etc. In human beings, chloroform and asphyxia must, in many instances, be the determining causes. There remain, however, other cases in which different factors are to be sought.     

THE INDUCED SUSCEPTIBILITY OF THE GUINEA-PIG TO THE TOXIC ACTION OF THE BLOOD SERUM OF THE HORSE

Following the divisions before used, the results presented in the preceding pages may be briefly stated. I. The particular method of sensitization and the place where the test injection is made have an important bearing on the results obtained by various workers. Comparing the results obtained by the various methods, we may conclude that the incubation period of the hypersensitive reaction is not sharply limited, but that there is a progressive increase in sensitiveness from the sixth day, and presumably before that, extending over a period of several weeks. It seems very probable that the degree of hypersensitiveness attained where the sensitizing dose consists of a mixture of diphtheria toxin and serum is greater than when a single dose of the same small quantity of serum is given alone. II. Our early experiments, the first in this field, are in thorough agreement with those first reported by Otto, and shortly after him by Rosenau and Anderson. III. This hypersensitive reaction is transmissible from mother to offspring. The transmission is probably not equally effective in all cases, and individual young guinea-pigs probably vary greatly in the rate with which they lose their ability to react. As a result not all of the young of a hypersensitive mother react to a subcutaneous dose of five cubic centimeters of serum given when they are four or five weeks old. The reaction in the young animals differs quite markedly from that in those actively sensitized. These differences are such as to indicate that in the mother there is a considerable localization of the reaction in tissues and organs whose destruction does not cause sudden death. This local reaction is a protective factor and is not transmitted to the same degree as the factors involved in the fatal acute reaction. IV. The hypersensitive reaction to horse serum depends on the development of a special anti-body during the incubation period, which anti-body may be passively transferred to a fresh animal. If the dose of hypersensitive serum be sufficient, and the intoxicating injection be given directly into the circulation, this passive hypersensitiveness may be enough so that the animal will die when tested. There is also in the serum of hypersensitive guinea-pigs an uneliminated horse serum element or "rest," which is distinct from this antibody, and probably without influence on the course of the acute reaction. V. The anti-body on which the hypersensitive reaction depends may be entirely neutralized by horse serum without causing symptoms. The gradual introduction of increasing doses over a total period of twenty-four hours suffices for this. The animal is then, properly speaking, neither immune nor refractory, but is essentially in the condition of a normal animal which has recently had a large dose of horse serum. This rapid neutralization is made possible by the great binding power which the subcutaneous and other relatively unimportant tissues have for the toxic element of the serum. The so-called "Phenomenon of Arthus" is probably the same reaction for the rabbit that we have here dealt with in the guinea-pig. The fact that the manifestation is more prominently a local one depends on racial differences. I have encountered cases in the guinea-pig in which the conditions in the rabbit are closely simulated.     

THE EXTENT OF THE CAPILLARY BED OF THE HEART

By means of injections made into the coronary arteries of beating hearts it has been possible to determine the number of capillaries in the normal heart muscle. This study has shown a very rich blood supply with an average of approximately one capillary for each muscle fibre in the ventricular walls and papillary muscles, and a less abundant supply in the auricular muscle and Purkinje system. The number of capillaries per sq. mm. of ventricular wall or papillary muscle is about twice that found by Krogh in skeletal muscle. Capillaries were not found constantly in the valves of hearts in which there was apparently a complete injection of the capillary bed. The method described for injecting the capillaries of the heart also provides a means of studying the blood supply to the muscle, valves and aortic wall in pathological hearts.     

THE INFLUENCE UPON THE SPLEEN AND THE THYROID OF THE COMPLETE REMOVAL OF THE EXTERNAL FUNCTION OF THE PANCREAS

The complete removal of the function of the pancreas concerned in digestion is followed by marked changes in the spleen and in the thyroid apparatus. Second, the spleen shows an extreme simple atrophy. Third, the thyroid apparatus exhibits a constant change shown by the macroscopic transparency of the gland, by the microscopic increase in the amount of colloid, by the chemical increase of the iodine content of the gland, and by the functional test of the delayed appearance of tetany after the complete removal of the thyroid apparatus.     

学生健康体检结果分析

在校学生进行健康体检,对常见病患病率进行统计分析,提议在体检 中应加入血液流变学指标。