慢性坐骨神经结扎神经痛大鼠脊髓nNOS阳性神经元的表达

目的:探讨慢性坐骨神经结扎(CCI)神经病理性疼痛大鼠脊髓背角nNOS阳性神经元的表达。方法:SD大鼠40只,随机分为五组,每组8只,即naive组、sham组、CCI5d组、CCI10d组;CCI15d组;测定各组大鼠机械缩腿阈值(MWT)和热缩腿潜伏期(TWL);同时sham组大鼠在术后5d、CCI各组分别在术后5d、10d、15d断头处死取脊髓腰膨大,采用免疫组化法测定脊髓背角nNOS阳性神经元的表达。结果:CCI大鼠在手术后形成稳定的痛敏,与naive组和sham组大鼠比较有显著差异;naive组大鼠和sham组大鼠脊髓背角仅见少量nNOS阳性神经元的表达,且两组间无统计学意义;CCI各组大鼠脊髓背角nNOS阳性神经元的表达明显增多,与naive组和sham组比较差异显著。结论:慢性坐骨神经结扎(CCI)神经痛大鼠脊髓背角nNOS阳性神经元的表达增加,脊髓背角nNOS可能参与了CCI大鼠神经病理性疼痛的形成。     

尼莫地平对大鼠坐骨神经损伤后脊髓神经元的保护作用

目的:研究尼莫地平对大鼠坐骨神经损伤后脊髓神经元的保护作用。方法:将50只成年SD大鼠,随机分成4组:假手术组(A组,n=5);坐骨神经切断未干预组(B组,n=15);生理盐水组(C组,n=15);尼莫地平组(D组,n=15)。B,C及D3组又按切断右侧坐骨神经后4,9及16d取材时间分为3个时间组,每组5只大鼠,各时间组取大鼠的脊髓L5段,以TUNEL法检测细胞凋亡数,以免疫组化技术检测Bax的表达,苏木精-伊红染色计算脊髓内神经元的数目。结果:坐骨神经损伤后4,9及16d,C组凋亡细胞数目为(8.4±1.8),(13.1±2.1),(15.4±1.8)个/前角视野,明显多于D组(2.3±1.3),(8.2±1.7),(10.1±2.2)个/前角视野(P<0.01);D组神经元存活率为(94.3±3.1)%,(85.4±3.1)%,(76.3±3.2)%,明显高于C组(84.1±4.5)%,(77.5±2.9)%,(67.0±3.5)%,差异有非常显著性意义(P<0.01);D组Bax表达(-～+,+～,+～)低于C组(+,+～,);B与C组凋亡细胞数目、神经元存活率及Bax表达差异无显著性意义(P>0.05)。结论:尼莫地平可以减少坐骨神经损伤后脊髓神经元的凋亡及Bax的表达,因此,对脊髓神经元具有保护作用。     

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。     

异氟醚对甲醛致痛诱发大鼠脊髓背角P物质释放的影响

目的:研究异氟醚对甲醛疼痛模型大鼠脊髓背角P物质(SP)释放的影响。方法:SD大鼠16只,随机数字表法分为4组,每组4只。通过向右侧后肢跖部皮下注射40g/L甲醛150μL建立疼痛模型。A组、B组跖部皮下注射注射生理盐水,C组、D组注射甲醛,B组、D组在皮下注射前10min吸入15g/L异氟醚,并持续60min,A组、C组吸纯氧。麻醉大鼠,取脊髓L3～5节段。用SP免疫组化方法结合图像分析测定脊髓背角SP样免疫反应阳性(SPLI)纤维及终末的光密度(A)。结果:4组大鼠两侧脊髓背角SPLI的A值无显著差别(P>0.05,t=1.065);与A组脊髓背角SPLI的A值比较,B组无明显变化(P>0.05,t=1.987),C,D组显著小于A组(P<0.01,t=14.342,t=7.514),有明显的纤维和终末脱失趋势;D组SPLI的A值较C组显著增加(P<0.01,t=8.263)。结论:异氟醚不影响髓背角浅层SP含量,但能明显减轻甲醛引起的疼痛反应和脊髓背角SP的释放,部分阻断脊髓痛觉的传导。     

鞘内注射AIP对神经病理性痛大鼠的镇痛作用及脊髓背角神经元磷酸化CREB的影响

目的:探讨钙/钙调素依赖性蛋白激酶Ⅱ(calcium/calmodulin-dependent protein kinaseⅡ,CaMKⅡ)在神经病理性疼痛中的作用。方法:坐骨神经选择性损伤(SNI)大鼠模型,随机分为4组(n=8):SNI组,Sham组,NS组,AIP组,后两组分别于术前20 min鞘内注射10μl的生理盐水或10%的AIP。于术后1d、2d、3d、4d、6d、8d、10d、14d测定大鼠机械缩足反射阈值(mechanical with-drawal threshold,MWT)。分别于术后1d、3d、7d取大鼠脊髓腰段,用免疫组织化学法(n=6)检测脊髓背角磷酸化pCaMKⅡ的表达;Western blot法(n=4)检测脊髓背角神经元细胞核内pCREB的表达。结果:术后1~3d AIP组大鼠MWT较NS组明显升高(P<0.01);术前鞘内注射AIP在术后1d、3d能够使脊髓背角pCaMKⅡ的表达减少,同时脊髓背角神经元细胞核内pCREB的表达也明显减少。结论:CaMKⅡ参与了神经病理性痛的形成,其作用部分通过CREB介导。     

脊髓背角神经元凋亡在炎性痛大鼠吗啡耐受中作用研究

目的:探讨NMDAR及谷氨酸转运体(GT)在阿片耐受机制中的作用,以及脊髓神经元凋亡在慢性阿片耐受形成机制中的意义.方法:将60只健康雄性SD大鼠行鞘内置管后随机分为6组(n=10),空白对照组(A)于左后足踝关节腔注射生理盐水50μl,其他5组注射CFA 50μl.3d后分别经鞘内给予生理盐水10μl(A和B)、吗啡10μg(C组)、吗啡20μg(D组)、吗啡10μg+NMDA受体抑制剂MK-801 10nM(E组)、吗啡10μg+GT抑制剂PDC10μg(F组),1日2次,连续给药7 d.检测大鼠左后肢机械缩爪阈值评价其痛行为学,于给药后第7天取出左侧腰膨大处脊髓进行TUNEL染色和Western blotting检测.结果:C、D两组关节炎大鼠在鞘内给予吗啡第7天形成较稳定的吗啡耐受,MK-801和PDC分别对吗啡耐受的机械痛敏具有抑制和促进作用.B组大鼠脊髓背角凋亡细胞数及凋亡相关蛋白表达与A组比较,差异无统计学意义.与B组比较,C、D组大鼠脊髓背角的凋亡细胞数显著增加(P＜0.01),Bax和caspase-3蛋白表达上调(P＜0.01),Bcl-2表达下调(P＜0.01).与C组比较,E组大鼠脊髓背角凋亡细胞显著减少(P＜0.05),Bax和caspase-3蛋白表达下调(P＜0.01),Bcl-2表达上调(P＜0.05);F组凋亡细胞增多(P＜0.01),Bax和caspase-3蛋白表达上调(P＜0.01),Bcl-2表达下调(P＜0.01).结论:脊髓神经元凋亡可能是慢性阿片耐受的神经基础,NMDAR及GT在阿片耐受的神经细胞凋亡过程中发挥作用.     

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。     

大鼠坐骨神经慢性压迫损伤后脊髓感觉神经元发生非凋亡性细胞程序性死亡

目的:观察大鼠坐骨神经慢性压迫损伤后脊髓背角感觉神经元是否发生非凋亡性细胞程序性死亡(paraptosis).方法:成年雄性SD大鼠20只,随机分为正常组,手术组.手术组建立慢性压迫性损伤(CCI)模型,又分为术后3d、7d、14d组.实验动物以4%多聚甲醛PBS液灌注内固定,取与坐骨神经相应的腰4～腰6节段脊髓,于2.5%戊二醛中固定,透射电镜观察左侧脊髓背角浅层神经元超微结构并摄片.结果:电镜下,术后3d组腰4～腰6节段脊髓左侧背角浅层神经元形态学典型变化为:细胞肿胀,胞膜完整,胞浆内大量空泡,核结构基本正常,符合paraptosis形式程序性细胞死亡表现;7d和14d组神经元形态变化:细胞皱缩、密度增大,核带增多及染色质浓聚贴附于核膜,符合凋亡表现.结论:坐骨神经损伤3d腰4～腰6节段脊髓背角浅层神经元发生了paraptosis形式程序性细胞死亡.     

重复经颅磁刺激对神经病理性疼痛大鼠脊髓内星形胶质细胞的抑制作用

目的观察低频和高频重复经颅磁刺激（rTMS）对大鼠神经病理性疼痛的疗效及其对大鼠脊髓内星形胶质细胞标志物胶质酸性纤维蛋白（GFAP）的影响,探讨rTMS治疗神经病理性疼痛的机制。 方法28只雄性SD大鼠,随机分为假手术组、假治疗组、低频rTMS组(1Hz)、高频rTMS组(20Hz),每组7只。假手术组仅暴露游离大鼠坐骨神经,不予结扎;其余3组经手术结扎坐骨神经制作神经病理性疼痛模型。术后第3天开始进行rTMS治疗,连续10d,刺激疼痛对侧大脑初级运动皮质。并于造模前、rTMS治疗前和治疗后测定疼痛行为学表现、机械痛觉和热痛觉,并于治疗结束后测定腰段脊髓内GFAP的表达。 结果造模后3d,假治疗组、低频rTMS组、高频rTMS组大鼠均出现明显的疼痛行为学表现,热痛潜伏时较假手术组均明显降低(P<0.05)。rTMS治疗后,高频rTMS组热痛潜伏时较假治疗组升高(P<0.05),而低频rTMS组无明显变化。与假手术组比较,假治疗组和低频rTMS组损伤侧脊髓背角内GFAP阳性表达细胞数量和染色强度均明显增加(P<0.05)。与假治疗组比较,高频rTMS组脊髓背角GFAP的表达显著下调（P<0.05）,而低频rTMS无此改变。高频rTMS组大鼠疼痛改善程度与脊髓背角中GFAP的表达呈负相关。 结论坐骨神经结扎导致的神经病理性疼痛伴有脊髓背角内星形胶质细胞增殖;高频rTMS可以通过抑制脊髓内星形胶质细胞的增殖和活性而缓解疼痛,低频rTMS则无明显效果。     

两种浓度阿霉素靶向治疗大鼠顽固性腹腔神经痛的实验研究

目的:观察两种浓度阿霉素靶向治疗大鼠顽固性腹腔神经痛的作用,为临床治疗上腹部顽固性疼痛提供依据。方法:健康SD雄性大鼠60只,随机分为生理盐水(A组),无水酒精(B组),0.33%阿霉素(C组),0.5%阿霉素(D组)。2天后,观察脊髓T6-9背根神经组织中阿霉素荧光表达。第8周末,制做胃痛模型,观察大鼠行为学变化和脊髓背角Fos样免疫反应神经元(Fos-like immunoreactive neuron,FLIN)阳性神经元含量分布变化。结果:C,D组背根神经节内显示阿霉素荧光表达,A,B组未见荧光。A组大鼠胃痛反应明显,B,C,D组都对胃痛有一定程度的抑制。A组FLIN神经元分布集中,区别于B,C,D组散在分布。A组脊髓背角FLIN阳性神经元出现率为33.43±6.52%,B组为23.30±7.82%,C组为27.80±10.02%,D组为23.27±6.99%,其中B,D组脊髓背角FLIN阳性神经元计数与A组比较有统计学意义(P<0.05)。结论:阿霉素靶向治疗大鼠顽固性腹腔神经痛能达到镇痛效果,0.33%的阿霉素副作用比0.5%的阿霉素小。     

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.     

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 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 EFFECT OF PULMONARY CONGESTION ON THE ON THE VENTILATION OF THE LUNGS

1. A method is described for producing pulmonary congestion, together with what may be termed a differential spirometer method for studying lung ventilation. 2. The method utilized permits an approximately accurate prediction of degrees of pulmonary edema in the living animal, and suggests avenues of approach for the very difficult problems of pulmonary capillary pressure. 3. It is shown that intravascular blood can encroach markedly upon the pulmonary air space. Although the methods used in these animal experiments do not resemble vital capacity measurements in man, their result is so definite that their applicability to clinical conditions may be considered. 4. The similarity between the experiments described and certain conditions of cardiac decompensation, of which mitral stenosis is the best example, is pointed out.     

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 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.