SD大鼠、Beagle犬和新西兰白兔主泪腺的解剖学特点及形态学观察

目的观察及比较SD大鼠、Beagle犬、新西兰白兔主泪腺的解剖学和形态学特点.方法 SD大鼠、Bea-gle犬和新西兰白兔的主泪腺剖取并用12%福尔马林固定,进行石蜡切片、HE染色和PAS染色,光学显微镜观察.结果SD大鼠的眶外泪腺和眶内泪腺均为管泡状的浆液性腺;Beagle犬的主泪腺属于管泡状混合性腺,腺组织被结缔组...     

PI3K和Akt蛋白在异丙肾上腺素所致大鼠心肌肥厚中的表达

目的研究异丙肾上腺素(ISO)致大鼠心肌肥厚中PI3K和Akt在心肌组织中的表达,为探讨心肌肥厚的信号转导机制和逆转心肌肥厚提供形态学资料.方法健康成年SD大鼠20只,随机分为实验组、对照组,每组10只.实验组给予异丙肾上腺素处理.1周后处死大鼠,取心肌组织,常规石蜡切片,HE染色,观察心肌组织的病理变化,测量心肌肥厚指标;免疫组织化学染色和免疫荧光染色,检测p-PI3K和p-Akt的表达及分布.结果实验组大鼠心肌肥厚指标与对照组相比均明显升高;免疫组织化学检测显示,实验组心肌组织p-PI3K和p-Akt蛋白表达面积和平均光密度较对照组高.免疫荧光检测实验组心肌组织p-PI3K和p-Akt蛋白表达较对照组高.结论小剂量持续给予 ISO 能建立大鼠心肌肥厚模型;p-PI3K和p-Akt蛋白表达均与心肌肥厚的发生和发展过程相关,PI3K/Akt信号通路激活,可能是导致心肌肥厚的机制之一.     

大天鹅肾组织显微结构及细胞凋亡相关蛋白的表达

为了解大天鹅(Cygnus cygnus)肾的结构特点和相关活性蛋白的表达情况,采用石蜡切片、H.E染色和免疫组化法,观察分析大天鹅的肾组织显微结构和Caspase-3、Bcl-2、Bax及AQP-3的表达。大天鹅的肾组织主要由肾单位、集合管和结缔组织构成;肾小球由一团盘曲的毛细血管构成,近端小管由单层立方上皮组成,游离面有刷状缘,细段由单层扁平上皮组成,远端小管由立方形上皮组成,腔面无刷状缘。近端小管上皮细胞中有Caspase-3、Bcl-2、Bax阳性表达,远端小管上皮细胞有Caspase-3阳性表达,集合管上皮细胞有Bcl-2、AQP-3阳性表达,在肾小球毛细血管内皮有Bcl-2阳性表达。Caspase-3、Bcl-2、Bax及AQP-3共同调节细胞的生长与凋亡过程,可能在稳定鸟类肾单位和集合管结构及调节肾组织水平衡等方面有重要的作用。     

脂褐素三种染色方法比较

目的比较显示脂褐素的3种特殊染色方法,探讨哪一种方法更适合应用于临床病理诊断。方法分别采用三氯化铁-铁氰化钾、醛品红和PAS 3种特殊染色方法,检测HE染色石蜡切片中呈浅棕色或金黄色的颗粒是否为脂褐素。三氯化铁-铁氰化钾染色的脂褐素呈蓝黑色颗粒,醛品红染色的脂褐素呈深紫色颗粒,PAS染色的脂褐素呈红色颗粒。结果通过3种染色结果的比较,发现三氯化铁-铁氰化钾染色对比清晰,定位准。结论三氯化铁-铁氰化钾、醛品红、PAS三种染色均可用于显示脂褐素,但三氯化铁铁氰化钾染色方法优于其他两种。     

卡维地洛对顺铂所致大鼠肾毒性预防作用的形态学研究

目的研究顺铂诱导大鼠肾损伤的组织病理学和超微结构变化以及卡维地洛对大鼠肾损伤的预防作用。方法雄性Wistar大鼠随机分为4组,给药后3～6d处死,取肾组织,常规石蜡切片,HE染色、PAS反应,超薄切片电镜观察。结果卡维地洛组较顺铂肾脏损伤模型组的组织病理学及超微结构变化明显减轻。结论卡维地洛预防性灌胃给与能明显减轻大鼠顺铂所致的肾损伤,其机制可能与其抗氧化和清除自由基活性有关。     

ABRA在不同年龄大鼠腰段脊髓中的表达变化

目的检测Actin binding Rho activator（ABRA）在不同年龄大鼠腰段脊髓中的表达变化。方法采用Western blot定量检测不同年龄大鼠腰段脊髓中ABRA蛋白水平表达变化,采用免疫荧光染色显示不同年龄大鼠腰髓中ABRA细胞定位。结果Western blot显示ABRA在新生鼠腰段脊髓中表达显著高于成年鼠及老年鼠。免疫荧光染色显示ABRA广泛表达于神经元的胞核、胞浆和突起,在腰髓前角,与前角运动神经元存在共定位,在腰髓后角,与小的NeuN阳性感觉神经元存在共定位。腰髓前角、后角的阳性细胞计数均显示新生鼠ABRA＋NeuN双阳性细胞占总ABRA阳性细胞百分比显著低于成年鼠及老年鼠。结论ABRA广泛表达于腰髓中的神经元,ABRA在新生鼠腰髓中表达最强,随年龄的增长呈现明显的时相变化,提示ABRA可能参与了腰髓中神经元的发育和成熟。     

两种切片方法检测肝糖原染色效果的比较

目的比较石蜡和冰冻两种不同切片用于检测牛蛙肝糖原的效果。方法采用石蜡和冰冻两种切片方法制作牛蛙肝脏切片,高碘酸希夫氏（periodicacid-Schiff＇s,PAS）染色法对肝糖原进行组织化学染色,光密度分析糖原含量。结果PAS染色显示肝糖原为紫红色或红色颗粒,冰冻切片中糖原颗粒明显大于石蜡切片,光密度分析显示,石蜡切片中糖原流失明显,与冰冻切片相比,糖原流失了约28％。结论两种切片均可用于糖原检测,冰冻切片制作环节较少、耗时短且染色过程中糖原不易流失。     

小熊猫肾脏和输尿管的组织学研究

小熊猫的肾脏呈蚕豆形,表面光滑不分叶,只有1个肾锥体和1个肾盏,无肾盂。肾脏皮质内可见皮质迷路和髓放线。皮质迷路内有近曲小管、远曲小管和肾小体等结构。髓放线内有近端小管直部和远端小管直部。髓质可分为外髓和内髓两个区域。外髓有较多的集合管断面,少量的远端小管直部和细段,较多的直小血管束。内髓部位有大量的细段和乳头管。各种泌尿小管之间有少量的疏松结缔组织构成的间质,间质内有丰富的毛细血管。输尿管横切面呈圆形或卵圆形,管腔呈不规则的裂隙状。管壁由粘膜、肌肉层和外膜组成。并与大熊猫肾脏和输尿管的组织结构作了比较研究。     

特殊染色及免疫组化双重染色在恒河猴糖尿病模型中的应用

目的在链脲佐菌素（STZ）诱导恒河猴糖尿病动物模型基础上,用特殊染色、免疫组化双染方法显示胰腺等组织中的特征性病变。方法健康恒河猴5只,小剂量（30mg／kg）多次静脉注射STZ,濒死状态时将动物安乐死。取胰腺、心脏、肾脏、脾脏、肝脏、眼球、脑等器官制成石蜡切片,用HE、PAS、Masson、天狼星红和甲苯胺蓝等方法进行特殊染色,用免疫组化双重染色法同时显示胰岛A、B细胞。结果模型动物的胰岛萎缩,数量减少。Masson染色见外分泌部间质内纤维增生。局灶性慢性肾炎,病变区肾间质纤维组织增生,部分肾小球及肾小管萎缩。天狼星红染色见脾脏中央动脉管壁增厚。免疫组化双染见胰岛A胰高血糖素表达增多,胞浆呈棕褐色。B胰岛素表达减少,胞浆粉红色。结论HE染色结合特殊染色和免疫组化双重染色可较好地对STZ诱导糖尿病动物模型进行组织学评价。     

NPR-A在压力超负荷性大鼠心肌重构过程中的表达变化

目的观察在A型利钠肽受体(NPR-A)表达压力超负荷性大鼠心肌重构过程中的变化,探讨其在高血压心肌重构过程中的可能作用。方法 40只清洁级SD大鼠随机分为对照组(20只)及模型组(20只),模型组采用"两肾一夹法"构建肾性高血压大鼠模型,术后每周测尾动脉收缩压(SBP),于术后4、8周后处死大鼠。计算左心室重量指数(LVMI),采用HE、天狼星红染色观察左心室病理形态学变化,采用ELLSA法测定血浆脑钠肽(BNP),免疫组化测定左心室NPR-A蛋白表达水平。比较两组SBP、LVMI、心肌细胞直径(MD)、心肌组织胶原容积分数(CVF)、BNP及NPR-A蛋白表达水平。结果模型组大鼠术后4、8周后SBP、LVMI、MD、CVF均明显高于对照组(均P0.05);模型组大鼠术后4周血浆BNP、左心室NPR-A表达水平均明显升高(P0.05),术后8周血浆BNP明显升高(P0.05),左心室NPR-A水平明显下降(P0.05)。与模型组术后4周相比,模型组大鼠术后8周尾动脉SBP无明显变化(P0.05),但LVMI、MD、CVF均显著升高,血浆BNP水平显著升高,左心室NPR-A表达显著下降,差异均有统计学意义(均P0.05)。结论压力超负荷性大鼠左心室NPR-A的表达呈动态变化趋势,可能参与高血压心肌重构发生发展的过程。     

Immunocytochemical demonstration of vasopressin binding in rat kidney

We investigated the immunoperoxidase demonstration of vasopressin (VSP) bound to paraffin-embedded sections of rat kidney and the effects of various fixatives. Slices of rat kidney from normal and 4-day water-deprived rats were incubated with 10(-7) M VSP, fixed, and embedded in paraffin. Hydrated sections of these tissues were again incubated with 10(-7) M VSP or 10(-7) M VSP and 10(-5) M oxytocin (OXY). VSP bound to the sections was demonstrated using rabbit anti-Arg8 VSP antiserum and peroxidase-labeled second antibody. In sections of kidney from both normal and water-deprived rats, immunoperoxidase labeling was most intense in the renal papilla and was restricted to the cells of the ducts of Bellini and loops of Henle. In the medulla, the collecting ducts and medullary thick ascending limbs of Henle were moderately stained. In the normal kidney sections there was no staining of the proximal tubules, distal convoluted tubules (DCT), and only slight staining of the cortical collecting ducts (CCD). However, in the water-deprived rats there was a considerable increase in the staining of the DCT and CCD. Simultaneous incubation in OXY and VSP resulted in reduced immunoperoxidase labeling of the tubules. Omission of VSP incubation led to a similar decrease in stain intensity, indicating a specificity for the sites of VSP binding. This technique allows the identification of cells responsible for the binding of VSP in the kidney.     

Focal expression of insulin-like growth factor I in rat kidney collecting duct

To address the question of insulin-like growth factor (IGF) I localization and synthesis in kidney, we used two complementary experimental approaches: immunohistochemistry of fixed paraffin-embedded rat kidney sections; and measurement of IGF I mRNA in isolated components of the rat nephron, using a highly sensitive and specific solution hybridization assay. Immunostainable IGF I was localized exclusively to principal cells of cortical and medullary collecting ducts. Administration of growth hormone to hypophysectomized rats for 8 d resulted in enhanced immunohistochemical staining of IGF I within collecting ducts, but no detectable IGF I in other portions of the nephron. The abundance of IGF I mRNA was 7-12-fold higher in isolated papillary collecting ducts than in proximal tubules or glomeruli, and was enriched 10-fold compared with whole kidney. Our data demonstrate colocalization of IGF I and IGF I mRNA in the collecting duct, consistent with focal expression of the IGF I gene at this site.     

Investigation of Tissue Preparation Conditions for Non-radioactive In Situ Hybridization: Localization of Transforming Growth Factor-α Message in Rat Kidney

A non-radioactive method of in situ hybridization was used to localize transforming growth factor- mRNA in epithelial cells of collecting ducts and tubules in rat kidney tissue sections. The intensity and specificity of staining were assessed under a variety of tissue preparation conditions, including a direct comparison of paraffin against frozen sections. Under optimal conditions, both the signal strength and the cellular localization of the growth factor message were superior in paraffin sections. The staining method could also be used to localize the message in lung tissue, indicating that the procedure is generally applicable to other tissues. Our results indicate that the use of paraffin sections for non-radioactive in situ hybridization affords a number of advantages for the localization of specific messages in tissue sections.     

Immunocytochemical localization of Na+, K+-ATPase in the rat kidney

To determine if rat kidney Na+, K+-ATPase can be localized by immunoperoxidase staining after fixation and embedding, we prepared rabbit antiserum to purified lamb kidney medulla Na+, K+-ATPase. When sodium dodecylsulfate polyacrylamide electrophoretic gels of purified lamb kidney Na+, K+-ATPase and rat kidney microsomes were treated with antiserum (1:200), followed by [125I]-Protein A and autoradiography, the rat kidney microsomes showed a prominent radioactive band coincident with the alpha-subunit of the purified lamb kidney enzyme and a fainter radioactive band which corresponded to the beta-subunit. When the Na+, K+-ATPase antiserum was used for immunoperoxidase staining of paraffin and plastic sections of rat kidney fixed with Bouin's, glutaraldehyde, or paraformaldehyde, intense immunoreactive staining was present in the distal convoluted tubules, subcapsular collecting tubules, thick ascending limb of the loops of Henle, and papillary collecting ducts. Proximal convoluted tubules stained faintly, and the thin portions of the loops of Henle, straight descending portions of proximal tubules, and outer medullary collecting ducts did not stain. Staining was confined to basolateral surfaces of tubular epithelial cells. No staining was obtained with preimmune serum or primary antiserum absorbed with purified lamb kidney Na+, K+-ATPase, or with osmium tetroxide postfixation. We conclude that the basolateral membranes of the distal convoluted tubules and ascending thick limb of the loops of Henle are the major sites of immunoreactive Na+, K+-ATPase concentration in the rat kidney.     

Expression of cytochrome P-450 4 enzymes in the kidney and liver: Regulation by PPAR and species-difference between rat and human

Members of the cytochrome P-450 4 (CYP4) family catalyze the ω-hydroxylation of fatty acids, and some of them have the PPAR response element in the promoter area of the genes. The localization of CYP4A and PPAR isoforms and the effect of PPAR agonists on CYP4A protein level and activity were determined in rat kidney and liver. Immunoblot analysis showed that CYP4A was expressed in the liver and proximal tubule, with lower expression in the preglomerular microvessel, glomerulus and thick ascending limb (TAL), but the expression was not detected in the collecting duct. PPARα was expressed in the liver, proximal tubule and TAL. PPARγ was expressed in the collecting duct, with lower expression in the TAL, but no expression in the proximal tubule and liver. The PPARα agonist clofibrate induced CYP4A protein levels and activity in the renal cortex and liver. The PPARγ agonist pioglitazone did not modulate them in these tissues. The localization of CYP4A and CYP4F were further determined in human kidney and liver by immunohistochemical technique. Immunostainings for CYP4A and CYP4F were observed in the hepatocytes of the liver lobule and the proximal tubules, with lower stainings in the TALs and collecting ducts, but no staining in the glomeruli or renal vasculatures. These results indicate that the inducibility of CYP4A by PPAR agonists in the rat tissues correlates with the expression of the respective PPAR isoforms, and that the localization of CYP4 in the kidney has a species-difference between rat and human.     

Immunocytochemical localization of a renal glycoprotein (gpCDI) synthesized by cultured collecting duct cells

A sulfated, proline-rich glycoprotein (gpCDI, apparent molecular weight 200,000 in column chromatography and 150,000 in SDS-PAGE) was isolated from cultured renal collecting duct epithelium by centrifugation. Triton X100 extraction and DEAE-cellulose ion exchange chromatography. A DEAE-cellulose ion exchange chromatography fraction with the enriched gpCDI was used for immunization of guinea pigs. The antiserum was prepared for antigen localization by indirect immunofluorescence in collecting duct cell cultures and in tissue sections of neonatal and adult rabbit kidneys. In the cultured collecting duct epithelium, antibody staining of the epithelium and structures of the extracellular matrix was age dependent. Cultures of dedifferentiated collecting duct monolayers revealed positive reaction in the cytoplasm. In neonatal and adult rabbit kidneys, the antibody was localized in the entire collecting duct system but not in the collecting duct ampullae of the newborn kidney. Staining of the cytoplasm was found only in medullary collecting ducts of the neonatal kidney; other portions revealed staining mostly at the basal circumference of the tubule and at the luminal cell borders. Apart from collecting ducts, no other tubular segments were reactive. The cortical and the medullary interstitium contained fluorescent fibres which were concentrated around vascular structures. A possible relation between gpCDI and collagenous compounds is discussed. Bowman's capsule reacted positively, whereas staining of the mesangial matrix was weak. The localization of the antigen, as revealed by indirect immunofluorescence, suggests that gpCDI occurs both in intracellular and extracellular (interstitial) location. Two main points are emphasized: Firstly gpCDI is considered an important constituent in different stages of collecting duct development, and secondly, the staining pattern of the antibody varies with the different portions of both young and adult kidney collecting ducts; this staining heterogeneity may correspond with the known regional differences of collecting duct functions.     

Expression and nephron segment-specific distribution of major renal aquaporins (AQP1-4) in Equus caballus, the domestic horse

Aquaporins (AQPs) play fundamental roles in water and osmolyte homeostasis by facilitating water and small solute movement across plasma membranes of epithelial, endothelial, and other tissues. AQP proteins are abundantly expressed in the mammalian kidney, where they have been shown to play essential roles in fluid balance and urine concentration. Thus far, the majority of studies on renal AQPs have been carried out in laboratory rodents and sheep; no data have been published on the expression of AQPs in kidneys of equines or other large mammals. The aim of this comparative study was to determine the expression and nephron segment localization of AQP1-4 in Equus caballus by immunoblotting and immunohistochemistry with custom-designed rabbit polyclonal antisera. AQP1 was found in apical and basolateral membranes of the proximal convoluted tubules and thin descending limbs of the loop of Henle. AQP2 expression was specifically detected in apical membranes of cortical, medullary, and papillary collecting ducts. AQP3 was expressed in basolateral membranes of cortical, medullary, and papillary collecting ducts. Immunohistochemistry also confirmed AQP4 expression in basolateral membranes of cells lining the distal convoluted and connecting tubules. Western blots revealed high expression of AQP1-4 in the equine kidney. These observations confirm that AQPs are expressed in the equine kidney and are found in similar nephron locations to mouse, rat, and human kidney. Equine renal AQP proteins are likely to be involved in acute and chronic regulation of body fluid composition and may be implicated in water balance disorders brought about by colic and endotoxemia.     

Cell-specific expression of epithelial sodium channel alpha, beta, and gamma subunits in aldosterone-responsive epithelia from the rat: localization by in situ hybridization and immunocytochemistry

A highly selective, amiloride-sensitive, epithelial sodium channel from rat colon (rENaC), composed of three homologous subunits termed alpha, beta, and gamma rENaC, has been cloned by functional expression and was proposed to mediate electrogenic sodium reabsorption in aldosterone- responsive epithelia. To determine whether rENaC could account for sodium absorption in vivo, we studied the cellular localization of the sodium channel messenger RNA subunits by in situ hybridization and their cellular and subcellular distribution by immunocytochemistry in the kidney, colon, salivary, and sweat glands of the rat. In the kidney, we show that the three subunit mRNAs are specifically co- expressed in the renal distal convoluted tubules (DCT), connecting tubules (CNT), cortical collecting ducts (CCD), and outer medullary collecting ducts (OMCD), but not in the inner medullary collecting ducts (IMCD). We demonstrate co-localization of alpha, beta, and gamma subunit proteins in the apical membrane of a majority of cells of CCD and OMCD. Our data indicate that alpha, beta, and gamma subunit mRNAs and proteins are co-expressed in the distal nephron (excepting IMCD), a localization that correlates with the previously described physiological expression of amiloride-sensitive electrogenic sodium transport. Our data, however, suggest that another sodium transport protein mediates electrogenic amiloride-sensitive sodium reabsorption in IMCD. We also localized rENaC to the surface epithelial cells of the distal colon and to the secretory ducts of the salivary gland and sweat gland, providing further evidence consistent with the hypothesis that the highly selective, amiloride-sensitive sodium channel is physiologically expressed in aldosterone-responsive cells.     

Immunocytochemical localization of a renal glycoprotein (gp CD I) synthesized by cultured collecting duct cells

Summary A sulfated, proline-rich glycoprotein (gp_CDI, apparent molecular weight 200,000 in column chromatography and 150,000 in SDS-PAGE) was isolated from cultured renal collecting duct epithelium by centrifugation, Triton X100 extraction and DEAE-cellulose ion exchange chromatography. A DEAE-cellulose ion exchange chromatography fraction with the enriched gp_CDI was used for immunization of guinea pigs. The antiserum was prepared for antigen localization by indirect immunofluorescence in collecting duct cell cultures and in tissue sections of neonatal and adult rabbit kidneys. In the cultured collecting duct epithelium, antibody staining of the epithelium and structures of the extracellular matrix was age dependent. Cultures of dedifferentiated collecting duct monolayers revealed positive reaction in the cytoplasm. In neonatal and adult rabbit kidneys, the antibody was localized in the entire collecting duct system but not in the collecting duct ampullae of the newborn kidney. Staining of the cytoplasm was found only in medullary collecting ducts of the neonatal kidney; other portions revealed staining mostly at the basal circumference of the tubule and at the luminal cell borders. Apart from collecting ducts, no other tubular segments were reactive. The cortical and the medullary interstitium contained fluorescent fibres which were concentrated around vascular structures. A possible relation between gp_CDI and collagenous compounds is discussed. Bowman's capsule reacted positively, whereas staining of the mesangial matrix was weak. The localization of the antigen, as revealed by indirect immunofluorescence, suggests that gp_CDI occurs both in intracellular and extracellular (interstitial) location. Two main points are emphasized: Firstly, gp_CDI is considered an important constituent in different stages of collecting duct development, and secondly, the staining pattern of the antibody varies with the different portions of both young and adult kidney collecting ducts; this staining heterogeneity may correspond with the known regional differences of collecting duct functions.     

Immunohistochemical localization of atrial natriuretic peptide in the developing and adult mammalian kidney

The discovery, within the last decade, of atrial natriuretic peptide (ANP), a family of peptides with natriuretic/diuretic and vasorelaxant properties, has prompted much research into the mechanisms and sites of action of ANP within the kidney. In the present study, ANP was localized in the kidneys of several mammalian species by immunohistochemical techniques 1) to identify possible sites of synthesis; 2) to compare the localization of ANP to known physiological effects; 3) to determine species differences, if any, in ANP localization; and 4) to study the development of ANP immunoreactivity in the fetal and neonatal rat kidney. Using an antibody against rat ANP, IV, ANP was localized exclusively on the proximal convoluted tubule (PCT) brush border and within intercalated cells of the outer medullary and cortical collecting tubules and ducts of adult mouse, rat, pig, monkey, and human kidneys. The development of ANP immunoreactivity paralleled the differentiation and maturation of collecting duct epithelium in rat fetal kidney. Atrial natriuretic peptide found within intercalated cells of the cortical and outer medullary collecting ducts may be the result of endogenous synthesis and, following secretion, may be available to receptors in the inner medullary collecting ducts.