早期股骨头缺血坏死中西医结合治疗进展

股骨头缺血性坏死（avascular necrosis of the femoral head,ANFH）是由于髋部外伤,长期应用激素类药物,酒精中毒等原因,引起股骨头血液供应障碍,股骨头组织不能得到正常的营养,使股骨头组织中的骨细胞,骨髓造血细胞,脂肪细胞发生坏死。由于坏死的骨组织脆弱,加之髋关节需要负重,日久就会发生股骨头塌陷,影响全部髋关节,     

类固醇激素影响骨髓间充质干细胞生物学特性的研究进展

类固醇诱生股骨头缺血性坏死（Steroid—induced Avascular Necrosis of Femoral Head, SANFH）,俗称激素性股骨头坏死，为进展性和致残性疾病。近年来采用移植患者自体骨髓治疗股骨头坏死的尝试已有报道，并见到初步疗效，最近也有动物实验证实向家兔骨髓腔内注射自体骨髓细胞可防止SANFH的报道。     

髓芯减压联合rhBMP-2复合人工骨移植修复兔股骨头坏死模型的实验研究

取健康新西兰白兔30只,分为A组：右侧股骨头内钻孔髓芯减压后填充rhBMP-2/β-TCP复合人工骨;B组：左侧股骨头内钻孔髓芯减压后填充β-TCP;C组：单纯激素性股骨头坏死模型;D组：正常对照组。A组术后股骨头X线下骨密度高于B组;A组术后骨小梁占修复面积的比例高于B组;A组术后兔股骨头CGRP阳性细胞数多于B组,两组间差异（P〈0.05）,具有统计学意义。利用rhBMP-2/β-TCP复合人工骨移植治疗兔SANFH,能有效提高股骨头坏死病理条件下骨修复能力,并能增加CGRP的表达,提示CGRP免疫阳性神经纤维参与的股骨头缺血坏死后修复过程。     

力学载荷下大鼠激素性股骨头坏死的改变

背景:力学载荷下大鼠激素性股骨头坏死表现更为典型.目的:建立大鼠激素性股骨头坏死正常负重和超负重模型,对使用激素后不同时段大鼠股骨头进行观察,探究在力学载荷下大鼠激素性股骨头的相关改变.方法:4月龄健康Wistar大鼠随机分成实验组和对照组.两组大鼠均臀肌注射地塞米松磷酸钠(20 mg/kg),1次/周,共计8周.实验组,置于1 km/h跑步机中,强迫其跑动,形成股骨头坏死超负重模型;对照组,生理状态下正常负重.分别于2,4,6,8周处死大鼠,取右侧股骨头标本,于Endura TEC ELF 3200生物力学材料动态力学性能测试系统行压力测定,计算样本纵向最大位移、刚度吸收等生物力学参数,计算空骨陷窝数,并行免疫组织化学法Bcl-2染色,比较不同组之间的累计吸光度值.结果与结论:实验组8周时病理学呈股骨头坏死表现,各个时期股骨头骨小梁宽度、刚度均低于同期对照组:最大形变均高于同期对照组(P＜0.05).实验组Bcl-2累计吸光度与对照组比较,第4周起差异有显著性意义(P＜0.05),实验组Bcl-2表达随激素注射时间增加有显著性意义(P＜0.05).结果提示,在激素性股骨头坏死中力学载荷使骨细胞凋亡明显,软骨细胞坏死修复增加,并增多骨小梁断裂,不利于保护骨小梁的完整性,成为引起激素性股骨头坏死塌陷的直接外部因素,并出现更显著的股骨头坏死表现.     

激素诱发兔股骨头坏死的组织学及超微结构变化

背景长期大量应用糖皮质激素可诱发股骨头坏死,其发病机制尚需深入研究.目的通过兔股骨头坏死模型,应用光、电镜观察,从形态学角度探讨其发病机制.设计随机对照观察.单位潍坊医学院形态学实验室,病理学教研室,外科学实验室.材料实验于2002-03/2003-03在潍坊医学院形态实验中心完成,成年新西兰白兔40只,随机分为生理盐水对照(10只)、氟美松组(10只)、马血清3组(20只).方法生理盐水对照组用生理盐水10 mL/(kg·d)静脉注射,连续7 d.氟美松组肌肉注射氟美松10 mL/(kg·d),连续7 d.马血清组静脉注射马血清10 mL/kg,间隔3周,重复注射同量的马血清一次并连续7 d肌肉注射氟美松10 mL/(kg·d).分别在第5周、第10周取实验动物的股骨头的软骨下区,用光镜、电镜观察组织学及超微结构变化.主要观察指标①各组动物的组织形态学观察.②超微结构变化.结果所有的实验动物均存活并纳入实验结果分析.①组织形态学观察生理盐水对照组股骨头软骨下骨细胞排列规则,骨细胞体积小,呈扁椭圆形,胞体位于骨陷窝内,骨髓腔内血管分布均匀.氟美松,马血清两组股骨病变特征相似骨髓腔内造血组织显著减少,脂肪组织明显增多;在股骨干骺端及软骨下区发现骨小梁萎缩,骨细胞核固缩,空骨陷窝数增多.②超微结构变化生理盐水对照组正常骨细胞呈扁椭圆形,位于骨陷窝内.细胞核位于细胞一端,核膜完整,细胞质内线粒体丰富.氟美松,马血清两组电镜发现骨细胞内有脂滴,骨髓腔毛细血管狭窄,血管内皮细胞受损.结论肾上腺糖皮质激素可诱发兔股骨头坏死,激素引起脂肪在骨髓腔内堆积,骨髓腔内压升高而导致股骨头缺血,诱发骨细胞坏死.     

桃红四物汤对激素诱导骨髓间充质干细胞成脂分化的干预作用

背景:临床组织学及病理学研究证明:激素性股骨头缺血性坏死的早期病理变化是股骨头骨髓内造血组织减少,脂肪组织增多,这可能与激素诱导下骨髓间充质干细胞的成脂分化有关.目前被很多学者用来解释激素性股骨头缺血性坏死的发病机制. 目的:观察桃红四物汤对激素诱导臂髓间充质干细胞成脂分化的干预作用. 方法:体外培养大鼠骨髓间充质干细胞,通过大剂量激素诱导体外培养的骨髓间充质干细胞成脂分化,在诱导成脂的同时给予桃红四物汤含药血清干预.检测干预6 d后细胞内成脂标志物三酰甘油、PPARy mRNA和aP2 mRNA的表达. 结果与结论:桃红四物汤含药血清可对抗激素诱导下的骨髓间充质干细胞三酰甘油、PPARy mRNA和aP2 mRNA表达的增加.提示桃红四物汤防治激素性股骨头缺血坏死的机制不仅是改善股骨头的微循环,同时还与其抑制激素诱导下的骨髓间充质干细胞成脂分化有关.     

激素诱发兔股骨头坏死的组织学及超微结构变化

背景：长期大量应用糖皮质激素可诱发股骨头坏死，其发病机制尚需深入研究。目的：通过兔股骨头坏死模型，应用光、电镜观察，从形态学角度探讨其发病机制。 设计：随机对照观察。 单位：潍坊医学院形态学实验室，病理学教研室，外科学实验室。 材料：实验于2002-03／2003-03在潍坊医学院形态实验中心完成，成年新西兰白兔40只，随机分为生理盐水对照（10只）、氟美松组（10只）、马血清3组（20只）。方法：生理盐水对照组用生理盐水10mL／（kg&;#183;d）静脉注射，连续7d。氟美松组肌肉注射氟美松10mL／（kg&;#183;d），连续7d。马血清组静脉注射马血清10mL／kg，间隔3周，重复注射同量的马血清一次并连续7d肌肉注射氟美松10mL／（kg&;#183;d）。分别在第5周、第10周取实验动物的股骨头的软骨下区，用光镜、电镜观察组织学及超微结构变化。 主要观察指标：①各组动物的组织形态学观察。②超微结构变化。 结果：所有的实验动物均存活并纳入实验结果分析。①组织形态学观察：生理盐水对照组股骨头软骨下骨细胞排列规则，骨细胞体积小，呈扁椭圆形，胞体位于骨陷窝内，骨髓腔内血管分布均匀。氟美松，马血清两组股骨病变特征相似：骨髓腔内造血组织显著减少，脂肪组织明显增多；在股骨干骺端及软骨下区发现骨小梁萎缩，骨细胞核固缩，空骨陷窝数增多。②超微结构变化：生理盐水对照组正常骨细胞呈扁椭圆形，位于骨陷窝内。细胞核位于细胞一端，核膜完整，细胞质内线粒体丰富。氟美松，马血清两组电镜发现骨细胞内有脂滴，骨髓腔毛细血管狭窄，血管内皮细胞受损。 结论：肾上腺糖皮质激素可诱发兔股骨头坏死，激素引起脂肪在骨髓腔内堆积，骨髓腔内压升高而导致股骨头缺血，诱发骨细胞坏死。     

普伐他汀修复激素性股骨头坏死免模型的超微结构评价

背景:研究表明普伐他汀可通过上调内源性骨形成蛋白2、核心结合因子α1和血管内皮生长因子等基因的表达从而产生促进激素性股骨头坏死兔模犁坏死股骨头修复的作用.目的:验证性观察普伐他汀干预激素性股骨头坏死兔模型的超微结构改变.方法:将80只新西兰白兔按随机数字表法分为对照组18只,实验组62只.实验组制各兔激素性股骨头缺血坏死模型.造模第5周,以随机数字表法分配36只模型兔至模型组和他汀组,每组18只.他汀组以普伐他汀(1.2 mg/kg)1次/d灌胃,模型组和对照组以等体积的蒸馏水灌胃.造模后8,12,16周,截取股骨头行透射电镜观察.结果与结论:透射电镜观察显示模型组骨细胞变性坏死严重,大部分骨陷窝内骨细胞消失,残存的骨细胞内脂肪沉积增多;与模型组比较,他汀组骨细胞受损程度较轻,形态基本正常,胞浆内脂肪沉积少.说明普伐他汀可有效促进早期激素性股骨头坏死兔模犁坏死股骨头的修复.     

高压氧对兔早期激素性股骨头缺血性坏死骨修复的影响

目的：建立激素性股骨头缺血性坏死（SANFH）动物模型，然后采用高压氧（HBO）治疗，探讨HBO治疗早期SANFH的病理变化及作用机制。方法：健康成年日本大耳白兔60只，随机分为模型组（n=42）与对照组（n=18）,造模成功后，再将模型组分为HBO组（n=16）及其对照组（n=16）。观察模型组、HBO组及其各自对照组X线、MRI、光镜、透射电镜的变化。结果：①模型组第6周股骨头软骨下区骨小梁稀疏、变细，甚至骨小梁断裂，死骨形成。部分关节软骨区域性坏死。髓腔内造血组织减少，肥大脂肪细胞增多，后期脂肪细胞液化坏死,基质水肿出血。透射电镜观察到骨细胞坏死及成骨细胞凋亡现象。②HBO组第4—6周时不同程度的造血功能恢复,肥大脂肪细胞数目逐渐减少,部分骨小梁周围出现少量梭形或成排的成骨细胞,髓腔内纤维组织增生,在部分坏死的关节软骨下区髓腔内见活跃的纤维组织及成骨细胞增生：第8周时坏死骨小梁周围出现多核破骨细胞及较肥胖的成骨细胞。造血细胞增生明显、肥大脂肪细胞明显减少。透射电镜观察到成骨细胞、骨细胞、胶原纤维再生、修复的证据。结论：HBO治疗对早期SANFH有明显的骨修复作用。     

脂联素与大鼠激素性股骨头坏死的修复

背景:动物实验表明脂联素影响成骨细胞和破骨细胞的增殖和分化,临床研究发现脂联素和骨密度存在着一定的相关性,但具体作用机制尚不清楚.目的:评价髋关节内直接注射重组大鼠脂联素对激素性股骨头缺血性坏死修复的影响.方法:将SD大鼠随机分成4组.空白对照组腹腔注射生理盐水,其余大鼠腹腔注射醋酸泼尼松龙诱导出早期激素性股骨头缺血坏死的模型.随后将低剂量组、高剂量组大鼠右侧髋关节内分别注入1 μg、10 μg的重组脂联素;生理盐水组注入等量的生理盐水;空白对照组不作任何处理.4周后行X射线平片观察,光镜下观察股骨头坏死和修复的程度.分析骨小梁面积、空骨陷窝数、软骨下骨血管数量,免疫组织化学检测股骨头肿瘤坏死因子α表达情况.结果与结论:空白对照组X射线片无异常发现,生理盐水组、低剂量组显示股骨头密度不均骨小梁模糊;高剂量组股骨头形态和骨密度均正常.大体所见空白对照组骨质硬,生理盐水组骨质明显疏松,低剂量组、高剂量组骨质硬度介于空白对照组、生理盐水组之间.光镜显示空白对照组股骨头软骨、骨小梁、骨细胞、空骨陷窝、毛细血管数及骨髓造血组织均正常,而生理盐水组明显异常,与空白对照组比较差异有显著性意义(P<0.01);高剂量组结果显著优于生理盐水组(P<0.01),低剂量组结果与生理盐水组差异无显著性意义(P>0.05),生理盐水组肿瘤坏死因子α的表达高于其余3组(P<0.01).结果表明,髋关节内直接注射重组脂联素可促进激素性股骨头缺血性坏死的修复,且具有剂量依赖性.     

Lung Structure and Function with Age in Normal Rats and Rats With Papain Emphysema

Intrapulmonary deposition of the proteolytic enzyme papain produces a lesion resembling emphysema in experimental animals. The natural history of this lesion has not been well defined. The present study was performed to evaluate changes in lung structure and function with aging in normal rats and rats exposed to an aerosol of papain at 2 mo of age. Groups of control and papain-exposed animals were studied at 4, 8, and 18 mo of age. The parameters of lung function studied were specific airways' conductance (G(aw)/TGV), diffusing capacity per unit of alveolar volume (D(Lco)/V(A)), diffusing capacity (D(Lco)), and functional residual capacity (FRC). Morphometric parameters were the postfixation lung volume (V(L)) and mean chord length (L(M)); internal surface area (ISA) and ISA extrapolated to both the mean V(L) of the corresponding papain group and a V(L) of 10 ml (ISA(10)) were calculated.At 4 mo of age L(M) and FRC were significantly increased and ISA, D(Lco)/V(A), and D(Lco) were significantly reduced in the papain group. At 8 mo of age L(M) was significantly increased and ISA was significantly decreased in the papain group: physiologic studies were not performed in this group. At 18 mo of age L(M) was significantly increased and D(Lco)/V(A), D(Lco), and ISA were significantly decreased. Neither progression nor healing of the lesion was observed despite similar lung growth in both groups.This study demonstrates that a single proteolytic lung injury produces a fixed deficit of lung parenchyma. Progressive lung destruction may require repeated or continuous lung injury.     

Comparative Nephrotoxicity of Gentamicin and Tobramycin in Rats

A rat model was utilized to compare the nephrotoxic potential of gentamicin and tobramycin. Gentamicin, 40 mg/kg per day, predictably produced renal failure and morphological evidence of proximal tubular necrosis over 14 days of treatment. An identical dosage of tobramycin was associated with only minimal morphological changes and normal concentrations of serum creatinine and blood urea nitrogen. Similar results were obtained even after the tobramycin dosage was tripled to 120 mg/kg per day. A decrease in urine osmolality, mechanism unknown, was observed in all aminoglycoside-treated rats, but the lowest osmolalities were found in the gentamicin-treated rats. According to both histological criteria and renal function measurements, gentamicin was more nephrotoxic than tobramycin in this animal model.     

Chondrotoxicity and Toxicokinetics of Sparfloxacin in Juvenile Rats

Sparfloxacin is a fluoroquinolone with improved antibacterial activity against gram-positive pathogens. Like other quinolones, use of this drug is contraindicated in children and adolescents because of its potential chondrotoxicity in juveniles. We performed histological and immunohistochemical studies on the knee joint cartilage in 5-week-old rats after treatment with 600 or 1,800 mg of sparfloxacin/kg of body weight. Treatment with single or multiple oral doses of 600 mg of sparfloxacin/kg was not sufficient to induce joint cartilage lesions. However, five of eight rats treated with a single oral dose of 1,800 mg of sparfloxacin/kg of body weight showed typical cartilage lesions in the femoral part of the knee joint. The concentrations of the drug in plasma measured 0.25, 0.75, 1.5, 3, 6, 12, and 24 h after the administration of an oral dose of 600 mg of sparfloxacin/kg were 6.3 ± 1.8, 9.2 ± 1.7, 9.6 ± 2.7, 13.0 ± 1.8, 12.3 ± 1.6, 3.4 ± 0.4, and 0.30 ± 0.20 mg/liter, respectively (mean ± standard deviation [SD]; n = 5 to 6 per group). The concentrations in plasma measured 0.75, 1.5, 3, 6, 24, and 48 h after the administration of an oral dose of 1,800 mg of sparfloxacin/kg were 10.9 ± 1.5, 15.9 ± 1.6, 19.1 ± 1.7, 14.9 ± 3.1, 4.1 ± 0.6, and 0.46 ± 0.37 mg/liter, respectively (mean ± SD; n = 3 to 4 per group). The concentrations of sparfloxacin in joint cartilage were significantly higher at all time points studied (114.8 ± 80, 99.4 ± 31.5, 84.9 ± 16.8, 44.4 ± 13.9, and 14.2 ± 4.8 mg of sparfloxacin/kg at 1.5, 3, 6, 24, and 48 h after the administration of 1,800 mg/kg, respectively). The range of concentrations in bone were similar to the range of concentrations in cartilage (peak, 115 ± 12 mg/kg after 3 h). Our data indicate that chondrotoxic doses of sparfloxacin in juvenile rats are approximately 300 times higher than the doses of sparfloxacin used therapeutically (1,800 versus approximately 6 mg/kg of body weight), but due to species differences in kinetics, concentrations in plasma differ by a factor of only approximately 15. More data on quinolone concentrations in cartilage from animals and humans could provide a better basis for a reasonable risk assessment.     

Comparative Nephrotoxicities of Netilmicin and Gentamicin in Rats

The relative nephrotoxicities of netilmicin (Sch 20569) and gentamicin were compared in rats at doses of 30, 60, 90, and 120 mg/kg per day for 15 days. Both drugs caused proteinuria and a decrease in urine osmolality; however, netilmicin produced significantly less changes at all doses than gentamicin. Whereas gentamicin resulted in a decline in creatinine clearance at all doses, netilmicin failed to cause a decline in creatinine clearance. Renal-cortical concentrations of antibiotic at sacrifice were similar in animals receiving either drug. Light-microscopic changes were less severe with netilmicin than gentamicin. Cytosegresomes with myeloid bodies were identified electron microscopically in the kidneys of animals receiving either netilmicin or gentamicin at all doses. Electron-microscopic manifestations were similar. The data indicate that in the rat, netilmicin is distinctly less nephrotoxic than gentamicin.     

环扁桃酯对大鼠血液流变性及血栓形成的影响

目的研究环扁桃酯对血流变和血栓形成的影响。方法测定环扁桃酯高、低剂量组大鼠的全血粘度、血小板聚集率、红细胞压积、血栓湿重,并与正常对照组、阿司匹林组进行比较。结果环扁桃酯的高剂量组降低全血粘度的效果最显著,高切变率80s~(-1)降低率为19．5％,低切变率20s~(-1)降低率为26．2％;环扁桃酯的高剂量组使血小扳聚集率明显降低,最大聚集率下降了29．2％;与空白对照组比较,环扁桃酯高剂量组使血栓湿重明显降低。结论提示环扁桃酯是改善血流变及血栓形成的有效药物。     

No Association between Hyponatremia and Rhabdomyolysis in Rats

Background

Rhabdomyolysis is an uncommon complication of hyponatremia, reported previously only in case reports and small retrospective studies, and its underlying mechanism is controversial. Some studies support the hypothesis that the rapid correction of hyponatremia is responsible for rhabdomyolysis, whereas others emphasize the severity of the hyponatremia as a predisposing factor for rhabdomyolysis.

Objectives

To test the association between hyponatremia and rhabdomyolysis and to demonstrate a causal association.

Methods

Hyponatremia was induced by administration of water and desmopressin acetate in rats during 3 days, followed by its rapid correction, using animal models established for the evaluation of central pontine myelinolysis. The plasma creatine phosphokinase levels, a marker for rhabdomyolysis, were monitored, and hematoxylin and eosin sections of the quadriceps and gastrocnemius muscles were evaluated for signs of rhabdomyolysis.

Results

The induction of hyponatremia and its correction were accompanied by the previously reported neurological sequelae, including signs of central pontine myelinolysis. However, no increase in plasma creatine phosphokinase levels was found, and histopathological examination of the quadriceps and gastrocnemius muscles revealed no sign of rhabdomyolysis.

Conclusions

The present study, which is the first to test the association between hyponatremia and rhabdomyolysis in an animal model, does not support any causal association between hyponatremia and rhabdomyolysis. Thus, other factors might be necessary for an association between hyponatremia and rhabdomyolysis, such as genetic factors or convulsions that are known to be associated with both hyponatremia and rhabdomyolysis. Further research in this important physiologic and clinical question is needed.     

Comparison of the Nephrotoxicity of Netilmicin and Gentamicin in Rats

The nephrotoxicity of netilmicin relative to that of gentamicin was examined in Sprague-Dawley rats. Balance studies were performed on rats injected with netilmicin or gentamicin (50 mg/kg per day for 14 days, 100 mg/kg per day for 8 days, and 150 mg/kg per day for 8 days). Control rats were injected with saline. Both drugs caused a dose-related decrease in urine osmolality and increases in urine volume, water intake, and serum creatinine; however, the magnitude of these changes was significantly less in netilmicin- than in gentamicin-injected rats. Light microscopy of renal tissue revealed less proximal tubular cell necrosis in netilmicin- than in gentamicin-injected rats. There was no significant difference between the renal cortical concentrations of the two drugs. Both drugs stimulated uptake of p-aminohippurate in rat renal cortical slices to the same degree. The data indicate that netilmicin is less nephrotoxic than gentamicin in rats, that the difference in nephrotoxicity cannot be explained by a difference in drug concentration in the renal cortex, and that the ability of aminoglycosides to stimulate the organic acid transport system of proximal tubular cells does not correlate with their nephrotoxic potential.     

Pathophysiology of Experimental Glomerulonephritis in Rats

Micropuncture, clearance, immunofluorescence and light microscopy techniques were used to study kidney structure and single nephron function in rats with autologous immune complex nephritis (AICN), a membranous glomerulonephritis developing over 5 to 20 mo, in the more acute and proliferative glomerular basement membrane (GBM) nephritis and in controls. Both models are known to have clinical counterparts in human disease. Kidney functional abnormalities correlated with the degree of architectural derangement. In both AICN and anti-GBM nephritis filtration fraction fell in direct proportion to the fall in glomerular filtration rate (GFR), renal plasma flow being unchanged. Fractional electrolyte excretion increased as the GFR fell. Despite marked heterogeneity of single nephron filtration rate (SNGFR) (AICN, 5-93 nl/min; anti-GBM, 0-50 nl/min) and of proximal tubular hydrostatic pressure (4-48 mm Hg), each nephron showed almost complete glomerulotubular balance, absolute reabsorption to the late proximal convolution varying directly with filtration rate. In addition SNGFR could be related both to proximal intratubular hydrostatic pressure and to calculated glomerular capillary pressure (Pg), being lowest in those nephrons with the highest intratubular pressure. Nephrons with very high filtration rates did not apparently reach filtration equilibrium. Mean SNGFR was significantly lower in the anti-GBM group, while calculated Pg was the same in both. This probably reflects the acute and diffuse involvement of the anti-GBM lesion with different filtration characteristics from the more chronic AICN disease. Tubular damage was more marked in AICN, and extraction of p-aminohippurate was reduced in this group.     

Pharmacokinetics of Itraconazole in Diabetic Rats

After intravenous or oral administration of 10 mg/kg itraconazole to rats with streptozotocin-induced diabetes mellitus and to control rats, the total area under the plasma concentration-time curve from time 0 to 24 h (AUC_0-24) for itraconazole and that for its metabolite, 7-hydroxyitraconazole, were similar between the two groups of rats. This may be explained by the comparable hepatic and intestinal intrinsic clearance rates for the disappearance of itraconazole and the formation of 7-hydroxyitraconazole in both groups of rats.Itraconazole is a prototype triazole antifungal agent. Superficial fungal infections of the feet among elderly patients with diabetes mellitus are common, and itraconazole has been shown to have acceptable cure rates (12). In humans, hepatic cytochrome P450 3A4 (CYP3A4) appears to be involved in the metabolism of itraconazole to form several metabolites, including 7-hydroxyitraconazole (9). No in vivo studies of itraconazole metabolism in rats have been reported. Hepatic CYP3A1 (5) and CYP3A2 (10) proteins and/or mRNA levels have been shown to increase in male Sprague-Dawley rats with diabetes mellitus induced by streptozotocin (DMIS rats), but there are no reports on the intestinal CYP3A subfamily in DMIS rats. Furthermore, the pharmacokinetics of itraconazole and 7-hydroxyitraconazole may differ between intravenously and orally administered itraconazole in DMIS rats.In the present study, itraconazole metabolism was examined in DMIS rats as an animal model of diabetes mellitus. We report the pharmacokinetics of itraconazole and 7-hydroxyitraconazole after intravenous or oral administration in DMIS rats compared with those in control rats. Our results show that hepatic CYP3A1/2 is responsible for the metabolism of itraconazole and the formation of 7-hydroxyitraconazole in rats and that the expression of the intestinal CYP3A1/2 protein was not altered in DMIS rats compared with that in control rats, based on Western blot analysis.Overall, the methods used in this study were similar to those described in previous reports. The chemicals used in addition to itraconazole, the methods of housing and handling the male Sprague-Dawley rats (7 to 9 weeks old, weighing 230 to 280 g), the intravenous and oral administration of itraconazole, the measurement of plasma protein binding values of itraconazole by equilibrium dialysis, and the high-performance liquid chromatographic analysis of itraconazole and 7-hydroxyitraconazole were all performed as described previously (1, 11). Dia-betes mellitus was induced with streptozotocin (5). Seven control rats and eight DMIS rats were used in the intravenous administration study. Nine control rats and nine DMIS rats were used in the oral study. Intravenous administration of itraconazole to control rats pretreated with dexamethasone and troleandomycin was performed as previously described (3). Hepatic and intestinal microsomes were prepared from control and DMIS rats (6). The protein expression of intestinal CYP3A1/2 was examined by Western blot analysis (7).The procedures for measuring V_max and K_m for the disappearance of itraconazole and the formation of 7-hydroxyitraconazole were similar to those used in a previous report (6). Microsomes (equivalent to 0.5 mg protein); 5 μl of dimethyl sulfoxide containing 2.5, 5, 10, 20, 30, or 50 μM itraconazole; and 50 μl of 0.1 M phosphate buffer (pH 7.4) containing 1 mM NADPH were mixed and incubated for 0, 15, 30, 45, or 60 min for hepatic microsomes or for 5, 15, 30, 45, 60, or 75 min for intestinal microsomes. All microsomal incubation conditions were within the linear range of the reaction. After incubation for 45 min (for hepatic microsomes) or 50 min (for intestinal microsomes), 100 μl of each reaction mixture was transferred to a test tube containing 100 μg/ml {"type":"entrez-nucleotide","attrs":{"text":"R51012","term_id":"812914","term_text":"R51012"}}R51012 (internal standard) in 50 μl of acetonitrile, 250 μl of 0.1 M carbonate buffer (pH 9.8), and 1 ml of methyl t-butyl ether. The kinetic constants (K_m and V_max) were calculated using a nonlinear regression method (4). Intrinsic clearance (CL_int) was calculated by dividing V_max by K_m.The total area under the plasma concentration-time curve from time 0 to infinity (AUC_0-∞) or from time 0 to the last measured time at 24 h (AUC_0-24) was calculated using the trapezoidal rule-extrapolation method (2). The peak plasma concentration (C_max) and time needed to reach C_max (T_max) were directly read from the experimental data. The percentage of the dose excreted in a 24-h urine sample (Ae_0-24) and that recovered from the gastrointestinal tract (including its contents and feces) sampled after 24 h (GI_0-24) were also measured (11). All results are expressed as mean values ± standard deviations, with the exception of values for T_max, which are expressed as median values with ranges. Unpaired t tests were performed, and P values of <0.05 were regarded as statistically significant.Plasma protein binding values of itraconazole at 5 μg/ml were similar between the control (97.9% ± 0.242%) and DMIS (97.9% ± 0.137%) rats (n = 4 for each). The protein expression of intestinal CYP3A1/2, as determined by Western blot analysis, did not differ between the two groups of rats (n = 3 for each) (data not shown). Furthermore, K_m, V_max, and CL_int values for the disappearance of itraconazole and the formation of 7-hydroxyitraconazole in both hepatic and intestinal microsomes (n = 4) were comparable between the control and DMIS rats (Table ​(Table11).

TABLE 1.

V_max, K_m, and CL_int values for the disappearance of itraconazole and formation of 7-hydroxyitraconazole in hepatic and intestinal microsomes from control and DMIS rats

Virulence in Rats of Gentamicin-Carbenicillin-Resistant Pseudomonas

Strains of Pseudomonas aeruginosa, with resistance to gentamicin and carbenicillin which is R factor mediated, showed no alteration in virulence as tested by intraperitoneal injection of rats.