原理:体内全肾脏主要解剖结构的介观可视化在肾积水的病理诊断和病因探讨中起着重要作用。然而,传统的成像方法在代表体内灌注的条件下无法实现微米分辨率的全肾成像。方法:我们使用体内冷冻固定术(IVCF)固定急性梗阻性肾积水(单侧输尿管梗阻,UUO),慢性自发性肾积水(db/db小鼠),和他们的对照小鼠肾脏用于低温显微光学切片断层扫描(cryo-MOST)自发荧光成像。我们定量评估了主要解剖结构的全肾脏病理变化,包括肾积水,肾亚区,动脉,静脉,肾小球,肾小管,和管周功能毛细血管。结果:通过与显微计算机断层扫描成像相比,我们证实IVCF可以维持体内肾脏的状态。Cryo-MOST自发荧光成像可以在没有造影剂的情况下显示具有细胞分辨率的主要肾脏解剖结构。UUO后3天和15周龄db/db小鼠肾脏的肾积水体积达到26.11±6.00mm3和13.01±3.74mm3,分别。在UUO小鼠肾脏中,外髓质(ISOM)的皮质和内部条纹的体积增加,而内部髓质(IM)的体积减少。Db/db小鼠还显示皮质体积和ISOM体积增加,但IM中没有萎缩。UUO和db/db小鼠肾脏的近曲小管和近曲小管的直径增加,表明近端小管受损。然而,一些肾小管在UUO小鼠中显示出异常的中央隆起突出,但是db/db小鼠的肾小管形态正常,提示两种模型之间肾积水的病理和严重程度存在差异。UUO小鼠肾脏也显示血管损伤,包括节段性动脉和静脉萎缩和弓形静脉扩张,皮质和IM的肾小管周围功能毛细血管密度分别降低了37.2%和49.5%,分别,提示肾脏缺氧.相比之下,db/db小鼠肾脏显示正常的血管形态和管周功能毛细血管密度。最后,我们发现db/db小鼠表现出膀胱输尿管反流和膀胱过度活动,这可能是肾积水形成的原因。结论:我们观察并比较了在UUO体内灌注条件下肾积水的主要肾脏结构变化,db/db,和对照小鼠通过cryo-MOST自发荧光成像。结果表明,与IVCF的冷冻MOST可以作为一个简单而强大的工具,定量评估体内的病理变化在三个维度。尤其是整个肾脏的体液分布.该方法可能适用于其他组织的三维可视化,器官,甚至整个身体,这可能为疾病的病理变化提供新的见解。
Rationale: Mesoscopic visualization of the main anatomical structures of the whole kidney in vivo plays an important role in the pathological diagnosis and exploration of the etiology of hydronephrosis. However, traditional imaging methods cannot achieve whole-kidney imaging with micron resolution under conditions representing in vivo perfusion. Methods: We used in vivo cryofixation (IVCF) to fix acute obstructive hydronephrosis (unilateral ureteral obstruction, UUO), chronic spontaneous hydronephrosis (db/db mice), and their control mouse kidneys for cryo-micro-optical sectioning tomography (cryo-MOST) autofluorescence imaging. We quantitatively assessed the kidney-wide pathological changes in the main anatomical structures, including hydronephrosis, renal subregions, arteries, veins, glomeruli, renal tubules, and peritubular functional capillaries. Results: By comparison with microcomputed tomography imaging, we confirmed that IVCF can maintain the status of the kidney in vivo. Cryo-MOST autofluorescence imaging can display the main renal anatomical structures with a cellular resolution without contrast agents. The hydronephrosis volume reached 26.11 ± 6.00 mm3 and 13.01 ± 3.74 mm3 in 3 days after UUO and in 15-week-old db/db mouse kidneys, respectively. The volume of the cortex and inner stripe of the outer medulla (ISOM) increased while that of the inner medulla (IM) decreased in UUO mouse kidneys. Db/db mice also showed an increase in the volume of the cortex and ISOM volume but no atrophy in the IM. The diameter of the proximal convoluted tubule and proximal straight tubule increased in both UUO and db/db mouse kidneys, indicating that proximal tubules were damaged. However, some renal tubules showed abnormal central bulge highlighting in the UUO mice, but the morphology of renal tubules was normal in the db/db mice, suggesting differences in the pathology and severity of hydronephrosis between the two models. UUO mouse kidneys also showed vascular damage, including segmental artery and vein atrophy and arcuate vein dilation, and the density of peritubular functional capillaries in the cortex and IM was reduced by 37.2% and 49.5%, respectively, suggesting renal hypoxia. In contrast, db/db mouse kidneys showed a normal vascular morphology and peritubular functional capillary density. Finally, we found that the db/db mice displayed vesicoureteral reflux and bladder overactivity, which may be the cause of hydronephrosis formation. Conclusions: We observed and compared main renal structural changes in hydronephrosis under conditions representing in vivo perfusion in UUO, db/db, and control mice through cryo-MOST autofluorescence imaging. The results indicate that cryo-MOST with IVCF can serve as a simple and powerful tool to quantitatively evaluate the in vivo pathological changes in three dimensions, especially the distribution of body fluids in the whole kidney. This method is potentially applicable to the three-dimensional visualization of other tissues, organs, and even the whole body, which may provide new insights into pathological changes in diseases.