大鼠腰骶神经离断后不同时间膀胱形态功能变化及其对上尿路扩张的影响

Changes of bladder morphology and function and effect on upper urinary tract dilatation at different time after lumbosacral nerve transecting in rats

目的探讨大鼠腰骶神经离断后, 膀胱形态功能的变化及其对上尿路扩张的影响。方法取45只雌性SD大鼠, 采用随机数字表法随机分为3组, 每组15只。其中2组行椎管内双侧第6腰神经(L6)+马尾神经离断建立神经源性膀胱(NB)模型, 分别为神经离断4周组(NB-4W组)和神经离断12周组(NB-12W组);另一组大鼠仅打开椎管, 不离断神经, 为假手术组。NB-4W组手术4周后, NB-12W组和假手术组手术12周后, 分别行膀胱测压和肾脏超声检查, 检查完成后处死大鼠, 收集肾脏和膀胱组织行HE、Masson染色检查观察组织学变化, 免疫组化染色检测转化生长因子-β1(TGF-β1)表达情况, 蛋白质印迹法检测α-平滑肌肌动蛋白(α-SMA)表达情况。结果 NB-4W组和NB-12W组大鼠均无逼尿肌收缩, 最大膀胱测压容量分别为(5.84±0.33)、(3.13±0.35)ml, 逼尿肌漏尿点压力分别为(25.41±0.86)、(27.36±2.04)cmH_(2)O(1 cmH_(2)O=0.098 kPa), 均显著高于假手术组[(0.98±0.14)ml和(7.13±0.90)cmH_(2)O, P<0.05]。NB-4W组和NB-12W组膀胱顺应性分别为(0.28±0.21)、(0.17±0.12)ml/cmH_(2)O, 显著低于假手术组[(0.34±0.26)ml/cmH_(2)O, 均P<0.05];NB-12W组与NB-4W组的最大膀胱测压容量和膀胱顺应性差异均有统计学意义(P<0.05)。膀胱组织HE染色结果示NB-4W组和NB-12W组较假手术组膀胱壁增厚, 炎症细胞浸润明显;Masson染色结果显示NB-4W组、NB-12W组的胶原容积分数分别为(30.5±1.5)%、(45.2±3.8)%, 显著高于假手术组的(20.7±2.2)%(均P<0.05), 且NB-12W组的胶原容积分数显著高于NB-4W组(P<0.05)。NB-4W组膀胱组织TGF-β1、α-SMA表达量较假手术组升高, NB-12W组TGF-β1、α-SMA表达量较NB-4W组升高。肾脏超声检查示NB-4W组和NB-12W组分别有3只(20.0%)和7只(46.7%)大鼠出现肾积水;HE染色结果示NB-4W组和NB-12W组的肾小管损伤程度和炎症细胞浸润数量高于假手术组;Masson染色结果示NB-4W组和NB-12W组肾脏胶原容积分数分别为(13.1±1.4)%和(21.6±1.9)%, 较假手术组[(4.6±0.7)%]显著增加(均P<0.05)。结论采用双侧L6+马尾神经离断能够建立大鼠NB肾积水模型, 随着神经离断时间增加, 膀胱纤维化程度逐渐加重, 上尿路扩张发生率和严重程度也逐渐增加。

Objective To investigate the changes in the morphology,structure and function of the bladders and their effects on the upper urinary tract dilatation(UUTD)after lumbosacral nerve transecting in rats.MethodsA total of 45 female SD rats were included,randomly divided into 3 groups with 15 rats in each group.Two groups were performed bilateral lumbar 6(L6)and cauda equina nerve shearing to establish neurogenic bladder(NB)model,which were nerve transected for 4 weeks(NB-4W)group and nerve transected for 12 weeks(NB-12W)group.Another group was performed bilateral L6 nerves and cauda equine exposing but not transecting,which was sham-operation(Sham)group.Cystometry and renal ultrasound examination were performed and rats in each group were killed to collect the kidney and bladder tissues in NB-4W group at 4 weeks,in Sham group and NB-12W group at 12 weeks after operation.HE,Masson staining,immunohistochemical staining and western blot were used to detect histological changes,expression of transforming growth factor-β1(TCF-β1)andα-smooth muscle actin(α-SMA).Results All rats in NB-4W and NB-12W group showed acontractile detusor.In the NB-4W and NB-12W group the maximum cystometric capacity[(5.84±0.33)ml and(3.13±0.35)ml],the detrusor leak point pressure[(25.41±0.86)cm H_(2)O and(27.36±2.04)cm H_(2)O](1 cm H_(2)O=0.098 kPa),were significantly higher than those in the Sham group[(0.98±0.14)ml,(7.13±0.90)cm H_(2)O,both P<0.05].Compliance in NB-4W group[(0.28±0.21)ml/cm H_(2)O]and NB-12W group[(0.17±0.12)ml/cm H_(2)O]were significantly lower than that of the Sham group[(0.34+0.26)ml/cm H_(2)O],and the compliance of NB-12W group was lower than that of NB-4W group significantly(all P<0.05).HE staining of the bladder showed that the inflammatory cell infiltration was obvious in the NB-4W and NB-12W group.Bladder collagen volume fractions in NB-4W group[(30.5±1.5)%]and NB-12W group[(45.2±3.8)%]were both higher than that of Sham group[(20.7±2.2)%,both P<0.05].The expression of TCF-β1 andα-SMA in the bladder tissue of NB-4W group were higher than those of sham group,and that of NB-12W group were higher than NB-4W group.In NB-4W group and NB-12W group,3(20.0%)and 7(46.7%)rats were found hydronephrosis,respectively.Aditionally,HE staining showed that the degree of renal tubule injury and the number of inflammatory cell infiltration in the NB-4W and NB-12W group were higher than those in the Sham group.Masson staining showed that the volume fraction of collagen in kidneys of NB-4W and NB-12W group were(13.1±1.4)%and(21.6±1.9)%,respectively,which were significantly higher than that in sham operation group[(4.6±0.7)%,both P<0.05].Conclusions Bilateral L6+cauda equina nerve transecting can induce NB with hydronephrosis in parts of rats.The degree of bladder fibrosis gradually increased with the time of nerve transection,and the incidence and severity of UUTD also increased with the time of nerve transection.