生物血管内送货到大鼠肾脏

摘要

药物对肾功能恢复的管理需要本地化和治疗化合物的分布控制。在这里，我们详细描述了大鼠肾内药物递送一个简单的技术。这个过程可以与无死亡率和高再现性容易地进行。

摘要

The renal microvascular compartment plays an important role in the progression of kidney disease and hypertension, leading to the development of End Stage Renal Disease with high risk of death for cardiovascular events. Moreover, recent clinical studies have shown that renovascular structure and function may have a great impact on functional renal recovery after surgery. Here, we describe a protocol for the delivery of drugs into the renal artery of rats. This procedure offers significant advantages over the frequently used systemic administration as it may allow a more localized therapeutic effect. In addition, the use of rodents in pharmacodynamic analysis of preclinical studies may be cost effective, paving the way for the design of translational experiments in larger animal models. Using this technique, infusion of rat recombinant Vascular Endothelial Growth Factor (VEGF) protein in rats has induced activation of VEGF signaling as shown by increased expression of FLK1, pAKT/AKT, pERK/ERK. In summary, we established a protocol for the intrarenal delivery of drugs in rats, which is simple and highly reproducible.

引言

The renal microvasculature is involved in a wide spectrum of kidney diseases. Depending on the pathophysiology of disease, the endothelial cells may present structural or functional impairment, which may play a pivotal role in propagating kidney damage by creating an ischemic microenvironment. This renal microvascular dysfunction may catalyze the onset of a progressive deterioration of renal function over time, leading to chronic kidney disease (CKD), end-stage renal disease, hypertension and cardiorenal syndrome. In fact, untreated hypertension may have implications in renal arterioles, causing nephrosclerosis or glomerulosclerosis with significant reduction in vascular volume fraction, increase in vascular resistance and development of tubulointerstitial fibrosis¹.

Loss of renal microvasculature may be due to altered vascular homeostasis induced by local angiogenic/anti-angiogenic factors imbalance. This correlates with attenuated Vascular Endothelial Growth Factor (VEGF) signaling as well as elevated thrombospondin-1^2-4. Thus, using different animal models (mice, rats and pigs), the therapeutic effect of exogenous administration of VEGF has been recently investigated in some forms of renal disease, showing reduced interstitial fibrosis and stabilized renal and cardiac function^3-5. This effect is likely due to actions of VEGF on endothelial cells of the microvascular bed and inflammatory monocyte phenotype switching⁶.

For some preclinical studies, the use of rodents, the most commonly used laboratory animals, provides a good animal model for high throughput studies due to relatively low costs and ease of handling. Moreover, the use of genetically-altered rats as models of human diseases, such as hypertension, has become more and more frequent in the scientific community. Therefore, the aim of this protocol is to describe a useful intrarenal VEGF delivery technique in rats that is easy to perform and highly reproducible. Moreover, the same method can be used to selectively deliver other drugs.

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研究方案

实验在雌性SD大鼠进行的，体重250〜300克与指南的关怀和实验动物的使用（实验动物资源研究所，国家科学院，贝塞斯达，MD，USA），并在规定的标准符合所有动物的程序是由医学机构动物护理的梅奥诊所医学院批准与使用委员会（IACUC）。

1.准备

1. 术前高压灭菌所有的手术器械。如果在不同的大鼠多次手术的同一天计划，冲洗仪器每只动物手术后，然后消毒用热珠灭菌。
2. 麻醉，用4％的异氟醚的大鼠以1升/分钟_的 O _2。
3. 大鼠转移到控制的加热垫在37℃下以保持体温。保持用1-2％异氟烷麻醉在1升/分钟_的 O _2。
4. 辖止痛药物（丁丙诺啡缓释0.6毫克/千克）苏bcutaneously。
5. 适用软膏的眼睛，以防止在手术过程中干燥。
6. 为了补偿体液由于剖腹的损失，它辖10毫升/公斤的0.9％的生理盐水皮下注射的术前是重要的。
7. 剃腹部区域和清洁用聚维酮碘和70％乙醇垫皮肤。

2.手术过程

1. 确保镇静的深度是通过监测物理反射，如退出脚趾捏，眼睑反射，下颌音，呼吸速率/模式足够。
2. 执行通过小中线切口使用外科手术刀刀片10号剖腹手术（2-2.5厘米长度）。
3. 通过使用棉签拉肠和结肠的腹部右侧，并与在0.9％生理盐水以维持器官湿润浸透无菌纱布覆盖它们。
4. 轻轻向上缩回脾，肝，胃和胰腺揭露AORta和左肾动脉。
5. 用外科显微镜的帮助下，小心地分离上述和左肾和从静脉，脂肪左肾动脉并用钝解剖弯钳和无菌棉签周围结缔组织以下腹主动脉。
   1. 使用带有一个反复开闭动作（钝器解剖）沿所述容器的长度钳子去除结缔组织和棉签与横向滚动运动以除去脂肪。
      注:围主动脉区的解剖是一个非常微妙的一步，神经和淋巴管可能被损坏。确保解剖过程中，以保持动脉湿润用生理盐水。
6. 将4-0丝线缝合主动脉下方。
7. 使用微血管剪辑，夹住主动脉上述（正下方肠系膜动脉）并低于肾动脉分叉处。
8. 穿刺主动脉在左侧kidn的水平安永动脉分叉处有一个24克静脉导管，推进导管进入肾动脉。
   注:这是因为爆胎的关键一步，通过肾动脉可能发生。
9. 连接填充有药物溶液或盐水（达500微升）注射器到导管和灌注肾脏。
10. 灌注之后立即用镊子左肾静脉和左侧输尿管与微血管剪辑并移除导管。然后将一块可吸收止血剂明胶海绵，与组织粘合剂的一小滴，过主动脉的穿刺区域，并轻轻施加压力用棉签。
11. 在同一时间，从腹主动脉松开夹具，左肾动脉分叉下方。 5分钟后，释放来自肾静脉和输尿管钳。
12. 小心地从主动脉松开夹具，左肾动脉分叉以上，并允许肾再灌注。总肾缺血应该持续时间不超过7分钟。
确保无活动性出血发生密切观察该地区10多分钟。
13. 关闭两层（肌肉和皮肤）的腹部切口，用4-0可吸收缝线和连续模式，以防止感染。除了连续图案缝合技术，另一种选择是使用一个简单的，中断的技术，尤其是对体壁封闭以防止裂开。
14. 在切口区域，以防止感染的局部应用抗生素软膏。
15. 转移大鼠成无寝具观测笼上的温暖垫直到与设置在35-37℃的温度范围内完全恢复。宽松的床上用品应覆盖（ 如用悬垂或纸巾）或从笼子里删除，直到动物完全恢复，以防止窒息或被褥的愿望。
16. 手术后，连续观察动物直至自主呼吸，然后每小时为几个小时。重剂量的镇痛丁丙诺啡SR 72小时以后如果不适的迹象观察，如嗜睡，驼背和邋遢，鬼脸，没有恢复正常活动。
17. 所有的研究完成后，安乐死动物_二氧化碳过量吸入和收获的肾组织进行体外分析，如组织学和免疫印迹^5。

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结果

我们注射两种不同剂量重组大鼠VEGF（rrVEGF，0.17微克/公斤和5微克/ kg）或PBS中。使动物安乐死8小时手术后检查的VEGF途径的活化。与对照相比（ 图1B）时，如图由H＆E染色的外科手术过程没有影响灌注肾脏（ 图1A）的形态。而与对照相比（ 图1D）时天狼红染色没有显示出响应于缺血时间和rrVEGF（ 图1C）的输...

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讨论

The increasing incidence of chronic kidney disease raises the need for novel therapeutic approaches that can promote functional kidney recovery^7,8. Traditional therapies include the systemic administration of anti-inflammatory, anti-fibrotic drugs⁹. However, these strategies are frequently characterized by unwanted side effects due to off-target distribution of the injected drug. Therefore, in this manuscript, we describe a simple procedure for delivering drugs into the renal artery of rats. This pr...

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材料

Name	Company	Catalog Number	Comments
Surgical Microscope	Leica	M125
Isoflurane 100 ml	Cardinal Healthcare	PI23238	Anesthetic
Buprenorphine HCL SR LAB 1 mg/ml, 5 ml	ZooPharm Pharmacy		Buprenorphine narcotic analgesic formulated in a polymer that slows absorption extending duration of action (72 hr duration of activity).                                                        Liquid is viscous, warming to RT aids in drawing into syringe.                                                           Recommended dosage: 1 - 1.2 mg/kg SC. DO NOT DILUTE.
Puralube Vet Ophthalmic Ointment	Dechra	NDC17033-211-38	Sterile ocular lubricant
Lactated Ringer's Injection, USP, 250 ml VIAFLEX Plastic Container	Baxter Healthcare Corp.	NDC0338-0117-02	For body fluids replacement
Sol Povidone-Iodine  Swabstick, 3'	Cardinal Heatlhcare	23405-010B
Sterile cotton tipped applicators	Kendall	8884541300
4-0 silk suture (without needle)	Cardinal Heatlhcare	A183H
Vessel Clip, Straight, 0.75 mm x 4 mm Jaw	World Precision Instruments	501779-G
I.V. Catheter, Straight Hub, Radiopaque, 24 g x 3/4", FEP Polymer	Jelco	4053
Phosphate Buffered Saline	Life Technologies	10010023
SURGIFOAM Absorbable Gelatin Sponge	Cardinal Healthcare	179082
4-0 VICRYL PLUS (ANTIBACTERIAL) VIOLET 27" RB-1 TAPER	Ethicon	VCP304H	For muscle layer suturing
4-0 VICRYL PLUS (ANTIBACTERIAL) UNDYED 18" PC-3 CUTTING	Ethicon	VCP845G	For skin layer suturing
Triple antibiotic ointment	Actavis	NDC0472-0179-56	For topical use on the site of the incision
Recombinant Rat VEGF 164 Protein	R&D Sytems	564-RV
Rabbit monoclonal VEGFA	Abcam	ab46154
Rabbit monoclonal FLK1	Cell Signaling	9698
Rabbit monoclonal AKT	Cell Signaling	4691
Rabbit monoclonal phosphoAKT (Ser 473)	Cell Signaling	4060
Rabbit monoclonal p44/42 MAPK (ERK1/2)	Cell Signaling	4695
Rabbit monoclonal phospho p44/42 MAPK (Thr202 and Tyr 204)	Cell Signaling	4370