作者:毛晓春,李贵刚,李彬,陈辉,杜龙庭,张虹 作者单位:1.华中科技大学同济医学院附属同济医院 眼科,湖北 武汉 430030;2.华中科技大学同济医学院附属襄樊医院 放射科,湖北 襄樊 44102
【摘要】 目的 使用磁共振成像(magnetic resonance imaging,MRI)评价兔结膜下药物注射后的渗透性和清除率,探讨一种能够在活体内动态观察的眼部药代动力学检测方法。方法 健康新西兰白兔24只,按随机数字表法分为3组,每组8只,取右眼作为实验眼,对侧眼作为对照眼。以MRI的造影剂钆喷酸葡胺(Gadolinium-diethylene triamine pentaacetic acids,Gd-DTPA)作为示踪剂,分别于结膜下注射0.5 mol/L、0.05 mol/L、0.005 mol/L造影剂0.1 ml,对照眼注射0.9%生理盐水0.1 ml。采用MRI扫描仪,于注药后1 h内每间隔15 min左右扫描1次,2~3 h内间隔30 min左右扫描1次,通过MRI观察药物的渗透性、眼内分布及结膜下的清除速度。0.5 mol/L组于术前和术后24 h进行角膜内皮镜检查,观察角膜内皮细胞密度、内皮细胞面积变异系数、六边形细胞百分数和中央角膜厚度。使用Excel绘制信号增强率对时间曲线,采用配对t检验分析角膜内皮细胞密度变化。结果 MRI扫描可见:0.5 mol/L组前房及睫状体信号明显增强,而眼后段则未探测到高信号;0.05 mol/L组前房信号无明显增强,接近注射位置的睫状体信号增强;0.005 mol/L组眼前、后段的药物渗透浓度均低于MRI探测阈值。睫状体部位对药物经巩膜的被动转运阻力最低,结膜下药物的体积和浓度随时间逐渐下降。结膜下Gd-DTPA用药前后,0.5 mol/L组的角膜内皮细胞密度、内皮细胞面积变异系数、六边形细胞百分数和中央角膜厚度差异均无统计学意义(P>0.05)。结论 MRI作为非侵袭、动态、实时的检查方法可于活体内定量分析结膜下药物注射后的渗透效率,是对传统药代动力学研究方法的有力补充。
【关键词】 磁共振成像;Gd-DTPA;结膜下注射;药代动力学;经巩膜药物转运
1Assessment of subconjunctival delivery of ionic permeants in rabbits with Gd-DTPA and magnetic resonance imaging in vivo
MAO Xiaochun, LI Guigang, LI Bin, et al.
Department of Ophthalmology, Tongji Hospital Affiliated to Tongji Medical College, Huazhong University of Science and Technology, Wuhan China, 430030
[Abstract] Objective To assess the permeability and clearance rate of a model for ionic permeants after subconjunctival injection using nuclear magnetic resonance imaging (MRI); to investigate a pharmacokinetic method for the ocular region in vivo. Methods Twenty-four rabbits were randomly divided into three groups according to a random numbers table, taking the right eye as the experimental eye and the left eye as the control eye. Experimental eyes in each group received subconjunctival injections of 0.5 mol/L,0.05 mol/L and 0.005 mol/L as a contrast agent. The control eyes received a subconjunctival injection of isotonic NaCl. All eyes were scanned once every 15 min for the first hour and once every 30 min for the next 2~3 hours. The permeability, distribution and subconjunctival clearance rate of the drugs were observed with MRI. The corneal endothelial cell density (ECD), coefficient of variance of the endothelial cell area (CV), percentage of hexagonal cells (6A) and central corneal thickness (CCT) were examined before and 24 h after the subconjunctival injection of the 0.5 mol/L contrast agent using a non-contact corneal endothelial microscope. The signal enhancement rate vs. time curve was plotted with Microsoft Excel software and statistical analysis of the changes in the corneal endothelium was done using a paired t-test. Results In the 0.5 mol/L group, there was an obvious strengthening of the signal in the anterior chamber and ciliary body, however, a strong signal was not detected in the posterior segments. In the 0.05 mol/L group, there was no obvious strengthening of the signal in the anterior chamber but the signal in the ciliary body close to the injection site strengthened. In the 0.005 mol/L group, the drugs and the osmotic concentration in the anterior and posterior segments of the eyes were below the detection threshold with MRI. The trans-scleral passive transport resistance of drugs was the lowest in the ciliary body. The subconjunctival pharmacological volume and concentration gradually decreased over time. There were no significant differences in ECD, CV, 6A and CCT before and after the experiment in the 0.5mol/L group (P>0.05). Conclusion Subconjunctival delivery of ionic permeants in vivo can be quantitatively analyzed by MRI. MRI is a noninvasive, dynamic and real-time complementary technique to traditional pharmacokinetic methods.
[Key words] magnetic resonance imaging; Gd-DTPA; subconjunctival injection; transscleral drug delivery; pharmacokinetics
结膜下注射是眼病治疗的常规手段,如激素、抗菌素结膜下注射后,药物直接通过组织持续渗透进入眼内。虽然结膜下注射在临床上已广泛使用,但在体结膜下注射的药代动力学研究还未完全明了。本研究以MRI的造影剂钆喷酸葡胺(Gadolinium-diethylene triamine pentaacetic acids,Gd-DTPA)作为示踪剂,通过MRI动态观察结膜下注射药物的渗透性、眼内分布及结膜下清除率,同时观察原浓度Gd-DTPA对角膜内皮细胞形态及角膜厚度的影响,以期探讨一种在活体内、动态、实时的眼部药代动力学检测方法。
1 资料和方法
1.1 实验动物
健康清洁级新西兰白兔24只,雌雄不限,体重2.0~2.6 kg(由华中科技大学同济医学院动物实验中心提供)。实验动物按随机数字表法分为3组,每组8只,取右眼作为实验眼,对侧眼作为对照眼。实验眼分别于结膜下注射0.5 mol/L、0.05 mol/L、0.005 mol/L Gd-DTPA造影剂,对照眼注射生理盐水。
1.2 仪器和试剂
Siemens Magnetom Avanto 1.5 T超导型MRI扫描仪(德国),非接触式角膜内皮照相仪(SP29000型,日本),Gd-DTPA(北京北陆药业股份有限公司)。
1.3 药物注射
以氯胺酮30 mg/kg、氯丙嗪15 mg/kg 肌肉注射使动物全身麻醉(扫描过程中每40 min重复肌肉注射同等剂量的麻醉剂)后,使用1 ml注射器,于12点位角巩缘后2~3 mm进针,术中应避免巩膜穿孔,之所以选择上方结膜是因为上方的可利用空间比下方大,注射药物分别为0.5 mol/L、0.05 mol/L、0.005 mol/L的Gd-DTPA 0.1 ml(以生理盐水稀释),对照组注射0.9%的生理盐水0.1 ml,冲洗结膜囊后,立即进行MRI扫描。
1.4 MRI检查方法
实验动物取仰卧位固定于自制扫描架上,采用膝关节专用线圈。MRI平扫与增强体位、序列及参数相同,采用SE T1WI(TR 480 ms,TE 14 ms)加脂肪抑制,横断位,层厚2 mm,间隔0.2 mm,视野(field of vision,FOV)13 cm×13 cm,像素256×256,激励次数3,扫描时间8 min 42 s。1 h内每间隔15 min左右扫描1次,2~3 h间隔30 min左右扫描1次;结膜下注射Gd-DTPA后扫描的体位、序列及参数与平扫相同。实验完成24 h后再次扫描,观察眼内有无Gd-DTPA残留。
1.5 感兴趣区信号增强率对时间曲线的绘制 影像分析由同一名MRI诊断医师应用MRI工作站完成,分别测量三个感兴趣区(睫状体、前房、后房)的平均信号强度,前房、睫状体、后房分别选择100、15、5个像素,选择的像素均在解剖结构之内,避开边界区以防止信号干扰。感兴趣区信号增强率(E)由以下公式计算:E=(St-S0)/S0。St是结膜下注射造影剂后某时间点的感兴趣区信号强度,S0是同一区域注射造影剂前的信号强度。
1.6 角膜内皮细胞形态及角膜厚度的测量
为了解Gd-DTPA对角膜内皮细胞的毒性,0.5 mol/L组结膜下注射前及注射后24 h采用非接触式角膜内皮镜测量角膜内皮细胞密度(corneal endothelial cell density,ECD)、内皮细胞面积变异系数(coefficient of variation of the endothelial cell’s area,CV)、六边形细胞的百分数(percentage of hexagonal cells,6A)及中央角膜厚度(central corneal thickness,CCT)。
1.7 统计学方法
本实验所有数据以x±s表示,信号增强率对时间曲线使用Excel绘制,角膜内皮分析采用SPSS 13.0软件进行统计学处理,统计方法为配对t检验。
2 结果
2.1 兔眼的MRI扫描
正常兔眼结构清晰,MRI平扫见图1。图2显示兔结膜下注射0.5 mol/L、0.05 mol/L、0.005 mol/L浓度的Gd-DTPA后的MRI影像。各组结膜下的低信号区和高信号区(上眼睑/结膜与巩膜/玻璃体之间的暗区和明亮区)分别为不同浓度Gd-DTPA的沉积处。结膜下注射0.5 mol/L Gd-DTPA后,在0.5 h内可见前房、睫状体信号增强,随时间延长,睫状体信号强度减低;前房信号强度逐渐升高,3 h时前房仍为高信号;0.05 mol/L组前房信号无明显增强,接近注射位置的睫状体信号增强;而0.005 mol/L组前房和睫状体信号均未见增强。在3 h的观察期内,结膜下注射3种浓度的Gd-DTPA,均未见后房、玻璃体的信号改变。对照组结膜下注射盐水,前房内未探测到高信号,45 min左右盐水基本吸收。在3 h观察期内实验组结膜下Gd-DTPA未完全消失。24 h后眼内无Gd-DTPA残留。
2.2 感兴趣区信号增强率对时间曲线
图3为各组平均信号增强率对时间曲线。感兴趣区分别为睫状体、前房、后房,睫状体信号在结膜下注射Gd-DTPA约15~25 min后快速增强,达到高峰后逐渐减弱;前房的信号在研究时限内缓慢增强,而后房未发现信号增强。
2.3 角膜厚度及角膜内皮细胞形态学改变
结膜下注射各种浓度造影剂并完成3 h的检查后,使用裂隙灯显微镜观察,角膜、晶状体保持透明,结膜、角膜无水肿,角膜内皮镜检查结果见表1。结膜下注射前及注射后24 h,角膜内皮细胞密度(ECD)、内皮细胞面积变异系数(CV)、六边形细胞的百分数(6A)及中央角膜厚度(CCT)比较,差异均无统计学意义。
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