微透析技术

微透析(Microdialysis)技术 微透析(Microdialysis)技术 (Microdialysis)灌流取样和透析技术 一种将灌流取样 透析技术 灌流取样 透析技术结合起来并逐渐 完善的一种从生物活体内进行动态微量生化取样 的新技术。具有活体连续取样、动态观察、定量 分析、采样量小、组织损伤轻等特点。

微透析系统装置主要由微量泵、微透析探头、收集器、连 接管及配套设备组成。 将一种具有透析作用的微细探针置于采样的 生物组织内，利用一部非常精细的注射泵，推 送溶液至探针处，以达到与组织内欲取出测量 的低分子量物质，进行透析交换，再将透析液 做进一步的分析。

脑微透析技术脑微透析技术是一种在体或离体脑化学采样 技术 ,其原理是脑细胞外液中可溶性分子可通过 一种半透膜管的微小径孔顺浓度梯度扩散。半 透膜管连接在经立体定位技术植入大脑特定部 位的探针上 ,探针则与灌注泵相连 ,向管内灌注 与管外组织间液近似的人工脑脊液或其他液体。

脑微透析探针

典型的同心圆型微透析探针包含连接有进 液和出液两根管子和一个圆柱状的透析膜 。

脑微透析与其他技术的比较项目 分析样品 脑损伤性 取样部位 操作复杂性 代谢产物 时间分辨率 空间分辨率 灵敏度 成本 脑微透析 无 有 组织的细胞外液 低 可检测 高 高 高 (10- 9~10- 3mol/L) 低 PET 正电子放射同位 素 无(但具有放射 性) 组织 高 不可检测 高 中 高 ( 10- 12mol/L ) 高 MRS 核素 无 组织 高 可检测 低 低 低 (10- 5~10- 3mol/L) 高

Intracerebral microdialysis during deep stimulation surgery1.BackgroundThis report describes the use of microdialysis in conjunction with deep stimulation (DBS) surgery to assess extracellular levels of neurotransmitters within the human basal ganglia(BG). Electrical stimulation of the subthalamic nucleus(STN) is an efficacious treatment for advanced Parkinson’s disease, yet the mechanisms ofSTN DBS remain poorly understood. Measurement of neurotransmitter levels within the BG may provide insight into mechanisms of DBS, but such an approach presents technical challenges.

2. Methods Five patients undergoing DBS surgery for advanced participated in our study.Four of the patients were male and one patient was female. The mean age at the time of surgery was 57 years, and the mean length of diagnosis was 8.7 years. Individual patient characteristics are reported in Table 1.

Table 1 Patient characteristicsPatient Age (years) 48 55 61 59 61 Length of diagnosis (years) 3 8 10 10 10 L-dopa equivalent medication before surgery (mg/day) 1050 625 500 625 675

1 2 3 4 5

All patients were asked to discontinue their antiparkinsonian medications at midnight prior to the morning of surgery. DBS surgery is typically performed in awake, conscious patients, although local a

nalgesia is provided at the surgical site.

2.1. Microdialysis baseline studiesPrior to the start of dialysis, this apparatus was perfused with a mock CSF solution for 5 min at 2.0 µL/min in order to discard the dead volume.The location of was confirmed using intraoperative microrecordings per routine. The standard DBS guide cannula was then replaced with a custom designed microdialysis guide cannula (Fig. 1), and the microdialysis probe was inserted so that its membrane was located in the (Fig. 2).

Fig 1

Fig 2

The microdialysis guide cannula was a modified 18gauge epidural needle. The needle was shortened such that the length from the top of the cuff to the tip of the needle was 60 mm. A polymethyl ring with outside diameter of 8 mm was added above the existing cuff. With these modifications, the needle could be mounted on the DBS microdrive in place of the standard guide cannula (see Fig. 1B). The catheter was perfused at a rate of 2.0 µL/min, and samples were collected every 15 min for 60 min in patients 1 and 2 (Fig. 3).

Fig 3

2.2. Microdialysis stimulation studiesAfter the location of was confirmed, the recording electrode was replaced with a stimulating electrode. We recorded along a track 2 mm anterior to the stimulating electrode until the was again localized. The microdialysis probe was then positioned in the anterior track so that the membrane was located in the (see Fig. 2). In patient 5 the microdialysis catheter was positioned in , 2 mm inferior to the bottom of . The catheter was perfused at a rate of 2.0 µL/min, and in these patients samples were collected every 10 min for a 40 min period.

2.3. Sample analysisDialysate samples were divided into two aliquots for measurement of , gamma-aminobutyric acid (GABA) and dopamine .①High-pressure liquid chromatography (HPLC) and fluorometric

detection were used for quantitation of and GABA . Samples were automatically derivatized with o-phthaldialdehyde and injected onto a reverse-phase C-18 column using a Model 717 plus Autosampler (Waters, Millipore, Milford, MA, USA). Derivatized amino acids were eluted with a stepped linear gradient using a buffer (50 mM, pH 6.0) with either 20 or 80% methanol and were detected with a McPherson Model FL-750 fluorescence detector (Acton, MA, USA) with a xenonmercury lamp set at 316 nm excitation. and GABA were identified by comparing their elution times to those of external standards. Levels were quantified by integrating the areas of the peaks and comparing these results with a linear regression analysis of a series of external standards.

② Dopamine was analyzed using HPLC with electrochemical detection. The HPLC mobile phase (12.42 citric acid, 39.85 mM monobasic, 0.25 mM , 0.74 mM 1decanesulfonic acid, 10.0 mM , 0.2% and 15–18% methanol, pH 4.3) was pumped through a reverse-phase 1mm × 100-mm ODS 3-µm microbore column (C-18; Bioanalytical Systems, West Lafayette, IN, USA) at a flow rate of 1 ml/min. was identified by compari

ng its elution time with that of reference standards. Unlike our procedure for quantification of and GABA, which used area under the curve, was quantified using peak heights and linear regression analysis of the peak heights obtained from a series of reference standards.

3. ResultsResults in the study are showed in the following tables and Fig.4

Note:

Patients 1–2 were dialyzed for a 60 min period and measurements of neurotransmitter concentration were performed every 15 min. In patients 3–5, microdialysis was performed for 40 min, with collections every 10 min.

Table 2 Concentrations of GlutamateTime (min) Patient 1a 10 15 20 30 40 45 60 0.810 0.672 2.018 1.443 0.947 5.068 17.78 6.440 14.072 11.15 3.807 5.224 4.405 3.501 6.021 3.446 3.530 Patient 2a GABA (µM) Patient 3 28.44 Patient 4 10.46 Patient 5 23.00

Table 3 Concentrations of GABATime (min) Patient 1a 10 15 20 30 40 45 60 0.338 0.233 0.032 0.030 0.313 0.042 1.048 0.098 0.132 0.156 0.103 0.065 0.211 0.095 0.102 0.110 0.201 Glutamate (µM) Patient 2a Patient 3 0.091 Patient 4 0.229 Patient 5 0.066

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