Following intrathecal injection of fluorescent tracers, ex vivo imaging of brain vibratome slices has been widely used to study the glymphatic system in the rodent brain. Tracer penetration into the brain is usually quantified by image-processing, even though this approach requires much time and manual operation. Here, we illustrate a simple protocol for the quantitative determination of glymphatic activity using spectrophotofluorometry. At specific time-points following intracisternal or intrastriatal injection of fluorescent tracers, certain brain regions and the spinal cord were harvested and tracers were extracted from the tissue. The intensity of tracers was analyzed spectrophotometrically and their concentrations were quantified from standard curves. Using this approach, the regional and dynamic delivery of subarachnoid CSF tracers into the brain parenchyma was assessed, and the clearance of tracers from the brain was also determined. Furthermore, the impairment of glymphatic influx in the brains of old mice was confirmed using our approach. Our method is more accurate and efficient than the imaging approach in terms of the quantitative determination of glymphatic activity, and this will be useful in preclinical studies.

鞘内注射荧光示踪剂后，离体脑振动切片的成像已被广泛用于研究啮齿动物大脑中的淋巴系统。示踪剂对大脑的渗透通常通过图像处理进行量化，尽管这种方法需要大量时间和手动操作。在这里，我们说明了使用分光光度法定量测定 glymphatic 活动的简单协议。在脑池内或纹状体内注射荧光示踪剂后的特定时间点，采集某些大脑区域和脊髓，并从组织中提取示踪剂。用分光光度法分析示踪剂的强度，并从标准曲线量化它们的浓度。使用这种方法，评估了蛛网膜下腔 CSF 示踪剂进入脑实质的区域和动态传递，并且还确定了从大脑中清除示踪剂。此外，使用我们的方法证实了老年小鼠大脑中glymphatic流入的损害。在定量测定淋巴活动方面，我们的方法比成像方法更准确和有效，这将在临床前研究中有用。