Dynamics of Evans blue clearance from cerebrospinal fluid into meningeal lymphatic vessels and deep cervical lymph nodes

Abstract

Objectives Recently, it has been confirmed, that excess fluid and waste products from the brain are drained into the cerebrospinal fluid (CSF) and afterwards cleared via the olfactory route and/or lymphatic vessels in the brain dura and corresponding extracranial lymphatic structures. Therefore, the aim of present study was to monitor time-dependent uptake of Evans blue (EB) tracer from subarachnoid space into the meningeal lymphatic vessels and extracranial lymph nodes in rats during 3 hours-12 days. Methods EB was injected into the cisterna magna of anesthetized rats and after required survival, plasma, brain dura matter and corresponding lymph nodes (cervical, thoracic and lumbar) were dissected and processed for lymphatic vessels analyses using immunofluorescence and immunohistochemistry. Furthermore, we have used sensitive ultra-high-performance liquid chromatography (UHPLC) method for the determination of EB concentrations in selected samples. Results Using a combination of imaging methods, we have detected two different types of the vascular structures in the brain dura and in deep cervical lymph nodes. The blood vessels, which were RECA-1 + positive and the lymphatic-like vessels, expressing bright intense red fluorescence of EB tracer. Subsequently, using UHPLC with UV detection, we have quantified the EB concentration in positive structures by 3 hours up to 12 days after tracer delivery. A significant increase of EB concentration was detected in deep cervical lymph nodes already at 3 hours with a peak at 1 day that decreased to about one-tenth of its peak value by 12 days. Similar pattern was detected in brain dura. On the contrary, the brain tissue and plasma were almost negative for EB tracer during all tested time periods. Conclusion Our results demonstrate the dynamic changes of EB in meningeal lymphatic vessels and in deep cervical lymph nodes, thus recapitulating the downstream outflow of intracisternally injected tracer during 3 hours-12 days via dura mater lymphatic vessels towards corresponding extracranial draining system, particularly the deep cervical lymph nodes.

Spinal cerebrospinal fluid flow is increased in rats with elevated intracranial pressure 18 hours after cortical ischaemic stroke

“…Experiments to elucidate physiological CSF dynamics are challenging and often involve some sort of intervention. In this study, we infused the CSF tracer, Evans blue, directly into the lateral ventricles of the animals [18,21,22]. This type of intervention has the potential to disturb normal CSF dynamics; however, this technique is widely used and we took care to limit the infusion rate to lower than that of CSF production (2.66-2.84 µl/min) to maintain the integrity of the system as much as possible [23].…”

“…Several CSF drainage hypotheses have been presented in recent years and we are yet to meet a consensus on the topic [24][25][26]. In recent years, lymphatic drainage of CSF from both the cranium and the spinal space have gained merit in the eld, with several groups demonstrating the presence of CSF tracers in the peripheral lymphatic system, primarily the cervical lymphatics [21,[27][28][29][30]. This may be an interesting direction of investigation to understand how CSF drainage is altered post-stroke.…”

The involvement of perivascular spaces or tissues in the facial intradermal brain-targeted delivery

“…In the present study, using the dye EB as tool, we demonstrated the dermal-trigeminal-brain perivascular spaces or tissues were main pathways for intradermal brain-targeted delivery, and the lymphatic network linked to perivascular spaces or tissues. EB was extensively used as lymphatic vessels tracer in CSF drainage and the BBB permeability; in addition, EB binds to extracellular matrix, such as collagen, elastin and AMPA receptors (Heinle and Lindner, 1984; Weiser et al., 1996; Marchi et al., 2010; Maloveska et al., 2018). It is easy to visualize the structures or tissues and the lymphatic vessels.…”

“…The dermal-trigeminal-CNS lymphatic systems might connect to the perivascular spaces or tissues. In the CNS, dural lymphatic vessels, perivascular spaces, and parenchymal paravascular microcirculation function as exchange channels, and the dural lymphatic vessels connected the perivascular spaces and the extra-cranial lymphatic systems (Durant and Yaksh, 1986; Iliff and Nedergaard, 2013; Rangroo Thrane et al., 2013; Aspelund et al., 2015; Maloveska et al., 2018). The trigeminal perineurium and the epineurium elongate from pia and dura mater, respectively.…”

Brain lymphatic drainage system – visualization opportunities and current state of the art

“…Maloveska и соавт. обнаружили, что краситель Evans blue, введенный в большую цистерну, в течение 3 часов -12 дней дренируется через менингеальные лимфатические сосуды в направлении экстракраниальной дренажной системы, в частности в глубокие шейные лимфоузлы [50]. В работе [51] продемонстрировано, что менингеальные лимфатические сосуды ответственны за дренирование макромолекул и иммунных клеток из ЦСЖ при нейровоспалительных состояниях, а менингеальные лимфатические узлы управляют воспалительными процессами и иммунным наблюдением за ЦНС.…”