Behind the barrier: cerebral open flow microperfusion to monitor macromolecules in the brain

Neurological disorders are one of the most common, yet also most poorly treated
illnesses of the 21st century. The burden can in large part be explained by the several barriers that are inherently present to protect the central nervous system (CNS), slowing down drug discovery processes in this area of research. More specifically, the barriers make it difficult for macromolecular therapeutics to enter the CNS, on the one hand, and make it difficult to obtain information from the CNS, on the other hand.
There are only a few techniques available that allow for the determination of exogenous and endogenous (macro)molecules and their alterations over time directly from the cerebral interstitial space. This PhD thesis frames within a preclinical research project, involving microdialysis and cerebral open flow microperfusion (cOFM) as sampling techniques for macromolecules to obtain real-time information from the brain.
Historically, classical microdialysis was used to sample neurotransmitters and other small molecules. Nowadays, new probe types are on the market containing membranes with larger cut-off values that permit the exchange of compounds with a higher molecular weight, so-called large pore microdialysis. However, sampling remains challenging based on limitations regarding recovery rates and their associated analytical considerations (low sample volumes and concentrations), aspecific adsorption, pressure fluctuations, and tissue trauma including gliosis and blood-brain barrier (BBB) integrity. cOFM is a more recent in vivo sampling technique that is based on microdialysis but tries to overcome its
limitations. The two main features of the patented cOFM probe body design itself are the replacement of the membrane by macroscopic openings and the biocompatible material it consists of.
In a first step, we compared the applicability of cOFM and large pore microdialysis as techniques to sample macromolecules in the mouse brain. We explored both techniques, since they emerged around the same time and both offered interesting features. More specifically, we assessed the best timeframe to perform future experiments and determined the BBB integrity and inflammatory reaction around the probe track.
Immunohistochemical stainings showed an astrocytic and microglial reaction in the immediate vicinity along the implantation track for both probe types. Coronal sections showed higher BBB leakage around the microdialysis probe track than after cOFM sampling experiments, however this leakage was clearly limited compared to a positive control where the BBB was disrupted. For cOFM, according to the manufacturer, replacement of the healing dummy with the sampling insert causes limited damage. We hypothesize that the BBB is disrupted again after insertion of the sampling insert and flushing with the perfusate. We therefore suggest, when performing BBB studies using the cOFM probe type, to implement a longer run-in phase than recommended by the manufacturer.
In a second step, we implemented both techniques as a tool to gain insight into the brain penetrating properties and the brain pharmacokinetic profile of an exogenous anti transferrin receptor nanobody that should be able to cross the BBB by receptor-mediated transcytosis. A custom AlphaScreen assay was validated and has been found suitable to quantify nanobody concentrations in the cOFM and microdialysis samples. Overall, although it seems promising that we were able to quantify the anti-transferrin receptor nanobody in the interstitial fluid (ISF) of the mouse brain, our work also demonstrates that it remains challenging. Indeed, with cOFM or microdialysis low sample volumes with
nanobody concentrations in the pM/nM range are obtained and small-scale CNS damage is created. Therefore, sampling conditions must be monitored carefully. As for an acute experiment, our results do not indicate that one of the sampling techniques is superior to the other.
In a final experimental chapter, we applied cOFM to sample an endogenous macromolecule in the mouse brain in a chronic setting. More specifically, our overall aim is to gain insight into neurofilament light chain (NfL) levels during the different phases of temporal lobe epilepsy in the kainic acid mouse model—namely the status epilepticus (SE) and the chronic phase with spontaneous seizures—and thus assess the potential of NfL as a diagnostic fluid biomarker for epilepsy. In the last decade there is an ongoing quest to discover new epilepsy biomarkers. The topic is especially timely given the poor sensitivity of current state-of-the-art tools for epilepsy diagnosis, more specifically electroencephalography combined with structural brain imaging. Additionally, biomarker analysis would be of great value to assess the presence, alleviation, or cessation of epileptogenesis; to evaluate the risk of seizure recurrence; and to choose or evaluate the efficacy of a treatment. NfL levels were determined directly in the cerebral ISF through sampling with cOFM, as well as in cerebrospinal fluid (CSF) and plasma. Lastly, we hypothesize that NfL levels are attenuated upon curtailing SE with diazepam and ketamine. Here, we aim to assess the value of NfL as a response biomarker. Our findings indicate that NfL levels are increased during SE in both cerebral ISF and plasma.
Additionally, NfL levels are attenuated upon treatment of SE. The coherent levels in the CNS and peripheral samples demonstrate the translational potential of NfL as a blood based fluid biomarker for SE. This is less evident for chronic epilepsy, as in this casehigher NfL levels could only be detected in ISF and CSF, and not in plasma.
All things considered, we believe cOFM can offer great value in preclinical research to obtain information from the CNS. More specifically, it is an interesting tool for monitoring of endogenous (macro)molecules at the site of action to gain insights into the physiology of the brain and its disease processes, as well as for the determination of concentrations of exogenous (bio)pharmaceutical compounds for pharmacokinetic and -dynamic studies.