Characterising epi-perineurial barrier function by microscale techniques including a miniaturised Ussing chamber

Barriers of peripheral nerves, like the sciatic nerve, are complex structures, consisting of the inner endoneurial capillary barriers and the outer epi‑ and perineurial layers. The latter two, collectively also known as epi‑perineurium (EPN), are necessary for maintenance of the nerve homeostasis. However, the involvement of the EPN in altered nerve conduction in neuropathy is not well-understood. To date, reliable data on barrier properties and ion permeabilities have been limited by the difficulty of accessing the barrier experimentally. For analysing the EPN of rat sciatic nerves, we developed a preparation technique and a miniaturised (area 0.6 mm²), though edge damage-free, Ussing chamber. Electrophysiological characterisation included measurement of transepiperineurial resistance, differentiation of para- and transcellular contributions to this by two-path impedance spectroscopy and determination of permeabilities for flux markers and for ions by dilution and bi-ionic potential measurements.We found the EPN being definable as tight and responsive to changes in the gradients between the endoneurial and the extra-nerval compartment. In a rat model of bortezomib (Bortezomib)-induced polyneuropathy, we demonstrate the EPN to be impaired with a specific increase in potassium permeability, which normalises with the recovery of the animals.In conclusion, we present an advanced, dependable method to analyse the EPN, which can be extended to other microscale epi‑ or endothelia. Functionally, we demonstrate with this technique that the EPN forms a crucial and specific barrier to maintain ion gradients within the sciatic nerve. STATEMENT OF SIGNIFICANCE: We developed a miniaturized Ussing chamber allowing precise electrophysiological analysis of microscale barrier tissues, avoiding edge damage and experimental interferences. Using this, we characterized the epi‑perineurium (EPN) barrier of sciatic nerves, demonstrating it to be a tight and responsive barrier, essential for maintaining ion balance within that nerve. In a neuropathy model, we identified impaired potassium permeability during hyperalgesia, which normalized with recovery. Beyond the EPN, this method is broadly applicable to other previously inaccessible microscale barriers, enabling advanced studies of barrier (patho)physiology. Our work bridges biomaterial development and tissue barrier research, providing detailed insights into ion and solute transport, and may be used to study regulatory mechanisms and the subsequent development of potential therapeutic strategies such as targeted drug delivery across these barrier tissues.