The deficit of organ donors has fueled the need for advances in tissue engineering and regenerative medicine. Microencapsulation in alginate immuno-isolation membranes has been used to treat many disabling metabolic disorders, namely, phenylketonuria, kidney failure and diabetes mellitus. Systematic nutrient flux determinations are hindered by the lack of experimental data on alginate-based membrane topography and the pore size thus preventing the full therapeutic potential of the bio-membranes to be reached. In this study, samples of cross-linked alginate membranes were subjected to the following analytical characterization: 1) pore size characterization using atomic force microscopy operated in contact mode to detect and measure pore size; 2) differential scanning calorimetry to confirm biopolymer cross-linking; and 3) diffusivity measurements using spectrophotometry and fluorescence microscopy to confirm the presence of through pores and to calculate reflection coefficients. The pore sizes for the pre-clinical standard formulation of 1.5% (w/v) medium viscosity alginate cross-linked with 1.5% CaCl 2 and 0.5% (w/v) alginate and chitosan cross-linked with 20% CaCl 2 are 5.2 nm ± 0.9 nm and 7.0 nm ± 3.1 nm, respectively. An increase in the glass transition temperatures as a function of cross-linker concentration was observed. Diffusivity values obtained from the inward diffusivity of creatinine into macrocapsules (d = 1000 µm ± 75 µm) and the outward diffusivity of FITC dextrans from macrocapsules (d = 1000 µm ± 75 µm) and microcapsules (d = 40 µm ± 5 µm) were shown to correlate strongly (R 2 = 0.9835) with the ratio of solute to pore sizes, confirming the presence of through pores. Reflection coefficients approaching and exceeding unity correlate with the lack of permeability of the membranes to MW markers that are 70 kDa and greater.
Key points Persistent inward currents (PICs) in spinal motoneurons are long‐lasting, voltage‐dependent currents that increase excitability; they are dramatically potentiated by serotonin, muscarine, and noradrenaline (norepinephrine). Loss of these modulators (and the PIC) during sleep is hypothesized as a major contributor to REM sleep atonia. Reduced excitability of XII motoneurons that drive airway muscles and maintain airway patency is causally implicated in obstructive sleep apnoea (OSA), but whether XII motoneurons possess a modulator‐sensitive PIC that could be a factor in the reduced airway tone of sleep is unknown. Whole‐cell recordings from rat XII motoneurons in brain slices indicate that PIC amplitude increases ∼50% between 1 and 23 days of age, when potentiation of the PIC by 5HT2, muscarinic, or α1 noradrenergic agonists peaks at <50%, manyfold lower than the potentiation observed in spinal motoneurons. α1 noradrenergic receptor activation produced changes in XII motoneuron firing behaviour consistent with PIC involvement, but indicators of strong PIC activation were never observed; in vivo experiments are needed to determine the role of the modulator‐sensitive PIC in sleep‐dependent reductions in airway tone. Abstract Hypoglossal (XII) motoneurons play a key role in maintaining airway patency; reductions in their excitability during sleep through inhibition and disfacilitation, i.e. loss of excitatory modulation, is implicated in obstructive sleep apnoea. In spinal motoneurons, 5HT2, muscarinic and α1 noradrenergic modulatory systems potentiate persistent inward currents (PICs) severalfold, dramatically increasing excitability. If the PICs in XII and spinal motoneurons are equally sensitive to modulation, loss of the PIC secondary to reduced modulatory tone during sleep could contribute to airway atonia. Modulatory systems also change developmentally. We therefore characterized developmental changes in magnitude of the XII motoneuron PIC and its sensitivity to modulation by comparing, in neonatal (P1–4) and juvenile (P14–23) rat brainstem slices, the PIC elicited by slow voltage ramps in the absence and presence of agonists for 5HT2, muscarinic, and α1 noradrenergic receptors. XII motoneuron PIC amplitude increased developmentally (from −195 ± 12 to −304 ± 19 pA). In neonatal XII motoneurons, the PIC was only potentiated by α1 receptor activation (5 ± 4%). In contrast, all modulators potentiated the juvenile XII motoneurons PIC (5HT2, 5 ± 5%; muscarine, 22 ± 11%; α1, 18 ± 5%). These data suggest that the influence of the PIC and its modulation on XII motoneuron excitability will increase with postnatal development. Notably, the modulator‐induced potentiation of the PIC in XII motoneurons was dramatically smaller than the 2‐ to 6‐fold potentiation reported for spinal motoneurons. In vivo measurements are required to determine if the modulator‐sensitive, XII motoneuron PIC is an important factor in sleep‐state dependent reductions in airway tone.
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