Viability Assessment of Primo-node Slices From Organ Surface Primo-vascular Tissues in Rats

Abstract

The primo-vascular system is a novel thread-like structure which is recently rediscovered, but its cellular properties are largely unknown. In this study, a slice preparation for primo-nodes was developed to facilitate study of the cellular properties of primo-node cells in vitro. Slices (4-8 slices; 200 μm thick) were sectioned from single primo-nodes collected from the abdominal organ surface of rats and incubated in oxygenated Krebs solution at 25°C or 31°C for up to 7 hours. Trypan blue staining and whole-cell patch-clamp recordings were performed to estimate the viability of cells in the slices. Viability was largely maintained during the first 3 hours, but subsequently decreased (from 80% to 21%, p < 0.001). In addition, the viability of slices incubated at 31°C was higher than those incubated at 25°C (80%vs. 47%, p < 0.001). In whole-cell patch-clamp experiments, high resistance seals readily formed and primo-node cells showed a mean resting membrane potential (-38 mV) comparable to that recorded with sharp electrodes and outwardly-rectifying current-voltage relationships. The results show that the primo-node slices developed in this study maintained viability for up to 4 hours in vitro.

Gross Morphological Features of the Organ Surface Primo-Vascular System Revealed by Hemacolor Staining

“…It has been consistently identified in various tissues including the surfaces of internal organs [5–8], blood vessels [9, 10], and lymphatic vessels [11] using special dyes, such as trypan blue [12, 13], acridine orange [9], and alcian blue [10, 11]. The organ-surface PVS has been primarily used in various PVS studies, as it is semitransparent, freely movable, and relatively easy to recognize with careful gross examination.…”

“…In addition, PVS cells have been categorized into four major types based on current-voltage (I-V) relations recorded from the cells in PN slices [7]. Some PVS cells are much larger and rounder (10–20  μ m) with granules, whereas others are much smaller and round or appear similar to red blood cells (RBCs) [7, 8]. …”

The Primo Vascular Structures Alongside Nervous System: Its Discovery and Functional Limitation

“…When measured using whole-cell patch clamp recordings, the cells found in the slices of primo nodes showed large variability in the cell populations, which was characterized by a lower resting membrane potential (−50 ~−10 mV), higher input resistance (200~1600 MΩ), and lower capacitance (5~20 pF) compared to the neurons in the hypothalamic paraventricular nucleus [9, 10]. In addition, no active components in the cell membrane properties were observed in the preparation of primo nodes [9, 10], suggesting that the cells found in the live slices of primo nodes are not excitable electrophysiologically [10].…”

“…When measured using whole-cell patch clamp recordings, the cells found in the slices of primo nodes showed large variability in the cell populations, which was characterized by a lower resting membrane potential (−50 ~−10 mV), higher input resistance (200~1600 MΩ), and lower capacitance (5~20 pF) compared to the neurons in the hypothalamic paraventricular nucleus [9, 10]. In addition, no active components in the cell membrane properties were observed in the preparation of primo nodes [9, 10], suggesting that the cells found in the live slices of primo nodes are not excitable electrophysiologically [10]. When cells in the primo nodes are classified based on the current-voltage relations and the kinetics of the activation and inactivation of currents generated by the voltage pulses (600 ms) in voltage-clamp mode, one type of cell was found to be sensitive to relatively high concentrations of tetraethylammonium (IC 50 = ~4.3 mM) [10], indicating that unidentified K + channels may be expressed in these cells.…”

Patch Clamp: A Powerful Technique for Studying the Mechanism of Acupuncture

“…Whole-cell recordings were performed on cells of a PN slice (200  μ m in thickness) isolated from the surface of abdominal organs in rats. The small round PN cells had a low resting membrane potential (−39 mV) and spontaneous activities [73, 74]. Of the four types of IV relationships and kinetics, most (69%) cells were type I and showed outward rectification.…”