Apparatus and method for measuring physiological characteristics of an intact trachea in vitro

Abstract

Apparatus and methods for measuring smooth muscles responses (relaxation and contraction), transepithelial potential difference, and/or transepithelial impedance of an intact trachea in vitro. In particular embodiments, the apparatus includes a perfusion device on which an extracted, intact trachea is mounted. The perfusion device and the trachea are immersed in an extraluminal bath, which is isolated from the perfusion liquid flowing through the trachea. A set of voltage-sensing electrodes is provided for measuring the transepithelial potential difference across the trachea wall. A set of current electrodes is provided for inducing an electrical current to flow across the trachea wall in order to determine transepithelial impedance.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic diagram of an apparatus for measuring physiological characteristics of an intact trachea in vitro, according to one embodiment.

FIG. 2 is a perspective view of the perfusion device used in the apparatus of FIG. 1 for mounting the trachea.

FIG. 3 is a cross-sectional view of the perfusion device taken along line 3-3 of FIG. 2.

FIG. 4 is a cross-sectional view of the perfusion device taken along line 4-4 of FIG. 2.

FIG. 5 is a “flattened” cross-sectional view of the perfusion device taken along section 5-5 of FIG. 3.

FIG. 6 is a “flattened” cross-sectional view of the perfusion device taken along section 6-6 of FIG. 4.

FIG. 7 is an enlarged, fragmentary view of the perfusion device, extraluminal voltage electrode, and extraluminal current electrode of the apparatus shown in FIG. 1.

FIGS. 8A and 8B are data plots showing the effects of amiloride and NPPB, respectively, on the transepithelial potential difference (V_t) of a trachea. The vertical lines extending above the data plots show the voltage excursions in response to the application of a calibrated current across the trachea wall.

FIGS. 9A and 9B are bar graphs summarizing the effects of amiloride and NPPB on V_t and the transepithelia impedance (R_t) of the trachea.

FIGS. 10A and 10B are data plots showing the effects of ouabain and bumetanide, respectively, on the differential pressure (ΔP) of the trachea.

FIGS. 10C and 10D are data plots showing the effects of ouabain and bumetanide, respectively, on V_t of the trachea. The vertical lines extending above the data plots show the voltage excursions in response to the application of a calibrated current across the trachea wall.

FIGS. 11A and 11B are bar graphs summarizing the effects of ouabain and bumetanide on V_t and R_t of the trachea.

FIGS. 12A and 12B are data plots showing the effects of histamine and terbutaline, respectively, on ΔP of the trachea.

FIGS. 12C and 12D are data plots showing the effects of histamine and terbutaline, respectively, on V_t of the trachea. The vertical lines extending above the data plots show the voltage excursions in response to the application of a calibrated current across the trachea wall.

FIGS. 13A-13D are bar graphs summarizing the effects of histamine and terbutaline on V_t and R_t of the trachea.

FIGS. 14A and 14B are data plots showing the effects of hyperosmolarity elicited with NaCl and D-M, respectively, on ΔP of the trachea.

FIGS. 14C and 14D are data plots showing the effects of hyperosmolarity elicited with NaCl and D-M, respectively on V_t of the trachea. The vertical lines extending above the data plots show the voltage excursions in response to the application of a calibrated current across the trachea wall.

FIGS. 15A-15D are bar graphs summarizing the effects of hyperosmolarity elicited with NaCl and D-M on V_t and R_t of the trachea.

Claims

1. An apparatus for measuring physiological characteristics of an intact trachea in vitro, comprising: a perfusion device configured to flow a perfusion liquid through the trachea;a differential pressure device operable to measure a pressure difference of the perfusion liquid flowing through the trachea;a set of current electrodes comprising at least a first current electrode and a second current electrode operable to pass an applied current through the epithelium of the trachea between the first and second current electrodes; anda set of voltage-sensing electrodes comprising at least a first voltage-sensing electrode and a second voltage-sensing electrode operable to detect a transepithelial potential difference between the voltage-sensing electrodes.

2. The apparatus of claim 1, further comprising: a vessel containing an extraluminal bath;wherein the trachea is disposed in the extraluminal bath;wherein the first voltage electrode is in electrical continuity with the perfusion liquid flowing and the second voltage electrode is disposed in the extraluminal bath; andwherein the first current electrode is in electrical continuity with the perfusion liquid and the second current electrode is disposed in the extraluminal bath.

3. The apparatus of claim 2, wherein the second current electrode completely surrounds the perfusion device and the second voltage electrode.

4. The apparatus of claim 3, wherein the second current electrode comprises a metal screen.

5. The apparatus of claim 1, wherein the perfusion device comprises: an inlet portion adapted to introduce the perfusion liquid into a first end of the trachea and an outlet portion adapted to receive the perfusion liquid flowing outwardly from a second end of the trachea;a first, inlet conduit in fluidic communication with the inlet portion and adapted to introduce the perfusion liquid into the inlet portion;a second conduit in fluidic communication with the inlet portion and fluidly connected to the differential pressure device for measuring the pressure of the perfusion liquid flowing from the inlet portion and into the trachea;a third, outlet conduit in fluidic communication with the outlet portion and adapted receive the perfusion liquid flowing from the outlet portion;a fourth conduit in fluidic communication with the outlet portion and fluidly connected to the differential pressure device for measuring the pressure of the perfusion liquid flowing from the trachea and into the outlet portion.

6. The apparatus of claim 5, further comprising: a vessel containing an extraluminal bath;wherein the trachea is disposed in the extraluminal bath;wherein the first current electrode is in electrical continuity with the perfusion liquid flowing through the first conduit and the second current electrode is disposed in the extraluminal bath; andwherein the first voltage electrode is in electrical continuity with the perfusion liquid in the second conduit and the second voltage electrode is disposed in the extraluminal bath.

7. The apparatus of claim 5, wherein the perfusion device further comprises: an inlet cannula having a lumen, a proximal end portion in the inlet portion and a distal end portion extending outwardly from the inlet portion and into the first end of the trachea, the distal end portion having at least one opening;an outlet cannula spaced from and aligned with the inlet cannula, the outlet cannula having a lumen, a proximal end portion in the outlet portion and a distal end portion extending outwardly from the outlet portion and into the second end of the trachea, the distal end portion having at least one opening;wherein the first conduit and the inlet portion define an inlet flow path for the perfusion liquid between the outer surface of the inlet cannula and the inner surface of inlet portion;wherein the second conduit is in fluidic communication with the lumen of the inlet cannula at the proximal end thereof and the differential pressure device;wherein the third conduit and the outlet portion define an outlet flow path for the perfusion liquid between the outer surface of the outlet cannula and the inner surface of the outlet portion;wherein the fourth conduit is in fluidic communication with the lumen of the outlet cannula at the proximal end thereof and the differential pressure device; andwherein the differential pressure device measures the pressure difference of the perfusion liquid between the opening in the distal end portion of the inlet cannula and the opening in the distal end portion of the outlet cannula.

8. The apparatus of claim 1, further comprising voltage-measuring means for measuring the transepithelial potential difference between the voltage electrodes.

9. The apparatus of claim 8, further comprising current-generating means for generating the applied current to the current electrodes.

10. The apparatus of claim 9, wherein the voltage-measuring means and the current-generating means comprise a voltage/current clamp amplifier.

11. An apparatus for measuring physiological characteristics of an intact trachea in vitro, comprising: means for perfusing a perfusion liquid through the trachea;means for measuring a pressure difference of the perfusion liquid flowing through the trachea;means for passing a current through the wall of the trachea;means for measuring a transepithelial potential difference of the trachea; andmeans for determining the impedance of the trachea based on the current and the transepithelial potential difference.

12. The apparatus of claim 11, wherein the means for measuring a transepithelial potential difference of the trachea comprises a first voltage-sensing electrode in electrical continuity with the perfusion liquid, a second voltage-sensing electrode disposed in a liquid surrounding the trachea, and a voltage-measuring device operable to measure the transepithelial potential difference by measuring the potential difference between the voltage-sensing electrodes.

13. The apparatus of claim 12, wherein the means for passing a current through the wall of the trachea comprises a first current electrode in electrical continuity with the perfusion liquid, a second current electrode disposed in a liquid surrounding the trachea, and a current generator operable to generate and cause the current to flow from the first current electrode through the trachea wall to the second current electrode.

14. The apparatus of claim 13, wherein the second current electrode comprises a metal screen completely surrounding the second voltage-sensing electrode.

15. The apparatus of claim 11, wherein the means for perfusing a perfusion liquid through the trachea comprises a perfusion circuit that circulates a perfusion liquid through the trachea, the perfusion circuit comprising a trachea-mounting mechanism that includes an inlet portion and an outlet portion, wherein a first end of the trachea is secured to the inlet portion and a second end of the trachea is secured to the outlet portion such that circulating perfusion liquid flows into the trachea via the inlet portion and out of the trachea via the outlet portion.

16. A method for measuring physiological characteristics of an intact trachea in vitro, comprising: flowing a perfusion liquid through the trachea;measuring a transepithelial potential difference of the trachea;passing an electrical signal through the wall of the trachea; andmeasuring an electrical impedance of the trachea.

17. The method of claim 16, further comprising measuring a pressure difference of the perfusion liquid flowing through the trachea.

18. The method of claim 17, wherein the transepithelial potential difference, the electrical impedance, and the pressure difference are measured simultaneously.

19. The method of claim 16, wherein: the trachea is placed in an extraluminal bath; andmeasuring the transepithelial potential difference comprises measuring the electrical potential between first and second voltage-sensing electrodes, the first voltage-sensing electrode being in electrical continuity with the perfusion liquid flowing into the trachea and the second voltage-sensing electrode is disposed in the extraluminal bath.

20. The method of claim 19, wherein passing an electrical signal through the wall of the trachea comprises placing a first current electrode in electrical continuity with the perfusion liquid flowing into the trachea, placing a second current electrode in the extraluminal bath, and applying the electrical signal to the first current electrode such that the electrical signal is transmitted through the trachea wall to the second current electrode.

21. The method of claim 19, further comprising: adding an agent to the perfusion liquid; anddetermining the change in the transepithelial potential difference and the electrical impedance caused by the addition of the agent.

22. The method of claim 21, further comprising: prior to adding the agent to the perfusion liquid, measuring a pressure difference of the perfusion liquid flowing through the trachea; anddetermining the change in the pressure difference caused by the addition of the agent.

23. The method of claim 19, further comprising: adding an agent to the extraluminal bath; anddetermining the change in the transepithelial potential difference and the electrical impedance caused by the addition of the agent.

24. The method of claim 23, further comprising: prior to adding the agent to the extraluminal bath, measuring a pressure difference of the perfusion liquid flowing through the trachea; anddetermining the change in the pressure difference caused by the addition of the agent.

25. An apparatus for measuring physiological characteristics of an intact trachea in vitro, comprising: a perfusion device configured to flow a perfusion liquid through the trachea;a vessel containing an extraluminal bath, the trachea being immersed in the extraluminal bath;a differential pressure device operable to measure a pressure difference of the perfusion liquid flowing through the trachea;a set of voltage-sensing electrodes comprising a first voltage-sensing electrode in electrical continuity with the perfusion liquid and a second voltage-sensing electrode disposed in the extraluminal bath;a voltage-measuring device operable to measure a transepithelial potential difference of the trachea by measuring electrical potential between the voltage-sensing electrodes;a set of current electrodes comprising a first current electrode in electrical continuity with the perfusion liquid and a second current electrode disposed in the extraluminal bath and completely surrounding the trachea and the second voltage-sensing electrode; anda current generator connected to the current electrodes and operable to generate a current that passes through the epithelium of the trachea between the first and second current electrodes.

26. The apparatus of claim 25, wherein the perfusion device comprises: an inlet portion secured to a first end of the trachea and an outlet portion secured to a second end of the trachea so that perfusion liquid can flow into the first end of the trachea via the inlet portion and out of the second end of the trachea via the outlet portion;a first, inlet conduit in fluidic communication with the inlet portion for introducing the perfusion liquid into the inlet portion and the trachea;a second conduit in fluidic communication with the inlet portion and the differential pressure device for measuring the pressure of the perfusion liquid at a first location in the trachea;a third, outlet conduit in fluidic communication with the outlet portion and adapted receive the perfusion liquid from the outlet portion;a fourth conduit in fluidic communication with the outlet portion and the differential pressure device for measuring the pressure of the perfusion liquid at a second location in trachea downstream of the first location.