U.S. patent application number 14/674638 was filed with the patent office on 2017-03-09 for methods to determine fluid filtration rates through mammalian luminal organs.
The applicant listed for this patent is DTherapeutics, LLC. Invention is credited to Ghassan S. Kassab, Xiao Lu.
Application Number | 20170067879 14/674638 |
Document ID | / |
Family ID | 42667484 |
Filed Date | 2017-03-09 |
United States Patent
Application |
20170067879 |
Kind Code |
A9 |
Kassab; Ghassan S. ; et
al. |
March 9, 2017 |
METHODS TO DETERMINE FLUID FILTRATION RATES THROUGH MAMMALIAN
LUMINAL ORGANS
Abstract
Methods to determine fluid filtration rates through mammalian
luminal organs. In one method, the method comprises the steps of
positioning a segment of a mammalian luminal organ within a device,
the device configured to prevent axial flow conditions through a
lumen of the segment from a first end of the segment to an opposite
second end of the segment, obtaining a first segment measurement at
a first time, obtaining a second segment measurement at a second
time, and determining a rate of fluid filtration through a wall of
the segment based upon a difference between the first segment
measurement and the second segment measurement and a difference in
time between the first time and the second time.
Inventors: |
Kassab; Ghassan S.; (La
Jolla, CA) ; Lu; Xiao; (San Diego, CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
DTherapeutics, LLC |
San Diego |
CA |
US |
|
|
Prior
Publication: |
|
Document Identifier |
Publication Date |
|
US 20150204856 A1 |
July 23, 2015 |
|
|
Family ID: |
42667484 |
Appl. No.: |
14/674638 |
Filed: |
March 31, 2015 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
12727909 |
Mar 19, 2010 |
8992444 |
|
|
14674638 |
|
|
|
|
11919469 |
Oct 29, 2007 |
8998816 |
|
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PCT/US2006/016523 |
May 1, 2006 |
|
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|
12727909 |
|
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60675908 |
Apr 29, 2005 |
|
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
G01N 3/12 20130101; A61B
5/4047 20130101; A61B 5/4052 20130101; A61B 2503/40 20130101; G01N
33/4833 20130101; A61B 5/036 20130101; A61B 5/02007 20130101; A61B
5/04882 20130101; A61B 5/418 20130101; G01N 33/5088 20130101 |
International
Class: |
G01N 33/50 20060101
G01N033/50 |
Claims
1. A method, comprising the steps of: positioning a segment of a
mammalian luminal organ within a device, the device configured to
prevent axial flow conditions through a lumen of the segment from a
first end of the segment to an opposite second end of the segment;
obtaining a first segment measurement at a first time; obtaining a
second segment measurement at a second time; and determining a rate
of fluid filtration through a wall of the segment based upon a
difference between the first segment measurement and the second
segment measurement and a difference in time between the first time
and the second time.
2. The method of claim 1, wherein the step of obtaining a second
segment measurement is performed after the segment is in fluid
communication with an agonist introduced external to the
segment.
3. The method of claim 1, wherein the first segment measurement
comprises a first segment diameter measurement, and wherein the
second segment measurement comprises a second segment diameter
measurement.
4. The method of claim 3, wherein the determining step is further
based upon the first segment diameter measurement.
5. The method of claim 4, wherein the determining step is further
based upon a length of the segment.
6. The method of claim 1, wherein the first segment measurement
comprises a first segment volume measurement, and wherein the
second segment measurement comprises a second segment volume
measurement.
7. The method of claim 1, wherein the determining step is performed
to determine the rate of fluid filtration per surface area of the
segment.
8. The method of claim 1, further comprising the step of: obtaining
a rate of fluid filtration per surface area of the segment by
dividing the rate of fluid filtration by two.
9. The method of claim 3, wherein the first segment diameter
measurement and the second segment diameter measurement are
obtained using a device selected from the group consisting of a
camera and a microscope.
10. The method of claim 1, wherein the first segment measurement
comprises a first segment pressure measurement, and wherein the
second segment measurement comprises a second segment pressure
measurement.
11. The method of claim 10, wherein the first segment pressure
measurement and the second segment pressure measurement are
obtained using a pressure transducer in fluid communication with
the lumen of the segment.
12. The method of claim 1, further comprising the step of:
operating a volume compensator in fluid communication with the
lumen of the segment so to adjust a quantity of luminal fluid
within the lumen of the segment.
13. A method, comprising the steps of: positioning a segment of a
mammalian luminal organ within a device, the device configured to
prevent axial flow conditions through a lumen of the segment from a
first end of the segment to an opposite second end of the segment;
obtaining a first segment measurement at a first time; introducing
an agonist into a reservoir of fluid surrounding the segment so
that the segment is in fluid communication with the agonist;
obtaining a second segment measurement at a second time; and
determining a rate of fluid filtration through a wall of the
segment in response to the agonist based upon a difference between
the first segment measurement and the second segment measurement
and a difference in time between the first time and the second
time.
14. The method of claim 13, wherein the first segment measurement
comprises a first segment diameter measurement, wherein the second
segment measurement comprises a second segment diameter
measurement, and wherein the determining step is further based upon
the first segment diameter measurement.
15. The method of claim 13, wherein the first segment measurement
comprises a first segment volume measurement, and wherein the
second segment measurement comprises a second segment volume
measurement.
16. The method of claim 13, further comprising the step of:
operating a volume compensator in fluid communication with the
lumen of the segment so to adjust a quantity of luminal fluid
within the lumen of the segment.
17. The method of claim 16, wherein the operating step is performed
to introduce additional fluid from the volume compensator into the
lumen of the segment.
18. The method of claim 17, further comprising the step of:
obtaining a third segment measurement at a third time, the third
segment measurement equal to the first segment measurement and
resulting from the additional fluid within the lumen of the
segment.
19. A method, comprising the steps of: positioning a segment of a
mammalian luminal organ within a device, the device configured to
prevent axial flow conditions through a lumen of the segment from a
first end of the segment to an opposite second end of the segment;
obtaining a first segment pressure measurement at a first time;
introducing an agonist into a reservoir of fluid surrounding the
segment so that the segment is in fluid communication with the
agonist; obtaining a second segment pressure measurement at a
second time; and determining a rate of fluid filtration through a
wall of the segment in response to the agonist based upon a
difference between the first segment pressure measurement, the
second segment pressure measurement, and difference in time between
the first time and the second time.
20. The method of claim 19, further comprising the step of:
operating a volume compensator in fluid communication with the
lumen of the segment so to introduce additional fluid from the
volume compensator into the lumen of the segment.
Description
PRIORITY
[0001] The present application is related to, claims the priority
benefit of, and is a U.S. continuation application of, U.S. patent
application Ser. No. 12/727,909, filed Sep. 19, 2010 and issued as
U.S. Pat. No. 8,992,444 on Mar. 31, 2015, which is related to,
claims the priority benefit of, and is a U.S. continuation-in-part
patent application of, U.S. patent application Ser. No. 11/919,469,
filed Oct. 29, 2007, which is related to, claims the priority
benefit of, and is a U.S. national stage patent application of,
International Patent Application Serial No. PCT/US2006/016523,
filed May 1, 2006, which is related to, and claims the priority
benefit of, U.S. Patent Application Ser. No. 60/675,908, filed Apr.
29, 2005. The contents of each of these applications are hereby
incorporated by reference in their entirety into this
disclosure.
BACKGROUND
[0002] The present disclosure relates to the measurement of
isometric and isotonic contraction of blood vessels and luminal
organs. More particularly, the disclosure of the present
application relates to devices, systems and methods for isometric
and isotonic contraction of blood vessels and the determination of
isometric and isotonic activity of luminal organs using an
isovolumic myograph.
[0003] Vascular smooth muscle coils (VSMCs) modulate the tone of a
blood vessel in response to neural, humoral or local hemodynamic
stimuli. The VSMCs are important for auto-regulation and largely
determine the spatial and temporal distribution of blood flow in an
organ. Thus, conditions that affect the proper function of VSMCs
cause a variety of medical problems.
[0004] Many diseases, including hypertension, diabetes, heart
failure and atherogenesis, show signs of impaired arterial
vasoactivity. Hypertension, for example, is identified in relation
to changes in the myogenic tone of the resistance arteries. The
vasoactivity may be attenuated due to physiological (normal growth,
exercise, pregnancy, etc.) or pathological remodeling
(hypertension, hypertrophy, heart failure, etc.). The
pressure-induced myogenic response (or tone) is initiated as a
consequence of pressure-dependent modification of vascular smooth
muscle wall tension and subsequent activation of mechanosensitive
ion channels. Steady-state myogenic tone accounts for a substantial
portion of the peripheral resistance and is an important
determinant of arterial blood pressure. Although vasoconstriction
and vasodilation are intrinsic properties of VSMC, they are often
modulated by endothelium-derived vasoactive factors.
[0005] Because of the importance of maintaining proper vasoactivity
in VSMC, various drugs are tested for their effects on such
vasoactivity. Two of the tools used in such tests to identify
vasoactivity in blood vessels include the wire and pressure
myographs. A Medline search with keyword "wire myograph" or
"pressure myograph" reveals 140 and 207 publications, respectively,
from 1990 to the present having at least some reference to these
conventional tools for testing vasoactivity. In pharmacology, these
methods are used to understand the vasoreactivity and the
dose-response relation of various agonists and antagonists.
[0006] Although the wire myograph method is used often for
pharmacological experiments, it has a number of drawbacks, one
being that it is far from physiological. The mechanical deformation
of the ring is non-physiological and the cutting of the vessel
produces some injury to the vessel which has a direct impact on the
response of the vessel to the testing. In addition, the excision of
rings and attachment to hooks cause injury and lead to a
non-physiological geometry and loading. Furthermore, the reference
length for the vessel ring is unknown and comparison between
various vessels at various conditions is difficult to
standardize.
[0007] The pressure myograph was developed to address some of the
limitations of the wire myograph. In the pressure myograph, the
vessel geometry and loading are typically more physiological. The
pressure myograph method involves changes in pressure while
recording the change in diameter under passive and active
conditions. The method is substantially isobaric because the
pressure is maintained constant during contraction. Since the
radius changes during the test, which can change the wall stress
(based on Laplace's equation), this method of mechanical testing is
neither isometric nor isotonic, which in turn affects
interpretation of the results. Unlike the high sensitivity of wire
myograph that records tension, the pressure myograph records the
diameter changes under isobaric conditions and hence is limited to
small vessels that have substantial vasoactivity. Hence, there is
currently no unified myograph that applies to small as well as
large vessels under identical geometry, loading and testing
protocols.
[0008] Vascular endothelial dysfunction is widely considered to be
a consequence, a biomarker and a mediator of the adverse effects of
cardiovascular risk factors. Endothelial dysfunction precedes the
development of morphological atherosclerotic changes and can also
contribute to lesion development and later clinical complications.
Endothelial dysfunction has also been shown to be a predictor of
adverse outcomes in patients with coronary artery disease. Ongoing
efforts to identify and develop new drugs for the treatment of
atherosclerosis depend on robust evaluation of vascular lesion
pathology in preclinical models, a time consuming approach
associated with significant variability of the data.
[0009] The stomach is largely dependent upon extrinsic nervous
inputs arising from the central nervous system. These inputs
regulate the smooth muscles and coordinate the digestive function
of stomach by parasympathetic and sympathetic pathways. The
excitatory neurotransmitters by efferent vagus fibers (mainly
acetylcholine and tachykinins) cause rhythmic contractions of
gastric smooth muscles. The gastric smooth muscles exhibit the tone
on which there is superimposition of rhythmic contractions driven
by cycles of membrane depolarization and repolarization.
[0010] In addition, it has been known for nearly three decades that
the gastric mechanoreceptors which respond to gastric muscular
distension and contraction are implicated in post-prandial satiety,
in sensing the effectiveness of a contraction to expel contents,
and in a variety of reflexes. Electrophysiological studies in
different species have shown that mechanosensitive afferent fibers
located in the antrum muscle wall respond to changes in smooth
muscle transmural and local tension with an increased firing rate.
Gastric distension is correlated with a firing of vagal
mechanosensitive afferent fibers, which play an important role in
satiety.
[0011] The physical forces that act on the intestinal wall during
the intestine contraction propels chyme. The intestinal tract is
abundantly innervated with mechanosensors in response to the
physical forces in intestinal wall when a meal transits through the
gut. The excitation of extrinsic sensory afferents provides clear
evidence on the intestinal mechanosensory endings in response to
distension, responding to mechanical stimulation arising during
distension and contraction. The level of mesenteric afferent firing
is a proportional increase when the intraintestinal pressure
increases. Brain-gut interactions are recognized as major players
the in physiological and pathpophysiological regulation of the
intestinal tract, as the intestinal tract possesses an intrinsic
nervous plexus (pacemaker) that allows the intestine to have a
considerable degree of independent control from central nervous
system.
[0012] Intestinal motility is one of the objectives of central
nervous system and local nervous regulation. Intestinal motility
disorders exist in a pathological state, such as intestinal
obstruction or ileus. Laparotomy and manipulation also interfere
with intestinal movements. The most widely accepted explanation of
postoperative ileus was based on the idea that manipulation
inhibited motor function through some sort of neurologic reflex
response. Experimental studies have identified central neural
influences that mediate ileus of the gastrointestinal tract. Three
main mechanisms are involved in its causation, namely neurogenic,
inflammatory and pharmacological mechanisms. In the acute
postoperative phase, mainly spinal and supraspinal adrenergic and
non-adrenergic pathways are activated. However, although the
mechanical sensory and afferent excitation in response to
mechanical stimulation have been extensively studied, the
alteration of intestinal motility in response to mechanical
stimulation is poorly understood since the response of the motility
experiences a cycle of the intestinal sensor to afferent nerve to
central nervous system to efferent nerve finally back to intestinal
smooth muscle.
[0013] Thus, although both of the above conventional methods are
widely in use, a need exists in the art for an alternative to the
conventional techniques for testing vasoactivity in blood vessels
such that the need addresses the setbacks and limitations of the
conventional techniques, while at the same time, is easy to use and
interpret and provides a more accurate measurement of vasoactivity.
In addition, a need also exists in the art for various devices,
systems, and methods to determine isotonic and isometric of
non-vascular luminal organs, such as the stomach and the
intestines.
BRIEF SUMMARY
[0014] In at least one embodiment of a method for detecting a
luminal organ response to mechanical stimulation of the present
disclosure, the method comprises the steps of maintaining a luminal
organ at a first internal pressure, increasing the first internal
pressure of the luminal organ, and measuring a first organ
parameter change in response to the increase in internal pressure.
In another embodiment, the luminal organ is positioned within a
chamber for receiving a fluid, and wherein the fluid is in contact
with the luminal organ. In yet another embodiment, the step of
maintaining a luminal organ at a first internal pressure comprises
the steps of positioning a conduit within an incision of the
luminal organ so that a lumen of the conduit is in fluid
communication with a lumen of the luminal organ, and introducing a
liquid through the conduit into the lumen of the luminal organ
until the luminal organ achieves the first internal pressure. In an
additional embodiment, the step of increasing the first internal
pressure of the luminal organ comprises the step of introducing a
fluid from the conduit into the lumen of the luminal organ.
[0015] In at least one embodiment of a method for detecting a
luminal organ response to mechanical stimulation of the present
disclosure, the step of maintaining a luminal organ at a first
internal pressure comprises the steps of positioning the luminal
organ within a system for detecting a luminal organ response,
introducing a fluid into a lumen of the luminal organ until a
desired first internal pressure is achieved, and closing at least
part of the system so that fluid is not permitted to escape the
luminal organ through a component of the system. In another
embodiment, the first organ parameter change is selected from the
group consisting of a decrease in luminal organ diameter, an
increase in luminal organ diameter, a decrease in internal luminal
organ pressure, an increase in internal luminal organ pressure, and
an increase in gastric contractility. In at least one embodiment,
the step of measuring a first organ parameter change is performed
using a device selected from the group consisting of a pressure
transducer, a microscope, and a camera.
In at least one embodiment of a method for detecting a luminal
organ response to mechanical stimulation of the present disclosure,
the step of maintaining a luminal organ at a first internal
pressure comprises the step of injecting additional fluid into a
lumen of the luminal organ in response to luminal organ leakage
through a wall of the luminal organ. In another embodiment, the
step of injecting additional fluid is performed using a volume
compensator. In another embodiment, the luminal organ is present
within a living mammal.
[0016] In at least one embodiment of a system for detecting a
luminal organ response to mechanical stimulation of the present
disclosure, the system comprises a first conduit having a proximal
end, a distal end, and a lumen therethrough, the distal end sized
and shaped to fit within a luminal organ, a pressure transducer,
and at least one pressurized vessel capable of introducing a fluid
into the lumen of the first conduit, wherein the first conduit, the
pressure transducer, and the at least one pressurized vessel are
either directly or indirectly coupled to one another so that a
pressure of a fluid present within the first conduit can be
measured using the pressure transducer. In another embodiment, the
system further comprises a chamber for receiving a fluid and the
luminal organ. In another embodiment, the luminal organ is selected
from the group consisting of a stomach, a trachea, a lymph vessel,
a lymph duct, a urinary bladder, a ureter, a gall bladder, a bile
duct, a hepatic duct, and an intestine. In yet another embodiment,
the distal end of the conduit is positioned within an incision of
the stomach so that the lumen of the first conduit is in fluid
communication with a lumen of the stomach. In an additional
embodiment, the distal end of the conduit is positioned within an
incision of the luminal organ while the luminal organ is present
within a living mammal so that the lumen of the first conduit is in
fluid communication with a lumen of the luminal organ. In at least
one embodiment, when the distal end of the first conduit is
positioned within the luminal organ, the system is operable to
detect a response of the luminal organ to an increase in internal
pressure of the luminal organ.
[0017] In at least one embodiment of a system for detecting a
luminal organ response to mechanical stimulation of the present
disclosure, the system further comprises a chamber for receiving a
fluid and the luminal organ, and wherein the chemical is introduced
into the chamber. In another embodiment, the system further
comprises a volume compensator in communication with the first
conduit, wherein the volume compensator is operable to inject a
liquid so that the liquid increases the pressure within the luminal
organ. In an exemplary embodiment, the volume compensator comprises
a syringe. In another embodiment, the system further comprises a
device capable of detecting a physical change to the luminal organ.
In yet another embodiment, the device is selected from the group
consisting of a camera and a microscope. In an additional
embodiment, the system farther comprises a pressure regulator in
communication with the at least one pressurized vessel, the
pressure regulator capable of regulating a vessel pressure.
[0018] In at least one embodiment of a method of detecting a
luminal organ response to one or more chemicals of the present
disclosure, the method comprises the steps of maintaining a luminal
organ at a first length and a first internal pressure within a
fluid bath, introducing a first chemical into the fluid bath, and
measuring a first organ parameter change in response to exposure of
the luminal organ to the first chemical. In at least one
embodiment, the chemical causes the luminal organ to constrict. In
another embodiment, the first organ parameter change is selected
from the group consisting of a decrease in luminal organ diameter
and an increase in internal luminal organ pressure. In yet another
embodiment, the chemical causes the luminal organ to expand. In an
exemplary embodiment, the first organ parameter change is selected
from the group consisting of an increase in luminal organ diameter
and a decrease in internal luminal organ pressure.
[0019] In at least one embodiment of a method of detecting a
luminal organ response to one or more chemicals of the present
disclosure, the first organ parameter change is detected using a
device selected from the group consisting of a camera, a pressure
transducer, and a microscope. In an exemplary embodiment, the step
of maintaining a luminal organ at a first length and a first
internal pressure comprises the steps of positioning the luminal
organ within a system for detecting a luminal organ response,
adjusting the length of the luminal organ until the first length is
achieved, introducing a fluid into a lumen of the luminal organ
until a desired first internal pressure is achieved, and closing at
least part of the system so that fluid is not permitted to escape
the luminal organ through a component of the system. In an
additional embodiment, the step of maintaining a luminal organ at a
first length and a first internal pressure comprises the step of
injecting additional fluid into a lumen of the luminal organ in
response to luminal organ leakage through a wall of the luminal
organ. In another embodiment, the step of injecting additional
fluid is performed using a volume compensator in fluid
communication with the lumen of the luminal organ.
[0020] In at least one embodiment of a method of detecting a
luminal organ response to one or more chemicals of the present
disclosure, the first length is substantially a length of the
luminal organ when the luminal organ was present within a mammal
prior to removal of the luminal organ and placement of the luminal
organ within the fluid bath. In another embodiment, the first
length is longer than a length of the luminal organ when the
luminal organ was present within a mammal prior to removal of the
luminal organ and placement of the luminal organ within the fluid
bath, and wherein the first organ parameter change is in part
related to an axial overstretch of the luminal organ. In an
additional embodiment, the method further comprises the steps of
stretching the luminal organ to a second length, measuring a second
organ parameter change in response to the exposure of the luminal
organ to the first chemical, and comparing the first organ
parameter change to the second organ parameter change to determine
a response indicative of axial overstretch.
[0021] In at least one embodiment of a method of detecting a
luminal organ response to one or more chemicals of the present
disclosure, the first pressure is substantially a pressure within
the luminal organ when the luminal organ was present within a
mammal prior to removal of the luminal organ and placement of the
luminal organ within the fluid bath. In another embodiment, the
first pressure is higher than a pressure within the luminal organ
when the luminal organ was present within a mammal prior to removal
of the luminal organ and placement of the luminal organ within the
fluid bath, and wherein the first organ parameter change is in part
related to a circumferential overstretch of the luminal organ. In
yet another embodiment, the method further comprises the steps of
introducing a fluid into a lumen of the luminal organ so that the
luminal organ has a second internal pressure higher than the first
internal pressure, measuring a second organ parameter change in
response to the exposure of the luminal organ to the first
chemical, and comparing the first organ parameter change to the
second organ parameter change to determine a response indicative
circumferential overstretch.
[0022] In at least one embodiment of a method of detecting a
luminal organ response to one or more chemicals of the present
disclosure, the luminal organ is selected from the group consisting
of a blood vessel and any mammalian organ having a lumen therein.
In another embodiment, the method comprises the steps of
introducing a second chemical into the fluid bath, and measuring a
second organ parameter change in response to exposure of the
luminal organ to the second chemical. In an exemplary embodiment,
the first chemical causes an increase in intraluminal pressure and
circumferential tension of the luminal organ, and wherein the
second chemical causes a decrease in intraluminal pressure and
circumferential tension of the luminal organ. In yet another
embodiment, the method further comprises the step of determining a
percent relaxation of intraluminal pressure and circumferential
tension based upon at least the increase in intraluminal pressure
and circumferential tension of the luminal organ in response to the
first chemical and the decrease in intraluminal pressure and
circumferential tension of the luminal organ in response to the
second chemical.
[0023] In at least one embodiment of a system for detecting a
luminal organ response to one or more chemicals of the present
disclosure, the system comprises a chamber for receiving a fluid, a
retaining device positioned at least partially within the chamber,
the retaining device capable of retaining a luminal organ
positioned therein at a first length and a first internal pressure,
a first conduit coupled to the first retaining wall and a second
conduit coupled to the second retaining wall, and at least one
pressurized vessel, the at least one pressurized vessel coupled to
at least one of the first conduit and the second conduit. In
another embodiment, and when a luminal organ is retained therein,
the system is operable to facilitate detection of a response of the
luminal organ to one or more chemicals introduced to the luminal
organ. In yet another embodiment, the chemical is introduced into
the chamber. In an additional embodiment, the chemical is
introduced into a lumen of the luminal organ via at least one of
the first conduit and the second conduit.
[0024] In at least one embodiment of a system for detecting a
luminal organ response to one or more chemicals of the present
disclosure, the system further comprises a volume compensator in
communication with at least one of the first conduit, the second
conduit, and the at least one pressurized vessel. In another
embodiment, the volume compensator is operable to inject a liquid
so that the liquid increases a pressure within the luminal organ.
In yet another embodiment, the volume compensator is operable to
inject a liquid into the luminal organ in response to a detected
loss of fluid from the luminal organ. In an additional embodiment,
the volume compensator comprises a syringe.
[0025] In at least one embodiment of a system for detecting a
luminal organ response to one or more chemicals of the present
disclosure, the system further comprises a device capable of
detecting a physical change to the luminal organ. In at least one
embodiment, the device is selected from the group consisting of a
camera, a pressure transducer, and a microscope. In another
embodiment, the at least one pressurized vessel is operable to
inject a solution present therein into the luminal organ. In yet
another embodiment, the system further comprises a pressure
regulator in communication with the at least one pressurized
vessel, the pressure regulator capable of regulating a vessel
pressure. In an additional embodiment, the retaining device
comprises a first retaining wall and a second retaining wall.
BRIEF DESCRIPTION OF THE DRAWINGS
[0026] FIG. 1 shows an exemplary embodiment of an isovolumic
myograph in the process of testing a blood vessel, according to the
present disclosure;
[0027] FIG. 2 shows an exemplary dosage-dependent myogenic response
to phenylephrine, according to the present disclosure;
[0028] FIG. 3 shows tension-diameter relationships for passive and
active properties of carotid artery and femoral artery and vein,
according to the present disclosure;
[0029] FIG. 4 shows a time course of pressure decrease during
vasodilation with sodium nitroprusside (SNP), according to the
present disclosure;
[0030] FIG. 5A shows an exemplary embodiment of an automated
isometric or isotonic myograph in the process of testing a blood
vessel, according to the present disclosure;
[0031] FIG. 5B shows a pressure or volume control feedback loop as
used in one or more exemplary embodiments of the present
disclosure;
[0032] FIG. 6 shows an exemplary isometric experiment on a swine
right coronary artery (RCA) reflecting a regulated pressure to
maintain a constant diameter during vasomotion, according to the
present disclosure;
[0033] FIG. 7 shows an exemplary isotonic experiment on a swine
right coronary artery (RCA) reflecting a regulated pressure to
maintain a constant tension (product of pressure and diameter)
during vasomotion, according to the present disclosure;
[0034] FIG. 8 shows an exemplary embodiment of an isovolumic
myograph having electrical stimulus and in the process of testing a
blood vessel, according to the present disclosure;
[0035] FIG. 9 shows an exemplary embodiment of an isovolumic
multi-vessel myograph, according to the present disclosure;
[0036] FIG. 10A shows an exemplary embodiment of an isovolumic
multi-pressure myograph, according to the present disclosure;
[0037] FIG. 10B shows a schematic perspective of an exemplary
myograph having multiple pulsatile pressure controls, according to
the present disclosure;
[0038] FIGS. 11A and 11B show exemplary embodiments of an
isovolumic myographs, according to the present disclosure;
[0039] FIG. 11C shows steps of an exemplary method of detecting a
luminal organ response to one or more chemicals, according to the
present disclosure;
[0040] FIG. 11D shows a schematic of the range of applicability
(size of vessels) of a wire myograph, a pressure myograph, and an
isovolumic myograph, according to the present disclosure;
[0041] FIG. 12 shows a typical tracing curve of intraluminal
pressure before and after volume compensation, and thereafter in
response to vasoconstriction, according to the present
disclosure;
[0042] FIG. 13A shows three typical tracing curves of intraluminal
pressure in response to pharmacological vasoconstriction and
vasorelaxation, according to the present disclosure;
[0043] FIG. 13B shows that the percent relaxations of aorta,
femoral artery, and mesenteric artery are calculated as the ratio
of pressure deference from the tracing curves of intraluminal
pressures, according to the present disclosure;
[0044] FIG. 14A shows the interrelationship of % Relaxation
resulted from circumferential tension and transmural pressure,
according to the present disclosure;
[0045] FIG. 14B shows a Bland-Altman plot of percent difference in
measurements vs. mean of % Relaxation obtained by the methods
referenced by FIG. 14A, according to the present disclosure;
[0046] FIGS. 15A and 15B show the effect of axial over-stretch
(FIG. 15A) and pressure-overload (circumferential over-stretch,
FIG. 15B) on the endothelium-dependent dose-response relation;
according to the present disclosure;
[0047] FIG. 16A shows an exemplary in vivo/ex vivo system for
detecting a luminal organ response to one or more chemicals,
according to at least one embodiment of the present disclosure;
[0048] FIG. 16B shows a graph of typical pressure waves relating to
gastric contraction obtained by an exemplary method and/or system
of the present disclosure;
[0049] FIG. 16C shows method for detecting a luminal organ response
to mechanical stimulation, according to the present disclosure;
[0050] FIG. 17 shows a graph of the relationship between gastric
capacity and inflation pressure for various conditions, according
to the present disclosure;
[0051] FIGS. 18A and 18B show graphs of intragastric pressure
waveforms during gastric contraction, according to the present
disclosure;
[0052] FIGS. 19A and 19B show graphs of amplitudes of the
contractile waves for various conditions, according to the present
disclosure;
[0053] FIGS. 20A and 20B show graphs of durations of gastric
contractility in vivo and ex vivo, according to the present
disclosure;
[0054] FIGS. 21A and 21B show graphs of the period of gastric
contractility in vivo and ex vivo, according to the present
disclosure;
[0055] FIG. 22 shows an exemplary in vivo/ex vivo system for
detecting a luminal organ response to one or more chemicals,
according to at least one embodiment of the present disclosure;
[0056] FIGS. 23A, 23B, and 23C show graphs of intraluminal
waveforms during duodenal contraction, according to the present
disclosure;
[0057] FIGS. 24A and 24B show graphs of amplitude and diameter,
respectively, for various conditions, according to the present
disclosure;
[0058] FIGS. 25A and 25B show graphs of intraluminal pressure
waveforms during colonic contraction, and FIG. 25C shows the
inflation protocol used to test the stretch-elicited contractility,
according to the present disclosure;
[0059] FIGS. 26A and 26B show graphs of amplitude and diameter,
respectively, for various conditions, according to the present
disclosure; and
[0060] FIGS. 27A and 27B show graphs of the relationship between
circumferential, axial, and radial stresses in the intestinal wall
in connection with the duodenum and the colon, respectively,
according to the present disclosure.
DETAILED DESCRIPTION
[0061] For the purposes of promoting an understanding of the
principles of the present disclosure, reference will now be made to
the embodiments illustrated in the drawings, and specific language
will be used to describe the same. It will nevertheless be
understood that no limitation of the scope of this disclosure is
thereby intended.
[0062] To understand and fully appreciate the advantages of the
disclosure of the present application, it is useful to first
consider the conventional techniques that are in use today, their
uses and their drawbacks, and to consider an engineering analysis
that leads to the devices, systems, and methods of the present
disclosure.
[0063] Conventionally, wire and pressure myographs are widely used
to study the vasoactive properties of blood vessels and other
luminal organs. As referenced herein, luminal organs may include
blood vessels, a stomach, and any number of other mammalian organs
having a lumen therethrough. In the wire myograph, the blood vessel
is cut into rings and each ring is mounted by two hooks in an
isometric myograph. Typically, one of the hooks is fixed while the
other is connected to a force transducer. The length of the ring is
maintained relatively constant (isometric) while the measured force
is recorded during vasoconstriction or vasodilatation. A useful
property of this model is that it tests isometric properties with
high sensitivity, but some drawbacks include the non-physiological
nature of the blood vessel geometry and the mechanical loading. To
remedy these drawbacks, the pressure myograph was developed.
[0064] In the pressure myograph, the blood vessel is cannulated to
a perfusion system and connected to a pressurized container which
can regulate the pressure. A microscope with a charge-coupled
device (CCD) camera is used to monitor the diameter of the vessel.
The increase or decrease of the diameter reveals the vasodilatation
or vasoconstriction, respectively. In comparison with the isometric
wire myograph, the measurement in the pressure myograph is more
physiological.
[0065] However, the sensitivity to detect vasoactivity in the
pressure myograph is lower than in the wire myograph. In other
words, the force change is much larger than the diameter change in
the blood vessel during vasoactivity, especially for elastic
vessels. For example, the force in the isometric myograph may
increase many fold during norepinephrine-induced vasoconstriction.
At similar conditions, the diameter changes about 10-20% in a
pressure myograph. The force in an isometric myograph may decrease
to zero during acetylcholine-induced vasodilatation while the
dimension changes less than 10% in a pressure myograph. Such
discrepancies and variations are just some of the drawbacks of
these conventional systems and must be kept in mind when
considering the following engineering analysis of the reaction of
blood vessels and other luminal organs in the body to determine an
improved technique of measuring vasoactivity.
[0066] Under homeostatic in vivo conditions, blood vessels are
arguably under more isometric than isotonic conditions. This is
supported by the observation that the variation in vessel diameter
is less than 10% during the cardiac cycle while the mean hoop
stress (.tau..sub..theta.), which can be estimated as the product
of pressure (P) and inner radius (r.sub.i) divided by the wall
thickness (h), varies much more than that. This follows from
Laplace's equation which can be stated as:
.tau. .theta. = Pr i h [ 1 ] ##EQU00001##
[0067] The inner radius and wall thickness are related, however,
through the incompressibility principle which can be given as:
A.sub.0L.sub.0=.pi.(r.sub.0.sup.2-R.sub.i.sup.2)L=H(r.sub.0-r.sub.i)L
[2a]
where A.sub.0 and L.sub.0 correspond to the wall area and length of
vessel in the no-load state (zero-transmural pressure), and r.sub.0
and L correspond to the outer radius and length of vessel in the
loaded state. Approximating the vessel as thin walled, i.e.,
r.sub.0.about.r.sub.i, Equation [2a] becomes:
h = A 0 2 .pi..lamda. z r i [ 2 b ] ##EQU00002##
where .lamda..sub.z is the axial stretch ratio given by L/L.sub.0
and h=r.sub.0-r.sub.i. If Equations [1] and [2b] are combined, the
following equation is obtained:
.tau. .theta. = 2 .pi..lamda. z A 0 Pr i 2 [ 3 ] ##EQU00003##
[0068] Since both pressure and radius change throughout the cardiac
cycle, the change in stress will be much larger than the change in
radius as shown by Equation [3]. Thus, the vessel experiences more
isometric than isotonic conditions in vivo.
[0069] Furthermore, the computation of tension or stress for the
cylindrical geometry using Laplace's equation requires that the
vessel or other luminal organ be under equilibrium conditions. This
occurs under isometric not isotonic conditions. For these reasons,
the devices, systems, and methods of the present disclosure were
devised to allow the determination of active mechanical properties
of blood vessels and other luminal organs under isometric
conditions while preserving the physiological geometry and pressure
loading.
[0070] An isovolumic myograph according to the present disclosure
has the advantages of both wire and pressure myographs while
avoiding their limitations. In an isovolumic myograph, a blood
vessel is cannulated and distended similarly to a pressure
myograph, and the vasoconstriction or vasodilatation response is
determined through pressure signals. Using the exemplary
embodiments of the disclosure of the present application, very
small pressure changes can be measured in a similar manner as the
wire myograph, while maintaining a physiological geometry and
loading of the blood vessel or other luminal organ similar to the
pressure myograph.
[0071] Furthermore, an exemplary method of to the present
disclosure is used to show that the pressure during
vasoconstriction may increase up to 3-fold or higher depending on
the initial pressure. Similarly, vasodilatation induces a
significant pressure drop, as much as from 80 mmHg to 0 mmHg, when
the vessel was pre-constricted by phenylephrine.
[0072] In an exemplary embodiment of a system of the present
disclosure, an isovolumic myograph system is disclosed as shown in
FIG. 1. As shown in FIG. 1, a stereomicroscope 2 is used to detect
and measure the changes in dimensions and geometry of a blood
vessel 3 under consideration. A micromanipulator 1 allows the
length of vessel 3 to be properly positioned within the unit and
connected to an axial force transducer 4. A constant and/or
continuous volume is maintained through the closed unit,
controllable by stopcocks 6 and 7 positioned in close proximity to
either end of the blood vessel, and adjacent to flasks 8 and 9,
respectively. Pressure regulators 10 and 11 are used to set and
control the gas pressure within the closed fluid path which in turn
controls the pressure within the lumen of vessel 3 while
solid-state pressure transducer 5 detects such pressure of the
fluid within the lumen of vessel 3.
[0073] In operation, the exemplary isovolumic myograph in FIG. 1
serves to maintain an isovolumic environment for vessel 3 under
consideration of and exposure to a particular drug, agonist, or the
like. The various components shown in FIG. 1 serve to allow the
introduction of fluid into the lumen of vessel 3, or alternatively,
allow the constant flow of fluid through vessel 3. Using either
method, the dimensions and stresses on vessel 3 is measured using
the pressure transducer 5 and microscope 2 and recorded through a
camera and recording system for later analysis. Alternatively, and
as shown in FIG. 5A, a computer system may be in real-time
communication with the microscope 2 and camera system such that the
measurements and stresses of vessel 3 are presented in a display in
real time.
[0074] To consider the measurements and analysis of the exemplary
embodiment of the present disclosure as shown in FIG. 1, an
experiment was conducted using arterial segments from rats. Six
Wistar rats weighing 300-350 g were used in the study. The animals
were anesthetized with sodium pentobarbital (60 mg/kg, ip). A
heating pad was used to maintain the body temperature of the animal
during anesthesia. The left carotid and common femoral arteries of
the rat were exposed and cannulated for blood pressure
measurements. This was done to measure the in vivo difference in
blood pressure between carotid and femoral arteries. Several 1 cm
segments of right carotid and femoral arteries were excised and
immediately stored in 4.degree. C. physiological saline solution
(PSS).
[0075] The samples were then prepared to be tested in the exemplary
isovolumic myograph according to the present disclosure as shown in
FIG. 1. To prepare the samples, PSS was first contained in an organ
bath with a controlled heating system and warmed to 37.degree. C.
The PSS in the organ bath was aerated by a mixture of 95% O.sub.2
and 5% CO.sub.2 throughout the experiment.
[0076] A micromanipulator 1 was mounted on the edge of organ bath
as shown in FIG. 1. An "arm" fixed on the micromanipulator 1 was
used to hold a connector to a cannulate on one end of vessel 3, and
a second arm held a connector to cannulate on the other end of
vessel 3. Both connectors were immersed into PSS in the organ bath
and vessel 3 was cannulated on the two ends. The back ends of the
two connectors were connected with thick-wall Tygon tubing to
individual two-way stopcocks 6 and 7. A solid-state pressure
transducer 5 was inserted into the tubing between the connector and
a two-way stopcock 6 to monitor the pressure in the blood vessel 3.
Each two-way stopcock 6 and 7 was connected to an individual flask
(approximately 50 ml) 8 and 9, respectively, with about 20 ml of
PSS (and/or another agonist) to fill the lumen of vessel 3. Each
flask 8 and 9 was pressurized by a mixed 95% O.sub.2 and 5%
CO.sub.2 gas tank, and the pressure in each flask was regulated by
an independent pressure regulator 10 or 11. Regulator 10 or 11
pressurizes the fluid in the flask 8 or 9, respectively, to any
desired pressure (the accuracy being to within about 1 mmHg).
[0077] Vessel 3 and pressure transducer 5 are isolated from the
pressure system when vessel 3 is pressurized and the two stopcocks
6 and 7 are closed to vessel 3. Since the two ends of vessel 3 are
closed off, contraction of vessel 3 causes an increase in
intravascular pressure. During vasodilatation, the vessel 3
expands, and hence results in a decrease of intravascular pressure.
The changes of the pressure are recorded. The diameter change,
however, is very small in the isovolumic system as confirmed by a
CCD camera mounted on a stereomicroscope 2 to record the diameter
change. Pharmacological agonists may be endothelium-dependent, and
hence could be introduced through the lumen, or may be
endothelium-independent, and hence could be applied externally to
vessel 3 in the bath.
[0078] With the aid of stereomicroscope 2, the adjacent loose
tissue of vessel 3 was dissected carefully and all of the branches
of the vessel 3 were ligated by suitable suture in 4.degree. C.
PSS. Vessel 3 was then cannulated onto the connectors in the organ
bath containing PSS in room temperature and gassed by 95%
O.sub.2/5% CO.sub.2 at 37.degree. C. Vessel 3 was then stretched to
its in vivo length and the two stopcocks 6 and 7 were opened to
vessel 3. The intravascular pressure was set at 10 mmHg to allow
vessel 3 to equilibrate for 40 minutes. The intravascular pressure
was then increased to 60 mmHg and the two stopcocks 6 and 7 were
simultaneously closed to vessel 3. Vessel 3 was challenged twice by
phenylephrine at 1 .mu.mole/L. The PSS was replaced and vessel 3
was allowed to equilibrate for 40 min. The vessel 3 segment was
then pressurized to 100 mmHg in the carotid artery while the
femoral artery was pressurized to 85 mmHg.
[0079] The dosage-dependent vasoconstriction in response to
phenylephrine was recorded. The dosage- and endothelium-dependent
vasodilatation in response to acetylcholine was also recorded in
phenylephrine pre-constriction. The dosage-dependent and
endothelium-independent vasodilatation in response to sodium
nitroprusside (SNP) was recorded in phenylephrine pre-constriction.
The maximum concentrations of agonists were then used in the
pressure-dependent myogenic contraction which induced maximum
vasoconstriction and vasodilation as outlined below.
[0080] Vessel 3 was then pressurized at 10 mmHg for 5 minutes and
the two stopcocks 6 and 7 were closed simultaneously. The PSS with
maximum concentration of phenylephrine caused vasoconstriction
compared to the PSS in vessel 3. The pressure in vessel 3 and
diameter of vessel 3 were recorded. The PSS with phenylephrine was
drained and PSS was refilled into the organ bath. Vessel 3 was
allowed to recover for 40 minutes and was then pressurized from 20
to 140 mmHg in increments of 20 mmHg. At every pressure,
vasoconstriction induced by phenylephrine was repeated as outlined
above. Vessel 3 was then allowed to recover for 40 minutes between
every phenylephrine administration.
[0081] After the vasoactivity experiment, calcium-free PSS with 2.5
mmole/L of ethylene glycol tetraacetic acid (EGTA) was used to
replace the PSS in the organ bath and flasks 8 and 9. After 20
minutes, the diameter of vessel 3 was recorded at every setting
pressure: 10, 20, 40, 60, 80, 100, 120, 140, and 160 mmHg. Vessel 3
was disconnected from the organ bath and three rings (0.5 mm in
length) were cut from vessel 3. The cross-section was videotaped
and wall area and inner and outer perimeters were measured. The
rings of vessel 3 were further cut radially and the inner and outer
lengths were measured at zero-stress state.
[0082] PSS used in these experiments was made of the following (in
mmole/L): 119 NaCl, 4.7 KCl, 25 NaHCO.sub.3, 1.17 KH.sub.2PO.sub.4,
1.17 MgSO.sub.4, 1.6 CaCl, and 5.5 glucose. Phenylephrine and
acetylcholine were made in 1 mmole/L in 0.1 mmol/L HCl stock
solution and stored at -20.degree. C. The solutions were diluted
and used immediately. Sodium nitroprusside was made in 1 mmole/L in
PSS instantly.
[0083] Data was presented as the arithmetic mean.+-.standard
deviation (SD), unless otherwise noted. Significant differences
between various parameters were determined by use of parametric
analysis of variance followed by the Student t-test. A probability
of p<0.05 was considered to be indicative of a statistically
significant difference.
[0084] The concentration-dependent contraction of vessel 3 to
Phenylephrine is presented in FIG. 2. The pressure in vessel 3
increased sequentially when phenylephrine was administered in
increasing concentrations, as shown by the arrow points. The
pressure reached a maximum when the concentration was 3 .mu.mole/L
as shown in FIG. 2. Dosage dependent vasodilation was observed by
the administration of acetylcholine.
[0085] FIG. 3 shows tension-diameter relationships for passive and
active properties of carotid artery and femoral artery and vein. In
comparison with active response to phenylephrine, the passive
tension was much smaller at the same diameter. Vasoconstriction
caused a large contractile force in the wall of vessel 3.
[0086] FIG. 4 shows a time course of pressure decrease during
vasodilation with SNP. There are spontaneous small amplitude
contractions during the vasodilatory process. This phenomena has
not been previously reported, as it is unlikely that the diameter
change is measurable with a traditional pressure myograph for these
small pressure changes.
[0087] The isovolumic myograph shown in FIG. 1 is just one
exemplary embodiment of a myograph of the present disclosure. Many
other variations are possible and within the purview of the present
disclosure. For example, the system shown in FIG. 5A is yet another
exemplary embodiment of the present disclosure. This exemplary
embodiment may be used for measuring isometric (FIG. 6) and
isotonic (FIG. 7) vasomotion, and although it is substantially
similar to the exemplary embodiment shown in FIG. 1, it also
includes a computer-controlled electronic pressure or volume
regulator as well as computer controlled measurement of vessel
diameter.
[0088] This embodiment also addresses the limitations of
conventional methods, namely, that although both isometric
(constant length) and isotonic (constant tension) mechanical
testing have been utilized extensively in skeletal muscle
preparations to understand muscle mechanics, to date, no similar
device that allows both isometric and isotonic experiments in
cylindrical vessels has been created, let alone with electronic
and/or computer control. The isovolumic method (constant volume)
can be extended to isometric and isotonic modes as well, as
described with respect to FIG. 5B and as shown in FIGS. 6 and 7,
respectively.
[0089] FIG. 5B shows a schematic feedback loop for the isometric
and isotonic measurements used in the exemplary embodiment of FIG.
5A. To better understand the feedback loop control of this
embodiment, first, isometric vasoactivity is considered. The
diameter of vessel 3 will increase or decrease during relaxation or
contraction, respectively. The isometric vasomotion requires that
vessel 3 diameter during vasoactivity is maintained constant by
regulating the pressure or volume. Therefore, pressure or volume is
regulated in a feedback loop to a set diameter. During
vasoconstriction, the reference diameter is decreased. The system
measures the decrease in diameter and responds by increasing the
pressure or volume to the set value. The feedback loop is
reiterated until the diameter is maintained within 1% of the set
value. Conversely, pressure or volume is decreased during
vasodilatation to decrease the diameter to the set value through a
negative feedback loop as shown in FIG. 6.
[0090] Next, isotonic vasoactivity is considered. Isotonic
vasomotion requires that circumferential tension of vessel 3 be
constant (e.g., the product of pressure and inner radius is
constant). During isotonic contraction, the circumferential tension
is maintained constant but both pressure and diameter change as
shown in FIG. 7. Briefly, the set point is computed as the product
of pressure and diameter and the system will vary the pressure or
volume to maintain a constant product similar to the isometric
test.
[0091] In performing isometric and isotonic tests, the vessel
diameter is measured. Typically, the smaller diameter of the
vessel, the more transparent it is. Hence, the inner and outer
diameters can be measured directly in smaller vessels. In the
present system, the inner diameter can be continuously measured
very well for vessels <600 .mu.m in diameter. For vessels
>600 .mu.m in diameter, only the outer diameter can be measured
directly. The inner diameter can be calculated from methods
established in the art based on measurements of no-load
cross-sectional area, axial stretch ratio and the incompressibility
assumption. Hence, the inner diameter may be computed from the
outer diameter and additional measurements as described above.
[0092] As discussed above, there are several modes of vessel smooth
muscle activation, including, for example: (1) physical, such as
increase in pressure during myogenic response; (2) chemical, such
as with various agonist and antagonist through pharmacological
agents; and (3) electrical, through current stimulation. The first
two are referenced generally within the present disclosure, and the
third type is discussed with respect to FIG. 8, which shows an
arrangement where an electrical current source 12 can be used to
stimulate the contraction of vessel by electrical field stimulation
(EFS). A variety of electrodes may be used to provide such a
stimulus. As a non-limiting example, two platinum wire electrodes
may be used to stimulate the vessel segment with an electronic
stimulator by 20 Hz with square wave pulses of 0.3-ms duration and
60 mV. This embodiment can be used to show various vasoactivity in
response to electrical stimulation.
[0093] FIG. 9 shows yet another exemplary embodiment according to
the present disclosure wherein multiple vessels may be tested in
the same system. In this particular example, a second vessel 13 may
be simultaneously measured in the same organ bath as the first
blood vessel 3. The tubing, force transducer 14 and pressure
transducer 15, stopcocks 16 and 17, flasks 18 and 19, and fine
pressure regulators 20 and 21 are similar to those used for the
first vessel 3, and as described in the aforementioned exemplary
embodiments. An additional manipulator 12 may be used to adjust the
length of second vessel 13 length independently of the first vessel
3. The second vessel 13 may be exposed to the same vasostimulators
or pressure loading as the first vessel 3. Using such a system,
different vessels from different parts of the body may be tested
for response to same or similar stimuli. Other uses are also
possible, as referenced below.
[0094] In yet another exemplary embodiment, as shown in FIG. 10A, a
system is provided that allows the testing of a vessel wherein the
internal and external pressures of a vessel may be controlled.
Further, a particular pulse pressure 15 may be electronically
produced by pulse pressure generator 14 and forwarded to pressure
transducer 13, leading to sealed external bath 12 containing vessel
3. Sealed external bath 12 is secured such that the external
pressure of the vessel 3 is controllable by the pressure pulse
system. Such an exemplary system allows an even more realistic
model of the actual vessel environment that may be used to test a
vessel as it experienced pulsatile pressure changes. Other tests
and configurations are possible and are within the scope of the
present disclosure.
[0095] In vivo, vessels experience pulsatile intravascular pressure
conditions. Furthermore, some vasculatures, such as the coronary
vessels, experience pulsatile external loading in addition to
pulsatile intravascular loading. Hence, it is very useful to mimic
both intravascular as well as external pulsatile loading conditions
shown in an exemplary embodiment of the present disclosure as
referenced in FIG. 10B. This exemplary embodiment shows a schematic
of an isovolumic system that enables internal and external
pulsatile pressure conditions. This can be readily done using the
disclosure of the present application by pressurizing the external
medium (solution bath) of the vessel with a pulsatile pressure
apparatus. To generate internal pulsatile pressures, a compliant
balloon is connected in series with the vessel. The balloon is then
loaded externally with a pulsatile pressure. The pressure pulse is
transmitted to the lumen of the vessel through the compliant
balloon.
[0096] Although the above examples show some of the advantages of
the present disclosure, additional benefits and abilities are also
inherent and apparent herein. For example, a myogenic response may
be measured through a pressure response after a sudden change in
pressure. In addition, axial force measurements may be made
allowing for measurement of simultaneous axial forces.
[0097] Yet another advantage of the present disclosure is that the
filtration rate in small vessels may also be determined, wherein
the filtration rate can be computed during an isovolumic
experiment. Consider a vessel of cylindrical geometry whose volume
is given by
V = .pi. 4 D 2 L [ 4 ] ##EQU00004##
[0098] A change in volume during an isovolumic contraction is due
to filtration and can be related to the diameter change as
follows:
.delta. V = .pi. 2 DL .delta. D [ 5 ] ##EQU00005##
[0099] The filtration rate, J.sub.V, can be given as
J V = .delta. V .delta. t = .pi. 2 DL .delta. D .delta. t [ 6 ]
##EQU00006##
[0100] The filtration rate per surface area, S, can be expressed
as
J V / S = 1 2 .delta. V .delta. t [ 7 ] ##EQU00007##
[0101] Hence, the filtration rate is equal to one half of the rate
of change of diameter which can be quantified during the
experiment.
[0102] In addition to the foregoing, the disclosure of the present
application includes a method through an on-line real-time
measurement of pressure which can be extended to the full range of
vessels (arterioles to aorta) using the various systems referenced
herein. Such methods demonstrate that the physiologic loading
(circumferential and axial) significantly affects endothelial
function and hence the preservation of physiological geometry and
loading conditions are essential for a functional endothelial
assay.
[0103] Animals and Tissue Preparation.
[0104] To demonstrate the foregoing, Wistar male rats were obtained
at 3 months of age (from Charles River, Wilmington, Mass., USA).
Six aorta, six femoral arteries, and six mesenteric arteries were
harvested from eighteen rats. The animals were acclimated to the
testing facility for approximately one week prior to the start of
the study. On the day of termination, each animal was first
anesthetized with sodium pentobarbital (80 mg/kg, i.p.) and
euthanized by overanesthesia with sodium pentobarbital (300 ng/kg,
i.p.), Either the aorta, common femoral artery, or mesenteric
artery were excised quickly and placed in ice-cold physiological
saline solution (PSS in mmole/L: 119 NaCl, 4.7 KCl, 25 NaHCO.sub.3,
1.17 KH.sub.2PO.sub.4, 1.17 MgSO.sub.4, 1.6 CaCl, 5.5 Dextrose,
solution gassed by 95% O.sub.2/5% CO.sub.2). The artery was
carefully cleaned from adjacent tissue with the aid of a
stereo-dissection microscope. The branches on the artery were
ligated and the artery was allowed to warm up to room temperature
(22.degree. C.) slowly in approximately 10-15 min. The artery was
transferred to the chamber of isovolumic system and cannulated with
connectors and secured with 8-0 suture twice to avoid any leakage.
The artery was warmed up to 37.degree. C. slowly (20-25 min) and
equilibrated for 40 min at a transmural pressure of 15 mmHg before
agonist and antagonist stimulation.
[0105] Isovolumic Myography.
[0106] An isovolumic myograph system, such as shown in FIG. 11A,
was used in the present study. As referenced herein, such an
isovolumic myograph system 100 comprises a chamber 102 for
receiving a fluid, and two connectors 104, 106 (exemplary retaining
devices) which bridge the bodily vessel 108 and various tubes of
system 100. One tube 110, as shown in the exemplary system 100
shown in FIG. 11A, connects to a pressurized vessel 112 (such as a
50 mL flask with PSS therein, for example), whereby vessel 112 is
pressurized with gas source 114 and a regulator 116 to
inflate/pressurize vessel 112 at the desired pressure. Another tube
118 connects to a solid state pressure transducer 120 to monitor
the transmural pressure. A volume compensator 123 (a syringe, for
example), may also be coupled to a tube of system 100 to compensate
for water transport across the wall of vessel 108. The outlet of
tube 110, for example, may be blocked to achieve isovolumic
conditions by way of stopcock 122 coupled thereto.
[0107] PSS (an exemplary fluid 124) fills the various tubes prior
to vessel 108 cannulation. A CCD camera 126 mounted on a
stereomicroscope 128, for example, may be used to transfers
image(s) of vessel 108 to a computer (as shown in FIG. 5A, for
example), that digitizes the external diameter of vessel 108. Since
the sample rate of digital conversion (200/sec in at least one
embodiment) is higher than the rate of change in vessel 108 during
vasoreactivity, the diameter is easily tracked using such
components.
[0108] To start the study, vessel 108 is inflated to a desired
pressure; e.g., physiologic pressure. Since the outlet is closed
off, there is no flow of fluid in vessel 108 and vessel 108 is
merely pressurized. To achieve isovolumic state, a clamp (not
shown) placed on a tube between the vessel 112 and connector 104,
and/or stopcock 122 is used, for example, to close the system 100
and to seal the PSS in the lumen of the vessel and tubes; volume is
constant. The vascular contraction or relaxation during chemical
stimulation is characterized with significant changes of
intraluminal pressure.
[0109] In addition to the foregoing, various other components of
systems 100 and 200 (referenced below) as disclosed herein may be
part of such an exemplary system 100. For example, an additional
tube 130 may be coupled to system 100 between connector 104 and
connector 132 so that tubes 110, 118, and 130 may couple to and be
in fluid communication with one another as shown in FIG. 11A. In
addition, and as shown in FIG. 11A, system 100 may comprise an
axial force transducer 134 to facilitate adjustment of a vessel
present within sustem 100. Furthermore, and as shown in the
exemplary system 100 of the present disclosure shown in FIG. 11B,
system 100 may further comprise a micromanipulator 136 to permit a
user to manipulate the length of vessel 108. In addition, an
exemplary system 100 may comprise a second tube 110A, a second
stopcock 122A, a second vessel 112A, a second pressure regulator
116A, and a second gas source 114A, as shown in FIG. 11B. Gas
sources 114 and 114A may comprise the same gas source, whereby the
same gas source is in communication with pressure regulators 116
and 116A.
[0110] Steps of an exemplary method of detecting a luminal organ
response to one or more chemicals of the present disclosure is
shown in FIG. 11C. As shown in FIG. 11C, an exemplary method 150
comprises the steps of maintaining a luminal organ at a first
length and a first internal pressure within a fluid bath (an
exemplary maintenance step 152), introducing a first chemical into
the fluid bath (an exemplary chemical introduction step 154), and
measuring a first organ parameter change in response to exposure of
the luminal organ to the first chemical (an exemplary parameter
change measurement step 156). In at least one embodiment, and if
the chemical causes the luminal organ to constrict, the first organ
parameter change may be a decrease in luminal organ diameter and/or
an increase in internal luminal organ pressure. In another
embodiment, and if the chemical causes the luminal organ to expand,
the first organ parameter change may be an increase in luminal
organ diameter and/or a decrease in internal luminal organ
pressure. Such changes may be detected using a camera, a pressure
transducer, and/or a microscope, for example, but are not limited
to those exemplary detection devices.
[0111] In at least one embodiment of an exemplary method 150 of the
present disclosure, maintenance step 152 comprises positioning the
luminal organ within a system for detecting a luminal organ
response, adjusting the length of the luminal organ until the first
length is achieved, introducing a fluid into a lumen of the luminal
organ until a desired first internal pressure is achieved, and
closing at least part of the system so that fluid is not permitted
to escape the luminal organ through a component of the system. In
addition, maintenance step 152 may also comprise injecting
additional fluid into a lumen of the luminal organ in response to
luminal organ leakage through a wall of the luminal organ. Such an
injection may be performed using a volume compensator in fluid
communication with the lumen of the luminal organ.
[0112] As referenced in further detail herein, the luminal organ
may be overstretched or inflated when performing an exemplary
method 150. In at least one embodiment, the first length is
substantially a length of the luminal organ when the luminal organ
was present within a mammal prior to removal of the luminal organ
and placement of the luminal organ within the fluid bath. In
another exemplary embodiment, the first length is longer than a
length of the luminal organ when the luminal organ was present
within a mammal prior to removal of the luminal organ and placement
of the luminal organ within the fluid bath, and wherein the first
organ parameter change is in part related to an axial overstretch
of the luminal organ.
[0113] An exemplary method 150 of the present disclosure, as shown
in FIG. 11C, may further comprise the steps of stretching the
luminal organ to a second length (an exemplary stretching step
158), measuring a second organ parameter change in response to the
exposure of the luminal organ to the first chemical (also an
exemplary parameter change measurement step 156), and comparing the
first organ parameter change to the second organ parameter change
to determine a response indicative of axial overstretch (an
exemplary comparison step 160).
[0114] In an exemplary embodiment of a method 150 of the present
disclosure, the first pressure is substantially a pressure within
the luminal organ when the luminal organ was present within a
mammal prior to removal of the luminal organ and placement of the
luminal organ within the fluid bath. In another embodiment, the
first pressure is higher than a pressure within the luminal organ
when the luminal organ was present within a mammal prior to removal
of the luminal organ and placement of the luminal organ within the
fluid bath, and wherein the first organ parameter change is in part
related to a circumferential overstretch of the luminal organ.
[0115] Another exemplary method 150 of the present disclosure, as
shown in FIG. 11C, may further comprise the steps of introducing a
fluid into a lumen of the luminal organ so that the luminal organ
has a second internal pressure higher than the first internal
pressure (an exemplary fluid introduction step 162), measuring a
second organ parameter change in response to the exposure of the
luminal organ to the first chemical (also an exemplary parameter
change measurement step 156), and comparing the first organ
parameter change to the second organ parameter change to determine
a response indicative circumferential overstretch (also an
exemplary comparison step 160). In at least one embodiment of a
method 150 of the present disclosure, method 150 may further
comprise the steps of introducing a second chemical into the fluid
bath (also an exemplary chemical introduction step 154), and
measuring a second organ parameter change in response to exposure
of the luminal organ to the second chemical (also an exemplary
parameter change measurement step 156) as shown in FIG. 11C.
[0116] In various embodiments of methods 150 and 250 (as referenced
in detail herein), the luminal organ is selected from the group
consisting of a blood vessel and any mammalian organ having a lumen
therein. In at least one embodiment, the first chemical causes an
increase in intraluminal pressure and circumferential tension of
the luminal organ, and wherein the second chemical causes a
decrease in intraluminal pressure and circumferential tension of
the luminal organ. In such an embodiment, and as shown in FIG. 11C,
method 150 may further comprise the step of determining a percent
relaxation of intraluminal pressure and circumferential tension
based upon at least the increase in intraluminal pressure and
circumferential tension of the luminal organ in response to the
first chemical and the decrease in intraluminal pressure and
circumferential tension of the luminal organ in response to the
second chemical (an exemplary percent relaxation step 164).
[0117] Volume Compensation Due to Fluid Filteration:
[0118] Although the various systems and methods of the present
disclosure can achieve a fairly constant volume of the solution in
the lumen of a vessel, it is not strictly constant since the PSS
may be transported across the vessel wall (water flux) drived by
the transmural pressure. Although the rate of water flux is very
small (<1 nl/min) and no visible reduction of diameter is seen
during the duration of experiment (<1 hr.), a pressure drop,
namely a drop in baseline pressure, is observable. In order to
stablize the baseline of pressure, a volume compensator may be
connected in parallel with pressure transducer. The volume
compensator comprises a gastight connector, a microsyringe (maxium
volume: 25 .mu.l), a microsyringe pump (UltraMicroPump III, World
Precision Instruments, USA) and a microsyringe pump controller
(Micro 4.TM., World Precision Instruments, USA). The criteria for
compensatory rate of microsyringe pump controller is to maintain
the transmural pressure at the desired baseline value. There is no
measurable change of vessel diameter during compensation. If the
leak rate was >1 .mu.l/min, the specimen was discarded as the
vessel wall was damaged.
[0119] Tension and Percent Relaxation:
[0120] The circumferential tension can be computed based on the
following:
T = P .times. r int 2 and [ 8 A ] r int = r ext 2 - A 0 .pi..lamda.
[ 8 B ] ##EQU00008##
[0121] wherein T is circumferential tension given by Laplace's
equation (Equation [8A], P is intraluminal pressure, and r.sub.int
is internal radius of blood vessel related to the external radius,
through the incompressibility assumption (Equation [8B]). A.sub.0
is the cross-sectional wall area of vessel at no-load state (zero
intraluminal pressure), and .lamda. is axial stretch ratio.
[0122] A dose-dependent vasoconstriction and vasodilatation in
response to phenylephrine (PE) and acetylcholine (ACh) were carried
out under isovolumic conditions. Briefly, the artery was stimulated
to contract with phenylephrine (PE) that was injected the PSS in
the chamber to increase PE concentration of the PSS step by step
from 10.sup.-10, 10.sup.-9, to 10.sup.-5 mole/l, as shown in FIG.
12, FIG. 12, shows a typical tracing curve of intraluminal pressure
before and after volume compensation, and thereafter in response to
vasoconstriction. The vessel segment was femoral artery which was
stretched to its in vivo length and inflated to .about.70 mmHg, and
closed the valve and compensated, and then stimulated with PE
(phenylephrine, mole/L). The compensatory rate was 38 nl/min. Then,
the artery was relaxed with acetylcholine (ACh) by a series of
doses: 10.sup.-10, 10.sup.-9, 10.sup.-5 mole/l in the PSS. The
relaxation results in the reductions of intraluminal pressure and
circumferential tensions which was computed using Equations [8A]
and [8B]. The calculation of percent relaxation (% R) was based to
both intraluminal pressure (% R.sub.P) and tension (% R.sub.T) for
comparison:
%R.sub.P=(P.sub.d-P.sub.i)/(P.sub.max-P.sub.i).times.100 [9A]
and
%R.sub.T=(T.sub.d-T.sub.i)/(T.sub.max-T.sub.i).times.100 [9B]
wherein P.sub.d, P.sub.i, and P.sub.max are the intraluminal
pressures at each dose (P.sub.d), inflation pressure (P.sub.i), and
maximum pressure (P.sub.max) at 0 mole/1 of ACh, respectively.
T.sub.d, T.sub.i, and T.sub.max are the circumferential tension at
every dose (T.sub.d), physiological level (T.sub.i), and maximum
tension (T.sub.max) at 0 mole/l of ACh, respectively.
[0123] Effects of Over-Stretch on Endothelial Finction of Femoral
Artery.
[0124] The loading perturbation was only superimposed on the
femoral artery. After the test of endothelium-dependent relaxation,
the femoral artery was incubated in fresh PSS for 90 minutes to
restore endothelial function. The femoral artery was, then, exposed
to mechanical perturbation with either axial over-elongation
(stretch ratio of 1.47 from 1.3, 118% increase) or over-inflation
(pressure of 120 from 70 mmHg, 170% increase). The stretch ratio of
1.3 is the length ratio of in vivo to ex vivo since the excised
vessel shrinks approximate 30%. In axial over-stretch, the vessel
was stretched to ratio 1.47 and inflated to physiologic pressure 70
mmHg. The vasoreactivity was performed according to the above
protocol. In over-inflation, the vessel was inflated to 120 mmHg
and stretched to physiologic stretch ratio of 1.3. Sodium
nitroprusside (SNP)-induced vasorelaxation was applied to evaluate
the endothelium-independent vasorelaxation.
[0125] Data Analysis and Statistics:
[0126] The relation of the % Relaxation between tension (% R.sub.T)
and transluminal pressure (% R.sub.P) measurements were expressed
by % R.sub.P=.alpha.% R.sub.T+.beta., where .alpha. and .beta. are
empirical constants that were determined with a linear least
squares fit and a corresponding correlation coefficient R.sup.2. In
a Bland-Altman scatter diagram, the percent differences between the
two measurements of diameter [(% R.sub.T-% R.sub.P)/%
R.sub.T.times.100] can be plotted against their means [(% R.sub.R+%
R.sub.T)/2.times.100] (1). In the scatter diagram the precision and
bias of the method can be quantified, and any significant
differences between two data points were determined by student
t-test. Significant differences between the dose-dependent groups
were determined by use of Analysis Of Variance between groups
(ANOVA). A probability of p<0.05 was considered to be indicative
of a statistically significant difference.
[0127] Results:
[0128] The isovolumic myograph was used to test mesenteric, femoral
and aortic vessels in a consistent manner as opposed to wire and
pressure myograph which invoke different methodologies as shown in
FIG. 11D. FIG. 11D shows a schematic of the range of applicability
(size of vessels) of a wire myograph, a pressure myograph, and an
isovolumic myograph of the present disclosure. Since the vessel
wall is permeable to water, intraluminal pressure results in a
water flux across the vessel wall which causes a gradual drop of
baseline pressure in the isovolumic myograph. A microsyringe was
used to compensate for the water flux to maintain a constant
baseline pressure. FIG. 12 shows the changes in intraluminal
pressure determined with isovolumic myography in a femoral artery
segment with volume compensation at 38 nl/min to offset the fluid
filteration. When a vessel is distended to a higher pressure, a
higher compensatory rate is needed to maintain a uniform baseline
pressure. Similarly, a thinner-walled vessel requires higher
compensatory rate because of increased filtration.
[0129] The typical tracing curves of aorta, femoral artery, and
mesenteric artery are shown in FIG. 13A. FIG. 13A shows three
typical tracing curves of intraluminal pressure in response to
pharmacological vasoconstriction and vasorelaxation. The vessel
segments were stretched to their in vivo length and inflated to
physiological pressure and then stimulated with phenylephrine
(mole/l) and acetylcholine (mole/l). The top panel corresponds to
the aorta, the middle panel corresponds to the femoral artery, and
the bottom panel corresponds to the mesenteric artery. The inflated
physiological pressure of aorta, femoral artery, and mesenteric
artery were .about.100, 70, and 50 mmHg, respectively. The
contraction generated from vascular smooth muscle in the three
types of arteries caused an increase in intraluminal pressure of 30
to 40 mmHg in response to agonist of PE at 10.sup.-5 mole/1. The
dose-dependent vasorelaxations in response to ACh were clearly
observed by the stepwise reduced intraluminal pressures as shown in
FIG. 13A. The % Relaxation of the three types of arteries was
summarized in FIG. 13B, showing that the maximal % Relaxation was
identical in the three types of arteries. FIG. 13B shows that
percent relaxations of aorta, femoral artery, and mesenteric artery
are calculated as the ratio of pressure deference from the tracing
curves of intraluminal pressures.
[0130] The % Relaxation can be calculated based on both
intraluminal pressure and tension. FIG. 14A shows a comparison of
the two measurements relative to an identity line. FIG. 14 shows
the interrelationship of % Relaxation resulted from circumferential
tension and transmural pressure, whereby the empirical relation is
expressed as % R.sub.P=1.02% R.sub.T-0.102(R.sup.2=0.99), wherein %
R.sub.P and % R.sub.T represent percentage of pressure and
percentage of tension measurements, respectively. A Bland-Altman
plot is shown in FIG. 14B, which is the average of two measurements
versus difference, and the data are seen to scatter randomly within
two standard deviations of the mean of the difference. As shown in
FIG. 14B, the Bland-Altman plot of percent difference in
measurements vs. mean of % Relaxation obtained by the two methods
referenced above. The mean.+-.SD for the data is 0.78.+-.5.0%, and
the top and bottom dotted lines represent mean+2SD (10.8%) and
mean-2SD (-9.2%), respectively.
[0131] The root mean square (rms) is 13.7% of the mean value of the
two methods. This analysis shows that pressure is an appropriate
surrogate of tension in the calculation of % Relaxation and
simplifies the measurements and analysis of vasorelaxation.
[0132] As shown in FIGS. 15A and 15B, the effect of loading
perturbation on endothelial-dependent vasodilation in both
circumferential and axial directions can be determined. The effect
of axial over-stretch (FIG. 15A) and pressure-overload
(circumferential over-stretch, FIG. 15B) on the
endothelium-dependent dose-response relation are shown. The # shown
in FIGS. 15A and 15B indicate a statistical difference (p<0.05)
of the dose-dependent curve between the physiologic loading and
over-physiologic loadings (axial over-stretched or circumferential
over-stretched), and the * shown therein indicates significant
difference (p<0.05) at single dose between physiologic and
over-physiologic loadings. As compared with physiologic loading,
ACh-induced relaxations in the arteries were attenuated during
acute over-inflation (170% of physiologic pressure) and axial
over-elongation (118% of physiologic stretch) as shown in FIG. 15.
The SNP (10.sup.-5 mole/l)-induced endothelium-independent
relaxations were 96%.+-.8% in physiological loading, 93%.+-.11% in
axial over-stretch loading, and 99%.+-.13 in over-inflation (NS).
Therefore, the attenuation of ACh-induced vasodilation may be
attributed to a decrease in the endothelium-released
vasodilators.
[0133] As referenced above, a volume-compensated isovolumic
myograph has equal sensitivity to aortic, femoral, and mesenteric
arterial segments in rats. Hence, the isovolumic myograph provides
a unified assay for a functional biomarker of endothelial function
(% Relaxation) of small, medium and large vessels as shown in FIG.
11. Such an approach provides consistent testing conditions of all
vessels sizes and allows comparisons of different vessels under
different pathological conditions as shown in FIG. 13B. Such
studies can also show that over-inflation and -elongation cause
immediate decrease of endothelium-dependent vasorelaxation. The
latter findings underscore the significance of physiological
loading on assessment of endothelial function.
[0134] In order for a vascular assay to garner utility, it must be
simple and easy to use. Accordingly, the following question was
addressed: can pressure replace tension to eliminate the need for
microscope and only require a pressure transducer? FIG. 14, as
referenced herein, shows that this is possible with a 13.7% rms of
the mean. Hence, the pressure can be used interchangeably with
tension which simplifies future experiments and allows multiple
parallel vessel testing. This will provide a higher throughput for
vascular physiopathology.
[0135] There is no doubt that physical loading influences the
reactivity of blood vessel and the response of the endothelium.
Ideally, the loading and geometry of vessel segment should mimic
physiologic conditions. In a wire myograph, a vessel ring is loaded
by hooks to make the loading uniaxial and planar in the
circumferential direction while the axial tension is zero. Although
the loading is clearly non-physiological, the wire myograph has
been a very popular method for vessel reactivity due to its
excellent tension measurement sensitivity. As demonstrated here,
the disclosure of the present application shows that the
endothelium-dependent vasorelaxation can be significantly affected
by axial loadings, w here axial over-elongation attenuates the
endothelium-dependent vasorelaxation as shown in FIG. 15. This
observation confirms that the wire myograph is not physiological
and may have methodological artifacts.
[0136] In contrast to the tension in wire myograph, the vessel
diameter is the measurement variable in pressure myograph. The
contractile tension of a muscle depends on the number of activated
actin-myosin filaments while the contractile dimension depends on
the movement between actin and myosin fiber. Based on Hill's
equation (tension-velocity relation), the relation between tension
and diameter is strongly nonlinear during contraction. Since
measurement of either tension or diameter is inadequate to
understand active properties of an artery, the wire myograph
(tension measurement) and pressure myograph (diameter measurement)
are inadequate and comparison between the two methods is difficult
due to the different loading patterns and measurement parameters.
In order to monitor the tension during vasoreactivity, the
isovolumic myograph of the present disclosure has been developed to
track the transient tension (pressure) as well as diameter. This
development allows both measurements of tension and diameter. In
practice, the isovolumic myograph provides consistent results with
previous studies but also leads to new observations like those
shown in FIG. 15.
[0137] The importance of maintaining the blood vessel at
physiological load is that the wall tension of an artery may
influence vasoreactivity in two ways: vascular smooth muscle and
endothelial cells. The alteration of wall tension may activate or
inactivate contraction of vascular smooth muscle and the signal
pathways of endothelial cells mediated by mechanotransductions such
as integrins and G-protein coupled receptors. As shown herein, the
effect of perturbations from physiological loading on the
vasorelaxation of blood vessel is substantial as shown in FIG. 15,
and can be verified The vessel segment in wire myograph is tension
free axially in comparison with an in vivo vessel which is
stretched axially; the extent of which can vary in hypertension,
aging and vessel disease. These investigations provide, for the
first time, direct evidence that either acute axial over-elongation
or intraluminal over-inflation causes immediate endothelial
dysfunction.
Gastric Study
[0138] Furthermore, and regarding gastric motility, the disclosure
of the present application provides various systems and methods to
measure the same.
[0139] The effect of gastric distension has important implications
for satiety. A hypothesis used leading up to the disclosure of the
present application was that distension affects the amplitude and
duration of gastric contraction, and that these parameters are
largely mediated by efferent vagus activation. A novel isovolumic
myograph was developed to test these hypotheses, with the
isovolumic myograph isolating the stomach and recording the
pressure generated by the gastric contraction in isovolumic
conditions. Accordingly, the phasic changes of gastric
contractility can be documented.
[0140] The animal experiments were performed as follows. Twelve
C571/B mice at twelve (12) weeks of age were obtained from an
off-site location and were acclimated to the testing facility for
approximately one (1) week prior to the start of the study. The
animals were housed at 22.degree. C. under a 12-hour light and dark
cycle and were given free access to tap water and standard rodent
chow. The animals the were anesthetized with xylazine (1 mg/kg,
i.p.) and ketamine (9 mg/kg, i.p.) and maintained with xylazine
(0.5 mg/kg) and ketamine (4.5 mg/kg) every half hour.
[0141] In Vivo Gastric Contractility:
[0142] Under anesthesia, the abdominal skin and muscle layers of
the animal were opened to expose the stomach. The stomach was
moistured with warm (37.degree. C.) physiological saline solution
(HEPES-PSS in mmole/L: 119 NaCl, 4.7 KCl, 25 NaHCO.sub.3, 1.17
KH.sub.2PO.sub.4, 1.17 MgSO.sub.4, 1.6 CaCl, 5.5 Dextrose). The
stomach was canulated with a HEPES-PSS prefilled catheter (ID: 1
mm, OD: 2 mm) which connected to the isovolumic system as shown in
FIG. 11A. A 2 mm incision was cut at fundus apex of stomach through
which the catheter (OD: 2 mm) was inserted into stomach lumen. The
fundus adjacent to the incision was tied on the tube with 6-0 silk
suture twice to ensure no leakage. Two ml of HEPES-PSS was gently
injected into stomach through the tube to wash out the content. The
lower esophageal sphincter and pyloric sphincter were ligated with
6-0 silk suture. The gastric mesentery was untouched to allow the
stomach to work in a physiological environment maintaining normal
circulation and vagal responses.
[0143] Ex Vivo Gastric Contractility.
[0144] The animals were euthanized by overanesthesia. The stomach
was excised quickly and placed in cold HEPES-PSS. The adjacent
tissue was dissected with the aid of a stereo microscope. The
stomach was allowed to warm up to room temperature (22.degree. C.)
slowly in 10-15 min and was transferred to a chamber with HEPES-PSS
(22.degree. C.) of the isovolumic myograph, A 2 mm incision was cut
at fundus apex of the stomach (fibrosic portion) and a catheter
(ID: 1 mm, OD: 2 mm) was inserted into the stomach lumen through
the incision. The fundus adjacent to the incision was tied on the
catheter with 6-0 suture twice to avoid leakage. A 2 ml HEPES-PSS
was gently injected into the stomach through the catheter to wash
out the gastric content. Following drainage, the lower esophageal
sphincter and pyloric sphinter were ligated with 6-0 silk suture.
The stomach in the chamber was warmed to 37.degree. C. slowly
(15-20 min) and equilibrated for 30 min at a basal intragastric
pressure of about 2 mmHg before distension.
[0145] Isovolumic System.
[0146] An exemplary isovolumic system used for the present study is
shown in FIG. 16A. As shown in FIG. 16A, an exemplary ex vivo
system 200 comprises a chamber 202 with a catheter 204 on one side
wall of the chamber which bridges the lumen 206 of a stomach 208 to
inflation flask 210 and pressure transducer 212. As shown in FIG.
16A, system 200 is connected to a stomach 208, but in various other
embodiments, system 200, and related methods as referenced herein,
may be connected to various other mammalian organs, such as the
trachea, lymph vessels, lymph ducts, urinary bladders, ureters,
gall bladders, bile ducts, hepatic ducts, intestines, and the like,
whereby contraction can be generated by surrounding smooth
muscle.
[0147] Chamber 202, in at least one embodiment, may contain
HEPES-PSS maintained at 37.degree. C. using a heater (not shown),
for example. Inflation flask 210, which in at least one embodiment
comprises a 50mL flask, having PSS 214 therein is connected to a
pressure regulator 216 so that a stomach 208, for example, can be
inflated/distended to the desired pressure. The catheter 204, a
solid state pressure transducer 212 (SPR-524, Microtip catheter
transducer, Millar Inc, Texas), a tube 218 to inflation flask 214,
and a compensatory microsyringe 220 were assembled using a four-way
connector 222. A compensatory microsyringe 220 (50 .mu.l gastight
microsyringe, UltraMicroPump III, and Micro 4.TM. microsyringe
control, World Precision Instruments, USA) was used to stablize the
baseline of the pressure since water transport across the gastric
wall reduces the intragastric pressure. The clamping of tube 218
between inflation flask 210 and four-way connector 222, by way of
stopcock 224, achieved isovolumic conditions, i.e., intragastric
volume was constant. In at least one embodiment, a CCD camera on a
microscope 2, such as shown in FIG. 1, and an image processing
system (such as the computer shown in FIG. 5A), may be used to
capture the gastric geometry. As stomach 208 was inflated to a
desired pressure (e.g., 5 mmHg, 10 mmHg, etc.), stopcock 224 was
closed and the gastric contraction or relaxation was reflected by
the variation of intragastric pressure recorded with solid state
pressure transducer 212. The isovolumic system 200 can also record
the periodic contractions of stomach 208 by the periodic variations
of pressure.
[0148] In at least an additional embodiment of a system 200 of the
present disclosure, various components of said system 200 may be
used in connection with measurements of in vivo gastric
contractility as referenced herein, including, but not limited to,
those components used in the various embodiments of system 100
referenced herein. For example, and as shown in FIG. 16A, catheter
204 may connect directly to a stomach of a mammal 226 under
anesthesia, so that measurements can be taken in a in physiological
environment maintaining normal circulation and vagal responses.
Furthermore, an exemplary system 200 may include any number of
other components useful for the same, such as various additional
tubes and clamps to facilitate connection of the various
components. Furthermore, chamber 202 may include any number of
solutions, including HEPES, HEPES-PSS, PSS, and/or any number of
other solutions to facilitate the aforementioned measurements.
[0149] Gastric Contractility in Response to Mechanical
Stimulation.
[0150] The mechanical stimulation of stomach 208 was induced by an
intragastric inflation pressure. The gastric contraction was
quantified by the intragastric pressure under isovolumic condition
and the contractility was characterized with the amplitude and
period of the pressure waveforms. Stomach 208 was inflated to a
desired pressure by a pressure regulator 216 connected to flask 210
as referenced above. The clamping of the tube 218 between the
inflation flask 210 and the four-way connector 222 maintained a
constant volume of solution in the gastric lumen (isovolumic
condition). The compensatory microsyringe 220 maintains isovolumic
conditions at, for example, an infusion rate of 1-3 .mu.l/min. The
data was discarded if the rate was larger than 10 .mu.l/min since
this implied damage (leakage) of the gastric wall. At isovolumic
conditions, the variations of intragastric pressure was recorded
with a data acquisition system (Biopac, MP100, Houston, Tex.). The
amplitude, frequency, and contractile duration of pressure
waveforms were analyzed to characterize the gastric
contractility.
[0151] Protocol of Mechanical Stimulation.
[0152] The intragastric pressure was increased stepwise to 2, 5,
10, 15, 20, 30, 40, and 50 mmHg at a rate of 0.06 ml/min,
respectively. The gastric contraction at isovolumic condition was
recorded as the variation of the pressure at each individual
inflation pressure. This protocol was applied to both in vivo and
ex vivo stomach 208. In the experiment of ex vivo stomach,
acetylcholine (10.sup.-6 mole/l) was used to elicit non-neuroactive
contraction of gastric smooth muscle at intragastric pressure of 50
mmHg to evaluate contractility of gastric smooth muscle.
[0153] Data Analysis and Statistics.
[0154] FIG. 16B illustrates the definition of the parameters used
in the analysis, showing typical pressure waves relating to gastric
contraction. The gastric contractile amplitude (A) was indicated by
the amplitude of the variation of pressure. The gastric contractile
period (.DELTA.t) was defined as the interval from one pressure
waveform to the next pressure waveform. The gastric contractile
duration (.DELTA.t.sub.d) was defined as the interval from pressure
rise to fall.
[0155] The data is presented as mean.+-.SD. Significant differences
between groups were determined by student t-test. Significant
differences between the in vivo and ex vivo groups were determined
by use of Analysis Of Variance (ANOVA) between groups. A
probability of p<0.05 was considered indicative of a
statistically significant difference.
[0156] Steps of an exemplary method for detecting a luminal organ
response to mechanical stimulation of the present disclosure is
shown in FIG. 16C. As shown in FIG. 16C, exemplary method 250
comprises the steps of maintaining a luminal organ at a first
internal pressure (an exemplary maintenance step 252), increasing
the first internal pressure of the luminal organ (an exemplary
pressure increase step 254), and measuring a first organ parameter
change in response to the increase in internal pressure (an
exemplary parameter change measurement step 256). In an exemplary
embodiment of method 250, the luminal organ is positioned within a
chamber for receiving a fluid, and wherein the fluid is in contact
with the luminal organ.
[0157] In an exemplary embodiment of a method 250 of the present
disclosure, maintenance step 252 comprises positioning a conduit
within an incision of the luminal organ so that a lumen of the
conduit is in fluid communication with a lumen of the luminal
organ, and introducing a liquid through the conduit into the lumen
of the luminal organ until the luminal organ achieves the first
internal pressure. In at least one embodiment of a method 250,
pressure increase step 254 comprises introducing a fluid from the
conduit into the lumen of the luminal organ.
[0158] In additional exemplary maintenance step 252 of an exemplary
method 250 of the present disclosure comprises the steps of
positioning the luminal organ within a system for detecting a
luminal organ response, introducing a fluid into a lumen of the
luminal organ until a desired first internal pressure is achieved,
and closing at least part of the system so that fluid is not
permitted to escape the luminal organ through a component of the
system.
[0159] In at least one embodiment of a method 250 of the present
disclosure, and as referenced in detail herein, the first organ
parameter change is selected from the group consisting of a
decrease in luminal organ diameter, an increase in luminal organ
diameter, a decrease in internal luminal organ pressure, an
increase in internal luminal organ pressure, and an increase in
gastric contractility. In an exemplary method 250, parameter change
measurement step 256 is performed using a pressure transducer, a
microscope, and/or a camera. In an exemplary embodiment,
maintenance step 252 comprises injecting additional fluid into a
lumen of the luminal organ in response to luminal organ leakage
through a wall of the luminal organ. The additional fluid may be
injected using a volume compensator.
[0160] In an exemplary method 250 of the present disclosure, the
luminal organ is present within a living mammal while various
method steps are being performed. In another exemplary embodiment,
and when the stomach is present within a living mammal, the
attenuation of gastric contractility is mediated by efferent vagus
activation as referenced in detail herein. In yet another exemplary
embodiment, such a method 250 may be used in connection with
intestinal studies as referenced below.
[0161] Results.
[0162] The intragastric pressure and gastric volume in both in vivo
and ex vivo are shown in FIG. 17, which reflects the global
distension (compliance) of the stomach. The in vivo gastric volume
was significantly different from the ex vivo volume. The ex vivo
gastric volume increased in low pressure range (5-20 mmHg) while in
vivo gastric volume increased almost linearly.
[0163] The intragastric pressure waveforms during gastric
contraction are shown in FIG. 18A. The variation of the pressure
reflects the gastric contractility which is characterized by three
parameters: contractile amplitude, duration, and period. The in
vivo amplitude of gastric contraction increased from 1.6 mmHg to
12.5 mmHg when the inflation pressure changed from 2 mmHg to 30
mmHg, respectively, as shown in FIG. 18A. FIG. 18B shows inflation
pressure vs. distension time in stepwise fashion.
[0164] The contractility relation is shown in FIGS. 19A and 19B
both as a function of inflation pressure (FIG. 19A) and volume
(FIG. 19B). The contractility increases with an increase in
inflation pressure of volume and reaches a maximum for the in vivo
condition. FIGS. 19A and 19B show the amplitude of the contractile
waves for various conditions. FIG. 19A shows the amplitude
represented as a function of intragastric pressure, and FIG. 19B
shows the amplitude represented as a function of inflation volume.
The in vivo amplitude then decreases from 12.5 to 3.3 mmHg (FIG.
19A) when the inflation pressure further increases from 30 to 50
mmHg. In ex vivo, the gastric contractile amplitude is
significantly lower than that in in vivo at every inflation
pressure. At 50 mmHg inflation pressure, the ex vivo contraction is
completely abolished, It was noted that the contraction stimulated
by the external administration of ACh (the right columns of FIGS.
18A and 19A, for example), however, is still high, which suggests
that the efferent nervous activated contraction fails under high
distension and may be the cause of the attenuation of
contractility.
[0165] The durations of the gastric contractility in vivo and ex
vivo and under two gastric banding conditions are presented in
FIGS. 20A and 20B as a function of intragastric pressure (FIG. 20A)
and inflation volume (FIG. 20B). The duration indicates the
sustained interval of a single contraction wave. The in vivo
duration was significantly larger than the ex vivo duration when
the inflation pressure was below 35 mmHg or inflation volume was
below 0.7 ml. The in vivo duration reached a maximum between 10 to
20 mmHg or 0.3 to 0.5 ml of inflation pressure or volume,
respectively. The ex vivo duration did not significantly change
with the inflation.
[0166] The period of the gastric contractility in vivo and ex vivo
are shown in FIGS. 21A and 21B as a function of intragastric
pressure (FIG. 21A) and volume (FIG. 21B). The period indicates the
contractile frequency. The results show that the period was similar
in both in vivo and ex vivo preparation and increases with
inflation.
[0167] As referenced above, and in summary, the isovolumic
myography system 200 was used to assess gastric contractility in
terms of amplitude, duration, and period. The in vivo preparation
was designed to detect the efferent neurogenic contraction and ex
vivo preparation was designed to measure the efferent-independent
contraction. The in vivo contractile amplitude and duration were
significantly larger than those in ex vivo, indicating that
contractile amplitude and duration may be efferent neurogenic. The
similar period in in vivo and ex vivo preparation indicates that
contractile period may be efferent-independent.
[0168] Gastric contractility is closely coupled to the
mechanosensitivity located in gastric wall. The myogenic response
of gastric smooth muscle and efferent neurogenic contraction are
regulated by mechanoreceptors and afferent and efferent vagus
nerves. The relation between afferent vagus signals and gastric
distension was identified decades ago. The mechanoreceptors in
gastric wall are primary sensors of mechanical stimulation. The
efferent (motor) vagus signals are responses of the central nervous
system to the afferent (sensory) vagus stimulation. One of the
physiological functions of efferent vagus signals is to regulate
the gastric contractility. Hence, the gastric contractility
reflects the activation of an efferent vagus nerve.
[0169] As referenced herein, gastric contractility was evaluated in
an ex vivo preparation which excludes efferent vagus regulation. In
the ex vivo stomach, the nerve fibers are excised and damaged, and
hence there is a loss of efferent vagus signals which appears to be
significant for mechanical distension-induced contractility. The ex
vivo contractility of gastric smooth muscle was significantly
attenuated due to the absence of efferent vagus signals. The strong
in vivo contractility reflects the efferent vagus activation in
response to mechanical stimulation sensed by afferent vagus nerves.
The role of duration is noted as it may reflect the efferent vagus
activation. The duration of in vivo contraction varies with
mechanical stimulation whereas the duration of ex vivo contraction
is largely unchanged in response to mechanical stimulation. The
latter implies that the duration is regulated by the central
nervous system. In contrast, the period seems to be independent of
vagus nerve activation since both in vivo and ex vivo periods
increase during the increase in distension.
[0170] An exemplary isovolumic myograph of the present disclosure
(system 200) was used to evaluate the gastric global contractility.
The regional contraction, however, was not measured. Since the
gastric contractile wave is generated in the lower stomach, the
gastric tone (basal pressure) in the fundus and upper body was not
characterized herein. The studies referenced in the present
disclosure introduced a novel isovolumic myograph to understand the
contractility of the stomach. The in vivo and ex vivo gastric
contractility in response to distension (inflation) provides
evidence that gastric motility can be regulated by the central
nervous system.
Intestinal Study
[0171] The disclosure of the present application also provides
various systems and methods for determining intestinal contraction
in response to the stimulation of inflation. In-vivo and ex-vivo
protocols were used to verify the effect of extrinsic nervous
system and intrinsic nervous regulation on the motility,
respectively. The duodenum and colon of mouse were involved in the
exemplary study detailed below.
[0172] The animal experiments were performed as follows, Twelve
C571/B mice at 24 weeks of age were obtained from Charles River.
The animals were acclimated to the facility for approximately one
(1) week prior to the start of the study. The animals were housed
at 22.degree. C. under a 12-hour light and dark cycle and were
given free access to tap water and standard rodent chow. The
animals were anesthetized with xylazine (1 mg/kg, i.p.) and
ketamine (9 mg/kg, i.p.) and maintained with xylazine (0.5 mg/kg)
and ketamine (4.5 mg/kg) every half hour. The animal experiments
were performed in accordance with the guidelines of Institute of
Laboratory Animal Research Guide, Public Health Service Policy,
Animal Welfare Act, and an approved IACUC protocol.
[0173] In-Vivo Intestinal Contractility.
[0174] Under anesthesia, the abdominal skin and muscle layers of
the animal were opened to expose either the duedenum or the colon.
The intestine was moistured with warm (37.degree. C.) physiological
saline solution (HEPES-PSS in mmole/L: 119 NaCl, 4.7 KCl, 3 HEPES
acid, 2.3 HEPES sodium salt, 1.17 MgSO.sub.4, 1.6 CaCl, 5.5
Dextrose). The intestine was canulated with a HEPES-PSS prefilled
catheter (ID: 1 mm, OD: 2 mm) which connected to the isovolumic
system as shown in FIG. 11A. A 2 mm incision was cut at the oral
intestine where the catheter (OD: 2 mm) was inserted into the
intestinal lumen. The incision was tied on the tube with 6-0 silk
suture twice to ensure no leakage. Two ml of HEPES-PSS was gently
injected into the intestine through the tube to wash away the
content. Another 6-0 silk suture was tied 11 mm away towards the
anal intestine from the cannulation. The intestinal mesentery was
untouched to allow the intestine to work in a physiological
environment maintaining normal circulation and vagal responses.
[0175] Ex-Vivo Intestinal Contractility.
[0176] The animals were euthanized by overanesthesia. Either the
deudenum or the colon was excised quickly and placed in cold
HEPES-PSS. The adjacent tissue was dissected with the aid of a
stereo microscope. The intestine was allowed to warm up to room
temperature (22.degree. C.) slowly in 10-15 min and was transferred
to a chamber with HEPES-PSS (22.degree. C.) of the isovolumic
myograph. The two ends of the intestine were cannulated to the
connectors (ID: 1 mm, OD: 2 mm) in the chamber of the isovolumic
myograph. The content in the intestine was gently rinsed with
HEPES-PSS. The intestine in the chamber was warmed to 37.degree. C.
slowly (15-20 min) and equilibrated for 30 min at a basal pressure
of about 1 mmHg before distension.
[0177] Isovolumic System.
[0178] An exemplary isovolumic system used for the present study is
shown in FIG. 22. As shown in FIG. 22, an exemplary ex vivo system
200 comprises a chamber 202 with a catheter 204 on one side wall of
the chamber which bridges the lumen 306 of an intestine 308 to
inflation flask 210 and pressure transducer 212. As shown in FIG.
16A, system 200 is connected to intestine 308, but in various other
embodiments, system 200, and related methods as referenced herein,
may be connected to various other mammalian organs, such as the
stomach, trachea, lymph vessels, lymph ducts, urinary bladders,
ureters, gall bladders, bile ducts, hepatic ducts, and the like,
whereby contraction can be generated by surrounding smooth
muscle.
[0179] Chamber 202, in at least one embodiment, may contain
HEPES-PSS maintained at 37.degree. C. using a heater (not shown),
for example. Inflation flask 210, which in at least one embodiment
comprises a 50 mL flask, having PSS 214 therein is connected to a
pressure regulator 216 so that intestine 308, for example, can be
inflated/distended to the desired pressure. The catheter 204, a
solid state pressure transducer 212 (SPR-524, Microtip catheter
transducer, Millar Inc, Texas), a tube 218 to inflation flask 214,
and a compensatory microsyringe 220 were assembled using a four-way
connector 222. A compensatory microsyringe 220 (50 .mu.l gastight
microsyringe, UltraMicroPump III, and Micro 4.TM. microsyringe
control, World Precision Instruments, USA) was used to stablize the
baseline of the pressure since water transport across the
intestinal wall reduces the intraluminal pressure. The clamping of
tube 218 between inflation flask 210 and four-way connector 222, by
way of stopcock 224, achieved isovolumic conditions, i.e.,
intragastric volume was constant. In at least one embodiment, a CCD
camera on a microscope 2, such as shown in FIG. 1, and an image
processing system (such as the computer shown in FIG. 5A), may be
used to capture the intestinal geometry, including intestinal
diameter. As intestine 308 was inflated to a desired pressure
(e.g., 5 mmHg, 10 mmHg, etc.), stopcock 224 was closed and the
intestinal contraction or relaxation was reflected by the variation
of intraluminal pressure recorded with solid state pressure
transducer 212. The isovolumic system 200 can also record the
periodic contractions of intestine 308 by the periodic variations
of pressure. Additional features/elements of isovolumic system 200
of the present disclosure may also apply to the exemplary system
200 shown in FIG. 22.
[0180] Intestinal Contractility in Response to Mechanical
Stimulation.
[0181] The mechanical stimulation of intestine 308 was induced by
an intragastric inflation pressure. The intestinal contraction was
quantified by the intraluminal pressure under isovolumic condition
and the contractility was characterized with the amplitude and
period of the pressure waveforms. Intestine 308 was inflated to a
desired pressure by a pressure regulator 216 connected to flask 210
as referenced above. The clamping of the tube 218 between the
inflation flask 210 and the four-way connector 222 maintained a
constant volume of solution in the intestinal lumen (isovolumic
condition). The compensatory microsyringe 220 maintains isovolumic
conditions at, for example, an infusion rate of 0.6-2.3 .mu.l/min.
The data was discarded if the rate was larger than 5 .mu.l/min
since this implied damage (leakage) of the intestinal wall. At
isovolumic conditions, the variations of intraluminal pressure was
recorded with a data acquisition system (Biopac, MP100, Houston,
Tex.). The amplitude, frequency, and contractile duration of
pressure waveforms were analyzed to characterize the intestinal
contractility.
[0182] Protocol of Mechanical Stimulation.
[0183] The intraluminal pressure was increased stepwise to 2, 5,
10, 15, 20, 30, 40, and 50 (colon only) mmHg at a rate of 0.05
ml/min, respectively. The intestinal contraction at isovolumic
condition was recorded as the variation of the pressure at each
individual inflation pressure. This protocol was applied to both in
vivo and ex vivo intestine 308. In the experiment of ex vivo
intestine, acetylcholine (10.sup.-6 mole/l) was used to elicit
non-neuroactive contraction of intestinal smooth muscle at
intraluminal pressure of 40 mmHg (duodenum) or 50 mmHg to evaluate
contractility of intestinal smooth muscle.
[0184] Data Analysis and Statistics.
[0185] The data is presented herein as mean.+-.SD. Significant
differences between groups were determined by student West.
Significant differences between the in vivo and ex vivo groups were
determined by use of Analysis Of Variance (ANOVA) between groups. A
probability of p<0.05 was considered indicative of a
statistically significant difference.
[0186] Results.
[0187] The intraluminal pressure waveforms during duodenal
contraction are shown in FIGS. 23A-23C, which clearly show that the
amplitudes of intraluminal pressure altered with the inflation
pressure. FIG. 23A shows in vivo contractile waves, while FIG. 23B
shows ex vivo contractile waves. FIG. 23C represents the inflation
protocol used to test the stretch-elicited contractility. The
amplitude reached to maximum in all conditions at 5 mmHg of
inflation pressure and suppressed down to minimum when inflation
pressure increased up to 40 mmHg. The amplitude of the intraluminal
pressure reflects the intestinal contractility.
[0188] The duodenal contractility is shown in FIGS. 24A and 24B as
a function of inflation pressure, with FIG. 24A relating to
amplitude (in mmHg) and FIG. 24B relating to diameter (in mm). The
in-vivo contractility increased from 0.9 mmHg to 1.4 mmHg while the
inflation pressure increased from 1 mmHg to 5 mmHg. Then, the
duodenal contractility decreased significantly down to 0.4 mmHg
when the inflation pressure further increased from 5 mmHg to 40
mmHg. The ex-vivo duodenal contractility linearly decreased with
the increase in inflation pressure. The ex-vivo duodenal
contractility was significantly attenuated in comparison with the
in-vivo contractility.
[0189] The results identified above indicate that the inflation
pressure higher than 5 mmHg may be considered an inhibitory role in
duodenal motility. The local neuro-regulation may be major role
since the trend lines of the in-vivo and ex-vivo contractility are
similar. The central nervous regulation in in-vivo contractility
seems superimposing a high baseline on the ex-vivo contractility.
The smooth muscle contraction stimulated by ACh, however, is still
similar to maximum contractility of the in-vivo state, which
suggests that neuroactive contraction fails under high
distension.
[0190] The in-vivo and ex-vivo relationship of inflation pressure
and duodenal diameter are shown in FIG. 24B, which reflects the
circumferential distensibility of the duodenum. The in-vivo
circumferential distensibility was not significantly different to
the ex-vivo one (p>0.05).
[0191] The intraluminal pressure waveforms during colonic
contraction are shown in FIGS. 25A-25C. FIG. 25A shows in vivo
contractile waves, while FIG. 25B shows ex vivo contractile waves.
FIG. 25C represents the inflation protocol used to test the
stretch-elicited contractility. The amplitudes of intraluminal
pressure altered with the inflation pressure. The amplitude reached
to maximum in all conditions at 5 mmHg of inflation pressure and
suppressed down to minimum when inflation pressure increased up to
50 mmHg.
[0192] The colonic contractility is shown in FIGS. 26A and 26B as a
function of inflation pressure, with FIG. 26A relating to amplitude
(in mmHg) and FIG. 26B relating to diameter (in mm). The in-vivo
contractility increased from 4.2 mmHg to 4.8 mmHg while the
inflation pressure increased from 1 mmHg to 5 mmHg. Then, the
colonic contractility decreased significantly down to 1.6 mmHg when
the inflation pressure further increased from 5 mmHg to 50 mmHg.
The ex-vivo colonic contractility was significantly attenuated in
comparison with the in-vivo contractility. The inflation pressure
higher than 5 to 10 mmHg may play an inhibitory role in colonic
motility. The local neuro-regulation may not be major role since
the ex-vivo colonic contractility was significantly lower than the
in-vivo contractility. The central nervous regulation largely
dominates the colonic contractility. The non-neuroactive
contraction stimulated by ACh, however, is still similar to maximum
contractility of the in-vivo state, which suggests that neuroactive
contraction fails under high distension.
[0193] The in-vivo and ex-vivo relationship of inflation pressure
and colonic diameter are shown in FIG. 26B, which reflects the
circumferential distensibility of the colon. The in-vivo
circumferential distensibility was not significantly different to
the ex-vivo one (p>0.05).
[0194] To examine the effect of the stresses on intestinal
contractility, the relationship between circumferential, axial, and
radial stresses in intestinal wall and inflation pressure were
plotted in FIGS. 27A and 27B. FIG. 27A represents data in
connection with the duodenum, and FIG. 27B represents data in
connection with the colon. Circumferential and axial stresses
increased when inflation pressure sent up, which means that the
intestine were stretched gradually at circumferential and axial
directions, respectively. In addition, radial stress negatively
increased when inflation pressure went up, which means that the
intestine was compressed gradually.
[0195] As referenced above, and in summary, the isovolumic
myography system 200 was used to assess intestinal contractility in
terms of amplitude of the variation of intraluminal pressure. The
in-vivo preparation was designed to detect the efferent neurogenic
contraction that efferent fibers were intact, and the ex-vivo
preparation was designed to measure the efferent-independent (local
regulatory) contraction that the efferent nervous signals were
eliminated. The in-vivo duodenal contractile amplitude is a little
larger than that in ex-vivo state, indicating that local regulatory
signal contributes the contractility. The in-vivo colonic
contractile amplitude is significantly larger than that in ex-vivo
state, indicating that efferent neurogenic signal contributes the
contractility. The variation of circumferential, axial, and radial
stresses in intestinal wall were analyzed to examine the role of
the stresses in the inhibition of intestinal contractility.
[0196] Intestinal contractility is closely correlated to the
mechanosensor located in the intestinal wall. The myogenic response
of intestinal smooth muscle and efferent neurogenic contraction are
regulated by mechanosensors, namely the afferent vagus nerve and
the efferent vagus nerve. The relation between afferent vagus
signals and intestinal distension was identified decades ago,
noting that the mechanosensors in the intestinal wall are primary
sensors of mechanical stimulation. The efferent (motor) vagus
signals are responses of central nervous system to the afferent
(sensory) vagus stimulation.
[0197] Intestinal motility is regulated by the extrinsic nervous
system (parasympathetic and sympathetic nervous systems) and the
intrinsic nervous system. The intrinsic nervous system is
structurally different in the colon than in the small intestine.
The contraction and motility of colonic cells are more dependent on
the extrinsic nervous system for regulation than in the small
intestine. One of the physiological functions of efferent vagus
signals is to regulate the intestinal contractility, as the
intestinal contractility reflects the activation of efferent vagus
nerve.
[0198] As referenced herein, intestinal contractility was evaluated
in an ex-vivo preparation which excludes efferent vagus regulation.
In the ex-vivo intestine, the nerve fibers are excised and damaged,
and hence there is a loss of efferent vagus signals which appears
to be significant for mechanical distension-induced contractility.
The ex-vivo colonic contractility of intestinal smooth muscle was
significantly attenuated due to the absence of efferent vagus
signals. The strong in-vivo contractility reflects the efferent
vagus activation in response to mechanical stimulation which is
sensed by afferent vagus nerves, which implies that the
contractility is regulated by the central nervous system.
[0199] Distension has been confirmed as a stimulator of intestinal
afferent sensors. The afferent nerve is excited to very high level
in response to inflation, which establishes the sensory and
transmission to central nervous system. This is the first part of
gut-brain cross-talk and the second part is the signal from central
nervous system to control the intestinal function. The in-vivo
intestinal contractility measured by an exemplary isovolumic
myograph of the present disclosure, a parameter of motility,
virtually reflects the resultant of gut-brain cross-talk on
motility, which indicates the final action of afferent sensory
central nervous system efferent action signal final action
(function, motility, etc). Since the distension excited afferent
signal is well established, the results referenced in the present
disclosure indicate that the efferent signal may be parasympathetic
excitation to attenuate the intestinal motility.
[0200] Furthermore, it is noted that the efferent vagus (motor)
inactivation may occur after abdominal surgery, postoperative
intestinal ileus. Ileus is the failure of the gastrointestinal
tract to provide timely, aboral movements of air and chyme from
esophagus to the anus. The intestinal ileus (obstruction) may be
mediated by central neural influences, neurologic reflex (sensitive
afferent nerves) response, the disturbances of myoelectrical
activity, humoral responses, and local or regional activation of
immune system function. The weakening or loss of intestinal
motility is the significant character of intestinal ileus. In the
studies referenced herein, the results implied that the central
nervous system and the neurologic reflex response involved in the
decrease in intestinal contractility. The humoral response and the
immune system may not involve in the mediation of the acute
decrease in intestinal contractility.
[0201] An exemplary isovolumic myograph of the present disclosure
(system 200) was used to evaluate the intestinal globe
contractility, and is therefore suitable to understand the effect
of stimulations of inflation pressure on intestinal contraction.
Although the regional contraction was not measured with such an
isovolumic myograph during the aforementioned study, the efferent
vagus activation was evaluated by the intestinal contractility with
the isovolumic myograph. In conclusion, the various isovolumic
myographs (systems 200) of the present disclosure facilitates the
understanding of intestinal contractility in response to mechanical
stimulation. The in-vivo and ex-vivo intestinal contractility in
response to inflation pressure provides evidence that intestinal
motility can be regulated by central nervous system and local
nervous regulation.
[0202] While various embodiments of iosvolumic myograph systems and
methods for using the same have been described in considerable
detail herein, the embodiments are merely offered by way of
non-limiting examples of the disclosure described herein. It will
therefore be understood that various changes and modifications may
be made, and equivalents may be substituted for elements thereof,
without departing from the scope of the disclosure. Indeed, this
disclosure is not intended to be exhaustive or to limit the scope
of the disclosure.
[0203] Further, in describing representative embodiments, the
disclosure may have presented a method and/or process as a
particular sequence of steps. However, to the extent that the
method or process does not rely on the particular order of steps
set forth herein, the method or process should not be limited to
the particular sequence of steps described. Other sequences of
steps may be possible. Therefore, the particular order of the steps
disclosed herein should not be construed as limitations of the
present disclosure. In addition, disclosure directed to a method
and/or process should not be limited to the performance of their
steps in the order written. Such sequences may be varied and still
remain within the scope of the present disclosure.
* * * * *