U.S. patent application number 16/291930 was filed with the patent office on 2019-12-26 for intraoperative determination of nerve location.
This patent application is currently assigned to Novadaq Technologies ULC. The applicant listed for this patent is Novadaq Technologies ULC. Invention is credited to Dragan GOLIJANIN.
Application Number | 20190388565 16/291930 |
Document ID | / |
Family ID | 37809582 |
Filed Date | 2019-12-26 |
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United States Patent
Application |
20190388565 |
Kind Code |
A1 |
GOLIJANIN; Dragan |
December 26, 2019 |
INTRAOPERATIVE DETERMINATION OF NERVE LOCATION
Abstract
The invention provides methods for intraoperatively determining
the location of nerves by use of fluorescent dyes. The methods are
particularly useful for locating the cavernous nerves innervating
the penis.
Inventors: |
GOLIJANIN; Dragan;
(Rochester, NY) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Novadaq Technologies ULC |
Burnaby |
|
CA |
|
|
Assignee: |
Novadaq Technologies ULC
Burnaby
CA
|
Family ID: |
37809582 |
Appl. No.: |
16/291930 |
Filed: |
March 4, 2019 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
11515419 |
Sep 1, 2006 |
10265419 |
|
|
16291930 |
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60713643 |
Sep 2, 2005 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61K 49/0034
20130101 |
International
Class: |
A61K 49/00 20060101
A61K049/00 |
Claims
1: A method of determining the location of a nerve or portion of a
nerve of interest in a subject during a surgical operation,
comprising: a) prior to said surgical operation, administering to
an organ or area of said subject innervated by said nerve or
portion of a nerve of interest a dye which fluoresces at an
emission wavelength when said dye is contacted with an excitation
wavelength, whereby said dye is taken up by or proceeds along the
path of said nerve; b) exposing said nerve or portion of nerve
during said operation to illumination comprising said excitation
wavelength, thereby causing said fluorescent dye in or along the
nerve or portion thereof to fluoresce; c) detecting the
fluorescence of said dye, thereby determining the location of said
nerve or portion of nerve during said surgical operation.
2-22. (canceled)
23: A method of claim 1, wherein said dye is injected into a
cavernous body of the penis.
24: A method of claim 23, wherein said injection into said
cavernous body is into a crus of said cavernous body.
25: A method of claim 1, wherein said dye is injected by epidural
injection.
26: A method of claim 1, wherein said nerve is transected during
the surgical operation, creating two ends, and the detection of
fluorescence of step (c) is used to determine the location of the
two ends.
27: A method of claim 26, wherein said determination of the
location of the two ends is used to guide grafting of nerve tissue
between the ends or to reconnect them.
28: A method of claim 1, wherein said determination of the location
of 2 said nerve is used to avoid transecting said nerve.
29: A method of claim 28, wherein said surgical operation is a
radical prostatectomy.
30: A method of claim 29, wherein said nerve is a cavernous
nerve.
31: A method of claim 1, wherein said nerve or said portion of said
nerve is visualized on a image display, thereby permitting
determination of the location of said nerve or portion of said
nerve.
32: A method of claim 1, wherein said exposing of said nerve to
excitation wavelength is by a laparoscopic instrument.
33: A method of claim 1 wherein said dye is a dye which fluoresces
when exposed to near infrared light.
34: A method of claim 1, wherein said dye is a tricarbocyanine dye
or an analog thereof.
35: A method of claim 34, wherein the tricarbocyanine dye is
indocyanine green.
36: A method of claim 1, wherein the subject is a human.
37: A method of claim 1, wherein said dye is administered between 1
hour and 30 hours before said surgical operation.
38: A method of claim 1, wherein said dye is administered between
about 18 hours and about 24 hours before said surgical
operation.
39: A method of claim 1, wherein said dye is administered between
about 6 hours and about 24 hours before said surgical
operation.
40: A method of claim 30, wherein the nerve is the small cavernous
nerve.
41: A method of claim 30, wherein the nerve is the large cavernous
nerve.
Description
CROSS-REFERENCES TO RELATED APPLICATIONS
[0001] This application claims priority from and the benefit of
U.S. Provisional Application 60/713,643, filed Sep. 2, 2005, and
U.S. patent application Ser. No. ______, Attorney Docket No.
021501-001710US, filed Aug. 31, 2006. The contents of both
applications are hereby incorporated by reference.
STATEMENT AS TO RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSORED
RESEARCH AND DEVELOPMENT
[0002] Not Applicable
REFERENCE TO A "SEQUENCE LISTING," A TABLE, OR A COMPUTER PROGRAM
LISTING APPENDIX SUBMITTED ON A COMPACT DISK
[0003] Not Applicable
BACKGROUND OF THE INVENTION
[0004] A variety of medical techniques suitable for imaging
biological tissues and organs are known. These include traditional
x-rays, ultra-sound, magnetic resonance imaging, and computerized
tomography.
[0005] A variety of dyes useful for medical imaging have been
described, including radio opaque dyes, fluorescent dyes, and
colorimetric dyes (see e.g., U.S. Pat. Nos. 5,699,798; 5,279,298;
6,351,663). Imaging techniques and systems using fluorescent dyes
have been described for the heart and eye (see, e.g., U.S. Pat. No.
5,279,298). Some dyes can serve both an imaging function and a
therapeutic function (see, e.g. U.S. Pat. No. 6,840,933). Some
specific neuronal imaging agents have been used to visualize tissue
in the central nervous system. Tracer uptake and transport has been
demonstrated in different studies using various routes of
administration including antegrade, retrograde and combined routes
(Jones et al. 1978. Annu Rev Neurosci.; 1:215; Rosina A., 1982,
Neurosci Lett. 33(3):217; Illing R B, et al., 1985, Neuroscience
14(2):455; Sloniewski P, et al., 1985, Neurosci Lett. 60(2):189;
and Schmued et al., 1986, Brain Res. 377(1):147). After appropriate
time for endo/pinocytosis, perineural lymphatic and axonal
transport, which generally measures 0.5-2 mm per hour, tracers were
visually detected using ultraviolet or visible light (Bentivoglio
et al., 1980, Neurosci Lett. 18(1):19; Minciacchi D et al., 1991, J
Neurosci Methods. 38(2-3):183). Non-toxic tracers such as
Indocyanine Green, Fast Blue, and Fluorogold, have been used in
mammals without evidence of neuronal toxicity several months after
the treatment (Thielert et al., 1993, J Comp Neurol. 337(1):113;
Yeterian et al., 1994, Exp Brain Res. 99(3):383; vogt Weisenhom et
al., 1995, J Comp Neurol. 362(2):233). Marangos et al. labeled the
auditory nerve using Fluorogold and Fast Blue in rats and monkeys
by suctioning out perilymph and filling the cochlea with neuronal
tracers to identify the nerve and cochlear brain stem nucleus for
the positioning of electrodes for an auditory neuroprosthesis
(Marangos N, et al., 2001, Hear Res. 162(1-2):48).
[0006] The prostate is an accessory sex gland in men. It is about
the size of a walnut, and surrounds the neck of the bladder and the
urethra, the tube that carries urine from the bladder. It is partly
muscular and partly glandular, with ducts opening into the
prostatic portion of the urethra. It is made up of three lobes: a
center lobe with one lobe on each side. The prostate gland secretes
a slightly alkaline fluid that forms part of the seminal fluid.
[0007] Prostate cancer is the most common type of cancer (excluding
skin cancer) among American men. It is found most often in men aged
50 and over, with an especially high prevalence rate among African
Americans. In men, it is second only to lung cancer as a cause of
cancer-related death. The American Cancer Society has estimated
that 220,900 new cases of prostate cancer will be diagnosed
annually and that 28,900 men annually will die of the disease
(Cancer Facts and Figures, American Cancer Society, 2003).
Treatment options include hormonal therapy aimed at lowering
testosterone levels, radiation therapy, chemo therapy and
surgery.
[0008] Surgical removal of the entire prostate gland is called
radical prostatectomy ("RP"). The aim of radical prostatectomy is
removal of early-stage prostate cancer, one that has not yet spread
locally or to distant organs. Radical prostatectomy complications
include incontinence and impotence. Most men experience urinary
incontinence after surgery. Many continue to have intermittent
problems with dribbling caused by coughing or exertion. Damage to
nerves which innervate both the prostate and the penis plays a
significant part in these unwanted side effects. Approximately 40
to 60% of men undergoing RP are impotent due to injury to the
cavernous nerves during the surgery.
[0009] Topographically, cavernous nerves are part of the
neurovascular bundle, which travels at the posterolateral border of
the prostate, outside the prostatic capsule and on the
anterolateral surface of the rectum. McNeal described large
superior and small inferior pedicles innervating the base and the
apex of the prostate respectively (McNeal J E., 1988, Am J Surg
Pathol.; 12(8):619). After reaching the apex at the 5 and 7-o'clock
positions, nerves travel posterolaterally to the urethra. At the
level of membranous urethra they divide into more superficial
branches to the sphincter muscle and finally at the level of the
hilum of the penis, together with the arteries, the nerves pierce
the cavernous bodies and innervate erectile tissue diffusely (Lue
et al., 1983, J Urol.; 130(6):1237; Breza et al., 1989, J Urol.
141(2):437).
[0010] The risk of impotence may be reduced by avoiding cutting or
stretching bundles of nerves and blood vessels that run along the
surface of the prostate gland and are needed for an erection.
Successful nerve sparing surgery, however, is often difficult to
achieve because of the difficulty in distinguishing between the
prostate tissue, in particular the cancerous prostate tissue, and
the innervating nerve tissue. Appropriate mapping of the nerves can
also lead to better understanding of cavernous nerves topography
and penile accessory innervation. Accordingly, a need exists for
improved methods of imaging peripheral nerves, such as the nerves
which innervate the prostate. The present invention fills these and
other needs.
BRIEF SUMMARY OF THE INVENTION
[0011] The invention provides methods of determining the location
of a nerve or portion of a nerve of interest in a subject during a
surgical operation. The methods comprise, prior to the surgical
operation, administering to an organ or area of the subject
innervated by the nerve or portion of a nerve of interest a dye
which fluoresces at an emission wavelength when the dye is
contacted with an excitation wavelength, whereby the dye is taken
up by or proceeds along the path of the nerve; exposing the nerve
or portion of nerve during said operation to illumination
comprising said excitation wavelength, thereby causing the
fluorescent dye in or along the nerve or portion thereof to
fluoresce; and detecting the fluorescence of the dye, thereby
determining the location of said nerve or portion of nerve during
said surgical operation. In some embodiments, the dye is injected
into a cavernous body of the penis. In some embodiments, the
injection into the cavernous body is into a crus of said cavernous
body. In some embodiments, said dye is injected into a cavernous
body of the clitoris, into the vaginal wall, or into both. In some
embodiments, the dye is injected by epidural injection. In some
embodiments, the nerve is transected during the surgical operation,
creating two ends, and the detection of fluorescence of step is
used to determine the location of the two ends. In some
embodiments, the determination of the location of the two ends of
the transected nerve is used to guide grafting of nerve tissue
between the ends or to reconnect them. In some embodiments, the
determination of the location of the nerve is used to avoid
transecting said nerve. In some embodiments, the surgical operation
is a radical prostatectomy. In some embodiments, the surgical
operation is a radical hysterectomy. In some embodiments, the nerve
is a cavernous nerve. In some embodiments, the nerve or aid portion
of the nerve is visualized on a image display, thereby permitting
determination of the location of the nerve or portion of the nerve.
In some embodiments, the exposing of the nerve or portion of nerve
to excitation wavelength is by a laparoscopic instrument. In some
embodiments, the dye is a dye which fluoresces when exposed to near
infrared light. In some embodiments, the dye is a tricarbocyanine
dye or an analog thereof. In some embodiments, the tricarbocyanine
dye is indocyanine green. In some embodiments, the subject is a
human. In some embodiments, the dye is administered between 1 hour
and 30 hours before the surgical operation. In some embodiments,
the dye is administered between about 18 hours and about 24 hours
before the surgical operation. In some embodiments, the dye is
administered between about 6 hours and about 24 hours before the
surgical operation.
[0012] In some embodiments, the nerve is the small cavernous nerve.
In some embodiments, the nerve is the large cavernous nerve.
BRIEF DESCRIPTION OF THE DRAWINGS
[0013] FIG. 1a shows the exposing of the penis of a rat. FIGS. 1b
and 1c show the injection of ICG into the crura of the left and
right cavernous bodies, respectively. FIG. 1d shows the exposed
penile crura and cavernous nerves under near infrared (NIRF)
illumination following injection of ICG. Arrows show the location
of the cavernous nerves.
[0014] FIG. 2 shows a cavernous nerve under NIRF illumination
following injection of ICG into the crura of the cavernous bodies.
The white arrow points to the nerve.
[0015] FIG. 3 shows a hook electrode (arrow) hooked around a
cavernous nerve under NIRF illumination, after ICG injection into
the crura of the cavernous bodies.
[0016] FIG. 4 shows NIRF illumination of a cavernous nerve (arrow)
from an animal injected with ICG as described above.
[0017] FIGS. 5a and 56b are photographs of a cavernous nerve
excised from an animal whose penile crura were injected with ICG.
Figure Sa shows the nerve under infrared and LED illumination. FIG.
5b shows the same nerve under NIRF alone.
DETAILED DESCRIPTION OF THE INVENTION
[0018] As set forth in the Background, some dyes and other agents
have been used to image nerve cells in the eye and nerves
connecting the ear to the central nervous system. Surprisingly,
dyes can also be used to image (that is, to permit visualization)
of nerves elsewhere in the body and can be used either to reduce
the chance that nerves will inadvertently be transected during
surgical procedures or to help guide neural grafts when unintended
transactions occur or when they cannot otherwise be avoided.
[0019] The original surgical treatment for prostate cancer was
radical perineal prostatectomy, which was followed, in the 1940's,
by radical retropubic prostatectomy. Both forms of surgery not only
typically led to incontinence and to impotence, but also resulted
in extensive bleeding, which made it difficult to detect and
preserve associated structures. The severity of the side effects
from these surgical procedures made radiation therapy for prostate
cancer a popular alternative once it was developed in the 1960's.
The development of less bloody surgical techniques in the 1970's,
however, resulted in the preservation of more structure and less
inadvertent damage to blood vessels and to nerves. In the early
1980's, Drs. Patrick Walsh and Pieter Donker were able for the
first time to determine that the nerves for the penis did not go
through the prostate. In 1982, Dr. Walsh showed it was possible to
remove the prostate without severing the nerves innervating the
penis. This so-called "nerve sparing" surgery, medically termed
"anatomic radical prostatectomy," properly done, permits retention
of potency in many patients. Nonetheless, surgeons still sever the
nerves in some patients, leading to loss of potency in those
patients. Thus, visualizing the nerves during surgery is highly
desirable.
[0020] In particular, the methods of the invention provide for the
visualization of cavernous nerve cells during radical prostatectomy
("RP") and other surgical procedures. The recovery of erectile
function is a significant concern for patients undergoing treatment
for localized prostate cancer. As noted in the Background,
impotence is a common side effect of RP resulting from damage to
the cavernous nerves that innervate the penis. Damage to the nerve
tissue surrounding the prostate usually results from the surgeon's
inability to distinguish between prostate tissue and nerve tissue.
The present invention increases the ability of the surgeon to
identify and avoid the cavernous nerves as they traverse the area
surrounding the prostate and other abdominal organs and thus to
reduce or to prevent damage to those nerves tissue during the
course of surgery.
[0021] The methods are useful not only to reduce the inadvertent
transection of these nerves during prostate surgery, but also to
aid in grafting the ends of the nerves if they are transected. In
the event of transection, genitofemoral or sural nerve grafts can
be applied directly to the ends to facilitate sprouting of
regenerative neural fibers. In this case, the light visible from
the fluorescence of the ends of transected nerves provides a target
to guide the anastomosis of the nerves by the nerve graft. The
methods can further be used to identify the cavernous nerves in
patients undergoing radical pelvic surgeries, such as low anterior
resection or abdominal perineal resection.
[0022] In the methods of the invention, a fluorescent dye that can
be taken up by nerve cells and that has low toxicity is injected
into the corpora cavernosa or one corpus cavernosum of the penis
(as persons of skill are aware, "corpora cavernosa" is the plural
form and "corpus cavernosum" is the singular form. Thus, the
practitioner may choose to inject the dye into one corpus
cavernosum or into both, depending on whether the practitioner
wishes to be able to image one or both of the subject's cavernous
nerves.) For convenience, the corpora cavernosa will sometimes be
referred to herein as the "cavernous bodies."
[0023] The injections may be made into the cavernous bodies as they
run parallel to each other along the length of the penis, or at the
base or root of the penis into the crura, which is the area where
the two corpora cavernosa diverge within the subject's body and
anchor to bone. (If the intent is to visualize just one of the
cavernous nerves, the injection will be made into the appropriate
crus of penis, "crus" being the singular form of "crura.") In
studies underlying the present invention, injections into the
cavernous bodies closer to the glans of the penis, and therefore
more distal from the body, resulted in less favorable visualization
of the nerves than did injections into the crura. Accordingly,
injection into the crura is preferred.
[0024] In humans, the injections are easily made by palpating the
pubic bone and making the injection about 1 cm distal (along the
penis, away from the body) from the pubic bone. This location is
the same as that at which patients are taught to self inject
erectile dysfunction drugs to cause erections, so it is well known
and relatively easy to find even for the layperson.
[0025] Following injection, the dye is transported along the nerves
supplying the organ, permitting visualization of the nerve and
determination of their location. Without wishing to be bound by
theory, it is surmised that at least some of the flow of the dye is
in lymphatic channels around the nerve rather than in the nerve
itself. This belief arises because, in studies underlying the
invention, it was noted both that the periphery of the nerve
fluoresced more strongly than the middle of the nerve that the
speed at which the dye moved along the nerve seemed faster than
might be expected from retrograde transport alone.
[0026] Since there are two sets of cavernous nerves, which run on
the left and right sides of the prostate, the surgeon will
generally choose to inject the dye into both the left and the right
cavernous bodies of the penis so that both sets of nerves can be
visualized. The dye is then transported along by the cavernous
nerves. Helpfully, accessory nerves may also be visualized by this
procedure. Since these nerves can also play some role in achieving
erections, the visualization of these nerves and consequent ability
to avoid transecting them, or to reconnect them if they are
transected, further improves the prospect that the patient will not
be impotent following the operation.
[0027] In some instances, which are expected to be relatively
uncommon, the surgeon may wish to visualize only one cavernous
nerve. In these instances, the surgeon may inject dye only into the
cavernous body innervated by the cavernous nerve whose
visualization is desired. It is contemplated, however, that the
surgeon will usually want the ability to visualize and thereby
locate, the nerves around both sides of the prostate, in part
because visualizing the transected ends will permit the nerves to
be bridged (interpositioned) with genitofemoral or sural nerve.
Additionally, in some prostate cancers, it is difficult for the
surgeon to determine where the prostate tissue ends. In such cases,
visualization of the nerves through the methods of the invention
will assist the surgeon determine the margin between prostate and
non-prostate tissue. Accordingly, injection of dye into both
cavernous bodies is preferred.
[0028] Imaging of the nerves can also be made by injecting the dye
by an epidural injection in the area of S2-S4 of the spinal cord
level to allow antegrade flow of the dye along the nerve. Tens of
thousands of epidural injections are made every year, for example,
to women undergoing childbirth, and techniques for making such
injections, and for positioning them at desired levels are well
known in the art. It is expected that dye injected by epidural
injection, will undergo transport along the nerves from the spine
towards the penis, and will permit visualization of the nerves in a
manner like that seen from injections of dye into the cavernous
bodies.
[0029] In recent years, it has also been recognized that women
undergoing gynecological operations, such as hysterectomies, can
suffer damage to nerves controlling vaginal erectile tissue in a
manner similar to that sustained by men under radical
prostatectomy. Nerve damage in the course of radical hysterectomies
can also result in post-operative bladder dysfunction due to damage
to the pelvic autonomic nerves during surgical resection, as well
as other pelvic morbidity. This recognition has led to attempts to
develop nerve-sparing techniques for gynecological procedures. See,
e.g., Possover, Gynecol Oncol. 2003; 90(2):245-7; Ito and Saito,
Eur J Surg Oncol. (2004), 30(10): 1137-40; Butler-Manuel et al.,
(2000), Cancer, 89(4):834-841; Trimbos et al., Int J Gynecol Cancer
(2001), 11:180-186.
[0030] Based on the results seen in visualizing the cavernous
nerves in male animals, it is expected that the same procedures can
be used to visualize the nerves innervating homologous structures
in women, thereby improving the ability to visualize and locate the
nerves during gynecological operations, such as simple and radical
hysterectomies. The clitoris contains a glans, shaft, and crura
containing cavernous bodies homologous to the structures of the
penis. The dye can be injected into the cavernous bodies in the
clitoris in a like manner to the injections described above with
regard to the penis. Specifically, the dye is injected into the
clitoris or into the anterior wall of the vagina just below the
clitoris or, preferably, both. Alternatively, or in addition, the
dye is administered as an epidural injection at the S2-S4 spinal
cord level, at the start of the parasympathetic outflow. It is
expected that the improved ability to visualize the nerves will
help the surgeon avoid transecting the nerves or, if the nerves are
transected, will help the surgeon identify the cut ends and assist
in bridging the gap with a nerve graft or, where possible,
reattaching the cut ends.
[0031] Based on the results seen in visualizing the cavernous
nerves following injection of dye into the tissue of the organ that
they innervate, it is believed that injection of dye into the
tissue of other organs will result in the uptake of dye by nerves
innervating those organs and will result in the ability to image
those nerves. It is not expected, however, that this method will be
useful in imaging the brain.
[0032] It is understood that fluorescent dyes have a particular
excitation wavelength which causes the dye to fluoresce and emit
light of a particular emission wavelength. As persons of skill are
aware, there is a considerable literature on the characteristics of
different dyes, including their excitation wavelength and emission
wavelength.
[0033] The methods described herein are suitable for use in
mammals. Examples of mammals for which the techniques can be used
include, but are not limited to, non-human primates, dogs, cats,
sheep, cows, pigs, horses, mice, rats, rabbits, and guinea pigs.
The methods are particularly useful in visualizing nerves in
humans, and particularly the cavernous nerves in humans.
Penile Anatomy
[0034] It is assumed that urologic surgeons and other persons of
skill are well familiar with the anatomy of the penis, the
prostate, and the surrounding areas, and that no detailed
discussion is needed here. For purposes of the present discussion,
it is noted that the penis can be thought of as comprising three
cylinders. Two, the corpora cavernosa, are disposed on either side
of the penis, and make up the bulk of the penis. The third, the
corpus spongiosum, which contains the urethra, is disposed in the
middle of the penis, in a cleft between the undersides of the
corpora cavernosa. A "deep artery" runs down the center of each
corpus cavernosum and provides blood to sinusoidal spaces in the
respective corpus. During erection, the deep artery is expanded and
the sinusoidal spaces are swollen, while the emissary veins that
drain blood from the corpora cavernosa are compressed. The
sinusoidal spaces within each corpus cavernosum communicate,
permitting a common intracavernosal pressure and a common penile
rigidity.
[0035] Preganglionic parasympathetic neurons originate from the
sacral spinal nucleus at levels S2-S4. The axons travel from the
anterior sacral roots and end as cavernous nerves giving
innervation to the cavernous bodies. Stimulation of pelvic and
cavernous nerves has been shown to result in an erection in animals
as well as in humans. Complete loss of erection is observed after
bilateral cavernous nerve resection. The sympathetic contribution
to the cavernous bodies originates at the T11-L2 level of the
spinal cord, and travels through the prevertebral pathway,
consisting of lumbar splanchnic nerves, hypogastric nerves, pelvic
plexus and cavernous nerves and through the paravertebral chain,
leading to the pelvic nerves, pelvic plexus, cavernous and pudendal
nerves.
[0036] Stimulation of the paravertebral sympathetic chain results
in detumescence. Topographically, cavernous nerves are part of the
neurovascular bundle, which travels at the posterolateral border of
the prostate, outside the prostatic capsule and on the
anterolateral surface of the rectum. Large superior and small
inferior pedicles innervate the base and the apex of the prostate,
respectively. After reaching the apex at the 5 and 7 o'clock
positions, nerves travel posterolaterally to the urethra. At the
level of membranous urethra, they divide into more superficial
branches to the sphincter muscle and finally at the level of the
hilum of the penis, together with the arteries, the nerves pierce
the cavernous bodies and innervate erectile tissue diffusely. A
description of the prostatic plexus and associated nerves,
including the cavernous nerves, can be found on-line by entering
"http://" followed by
"education.yahoo.com/reference/gray/subjects/subject?id=220".
Instrumentation
[0037] For convenience, the following discussion will refer to
instrumentation optimized for use with the exemplar dye ICG.
Persons of skill are, of course, aware of the excitation and
emission frequencies of other fluorescent dyes and can adjust the
device as needed for use with respect to other dyes as desired.
[0038] Conveniently, the device used for visualization of the
nerves in the area of interest comprises both a laser and a camera.
For use with ICG, the laser preferably consists of a laser diode
providing a maximum of 3 W output at 806 nm. For other dyes, the
laser diode is selected to provide a light with a wavelength at an
excitation frequency appropriate for the dye selected. For
convenience of reference, the discussion below refers to the
exemplar dye ICG. Persons of skill will recognize that the other
dyes mentioned herein as suitable for use in the inventive methods
and procedures could be substituted for ICG, with the light source
selected or adjusted to provide illumination optimized for the
excitation frequency suitable for the particular dye chosen and the
device for capturing the light emitted by the dye being selected or
adjusted to be able to receive light of the appropriate
frequency.
[0039] The laser output is decollimated (i.e. optics are used to
spread out the laser light from a tight beam) to provide even
illumination over a field of view, for example, 7.6 cm by 7.6 cm at
a working distance of 30 cm. The unit typically contains a
charge-coupled device ("CCD") video camera sensitive into the near
infrared spectrum and, for use with ICG, is equipped with an 815 nm
edge filter. An articulated arm, connected to a wheeled base,
supports the laser/camera device. This allows the imaging head to
be moved into close proximity to the surgical table and for
vertical movement of the head to attain the correct focal distance
above the area of interest. The imaging head and extension arm that
protrudes over the surgical field is typically covered with an
optically transparent sterile drape. The laser can be activated by
means of a computer command or by foot pedal. Such laser/camera
devices are commercially available. Laser/camera devices suitable
for intra-operative imaging are commercially available. In a
preferred embodiment, the laser/camera device is a SPY.RTM.
Intra-operative Imaging System (Novadaq Technologies, Inc.,
Mississauga, Ontario, Canada).
[0040] For visualizing the cavernous nerves, the ICG is
administered by injection into one of the corpus cavernosum of
interest, preferably into the crus, permitting the dye to be taken
up by the nerve serving that corpus cavernosum, and transported by
retrograde transport back towards the pelvic plexus. Generally, the
practitioner will want to visualize both cavernous nerves and will
inject the dye into both cavernous bodies. Following an interval
sufficient for the dye to be transported throughout the area in
which the nerve is to be visualized, a 806 nm excitation light
causes the dye to fluoresce, emitting light at 830 nm. The emitted
light can then be imaged directly or, preferably, is captured using
an imaging system. Typically, the capture system is a
charge-coupled device (CCD) camera or CMOS (complementary symmetry
metal oxide semiconductor) image sensor, which feeds the image to a
monitor so that the surgeon can visualize the fluorescence of the
dye in the nerves in the area of interest in real time. Optionally,
the camera is also attached to a computer and the image is saved,
which not only permits documentation of the nerve's location and
path in the area of interest, but also can be used for training
urologic surgeons, nurses, and other medical staff. Typically, the
time required for positioning the device is 2 minutes, while the
total time that the nerve or nerves are illuminated with laser
light is 30 seconds.
[0041] The methods described herein are suitable for use in
mammals. Examples of suitable mammals include, but are not limited
to, humans, non-human primates, dogs, cats, sheep, cows, pigs,
horses, mice, rats, rabbits, and guinea pigs. Use in humans is
primates, and particularly in humans, is preferred.
Dyes for Imaging
[0042] As persons of skill are aware, fluorescent dyes have a
particular excitation wavelength which causes the dye to fluoresce
and emit light of a particular emission wavelength. Persons of
skill will appreciate that a considerable literature is available
in the art on the characteristics of different dyes, including
their excitation wavelength and emission wavelength. This
literature is well known, and will not be set forth in detail
herein.
[0043] The dye is imaged by exciting it with a light that has an
excitation wavelength appropriate for the particular dye used.
Persons of skill are aware that a variety of dyes exist, and that
each dye has an excitation wavelength and an emission wavelength.
Some dyes, for example, fluoresce under ultraviolet ("UV")
illumination while others fluoresce under incandescent
illumination. There is a large literature on the use of fluorescent
dyes and probes in biological assays, such as Dewey, T. G., Ed.,
Biophysical and Biochemical Aspects of Fluorescence Spectroscopy,
Plenum Publishing (1991), Guilbault, G. G., Ed., Practical
Fluorescence, Second Edition, Marcel Dekker (1990), Lakowicz, J.
R., Ed., Topics in Fluorescence Spectroscopy: Techniques (Volume 1,
1991); Principles (Volume 2, 1991); Biochemical Applications
(Volume 3, 1992); Probe Design and Chemical Sensing (Volume 4,
1994); Nonlinear and Two-Photon Induced Fluorescence (Volume 5,
1997); Protein Fluorescence (Volume 6, 2000); DNA Technology
(Volume 7, 2003); Plenum Publishing, and Lakowicz, J. R.,
Principles of Fluorescence Spectroscopy, Second Edition, Plenum
Publishing (1999). Invitrogen Inc. (Carlsbad, Calif.) makes a
comprehensive handbook on fluorescence and its use in biological
assays available on its website. The reference is entitled "The
Handbook--A Guide to Fluorescent Probes and Labeling Technologies"
and is currently in its tenth edition. The Handbook can be found
on-line by entering "http://" followed by
"probes.invitrogen.com/handbook/".
[0044] Fluorescent dyes suitable for use in the methods of the
invention are non-toxic dyes which fluoresce when exposed to
radiant energy, e.g. light. Preferably, the dyes are near infrared
fluorochromes, or "NIRF" that emit light in the near infra red
spectrum. In some embodiments, the dye is a tricarbocyanine dye,
and in particularly preferred embodiments, is indocyanine green
("ICG"). ICG is commercially available from, for example, Akorn,
Inc. (Buffalo Grove, Ill.), which sells it under the name
IC-GREEN.TM.. In other embodiments the dye is selected from
fluorescein isothiocyanate, rhodamine, phycoerythrin, phycocyanin,
allophycocyanin, o-phthaldehyde, fluorescamine, Rose Bengal, trypan
blue, and fluoro-gold. The dyes may be mixed or combined. In some
embodiments, dye analogs may be used. A "dye analog" is a dye that
has been chemically modified, but still retains its ability to
fluoresce when exposed to radiant energy of an appropriate
wavelength. ICG, Fast Blue and Fluorogold have all been used in
mammals with low evidence of neuronal toxicity and are
preferred.
[0045] ICG is particularly preferred both because it has low
toxicity and because it has been approved by the Food and Drug
Administration for several diagnostic purposes in humans. Its
absorption (excitation) and emission peaks (805 and 835 nm,
respectively) lie within the "optical window" of tissue, where
absorption due to endogenous chromophores is low. Near infrared
light can therefore penetrate tissue to a depth of several
millimeters to a few centimeters. After intravenous injection, ICG
is bound within 1 to 2 seconds, mainly to globulins
(1-lipoproteins), and remains intravascular, with normal vascular
permeability. ICG is not metabolized in the body and is excreted
exclusively by the liver, with a plasma half-life of 3 to 4
minutes. It is not reabsorbed from the intestine and does not
undergo enterohepatic recirculation. Commercially available ICG
contains iodine and should not be administered to persons with a
history of reaction to iodine. The recommended dose for ICG video
angiography is 0.2 to 0.5 mg/kg; the maximum daily dose should not
exceed 5 mg/kg.
[0046] For intraoperatively visualizing the cavernous nerves, the
surgical field, or the portion of the surgical field in which
imaging is desired, is illuminated with a light of the excitation
wavelength or wavelengths suitable for the dye or dyes used. Since
the nerves are quite thin (accounting in part for the difficulty in
discerning them with the unaided eye), ambient light may need to be
dimmed to permit the fluorescence to be seen. Observation will
typically also require magnification. Where the excitation
wavelength is outside of the visible range (where, for example, the
excitation wavelength is in the ultraviolet or near infrared
range), the light source may be designed to permit switching or
"toggling" between the excitation wavelength and visible light.
This permits the practitioner to note the position of the nerves
using the fluorescent property in relation to the rest of the
surgical field and surrounding (but non-fluorescent)
structures.
[0047] In some embodiments, an instrument having an optical
configuration similar to a fluorescence microscope may be used, in
which a dichroic mirror is used to split the paths of the
illumination (the excitation light). The excitation light reflects
off the surface of the dichroic mirror into the objective, while
the fluorescence emission passes through the dichroic mirror to the
eyepiece or is converted into a signal to be presented on a screen.
The instrument may further have an excitation filter or an emission
filter, or both, to select the wavelengths appropriate for each
function. Conveniently, the filters are interference filters, which
block transmission of frequencies out of their bandpass.
[0048] The dye is typically administered by an injection into one
or both of the corpora cavernosa. Typically, the dye will be
administered some hours preoperatively, to permit the dye to be
taken up by the nerves and transported throughout the area of
interest prior to commencing the radical prostatectomy or other
surgical operation.
[0049] Conveniently, the dye may be administered in the patient's
room. Typically, the dye is administered sufficiently before the
intended surgery to permit the nerves to take up the dye and to
transport it over the length of their axons, but not so long before
the surgery that the dye has been transported in large part to the
cell body. Preferably, the dye is administered more than 2 hours,
but less than 48 hours, before the intended surgery. More
preferably, the dye is administered at least about 5 hours but not
more than 40 hours before the intended surgery. In some
embodiments, the dye is administered at least about 10 hours but
not more than 36 hours before the intended surgery. In other
embodiments, the dye is administered at least about 14 hours but
not more than 30 hours before the intended surgery. In preferred
embodiments, the dye is administered between about 16 hours to 26
hours before the intended surgery. In still preferred embodiments,
the dye is administered between about 18 hours to about 24 hours
before the intended surgery, with "about" meaning an hour on either
side.
[0050] The maximum daily dosage of ICG for adults is 2 mg/kg. There
is no data available describing the signs, symptoms, or laboratory
findings accompanying an overdose of ICG. The LD.sub.50 after IV
administration ranges between 60 and 80 mg/kg in mice, 50 and 70
mg/kg in rats, and 50 to 80 mg/kg in rabbits.
EXAMPLES
Example 1
[0051] Intraoperative video angiography is performed with a
laser-fluorescence imaging device (Novadaq Technologies, Inc.,
Mississauga, Ontario, Canada) consisting of a near infrared (NIR)
laser light source and a NIR-sensitive digital camcorder. For
measurements, the unit is positioned 30 to 40 cm from the area of
interest. ICG, dissolved in water, is then injected as a bolus. The
NIR light emitted by the laser light source induces ICG
fluorescence. The fluorescence is recorded by a digital video
camera, with optical filtering to block ambient and laser light so
that, when desired, only ICG fluorescence is captured. Images can
be observed on screen in real time (25 images/sec). The images can
be reviewed and stored on the digital video camera or transferred
to a computer or to storage media.
Example 2
[0052] The rat cavernous nerve model is well-recognized as a model
for radical retropubic prostatectomy-associated neurogenic erectile
dysfunction and has distinctive advantages over the other animal
models. The rat cavernous nerves are single neural bundles that run
alongside the prostate on either side. They are easily identified
and are ideal neural bundles to evaluate the ability of neuro
tracer to highlight pelvic nerves. Further, electrical stimulation
is easily accomplished and yields reliable and reproducible
results. Additionally, neurophysiological studies are feasible, and
animal purchase, housing, and maintenance costs are low. The
medical literature describes the successful use of Sprague-Dawley
rats for the assessment of erectile dysfunction after cavernous
nerve injury.
[0053] The animals are divided into three groups: Group I, placebo
injection; group II intracavernous ICG injection; group III
Fluorogold injection. Groups I-III are subdivided into 3
sub-groups, according to the timing for intraoperative evaluation
of axonal fluorescent staining in cavernous nerves. Retrograde
injection of placebo (distilled water) or fluorochromes, ICG or
Fluorogold, is administered by intra-penile, sub-albugineal
injection of 25 ul of ICG diluted in 100 .mu.l of water for
injection, per cavernous body.
[0054] Sprague-Dawley rats, 60 to 100 days old, weighing 275-325
grams are used. All animals are anesthetized using intraperitoneal
injection of Ketamine/Xylazine (40-80 mg/kg and 5/10 mg/kg,
respectively). No pre-anesthetic medications are used. When
appropriate depth of anesthesia is reached, positioning of the
animal takes place. All animals are fastened to a padded rack in
the supine position using gauze knots to fix all four extremities
to the rack equipped with heating device. Depth of anesthesia,
regularity of respirations, and heart beat palpation are repeatedly
checked. A pulse oximeter is used to monitor the animal.
[0055] Surgery/Procedure starts after appropriate preparation of
surgical field by Povidone-Iodine scrub, 70% Isopropyl Alcohol and
Povidone-Iodine solution. The surgical field includes the genital
area, lower abdomen and perineum. The penis is squeezed out from
prepuce, then stretched using finger grip at the glans and, when
maximally stretched, a clamp used for atraumatic clamping in
neurosurgical operation on brain aneurysms is placed at the root of
the penis. This allows blood to pool inside cavernous bodies, an
erection, and easier application of a 27 Gauge butterfly needle to
sub-albugineal space bilaterally. Adequate placement is assured
when blood is easily aspirated. 0.5 mg/kg of ICG and 1 mg/kg of
Fluorogold diluted in distilled water to total volume of 50 .mu.l
is injected, 25 .mu.l per cavernous body. A placebo group is
injected using 25 .mu.l of distilled water per cavernous body.
Animals in all groups have an exploratory laparotomy through
midline lower incision, with intraoperative identification of
cavernous nerves. Midline incisions are made from umbilicus to
pubis. Bowels and testicles, after their release from scrotal
attachments, are packed back to upper abdomen. For better
visualization of the pelvic structures, surgical loops with
3.8.times. magnification are used. In all animals, pelvic ganglion
and cavernous nerves are identified bilaterally. The pressure at
the level of penile base is held for 15 minutes to allow better
penetration of injected fluorochromes into neural endings in
cavernous bodies. After release of the clamps, the 27 Gauge needles
are removed, but a slight pressure by fingers at the injection
sites is maintained for 3-5 minutes to prevent extravasation.
Buprenorphine (0.01-0.05 mg/kg) is administered intraoperatively
and then as needed to control pain.
[0056] Post-procedurally, optimal recovery is routinely performed
in all animals: animals are kept warm using warming packs, pads and
lamps, the animals are placed on a paper towel, and are rotated
from side to side every 15 minutes until they are able to maintain
sternal recumbence. 3-4 ml of Lactated Ringer solution, warmed to
37.degree. C., is administered subcutaneously, unilaterally in the
flank region. This prevents postoperative dehydration of the
animal. Animals are attended at all times during postoperative
recovery. The animals are then returned to cages, and hydration is
assessed on a daily basis with desired volume of 60-80 ml/kg/day,
which translates to approximately 30 ml of fluids per 300 g animal,
per day. Similarly, a 300 gram rat which is estimated to be 10%
dehydrated would need to have 300 g.times.0.10=30 ml of fluids
replaced per day. Analgesia is used for all animals for
postoperative pain. Animals are checked for signs of pain every 6
hours during first 24 hours post-surgery and then every 12 hours
until sacrifice surgery. Signs of acute pain are guarding
(protecting the painful area), vocalization, especially when the
animal moves or the painful area is touched, licking, biting,
scratching or shaking a particular area, restlessness, such as
pacing and repeatedly lying down and getting up again
(stereotypic-like behavior), lack of mobility as seen with joint,
colic or gut pain or an unusual gait posture during movement,
failure to groom, causing an unkempt appearance (rats accumulate
red porphyrin around the eyes when they fail to groom properly),
abnormal resting postures in which the animal appears to be
sleeping or is hunched up and cannot get comfortable, failure to
show normal patterns of inquisitiveness or alertness, loss of
appetite or reduction in water consumption, and changes in behavior
or signs of aggression.
[0057] To perform neurostimulation and harvesting of cavernous
nerves and cavernous bodies, the rats undergo a second surgery
(non-survival) and postmortem harvesting at chosen timepoints after
fluorochrome injection.
[0058] Anesthesia identical to the first procedure is administered
and then appropriate skin preparation for surgery is performed. The
same level of incision is used in second surgery/harvesting
procedure. The bladder and prostate are exposed as in the first
surgery. The penis is denuded of skin and the prepuce circumcised.
Cavernous nerves, located lateral to urethra and prostate
bilaterally, are identified using the SPY.RTM. Imaging System
(Novadaq Technologies, Inc. Toronto, Canada) and isolated for
electrode placement. A stainless-steel bipolar electrode with
parallel hooks (1 mm apart) is placed around a cavernous nerve and
positioned by micromanipulator. The electrode cable is attached to
a Grass S48 stimulator (Quincy, Mass.), with stimulation parameters
16 Hz, 5 msec duration, 0.5 to 4 volts. To monitor the
intracavernous pressure (ICP), a heparinized (100 units per 1 ml
NS) 24 gauge angiocath (Insyte-N, Becton Dickinson Vascular Access,
Sandy, Utah), attached to polyethylene-50 tubing, is inserted in
the rat's right cavernous body. A cannula inserted to the right
cavernous body is connected to a pressure transducer and an
amplifier unit (Harvard Apparatus, Holliston, Mass.). The amplifier
is connected to a data acquisition module (DI-190, Dataq
Instruments, Akron, Ohio). The data is recorded on a computer with
Windaq/Lite recording software (Dataq). Similarly, Central Arterial
Pressure (CAP) is measured after appropriate placement of silicone
tube into common carotid artery identified just lateral to the
trachea. This allows measurement of the cavernous/central arterial
pressure ratio and accurate assessment of erection in animal after
cavernous nerve stimulation. When ICP measurements are completed,
the animal is euthanized by performing elongation of abdominal
incision with opening of the chest and use of large vascular clip
to ligate aorta and vena cava at the exit/entrance from/to the
heart. Death of the animal is confirmed using pulse oximeter and
observation of complete absence of heart contractions. Both
cavernous nerves are harvested in full length after appropriate
exposure of the pelvic structures bilaterally, close to the pubis.
Nerves are transected and an excised segment is fixed in 1.5 ml, 5%
formalin, and contralateral segment is frozen in liquid nitrogen
for further analysis.
[0059] Already denuded cavernous bodies are transected close to
their roots and divided tangentially. Identical fixation and
storage are used for cavernous bodies as for cavernous nerves. At
the end of non-survival surgery cavernous nerves are harvested as
well as cavernous bodies using NS and 10% Formaldehyde adequately
marked tubes. Half of the specimen is placed into Formalin Aldehyde
and half into NS. These are sent for pathological assessment of the
structures for presence of fluorochrome in cavernous bodies and
presence of axonal tracer/fluorochrome in cavernous nerves.
Electronic microscopy is used to assess subcellular integrity of
the neural and myovascular structures.
Example 3
[0060] To determine whether crural injections of dye would also
result in the ability to image the cavernous nerves, 0.22 ml 1f
0.25 mg/ml of ICG was injected subalbugineally into both crura of
22 rats. The area was examined for retrograde axonal transport at
different time points, using a NIRF intraoperative imaging system
(SPY.RTM., Novadaq Technologies Inc., Mississauga, Ontario, Canada)
that illuminates tissues with a 805 nm laser and captures images
with a camera sensitive to infrared fluorescence. Near infrared
microscopy and conventional histology were used to confirm the
macroscopically identified fluorescent structures.
[0061] Intracrural injection of ICG permitted identification of the
nerves at 6, 8, 12, 18, 24 and 36 hours post-operatively. (These
were specific time points at which examinations were made. It can
be assumed that the nerves would have also fluoresced and therefore
permitted identification of the nerves at times, such as 20 hours
and 30 hours, between those at which the nerves were examined.)
Although other penile structures fluoresced for extended periods,
fluorescence of the cavernous nerves was not detectable at longer
post-injection periods (e.g., 30 days). The highest intensity was
achieved at 18 and at 24 hours post-injection. NIRF and hematoxylin
and eosin (H&E) staining were used to confirm that the
fluorescence observed macroscopically coincided with the cavernous
nerves.
Example 4
[0062] The cavernous nerves of rats were imaged using ICG following
the protocol set forth in the preceding Examples. FIGS. 1a, b, and
c show the exposing of the penis and injection of ICG into the
crura of the cavernous bodies. FIG. 1d shows the penile crura and
cavernous nerves under near infrared (NIRF) illumination following
injection of ICG.
[0063] FIG. 2 shows a cavernous nerve under NIRF. The white arrow
points to the nerve. In a series of studies, nerves were identified
in situ at 6, 8, 12, 18, 24, and 36 hours post ICG injection. The
highest fluorescent intensity was noted at 18 and at 24 hours post
injection. The fluorescent nerves were then excised. NIRF and
hematoxylin and eosin (H&E) staining were used to confirm that
the fluorescence observed macroscopically coincided with the
cavernous nerves. FIG. 3 shows a hook electrode hooked around a
cavernous nerve under NIRF, after ICG injection as described above.
FIG. 4 shows NIRF illumination of a cavernous nerve (arrow) from an
animal injected with ICG as described above.
[0064] FIGS. 5a and 5b are photographs of a cavernous nerve excised
from an animal injected with ICG as described above. FIG. 5a shows
the nerve under infrared and LED illumination. FIG. 5b shows the
same nerve under NIRF alone. The surgeon can alternate at will
between visualizing the nerve under normal illumination (with or
without infrared illumination) and by fluorescence induced by NIRF
illumination.
[0065] It is understood that the examples and embodiments described
herein are for illustrative purposes only and that various
modifications or changes in light thereof will be suggested to
persons skilled in the art and are to be included within the spirit
and purview of this application and scope of the appended claims.
All publications, patents, and patent applications cited herein are
hereby incorporated by reference in their entirety for all
purposes.
* * * * *