Ci a ion: Okamo o, N.; Al-Di aie, Z.;
Scheepe s, M.H.M.C.; Heu elings,
D.J.I.; Rod íguez-Luna, M.R.;
Ma escaux, J.; Diana, M.; S assen,
L.P.S.; Bou y, N.D.; Al-Tahe , M.
Simul aneous, Mul i-Channel,
Nea -In a ed Fluo escence
Visualiza ion o Mesen e ic Lymph
Nodes Using Indocyanine G een and
Me hylene Blue: A Demons a ion in
a Po cine Model. Diagnos ics 2023,13,
1469. h ps://doi.o g/10.3390/
diagnos ics13081469
Academic Edi o : Joaquin Cubiella
Recei ed: 17 Feb ua y 2023
Re ised: 17 Ma ch 2023
Accep ed: 24 Ma ch 2023
Published: 18 Ap il 2023
Copy igh : © 2023 by he au ho s.
Licensee MDPI, Basel, Swi ze land.
This a icle is an open access a icle
dis ibu ed unde he e ms and
condi ions o he C ea i e Commons
A ibu ion (CC BY) license (h ps://
c ea i ecommons.o g/licenses/by/
4.0/).
diagnos ics
A icle
Simul aneous, Mul i-Channel, Nea -In a ed Fluo escence
Visualiza ion o Mesen e ic Lymph Nodes Using Indocyanine
G een and Me hylene Blue: A Demons a ion in
a Po cine Model
Na iaki Okamo o 1,2,*,† , Zaid Al-Di aie 3,4,†, Max H. M. C. Scheepe s 3,4, Danique J. I. Heu elings 4,5 ,
Ma ía Ri a Rod íguez-Luna 1,2 , Jacques Ma escaux 1, Michele Diana 1,2 , Lau en s P. S. S assen 4,5,
Nicole D. Bou y 4and Mahdi Al-Tahe 1,4,*
1IRCAD, Resea ch Ins i u e agains Diges i e Cance , 67091 S asbou g, F ance
2ICube Labo a o y, Pho onics Ins umen a ion o Heal h, 67081 S asbou g, F ance
3GROW School o Oncology and De elopmen al Biology, Maas ich Uni e si y,
6229 ER Maas ich , The Ne he lands
4Depa men o Su ge y, Maas ich Uni e si y Medical Cen e , 6229 ER Maas ich , The Ne he lands
5NUTRIM School o Nu i ion and T ansla ional Resea ch in Me abolism, Maas ich Uni e si y,
6229 HX Maas ich , The Ne he lands
*Co espondence: [email p o ec ed] (N.O.); [email p o ec ed] (M.A.-T.)
† These au ho s con ibu ed equally o his wo k.
Abs ac :
Nea -in a ed luo escence (NIRF) image-guided su ge y is a use ul ool ha can help
educe pe iope a i e complica ions and imp o e issue ecogni ion. Indocyanine g een (ICG) dye
is he mos equen ly used in clinical s udies. ICG NIRF imaging has been used o lymph node
iden i ica ion. Howe e , he e a e s ill many challenges in lymph node iden i ica ion by ICG. The e
is inc easing e idence ha me hylene blue (MB), ano he clinically applicable luo escen dye, can
also be use ul in he in aope a i e luo escence-guided iden i ica ion o s uc u es and issues. We
hypo hesized ha MB NIRF imaging could be used o lymph node iden i ica ion. The aim o his
s udy was o e alua e he easibili y o in aope a i e lymph node luo escence de ec ion using
in a enously (IV) adminis e ed MB and compa e i o ICG ia a came a ha has wo dedica ed
nea -in a ed (NIR) channels. Th ee pigs we e used in his s udy. ICG (0.2 mg/kg) was adminis e ed
ia a pe iphe al enous ca he e ollowed by immedia e adminis a ion o MB (0.25 mg/kg). NIRF
images we e acqui ed as ideo eco dings a di e en ime poin s (e e y 10 min) o e an hou
using he QUEST SPECTRUM
®
3 sys em (Ques Medical Imaging, Middenmee , The Ne he lands),
which has wo dedica ed NIR channels o simul aneous in aope a i e luo escence guidance. The
800 nm channel was used o cap u e ICG luo escence and he 700 nm channel was used o MB. The
a ge (lymph nodes and small bowel) and he backg ound ( essels- ee ield o he mesen e y) we e
highligh ed as he egions o in e es (ROIs), and co esponding luo escence in ensi ies (FI) om
hese ROIs we e measu ed. The a ge - o-backg ound a io (TBR) was hen compu ed as he mean FI
o he a ge minus he mean FI o he backg ound di ided by he mean FI o he backg ound. In all
included animals, a clea iden i ica ion o lymph nodes was achie ed a all ime poin s. The mean TBR
o ICG in lymph nodes and small bowel was 4.57
±
1.00 and 4.37
±
1.70, espec i ely o he o e all
expe imen al ime. Rega ding MB, he mean TBR in lymph nodes and small bowel was
4.60 ±0.92
and 3.27
±
0.62, espec i ely. The Mann-Whi ney U es o he lymph node TBR/small bowel TBR
showed ha he TBR a io o MB was s a is ically signi ican ly highe han ICG. The luo escence
op ical imaging echnology used allows o double-wa eleng h assessmen . This easibili y s udy
p o es ha lymph nodes can be disc imina ed using wo di e en luo opho es (MB and ICG)
wi h di e en wa eleng hs. The esul s sugges ha MB has a p omising po en ial o be used o
de ec lympha ic issue du ing image-guided su ge y. Fu he p eclinical ials a e needed be o e
clinical ansla ion.
Diagnos ics 2023,13, 1469. h ps://doi.o g/10.3390/diagnos ics13081469 h ps://www.mdpi.com/jou nal/diagnos ics
Diagnos ics 2023,13, 1469 2 o 13
Keywo ds:
colo ec al su ge y; image-guided su ge y; op ical imaging; simul aneous; mul i-channel;
nea -in a ed luo escence image; lymph node; indocyanine g een; me hylene blue
1. In oduc ion
Colo ec al cance (CRC) is he hi d leading cause o cance and he second leading
cause o cance - ela ed mo ali y wo ldwide. I is es ima ed ha he e we e app oxima ely
1 million cases o colo ec al cance and 88,000 dea hs wo ldwide in 2018 [
1
]. The su gical
app oach o colo ec al cance has changed signi ican ly o e he las ew decades, including
he in oduc ion o o al meso ec al excision (TME) and comple e mesocolic excision (CME),
ad ances in minimally in asi e p ocedu es, p eope a i e chemo adio he apy, and su gical
op ical imaging echnologies.
O e he pas ew decades, one o he mos signi ican inno a ions in he his o y
o colo ec al cance ea men has been o al meso ec al excision (TME) p oposed by
Heald e al. in 1982 [2]
. Due o he emb yologic de elopmen , he ec um and meso ec um
a e conside ed pa o a single ana omical s uc u e which su ounded by he isce al
pel ic ascia. This ho ough unde s anding o ana omical ea u es allow TME o p o ide a
sa e “en bloc” esec ion o he ec um and i s meso ec um, which include he majo i y o
egional lymph nodes, lympha ic essels, and su ounding a issue [
3
]. The TME “en bloc”
esec ion was pi o al in signi ican ly educing he local ecu ence a e a e ec al cance
su ge y, dec easing he incidence o pe manen s omas, imp o ing he cu a i e esec ion
a e, and imp o ing su i al and umo - ee su i al a es. TME has become he “gold
s anda d” su gical ea men o ec al cance wo ldwide [4,5].
CME p oposed by Hohenbe ge e al. consis s o esec ing he umo -bea ing bowel
segmen s wi h “en bloc” esec ion o he mesocolon along i s emb yonic ascial planes based
on he concep o TME [
6
,
7
]. CME, which is cha ac e ized by a cen al ascula liga ion and
a la ge mesen e ic specimen, esul s in a g ea e numbe o lymph nodes han non-CME
esec ions. This supe io lymphadenec omy may accoun o he lowe epo ed isk o
local ecu ence in some s udies [
6
–
9
]. Howe e , se e al a icles ha e shown ha wide
excisions did no imp o e oncologic bene i s and CME has been associa ed wi h inc easing
conce ns ega ding he ele a ed mo bidi y and isk o pos ope a i e complica ions such as
inju ies o he splenic and supe io mesen e ic eins [10–14].
In oncological su ge y, accu a e lymph node assessmen enables pa hological s aging
and p ognosis-guiding pos ope a i e he apies. Impo an ly, adequa e lymphadenec omy
has shown o imp o e o e all su i al [
15
]. In colo ec al cance , he main goal o oncologic
esec ion is esec ing he p ima y umo along wi h i s blood supply and lymph node
d ainage [
6
,
16
–
20
]. Nume ous s udies ha e been conduc ed on su gical imaging echnolo-
gies, in diges i e su ge y, nea -in a ed luo escence (NIRF) image-guided su ge y has been
s udied ex ensi ely o unde s and i s in luence in su gical ou comes, such as he p e en ion
o complica ions, diagnosis, s aging, and imp o ed adical esec ion o cance all o e he
wo ld [
21
]. NIRF imaging is a ela i ely new echnology ha uses a nea -in a ed ligh ,
allowing su geons o see beyond he no mal whi e ligh spec um, helping hem isualize
no only he su ace o he s uc u e bu also he inside o he s uc u e [
22
]. I equi es a
locally o sys emically adminis a ion o a luo escen dye and an op ical imaging sys em
able o exci e, de ec s, and measu es he luo escen dye spec a.
Indocyanine g een (ICG), he mos equen ly used clinically app o ed luo escence
dye, abso bs ligh a 790 o 805 nm and has a peak emission a 835 nm when i is bound
o
α
1 lipop o ein in he body [
23
]. Since ICG was app o ed by Food and D ug Admin-
is a ion (FDA) in 1959, i has been used p ima ily o he e alua ion o blood essels in
con as s udies such as blood low assessmen in he e ina [
24
], and since hen, i s ield o
applica ions ha e b oadly expanded. ICG has a hal -li e o 150 o 180 s, and i is emo ed
om ci cula ion apidly and exclusi ely by he li e o bile juice. Thanks o he hepa ic
Diagnos ics 2023,13, 1469 3 o 13
clea ance, ICG has been used o he assessmen o li e unc ion, measu emen o blood
low in he li e , and ex ahepa ic bile duc di e en ia ion [16].
ICG is a non- oxic dye and he epo ed p obabili y o alle gy is e y low
(1:40,000–1:60,000 cases) [
25
–
28
]. In a enous adminis a ion o ICG has been used in,
amongs o he s, ca diac ou pu assessmen [
23
], in aope a i e umo dema ca ion [
29
],
and pe usion assessmen a he le el o he anas omosis [
30
–
34
] as a luo escen dye in
colo ec al su ge y. Since i is known ha ICG has a endency o mig a e o lympha ic
issues, ICG NIRF imaging has been used o sen inel node iden i ica ion [
35
–
37
], and
in aope a i e eal- ime lympha ic low isualiza ion [
15
,
38
]. Howe e , one o he main
d awbacks o ICG is i s high a ini y o albumin and p og essi e di usion in issues, which
educes i s capaci y o accu a ely loca e a ec ed a eas [39].
Me hylene blue (MB) is ano he clinically app o ed dye and has a peak exci a ion
wa eleng h o ~700 nm and an emission wa eleng h o 688 nm [
40
,
41
]. Due o i s enal
clea ance, NIRF imaging wi h MB has mos ly been used in colo ec al cance su ge y
o in aope a i e isualiza ion o he u ina y ac [
41
–
43
]. Mo eo e , he e ha e been
se e al s udies using MB o de ec sen inel lymph nodes in colo ec al cance [
44
] and o
pos ope a i e lymph node ha es ing [
45
,
46
]. I s use is limi ed in a ew op ical imaging
de ices since mos NIRF imaging sys ems a e designed o imaging wi h ICG and dyes
wi h smilia op ical p ope ies. An example o a dual imaging sys em is he QUEST
SPECTRUM
®
3 (Ques Medical Imaging, Middenmee , The Ne he lands) came a which has
wo dedica ed nea -in a ed (NIR) channels o simul aneous in aope a i e luo escence
guidance, which hus enables imaging o bo h MB and ICG. As p e iously s a ed, he
di e ences in peak exci a ion and pha macodynamics be ween MB and ICG e lec hei
di e en p incipal applica ions in he clinical se ing. In colo ec al su ge y, Polom W e al.
we e able o iden i y he loca ion o he u e e using MB while assessing in es inal pe usion
o de e mine he op imal anas omo ic si e wi h ICG using a single came a sys em. Thei
esul s highligh he easibili y o mul i-wa e imaging o aid in p e en ing wo o he mos
common complica ions in colo ec al su ge y, namely anas omo ic leak, and u e e al inju y.
The ele ance o simul aneous, mul i-channel, nea -in a ed luo escence imaging may
ep esen a pa h owa d inc eased su gical sa e y [47].
This s udy’s p ima y aim was o alida e whe he in a enously adminis e ed MB
and ICG ac simul aneously as luo escence dyes o lymph node de ec ion. As a seconda y
aim, we compa ed he luo escence pe o mance o MB and ICG in lymph node de ec ion.
The in- i o p eclinical e alua ion o in a enous (IV) injec ed MB and ICG simul a-
neously shows easibili y in de ec ing lymph nodes du ing diges i e su ge y. We discuss
ele an esul s including he as e washou o MB as well as less backg ound luo escence
p o iding easie de ec ion o lymph nodes.
2. Ma e ials and Me hods
This s udy was pe o med a he animal labo a o y o he Maas ich Uni e si y Medi-
cal Cen e (MUMC+, Maas ich , The Ne he lands). The expe imen s we e pe o med in
h ee emale Du ch land ace pigs (mean weigh : 40 kg). Animals we e used in compliance
wi h Du ch egula ions and legisla ion conce ning animal esea ch, and he s udy was
pe o med acco ding o a p o ocol app o ed by he Expe imen al Animal Commi ee o
Maas ich Uni e si y (DEC-UM) (app o al code: 2017-021-001). All su gical p ocedu es
we e pe o med unde gene al anes hesia. In amuscula injec ion o azape one 3 mg/kg,
ke amine 10 mg/kg, a opine 0.05 mg/kg, hiopen al 10–15 mg/kg, iso lu ane (dose de-
pending on he e ec ), and oxygen 20–40 mL/kg/min was used as a s anda d medica ion
o ensu e app op ia e seda ion and analgesia. All a ia ions in i al pa ame e s we e
con inuously moni o ed. A he end o he p o ocol, animals we e eu hanized wi h a le hal
dose o pen oba bi al (40 mg/Kg).
Diagnos ics 2023,13, 1469 4 o 13
2.1. Fluo escence Imaging Sys em
The QUEST SPECTRUM
®
3 sys em was de eloped o NIRF image-guided su ge y in
an open su gical se ing. This luo escence imaging sys em has wo dedica ed NIR channels
in he elec omagne ic spec um (700 and 800 nm), which allows o simul aneously isual-
iza ion o ICG and MB. The channels we e changed di ec ly on he moni o ouchsc een by
simply clicking on he p e e ed channel. Sc een se ings included he Red/G een/Blue
image, NIRF esponse in g ayscale image, o e lay o luo escen image in g een wi h he
colo image, and o e lay o he luo escen esponse image in Colo (Blue o Red) on he
Colo image in g ayscale.
2.2. P epa a ion o he Dyes
ICG (Ve dye, Diagnos ic G een GmbH, Aschheim, Ge many) was dilu ed in a s e ile
H
2
O solu ion o a concen a ion o 2.5 mg/mL and was injec ed in a enously a a concen-
a ion o 0.2 mg/kg body weigh ia a pe iphe al enous line. The dose is based on he
indings o ou p e ious publica ion in which his was he mos equen ly used dose in
clinical se ings [8].
MB (P o epha m Li e Solu ions, Ma seille, F ance) was dilu ed in a s e ile phospha e-
bu e ed saline solu ion o a concen a ion o 1 mg/mL and was injec ed in a enously a a
concen a ion o 0.25 mg/kg body immedia ely ollowing he injec ion o ICG. This dosage
was de e mined based on p e ious li e a u e [42].
2.3. Su gical P ocedu e
To expose he ROI mesen e ic lymph nodes and small bowel, he abdominal ca i y
was accessed ia a midline lapa o omy, and a sel - e aining abdominal wall e ac o was
placed. The came a ip was posi ioned a 15 cm abo e he ROI. Du ing NIRF imaging,
en i onmen al ligh s we e u ned o p e en ing ambien ligh in e e ence, and he a -
e age cap u e ime was 20 s pe image. The expe imen al su gical se ing is ep esen ed
in Figu e 1.
2.4. Image Acquisi ion and S a is ical Analysis
NIRF images we e acqui ed om ideo eco ding a e 1, 10, 20, 30, 40, 50, and 60 min
o dye adminis a ion, espec i ely, using he QUEST SPECTRUM®3.
Fi s , he a ge (mesen e ic lymph nodes and small bowel) and he backg ound
( essels- ee ield o he mesen e y) we e highligh ed as ROI, and co esponding luo es-
cence in ensi ies (FI) which we e exp essed in a bi a y uni s (a.u.) om hese ROIs we e
measu ed. Fluo escence in ensi y measu emen was pe o med using he Ques A emis
(Ques Medical Imaging, Middenmee , The Ne he lands) so wa e (TBR ool 1.0.). The
a ge - o-backg ound a io (TBR) was hen compu ed as he mean FI o he a ge di ided
by he mean FI o he backg ound.
To iden i y any po en ial supe io i y in e ms o dye pe o mance, a ios we e used
di iding lymph node TBR/small bowel TBR pe dye. A a io compa ison o MB and ICG
was made using a Mann-Whi ney U es o con inuous a iables. A wo- ailed analysis
wi h a p alue < 0.05 was conside ed s a is ically signi ican . These analyses we e pe o med
using comme cially a ailable da abase so wa e (Excel e sion 2021, Mic oso Co po a ion,
Redmond, WA, USA).
Diagnos ics 2023,13, 1469 5 o 13
Diagnos ics 2023, 13, x FOR PEER REVIEW 5 o 13
Figu e 1. Se -up du ing expe imen s: The abdominal ca i y was accessed ia a midline lapa o omy
o expose he egions o in e es (i.e., small bowel and mesen e ic lymph nodes). The dis al lens o
he QUEST came a
®
3 (Ques Medical Imaging, Middenmee , The Ne he lands) was posi ioned 15
cm abo e he egions o in e es . Ex e nal ligh in e e ence was a oided du ing he acquisi ion o
images.
3. Resul s
In all h ee animals included, i was possible o clea ly isualize he lymph nodes
unde NIRF imaging using ei he o he dyes adminis e ed du ing he o e all su gical
p ocedu e (Figu e 2). Subjec i e e alua ion o lymph node isualiza ion wi h wo dyes by
wo expe ienced su geons was pe o med on a ou -poin scale
(Excellen /Good/A e age/Poo ), wi h Good o Excellen a all in aope a i e ime poin s.
The TBR o each ime poin is ep esen ed in Figu e 3. The TBRs o MB and ICG we e
g ea e han 2.0 a all measu ed ime poin s. The mean TBR o ICG in lymph nodes and
small bowel was 4.57 ± 1.00 and 4.37 ± 1.70, espec i ely, o he o e all expe imen al ime.
Rega ding MB, he mean TBR in lymph nodes and small bowel was 4.60 ± 0.92 and 3.27 ±
0.62, espec i ely. The Mann-Whi ney U es o lymph node TBR/small bowel TBR
compa ing ICG o MB showed ha he alue o MB was s a is ically signi ican ly highe
(p = 0.012). No complica ions ela ed o dye adminis a ions occu ed.
Figu e 1.
Se -up du ing expe imen s: The abdominal ca i y was accessed ia a midline lapa o omy
o expose he egions o in e es (i.e., small bowel and mesen e ic lymph nodes). The dis al lens
o he QUEST came a
®
3 (Ques Medical Imaging, Middenmee , The Ne he lands) was posi ioned
15 cm
abo e he egions o in e es . Ex e nal ligh in e e ence was a oided du ing he acquisi ion
o images.
3. Resul s
In all h ee animals included, i was possible o clea ly isualize he lymph nodes unde
NIRF imaging using ei he o he dyes adminis e ed du ing he o e all su gical p ocedu e
(Figu e 2). Subjec i e e alua ion o lymph node isualiza ion wi h wo dyes by wo expe-
ienced su geons was pe o med on a ou -poin scale (Excellen /Good/A e age/Poo ),
wi h Good o Excellen a all in aope a i e ime poin s. The TBR o each ime poin is
ep esen ed in Figu e 3. The TBRs o MB and ICG we e g ea e han 2.0 a all measu ed
ime poin s. The mean TBR o ICG in lymph nodes and small bowel was 4.57
±
1.00
and
4.37 ±1.70
, espec i ely, o he o e all expe imen al ime. Rega ding MB, he mean
TBR in lymph nodes and small bowel was 4.60
±
0.92 and 3.27
±
0.62, espec i ely. The
Mann-Whi ney U es o lymph node TBR/small bowel TBR compa ing ICG o MB showed
ha he alue o MB was s a is ically signi ican ly highe (p= 0.012). No complica ions
ela ed o dye adminis a ions occu ed.
Diagnos ics 2023,13, 1469 6 o 13
Diagnos ics 2023, 13, x FOR PEER REVIEW 6 o 13
Figu e 2. Pic u e o mesen e ic lymph nodes and bowel a e 20 min o in a enous MB and ICG
adminis a ions. (A) The RGB image. (B) O e lay luo escence pic u e a 700 nm ( luo escence o
MB). (C) O e lay luo escence pic u e a 800 nm ( luo escence o ICG). MB, me hylene blue; ICG,
indocyanine g een.
(A) (B)
Figu e 3. The TBR o e ime, (A) MB (B) ICG. TBR, a ge - o-backg ound a io; MB, me hylene blue;
ICG, indocyanine g een.
4. Discussion
In his s udy, we alida ed ha he wo dyes (MB and ICG) can be used in pa allel in
an animal model o he iden i ica ion o mesen e ic lymph nodes. A e simul aneous
in a enous adminis a ion o he dyes, he NIRF imaging o lymph nodes and bowel was
succes ull in bo h imaging modali ies. The TBR o MB in mesen e ic lymph nodes was no
in e io o ha o ICG. A possible explana ion o hese esul s is ha he e was a lowe
MB FI in he small bowel in compa ison o ICG FI which was highe in he in es ines. ICG
possesses a highe FI backg ound e en in he essels- ee ield o he mesen e y.
Addi ionally, since MB has a sho e washou ime om he bowel han ICG, i may o e
an enhanced po en ial o mesen e ic lymph node de ec ion as he FI backg ound
dec eases mo e apidly han when using ICG.
In CRC, lymph node me as asis modi ies s aging, and he s age de e mines whe he
pos ope a i e adju an chemo he apy is indica ed. Resul s o a seconda y analysis o he
In e g oup ial INT-0089 showed ha an inc eased numbe o lymph nodes e ie ed
co ela es wi h a p olonged su i al a e in bo h nega i e and posi i e cases o lymph
node me as asis [48]. Swanson e al. showed ha he numbe o lymph nodes examined,
e en in T3N0 colon cance wi hou p eope a i e lymph node me as asis, linea ly
co ela ed wi h 5-yea su i al [49]. In e na ional guidelines ecommend he e alua ion
o 12 o mo e lymph nodes [50–52], Howe e , scien i ic e idence is s ill lacking and i is
s ill unclea i he e is an absolu e numbe ha is conside ed as a gold s anda d o ou ine
analysis [53]. Golds ein s a ed ha a minimum o 30 lymph nodes in a hypo he ical
specimen was equi ed o each a p obabili y o 80% o iden i y a single me as asis [54].
In o he wo ds, in aope a i e lymph node de ec ion and esec ion ep esen an essen ial
Figu e 2.
Pic u e o mesen e ic lymph nodes and bowel a e 20 min o in a enous MB and ICG
adminis a ions. (
A
) The RGB image. (
B
) O e lay luo escence pic u e a 700 nm ( luo escence o
MB). (
C
) O e lay luo escence pic u e a 800 nm ( luo escence o ICG). MB, me hylene blue; ICG,
indocyanine g een.
Diagnos ics 2023, 13, x FOR PEER REVIEW 6 o 13
Figu e 2. Pic u e o mesen e ic lymph nodes and bowel a e 20 min o in a enous MB and ICG
adminis a ions. (A) The RGB image. (B) O e lay luo escence pic u e a 700 nm ( luo escence o
MB). (C) O e lay luo escence pic u e a 800 nm ( luo escence o ICG). MB, me hylene blue; ICG,
indocyanine g een.
(A) (B)
Figu e 3. The TBR o e ime, (A) MB (B) ICG. TBR, a ge - o-backg ound a io; MB, me hylene blue;
ICG, indocyanine g een.
4. Discussion
In his s udy, we alida ed ha he wo dyes (MB and ICG) can be used in pa allel in
an animal model o he iden i ica ion o mesen e ic lymph nodes. A e simul aneous
in a enous adminis a ion o he dyes, he NIRF imaging o lymph nodes and bowel was
succes ull in bo h imaging modali ies. The TBR o MB in mesen e ic lymph nodes was no
in e io o ha o ICG. A possible explana ion o hese esul s is ha he e was a lowe
MB FI in he small bowel in compa ison o ICG FI which was highe in he in es ines. ICG
possesses a highe FI backg ound e en in he essels- ee ield o he mesen e y.
Addi ionally, since MB has a sho e washou ime om he bowel han ICG, i may o e
an enhanced po en ial o mesen e ic lymph node de ec ion as he FI backg ound
dec eases mo e apidly han when using ICG.
In CRC, lymph node me as asis modi ies s aging, and he s age de e mines whe he
pos ope a i e adju an chemo he apy is indica ed. Resul s o a seconda y analysis o he
In e g oup ial INT-0089 showed ha an inc eased numbe o lymph nodes e ie ed
co ela es wi h a p olonged su i al a e in bo h nega i e and posi i e cases o lymph
node me as asis [48]. Swanson e al. showed ha he numbe o lymph nodes examined,
e en in T3N0 colon cance wi hou p eope a i e lymph node me as asis, linea ly
co ela ed wi h 5-yea su i al [49]. In e na ional guidelines ecommend he e alua ion
o 12 o mo e lymph nodes [50–52], Howe e , scien i ic e idence is s ill lacking and i is
s ill unclea i he e is an absolu e numbe ha is conside ed as a gold s anda d o ou ine
analysis [53]. Golds ein s a ed ha a minimum o 30 lymph nodes in a hypo he ical
specimen was equi ed o each a p obabili y o 80% o iden i y a single me as asis [54].
In o he wo ds, in aope a i e lymph node de ec ion and esec ion ep esen an essen ial
Figu e 3.
The TBR o e ime, (
A
) MB (
B
) ICG. TBR, a ge - o-backg ound a io; MB, me hylene blue;
ICG, indocyanine g een.
4. Discussion
In his s udy, we alida ed ha he wo dyes (MB and ICG) can be used in pa allel
in an animal model o he iden i ica ion o mesen e ic lymph nodes. A e simul aneous
in a enous adminis a ion o he dyes, he NIRF imaging o lymph nodes and bowel was
succes ull in bo h imaging modali ies. The TBR o MB in mesen e ic lymph nodes was no
in e io o ha o ICG. A possible explana ion o hese esul s is ha he e was a lowe
MB FI in he small bowel in compa ison o ICG FI which was highe in he in es ines.
ICG possesses a highe FI backg ound e en in he essels- ee ield o he mesen e y.
Addi ionally, since MB has a sho e washou ime om he bowel han ICG, i may o e
an enhanced po en ial o mesen e ic lymph node de ec ion as he FI backg ound dec eases
mo e apidly han when using ICG.
In CRC, lymph node me as asis modi ies s aging, and he s age de e mines whe he
pos ope a i e adju an chemo he apy is indica ed. Resul s o a seconda y analysis o he
In e g oup ial INT-0089 showed ha an inc eased numbe o lymph nodes e ie ed
co ela es wi h a p olonged su i al a e in bo h nega i e and posi i e cases o lymph
node me as asis [
48
]. Swanson e al. showed ha he numbe o lymph nodes examined,
e en in T3N0 colon cance wi hou p eope a i e lymph node me as asis, linea ly co ela ed
wi h
5-yea
su i al [
49
]. In e na ional guidelines ecommend he e alua ion o 12 o
mo e lymph nodes [
50
–
52
], Howe e , scien i ic e idence is s ill lacking and i is s ill
unclea i he e is an absolu e numbe ha is conside ed as a gold s anda d o ou ine
analysis [
53
]. Golds ein s a ed ha a minimum o 30 lymph nodes in a hypo he ical
specimen was equi ed o each a p obabili y o 80% o iden i y a single me as asis [
54
].
Diagnos ics 2023,13, 1469 7 o 13
In o he wo ds, in aope a i e lymph node de ec ion and esec ion ep esen an essen ial
componen in p edic ing he long- e m p ognosis o CRC and in de e mining pos ope a i e
ea men s a egies.
A sys ema ic e iew and me a-analysis by Emile e al. showed ha s udies on luo-
escen lymph node imaging di e wi h espec o ICG concen a ion, dose, injec ion si e
(submucosal, subse osal, combined submucosal and subse osal, in a enous), and iming
o injec ion (p eope a i e, in aope a i e, bo h p eope a i e and in aope a i e) [55].
Fo luo escence lymph node de ec ion and lympha ic mapping using ICG, he dye is
o en injec ed in he subse osal o he submucosal laye s whe e he main lympha ic ne wo k
is loca ed a ound he cance ous issue [
38
,
55
,
56
]. Howe e , his echnique equi es high
echnical p ecision du ing injec ion o p e en dye spillage, since abdominal ca i y leakage
al e s he su gical ield wi h alse-posi i e luo escence egions [
57
]. Rega ding echnical
de ails du ing lapa oscopic subse osal ICG injec ion, he lack o ac ile eedback could
cause he needle ip o each he muscle laye , hence injec ing he w ong laye esul ing in
inadequa e dye abso p ion in o he lympha ics [
55
]. Nishigo i e al. s udied 21 pa ien s who
unde wen lapa oscopic CRC su ge y o e alua e bo h lympha ic and blood low wi h ICG
luo escence imaging. To e alua e lymph low, au ho s injec ed ICG in o he submucosa
a ound he umo unde colonoscopic guidance, wi h a iming o injec ion comp ised ei he
be ween 1 and 3 days be o e su ge y o in aope a i ely. To e alua e blood low, ICG
was injec ed in a enously du ing he p ocedu e. Inadequa e lympha ic low delinea ion
esul ed in non-de ec ion in h ee cases. In he wo cases in ol ing in aope a i e IV
injec ion, ICG leakage was a p oblem in bo h [15].
A s udy by Libe ale e al. epo ed ha in aope a i e IV adminis a ion o ICG o
lymph node de ec ion was easible. Au ho s showed ha in aope a i e and pos ope a i e
luo escence imaging migh be a new ool o de ec me as a ic lymph nodes in pa ien s
wi h CRC, allowing o a mo e accu a e s aging [
58
]. In he p e iously quo ed sys ema ic
e iew, he IV adminis a ion o ICG showed he second highes sensi i i y, speci ici y,
and accu acy o lymph node de ec ion behind he combined subse osal and submucosal
injec ion [
59
]. Howe e , pooled esul s we e based on only wo lymph node mapping
s udies using IV ICG. A p esen , he p e e ed me hod o adminis a ion and dosage o
ICG o luo escence lymph node mapping is s ill unde deba e, and u he clinical ials
a e highly necessa y o es ablish he bes eliable echnique. ICG also di uses in o asci es
and edema because o i s albumin binding. In his expe imen , some animals de eloped a
deg ee o asci es o mesen e ic edema a e lapa o omy. In hose ha did, ICG luo escence
images showed compa able FI in edema ROIs o ha o he lymph nodes (Figu e 4). Asci es
ha inc eased o e ime du ing su ge y also showed simila FI. (Figu e 5). As a esul , he
ICG luo escence p esen in ei he asci es o edema may educe he sensi i i y o mesen e ic
lymph node de ec ion du ing NIRF-guided su ge y.
Diagnos ics 2023, 13, x FOR PEER REVIEW 7 o 13
componen in p edic ing he long- e m p ognosis o CRC and in de e mining
pos ope a i e ea men s a egies.
A sys ema ic e iew and me a-analysis by Emile e al. showed ha s udies on
luo escen lymph node imaging di e wi h espec o ICG concen a ion, dose, injec ion
si e (submucosal, subse osal, combined submucosal and subse osal, in a enous), and
iming o injec ion (p eope a i e, in aope a i e, bo h p eope a i e and in aope a i e)
[55].
Fo luo escence lymph node de ec ion and lympha ic mapping using ICG, he dye
is o en injec ed in he subse osal o he submucosal laye s whe e he main lympha ic
ne wo k is loca ed a ound he cance ous issue [38,55,56]. Howe e , his echnique
equi es high echnical p ecision du ing injec ion o p e en dye spillage, since abdominal
ca i y leakage al e s he su gical ield wi h alse-posi i e luo escence egions [57].
Rega ding echnical de ails du ing lapa oscopic subse osal ICG injec ion, he lack o
ac ile eedback could cause he needle ip o each he muscle laye , hence injec ing he
w ong laye esul ing in inadequa e dye abso p ion in o he lympha ics [55]. Nishigo i e
al. s udied 21 pa ien s who unde wen lapa oscopic CRC su ge y o e alua e bo h
lympha ic and blood low wi h ICG luo escence imaging. To e alua e lymph low,
au ho s injec ed ICG in o he submucosa a ound he umo unde colonoscopic guidance,
wi h a iming o injec ion comp ised ei he be ween 1 and 3 days be o e su ge y o
in aope a i ely. To e alua e blood low, ICG was injec ed in a enously du ing he
p ocedu e. Inadequa e lympha ic low delinea ion esul ed in non-de ec ion in h ee
cases. In he wo cases in ol ing in aope a i e IV injec ion, ICG leakage was a p oblem
in bo h [15].
A s udy by Libe ale e al. epo ed ha in aope a i e IV adminis a ion o ICG o
lymph node de ec ion was easible. Au ho s showed ha in aope a i e and
pos ope a i e luo escence imaging migh be a new ool o de ec me as a ic lymph nodes
in pa ien s wi h CRC, allowing o a mo e accu a e s aging [58]. In he p e iously quo ed
sys ema ic e iew, he IV adminis a ion o ICG showed he second highes sensi i i y,
speci ici y, and accu acy o lymph node de ec ion behind he combined subse osal and
submucosal injec ion [59]. Howe e , pooled esul s we e based on only wo lymph node
mapping s udies using IV ICG. A p esen , he p e e ed me hod o adminis a ion and
dosage o ICG o luo escence lymph node mapping is s ill unde deba e, and u he
clinical ials a e highly necessa y o es ablish he bes eliable echnique. ICG also di uses
in o asci es and edema because o i s albumin binding. In his expe imen , some animals
de eloped a deg ee o asci es o mesen e ic edema a e lapa o omy. In hose ha did,
ICG luo escence images showed compa able FI in edema ROIs o ha o he lymph nodes
(Figu e 4). Asci es ha inc eased o e ime du ing su ge y also showed simila FI. (Figu e
5). As a esul , he ICG luo escence p esen in ei he asci es o edema may educe he
sensi i i y o mesen e ic lymph node de ec ion du ing NIRF-guided su ge y.
Figu e 4. Pic u e o asci es a e 40 min o IV ICG adminis a ion. (A) The RGB image. (B)
Fluo escence pic u e a 800 nm (C) O e lay luo escence pic u e a 800 nm.
Figu e 4.
Pic u e o asci es a e 40 min o IV ICG adminis a ion. (
A
) The RGB image. (
B
) Fluo es-
cence pic u e a 800 nm (C) O e lay luo escence pic u e a 800 nm.
Diagnos ics 2023,13, 1469 8 o 13
Diagnos ics 2023, 13, x FOR PEER REVIEW 8 o 13
Figu e 5. Pic u e o mesen e ic edema a e 40 min o IV ICG adminis a ion. (A) The RGB image.
(B) Fluo escence pic u e a 800 nm (C) O e lay luo escence pic u e a 800 nm.
MB is hyd ophobic and does no bind o albumin as opposed o ICG. Jun Li e al.
epo ed NIRF imaging using MB o he iden i ica ion o lympha ic essels, sen inel
lymph node loca ion, and b eas lympha ic low pa e ns in b eas cance . They epo ed
ha MB causes less backg ound luo escence con amina ion han ICG, and MB could be
sui able o obse a ion due o i s highe abso p ion and o he ac ha i can be clea ly
de ec ed ac oss he skin and adipose issue [59].
The luo escence o MB injec ed simul aneously wi h ICG could be used o con i m
whe he he luo escen ROIs a e ei he mesen e ic lymph nodes o mesen e ic edema.
The in a enous injec ion o wo dyes may inc ease he sensi i i y o mesen e ic lymph
node de ec ion, he eby a oiding complex p ocedu es such as subse osal and submucosal
injec ions. In addi ion, his may p e en any excessi e mesen e ic inju y o inadequa e
lymph node esec ion ha could occu when ICG is adminis e ed alone.
Addi ionally, MB in he lympha ic ne wo k and lymph nodes s ains a blue colo in
he specimen. The numbe o de ec able lymph nodes depends on age, gende , ype o
malignancy, and umo loca ion [60]. Besides, p eope a i e adju an chemo adia ion has
become he s anda d o ca e o ad anced ec al cance in ecen yea s. Neoadju an
chemo adio he apy educes lymph node size, which causes a dec ease in lymph node
ha es ing [61–63]. Mä kl e al. epo ed a s udy in which MB was injec ed in o he
supe io ec al a e y ex i o, o iginally pe o med o e alua e he accu acy o TME [64].
O he au ho s ound ha injec ing MB pos ope a i ely di ec ly in o he essels o ec al
esec ion specimens imp o ed he lymph node de ec ion a e [45]. Al hough i mus be
men ioned ha his is an a e ial injec ion and he concen a ion adminis e ed is high, hey
ha e shown ha MB ans e ed om he blood essels in o he lympha ic low s ains he
lymph nodes. Se e al s udies ha e shown ha highe doses o MB imp o ed
pos ope a i e lymph node ha es ing om ec al cance specimens [45,46].
Ano he po en ial applica ion o he p esen ed imaging se -up, apa om he es ed
lymph node and bowel de ec ion, is o assess blood low and u e e al ecogni ion as hese
a e wo o he p ope ies o he ICG and MB dyes. U e e al inju y is a se ious complica ion
du ing in apel ic su ge y, and he majo i y o ia ogenic u e e al inju ies may lead o
se e e mo bidi y. The incidence o u e e al inju y in colo ec al su ge y is no ha high
(0.25–1.1%). Howe e , se e al s udies showed ha only 15% o 30% o in aope a i e
u e e al inju ies a e ecognized. I mos equen ly occu s du ing p oc ec omy and
sigmoidec omy colo ec al cance su ge y [65]. Ande sen e al. e iewed 18,474 colo ec al
esec ions o he Danish Na ional Colo ec al Cance da abase and epo ed ha
lapa oscopic p ocedu es co ela e wi h he isk o ia ogenic u e e al inju y [66]. The
su geon should be amilia wi h he ana omy o he u e e and he isk ac o s o inju y
[67]. Visualiza ion o he u e e is e y impo an , especially a e eope a ion o adia ion
he apy. In aope a i e u e e al iden i ica ion also con ibu es o he educa ion o young
su geons in ana omy. As e e ed o ea lie , ICG is clea ed ia he bile juice. The
luo escen u ina y ac isualiza ion wi h ICG equi es he inse ion o one o se e al
u e e al ca he e (s) ia cys oscopy equi ing an addi ional in asi e p ocedu e which may
Figu e 5.
Pic u e o mesen e ic edema a e 40 min o IV ICG adminis a ion. (
A
) The RGB image.
(B) Fluo escence pic u e a 800 nm (C) O e lay luo escence pic u e a 800 nm.
MB is hyd ophobic and does no bind o albumin as opposed o ICG. Jun Li e al.
epo ed NIRF imaging using MB o he iden i ica ion o lympha ic essels, sen inel lymph
node loca ion, and b eas lympha ic low pa e ns in b eas cance . They epo ed ha MB
causes less backg ound luo escence con amina ion han ICG, and MB could be sui able
o obse a ion due o i s highe abso p ion and o he ac ha i can be clea ly de ec ed
ac oss he skin and adipose issue [59].
The luo escence o MB injec ed simul aneously wi h ICG could be used o con i m
whe he he luo escen ROIs a e ei he mesen e ic lymph nodes o mesen e ic edema.
The in a enous injec ion o wo dyes may inc ease he sensi i i y o mesen e ic lymph
node de ec ion, he eby a oiding complex p ocedu es such as subse osal and submucosal
injec ions. In addi ion, his may p e en any excessi e mesen e ic inju y o inadequa e
lymph node esec ion ha could occu when ICG is adminis e ed alone.
Addi ionally, MB in he lympha ic ne wo k and lymph nodes s ains a blue colo in
he specimen. The numbe o de ec able lymph nodes depends on age, gende , ype o
malignancy, and umo loca ion [
60
]. Besides, p eope a i e adju an chemo adia ion has
become he s anda d o ca e o ad anced ec al cance in ecen yea s. Neoadju an
chemo adio he apy educes lymph node size, which causes a dec ease in lymph node
ha es ing [
61
–
63
]. Mä kl e al. epo ed a s udy in which MB was injec ed in o he
supe io ec al a e y ex i o, o iginally pe o med o e alua e he accu acy o TME [
64
].
O he au ho s ound ha injec ing MB pos ope a i ely di ec ly in o he essels o ec al
esec ion specimens imp o ed he lymph node de ec ion a e [
45
]. Al hough i mus be
men ioned ha his is an a e ial injec ion and he concen a ion adminis e ed is high, hey
ha e shown ha MB ans e ed om he blood essels in o he lympha ic low s ains he
lymph nodes. Se e al s udies ha e shown ha highe doses o MB imp o ed pos ope a i e
lymph node ha es ing om ec al cance specimens [45,46].
Ano he po en ial applica ion o he p esen ed imaging se -up, apa om he es ed
lymph node and bowel de ec ion, is o assess blood low and u e e al ecogni ion as hese
a e wo o he p ope ies o he ICG and MB dyes. U e e al inju y is a se ious complica ion
du ing in apel ic su ge y, and he majo i y o ia ogenic u e e al inju ies may lead o
se e e mo bidi y. The incidence o u e e al inju y in colo ec al su ge y is no ha high
(0.25–1.1%)
. Howe e , se e al s udies showed ha only 15% o 30% o in aope a i e
u e e al inju ies a e ecognized. I mos equen ly occu s du ing p oc ec omy and sig-
moidec omy colo ec al cance su ge y [
65
]. Ande sen e al. e iewed 18,474 colo ec al
esec ions o he Danish Na ional Colo ec al Cance da abase and epo ed ha lapa o-
scopic p ocedu es co ela e wi h he isk o ia ogenic u e e al inju y [
66
]. The su geon
should be amilia wi h he ana omy o he u e e and he isk ac o s o inju y [
67
]. Visu-
aliza ion o he u e e is e y impo an , especially a e eope a ion o adia ion he apy.
In aope a i e u e e al iden i ica ion also con ibu es o he educa ion o young su geons
in ana omy. As e e ed o ea lie , ICG is clea ed ia he bile juice. The luo escen u ina y
ac isualiza ion wi h ICG equi es he inse ion o one o se e al u e e al ca he e (s)
ia cys oscopy equi ing an addi ional in asi e p ocedu e which may inc ease he isk
o pe iope a i e complica ions [
68
]. Se e al s udies ha e been published on u e e al lu-
Diagnos ics 2023,13, 1469 9 o 13
o escence using p eclinical luo opho es wi h emission peaks simila o ICG. Since hese
no el luo opho es (including IRDye
®
800CW, IRDye
®
800BK, IRDye
®
800NOS [
69
], and
IS-001 [
70
]) con ibu e o u ina y clea ance, hey ha e shown p omising esul s in de ec ing
he u e e . Howe e , u he clinical ansla ion is necessa y since hese dyes a e cu en ly
no a ailable in he ma ke place.
As a esul , since MB is exc e ed h ough he u ine, i may ep esen he bes way
o in aope a i ely iden i y u e e s du ing colo ec al su ge y. Se e al s udies ha e been
published in which MB is injec ed in a enously and he u e e is isualized wi h luo es-
cence [
41
–
43
]. The i s s udy o NIRF isualiza ion o he u e e using MB was p oposed
by Ve beek e al. in 12 pa ien s. In his s udy, he au ho s showed co ec isualiza ion
o bo h u e e s a e in a enous injec ion o MB [
43
]. La e on, Polom e al. published
a s udy on simul aneous luo escen isualiza ion o u e e and bowel pe usion ia he
adminis a ion o MB and ICG espec i ely using he lapa oscopic came a sys em ha was
used in ou s udy. The luo escen isualiza ion o he u e e was success ul in 11 ou o
12 pa ien s (96.1%), and in all pa ien s, bowel pe usion by means o ICG was success ully
con i med [
47
]. As a esul , simul aneous assessmen using MB and ICG is easible and
ep esen a comp ehensi e NIR guidance, helping su geons o de ec lymph nodes, o
educe he isk o anas omo ic leakage by assessing local issue pe usion, and o educe
he isk o complica ions by de ec ing u e e s.
Howe e , he e a e some d awbacks ega ding he adminis a ion o MB ha should
be discussed. MB is con aindica ed in pa ien s wi h glucose-6-phospha e dehyd ogenase
(G6PD) de iciency and Heinz body anemia. MB is a sa e agen when used a he apeu ic
doses o 2mg/kg o less [
71
]. In high doses o in pa ien s wi h enal ailu e, MB may induce
se e e ad e se e ec s, such as co ona y asocons ic ion, a hy hmias, and hemoly ic
anemia [
71
]. While i is p uden o use he lowes dose o MB necessa y o a gi en case o
minimize he po en ial o oxic mani es a ions [
72
], se e al s udies o luo escence u e e al
isualiza ion wi h he adminis a ion o MB showed ha highe doses o MB esul ed
in be e u e e al isualiza ion [
42
,
43
]. MB adminis a ion may cause alse ele a ions in
me hemoglobin le els wi h CO-oxime y and pulse oxime y [
73
]. Finally, excess doses o
MB may pa adoxically inc ease oxidan s ess and me hemoglobinemia [
74
]. Du ing his
expe imen , we did no obse e any such changes in i al signs a e MB adminis a ion.
Fu he expe imen s a e necessa y o de e mine he op imal me hod o adminis a ion,
iming, and dosage o MB dye, pa icula ly o lymph node mapping based on hese
p e ious s udies.
The limi a ion o he p esen s udy lies in he ac ha i is a small-numbe pilo s udy
using a po cine model. The mesen e y o an adul human is no as hin as ha o a pig. The
mesen e ic lymph nodes o humans, which a e usually embedded in adipose issue, a e
gene ally mo e di icul o de ec , an he o e, he cu en esul s should be in e p e ed wi h
causion o p edic whe he his me hod can be gene alized o humans. Addi ionally, he
cu en s udy and he imaging sys em we e used in an open su ge y se ing, and lapa o omy
was equi ed o image acquisi ion while in clinical se ing, minimally in asi e lapa oscopic
su ge y is he s anda d ea men in CRC su ge y. A p esen , a lapa oscopic came a
equipped wi h a double-wa eleng h imaging sys em is a ailable, enabling u he esea ch
o es ablish he accu acy o his imaging sys em in minimally in asi e su ge y se ings.
5. Conclusions
This easibili y s udy p o es ha mesen e ic lymph nodes can be disc imina ed using
wo di e en luo opho es (MB and ICG) a di e en wa eleng hs. Based on supe io
disc imina ion om he backg ound, MB has a p omising po en ial o be used o de ec
lympha ic issue du ing image-guided su ge y. Fu he p eclinical ials a e needed be o e
clinical ansla ion.
Au ho Con ibu ions:
Concep ualiza ion, N.O.; Da a cu a ion, N.O., Z.A.-D., M.H.M.C.S. and
D.J.I.H.; Fo mal analysis, N.O.; Funding acquisi ion, M.A.-T.; Me hodology, N.O. and M.A.-T.; P ojec
adminis a ion, N.O., Z.A.-D., M.H.M.C.S., D.J.I.H., N.D.B. and M.A.-T.; Resou ces, N.O., Z.A.-D.,
