Effects of temporary vascular occluder poloxamer 407 Gel on the endothelium.

1.8 MB·English

by  Gucu, Arif

Gucuetal.JournalofCardiothoracicSurgery2013,8:16
http://www.cardiothoracicsurgery.org/content/8/1/16
RESEARCH ARTICLE Open Access
Effects of temporary vascular occluder poloxamer
407 Gel on the endothelium
Arif Gucu1*, Ilkin Cavusoglu2, Cuneyt Eris1, Faruk Toktas1, Tugrul Goncu1 and Ahmet Ozyazicioglu1
Abstract
Background: Coronary occlusion techniques during OPCABmay lead to anendothelial damage to thetarget
vessel. The adverse effects of thesetechniques are well-known, and researches have been trying to find out new
materials to occlude thecoronaryartery without an endothelial damage. Inthe present study, we investigate to the
endothelial damage intherat aorta which is occluded by Poloxamer 407 gel.
Methods: Forty-five rats were randomized inthreegroups: (1) segment ofthe aorta was occluded withPoloxamer
407gel in P407 group; (2)segment oftheaorta was occluded with microvascular clamp in MV clamp group; and
(3)no onclusion was available intheControl group. The rats were sacrificed of observation,and a 15mm segment
ofthe aorta was obtained as a specimen. Integrity ofthe endothelial lining was observed witha scanning electron
microscopy.
Results: Scanning electron microscopyrevealed a statistically significant difference among the 3groups (p<0,001)
using the SPSS 13.0 test. No difference was found between the Control group and theP 407group (p=0,059). The
differences between MV clamp–Control group (p<0,001) and MV clamp–P407 group were statistically significant
(p<0,002).
Conclusions: We suggest that Poloxamer 407 gel occlusion may be a safer and more effective method compared
to themicrovascular clamp occlusion.
Keywords: Endothelial damage, Poloxamer 407, Scanningelectron microscopy, OPCAB
Background have atherosclerotic coronary arteries; therefore, bear
Coronary artery occlusion techniques during OPCAB endothelial damage to some extent [8]. Using the afore-
often result in suboptimal visualization due to bleeding mentioned methods for optimal visualization during
fromarteriotomysite.Thisleadstoanincreaseinsurgical OPCABmayevenincreasethisendothelialdamage.Thus,
trauma and risk the quality of the anastomosis. Recently, it is crucial to use the safest occlusion technique during
techniques and devices have been employed to improve OPCAB.
the visibility during OPCAB surgery, including elastic Poloxamersareabroadgroupofsurfactantswidelyused
snaresutures,microvascularclamps, intracoronaryshunts in various industrial applications. These water-soluble,
and high flow gas insufflation materials. However, these nontoxic and inert surfactants are tribloc copolymer of
techniquesanddevicesallhaveapotentialformechanical polyethylene oxide –polypropylene oxide –polyethylene
a b
damagetothecoronaryendothelium[1-7]. oxide .Differenthydrophilic/hydrophobicratiosandphys-
a
Despite improvements in hemostatic devices used in ical characteristics can be obtained by varying the block
OPCAB surgery, the perfect device which allows a size and total molecular weight of the poloxamers [9]. In
complete control of bleedingwithoutgeneratingcoronary particular, Poloxamer 407 - a polymer witha ratio of70%
endothelial injury is yet to be introduced for use. On the polyoxyethylene and 30% polyoxypropylene - shows in-
other hand, patients undergoing coronary surgery already verse thermo sensitivity [10]. Therefore, aqueous polymer
solutions - with greater than 12% critical concentration -
*Correspondence:[email protected] are liquid at low temperatures.However, it isnotable that
1DepartmentofCardiovascularSurgery,BursaYuksekIhtisasTrainingand
they turn into gel form at higher temperatures. The
ResearchHospital,Bursa,Turkey
Fulllistofauthorinformationisavailableattheendofthearticle
©2013Gucuetal.;licenseeBioMedCentralLtd.ThisisanOpenAccessarticledistributedunderthetermsoftheCreative
CommonsAttributionLicense(http://creativecommons.org/licenses/by/2.0),whichpermitsunrestricteduse,distribution,and
reproductioninanymedium,providedtheoriginalworkisproperlycited.
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http://www.cardiothoracicsurgery.org/content/8/1/16
Table1Theclassificationofhistomorphologicalchangesoftheendotheliallayer
Damagetype Histomorphologicalfeatures
Nodamage Theendothelialcellsarecontactwitheachother,neitherchangeinthecellularmorphologynoranydecreaseinsize.
Type1 Cellularintegrityofendothelialcellsisunchanged.Endothelialcellsareincontactwitheachother,buttheflatteningofthe
damage endothelialcells,changesindiameterandtheoccurrenceofthenuclei.Inaddition,theadhesionofbloodcellsontheendothelium.
Type2 Detachmentofthecellsattheirjunctionpointsand/orisolatedabsenceofendothelialcells.
damage
Type3 Appearanceofthesubendothelialtissuewithdesquamationofendothelialcells.
damage
polymer is highly soluble; therefore, gel plug erodes in olive-shaped ending taken as atraumatic for the endo-
blood, and the dissolution time depends on how much thelium. After the introduction of the catheter 15–20
polymer is injected, the concentration of the polymer in mm into the proximal segment of rat abdominal aorta,
W
the solution, and the surrounding temperature [11]. This 0.15 cc of Poloxamer 407 gel (LeGoo , 22,5%, Pluromed
nontoxicpolymerisnotabsorbedormetabolizedanddoes Inc, Lincoln, MA, USA) was injected rapidly in order to
notleadtoanembolism.Itisexcretedurinarywithahalf- avoid uneven coating of the gel, which could comprom-
lifeofapproximately25hours[12].TheuseofPoloxamer ise the effectiveness of the occlusion. Lack of blood flow
407invascularocclusionduringOPCABsurgeryhasbeen from the proximal direction in a few seconds meant that
previously reported [11,13-15]. In this study, we aimed to Poloxamer 407 gel became solid, and the plug occluded
investigate the endothelial damage caused by Poloxamer the vessel successfully. Ten minutes following complete
407 gel occlusion in the rat aorta using scanning electron occlusion, we irrigated the vessel gently with a cold sa-
microscopy. line solution to dissolve the plug in a few seconds. Sub-
sequently, the vessel was carefully dissected free from
the adherent tissue in a no-touch technique and was cut
Methods in transverse fashion 5 mm away from the occlusion
Animalsandsurgicaltechnique area on each side. Hence, approximately 15-mm seg-
Forty-five Wistar Albino female rats (3–4 months old, ment of the aorta was excised and taken as a specimen
weighing300–400grams)wereusedinthisexperimental from theP407group.
study. Following Uludag University Animal Experiments 2) MV clamp group: Following arteriotomy of the ab-
Local Ethic Committee approval (15.02.2011, ethics dominal aorta, the proximal segment of arteriotomy was
committee decision no: 2011-02-01), the study was con- clamped with a microvascular clamp (Micro DeBakey
ducted in accordance with the ‘Guide for the Care and bulldog clamp, Malden, MA, USA). After waiting for ap-
Use ofLaboratoryAnimals2010’. proximately 10 minutes, the vessel was harvested in the
All of the rats were anesthetized with ketamine same fashion as in P 407 group and was taken as a spe-
W
hydrochloride, 20 mg/kg IM (Ketalar 500 mg, Pfizer, cimenfrom theMVclampgroup.
New York, USA) and xylazine hydrochloride, 15 mg/kg 3) Control group: Following exploration, the vessel
W
IM (Rompun Inj. Solution 2%, Bayer, Leverkusen, was harvested in the same fashion as in both groups,
Germany). In light of median laparotomy and exploration andwastaken asa specimenfrom theControl group.
anddissectionoftheabdominalaortathefollowingproce-
dureswereperformed:
1) P 407 group: after arteriotomy of the abdominal TissueprocurementandpreparationforSEM
aorta, we placed a cardiac control syringe (Pluromed The specimens of aorta were dissected longitudinally,
Inc, Lincoln, MA, USA) into the proximal part of the pinned on cork plates, and fixed with 5% glutaraldehyde
aorta. The distal part of the catheter (made of polypro- (in0.13Mphosphatebuffer,pH7,2)at+4°Cfor4hours.
pylene and styrene butadiene rubber) is smooth with an Secondary fixation was performed with 1% OsO
4
Table2Numberofsampleswithdifferenttypesofendothelialdamageincontrol,Poloxamer407geland
microvascularclampgroups
Control(n:15)group MVClamp(n:15)group Poloxamer407Gel(n:15)group
Nodamage 15 - 10
TypeIdamage - 10 4
TypeIIdamage - 4 1
TypeIIIdamage - 1 -
Gucuetal.JournalofCardiothoracicSurgery2013,8:16 Page3of7
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Figure1ScanningelectronmicrographoftheP407group.Theendothelialcellswereintact.Bloodcellsandfibrinwereobservedadhered
totheendothelialsurface.Thenucleioftheendothelialcellswereprominent.
Figure2ScanningelectronmicrographoftheP407group.In4samples,endothelialcellswereincontactwhicheachother,butthe
flatteningendothelialcellsanddecreasedindiameterwasdetected.Otherbloodcellsobservedinadhesiontoendothelium,damagewas
evaluatedastype1.
Gucuetal.JournalofCardiothoracicSurgery2013,8:16 Page4of7
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Figure3ScanningelectronmicrographoftheP407group.Inonesample,minimaldetachmentbetweentheendothelialcellswas
observed.Damagewasevaluatedastype2.
(in 0,13 M phosphate buffer, pH 7,2) at +4°C for 1 hour. Results
Thespecimensweredehydrated ingraded alcohol series, The specimens were examined with a scanning electron
treated with 1/3, 1/1 and 3/1 alcohol/amylacetate mix- microscope and scored in accordance with the afore-
tures and transferred to pure amylacetate. The tissue mentioned classification criteria. Number of samples
samples were kept in pure amylacetate for 2 days and with different types of endothelial damage among con-
then dried in critical point dryer (Baltec CPD Critical trol, Poloxamer 407 gel and microvascular clamp groups
Point Dryer) and, coated with gold-palladium (Baltec were described inTable2.
SCD 005 Spotter Coater). The endothelial surfaces of Freedman test revealed a statistically significant differ-
the samples were investigated through a scanning ence among the 3 groups (p<0,001). However, there was
electron microscope (Carl Zeiss EVO 40, Carl Zeiss not any difference between the Control group and P 407
SMT AG, Germany). The specimens were examined by group (p=0,059) using the Wilcoxon test. The differences
a histologist who was experienced in scanning electron between MV clamp–Control group (p<0,001) and MV
microscopyand,wasblinded toalloftheotherdata. clamp–P407groupwerestatisticallysignificant(p<0,002).
Histologicalinvestigation Discussion
Histomorphologic changes of the endothelial layer were One of the basic principles of the OPCAB is to establish
classifiedintofourgrades,andweredescribedinTable1. a perfect anastomosis. On the other hand, one of the
most important problems in OPCAB is the suboptimal
visualization of the anastomosis site because of the
Statisticalanalysis bleeding through arteriotomy site despite the occlusion
Data were analyzed using SPSS 13.0 (SPSS, Inc, Chicago, of the proximal coronary artery. The bleeding in the
USA). Three dependent groups were compared by anastomosis sitedecreasesthequalityoftheanastomosis
Freedman Test, and the dual groups were compared by and increases the probability of the surgical trauma [1].
theWilcoxonTest.Resultswhichwerep<0,05were con- For the improvement of the optimal visualization, the
sidered as significant. Descriptive values were noted as native coronary artery blood flow is usually occluded by
Median(Max-Min). the elastic snare suture or microvascular clamp. In spite
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Figure4ScanningelectronmicrographoftheMVclampgroup.Inthissample,detachmentoftheendothelialcellsandpatchyareasof
peelingsubendothelialtissuewereobserved.Damagewasevaluatedtype3.
of the occlusion of the proximal coronary artery, collat- inhibits cellular growth and migration. Prostacyclin and
eralflowimpairsthe visualizationoftheanastomosissite nitricoxidebothexertspotentantiatherogenicandthrom-
and distal portion of the coronary artery occlusion may boresistant properties by preventing platelet aggregation
be required. This is not recommended to avoid the and cell adhesion. Endothelial vasoconstrictors, such as
possibility of the formation of the late term vascular angiotensin II and endothelin-1, both of which exert
stenosis and intimal damage [1]. Additional methods are prothromboticandgrowth-promotingpropertiescounter-
employed to have an optimal visualization of the anasto- balancestheseeffects[17,18].
mosis site. These methods include intracoronary shunt Ip et al. [19] classified the coronary endothelial damage
or occluders and using high flow gas insufflation. into three groups and reported that type 3 damage may
Adverse effects of these methods are well reported [1-6]. cause restenosis of the coronaryartery. Similarly, Okazaki
Inanexperimentalstudy,Gucu etal. [4]havefound that etal.[2]haveclassifiedtheendothelialdamagein5stages
filtrated high flow room air insufflation to the coronary andstatedthat,particularly,type3orstage5damageshad
anastomotic area may frequently cause type 1 and rarely resulted in the development of platelet aggregation and
type 2 endothelial damage. Intracoronary shunts may thrombus formation. Factors, such as PDGF, initiate
posestructurallyandafunctionallypermanentendothelial smoothmuscleproliferationandmigrationandasaresult,
damage [5] and high-flow gas insufflation may bear the these factors could lead to an early and a late period
riskofgasembolismand/oranendothelialdamage[16]. restenosis[18].
The endothelium is a barrier between the vascular Previous studies have shown that Poloxamer 407 can
smoothmuscleandbloodthatproducesenzymesforacti- be utilized to occlude blood vessels during OPCAB
vating and deactivating cardiovascular hormones and also [11,15,20,21]. Bouchot et al. [14] have reported that,
the producer of relaxing and contracting factors. Vascular Poloxamer 407 was effective as a temporary vascular
endothelium is located between the circulating the vascu- occluder during OPCAB anastomoses and the endothe-
lar smooth muscle and blood. Endothelial cells release lium of the coronary arteries revealed no functional or a
various vasoactive substances regulating the function of structural damage and the myocardium exhibited no
vascular smooth muscle and trafficking blood cells. There ischemic injury attributable to the polymer plug. In a
are important endothelium-derived vasodilators such as prospective multicenter randomized clinical study,
bradykinin, prostacyclin, nitric oxide and, endothelium- Wimmer-Greineckeretal.[21]evaluatedthatPoloxamer
derived hyperpolarizing factor. Among these, nitric oxide 407 could be used safely and effectively in the OPCAB
Gucuetal.JournalofCardiothoracicSurgery2013,8:16 Page6of7
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surgery. Poloxamer 407 provided a dry surgical anasto- As a result, the finding of the present study clearly
motic field andsurgical comfortmorefrequently thanthe demonstrates the safety of Poloxamer 407 gel occlusion
conventional vessel loop. In addition, anastomotic times and shows lower damage to the endothelium when ana-
were shorter with Poloxamer 407 and major cardiac lyzed atthescanningelectron microscopical level.
eventsweresimilarinthePoloxamer407andvesselloop.
Gertz et al. [6] studied the endothelial cell damage Competinginterests
Theauthorsdeclarethattheyhavenocompetinginterests.
caused by temporary arterial occlusion with surgical clips
using scanning and transmission electron microscopy. Authors’contributions
They have occluded the right carotid artery for 5, 15 and Conceptionanddesigned:AG,CE,IC.Analysisandinterpretation:AG,CE,FT,
TG.DataCollection:AG,CE.Writingthearticle:AG,CE.Criticalrevisionofthe
30 minutes with a surgical clip. They reported that the
article:AO.Finalapprovalofthearticle:AG,CE,FT,TG,AO.Optioned
endotheliumof the compressedsegments revealedcraters funding:notapplicable.Overallresponsibility:AG.Allauthorsreadand
and balloons, endothelial cell flattening, discontinuity and approvedthefinalmanuscript.
desquamation exposingthe sub endothelial tissueafter 15
Acknowledgements
or 30 minutes of occlusion. In this study, there was not
WethanktoDr.OzerYilmazforscanningelectronmicroscopicallyanalyses
any crater or balloons, but endothelial cell flattening, dis- andDr.IlkerErcanforstatisticalanalyses.
continuity and desquamation were observed especially in
Authordetails
the MVclamp group samples. The duration of occlusion 1DepartmentofCardiovascularSurgery,BursaYuksekIhtisasTrainingand
wasnot morethan10minutesin ourstudy since wepre- ResearchHospital,Bursa,Turkey.2DepartmentofHistologyandEmbryology,
UludagUniversityFacultyofMedicine,Bursa,Turkey.
feredtheoptimaltimeforanastomosisduringOPCAB.
According to the literature, in type 1 damage, integrity
Received:24September2012Accepted:3January2013
of the endothelial cells is reversible and complete recov- Published:22January2013
ery is expected in this process [22,23]. In our study, no
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doi:10.1186/1749-8090-8-16
Citethisarticleas:Gucuetal.:Effectsoftemporaryvascularoccluder
poloxamer407Gelontheendothelium.JournalofCardiothoracicSurgery
20138:16.
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