research article gastric tissue damage analysis generated by ischemia: bioimpedance...

Hindawi Publishing CorporationBioMed Research InternationalVolume 2013 Article ID 824682 8 pageshttpdxdoiorg1011552013824682

Research ArticleGastric Tissue Damage Analysis Generated by IschemiaBioimpedance Confocal Endomicroscopy and Light Microscopy

Nohra E Beltran12 Laura E Garcia3 and Mario Garcia-Lorenzana4

1 Departamento de Procesos y Tecnologia Universidad Autonoma Metropolitana Cuajimalpa Artificios 40Colonia Hidalgo 01120 Ciudad de Mexico DF Mexico

2 Centro Nacional de Investigacion en Instrumentacion e Imagenologia Medica Universidad Autonoma MetropolitanaIztapalapa Avenida San Rafael Atlixco 186 Colonia Vicentina 09340 Ciudad de Mexico DF Mexico

3 Departamento de Ingenieria Electrica Universidad Autonoma Metropolitana Iztapalapa Avenida San Rafael Atlixco 186Colonia Vicentina 09340 Ciudad de Mexico DF Mexico

4Departamento de Biologia de la Reproduccion Universidad Autonoma Metropolitana Iztapalapa Avenida San Rafael Atlixco 186Colonia Vicentina 09340 Ciudad de Mexico DF Mexico

Correspondence should be addressed to Nohra E Beltran nbeltrancorreocuauammx

Received 2 May 2013 Accepted 4 June 2013

Academic Editor Andrea Vecchione

Copyright copy 2013 Nohra E Beltran et al This is an open access article distributed under the Creative Commons AttributionLicense which permits unrestricted use distribution and reproduction in any medium provided the original work is properlycited

The gastricmucosa ischemic tissular damage plays an important role in critical care patientsrsquo outcome because it is the first damagedtissue by compensatory mechanism during shock The aim of the study is to relate bioimpedance changes with tissular damagelevel generated by ischemia by means of confocal endomicroscopy and light microscopy Bioimpedance of the gastric mucosa andconfocal images were obtained fromWistar male rats during basal and ischemia conditions They were anesthetized and stain wasapplied (fluorescein andor acriflavine) The impedance spectroscopy catheter was inserted and then confocal endomicroscopyprobe After basal measurements and biopsy hepatic and gastric arteries clamping induced ischemia Finally pyloric antrum tissuewas preserved in buffered formaldehyde (10) for histology processing using light microscopy Confocal images were equalizedbinarized and boundary defined and infiltrations were quantified Impedance and infiltrations increased with ischemia showingsignificant changes between basal and ischemia conditions (119875 lt 001) Light microscopy analysis allows detection of generalalterations in cellular and tissular integrity confirming gastric reactance and confocal images quantification increments obtainedduring ischemia

1 Introduction

Shock is a critical condition in which oxygen availabilitybecomes restricted and tissues will consume asmuch oxygenas is available Compensated shock occurs when systemicdelivery of oxygen (DO

2) decreases and the tissues turn

to anaerobic sources of energy Under these conditionscellular function is maintained as long as the combinedyield of aerobic and anaerobic sources of energy providessufficient ATP for protein synthesis and contractile processesSome tissues are more resistant to hypoxia than others[1] The intestinal and gastric mucosae show evidence ofanaerobic metabolism before decreases in systemic oxygen

consumption (VO2) is detected [2] Uncompensated shock

resulting in irreversible tissue damage occurs when thecombined aerobic and anaerobic supplies of ATP are notsufficient tomaintain cellular function Failure ofmembrane-associated ion transport pumps in particular those associatedwith the regulation of calcium and sodium results in theloss of membrane integrity and in cellular swelling [3 4]Among other mechanisms that lead to irreversible cellularinjury during hypoxia are depletion of cellular energy cellularacidosis and oxygen free radical generation [1]

An important objective in the care of critically ill patientsis to ensure the adequacy of tissue oxygenation since tis-sue hypoxia may result in anaerobic metabolism cellular

2 BioMed Research International

acidosis and death [5] Emerging data suggest that earlyaggressive resuscitation of critically ill patients may limitandor reverse tissue hypoxia progression to organ failureand improve outcome [6] In critical illness blood flow tothe splanchnic tissues is frequently reduced and redirectedto other vital organs such as the brain heart and kidneysInadequately oxygenated the splanchnic tissues may becomeprone to ischemia-related complications [7] However atpresent there is no clinically useful method to directlymonitor the level of gastric tissue dysoxia injury

After a certain period of ischemia the gastrointestinaltissue becomes vulnerable to damage due to reperfusion inwhich restored oxygen supply produces free radical specieswhich lead to further tissue injury [8] For an effective andcorrectly timed therapy it may be very useful to know theischemic level and the tissue damage in a continuous andsimple manner [9]

Electric impedance measurements in tissues and biolog-ical systems have been used for decades in a wide variety ofapplications [10] Impedance spectroscopy is the study of thepassive electrical properties of biological tissues as a functionof frequencyThe impedance of biological tissues results fromthe interaction of an electrical current with the tissue at thecellular and molecular level Impedance is the total effect oftwo separate orthogonal dimensions the electrical resistancewhich restricts the flow of electrons and dissipates energyand the electrical reactance which is the capacity to store andrelease energy

On a conceptual level the cell cytoplasm and extracellularspace act as conductivemedia isolated from each other by thecell membrane The conductivity of the extra- and intracel-lular space contributes to the overall resistance of the tissuewhile the cell membrane contributes to the capacitive effect[10 11] For instance in a normoxic condition a significantamount of low frequency current is able to flow through theextracellular spaces when dysoxia occurs the cells are notcapable to generate enough energy to feed the ion pumps andextracellular water penetrates into the cell As a consequencethe cells grow and invade the extracellular space causing areduction of the current in extracellular fluids and are seenas a low frequency impedance increase [12] Also the closureof gap junctions contributes to the impedance incrementat these low frequencies At high frequencies impedancechanges are influenced by intracellular and extracellular fluidimpedances and ion permeability of cellular membranes[11 13] The electrical impedance of a living tissue can becontinuously measured to determine its pathophysiologicalevolution Somepathologies like ischemia infarct or necrosiscause cellular alterations that are reflected as impedancechanges [14]

Our group developed an instrument that measures theimpedance spectrum of the gastric mucosa in the range of215Hz to 1MHz [15] Alongwith the technique an impedancespectrometry probe and nasogastric tube (ISPNGT) [16]allow the direct acquisition of an electric impedance spec-trum of themucosa Most of the authors in the bioimpedancefield use the Cole-Cole equation to describe their experi-mental results characterizing the tissue bioimpedance withfour parameters For our device an adaptation of Cole-Cole

model was performed to develop an algorithm to calculatethe 6 characteristic electrical values that best describe humangastric impedance measurements [17] The device has beentested and validated during the last 12 years The resultsconfirm the potential of this technology for critical caremonitoringHowever some studies are required to determineactual tissue damage levels via impedance spectroscopyin critical care patients in order to guide therapy andimprove outcome The objective of the study was to relatebioimpedance changes with tissular damage level generatedduring induced hypovolemic shock in a rat model usingconfocal endomicroscopy and light microscopy

2 Materials and Methods

21 Animals Healthy adult male rats of Wistar strain aged3 months and weighting 350ndash400 g at the beginning of theexperiment were used Animals were bred and kept at theAnimal Facility of the Autonomous Metropolitan Universityin a temperature-controlled environment on a 12 12 h light-dark cycle and were fasted 12 h before experiment startedAll procedures used in the study complied with the guidefor care and use of laboratory animals The experimentalprotocol was approved by the National Center for MedicalInstrumentation and Imaging Research Ethical Committee

22 Ischemia Induced Procedure The surgical procedureswere performed under clean conditions Rats were deeplyanesthetized using 15mL intraperitoneal anesthesic cocktail(005mL ketamine 025mL propionylpromazine and 01mLxylazine in 06mL saline solution per mL) Another 1mLof the ketaminepropionylpromazinexylazine combinationwas kept for further maintenance anesthesia as needed

The animals were mounted on a dissection frame withattached limbs After the onset of general anesthesia 10 120583Lgof fluorescein (Alcon Pharma Novartis PharmaceuticalsMexico) was injected in the tail vein The rats underwent a5 cm midline laparotomy from the xiphoid process to justabove the penis After opening the abdomen the abdominalcavity was carefully inspected and stomach was exposed(Figure 1)

Using a 05mL syringe containing heparin blood sampleswere taken from the femoral artery immediately after open-ing the abdomen and 30min after isquemia pH and lactateconcentration were assessed with a blood gas analyzer (GEMPremier 3000 Instrumentation Laboratory Lexington MA)

Based on duodenum a 1 cm incision was made overthe greater curvature Then a biopsy was obtained andimpedance measurements were taken during 15 minutesWhen it was necessary for topical staining some drops ofa 002 solution of acriflavine (Merck KGaA DarmstadtGermany) in saline were applied to the tissue surface andexcess dye was washed away with phosphate buffered salineThen a collection of endomicroscopy images was storedAfterwards hepatic and gastric arteries were isolated andclamped (Figure 2) Bioimpedance measurements confocalimages and biopsy were taken After 30min of ischemiaEvanrsquos blue solution (15mL of 01) was injected into the

BioMed Research International 3

Figure 1 Stomach exhibition for greater curvature incision

Figure 2 Hepatic and gastric arteries occlusion for ischemiageneration

celiac artery to evaluate perfusion Once the experiment wasconcluded a humanitarian sacrifice was developed accordingto Mexican Official Standard (NOM-033-ZOO-1995)

23 Impedance Spectra Acquisition The impedance spec-troscopy system consists of three elements the catheter theimpedance spectrometer and the control and analysis systemThe catheter is a flexible plastic tube that can be inserted inany hollow viscous organ At the distal tip four electrodes arelocated that function as ionic current to electronic currenttransducers such as AgAgCl electrodes The electrodes areconnected to leads that provide an electrical connection tothe other end of the catheter along the wall of the tubingor in the lumen At the proximal end the leads end in anelectrical multichannel connector that can be plugged intothe impedance spectrometer [15] The prototype impedancespectrometer meets the international regulation standardsas the BS EN 60601-11990 and ANSIAAMI ES11993 APC with special software developed by our research groupcontrols the impedance spectrometer operations and dataacquisition storage and analysis Impedance measurements

obtained from these animals were automatically recordedevery 30 seconds during 15 minutes for each condition

24 Confocal Endomicroscopy Images Acquisition Confo-cal endomicroscopy (EC-3870CIFK Pentax Tokyo Japan)provides a 3-dimensional optical biopsy in vivo withoutphysically disrupting epithelial integrity For labeling oftissues fluorescent dyes (fluorescein and acriflavine) are usedConfocal gray scale images with a field of view of 475 times475 120583m can be obtained by gently pressuring the confocalwindow onto the surface mucosa The optical slice thicknessof a single image is 7 120583m with a lateral resolution of 07 120583mThe range of the 119911-axis can be varied from the surface to0ndash70120583m below the surface layer of the gastric mucosa aftertopical staining with acriflavine A single point within thetissue is scanned in a raster pattern andmeasurement of lightreturning to the detector from successive points is digitizedto construct an image of the scanned region Each resultantimage is a transverse optical section 500 times 500mm in sizeSerial images are collected at a scan rate of 08 frames persecond at a resolution of 1024 times 1024 pixels approximating a1000xmagnification on a 19-inch screen Five sets of confocalendomicroscopy images were obtained for each condition

25 Tissue Collection and Histological Analysis Chemicalfixation of the pyloric antrum tissue for light microscopywas developed using buffered formaldehyde (10) [18] Thepyloric antrum tissue was processed with the conventionalhistological techniques and included in Paraplast (OxfordLabware St Louis Mo USA) The pyloric antrum tissuewas cut longitudinally and transversally in each case 5-micrometer serial sections were obtained and stained usinghematoxylin-eosin [19] The tissues sections were analyzedunder a clear field light microscope (Axioskop II CarlZeiss) and image analyzer (Axiovision 48 Carl Zeiss)Thirtymicroscopic fields by condition were chosen at randomMicrographs were taken with an AxioCam MRc5 (CarlZeiss)

26 Data Analysis Impedance measurements were pro-cessed to calculate the central resistance and reactance atlow frequencies (119877

119871 and 119883

119871 resp) and central resistance

and reactance at high frequencies (119877119867

and 119883119867

resp) asthe characteristic gastric impedance parameters of interestas reported by Beltran et al [17] To analyze differencesin impedance parameters between the different conditions(basal and ischemia) we calculated the average for eachparameter over the monitoring time for each animal A totalof 50 to 100 images were collected per condition with thehelp of a foot pedal and digitally stored as gray-scale imagesAnalysis of images was performed using a specific algorithmdesigned by our group Confocal endomicroscopy imageswere grouped by condition and staining type Differencein the mean of all the values of experimental groups wasanalyzed using the Studentrsquos 119905-test A 119875 value lt 001 wasconsidered statistically significant Data are presented asmean plusmn standard deviation (SD)


Table 1 Impedance parameters for acriflavine staining Differencein the mean parameters by condition was analyzed using theStudentrsquos 119905-test Data are presented as mean plusmn standard deviation(SD)

Impedance parameter Basal Ischemia 119875

119877119871[Ohms] 754 plusmn 17 919 plusmn 14 lt001lowast

119877119867[Ohms] 343 plusmn 07 359 plusmn 05 0053119883119871[-jOhms] 74 plusmn 05 115 plusmn 04 lt001lowast

119883119867[-jOhms] 89 plusmn 02 185 plusmn 01 0037

lowastStatistically significant (119875 lt 001)

Table 2 Impedance parameters for fluorescein staining Differencein the mean parameters by condition was analyzed using theStudentrsquos 119905-test Data are presented as mean plusmn standard deviation(SD)




119883119871[-jOhms] 31 plusmn 03 67 plusmn 04 lt001lowast



Table 3 Impedance parameters for the combination of fluoresceinand acriflavine Difference in themean parameters by condition wasanalyzed using the Studentrsquos 119905-test Data are presented as mean plusmnstandard deviation (SD)






3 Results

The arteries clamping resulted in a significant decrease inblood pH (736 plusmn 004 for basal condition versus 718 plusmn 001for ischemia condition) Lactate increased significantly to523 plusmn 051mmolL indicating metabolic acidosis and tissueischemia

In order to obtain impedance spectroscopy measure-ments and evaluate staining effects in spectra measurementsdata were obtained using just acriflavine just fluorescein andthe combination of fluorescein and acriflavine Impedanceparameters 119877

119871(central resistance at low frequencies) 119883

119871

(central reactance at low frequencies) 119877119867(central resistance

at high frequencies) and 119910119883119867

(central reactance at highfrequencies) were calculated and Studentrsquos 119905-test was used inorder to evaluate changes between basal and isquemia con-ditions Table 1 shows impedance parameters for acriflavinestaining Table 2 for fluorescein staining and Table 3 for thecombination of fluorescein and acriflavine No immediateadverse reactions were noted following topical or systemicdye application

Table 4 Relationship between impedance parameters and confocalimages infiltration numbers Data are presented as mean plusmn standarddeviation (SD)

119877119871[Ohms] 119883

119871[-jOhms] Infiltration

numberAcriflavine

Basal 754 plusmn 17 74 plusmn 05 4294Ischemia 919 plusmn 14 115 plusmn 04 4423

FluoresceinBasal 629 plusmn 31 31 plusmn 03 2349Ischemia 991 plusmn 34 67 plusmn 04 4150

Acriflavine and fluoresceinBasal 703 plusmn 21 76 plusmn 04 691Ischemia 913 plusmn 16 117 plusmn 03 4169

Confocal endomicroscopy images were grouped accord-ing to staining type a set of 6 images was used for eachstaining and 2 cut series (basal and ischemia) for the combi-nation of fluorescein and acriflavine Images were processedusing an algorithm developed by our group Equalizationfiltering and binarization processing were applied in orderto enhance image features (Figure 3) The number of imag-ing infiltrations was calculated in Figure 4 (right corner)which indicate oedema alterations Table 4 illustrates therelationship between impedance parameters (119877

119871and119883

119871) and

number of infiltrations grouped by staining and conditionImpedance parameters increase during ischemia and arerelated to more infiltrations especially for the combinationof fluorescein and acriflavine staining

31 Light Microscopy In basal condition the gastric mucosapresents simple columnar epithelium with invaginations toform foveolae and glands A thin lamina propria layer formspacking among many gastric glands (Figures 5(A) and 5(E))

In ischemic animals evidence of sub-lethal (pale stained)and lethal cell injury (cytoplasmic eosinophilia and nuclearcondensation) are observed in Figures 5(C) and 5(F) respec-tively The lethal damage generates cellular death Fewscattered mononuclear cells (neutrophils) mainly localizedbetween the crypts and diffusely distributed through thelamina propria and vessels dilatation are observed (Figures5(B) 5(C) and 5(D)) These are events during the initialphases of the acute inflammatory response Also slightlyfoveolar epithelium erosion is illustrated in Figures 5(B) and5(C)

4 Discussion and Conclusion

Up to now no clinically useful method to directly monitorthe level of tissue dysoxia injury is available We proposedthat bioimpedance and particularly gastric reactance is anearly indicator of ischemia and we developed some studiesthat confirm the prognostic and diagnostic value of thesemeasurements [20 21] However before wemay advocate theclinical use of this technology we need to first answer the


Nonequalized Equalized

11 12

21 22

31 32

41 42

51 52

61 62

Acrifl

avin

eBa

sal

Acrifl

avin

eIs

chem

iaFl

uore

scei

nBa

sal

Fluo

resc

ein

Isch

emia

Basa

l

Acrifl

avin

e and

fluor

esce

inIs

chem

ia

Acrifl

avin

e and

fluor

esce

in

Figure 3 Confocal images comparison between nonequalized and equalized images after edge enhancement for different staining

following questions (1) Can we quantify oedema and tissuedamage from impedance parameters (2) Can we use thisinformation to improve patient management and treatmentThe second question is the hardest to answer and will requirefurther research and experience This study was designed toanswer the first question and relate impedance parameterschanges with tissular damage level generated during inducedischemia in a rat model using confocal endomicroscopy andlight microscopy

This is the first animal model designed to quantify gastricinjury generated by ischemia by confocal endomicroscopyimaging and evaluate its relationship with bioimpedancemeasurements In this hypovolemic shock model impedanceparameters statistically increase during ischemia as Tables 12 and 3 showed The greater changes are observed in centralresistance at low frequencies that reflects tissue oedemacaused by prolonged ischemia causing a net increase inintracellular to extracellular volume ratio Accumulation of



4294

4423

2349

4150

691

4169

11

189

12

21

81

22

31

135

32

41

444

42

51 52

61

402

9

62

Acrifl

avin

eBa

sal

Acrifl

avin

eIs

chem

iaFl

uore

scei

nBa

sal

Fluo

resc

ein

Isch

emia

Basa

l

Acrifl

avin

e and

fluor

esce

inIs

chem

ia

Acrifl

avin

e and

fluor

esce

in

Figure 4 Number of imaging infiltrations according to staining type (right square) after binarization processing

metabolic products cell swelling caused by osmosis andclosing gap junctions are important effects which can bedetected by means of electrical impedance spectroscopyat low frequencies (lt1 kHz) tissue impedance is influencedmostly by extracellular fluid impedance (extracellular path-way narrowing (caused by cell swelling) and gap junctionstatus can be ascertained) while at high frequencies itis influenced by both intercellular and extracellular fluidimpedances [11 13] According to confocal endomicroscopy

imaging processing we developed an algorithm to quan-tify indirectly cell swelling by infiltration accumulationAlthough acriflavine has been used to highlight nuclei whenit is used alone no differences in infiltrations were foundbetween basal and ischemia conditions (Table 4) Nuclearstaining was diminished in necrotic areas generated duringischemia Because the inflammatory process was focal ratherthan homogeneous in vivomicroscopy helped to localize theinflammatory infiltrate and necrotic areasThe inflammatory


e

L

G

(A) (B)

(C) (D)

(E) (F)

lowast

lowast

lowast

Figure 5 Hematoxylin and eosin stained histological sections of rat pyloric antrum tissue under basal and ischemia conditions (A) and (E)basal condition normal epithelium (e) lamina propria (L) and gastric glands (G) (B) (C) (D) and (F) ischemia condition some epithelialcells show histological features of necrosis (998771) and the initial phases of the acute inflammatory response leukocyte infiltration (rarr ) vascularcongestion (lowast) and epithelium integrity lost (nabla) ((A) (B) (C) and (D) magnification times100 Bar = 100 120583m H-E) ((E) and (F) magnificationtimes400 bar = 25 120583m)

infiltration and weak nuclear pyloric antrum tissue stainingof the foci corresponded well with ex vivo histology usinghematoxylin and eosin staining as reported in other confocalendomicroscopy studies [22]

Fluorescein staining exhibits significant differences (119875 lt001) between basal and ischemic groups however there werea large number of infiltrations for basal conditions that arenot related with the normal pyloric antrum tissue evidencedby histology analysis (Figure 5)The staining that best reflectsischemia condition and that can be quantified properly by ouralgorithm is the combination of fluorescein and acriflavine Inthis case our algorithm made a clear differentiation betweennormal and inflammatory infiltrations in pyloric antrum

tissue as observed in Table 4 Relatively few studies havetherefore successfully used this technique for imaging inanimal diseasemodels or in humans in vivo [22] On the otherhand targeted fluorescence technology has been thoroughlyexploited but has not been linked to in vivo microscopicimaging [23]

Impedance parameters increased during ischemia how-ever there is not a clear effect of staining type in these mea-surements It seems like fluorescein alone lightly decreases119877119871 but we did not find a clear relationship between

impedance parameters and inflamatory infiltration num-bers Nevertheless there was a relation between impedanceincrease and cellular damage observed by light microscopy


(Figure 5) indicating that the impedance spectrometerdevice proposed will be useful to reflect tissue oedema incritically ill patients Further studies are necessary in orderto understand cellular function and interactions in humandiseases to improve patient management and treatment Theresults demonstrated that the infiltration number calculatedis associated with the tissue injury observed histologicallyThis method could be a quick evaluation for the tissue statusthat realizes the concept of electrical biopsy [24]

Conflict of Interests

The authors declare that they have no conflict of interestsassociated with this paper

Acknowledgments

The authors wish to thank Saul Gaona for technical assitanceThis work was developed in the National Center for MedicalInstrumentation and Imaging Research and the TissularNeurobiology Laboratory at AutonomousMetropolitan Uni-versity in Mexico City

References

[1] G Gutierrez H D Reines and M E Wulf-Gutierrez ldquoClinicalreview hemorrhagic shockrdquo Critical Care vol 8 no 5 pp 373ndash381 2004

[2] ADubin E Estenssoro GMurias et al ldquoEffects of hemorrhageon gastrointestinal oxygenationrdquo Intensive Care Medicine vol27 no 12 pp 1931ndash1936 2001

[3] S A Oakes J T Opferman T Pozzan S J Korsmeyerand L Scorrano ldquoRegulation of endoplasmic reticulum Ca2+dynamics by proapoptotic BCL-2 familymembersrdquoBiochemicalPharmacology vol 66 no 8 pp 1335ndash1340 2003

[4] R G Boutilier ldquoMechanisms of cell survival in hypoxia andhypothermiardquo Journal of Experimental Biology vol 204 no 18pp 3171ndash3181 2001

[5] G Gutierrez H Bismar D R Dantzker and N Silva ldquoCom-parison of gastric intramucosal pH with measures of oxygentransport and consumption in critically ill patientsrdquo CriticalCare Medicine vol 20 no 4 pp 451ndash457 1992

[6] MM LevyW LMacias J A Russell et al ldquoFailure to improveduring the first day of therapy is predictive of 28-day mortalityin severe sepsisrdquo Chest vol 124 supplement p 120S 2003

[7] J Arnold J Hendriks C Ince and H Bruining ldquoTonometry toassess the adequacy of splanchnic oxygenation in the criticallyill patientrdquo Intensive Care Medicine vol 20 no 6 pp 452ndash4561994

[8] D N Granger ldquoRole of xanthine oxidase and granulocytes inischemia-reperfusion injuryrdquo American Journal of Physiologyvol 255 no 6 p 246 1988

[9] G Gutierrez F Palizas G Doglio et al ldquoGastric intramucosalpH as a therapeutic index of tissue oxygenation in critically illpatientsrdquoThe Lancet vol 339 no 8787 pp 195ndash199 1992

[10] A H Kyle C T O Chan and A I Minchinton ldquoCharac-terization of three-dimensional tissue cultures using electricalimpedance spectroscopyrdquo Biophysical Journal vol 76 no 5 pp2640ndash2648 1999

[11] E Gersing ldquoImpedance spectroscopy on living tissue fordetermination of the state of organsrdquo Bioelectrochemistry andBioenergetics vol 45 no 2 pp 145ndash149 1998

[12] J Flores D R DiBona C H Beck and A Leaf ldquoThe role of cellswelling in ischemic renal damage and the protective effect ofhypertonic soluterdquo Journal of Clinical Investigation vol 51 no1 pp 118ndash126 1972

[13] B Rigaud L Hamzaoui N Chauveau M Granie J-P ScottoDi Rinaldi and J-P Morucci ldquoTissue characterization byimpedance amultifrequency approachrdquo Physiological Measure-ment vol 15 no 2A pp A13ndashA20 1994

[14] A Ivorra M Genesca A Sola et al ldquoBioimpedance dispersionwidth as a parameter to monitor living tissuesrdquo PhysiologicalMeasurement vol 26 no 2 pp S165ndashS173 2005

[15] SOthman andE Sacristan ldquoComplex impedance spectrometerusing parallel demodulation and digital conversionrdquo UnitedStates Patent US 6970738 B1 2005

[16] E Sacristan ldquoImpedance spectroscopy system and catheterfor ischemic mucosal damage monitoring in hollow viscousorgansrdquo United States Patent US 6882879 B2 2005

[17] N E Beltran J J M De Folter MM Godinez and E SacristanRock ldquoSystems and methods for characteristic parameter esti-mation of gastric impedance spectra in humansrdquo United StatesPatent US 20100268110 A1 2010

[18] E B Prophet B Mills J B Arrington and L H Sobin AFIPLaboratory Methods in Histotechnology American Registry ofPathology 1992

[19] J K Presnell and M P Schreibman Animal Techniques TheJohn Hopkins University Press Baltimore Md USA 1997

[20] N E Beltran G Sanchez-Miranda M Godinez U Diazand E Sacristan ldquoGastric impedance spectroscopy in electivecardiovascular surgery patientsrdquo Physiological Measurementvol 27 no 3 pp 265ndash277 2006

[21] N E Beltran G Sanchez-Miranda M M Godinez U Diazand E Sacristan ldquoThe predictive value of gastric reactancefor postoperative morbidity and mortality in cardiac surgerypatientsrdquo Physiological Measurement vol 31 no 11 pp 1423ndash1436 2010

[22] M Goetz C Footner E Schirrmacher et al ldquoIn vivo confocalreal-time mini-microscopy in animal models of human inflam-matory and neoplastic diseasesrdquo Endoscopy vol 39 no 4 pp350ndash356 2007

[23] R Weissleder C-H Tung U Mahmood and A BogdanovJr ldquoIn vivo imaging of tumors with protease-activated near-infrared fluorescent probesrdquo Nature Biotechnology vol 17 no4 pp 375ndash378 1999

[24] Y-J Huang E-Y Huang and K-S Cheng ldquoThe correlationbetween extracellular resistance by electrical biopsy and theratio of optical low staining area in irradiated intestinal tissuesof ratsrdquo Biomed Eng Online vol 12 p 23 2013

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acidosis and death [5] Emerging data suggest that earlyaggressive resuscitation of critically ill patients may limitandor reverse tissue hypoxia progression to organ failureand improve outcome [6] In critical illness blood flow tothe splanchnic tissues is frequently reduced and redirectedto other vital organs such as the brain heart and kidneysInadequately oxygenated the splanchnic tissues may becomeprone to ischemia-related complications [7] However atpresent there is no clinically useful method to directlymonitor the level of gastric tissue dysoxia injury

After a certain period of ischemia the gastrointestinaltissue becomes vulnerable to damage due to reperfusion inwhich restored oxygen supply produces free radical specieswhich lead to further tissue injury [8] For an effective andcorrectly timed therapy it may be very useful to know theischemic level and the tissue damage in a continuous andsimple manner [9]

Electric impedance measurements in tissues and biolog-ical systems have been used for decades in a wide variety ofapplications [10] Impedance spectroscopy is the study of thepassive electrical properties of biological tissues as a functionof frequencyThe impedance of biological tissues results fromthe interaction of an electrical current with the tissue at thecellular and molecular level Impedance is the total effect oftwo separate orthogonal dimensions the electrical resistancewhich restricts the flow of electrons and dissipates energyand the electrical reactance which is the capacity to store andrelease energy

On a conceptual level the cell cytoplasm and extracellularspace act as conductivemedia isolated from each other by thecell membrane The conductivity of the extra- and intracel-lular space contributes to the overall resistance of the tissuewhile the cell membrane contributes to the capacitive effect[10 11] For instance in a normoxic condition a significantamount of low frequency current is able to flow through theextracellular spaces when dysoxia occurs the cells are notcapable to generate enough energy to feed the ion pumps andextracellular water penetrates into the cell As a consequencethe cells grow and invade the extracellular space causing areduction of the current in extracellular fluids and are seenas a low frequency impedance increase [12] Also the closureof gap junctions contributes to the impedance incrementat these low frequencies At high frequencies impedancechanges are influenced by intracellular and extracellular fluidimpedances and ion permeability of cellular membranes[11 13] The electrical impedance of a living tissue can becontinuously measured to determine its pathophysiologicalevolution Somepathologies like ischemia infarct or necrosiscause cellular alterations that are reflected as impedancechanges [14]

Our group developed an instrument that measures theimpedance spectrum of the gastric mucosa in the range of215Hz to 1MHz [15] Alongwith the technique an impedancespectrometry probe and nasogastric tube (ISPNGT) [16]allow the direct acquisition of an electric impedance spec-trum of themucosa Most of the authors in the bioimpedancefield use the Cole-Cole equation to describe their experi-mental results characterizing the tissue bioimpedance withfour parameters For our device an adaptation of Cole-Cole

model was performed to develop an algorithm to calculatethe 6 characteristic electrical values that best describe humangastric impedance measurements [17] The device has beentested and validated during the last 12 years The resultsconfirm the potential of this technology for critical caremonitoringHowever some studies are required to determineactual tissue damage levels via impedance spectroscopyin critical care patients in order to guide therapy andimprove outcome The objective of the study was to relatebioimpedance changes with tissular damage level generatedduring induced hypovolemic shock in a rat model usingconfocal endomicroscopy and light microscopy

2 Materials and Methods

21 Animals Healthy adult male rats of Wistar strain aged3 months and weighting 350ndash400 g at the beginning of theexperiment were used Animals were bred and kept at theAnimal Facility of the Autonomous Metropolitan Universityin a temperature-controlled environment on a 12 12 h light-dark cycle and were fasted 12 h before experiment startedAll procedures used in the study complied with the guidefor care and use of laboratory animals The experimentalprotocol was approved by the National Center for MedicalInstrumentation and Imaging Research Ethical Committee

22 Ischemia Induced Procedure The surgical procedureswere performed under clean conditions Rats were deeplyanesthetized using 15mL intraperitoneal anesthesic cocktail(005mL ketamine 025mL propionylpromazine and 01mLxylazine in 06mL saline solution per mL) Another 1mLof the ketaminepropionylpromazinexylazine combinationwas kept for further maintenance anesthesia as needed

The animals were mounted on a dissection frame withattached limbs After the onset of general anesthesia 10 120583Lgof fluorescein (Alcon Pharma Novartis PharmaceuticalsMexico) was injected in the tail vein The rats underwent a5 cm midline laparotomy from the xiphoid process to justabove the penis After opening the abdomen the abdominalcavity was carefully inspected and stomach was exposed(Figure 1)

Using a 05mL syringe containing heparin blood sampleswere taken from the femoral artery immediately after open-ing the abdomen and 30min after isquemia pH and lactateconcentration were assessed with a blood gas analyzer (GEMPremier 3000 Instrumentation Laboratory Lexington MA)

Based on duodenum a 1 cm incision was made overthe greater curvature Then a biopsy was obtained andimpedance measurements were taken during 15 minutesWhen it was necessary for topical staining some drops ofa 002 solution of acriflavine (Merck KGaA DarmstadtGermany) in saline were applied to the tissue surface andexcess dye was washed away with phosphate buffered salineThen a collection of endomicroscopy images was storedAfterwards hepatic and gastric arteries were isolated andclamped (Figure 2) Bioimpedance measurements confocalimages and biopsy were taken After 30min of ischemiaEvanrsquos blue solution (15mL of 01) was injected into the










119871 and 119883



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3 Results




119871





119877119871[Ohms] 119883


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119871 and 119883



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3 Results




119871





119877119871[Ohms] 119883


numberAcriflavine





119871and119883

119871) and








11 12

21 22

31 32

41 42

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61 62

Acrifl

avin

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chem

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Acknowledgments


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3 Results




119871





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OncologyJournal of





PPAR Research



Journal of

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Acknowledgments


References






























of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of





















of






Disease Markers



OncologyJournal of





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Journal of

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research article gastric tissue damage analysis generated by ischemia: bioimpedance...

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