role of complement inhibitory

8/3/2019 Role of Complement Inhibitory

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Role of Complement Inhibition in a IschemicReperfusion Induced Regeneration Model in Liver

Background: Assessment of C1-INH affect on regeneration and apoptosisin ischemia and reperfusion(IR) damage in liver tissue.

Materials and Methods: 48 rats were included this study. Subjects weredivided into 8 groups. Partial hepatectomy (PH) was performed in Group Irats. IR was constituted in Group II rats and followed by PH. Group IIIrats were exposed to only IR. Group IV was control group. Group V ratsreceived only C1-INH. Group VI rats received C1-INH prior to IR; PH wasperformed after IR. Group VII rats received C1-INH and followed by PH.Group VIII rats received intravenous C1-INH, followed by IR.

Immunohistochemical analysis, liver function test, cellular assessment byelectron microscopy, malondialdehyde(MDA) and reducedglutathione(GSH) evaluation were performed.

Results: Statically significant difference was observed for apoptosisbetween group I and VII, group III and VIII, group IV and V. There wasstatically significant difference for PCNA between group II and VI, groupIII and VIII, group IV and V. Alanine aminotransferase levels weredecreased in all drug received groups. Statically significant difference wasfound in MDA and GSH tissue analysis between group II and VI, group IIIand VIII.

Conclusion: C1-INH increases the cell generation and significantlyreduces apoptosis

Introduction:

Ischemia is lack of supplying

oxygen and other metabolites totissues by blood circulation andlack of waste product removal byblood circulation(1). Reperfusion isdefined as reassuring bloodcirculation to these tissues.Reperfusion supplies the oxygenand metabolite demand of theischemic tissue but on thecontrary it causes tissue damageat the same time(2).Ischemia andreperfusion(IR) causes much more

damage than ischemia itself(3).Reperfusion damage followingischemia has been investigated inmany studies for several organssuch as lung, brain, liver, hearthand intestines (4-7). Butphysiopathology of the IR damagestill remains unclear. It is knownthat major components like freeradical formation, PNL lymphocyteactivation, endothelial system andcomplement system play role in IRdamage(8-9). This process leadsto systemic inflammatory responseand thus end organ damage.Major causes of liver ischemia are;

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liver resection, hemorrhagic shockand traumatic liver damage. Allthree pathways of complementsystem are activated in IRdamage(classical, alternative and

lectin pathways). Complement-1inhibitory(C1-INH) belongs toserine protease family and it is themajor inhibitory of classicalcomplement pathway(10). Itprotects the tissue fromreperfusion damage. Reperfusionincreases the complementactivation in ischemic liver tissue.Inhibition of complement cascade

decreases the post ischemicinflammation in liver tissue. In thisstudy we aimed to investigate theaffect of complement inhibition onregeneration and apoptosis viainhibition of complement systemby C1-INH in liver tissue.

Materials and Methods:

This study was performed inBaşkent University ExperimentalResearch Center. The project hadbeen approved by EthicalCommittee of Animal Experimentsprior to the study. All the surgicalprocedures performed during thestudy were proper with “NationalInstitutes of Health, Guide for theCare and Use of Laboratory

Animals” rules. 48 Wistar Albino,200-250 g, female, randomlyselected rats were used in thisstudy. Animals were divided into 8groups equally as 6 rats for eachgroup. 50 mg/kg KetamineHydrochloride(Ketalar, EczacıbaşıWarner-Lambert pharmaceuticalindustry, Levent, İstanbul) and 7mg/kg Xylazine Hydrochloride

(Rompun, Bayer Şişli, İstanbul)

were used for anesthesia underaseptic conditions.

Partial hepatectomy wasperformed in Group I rats. IR wasconstituted in Group II rats via

clamping the portal hilus for 45minutes, followed by partialhepatectomy. In group III rats IRwas constituted only. Group IVdefined as control group. In GroupV only C1-INH was administered.In Group VI rats received C1-INHprior to IR and after IR partialhepatectomy was performed. InGroup VII rats received C1-INH

and followed by partialhepatectomy. In group VIII IR wasperformed after 20 minutes of intravenous C1-INH injection. Inorder to compare the drugreceived and surgery performedgroups with each other; the eightgroups were compared as follow:Group I with Group VII, Group IIwith Group VI, Group III with

Group VIII, Group IV with GroupV.C1-INH 200IU/kg was

administered to rats 10 minutesbefore to surgery. In order tocompensate the fluid deficitcaused by surgical procedure anddrug administration each ratreceived 2 cc ringer lactate and 3cc 5% dextrose intraperitoneally.

Single dose 0.02 mg fentanyl wasadministered to all ratssubcutaneously for post-operativepain.

Left lateral segmentectomywas performed for partialhepatectomy. Ischemia wasprovided by microvascular clampplacing to portal peduncle andmaintained for 45 minutes. Portalpeduncle was clamped withbleeding control by dissection

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microscope and ischemia wasperformed. Clamp was removedafter 45 minutes of waiting periodand reperfusion was observed bydissection microscope.

All rats were sacrificed at theend of 5th post-surgical day.Before the sacrifice procedure 5 ccblood was taken from the inferiorvena cava of all rats for cytokineanalysis. Latter liver was excisedand preserved forimmunohistochemical andbiochemical analysis. Reperfusiondamage in liver tissue was

analyzed with reduced glutathione(GHS) and malondialdehyde(MDA) levels. Liver function wasassessed by aspartateaminotransferase (AST) andalanine aminotransferase (ALT)levels. Apoptosis and regenerationwere assessed with electronmicroscopy andimmunohistochemical methods.

TUNEL (Transferase - mediated d-UTP - biotin Nick End Labeling)method was used forhistochemical assessment. 200cells were counted in the lightmicroscopy at the maximummagnification level(x40 HBF).Positive stained cells noted aspercentage (%). An indicator of the cell proliferation PCNA

(Proliferating Cell Nuclear Antigen)was assessed by light microscopyvia 1000 cell counting under themaximum magnification (x40HBF); positive cells were noted aspercentage(%).SPSS 9.0 box program was usedfor data analysis. Standarddeviation and mean values werecalculated. For analyzing the nonparametric values IndependentSamples T test was used to

evaluate the differences of nominal values between groups.p<0.05 value was accepted asstatically significant.Results:

Same surgery procedureperformed groups were comparedto each other for apoptosis andstatically significant differenceswere observed between Group Iand VII, group III and VIII, groupIV and V(p<0.05).

Drug received and samesurgery procedure performed

groups were compared to eachother for PCNA and staticallysignificant differences wereobserved between group II and VI,group III and VIII, group IV andV(p<0.05). There was no staticallysignificant difference betweengroup I and VI, partial liverresection performed without IR.There was no statically significant

difference for AST levels; but ALTlevels were significantly lower indrug received groups(p<0.05)There were statically significantdifferences for MDA levels betweengroup II and VI, group III andVIII(p<0.05)(Graphic 1).Statically significant differenceswere observed for GSH analysisbetween group II and VI, group III

and VIII (p<0.05)(Graphic 2).

Apoptosis, PCNA, ALT, MDA andGSH mean values weresummarized in Table 1 for GroupI/VII, Group II/VI, Group III/VIIIand Group IV/V.

Electron Microscopy Findings:

Group I and VII: Partialhepatectomy performed groups

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and drug received + partialhepatectomy performed groupswere assessed and compared witheach other. Nuclear structure,chromatin distribution,

mitochondrion, granularendoplasmic reticulum (GER) andside-link units of cells wereevaluated as normal in bothgroups.

Figure 1: Deterioration in mitochondria

membrane

Group II and VI: In the ischemia

and partial liver resectionperformed group someobservations were noted asincrease in cytoplasmic lipiddroplets in some hepatocytes,deterioration in mitochondriamembrane (Figure 1) and increasein intracellular connective tissue.In drug received latter IR andpartial liver resection performed

group it has been noted thathepatocytes were not affected byischemia and reperfusion, and cellstructure was normal like (Figure2).

Figure 2: Hepatocyte unaffected from IR

Group III and VIII: Ischemiaand reperfusion resulted inincrease of cytoplasmic lipiddroplets (Figure 3), mitochondrial

degeneration and vacuolization,GER dilatation and intracellularconnective tissue increase in allhepatocytes. Controversially noneof these findings were observedand intracellular connective tissuewas normal in drug received andIR constituted group(Figure 4).

Figure 3: Lipid droplets in hepatocytes

Group IV and V: Polygonalstructure of the hepatocytes wasmaintained in both groups.Nuclear structure, chromatindistribution, nucleolus,mitochondrion, GER andintercellular connective tissuewere normal in both groups.

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Figure 4: Normal Hepatocyte

Discussion:

IR damage is characterized bytemporary loss of tissue bloodcirculation and developing animportant inflammatory responseafter reassuring the blood flow.Major liver ischemia causes are asfollow: liver resection, traumaticliver damage and hemorrhagicshock. Reasons of developing

organ dysfunction after the bloodcirculation loss are cell damagecaused by reperfusion andincreased organ sensitivity (11-14). Factors that causes damageare; free oxygen radicals (ROS),leukocyte migration, leukocyteactivation, sinusoidal endothelialcell damage, microcirculatoryimbalances, coagulation system

activation and complement systemactivation.Both classical and alternativecomplement pathways areactivated in IR damage. “Decayaccelerating factor” located in thecell membrane and membranecofactor protein protect the cell toa complement attack. Complementdepended proinflammatorypeptides(C3a-C5a) are releasedduring the reperfusion. Thesepeptides leads to neutrophil

accumulation, smooth musclecontractions, increase in vascularpermeability, Kupffer cellactivation (11,15-16).

At the beginning phase of IR

damage; oxidative stress induceddamage in Kupffer cells leads toproduction and release of reactiveoxygen radicals. Complementproducts are required in this phasefor Kupffer cell activation (17-20).Plasma levels of activatedcomplement components increasesin the late phase. Complementactivation products

(Anaphylatoxins and membraneattack complex components) arecharacterized by massiveneutrophil infiltration and it canmaintain for 12-24 hours afterreperfusion. Kupffer cells andneutrophils causes increase inreactive oxygen radicals andproinflammatory cytokines,sinusoidal concession and

cytoplasmic vacuolization inhepatocytes, deterioratesmicrocirculation viavasoconstriction, facilitates theopsonization of damaged cells andincreases their sensitivity tophagocytosis. They lead to variousresults up to irreversible tissuedamage such as apoptosis andnecrosis(17,19-22).

There are many studiesindicating that inhibition of complement cascade decreases IRdamage and prevents fatal organdamage. In a study indicatingcomplement activation duringmyocardial reperfusion; classicalpathway of complement systeminhibited by C1 inhibitor andischemic myocardium wasprotected from reperfusiondamage effectively (21). Also in

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following studies about intestinesand kidney IR damage models ithas been noted that complementsystem plays the key role(21,23-34).

C1-INH belongs to serineinhibitor protease family and it isthe major inhibitory of classicalcomplement pathway (35-36).Many studies show that C1-INHdecreases the reperfusion relatedmicrocirculatory irregularity tominimum. C1-INH has been usedin several animal trials such assepsis and myocardial infract due

to its anti-inflammatory affects(35-41). In various IR models ithas been observed that 100-400IU/kg dose of C1-INH has aprotective affect on tissues(35). Inthe pilot study that we conductedprior to main study in order toshape the experimental model; wefound that >400 IU/kg C1-INHdose increases the subject

mortality. For that reason C1-INHdose was decided as 200 IU/kg. Inearlier studies it has been notedthat administration of C1-INH priorto ischemia had a better result inpreventing the tissue damagecaused by IR(39-42). For thatreason we administrated C1-INH(intravenous, 10min infusion) 20minutes before to ischemia. In a

study investigating therelationship between inhibition of classical pathway of complementsystem and liver damage; groupswere compared for albumin andC1-INH administration. In thestudy it has been noted that C1-INH administration leads toconstriction in all livermicrocirculatuary system and C1-INH prevents the affect of complement by attaching to

sinusoidal endothelium.Hepatocyte functions wereassessed at the 24th hour of reperfusion by ALT levels and bilesecretions; they were significantly

better in C1-INH receivedgroup(35). In anotherexperimental study ringer solutionand C1 esterase inhibitoryreceived(prior to ischemia) groupswere compared. Increased acinarcell perfusion, decreased sinusoidattached leukocytes andsignificantly decreased liverfunction tests (AST,ALT) were

observed in the study(41). In ourstudy there was no significantdifference in AST levels betweengroups(Graphic 3). ALT levelswere significantly lower in bothdrug received and non receivedgroups(Graphic 4)(p<0.05). Thisresults show that ALT is a betterindicator for assessing thehepatocyte functions when it

comes to decreasing the liverdamage via complement inhibitionafter IR.

There is no earlier studyinvestigating the anti-apoptoticaffect of C1-INH in the literature.All prior studies were experimentalstudies, performed on myocardialcells, vessel endothelium cells andskeletal muscle cells. In patients

with ischemic heart disease,particularly in patients who hadrecanalization treatment, it hasbeen showed that inflammatoryresponse leads to myocardial celldamage increasingly. In studiesinvestigating affect of complementsystem on cell damage andapoptosis; it has been showed invascular endothelial cell culturesthat C1-INH protects the

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endothelial cells to apoptosis(43-44).

In our study; there weresignificant differences betweengroup III and VIII, group I and VII

in TUNEL stains performed groupsto assess the apoptosis. Especiallyin Group VIII (C1-INH receivedprior to IR damage) hadsignificantly lower apoptosispercentage than group III(IRdamage only). This result is areflection of the drug’sefficacy(table 1 an Graphic 5).

Especially ischemia groups

had higher proportions for PCNAstaining(Group II and III). Thiscould be considered asregeneration response of cells toIR damage. Regeneration is aninflammatory process and resultsin an anti-inflammatory response.Lower proportions of PCNA in drugreceived groups might caused byanti-inflammatory effect of the

drug. In electron microscopyexaminations in groups without IRdamage, only partial liverresection performed group(groupI) and control group(group IV),there was no difference betweenC1-INH received and not receivedgroups for ultra-structuralappearance of hepatocytes. But in

IR damaged groups(II and III)increase in cytoplasmic lipiddroplets, significant mitochondrialdegeneration and vacuolization,granular endoplasmicreticulum(GER) dilatation andincrease in intracellular connectivetissue were observed in allhepatocytes. Cluster formationwas observed in nuclear chromatin(Figure 1,3). It is ascertained thathepatocytes were moving forward

to apoptosis. In complementinhibition provided groups via C1-INH administration; lack of cellularalterations as noted above wasconsidered as efficient protection

of nucleus and other organelles of hepatocyte. Evaluation of MDA inliver tissue indicates to freeoxygen radicals related lipidperoxidation thus cell damage(45-48). MDA results were assessedand it was found that MDA levelswere lower in drug receivedgroups than groups without drugadministration(p<0.05). This

result indicates that complementinhibition prevents or reduces thefree oxygen radicals relatedhepatocyte damage during IR.

One of intracellular GSHreduction reason is glutathionedepletion by conjugation during IRdamage. Glutathione demand iscovered by either biosynthesis inhepatocytes or exogenous GSH

intake. Reduction of intracellularGSH levels causes a cellularadaptive response, thus GSHbiosynthesis increases (49-51).

Lower levels of GSH levels inliver tissue in ischemiagroup(group II and III) may beconsidered as an indicator of mitochondrial damage due toincrease in intracellular reactive

oxygen metabolites at the latephase of IR. Furthermore;decreased GSH levels, depletion of mitochondrial and cytoplasmicglutathione are constitute theearly signal of apoptotic cell death(Graphic 1, Figure 1,3). Incomplement inhibition constitutedgroups prior to IR damage(GroupVI and Group VIII) GSH levelswere found higher. This resultmight arise from the protective

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affect of C1-INH on cells andparticularly on mitochondria viareducing the lipid peroxidationcaused by neutrophils andreducing the oxidative

stress(Graphic 1, Figure 2,4).

In conclusion; our results indicatesthat prophylactic C1-INHadministration (prior to IR) mightprovide hepatocyte damage viareducing the lipid peroxidation andoxidative stress. Furthermore thiseffect is provided by anti-inflammatory process; it increases

the cell generation and mostimportantly reduces the apoptosissignificantly.

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role of complement inhibitory

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