Role of Complement Inhibition in a Ischemic Reperfusion Induced Regeneration Model in Liver

Background: Assessment of C1-INH affect on regeneration and apoptosis in ischemia and reperfusion(IR) damage in liver tissue. Materials and Methods: 48 rats were included this study. Subjects were divided into 8 groups. Partial hepatectomy (PH) was performed in Group I rats. IR was constituted in Group II rats and followed by PH. Group III rats were exposed to only IR. Group IV was control group. Group V rats received only C1-INH. Group VI rats received C1-INH prior to IR; PH was performed 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 by electron microscopy, malondialdehyde(MDA) and reduced glutathione(GSH) evaluation were performed. Results: Statically significant difference was observed for apoptosis between group I and VII, group III and VIII, group IV and V. There was statically significant difference for PCNA between group II and VI, group III and VIII, group IV and V. Alanine aminotransferase levels were decreased in all drug received groups. Statically significant difference was found in MDA and GSH tissue analysis between group II and VI, group III and VIII. Conclusion: C1-INH increases the cell generation and significantly reduces apoptosis

Introduction:
Ischemia is lack of supplying oxygen and other metabolites to tissues by blood circulation and lack of waste product removal by blood circulation(1). Reperfusion is defined as reassuring blood circulation to these tissues. Reperfusion supplies the oxygen and metabolite demand of the ischemic tissue but on the contrary it causes tissue damage at the same time(2).Ischemia and reperfusion(IR) causes much more

damage than ischemia itself(3). Reperfusion damage following ischemia has been investigated in many studies for several organs such as lung, brain, liver, hearth and intestines (4-7). But physiopathology of the IR damage still remains unclear. It is known that major components like free radical formation, PNL lymphocyte activation, endothelial system and complement system play role in IR damage(8-9). This process leads to systemic inflammatory response and thus end organ damage. Major causes of liver ischemia are; 1

liver resection, hemorrhagic shock and traumatic liver damage. All three pathways of complement system are activated in IR damage(classical, alternative and lectin pathways). Complement-1 inhibitory(C1-INH) belongs to serine protease family and it is the major inhibitory of classical complement pathway(10). It protects the tissue from reperfusion damage. Reperfusion increases the complement activation in ischemic liver tissue. Inhibition of complement cascade decreases the post ischemic inflammation in liver tissue. In this study we aimed to investigate the affect of complement inhibition on regeneration and apoptosis via inhibition of complement system by C1-INH in liver tissue.

Materials and Methods:

This study was performed in Bakent University Experimental Research Center. The project had been approved by Ethical Committee of Animal Experiments prior to the study. All the surgical procedures performed during the study were proper with National Institutes of Health, Guide for the Care and Use of Laboratory Animals rules. 48 Wistar Albino, 200-250 g, female, randomly selected rats were used in this study. Animals were divided into 8 groups equally as 6 rats for each group. 50 mg/kg Ketamine Hydrochloride(Ketalar, Eczacba Warner-Lambert pharmaceutical industry, Levent, stanbul) and 7 mg/kg Xylazine Hydrochloride (Rompun, Bayer ili, stanbul)

were used for anesthesia under aseptic conditions. Partial hepatectomy was performed in Group I rats. IR was constituted in Group II rats via clamping the portal hilus for 45 minutes, followed by partial hepatectomy. In group III rats IR was constituted only. Group IV defined as control group. In Group V only C1-INH was administered. In Group VI rats received C1-INH prior to IR and after IR partial hepatectomy was performed. In Group VII rats received C1-INH and followed by partial hepatectomy. In group VIII IR was performed after 20 minutes of intravenous C1-INH injection. In order to compare the drug received and surgery performed groups with each other; the eight groups were compared as follow: Group I with Group VII, Group II with Group VI, Group III with Group VIII, Group IV with Group V. C1-INH 200IU/kg was administered to rats 10 minutes before to surgery. In order to compensate the fluid deficit caused by surgical procedure and drug administration each rat received 2 cc ringer lactate and 3 cc 5% dextrose intraperitoneally. Single dose 0.02 mg fentanyl was administered to all rats subcutaneously for post-operative pain. Left lateral segmentectomy was performed for partial hepatectomy. Ischemia was provided by microvascular clamp placing to portal peduncle and maintained for 45 minutes. Portal peduncle was clamped with bleeding control by dissection

microscope and ischemia was performed. Clamp was removed after 45 minutes of waiting period and reperfusion was observed by dissection microscope. All rats were sacrificed at the end of 5th post-surgical day. Before the sacrifice procedure 5 cc blood was taken from the inferior vena cava of all rats for cytokine analysis. Latter liver was excised and preserved for immunohistochemical and biochemical analysis. Reperfusion damage in liver tissue was analyzed with reduced glutathione (GHS) and malondialdehyde (MDA) levels. Liver function was assessed by aspartate aminotransferase (AST) and alanine aminotransferase (ALT) levels. Apoptosis and regeneration were assessed with electron microscopy and immunohistochemical methods. TUNEL (Transferase - mediated dUTP - biotin Nick End Labeling) method was used for histochemical assessment. 200 cells were counted in the light microscopy at the maximum magnification level(x40 HBF). Positive stained cells noted as percentage (%). An indicator of the cell proliferation PCNA (Proliferating Cell Nuclear Antigen) was assessed by light microscopy via 1000 cell counting under the maximum magnification (x40 HBF); positive cells were noted as percentage(%). SPSS 9.0 box program was used for data analysis. Standard deviation and mean values were calculated. For analyzing the non parametric values Independent Samples T test was used to

evaluate the differences of nominal values between groups. p<0.05 value was accepted as statically significant.

Results:
Same surgery procedure performed groups were compared to each other for apoptosis and statically significant differences were observed between Group I and VII, group III and VIII, group IV and V(p<0.05). Drug received and same surgery procedure performed groups were compared to each other for PCNA and statically significant differences were observed between group II and VI, group III and VIII, group IV and V(p<0.05). There was no statically significant difference between group I and VI, partial liver resection performed without IR. There was no statically significant difference for AST levels; but ALT levels were significantly lower in drug received groups(p<0.05) There were statically significant differences for MDA levels between group II and VI, group III and VIII(p<0.05)(Graphic 1). Statically significant differences were observed for GSH analysis between group II and VI, group III and VIII (p<0.05)(Graphic 2). Apoptosis, PCNA, ALT, MDA and GSH mean values were summarized in Table 1 for Group I/VII, Group II/VI, Group III/VIII and Group IV/V. Electron Microscopy Findings: Group I and VII: hepatectomy performed Partial groups

and drug received + partial hepatectomy performed groups were assessed and compared with each other. Nuclear structure, chromatin distribution, mitochondrion, granular endoplasmic reticulum (GER) and side-link units of cells were evaluated as normal in both groups. Figure 2: Hepatocyte unaffected from IR Group III and VIII: Ischemia and reperfusion resulted in increase of cytoplasmic lipid droplets (Figure 3), mitochondrial degeneration and vacuolization, GER dilatation and intracellular connective tissue increase in all hepatocytes. Controversially none of these findings were observed and intracellular connective tissue was normal in drug received and IR constituted group(Figure 4).

Figure 1: Deterioration in mitochondria membrane Group II and VI: In the ischemia and partial liver resection performed group some observations were noted as increase in cytoplasmic lipid droplets in some hepatocytes, deterioration in mitochondria membrane (Figure 1) and increase in intracellular connective tissue. In drug received latter IR and partial liver resection performed group it has been noted that hepatocytes were not affected by ischemia and reperfusion, and cell structure was normal like (Figure 2).

Figure 3: Lipid droplets in hepatocytes Group IV and V: Polygonal structure of the hepatocytes was maintained in both groups. Nuclear structure, chromatin distribution, nucleolus, mitochondrion, GER and intercellular connective tissue were normal in both groups.

Figure 4: Normal Hepatocyte

Discussion:
IR damage is characterized by temporary loss of tissue blood circulation and developing an important inflammatory response after reassuring the blood flow. Major liver ischemia causes are as follow: liver resection, traumatic liver damage and hemorrhagic shock. Reasons of developing organ dysfunction after the blood circulation loss are cell damage caused by reperfusion and increased organ sensitivity (1114). Factors that causes damage are; free oxygen radicals (ROS), leukocyte migration, leukocyte activation, sinusoidal endothelial cell damage, microcirculatory imbalances, coagulation system activation and complement system activation. Both classical and alternative complement pathways are activated in IR damage. Decay accelerating factor located in the cell membrane and membrane cofactor protein protect the cell to a complement attack. Complement depended proinflammatory peptides(C3a-C5a) are released during the reperfusion. These peptides leads to neutrophil

accumulation, smooth muscle contractions, increase in vascular permeability, Kupffer cell activation (11,15-16). At the beginning phase of IR damage; oxidative stress induced damage in Kupffer cells leads to production and release of reactive oxygen radicals. Complement products are required in this phase for Kupffer cell activation (17-20). Plasma levels of activated complement components increases in the late phase. Complement activation products (Anaphylatoxins and membrane attack complex components) are characterized by massive neutrophil infiltration and it can maintain for 12-24 hours after reperfusion. Kupffer cells and neutrophils causes increase in reactive oxygen radicals and proinflammatory cytokines, sinusoidal concession and cytoplasmic vacuolization in hepatocytes, deteriorates microcirculation via vasoconstriction, facilitates the opsonization of damaged cells and increases their sensitivity to phagocytosis. They lead to various results up to irreversible tissue damage such as apoptosis and necrosis(17,19-22). There are many studies indicating that inhibition of complement cascade decreases IR damage and prevents fatal organ damage. In a study indicating complement activation during myocardial reperfusion; classical pathway of complement system inhibited by C1 inhibitor and ischemic myocardium was protected from reperfusion damage effectively (21). Also in

following studies about intestines and kidney IR damage models it has been noted that complement system plays the key role(21,2334). C1-INH belongs to serine inhibitor protease family and it is the major inhibitory of classical complement pathway (35-36). Many studies show that C1-INH decreases the reperfusion related microcirculatory irregularity to minimum. C1-INH has been used in several animal trials such as sepsis and myocardial infract due to its anti-inflammatory affects (35-41). In various IR models it has been observed that 100-400 IU/kg dose of C1-INH has a protective affect on tissues(35). In the pilot study that we conducted prior to main study in order to shape the experimental model; we found that >400 IU/kg C1-INH dose increases the subject mortality. For that reason C1-INH dose was decided as 200 IU/kg. In earlier studies it has been noted that administration of C1-INH prior to ischemia had a better result in preventing the tissue damage caused by IR(39-42). For that reason we administrated C1-INH (intravenous, 10min infusion) 20 minutes before to ischemia. In a study investigating the relationship between inhibition of classical pathway of complement system and liver damage; groups were compared for albumin and C1-INH administration. In the study it has been noted that C1INH administration leads to constriction in all liver microcirculatuary system and C1INH prevents the affect of complement by attaching to

sinusoidal endothelium. Hepatocyte functions were th assessed at the 24 hour of reperfusion by ALT levels and bile secretions; they were significantly better in C1-INH received group(35). In another experimental study ringer solution and C1 esterase inhibitory received(prior to ischemia) groups were compared. Increased acinar cell perfusion, decreased sinusoid attached leukocytes and significantly decreased liver function tests (AST,ALT) were observed in the study(41). In our study there was no significant difference in AST levels between groups(Graphic 3). ALT levels were significantly lower in both drug received and non received groups(Graphic 4)(p<0.05). This results show that ALT is a better indicator for assessing the hepatocyte functions when it comes to decreasing the liver damage via complement inhibition after IR. There is no earlier study investigating the anti-apoptotic affect of C1-INH in the literature. All prior studies were experimental studies, performed on myocardial cells, vessel endothelium cells and skeletal muscle cells. In patients with ischemic heart disease, particularly in patients who had recanalization treatment, it has been showed that inflammatory response leads to myocardial cell damage increasingly. In studies investigating affect of complement system on cell damage and apoptosis; it has been showed in vascular endothelial cell cultures that C1-INH protects the

endothelial cells to apoptosis(4344). In our study; there were significant differences between group III and VIII, group I and VII in TUNEL stains performed groups to assess the apoptosis. Especially in Group VIII (C1-INH received prior to IR damage) had significantly lower apoptosis percentage than group III(IR damage only). This result is a reflection of the drugs efficacy(table 1 an Graphic 5). Especially ischemia groups had higher proportions for PCNA staining(Group II and III). This could be considered as regeneration response of cells to IR damage. Regeneration is an inflammatory process and results in an anti-inflammatory response. Lower proportions of PCNA in drug received groups might caused by anti-inflammatory effect of the drug. In electron microscopy examinations in groups without IR damage, only partial liver resection performed group(group I) and control group(group IV), there was no difference between C1-INH received and not received groups for ultra-structural appearance of hepatocytes. But in IR damaged groups(II and III) increase in cytoplasmic lipid droplets, significant mitochondrial degeneration and vacuolization, granular endoplasmic reticulum(GER) dilatation and increase in intracellular connective tissue were observed in all hepatocytes. Cluster formation was observed in nuclear chromatin (Figure 1,3). It is ascertained that hepatocytes were moving forward

to apoptosis. In complement inhibition provided groups via C1INH administration; lack of cellular alterations as noted above was considered as efficient protection of nucleus and other organelles of hepatocyte. Evaluation of MDA in liver tissue indicates to free oxygen radicals related lipid peroxidation thus cell damage(4548). MDA results were assessed and it was found that MDA levels were lower in drug received groups than groups without drug administration(p<0.05). This result indicates that complement inhibition prevents or reduces the free oxygen radicals related hepatocyte damage during IR. One of intracellular GSH reduction reason is glutathione depletion by conjugation during IR damage. Glutathione demand is covered by either biosynthesis in hepatocytes or exogenous GSH intake. Reduction of intracellular GSH levels causes a cellular adaptive response, thus GSH biosynthesis increases (49-51). Lower levels of GSH levels in liver tissue in ischemia group(group II and III) may be considered as an indicator of mitochondrial damage due to increase in intracellular reactive oxygen metabolites at the late phase of IR. Furthermore; decreased GSH levels, depletion of mitochondrial and cytoplasmic glutathione are constitute the early signal of apoptotic cell death (Graphic 1, Figure 1,3). In complement inhibition constituted groups prior to IR damage(Group VI and Group VIII) GSH levels were found higher. This result might arise from the protective

affect of C1-INH on cells and particularly on mitochondria via reducing the lipid peroxidation caused by neutrophils and reducing the oxidative stress(Graphic 1, Figure 2,4). In conclusion; our results indicates that prophylactic C1-INH administration (prior to IR) might provide hepatocyte damage via reducing the lipid peroxidation and oxidative stress. Furthermore this effect is provided by antiinflammatory process; it increases the cell generation and most importantly reduces the apoptosis significantly.