Orientadores: Ilka de Fátima Santana Ferreira Boin, Artur Udelsmann / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas / Made available in DSpace on 2018-08-27T00:05:18Z (GMT). No. of bitstreams: 1 Jorge_GracindadeLourdes_D.pdf: 6636878 bytes, checksum: 9167ec95670521d52d8c88a19fc3ceb9 (MD5) Previous issue date: 2015 / Resumo: A oclusão vascular temporária do fluxo hepático é um dos procedimentos essenciais nas cirurgias hepáticas. Muitas pesquisas relacionadas à isquemia e reperfusão (I/R) em ratos Wistar, seja através de interrupção por pouco tempo, ou período maior, têm demonstrado sérias complicações causadas por lesões de I/R. Pesquisas demonstram grandes alterações metabólicas e fisiológicas quando analisadas durante, ou nas primeiras horas após o experimento. O objetivo deste trabalho foi avaliar as alterações hepáticas morfológicas e bioquímicas tardias, ocorridas após pinçamento total ou parcial do hilo hepático em ratos Wistar. Um total de 26 ratos Wistar, machos, peso médio 315,5 ± 61,5g foram utilizados em dois estudos. No primeiro estudo, 12 ratos foram divididos em dois grupos: Grupo Pinçamento do Ducto Biliar Comum (PDBC; n=6) foram submetidos à anestesia com tiopental sódico iv, à incisão abdominal até 2cm, tendo o ducto biliar isolado, dissecado e pinçado por 10 minutos. Após este tempo, a pinça foi retirada e a incisão fechada. Grupo Operação Simulada I (OSI; n=6) em condições de normalidade, os animais foram submetidos unicamente à anestesia e laparotomia e, posteriormente, a exames de controle. Nos dois grupos após o 28ºdia foram realizadas biópsias hepáticas e exames bioquímicos seguidos de eutanásia dos animais. Observamos que 83% dos animas do grupo PDBC apresentaram dilatação do colédoco, com alterações histológicas hepáticas: proliferação ductular, formação de septos, focos de necrose do parênquima, com formação de micro abcessos e alterações dos exames bioquímicos, quando comparados aos animais do grupo OSI (p < 0,05). No segundo estudo, 14 animais foram anestesiados com ketamina 5% (30mg/Kg) e xylazina 2% (30mg/Kg) via intraperitoneal. No grupo clampeamento intermitente do pedículo hepático (CIPH; n=7) os animais foram submetidos à incisão em U no abdome; o pedículo hepático foi isolado, dissecado e submetido a pinçamento, com micro pinça, intermitente por 4 ciclos de 5 minutos de isquemia, seguidos de 5 minutos de reperfusão, e a incisão foi fechada. No Grupo Operação Simulada II (OSII; n=7) os animais foram submetidos à anestesia, laparotomia e manipulação do pedículo hepático e,posteriormente, ao controle dos exames. Em todos os animais no 35o dia, após jejum de 12 horas, foi realizada nova anestesia para coleta da biópsia hepática e sangue para dosagem de alanina amino transferase (ALT) e de aspartato amino transferase (AST). A análise estatística foi realizada pelo teste Mann-Whitney para comparação de médias com significância de 5%. Observamos que todos os animais do grupo CIPH apresentaram dilatação do colédoco e aumento significativo nas enzimas hepáticas (p<0,05). Na avaliação histológica constatamos proliferação ductular (100% dos casos), septos porta-porta (42,8%), formação de nódulos (42,8%), focos de necrose (14,2%) e rolhas de bile (14,2%). No grupo OSII estas alterações não foram encontradas. Concluímos que o pinçamento do ducto biliar (10 minutos) foi suficiente para gerar importantes alterações morfológicas hepáticas e do colédoco, confirmadas através de análise enzimática e histológica, podendo, portanto, ser utilizado como modelo de obstrução biliar, visando estudos semelhantes. Constatamos, também, que o pinçamento intermitente do pedículo hepático provocou lesões semelhantes na árvore biliar e no parênquima hepático / Abstract: The temporary vascular occlusion of the hepatic blood flow is one of the essential procedures in hepatic surgery. Many researches are related to ischemia and reperfusion (I/R) in rats, either through interruption for a short time period or higher and all has shown serious complications caused by I/R injury. Researches have demonstrated, in the early analysis severe metabolic and physiological disorders but there are not reports about hepatic injuries after late analysis. The objective was to assess the morphological and biochemical hepatic late alterations occurring after total or partial hepatic pedicle clamping, in Wistar rats. A total of 26 male Wistar rats, weighting 315.6 ±61.9g were used in two studies. In the first, 12 rats were distributed into two groups: bile duct clamping group (BDCG; n = 6); anesthetized with thiopental sodium; with bile duct dissection, isolation and clamped for 10 minutes. After this time, the clamp was removed and the incision was closed. In simulated operation group I (SOGI; n = 6) the animals were submitted solely to anesthesia and laparotomy and subsequently control exams.On the 28th day, liver biopsies and biochemical exams were performed. All animals were sacrificed while still under anesthesia. We observed that 83% of the animals of BDCG group showed dilation of the common bile duct with hepatic histological changes such as ductular proliferation, septa formation, parenchymal foci of necrosis with formation of micro abscesses and changes of biochemical tests when compared to SOGI (p<0.05).In the second study, 14 animals were anesthetized with ketamine 5% (30mg/kg) and xylazine 2% (30mg/kg)intraperitoneally. In intermittent Hepatic Pedicle Group (IHPC; n = 7) the animals had the hepatic pedicle isolated, dissected and submitted to clamping applying 4 cycles of 5 minutes of ischemia followed by 5 minutes of reperfusion, and the incision was closed.In group operation simulated II (SOGII; n = 7), the animals were anesthetized, laparotomy and manipulation of the hepatic pedicle were performed. On the 35th day, after 12 hours fasting, anesthesia wasinduced for liver biopsy and serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) collection. The Mann-Whitney test for comparison of means with significance of 5% was used. In result all the animals of IHPC group had a dilated common bile duct and the significant increase in hepatic enzymes (p <0.05). In histological evaluation we found ductular proliferation (100% of cases), septa port-portal (42.8%), nodules formation (42.8%), foci of necrosis (14.2%) and bile plugs (14.2%). In the group SOGII these changes were not found. We conclude that the short clamping time of the bile duct (for 10 minutes) was enough to cause major hepatic and bile duct morphological changes confirmed by enzymatic and histological analysis, and may therefore be used as biliary obstruction model in order to similar studies. We also noted that the intermittent clamping of the hepatic pedicle caused similar injuries in the biliary tree and in the hepatic parenchyma / Doutorado / Fisiopatologia Cirúrgica / Doutora em Ciências

Lesão hepática é uma das complicações decorrentes de pancreatite aguda (PA) e mostra uma correlação positiva com a gravidade da doença. Recentemente foi demonstrado que tratamento com solução hipertônica (SH) reduz inflamação e mortalidade na PA. O presente trabalho investigou os efeitos de SH na modulação do estresse oxidativo, apoptose e remodelamento tecidual no fígado durante a PA. Ratos machos foram divididos em quatro grupos: C animais que não foram submetidos à PA ou tratamento; past animais submetidos à indução de PA e que não receberam tratamento; pas animais submetidos à PA e tratados com solução fisiológica (NaCl 0.9%); pah animais submetidos à indução de PA e que receberam SH (NaCl 7.5%). PA foi induzida pela injeção retrógrada de ácido taurocólico (2.5%) no ducto bilo-pancreático. Após 4, 12 e 24 horas os animais foram sacrificados para coleta das amostras de interesse (fígado e plasma) submetidas a ensaios para análise de expressão (western blot e PCR) de Óxido Nítrico Sintase induzível (iNOS), metaloproteinases (MMP) -2 e -9, Heat Schock Protein (HSP) 47, 60, 70 e 90, colágenos tipos I e III, caspases 2 e 9, APAF-1 e AIF, formação de nitrotirosina (imunohistoquímica), peroxidação lipídica (TBARs), atividade de alaninoaminotransferase (ALT), produção de nitrito/nitrato (Reação de Griess) e das citocinas TNF-, IL-1, IL-6 e IL-10 (ELISA). O tratamento com SH reduziu significantemente o estresse oxidativo hepático com a redução da expressão gênica de iNOS (p<0,01 vs. pas), dos níveis de nitrito/nitrato (P<0,01 vs. pas), liberação de ALT (p<0,01 vs. pas) e inibição da formação de peroxinitrito após 12 horas da indução de PA. Conseqüentemente, a expressão de HSP70 não foi ativada devido à proteção hepática causada pela administração de SH. A expressão e atividade de MMP-9 aumentaram significativamente nos grupos pas e past, entretanto o tratamento com SH manteve os níveis basais (p<0,05 vs. past, pas). Aumento da expressão de HSP47 e alterações na expressão de colágeno indicaram intenso remodelamento de matriz extracelular nos grupos não tratados ou tratados com solução fisiológica, permanecendo semelhante ao controle no grupo pah. Não ocorreram mudanças significativas na expressão das proteínas envolvidas no processo apoptótico. A SH diminui o estresse oxidativo no período crítico da PA, resultando na diminuição da lesão hepática e do remodelamento tecidual, mantendo dessa forma a integridade de matriz extracelular / It has been shown an hepatic injury following pancreatitis and a positive correlation with severity of the disease. Hypertonic Solution (HS) reduced morbidity and mortality in experimental pancreatitis. We hypothesize that hypertonic solution resuscitation of acute pancreatitis (AP) may exert antiinflammatory effects by modulating hepatic oxidative stress, apoptosis and matrix extracellular remodeling in liver. Wistar rats were divided in four groups: C- control animals not subjected to insult or treatment; NT- subjected to pancreatitis induction and receiving no treatment; NS- subjected to pancreatitis induction and receiving normal saline (0.9% NaCl); HSsubjected to pancreatitis induction and receiving hypertonic saline (7.5% NaCl). AP was induced by retrograde infusion of 2,5% sodium taurocholate into the pancreatic duct transduodenally. At 4, 12 and 24 h following pancreatitis induction, liver tissue samples were assayed in order to analyse expression of metalloproteinases (MMPs) -2 and -9, iNOS, collagens (type I and III), Heat Shock Proteins (HSPs) 47, 60, 70 and 90, caspases -2 and -7, APAF-1 and AIF, production of the cytokines TNF-, IL-1, IL-6 and IL-10, Nitrite/nitrate and ALT, Lipid peroxidation and formation of Nytrotirosine. Hypertonic solution resuscitation significantly modulates the oxidative stress in liver by reduction of iNOS gene expression (p<0,01 vs. NS), nitrite and nitrate levels (p<0,01 vs. NS), lipid peroxidation (p<0,05 vs. NT), ALT release (p<0,01 vs. NS) and peroxinitrite inhibition after 12 hours of pancreatitis induction. Consequently, the HSP70 production has not been activated due to the hypertonic solution effect in hepatic protection. At 4 h and 12 h, MMP-9 expression and activity increased in the NS and NT groups, although remaining at basal levels in the HS group (p<0.05 vs. past, pas). At 12 h, MMP-2 expression increased in the NS group (p<0.05 vs. c) but not in the HS group. At 4 h after pancreatitis induction, HSP47 expression increased in the NS and NT groups. Greater extracellular matrix remodelling occurred in the NS and NT groups than in the HS group, probably as a result of the hepatic wound-healing response to repeated injury. However, the collagen content in hepatic tissue remained at basal levels in the HS group. The proteins involved in apoptosis remained unchanged in all groups. Hypertonic saline is hepatoprotective, since it decreases oxidative stress in the critical time resulting in diminished liver damage, reducing hepatic remodelling, maintaining the integrity of the hepatic extracellular matrix during pancreatitis. Hypertonic saline-mediated regulation of MMP expression might have clinical relevance in pancreatitis-associated liver injury

Lesão hepática é uma das complicações decorrentes de pancreatite aguda (PA) e mostra uma correlação positiva com a gravidade da doença. Recentemente foi demonstrado que tratamento com solução hipertônica (SH) reduz inflamação e mortalidade na PA. O presente trabalho investigou os efeitos de SH na modulação do estresse oxidativo, apoptose e remodelamento tecidual no fígado durante a PA. Ratos machos foram divididos em quatro grupos: C animais que não foram submetidos à PA ou tratamento; past animais submetidos à indução de PA e que não receberam tratamento; pas animais submetidos à PA e tratados com solução fisiológica (NaCl 0.9%); pah animais submetidos à indução de PA e que receberam SH (NaCl 7.5%). PA foi induzida pela injeção retrógrada de ácido taurocólico (2.5%) no ducto bilo-pancreático. Após 4, 12 e 24 horas os animais foram sacrificados para coleta das amostras de interesse (fígado e plasma) submetidas a ensaios para análise de expressão (western blot e PCR) de Óxido Nítrico Sintase induzível (iNOS), metaloproteinases (MMP) -2 e -9, Heat Schock Protein (HSP) 47, 60, 70 e 90, colágenos tipos I e III, caspases 2 e 9, APAF-1 e AIF, formação de nitrotirosina (imunohistoquímica), peroxidação lipídica (TBARs), atividade de alaninoaminotransferase (ALT), produção de nitrito/nitrato (Reação de Griess) e das citocinas TNF-, IL-1, IL-6 e IL-10 (ELISA). O tratamento com SH reduziu significantemente o estresse oxidativo hepático com a redução da expressão gênica de iNOS (p<0,01 vs. pas), dos níveis de nitrito/nitrato (P<0,01 vs. pas), liberação de ALT (p<0,01 vs. pas) e inibição da formação de peroxinitrito após 12 horas da indução de PA. Conseqüentemente, a expressão de HSP70 não foi ativada devido à proteção hepática causada pela administração de SH. A expressão e atividade de MMP-9 aumentaram significativamente nos grupos pas e past, entretanto o tratamento com SH manteve os níveis basais (p<0,05 vs. past, pas). Aumento da expressão de HSP47 e alterações na expressão de colágeno indicaram intenso remodelamento de matriz extracelular nos grupos não tratados ou tratados com solução fisiológica, permanecendo semelhante ao controle no grupo pah. Não ocorreram mudanças significativas na expressão das proteínas envolvidas no processo apoptótico. A SH diminui o estresse oxidativo no período crítico da PA, resultando na diminuição da lesão hepática e do remodelamento tecidual, mantendo dessa forma a integridade de matriz extracelular / It has been shown an hepatic injury following pancreatitis and a positive correlation with severity of the disease. Hypertonic Solution (HS) reduced morbidity and mortality in experimental pancreatitis. We hypothesize that hypertonic solution resuscitation of acute pancreatitis (AP) may exert antiinflammatory effects by modulating hepatic oxidative stress, apoptosis and matrix extracellular remodeling in liver. Wistar rats were divided in four groups: C- control animals not subjected to insult or treatment; NT- subjected to pancreatitis induction and receiving no treatment; NS- subjected to pancreatitis induction and receiving normal saline (0.9% NaCl); HSsubjected to pancreatitis induction and receiving hypertonic saline (7.5% NaCl). AP was induced by retrograde infusion of 2,5% sodium taurocholate into the pancreatic duct transduodenally. At 4, 12 and 24 h following pancreatitis induction, liver tissue samples were assayed in order to analyse expression of metalloproteinases (MMPs) -2 and -9, iNOS, collagens (type I and III), Heat Shock Proteins (HSPs) 47, 60, 70 and 90, caspases -2 and -7, APAF-1 and AIF, production of the cytokines TNF-, IL-1, IL-6 and IL-10, Nitrite/nitrate and ALT, Lipid peroxidation and formation of Nytrotirosine. Hypertonic solution resuscitation significantly modulates the oxidative stress in liver by reduction of iNOS gene expression (p<0,01 vs. NS), nitrite and nitrate levels (p<0,01 vs. NS), lipid peroxidation (p<0,05 vs. NT), ALT release (p<0,01 vs. NS) and peroxinitrite inhibition after 12 hours of pancreatitis induction. Consequently, the HSP70 production has not been activated due to the hypertonic solution effect in hepatic protection. At 4 h and 12 h, MMP-9 expression and activity increased in the NS and NT groups, although remaining at basal levels in the HS group (p<0.05 vs. past, pas). At 12 h, MMP-2 expression increased in the NS group (p<0.05 vs. c) but not in the HS group. At 4 h after pancreatitis induction, HSP47 expression increased in the NS and NT groups. Greater extracellular matrix remodelling occurred in the NS and NT groups than in the HS group, probably as a result of the hepatic wound-healing response to repeated injury. However, the collagen content in hepatic tissue remained at basal levels in the HS group. The proteins involved in apoptosis remained unchanged in all groups. Hypertonic saline is hepatoprotective, since it decreases oxidative stress in the critical time resulting in diminished liver damage, reducing hepatic remodelling, maintaining the integrity of the hepatic extracellular matrix during pancreatitis. Hypertonic saline-mediated regulation of MMP expression might have clinical relevance in pancreatitis-associated liver injury

Yang Lei. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2001. / Includes bibliographical references (leaves 141-148). / Abstracts in English and Chinese. / Acknowledgements --- p.i / Abstract --- p.ii / Abstract (Chinese Version) --- p.iv / Table of Contents --- p.vi / List of Abbreviations --- p.xii / List of Figures --- p.xiii / List of Tables --- p.xviii / Chapter Chapter 1 --- Literature review --- p.1 / Chapter 1.1 --- Carbon tetrachloride (CC14) --- p.1 / Chapter 1.2 --- Major uses of CC14 --- p.1 / Chapter 1.3 --- Potential human exposure pathways to CC14 --- p.2 / Chapter 1.4 --- Toxicity of CC14 --- p.3 / Chapter 1.5 --- Mechanism of CCl4-induced hepatotoxicity --- p.5 / Chapter 1.6 --- Role of CYP2E1 involved in CCl4-induced hepatotoxicity --- p.7 / Chapter 1.7 --- Definite proof of the involvement of CYP2E1 in CCl4-induced hepatotoxicity by CYP2El-null mouse in vivo model --- p.10 / Chapter 1.8 --- Identification of CYP2E1 -dependent genes involved in CCl4-induced hepatotoxicity by fluorescent differential display (FDD) --- p.11 / Chapter 1.9 --- Objectives of the study --- p.14 / Chapter Chapter 2 --- Materials and methods --- p.16 / Chapter 2.1 --- Animals and treatments --- p.16 / Chapter 2.1.1 --- Materials --- p.16 / Chapter 2.1.2 --- Methods --- p.16 / Chapter 2.2 --- Serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) analyses --- p.17 / Chapter 2.2.1 --- Materials --- p.17 / Chapter 2.2.2 --- Methods --- p.17 / Chapter 2.2.2.1 --- Serum preparation --- p.17 / Chapter 2.2.2.2 --- Activity determination --- p.18 / Chapter 2.3 --- Tail-genotyping by PCR --- p.18 / Chapter 2.3.1 --- Materials --- p.18 / Chapter 2.3.2 --- Methods --- p.20 / Chapter 2.3.2.1 --- Preparation of genomic DNA from mouse tail --- p.20 / Chapter 2.3.2.2 --- PCR reaction --- p.20 / Chapter 2.4 --- Total RNA isolation --- p.21 / Chapter 2.4.1 --- Materials --- p.21 / Chapter 2.4.2 --- Methods --- p.21 / Chapter 2.5 --- DNase I treatment --- p.23 / Chapter 2.5.1 --- Materials --- p.23 / Chapter 2.5.2 --- Methods --- p.23 / Chapter 2.6 --- Reverse transcnption of mRNA and amplification by fluorescent PCR amplification --- p.26 / Chapter 2.6.1 --- Materials --- p.27 / Chapter 2.6.2 --- Methods --- p.27 / Chapter 2.7 --- Fluorescent differential display (FDD) --- p.28 / Chapter 2.7.1 --- Materials --- p.28 / Chapter 2.7.2 --- Methods --- p.28 / Chapter 2.8 --- Excision of differentially expressed cDNA fragments --- p.29 / Chapter 2.8.1 --- Materials --- p.29 / Chapter 2.8.2 --- Methods --- p.29 / Chapter 2.9 --- Reamplification of differentially expressed cDNA fragments --- p.34 / Chapter 2.9.1 --- Materials --- p.34 / Chapter 2.9.2 --- Methods --- p.34 / Chapter 2.10 --- Subcloning of reamplified cDNA fragments --- p.36 / Chapter 2.10.1 --- Materials --- p.36 / Chapter 2.10.2 --- Methods --- p.37 / Chapter 2.11 --- Purification of plasmid DNA from recombinant clones --- p.39 / Chapter 2.11.1 --- Materials --- p.39 / Chapter 2.11.2 --- Methods --- p.39 / Chapter 2.12 --- DNA sequencing of differentially expressed cDNA fragments --- p.40 / Chapter 2.12.1 --- Materials --- p.40 / Chapter 2.12.2 --- Methods --- p.40 / Chapter 2.12.3 --- BLAST search against the GenBank DNA databases --- p.41 / Chapter 2.13 --- Northern blot analysis of differentially expressed cDNA fragments --- p.41 / Chapter 2.13.1 --- Formaldehyde gel electrophoresis of total RNA --- p.41 / Chapter 2.13.1.1 --- Materials --- p.42 / Chapter 2.13.1.2 --- Methods --- p.42 / Chapter 2.13.2 --- Preparation of cDNA probes for hybridization --- p.42 / Chapter 2.13.2.1 --- EcoRI digestion of cDNA inserts from plasmid DNA --- p.42 / Chapter 2.13.2.1.1 --- Materials --- p.42 / Chapter 2.13.2.1.2 --- Methods --- p.43 / Chapter 2.13.2.2 --- Purification of DNA from agarose gel --- p.43 / Chapter 2.13.2.2.1 --- Materials --- p.43 / Chapter 2.13.2.2.2 --- Methods --- p.43 / Chapter 2.13.2.3 --- DIG labeling of cDNA --- p.44 / Chapter 2.13.2.3.1 --- Materials --- p.44 / Chapter 2.13.2.3.2 --- Methods --- p.44 / Chapter 2.13.3 --- Hybridization --- p.45 / Chapter 2.13.3.1 --- Materials --- p.45 / Chapter 2.13.3.2 --- Methods --- p.45 / Chapter Chapter 3 --- Results --- p.47 / Chapter 3.1 --- Liver morphology --- p.47 / Chapter 3.2 --- Serum ALT and AST activities --- p.47 / Chapter 3.3 --- Tail-genotyping by PCR --- p.51 / Chapter 3.4 --- DNase I treatment --- p.51 / Chapter 3.5 --- FDD RT-PCR and excision of differentially expressed cDNA fragments --- p.51 / Chapter 3.6 --- Reamplification of excised cDNA fragments --- p.61 / Chapter 3.7 --- Subcloning of reamplified cDNA fragments --- p.61 / Chapter 3.8 --- DNA sequencing of subcloned cDNA fragments --- p.69 / Chapter 3.9 --- Confirmation of differentially expressed patterns by Northern blot analysis --- p.106 / Chapter 3.10 --- Temporal expression of differentially expressed genes --- p.113 / Chapter 3.11 --- Tissue distribution of differentially expressed genes --- p.117 / Chapter Chapter 4 --- Discussion --- p.125 / Chapter 4.1 --- Liver morphology and serum ALT and AST activities --- p.126 / Chapter 4.2 --- Identification of CYP2E1 -dependent genes involved in CCl4-induced hepatotoxicity --- p.127 / Chapter 4.3 --- Functional roles of the identified differentially expressed genes --- p.129 / Chapter 4.3.1 --- Fragment B4 --- p.129 / Chapter 4.3.2 --- Fragment C12 --- p.130 / Chapter 4.3.3 --- Fragment B13 --- p.131 / Chapter 4.3.4 --- Fragment A5 --- p.132 / Chapter 4.4 --- Temporal expression of differentially expressed genes --- p.133 / Chapter 4.4.1 --- Fragment B4 --- p.133 / Chapter 4.4.2 --- Fragment C12 --- p.134 / Chapter 4.4.3 --- Fragment B13 --- p.134 / Chapter 4.4.4 --- Fragment A5 --- p.135 / Chapter 4.5 --- Tissue distribution of differentially expressed genes --- p.136 / Chapter 4.5.1 --- Fragment B4 --- p.136 / Chapter 4.5.2 --- Fragment C12 --- p.136 / Chapter 4.5.3 --- Fragment B13 --- p.137 / Chapter 4.5.4 --- Fragment A5 --- p.137 / Chapter 4.5.5 --- Roles of the identified genes involved in CCl4-induced hepatotoxicity --- p.138 / Chapter 4.6 --- Normalization of Northern blot analysis --- p.13 8 / Chapter 4.7 --- Limitations of FDD technique to identify differentially expressed genes --- p.138 / Chapter 4.8 --- Future studies --- p.139 / Chapter 4.8.1 --- Investigation of the differential expression patterns of the identified genes in acetaminophen-induced liver injury --- p.139 / Chapter 4.8.2 --- Dot blot analysis --- p.140 / Chapter 4.8.3 --- DNA microarray --- p.140 / References --- p.141