Hypothermic Treatment Attenuates Liver Damage Following Asphyxial Cardiac Arrest In Rats Through Regulation Of Inflammation And Oxidative Stress = 치료적 저체온 요법을 통한 질식성 심정지 랫드 모델에서 염증 및 산화적 스트레스 조절을 통한 간 손상 완화

다국어 초록 (Multilingual Abstract)

Hypothermic treatment is known to protect against cardiac arrest (CA) including improving survival rate. However, little study has evaluated CA-induced liver damages and effects of hypothermia on the damages. This study, therefore, was designed to determine possible protective effects of hypothermia on the liver after asphyxial CA (ACA). Rats were subjected to 5-minute ACA followed by return of spontaneous circulation (ROSC). Body temperature was controlled at 37 ± 0.5℃ (normothermia group) or 33 ± 0.5℃ (hypothermia group) for 4 h after ROSC. Livers were examined at 6 h, 12 h, 24 h and 2 days, respectively, after ROSC. Histopathological examination was performed by hematoxylin and eosin staining, and changes of pro-inflammatory cytokines (tumor necrosis factor (TNF-α) and interleukin (IL)-2) and anti-inflammatory cytokines (IL-4 and IL-13) expressions were investigated by immunohistochemistry. For oxidative stress and antioxidant effects, superoxide anion radical production was evaluated using dihydroethidium fluorescence straining, and endogenous antioxidants (superoxide dismutase 1 (SOD1) and superoxide dismutase 2 (SOD2)) were examined using immunohistochemistry. In histopathology examination, sinusoidal dilatation and vacuolization were shown after ACA, however, the CA-induced structural alterations were prevented by hypothermia. In immunohistochemical examination, expressions of pro-inflammatory cytokines were reduced in the hypothermia group compared with those in the normothermia group, and expressions of anti-inflammatory cytokines have been increased in the hypothermia group compared with the normothermia group. In conclusion, hypothermic treatment for 4 h following ACA in rats inhibited increases of pro-inflammatory cytokines and stimulated anti-inflammatory cytokines expression. Increased numbers of mast cells in the normothermia group with ACA were significantly reduced in the hypothermia group with ACA. Gradual increase in superoxide anion radical production was found with time in the normothermia group with ACA, but the production was significantly suppressed in the hypothermia group with ACA. Immunoreactivities of SOD1 and SOD2 were increased in the hypothermia group with ACA compared to those in the normothermia group. This study suggests that hypothermic treatment after ACA events has an ability to reduce liver damages through regulation of inflammation, oxidative stress and expressions of antioxidant enzymes.

□ Keywords
Antioxidants, Anti-inflammatory cytokines, Asphyxial cardiac arrest, Liver, Oxidative stress, Therapeutic hypothermia, Pro-inflammatory cytokines

국문 초록 (Abstract)

치료적 저체온 요법은 생존율 개선을 포함하여 심정지로 인한 손상을 보호하는 것으로 알려져 있다. 하지만, 치료적 저체온 요법 적용함으로써 심정지에 따른 간 손상 완화 기전에 관한 연구는 미비한 실정이다. 이에 따라, 본 연구에서는 심정지 랫드 모델에서 치료적 저체온 요법을 적용하여 간 손상이 완화되는 것을 확인하였다. 본 연구에서는 랫드를 이용하여 5분 동안 질식성 심정지 유발 후, 자발적 순환 회복 (return of spontaneous circulation, RoSC)을 유도하는 방법으로 심정지 모델을 정립하였다. 치료적 저체온 요법은 RoSC 직후 4시간 동안 실험동물의 체온을 33 ± 0.5℃로 유지함으로써 처치하였고, 간 조직의 조직학적 분석은 RoSC 후 각각 6시간, 12시간, 1일 및 2일에 수행하였다. 조직 병리학적 분석을 위해 hematoxylin and eosin 염색하였고, 전염증성 사이토카인 (tumor necrosis factor α, TNF- α; interleukin 1β, IL-1 β) 및 항염증성 사이토카인 (interleukin 4, IL-4; interleukin 13, IL-13)을 관찰하기위하여 면역조직 화학 염색을 수행하였다. 또한, 산화적 스트레스 및 항산화 효소의 변화를 확인하기 위하여 dihydroethidium (DHE) 조직 형광 염색 및 초과산화물 불균등화 효소 (superoxide dismutases, SODs)에 대한 면역조직 화학 염색을 수행하였다. 심정지 유발후에 조직 병리학적인 변화로 간 조직의 혈관 팽창 및 공포 형성이 관찰되었고, 이들은 치료적 저체온 처치로 완화되는 것이 확인되었다. 더 나아가, 정상 체온 군에 비하여, 저체온 처치를 한 실험동물의 간 조직에서 심정지 유발에 따른 전염증성 사이토카인의 면역반응성이 유의하게 감소하였으며, 항염증성 사이토카인의 면역반응성은 유의하게 증가한 것을 확인하였다. 또한, 정상 체온 군에 비하여, 저체온 처치를 한 실험동물의 간 조직에서 심정지 유발에 따른 초과산화물 음이온 생성이 유의하게 감소하고, 내인성 항산화 효소의 면역반응성은 유의하게 증가한 것을 확인하였다. 본 연구 결과를 토대로, 치료적 저체온 요법 적용은 염증성 사이토카인의 조절, 산화적 스트레스 완화 및 항산화 효소 증진을 통하여 심정지로 인한 간 손상을 완화할 수 있는 치료적 전략으로 사용될 수 있음을 시사한다.

□ 핵심주제어
항산화, 항염증성 사이토카인, 질식성 심정지, 간, 산화적 스트레스, 치료적 저체온 요법, 전염증성 사이토카인

목차 (Table of Contents)

• CONTENTS
• Part. I: Effects of hypothermia on inflammatory cytokine expression in rat liver following asphyxial cardiac arrest 1
• 1. INTRODUCTION 1
• 2. MATERIALS AND METHODS 4
• 2.1. Experimental animals and groups 4
• 2.2. CA induction and cardiopulmonary resuscitation (CPR) 6
• 2.3. Hypothermic treatment 8
• 2.5. Hematoxylin and eosin (H and E) staining 10
• 2.6. Immunohistochemistry 11
• 2.7. Statistical analysis 13
• 3. RESULTS 14
• 3.1. Physiological variables and survival rate 14
• 3.2. H and E staining 16
• 3.3. TNF-α immunoreactivity 19
• 3.4. IL-2 immunoreactivity 22
• 3.5. IL-4 immunoreactivity 25
• 3.5.2. IL-13 immunoreactivity 28
• 4. DISCUSSION 31
• 5. REFERENCES 36
• Part. II: Therapeutic hypothermia reduces inflammation and oxidative stress in the liver following asphyxial cardiac arrest in rats 43
• 1. INTRODUCTION 43
• 2. MATERIALS AND METHODS 47
• 2.1. Experimental Animals and Groups 47
• 2.2. ACA Induction and Cardiopulmonary Resuscitation (CPR) 49
• 2.3. Hypothermia Treatment 52
• 2.4. Liver Tissue Preparation 53
• 2.5. Toluidine Blue Staining 54
• 2.6. Measurement of Superoxide Anion 55
• 2.7. Immunohistochemistry 56
• 2.8. Statistical Analysis 57
• 3. RESULTS 58
• 3.1. Mast Cells 58
• 3.2. DHE Fluorescence 61
• 3.3. SOD1 Immunoreactivity 64
• 3.4. SOD2 Immunoreactivity 67
• 4. DISCUSSION 70
• 5. REFERENCES 75
• FIGURE CONTENTS
• Part. I: Effects of hypothermia on inflammatory cytokine expression in rat liver following asphyxial cardiac arrest 1
• Fig. 1. 18
• Fig. 2. 21
• Fig. 3. 24
• Fig. 4. 27
• Fig. 5. 30
• Part. II: Therapeutic hypothermia reduces inflammation and oxidative stress in the liver following asphyxial cardiac arrest in rats 43
• Fig. 1. 60
• Fig. 2. 63
• Fig. 3. 66
• Fig. 4. 69
•  
• TABLE CONTENTS
• Part. I: Effects of hypothermia on inflammatory cytokine expression in rat liver following asphyxial cardiac arrest 1
• Table 1. 15
• Part. II: Therapeutic hypothermia reduces inflammation and oxidative stress in the liver following asphyxial cardiac arrest in rats 43
• Table 1. 51
•