الفهرس 
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Abstract
Summary and Conclusion
The present work was designed to investigate the possible hepatic and cardiac functional and structural changes, as well as the cardiac responses to its in vitro ischemia-reperfusion injury (IR) in a rat model of hepatic IR injury. Also, to evaluate the potential effect and mechanism(s) of exercise training and oxytocin, either each alone or together, in comparison to, L-arginine, the nitric oxide donor, in protecting the liver and heart function and structure against the assumed liver IR-induced impairment.
The study was carried out on 62 adult male albino rats, which were allocated into the following 6 groups:
group I: Sham-Operated Control group (Sham group) (n=10): Rats in this group were kept undisturbed in their cages for 3 weeks. In the fourth week, rats received 2 ml/kg distilled water (L-Arg solvent) daily, by oral gavage. At the end of the fourth week, rats were subjected to the same surgical procedures as hepatic IR group without portal triad clamping, then were studied after 2 hours.
group II: Hepatic ischemia-reperfusion group (IR group) (n=10): Rats in this group were kept undisturbed in their cages for 3 weeks. In the fourth week, rats received distilled water as sham-operated group. At the end of the fourth week, rats underwent
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hepatic IR (30 minutes of partial hepatic ischemia (70%) followed by 2 hours of reperfusion), then studied.
group III: L-arginine-treated hepatic ischemia-reperfusion group (L-Arg + IR group) (n=10): Rats in this group were kept undisturbed in their cages for 3 weeks. In the fourth week, rats received L-Arg daily, in a dose of 100 mg/kg by oral gavage. At the end of the fourth week, rats underwent hepatic IR, then were studied.
group IV: Exercise-trained hepatic ischemia-reperfusion group (Ex + IR group) (n=10): Rats in this group were subjected to swim exercise 2 hours daily, 6 days/week for 4 weeks. In the fourth week, rats, received distilled water as in sham-operated group. At the end of the fourth week, rats underwent hepatic IR, then were studied.
group V: Oxytocin-treated hepatic ischemia-reperfusion group (Oxy + IR group) (n=10): Rats in this group were kept undisturbed in their cages for 3 weeks. In the fourth week, rats received distilled water as in sham-operated group and were subjected daily to subcutaneous injection of Oxy, in a dose of 3.6 μg/100 g BW. At the end of the fourth week, rats underwent hepatic IR, then were studied.
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group VI: Exercise-trained and oxytocin-treated hepatic ischemia-reperfusion group (Ex + Oxy + IR group) (n=12): Rats in this group were subjected to swim exercise as in Ex + IR group. In the fourth week, rats received distilled water and Oxy daily as in Oxy + IR group. At the end of the fourth week, rats underwent hepatic IR, then were studied.
Rats in all groups were subjected to determination:
- Initial and final body weight (BW).
- Liver weight (LW) and left ventricle weight (LV).
- Serum levels of alanine transferase (ALT), aspartate transferase (AST), creatine kinase-MB (CK-MB) and cardiac troponin I (cTnI).
- Plasma levels of malondialdehyde (MDA), total antioxidant capacity (TAC), tumor necrosis factor-alpha (TNF-α) and nitrite.
- In vitro intrinsic activity of the isolated hearts perfused in a Langendorff preparation, under baseline conditions and its responses during 30 minutes of reperfusion following 30 minutes of total global ischemia.
- Liver and heart tissues histopathology.
Rats in Hepatic IR group revealed significant increase in serum levels of ALT, AST, CK-MB and cTnI and plasma levels of MDA
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and TNF-α together with significant decrease in plasma levels of TAC and nitrite. Hepatic tissue histopathology showed corruption of the hepatic architecture in the form of hepatocytes vacuolization, necrosis, nuclear pyknosis, lymphocytic infiltration and congestion of blood sinusoids.
Isolated hearts showed significant decrease in basal peak developed tension (PT) and peak developed tension per left ventricle weight (PT/LV), together with decreased basal myocardial flow rate (MFR) and myocardial flow rate per left ventricle weight (MFR/LV). Following in vitro cardiac IR, hearts showed significant decrease in heart rate (HR) and significant increase time to peak tension (TPT) and half relaxation time (HRT) at the start (5 min) of reperfusion. This was associated with significant decrease in PT, PT/LV, MFR and MFR/LV throughout the whole reperfusion periods. Such data indicate that these hearts suffered from systolic dysfunction and myocardial ischemia under basal conditions and following reperfusion.
Heart tissue histopathology revealed cardiac structural injury in the form of necrosis, inflammatory cells infiltration, loss of cross striation, interstitial edema and interstitial hemorrhage together with appearance of contraction bands necrosis.
L-Arg + IR group exhibited significant reduction in BW % change, serum levels of ALT and AST and plasma levels of MDA
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and TNF-α together with significant increase in plasma levels of TAC and nitrite as compared to IR group. Hepatocellular damage was decreased as evidenced by regression of hepatocytes necrosis, vacuolization and nuclear pyknosis.
In addition, L-Arg pretreatment offered cardioprotection against hepatic IR-induced damage, evidenced by the significant reduction in the serum levels of CK-MB and cTnI together with improvement of myocardial injury score, that showed reduction of necrosis, inflammatory cells infiltrate, interstitial edema and interstitial hemorrhage.
L-Arg treatment increased basal and postischemic PT and PT/LV, though insignificant, reflecting partial improvement in cardiac systolic function. This was associated with enhancement of postischemic recovery of contraction rate as denoted by the significant decrease of TPT throughout whole period of reperfusion. Improvement of coronary flow basal and postischemic recovery was evidenced by significant increase in both MFR and MFR/LV values at basal conditions and during all reperfusion values.
Ex + IR group displayed significant reduction in FBW, BW % change and LW together with significant increase in LV and LV/BW as compared to IR group. Also, exercise training ameliorated the hepatic injury and dysfunction induced by hepatic
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IR, as revealed by significant decrease in serum levels of ALT and AST, and plasma levels of MDA and TNF-α, and significant increase in TAC and nitrite levels, as well as improvement of the liver histopathological data, and regression of necrosis, vacuolization and congestion.
Exercise training attenuated the cardiac damage induced by hepatic IR as evidenced by the significant decrease in serum levels of CK-MB and cTnI together with regression of cardiac morphological changes and histopathological injury scores of necrosis, inflammatory cells infiltrate, cross striation loss, interstitial edema and interstitial hemorrhage.
Moreover, Ex + IR group showed significant increase in basal and postischemic values of PT and PT/LV as well as shortened postischemic values of TPT at 5 minutes of reperfusion, reflecting systolic function improvement, reaching the control values. Also, myocardial coronary flow rate improved as shown by the significant increase in basal and postischemic values of MFR and MFR/LV that reached the control values, except for MFR basal and 5 minutes of reperfusion that were even significantly higher than control values. However, at 30 minutes of reperfusion, HR was slower than both hepatic IR and control groups and HRT was longer than control group and preischemic values, which reflects diastolic dysfunction.
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As compared to L-Arg + IR group, Ex + IR group displayed significant reduction in FBW, BW % change, and plasma levels of TNF-α and nitrite, insignificant decrease in ALT and AST serum levels and MDA plasma level and significant increase in LV and LV/BW. Meanwhile, the increase in plasma TAC reached control values. These results reflect better anti-inflammatory and antioxidant effects but lower NO producing effect of exercise than L-Arg. Also, hepatic histopathological data was better by exercise training as shown by more regression of scores of vascular congestion, hepatocytes necrosis and vacuolization.
In addition, as compared to L-Arg pretreatment group, exercise training group revealed better basal and post-ischemic recovery of cardiac contractile function and coronary flow, as shown by the significantly increased basal values of PT and PT/LV and increased reperfusion values that were significant for PT and insignificant for PT/LV. MFR basal and reperfusion values at 5 and 15 minutes and MFR/LV at 5 minutes of reperfusion were significantly increased. However, reperfusion values of TPT at 15 minutes and HRT at 30 minutes were significantly more prolonged. Moreover, the cardiac histopathology showed better improvement as evidenced by the more regression of scores of necrosis, inflammatory cell infiltration, cross striation loss and interstitial hemorrhage.
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Oxytocin pretreatment in Oxy + IR group significantly decreased FBW and BW % change as compared to both IR and sham groups and improved hepatic dysfunction induced by hepatic IR, as denoted by the significant reduction in serum levels of ALT and AST, and plasma levels of MDA and TNF-α together with significant increase in TAC and nitrite, however, these improvements did not reach the control levels.
In addition, regression of histopathological injury score was evidenced by the reduction in necrosis and vacuolization scores, together with slight improvement in congestion score.
Also, cardiac damage induced by hepatic IR was attenuated by Oxy pretreatment as denoted by the significant reduction in serum levels of CK-MB and cTnI, together with improvement of cardiac damage and regression of scoring of necrosis, inflammatory cell infiltrate, cross striation loss and interstitial hemorrhage. However, interstitial edema score was slightly improved.
In addition, Oxy + IR group exhibited improvement of cardiac dysfunction induced by hepatic IR as shown by the significant increase of PT and PT/LV at 15 and 30 minutes of reperfusion that approached the control values together with increase in basal values of PT and PT/LV, though insignificant. Oxy treated group exhibited significant bradycardia and prolongation of HRT at the start of reperfusion, however, at 15 and 30 minutes of reperfusion
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both HR and HRT values were restored and even normalized as they reached their pre-ischemia and sham group values. Moreover, Oxy + IR group revealed significant increase in the basal MFR/LV as well as postischemic values of MFR and MFR/LV at 5 and 15 minutes of reperfusion.
As compared to L-Arg + IR group, Oxy + IR group displayed significant reduction in BW % change and plasma levels of both TNF-α and nitrite that was associated with the insignificant decrease in serum levels of ALT and AST and plasma levels of MDA and TAC. These findings point to better anti-inflammatory effect but lower NO producing effect of Oxy than L-Arg. As regards the improvement of hepatic histopathology, slight differences were observed between both groups, with Oxy + IR group showing slight greater improvement of congestion and necrosis scores, than the L-Arg + IR group that was more superior in the improvement of vacuolization score.
Also, in comparison to L-Arg + IR group, Oxy + IR group showed significantly lower basal values of MFR and MFR/LV as well as values of MFR throughout reperfusion period and MFR/LV at 15 and 30 minutes of reperfusion. This was associated with significantly increased PT/LV at 30 minutes of reperfusion, together with significantly prolonged HRT and bradycardia at 5 minutes of reperfusion.
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Meanwhile, the improvement of cardiac histopathological injury was better in Oxy + IR group than L-Arg + IR group as evidenced by regression of scores of necrosis, cross striation loss and interstitial hemorrhage, however, the interstitial edema score was aggravated in the Oxy treated group.
Combination of both exercise training and Oxy pretreatment revealed significant decrease in FBW, BW % change and LW, and significant increase in LV/BW as compared to both IR and shamgroups. This was associated with amelioration of hepatic IR-induced damage, evidenced by significant decrease in serum levels of ALT and AST, and plasma levels of MDA and TNF-α and significant elevation in plasma levels of TAC and nitrite. Liver histopathology showed reduction of the hepatic damage induced by hepatic IR, evidenced by the decrease in scores of congestion, vacuolization and necrosis.
Also, Ex + Oxy + IR group, showed improvement of cardiac damage induced by hepatic IR, evidenced by significant decrease in serum levels of CK-MB and cTnI, the latter reached control values, together with improvement of myocardial injury score denoted by reduction in necrosis, inflammatory cell infiltrate, interstitial edema, and interstitial hemorrhage.
In addition, Ex + Oxy + IR showed abrogation of the hepatic IR-induced systolic dysfunction as shown by significant increase
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in baseline and the whole reperfusion values of PT and PT/LV together with significant decrease in reperfusion values of TPT; all values being normalized approaching the control values.
Baseline values of MFR and MFR/LV were significantly increased and reached the control values. Postischemic values of MFR were significantly increased throughout whole periods of reperfusion, and postischemic values of MFR/LV were increased; significantly at 15 minutes and insignificantly at 5 and 30 minutes.
On comparison with L-Arg + IR group, Ex + Oxy + IR group exhibited significant reduction in FBW, BW % change and LW, and significant increase in LV/BW. Ex + Oxy + IR group exhibited greater hepatoprotection than L-Arg + IR group as evidenced by the significant lowering of serum ALT and AST levels together with the more regression of liver histopathological injury score. Plasma levels of MDA, TNF-α and nitrite were significantly lower whereas TAC was insignificantly higher.
Serum levels of CK-MB and cTnI were significantly lower and cardiac damage scores of necrosis and interstitial hemorrhage were more attenuated by combined treatment than L-Arg. Also, improvement of basal and post-ischemic inotropic activity were better by combined treatment than L-Arg as evidenced by significant increase of baseline and reperfusion values of PT and PT/LV, except for PT/LV at 30 minutes of reperfusion where the
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increase did not reach the statistical significance. However, the improvement in pre-ischemia baseline and reperfusion values of MFR/LV were significantly lower than L-Arg.
In conclusion, hepatic IR, mediated impairment of the liver functional and structural integrity and, also, promoted heart insult as a remote organ, which presented as deterioration of basal and postischemic recovery of systolic function and myocardial flow together with cardiac structural damage.
Hepatic IR, not only induced basal cardiac contractile dysfunction and myocardial ischemia, but it, also, hypersensitized the heart rendering it more susceptible to the detrimental effects of in vitro cardiac IR. Such hepatic IR-induced hepatic and cardiac injurious effects could be attributed to enhancement of oxidative and inflammatory states and reduction of NO availability.
All treatment modalities applied; L-Arg, Ex, Oxy and combined Ex + Oxy were effective in abrogation of hepatic and cardiac functional and structural derangement induced by hepatic IR, owing to their antioxidant, anti-inflammatory and NO producing effects.
In addition, the hepatic and cardiac favorable effects of Ex, Oxy and combined Ex + Oxy were better than that of L-Arg, with the combined treatment offering the superior beneficial effect that
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almost approached control values. This superiority of combined treatment could be attributed to its greater and more prominent role in dampening the inflammatory response and lipid peroxidation together with enhancing antioxidant activities.
Meanwhile, the effect of L-Arg in improvement of basal and postischemic myocardial flow, surpassed, all treatment modalities, which could be explained by its superior role in NO production.
from this study we could recommend that:
1. Any patient undergoing liver transplantation, heart function should be evaluated
2. Hearts susceptible to cardiac IR injury should be protected for 2 hours following hepatic transplantation.
3. Before liver transplantation, L-Arg and aerobic exercise could be used to increase tolerance of liver to IR and to decrease remote effect of IR on heart.
4. Further studies are needed using combination of L-Arg + Oxy, Ex + L-Arg, and Ex + L-Arg + Oxy, also, evaluating the effects of shorter periods of exercise.