Investigación experimental aplicada a la clínica: isquemia-reperfusión hepática / Experimental research applied to the clinical setting: liver ischemic- reperfusion

Existen numerosas situaciones fisiopatológicas en la actividad clínica en las que el hígado se somete a una situación de isquemia transitoria, total o parcial (hepatectomías parciales, trasplante hepático, donantes en asistolia, estados de shock, situaciones de parada cardiorrespiratoria reversibles), pudiendo desarrollarse una lesión por la isquemia-reperfusión, que no se ha podido prevenir totalmente. Por tanto, es importante investigar, utilizando modelos experimentales, los mecanismos que están implicados en su producción, así como el efecto potencialmente beneficioso que puedan tener sobre la misma diferentes fármacos que interfieren sobre los mecanismos de producción, evitando la aparición posterior de la lesión, parcial o totalmente.En este estudio revisamos, en primer lugar, los fundamentos patogénicos de las lesiones por isquemia-reperfusión, analizando los factores más importantes que se han implicado en la misma hasta la actualidad: calcio, endotelio, neutrófilos, activación del complemento, metales pesados, radicales libres de oxígeno y óxido nítrico. En segundo lugar se revisa el papel beneficioso para prevenir las lesiones por isquemia-reperfusión de diversos antioxidantes (superóxido-dismutasa, alopurinol, vitamina E, captopril, propanolol y N-acetilcisteína).Finalmente, se subrayan las perspectivas de futuro e implicaciones clínicas del estudio patogénico de la isquemia-reperfusión hepática, y cómo recientemente se ha implicado el fallo de los mecanismos enzimáticos reparadores del ADN, lesionado por los radicales libres, como polo de investigación para prevenir las lesiones por isquemia-reperfusión hepática (AU)

Free radical scavengers to prevent reperfusion injury following experimental warm liver ischaemia. Is there a real physiological benefit?

Free radical scavengers have been utilized to prevent the consequences of ischemia, however, results do not seem conclusive. In our study we analyzed the blood flow, function, and histology of rat liver tissue after warm liver ischemia, in order to assess the effect of free radicals in liver reperfusion injury. N-acetyl cysteine (NAC), tocopherol, allopurinol, and superoxide dismutase (SOD), pharmacological agents expected to protect from injury mediated by free radicals, were investigated. Laser Doppler flowmetry and photometry were utilized to measure post-ischemic microcirculatory changes as an expression of ischemia-reperfusion injury in a model of segmental liver ischemia in the rat, with an ischemic time of 45 min. Galactose elimination capacity, ALT and histology were used to assess the functional and morphological consequences of ischemia after 24 h of reperfusion. The overall mean blood flow over 1 hour after reperfusion was of 33.9% (SD 11.2) of the normal, non-ischemic control. NAC (31.2% SD 10.9) did not show any protective effect and in some cases the effect seemed to be negative. Tocopherol (41.7% SD 5.1) marginally improved post ischemic liver tissue blood flow. Treatment with allopurinol did not show any beneficial effects (37.5% SD 14.2). Only animals treated with SOD showed an improvement of the post ischemic liver microcirculation (57.9% SD 14.4)(P < 0.001) and function. Only SOD produced statistically significant differences in galactose elimination capacity, compared with those of the ischemic control group. This moderately protective effect of SOD is encouraging, however, the relevance of all these compounds in a broader pathophysiological setting remains unproven.

Blood clearance of 99mTc-trimethyl-Br-IDA discriminates between different degrees of severe liver ischaemia--reperfusion injury in the rat.

The kinetics of 99mTc-Trimethyl-Br-IDA blood clearance was analysed in the rat 24 h after warm ischaemia and reperfusion of the liver. There were changes in the elimination of 99mTc-Trimethyl-Br-IDA depending on the length of the ischaemic period and the dose given. Statistically significant differences were found between the various periods of ischaemia when higher doses of the radionuclide were utilised. At lower doses, the clearance was not capable to discriminate between control rats and rats submitted to 45 min of ischaemia, but it did discriminate more severe degrees of ischaemic liver injury. Instead, galactose elimination capacity discriminated between ischaemic and control rats, but not between 45 and 90 min or between 90 and 120 min of ischaemia. Alanine aminotransferase was able to discriminate between control and ischaemic rats and between 45 and 90 min of ischaemia, but not between 90 and 120 min of ischaemia. The response of 99mTc-Trimethyl-Br-IDA clearance under extreme conditions of ischaemia and reperfusion is consistent and opens a possible window for the application of this test in the quantification of liver function in severely damaged livers and in decision making and prognosis in liver disease.

Ischemia and reperfusion injury of the rat liver: the role of nimodipine.

The protective effect of the calcium channel blocker nimodipine on liver ischemia and reperfusion was studied in the rat. The homeostasis of intracellular calcium ions seems to be a determinant factor in the cell injury that appears after ischemia and reperfusion. Nimodipine was used to downregulate the calcium levels in the cytosol of the ischemic cell, the hypothetical role of Ca2+ in the pathogenesis of ischemia and reperfusion injury. The experimental procedure consisted of the temporary interruption of blood flow to the left lateral and medial lobes of the rat liver and subsequent reperfusion after a period of 45 min of ischemia. Nimodipine (10 micrograms/kg body wt) was administered either before or after the onset of ischemia. The postischemic liver blood flow and liver oxyhemoglobin saturation were recorded using a He-Ne laser Doppler flowmeter and photometer, which showed, in the pretreated group, a recovery of reperfusion blood flow (58.1%) and liver reflectance (85.5%) significantly better (P < 0.01 and P < 0.001) than those in the respective untreated controls of flow (32.8%) and reflectance (70.5%). In the group that received nimodipine after ischemia, the recovery of the blood flow and the postreperfusion liver reflectance were not significantly better than those in the untreated control group. ALT levels (P < 0.05), galactose elimination capacity (P < 0.001), and histological studies also showed a protective effect of calcium antagonist nimodipine when administered before ischemia.

Regulation of the complement cascade by soluble complement receptor type 1. Protective effect in experimental liver ischemia and reperfusion.

The complement cascade was inactivated in a model of rat liver ischemia with the purpose of studying the role of complement in tissue injury after ischemia and reperfusion. Soluble human complement receptor type 1 (sCR1) was administered either in a single dose of 25 mg/kg or in 2 doses of 50 mg/kg i.v. over 24 hr after vascular occlusion. Sham-operated rats, nontreated rats submitted to liver ischemia, and rats pretreated with cobra venom factor and submitted to liver ischemia were used as controls. This experiment consists of the temporary interruption of arterial and portal blood flow to the left lateral and medial lobes of the liver for 45 min, followed by a 24-hr period of follow-up after reperfusion. Liver blood flow and hemoglobin saturation were recorded for 1 hr after declamping, with statistically significant differences between the experimental groups and the untreated control group, which received liver ischemia (P < 0.001). At 24 hr, galactose elimination was assayed as a liver function test; it was significantly better in the sCR1-treated rats when compared with control rats submitted to ischemia (P < 0.01). Alanine aminotransferase levels were also significantly lower in the sCR1-treated rats at 6 and 24 hr (P < 0.05). Complement activity was reduced to 25% and 12.5% of normal rats with the respective doses of sCR1. Immunoperoxidase stainings for C3 and C9 were performed on liver sections; they showed endothelial deposits of C3 and C9 in the control group subjected to ischemia. Few C3 deposits were present in the sCR1 (25 mg/kg)-treated rats, but not in the cobra venom factor or sCR1 (50 mg/kg) groups. These results confirm that complement is inactivated by sCR1 with amelioration of reperfusion injury in the rat liver.

Haemoglobin saturation in the rat liver after ischaemia and reperfusion: study using a laser photometry technique and correlation with changes in liver tissue blood flow.

Tissue oxygenation depends on the volume of oxygen consumed by the tissue and the volume of oxygen supply. This is particularly important in the liver after ischaemia and reperfusion, due to the relatively low oxygen saturation of the portal blood flow, the main source of oxygen to the liver. In this study we established a correlation between the postischaemic liver blood flow and tissue haemoglobin saturation, measured by laser Doppler flowmetry and laser surface photometry, with the purpose of investigating the possible role of a postischaemic imbalance of oxygen delivery/uptake in reperfusion injury. The experimental procedure consisted of the temporary interruption of blood flow to the left lateral and medial lobes of the rat liver in vivo, and subsequent reperfusion after defined periods, recording the postischaemic liver blood flow and liver oxyhaemoglobin saturation. Changes were found in the postischaemic liver blood flow and haemoglobin saturation in all the groups when compared to control values, showing a correlation between the length of the period of ischaemia and the magnitude of the alteration in the reperfusion blood flow and oxygenation. These alterations may be considered as a prolongation of the metabolic condition of ischaemia and may be part of an additional tissue damage upon reperfusion.

Rat liver blood flow after ischemia and reperfusion. The effects of the platelet-activating factor antagonist WEB-2170 and of removing circulating leukocytes.

The inflammatory response to trauma induces release of platelet activating factor (PAF), which promotes leukocyte adherence to the vascular endothelium. Ischemia and reperfusion induces inflammatory reactions that play a role in reperfusion injury, and here we investigate the role of both PAF and of leukocytes in damage to reperfused rat liver. The experimental procedure consisted of the temporary interruption of blood flow to the left lateral and medial lobes of the rat liver in vivo, and subsequent reperfusion after defined periods. Rats were pretreated either with the PAF-antagonist WEB-2170 or with vinblastine to induce leukopenia, and compared with controls. The postischemic liver blood flow and liver oxyhemoglobin saturation were recorded using an He-Ne Laser doppler flowmeter and photometer. Reperfusion after 30 and 45 min of ischemia was associated with partial recovery to normal values and was inversely proportional to the duration of ischemia. In the WEB-2170-treated group, liver flow and hemoglobin saturation upon reperfusion did not show significant differences when compared with the untreated control groups, suggesting that inhibition of PAF activity did not protect against the microcirculatory disturbance induced by ischemia and reperfusion in the liver. In contrast, rats made leukopenic by treatment with vinblastine showed significantly better recovery of blood flow and hemoglobin saturation than the control group after 45 min of ischemia. Thus, we found that although PAF alone did not appear to have a pivotal role in the cascade of reperfusion injury, the effect of leukocytes is critical.

Laser-Doppler flowmetry in the study of in vivo liver ischemia and reperfusion in the rat.

Estimation of blood flow in the microcirculation is essential in the analysis of the events that occur during organ ischemia and subsequent reperfusion. In this study the use of laser-Doppler flowmetry is evaluated as a method for studying liver blood flow in the rat using an in vivo ischemia-reperfusion model. The experimental procedure consists of the temporary interruption of blood flow to the left lateral and medial lobes of the liver and subsequent recording of the postischemic liver blood flow using a laser-Doppler flowmeter. Flow was recorded for 60 min after periods of ischemia of 30 sec (control), 30, 45, and 60 min. The reperfusion records showed a biphasic curve with a mean flow of 59.6% of the baseline after 30 min of ischemia and of 38.3 and 41.1% after 45 and 60 min of ischemia, respectively. There were statistically significant differences between all the groups and the controls, and between 45 and 30 min of ischemia but not between 45 and 60 min. This is a useful model for studying ischemic liver injury in the rat.

Adjustable nylon ties for abdominal wall closure.

A study in 64 patients with a high risk of wound dehiscence or eviscerated wounds or in whom repeat laparotomies for peritoneal lavages due to sepsis were planned was accomplished using adjustable nylon ties for the closure of the abdominal wall, either as retention sutures or as one-layer sutures. The adjustable nylon ties are described, as well as the technique for placing and adjusting them. It is concluded that the adjustable nylon ties can substitute advantageously for the conventional retention sutures since the former are wider, easy to place, and can be readjusted to the point that they can be opened completely if it is necessary and then closed again. This is especially useful when repeat laparotomies for peritoneal lavages are required in patients with intra-abdominal sepsis.