A reproducible method for biochemical, histological and functional assessment of the effects of ischaemia–reperfusion syndrome in the lower limbs

Abstract

Current methodology described to mimic lower limb ischaemia–reperfusion injury (LL-IRI) does not accurately define the procedures and pressures exerted to induce and maintain ischaemia. In this piece of work, we propose a well-defined and detailed rat model that simulates the conditions established in clinical practice guidelines for tourniquet application and allows us to test treatments that aim to prevent/reduce LL-IRI. Eighty-six male WAG/RijHsd rats were subjected to hind limb IRI (LL-IRI), using a mechanical system applying a 1 kg tension to induce and maintain ischemia for 2 or 3 h, and assessed the damage caused by reperfusion at biochemical and muscular levels at different time points. At the biochemical level, both 2 and 3 h of ischemia induced changes (except for electrolyte levels); 3 h of ischemia induced greater changes in specific markers of muscular damage: creatine kinase (CK) and lactate dehydrogenase (LDH). At the histopathological level, 3 h of ischemia and 24 h of reperfusion was associated with an increase in hind limb girth, cross-sectional area, and weight and presence of neutrophils, as well as histological damage in more than 60% of muscle fibres. Our model allows to reliably reproduce the damage associated with the use of a pneumatic tourniquet. CK and LDH, as well as measures of tissue damage, allow to define and characterize the response to LL-IRI-related damage. A period of 3 h of ischemia followed by 3 h of reperfusion caused only local damage but showed greater sensitivity to detect differences in future studies on prophylactic treatments against LL-IRI.

Effects of Folinic Acid Administration on Lower Limb Ischemia/Reperfusion Injury in Rats

“…This time, although longer than that usually applied in clinical reality [ 33 , 34 ], has been chosen to ensure that, even in those long-term surgeries, FA could be indicated as a preventive treatment for IRI-LL. Furthermore, experimental and clinical evidence at this period of ischemia indicates that the damage remains located in the limb [ 24 , 35 , 36 , 37 ].…”

“…Ischemia was induced and maintained by an in-house designed and built mechanical device. Briefly, it consists in an adjustable support that allows placing the anesthetized animal in the supine decubitus position and a nylon cable, connected to a dynamometer and a plunger system, which slides around the limb at its base exerting a tension of 1 kg, the whole assembly being stabilized by two articulated arms [ 24 ].…”

“…In most of the studies, the effectiveness of the abovementioned treatments is assessed by considering only immediate indicators of oxidative stress, such as MDA; there are very few experiments including biochemical, anatomopathological or clinical markers of muscle damage with a later onset [ 13 , 14 , 16 , 17 , 23 ]. We have recently validated a rat model in which, by exerting a controlled pressure to achieve 3 h of ischemia in the lower limbs, we are able to assess the response to IRI from the biochemical, histological and functional point of view [ 24 ].…”

The Prevention of Ischemia-Reperfusion Injury in Elderly Rats after Lower Limb Tourniquet Use

“…The functional recovery after IRI-LL was assessed using a rotarod apparatus (Panlab Harvard Apparatus, Barcelona, Spain). The rotation speed and acceleration, as well as the time for acclimatization to the rod, were the same as those indicated in previous works of our group [ 51 , 52 ]. The latency time to fall, as well as the speed of the rod at the time of the fall, were automatically recorded using the SeDaCom v2.0.03 software (Panlab Harvard Apparatus, Barcelona, Spain).…”

“…A self-designed device was used for the induction of ischemia and its maintenance for 3 h [ 51 ]. In order to properly position the animal in the device, the animal was sedated with an intraperitoneal injection of diazepam (15 mg/kg).…”