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

Abstract

Surgery under ischemic conditions, lasting up to 3 h, is routinely performed in orthopedic surgery, causing undesirable injury due to ischemia-reperfusion syndrome, with short and medium-term functional repercussions. To date, there is no established prophylactic treatment. In this work we evaluated folinic acid (FA) in a rodent model of lower limb ischemia-reperfusion (IRI-LL). 36 male WAG rats underwent 3 h of lower limb ischemia. In the saline group, rats received intraperitoneal administration of saline (used as vehicle for treatment). In the experimental group, rats were pretreated with FA (2.5 mg/kg) before the end of ischemia. After ischemia, animals were sacrificed at 3 h, 24 h or 14 days (for biochemical determination (Na+, K+, Cl-, urea, creatinine, CK, LDH, ALP, ALT, and AST), pathological assessment, or functional study using the rotarod test; respectively). Another six animals were used to establish the reference values. The prophylactic administration of FA significantly reduced the elevation of biochemical markers, especially those that most directly indicate muscle damage (CK and LDH). In addition, it also improved direct tissue damage, both in terms of edema, weight, PMN infiltrate and percentage of damaged fibers. Finally, the administration of FA allowed the animals to equal baseline values in the rotarod test; what did not occur in the saline group, where pre-ischemia levels were not recovered. Following 3 h of lower limb ischemia, FA minimizes the increase of CK and LDH, as well as local edema and leukocyte infiltration, allowing a faster recovery of limb functionality. Therefore, it could be considered as a prophylactic treatment when tourniquet is used in clinics.

Keywords: folinic acid; functional recovery; ischemia-reperfusion injury; lower limb; prophylactic treatment; tourniquet.

Figure 1

Serum electrolytes and enzymes of control animals (control, white bar); animals exposed to 3 h of ischemia & 3 h of reperfusion, and treated with saline (saline 3 h, gray bar), or treated with folinic acid (FA 3 h, dark gray bar); animals exposed to 3 h of ischemia & 14 days of reperfusion, and treated with saline (saline 14 D, gray bar with horizontal striped pattern), or treated with folinic acid (FA 14 D, gray bar with vertical striped pattern. Na⁺ (A), K⁺ (B), Cl⁻ (C), urea (D), creatinine (E), alkaline phosphatase (ALP (F)), aspartate transaminase (AST (G)), alanine transaminase (ALT (H)), creatine kinase (CK (I)), and lactate dehydrogenase (LDH (J)). Units are expressed in international units per liter (IU/L), milligrams per deciliter (mg/dL) or in milliequivalents per liter (mEq/L). The asterisks show the statistically significant differences compared to the control (* p < 0.05; ** p < 0.01; *** p < 0.001), and the pads show the statistically significant differences between the groups indicated by the upper box (### p < 0.001); ns: p > 0.05.3.3. Effect of FA on histological damage induced by IRI.

Figure 2

Perimeter (a) and percentage of increase in the cross-sectional area of the ischemic limb with respect to the contralateral healthy limb (b). The asterisks show the statistically significant differences compared to the control (** p < 0.01; *** p < 0.001), and the pads show the statistically significant differences between the groups indicated by the upper box (# p < 0.05); ns: p > 0.05.

Figure 3

Fresh weight of the gastrocnemius muscle of both limbs: left limb (healthy, bars without pattern) and right limb (ischemic, bars with vertical striped pattern), from animals not subjected to ischemia (white bars), treated with saline (Saline 24 h, light gray bars), or treated with folinic acid (FA 24 h, dark gray bars). The asterisks show the statistically significant differences compared to the control (* p < 0.05), and the pads show the statistically significant differences between the groups indicated by the upper box (# p < 0.05); ns: p > 0.05.

Figure 4

Polymorphonuclear neutrophil (PMN) cell count (left axis, bars without pattern) and percentage of damaged fibers in each field at 20 × (right axis, bars with vertical striped pattern) of control healthy animals (Control, white bars), and animals undergoing 3 h of ischemia and 24 h of reperfusion and treated with saline (Saline 24 h, light gray bars), or treated with folinic acid (FA 24 h, dark gray bars). The asterisks show the statistically significant differences compared to the control (*** p < 0.001), and the pads show the statistically significant differences between the groups indicated by the upper box (## p < 0.01; ### p < 0.001); ns: p > 0.05.

Figure 5

Assessment of limb functional status over a 14-day period using the Rotarod test. Data show the mean and standard deviation in the fall time (expressed in seconds) of animals subjected to 3 h of ischemia period, treated with folinic acid (blue line) or with saline (red line). The asterisks show the statistically significant differences compared to the control (* p < 0.05; ** p < 0.01). The dashed red line box indicates the differences between the 14th day of the study and the baseline values determined on the day before ischemia induction (day −1) (# p < 0.05; ns: p > 0.05).