Poloxamer 188 Significantly Decreases Muscle Necrosis in a Murine Hindlimb Model of Ischemia Reperfusion Injury
Adrian D. Murphy, MD MRCSI, Michael C. McCormack, BSc, David Bichara, MD, John T. Nguyen, MD, Mark A. Randolph, MAS, William G. Austen, Jr., MD FACS.
Massachusetts General Hospital, Boston, MA, USA.
Ischemia-reperfusion injury is the inflammatory response to restoration of blood flow after an ischemic event. This is associated with significant morbidity and mortality in many clinical settings, such as free flap transfers, transplantation and replantation surgery, and crush injuries. Locally, this can lead to tissue loss and muscle necrosis.
Complement is a key mediator of reperfusion injury. Complement activation releases chemotactic agent (C5a) and anaphylatoxins (C3a, C5a) that induce degranulation of mast cells. The end result is the formation of membrane-attack complexes that damage cells by creating pores in cell membranes.
Poloxamer 188 (P188), which is a biocompatible polymer consisting of two hydrophilic side-chains attached to a hydrophobic center core, has been shown capable of sealing stable defects in cell membranes after various types of trauma. This substance, which is FDA approved, has been used previously in the treatment of sickle cell crisis.
We hypothesized that administration of P188 would seal membrane defects created by the membrane-attack complexes formed during IR injury and would prevent myocyte necrosis.
Male C57/BL6 mice were used for this study. Three experimental groups were used; animals received an intra-venous injection of 150 μL P188 (n=20), Dextran (n=10) or Saline (n=14) ten minutes prior to bilateral hind-limb ischemia. Hind-limb ischemia was achieved with orthodontic rubber bands applied above the greater trochanter using a McGivney Hemorrhoidal Ligator. Limbs were rendered ischemic for two hours and then reperfused for 24 hours before the animals were euthanized and their hind limbs harvested for histological analysis.
The gastrocnemius muscle was embedded cut in cross-section and stained with Masson Trichrome. The stained slides were examined under microscopy. Using a validated scoring system, the photographed slides were analyzed to produce a percentage injury score for each limb.
There was no difference between the groups that received Dextran and Saline; average injury scores of 51% and 55% respectively. The group that received P188 prior to ischemia had an average injury score of 8%. This was a highly significant difference compared to the other two groups (p<0.001).
Single dose P188 administered prior to the onset of hind-limb ischemia causes a dramatically increased myocyte survival rate following ischemia reperfusion injury. We hypothesize that this effect is most likely due to P188 inserting into membrane defects and allowing membrane repair. This degree of protection from IR injury is vastly superior to that seen in most other studies. P188 is FDA approved and may provide a useful clinical adjunct in preventing and treating ischemia reperfusion injury.