These experiments demonstrate that post ischemic treatment with PJ34 results in decreased skeletal muscle fiber injury, preservation of tissue ATP (i.e. metabolic rescue), decreased inflammation (levels of tissue cytokines, leukocyte activation) in a model of skeletal muscle ischemia and reperfusion (I/R). These results were obtained in a post-ischemic treatment model, where PJ34 was administered systemically (via intraperitoneal injections) starting immediately before reperfusion.
Previously, our laboratory reported on the beneficial effects of PJ34 in a pre-ischemic treatment model of hind-limb I/R10
. In a follow-up report using a treatment protocol where PJ34 was administered intraperitoneally only during the reperfusion period failed to show preserved mitochondrial activity or anti-inflammatory action 9
. Local injection of PJ34 into skeletal muscle during the ischemic period resulted in preserved mitochondrial activity after I/R without affecting local cytokine production. Since intramuscular injection of PJ34 did not alter the local levels of pro-inflammatory skeletal muscle cytokines, intraperitoneal injection prior to reperfusion, early reperfusion and at an additional interval of reperfusion was chosen with the hope of establishing a steady state level of PJ34 in the serum similar to one might see with intravenous bolus or infusion. The desire to reassess intraperitoneal injection was also driven by concerns for potential bleeding complications associated with direct intramuscular administration of PJ34 into fragile, reperfused tissue in a clinical scenario where anticoagulants and/or anti-platelet therapies are routinely utilized.
In this protocol, PJ34 was administered 15 minutes prior to reperfusion, 15 minutes and two hours after reperfusion at a dose (30 mg/kg) known to provide protection against inflammation in rodent models31
. In preliminary experiments, 15mg/kg dose administered at the same time intervals did not salvage skeletal muscle ATP after 1.5 hours of ischemia followed by 24 hours reperfusion(data not shown). A dose of PJ34 was administered immediately prior to reperfusion so that PJ34 would be delivered to the limb during the earliest phase of reperfusion. Since the half life of PJ34 in plasma has been reported to be as short as 30 minutes 16
, additional doses of PJ34 were administered 15 minutes and 2 hours into reperfusion because previous work from our laboratory indicated that the intense expression of mRNA for MIP-2 and KC occurred during the first 4 hours of reperfusion28
. Additional rationale for the additional doses of PJ34 during reperfusion is provided by previous work from our laboratory which demonstrate stabilization of tissue flow (documented using quantitative laser Doppler imaging) after two hours reperfusion22
The current experimental protocol involves several changes from our previous work. First, the model used to induce complete ischemia from the controlled tension tourniquet (CTT) model 22
to an orthodontic rubber band (ORB) model 21
. The CTT model limited our assessment to 5 mice at a time because each there was only 5 tourniquets on the device. Thus the CTT prevented evaluation of treated and untreated mice simultaneously. Using ORB, the control and experimental mice could be analyzed concurrently since the number of mice evaluated at any time interval was not limited by the number of tourniquets. Prior assessment of skeletal muscle tissue injury with the indirect, semi-quantitative mitochondrial activity assay (MTT) was replaced with a direct quantitative histological assessment of fiber morphology 20
. In addition, the current treatment protocol involved administration of PJ34 immediately prior to reperfusion and two additional doses during reperfusion (15 minutes and 2 hours). By administering this drug during three intervals, the protocol provided a more clinically relevant treatment scenario that reflects ongoing treatment during early reperfusion.
At 24 hours reperfusion, the percent-injured fibers were significantly lower in mice treated with PJ34 vs. LR treated animals (). These experiments were terminated at 24 hours because the level of muscle fiber injury and the cytokine response associated with hind limb ischemia reperfusion is known to stabilize at 24 hours reperfusion 20, 27, 28
. It remains to be determined whether the substantial degree of muscle fiber salvage correlates with a substantial preservation of skeletal muscle function.
Treatment with PJ34 also rescued the absolute amount of skeletal muscle levels of ATP compared to mice treated with LR alone during hind limb ischemia reperfusion (). ATP is essential to maintain homeostatic functions essential to maintenance of membrane stabilization. Despite the ability of skeletal muscle to withstand periods of ischemia and utilize alternate energy substrates (derived from glycogen and free fatty acid metabolism) during times of stress, ATP becomes depleted during ischemia and skeletal muscle necrosis is a direct consequence of ATP depletion 32
. The mechanism by which PARP inhibition preserves ATP during I/R involves the glycolytic pathway and mitochondrial function 16, 33
. Inhibition of PARP activity decreases cleavage of NAD+ into nicotinamide and ADP-ribose during periods of ischemia and early reperfusion. PJ34’s inhibition of PARP, keeps the pool of NAD+
available for energy generation via glycolysis. While we have not directly evaluated mitochondrial function in skeletal muscle using this post ischemic treatment protocol, previous work in our lab had documented that PJ34 treatment preserved mitochondrial activity in reperfused skeletal muscle10
. Evidence to support an effect of PJ34 on mitochondria is provided by experiments where PJ34 treatment decreased bax translocation in HELA cell mitochondria34
and mitochondrial superoxide production in the aorta of diabetic mice35
Skeletal muscle ischemia reperfusion is a form of acute inflammation in which reactive oxygen species promote the activation of pro-inflammatory cytokines and leukocytes that lead to microvascular injury, membrane dysfunction and edema formation36
. In these experiments, post ischemic treatment with PJ34 markedly decreased tissue levels of pro-inflammatory cytokines which promote influx of inflammatory cells during reperfusion. KC (Keratinocyte-Derived Cytokine) and MIP-2 (Macrophage Inflammatory Protein) are both members of the CXC Chemokine family of proteins implicated as neutrophil chemoattractants that regulate the migration of leukocytes to injured tissue. KC and MIP-2 are both murine equivalents of human IL-8, which is implicated in the pathogenesis of ischemia reperfusion injury37
. The decreased levels of pro-inflammatory cytokines detected in skeletal muscle where treatment was administered in a post hoc scenario is consistent with findings detected when PJ34 was administered prior to the onset of ischemia/reperfusion10
Myeloperoxidase (MPO) is an enzyme secreted by activated neutrophils and is involved in the production of tissue oxidants that contribute to reperfusion injury. MPO catalyzes the reaction of hydrogen peroxide with chloride ions that yields the toxic metabolite, hypochlorous acid (HOCl). It is not clear whether PARP inhibition with PJ34 directly or indirectly inhibited the activation of neutrophils and hence the release of MPO, or alternatively that the inhibition of KC by PJ34 administration resulted in decreased numbers of neutrophils entering the injured area. The absolute decrease in myeloperoxidase protein concentration in the muscle of PJ34 treated mice () is consistent with less tissue injury detected during histologic evaluation of skeletal muscle during reperfusion ().
Quantitative analysis of PAR expression using immunoblotting following treatment with PJ34 has been used to document the effect of PJ34 treatment on diabetic mice 30, 38
. PAR expression in the hind limbs of ischemia reperfused mice was markedly increased in LR treated mice at 3 and 24 hours of reperfusion. In PJ34 treated mice, PAR expression was less than LR at 3hours but not 24 hours of reperfusion (). These findings indicate that the benefits of PARP inhibition are reflected in decrease poly-ADP ribosylation of proteins during the early, i.e. three hours of reperfusion, and remain durable at 24 hours of reperfusion even though the level of protein ADP-ribosylation eventually increases to untreated levels by 24 hours reperfusion. These findings suggest that early post ischemic therapy is effective in decreasing PARP expression, and the rebound in PAR expression at 24 hours does not appear to have a deleterious effect on skeletal muscle fiber histology, nor does it promote an ongoing inflammatory response.