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Topical Combinations to Treat Microvascular Dysfunction of Chronic Postischemia Pain

Laferrière, André BA*; Abaji, Rachid BSc*; Tsai, Cheng-Yu Mark BSc; Ragavendran, J. Vaigunda PhD*; Coderre, Terence J PhD‡§

doi: 10.1213/ANE.0000000000000141
Pain and Analgesic Mechanisms: Research Report

BACKGROUND: Growing evidence indicates that patients with complex regional pain syndrome (CRPS) exhibit tissue abnormalities caused by microvascular dysfunction in the blood vessels of skin, muscle, and nerve. We tested whether topical combinations aimed at improving microvascular function would relieve allodynia in an animal model of CRPS. We hypothesized that topical administration of either α2-adrenergic (α2A) receptor agonists or nitric oxide (NO) donors given to increase arterial blood flow, combined with either phosphatidic acid (PA) or phosphodiesterase (PDE) inhibitors to increase capillary blood flow, would effectively reduce allodynia and signs of microvascular dysfunction in the animal model of chronic pain.

METHODS: Mechanical allodynia was induced in the hindpaws of rats with chronic postischemia pain (CPIP). Allodynia was assessed before and after topical application of vehicle, single drugs or combinations of an α2A receptor agonist (apraclonidine) or an NO donor (linsidomine), with PA or PDE inhibitors (lisofylline, pentoxifylline). A topical combination of apraclonidine + lisofylline was also evaluated for its effects on a measure of microvascular function (postocclusive reactive hyperemia) and tissue oxidative capacity (formazan production by tetrazolium reduction) in CPIP rats.

RESULTS: Each of the single topical drugs produced significant dose-dependent antiallodynic effects compared with vehicle in CPIP rats (N = 30), and the antiallodynic dose-response curves of either PA or PDE inhibitors were shifted 5- to 10-fold to the left when combined with nonanalgesic doses of α2A receptor agonists or NO donors (N = 28). The potent antiallodynic effects of ipsilateral treatment with combinations of α2A receptor agonists or NO donors with PA or PDE inhibitors were not reproduced by the same treatment of the contralateral hindpaw (N = 28). Topical combinations produced antiallodynic effects lasting up to 6 hours (N = 15) and were significantly enhanced by low-dose systemic pregabalin in early, but not late, CPIP rats (N = 18). An antiallodynic topical combination of apraclonidine + lisofylline was also found to effectively relieve depressed postocclusive reactive hyperemia in CPIP rats (N = 61) and to increase formazan production in postischemic tissues (skin and muscle) (N = 56).

CONCLUSIONS: The present results support the hypothesis that allodynia in an animal model of CRPS is effectively relieved by topical combinations of α2A receptor agonists or NO donors with PA or PDE inhibitors. This suggests that topical treatments aimed at improving microvascular function by increasing both arterial and capillary blood flow produce effective analgesia for CRPS.

From the *Alan Edwards Centre for Research on Pain, Department of Anesthesia, Department of Psychology, Alan Edwards Centre for Research on Pain, Department of Anesthesia, Neurology and Neurosurgery, and Psychology, and §Anesthesia Research Unit, McGill University Health Centre Research Institute, Montreal, QC, Canada.

Funding: Louise and Alan Edwards Foundation to TJC, Canadian Institutes of Health Research (MOP-143406 to TJC), and Natural Sciences and Engineering Research Council of Canada (194525-09 to TJC).

Conflict of Interest: See Disclosures at the end of the article.

Address correspondence and reprint requests to Terence J. Coderre, PhD, Anesthesia Research Unit, McGill University, 3655 Promenade Sir William Osler, Montreal, QC, H3G 1Y6, Canada. Address e-mail to

Complex regional pain syndrome (CRPS) typically follows fracture, crush, or soft tissue injury, with (type II) or without (type I) accompanying injury of a major nerve. CRPS-I (formerly known as reflex sympathetic dystrophy) and CRPS-II (formerly known as causalgia) have historically been viewed to depend on pathology in the sympathetic nervous system. However, there is evidence to suggest that a key mechanism underlying the pathology of both CRPS-I and CRPS-II is persistent deep tissue ischemia associated with microvascular dysfunction.1–5 We have generated evidence that pain in an animal model of CRPS also depends on microvascular dysfunction.6–9 In rats with chronic postischemia pain (CPIP), whose symptoms parallel those of patients with CRPS, ischemic tissue injury leads to the generation of oxygen free radicals and proinflammatory cytokines, which causes arterial vasospasm and capillary slow flow/no-reflow (signs of microvascular dysfunction) in hindpaw blood vessels.6–9 Vasospasm and capillary slow flow/no-reflow lead to reduced nutritive blood flow, poor muscle oxygenation, and the build up of muscle lactate, all of which contribute to the pain.7,8 Thus, CPIP animals have pain, allodynia, vasospasm, poor tissue perfusion, and oxidative stress in their affected hindpaw.8 It is important to note that we showed that pain/allodynia and microvascular dysfunction in CPIP rats are attenuated by systemic treatments with an α2A receptor agonist and nitric oxide (NO) donors, which improve arterial blood flow (relieving vasospasm), and by a phosphodiesterase (PDE)4 inhibitor (pentoxifylline), which improves capillary blood flow (relieving capillary slow flow/no-reflow).7–9 Therefore, we hypothesized that treatments aimed at enhancing tissue oxygenation by reducing arterial vasospasm and capillary slow flow/no-reflow will effectively relieve pain in this animal model of CRPS and potentially in CRPS patients.

Regional blood flow is regulated by the vasoconstrictive transmitter norepinephrine (NE) released from sympathetic postganglionic neurons, which contracts vascular smooth muscles.10,11 Blood flow is also regulated by the vasodilatory substance NO that is released from vascular endothelial cells and relaxes vascular smooth muscles.12,13 Thus, drugs that reduce NE release or binding, or drugs that increase NO, can be used used to alleviate conditions with poor blood flow,14,15 including microvascular dysfunction. Various isoforms of PDE enzymes (types I–II) also regulate blood flow, and affect various rheological properties, by influencing the intracellular messengers cAMP and cGMP.16 Thus, PDE4,7 and 8 hydrolyse cAMP, PDE5,6 and 9 hydrolyse cGMP, and PDE1,2,3,10 and 11 hydrolyse both cAMP and cGMP.17 While PDE inhibitors that reduce cGMP hydrolysis enhance blood flow,18 PDE inhibitors that reduce cAMP hydrolysis also reduce platelet aggregation,19 decrease blood viscosity,20 and increase the flexibility of red blood cells,20 all of which relieve microvascular dysfunction. Thus, PDE inhibitors have been developed for conditions such as peripheral arterial disease21 and intermittent claudication,20 where microvascular injury leads to capillary slow flow/no reflow and reduced tissue oxygenation. Pentoxifylline, a PDE4 inhibitor, has been specifically developed to treat these ischemic disorders.20,21 Lisofylline, a major metabolite of pentoxifylline, shares many of the actions of its prodrug22 but also inhibits the conversion of lysophosphatidic acid to phosphatidic acid (PA), a pathway critical to the production of various cytokines, including interleukin 1β and tumor necrosis factor-α.23

Since systemic use of drugs that regulate blood flow may seriously affect arterial blood pressure and cardiac function, the topical use of these drugs could enhance both their efficacy and safety. Also, since the pain of CRPS may depend not only on reduced arterial blood flow but also microvascular dysfunction at the capillary level, pain relief may be optimized by the simultaneous use of drugs that increase both arterial and capillary blood flow, that is, by combining α2A receptor agonists or NO donors with PDE or PA inhibitors. Although α2A receptor agonists24,25 and NO donors26,27 have been used topically or regionally to alleviate chronic pain, the topical pain-relieving effects of PDE and PA inhibitors have not been previously assessed. Furthermore, these drugs have not been combined systemically or topically for the treatment of chronic pain. However, as single drugs, α2A receptor agonists, NO donors, or PDE inhibitors have been used systemically to treat pain associated with angina,28–30 peripheral arterial disease,31,32 and neuropathic pain,33–35 indicating their usefulness in these syndromes. PA inhibitors share the vasodilator,36 anti-ischemic37 and antiplatelet aggregation,38 effects of PDE inhibitors, but their added antioxidant,39 anticytokine,40 antileukocyte attractant,41 immunosuppressant,42 and mitochondrial protective43 effects suggest they may be more effective at relieving chronic pain.

In the present study, we have tested the hypothesis that combinations of drugs aimed at reducing microvascular dysfunction (including α2A receptor agonists or NO donors, combined with PA or PDE inhibitors) can be used topically on the hindpaw to reduce allodynia in an animal model of CRPS. One combination was also tested for its ability to restore compromised postocclusive reactive hypermia (PORH) that is observed after ischemia-reperfusion (I/R) injury in CPIP rats.

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Male Long Evans rats (225–250 g, Charles River, St. Constant, QC) were received at our animal housing facility at least 7 days before the experiments started. Our study methods were approved by the Animal Care Committee of McGill University and conformed to ethical guidelines of the Canadian Council on Animal Care and the International Association for the Study of Pain.

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Drugs used for topical administration included linsidomine (Tocris, Ellisville, MO), lisofylline (Cayman Chemical, Ann Arbor, MI), apraclonidine and pentoxifylline (Sigma-Aldrich, St. Louis, MO). Pregabalin, used for systemic treatment, was obtained as a gift from Pfizer Corporation.

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Analgesic Formulations

Ointment-type analgesic formulations containing the above-mentioned drugs were formulated by using a composite, water-soluble polyethylene glycol (PEG) base system by using carbowax (PEG 3350) and PEG 400 (both from Sigma-Alrich, St. Louis, MO) in the ratio of 60:40, respectively. The water-soluble base system was selected due to its nonsticky nature. Briefly, the required amounts of the active ingredients were first weighed and then added to the already molten base in decreasing order of their melting points and stirred well. After uniform melting, the formulation was brought to room temperature to ensure proper solidification. A standard amount of 150 mg (mean ± SEM = 150.88 ± 2.7 mg) ointment was used for all rat hindpaw applications throughout the experimental procedure.

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CPIP was generated after prolonged hindpaw ischemia and reperfusion, as previously described by Coderre et al.6 Briefly, rats were anesthetized over a 3-hour period with a bolus (55 mg/kg, intraperitoneal), followed by intraperitoneal infusion (0.15 mL/h) of sodium pentobarbital (Ceva Santé Animale, Libourne, France) for 2 hours. After induction of anesthesia, a Nitrile 70 Durometer O-ring (O-rings West, Seattle, WA) with 5.5 mm internal diameter was placed around the rat’s left hindlimb proximal to the ankle joint. The tight-fitting O-ring produces a complete blockade of arterial blood flow as measured by using laser Doppler flowmetry.8 The ring was then left in place for 3 hours, and the rats recovered from anesthesia 30 to 60 minutes after reperfusion. Sham rats were anesthetized for the same period and had a cut (loose) O-ring placed around their ankles. Rats were tested either between 2 and 14 (early) or 18 and 26 days (late) post-I/R or sham-injury.

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Mechanical Sensitivity Testing

The plantar surface of the ipsilateral hindpaw was tested for mechanical allodynia (paw withdrawal thresholds, PWTs) in CPIP rats. Semmes Weinstein nylon monofilaments were applied in either ascending (after negative response) or descending (after positive response) force as necessary to determine the filament closest to the threshold of response. Six different filaments were used: the minimal stimulus intensity was 1 g, and the maximum was 15 g. Individual threshold determinations could thus require from 2 to 6 filaments and from 6 to 9 stimuli, depending on the pattern of responses obtained. Each filament was applied for 10 seconds or until a flexion reflex occurred. Based on the response pattern and the force of the final filament (fifth stimulus after first direction change), the 50% threshold (grams) was calculated as (10[Xf+kδ])/10,000 where Xf = filament number of the final von Frey hair used, k = value for the pattern of positive/negative responses, and δ = mean difference in log unit between stimuli (here, δ = 0.220, for more details see Chaplan et al.44).

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Pharmacological Treatments

The topical formulations containing an NO donor, α2A receptor agonist, or a PA/PDE inhibitor were tested for their antiallodynic effects, either singly, or in combination with each other, at concentrations selected from the published literature for use as topical drugs. In the single drug pharmacological trials, apraclonidine was tested at 0.005 %, 0.01%, 0.02%, and 0.04% W/W (N = 6); linsidomine at 0.2, 0.4, 0.8, and 1.6% W/W (N = 8); lisofylline at 0.063, 0.09, 0.125, and 0.25% W/W (N = 10); and pentoxifylline at 0.6, 1.2, 2.5, and 5% W/W (N = 6). Each drug was tested in a separate group. Doses were tested in ascending fashion with a minimum of 24 hours washout period between each successive test. The absence of carryover drug effects was confirmed by comparing predrug measurements for each group to those of the preceding trial.

In separate groups of rats, formulations containing lisofylline or pentoxifylline were tested in drug combinations with noneffective concentrations of apraclonidine or linsidomine. Accordingly, the formulations tested in the combination trials included apraclonidine (0.005% W/W) + lisofylline (0.0078, 0.0156, and 0.0313% W/W) (N = 6) and linsidomine (0.4% W/W) + pentoxifylline (0.075, 0.15, and 0.3% W/W) (N = 8).

A third cohort of rats was used to confirm the local site of action of the topical treatments. For this study, the formulations were applied to the contralateral paw (noninjured), and the ipsilateral paw (injured) was tested for antiallodynic effects. The most effective drug combinations were tested in this manner. Accordingly, the combinations included apraclonidine (0.005% W/W) + lisofylline (0.03125% W/W) (N = 6), and linsidomine (0.4% W/W) + pentoxifylline (0.3% W/W) (N = 8). In addition, vehicle (ointment base) application to the ipsilateral paw was also evaluated. All the rats underwent initial baseline PWT assessment before application of the ointment followed by testing at 45 minutes after application. CPIP rats treated with apraclonidine (0.005% W/W) + lisofylline (0.0156% W/W) or linsidomine (0.4% W/W) + lisofylline (0.0318% W/W) were also assessed to compare predrug PWTs and the time course of drug effect on PWTs between 20 minutes and 24 hours after treatment. Also, pre- and postdrug PWTs were assessed in either early or late CPIP rats that were treated with systemic pregabalin (10 mg/kg in sterile saline, intraperitoneal), topical apraclonidine (0.005% W/W) + lisofylline (0.03% W/W), or both systemic pregabalin and the topical combination at the same doses at 120 minutes postdrug for all treatments.

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Laser Doppler Flowmetry

Plantar blood flow (flux) was assessed by using a laser Doppler perfusion and temperature monitor (DRT4, Moor Instruments, Wilmington, DE) with the laser-emitting probe (7.0 mm in diameter) placed between the tori pads along the midline of the plantar hindpaw. Briefly, rats were anesthetized by intraperitoneal administration of a bolus of urethane (30% w/v in saline at a dose of 0.3 mL/100 g body weight) and α-chloralose (1% w/v in water at a dose of 0.125 mL/100 g body weight). α-chloralose was initially dissolved at 55°. Body temperature was monitored throughout the experiment by a rectal thermometer coupled to a heating pad (FHC, Bowdoinham, ME) and was maintained between 36.0° and 39.0°. Deep anesthesia, defined by the absence of spontaneous vibrissae movement, pedal reflexes, and blink responses, was achieved at normal heart rate (monitored by the flux probe) and normal respiratory rate (range: 240–310 bpm and 80–110 breaths/min, respectively). Anesthesia level was maintained throughout the experiment by the intraperitoneal administration of additional anesthetic mixture at half the original dose, as required.

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Plantar blood flow of the hindpaw was monitored in the left hindpaw of rats previously subjected to the I/R injury (CPIP) or of the left paw of sham-injured control animals (Sham CPIP). All CPIP animals were tested between 5 and 10 days after the initial I/R or sham-injury. Before recording plantar blood flow, each rat underwent a period of stabilization lasting 25 to 45 minutes. An initial 2-minute period of baseline blood flux was recorded, followed immediately by a 2-minute occlusion induced by pressurizing and inflating a tight-fitting loop of Tygon® R 3603 tubing (outside diameter = 2.38 mm, inside diameter = 0.79 mm, wall thickness = 0.79 mm) (Sigma-Aldrich, St. Louis, MO) connected to an air-filled pump-driven 60 mL syringe (Model 11, Harvard Apparatus, Montreal, QC), creating a tourniquet around the ankle joint. Ischemia was confirmed by observing a flux reduction >95% of preocclusion value. Two minutes later, the pressure was released quickly, and the tourniquet loosened to allow reperfusion to occur. Predrug PORH was monitored by sampling flux at the rate of 1 per second for 2 minutes after the onset of reperfusion (Fig. 1 for a sample trace). The laser Doppler probe was then removed, and a topical combination of 0.01% apraclonidine and 0.06% lisofylline was applied to the hindpaw, as above. The plantar probe was then replaced and recording resumed. Beginning 45 minutes after ointment application, a second series of baseline recording, 2 minutes occlusion, reperfusion, and 2 minutes recording of the postdrug PORH was performed. Rats were euthanized immediately after the second PORH trial.

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Analysis of Oxidative Capacity in Muscle and Skin by Spectrophotometric Measurement of Tetrazolium Reduction

After pentobarbital overdose, muscle samples of the superficial plantar layer (one each from the flexor hallucis brevis, flexor digiti minimi brevis, and flexor digitorium brevis) weighing between 44 and 70 mg (mean 55.8 ± 2.2 mg), and approximately, 0.7 × 0.8 cm samples of the glabrous plantar skin weighing between 39 and 47 mg (mean 44.8 ± 4.7 mg) were collected from the postischemic and contralateral hindpaws of treated and from the left and right hindpaws of sham-injured rats 60 minutes after the application of either vehicle (ointment base) or a topical combination of 0.01% apraclonidine and 0.06% lisofylline to the postischemic hindpaw of treated animals or the left hindpaw of sham-injured rats. Samples were collected on ice, sliced longitudinally with a scalpel blade, and immediately incubated in a solution of 10% 2,3,5-triphenyltetrazolium chloride in phosphate-buffered saline pH 7.4 for 60 minutes at 37°. The samples were then mechanically homogenized in 0.25 M sucrose in phosphate-buffered saline (100 µL/mg wet weight) and centrifuged at 1000g for 5 minutes. The supernatant was collected, its protein content was assayed by the Bradford method, and was then incubated in acetone (2:1 V/V) at 37° for 40 minutes with occasional agitation to extract the formazan produced by reduction of the tetrazolium salt. After a final centrifugation at 1000g for 10 minutes, 200 μL supernatant was collected from each processed sample, and its absorbance at 485 nm was measured in a spectrophotometer. Background was measured from samples processed without tetrazolium. Net absorbance was calculated by background subtraction and divided by total sample protein.

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Data Analysis

Calculated (von Frey) PWTs were averaged by group, treatment time, and dose. The data were first assessed for normality (Shapiro-Wilk test) and variance homogeneity (Levene test) and subjected to analysis of variance (ANOVA) by using repeated measures (pre- versus postdrug being the first repeated factor and drug dose being the second repeated factor). For single drug trials, drug effects were assessed by pairwise comparisons of group means by using Bonferroni tests after the observation of significant interaction effects of drug by dose in the repeated-measures ANOVA (pre- versus postdrug by dose). For control trials, testing the effects of applying effective topical combinations to the untreated, contralateral paw, pairwise comparisons of mean PWTs were made by using Bonferroni tests after the observation of significant drug by dose interaction effects.

Shifts in drug antiallodynic potency obtained by the use of combination treatments were illustrated by first calculating the difference between pre- and postdrug measures for each rat (ΔPWT), then averaging these differences by treatment group. Mean differences were then plotted on a semilog scale of the amount of drug used per application. For each drug condition tested, unweighted linear regressions of ΔPWT versus log dose were calculated for individual subjects, and the regression x-intercept estimates were then compared between drug alone and drug combination groups by 1-way ANOVA to assess shifts in the dose-response profiles.

The flux measurements recorded from each animal during the PORH period were first normalized to baseline (expressed as a % change from the mean value of the baseline period preceding each occlusion) and then averaged into successive 10-second periods spanning the 120 seconds of reperfusion. These binned data were then grouped by injury and topical drug treatment (sham-vehicle; N = 8, sham-drug; N = 7, CPIP-vehicle; N = 9, and CPIP-drug; N = 10). To compare the magnitude of drug effects among the treatment groups, the total area under the normalized hyperemic response curve for each rat was calculated for each reperfusion period by using the trapezoidal rule. The area under the curve (AUC) measurements were then averaged by treatment group and drug and subjected to a 2-way ANOVA with 1 repeated measure (treatment and pre- versus postdrug as the second repeated factor), followed by pairwise orthogonal contrasts. The effect of drug or vehicle application within each treatment group over the reperfusion time was assessed by subjecting the binned flow data from each group to a 2-way ANOVA with 1 repeated measure, followed by post hoc pairwise comparisons by using the Bonferroni test, when appropriate.

Absorbance measurements were likewise grouped by injury and topical drug treatment (sham-vehicle; N = 8, sham-drug; N = 6, CPIP-vehicle; N = 9 and CPIP-drug; N = 9). The grouped data were first assessed for normality (Shapiro-Wilk test) and variance homogeneity (Levene test) and analysed by 3-way ANOVA with 1 repeated factor: injury (I/R versus sham), drug treatment (drug versus vehicle), and ipsilateral versus contralateral paw (the repeated factor). The observation of a significant interaction effect was followed by post hoc pairwise comparisons by using a Bonferroni test, when appropriate.

Statistical computations were performed with Statistica, version 6 (Statsoft, Tulsa, OK) or Prism 5 (GraphPad Software, La Jolla, CA). Significance was defined as P < 0.05.

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Effects of Topical Treatments on Mechanical Allodynia in CPIP Rats

In the single drug pharmacological trials, topical hindpaw application produced significant, dose-related antiallodynic effects in CPIP rats for each of the 4 drugs tested (Fig. 2, A–F). Apraclonidine significantly elevated PWTs above predrug levels at 0.02% and 0.04% W/W (P = 0.023 and P = 0.009, respectively; drug by dose interaction P = 0.019); linsidomine at 1.6% W/W (P = 0.005; drug by dose interaction P = 0.025); lisofylline at 0.125 and 0.25% W/W (P = 0.0008 and P = 0.044, respectively; drug by dose interaction P = 0.038); and pentoxifylline at 5% W/W (P = 0.0003; drug by dose interaction P = 0.002). Application of ointment base alone (vehicle) was without effect on ipsilateral PWTs for every drug tested (data not shown).

The combination of apraclonidine 0.005% W/W with lisofylline significantly elevated PWTs above predrug values at 0.0313% W/W (P = 0.037; pre- versus postdrug by dose interaction P = 0.012; Fig. 3A) and shifted the x-intercept value of the log dose-response curve for lisofylline from (mean ± SEM) 0.154 ± 0.046 to 0.012 ± 0.003 mg (P = 0.0018; Fig. 3B). When combined with 0.4% W/W linsidomine, pentoxifylline significantly increased PWTs at 0.30% W/W (P = 0.0232; pre- versus postdrug by dose interaction P = 0.0046; Fig. 3C). The pentoxifylline log dose-response curve was shifted to the left, and the x-intercept dose decreased from 1.318 ± 0.072 to 0.542 ± 0.127 mg (P = 0.001; Fig. 3D).

Application of drug combinations to the contralateral paw was without effect on the PWTs measured from the injured paw, when compared with predrug values. PWTs were thus significantly lower after contralateral paw treatment than after ointment application to the injured paw after treatment with apraclonidine 0.005% W/W + lisofylline 0.03125% W/W (P = 0.014; treatment by drug [pre- versus postdrug] interaction P =0.117; Fig. 4A) or linsidomine 0.4% W/W + pentoxifylline 0.3% W/W (P = 0.047; treatment by drug [pre- versus postdrug] interaction P = 0.005; Fig. 4B). In addition, for all combinations tested, application of vehicle to the ipsilateral paw was without effect on the PWTs measured from the injured paw, when compared with predrug values (for all treatments, treatment by drug [pre- versus postdrug] interaction P = 0.473).

In time course trials, 2 low-dose combinations produced prolonged reductions in mechanical allodynia in the treated (ipsilateral) hindpaw of CPIP rats. Thus, apraclonidine (0.005% W/W) + lisofylline (0.0156% W/W) increased PWTs above predrug baseline values when measured at 45 minutes after drug application (P = 0.002; effect of time after drug administration P = 0.0003; Fig. 5A). A combination of linsidomine (0.4% W/W) + lisofylline (0.0318% W/W) increased PWTs above predrug baseline values when measured at 45 minutes and 3 hours after drug application (P = 0.002; P =0.021, respectively; effect of time after drug administration P = 0.0004; Fig 5B).

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Effects of Systemic Administration of Pregabalin Along with Topical Treatments on Mechanical Allodynia in Early Versus Late CPIP Rats

The effect of pregabalin in early CPIP rats was found to depend on the administration of topical drugs (drug by group interaction P = 0.0073). Thus, systemic administration of 10 mg/kg intraperitoneal pregabalin did not significantly affect allodynia (Fig. 6), with postpregabalin PWTs remaining unchanged from predrug baseline or postdrug vehicle PWTs (P = 0.996 and P = 0.994, respectively). A higher dose of pregabalin (30 mg/kg intraperitoneal) was found to be significantly antiallodynic at 1 (P = 0.0007) and 3 hours (P = 0.011) after injection (data not shown). However, the noneffective 10 mg/kg dose of systemic pregabalin significantly enhanced the antiallodynic effect of the topical combination of apraclonidine + lisofylline (P = 0.046). In early CPIP animals, topical apraclonidine + lisofylline significantly elevated PWTs compared with predrug baseline PWTs (P = 0.025), and systemic pregabalin given along with the topical treatment significantly elevated PWTs above both predrug baseline PWTs (P = 0.0007) and postdrug PWTs of the topical treatment alone (P = 0.025). In late CPIP animals, pregabalin and the topical treatment had no comparable interaction (P = 0.104). Pregabalin was very effective when used alone at 10 mg/kg (P =0.010), but was no more effective when given in addition to the topical treatment (P = 0.261 versus predrug baseline).

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Effects of Topical Combination Treatment on PORH in CPIP and Sham Rats

In early CPIP rats, all 4 treatment groups exhibited PORH at the end of each ischemia period, as indicated by a rapid elevation in flow gradually abating toward preocclusion levels over the course of the measurement period (Fig. 7, A–D). However, the magnitude of the flow increase (Fig 7, A–D) and the changes in AUC (Fig. 8) varied depending on treatment group and topical drug application, as indicated by a significant interaction between these 2 variables (P = 0.019). This occurred in part because both CPIP groups displayed significantly weaker hyperemic responses to the predrug occlusion compared with their controls (CPIP-drug versus sham-drug: P = 0.004 and CPIP-vehicle versus sham-vehicle: P = 0.029; Fig. 8). In addition, drug-treated CPIP animals but not vehicle-treated CPIP rats displayed significantly increased hyperemic responses to the second ischemic challenge (pre- versus postdrug: P = 0.0001 vs P = 0.675, respectively; Fig. 8). The PORH AUC measured from sham-injured animals after drug or vehicle application did not change (P = 0.621 and P = 0.676, respectively; Fig. 8). A similar pattern was seen in the time course data: only drug-treated CPIP animals displayed a significant increase in flow after drug application (P = 0.508, P = 0.0003, P = 0.064, and P = 0.174 for the CPIP-vehicle (Fig. 7A), CPIP-drug (Fig. 7B), sham-drug (Fig. 7C), and sham-vehicle (Fig. 7D) groups, respectively). The increase in flow in CPIP animals given apraclonidine and lisofylline was detected from the onset to 80 seconds of the reperfusion period. (Fig. 7B).

In contrast to the decreased PORH in early CPIP rats, flux measures displayed a large PORH in late CPIP rats (effect of time since start of reperfusion P < 0.00001, data not shown). Also, in late CPIP rats, there were no effects of topical drug application compared with vehicle treatment (P = 0.721), no effects of injury (CPIP or sham-injury; P = 0.127), and no interactions between drug and injury (P = 0.509).

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Effects of Topical Combination Treatment on Skin and Muscle Oxidative Capacity in CPIP and Sham Rats

In early CPIP and sham groups, formazan production in skin was affected by the presence of an I/R injury and the topical drug treatment (injury by drug by side interaction P = 0.0182). Thus, skin from the injured hindpaw of CPIP animals treated with vehicle displayed lower levels of formazan than that of their uninjured contralateral hindpaw (P = 0.0067) and that of the vehicle-treated hindpaw of sham-injured animals (P = 0.0179). Topical application of apraclonidine and lisofylline to the CPIP hindpaw resulted in higher levels of formazan in the skin compared with that in skin samples from vehicle-treated CPIP rats (P = 0.0039). Formazan levels measured after drug combination treatment in CPIP hindpaw skin did not differ from those obtained from the uninjured contralateral hindpaw skin or from drug- or vehicle-treated hindpaw skin from sham-injured animals (P = 0.556). Drug application to the uninjured hindpaw of CPIP rats or to either hindpaw of sham-injured animals was similarly without effect compared with vehicle treatment (P =0.992). Vehicle application was without effect (compared with the contralateral untreated side).

A similar pattern of effects was noted in muscle tissue (injury × drug × side interaction P = 0.0042; Fig. 9). Formazan levels were lower in muscle samples from the I/R-injured hindpaw of CPIP rats compared with those from the uninjured contralateral hindpaw (P = 0.001), or to those obtained from sham-injured animals (P = 0.0146 and P = 0.0263 for sham-injured and contralateral hindpaw, respectively). Topical application of apraclonidine and lisofylline increased formazan levels in CPIP hindpaw muscle to a level similar, not different, from sham-injured hindpaws receiving the topical combination (P = 0.1061) or vehicle (P = 0.3003). Topical application of apraclonidine and lisofylline to the contralateral hindpaw was without effect in samples from the allodynic hindpaw (P = 0.8969), or in hindpaw muscle samples obtained from sham-injured rats (P = 0.4599). Vehicle application was without effect (compared with the untreated contralateral side, P = 0.9901; Fig. 9).

In late CPIP and sham groups, the effects of I/R injury were markedly different: formazan levels in hindpaw skin and muscle did not differ either between vehicle-treated CPIP and contralateral samples or between vehicle-treated CPIP and sham samples (P = 0.368 for all interactions and main effects). Application of an apraclonidine and lisofylline combination at the same concentrations used in early CPIP rats had no effect on late CPIP, vehicle-treated, or contralateral samples (Fig. 9).

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In the present study, we show that topical combinations of drugs aimed at reducing microvascular dysfunction synergistically relieve allodynia in CPIP rats. Thus, a combination of α2-adrenergic agonists or NO donors with either PDE or PA inhibitors produce antiallodynic effects that are much more potent than the single drugs alone. In CPIP rats, these topical combinations have an antiallodynic efficacy greater than systemic acetaminophen, ibuprofen, dexamethasone, or amitriptyline.45 The topical combinations also produced maximal effects that are equivalent to those produced by systemic doses of morphine and pregabalin.45,46 The concentrations of topical drugs used in the present combinations are much lower than the recommended concentrations used for neuropathic/ischemic pain or other clinical uses, which typically use apraclonidine (0.5%–1.0%), lisofylline (0.5%–5%), pentoxifylline (5%–15%), and linsidomine (2%). Thus, the combinations of these drugs resulted in significant antiallodynic effects at topical concentrations that are 5 to 640 times lower than those used typically for the single drugs. Thus, by using combinations that greatly enhance antiallodynic effects, it is possible to use even lower topical drug concentrations that are less likely to produce adverse side effects. Also, combinations that use apraclonidine, which does not cross the blood-brain,47 decrease arterial blood pressure largely by actions at presynaptic α2A and/or imidazoline receptors in the brainstem.48

We also show that one of these combinations (apraclonidine + lisofylline) produced a significant increase of PORH and improved oxidative capacity in the hindpaw skin and muscle of early CPIP rats, which normally have depressed PORH, as well as exhibit poor microvascular perfusion and reduced oxidative capacity in hindpaw muscle.7,8 Although topical treatment with apraclonidine + lisofylline significantly increased PORH in CPIP rats, it had no effects in sham rats, while vehicle treatment had no effect in either group. The current results support our earlier findings that systemic administration of the PDE4 inhibitor pentoxifylline reduces both allodynia and microvascular dysfunction, as evidenced by reduced PORH, in CPIP rats.9 PORH has been shown to be a robust measure to examine microvascular function49 and has been shown to be reduced in various conditions in which microvascular dysfunction has been suspected.50 PORH has also been used to assess microvascular function in CRPS patients. Koban et al.5 reported that PORH was reduced in the affected arms of CRPS patients, compared with the contralateral limb or the limbs of control subjects. These results were confirmed by Dayan et al.4 who found reduced PORH in both affected arms and legs of CRPS patients. Moreover, Kurvers et al.1 also showed that PORH was reduced in the affected limbs of cold (stage II and III) CRPS patients.

Previously, studies exhibiting analgesic effects of topical/regional clonidine in CRPS patients24,25 or in rat models of neuropathic pain51 did not address the role of blood flow or microvascular dysfunction. However, studies27,52,53 that used either a topical NO donor or a systemic PDE inhibitor have compared the analgesic effects of these treatments and their effects on blood flow in CRPS patients. Thus, Groeneweg et al.27 showed that a topical ointment of the NO donor isosorbide dinitrate that caused vasodilation, and a 4° to 6° increase in skin temperature, produced analgesia in the most cold CRPS patients. However, in a second study,52 these authors reported that the same NO donor applied topically in Vasoline®, which did not affect vasodilation or skin temperature, was unable to reduce pain scores in CRPS patients, suggesting that the topical formulation was suboptimal. In a separate study, Groeneweg et al.53 reported a small 15% reduction in pain scores with a systemic treatment with the PDE inhibitor tadalafil that did not significantly alter skin temperature, relative to placebo, in CRPS patients. While not particularly impressive, none of these studies used combinations of α2A receptor agonists or NO donors with PDE or PA inhibitors as we have here, to target both reduced blood flow and microvascular dysfunction.

In the present study, we also found that 10 mg/kg systemic pregabalin was ineffective at relieving allodynia in early CPIP rats, for which hindpaw PORH and oxidative capacity was suppressed, but systemic pregabalin enhanced the efficacy of low-dose apraclonidine + lisofylline in early CPIP rats. In late CPIP rats (i.e., >18 days after I/R injury), which show normal hindpaw PORH and oxidative capacity, 10 mg/kg pregabalin was maximally effective, and its antiallodynic effect could not be further enhanced by the topical combination, which itself lacked efficacy in late CPIP rats. These findings suggest that CPIP rats evolve from a phase in which hindpaw microvascular dysfunction is critical, and our topical combinations are most effective at relieving allodynia, to a phase in which hindpaw microvascular dysfunction has resolved and systemic pregabalin is more effective. We expect that allodynia during this second phase is dependent on central sensitization, since allodynia in late CPIP, but not early CPIP rats, is also relieved by intrathecal treatment with an inhibitor of protein kinase M-zeta, an isoform of protein kinase C that has been shown to be critical for the maintenance of spinal nociceptive plasticity.53,54

While our studies suggest that the topical combinations reduce allodynia by improving microvascular function, we cannot exclude other anti-inflammatory or nonspecific effects. In the case of pentoxifylline or lisofylline, these drugs produce multiple anti-inflammatory effects including anticytokine, and immune system modulator effects.40–42 However, most of the anti-inflammatory effects of pentoxifylline and lisofylline stem from their actions to reduce microvascular dysfunction: by reducing the chemotactic signals from damaged endothelial cells that attract immune cells, and by reducing the production of oxygen free radicals and cytokines after improving the oxygenation of tissues affected by poor capillary blood flow. Since we only measured the effects of our treatments on blood flow and oxidative capacity, we cannot speculate on additional anti-inflammatory effects. However, additional studies show that our combinations are not effective in late CPIP rats, for which microvascular dysfunction has resolved (when we have shown here that PORH and oxidative capacity, and previously that muscle perfusion and oxidative capacity are normal).

It could also be argued that both clonidine and apraclonidine produce nonspecific effects at α1-adrenoceptors. Like most drugs, the specificity of clonidine and apraclonidine depends on its tissue concentration. For example, evidence suggests that apraclonidine is a relatively selective α2-adrenergic agonist, and that its affinity at α2-adrenoceptors is 100- to 528-fold greater than its affinity at α1-adrenoceptors.55 In most tissues, α2-adrenoceptors are predominantly prejunctional receptors, producing negative feedback on NE release from sympathetic postganglionic neurons. They may also be present on smooth muscle cells of resistance vessels and cause vasoconstriction, although they are typically extrajunctional and do not normally respond to NE released from sympathetic postganglionic neurons.56,57 In some tissues, such as the anterior segment of the eye (conjunctiva, iris, ciliary body), α2-adrenergic agonists produce significant vasoconstriction, since there is a much higher proportion of postjunctional vasoconstrictive α2-adrenoceptors.58 Thus, apraclonidine produces vasoconstriction in the anterior segment of the eye, while producing no vasoactive effects in the posterior segment where postjunctional α2-adrenoceptors are less abundant (retina, choroid, and optic nerve).59

In conclusion, combining topical drugs designed to enhance local arterial and capillary blood flow to overcome microvascular dysfunction produce effective antiallodynic effects in an animal model of CRPS. We confirmed that one of these combinations reduced microvascular dysfunction, as it increased PORH and oxidative capacity in early CPIP rats. Furthermore, the dramatic enhancement of antiallodynic effects of PA/PDE inhibitors when combined with α2A receptor agonists or NO donors should reduce the potential systemic side effects of these low-concentration topical treatments.

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Name: André Laferrière, BA.

Contribution: This author helped in animal testing, statistical analysis, and figure and manuscript preparation.

Attestation: André Laferrière attests to the integrity of the original data and the analysis reported in this manuscript and is the archival author.

Conflicts of Interest: André Laferrière has interest in patent, potential commercialization.

Name: Rachid Abaji, BSc.

Contribution: This author helped in animal testing.

Conflicts of Interest: Rachid Abaji has no conflicts of interest to declare.

Name: Cheng-Yu Mark Tsai, BSc.

Contribution: This author helped in animal testing.

Conflicts of Interest: Cheng-Yu Mark Tsai has no conflicts of interest to declare.

Name: J. Vaigunda Ragavendran, PhD.

Contribution: This author helped in animal testing and manuscript preparation.

Conflicts of Interest: J. Vaigunda Ragavendran has interest in patent, potential commercialization.

Name: Terence J. Coderre, PhD.

Contribution: This author helped study design and figure and manuscript preparation.

Attestation: Terence J. Coderre attests to the integrity of the original data and the analysis reported in this manuscript.

Conflicts of Interest: Terence J. Coderre has interest in patent, potential commercialization.

This manuscript was handled by: Martin S. Angst, MD.

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The authors would like to thank Pfizer Corporation for their generous donation of pregabalin.

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