Poly- l -Arginine Topical Lotion Tested in a Mouse Model for Frostbite Injury

Abstract

Background

Frostbite injury occurs when exposure to cold results in frozen tissue. We recently reported a novel mouse model for frostbite injury to be used in screening potentially therapeutic drugs and other modalities.

Objective

We used the mouse skin frostbite model to evaluate the effect of poly-l-arginine contained in lotion (PAL) applied topically to involved skin.

Methods

Sixty mice were studied in a randomized, double-blind method. Standardized 2.9-cm-diameter circles were tattooed on the mouse dorsum. Magnets snap frozen in dry ice (–78.5°C) were used to create a frostbite injury on skin within the circle as a continuous 5-minute freeze. Mice were treated with prefreeze placebo, postthaw placebo, combined prefreeze and postthaw placebo, prefreeze with PAL, postthaw with PAL, or combined prefreeze and postthaw with PAL. Appearance, healing rate, tissue loss, and histology were recorded until the wounds were healed.

Results

Application of PAL before inducing frostbite injury resulted in decreased tissue loss as compared with other treatment conditions.

Conclusions

Applying PAL topically to frostbitten mouse skin caused decreased tissue loss. Poly-l-arginine should be studied further to determine whether it is a beneficial therapeutic modality for frostbite injury.

Introduction

We recently invented a novel mouse skin model of induced frostbite injury, with the intention of using the model to allow quantification of the surface area of involved skin, histology of the wound, rate of wound healing, and skin loss in a standardized fashion after frostbite injury. ¹ One purpose for this model is to facilitate efforts to establish or refute efficacy of preventive techniques or therapeutic interventions in a screening manner. To this end, we selected a compound containing poly-l-arginine, which supports nitric oxide synthesis, for testing. Our hypothesis was that we might see a beneficial effect on induced frostbite injury.

Nitric oxide synthase (NOS) is a group of enzymes that helps catalyze production of nitric oxide (NO) from l-arginine, NADPH (the reduced form of nicotinamide adenine dinucleotide phosphate), and molecular oxygen. The signaling molecule NO plays conflicting physiological roles. It helps modulate vascular tone and angiogenesis, but is also a free oxygen radical and can be toxic at high concentrations, particularly in inflammatory disorders. ² At normal NO levels, there is protection against vascular inflammation and oxidative injury. But when NO rises above physiological levels, it reacts with superoxide anions to produce peroxynitrite anion (ONOO–). This compound combines with lipids, proteins, and DNA to form nitrosoperoxocarbonate, leading to oxidative damage of tissues. ³ Normally not present in resting cells, the inducible form of NOS (iNOS) is part of an immune response induced mainly as a result of the presence of cytokines, producing NO as a defense mechanism. Activation of iNOS raises NO independent of cellular calcium levels, which can have protective or destructive implications.

l-Arginine is one of the 20 most common amino acids and is manufactured by the body when needed. The amino acid plays a role in cell division and tissue repair, and reduces the healing time of bone injuries. ⁴ l-Arginine is a substrate for NOS, but is limited inside cells. Poly-l-arginine effectively transfers l-arginine across cell membranes to serve as an intracellular reservoir of l-arginine. ^{5 –}9 In an oxidative environment and without sufficient l-arginine, iNOS generates superoxide radicals in a manner that injures endothelium and can cause intimal hyperplasia and smooth muscle accumulation within arteries, which unfavorably thickens their walls, resulting in a form of vasculopathy. ¹⁰ With sufficient l-arginine, endothelial NOS produced by vascular tissue generates NO, which inhibits intimal hyperplasia and monocyte adherence and infiltration, slowing smooth muscle cell proliferation and inducing smooth muscle cell apoptosis. ¹⁰ Nitric oxide enables veins to strengthen their walls by allowing intimal cells to lengthen and thicken. ¹¹ It also promotes vasodilation by a variety of mechanisms.

Poly-l-arginine also has been shown to penetrate skin and to have the ability to deliver, via covalent attachment, cargo into skin. ^{12 –}14 Because reperfusion injury might be a component of frostbite injury and NO can be protective from ischemia–reperfusion injury, we elected to observe the impact on a mouse frostbite injury of topical application of poly-l-arginine contained in a lotion. ¹⁵

Methods

Frostbite Model

The method of Auerbach et al ¹ was used and is briefly recapitulated here. Sixty 8- to 12-week-old C57Bl/6J male mice (Jackson Laboratories, Bar Harbor, ME) were used in a study approved by the Stanford Institutional Review Board and in accordance with Stanford University Institutional Animal Care and Use Committee Guidelines. The animals were housed 5 per cage before and after surgery in a temperature-controlled facility under a 12-hour light/dark cycle.

Skin preparation

Twenty-four hours before day 0, mice were individually anesthetized using isoflurane (Baxter, Deerfield, IL). The dorsal skin surface from the base of the neck to the top of the rear haunches was shaved with an electric clipper, after which a depilatory cream (Nair, Church & Dwight, Princeton, NJ) was applied for 2 minutes to remove any remaining hair. After depilation, the skin was cleansed with a 70% isopropyl alcohol swab. A circle of uniform diameter 2.9 cm was gently traced on the skin with a permanent marker using a silicone sheet template (Invitrogen, Grand Island, NY). The marked circle was then permanently darkened by using an animal tattoo machine (Animal Identification and Marking Systems Inc, Hornell, NY).

Freeze method

Twenty-four hours after skin preparation, ceramic (ferrite) magnets (diameter 0.5 inches, thickness 0.219 inches, weight 3.5 g) were placed in crushed dry ice (–78.5° C) and allowed to freeze for 15 minutes. Using fingers, the back skin of the mouse was lifted into a skin fold and then 2 frozen magnets were placed so that they adhered from opposite sides of the intervening skin fold. Sets of 2 cooled magnets were left in place for 1 minute, then removed to allow new magnets to be immediately placed in the same location against the frozen tissue. The magnet exchange was repeated for a total of 5 applications, resulting in a freeze time slightly longer than 5 minutes. After 5 magnet placements and removals, the skin was allowed to completely thaw. Mice were given subcutaneous injections of buprenorphine (0.05 mg/kg) for analgesia after the thaw. No dressings were applied to the wounds.

Application of poly-l-arginine suspended in AmLactin lotion

One hundred micromolar poly-l-arginine (Lumen Therapeutics, LLC, Menlo Park, CA) was homogenized in 15 mL of AmLactin (Upsher-Smith Laboratories, Maple Grove, MN), a commercially available skin-moisturizing lotion. Placebo lotion was AmLactin absent poly-l-arginine. The mice were divided into 6 groups: poly-l-arginine in AmLactin (PAL) applied prefreeze; PAL applied postfreeze; PAL applied both prefreeze and postfreeze; AmLactin alone applied prefreeze; AmLactin applied postfreeze; and AmLactin applied both prefreeze and postfreeze. For groups consisting of pretreatment only, 1 hour before induced freeze injury 0.5 mL of room temperature AmLactin or PAL was rubbed into the skin with fingers until it was uniformly distributed. For groups receiving posttreatment only, 0.5 mL of room temperature AmLactin or PAL was rubbed into thawing skin with fingers until uniformly distributed. For groups receiving both pretreatment and posttreatment AmLactin or PAL, both actions described above were performed. All mice were monitored until the AmLactin or PAL was visually noted to be completely absorbed.

Wound Surface Analysis and Tissue Loss Quantification

Digital photographs of the wounds were taken after the thaw on day 1 and every other day thereafter until day 27, at which time the wounds were completely healed by visual inspection by an observer blinded to the treatment conditions. Wound surface area was determined by tracing the wound margin displayed by photograph with a fine-resolution computer mouse. The number of pixels corresponding to a surface area measurement was calculated using ImageJ software (National Institutes of Health, Bethesda, MD). Wound area was reported as percent area of the original maximum frostbite injury ([original wound area – wound area on day “x”] / original wound area). Tissue loss and contraction was reported as area of the original maximum skin area inside the tattooed circle ([original surface area within the tattooed boundary – surface area within the tattooed boundary on day “x”] / original surface area within the tattooed boundary).

Histopathology Examination

The animals were humanely killed, and the entirety of the frostbite wounds were harvested from 2 mice per group on each of days 3, 7, 14, 21, and 27. The tissue samples were fixed overnight in 4% paraformaldehyde, and then embedded in paraffin. Sections (7 µm) were cut and stained with hematoxylin and eosin. Analysis was performed by a board-certified dermatopathologist. Tissue necrosis, reepithelialization, healing rate, acute inflammation, chronic inflammation, fibroblast maturation, collagen deposition, neovascularization, and the overall healing score were analyzed based on a modified version of a scoring system suggested by Abramov et al. ¹⁶ All measurements were recorded on a scale of 0 to 3, except for the overall healing score.

Statistical Analysis

Surface area of the wound, tattooed region, and aforementioned silicone stencil were measured in pixels and presented as a mean ± SD. Statistical significance was accepted at P < .05. Statistical analyses were performed using SPSS (Version 17.0; SPSS Inc, Chicago, IL).

Results

Wound Closure and Analysis

The placement of 2 frozen magnets on opposite sides of the dorsal fold resulted in a single frostbite injury spanning both circles and the intervening tissue. The skin between the magnets, as well as a small portion of the surrounding area, was affected by the cold. This single frostbite injury was subsequently monitored (Figure 1).

Figure 1

Gross appearance of wound progression at various times after induced frostbite injury under different treatment conditions. Variations among treatments can be seen at intermediate times, although all wounds were completely healed by day 27.

Wound healing

Mice pretreated with placebo AmLactin were completely healed by day 21. Mice pretreated with PAL were completely healed by day 25. Mice posttreated with placebo AmLactin were completely healed by day 17. Mice posttreated with PAL were completely healed by day 26. Mice both pretreated and posttreated with placebo AmLactin were completely healed by day 27. Mice both pretreated and posttreated with PAL were completely healed by day 21. The mice left untreated (control) were completely healed by day 27 (Figure 2).

Figure 2

Wound healing showing the percentage of original wound area healed by day. Each data point used to create a curve is the mean healed percentage (confidence interval = 97%) of the original wound area for all mice treated for that condition.

Tissue loss

Tissue loss is the amount of skin area within the bounded area of the tattoo that disappears during the healing process. Mice pretreated with placebo AmLactin lost a mean of 43% of the original skin surface area bounded by the tattoo. Mice pretreated with PAL lost a mean of 39% of the original tattooed skin surface area bounded by the tattoo. Mice posttreated with placebo AmLactin lost a mean of 56% of the original skin surface area bounded by the tattoo. Mice posttreated with PAL lost a mean of 56% of the original tattooed skin surface area bounded by the tattoo. Mice pretreated and posttreated with placebo AmLactin lost a mean of 43% of the original skin surface area bounded by the tattoo. Mice pretreated and posttreated with PAL lost a mean of 37% of the original skin surface area bounded by the tattoo. Mice left untreated (control) lost a mean of 46% of the original skin surface area bounded by the tattoo (Figure 3).

Figure 3

Percentage surface area of tissue lost within the tattooed boundary compared with the original surface area within the boundary. Mice pretreated with poly-l-arginine before the frostbite injury lost less tissue than did mice under any other treatment condition. Each data point used to create a curve is the mean percentage (confidence interval = 95%) loss of the original tattooed area for all mice treated for that condition.

Histology

Histological analysis revealed no significant trend among the treatment conditions (online Supplementary Figure).

Discussion

One goal of establishing our mouse frostbite model was to allow the model to be used for evaluation of pharmacological and other approaches to frostbite prevention and treatment. We used the model to evaluate the effect of topical PAL on a controlled frostbite injury.

Regardless of treatment applied, all wounds were completely healed by day 27. However, certain treatments resulted in shorter healing times than others. The treatments involving poly-l-arginine (pretreatment and posttreatment only) resulted in a slower healing time than was observed in untreated mice. Preapplication and postapplication only of the placebo AmLactin allowed the wounds to heal faster than did the other treatment groups. Moisturizing would be the presumed action. It is traditionally taught that moisturizing human skin before cold exposure might increase the propensity for frostbite, so perhaps the beneficial effect we observed in mice treated with AmLactin can be attributed to the postapplication aspect of treatment. More likely, the traditional empiric advice to avoid skin hydration might be incorrect or not apply to mice, because mice receiving a pretreatment of any sort, whether placebo or poly-l-arginine, lost less tissue than did control mice.

Our most encouraging finding was that mice that received poly-l-arginine as a pretreatment (both pretreatment only and pretreatment and posttreatment) showed less tissue loss than did mice under all other treatment conditions. The importance of pretreatment might indicate that after tissue injury has occurred as a result of frostbite, our topical compound could not effectively be absorbed through skin, as compared with absorption through uninjured skin.

Histologically, no themes were observed among the treatment conditions. This could be owing to the small sample sizes (n = 2) taken at each time. An increased sample size centered around approximately days 7 to 14 might show changes in the degree or rate of chronic inflammation, amount of granulation tissue, and neovascularization. ¹

As previously, we note certain limitations with the mouse frostbite model. ¹ Notably, there is not a blistering phase in our model. We did not study a deep tissue or comprehensive limb injury. Mice are not humans, and we do not expect to find absolute comparability in clinical or pathologic presentation. For these and other reasons, it may be necessary to advance PAL to another animal model, such as rabbit limb (which demonstrates deep tissue loss), before undertaking human investigation. However, to provide a counterpoint, given the known human safety profile of poly-l-arginine, it may be possible to take this drug directly to a human trial for prevention or treatment of frostbite. Also, it may be that another method of drug administration, such as parenteral injection, may be more efficacious than topical application. Finally, further work will hopefully better elucidate the intersection of this specific drug’s entry into tissue and therapeutic effectiveness with the pathophysiology of frostbite injury to optimize the method and timing of administration.

Conclusions

We describe application of a poly-l-arginine-containing topical cream in a mouse model of frostbite. We were able to demonstrate that pretreatment only and pretreatment and posttreatment application of PAL showed decreased total tissue loss compared with placebo. The specific placebo treatment we used may aid in treatment or prevention of frostbite. We propose that poly-l-arginine warrants further evaluation as an effective preventive or therapeutic pharmacological intervention for larger animal deep tissue frostbite, and perhaps eventually for human frostbite.