A novel murine model of cyclical cutaneous ischemia-reperfusion injury

Journal of Surgical Research 116, 172–180 (2004) doi:10.1016/S0022-4804(03)00227-0

A Novel Murine Model of Cyclical Cutaneous Ischemia-Reperfusion Injury Russell R. Reid, M.D, PhD., Alan C. Sull, B.S., Jon E. Mogford, Ph.D., Nakshatra Roy, Ph.D., and Thomas A. Mustoe, M.D., F.A.C.S. 1 Division of Plastic and Reconstructive Surgery, Northwestern University, Chicago, Illinois, USA Submitted for publication May 21, 2003

Background. Increasing evidence points to a principal role of ischemia-reperfusion in the pathogenesis of chronic skin ulceration, including pressure sores, diabetic ulcers, and venous ulcers. An incomplete understanding of this process and the limitations of current animal models of chronic wounds mandate a reproducible model in mice, in which transgenic and knockout technology are continually evolving. Materials and methods. A murine model of chronic skin ulceration based on cyclical magnetic compression is presented. Forty-three C57BL/6J mice underwent varying degrees of cyclical compression with defined periods of reperfusion. Injury was measured grossly as regional necrosis, and tissue was harvested for histology, DNA electrophoresis, and reverse transcription polymerase chain reaction. Results. Skin necrosis became apparent only 12 h post cycling, and was cycle-responsive and quantitative in cycled subjects. Histopathologic analysis revealed a statistically significant doubling of the leukocyte count in sections from compressed skin versus sham controls. Moreover, apoptotic DNA laddering was evident in post ischemic skin and absent in controls. Real-time PCR analysis revealed a 300-fold higher expression in iNOS mRNA from cyclically compressed skin compared with normal skin: such expression was temporal in nature. Conclusions. A murine model of pressure necrosis, which bears all of the gross, histological, and molecular features of ischemia-reperfusion injury, has been established. Application of this model to the vast number of transgenic mice available will further our understanding of the mechanism of pressure sore development. © 2004 Elsevier Inc. All rights reserved. 1 To whom correspondence should be addressed at Division of Plastic and Reconstructive Surgery, Northwestern University/ Feinberg School of Medicine, 675 North St. Clair, Suite 19-250, Chicago, IL 60611. E-mail: [email protected].

0022-4804/04 $30.00 © 2004 Elsevier Inc. All rights reserved.

Key Words: ischemia reperfusion; wound healing; apoptosis; mouse; recurrent ischemia. INTRODUCTION

The chronic pressure sore has been a clinical problem for years, amounting in more than 1 billion dollars of medical resources annually [1]. Yet the precise pathophysiologic basis for the development of such troublesome wounds remains unclear. Tissue ischemia has long been thought to be the key event in pressure sore development, dating back to the pioneering work of Kosiak [2]. From these sentinel studies, an external pressure of ⬎35 mmHg was engendered as a prerequisite for necrosis. Curiously, in the initial canine model described, prolonged ischemia alone (⬎100 mmHg for at least 10 h) did not invoke skin necrosis, suggesting other factors at play. Since then, several other etiologies have been hypothesized, from direct mechanical damage [3], to pressure and friction [4], to differences in soft tissue coverage in different species [5], and the combination of ischemia and paraplegia [6]. More recently, the phenomenon of ischemiareperfusion (I-R) has been postulated and shown to be an inciting event in not only pressure ulceration [7, 8] but also in other chronic wounds such as venous stasis and diabetic foot ulcers [9, 10]. The mechanism of I-R injury, however, is quite complex because several soluble and cellular components have been shown to be causative. Along with this limited knowledge comes the arduous search for a reproducible animal model that is clinically relevant. Early models of pressure necrosis relied upon the application of large mechanical compression devices on the femoral trochanters of dogs [2] or pigs [5, 6], which, in turn, required an anesthetized, laterally recumbent subject. A major drawback to these models was the confounding effect of anesthesia on local tissue hemodynamics. With the use

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FIG. 1. Dimensional illustration of ring magnets used for recurrent I-R injury and calculation of magnetic flux density (B). The magnetic flux density was calculated from the relationship B ⫽ (F ⫻ 2 ⫻ ␮ 0/A) 1.2, where F is the compressive force and the product of the hydrostatic pressure (P) and the area (A) of the magnetic surface, ␮ 0 is the magnetic permeability of the skin. (Color version of figure is available online.)

of an implantable steel sheet, Peirce et al. [11] introduced magnetic compression as a safe, effective, and reproducible way to study chronic pressure ulceration in the rat. The rat, however, is limited in genetic variation and so the ultimate utility of this species in understanding I-R mediated chronic skin necrosis on a molecular basis is limited. In this work, we extend the concept of cyclical magnetic compression to the mouse, and for the first time demonstrate its validity in studying I-R-mediated events via gross, histological, and molecular markers. Our work points to the importance of apoptosis and inducible nitric oxide synthase as early indicators of recurrent I-R injury of the skin, which in turn may serve as a guideline as to when to intervene prior to irreversible damage. MATERIALS AND METHODS Murine Subjects C57BL6/J mice were obtained from Harlan (Indianapolis, IN), housed in a separate animal facility, and fed ad libidum. They were maintained by standard Northwestern University AUC protocols. At the end of all experiments, mice were euthanized by xylazine: ketamine overdose intraperitoneally and cervical dislocation.

Magnets and Calculation of Magnetic Flux Density (B) Ceramic ring magnets (outer diameter 1⁄2 ”; inner diameter 3/16”; thickness 1/8”; weight 1.6 g) were obtained from McMaster-Carr, Inc. (Long Island, NY). The determination of the theoretical magnetic flux density B, based on the desired hydrostatic pressure to induce

skin necrosis has been previously described [11]. It has been demonstrated that an external pressure exceeding the microcapillary pressure of the local tissue (12–32 mmHg 2) is sufficient to induce necrosis. Incorporating 50 mmHg as the desired pressure [11], a ceramic ring magnet of the dimensions described would need to have a magnetic flux density of 1296 Gauss to induce necrosis (Fig. 1). All magnets were autoclaved prior to implantation.

Implantation Procedure Briefly, 20 –30 g C57BL6/J mice were anesthetized with an intraperitoneal injection of ketamine hydrochloride and xylazine hydrochloride followed by chemical depilation of the back. The back skin was marked with a template that resulted in a skin paddle bearing 4-cm cranio-caudad and 3-cm transverse dimensions. Following sterile preparation of the back skin and prophylactic treatment of the mice with penicillin intramuscularly a dorsal midline incision was carried down to the fascia. Using blunt dissection, a ceramic ring magnet (outer diameter 1/2”; inner diameter 3/16”; thickness 1/8”; weight 1.6 g) was tunneled under the skin to a point 0.5 cm inferior to the left or right shoulder. A second midline incision was generated caudally and a second identical magnet placed in the contralateral flank in relation to the first magnet. The incisions were closed with 6-0 Dexon in running fashion and post-implantation weights were recorded. The mice were left to properly emerge from anesthesia and recover at least 18 h prior to magnetic compression cycling.

Cyclical Magnetic Compression Protocol A factorial design based on varying ischemic times, reperfusion times, cycles per day, and number of days of cycling was implemented (see Table 1). In this way, we could assess whether the length of the ischemic period (e.g., Group III) or the number of cycles (e.g., Groups I and II) was a key determinant in injury production. Ischemia was induced by the simple addition of an extracorporeal

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TABLE 1 Magnet Application Protocol Treatment groups (N) Group Group Group Group Group

I* (7) II (9) III (5) IV (3) V (3)

Ischemic cycle time (h)

Rep. cycle time (h)

#Cycles/day

#Days

Total hours of ischemia

1 1 5 2 2

0.5 0.5 — 0.5 0.5

5 5 1 2 2

1 3 2 2 1

5 15 10 8 4

*Group IB not shown above is comprised of animals subject to the same I-R cycles as Group I with long-term analysis (to day 10) for skin survival.

magnet over the skin bearing the implanted magnet of opposite pole; a defined period of reperfusion ensued by magnet removal. Mice were housed singly or in pairs to minimize cross-reactivity or dislodging of magnets.

Purification of RNA from Mouse Skin and Real-time RT-PCR of iNOS

Back skin from implanted subjects was harvested at designated time points after cycling, fixed in 10% zinc formalin, processed, and paraffin-embedded. Hematoxylin-eosin stained paraffin sections were used for histopathologic assessment (10⫻ magnification) and leukocyte counts (40⫻ magnification). The latter was achieved by counting the number of extravasated leukocytes per high-power field (25 ␮m 2). Skin adjacent to or on the border of frankly necrotic skin was used for this measurement because the architecture was retained. Three high-power fields were counted per section.

Total RNA was column-purified from liquid nitrogen snap-frozen mouse skin using RNeasy Mini Kit (Qiagen, Inc., Valencia, CA) with intermediary proteinase K and RNAse-free DNAse steps. Extracted RNA was initially quantitated using either a microplate reader (␮quant, Biotek Instruments, Inc., Winooski, VT) or a standard spectrophotometer (GeneQuant RNA/DNA calculator, Pharmacia, Inc., Mississauga, Ontario) at A260/280. A two-step method was performed to quantitate inducible nitric oxide synthase (iNOS) mRNA expression relative to 18S RNA. Isolated RNA was converted to cDNA by an initial reverse transcriptase reaction with custom-designed primers (ABI Biosystems, Foster City, CA). The forward and reverse primers for the mouse iNOS gene were 5⬘-GGC AGC CTG TGA GAC CTT TG (iNOS 2200 NCBI accession number 010927) and 5⬘-GCA TTG GAA GTG AAG CGT TTC-3⬘ (iNOS 2271-NCBI accession number 010927), respectively. The conditions for the reverse transcriptase (RT) step consisted of 48°C for 30 min, followed by a 2-min incubation at 50°C to prevent any carryover reactivity. Termination of the initial reaction ensued for 10 min at 95°C. Real-time polymerase chain reaction (PCR) in an ABI Prism 7700 SDS (ABI Biosystems) consisted of 40–50 cycles of 95°C for 15 s, followed by 60°C for 1 min. Probes used to detect the cycle threshold (Ct) of amplification were TaqMan probes mouse iNOS 2221T-TGT CCG AAG CAA ACA TCA CAT TCA GAT CC and ribosomal RNA VIC™ (ABI Biosystems) for iNOS and 18S RNA. respectively. The fluorescent dye 6-carboxymethylfluorescein (FAM) was covalently linked to the 5⬘ end of iNOS, whereas VIC was linked to the 5⬘ end of the 18S RNA probe for differential fluorescence. 3⬘-Linked 6-carboxy-tetramethylrhodamine (TAMRA) served as the quencher for both probes. All RNA samples were performed in triplicate and results were expressed as delta Ct (comparing target RNA expression to 18S) or delta-delta Ct (comparing target RNA expression in experimental samples to that of untreated, normal skin from the same mouse).

Apoptotic DNA Laddering Assay

Long-Term Analysis of Skin Necrosis

Genomic DNA from mouse skin was isolated by methods previously described for DNA fragmentation analysis [12]. Briefly, harvested tissue, previously snap-frozen in liquid nitrogen and stored at ⫺80°C, was incubated overnight at 55°C in digest buffer (50 mM Tris-HCl, pH 8.0, 1% SDS, 1 mM EDTA) containing proteinase K (final concentration 100 ␮g/ml). After centrifugation (5 min at 10,000 rpm), the supernatant was collected and genomic DNA isolated by a commercially available kit (Apoptotic DNA Ladder Kit, Roche Molecular Biochemicals, Indianapolis, IN) per manufacturer’s instructions. One to two micrograms of purified DNA per sample was loaded onto a 0.8% agarose gel containing ethidium bromide and the lanes imaged via an ultraviolet digital camera (Epi Chemi II Darkroom, UVP Inc., Upland, CA).

After compression cycles were complete, designated subjects were grossly examined over a period of 7–10 days to observe the natural history of compressed skin. Daily tracings were obtained and percent necrosis was determined as described above. The mice were sacrificed at the end of the experiment and skin harvested for RNA analysis.

Evaluation of Skin Necrosis Necrotic areas of compressed skin were measured by tracing the visualized area on transparency paper bearing the magnet template. Necrosis was defined grossly by black discoloration, induration, and the absence of bleeding of skin upon needle puncture. Tracings were scanned and measured on a Model GS-700 Densitometer (Bio-Rad Laboratories, Hercules, CA) via manufacturer software (Molecular Analyst, Bio-Rad). Values were exported to spreadsheet software (Microsoft Excel, Microsoft Corporation, Seattle, WA) and the area of necrosis was determined by the following equation:

% Necrosis ⫽ (area of nonviable tissue/total compressed area) ⫻ 100

Histochemistry and Leukocyte Counts

Statistical Analysis Student’s t test from Microsoft Excel (Microsoft Corp.) was used when appropriate. Statistical significance was achieved with P value ⬍ 0.05.

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TABLE 2 Morbidity and Mortality of Implanted Mice Total mice implanted (total magnets)

Deaths (% total)

Post-Op migration (% total)

Dehiscence/magnet exposure (% total)

Infections (% total)

43 (86)

7 (16.3)

6 (13.9)

3 (6.9/3.5)

0 (0)

RESULTS

Implantation Procedure

Measurement of Magnetic Flux Density (B)

A total of 43 wildtype mice underwent magnetic implantation, which equals 86 total magnets implanted. There were seven deaths, the majority of which were anesthesia related. Importantly, only six total magnets relocated to their original site of implantation (13.9% of mice, 7% of total magnets implanted), and only three magnets dehisced through the midline incision (6.9% of mice, 3.5% of total magnets implanted). Lastly, no mice

The magnetic flux density of the selected ring magnet 1 mm from its surface was determined to be 2300 Gauss by the manufacturer (Eneflux, Inc., Bethpage, NY), 77% higher actual flux density than calculated (see Materials and Methods). We were therefore well above the compressive force required to induce necrosis.

FIG. 2. Skin necrosis is clinically evident in cyclically compressed subjects. Representative digital photographs were taken of murine subjects who underwent either 3 days of recurrent I-R insults (Fig. 3A, Group II) or 2 days of prolonged treatment (Fig. 3B, Group III). (b) Skin necrosis plotted over time. The percentage of necrotic area was calculated for each subject (see Materials and Methods) and plotted over time for Group I (clear bars), Group II (striped bars), and Group III (shaded bars). Error bars represent the S.E.M.

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suffered from cellulitis or sepsis from foreign body placement (Table 2). One initial concern from this model was the ability of the mice to tolerate implantation of 2 magnets. Postimplantation weights demonstrated only a modest increase (14%) in all subjects (data not shown). Additionally, the mice were observed to be quite active postoperatively and did not appear hindered by the additional weight or anatomic placement of the magnets. Magnetic Cyclical Compression Invokes Skin Necrosis in Reliable Fashion

To achieve optimal necrosis, several combinations of ischemic times, reperfusion times, cycles/day, and days of cycling were implemented. Consistent with pressure necrosis, the clinical progression of blanchable to nonblanchable hyperemia, pallor, and eventual necrosis was apparent in all experimental subjects. Overall, necrotic tissue was visualized as early as 2 days after repetitive cycling (Group II, n ⫽ 9) and 1 day after cycling for a prolonged period of time (Group III, n ⫽ 5). The magnitude of necrosis appeared to be cycleresponsive, because a consistent daily progression was seen (Fig. 2a and b). Although the onset of necrosis was faster for Group III, the percentage of necrosis for both groups at the end of the experiment (3 days) was equivalent (approximately 34%, Fig. 2b). Of note, necrosis of the central portion, i.e., the middle of the ring magnet where theoretically no compression is occurring, was present in the majority of mice analyzed. Negligible amounts of necrosis were detected in animals repetitively cycled for just 1 day (0% for Group I, n ⫽ 7; 1.8% for Group II, n ⫽ 9). As expected, necrosis was absent in sham (noncompressed, implanted) skin. Long-Term Analysis Reveals Necrosis is Sustained Over Time

To mimic chronic wounds, we evaluated the ability to maintain necrosis in our model after the compressive insult. Long-term skin survival was therefore assessed 7–10 days after the end of cyclical compression. Indeed, the necrotic area increased from 0-12% in 10 days in animals cycled repetitively on day 0 (Group IB, n ⫽ 3: Fig. 3. top panel). For those with longer periods of ischemia (2 h), postischemic skin survival was even more reduced, because subjects cycled repetitively (2 cycles/day) demonstrated approximately 30 –35% necrosis on day 8; this finding was irrespective of whether the animals were cycled on day 0 only (Group IV, n ⫽ 3; Fig. 3, middle panel) or day 0 and day 1 (Group V, n ⫽ 3; Fig. 3, bottom panel). These results point to the significance of the reperfusion phase of injury and its sustained effects over time.

FIG. 3. The effects of recurrent cutaneous I-R injury are sustained over time. Daily necrotic areas were determined from cyclically compressed skin several days after the last ischemic insult for mice subjected to 1 h of ischemia, 0.5 h of reperfusion. 5 cycles for 1 day (Group IB, top panel), 2 h of ischemia, 0.5 h of reperfusion, 2 cycles/day for 1 (Group IV, middle panel) or 2 (Group V, bottom panel) days. Error bars represent the S.E.M.

Histological Analysis

Paraffin-embedded skin harvested from cycled animals was sectioned for hematoxylin-eosin staining. At lower magnifications (10⫻), the characteristics of tissue injury were apparent, including hyperchromatism, nuclear pyknosis, vascular congestion, and endothelial cell swelling (Fig. 4B). These features were not present in sham controls (Fig. 4A). Furthermore, at higher magnification (40⫻), the acute inflammatory phase was obvious in treated samples, captured by the classic process of leukocyte margination and diapedesis in selected samples (Fig. 4C). Indeed, one of the hallmark cell types of I-R injury is the polymorphonuclear cell, and its critical role for the

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FIG. 4. Histologic analysis reveals hallmarks of inflammatory process in postischemic tissue compared with controls. Panel A, sham control skin (10⫻ magnification); Panel B, Group II treated skin (10⫻ magnification); Panel C, Group II treated skin (40⫻ magnification); Panel D, leukocyte counts. Asterisk denotes P ⬍ 0.05 (P ⫽ 0.011).

generation of free-oxygen radicals in this process has been well characterized in a number of tissue types [13]. We thus sought to quantitate the leukocytic infiltrate in experimental samples versus controls. After 1 day of repetitive cycling (Group I ⫽ 1 h ischemia/0.5 h reperfusion/5 cycles, n ⫽ 5), there was a virtual doubling of the leukocyte count in postischemic tissue compared with sham controls, a difference that approaches statistical significance (Fig. 4D). This is consistent with the data from the animals after 3 days of cyclical compression, which, when compared with controls, is statistically significant (P ⫽ 0.011). In sum, histological analysis of prenecrotic skin reveals early signs of acute inflammation.

trophoresis of genomic DNA. Genetic material isolated from pooled tissue (n ⫽ 3) for each group was purified and fractionated on a 0.8% agarose gel. As shown in

Molecular Analysis

Apoptotic DNA Laddering On a molecular level, cell injury and subsequent apoptosis has been characterized by fragmentation of genetic material, a process known as DNA laddering [14, 15]. It followed that another way to characterize I-R injury in this model was through agarose gel elec-

FIG. 5. Gel electrophoresis demonstrates apoptotic DNA present in postischemic tissue compared with control tissue.

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Fig. 5, laddering was inherent in the DNA from postischemic skin, but not characteristic of sham control tissue (lane 2). Of note, this fragmentation of DNA in our experimental sample resembled that of 2 ⫻ 10 6 U937 epithelial cells chemically treated with camptothecin, which yields 30% apoptotic cells (positive control supplied by manufacturer). Our data suggests that active cell injury is present on a molecular level and, more importantly, is absent in noncompressed skin overlying a magnetic implant. Real-Time PCR Quantitation Demonstrates CycleResponsive Upregulation of iNOS mRNA It is well established that I-R injury is a multifactorial process, stemming from several cellular and soluble mediator interactions. In turn several genes are upregulated, including free-radical related species xanthine oxidase [16] and superoxide dismutase [17], as well as those governing the L-arginine–nitric oxide pathway. Recently, inducible nitric oxide synthase (iNOS or NOS2) has been characterized in several forms of cutaneous injury, namely wounding [18], thermal injury [19], as well as I-R injury [20, 21]. We therefore sought to determine iNOS mRNA levels quantitatively and therefore assay the effectiveness of this gene as a marker for injury. Two-step real-time RT-PCR served to answer these questions. Messenger RNA is clearly upregulated in our model of chronic pressure skin necrosis, because skin cyclically compressed for 1 day (Group I, n ⫽ 2, total ischemia time ⫽ 5 h) demonstrated a 5-fold increase compared with normal skin (Fig. 6, panel A). Interestingly, the upregulation was cycle-responsive and exponential because mRNA from skin cycled for 3 days (Group II, n ⫽ 3, total ischemia time ⫽ 15 h) was ⬎300-fold supranormal (Fig. 6, panel B). Levels of iNOS mRNA remain elevated even out to 10 –11 days post-cycling, exhibited by the near 20-fold greater transcription of the gene above normal skin (Fig. 6, panel B). This is consistent with studies demonstrating that iNOS can be active for several hours or days [22]. By this molecular analysis, our model is physiologic and demonstrates that iNOS may be a critical marker for the detection of injury in prenecrotic skin. DISCUSSION

Ischemia-reperfusion injury has been implicated in a number of processes and has been described in a plethora of tissue types, including liver, kidney, heart, muscle, brain, and lung [23]. Only recently has this form of injury been postulated and demonstrated to play a role in chronic wounds induced by venous stasis, diabetic, and, of interest to this study, pressure [7]. The testing of this hypothesis is hindered by the limitations of existing models to study chronic cutaneous and more

FIG. 6. Real-time PCR assay reveals temporal and cycleresponsive upregulation of iNOS in recurrently injured skin.

specifically, pressure wounds in a reliable fashion. From the pressure induced on the canine trochanter described by Kosiak [2], to the pressure induced on rabbit ischii by Dinsdale [4], to the porcine trochanter model of Daniel [5, 6], past models of chronic pressure necrosis were plagued by the delivery of pressure to an anesthetized subject as well as the inability to mimic a repeated I-R insult. The physiologic aberration inherent in these early models clouded accurate determination of the etiology of disease. Furthermore, as described [11], these early models based findings upon a single ischemic insult, which is not so relevant when the clinical scenario of pressure sore development is considered. Other chronic wounds, such as venous leg ulcers and diabetic foot ulcers, are also clinically characterized by alternating periods of ischemia and reflow because the patient is either recumbent or ambulatory. A model of recurrent I-R injury of the skin would therefore be valuable in many ways. Along these lines, we put forth a murine model of recurrent pressure induced necrosis based upon cyclical magnetic compression. Our data demonstrate the ability to create a pressure wound in a relatively short period of time with a minimum number of cycles. In this regard, the findings of this model differ from the results of its precedent, the rat model of cyclical magnetic compression of Peirce and colleagues [11]. In their study, maximal necrosis was achieved after repetitive compression totaling 50 h of ischemia. Sub-

REID ET AL.: INDUCTION OF RECURRENT CUTANEOUS I-R INJURY IN MICE

stantial (⬎20%) necrosis was achieved only after 25 total h of ischemia for 1-h ischemic time cycles (total % necrosis ⫽ 35), or after 30 h of ischemia for 2-h ischemic time cycles (total % necrosis ⫽ 28). In comparison, substantial necrosis was accomplished in this study in approximately one half the total hours of ischemia used in the rat model (Group II: total ischemic time 15 h, % necrosis ⫽ 34). Equivalent degrees of necrosis were also found in subjects who underwent more prolonged ischemic times with minimal cycling (Group III, ischemia time ⫽ 5 h, 1 cycle/day, 2 days, % necrosis ⫽ 34). This argues against the frequency of cycling or traumatic reapplication of the magnet as a factor in necrosis production. Differences in the species-specific anatomic differences in blood supply of the back skin, the placement of the implants, and the magnets themselves could account for the differences between these outcomes. One notable limitation in this study is the failure to characterize the regional hemodynamic effect of the ring magnet system and whether or not this effect is uniform throughout the distribution of pressure. Even though our studies lack definitive measurements of blood flow via Doppler and transcutaneous oxygen tension, our measured flux density value (2300 Gauss) well exceeds the theoretical B required to achieve an external pressure of 50 mmHg or 80 –100% reduction of blood flow (12% Gauss). It should be noted that our experiments involve the use of magnet-magnet interaction, which should theoretically enhance the uniformity of this system. In addition to the effective necrosis produced, both histologic and molecular features of I-R injury were demonstrated. Leukocytic infiltrates were clearly doubled in both 1-day and 3-day cyclically compressed groups. The neutrophil has been shown to be a critical cellular mediator in postischemic tissue through the generation of free oxygen radicals. In fact, one of the therapeutic strategies to abrogate injury is directed at leukocyte blockade, either through depletion or antiadhesion [24]. Consistent with the literature, histological analysis of harvested tissue reveals infiltrating neutrophils not only in skin bearing necrosis (Group II) but also in skin that is prenecrotic (Group I). Thus, the infiltration of neutrophils can be seen as a cause of and not an effect of the injury in our model. Molecular harbingers of injury corroborated these cellular findings. DNA laddering of postischemic tissue is suggestive of active apoptosis in this study. It is important to note that skin that was manipulated by implantation alone, was not compressed, and had no apoptotic features present. From this and the relatively low leukocyte count determined from sham tissue, we can surmise that magnetic implantation is a relatively safe, nontoxic, and noninflammatory process on both the cellular and molecular levels.

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Nitric oxide is a potent free radical that has been implicated in a variety of wounding and healing processes. Its role in I-R injury has been well documented in a variety of tissue types [21] and the 3⬘ location of iNOS to the transcription factor hypoxia-response element (HRE) in human [25], rat [22], and murine [26] clones indicates its importance in ischemic processes. Our choice of iNOS as a marker gene for injury arose from the fact that once transcriptionally induced, iNOS is long-acting and produces nitric oxide at 100- to 1000fold greater amounts than its constitutive analogues [22]. To this end, iNOS mRNA was found to be five times more prevalent in postischemic prenecrotic tissue (Group I) than in normal skin (Fig. 6). With two additional days of cyclical compression, the level was profoundly elevated to 300-fold. Therefore, iNOS mRNA levels appear to be a sensitive marker for injury. Several studies have implicated nitric oxide as a key modulator in the process of I-R injury. However, its exact role is controversial, and appears to be speciesand organ-specific. Targeted disruption of iNOS in mice has resulted in marked resistance to intestinal I-R injury and bacterial translocation [27] and mitigation of renal postischemic injury [28] in two recent studies. Contrary to this negative role, Zingarelli and colleagues [29] found that iNOS⫺/⫺ mice had significantly enhanced myocardial cell apoptosis and necrosis, suggesting a cardioprotective role for the enzyme. Moreover, absence of iNOS led to a depression of signal transduction mechanisms as 1␬B␣ degradation, nuclear factor-␬B nuclear translocation and JNK activity were all significantly reduced in mutant mice. Therefore, nitric oxide produced by iNOS appears to be a critical cell signaling molecule in oxidative stress. Consistent with our findings. iNOS-mediated events play an essential role early in the process, just 15 min after a single ischemic insult [25]. To our knowledge, our study is the first demonstration of a putative role of iNOS in recurrent pressure-induced I-R injury. Further investigation of the role of downstream signaling elements in our model is warranted. Lastly, long-term analysis was performed to show that the postischemic insult is sustained in cyclically compressed mice even 10 days after the last I-R cycle, because these subjects demonstrated long-term skin necrosis of 25–35% (Fig. 3; Group IV and Group V). iNOS levels were still elevated at this time, specifically 20-fold above normal levels. This postischemic “memory” makes this model unique and ideal for the study of the chronic fate of pressure wounds several days after the insult. Given these data, we conclude that cyclical magnetic compression in the mouse is a reliable, effective, and physiologic means to study I-R-mediated pressure skin necrosis of both acute and chronic variety. This exciting model will be applied to several ge-

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netically altered mice to comprehensively define the process of pressure sore development. ACKNOWLEDGMENTS This work was supported by NIH grant GM41303-13 and a grant from the 3M Wound Healing Foundation.

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