Vasodilation Study

Cassandra E. Morris and Thomas C. Skalak J Appl Physiol 103:629-636, 2007. First published May 3, 2007; doi:10.1152/japplphysiol.01133.2006 You might find this additional information useful...

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J Appl Physiol 103: 629–636, 2007.

First published May 3, 2007; doi:10.1152/japplphysiol.01133.2006.

Chronic static magnetic field exposure alters microvessel enlargement resulting from surgical intervention Cassandra E. Morris and Thomas C. Skalak Department of Biomedical Engineering, University of Virginia Health Sciences Center, Charlottesville, Virginia Submitted 6 October 2006; accepted in final form 28 April 2007

microvascular remodeling

recently become a widely used complementary or alternative medicine for the treatment of vascular, as well as other musculoskeletal pathologies, including soft tissue injuries. Ongoing research to investigate the specific effects of magnetic fields in the areas of wound healing, inflammation, and the microcirculation increasingly support the therapeutic efficacy of pulsed electromagnetic fields (PEMF) (2, 3, 10, 14, 27, 30, 31, 40), but evidence concerning the efficacy of static magnetic fields (SMF) is less established. This study aimed to investigate, for the first time, the adaptive microvascular response to chronic magnetic field exposure.

Recent studies in our laboratory and others have suggested that acute SMF exposure can have a modulatory influence on the microvasculature, acting to normalize vascular function.

Specifically, acute SMF application can modulate microvascular tone (21–23), blood pressure (25), and blood flow (47).

MAGNETIC FIELD THERAPY HAS

Address for reprint requests and other correspondence: T. C. Skalak, Dept. of Biomedical Engineering, Univ. of Virginia Health Sciences Center, P.O. Box 800759, Health System, Charlottesville, VA 22908 (e-mail: tskalak @virginia.edu). http://www. jap.org

These results may wholly, or in part, account for the observed beneficial effects of SMF application on wound healing, inflammation (15, 44), collateral vessel development (6), and decreased tumor angiogenesis (8, 42), but a direct relationship has not been established. Variations in magnetic field intensity, site of application, and duration of exposure make these studies difficult to compare, although the reported positive therapeutic results warrant careful investigation of the vascular effects.

Studies addressing the potential therapeutic effects of chronic exposure to SMFs are limited, as most research has been aimed at studying the adverse effects of chronic exposure to extremely low-frequency and radio-frequency electromagnetic fields, similar to those emitted by power lines and cellular telephones (9). Only a few detailed in vivo studies utilizing sustained chronic SMF exposure have been completed to date, but have shown measurable effects. A direct vascular effect was observed by Xu et al. (46) as a long-lasting vasodilation of a single arteriole after 1–3 wk of an 180-mT SMF exposure.

Additionally, Okano et al. reported a suppressed and delayed onset of hypertension in spontaneously hypertensive rats exposed to a 5-mT gradient field for 2–12 wk (24, 26) that is possibly related to the measured decrease in angiotensin II (ANG II) and aldosterone levels. While these studies suggest chronic SMF exposure may directly or indirectly influence the microvasculature, no assessment of the effects of a localized magnetic field on microvascular structural remodeling has been completed to date.

Chronic alterations in microvascular tone resulting from hemodynamic and/or inflammatory stimuli have been shown to induce microvascular remodeling (36, 38). Based on the existing evidence that SMF application may influence microvascular tone (21), these experiments were designed to investigate the effect of a locally applied chronic SMF on microvascular structural remodeling induced by surgical intervention. The objective of this study was to characterize the effects of a chronic, localized SMF on microvascular network adaptations.

These results will be important for elucidation of the vascular response to chronic, localized SMF exposure and the development of efficacious therapeutic modalities.

METHODS

Experimental protocol. All experiments were conducted in accordance with an animal protocol approved by the University of Virginia Animal Care and Use Committee. Male c57Bl/6 mice (Harlan, Indianapolis, IN) weighing 24 –30 g were anesthetized with an intraperitoneal injection of ketamine (0.00156 ml/g), xylazine (0.00052 ml/g), and sterile saline (0.00312 ml/g), and dorsal skinfold chambers were

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Morris CE, Skalak TC. Chronic static magnetic field exposure alters microvessel enlargement resulting from surgical intervention.

J Appl Physiol 103: 629–636, 2007. First published May 3, 2007; doi:10.1152/japplphysiol.01133.2006.—Magnetic field therapy has recently become a widely used complementary/alternative medicine for the treatment of vascular, as well as other musculoskeletal pathologies, including soft tissue injuries. Recent studies in our laboratory and others have suggested that acute static magnetic field (SMF) exposure can have a modulatory influence on the microvasculature, acting to normalize vascular function; however, the effect of chronic SMF exposure has not been investigated. This study aimed to measure, for the first time, the adaptive microvascular response to a chronic 7-day continuous magnetic field exposure. Murine dorsal skinfold chambers were applied on day 0, and neodymium static magnets (or size and weight-matched shams) were affixed to the chambers at day 0, where they remained until day 7. Separate analysis of arteriolar and venular diameters revealed that chronic SMF application significantly abrogated the luminal diameter expansion observed in sham-treated networks. Magnet-treated venular diameters were significantly reduced at day 4 and day 7 (34.3 and 54.4%, respectively) compared with sham-treated vessels. Arteriolar diameters were also significantly reduced by magnet treatment at day 7 (50%), but not significantly at day 4 (31.6%), although the same trend was evident. Venular functional length density was also significantly reduced (60%) by chronic field application. These results suggest that chronic SMF exposure can alter the adaptive microvascular remodeling response to mechanical injury, thus supporting the further study of chronic application of SMFs for the treatment of vascular pathologies involving the dysregulation of microvascular structure.

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Probes) and 1:200 CY3-conjugated mouse monoclonal anti-smooth muscle ␣-actin (SMA) (Sigma) in a staining solution (PBS, 0.1% saponin, and 2% bovine serum albumin) at 4°C overnight. Sections were then washed in PBS and 0.1% saponin solution every 10 min for 30 min and finally mounted in a 50% PBS and glycerol solution. The sections were analyzed with a scanning confocal microscope (Nikon D-Eclipse-C1) and EZ.C1.22 software. Eight to twelve SMA-positive vessels were randomly selected from each tissue, and the inner and outer perimeter of the actin-positive area of each vessel was traced.

The area encompassed by each perimeter was calculated, and the difference between the two was calculated to represent the medial wall area. All measurements were made in a blinded fashion, with the treatment groups revealed after all measurements had been made.

Magnetic field distribution. The strength and uniformity of the magnets used in these experiments was established by utilizing a magnetic calibration system that generates a three-dimensional map of the magnetic flux density (21). In summary, the system collects three-dimensional flux density measurements by moving a gaussmeter probe (model STD61-0202-15, Ultra Thin Transverse Probe; F. W. Bell, Orlando, FL, linear accuracy of 0.5%) through a specified three-dimensional volume via a computer, controller (VP9000 Series, Velmex, Bloomfield, NY), and three coupled motorized stages (BiSlide Assemblies, Velmex), one corresponding to each axis. The user specifies the resolution of measurement, and the computer system (Labview version 6.0, National Instruments, Austin, TX) records the acquired flux density measurements, as measured by the gaussmeter (model 6010 Hall-effect gaussmeter, F. W. Bell, accuracy of 0.25%).

Spatial representation of the magnetic flux density is displayed graphically, utilizing Matlab software (The MathWorks, Natick, MA), allowing for interpretation of the flux penetration and strength at the target tissue site. The magnet utilized in these experiments (www. painreliver.com) was scanned with a 2-mm resolution over a 2-cm2 area, 3 mm from the surface of the magnet. This separation distance corresponds to the distance between the surface of the magnet and the underlying microvasculature. Graphical representation of the threedimensional surface (B) and two-dimensional projection (A) of the flux distribution at the tissue level is given in Fig. 1. The area corresponding to the tissue within the window chamber itself is demarcated by the inscribed square. It is noted that the field the tissue is exposed to is a gradient, with a maximum of ⬃20 – 60 mT/0.7 cm or 25– 85 mT/cm. The origin of the gradient, however, is inherent to the magnet itself; the tissue thickness in this case (⬃400 ␮m) does not contribute significantly to the gradient effect. Sham exposure in these experiments was achieved by application of a size- and weightmatched copper sham to the external surface of the chamber in lieu of the magnet.

Statistical analysis. All diameter changes were compared with a two-way ANOVA and post hoc comparisons with Tukey’s test, assuming a P value of 0.05. Vessel densities, medial wall area, and comparisons of average maximally dilated day 0 and day 7 diameters were completed with Student’s t-test, assuming a P value of 0.05 (Sigma-Stat Software, SPSS, Chicago, IL).

RESULTS

Seven-day SMF exposure abrogates vessel enlargement.

Qualitative analysis revealed a visually identifiable difference in the network morphology between the sham- and magnettreated tissues (Fig. 2). Untreated tissues were visually indistinguishable from sham tissues (data not shown). Quantification of vessel diameters and comparison back to day 0 values revealed marked venular enlargement in the sham-treated networks (n ⫽ 12), 59.2 ⫾ 5.6% on day 4 and 91.2 ⫾ 8.3% on day 7, which was significantly abrogated by magnet treatment (n ⫽ 11), 38.9 ⫾ 4.0% on day 4 and 41.6 ⫾ 4.9% on day 7 (Fig. 3A).

Stratification of the data into diameter bins demonstrated that

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applied according to the procedure outlined previously (13). Briefly, the dual layer of dorsal skin was raised, and a 7-mm ⫻ 7-mm square area of one layer of the epidermis was removed from one side of the raised skinfold, leaving the underlying dermis (containing the microvasculature), superficial fascia, and panniculus carnosus (striated skin muscle) intact. The full thickness of the skin on the contralateral side of the chamber was not altered. Sterile saline (0.9%) was applied during the excision of the skin to ensure that the tissue remained hydrated. Following excision, the skinfold was then sandwiched perpendicular to the spine between two aluminum frames, and the remaining tissue was covered by a sterile coverslip. A neodymium magnet (1.39 ⫾ 0.01 g) or a size- and weight-matched copper sham (1.44 ⫾ 0.01 g) was applied directly to the chamber after chamber application and imaging on day 0 and removed only for imaging purposes throughout the duration of the experiment.

Data acquisition and analysis. Intravital observation was facilitated with a Zeiss microscope (Axioskop, Carl Zeiss, Thornwood, NY) utilizing ⫻2.5 (0.075 numerical aperture, Zeiss Plan Neofuar) and ⫻10 (0.25 numerical aperture, Zeiss Plan Neofuar) objectives under transillumination. Images of the microvasculature were taken on day 0 following surgery and subsequently on days 4 and 7 (under isoflurane anesthesia, 2.5% isoflurane, 97.5% oxygen at 0.4 l/min) with an Olympus Microfire digital camera and Picture Frame Software (Optronics, Muskogee, OK) or a charge-coupled device camera (CCD-72, DAGE MTI, Michigan City, IN) and video recorder (S-VHS recorder; Panasonic, Osaka, Japan). Digital images from the charge-coupled device camera were acquired via frame grabbing sequential images from the experimental video corresponding to the points along the vessel to be measured. Quantification of vessel diameters and densities was completed with the aid of Image J (NIH Image) and Scion Image version 4.0.2 (Scion, Frederick, MD) software packages. Three diameter measurements were taken along the length of each vessel at each time point and were averaged to represent the vessel diameter of that particular vessel at that given time point. All diameters were measured from the inner lumen. Vessel density measurements were acquired by tracing all visible vessels in the entire window at a magnification of ⫻100 (⫻10 objective) and calculating the length of the combined tracings to yield a measurement of total length per tissue area. To establish maximal microvascular diameter, the dorsal skinfold tissue was continually suffused for 30 min before placement of the coverslip at day 0 and after removal of the coverslip at day 7 with adenosine (10⫺4 M) (4) dissolved in a bicarbonate-buffered saline drip (137.9 mM NaCl, 4.7 mM KCl, 1.2 MM MgSO4, 1.9 mM CaCl2, 23 mM NaHCO3), maintained at 37°C and bubbled with 5% CO2 in N2 to maintain a physiological pH of 7.4.

All measurements were made in a blinded fashion, with the treatment groups revealed after all measurements had been made.

Tissue fixation, sectioning, and immunohistochemistry. Immediately following dilation of the vasculature at day 7, the dilated morphology of the tissue was preserved by perfusion fixation of the animal followed by excision of the chamber tissue. Animals were euthanized with pentobarbital sodium (60 mg/kg ip, Abbot Laboratories, North Chicago, IL), and the entire vasculature was thoroughly perfused with heparinized saline followed by 4% paraformeldehyde (Sigma) in PBS solution. The paraformeldehyde solution was also dripped on the tissue to preserve the in vivo orientation of the tissue.

Following 40 min of fixation, the chamber tissue was carefully excised and placed in a sucrose wash solution composed of 20% sucrose and 0.02% azide in PBS at room temperature for 1 h. The wash solution was then replaced with a gelatin gel solution composed of 20% sucrose, 7.5% gelatin, and 0.02% azide, and placed in a 37°C water bath overnight. The tissues were then aligned vertically in sectioning dishes along the axis of the main feeder vessels and snap frozen in liquid nitrogen. Ten-micrometer-thick cross sections were taken at 100-␮m intervals throughout the tissue and placed on gelatincoated slides. These sections were washed with PBS and then incubated in 1:100 Alexa Fluor 488-conjugated BSI-Lectin (Molecular

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Fig. 1. Graphical representation of the magnetic field utilized for chronic application.

A: two-dimensional projection of the magnetic flux distribution applied to the tissue.

B: three-dimensional surface plot. Square in A represents the window chamber area and, therefore, the portion of the distribution to which the tissue in the window is exposed.

Units are in Gauss, 10 G ⫽ 1 mT.

day 7. Chronic magnet application (n ⫽ 10) significantly reduced arteriolar enlargement at day 7 (20.5 ⫾ 5.1%), but not at day 4 (11.35 ⫾ 3.6%), although the same trend was evident (Fig. 3E). Separation into initial vessel diameter bins showed that the most significant reduction in arteriolar enlargement was manifested in the smallest vessels, with initial diameters

Fig. 2. Photomicrographic images of the microvasculature treated with a chronic magnet or sham application at day 0. Low magnification of entire window (A and C) and high magnification of a selected portion of the window (B and D) of chronic sham- (A and B) and magnet-treated (C and D) microvascular networks is shown. Bar ⫽ 200 ␮m.

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this significant abrogation was evident in all but the largest (⬎75 ␮m) initial diameter vessels (Fig. 3B). The number of vessels measured at each time point and each initial vessel diameter bin are given in Fig. 3, C and D, respectively.

Arteriolar enlargement was also observed in sham-treated networks (n ⫽ 10), 16.6 ⫾ 4.1% on day 4 and 40 ⫾ 4.2% on

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10 –24 ␮m (Fig. 3F). Again, the number of vessels measured are given in Fig. 3, G and H. Seven-day SMF exposure reduces venular functional length density. Understanding that quantification of changes in microvascular diameter limits the analysis to vessels that are visible under the set magnification conditions at day 0 and also provides no information about the pattern of the network, quantification of total functional length density of sham- (n ⫽ 8) and magnet-treated (n ⫽ 9) window chambers was completed. Functional length density in this context was defined as all perfused vessels (including visible capillaries) in the network under anesthetic conditions at day 0 and day 7. At these time points, the length of all vessels in the entire visible network was traced and quantified (Fig. 4).

Chronic application of a SMF significantly reduced venular functional length density compared with sham-treated networks (33.3 ⫾ 5.7 vs. 83.4 ⫾ 20.5%, P ⬍ 0.025). Arteriolar functional length density was also reduced by chronic magnet application, but not to a significant degree (Fig. 4), in part due to the large variability of the sham-treated arteriolar densities.

It is also interesting to note from this data that there was a twofold greater increase in sham-treated venular length density (83.4 ⫾ 20.5%) over arteriolar length density (36.1 ⫾ 30.8%), suggesting that the increase in density is preferentially J Appl Physiol • VOL

weighted to the venular side of the vascular network. Specific conclusions concerning the mechanistic origin of the observed increase in density (formation of new vessels or enlargement of existing vessels that were not originally visible at day 0) cannot be directly ascertained from this data, but it is still noteworthy that application of the static field can have a significant effect on the resting, functional density of this network.

In an attempt to eliminate the possibility that the increase in sham venular density was not primarily due to dilation of the vasculature alone, dilated day 0 and day 7 (n ⫽ 5) venular density was quantified (data not shown). The percent change in dilated, sham-treated, day 7 vs. day 0 length density (90.7 ⫾ 24.1%) was not significantly different from undilated, shamtreated density (83.4 ⫾ 20.5%). This lends support to the assertion that the measurements made for Fig. 4, although not dilated, represent all venules in the network, and we can assume that no venular lumens are completely abolished under normal anesthetic conditions. Therefore, our assessment of nondilated diameters can be interpreted as not only a reduction in enlarged venules, but also a reduction in new venule formation upon chronic magnetic field application. Consequently, magnet application cannot modulate only the caliber but also the density of microvascular networks.

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Fig. 3. Percent change in venular (A and B) and arteriolar (E and F) diameters compared with day 0 for all vessels (A and E) or separated into bins by initial vessel diameter (B and F). C, D, G, and H: number of vessels measured corresponding to each bar in A, B, E, and F, respectively. Values are means ⫾ SE. *P ⬍ 0.05.

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Decreased structural wall remodeling is not via reduction of medial wall area. To further assess the nature of the observed increase in vessel diameter in sham-treated networks (an increase in diameter via dilation vs. structural wall remodeling), sham-treated networks were dilated and imaged at days 0 and 7 (n ⫽ 9 and 8, respectively). Maximal dilation of the vasculature and comparison at these time points allowed for a more direct assessment of structural remodeling by forcing the network to have uniform tone, and therefore any significant increase in maximal diameter may be attributed to a structural remodeling of the vascular wall.

Comparison of the average day 0 maximal vessel diameters to their average day 7 maximal diameters (Fig. 5) revealed a significant increase in both venular (49.7 ⫾ 3.2 vs. 80.2 ⫾ 5.0 ␮m) and arteriolar (46.7 ⫾ 2.8 vs. 58.2 ⫾ 3.7 ␮m) diameter by day 7. These results suggest that the sham-treated networks had undergone some structural remodeling in response to application of the window chamber, and it was this remodeling that was prevented by application of the magnetic field.

Quantification of medial wall cross-sectional area is a commonly used metric to assess vascular wall structural remodeling (38). Therefore, to investigate the nature of the observed structural wall remodeling in sham vs. magnet networks, 10␮m-thick cross sections of dilated day 7 tissues were taken and stained with an antibody to SMA to demarcate the medial layer. Calculation of the maximally dilated medial crosssectional area from magnet- (n ⫽ 6) and sham-treated (n ⫽ 5) tissues revealed no significant difference for venules (633.7 ⫾ 80.1 vs. 777.6 ⫾ 126.2 ␮m2, not significant) or arterioles (411.6 ⫾ 52.3 vs. 394.6 ⫾ 92.6 ␮m2), respectively (Fig. 6).

This suggests that the remodeling that occurs in sham-treated networks is not due to medial tissue hypertrophy, leaving the hypothesis that the structural remodeling could be attributed to the rearrangement of existing wall constituents, resulting in larger, remodeled vessels with a decreased wall thickness.

response to chronic external stimuli, such as injury and inflammation, is critical for maintenance of a healthy vasculature.

These experiments aimed to investigate the hypothesis that chronic SMF application could modulate vascular dilation and thus structural microvascular adaptations in a murine dorsal skinfold chamber.

The magnets used in these studies were carefully calibrated utilizing a calibration system designed specifically for this purpose, allowing for precise determination of the local field strength acting at the target tissue location. The range of field strengths utilized was 20 – 60 mT (Fig. 1). These values fall into the range generally advertised for magnetic field therapy products (7) and are on the same order of magnitude as those reported in other investigations into the efficacy of SMF application. The dependency of dose, duration, and time of application on significant outcomes has not been thoroughly

DISCUSSION

Fig. 5. A: average, sham-treated, maximal venular, and arteriolar vessel diameters at day 0 and day 7. Representative photomicrographic images are shown of maximally dilated day 0 (B) and day 7 (C) vessels in the murine skinfold chamber. Values are means ⫾ SE. **P ⬍ 0.002, *P ⬍ 0.015.

The ability of the microvasculature to remodel both in network topology and in single-vessel wall morphology in J Appl Physiol • VOL

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Fig. 4. Representative photomicrographic montages of sham- (A and B) and magnet-treated (C and D) tissues at day 0 (A and C) and day 7 (B and D). E: percent change in functional length density, day 7 vs. day 0, for 7-day sham- and magnet-treated skinfold chambers. Values are means ⫾ SE. a, Arterioles; v, venules. *P ⬍ 0.025.

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Fig. 6. 20⫻ confocal images of BSI-Lectin (green) and smooth muscle ␣-actin (SMA; red; A) and 60⫻ confocal images of SMA alone (B) labeled cross sections of sham(top) and magnet-treated (bottom) day 7 tissues. Bar ⫽ 200 ␮m. C: quantification of medial wall area for sham- and magnettreated arterioles and venules. Values are means ⫾ SE.

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a decrease in the hemodynamic stimuli that induce microvessel remodeling. While other researchers have found that acute SMF exposure can affect blood flow (12, 18, 47), additional experiments are needed to quantify the flow dynamics in response to chronic SMF exposure.

Further characterization of the response revealed that SMF application also significantly reduced venular functional length density, suggesting that magnet application cannot only modulate the caliber, but can also modify the density of microvascular networks. Whether the modulation of diameter leads to modification of the density remains to be determined. As previous research has shown that elevated hemodynamic stimuli resulting from chronic dilation increases arterialization of capillaries (1, 5, 11, 32), the increase in functional arteriolar length density is expected, but a twofold increase in venular density over the arteriolar density in sham-treated networks has not been previously observed. This result is consistent, however, with previous observations that new vessels preferentially originate on the venular side of the network (20). Whether the magnet affects endothelial cell proliferation directly, resulting in decreased vessel density, or if the decrease in density is a result of a chronic alteration of vascular tone, is yet to be determined.

Interestingly, this result is consistent with the finding that application of a PEMF to murine 16/C mammary adenocarcinoma tumors (45), 10 min a day for 12 days, resulted in a significant decrease in tumor volume, as well in angiogenesis, measured as CD31-positive tumor area (40 –70%). Similar results were observed in a nude mouse model infected with a WiDr colon adenocarcinoma. Significant tumor growth inhibition (up to 50%), significant reduction of p53 expression (58%), significant increase (98%) in apoptotic index, and an overall 40% reduction in tumor growth in treated animals resulted from a 70-min daily whole body exposure for 4 wk to a modulated (static with superimposed 50-Hz extremely low frequency) magnetic field with an average intensity of 3.59 mT (41, 42). Furthermore, in a series of experiments using a murine model with transplanted mammary adenocarcinomas,

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investigated to date. Observations in our laboratory suggest, however, that the application of the field at the time of injury (day 0 in this case) is important for affecting a significant physiological change (unpublished observations). Additionally, it had been suggested in acute experiments that a traditional dose-response relationship does not apply to magnetic field exposure [Ref. 47, unpublished observations], rather that there may exist an optimal therapeutic window of magnetic field strength. However, no such data have been collected with respect to chronic experiments in general and microvessel remodeling in particular.

Analysis of sham-treated tissues revealed a quantifiable increase in arteriolar and venular diameters 4 and 7 days after window chamber placement. While the exact mechanism(s) underlying this response is unknown, we postulate that the surgical manipulation of the tissue required for chamber application produced a local inflammatory response, as mechanical injury is a known stimulus for acute inflammation (33), resulting in chronically dilated vessels. As chronic vasodilation and increased hemodynamic stimuli, such as wall shear stress and circumferential stress, are known to induce microvascular remodeling (36, 38, 43), the relative contribution of dilation vs. structural remodeling to the observed increase in diameter was established by comparison of maximally dilated diameters at day 0 and day 7. As we found that the day 7 diameters were significantly larger than those of day 0, we conclude that a portion of the measured increase in arteriolar and venular diameters in sham-treated networks may be a result of structural wall remodeling. Application of the SMF immediately after placement of the window chamber was found to significantly alter the formation of enlarged venules at day 4 and day 7, as well as enlarged arterioles at day 7. Based on the conclusion that the sham networks undergo some degree of structural remodeling, these results demonstrate for the first time that magnet application can reduce the enlargement of microvessels in the network. We suggest that this is due to modulation of vessel diameter, consistent with our previous results (21), resulting in modulated blood flow and, therefore,

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applications for magnetic field therapy, including pain management, edema reduction, modulation of tissue perfusion, and apoptotic or anti-angiogenic treatment of tumors. The mechanisms for these effects have not been proven unequivocally, but are generally hypothesized to be related to the activation of enzymatic reactions and/or alterations in ion transport, specifically Ca2⫹, based on several in vitro findings.

Application of a 120-mT field to cultured GH3 cells resulted in a reduced calcium current and activation rate of calcium channels (19, 23, 25), as well as calcium-dependent changes in acetylcholine release at the neuromuscular junction (34). These results were proposed to be due to lipid reorganization in the membrane following magnetic field-induced increases in membrane fluidity. More recent investigations have observed decreased proliferation, inositol phosphate, and diacylglycerol signaling in human skin fibroblasts (28) and FNC-B4 neuronal cells (29) exposed for 1 h and 15 min, respectively, to a 200-mT SMF. These results further suggest that SMF may result in decreased intracellular Ca2⫹ levels. This same group also found that a 200-mT SMF applied for 3 h significantly inhibited PGE1 and fetal calf serum-induced angiogenesis in the chick embryo chorioallantoic membrane (35), further suggesting a potential anti-angiogenic role of SMF exposure.

These findings support the hypothesis that SMF exposure alters Ca2⫹ dynamics, and this may relate to the change in vascular tone. While these results are intriguing, the explanation as to precisely how the magnetic field is affecting these changes is elusive. Several studies with proteins in solution have indicated changes in chemical kinetics, such as phosphorylation events (16, 17), which may be due to magnetically induced changes in protein (e.g., ion channel) configuration; however, further research is needed to elucidate the specific mechanism of action and, therefore, design the most efficacious therapy.

ACKNOWLEDGMENTS We thank Dr. Shigeru Ichioka for donating the backpacks utilized in these experiments.

GRANTS This work was supported by National Institutes of Health (National Center for Complementary and Alternative Medicine) Grant AT-00582.

REFERENCES 1. Brown MD, Kent J, Kelsall CJ, Milkiewicz M, Hudlicka O. Remodeling in the microcirculation of rat skeletal muscle during chronic ischemia. Microcirculation 10: 179 –191, 2003.

2. Canedo-Dorantes L, Garcia-Cantu R, Barrera R, Mendez-Ramirez I, Navarro VH, Serrano G. Healing of chronic arterial and venous leg ulcers through systemic effects of electromagnetic fields [corrected]. Arch Med Res 33: 281–289, 2002.

3. Ciombor DM, Aaron RK, Wang S, Simon B. Modification of osteoarthritis by pulsed electromagnetic field–a morphological study.

Osteoarthritis Cartilage 11: 455– 462, 2003.

4. Damon DN, Duling BR. Venular reactivity in the hamster cheek pouch and cremaster muscle. Microvasc Res 31: 379 –383, 1986.

5. Dawson JM, Hudlicka O. The effects of long term administration of prazosin on the microcirculation in skeletal muscles. Cardiovasc Res 23: 913–920, 1989.

6. Durdyeva TM. [Cerebral hemodynamics as affected by an alternating magnetic field in patients with vascular failure of the vertebrobasilar system.] Vopr Kurortol Fizioter Lech Fiz Kult 3: 59, 1983.

7. Eisenberg DM, Kessler RC, Foster C, Norlock FE, Calkins DR, Delbanco TL. Unconventional medicine in the United States. Prevalence, costs, and patterns of use. N Engl J Med 328: 246 –252, 1993.

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application of static electric fields and SMFs applied 8 –12 h/day for 13 days was found to enhance the efficacy of the chemotherapy agent adriamycin in inducing tumor regression (8). In this study, vascular density was not assessed, but, taken together with the PEMF results as well as our findings, we can postulate that SMF exposure may be effective in reducing new vessel growth in conditions involving pathological vessel growth.

To additionally evaluate the modulation of structural remodeling by application of the SMF, medial wall area was assessed and compared between sham- and magnet-treated networks at day 7. As no significant difference in wall area for arterioles or venules was measured, we can argue that, based on the significant decrease in arteriolar and venular luminal diameter measured after magnet application, although medial wall crosssectional area remains constant, medial wall thickness may differ between sham- and magnet-treated vessels. This would suggest that the remodeling of the vascular wall in the sham networks could be the result of eutrophic remodeling, the rearrangement of existing wall material into a larger lumen with a decreased wall thickness maintaining constant medial wall cross-sectional area. This suggests that the application of the magnetic field may have altered the sham-induced remodeling response.

Reconciliation of these findings with existing data is difficult, as there are only a few experiments that have investigated the role of sustained chronic SMF application. Contrary to our findings that chronic SMF application results in a reduction in vessel diameter, others observed a long-lasting dilation of a single arteriole after 1–3 wk of SMF exposure, as measured by microphotoplethysmography (MPPG) (46) in a rabbit ear chamber. This observed vasodilation could be attributed to the initial tone of the treated vessel. Our previous studies have shown that initially vasoconstricted vessels dilate in response to SMF exposure (21), and, therefore, we could conclude that the vessels were initially vasoconstricted, resulting in a vasodilatory response to the SMF exposure. The prolonged vasodilation and the resultant change in hemodynamics in this study may have resulted in a remodeling response; however, this is impossible to discern utilizing MPPG techniques, as MPPG measures changes in volume, not blood flow or velocity.

Additionally, application of a whole body 5-mT gradient SMF to young, spontaneously hypertensive rats was shown to suppress and delay the onset of hypertension, as measured by mean arterial pressure (24, 26) and characterized by decreased ANG II and aldosterone levels 6 wk after the onset of magnetic field treatment. As elevated ANG II levels have been found to enhance inward microvascular remodeling due to chronic constriction (37, 39), we would expect that decreased levels would delay the remodeling prevalent in hypertension. These results suggest that whole body SMF exposure may have a beneficial role in the prevention of microvascular remodeling via an indirect influence on circulating factors that are involved in tone regulation. The causal dependence with regard to the SMF application, however, is difficult to assess due to the application of the field globally to the whole body. This result is consistent with ours, if we deduce that the reduction in ANG II levels corresponds to a delay in the inward remodeling.

The physiological influence of therapeutic magnetic field application has been the focus of a range of in vivo, in vitro, and clinical studies. These studies imply several possible

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28. Pacini S, Gulisano M, Peruzzi B, Sgambati E, Gheri G, Gheri BS, Vannucchi S, Polli G, Ruggiero M. Effects of 0.2 T static magnetic field on human skin fibroblasts. Cancer Detect Prev 27: 327–332, 2003.

29. Pacini S, Vannelli GB, Barni T, Ruggiero M, Sardi I, Pacini P, Gulisano M. Effect of 0.2 T static magnetic field on human neurons: remodeling and inhibition of signal transduction without genome instability. Neurosci Lett 267: 185–188, 1999.

30. Patino O, Grana D, Bolgiani A, Prezzavento G, Mino J, Merlo A, Benaim F. Pulsed electromagnetic fields in experimental cutaneous wound healing in rats. J Burn Care Rehabil 17: 528 –531, 1996.

31. Pilla AA. Low-intensity electromagnetic and mechanical modulation of bone growth and repair: are they equivalent? J Orthop Sci 7: 420 – 428, 2002.

32. Price RJ, Skalak TC. Arteriolar remodeling in skeletal muscle of rats exposed to chronic hypoxia. J Vasc Res 35: 238 –244, 1998.

33. Rang HP, Dale MM, Ritter JA, Gardner P. Pharmacology. Philadelphia, PA: Churchill Livingstone, 2001, p. 274 –276.

34. Rosen AD. Magnetic field influence on acetylcholine release at the neuromuscular junction. Am J Physiol Cell Physiol 262: C1418 –C1422, 1992.

35. Ruggiero M, Bottaro DP, Liguri G, Gulisano M, Peruzzi B, Pacini S. 0.2 T magnetic field inhibits angiogenesis in chick embryo chorioallantoic membrane. Bioelectromagnetics 25: 390 –396, 2004.

36. Schaper W, Scholz D. Factors regulating arteriogenesis. Arterioscler Thromb Vasc Biol 23: 1143–1151, 2003.

37. Schmieder RE. Mechanisms for the clinical benefits of angiotensin II receptor blockers. Am J Hypertens 18: 720 –730, 2005.

38. Skalak TC, Price RJ. The role of mechanical stresses in microvascular remodeling. Microcirculation 3: 143–165, 1996.

39. Stassen FR, Raat NJ, Brouwers-Ceiler DL, Fazzi GE, Smits JF, De Mey JG. Angiotensin II induces media hypertrophy and hyperreactivity in mesenteric but not epigastric small arteries of the rat. J Vasc Res 34: 289 –297, 1997.

40. Stiller MJ, Pak GH, Shupack JL, Thaler S, Kenny C, Jondreau L. A portable pulsed electromagnetic field (PEMF) device to enhance healing of recalcitrant venous ulcers: a double-blind, placebo-controlled clinical trial.

Br J Dermatol 127: 147–154, 1992.

41. Tofani S, Barone D, Cintorino M, de Santi MM, Ferrara A, Orlassino R, Ossola P, Peroglio F, Rolfo K, Ronchetto F. Static and ELF magnetic fields induce tumor growth inhibition and apoptosis. Bioelectromagnetics 22: 419 – 428, 2001.

42. Tofani S, Cintorino M, Barone D, Berardelli M, de Santi MM, Ferrara A, Orlassino R, Ossola P, Rolfo K, Ronchetto F, Tripodi SA, Tosi P. Increased mouse survival, tumor growth inhibition and decreased immunoreactive p53 after exposure to magnetic fields. Bioelectromagnetics 23: 230 –238, 2002.

43. Tulis DA, Unthank JL, Prewitt RL. Flow-induced arterial remodeling in rat mesenteric vasculature. Am J Physiol Heart Circ Physiol 274: H874 – H882, 1998.

44. Weinberger A, Nyska A, Giler S. Treatment of experimental inflammatory synovitis with continuous magnetic field. Isr J Med Sci 32: 1197– 1201, 1996.

45. Williams CD, Markov MS, Hardman WE, Cameron IL. Therapeutic electromagnetic field effects on angiogenesis and tumor growth. Anticancer Res 21: 3887–3891, 2001.

46. Xu S, Okano H, Ohkubo C. Subchronic effects of static magnetic fields on cutaneous microcirculation in rabbits. In Vivo 12: 383–389, 1998.

47. Xu S, Okano H, Ohkubo C. Acute effects of whole-body exposure to static magnetic fields and 50-Hz electromagnetic fields on muscle microcirculation in anesthetized mice. Bioelectrochemistry 53: 127–135, 2001.

103 • AUGUST 2007 •

www.jap.org

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8. Gray JR, Frith CH, Parker JD. In vivo enhancement of chemotherapy with static electric or magnetic fields. Bioelectromagnetics 21: 575–583, 2000.

9. Habash RW, Brodsky LM, Leiss W, Krewski D, Repacholi M. Health risks of electromagnetic fields. II. Evaluation and assessment of radio frequency radiation. Crit Rev Biomed Eng 31: 197–254, 2003.

10. Hannan CJ Jr, Liang Y, Allison JD, Pantazis CG, Searle JR. Chemotherapy of human carcinoma xenografts during pulsed magnetic field exposure. Anticancer Res 14: 1521–1524, 1994.

11. Hansen-Smith F, Egginton S, Hudlicka O. Growth of arterioles in chronically stimulated adult rat skeletal muscle. Microcirculation 5: 49 – 59, 1998.

12. Ichioka S, Iwasaka M, Shibata M, Harii K, Kamiya A, Ueno S. Biological effects of static magnetic fields on the microcirculatory blood flow in vivo: a preliminary report. Med Biol Eng Comput 36: 91–95, 1998.

13. Ichioka S, Minh TC, Shibata M, Nakatsuka T, Sekiya N, Ando J, Harii K. In vivo model for visualizing flap microcirculation of ischemiareperfusion. Microsurgery 22: 304 –310, 2002.

14. Linovitz RJ, Pathria M, Bernhardt M, Green D, Law MD, McGuire RA, Montesano PX, Rechtine G, Salib RM, Ryaby JT, Faden JS, Ponder R, Muenz LR, Magee FP, Garfin SA. Combined magnetic fields accelerate and increase spine fusion: a double-blind, randomized, placebo controlled study. Spine 27: 1383–1389, 2002.

15. Man D, Man B, Plosker H. The influence of permanent magnetic field therapy on wound healing in suction lipectomy patients: a double-blind study. Plast Reconstr Surg 104: 2261–2266, 1999.

16. Markov MS, Pilla AA. Static magnetic field modulation of myosin phosphorylation: calcium dependence in two enzyme preparations.

Bioelectrochem Bioenerg 35: 57– 61, 1994.

17. Markov MS, Pilla AA. Weak static magnetic field modulation of myosin phosphorylation in a cell-free preparation: calcium dependence. Bioelectrochem Bioenerg 43: 233–238, 1997.

18. Mayrovitz HN, Groseclose EE. Effects of a static magnetic field of either polarity on skin microcirculation. Microvasc Res 69: 24 –27, 2005.

19. Miura M, Takayama K, Okada J. Increase in nitric oxide and cyclic GMP of rat cerebellum by radio frequency burst-type electromagnetic field radiation. J Physiol 461: 513–524, 1993.

20. Monos E, Berczi V, Nadasy G. Local control of veins: biomechanical, metabolic, and humoral aspects. Physiol Rev 75: 611– 666, 1995.

21. Morris C, Skalak T. Static magnetic fields alter arteriolar tone in vivo.

Bioelectromagnetics 26: 1–9, 2005.

22. Ohkubo C, Xu S. Acute effects of static magnetic fields on cutaneous microcirculation in rabbits. In Vivo 11: 221–225, 1997.

23. Okano H, Gmitrov J, Ohkubo C. Biphasic effects of static magnetic fields on cutaneous microcirculation in rabbits. Bioelectromagnetics 20: 161–171, 1999.

24. Okano H, Masuda H, Ohkubo C. Decreased plasma levels of nitric oxide metabolites, angiotensin II, and aldosterone in spontaneously hypertensive rats exposed to 5 mT static magnetic field. Bioelectromagnetics 26: 161–172, 2005.

25. Okano H, Ohkubo C. Modulatory effects of static magnetic fields on blood pressure in rabbits. Bioelectromagnetics 22: 408 – 418, 2001.

26. Okano H, Ohkubo C. Effects of static magnetic fields on plasma levels of angiotensin II and aldosterone associated with arterial blood pressure in genetically hypertensive rats. Bioelectromagnetics 24: 403– 412, 2003.

27. Ottani V, De P, V, Govoni P, Franchi M, Zaniol P, Ruggeri A. Effects of pulsed extremely-low-frequency magnetic fields on skin wounds in the rat. Bioelectromagnetics 9: 53– 62, 1988.