extfieldsofstudy:
Materials Science
Medicine

text:
Polymer Multilayers that Promote the Rapid Release and Contact Transfer of DNA

We report a layer-by-layer approach to the fabrication of thin polymer-based multilayers that release DNA rapidly in physiologically relevant environments. This approach exploits the properties of a weak anionic polyelectrolyte [poly(acrylic acid); PAA] to disrupt ionic interactions and promote disassembly in coatings that otherwise erode slowly. We investigated this approach using multilayers fabricated from plasmid DNA and linear poly(ethylenimine) (LPEI), a model synthetic cationic polymer used widely for DNA delivery. LPEI/DNA multilayers erode and release DNA slowly over ∼4 days when incubated in PBS buffer. In contrast, substitution of every other layer of DNA with PAA lead to thin films that released DNA rapidly, with >60% being released in the first 5 min. These quick-release coatings release bioactive DNA and can be used to fabricate uniform coatings on a variety of objects, including the tips of inflatable balloon catheters. We demonstrate that these coatings can promote high levels of cell transfection in vitro and the robust contact transfer and expression of DNA in vascular tissue in vivo using a rat model of vascular injury. These materials provide useful alternatives to multilayers and other coatings that promote the prolonged release of DNA. More broadly, approaches that depart from the use of degradable polymers to promote film erosion create opportunities to design new gene delivery coatings using a broader range of polymer-based building blocks designed for other gene delivery applications. With further development, this approach could thus provide a new and useful platform for the rapid contact transfer of DNA to cells and tissues of interest in a range of fundamental and applied contexts.

■ INTRODUCTION
Thin films, coatings, and matrices that provide control over the release of DNA from surfaces have the potential to serve as platforms for the local delivery of DNA in therapeutic contexts and are also useful as tools for basic biomedical research. Many different approaches, including (i) the encapsulation of DNA or polymer/DNA complexes (polyplexes) into bulk polymer 1−3 and (ii) the physical adsorption or tethering of DNA or polyplexes onto surfaces, 4−9 have been developed for this purpose. These methods can provide spatial and temporal control over the release, delivery, and expression of DNA in vitro and in vivo, but they often provide limited control over the extent to which these or other important parameters and properties can be manipulated or tuned to meet the needs of specific applications.
Our group and others have pursued alternative approaches to the surface-mediated delivery of DNA by exploiting "layer-bylayer" methods of assembly 10−13 that permit the incorporation of plasmid DNA into ultrathin polymer-based "multilayers". 14 These DNA-containing multilayers are fabricated by the alternating deposition of DNA with thin layers of cationic polymers and are, therefore, comprised of intimate mixtures of DNA and cationic polymer agents that can promote the internalization and processing of DNA by cells. This approach also offers other practical advantages when compared with methods mentioned above, including (i) the ability to precisely tune the loading of DNA by changing the number of DNA layers deposited, 15,16 (ii) precise control over the amounts and locations of different DNA constructs by sequential deposition of multiple different plasmids, 17−22 and (iii) the ability to faithfully and conformally coat the surfaces of topologically complex substrates, including interventional medical devices 23,24 with ultrathin and mechanically compliant coatings.
One challenge confronting the design of DNA-containing multilayers for potential gene delivery applications lies in designing assemblies that undergo physical erosion and release their DNA on appropriate time scales (e.g., to achieve sustained release, rapid release, sequential release, or exert tunable temporal control). Many different types of multilayers have been developed to address these needs, with most approaches placing an emphasis on the incorporation of degradable groups (e.g., hydrolytically or reductively degradable groups) that can be used to disrupt ionic interactions, promote film disassembly, and enable DNA release. 14,25 These approaches and other biomedical applications of degradable and DNA-containing multilayers have been reviewed recently. 14,23−28 Many of these past studies have focused on the design of coatings that promote the gradual and continuous release of DNA for prolonged periods. The work reported here was motivated by the potential clinical utility of conformal multilayer coatings that promote the rapid release or rapid transfer of DNA in contexts that are inherently time-limited, such as in vascular interventions 29,30 or where short-duration transfer is otherwise useful or desirable. 31 Here, we report DNA-containing multilayers that release DNA rapidly upon exposure to physiologically relevant media. This approach builds from recent work in our group demonstrating that depositing layers of the weak polyelectrolyte poly(acrylic acid) (PAA; a biocompatible polyanion; Figure 1A) during assembly can be used to design DNAcontaining multilayers that disassemble rapidly at physiological pH (through a mechanism that involves increases in the ionization of the PAA layers and an increase in net anionic charge upon exposure to physiological pH; this change in ionization results in changes in ionic interactions within the films that can promote more rapid film disassembly). 32 In that study, we used films fabricated from DNA, PAA, and a hydrolytically degradable cationic poly(β-amino ester) (PBAE) to demonstrate proof of concept. Those PAA-containing coatings were found to release DNA up to 24× faster than films designed using DNA and degradable PBAEs alone (over ∼3 h as compared to ∼3 days). 32 We note, however, that exploiting changes in the ionization of a weak polyelectrolyte to drive film disruption, rather than relying on the incorporation of a degradable polymer, also creates opportunities to integrate a broader range of functional polymers into these assemblies, including many non-degradable cationic polymers and other anionic weak polyelectrolytes commonly used to promote DNA delivery, into "quick-release" coatings that could promote more rapid and efficient cell transfection.
As a step toward these goals, we sought to characterize the ability of this "weak polyelectrolyte approach" to destabilize multilayers formed from DNA and linear poly(ethylenimine) (LPEI), a model non-degradable polyamine used widely as a basis for the design of DNA delivery systems ( Figure 1A) 33,34 and investigated as a potential film component in preliminary experiments described in our past study on degradable polymers. 32 We report here that multilayers fabricated from alternating layers of plasmid DNA, PAA, and LPEI erode rapidly upon exposure to physiological media, and that substrates coated with these quick-release coatings can be used to promote the efficient surface-mediated transfection of cells in vitro. With a view to developing new device/coating combinations that can promote the rapid transfer of DNA from the surfaces of interventional devices, we also investigated the ability of these coatings to promote the contact-transfer of DNA to vascular tissue in vivo using film-coated inflatable balloon catheters. Our results reveal this approach to promote the robust transfer and local expression of plasmid DNA encoding the reporter gene β-galactosidase (β-gal) in a rat model of carotid artery injury. . For experiments requiring fluorescently labeled DNA, a tetramethylrhodamine (TMR) Label-IT nucleic acid kit was purchased from Mirus Bio Corporation (Madison, WI) and used according to the manufacturer's instructions. X-Gal staining kits were purchased from Genlantis (San Diego, CA). Solutions of sodium acetate buffer (VWR, West Chester, PA) and phosphate-buffered saline (PBS; EM Science, Gibbstown, NJ) were prepared by diluting commercially available concentrate. Sodium acetate buffer (100 mM, pH = 4.9) was used for all rinsing steps during film fabrication and the preparation of polymer and DNA solutions. Dulbecco's modified Eagle medium (DMEM) culture medium used in in vitro cell culture experiments was purchased from Invitrogen (Carlsbad, CA). COS-7 cells used for in vitro transfection experiments were purchased from the American Type Culture Collection (ATCC, Manassas, VA). All materials were used as received unless otherwise noted. Solutions of LPEI and PAA used to fabricate polymer multilayers were filtered through a 0.2 μm nylon membrane syringe prior to use.

■ MATERIALS AND METHODS
General Considerations. Silicon and mesh substrates (∼3.5 cm × 0.5 cm) were cleaned with acetone, ethanol, methanol, and deionized water for 5 min prior to film fabrication. The optical thicknesses of films fabricated on silicon substrates were determined using a Gaertner LSE Stokes ellipsometer (632.8 nm, incident angle = 70°). Thicknesses were measured in at least five locations and the data were processed using the Gaertner Ellipsometer Measurement Program software package to calculate relative thicknesses by assuming an average refractive index of 1.58 for the multilayered films. Prior to ellipsometric measurements the films were dried with filtered, compressed air using a 0.2 μm membrane syringe filter. The amount of DNA released from the multilayered films during incubation in PBS was quantified by recording UV−vis absorbance values at a wavelength of 260 nm (corresponding to the absorbance maximum of doublestranded DNA) using a DU 520 UV−vis spectrophotometer (Beckman Coulter, Fullerton, CA). Fluorescence microscopy and phase contrast microscopy images were acquired using either an Olympus IX70 fluorescence microscope, Nikon Eclipse E600 micro-  Preparation of Polyelectrolyte Solutions. Solutions of LPEI and PAA (5 mM with respect to the repeat unit molecular weight of the polymer) were prepared using 100 mM sodium acetate buffer (pH 4.9). Solutions of plasmid DNA were prepared at 1 mg/mL in 100 mM acetate buffer (pH 4.9) but were not filtered prior to use. For experiments requiring fluorescently labeled DNA, labeled DNA was added to a solution of unlabeled DNA to give a 20% (w/w) labeled/ unlabeled plasmid solution.
Fabrication of Multilayered Films. Films were deposited layerby-layer on planar silicon substrates, stainless steel mesh substrates, and inflatable embolectomy catheter balloons according to the following protocol: (1) substrates were immersed in a solution of LPEI for 5 min, (2) substrates were removed and immersed in two wash baths of 100 mM acetate buffer for 1 min each, (3) substrates were then immersed in a solution of anionic polymer (either PAA or DNA; as appropriate, see below) for 5 min, and (4) substrates were rinsed again in the manner described above. This cycle was repeated until the desired number of cationic and anionic polymer layers were deposited. This general procedure was used to fabricate films having the following general structure: [LPEI/(PAA or DNA)/LPEI/DNA] x , where "x" denotes the number of polymer "tetralayers" deposited. For films fabricated on silicon substrates coated with base layers (see text), thin multilayers composed of 10 bilayers of LPEI and SPS were deposited prior to the assembly of DNA-containing tetralayers using methods described previously. 15,32 For experiments using balloon catheters, film-coated balloons were rinsed with 18 MΩ deionized water, allowed to air-dry following the final rinse step, and then stored in their original packaging in a dry, dark location prior to use. All films were fabricated and stored at ambient room temperature.
Characterization of Film Stability and DNA Release Profiles. Experiments designed to investigate film stability and characterize the release of DNA from multilayered films were performed in the following general manner. Film-coated substrates were submerged in PBS (1 mL, pH = 7.4, 137 mM NaCl) in plastic UV-transparent cuvettes. These samples were incubated at 37°C and removed at predetermined intervals for characterization by ellipsometry and UV−vis spectroscopy. For experiments designed to characterize time-dependent changes in film thickness, optical thickness was measured in at least five different pre-determined locations on each substrate. The concentration of DNA released from the films into solution over time was characterized by measuring the UV absorbance of the PBS incubation buffer (at a wavelength of 260 nm, the absorbance maximum of DNA). After each set of measurements, the coated substrates were placed into new cuvettes with fresh aliquots of PBS and returned to the incubator at 37°C.
Cell Transfection Experiments. COS-7 cells were grown in 24well plates at an initial seeding density of 100000 cells/mL in 0.5 mL of growth medium (90% Dulbecco's modified Eagle's medium, 10% fetal bovine serum, 100 units/mL of penicillin, and 100 μg/mL of streptomycin). Cells were allowed to grow overnight to approximately 80% confluence, at which point the growth medium was replaced by 0.5 mL of fresh serum-containing growth medium. Stainless steel mesh substrates coated with polymer multilayers were then placed manually into the wells and incubated for 5 h. The meshes were then removed, and growth medium was replaced by 0.5 mL of new serum-containing growth medium. Cells were incubated for an additional 48 h and both fluorescence and phase contrast images were recorded directly using an Olympus IX70 fluorescence microscope. To quantify transfection efficiency, the percentage of cells expressing EGFP was determined by flow cytometry. For these experiments, cells were incubated for 48 h as described above and then washed twice with PBS. Cells were then harvested by treatment with trypsin, collected in microcentrifuge tubes, washed with PBS, and finally resuspended in PBS containing 1% bovine serum albumin (BSA). Cell samples were characterized using a FACSCalibur flow cytometer (Becton Dickinson, Franklin Lakes, NJ). Data were collected for populations of at least 10000 cells and analyzed using WinMDI software (Joe Trotter, Scripps Institute).

Results are expressed as the percentage of cells expressing EGFP relative to all cells observed.
General Surgical Procedures. After induction of anesthesia with 2.5% isoflurane, arterial injury was induced in male Sprague−Dawley rats (∼2−3 months old, ∼350 g) by means of carotid balloon angioplasty, as described previously. 29,30,35 Briefly, a longitudinal incision was made in the neck of the rat in order to isolate the left external, internal, and common carotid arteries. To denude the common carotid artery of the endothelial layer, an uncoated angioplasty balloon was inserted into the common carotid artery, inflated to a pressure of 2 atm, and passed three times. Next, a balloon coated with a LPEI/PAA/LPEI/DNA film (either 8 or 32 tetralayers thick, see below) was inserted and inflated until it was observed to expand against the arterial wall (∼2 atm). After a 20 min incubation period, the balloon was deflated and removed from the artery. The external carotid artery was then ligated, blood flow was restored to the common and internal carotid arteries, and the surgical wound was closed layer-to-layer. Animals were sacrificed post-operatively at a predetermined time depending on the time scale of the experiment (see below). All experimental protocols were approved by the Institute Animal Care and Use Committee at University of Wisconsin− Madison (#M002285) and conformed to the Guide for the Care and Use of Laboratory Animals published by the NIH Publication No. 85− 23, 1996 revision.
Characterization of in vivo Delivery and Expression of DNA in Rat Carotid Arteries. For fluorescence-based experiments designed to characterize the extent of delivery of DNA to rat arterial tissue immediately following balloon-mediated delivery, the common carotid artery was harvested immediately (i.e., prior to the restoration of blood flow) after treatment with balloons coated with films having the general structure (LPEI/PAA/LPEI/pCMV-Luc TMR ) 8 . For these experiments, films were fabricated according to protocols outlined above using solutions of DNA containing 20% (w/w) TMR-labeled DNA. Following sacrifice, the common carotid arteries were embedded and frozen in OCT compound and cut into 5 μm sections for analysis by fluorescence microscopy. For experiments designed to characterize β-galactosidase expression, the common carotid artery was treated with balloons coated with films having the general structure (LPEI/PAA/LPEI/pCMV-β) 32 . Balloons coated with films having the general structure (LPEI/PAA/LPEI/pEGFP) 32 were used as controls. Rats were sacrificed at 3 days and arteries were immediately embedded and frozen in OCT compound and cut into 5 μm sections for X-Gal staining following the manufacturer's instructions. Slides were then characterized using a Nikon Eclipse E800 upright microscope equipped with appropriate filters.

■ RESULTS
Fabrication of Multilayers Containing DNA, PAA, and LPEI. To fabricate multilayers containing both DNA and PAA (both anionic polyelectrolytes), we adopted a "tetralayer" approach used previously to design films containing layers of DNA, PAA, and a degradable cationic PBAE. 32 This tetralayer approach permits fabrication of films having the general structure (LPEI/PAA/LPEI/DNA) x , where "x" denotes the number of repeating tetralayers, by the sequential immersion of substrates into dilute solutions of LPEI, PAA, LPEI again, and then DNA ( Figure 1B). We note that this approach also provides a straightforward approach to vary the relative numbers or locations of layers of PAA and DNA deposited in a film (for example, by depositing intermittent tetralayers having the structure (LPEI/PAA/LPEI/PAA) or (LPEI/DNA/ LPEI/DNA) during film assembly). For the work reported here, we focused on films having the structure (LPEI/PAA/ LPEI/DNA) x , for which the number of layers of PAA and DNA deposited was equal.
In our past study, we reported the behaviors of (LPEI/PAA/ LPEI/DNA) x films fabricated on silicon substrates that were first pre-coated with a polyelectrolyte multilayer "base layer" film composed of 10 bilayers of LPEI and the synthetic anionic polymer sodium polystyrenesulfonate (SPS). 32 This thin base layer film [denoted (LPEI/SPS) 10 ; ∼25 nm thick] was used to permit comparison to the behaviors of hydrolytically degradable (PBAE/PAA/PBAE/DNA) x films that were the primary focus of that past study. We note here that while DNA-containing multilayers fabricated using PBAEs cannot generally be fabricated on surfaces that are not pre-treated in this way, 15 the presence of these base layers adds substantial complexity to these assemblies and they have been found, in some cases, to participate in, and perhaps promote, the evolution of nanoscale rearrangements that can impact the behaviors of these materials and complicate physicochemical characterization. 36 With the potential for this additional complexity in mind, we performed a first series of experiments to characterize (LPEI/PAA/LPEI/ DNA) x films fabricated on planar silicon substrates (i) with and (ii) without these LPEI/SPS base layers to determine whether they were required to promote robust and uniform film growth in this LPEI/DNA/PAA system. Figure 2 shows a plot of optical film thickness versus the number of polymer tetralayers deposited for films having the structure (LPEI/PAA/LPEI/pEGFP) 8 (closed circles; fabricated with PAA layers) or (LPEI/pEGFP/LPEI/pEGFP) 8 (open circles; fabricated without PAA layers) on LPEI/SPS base layers 25 nm thick. Inspection of these data reveals both films to exhibit similar and approximately linear growth profiles, and that both films reach thicknesses of ∼300 to 350 nm after the deposition of 8 tetralayers (or 32 individual polymer layers). These results demonstrate that substitution of eight layers of DNA with eight layers of PAA does not change film growth profiles or overall film thickness substantially. For comparison, Figure 2 also shows growth profiles for (LPEI/ PAA/LPEI/pEGFP) 8 films (closed squares) and (LPEI/ pEGFP/LPEI/pEGFP) 8 films (open squares) fabricated on bare silicon substrates (no base layers). These films increased in thickness more slowly than films fabricated on base layers for the first 2−3 tetralayers and then grew in a manner that was approximately linear to yield films with overall thicknesses of ∼200 to 250 nm after the deposition of 8 tetralayers. These results again suggest that "swapping" every other layer of DNA with PAA does not have a substantial impact on overall film growth. These results also demonstrate that LPEI/SPS base layers are not required to promote robust film growth.
Characterization of Film Stability and the Release of DNA. To characterize the impact of PAA and the influence of base layers on the stability and DNA release profiles of these PAA-containing films in physiologically relevant media, we incubated silicon substrates coated with (LPEI/PAA/LPEI/ pEGFP) 8 and (LPEI/pEGFP/LPEI/pEGFP) 8 films (fabricated with or without base layers) in PBS at 37°C. Figure 3A shows a plot of the amount of DNA released into solution, normalized to the total amount of DNA released from each film, as a function of time. Inspection of these data reveals (LPEI/ pEGFP/LPEI/pEGFP) 8 films (open symbols) to release DNA into solution gradually over a period of 4−5 days and that the presence of base layers does not influence release profiles significantly (compare open circles and open squares). In contrast, films containing PAA released DNA much more rapidly (with >60% of DNA released in the first 5 min and the remainder being released over the following ∼24 h; Figure 3A, closed symbols). This characteristic release profile, consisting of a period of rapid release followed by a short period during which the remaining DNA was released more slowly, differed substantially from that of films that did not contain PAA (open symbols) and was unaffected by the presence or absence of base layers applied prior to fabrication (compare closed circles and closed squares).
The influence of film architecture and the presence of base layers on the total amount of DNA released from these films is shown in Figure 3B. In general, films fabricated with layers of PAA released less DNA than films fabricated without PAA (for example, for films fabricated on base layers, (LPEI/PAA/LPEI/ pEGFP) 8 films released 14.10 ± 2.01 μg/cm 2 of DNA, while (LPEI/pEGFP/LPEI/pEGFP) 8 films, which contained eight additional layers of DNA, released 25.55 ± 2.47 μg/cm 2 of DNA). The presence of base layers had a measurable impact on the amount of DNA released from (LPEI/pEGFP/LPEI/ pEGFP) 8 films ( Figure 3B, right), but the amount of DNA released from PAA-containing films did not vary significantly regardless of the presence or absence of base layers ( Figure 3B, left).
PAA-Containing Rapid-Release Coatings Promote Surface-Mediated Transfection in vitro. We performed a series of experiments to characterize the ability of objects coated with the rapid-release PAA-containing films characterized above to promote the transfection of mammalian cells in vitro. In these and all other studies described below, we used films fabricated in the absence of base layers (see Discussion). For these experiments, films were fabricated on the surfaces of stainless steel wire mesh substrates suitable for direct placement into cultures without harming cells. 37 Film-coated substrates were placed in cultures of COS-7 cells growing in serumcontaining medium for 5 h and then removed; cells were incubated for another 2 days before characterization of EGFP . Figure 4A,B shows representative fluorescence and phase contrast micrographs of COS-7 cells treated with a mesh substrate coated with a (LPEI/PAA/LPEI/pEGFP) 8 film. The image in Figure 4A shows high levels of green EGFP-associated fluorescence, demonstrating that DNA is released from these films in a form that is transcriptionally active and that these rapid release coatings can promote significant levels of transgene expression in vitro without the use of additional transfection agents (at levels of ∼30%; vide infra). Figure 4C,D shows the results of a similar experiment using mesh substrates coated with PAA-containing films 16 tetralayers thick [(LPEI/ PAA/LPEI/pEGFP) 16 ]. These thicker films, which contained twice as many layers of DNA as the eight-tetralayer films used in Figure 4A,B, promoted higher levels of transfection (∼50% of cells transfected; vide infra). Expression of EGFP was not confined or localized to cells in direct contact with (or growing in the immediate vicinity of) the film-coated substrates, as observed in past studies on the substrate-mediated transfection of cells using glass slides coated with hydrolytically degradable multilayers. 17 Rather, we observed the expression of EGFP to be well distributed across the culture well ( Figure 4E), consistent with the rapid release and dissemination of DNA from these substantially more porous and open mesh substrates. 16,37 To quantify the relationship between the number of DNA layers deposited and levels of transfection promoted by filmcoated mesh substrates, we performed a series of otherwise identical in vitro experiments using mesh substrates coated with (LPEI/PAA/LPEI/pEGFP) x films 0, 2, 4, 8, and 16 tetralayers thick and used flow cytometry to quantify differences in transgene expression in larger populations of cells. Figure 5 shows a plot of the average percentages of cells expressing EGFP relative to the total number of cells as a function of the number of LPEI/PAA/LPEI/pEGFP tetralayers deposited. These results reveal a correlation between levels of transfection and the number of tetralayers, demonstrating that transfection efficiency can be modulated or tuned by control over the amount of DNA deposited during fabrication. For films 4, 8, and 16 tetralayers thick, the percentage of cells transfected increased linearly with the number of tetralayers deposited. Mesh substrates coated with films 16 tetralayers thick promoted surface-mediated transfection in ∼50% of cells using this model in vitro system.
Characterization of Film-Coated Balloon Catheters and Transfer of DNA to Arterial Tissue. Figure 6A shows a fluorescence microscopy image of a portion of the inflatable tip of an embolectomy balloon catheter coated with an LPEI/ PAA/LPEI/DNA film eight bilayers thick. The films in these experiments were fabricated using a plasmid construct encoding firefly luciferase (pCMV-Luc) fluorescently labeled with the fluorophore tetramethylrhodamine (pCMV-Luc TMR ) to permit characterization of film uniformity and the transfer of DNA to arterial tissue in subsequent experiments. Inspection of the image in Figure 6A reveals fluorescence to be distributed uniformly over the surface of the balloon. Figure 6B shows an image of the same balloon after insertion and inflation in the carotid artery of a rat for 20 min (the balloon was removed prior to imaging; dotted white lines are shown to guide the eye and identify the location of the non-fluorescent balloon). This image reveals a striking decrease in fluorescence intensity on the surface of the balloon, consistent with the substantial and nearly complete removal of DNA upon the insertion, inflation, and contact-transfer of DNA to arterial tissue. Further evidence in support of the contact-transfer of DNA is shown in Figure  6C,D, which shows low and high magnification fluorescence microscopy images of cross sections of the artery treated in this experiment (tissue was harvested after removal of the balloon and prior to restoring blood flow). Inspection of these images reveals bright red DNA-associated fluorescence around the circumference of the inner surface of the arterial wall.
Film-Coated Balloon Catheters Promote Contact-Mediated Transfection in Arterial Tissue. We conducted a final series of experiments to characterize the ability of our PAA-containing coatings to promote localized transgene

Biomacromolecules
Article expression in arterial tissue using the rat model of carotid artery injury used above and methods used in past studies 29,30 to characterize the balloon-mediated transfer of DNA using hydrolytically degradable multilayer coatings. For these experiments, we used catheter balloons coated with films having the general structure (LPEI/PAA/LPEI/pCMV-β-gal) 32 . These films were fabricated using plasmid DNA encoding β-gal to facilitate characterization of transgene expression in arterial tissue using X-gal staining; we used films 32 tetralayers thick to maximize the amount of DNA available for transfer in these proof of concept experiments (balloon catheters coated with films having this structure released ∼23 μg of DNA when incubated in PBS buffer). For these in vivo experiments, balloon catheters coated with (LPEI/PAA/LPEI/pEGFP) 32 films containing the pEGFP plasmid were used as controls. Film-coated balloons were inserted into rat carotid arteries denuded of endothelium for 20 min and then deflated and removed. Rats were sacrificed 3 days after surgery, and cross sections of balloon-treated tissue were characterized using X-gal staining and optical microscopy (see Materials and Methods and other past studies for details of surgical procedures and tissue characterization). Figure 7A,B shows low and high magnification microscopy images of cross sections of arterial tissue treated with a balloon coated with a (LPEI/PAA/LPEI/pCMV-β-gal) 32 film. The blue precipitate observed in this tissue indicates the expression of βgal as revealed by X-gal staining. These images reveal intense blue staining to be located uniformly around the circumference of the artery and present in both the medial layers and a portion of the adventitial layers of the tissue. Figure 7C,D shows cross sections of a control artery treated with a balloon coated with a (LPEI/PAA/LPEI/pEGFP) 32 film (the image was also acquired after treatment with X-gal). No significant blue staining was observed in the medial or adventitial layers of this control artery, confirming that the localized expression of β-gal in arteries treated with balloons coated with the (LPEI/ PAA/LPEI/pCMV-β-gal) 32 films does not arise from nonspecific effects associated with the LPEI or PAA components of these coatings.

■ DISCUSSION
Our group and others have demonstrated that polyelectrolyte multilayers containing DNA can be used to exert both spatial and temporal control over the release or contact-transfer of DNA [e.g., by placing film-coated objects directly in contact with cells (spatial control) or by changing the structures of degradable polymer components to tune film erosion rates (temporal control)]. 14,23−28 These past studies have demonstrated that fabrication using hydrolytically degradable cationic polymers is a useful approach to design multilayers that release DNA gradually (e.g., over days, weeks, or months). 14,27,28 We

Biomacromolecules
Article have found it more difficult, in general, to use this "degradable polymer approach" alone to design coatings that release DNA rapidly (e.g., over several seconds or several minutes). 38 Multilayers that promote the rapid release or transfer of DNA from film-coated devices would be broadly useful and, in many cases, critical in contexts for which dwell time is restricted (such as in balloon-mediated vascular interventions) 29,30 and in other applications (such as microneedle-base delivery to the

Biomacromolecules
Article skin) 31 where short-duration transfer is desirable. The 'weak polyelectrolyte approach reported here departs from the use of degradable polymers and provides alternative, pH-based means to promote the short-term instability of DNA-containing multilayers in physiological media. 32 This approach thus provides new opportunities to design films that promote the rapid release and rapid contact-transfer of DNA.
We used the synthetic polyamine LPEI as a model cationic polymer for this study for several reasons. First, this polymer is commercially available and used widely for the design of nonviral gene delivery systems; 33,34 the presence of LPEI in DNAcontaining multilayers could thus, in principle, be leveraged to address intracellular barriers to cell transfection and improve levels of contact-mediated gene expression. Second, and more relevant to the specific goals of this study, multilayers fabricated from plasmid DNA and this non-degradable polyamine do not themselves release DNA rapidly when incubated in physiological media (e.g., Figure 3A). Finally, initial results communicated in our past study demonstrated that integration of PAA into LPEI/DNA multilayers could be used to promote rapid release of DNA upon exposure of these materials to physiologically relevant media. 32 In this current study, we sought to (i) characterize factors that influence the fabrication and stability of LPEI/DNA multilayers containing intermittent layers of PAA and (ii) evaluate the utility of these PAAcontaining films as coatings for the rapid release and surfacemediated transfer of DNA in vitro and in vivo. To facilitate characterization of these materials in the context of these broader biological goals, we used films fabricated using transcriptionally active reporter plasmids encoding either EGFP or β-gal.
Our results demonstrate that the substitution or "swapping" of every other layer of DNA in a LPEI/DNA multilayer with PAA during layer-by-layer assembly (i.e., films having a repeating (LPEI/PAA/LPEI/DNA) x "tetralayer" structure; Figure 1) leads to coatings that release DNA rapidly, with ∼60% being released within the first 5 min; Figure 3A). This behavior contrasts starkly to that of films fabricated exclusively from LPEI and DNA, which release DNA gradually over a period of ∼4 days ( Figure 3A). These PAA-containing coatings release DNA that is transcriptionally active and can be used to promote high levels of transfection in mammalian cells in vitro in the presence of serum (e.g., Figure 4) without the addition of auxiliary cell transfection agents. One merit of this overall approach, relative to other methods for the encapsulation of DNA in thin degradable films, is that the amount of DNA incorporated into (and subsequently released from) these coatings can be controlled precisely by controlling the number of layers of DNA deposited during layer-by-layer assembly. Our results demonstrate that this feature can be used to modulate levels of cell transfection in vitro, with the number of transfected cells increasing monotonically from ∼4% for coatings two tetralayers thick, to ∼50% for films containing 16 tetralayers (the thickest films characterized quantitatively in this study; Figure 5).
Results shown in Figure 7 demonstrate that inflatable balloon catheters coated with LPEI/PAA/LPEI/DNA coatings 32 tetralayers thick can promote high levels of contact-mediated transgene (β-gal) expression in the arterial walls of rats. We used balloon inflation and tissue contact times of 20 min to maximize film transfer in this proof of concept study and to permit comparison to the results of past studies characterizing the contact-transfer of DNA using balloons coated with hydrolytically degradable multilayer coatings. 29,30 However, these new "quick release" coatings provide principles useful for the design of coatings that could enable more rapid contacttransfer of DNA to vascular tissue (e.g., over periods of seconds or minutes) than the degradable coatings used in those past studies. We therefore anticipate that these rapid-release coatings will be useful for the development of new approaches to the contact-transfer and delivery of therapeutic DNA constructs (or other agents) that could mitigate the consequences of arterial injury that occur during angioplasty procedures (e.g., to reduce the occurrences of intimal hyperplasia and restenosis, etc.). As noted above, rapid release of DNA from film-coated objects and devices would also be useful in a broad range of other contexts.
Multilayer coatings fabricated using DNA and hydrolytically degradable cationic polymers investigated in past studies generally require the deposition of thin polyelectrolyte multilayer-based "base layers" on substrates to promote robust film growth. [15][16][17]29,32 Our current results reveal that this form of substrate preparation or pre-treatment is not necessary to promote robust film growth in this LPEI/PAA/DNA system ( Figure 2A). The presence of LPEI/SPS base layers facilitates initial film growth (presumably by providing a more uniform density of anionic charge that promotes the initial adsorption of LPEI) and leads to films that are thicker than those grown on pristine substrates. The presence of base layers does not impact DNA release profiles when these films are subsequently transferred to physiological media ( Figure 3). However, in view of the additional complexity that these base layers add to these macromolecular assemblies, we opted to eliminate them in our in vitro and in vivo studies. Our results demonstrate that (LPEI/PAA/LPEI/DNA) x coatings can be fabricated on a variety of different silicon, stainless steel, and flexible plastic substrates (Figures 2, 4, and 6) and used to promote high levels of transfection in cells and tissue without the use of base layers or other substrate pre-treatment protocols.
Finally, we note again that we used LPEI as a model polymer for these studies based on the fact that it is (i) non-degradable and (ii) used widely as a basis for the design of non-viral DNA delivery systems. 33,34 While our results demonstrate that LPEI/ PAA/DNA-based coatings can promote high levels of cell transfection in vitro and in vivo (Figures 4 and 7), this current study does not establish the extent to which the presence of LPEI may (or may not) help promote the transport of DNA or address common intracellular barriers to transfection. Additional studies will be required to characterize the extent to which LPEI may form complexes or aggregates with DNA in solution or promote endosomal escape or other important processes more effectively than other DNA-containing multilayer systems. We note further, however, that the "weak polyelectrolyte approach" used here promotes rapid film disruption through an alternate mechanism (pH-induced changes in the ionization of PAA that promote destabilizing ionic interactions) that does not require the integration of hydrolytically degradable polymers, yet remains compatible with their use. As such, this framework opens the door to new film designs that could leverage the physical properties and biological behaviors of a broad range of other cationic polymers used conventionally to promote the transfer of DNA to cells. We anticipate that the work reported here will help guide the development of more advanced DNA-containing coatings, including films fabricated using other weak polyanions, that could further enhance contact-mediated gene expression.

■ SUMMARY AND CONCLUSIONS
We have demonstrated a layer-by-layer approach to the fabrication of thin polyelectrolyte multilayer coatings that release plasmid DNA rapidly from the surfaces of film-coated objects. This approach exploits the pH-dependent ionization of a weak anionic polyelectrolyte to disrupt ionic interactions and promote disassembly in films that otherwise erode slowly in physiological media. Our results demonstrate that the substitution of every other layer of DNA with PAA in LPEI/ DNA multilayers leads to thin films that release DNA rapidly, with >60% of DNA being released within the first 5 min of introduction to physiological buffer. These coatings release DNA in a transcriptionally active form and can be used to fabricate uniform coatings on a variety of objects, including the flexible tips of inflatable balloon catheters. The layer-by-layer approach used here can be used to tune the amount of DNA incorporated within a film and, thereby, control levels of surface-promoted transfection that can be achieved. Although not investigated as a part of this study, this approach also provides a straightforward means to control the relative numbers and locations of the layers of PAA that are incorporated. This approach should therefore also provide a useful means to tune film erosion and DNA release profiles further. Finally, our results demonstrate that these quick-release coatings can be used to promote high levels of surfacepromoted cell transfection in vitro, as well as the robust contact-transfer and localized expression of DNA in vascular tissue in vivo using a rat model of vascular injury. Promoting rapid film erosion through a mechanism that does not require the use of a degradable cationic polymer creates future opportunities to design DNA-releasing multilayers using a much broader range of polymer-based building blocks, including non-degradable cationic polymers that could promote more efficient cell transfection or improve overall biocompatibility. The materials reported here provide useful alternatives to multilayers and other surface coatings that promote the gradual or prolonged release of DNA. With further development, this approach could thus provide a new and useful platform for the rapid release or contact-transfer of DNA to cells and tissues from objects of interest and potential utility in a range of fundamental and applied contexts.


extfieldsofstudy: Biology Medicine

text:
On the function of the ultimate legs of some Scolopendridae (Chilopoda, Scolopendromorpha)

Abstract The function of the variously shaped ultimate legs of Scolopendridae is briefly reviewed. Their function in Scolopendra heros Girard, 1853, Scolopendra subspinipes Leach, 1815, Scolopendra morsitans (Linnaeus, 1758), Scolopendra galapagoensis Bollman, 1889, Scolopendra hainanum Kronmüller, 2012, Scolopendra spinosissima Kraepelin, 1903 Cormocephalus aurantiipes (Newport, 1844) and Ethmostigmus trigonopodus (Leach, 1817), in which they are least specialised has been investigated. Specimens were tapped with forceps on different parts of the trunk to simulate the attack of a predator. When tapped on the first third of the trunk (near the head), the centipedes attacked the forceps with their forcipules. When tapped on the last third or the ultimate legs, they adopted a warning position, raising the ultimate legs to display the ventral and medial prefemoral spines as well as the spined coxopleural processes. In some cases the centipedes attacked the forceps with the claws of the ultimate legs by chopping down on them after lifting the legs high into the warning position. When tapped in the mid part of the trunk, the centipedes curled sideways to reach the forceps with their forcipules and ultimate legs simultaneously. Scolopendra galapagoensis not only lifted the ultimate legs into the warning position but also the last 3-4 pairs of locomotory legs, presenting their distodorsal prefemoral spines. This resembles the warning posture of some spiders. In addition to their function in warning behaviour, defensive stabbing, ritualised meeting reactions and during courtship behaviour, the ultimate legs may in addition act as hooks and perhaps be involved in species recognition. No evidence was found that the ultimate legs are used to catch prey, nor of prey or predators being held between the prefemora.

Introduction
The ultimate legs of scolopendrids exhibit a variety of shapes, the majority being what Schileyko (2009) called the common shape, the least specialised and most like locomotory legs ( Figure 1A). They are pincer-shaped in Scolopendropsis, Edentistoma, and Arthrorhabdus and this type Schileyko stated are adapted for capturing prey ( Figure 1B). The African scolopendrid Asanada socotrana Pocock, 1899, also has pincer-like legs. These lack prefemoral spines and are probably involved not in the capture of prey but in the distraction of a would-be predator as they are readily autotomised and when detached perform wriggling movements (Lewis, 1981). Species of the genus Alipes have large leaf-like ultimate legs ( Figure 1C). Alipes grandidieri Lucas, 1864 when irritated swings these from side to side and stridulates perhaps to frighten potential mammalian predators (Skovmand and Enghoff 1980). The legs are sometimes autotomised and when this happens the legs continue to stridulate. Cloudsley-Thompson (1961) reported that the long ultimate legs of the West African Rhysida nuda togoensis Kraepelin, 1903 (now R. immarginata togoensis) ( Figure 1D) slowly bend and straighten when detached and emit a faint creaking sound.
In this paper we review the functions of ultimate legs in those species that have the least modified ultimate legs and report the results of laboratory experiments on species of Scolopendra, Cormocephalus and Ethmostigmus.

Anchoring
Field observations have shown that the ultimate legs of scolopendrids are used as hooks from which the animal can hang. Thus Remington (1950) reported that S. subspinipes Leach, 1815, fastened onto a tent near the ventilator hole with their ultimate legs and swung their bodies to one side or the other to seize insects that had alighted nearby. On 4.xi.95 Peter Daszak and Janet Cottingham found a female Scolopendra abnormis Lewis & Daszak, 1996, hanging by her ultimate legs in a hollowed out cavity beneath a slab of tuff on Serpent Island, Mauritius. The anterior part of the body was curled around 40 oval yellow eggs (Lewis unpublished data). Molinari et al (2005) observed two specimens of Scolopendra gigantea L., 1758, feeding on bats whilst hanging from the roof of a Venezuelan cave to which they anchored using the last five or eight pairs of legs.
Kronmüller (2013, unpublished data) reported and photographed a Scolopendra dehaani that escaped the plastic container in the laboratory and was later found hanging on the camera tripod just using the ultimate legs ( Figure 3A).

Defence reactions
Warning posture Lewis (unpublished data) noted in Nigeria that a small Scolopendra morsitans L., 1758, raised and splayed its ultimate legs when approached from behind by a large Mecistocephalus (Geophilomorpha). Neck (1985) described such threat display in Scolopendra viridis Gervais, 1847, the ultimate legs being spread apart and in Scolopendra heros Girard, 1853, where the legs were raised and waved back and forth, active defence involving biting or "holding" with the ultimate legs. A photo of an Ethmostigmus sp. sent to one of us (J G E L) by Chris Pennington from Krambach, New South Wales, Australia, showed the ultimate legs lifted and splayed revealing the spines on the median surface of the prefemora and on the coxopleural processes. A predator approaching from behind would come into contact with a battery of spines.

Autotomy and sound production
As noted above, in case of danger the members of the genus Alipes swing the ultimate legs from side to side and stridulate perhaps to distract or frighten potential predators.. They also use autotomy to detract potential predators like birds, lizards or mammals. In case of an attack, the centipede starts to stridulate which directs the attention of the predator to the ultimate legs which can be autotomised to make the escape easier. When detached with forceps the leaf-like ultimate legs of an Alipes grandidieri from Tanzania continued to stridulate for more than half a minute.
Cloudsley- Thompson's (1961) observed that the long ultimate legs of the West African Rhysida nuda togoensis (now R. immarginata togoensis) slowly bend and straighten when detached and emit a faint creaking sound. This observations were repeated in Northern Nigeria in 1967 by Lewis (unpublished data): an ultimate leg of the same species detached with forceps produced very quiet squeaking for about 75 seconds, flexing at 2-3 second intervals and Kronmüller (2009, unpublished data) observed that the long slender ultimate legs of Rhysida immarginata immarginata (Porat, 1876) from the Philippines ( Figure 1D) as well as Rhysida longipes (Newport, 1845) from various countries in Africa bend and straighten for a few minutes presumably to enable the centipede to flee while potential predators become distracted. In our experiments, when the ultimate legs were detached with forceps,they did not squeak as did those of Rhysida immarginata togoensis..
Video clips of Alipes sp. showing the typical defence behaviour including the sound of the stridulation as well as the bending movements of the detached ultimate legs of Rhysida sp. can be seen on the website http://www.scolopendromorpha.com.

Meeting reactions
Ritualised meeting reactions have been described for Scolopendra cingulata Latreille, 1829 (Klingel 1960), Cormocephalus anceps anceps Porat, 1871 (Brunhuber 1969) and observed in Scolopendra galapagoensis Bollman, 1899 (Kronmüller 2010, unpublished data). When two specimens meet, each attempts to grasp the posterior region of the trunk of the other with its last pair of legs ( Figure 2C).
In head to head contact both rapidly swing round the posterior part of body and attempts to grip the other with the ultimate, followed by the more posterior legs.
Brunhuber's plate 1A shows that the legs are parted and hooked over the body of the other animal. In this reaction the median spines on the ultimate legs seem to aid the grip. Kronmüller (unpublished data) observed that when two Scolopendra dehaani meet and they grasp each other in the previous described way, they hold this position for several minutes and up to more than half an hour. This ritual may be to avoid any aggressive behaviour.
A short video clip of this meeting reaction can also be seen on the website http:// www.scolopendromorpha.com.

Courtship behaviour
During courtship behaviour in Cormocephalus anceps anceps the "defence posture" is adopted both partners use their antennae to tap the posterior part of the body especially the ultimate legs of the opposite sex ( Figure 2D). Tapping and/or stroking with the ultimate legs is carried out by both sexes. The ultimate legs are waved sideways and tapped irregularly up and down and quite quickly (Brunhuber 1969) [for a recent review see Rosenberg (2009)]. Lewis (1985) argued that as the prefemoral spines on the ultimate legs are directed backwards (posteriorly) it is unlikely that they are used to facilitate gripping for which they should be directed forwards (anteriorly). He suggested that the function of the spines was that of specific discrimination during courtship prior to sperm transfer. If this were the case then one would not expect sympatric species to have a similar spine arrangement. There followed a lengthy discussion to justify this hypothesis. However no evidence has been found in this investigation to support this suggestion.

Material and methods
Laboratory experiments were carried out to further investigate behaviour in prey capture as well as possible defence reactions in case of a predator attack. To simulate a the attack of mammal or bird predator, a piece of foam held with 25cm forceps was used to tap the centipedes from above.
The specimens were kept for at least 12 weeks before the experiments started in plastic containers of different sizes depending on the size of the specimen, with an air temperature of 25 degrees Celsius in day-time (around 10 hours illumination with a neon lamp) and 20-22 degrees Celsius at night-time. The average air humidity was around 70%. All plastic containers had a layer of humus and a piece of bark as a hiding-place. Once a week, the humus was sprayed with water to keep it moist and the specimen was fed one adult cricket (Gryllus assimilis).
For the experiments, the centipedes were moved to a 20 × 20 cm plastic container with a layer of about 2cm humus and left for at least one hour without disturbance before the experiments started. After each experiment, the centipedes were left for a further hour to recover before the next experiment.
The first experiment involved tapping the centipedes at different parts along the length of the body (anterior third, middle third and posterior third) to observe possible differences in defense reactions and to compare the responses of the investigated species.
In a second experiment a piece of bark was placed in the container as a refuge and the layer of humus was increased to 10cm. A centipede was placed in the container and as soon as the first third of the trunk was hidden either by the centipede crawling under the bark or by it digging into the humus, the tapping experiments described above were repeated.
Photos and short video clips are made with a Canon EOS 60D and a Canon EOS 6D, connected either to a Canon standard lens (18-55mm) or a Canon macro lens (100mm), three Canon Speedlight EX550/EX430 flashes (photos only), a tripod and a remote control (video clips).

Results of the laboratory experiments
Initial experiments were carried out on an adult Ethmostigmus trigonopodus (Leach, 1817) from Tanzania, length 13 cm. When tapped on the first third of the trunk (near the head), the centipede attacked the forceps with the forcipules. When tapped on the posterior third, or even at the ultimate legs, it adopted the warning position, raising the ultimate legs to display the ventral and medial prefemoral spines as well as the coxopleural processes which are also equipped with spines. In the most cases the centipede directed the warning position towards the forceps by turning the posterior segments. In some cases, depending on the strength and speed of the simulated attack, the centipede counter-attacked the forceps with the claws of the ultimate legs by chopping down on them after lifting them high into the warning position.
In addition Scolopendra galapagoensis lifted not only the ultimate legs into the warning position but also the last 3-4 pairs of locomotory legs ( Figure 2B). This may be to present the distodorsal prefemoral spines on these legs. This behaviour was not seen in S. heros var. arizonensis, the other New World species investigated which lacks these spines.
In the experiments with bark and a thicker layer of humus, no difference was observed in the behaviour depending on whether the specimen was tapped on the mid part of the trunk or on the last third. The result was the same when the centipede was fed (Schistocerca sp.) and the forcipules as well as the anterior pairs of locomotory legs were involved in feeding. In this situation the centipede could not use the forcipules to carry out a warning bite and the only defence behaviour was hitting with the ultimate legs and stabbing with the ultimate leg pretarsi (claws). The experiment was repeated with Ethmostigmus trigonopodus, Scolopendra galapagoensis, Cormocephalus aurantiipes and Scolopendra subspinipes, which all showed the same results.

Discussion
The scolopendrid species investigated here have unmodified and relatively robust ultimate legs. Species with very long thin ultimate legs such as some Otostigmus (Otostigmus) and Rhysida species, have not been investigated. Bücherl (1971) stated that "The specimens of Scolopendra and Otostigmus, Cryptops and others attack their prey with the last prehensorial anal legs, then the head is rapidly curved behind and the venom claws deeply and firmly buried in the body of the prey." Lewis (2010) concluded that there was no evidence to support the suggestion that the ultimate legs of Cryptops were involved in the capture of prey, instead suggesting that they are defensive; trapping some part of a potential predator and then being autotomised as the centipede makes good its escape. Dugon and Arthur (2012) stated that several species of scolopendrids had been observed seizing prey with the terminal pair of legs and then flicking the anterior half of the body to inject the prey with venom but in experiments with Scolopendra subspinipes mutilans Koch, 1878 (now S. subspinipes Leach, 1815) prey was only attacked with the forcipules.
We conclude that in the case of the species investigated the use of the ultimate legs is primarily for defence, however, should potential prey make contact with the centipede, the defensive response and defensive biting will lead to feeding whereas in the case of a predator, the centipede will try to escape after one or more warning bites.
Rowland Shelley (email dated 03 January 2011) notes "that there are frequently spines on the medial surface of the scolopendrid (ultimate leg) prefemora and I've always presumed that these must function to hold something steady I had always thought this would be prey ... " We were unable to confirm that scolopendrid centipedes hold prey or predators between the ultimate leg prefemoral, although in the ritualised meeting reaction in some species, the median spines on the ultimate legs seem to aid the grip.
The four main functions in species in which the ultimate legs are little specialised are: 1) Acting as hooks to suspend the animal for example from cave ceilings.
2) Warning position, when the legs are raised and splayed to reveal the prefemoral and coxopleural spines. 3) Stabbing as a defence, especially when the front part of the trunk and the head are unable to carry out any defensive action. 4) Grasping, the legs being flexed as when two animals assume the defence posture or as part of the mating ritual. In this case posteriorly directed spines would aid the grip. No evidence was found of objects being held between the prefemora.
The investigated centipedes (S. heros, S. subspinipes, S. morsitans, S. hainanum, S. galapagoensis, S. spinosissima, C. aurantiipes, E. trigonopodus) are from different genera, different continents and different types of habitat. Nevertheless, their behaviour in prey capture and defense reaction do not differ. Also the meeting reaction seems to be very similar the same in all observed species.
In addition to the behavioural functions of the ultimate legs, two visual signals should be considered:

Auto mimicry
Some scolopendrid centipedes, especially the giant Scolopendra sp. have striking colour patterns. In several species, the colouration of the last trunk segments and the ultimate legs is the same as the colouration of the head and antennae (Figure 3 B and C). Predators may not be able to differentiate between the head and the posterior part of the trunk.

Possible signal co-evolution
The warning posture, especially that of Scolopendra galapagoensis, where the last three or four pairs of locomotory legs are raised in addition to the ultimate pair, resembles that of spiders such as Phoneutria sp. or tarantulas and may be a case of possible signal co-evolution.


s2fieldsofstudy: Biology Psychology

text:
Myelinated Afferents Are Involved in Pathology of the Spontaneous Electrical Activity and Mechanical Hyperalgesia of Myofascial Trigger Spots in Rats

Myofascial trigger points (MTrPs) are common causes for chronic pain. Myelinated afferents were considered to be related with muscular pain, and our clinical researches indicated they might participate in the pathology of MTrPs. Here, we applied myofascial trigger spots (MTrSs, equal to MTrPs in human) of rats to further investigate role of myelinated afferents. Modified pyridine-silver staining revealed more nerve endings at MTrSs than non-MTrSs (P < 0.01), and immunohistochemistry with Neurofilament 200 indicated more myelinated afferents existed in MTrSs (P < 0.01). Spontaneous electrical activity (SEA) recordings at MTrSs showed that specific block of myelinated afferents in sciatic nerve with tetrodotoxin (TTX) led to significantly decreased SEA (P < 0.05). Behavioral assessment showed that mechanical pain thresholds (MPTs) of MTrSs were lower than those of non-MTrSs (P < 0.01). Block of myelinated afferents by intramuscular TTX injection increased MPTs of MTrSs significantly (P < 0.01), while MPTs of non-MTrSs first decreased (P < 0.05) and then increased (P > 0.05). 30 min after the injection, MPTs at MTrSs were significantly lower than those of non-MTrSs (P < 0.01). Therefore, we concluded that proliferated myelinated afferents existed at MTrSs, which were closely related to pathology of SEA and mechanical hyperalgesia of MTrSs.

Introduction
Myofascial trigger points (MTrPs), which are critical for Myofascial Pain Syndrome, are hyperirritable nodules of spot tenderness in muscle taut bands (strip cords along the muscle fibers) [1]. When stimulated manually or with a needle, MTrPs induce sharp localized tenderness, spontaneous electrical activity (SEA), local twitch response (LTR) in the taut band, and a referred pain, accompanied with autonomic symptoms such as vasoconstriction and dizziness [1,2].
SEA is the combination of endplate noise and endplate spikes with action potential [3,4]. It consists of 2 parts: a high frequency but low amplitude persistent background current (<50 V) and a low frequency but high amplitude intermittent activity (100∼700 V) [5]. Local pain intensity related to the duration and amplifies muscle cramp episodes [2]. According to the Integrated Hypothesis, muscle contractions/cramps within taut bands induced ischemia and gave rise to local pain and higher sensitivity of MTrPs [2,6,7]. The irritability of MTrPs affected SEA. A higher amplified SEA was usually accompanied with a lower pressure pain threshold (PPT) [8].
Myelinated afferents were considered to mediate electrically induced muscle pain [9]. Clinical researches showed sympathetic outflow modulated myelinated afferents [10] and affected pain as well as hyperalgesia at MTrPs [11]. Moreover, our previous human experiment reported, after ischemic compression block (ICB) of myelinated afferents, PPT increased while the mean amplitude and frequency of SEA decreased reversibly [12][13][14]. All these clinical researches indicated myelinated afferents might participate in the pathology of SEA and hyperalgesia of MTrPs. However, current proofs were almost clinical ones and were more subjective other than objective. And some further investigations, especially invasive tests, were hardly to be applied to human beings. In order to get a more definitive conclusion about the role of myelinated afferents in peripheral changes of MTrPs with more objective and morphological proofs, we designed this animal study. Modified pyridine-silver staining method and immunohistochemistry with Neurofilament 200 (NF 200, a specific marker for myelinated afferents [15]) were firstly used to analyze nerve endings in myofascial trigger spots (MTrSs, equivalent to the human MTrPs in many aspects [16]) and non-MTrSs. SEA and behavioral changes were analyzed before and after block of myelinated afferents to explore the function of myelinated afferents in SEA and mechanical hyperalgesia of MTrSs in biceps femoris muscle of rats.

Experimental Animals.
Fifty-four adult male Wistar rats (specific pathogen free (SPF) level, weight about 300∼400 g) bought from Shandong University Lab Animal Center (Jinan, China) were used in the study. All the rats were fed with food and water ad libitum in the animal house (26 ∘ C) with 12 : 12 hours of reversed light : dark cycle. This study was approved by Shandong University Animal Use Committee. Considering some gender differences, such as concentration of estrogen [17], may affect the pain pathology of rats, only male rats were used in this study.

Localization of MTrSs.
Rats were lightly anesthetized with most of the spinal reflexes reversed and immobilized in prone position. Fur and skin covering the biceps femoris were shaved and cleared. As myofibers of taut band were stiffer than the surrounding tissues, an experienced investigator could identify the taut band (diameter of 2∼3 mm) by palpating the biceps femoris. Next, squeezing the muscle along the taut band was done until an LTR existed. An LTR is brisk contraction of a group of muscle fibers in a taut band when MTrSs were stimulated. According to our experience and the current literature, usually only one most sensitive spot in the taut band can induce LTR [4], and this spot was defined as an MTrS [4,6]. According to Integrated Hypothesis, muscle contractions/cramps within taut bands induced crisis of energy, and this vicious circle led to MTrSs finally [2,6,7]. As the activities of rats were not the same, not all rats exhibited MTrSs, and the active rats tend to exhibit MTrSs. Besides, individual differences should not be ignored. We chose rats with/without MTrSs as needed (described later in the Methods). Non-MTrSs were healthy spots outside the taut band without SEA or LTR.

Electromyography (EMG) Unit Settings for SEA Recordings and EMG-Guide Injection.
A hollow-needle electrode (Ambu Neuroline Inoject, 25 × 0.30 mm, Denmark) was used as an active needle electrode inserted into MTrS region while the control needle electrode was inserted into the normal tissue, and the ground electrode was placed in the tail.
The active needle electrode was advanced about 1 mm per step until there was SEA (a high frequency but low amplitude persistent background current (<50 V) and a low frequency but high amplitude intermittent activity (100∼700 V) [5]) existing on the screen and the specific electrical activity sound "Da-Da-Da" was heard. If the control electrode recorded no SEA at the same time, an MTrS was reconfirmed at the spot the active needle electrode penetrated [3,4,6,18]. This spot was marked for SEA recording and injection of tetrodotoxin (TTX).
Settings of the EMG unit were as follows: the high frequency filter was 1000 Hz, while the low frequency filter was 100 Hz, the amplitude for recording was 20 V/division, and the sweep speed was 100 ms/division.

Section 1: Modified Pyridine-Silver
Staining and Immunohistochemistry. Twenty rats were used in this section, and the MTrS/non-MTrS tissues in biceps femoris were localized and removed. 10 MTrSs and 11 non-MTrSs were used for modified pyridine-silver staining to show the nerve endings in these tissues. All the tissues were fixed in a freshly prepared fixative (consisted of ethyl alcohol, 4.5 mL, concentrated HNO 3 , 0.1 mL, and distilled water, 5 mL) for 24 hours. Then the tissue blocks were immersed in 10 mL of absolute ethyl alcohol containing 0.1 mL of ammonia solution for another 24 hours and were washed with distilled water for 30 min. After that, the blocks were stained in 2% AgNO 3 for 3 days away from light (25 ∘ C) and were put into 10 mL of 5% formic acid containing 0.4 mg of pyrogallol for 6-24 hours [19]. After washing with distilled water, routine dehydration, transparent, paraffin immersing, and paraffin embedding of tissue blocks were carried out in sequence to prepare the paraffin blocks. Finally, both MTrSs and non-MTrSs were sectioned transversely and longitudinally at 4 m by a rotary microtome (Leica), collected, and made into paraffin sections [20]. After being dried and dewaxing, the black or dark brown nerve endings could be seen in the sections.
12 MTrSs and 8 non-MTrSs were used for the immunochemistry with NF 200 (CST) to show myelinated afferents specifically [15]. The tissue blocks were fixed in 4% paraformaldehyde overnight and were embedded in paraffin after being washed with distilled water. The paraffin blocks were cut into 4 m transverse and longitudinal sections on a rotary microtome (Leica). After that, dewaxing and antigen retrieval were carried out. After that, routine immunohistochemistry with NF 200 was carried out. NF 200 antibody was diluted to a proper concentration (1 : 100, confirmed by the preliminary tests) with phosphate-buffered saline (PBS). Peroxidase histochemistry was carried out with diaminobenzidine (DAB). Then the NF 200 positive myelinated afferents were stained brown.
In this section, for both modified pyridine-silver staining and immunochemistry, longitudinal sections were used to show location and morphology of stained fibers, while 100 nonoverlapping transverse views for MTrSs/non-MTrSs, respectively, were analyzed to get the mean number of stained fibers with light microscope equipped with a digital camera (Olympus). In order to limit the error, all measurements were performed by the same investigator.

Section 2: SEA Changes after Block of Myelinated Afferents in Sciatic
Nerve. 24 rats with MTrSs were randomly divided into TTX/isotonic saline group. After MTrS was located in the biceps femoris, a diagonal incision was made at the midpoint of ischial tuberosity and knee of the same side with MTrS. The skin, subcutaneous tissue, and fascia were dissected in sequence, and gluteus maximus was blunt dissected to expose sciatic nerve. The skin and fascia around the incision were lifted to make a bath, and artificial cerebrospinal fluid (ACSF) was used to immerse the nerve. ACSF was made up of NaCl 150 mM, KCl 5 mM, MgCl 2 1 mM, glucose 10 mM, CaCl 2 2 mM, and Tris 5 mM, and had been adjusted to pH 7.4 with HCl.
For rats in TTX group, TTX solution (1 M, Aladdin) was added into the bath to block myelinated afferents selectively [21,22]. And for rats in isotonic saline group, isotonic saline of the same volume as TTX in the TTX group was added into the bath as the blank control. Before and ten minutes after the addition of TTX/isotonic saline, SEA was recorded stably for 3 min at MTrSs of all rats. An MTrS region was a tiny area for about 1∼2 mm 2 [23]; so after locating it, the operator had to be very careful to avoid the movement of needle electrode and make sure the electrode stays in the MTrS region all the time. According to our previous research, ischemic compression block of myelinated afferents of MTrPs resulted in a 50% decrease of the average amplitude and frequency of SEA [14]. Therefore, in this study, if the average amplitude or frequency of the high amplitude wave of SEA was 50% lower than before, it was considered as a significant change. Then the incidence rates of SEA significant changes in the two groups were analyzed. In order to limit the error, all measurements in this section were performed by the same investigator.

Section 3: Behavioral Assessment of Mechanical Hyperalgesia of MTrSs.
Animal study revealed that stimulating masseter muscle of rats with mechanical stimulation evoked the bilateral limb nociceptive withdrawal reflex (NWR), and this model was confirmed to be an effective one to test the muscular sensitivity of lightly anaesthetized rats, and the results could been treated as "objective" ones [24][25][26][27]. Therefore, we assessed the mechanical hyperalgesia of MTrSs in the same way.
In this section, 10 rats were used. An electronic von Frey (VF) analgesiometer with a hard tip was used to stimulate the skin above the MTrSs, until ipsilateral hind-limb NWR existed. The minimum stimulus intensity that just evoked the NMR was mechanical pain threshold (MPT) which can represent the mechanical sensitivity of MTrSs/non-MTrSs [24,25].
After MTrS was located in biceps femoris muscle of rat, a non-MTrS, a non-taut band spot (normal healthy tissue), was located in the contralateral biceps femoris muscle. First, MPTs of MTrSs and non-MTrSs were assessed, respectively. Then, 0.1 mL 1 M TTX was injected into MTrSs as well as non-MTrSs to block myelinated afferents [21]. And MPTs of MTrSs/non-MTrSs were reassessed 5 min, 15 min, and 30 min after the injection. The thresholds were measured twice with an interval of 1 min at each proper time, and the average of the two values was used as the result. In order to limit the error, all measurements in this section were performed by the same investigator. As the area of MTrS was quite small, just about 1∼2 mm 2 [23], small dose (about 0.1 mL) of liquid was used so that all of the drug could be injected [6,18].

Analysis and Statistics of Data.
The "a priori" sample size estimations for the rats needed in three sections were made according to the results of our preliminary experiments and the sample size estimations formulae. And the minimum of rats needed in each section was shown as follows: four rats for MTrS/non-MTrS group in modified pyridinesilver staining/IHC with NF200 in Section 1; eleven rats for TTX/isotonic saline group in Section 2; and four rats for MTrS/non-MTrS group in behavioral assessment in Section 3. Considering there might be unexpected conditions, we used a few more rats in the formal experiments. Fisher's exact test was applied to analyze the incidence rate differences of the significant SEA changes between TTX and isotonic saline groups in Section 2. All data in Sections 1 and 3 were presented as mean ± SD and analyzed with SPSS 13.0 for Windows (SPSS Inc., Chicago, IL, USA). And the results indicated that data of each variable fitted normal distribution. Student's -test was used to analyze the differences of silver/NF 200 marked fibers between MTrS and non-MTrS tissues. And a two-way ANOVA followed with Student Newman-Keuls Test (SNK) was used to analyze the differences of MPTs at different time after the TTX injection between MTrS and non-MTrS. For all data, it was considered to be significant if < 0.05.

SEA at MTrS Region Decreased Significantly after Specific
Block of Myelinated Afferents in Sciatic Nerve. For Section 2, in the TTX group, 7 rats showed significantly decreased SEA, while the other 5 rats had decreased, but not significantly changed, SEA after TTX addition. In the isotonic saline group, 2 rats had significantly decreased SEA after the addition of isotonic saline and the rest of the rats just had no significant changes (see Figure 2). Incidence rates of SEA significant changes between the two groups were significantly different ( = 0.0447).

MPT of MTrS Was Lower Than That of Non-MTrS, and MTrS Was More Sensitive to Block of Myelinated Afferents.
For Section 3, a two-way ANOVA revealed, 5, 15, and 30 min after the block, there were significant differences between MPTs of MTrSs and non-MTrSs, and there was significant interaction between these two factors (5 min after the block:  "#" represents, before the TTX injection, MPTs at MTrSs were significantly lower than those at non-MTrSs ( < 0.01). "@" represents, 5 min, 15 min, and 30 min after the TTX injection, MPTs at MTrSs increased significantly ( < 0.01). " * " represents, 5 min after the TTX injection, MPTs at non-MTrSs were lower than those before the injection ( < 0.05). "!" represents, 15 min and 30 min after the TTX injection, MPTs at non-MTrSs were higher than those at non-MTrSs before injection, but the differences did not reach the significant level ( > 0.05). "&" represents, 30 min after TTX injection, MPTs at MTrSs were significantly lower than those at non-MTrSs ( < 0.01).

Discussion
This study further investigated the function of myelinated afferents in pathology of MTrSs. We found more nerve endings, especially more myelinated afferents, existed in MTrS regions than normal tissue. And specific block of myelinated afferents with TTX resulted in decreased SEA as well as higher MPTs of MTrSs. Thus, we concluded proliferated myelinated afferents were closely related with SEA and mechanical hyperalgesia of MTrSs.
Modified pyridine-silver staining method was used to indicate sensory nerve endings in skeletal muscles, and these nerve endings were stained as dark brown or black [19]. In this current study, we applied this method to show nerve endings in MTrS regions for the first time and found there were more nerve endings in MTrS region than non-MTrS region. Previous study revealed similar results by applying iron deposition method, and they indicated these nerve endings were nociceptive ones [28,29]. Additionally, in this study, immunohistochemistry for NF 200 was performed [15] and revealed more myelinated afferents in MTrS region than normal tissues. "#" represents, before the TTX injection, MPTs at MTrSs were significantly lower than those at non-MTrSs ( < 0.01). "@" represents, 5 min, 15 min, and 30 min after the TTX injection, MPTs at MTrSs increased significantly ( < 0.01). " * " represents, 5 min after the TTX injection, MPTs at non-MTrSs decreased ( < 0.05). "!" represents, 15 min and 30 min after the TTX injection, MPTs at non-MTrSs were higher than those at non-MTrSs before injection, but the differences did not reach the significant level ( > 0.05). "&" represents, 30 min after TTX injection, MPTs at MTrSs were significantly lower than those at non-MTrSs ( < 0.01).
The reason for proliferated myelinated afferents was not clear yet. It was reported that, after nerve injury, membrane properties of myelinated afferents changed which induced sprout of axon endings [30]. We thought local environment changes at MTrS regions, including changes of electrolytes, pH, muscle metabolites, inflammatory mediators, and neurotransmitters [3,7], might induce the sprout of myelinated afferents as well and lead to increased number of them.
In order to investigate the function of myelinated afferents, we selected TTX to block them specifically [21,22,31]. TTX is a neurotoxin, which can block the rapid sodium channels in fast conducted myelinated afferents with little effect to slow sodium channels in unmyelinated afferents [32]. After blocking myelinated afferents, SEA and MPT of MTrSs changed accordingly, which indicated the relations between myelinated afferents and MTrSs. And our previous clinical study supported this; ischemic compression blockage (ICB) of myelinated afferents changed SEA and pressure pain threshold (PPT) as well as pressure threshold for eliciting referred pain (PTRP) [13,14].
SEA is one of the characteristics of MTrSs. It is a combination of endplate noise and endplate spikes with action potential [3,4,23,33] and can be recorded at MTrPs/MTrSs with EMG equipment. Nociceptive stimuli, such as inflammation, induced higher sensitivity of myelinated afferents and raised SEA [34]. TTX blocked the action activity of fast conducted 6 Evidence-Based Complementary and Alternative Medicine myelinated afferents [32], and then amplitude and frequency of SEA decreased.
As no criterion about SEA changes was generally recognized, in Section 2, we chose 50% decrease of amplitude or frequency of the high amplitude wave as a significant change (according to our previous clinical results [14]). Any SEA change that did not reach this level was considered to be no significant one, even if their amplitude/frequency did decrease.
And it was worth noticing that although incidence rates of SEA significant changes caused by TTX/isotonic saline were significantly different ( < 0.05), there were still 5 rats that had decreased but not significantly changed SEA after applying TTX. We inferred the reasons might be as follows. Individual differences of rats, such as numbers of sodium channels on myelinated afferents, led to different sensitivity to TTX [35,36]. And myelinated afferents of these 5 rats might be blocked to a lesser extent when compared with the other 7 rats in TTX group; thus, their amplitude or frequency of SEA decreased, but did not reach significant level according to our criterion [14]. In addition, C fibers and sympathetic nerves, which were less sensitive to TTX [32,37], were inferred to innervate muscle spindles and modify SEA of MTrSs as well [38][39][40][41], and they might influence the results. Besides, our criterion for significant changes might be a little too strict, and these not significantly changed SEAs in the 5 rats were possible to be significant ones according to a looser criterion. Further studies were needed to find an improved criterion and better understand SEA.
Noxious cutaneous stimulations elicit nociceptive withdrawal reflex (NWR), which protects the subject from nociceptive damage. NWR is a spinal polysynaptic reflex, and it has been used to investigate pain processing as well as pain pathways at spinal levels [26]. Moreover, the results of behavior with NWR can be used as an "objective" measure of experimental pain [24,25,27]. Therefore, we assessed mechanical hyperalgesia of MTrSs/non-MTrSs in the same way. MPT at non-MTrS regions increased, 15 min or 30 min after the TTX injection, but not significantly, while that of MTrS regions increased significantly at any time after the injection. It seemed that block of myelinated afferents affected the mechanical hyperalgesia of both MTrSs and non-MTrSs, and MTrSs were more sensitive to the block of myelinated afferents. An explanation for this was that there were more myelinated afferents existing in MTrSs than in non-MTrSs, which could be proved by Section 1 of this study.
Besides, 30 min after the injection, although the MPT of MTrS was obviously increased, it was still lower than that of non-MTrS. We inferred C fibers and sympathetic nerves were responsible for this. C fibers participated in the conduction of nociceptive stimuli, increased the sensitivity of MTrSs [29,[38][39][40], and led to decreased MPT of MTrSs [8]. Moreover, sympathetic nerves were indicated to modify MTrS related pain via sympathetic-sensory interactions [10]. Both of them were much less sensitive to TTX than myelinated afferents [32,37], and therefore, they might be potential reasons for the MPT differences between MTrS and non-MTrS after blocking myelinated afferents. Additionally, MPT of non-MTrS decreased 5 min after the TTX injection, and a likely explanation was that the injection operation might stimulate C fibers.
Although this study demonstrated myelinated afferents affected SEA and mechanical hyperalgesia of MTrSs, the mechanism was still mysterious. We hypothesized the reactions between myelinated afferents and sensitization (both central and peripheral) of MTrSs [7] were responsible for this process. We found that muscle fibers in MTrSs were especially prone to fatigue [42,43], and these fatigue muscle fibers could enhance input impulses of myelinated afferents [44]. According to the Integrated Trigger Point Hypothesis [1,2,6,7], muscle contractions/cramps within taut bands led to ischemia and energy crisis and induced release of neural active substances and metabolites and resulted in higher sensitivity of MTrSs [2,6,7] and higher sensitivity and proliferation of myelinated afferent terminals [1,7,30]. The irritability of MTrS is related to SEA [8], while proliferated sensitive myelinated afferents conducted more peripheral nociceptive stimuli to the central nerve system, participated in the development of central sensitization by sprout and phenotypic switch, and induced hyperalgesia of MTrSs [45][46][47]. In addition, increased sympathetic activity and pain intensity were reported at MTrPs, and the sympathetic nerves in muscle spindles were consisted to be related with sensitized myelinated afferents and gave rise to lower pain threshold, higher pain intensity, and increased SEA at MTrPs [10,11,13,14,41].
The mechanism of MTrSs is mysterious, and in this study, we found proliferated myelinated afferents modified SEA and mechanical hyperalgesia of MTrSs and proposed a hypothesis of the modulatory mechanism. More studies, such as investigating central sensitization with immunohistochemistry about the nociceptive related receptors in spinal cord, afferent nerve tracing to show their connections and conduction, were already in operation to reveal the exact mechanism.

Conclusion
This study demonstrated that MTrSs contained more myelinated afferents than normal healthy tissues, which were associated with pathology of SEA and mechanical hyperalgesia of MTrSs. Nevertheless, how myelinated afferents affect SEA and mechanical hyperalgesia was still uncertain. We hypothesized myelinated afferents affected SEA and mechanical hyperalgesia of MTrSs through central and peripheral sensitization. More studies were already in operation to reveal the exact mechanism.