Tissue Necrosis Associated with Chemical Ablations1

Chemical ablations (CHAs) have become an active area of research. Although still in the investigational stage, CHAs offer low cost procedure with significantly lower preparation and procedure times as compared to other ablation modalities. Although numerous human and animal studies have been performed, feasibility, predictability, repeatability, and reproducibility of tissue necrosis as a result of needle injections still remain to be better understood and characterized. CHA is considered as a nonsurgical technique that uses chemical agents at lethal concentrations that are directly injected into the target tissue site to induce desired ablations. Its proposed mechanisms of action are by denaturing proteins, creating focal tissue damage, and ultimately leading to sustained tissue necrosis at the site of injection. We describe here the methodology to quantify the relative amounts of tissue damage/necrosis as a result of CHA so that: (1) injected dose can be optimized; (2) collateral injury to surrounding structures/organs can be minimized; and/or (3) procedural safety and effectiveness can be enhanced to achieve better outcomes.All the animal studies were approved by the University of Minnesota Institutional Animal Care and Use Committee. Muscle bundles of freshly excised swine cardiac trabeculae (n = 16 experimental and 8 control) and respiratory diaphragm (n = 48 experimental and 32 control) were prepared as cylindrical shape muscle bundles (lengths: 15–25 mm and diameters: 2–4 mm) as described previously [1].Four different chemical ablative agents were used which included: (1) 97% glacial acetic acid (Item 320099-500 ml, mf: C2H4O2, mw: 60.05 gm/mol, Sigma-Aldrich, St. Louis, MO); (2) 200 proof ethanol (Item 2716, mf: C2H5OH, mw: 46.07 gm/mol, Decon Laboratories, Inc., King of Prussia, PA); (3) 30% hypertonic sodium chloride solution (Item 7581-06, mf: NaCl, mw: 58.44 gm/mol, Macron Fine Chemicals, Center Valley, PA); and (4) 8 M urea (Item U4883-6 × 25 ml, mf: CH4N2O, mw: 60.06 gm/mol, Sigma-Aldrich, St. Louis, MO). A special syringe with an ultrafine short needle and resolution of 5 μL was used for injections of chemical agents within the muscle bundles (Item 328438, 31 G × 5/16 in., 3/10 mL Lo-Dose™ Ultra-Fine™ short needle with permanently attached needle, BD Syringes, Franklin Lakes, NJ).Four chemical agent volumes (doses) of 10 μl, 25 μl, 40 μl, and 50 μl were investigated because these volumes match relative dose levels that are administered clinically. The control muscle bundles were injected with Krebs-buffer solution at the same dose volumes. A given muscle bundle was injected with only one chemical agent or Krebs buffer at a given dose volume. All the administrations were near the center of the muscle bundle (Fig. 1). Extreme care was exercised during injections, especially to localize the entire volume of the agent within the muscle bundle itself (i.e., prevent agent from oozing out) to maximize ablation efficiency and repeatability. The rate of injection was approximately 5 μl/s, and all the injections were typically completed within 30 s. Following injections and to subsequently assess relative degrees of tissue viability and/or the extent of necrosis, muscle bundles were soaked and stained in individual tubular compartments containing triphenyltetrazolium chloride (TTC) solution in Trizma buffer (7.4 pH), while being immersed in a water bath maintained at constant temperature of 37 °C for at least 3 hrs. TTC differentiates between metabolically active and inactive tissues by utilizing various dehydrogenase enzymes within the cell. These enzymes are involved in oxidation and cellular metabolism that convert tetrazolium salt to a formazan pigment within viable tissue (formazan derivative). Thus, cells with preserved enzymatic activity undergo a cytochemical color change to red–maroon. Whereas, cells that lack the enzymatic activity (necrosis) are unable to metabolize the tetrazolium salt, do not stain, and remain pale or white.Following staining with TTC, the sutures on both ends of the muscle bundle were cut and thin slices of the cylindrical muscle bundle were prepared using a #11 surgical blade (Fig. 2). Slices were made as thin as possible along the entire length of the muscle bundle with approximately 3 mm interslice distance. Each slice was placed sequentially from top to bottom on gauze with the cross-sectional surface visible. Slices were then photographed with a high-resolution camera, which were then used for analysis using the imagej software.The cross-sectional images were analyzed using imagej software to calculate the ablated area as shown in Fig. 3. These analyses were performed at the same magnification for all the images within a given group. Two measurements were made for each cross-sectional surface for each sample: (1) total circumferential surface area and (2) estimated necrotic tissue area. The surface areas were calculated by selecting the circumferential surface of the muscle bundle using the “Freehand selections” tool in imagej, thus measuring the areas within that selection. Again, the TTC staining distinguished viable tissue as red, while necrotic tissue as pale-white as shown in Fig. 3. The necrotic area was identified by the distinct pale-white color. If a transition zone was observed that was questionably part of a lesion, it was included as such; therefore, our results include areas that may have subsequently elicited apoptosis. The resulting necrotic tissue areas for all the sections within a tissue bundle were summed, and the percent ablated areas were calculated by taking the percent ratio of the area of necrotic tissue to that considered viable (data presented as μ + σ).Figure 4 displays the calculated percent ablated areas of tissues for all the tissue types, i.e., relative to each ablation modality at various doses. The control Krebs-injections elicited no necrosis for any injected volume (0% ablated area, Fig. 3—left), suggesting that the necrosis observed in the ablated tissue samples was indeed a result of injury from a given CHA. For every chemical agent, the percent ablated area was found to be dose dependent, that is, larger necrotic areas were observed at higher ablation doses. In general, as expected, consistent increases in percent ablated areas were observed with increasing ablation doses. CHA employing acetic acid displayed the greatest percentages of ablated tissue areas (p < 0.05), followed by ethanol, urea, and sodium chloride (in this order), although statistically significant differences were not observed with these three agents. Ablations with hypertonic sodium chloride showed the least necrotic areas as compared to other ablative agents. It should also be noted that the extent of necrosis induced by these ablative agents also depends on the tissue type. Figure 5 displays the percent ablated tissue for diaphragm and trabeculae tissue types for two chemical ablative agents (acetic acid and ethanol). A larger ablated area was observed for trabeculae as compared to the diaphragm muscle bundles. These differences were more profound with acetic acid than ethanol. For example, at a dose of acetic acid above 40 μL, the trabeculae muscle bundles were observed to be 100% ablated (Fig. 3—right), while the diaphragmatic muscle bundles were ∼70% ablated. This is considered due to the morphology of cardiac tissue which has higher compactness from excessive branching of muscle fibers that facilitates localization and prevents the chemical ablative agent from oozing out resulting in higher levels of tissue necrosis.We describe here in vitro reproducible methodologies to study tissue necrosis induced by chemical ablative therapies. One of the biggest challenges of CHA is the ability of the agent to remain localized at the injection site, making the spread of the agent through the tissue irregular and unpredictable. This is even more profound when the injection site is within or near an area of high blood perfusion. We consider that in order to better understand this complex phenomenon, it is important to divide the response mechanisms into multiple steps, i.e., where each behavioral process can be investigated in a controlled manner. In this investigation, we characterized two tissue types which were exposed to four different CHA agents at various doses. These results will be utilized to design the next set of experiments that will aid in better understanding of tissue necrosis in various tissue types and under different physiological and ablative conditions. In the future, CHAs may become a more effective modality by combining it with other energy sources, such as radiofrequency, cryotherapy, or microwave ablation to name a few.