Criteria for Qualitative and Quantitative Pathologic Evaluation

Useful qualitative pathologic assessment of thermal lesions (or any pathologic lesion) requires 1) the identification of an abnormality and 2) a precise description of the abnormality relative to the surrounding tissues. Then, 3) the abnormality has to be proven to be due to the energy deposition and the subsequent generation of heat and not to artifact. Histologic artifacts resulting from poor fixation, rough tissue handling, and/or inadequate specimen processing have led to erroneous interpretations since many processing artifacts resemble thermal lesions.

Quantitative pathologic evaluation of thermal lesions encompasses all the criteria for qualitative pathology, but in addition, 4) the abnormality has to be measurable.

Anatomy and Tissue Composition

The cell with its nucleus and cytoplasm is the basic living unit. Tissues are complex assemblages of cells supported by extracellular matrix usually arranged in layers, nodules, masses and bands. Organs are composed of particular tissue types that have defined functions such as the bones for support, the heart to pump blood, the kidneys to maintain water, salt and nitrogen balance and the liver to metabolize many endogenous and exogenous substances. [10-14]

Tissues and organs are visually distinct because of their composition, color, function and boundaries. Likewise, the components of tissues and organs and their boundaries can be defined by other physical properties such as 1) elasticity, viscosity and density 2) electromagnetic conductivity and resistance, 3) heat transport and specific heat capacity and 4) mass.

Some tissues and organs such as the human liver or the thick subcutaneous adipose tissue of the sumo wrestler are sufficiently large to be considered as targets with semi-infinite boundaries. However, most animals and people parts are composed of closely packed layers with many well defined physical and physiologic boundaries. For example, the muscles of the legs and arms are composed primarily of skeletal muscle cells supported by a strong, relatively inelastic collagenous network. This network conveys the muscle contractile force to the rigid bones to produce work across the moveable joints. Muscle cell myoglobin and blood hemoglobin produce the distinctive red color of the muscle that contrasts with the light tan collagenous membranous coverings (fascia) that separate the individual muscle groups. The muscle cells and nerve conduct electrical currents to coordinate the extremity movements. The muscle groups are covered by a layer of fat tissue (subcutaneous tissue) whose thickness varies depending on the nutritional status, the habitus and sex of the individual. The subcutaneous tissue is covered by the distinctively layered skin composed of the vascular dermis and surface epithelium. The skin is important for temperature regulation, secretion of water, salts and C02 and protection from desiccation and outside elements.

Then, to make things more complex, the anatomy and physiology of living organisms can change in response to age, sex, hormonal status, physical environment, metabolism, nutrition and reproduction. And, the organism is not even sick!

Diseases

The ancient Greek and Hindu physicians advocated cautery as a treatment of last resort after all their herbs, salves, prayers and incantations failed. [3-4]

“The diseases that medicine cannot cure, excision cures: those which excision cannot cure, are cured by the cautery; but those which the cautery cannot cure, may be deemed incurable.” [15]

Nowadays, we can look at thermal cautery, cutting and coagulation as treatments for cure or palliation (relief of symptoms without total disease removal) depending on the disease process itself and the health status of the patient. The desired therapeutic goal of thermal medical applications is the modification or removal of a diseased part of the target tissue without creating undue harm to the surrounding normal tissues and to the patient himself.

Thermal Treatment Criteria

The diseases that are most amenable to ablative and coagulative thermal treatments are localized lesions in which the thermal treatments and their particular energy sources and instrumentation offer greater advantages than other available therapies. An excellent example is coagulation of detached retinas or retinal hemorrhages using laser light. This non-invasive, relatively cheap, outpatient procedure has stabilized the sight of thousands without requiring extensive eye surgery with its attendant morbidity.

New thermal treatments and new instrument development are being developed to exploit the advantages using minimally invasive surgical procedures. In particular, these procedures are beneficial for those patients who have other medical conditions that preclude major surgery, systemic chemotherapy of ionizing irradiation. The need to destroy isolated tumors such as uterine myomas (fibroids), metastatic cancers to the liver or lung or small primary breast cancers have led to development of new instruments for superficial and deep interstitial thermal coagulation. Superficial thermal fulgeration (ablation and coagulation) continues to be a useful approach for removal of superficial precancers and minimally invasive cancers of the larynx, uterine cervix and rectum.

Effective thermal treatments have to be designed paying strict attention to the anatomy, physiology and composition of the lesion as well as of the surrounding non-diseased tissue. Keeping this in mind, investigators are urged strongly to keep up-to-date anatomy, histology, pathology and physiology books on their desks. In addition, engineers and physicists should include motivated and interested pathologists, physiologists and clinicians on their lists of their closest and dearest collaborators.

Laser Light:

At the present time, therapeutic laser light irradiation is limited to the near-ultraviolet, visible and near-infrared wavelengths. Light energy is transformed to heat energy upon the light absorption by various tissue constituents. Therefore, the absorption, scattering and anisotropy of the target tissues and the delivery systems (air, fiber optics, wave guides, lenses, etc.) will be major determinants for energy deposition into a desired geometry. [16] The thermal lesion size and geometry can modified by using one or more irradiation beams directed to the target tissues using multiple lasers and/or multiple lenses, fiber optics or wave guides. Thus, the thermal lesion size and geometry can be refined by beam placement via the delivery system. The optical and thermal properties of the irradiated tissues usually change dynamically during the light exposure due to water loss and thermal tissue coagulation. [17]

Radiofrequency Fields

Radiofrequency electromagnetic fields (typically 500 kHz to 500 MHz) are delivered to tissues by contact electrodes or antennas of various geometries to obtain useful current fields and heat lesion size. [1] Heat is generated as resistive dissipation (resistive or joule heating) due to collisions occurring during translational motion of charged carriers in the tissue located in the electric field. [18, 19] Therefore, the heat lesion will be greatly influenced by 1) the electrical properties of the tissue, 2) the use of monopolar vs bipolar electrode designs, 3) the current density, 4) the tissue anatomy and 5) tissue desiccation and thermal coagulation.

Microwave Irradiation

Microwaves (500 MHz-5 GHz and higher) produce heating by rotational motion of water molecule dipoles present in the tissue. [19] Like radiofrequency lesions, the thermal lesion produced by microwaves is markedly influenced by the configuration of the delivery antennas (wave guides) since heat generation occurs only in the electric field. Water is a major constituent of tissues, therefore heat generation will be dominated by the original tissue water content and the dynamic changes of water concentration due to vaporization during heating.

Ultrasound

Therapeutic ultrasound (typically 0.5-2.0 MHz) generates heat from viscous dissipation the acoustic waves propagating through biological tissues. [18, 20] Therefore, the tissue mechanical properties including the tissue viscosity, elasticity, specific heat and tissue density will be major factors in the production of thermal lesions. Tissue contact with the ultrasound probe and the probe configuration can also influence the lesion size. Ultrasound waves are easily scattered by the complex echogenic interfaces of tissues. Therefore, disease lesions close to the contact surface are most amenable to treatments using ultrasound energy sources. However, newer techniques of ultrasound focussing could allow deeper penetration for interstitial thermal treatments. Since piezoelectric probes can be fitted to flexible cables, the deeper cavities of the body can be accessed for diagnostic as well as therapeutic ultrasound applications.

Heat Transfer in Living Tissues

The major determinant of heat transport in biological tissues is their water content (around 80% for soft tissues) therefore, their thermal properties will change as the tissue desiccates during the heating process. As desiccation continues on, the residual tissue constituents such as coagulated proteins, trapped vaporized water and melted fats will assume larger roles in heat transport. Convection heat loss is very important in living patients being treated by relatively low temperature (below 100°C) and prolonged (seconds to hours) heat exposures. Convection heat transfer is produced by physiologic functions and environmental conditions. Blood and, to a lesser extent, lymph flow in vascular channels and air flow through the trachea and lung bronchi during respiration are the important intrinsic physiologic factors. Environmental causes of significant convection heat loss include 1) exposure of tissues to ambient air temperatures and room air circulation (operating rooms have significant air exchange volumes and are always too cold!), 2) irrigation of the surgical field, 3) exogenous organ perfusion such as the use of respirators or the heart-lung machines and 4) stoppage of blood and lymph flow as the blood and lymph vessels are tied off during surgery. [16]

Pathologic Markers of Thermal Damage in Biologic Tissues

The geometric volume of energy deposition and heat transformation can be considered as a single volume heat source for most clinical thermal applications. As heat is transported along gradients from the hot center to the cooler periphery, concentric zones of thermal damage can be detected using gross and microscopic pathologic techniques. Likewise, a heat source slab placed on the target tissue surface creates layered thermal damage zones extending from the hotter surface to the cooler deep tissues. [Figure 2]

Figure 2

Thermal ablation and coagulation lesions: Examples of single volume heat source and slab heat source lesions.

A. Coagulation (upper) and coagulation/thermal lesions were produced in vivo in rats and the liver harvested 30 minutes after treatment using a CW Nd:Y laser. The concentric zones of thermal damage extend from the center point of light irradiation which in these lesions are represented by the white central coagulum (structural protein thermal coagulation) in the upper lesion and the dark hole in the lower lesion.

B. A flat heat source (water-filled balloon) was expanded within the uterus cavity of a woman undergoing hysterectomy. The blood supply was cut off and the uterus was removed about 30 minutes after heating. The thermal damage zones, surface white coagulum and subjacent red damage zone have distinct, measurable borders.

The boundaries between some of these zones can be distinct and measurable thus allowing mapping of thermal lesion. [9] These measurements also allow comparisons of different treatment effects, energy sources and instrument designs. Accurate comparisons require identification of useful treatment end points (markers) that can be measured and followed over time. In addition, pathologic observation techniques should be chosen that allow meaningful qualitative and quantitative evaluations of the treatment and its resolution. And, multiple experiments should be performed under the same controlled conditions using the same energy and delivery parameters to obtain statistically significant data.

Thermal Damage Zones

Beginning at the hotter center and progressing to the cooler periphery, the thermal damage zones can include 1) tissue ablation, 2) carbonization, 3) water-vaporization thermal damage including steam vacuole formation, explosive fragmentation and tissue desiccation, 4) structural protein dénaturation (thermal coagulation) including collagen hyalinization, collagen and muscle birefringence changes, tissue whitening and cell shrinkage, 5) vital enzyme protein denaturation, 6) hemorrhage, hemostasis and hyperhemia, 7) tissue necrosis and 8) wound organization and healing. Some of these zones can be seen during and immediately after heating in vitro or in vivo applications but others can only develop in the living and surviving subject. [Table 1]

Table 1

Thermal Damage Zones

Ablation:

We have arbitrarily defined ablation to be the physical removal of the solid components of tissue. Chunks, fragments, layers, cells, molecules and ionized atoms can be removed from tissue depending on the energy delivered in each exposure. In the realm of current thermal therapies, holes, defects, craters or cuts can be produced by any or all of the following 1) thermal tissue vaporization, 2) combustion and/or 3) explosive fragmentation. [Figure 3]

Figure 3

Thermal ablation and coagulation lesion in rat liver produced with CW argon laser 9W, 9Sec, Spot Size 1 mm. The central ablation hole is lined by scattered black carbon granules covering the subjacent vacuolated water vapor damage zone. Beyond this zone, the structural protein coagulation zone extends to the inner boundary of the red damage zone (arrows). The outer boundary of the red damage zone (arrow heads) is faint in this black and white image. The lower picture is a higher magnification of the upper image. [H&E. Original Mag. 3X and 8X]

Carbonization:

Carbonization is the thermal reduction of organic tissue components to carbon. In ablation lesions, the black granular carbon residues (<1 to 10-μm) form a thin lining of the ablation site. Although this layer can be measured microscopically, the actual identification of carbon formation probably is more important than the measurements when evaluating dosimetry and/or instrument design. Carbon formation is undesirable in laser interstitial thermal therapy as the carbon absorbs the incident light preventing its transport into the surrounding tissues. In addition, carbonization can signal unwanted arcing across electrodes or tissue combustion. [Figures 3 & 4]

Figure 4

Part of thermal ablation hole and water vapor damage zone in dog aorta. Irregular black carbon granules are scattered on the surface of the lesion. The water vapor (steam) vacuoles form a distinct zone with a measurable boundary. The structural protein thermal coagulation damage is not visible at this magnification. [H&E. Original Mag. 25X]

Water Vaporization:

Nearly all biological animal tissues contain about 80% water (except bone, tooth enamel and dentine, finger and toe nails, hair and the outer layer of the skin, the stratum corneum). Water is always sublimating from tissue surfaces and, as the tissue temperature increases, more water vaporizes. At temperatures below 100°C, water vapor formed close to the surface usually can diffuse out of the tissue. But, as the temperature approaches and passes 100°C, water vapor (steam) formation accelerates. The amount of steam formed is greater than that which can escape from the tissue by diffusion. Therefore, small vapor pockets form within the tissue and grow bigger as the superheated steam rapidly expands, the so-called “popcorn” effect. The thin, desiccated, rigid walls of the vacuoles rupture as the vapor pressure increases allowing the vacuoles to form larger steam pockets deeper in the tissues. But, closer the surface, the rapidly expanding pockets burst open blowing tissue fragments into the air thus producing an ablation cavity with tissue mass loss due to explosive fragmentation. [Figure 3]

Not infrequently, the zone of water vacuolization has a fairly distinct, measurable border. [Figure 4] However, depending on the tissue anatomy and its mechanical properties, the vapor can dissect along weak planes to form a recognizable, but not measurable, thermal damage zone.

The problem of distinguishing true water vacuoles from fixation or tissue processing artifact in microscopic sections can be difficult in certain tissues. For example, slit-like spaces are frequent in light microscopic sections of arteries and cornea. These artifacts are due to uneven shrinkage and contraction of collagen layers produced by fixation and heat applied during the preparation of the tissues for paraffin sectioning. Thermal vacuoles can be distinguished from histologic artifact by the following observations: 1) heat lesion vacuoles will be clustered and more numerous in the lesion than in the surrounding tissue, 2) the lesion vacuoles will have some regular distribution reflecting the geometry of energy deposition and heat transport and 3) other signs of thermal damage such as collagen hyalinization and birefringence changes will be associated with the vacuoles. Of course, the knowledge that a thermal lesion was made in the location under observation is an additional diagnostic bonus.

Structural Protein Thermal Coagulation (Denaturation)

Structural proteins are the proteins that form the extracellular matrix and cytoskeleton of all cells. The formed extracellular structural proteins such as the collagens and elastins form the dermis, tendons and general fibrous supporting structures of all tissues. These formed elements are intimately related to various proteoglycans that generally act as an amorphous matrix that binds considerable amounts of water. The cytoskeleton structural proteins are the microtubules, microfilaments and intermediate filaments that form 1) the three-dimensional scaffolding of the cytoplasm and nucleus, 2) the contractile proteins of the muscle cells, 3) the mitotic spindles and 4) intracellular and extracellular communication transport networks. [10, 11,14, 22,23]

Grossly, most thermally coagulated tissues become lighter colored and more opaque as is seen when red steak is boiled and eggs are fried. Cooked meat and egg whites are good examples of protein thermal coagulation. On the other hand, fat (adipose) tissues and collagen-rich tissues such as tendon and sclera become more translucent with heating. The pale borders of thermally coagulated tissue are usually measurable grossly.

Microscopically, within the coagulum, there are several concentric thermal damage zones with distinct boundaries that can be measured. Beginning from the hotter center and going to the cooler periphery these zones include 1) collagen (Type 1) denaturation manifest by birefringence changes and hyalinization, 2) muscle cell birefringence changes and 3) cell shrinkage. All of these changes are due to thermal denaturation of structural proteins.

Collagen and Muscle Birefringence Changes.

Native Type 1 fibrillar collagen is composed of macromolecules arranged in a semicrystalline array which is responsible for the formation of a bright birefringent image when observed with polarizing light microscopy. [10, 11, 23-26,] In addition, the α-helical arrangements of the polypeptide molecules that form the basic collagen molecule, tropocollagen, confer birefringence intrinsic to the molecule. In a similar fashion, the contractile proteins of skeletal, cardiac and smooth muscle are also brightly birefringent. Roughly, 80% of the collagen and muscle birefringence is form birefringence due to the crystalline array of the macromolecules while the rest is the intrinsic molecular birefringence. [Figures 5 & 6]

Figure 5

Thermal coagulation lesion in pig cornea in vtro.

Several structural protein thermal coagulation changes can be seen in the routine H&E stained paraffin section examined through a diffuse light microscope (upper) and a transmission polarizing microscope (lower). Thermally induced collagen hyalinization (upper right) forms a dark, amorphous area with a distinct boundary (arrows). In this case, the boundary of partial birefringence loss coincides with that of hyalinization. The characteristic fibrous profiles of native collagen (left) are well illustrated in the TPM image. The corneal epithelial cells covering the coagulated collagen are spindled due to thermal denaturation of their cytoskeletons. The continuous bright white line in the TPM is a drying artifact of the epithelial surface. (H&E. Org. Mag. 40X)

Figure 6

Birefringence Loss in Thermal Ablation/Coagulation in Rabbit Heart

Structural protein thermal coagulation is not apparent in the light microscopic image of heart muscle (A) but the boundary of birefringence image intensity loss (B) in the TPM of the same field is definite. Transmission electron microscopy of the native heart muscle (C) reveals the normal semicrystalline array of the fibrillar contractile proteins and the layered membranes of the mitochondria. In the thermally coagulated area of birefringence loss (D), the contractile proteins are dissociated and the mitochondria have been reduced to relatively large dark globules with no internal structure. [A and B. H&E. Org. Mag. 12.5X. C and D. Uranyl acetate and lead citrate stains. Org. Mag. About 15,000X]

Thermal denaturation of the collagen and muscle proteins disrupts their crystalline arrays and tertiary molecular structures leading to a loss of their birefringent properties. [Figure 6 and ref. 24]

Depending on the temperature and time of heating, different zones of partial and complete birefringence loss of collagen can be seen and their boundaries measured. The birefringence changes of the proteins that form the contractile elements of muscle cells occur at slightly lower temperatures. The boundary of collagen birefringence loss is closer to the hot heat source than that of muscle loss. Therefore, if the target tissue contains muscle and collagen and the heating is relatively slow (several seconds to few minutes), four different boundaries of total and partial birefringence loss can be potentially identified and measured.

The development of the thermal damage end point of total birefringence loss observed in stained paraffin sections using the transmission light microscope (TPM) is related exponentially to temperature and linearly to time. [24, 27-30] In this case, the endpoint is precisely defined, total loss of the birefringent image as observed at the magnification levels offered by the TPM in tissues heated at constant temperatures over several minutes. The first-order kinetics of this specific change from native to thermally denatured collagen do not consider the numerous intervening episodes of chemical bond breakages that occur before the desired end point, birefringence intensity loss is obtained. [24]

Transmission electron microscopy (TEM) of totally hyalinized and partialy and totally non-birefringent collagen tissue give some idea of the numerous dissociation processes that are taking place. In areas of total hyalinization and birefringence loss, TEM shows monotonous fields of small granules in stained plastic sections. The granules probably represent totally dissociated collagen molecules that have lost their linear and macromolecular structure. Some of the granules may also represent other denatured extracellular matrix proteins and proteoglycans. In areas of partial birefringence, TEM shows varying degrees of collagen macromolecular dissociation with disruption of the crystalline linear array of the macromolecules. As the dissociation and birefringence loss becomes more prominent, more granules appear mixed with the more loosely formed macromolecules.

There are many types of collagens and not all are birefringent when observed with the polarizing microscope. [10, 11] Some collagen fibers are thin and loosely distributed in tissues thus are difficult to see. Sometimes, certain stains such as Sirius red can enhance their birefringence. However, the thermally induced birefringence changes cannot be distinguished easily with this stain. Other collagens have a netlike macromolecular configuration formed of non-fibrillar collagen molecules therefore are not birefringent.

Vital Enzyme Denaturation and Cellular Membrane Dissociation

The thermal denaturation of the enzymes required for basic life processes is similar to that of the structural proteins but occurs at lower temperatures and heating times than structural protein coagulation. Therefore, these lethal effects occur beyond the zones described above and are found at the periphery of the lesion.

Several of the vital enzymes, if still functional, can reduce certain dyes to colored products thus supposedly demarcate viable from non-viable tissues. [31-33] However, in very acute lesions, some cells may be severly damaged nigh onto death but their enzymes will continue to work for awhile. Therefore, the colored (“viable”) / non-colored(“dead”) boundary may be distinct but not show the ultimate lethal thermal damage boundary. Vital staining can be useful for comparisons of lesion extent or size but cannot be relied upon to indicate the ultimate extent of cell death and effective treatment.

Red Thermal Damage Zone

A grossly apparent red band or zone develops within 30 seconds to 2-3 minutes just peripheral to the central white coagulation zone in thermal lesions produced in living organs with an intact blood supply. This red zone forms as a consequence of thermal damage to blood vessels and a complex series of physiological vascular responses to heating that produce 1) hemostasis (blood flow stasis), 2) hemorrhage and 3) hyperhemia (increase blood flow). The three pathologic changes are intermixed and do not form distinct microscopic zones within the red zone but hemostasis tends to be closer to the white coagulum and the hemorrhage and hyperhemia are more peripheral. [20, 24, 26-30]

The hemostasis is a result of direct thermal coagulation of red blood cells with small blood vessels stopping blood flow. Hemorrhage, the escape of blood from thermally damaged blood vessels, requires flow of incoming blood with a pressure gradient that pushes the blood in the surround tissues. Increased blood flow produced by local release of vasoactive substances that stimulate dilatation of small blood vessel is a classic vascular reaction to increased temperature of time. [20, 28, 29] This response is familiar to anyone who has burned his hand while cooking. The vasoactive response has been shown to be exponentially related to temperature and linearly to time at temperature. 1, 27-30]

If the single heat source energy delivery remains constant, then the red thermal damage zone stabilizes and stops growing because of the equilibrium established between heat generation and convection heat loss due to blood flow. [17] The red thermal damage zone persists for various intervals due differences of mechanisms of resolution, the organ site and the species. [Table II]

Table II

Red Thermal Damage Zone Resolution Intervals

Necrosis:

The full extent of lethal thermal damage cannot be determined until all injured cells that are going to die are dead and undergo necrosis. Depending on the mammalian species and the tissue, the full extent of lethal thermal damage as revealed by necrosis takes from 1 to 5 days to develop. There are four general mechanisms of necrosis in thermal lesions. Thermal coagulative necrosis occurs immediately upon heating. Heat-related lytic necrosis, ischemic necrosis and apoptosis, are delayed responses to lethal thermal injury. [14,20]

Apoptosis:

On the other hand, apoptosis (programmed cell death) is an entirely different mode of cell death that usually occurs within 24 hours of treatment. Heat as well as several other chemical, biological and physical insults can trigger apoptosis, a series of energy-requiring events leading to cellular and nuclear fragmentation and death in specifically genetically programmed cells. Not all cells can undergo apoptosis. Endothelial cells, lymphocytes and some tumor cells can show the characteristic apoptotic degenerative changes at the periphery of thermal lesions around 24 hours after heating. Apoptosis is a phenomenon initiated in living cells therefore apoptosis will not be seen in the central thermal coagulum nor later than 24-36 hours unless there is further insult to still viable cells in the periphery. [Cotran]

The outer boundary of the red thermal damage zone corresponds to the boundary of tissue necrosis in thermal lesions of several organs including rat liver, goat breast, pig lung and human endometrium and myometrium. [Figure 7 and ref. 34] Therefore, the red zone can be used to predict the full extent of treatment effect in some thermal coagulation lesions. However, the necrosis and scarring can extent beyond the thermal lesion when occlusive thrombosis and regional ischemia occur. In this case, the treatment effect will involve not only the thermal lesion but also the tissue downstream from the blockage. The ischemia can result in infarction, or, in the case of the pig lung, collapse and chronic fibrosis of functioning lung tissue.

Figure 7

Thermal Coagulation Lesions in Rat Liver: Comparisions of Diameters of Outer Boundary of Red Zone in the Acute Lesions and Necrosis at Three Days.

The outer boundary (arrows) of the red thermal damage zone in the acute lesion and the boundary (arrows) of necrosis are the same as shown in the graph. The measurements were made in the same surviving animals at the time of treatment (red zone) and at time of maximal necrosis at three days. The liver photos are of rat 94RL-3.

Wound Healing

Wound healing involves a series of steps that can be divided into three general categories: 1) organization of the necrotic debris, 2) tissue regeneration and 3) scar formation and contraction. [20, 35]

Scar Tissue Formation:

Depending on the size of the thermal lesions and the general physiology of the tissue/organ, the rate of vascular and fibroblast proliferation can be greater than the rate of tissue regeneration. This proliferation leads to the formation of vascular and fibrous granulation tissue, the early forms of scar tissue. Granulation tissue originates form the peripheral viable tissues and invades into the necrotic tissues hard on the tail of the organization processes. Depending on the lesion size and vascularity of the host organ, complete replacement of the necrotic tissue can take several weeks to months.

New scar tissue tends to be very vascular and formed of loose collagenous fibers and bands. However, as the scar ages, many blood vessels are resorbed, the collagen fibers become larger and more densely packed and the entire scar shrinks. Scar tissue is usually pale tan reflecting its collagen content and can be readily distinguished from surrounding tissues. However, as the scar ages and contracts, the boundary between the scar and normal tissue can become obscured particularly in normally pale tissues such as breast. [Figure 8] Then, microscopic evaluation sometimes with the use of special stains will be needed to discover the thermal lesion. It is to be remembered that most thermal lesions used to treat symptomatic diseases are too large to be replaced by the original tissue. The scar will always be there no matter how small.

Figure 8

Healing Thermal Interstitial Coagulation Lesions in Goat Breast.

The red thermal damage zone is prominent in the acute lesion. The red zone as been replaced by scar tissue at 13 days however the central darker coagulum remains. At 84 days, the pale residual coagulum (necrotic fat) is outlined by the slightly darker fibrous scar tissue. The bars equal 1 cm.

Choosing the Right Marker and the Right Tissue

Comparisons require choosing markers that are measurable and reliably reproducible in the target tissue. The marker(s) should be relevant to your experimental goals. For example, comparisons of treatment effects require choosing markers that indicate the full extent of lethal thermal damage such as necrosis and wound healing in survival animals. On the other hand, comparisons of different experimental parameters or instruments can be done using a variety of thermal markers of immediate effect such as birefringence loss, collagen hyalinization, red zone formation, etc. that do not require survivial studies. [Table III]

Table III

Useful Thermal Markers for Comparison Studies

*

Vital Dyes: Gross or microscopic oxidation/reduction dyes. Can be used only on fresh gross specimens or on frozen, unfixed tissue sections.

Just as important to a successful outcome is choosing the best tissue to match the marker. Some investigations will be driven by 1) the particular disease process and 2) a specific organ or tissue in which the disease occurs. In those cases, the best thermal marker to use will be the one(s) that appears most reliably in that tissue. In other experiments involving 1) thermal lesion model testing and validation or 2) testing of energy sources or instrument design, the choice of tissue is determined by which marker will give you the best measurements.

Statistically Valid Experiments:

Biological tissues do not respond to heating or any other exogenous injury in a precisely predictable fashion as compared to metals or plastics. This is referred to as “biologic variation” and any investigator who works with living or dead biologic tissues has to compensate for the “variation” in her/his experiments. This is accomplished by 1) establishing strict controls to limit the number of experimental variables, 2) producing multiple lesions using exactly identical experimental conditions, 3) making multiple measurements of specific end points using consistent planes, reliable instruments and comparable locations to avoid measurement artifact.

Biologic and medical research is statistical research. Therefore, one experiment will not reveal truth but the average of multiple (preferably odd numbered) experiments will show important statistical probabilities. Statistically significant probabilities derived from appropriately controlled experiments are appropriate results to satisfy the clinicians, journal and grant reviewers and the Food and Drug Administration (FDA).

Measurement of Ablation Lesions:

Thermal ablation lesions can be made in all biologic tissues as long as the energy parameters fulfill the criteria for tissue ablation. Therefore, cheap, plentiful and clinically relevant target tissues, such as chicken breast and liver, can be used as long as they are not rotten (i.e. stored in the refrigerator for a month!) and their limitations as target tissues are understood.

Ablation energies produce holes, cuts and defects that can be measured grossly in either fresh or fixed specimens or microscopically in well-oriented tissue sections. Linear, volumetric or mass measurements are good estimates of tissue removal and, when done under well-controlled conditions, are very useful for comparisons.

Measurements of ablation depths and diameters can be obtained by using a little forethought and consistent techniques. Routine formalin fixation of biologic tissues and tissue processing techniques can cause swelling or shrinkage depending on the tissue. In addition, in musclular tissues, formalin fixation induces muscle contraction further distorting the lesion and the tissue precluding accurate measurements. Also, removal of fresh tissue from the animal releases normal tissue tensions that can also lead to artifactual collapse. Therefore, it is important to orient and support the tissues during the fixation and measurement processes. Fixation by organ perfusion may be necessary to prevent unwanted contraction and distortion of the holes. A good example is perfusion of the intact heart with saline to remove blood then fixative at systolic pressures to minimize the contraction artifact in transmyocardial recanalization experiments. Tissue slabs or skin pelts can be stabilized for fixation by pinning them down on a cork board before putting them into formalin. [Figure 9]

Figure 9

Pointers on Preparing Tissues for Fixation and Microscopic Section Preparation

Plastic refrigerator containers with air-tight lids make excellent containers for formalin fixative. A layer of melted paraffin can be poured into a clean container to form a base to support the tissue and the pins. The containers with the paraffin will last for years.

The rigid rod can be marked at the surface tissue level (arrow) then measured. This procedure is not useful for small ablation holes which should be subjected to microscopic examination.

Many experiments are ruined because too many lesions are placed too close together on a small target tissue. Allow at least 0.5 cm. between all thermal ablation and coagulation lesions. Frequently, excess tissue is trimmed from the paraffin block containing the tissue before sections are collected. If you cut through the exact center, then the first collected sections will not be representative of the lesion and measurements are not accurate.

Single holes drilled into tissues will have a center of maximum depth. Therefore the lesion surface and bottom centers have to be identified to obtain accurate measurements. This requires careful dissection and orientation of the lesion and, in the case of small lesions, preparation of multiple serial histologic sections to obtain the exact section that represents the center of the lesion. [Figure 9] Larger lesions can be cut through the center freehand but it is difficult to make a perfectly perpendicular cut particularly in relatively fresh soft tissues as skeletal muscle brain or liver. A rigid, blunt-ended rod smaller than the hole diameter can be inserted gently into the hole and used as a guide to cut through the hole and/or marked to show the depth relative to the tissue surface. This gross measurement is best done in the fresh tissue since fixation can produce distortion leading to blockage to the passage of the rod. However, care needs to be taken to not push the probe past the bottom of the lesion: soft tissues such as brain and liver can be easily penetrated. Needless to say, sharp-pointed hypodermic needles are not appropriate measuring rods in any tissue.

Ablation hole diameters can vary because of 1) probe configuration, 2) laser beam profile (Gaussian vs “top hat) 2) mechanism of tissue removal and 3) tissue spreading, contraction and/or collapse during dissection or processing. This variation is particularly prominent at the upper portions of the lesion and is particularly bothersome in histologic sections of skeletal and heart muscle. Therefore, to obtain consistent measurements, multiple diameters should be measured in the straight portion of the hole and averaged for each lesion.

Special care should be taken to orient small lesions to obtain the best paraffin sections for measurement. Small coagulation and ablation lesions should be removed en bloc (whole lesion in center surrounded by tissue) and submitted for serial sectioning through the block. All sections should be examined to locate those few sections (usually two or three that represent the full depth and center of the hole. [Figure 9]

Transverse sections of long cuts frequently have a V profile with splaying of the cut edges above the tissue surface. [Figure 10] This splaying is particularly evident in ablations in muscle tissues. When the edges splay out above the surface of the surrounding tissues, various approaches for measurements can be made. In general, surface cuts, particularly those made free-hand, will have variable depths therefore, multiple depth measurements should be made along the cut by looking at least 3 different points along the cut. The measurements will be best done in transverse sections of either fresh or fixed tissues observed under a dissection microscope (Mag. 2X-40X) or in paraffin sections representative of the entire lesion using a calibrated image analysis system or ocular micrometers. Either method requires using the same magnification for all measurements and measuring at the same locations for consistency.

Figure 10

The depths should be measured from a perpendicular dropped from a line defining the desired surface reference point. Whenever possible, the recommended reference point would be the tissue surface just beyond the splayed edges. Multiple measurements (at least 3/histologic specimen) can be averaged with standard deviations calculated for comparison purposes.

Structural Protein Thermal Coagulation: