Pathological Effects of Hyperthermia in Normal Tissues1

[CANCER RESEARCH (SUPPL.) 44, 4826s-4835s, October 1984] Pathological Effects of Hyperthermia in Normal Tissues1

Luis Felipe Fajardo L-G

Department ol Pathology, Stanford University School of Medicine, Stanford, California 94305, and Veterans Administration Medical Center, Palo Alto, California 94304

Abstract alterations, serum enzyme abnormalities, and symptoms of var iable severity have been described (7, 44) [see also article by L. This is a brief review of the major pathological alterations H. Cronau in this issue (11)]. It appears that, by the current produced by hyperthermia in normal tissues of humans and other methods used for TBH, fatalities directly attributed to the hyper mammals. Whole-body hyperthermia, spontaneous or artificially thermia are rare; autospy descriptions are rather difficult to find. induced, can produce severe lesions that have been best de What we know about the pathology of systemic hyperthermia scribed in humans: necropsies, of fatal cases of heatstroke or of comes mostly from observations made in fatal cases of heat individuals treated in the 1940s by hyperpyrexia, have demon stroke (30, 36), or from deaths in patients treated by "hyperpy strated important lesions in the central nervous system, liver, rexia" for conditions other than cancer (23) (see Table 1). These kidney, heart, adrenal, testis, and bone marrow. All cases have 2 situations are somewhat different from current TBH (because shown hemorrhagic diathesis affecting many tissues, and in of the methodology, intensive care, and careful monitoring in some the hemorrhages may have directly contributed to death. TBH), but the pathology of the fatal cases is probably comparable The information on the pathology of localized hyperthermia and, at the present, is the best material available. One of the comes mainly from experimental studies in mammals. Pathology most informative morphological studies was made during the descriptions are available mainly for skin, mesenchymal tissues 1940s in patients subjected to "systemic hyperpyrexia." In their (skeletal muscle and adipose tissue), liver, small intestine, brain, publication, Gore and Isaacson (23) described meticulously the kidney, urinary bladder, prostate, and cartilage. In several of findings in 17 necropsies of individuals who died of TBH induced these tissues, however, the morphological data are incomplete, (by the Kettering cabinet, by i.v. injection of typhoid vaccine, by and very few have sequential observations. Thus, the ultimate hot baths, etc.) in an attempt to treat gonorrhea, nonsuppurative (delayed) result of the acute lesions of focal hyperthermia is arthritis, etc. The temperatures (p.o. or rectal) reached were unknown for most tissues. 40.5-43Â°, and the duration of hyperthermia was 3 to 11 hr (23). Clearly, more information is needed in order to define the Unfortunately, the total number (the denominator) of patients range of safety for clinical hyperthermia. treated by such a method was not stated, and probably was not available to the authors. Thus, although stated as low (23), one Introduction cannot decide from their paper the incidence of complications or fatalities of that unusual treatment. Unquestionably, hyperthermia has an important role in cancer One other thorough and extensive clinicopathological study therapy today. Also, unquestionably, hyperthermia can affect the was made by Malamud et al. (36) on fatal cases of heatstroke.3 normal tissues adjacent to the treated tumor or, in the case of These pathologists described the findings in 125 necropsies of TBH,2 many normal tissues. Therefore, to apply this modality United States Army personnel who died between 1941 and 1944. judiciously one must know, and weigh, these possible deleterious In their series, the p.o. or rectal temperatures registered at effects. admission to hospitals were 38-44Â°; in 107 of these patients the In this article I describe the pathological effects of both whole- temperature range was 41.5-44Â° (36). In 70% of these cases body (systemic), and localized hyperthermia on tissues of several death occurred in less than 24 hr, and in the rest between 1 and mammals, including humans. The scope of this publication does not allow an extensive review of "thermal pathology." Therefore, 12 days from initiation of illness (36). The following is a description of the alterations observed in I limit it to outlines of the morphological findings in selected the most affected organs or tissues (in order of severity), follow tissues. The descriptions of organs and tissues are organized ing spontaneous (30, 36) or induced hyperthermia. not along conventional organ systems but according to the quantity of pathology data available (Tables 1 and 2). Liver

Whole-Body Hyperthermia The most severe and consistent injuries observed in the Gore This was the initial form of therapeutic hyperthermia. It was and Isaacson (23) series occurred in the liver: initially there was based on reports of spontaneous cures of tumors following high vacuolization of hepatocytes (8 to 16 hr after the end of therapy); (erysipelas-associated) fever, in the mid-19th century. Clinical then progressive necrosis of cells in the center of the lobules occurred, reaching a maximum ("60% of the central part of the TBH is used nowadays only in carefully controlled trials at a few lobule") by 60 hr. At 7 days necrotic debris was removed by institutions (8, 43). In such clinical trials, fluid loss, hemodynamic macrophages. Regeneration from peripheral hepatocytes, and 1 Presented at the Workshop Conference on Hyperthermia in Cancer Treatment, proliferation of bile ducts was active in those who died late; March 19 to 21, 1984, Tucson, AZ. The experiments described here have been supported by Veterans Administration Research Funds (MRIS 2735-01), and 3 Heatstroke must be distinguished from malignant hyperthermia, a genetically USPHS Grants CA 04542, CA 19386, and CA 34686 of the National Cancer determined myopathy with fever. The latter is often accompanied by metabolic Institute, NIH. acidosis, hyperkalemia, muscle rigidity, etc. This condition may be precipitated by 2 The abbreviations used are: TBH, total-body hyperthermia; TER, thermal anesthesia and, if unrecognized, is often fatal. The pathology of malignant hyper enhancement ratio; LDay,, 50% lethal dose at 7 days. thermia is not within the scope of this article.

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Table 1 Heart Whole-body hyperthermia Tissues most affected in fatal cases of heatstroke or therapeutic hyperpyrexia8 The most conspicuous lesions in spontaneous (36) or induced Liver Adrenal (23) hyperthermia have been hemorrhages: subepicardial, intra Central nervous system Testis muscular, subendocardial, or even intravalvular. Some hemor Kidney Bone marrow Heart rhages are massive (36), enough to produce ventricular failure. * Hemorrhages in many tissues and cavities. Various degrees of individual myocytolysis and myocyte necrosis have been described, from mild to extensive, but generally focal (23,36). Fragmentation and fatty degeneration of myocytes have fibrosis, however, was not seen in the longest (14 days) survivor. been observed (36). Jaundice (interpreted as a sign of liver failure) was observed in all patients surviving more than 49 hr (23). Similar but less striking liver alterations were described by Adrenal Malamud ef al. (36). Additional information comes from Kew ef Both heatstroke and induced hyperthermia produce adrenal al. (30), who studied 18 liver biopsies and 8 necropsies from 39 lesions, limited to the cortex. Pericortical hemorrhage is common Bantu miners afflicted by heatstroke. The biopsies showed usu (36), but intraparenchymal hemorrhage is rare. Separation of ally mild lesions; 6 of the necropsies revealed structural altera cortical cells (artifactual?) from their basement membrane and tions similar to those described above. Portal inflammatory infil from each other results in a tubular-like arrangement (23, 36). trate and fatty changes were also noted (30). Focal necrosis is variable (23, 36). Gore and Isaacson (23) Pettigrew ef a/. (44) have described alterations of serum liver describe an early coalescence of lipid droplets in the zona function tests in patients subjected to TBH by the paraffin bath method: patients who reached temperatures of 41.8-42.2Â° fasciculata which results in large irregular vacuoles as early as 3 hr after heat induction. showed elevation of lactic dehydrogenase (2-fold), aspartate Interestingly enough, no alterations of other endocrine organs aminotransferase (25-fold), alanine aminotransferase (8-fold), are described in whole-body hyperthermia. and bilirubin (modest, to 1.56 mg/dl) (44). Higher levels of bilirubin have been reported by Wills ef al. (54), up to 18.5 mg/dl 5 days after treatment of 42Â°for 8 hr. In vitro studies have indicated 7esf/s that increase in ammonia production by liver tissue (accumulation in the medium) is a sensitive indicator of hyperthermic injury and As expected, the seminiferous tubules are affected, although becomes detectable at 42Â°(9). only in patients dying after 8 hr of induction of hyperthermia (23). The lesions consist of severe decrease in spermatogenesis, with formation of multinucleated cells in the germinal layers, Central Nervous System which desquamate into the lumen. Later there is loss of germinal cells. Interstitial cells are not affected and there is no inflamma The entire brain was available for study in one-third of the tory exÃºdate (23). These changes are similar to those seen after cases of Malamud ef al. (36). It showed strikingly severe lesions: radiation (17), or in starvation, or in deficiencies of vitamins A or congestion, edema (with increase in weight by several hundred E (23). g), extensive neuronal loss, and gliosis. The authors considered the neuronal damage to be a direct heat effect and the congestion and edema to be secondary to shock. Multiple hemorrhages, in Bone Marrow the cerebral parenchyma and meninges, were interpreted as Malamud ef al. (36) studied 15 samples from their fatal cases secondary to thrombocytopenia (36). of heatstroke. Depletion of granulocytes and erythrocytic pre Almost identical lesions were noted in the Gore and Isaacson cursors was present in patients surviving 35 hr or longer, but description of 16 brains (23). They observed the most severe only in one was there significant hypocellularity. The most con alterations in the cerebellum, often with complete loss or damage sistent changes occurred in megakaryocytes: karyopyknosis, of Purkinje cells (23). karyorrhexis, loss of nuclei, and reduction in number of mega karyocytes (in one-half of the cases). In 3 cases there was Kidney evidence of regeneration, with many megakaryoblasts (36). These megakaryocyte alterations do not appear to be artifac Renal lesions of variable severity were seen in 9 of 17 patients tual and were not seen in appropriate controls, including cases who died following therapeutic hyperpyrexia (23). Three of these of anoxia (36). Furthermore, thrombocytopenia was present in patients, who survived 4, 7, and 14 days, died of renal failure. the majority of the patients having platelet counts (36), and it is The lesions observed were progressive necrosis of tubular cells, known to occur in induced hyperthermia (23). edema and lymphocytic, rather than granulocytic, infiltrate (23). Thrombocytopenia, therefore, could be explained by what may Following heatstroke, renal lesions are also frequent and im be a selective effect of hyperthermia on megakaryocytes. This, portant. These consist of edema and congestion, and tubular however, does not explain the severe and widespread hemor necrosis (36). Of the 125 fatal cases reviewed in 1946, 19 had rhages seen on multiple sites in fatal cases; the recorded platelet enough tubular damage to be classified as "lower nephron counts were not below 22,000/ml and most were above 40.000/ nephrosis," the old term used to describe acute tubular necrosis. ml (36). Such levels ordinarily do not lead to hemorrhagic di The incidence of tubular necrosis increased with time of survival athesis, unless other factors contribute to it. Such factors may in these series (36). include deficiencies in soluble coagulation factors, especially if

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there is severe liver injury. An important mechanism of hemor Table 2 Effects of therapeutic localized hyperthermia' rhage may be injury to capillary endothelial cells by heat. Evi dence for thermal sensitivity of endothelial cells has been found Skin nervous system recently in our laboratory.4 Adipose tissue Kidney Skeletal muscle Urinary bladder Liver Prostate Small intestine Growing cartilage Other Tissues Embryonal tissueCentral * List of tissues for which information is most available The striking alteration common to most organs and tissues, including serosal membranes, skin and mucosae, both in spon taneous (36) and induced (23) hyperthermia, is hemorrhage. The the effects of burns, and therefore included a range of tempera tures (44-52Â°) and exposure times (e.g., 6 hr) not used in importance of this finding has been discussed for the individual organs, and its possible mechanisms have been mentioned under therapeutic hyperthermia. They devised a grading system (hy- "Bone Marrow." peremia, focal necrosis, and complete epidermal necrosis) based on gross changes (41). They found similarities in the responses of human and pig skin: complete epidermal necrosis occurred in Localized Hyperthermia both at 45Â°for 180 min, but times were different for higher or lower temperatures (e.g., 30 versus 45 min, respectively, at 47Â°) On the basis of human and experimental observations, one would expect that local hyperthermia at 40-50Â°/30 min would (24,41). cause major injury in skin (42, 52), mesenchymal tissues (40), One of the few studies including systematic histological ex liver (23),5 intestine (28), testis (23), and tissues of the embryo. amination of the skin was performed by Thomson ef al. in swine In addition, important lesions may occur in tissues (organs) such (48). These authors compared the lesions produced by electricity as kidney (36), endocrine glands (especially adrenal) (23, 36), (59 to 100,000 Hz) with those produced by heat, in sequential lymphopoietic organs, and various structures of the eye. The biopsies. They noted detachment of epidermis after heat, but central nervous system and the bone marrow, which are affected not after electrical injury, with fibrillary or granular cytoplasm in in systemic hyperpyrexia (see above), are less likely to suffer the necrotic epidermocytes. Thomsen ef a/. (48) were successful from local hyperthermia because of the poor heat conductivity in distinguishing thermal burns from electric lesions (their aim of bone, unless the hyperthermia is designed to treat precisely was to diagnose the effects of electrical torture), but unfortu such organs (see "Central Nervous System" below). The heart, nately their findings are difficult to apply to the different levels of major blood vessels, and lung would be least affected by local heat used in clinical hyperthermia. hyperthermia because of very effective heat dissipation by con The skin of mice appears to be more thermosensitive than human or porcine skin; in the latter, 45Â°for 1 hr does not seem vection. Clearly, there is a need to study systematically the effects of to cause permanent damage (24, 41 ). The mouse ear has been therapeutic hyperthermia in many of the tissues mentioned above used to quantitate thermal skin response (19, 31). From various experiments it appears that injury occurs after treatment at 44Â° (and in others not mentioned), as well as in normal cell lines. Radiobiologists have already begun this task. The thermal sen for 1 hr, or higher. Complete epidermal necrosis is seen in all animals with doses of 45.5Â°for 45 min, and this occurs in 4 days sitivity and (conversely), the induced thermotolerance of several normal cell lines is already known (21). (19, 24). These lesions appear earlier than those produced by X- The scope of this publication does not allow an extensive radiation (e.g., 3000 rads) (19). Recovery from this thermal review of "thermal pathology." Therefore, I limit it to brief descrip necrosis is slow or may not occur. Below 44Â°for 1 hr, lesions tions of the morphological findings in selected tissues (see Table are mild (mostly hyperemia), and recovery is usually complete within 2 weeks. These time-dose data generally agree with the 2). Some descriptions are based on studies performed in our institution (38, 40).4~6 Many have been obtained from data of observations of Okumura and Reinhold (42). Anderson ef a/. (1) other investigators. Notice that most morphological observations have shown that the mouse ear lesions can be potentiated by of the effects of localized hyperthermia have been made in exposure of the animals to ethanol. mammalian species other than humans. In some instances, only A popular method for the study of hyperthermic injury has been the immersion of a mouse foot in a water bath (52). The gross pathology is available, and sequential observations are subsequent "foot reaction" has been quantitated by a scoring lacking for most tissues. system of gross changes that include edema, epilation, wet desquamation, and may be as severe as complete loss of the Skin foot (52). This foot reaction includes, of course, damage to Although the skin has been perhaps the normal tissue most several tissues aside from skin, and hopefully it has been inter preted as such (49-51). often studied in therapeutic hyperthermia (there are many more publications than those listed here), relatively few clinical or The mouse foot reaction has been quite useful in several experimental protocols have included detailed morphological de respects. Urano ef a/. (51) have used it to demonstrate enhance scriptions. ment of the hyperthermia effect: Corynebacterium parvum given The careful sequential observations by Moritz and Henriquez to the animals several days before hyperthermia enhanced the (41) in 1947, in porcine and human skin, were aimed at studying effect of heat in tumors and, to a lesser degree, in the normal tissues. Glucose (i.p.) also enhanced the tumor response and ' L. f. Fajardo, A. Schreiber, N. Kelly and G. M. Hahn, unpublished observations. produced a lesser enhancement in the foot (50). The phenome 5 S. D. Frionas, M. A. Taylor, L. F. Fajardo, N. I. Kelly, T. S. Nelson, and G. M. non of "step-down heating" (sensitization to low-temperature Hahn. Thermal sensitivity of normal canine liver, submitted for publication. â€¢L.F.Fajardo, S. Frionas, and N. I. Kelly, unpublished observations, 1983. heat by a prior shock of high temperature) was shown to occur

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Downloaded from cancerres.aacrjournals.org on September 27, 2021. © 1984 American Association for Cancer Research. Pathological Effects of Hyperthermia in the mouse foot as well as in a transplanted tumor (49). There in the deep muscle layers; and fibrosis. The latter lesion was by was no therapeutic advantage in this model (49). far the most important in severity (extent) and affected especially The enhancement effect (TER) of hyperthermia on the effect the adipose tissue; thick bands of collagen and fibroblasts re of ionizing radiation has been studied in skin, as well as in other placed and deformed many of the normal adipocyte lobules (40). tissues. One of such studies (with ultrasound-induced hyper There was a clear dose response in these delayed lesions (38, thermia and X-radiation), performed by Baker ef al. (2), has 40). Thermotolerance was demonstrated in both adipose tissue shown that the TER was independent of the sequence of the 2 and muscle, and it was most remarkable in the former (38); in treatments for intervals up to 1 hr in rodent skin. the sites that received the initial conditioning dose, the scores Recently, a modified Fowler scale has been used to quantitate were reduced by 76 to 86% at 45-48Â°. This induction of ther- the gross skin reaction in 80 patients treated by radiation (3395 motolerance in vivo provided an overall advantage of 2Â°for the rads, mean total dose) and microwave hyperthermia (mean value, protection of these normal tissues (38). 42.3Â°)(33); 17% of patients developed only desquamation; ery Although fibrosis and possibly deep abscesses are potentially thema occurred in 24%; blisters occurred in 7%; and ulcÃ©ration serious, chronic complications of therapeutic hyperthermia, these occurred in 18% (33). lesions were usually significant at 46Â°and above. Therefore, by Perhaps the thermal sensitivity of skin is only of minor impor extrapolation, serious fibrosis and abscesses should not be tance in clinical hyperthermia, especially when using electromag expected in treatment protocols using doses of 45Â°for 30 min netic sources; the skin can be effectively cooled during therapy, or less. Furthermore, the demonstration of thermotolerance in sparing it from any thermal damage (38, 40). vivo provides a possible therapeutic advantage.

Soft Tissues Liver Localized radio frequency-induced hyperthermia for superficial From the data of TBH (see above), the liver appears to be at or deep-seated tumors often requires deposition of energy in risk for significant injury from localized hyperthermia. Indeed, skin or in mesenchymal tissues. Damage to skin can be mini mized by cooling of the electrodes (38). The mesenchymal (soft) tissues, such as skeletal muscle and adipose tissue, cannot be effectively cooled by external means. The adipose tissue has a relatively high electrical resistivity which causes greater power absorption from radio frequency sources. The muscle has greater electrical conductivity and will absorb energy from micro wave heating sources. Because of its low blood perfusion rate, adipose tissue removes heat poorly. Additionally, the fat-muscle interface causes wave reflections leading to high-energy absorp tion in the adipose tissue at some frequency ranges. All of the above, and other considerations (38), indicate that damage to adipose tissue and skeletal muscle may be a limiting factor in hyperthermia. A systematic study of thermal injury has been performed in the superficial skeletal muscle and adipose tissue of swine (38, 40). Hyperthermia was induced by radio frequency currents passed through electrodes placed symmetrically on the skin of opposite sites of the flanks of pigs. Water circulating through the hollow electrodes cooled the skin. Fifteen sites were sham treated (controls), while in 102 sites the temperature was in creased, during 30 min, to levels of 40-48Â° (40). Some sites were subjected to 43Â°for 30 min, 4 hr before the final, prese lected treatment, testing for thermotolerance (40). Large samples of the adipose and muscle layers were obtained 18 to 24 hr later in some sites, and 28 to 31 days later in other sites (40). The lesions were examined histologically, using a specially developed scoring system (40). The early (acute) samples revealed edema, focal hemorrhage, necrosis, and granulocytic infiltrate. The latter 2 (particularly necrosis) were more severe in muscle. In general (but not in all samples), the severity of these acute lesions increased with dose, but it never reached the levels of the delayed lesions (40). Thermotolerance was not observed in these eaiiy samples (38). The delayed samples revealed important lesions: chronic lym- Fig. 1. Normal canine liver architecture, shown here for comparison with the phohistiocytic infiltrate in both fat and muscle; evidence of fat subsequent illustrations. A lobule occupies most of the central portion of the photograph and is limited by 3 portal areas (arrows). Liver cell plates converge necrosis, with lipophages; persistent muscle necrosis and foci of radially toward the delicate central vein (clear oval in center). Human liver lobules muscle regeneration; abscesses (only at highest temperatures) have a similar structure. Gomori's trichrome, x 80.

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lesion, or one that will affect liver function. Thermotolerance was not induced in this study,5 but this does not rule out its very likely occurrence. The ultrastructural studies in our experiments (at 28 days) did not add to the information already obtained from the light mi croscopy. The earlier stages of these lesions, however, might be better observed by electron microscopy. Wills ef al. (54) have studied by electron microscopy biopsies of human liver obtained before, and within 2 days after 40-42Â° for 65 to 495 min. Evidence of ultrastructural damage in some hepatocytes was observed immediately after therapy (when no histolÃ³gica! lesions were evident), and became progressively worse. By 2 days, there were numerous autophagic vacuoles, dilatation of Golgi cisternae, massive distension of rough endoplasmic reticulum, flocculent deposits in mitochondria, dilatation of bile canaliculi with loss of villi, and increase in bile granules. Most prominent were large vacuoles (as much as 12 Â¿Â¿mindiameter) (54). Presumably, these alterations result in necrosis of the damaged cells and extrusion from their plates in less than 4 weeks. In mice treated for tumors located in the flank by radio fre quency (44Â°for 30 min), we have observed focal necrosis of liver, kidney, and intestine adjacent to the treated area (37). Additionally, we have observed foci of hepatocyte necrosis in mice subjected to TBH for 1 hr (rectal temperature of 41 -41.5Â°) within 3 hr of termination of treatment.6 Long-term studies are needed to determine if these alterations will result in permanent or even progressive, physiologically important lesions, such as postnecrotic cirrhosis, with portal hypertension. Hopefully, fibrosis will be limited to the portion of the liver parenchyma within the area of energy deposition.

Flg. 2. Distortion of the lobular architecture 28 days after a local (conditioning) Alimentary Tract dose of 43.3Â°for 30 min, followed 4 hr later by a challenging dose of 47.5Â°for 30 min. The following figures also refer to specimens obtained 28 days after consec utive conditioning and challenging doses. Notice irregular bands of dark collagen Potentially all segments of the digestive tube could be dam in the lower half of the photograph, with loss of the lobular outline. Probably one aged by therapeutic heat. Morphological studies, however, have or 2 lobes have collapsed in this area. Two slightly thickened central veins appear near the right border (arrows). Gomori's trichrome, x 80. been almost exclusively limited to the small intestine. Indeed, the enteric mucosa appears to be rather thermosensitive (26, 28, 29). Field ef al. (20) and Hume ef al. (27-29) have shown a dose-dependent loss of epithelial cells in crypts and lesions are consistently found, but their ultimate clinical effect villi, following hyperthermia (by immersion) of exteriorized loops may not be as important as expected. of jejunum. Following doses of 42.3-44.5Â° for 30 min, the lesions A systematic study of liver lesions produced by localized are apparently characterized by necrosis of epithelial cells of hyperthermia has been completed recently.5 Using radio fre both villi (first) and crypts (later), with cessation of mitotic activity quency currents, individual liver lobes in dogs were subjected to (28). These authors observed an all-or-none effect; either the temperatures of 43-47.5Â° for 30 min. Several sites were heated crypts were well preserved, with dividing cells, or the crypts first with a conditioning dose of 41-43Â° for 30 min, 4 hr before were totally necrotic (28). Edema was also noted, immediately a final predetermined dose, in order to test for thermotolerance. after cessation of hyperthermia. Crypt loss was always more Temperatures were measured with thermocouple sensors. severe, and sometimes occurred only, at the wall of the intestine Twenty-eight days later, large samples were obtained for light opposite to the mesentery. These authors have used the "crypt survival assay" devised (for radiation effect) by Withers and and electron microscopic examination of the treated and control lobes. Elkind (55) to quantitate hyperthermic injury in the small intestine Among multiple histologically graded parameters of injury, the (28). ones that correlated best with dose were hepatocyte loss, focal More recently, experiments using incorporation of [3H]thymi- fibrosis, and distortion of lobular architecture (Figs. 1 to 4). dine indicate that the nonproliferative epithelial enteric cells, Striking, large areas of necrosis were observed, but their inci closer to the lumen, are more sensitive to thermal injury than are dence and severity did not correlate with dose (Figs. 5 and 6). In the crypt cells (29). this canine study, the dose required to produce 50% of maximal The effects of thermal injury in the intestine have been com tissue damage was 43.6 Â± 1.0Â°for 30 min (S.D.). "Maximal pared with those of ionizing radiation (10, 28, 45). One study tissue damage" in this context, however, refers only to the most used transmission and scanning electron microscopy to show severe damage observed in this experiment, at a given site by that while radiation (X-rays; 1000 rads) injures mainly the prolif- 28 days, and does not imply necessarily a total, or a permanent erative pool of crypt cells, an already well known fact, heating

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Downloaded from cancerres.aacrjournals.org on September 27, 2021. © 1984 American Association for Cancer Research. Pathological Effects of Hyperthermia (43Â°for 20 min) affects mainly the villi (10). Both agents produced 42.5Â°(4). Such lesions were sharply demarcated and consisted conical villi (10). of edema of the white matter, pyknosis and loss of neurons As could be suspected, hyperthermia enhances the effect of (especially at 44-45Â°), and destruction of myelin. Coagulative radiation on the small intestinal mucosa (20). This has been well necrosis was observed at 47Â°(4). demonstrated in the mouse by a study of Merino ef al. (39). The Single or multiple intracerebral microwave antennas were used maximum TER was quite significant, 4.7 for a dose of 44Â°for to generate temperatures of 40-47Â° during 50 to 75 min in 3 30 min prior to radiation (total body single dose of 400 to 2000 groups of dogs: normal adults, normal growing animals (5 to 6 rads) (39). weeks old) and dogs with SR-Rous sarcoma virus brain tumors Thermotolerance has been demonstrated in the mouse je (34). The animals were terminated at the end of the thermal junum, both by crypt-survival assay (27), and by using death exposure. As in the cats, the acute lesions consisted of edema (LDso/7) as the end point (26). Heating of exteriorized enteric of white matter, pyknosis of neurons, and damage of white loops is often fatal to mice because septicemia develops, pre matter tracts. Neuronolysis was seen above 43Â°.Necrosis and sumably due to penetration of bacteria through the denuded hemorrhage of tumor were noted (no control tumors were de mucosa. The LDso/7is around 6 min of heating at 45Â°(26). This scribed) (34). It was concluded that the threshold of irreversible LDso/7is independent of the length of the intestinal loop, prior damage occurred at 42.2Â°for 50 to 60 min. feeding of animals, and fluid replacement postheating (26). Gen- Earlier observations of Harris ef al. (25) in regionally perfused tamicin administration increases the LDso/?to more than 9 min/ dogs also indicated absence of lesions below 42Â°and significant 45Â°(26). lesions above 44Â°,edema, focal hemorrhage, and infarcÃ¬. The membrane labilizer retinol did not enhance gross damage A physiological study in cats subjected to TBH (5) (via cardi- to mouse small intestine, although it did enhance lysosomal opulmonary bypass) confirmed the above morphological obser membrane permeability in the spleen, as measured by histo- vations: the critical temperature for evoked potential responses chemistry (45). These findings were interpreted by Rogers ef al. was calculated, by Arrhenius equations, to be 42.2Â°per 1 hr (5). (45) as suggesting that lysosomal membrane injury is not a Physiological observations in humans during TBH (electroen primary event in hyperthermal cell killing. Our morphological cephalogram and somatosensory-evoked responses) suggest study of a thermosensitive mouse neoplasm supports the same functional impairment at 41.8-42Â°, which is reversible (13). There view (18). is concern about the combined effects of ionizing radiation and Since microwave heating is a more likely method than water hyperthermia on the spinal cord (22). Sudden myelopathy oc bath for abdominal neoplasms in clinical hyperthermia, one curred in 3 patients treated by spinal irradiation and TBH (12). should also be concerned with the possible effects of micro Studies in mice suggest enhancement of the functional deficit waves on intestinal motility. A study of the effects of low-level when the 2 modalities are applied to the spinal cord within a microwave irradiation (5 and 7.5 milliwatts/sq cm at 2.45 GHz) short time interval (22). was made in the intestine of rats (47). Acceleration of slow Hopefully, brain neoplasms may respond to hyperthermia waves and inhibition of action potentials were shown in this doses below the apparent threshold of 42Â°for 60 min. model, using chronically implanted electrodes (47). Most experiments have ignored the possibility of delayed injury Kidney in the intestine. In one study, the rectum of rabbits was examined histologically up to 3 months following 43Â°for 30 min delivered Because of its high blood flow rate the renal parenchyma has by a coaxial probe (56). In these experiments the authors found not been considered by some investigators as a likely site of no injury of the rectum in most of the animals (56). hyperthermic injury. Some studies, however, suggest that this is Hahn (24) has suggested that intestinal heat lesions may be not the case. more severe when there are lumenal masses of undigested A well-described histological study in mice was published by material that may overheat and do not cool effectively. If so, Elkon ef al. (15). After unilateral nephrectomy, the contralateral colonie lesions would be worse in the presence of fecal masses, kidney was shielded from adjacent organs and heated intraoper- and these could be ameliorated by fasting (24). atively by ultrasound, reaching temperatures of 40.5, 42.5, and We have not seen morphological descriptions of intestinal 44.5Â°for 35 min, as measured by 4 thermocouples inserted in lesions following therapeutic localized hyperthermia in humans. the kidney. The kidneys were systematically examined by a Considering the increasing clinical use of this modality, and the pathologist 1, 7, or 28 days later (15). obvious sensitivity of the small intestine, we would not be sur Sharply demarcated, subcapsular areas of necrosis occurred, prised to find such lesions. involving only the tubules in minimal lesions, or tubules, glomeruli, and even vessels in large lesions. The extent of the necrosis did Central Nervous System not increase in time (i.e., it had reached maximum by 24 hr), but segmented neutrophils were maximal at 7 days and had disap The possibility of treating cerebral neoplasms by local hyper peared by 28 days. Calcium deposits increased with time. Even thermia (either microwave or ultrasound induced) has great at 28 days, there was no collapse of the framework of the potential (4, 6, 34, 46). In preparation for clinical trials, studies of tubules, although the epithelial cells had disappeared within the the thermal response of various normal brain structures have first day, and fibrosis was only minimal and peripheral (15). been made by several investigators (3,25), particularly the group In kidneys heated to 40.5Â°,lesions did not reach 5% of a given of Britt ef al. (4, 6, 34). section of the renal parenchyma; of those kidneys receiving the Ultrasound, applied to the exposed durai surface of cats during higher doses (42.5Â°and 44.5Â°), one-third had lesions >5% of 50 min, caused acute lesions detectable at temperatures above the parenchyma. Based on these observations, the authors

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conclude that the mean renal temperature at which necrosis the rectum (focal karyopyknosis, edema, etc.) were observed in developed was 43Â°for 20 min (15). a few animals (56). The authors found no prostatic lesions after In a different study, exteriorized rabbit kidneys were treated one or two 43Â°/30-min exposures (56). by brief (15 sec) exposures to ultrasound beam of total acoustic The above report contrasts sharply with (but does not neces power of 30 watts (approximately 900 watts/sq cm at center) sarily contradict) that of Magin ef al. (35). These authors treated (14). The kidneys of 45 rabbits so treated were examined 10 min intrasurgically the prostate of 8 dogs with microwaves (60Â°for to 1 year later. The progression of the sharply defined histological 15 to 22 min, controlled by thermistor) after shielding the rectum lesions ranged from acute injury at 10 min (lysis of erythrocytes, and some of the adjacent muscles. At the end of this severe early necrosis of tubular cells, karyolysis, edema, etc.) to estab therapy, the prostate was edematous and dark; 1 week later (4 lished, progressive necrosis between 1 and 10 days: tubulor- dogs), although its volume was not decreased, there was coag rhexis, glomerular necrosis, areas of coagulation necrosis, neu- ulation necrosis of the entire organ with some foci of neutrophilic trophilic infiltrate, and later calcification. Between 1 week and 1 infiltrate and/or liquefaction necrosis. Six months later (4 dogs) year, there was removal of cell debris by macrophages and there was no recognizable prostate; the organ had been replaced progressive fibrosis which eventually resulted in a scar. Appar by a fibrous scar. Minor lesions were seen in the bladder and ently some tubular regeneration occurred (14). Unfortunately, no the vasa deferentia, but not in the (shielded) rectum (35). temperature measurements were obtained in the kidneys, al These separate observations may, or may not, be pertinent to though the authors estimate that these exposures could have the human prostate. If applicable, they would indicate a threshold resulted in temperatures of 60-70Â° (14). Such exposures are of thermal injury in the normal prostate, somewhere between difficult to compare with those used in clinical hyperthermia. The 43Â°for 30 min and 60Â°for 15 min. Hopefully, carcinomas will histological observations, however, serve to corroborate the respond at lower doses. findings of sharply defined lesions observed later by Elkon ef al. As indicated above, the systemic temperatures reached by (15). Furthermore, the long sequential study indicates that ulti these patients were not always documented, and obviously the mately fibrous scars should be expected from early lesions, quite renal temperatures were unknown (1). similar to those occurring at lower temperatures (15).

Skeletal System Lower Urinary Tract

Damage to these structures could occur in the course of It is rather difficult to find studies of the osseous pathology therapy, either to adjacent organs or to the urinary tract itself. produced by therapeutic hyperthermia. There seems to be more In rabbits, heating of the rectum at 43Â°for 30 min by means interest in the effects of high-speed drills and other surgical of a coaxial probe did not produce gross or histological damage instruments. of the bladder examined sequentially at 16 time points, 1 day to An interesting device, designed for vital microscopic observa tions during local bone heating, is the "thermal chamber" (16). 3 months following exposure (56). Hyperthermia of the bladder wall can be achieved by continu This titanium chamber is implanted for an indefinite time in a ous irrigation with warm fluid. Using water, Linke ef al. (32) bone, allowing multiple observations of various in vivo responses, treated 20 dogs with exposures at 35-69Â° (measured by therm especially vascular, to a thermal insult. Using this device in the istors) during 8 min. The urine was diverted and the animals tibia of rabbits, Eriksson ef al. (16) studied the effects of 53Â°for were killed 6 weeks later. Neither gross nor microscopic altera 1 min; blood flow became sluggish and stopped 2 days later. tions were noted between 35 and 44.5Â°. Patchy destruction of The original vessels gradually disappeared and were replaced by the mucosa, and focal necrosis of the muscularis were seen new ones. Adipocytes decreased in number, and bone remod between 46.5 and 61.5Â°. The muscularis lesions were often eling started 3 to 5 weeks postinjury (16). Although these data severe, of full thickness, and did not necessarily coincide with are not too pertinent to clinical hyperthermia, the thermal cham the mucosa! lesions. ber is potentially a very useful tool for the study of the effects of Complete destruction of the bladder wall occurred between therapeutic heat, ionizing radiation, etc. 59 and 69Â°;by 6 weeks the bladder was replaced by a fibrous The effect of hyperthermia on cartilage has been measured in scar. Occasional thrombosis of small vessels was noted but no terms of its ability to inhibit the growth of the tail in baby rats damage was observed in the adjacent organs (32). and mice (24). The tails are partially immersed in heated water Thus, it appears that, at least in dogs and rabbits, the urinary at 7 days of age. Several months later, the heated caudal bladder can tolerate temperatures of up to 44.5Â°for 8 min (and vertebrae are compared radiographically with the unheated ones, perhaps longer) without significant damage. and a quantitative index of stunting is obtained (24). In addition, necrosis occurs within a few days after exposures in the higher Prostate range. From these observations, Hahn ef al. (24) and Field ef al. (19) In 2 interesting experiments (35, 56), the thermal sensitivity of have shown a dose-related stunting effect; 43Â°for 1 hr resulted the normal prostate has been assayed histologically; by means in 10% growth inhibition, but did not cause necrosis; 44Â°for 1 of a coaxial probe located in the rectum, microwave hyperthermia hr caused necrosis in 50% of the animals; if the time at 44Â°was was induced, reaching temperatures of 42.6-43Â° for 30 min in reduced to 50 min or less, the necrosis was prevented (24). the prostate, as measured by thermocouples (56). The 32 rabbits Thus, in this system the limit of tolerance for growth inhibition is thus treated were examined histologically at multiple times from below 43Â°for 1 hr, and the limit of tolerance for acute damage 1 day to 3 months after exposure(s). Only minimal alterations of in the various soft tissues of the tail seems to be 44Â°for 1 hr.

4832s CANCER RESEARCH VOL. 44

for the Improvement of Radiotherapy. Vienna: International Atomic Energy Embryo Comission, 1976. 20. Field, S. B., Hume, S. P., Law, M. P., and Myers, R. The response of tissues Of great concern are the effects of temperature elevation on to combined hyperthermia and x-rays. Br. J. Radiol., 50: 129-134,1977. the embryo. Retardation of brain growth has been documented 21. Freeman, M. L., Raaphorst, G. P., Hopwcod, L. E., and Dewey, W. C. The effect of pH on cell lethality induced by hyperthermic treatment. Cancer (Phila.), in guinea pig embryos exposed to maternal temperatures of 45:2291-2300,1983. 41.8-43.9Â°for 1 hr (in an incubator) on the 21st day of gestation 22. Goffinet, D. R., Choi, K. Y., and Brown, J. M. The combined effects of (53). Such exposures resulted in a dose-related microcephaly of hyperthermia and ionizing radiation on the adult mouse spinal cord. RadiÃ¢t. the offspring (53). A comparison with 7-radiation in the same Res., 72:238-245, 1977. 23. Gore, I., and Isaacson, N. H. The pathology of hyperpyrexia. Am. J. Pathol., experimental system showed that an elevation of 1Â°produceda 25:1029-1060,1949. deficit in brain weight equivalent to the effect caused by a dose 24. Hahn, G. M. Hyperthermia and Cancer. New York: Plenum Press, 1982. 25. Harris, A. B., Erickson, L., Kendig, J. H., Mingrino, S., and Goldring, S. increment of 42.5 rad (53). Obviously, this information refers only Observations on selective brain heating in dogs. J. Neurosurg., 79: 514-521, to maternal temperatures; the embryonal temperatures are as 1962. 26. Hente, K. J. Thermotolerance in the murine jejunum. J. Nati. Cancer Inst., 68: sumed to be equal, or at least to change parallel,to the maternal 1033-1036,1982. ones, but this assumption may be wrong. 27. Hume, S. P., and Marigold, J. C. L. Transient, heat induced, thermal resistance Other studies of the effects of systemic or local hyperthermia in the small intestine of mouse. RadiÃ¢t.Res., 82: 526-535, 1980. 28. Hume, S. P., Marigold, J. C. L., and Field, S. B. The effects of local hyperthermia on the embryo are available, but their review is beyond the scope on the small intestine of mouse. Br. J. Radiol., 52: 657-662,1979. of this article. In any case, the possible teratogenic effects of 29. Hume, S. P., Marigold, J. C., and Michalowski, A. The effect of local hyper thermia on nonproliferative, compared with proliferate, epithelial cells of the hyperthermia should be a consideration in the therapy of abdom mouse intestinal mucosa. RadiÃ¢t.Res., 94: 252-262, 1983. inal neoplasms, and even in the diagnostic use of ultrasound. 30. Kew, M., Bersohn, I., Seflel, H., and Kent, G. Liver damage in heatstroke. Am. J. Med., 49:192-202,1970. 31. Law, M. P., and Field, S. B. The response of the mouse ear to heat applied References alone or combined with x-rays. Br. J. Radiol., 57:132-138,1978. 32. Unke, C., Elbadawi, A., Netto, V., Roberts, A., and Russo, M. Effect of marked 1. Anderson, R. L, Ahier, R. G., and Littleton, J. M. Observations on the cellular hyperthermia upon the canine bladder. J. Ural., 707: 599-602,1972. effects of ethanol and hyperthermia in vivo. RadiÃ¢t.Res., 94: 318-325,1983. 33. Luk, K. H., Francis, M. E., Perez, C. A., and Johnson, R. J. Radiation therapy 2. Baker, D. G., Sager, H., Constable, W., and Goodchild. N. The response of and hyperthermia in the treatment of superficial lesions: preliminary analysis: previously irradiated skin to combinations of x-irradiation and ultrasound- treatment efficacy and reaction of skin, subcutaneous tissues. Am. J. Clin. induced hyperthermia. RadiÃ¢t.Res., 96: 367-373, 1983. Oncol., 6:399-406,1983. 3. Barnard, J. W., Fry, W. J., Fry, F. J., and Brennan, J. F. Small localized 34. Lyons, B. E., Britt, R. H., and Strohbehn, J. W. Localized hyperthermia in the ultrasound lesions in the white and gray matter of the cat brain. Arch. Neural. treatment of malignant brain tumors using an interstitial microwave antenna Psychiatry, 75. 15-35, 1956. assay. IEEE Trans. Biomed. Eng., 37: 53-62,1984. 4. Britt, R. H., Lyons, B. E., Pounds, D. W., and Prionas, S. D. Feasibility of 35. Magin, R. L., Fridd, C. W., Bonfiglio, T. A., and Linke. C. A. Thermal destruction ultrasound hyperthermia in the treatment of malignant brain tumors. Med. of the canine prostate by high intensity microwaves. J. Surg. Res., 29: 265- Instr, 77. 172-177, 1983. 275, 1980. 5. Britt, R. H., Lyons, B. E., Ryan, T., Saxer, E., Obana, W., and Rossi, G. Effect 36. Malamud, N., Haymaker, W., and Custer, R. P. Heat stroke. A clinico-patho- of whole body hyperthermia on auditory brainstem, somatosensory and visual logic study of 125 fatal cases. Mil. Surg., 97: 397-449,1946. evoked potentials. In: J. R. S. Hates (ed.), Thermal Physiology, pp. 519-523. 37. Marmor, J. B., Hahn, N., and Hahn, G. M. Tumor cure and cell survival after New York: Raven Press, 1983. localized radtofrequency heating. Cancer Res., 37:879-883,1977. 6. Britt, R. H., Pounds, D. W., and Lyons, B. E. Feasibility of treating malignant 38. Martinez, A. A., Meshorer, A., Meyer, J. L., Hahn, G. M., Fajardo, L. F., and brain tumors with focused ultrasound. Prog. Exp. Tumor Res., in press, 1984. Prionas, S. D. Thermal sensitivity and thermotolerance in normal porcine 7. Bull, J. M. Whole body hyperthermia as an anticancer agent. Cancer (Am. tissues. Cancer Res., 43: 2072-2075,1983. Cancer See.), 32:123-128, 1982. 39. Merino, 0. R., Peters, L. J., Mason, K. A., and Withers, H. R. Effect of 8. Bull, J. M., Lees, D., Schuette, W., Whang-Peng, J., Smith, R., Bynum, G., hyperthermia on the radiation response of the mouse jejunum. Int. J. RadiÃ¢t. Atkinson, E. R., Gottdiener, J. S., Gralnick, H. R., Shawker, T. H., and Devita, Oncol. Bicrf. Phys., 4: 407-414,1978. V. T., Jr. Whole body hyperthermia: a phase-l trial of a potential adjuvant to 40. Meshorer, A., Prionas, S. D., Fajardo, L. F., Meyer, J. L., Hahn, G. M., and chemotherapy. Ann. Intern. Med., 90: 317-323, 1979. Martinez, A. A. The effects of hyperthermia on normal mesenchymal tissues. 9. Burger, F. J., and Fuhrman, A. Evidence of injury by heat in mammalian tissues. Arch. Pathol. Lab. Med., 707: 328-334, 1983. Am. J. Physiol., 206: 1057-1061, 1964. 41. Moritz, A., and Henriquez, F. Studies of thermal injury. II. The relative impor 10. Carr, K. E., Hume, S. P., Marigold, J. C., and Michalowski, A. Scanning and tance of time and surface temperature in the causation of cutaneous bums. transmission electron microscopy of the damage to small intestinal mucosa Am. J. Pathol., 23: 695-720,1947. following x-radiation or hyperthermia. Scanning Electron Microsc., Part 1,393- 42. Okumura, Y., and Reinhold, H. Heat sensitivity of rat skin. Eur. J. Cancer, 74: 402, 1982. 1161-1166,1978. 11. Cronau, L. H., Jr., Bourke, D. L., and Bull, J. M. General anesthesia for whole- 43. Pettigrew, R. T., Galt, J. M., Ludgate, C. M., and Smith, A. N. Clinical effects body hyperthermia. Cancer Res. (Suppl.), 44: 4873s-4877s, 1984. of whole-body hyperthermia in advanced malignancy. Br. Med. J., 4:679-682, 12. Douglas, M. A., Parks, L. C., and Bebin, J. Sudden myelopathy secondary to 1974. therapeutic total-body hyperthermia after spinal cord irradiation. N. Engl. J. 44. Pettigrew, R. T., Galt, J. M., Ludgate, C. M., Horn, D. B., and Smith, A. N. Med., 304: 583-585, 1981. Circulatory and biochemical effects of whole body hyperthermia. Br. J. Surg., 13. Dubois, M., Coppola, R., Buchsbaum, M. S., and Lees, D. E. Somatosensory 6Ã•:727-730,1974. evoked potentials during whole body hyperthermia in humans. Etectroence- 45. Rogers, M. A., Marigold, J. C., and Hume, S. P. The effect of retino! on the phalogr. Clin. Neurophysiol., 52:157-162,1981. hyperthermal response of normal tissue in vivo. RadiÃ¢t.Res., 95: 165-174, 14. Elbadawi, A., Unke, C. A., Carstensen, E. L., and Fridd, C. W. Histomorpho- 1983. logic features of ultrasonic renal injury. Arch. Pathol. Lab. Med., 700: 199- 46. Salcman, M., and Samaras. G. M. Hyperthermia for brain tumors: biophysical 205, 1976. rationale. Neurosurgery, 9: 327-335, 1981. 15. Elkon, D., Fechner, R. E., Homzie, M. J., Baker, D. J., and Constable, W. C. 47. Santini, R., and Deschaux, P. Incidence of low-level microwave irradiation on Response of mouse kidney to hyperthermia. Pathology and temperature- intestinal myelectrical activity in the rat. Health Phys., 45: 775-778,1983. dependence of injury. Arch. Pathol. Lab. Med., 704. 153-158,1980. 48. Thomsen, H. K., Danielsen, L., Nielson, 0., Aalund, O., Nielsen, K. G., Karls 16. Eriksson, A., Albrektsson, T., Grane, B., and McQueen, D. Thermal injury to mark, T., Genefke, I. K., and Christoffersen, P. Epidermal changes in heat and bone. A vital microscopic description of heat effects. Int. J. Oral Surg., 77: electrically injured pig skin. A light microscopic study of the influence of heat 115-121, 1982. energy intensity and electrical current frequency. Acta Pathol. Microbiol. Im- 17. Fajardo, L. F. Pathology of Radiation Injury. New York: Masson Publishing munol. Scand. (A), 97: 297-306,1983. U.S.A., Inc., 1982. 49. Urano, M., and Kahn, J. The effect of step-down heating on murine normal 18. Fajardo, L. F., Egbert, B., Marmor, J., and Hahn, G. M. Effects of hyperthermia and tumor tissues. RadiÃ¢t.Res., 94: 350-358,1983. in a malignant tumor. Cancer (Phila.), 45: 613-623, 1980. 50. Urano, M., Montoya, V., and Booth, A. Effect of hyperglycemia on the thermal 19. Field, S. B., Hume, S., Law, M. P., Morris, C., and Meyers, R. Some effects response of murine normal and tumor tissues. Cancer Res., 43: 453-455, of combined hyperthermia and ionizing radiation on normal tissues. In: Pro 1983. ceedings of the International Symposium on Radiobiology Research Needed 51. 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by Corynebaeterium para/m of the normal skin and tumor tissue response to 54. Wills, E. J., Findlay, J. M., and McManus, J. P. A. Effects of hyperthermia hyperthermia. Cancer Res., 38: 862-864,1978. therapy on the liver. II. Morphological observations. J. Clin. Pathol. (Lond.), 29: 52. Urano, M., Rice, L., Kahn, J., and Sedlacek, R. S. Studies on fractionated 1-10,1976. hyperthermia Â¡nexperimental animal systems. I. The foot reaction after equal 55. Withers, H. R., and Elkind, M. M. Microcolony survival assay for cells of mouse doses: heat resistance and repopulation. Int. J. RadiÃ¢t.Oncol. Btol. Phys., 6: intestinal mucosa exposed to radiation. Int. J. RadiÃ¢t.Btol. Relat. Stud. Phys. 1519-1523,1980. Chem. Med., 17: 261-267,1970. 53. Wanner, R. A., and Edwards, M. J. Comparison of the effects of radiation and 56. Yerushalmi, A., Shpirer, Z., Hod, l., Gottesfeld, F., and Baos, D. D. Normal hyperthermia on prenatal retardation of brain growth of guinea pigs. Br. J. tissue response to localized deep microwave hyperthermia in the rabbit's Radiol., 56: 33-39,1983. prostate: a preclinical study. Int. J. RadiÃ¢t.Oncol. Biol. Phys., 9:77-82,1983.

Fig. 3. Focal loss of hepatocytes is illustrated in the midzonal area of a lobule whose thickened central vein appears in the left upper corner. Normal, continuous plates of liver cells are present in the right upper and left lower areas, while in the center the hepatocytes do not form plates but are separate from each other, due to the loss of multiple intervening liver cells. Treatments were at 43Â°for 30 min and 47.5Â°for 30 min. Gomori's trichrome, x 176. Fig. 4. Focal toss of liver cells is shown in the center of this photograph from a canine liver treated at 43Â°for 30 min and 47.5Â°for 30 min. In the center, and below a portal area, hepatocytes have disappeared, leaving delicate reticular-vascular framework. Compare with the normal hepatocytes plates and sinusoids in the right and left portions of the field. Neither this nor Fig. 3 show cells in the process of necrosis, such as "acidophilic bodies." In this experiment, individual cell necrosis presumably occurred during the first weeks after hyperthermia, followed by removal of cell debris by macrophages. Gomori's trichrome, x 320. Fig. 5. Massive area of liver necrosis (after 41.2" for 30 min and 45.3" for 30 min), involving more than one lobule. With this stain necrotic hepatocytes are pale and devoid of nuclei, although the continuity of the plates is preserved. A large portal area, with thrombosis of the vein and artery, is present in the right upper corner. A margin of inflammatory reaction (neutrophils) is seen at the lower edge. Unlike individual cell necrosis, massive necrosis did persist to 28 days. H & E, x 100. Fig. 6. Early scar in area of massive necrosis, after 41 Â°for 30 min and 44.5Â°for 30 min. The persistent necrotic liver plates appear dark with this stain, at the bottom of the figure. Invading collagen bands occupy the top. Although the ultimate result of these areas of fibrosis is unknown, probably localized hyperthermia affecting portions of the liver will not result in significant functional impairment of this organ. Gomori's trichrome, x 100.

4834s CANCER RESEARCH VOL. 44

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