Effects of Hypothalamic and Amygdaloid Lesions on Development and Growth of Carcinogen-induced Mammary Tumors in the Female Rat

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1 [CANCER RESEARCH 29, , August 1969] Effects of Hypothalamic and Amygdaloid Lesions on Development and Growth of Carcinogen-induced Mammary Tumors in the Female Rat Clifford W. Welsch a, James A. Clemens 4, and Joseph Meites Department of Physiology, Michigan State University, East Lansing, Michigan SUMMARY The effects of median eminence and preoptic and amygdaloid lesions on normal and 7,12-dimethylbenzanthraceneinduced neoplastic mammary tissue growth were investigated in female Sprague-Dawley rats. Marked stimulation of normal and neoplastic mammary tissue growth was observed in intact rats 10 and 25 days after placement of median eminence lesions. When median eminence lesions and ovariectomy were combined, a striking stimulation of normal and neoplastic tissue was noted 10 days but not 25 days later. f ovariectomy preceded the median eminence lesions by 10 days, no effect was observed on neoplastic mammary growth, and only a slight stimulation of normal mammary growth was observed 10 days after lesion placement. Since lesions in the median eminence result in enhanced prolactin secretion and reduced secretion of all other anterior pituitary hormones, these observations indicate that prolactin is the principal hormone responsible for growth of the 7,12-dimethylbenzanthraceneinduced mammary tumors. Lesions in the preoptic or amygdaloid complex of intact rats induced marked tumor regression 10 or 25 days after lesion placement, but they apparently had no effect on normal mammary development 10 days after lesion placement. The reduction in mammary tumor growth was accompanied by decreased ovarian and uterine weights, suggesting that estrogen secretion was lowered. Since the latter can result in diminished prolactin secretion, it is possible that reduced secretion of estrogen and prolactin was responsible for the mammary regression observed in the lesioned rats. 1Supported in part by NH research Grants AM and CAo10771 and Grant TA from the Michigan Cancer Foundation. 2Reported in part at the Annual Meeting of the American Association for Cancer Research, April Special Research Fellow, National Cancer nstitute, NH, USPHS. Present address: Department of Anatomy, Michigan State University, East Lansing, Michigan NH predoctoral trainee (GM-1121) during tenure of his work. Present address: Department of Anatomy, University of California at Los Angeles, Los Angeles, California Received September 30, 1968; accepted April 1, NTRODUCTON There are many indications that the central nervous system can influence development and growth of tumors (19). Several investigators have attempted to influence mammary tumorigenesis by administering tranquilizing drugs. Lacassagne and Duplan (18) observed that mice of the C3H strain, when treated with reserpine, developed mammary cancers earlier and in greater numbers than untreated controls. Welsch and Meites (39) also reported that reserpine significantly stimulated development of carcinogen-induced mammary tumors in rats. Only a few attempts have been made to determine the role of specific areas of the central nervous system on mammary tumorigenesis (1, 26, 36, 37). The purpose of this investigation is the evaluation of the effects of lesions of the median eminence and preoptic areas of the hypothalamus, and the amygdaloid complex, on the development and growth of carcinogen-induced rat mammary tumors. MATERALS AND METHODS All animals used in this investigation were virgin female Sprague-Dawley rats, obtained from Holtzman Co., Madison, Wise. They were housed in a temperature-controlled (75 ~ -+ 2~ and light-controlled (14 hr/day) room and were given a diet of Wayne Lab Blox (Allied Mills, Chicago, U.) and water ad libitum. Placement of Lesions The electrodes were prepared from size No. 1 steel insect pins, insulated with four coatings of epoxylite, and oven-baked after each coating at 140~ for three hours. The electrodes used for median eminence lesions were ground flat at the tip in order to produce lesions which extended horizontally and not vertically. Electrodes used for preoptic and amygdaloid lesions were ground to a point, producing a bare tip approximately 1.5 mm long which resulted in lesions extending horizontally and vertically. The median eminence and preoptic and amygdaloid lesions were produced bilaterally by passing a direct current of 2 ma for 7 sec, 1.8 ma for 7 sec, and 10 ma for 10 sec respectively through the electrodes. These areas were located with the aid of a Stoehing stereotaxic instrument and de Groot's (6) atlas of the rat brain. AUGUST

2 C W. Welsch, J. A. Clemens, and J. Meites Representative rats from each group were sacrificed and the brains were removed, flxed in neutral buffered formalin, sectioned at 10 microns, and stained with cresyl violet and luxol fast blue. The location of the lesions was confirmed. f any doubt existed regarding the site of the lesion, the rat was not included in the computation of the data. Properly placed lesions in the median eminence and preoptic areas resulted in almost total destruction of the arcuate nucleus and suprachiasmatic nucleus respectively (Figs. 1-3). No evidence of pituitar/ stalk damage was observed as a result of the median eminence lesions. Sham lesions were placed in rats by making bilateral lesions (2 ma for 7 sec) on the skull at the midline where the frontal and parietal bones meet (bregma). Prior to placement of the lesions, the rats were anesthetized with a small intravenous dose of pentobarbital; they were maintained in an anesthetized state with ether during the stereotaxic procedures. Bilateral ovariectomy was performed under ether anesthesia. Effects of Median Eminence and Preoptic and Amygdaloid Lesions on Mammary Tumor Development One hundred and fifty rats, 55 days of age, were given a single intravenous injection of a lipid emulsion containing 5 mg of 7,12-dimethylbenzanthracene (DMBA). Seventy to 90 days after carcinogen treatment, when all rats had at least one palpable mammary tumor, they were divided into groups and treated as follows: (a) intact controls, sham operated; (b) intact and lesions placed in the median eminence; (c) intact and lesions placed in the preoptic area; (d) intact and lesions placed in the amygdaloid complex; (e) ovariectomized controis, sham operated; and (Jr) ovariectomized and lesions placed in the median eminence. Median eminence lesions were made in intact rats on the day of estrus or the first day of diestrus. At 0, 10, and 25 days after placement of the lesions, all rats were examined for the number of palpable mammary tumors. All tumors one cm or larger were measured with a vernier caliper at the largest diameter. Twenty-five days after placing the lesions, the rats were sacrificed, the brains were removed, and the location of each lesion was confirmed with the aid of a dissecting microscope. Mammary tumors were excised from rats of each group, fixed in Bouin's fluid, sectioned at 3-5 microns, and stained with hematoxylin and eosin for histologic evaluation. Where possible, gross examination of the mammary glands was performed to determine the degree of mammary development. Daily vaginal smears were taken from rats of each group until a pattern was established. Statistical analysis within each group was prepared according to the mean increase or decrease in the number of palpable mammary tumors and in the tumor diameter (mm) at 0-10 and 0-25 days after lesion placement. The significance of differences between means was calculated by Student's "t" test. Effects of Median Eminence and Preoptic and Amygdaloid Lesions on Normal Mammary Development One hundred and twenty rats, approximately 140 days of age, were divided into groups and treated as follows: (a) intact controls, sham operated; (b) intact and lesions placed in the median eminence; (c) intact and lesions placed in the preoptic area; (d) intact and lesions placed in the amygdaloid complex; (e) ovariectomized controls, sham operated; and (f) ovariectomized and lesions placed in the median eminence. Median eminence lesions were made in intact rats on the day of estrus or the first day of diestrus. These rats were sacrificed 10 and 25 days after placement of lesions. ntact rats with lesions in the preoptic area and in the amygdaloid complex were sacrificed 10 days after the placement of lesions. Ovariectomized rats were divided into 4 groups. One group was sham operated, the second group was ovariectomized immediately after the lesion placement, and the third and fourth groups were ovariectomized 10 and 25 days respectively before the placement of lesions. All ovariectomized rats were sacrificed 10 days after the placement of lesions. At the time of sacrifice, pituitaries, thyroids, adrenals, ovaries, and uteri were excised and weighed. nguinal mammary glands were excised, spread flat on cork, and fixed in Bouin's fluid for whole-mount evaluation. Whole-mount preparations of mammary glands were stained by a standard procedure (27) and were rated for development according to the following: (a) few ducts, few or no end buds; (b) moderate duct growth, moderate number of end buds; (c) numerous ducts and branches, many end buds; (d) numerous ducts and branches, minimum lobulo-alveolar growth; (e) numerous ducts and branches, moderate lobulo-alveolar growth; (J) numerous ducts and branches, dense lobulo-alveolar growth as in late pregnancy. The significance of differences between the mean organ weights and the mammary development in each group was calculated by Student's "t" test. RESULTS Effects of Median Eminence and Preoptic and Amygdaloid Lesions on Mammary Tumor Development ntact rats bearing mammary tumors responded 10 and 25 days after lesions were placed in the median eminence with a 130% and 200% increase respectively in number of palpable mammary tumors/rat in contrast to an increase of only 23% and 27% respectively in the intact controls (Table 1, Groups and ). n addition, a slight but significant increase in mean tumor diameter was observed at 10 days but not at 25 days after placement of lesions in these rats (Chart 1). ntact rats with lesions in the preoptic area showed significant tumor regression 10 and 25 days after surgery as indicated by a decrease in the number of mammary tumors/rat (Table 1, Groups and ) and a marked decrease in the mean tumor diameter (Chart 1). The mean tumor diameter in rats bearing amygdaloid lesions was significantly decreased 10 and 25 days after lesion placement (Chart 1), but this treatment had no significant effect on the number of mammary tumors/rat (Table 1, Groups and V). Generally, the mammary tumors of the amygdaloid-lesioned rats were atrophic, although two rats in this group showed marked tumor stimulation comparable to that observed in rats with lesions in the median eminence. Rats that had lesions placed in the median eminence and 1542 CANCER RESEARCH VOL. 29

3 Brain Lesions and Mammary Tumorigenesis Table 1 Average number of palpable mammary tumors No. of At time 10 days % 25 days % Group Treatment a rats of lesion after lesion change after lesion change ntact + sham lesion /~ a +23% d +27% ntact + median eminence lesion b +138% e +200% ntact + preoptic lesion e --14% f --32% V ntact + amygdaloid lesion % % Effect of median eminence and preoptic and amygdaloid lesions on mammary tumor development and growth in intact female rats treated with 7,12-dimethylbenzanthracene (DMBA). a/b, d/e = P < 0.001; d/f = P < 0.002; a/c = P < admba was administered at 55 days of age. Bilateral lesions were made days after DMBA treatment. ~Standard error of the mean. that were ovariectomized immediately thereafter responded 10 days later with a 25% increase, and 25 days later with a 35% decrease in number of palpable mammary tumors/rat, in contrast to a 40% and 58% decrease at 10 and 25 days respectively in the ovariectomized controls (Table 2, Groups and ). n addition, a highly significant increase in the mean tumor diameter was seen at 10 days, followed by a marked decrease in tumor diameter 25 days after placement of the lesions (Chart 1). By contrast, when ovariectomy preceded the median eminence lesions by 10 days, no significant effect of the lesions was observed on the number of mammary tumors/rat or mean tumor diameter (Table 2, Groups and V). Frequently, when the mammary glands were not completely tumorous, gross evaluation of mammary development was attempted at the time of sacrifice. Generally, parallel growth responses were observed between normal and neoplastic mammary tissue in rats with lesions in the median eminence, with one exception. When lesions were placed in the median eminence and the rats were ovariectomized immediately thereafter, a small proportion of the rats still retained an extensive mammary structure 25 days later, but tumor regression was apparent at this time. The predominant tumor type observed in these rats was classified histologically as an adenocarcinoma, as described previously by Huggins et al. (13). Medion Eminence/Pre~ptic Lesion v Meclion Eminence Lesion/Ovariectomy ::: / Amygdoloid Lesion _z L.-~rote~..~i,b 15 DAYS DAYS Ov~176 Controls Chart 1. Changes in mean tumor diameter 10 and 25 days after lesion placement. Top left, intact rats with lesions in the median eminence (ME) or preoptic area and intact controls. Bottom left, intact rats with lesions in the amygdaloid complex and intact controls. Top right, ovariectomized rats with lesions in the ME and ovariectomized controls. Bottom right, ovariectomized rats with lesions placed in the ME 10 days later and ovariectomized controls. N. S., not significantly different. AUGUST

4 C W. lqelsch, J. A. Clemens, and J. Meites Table 2 Average number of palpable mammary tumors No. of At time 10 days % 25 days % Group Treatment a rats of lesion after lesion change after lesion change Ox + sham lesion a -40% % Ox + ME lesion b +25% % Ox 10 days, sham lesion % % V Ox 10 days ~ ME lesion % % Effect of median eminence lesion (ME) on mammary tumor development and growth in ovariectomized (Ox) rats treated with 7,12-dimethylbenzanthracene (DMBA). a/b = P < admba was administered at 55 days of age. Bilateral lesions were placed in the median eminence area of the hypothalamus approximately 80 days after DMBA treatment. gstandard error of the mean. Effects of Median Eminence, and Preoptic and Amygdaloid Lesions on Normal Mammary Development Mammary growth was significantly enhanced in intact rats 10 or 25 days after placing lesions in the median eminence area (Table 3, Groups,, A) (Figs. 4, 5). Many of these rats had mammary development comparable to that seen in rats in late pregnancy, with an extensive lobulo-alveolar system and alveoli occasionally distended with secretion. No apparent effect on mammary growth was observed in intact rats 10 days after lesions were placed in the preoptic area or amygdaloid complex (Table 3, Groups and V). Vaginal smears of intact rats with lesions in the median eminence and amygdaloid complex indicated that these rats were in prolonged diestrus in contrast to the regular 4- to 5-day cycles in the controls. Rats with lesions in the preoptic area initially showed prolonged diestrus followed by a tendency toward irregular estrus. Diuresis was observed in all rats with lesions in the median eminence. Rats with lesions in the median eminence followed by immediate ovariectomy showed a striking increase in mammary development ten days later (Table 4, Group V) (Fig. 6) in contrast to the regression seen in the mammary glands of the ovariectomized (Table 4, Group V) or the normal intact controis (Table 3, Group ) (Fig. 4). Several of the ovariectomized rats with median eminence lesions showed extensive lobulo- alveolar development comparable to that seen in intact rats beating lesions in the median eminence. n contrast, rats ovariectomized either 10 or 25 days before placement of lesions in the median eminence showed only a small but nonetheless significant stimulation of mammary growth 10 days after lesion placement (Table 4, Groups V and X). This was in contrast to the atrophied mammary glands of the ovariectomized controls (Table 4, Groups V and X). Table 4 No. of Average mammary Group 3' Treatment" rats gland ratings V Ox + sham lesion a V Ox + ME lesion b V Ox 10 days., sham lesion c V Ox 10 days ME lesion d X Ox 25 days, sham lesion e X Ox 25 days, ME lesion f Effect of median eminence lesion (ME) on mammary gland development in mature ovariectomized (Ox) rats. a/b = P < 0.001; c/d = P < 0.005; e/f = p < aall rats were sacrificed 10 days after placement of the lesion. ~Mean + standard error. ~The rats represented in these groups are the same animals as in Table 5. Table 3 No. of Average mammary Group qt Treatment a rats gland ratings3 ntact + sham lesion a ntact + median eminence lesion b A ntact + median eminence lesion c ll ntact + preoptic lesion V ntact + amygdaloid lesion Effect of median eminence and preoptic and amygdaloid lesions on mammary gland development in mature intact female rats. a/b, a/c = P < a Rats of Groups,,, and V were sacrificed 10 days after placement of the lesion. Rats of Group A were sacrificed 25 days after placement of the lesion. BMean + standard error. "/The rats represented in these groups are the same animals as in Table 5 with the exception of the animals of Group A. Effect of Median Eminence and Preoptic and Amygdaloid Lesions on Organ Weights (Table 5) Bilateral lesions placed in the median eminence and preoptic and amygdaloid complex of intact rats significantly reduced ovarian and uterine weights. Median eminence lesions in intact or ovariectomized rats significantly reduced thyroid weight, but no effect on thyroid weight was observed in rats with lesions in the preoptic or amygdaloid complex. Median eminence lesions in ovariectomized rats significantly reduced anterior pituitary weight, but no effect on pituitary weight was noted in intact rats with lesions in the median eminence and preoptic or amygdaloid complex. No effect on adrenal weight was observed in rats with median eminence lesions, except for rats ovariectomized 25 days before placing the lesions; these 1544 CANCER RESEARCH VOL. 29

5 Brain Lesions and Mammary Tumorigenesis. Table 5 Average Beginning of End of anterior treatment treatment pituitary No. of Average body wt. Average body wt. wt. Group Treatment" rats (gm) (gm) (rag) ntact + sham lesion g g ~ ntact + ME lesion h ntact + preoptic lesion P V ntact + amygdaloid lesion V Ox + sham lesion a i V Ox + ME lesion b J V Ox 10 days > sham lesion c k Average Average Average Average thyroid adrenal ovarian uterine wt. wt. wt. wt. {mg) (mg) (mg) (mg) V 0x10 days ~ ME lesion a X Ox25 days 9 ~ sham lesion e m q X Ox 25 days > ME lesion f n r s w t x u y v z Organ weights of mature ovariectomized (Ox) and intact female rats after placement of lesions in the median eminence (ME) and the preoptic and amygdaloid areas, a/b, e/f, g/h, m/n, q/r, s/u, s/v, w/x, w/z = P < 0.001; i/j, s/t = P < ; c/d = P < 0.01; k/l, o/p, w/y = P < aall rats were sacrificed 10 days after placement of the lesion. flstandard error of the mean. showed increased adrenal weight. Lesions in the preoptic area significantly increased adrenal weight, whereas lesions in the amygdaloid complex had no significant effect. A slight loss in body weight of about 4% was observed in rats 10 days after placing lesions in the preoptic or amygdaloid complex, whereas all groups with lesions in the median eminence area showed a mean increase in body weight comparable to that of the controls. DSCUSSON Bilateral lesions placed in the median eminence of intact female rats resulted in marked enhancement of growth of DMBA-induced mammary tumors. When lesions were placed in the median eminence and ovariectomy was performed immediately thereafter, initial stimulation with subsequent regression of mammary tumors was observed. f ovariectomy preceded the median eminence lesions by 10 days, no effect of the lesions on mammary tumor growth was noted. Significant stimulation of normal mammary growth was also observed in intact and ovariectomized rats as a result of placing lesions in the median eminence. The effects of median eminence lesions on pituitary function are qualitatively similar to those seen after stalk section (20, 22) or transplantation of the pituitary (20, 22, 41), resulting in increased secretion of prolactin and significantly reduced secretion of all other anterior pituitary hormones. The hypothalamus stimulates secretion of five anterior pituitary hormones by releasing growth hormone releasing factor, luteinizing hormone releasing factor, follicle stimulating hormone releasing factor, corticotropin releasing factor, and thyrotropin releasing factor into the hypothalamo-pituitary portal vessels and inhibiting prolactin secretion by releasing prolactin inhibiting factor (20, 22). Although the median eminence contains all of these factors (20), it is not clear whether they are synthesized or only stored and released from this area. Lesions in the median eminence are believed to act, at least in part, by disrupting the hypothalamic-hypophyseal portal system, thus preventing the neurohormones from reaching the anterior pituitary gland. The general reduction in pituitary target organ weights (adrenals were an exception) and pronounced mammary development observed in this study agree with this concept. The marked stimulation of normal and neoplastic mammary growth as a result of median eminence lesions, particularly in the ovariectomized rats, indicates that prolactin was mainly responsible for these effects. Several investigators have reported that pituitary hormones alone can elicit full mammary lobulo-alveolar development in rats. Thus adreno-ovariectomized rats bearing transplantable Furth F 4 pituitary tumors that secrete prolactin, growth hormone (GH), and adrenocorticotropic hormone (ACTH), showed full mammary duct and lobulo-alveolar growth (33). Similar mammary development was observed in adreno-ovariectomized-hypophysectomized rats (32) or adreno-orchidectomized rats (21) given injections of prolactin and GH. Recently, an in vitro study indicated that only insulin and prolactin were necessary to induce lobulo-alveolar development of rat mammary tissue (8). Other studies have indicated that pituitary hormones alone can stimulate growth of carcinogen-induced mammary tumors in rats. Kim and Furth (17) reported that in rats with mammary tumor regression after ovariectomy or hypophysectomy, grafts of pituitary tumors which secreted prolactin, GH, and ACTH caused resumption of tumor growth. O. H. Pearson (personal communication) observed that injections of prolactin alone into adreno-ovariectomized-hypophysectomized rats bearing carcinogen-induced mammary tumors resulted in maintenance of tumor growth. A previous study had also indicated that injections of prolactin and GH could induce growth of mammary tumors in ovariectomized DMBA-treated rats (34). The results of the present study suggest that after removal of ovarian steroids, prolactin alone can at least initially promote the growth of mammary tumors. AUGUST

6 C. W. Welsch, J. A. Clemens, and J. Meites The absence of continued growth of mammary tumors observed in ovariectomized rats 25 days after placement of median eminence lesions, and the lack of effect in rats ovariectomized 10 days before placing of the lesion, may be the result of inadequate pituitary prolactin and/or GH secretion. t is probable that the stimulating effect of a median eminence lesion on prolactin secretion is not as pronounced and does not persist as long in ovariectomized as it does in intact rats. t is well established that estrogen can promote the synthesis and release of pituitary prolactin both by a direct action on the pituitary (28) and via the hypothalamus (23). Progesterone can also increase prolactin secretion (29), although it is not as effective as estrogen. These 2 steroids may therefore act through the pituitary as well as directly on the mammary tissues to promote mammary tumor growth. The progressive growth of.mammary tumors in intact rats as a result of median eminence lesions suggests that GH may have only a minimal role in the growth of mammary tumors since GH secretion is reduced by these lesions. The possibility cannot be excluded that median eminence lesions may also contribute to the growth of mammary tumors by depressing the secretion of anterior pituitary hormones such as ACTH. t has been reported that administration of adrenal corticoids reduced growth of mammary tumors (30). Kim (16) and Daniel and Prichard (3) observed that removal of the adrenals of rats bearing DMBA-induced mammary tumors resulted in enhanced growth of the tumors. Other studies have indicated that adrenalectomy does not significantly influence the incidence of carcinogen-induced rat mammary tumors (14, 31). The degree to which the ACTH-adrenal cortical system participates in mammary tumor growth remains to be determined. Suppression of hypothalamic functions by administration of tranquilizers such as reserpine and chloropromazine has been shown to result in increased pituitary prolactin release and enhanced mammary development (24). The stimulatory effect of reserpine on carcinogen-induced mammary tumors in rats was recently reported by Welsch and Meites (39). Reserpine stimulated development of normal and neoplastic mammary tissue in intact rats but had no effect in ovariectomized rats. The present work suggests that lesions in the median eminence area of the hypothalamus result in quantitatively greater pituitary prolactin secretion than suppression of the hypothalamus by the administration of tranquilizers such as reserpine. t is of interest that placement of median eminence lesions before DMBA administration was previously found to inhibit mammary tumorigenesis in the rat (1). This was associated with increased stimulation of mammary growth. Apparently stimulation of a predominantly lobulo-alveolar structure in the rat induces a refractoriness of the mammary tissue to the action of the carcinogen (5, 38, 40). Recendy Montemurro and Toh (26) reported that hypothalamic lesions, regardless of their specific location, increased the incidence and decreased the latency period of spontaneous mammary tumors in two mains of mice. They concluded that hypothalamic damage stimulated mammary tumorigenesis by a hormonal imbalance related to an elevated secretion of prolactin. Daniel and Prichard (2) studied the comparative effects of pituitary stalk section and hypophysectomy on the growth of carcinogen-induced mammary tumors in rats. After stalk section, slight regression of mammary tumors was observed in approximately two-thirds of the rats; no effect was seen in the remaining rats. After hypophysectomy, nearly all of the rats showed complete regression of tumors. These results appear to be at variance with the stimulatory effects observed on mammary tumors after placement of median eminence lesions, reserpine treatment, or transplantation of pituitary tissue (36-39). However, extensive infarction (29-78%) of the pituitary was noted as a result of stalk section, and this is the probable explanation for the slight regression seen in the mammary tumors. Transection of the pituitary stalk in women with breast cancer has also been reported to result in tumor regression (9), but the prognosis for pituitary infarction after such surgery was reported to be approximately 90% (2). Lesions in the median eminence, under the conditions set forth in this study, do not produce pituitary infarction. More extensive electrolytic lesions of the median eminence, including the pituitary stalk, could conceivably result in pituitary infarction and tumor regression. Lesions in the preoptic area of the hypothalamus, including the suprachiasmatic area, resulted in pronounced mammary tumor atrophy, as indicated by a significant decrease in the number of mammary tumors/rat and a highly significant decrease in the mean tumor diameter. n addition, there was a significant reduction in ovarian and uterine weights and cessation of normal vaginal cycles as indicated by an initial prolonged diestrus followed by a tendency toward irregular estrus. Preoptic lesions in rats have been reported to stimulate folliclestimulating hormone and inhibit leuteinizing hormone secretion, resulting in constant vaginal estrus and small hyperfollicular ovaries (11). Continuous vaginal estrus in rats induced by constant light has been shown to induce regression (unpublished data) and to alter carcinogen-induced mammary tumor morphology (15). However, the decrease in ovarian and uterine weights observed in the present study suggests that estrogen secretion may have been reduced. The effects of the lesions on prolactin secretion were not determined but this too may have been decreased. A reduction in secretion of either of these hormones could have resulted in mammary tumor regression. The amygdaloid complex is connected to the hypothalamus by the stria terminalis and more directly by the amygdalopreoptic and amygdalo-hypothalamic fibers. Lesions in the amygdaloid complex have been reported to result in altered hypothalamic-pituitary function in several species (7). n the immature rat, lesions in this area promoted precocious ovarian stimulation (10). n the mature rat, bilateral lesions of this area were reported to result in reduced gonadotropin secretion (42). The amygdaloid complex may also be associated with pituitary prolactin secretion, since a recent study by Tindal and Knaggs (35) indicates that implantation of estrogen into the amygdaloid complex of rabbits resulted in lactogenesis. Altered hypothalamic-pituitary function as a resuk of amygdaloid lesions is indicated in our study by the significant atrophy of mammary tumors, ovaries and uteri, and the transformation from normal 4- to 5-day vaginal cycles to prolonged diestrus. The reduction in ovarian and uterine weights suggests a decrease in estrogen secretion, which in turn could lead to a fall 1546 CANCER RESEARCH VOL. 29

7 Brain Lesions and Mammary Tumorigenesis in prolactin secretion. A decreased secretion of either or both of these two hormones could account for the mammary tumor regression observed in the rats with lesions in their amygdaloid complex. t is of interest that two rats in this group showed marked tumor stimulation comparable to that observed in rats with lesions in the median eminence. Amygdaloid lesions as well as preoptic lesions did not induce any obvious regression of normal mammary tissue under the conditions of this study. This suggests a possible difference in responsiveness of normal and neoplastic mammary tissues to the altered hormonal balance induced by these lesions. The slight reduction in body weight (4%) observed in rats with amygdaloid and preoptic lesions is believed to be insufficient to account for the marked mammary tumor regression. Gropper and Shimkin (12) reported that a 12-24% body weight reduction was necessary to significantly reduce the growth of carcinogen-induced mammary tumors in the rat. Further studies are required to elucidate the role of the amygdaloid complex and the preoptic area on the development and growth of normal and neoplastic mammary tissues. The results of this study, as well as other preliminary reports from this laboratory (1, 36, 37), indicate that lesions in specific areas of the central nervous system can significantly influence development and growth of carcinogen-induced mammary tumors in the rat, a tumor system which closely resembles human mammary cancer (4, 13, 25). Alterations in pituitary prolactin secretion appear to be mainly responsible for these effects. t is conceivable that other hormonedependent tumors may originate or may at least be influenced by altered functions of central nervous system areas which influence anterior pituitary function. Additional investigations of the complex central nervous system-endocrine interrelationships are necessary to further elucidate their role in tumorigenesis. ACKNOWLEDGMENTS The authors wish to express their appreciation to Dr. J. Hinman, The Upjohn Company, Kalamazoo, Michigan, for providing the 7,12-dimethylbenzanthracene. REFERENCES 1. Clemens, J. A., Welsch, C. W., and Meites, J. Effects of Hypothalamic Lesions on ncidence and Growth of Mammary Tumors in Carcinogen-Treated Rats. Proc. Soc. Exptl. Biol. Med., 27: , Daniel, P. M., and Prichard, M. M. L. The Response of Experimentally nduced Mammary Tumours in Rats to Hypophysectomy and to Pituitary Stalk Section. Brit. J. Cancer, 17: , Daniel, P. M., and Prichard, M. M. L. The Effect of Adrenalectomy on the Growth of Mammary Tumours nduced by 3-methylcholanthrene in Rats. ntern. J. Cancer, 2: , Dao, T. L. Carcinogenesis of Mammary Gland in Rat. Prog. Exptl. Tumor Res., 5: , Dao, T. L., Bock, F. G., and Greiner, M. J. Mammary Carcinogen e- sis by 3-methylcholanthrene.. nhibitory Effect of Pregnancy and Lactation on Tumor nduction. J. Natl. Cancer nst., 25: , de Groot, J. The Rat Hypothalamus in Stereotaxic Coordinates. J. Comp. Neurol., 13: , de Groot, J. Limbic and other Neural Pathways that Regulate Endocrine Function. n: L. Martini and W. F. Ganong (eds.), Neur- ' oendocrinology, pp New York: Academic Press, nc., Dilley, W. J., and Nandi, S. Rat Mammary Gland Differentiation in vitro in the Absence of Steriods. Science, 161: 59-60, Ehni, G., and Eckles, N. E. nterruption of the Pituitary Stalk in the Patient with Mammary Cancer. J. Neurosurg., 16: , Elwers, M., and Critchlow, V. Precocious Ovarian Stimulation Following nterruption of Stria-Terminalis. Am. J. Physiol., 201: , Flerko, B. Control of Gonadotrophin Secretion in the Female. n: L. Martini and W. F. Ganong (eds.), Neuroendocrinology, pp New York: Academic Press, nc., Gropper, L., and Shimkin, M. B. Combination Therapy of 3-methylcholanthrene-nduced Mammary Carcinoma in Rats: Effect of Chemotherapy, Ovariectomy, and Food Restriction. Cancer Res., 27: , Huggins, C., Braiziarelli, G., and Sutton, H. Rapid nduction of Mammary Carcinoma in the Rat and the nfluence of Hormones on the Tumors. J. Exptl. Med., 109: 25-42, Huggins, C., Grand, L. C., and Brillantes, F. P. Critical Significance of Breast Structure in the nduction of Mammary Cancer in the Rat. Proc. Natl. Acad. Sci. U. S., 45: , Jull, J. W. The Effect of nfection, Hormonal Environment, and Genetic Constitution on Mammary Tumor nduction in Rats by 7,12-Dimethylbenz(a)anthracene. Cancer Res., 26: , Kim, U. Pituitary Function and Hormonal Therapy of Experimental Breast Cancer. Cancer Res., 25: , Kim, U., and Furth, J. Relation of Mammary Tumors to Mammotropes.. Hormone Responsiveness of 3-methylcholanthrene nduced Mammary Carcinomas. Proc. Soc. Exptl. Biol. Med., 103: , Lacassagne, A., and Duplan, J. F. Le Mecanisme de la Cancerisation de la Mamelle Chez la Souris, Considere d'apres les Resultats d'experiences au Moyen de la Reserpine. Compt. Rend. Acad. Sci., 249: , LeShan, L. Psychological States as Factors in the Development of Malignant Disease: A Critical Review. J. Natl. Cancer nst., 22: 1-13, McCann, S. M., and Dhariwal, A. P. S. Hypothalamic Releasing Factors and the Neurovascular Link Between the Brain and the Anterior Pituitary. n: L. Martini and W. F. Ganong (eds.), Neuroendocrinology, pp New York: Academic Press, nc., Meites, J. Maintenance of the Mammary Lobulo-Alveolar System in Rats After Adreno-Orchidectomy by Prolactin and Growth Hormone. Endocrinology, 76: , Meites, J. Control of Mammary Growth and Lactation. n: L. Marthai and W. F. Ganong (eds.), Neuroendocrinology, pp New York: Academic Press, nc., Meites, J., and NicoU, C. S. Adenohypophyses: Prolactin. Ann. Rev. Physiol., 28: 57-88, Meites, J., Nicoll, C. S., and Talwalker, P. K. The Central Nervous System and the Secretion and Release of Prolactin. n: A. V. Nalbandov (ed.), Advances in Neuroendocrinology, pp Urbana, llinois: University of llinois Press, Middleton, P. J. The Histogenesis of Mammary Tumors nduced in the Rat by Chemical Carcinogens. Brit. J. Cancer, 19: , Montemurro, D. G., and Toh, Y. C. Effect of Hypothalamic Lesions on the Genesis of Spontaneous Mammary Gland Tumors in AUGUST

8 C W. Welsch, J. A. Clemens, and J. Meites Mice. Excerpta Med., 57: 136, 196" Nandi, S. Hormonal Control of Mammogenesis and Lactogenesis in the C3H/He Crgl Mouse. University Calif. (Berkeley) Pub. Zool., 65: Nicoll, C. S., and Meites, J. Estrogen Stimulation of Prolactin Production by Rat Adenohypophysis in vitro. Endocrinology, 70: , Sar, M., and Meites, J. Effects of Progesterone, Testosterone, and Cortisol on Hypothalamic Prolactin-nhibiting Factor and Pituitary Prolactin Content. Proc. Soc. Exptl. Biol. Med., 127: , Segaloff, A. Hormones and Breast Cancer. Rec. Progr. Hormone Res., 22: , 1966, 31. Shay, H., Harris, C., and Gruenstein, M. Further Studies in Prevention of Experimentally nduced Breast Cancer in the Rat. Some Endocrine Aspects. Acta Un. nt. Cancer, 16: , Talwalker, P. K., and Meites, J. Mammary Lobulo-Alveolar Growth nduced by Anterior Pituitary Hormones in Adreno-Ovariectomized and Adreno-Ovariectomized-Hypophysectomized Rats. Proc. Soc. Exptl. Biol. Med,, 107: , Talwalker, P. K., and Meites, J. Mammary Lobulo-Alveolar Growth in Adreno-Ovariectomized Rats Following Transplantation of "Mammotropic" Pituitary Tumor. Proc. Soc. Exptl. Biol. Med., 117: , Talwalker, P. K., Meites, J., and Mizuno, H. Mammary Tumor nduction by Estrogen or Anterior Pituitary Hormones in Ovariectomized Rats Given 7, 12-Dimethylbenzanthracene. Proc. Soc. Exptl. Med., 116: , Tindal, J. S., and Knaggs, G. S. Lactogenesis in the Pseudopregnant Rabbit after the Local Placement of Oestrogen in the Brain. J. Endocrinol., 34: ii, Welsch, C. W., Clemens, J. A., and Meites, J. Effects of Hypothalamic Lesions on Growth and Development of 7, 12-Dimethylbenzanthracene (DMBA)-nduced Mammary Tumors in Rats. Proc. Am. Assoc. Cancer Res., 9: 76, Welsch, C. W., Clemens, J. A., and Meites, J. Growth and Development of Carcinogen-nduced Mammary Tumors in Rats as nfluenced by Placement of Median Eminence Lesions in the Hypothalamus. Excerpta Med., 57: 203, Welsch, C. W., Clemens, J. A., and Meites, J. Effects of Multiple Pituitary Homografts or Progesterone on DMBA-nduced Mammary Tumors in Rats. J. Natl. Cancer nst., 4: , Welsch, C. W., and Meites, J. Effects of Reserpine on Development of Carcinogen-nduced Mammary Tumors in Rats. 24th ntern. Congr. Physiol. Sci., Vol. 6, p Washington, D. C., Welsch, C. W., and Meites, J. Effects of a Norethynodrel-Mestranol Combination (Enovid) on Development and Growth of Carcinogen- nduced Mammary Tumors in Female Rats. Cancer, 23" , Welsch, C. W., Negro-Vilar, A., and Meites, J. Effects of Pituitary Homografts on Host Pituitary Prolactin and Hypothalamic PF Levels. Neuroendocrinology, 3" , Yamada, T., and Greer, M. A. The Effect of Bilateral Ablation of the Amygdala on Endocrine Function in the Rat. Endocrinology, 66: , Figs Diagrammatic cross sections of rat brain. Solid black areas represent the approximate region of lesions. The abbreviations used are: AHA, anterior hypothalami area; AM, anteromedialis thalmi nucleus; ARH, arcuatus hypothalami nucleus; BST, proprius striae terminalis nucleus; CA, commissura anterior; C, capsula interna; CO, chiasma opticum; DMH, dorsomedialis hypothalami nucleus; EP, entopeduncularis nucleus; FX, fornix; GP, globus pauidus; LHA, lateralis hypothalami area; MFB, medialis forebrain bundle; MT, mammillo thalamicus tractus; OT, tractus opticus; PVH, paraventricularis hypothalami nucleus; RH, rhomboideus thalami nucleus; RT, reticularis thalami nucleus; SM, stria medullaris thalami; SO, supraopticus hypothalami nucleus; VE, ventralis thalami nucleus; VMH, ventromedialis hypothalami nucleus; Z, zona incerta. Fig. 1. Median eminence area. Fig. 2. Preoptic area. Fig. 3. Amygdaloid area. Figs Representative whole mounts of mammary glands. 10. Fig. 4. ntact control. Fig. 5. ntact and median eminence lesions. Fig. 6. Ovariectomized and median eminence lesions CANCER RESEARCH VOL. 29

9 Brain Lesions and Mammary Tumorigenesis -2 O o,.,r174,"- LHA VMH // l\ ~ MFB t -3 " t / \,,. i AR~] \ -4/A 6. '4 l 3 2 J 0 A i AUGUST 1969 Downloaded from cancerres.aacrjournals.org on October 12, American Association for Cancer 1549

10 Effects of Hypothalamic and Amygdaloid Lesions on Development and Growth of Carcinogen-induced Mammary Tumors in the Female Rat Clifford W. Welsch, James A. Clemens and Joseph Meites Cancer Res 1969;29: Updated version Access the most recent version of this article at: alerts Sign up to receive free -alerts related to this article or journal. Reprints and Subscriptions Permissions To order reprints of this article or to subscribe to the journal, contact the AACR Publications Department at To request permission to re-use all or part of this article, use this link Click on "Request Permissions" which will take you to the Copyright Clearance Center's (CCC) Rightslink site.