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The histochemical measurements of the lipid content of the pineal gland in rats suffering from malignancy
SHORT COMMUNICATIONS
123
The histochemical measurements of the lipid content of the pineal gland in rats suffering from malignancy The possibility that the pineal gland has an inhibitory effect on tumours elsewhere in the body has been suspected for many years. Acceleration in the growth of various tumours has been found after pinealectomy in rats and mice 1°,11 and more recently Das Gupta and Terz a described more rapid growth and spread of a transplantable melanoma in pinealectomized hamsters. Moreover some pineal extracts have been shown to have an inhibitory effect on the growth of tumours 4,6,7. If the pineal gland plays a significant role in the defence mechanism against cancer it is possible that some signs of a reactive nature might be found in patients dying of malignancy. With this in mind the pineal gland has been examined in such patients by two groups of workers with conflicting results. Rodin and OveralP 4 found that the pineal gland was larger in patients dying of malignancy compared with patients dying of a variety of other diseases, whilst the present workers showed the reverse to be the caselL In rats no significant differences could be found in the weight of pineal glands or in the size of the pineal parenchymal cells and their nuclei from animals suffering from malignancy and normal controls 9. However, the lipid content of the pineal gland is known to fluctuate under physiological conditions and in view of this the following experiment was carried out. Fibrosarcomas were induced in the hind leg of 17 Sprague-Dawley rats of both sexes by the subcutaneous injection of 2 mg 9 : 10 dimethyl-1 : 2 benzanthracene in 0.2 ml arachis oil. A larger number of control animals received arachis oil only. At the time that a tumour-bearing rat was to be killed a control rat of the same sex and weight was selected and killed along with the tumour animal. The pineals from the control rats were then treated in exactly the same way as those from the tumour-bearing animals. The rats were killed when the tumours reached about 6 cm in diameter, at which time the animals did not appear to be suffering from any systemic effects of the tumours. Post mortem examination, however, showed metastases in the lungs and abdominal lymph nodes of 8 animals. All the animals were killed at midday. The pineal gland was removed as quickly as possible and subjected to controlled chromation 5before being embedded in paraffin wax. Sections cut at 5 #m were then stained with Sudan Black B. The lipid content of the central part of these secions was calculated using an integration eye piece containing a grid showing 25 testing points. The number of lipid droplets hit by these testing points was counted and from this number the surface percentage could be calculated. Three median sections were examined from each gland and 200 points counted on each section. Calculations of the pineal lipid content done in this way may be regarded as semi-quantitative 16. It should be noted, however, that whilst Prop 12 has shown that Sudan Black after controlled chromation stains both triglycerides and phospholipids, the lipid quantitated in the way outlined above will only include that in the form of histologically visible droplets and not that which is bound to membranes in the cell. The surface percentages of lipid in the sections are given in Table I. The difference between the lipid content of the pineal glands from tumour-bearing animals and Brain TResearch, 37 (1972) 123-125
124
SHORT COMMUNICATIONS
TABLE I LIPID MEASUREMENTS
Values expressed as means 4- S.E.M. Numbers in parentheses indicate numbers of animals. Surface % lipid
All animals (17) Males (7) Females (10)
Tumour-bearing rats
Control rats
39 ± 1.1 40 4- 1.5 39 4- 1.5
31 4- 1.6 29 4- 2.3 32 ± 2.0
their controls is statistically highly significant (P < 0.001). Similar differences in the amount of lipid are found in the pineal glands of both male and female rats. A number of factors have already been shown to affect the amount of lipid detectable in the pineal gland with techniques similar to those described above. The level in female rats varies with the oestrous cycle 16 and is influenced by circulating gonadotrophins 17. It is unlikely, however, that this was responsible for the increased pineal lipid in the tumour-bearing animals in the present experiments. The lipid content of the pineal glands of both male and female tumour-bearing rats was raised. Cyclical variations of the type found in females at different stages of the oestrous cycle do not appear to occur in males and it is unlikely therefore that the results were due to coincidental fluctuations of this nature. The lipid content of the gland also depends on the age of the animal 12 and on environmental lighting conditionsaL These factors were avoided in the present experiments by using tumour-bearing animals and controls of the same age and by killing them all at midday to avoid diurnal variations. Finally, there is evidence that the pineal lipid is implicated in the sodium regulating processes e f the body 8. It is impossible to be certain that the tumour-bearing animals did not have electrolytic imbalances or were not dehydrated, but this is unlikely as they were invariably killed before any effect was observed on eating or drinking habits of the animal. It does not appear, therefore, that any of the factors knownPto influence pineal lipids were important in the present experiments, and whilst the possibility that the greater amount of lipid in the pineal gland of the tumour-bearing rats is a nonspecific manifestation cannot be excluded, there is increasing evidence that accumulation of lipid indicates increased activity of the pineal gland. As a result of this Prop 1~ has suggested that pineal lipid content could be used as a parameter for pineal secretory activity. Supporting evidence for this has come more recently from studies with the electron microscope, in histochemical investigations 2 and from Basinska et al. 1 who found evidence of a correlation between pineal lipids and the physiological activity of the gland. It is clear therefore that whilst the exact significance of pineal lipid accumulation in the tumour-bearing rats requires further investigation, this finding does afford Brain Research, 37 (1972) 123-125
SHORT COMMUNICATIONS
125
indirect evidence that the pineal gland m a y be involved in the c o n t r o l o f m a l i g n a n c y in the rat. This work was supported by a g r a n t from the Research C o m m i t t e e of the Manchester Regional Hospital Board. Department of Pathology, University Hospital of South Manchester and the University of Manchester, Withington Hospital West Didsbury, Manchester (Great Britain)
MARIANNE HUXLEY E. TAPP
1 BASINSKA,J., SASTRY,P. S., AND STANCER,H. C., Lipid composition of human, bovine and sheep pineal glands, J. Neurochern., 16 (1969) 707-714. 2 BOSTELMANN,W., The ultrastructural and enzyme histochemical behaviour of the pineal gland of the rat after alteration of the functional phase by continuous illuminationand continuous darkness, Endokrinologie, 53 (1968) 365-384. 3 DAS GUPTA,T. K., ANDTERZ,J., Influence of pineal gland on the growth and spread of melanoma in the hamster, Cancer Res., 27 (1967) 1306-1311. 4 DOBROVOLSKAIA-ZAVASKAIA,N., ET ZEPHIROEF, P., Effet des produits d'origine 6piphysaire et d'origine h6patique sur la croissance des tumeurs chez la souris, C.R. Soc. Biol. (Paris), 134 (1940) 60-63. 5 ELFTMAN,H., Controlled chromation, J. Histochem. Cytochem., 2 (1954) 1-8. 6 ENGEL,P., Untersuchungen tiber die Wirkung der Zirbeldriise, Z. ges. exp. Med., 93 (1933) 69-78. 7 HOFSTATTER,R., Versuche der postoperativen Krebsbehandlung mit Zirbelstoffen, Krebsarzt, 14 (1959) 307-316. 8 HUNGERFORO,G. F., AND PANAGIOTIS,N. M., Response of pineal lipid to hormone imbalances, Endocrinology, 71 (1962) 936-942. 9 HUXLEY,M., AND TAPP, E., Unpublished observations. 10 KATAGIRI,E., Shoka-sen ni kan-sura kenkyu ni shuyo Z6shoku to shoka-sen (Studies on the pineal gland; turnout proliferation and the pineal gland), Osaka Igakkai Zasshi, 43 (1944) 315-320. 11 NAKATANI,M., OHARA,Y., KATAGIRI,E., AND NAKANO,K., Shoka-sen tekishutsu shi-sei shironezumi no kenkyu (Studies on pinealectomized white rats), Acta Soc. path. Jap., 30 (1940) 232-236. 12 PROP, N., Lipids in the pineal body of the rat. In J. ARIENSKAPPERSAND J. P. SCHADI~(Eds.), Structure and Function of the Epiphysis Cerehri, Vol. 10, Elsevier, Amsterdam, 1965, pp. 454-464. 13 QUAY, W. B., Reduction of mammalian pineal weight and lipid during continuous light, Gen. comp. Endocrinol., 1 (1961) 211-217. 14 RODIN,A. E., AND OVERALL,J., Statistical relationships of weight of the human pineal to age and malignancy, Cancer (Philad.), 20 (1967) 1203-1214. 15 TAPP, E., AND BLUMFIELD,M., The weight of the pineal gland in malignancy, Brit. J. Cancer, 24 (1970) 67-70. 16 ZWEENS, J., Influence of the oestrous cycle and ovariectomy on the phospholipid content of the pineal gland in the rat, Nature (Lond.), 197 (1963) 1114-1115. 17 ZWEENS,J., Alterations in pineal lipid content in the rat under hormonal influences. In J. ARIi~NS KAPPERS AND J. P. SCHADIS(Eds.), Structure and Function of the Epiphysis Cerebri, Vol. 10, Elsevier, Amsterdam, 1965, pp. 540-551.
(Accepted November 23rd, 1971)
Brain Research, 37 (1972) 123-125
123
The histochemical measurements of the lipid content of the pineal gland in rats suffering from malignancy The possibility that the pineal gland has an inhibitory effect on tumours elsewhere in the body has been suspected for many years. Acceleration in the growth of various tumours has been found after pinealectomy in rats and mice 1°,11 and more recently Das Gupta and Terz a described more rapid growth and spread of a transplantable melanoma in pinealectomized hamsters. Moreover some pineal extracts have been shown to have an inhibitory effect on the growth of tumours 4,6,7. If the pineal gland plays a significant role in the defence mechanism against cancer it is possible that some signs of a reactive nature might be found in patients dying of malignancy. With this in mind the pineal gland has been examined in such patients by two groups of workers with conflicting results. Rodin and OveralP 4 found that the pineal gland was larger in patients dying of malignancy compared with patients dying of a variety of other diseases, whilst the present workers showed the reverse to be the caselL In rats no significant differences could be found in the weight of pineal glands or in the size of the pineal parenchymal cells and their nuclei from animals suffering from malignancy and normal controls 9. However, the lipid content of the pineal gland is known to fluctuate under physiological conditions and in view of this the following experiment was carried out. Fibrosarcomas were induced in the hind leg of 17 Sprague-Dawley rats of both sexes by the subcutaneous injection of 2 mg 9 : 10 dimethyl-1 : 2 benzanthracene in 0.2 ml arachis oil. A larger number of control animals received arachis oil only. At the time that a tumour-bearing rat was to be killed a control rat of the same sex and weight was selected and killed along with the tumour animal. The pineals from the control rats were then treated in exactly the same way as those from the tumour-bearing animals. The rats were killed when the tumours reached about 6 cm in diameter, at which time the animals did not appear to be suffering from any systemic effects of the tumours. Post mortem examination, however, showed metastases in the lungs and abdominal lymph nodes of 8 animals. All the animals were killed at midday. The pineal gland was removed as quickly as possible and subjected to controlled chromation 5before being embedded in paraffin wax. Sections cut at 5 #m were then stained with Sudan Black B. The lipid content of the central part of these secions was calculated using an integration eye piece containing a grid showing 25 testing points. The number of lipid droplets hit by these testing points was counted and from this number the surface percentage could be calculated. Three median sections were examined from each gland and 200 points counted on each section. Calculations of the pineal lipid content done in this way may be regarded as semi-quantitative 16. It should be noted, however, that whilst Prop 12 has shown that Sudan Black after controlled chromation stains both triglycerides and phospholipids, the lipid quantitated in the way outlined above will only include that in the form of histologically visible droplets and not that which is bound to membranes in the cell. The surface percentages of lipid in the sections are given in Table I. The difference between the lipid content of the pineal glands from tumour-bearing animals and Brain TResearch, 37 (1972) 123-125
124
SHORT COMMUNICATIONS
TABLE I LIPID MEASUREMENTS
Values expressed as means 4- S.E.M. Numbers in parentheses indicate numbers of animals. Surface % lipid
All animals (17) Males (7) Females (10)
Tumour-bearing rats
Control rats
39 ± 1.1 40 4- 1.5 39 4- 1.5
31 4- 1.6 29 4- 2.3 32 ± 2.0
their controls is statistically highly significant (P < 0.001). Similar differences in the amount of lipid are found in the pineal glands of both male and female rats. A number of factors have already been shown to affect the amount of lipid detectable in the pineal gland with techniques similar to those described above. The level in female rats varies with the oestrous cycle 16 and is influenced by circulating gonadotrophins 17. It is unlikely, however, that this was responsible for the increased pineal lipid in the tumour-bearing animals in the present experiments. The lipid content of the pineal glands of both male and female tumour-bearing rats was raised. Cyclical variations of the type found in females at different stages of the oestrous cycle do not appear to occur in males and it is unlikely therefore that the results were due to coincidental fluctuations of this nature. The lipid content of the gland also depends on the age of the animal 12 and on environmental lighting conditionsaL These factors were avoided in the present experiments by using tumour-bearing animals and controls of the same age and by killing them all at midday to avoid diurnal variations. Finally, there is evidence that the pineal lipid is implicated in the sodium regulating processes e f the body 8. It is impossible to be certain that the tumour-bearing animals did not have electrolytic imbalances or were not dehydrated, but this is unlikely as they were invariably killed before any effect was observed on eating or drinking habits of the animal. It does not appear, therefore, that any of the factors knownPto influence pineal lipids were important in the present experiments, and whilst the possibility that the greater amount of lipid in the pineal gland of the tumour-bearing rats is a nonspecific manifestation cannot be excluded, there is increasing evidence that accumulation of lipid indicates increased activity of the pineal gland. As a result of this Prop 1~ has suggested that pineal lipid content could be used as a parameter for pineal secretory activity. Supporting evidence for this has come more recently from studies with the electron microscope, in histochemical investigations 2 and from Basinska et al. 1 who found evidence of a correlation between pineal lipids and the physiological activity of the gland. It is clear therefore that whilst the exact significance of pineal lipid accumulation in the tumour-bearing rats requires further investigation, this finding does afford Brain Research, 37 (1972) 123-125
SHORT COMMUNICATIONS
125
indirect evidence that the pineal gland m a y be involved in the c o n t r o l o f m a l i g n a n c y in the rat. This work was supported by a g r a n t from the Research C o m m i t t e e of the Manchester Regional Hospital Board. Department of Pathology, University Hospital of South Manchester and the University of Manchester, Withington Hospital West Didsbury, Manchester (Great Britain)
MARIANNE HUXLEY E. TAPP
1 BASINSKA,J., SASTRY,P. S., AND STANCER,H. C., Lipid composition of human, bovine and sheep pineal glands, J. Neurochern., 16 (1969) 707-714. 2 BOSTELMANN,W., The ultrastructural and enzyme histochemical behaviour of the pineal gland of the rat after alteration of the functional phase by continuous illuminationand continuous darkness, Endokrinologie, 53 (1968) 365-384. 3 DAS GUPTA,T. K., ANDTERZ,J., Influence of pineal gland on the growth and spread of melanoma in the hamster, Cancer Res., 27 (1967) 1306-1311. 4 DOBROVOLSKAIA-ZAVASKAIA,N., ET ZEPHIROEF, P., Effet des produits d'origine 6piphysaire et d'origine h6patique sur la croissance des tumeurs chez la souris, C.R. Soc. Biol. (Paris), 134 (1940) 60-63. 5 ELFTMAN,H., Controlled chromation, J. Histochem. Cytochem., 2 (1954) 1-8. 6 ENGEL,P., Untersuchungen tiber die Wirkung der Zirbeldriise, Z. ges. exp. Med., 93 (1933) 69-78. 7 HOFSTATTER,R., Versuche der postoperativen Krebsbehandlung mit Zirbelstoffen, Krebsarzt, 14 (1959) 307-316. 8 HUNGERFORO,G. F., AND PANAGIOTIS,N. M., Response of pineal lipid to hormone imbalances, Endocrinology, 71 (1962) 936-942. 9 HUXLEY,M., AND TAPP, E., Unpublished observations. 10 KATAGIRI,E., Shoka-sen ni kan-sura kenkyu ni shuyo Z6shoku to shoka-sen (Studies on the pineal gland; turnout proliferation and the pineal gland), Osaka Igakkai Zasshi, 43 (1944) 315-320. 11 NAKATANI,M., OHARA,Y., KATAGIRI,E., AND NAKANO,K., Shoka-sen tekishutsu shi-sei shironezumi no kenkyu (Studies on pinealectomized white rats), Acta Soc. path. Jap., 30 (1940) 232-236. 12 PROP, N., Lipids in the pineal body of the rat. In J. ARIENSKAPPERSAND J. P. SCHADI~(Eds.), Structure and Function of the Epiphysis Cerehri, Vol. 10, Elsevier, Amsterdam, 1965, pp. 454-464. 13 QUAY, W. B., Reduction of mammalian pineal weight and lipid during continuous light, Gen. comp. Endocrinol., 1 (1961) 211-217. 14 RODIN,A. E., AND OVERALL,J., Statistical relationships of weight of the human pineal to age and malignancy, Cancer (Philad.), 20 (1967) 1203-1214. 15 TAPP, E., AND BLUMFIELD,M., The weight of the pineal gland in malignancy, Brit. J. Cancer, 24 (1970) 67-70. 16 ZWEENS, J., Influence of the oestrous cycle and ovariectomy on the phospholipid content of the pineal gland in the rat, Nature (Lond.), 197 (1963) 1114-1115. 17 ZWEENS,J., Alterations in pineal lipid content in the rat under hormonal influences. In J. ARIi~NS KAPPERS AND J. P. SCHADIS(Eds.), Structure and Function of the Epiphysis Cerebri, Vol. 10, Elsevier, Amsterdam, 1965, pp. 540-551.
(Accepted November 23rd, 1971)
Brain Research, 37 (1972) 123-125