171
Archives Internationales de Physiologie, de Biochimie et de Biophysique, 1992, 100, 17 1- 173
Requ le 6 aoct 1991.
Influence of thyroid hormone deficiency in DNA contents in different areas of adult rat brain. BY
A. SERRANO-LOZANO, P. MORATA, M. MONTIEL, M. T. MIRANDA (') and M. MORELL
Archives of Physiology and Biochemistry Downloaded from informahealthcare.com by V U L Periodicals Rec on 12/29/14 For personal use only.
[Departamentode Bioquimica y Biologia Molecular, ( I ) Catedra de Bioestadktica, Facultad de Medicina, Universidad de Malaga, Malaga, Spain]
We have studied DNA concentrations in various regions of the adult rat brain in prolonged hypothyrodism of different origin. In both normal and hypothyroid rats hypophysis, cerebellum and hypothalamus contained the highest concentrations of DNA. Hypothyroidism produced a slight decrease in DNA content but this effect varied according to the different regions. These variations were due to changes in their maturation period, a critical and specific target for thyroid hormone action after birth.
Introduction
Materials and Methods
Thyroid hormones deficiency modify the development of the nervous system : growth retardation which modifies tissue respiration, cells formation rate, DNA, protein synthesis and enzymatic system maturation (EAYRS, 1971; BALMS et al., 1975; FORD & CRAMER, 1977; MORREALE DE ESCOBAR & ESCOBAR DEL REY, 1980; Mc INTOSH et al., 1981). The tissue DNA concentration is an indication of the cell number, while the ratio of protein/DNA provides an index of cell size; so DNA content represents a formation and destruction cell balance (POTTER et al., 1986). Numerous studies of the development patterns measuring changes in weight, DNA, RNA and protein contents indicate that cell maturation is permanently affected in the hypothyroid brain. Those regions which mature late are most severely affected by early hypothyroidism. Furthermore these regions : cerebellum, hippocampus and olfatory bulb, show significant postnatal neurogenesis : thyroid hormone deficiency leads to a decrease in the rate of cell acquisition. It also provokes a lengthening of the proliferative phase in the germinal zones of the brain (LEGRAND, 1985). Therefore, the thyroid hormones induce different effects on the various regions of the brain, modifying their morphology (MEISAMI, 1983). We have studied tissue DNA concentrations in different areas of the adult brain under prolonged pharmacological hypothyroidism (methimazole and propylthiouracil), low iodine diet and surgical thyroidectomy. The whole brain and the following regions were analyzed :cortex, cerebellum, brain-stem, hypothalamus, hypophysis.
Male Wistar rats, fed on a standard laboratory diet and tap water ad libitum, were used. All the animals were weaned on day 21, the treatments beginning on day 28, and killed by decapitation 90 days after birth.
Experimental groups Six groups and 2 subgroups were studied (1) Euthyroid rats : Control (C). (2) Propylthiouracil (PTU) : 0.05% (wt/vol), administered in the drinking water. (3) Methimazole (MMI) : 2-methylmercaptoimidazole 0.02% (wthol), was administered in the drinking water. (4) Low iodine diet (LID). Rats were given 1% KCIOs as drinking water and fed with a low iodine diet (containing 25 pg iodine/kg diet PANLAB Spain) for 3 days after which no KC10, was given. The rats were divided into two groups : (4a) Receiving LID and distilied water until completion of the experiment. (4b) Control low iodine diet (LID + I). Rats were fed on the same diet and distilled water containing KI at 0.65 pg/ml. (5) Thyroidectomy (Tx):Animals were thyroidectomized swgically on day 28 after birth, followed by an injection of 100 pCi l3II after 7 days, in order to ensure complete removal of any remaining thyroid tissue et al., 1981). (OBREGON (6) Control thyroidectomy (CTx) : Rats of this group underwent sham-operation using the same surgical manipulations without removing the thyroid gland. After 7 days, each animal was injected i.p. with 100 pl of phosphate buffered saline solution, containing 1 mg/ml of sodium thiosulphate.
172
A.
SERRANO-LOZANO, P. MORATA, M. MONTJEL, M.
T. MIRANDA AND M. MORELL
Archives of Physiology and Biochemistry Downloaded from informahealthcare.com by V U L Periodicals Rec on 12/29/14 For personal use only.
Hornogenate preparation. Animals were decapitated between 10-12 a.m.. The brains were rapidly removed and weighed. Areas were dissected and individually weighed at 0-4 "C. Tissues were homogenized in 10 vol50 mM. Tris-HC1, pH 7.4 with a motor-driven Teflon pestle. Serum was obtained from blood collected at the killing time by centrifugation at lo00 x g for 20 min and stored at -40°C for T, and T4 determination. Total T, and T, in serum were determined by RIA after extraction with ethanol using the method described by et al. (1979). TAKAISHIet al. (1978) and OBREGON Deoxyribonucleic acid (DNA) content was determined using the method of BURTON(1968).
Statistical analysis The results are expressed as mean k SEM. The levels of statistical significance were analyzed by a Student t-test. Results
T, and T, serum levels and brain weights were used for screening hypothyroid state. Brain weights in the different groups are shown Table I. Both pharmacological induced (MMI and PTU) and surgical hypothyroidisms produced a significant decrease in brain weight as compared to the controls. However no difference was observed after low iodine diet. TABLE I. Whole brain weight, (T,) and (T,) serum levels of controls and hypothyroid rats. T, &g/lOO ml) T, (ng/lOO ml C PTU MMI LID + I LID CTx Tx
1.84f 0.02 1.68k 0.01* 1.70k 0.02* 1.83 f 0.02 1.81 f 0.02 1.80f 0.02 1.60f0.01*
3.01 k0.22 0.90+0.08* 0.73 fO.lO* 3.17 k0.12 1.24k0.05* 4.69k 0.24 1.09f 0.10*
24.83k2.18 1.91 f0.35* 9.83 f 1.35* 31.58f2.54 48.08k 6.35* 26.75 f I .47 4.91 k 1.49*
I
The results are means k SEM of twelve experiments. * In comparison with control animals, P
Archives Internationales de Physiologie, de Biochimie et de Biophysique, 1992, 100, 17 1- 173
Requ le 6 aoct 1991.
Influence of thyroid hormone deficiency in DNA contents in different areas of adult rat brain. BY
A. SERRANO-LOZANO, P. MORATA, M. MONTIEL, M. T. MIRANDA (') and M. MORELL
Archives of Physiology and Biochemistry Downloaded from informahealthcare.com by V U L Periodicals Rec on 12/29/14 For personal use only.
[Departamentode Bioquimica y Biologia Molecular, ( I ) Catedra de Bioestadktica, Facultad de Medicina, Universidad de Malaga, Malaga, Spain]
We have studied DNA concentrations in various regions of the adult rat brain in prolonged hypothyrodism of different origin. In both normal and hypothyroid rats hypophysis, cerebellum and hypothalamus contained the highest concentrations of DNA. Hypothyroidism produced a slight decrease in DNA content but this effect varied according to the different regions. These variations were due to changes in their maturation period, a critical and specific target for thyroid hormone action after birth.
Introduction
Materials and Methods
Thyroid hormones deficiency modify the development of the nervous system : growth retardation which modifies tissue respiration, cells formation rate, DNA, protein synthesis and enzymatic system maturation (EAYRS, 1971; BALMS et al., 1975; FORD & CRAMER, 1977; MORREALE DE ESCOBAR & ESCOBAR DEL REY, 1980; Mc INTOSH et al., 1981). The tissue DNA concentration is an indication of the cell number, while the ratio of protein/DNA provides an index of cell size; so DNA content represents a formation and destruction cell balance (POTTER et al., 1986). Numerous studies of the development patterns measuring changes in weight, DNA, RNA and protein contents indicate that cell maturation is permanently affected in the hypothyroid brain. Those regions which mature late are most severely affected by early hypothyroidism. Furthermore these regions : cerebellum, hippocampus and olfatory bulb, show significant postnatal neurogenesis : thyroid hormone deficiency leads to a decrease in the rate of cell acquisition. It also provokes a lengthening of the proliferative phase in the germinal zones of the brain (LEGRAND, 1985). Therefore, the thyroid hormones induce different effects on the various regions of the brain, modifying their morphology (MEISAMI, 1983). We have studied tissue DNA concentrations in different areas of the adult brain under prolonged pharmacological hypothyroidism (methimazole and propylthiouracil), low iodine diet and surgical thyroidectomy. The whole brain and the following regions were analyzed :cortex, cerebellum, brain-stem, hypothalamus, hypophysis.
Male Wistar rats, fed on a standard laboratory diet and tap water ad libitum, were used. All the animals were weaned on day 21, the treatments beginning on day 28, and killed by decapitation 90 days after birth.
Experimental groups Six groups and 2 subgroups were studied (1) Euthyroid rats : Control (C). (2) Propylthiouracil (PTU) : 0.05% (wt/vol), administered in the drinking water. (3) Methimazole (MMI) : 2-methylmercaptoimidazole 0.02% (wthol), was administered in the drinking water. (4) Low iodine diet (LID). Rats were given 1% KCIOs as drinking water and fed with a low iodine diet (containing 25 pg iodine/kg diet PANLAB Spain) for 3 days after which no KC10, was given. The rats were divided into two groups : (4a) Receiving LID and distilied water until completion of the experiment. (4b) Control low iodine diet (LID + I). Rats were fed on the same diet and distilled water containing KI at 0.65 pg/ml. (5) Thyroidectomy (Tx):Animals were thyroidectomized swgically on day 28 after birth, followed by an injection of 100 pCi l3II after 7 days, in order to ensure complete removal of any remaining thyroid tissue et al., 1981). (OBREGON (6) Control thyroidectomy (CTx) : Rats of this group underwent sham-operation using the same surgical manipulations without removing the thyroid gland. After 7 days, each animal was injected i.p. with 100 pl of phosphate buffered saline solution, containing 1 mg/ml of sodium thiosulphate.
172
A.
SERRANO-LOZANO, P. MORATA, M. MONTJEL, M.
T. MIRANDA AND M. MORELL
Archives of Physiology and Biochemistry Downloaded from informahealthcare.com by V U L Periodicals Rec on 12/29/14 For personal use only.
Hornogenate preparation. Animals were decapitated between 10-12 a.m.. The brains were rapidly removed and weighed. Areas were dissected and individually weighed at 0-4 "C. Tissues were homogenized in 10 vol50 mM. Tris-HC1, pH 7.4 with a motor-driven Teflon pestle. Serum was obtained from blood collected at the killing time by centrifugation at lo00 x g for 20 min and stored at -40°C for T, and T4 determination. Total T, and T, in serum were determined by RIA after extraction with ethanol using the method described by et al. (1979). TAKAISHIet al. (1978) and OBREGON Deoxyribonucleic acid (DNA) content was determined using the method of BURTON(1968).
Statistical analysis The results are expressed as mean k SEM. The levels of statistical significance were analyzed by a Student t-test. Results
T, and T, serum levels and brain weights were used for screening hypothyroid state. Brain weights in the different groups are shown Table I. Both pharmacological induced (MMI and PTU) and surgical hypothyroidisms produced a significant decrease in brain weight as compared to the controls. However no difference was observed after low iodine diet. TABLE I. Whole brain weight, (T,) and (T,) serum levels of controls and hypothyroid rats. T, &g/lOO ml) T, (ng/lOO ml C PTU MMI LID + I LID CTx Tx
1.84f 0.02 1.68k 0.01* 1.70k 0.02* 1.83 f 0.02 1.81 f 0.02 1.80f 0.02 1.60f0.01*
3.01 k0.22 0.90+0.08* 0.73 fO.lO* 3.17 k0.12 1.24k0.05* 4.69k 0.24 1.09f 0.10*
24.83k2.18 1.91 f0.35* 9.83 f 1.35* 31.58f2.54 48.08k 6.35* 26.75 f I .47 4.91 k 1.49*
I
The results are means k SEM of twelve experiments. * In comparison with control animals, P
