- Email: [email protected]
Effect of undernutrition on cell generation in the rat hippocampus
186
Brain Research, 168 (1979) 186-189 ~ Elsevier/North-HollandBiomedicalPress
Effect of undernutrition on cell generation in the rat hippocampus
P. D. LEWIS, A. J. PATELand R. BAL/f,ZS Department of Histopathology, Royal Postgraduate Medical School, Hammersmith Hospital, London, WI2 0HS and ( A.J.P. and R.B.) M.R.C. Developmental Neurobiology Unit, Institute of Neurology, London, WCIN 2NS (U.K.)
(Accepted January 18th, 1979)
Undernutrition early in life is believed to have irreversible adverse effects on human brain functionS, 16. Investigation of possible mechanisms has revealed a vast array of biochemical and anatomical changes in experimentally undernourished animals4,L Amongst these, effects on cell acquisition in the postnatally developing brain have been shown and explored in some detail. Previous studies10,~1 have examined changes both in whole forebrain and cerebellum and in specific areas where replicating cells are concentrated, viz. the lateral ventricular subependymal layer and the cerebellar external granular layer. We here report the effects of undernutrition on a cell population, the granule cells of the hippocampus, that is largely acquired postnatally and has been intensively studied in recent years1,3,6,9,13,14. Porton rats were undernourished by halving the normal diet of mother rats from the sixth day of pregnancy onwardslL Groups of 16-24 undernourished animals aged 1, 6 and 12 days, together with groups of similarly aged control rats from normally fed mothers, were given an intrapcritoneal injection of [3H]thymidine (spec. radioact. 22 Ci/mmol, Radiochemical Centre, Amersham), 2.5 #Ci/g body weight, and killed by aortic perfusion fixation with formol-acetic acid (1 ~o glacial acetic acid in 10% neutral formalin) at various times between 1 and 32 h later. After 24 h postfixation in neutral formalin the cerebral hemispheres were bisected and 7 #m paraffin sections were cut at levels AA1 and BB1is, mounted on chrome gelatin slides and autoradiographs prepared, using Kodak AR-10 plates. Slides were developed in Kodak D19 after 3 weeks exposure, fixed and stained with Mayer's haemalum. The caudal (temporal) region of the dentate gyrus was examined under a × 100 oil immersion objective. Nuclei were scored as labelled if associated with more than 3 silver grains. In sections from 1- and 6-day-old animals killed 1 h after injection of [aH]thymidine, labelling indices were derived from counts of 800-1200 nuclei in the hilus, in an area distal to the termination of CA4, and adjacent to the granular layer and equidistant between its medial and lateral blades. Between 15 and 50 metaphases and anaphases were counted in the dentate hilus and granular layer of each animal, and the percentage of mitoses labelled (PLM) was plotted against time after injection of [SH]thymidinetL Data were analyzed by the method of BarretO as modified by Steel
187 TABLE I Effect of undernutrition (UN) on generation cycle in rat dentate gyrus
Median values (mean 4- S.D. in parentheses). All values in hours. Day l
Day 6
Control
Cell cycle time 15.1 Length of S-phase 10.1 (10.24-1.5) Length of Gz-phase 3.3 (3.44-0.7) Length of Gl-phase 1.1 (2.84-6.8)
Day 12
UN
Control
UN
Control
UN
19.5 15.4 (15.7±3.3) 3.8 (3.84-0.4) 0.1 (0.5 :t:2.9)
17.7 11.7 (12.1-t-3.1) 2.7 (2.8±0.6) 2.4 (3.84-4.7)
18.7 14.4 (14.64-2.6) 3.9 (4.0:[:1.1) 0.1 (0.7±8.3)
15.3 11.2 (11.7i3.7) 2.5 (2.54-0.6) 1.2 (1.6il.4)
24.9 18.8 (18.94-1.9) 5.5 (5.64-1.0) 0.1 (1.04-10.0)
and Hanes 15, in which computer-generated curves, based on independent, lognormally distributed values of G1, S and G2 are fitted to the experimental findings. In 12-day-old animals, the width of the dentate fascia was measured at its most caudal point under a ×25 objective in 10 animals in each group. Mitoses and labelled nuclei were observed in the dentate hilus and granular layer of both control and undernourished rats at all ages examined. At 1 and 6 days, treatment had no obvious effect of the anatomical development of the dentate gyrus. At 12 days, however, the granular layer of undernourished animals was reduced in thickness (maximally 6 cells thick, and measuring 66.5 4- 3.8 /~m in width) in comparison with controls (7 cells thick, and 77.8 4- 7.1 # m in width) (S.D.; P <
0.001). Cell cycle parameters derived from curves fitted by computer to percentage labelled mitoses data are given in Table I. It appears that undernutrition moderately prolongs cell cycle time and Gz, substantially lengthens the D N A synthesis phase, and severely curtails the G~ phase. These changes are maximal at 12 days.
TABLE II Effect of undernutrition on cell acquisition in rat dentate gyrus
Figures for labelling index are means 4- S.D., with numbers of animals in parentheses. Turnover time = S-phase duration/labelling indexlL
Day 1 Day 6
Labelling index (%)
Turnover time (h)
Cell acquisition rate ( %/24 h)
Control
UN
Control
UN
Control
UN
3.3 4- 0.5 (6) 4.0 4- 0.7 (6)
3.4 d- 0.8 (7) 3.7 4- 0.7 (5)
310 290
450 390
7.8 8.2
5.3 6.2
188 Labelling indices (L.I.) in the hilus of the dentate gyrus in 1- and 6-day-old rats at 1 h after injection of [ZH]thymidine, together with turnover times and fractional increase in cell numbers in 24 h are shown in Table II. It is apparent that both at day 1 and day 6, the effect of undernutrition is markedly to diminish the cell acquisition rate: the rate is reduced by 32 ~ at day 1 and 24)o at day 6. In their response to undernutrition, the proliferating cells studied in the dentate gyrus (which are taken in the main to be granule cell precursors 13) behave in the same way as those in other areas of the brain, for the reduced rate of cell acquisition and cell cycle changes parallel what has been previously observed in the forebrain subependymal layer and cerebellar external granular layer 10,11. An estimate for the accumulated deficit in hippocampal granule cell number at the end of the second postnatal week can be arrived at, assuming: (1) that undernutrition reduces cell acquisition by 25 ~ on average throughout the period under study (Table II), and (2) the data of Schlessinger et al. 13 for the daily fractional output of granule cells. The estimated deficit in cell number at 12 days, 18 ~, is in keeping both with the calculated reduction in total forebrain D N A content (cell number) at this age 11 and with the present observation of reduced granular layer width. Hippocampal lesions caused by a variety of means may produce lasting behavioural abnormalities 2, and a reduction of this order could significantly disturb the normal afferent and efferent relationships of the dentate gyrus and underlie some of the functional changes seen in undernutrition. The authors are grateful to Miss M. Lai for technical assistance and to Dr. G. G. Steel for computation of cell cycle parameters. This work is supported, in part, by a grant to P.D.L. from Action Research for the Crippled Child.
1 Altman, J., DNA metabolism and cell proliferation. In A. Lajtha (Ed.), Handbook of Neurochemistry, Vol. 2, Plenum Press, New York, 1969, pp. 137-182. 2 Altman, J., Brunner, R. L. and Bayer, S. A., The hippocampus and behavioural maturation, Behav. Biol., 8 (1973) 557-596. 3 Angevine,J. B., Time of neurone origin in the hippecampal region. An autoradiographic study in the mouse. Exp. Neurol., Suppl., 2 (1965) 1-70. 4 Bal~zs, R., Lewis, P. D. and Patel, A. J., Nutritional deficiencies and brain development. In F. Falkner and J. Tanner (Eds.), Human Growth, Vol. 3, Plenum Press, New York, 1978, pp. 415-480. 5 Barrett, J. C., A mathematical model of the mitotic cycle and its application to the interpretation of percentage labelled mitoses data, J. nat. Cancer Inst., 37 (1966) 443-450. 6 Bayer, S. A. and Altman, J., Hippocampal development in the rat: cytogenesis and morphogenesis examined with autoradiography and low-level x-irradiation, J. comp. Neurol., 158 (! 974) 55-80. 7 Dodge, P. R., Prensky, A. L. and Feigin, R. D., Nutrition and the Developing Nervous System, Mosby, St. Louis, 1975. 8 Hoorweg, J. and Stanfield, J. P., The effects of protein energy malnutrition on intellectual and motor abilities in later childhood and adolescence, Develop. Med. Child Neurol., 18 (1976) 330--350. 9 Lewis, P. D., Kinetics of cell proliferation in the postnatal rat dentate gyrus, Neuropath. appl. NeurobioL, 4 (1978) 191-195. 10 Lewis, P. D., Bahizs, R., Patel, A. J. and Johnson, A. L., The effect of undernutrition in early life on cell generation in the rat brain, Brahz Research, 83 (1975) 235-247.
189 11 Patel, A. J., Bai,-izs, R. and Johnson, A. L., Effect of undernutrition on cell formation in the rat brain, J. Neurochem., 20 (1972) 1151-1165. 12 Quastler, H. and Sherman, F. G., Cell population kinetics in the intestinal epithelium of the mouse, Exp. Cell Res., 17 (1959) 420--438. 13 Schlessinger, A. R., Cowan, W. M. and Gottlieb, D. I., An autoradiographic study of the time of origin and the pattern of granule cell migration in the dentate gyrus of the rat, J. comp. Neurol., 159 (1975) 149-176. 14 Schultze, B., Nowak, B. and Maurer, W., Cycle times of the neural epithelial cells of various types of neuron in the rat. An autoradiographic study, J. comp. Neurol., 158 (1974) 207-218. 15 Steel, G. G. and Hanes, S., The technique of labelled mitoses: analysis by automatic curve-fitting, Cell Tiss. Kinetics, 4 (1971) 93-105. 16 Stoch, M. B. and Smyth, P. M., 15-year developmental study on effects of severe undernutrition during infancy on subsequent physical growth and intellectual functioning, Arch. Dis. Childh., 51 (1976) 327-336. 17 Thrasher, J. D., Analysis of renewing epithelial cell populations. In D. M. Prescott (Ed.), Methods in Cell Physiology, Vol. 2., Academic Press, New York, 1966, pp. 323-357. 18 Zeman, W. and Innes, J. R. M., Craigie's Neuroanatomy of the Rat, Academic Press, New York, 1963, pp. 216--217.
Brain Research, 168 (1979) 186-189 ~ Elsevier/North-HollandBiomedicalPress
Effect of undernutrition on cell generation in the rat hippocampus
P. D. LEWIS, A. J. PATELand R. BAL/f,ZS Department of Histopathology, Royal Postgraduate Medical School, Hammersmith Hospital, London, WI2 0HS and ( A.J.P. and R.B.) M.R.C. Developmental Neurobiology Unit, Institute of Neurology, London, WCIN 2NS (U.K.)
(Accepted January 18th, 1979)
Undernutrition early in life is believed to have irreversible adverse effects on human brain functionS, 16. Investigation of possible mechanisms has revealed a vast array of biochemical and anatomical changes in experimentally undernourished animals4,L Amongst these, effects on cell acquisition in the postnatally developing brain have been shown and explored in some detail. Previous studies10,~1 have examined changes both in whole forebrain and cerebellum and in specific areas where replicating cells are concentrated, viz. the lateral ventricular subependymal layer and the cerebellar external granular layer. We here report the effects of undernutrition on a cell population, the granule cells of the hippocampus, that is largely acquired postnatally and has been intensively studied in recent years1,3,6,9,13,14. Porton rats were undernourished by halving the normal diet of mother rats from the sixth day of pregnancy onwardslL Groups of 16-24 undernourished animals aged 1, 6 and 12 days, together with groups of similarly aged control rats from normally fed mothers, were given an intrapcritoneal injection of [3H]thymidine (spec. radioact. 22 Ci/mmol, Radiochemical Centre, Amersham), 2.5 #Ci/g body weight, and killed by aortic perfusion fixation with formol-acetic acid (1 ~o glacial acetic acid in 10% neutral formalin) at various times between 1 and 32 h later. After 24 h postfixation in neutral formalin the cerebral hemispheres were bisected and 7 #m paraffin sections were cut at levels AA1 and BB1is, mounted on chrome gelatin slides and autoradiographs prepared, using Kodak AR-10 plates. Slides were developed in Kodak D19 after 3 weeks exposure, fixed and stained with Mayer's haemalum. The caudal (temporal) region of the dentate gyrus was examined under a × 100 oil immersion objective. Nuclei were scored as labelled if associated with more than 3 silver grains. In sections from 1- and 6-day-old animals killed 1 h after injection of [aH]thymidine, labelling indices were derived from counts of 800-1200 nuclei in the hilus, in an area distal to the termination of CA4, and adjacent to the granular layer and equidistant between its medial and lateral blades. Between 15 and 50 metaphases and anaphases were counted in the dentate hilus and granular layer of each animal, and the percentage of mitoses labelled (PLM) was plotted against time after injection of [SH]thymidinetL Data were analyzed by the method of BarretO as modified by Steel
187 TABLE I Effect of undernutrition (UN) on generation cycle in rat dentate gyrus
Median values (mean 4- S.D. in parentheses). All values in hours. Day l
Day 6
Control
Cell cycle time 15.1 Length of S-phase 10.1 (10.24-1.5) Length of Gz-phase 3.3 (3.44-0.7) Length of Gl-phase 1.1 (2.84-6.8)
Day 12
UN
Control
UN
Control
UN
19.5 15.4 (15.7±3.3) 3.8 (3.84-0.4) 0.1 (0.5 :t:2.9)
17.7 11.7 (12.1-t-3.1) 2.7 (2.8±0.6) 2.4 (3.84-4.7)
18.7 14.4 (14.64-2.6) 3.9 (4.0:[:1.1) 0.1 (0.7±8.3)
15.3 11.2 (11.7i3.7) 2.5 (2.54-0.6) 1.2 (1.6il.4)
24.9 18.8 (18.94-1.9) 5.5 (5.64-1.0) 0.1 (1.04-10.0)
and Hanes 15, in which computer-generated curves, based on independent, lognormally distributed values of G1, S and G2 are fitted to the experimental findings. In 12-day-old animals, the width of the dentate fascia was measured at its most caudal point under a ×25 objective in 10 animals in each group. Mitoses and labelled nuclei were observed in the dentate hilus and granular layer of both control and undernourished rats at all ages examined. At 1 and 6 days, treatment had no obvious effect of the anatomical development of the dentate gyrus. At 12 days, however, the granular layer of undernourished animals was reduced in thickness (maximally 6 cells thick, and measuring 66.5 4- 3.8 /~m in width) in comparison with controls (7 cells thick, and 77.8 4- 7.1 # m in width) (S.D.; P <
0.001). Cell cycle parameters derived from curves fitted by computer to percentage labelled mitoses data are given in Table I. It appears that undernutrition moderately prolongs cell cycle time and Gz, substantially lengthens the D N A synthesis phase, and severely curtails the G~ phase. These changes are maximal at 12 days.
TABLE II Effect of undernutrition on cell acquisition in rat dentate gyrus
Figures for labelling index are means 4- S.D., with numbers of animals in parentheses. Turnover time = S-phase duration/labelling indexlL
Day 1 Day 6
Labelling index (%)
Turnover time (h)
Cell acquisition rate ( %/24 h)
Control
UN
Control
UN
Control
UN
3.3 4- 0.5 (6) 4.0 4- 0.7 (6)
3.4 d- 0.8 (7) 3.7 4- 0.7 (5)
310 290
450 390
7.8 8.2
5.3 6.2
188 Labelling indices (L.I.) in the hilus of the dentate gyrus in 1- and 6-day-old rats at 1 h after injection of [ZH]thymidine, together with turnover times and fractional increase in cell numbers in 24 h are shown in Table II. It is apparent that both at day 1 and day 6, the effect of undernutrition is markedly to diminish the cell acquisition rate: the rate is reduced by 32 ~ at day 1 and 24)o at day 6. In their response to undernutrition, the proliferating cells studied in the dentate gyrus (which are taken in the main to be granule cell precursors 13) behave in the same way as those in other areas of the brain, for the reduced rate of cell acquisition and cell cycle changes parallel what has been previously observed in the forebrain subependymal layer and cerebellar external granular layer 10,11. An estimate for the accumulated deficit in hippocampal granule cell number at the end of the second postnatal week can be arrived at, assuming: (1) that undernutrition reduces cell acquisition by 25 ~ on average throughout the period under study (Table II), and (2) the data of Schlessinger et al. 13 for the daily fractional output of granule cells. The estimated deficit in cell number at 12 days, 18 ~, is in keeping both with the calculated reduction in total forebrain D N A content (cell number) at this age 11 and with the present observation of reduced granular layer width. Hippocampal lesions caused by a variety of means may produce lasting behavioural abnormalities 2, and a reduction of this order could significantly disturb the normal afferent and efferent relationships of the dentate gyrus and underlie some of the functional changes seen in undernutrition. The authors are grateful to Miss M. Lai for technical assistance and to Dr. G. G. Steel for computation of cell cycle parameters. This work is supported, in part, by a grant to P.D.L. from Action Research for the Crippled Child.
1 Altman, J., DNA metabolism and cell proliferation. In A. Lajtha (Ed.), Handbook of Neurochemistry, Vol. 2, Plenum Press, New York, 1969, pp. 137-182. 2 Altman, J., Brunner, R. L. and Bayer, S. A., The hippocampus and behavioural maturation, Behav. Biol., 8 (1973) 557-596. 3 Angevine,J. B., Time of neurone origin in the hippecampal region. An autoradiographic study in the mouse. Exp. Neurol., Suppl., 2 (1965) 1-70. 4 Bal~zs, R., Lewis, P. D. and Patel, A. J., Nutritional deficiencies and brain development. In F. Falkner and J. Tanner (Eds.), Human Growth, Vol. 3, Plenum Press, New York, 1978, pp. 415-480. 5 Barrett, J. C., A mathematical model of the mitotic cycle and its application to the interpretation of percentage labelled mitoses data, J. nat. Cancer Inst., 37 (1966) 443-450. 6 Bayer, S. A. and Altman, J., Hippocampal development in the rat: cytogenesis and morphogenesis examined with autoradiography and low-level x-irradiation, J. comp. Neurol., 158 (! 974) 55-80. 7 Dodge, P. R., Prensky, A. L. and Feigin, R. D., Nutrition and the Developing Nervous System, Mosby, St. Louis, 1975. 8 Hoorweg, J. and Stanfield, J. P., The effects of protein energy malnutrition on intellectual and motor abilities in later childhood and adolescence, Develop. Med. Child Neurol., 18 (1976) 330--350. 9 Lewis, P. D., Kinetics of cell proliferation in the postnatal rat dentate gyrus, Neuropath. appl. NeurobioL, 4 (1978) 191-195. 10 Lewis, P. D., Bahizs, R., Patel, A. J. and Johnson, A. L., The effect of undernutrition in early life on cell generation in the rat brain, Brahz Research, 83 (1975) 235-247.
189 11 Patel, A. J., Bai,-izs, R. and Johnson, A. L., Effect of undernutrition on cell formation in the rat brain, J. Neurochem., 20 (1972) 1151-1165. 12 Quastler, H. and Sherman, F. G., Cell population kinetics in the intestinal epithelium of the mouse, Exp. Cell Res., 17 (1959) 420--438. 13 Schlessinger, A. R., Cowan, W. M. and Gottlieb, D. I., An autoradiographic study of the time of origin and the pattern of granule cell migration in the dentate gyrus of the rat, J. comp. Neurol., 159 (1975) 149-176. 14 Schultze, B., Nowak, B. and Maurer, W., Cycle times of the neural epithelial cells of various types of neuron in the rat. An autoradiographic study, J. comp. Neurol., 158 (1974) 207-218. 15 Steel, G. G. and Hanes, S., The technique of labelled mitoses: analysis by automatic curve-fitting, Cell Tiss. Kinetics, 4 (1971) 93-105. 16 Stoch, M. B. and Smyth, P. M., 15-year developmental study on effects of severe undernutrition during infancy on subsequent physical growth and intellectual functioning, Arch. Dis. Childh., 51 (1976) 327-336. 17 Thrasher, J. D., Analysis of renewing epithelial cell populations. In D. M. Prescott (Ed.), Methods in Cell Physiology, Vol. 2., Academic Press, New York, 1966, pp. 323-357. 18 Zeman, W. and Innes, J. R. M., Craigie's Neuroanatomy of the Rat, Academic Press, New York, 1963, pp. 216--217.