The effect of undernutrition in early life on cell generation in the rat brain

235

Brain Research, 83 (1975) 235-247 © Elsevier Scientific Publishing Company, Amsterdam - Printed in The Netherlands

THE EFFECT OF U N D E R N U T R I T I O N GENERATION IN THE RAT BRAIN

IN

EARLY

LIFE

ON

CELL

P. D. LEWIS, R. BAL/kZS, A. J. PATEL AND A. L. JOHNSON

Department of Histopathology, Royal Postgraduate Medical School, Hammersmith Hospital, London W. 12, Medical Research Council Neuropsychiatry Unit, Woodmansterne Road, Carshalton, Surrey and Medical Research Council Statistical Research and Services Unit, University College Hospital Medical School, London WC1E 6AS (Great Britain) (Accepted September 13th, 1974)

SUMMARY

In undernourished rats aged up to 21 days, the DNA synthesis period in dividing cells of the subependymal and external granular layers is consistently and markedly prolonged, while rates of cell production from these layers are only slightly altered. Cell cycle times are unchanged up to the end of the first week of life and prolonged from day 12. The G1 phase is markedly shortened at 1, 6 and 12 days of age. It would appear that, in comparison with controls, disappearance of the external granular layer is delayed, and cell numbers in both germinal layers may be reduced.

INTRODUCTION

Evidence is accumulating that malnutrition in early life may have a prolonged effect on the human brain. The interpretation of available data is beset with great difficulties; nevertheless, careful studies clearly suggest that the intellectual performance of children who were malnourished in early life may be permanently impaired la,~0,28. The biological mechanisms underlying such a phenomenon are amenable to study in animal experiments. Several laboratories have shown undernutrition to have significant adverse effects on the functional development of the brain, in association with retardation of morphological and biochemical maturation 4,9,a°. It has been observed that cell numbers are permanently reduced in the brains of rats and mice which were underfed during the suckling period 14,2x,a°. In the rat, cells formed after birth account for about 50 9/0 and 97 ~o of the total cell number in the forebrain and cerebellum, respectively4. Although glial cell formation predominates in the postnatal period, neurogenesis is significant in certain parts of the brain including the hippocampus, the olfactory lobes and especially the cerebellum, where most of the

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v.D. LEWISet al.

interneurones are formed during this period 1. Thus these cells might be vulnerable to nutritional deprivation in the first weeks of life. Our recent studies zl on cell numbers in the brains of undernourished rats confirmed previous observations14, 3°. However, it was found that during the period of most active cell proliferation in the postnatal brain (the first 3 weeks of life), the cell number in undernourished rats was only moderately reduced in comparison with controls, whereas the rate of labelled thymidine incorporation into DNA was much more severely depressed 21. We have suggested some possible explanations for this apparent discrepancy 4. The present experiments were aimed to test one of the hypotheses. When a relatively long period - - such as the first 3 weeks of life - - is considered, the rate of cell acquisition in an asynchronously replicating cell population is related to the doubling time of the proliferating cells, whereas the rate of incorporation of a precursor into DNA is primarily a function of the length of the DNAsynthesis phase (S-phase). Thus, if the S-phase were prolonged by undernutrition to a greater extent than the doubling time, the rate of DNA synthesis would be more depressed than the rate of cell acquisition. We have measured the parameters of cell division in the germinal layers of the brain of undernourished rats in an attempt to investigate this possibility. METHODS

Porton rats were undernourished by halving the normal diet of mother rats from the 6th day of pregnancy continuing through lactation 21. Groups of 16 undernourished animals aged 1, 6, 12 and 21 days, together with groups of similarly aged control rats from normally fed mothers, were given an intra-peritoneal injection of 2.5/~Ci/g body wt [3H]thymidine (specific radioactivity 22 Ci/mmole, Radiochemical Centre, Amersham). Altogether 128 animals were thus studied. At various times between 1 and 32 h after the injection of [~H]thymidine, rats were killed under chloroform anaesthesia by aortic perfusion-fixation with formolacetic acid (1 ~ glacial acetic acid in 10 ~ neutral formalinT). After 24-h postfixation in neutral formalin, brains were cut through the mesencephalon, the cerebrum bisected horizontally through the lateral ventricles and the cerebellum and pons transected. Paraffin sections of cerebral hemispheres at levels AA1 and BB131 and of cerebellum and midpons were cut at 7/zm, mounted on chrome-gelatin slides, and autoradiographs prepared, using Kodak AR-10 plates. After exposure for 3 weeks at 4 °C in light-tight boxes containing desiccant, slides were developed for 5 min at 20 °C in Kodak D-19, fixed, and stained with Mayer's haemalum. Duplicate nonautoradiographic sections were stained with haematoxylin and eosin. The external granular layer of the cerebellum and the subependymal zone in the anterior region of the lateral ventricles were examined under a × 100 oil-immersion objective. Mitotic indices were obtained by counting 1000 cells in haematoxylin and eosin stained sections of each germinal layer in 10 brains of animats in each age group. In autoradiographs, nuclei were scored as labelled if associated with more than 3 silver grains. Labelling indices were derived from counts of 1000 nuclei in animals killed 1 h

UNDERNUTRITION AND RAT BRAIN CELL GENERATION

237

after injection of [aH]thymidine. Between 80 and 150 mitoses (metaphases and anaphases) were counted in each germinal layer of each brain and percentages of labelled mitoses plotted against time2L

Mathematical methods Mitotic indices were subjected to analysis of variance. The data were such that the standard deviation within a subclass was proportional to the mean. Consequently the raw data were transformed by taking square roots (after multiplication by a factor of 10) to stabilise the subclass variances. The transformed data were then analysed as a 3-factor (age x brain region x treatment) factorial experiment with replication. Percentage labelled mitoses data were analysed by the method of Barrett 5 as developed by Steel and Hanes 27, in which computer-generated curves, based on independent, log-normally distributed values of G1, S and G~ are fitted to experimental findings. The estimates obtained from the computer analysis of cell cycle time, length of S-phase and length of G2-phase, were analysed separately without transformation. The estimate of length of Gl-phase was transformed logarithmically before analysis (after multiplication by a factor of 10). In each case the data were analysed as a 3factor (age x brain region x treatment) factorial experiment with one observation per subclass. Since there was no independent estimate of residual variation, the sums of squares in the analysis of variance for all interactions involving 'brain region' were summed and the resultant mean square with 7 degrees of freedom (df) was taken as an estimate of residual variation. Further, since the main effect of 'brain region' was not significant in any of the analyses, the data for the cerebellum and cerebrum were combined in the 'treatment' comparisons given in Table III. RESULTS

Mitotic activity (Fig. 1) The mitotic index in the external granular layer fell with age throughout the period studied in both control and treated rats. At this germinal site, the only difference, compared with controls, was the higher mitotic activity (P < 0.001) in the 21day-old undernourished rats. This finding was associated with a retarded disappearance of the external granular layer, for in control animals aged 21-22 days this layer was only one cell thick and discontinuous, while in undernourished rats it was uniformly continuous and 2-4 cells in thickness. In the cerebral subependymal layer of undernourished animals the mitotic index was consistently less than in controls and the difference between the two age curves was significant (P < 0.05).

A utoradiographic findings The percentages of mitoses labelled at different times after injection of [aH]thymidine into control and undernourished rats aged 1, 6, 12 and 21 days are shown in Figs. 2 and 3, and computer-generated curves are superimposed on the observed

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points. The parameters of these curves are set out in Tables I and II; the results o f the statistical analysis are given in Table III. The data showed that in both the external granular and the subependymal layer undernutrition consistently prolonged the D N A synthesis (S) phase, The prolongation was 28-79 % in external granular layer and 13-86 % in subependymal layer, depending on age. Further, values for undernourished animals were over 12 h, while all but one control values (21 days subependymal layer: 12.4 h) were under 12 h. The median values of the G~-phase were also consistently larger in the dividing cells o f the undernourished brain than in controls, the subependymal layer o f 21-day-old rats again be ing the exception. The Gt-phase was markedly shortened by undernutrition in rats younger than 21 days old, and it was virtually eliminated at days 6-7 and 12-13. Cell cycle times were unaffected by undernutrition up to the end of the first week of life, and prolonged thereafter. This prolongation, as well as changes observed in other parameters, appeared to be maximal at days 12-13.

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242 TABLE 111

COMPARISON OF LENGTHS OF CELL CYCLE PHASES 1N CONTROL AND UNDERNOURISHED ( U N ) RATS (DATA FOR CEREBRUM AND CEREBELLUM COMBINED)

Estimate

Treatment

Length of S-phase (S) Length of Gl-phase (GI) Length of Gz-phase (G~) Total cell cycle time (T)

Control UN Control UN Control UN Control UN

Age group (days) 1-2

6-7

12-13

21-22

9.6 13.1'* 5.7 1.3" 3.3 4.3 18.7 20.1

10.2 14.1"* 3.4 0.2*** 2.1 2.8 16.8 17.4

10.8 19.7'** 2.9 0.2** 1.5 3.6' 16.1 23.9**

11.1 13.2 5.0 5.7 2.1 2.4 18.7 21.9

Residual variation§

0.83 0.06§§ 0.50 1.74

§ With 7 degrees of freedom for each estimate (see Methods). §§ Residual variation refers to logarithmically transformed data. Significance of difference between UN and corresponding control : * P -< 0.05, ** P -< 0.01, * * * P < 0.001.

TABLE IV EFFECT OF UNDERNUTRITION ON CELL FORMATION IN THE CEREBELLAR EXTERNAL GRANULAR LAYER

Day 1 Day 6 Day 12 Day 21

Labelling index (%)

Doubling time (h)

Cell acquisition rate (°/o/day)

Growth fraction

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Control

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0.46 0.31 0.36 0.35

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Growth fraction

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79 111 118 105

83 98 140 101

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UNDERNUTRITION AND RAT BRAIN CELL GENERATION

243

Labelling indices in the external granular layer and subependymal layer 1 h after injection of [SH]thymidine are shown in Tables IV and V. From these indices, potential doubling times for the dividing cells in the germinal layer can be calculated by assuming that their population is in a steady state, according to the formula: doubling time = (100 × length of S-phase)/labelling index; growth fractions (cell cycle time/doubling time) can in turn be derived. The results indicated that in the case of the external granular layer, undernutrition consistently prolonged doubling times in rats aged I, 6 and 12 days, with a moderate fall in the rate of cell acquisition (expressed as a fraction of the dividing cell population). In the case of the subependymal layer, undernutrition has no clear cut effects on cell acquisition rates. DISCUSSION

Comparison of mitotic indices in control and undernourished animals shows certain differences between the two groups. The finding that in the external granular layer of 21-day-old experimental rats both the proportion of mitotic cells and the numbers of proliferating cells were increased suggests that undernutrition may have resulted in a retardation of cerebellar maturation. Rebi~re and Legrand z4 have also observed that there is a tendency in the cerebellum of undernourished animals for the persistence of this germinal site for a longer time than in controls. However, it would appear that this effect is slight in comparison with that seen in neonatally-induced thyroid deficiency11,15. There is evidence indicating that undernutrition may lead to a decrease in thyroid function 19, although the situation may be different in chronic experiments as in the present studyl6, 28. It is perhaps more likely that the observed changes in the external granular layer are non-specific, for the cerebellum of young mammals seems to respond similarly to other insults, including treatment with corticosteroids for a short period after birth 8, and surgical deafferentation 12. The other statistically significant difference between mitotic indices of control and undernourished rats was observed in the subependymal layer. Here, the relatively small reduction in mitotic indices in undernourished animals, throughout the period studied, is consistent with the mild decrease in the rate of cell acquisition in the forebrain reported by Patel et al. 2t. Data from percentage labelled mitoses (PLM) curves show that undernutrition in early life caused a marked prolongation of DNA synthesis time, and thus slowing of the DNA synthesis rate, in the cells of the germinal layers of the brain (Figs. 2 and 3 and Tables I and II). Parallel biochemical studies (Patel, Bal6.zs and Lewis, to be published) indicate that the effect cannot be accounted for by an altered availability of DNA-precursors. At day 12, when the prolongation of S-phase was most pronounced in the undernourished brain, the concentration of acid-soluble ~4C diminished rapidly, in both the control and experimental animals, after a subcutaneous injection of [2-t4C]thymidine. Although, in comparison with controls, at 0.5-2 h after the injection the decay curve in the undernourished rats was displaced to the right along the

244

e.D. LEWISe t a / .

time axis by about 0.5 h, the acid-soluble 14C concentrations were similar from 3 h onwards. The labelling of DNA reached a plateau in both control and experimental animals by 2 h after the injection of [14C]thymidine. Thus, the effect of prolonged availability of DNA-precursor on the first wave of the PLM curve could be a lengthening by 0.5 h at most, which, compared with the observed prolongation of the S-phase, would be insignificant. It has been observed previously that food deprivation also results in slowing of D N A synthesis in organs other than brain 14,17,29. For example, Wiebecke et al. 29 have reported that, in comparison with controls, the length o f the S-phase is doubled in the intestinal mucosa of starved mice, but, in contrast to the present observations on the brain, the duration of the cell cycle is increased to an even greater extent. In developing organs the reduction in the acquisition of cells is usually more severe than that observed in the brain 14,17, implying that in these organs undernutrition effected a marked prolongation of both the cell cycle time and the DNA synthesis period. The finding that the prolongation of the cell cycle time in the brain was much less than that of the S-phase, was due to a marked shortening of the length of the G~-phase of the cell cycle in undernourished rats aged 1, 6 and 12 days. While the functional significance of the G1-phase is unknown (cf. ref. 18), it is tempting to speculate that the virtual elimination of this phase might have some effect on the progeny of the dividing cells. Whether or not the retarded biochemical maturation4,22, 25, various persistent physical changes 6 in the undernourished brain, and defective behavioural patterns2, 9 can ultimately be linked to our findings is a matter of conjecture. The rates of cell acquisition were calculated from the observed labelling indices and the calculated D N A synthesis times (Tables IV and V). Both estimates are subject to error if, in comparison with control, more DNA-precursor is available and for a longer time in the undernourished brain. As discussed above, the influence of this factor on the computed S-phase length is slight, and it would appear that the effect on the labelling indices, which are estimated 1 h after the injection of labelled thymidine, is also small. An increase in the concentration of labelled DNA-precursors during that period would result in a rise in the radioactivity content of DNA produced per cell, rather than in an increase in the number of cells labelled. The latter would only apply to that relatively small fraction of cells which in the control were synthesizing DNA for too short a time to accumulate enough radioactivity to be counted under the experimental conditions (i.e. during the 1-h period of exposure to labelled precursor, these cells either entered into S-phase too late or were too near to the termination of D N A synthesis). It was observed that the rate of cell acquisition was normal in the subependymal layer (Table V) and it was only slightly reduced in the external granular layer (Table IV). At first sight, the marked depression of D N A synthesis rates (Tables I-I!I), without a comparable effect on cell acquisition rates (Tables IV and V) offers an immediate explanation for the 'discrepant' biochemical findings in the undernourished brain 2~ referred to earlier. However, on reflection the situation is more complicated. The slowing of D N A acquisition in the undernourished brain (Fig. 1 of Patel et al. 21)

UNDERNUTRITION AND RAT BRAIN CELL GENERATION

245

implies - - in the face of more or less unchanged germinal cell doubling times - - that numbers of germinal cells are reduced. Using both biochemical and autoradiographic data, it is possible to estimate approximately the fraction of cells in the cerebrum and cerebellum which belong to the germinal layers, and thus to evaluate whether or not undernutrition did in fact result in lower than normal cell numbers in these zones. If it is assumed, for the sake of such a calculation, that in the forebrain cell proliferation is confined to the subependymal layer, then the fraction of total cells resident in this layer can be computed from the slopes of the cerebral DNA content curves at various ages (Fig. 1 of Patel et al. 21) and the germinal cell doubling times (Table V). This calculation inevitably leads to an overestimation of this fraction, since mitotic figures and labelled nuclei are not infrequently encountered away from the subependymal layer. For control rats aged 1, 6 and 12 days the total cerebral DNA doubling times were about 400 h, 540 h and 670 h, respectively, and thus the maximal estimate of cell numbers in the subependymal layer, as a percentage of the total cell population in the forebrain, was about 3 ~ at day 1 and 6, and 2 ~ at day 12. Throughout this period, undernutrition was estimated to produce a decrease in the fraction of total cells in the subependymal layer (0.9-1.4 ~). By using a similar approach, the proportion of the cerebellar cell population resident in the external granular layer can be calculated. Here, further assumptions need to be made: (a) cell proliferation takes place both in the external granular layer and in white matter; (b) this leads respectively to the formation of neurones and glial cells primarily; and (c) a glia to nerve cell ratio of 2:1, which has been calculated as a maximal value for the adult cerebellum3, also applies to the immature brain. In the 12-day-old normal cerebellum, the doubling time for total DNA was about 200 h (from ref. 21), for the external granular layer 49 h (Table IV), and for the white matter 115 h (labelling index 9.2~). These values are consistent with about 4.3 ~ of total cerebellar cell numbers being resident in the external granular layer. Similar calculations indicated that in the undernourished cerebellum, at that age, the fraction of total cell population in this germinal site was slightly higher, about 5.8 ~o (doubling times for total DNA 230 h (see ref. 21), for the external granular layer 61 h (Table IV) and for white matter, where the labelling index was 6.6 ~, 290 h). Total cell numbers were determined simultaneously in the cerebella of undernourished rats: the values were 80-85 ~ of control, and thus it would appear that, at 12 days of age and by using this method of calculation, undernutrition did not cause appreciable change in cell numbers in the external granular layer. However, for the cerebellum, an alternative and less circumstantial estimate of germinal cell numbers can be derived from a comparison of cross-sectional area of whole cerebellum, and the external granular layer, together with the packing density of cells in that layer. In contrast to the calculations described above, these results indicated that there was a deficit in germinal cells in the undernourished cerebellum of about 20 ~. However, rigorous quantitation of cells in the germinal layers presents formidable problems, and reduction of their total numbers is a possibility which awaits confirmation. Slowing of DNA synthesis and a probable reduction in germinal cell numbers may not be the only effects of undernutrition on cell proliferation in the brain. Since,

246

P.D. LEWIS et al.

in comparison with controls, the rate of increase in cell numbers in the undernourished brain is less severely depressed than the rate of D N A synthesis, we proposed that one of the factors involved might be a reduction in the rate of cell loss which normally accompanies cell production (histogenetic degeneration10). Preliminary investigation of this possibility by counting degenerate nuclei in the subependymal and the external granular layers suggests that this is not the case. At day 1, 6 and 21 there is no marked difference in numbers of degenerate nuclei between control and experimental rats. However, in animals aged 12-13 days the pyknotic indices are higher in the germinal sites of the undernourished rats than in controls. In conclusion, the results of both our previous and present studies are consistent with the view that the decrease, in comparison with normal, in the deposition of cells in the undernourished brain is primarily due to depressed cell formation, which may involve a small decrease in germinal cell numbers. The relatively more marked depression of D N A synthesis rate can be accounted for, in part, by the prolongation of the S-phase, which is greater than the prolongation of the doubling time of the germinal cells (Tables Ill-V). However, it would appear that the slowing of the cerebral D N A synthesis rate observed previously 21 cannot be accounted for exclusively by prolongation of the S-phase. Thus, factors other than the slowing of D N A synthesis, such as the conversion rate of thymidine into the proper DNA-precursor, may also be involved in the depressed incorporation of labelled thymidine into D N A in the undernourished brain. The effect of undernutrition on the rate on thymidine nucleotide synthesis is currently under investigation in our laboratory. ACKNOWLEDGEMENTS

We are indebted to Dr. G. G. Steel for computer analysis of percentage labelled mitosis data, and to Miss M. Lai for skilled technical assistance. This work was supported, in part, by a grant to P.D.L. from the National Fund for Research into Crippling Diseases.

REFERENCES 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 BAIRD,A., WIDDOWSON,E. M., AND COWLEY,J. J., Effects of calorie and protein deficiencies early in life on the subsequent learning ability of rats, Brit. J. Nutr., 25 (1971) 391-403. 3 BAL~.Z8,R., HAJ6S,F., JOHNSON,A. L., TAPIA,R., AND WILKIN, G., Biochemical dissection of the cerebellum, Biochem. Soc. Trans., 2 (1974) 682-687. 4 BALAZS,R., ANDPATEL,A. J., Factors affecting the biochemical maturation of the brain. Effect of undernutrition during early life. In D. H. FORD(Ed.), Neurobiological Aspects of Maturation and Ageing, Progr. Brain Res., Vol. 40, Elsevier, Amsterdam, 1973, pp. 115-128. 5 BARR~XT,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 BASS,N. H., NE'rSKY,M. G., ANDYOUNG,C., Effect of neonatal malnutrition on developing cerebrum. I. Microcbemical and histologic study of cellular differentiation in the rat, Arch. Neurol. (Chic.}, 23 (1970) 289-302.

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