AMERICAN JOURNAL OF PHYSICAL ANTHROPOLOGY 72:67-75 (1987) ... Nutritional stresses during lactation affected orthocephalization through an.
AMERICAN JOURNAL OF PHYSICAL ANTHROPOLOGY 72:67-75 (1987)
Effects of Maternal Food Restriction During Lactation on Craniofacial Growth in Weanling Rats HECTOR M. PUCCIARELLI AND EVELIA E. OYHENART Lahoratorio de Investiguciortes Morfoldgicas, Catedra IIIu de Anatomia, Facultad de Ciencias Medicas, Universidad Nacional de La Plata, La Plata, Buenos Aires, Argentina
KEY WORDS Skull growth, Experimental malnutrition, Functional craniology, Multivariate analysis.
ABSTRACT Adult Holtzman rats were submitted during suckling period to a food restriction with or without protein or carbohydrate restoration. Twenty-one-day-old weanling pups were compared with controls of 9, 13, 17, and 21 days of age. Lateral craniofacial roentgenographies were taken. The length in midsagittal plane of each bone and its angle with respect to the vestibular line were measured in males. In females, the brain and the left masseter muscle were weighed, and the musclebrain ratios (neuromuscular index) were calculated. Food restriction altered skull size and shape. Size changes were due to arrested lengths in all studied skull bones. Shape variation was evident by orthocephalization changes, reflected in angulation changes of bones belonging to the frontoethmofacial (frontal, nasal, and maxillary bones) and to the occipitointerparietal (interparietal bone) complexes. Partial restorations by both protein or carbohydrate supplementation were found. Nutritional stresses during lactation affected orthocephalization through a n altered growth ratio between two soft tissues functionally associated to the craniofacial complex: brain and masticatory muscles. Head variation between human populations is a process determined by action of genetic and nongenetic factors. This concept was inferred from human comparative studiesand demonstrated by means of experimentation. It is relevant to physical anthropology to enquire experimentally about the ways some kind of nongenetic factors could actually influence cranial differentiation. Nutritional deficiencies are considered to be important nongenetic factors, because they evoke outstanding deviations from normal growth patterns. Nutritional factors can easily be studied on the growing rat skull, because it is known that the orthocephalization of the skull of the growing rat is a n appropriate model system for the study of adjustive cranial growth processes (Moss and Vilmann, 1978). Orthocephalization is due mainly to two developmental systems in mammalian skeletons: (a) The neurocranial system, which shows a fast initial growth of short duration
(C 1987 ALAN R. LISS, INC
and (b) The splanchnocranial system, which shows initial growth but sustained and of long duration. At birth the cranial vault is globular, the splanchnocranium is deflected downward relative to the neurocranium, and the cranial base is flexed dorsally. During postnatal development and after near completion of brain and vault growth, growth results in a flattening of the cranial vault, an elevation of the nasal portion anteriorly, and a n elevation of the occipital complex posteriorly. Orthocephalization studies based on the vestibular orientation of the skull were carried out during normal growth (Destombes and Fenart, 1974; Pucciarelli, 1978a; Vilmann and Moss, 1979) and under different experimental conditions, such as hydrocephalus (Destombes and Fenart, 1970), cranial
Received May 13, 1985;revision accepted June 27, 1986.
68
H.M. PUCCIARELLI AND E.E. OYHENART
deformation (Pucciarelli, 1978b), and postweaning malnutrition (Pucciarelli and Niveiro, 1981). When malnutrition was imposed between 21 and 49 days of age in the rat, cranial growth retardation without altered bone rotation was found. This indicated that a postweaning malnutrition evoked inhibitory effect in size but not in shape, and consequently orthocephalization would have not been affected. The present study was intended to find out whether: (a) a maternal food restriction imposed during the nursing period of the rat would bring about a delayed orthocephalization in the offspring; (b)such inhibitory effect would be linked to variations in the relative growths of functionally related soft tissues, such as the encephalon and the masticatory muscular mass; and (c) some kind of restoration might take place after protein or carbohydrate supplementation was administered to restricted dams. MATERIALS AND METHODS
Pregnant Holtzman rats, body weight 200250 g, were caged individually and fed on stock diet ad libitum. At delivery the offspring were reduced to four males and four females. Food intake and body weight of dams and pups were registered three times a week. Experimental nutritional treatments began a t delivery and finished a t weaning when the offspring were 21 days old. The experiment treatments were: Control (C): Dams were fed on stock diet ad libitum throughout. Pups were killed a t 9 (C9), 13(C13),17 (C17),or 21 (C21)days of age. Restriction (R): Each dam was given half the amount of stock diet taken in daily by a control dam of similar body weight at the beginning of the experiment. Protein supplementation (PS): Some restricted dams received a daily protein supplement (Table 1) throughout the nursing period. This supplementation was equivalent to the difference in daily protein intake between a control and a restricted dam of similar body weight a t the beginning of the experiment. Carbohydrate supplementation (CS): Some restricted dams received a daily carbohydrate supplement (Table 1) throughout the nursing period. This supplementation was equivalent to the difference in daily carbohydrate intake between a control and a restricted dam of similar body weight a t the beginning of the treatment: ResGicted and
TABLE 1. Percent composition of the supplements given to nutritionally restricted rats Components Casein d-I-Methionine Gelatine Corn starch Glucose
Protein supplement
Carbohydrate supplement
88.6 1.4 10.0
-
-
65.6
34.4
supplemented animals were killed a t weaning. One hundred and forty animals were sampled (ten males and ten females in each group). Males-except for these 9-day-oldcontrols-were used for the craniometrical study. A midsagittal X-ray of each skull was taken. Anatomical points and the left external semicircular canal (vestibular line) were indicated by metallic markers. Radiographic technique was detailed previously (Pucciarelli and Niveiro, 1981). Measurements were made of the midsagittal lengths of the bones belonging to the neurocranium and the splanchnocranium and the diameters of the foramen magnum and the anterior nasal opening. The angles subtended between each projected bone length and the vestibular line were also measured (Fig. 1). Radiographs were obtained a t the Instituto de Radiologia y Fisioterapia (La Plata, Buenos Aires). In females, the brain (BW) and the left masseter muscle (MW) were weighed, and the neuromuscular index (NMI) was calculated thereof: NMI= 100 (MWIBW). The amount of cranial differentiation was estimated among control and experimental males by multivariate Mahalanobis D2-distances. The significance of differences among variate means were made by analysis of variance (ANOVA). All the statistical work was carried out at the Centro de Estudios Superiores Para el Procesamiento de la Informacion (CESPI) of the Universidad Nacional de la Plata. RESULTS
Body weight Experimental dams showed greater body weight loss than controls. Losses were proportional to the intensity of restriction. Accordingly, the lowest calorie intake belonged to the restricted group, which was followed, in increasing order by protein-supplemented, carbohydrate-supplemented, and control _dams. (Fig: 2).
MATERNAL FOOD RESTRICTION ON CRANIOFACIAL GROWTH
Fig. 1. Lateral roentgenography from a growing rat skull. Upper case letters: bone name; lower case letters: angle with respect to vestibular line GV). Splanchnrr cranial component: N: nasal; PM: premaxillary; M: maxillary; PL: palatine bones; AN: anterior nasal opening. Neurocrunzal component: (1) vault region: F: frontal (the
69
anterior portion from nasion to frontoethmoidal junction was included here); P parietal, I: interparietal, and S: supraoccipital bones; (2) Basicranial region: E: cribriform plate of the ethmoid; PE: presphenoid, B: basisphenoid, and BO: basioccipital bones; and FM: foramen magnum. Enlargement, x3.
while D2-value evoked by carbohydrate supplementation was the only nonsignificant distance (Table 2).
240
200
160
120
80
40
4,I
3
I
6
I
9
I
I
12
15
AGEIMVS) I I 18 21
Fig. 2. Daily caloric intake in control (circles), carbohydrate-supplemented (closed squares), protein-supplemented (open squares),and restricted (triangles)mothers.
Cranial distances In controls of 13-17 days of age, cranial differentiation was similar to 17-21 days. The differentiation evoked by restriction was greater than each control comparison and slightly lower than that of the overall control period (13-21 days). Protein supplementation evoked similar distance to 13-17 controls,
Bone measurement In the control rats the greatest increment in length between 13 and 21 days of age were recorded for the nasal, maxillary and basisphenoid bones, and the nasal opening (Fig. 31, whereas the lowest increments in length were recorded for the premaxillary and interparietal bones. The presphenoid, basisphenoid, supraoccipital, frontal, and ethmoid bones showed length increments between those extremes. On the other hand, the length of the parietal bone and foramen magnum did not change significantly in that interval. Restriction arrested the elongation of all bones. The supply of protein or carbohydrate restored the growth in length of all bones, except for the interparietal and basisphenoid bones (protein supplementation) and the ethmoid bone (carbohydrate supplementation) (Fig. 3). The control rats showed two kinds of bone rotation: (a) anterior and superior (the frontal, nasal, premaxillary, maxillary, and the ethmoid bones), and (b) posterior and superior (the interparietal and the basioccipital,
70
H.M. PUCCIARELLI AND E.E. OYHENART
TABLE 2. Mahalanobis D‘-distances between controlcontrol and control-experimental groups ComDarisona Between controls C13-Cl7 C17-C21 C13-C21 Control-experimental C21-R c21-PS c21-cs
D2
F
5.45 6.66 15.67
5.81‘* 7.10** 16.04**
12.93 5.96 1.16
13.12**’ 6.35** 1.24
= controls of 13, 17, and 21 days of age; R = restricted group; PS = protein-supplemented group; CS = carhahydrate-supplemented group. **P < 0.01.
“C13,C17, C21
as well as the foramen magnum). The parietal, supraoccipital, presphenoid, and the basisphenoid bones did not rotate significantly. Individual rotations of the frontal, nasal, maxillary, and the interparietal bones, and the nasal opening were decreased by restriction. Protein supplementation restored the rotation of the frontal bone and carbohydrate supplementation restored that of the nasal opening. Both supplementations restored the rotation of the interparietal bone (Fig. 4).
Neuromuscular index Brain weight increased from 9-17 days, and showed nonsignificant differences throughout. The brains in the restricted group weighed the same as those in 13-day-oldcontrols. Protein- or carbohydrate-supplemented groups yielded similar weights to those in 17and 21-day-old controls. Muscular weight increased from 9 days throughout, and decreased by both restriction and supplementation, to similar values to those of 17-dayold controls. Neuromuscular index in control rats increased throughout. Restriction and carbohydrate supplementation elicited NMI values similar to 17-day-old controls. NMI values from protein-supplemented group fell between the 17- and 21-day-old controls (Tables 3 and 4). DISCUSSION
Bone rotation About 70% of the bones showed significant rotations during normal growth period. The present study confirmed the existence of three dynamic regions in the orthocephalization of the rat, i.e., the frontoethmofacial rotating in a n anterosuperior direction, the
occipitointerparietal rotating in a posterosuperior direction, and the nonrotating sphenoparietal region (Pucciarelli, 1978a). Vilmann and Moss (1981) observed in normally growing rats a n increase in the frontoparietal angle together with nonuniform changes in frontonasal angulation. The rotation of the frontal bone (linked to the facial elements) observed in the present experiment corroborated these observations. Also the basioccipital rotation (linked to the occipitointerparietal complex) found here was in accordance with the occipital lordosis of the basisphenoid bone and with the pre- and postsellar changes found in normal rat skulls (Vilmann, 1969; Moss and Vilmann, 1978; Vilmann and Moss, 1979). The invariability of the sphenoparietal region indicated that the angle subtended between the basisphenoid bone and the projected external semicircular canal did not change, as it was previously found (Pucciarelli, 1978a; Vilmann and Moss, 1979).
Food restriction Many studies have been carried out on the effects of some nutritional deficiencies on bone growth during gestation (Warkany and Nelson, 19411, lactation (Williams and Hughes, 19781, gestation and lactation (Shaw, 1970; Toews and Lee, 1975a,b), and postweaning (Riesenfeld, 1967, 1973; Pucciarelli, 1980,1981; Pucciarelli and Niveiro, 1981)periods. Different kinds of cranial growth delays were reported depending upon the type of malnutrition and/or its intensity as well as on the period in which the stress was applied. Skull variation due to a restriction during lactation differed from that obtained by postweaning malnutrition, in which size but not shape changes were seen (Pucciarelli and Niveiro, 1981). In the present study, multivariate analysis indicated that pups from restricted mothers had skulls about 30% lower in size and shape than those of controls a t the same age. This altered skull growth was due to both a n inhibitory effect in bone length and to delayed rotation in four out of the seven bones involved in the orthocephalization process. The fact that the three bones (nasal, maxillary, and frontal) that rotated less belonged to the frontoethmofacial complex meant that most of the variability in shape evoked by suckling malnutrition was reflected in the face. Shape changes led to the permanence of a “klinorynchal trend”
71
MATERNAL FOOD RESTRICTION ON CRANIOFACIAL GROWTH 50
FORAMEN MAGNUM
,>
I
3
26
17
13
0
0 INTERPARIETAL BONE
PAFIETAL BONE
70
70
II 1 L6
46
23
23
0
0
FRON
L BONE
NASAL BONE
11( 71
37
0
SUPRAOCCIPITAL BONE
II
I
110 71 37
0
NASAL OPENING
REMAXILLARY BONE
6
50
1
3L
2
17
0
0
MAXILLARY
II
[I
RESPHENOID BONE
BONE
7c
L
L6
22
0
it
I
31
17
0 BASIOCCIPITAL BONE
BASISPHENOID BONE 70
6
I
46
23
2
0
C
CU 0 7
C n 03
C17 R
R C.5
R
R CS
R CS
ETHMOID BONE
0 3 CR
C n Cl3 C1J
R
R CS
R
K C5
K CS
Fig. 3. Bone length measurements. Mean, standard error and analysis of variance. Black: reference group; hatched: p g0.05; quadriculated p 40.01; white: nonsignificant difference. C13, C17, C21=13-, 17-, and 21-day-old controls; R: restricted PS: protein-supplemented group; CS: carbohydrate-supplemented group. Vertical lines: standard errors.
72
H.M. PUCCIARELLI AND E.E. OYHENART SUPRAOCCIPITAL BONE
FORAMEN MAGNUM 110
III
66.
93
17
33-
0-
3
INTERPARIETAL BONE
PARIETAL BONE
20
"3
'3
7;,
7
0
13
a
p1
13
c NASAL BONE
FRONTAL BONE 53
33
LO
?7
20
0
s NASAL OPENING
PREMAXliLARY BONE
150
50
?06
3:
53
17
3
s
50
PRESPHENOID BONE
MAXILLARY BONE
20 ?:
31 7
I
0
3
BASISPHENOID
BONE
BASIOCCIPITAL BONE 30
:j fl 0
CI3 Cl7
180 -
C2I Ci3 C17 R
K CS
R Pi CS
R
r~
ETHMOID BONE
120 ..
60 ..
C13 07
C21 0 3 C l l R
R CS
R
R CS
R CS
Fig. 4. Vestibular angles, Mean, standard error, and analysis of variance. Black: reference groups, hatched: pGO.05; quadriculated: p GO.01; white: nonsignificant differences. C13, C17, C21= 13-,17-,and 21-day-old controls; R: restricted; PS: protein-supplemented group; CS: carbohydrate-supplemented group. Vertical lines: standard errors.
MATERNAL FOOD RESTRICTION ON CRANIOFACIAL GROWTH
73
TABLE 3. Mean f standard error for body, Left masseter muscle, and brain weights. and neuromuscular index Groups Controls (days) 9 13 17 21 Experimentals Restricted Proteinsupplemented Carbohydratesuuulemented
Body weight
Masseter weight
Brain weight
Neuromuscular index
15.0 f 0.8 24.3 + 0.6 31.1 0.9 39.5 f 1.3
0.038 0.075 0.136 0.195
I 0.001 f 0.001 f 0.000
0.770 f 0.008 1.120 f 0.004 1.290 f 0.001 1.328 f 0.002
4.810 f 0.284 6.683 f 0.221 10.490 +_ 0.531 14.648 k 0.408
20.9 f 0.7 28.0 f 0.9
0.120 f 0.001 0.153 f 0.001
1.147 f 0.005 1.264 f 0.003
10.427 f 0.465 12.100 f 0.525
34.4 f 0.5
0.153 f 0.001
1.305 f 0.001
11.677 I 0 . 3 2 6
7
f 0.001
TABLE 4. Calculated F-values for body weight, masseter weight, brain weight, and neuromuscular index Body weight
Masseter weight
Brain weight
Neuromuscular index
53.8** 28.7** 43.9** 373.1**
20.8** 39.0** 52.3** 422.8**
167.0** 39.0** NS 366.3**
10.4* 43.2** 51.5** 288.5**
21.6** 7.2* 64.7** 215.0**
100.0** 29.6**
193.1* * NS 27.8** 83.5**
93.9** 41.8** NS 53.1**
105.0** 8.5* NS 82.2**
196.2** 89.2** NS 26.3**
331.9** 28.0**
158.4** 87.5** 7.7* 19.4**
c13-cs
233.9** 63.4**
c17-cs c21-cs
NS 16.2**
195.6** 88.8** NS 26.6**
388.7** 46.1** NS NS
Comparisiona Control-control C9-Cl3 C13-Cl7 C17-C21 C9-C21 Control-restricted C9-R C13-R C17-R C21-R Control-proteinsupplemented c9-PS c13-PS c17-PS c21-PS Control-carbohydratesupplemented c9-cs
NS 44.4**
NS NS
"C9, C13. C17. C21 = controls of 9, 13, 17, and 21 days of age; R = restricted group; PS CS = carbohydrate-supplemented group. *P 4 0.05. **P < 0.01.
during the orthocephalization process (Fig. 5). This effect may be explained by the fact that the neurofacial flexion finishes shortly after weaning (Moss, 1958; Pucciarelli, 1978a), when much of the orthocephalization has been completed. A functional relationship between orthocephalization and growth of some head soft tissues was evident by the behavior of the neuromuscular index, which measures the relative growth of the brain-functionally associated with the neurocranial region-and the masseter muscle-functionally associated
=
140.4** 74.3** ._
NS 26.3**
protein-supplemented group;
with the facial skeleton. In controls, while the muscle steadily grew during lactation, there was a period (between 17 and 21 days) in which brain weight did not change significantly. The neuromuscular index for restricted animals was equivalent to that of 17day-old controls, meaning a growth delay of about 20%. This effect was mainly due to a greater reduction in masseter muscle growth compared to brain growth. That reduction was equivalent to that found in the craniofacia1 skeleton, as measured by D2-distances (Table 2).
74
H.M. PUCCIARELLI AND E.E. OYHENART
Fig. 5. Lateral roentgenographies of 21-day-old control (C) and malnourished (M) rat skulls superimposed along the vestibular line (LV). The smaller size and the shifted splanchnocranial and vault regions indicates “klinorynchal retention” in malnourished skull. Enlargement, x3.
out, since D2-values of both supplemented groups showed at least 50% of craniofacial improvement with respect to the controls. Cerebral and muscular recuperation through supplementation also took place as indicated by increasing values in NMI. The effects of supplemented diets suggested that recuperation was not only due to a restoration of protein level but rather to relatively larger caloric intake associated with those supplementations. CONCLUSIONS I
1-4
3
6
9
12
15
AGE IMYSI
18
21
Fig. 6. Daily caloric intake per gram of body weight in controls (circles), carbohydrate-supplemented (closed squares), protein-supplemented (open squares), and restricted (triangles) mothers.
Effectsof supplementation Carbohydrate-supplemented mothers ate more than protein-supplemented ones and the former showed caloric intake values intermediate between protein-supplemented and 21-day-old control groups (Fig. 2). However, the difference became nonsignificant when a caloric intake per gram of body weight was calculated (Fig. 6). Very likely most of the energetic supply was mainly used for body weight recovery in dams. Some body weight recovery in pups could not be ruled
Orthocephalization in the rat partly results from neurofacial adjustive growth during lactation. Maternal food restriction alters both skull growth and orthocephalization in the offspring. An altered growth ratio between two soft components (brain and masticatory muscles), that are functionally related to the neurocranium and facial skeleton, respectively, can evoke a n underdeveloped architecture in restricted nursing rats. On the other hand, that developmental delay does not mean a nonreversible process, since a n adequate protein-calorie supplementation allowed a high degree of restoration. A cause-and-effect relationship between functionally associated hard and soft tissues of the head may be concluded. Nursing restriction affected craniofacial structure through a n alteration in the growth ratio between brain and masticatory mass, which
MATERNAL FOOD RESTRICT101V ON CRANIOFACIAL GROWTH
was partially restored by protein-calorie supplementation.
75
LITERATURE CITED
tion on craniofacial differentiation in rats. A multivariate analysis. Am. J.Phys. Anthropol. 53:359-368. Pucciarelli, HM (1981)Growth of the functional components of t h e rat skull and its alterations by nutritional effects. A multivariate analysis. Am. J. Phys. Anthropol. 56:33-41. Pucciarelli, HM, and Niveiro, MH (1981) Effet de la malnutrition sur le developpement de l’ontogenese cranio-faciale. Cahiers D’Anthropologie. 297-109. Riesenfeld, A (1967) Biodynamics of head form and craniofacial relationships. Homo. 18:233-251. Riesenfeld, A (1973) The effect of extreme temperatures and starvation on t h e body proportions of t h e r a t . Am. J. Phys. Anthropol. 39:429-460. Shaw, JH (1970) Marginal protein deficiency during the reproductive cycle in rats: Influence in body weight and development of skulls and teeth of offspring. J. Dent. Res. 49:350-359. Toews, J G , and Lee, M (1975a) Permanent skeletal growth retardation in the progeny of r a t s malnourished during pregnancy and lactation. Nutr. Rep. Int.
Destombes, P, and Fenart, R (1970) Modifications craniennes chez le rat rendu hydrocephale. C. R. Assoc. Anat. 147235-241. Destombes, P, and Fenart, R (1974) Le crane du rat en orientation vestibulaire. Bull. Assoc. Anat. 58:287290. Moss, ML (1958) Rotations of the cranial components in the growing r a t and their experimental alteration. Acta Anat. 32:65-86. Moss, ML, and Vilmann, H (1978) Studies on orthocephalization of t h e rat head. 1. A model system for t h e study of adjustivc cranial skeletal growth process. Gegenbaurs morphol. Jahrb. 124:559-579. Pucciarelli, HM (1978a) Craniofacial development of t h e r a t with respect to vestibular orientation. Acta Anat. 100:101-110. Pucciarelli, HM (1978b3 The influence of experimental deformation on craniofacial development in rats. Am. J. Phys. Anthropol. 48:455-462. Pucciarelli, HM (1980) The effects of race, sex and nutri-
Toews, JG, and Lee, M (1975b) Retarded skeletal maturation i n the progeny of r a t s malnourished during pregnancy and lactation. Nutr. Rep. Int. 11:223-230. Vilmann, H (1969) The growth of the cranial base in the albino r a t revealed by roentgenocephalometry. J. 2001. Lond. 159:283.-291. Vilmann, H, and Moss, ML (1979) Studies on orthocephalization. 2. Flexion of the r a t hsad in the period hetween 14 and 60 days. Gegenbaurs morphol. Jahrh. 125:577-582. Vilmann, H, and Moss, ML (1981) Studies on orthocephalization. 7. Behavior of t h e r a t cranial frame in the period between 1 day before birth and 14 days after birth. Acta Anat. 109:157-160. Warkany, J, and Nelson, RC (1941) Skeletal abnormalities in t h e offspring of r a t s reared on deficient diets. Anat. Rec. 79:83-100. Williams, JPG, and Hughes, PCR (1978)Catch-up growth in the r a t skull after retardation during suckling period. J. Embryol. Exp. Morphol. 45:229-235.
ACKNOWLEDGMENTS
The authors are grateful to Prof. Dr. Mario H. Niveiro (Catedra IIIa de Anatomia), Dr. Jorge R. Da Silva (Instituto de Radiologia y Fisioterapia), Ing. Agr. Hector D. Ginzo (Centro de Ecofisiologia Vegetal), Mrs. Maria C Mune (Laboratorio de Investigaciones Morfologicas) and Mr. Hector J. Alvarez (Departmento de Tecnologia Educativa), for their highly valuable assistance. This study was supported by a grant from the Consejo Nacional de Investigaciones Cientificas y Tecnicas (CONICET).
11:213-222.
