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The effects of alizarin red S on weight gain and skull growth in the rat
THE EFFECTS OF ALIZARIN RED S ON WEIGHT GAIN AND SKULL GROWTH IN THE RAT fi. Department
of Anatomy.
Royal
Department of Orthodontics,
VILMANN Dental
and University
College.
Copenhagen.
of Nymegen.
Denmark
The Netherlands
Summary~~~Reliability of conclusions drawn from growth studies combining X-ray cephalometry and vital staining with alizarin red S is dependent on the knowledge of the method errors involved. The significance of the method errors in X-ray cephalometric studies arc well known, whereas expressions of method errors in studies using alizarin red S are sparse. Therefore. a stud! which might throw light upon this problem was carried out. Intraperitoneal injections of 100 mg/kg body weight of alizarin red S retarded weight gain of both 14 and 30-day-old rats for 2 days. The 30-day rats recovered quickly, while the 14-day rats. after a quick recovery. gained less weight than normal rats. These rats cndcd ~113 as smaller anmals. Significant differences in cranial bone measurements between rats injected with ali/arin I-cd S at 14 days and control rats were also present at 30 days.
INTRODUCTION
The irkriro staining of bone with alizarin red S is much used in the study of the growth of the vertebrate skull. Studies dealing with possible toxic effects of the dye on the growth of different animal species suggest that bone formation may be retarded or even stopped for some days after injection (Hoyte, 1960; Hong, Yen and Shaw, 196X:-Running, 197 1). Recent investigations (Vilmann, 1968, 1972) have compared results from X-ray cephalometric studies with results from histological studies by means of vital staining. The r&ability of the conclusions drawn from such investigations is dependent on the knowledge of the method errors involved. The method errors in the X-ray ccphalometric parts of the studies performed are well-known and quantitatively expressed. The significance of the method error introduced by intraperitoncal injection of alizarin red S into rats at various ages is. however. unknown. The aim of the present study was to acquire information on this topic.
MATERIAL
AND
METHOD
The study was based on comparisons of gain in weight and gain or change in several linear and angular neurocranial parameters of untreated rats and rats which have received one intraperitoneal injection of 100 mg/kg body weight of a 2 per cent solution of ali/at-in red S in a 0.45 per cent sodium chloride solution.
One experimental group consisted of 57 animals injected with alizarin red Sat 14 or 30 days and successively sacrificed at 15. 18.2 I and 30 days: and 3 I. 35. 40 and 60 days. respectively. The weight gain of this mixed longitudinal group was compared to the weight gain ofanother mixed longitudinal group consisting of 53 control animals.
Another experimental group consisted of 16 female rats. This group was formed as a consequence of the results from the part of the study just mcntioncd. The rats were at 14 days weighed. radiographcd and injected with alizarin red S. They wcrc succcssivclh weighed and radiographed at 21 and 30 dabs. The! were kept with their mothers until the termination 01 the experiment at 30 days. The corresponding control group consisted of 25 female rats. This group has been described in my carlier papers as have the X-ray ccphalomctric tcchniclue. the equipment used and the X-ray ccphalomctric t-cfercnce points (Vilmann, 1968, 1972). For an evaluation of the growth changes in the skull between 14 and 30 days the following distances and angles were measured. Disturlc.c,.s. Length of nasal bone. length OF parietal bone, height of foramen magnum, length of hasi-occipital bone. length of basisphenoid bone. length of cl-ibriform plate of ethmoid bone. length of cranium. height of neurocranium. length of neurocranium.
H. Vilmann
IOOX
A~~lus. Nasal angle, basi-occipital-basisphenoid angle, basisphenoid--parietal angle. RESULTS
Figure 1 presents the velocity ofweight gain between 14 and 30 days in the normal rats and in the rats injected with alizarin at 14 days. The weight curve of the injected rats is at a lower level than the weight curve of the untreated animals the first 3 days after injection. Thereafter the curve rises above the normal curve for some 5 days. From about 22 days of age, the curve of the experimental rats is situated below the curve of the untreated rats, Figure 2 presents the conditions between 30 and 45 days in the rats injected at 30 days. It is seen that the weight curve of the injected rats is situated below that of the normal rats the 1st day after injection. Thereafter the former curve rises above the normal. With increasing time, the two curves approach each other.
Figure 3 shows the weight curves of the two groups of rats from 14 to 30 days. The curve of the rats injected at 14 days is below that of the normal rats. However. the weight differences between the two groups are statistically significant only at 30 days. Table I shows the differences in the length of the measured bones and bone units between 14 and 21 days and between 21 and 30 days in the two groups of rats together with the angular changes between 14 and 30 days. Significant differences between the groups are present only in the gain in length of the nasal bone and of the total skull, and only between 21 and 30 days. Howcvcr, a systematic difference in the measurements of length gain is present both between 14 and 21 days and between 21 and 30 days, being more marked in the latter age period than in the former. During both periods, the measured units of the controls usually have increased more in length than those of the experimental group.
Fig. 2. Weight velocity of the two groups of rats from 30 to 45 days. Normal rats -~ Experimental rats ~.
porary weight loss for some days in rabbits. Hoyte (1960) arrived at the same result both in rabbits and guinea-pigs. In the rat, Schour et (I/. (1941) recommended a dose of 100 mg alizarin red S per kg body weight and found a temporary retardation of gain in weight followed by a quick recovery after injection of doses of this magnitude. Similar findings have been published by Hoyte (1960). whereas Cleall. Perkins and Gilda (1964) reached the conclusion that doses less than 520 mg/kg body weight cause no retardation of weight gain in rats at about 3540 days of age. However, Ronning (1971). in a study of the growth of rats from the age of 4 days, found a temporary retardation in weight gain after injection of 400 mg alizarin per kg body weight. The observations from the present study add new information to that formerly obtained. It seems that the response to injection of dye in some way is age dependent. The weight of rats injected at 14 days is initially little affected. Nevertheless, the injected rats are finahy smaller than the untreated rats. The weight of rats injected at 30 days is initially slightly affected. However. these rats recover and reach normal weights within a few days. The explanation of this phenomenon might, as suggested by Running (1971). be that the effect of the dye on the growth may be comparable to effects observed after induction of various stress conditions. such as cold, isolation and undernutrition. As such gms
50.
DISCUSSION
The effect of injections of alizarin red S on weight gain and bone formation has been discussed thoroughly in the literature. Hong et nl. (1968)have shown that doses of only 50 mg/kg body weight cause a tem-
LO-
30.
20.
‘i IL
2;
I 30
days
Fig. 1. Weight velocity of the two groups of rats from 14 to 30 ddys. Normal rats ~Experimental rats ~~
, I&
21
30
days
Fig. 3. Weight of the two groups of rats from I4 to 30 days. Normal rats -_ Experimental rats -
EfTccts ofahzarin
red S
IO09
T:~hlc I. Means (XI ;~nd standard deviations (s,) of the differences in distances and angles in the skull of the control rats (c) and the alizarin-injected I)i~l2llcc~
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Y
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03; 0.x 0.16 0~360. I3 0 I7 0.2 I 0.X 0. I7 0 I5 0, IS O-30 0.3 0. I x 0.3: 0.27 _‘m*1~75 0.17 0. I .: 0 I s 0.20 o-51 o-47 0.5, 047 03x o-34 0. I Y 0. I’) 34x*‘~s?
‘~
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from the two groups differ significantI> at the I per cent Ic\cl.
\trcs\ conditions
influence the rat more seriously lht’lorc weaning than after weaning (Widdowson and McCancc. 1963). the observed differences in weight
the morphologic appearances of radioactlvc calcium andalizarinredSconcluded that a single intrapcritoncal inlcction of alizarin red S does not disturb the normal
gain hctvx~n the‘txo
growth pattern of the single bones in the rat &III. Running (I971 ). in his stud) of the gro\\th of skull bones in young rats. quantified the retarding cllecl 01 400 mg,!kg body weight of the dye on bone formation. He found differences in several length and height measurements at IO days between animals inlcctcd at 4 days and control animals. Some of these d&crcnccs had. however. disappeared b\ I5 da>\ of age nhilc others remamed over a longer period. III the prsscnt stud). it was not possible to detnonstratc an? signilicant retarding effect in any of the nourocranial bones. Only the growth of the: nasal bone. i.c. :I hone rcpresentmg a unit of the face, was significantly afTcctcd and only at 30 days. However. there was ;I systematic dilkrence of increasing significance with incrcaGng age in the growth rate of the bones of the ali/arin-iri,jcctcil rats. It may be thus concluded that ~hcrc arc limtations in the rcliabilit\ of result\ from >tudics using alicarin red S injections at I4 day\. The importance C; these limitations increases with increaGng lxriod 01 sur\i\al.
~>LI~S
studied ma? be related to
Ihi\ condition. ~2lthounh the re\ults I-cparding the severit) of the inIlu~wx olrtlie d!c 011 young animals arc in conformity \\ith the results of studies on the influence of stress. an Initial catch-up growth followed by a subnormal NCI&I gain ;I< hnd in the present study. has not been d&ibcd before. ‘This phenomenon may have several ~\pl;lnations. One possihitity is that the rcstllts have
arixx as ;I conscqucnce of selection. Thcrcfore the uond e\pcrmicntal tYOLI~ of Il-da)-old rats was wormed and given alizarin. with the same result. :Znoth~‘r po\siblc explanation of the phenomenon InighC he that. in suckling rats. certain age levels are more critical th;ln c,tlw~-s. Moss and BXI- (1956). in a ad\ of tliflicrsntial growth of the rat skull. observed J cl-itical ~lc\,cl~)l~rncn~athoriron in the skull bones at .~hout IO &I!\ in ~11~‘ Long-Evans strain and at about IS (Iam in the M’istu alh~no strain. This might in some \I :I\ Ihc rstatcd 10 I tic‘ t~tw~omenon mcntioncd dx~~c. i-11~ wtxght pin of the rats injected at 30 da!s \ras ~ud~cd to lx onI\ slightly afIccted. although Fig. 2 Iu\cs the impi-cs\ioii of ;I scvcrt‘ atliiction. It is neccssx1-1 to heat- ill ~niml that UK rats in fhc prcscnt stud) \\L’IC tahcn li-om (hail- mother on the da> of initiation 01 Ihc’ cuperimcnt. NOI-nial rats react b! gainin! less \\crght for 2 d;~\\. 131adding another stress condition, the 11oriii;~l appcar;inc~ 01 the \clocit! curve onl) Ihccome~xccntuatcd. not changed. I Iong (‘I rrl. ( I W) found ;I retardation of bone forI~;IIKW in the‘ r;lbhit ;rftcr injection of 50 I00 mg, kg hotI! weight 01’ ;Iti/;lt-In I-cd S. Hoqtc (1960) found I ~tartlatlon both in rhc rabbit and in the rat. 14hercas 111 t tic rat both Scllour ( 1936) and Cleall cf rrl. ( 1964) louml no retarding clfcct of the dye on bone grou th. Ilouc~cr, both Ho)r~ (1960) and Dixon and tioytc
Cleall J. F‘.. Perkins R. E. and Gilda _I. t. I9hJ. Bone marhing agents for the longitudinal stud! ofgrowth in animals. ./Ir<~h.\ ortrl Biol. 9, 627 -646. Dixon A. D. and Hoyte D. A. N. 1963. A compar~n 01 autoradiographic and alirarin technique\ in ~ht’ \tud) 01 bone growth. .%trrr. Rw. 145. IO1 I I?. Hong Y. C.. Yen P. K.-J. and Shaw .I H. 19hX. M~oscop~c evaluation of the effects of some bital staining agents on growing bone in rahblts. Calc. Tis.\. KC,\.2, 2~6 295. Ho!te D. A. N. 1960. Alizarm as an indicator of hone growth. J. 41lur. 94, 432~ 443. Moss M. L. and Bacr M. J. 1956. Differential ~rov, th of the
H.
1010
Vilmann
Renning 0. 1971. Alterations in craniofacial morphogenesis induced by parenterally administered papain. Academic Dissertation. University of Turku. Finland. Schour 1. 1936. Measurements of bone growth bq alirarinc injections. PJYK. Sot,. rup. Biol. Mrrl. 34, 140 141. Schour I., Hotmmann M.. Sarnat B. and Engel B. 1941. Vital staining of growing bones J. tlr,jr. Rex 20, 41 I-418.
and teeth with ali7arin
red S.
Vilmann H. 196X. The growth of the parietal bone in the albino rat studied by roentgenocephalometry and vital staining. 3w/1,5 ortrl Biol. 13, X87-901. Vilmann H. 1971. The growth of the cranlal vault m the albino rat. 3r,c/1.\ wtrl Biol. 17, 399 414. Widdowson E. M. and McCance R. A. 1963. The effect of linite periods of undernutrition at different ages on the composition and subsequent development of the rat. Proc~. R. Sot,. Series B. 158, 329~ 342.
R&sum&La rkgularitk des conclusions tirCes des Ctudes sur la croissance, en combinant la &phalom&rie aux rayons-X et la coloration vitale avec le rouge d’alizarine S est dt-pendante de la connaissance des erreurs de mt-thode impliqut-es. La signification des erreurs de mtthode dans les i-tudes cCphalom6triques aux rayons-X est bien connue, tandis que les expressions des erreurs de mkthode dans les Ctudcs utilisant le rouge d’alizarine S sont rares. Par consCquent, on a entrepris une Ctude qui pourrait kclairer ce problkme. Les injections intrapCriton6ales de lOOmg/kg de poids corporel de rouge d’alizarine S ont retard&es le gain de poids des rats 2g6s de 14 et 3Ojours, pendant 2 jours. Les rats Lg& de 30 jours ont recouvrk vite, tandis que les rats Bgt-s de 14 jours apris un recouvrement rapide, ont gag& moins de poids que les rats normaux. Ces rats finirent comme animaux plus petits. Des difft-rences significatives dans les mesurages de l’os crinien, entre les rats inject&s avec du rouge d’alizarine S apr&s 14 jours et les rats tCmoins ktaient kgdlement prksentes apr&s 30 jours.
Zusammenfassung-Die Verl%Blichkeit von Schliissen, die aus Wachstumsuntersuchungen gezogen werden, welche Rantgenstrahl-Kraniometrie und grundlegende FLrbung mit Alizarinrot S kombiniert, hlngt von der Kenntnis der verbundencn Verfahrensabwcichungen ab. Die Bedeutung von Verfahrensabweichungen in Riintgenkraniometrieuntersuchungen sind allgemein bekannt. dagegen sind Ausdriickc fiir Abweichungen itn Verfahren in Untersuchungen mit Alizarin-Rot S splrlich. Daher wurde eine Untersuchung durchgefiihrt. welche dieses Problem aufklgren kiinnte. Interperitoneale Injektionen von 100 mg/kg Kiirpergewicht von Alizarinrot S verziigerte den Gewichtszuwachs von 14 und 30 Tage alten Ratten urn 2 Tage. Die 30 Tage alten Ratten erholten sich schnell, wlhrend die 14 Tage alten Rattcn nach schnellcr Erhoiung an Gewicht wcniger als normale Ratten zunahmen. Diese Ratten wuchsen zu kleineren Tieren heran. Bedeutende Unterschiede in kranialen Knochenmessungcn rwischcn Ratten. mit Alizarinrot S injizierten 14 Tage- und Kontrollrattcn. warcn such bci 30 Tagcn \#orhanden.
of Anatomy.
Royal
Department of Orthodontics,
VILMANN Dental
and University
College.
Copenhagen.
of Nymegen.
Denmark
The Netherlands
Summary~~~Reliability of conclusions drawn from growth studies combining X-ray cephalometry and vital staining with alizarin red S is dependent on the knowledge of the method errors involved. The significance of the method errors in X-ray cephalometric studies arc well known, whereas expressions of method errors in studies using alizarin red S are sparse. Therefore. a stud! which might throw light upon this problem was carried out. Intraperitoneal injections of 100 mg/kg body weight of alizarin red S retarded weight gain of both 14 and 30-day-old rats for 2 days. The 30-day rats recovered quickly, while the 14-day rats. after a quick recovery. gained less weight than normal rats. These rats cndcd ~113 as smaller anmals. Significant differences in cranial bone measurements between rats injected with ali/arin I-cd S at 14 days and control rats were also present at 30 days.
INTRODUCTION
The irkriro staining of bone with alizarin red S is much used in the study of the growth of the vertebrate skull. Studies dealing with possible toxic effects of the dye on the growth of different animal species suggest that bone formation may be retarded or even stopped for some days after injection (Hoyte, 1960; Hong, Yen and Shaw, 196X:-Running, 197 1). Recent investigations (Vilmann, 1968, 1972) have compared results from X-ray cephalometric studies with results from histological studies by means of vital staining. The r&ability of the conclusions drawn from such investigations is dependent on the knowledge of the method errors involved. The method errors in the X-ray ccphalometric parts of the studies performed are well-known and quantitatively expressed. The significance of the method error introduced by intraperitoncal injection of alizarin red S into rats at various ages is. however. unknown. The aim of the present study was to acquire information on this topic.
MATERIAL
AND
METHOD
The study was based on comparisons of gain in weight and gain or change in several linear and angular neurocranial parameters of untreated rats and rats which have received one intraperitoneal injection of 100 mg/kg body weight of a 2 per cent solution of ali/at-in red S in a 0.45 per cent sodium chloride solution.
One experimental group consisted of 57 animals injected with alizarin red Sat 14 or 30 days and successively sacrificed at 15. 18.2 I and 30 days: and 3 I. 35. 40 and 60 days. respectively. The weight gain of this mixed longitudinal group was compared to the weight gain ofanother mixed longitudinal group consisting of 53 control animals.
Another experimental group consisted of 16 female rats. This group was formed as a consequence of the results from the part of the study just mcntioncd. The rats were at 14 days weighed. radiographcd and injected with alizarin red S. They wcrc succcssivclh weighed and radiographed at 21 and 30 dabs. The! were kept with their mothers until the termination 01 the experiment at 30 days. The corresponding control group consisted of 25 female rats. This group has been described in my carlier papers as have the X-ray ccphalomctric tcchniclue. the equipment used and the X-ray ccphalomctric t-cfercnce points (Vilmann, 1968, 1972). For an evaluation of the growth changes in the skull between 14 and 30 days the following distances and angles were measured. Disturlc.c,.s. Length of nasal bone. length OF parietal bone, height of foramen magnum, length of hasi-occipital bone. length of basisphenoid bone. length of cl-ibriform plate of ethmoid bone. length of cranium. height of neurocranium. length of neurocranium.
H. Vilmann
IOOX
A~~lus. Nasal angle, basi-occipital-basisphenoid angle, basisphenoid--parietal angle. RESULTS
Figure 1 presents the velocity ofweight gain between 14 and 30 days in the normal rats and in the rats injected with alizarin at 14 days. The weight curve of the injected rats is at a lower level than the weight curve of the untreated animals the first 3 days after injection. Thereafter the curve rises above the normal curve for some 5 days. From about 22 days of age, the curve of the experimental rats is situated below the curve of the untreated rats, Figure 2 presents the conditions between 30 and 45 days in the rats injected at 30 days. It is seen that the weight curve of the injected rats is situated below that of the normal rats the 1st day after injection. Thereafter the former curve rises above the normal. With increasing time, the two curves approach each other.
Figure 3 shows the weight curves of the two groups of rats from 14 to 30 days. The curve of the rats injected at 14 days is below that of the normal rats. However. the weight differences between the two groups are statistically significant only at 30 days. Table I shows the differences in the length of the measured bones and bone units between 14 and 21 days and between 21 and 30 days in the two groups of rats together with the angular changes between 14 and 30 days. Significant differences between the groups are present only in the gain in length of the nasal bone and of the total skull, and only between 21 and 30 days. Howcvcr, a systematic difference in the measurements of length gain is present both between 14 and 21 days and between 21 and 30 days, being more marked in the latter age period than in the former. During both periods, the measured units of the controls usually have increased more in length than those of the experimental group.
Fig. 2. Weight velocity of the two groups of rats from 30 to 45 days. Normal rats -~ Experimental rats ~.
porary weight loss for some days in rabbits. Hoyte (1960) arrived at the same result both in rabbits and guinea-pigs. In the rat, Schour et (I/. (1941) recommended a dose of 100 mg alizarin red S per kg body weight and found a temporary retardation of gain in weight followed by a quick recovery after injection of doses of this magnitude. Similar findings have been published by Hoyte (1960). whereas Cleall. Perkins and Gilda (1964) reached the conclusion that doses less than 520 mg/kg body weight cause no retardation of weight gain in rats at about 3540 days of age. However, Ronning (1971). in a study of the growth of rats from the age of 4 days, found a temporary retardation in weight gain after injection of 400 mg alizarin per kg body weight. The observations from the present study add new information to that formerly obtained. It seems that the response to injection of dye in some way is age dependent. The weight of rats injected at 14 days is initially little affected. Nevertheless, the injected rats are finahy smaller than the untreated rats. The weight of rats injected at 30 days is initially slightly affected. However. these rats recover and reach normal weights within a few days. The explanation of this phenomenon might, as suggested by Running (1971). be that the effect of the dye on the growth may be comparable to effects observed after induction of various stress conditions. such as cold, isolation and undernutrition. As such gms
50.
DISCUSSION
The effect of injections of alizarin red S on weight gain and bone formation has been discussed thoroughly in the literature. Hong et nl. (1968)have shown that doses of only 50 mg/kg body weight cause a tem-
LO-
30.
20.
‘i IL
2;
I 30
days
Fig. 1. Weight velocity of the two groups of rats from 14 to 30 ddys. Normal rats ~Experimental rats ~~
, I&
21
30
days
Fig. 3. Weight of the two groups of rats from I4 to 30 days. Normal rats -_ Experimental rats -
EfTccts ofahzarin
red S
IO09
T:~hlc I. Means (XI ;~nd standard deviations (s,) of the differences in distances and angles in the skull of the control rats (c) and the alizarin-injected I)i~l2llcc~
~~~
I
-..._
2
C I4
2 I da!
2I
\
30&I>5
45 u
I
C
7x
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C
;I
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rats (a)
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c
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C
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C
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;I
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Y
245
\ \
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‘~
0~.3h 0.x
0.21 0.1-1 0, 13 0. 18 0.1’)
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~_.____ c
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-6.7 30
0I I3
-0.3 I -.?
- .:a 3.2
~ 3.3 -7.3
from the two groups differ significantI> at the I per cent Ic\cl.
\trcs\ conditions
influence the rat more seriously lht’lorc weaning than after weaning (Widdowson and McCancc. 1963). the observed differences in weight
the morphologic appearances of radioactlvc calcium andalizarinredSconcluded that a single intrapcritoncal inlcction of alizarin red S does not disturb the normal
gain hctvx~n the‘txo
growth pattern of the single bones in the rat &III. Running (I971 ). in his stud) of the gro\\th of skull bones in young rats. quantified the retarding cllecl 01 400 mg,!kg body weight of the dye on bone formation. He found differences in several length and height measurements at IO days between animals inlcctcd at 4 days and control animals. Some of these d&crcnccs had. however. disappeared b\ I5 da>\ of age nhilc others remamed over a longer period. III the prsscnt stud). it was not possible to detnonstratc an? signilicant retarding effect in any of the nourocranial bones. Only the growth of the: nasal bone. i.c. :I hone rcpresentmg a unit of the face, was significantly afTcctcd and only at 30 days. However. there was ;I systematic dilkrence of increasing significance with incrcaGng age in the growth rate of the bones of the ali/arin-iri,jcctcil rats. It may be thus concluded that ~hcrc arc limtations in the rcliabilit\ of result\ from >tudics using alicarin red S injections at I4 day\. The importance C; these limitations increases with increaGng lxriod 01 sur\i\al.
~>LI~S
studied ma? be related to
Ihi\ condition. ~2lthounh the re\ults I-cparding the severit) of the inIlu~wx olrtlie d!c 011 young animals arc in conformity \\ith the results of studies on the influence of stress. an Initial catch-up growth followed by a subnormal NCI&I gain ;I< hnd in the present study. has not been d&ibcd before. ‘This phenomenon may have several ~\pl;lnations. One possihitity is that the rcstllts have
arixx as ;I conscqucnce of selection. Thcrcfore the uond e\pcrmicntal tYOLI~ of Il-da)-old rats was wormed and given alizarin. with the same result. :Znoth~‘r po\siblc explanation of the phenomenon InighC he that. in suckling rats. certain age levels are more critical th;ln c,tlw~-s. Moss and BXI- (1956). in a ad\ of tliflicrsntial growth of the rat skull. observed J cl-itical ~lc\,cl~)l~rncn~athoriron in the skull bones at .~hout IO &I!\ in ~11~‘ Long-Evans strain and at about IS (Iam in the M’istu alh~no strain. This might in some \I :I\ Ihc rstatcd 10 I tic‘ t~tw~omenon mcntioncd dx~~c. i-11~ wtxght pin of the rats injected at 30 da!s \ras ~ud~cd to lx onI\ slightly afIccted. although Fig. 2 Iu\cs the impi-cs\ioii of ;I scvcrt‘ atliiction. It is neccssx1-1 to heat- ill ~niml that UK rats in fhc prcscnt stud) \\L’IC tahcn li-om (hail- mother on the da> of initiation 01 Ihc’ cuperimcnt. NOI-nial rats react b! gainin! less \\crght for 2 d;~\\. 131adding another stress condition, the 11oriii;~l appcar;inc~ 01 the \clocit! curve onl) Ihccome~xccntuatcd. not changed. I Iong (‘I rrl. ( I W) found ;I retardation of bone forI~;IIKW in the‘ r;lbhit ;rftcr injection of 50 I00 mg, kg hotI! weight 01’ ;Iti/;lt-In I-cd S. Hoqtc (1960) found I ~tartlatlon both in rhc rabbit and in the rat. 14hercas 111 t tic rat both Scllour ( 1936) and Cleall cf rrl. ( 1964) louml no retarding clfcct of the dye on bone grou th. Ilouc~cr, both Ho)r~ (1960) and Dixon and tioytc
Cleall J. F‘.. Perkins R. E. and Gilda _I. t. I9hJ. Bone marhing agents for the longitudinal stud! ofgrowth in animals. ./Ir<~h.\ ortrl Biol. 9, 627 -646. Dixon A. D. and Hoyte D. A. N. 1963. A compar~n 01 autoradiographic and alirarin technique\ in ~ht’ \tud) 01 bone growth. .%trrr. Rw. 145. IO1 I I?. Hong Y. C.. Yen P. K.-J. and Shaw .I H. 19hX. M~oscop~c evaluation of the effects of some bital staining agents on growing bone in rahblts. Calc. Tis.\. KC,\.2, 2~6 295. Ho!te D. A. N. 1960. Alizarm as an indicator of hone growth. J. 41lur. 94, 432~ 443. Moss M. L. and Bacr M. J. 1956. Differential ~rov, th of the
H.
1010
Vilmann
Renning 0. 1971. Alterations in craniofacial morphogenesis induced by parenterally administered papain. Academic Dissertation. University of Turku. Finland. Schour 1. 1936. Measurements of bone growth bq alirarinc injections. PJYK. Sot,. rup. Biol. Mrrl. 34, 140 141. Schour I., Hotmmann M.. Sarnat B. and Engel B. 1941. Vital staining of growing bones J. tlr,jr. Rex 20, 41 I-418.
and teeth with ali7arin
red S.
Vilmann H. 196X. The growth of the parietal bone in the albino rat studied by roentgenocephalometry and vital staining. 3w/1,5 ortrl Biol. 13, X87-901. Vilmann H. 1971. The growth of the cranlal vault m the albino rat. 3r,c/1.\ wtrl Biol. 17, 399 414. Widdowson E. M. and McCance R. A. 1963. The effect of linite periods of undernutrition at different ages on the composition and subsequent development of the rat. Proc~. R. Sot,. Series B. 158, 329~ 342.
R&sum&La rkgularitk des conclusions tirCes des Ctudes sur la croissance, en combinant la &phalom&rie aux rayons-X et la coloration vitale avec le rouge d’alizarine S est dt-pendante de la connaissance des erreurs de mt-thode impliqut-es. La signification des erreurs de mtthode dans les i-tudes cCphalom6triques aux rayons-X est bien connue, tandis que les expressions des erreurs de mkthode dans les Ctudcs utilisant le rouge d’alizarine S sont rares. Par consCquent, on a entrepris une Ctude qui pourrait kclairer ce problkme. Les injections intrapCriton6ales de lOOmg/kg de poids corporel de rouge d’alizarine S ont retard&es le gain de poids des rats 2g6s de 14 et 3Ojours, pendant 2 jours. Les rats Lg& de 30 jours ont recouvrk vite, tandis que les rats Bgt-s de 14 jours apris un recouvrement rapide, ont gag& moins de poids que les rats normaux. Ces rats finirent comme animaux plus petits. Des difft-rences significatives dans les mesurages de l’os crinien, entre les rats inject&s avec du rouge d’alizarine S apr&s 14 jours et les rats tCmoins ktaient kgdlement prksentes apr&s 30 jours.
Zusammenfassung-Die Verl%Blichkeit von Schliissen, die aus Wachstumsuntersuchungen gezogen werden, welche Rantgenstrahl-Kraniometrie und grundlegende FLrbung mit Alizarinrot S kombiniert, hlngt von der Kenntnis der verbundencn Verfahrensabwcichungen ab. Die Bedeutung von Verfahrensabweichungen in Riintgenkraniometrieuntersuchungen sind allgemein bekannt. dagegen sind Ausdriickc fiir Abweichungen itn Verfahren in Untersuchungen mit Alizarin-Rot S splrlich. Daher wurde eine Untersuchung durchgefiihrt. welche dieses Problem aufklgren kiinnte. Interperitoneale Injektionen von 100 mg/kg Kiirpergewicht von Alizarinrot S verziigerte den Gewichtszuwachs von 14 und 30 Tage alten Ratten urn 2 Tage. Die 30 Tage alten Ratten erholten sich schnell, wlhrend die 14 Tage alten Rattcn nach schnellcr Erhoiung an Gewicht wcniger als normale Ratten zunahmen. Diese Ratten wuchsen zu kleineren Tieren heran. Bedeutende Unterschiede in kranialen Knochenmessungcn rwischcn Ratten. mit Alizarinrot S injizierten 14 Tage- und Kontrollrattcn. warcn such bci 30 Tagcn \#orhanden.