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Alteraciones morfologicas y morfometricas inducidas por acido valproico en cartilago de Meckel, musculatura lingual y glandula submandibular de fetos de ratas.


RESUMEN: El acido valproico es una droga antiepileptica, bien conocida como agente teratogenico; su principal organo blanco es el tubo neural, aunque malformaciones de otros organos tambien han sido descritas. El proposito del trabajo fue analizar los efectos del acido valproico en las estructuras orales embriofetales: Cartilago de Meckel, musculos de la lengua y glandula submandibular. Las ratas recibieron una inyeccion diaria intraperitoneal de acido valproico (300 mg/Kg), durante 10 dias de su gestacion. Las ratas hembras fueron sacrificadas al 20 dia post coito y fueron examinados los fetos. No fueron observadas malformaciones macroscopicas. Fueron observadas las siguientes malformaciones: bajo peso del cuerpo y de la placenta; cordon umbilical corto; parenquima de la glandula submandibular menos diferenciada con gran cordon celular y ramificaciones terminales e incremento del volumen nuclear; las fibras de los musculos de la lengua desorganizados y de menor tamano y de volumen nuclear pequeno; cartilago de Meckel rudimentario, con pequenos condrocitos y muy abundante matriz. Estos datos sugieren que alteraciones en el tejido oral, inducidos por acido valproico, como resultado de un efecto directo de la droga en los tejidos, provoca una diferenciacion retardada de ellos.

PALABRAS CLAVE: 1. Acido valproico; 2. Rata; 3. Cartilago de Meckel; 4. Musculatura lingual; 5. Glandula submandibular.

SUMMARY: Valproic acid, an antiepileptic drug, is a well-known teratogenic agent; its main target organ is the neural tube, though organ malformations have also been described. The aim of the present work was to analyze the effects of valproic acid on embryo/ fetal oral structures: Meckel's cartilage, muscles of the tongue, and submandibular gland. Rats received a intraperitoneal injected of Valproic acid (300mg/Kg) on the 10th day of their gestation. Females were sacrificed on day 20 post coitus, and fetuses were examined. No gross malformations were observed. The following morphological alterations were observed: lower body and placental weight; shorter umbilical cord length; submandibular gland parenchyma less differentiated with larger cellular cord and terminal buds, and increased nuclear volume; lingual muscles fibers disorganized and of smaller size, and smaller nuclear volume; rudimentary Meckel's cartilage with small chondrocytes and more abundant matrix. These data suggest that alterations in oral tissue, induced by valproic acid, result from a direct effect of the drug on tissues causing a delayed differentiation.

KEY WORDS: 1. Valproic acid; 2. Rat; 3. Meckel's cartilage; 4. Lingual musculature; 5. Submandibular gland.


Valproic acid (VPA) has been shown to be a teratogenic in animals and man. In man, VPA exposure during pregnancy may be related to an increased incidence of ocular abnormalities (Glover et al., 2002), lumbosacral neural tube defects (bifid spine); as well as a spectrum of major malformations of the skeleton and at the level of the cardiovascular and urogenital system (Koch et al., 1983; Winter et al., 1987; Rodriguez-Pinilla et al., 2000; Kozma, 2001).

Multiple congenital malformations, including generalized hypertrichosis with gum hypertrophy, were observed in a child that had been exposed to valproic acid while in the womb (Stoll et al., 2003).

There are records of neural tube defects (bifid spine and exencephaly) in mice (Ehlers et al., 1992), and occult bifid spine in rats (Ceylan et al., 2001).

Skeletal defects, both facial and in the axial skeleton, have been observed in all tested species: mice, rats, and rabbits (Ong et al., 1983; Nau, 1985; Petrere et al., 1986; Ehlers et al.; Menegola et al., 1996).

The goal of the present study was to investigate the histopathological alteration caused by VPA in rats, on their submandibular gland, Meckel's cartilage, and tongue musculature, using appropriate karyometric and stereologic techniques.


Virgin female Wistar rats were housed in plastic cages and maintained in climate-controlled room under an alternating 12-hr light/dark cycle. Timed pregnancies were obtained by placing 2 females with singly caged males over night. Detection of vaginal spermatozoids was used to designate gestational day 1.

Pregnant females were treated intraperitoneally with saline (control) and with 300mg/Kg of VPA (3 times a day) on the 10th day of gestation. All females were killed on day 20 of gestation by ether inhalation, the abdomen was opened, and uterine contents were removed. The fetuses were collected, which were immediately immersed in a fixative solution consisting of alcohol (85ml), formalin (10ml), and acetic acid (5ml), and fixed for as long as 24 hours. After fixation, the heads were separated from the bodies, embedded in paraffin, cut into serial 6[micron]m sections and stained with hematoxylin and eosin. The following karyometric parameters were measured in the drawing of each nucleus of submandibular acini and ducts, and of lingual muscular fibers: longest, shortest, and mean diameters, the relation between longest and shortest diameters, perimeter, area, volume, the relation between volume and area, form coefficient, contour index and eccentricity (Sala et al., 1994).

The outlines of 50 chondroplasts projected with a camara lucida (Zeiss Jena) at a final magnification of 1000X, were drawn with soft pencil. The following morphometric parameters were used: axis, perimeter, area, and volume. Data were analyzed statistically by Mann-Whitney non-parametric test.


The fetuses that received valproic acid during pregnancy showed significantly lower body weight (1.32g), placental weight (320.6mg), and umbilical cord length (1.9cm) than control fetuses (4.78g, 541.9mg, and 2.4cm, respectively), but no gross malformations were observed in this work.

Histopathological analysis revealed submandibular gland parenchyma less differentiated in the valproic acid-treated fetuses. The submandibular glands of treated-fetuses consisted of cellular cord and larger terminal buds and increased nuclear volume (Fig. 1). Table I gives mean values for the nuclear parameters: it is possible to observe that all parameters studied showed significant differences between control and treated group. All these findings showed a delayed differentiation of the analyzed gland in treated animals.


The lingual muscles fibers of treated fetuses were disorganized and of smaller size; the round nuclei were of smaller volume (Fig. 2). The mean values of karyometric parameters are seen in Table I. It is possible to observe that all parameters studied showed significant differences between control and treated group.


The Meckel's cartilage of VPA treated fetuses was rudimentary, with small chondrocytes with smaller nuclei. The cartilaginous matrix is more abundant (Fig. 3). Table II gives mean values for chondroplast morphometric parameters: it is possible to observe smaller values for axis (longest, shortest, and geometric), perimeter, area, and volume. These findings showed a delayed differentiation of the analyzed cartilaginous tissue in the treated fetuses.



The VPA treated fetuses were developmentally delayed as compared to the controls, with all fetuses having lower body weight, placental weight, and umbilical cord length. Decreased fetal body weight was also observed in mice (Hansen et al., 1995; Menegola et al.) as well as in rats (Hansen et al.).

The concentration of VPA in placental and embryonic tissues were higher than those observed in maternal blood, as observed in monkeys (Dickinsson et al., 1979), suggesting that this drug affects the placental tissues, resulting in a smaller placenta, as observed in this work. Smaller placenta has a decreased blood flow, resulting in significant fetal hypoxia and delayed development (Emmanouilides et al., 1972).

VPA probably has a direct effect on the organization of the cartilaginous tissue of Meckel's cartilage, resulting in delayed differentiation, as observed in this work, with a reduction in the collagenous concentration and a decrease in the synthesis of proteoglycans (Aulthouse & Daron, 1994).

The mechanism for the drug's muscular and glandular effects is unknown. Probably the VPA exert its effect directly on lingual and submandibular structures. Our results confirm the VPA induced oral alterations previously described in rat fetuses' floor of the mouth (Freitas et al., 1999), tongue (Iyomasa et al., 1999) and palate mucous (Sousa et al., 1999), i.e., a picture of delayed differentiation tissues.

Several theories on the mechanism of VPA teratogenesis have been presented; the theory receiving the most experimental attention is that VPA acts through a deficiency of the folic acid vitamin (Lammer et al., 1987; Finnel & Dansky, 1991). Padmanabhan & Shafiullah (2003) suggest that plasma levels of folic acid and B 12 have to be kept substantially elevated and maintained high throughout the organogenesis period to protect embryos against VPA--induced neural tube defects. A number of studies have noted that VPA doesn't affect glycolysis, DNA, or protein synthesis (Turner et al., 1990). In 1996, Barnes et al. hypothesize that a candidate set of molecular targets of teratogens are the Pax family of pattern-forming genes, specifically Pax-a, which has been previously demonstrated to be an important regulator of axial skeletal patterning at the somite level.

ACKNOWLEDGMENTS: We would like to thank Luisa Caliri Juzzo for translating this paper to the English Language.


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Correspondence to:

Prof. Dra. Marisa Semprini

Faculdade de Odontologia de Ribeirao Preto--USP

Departamento de Morfologia, Estomatologia e Fisiologia

Avenida do Cafe, s/n Bairro: Monte Alegre

CEP: 14040-904

Ribeifao Preto--SP--BRASIL


Received: 22-02-2004

Accepted: 12-04-2004

*,** Selma Siessere; * Marisa Semprini; * Ruberval A. Lopes; * Miguel A. Sala; & * Maria G. C. Mattos

* College of Dentistry of Ribeirao Preto / Sao Paulo University, Ribeirao Preto, SP, Brazil. ** College of Dentistry of Uberaba / Uberaba University, Uberaba, MG, Brazil. This work is supported by grants from FAPESP (97/11738-6).
Table I. Mean values of karyometric parameters of submandibular
gland acinus, duct, and muscle fiber cell nuclei for control (C)
and valproic acid treated (T) Wistar rat fetuses. Mann-Whitney test.

Parameter                            Acinus              Duct

                                   C         T        C         T

Longest diameter ([micron]m)      5,50     7,44 *     5,25    6,80 *
Shortest diameter ([micron]m)     4,25     5,18 *     4,06    4,66 *
Mean diameter ([micron]m)         4,82     6,18 *     4,60    5,60 *
Ratio D/d                         1,32     1,45 *     1,31    1,48 *
Perimeter ([micron]m)            15,42    20,03 *    14,71   18,19 *
Area ([micron][m.sup.2])         18,43    30,49 *    16,87   24,97 *
Volume ([micron][m.sup.3])       60,44   129,29 *    53,34   95,43 *
Ratio V/A                         3,21     4,12 *     3,06    3,74 *
Form Coefficient                  0,96     0,94 *     0,96    0,93 *
Contour index                     3,61     3,65 *     3,61    3,66 *
Eccentricity                      0,54     0,69 *     0,53    0,69 *

Parameter                        Muscle fibers

                                  C         T

Longest diameter ([micron]m)    11,18    9,67 *
Shortest diameter ([micron]m)    3,77    3,93 **
Mean diameter ([micron]m)        6,44    6,13 **
Ratio D/d                        3,08    2,54 *
Perimeter ([micron]m)           29,31   16,87 *
Area ([micron][m.sup.2])        33,28   29,86 **
Volume ([micron][m.sup.3])      87,61   71,40 **
Ratio V/A                        2,52    6,45 *
Form Coefficient                 0,49    1,30 *
Contour index                    5,14    3,10 *
Eccentricity                     0,93    0,90 **

* Statistically different from the respective control ([infinito]=0,01)

** Statistically different from the respective control

Table II. Mean values of morphometric parameters of Meckel's
cartilage chondroplast for control (C) and valproic acid treated (T)
Wistar rat fetuses. Mann-Whitney test.

Parameter                           C           T

Longest diameter ([micron]m)       29,64      13,71 *
Shortest diameter ([micron]m)      22,35       9,21 *
Mean diameter ([micron]m)          25,65       8,93 *
Perimeter ([micron]m)              82,20      36,65 *
Area ([micron][m.sup.2])          536,85     105,95 *
Volume ([micron][m.sup.3])       9880,41     934,97 **

* Statistically different from the respective control ([infinito]=0,01)
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Author:Siessere, Selma; Semprini, Marisa; Lopes, Ruberval A.; Sala, Miguel A.; Mattos, Maria G.C.
Publication:International Journal of Morphology
Date:Jun 1, 2004
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