TEIZATOLOGY 461147-157(1992)
Inner Ear Malformations Induced by lsotretinoin in Hamster Fetuses DOROTHY T. BURK AND CALVIN C. WILLHITE Department of Anatomy, University of the Pacific School of Dentistry, Sun Francisco, California 941 15 (D.T.B.1; State of California Department of Toxic Substance Control, Berkeley, California 94710 fC.C.W.1
ABSTRACT
Inner ear malformations induced in anotic hamster fetuses following maternal treatment with 50 mgkg isotretinoin (13-cis-retinoic acid) on gestational day 8 are described. Computer-assisted three dimensional reconstruction was used. Two general types of defective vestibulocochlear development were seen. Defects were bilateral and correlated with extent of middle ear deficiency and severity of mandibular defects. In the more severely affected fetuses the inner ear was limited to an epithelial sac with occasional small projections, no apparent innervation and a correspondingly reduced otic capsule. In most of the fetuses examined the inner ear was less severely affected and was characterized by a reduction in the number of semicircular ducts and alterations in the size and shape of the cochlear duct. These defects are similar to those seen in a child with the isotretinoin embryopathy. Pathogenesis may result from a direct effect on otic epithelium or from faulty inductive interactions with the rhombencephalon or with periotic neural crest cells. 0 1992 Wiley-Liss, Inc.
The use of the vitamin A derivative, 13cis-retinoic acid (Accutane, isotretinoin), for treatment of severe acne by pregnant women has been associated with a malformation syndrome in the offspring. The chief defects include malformations of the central nervous system, such as hydrocephalus and cerebellar hypoplasia; cardiovascular defects, primarily conotruncal; craniofacial malformations, primarily microtia, orbital hypertelorism and micrognathia; and congenital thymic hypoplasia reminiscent of the DiGeorge syndrome (Lammer et al., '85; Rosa et al., '86). Animal models of the isotretinoin embryopathy, exhibiting most if not all of these anomalies, have been developed in the hamster (Willhite et al., '86), mouse (Webster et al., '86), chick (Hart et al., '90) and macaque (Hummler et al., '90). The human, however, appears to be more sensitive to this agent than any of the animal models (Teratology Society, '91). While malformation of the external ears (anotia, microtia, agenesis or hypoplasia of the ear canals), which is generally but not always bilateral, is one of the hallmarks of human isotretinoin embryopathy (Lott et 0 1992 WILEY-LISS. INC.
al., '84; Webster et al., '86), it seems likely that defects of the middle and/or inner ear and its innervation contribute to the variable degree of hearing loss and congenital deafness. An autopsy of a 4-month-old girl born to a mother who used Accutane during early pregnancy indicated the presence of an inner ear malformation associated with aural atresia on one side (E.J. Lammer: personal communication). Furthermore, anomalies of the sphenoid and temporal bones, suggestive of inner ear anomaly, accompanied lowset, hypoplastic or absent external ears and other characteristic craniofacial terata in three Accutane-exposed children who died between 19 weeks and three years of age (Siebert and Lammer '90). Temporal bone and ossicular abnormalities accompanied microtia in mouse embryos exposed to isotretinoin (Jarvis et al., '90; Louryan et al., '91). Since most aspects of the human isotretinoin embryopathy can be reproduced in hamsters, we describe here malformaReceived January 8, 1992; accepted March 17, 1992.
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tions of the inner ear in hamster fetuses recovered from dams treated with isotretinoin a t a dose that results in the consistent presence of anotialmicrotia as well as other craniofacial terata in the offspring (Willhite et al., '86).The results are discussed with regard to previous reports on paradigms of retinoid-induced aberrant morphogenesis and the role of neural crest cells in ear development.
with each digitized polygon (structure to be reconstructed), assigned a number code, and entered into the computer. In order to provide fiducial points for correct alignment of sections, we attempted to embed reference structures into paraffin with the specimen (Ongaro et al., '91). When this method was not successful, we relied on the best-fit method. Structures of the inner ear were reconstructed using the PC3D software, which allows the user to create three-dimensional MATERIALS AND METHODS images that can be rotated and viewed from Timed-pregnant Golden Syrian hamsters any perspective (Prothero and Prothero, were purchased from the Charles River '86). The images displayed are composed of Breeding Laboratories (Wilmington, MA) superimposed line tracings. Black and and cared for as previously described (Will- white plots were printed with the assistance hite and Book, '90). The day following the of Pizazz Plus print enhancement software evening of breeding was day 1 of gestation. (Application Techniques, Inc.). Isotretinoin (96%)was purchased from the RESULTS Eastman Kodak Company (Rochester, NY) Control fetuses and stored at -80°C. in the dark under argon. The drug was dissolved in acetone and At 14 days of gestation the vestibular pordiluted with polyoxyethylenesorbitan mono- tion of the inner ear of control fetuses exlaurate (Tween 20, Sigma Chemical Co., St. hibits three complete semicircular ducts Louis, MO), giving a final acetone concen- (anterior, posterior, and lateral), each lined tration of 5%. The isotretinoin solution was with squamous epithelium and oriented at prepared under yellow light with as little right angles to one another (Fig. la). Each exposure to air as possible. At 1O:OO A.M. on semicircular canal has a crista ampullaris, day 8 of pregnancy, hamsters were given a a ridge of tissue composed of tall columnar single intubation of 50 mglkg isotretinoin or epithelium with fine apical projections (Fig. an equivalent volume of the vehicle at 0.5 2a). Branches of the vestibular portion of mlilOO g body weight (Irving et al., '86).An- the 8th cranial nerve are visible in the mesimals were killed in excess C02 on day 14 enchyme underlying the cristae. The cristae and fetuses exhibiting anotia but not exen- of the anterior and lateral canals are located cephaly were fixed in Bouin's solution. near the junction of these two canals with Heads were embedded in paraffin and seri- the utricle, i.e., anteriorly, while the crista ally sectioned at 5 pm in the transverse or of the posterior canal is located near its in the coronal plane. Sections were stained opening into the caudal end of the utricle. with hematoxylin and eosin and examined Also comprising the vestibular apparatus with the light microscope. A total of 10 fe- are the utricle into which all the semicircutuses from 5 isotretinoin-treated litters and lar ducts empty and the more anteriorly lo4 control fetuses from 3 litters were evalu- cated saccule. Both the utricle and saccule contain sensory areas (maculae), which at ated. To aid in understanding the complex in- this stage of development are recognized by ner ear morphology, the inner ears and as- the presence of tall epithelial cells with apisociated structures of two control and five cal microvilli and associated subepithelial isotretinoin-treated fetuses were recon- nerve fibers. Otoliths are not yet visible. structed using a computer-assisted three di- Projecting from the medial side of the utrimensional reconstruction program (PC3D, cle and saccule is the narrow endolymphatic Jandel Scientific, Corte Madera, CAI. Cam- duct which is directed dorsally and ends in a era lucida drawings were made of sections dilated sac adjacent to the brain (Fig. 2a). at constant intervals (typically every third Anteriorly, the saccule is continuous with section) by projecting sections at constant the cochlear duct. When fully developed the hamster coknown magnification using a Bausch and Lomb microprojector and tracing structures chlear duct exhibits two and one-half turns of interest onto white paper. The drawings or coils (Stephens, '72), however, at day 14 were then digitized (Numonics model 2210) only one and one-half to two turns are
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A
B
I C
C
c Fig. 1. A Reconstruction of the vestibular apparatus of a control fetus observed from the medial side and 15" above the horizontal plane. Anterior is to the left. Anterior semicircular duct (a); posterior semicircular duct (p); lateral semicircular duct (1); utricle (u);saccule (s). B: Reconstruction of the cochlear duct (c) of a control fetus viewed from anterolateral and 15" above the horizontal plane. At least one and one-half turns in the cochlear duct are apparent. C: Reconstruction of a severely malformed inner ear from an isotretinoin-treated fetus viewed from the medial side and 15" above the
horizontal plane. Anterior is to the right. The entire inner ear is composed of a saclike epithelial structure (otocyst). A small projection of epithelium is present (arrow). D: Reconstruction of a moderately malformed inner ear of a n isotretinoin-treated fetus viewed from anterolateral and 15" below the horizontal plane. The anterior semicircular duct (a) is complete and there is a broad lateral projection (1) from the utricle in a location suggesting the lateral semicircular duct. The cochlear duct (c) in this particular fetus was reduced in length and makes approximately one complete turn.
present (Fig. lb). Perilymph spaces, which will coalesce into the scala vestibuli and scala tympani, are beginning to appear within the mesenchyme surrounding the duct (Fig. 2b). The organ of Corti is not yet differentiated, but spiral ganglion cells and associated nerve fibers are prominent. The entire inner ear is encased in the cartilaginous otic capsule with openings through the capsule located on the medial side for entrance of the vestibulocochlear nerve and on the lateral side in association with the middle ear region. Although not reconstructed or studied in detail, external and middle ear structures were easily observed in sectioned control specimens. The pinna of the external ear is prominent and a solid, ie., not yet cana-
lized, cord of epithelium represents the ear canal. This epithelial cord ends in the vicinity of the middle ear cavity (Fig. 2b), which in turn exhibits a connection to the pharynx (Eustachian tube). Cartilaginous ear ossicles are prominent, and the facial nerve and stapedial artery are present in the middle ear region.
Isotretinoin-treated fetuses All of the inner ears of day 14 isotretinoin-treated fetuses examined were malformed to some degree, but the severity of the malformation varied. In general, there were two basic degrees of malformations with the degree of severity consistent bilaterally within a given fetus and generally consistent within littermates. One group
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Fig. 2. A: Low magnification of a transverse section through the vestibular apparatus of a day 14 control fetus. The lateral semicircular duct (1) is entering the utricle (u). Nerve fibers of the vestibular nerve (v) are visible between the vestibular ganglion (vg) and the crista of the lateral semicircular duct (arrow). Posterior
semicircular duct (p); crus communis (cr); endolymphatic duct (e). ( x 80) B: Low magnification of a section through the cochlea of a control fetus. Cochlear duct (c); spiral (cochlear) ganglion and nerve (sg); middle ear cavity (me); stapedial artery (sa); facial nerve (fn) ( x 80).
had severely retarded inner ear development characterized by an inner ear resembling a simple epithelial otocyst encased within a cartilaginous capsule. In the other group the inner ears showed a more ad-
vanced state of development but still displayed some abnormalities. In the more severely affected fetuses the inner ear structure was no more than rudimentary with the inner ear consisting of an
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ovoid epithelially lined sac often only in- spite gross morphologic differences, cocompletely encased in cartilage (Fig. lc). Al- chlear histology appeared similar to the though, in one instance, an epithelial sac controls. Spiral ganglion cells and nerve fiwas present with no cartilage lateral to it bers were observed in association with the (Fig. 3b), more often the cartilage capsule cochlear ducts as were small perilymph was open on the medial side. In most cases, spaces. As in controls, the organ of Corti had short epithelial projections, suggestive of an not yet differentiated within the cochlear abortive endolymphatic duct or a rudimen- duct at this stage. The cartilaginous capsule tary semicircular canal flange, were ob- appeared to reflect the anatomy of the unserved (Fig. 3a). All in all, inner ear devel- derlying membranous labyrinth. opment in these severely malformed fetuses In this second group of embryos no exterappeared not to have progressed morpholog- nal ear structures, pinna or external ear caically beyond the otocyst stage (comparable nal were observed. The facial nerve and stato approximately day 9 of gestation). Inter- pedial artery were seen in the middle ear estingly, in a few of these severely mal- region, but, although the pharynx someformed inner ears there were areas of epi- times displayed a distinct lateral projection thelium with the histologic features of (Fig. 4b), no middle ear cavity was ever sensory tissue, i.e., a folded or raised area identified. Numerous cartilaginous nodules composed of tall epithelial cells with hair- were observed in the area of the middle ear like apical projections. (Fig. 312).No nerve including some in close association with the fibers were observed in association with stapedial artery, but their exact form and these areas, however. In fact, neither the identity is difficult to surmise without re8th nor the 7th cranial nerves were appar- construction. Meckel's and Reichert's cartient in any of these fetuses. In contrast, gan- lages could be easily identified and followed glia and nerve fibers of the 5th, 9th, and into the first and second pharyngeal arches. 10th cranial nerves were prominent. FeDISCUSSION tuses with severe inner ear abnormalities also had severe middle and external ear deThe two basic types of inner ear defects fects as well as micrognathia and tongue de- seen in the isotretinoin exposed fetuses defects. They exhibited no sign of an external scribed above are consistent with the huear or ear canal epithelium. A middle ear man ear anomalies known as Michel aplasia cavity was lacking and it was difficult to and the Mondini-Alexander defect. These distinguish whether any of the ossicles were conditions are two of the four main categopresent. ries of human congenital deafness due to There was a second population of iso- failure of the inner ear to reach full develtretinoin-treated fetuses that exhibited sig- opment (Ormerod, '60; Lim, '72). Michel nificant, but less severe, abnormality of the aplasia, originally defined as total lack of inner ear than those just described. In this development of the inner ear and petrous group, inner ears were characterized by a temporal bone, also describes a lesser dereduction in the number of semicircular gree of dysplasia in which a petrous bone is ducts and in the size and shape of the co- present and inner ear components are repchlea (Fig. ld). For example, in the speci- resented by vestigial membrane structures. men illustrated in Figure 4a, the anterior This would seem to describe as well the semicircular duct was complete but the pos- hamster fetuses considered to be most seterior and lateral ducts were represented verely affected by isotretinoin treatment. only by small bulges or flanges projecting Inner ear defects in the moderately affected from the utricle. Innervated cristae were ob- fetuses are best described as the Mondiniserved in complete canals and sometimes in Alexander defect, which is characterized by incomplete ones and the vestibular ganglion a flattened cochlea with development of the was identifiable. An endolymphatic duct basal coil only and with a comparable unwas consistently observed on the medial derdevelopment of the vestibular strucside passing through the cartilaginous otic tures. This defect may be unilateral or bicapsule and into the utricle and saccule. The lateral and a number of variations have cochlear ducts showed tighter coiling pat- been described (Lim, 1972). Sensory epitheterns than controls and reductions in the lium may be present either in the cochlea or number of turns, which varied from one- in the sacculovestibular cavity. half to one and one-half (Figs. Id, 4b). DeThe inner ear defect reported in the hu-
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Fig. 3. Transverse sections through the severely malformed inner ears of three day 14 isotretinointreated fetuses. Anterior is to the right and lateral toward the top. ( x 100)A: A simple sac-like otocyst with a small epithelial projection on the medial side (arrow).
B: Inner ear is represented by an epithelial sac with a lateral projection herniating through the otic capsule (oc). C: Otocyst contains tissue with features of a sensory area (arrow) but lacking innervation.
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Fig. 4. A: Low magnification of a transverse section through the inner ear (vestibular apparatus and a portion of the cochlea) of the isotretinoin-treated fetus reconstructed in figure Id. Utricle (u), lateral extension from utricle (1); anterior semicircular duct (a); endolymphatic duct (e); saccule (s).( x 80) B: Low magnification
of a section through the cochlea of an isotretinointreated fetus. In this specimen the cochlear duct (c) ifi small and tight but at least two coils are evident. Saccule (s); pharynx (p), facial nerve (fn);Meckel's cartilage (m) ( x 80).
man postmortem of a child whose mother used Accutane during pregnancy was diagnosed as a Mondini defect (Lammer: personal communication). The child, who died at four months of apnea secondary to struc-
tural brain abnormalities, had a cochlea characterized by one and one-half turns (normal = 2 VZ), near total absence of cochlear neurons and an abnormally large utricle and saccule. This defect was found on
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the left side only and was associated with a left aural atresia. Findings were considered to be consistent with normal hearing on the right and severe hearing loss on the left. In the present study of anotic fetuses induced by 50 mgkg isotretinoin, inner ear malformations were identified in all fetuses studied. The defects were always bilateral and the severity of inner ear maldevelopment appeared to correlate with the extent of middle ear deficiency and degree of micrognathia. Similarly, in a study of isotretinoin-treated mouse fetuses, inner ear anomalies (small deformed labyrinth, abnormalities of cochlear shape or size, abnormalities of internal auditory meatus, or oval window) were most often associated with severely affected external ears (Jarvis, '90). Other active retinoids have also been reported to cause inner ear anomalies in association with anomalies of the external and middle ear. A correlation between the severity of the malformations of the external auditory meatus, middle, and inner ear and the degree of malformation of the auricle was noted in mouse offspring treated with hypervitaminosis A (Geelen, '79). Padmanabhan ('90) found a similar relation in the offspring of mice treated with all-trunsretinoic acid on day 7 of gestation. The anotic fetuses lacked a cochlea and spiral ganglia, while the microtic fetuses had a maximum of 1.5 turns of the cochlea with extreme dilation of the utricle and saccule and severe hemorrhage in the perilymphatic space. Inner ear defects similar to those seen in this study with isotretinoin have also been reported in both the human and primate thalidomide embryopathy and in Treacher Collins cases (Livingstone, '65; Jorgensen, '64; Newman and Hendrickx, '81). Both the retinoids and thalidomide (Newman and Hendrickx, '81) seem to have a specific critical period for induction of inner ear malformations that appears to involve the otic placode stage. For hamsters the critical time for all-trans-retinoic acid-induced inner ear malformations has been determined to be between days 7?h and 8% (0-18 somites) and for external ear defects slightly longer, between 7Y4 and 8V4 (Schenefelt, '72). At the time of administration of isotretinoin to the embryos in this study (day 8, 1O:OO A.M.), the otic placodes are present and the 2nd visceral arch is forming. Shortly thereafter (day 8Vz) the
otic pits will form and approach closure (Schenefelt, '72; Stephens, '72). On the other hand, when mice were treated with isotretinoin a t four gestational stages, the most severe inner ear defects resulted from treatment a t the time of gastrulation and were associated with exencephaly. However, inner ear defects were also produced in the absence of exencephaly by treatment at the time when the otic placode begins programmed cell death (Jarvis, '90). The nature of the defects produced in isotretinoin-treated fetuses suggests that development continued but was subsequently interrupted some time after the time of treatment. On the one extreme are the more severely malformed inner ears, characterized by simple epithelial sacs with or without small projections and an apparent absence of innervation, in which the morphogenesis of the otocyst appears to have been interrupted fairly shortly after the time of treatment at the equivalent of day 9 of gestation. On the other hand, other specimens show more development in which one complete semicircular duct is found (typically the anterior, which forms first embryologically) and coiling of the cochlea has occurred at the morphologic equivalent of approximately day 13 (Stevens, '72). Since cochlear coiling and histodifferentiation are not complete a t the time of birth in the hamster, we cannot rule out the possibility that continued inner ear morphogenesis may still occur, particularly in the more moderately malformed. We would expect that histodifferentiation of the organ of Corti and other sensory structures would continue. Nevertheless, it seems unlikely that postnatal growth could result in morphologic normality. It is also unlikely that the teratogenic effect of isotretinoin is actually delayed or extended. Pharmacokinetic studies in hamsters and mice have shown that isotretinoin is rapidly isomerized to alltrans-retinoic acid and that peak concentrations of both of these retinoids occur within four hours after treatment (Kochhar et al., '87; Howard et al., '89). This would suggest that the teratogenic effect is, in fact, initiated within a few hours after administration of the isotretinoin on day 8. It is of interest that in the severely malformed inner ears (comparable to Michel aplasia) areas with the morphological appearance of sensory tissue (cristae, maculae) appeared to differentiate in the appar-
ISOTRETINOIN-INDUCED INNER EAR MALFORMATIONS
ent absence of innervation by branches of the 8th cranial nerve. In fact, experimental studies support the hypothesis that the presence of the statoacoustic ganglion and possible trophic interaction with otic epithelium is not required for the differentiation of sensory areas in the inner ear, but rather that attractant fields produced by areas of differentiating sensory cells act to guide the nerve growth cones of ingrowing neurites to the appropriate tissues (Van De Water, 1988). The fact that sensory areas are occasionally seen in our severely malformed specimens is consistent with these findings.
Possible Pathogenesis One hypothesis put forward to explain the pattern of defects observed after retinoid treatment early in gestation has been that of interference with neural crest cell populations (Wiley '83; Webster et al., '86; Pratt et al., '87; Sulik et al., '88). The present data, however, beg the question as to whether the inner ear defects seen in isotretinoin-damaged individuals result from an effect on neural crest cell development or from some other fundamental mechanism, such as direct cytotoxic effects on the otic vesicle or its derivatives or possibly effects on otocyst development secondary to altered inductive interactions. Excessive necrosis in areas of normal programmed cell death may result in teratogenesis by depleting an area of cells for which the embryos cannot compensate (Sulik et al., '88). Programmed cell death has been observed in otic placodes of day 8 mouse embryos (Sulik et al., '88; Jarvis, '90) and in the ventromedial area of the rat otocyst (Marowitz et al., '77). Additionally, evidence suggests that, at least in chicks, cells derived from the otic placodesivesicle contribute most of the neurons in the vestibuloacoustic ganglion with neural crest cells giving rise only to supporting cells (D'Amico-Martel and Noden, '83; Noden, '84). Assuming a similar process in mammals, the absence of the 8th cranial nerve in the more severely malformed inner ears examined in this study indicates a lack of contribution of neuroblastic cells from the otic vesicle itself, suggestive of direct cytotoxicity to otic cells. Although the epithelium of the inner ear is derived from the otic placode, associated ear structures, such as the otic capsule, middle ear ossicles, and cartilage of the auricle
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differentiate from periotic mesenchyme derived from cells of neural crest origin along with a contribution from the paraxial mesoderm (Noden and Van De Water, '86). During development of the otocyst the periotic mesenchyme is thought to participate in inductive interactions critical to normal cochlear and labyrinthine morphogenesis, and, conversely, the periotic mesenchyme relies upon the otocyst for reciprocal induction of the otic capsule and stapedial footplate (Orr and Hafft, '80; Anniko and Schacht, '84; Noden and Van De Water, '86). Thus, we cannot rule out the possibility of a primary effect of isotretinoin on neural crest cells with consequent effects on interactions with the otocyst. In addition to the periotic mesenchyme, the rhombencephalon is also thought to play an inductive role in otic vesicle development. During early development the basement membranes of the otic vesicle and rhombencephalon are in direct contact (O'Rahilly, '63). Experimental studies in amphibians and avians have shown that disturbance of the normal embryonic relationship between the otic vesicle and the rhombencephalon results in abnormal development of the otic vesicle with defects ranging from an undifferentiated cyst-like stage to a malformed labyrinth with an imperfect otic capsule (Van de Water, '80). In studies of mouse mutants anomalies of the rhombencephalon, such as faulty segmentation and degeneration of the 4th rhombomere, have been correlated with abnormalities of the inner ear and otic capsule (Deol, '64, '66). Isotretinoin-treated cultured rat embryos exhibited a diminished second arch and a cranial shift in otic vesicle location, suggestive of hindbrain abnormality (Webster et al., '86). Inner ear defects are also common in the presence of exencephaly (Padmanabhan, '87; Jarvis et al., '90). Thus, an adverse effect of isotretinoin on the neuroepithelium of the rhombencephalon or on its segmentation pattern might result in brain defects as well as inner ear defects as a result of faulty inductive interactions. Specific localization of cellular retinoic acid binding proteins (CRABP) and nuclear retinoic acid receptors (RAR), taken together with observations on homeobox genes encoding segmentation of the rhombomeres may provide future insight into the molecular basis of isotretinoin craniofacial teratogenesis. Cells that express CRABP or
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nuclear RARs appear to be target cells for the teratogenic exogenous retinoids (Dencker, '90; Ruberte et al., '91). CRABP or its gene transcript has been detected in the hindbrain and in neural crest and placodederived tissues (Dencker et al., '90; Vaessen et al., '90; Ruberte et al., '91; Maden et al., '91) and, in the chick, in cells of the open auditory pit and later in the auditory vesicle (Vaessen et al., '90). RAR-P transcripts have been found in craniofacial mesenchyme, including mesenchyme surrounding the inner ear epithelium (Ruberte et al., 'go), and in hindbrain neuroectoderm (Smith and Eichele, '91), while RAR-y is prominent in cartilages, including the otic (Ruberte et al., '90)). Specification of regional identity in the central nervous system is thought to be a function of homeobox genes. Additionally, each area of neural crest derived tissue has been found to have a unique code of Hox 2 gene expression related to the rhombomeric origin of the crest. This suggests that the Hox 2 genes also provide part of the positional information to the neural crest and hence are involved in patterning the structures of the branchial arches as well (Hunt et al., '91). The fact that distribution of CRABP and Hox-2.9 are coincident in the hindbrain suggests the possibility that retinoic acid could also be used to establish gradients of homeobox gene expression, and thus play a role in determination of normal segmentation pattern in developing central and peripheral nervous systems (Maden et al., '91). ACKNOWLEDGMENTS
This work was supported in part by a grant from the Pacific Dental Research Foundation. LITERATURE CITED Anniko, M., and J. Schacht (1984) Inductive tissue interactions during inner ear development. Arch. Otorhinolaryngol., 24Ot27-33. DAmico-Martel, A., and D.M. Noden (1983) Contributions of placodal and neural crest cells to avian cranial peripheral ganglia. Am. J. Anat., 166t445-468. Dencker, L., E. Annerwall, C. Busch, and U. Eriksson (1990) Localization of specific retinoid-binding sites and expression of cellular retinoic-acid-binding protein (CRABP) in the early mouse embryo. Development, IlOt343-352. Deol, M.S. (1964) Abnormalities of the inner ear in kreisler mice, J. Embryol. Exp. Morphol., 12t475-490. Deol, M.S. 11966) Influence of the neural tube on the differentiation of the inner ear in the mammalian embryo. Nature, 209t219-220.
Geelen, J.A.G. (1979) Hypervitaminosis A induced teratogenesis. CRC Crit. Rev. Toxicol., 6:351-376. Hart, R.C., P.A. McCue, W.L. Ragland, K.J. Winn, and E.R. Unger (1990) Avian model for 13-cis retinoic acid embryopathy: Demonstration of neural crest related defects. Teratology, 41 t463-472. Howard, W.B., C.C. Willhite, S.T. Omaye, and R.P. Sharma (1989) Comparative distribution, pharmacokinetics and placental permeabilities of all-transretinoic acid, 13-cis-retinoic acid, all-trans-4-0~0retinoic acid, retinyl acetate and 9-cis-retinal in hamsters. Arch. Toxicol., 63t112-120. Hummler, H., R. Korte, and A.G. Hendrickx (1990) Induction of malformations in the cynomolgus monkey with 13-cis retinoic acid. Teratology, 42:263-272. Hunt, P., D. Wilkinson, and R. Krumlauf (1991) Patterning the vertebrate head Murine Hox 2 genes mark distinct subpopulations of premigratory and migrating cranial neural crest. Development, 112t4350. Irving, D.W., C.C. Willhite, and D.T. Burk (1986) Morphogenesis of isotretinoin-induced microcephaly and micrognathia studied by scanning electron microscopy. Teratology, 34t141-153. Jarvis, B.L., M.C. Johnston, and K.K. Sulik (1990)Congenital malformations of the external, middle and inner ear produced by isotretinoin exposure in mouse embryos. Otolaryngol. Head Neck Surg., 102r391401. Jorgensen, M.B., H.K. Kristensen, and N.H. Buch (1 9641 Thalidomide-induced anlania of the inner ear. J. Laryngol. Otol., 78t1095-1fOl. Kochhar, D.M., J . Craft, and H. Nau (1987) Teratogenicity and disposition of various retinoids in vivo and in vitro. In: Pharmacokinetics in Teratogenesis, Vol. 2. H. Nau and W.J. Scott. eds. CRC Press. Boca Raton. Florida, pp. 173-186. Lammer, E.J., D.T. Chen, R.M. Hoar, N.D. Agnish, P.J. Henke, J.T. Braun, C.J. Currv. P.M. Fernhoff, A.W. Grix, I.T. Lott, J.M. Richard and S.C. Sun (1985) Retinoic acid embryopathy. N. Engl. J . Med., 313: 837-841. Lim, D.J. (1977) Histology of the developing inner ear. Normal anatomy and developmental anomalies. In: Hearing Loss in Children. B.F. Jaffee, ed. University Park Press, Baltimore, pp. 27-50. Livingstone, G. (1965) Congenital ear abnormalities due to thalidomide. Proc. R. SOC. Med., 58t493-497. Lott, LT., M. Bocian, H.W. Pribram, and M. Leitner (1984) Fetal hydrocephalus and ear anomalies associated with maternal use of isotretinoin. J. Pediatr., 105t595-597. Louryan, S., and R. Glineur (1991) Mouse middle ear abnormalities following retinoic acid administration. Comparison with fadfar embryos features. Teratology, 44:25A. Maden, M., P. Hunt, U. Eriksson, A. Kuroiwa, R. Krumlauf, and D. Summerbell (1991) Retinoic acid-binding protein, rhombomeres and the neural crest. Development, 111t35-44. Marovitz, W.F., K.M. Khan, and T. Schulte (1977) Studies in otic embryogenesis. 2. Ultrastructural development of the early rat otocyst. Ann. Otol. Rhinol. Laryngol., 86(Suppl. 35):9-28. Newman, L.M., and A.G. Hendrickx (1981) Fetal ear malformations induced by maternal ingestion of thalidomide in the bonnet monkey (Macaca radiata).Teratology, 23t351-364. Noden, D.M. (1984) Craniofacial development New views on old problems. Anat. Rec., 208:1-13. Noden, D.M., and T.R. Van De Water (1986) The devel-
ISOTRETINOIN-INDUCED INNER EAR MALFORMATIONS oping ear: Tissue origins and interactions. In: The Biology of Change in Otolaryngology. R.W. Ruben, T.R. Van De Water, and E.W. Rubel, eds. Elsevier, New York, pp. 15-46. Ongaro, I., G.H. Sperber, G.A. Machin, and C.A. Murdoch (1991) Fiducial points for three-dimensional computer-assisted reconstruction of serial light microscopic sections of umbilical cord. Anat. Rec., 229~285289. ORahilly, R. (1963) The early development of the otic vesicle in staged human embryos. J Embryol. Exp. Morphol., 11t741-755. Ormerod, F.C. (1960) The pathology of congenital deafness. J. Laryngol. Otol., 74:919-950. Orr, M.F., and L.P. Hafft (1980) The influence of mesenchyme on the development of the embryonic otocyst An electron microscopic study. J. Cell Biol., 87: 27a. Padmanabhan, R. (1987) Abnormalities of the ear associated with exencephaly in mouse fetuses induced by maternal exposure to cadmium. Teratology, 35:9-18. Padmanabhan, R., H.R. Vaidya, and A.A.F. Abu-Alatta (1990) Malformations of the ear induced by maternal exposure to retinoic acid in the mouse fetuses. Teratology, 42r25A. Pratt, R.M., E.H. Goulding, and B.D. Abbott (1987) Retinoic acid inhibits migration of cranial neural crest cells in the cultured mouse embryo. J. Craniofac. Gen. Dev. Biol., 7t205-217. Prothero, J.S., and J.W. Prothero (1986) Three-dimensional reconstruction from serial sections. IV. The reassembly problem. Computers Riomedic. Res., 19: 361-373. Rosa, F.W., A.L. Wilk, and F.O. Kelsey (1986) Teratogen update: Vitamin A Congeners. Teratology, 33: 355-364. Ruberte, E., P. Dtolle, A. Krust, A. Zelent, G. MorrissKay, and P. Chambon (1990) Specific spatial and temporal distribution of retinoic acid receptor gamma transcripts during mouse embryogenesis. Development, 108r213-222. Ruberte, E., P. Dolle, P. Chambon, and G. Morriss-Kay (1991) Retinoic acid receptors and cellular binding proteins. 11. Their differential pattern of transcription during early morphogenesis in mouse embryos. Development, 1llr45-60. Schenefelt, R.E. (1972) Morphogenesis of malformations in hamsters caused by retinoic acid: Relation
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to dose and stage at treatment. Teratology, 5;103118. Siebert, J.R., and E.J. Lammer (1990) Craniofacial anatomy of retinoic acid embryopathy. Teratology, 41: 592. Smith, S., and G. Eichele (1991) Temporal and regional differences in the expression pattern of distinct retinoic acid receptor-B transcripts in the chick embryo. Development, 1Ilr245-252. Stephens, C.B. (1972) Development of the middle and inner ear in the golden hamster (Mesocricetus auratus). A detailed description to establish a norm for pathophysiopathological study of congenital deafness. Acta Otolaryngol. Suppl., 296:l-51. Sulik, K.K., C.S. Cook, and W.S. Webster (1988) Teratogens and craniofacial malformations: Relationships to cell death. Development Suppl., 103:213-232. Teratology Society (1991) Recommendations for isotretinoin use in women of childbearing potential. Teratology, 44:l-6. Vaessen, M.-J., J.H.C. Meijers, D. Bootsma, and A.G. van Kessel (1990) The cellular retinoic-acid binding protein is expressed in tissues associated with retinoic-acid-induced malformations. Development, 110: 371-378. Van De Water, T.R. (1988) Tissue interactions and cell differentiation: Neurone-sensory cell interaction during otic development. Development Suppl. 103:185193. Van De Water, T.R., C.W. Li, R.J. Ruben, and C.A. Shea (1980) Ontogenic aspects of mammalian inner ear development. In: Morphogenesis and Malformation of the Ear. R.J. Gorlin, ed. Alan R. Liss, New York, pp. 5-45. Webster, W.S., M.C. Johnston, E.J. Lammer, and K.K. Sulik (1986) Isotretinoin embryopathy and the cranial neural crest: An in vivo and in vitro study. J. Craniofac. Genet. Dev. Biol., 6.211-222. Wiley, M.J., P. Cauwenbergs, and I.M. Taylor (1983) Effects of retinoic acid on the development of the facial skeleton in hamsters: Early changes involving cranial neural crest cells. Acta. Anat., 116:180-192. Willhite, C.C., and S.A. Rook (1990) Utility of disposition data in evaluating retinoid developmental toxicity. Methods Enzymol., 19Or406-417. Willhite, C.C., R.M. Hill, and D.W. Irving (1986) Isotretinoin-induced craniofacial malformations in humans and hamsters. J. Craniofac. Gen. Dev. Biol. Suppl., 2r193-209.
Inner Ear Malformations Induced by lsotretinoin in Hamster Fetuses DOROTHY T. BURK AND CALVIN C. WILLHITE Department of Anatomy, University of the Pacific School of Dentistry, Sun Francisco, California 941 15 (D.T.B.1; State of California Department of Toxic Substance Control, Berkeley, California 94710 fC.C.W.1
ABSTRACT
Inner ear malformations induced in anotic hamster fetuses following maternal treatment with 50 mgkg isotretinoin (13-cis-retinoic acid) on gestational day 8 are described. Computer-assisted three dimensional reconstruction was used. Two general types of defective vestibulocochlear development were seen. Defects were bilateral and correlated with extent of middle ear deficiency and severity of mandibular defects. In the more severely affected fetuses the inner ear was limited to an epithelial sac with occasional small projections, no apparent innervation and a correspondingly reduced otic capsule. In most of the fetuses examined the inner ear was less severely affected and was characterized by a reduction in the number of semicircular ducts and alterations in the size and shape of the cochlear duct. These defects are similar to those seen in a child with the isotretinoin embryopathy. Pathogenesis may result from a direct effect on otic epithelium or from faulty inductive interactions with the rhombencephalon or with periotic neural crest cells. 0 1992 Wiley-Liss, Inc.
The use of the vitamin A derivative, 13cis-retinoic acid (Accutane, isotretinoin), for treatment of severe acne by pregnant women has been associated with a malformation syndrome in the offspring. The chief defects include malformations of the central nervous system, such as hydrocephalus and cerebellar hypoplasia; cardiovascular defects, primarily conotruncal; craniofacial malformations, primarily microtia, orbital hypertelorism and micrognathia; and congenital thymic hypoplasia reminiscent of the DiGeorge syndrome (Lammer et al., '85; Rosa et al., '86). Animal models of the isotretinoin embryopathy, exhibiting most if not all of these anomalies, have been developed in the hamster (Willhite et al., '86), mouse (Webster et al., '86), chick (Hart et al., '90) and macaque (Hummler et al., '90). The human, however, appears to be more sensitive to this agent than any of the animal models (Teratology Society, '91). While malformation of the external ears (anotia, microtia, agenesis or hypoplasia of the ear canals), which is generally but not always bilateral, is one of the hallmarks of human isotretinoin embryopathy (Lott et 0 1992 WILEY-LISS. INC.
al., '84; Webster et al., '86), it seems likely that defects of the middle and/or inner ear and its innervation contribute to the variable degree of hearing loss and congenital deafness. An autopsy of a 4-month-old girl born to a mother who used Accutane during early pregnancy indicated the presence of an inner ear malformation associated with aural atresia on one side (E.J. Lammer: personal communication). Furthermore, anomalies of the sphenoid and temporal bones, suggestive of inner ear anomaly, accompanied lowset, hypoplastic or absent external ears and other characteristic craniofacial terata in three Accutane-exposed children who died between 19 weeks and three years of age (Siebert and Lammer '90). Temporal bone and ossicular abnormalities accompanied microtia in mouse embryos exposed to isotretinoin (Jarvis et al., '90; Louryan et al., '91). Since most aspects of the human isotretinoin embryopathy can be reproduced in hamsters, we describe here malformaReceived January 8, 1992; accepted March 17, 1992.
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tions of the inner ear in hamster fetuses recovered from dams treated with isotretinoin a t a dose that results in the consistent presence of anotialmicrotia as well as other craniofacial terata in the offspring (Willhite et al., '86).The results are discussed with regard to previous reports on paradigms of retinoid-induced aberrant morphogenesis and the role of neural crest cells in ear development.
with each digitized polygon (structure to be reconstructed), assigned a number code, and entered into the computer. In order to provide fiducial points for correct alignment of sections, we attempted to embed reference structures into paraffin with the specimen (Ongaro et al., '91). When this method was not successful, we relied on the best-fit method. Structures of the inner ear were reconstructed using the PC3D software, which allows the user to create three-dimensional MATERIALS AND METHODS images that can be rotated and viewed from Timed-pregnant Golden Syrian hamsters any perspective (Prothero and Prothero, were purchased from the Charles River '86). The images displayed are composed of Breeding Laboratories (Wilmington, MA) superimposed line tracings. Black and and cared for as previously described (Will- white plots were printed with the assistance hite and Book, '90). The day following the of Pizazz Plus print enhancement software evening of breeding was day 1 of gestation. (Application Techniques, Inc.). Isotretinoin (96%)was purchased from the RESULTS Eastman Kodak Company (Rochester, NY) Control fetuses and stored at -80°C. in the dark under argon. The drug was dissolved in acetone and At 14 days of gestation the vestibular pordiluted with polyoxyethylenesorbitan mono- tion of the inner ear of control fetuses exlaurate (Tween 20, Sigma Chemical Co., St. hibits three complete semicircular ducts Louis, MO), giving a final acetone concen- (anterior, posterior, and lateral), each lined tration of 5%. The isotretinoin solution was with squamous epithelium and oriented at prepared under yellow light with as little right angles to one another (Fig. la). Each exposure to air as possible. At 1O:OO A.M. on semicircular canal has a crista ampullaris, day 8 of pregnancy, hamsters were given a a ridge of tissue composed of tall columnar single intubation of 50 mglkg isotretinoin or epithelium with fine apical projections (Fig. an equivalent volume of the vehicle at 0.5 2a). Branches of the vestibular portion of mlilOO g body weight (Irving et al., '86).An- the 8th cranial nerve are visible in the mesimals were killed in excess C02 on day 14 enchyme underlying the cristae. The cristae and fetuses exhibiting anotia but not exen- of the anterior and lateral canals are located cephaly were fixed in Bouin's solution. near the junction of these two canals with Heads were embedded in paraffin and seri- the utricle, i.e., anteriorly, while the crista ally sectioned at 5 pm in the transverse or of the posterior canal is located near its in the coronal plane. Sections were stained opening into the caudal end of the utricle. with hematoxylin and eosin and examined Also comprising the vestibular apparatus with the light microscope. A total of 10 fe- are the utricle into which all the semicircutuses from 5 isotretinoin-treated litters and lar ducts empty and the more anteriorly lo4 control fetuses from 3 litters were evalu- cated saccule. Both the utricle and saccule contain sensory areas (maculae), which at ated. To aid in understanding the complex in- this stage of development are recognized by ner ear morphology, the inner ears and as- the presence of tall epithelial cells with apisociated structures of two control and five cal microvilli and associated subepithelial isotretinoin-treated fetuses were recon- nerve fibers. Otoliths are not yet visible. structed using a computer-assisted three di- Projecting from the medial side of the utrimensional reconstruction program (PC3D, cle and saccule is the narrow endolymphatic Jandel Scientific, Corte Madera, CAI. Cam- duct which is directed dorsally and ends in a era lucida drawings were made of sections dilated sac adjacent to the brain (Fig. 2a). at constant intervals (typically every third Anteriorly, the saccule is continuous with section) by projecting sections at constant the cochlear duct. When fully developed the hamster coknown magnification using a Bausch and Lomb microprojector and tracing structures chlear duct exhibits two and one-half turns of interest onto white paper. The drawings or coils (Stephens, '72), however, at day 14 were then digitized (Numonics model 2210) only one and one-half to two turns are
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A
B
I C
C
c Fig. 1. A Reconstruction of the vestibular apparatus of a control fetus observed from the medial side and 15" above the horizontal plane. Anterior is to the left. Anterior semicircular duct (a); posterior semicircular duct (p); lateral semicircular duct (1); utricle (u);saccule (s). B: Reconstruction of the cochlear duct (c) of a control fetus viewed from anterolateral and 15" above the horizontal plane. At least one and one-half turns in the cochlear duct are apparent. C: Reconstruction of a severely malformed inner ear from an isotretinoin-treated fetus viewed from the medial side and 15" above the
horizontal plane. Anterior is to the right. The entire inner ear is composed of a saclike epithelial structure (otocyst). A small projection of epithelium is present (arrow). D: Reconstruction of a moderately malformed inner ear of a n isotretinoin-treated fetus viewed from anterolateral and 15" below the horizontal plane. The anterior semicircular duct (a) is complete and there is a broad lateral projection (1) from the utricle in a location suggesting the lateral semicircular duct. The cochlear duct (c) in this particular fetus was reduced in length and makes approximately one complete turn.
present (Fig. lb). Perilymph spaces, which will coalesce into the scala vestibuli and scala tympani, are beginning to appear within the mesenchyme surrounding the duct (Fig. 2b). The organ of Corti is not yet differentiated, but spiral ganglion cells and associated nerve fibers are prominent. The entire inner ear is encased in the cartilaginous otic capsule with openings through the capsule located on the medial side for entrance of the vestibulocochlear nerve and on the lateral side in association with the middle ear region. Although not reconstructed or studied in detail, external and middle ear structures were easily observed in sectioned control specimens. The pinna of the external ear is prominent and a solid, ie., not yet cana-
lized, cord of epithelium represents the ear canal. This epithelial cord ends in the vicinity of the middle ear cavity (Fig. 2b), which in turn exhibits a connection to the pharynx (Eustachian tube). Cartilaginous ear ossicles are prominent, and the facial nerve and stapedial artery are present in the middle ear region.
Isotretinoin-treated fetuses All of the inner ears of day 14 isotretinoin-treated fetuses examined were malformed to some degree, but the severity of the malformation varied. In general, there were two basic degrees of malformations with the degree of severity consistent bilaterally within a given fetus and generally consistent within littermates. One group
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Fig. 2. A: Low magnification of a transverse section through the vestibular apparatus of a day 14 control fetus. The lateral semicircular duct (1) is entering the utricle (u). Nerve fibers of the vestibular nerve (v) are visible between the vestibular ganglion (vg) and the crista of the lateral semicircular duct (arrow). Posterior
semicircular duct (p); crus communis (cr); endolymphatic duct (e). ( x 80) B: Low magnification of a section through the cochlea of a control fetus. Cochlear duct (c); spiral (cochlear) ganglion and nerve (sg); middle ear cavity (me); stapedial artery (sa); facial nerve (fn) ( x 80).
had severely retarded inner ear development characterized by an inner ear resembling a simple epithelial otocyst encased within a cartilaginous capsule. In the other group the inner ears showed a more ad-
vanced state of development but still displayed some abnormalities. In the more severely affected fetuses the inner ear structure was no more than rudimentary with the inner ear consisting of an
ISOTRETINOIN-INDUCED INNER EAR MALFORMATIONS
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ovoid epithelially lined sac often only in- spite gross morphologic differences, cocompletely encased in cartilage (Fig. lc). Al- chlear histology appeared similar to the though, in one instance, an epithelial sac controls. Spiral ganglion cells and nerve fiwas present with no cartilage lateral to it bers were observed in association with the (Fig. 3b), more often the cartilage capsule cochlear ducts as were small perilymph was open on the medial side. In most cases, spaces. As in controls, the organ of Corti had short epithelial projections, suggestive of an not yet differentiated within the cochlear abortive endolymphatic duct or a rudimen- duct at this stage. The cartilaginous capsule tary semicircular canal flange, were ob- appeared to reflect the anatomy of the unserved (Fig. 3a). All in all, inner ear devel- derlying membranous labyrinth. opment in these severely malformed fetuses In this second group of embryos no exterappeared not to have progressed morpholog- nal ear structures, pinna or external ear caically beyond the otocyst stage (comparable nal were observed. The facial nerve and stato approximately day 9 of gestation). Inter- pedial artery were seen in the middle ear estingly, in a few of these severely mal- region, but, although the pharynx someformed inner ears there were areas of epi- times displayed a distinct lateral projection thelium with the histologic features of (Fig. 4b), no middle ear cavity was ever sensory tissue, i.e., a folded or raised area identified. Numerous cartilaginous nodules composed of tall epithelial cells with hair- were observed in the area of the middle ear like apical projections. (Fig. 312).No nerve including some in close association with the fibers were observed in association with stapedial artery, but their exact form and these areas, however. In fact, neither the identity is difficult to surmise without re8th nor the 7th cranial nerves were appar- construction. Meckel's and Reichert's cartient in any of these fetuses. In contrast, gan- lages could be easily identified and followed glia and nerve fibers of the 5th, 9th, and into the first and second pharyngeal arches. 10th cranial nerves were prominent. FeDISCUSSION tuses with severe inner ear abnormalities also had severe middle and external ear deThe two basic types of inner ear defects fects as well as micrognathia and tongue de- seen in the isotretinoin exposed fetuses defects. They exhibited no sign of an external scribed above are consistent with the huear or ear canal epithelium. A middle ear man ear anomalies known as Michel aplasia cavity was lacking and it was difficult to and the Mondini-Alexander defect. These distinguish whether any of the ossicles were conditions are two of the four main categopresent. ries of human congenital deafness due to There was a second population of iso- failure of the inner ear to reach full develtretinoin-treated fetuses that exhibited sig- opment (Ormerod, '60; Lim, '72). Michel nificant, but less severe, abnormality of the aplasia, originally defined as total lack of inner ear than those just described. In this development of the inner ear and petrous group, inner ears were characterized by a temporal bone, also describes a lesser dereduction in the number of semicircular gree of dysplasia in which a petrous bone is ducts and in the size and shape of the co- present and inner ear components are repchlea (Fig. ld). For example, in the speci- resented by vestigial membrane structures. men illustrated in Figure 4a, the anterior This would seem to describe as well the semicircular duct was complete but the pos- hamster fetuses considered to be most seterior and lateral ducts were represented verely affected by isotretinoin treatment. only by small bulges or flanges projecting Inner ear defects in the moderately affected from the utricle. Innervated cristae were ob- fetuses are best described as the Mondiniserved in complete canals and sometimes in Alexander defect, which is characterized by incomplete ones and the vestibular ganglion a flattened cochlea with development of the was identifiable. An endolymphatic duct basal coil only and with a comparable unwas consistently observed on the medial derdevelopment of the vestibular strucside passing through the cartilaginous otic tures. This defect may be unilateral or bicapsule and into the utricle and saccule. The lateral and a number of variations have cochlear ducts showed tighter coiling pat- been described (Lim, 1972). Sensory epitheterns than controls and reductions in the lium may be present either in the cochlea or number of turns, which varied from one- in the sacculovestibular cavity. half to one and one-half (Figs. Id, 4b). DeThe inner ear defect reported in the hu-
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Fig. 3. Transverse sections through the severely malformed inner ears of three day 14 isotretinointreated fetuses. Anterior is to the right and lateral toward the top. ( x 100)A: A simple sac-like otocyst with a small epithelial projection on the medial side (arrow).
B: Inner ear is represented by an epithelial sac with a lateral projection herniating through the otic capsule (oc). C: Otocyst contains tissue with features of a sensory area (arrow) but lacking innervation.
ISOTRETINOIN-INDUCED INNER EAR MALFORMATIONS
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Fig. 4. A: Low magnification of a transverse section through the inner ear (vestibular apparatus and a portion of the cochlea) of the isotretinoin-treated fetus reconstructed in figure Id. Utricle (u), lateral extension from utricle (1); anterior semicircular duct (a); endolymphatic duct (e); saccule (s).( x 80) B: Low magnification
of a section through the cochlea of an isotretinointreated fetus. In this specimen the cochlear duct (c) ifi small and tight but at least two coils are evident. Saccule (s); pharynx (p), facial nerve (fn);Meckel's cartilage (m) ( x 80).
man postmortem of a child whose mother used Accutane during pregnancy was diagnosed as a Mondini defect (Lammer: personal communication). The child, who died at four months of apnea secondary to struc-
tural brain abnormalities, had a cochlea characterized by one and one-half turns (normal = 2 VZ), near total absence of cochlear neurons and an abnormally large utricle and saccule. This defect was found on
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the left side only and was associated with a left aural atresia. Findings were considered to be consistent with normal hearing on the right and severe hearing loss on the left. In the present study of anotic fetuses induced by 50 mgkg isotretinoin, inner ear malformations were identified in all fetuses studied. The defects were always bilateral and the severity of inner ear maldevelopment appeared to correlate with the extent of middle ear deficiency and degree of micrognathia. Similarly, in a study of isotretinoin-treated mouse fetuses, inner ear anomalies (small deformed labyrinth, abnormalities of cochlear shape or size, abnormalities of internal auditory meatus, or oval window) were most often associated with severely affected external ears (Jarvis, '90). Other active retinoids have also been reported to cause inner ear anomalies in association with anomalies of the external and middle ear. A correlation between the severity of the malformations of the external auditory meatus, middle, and inner ear and the degree of malformation of the auricle was noted in mouse offspring treated with hypervitaminosis A (Geelen, '79). Padmanabhan ('90) found a similar relation in the offspring of mice treated with all-trunsretinoic acid on day 7 of gestation. The anotic fetuses lacked a cochlea and spiral ganglia, while the microtic fetuses had a maximum of 1.5 turns of the cochlea with extreme dilation of the utricle and saccule and severe hemorrhage in the perilymphatic space. Inner ear defects similar to those seen in this study with isotretinoin have also been reported in both the human and primate thalidomide embryopathy and in Treacher Collins cases (Livingstone, '65; Jorgensen, '64; Newman and Hendrickx, '81). Both the retinoids and thalidomide (Newman and Hendrickx, '81) seem to have a specific critical period for induction of inner ear malformations that appears to involve the otic placode stage. For hamsters the critical time for all-trans-retinoic acid-induced inner ear malformations has been determined to be between days 7?h and 8% (0-18 somites) and for external ear defects slightly longer, between 7Y4 and 8V4 (Schenefelt, '72). At the time of administration of isotretinoin to the embryos in this study (day 8, 1O:OO A.M.), the otic placodes are present and the 2nd visceral arch is forming. Shortly thereafter (day 8Vz) the
otic pits will form and approach closure (Schenefelt, '72; Stephens, '72). On the other hand, when mice were treated with isotretinoin a t four gestational stages, the most severe inner ear defects resulted from treatment a t the time of gastrulation and were associated with exencephaly. However, inner ear defects were also produced in the absence of exencephaly by treatment at the time when the otic placode begins programmed cell death (Jarvis, '90). The nature of the defects produced in isotretinoin-treated fetuses suggests that development continued but was subsequently interrupted some time after the time of treatment. On the one extreme are the more severely malformed inner ears, characterized by simple epithelial sacs with or without small projections and an apparent absence of innervation, in which the morphogenesis of the otocyst appears to have been interrupted fairly shortly after the time of treatment at the equivalent of day 9 of gestation. On the other hand, other specimens show more development in which one complete semicircular duct is found (typically the anterior, which forms first embryologically) and coiling of the cochlea has occurred at the morphologic equivalent of approximately day 13 (Stevens, '72). Since cochlear coiling and histodifferentiation are not complete a t the time of birth in the hamster, we cannot rule out the possibility that continued inner ear morphogenesis may still occur, particularly in the more moderately malformed. We would expect that histodifferentiation of the organ of Corti and other sensory structures would continue. Nevertheless, it seems unlikely that postnatal growth could result in morphologic normality. It is also unlikely that the teratogenic effect of isotretinoin is actually delayed or extended. Pharmacokinetic studies in hamsters and mice have shown that isotretinoin is rapidly isomerized to alltrans-retinoic acid and that peak concentrations of both of these retinoids occur within four hours after treatment (Kochhar et al., '87; Howard et al., '89). This would suggest that the teratogenic effect is, in fact, initiated within a few hours after administration of the isotretinoin on day 8. It is of interest that in the severely malformed inner ears (comparable to Michel aplasia) areas with the morphological appearance of sensory tissue (cristae, maculae) appeared to differentiate in the appar-
ISOTRETINOIN-INDUCED INNER EAR MALFORMATIONS
ent absence of innervation by branches of the 8th cranial nerve. In fact, experimental studies support the hypothesis that the presence of the statoacoustic ganglion and possible trophic interaction with otic epithelium is not required for the differentiation of sensory areas in the inner ear, but rather that attractant fields produced by areas of differentiating sensory cells act to guide the nerve growth cones of ingrowing neurites to the appropriate tissues (Van De Water, 1988). The fact that sensory areas are occasionally seen in our severely malformed specimens is consistent with these findings.
Possible Pathogenesis One hypothesis put forward to explain the pattern of defects observed after retinoid treatment early in gestation has been that of interference with neural crest cell populations (Wiley '83; Webster et al., '86; Pratt et al., '87; Sulik et al., '88). The present data, however, beg the question as to whether the inner ear defects seen in isotretinoin-damaged individuals result from an effect on neural crest cell development or from some other fundamental mechanism, such as direct cytotoxic effects on the otic vesicle or its derivatives or possibly effects on otocyst development secondary to altered inductive interactions. Excessive necrosis in areas of normal programmed cell death may result in teratogenesis by depleting an area of cells for which the embryos cannot compensate (Sulik et al., '88). Programmed cell death has been observed in otic placodes of day 8 mouse embryos (Sulik et al., '88; Jarvis, '90) and in the ventromedial area of the rat otocyst (Marowitz et al., '77). Additionally, evidence suggests that, at least in chicks, cells derived from the otic placodesivesicle contribute most of the neurons in the vestibuloacoustic ganglion with neural crest cells giving rise only to supporting cells (D'Amico-Martel and Noden, '83; Noden, '84). Assuming a similar process in mammals, the absence of the 8th cranial nerve in the more severely malformed inner ears examined in this study indicates a lack of contribution of neuroblastic cells from the otic vesicle itself, suggestive of direct cytotoxicity to otic cells. Although the epithelium of the inner ear is derived from the otic placode, associated ear structures, such as the otic capsule, middle ear ossicles, and cartilage of the auricle
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differentiate from periotic mesenchyme derived from cells of neural crest origin along with a contribution from the paraxial mesoderm (Noden and Van De Water, '86). During development of the otocyst the periotic mesenchyme is thought to participate in inductive interactions critical to normal cochlear and labyrinthine morphogenesis, and, conversely, the periotic mesenchyme relies upon the otocyst for reciprocal induction of the otic capsule and stapedial footplate (Orr and Hafft, '80; Anniko and Schacht, '84; Noden and Van De Water, '86). Thus, we cannot rule out the possibility of a primary effect of isotretinoin on neural crest cells with consequent effects on interactions with the otocyst. In addition to the periotic mesenchyme, the rhombencephalon is also thought to play an inductive role in otic vesicle development. During early development the basement membranes of the otic vesicle and rhombencephalon are in direct contact (O'Rahilly, '63). Experimental studies in amphibians and avians have shown that disturbance of the normal embryonic relationship between the otic vesicle and the rhombencephalon results in abnormal development of the otic vesicle with defects ranging from an undifferentiated cyst-like stage to a malformed labyrinth with an imperfect otic capsule (Van de Water, '80). In studies of mouse mutants anomalies of the rhombencephalon, such as faulty segmentation and degeneration of the 4th rhombomere, have been correlated with abnormalities of the inner ear and otic capsule (Deol, '64, '66). Isotretinoin-treated cultured rat embryos exhibited a diminished second arch and a cranial shift in otic vesicle location, suggestive of hindbrain abnormality (Webster et al., '86). Inner ear defects are also common in the presence of exencephaly (Padmanabhan, '87; Jarvis et al., '90). Thus, an adverse effect of isotretinoin on the neuroepithelium of the rhombencephalon or on its segmentation pattern might result in brain defects as well as inner ear defects as a result of faulty inductive interactions. Specific localization of cellular retinoic acid binding proteins (CRABP) and nuclear retinoic acid receptors (RAR), taken together with observations on homeobox genes encoding segmentation of the rhombomeres may provide future insight into the molecular basis of isotretinoin craniofacial teratogenesis. Cells that express CRABP or
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D.T. BURK AND C.C. WILLHITE
nuclear RARs appear to be target cells for the teratogenic exogenous retinoids (Dencker, '90; Ruberte et al., '91). CRABP or its gene transcript has been detected in the hindbrain and in neural crest and placodederived tissues (Dencker et al., '90; Vaessen et al., '90; Ruberte et al., '91; Maden et al., '91) and, in the chick, in cells of the open auditory pit and later in the auditory vesicle (Vaessen et al., '90). RAR-P transcripts have been found in craniofacial mesenchyme, including mesenchyme surrounding the inner ear epithelium (Ruberte et al., 'go), and in hindbrain neuroectoderm (Smith and Eichele, '91), while RAR-y is prominent in cartilages, including the otic (Ruberte et al., '90)). Specification of regional identity in the central nervous system is thought to be a function of homeobox genes. Additionally, each area of neural crest derived tissue has been found to have a unique code of Hox 2 gene expression related to the rhombomeric origin of the crest. This suggests that the Hox 2 genes also provide part of the positional information to the neural crest and hence are involved in patterning the structures of the branchial arches as well (Hunt et al., '91). The fact that distribution of CRABP and Hox-2.9 are coincident in the hindbrain suggests the possibility that retinoic acid could also be used to establish gradients of homeobox gene expression, and thus play a role in determination of normal segmentation pattern in developing central and peripheral nervous systems (Maden et al., '91). ACKNOWLEDGMENTS
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