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Magnetic Resonance Imaging and Surgical Repair of Cleft Palate in a Four-Week-Old Canine (Canis familiaris): An Animal Model for Cleft Palate Repair RAVI J. TOLWANI, DVM, PHD,1* CATHERINE E. HAGAN, DVM,1 JONATHAN A. RUNSTADLER, DVM, PHD,1 HEATHER LYONS, VMD,1 SHERRIL L. GREEN, DVM, PHD,1 DONNA M. BOULEY, DVM, PHD,1 LUIS F. RODRIGUEZ, MD,2 STEPHEN A. SCHENDEL, MD, DDS,2 MICHAEL E. MOSELEY, PHD,3 DAVID A. DAUNT, DVM,1 GLEN OTTO, DVM,1 AND LINDA C. CORK, DVM, PHD1 Successful cleft palate repair (palatoplasty) was accomplished in a male canine pup from a kindred with autosomal recessive transmission for a complete cleft palate phenotype. This case represents the potential application of a new animal model for cleft palate repair. This reproducible congenital defect provides a clinically relevant model to improve research into the human anomaly, as compared with previous iatrogenic or teratogenically induced animal models. This case report presents the basis for new repair techniques and for studying the genetic basis of the cleft palate defect. Cleft lip and palate (CLP or orofacial cleft) deformities are common congenital anomalies in both human and animal populations. The condition derives from a developmental defect resulting in failure of fusion of the palatine shelves arising from the maxillary processes (1). CLP comprises two morphologic entities: cleft lip with or without cleft palate and cleft palate alone (1). Among humans, the incidence of cleft palate varies with ethnicity, (2) and has been estimated to be in the range of 1 in 500 to 1 in 2,500; the defect is more prevalent in females (3, 4). The incidence and prevalence of cleft palate in animal populations is unknown, but estimates have been attempted. In 1994 Natsume et al. reported that the incidence of cleft palate in breeding colonies of beagles was < 2% (5). Data on 5,635 male and 4,974 female Boxer pups from 1,327 litters, born in the German Democratic Republic in 1977 to 1984, showed an overall incidence of cleft lip plus cleft palate of 0.6% (6). Among 331 animals with cleft palate in a veterinary hospital population, cats, mixed breed dogs, and German Shepherd dogs had low risk for cleft palate; high rates were seen in English Bulldogs, some small purebred dogs, and Charolais cattle, in which cleft palate occurred as part of a syndrome of multiple malformations (7). A keyword search for cleft palate in the electronic case records of the University of California at Davis Veterinary Medical Teaching Hospital covering the period from 1987 through 2003 returned a total of 472 animals, including 130 horses, 29 cows, and 235 dogs representing over 40 different breeds. In animals, the primary etiology of CLP is hereditary, although nutrition, drugs or chemicals, mechanical interferences, and even viral infections also are implicated (8). In addition to being an easily noticed deformity, cleft palate can cause physiological dysfunction. Affected animals are unable to nurse successfully, which may lead to aspiration pneumonia and subsequent respiratory infection (8). Treatment in animals involves intensive nursing care and a variety of surgical interventions (8). In humans, treatment of cleft palate hinges primarily on surgical interventions with the goals of both improved aesthetics and return Department of Comparative Medicine,1 Department of Surgery,2 Department of Radiology,3 Stanford University School of Medicine, Stanford, California 94305 * Corresponding author Volume 43, No. 6 / November 2004

of function (9, 10). Controversy revolves around the timing of the surgery, with advocates of early surgery (prior to 1 year of age) claiming improved development of early speech function and advocates of late intervention emphasizing the importance of allowing maximal growth of the maxilla (10). Understanding the basis of CLP in humans has been the subject of a great deal of research. Developments in this field have been considerable, including identification of various genetic (11-17) and environmental factors, such as alcohol (18-21), smoking (22-27), nutrition (28-30), and drugs such as glucocorticoids (31-33) and anticonvulsants (34-38). Current research on surgical intervention investigates the appropriate timing and technique, as well as the possibility of fetal surgery (10, 39-43). This research has been driven by a wide variety of animal models including mice, rats, rabbits, guinea pigs, swine, goats, sheep, primates, dogs, and even alligators (44). Some studies aim to illuminate the embryogenesis and pathogenesis of cleft palate whereas others are specifically targeted at developing repair techniques. Different species offer different advantages and limitations depending on the study for which they are being used. Although many rodent models offer much in the way of gaining an understanding of the genetic and molecular factors at work (45-50) which likely will help reveal the embryologic sequence of morphogenesis (51-54), the small size of these animals limits their utility as a means of developing surgical interventions. Larger animal models for this area of research have been developed by using iatrogenic (mechanical or chemical) means (55-63). Environmental factors contributing to cleft palate have been investigated using a wide variety of species and teratogenically induced models (12, 64, 65). The mechanisms for teratogenically induced cleft palate are poorly understood. Despite this vast assortment of animal models, the majority are teratogenically and iatrogenically produced. A need exists, therefore, for a reliably reproducible, naturally occurring model that offers a more clinically relevant condition for the extrapolation of research data to the human syndrome. Some mouse models meet these criteria (53, 66, 67), but again, their small size limits their utility for surgical studies. Other animals have produced what was concluded to be an inherited phenotype (5, 6, 68), but the phenotype emerged too sporadically. CONTEMPORARY TOPICS © 2004 by the American Association for Laboratory Animal Science

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The cleft palate research community has called for a reproducible, clinically relevant, naturally occurring animal model (59, 69). We performed our study to address the need for an appropriate model of cleft palate. A cleft palate phenotype was identified in a large kindred of Brittany Spaniel-Beagle crossbreds maintained for a dominantly inherited motor neuron trait, a model for Lou Gehrig disease (70). All of these dogs had clearly visible, severe palatine deformities, and this trait was not linked to the motor neuron disease. Pedigree analysis suggests that the cleft palate trait was inherited as an autosomal recessive in simple Mendelian fashion, occurring in about one-fourth (26.9%) of the offspring. However, because affected female neonates outnumber males by a ratio of 9:2, alternative modes of inheritance may be possible (70). A male canine pup from the described affected kindred was selected for what we consider to be an early (prior to weaning) surgical repair of the defect. We documented the defect before surgery by precise characterization with magnetic resonance imaging (MRI) and the repair by postsurgical radiography. This case study provides a foundation for developing this animal model of naturally occurring cleft palate.

Materials and Methods

Subject. This study was reviewed and approved by our Institutional Animal Care and Use Committee. A palatognathous disorder was identified on postnatal day 1 in a male pup. The pup had difficulty nursing and was mildly dehydrated, lethargic, and hypothermic. Postnatal care. The dog was immediately weaned and hand-reared. All meals were fed via orogastric tube to curtail aspiration. The pup was maintained on Esbilac (Pet Ag, Hampshire, Ill.) milk replacer supplemented with additional milk from the bitch. The pups feed intake and weight gain were recorded. Starting on postnatal day 4, the pup was maintained entirely on Esbilac fed in appropriate amounts and frequency for his ideal weight. He was maintained in a thermal unit in Stanfords veterinary service center intensive care unit to facilitate constant monitoring by the veterinary staff. Imaging and diagnostics. Because the full extent of the palate defect could not be readily visualized grossly, an MRI of the dog was performed on postnatal day 24 to quantify the size of the cleft and evaluate it for possible surgery. Isoflurane anesthesia of the pup was induced and maintained via face cone. Serial images in a vertical transverse plane were obtained in a 1.5-T General Electric Signa magnet (GEMS, Waukesha, Wis.) at Stanford Universitys Lucas Imaging Center. Both T1- and T2-weighted 3-mm slices were acquired and evaluated. A complete blood count (CBC) and clinical chemistry panel were obtained prior to surgery to evaluate the animals overall health status. Radiographs of the skull and thorax were taken to identify the existence of any other potential craniofacial abnormalities or medical issues, such as aspiration pneumonia. Surgery. At 4 weeks of age, the pup underwent surgical repair of both hard (palatum durum) and soft (palatum molle) palate defects. The animal was treated with atropine (0.04 mg/kg subcutaneously), underwent induction of isoflurane anesthesia via face mask, was intubated, and was maintained under general anesthesia with isoflurane. The surgical field was prepared and draped aseptically. The entire hard and soft palate was infiltrated with 1% lidocaine with 1:100,000 epinephrine in a submucosal plane. A von Langenbeck-type procedure was performed (10, 41, 43). The edges of the cleft were incised from the alveolus to the base of the uvula, the sides of which were excised sharply with a scalpel. Long relaxing incisions were made from the anterior tonsillar pillar at the lateral edge of the soft palate to a point lateral to the posterior maxillary tubercle. The incisions were carried through the periosteum, and mucoperiosteal flaps were elevated bilaterally off the bone with blunt periosteal elevators. The lateral relaxing incisions were opened widely in both the 18

CONTEMPORARY TOPICS © 2004 by the American Association for Laboratory Animal Science

Figure 1. Representative view of the cleft palate defect.

soft and hard palate areas. Scissors were used for blunt dissection posteriorly. Once the hard palate mucoperiosteum had been elevated, a small amount of elevation was performed on the nasal side of the palate by using an angled periosteal elevator. In the soft palate, the elevation was performed between the nasal, and similarly, the oral mucosa and the soft palate musculature. The levator muscle was detached from the posterior edge of the hard palate. Electrocautery was used throughout the procedure to maintain meticulous hemostasis. The palatal cleft was closed in two layers in the hard palate and three in the soft palate by using 4-0 Vicryl (Ethicon, Somerville, N.J.) sutures in a horizontal mattress pattern. In the hard palate, the nasal mucosa was closed first; the mucoperiosteal flaps were approximated at the midline and closed with interrupted sutures. In the soft palate, the nasal mucosa was closed first as well. The muscle was reapproximated in the midline after reorienting the fibers posteriorly. The oral mucosa of both the hard and soft palates was then closed at midline. This technique resulted in the tension-free closure necessary to prevent dehiscence and avoid surgical failure. Surgicel (Johnson & Johnson, Piscataway, N.J.) was packed into the site of the relaxing incisions to aid in hemostasis. The mouth was irrigated with saline and suctioned, and the throat pack removed. The subject recovered from anesthesia and was extubated without complications. Recovery. Buprenorphine (0.01 to 0.02 mg/kg subcutaneously) was provided for postoperative analgesia for 24 h. Enrofloxacin (5 mg/kg intramuscularly) was administered twice daily for 10 days for infection prophylaxis. Warmed Esbilac milk replacer in a bowl was offered for the first 4 days postoperatively. Two days later the puppy consumed semi-solid gruel from a bowl without difficulty. Follow-up. When the pup was 4 months of age, the oral and nasal cavities and surgical site were examined grossly. Skull radiographs were taken to evaluate postsurgical healing. At 6 months of age, the animal was euthanized with an overdose of intravenous sodium pentobarbital (200 mg/kg) and rinsed intracardially with intravenous 0.9% saline. After the saline rinse, the dog was perfused with 4% paraformaldehyde. After fixation was complete, the skull minus the mandibles but including the hard and soft palates were decalcified in Cal-Ex (Fisher Scientific, Bridgewaters, N.Y.) and sectioned. Histologic sections were obtained in a transverse plane across the areas of surgical repair in the hard and soft palates. Sections were stained with hematoxylin and eosin and examined under light microscopy to evaluate the structure and scar tissue at the surgical sites.

Results

Prior to surgery, the pups cleft palate defect was visible grossly (see Fig. 1 for representative view of a cleft palate defect). In light of Volume 43, No. 6 / November 2004

Figure 2. Transverse views from a series of magnetic resonance images from rostral to caudal characterizing the complete midline cleft palate in the 3-week-old pup. Arrows indicate the cleft in the hard palate (A) and soft palate (BD).

the pre-operative CBC, chemistry profile, and chest radiographs, the animal had no additional recognizable medical abnormalities. T1weighted MRI images revealed a gap in the hard palate which opened at the rostral point of the hard palate and widened gradually to a 7mm gap at the distal aspect of the soft palate (Fig. 2). After surgical repair of the cleft, the animal was able to eat and drink without observable functional deficit. Radiographs of the skull revealed no noteworthy developmental distortion of the skull relative to the normal limits of nonpathologic canine skull anatomy (Fig. 3). Histologic sections of the repaired defect revealed a solid fibrous connective tissue bridge between the two sides of the palate, with invagination and slight puckering of both the oral and nasopharyngeal mucosa. There was minimal boney remodeling consisting of periosteal spicules of woven bone, focal medullary fibrosis, and a rare remnant of refractile suture material. No marked inflammation was noted in the surgical repair site (data not shown).

Discussion

The purpose of this study was to investigate whether this naturally occurring cleft palate phenotype may provide a suitable animal model for investigating the human defect. The morphologic similarity of the cleft palate phenotype in this canine kindred to human clefts and the demonstrated repairability offers a new opportunity for cleft palate research. Cleft palate research can take advantage of several aspects of this model: heritability, reproducibility, clinical relevance as a naturally occurring defect, and the size and ease of management of the animal involved. The heritability has been documented at approximately 26%, suggesting that a simple Mendelian mode of inheritance is most likely, and reproducibility of the lesion was evident in that all affected dogs in the colony had clearly visible, severe defects. Anatomically, variants of cleft palate in humans include lesions of the soft palate only and those of the hard and soft palate with or without cleft lip; our dog had clefting of both palates without any lip involvement. Although the impairment of speech in humans due to CLP is not a problem in canines, aspiration associated with CLP is a serious side effect in both humans and dogs and dictates the need for appropriate surgical repair techniques. Volume 43, No. 6 / November 2004

Figure 3. Postoperative (4 months) ventrodorsal radiographs of the maxilla.

The heritability of this cleft palate trait is advantageous in that consistently affected dogs can be produced in numbers suitable for scientific study, provided that the affected animals are able to survive and be bred as homozygous recessive individuals. A considerable investment in neonatal care may be required. Because cleft palate is a congenital, heritable defect, there may be some aspects of the underlying molecular basis that could illuminate the complex genetics involved in the human anomalyparticularly in view of the rapid DNA sequencing of the canine genome. Current cleft palate research is aimed at assessing appropriate surgical interventions and their timing. Harling et al. (69) reviewed animal models for in utero craniofacial surgery and discussed benefits and limitations of a variety of animal models. Recently published articles on in utero models highlight issues and limitations that are relevant for ex utero surgical research as well. Small animal models with short gestation periods, such as the rat, mouse, and rabbit, allow fetal manipulation only late in gestation, with decreased post-surgical intrauterine periods. When fetal manipulation is close to the birth, wounds may heal with an adult (scarring) phenotype (69). Small animals also pose more of a technical challenge but are relatively inexpensive and easier to manage. Larger animals, such as sheep and cows, offer a longer postoperative, intrauterine period; this attribute can be crucial to assessing fetal wound healing. Although the technical manipulation of these animals is easier, these models have longer gestations, are difficult to repair, and require a greater management investment. Harlings review of in utero models culminates in an assessment that the ideal model for evaluating fetal cleft repair would be a large animal model that intrinsically forms clefts. One of the more recently developed models is Weinzweigs ovine model (59-61, 71), in which the congenital cleft palate is induced by gavaging pregnant dams with Nicotiana glauca, which has the piperidine alkaloid anabasine. This model currently is the closest approximation to Harlings ideal model, falling short only in that it is still an induced model. The model represented by this canine kindred offers fewer technical challenges than rodent models. The potential for in utero CONTEMPORARY TOPICS © 2004 by the American Association for Laboratory Animal Science

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manipulation of this canine model has not been explored, and thus its utility for fetal cleft repair has not been compared with that of Weinzweigs ovine model. Further development of this animal model could begin by assessing any degree of variability in trait manifestation among affected animals or elucidating the genes involved. We have established the feasibility of this model and have explored the potential benefits of its application. Because CLP in these dogs is a naturally occurring congenital defect, the model offers a more clinically relevant condition for extrapolation to the human anomaly than do iatrogenically induced models and is an exciting opportunity for the orofacial research community.

Acknowledgment

This study was supported in part by a grant to Linda C. Cork and Martin J. Pinter from the National Institute of Neurological Disorders and Stroke.

References

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47. Bienengraber, V., F. Malek, J. Fanghanel, et al. 1999. Disturbances of palatogenesis and their prophylaxis in animal experiments. Anat. Anz. 181:111-115. 48. Juriloff, D. M., M. J. Harris, and C. J. Brown. 2001. Unravelling the complex genetics of cleft lip in the mouse model. Mamm. Genome 12:426-435. 49. De Felice, M., C. Ovitt, E. Biffali, et al. 1998. A mouse model for hereditary thyroid dysgenesis and cleft palate. Nat. Genet. 19:395398. 50. Bush, J. O., Y. Lan, K. M. Maltby, et al. 2002. Isolation and developmental expression analysis of Tbx22, the mouse homolog of the human X-linked cleft palate gene. Dev. Dyn. 225:322-326. 51. Diehl, S. R. and R. P. Erickson. 1997. Genome scan for teratogeninduced clefting susceptibility loci in the mouse: evidence of both allelic and locus heterogeneity distinguishing cleft lip and cleft palate. Proc. Natl. Acad. Sci. U.S.A. 94:5231-5236. 52. Juriloff, D. M. and D. G. Mah. 1995. The major locus for multifactorial nonsyndromic cleft lip maps to mouse chromosome 11. Mamm. Genome 6:63-69. 53. Juriloff, D. M. 1995. Genetic analysis of the construction of the AEJ.A congenic strain indicates that nonsyndromic CL(P) in the mouse is caused by two loci with epistatic interaction. J. Craniofac. Genet. Dev. Biol. 15:1-12. 54. Trotman, C. A., D. Hou, A. R. Burdi, et al. 1995. Histomorphologic analysis of the soft palate musculature in prenatal cleft and noncleft A/ Jax mice. Cleft Palate Craniofac. J. 32:455-462. 55. Bardach, J. and K. M. Kelly. 1988. Role of animal models in experimental studies of craniofacial growth following cleft lip and palate repair. Cleft Palate J. 25:103-113. 56. Canady, J. W., S. K. Landas, H. Morris, et al. 1994. In utero cleft palate repair in the ovine model. Cleft Palate Craniofac. J. 31:37-44. 57. Canady, J. W., S. A. Thompson, and A. Colburn. 1997. Craniofacial growth after iatrogenic cleft palate repair in a fetal ovine model. Cleft Palate Craniofac. J. 34:69-72. 58. Longaker, M. T., M. Stern, P. Lorenz, et al. 1992. A model for fetal cleft lip repair in lambs. Plast. Reconstr. Surg. 90:750-756. 59. Weinzweig, J., K. E. Panter, M. Pantaloni, et al. 1999. The fetal cleft palate. I. Characterization of a congenital model. Plast. Reconstr. Surg. 103:419-428.

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60. Weinzweig, J., K. E. Panter, M. Pantaloni, et al. 1999. The fetal cleft palate. II. Scarless healing after in utero repair of a congenital model. Plast. Reconstr. Surg. 104:1356-1364. 61. Weinzweig, J., K. E. Panter, A. Spangenberger, et al. 2002. The fetal cleft palate. III. Ultrastructural and functional analysis of palatal development following in utero repair of the congenital model. Plast. Reconstr. Surg. 109:2355-2362. 62. Ascherman, J. A., V. P. Marin, L. Rogers, et al. 2000. Palatal distraction in a canine cleft palate model. Plast. Reconstr. Surg. 105:1687-1694. 63. Liang, L., C. Liu, and M. Hou. 2002. Osteo-distractive technique for bony repair of cleft palate in a dog model. Zhonghua Zheng Xing Wai Ke Za Zhi 18:360-362. 64. Bianchi, F., E. Calzolari, L. Ciulli, et al. 2000. [Environment and genetics in the etiology of cleft lip and cleft palate with reference to the role of folic acid.] Ambiente e genetica nell'eziologia delle labioschisi e palatoschisi con particolare riferimento al ruolo dell'acido folico. Epidemiologia E Prevenzione (Milano) 24:21-27. 65. Murray, J. C. 2002. Gene/environment causes of cleft lip and/or palate. Clin. Genet. 61:248-256. 66. Nagata, M., N. Amin, Y. Kannari, et al. 1997. Isolated maxillary bending in CL/FR strain mice: observation of craniofacial deformity and inheritance pattern. Cleft Palate Craniofac. J. 34:101-105. 67. Matsushima, Y., M. Ohne, T. Irino, et al. 1992. Spontaneous cleft palate in CF#1/Ohu mice. Jikken Dobutsu 41:83-85. 68. Bail, R. J. L., J. J. Pasquet, J. Y. Detaille, et al. 1983. Malformations of genetic origin in teratology studies: cases of cleft palate in rabbits, p. 101-114. In E. C. Melby, Jr. and M. W. Balk (ed.), The importance of laboratory animal genetics, health, and the environment in biomedical research. Academic Press Inc., Orlando, Fla. 69. Harling, T. R., E. J. Stelnicki, M. H. Hedrick, et al. 2003. In utero models of craniofacial surgery. World J. Surg. 27:108-116. 70. Richtsmeier, J. T., G. H. Sack, Jr., H. M. Grausz, et al. 1994. Cleft palate with autosomal recessive transmission in Brittany spaniels. Cleft Palate Craniofac. J. 31:364-371. 71. Weinzweig, J., K. Panter, A. Spangenberger, et al. 2001. Toward an understanding of the cleft palate anomaly using a congenital model. Med. Health R. I. 84:128-131.


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