[Methods in Molecular Biology] Teratogenicity Testing Volume 947 || Fetal Soft Tissue Examination by Serial Sectioning

233 Paul C. Barrow (ed.), Teratogenicity Testing: Methods and Protocols, Methods in Molecular Biology, vol. 947, DOI 10.1007/978-1-62703-131-8_19, © Springer Science+Business Media, LLC 2013 Chapter 19 Fetal Soft Tissue Examination by Serial Sectioning Karon Critchell Abstract This chapter describes the method used for serial sectioning and soft tissue examination of the Bouin’s fi xed fetus (mainly whole rat and or mouse fetuses or rabbit fetal heads) for the assessment of developmen- tal and structural abnormalities. Fetuses are examined externally, together with the internal structures of the head as well as the thoracic and abdominal organs. Key words: Serial sectioning , Fetal soft tissue , Bouin’s , Wilson technique , Examination , Abnormalities As part of the assessment of reproductive toxicity testing there is a regulatory requirement that fetuses from treated dams are exam- ined for developmental and structural abnormalities by soft tissue examination. Current guidelines ( 1, 2 ) stipulate that this is per- formed in two laboratory species, one rodent (rat or mouse) and one non-rodent (routinely rabbit), in order to assess the safety of a test compound before, or in case, humans are exposed. This technique facilitates soft tissue examination by serial sec- tioning (Modi fi ed Wilson technique ( 3 ) ), as it is performed on preserved whole rat and mouse fetuses/rabbit heads (see Note 1 ). During the assessment all fetuses are examined both externally and internally. The principal. De fi nition main/primary of the proce- dure is to be able to distinguish between what is “normal” and “abnormal,” both structurally and developmentally, according to the stage of gestation. 1. The technique is not destructive, unlike fresh visceral microdissection. 2. Fixation in Bouin’s is rapid. 1. Introduction 1.1. Advantages 234 K. Critchell 3. Facilitates examination of internal structures, particularly of the head. 4. As the specimens are fi xed, examination is at the convenience of the evaluator. 5. Allows retention of tissue for secondary or con fi rmation analy- ses and future reference. 1. Time consuming. 2. Heavily dependent on thorough training. 3. Can be dif fi cult to visualize some structures in this plane. 1. Bouin’s: Picro-formol, 10% formaldehyde. 2. Water or Neutral Buffered Formaldehyde (NBF). 3. Industrial Methylated Spirits (IMS). S.G. 0.790–0.80. 95% ethanol and 5% wood naptha. 4. Wax block/dental wax. 5. Thin (razor type) blade and holder. 6. Suitable, clear shallow container, such as petri dish. 7. Forceps. 8. Protective gloves. 9. Laboratory coat. 10. Cold light source. 11. Low-power microscope, minimum ×6 magni fi cation. As soon as possible after removal from the uterus, the fetuses are euthanized and fi xed in Bouin’s solution. Fixation takes 7–14 days (see Note 2 ). Bouin’s is the preferred fi xative as it is suf fi ciently acidic to act as a decalcifying agent, which facilitates sectioning through bone, particularly the skull. Specimens may require hardening, by immersion in IMS for at least 24 hours to aid in sectioning. 1. It is recommended that specimens are washed in either IMS or water prior to sectioning, to help minimize the fumes from the Bouin’s. 2. A petri dish is labeled with the appropriate identi fi cation, to store all the sections (see Note 3 ). 1.2. Disadvantages 2. Materials 3. Methods 3.1. Preparation of Samples 3.2. Serial Sectioning Procedure 23519 Fetal Soft Tissue Examination by Serial Sectioning 3. The fetal head or whole body is sectioned on a slab of dental wax or equivalent, using a sharp razor blade. The specimen is placed onto the wax block and the razor blade inserted between the upper and lower jaws and, with fi rm even pressure, the upper part of the head is cut in a plane (along the rostro- occipital axis) just below the ears. 4. The tongue is removed from the head section and the palate examined for clefts. 5. The cut surface of the head is placed cut side down. A series of transverse slices are made through the head (approximately 7–12 sections), beginning at the nose and proceeding back- wards towards the ears. One of the cuts should be made between the nose and eyes, one centrally through the eyes, and one through the head at its largest transverse diameter and in the region of the pineal and pituitary glands. The cerebellum should be left unsectioned in the remaining portion of the head. Care must be taken to keep all cuts parallel, vertical, and at right angles to the rostro-occipital axis. 6. For whole body specimens, position the razor blade just below the lower jaw and cut to remove the mandibles whilst preserv- ing as much of the cervical region as possible. 7. Remove each forelimb from the torso with a single cut, taking care not to damage any adjacent structures. 8. Transverse serial sections are now cut (consistently up to 1 mm thickness—see Note 4 ) through the neck and thorax down to the apex of the heart, just above the diaphragm (approximately 15 sections) (see Note 5 ). 9. Further sections are then made through the liver—cut two thicker sections, the fi rst to include the complete diaphragm, the second to include a cross section of liver lobes and stom- ach, leaving the remainder of the abdominal region intact (see Note 6 ). (Diagram of serial sections—see Fig. 1 .) 10. All the sections are placed in a petri dish and moistened with water or NBF and the dish covered, to prevent the sections from drying out. The tissue can be stored in this way for sev- eral weeks. 1. Fetuses are examined externally at fresh necropsy and during serial sectioning. Any abnormalities reported may be con fi rmed/ supported at serial sectioning evaluation (see Note 7 ). 2. Using a low-power microscope examine the structures on both surfaces of each section in cranial–caudal order, looking for size, form, shape, symmetry, and position of the internal struc- tures (see Notes 8 and 9 ). 3.3. Soft Tissue Examination 236 K. Critchell Fig. 1. Serial sections through a whole rat fetus. 3. Examine the head, upper and lower jaws and lips, snout, naris, diagrams and relevant descriptions correspond. Nasolabial sul- cus/cleft, nasal cavity and septum, oral cavity, palate, palatine ridges, incisors, cranium, pinna, eyelid, eye/lens, retina, cor- nea, vitreous and aqueous chambers, nasopharynx, olfactory lobe, cerebral hemispheres, lateral ventricles, cranial nerves, third ventricle, pituitary, pineal gland, thalamus, perimeningeal space, and internal ear. Possible abnormalities that can be observed include microphthalmia/anophthalmia, retinal fold, dilated lateral ventricles, and hemorrhages affecting the brain. 23719 Fetal Soft Tissue Examination by Serial Sectioning 4. Detach the cerebellum and other neural tissue from the unsec- tioned portion of the head and examine the midbrain, cerebel- lum, medulla oblongata, cerebral aqueduct, and fourth ventricle. 5. Examine the cut surface of lower jaw section, lower incisors, and tongue and genioglossus muscle (see Note 10 ). 6. Examine the upper thorax and heart: Larynx, spinal cord, tra- chea, esophagus, salivary glands, thyroid gland, thymus gland, lung lobes, azygos vein, and diaphragm. First level of thoracic vessels: Aorta, pulmonary trunk, ductus arteriosus, atria, bronchus, pulmonary vein and arter- ies, and vena cava (see Note 11 ). Second level of thoracic vessels—aortic arch arteries, com- mon carotid and subclavian arteries, and innominate/brachio- cephalic artery. Then heart: Pericardium, atrium, ventricle, ventricular septum, atrioventricular valve, and semilunar valve. Possible abnormalities that can be observed include ret- roesophageal aortic arch, ventricular septal defects, and par- tially undescended thymus. 7. Unsectioned abdominal region; externally check patency of the anus and structure of genital tubercle. 8. The liver and intestines are then removed to allow examination of the organs contained within the abdomen. 9. Subsection kidneys to reveal renal papillae. 10. Examine liver, gallbladder (mouse), stomach, spleen, pancreas, intestines, kidneys, adrenal gland, ureters, bladder, umbilical artery, genital organs, dorsal aorta, and caudal vena cava. Possible abnormalities that can be observed include absent renal papilla, dilated ureter(s), and displaced testis. 11. Examine the external torso, body surface/skin, body wall, umbilical cord, tail, limbs, and digits (fore and hind). 12. Any structures not revealed directly from sectioning should be sub-sectioned with a razor blade or probed by use of a fi ne hair. 13. There are many other abnormalities that can be identi fi ed in these sections, ranging from abnormalities considered as mal- formations (e.g., microphthalmia) through to minor abnor- malities (e.g., partially undescended thymus). 14. A peer review is then performed on a percentage of examined litters for consistency (Figs. 2– 9 ). 15. After examination, the fetal specimen can be placed in tubes, covered with NBF, and stored for a long term. Sectioned fetal preparations are stable and not subject to short-term loss or degradation if stored correctly. Fig. 2. ( a ) Section through a normal rat head. ( b ) Section through a rat head showing microphthalmia (and oval left lens. ). Fig. 3. ( a ) Section showing the normal palate of a rat. ( b ) Section showing cleft palate on a rat. Fig. 4. ( a ) Section through a rat heart showing a normal septum. ( b ) Section through a rat heart showing a ventricular septal defect. Fig. 5. ( a ) Section through the cervical region of a rat showing normal thyroids. ( b ) Section through the cervical region of a rat showing absent thyroid (and partially undescended left lobe of thymus). Fig. 6. ( a ) Section showing a normal rat diaphragm. ( b ) Section showing rat diaphragmatic hernia. Fig. 7. ( a ) Section through rat kidneys showing normal renal papilla. ( b ) Section through rat kidney showing dilated renal pelvis. 240 K. Critchell Fig. 8. ( a ) Section through a normal rabbit head. ( b ) Section through a rabbit head showing partially open eyelids. 16. To conduct fetal examinations adequately, it is important to have a clear idea of the normal anatomy. Every effort should be made to assess the anatomy in exactly the same way for every fetus, applying identical criteria to distinguish “normal” from “abnormal.” The priority should be ensuring consistency within each laboratory so that valid comparisons can be made between treated and control animals, both within and between studies conducted in that laboratory. It takes a lot of experi- ence to be con fi dent that you can “spot the difference.” Fig. 9. ( a ) Section showing a normal rabbit cerebellum. ( b ) Section showing subdural hemorrhage cerebellum. 24119 Fetal Soft Tissue Examination by Serial Sectioning 1. Successful results have been achieved using this technique with—rat whole body from fetuses at Day 20 of gestation; rat heads only or whole body from fetuses at Day 21 of gestation; mouse whole body from fetuses at Day 17, 17.5, and 18 of gestation; and rabbit heads only from fetuses at Day 29 of gestation. 2. Generally the larger the specimen the longer the time required for fi xation, e.g., rabbit heads. There are alternative fi xation techniques that may be used, e.g., fi xation in Davidson’s prior to fi xation in Bouin’s ( 4 ) . 3. Pencil is used to record the litter and fetal identi fi cation, on a small label, so that the petri dishes can be recycled. An alterna- tive is a permanent marker but apply caution when using IMS as the ink can be washed off. 4. Sectioning procedures may vary slightly between laboratories; however the critical part of this procedure is to have uniform, straight, thin sections. 5. Care must be taken not to apply excess lateral pressure, which could displace the viscera and distort the sections. 6. Care must be taken not to slice through the diaphragm so that this structure can be examined intact for herniations and posi- tioning of the major vessels that penetrate it. 7. Other considerations during examination are the recognition of artifacts, assessing mechanical damage at necropsy and/or during sectioning, and processing/ fi xation artifacts. 8. It may be necessary to rebuild the sections to examine whole structures, e.g., eyes and palate. 9. Structures may be recorded in less or more detail depending on the strain/source of the species used and in-house training guides. 10. Manipulate the tongue to check that it is not attached anteriorly. 11. Check the orientation of the trachea, centrally below the spinal cord, esophagus orientated to the left. Acknowledgments Thanks to the Reproductive Necropsy and Fetal pathology Departments who are involved with this work. 4. Notes 242 K. Critchell References 1. International Conference on Harmonisation of technical requirements for the registration of Pharmaceuticals for Human use. ICH guide- lines on Male Fertility Studies S5 (R2) November 2005 2. OECD Guideline 416 two-generation repro- ductive toxicity study January 2001 3. Wilson JG (1965) Methods for administering agents and detecting malformations in experi- mental animals. In: Wilson JG, Warkany J (eds) Teratology principles and techniques. University of Chicago Press, Chicago, pp 262–277 4. French J (2008) Retinal folding in the term rabbit fetus: developmental abnormality or fi xation artifact. Reprod Toxicol 26:262–266 Chapter 19: Fetal Soft Tissue Examination by Serial Sectioning 1. Introduction 1.1. Advantages 1.2. Disadvantages 2. Materials 3. Methods 3.1. Preparation of Samples 3.2. Serial Sectioning Procedure 3.3. Soft Tissue Examination 4. Notes References