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Recommendations for Routine Sampling, Trimming, and Paraffin-Embedding of Female Reproductive Organs, Mammary Gland, and Placenta in the Cynomolgus Monkey
1 Principal Pathologist, Department of Toxicology and Drug Disposition, Schering-Plough, Oss, the Netherlands Correspondence: Eric Van Esch, Schering-Plough, Department of Toxicology and Drug Disposition, P.O. Box 20, 5340 BH Oss, the Netherlands; e-mail:eric.van.esch{at}spcorp.com
In toxicity studies, the nonhuman primate is often the species of choice to evaluate the toxicologic potential of chemicals and drugs. Especially in the case of effects on female reproductive organs and mammary glands, other animal species are less predictive for man. To enable reliable histopathologic interpretation allowing a solid safety assessment, it is a prerequisite to obtain material of consistently high quality. Standardization of autopsy techniques, tissue sampling, and fixation and staining procedures will help significantly to obtain the quality that is needed. For this purpose, a detailed description of the procedures from necropsy to microscopic slide preparation of the female reproductive organs of the cynomolgus monkey is given. Procedures to sample and process the placenta are included. These recommendations can be used to achieve consistent, high-quality tissue preparations, allowing pathologists to conduct sensitive, accurate, and meaningful evaluations of the study material. Competing Interests: This article was sponsored by Covance Inc. and Schering- Plough. Martina Zölle and Eberhard Buse are employed by Covance Inc. Eveline P. C. T. de Rijk and Eric Van Esch are employed by Schering-Plough. No other competing interests were declared.
Key Words: dissection • female reproductive organs • placenta • tissue sampling • histotechnique • nonhuman primates • cynomolgus monkey
In general, regulatory guidelines, intending to aid the approval and registration of pharmaceutical and other chemical products, specify only the organs and tissues that must be evaluated grossly, weighed, and microscopically examined. Such guidelines do not specify the sample sizes and numbers, nor do they give indications of the parts of the appropriate organs and tissues that have to be examined by the pathologist. As a consequence, possible regional intratissue differences in susceptibility of the cellular (sub)components within certain organs may be overlooked. Sample size and number are more critical when processing small organs/tissues, since when not correctly oriented, essential structures might be lost, for example, when dealing with small laboratory animals. In larger laboratory animals such as dogs and monkeys, the reproductive organs and mammary glands are greater in volume, and therefore sample size is not limiting when the tissues are adequately dissected and trimmed. Nevertheless, to reduce intra- and interstudy variability, and to fulfill quality assurance requirements (good laboratory practice, or GLP), it is important to standardize sampling, trimming, and embedding procedures as much as possible. This paper is intended to be a guide for the toxicologic pathologist involved in studies with nonhuman primates. The procedures are focused on the female reproductive organ system and mammary glands and ensure the preparation of well-oriented and standardized microscopic slides that facilitate thorough routine histopathologic examination of these complex tissues by the pathologist. Because nonhuman primates are, to an increasing extent, used in reproductive toxicity studies or in other studies with pregnant animals, it can be useful to make slides of the placenta. For this reason, sampling, trimming, and embedding procedures for the placenta have also been included. For illustration, the cynomolgus monkey is chosen as an example of a typical nonhuman primate.
For fixation, we generally recommend 4% formaldehyde made fresh from paraformaldehyde if immunostaining is intended (Williams 1977), otherwise commercial 10% neutral buffered formalin is acceptable.
Mammary Glands Prior to opening the thoracic cavity, the mammary glands should be sampled. If possible, the animal should be clipped to make mammary gland manipulation easier. Both mammary glands, including the overlying skin, should be collected. This can be done in the following way:
Abdominal Reproductive Organs When the abdominal cavity is opened and the organs inspected, the gastrointestinal tract and associated organs can be removed. To be able to inspect and dissect the whole genital tract, first locate the obturator foramina on one side of the pelvis. Place the tip of a bone-cutting shear into one foramen and cut the pubis (caudal) and ischium (cranial). Repeat this procedure on the opposite side, and remove the pubic bone to expose the pelvic canal. Alternatively, the pubic symphysis can be separated in younger animals by cutting with a sharp scalpel and pushing both pubic bones apart. The whole genital tract now can be inspected in situ (Figure 2). (Alternatively, the ovaries and oviducts can be sampled before the pelvic bone is removed.)
After recording the findings, first the ligaments are dissected from the uterus visible and cervix and then the whole tract, including ovaries, oviducts, cervix, bladder, and vagina, can be further dissected from its surrounding tissues. To prevent loss of small or fragile tissues, some tissues are placed in a prelabeled histology (or embedding) cassette that is put into the fixative jar.
Ligaments
Because the ligaments are rather thin structures, the preferred technique for fixation is the Swiss-roll technique (Moolenbeek and Ruitenberg 1981). This means that (samples of) the ligaments are rolled on a toothpick or cotton swab (or comparable tool) and placed, coiled, into the fixative. The Swiss-roll technique is recommended to be sure that a considerable amount of tissue can be obtained. After removal of the ligaments, the ovaries and oviducts can be removed.
Ovaries
Oviducts
The remaining reproductive organs can now be taken from the abdominal cavity (uterus, cervix, and vagina, with urinary bladder still adherent) by pulling while cutting them free from the surrounding tissues using a sharp scalpel. Keep the bladder intact with the vaginal wall. The different components can now be sampled as described below and depicted in Figure 1C.
Uterus and Cervix
Vagina
Vulva and Clitoris
Placenta
Before the tissues are processed, they are trimmed and put into histology cassettes. After processing (dehydrated and saturated with liquid paraffin), the trimmed tissues are manually put into paraffin blocks. Both trimming and embedding are essential steps, because proper tissue orientation enables the pathologist to examine the most interesting parts of the tissue and ensures standardization. Standardization is a necessity when comparing tissues from control versus treated animals and drawing the right conclusions on treatment-related findings.
Mammary Glands
Ligaments Cut transverse through the fixed Swiss-roll and embed the cut roll of tissue flat in paraffin.
Ovaries
Oviduct
Uterus and Cervix
Vagina
Placenta Slides prepared from the paraffin-embedded tissues routinely are stained using hematoxylin and eosin, although all kinds of other staining techniques can be applied to visualize specific structures or substances within these tissues (e.g., reticulum fibers, glycogen, mucopolysaccharides). Examples of routinely sampled and stained paraffin sections from the reproductive organs and mammary glands of a mature cynomolgus monkey are illustrated in Figure 4. Also, immunohistochemical staining methods for the detection of the estrogen receptors, progesterone receptors, and the KI67-protein (a marker for cell proliferation) can be very helpful. (Nowadays, such immunohistochemical techniques can well be performed on formalin-fixed tissues). Examples of such techniques are given in the different papers within this monograph.
Pituitary Gland After removal of the brain, the pituitary should be carefully dissected from the sella turcica by cutting the overlying dura, fixed in toto, weighed, and trimmed coronally to include the neurohypophysis and adenohypophysis (a low-power microscopic image of a pituitary gland prepared in this way is given in the paper by Weinbauer et al., this monograph).
Adrenal Glands
Endometrial Biopsies Endometrial biopsies must be taken laproscopically under deep anesthesia, because the cervical canal of the macaque is too coiled to be used for transvaginal biopsy techniques. A wedge-shaped biopsy is carefully cut from the uterine body. One should be certain that the biopsy includes a significant amount (preferably the full thickness) of the endometrium, including glands of the zona functionalis. The biopsies are placed in 4% paraformaldehyde in phosphate-buffered saline (PBS) (pH 7.4) (Lobie 1997) for fixation in a prelabeled histology cassette. After processing, the biopsy is embedded in a paraffin block. Orientation of the tissue in the block is of utmost importance, since slides prepared from the block need to contain a significant amount of endometrium. Ideally, the full thickness of the endometrium, including luminal epithelium, is present in the slide (Figure 5A).
Incorrectly taken biopsies often consist mainly of myometrium or include the zona basalis of the endometrium only (Figure 5B). The presence of a significant amount of zona functionalis in the final slide is a prerequisite for a sound microscopical judgment. Biopsies always have to be handled with care to avoid squeezing artifacts, which can hamper microscopic evaluation. In Figure 6, two examples are given of the microscopic appearance of such an artifact.
Vaginal/Cervical Cytology Collection (Smears) The vaginal vault is to be swabbed, and the cotton swab then rolled onto two prelabeled glass slides. After drying, the slides need to be fixed, which can be done using Spray-cyte fixative or 95% ethanol. It is important that these vaginal swabs are collected from approximately the same site in each animal. Smears are best stained using the Papanicolaou (Pap) stain (Lillie and Fullmer 1976), although a modified Wright’s stain (Lillie and Fullmer 1976) may suffice.
In this paper we describe the standardized procedures, from necropsy techniques to microscopic slide preparation of cynomolgus monkey female reproductive organs, mammary glands, and placenta. The procedures described herein help to obtain representative, high-quality microscopic specimens necessary for reliable histopathologic evaluation by the pathologist. High-quality specimens also facilitate the comparison of changes and/or findings among animals within a study (or between different studies). Nevertheless, the qualifications, training, and experience of the pathologist represent the most essential factors in the quality of the histopathology evaluation, interpretation (Crissmann et al. 2004), and risk assessment.
Crissman, JW, Goodman, DG, Hildebrandt, PK, Maronpot, RR, Prater, DA, Riley, JH, Seaman, WJ, & Thake, DC. (2004). Best Practices Guideline: Toxicologic Histopathology. Toxicol Pathol, 32, 126-31 Lillie, RD, & Fullmer, HM. (1976). Histopathologic Technic and Practical Histochemistry. (4th ed). New York: McGraw-Hill Lobie, PE. In Morel, G (Ed.). (1997). Light microscopic visualization of polypeptide ligand receptors. Visualization of Receptors: Methods in Light and Electron Microscopy (pp.181-96). Boca Raton, FL: CRC Press Mazur, MT, & Kurman, RJ. In Mazur, MT, & Kurman, RJ (Eds.). (1995). Artifacts and Contaminants. Diagnosis of Endometrial Biopsies and Curettings. A Practical Approach (pp.23-24). New York: Springer Moolenbeek, C, & Ruitenberg, EJ. (1981). The ‘Swiss roll’: a simple technique for histological studies of the rodent intestine. Lab Anim, 15, 57-59 Williams, MA. In Glauert, AM (Ed.). (1977). Autoradiography and immunocytochemistry. Part I in the series: Practical methods in electron microscopy, 6, Amsterdam, New York, Oxford: North Holland Publishing Company
Toxicologic Pathology, Vol. 36, No. 7 Suppl,
164S-170S (2008)
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Abstract
Introduction
