大鼠小鼠器官取材与切面方向速查表

使用说明：本表依据 RITA/NACAD 指南整理，适用于大鼠与小鼠的常规毒理病理取材。原文为英文，此处保留英文术语（如 lobe、transverse）以便与国际规范对照。实际取材请结合具体试验方案与实验室 SOP，本表仅作参考。

体被与皮肤腺

皮肤、乳腺及皮肤相关腺体的取材规范

1.1

乳腺与皮肤

Mammary gland & Skin

取材部位: Inguinal region
切片数量: 1
切面方向: a) Transverse b) Longitudinal vertical to the direction of the hair flow
样本大小: 1 cm × 3 cm
注意事项: Transverse section: includes the nipple and the lateral iliac lymph node. Longitudinal section: the nipple is not included if the lymph node is enclosed. Both sections: ensure a high amount of mammary gland tissue. The mammary gland is a paired organ. Due to the diffuse distribution of mammary gland tissue it is of no concern whether one or both sides are in the section. The inguinal region is the recommended area for harvesting mammary gland. Sections of mammary gland should be taken with associated nipple and skin. The result of histotechnique may be improved by shaving the skin at necropsy or removing the hair with scissors at trimming. Orientation of a shaved skin specimen is possible by the nipples in female animals. In male animals, the inguinal region is also preferred to examine skin and mammary gland tissue. The section will be embedded on the cut edge so that it reveals skin, subcutis and mammary gland close to the nipple. In the longitudinal section, the hair follicles will be visible in full length. Relevant differences between rats and mice Rats have 6 pairs of mammary glands while mice have only 5 pairs. This difference is not of practical importance since mammary tissue is abundant in the inguinal region of both species. In females, the mammary tissue extends from the salivary gland region to the base of the tail. Related references BOORMAN et al. 1990b, DEERBERG et al. 1986, ELWELL et al. 1990, KOVATCH 1990a, KOVATCH 1990b, MILITZER et al. 1990, RUSSO et al. 1989 hair flow. Exp Toxic Pathol 55 (2003) 2–3 95 1 Integumentary system

1.2

Zymbal 腺

Zymbal's gland

取材部位: Adjacent to the auditory canal
切片数量: 1
切面方向: Transverse
注意事项: A preparation of the Zymbal’s gland is not advisable, instead a transverse section across the base of the decalcified skull at both ethmoidal bullae is performed. The Zymbal’s glands are made up of several lobules of modified sebaceous glands which are located at the base of the external ear (anterio-ventral). A section through the base of the skull at the level of the external ears generally results in a section plane through one or more lobules of Zymbal’s glands tissue. Related references ALTMAN and GOODMAN 1979, COPELAND-HAINES and EUSTIS 1990 and brain: Zymbal’s gland (Ma: meatus acusticus externus, P: pituitary gland, Eb: ethmoidal bullae). 1 Integumentary system

1.3

阴蒂腺 / 包皮腺

Clitoral / Preputial gland

取材部位: Subcutaneous adipose tissue, lateral to penis/cranial to vulva
切片数量: 1
切面方向: Longitudinal horizontal Clitoral/preputial glands are modified sebaceous glands. The whole organs are removed at necropsy and embedded in toto. Related references glands (P), testis (T), penis (Pe). (right). (T), penis (Pe). ducts. glands (C) and vulva (V). glands. Exp Toxic Pathol 55 (2003) 2–3 97 2 Digestive system

消化系统

口腔至胰腺的消化道各段取材与切面

2.1

舌

Tongue

切片数量: 1
切面方向: Longitudinal vertical
可选切片: transverse section of mid-portion
注意事项: Tip removed if organ does not fit into the cassette The longitudinal vertical section of the tongue covers a large part of the dorsum including the dorsal prominence. Well developed papillae are found rostral to the dorsal prominence. The section also includes the lingual lesser salivary glands and should be slightly lateral to the median sulcus. The transverse section is recommended if blood sampling from the tongue is performed. Related references BROWN and HARDISTY 1990, KOCIBA and KEYES 1985 2 Digestive system

2.2

唾液腺

Salivary glands

取材部位: Cranio-ventral throat region
切片数量: 1
切面方向: Longitudinal horizontal
可选切片: together with mandibular lymph nodes The three salivary glands and the mandibular lymphatic center, which consists of two or three lymph nodes are removed in one piece. At necropsy it is not necessary to prepare each organ individually. The extraorbital lacrimal gland may be removed separately or in conjunction with the other glands. All three salivary glands should be present in the section. Related references HEBEL and STROMBERG 1986 glands, extraorbital lacrimal gland and mandibular lymph nodes. throat region: mandibular, parotid and sublingual glands and mandibular lymph nodes. gland. glands. PG: parotid gland, SG: sublingual gland, MG: mandibular gland). Exp Toxic Pathol 55 (2003) 2–3 99 2 Digestive system
注意事项: Mandibular gland through largest surface

2.3

咽与喉

Pharynx & Larynx

取材部位: Middle of the larynx
切片数量: 1
切面方向: Transverse
注意事项: If necessary, decalcified for one day. Nasopharynx is included in the most caudal localization of the nasal cavity Section contains larynx and pharynx. See also: Larynx (inhalation study) Related references BOORMAN et al. 1990a 2 Digestive system

2.4

食管与气管

Esophagus & Trachea

取材部位: Region of thyroid gland
切片数量: 1
切面方向: Transverse
注意事项: Together with trachea This procedure is recommended if the thyroids are removed for weighing or for performing a longitudinal section of the thyroid gland. See also: Thyroid gland Trachea (inhalation study) Related references BROWN and HARDISTY 1990 view) indicating the level in the region of the ventral pouch. (with ventral pouch and vocal processes) and pharynx (P). esophagus (E) and trachea (T) with tongue (To), hyoid bone (H) and thyroid gland (Th). glands. 2 Digestive system

2.5

胃

Stomach

取材部位: 1) From cardiac region through pyloric sphincter to the duodenum 2) Across the limiting ridge with forestomach and the fundic part of the glandular stomach 3)
切面方向: Longitudinal vertical
可选切片: section through the fundus Number of samples: 2 (3)
注意事项: Opened along the greater curvature, mounted and fixed The stomach of the rat is opened along or paramedian to the greater curvature and placed on cardboard or a flat piece of styrofoam. The ingesta are removed and, if necessary, the mucosa is cleaned carefully with saline solution or fixative. The stomach is spread out and fixed with about six pins. This is a prerequisite for the macroscopic orientation and the reproducible microscopic evaluation of the gastric mucosal height or actually the measurement of the mucosa by morphometric means as it avoids folds in the mucosa. The first section is cut from the cardiac region of the stomach across fundus and antrum and the pyloric sphincter to the duodenum. If the piece is too large for the normal cassette, the tissue can be cut in halves. The second section is taken from the forestomach through the fundic area, allowing the best evaluation of the limiting ridge between the forestomach and the glandular stomach. Optionally, a third section can be made through the fundic area, where the fundic glands are most bulky. The fundic area is the thickest part of the mucosa and can vary under acid suppressing conditions or other drug-induced influences. ECL-cell hyperplasia and neuroendocrine tumors are found in the fundic mucosa. Related references BETTON et al. 1988, BROWN and HARDISTY 1990, HAKANSON et al. 1984, MATSUKURA and ASANO 1997 spread out before fixation. fundus (GF), antrum (A), pylorus (P). fundus (GF). fundus. Exp Toxic Pathol 55 (2003) 2–3 101 2 Digestive system

2.6

肠

Intestine

取材部位: 1) Duodenum: 1 cm distal to the pyloric sphincter 2) Jejunum: central section 3) Ileum: 1 cm proximal to cecum 4) Cecum 5) Colon: central section 6) Rectum: 2 cm proximal to the anus
切片数量: 6
切面方向: Transverse
可选切片: additional longitudinal vertical and/or transverse section through Peyer’s patches Cecum: due to the large diameter it is advisable to open the specimen Rectum longitudinal vertical section to include the anus During necropsy or after fixation, the intestine is carefully separated from the mesentery. Peyer’s patches of the jejunum are mostly visible as slightly elevated lighter fields in the intestine’s wall or are even discernible as prominent areas when activated. One transverse section from each part of the unopened bowel is taken. The remaining intestine should be opened and examined for abnormalities. At necropsy, the ingesta should not be removed vigorously but only gently rinsed with physiological saline if necessary. Swiss roll technique: This technique is sometimes required for examination of the whole intestine and the gut associated lymphatic tissue (GALT). The intestine is stripped off the mesentery, opened with a pair of scissors and gently rinsed. The intestine except cecum is recoiled on cotton swabs and fixed. After fixation, the spooled intestine is detached and embedded. This procedure is technically challenging and not recommended for routine purposes, as the intestinal mucosa and the lymph follicles will often be found cut tangential. However, transverse sections as described above will often provide a better histoanatomy. The jejunum and ileum or the distal colon and rectum cannot readily be differentiated microscopically. For consistency in routine examination of each required site, accurate sampling is necessary. In this case, the colon differs from the rectum by a thinner muscle layer and a larger lumen. For dehydration and embedding, cassettes with a subdivision are helpful. Please note that the magnification of the histological images is not the same for all parts of the intestine. Related references ELWELL and MCCONNELL 1990, KUPER et al. 2000, MOOLENBEEK and RUITENBERG 1981, SMINIA and JEURISSEN 1990, SOUL 1987 lymph nodes. mesentery is removed (explanations of numbers see fig. 2.6a). Exp Toxic Pathol 55 (2003) 2–3 103 2 Digestive system
注意事项: Duodenum in conjunction with an adjacent piece of pancreas Jejunum containing Peyer’s patch or lymph follicle.

2.7

肝与胆囊

Liver & Gall bladder

取材部位: 1) Left lateral lobe 2a) Rat: right medial lobe 2b) Mouse: left and right medial lobe including gall bladder 3)
切片数量: 2 (3)
切面方向: 1, 2a, 3) Transverse, 2b) longitudinal-vertical
可选切片: caudate lobe
注意事项: Sample sizes should be as large as possible but can be adapted so that all pieces fit into one cassette. For identification purposes, standardized shaping of one of the larger lobes can be performed. Sampling from other locations is also appropriate, if consistency is provided. If major bile duct is required, the optimal section is the one through the left lateral lobe. Relevant differences between rats and mice Gall bladder in mice: the longitudinal section is preferred to the transverse one. In the longitudinal section, the gall bladder will be in anatomical conjunction with the liver lobes, whereas in the transverse section, the slice of the gall bladder is prone to lose its connection to the liver tissue. Related references GARCIA-MORENO et al. 1994, POPESKO et al. 1992, RUBARTH 1958, FRITH et al. 1994, YOSHITOMI and BOORMAN 1994 for rats and mice. and 2b. 2 Digestive system

2.8

胰腺

Pancreas

取材部位: Left lobe anatomical site or part of an organ from which a sample should be taken (i.e. lobe). 2. Number of samples: number of organs (i.e. both for bilateral organs) or organ pieces prepared for evaluation (not necessarily identical with the number of slides/blocks). 3.
切片数量: 1
切面方向: Longitudinal horizontal direction (plane of section) in which an organ should be cut at trimming or microtome sectioning. The proposed direction is shown in green color and optional sections (if defined) are shown in blue (see fig. 1 for an explanation of the symbols used). 4.
样本大小: the size (area) of an organ or part of an organ which is sampled in a cassette for processing. The sample size is determined by the size of an organ or the cassette. For optimal fixation, sample thickness should not exceed 3–5 mm. In general, the examined area should be as large as possible and should contain the relevant anatomical structures. The tissue can be adapted to the size of cassettes by trimming the margins off. 5. Optional remarks are used to present additional information, as recommended by the RITA/NACAD groups, such as the instillation of fixative into the lung or the urinary bladder, reasons for optional recommended sections, placements of organs in cassettes, etc. 6. Schematic drawings and/or gross photographs are shown indicating the plane of section. Some of the gross photographs show the organ and trimming direction in situ. However, this is just for orientation purposes and it is recommended to remove the organ parallel to the plane of the picture, b: cutting level perpendicular to the plane of the picture, c: cut level, 3-D. or tissue first. Trimming is performed as the next step, either on the fresh wet tissue or, in most cases, after fixation of the organ. Most of the gross photographs were taken from fresh unfixed organs; shape and color may be slightly different after fixation. 7. An image of a Hematoxylin and Eosin (H&E) stained slide is shown for the recommended section level (sometimes also for optional levels). Typical structures included in this section are indicated as necessary. If not otherwise specified, 10% buffered formalin is recommended as the fixative. In the descriptions the following terms are used for the determination of the trimming directions (see also fig. 2 with a schematic presentation of the related cut levels): • transverse: perpendicular to the long axis of an organ or part of an organ • longitudinal vertical: in the direction of the long axis of the body, an organ or part of an organ in the dorsoventral axis or parallel to it (in the text also referred to in short as “longitudinal”) • longitudinal horizontal: in the direction of the long axis of the body, an organ or part of an organ, perpendicular to the dorsoventral axis (in the text also referred to in short as “horizontal”) By defining either the “body”, the “whole organ” or a “part of an organ” (for example a liver lobe or a certain part of the brain), as a unit of reference, it is relatively simple to precisely characterize a trimming direction by using only the three above defined terms and avoiding therefore the vast amount of anatomical terms and confusing synonyms present in literature. Acknowledgements: The authors would like to thank Mr. Theodor Laforme and Mrs. BRIGITTE POSCHMANN (Bayer HealthCare AG) for the gross preparation of organs and histological slides; Mrs. PETRA HARTMANN, Mrs. MEIKE PETERSEN and Mrs. MARTINA NEUENHAUS (Bayer HealthCare AG) for scanning the histological slides, Dr. GERDPETER FEHLERT for his help in image processing; Mr. PETER KOCH and Mr. MIRCO JAHNKE (BASF AG) for the photo documentation on prostate preparation; Dr. MARTIN ROSENBRUCH (Bayer HealthCare AG), Dr. SUSANNE RITTINGHAUSEN and Dr. HEINRICH ERNST (Fraunhofer ITEM) for their scientific advice for organ preparation in inhalation studies and Dr. PAUL DESLEX (Pfizer Centre Recherche) for providing additional photographs and slides. transverse, b: longitudinal vertical, c: longitudinal horizontal. 3 Respiratory system
注意事项: A cut surface as large as possible The left lobe of the pancreas represents a major part of pancreatic tissue being located close to the spleen in the greater omentum. For trimming, the whole left lobe is removed and fixed. A large part of the left lobe is taken and embedded, in order to achieve a cut surface as large as possible. The right lobe is removed together with the adjacent small intestine. Related references EUSTIS and BOORMAN 1997, POPESKO et al. 1992, RUBARTH 1 References for organ guides ALTMAN NH, GOODMAN DG: Neoplastic diseases. In: BAKER HJ, LINDSEY JR, WEISBROTH SH (eds) The laboratory rat. Vol. 1, Biology and diseases. Academic Press, New York, 1979; pp 334–377. BETTON GR, DORMER CS, WELLS T, et al.: Gastric ECLCell hyperplasia and carcinoids in rodents following chronic administration of H2-antagonists SK&F 93479 and oxmetidine and omeprazole. Toxicol Pathol 1988; 16: 288–298. BOORMAN GA, MORGAN KT, URIAH LC: Nose, larynx and trachea. In BOORMAN GA, EUSTIS SL, ELWELL MR, et al. (eds): Pathology of the Fischer rat, reference and atlas. Academic Press, San Diego, New York, Boston, 1990a; pp 315–337. BOORMAN GA, WILSON JT, VAN ZWIETEN MJ, et al.: Mammary gland. In: BOORMAN GA, EUSTIS SL, ELWELL MR, et al. (eds): Pathology of the Fischer rat. Reference and atlas. Academic Press, San Diego New York London, 1990b; pp 295–313. BROWN HR, HARDISTY JF: Oral cavity, esophagus and stomach. In: BOORMAN GA, EUSTIS SL, ELWELL MR, et al. (eds) Pathology of the Fischer rat. Reference and atlas. Academic Press, San Diego New York London, 1990; pp 9–30. COPELAND-HAINES D, EUSTIS SL: Specialized sebaceous glands. In: BOORMAN GA, EUSTIS SL, ELWELL MR, et al. (eds) Pathology of the Fischer rat. Reference and atlas. Academic Press, San Diego New York London, 1990; pp 279–294. DEERBERG F, KNÜP F, REHM S: Spontaneous epithelial tumors of the skin in Han:WIST- and DA/Han rats. Z Versuchstierkd 1986; 28: 45–57. ELWELL MR, MCCONNELL EE: Small and large intestine. In: BOORMAN GA, EUSTIS SL, ELWELL MR, et al. (eds) Pathology of the Fischer rat. Reference and atlas. Academic Press, San Diego New York London, 1990; pp 43–61. ELWELL MR, STEDMAN MA, KOVATCH RM: Skin and subcutis. In: BOORMAN GA, EUSTIS SL, ELWELL MR, et al. (eds) Pathology of the Fischer rat. Reference and atlas. Academic Press, San Diego New York London, 1990; pp 261–277. EUSTIS SL, BOORMAN GA: Embryology, histology and ultrastructure of the exocrine pancreas. In: JONES TC, POPP JA, MOHR U (eds) Monographs on pathology of laboratory animals. Digestive system, 2nd edition. Springer, Berlin Heidelberg New York Tokyo, 1997; pp 257–267. FRITH CH, WARD JM, TURUSOV VS: Tumours of the liver. In: TURUSOV VS, MOHR U (eds) Pathology of tumours in laboratory animals. Vol 2. Tumours of the mouse, 2nd edition. IARC Scientific Publications No. 111, Lyon, 1994; pp 223–248. GARCIA-MORENO L, VALLEJO G, ARIAS JL, et al.: Behaviour of the nucleolar organizer regions on the different Wistar rat liver lobes. Lab Anim 1994; 28: 50–54. HAKANSON R, EKLUND M, SUNDLER F: Activation and proliferation of gastric endocrine cells. In: HAKANSON R, SUNDLER F (eds) Evolution and tumor pathology of the neuroendocrine system. Elsevier, Amsterdam, 1984; pp 371–398. HEBEL R, STROMBERG MW: Anatomy and embryology of the laboratory rat. BioMed, Wörthsee, 1986; pp 48–49. KOCIBA RJ, KEYES DG: Squamous cell carcinoma, tongue rat. In: JONES TC, MOHR U, HUNT RD (eds) Monographs on pathology of laboratory animals. Digestive system. Springer, Berlin Heidelberg New York, 1985; pp 255–259. KOVATCH R: Neoplasm of the integument. In: STINSON SF, SCHÜLLER HM, REZNIK GK (eds) Atlas of tumor pathology of the Fischer rat. CRC Press, Boca Raton, 1990a; pp 19–32. KOVATCH R: Neoplasms of the mammary gland. In: STINSON SF, SCHÜLLER HM, REZNIK GK (eds) Atlas of tumor pathology of the Fischer rat. CRC Press, Boca Raton, 1990b; pp 473–484. KUPER CF, HARLEMAN JH, RICHTER-REICHELM HB, et al.: Histopathologic approaches to detect changes indicative of immunotoxicity. Toxicol Pathol 2000; 28: 454–466. MATSUKURA N, ASANO G: Anatomy, histology, ultrastructure, stomach, rat. In: JONES TC, POPP JA, MOHR U (eds) Monographs on pathology of laboratory animals. Digestive system, 2nd edition. Springer, Berlin Heidelberg New York Tokyo, 1997; pp 343–350. MILITZER K, HIRCHE H, MOOG E: The ontogenesis of skin and organ characteristics in the Syrian golden hamster. 1. Skin compartments and subcutaneous adipocytes. Exp Pathol 1990; 40: 77–93. MOOLENBEEK C, RUITENBERG EJ: The “Swiss roll”: a simple technique for histological studies of the rodent intestine. Lab Anim 1981; 15: 57–59. POPESKO P, RAJTOVA V, HORAK J: A colour atlas of the anatomy of small laboratory animals, Vol. 2. Wolfe Publishing, Bratislava, 1992; pp 11–104. REZNIK G, WARD JM: Morphology of hyperplastic and neoplastic lesions in the clitoral and preputial gland of the F344 rat. Vet Pathol 1981a; 18: 228–238. REZNIK G, WARD JM: Morphology of neoplastic lesions in the clitoral and prepucial gland of the F344 rat. J Cancer Res Clin Oncol 1981b; 101: 249–263. RUBARTH: Leber und Gallenwege. In: COHRS P, JAFFE R, MEESSEN H (eds) Pathologie der Laboratoriumstiere. Springer, Berlin Göttingen Heidelberg, 1958; pp 155–175. RUSSO IH, TEWARI M, RUSSO J: Morphology and development of the rat mammary gland. In: JONES TC, MOHR U, HUNT RD (eds) Monographs on pathology of laboratory animals. Integument and mammary glands. Springer, Berlin Heidelberg New York Tokyo, 1989; pp 233–252. SMINIA T, JEURISSEN SHM: Gut-associated lymphoid tissue, rodent, normal structure. In: JONES TC, WARD JM, MOHR U, et al. (eds) Monographs on pathology of laboratory animals. Hemopoietic system. Springer, Berlin Heidelberg New York Tokyo, 1990; pp 307–315. SOUL NW: Gut rolls: a better technique for GI tumor assessment. Toxicol Pathol 1987; 15: 374. YOSHITOMI K, BOORMAN GA: Tumours of the gallbladder. In: TURUSOV VS, MOHR U (eds) Pathology of tumours in laboratory animals. Vol 2. Tumours of the mouse, 2nd edition. IARC Scientific Publications No. 111, Lyon, 1994; pp 271–279. Exp Toxic Pathol 2004; 55: 413–431 http://www.elsevier.de/etp 1 Department of Product Safety, Regulations, Toxicology and Ecology, BASF AG, Ludwigshafen, Germany 2 Department of Toxicologic Pathology, Bayer HealthCare AG, Wuppertal, Germany 3 Department of Information Technology and Databases, Fraunhofer Institute of Toxicology and Experimental Medicine, Hannover, Germany 4 Department of Pathology, Pharmacia Italia S.p.A., Gruppo Pfizer Inc., Nerviano, Italy 5 Department of Pathology, Pfizer, Inc., Groton, CT, USA 6 Department of Toxicology, Hoffmann-LaRoche AG, Basel, Switzerland 7 Department of Pathology/Toxicology, 3M Pharmaceuticals, St. Paul, MN, USA 8 Department of Pathology, Novartis Pharma AG, Basel, Switzerland 9 Department of Pathology, AstraZeneca UK, Alderley Park, Macclesfield, England Revised guides for organ sampling and trimming in rats and mice – Part 2 A joint publication of the RITA*) and NACAD**) groups BIRGIT KITTEL1, CHRISTINE RUEHL-FEHLERT2, GERD MORAWIETZ3, JAN KLAPWIJK4, MICHAEL R. ELWELL5, BARBARA LENZ6, M. GERARD O’SULLIVAN7, DANIEL R. ROTH8, and PETER F. WADSWORTH9 With 65 figures Received: February 24, 2004; Revised: March 20, 2004; Accepted: April 16, 2004 Address for correspondence: GERD MORAWIETZ, Department of Information Technology and Databases, Fraunhofer Institute of Toxicology and Experimental Medicine, Nikolai-Fuchs-Str. 1, 30625 Hannover, Germany; Fax: ++49 511 5350 155, E-mail: morawietz@item.fraunhofer.de Key words: Trimming; RITA; NACAD; rat; mouse; standardization; guidelines; nasal cavity; nasopharynx; paranasal sinus; larynx; trachea; bronchus; bronchiole; lung; testis; rete testis; epididymis; prostate; coagulating gland; seminal vesicle; ovary; oviduct; uterus; uterine cervix; vagina; pituitary gland; thyroid gland; parathyroid gland; adrenal gland. *) RITA: Registry of Industrial Toxicology Animal-data. Members: Abbott GmbH & Co KG, Ludwigshafen, Germany; ALTANA Pharma AG, Hamburg, Germany; AstraZeneca, Södertälje, Sweden and Macclesfield, England; Aventis Pharma Deutschland GmbH, Hattersheim, Germany; BASF AG, Ludwigshafen, Germany; Bayer HealthCare AG, Wuppertal, Germany; Boehringer Ingelheim Pharma GmbH & Co KG, Biberach, Germany; Fraunhofer Institute of Toxicology and Experimental Medicine, Hannover, Germany; Hoffman-LaRoche AG, Basel, Switzerland; Merck KGaA, Darmstadt, Germany; Novartis Pharma AG, Basel, Switzerland; Pfizer, Amboise, France; Pharmacia, Nerviano, Italy; Syngenta CTL, Macclesfield, England **) NACAD: North American Control Animal Database. Members: 3M Pharmaceuticals, St. Paul, MN, USA; Adolor Corporation, Malvern, PA, USA, Bayer CropScience, Stillwell, KS, USA; Pfizer, Inc., Groton, CT, USA; Pfizer, Inc., Ann Arbor, MI, USA; Pharmacia, Inc., Kalamazoo, MI, USA; R.W. Johnson Pharmaceutical Research Institute, Spring House, PA, USA; Schering-Plough Research Institute, Lafayette, NJ, USA Summary This is the second part of a series of three articles on trimming instructions of rat and mouse protocol organs and tissues in regulatory type toxicity studies, covering the respiratory, male and female genital, and the endocrine systems. The article is based on the experience of the European RITA and American NACAD working groups and is an extended revision of trimming guides published in 1995 (BAHNEMANN et al.). The optimum localization for tissue preparation, the sample size, the direction of sectioning and the number of sections to be prepared is described organ by organ. These descriptions are illustrated for each organ by a schematic drawing and/or a macro-photograph showing the plane of section as well as a low magnification of the H&E stained slide demonstrating the optimum “end-product”. The objectives of this work, as addressed in detail in the first part (RUEHL-FEHLERT et al. 2003), are to stan0940-2993/04/55/06-413 $ 30.00/0 413 dardize tissue sampling and trimming, to improve the comparability of historical data obtained from different studies and different laboratories, ensure the presence of all relevant target sites for histopathological evaluation and provide technical advice for preparatory techniques during necropsy, fixation and trimming. Brief introduction to the use of the individual organ guides For each organ the following information is usually presented (for more details see part 1: RUEHL-FEHLERT et al. 2003): 1.

呼吸系统

鼻腔、喉、气管与肺，含吸入给药研究要点

3.1

鼻腔与鼻咽

Nasal cavity & Nasopharynx

取材部位: 1) Posterior part of upper incisors 2) Incisive papilla 3) Second palatine crest 4) First molar teeth
切片数量: 1 (oral toxicity study: third level) 4 (inhalation study)
切面方向: Transverse
注意事项: Embedded with the rostral faces down Decalcified The structures of the palate and the teeth are used for orientation to achieve transverse sections through the nasal cavity at certain levels. In inhalation studies, four transverse tissue levels should be taken, because the examination of these sections at defined levels assures consistent recognition of degenerative and proliferative lesions of all different epithelial cell types of the nasal cavity and paranasal sinus. Typically in oral toxicity studies, only the third level is examined. Neoplastic lesions occur more frequently in the anterior and middle portions of the nasal cavity, whereas some non-neoplastic and neoplastic lesions are observed exclusively in the olfactory epithelium. The third level includes respiratory and olfactory epithelial cells. The resulting slices of tissue are embedded with the rostral face down, because non-neoplastic lesions have been found to be most severe at the more rostral borders of the affected epithelium. Slight differences of the cut level may occur depending on anatomical variations in different strains. If more squamous epithelium is required for examination, a section rostral to level 1 should be performed. For examination of the olfactory bulb, a section caudal to level 4 is recommended. Relevant differences between rats and mice Mice should be trimmed in the same manner as rats. However, in inhalation studies with very young or very small mice (e.g., transgenic strains), it can be difficult to cut the nose in four levels. For those exceptions, the following three level-procedure is recommended: 1) Immediately posterior to the incisors, 2) At the level of the incisive papilla, 3) Through the middle of the second molar tooth. For non-inhalation studies level 3 is recommended for examination. Related references BROWN 1990, HARDISTY et al. 1999, HARKEMA 1990, HARKEMA and MORGAN 1996, MORGAN 1991, POPP and MONTEIRO-RIVIERE 1985, URAIH and MARONPOT 1990, YOUNG 1981 location 1. rat, location 2. Nd: nasopalatine (incisive) duct, Nl: Nasolacrimal duct. Exp Toxic Pathol 55 (2004) 6 415 (nasopharynx). sinus. 3 Respiratory system

3.2

喉

Larynx

取材部位: 1) Base of epiglottis 2) Ventral pouch 3) Cricoid cartilage (rats only)
切片数量: Inhalation studies: rats 3, mouse 2 Optional for rats and mice: if necessary, the larynx can also be embedded in one block and step sections are taken at the predilection sites. Oral studies: 1
切面方向: Transverse
注意事项: Since the larynx of mice is very small, only two pieces (level 1 and 2) are trimmed. For oral studies the organ is cut at level 2 to include the most sensitive parts: ventral pouch and vocal processes (medial surfaces) of arytenoid cartilages. Only the cranial portion of the epiglottis is removed to ensure inclusion of the major predilection site for induced lesions. This site is primarily represented by the epithelial lining of the ventral and ventrolateral luminal surface of the larynx (cranial to the ventral laryngeal pouch). The remaining larynx is trimmed according to the proposed scheme at three levels including base of epiglottis, ventral diverticulum and cricoid cartilage. The three pieces are embedded with the cranial cut surface downwards. The three levels assure recognition of all different epithelial cell types of the larynx and underlying seromucinous glands. See also: Thyroid gland Related references LEWIS 1991, RENNE et al. 1992, SAGARTZ et al. 1992 levels 1–3, mice: levels 1 and 2. base of the epiglottis. of the arytenoid cartilages, U: u-shaped cartilage. Exp Toxic Pathol 55 (2004) 6 417 3 Respiratory system

3.3

气管

Trachea

取材部位: Including the bifurcation
切片数量: 1 (2)
切面方向: Longitudinal horizontal
可选切片: transverse
注意事项: Embedded in toto; careful microtome sectioning until recommended cutting level is obtained. In inhalation studies, tracheal epithelium including the epithelial lining of the bifurcation should be examined, because this is known as the most sensitive area to respond to inhaled particulate irritants. For this purpose, a longitudinal horizontal section should provide a long distance of the epithelial surface and the tip of the carina. For optimal estimation of e.g. mild hyperplasia, it can be helpful to have an optional transverse section (see alternative trimming technique for oral toxicity study under thyroid gland/trachea/parathyroid gland). Related references GOPINATH et al. 1987, SCHWARTZ et al. 1991 3 Respiratory system

3.4

肺

Lung

取材部位: Recommended procedure: 1) Left lobe 2) 1) Left lobe 2) Right caudal lobe 3) Right cranial lobe 4) Right middle lobe 5) Accessory lobe 1) Left lobe 2) Right caudal lobe 3) Right cranial lobe 4) Right middle lobe 5) Accessory lobe
切片数量: 1 (3) 5 5
切面方向: Longitudinal horizontal Sections 1, 2: longitudinal horizontal Sections 3, 5: transverse Section 4: longitudinal vertical Sections 1, 2, 4, 5: longitudinal horizontal Section 3: transverse
可选切片: right caudal lobe 3) right cranial lobe transverse whole lung in toto (ventral surface down) without removal of the trachea. Microtome sectioning of left lobe and right caudal lobe until lobar bronchus and its main branches are visible (longitudinal-horizontal axis). Spontaneous neoplastic pulmonary lesions are rare in rats and arise mostly in the lung periphery whereas regenerative hyperplasia and squamous metaplasia occur mainly in the centroacinar region. Therefore tissue of the lung including parenchyma, bronchiolo-alveolar junctions and main bronchi should be investigated. In oral toxicity studies, at minimum, one longitudinal section of the left lobe should be examined. Additionally, transverse sections of the right cranial and caudal lobes may be examined. In these sections, the epithelium of the major bronchioles, which is one important site of lesions, Exp Toxic Pathol 55 (2004) 6 419 can be examined at its widest diameter. In inhalation studies, sections of all five lobes should be examined according to the proposed scheme, which facilitates unambiguous identification of individual lung lobes. For histological identification of proliferative lesions in the lung, careful fixation by intratracheal instillation is recommended, even for oral studies. Related references DUNGWORTH et al. 1992, GOPINATH et al. 1987, PLOPPER 1996, RENNE et al. 2001, RITTINGHAUSEN et al. 1992, SCHWARTZ et al. 1991, SMINIA et al. 1990 left lobe. location 2, right caudal lobe. location 3, right cranial lobe. location 4, right middle lobe. location 5, accessory lobe. 4 Male genital system
吸入研究: Rats Mice
注意事项: Instillation strongly recommended. Sectioning to the axis of the lobar bronchus. Longitudinal section comprising the lobar bronchus and its main branches. Sample size(s) adapted to the size of the cassette(s). Alternative procedure: Rat: right lobes embedded ventral surface down. Mouse: whole lung embedded, ventral surface down. Instillation obligatory. Longitudinal horizontal section comprising the lobar bronchus and its main branches. Sample size(s) adapted to the size of the cassette(s); preferentially, the diaphragmatic margin is trimmed off. Alternative procedure: right and left lobes (separate blocks) embedded ventral surface down. Instillation obligatory. Similar procedure as in rats, but lobes are embedded in toto, ventral surface down and detached from the trachea. The five lobes normally fit into one cassette.

雄性生殖系统

睾丸、附睾、精囊与前列腺

4.1

睾丸与睾丸网

Testis & Rete testis

取材部位: Transverse, close to the rete testis
切片数量: 2 (1 per side)
切面方向: Transverse
可选切片: longitudinal vertical section containing the rete testis. Near the capsule, focal tubules with flat or incomplete epithelium can be found. These should not be confused with atrophic or degenerating tubules, as they represent tubules of the rete testis. Generally, the rete testis is very small in rodents and not well visible in the histological section. In mice it is slightly more prominent than in rats and hyperplasia of the rete testis can be observed in older animals. For inclusion of the rete testis in the histological section orientation is given by the vasculature. In short-term studies, fixation with Davidson’s or Bouin’s solutions is highly recommended to detect less extensive toxicity. Leydig cells (interstitial cells) are present in small groups in the interstitium between the seminiferous tubules. They are found in a similar distribution in all sections proposed. Related references BOORMAN et al. 1990a, CHAPIN and HEINDEL 1993, CREASY and FOSTER 2002, FERM 1987, FOLEY 2001, LANNING et al. 2002, LATENDRESSE et al. 2002, RUSSELL et al. 1990 optional section (blue). cells). Exp Toxic Pathol 55 (2004) 6 421 4 Male genital system
注意事项: A transverse section containing the area of the rete testis provides good histology of seminiferous tubules as well as of the rete testis.

4.2

附睾

Epididymis

切片数量: 2 (1 per side)
切面方向: Longitudinal vertical
样本大小: Whole organ It appears that the epithelium in the body of the organ is sometimes more sensitive than in the head and tail. Therefore, the body should not be excluded from the investigation. The whole organ should be fixed and embedded. It should be noted that some toxicants affect in particular the efferent ducts, which are located between testis and epididymal head. Care should be taken that these structures are not destroyed during preparation. Related references BOORMAN et al. 1990a, CARDY 1987, FERM 1987, HESS 1998 Epididymis. 4 Male genital system

4.3

精囊与凝固腺

Seminal vesicle & Coagulating gland

取材部位: In the mid portion
切片数量: 2 (1 per side)
切面方向: Transverse
注意事项: Together with coagulating gland The coagulation gland represents the dorsocranial part of the prostate. A transverse section should be made through the widest part of seminal vesicle together with coagulating gland. Related references BOORMAN et al. 1990b, CREASY and FOSTER 2002, FERM 1987, SUWA et al. 2002 4 Male genital system

4.4

前列腺

Prostate

取材部位: Dorsolateral and ventral lobe
切片数量: 1
切面方向: Longitudinal horizontal after special preparation (see below). dorsal lobe: only caudal part visible. The dorsolateral and ventral lobes that normally lie in a vertical axis above each other (with urinary bladder and seminal vesicle in between) are spread in a horizontal axis and embedded with the “outer” aspect down into the cassette. Preparation: The group of adjacent organs consisting of prostate, urinary bladder, seminal vesicles and coagulation glands is removed (see figures 4.4.d through 4.4.f) and (if weights are not required) fixed in toto to prevent leakage of the glandular secretions. After fixation, the ventral lobe is detached from the urinary bladder and is flipped back. The urinary bladder and seminal vesicles with coagulation glands are removed. The two ventral lobes are separated from each other, but are left attached to the dorsolateral parts. The dissected prostate is put into a cassette with the “outer” surfaces down; i.e. ventral face of the ventral lobes down and dorsal face of the dorsolateral lobes down (see figures 4.4.g through 4.4.i). After histotechnical processing, a section at the mid level of the ventral lobes is made. The dorsocranial lobe of the prostate (i.e. coagulating gland) is processed with the seminal vesicle. Chemically induced or spontaneous proliferative lesions of the rat prostate can be found in all three lobes. The dorsal and lateral lobes exhibit the same spectrum of proliferative lesions. These differ from spontaneous and induced lesions in the ventral lobe. Additionally, some strain specific deviations in the interlobular distribution of benign and malignant neoplasms consequently require the assessment of all compartments. Accordingly, a longitudinal-horizontal section through the prostate complex, including dorsolateral and ventral lobes, urethra and, optionally, ureter and ductus deferens represents a less time consuming method, applicable to routine histological processing and examination. Related references BOORMAN et al. 1990b, FERM 1987, LEE and HOLLAND 1987, MITSUMORI and ELWELL 1988, SUWA et al. 2001, SUWA et al. 2002 Abbreviations used in figures 4.4c to 4.4h: Cg: Coagulation gland Dd: deferent duct Dl: dorsolateral lobe of prostate Dsv: Duct of seminal vesicle Sv: Seminal vesicle Ub: Urinary bladder Ur: Urethra Vl: ventral lobe of prostate Exp Toxic Pathol 55 (2004) 6 423 calization of prostate and attached organs. fixed (right) prostate. living animal (in situ), the ventral prostate is flipped back. nal vesicles as a unit. “outer” aspects down, i.e. the dorsolateral lobes with the dorsal surface down, and the ventral prostate with the ventral surface down, because this part was flipped back. 5 Female genital system

雌性生殖系统

卵巢、输卵管、子宫与阴道

5.1

卵巢与输卵管

Ovary & Oviduct

切片数量: 2 (1 per side and organ)
切面方向: Ovary: longitudinal Oviduct: transverse
注意事项: If ovaries are not weighed: Ovary processed along with the oviduct. Longitudinal sections are made, resulting in multiple transverse sections of the oviducts. If ovaries are weighed: Ovaries and oviducts are separated at necropsy. In rats, the ovary is embedded together with the oviduct and a central section is cut. Larger ovaries or ovaries with masses are halved longitudinally or a slice from the middle of the organ is taken at trimming. In mice, the ovary is very small and therefore removed and fixed together with the bursa to avoid preparation artifacts. This procedure allows the detection of bursa cysts and cystadenomas in mice. Ovarian cysts should remain intact if possible. If the oviduct is prepared attached to the ovary, the longitudinal cut through the ovary will result in multiple transverse sections through the oviduct. If the ovaries are weighed, attached tissues (ovarian bursa, oviduct) have to be removed. In this case, the oviduct together with the tip of the uterine horn is dissected from the ovary during necropsy. The whole oviduct in conjunction with the tip of the uterine horn is fixed and embedded. Longitudinal sections through the tip of the uterine horns are made, resulting in transverse sections of the oviduct. If the uterus is also weighed, the oviducts are detached from the horns and put into a cassette with the ovaries. Related references FERM 1987, HEINDEL and CHAPIN 1993, U.S. Food and Drug Administration 2000, YUAN and FOLEY, 2002 and uterine horn (H), if ovaries are weighed. bursa removed, oviducts separated from ovaries and uterine horns. Exp Toxic Pathol 55 (2004) 6 425 5 Female genital system

5.2

子宫与阴道

Uterus & Vagina

取材部位: 1 + 2) Middle region: uterine horns 3) Whole organ: uterine body and cervix 4) Whole organ or anterior portion: vagina
切片数量: 3, 4 if uterus is separated from vagina
切面方向: 1 + 2) Transverse: uterine horns 3) Longitudinal horizontal: uterine body (corpus), cervix and vagina
可选切片: oviducts together with the tip of the uterine horns; other options see ovary. The uterine body (fused part of the uterus) together with the vagina should be placed with its dorsal aspect on cardboard before fixation. If uterus is weighed, uterine cervix and vagina are two separate specimens which are cut longitudinally. A horizontal section is made through the cervix and vagina. A transverse section is made through the middle portion of both uterine horns. These sections cover the relevant anatomical and functional structures of these organs. In most cases, the uterine body and most of the vagina will fit in one cassette, facilitating the interpretation of findings in the female genital tract. The oviducts are fixed attached to the uterus when the ovaries are weighed. Related references FERM 1987, HEBEL and STROMBERG 1986, HEINDEL and CHAPIN 1993, U.S. Food and Drug Administration 2000, YUAN and CARLSON 1987, YUAN and FOLEY, 2002 H: uterine horn, Od: oviduct, Ov: ovary). Uterine cervix. 6 Endocrine system

内分泌系统

垂体、甲状腺、甲状旁腺与肾上腺

6.1

垂体

Pituitary gland

取材部位: Sella turcica of sphenoid bone
切片数量: 1
切面方向: Transverse, parallel to the caudodorsal surface of the gland.
可选切片: trimmed in situ, closely caudal to the pituitary, caudal surface embedded down. Transverse section of decalcified skull. The pituitary consists of three portions: pars distalis, pars intermedia, and pars nervosa. All three parts should be present in one histologic section with the largest possible area. Relevant differences between rats and mice In mice, in situ fixation may be particularly useful to avoid preparation artifacts and to obtain consistent planes of section (Mahler and Elwell, 1999). Related references CAPEN 1996a, HEBEL and STROMBERG 1986, MAHLER and ELWELL 1999, OSAMURA 1996, SATOH et al. 1997 trimming. intermedia, Pd: pars distalis, Sb: sphenoid bone). staining for prolactin: depending on the level of section, the positive cells are not evenly distributed. Exp Toxic Pathol 55 (2004) 6 427 6 Endocrine system
注意事项: To avoid destruction of the pituitary, fixation is recommended before removal from the skull and/or weighing. The organ is embedded in toto with its caudodorsal surface down, so that the section will include all three parts.

6.2

甲状腺与甲状旁腺

Thyroid & Parathyroid

取材部位: In the area of the parathyroid gland
切片数量: 1
切面方向: If thyroid glands are not weighed: Transverse section of trachea, esophagus, thyroid and parathyroid glands.
可选切片: longitudinal horizontal section of thyroid glands in conjunction with trachea. A separate transverse section of the esophagus is made. If thyroid glands are weighed: Longitudinal horizontal (largest cut surface), section of thyroid and parathyroid glands. Transverse section of trachea and esophagus.
注意事项: Recuts are sometimes required to consistently include the parathyroid in the section. The number of focal lesions observed depends on the area of thyroid examined. Relevant differences between rats and mice The rat possesses only one pair of parathyroids. They are located on the anterior and lateral aspect of the thyroid lobes but may vary in position. In the mouse, the position and the number of parathyroids is variable. Usually, there are two parathyroid glands located bilaterally just under the capsule near the dorsolateral border of each thyroid lobe. They are rarely found at the same level, sometimes one or both may be posterior to the thyroid; they may be deeply embedded in the thyroid tissue and there may be more than two. Related references BOORMAN and DELELLIS 1983, BOTTS et al. 1991, BOTTS et al. 1994, CAPEN 1996b, CAPEN 1996c, KITTEL et al. 1996a, KITTEL et al. 1996b, POZHARISSKI 1990 Pg: parathyroid gland, E: esophagus). 6 Endocrine system

6.3

肾上腺

Adrenal gland

取材部位: Through cortex and medulla anatomical site or part of an organ from which a sample should be taken (i.e. lobe). 2. Number of samples: number of organs (i.e. both for bilateral organs) or organ pieces prepared for evaluation (not necessarily identical with the number of slides/blocks). 3.
切片数量: 2 (1 per side) The number of sections per specimen is usually one. If in some instances more than one section has to be taken, it should be borne in mind to evaluate the same amount of sections in all animals/groups to obtain comparable results. Staining: The H&E stain is regarded as the standard in toxicological studies. Other histological stains and immunohistochemistry can be applied as a routine or on a case by case basis in addition to the H&E stained sections. Fixation: In literature a volume ratio of tissue to fixative of 1:20 is often mentioned. However, much less fixative is sufficient, especially if a shaking device is used for freshly fixed tissues and/or fixative is replaced once. Tissues must be promptly and appropriately fixed by immersion. Adequate fixation time is necessary before tissue processing commences (CRISSMAN et al. 2004). Internet: With kind permission of Elsevier Publishing Jena, an extended version of these guides for organ sampling and trimming will be available in the Internet under the URL www.item.fraunhofer.de/reni/trimming. Acknowledgements: The authors would like to thank Mr. THEODOR LAFORME and Mrs. BRIGITTE POSCHMANN (Bayer HealthCare AG) for the gross preparation of organs and histological slides; Mrs. PETRA HARTMANN, Mrs MEIKE PETERSEN and Mrs. MARTINA NEUENHAUS (Bayer HealthCare AG) for scanning the histological slides, Dr. GERDPETER FEHLERT for his help in image processing; Dr. WOLFGANG KAUFMANN (BASF AG) for his scientific advice regarding the nervous system, Dr. PAUL DESLEX (Pfizer Centre Recherche) for providing additional photographs and slides, Dr. THOMAS NOLTE (Boehringer Ingelheim Pharma GmbH & Co KG) and Dr. ANNE PROVENCHER BOLLIGER (Novartis Pharma AG) for help in establishing the bone marrow manuscript and Dr. MATTHEW JACOBSEN (Syngenta CTL) and Dr. RICHARD DOUGHTY (AstraZeneca) for their helpful input during the editing process. References BAHNEMANN R, JACOBS M, KARBE E, et al.: RITA – Registry of Industrial Toxicology Animal-data – Guides for organ sampling and trimming procedures in rats. Exp Toxic Pathol 1995; 47: 247–266. CRISSMAN JW, GOODMAN DG, HILDEBRANDT PK, et al: Best practice guideline: toxicologic histopathology. Toxicol Pathol 2004; 32: 126–131. KITTEL B, RUEHL-FEHLERT C, MORAWIETZ G, et al.: Revised guides for organ sampling and trimming in rats and mice – Part 2. Exp Toxic Pathol 2004; 55: 413–432. KRINKE GJ (ed): The laboratory rat. Academic Press, San Diego San Francisco New York, 2000. RUEHL-FEHLERT C, KITTEL B, MORAWIETZ G, et al.: Revised guides for organ sampling and trimming in rats and mice – Part 1. Exp Toxic Pathol 2003; 55: 91–106. Exp Toxic Pathol 55 (2004) 6 435 7 Urinary system
切面方向: Longitudinal (largest cut surface) direction (plane of section) in which an organ should be cut at trimming or microtome sectioning. The proposed plane of section is shown in green color and optional sections (if defined) are shown in blue (see fig. 1 for an explanation of the symbols used). 4.
样本大小: the size (area) of an organ or part of an organ which is placed in a cassette for processing. The sample size is determined by the size of the organ. For optimal fixation, sample thickness should not exceed 3–5 mm. In general, the examined area should be as large as possible and should contain the relevant anatomical structures. The tissue can be adapted to the size of cassettes by trimming the margins off. 5. Optional remarks are used to present additional information, as recommended by the RITA/NACAD groups, such as the instillation of fixative into the lung or the urinary bladder, reasons for optional recommended sections, placing of organs in cassettes, etc. 6. Schematic drawings and/or gross photographs are given indicating the plane of section. Some of the gross photographs show the organ and trimming di rection in situ for orientation purposes. However, it is recommended to remove the organ or tissue first. Trimming can then be performed on the fresh wet tissue or, in most cases, after fixation of the organ. Most of the gross photographs were taken from fresh unfixed organs; shape and color may be slightly different after fixation. 7. An image of a Hematoxylin and Eosin (H&E) stained slide is shown for the recommended section level (sometimes also for optional levels). Typical structures included in this section are indicated as necessary. If not otherwise specified, 10% buffered formalin is recommended as the fixative. The following terms are used to describe the trimming directions (see also fig. 2 with a schematic presentation of the related cut levels): • transverse: perpendicular to the long axis of an organ or part of an organ • longitudinal vertical: in the direction of the long axis of the body, an organ or part of an organ in the dorsoventral axis or parallel to it (in the text also referred to in short as “longitudinal”) • longitudinal horizontal: in the direction of the long axis of the body, an organ or part of an organ, perpendicular to the dorsoventral axis (in the text also referred to in short as “horizontal”) By defining either the “body”, the “whole organ” or a “part of an organ” (for example a liver lobe or a certain part of the brain), as a unit of reference, it is relatively simple to precisely characterize a trimming direction by using only the three above defined terms and avoiding therefore the vast amount of anatomical terms and confusing synonyms present in literature. Final technical remarks Study types: Although carcinogenicity studies in rodents are the focus of this publication, the same trimming procedures are also recommended for short-term rodent studies, unless specific target tissues or organs require an adaptation. In general, it is advisable to follow one standard trimming procedure in the laboratory for all studies to avoid technical inconsistencies and to facilitate interstudy comparison. transverse, b: longitudinal vertical, c: longitudinal horizontal. Additional organs: Besides the organs addressed in these guides, special investigations or guidelines may require additional organs. Some can be found in the specimens described here, such as teeth in the transverse sections of the nasal cavity, brown adipose tissue at the renal hilus and heart base, white adipose tissue in the subcutis and the urethra of males in the prostate section. Others, however, may need adaptation of sampling procedures or collection of additional specimens. Blocking: The following recommendations should be taken into account: 1. For reasons of economy it is desirable to have a small number of blocks per animal. 2. Unduly large numbers of specimens in one block can lead to a loss of quality. Besides the obvious limitation of specimen size, difficulties may arise when cutting tissues with different physical properties in the same block. 3. Small organs often benefit from being embedded by themselves which makes it easier to cut accurately the level of interest. Examples are the pituitary gland, the adrenal gland and the thyroid gland with parathyroid glands. 4. Organs with similar cutting properties are often combined in one block. 5. The pathologist benefits from being able to examine organs in functional groups, e.g. stomach together with intestine or a combination of the lymphoid organs in one block. In order to reduce the number of slides, more than one block may be placed on a slide. Some laboratories see an advantage in collecting the adrenal glands, pituitary gland and thyroid gland with parathyroid glands on one slide which were processed in different blocks. An example of a blocking scheme reflecting the above mentioned considerations was given by KRINKE (2000).
注意事项: Embedded in toto; careful microtome sectioning until recommended cutting level is obtained. Median sections of the adrenal glands (one per side) are required in order to demonstrate a representative part of both cortical and medullary tissues. Relevant differences between rats and mice The mouse adrenal differs from that of the rat by the absence of a zona reticularis in the inner cortex and an additional “X-zone” at the junction between cortex and medulla in females which regresses with age (Nyska and Maronpot, 1999). The adrenal gland in male mice is very small. Therefore, careful cutting is required to obtain medullary tissue in the section. Related references FRITH 1996, HEBEL and STROMBERG 1986, NYSKA and MARONPOT 1999, PAGET and THOMSON 1979 medulla). months (C: cortex, M: medulla, X: X-zone). Exp Toxic Pathol 55 (2004) 6 429 References BAHNEMANN R, JACOBS M, KARBE E, et al.: RITA – Registry of Industrial Toxicology Animal-data – Guides for organ sampling and trimming procedures in rats. Exp Toxic Pathol 1995; 47: 247–266. BOORMAN GA, CHAPIN RE, MITSUMORI K: Testis and epididymis. In: BOORMAN GA, EUSTIS SL, ELWELL MR, et al. (eds) Pathology of the Fischer rat. Reference and atlas. Academic Press, San Diego New York London, 1990a; pp 405–418. BOORMAN GA, DELELLIS RA: C-cell hyperplasia, thyroid, rat. In: JONES TC, MOHR U, HUNT RD (eds) Monographs on pathology of laboratory animals. Endocrine System. Springer, Berlin Heidelberg New York, 1983; pp 192–204. BOORMAN GA, ELWELL MR, MITSUMORI K: Male accessory sex glands, penis, and scrotum. In: BOORMAN GA, EUSTIS SL, ELWELL MR, et al. (eds) Pathology of the Fischer rat. Reference and atlas. Academic Press, San Diego New York London, 1990b; pp 419–428. BOTTS S, CAPEN CC, DELELLIS RA, et al.: 6. Endocrine System. In: MOHR U, CAPEN CC, DUNGWORTH DL, et al. (eds) International classification of rodent tumours. Part I, The rat. IARC Scientific Publications No. 122, Lyon, 1994. BOTTS S, JOKINEN NP, ISAACS DJ, et al.: Proliferative lesions of the thyroid and parathyroid glands. E-3. Guides for Toxicologic Pathology, STP/ARP/AFIP, Washington, 1991. BROWN HR: Neoplastic and potentially preneoplastic changes in the upper respiratory tract of rats and mice. Environ Health Perspect 1990; 85: 291–304. CAPEN CC: Functional and pathologic interrelationships of the pituitary gland and hypothalamus in animals. In: JONES TC, CAPEN CC, MOHR U (eds) Monographs on pathology of laboratory animals. Endocrine system, 2nd edition. Springer, Berlin Heidelberg New York Tokyo, 1996a; pp 3–33. CAPEN CC: Hormonal imbalances and mechanisms of chemical injury of thyroid gland. In: JONES TC, CAPEN CC, MOHR U (eds) Monographs on pathology of laboratory animals. Endocrine system, 2nd edition. Springer, Berlin Heidelberg New York Tokyo, 1996b; pp 245–238. CAPEN CC: Pathobiology of parathyroid structure and function in animals. In: JONES TC, CAPEN CC, MOHR U (eds) Monographs on pathology of laboratory animals. Endocrine system, 2nd edition. Springer, Berlin Heidelberg New York Tokyo, 1996c; pp 293–327. CARDY RH: Segmental degeneration of the epididymis in aged F344 rats. Vet Pathol 1987; 24: 361–363. CHAPIN RE, HEINDEL JJ: Methods in Toxicology. Volume 3. Part A: Male Reproductive Toxicology. Academic Press, San Diego, 1993. CREASY DM, FOSTER PMD: Male reproductive system. In: HASCHEK WM, ROUSSEAUX CG, WALLIG MA (eds) Handbook of toxicologic pathology, Vol 2. Academic Press, San Diego New York Boston, 2002; pp 785–846. DUNGWORTH DL, ERNST H, NOLTE T, et al.: Nonneoplastic lesions in the lungs. In: MOHR U, DUNGWORTH DL, CAPEN CC (eds) Pathobiology of the aging rat. Vol I. ILSI Press, Washington, 1992; pp 143–160. FERM VH: Embryology and comparative anatomy, rodent reproductive tract. In: JONES TC, MOHR U, HUNT RD (eds) Monographs on pathology of laboratory animals. Genital system. Springer, Berlin Heidelberg New York Tokyo, 1987; pp 3–7. FOLEY GL: Overview of male reproductive pathology. Toxicol Pathol 2001; 29: 49–63. FRITH CH: Histology, adrenal gland, mouse. In: JONES TC, CAPEN CC, MOHR U (eds) Monographs on pathology of laboratory animals. Endocrine system, 2nd edition. Springer, Berlin Heidelberg New York Tokyo, 1996; pp 386–391. GOPINATH C, PRENTICE DE, LEWIS DJ: The respiratory system. In: Atlas of experimental toxicological pathology. MTP Press, Lancaster Boston The Hague, 1987; pp 22–42. HARDISTY JF, GARMAN RH, HARKEMA JR, et al.: Histopathology of nasal olfactory mucosa from selected inhalation toxicity studies conducted with volatile chemicals. Toxicol Pathol 1999; 27: 618–627. HARKEMA JR, MORGAN KT: Normal morphology of the nasal passages in laboratory rodents. In: JONES TC, DUNGWORTH DL, MOHR U (eds) Monographs on pathology of laboratory animals. Respiratory system, 2nd edition. Springer, Berlin Heidelberg New York Tokyo, 1996; pp 3–17. HARKEMA JR: Comparative pathology of the nasal mucosa in laboratory animals exposed to inhaled irritants. Environ Health Perspect 1990; 85: 231–238. HEBEL R, STROMBERG MW: Anatomy and embryology of the laboratory rat. BioMed, Wörthsee, 1986. HEINDEL JJ, CHAPIN RE (eds.): Methods in reproductive toxicology: Female reproductive toxicology, Vol. 3B. Academic Press, Orlando, 1993. HESS RA: Effects of environmental toxicants on the efferent ducts, epididymis and fertility. J Reprod Fertil 1998; Suppl 53: 247–259. KITTEL B, ERNST H, KAMINO K: Anatomy, histology and ultrastructure, parathyroid, mouse. In: JONES TC, CAPEN CC, MOHR U (eds) Monographs on pathology of laboratory animals. Endocrine system, 2nd edition. Springer, Berlin Heidelberg New York Tokyo, 1996a; pp 328–329. KITTEL B, ERNST H, KAMINO K: Anatomy, histology and ultrastructure, parathyroid, rat. In: JONES TC, CAPEN CC, MOHR U (eds) Monographs on pathology of laboratory animals. Endocrine system, 2nd edition. Springer, Berlin Heidelberg New York Tokyo, 1996b; pp 330–332. LANNING LL, CREASY DM, CHAPIN RE, et al.: Recommended approaches for the evaluation of testicular and epididymal toxicity. Toxicol Pathol 2002; 30: 507–520. LATENDRESSE JR, WARBRITTION AR, JONASSEN H, et al.: Fixation of testes and eyes using a modified Davidson’s fluid: comparison with Bouin’s fluid and conventional Davidson’s fluid. Toxicol Pathol 2002; 30: 524–533. LEE CH, HOLLAND JM: Anatomy, histology, and ultrastructure (correlation with function), prostate, rat. In: JONES TC, MOHR U, HUNT RD (eds) Monographs on pathology of laboratory animals. Genital system. Springer, Berlin Heidelberg New York, 1987; pp 239–251. LEWIS DJ: Morphological assessment of pathological changes within the rat larynx. Toxicol Pathol 1991; 19: 352–358. MAHLER JF, ELWELL MR: Pituitary Gland. In: MARONPOT RR, BOORMAN GA, GAUL BW (eds) Pathology of the mouse. Reference and atlas. Cache River Press, Vienna, 1999; pp 491–507. MITSUMORI K, ELWELL MR: Proliferative lesions in the male reproductive system of F344 rats and B6C3F1 mice: incidence and classification. Environ Health Perspect 1988; 77: 11–21. MORGAN KT: Approaches to the identification and recording of nasal lesions in toxicology studies. Toxicol Pathol 1991; 19: 337–351. NYSKA A, MARONPOT RR: Adrenal Gland. In: MARONPOT RR, BOORMAN GA, GAUL BW (eds) Pathology of the mouse. Reference and atlas. Cache River Press, Vienna, 1999; pp 509–536. OSAMURA RY: Histology, ultrastructure and immunohistochemistry, pituitary gland, rat. In: JONES TC, CAPEN CC, MOHR U (eds) Monographs on pathology of laboratory animals. Endocrine system, 2nd edition. Springer, Berlin Heidelberg New York Tokyo, 1996; pp 33–41. PAGET EG, THOMSON R: Standard operating procedures in pathology. MTP Press, Lancaster, 1979; pp 134–139. PLOPPER CG: Structure and function of the lung. In: JONES TC, DUNGWORTH DL, MOHR U (eds) Monographs on pathology of laboratory animals. Respiratory system, 2nd edition. Springer, Berlin Heidelberg New York Tokyo, 1996; pp 135–150. POPP JA, MONTEIRO-RIVIERE NA: Macroscopic, microscopic, and ultrastructural anatomy of the nasal cavity, rat. In: JONES TC, MOHR U, HUNT RD (eds) Monographs on pathology of laboratory animals. Respiratory system. Springer, Berlin Heidelberg New York, 1985; pp 3–10. POZHARISSKI KM: Tumours of the oesophagus. In: TURUSOV V, MOHR U (eds) Pathology of tumours in laboratory animals. Vol I. Tumours of the rat, 2nd edition. IARC Scientific Publications No. 99, Lyon, 1990; pp 109–128. RENNE R, FOUILLET X, MAURER J, et al.: Recommendation of optimal method for formalin fixation of rodent lungs in routine toxicology studies. Toxicol Pathol 2001; 29: 587–589. RENNE RA, GIDEON KM, MILLER RA, et al.: Histologic methods and interspecies variations in the laryngeal histology of F344/N rats and B6C3F1 mice. Toxicol Pathol 1992; 20: 44–51. RITTINGHAUSEN S, DUNGWORTH DL, ERNST H, et al.: Primary pulmonary tumours. In: MOHR U, DUNGWORTH DL, CAPEN CC (eds) Pathobiology of the aging rat. Vol I. ILSI Press, Washington, 1992; pp 161–172. RUEHL-FEHLERT C, KITTEL B, MORAWIETZ G, et al.: Revised guides for organ sampling and trimming in rats and mice – Part 1. Exp Toxic Pathol 2003; 55: 91–106. RUSSELL LD, ETTLING LA, SINHAHIKIM AP, et al.: Histological and histopathological evaluation of the testis. Cache River Press, Vienna, 1990. SAGARTZ JW, MADARASZ AJ, FORSELL MA, et al.: Histological sectioning of the rodent larynx for toxicity testing. Toxicol Pathol 1992; 20: 118–121. SATOH H, KAJIMURA T, CHEN CJ, et al.: Invasive pituitary tumors in female F344 rats induced by estradiol dipropionate. Toxicol Pathol 1997; 25: 462–469. SCHWARTZ LW, HAHN FF, KEENAN KP, et al.: Proliferative lesions of the rat respiratory tract. R-1. In: Guides for toxicologic pathology. STP/ARP/AFIP, Washington, 1991. SMINIA T, VAN DER BRUGGE-GAMELKOORN G, VAN DER ENDE MB: Bronchus-associated lymphoid tissue, rat, normal structure. In: JONES TC, WARD JM, MOHR U, et al. (eds) Monographs on pathology of laboratory animals. Hemopoietic system. Springer, Berlin Heidelberg New York Tokyo, 1990; pp 300–307. SUWA T, NYSKA A, HASEMAN JK, et al.: Spontaneous lesions in control B6C3F1 mice and recommended sectioning of male accessory sex organs. Toxicol Pathol 2002; 30: 228–234. SUWA T, NYSKA A, PECKHAM JC, et al.: A retrospective analysis of background lesions and tissue accountability for male sex organs in Fischer-344 rats. Toxicol Pathol 2001; 29: 467–478. URAIH LC, MARONPOT RR: Normal histology of the nasal cavity and application of special techniques. Environ Health Perspect 1990; 85: 187–208. U.S. Food and Drug Administration: IV.C.9.a. Guidelines for Reproduction Studies. In: Office of Food Additive Safety, Redbook 2000, Toxicological principles for the safety assessment of food ingredients, 2000. YOUNG JT: Histopathologic examination of the rat nasal cavity. Fund Appl Toxicol 1981; 1: 309–312. YUAN YD, CARLSON RG: Structure, cyclic change, and function, vagina and vulva, rat. In: JONES TC, MOHR U, HUNT RD (eds) Monographs on pathology of laboratory animals. Genital system. Springer, Berlin Heidelberg New York Tokyo, 1987; pp 161–168. YUAN YD, FOLEY GL: Female reproductive system. In: HASCHEK WM, ROUSSEAUX CG, WALLIG MA (eds) Handbook of toxicologic pathology, Vol 2. Academic Press, San Diego New York Boston, 2002; pp 847–894. Exp Toxic Pathol 55 (2004) 6 http://www.elsevier.de/etp 1 Department of Information Technology and Databases, Fraunhofer Institute of Toxicology and Experimental Medicine, Hannover, Germany 2 Department of Toxicologic Pathology, Bayer HealthCare AG, Wuppertal, Germany 3 Department of Product Safety, Regulations, Toxicology and Ecology, BASF AG, Ludwigshafen, Germany 4 Drug Safety Evaluation Pathology, Aventis Pharma Deutschland GmbH, Hattersheim, Germany 5 Schering-Plough Research Institute, Lafayette, NJ, USA 6 Department of Toxicology/Pathology/Animal Care, Abbott GmbH Co. KG, Ludwigshafen, Germany 7 Institute of Pathology and Toxicology, ALTANA Pharma AG, Hamburg, Germany 8 Institute of Toxicology, Merck KGaA, Darmstadt, Germany Revised guides for organ sampling and trimming in rats and mice – Part 3 A joint publication of the RITA*) and NACAD**) groups GERD MORAWIETZ1, CHRISTINE RUEHL-FEHLERT2, BIRGIT KITTEL3, AXEL BUBE4, KEVIN KEANE5, SABINE HALM6, ANKE HEUSER7, and JÜRGEN HELLMANN8 With 48 figures Received: March 25, 2004; Revised; May 17, 2004; Accepted: May 25, 2004 Address for correspondence: GERD MORAWIETZ, Department of Information Technology and Databases, Fraunhofer Institute of Toxicology and Experimental Medicine, Nikolai-Fuchs-Str. 1, 30625 Hannover, Germany; Fax: ++49 511 5350 155, E-mail: morawietz@item.fraunhofer.de Key words: Trimming; RITA; NACAD; rat; mouse; standardization; guidelines; kidney; ureter; urinary bladder; brain; spinal cord; spinal nerve root; eye; optic nerve; Harderian gland; skeletal muscle; peripheral nerve; bone; cartilage; femur; joint; heart; aorta; thymus; spleen; bone marrow; sternum; lymph nodes. *) RITA: Registry of Industrial Toxicology Animal-data. Members: Abbott GmbH & Co KG, Ludwigshafen, Germany; ALTANA Pharma AG, Hamburg, Germany; AstraZeneca, Södertälje, Sweden and Macclesfield, England; Aventis Pharma Deutschland GmbH, Hattersheim, Germany; BASF AG, Ludwigshafen, Germany; Bayer HealthCare AG, Wuppertal, Germany; Boehringer Ingelheim Pharma GmbH & Co KG, Biberach, Germany; Fraunhofer Institute of Toxicology and Experimental Medicine, Hannover, Germany; Hoffman-LaRoche AG, Basel, Switzerland; Merck KGaA, Darmstadt, Germany; Novartis Pharma AG, Basel, Switzerland; Pfizer, Amboise, France; Pharmacia, Nerviano, Italy; Syngenta CTL, Macclesfield, England **) NACAD: North American Control Animal Database. Members: 3M Corporate Toxicology, St. Paul, MN, USA; Adolor Corporation, Malvern, PA, USA, Bayer CropScience, Stillwell, KS, USA; Pfizer, Inc., Groton, CT, USA; Pfizer, Inc., Ann Arbor, MI, USA; Pfizer, Inc., Kalamazoo, MI, USA; Schering-Plough Research Institute, Lafayette, NJ, USA Summary This is the third part of a series of three articles on trimming instructions of rat and mouse protocol organs and tissues in regulatory type toxicity studies, covering the urinary, nervous, musculoskeletal, cardiovascular, and lymphoreticular systems. The article is based on the experience of the European RITA and American NACAD working groups and is an extended revision of trimming guides published in 1995 (BAHNEMANN et al.). The optimum localization for tissue preparation, the sample size, the direction of sectioning and the number of sections to be prepared is described organ by organ. These descriptions are illustrated for each organ by a schematic drawing and/or a macro-photograph showing the plane of section as well as a low magnification of the H&E stained slide demonstrating the optimum “endproduct”. 0940-2993/04/55/06-433 $ 30.00/0 433 The objectives of this work, as addressed in detail in the first part (RUEHL-FEHLERT et al. 2003), are to standardize tissue sampling and trimming for comparison of historical data obtained from different studies and different laboratories, ensure the presence of all relevant target sites for histopathological evaluation and provide technical advice for preparatory techniques during necropsy, fixation and trimming (CRISSMAN et al. 2004). Brief introduction to the use of the individual organ guides For each organ the following information is usually given (for more details see part 1: RUEHL-FEHLERT et al. 2003): 1.

泌尿系统

肾、输尿管与膀胱

7.1

肾与输尿管

Kidney & Ureter

取材部位: Kidney: both in the median, through the tip of papilla and renal pelvis Ureter: transverse section midway between kidneys and bladder.
切片数量: 2 (1 per side)
切面方向: Kidney: one side longitudinal, other side transverse Ureter: longitudinal adjacent to kidney or transverse with adipose tissue Remarks kidney: Capsule should not be removed. Fixation can be improved by an incision at necropsy. The transverse section of the kidney from the middle portion allows optimal examination of the renal pelvis, renal papilla and the junction with the ureter. The longitudinal section permits histological evaluation of a relatively large area of tissue which includes both renal poles. This is advantageous for the evaluation of any focal lesions. In addition, the regions of the renal pelvis close to the poles are of interest with respect to concretions and urothelial changes. A slightly paramedian cut at trimming is helpful to get the full length of the renal papilla in section. The ureter can be fixed on a cardboard together with a small amount of attached adipose tissue. Related references BACHMANN and KRIZ 1998, BANNASCH et al. 1974, COHEN et al. 2002, CORMAN and OWEN 1992, FRITH et al. 1998, KHAN and ALDEN 2002, LIEBELT 1998, SHORT and GOLDSTEIN 1992 renal pelvis). 7 Urinary system
可选切片: adjacent to the renal pelvis (not shown in the image)

7.2

膀胱

Urinary bladder

切片数量: 1
切面方向: Longitudinal vertical
注意事项: Instillation 0.2 ml rat, 0.05 ml mouse For instillation, the needle is inserted via the urethra into the urinary bladder. For the mouse, fixative can be injected through the urinary bladder wall after ligation of the urethra. The organ is filled by injection of approximately 0.2 ml (rat) or 0.05 ml (mouse) of the fixative. Do not infuse if distended with urine. Ligation is performed with a ventral knot. In addition, it may be helpful to mark the ventral side of the bladder with a stick of silver nitrate. After removal of the bladder, fixation is continued in a suitable container. The bladder is cut vertically through the ventral knot to access the following regions: 1. Vertex and ventral body: The vertex is the area generally most prone to develop neoplasms; deposition of sediments and calculi occurs mainly in the ventral aspect, which can lead to urothelial alterations. 2. Dorsal part of the bladder. 3. Bladder neck with trigone: Whilst most of the urinary bladder in the embryo is of endodermal origin, the trigone is derived from the mesoderm of the Wolffian ducts. Related references COHEN at al. 2002, FRITH 1980, KUNZE 1992, PAULI et al. 1998 prostate, R: rectum, S: seminal vesicle, U: urinary bladder. Exp Toxic Pathol 55 (2004) 6 437 8 Nervous system

神经系统与特殊感官

脑、脊髓、眼与周围神经

8.1

脑

Brain

取材部位: 1) Cerebrum at the optic chiasm 2) Cerebrum at the base of the posterior hypothalamus 3) Midcerebellum and medulla oblongata
切片数量: 3 (4)
切面方向: Transverse
可选切片: 4) Pons at the middle of its protrusion 5) cranial cervical cord
注意事项: Embedded with the anterior faces down. To achieve accurate brain weights, the spinal cord should be cut off at a consistent level. Unless the scope of examinations in neurotoxicity studies is extended, it is advised to use the above mentioned brain sections for the morphological screening in all rodent studies concerned. Transverse sections of the brain are required to assess whether the findings are distributed uni- or bilaterally and symmetrically or asymmetrically. The areas of the brain known to be susceptible to neurotoxicity, including oncogenicity, are: the cerebral and cerebellar cortex, hippocampus, basal ganglia, thalamus, hypothalamus, midbrain, pons and medulla oblongata. Routinely, three transverse sections are obtained: 1. the first section at the level of optic chiasm including the basal ganglia, septum, cortex, anterior hypothalamus; 2. the second section at the level of hippocampus containing the cortex and brain stem at the transition of diencephalon to mesencephalon and 3. the third section containing the cerebellum and brain stem (medulla oblongata). Location 4 is recommended for special investigations of the pons. To achieve precise vertical cuts, brain matrix moulds can be used. Different sizes are available for different laboratory species. For rodents, the brain can be sliced at 1 mm intervals. A sample of the cranial cervical cord can be removed together with the brain (location 5). Alternatively, it can be taken together with the cranial vertebral column to avoid artifacts (see 8.2: spinal cord). Trimmed specimens should not be stored in alcohol for extended periods during routine processing to avoid artifactual vacuolation. Related references DORMAN et al. 2002, KRINKE 1989, PAXINOS and WATSON 1997, SCHÄPPI and KRINKE 1991, WHO 1986 Abbreviations used in figures 8.1d to 8.1g: Ac: anterior commissure C: cerebellum Cc: cerebral cortex Cca: corpus callosum H: hypothalamus Hc: hippocampus Mo: medulla oblongata O: optic chiasm P: pons Sr: striatum T: thalamus oblongata. Exp Toxic Pathol 55 (2004) 6 439 8 Nervous system

8.2

脊髓与脊神经根

Spinal Cord & Spinal nerve root

取材部位: 1) Cervical cord at upper cervical segment.
切片数量: 3
切面方向: Transverse
可选切片: cervical cord cut at the level of the first cervical nerve (see 8.1 brain) 2) Thoracic cord at mid-thoracic segment 3) Lumbar cord at 4th lumbar segment close to last rib without bone to avoid decalcification. Embedded with anterior faces down. Transverse sections of the spinal cord are required to assess whether the findings are distributed uni- or bilaterally, and symmetrically or asymmetrically. Sections should be obtained at the upper cervical, mid-thoracic and lumbar levels. The upper cervical area positioned at the transition of medulla oblongata to cervical spinal cord (level of the first cervical nerve) is important for the detection of distally accentuated neuropathy affecting the long spinal tracts. This region is known to produce the earliest and most prominent lesions in the dorsal cervical column. At necropsy, the spinal cord should be cut off directly posterior to the cerebellum and can be removed with the vertebral bone as with the thoracic and lumbar spinal cord. In the lumbar spinal cord, the area of L4 segment should preferably be examined, as this segment provides the main contribution to the peripheral sciatic nerve. The L4 segment of the spinal cord is located at the junction of the thoracic and lumbar spine which is indicated by the last rib. If the lumbar specimen is taken more caudally, it will contain only the cauda equina. It is advantageous to remove the spinal cord from the vertebrae before processing to avoid decalcification. However, processing in situ has the advantage to include dorsal root ganglia and nerve roots to assess radiculoneuropathy. In neurotoxicity studies, sections from the cervical and lumbar swellings are recommended in current guidelines (OECD 424). The cervical swelling is located at the C3–C6 segments of the spinal cord. This section is not prioritized but can be taken as additional option in routine rodent studies. To avoid artifactual vacuolation in the white matter, the specimens should not be stored in alcohol. Related references GELDERD and CHOPIN 1977, KRINKE 1989, KRINKE and FITZGERALD 1988, OECD 1997, SCHÄPPI and KRINKE 1991 cord, Lc: lumbar cord). Option without bone shown for the cervical cord. 8 Nervous system
注意事项: In conjunction with vertebral body (decalcified).

8.3

眼、视神经与副泪腺

Eye, Optic nerve & Harderian gland

取材部位: In the plane of the optic nerve
切片数量: 2 (1 per side)
切面方向: Longitudinal vertical
可选切片: together with Harderian gland. together with eye lids for better orientation. For long-term studies, formalin fixation is generally sufficient. For other study types, fixation in Davidson’s fixative is recommended to avoid detachment of the retina. Each eye can be removed from the socket together with the optic nerve and the Harderian gland (optional) after fixation of the head. These organs are embedded in such a way that the section plane includes the anterior pole of the eye, the lens, the optic nerve and the gland. Alternatively, each eye with the optic nerve can be taken at necropsy followed by the removal of the Harderian gland; in this case, both samples are fixed and embedded separately. As an option, the optic nerve specimen can be taken at the base of the brain. Relevant differences between rats and mice The rodent eye has no areas of increased visual acuity (fovea and macula), therefore orientation is not as critical as in the non-rodent eye. The Harderian gland lies intraorbitally and is cone-shaped in the rat and horseshoeshaped in the mouse. See also: 2.2 Salivary glands (extraorbital lacrimal gland) Related references LATENDRESSE et al. 2002, MOLON-NOBLOT and DUPRAT 1991, WHITELEY and PEIFFER 2002 eye lid. Harderian gland). Harderian gland. chiasm. Exp Toxic Pathol 55 (2004) 6 441 8 Nervous system
注意事项: Optic nerve included.

8.4

骨骼肌与周围神经

Skeletal muscle & peripheral nerve

取材部位: Biceps femoris muscle Sciatic nerve
切片数量: 3 (2)
切面方向: Skeletal muscle: longitudinal and/or transverse Sciatic nerve: longitudinal;
可选切片: transverse skeletal muscle and sciatic nerve sampled attached to each other. Longitudinal and transverse sections in one cassette. If only one sample of muscle is processed, a transverse section is preferred. muscle and sciatic nerve from the hind leg. longitudinal horizontal sections. If skeletal muscle and sciatic nerve are sampled together, the gracilis, adductor, semimembranous and semitendinous muscles are removed from the medial aspect of the thigh to get access to the sciatic nerve running along the medial surface of the biceps femoris muscle. The sample is taken by proximal and distal transverse cuts. After fixation, transverse and longitudinal sections are prepared. The sciatic nerve can be fixed on a card board, if not sampled together with muscle. Related references MCGAVIN 1991, POPESKO et al. 1992 transverse section. 9 Musculoskeletal system
注意事项: Muscle and nerve are sampled separately.

骨骼系统

骨、软骨、股骨与关节，含脱钙处理

9.1

骨、软骨、股骨与关节

Bone, Cartilage, Femur & Joint

取材部位: Knee joint with distal femur and proximal tibia
切片数量: 1
切面方向: Longitudinal
注意事项: Decalcified For routine histological examination of bone (and bone marrow), the distal portion of one femur with the knee joint and proximal portion of the adjacent tibia are removed at necropsy, fixed and decalcified. A longitudinal section is then made through femur, knee joint and tibia, possibly including patella and/or menisci. Both long bones should be cut at similar length with inclusion of parts of their diaphyses. The section should be slightly lateral to the center of the joint to ensure that articular cartilage is present rather than ligaments. The use of large sections of bone with the joint has the advantage of maintaining anatomic integrity and allows separation of systemic pathologic conditions from reactive processes. With the proposed technique, the epiphyses, metaphyses, growth plates and articular cartilages of femur and tibia are also sectioned allowing assessment of growth, modeling, and remodeling parameters. In old rats, the femur contains a high proportion of fat marrow. Related references WOODARD et al. 2002 young rat. Exp Toxic Pathol 55 (2004) 6 443 10 Cardiovascular system

心血管系统

心脏与主动脉

10.1

心脏

Heart

取材部位: Through ventricles and atria with auricles
切片数量: 1
切面方向: Longitudinal
注意事项: One half that contains the main vascular trunks A longitudinal section through both ventricles should be made from the base to the apex of the heart. Do not open the heart at necropsy. The half with the main vessel trunks is blocked to get a section through the opened ventricles and atria with auricles as well as through base, septum, apex, papillary muscle and main vessels of the heart. Related references HEBEL and STROMBERG 1986, PIPER 1981, VAN VLEET et al. 2002 10 Cardiovascular system

10.2

主动脉

Aorta

取材部位: Thoracic region
切片数量: 1
切面方向: Transverse The section should be taken from the middle of the last 1 cm caudal segment of the thoracic aorta. This region is closely attached to the thoracic vertebrae and can easily be removed during necropsy. Induced lesions of the vascular system are rare in rats except for some pharmaceutical compounds such as vasodilators. Most of the induced changes are observed in smaller arteries and arterioles of various organs but not in thick-walled major blood vessels such as the aorta. Related references VAN VLEET et al. 2002 La: left auricle, Lv: left ventricle, Ra: right auricle, Rv: right ventricle). aorta, At: atrium, Lv: left ventricle, Rv: right ventricle). ↑ adjacent brown adipose tissue (At). 11 Lymphoreticular system

免疫与造血系统

胸腺、脾、骨髓与淋巴结

11.1

胸腺

Thymus

取材部位: Along the length of one lobe
切片数量: 1
切面方向: Longitudinal
可选切片: whole organ
注意事项: Largest area Thymic region in old animals The whole thymus is fixed and trimmed along the length of one lobe. This gives a standardized longitudinal section showing all anatomical structures of this organ. In immuno-toxicological assays, it is advisable to embed the whole organ, ventral aspect down, and to cut both lobes. In case of thymic atrophy or involution, the whole organ/thymic region should be embedded. Related references DJIKSTRA and SMINIA 1990, KUPER et al. 2000, KUPER et al. 2002, SCHUURMAN et al. 1994, U.S. Food and Drug Administration 2 11 Lymphoreticular system

11.2

脾

Spleen

取材部位: At largest extension
切片数量: 1
切面方向: Transverse
可选切片: whole organ longitudinal horizontal (not shown in the image) A transverse section is made at the largest extension of the organ, showing red and white pulp. This plane of section guarantees the presence of all relevant anatomical structures and hallmarks of the white pulp, e.g. PALS (periarteriolar lymphatic sheath), marginal zone and follicles. Related references DJIKSTRA and VEERMAN 1990, KUPER et al. 2000, KUPER et al. 2002, SCHUURMAN et al. 1994, U.S. Food and Drug Administration 2002 11 Lymphoreticular system

11.3

骨髓（胸骨）

Bone marrow (Sternum)

取材部位: Sternum
切片数量: 1
切面方向: Longitudinal horizontal
样本大小: 2–3 sternebrae
注意事项: Decalcified The bone marrow is generally examined concurrently with the bone tissue. From this decalcified section it is possible to evaluate the cellularity, number of megakaryocytes and the stromal compartment. Examination of a bone marrow smear of a core sample from the femur may be useful for evaluation of iron content and more precise cytology. Related references KUPER et al. 2000, MACKENZIE and EUSTIS 1990, VALLI et al. 1990, VALLI et al. 2002, WICKRAMASINGHE 1992 11 Lymphoreticular system

11.5

淋巴结

Lymph nodes

取材部位: Mesenteric lymph node
切片数量: 1 (per lymph node)
切面方向: Longitudinal (largest cut surface)
样本大小: Whole organ
可选切片: mandibular lymph node, axillary lymph node, popliteal lymph node, auricular lymph node, inguino-femoral lymph node
吸入研究: lung associated lymph nodes
注意事项: In case of parenteral application, one lymph node draining the application site and another distant from it should be chosen The peripheral lymph nodes that are most often examined are the mandibular, axillary and/or popliteal lymph nodes. The lymph nodes are often embedded untrimmed as whole organ due to their small size. It is important that a section is taken through the middle of the longitudinal axis of the lymph node in order to be able to examine all major areas, such as cortex, paracortex and medulla. Related references KUPER et al. 2000, KUPER et al. 2002, SCHUURMAN et al. 1994, U.S. Food and Drug Administration 2002, WARD 1990 References for organ guides BACHMANN S, KRIZ W: Anatomy, cytology, ultrastructure nephron and collecting duct structure in the kidney, rat. In: JONES TC, HARD GC, MOHR U (eds) Monographs on pathology of laboratory animals. Urinary system, 2nd edition. Springer, Berlin Heidelberg New York Tokyo 1998, pp 3–36. BANNASCH P, SCHACHT U, STORCH E: Morphogenese und Mikromorphologie epithelialer Nierentumoren bei Nitrosomorpholin-vergifteten Ratten. I. Induktion und Histologie der Tumoren. Z Krebsforsch 1974; 81: 311–331. COHEN SM, WANIBUCHI H, FUKUSHIMA S: Lower urinary tract. In: HASCHEK WM, ROUSSEAUX CG, WALLIG MA (eds) Handbook of toxicologic pathology, 2nd edition, Vol 2. Academic Press, San Diego New York Boston, 2002; pp 337–362. CORMAN BJ, OWEN RA: Normal development, growth, and aging of the kidney. In: MOHR U, DUNGWORTH DL, P: paracortex). CAPEN CC (eds) Pathobiology of the aging rat. Vol I. ILSI Press, Washington 1992, pp 195–209. DJIKSTRA CD, SMINIA T: Thymus. Structure and function. Normal anatomy, histology, immunohistology, ultrastructure, rat. In: JONES TC, WARD JM, MOHR U, et al. (eds) Monographs on pathology of laboratory animals. Hemopoietic system. Springer, Berlin Heidelberg New York Tokyo, 1990; pp 249–256. DIJKSTRA CD, VEERMAN AJP: Spleen. Structure and function. Normal anatomy, histology, ultrastructure, rat. In: JONES TC, WARD JM, MOHR U, et al. (eds) Monographs on pathology of laboratory animals. Hemopoietic system. Springer, Berlin Heidelberg New York Tokyo, 1990; pp 185–193. DORMAN DC, BRENNEMAN KA, BOLON B: Nervous system. In: HASCHEK WM, ROUSSEAUX CG, WALLIG MA (eds) Handbook of toxicologic pathology, 2nd edition, Vol 2. Academic Press, San Diego New York Boston, 2002; pp 509–537. FRITH CH: Biologic and morphologic characteristics of urinary bladder neoplasms induced in BALB/c female mice with 2-acetylaminofluorene. J Environ Pathol Toxicol 1980; 3: 103–119. FRITH CH, TOWNSEND JW, AYRES PH: Histology, ultrastructure, urinary tract, mouse. In: JONES TC, HARD GC, MOHR U (eds) Monographs on pathology of laboratory animals. Urinary system, 2nd edition. Springer, Berlin Heidelberg New York Tokyo 1998, pp 319–322. GELDERD JB, CHOPIN SF: The vertebral level of origin of spinal nerves in the rat. Anat Rec 1977; 188: 45–48. HEBEL R, STROMBERG MW: Anatomy and embryology of the laboratory rat. BioMed, Wörthsee, 1986; pp 97–116. KHAN KNM, ALDEN CL: Kidney. In: HASCHEK WM, ROUSSEAUX CG, WALLIG MA (eds) Handbook of toxicologic pathology, 2nd edition, Vol 2. Academic Press, San Diego New York Boston, 2002; pp 255-336. KRINKE GJ: Neuropathologic screening in rodent and other species. J Am Coll Toxicol 1989; 8: 141–146. KRINKE GJ, FITZGERALD R: The pattern of pyridoxine-induced lesion: difference between the high and the low toxic level. Toxicology 1988; 49: 171–178. KUNZE E: Nonneoplastic and neoplastic lesions of the urinary bladder, ureter, and renal pelvis. In: MOHR U, DUNGWORTH DL, CAPEN CC (eds) Pathobiology of the aging rat. Vol I. ILSI Press, Washington 1992; pp 259–284. KUPER CF, DE HEER E, VAN LOVEREN H, et al.: Immune system. In: HASCHEK WM, ROUSSEAUX CG, WALLIG MA (eds) Handbook of toxicologic pathology, 2nd edition, Vol 2. Academic Press, San Diego New York Boston, 2002; pp 585–646. KUPER CF, HARLEMAN JH, RICHTER-REICHELM HB, et al.: Histopathologic approaches to detect changes indicative of immunotoxicity. Toxicol Pathol 2000; 28: 454–466. LATENDRESSE JR, WARBRITTION AR, JONASSEN H, et al.: Fixation of testes and eyes using a modified Davidson’s fluid: comparison with Bouin’s fluid and conventional Davidson’s fluid. Toxicol Pathol 2002; 30: 524–533. LIEBELT AG: Unique features of anatomy, histology and ultrastructure, kidney, mouse. In: JONES TC, HARD GC, MOHR U (eds) Monographs on pathology of laboratory animals. Urinary system, 2nd edition. Springer, Berlin Heidelberg New York Tokyo 1998, pp 37–57. MACKENZIE WF, EUSTIS SL: Bone marrow. In: BOORMAN GA, EUSTIS SL, ELWELL MR, et al. (eds) Pathology of the Fischer rat. Reference and atlas. Academic Press, San Diego New York London, 1990; pp 421–488. MCGAVIN MD: Procedures for morphological studies of skeletal muscle, rat, mouse, and hamster. 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