小鼠淋巴结位置

Anatomy and nomenclature of murine lymph nodes:Descriptive study and nomenclatory standardization in BALB/cAnNCrl mice

Wim Van den Broeck a,⁎,Annie Derore b,c ,Paul Simoens a

a

Department of Morphology,Faculty of Veterinary Medicine,Ghent University,Salisburylaan 133,B-9820Merelbeke,Belgium

b

Innogenetics NV ,Industriepark Zwijnaarde 7,B-9052Ghent,Belgium

c

Flanders Interuniversity Institute for Biotechnology (VIB),Technologiepark 927,B-9052Ghent,Belgium

Received 21November 2005;received in revised form 10January 2006;accepted 26January 2006

Available online 6March 2006

Abstract

Murine lymph nodes are intensively studied but often assigned incorrectly in scientific papers.In BALB/cAnNCrl mice,we characterized a total of 22different lymph nodes.Peripheral nodes were situated in the head and neck region (mandibular,accessory mandibular,superficial parotid,cranial deep cervical nodes),and at the forelimb (proper axillary,accessory axillary nodes)and hindlimb (subiliac,sciatic,popliteal nodes).Intrathoracic lymph nodes included the cranial mediastinal,tracheobronchal and caudal mediastinal nodes.Abdominal lymph nodes were associated with the gastrointestinal tract (gastric,pancreaticoduodenal,jejunal,colic,caudal mesenteric nodes)or were located along the major intra-abdominal blood vessels (renal,lumbar aortic,lateral iliac,medial iliac and external iliac nodes).Comparative and nomenclative aspects of murine lymph nodes are discussed.The position of the lymph nodes of BALB/cAnNCrl mice is summarized and illustrated in an anatomical chart containing proposals for both an official nomenclature according to the Nomina Anatomica Veterinaria and English terms.©2006Elsevier B.V .All rights reserved.

Keywords:Mouse;Lymph node;Nomenclature

1.Introduction

Rodents,and mice in particular,have long been used as laboratory animals in various scientific experiments.The possibility to produce different murine strains and a variety of knock-out mice,the high reproductive rate of these animals,and the ease of their handling have made them the preferential laboratory animal.In immunolog-ical sciences,murine lymph nodes (lnn.)are often used to isolate lymphocytes in order to study fundamental

aspects of immunology and immunopathology.The methodology to recognize and dissect these lymph nodes requires at least a basic anatomical knowledge.In numerous studies,however,inaccurate,misleading or even enigmatic terms such as genital nodes (Cain and Rank,1995)or tonsillar nodes (Deaglio et al.,1996)have sometimes been assigned to murine lymph nodes.The ambiguity of murine lymph node (ln.)nomenclature is illustrated by the lymph node at the ear base of mice which has been variably designated by various terms such as parotid ln.(Cuq,1966;Grassé,1972;Popesko et al.,1992),lateral mandibular ln.(Cuq,1966),and facial ln.(Wolvers et al.,1999),while numerous recent studies refer to an allegedly auricular ln.(Anjuère et al.,1999;Dearman et al.,1996;Sailstad et al.,1995)or pre-

Journal of Immunological Methods 312(2006)12–

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⁎Corresponding author.Tel.:+3292747716;fax:+32927790.E-mail address:wim.vandenbroeck@UGent.be (W.Van den Broeck).

0022-1759/$-see front matter ©2006Elsevier B.V .All rights reserved.doi:10.1016/j.jim.2006.01.022

auricular ln.(Hendrickx et al.,1992)in this region.Given this confusion,it becomes very difficult to reproduce the experimental reports or compare different scientific results.Nevertheless,the localization of the different lymph nodes with their respective names in mice has been thoroughly described in a number of anatomical publications (Barone et al.,1950;Cuq,1966;Kawashima et al.,19),but these papers are seldom referred to.A sample bibliographic (Medline)search from 19to 1999demonstrated that of 293randomly chosen papers in which the words “mouse lymph node(s)”are used,citations (i.e.30%)used only vague terms such as “lymph node ”,“peripheral lymph node ”,“draining lymph node ”,“local lymph node ”,or “regional lymph node ”instead of the precise anatomical names.In the remaining 204publications,at least 42different specific names were given to the lymph nodes that were studied.Only 1article,however,contained some figures illustrating the anatomical position and identification of the lymph nodes in question (Wolvers et al.,1999).In contrast,in the remaining 203studies the exact scientific identification of the node was lacking:59of these investigations referred to previous publica-tions in which the nomenclature used was not based on a

sufficiently scientific anatomical support,while in the remaining 144articles no anatomical reference was given at all.

In an attempt to rectify this situation,we first characterized the lymph nodes in BALB/cAnNCrl mice and then summarized our findings in an anatomical chart.

2.Materials and methods 2.1.Animals

Seventy female BALB/cAnNCrl mice (Iffa Credo N.V .,Brussels,Belgium)aged 8to 32weeks were housed in groups of 3to 6animals in conventional type II cages containing nesting material as environmental enrich-ment (Brain et al.,1994)along with water and food supply ad libitum.At the end of the experiments,all animals were euthanized by intraperitoneal (IP)injec-tion of 30μl T61(Hoechst Roussel Vet,Brussels,Belgium).All experimental studies described in this paper were approved by the Institutional Animal Welfare Committee of Innogenetics (September 15,1999).

Table 1

Protocols used for demonstrating murine lymph nodes Protocol Route of administration

Sedation/anaesthesia Product Quantity (μl)Incubation (days)Number of animals I Intravenous (lateral caudal vein)–Ink+RAS a 200b 103II Subcutaneous,mental region –Ink+CFA c 60b 286III Subcutaneous,mental region –Ink+RAS 10b 214IV Subcutaneous,frontal region –Ink+CFA 60b 286V Subcutaneous,auricular base

–Ink+RAS 10b 216VI Subcutaneous,palmar metacarpal region –Ink+CFA 40b 183426VII Subcutaneous,plantar metatarsal region –Ink+CFA 40b 183426VIII Intranasal instillation Sedation Ink+RAS 2×30b,d 10e 317e 3IX Intraperitoneal –

Ink+tR f

2000g 143X Peroral

Sedation Ink+RAS or CFA 500b 216XI Intrahepatic h Anaesthesia Ink+RAS 30b 216XII

Intralienal i

Anaesthesia

Ink+RAS

50b

21

6

a RAS:Ribi Adjuvant System®,RIBI Immuno Chem Research,Inc.,Hamilton,USA.b

Equal quantities ink/RAS or CFA.c

CFA:Complete Freunds Adjuvant®,Difco Laboratories,Detroit,Michigan,USA.d

Two administrations of 30μl with 21-day interval.e

Days after the last administration.f

tR:Thioglycollate+Resazurin®,Sanofi Diagnostics Pasteur,Marnes-la-Coquette,France.g

50μl ink+1950μl Thioglycollate +Resazurin®.h

After anaesthesia,the abdominal wall was incised 5mm caudal to the xiphoid process under surgical conditions;after the injection of the solution into the left and right hepatic lobes,the abdominal incision was closed.i

After anaesthesia,the left abdominal wall was incised under surgical conditions;after the injection of the solution into the spleen,the abdominal incision was closed.

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Table 2

List of lymph nodes observed in the present study of BALB/cAnNCrl mice #English name Official name Protocol Occurrence

a Topography

2Accessory mandibular ln.Ln.mandibularis accessorius I,II,IV ,V Constant (21/21)Dorsolateral to the mandibular lymph node

Superficial parotid ln.

Ln.parotideus superficialis

I,II,IV ,V

Constant (21/21)

Ventral to the external acoustic pore,caudal to the extraorbital lacrimal gland,cranioventral to the parotid salivary gland,dorsal to the junction between the superficial temporal vein (v.)and the maxillary v.

4

Cranial deep cervical ln.Ln.cervicalis

profundus cranialis I,II,IV ,VI

Constant (24/24)Medial to the external jugular vein and sternocephalic muscle (m.),lateral to sternohyoid m.,caudal to digastric m.,dorsal to the trachea

5Proper axillary ln.

Ln.axillaris proprius

I,VI

Constant (12/12)Medial to the shoulder,dorsolateral to ascending pectoral m.,at the junction between the lateral thoracic vein and the axillary vein

6Accessory axillary ln.

Ln.axillaris accessorius I,VI Constant (12/12)Caudal to triceps brachii m.,lateral to cutaneous trunci m.,in subcutaneous adipose tissue

7Subiliac ln.Ln.subiliacus

I,VII

Constant (12/12)

In the fold of the flank (plica lateralis)cranial to thigh musculature,near the deep circumflex iliac artery (a.)and v.

8

Sciatic ln.Ln.ischiadicus I,VII

Constant (12/12)

Medial to gluteus superficialis m.,caudal to gluteus medius m.and sciatic nerve

9Popliteal ln.Ln.popliteus I,VII Constant

(12/12)

In the popliteal fossa between biceps femoris m.and semitendinosus m.

10Cranial mediastinal lnn.Lnn.mediastinales craniales I Constant

(3/3)Bilaterally 2lymph nodes located lateral to the thoracic thymus and along the internal thoracic a.and v.

11Tracheobronchal ln.Ln.tracheobronchalis VIII Constant

(6/6)

Single (unpaired)lymph node at the tracheal bifurcation 12Caudal mediastinal ln.Ln.mediastinalis caudalis I Constant

(3/3)Single (unpaired)lymph node in the caudal mediastinum,ventral to the esophagus,along the ventral vagal trunk 13Gastric ln.Ln.gastricus I,IX,X,XI,XII Constant

(24/24)

Single (unpaired)lymph node in the lesser omentum at the minor curvature of the stomach

14Pancreaticoduodenal ln.Ln.pancreaticoduodenalis I,IX,X,XI,XII Constant

(24/24)Single (unpaired)lymph node in the mesoduodenum,dorsal to the portal vein,surrounded by pancreatic tissue 15Jejunal lnn.Lnn.jejunales I,IX,X,XI,XII Constant

(24/24)

Large cluster of lymph nodes in the mesojejunum along the cranial mesenteric a.

16Colic ln.Ln.colicus I,IX,X,XI,XII Constant

(24/24)

In the mesocolon at the transition between ascending colon and transverse colon

17Caudal mesenteric ln.Ln.mesentericus caudalis I,IX,X,XI,XII Constant

(24/24)Single (unpaired)lymph node in the caudal mesentery at the origin of the caudal mesenteric a.

18Renal ln.Ln.renalis I,VII,IX,X,XI,XII Constant

(33/33)

Dorsal to the ipsilateral kidney nearby the renal blood vessels,caudal to the adrenal gland

19Lumbar aortic ln.Ln.lumbalis aorticus VII b

Inconstant

(4/6bilateral,2/6only left)

Lateral to (and adjacent with)the abdominal aorta,halfway between the origin of the renal and common iliac arteries

20Lateral iliac ln.Ln.iliacus lateralis I Inconstant

(2/3only right,1/3absent)

In adipose tissue caudolateral to the kidney along the deep circumflex iliac a.

21Medial iliac ln.Ln.iliacus medialis I,VII,IX,X Constant

(21/21)

Major bilateral lymph node at the terminal segment of the abdominal aorta and the origin of the common iliac a.22External iliac ln.Ln.iliacus externus I Constant

(3/3)

Small lymph node along the initial (intra-abdominal)segment of the external iliac a.,before the latter enters the femoral canal

English and official Latin names of each node are given together with their frequency and a short description of their topography.a

F :number of animals in which lymph nodes were found,E :number of animals in which these particular lymph nodes were examined.b

Protocol VII with 42incubation days.

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As murine lymph nodes are hardly distinguishable from the surrounding fat and connective tissue(Cuq, 1966),they were stimulated and colored in vivo by an injection of Indian ink in combination with an adjuvant prior to euthanasia and subsequent dissection of the animals.Intravenous injections were performed in three mice to obtain a general overview(protocol I),whereas different additional stimulation protocols were used to demonstrate the presence of particular nodes in various body regions(protocols II–XII).In some protocols,a previous sedation of the mice by intramuscular injection of1μl/g body weight of a solution of200μl ketamine (Ketalar,Parke Davis,Dublin,Ireland)and30μl xylazine(Rompun2%,Bayer,Brussels,Belgium)was required.In a few cases,anaesthesia was induced by injecting the mice intraperitoneally with220μl of a solution containing200μl ketamine,100μl xylazine and 700μl physiological salt solution.The different protocol details are listed in Table1.The specific protocols that have been used to identify the particular nodes are listed in Table2.

2.3.Histological examination

The lymphoid architecture of the in vivo colored structures was verified by histological examination. Dissected lymph nodes were fixed in3.5%phosphate-buffered formaldehyde immediately after necropsy. Paraffin sections were made and stained with eosin–haematoxylin.

3.Results

Based on their topography,the murine lymph nodes were divided into peripheral(head and neck region, forelimb,hindlimb),intrathoracic,and intra-abdominal lymph nodes.A precise nomenclature based on the Nomina Anatomica Veterinaria(2005),equivalent English terms,and the topography of the lymph nodes are described in Table2.The anatomical position of

the Fig.1.Peripheral lymph nodes in the mouse.(1a)Ventro-lateral view of the head and throat region with sublingual(a),mandibular(b)and parotid (c)salivary gland and the extraorbital lacrimal gland(d),(1b)ventral view of the axillary region,(1c)lateral view of the thorax and forelimb,(1d) dorsal view of the sacral region with the sciatic nerve(a),and(1e)ventral view of the spread hindlimbs;numbers(1–9)according to the description in Table2.

15 W.Van den Broeck et al./Journal of Immunological Methods312(2006)12–19

exposed lymph nodes is illustrated in 14photographs (Figs.1–3)and 2drawings (Fig.4).

Nine peripheral lymph nodes are constant and bilaterally present,namely the mandibular,accessory mandibular,superficial parotid,and cranial deep cervical ln.in the head and neck regions,the axillary and accessory axillary ln.in the forelimb,and the subiliac,sciatic and popliteal ln.in the hindlimb region.Intrathoracic nodes are few in number and consist of the cranial mediastinal lnn.,tracheobronchal ln.and the caudal mediastinal ln.Intra-abdominal lymph nodes are either associated with the gastroin-testinal tract or lie along the major abdominal arteries.The former group consists of the gastric and pancrea-ticoduodenal ln.,the jejunal lnn.and colic ln.which together represent the cranial mesenteric lnn.,and the caudal mesenteric ln.The other intra-abdominal lymph nodes include the bilateral renal,medial iliac and external iliac ln.,as well as the inconstant lumbar aortic and lateral iliac ln.The latter lymph node was observed in 2out of 3mice that were stimulated by intravenous injection.

Other lymph nodes such as the facial (Wolvers et al.,1999),auricular or pre-auricular (Anjuère et al.,1999;Dearman et al.,1996;Hendrickx et al.,1992;Sailstad et al.,1995),superficial cervical (Barone et al.,1950;Cuq,1966),caudal deep cervical (Barone et al.,1950),pulmonary (Teitelbaum et al.,1999),hepatic and lienal (Barone et al.,1950),(ileo)cecal (Barone et al.,1950;Cuq,1966),sacral (Popesko et al.,1992),and femoral (Björkdahl et al.,1999;Mishell et al.,1980)lymph nodes were not observed in any of the BALB/cAnNCrl mice that were examined in the present study.Furthermore,there was no evidence of the presence of a submental lymph node (Cook,1983;Jacoby and Fox,1984),but a number of subcutaneous submental lymph nodules were demonstrated just caudal to the inter-mandibular synchondrosis by histological examination.4.Discussion

We sought to definitely localize lymph nodes in mice and to provide an up-to-date anatomical determination chart to identify the different nodes.Most of

these

Fig.2.Intrathoracic and intra-abdominal lymph nodes in the mouse.(2a)Ventral view of the thoracic cavity with the right lung (a)and thymus (b),both turned over to the left side,(2b)ventral view of the thoracic cavity with oesophagus (a),heart (b)and thymus (c),(2c)ventral view of the abdominal cavity with stomach (a),liver (b)and spleen (c),(2d)exposed mesentery,and (2e)ventral view of the abdominal cavity with the left uterine horn (a)and the caudal mesenteric artery (b);numbers (10–17)according to the description in Table 2.

16W.Van den Broeck et al./Journal of Immunological Methods 312(2006)12–19

lymph nodes have already been described in anatomical papers (Barone et al.,1950;Cuq,1966;Kawashima et al.,19),but bibliometric analysis indicates that contemporary investigators are often not familiar with these publications.As a consequence,the nomenclature of murine lymph nodes used in recent literature lacks uniformity and is sometimes inadequate or even incorrect.

By using different conventional in vivo staining techniques,22lymph nodes could be demonstrated in BALB/cAnNCrl mice.They were named by analogy to the terms listed in Nomina Anatomica Veterinaria (2005).This terminology is based on precise nomen-clatory principles leading to short and simple terms with instructive and descriptive value.Several lymph nodes that were observed in BALB/cAnNCrl mice could be identified because of their comparative and topographic similarities with analogous lymph nodes in domestic carnivores,pigs,and herbivores,and they were named accordingly.However mice lack several lymph nodes that are present in other mammals,such as the deep parotid or proper lumbar lymph nodes.Despite the absence of these complementary structures in mice,the terms superficial parotid and lumbar aortic lymph nodes were retained because the pertaining adjectives have useful descriptive value.This was also the case for the term cranial deep cervical lymph node,although the superficial cervical and caudal deep cervical lymph nodes were not observed in BALB/cAnNCrl mice.No additional topographic adjective was used for the single tracheobronchal lymph node because a precise homol-ogy with either the right,left,or middle tracheobronchal lymph node of domestic animals could not be ascertained in the present study or by data from the literature (Cuq,1966;Kawashima et al.,19).

A number of lymph nodes that has been described in murine species by other authors were not found in the present study.The facial lymph node as mentioned by Wolvers et al.(1999),and the auricular (Anjuère et al.,1999;Dearman et al.,1996;Sailstad et al.,1995)and pre-auricular lymph node (Hendrickx et al.,1992)probably correspond with the superficial parotid ln.described in our study.The submental ln.,illustrated as bilateral lymph nodes in two papers (Cook,1983;Jacoby and Fox,1984),were not observed as nodes as such,but subcutaneous median lymph nodules

were

Fig.3.Intra-abdominal lymph nodes in the mouse (ventral view).(3a,3b,3c,3d)Ventral views of the abdominal cavity with the right kidney (a)(turned over to the left side in 3a),the right adrenal gland (b),the descending colon (c)(displaced in 3c)and the deep circumflex iliac artery (d);numbers (7,17–22)according to the description in Table 2.

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present just caudal to the intermandibular synchondro-sis.Furthermore,there was no evidence of the caudal deep cervical ln.which has been described ventral to the trachea and dorsal to the sternum at the level of the first two ribs (Barone et al.,1950).Similarly,the existence of the superficial cervical ln.which has inconstantly be seen medial to the cervical part of the trapezius muscle and cranial to the supraspinatus muscle (Barone et al.,1950;Cuq,1966),and the presence of the femoral ln.which has been described in the inguinal region (Björkdahl et al.,1999;Mishell et al.,1980)could not be demonstrated.An intrathoracic pulmonary lymph node (Teitelbaum et al.,1999)was also absent in all mice examined in the present study.The (ileo)cecal lnn.,described in the ileocecal mesentery as accessory nodes (Barone et al.,1950;Cuq,1966),were not observed in

our study,whereas the sacral ln.which has been illustrated by Popesko et al.(1992)most likely refers to the caudal mesenteric ln.as defined by Kawashima et al.(19).Despite the minute dissections and the use of specific intrahepatic and intralienal stimulation techni-ques,our study failed to demonstrate the existence of hepatic and lienal lymph nodes in BALB/cAnNCrl mice.The presence of these nodes in mice has been discussed by Barone et al.(1950).According to these authors,murine lienal nodes are absent,which corre-sponds with the present findings in BALB/cAnNCrl mice.On the other hand,they observed a (retro)hepatic or portal lymph node which could hardly be discerned from the lymph nodes adjacent to the stomach and the pancreas.This lymph node corresponds most likely with the pancreaticoduodenal lymph node described in the present study.A novel finding in our study was the presence of a small and inconstant lateral iliac lymph node in BALB/cAnNCrl mice.The presence and lymphoid nature of the latter lymph node were verified by histological examination.It is not unlikely that this structure,along with other lymph nodes,might also be demonstrated in other murine species and breeds.

To date,no precise nor conclusive data are available concerning the presence of hemal lymph nodes in mice.The exact function of these nodes,which are very obvious in some domestic animal species such as oxen and sheep,has still to be elucidated,but probably they perform a spleen-like function,as suggested by their morphology (Bassan et al.,1999).In the present study,the presence of hemal lymph nodes could not be demonstrated neither by macroscopic nor by micro-scopic examination in any of the stimulated or unstimulated regions in BALB/cAnNCrl mice.

In summary,we recommend that scientific papers on laboratory animals,and on mice in particular,should carefully observe universally accepted rules of nomen-clature for the identification of all lymphatic organs that are described and investigated.References

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