`The 0use“’ B’
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`
`
`
`ese“ (2
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`Volume III
`Normative Biology,
`Immunology, and Husloanclry
`
`EDITED BY
`
`Hemry L. Foster
`The Charles River Breeding Laboratories. Inc.
`Wiimington, Massachusetts
`
`] . David Small
`Conmarative Medicine Branch
`National Institute of Environmental Health Sciences
`
`Reseamh Triangle Park, Noni: Carolina
`
`james G . Fox
`Division of Comparative Meciicine
`Massachusetts Institute of Technology
`Cambridge, Massachusetts
`
`ACADEMIC PRESS 1988
`
`A SUBSIDEARY OF HARCOURT BRACE IOVANOVICR PUBLISHERS
`
`New York Lonclon
`Paris San Diego San Fn:mci.sc0
`
`5&0 Ptmlo
`
`Sggclneg; Takyo Tommfa)
`
`|nnoPharma Exhibit 1100.0001
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`

`

`CiZ}l’YRIGl~1T © 1983, BY ACADEMIC P3533, INC.
`ALL RIGHTS RESERVED.
`NO mg? or THIS PUBLICATION MAY BE RE}?-’RODUCED <32:
`’I'R.9~,N32s~i1’I"I‘I3D IN my ream OR BY ANY MEANS, ELECTRONIC
`on MECH.«’&NI{3!sL, INCLUDING morccoyy, RECORDING, on ANY
`ENFORMATION 510:2.-ass AND RE'Il{IE¥AL SYSTEM, wmzovr
`PERMISSION IN wnumo FROM THE PUBLISHER.
`
`x
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`ACADEMIC PRESS, ENC.
`111 Fifth Avenue. New York, New York 10003
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`ACADEMIC PRESS, INC. {LONDON} LTX}.
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`GTDX
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`Library of Ccrgress Cataloging in Publication Data
`Vain entry under title:
`
`The Mouse in biomedical resaarch.
`
`{iimerican College :3? Laboratory éinisnal I-iedicine
`..-Series)
`Includes indexes.
`Contents: v. 1. Histery, genetics, and wild
`mice -~ [etc.3 ~- V‘. 3. husbandzy.
`1. Foster,
`1. Rice as labcratory animals.
`Henry 1..
`11. Small. 3. Ctavid.
`Iii. Fox, James (3.
`Z55. Series.
`DZINLM: 1. Nice.
`2. Research.
`3. animais. Laboratory.
`[111 60.86 H9321]
`QL733’. R633468
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`PRINTED IN THE UNITED STATES OF AMERICA
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`83348536
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`987654331
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`|nnoPharma Exhibit 1100.0002
`
`

`

`Chapter 18
`
`Biomethodology and Surgical Techniques
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`Ter‘2‘fe L. Cunlzfie-865111281‘
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`Introduction .
`I.
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`11. Handling. Identificaiitm, and Restraint .
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`A. Handling .
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`B.
`Idemificaticm .
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`C. Rrsstraint .
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`IE1. Administration of Drugs or Other Compounds .
`A. Topical .
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`13. Per 05 .
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`C.
`Subcutaneous Injection .
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`D.
`Featpact Itzjcction .
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`E.
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`F.
`Inlramusctular Injection .
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`G.
`lntraperitozzeai Injrzction .
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`H.
`Intmthoracic Injection .
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`Imrzwasculzlr Injection and Czmnulation .
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`IV‘. Cullection of Biological Specimens .
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`A. Bile .
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`8. Blood .
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`C. Bone Marrow .
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`Lyimph .
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`In Milk .
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`I. Ova and Sperm .
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`J. Urine...'....,V....I.
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`V. Assessment of Physroiogtcal Status .
`A. Blood Pressure, Heart Rate, and Respiratory Rate .
`B.
`Fused and Water Consumptimt .
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`C. Neurological Examination .
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`D. Miscellaneous Techniques .
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`403
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`406
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`40’?
`40’?
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`!E\' Bl{,}.\1}£{)t(.‘.~\L Rb'.$E;kI<:(,ftL mt- us
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`‘
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`C0;)§.'rigl|[ 2: 1933 by.>Xc:aden1i<: Press. Inc.
`Ail rights of rcgmxluclinn in any Rm}: reserved.
`ISBN {)~E2r262503~X
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`|nnoPharma Exhibit 1100.0003
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`

`

`402
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`TERRIE L. CUNLIFFE-BEs\h»’IEIil
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`‘fl. Anesthetics .
`A. Analgesics, Scdatives. and Other Preanesrherie in-Ietiicatioris .
`B.
`H}'}}(}!l‘t€l’n‘tla .
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`C.
`Local Anesthetics .
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`D.
`liilxalent Aiiesthetics .
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`E.
`Injectable Anesthetics .
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`F. Neuroleprarialgesics .
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`Surgical Procedures .
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`3%.. Basic Teeliniques .
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`8.
`z‘5>.dr'cnaleetomy .
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`D. Embryos and Embryo Transplants .
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`E. Hepatcetomjr .
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`F.
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`G. Hystereetorm; and lriysterotomy .
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`H.
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`l, Lyniphnotlectoniy .
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`J. Mammary Gland Excision and 'l‘ran:spla:it .
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`K. Miscellaneous Transplant Teclisiiqlres .
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`L. Nephrectomy and Kidney Transplant .
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`M. Glfactory Bulb Ablation .
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`N. Orelaideetorny, ’l‘esti<:ulztr Biopsy .
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`0. Ovariectoinjr, Omrizrit Transplant, and f}&'ariohys.£erec£on1j; .
`P.
`Paralaiosis .
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`Q.
`13zkratli};r()idectomy .
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`3.
`Skin Grafts .
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`Splenectomy .
`'1”.
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`U. Tltyrnectomy and Thorzieotoniy .
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`‘v’. Tliyroiciectorny .
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`W. Vaseetorny .
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`Postaiiesthetie and Postogierative Care .
`VIII.
`IX. Euthanasia .
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`X. Diagnostic Procedures and Necropsy .
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`A. Diagnostic Procedures .
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`B. Necropsy .
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`C. Histirpatholiigical Exarnination .
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`References .
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`
`413
`414
`at-M
`414
`tllsil
`416
`418
`ell‘)
`ill‘)
`420
`421
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`421
`421
`43}
`42?.
`423
`423
`423
`-423
`424
`424
`£124
`425
`42:3
`426
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`42'?
`6127
`£128
`L428
`428.
`429
`429
`429
`430
`‘$30
`
`‘
`
`I.
`
`INTRODUCTION
`
`The mouse’s small size, relatively short life span, proficierit
`reproductive capabilities, arid susceptibility to microbiological
`or eliemical agents make it an appropriate animal model to
`irwestigate problems
`in rriariy
`di'~.-*erse disciplines,
`such
`as enibryology, ethology, genetics, gerontology, microbiol-
`ogy, and oncology. The menses small body size makes it pos-
`sible to maintain marry miee efficiently and economically;
`however‘,
`this characteristic makes administration of drugs,
`collection of biological specimens, or performance of surgical
`procedures a challenge.
`The objectives of this chapter are to (1) describe procedures
`for restraint, administration of drugs, and collection of biolog-
`
`ical specimens or physiological data; (2) outline surgical pro-
`cedures and appropriate anesthetic and postoperative care; (3)
`discuss advantages and rlisadvaittages of alternative ways to
`perforrri the same procedure; and (4) provide references that
`contain detailed descriptions of umrsual procedures.
`Commonly used procedures and less common procedures for
`which tieseriptioris eoulti not be found in the literature are
`described in detail.
`in cases where good descriptioris of un~
`usual procedures are available in the literature, the reader will
`be referred to the original articles. Before attempting marry of
`the procedures described in this chapter, one should review the
`anatomy of the area in question and practice the proeeclure on
`an anesthetized or dead mouse. Even though laboratory mice
`(Mics mrrrcrttzrs o‘ozizesr£cz¢.rj! can be handled or restraineti witi1~
`out the administratior: of drugs, one should not stibstitute physw
`
`|nnoPharma Exhibit 1100.0004
`
`

`

`18. BIOMETHODOLOGY AND SURGICAL TECHNIQUES
`
`403
`
`ical restraint for analgesia during procedures that result in more
`than momentary pain.
`‘
`
`II. HANDLING, IDENTIFICATION, AND
`RESTRAINT
`
`A. Handling
`
`Juvenile and adult niiee are caught and picked up hy grasping
`the base or middle third of the tail with fingers or smooth
`tipped forceps. Aggressive mice often climb their tails. in order
`to bite fingers or forceps. Once caught by the taili the mouse
`can be restrained for examination or injection by placing it on a
`table or cage lid, grasping the loose skin behind the neck and
`
`ears with thumb and forefingers and holding the tail against the
`palm of the hand using the fourth and fifth fingers {Fig 1). if
`the mouse can move his head from side to side, fingers may be
`bitten; however, by pulling the skin on the neck too tight, the
`inouse’s airway is compromised.
`Forceps {9—lO~inch smooth dressing forceps} are an excel»
`lent way to manipulate wild, aggressive mice. Adult mice are
`caught by grasping the cranial third of the tail (Fig. 2)‘ Mice
`should be lowered, not dropped, into a cage and released as
`soon as their front feet touch the bedding. Pregnant female
`mice approaching parturition or very large mice, e.g., homo-
`zygous obese (obtoo) or diabetic (zibfrlb) mice, should be han-
`dled gently and supported, if necessary, with a hand under
`their feet. Young rnice {less than 2 weeks of age) are picked up
`by grasping the loose skin over the neck and shoulders witli
`forceps or thumb and forefinger, or by scooping the litter into
`
`Fig. 1.
`Restraining a mouse by hand. (A) and ([3) Proper finger placement. (C) Fingers are located over the mouses shoulders, rather than behind neck and
`ears. The mouse can turn around and bite. (D) Excessive traction on the‘ skin can c inke the mouse. Note the protmding eyes.
`
`|nnoPharma Exhibit 1100.0005
`
`

`

`434
`
`TERRIE L. CUNLIFFE—BEAMER
`
`
`
`A
`
`B
`
`
`
`C
`
`Fig. 2.
`Restraining a mouse using forceps. (A) Proper placement of forcepia. {B} This mouse is too big to pick up over the shoulders. Forceps are gmsping
`hair, not skin, and the hair may he plucked out by the weight of the mouse. {C} The tail slides through forceps if they are placed too close to the tip of the tail.
`
`the palm of groin‘ hand. Newborn litters should he returned to a
`nest. not scattered throughout the cage, after they have been
`handled.
`
`B.
`
`Identification
`
`A variety of methods have been devised for permanent, or
`temprirary identification of lt‘l(il'v‘l(lLl8l mice. Permanent identi-
`fi€:'ation methods include metal ear tags, notches; (Dickie,
`19275), toe clipping (Kumar,
`l9"?9; Dickie. 1975), tail clipping
`(Dickie, 19335),
`tattooing {Schoenhorne at at,
`l9":"}'; Green-
`ham,
`l9’?8; Avery and Spyker, 1925’), and freeze marking
`(Farrell and iohnston,
`l9’?3l. Duberte {I968} devised a binary
`number system of toe clipping and ear notching that permitted
`individual identification of over 1,000,000 mice. ‘l‘emporz;u‘y
`identification of individual eagemates can be achieved by dye-
`ing for of albino or dilute mice with food coloring, clipping or
`plucking unique patterns in the for, or marking tails with indel-
`ible markers. The first two methods permit identification for
`l—2 weeks; ink marks disappear in [M2 days.
`
`C. Restraint
`
`shipping containers {Boutelle and Oper, 196?), scizssor handles
`(Liebenberg es’ ai., 1980}, and Plexiglas or metal (Archuleta,
`l9'i"?; Billings, 196?; Boggs,
`l9'?8; Chamgrlin and McGill,
`196?; Kaplun and Wolf, l9?2; Keighley, 1966; Mulder, 19??)
`(Fig. 3). Most of these devices were designed to facilitate tail
`vein injection, collection of bloodt or irradiation. Regardless
`of the material,
`the device should prevent the mouse from
`turning around, have adequate air holes for ventilation, and
`should be easily cleaned and disinfected.
`Wild mice (Per<‘;m_3>sczes sppq M£t.$ c:::i‘oi:', etc.) present spe~
`Ciitl challenges for even the most routine animal care pro-
`ccc.1ui'es. Forceps are mandatory for handling wild mice.
`in
`addition, one should consider using red light (Fall. 1974),
`worldng at odd hour:«; when the animal room is quiet, and
`building a high-sitled box or eluzte (Wallace,
`l968).
`
`III. ADMINISTRATION OF DRUGS OR UTHER
`
`CONIPOUNDS
`
`A. Topical
`
`Restraint devices for mice have been made in minty shapes
`using a wide variety of materials:
`lia:‘dwa1'e cloth {Odo and
`Mirancla, l9?6; Crispens and Kaliss, 1961), leather {Lawson at
`(25,.
`1966), plastic tubing (Mylrea and Abb1‘et:ht, 196?),
`3{l——50n1l plastic syringes (Lul<asewycz, 1976; Farmer and
`Mellett, 1935), metal dose syringe (Jones, 1965), I'2idi{)iSOl()pe
`
`Topical application of‘ compounds to depilated skin of the
`tail, ear, or the body of normal or genetically hairless mice,
`e.g., f2rfI2:',
`izzifrzsi,
`is an easy procedure. It is more difficult to
`prevent the mice from licking the area and ingesting the com-
`pound. Various: devices. have been developed for this purpose:
`Elizabethan collar (Eiuhcber at 541., 196?‘), Plexiglas box ap»
`plied with collodian {l\iixon andiieer, IS-W3), glass tube over‘
`
`|nnoPharma Exhibit 1100.0006
`
`

`

`13. 8§f);\~I[ETH(,)I}OL(}(}Y ,»\:\ED S{7R{§I{,"/XI, 'i"iiCiE;\‘lQL-‘ES
`
`:,f(};T
`
`A
`
`.
`
`‘
`
`
`
`
`
`B
`
`
`
`21 2:23:11-
`If 21-:;r:u1'2:te 011:1 z:e;11ati:“:ié;tI‘aIi<m :31’
`bztctmietl gruwih.
`pousicl is; requirserzi, :1 fozzrciing nceciiét (Clam; and H:1rEz::1cE. 1969}.
`dose $$js“E‘iI1g€ {Shani 2»: mi. 19?f3L :31‘ cmatimlous int1*;1gast:"c
`infu.~;i{:m :5}-‘stem (W21j;:1f<):‘tI1 :21’ m’.. 19??) should be used. SLED
`rccssfui per‘ ax admini.<;tratis:m of C<1rn;:»()L:|1ch; 1'equir::s thm‘0ug.h
`knowledge: of the zmalomical 1'cl21tionsl1ip:s of the o1‘0phm‘y1 X
`and deft much because the csophagcral orifice:
`‘smut:-t be easi jg
`observmi in the living 1nr:su:;e {I*vilz:ced2:~S0brir:ho e: mi,
`i9?E~§}
`(Fig. 4}. The mause is :'€Sf1'2ii11€3fi£!?$
`shmvn in Fig. IA. and the
`fee«:1i:‘sg azamiie is immmtced into the left dias;t<:me1 and gum y
`<iirectv:<“£ czmtizzliy {(}W2lI‘{i the right rami of the rmmdihlc. At thix
`paint,
`the mGlt.‘:1{.‘ uszzieiiiy begins to swaiiicww and the ikzeziiis g
`ncadle can be gently inserted into the »;:sophagLI:; (Fig. 5).
`1
`i:1t1'atg2tst3'ic adiniitistiutiotx of the c<>mp0uI1:.l is desimcl. thit <ii~~
`amc:t<::1' of the feeding net-:rJ1r: or the tube should be smzi
`crmugh to pass through the §:&i(}ph23.gU:~3 where its diameter mar»
`rows mean’ the heart. The length of mi: ftzedixlg needle or tube:
`Can he ezztimated by n1ea3L::'ir1g the ciistamteé fmm the msze
`as
`111:: last rib. Extending the n1uu:~‘.r3’§; ties}: to form 21 straight rim:
`between esophageal orifice: and the cardiac sphincter alga facil-
`itates intrzzgastm: a<iminisn'ario:1 <:ef<:or11poun<:!s‘
`
`C. Subcutaneous Iujectiun
`
` T inserted into the skin '/iw ‘X2-inch caudal to the injection site zmd
`
`"M" *9‘
`'%
`
`'
`
`Rf3.\iSI’2tit1-E devices.‘ 62%) Plexigtans hm for tail htecding. EB) H:1:':i—
`!4‘r't:. 3.
`wztm ciush and cork nmusc hcrldcr. (C1 l’icxiglas C}‘ii!3r:l::°r for irrzutizitimi nr mil
`vim: injection.
`
`the tail fJeIming:s at m’.. 1973). or at body htllidéigfi (Brj,«'zmI and
`13::rnard.
`I955; S£‘:ib:3I't and Pnllanl, 19?}; Sediacek car
`.»:z{..
`
`19701
`
`B.
`
`Per('}S
`
`The easiest, but least €IC{.‘LEI'£l{€. way to £i(11”!‘iiI1iSt€I‘ coinpounds
`gm‘ as is 11> mix them in the fomi or c;!rin}<iu_;_z_ water. HCWGVGI’.
`if the compmxnd imparts an unpIeas2Ir1t flzwczr to the fond er
`wzzter. food or water consumptian is often dras;ti<:aH},2 reducml.
`In one smciy ~.vaater consumption Lie<::'ea:sed 436;? f‘uH<>wi:1g the
`znzltliticm (‘sf 0xytett‘aC}=c1i:1c to the drinking water {'StL1n1»:arc1 :3:
`mi, 19391): the pr<:>bIt:n1 was izliminatésni by Fizm)1‘i:1g the water
`with .*il1{.‘l‘£)Si;‘., However, h0tih—:.<; of drinking wzttr5:r flax!<)1'::~:d with
`sugar shoulci be rezpiacai at least twice at week because cf rapid
`
`Suhcutzmeous injections (SC) of l.U—.?:.(3 ml per adult m<:+us;e
`are made into the 1(mse skin over the neck or flank us‘mg 21 20-
`tca ;"6«gauge '/3~l-inch zxeedle (Fig. {S}. The nsedle should be
`
`th+::na<ivaa:1cc—:ci through the subcutanmus iisstaes to the injection
`suite in Carder in m'mimi;.:c Eczzkzigc of the injeisicii :11a.t:::‘i;i! 0:11:74
`the peiage. Szihcutanezous implants h.sw:~: heezz tisfid ta mzzimain
`ErEmSF)E9‘mab}‘3 mmom‘ Creme Cuhum Chamberg fiéfiko‘ E9133)‘
`
`_
`_
`epaglofhs
`
`esophagus
`
`diosfemo
`
`diosfemc
`
`tongue
`
`E‘}_:;. 4.
`
`Anzztmnisszii rc!aEie)m;hips of the m-npma-;.«.a,»;.
`
`|nnoPharma Exhibit 1100.000?
`
`

`

`4&6
`
`TFLRRIE L. CUNLIFFE—BEAMER
`
`iv
`
`56
`
`im
`
`Injection sites in the meme. iv, intravenous; ip, itttraperitoneal; se.
`Hg. 6.
`suhtrutaneous; im, intmnitnseuiar.
`
`I). Fnotpad Injeetiun
`
`injection into the voittr aspect of the foot pad is used tu elicit
`iznmuttelogieai respcnses. Up to 0.05 ml of i[1OCtli2i can be
`itijecteti into a hindpaw (Nelson, 19733}, and the response can
`be easily measured (Pear:~;0:1 3: ::'ti.. 19?!) (Fig. 6).
`
`E.
`
`Intracranial Injection
`
`itttraeranial (Tic) injection of stispect material into infant Qt‘
`weanling mice is used as an in mm assay for neumtmpltie
`viruses. Netmates are iestrttinerl with forceps; wettntirig mice
`shouts} be anesthetized. The needle (2?-gauge fur neonate, 22-
`to Eébgauge far weanling) is inserted through the :-skin over the
`midsection of the parietat bone slightiy laterai to the eeutrai
`suture; this awsids puueture ed’ the sagittal or tt'at1sverse veneus
`siiittses. The needie is gently rotated until the bone is pens»
`trated. ‘Then the needle is advanced to a depth of 1M4 mm,
`depending upon the size of the mouse. Apprdximately 0.015
`and 0,03 ml can be injected ititraeraniaiiy into neonatal and
`weanling mice, respectively (Johnson, £914; Murine Vi1'us Di-
`agnostic L£1E)0l'a{0§'}‘, £9238}. Soititiuns injected 'mtracrania11y
`should be as near body temperature as possible, and after injec~
`tier: mice should be kept warm to reduce the p{)SSii}i1it}‘ of
`shoctt.
`
`A technique for intracistemai injection into the cisterna mag-
`ma of conscious mice was deseribe<:i by Ucde tar as’. (1909). A
`specially modified 2?-gauge needle was used to inject I0-20
`p.l. httracerebroventricular injection of hormones or other
`pharniaeologic agents into specific areas of the "t'enti'icIes re—
`quires stereetaxic placement of" the needle as described by
`Delaney et at’. (197%) and Holman (1980). Severe] stereotoxic
`
`|nnoPharma Exhibit 1100.0008
`
`ms. 5.
`(21) Feectittg needle and restrairtetl nmtxse
`Iittragastric intuttzztitm.
`pricr to insertioit of the feeding needte. {I3} Inserting the feeding needie "mm
`the left diastema.
`(C) Conzpietirig il1$€l'tt0i"t of the feeding needle into the
`stomach.
`
`induce fume-rs (Prehn and Kzamik. 19?9). culture en»:ic.:critte
`organs in \*i\9{} (Krohn, 1963), or test materials for dental pres-
`thesis {Russell 3: a1’., 19259). étnesstliesia is 21{1mit3isfel‘€(i
`if the
`
`implant requires; incisitm of the skin with scissors or use cd‘ 21
`targe 14—gaL:ge tr<:~eat'.
`
`

`

`18. BIOME‘{‘HOl}{)L{)G‘x’ AND SURGICAL TECHNIQUES
`
`«Kl?
`
`l9?4;
`l97l; Leliniami,
`atlases of the mouse brain (Krueger,
`Montenmrro and Btikelew,‘ l9'}2; Sidman et‘ a:’., 192%} are
`available.
`'
`
`F.
`
`Intramuscular Injection
`
`injections {imj are usually avoided in the
`ltttramuseulai'
`mouse because of the small muscle masses. Tltie rate of al>s0r1:a~
`lien of aqueous solutiens is similar following lt}li’Z1m1ISCL!l€il'
`and subcutaneous injections {Bagge-tt, 19%’). lf necessary, Em
`iztjcctioras of {L05 ml or less may be mettle inte the anterulateral
`thigh musclezs (quadriceps femoris group) using a 22» to 26»
`gauge ‘xi:-inch needle (Fig. 6). '1“he needle should be directed
`away from the femur and sciatic meme.
`
`G.
`
`Intraperitoneal Injection
`
`T0 amid puncturing the smniaeh, spleen, or liver inim-
`peritoneal {ip} injections of up to 1.0 ml are made inte the
`lower right quadrant of the ventral abclemen (Fig. 6). The
`mouse is restrained as shown in Fig. 1A, and the hez1dlet"s
`wrist is rotated until the meuse’3 head and body are tilted in 3
`clownward tlirectiorl, allowing the m0use‘s abtlomiraal ‘r.s‘i:5<:£3I'%1
`re shift crariielly. The needle (23- to 26$-gauge ‘/i—’/3-inch) is
`then inserted through the skin slightly medial to the flzmk zmtl
`cranial to the inguinal canal, advanced eranially through sub-
`cutaneous tissue for 2-3 mm, and then inserted througlt the
`abdominal muscles. Care should be taken tea avoid penetrating
`the preputial glands in the male mouse. The needle and syringe
`should be lieltl pzzmllel
`to the mottstfs vertebral column in
`order to amid accidental retroperitoneal or intrarenal injection.
`Further sources of €3i"I.‘{}I' using intraperitoneal injections have
`been outlined by Lewis at as’. {l9f:36} and Hamilton as at’.
`f 196?}.
`
`H.
`
`Intrathoracic: Injection
`
`Unless experimental objectives mandate intmtheracie injec-
`tion, intraperiteneal injection is prefe1‘rec:i because it is easier
`and less lmzatrclous (no risk of pneuniethorax or punctured
`lungs), and abseiption rates are similar.
`lntrathoracit: injec~
`lions, if necessary, are made at appmxirtiately the midpoint of
`the chest using a slightly lieztt 3/¥:~in<:h 22-gauge needle inserted
`at an angle between the ribs {Simmons and Brick,
`l9?G).
`
`I.
`
`Intravascular Injection and Canltulaticrn
`
`1.
`
`Intraarterial Injection
`
`ln certain procedures such as angiography, intraarterial in-
`jection may be necessary.
`ln_jec;ti(m into the femoral artery
`
`using a l/Ewlllfth 24-gauge needle has been tlescrllzaecl (Simmons
`and B:'iCl<.,
`I930}.
`zmtl
`teelmiqttes for carotid cazmtzlation
`(M<:lS<laster; I941; Sugzmo and Nemura, @633) can be adapted
`fer int1'aa1‘tet‘ial
`injection. To ttszslure
`intt'aaI'Ee1'ial
`injection,
`anesthesia is performed and the artery is expoeed tlirouglt 8.
`skin inciezien.
`
`2.
`
`Intravenous Injeetitm
`
`lhe lateral or Cl(}l'Sal tall veins are the usual sites for intra-
`
`veneus injection in mice {Gt'it:e, 1964) (Fig. 6}. Devices to
`1‘(i§$l;£'.‘:tl|”1 mice for tail vein lI’Jjt3Cli<2sI“tS are €lt?SC1‘ll?{2£l by C‘t'i5pem;
`and Kitlllié‘. U961), Champlin and McGill
`(I963), Boggs,
`{I938}. fiillings (19:93? Ft1z'net' and Mallet: (I915), Lul«:ase~—
`Wye: {l9?6}, Mylrea and Abbreclit {I963}, and l\llCl{%§0I'c and
`Barltulis {l94’3). Tail vein injection is ea:~:ier if lllfi veins are
`dilated by warming the tail f‘er5-10sec. in ajar of warm water
`{Bar1'0w, W68}. er warming the 1”l}{)L1S£3 for 5-13 min. in a jar
`liealetl by 2: 4{}wl0f} W light bulb {Simm(:sr1s; and Briclt.
`l9'?{}).
`If necessary, tourniquets tlevised fit) :1 2: weuncl clip ap;:»IicaIez~
`(Bergstt'é$m, l9':'I} or 2: hypodermic syrizzge and tltreacl {lV.llI1£l~
`slam. 1980) can be used re aeelticle ti il veins. In albino or gray
`mice, the tail veins are vistualized as thin reel-lzdut: lines coats»
`
`ing along the top (dorsal tail vein) and bottom {ventral tail
`vein). Tail vein injection of mice wl la pigtiientetl tails: is mere
`difficult than ittjeetien of mice wit nonpignientecl tails. De-
`pending upon thesizc of the mouse 11 26~ tn 3(}~gat1ge l/fz~ to ‘/2-
`inch z1ee~:lle is used. Other sites for izltrzn-enotts injectitmsg in-‘
`elude the external jugular vein ilicssel and Lexsitail. W53‘).
`Cl0I'S£tl metatarsal vein {Nobunaga at «:1, 1966), stzblingtzal
`vein {Wa3,rnfortl1 and Parkiit. 1969), and ophtltztlmie plerxzus
`{Pinl<ert0n and Webbet‘,
`I964). Strgicatl
`€J*{pOSLIt‘€
`til‘ these
`Veins is not required.
`
`3. Vascular Camiulatima
`
`The dorsal tail vein has been the ttsual site fat’ lnt:'2wen::.ms;
`
`canmilation in mice {C{‘mne1' ex 52]., W80; Martin anal Sttaus,
`1980; Rhodes and Pattetzsbzi. l9?9); depending upon the teel -
`ique, anesthesia may or may not be required. Tail vein can-
`iulas sheultl be protectecl by bandages: or splints.
`The jugular vein is also accessible fur ll1ll'£W'vE3l”1CIl.1$ <:Eil1I1Ul2’i~
`ion after the "mouse is anesthetized and placed it: ciorsal er
`dorsolateral fCCllI1’ll}2i[lC}{. After the skin is prepared for sur-
`gery, 3 l-cm paramedian incixion is made from the manubrium
`0 the rami of the mandible. The cattclomedial edge of the
`arotid E5$lll7>z'Eil'y gland is dissected free, exposing the jugular
`vein and its fascial sheath. Incision of the fascial sheath e>:.—
`
`oses the jugular vein. The cannula can be inserted or direct
`injectiens can be made into the jugular vein using a 30~gauge
`needle. The volume injected should not exceed O.l to 0.2 ml.
`7’ost«injection hetnerrltzage is centmllecl by gently compressing
`he jugular vein with the end of the sztlivziiy gland as the needle
`is withdmwn from the vein.
`
`|nnoPharma Exhibit 1100.0009
`
`

`

`4708
`
`TERRIE L. CUNLIFFE-BEAMER
`
`Procedures for cannulation of the Common carotid artery
`have been described by luiclylaster (1941) and Sugano and
`Nomura (19633). After insertion, chronic carotid or jugular
`cannulas are routed subcutaneously across the lateral surface of
`the neck and exteriorized on the dorsal inidlirie between dorsal
`
`borders of the scapulae. The abdominal aorta can be cannu-
`lated using the technique described by Weeks and Jones
`(1960). lixteriorized cannulas should be protected by a light
`weight body bandage. In some instances, a stanchion-lilte cage
`may be advised.
`
`J. Medication of Neonatal Mice
`
`hdedicatiori of neonatal mice is complicated because of their
`small size and the danfs tendency to reject or cannibalize
`offspring that have been handled excessiyely. Up to (}.l ml
`may be administered orally through a piece of plastic tubing
`inserted over a 30-gauge needle. Subcutaneous injections of
`approximately 0.1 ml can be made over the rieclr and shoulders
`using a E/4-inch 30-gauge needle (Gibson and Becker. 196?).
`l..eal<age from intraperitoneal injections (0.05----{}.l ml) is mini-
`mized if the ‘/5z- to ~l/9-inch 30-gauge needle is inserted into the
`skirt parallel to the right femoral vessels and advanced sub»
`cutancously until the lower right abdominal muscles are pene-
`trated. intravenous injection of the neonatal mouse is difficult.
`Several authors have recommended the anterior facial vein at
`
`the level of the lateral canthus of the eye (Anderson at aii,
`1959; Barnes ct mi. l963; Billingham and Brent, 1956} or the
`transverse (sigmoid) sinus (Barnes at m’..
`i963). The latter
`injection site may be used until the mice reach l8—2(} days of
`age. lntrncardiac injection of newborn mice with up to (3.05 ml
`using 30~gauge needle has been described by Grazer H958)
`and Poatniltoya (1960). lntracranial injection of neonates has
`been described previously (Section ll,l3}.
`The best defense against rejection or cannibalism of experi-
`mentally manipulated newborn mice is gentle handling of both
`neonate and dam.
`It
`is also helpful
`to select rnultiparous
`females that have successfully reared a litter and have demon-
`strated satisfactory maternal behavior, to select docile strains
`or stocks. and to separate dam and litter while the litter is being
`hszutrlled. Plastic gloves should be worn or an odor~nia:~;l<ing
`agent {perfume} may be placed on the danfs nose and on the
`neonates to prevent them from acquiring or recognizing human
`scent. After‘ injection or surgical manipulation, any extrava-
`sated blood is removed and the neonates are returned to their
`
`nest. East and Parrott (1962) described several surgical and
`poatsurgical procedures for neonatal mice. Additional sug-
`gestions made by Libbin and Person { 1979} for neonatal rat
`surgery can be applied to the mouse.
`
`IV. C{}LLECTI0l"vl OF BIOLOGICAL SPECIMENS
`
`Sections VI, Vll, and Vflll should be consulted before at-
`
`tempting some of the more complex procedures described
`below.
`
`:1. Bile
`
`Chronic cannulatinn of the bile duct of mice has been de-
`
`in detail, by Becker and Plan (19633). Atlaption of
`scribed,
`routine liver function tests for use in mice was described by
`Anonymous H962) and by Casals and Olitsky (1946).
`
`B. Blood
`
`lylany techniques for collecting large or small amounts; of
`blood from mice have been developed.
`
`1. Orbital Sinus
`
`‘Venous blood can be easily obtained from the orbital sinus.
`The mouse is placed on a table or cage lid in lateral recumban-
`cy, and its body is restrained against the table using the palm of
`one hand while the thumb and forefingers of the same hand
`restrain the head and gently open the eyelids to expose the eye.
`A microhematocrit tube or small bore Pasteur pipette is insert-
`ed through the conjunctiva of the medial canthus and is di-
`rected medially into the orbital sinus by quickly rotating the
`tube frorn side to side (Fig.
`'3}.
`'1‘he eye is not danianged
`because the tube passes under the eye. Reluctant blood tlow
`can be lmpI'()X-‘€Z(l by raising or lowering the tube. This tech-
`nique is usually performecl on anesthetized mice.
`After the required amount of blood is obtained, the tube is
`withdtmyn and bleeding sually ceases. If necessary, hemor-
`rhage can be controlled by direct pressure applied over the
`eyelids. Small amounts o" blood (39-80 til) can be obtained
`‘from orbital sinuses of nice as young as 14-15 days of age.
`Larger amounts of blood {G5 inll can be obtained from orbital
`sinues of older mice if tubes containing anticoagulant are used.
`Orbital bleeding can be re outed within hours it’ the arnount of
`blood removed at any one time is relatively small. Art alterna-
`tiye approach to orbital bleeding involves; restraining the mouse
`in an upright position, as s own in Fig. IA and B, and entering
`the venous sinus via the lateral cnnthus. This method provides
`less control over sudden ioycrnenta of the mouse’s head and
`
`increases the risk of corneal lacerations. Further descriptions
`of the technique can be found in articles by Cate (1969). Ril