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`Volume III
`Normative Biology,
`Immunology, and Husbondmy
`
`EDITED BY
`
`Henvy L. Foster
`The Charles River Breeding Laboratories. Inc.
`Wiimington, Massachusetts
`
`] . David Ssssoll
`Comparative Medicine Branch
`National Institute of Environmental Health Sciences
`
`Research Triangle Park, North Carolina
`
`James G. Fox
`Division of Comparative Medicine
`Massachusetts Institute of Technology
`Cambridge, Massachusetts
`
`ACADEMIC PRESS 1988
`
`A SUBSIDEARY OF HARCOURT BRACE IOVANOVICR PUBLISHERS
`
`New York London
`Porés San Diego San Francisco Séo Paulo
`
`Sydney Tokyo Toronto
`
`InnoPharma Exhibit 1100.0001
`
`

`

`(lemmas? © 1983, BY ACADEMIC PRESS, INC.
`ALL RIGHTS 11332111920.
`NO mm OF THIS PUBLICATION MAY BE REE’RODUCED GR
`mmmmav IN My mam OR BY ANY MEANS, ELECTRONIC
`(m MECHANImL, INCLUDING Primacow, RECORDING, on ANY
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`PERMISSION m wamm $30.31 THE PUBLISHER.
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`x
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`ACADEMIC PRESS, ENC.
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`United Kfésgdom £58502: pathlishea’ by
`ACADEMIC PRESS, INC. {LONDON} KIT).
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`Library of Congress Cataloging in Publication Data
`Rain entry under title:
`
`The Mouse in biomersical restmmh.
`
`{Merimn College a? Laboratory animal l-iedicine
`series)
`Imludes indexes.
`Contents: v. i. Histary, genetics, and wild
`mice —~ [etc.3 ..- v. 3. msmndry.
`1. Faster,
`1. Rice as labcratory animals.
`Henry L.
`1:. Small.
`.3. Cavid. Hi. Fox, James (3.
`IV. Series.
`[fiNLMr 1. Nice.
`2. Reswmh.
`3. Animis, Laboratory.
`[LII 60.86 H9321]
`QLKW. R63§fl68
`619‘ .93
`80—?0669
`ESBN 0-12-2625ny (V.
`3:3
`£31892
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`PRINTED IN THE UNITED STATES OF AMERICA
`
`83348536
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`987654331
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`InnoPharma Exhibit 1100.0002
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`

`

`Chapter 18
`
`Biomethodology and Surgical Techniques
`
`
`Terrie L. Cnniafiefieamgr
`
`lutmductinn ................................................
`I .
`11. Handling. Identificaiiflm and Restraint ...........................
`A. Handling ...............................................
`B.
`ldentificatiun ...........................................
`C. Restraint ..............................................
`Ill, Administfation of Drugs or Other Compounds ....................
`A . Topical ................................................
`8. Per 03 .................................................
`C.
`Subcutaneous Injection ...................................
`D.
`Feutpacl Injectinn ........................................
`E.
`lntracranial Injectitm .....................................
`F.
`Intramuscular Injection ...................................
`G.
`lntraperitoneal Injection ..................................
`HA
`lntmtlwracic Injection ....................................
`I.
`Imrzwascular lnjectiun and Cannulation ......................
`J. Medication of Neonatal Mice ..............................
`IV. Cullection of Biological Specimens .......................» ......
`A. Bile ..................................................
`B. Bland .................................................
`C. Bone Marrow ..........................................
`
`:1 Faces .................................................
`El Lymph ....................................., ...........
`E Milk .................................................
`G.
`Peritoneal Cells .........................................
`H.
`Pulnmnary Cells ........................................
`I. Ova and Sperm ........................................
`
`J. Urine ..................................................
`V. Assessment of Physiological Status .............................
`A Blood Pressura, Heart Rate, and Respirator}; Rate .............
`B.
`Fnod and Water Consumption ..............................
`C. Neurological Examination .................................
`D. Miscellaneaug Techniques .................................
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`‘
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`Copyright 2: )933 by academic Press. Inc.
`All rights of rcpwxluclinn in an)“ {mm reserved.
`ISBN {Hzrzézsmx
`
`InnoPharma Exhibit 1100.0003
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`

`

`402
`
`TERRIE L. CUNLIFFE-BEAMER
`
`‘3’}. Anesthetics; .................................................
`A. Analgesics, Scdatives. and Other Preanextlietie Medications .....
`B . Hypothermia ............................................
`C.
`Local Anesthetics ........................................
`D.
`Inhalent Anesthetics ......................................
`E.
`Injectable Anesthetics ....................................
`F. Neuroleptanalgesics ......................................
`Surgical Procedures ..........................................
`3%.. Basic Techniques ........................................
`B. Adrenaleetomy ..........................................
`C. Artificial lneeminntion ....................................
`
`Vll.
`
`D. Embryos and Embryo Transplants ..........................
`E, Hepateetomy ............................................
`F. Hypophysectomy ........................................
`G. Hysterectomy and liysrerotomy ............................
`ll.
`Isolated Intestinal Loops ..................................
`l, Lymphnodectomy ........................................
`J. Mammary Gland Excision and 'l‘ransplant ....................
`K. Miscellaneous ’l‘ransplam Techniques ........................
`L. Nephrectomy and Kidney Transplant ........................
`M. Glfnctory Bulb Ablation ..................................
`N. Orelaidcctorny, Testicular Biopsy ...........................
`0. Ovariectomy, Ovarian Transptant, and Qvuriohysterecromy ......
`P.
`Parahiosis ..............................................
`Q.
`l’orathyroidectomy .......................................
`R.
`Pinealectomy ...........................................
`3.
`Skin Grafts .............................................
`
`Splenectomy ............................................
`'1”.
`l}. Thymectomy and Thoracotomy .............................
`”if. Thyroidectomy ..........................................
`W. Vasectomy .............................................
`Poetanesthetic and Postoperative Care ...........................
`Vlll.
`IX. Euthanasia .................................................
`X. Enigmatic Procedures and Necropsy ............................
`A. Diagnostic Procedures ....................................
`B. Necropsy ...............................................
`C. Hixtopathologicai Examination .............................
`References .
`.
`.i ..............................................
`
`413
`414
`at l «l
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`4M
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`418
`CH9
`rill}
`420
`421
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`43 l
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`4235
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`:12?
`£28
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`‘
`
`I.
`
`INTRODUCTION
`
`The mouse’s small size, relatively short life span, proficient
`reproductive capabilities, and susceptibility to microbiological
`or chemical agents make it an appropriate animal model to
`investigate problems
`in many diverse disciplines,
`such
`as embryology, ethology, geneticsi gerontology, microbiol—
`ogy, and oncology. The mouse‘s small body size makes it pos-
`sible to maintain many mice 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)
`{licence advantages and disadvantagee of alternative ways to
`perform the same procedure; and {4) provide references that
`contain detailed descriptions of unusual procedures.
`Commonly used procedures and lost; common procedures. for
`which descriptions could not he found in the literature are
`described in detail. In cases where good descriptions of un«
`usual procedures are available in the literature, the reader will
`he referred to the original articles. Before attempting many of
`the procedures described in this chapter, one should review the
`anatomy of the area in question and practice the procedure on
`an anesthetized or dead mouse. Even though laboratory mice
`(Mics numerous doriresrs‘czisl can be handled or restrained with~
`out the administration of drugs, one should not substitute physw
`
`InnoPharma Exhibit 1100.0004
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`

`

`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 mice are caught and picked up by 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 forcfingers 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—10~inch smooth dressing forceps} are an excelm
`lent way to manipulate wild, aggressive mice. Adult mice are
`caught by grasping the cranial third of the tail (Fig. 21 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 (some) or diabetic (dares) mice, should be han—
`dled gently and supported, if necessary, with a hand under
`their feet. Young mice {less than 2 weeks of age) are picked up
`by grasping the loose skin over the neck and shoulders with
`forceps or thumb and forefinger, or by scooping the litter into
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`Fig, I.
`Restraining a mouse by hand. (A) and (B) Proper finger placement. (C) Fingers are located over the mouse‘s shouldersc rather than behind neck and
`ears. The mouse can turn around and bite. (D) Excessive traction on the skin can c take the mouse. Note the protmding eyes.
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`

`434
`
`TERRIE L. CUNLIFFE—BEAMER
`
`
`
`A
`
`B
`
` C
`
`Fig. 2.
`Restraining a mouse using forceps. (A) Proper placement of forcepla. {B} This mouse is too big to pick up over the shoulders. Forceps are grasping
`hair, not skin, and the hair may be 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 your hand. Newborn litters should be returned to a
`nest. not scattered throughout the cage, alter they have been
`handled.
`
`Bi
`
`identification
`
`A variety of methods have been devised for permanent, or
`temporary identification of individual mice. Permanent identi—
`fié‘ation methods include metal ear tags, notches (Dickie,
`19’35), toe clipping (Kumat, i939; Dickie. 1975), tail Clipping
`(Dickie, 193’5),
`tatooing {Schoenoome at at,
`l97?; Green-
`liam,
`[9?8; Avery and Spyket‘,
`l9??), and freeze marking
`(Farrell and inlinston, 19’?3l. Duben U968} deviated a binary
`number system of tee clipping and ear notehing that permitted
`individual identification of over l,000,000 mice Temporary
`identification of individual eugemates 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 [“2 days.
`
`C. Restraint
`
`shipping containers {Boutelle and Oper, 196?), scissor handles
`(Liebenberg er a!” 1980}, and Plexiglas or metal (AI-elitileta,
`l9??; Billings,
`i96’i; Boggs, 19%; Chamolin and MCGiIl,
`196?; Kaplun and Wolf, l9?‘2; Kelghley, 1966; Mulder, 19239)
`(Fig. 3). Most of these devices; were designed to facilitate tail
`vein injection, collection of blood 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 (Peromyscns sppq Mos coroii, etc.) present spe—
`cial challenges for even the moat routine animal care pro-
`cedures. Forceps are mandatory for handling wild mice.
`in
`addition? one should consider using red light (Fall. 193’4),
`working at odd hours when the animal room is quiet, and
`building a high-aided box or ehote (Wallace,
`l968).
`
`III. ADMINISTRATION OF DRUGS OR OTHER
`
`COMPOUNDS
`
`A. Topical
`
`Restraint devices for mice have been made in many shapes
`using a wide 1tiariety of materials: hardware cloth {Oda and
`Miranda, l9?6; Ci‘ispens and Kaliss, 1961), leather {Lawson at
`(25..
`1966), plastic tubing (Mylrea and Abbreoht, 196?),
`30-501nl plastie syringes {Lukasewyez, 1976; Fumei‘ and
`Mollett, 19%), metal dose ayringe (Jones, 1963* radioisotope
`
`Topical application of compounds to depilated skin of the
`tail, ear: or the body of normal or genetically hairless mice,
`eg, Kirstin, menu,
`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 (Eiuheber et- nl., 196?“), Plexiglas box an
`plied with collodian {Nixon andReer, 1W3), glass tube over
`
`InnoPharma Exhibit 1100.0006
`
`

`

`13. MOM[ETHODOLOGY AND Stiltiiliizkl, 'l‘iiCiENlQL-‘ES
`
`405
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`A
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`B
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`C
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`W
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`M. W.
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`“h
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`Fig, 3. Restraint device» (A) Plexigtan hint for tail bleeding. EB) Haird-
`wnre cloth and cork mouse holder. (C1 Plexiglas cylinder for irratlintinn in tail
`vim: injection.
`
`the tail {Jennings er (ii. HEEL or at hotly bandage (Bryant and
`Bernard. 1955: Seihert and PnllartL 19?}; Sedlat‘ek er all.
`193’0).
`
`13‘ Per 05?
`
`The easiest, but least accurate. way to administer compounds
`pm“ as is In mix them in the food or drinking water. However.
`it‘ the compound impartn an unpleasant flavor to the food ur
`Willi?!“ loud or water eonsumptinu is often drastically reduced.
`In one study water consumption decreased 43% following the
`adrlitim (if oxytetrncyclino to the drinking water {Stunltartl r3:
`nit, 19?} l; the problem was eliminated by Flavoring the water
`with unernne, However; bottles; of drinking water flavored with
`sugar should be replaced at least twice at week because of rapid
`
`If accurate drill administration til" a num-
`bacterial gruwth.
`ponnd is; required. a feeding needle (Clark and Harland 1909}.
`dose syringe {Shani a»: a?” 197tlli or continuous intragnstr’e
`intitixinn system (Waynl‘orth er a!” l9???) should be used. Suew
`ecsslul per 0); administratinn of compounds requires thorough
`knowledge of the anatomical relationships of the orophm‘yt x
`and deft touch because the esophageal orifice ‘unnnt bi: earn 3*
`observed in the living mouse €Maceda~$obrinho e: at, 39%;}
`(Fig. 4’}. The mouse is restrained 83 shown in Fig, 1A. and the
`feeding needle is; introduced into the left diastemn and gent v
`directed caudally toward the right rnrni of the mandihlc. At thin
`pnim,
`the mutant: usually begins to swallow and the teeth: g
`lififldlti can be gently inserted into the esophagus (Fig. 5).
`l
`intrngttstrie administration of the compound is desired. the di~~
`umeter of the feeding needle ur the tube shntlld be smal
`enough to pass through the esophagus where its diameter mm
`rows near the heart. The length nl‘ the feeding needle or tube
`Can be estimated by manning the distance l’rnm the hunt: n
`the lasst rib. Extending the mnuse’s neck to form a straight line
`between esophageal orifice and the cardiac sphincter algn facil-
`itates; intrugastrie administration of compoundsi
`
`
`
`C. Subcutaneous Injectiun
`
`Subcutaneous injections (SC) 01" Lil—2.0 ml per adult mouse
`are made into the loose skin over the neck or flank using at 20-
`to Eowgauge l/yl-ineh needle (Fig, {3). The needle should be
`inserted into the skin 'xitw Viz-inch caudal to the injection site and
`then advanced through the subcutaneous; tissues to the injection
`Site in order to minimize leakage ui‘ the injected material onto
`the pelage. Subcutaneous implants; have been used to maintain
`trzutsplantahle tumors. create culture chamberu (Arinx IQBL
`
` e pi g In Hi 5
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`9 so 1;) h n g u s
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`dinetemo
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`to n 9 tin
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`d to st em <3
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`Fig. 4. Anatomical relatinnnhips of the ot‘ophurym.
`
`InnoPharma Exhibit 1100.0007
`
`

`

`4&6
`
`TERRIE L. CUNLIFFEwBEAMER
`
`
`
`Fig. 5.
`(n) Feeding needle and restrained mouse
`littragantric intimation.
`prior to insertion of the feeding needle. {13} Inserting the feeding needle into
`the left (liastema. (c) Completing insertion of the feeding needle into the
`fitomach.
`
`induce minors (Prelim and Knrnik. 1W9). culture endocrine
`organs in vii-e (Krohn, 1963), or test materials for dental pros»
`thesis {Russell et (33., 1939). Anesthesia is administered if the
`
`implant requires incision of the skin with sciesors or use of a
`large iii—gauge trocar.
`
`
`
`
`foot pact
`
`sc
`
`so
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`.
`um
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`.
`w
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`it:
`
`it:
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`Injection Sites in the meme. iv, intravenous; ip‘ intrapcritoneel; sc.
`ngt 6.
`subcutaneous; im, intramusculun
`
`I). Footpad Injection
`
`injection into the velar aspect of the foot pad is; used to elicit
`immunological responses. Up to 0.05 ml of inocola can be
`injected into a hindpaw (Nelson, 19?3}, and the response can
`be easily measured (Pearson ct 6:1,.
`[971) (Fig. 6).
`
`E.
`
`Intracranial Injection
`
`lntrncranial {ic} injection of suspect material into infant or
`weanling mice is used as an in viva assay for neurotrophio
`viruses. Neonates are restrained with forceps; weanling mice
`Should he anesthetized. The needle {El-gauge for neonate, 22—
`to 24Mgauge for weanling) is inserted through the eltin over the
`midsection of the parietal bone slightly lateral to the central
`suture; this; avoids puncture cf the sagittal or transverse venous
`sinuses. The neetlle is gently rotated until the bone is pene»
`(rated. Then the needle is advanced to a depth of 1M4 mm,
`depending upon the size of the mouse. Approximately 0.015
`and 0333 ml can be injected intracranially into neonatal and
`weanling mice, respectively (Johnson, l9?4; Murine Virus Di-
`agnoetic Laboratory, $9338}. Solutions injectccl intrncranially
`should be as near body temperature as possible, and after injec~
`tion mice should be kept warm to reduce the possibility of
`shock.
`
`A technique for intracistemal injection into the cistema mag
`:12: of conscious mice was described by Uede er all. (19?9). A
`specially modified 2?-gauge needle was used to inject 10-90
`ul.
`lntraeet‘ehroventricular injection of hormones or other
`pharmacologic agents into specific areas of the ventricles re—
`quires stercotaxic placement of“ the neeclle as described by
`Delaney et all {19?8) and Holman {1980). Several Stereotoxic
`
`InnoPharma Exhibit 1100.0008
`
`

`

`18. BIOMETI‘IODGLQGY AND SURGICAL TECHNIQUES
`
`dill?
`
`l97l; Lcllninnm 19M;
`atlases of the mouse brain (Krueger,
`Montemurro and Bukelow,‘ 19'32; Sidinan er «3., 19'31} are
`available.
`'
`
`F.
`
`Intramuscular Injection
`
`intramuscular injections {im} are usually avoided in the
`mouse because of the small muscle masses. The rate of absurp~
`lieu of aqueous solutions is similar following intramuscular
`and subcutaneous injections {Bagged1 Will). lf necessary, im
`injections of {M35 ml or less may be made into the nnterulateral
`thigh muscle}; (quadriceps femoris grnup) using a 22« to 26»
`gauge iii-inch needle (Fig. 6). '1“he needle should he directed
`away from the femur and sciatic nerve.
`
`G.
`
`Intraperiluneal Injection
`
`To avoid puncturing the stomach, spleen. or liver intre-
`peritoneal {in} injections at" up to 1.0 ml are made intn the
`lower right quadrant of the ventral abdomen (Fig. 6). The
`mouse is restrained as shown in Fig. 1A, and the hendler’s
`wrist is rotated until the mouse’s head and body are tilted in a
`downward direction, allewing the mouse‘s abdominal viscera
`tn shift cranielly. The needle (23- tn Zn-gauge ‘xfi—‘fz—inch) is
`then inserted through the skin slightly medial tn the flank and
`cranial to the inguinal canal, advanced cranially through suh-
`cutaneous tissue for 2—3 mm, and then inserted through the
`abdominal muscles. Care should be taken to amid penetrating
`the prepntial glands in the male mouse. ’l‘he needle and syringe
`should be held parallel
`to the mouse‘s vertebral column in
`order to amid accidental retroperltoneal or intrarenal injection.
`Further snurees of errer using intraperitoneal injections have
`been outlined by Lewis er al. U966} and Hamilton e: nz’.
`{196?}.
`
`H.
`
`Intrathoracic Injection
`
`Unless experimental nbjecrires mandate intratheraeic injec-
`tion, intraperitoneal injection is preferred because it is easier
`and less lineardous (no risk of pneumothorax or punctured
`lungs), and absorption rates are similar.
`lntrathoracie lnjeo
`lions, if necessary are made at approximately the midpoint of
`the chest using a slightly bent Vadneh LEE-gauge needle inserted
`at an angle between the ribs {Simmons and Brick,
`l93’G).
`
`I.
`
`Intravesenlar Injectiun and Cannulation
`
`1.
`
`Intraarterial Injection
`
`In certain procedures such as angiography, intrnarterial in‘
`jection may be necessary.
`lnjectinn into the femoral artery
`
`using a Vzwineh 24—gauge needle has been described (Simmons
`and Bricle
`[9?0}. and techniques for carotid cannulation
`(McMaster; [941; Sugnuo and Nnmura, l963a) can be adapted
`for intraarterial
`injection. To assure intraarterial
`intention,
`anesthesia is performed and the artery is exposed through a
`skin incision.
`
`2.
`
`Intravenous Injection
`
`lhe lateral or dorsal tell veins are the usual sites for intra—
`
`venous injection in mine (Grim, 19nd) (Fig. 6}. Devices to
`restrain mice for tail vein injections are described by Crlspens
`and Kaliss £3961), Champlin and McGill
`(196?). Boggs.
`{1938}. hillings {1967}? Pruner and Mellett (£913), Ltlksse~
`wycz {19?6}, Mylrea and Abbrecht {196?}, and Nickson and
`Barhulis 0948). Tail vein injection is easier if the veins are
`dilated by warming the tail for 5_ 10 sec. in ajar of warm water
`{Barrow 1968}. or warming the mouse for SWIS min. in a jar
`heated by a dfiwlOf} W light hull) {Simmons and Brick. l9'llll.
`If necessary, tourniquets devised fro n a wound clip applicator
`(Bez‘gstriim, 197%} or a hypodermic syringe and thread {Mime
`sian. 1980) can be used to ecclude t2 il veins. In albino or gray
`mice. the tail veins are visualized as thin red-blue lines cents
`
`ing along the top (dorsal tail vein) and bottom {ventral tail
`vein). Tail vein injection of mice wi h pigmented tails is mere
`difficult than injection ol‘ mice wit nonpigmented tails. Se—
`pending upon the‘sizc of the mouse a 26« In 3ll~gauge 1/53“ to {/2—
`inch needle is used. Other sites for intravenous injections ini
`clude the external jugular vein {K‘ssel and Leuitau.
`l953l.
`dorsal metatarsal vein {Nnhunaga at at, 1966), sublingual
`vein {Wayni‘orth and Perkin. 1969), and ophthalmic: plexus
`{Pinkerton and Webber, 1964). Strgicul exposure of these
`veins is not required.
`
`
`
`3. Vascular Cannulatinn
`
`The dorsal tail vein has been the usual site for intrareuuus
`
`l980; Moran and Strauss,
`cannulation in mice {Cdnnér et 51].,
`1980; Rhodes and Patterson. 19759); depending upon the reel -
`ique, anesthesia may or may not be required. Tail rein can»
`wins should be protected by bandages or splints.
`The jugular rein is also aceessihle for intravenous cannula»
`ion after the "mouse is anesthetized and placed in dorsal or
`dorsolateral tecumbancg. After the skin is prepared for sur-
`gery, n l~crn paratnedian incision is made from the mauuhrium
`o the rami of the mandihle. The eaudomedial edge of the
`arotid salivary gland is dissected free, exposing the jugular
`vein and its fascial sheath. Incision of the fascial sheath ex.—
`
`oses the jugular vein. The cannula can be inserted or direct
`injectinns can he made into the jugular vein using a 30«gauge
`needle. The volume injected should not exceed 0.1 to 0.2 ml.
`Dostainjection hernerrhage is controlled by gently compressing
`he jugular vein with the end ol‘ the salivary gland as the needle
`is withdrawn from the vein.
`
`
`
`InnoPharma Exhibit 1100.0009
`
`

`

`4708
`
`TERRIE L. CUNLIFFE-BEAMER
`
`Procedures for cannulation of the common carotid artery
`have been described by McMsster (19413 and Sugano and
`Nomurs {1963a). After insertion, chronic carotid or jugular
`cannulas are routed subcutaneously across the lateral surface of
`the neck and exteriorized on the dorsal inidline between dorsal
`
`borders of the scapulac. The abdominal aorta can be cannu-
`lated using the technique described by Weeks and Jones
`{1960). Exteriorized cannulss should be protected by a light
`weight body bandage. In some instances, a stanchion-like cage
`may be advised.
`
`J. Medication of Neonatal Mice
`
`Medication of neonatal mice is complicated because of their
`small size and the dsm’s tendency to reject or cannibalize
`offspring that have been handled excessiyely, Up to 0.1 ml
`may be administered orally through a piece of plastic tubing
`inserted over a 3G~gauge needle. Subcutaneous injections of
`approximately 0.1 ml can be made over the neck and shoulders
`using a Exit-inch 30-geuge needle (Gibson and Becker; 1967’).
`leakage from intrsperitoneul injections (Oflfiwfll ml) is ntiniu
`mined if the l/Sw to iii—inch 30—gauge needle is inserted into the
`skin parallel to the right femoral vessels and advanced sub»
`cuteneously until the lower right abdominal muscles are pene-
`trated. intravenous injection of the neonatal mouse is difficult.
`Several authors have recommended the anterior facial yein at
`
`the level of the lateral canthus of the eye (Anderson or at,
`1959: Barnes et (33.. l963; Billingham and Brent, 1956} or the
`transverse (sigmoid) sinus (Barnes er m2,
`l963). The latter
`injection site may be used until the mice reach lS—ZG 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 Postnikova (1960}. lntrscraninl injection of neonates has
`been described previously (Section RE}.
`The best defense against rejection or cannibalism of experi~
`mentally manipulated newborn mice is gentle handling of both
`neonate and dnm.
`It
`is also helpful
`to select multipnrous
`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
`handled. Plastic gloves should be worn or an odor~rnusl<ing
`agent {perfume} may he placed on the dsm’s nose and on the
`neonates to prevent them from acquiring or recognizing human
`scent. After injection or surgical manipulation any extravsu
`sated blood is removed and the neonates are returned to their
`
`nest. East and Parrott {1962) described several surgical and
`postsurgical procedures for neonatal mice. Additional sug-
`gestions made by Libbin and Person {1979} for neonatal rat
`surgery can be applied to the mouse.
`
`IV. COLLECTION OF BIOLOGICAL SPECIMENS
`
`Sections ‘v’l, Vll, and Vlll should be consulted before at-
`
`tempting some of the more complex procedures described
`below.
`
`A. Bile
`
`Chronic cannulatinn of the bile duct of mice has been de-
`
`in detail, by Becker and Plan (196?). Adoption ol‘
`scribed,
`routine liver function tests for use in mice was described by
`Anonymous {l962} and by Casals and Olltsky (1946).
`
`B. Blood
`
`Many techniques for collecting large or small amounts of
`blood from mice have been developed.
`
`1. Orbital Sinus
`
`lv’enous blood can be easily obtained from the orbital sinus.
`The mouse is placed on a table or cage lid in lateral recumben—
`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 from side to side (Fig.
`'3}. The eye is not danianged
`because the tube passes under the eye. Reluctant blood llow
`can be improved by raising or lowering the tube. This tech—
`niquc is usually performed on anesthetized mice.
`After the required amount of blood is obtained, the tube is
`withdrawn and bleeding sunlly ceases. If necessary, hemor~
`rhage can be controlled by direct pressure applied over the
`eyelids. Small amounts o“ blood (39-80 ul) can be obtained
`from orbital sinuses of nice as young as 14—16 days of age.
`Larger amounts of blood {0.5 ml) can be obtained from orbital
`sinues of older mice if tubes containing anticoagulant are used.
`Orbital bleeding can be re entcd within hours if the amount of
`blood removed at any one time is relatively small An alterna-
`tive 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 ennthus. This method provides
`less control over sudden iovcments 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). Riley
`{ lgfillli Stone (1954) and Simmons and Brick (19’303.
`
`InnoPharma Exhibit 1100.0010
`
`

`

`£3. BIQMETHQDOLQQY AND SURGICAL TECHNiQUES
`
`469
`
`2. Tail Veins and Arteries
`
`’l‘ail veins; and arteries may also he used as sources of blood.
`Tail bleeding is facilitated by immersing the rail in warm water
`or warming the mouse for 5—H} min in a cage covered by a
`goose neck lamp with a 50~100 W light bulb. Hepztrinization
`of the mouse prior to tail bleeding also increases the yield of
`hlooacl {Lewis e! 523., 1936}. One technique involves amputating
`the lip of the rail of an anesthetized mouse with a scalpel blade
`(Stoltz and Bentlall, 19%). Another technique involves; incis-
`ing the skin and ventral artery and veins; of the rail rtpprox~
`imately 0.5—2 em from the base of the tail with a razor blade
`(Fields and Cunningham,
`IETNJ; Lewis. er (15., 19%). One-half
`to 1 ml blood can be obtained using this; technique. Small
`amounts of blood can be aspirated from tail veins following
`insertion of 30-gauge needle attached to 0.5- to 1.0-ml syringe
`(Gl‘ice, 3964}. The latter technique is; time~eonsuming com»
`pared to previously described methods.
`Blood samples obtained from the orbital sinus and the tail are
`significantly different with respect to hematoeril and red and
`white blood cell counts but are not Significantly different with
`respect to differential leukocyte count or polychromntic red
`blood cells. Less sample to sample variation in the above hern-
`atologienl parameters is observed in blood obtained from the
`orbital sinus compared to blood from the tail (Sakaki :3: 03..
`1961).
`
`3.
`
`Jugular Vein
`
`Unadulrel‘ated venous bloorl can he obtained from the ingulnr
`vein by modifying the jugular injection teehnique of Kassel
`and Levitan {1953), or by surgically exposing and severing the
`jugular vein {Ambrnn :2! (£23., 1951).
`
`4. Abdominal Aorta or Brnchial or Carotid Arteries
`
`Unadulternted arterial blood can he obtained from the ab—
`
`dominal aorta {Lushhough and Moline, 1961), hraehial emery
`(Young and Chambern, WW), or carotid artery {Ambrus :2:
`nl’., 1951). All of the above procedures require anesthesia and
`result in the death of the mouse with the possible exception of
`carotid artery bleeding as cleserihed by Ambrns er of. (1951}.
`
`5. Heart
`
`Large amounts of blood can be obtained directly from the
`heart using any of several different techniques. The technique
`described by Falahella {996?} utilizes manual restraint of the
`unanestheiized mouse and insertion of a 20-gauge needle at-
`
`InnoPharma Exhibit 1100.0011
`
`C F
`
`ig. 3’. Orbital sinus bleeding. (2!) Correct angle for ingenion oflhe micro
`hematocrit nine. (h) Incorrect angle: Mierohemntoerit lube will lnccrate the
`eye. {0)
`incorrect angle: Micrnhematoerit
`tube presses against
`the orbital
`boners am} (lees not enter the orbital ainus.
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