5.2 C HAPTER Routes of Administration Jiro Hirota, Shinya Shimizu National Institute of Animal Health, National Agriculture and Food Research Organization (NARO), Tsukuba, Japan Introduction Mice are the most widely used animals for a range of experiments including medical, chemical, pharmacological, toxicological, biological and genetic. The administration of test substances, such as chemical elements, compounds, drugs, antibodies, cells or other agents to mice is one of the major methods for evaluating their biological activity. A knowledge of available methods and techniques of administration, as well as knowl- edge of the deposition and fate of the adminis- tered substance, will help scientists to select the most appropriate route for their purpose. The administration route is largely dependent on the property of the test substance and the objective of the experiment. All administration should be performed with knowledge of the chemical and physical characteristics of the substance. All routes have both advantages and disadvantages, such as the absorption, bioavailability and metab- olism of the substance. Consideration should be given to the pH, viscosity, concentration, sterility, pyrogenicity, irritancy and toxicity, as well as the existence of hazardous substances. In addition, animal welfare must be taken into consideration in deciding on the administration route, and the route must be selected before the start of any experiment. Proper restraint is the most important tech- nique when mice are treated as this decreases stress and increases successful treatment. Personnel using experimental animals should be well trained in handling and restraint; they should be qualified in the responsible use of experi- mental animals and attain a scientifically high standard [1, 2]. Further experience will lead to repeatable and reliable results (see Chapter 5.1). The foundation of animal welfare is that during administration mice should be protected from pain, suffering, distress or lasting harmdor at least pain and distress must be kept to a minimum [1]. Some injection sites (such as footpad injection) are strongly discouraged and if required must be justified on a case-by-case The Laboratory Mouse Ó 2012 Elsevier Ltd. All rights reserved. ISBN 978-0-12-382008-2 DOI: 10.1016/B978-0-12-382008-2.00030-1 ROUTES OF ADMINISTRATION 709 PROCEDURES
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C H A P T E R
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Jiro Hirota, Shinya Shimizu
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National Institute of Animal Health, National Agricultureand Food Research Organization (NARO), Tsukuba, Japan
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IntroductionMice are themost widely used animals for a rangeof experiments including medical, chemical,pharmacological, toxicological, biological andgenetic. The administration of test substances,such as chemical elements, compounds, drugs,antibodies, cells or other agents to mice is one ofthe major methods for evaluating their biologicalactivity. A knowledge of available methods andtechniques of administration, as well as knowl-edge of the deposition and fate of the adminis-tered substance, will help scientists to select themost appropriate route for their purpose. Theadministration route is largely dependent on theproperty of the test substance and the objectiveof the experiment. All administration should beperformed with knowledge of the chemical andphysical characteristics of the substance. Allroutes have both advantages and disadvantages,such as the absorption, bioavailability and metab-olism of the substance. Consideration should be
The Laboratory Mouse� 2012 Elsevier Ltd. All rights reserved.ISBN 978-0-12-382008-2
given to the pH, viscosity, concentration, sterility,pyrogenicity, irritancy and toxicity, as well as theexistence of hazardous substances. In addition,animal welfare must be taken into considerationin deciding on the administration route, and theroute must be selected before the start of anyexperiment.
Proper restraint is the most important tech-nique when mice are treated as this decreasesstress and increases successful treatment.Personnel using experimental animals should bewell trained in handling and restraint; they shouldbe qualified in the responsible use of experi-mental animals and attain a scientifically highstandard [1, 2]. Further experience will lead torepeatable and reliable results (see Chapter 5.1).The foundation of animal welfare is that duringadministration mice should be protected frompain, suffering, distress or lasting harmdorat least pain and distress must be kept toa minimum [1]. Some injection sites (such asfootpad injection) are strongly discouraged andif required must be justified on a case-by-case
DOI: 10.1016/B978-0-12-382008-2.00030-1
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Figure 5.2.1 Manual restraint of a mouse using both hands. (a) The mouse is placed on the cage lid with thepreferred hand. The hand pulls the tail gently back. (b) The mouse is quickly and firmly picked up by the scruffof the neck behind ears with thumb and index finger of the other hand. (c) The tail is transferred from thepreferred hand to between palm and little or ring finger of the other hand, then held firmly. (d) The mouse isrestrained.
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basis [3]. The regulation of administration sites,methods, kind of substances and amount to beadministered all have to be reconciled with theanimal welfare board and permission has to beobtained.
Principles ofadministrationHandling and restraintGood handling and restraint is themost importanttechnique for correct administration. Properrestraint leads to successful administration andvaries with the routes of administration. Dispos-able gloves must be worn, as manual restraint isfrequently used for injections. There are two stylesof manual restraint, one using both hands and theother single-handed (see Chapter 5.1; [4, 5]).
Two-handed manual restraint
The mouse is lifted by the base of the tail andplaced on the cage lid or other solid surfacewith one hand and then its tail is pulled gently
back (Figure 5.2.1a). It is then quickly and firmlypicked up by the scruff of the neck behind theears with the thumb and index finger of theother hand (Figure 5.2.1b). The tail is transferredfrom the first hand to between the palm and littleor ring finger of the other hand, then fixed(Figure 5.2.1c). The mouse is now restrained(Figure 5.2.1d).
Single-handed restraint
The tail is picked up using thumb and indexfinger of the chosen hand (Figure 5.2.2a), thenthe mouse is placed on the cage lid or other solidsurface (Figure 5.2.2b). The tail is immediatelygrasped by the palm and middle finger, ringfinger and/or little finger, and the thumb andindex finger released (Figure 5.2.2c). The foldof skin from the scruff of neck down the backis immediately gripped using the thumb andindex finger (Figure 5.2.2d, e). The mouse isthen restrained (Figure 5.2.2f).
To prevent kicking by the hind legs, the tail isfixed using the palm and index finger and thenthe left hind leg is held firmly between the ringand little finger (where the mouse is restrainedby the left hand) (Figure 5.2.3).
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Figure 5.2.2 Single-handed restraint of the mouse. (a) The tail is picked up using thumb and index finger ofpreferred hand. (b) The mouse is placed on the cage lid or other solid surface pulling gently back by the hand.(c) The tail is immediately grasped by the palm and middle finger, ring finger and/or little finger and then thetail held between thumb and index finger is released. (d) and (e) The fold of skin from the scuff of the neckdown the back is immediately gripped using the thumb and index finger. (f) The mouse is restrained.
Figure 5.2.3 Manual restraint of a mouse to prevent kicks from hind leg. The tail is held using the palm andindex finger and then left hind leg is fixed between the ring and little finger (when the mouse is restrained bythe left hand).
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Site of administrationAmong several possibilities for the administra-tion of substances to mice, the most commonare subcutaneous, intraperitoneal or intrave-nous injection. Intramuscular administration isnot recommended, as the muscle of the mouseis too small. Some sites, such as footpad injectionof Freund’s complete adjuvant, intrasplenicinjection and intralymph node injection areunacceptable nowadays [3], and should berestricted to cases where they are absolutelynecessary.
Preparation of the site
The area for administration is clipped(Figure 5.2.4) or cleaned with warm water ifnecessary before cleaning the skin with cottonwool moistened with alcohol or disinfectant.Where aseptic skin is necessary the fur must beclipped, followed by a three-stage surgical prepa-ration: surgical soap, alcoholic rinse and surgicalpreparation solution. The skin is dried immedi-ately before administration [3]. In some casesa local anaesthetic may be applied first to preventpain.
Preparation, solubility andsafety of solutionsTest substances, solutions and equipment shouldbe prepared aseptically and free from pyrogens,especially for parenteral injections. Solutionscan be sterilized by filtration (0.22 mm). Livingorganisms or cells must be free from contami-nants when administered. The toxicity of thesubstance, the volume and the route of adminis-tration should be considered to prevent tissue
Figure 5.2.4 Clipping of hair on the b
damage and to give precise dosage. Thefollowing solvents or vehicles have been foundsuitable in most instances and do not greatlyaffect drug action because of their own inherentproperties:
• water• water with 0.85% sodium chloride• water with up to 50% polyethylene glycol• water with not over 10% Tween 80• water with up to 0.25% methylcellulose or
carboxymethylcellulose• corn oil, vegetable oil or peanut oil (oral and
intramuscular route only).
A small percentage of the lower alcohols,glycols and acetone can also be used, providedthe volume administered is kept small [6]. Phos-phate buffered saline (PBS) or various culturemedia are also suitable vehicles [2]. Lipid-solublesubstances can only be dissolved in oil but thisdelays absorption. Oil-soluble drugs have beensuccessfully given intravenously in 15% oil–wateremulsions using lecithin as an emulsifier [6].Until experience indicates otherwise, solutionsor suspensions should be prepared as near to thetime of use as possible because some substanceswill deteriorate in solution within a few hours[6]. When administering drugs, the solvent shouldideally be the same as the one in which the drug isnormally formulated [2]. Although distilled watercan be used under certain conditions, saline ispreferable because water for injections injectedsubcutaneously causes pain and intravenousinjection produces haemolysis. Oil and viscousfluids cannot be injected intravenously [2]. If sus-pended material is to be used for intravenousinjection, the particles should be removed byfiltration to prevent embolism [6]. The tempera-ture of fluids must be raised at least to room
temperature or better still up to body tempera-ture before use, because the injection of coldfluids is painful [7].
Intravenous cell injection has been per-formed in many experiments, such as immuno-logical or cancer research. In these cases, cellshave to be suspended singly in solution becausecell clumps cause embolism, and in some casesthe mice would die. Consequently, cell suspen-sions for injection should be filtered using a cellstrainer just before administration (Figure 5.2.5).Meanwhile, preintravenous administration ofheparin before tumour cell injection is reportedto be an effective method to decrease mortalitycaused by thromboembolism [8].
TABLE 5.2.1: Guidelines for maximal administra-tion volumes and needle size
Administrationroute
Maximaladministrationvolumes (mL) Needle size
Oral 0.2 22G
Subcutaneous 2e3 (scruff)0.2 (inguinal)
25G
Intraperitoneal 2e3 23G
Intravenous 0.2 25G
Intradermal 0.05 26G
Intramuscular 0.05 25e27G
Intracerebral <0.03 27G
Intranasal <0.02
Source: Flecknell [10], Reeves et al. [11], Wolfensohn andLloyd [12].
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Concentration of substances
The concentration can vary over a fairly widerange without greatly influencing the end resultof the experiment. Lower concentrations areclearly desirable [9]. Factors limiting the use ofaqueous solutions for parenteral administrationare probably related to their osmotic pressure.Low concentrations can be corrected by the addi-tion of sodium chloride but ought not to be sohigh as to materially exceed the osmotic pressureof 0.15M sodium chloride [6]. Highly concen-trated solutions can be administered intrave-nously provided the rate of injection is keptslow and precautions are taken to avoid gettingthe solution outside the vein.
pH of the injected solutionFor most administration routes, providing thesolutions are not highly buffered, a pH rangeof 4.5–8.0 is satisfactory. For oral administrationa pH as low as 3 can be tolerated, but alkalinesolutions are very poorly tolerated. A ratherwide range of pH is indicated for intravenousadministration, because of the buffering effectof blood and dilution by blood flow followinguse of the intramuscular and subcutaneousroutes. When solutions of low or high pH areinjected intravenously the injection rate is keptslow and again precautions are taken to avoidgetting solution outside the vein [6].
Volume and frequencyof administrationThe injection volume is limited by any toxicity ofthe substance and by the size of the mouse. Itshould be kept as small as possible. Excess volumesof solution can startle the animal. The frequencyof administration should be kept to a minimum,to avoid unnecessary stress. If solutions are admin-istered intravenously, haemodynamic changesand pulmonary oedema may occur, while veryrapid injections can produce cardiovascularfailure and be lethal [2]. Maximum volumes areshown in Table 5.2.1 [10–12]. For immunization,the maximum is lower still, because of the mixing
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with adjuvant. Maximum volumes for injection ofantigen with or without adjuvant per route areindicated in the section on ‘Immunization ofmice’ in this chapter.
Rate of absorption anddistribution of administeredsubstancesThe blood flow to the site of administration, thenature of the substance and its concentrationinfluence the rate of absorption [2, 12]. Thetime-course of the effect of the substance is animportant factor in determining the dosage andis influenced by the rate of absorption [9]. Nor-mally, injected substances must be absorbedfrom the site of administration into the blood.Therefore, the rate of absorption will be deter-mined by the size of the absorbing surface, theblood flow and the solubility of the substance inthe tissue fluid. The rate of absorption is alsoinfluenced by lipid solubility, physicochemicalproperties, degree of ionization and molecular
size of the substance [2]. Compounds that arehighly soluble in the body fluids, will be absorbedquickly. Substances that are ionized and are notlipid soluble can only be absorbed if a specificcarrier exists. In general, the rate of absorptionis in the following order [12]:
iv > ip > im > sc > po:
Needles andsyringesUsually, 26–27G, 12.5–15.6mm (1/2 to 5/8 inch)needles are satisfactory for injection. The smallestgauge should be selected, as a fine needle preventsleakage of fluids and will help to minimizediscomfort to the animal [2]. A 1–2mL syringe isadequate for most injections. When a smallvolume (<1.0mL) is administered, an insulinsyringe plus needle is convenient (Figures 5.2.6and 5.2.7; 27–30G, 8.0–12.5mm (5/16 to 1/2 inch)).
These syringes can be obtained from variouscompanies (e.g. Terumo, Japan; Becton Dickinson,USA). Intradermal needles are practical for intra-cerebral injections (Figure 5.2.8; Top, Tokyo,Japan). Plastic syringes cannot be used withsolvents such as acetone.
The withdrawal of hazardous substancesfrom bottles requires great care. An alcohol-moistened cotton pledget can be kept at the pointwhere the needle enters the stopper in order tominimize the inadvertent formation of aerosols
[13]. Because of the risk of embolism, air bubblesin fluid, syringe and needle must be purged.Gently tapping the side of the syringe and slowlyexpelling the air into absorbent tissue to preventany dispersal of the contents until fluid appearsat the tip of the needle can purge air bubbles.
The needle size will vary with the viscosity ofthe substance being used; the greater theviscosity, the larger the needle required [11]. Ifblood or body fluid flows back into the needle,it must be discarded and a fresh attempt made.
EnteraladministrationEnteral administration has the advantages that itis possible to give quite large amounts of non-sterile substances or solution and that a pH aslow as 3 can be administered by this route. Onthe other hand, alkaline solutions are very poorlytolerated by this route [6]. When using the oralroute it should be understood that substancescan be destroyed by the gastric juices and thatthe food content of the stomach influences bothrate and order of the gastric emptying. The rateof absorption is markedly influenced by its timeof residence in the stomach and is also directlyrelated to the rate at which substances are passedfrom the stomach into the intestine [14]. Enzymesof the host and microflora of the digestive tractcan also metabolize the substance. On the otherhand, some insoluble substances may becomesolubilized as the result of enzymatic activityduring their passage through the stomach andintestine, making absorption possible [2]. Thetwo major methods for enteral administrationare mixing the substance with food or water ordirect administration using gavage. Rectal admin-istration is also possible [6].
Oral administrationThe simplest method for administration is givingthe substance with food or drinking water.However, this is not practicable with substancesthat are unpalatable, insoluble or chemicallyunstable in drinking water or when they irritatethe mucosa of the gastrointestinal tract [2]. The
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daily food and water intake of the mice should beknown before the experiment, to calculate thequantity of substance to be added. Becausewastage of food and water happens all the time,it is difficult to determine the precise amountof food and water intake and therefore theprecise intake of the substance. The only waythis can be done is by keeping mice in metaboliccages and recording the wastage.
Intragastric administrationDirect administration by oral gavage is preferredto mixing substances with food or drinking waterbecause the intake of the substances is preciselymeasured. A ball-tip needle is used to preventdamaging the oesophagus or passing through theglottal opening into the trachea (Figure 5.2.9). A22G ball-tip needle is suitable for administrationto adult mice and can be obtained from supplierssuch as Cadence Inc., or Fine Science Tools Inc.The conscious mouse is manually restrained byfirmly gripping a fold of skin from the scruff ofthe neck down the back (Figure 5.2.10a); immobili-zation of the head is essential for this procedure(Figure 5.2.10b). When the neck is extended, theposition is vertical and there is a straight linefrom the mouth to the cardiac sphincter throughtheoesophageal orifice (Figure5.2.10b).Theneedleis passed gently through the mouth and pharynxinto the oesophagus (Figure 5.2.10c). The mouseusually swallows as the feeding needle approachesthe pharynx, and these swallowingmovements canhelp the probe to slip through the oesophagealopening. The substance is then administeredslowly. If any obstruction is felt, if the mouse
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Figure 5.2.9 Syringe with a gavage needle. (a) 1.0 mLsyringe with 22G � 1.0 in. feeding needle; (b) 1.0 mLsyringe with 22G � 11/2 in. feeding needle; (c) 1.0 mLsyringe with 20G� 11/2 in. disposable feeding needle.
coughs, chokes or begins to struggle vigorouslyafter the gavage begins, or if fluid is seen comingout of the nose, it may indicate that the needlehas entered the lungs. Any of these signs necessi-tates immediate withdrawal of the needle,and the mouse must be observed very carefully.If there is any sign that fluid has got into thelungs, the mouse should be euthanized. As soonas administration is finished, the needle must bewithdrawn [5, 15]. A volume of less than 0.2mL isrecommended.
ParenteraladministrationAdministration of substances to the body otherthan via the alimentary canal includes injection,infusion, topical application, inhalation andimplantation of an osmotic pump ora controlled-release drug delivery pellet. Smallamounts of solution are injected, and largevolumes are infused. In both cases a needlemust penetrate the skin. Subcutaneous, intraperi-toneal and intravenous administration are themost common and important routes to injectsubstances in solution or suspension into themouse. The rate of absorption is dependent onthe administration route. Following intravenousinjection the substance will disperse immediately;this route therefore achieves the most rapidabsorption. The large surface area of the abdom-inal cavity and its abundant blood supply alsofacilitate rapid absorption; absorption from thisroute is usually 25–50% as rapid as that fromthe intravenous route [6].
SubcutaneousadministrationSubcutaneous administration is easy. As it is rarelypainful [12], a consciousmouse can usually be used.The rate of absorption is lower than with intraper-itoneal or intramuscular injections [16]. Subcuta-neous injections are made into the loose skin overthe interscapular (Figure 5.2.11a) or inguinal area(Figure 5.2.11b). Subcutaneous administration
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Figure 5.2.10 Procedure for intragastric administration using ball-tip needle. (a) Before extending mouse’sneck; (b) A straight line is formed between mouth and stomach; (c) Intragastric injection is made using 1.0mLsyringe with 22G� 1.0 in. feeding needle.
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over the interscapular area is performedas follows.Themouse is manually restrained and then placedon a clean towel or solid surface. The needle isinserted under the skin of the interscapular areatented by the thumb and index finger and thesubstance is then injected. A volume of less than3mL is recommended. Subcutaneous administra-tion over the inguinal area is done as follows. Themouse is restrained manually and the head tilteddownwards. Holding the hind leg firmly helpsthis procedure (Figure 5.2.3). The needle is insertedinto the lower left or right quadrant of theabdomen, avoiding the abdominal midline, andthe substance is injected. A volume of less than0.2mL per site is recommended. To minimize
leakage, the needle should be advanced severalmillimetres through the subcutaneous tissue [5, 15].
IntraperitonealadministrationThis is the most common route, being technicallysimple and easy. It allows quite long periods ofabsorption from the repository site. The rate ofabsorption by this route is usually 25–50% asrapid as by the intravenous route [6]. Its limita-tions are the sensitivity of the tissue to irritating
(b)
injection at the base of a fold of loose skin (area at thebcutaneous injection at the lower left quadrant using
Figure 5.2.12 Intraperitoneal injection to lower leftquadrant using insulin syringe: 27G � 1/2 in., 1.0mL.
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substances and less tolerance to solutions of non-physiological pH. Solutions should be isotonicand quite large volumes can be administeredvia this route.
The conscious mouse is manually restrained[16] and held in a supine position with its posteriorend slightly elevated, or the head can be tiltedlower than the body (Figure 5.2.12). The needleand syringe should be kept almost parallel tothe mouse’s vertebral column in order to avoidaccidental penetration of the viscera [17]. Theneedle is pushed in at an approximately 10� anglebetween the needle and the abdominal surface inthe lower left quadrant of the abdomen [16]. Toavoid leakage from the puncture point, the nee-dle is run through subcutaneous tissue in a cranialdirection for 2–3mm and then inserted throughthe abdominal wall [15]. The recommendedvolume is less than 2.0 mL for a 40 g mouse.
IntravenousadministrationIntravenous injection has advantages over otherroutes. There is one primary route, the tail vein;another possibility is the ophthalmic plexus route.Solutions that are highly concentrated, high orlow pH, or irritating, can be administered intrave-nously provided that the rate of injection is keptslow and precautions are taken to avoid gettingthe solution outside the vein. Compounds that arepoorlyabsorbedby thedigestive tractmaybegivenintravenously but intravenous administrations
require technical expertise and skill. The syringeplus needle or the catheter must first be filledwith the solution to remove air bubbles. Adminis-tration is usually into the lateral tail vein, not thedorsal tail vein (Figure 5.2.13a), as it is not straight.The lateral veins are readily visualized, but arequite small in diameter. If anaesthesia is not used,a restraining device is usually necessary (seeChapter 5.1; [5, 11, 18]).
Themouse is either placed in the restrainer oranaesthetized and the tail is then warmed witha lamp orwarm towel, or immersed inwarmwater(40–45 �C) in order to dilate the vessels [10]. The tailis swabbed with 70% alcohol on a gauze sponge orswab.Theneedle is insertedparallel to the tail vein,penetrating 2–4mm into the lumen,while keepingthe bevel of the needle face upwards(Figure5.2.13b).The solution is then injected slowlyand no resistance should be felt if the solution isproperly administered (Figure 5.2.13c). Theinjected solution temporarily replaces the blood,but should then be washed away by the bloodstream. If this does not happen, the position ofthe needle is certainly not in the vein but in thesurrounding tissue, so it must be either moved inthe surrounding tissue in such a way that it thenenters the vein, or a new attempt must be made.When the intravenous administration is finishedor the cannula is pulled out, the injection sitemust be pressed firmly with a swab or fingers toprevent backflow of the administered solutionand/or blood [2, 5]. If the same vein must be usedseveral times the first administration should bemade as distal as possible in relation to the heartand subsequent administrations should be placedprogressively more proximally. Because vene-puncture and the administration of substancescan damage and/or block the vein, the distal partof the vein may no longer be used [2]. The recom-mended volume is less than 0.2mL.
The ophthalmic plexus route is also used forfluid administration [19, 20], but this method ofapplication is controversial and is under discussionor even forbidden in various countries for reasonsof animal welfare. This route may be suitable incaseof emergency for rescuing amouse that showssigns of anaphylactic shock. Details of the tech-nique are described in the section on ‘Rescuefrom anaphylactics’ later in this chapter.
Other routes for intravenous administrationvia the external jugular vein [21], the dorsal
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Figure 5.2.13 Intravenous administration. (a) Transverse section view of the mouse tail; (b) Sagittal view of themouse tail (the tail is turned 90�); (c) Intravenous injection to lateral tail vein of an anaesthetized mouse usingan insulin syringe: 27G � 1/2 in., 1.0mL.
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metatarsal vein [22] and the sublingual vein [23]have been reported.
IntramuscularadministrationThe intramuscular route should usually beavoided, as mouse muscles are small. If necessary,
Figure 5.2.14 Intramuscular injection into the legmuscle.
injections may be given into the thigh musclewith injection volumes of less than 0.05mL. Thetip of the needle should be directed away fromthe femur and sciatic nerve (Figure 5.2.14). Themouse is anaesthetized or is manually restrainedby another person. The needle tip is insertedthrough the skin and into the muscle and aspi-rated briefly with the syringe before injection.If there is backflow of blood or body fluid, theprocedure should be stopped; the needle mustbe moved or a fresh attempt must be made.Good technique and restraint are necessary andthis method should only be performed by well-trained personnel [2, 4, 6, 15].
IntradermaladministrationThis route is not recommended in general andshould be restricted to cases of absolute necessity[3, 24]. It is a very difficult route in the mousebecause of the animal’s very thin skin. A fine
Figure 5.2.15 Intradermal injection into the skin ofthe back.
Figure 5.2.17 Stereotaxic alignment system (Model940, David Kopf Instruments).
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needle (29Gor less) is recommended. Themouse isanaesthetized, the fur clipped or hair removedfrom an area on the back, ventral abdomen, orhind footpad, which is wiped with 70% ethanolon a gauze sponge or swab. The skin is held tautwith thumb and index finger and the needleinserted, bevel up and at a shallow angle, just underthe superficial layer of epidermis. The volumeshould be less than 0.05mL per site. Resistanceshould be felt both as the needle is advanced andas the compound is injected. A hard bleb will beseen upon successful intradermal injection ofeven a small quantity of fluid (Figure 5.2.15) [5]. Ifmultiple sites are injected, adequate separation isnecessary to prevent coalescing of lesions.
IntracerebraladministrationFor this procedure the mouse is anaesthetized andthen restrainedmanually on a solid surface [25, 26].
Ear line Eye line (a) (b
Figure 5.2.16 Intracerebral injection. (a) Injection site ofinjection into an anaesthetized mouse using an intrade
The site of injection is approximately half waybetween the eye and ear and just off the midline(Figure 5.2.16a). The recommended maximumvolume per suckling mouse is 0.01mL and thatfor weanling or older mice is up to 0.03mL. Theneedle directly pierces the cranium(Figure 5.2.16b). An intradermal needle(Figure 5.2.8) is convenient in order to prevent theneedle from extending too deeply into the brain.
To inject into a specific region of brain, micehave to be restrained using a small animal stereo-taxic instrument (Figure 5.2.17), as supplied forexample by David Kopf Instruments, NARISH-IGE or Stoelting. The injection region is decidedwith reference to a brain atlas [27]. This atlas
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the head for intracerebral injection; (b) Intracerebralrmal needle.
relates to the adult male C56BL/6J mouse, there-fore some experiment is needed if other mousestrains are to be used.
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IntrathoracicadministrationIntrathoracic injection is restricted to specialexperiments. It can bemade inmice with a slightlybent or curved needle, which should be insertedbetween the ribs at approximately the midpointof the rib cage. Caution must be taken to insertthe needle at an angle, thus preventing injectiondirectly into lung tissue. The speed of absorptionis similar to the intraperitoneal route [16].
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IntranasaladministrationThis is usually done with the mouse lightly anaes-thetized. The animal is manually restrained andthe tail anchored between the small finger andthe palm [16]. The mouse is held in a supine posi-tion with the head elevated. The end of themicropipette is placed at or in the external nares,and then the solution is poured in slowly(Figure 5.2.18) [25, 28, 29]. The volume should beless than 0.02mL. Excess volume or rapid injec-tion will induce suffocation and death.
Figure 5.2.18 Intranasal instillation into an anaes-thetized mouse using a pipette (Gilson P-20).
Topical applicationIt is not often realized that the skin is the largestorgan of the body and survival depends on itspatency perhapsmore than formost other organs.An animal (or human) can survive with onlyabout one-seventh of its liver or one-fourth ofits kidney functioning, but destruction of morethan 50% of the skin usually results in death [6].The skin is also a convenient site for the adminis-tration of drugs. Numerous factors, such as thephysicochemical properties of the substance, theattributes of the vehicle and the permeability ofthe skin, can affect the degree of percutaneousabsorption [30, 31]. The ability of a substance tobe absorbed through the skin and enter thesystemic circulation is determined by its abilityto partition into both lipid and aqueous phases [2].
The usual site is the skin of the back or theabdomen. After clipping the hair for topicaladministration (Figure 5.2.4), the hairless areashould be cleaned of any fat and grease andother debris. The substance to be administeredshould be dissolved in a volatile solvent or mixedin a suitable cream before application and thenapplied with a dropper or smeared onto theskin with a swab [2]. Some precautions are usuallynecessary to prevent the animal from licking orscratching the application sites [6].
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InhalationThis route is used for experiments on asthma, airpollution or respiration [32, 33]. The inhalationroute is, incidentally, the most akin to an intrave-nous injection because of the relatively largearea presented for absorption by a membranethat is separated from the blood by only one ortwo cell layers. Consequently, absorption of gasesand aerosols that reach the alveoli is virtuallycomplete. The greatest problems surroundingthe use of the inhalation route are the generationof a suitable aerosol of the test substance, if it is notsufficiently volatile, a constant and suitable airlevel of thematerial under study and the determi-nation of the dosage given. Particle sizes that aretoo small or too largearenot suitable; it is generallybelieved that a particle size of 0.5–2.0 mmdiameter
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is optimum [6]. Equipment is available to purchasee.g. from Omuron or Buxco Electronics.
Other routesOther routes of administration have beenreported such as intra-arterial administrationusing the femoral artery [16] or the carotid artery[34], intrathymic injection [4], intraspinal injec-tion [35], intrathecal injection [36] or intracardiacinjection [8].
The dosing and treatment of newborn miceprovides special problems not only because oftheir size or but also because the dam is apt toreject or cannibalize neonates that have beenhandled. Subcutaneous injections can be madeover the neck and shoulders using a less than30G � 5/16 inch needle. Up to 0.1mL (dependingon the age of the infant mice) may be adminis-tered orally using a piece of plastic tubing insertedover a needle [15, 37]. Direct injection into thestomach of infant mice can be made throughthe abdominal wall [38]. Intravenous injectioninto infant mice has also been reported [15, 39, 40].
Implantable pumps,controlled-releasedrug delivery pelletsand cannulasThe delivery of substances at a slow, steady rateover a period of days, weeks or months withoutthe need for external connection or frequentanimal handling can be accomplished by usingan osmotic pump or controlled-release drugdelivery pellets.
Osmotic pumps (ALZET pumps) can be usedfor systemic administration when implantedsubcutaneously or intraperitoneally, or can beattached to a catheter for intravenous, intracere-bral or intra-arterial infusion. The pumps havebeen used to target delivery to a wide variety ofsites including the spinal cord, spleen, liver, organor tissue transplants and wound healing sites.ALZET pumps are supplied by DURECT.
Controlled-release drug delivery pelletseffectively and continuously release the activeproduct into the animal. The pellets are intendedfor, although not limited to, simple subcutaneousimplantation in laboratory animals. Pellets areavailable from Innovative Research of America.The dosage can be selected (from 1 mg to 200mgper pellet), and the release period is also select-able (21, 60 or 90 days).
Implantable cannulas permit continuousaccess to the venous or arterial system for eitherintravenous substance administration or bloodwithdrawal. Using strict aseptic techniques, thecannula is inserted into a vein or artery (thefemoral vessels, jugular vein and carotid arteryare common sites) and secured in place. The otherend of the cannula is attached to a small port thatis secured in a subcutaneous location, most oftenover the shoulders [5]. See Desjardins [41] formore information on implantable cannulas.
ImmunizationMice arenot used for theproduction ofpolyclonalantibodies because of the small amountsproduced. On the other hand, they are a goodsource of antibody-producing lymphoid cells orhybridomas [42]. Ingeneral, immunizationconsistsof two stages; primary and booster. The primaryantigen is usually injected with adjuvant. Boostersare injectedonce ormorewith orwithout adjuvantdepending on the immunogen. Footpad, intra-splenic [43] or intralymph node [44] injection isnot recommended in general. If required, theinvestigators shouldprovide scientific justificationto ethics committees for suchprotocols (suchas theneed to use valuable, unique and irreplaceableantigens, or extremely small quantities of antigen).The injectionof immunogens at thebaseof the tailor in the popliteal area substitutes for footpadinjections with much less distress to the animal,because immunogens injected into the footpadare processed by the popliteal lymph node [3, 45].The intraperitoneal route for injectionofFreund’scomplete adjuvant (FCA) is permitted in smallrodents only. FCA should be administered onlyonce, and be limited to minimal volumes of up to0.1mL. In the mouse, up to 0.1mL with adjuvantmay be administered subcutaneously in the neck
TABLE 5.2.2: Maximum volumes of antigen, with or without adjuvant, per route
Administration routeMaximum volume (mL)
With adjuvant Without adjuvant
Subcutaneous 0.1 0.5
Intramuscular Not recommended 0.05
Intraperitoneal 0.2 1
Intravenous Not recommended 0.2
Intradermal Not recommended 0.05
The intradermal route should be restricted to cases where it is absolutely necessary [3].Source: Warsson et al. [48]; Van Zutphen et al. [49].
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region. Oil-based or viscous gel adjuvants shouldnot be injected by the intramuscular route [46].The intravenous route should also not be usedfor oil-based adjuvants, viscous gel adjuvants orlarge-particle antigens, due to the risk of pulmo-nary embolism [47]. Though FCA is the strongestadjuvant, use of other adjuvants can be recom-mended. Micemust be closelymonitored immedi-ately after injection for any anaphylacticreactions, both after the primary and any boosterinjections [3]. The recommended route andvolumes are shown in Table 5.2.2.
Rescue fromanaphylaxisIn some cases, anaphylactic shock happens inhyperimmunized mice. Intravenous fluid replace-ment of isotonic solution is effective to rescuemice, but the tail veinofmice inanaphylactic shockis often collapsed by the low blood pressure. In thiscase, peripheral intravenous access via the sublin-gual vein is the preferred route [23]. Alternatively,fluid replacement may also be achieved using theretro-orbital sinus route [19, 20]. In a normal-sizedmouse, the injection of 0.5–1.0mL of warmedisotonic solution is enough for rescue. In detail,the technique resembles blood collection byretro-orbital sinus puncture (see Chapter 5.3). Themouse is manually restrained on a solid surface,being held gently but firmly by the nape of theneck. By pressing down with the thumb and fore-finger in the occipital area and pulling back theskin, thepoint of theneedle canbedirected towardthebackof theorbit at a 20–40� angle.Theneedle is
inserted medially through the conjunctiva on theinner side of the ocular cavity. If entry is blockedby bone, the needle is withdrawn slightly. Fluid isinjected slowly, loosening the neck skin slightly.Mice in anaphylactic shock are unable to maintaintheir body temperature, therefore warming themis an effective means of recovery.
AcknowledgementWe are grateful to Mr T. Fujisawa for his skil-
ful photography.
References
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