List of Experiments in Animals

8/12/2019 List of Experiments in Animals

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POST GRADUATE EXPERIMENTS IN PHARMACOLOGY

Index

No Name of Experiment Pa!e N"m#er

1. Bioassay on isolated tissues:

- Experiments in rats

- Experiments in guinea pig

- Experiments in rabbit

2 7

2-4

5-6

6-7

2. Qualitatie analysis o! drugs in inta"t

animals # biologi"al tests$

% &

'. (emonstration o! simple te")ni*ues inExperimental animals.

1+

4. (rug ,"reening met)ods 11-15

I$ %IOASSAY

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Experiment in Rat

&$ Tit'e( Rat )o'on preparation

Objective:

. o determine un/no0n "on"entration o! a"etyl")oline #")$ using rat

as"endingdes"ending "olon.

B. o determine blo"/ing e!!e"t o! atropine on isolated rat "olon.

. o determine potentiating e!!e"t o! p)ysostigmine using rat "olon.

Requirements:

nimal tissue rat as"endingdes"ending "olon

3)ysiologi"al salt solution#3,,$ erbs#$ dealon#($

eer !rontal 0riting

(rug "etyl")oline ")loride trop)in p)ysostigmine.

Procedure:

. o determine un/no0n "on"entration o! a"etyl")oline:

(Rat ascending colon)

eep t)e animal #rat$ !asting !or at least 24 )ours .)en sa"ri!i"e t)e rat by stunning #a

strong blo0$ on t)e )ead. !ter sa"ri!i"e /eep t)e animal on t)e disse"ting board

#(B$.8at s)ould be !ixed on t)e (B by tying its legs 0it) t)read.

ut open abdomen o! rat by a erti"al midline in"ision and t)en expose abdominal

is"era. 9denti!y t)e as"ending "olon t)roug) "e"um. 9denti!y t)e "e"um and t)en go

to0ards t)e re"tum at least 5-7 "m

ut small pie"e #1.5-' "m$ as per t)e inner organ bat) olume and "lean it 0it) a

lu/e0arm 0ater or 3,, 0it) t)e )elp o! a syringe.)en tie bot) t)e ends 0it) t)e )elp o!nylon or "otton t)read and tie it to t)e balan"ed )oriontal leer.

eae t)e tissue at least !or '+ min !or relaxation.)en ta/e t)e response 0it) t)e

standard and test drug #prepared by serial dilution$.,ele"t t)e experimental design and

ta/e t)e responses o! t)e drug. al"ulate re"orded responses a""ording to t)e sele"tedexperimental design and ma/e a grap).

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(Rat descending colon)

eep t)e animal #rat$ !asting !or at least 24 )ours. ,a"ri!i"e t)e rat by stunning #a strong

blo0$ mi l)e )ead. !ter sa"ri!i"e /eep t)e animal on t)e disse"ting board #(B$. 8at is

!ixed on t)e (B by tying it legs 0it) t)e )elp o! t)read.

ut open abdomen o! rat by a erti"al midline in"ision and t)en expose abdominal

is"era. 9denti!y t)e re"tum and t)en go to0ards t)e re"tum at least 5-7 "m !or "olle"tion

o! des"ending "olon #($.

ut small pie"e #1.5-' "m$ o! ( as per t)e inner organ bat) olume and "lean it 0it) a

lu/e0arm 0ater or 3,, 0it) t)e )elp o! a syringe.)en tie bot) t)e end 0it) t)e )elp o!

nylon or "otton t)reads and tie it to t)e balan"ed )oriontal leer. eae tissue !or atleast'+ min !or relaxation.

,ele"t t)e experimental design and ta/e t)e responses o! t)e drug. al"ulate and ma/e

grap) o! re"orded responses.

;ote: )e "al"ulation and grap) 0ill be done depending on type o! design used< eit)er '-point 4

point or mat")ing assay 0ill be used.

B. o determine blo"/ing e!!e"t o! atropine on isolated rat "olon.

9solated rat "olon is tied to !rontal leer and gien '+ minutes !or relaxation. ")

is t)en added to bat) in "umulatie doses !ollo0ing t)e se*uen"e 1 =g > 2 =g > 7=g >2+ =g > >7+ =g > 2++ =g.et" so t)at drug "on"entration in t)e bat)

in"reases step0ise in t)e series 1 ' 1+ '+ 1++ '++ =g at +.5 log unit interals.

long 0it) it "ontra"tion o! tissue in"reases step by step 0it)out relaxation. 9!next "on"entration does not "ause !urt)er in"rease in "ontra"tion it is assumedt)at maximum e!!e"t !or t)e drug is rea")ed and drug is t)en 0as)ed out.

!ter re"ording t)e "umulatie dose response 0it) ") tissue is 0as)ed 0it)

nutrient solution. )en tropine is added to t)e inner bat).

") is t)en added in t)e se*uen"e o! 1 =g > 2 =g > 7 =g >2+ =g > >7+ =g >

2++ =g.

)e "ontra"tion o! t)e tissue is re"orded on t)e drum and "ompared 0it) t)eresponse o! ") alone.

. o determine potentiating e!!e"t o! p)ysostigmine using rat "olon.

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o 9solated rat "olon is tied to !rontal leer and gien '+ minutes !or

relaxation. ") is t)en added to bat) in "umulatie doses !ollo0ing t)e

se*uen"e 1 =g > 2 =g > 7 =g >2+ =g > >7+ =g > 2++ =g.et" so t)at

drug "on"entration in t)e bat) in"reases step0ise in t)e series 1 ' 1+ '+1++ '++ =g at +.5 log unit interals. long 0it) it "ontra"tion o! tissue

in"reases step by step 0it)out relaxation. 9! next "on"entration does not"ause !urt)er in"rease in "ontra"tion it is assumed t)at maximum e!!e"t!or t)e drug is rea")ed and drug is t)en 0as)ed out.

o !ter re"ording t)e "umulatie dose response 0it) ") tissue is 0as)ed

0it) nutrient solution. )en 3)ysostigmine is added to t)e inner bat).

o ") is t)en added in t)e se*uen"e o! 1 =g > 2 =g > 7 =g >2+ =g > >7+

=g > 2++ =g.

o )e "ontra"tion o! t)e tissue is re"orded on t)e drum and "ompared 0it)t)e response o! ") alone.

?

*$ Tit'e( Rat toma)+ ,f"nd"-

Objective: o determine un/no0n"on"entration o! serotonin #5@$ using rat stoma") #!undus$

Requirements:

nimaltissue 8at ,toma")!undus

3,, yrode (e alonrebs

eer Arontal 0riting simple leer

(rug ,erotonin#5@$ "etyl")oline ")loride #")$

3ro"edure:

1. ,a"ri!i"e t)e rat by a blo0 on t)e )ead and "arotid bleeding.

2. ut open t)e abdomen and expose t)e stoma").

'. 9denti!y t)e !undus o! t)e stoma") #upper part-greyis) in "olour$. 9n"ise it !rom t)e un"tiono! pylori" part #pin/ in "olour$ and put it in t)e dis) "ontaining rebs solution.

4. 9n"ise t)e !undus !rom t)e lesser "urature and open it longitudl nally #as s)o0n in t)e!igure$. Cie alternate ig-ag "uts to ma/e it !undal strip preparation. ie bot) t)e ends 0it) t)e

t)read and mount in t)e organ bat) "ontaining rebs solution at '7D. erate t)e tissue

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5. pply 1 g load and allo0 t)e preparation to e*uilibriate !or '+ min. sing !rontal 0riting

leer 0it) 1+-12 magni!i"ation re"ord t)e "ontra"tions due to in"reasing "on"entrations o!

serotonin. ,in"e t)e mus"le "ontra"ts slo0ly and relaxes slo0ly a "onta"t time o! &+ se" and 5min time "y"le are !ollo0ed !or proper re"ording o! t)e "on"entration - response "ure.

6. abel and !ix t)e tra"ing. 3lot t)e "on"entration response "ure.

.$ Tit'e( Rat Uter"

Fbe"ties :

a. o re"ord t)e "on"entration response "ure o! oxyto"in using rat uterus preparation.

b. o determine un/no0n "on"entration o! a"etyl")oline #")$ using rat uterus.

Requirements:

nimaltissue !emale rat uterus

3,, (e alonrebs G"E0en

eer Arontal 0riting

(rug oxyto"in "etyl")oline ")loride #")$

3ro"edure:

a$ o re"ord t)e "on"entration response "ure o! oxyto"in using rat uterus preparation.

Examine t)e aginal smear under mi"ros"ope to /no0 about t)e proper stage

o! oestrus "y"le. 9! t)e rat is not in !ran/ oesterous ine"t +.1 mg/g o!

stilbesterol and 0ait !or 24 )r.

,a"ri!i"e t)e animal by a blo0 on t)e )ead and "arotid bleeding. ut open t)e

peli" region and expose bot) t)e )orns o! uterus. ,eparate t)em gently !rom

t)e surrounding !atty material and trans!er t)em to a dis) "ontaining (e

alonHs solution. I)en t)e rat is in oestrus generally t)e uterus is !les)y and

pin/ in "olour. 0o separate pie"es #2-' "m long$ o! uterine preparations "an

be made !or experimental use.

Gount t)e uterine preparation in t)e organ bat) "ontaining (e alon solution at

'+-'2D. pply a tension o! +.5 g and allo0 t)e tissue to e*uilibrate !or '+ min.

8e"ord "ontra"tions due to di!!erent "on"entrations o! oxyto"in using !rontal

0riting leer. onta"t time o! '+ se"onds and ' min time "y"le is used !or proper

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re"ording o! t)e responses. 8e"ord at least !our responses due to di!!erent

"on"entrations o! oxyto"in.

abel and !ix t)e tra"ing and dra0 t)e "on"entration-response "ure.

b$ o determine un/no0n "on"entration o! a"etyl")oline #")$ using rat uterus.

Aemale rat #A8$ 0eig)ing around 15+-'++ g is ta/en and 24 )r prior to t)eexperiment t)e rat is primed 0it) 0.1 mg/kg stilbestrol 9G.,a"ri!i"e t)e A8 by stunning#a strong blo0$ on t)e )ead. !ter sa"ri!i"e /eep t)e A8 on t)e disse"ting board #(B$.

A8 is !ixed on t)e (B by tying its leg 0it) t)e )elp o! t)read. ut open t)e abdomen o!A8 by a erti"al midline in"ision a!ter a small )oriontal "ut and t)en exposed t)eabdominal "ontains

9denti!y t)e aginal ori!i"e and tra"e t)e aginal ori!i"e interiorly to !ind t)e t0o )orns o!

t)e uterus. ut t)e t0o )orns and no0 you )ae t0o samples o! t)e uterus. ut s)ort t)e

tissue a""ording to t)e inner organ bat) #)andle t)e tissue minimally at t)e middle part

0)i") "an redu"e t)e sensitiity o! t)e tissue$.

,ele"t t)e experimental design #mat")ing bra"/eting '-point or 4point assay$ and

"al"ulate t)e grap).

/$ Tit'e( Rat ano)o))0!e" m")'e

Objective:o determine un/no0n "on"entration o! a"etyl")oline #")$ using rat ano"o""ygeus

mus"le preparation.

Requirements:

nimaltissue 8at ano"o""ygeus mus"le

3,, rebs

eer Arontal 0riting

(rug "etyl")oline ")loride #")$

3ro"edure:

,a"ri!i"e t)e rat by a blo0 on )ead and "arotid bleeding. ut open t)e abdomen in mid-

line and split t)e pelis. 8emoe t)e bladder and uret)ra and "ut t)roug)t )e "olon at t)e

peli" brim. 3ull !or0ard t)e peli" portion and "lear t)e deli"ate "onne"tie tissue

be)ind until ano"o""ygeus mus"le "omes in ie0. are!ully separate t)e t0o

ano"o""ygeus mus"les.

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ie t)read at bot) ends o! ea") mus"le. 9solate ea") mus"le and mount it in a 1+ ml organ

bat) "ontaining rebs solution maintained at '7 J . erate t)e tissue and allo0 it to

e*uilibrate !or '+ min under 1g tension.

8e"ord "ontra"tions-dependent responses due to noradrenaline using !rontal 0riting leer

0it) 1+ !old magni!i"ation. onta"t time o! 6+ se" and a 5 minute time "y"le are used !or

proper re"ording o! t)e response.

abel and !ix t)e tra"ing and plot "on"entration-response "ure !or a"etyl")oline.

1$ Tit'e( Rat Heart

Objective: o obsere t)e e!!e"t o! arious drugs on t)e isolated )eart #angendor!!Ks

preparation$.

Requirements:

nimaltissue lbino 8ats#'++ g and at least 1year o! age$

3,, rebs

(rug stimulant drugs: epinep)rine norepinep)rine isoprenaline

(epressant drugs: a"etyl")oline potassium.

ntagonists: propranolol atropine.

3ro"edure:

)e Lan!endorff io'ated +eart preparation assay is a predominant in vitrote")ni*ue

used in p)arma"ologi"al and p)ysiologi"al resear").9t allo0s t)e examination o!

"ardia" "ontra"tile strengt) and )eart rate 0it)out t)e "ompli"ations o! an inta"t animal.

9n t)e angendor!! preparation animal is pre treated 0it) )eparin #5++-1+++ 9/g$ i..

nimal is t)en stunned and t)e t)orax is opened and t)e )eart exposed. )e )eart is

remoed !rom t)e ratKs body seering t)e blood essels < it is t)en per!used in a reerse

!as)ion ia t)e aorta usually 0it) a nutrient ri") oxygenated solution #"old rebs

solution$. )e )eart is gently s*ueeed seeral times to remoe blood !rom t)e )eart and

preent !ormation o! t)rombus.

)e ba"/0ards pressure "auses t)e aorti" ale to s)ut !or"ing t)e solution into t)e

"oronary essels 0)i") normally supply t)e )eart tissue 0it) blood. )is !eeds nutrients

and oxygen to t)e "ardia" mus"le allo0ing it to "ontinue beating !or seeral )ours a!ter

its remoal !rom t)e animal.

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Recording

8eading o! t)e rate o! )eating and o! t)e "oronary !lo0 "an most "ommonly be ta/en oer a

period o! '+ se"onds. 9! t)e moements are too !ast to "ount t)e /ymograp) "an be run at a )ig)

speed !or 15-'+ se"onds be!ore t)e drug rea")es t)e )eart and in most instan"es t)e e!!e"ts

passes in a !e0 minutes as t)e drugs are 0as)ed t)roug). 8e"ording o! t)e rate o! )eating o! !lo0s)ould be ta/en on"e eery 2 minutes. Aurt)er doses s)ould not be gien until t)e preparation

)as re"oered "ompletely and t)e rates are steady or until t)ey )ae settled do0n steadily at ne0

"ontrol leels.

Note: 9nitially t)e experiment 0ill be started 0it) t)e stimulant drugs #epinep)rine

norepinep)rine isoprenaline et". in in"reasing doses$ !ollo0d by depressants #a"etyl")oline

potassium$ drugs and !inally 0it) antagonists #propanolol and atropine et".$.

2$ Tit'e( Rat P+reni) ner3e

Objective:o determine un/no0n "on"entration o! a"etyl")oline #")$ using rat p)reni" nere

diap)ragm

Requirements:

nimaltissue 8ats p)reni" nere diap)ragm

3,, rebstyrode

eer pre!erably spring loaded leer or simple leer

(rug a"etyl")oline #")$

3ro"edure:

dult rat is sa"ri!i"ed and t)e !rontal part o! t)e rig)t t)ora"i" 0all is remoed a!ter

"utting along t)e rig)t side o! t)e stern-m. Gediastinum is "ut ust aboe t)e !rontal

insertion o! t)e diap)ragm ta/ing "are not to damage t)e p)reni" nere. Arontal part o!

t)e le!t t)ora"i" 0all is t)en remoed so as to expose t)e p)reni" nere and t)e le!t lung

remoed.

0o "onerging "uts are made t)roug) t)e rib #12 mm apart$ to0ards t)e teninous part o!

t)e diap)ragm #'mm 0ide$ parallel to its mus"le !ibres '' mm on eit)er side o! t)e point

0)ere t)e p)reni" nere enters t)e diap)ragm. ,trip is t)en "ut out beyond t)e tendinous

part 0it) about 2.5 "m o! p)reni" nere atta")ed to it.

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)e !an s)aped preparation is !ixed by inserting t)e rib inside a slit in t)e platinum

ele"trode. t)read tied around t)e tendinous end is atta")ed to a lig)t isotoni" spring

leer. )e p)reni" nere is laid on a slit a"ross t)e t0o 0ires and "oered 0it) a moist

pie"e o! "otton 0ool.

8e"ord t)e "on"entration-dependent "ontra"tile responses o! a"etyl")oline on t)e smo/ed

drum 0it) at time "y"le o! 5 minutes. Ainally !ix t)e tra"ings and dra0 t)e grap).

4$ Tit'e( Rat 3a deferen

Objective:

a. o determine un/no0n "on"entration o! a"etyl")oline#")$ using rat as de!erens

b. o determine un/no0n "on"entration o! p)enylep)erine using rat as de!erens

Requirements:

nimaltissue 8ats as de!erens

3,, rebs 8inger yrode G"E0en

eer !rontal leer

(rug a"etyl")oline #")$ @istamine

3ro"edure: a$ o determine un/no0n "on"entration o! a"etyl")oline using rat as de!erens

,a"ri!i"e t)e rat by spinal dislo"ation open t)e abdomen and expose t)e tested and asde!erns.)e as de!erens o! t)e rat are easily identi!iable as 0)ite t)in tubular stru"tures.

Bot) )ae blood essels running along t)eir lengt)s and t)ey emanate !rom t)e

epididymis "lose to t)e testes.

ut one as de!erns !ree !rom t)e epididymis and disse"t it as "lose to t)e un"tion 0it)

t)e uret)ra as possible. ut it at t)is point and trans!er it to a petri-dis) "ontaining rebKs

solution.are!ully remoe all t)e "onne"tie tissue and t)e blood essels and "losely

ad)ere to t)e mus"le and expel any semen !rom t)e as de!erns by gently pressing t)e

tissue 0it) a !inger.

Gount t)e tissue into t)e organ bat) by tying a !ine t)read at t)e tip o! one end o! t)e

mus"le !or atta")ment to t)e !ul"rum 0)ile t)e ot)er end o! t)e preparation to a tissue

)older. E*ulibrate t)e tissue !or '+ min under a tension o! +.5 g 0it) su!!i"ient 0as)ings

on"e eery 5 minutes.

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8e"ord t)e "on"entration-dependent "ontra"tile responses o! a"etyl")oline 0it) at time

"y"le o! 5 minutes. Aix t)e tra"ings and dra0 t)e grap).

b$ o determine un/no0n "on"entration o! p)enylep)erine using rat as de!erens

,a"ri!i"e t)e rat by spinal dislo"ation open t)e abdomen and expose t)e tested and asde!erns.)e as de!erens o! t)e rat are easily identi!iable as 0)ite t)in tubular stru"tures.

Bot) )ae blood essels running along t)eir lengt)s and t)ey emanate !rom t)e

epididymis "lose to t)e testes.

ut one as de!erns !ree !rom t)e epididymis and disse"t it as "lose to t)e un"tion 0it)

t)e uret)ra as possible. ut it at t)is point and trans!er it to a petri-dis) "ontaining rebKs

solution.are!ully remoe all t)e "onne"tie tissue and t)e blood essels and "losely

ad)ere to t)e mus"le and expel any semen !rom t)e as de!erns by gently pressing t)e

tissue 0it) a !inger.

Gount t)e tissue into t)e organ bat) by tying a !ine t)read at t)e tip o! one end o! t)e

mus"le !or atta")ment to t)e !ul"rum 0)ile t)e ot)er end o! t)e preparation to a tissue

)older. E*ulibrate t)e tissue !or '+ min under a tension o! +.5 g 0it) su!!i"ient 0as)ings

on"e eery 5 minutes.

8e"ord t)e "on"entration-dependent "ontra"tile responses o! p)enylep)rine 0it) at time

"y"le o! 5 minutes. Aix t)e tra"ings and dra0 t)e grap).

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Documents

List of Experiments in Animals