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 in
Experimental animals.
1+
4. (rug ,"reening met)ods 11-15


I$ %IOASSAY
1
Experiment in Rat
&$ Tit'e( Rat )o'on preparation
Objective:
-. .o determine un/no0n "on"entration o! a"etyl")oline #-")$ using rat
as"ending1des"ending "olon.
B. .o determine blo"/ing e!!e"t o! atropine on isolated rat "olon.
2. .o determine potentiating e!!e"t o! p)ysostigmine using rat "olon.
Requirements:
-nimal1 tissue rat as"ending1des"ending "olon
3)ysiologi"al salt solution#3,,$ 4erbs#-2$ 1 de5alon#(2$
6eer !rontal 0riting
(rug -"etyl")oline ")loride 7-trop)in 7 p)ysostigmine.
Procedure:
-. .o determine un/no0n "on"entration o! a"etyl")oline:
(Rat ascending colon)
4eep 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"e7 /eep t)e animal on t)e disse"ting board
#(B$.8at s)ould be !ixed on t)e (B by tying its legs 0it) t)read.
2ut open abdomen o! rat by a erti"al midline in"ision and t)en expose abdominal
is"era. 9denti!y t)e as"ending "olon t)roug) "e"um. 9denti!y t)e "e"um and t)en go
to0ards t)e re"tum at least 5-7 "m
2ut small pie"e #1.5-' "m$ 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 )ori:ontal leer.
6eae t)e tissue at least !or '+ min !or relaxation..)en7 ta/e t)e response 0it) t)e
standard and test drug #prepared by serial dilution$.,ele"t t)e experimental design and
ta/e t)e responses o! t)e drug. 2al"ulate re"orded responses7 a""ording to t)e sele"ted
experimental design and ma/e a grap).
2
(Rat descending colon)
4eep t)e animal #rat$ !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"e7 /eep t)e animal on t)e disse"ting board #(B$. 8at is
!ixed on t)e (B by tying it legs 0it) t)e )elp o! t)read.
2ut open abdomen o! rat by a erti"al midline in"ision and t)en expose abdominal
is"era. 9denti!y t)e re"tum and t)en go to0ards t)e re"tum at least 5-7 "m !or "olle"tion
o! des"ending "olon #(2$.
2ut small pie"e #1.5-' "m$ o! (2 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..)en7 tie bot) t)e end 0it) t)e )elp o!
nylon or "otton t)reads and tie it to t)e balan"ed )ori:ontal leer. 6eae tissue !or atleast
'+ min !or relaxation.
,ele"t t)e experimental design and ta/e t)e responses o! t)e drug. 2al"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 '-point7 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 doses7 !ollo0ing t)e se*uen"e 1 =g > 2 =g7 > 7
=g7 7 >2+ =g7 > >7+ =g 7 > 2++ =g.et" so t)at drug "on"entration in t)e bat)
in"reases step0ise in t)e series 17 '7 1+7 '+7 1++7 '++ =g at +.5 log unit interals.
-long 0it) it7 "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"tion7 it is assumed
t)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) -")7 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 =g7 > 7 =g7 7 >2+ =g7 > >7+ =g 7 >
2++ =g.
.)e "ontra"tion o! t)e tissue is re"orded on t)e drum and "ompared 0it) t)e
response o! -") alone.
2. .o determine potentiating e!!e"t o! p)ysostigmine using rat "olon.
3
o 9solated rat "olon is tied to !rontal leer and gien '+ minutes !or
relaxation. -") is t)en added to bat) in "umulatie doses7 !ollo0ing t)e
se*uen"e 1 =g > 2 =g7 > 7 =g7 7 >2+ =g7 > >7+ =g 7 > 2++ =g.et" so t)at
drug "on"entration in t)e bat) in"reases step0ise in t)e series 17 '7 1+7 '+7
1++7 '++ =g at +.5 log unit interals. -long 0it) it7 "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"tion7 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) -")7 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 =g7 > 7 =g7 7 >2+ =g7 > >7+
=g 7 > 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:
-nimal1tissue 8at ,toma")1!undus
3,, .yrode1 (e 5alon14rebs
6eer Arontal 0riting 1 simple leer
(rug ,erotonin#5@.$7 -"etyl")oline ")loride #-")$
3ro"edure:
1. ,a"ri!i"e t)e rat by a blo0 on t)e )ead and "arotid bleeding.
2. 2ut open t)e abdomen and expose t)e stoma").
'. 9denti!y t)e !undus o! t)e stoma") #upper part-greyis) in "olour$. 9n"ise it !rom t)e Bun"tion
o! pylori" part #pin/ in "olour$ and put it in t)e dis) "ontaining 4rebs solution.
4. 9n"ise t)e !undus !rom t)e lesser "urature and open it longitudl nally #as s)o0n in t)e
!igure$. Cie alternate :ig-:ag "uts to ma/e it !undal strip preparation. .ie bot) t)e ends 0it) t)e
t)read and mount in t)e organ bat) "ontaining 4rebs solution at '7D2. -erate t)e tissue
4
5. -pply 1 g load and allo0 t)e preparation to e*uilibriate !or '+ min. Esing !rontal 0riting
leer 0it) 1+-12 magni!i"ation re"ord t)e "ontra"tions due to in"reasing "on"entrations o!
serotonin. ,in"e t)e mus"le "ontra"ts slo0ly and relaxes slo0ly7 a "onta"t time o! &+ se" and 5
min time "y"le are !ollo0ed !or proper re"ording o! t)e "on"entration - response "ure.
6. 6abel and !ix t)e tra"ing. 3lot t)e "on"entration response "ure.
.$ Tit'e( Rat Uter"
FbBe"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:
-nimal1tissue !emale rat uterus
3,, (e 5alon14rebs1 G"E0en
6eer Arontal 0riting
(rug oxyto"in7 -"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/ oesterous7 inBe"t +.1 mg1/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. 2ut 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
5alonHs 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 5alon solution at
'+-'2D2. -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. 2onta"t time o! '+ se"onds and ' min time "y"le is used !or proper
5
re"ording o! t)e responses. 8e"ord at least !our responses due to di!!erent
"on"entrations o! oxyto"in.
6abel 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/en7 and 24 )r prior to t)e
experiment7 t)e rat is primed 0it) 0.1 mg/kg stilbestrol7 9G. ,a"ri!i"e t)e A8 by stunning
#a strong blo0$ on t)e )ead. -!ter sa"ri!i"e7 /eep t)e A8 on t)e disse"ting board #(B$.
A8 is !ixed on t)e (B by tying its leg 0it) t)e )elp o! t)read. 2ut open t)e abdomen o!
A8 by a erti"al midline in"ision a!ter a small )ori:ontal "ut7 and t)en exposed t)e
abdominal "ontains
9denti!y t)e aginal ori!i"e and tra"e t)e aginal ori!i"e interiorly to !ind t)e t0o )orns o!
t)e uterus. 2ut t)e t0o )orns and no07 you )ae t0o samples o! t)e uterus. 2ut s)ort t)e
tissue a""ording to t)e inner organ bat) #)andle t)e tissue minimally at t)e middle part
0)i") "an redu"e t)e sensitiity o! t)e tissue$.
,ele"t t)e experimental design #mat")ing7 bra"/eting7 '-point7 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:
-nimal1tissue 8at ano"o""ygeus mus"le
3,, 4rebs
6eer Arontal 0riting
(rug -"etyl")oline ")loride #-")$
3ro"edure:
,a"ri!i"e t)e rat by a blo0 on )ead and "arotid bleeding. 2ut 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. 2are!ully separate t)e t0o
ano"o""ygeus mus"les.
6
.ie t)read at bot) ends o! ea") mus"le. 9solate ea") mus"le and mount it in a 1+ ml organ
bat) "ontaining 4rebs solution maintained at '7 J 2. -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. 2onta"t time o! 6+ se" and a 5 minute time "y"le are used !or
proper re"ording o! t)e response.
6abel 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 #6angendor!!Ks
preparation$.
Requirements:
-nimal1tissue -lbino 8ats#'++ g and at least 1year o! age$
3,, 4rebs
(rug stimulant drugs: epinep)rine7 norepinep)rine7 isoprenaline
(epressant drugs: a"etyl")oline7 potassium.
-ntagonists: propranolol7 atropine.
3ro"edure:
.)e Lan!endorff io'ated +eart preparation assay is a predominant in vitro te")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 6angendor!! preparation7 animal is pre treated 0it) )eparin #5++-1+++ 9E1/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 body7 seering t)e blood essels < it is t)en per!used in a reerse
!as)ion ia t)e aorta7 usually 0it) a nutrient ri")7 oxygenated solution #"old 4rebs
solution$. .)e )eart is gently s*uee:ed 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)ut7 !or"ing t)e solution into t)e
"oronary essels7 0)i") normally supply t)e )eart tissue 0it) blood. .)is !eeds nutrients
and oxygen to t)e "ardia" mus"le7 allo0ing it to "ontinue beating !or seeral )ours a!ter
its remoal !rom t)e animal.
7
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 "ount7 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"es7 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! !lo0
s)ould be ta/en on"e eery 2 minutes. Aurt)er7 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)rine7
norepinep)rine7 isoprenaline et". in in"reasing doses$7 !ollo0d by depressants #a"etyl")oline7
potassium$ 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:
-nimal1tissue 8ats1 p)reni" nere diap)ragm
3,, 4rebs1tyrode
6eer 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 Bust 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.
8
.)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. Ainally7 !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:
-nimal1tissue 8ats1 as de!erens
3,, 4rebs1 8inger 1.yrode 1 G"E0en
6eer !rontal leer
(rug a"etyl")oline #-")$7 @istamine
3ro"edure: a$ .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"ation7 open t)e abdomen and expose t)e tested and as
de!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.
2ut one as de!erns !ree !rom t)e epididymis and disse"t it as "lose to t)e Bun"tion 0it)
t)e uret)ra as possible. 2ut it at t)is point and trans!er it to a petri-dis) "ontaining 4rebKs
solution.2are!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.
9
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 7 open t)e abdomen and expose t)e tested and as
de!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.
2ut one as de!erns !ree !rom t)e epididymis and disse"t it as "lose to t)e Bun"tion 0it)
t)e uret)ra as possible. 2ut it at t)is point and trans!er it to a petri-dis) "ontaining 4rebKs
solution.2are!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).
10