The document describes several in vitro and in vivo methods used to study anti-allergic and anti-inflammatory drugs. In vitro methods include inhibition of histamine release from mast cells and inhibition of T cell proliferation. In vivo methods include a rat anaphylaxis model, guinea pig Schultz-Dale reaction, and passive cutaneous anaphylaxis in rats. One method involves sensitizing rats with ovalbumin, then challenging them to induce shock, which can be counteracted by test drugs. Another involves sensitizing guinea pigs to egg albumin to study contractions in response to ovalbumin.

1. Submitted by,
Miss. Prajitha.P
First M.Pharm, Pharmacology
Devaki Amma Memmorial College
of Pharmacy, Malappuram
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5. IN VITRO METHODS
 Inhibition of histamine release from mast cells cell
 Mitogen induced lymphocyte proliferations
 Inhibition of T cell proliferation
 PFC (Plague forming colony) test in vivo
 Inhibition of dihydro-orotate dehydrogenase
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6.  IN VIVO METHODS
 Acute systemic anaphylaxis in rats
 Anti-anaphylactic activity in guinea pigs
( Schultz- dale reaction)
 Delayed type of hypersensitivity
 Passive cutaneous anaphylaxis in rats
 Arthus type immediate hypersensitivity
 Spontaneous autoimmune diseases in animals
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7.  Reversed passive arthus reaction
 Adjuvant arthritis in rats
 Collagen type 2 induced arthritis in rats
 Proteoglycan induced progressive polyarthritis in
mice
 Pristance induced arthritis in mice
 Experimental autoimmune thyroiditis
 Porcine cardiac myosin induced autoimmune
myocarditis in rats
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9.  Rats are sensitized by ovalbumin and bordetella
pertusis
 After 11 days they are challenged by i.v. ovalbumin
leading to shock
 It is counteracted by corticosteroids and i.v. disodium
cromoglycate
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10. Methodology:
Female sprague dawley rats (120-150g)
Injected i.m with 10mg/kg of ovalbumin
simultaneously
With bordetella pertusis suspension i.p
IgE Ab migrates to surface of mast cells & basophilic
granulocytes 11days
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11. Iv injection of 25mg/kg ovalbumin
18 hrs before given with corticosteroids and
disodium cromoglycate
Ag-Ab complex forms on the surfaceof mast cell
release histamine, 5-HT, etc
SHOCK MORTALITY(80%)
counteract
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12.  Symptoms of shock scored and mortality recorded,
compared with untrated animals.
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13.  Guinea pigs are sensitized against egg albumin
 3week
release histamine
Contraction of smooth muscle
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14.  Methodology
guinea pigs(f/m),300-400g
alum precipitated egg albumin
egg albumine(1mg/ml) in saline
stirred
kept in room temp
0.125 ml alum egg albumine to foot pad and 0.5ml SC
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15. 4 weeks
Killed using anaesthetic ether
Ileum dissected out & cleaned
Mounted on organ bath(tyrode soln @ 37°C (stabilize
for 15mnts)
barium chloride
The tissue respond to spasmogen are selected
3 bath selected
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16. Test gps std gps Control gp
Administered ovalbumin
Contractions recorded on physiograph
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17. 3.Passive cutaneous anaphylaxis in rats
it is Ag-Ab reaction: immediate type
skin is passively immunized with rat ovalbumin serum &
challenged after 2 days with ovalbumin at same site.
Ag-Ab complex in mast cell release
histamine and other mediators
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18.  Cause vasodilation ,increased vascular permeability,
leakage of plasma.
 To make the allergic reaction clear, inject Evans blue
dye with the Ag with get attached to the albumin
fraction of the plasma.
 Ag –Ab reaction spot appears as blue spot, so can
confirm the occurrence of allergy
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19. methodology:
male wistar rats (250g)
they are adrenalectomized
3 days
Egg albumin 1mg/ml injected on foot pad
8days
Animals are bled and antiserum is collected
Diluted such a way that it should produce a wheal of
15-20mm diameter on rat skin in preliminary test
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20.  It is injected intradermaly shaved dorsal skin of rts
100g.
24 hrs
0.1ml 2.5% Evans blue dye containing 25mg/ml egg
albumin
Test drug introduced
IV: simultan.with Ag- 1 hr before challenging
Dye soln.
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21.  30 min sacrificed using ether anaesthesia.
 At Passive cutaneous anaphylactic reaction site Evans
blue is leaked
 Extracted and determined colorimetrically 620nm
Amount of Evans blue leaked for control gp taken as 100%,
and calculating the % inhibition in the extravasation of
dye in the rats in treated animals. Compared with std &
control gps
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22. 3.Arthus type immediate hypersensitivity reaction
There is an Ag-Ab complex after the injection of
antigen. But if the person or the animal is previously
immunized with the antibody( i.e, it have circulating
Ab) there is precipitation of allergic reaction.
It is due to the activation of complement and PMN
cell get activate and leads to inflammation
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23. Methodology:
 in this animals are challenged with ovalbumin
suspension(in paraffin oil)+pertusis vaccine in saline
soln. mixed together to get emulsion.
 Wistar rats of 250-300g are selected
 Injected with emulsion
 After 7 days a gp of 8 animals (s,t c) are housed in a
cage with diet and optional water till 1 hr before the
arthus reaction
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24.  Each gp then injected with purified ovalbumin in paw
 Swelling reaches max in few hrs
 Thickness measured with calipers or plethymometric
method
 % reduction inthickness of foot pad compared with
std and control gp
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25. In vitro methods
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26. Inhibition of histamine release from mast cells.
Asthma hypersensitivity reactions
Mediators are released from mast cells (histamin)
Here; calcium ionophore induces mast cell to
release histamin
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27.  Procedure:
wistar rat are decapitated and exsanguinated
50ml Hank’s balanced salt soln (HBSS) injected to
peritonial cavity
Abdominal wall opened and collected fluid containing
peritonial cell
centrifuge at 2000rpm
Resuspend in HBSS
1ml test drug added to mast cell suspension
incubate @ 37ºC
Cell made to 3ml with HBSS
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28. Equal vol.of cacium ionophore ia added
Control soln
1. Spontaneous histamin release mast cells & soln used to
determine base line
2. Histamin release: mast cell+ calcium ionophpre
3. Test compound control: soln+ compound to test the
compound for native fluorescence
4. Solution control: only soln to determine the base line
incubate @ 37ºC
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29. From the suspension: 1mi of top layer transferred to
a tube containing 300mg Nacl+1.25ml butanol
+
NaOH
mechanical shaking
1ml of the top layer pipetted to 5ml tube
containing n-heptane +0.12N HCL
mixed by inverting
Organic layer aq.layer
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30.  To the two layer add 100µl 0.2% phthaldehyde
soln. + 100µl NaOH
2mnts
reaction is stopped by the addition of 50µl 3N Hcl
Total sample added to auto sample vial
Histamine concentration is determined by
fluorescence detector using excitation wave
length 350nm & emission wavelength 450nm
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