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This was tested in rabbits whose body temperature was increased by intravenously administered bacterial
pyrogen as described in the British Pharmacopoeia (1958).
Healthy adult rabbits (of either sex) each weighing not less than 1.5 kg and having a basal rectal temperature
ranging from 39.4 - 39.9 ° c were used. They were kept in the loboratory overnight to adjust to the rOO"
temporature (20 - 22 ° c). Food was withheld from the rabbits overnight and water during the experiment only.
Rectal tCJT1perotures were measured with a clinical thermometer (clinical maximum thermometer) lubric3ted with paraffin oil and inserted into the rectum to a
depth of about 6.0 cm.
Temperatures were recorded at 30 minutes intcrv�ls for 60 minutes before the intravenous injection of 1.0 ug bactcri'll pyrogen. Temperature recording continued for
,.� hours aft,r pyrogon injection. At the end of 2½ h,
test rabbits \-:?re given drugo intraperitoneally !.n 2.0 ml propylene -glycol only intraperitoneally. Tcmperr,ture
recording continued for another 2 hours oftcr drug •
adr.iinis tra t.lon . •
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-2.'12 Inhibition of Prostaalandin Synthesis in vivo It will be seen from the chapter on results that both OBA and Fagaramide inhibited rat paw oedema and
prostaglandin synthetase of guinea-pig lung and rabbit brain. Inhibition of prostaglandin synthesis could
account for the observed anti-phlogistic activity of OBA and Fagarrunide if it can be shown that these compounds
are active against prostaglandin synthesis in vivo.
This possibility was tested using the sponge exudate technique of Higg9 1 Harvey, Ferreira and vane (1976).
This model also afforded an opportunity to exllJlline the
effect of the drugs on leucocyte migration, histamine, 5-hydroxytryptamine content and protein exudation at the site of inflammation.
Procedure
Nylon foam sponges (2.5 x 1.2 x 1.2 cm) were cut from packing material. These were washed in distilled water dried 1nd sterilized by autoclaving. Fcmalo
albino c-ats 11eighlng between 180 g ond 250 g were
anilesthetizod with sodium pentobarbltone (30.0 m�/kg).
The flanks w,,re shaved and swabbed �,ith methyl .. t_d
spirit. An •. nclssion was made on one :.lcJc of the onimnli::'
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-flank and the subcutaneous space cleared. Sterile foam sponges were soaked in 2� (w/v) carrageenan
suspension in sterile saline. These were implanted, (one sponge into each animal) into the subcutaneous space. The would was closed up with silk thread and
('\dusted with hibitane powder. The animals were allowed to recover from the pentobarbitone anaesthesia.
Eight hours aft�r implantation, the sponges were
removed under light ether anaesthesia. The sponges were immediately immersed in 10.0 ml heparinized saline
contained in a centrifuge tube and gently squeezed .
The sponges were then pinned across the top of tne centri-fuge tubes by means of a short piece of wire and centri
fuged at 1,000 r.p.m. for 10 minutes. After centrifu-gation, the 'dry' sponges were re�ov�d and the volume of the remaining fluid was measured.
The total number of leucocyteo in the sponge exudate was counted with "Improved" ueubauer hi,emocytometcr .
The percentage of poly-morphonuclcar cells was deter
rained by scanning a thin film of the exudate stnincd with Lcishmann stain and counting one hundred calls • • •
Prostngloncin content of the exudate was d,:tt..rmincd by subjecting the exucate to acid lipid extract-on �nd assaying the c�troct over supcrfuscd rat otomnch �trip
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-as previou�ly described.
Extraction of Prostaolandin •
The exudate was acidified to pH 3 with hydr�chloric acid and extracted twice with equal volumes of ethyl
acetate. The combined ethyl acetate fractions ,,,ere evaporated under reduced pressure at 40 - so 0 c. The
residue wus either taken up in Tyrode solution for
bioassoy or 1n 0.5 ml ethanol for thin layer chromato
graphy.
Eff1cicncv of extraction:
The efficiency of the extraction procedure was estimated by determining the percentuge recovery of
trltiated PGP2a added to the fluid sample before extrac- • tion.
o.os ml of 8 ng/ml solution of tritium labelled PCP2a wa� added to 10.0 ml of th� fluid sample. It
was well shaken and an aliquot of 0.5 ml was withdrawn.
The rest was then extracted os earlier described.
After �vaporating the ethyl acetat�, the residu� was then token up in 10.0 ml Tyrode solution and anc,thcr
aliquot o:: 0.5 ml '-<u& again withdrawn. To tho !epar, Le aliquots was added 5.0 ml of liquid scintillont (PFO:
- SO : 1 in toluene) and the radiooctivJ ty ,�of.
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-counted in a Packard Tr1-Carbl1quid Scintillation Spectrometer for 2 minutes. Efficiency of •
extraction waa cstirnftted by comphring lho count
in the extracted onmplo �ith thg count in tho porcnt solution of tritlntcd PGF2a.
Thin lnyer Chro::\atoarnphy
Prostaglandin-likc activity waa separated on glooo plates (S c:i x 20 C?:l) coated with ailico gel (Camo9,
Sv it:erland). Test and standard (PGE + PGF2 o) were
app11� on the sW-\e plates ond developed in A1 solvent
systeo (Ben:ene-Dioxane-�cetic acid in the rotio 20 : 20 : � of Green and S�uels�on (�964). After developing the
plates, �.O o:1 strips were scrapped off and extracted vi�h ':"yrodc ·�olution and assayed biologically on the
��e:fu�c� rot stcocch atcip.
Ar.' 1 o� h!c�--1nc
-Hi�t �!ne content of the opongo exudate wa& oasayed
!1ugrooc::r1colly uo1ng the �cthod oC Honkonaon ond
�·r..bec9 (1'l7<:).
f cov.:in wot.. J,r cl pi to tcd Hi t;}J S:C. Tr 1-ch loroacL t..lc --1d � v .. , , l h �"'n) . The mlxtur1.1 wno l• rt. in thr :f rid !ur 2 h ror !vll re c1p1t. l1on of I rot:oin. lt
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