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    Experiment No. 03 M : ‘Aim: To study Common Laboratory Animals “| ; " References: 2 4 ey. Hilton & Company, Kolkata. 1.Ghosh MN. Fundamentals of Experimental Pharmacology. di | yt VallabhPrakashan, Delhi, Page’ 2.Kulkamni SK. Handbook of experimental pharmacology. 4" edition, i No. 1-2, : he ot Introduction: re putational Simulation . re! m ‘The most recent development in experimental pharmacology is the sophisticated computational of simulation of animal experiments, both in vitro and in vivo, referred to asin silicon test. It is so fe named because ofits development in silicon on computer chip. It is simplified ecological model that iz reduces a lot of animal testing, and considered to be more predictable regarding the success of the z developing drug in clinical trial. Despite these advantages, the animal experimentation cannot be done yo away with completety, and the drug in question has to be administered at some stage or the other. into an an animal before it can be passed on safely to humans. m ex Care and Handling of the Laboratory Animals ig ‘The handling of the laboratory animals involves two most im portant responsil ies on the part of the z : Ps e Fi experimenter. first, the animal is handled with utmost care so that it does not suffer any pain, and aa secondly. a due regard is paid towards the health and wellbeing of the animal colony. Even when they int are killed at the end of the experiment, it should be done by a humane method, ic. euthanasia. which i means painless killing, The Indian National Science Academy (INSA)* provides the following guidelines to be followed by fe all the research institution in the country engaged in animal experimentation ta Haine: care, breeding and maintenance of experimental animals to keep them in physical comfort Se and good health, and to permit them to grow, reproduce and behave nor mally. ch : The sourees of experinental animals of known geneties, health and nutritional status, the sacl ing Development of training facilities for sci i : ientists, technicians and o} ive : the animals and their use newman iclans and other supportive staff for the care of che Acceptable experi mental techniques and procedures foe anaesthesia and cuthanasia. Developing alternate in vtto systems to replace animal experiments The construction of institutional ethics committees, © co u the Obligations to ensure the ethical use of animals. a functions and the legal and ethical Committee for the purpose of the control an id super E (CPCSEA)* has also set guidelines for laborat al emi re tory animal faci promote the humane care of animals used in biomedical and behi basic objectives of providing specification that will enh Pursuit of the advancement of biological knowledge that is Periments on the Animals lity. The goal of the guidelines is to avioural research and testing with the rete nna! wellbeing, and quality in the Felevant to humans and animals, at one nto the ind ey ich Is to he he ofthe common laboratory animals such as mice, rats, guinea pigs and rabbits can be bred and Me under ideal conditions to provide a reliable supply of uniform and healthy animals for the beet ise of the experimentation. Dogs and cats, however, can be obtained from outside sources as and meme eauired but it is always better to keep them under quarantine for a few weeks before they are utilized. preding Types inbred strains, The primary purpose of inbreeding is to reduce the number of individuals that are peerozygous Tor any one gene pair, and to inerease the number that are homozygous for poe oat ater member ofthe gene pair Coefficient of inbreeding is the degree of inbreeding measured by the reduction in the number of heterozygotes and increase in the number of homozygotes. A strain is regarded as inbred When it has been mated full brothers and sisters bx s or full-sib mating) for 20 or mare consecutive generations. By this method most rapid and a maximum increase in the coefficient ofiabeeding can be achieved. Even then itis not completely inbred in the true sense, because it gives am inbreeding coefficient of only 98.6%, which means that on average 1.4% of the originally heeoz)gous genes are stil heterozygous. Parent and the offspring mating may be substituted for b x Satin provided in the ease of consecutive parent x offspring mating, the mating in each case isto younger of the two parents. The laboratory animals most commonly used their inbred states are mice and ras, The chief drawbacks of inbred strains are thatthe animals become generally less healthy and more susceptible to diseases, and their reproductive capacity is reduced(inbréeding depression). In experiments on the inbred strains the environment need to be more closely controlled than in test using hybrids or random-bred animals (Falconer, 1976) Fines the first generation (Fi) of a cross between two different inbred strains consist of animals that a genetically uniform but not inbred. The Fi animals, unlike inbred strains, do not suffer from iteeeding depression and they are improved in viability. Hence, they are less costly to produee than the inbred strains. They also exhibit increased resistance to diseases, Random breeding, It means that the young animals for breeding are chosen without regard to their Peper ie and mated together without regard to ther relationships: the breeding is thus ramowy wah Fespect to pedigree. Selective breeding, It consists of choosing the individuals to be parents according to the shattcterstics that itis desired to change. If, for example, itis desired to increase the body weight ie the heaviest individuals are chosen to be the parents of the nen generation; or if it is desired to Chica’ the response to certain drugs, then the individuals showing the greatest response to it are hosen as the parents, Breedine methods | Hand mati atin ating, the male and female are brought together fod brief reriod and land th e Mathes over. Rabbit and hamster are mated by thie method. \ 2 Pair mati Salina, In the case of mice, one mal thet ating, n One male is mated with one feinale, and left too. fe "breeding life. Inthe case of rats, Pregnant females should be is Lea ; ‘Solated prior to delivery. Tomaing, one male is mated with two females, \ 4 ure iti \ sepettig Up ac tena are regularly mated with one male. The female is isolated soon after established, and replaced by fresh females. Guinea pig is ‘nated by this method.” ee ee it f animal model is one of the most impor clog sutable animal model should be armacologi mn elected which follows three main s ai cological study. Hence, pharmacological objectives: 1 Use ofan animal phylogenetically closer to men, oF i sible to that in men vestigation is as closer as pos 2 Use ofan animal in which the process under dered to be similar 3 Anatomy. physiology, and biochemistry are cor vadly experimental animals are divided into thr Experimental animals (house, Rat, Guinea pio, Gerbil, Hamster, etc.) Non-rodents (Rabbit, Monkey, Dog, Cat, Pig ele.) (frog Pigeon, Zebra fish, Chicken, ete.) | | | | THE RAT (RATTUS NORVEGICUS) | Albino rat is one of the commonest laboratory animais suitable for experimental work because of its small size and greater sensitivity to most drugs. It is also the most standardized of all animals. It can be bred to obtain pure and uniform strains, and is found to be very sturdy to withstand long period of experimentation under anaesthesia. There are two original strains of albino rats, Wistar and Sprague-Dawley that have been widely used throughout the world (Lane-petter, 1976), Wistar rat is a quiet, moderately prolific strain rat spontaneous tumours. The head is wide, especiall is alwoys less than the body —length, her resistant to infection, and has a low incidence of in the male, and the ears are long. The tail length Spague-Dawley rat id more rapidly g1 narrower head, and a longer tail, wh Se Tae eae Betlig than seintar mgs art has longer and especially to respiratory disease. ich may equal the body-length. It is also resistant to infections, There are certain anatomical peculiarities that should be i last vomiting centre. Rats do not have any ae fee eae feauss they Pancreas, thus pancreatectomy is difficult to perform in < te inte oot eee ares an obvious division into two parts by prominy ia Citing ridge), The upper rece st curved transverse ridges lower three- fifth glandular secretary portion, Histologi eat and thinner than ferences. The glandular portion consists of two elec k Lean ets shows distinct ~ by naked eyes as well as under a mio lesser curvature below irregular inner ining, straddle the entre the thick portion of the st that this part of the both anatomically a noted. Rodents do (rumen) is t step in any of the experimental _— ( i tk am Ou ens Mey en ™ © wey ais eyele, The eycle makes its appearance at puberty at the age of two to three months, and the Yo, lasts for about four to five days being divided into four stages according to the cell type whole cycle and in vaginal smear. These are as follows (Hafez, 1970): esis (90 15 h) is characterised by sexual receptivity when the female will allow copulation. a During this period there are increased running activity, quivering of the ears, and lordosis in the presence ‘of another rat. The vaginal smear shows 100% cornified epithelial cells. Met-oestrus (about 20 h) follows oestrus and occurs. shortly after ovulation. The vaginal smear is characterised by many leucocytes with few cornified cells. Di-cestrus (60 to 70 h) is the longest of the phases, and the vaginal smear consists of mainly leucocytes. Pro-oestrus (about 12 h) that follows di-oestrus is preparatory to the next oestrus phase. The vaginal smear is characterised by nucleated epithelial cells either singly or in sheets. Vaginal smears may be taken in the following way to confirm the actual stage. Cotton swab made with toothpick is moistened with saline and gently inserted and slightly rotated within vagina. The swab is then pressed in drop saline on a microscope slide and examined under low power. An alerative and better method is to take the vaginal wash with few drops of saline with the help of capillary pipette fitted with a rubber teat. of its Experi a ce Rat as an Experimental Animal >d of —_Ratsare particularly suitable for testing of psychopharmacological agents because they can be trained properly for various types of work performances including development of conditioned reflexes. They are also utilised for the assay of different hormones and for the study of sa) wes and for the study of oestrus cycle, mating used behaviour and lactation : and ons. The, ae si nals are also employed for the study of drugs on the blood pressure ing experiments, both in acute as well as not clear. Since there is of gall bladder in the rat ® partial explanation. For the study from 24 to 72 ste secret eS emaNCE Of bie into inst ' animals are alwa; 8 fasted (water allo a wed) for a i eriod varyin ae Pe ) Sa a7) ei ; because they are in Tours Special care should be taken s0 that they do not get aecess 10 their stools, bet jours. Special care shi cir ow" oprophagy) the habit of eating their own stools (coprophas: , je secretion has been developed (Ghosh, 9 the assay ofthe inhibitors of rat (Ghosh and Schild, 1958). itative method for c A quantitative metho secretion int 1958) based on the continuous recording of acik ‘ ean Rats are also employed for the study of analgesic drugs by the method clade mania a the tail, They have been employed routinely in toxicity studies, both acute ani latter, because the drug can be administered in infants of this age group. jology of the liver, since following partial hepatectomy rates almost completely in course of a week It is particularly suitable for the study of phy (60 6 70% liver tissue removed) the organ regenerates al ry and carcinogenicity. Rats are suitable for the testing of drugs for teratogeni also routinely employed for the study of Various isolated tissues as uterus, stomach, colon, etc. are r s of one of the bioassay drug actions. ‘The uterus of rat is inhibited by adrenaline, which forms a bas ‘methods of adrenaline. THE |OUSE (MUS MUSCULUS) Albino mice are the smallest laboratory animals that can be bred uniformly, are cheap and easy to handle. and being very small are the sensitive to a small doses of a substances. Swiss albino mouse is the most widely used strain for laboratory investigations. .as.an Experimental Animal * Mice are employed widely in acute toxicity cases, They are also used for the a: 7 al issay of insulin and a induced pain. General screenii i ing of chemotherapeutics Mice are most frequently used for the testing of drugs gesics, the latter es i 1 pecially against chemi Agents is undertaken in this species, *lY for teratogenicity, Possess normal nu t imbers of iruses (Marx, 1986). Biege mi ble to cancer, 7 Mimour cells and those infected by vi onthe other hand. which lack Killer cells are unusually u Isa uscepi Being very small and delicate the isi Being “olated tissues of mice ‘are rarely used excepting vas deferens and Gu ke' su to the pi It It Tr te co Osh, non the omy Yor Ssay y to se is cally THE GUINEA PIG (CAVIA PORCELLUS) laboratory animal ike other rodents, it is easy to g docile in a are ver} en in captivity. They differ from other lab rodent, being docile in nature, an y rile to T.B. and anaphylactic shock, also very sensitive to histamine. “NEE -s usually due isceptible iylactic shock, also v ve to histami Animal - w asphyxia, in histamine or anaphylactic reaction. Penicitin 00 (0 times more toxic to them histamine o1 yl ; ais opamine causes @ fall in blood pressure. The uterus is inhibited by adrenaline. Guinea fhan mouse. Dopa pig serum contains an enzyme asparaginase tha shows some anti-leukemic action. Guinea pig or cavy are proved to be most use They are used for evaluation of bronchodilator compound against experimentally induced asthma. Widely used in imniunology, particularly in study of delayed hypersensitivity, which are conducted using different antigens like egg albumin, horse serum, ete. Employed in study of local anaesthesia and bioassay of Digitalis Suitable for hearing experiments as having sensitive cochlea Suitable for experiments on O2 consumption as they are more resistant 10 hypoxia Hisused in ascorbic acinl metabi m, is suitable host for mycobacterium infection, and used in study of TB. They are closer to humans than rats for study of isoniazid toxicity Various isolated tissues such as ileum, vas deferens, etc. a ferminal ileum is the most common and standard prepara Compounds, and is sensitive and suitable for detection and assay o i al studies. The nof spasmodic and antispasmodic histamine and related compounds. THE RABBIT (ORYCTOLAGUS CUNICULUS) studies. Most interesting finding is the linkage ‘nee the colour of fur. In atropinesterase containing red or it recovers within 10 to 15 minutes, while in Rabbit as an a Experimental animal ‘They are used for the pyrogen testing for the intravenous fluid Insulin and other diabetic drugs, curare and sex hormones are tested in rabbit ‘As having sensitive skin to irritants, widely used in experimentation on topical agents. : Rabbit is routinely used in serological work, screening embryotoxic agents and teratogens. Its especially suitable for study on reproduction research. Isolated heart, duodenum and ileum are some preparation commonly used for testing of drugs. THE HAMSTER Two species of hamster are commonly used as laboratory animals, the Syrian or golden (Mesocricetusauratus) and Chinese hamster (Cricetulusgriscus) ‘They have chunky body with short legs, fluffy tail. There are 4 toes on front foot and 5 on the back fooipad are hairless. Prominent cheek pouches extending up to shoulder region Hamster as an Experimental Animal Golden hamsters are used for investigation of number of fields such as virotu: A BY, cancer,” autridion research, genetics, pharmacology, toxicology, and reproductive physiology. Chinese hamsters are most commonly used in researc! h on diabetics, due to high inci spontaneous diabetes mellitus. = sere This species also used as host in certain parasitological inves igation. Chinese hamster shows low chromosome number (22) this make: ae Wwe Io s them excell . a investigations, genetics, tissue culture and radiation research, lent tool for eryptologic Strips cut from pouch of the Golden hamster stomach used in vit a assay of prostaglandin E & f 8 useful in vitro test preparation for the Ca ext hamster pe back. puto jence © THE CA’ i use for Cats are the common among the carnivores that are relatively easy to obtain ant to experimental purpose. pial Animal Catsare used in acute experiments for study of drugs affecting blood pressure. Both anaesthetized and spinal aration are us calechojamine, P 'd, the latter being suitable for the assay of asare essential in study of nerve centers in brain, Most suitable for the toxicity studies of compound like acetanilide, THE DOG Dogs ‘raneg MO*8F, Beagles) are useful among the lab animals because they can be tamed as well as the nit tMOut much difficulty. Small stomach and short intestinal tract resembling those of man, it is: "mal of choice for studies on gastric secretion and digesti ion. aid _ = perimental Animal y used in study of drugs on blood pressure in acute experiments ao pd lation that needs unanaesthetized animals. Dogs.asan ‘Anaesthetized dogs are routinel employed for experiments on circul neous diabetes mellitus resembling man, hypoglycemic drugs can be Since dog develops spontai studied in this animal. THE MONKEY Both structurally and functionally monkeys and apes closely resemble man. The high neurologic! development makes them suitable for studies of psychopharmacological agents. Monkey as an Experimental Animal Primates are used in the fields of virology, parasitology, immunology and immunosuppression, g-2 nutrition, reproduction, ete THE FROG AND TOAD In the frog and toad adrenaline isthe transmitter in sympathetic system. Hence, the frog heart falls in range of the predominant B adrenergic organs, and more sensitive to adrenaline. g ‘The Frog and Toad as an Experimental Animals They are commonly used in studi F m, y mmonly used in studies of the action of drugs on th i n a of drugs on the central nervous sys hear on the neuromuscular junction, as well as the diagnosis of pregnancy. pete. on he heat Frogs have been used for determining retinal toxicity of drugs. Result: “Fhe Commen , Labssatony animal was dhuditd ae = c Mowing questions Ents ae Answer the following questions ae S alsg J vuhat are the oftFerent obsective ee sales oo imemtal pharmacology Sticly. § Scan 4, Animal 0 Ea P ee tegen te AM. . 4 ‘0 Lag : 6 iy use oF av animal r Mere nthe lprocess under Inver iy use oF arm antmal eee hak men ' loving ro clon as PO se consider re fochemn shy o& WS Antemy , physfology emd efochemt ty similar gmaf used ("0 = (mental anim Fession, a Jarre clown the Cemmen Exper elegy SHidy, a Ea iy mite « eins ius Rabbit ¥? Hameln vid c ‘4 i iiyme t falls in Mild Freeg, Xi) Toad Ga-luhat ave the diferent breeding method. 3 : iy Hand making t- “The mate ond Fema AVE breve wo. Jd one Together foe boner pooled and thom Earls eet the meeking t's over Gabbit and #4™ ster on a ar meted) ath am Femate and leAr poget hr fer the rresnoP qneie lwrerding -tiFe . ity Peas making: - Bn Case of Ate, ope Male 1's mnatked with am ‘Female Wh together For phe mesh oF thedy Inrbeol'ng URE Wi) srvfomebing!) Up re 4 Remote om caegularty mated with one male Dy. rereyke down the common Speaeig of Following primal Faibable for not suffer from hypovolemia Basic consideration prior to the blood (a) Selection of Needle « Newdle selection is based on the size ofthe animal and the site of the venipuncture. The larger the bore of the needle. the morerapidly the sample can be collected. © ess damage tothe-blood-cells-is another benefit to larger-needles. Proce + "The main -disadvantageto large-bore needles is-the potential damage-to the vessel. © The choice of size range from 17-27 gauge needles that are 10-15 mm in length Requi need (b)Handling and restraint + Itis for to make the procedure easy and comfortable for both animal and experimenter i + Use of anaesthesia is preferred for the small animals as compared to large animals because handing of animals with anaesthesia it increases the accuracy and quality of the outcome of . the experinients p + Hethe-handling-and_cestraining of the experimentat-animal is not proper it-maysinterfere.with : the parameters.of the study. (©)Dilation of veins # This done by the warming or use of irritant. Anil species —__Shoraneshesia Medium anesthesia Long anesthesia Proc’ Mice =e 32 = ° (se Yyfaze + Ketamine ylacine = ketamine Rea - Halter intl (61mg + 100 gi) ‘6 mge60 maim.) need : Xylazne + ketamine Xylene + ketamine (5mg+ 100 ; (ings t00mgim ip)or i Urea 1200 mepkg ip) : ie pig Isofe i r . ~ ae de aie +tetanine ylazine + hetanine d (2g + 80 mg i.) $ . Fats sore inalation ee : yacine + ketamine ylaine + ketamine enter mals becaus > Outcome of nterfere with 4} por y ip.) irom vagal Temporary cannula (rat. mice) Blood vessel cannulation (rat, guinea pig, ferret) pig) vartery (rabbit) co Tarsal vein (guin o Marginal ear vei ‘Terminal procedure ‘© Cardiac puncture (rat, miei © Orbital sinus (rat, mice) ‘© Posterior vena cava (rat, mice) guinea pig, rabbit, ferret) procedure for Saphenous Vein [3lood Sample Collection Requirements include animal, rodent handling gloves, towel, cotton, sample collection tubes and 206 needle. Lateral saphenous vein is used for sampling while taking aseptic precautions. + The back of the hind leg is shaved with electric trimmer until saphenous vein is visible. Hair removal cream can also be used. + The animal is restrained manually or using a suitable animal restrainer. + Hind leg is immobilized and slight pressure may be applied gently above the knee joint. +The vein is punctured using a 20G needle and enough volume of blood is collected with a capillary tube or a syringe with a needle. The punctured site is compressed to stop the bleeding. While collecting blood: © the local anesthetic cream may be applied on the collection site © no more than three attempts are made © continuous sampling should be avoided and © collecting more than four samples in a day (24-hour period) is not advisable. Procddure for Dorsal Pedal Vein Bidod Sample Collection Requi ts include animal (rat or mie), rodent handling Bloyes, cotton, capillary tube, 23G/27G needle and‘bjood sample collection tubes \ +The aninta is kept ina restrainer \ * The hind fot around ankle is held and medialdorsal pedal vessel is lotated ‘on top of the foot. + The foot is clegned with absolute alcohol and dorsal pedal vein is puncitwed with 236/27 needle. see * Drops of blood thaNwyould appear on the skin surface xg collected ina capillary Tobe and a little pressure is appliethto stop the bleeding : Requi ———— Procedure for Ta igements include animal, rodent handling gloves, towel, cotton. sample collection tube and animal warming chamber, lume of blood sample (up t0 2ml ‘This method is recommended for collecting ® large Vv‘ aintaining the temperature around at (withdrawal) ‘The animal is made comfortable in a restraint while mi 24 to 27°C ; as it will result in leukocytosis. If the subbed from the base to the tip vein is not visible, the tail is dipped into warm water (40°C). dain aesthetic eream must be applied on the surface tf the tail 30 min before the experiment, 936 needle is inserted into the blood vessel and blood is collected using a capillary tube or srvcdle, In case of difficulties, 0.5 to 1 cm of surface of the skin is cut open Tanegt ne needa and blood is callented with eepillety ‘The tail should not be r a syringe with a and the vein is pricked with bleeding tube or a syringe with a needle. Having completed blood collection, the bleeding + LEmultiple samples are neede © Restrainer—is~washed—frequentlyto-avoid/prever infection prescure/silver nitrate ointment/solution is applied to stop be-used collect blood ls the needle slowly rembved and the site is mohitored for bleeding. If thehe is no bleeding. one more attempt can be thade. Further attempts Should be avoided in case df bleeding as it may collapse the vein Procedure for Blood Sample Collection with Temporary Cannula Requirements include animal, anesthetic agent, cotton, 25G needle, animal warming chamber and blood sample collection tubes. + Usually a temporary cannulation is made + The animal is restrained and local anestheti tail tip). + The tail is either cannulated or a 25G needle is used. + Tail bleeding normally requires the animal to be warmed in order to dilate the blood vessels (37 - 39°C for 5 - 15 min). ‘+ After cannulation, animal has to be housed individually in large cages. the tail veig and used for a few hours. cream is applied on the tail (1 - 2 cm above the Protocol for BloNd Vessel Cannulation Requirements incldde animal, anesthetic gent, cotton, 25G needle, i.v. cannula, surgical blade heparin (or any anticyagulant) and blood mine collection tubes. \ This method in\olves continuous and multiple sampling in the experiRental animal + This method req\jires close and continuou’\monitoring of the animal. + Usually blood véfsel cannulation is done ih the femoral artery, femorah vein, carotid artery jugular vein, vena\gava and dorsal aorta. 7 Surgery is required|for this method and appropiate anesthesia and analges} ‘minimize the pain. + Afr surgical cannul + Blood sample may be + After withdrawing the should be used to ion, animal should be house singly in a large and spacleus cage. lected over 24 hour at the Mlume of 0.1 to 0.2 mi/samble. (ood, the cannula is flushed Nith an anticoagulant and the withdrawn if required) with LRS and cdynula should be closed tightly [Figure * Caution: The experiment Yas to be conducted fully uNer aseptic precautions. - hemorrhage, blockage of cahnula and swelling around the ¥annulation site should be jon for. The needle size and maximum blood volume to be collecded are given in [Table 2] 49 Needle to be used Maximum collection Species mmvelume é 1m —— =26G Ye ‘0 21G 40-15 mI ‘ a 60 - 200 ml i 19-21G € ; aa Guinea pig 20-21G — “Table 2: Needle size used for blood vessel cannulation in ifferen + Requirements inchNe animal, anesthetid aver 2G loo La agent, cotton, 22G needle, hair remover imple collection tubes, dle. hair remover and bloot sal vein is identi legs of large animals. This method is commotl) Tarsal vein may 1 be visible in blue coloi table hair rem fe ‘over. A local anesthetic cream lowly by using 226 needle. 1 t0 0.3 ml of blood can be collected pe! Maximum three samples cd be taken per leg and sample. +” “After the sample collection, bleeding [Figure 6] gentle pressure is applied with finger for 2 minutes to sto? tion cant ot more than six samples from both hind legs are taken. The number of attempts is three or less / jon al, anesthetic agent, cotton,\26G needle, 95% viv alcohol, o-Xylene, Requirements include an Je collection tube. \ surgical blade and blood sai adopted for rabbits. 4 in a restrainer. iv alcohol and local anesthetic\gream is applied on the collection site 10 min prior to samplgg. (If required, the o-Xylene/tdpical vasodilator may be applied topically on the collection site to dilate blood vessels). \ + Size 11 surgical blade is uSed to cut the marginal ear vein\and blood is collected collecting tube. Otherwise, a 2G needle may be-used to collect\plood from animal mar vein, \ + After collecting blood, clean sterile cotton is kept on the collection site and finger pressure is applied to stop the bleeding [Figure\Z] and [Figure 8] + This method is commonly + The animal should be pld + Ear is cleaned with 95% SL Figure 8: Blood sample collection from rabbit marginal ear voin using incision method. Protocol for Blood Sample Collection through Posterior Vena Cava rgical scissor, 21 to | Requirements i ude animal anette agent, surgica\blade, small 25G needle with | t0 5 ml syr we and blood sample collection tube. \ recommended for terminal stage of the study. ‘sped cut in the abdomen is made and the cava blood sample etized and 'Y'- ot + The liver is pushed forward dgd the posterior vena cava \hetween the kidneys) is identified. + 21 t0 25G needle is inserted tdgollect blood from the posttyior vena cava. more volume of blood sample. + This procedure will be repeated\hiree to four times to coll Protocol for Cardiae Puncture Requirements include animal. anesthetic agent, towel, cotton, 19 to 25G needle with | to 5 ml Eringe; surgical blade, tube (internal diameter of 01 to 0.3 mm) for thoracotomy, plastic disposable bag and blood sample collection tubes.n general, cardiac puncture is recommended for terminal stage of, the study to collect a single. good quality and large volume of blood from the experimental animals + During blood sample collection, animal will be in terminal anesthesi + Appropriate needle is used for blood sample collection with or without thoracotomy. Blood sample wil be taken from the heart, preferably from the ventricle slowly to avoid woth of heart [Figure 9}. rane * Caution: If animal has dextrocardia, sampling may fail SS0F, 21 he study de and the ntified. sample. 1050 dispostbl Figure 9: Blood sample collection through cardiac puncture in rat Species Fa ciainn mo | ‘Mouse/Rat =| Tail vein, Saphenous vein, Retro-orbital sinus _ | Distal tail transection, Cardiac (terminal only} Tanase Sas haus vin Taralvein, Cardiac erminal | only) Tabb ier ein,Cariacterainal oni Doglat | Sopher filer ves | cei ane? —[iatiar vets Reto orbtaFanuS faa | Coohaks Saphenous Femoral, radial lugalor Swine Jugular vein, Ear veins _ Chicken Brachial wing vein, Jugular vein, Cardiac (anesthetized only) Table: Sites for Blood collection by species How to separate serum and plasma from blood Serum is the liquid fraction of whole blood that is collected after the blood is allowed to clot. The clot is removed by centrifugation and the resulting supernatant, designated serum, is carefully removed using a Pasteur pipette. Plasma is produced when whole blood is collected in tubes that are treated With an anticoagulant. The blood does not clot in the plasma tube. The cells are removed by Centrifugation. The supernatant, designated plasma is carefully removed from the cell pellet using @ Pasteur pipette ‘Serum preparation | Collect whole blood in a covered test tube. If commercially available tubes are £0 be wee NS researcher should use the red topped tubes. These are available from Becton Dickinson ED a trade name for the blood handling tubes is Vacutainer. After collection of the whole blood. allem he blood to clot by leaving it undisturbed at room temperature. This usually takes 15-30 minetes. Remove the clot by centrifuging at 1,000-2,000 x g for 10 minutes in a refrigerated centrifuge used. the The resulting supernatant is designated serum, Following centrifugation. it is imponsnt to immediately transfer the liquid component (Serum) into a clean polypropylene tube using & Pasteur pipette. The samples should be maintained at 2-8°C while handling. If the serum is not analyzed immediately, the seram should be apportioned into 0.5 ml aliquots, stored. and transported at - 20°C or lower. It is important to avoid freeze-thaw cycles because this is detrimental to many serum components. Samples which are hemolyzed, ieterie or lipemie ean invalidate certain tests. Plasma preparation | eg, EDTA-treated )) are indicated for Collect whole blood into commercially available anticoagulant-ireated tl (lavender tops) or citrate-treated (light blue tops). Heparinized tubes (green tops) some applications; however, heparin can often be contaminated with endotoxin, which can stimulate white blood cells to release cytokines. Cells are removed from plasma by centrifugation for 10 minutes at 1,000-2,000 x ¢ using a refrigerated centrifuge. Centrifugation for 15 minutes at 2,000 x ¢ depletes platelets in the plasma ‘sample, The resulting supernatant is designated plasma. Following centrifugation. it is important to immediately transfer the liquid component (plasma) into a clean polypropylene tube using a Pasteur pipette. The samples should be maintained at 2-8°C while handling. If the plasma is not analyzed immediately, the plasma should be apportioned into 0.5 ml aliquots, stored. and transported at -20°C or lower. It is important to avoid freeze-thaw cycles. Samples which are hemolyzed, icteric, ot lipemic can invalidate certain tests. There are other commercially available tubes for blood sample ISA kits collection. Invitrogen has not evaluated some of these tubes for compatibility with our El Serum and plasma tubes, The commercially available serum tubes are as follows: Red No anticoagulant Red with black Treated with gel to help to separate the clot (not evaluated). ‘The commercially available plasma tubes are as follows: Lavender ‘Treated with EDTA, Aus thaws Aes Treated with citrate. Blue Go ‘Treated with heparin, Treated with potassium oxalate/sodium fluoride (not evaluated). Grey Yellow Treated with potassium oxalate/sodium fluoride (not evaluated). esthesia and Euthanasia ful to humans are considered to be painful to animals. Always use the jes when performing surgeries on animals. Procedures that are pi appropriate anesthetics and analg Definitions: Pain: An unpleasant sensory or emotional experience associated with actual or potential tissue dam Anesthesia: A total loss of sensation in a part of or in the entire body Analgesia: A complete loss of sensation to pain Euthanasia: Causing death without pain Anesthesia Classification of Anesthetics |. Injectables: Effects of these agents cannot be reversed quickly. The drug must be metabolized, excreted, or counteracted by another drug to terminate anesthetic action, Some are controlled substances. Logs must be kept of usage and are subject to audit by the DEA (Drug Enforcement Agency). Examples include sodium pentobarbital, ketamine/xylazine cocktail, Medetomidine ete. Inhalants: Effects of these agents can be reversed quickly. The agent is eliminated when the administration is discontinued as the animal exhales. The most common inhalant is isoflurane. 5 Dissociatives: Agents that depress the central nervous system and produce a state of catalepsy (64. ketamine). These are most effective when combined with tranquilizers and sedatives (€ xylazine, diazepam), 4. Local Anaesthetics: Theses agents are injected at the site of incision. These agents are used as a Supplements to either inhalant or injectable anesthetics. (e.g, Lidocaine, Bupivacaine) Points to Remember Aaah an wud analgesic thadis listed in Sour protocol. en as a a a * Calculate the dyse by body weight, * Test the animal" reflexes (pedal and palpbyal) after the anesthetic has taken effect and betiye ri ge Monitor the dept anesthesia) Monitor body tempelature when the animal is abesthetized — temperature falls, especially jn small species. Perform\surgeries on a heated surface when available Drugs under the control\pf the Drug Enforcement Aency must be =“ in a locked cabinet and rate of respiration (increase in depth and decrease in rate signify a secure area, A written record is requirdtl when controlled drugs undby the contro! of\the DEA are used (how much of the drug you haye, how much was used and thy what purpose AAn inventory list of anesthetics, analgesics, tranquilizers, sedatives and othe drugs shouldbe kept. \ esthetic agents for laboratory animals T a (mgtis) ms JF POM Battate | Eeehe hays fl + Adkquestic Sine sag 228% lo agen + Flow a rage |. abated ating E08? |2¢35ng TP ee 7 tui = | arty | conbesen % si) slain | cian | s0-4tag i |, v oe Ougig P | some ig Po mete yj ] sngig ]omgke | LP [Smee Somes | 1000p | 1500mgreg 1080 P| Pe onomerTP" sscomtg hap I" 2 a “(prolonged amsesthesi: emia procedures ol) : ued tree slvr and bronchi NE: ose 002 205mg i seer storie yn ce ATROPINE: Des rains and petber fen ap Tainie oe aero mean * ifm = intramuscular, i= intravenous, ifp= intraperitoneal, f= subcutaneous tis a Greek word which means (Eu- good, Thanatos- death). It is a humane killing (sacrifice) of an animal which produce rapid unconsciousness and subsequent death without or minimal pain or distress of animal. Methods of Euthanasia |. Barbiturate overdose 2, CO2 asphyxiation: Must always be followed by a physical means fo ensure death, such as cervical dislocation or pneumothorax. Use CO2 from tanks, not from dry ice. 3, Cervical dislocation: Must be preceded by anesthesia or CO2 asphyxiation unless scientifically justified in the protocol. If cervical dislocation alone is done, it must be performed by well-trained personnel, 4. Decapitation: Must be preceded by anesthesia or CO2 asphyxiation unless scientifically justified in the protocol. Inhalation of gases: e.g. Carbon dioxide, carbon mono? and halothane. Ne fe, carbon dioxide plus chloroform Techniques should result in: ‘© Rapid unconsciousness © Cardiac arrest © Respiratory arrest «Loss of brain functions ie Euthanasia Procedures «Always follow the methods of euthanasia that are outlined in your protocol. © Ask questions if you are ever unsure of a procedure. + Fill out a green euthanasia sheet and mark the cage with an “X” if you need DLAM to euthanize animals for you (follow procedure in Policy Manual). Result: Cornmen Latlosy \ i A plasma Blood widrraus Baerrum ond P F ie ete euthanas 4 used For om studies was olueiied: sapere” Prof til ver the followir stions ae [ou lahat 1°s general privaple of Bt Se Bivod. tallecHannin i c Yo ee by the InsHlute anrmal ephics Sepmitee ef, pe ee Blood sample may be collected UndeT anesthesiy lt ot withoub amesthesiy - 2 Yale! © Adequike traming. Le Requeney oF blasd colled'on ine G2. \erni'te the site of copechon oF bleool Fam whe Followlng omimal oS ; ore } Mouse/ pop 7 Tatlvein, saphemoyys vetin , carole a fi tl i fe . ‘ + Uc Cteeminal on a iy Rabbit r= mangtnal etn cardite C M aso Til» Hamster: Jugular vern, Reb ovbrtal consi sparing : 7 - sas ous ven Taasal Vein Cardl’qc oa tal i> guinea pig:- Saphenous mi > % d ; tae ce 9-3.De Fine anesthesfq and Euthanes tq sists. A Ko fon (ng O Anesthesia'-A total loss of Sensattan PIE -OF os IN the embivre body ny Euthenesiq :- Causing death wrth out pair jP4 what are differnt methed oF euthanas! the mt propriate eerie Ovi sini ‘east pain and distres: isn a strong > Bobi burae overdose Pic ot Admin ity coq eSPhyxianin | ld ang iiiy- cervical dit lecabien stein \v) pe CoapitaHian bai 0There V? Inholapin of gases. 2G, a aR wag I Mae | vies tam | eu ase Ferlormanceand shan e Distribution Shas amy | Mendance@ ih Marks we om mT sf Experiment No. 07 — Aim: To study the effect of hepatic microsomal enzyme inducers on the phicnobarbitone sodium sleeping time in mice Reference: ') Kulkarni S.K.. Handbook of experimental pharmacology.4" edition. VallabhPrakashan, Deli Page No. 128-129 Principle ‘he drugs which induce hepatic microsomal oxidative enzyme system enhance the metabolism of other drugs. As a result in the presence of an enzyme inducer the duration of action of second drug will be reduced. This has significant relevance because when more than ‘one drug is administered at a Hie one drug may modify the action of another through the microsomal inducing property, me common drugs which induce the hepatic microsomal system are phenobarbitone. and meprobamate. Co-administration of any drug with either of these drugs may affect the disposition of the second drug and therefore, the desired pharmacological effects, Requirements Animal: Mice (20-25 x) Drugs: 1. Phenobarbitone sodium (Dose: 50 mg/ solution containing 5 1 solution every day) 5 Phenobarbitone sodium (Dose: 45 mg/kg jp, prepare a stock solution «staining 4.5 my/ml of the drug and inject 1 ml/100 g of body weight of animal) ip, once a day for 5 days) (Prepare a stock nl ofthe drug and inject 1 mV/100 g of body weight of animal, Prepare tiesh Procedure 1. Weigh and nuniber the animals. Divide them into two groups, exch « mice. 2. To the first group inject phenobarbitone once dally for $ days. Io the second distilled water. similarly for 5 days. 3, One hour after the lst dose of phenobarbtone on the 5" day. inject the phenobarbital To bth the groups 4. Note the time of injection and time of onset of slep (loss of righting ree), When the animal sleep, put it on ils back so that when i regains consciousness i ill wn over wy = Bosture (end point), Animals should be placed apart so that no mosse disturbs the eae whi it recovers from barbiturates induced sleep. 5, Note the time of recovery and duration of a rising Of at least 6 group inject al en Inference ¢ sleep for shorter duration of ime &s compare mat pre-treated with phenobarbitone sleep for shorter dura Pad t0 animals imal 7 The arith distilled water treated with ans — le “ety, =. 7 Result The ePpeet oP hep adeh c py) ovo orn of ena me Mau cer on fhe phoash ob} pore, : an sodjum on Sleeping time ofr ce Was Suckhed \ Answer the follo juestions Quhat & the hepahe mieosomal Mneluctng bol, ang: The mechanism of aductjon of hepan'c mere somal ncaa Dwg metabolised enzyme by a Sested oF barbiburatey y. ered! The eattond oF fnductton was related fo the plavna hal rbitone LIVE OF baabitembe. sposin ng ® umite the principle of hepatte microsomal eneyme induciiy on the phenobavbitene, ea ms!-—Cha dug whith tnduce hepakic mierosemal oxidakee ATE a siyg Enzyme syskem enchane the mibabot/sm oP other epare fs org AS result th presence OF An enzyme Induay L/tml of ty the climmeh'n of acHtn OF seconol olrug, WH Reduce the aontinhShis signet cank velevance beacure 1AM more hon one Arig admin shred at AL Hime ond, om mm meet Fy the ach» “at least é OF om other Sheroughithe yn|'er0s0ma] Mrdtang. is mre party, Pulp see —vthe common nag whith tnduee the hupatt's al toto Sysiem are pentoladibene.omd meprobavtitone a ® whar fs the sedative and typo & EMmistthe her whe Die Rerent lug oF Seclal’ve and Hypmott’c olotig Ms:@ Gedahvet- th Ps a CPTESSIEN OF CNE without! Causing crows “Use| Uy emotional enellater , Aypertension ConVvolston, and preanesthesta ete. hypnotics $- Th &s a stronge cbypressian oF ons causing éleep and as used rn thsominitg the used as hyprot’: oo Can be used os hupnollc as a sedahire and aryibly H% an Out nok onaioly he cust sedahtre ( Low close) [ Example + Secla live and RypnoHes- @ tong acting 1 phenobadbitea| | me probabil tal, barbjtay Seco basbi fof r ' Marks Viva (02M) | GLP (03 my | Performance and | Distribution [= o | FP ef oe | Sass iment No. 08 nt on frog oesophagus! buck a | Erp ags on ciliary motility/moveme! cal cavity. im: To study the effect of du , Kulka d € y,4"" edition, VallabhPrakashat |. Delhi, Handbook of experimental pharmacologys4 1) Kulkarni S.K., Handbook © Page No. 188 2) Principle icles, Similarly, the : agus help in the movement of food particles. Similarly, « iti inthe buccal cavity and in the oesophas ithe respiratory tract and of p funct s been established it (patna Sa } wil in cystic fibrosis. The integrity of mu y ronchitis,asthina and in cy e in that ay diseases, Cilia exhibit a great degree of autonomy 1) Ht air-way diseas ‘ degree of auton cI ervous innervation. It has al ce le of functioning in the absence of nerv a tips io the they ar aPeehotine present in the mucous membranes of trachea and buccal cavity helps he ee ae e neat Icholine serves as a local hormone and the presence of ry ‘iliary movement. Acetylcholine a sence of coral tes fact that acetylcholine is locally sy nnthesised in the mucous mem importance mucocili diseases such as chrot function is very important in these Requirements snimals: Frog Drugs: 1, Physostigmine stock solution (1 y2/ml). 2. Atropine stock solution (1 g/ml) Physiological solution: Normal saline : , Equipment: Frog board, poppy seeds or tine pieces of cork, stop watch, surgical instruments Procedure I. Decapitate the frog and pin the fio: the frog board on its back Pin the lower jaw to the abdomen cutting sufficiently the buccal cavity esophagus. Keep the buccal cavity and the o} saline To assess the distance travelled by th Jawand other at beginning of the oeso) taken by the particles to move from oesophagus. Place a poppy seed or a small stop-watch and note the time Repeat this several times. 5. Puta few drops of and exposing the | pening of the wet by irrigating it with normal Particles, fix two points i.e. one at the start of the lower phagus. Keep this distance constant to measure the time 4 fixed point in the lower jaw to the beginnit of the Dicce of cork at the pre-marked spot in the j a jaw. Turn on the taken by the object to reach the beginning of the oesophagus. Physostigmine on the buccal cavity and Pata afier 10 min repeat step 4. Note the 6. Wash the buccal cavit i 'y with normal saline, Put a few i 7. fut 10 min repeat the step 4. Note he time, S*9PS OF atropine on the buccal cavity. - Find out the difference the time taken by the obj aken by the object in presence of aie distance in the buccal cavity saline, physostigmi + Physostigmine Inference: Sarnih + Phisostigmin x ‘ wes = mobi eG Hime ond Ateoptiy ———_. © between the pre-marked and atropine. Ny corel dea crespe dered thre aac 25} Physostigmine reduces and atropine enhances the time taken by the object to move from Point in the lower jaw to reach the oesophagus, respectively. Result: The OF AmQS oy Sf; mon lh ty an On frog mere phagus of hu coay lye Answer the following questions QA Write the principle oP eppest of drugs o ayy moh] Ars cilia aes buces} can} ond fr tte oes phagUS help the mevlment of Food park cle. Shs Tody fhe reer mucedh Fem cht) hos been ‘eg pal} sheg ry Tespirahyy dea th OF muse Useare SU aA chewed c ci tainty ond opt Abos! s+ The inten'yvy of ailage | mucoct lay FoncHan has been v Pestant in these aie way piseases che exhibit a greece deg ree +t dtatorany are capable of. Fanatitrnng inthe Absence of pervory -(nnenvar’m 4 has alga Been clumorsbretedl ther the acety\cholitne peesent in the mucoes ynern brane Leachea pnd bo ccad canity bp tn the Ci (avery Verney Acebj|cheline Serves gg focal formers ond the choline acehjlese Sopport the Pact locally synthesized In +he macouy Pre-many esemce of Ace} Jcholine fs mem brrene. L °| the a ON ecb ond buccel cute 2 orton oF aceby|cholt*he 69 MULES Mem dean, } avnsie Ib Ts clemonstrotedl phat the ecety) choline peesemt in the mucot ‘Waar besiea roche q And buccal cowl ty help inthe erlfary movernent- Atehcholine serves at a Letal hawmmng and the presence af chletine acetylare support ee Phat atcelyfcheline 1 Lecady synth LOFhe mecoug mem brong Daily Practical Assessment sheet 20_~__ Date: Marks Performance and Total | Viva (02M) | GLP (03 M) ‘Attendance (2 M) Sign Distribution Skills (3M) (10) Marks Obtained Oe] ot] joc] om Je we Heo} ent No. 09 Expe | Aim: To study the effect of drugs on Rabbit Eye. Reference : 1. Kulkarni S.K., Handbook of experimental pharmacology." edition, VallabhPrakashan, Dei, Page No. 193-195. 2. Principle: A large number of drugs are used for their local action in the eye as eye drops, or eye ointments. Most of these drugs belong to antimicrobial, autonimic or local anaesthetic groups. The ye is supplied both by sympathetic and parasympathetic nerves. The superior palpebral muscle and the dilator pupillae of the iris have sympathetic supply. The sphincter paplillae of the iris has parasympathetic supply which exercises dominant control. The ciliary muscle is also supplied by the parasympathetic nerve and when it contracts, the ciliary body is moved inwards and forwards, Because of this the lens bulges forward and the eye is accommodated for near vision. The opposite effect is produced by the relaxation of ciliary muscle resulting in paralysis of accommodation. When the pupil dilates, the iris folds back near the opening of the canal of schlenzn and the drainage of aqueous humor is decreased thereby increasing in intraocular pressure. Constriction of pupil by the opposite action, will increase drainage and reduce intraocular pressure. Atropine, an antimuscarnic agent, blocks the effect of endogenously released acetylcholine on the circular muscles ofthe iris and the muscles ofthe ciliary bed to produce mydriasis and spasm of accommodation izading to cycleplegia but without producing loss of corneal reflex. The mydriatic Gucet of atropine eab be easily demonstrated in abbits. AS per the CPCSEA guidelines testing of Geass on e¥e has to be done with utmost care and prior permission. The drug or the solution re by {ested should not hurt or cause permanent damage to the eye of the animal, Requirements: Animal: Rabbits (2-5 kg) Drugs: Atropine (1% w/v). physostigmine (I%w/v) Equipment: Rabbit holder, torch light Procedure '. Place the rabbit in the rabbit box keeping the head outside, 2. Observe the size of pupil in both the eyes, 4, Examine the corneal reflex by touching a side of the Cornea with a cotton swab of ti of tip. 5. Instill a few drops of atropine solution in the con} i : ; s iunetiva (4. i P the right eye of the rabbit the left eye of the rabbit would serve eons porte us | Instill normal saline in left 6, Record the pupillary size, light reflex and corneal reflex tabulate observations. alter 10 min of drug instillation and ee N Po OF the fy “UDpligg 8nd ft Te aN Odation et he daw F Pupils, ety lcholig. and spay The myx Mes testy solution i ght be 10 sine toe” 71 me F Repeat the experiment with physostizm remit: The eprer oF dg on crabbit eye was sHidied GA. tehgts* what os mMyadefasi Ans ! - Pao ANS: 9-8. ‘- Cycloplegia : - Beacumeee Myadrites 417 Ser" Answer the following questions re and rmtosts- fs cath ar wyadersts up| Ste tle oF hith ane Fer awely hich acre nearee, Increases ty Pupt] Ste KS and ebug whieh inness P myadrihes doug Beause Y & Bur Fa) fo cobseree bse mivesi’s’- Peawease in puss!) U's Called oa and dug whith theses Pup) 2 1 mniokecs | Braue af mioste OO (ndividual Con see ob) er od G8 nearce Bt Cat fo observe whivh ace Far away fF Became dear Pnclividedy Can see objeck whith are Rarr.cus but Purl : Se whith ave Teaver Thi abit ps called oyclorley?4 Pavolysts OF ciccomedatien >— Beacuse 4 mfesfs 7 Indiviclual ar be See the object whith ave Nearee Bub fall Jo observe chfece whith are Fax ausay this (°s~ Paralysis. oF dahon , what s he principle of study the 2 Fred of Pry on rabbit eye § ThS Qnuolyed the shicly °F Sympathere and Part-sympathekie coug action on -2ye . sympattohc PYUd produce doit ‘Geb 5 . myacdotHe ‘Ach n ashile para Sympathen ® etna Produce Moke cdetin when Ppt) what 1s cyctoplag ia Paralysis ° Schema ANS. Bimpathelle Supply. ye Body IS moved Bury ets Os. write the example OF ees aoug's.2 2-¥) Parvasympathelt’: atu FS Daily Practical Assessment a Marks Distribution Marks Obtained Viva (02M) | GLP (03M) Tr het 20_- as! Performance and Skills (3m) oilakes rts Add hace Neus the opening “OF camal oF 94. Oescsibe about Nerve supply fo - the spicfer Pupiikte of the fais has para ciliary $ d crt Parvcsympattel3 ewe Khir dmirrote ; Coutond Barbiuraa alcoho, Benzodiziping Lepressent- aus Ans: what i prindple oF ocrephoromebee g ep chek vy Cherrzonta) actiuhy) Qan be easily measusecl using a7 Ssehee vet ah Which ost whitch spay or Phojoeleetc Ce : Te witha Coanter. comneded tn ctucd™ fc :, “thin fi ncn a Light Feuuing on the phrto COU Out off by the animal and the Caunt is @ecorded Ge Ans--The — Lecomel Gg. wrile the mechomitm of adr of chleracpreatite Hydrochloride. Apss- chlevpromert ne hyetrochleride of ch levopromearine ong chlavjreparniasne (0 the typical OM'sychottc class 1K mechani f achin G& nok enbisely clea but bie te da derivate jae amnbegen BF Thalgo het nH’ sevoleadthog: 2 Mein nihislornin ke. prreperh's nen ‘y ile > the ydaechlenele Sap 2 = ee 4 ee abe aC em ; > . fe cael bragtiHimal amtipsycheH’. egemr Daily Practical Assessment sheet 20_-_ Date: | Pert a rks ’erformance ant T ed [viva (o2m) | GLP (03m) Z Attendance (2m) | TT ion Distribution | Skills (3m) ay Marks | | Obtained | o| | BS oe | oe TTP ‘xperiment No. 12 “Aim:To study the anticonvulsant effect of drug by MES and PTZ method. Reference: 1. Kulkarni $.K., Handbook of experimental pharmacology, 4” edition. VallabhPrakashan, Delhi, Page No. 142-146 Maximal electroshock method (MES) Requirement: Animal: Rat Drugs: Phenyto' Equipment: Electro-convulsometer, comeal electrode (apply150 mA current for 0 2 sec), stop watch Principle: or psychomotor type, can be studied in laboratory ‘Marks Viva ( Performance and a yng, | Viva (02M) | GLP (03 op m ot Distribution 2 Skills (3M) [sete em] ee | se Marks “ oomina | Of 7 & a et Ansett elecposhede, — a a aim Refer Requi Animal Drugs?) Chlorpt Equipn Princi Compu is supp purpose activity Apomo behavio sniffing behavio Chiorpr blockin, Procec 1 a4 1 sigh x LK rr Experiment No. 13 ‘Aim:To study of stereotype and anti-catatonie activity of drugs on rats/mice Reference: 1, Kulkami $.K., Handbook of experimental pharmacology, Delhi, Page No. 134 4" edition, VallabhPrakashan, Stereotype activity Requirements: inimal: Mice (20-25 @ Drugs: Apomorphine Chlorpromazine Equipment: 250ml clean beakers with proper plastic lids to cover the beakers. Principle: Compulsive behavior is defined as purposeless activity exhibited by animal, The purposeless activity is supposed to be identical to the behavioral disorder seen in schizophrenic patients who shows purposeless activity. This behavioral abnormality in schizophrenia is due to excessive neuronal activity of dopamine in the limbic system. ‘Apomorphine, a receptor agonist through its dopaminergic activity induces compulsive stereotyped behavior in rats and mice. The stereotyped behavior includes repetitive standing (rearing), continous sniffing (touching the nose to the wall of the container) and licking of the wall of the container. These ehaviors can be easily observed and subjectively scored also. Chlorpromazine an antipsychotic agents, inhibit apomorphine induced compulsive behavior by blocking the dopamine receptors. Procedure: 1. Weigh and number the animals. Divide them into two groups, each comp: animals. 2. Inject apomorphine to 5 animals and place them individually into separate beaker and observe. Note the onset and intensity of repetitive standing (rearing), continuous Sniffing (touching the nose to the wall of the container) and licking of the wall of the container at 0 times, 15, 30, 45 and 60 min after apomorphine. Calculate cumulative score at each time interval. 3. To the second group inject chlorpromazine and 30 min later inject apomorphine to these animals. Place them in individual beakers and score as in step 2. Calculate cumul behavioral scores at each time interval, 4. Compare the actions of apomorphine in normal and chlorpromazine treated animals. of five Inference: Apomorphine induces compulsive rearing, sniffing and liking behavior in mice. The elect is blocked by pretreatment with chlorpromazine. — - 391 | : app mice Result: Stewahyped activity of eng on / wos udled. ae ‘Anti-catatonie activity Requirement: Animal: Rats (150-200 2) Drugs:Perphenazine Scopolamine Equipment: Two wooden blocks, one is being 3 cm high and other 9 cm high. Principle: Phenothiazine and butyrophenone type of antipsychotic drugs are known to produce extra pyramidal side effects in man. These effects such astakinesia, rigidity and tremors are called Parkinsons ike because in Parkinson disease the major clinical symptoms include difficulty t0 move and change posture (akinesia and rigidity) and tremors. These e4ffects of antipsychotic drugs are due to excessive blockade of dopamine receptors in extra pyramidal motor system. Therefore Phenothiazine (chlorpromazine or Perphenazine) are commonly used to produce Parkinsons like extra pyramidal symptoms in laboratory animals and to study anti-Parkinson drugs. Procedure: 1. Weigh and number the animals. Divide the animals into two groups. one far control and the other for studying effect of scopolamine, Each group should consist of at least 4 animals, 2. Inject Perphenazine to control animals, Observe severity of eatatonie response as folloves Stage I: Rat moves normally when placed on the table, score = 0 Stage II: Rat moves only when touched or pushed, score = 0.5 Stage Ill: Rat placed on the table with front paws set alternately on correct the posture in 10 seconds, score = 0.5 for each paw with a total of | for this stax Stage IV: Rat falls 10 remove when the font pas are placed sherman, fio ee bo * score= I for each paw with a total score of 2 for this stase ane | Thus for a single ra,the maximum possible score would be 3.5 revealing the cetat Observe the severity of catatonia at 5; 15;30; 45; 60:90 min after Penman 4. To the second group inject scopolamine and afler 30 min inject Peron already treated with scopolamine, Observe and se 5. Compare the results of both groups. a3 cm high block fails Phenazine tw these animals ‘ore severity of catatonia, Results GHeBeohypeand ante catakenie acHV) pug. on excite [mice wos SHclied Lge "2 pyran

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