Animal Experiment on CNS Part 3 PDF

Part 3 Experimental (In Vivo Studies)  Animal Experiment on 18 Central Nervous System (CNS) EXPERIMENT NO.: 18A Aim To demonstrate the effect of pentobarbital on righting reflex (Hypnosis) in mouse. Background Hypnosis is the process of...

Part 3 Experimental (In Vivo Studies)  Animal Experiment on 18 Central Nervous System (CNS) EXPERIMENT NO.: 18A Aim To demonstrate the effect of pentobarbital on righting reflex (Hypnosis) in mouse. Background Hypnosis is the process of natural sleep and the drugs or agents inducing it are known as the hypnotic agents. This concept is applicable to the patient or human use, but in the animal experiments, the term “hypnotic” means deeper stage of central depression which induces Fig. 18.1: Loss of righting reflex in rat (unable to correct unconsciousness, associated with loss of righting body posture) reflexes and muscle tone. The “loss of righting reflex” is the term mainly used to denote the ‘sleep’ Hexabarbital (60 mg/ kg, of an animal and it is defined as the loss of postural i.p), Diazepam (5-20 mg/kg, reaction which can’t be corrected when the animal i.p or s.c) is kept on its back. In the righting reflex animal turns body in such a way that, its paws or feet are Precautions before Experimentation pointed at the ground. Righting reflex reaction is Laboratory should be dim lighted and noise dependent on normal vestibular, visual and free proprioceptive functions (Fig. 18.1). Animal should be marked properly, to avoid mixing in two groups Materials and Methods Handle the animals with care (minimize the Materials stress and pain to animal) Observe the animals in a plexi glass chamber Animal/species : Mouse/Swiss albino Sex/Body weight : Either sex/ 20-30 g Methods Syringe/needle : 1ml/ preferably 24G on- wards Step 1 Drug : Pentobarbital (50 mg/kg, i.p) Weigh the animals and mark them properly to Other drugs : Barbital (180 mg/kg, i.p), distinguish from one another 184  Practical Manual of Experimental and Clinical Pharmacology Divide animals into two groups (n = 6 in each Step 3 group) Observe the animals for 45 min to 1 hour Observe for the onset of loss of righting reflex Step 2 and duration of loss of righting reflex Group 1: Control group (n = 6); mice are given saline at the equivalent dose of drug Note: Observe the animals for any other behavior Group 2: Treatment group (n = 6); mice are changes during the observation time (sniffing, given Pentobarbital 50 mg/kg, i.p. rearing, exploratory behavior, etc.) Observation Table Sl. Righting reflex Duration of loss of righting No. Onset (min) (O) Recovery (min) (R) reflex (min) (D= (R) – (O)) Group 1 Group 2 Group 1 Group 2 Group 1 Group 2 1. 2. 3. 4. 5. 6. Note: Cat is not a suitable animal for the experiment on “loss of righting reflex”. This is because; they have flexible backbone and no functional clavicle. Discussion SUGGESTED READING Evaluation of the righting reflex is an ideal method 1. Liu X, Lee TL, Wong PT. Cyclooxygenase-1 Inhibition to evaluate drug acting as CNS depressants, since Shortens the Duration of Diazepam-Induced Loss the entire CNS depressant drugs cause loss of of Righting Reflex in Mouse. Anesth Analg 2006; righting reflex in animals at certain dose. e.g.: 102:135-40. 2. Simon P, Chermat R, Doaré L, Bourin M, Farinotti R. Mouse and rat are the ideal models for studying Interactions imprévues de divers psychotropes avec righting reflex whereas the cat is not a suitable les effets du barbital et du pentobarbital chez la animal for the experiment “on righting reflex”. souris. J Pharmacol (Paris) 1982:13;241-52. This is because; they have flexible backbone and no functional clavicle. There are several methods EXPERIMENT NO: 18B of doing this experiment, generally putting animal on its back (distal portion) to correct the position Aim on its four legs or dropping the animal from a To demonstrate muscle relaxant property of certain height and observe either animal touches diazepam in mouse using rotarod apparatus ground on its four paws or not, is very commonly in use. Drugs used in such experiment have CNS depressant properties such as anesthetics like Background pentobarbital, thiopental etc. or long of short acting hypnotics like diazepam, nitrazepam or zolpidem, Rota rod test is used to evaluate fore and hind zopiclone, triazolam etc. respectively. Studies limb motor coordination of rodents. The apparatus suggest that when short or long acting hypnotics consists of a horizontal metal rod (coated with are added with the anaesthetics, it significantly rubber) of 3 cm diameter attached to a motor with increases the duration of the loss of righting reflex. the speed adjusted to 2 rotations/minute to 6  Animal Experiment on CNS  185 Fig. 18.2: Rota rod apparatus rotation/min. The rod is 75 cm in length and is Animal should be marked properly, to avoid divided into 6 sections by plastic discs, thereby mixing in two groups allowing the simultaneous testing of 6 mice. The Handle the animals with care (minimize the rod is at a height of about 50 cm above the tabletop stress and pain to animal) in order to discourage the animals to escape from Precondition the mouse with the procedure the instrument (Fig. 18.2). The cut off time for the Methods test is 2 min. The retention time (sec) for each mouse/rat is recorded. Step 1 Weigh the animals and mark properly to distinguish from one another Materials and Methods Divide animals into two groups (n = 6 in each Materials group) Animal/species : Mice/ Swiss albino Step 2 Group 1: Control group (n = 6); mice are given Sex/Body weight : Either sex/ 20-30 g saline at the equivalent dose of drug Syringe/needle : 1ml/ preferably 24G on- Group 2: Treatment group (n = 6); mice are wards given diazepam at the dose of 3 mg/kg, ip Drug : Diazepam (3-5mg/kg, i.p) Step 3 Observe the animals for 2 min on the rota rod Precautions before Experimentation apparatus Laboratory should be dim lighted and noise Observe the animals, to fall down and note free the time of falling down 186  Practical Manual of Experimental and Clinical Pharmacology Note: For mouse: Mouse is placed on a 2.5 cm SUGGESTED READING diameter rod rotating at 3 rev/min. The animal’s 1. Cartmell SM, Gelgor L, Mitchell D. A revised rotarod muscle coordination is considered to be impaired, procedure for measuring the effect of antinociceptive if it falls from the rota rod within 120 sec drugs on motor function in the rat. J Pharmacol observation period. Methods 1991;26(2):149-59. 2. Dunham NW, Miya T S. A note on a simple approach Dimensions of Rota Rod for determining neurological deficits in rats and mouse. J Am Pharmacol Assoc 1957;46:208. Mouse: Rod is 75 cm in length, 30 mm diameter 3. Liu X, Lee TL, Wong PT. Cyclooxygenase-1 Inhibition and is divided into 6 sections by plastic discs and Shortens the Duration of Diazepam-Induced Loss of about 50 cm above the table top. of Righting Reflex in Mouse. Anesth Analg 2006; 102:135-40. Rat: Rod is 75 cm in length, 60 mm diameter and 4. Stanistaw J. Czuczwar, Kinga K. Borowicz, Zdzistaw is divided into 6 sections by plastic discs and of Kleinrok, Piotr Tutka, Tomasz Zarnowski, about 50 cm above the table top. Waldemar A. Turski. Influence of combined treatment with NMDA and non-NMDA receptor Observation Table antagonists on electroconvulsions in mouse. Eur J Pharmacol 1995;281:327-33. Sl. No. Retention time on Rota rod (cut off time 120 sec) Pre-drug Post-drug EXPERIMENT NO: 18C 1. 2. Aim 3. To demonstrate muscle relaxant property of 4. 5. diazepam in mouse using chimney test. 6. Background Discussion: Motor coordination is a physiological Mouse is made to climb backward up a plastic process to achieve movement which is an essential Perspex tube (3 cm inner diameter and 25 cm interaction between neural processes involved in moving a limb, and the actual limb in movement. length). The animals usually unable to perform This is mainly processed through peripheral the test within 60s are considered to be positive nervous system (PNS). It is responsible for both for motor impairment. The internal diameter the transmission of the efferent to the CNS and varies with the animal’s weight such as for low the movement of the limb. CNS also plays a vital weight mouse, it requires less diameter and for role in integrating the afferent feedback and higher weight mouse, it require larger inner efferent signals. There are several experimental diameter of tube (22-28 mm). models which are established to study the motor co-ordination in the rodents such as rotarod Materials and Methods method, chimney test, grip strength, treadmill Materials performance etc. In the either tests, rodents (mouse Animal/species : Mouse/Swiss albino or rat) are tested for their ability to retain the muscle co-ordination such as in rotarod test. Mouse/rat Sex/Body weight : Either sex/ 20-30g should remain on the revolving rod or in chimney Syringe/needle : 1ml/ preferably 24G on- test; it should climb backward to show the muscle wards coordination. The drugs which can be screened Drug : Diazepam (3mg/kg, i.p) through these tests are anesthetics like Pheno- Precautions before Experimentation barbital, etc. centrally active skeletal muscle relaxants such as benzodiazepam, chlordia- Laboratory should be dim lighted and noise zepoxide or diazepam, zolpidem, zopiclone, etc. free  Animal Experiment on CNS  187 Animals should be marked properly, to avoid 2. Stanistaw J Czuczwar, Kinga K. Borowicz, Zdzistaw mixing in two groups Kleinrok, Piotr Tutka, Tomasz Zarnowski, Handle the animals with care (minimize the Waldemar A Turski. Influence of combined treatment with NMDA and non-NMDA receptor antagonists stress and pain to animal) on electroconvulsions in mouse. Eur J Pharmacol Precondition the mouse with the procedure 1995;281:327-33. one day prior to the experiment day. EXPERIMENT NO.: 18D Methods Aim Step 1 Weigh the animals and mark properly to To demonstrate anti-anxiety effect of diazepam in distinguish from one another rat using elevated plus maze apparatus Divide animals into two groups (n = 6 in each group) Background Step 2 It is a novel test for the selective identification of Group 1: Control group (n = 6); mice are given ‘anxiogenic and anxiolytic’ drug effects in rodents. saline at the equivalent dose of drug The plus maze apparatus consists of two open Group 2: Treatment group (n = 6); mice are (16 × 5 cm for mouse and 50 × 10cm for the rats) given diazepam at the dose of 3 mg/kg, ip and two closed arms (16 × 5 × 12 cm for mouse and 50 × 10 × 40 cm for rats), and an open roof of the Step 3 entire maze elevated (25 cm for mouse and 50 cm Force mice backwards in a plastic perspex tube for rats) from the floor. The animals are placed (3 cm inner dimeter, 25 cm length), and make individually at the centre of the elevated plus maze tube slightly tilted. with their head facing towards the open arm during Observe the animal performance for 60 sec, the 90 sec-5 min test. The preference of the animal (climbing backward into the perspex tube). for the first entry, the number of entries into the open and closed arms reflect the relative safety of Observations and Results closed arms as compared with the relative fearfulness of open arms. Rat/mouse are rodents Observation Table and feel safe in dark, hence normal rodents prefer Sl. Motor Co-ordination (cut off time 60s) dark arm first. Anxiolytics would be expected to No. Group 1 Group 2 increase the proportion of entries into and time Present Absent Present Absent spent in open arms (Figs 18.3 and 18.4). 1. 2. Materials and Method 3. Materials 4. 5. Animal/species : Rat/Wistar 6. Sex/Body weight : Either sex/150-250g Syringe/needle : 1ml/preferably 23G Discussion Drug : Diazepam (0.5-1 mg/kg, i.p) Refer to the exp 18b Precautions before Experimentation SUGGESTED READING Laboratory should be dim lighted and noise 1. Boissier JR, J Tardy, JC Diverres. Une nouvelle method free simple pour explorer l’action ‘tranquilisante’: le test Animal should be marked properly, to avoid de la chimin6e, Med Exp (Basel) 1960;3:81. mixing in two groups 188  Practical Manual of Experimental and Clinical Pharmacology Fig. 18.3: Rat facing towards the open arm in an elevated plus maze (For color version see Plate 3) Fig. 18.4: Diagram of rat elevated plus maze with dimensions of the arm and height  Animal Experiment on CNS  189 Handle the animals with care (minimize the Step 2 stress and pain to animal) Group 1: Control group (n = 6); rats are given Place the rat at the centre of the plus maze, saline at the equivalent dose of drug facing towards open field Group 2: Treatment group (n = 6); rats are given Expose rat to the experiment procedure 1-2 diazepam at the dose of 1 mg/kg, ip days prior to the experiment Video recorder is placed to record the experi- Step 3 ment in calm and dim lighted room Observe the animals for 5 minutes (cut off time) Observation parameters: (1) First arm Methods preference, (2) No. of entries into the open and closed arm, (3) Time spent in the open and Step 1 closed arm and (4) Total no. of entries in the arm Weigh the animals and mark properly Divide animals into two groups (n = 6 in each Note: Dimensions of mice elevated plus maze are group) explained in introduction Observations and Results Observation Table Group 1 Sl.no. No. of entries Time spent in the arm (sec) Total no. of entries 1st arm preference Closed Open Closed Open Closed Open (open/closed) 1. 2. 3. 4. 5. 6. Group 2 Sl.no. No. of entries Time spent in the arm (sec) Total no. of entries 1st Preference Closed Open Closed Open Closed Open (open/closed) 1. 2. 3. 4. 5. 6. Discussion whereas anxiogenic compounds increase time spent in the closed arm. The judgement parameters There are several methods available for screening during the experiment are time spent in open arm, the anxiolytic compounds such as elevated plus latency to enter in open arm, total number of entries maze, Z-maze, O-maze, Y-maze or radial maze in open arm, and first preference of arm by the etc. Elevated plus maze is most popularly used in animal (open/closed). The principle is same for the screening and evaluation of the drug by all above mentioned mazes. Drugs like decreasing anxiety. It is evaluated by increase in benzodiazepine, alcohol, barbiturates etc. may be the time spent and exploration time in open arm screened through this method. 190  Practical Manual of Experimental and Clinical Pharmacology SUGGESTED READING four quadrants. A different starting point is randomly used on each trial. The rats are allowed 1. Brett RR, Pratt JA. Chronic handling modifies the to swim freely until they find the escape platform. anxiolytic effect of diazepam in the elevated plus- The latency to find the hidden platform is recorded maze. Eur J Pharmacol 1990;178:135-38. 2. Cao BJ, Rodgers RJ. Comparative effects of novel 5- and used as a measure of acquisition of the task. If HT1A receptor ligands, LY293284, LY315712 and a rat fails to locate the hidden platform within 60 LY297996 on plus-maze anxiety in mouse. sec, it is then manually guided to escape platform Psychopharmacology 1998;139:185-94. by the experimenter. The rat remains on the 3. Kauppila T, Tanila H, Carlson S, Taira T. Effects of platform for at least 20 sec to orient itself to the atipamezole, a novel α2-adrenoreceptor antagonist, visual cues. Rats are then turned to their home in open-field, plus-maze, two compartment cage for a fixed interval (3-10 min), until the series exploratory, and forced swimming tests in rats. Eur of trials are completed. J Pharmacol 1991;205:177-82. 4. Pellow S, Chopin P, File SE and Briley M. Validation Note: The water maze test is ideal to measure of open:closed arm entries in an elevated plus-maze learning and memory rather than the anxiolytic as a measure of anxiety in the rat. J Neurosci Methods activity whereas elevated plus maze is ideal for 1985;14:149-67. screening anxiolytic activity rather than learning and memory. EXPERIMENT NO: 18E Materials and Methods Aim Materials To demonstrate amnesic effect of diazepam in rat using Morris water maze apparatus. Animal/species : Rat/ Wistar Sex/Body weight : Either sex/ 150-250g Background Syringe/needle : 1ml/ preferably 23G Drug : Diazepam (1-2 mg/kg, i.p) The test apparatus consists of a circular fiberglass tank (130 cm in diameter, 50 cm in depth). The Precautions before Experimentation pool is filled to a height of 30 cm with water at room temperature, 21-22°C. The pool is divided Laboratory should be dim lighted and noise into four quadrants (Q1, Q2, Q3 and Q4) of equal free surface area. A transparent escape platform (10 Animal should be marked properly, to avoid mixing in two groups cm in diameter, 29 cm in height) is placed in a Handle the animals with care (minimize the fixed location in the tank in the centre of one of the stress and pain to animal) quadrants, 1cm below the water surface. So, that Proper training should be given to animals for platform is not visible. Several clues arround the the swimming and for the location of the maze are available for the rats to use in locating platform for at least 4-5 days the escape platform (Fig. 3.6). Animals who do not float on the water and Training of animals: The platform remains in a search for the platform should be excluded constant location in the centre of one quadrant, from the study equidistant from the centre and the edge of the Check for the pregnancy if the female pool. Each training trial involves placing the rats are included into the experiment (Pre- animal into the pool facing the wall at one of the gnant animals should not be included in  Animal Experiment on CNS  191 the study; true for every experiment until it Step 2 requires) Group 1: Control group (n = 6); rats are given saline at the equivalent dose of drug Group 2: Treatment group (n = 6); rats are given Methods diazepam at the dose of 1.5 mg/kg, ip Step 1 Step 3 Weigh the animals and mark properly Observe the animal for 90 sec/5 min (cut off time) Divide animals into two groups (n = 6 in each Time to reach at the platform is recorded and group) the movement in the quadrant of the pool Observations and Result Sl. no. No. of entries/time spend (sec) Time to reach to Group 1 Group 2 the platform Q1 Q2 Q3 Q4 Q1 Q2 Q3 Q4 Group 1 Group 2 1. 2. 3. 4. 5. 6. Discussion 2. D’Hooge Rudi, Deyn Peter P. De. Applications of the Morris water maze in the study of learning and Morris water maze is mainly designed to evaluate memory. Brain Research Reviews 2001;36:60-90. the drugs acting on learning and memory and 3. Kant G J, Wylier R M, Vasilakis AA, Ghosh S. Effects was devised by Prof Richard Morris. Morris water of triazolam and diazepam on learning and memory maze has two advantages over other mazes, (1) as assessed using a water maze. Pharmacol Biochem rat searches for the escape, so there is no waiting Behav 1996;53(2):317-22. 4. Kant GJ, Wylie RM, Vasilakis AA, Ghosh S. Effects time and (2) there are no local cues (e.g. olfactory of triazolam and diazepam on learning and memory or auditory). Drugs like NMDA receptor as assessed using a water maze. Pharma-co-logy, antagonists which improve the learning and biochemistry and behavior 1996;53 (2):317-22. memory whereas benzodiazepines that impair the 5. McNamara RK, Skelton RW. Diazepam impairs memory, can also be evaluated in the experiment. acquisition but not performance in the Morris water Study reported that it impairs acquisition but not maze. Pharmacol Biochem Behav 1991;38(3): retrieval of spatial information and is not due to 651-58. the sedative, hypothermic or state-dependent learning effects of diazepam. EXPERIMENT NO: 18F SUGGESTED READING Aim 1. Arolfo MP, Brioni JD. Diazepam impairs place To demonstrate the anticonvulsant property of learning in the Morris water maze. Behav Neural Biol diazepam against pentylenetetrazole (PTZ) 1991;55(1):131-36. induced convulsions in mice. 192  Practical Manual of Experimental and Clinical Pharmacology Background equal distribution in each group (Check for non-pregnancy) Seizures are finite episodes because of abnormal discharge of cerebral neurons and are broadly Methods divided into two groups (1) Epileptic seizure: seizures which are periodic and unpredictable Step 1 associated with disorder of brain function and (2) Weigh the animals and mark properly Non-epileptic: seizures which are evoked in a Divide animal into two groups (n = 6 in each normal brain by treatment with electroshock or group) chemicals. That means animal model which is Step 2 developed to assess the effect of the drug is a non- Group 1: Control group (n = 6); mice are given epileptic seizure model. Most animal models saline at the equivalent dose of drug cannot show all the pathophysiological, Group 2: Treatment group (n = 6); mice are behavioral, electrophysiological and neuro- given diazepam at the dose of 4 mg/kg, ip, chemical alteration of the spontaneous human then after 15-30 min, give PTZ in a dose of 85 epileptic syndrome. However, PTZ induced mg/kg, ip seizures represent the absence seizures and regarded as a good chemical model. The seizure Step 3 induced is characterized by generalized spike and Observe the animal for 1 hour wave discharges on the EEG. PTZ mainly acts Assessment: (1) onset of seizure (2) severity of through inhibition of GABAB coupled chloride seizure (score) (3) No. of seizures in an hour (Cl–) channel. (4) percentage of positive responder (if seizures score ≥3) and (5) total duration of seizure Materials and Methods Observations and Result Materials Seizures are recorded in a seven point score Animal/species : Mice/ albino Swiss according to the following scale. Sex/Body weight : Male/ 20-30 g Syringe/needle : 1ml/ preferably 24G on- Score wards Drug : Pentylenetetrazole (PTZ; 80- 0 = no behavioral changes; 0.5 = atypical behavior 85mg/kg, i.p.) (e.g. intensive grooming, sniffing, and moving Diazepam (4 mg/kg, i.p) arrests); 1 = isolated myoclonic jerks, ear and facial twitching; 2 = atypical minimal seizures, Precautions before Experimentation convulsive wave through the body; 3 = fully Laboratory should be dim lighted and noise developed minimal seizures, clonus of the head free muscles and forelimbs, righting reflex present; Animals should be marked properly, to avoid 4 = major seizures (generalized without the tonic mixing in two groups phase); 5 = generalized tonic-clonic seizures Handle the animals with care (minimize the beginning with running. stress and pain to animal) Followed by the loss of righting reflex, then Observe the animals in a plexi glass chamber short tonic phase (flexion or extension of fore- and Female have been more sensitive to seizure, hind limbs) progresses to the clonus of all four limbs hence should be avoided or if added, make leading sometimes to the death of the animal.  Animal Experiment on CNS  193 Observations and Result Observation Table Group 1 Sl. no. Onset of Severity of No. of seizure in % of positive Duration of seizures (sec) seizure (score) 60 min responder* seizure 1. 2. 3. 4. 5. 6. *Score ≥3 is considered as positive responder Group 2 Sl. no. Onset of Severity of No. of seizure in % of positive Duration of seizures (sec) seizure (score) 60 min responder* seizure 1. 2. 3. 4. 5. 6. Discussion evaluated are phenytoin, valproate, pheno- barbitone for the generalized seizures, but pheny- There are several methods for induction of toin is not be evaluated by the PTZ model (Model seizures in animals which resembles human for absence seizure). seizures such as pentylenetetrazole (PTZ), a chemical method which resembles human absence SUGGESTED READING seizure whereas other method like maximal electroshock seizure (MES) which resembles 1. Fradley RL, Guscott MR, Bull S, Hallett DJ, Goodacre human generalized tonic clonic seizure (GTCS). SC, Wafford KA, Garrett EM, Newman RJ, O’Meara Diazepam shows protective effect on both the GF, Whiting PJ, Rosahl TW, Dawson GR, Reynolds models and it is found to enhance GABAA binding DS, Atack JR. Differential contribution of GABA(A) receptor subtypes to the anticonvulsant efficacy of to receptors, increases chloride channel opening benzodiazepine site ligands. J Psychopharmacol and indirectly blocks T-type Ca ++ channels. 2007;21(4):384-91. Whereas kindling model is induced by the 2. Scarlatelli-Lima AV, Magalhães LHM, Doretto MC, continuous administration of sub-maximal Moraes MFD. Assessment of the seizure chemical dose or electrical stimuli which evokes susceptibility of Wistar Audiogenic rat to the GTCS, but its resembling condition in human electroshock, pentylenetetrazole and pilocarpine. is still controversial. Other drugs which may be Brain Res 2003;960:184-89. 194  Practical Manual of Experimental and Clinical Pharmacology 3. Velisek L, Kubova H, Pohl M, Stankova L, Mares P, Observe the animals in a plexiglass chamber Schickerova R. Pentylenetetrazol-induced seizures Female have been more sensitive to seizure, in rats: an on to genetic study, Naunyn Schmiedebergs hence should be avoided Arch. Pharmacol 1992;346:588-91. Methods EXPERIMENT NO.: 18G Step 1 Weigh the animals and mark properly Aim Divide animals into three groups (n = 6 in each To demonstrate the anticonvulsant property of group) diazepam against pentylenetetrazole (PTZ) Step 2 induced kindling in rats Group 1(A): Control group (n = 6); rats are given saline at the equivalent dose of drug Background Group 2(B): Vehicle group (n = 6); rats are Kindling is another experimental model to given vehicle at the equivalent dose of drug 40 develop seizures which involves the delivery of min before PTZ (30-40 mg/kg, i.p.), 3 times/ submaximal electrical or chemical stimuli. The week for 9 weeks. repeated administration of the stimuli lowers the Group 3(C): Treatment group (n = 6); rats are seizure threshold and produces behavior changes given Diazepam (3 mg/kg, i.p), 40 min before in the animal. Its exact mechanism is not clear but PTZ (30-40 mg/kg, i.p. 3 times/week for 9 some studies demonstrate that brainstem, the week. substantia nigra (SN), can regulate the kindled Step 3 seizure threshold. However, its applicability to Observe* the animals for 1 hour, after the PTZ human epilepsy is still controversial. dose 3 times/week for 9 weeks. Assessment: (1) Onset of seizure (2) severity of Materials and Methods seizure (score) (3) No. of seizure in an hour (4) Materials percentage of positive responder (if seizures score ≥3) and (5) total duration of seizure Animal/species : Rat/ Wistar Sex/Body weight : Male/ 150-250g Note: *Observe the animal for any other behavioral Syringe/needle : 1ml/ preferably 24G on- changes such as sniffing, rearing, excitement, wards aggressiveness etc. Drug : Pentylenetetrazole (PTZ; 30- 40mg/kg, i.p) Observation and Results Diazepam (3 mg/kg, i.p) Kindling model scoring Precautions before Experimentation 0 – No response; 1 – Ear and facial twitching; 2 – One to 20 myoclonic body jerks in 10 min; 3 – Laboratory should be dim lighted and noise More than 20 body jerks in 10 min; 4 – Clonic free forelimb convulsions; 5 – Generalized clonic Animal should be marked properly, to avoid convulsions with rearing and falling down mixing in two groups episodes; 6 – Generalized convulsions with tonic Handle the animals with care (minimize the extension episodes (same score may be used to stress and pain to animal) score the PTZ induced seizure also. (Exp. 18F)  Animal Experiment on CNS  195 Sl no. Onset of Severity of No. of seizure % of positive Duration of seizure (sec) seizure (score) in 60 min responder seizure (sec) A B C A B C A B C A B C A B C 1. 2. 3. 4. 5. 6. Discussion EXPERIMENT NO: 18H Same as Experiment 18f Aim SUGGESTED READING To demonstrate the anti-convulsant activity of phenytoin against maximal electroshock (MES) 1. Giorgi O, Orlandi M, Lecca D, Corda MG. MK-801 prevents chemical kindling induced by induced convulsions in rats pentylenetetrazol in rats. Eur J Pharmacol 1991;193: 363-65. Background 2. Goddard GV, McIntyre DC, Leech CK. A permanent change in brain function resulting from daily electrical The maximal electroshock (MES) model is a model stimulation. Exp Neurol 1969;25:295-330. for grand mal epilepsy and the end point 3. Kodama M, Yamada M, Sato K, Kitamura Y, considered as tonic hind limb extension (THLE) Koyama F, Sato T, Morimoto K, Kuroda S. Effects of which are evoked by electric stimuli. The agents YM90K, a selective AMP receptor antagonist, on screened through this model considered an anti amgdala-kindling and long-term hippocampal potentiation in rats. Eur J Pharmacol 1999;374: epileptic drug if it corrects or suppresses THLE in 11-19. rat (Figs 18.5A and B). The maximal electroshock 4. Löscher W. Animal models of epilepsy for the is induced through corneal electrode and ear electrode. development of antiepileptic and disease-modifying Corneal electrode has the limited use due to the drugs. A comparison of the pharmacology of kindling risk of blindness and other harmful nerve effects and poststatus epilepticus models of temporal epilepsy. Epilepsy Res 2002b;50:105-23. to the animal used. Ear electrode is commonly 5. McNamara JO. Kindling model of epilepsy. Adv used in the experiment which is easy to use and Neurol 1986;44:303-18. less harmful compared to corneal electrodes. Figs 18.5A and B: (A) Showing positive THLE (score 3) and (B) Postictal depression after the THLE (score 4): it remains for few seconds then rat/mouse recovers 196  Practical Manual of Experimental and Clinical Pharmacology Materials and Methods Step 3 Observe the animals after MES Materials Record whether THLE “present or absent” Animal/species : Rat/ Wistar Calculate percentage protection Sex/Body weight : Male/ 150-250 g Note: Syringe/needle : 1ml/ preferably 23G 1. All the procedure and scoring is the same for Drug : Phenytoin (20-25mg/kg, i.p mouse. The only difference is the selection of or po) electrical stimuli and duration of electrical stimuli through ear/corneal electrodes Instrument 2. One can also videotape the whole seizure Electro convulsiometer: events in rat/mouse and can use the scale to (For rat: Intensity of stimulus: (150mA, 50Hz score the seizure events in the slow motion for 0.2sec) video run (Events takes place in the millisecond (For mouse: Intensity of stimulus: (12mA, 50 of time, hence not possible to observe by the Hz for 0.2 sec) naked eye; only THLE is seen as end point. Other drugs: Diazepam (3-4 mg/kg, i.p. for mouse) Seizure score Precautions before Experimentation 0 = no seizure; 1 = forelimb extension without hind limb extension; 2 = complete forelimb Laboratory should be dim lighted and noise extension and partial hind limb extension, 3 = free complete tonic hind limb extension (THLE) (hind Animals should be marked properly, to avoid limb become parallel to the tail) and 4 = post ictal mixing in two groups depression; See Figs 18.5A and B. Handle the animals with care (minimize the stress and pain to animal) Observations and Results Animals should be screened 1 week prior to the Percentage protection (%) = No. of animals with experiment THLE absent / total no. of animal × 100 Check the instrument and ear electrode for its proper working before experiment Observation Table Ear should be lubricated with gel or water to enhance the conductivity of electricity. Sl. Tonic hind limb extension (THLE) % Pro- no. Present (Yes) Absent (No) tection Methods Group 1 Group 2 Group 1 Group 2 Step 1 1. Weigh the animals and mark properly 2. Divide animals into two groups (n = 6 in each 3. group) 4. 5. Step II 6. Group 1: Control group (n = 6); rats are given the saline as per body weight Group 2: Treatment group (n = 6); rats are given Discussion phenytoin 20-25 mg/kg, i.p., thereafter 30 min The end point of the experiment is considered as rats are given electro shock at the intensity of the absence/presence of THLE following a drug 150 mA, 50 Hz for 0.2 sec. (in case of oral test treatment which is a position during the GTCS in drug, MES induced after 60 min) rodents when tail and both hind limbs are parallel  Animal Experiment on CNS  197 to each other. It is a subjective measure hence, and D1 to some extent. In the experiment, extra- mouse/rat should be screened 1 week prior to the pyramidal side effects are identified by catalepsy. experiment. The animal present with the positive It is an extreme tonus, muscular rigidity which is THLE is included in the study. One week time is characterized by a tendency to remain in a fixed given to animal to recover from the excitatory position for long period, hence unable to correct neuronal discharge in the brain. an externally imposed, unusual posture over a pro- (Also refer to discussion of experiment 18f) longed period of time (Figs 18.7A to C). SUGGESTED READING Materials and Methods 1. Blanco MM, Dos Santos Jr JG, Perez-Mendes P, Kohek Materials SR, Cavarsan CF, Hummel M, Albuquerque C, Mello LE. Assessment of seizure susceptibility in Animal/species : Rat/ Wistar pilocarpine epileptic and nonepileptic Wistar rats Sex/Body weight : Male/ 150-250g and of seizure reinduction with pentylenetetrazole Syringe/needle : 1ml/ preferably 23G and electroshock models. Epilepsia 2008 Nov 19. Drug : Haloperidol (0.4-4 mg/kg, 2. Rastogi SA, Ticku MK. Involvement of a GABAergic i.p) mechanism in the anticonvulsant effect of Instrument : Two Wooden or cardboard Phenobarbital against maximal electroshock-induced seizures in rats. Pharmacol Biochem Behav pillar, 10cm apart with a rod 1985;222:141-46. and rod at the height of 3. Stanistaw J, Czuczwar, Kinga K, Borowicz, Zdzistaw 10-20 cm (for mice, height = Kleinrok, Piotr Tutka, Tomasz Zarnowski, 7-10 cm) (Fig. 18.6) Waldemar A. Turski. Influence of combined treatment with NMDA and non-NMDA receptor Precautions before Experimentation antagonists on electroconvulsions in mouse. Eur J Pharmacol 1995;281:327-33. Laboratory should be dim lighted and noise 4. Stephen H Koslow, Lloyd J Roth. Reserpine and free acetazolamide in maximum electroshock seizure in Animal should be marked properly, to avoid the rat. J Pharmacol Exp Therap 1971;176(3):711-17. 5. Woodbury LA, Davenport VO. Design and use of a mixing in two groups new electroshock seizure apparatus and analysis of Handle the animals with care (minimize the factors altering seizure threshold and pattern. Arch stress and pain to animal) Int Pharmacodyn 1952;92:97-107. Condition the animal to the experimental box for at least 2min, before the experiment (Fig. EXPERIMENT NO.: 18I 18.6). Aim To demonstrate effect of phenothiazine (halo- peridol) induced catatonia in rat. Background Anti-psychotics drugs are well known for their Fig. 18.6: Dimensions of instrument used to evaluate extrapyramidal side effects. Present experiment catalepsy in rat demonstrates extra-pyramidal side effects like tardive dyskinesia in animal followed by the Methods phenothiazine (Haloperidol) treatment. Pheno- Step 1 thiazine and butyrophenone neuroleptics act Weigh the animals and mark properly for through the blockade of both β and D2 receptors identification 198  Practical Manual of Experimental and Clinical Pharmacology Figs 18.7A to C: Different postures of catatonia produced in the rat Divide animals into two groups (n = 6 in each Observation Table group) Sl. Cataleptic (Abnormal) Posture (60s) Step II % of no. Present Absent Group 1: Control group (n = 6); rats are given cataleptic Group 1 Group 2 Group 1 Group 2 animal the saline as per body weight Group 2: Treatment group (n = 6); rats are given 1. haloperidol 4 mg/kg, i.p., thereafter 10-15 min 2. rats expose to the test 3. 4. Step 3 5. Observe the animals behaviour and correction 6. of its externally induced abnormal posture (cutoff time 60s) Time of posture correction is noted down at Discussion 30, 60, 120 and 360 min Extrapyramidal adverse effects are commonly Calculate percentage of responders reported with the antipsychotic drugs like Note: If rat remains on the bar for 60s, then chlorpromazine, flufenazine, haloperidol etc. catalepsy is positive They are mediated through dopamine D 2 - receptor blocked mainly in the nigrostriatal Observations and Resulte pathway, which leads to dystonia, bradykinesia, akathisia, dyskinesias etc. These behavioral Calculation of percentage of cataleptic animals: changes are well studied in rodents by failure No. of animals showing cataleptic posture to correct the externally exposed abnormal 100 Total number of animals position.  Animal Experiment on CNS  199 SUGGESTED READING is the presence of the dorsal sacro coccygeus muscle on the either side of tail originating at the 1. de Sousa Moreira LF, Pinheiro MC, Masur J. base of spinal cord (Fig. 18.8D). In the previous Catatonic behavior induced by haloperidol, increased by retesting and elicited without drug in rats. study, it showed that either side cut of the muscle Pharmacology 1982;25(1):1-5. deviate the tail towards the side of attached 2. Cerbo R, Carchedi F, Casacchia M. Role of serotonin muscle. The mode of action is not clear. But various in catatonia induced with haloperidol and reserpine. studies suggest that straub tail results because of Boll Soc Ital Biol Sper 1976;52(4):245-50. direct action on the opioids μ-receptor, most 3. Costall B, Naylor RJ. On catalepsy and catatonia importantly μ2–receptor. There are some studies and the predictability of the catalepsy test for which suggest that, it also results through 5-HT neuroleptic activity. Psychopharmacologia 1974; receptors, α 2-(alpha-2) receptors, dopamine 34(3):233-41. receptors and glucocorticoid receptors as well. 4. Casey DE. Serotoninergic aspects of acute extra pyramidal syndromes in nonhuman primates. Psychopharmacol Bull 1989;25:457-59. Materials and Methods 5. Casey DE. Serotonergic and dopaminergic aspects Materials of neuroleptic-induced extrapyramidal syndromes in nonhuman primates. Psychopharmacology 1993; Animal/species : Mice/ albino Swiss 112:S55-S59. Sex/Body weight : Male/ 20-30 g Syringe/needle : 1ml/ preferably 24G on- EXPERIMENT NO.: 18J wards Aim Drug : Morphine (10-40mg/kg, s.c or ip) To demonstrate the Straub tail reaction/pheno- menon induced by morphine Precautions before Experimentation Animals should be marked properly, to avoid Background mixing in two groups Simply straub tail means erected tail. The Handle the animal with care (minimize the phenomenon was used to check doping i.e. opioid stress and pain to animal) level in the horses which took part in the race. A Observe the animal in a plexiglass chamber subcutaneous injection of morphine hydro- Laboratory should be noise free (noise may chloride (10-40 mg/kg, i.p./s.c) into the mouse delay the response) flank produces a curvature of the tail in the form of an ‘S’ , following 2 to 15 minutes after the Methods injection, depending on the dose. Finally, the tail Step 1 curves back along the body of the animal, the tip Weigh the animals and mark properly to touching the center of the head (Figs 18.8A to C). distinghish from one another Straub tail phenomenon occurs as a result of Divide animals into two groups (n = 6 in each injection of the centrally acting analgesics such group) as morphine. But, there are no such studies which have supporting data for the peripheral analgesic Step II induced straub tail. Group 1: Control group (n = 6); mice are given As straub tail reaction is thought to be due to the saline at the equvalent dose of drug mechanical contraction of the dorsal sacro coccygeus Group 2: Treatment group (n = 6); mice are muscle and electrical stimulation of spinal cord. There given morphine at the dose of 40 mg/kg, s.c. 200  Practical Manual of Experimental and Clinical Pharmacology Figs 18.8A to D: (A) Positive straub tail phenomenon, (B) “S” shaped positive straub tail phenomenon, (C) Extreme positive straub tail phenomenon, sometimes tail touches the head of mouse and (D) Showing dorsal sacro coccygeus muscle on the either side of tail root (pointed in the picture) (For color version of Figures 18.8A to C see Plate 3) Step 3 Note: Straub tail is said to be positive, when the Observe the animals for 45 min in plexiglass tail rises more than 30° of angle with tail base chamber Observe the animals behavior carefully, for (1) Numerical score for straub tail reaction/ onset of straub tail reaction/phenomenon (2) phenomenon; duration of straub tail reaction and (3) any 0 = 0 °, 0.5 = 1-30° ,1 = 31-45° , 1.5 = 46-60° , 2.0 = additional behavior changes 61-90°, 2.5 = more than 90 ° Observations and Results Observation Table Sl. No. Straub Tail Reaction Straub tail Group1 Group 2 reaction(Score) Present Absent Present Absent Group 1 Group 2 1. 2. 3. 4. 5. 6. Discussion α2-(alpha-2) receptors, dopamine receptors and Straub tail reaction or phenomenon is the glucocorticoid receptor are well studied in several characteristic contraction of the dorsal sacro- studies. The tail rise at an angle >30° is considered coccygeus muscle and electrical stimulation of to be the positive straub tail reaction. There is no spinal cord in which tail rises than its normal clinical condition resembling this condition hence position. The role of the μ2–receptor, 5-HT receptors, have only experimental implication.  Animal Experiment on CNS  201 SUGGESTED READING rat are placed on the hot plate and observed for either paw licking or jumping reaction. The 1. Anna Capasso, Carmela Casciano and Alberto reaction time is recorded by a stop-watch. Loizzo. Dexamethasone reduces morphine induced straub reaction in mouse. J Pharmacy Pharmacol Repeated reading is taken at 20, 60, and 90 2002;54:983-87. minutes after the drug administration. Cut off time 2. Bilbey D LJ, Salem H, Grossman MH. The anatomical for rat is 20-30 sec and for mice it is 15-20 sec. basis of the straub phenomenon. Br J Pharmacol 1960;15:540-43. Tail Flick Method 3. Kameyama T, Ukai M, Nabeshima T. Effects of catecholaminergic or tryptaminergie agents on the Animal is placed into restrainer and leaving the morphine-induced Straub tail reaction. Jpn J tail exposed outside the restrainer. Clean the tail Pharmacol 1978:28;249-57. with the help of cotton soaked in water or ethanol. 4. Schwe Fang Pong, Janet Mary Sweetman, Amy Sue Then, leave the tail for drying and also to settle Pong, John Franklin Carpenter. Evaluation of oral down the rat/mouse in the restrainer. When rat/ skeletal muscle relaxants in the morphine-induced straub tail test in mouse. Drug Development mouse setted, then keep restrainer on the “tail flick Research 2004;11(1):53-57. analgesiometer”. 1/3rd tail proximally left due to 5. Tsutomu Kameyama, Makoto Ukai and Toshitaka the thick and keratinized skin and then keep tail Nabeshima. Effect of catecholaminergic or trypta- on the place made for tail above hot wire (measure minergic agents on the morphine induced straub tail the height of tail from wire) of the analgesiometer. reaction. Japan J Pharmacol 1978;28:249-57. The time of tail flick is measured and recorded. The 6. Zarrindast MR, Alaei-Nia K, Shafizadeh M. On the cut off time is set up 15-20 sec in case of mouse mechanism of tolerance to morphine-induced Straub tail reaction in mouse. Pharmacol Biochem Behav whereas in the case of rat, cut off time is 20-30 sec 2001;69(3-4):419-24. to avoid any further injury to the tail (Fig. 18.9). EXPERIMENT NO: 18K Materials and Methods Aim Materials To demonstrate the analgesic effect of morphine Animal/species : Mice/ albino Swiss in mouse using hot plate/tail flick method Sex/Body weight : Either sex/ 20-30 g Syringe/needle : 1ml/ preferably 24G on- Background wards Drug : Morphine (5-7.5 mg/kg, i.p The method deals on the principle of the thermal or s.c.) radiation heat. This principle is used in the animal experiment for the evaluation of the centrally acting analgesics, and hence this method found to be the differentiating between the centrally acting opiates and non-opiates analgesics. Hotplate Method The instrument involved is known as “hotplate analgesiometer”. Instrument consists of an electrically heated surface (made up of iron, aluminum or copper) whose temperature is maintained by the thermostat ‘Knob’ at 55° to 56 °C. After maintaining the temperature mouse/ Fig. 18.9: Tail-flick test in rat 202  Practical Manual of Experimental and Clinical Pharmacology Other drugs : Codeine hydrochloride (30 Observe for the licking of the paw or jumping mg/kg s.c.), Pethidine hydro- in case of hot plate or the tail flick in tail flick chloride (30 mg/kg s.c.) and test and record the time Phenazone (400 mg/kg s.c.) Note: Precautions before Experimentation 1. Record the response at 20, 60 and 90 min after the saline/drug treatment (Hot Plate test) Animals should be marked properly, to avoid 2. Record the response at 30, 60 and 120 min mixing in two groups after the saline/drug treatment (Tail flick Handle the animals with care (minimize the test) stress and pain to animal) Check the instrument carefully for any error Observations and Results and electricity connection Carefully check the temperature within the Centrally acting analgesics can be evaluated by range (chances of burning of paw or tail) [hot the hot plate method where as peripherally acting plate] analgesics are not effective. e.g: Aspirin showed Clean the paw and hot plate for uniform no effect in hot plate method even at very high temperature distribution doses In tail flick method, clean the tail properly, to False positive results: sedatives and muscle avoid interference with result relaxants (Woolfe and MacDonald, 1944) or The time of wire getting red must be substracted psychotomimetics (Knoll, 1967) may give the from the total time recorded [tail flick] increase reaction time. Screening is must for the both tests before the experiment, if mouse show reaction time more Discussion than 6 sec, should be excluded from the study Hot-plate and tail-flick test mimic acute thermal (selection reaction time for rat is 10 sec). pain and persistent pain model by the formalin Methods test. Hot-plate and tail-flick test are two different methods for evaluation of nociception. These Step 1 experimental models of pain commonly used to Weigh the animals and mark properly tests for response thresholds to high intensity Divide animals into two groups (n = 6 in each stimuli (acute pain tests) or persistent pain group) models. Tail-flick test is predominantly a spinal Step II response and Hot-plate is mostly at supraspinal Group 1: Control group (n = 6); mice are given level. Studies have conducted to evaluate the saline at the equvalent dose of drug participation of nitric oxide in the agmatine- Group 2: Treatment group (n = 6); mice are mediated potentiation of morphine-induced given morphine at the dose of 5 mg/kg, s.c. analgesia in mice indicate that agmatine Step 3 potentiates morphine-induced spinal but not Hot plate/tail flick: Observe the animal for 15- supraspinal analgesia, and this effect is 20 seconds (cut off time for mice) 20-30 seconds not mediated by a nitric oxide-dependent (cut off time for Rat) mechanism.  Animal Experiment on CNS  203 Observation Table For hot plate Sl. Time of paw licking or jumping No. 20 min 60 min 90 min Group 1 Group 2 Group 1 Group 2 Group 1 Group 2 1. 2. 3. 4. 5. 6. For tail flick Sl. Time of tail flick No. 30 min 60 min 120 min Group 1 Group 2 Group 1 Group 2 Group 1 Group 2 1. 2. 3. 4. 5. 6. SUGGESTED READING in Drug Evaluation. Yearbook Med Publ. Inc., Chicago 1967;305-21. 1. Aanonsen LM, Wilcox GL. Nociceptive action of 6. Luszczki JJ, Czuczwar SJ. Dose-response relationship excitatory amino acids in the mouse: effects of analysis of vigabatrin doses and their antinociceptive spinally administered opioids, phencyclidine and effects in the hot-plate test in mouse. Pharmacol Rep sigma agonists. J Pharmacol Exp Ther 1987;243(1):9- 2008;60(3):409-14. 19. 7. Ung D, Cowan A, Parkman HP, Nagar S. Lack of 2. Abbott FV, Melzack R, Leber BF. Morphine analgesia interaction between metoclopramide and morphine and tolerance in the tail-flick and formalin tests: dose- in vitro and in mouse. Xenobiotica 2008;38(11):1365- response relationships. Pharmacol Biochem Behav. 76. 1982;17(6):1213-19. 8. Woolfe G, MacDonald AD. The evaluation of the 3. Abbott FV, Melzack R, Samuel C. Morphine analgesia analgesic action of pethidine hydrochloride (DE- in tail-flick and formalin pain tests is mediated by MEROL) J Pharmacol Exper Ther 1944;80:300-07. different neural systems. Exp Neurol 1982;75(3): 9. Zimer PO, Wynn RL, Ford RD, Rudo FG. Effect of 644-51. hot plate temperature on the antinociceptive activity 4. Kambur O, Männistö PT, Viljakka K, Reenilä I, of mixed opioid agonist antagonist compounds. Lemberg K, Kontinen VK, Karayiorgou M, Gogos Drug Dev Res 1986;7:277-80. JA, Kalso E. Stress-induced analgesia and morphine responses are changed in catechol-O-methyl- EXPERIMENT NO.: 18L transferase-deficient male mouse. Basic Clin Pharmacol Toxicol 2008;103(4):367-73. Aim 5. Knoll J. Screening and grouping of psycho- pharmacological agents. In: Siegler PE,Moyer HJ To demonstrate partial global cerebral ischemia (Eds) Animal and Clinical Pharmacologic Techniques in mice 204  Practical Manual of Experimental and Clinical Pharmacology Background Step 2 Group 1: Control group (n = 6); mice are given Cerebral ischemia leads to a cascade of patho- saline at the equivalent dose of drug physiological processes which contribute to Group 2: Treatment group (n = 6); mice are ischemic cell damage, reduction in oxygen and given dizocilpine (MK-801) at the dose of 0.1 glucose availability to brain leading to cellular mg/kg, i.p. 1st dose is given 30 min before the energy crisis which interrupts the activity of surgery thereafter, once every day at 24 hr, 48 cellular ion pumps thus disturbing the ionic hr and 72 hr gradients homeostasis resulting ultimately to an increased release of neurotransmitters (mainly Step 3 glutamate) within 1-2 minutes of ischemia. Brain is removed after 72 hr of cerebral Glutamate release causes excitation and results ischemia and then following observations are in early onset seizure. The contribution of over- carried out stimulation by excitatory amino acids leading to The infarct area neurotoxicity and cell death following cerebral Brain edema hypoxemia induced by ischemia is well estab- lished. Activation of NMDA-glutamate receptors Measurement of Brain Edema results in an increase in free Ca2+ ion which Brain edema is measured with the wet-dry triggers a number of potential cytotoxic cascades method. After the animal is sacrificed by decapi- including activation of protein kinase-C (PKC), tation under euthanasia, their brain is removed, release of platelet activating factor (PAF), weighed immediately to yield wet weight. After generation of free radicals and production of drying in a desiccating oven for 48 h at 70oC, the nitric oxide. tissue is reweighed to yield dry weight. The percentage of water in the tissues is calculated Materials and Methods according to the formula: Materials Wet weight – dry weight Animal/species : Mice/albino Swiss 100 Wet weight Sex/Body weight : Either sex/ 20-30 g Syringe/needle : Aneurysm clip/bull dog clip Measurement of Cerebral Infarct Size Precautions before Experimentation After 72 hr, the animal is sacrificed by decapi- tation, their brain is removed and infarct size is The area of surgery is thoroughly cleaned with calculated. The brain is kept overnight at (–4°C). 70% ethanol with the sterile cotton swab Frozen brain is sliced into uniform sections (7-8 Use sterilized instruments during the surgery in number per brain) of 1 mm thickness. The slices While performing the surgical procedure, the are immersed in 1% triphenyltetrazolinium animals are kept warm with the help of Infra- chloride (TTC) at 37°C in 0.25 M phosphate buffer red lamp (pH 8.5) for 5min; tissue sections are dipped in 10% formaldehyde solution for 5 min. Triphenyl- Method of Cerebral Ischemia Induction tetrazolinium chloride (TTC) is converted to red Method formazone pigment and therefore stained the viable cells deep red. The infarcted cell have lost Refer Flow Chart 18.1. the enzyme and cofactor and thus remained Step 1 unstained dull yellow. The brain slices are After ischemia induction, divide animals into placed in between two glass slides. A transparent two groups (n = 6 in each group) plastic grid with 100 squares per cm2 is placed  Animal Experiment on CNS  205 Flow Chart 18.1: Procedure for producing cerebral ischemia over slides. Number of squares falling over non- Observations and Results stained dull yellow area and total number Sl. no. Cerebral infarct size Brain edema of squares covered by each brain slice is counted. Control Treatment Control Treatment Infarcted area is expressed as a percentage of total brain volume. For calculation of infarcted area of a brain, all the brain sections are studied with the help of magnifying glass (40 X). 206  Practical Manual of Experimental and Clinical Pharmacology Result is expressed in mean ± SD and is on the duration of ischemia. Therefore, the severity compared by unpaired t-test (if treatment groups of ischemia has two components: degree of CBF are more than one and total mean is >2 then one reduction and duration of the ischemic episode. way ANOVA should be preferred) SUGGESTED READING Discussion There are several other models are used to study 1. Aggarwal R, Medhi B, Pathak A, Dhawan V, cerebral ischemia or stroke in the published Chakrabarti A. Neuroprotective effect of progesterone on acute phase changes induced by partial global literature such as middle cerebral artery occlusion cerebral ischaemia in mice. J Pharm Pharmacol 2008; model (MCAO) or unilateral carotid artery ligation 60(6):731-37. (UCAL). Four vessel occlusion models are also 2. Bochelen, D., Rudin, M. and Saute, A. Calcineurin used in the number of studies for the producing inhibitors FK506 and SDZASM 981 alleviate the global cerebral ischemia. The development of outcome of focal cerebral ischemic/reperfusion ischemic brain damage depends on the reduction injury. J Pharmacol Exp Ther 1999;288:653-59. of cerebral blood flow (CBF) below critical 3. Dempsey RJ, Baskaya, MK and Doglan, A. Attenu- ation of brain edema, blood-brain barrier breakdown, threshold level. So, the decrease of CBF in ischemic and injury volume by ifenprodil, a polyamine-site regions may result in an energy failure and further NMDA receptor antagonist, after experimental lead to an activation of the toxic intracellular traumatic brain injury in rats. Neurosurgery pathway, additionally the infarct volume depend 2000;47(2):399-406.