Screening Methods of Anti Hypertension Agents on Animal Model

High blood pressure - Blood forced through the arteries at an increased rate. It is measured as two numbers. 
e.g. 120/80 mmHg. 
Normal blood pressure is below this value. The first number is the systolic blood pressure. This is the maximum pressure in the arteries when heart contracts/beats. The second number is the diastolic blood pressure. This is the minimum pressure in the arteries when heart is at rest between beats. Until it becomes extreme, there are no symptoms. Hence, it is called the “Silent Killer.”

Severe hypertension may cause headache, sleepiness, confusion or coma.

Blood Pressure and Its procedure For Measurement 

Mechanism of hypertension 

In vivo models for anti-hypertension 

1. Angiotensin converting enzyme inhibition in rat (ACE Inhibition in rat)
2. Goldblatt Hypertension
3. Chronic renal hypertension in rats
4. Tail cuff method
5. Salt- sensitive Dahl Rats
6. Renin inhibition in Monkeys
7. Chronic renal hypertension in Dogs.
8. Antihypertensive and vasodilator action in rats

Serotonin - Types, Receptors, Functions, Pharmacology, Agonist, Antagonist & Uses

Angiotensin converting enzyme inhibition in rat  

Mechanism of angiotensin converting enzyme inhibitor

Angiotensin converting enzyme inhibition in rat

• Male Sprague- Dawley rats (200-225gm)  are selected.
• They are anesthetized with 60mg/kg of phenobarbitone sodium i.v.
• The intubated trachea is artificially respirated  with 30strokes/min and a stroke volume of  6-8ml.
• The right artery is cannulated for recording the pressure.
• The jujular vein is cannulated for i.v. test injection.
• The B.P is diminished by the administration of 5 mg/kg of pentolinium i.p.
• Atropine(40 µg/kg) is also injected i.m to inhibit the mucous secretion.
• Now 310 ng/kg of Angiotensin I is injected i.v. in 0.1ml saline.
• The injection is repeated in 5 minute interval until an identical pressure is attained.
• The test drug is administered at a dose of  10 mg/kg intravenously or 25 mg/kg intradermally.
• Again Angiotensin I is injected as the similar dose above.
• The diminution of the pressure after the administration of potent  ACE inhibitors is compared .

Screening Methods Of Diabetes Mellitus On Animal Model

Goldblatt hypertension ( 2 kidney 1 clip method) 

Acute hypertension 

Procedure of goldblatt hypertension 

• Sprague – Dawley Rats (300gm) are anesthetized Hexobarbital sodium (100mg/kg) Intra peritonially
• Cannulate the trachea for respiration and the Jugular vein for test compound administration.
• A transducer is connected to the carotid artery for recording the pressure.
• A PVC coated clip is placed in the left hilum of the kidney by fixing with the back muscle for 3.5 - 4 hour.
• Pentolinium is administered for ganglionic blockage.
• Release the clip and record the rise in blood pressure. 
• Administer the test drug through I.V. and monitor the pressure continuously.
• Increase in blood pressure after releasing the clip and reduction after the drug administration is determined. 
• Compare using percentage values

Screening Methods Of Antioxidant Activity On Animal Model

Chronic renal hypertension (1 kidney 1 clip method)

• Sprague – Dawley Rats (200 - 300gm) are anesthetized phenobarbitone sodium (100mg/kg) Intra peritonially.
• A flank parallel incision is made in the left lumbar area.
• Renal artery is dissected, cleaned and ‘U’ shaped silver clip is slipped around near the aorta.
• The internal gap b/w the clip is adjusted to 0.25 – 0.38 nm.
• The right kidney is removed after tying off the renal pedicel.
• After 4-5 weeks the B.P is measured and the animals are divided into groups of different doses.
• Test drug is administered for 3 days
• Pressure before and after drug administration(3minute) are recorded.
• Percent reduction in pressure is calculated and compare to the Standard. 

Screening Methods Of Diuretic Activity On Animal Model

Tail Cuff Method

Tail cuff method also know as Blood Pressure in Conscious rats.

• This method without any surgical procedure, Similar to sphygomanometry in humans (systolic pressure).
• Widely used to evaluate the anti-hypertensive drugs in experimentally induced animals.Charles River male rats(300-350 g) are anesthetized using I.P injection of 0.8 ml of 4% of chloral hydrate.
• Both the kidneys are exposed, hypertension is induced by placing a silver clip on both renal arteries,(0.2 mm dm & 4 mm l).
• After 5 to 6 weeks operated animals attain renal hypertension with systolic B.P of 170 to 200mmHg.
• A tubular inflatable cuff is placed around the base of tail and a Pizo- electric pulse detector is positioned distal to the cuff.
• The cuff is inflated to approximately 300mmHg.
• As the pressure in the cuff is released slowly, the systolic pressure is detected and recorded in the poly graph.
• Test substance is administered intra-peritonially  for alternative days in three times.
• Decrease in the systolic pressure is determined by the following steps.    

                     Day 1 : Predose & 2 hours postdrug

                     Day 3 : Predose & 2 hours postdrug

                     Day5  : Predose , 2 hours post drug & 4 hours postdrug

Salt Sensitive Dahl Rats

These rats Develop severe fatal hypertension when fed high – salt diets, whereas salt resistance rats cant.

• Sprague – Dawley Rats (250 - 300gm) are used in this study.
• Drinking water is replaced with 8% NaCl solution high salt diet is prepared by mixing salt with regular diet.
• The animals are fed with the above diet and saline.
• The test group rats are administered the drug orally for 1 month.
• Blood pressure changes are recorded.
• After the experiment the animals (both control & test) are sacrificed.
• The hearts are isolated, the mass, weight of  left ventricle & right ventricle is compared and measured.
• Upon salt feeding animals B.P rises steeply (up to 36%).
• The ability of the drug to reverse these changes is studied.
• Cardiac failure occurs at 4 to 5 months of age in these kind of rats.

Angiotensin - II antagonism 

• Male Sprague- Dawley rats (200-225gm)  are selected.
• They are anesthetized with 60 mg/kg of phenobarbitone sodium i.v.
• One carotid artery is cannulated & connected with a Statham transducer and the B.P is recorded in a polygraph.
• Both the jujular veins are cannulated to administer the test drug.
• Pentolinium (10mg/kg) is injected to block the ganglionic activity.
• Atleast 5 animals are used  for the evaluation of test drug.
• In 10 min interval doses of  0.5,1.0, and 2µg/kg of angiotensin II are injected to establish the dose response curves.
• After 10 min a continuous infusion is started of the Ang II blocker in a dose of  10µg/kg/ 0.1ml/ml.
• Again doses of 0.5,1.0, and 2µg/kg of angiotensin II are injected .
• Intensity and duration of fall in B.P. is recorded.
• The results are compared with the known std. drug.

Antihypertensive and vasdilator activity in ganglion blocked angiotensiongen II supported rats 

• To demonstrate the direct vasodilator activity of antihypertensive agent.
• This method is an anesthetized ganglion blocked rat whose B.P is maintained by an i.v. infusion of Ang II. 
• 7-9 nos of Wister Rats (250-300g) are anesthetized using a combination of Urethrane & Chlorolase (60mg/kg).
• Chlorisondamine is administered to block the ganglion (both sympathetic and para sympathetic nerve activity i.p)
• The right faemoral artery is cannulated to record the blood pressure. 
• Artificial respiration is supported during the experiment.
• Ang II is infused at a rate of  3.5µg/min in a volume equivalent to 0.05ml/min using Harvard  infusion pump.
• The steady state pressure is established with in 15-20 minute.
• Test drugs are injected i.v. at an interval of 3 minute (2ml/kg).
• Arterial pressure is recorded on a polygraph at 5,10, 15, 20 & 30 min.
• Blood pressure response for graded dose of phenylephrine can be obtained 15min before test administration.
• Data is obtained from 5-6 animals.
• A fall in blood pressure indicates the vasodilative action in turn anti hypertensive action of the test drug.

In vitro models of antihypertension agents 

1. Monocrotaline induced pulmonary hypertension 
2. Angiotensin converting enzyme inhibition in guinea pig ileum 
3. Beta 1 sympatholytic activity in guinea pig atria 

Monocrotaline induced pulmonary hypertension 

MONOCROTALINE, a pyrrolizidine alkaloids derived from Crotaloria spectabillis.

• Sprague- Dawley rats (200-225gm) of either sex are selected.
• Animals are fed with test drug for one week prior to S.C injection of MONOCROTALINE 100mg/kg.
• Animals are sacrificed 7 or 14 days later and their hearts and lungs are excised.
• Left ventricle and lung are weighed, along with main, extra and intrapulmonary, artery are isolated.
• After 1 hour arteries are made to contract with KCl.
• Contractions are recorded using lever transducer.
• Maximum force generated by an artery is plotted as a function of applied force and recorded on polygraph.
• Contraction and relaxation of agonist responses of artery are assessed. 
• Cumulative concentration. response of KCl, Angiotension II, and NOR-Epinephrine are plotted.
• Both the responses are plotted as a function of negative log of agonist concentration. 
• To compare the differences in mean responses, t-test is applied. 

Angiotensin converting enzyme inhibition in guinea pig ileum

The Guinea Pig Ileum responds powerfully for both Angiotensin II, and Bradykinin.

Mechanism of angiotensin converting enzyme 

Procedure of angiotensin converting enzyme inhibition in guinea pig ileum

• Guinea Pig of either sex (300 – 500gm) is selected.
• Animals are sacrificed by stunning, and abdomen is opened.
• A chord is tied around the starting of intestine.
• The intestine is gradually removed from the bottom and the mesentry is cut away.
• When reached the colon, the intestine is cut halfway by passing Tyrode solution to clean the surface.
• The distal pieces (more sensitive)  are fixed in the tissue clamp and brought into organ bath of 37 degree centigrade (oxygenated).
• Angiotensin I is added (10ng/ml) after 30mts of equilibrium in bath solution and contraction is recorded.
• 5 minute after the addition of ACE Inhibitor (test drug), the diminished contraction is recorded.
• The opposite response can be observed while using Bradykinin.

β1 sympatholytic activity in guinea pig atria

β1 receptor blocking activity can be evaluated by this technique.

Beta sympatholytic activity

Procedure of β1 sympatholytic activity in guinea pig atria

• Guineapig of either sex (250-300gm) is used.
• The animal is sacrificed by stunning and exsanguination.
• Heart is removed and right/ left atrium is mounted in 50ml organ bath containing Krebs – Henseleit buffer with aeration (95%O2, 5% CO2) at 37 degree centigrade.
• Contractions are recorded using lever transducer.

Right atrium

• Isoprenaline is administered in the organ bath after 30 minutes to induce ionotropy.
• Cumulative dose is maintained starting from 0.5µgm/ml and consecutive doses at 3 minute intervals.
• The bath is flushed for 3-5 times after the stable max plateau is achieved. 
• The test drug is added into the organ bath.
• 5 minute later, again isoprenaline is added in above Concentrations & observe the response.

Observation

Beta receptor blocking activity 

Left  atrium (Electricaly stimulated)

• Left Atrium is stimulated by square wave stimulator (2 impulses at 15V, duration - 1 minute).
• After equilibrium, repeat the same as above using Isoprenaline at concentrations of   0.05-0.1 5µgm/ml.
• The bath is flushed for 3-5 times after the stable max. plateau is achieved.
• The test drug is added into the organ bath, 3 minute later again isoprenaline is added in above same doses.
• If it has β1 receptor blocking activity, the ISP induced activity is inhibited.
• IC50 values are determined from the individual dose response.

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Screening Method of Anti Ulcer Drugs on Animal Model

Peptic ulcer  is one of the most prevalent  chronic  gastrointestinal disorder.

Ulcer imbalance chart 

Regulation of Acid Secretion

Regulation of acid secretion

Screening Methods Of Antioxidant Activity On Animal Model

Approaches to Treatment Ulcer 

• Reduction of gastric acid secretion
• Neutralization of gastric acid 
• Ulcer protective
• Anti H. Pylori drugs

Screening of Anti Ulcer Agents

Requirements of an ideal model

• Should be simple, reproducible & allow for easy quantification of results.
• Should induce characteristic ulceration in specific locations.
• Should involve different mechanisms by which ulceration is produced.
• Ulcers produced should not spontaneously heal during observation period. 
• Ideal animal for screening anti ulcer agents…..?
• Continuous secretion of acid
• Glandular portion of rat stomach analogous to body of stomach in man both anatomically and functionally.
• Being omnivorous resembles man nutritionally

Guinea pigs are used when histamine is used to induce ulcers.

Screening Methods Of Diabetes Mellitus Activity On Animal Model

Animal Models for Screening

1. Pylorus ligation induced gastric ulcers in rats (Shay rat)
2. Drug (NSAID ) induced gastric ulcers in rats
3. Ethanol induced gastric ulcers in rats
4. Acetic acid induced  gastric ulcers in rats
5. Stress induced ulcers in rats
6. Histamine induced duodenal ulcers in guinea pigs.

Pylorus Ligation Induced Ulcers in Rats

Pyloric ligation  in rats leads to accumulation of gastric acid in the stomach leading to acute gastric ulceration. First demonstrated by Shay and co-workers in 1945.

Requirements

• Albino Wistar Rats (150-200 g)

• Drugs :       

                           Ether (Anesthetic)

                          Saline (control) 

                          Omeprazole (standard) 

                          Test drug (3 different concentrations x, 2x, 4x)

• Reagents :  

                          NaOH (0.01N)

                          Topfer’s reagent (dimethyl amino azo benzene)

                          Phenolphthalein

• Dissecting microscope (10x magnification), pH meter, burette, surgical instruments.

Respiratory System - Organs, Structure & Functions

Procedure of pylorus ligation 

• Rats fasted for 24 hrs prior to pyloric ligation. 
• Randomly divided into 5 groups of 3 animals each.

                                     Group I   : Control vehicle

                                     Group II  : Standard drug (Omeprazole)

                                     Group III : ‘x’ concentration of test drug

                                     Group IV : ‘2x’ concentration of test drug

                                     Group V : ‘ 4x’ concentration of test drug

• Drugs administered once for 2 days and 30 mins prior to ligation
• Rats anesthetized with ether.
• Pyloric ligation procedure done.
• Rats  placed in separate cages and allowed to recover.
• 19 hrs after pyloric ligation, animals sacrificed by decapitation.

Isolated rat stomach 

• Ulcer index is calculated.

Screening Methods Of Hypertensive Activity On Animal Model 

Ulcer index formula 

Intensity of ulcers is scored as below :-

          0 – normal stomach

          1 – superficial mucosal erosion

          2 – deep ulcer

          3 – penetrated or perforated ulcer

Contents of the stomach analyzed for ulcer  

• Volume 
• pH : pH of it is noted with pH meter.
• Free acidity  and total acidity : Titration of the solution against 0.01N NaOH done using Topfer’s reagent and phenolphthalein as indicators. Volume of NaOH which turns the solution to yellowish orange corresponds to free acidity. Titration is continued  till solution turns pink. The total volume of NaOH used up corresponds to total acidity. 

                              Acidity(meq/l/100g) =  (Vol of NaOH X Normality x 100)/0.1

• Mucin and prostaglandin levels can be estimated to detect cytoprotective effects.           

Inference

Ulcer index of test drug  compared with control group to detect anti-ulcer effect of test drug. If present, it is compared with that of standard group.

Other parameters help to infer the mechanism of ulcer protection. Examples are given below - 

•  Decrease in vol, free & total acidity    :  antisecretory action 
•  Rise in pH                                           :    acid neutralizing action
•  Increase in mucin, PGs                      :   cytoprotective effect.

Drug induced ulcers in rats

Gastric ulceration is produced in rats by certain drugs. The ability of the test drug to protect against the ulceration is observed.

NSAIDs  like Aspirin, Indomethacin, Ibuprofen

Inhibition of endogenous prostaglandin production and consequent loss of gastric mucosal defence. Important model for identifying drugs that could be effective in NSAIDs induced gastropathy.

Procedure of drug induced ulcer 

• Rats fasted for 24 hours in separate cages and randomly divided into 5 groups
• The control, standard, test drug are administered once daily for 2 days and 30 mins prior to administration of ulcerogenic agent.
• Ulcerogens administered by oral gavage. 

Oral gavage

• Rats sacrificed  4 - 6 hours later
• Ulcer index, analysis of stomach contents done.

Ethanol induced gastric ulcer in rats

Ethanol damages superficial epithelial layers & inhibits prostaglandin release. An agent that protects against ethanol induced ulcers might be cytoprotective & exert its action by stimulating release of endogenous prostaglandins & mucin. However, anti secretory agents also are effective in this model.

Acetic acid induced gastric ulcers in rats

Model mimics the chronic pattern seen with peptic ulcer.

Procedure of acetic acid induced ulcer

• Overnight fasted rats operated under ether anesthesia
• Anterior & posterior walls of stomach clamped with forceps
• 0.2 ml of 40% acetic acid injected into clamped portion.
• After 45 secs , acid removed.
• Deep round ulcers develop on the anterior & posterior walls.
• Respective treatments( control, standard, test) started from 3rd to 10 th day. Rats sacrificed on 10th day.
• Ulcers respond well to most  anti ulcer drugs like PPI, H2 blockers, cytoprotectives.

Stress induced ulcer in rats

Model of restraint ulcer - 

Drugs (control, standard, test) administered once daily for 2 days & 30 mins prior to applying restraint. Fasted & lightly anesthetized rats placed on galvanized steel window screen. Limbs are held together in pairs & tightened with adhesives. After 24 hrs, animal removed, sacrificed and degree of ulceration noted.

Modified method - Water immersion induced restraint ulcer.

Histamine induced duodenal ulcers in guinea pigs

• Guinea pigs fasted for 48 hrs.
• Drugs (control, standard, test) once daily for 2 days & 30 mins before administration of histamine.
• Ulceration induced by injecting 1 ml of histamine solution intraperitoneally.
• Promethazine 5mg injected intraperitoneally  15 mins before and after histamine .
• Animals sacrificed after 4 hrs
• Degree of ulceration & gastric contents assessed. 

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Screening Methods for Diabetes Mellitus on Animal Model

Diabetes mellitus  Known since ages as “Madhumeha” i.e. sweetness of disease urine in Ayurveda by  ‘Sushruta’

• During first century Greek physician ‘Aeretaus’ gives term Diabetes as “to flow through”
• In 1755 by ‘Dobson’ demonstrated Diabetes as by presence of sugar in urine.
• In 1909 by ‘De Mayer’ gives statement about insulin is the non-digestive part of pancreas & islet cell was responsible for pancreatic diabetes.
• It is chronic metabolic disorder, resulting from insulin deficiency characterized by hyperglycemia, altered metabolism of carbohydrate, protein , lipid &  increases the risk of vascular complications.
• Diabetes is not a single disease, it is heterogeneous group of syndrome, characterized by an evaluation of Blood Glucose caused by a relative deficiency of Insulin.   

Screening Methods Of Hypertensive Activity On Animal Model

Glucose mechanism

Classification of Oral Hypoglycemic Agents OR classification of anti-diabetic Drugs  

• Insulin secretagogus – they promote the insulin release from β-cell of pancreas. 

       e.g.. Sulfonylurea :- Tolbutamide, Acetohexamide 

• Insulin sensitizer 

           - It increases insulin uptake & utilization by target tissue
                                            e.g.Thiazolidinediones-Rosiglitazone, Pioglitazone  
           - It reduces hepatic glucose output, largely by  inhibiting hepatic gluconeogenesis
                                            e.g. Biguanides - Metfomin, Phetformin 

• α-Glycosidase inhibitor – they act by delaying the digestion of carbohydrate thereby decreasing glucose absorption 

         e.g. Acarbose, Miglitol 

Screening Methods Of Antioxidant Activity On Animal Model

How to Oral hypoglycemic agents work 

Mechanism of anti-diabetic agents

Screening models of anti-diabetics 

Screening methods for anti-diabetics 

Screening Methods Of Depressant Activity On Animal Model

Models for insulin dependence diabetic mellitus 

Screening model for insulin dependence diabetes mellitus

Blood Pressure & Its Measurement Procedure

Chemical used in insulin dependence diabetes mellitus 

IRREVERSIBLE Chemical 

1. Alloxan 
2. Streptozotocin
3. Diphenyl thiocarbazine 
4. Oxine-9- Hydroxyquinolone 
5. Vacor 
6. Somatostatins 
7. Catecholamines 
8. Glucocorticoids 

REVERSIBLE Chemical 

1. 6- amino l-asparginase 
2. Azide 
3. Cyproheptadine 
4. Malonates 
5. Glucagoan

Alloxan induce Diabetes 

Alloxan Induces permanent diabetes.  

Mechanism of action of alloxan 

• Directly toxic to beta  cells.
• Interacts with sulfhydral enzyme and inhibits it. 
• Hexokinase activity.
• Protein kinase activity.
• Induces mitochondrial abnormalities.
• Damages the DNA (fragmentation).

Respiratory System - Organs, Structure & Function

Type of animal used in Screening model for insulin dependence diabetes mellitus

Glucose Estimation Method

Glucose Oxidase method - Glucose is oxidized in Gluconic Acid by glucose oxidize. The hydrogen peroxide liberated is reduced by peroxides & Oxygen transferred to an accepter, which is color less in the reduced form but colored  in oxidized form. 

• Glucometer

Urine analysis - Qualitative,Cheap,Convenient. Benedict Reagent are used but Diagnosis can’t based- may show false positive or false negative. 

Hexokinase enzyme method

The procedure is a fluorometric rate method measuring the formation of NADPH catalyzed by immobilized glucose-6-phosphate dehydrogenase and hexokinase held within a tiny stirrer. The enzyme stirrer is stable for at least two months and can be used over eight-hundred assays without any loss of activity. 

Glycosylated Haemoglobin (HbA1c)

Measurement of blood glucose level in diabetic suffer from variation due to dietary intake of previous day. Long term objective assessment degree of diabetic control is better done by measurement of glycosylated hemoglobin HbA1c,a minor hemoglobin component present in normal person. This is because non-enzymatic glycosylation of hemoglobin takes place over 120 day’s. This gives an estimated of diabetic control for the Preceding 6-10 day’s.

Glucose Tolerance Test (GTT) 

Fasting Blood glucose level is first determined & then blood glucose level are sampled minutes to hour after an oral dose of glucose(75gm in 300 ml water) . Blood and urine sample are collected at half hourly interval.

Procedure of alloxan induced diabetes mellitus 

Produce for alloxan induce diabetes mellitus 

Limitations of alloxan induced diabetes mellitus 

• High mortality
• Ketosis 
• Some species are resistant to alloxan e.g. guinea pig 
• Streptozocin has almost completely replaced alloxan 

Streptozotocin induced diabetic mellitus 

Broad spectrum anti biotic – 200 mg/kg i.p.

Mechanism of action of streptozotocin

• β cell damage by free radical injury
• Fragmentation of DNA 
• Nitric oxide generation 
• Induces diabetes in all species 

Procedure of streptozotocin induced diabetic mellitus

Procedure of streptozotocin induced diabetic mellitus

Modifications in streptozotocin induce diabetes mellitus

• Multiple low dose over three weeks 
• Cyclosporin A given with streptozotocin 

Advantages of streptozotocin induced diabetic mellitus

• Greater selectivity towards beta cells 
• Low mortality
• Longer and irreversible diabetes 
• Guinea pigs and rabbits are resistant 

Hormone induced diabetes mellitus 

Dexamethasone is a long acting glucocorticoid possessing immunosuppressant action in the islets and produces type 1 diabetes. 

Hormone induced diabetes mellitus 

Limitations for Hormone induced diabetes mellitus 

• Long standing procedure 

Viral induced diabetes mellitus 

Following virus are involve in developing diabetes mellitus -

• RNA picorna virus
• Coxsackie virus
• Encephalomyocarditis
• Mengo-2t
• Renovirus
• Lymphocytic choriomeningitis 

Procedure of viral induced diabetes mellitus 

Viral induced diabetes mellitus

Surgically induced diabetes mellitus methods 

Surgical removal of pancreas result in insulin dependent form of diabetes mellitus state.

Surgery induced diabetes mellitus 

Surgically induced animals

• Partial pancreatectomized animals

                               e.g. dog, primate, pig & rats


Advantage of surgically induced diabetes mellitus method 

• Avoids cytotoxic effects of chemical diabetogens on other body organs.
• Resembles human type 2 diabetes due to reduced islet beta cell mass.

Disadvantage of surgically induced diabetes mellitus method

• cumbersome technical and post operative procedure.
• digestive problems due to excision of exocrine portion of the pancreas.
• loss of counter regulatory response to hyperglycemia.
• high mortality

Insulin antibody induced diabetes mellitus

A transient diabetic syndrome can be induced by injecting guinea pigs with anti insulin serum.

Preparation of antibody for inducing diabetes mellitus

Preparation of antibody for inducing diabetes mellitus 

Procedure of antibody induced diabetes mellitus

Procedure of antibody induced diabetes mellitus

Limitations for antibody induced diabetes mellitus

• Effect persist as long as antibodies remain in the circulation.
• Large doses and prolonged administration- ketonemia,  ketonuria, glycosuria and acidosis are fatal to animals.

Genetic models for induced diabetes mellitus 

• Autoimmune destruction of pancreatic β cells in association with autoantibody production.
• Shares many characteristics with human insulin dependence diabetes mellitus.
• Spontaneously diabetes mellitus on hereditary basis.

Animal for genetic model induced diabetes mellitus

• May provide more insight into pathogenesis of diabetes in humans
• Discovered in 1974 by Drs Reignald and Clifford Chappel in a commercial rodent breeding company (Bio-breeding laboratories Ltd.) in Ottawa hypoinsulinemia is developed which is caused by autoimmune destruction of pancreatic beta cells in association with autoantibody production. 

Procedure for genetic models induced diabetes mellitus  

Procedure for genetic models induced diabetes mellitus

Limitation for genetic models induced diabetes mellitus

• Ketosis
• Glycosuria
• Weight loss
• High mortality

Models for non-insulin dependence diabetes mellitus 

1. Neonatal streptozotocin model 
2. Genetic model

Neonatal streptozotocin model for non-insulin dependence diabetes mellitus 

• Pancreatic beta cells destruction
• Decrease in pancreatic insulin  stores 

procedure for neonatal streptozotocin model for non-insulin dependence diabetes mellitus

Genetic models for non-insulin dependence diabetes mellitus  

Three type of genetic models are involve in developing non-insulin dependence diabetes mellitus.

• Monogenic models of obesity and NIDDM
• Polygenic models of obesity and NIDDM
• Animal models of NIDDM with unknown hereditary and environmental component

Monogenic models of obesity and non-insulin dependence diabetes mellitus 

• Obesity
• Hyperinsulinemia
• Hyperglycemia
• Hyperlipidemia

Polygenic models of obesity and non-insulin dependence diabetes mellitus 

• No single gene implicated.
• Interaction between environment and several genetic defects.
• Polygenic animal model represents human condition more closely.

Animal model of non-inslin dependence diabetes mellitus with unknown hereditary & environment component

Animals taken from natural environment developed DM when fed normal laboratory diet 

Diet and nutrition induced non-insulin dependence diabetes mellitus 

They remain normal in its natural habitat but develop obesity and diabetes in captivity when fed on standard laboratory chow (high energy diet) instead of its usual low energy vegetable diet. 
Sand rats are used extensively in the drug testing such as protein tyrosine phosphatase inhibitor 65 and glucogan like peptide-1 (GLP-1) analogues.

Transgenic or knockout animals 

• Genes – insulin resistance

                        Insulin receptor
                        Glucose tranporters
                        Hexokinase II
                        Tumour necrosis factor α

• Genes –defective insulin secretion

                        GLUT-2
                        Glucokinase
                        Islet amyloid polypeptide

• Genes- increases body fat

                        β3 receptors knockout mouse
                        Uncoupling protein knockout mouse

Transgenic technique 

Single gene is identified in embryonic stem cell, Female rat/mouse is allowed to mate. Next day single cell zygote is collected and maintained in culture for few hours. The desired gene is then injected into pro-nucleus of zygote and the construct integrates itself into the genome of the zygote.  

The progeny has the desired characters.

Transgenic technique 

Advantage and disadvantage of transgenic technique

Advantage and disadvantage of transgenic technique

Normoglycemic animal models 

• Rabbit Model 
• Rat Model
• Dog Model

Normoglycemic rabbit model 

Normoglycemic rabbit model

Normoglycemic Rat model

Normoglycemic rat model

Normoglycemic dog model  

• Beagle dog 15-20 kg 
• Food is stopped 18 hours prior to administration of test compound 
• Blood is collected up to 48 hours

Modification for norglycemic dog model 

• Dogs are pancreatectomized 2-3 years prior 
• Given pancreatic enzymes orally 
• Test compound is given with an oral suspension  

Insulin Assays 

Insulin assay

insulin assay 

In vitro methods on isolated organs and cells 

To study the effect of the drug on insulin, glucagon and somatostatin secretion without interference from other organs

In Vitro Methods 

1. Assays of insulin &of insulin like activity
2. Isolated organs, cell and membranes  
3.  Insulin receptor Binding assay
4. Assays of other glucose regulating peptide  hormones
5. Inhibition of polysaccharide degrading enzyme
6. Effect on secondary diabetes symptoms
7. Isolated pancreas of rat 
8. Isolated pancreatic islets of rat
9. Isolated rat liver 
10. Isolated hepatocytes of rat
11. Fructose 2,6-bi phosphate in rat hepatocytes 
12. Isolated target tissue Perfused hind limb in rats 
13. Muscle cell lines 
14. Assays for insulin or insulin like substances on adipocytes 
15. Assays for lipid synthesis
16. Assays for glucose transport 
17. Glucose uptake by the isolated diaphragm from mice and rats
18. Insulin receptor binding assays

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