Animal models and pharmacology

General methodology to study the therapeutic potential of newly synthesized drug

1. Choose the species

2. Model of the disease:

   1. Transgenic/genetically modified

   2. Pharmacological: chemical/induced

   3. Spontaneous: selective breeding over many generations

3. Groups:

   1. Healthy: vehicle and drug

   2. Pathology: vehicle and drug

4. Validation of the model

5. Choose the test/parameter

6. Choose the reference (positive/negative control)

7. Route of administration, doses, acute vs chronic

8. Toxicology testing

DSM criteria

2 individuals with the diagnosis of major depression with no symptoms in common

1. P1: depressed mood, weight loss, insomnia, psychomotor agitation, and suicidal thoughts

2. P2: diminished pleasure, weight gain, hypersomnia, psychomotor retardation, and fatigue

DSM V criteria

At least 5 of the following are present simultaneously for at least 2 weeks (symptom 1 or 2 is necessary):

1. Depressed or irritable mood

2. Markedly diminished interest or pleasure in all, or almost all, daily activities

3. Significant weight loss or weight gain

4. Insomnia or hypersomnia nearly every day

5. Psychomotor agitation or retardation nearly every day

6. Fatigue or loss of energy nearly every day

7. Feelings of worthlessness or inappropriate guilt nearly every day

8. Diminished ability to think or concentrate nearly every day

Behavioural endpoints

Animal models of depression

In early life or adulthood

• Based on application of stressors

• Biological causation models: induce the biological and chemical imbalances that are believed to cause depression

Early life adversity

• Separation of pups and mothers

• Support findings from human research maternal neglect or a history of childhood abuse increased MDD

• Limitation: newborn pups development stage corresponds to a pre-natal human stage

Social Defeat Stress Model

• Adult stress induced by aggressive peers, leading to social withdrawal

• Resilient vs susceptible rodent

• Sensitive to chronic SSRIs

• Difficult to carry in female due to lower aggressiveness

• Dysregulation of the prefrontal cortex and changes in neurotrophins

Unpredictable chronic mild stress (UCMS)

• Responsive to chronic antidepressants

• Strain differences in susceptibility

Biological causation models

• Lipopolysaccharide injection

  • Understand the role of inflammation in brain diseases

  • Induce sickness behavior and then a depressive behavior

  • Reversed by antidepressants

• Olfactory bulbectomy: a surgical procedure involving the removal of the olfactory bulbs, which are involved in the sense of smell

GR (Glucocorticoid Receptor) knock-out mice:

Study the cortisol hypothesis and HPA axis feedback loops.

Test of GABA-R subunits function

1. Elevated plus maze

2. Behavioural assessment of anyolitic-like actions of diazepam regulated by alpha 2 subunit

3. Open field test

Buspirone test

1. Measure the time spent in open arms reflect anxiolytic activity of a molecule

2. Buspirone has an anxiolytic effect in WT, the effect is lost in Ko mice. 5-HT1A is involved in the mechanism of the anxiolytic effect of buspirone in this model. Can hypothesize that buspirone is a ligand for this R. The absence of R.5HT1A is anxiogenic, therefore the activity of R is anxiolytic, buspirone is believed to be the R.5HT1 agonist

3. Measure the number of entries that assess the locomotor activity of animals, variations in this parameter could also influence the time spent in open arms

Parkinson’s disease

Progressive movement disorder of the nervous system. It causes nerve cells (neurons) in parts of the brain to weaken, become damaged, and die

Animal models in Parkinson disease

• Toxin models: 6-OHDA, MPTP

• Genetic: PARKIN-KO, PINK1, TG –AAV Alpha synuclein (mutatedor human form)

Variation between models regarding: danergic damage, mitochondrial respiratory deficit, oxidative stress, alpha-synuclein and levy bodies aggregates, locomotor and non-motor deficit

MPTP and stepping deficit

1. MPTP in neurotoxin-induced model of Parkinson disease inducing akinesia and rigidity

2. Stepping test used to evaluate motor function, specifically forelimb akinesia (difficulty initiating movement) and rigidity. Investigator holds animals so that both hind limbs and one forepaw are raised just off the surface of a wide table. The animal is moved laterally across the surface of the table (over ~80-90 cm for ~10 sec) in such a way that it must bear weight on its remaining forepaw. Increasing the number of steps is the measurable proof that the drug is working.

3. MTPT treatment is able to decrease stepping performance over time, so limb akinesia as in Parkinson disease. L-DOPA treatment improved the stepping performance of the MPTP-treated mice. Stepping test in mice seems to be a reliable and sensitive behavioral mesure for assessing forelimb akinesia of translational value for Parkinson disease

6‐OHDA model

1. 6-OHDA is injected directly into the medial forebrain bundle to unilaterally destroy dopaminergic neurons. Disrupt complex I of the mitochondrial electron transport chain and increase generation of reactive ROS that contributes to an apoptotic form of cell death. Limitations include being an acute, unilateral, and non-progressive model.

   1. Predictive validity: partial, symptoms only appear on one side of the animal’s body.

   2. Theoretical validity: Parkinson’s is chronic, slow and naturally progressive disease, in animals is an acute damage to the dopamine system.

   3. Predictive validity: 6-OHDA rats develop the same side effects (dyskinesias) and improves the movement symptoms

2. A significant decrease in locomotor activity was found in the 6-OHDA group 1 day after surgery compared with the sham groups. 21 days after, no difference were found between the neurotoxic and the sham groups, the animals’ locomotor activity recovers over time. 6-OHDA animals spent less time in the open arms of the EPM compared with the sham group. 6-OHDA is able to produce anxiety-like behaviour.

Cardiovascular system

The system is regulated by the autonomic nervous system and the renin-angiotensin system

Functional exploration of cardiac function

1. Echocardiograph is a non-invasive technique ro explore the cardiac morphology and function using ultrasounds. A probe emits ultrasounds at high frequency (15-40 MHz) toward the organs. Th probe receives back the echoes, which are translated in electrical signal and amplifies. Used for morphological exploration of the heart chamber and large vessels. Conducted on anaesthetized, unconscious animals.

2. Morphology: LV end systolic and diastolic volume, LV mass. Function: strike volume, ejection fraction and cardiac output.

3. Fractional shortening is significantly reduced at 4 and 9 weeks → systolic (heart contract) dysfunction. Increase left ventricle wall thickness and increased dilatation → hypertrophy (increased size of existing cells) and dilatation. The treatment allows to reduce the cardiac function and remodelling.

Hypertension test

1. Choose animal model for hypertension:

   1. Genetic model: SHR and Wistar Kyoto rat, to test anti-hypertensive drugs

   2. Genetic + environment: Dahl salt-sensitive and normal-high-salt-diet, if the compound targets salt-retention

   3. Transgenic rats: overexpression of gene regulating BP, if the drug targets a very specific pathway

2. Test to monitor blood pressure:

   1. Radiotelemetry

   2. Catheterism

   3. Tail cuff method

3. Experimental conditions: dose-response, non-selective effects, efficacy vs known effective drugs (valsartan, losartan)

Valsartan regulation of cardiovascular system

1. Radiotelemetry: implant of a pressure probe in the animal by surgery. Mesurement of electrical activity by ECG recording: heart rated, ECG anormalies, arrytmia (long QT).

2. The graph shows that a high-sodium (High Na) diet induces a steady increase in systolic blood pressure in the control (Vehicle) group. Both drug treatments (Valsartan and LCZ) significantly attenuate this rise in blood pressure compared to the vehicle. LCZ also appears more effective than Valsartan at keeping the blood pressure lower.Left Ventricular End Diastolic Pressure (LVEDP) is significantly lower in both the Valsartan and LCZ696 groups compared to the vehicle group. Lowering LVEDP indicates that the drugs are effectively protecting the heart against the increased filling pressures and dysfunction associated with hypertension.

3. Arrhythmic effects: You would use ECG (Electrocardiogram) recordings to measure the heart's electrical activity. This allows researchers to monitor heart rate, detect ECG anomalies, and identify specific arrhythmias.

4. To further explore the cardiovascular effects, you could use Echocardiography (ultrasound) to non-invasively assess cardiac morphology (like left ventricular hypertrophy or chamber dimensions) and function (like ejection fraction and stroke volume). Other tests could include measuring plasma biomarkers (like BNP), assessing endothelial dysfunction, or examining vessel wall remodeling through histology.