Preclinical ANS Screening Models
The Autonomic Nervous System (ANS) controls every involuntary function in the body—heart rate, blood pressure, smooth muscle contraction, glandular secretion, and pupil diameter. This unit covers the complete preclinical screening models used to identify and evaluate drugs that act on sympathetic (adrenergic) and parasympathetic (cholinergic) divisions of the ANS, along with screening methods for skeletal muscle relaxants, local anesthetics, and drugs acting on the eye.
Syllabus & Topics
- 1Sympathomimetic Screening Models: Sympathomimetics mimic the effects of sympathetic nervous system stimulation (norepinephrine/adrenaline). In-vivo Blood Pressure Measurement (Anesthetized Rat/Cat Preparation): The carotid artery is cannulated and connected to a pressure transducer. IV injection of the test drug, and the blood pressure response (rise/fall) is recorded on a polygraph/PowerLab. Comparison with standard Norepinephrine/Adrenaline dose-response curves. Isolated Tissue Preparations: Rabbit Aortic Strip (α1-adrenergic contraction), Guinea Pig Tracheal Chain (β2-adrenergic relaxation), Isolated Rat Vas Deferens (sympathetic nerve stimulation). These identify receptor selectivity (α1, α2, β1, β2).
- 2Sympatholytic Screening Models: Sympatholytics block adrenergic receptors or reduce sympathetic outflow. α-Adrenergic Blockade: Demonstrate the reversal of Adrenaline-induced pressor response on anesthetized rat BP preparation. Before α-blocker (Phentolamine): Adrenaline causes a rise in BP. After α-blocker: Adrenaline causes a FALL in BP (‘Adrenaline Reversal’—the β2 vasodilatory component is unmasked). β-Adrenergic Blockade: Propranolol blocks Isoprenaline-induced tachycardia (on isolated guinea pig heart) and bronchodilation (guinea pig tracheal chain). Demonstrate shift in the Isoprenaline dose-response curve to the right (competitive antagonism).
- 3Parasympathomimetic & Parasympatholytic Screening Models: Parasympathomimetics: Mimic acetylcholine’s muscarinic effects. Isolated Guinea Pig Ileum (Organ Bath): The gold standard for cholinergic drug screening. Acetylcholine causes dose-dependent contraction of the smooth muscle. The test parasympathomimetic’s potency is compared to Acetylcholine. Pilocarpine-Induced Salivation (in vivo in mice): Pilocarpine (muscarinic agonist) stimulates massive salivation. Quantified by weighing a pre-placed cotton ball in the mouth. Parasympatholytics (Anticholinergics): Atropine-Induced Mydriasis: Atropine eye drops applied to the rabbit eye cause measurable pupil dilation (mydriasis). The test drug’s ability to block Pilocarpine-induced miosis confirms anticholinergic activity. Reversal of ACh-induced contraction on isolated guinea pig ileum by the test drug (rightward shift of ACh dose-response curve).
- 4Skeletal Muscle Relaxant Screening Models: Skeletal muscle relaxants reduce muscle tone, spasm, or paralysis via central or peripheral mechanisms. Rota-Rod Test: The animal is placed on a rotating rod. Muscle relaxants impair the animal’s ability to maintain balance, causing it to fall off. The fall-off time is recorded. Grip Strength Test: The mouse/rat grasps a wire grid connected to a force transducer. The maximum grip force is recorded. Muscle relaxants significantly reduce grip strength. Traction Test: The mouse is hung by its forepaws from a horizontal wire. A normal mouse quickly pulls itself up. A muscle relaxant impairs this traction ability. Head Drop Method (Rabbits): Used specifically for peripherally acting NM blockers (like Tubocurarine). The rabbit’s head droops when skeletal muscles are paralyzed.
- 5Local Anesthetic Screening Models: Local anesthetics block nerve conduction by inhibiting voltage-gated sodium channels, preventing pain signal transmission. Intradermal Wheal Method (Guinea Pig): The test drug is injected intradermally to form a small bleb (wheal). Pin-prick stimulation of the wheal area is performed at timed intervals. The duration of loss of pain sensation (analgesia) is measured. Compared with standard Lidocaine. Rabbit Corneal Reflex Test: A drop of the test local anesthetic is applied to the rabbit’s cornea. The corneal reflex (blinking response to touch) is tested at intervals. Duration of corneal anesthesia is recorded. Sciatic Nerve Block (Rat/Frog): The test drug is injected around the sciatic nerve. Loss of hind-limb withdrawal reflex to painful stimulation confirms nerve block efficacy and duration.
- 6Drugs Acting on the Eye: Mydriasis (Pupil Dilation): Measured after topical application of sympathomimetics (Phenylephrine—α1 agonist) or parasympatholytics (Atropine—muscarinic blocker) to the rabbit eye. Pupil diameter is measured at timed intervals using calipers or a millimeter ruler under standardized lighting. Miosis (Pupil Constriction): Measured after topical Pilocarpine (muscarinic agonist) or Physostigmine (AChE inhibitor). Intraocular Pressure (IOP) Measurement: Using a Schiotz tonometer on the anesthetized rabbit eye. Anti-glaucoma drugs (Timolol—β-blocker, Pilocarpine—miotic, Latanoprost—prostaglandin analog) reduce elevated IOP.
Learning Objectives
Exam Prep Questions
Q1. What is “Adrenaline Reversal” and why does it occur?
Adrenaline typically increases blood pressure by stimulating α₁-adrenergic receptors, causing vasoconstriction. It also has a weaker action on β₂-adrenergic receptors, which cause vasodilation. Under normal conditions, the α₁ effect dominates, resulting in a rise in blood pressure. However, when an α-blocker (such as phentolamine) is administered, the α₁ receptors are blocked. As a result, only the β₂-mediated vasodilation remains active, causing a fall in blood pressure instead of a rise. This phenomenon is known as adrenaline reversal.
Q2. Why is the Isolated Guinea Pig Ileum the “gold standard” for cholinergic drug screening?
The guinea pig ileum is highly sensitive to cholinergic stimulation due to the presence of abundant muscarinic (M₃) receptors. When placed in an organ bath with physiological solution, it remains viable and responds strongly to even very small doses of acetylcholine. These responses produce clear, dose-dependent contractions that can be easily measured using recording devices. Its high sensitivity, reproducibility, and ease of use make it the preferred model for evaluating cholinergic drugs.
Q3. How does the Rota-Rod Test specifically screen for muscle relaxant activity?
The Rota-Rod test assesses motor coordination and muscle strength using a rotating rod. Normal animals can maintain balance and remain on the rod for a set duration. When a muscle relaxant drug is administered, it reduces coordination and muscle tone, causing the animal to fall off earlier. The fall-off time is recorded, and a significant reduction compared to control indicates muscle relaxant activity.