Models and Assays

In Vitro

Neurofit offers a large range of models and assays for evaluating neurotoxicity, neuroprotection or neurotrophic activity of new test compounds. We can also develop custom protocols to determine mechanism of action, or define therapeutic indication of new drug candidates.

Rodent Primary Culture:

• Cortical, hippocampal, mesencephalon neurons
• Spinal cord motor neurons
• Sensory neurons (dissociated DRG)
• Schwann cells – sensory neurons co-culture
• Rat nerve – human muscle co-culture

Models:

• Basal neuronal survival: cortical neurons and spinal motor neurons
• Exitotoxicity: cortical neurons or spinal motor neurons intoxicated with glutamate or NMDA
• Apoptosis: staurosporine intoxication
• Anti-mitotic intoxication: Purified sensory neurons intoxicated with cisplatin, vincristine or taxol, sensory neurons and Schwann cells co-culture intoxicated with cisplatin, vincristine or taxol, spinal motor neurons intoxicated with vincristine
• Alzheimer’s models: Intoxication of neuronal cultures with fibrillar amyloid beta peptide
• Parkinson’s models: mesencephalon neurons intoxicated with MPP+
• Proliferation of Schwann cells
• Neurotrophic effect: effects of test compounds on neurite outgrowth on cortical neurons, hippocampal neurons, spinal motor neurons
• Co-culture nerve / muscle: effects of test compounds on co-culture innervation

Assays:

• Apoptosis evaluation: measurement of caspase 3 activity
• Co-culture: length of neuritis, surface of innervated muscle fibre area, video samples
• Immunocytology: measurements of tyrosine hydroxylase, neurofilaments, Schwann cells (S-100 antibody), Hoechst nucleus
• Neurotrophic effect: neurite length, percentage of cells displaying neuritis – cortical neurons, hippocampal neurons, spinal motor neurons
• Viability and survival tests: LDH, AP, MTT assays
• Western blotting of proteins

Oncology:

• Cancer cell lines proliferation assay

Histology

Neurodegeneration investigation on peripheral nerves (computer-assisted analysis of nerve fibre morphometry):

• Axonal area
• G-ratio (relative myelin thickness)
• Fibre size distribution
• Fibre density
• Percentage of degenerated fibres (manual counting of axonal degeneration)

Standard colouration on paraffin and cryosections:

• HE staining
• Toluidin blue staining

In vivo – neurology

Neurofit has developed powerful testing methods in rodents for assessing the neurotoxicity, neuroprotective or neurotrophic effects of drug candidates. Studies may include quantitative behavioural measurements or analyses of effects upon neurodegenerating nerve fibres (axonopathy) or nerve cells (neuronopathy).

Neurological and neurodegenerative models:

• Alzheimer’s disease: Scopolamine induced cognitive deficits
• Multiple sclerosis: EAE rats, DTH rats
• Parkinson’s disease: Fri/fri mouse for late stage symptomatic treatment
• Epilepsy : Epi-mouse programme
• Pain and neuropathic pain
• Peripheral neuropathies
• Retinal and optic nerve degeneration

Tests:

• Cognitive deficits
• Motor coordination
• Muscular strength, muscle endurance
• Sedation: Spontaneous activity during open-field test
• Sensitivity tests
• EMG test

In vivo – psychiatry

Neurofit offers a variety of well-accepted behavioural tests to assess the potential anxiolytic, antidepressant, antipsychotic or effects upon memory or learning of test compounds.

Anxiety:

• Light/dark test in mice
• Marble burying in mice
• Elevated plus maze in rodents

Depression:

• Forced swimming test
• Chronic mild stress in mice
• Tail-suspension test in mouse

Schizophrenia:

• PCP or MK801-induced hyperactivity test
• Catalepsy test

Cognitive dysfunction:

• Dark avoidance
• Object recognition test
• T-maze
• Conditioned place preference test