Inhibition of acetylcholinesterase (AChE) activity in rat striatum serves as a valuable method for screening potential treatments for Alzheimer’s disease. Here’s a detailed procedure:
Materials
- Male Wistar rats (weight: 200-250g)
- AChE inhibitors (e.g., donepezil, rivastigmine)
- Acetylcholine (ACh) solution
- Phosphoric acid buffer (pH 8)
- Ellman’s reagent (5,5′-dithiobis(2-nitrobenzoic acid), DTNB)
- Spectrophotometer
- Microcentrifuge tubes
- Homogenization equipment
- Ice bath
- AChE activity assay kits (optional)
Procedure
- Animal Preparation
- Acclimate rats for at least a week in the lab environment.
- Maintain a 12-hour light/dark cycle and provide food and water ad libitum.
- Dosing
- Randomly assign rats to control and treatment groups.
- Administer the AChE inhibitor orally or via injection at predetermined doses. Control group receives a saline solution.
- Tissue Collection
- After 1-24 hours (as per design) post-dosing, euthanize the rats humanely using approved methods.
- Dissect the brain and isolate the striatum quickly.
- Store the striatal tissue in ice-cold buffer to maintain enzymatic activity until further processing.
- Homogenization
- Weigh the striatal tissue and homogenize it in ice-cold buffer (1:10 w/v).
- Centrifuge the homogenate at 10,000 rpm for 10 minutes at 4°C to remove debris.
- AChE Activity Assay
- Prepare reaction mixture: add the supernatant to a cuvette containing buffer, ACh, and Ellman’s reagent.
- Incubate the mixture for 30 minutes at room temperature.
- Measurement
- Measure absorbance at 412 nm using a spectrophotometer.
- Determine AChE activity based on the change in absorbance, which corresponds to the rate of ACh hydrolysis.
- Data Analysis
- Compare AChE activity between treated and control groups.
- Use appropriate statistical methods (e.g., t-test or ANOVA) to analyze the significance of the results.
- Interpretation
- A significant decrease in AChE activity in treated rats indicates successful inhibition, supporting the potential of the compound as an Alzheimer’s treatment.
Conclusion
This protocol offers a straightforward approach to screen AChE inhibitors in the striatum of rats, providing insights into therapeutic candidates for Alzheimer’s disease. Always ensure ethical considerations and appropriate approvals are in place prior to animal studies.