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A photograph of a male RNU Rat against a white wall.

Protocol: 6-OHDA Lesion of Medial Forebrain Bundle in RNU Rats

Unilateral 6-OHDA lesion to medial forebrain bundle (MFB) is used for model induction of Parkinson’sDisease in rodents. The lesion degenerates dopaminergic neurons within the nigrostriatal pathway causing a loss of 70-90% of dopaminergic neurons in the striatum, severe motor deficits, and non-motor behavioral symptoms.

Preparation of Pargyline

  1. Calculate the corrected factor for pargyline hydrochloride.
  2. Determine concentration of pargyline HCl solution to get 5mg/mL (see calculations below)
  3. Weigh out the amount of pargyline required for injection of total number of animals within 30 days of preparation date.
  4. Record actual weight of powder.
  5. Transfer pargyline powder to dark amber vial.
  6. Dissolve powder with appropriate volume of 0.9% NaCl (pH=7.4).

Preparation of 6-OHDA

  1. CAUTION: PPE Requirements → Double glove, Safety Goggles, and a mask
  2. Weigh out 6-OHDA (HBr or HCl) powder into a weigh boat and record actual weight.
  3. Transfer powder to a 1.5mL dark amber tube and cover with aluminum foil.
  4. Dissolve the powder in 1mL of 0.9% NaCl + 0.05% Ascorbic Acid
  5. Vortex the sample.
  6. Label tubes with 6-OHDA + Current Date + Initials
  7. Fill each tube with 50 uL of 6-OHDA solution.
  8. Store the tubes at -80*C and use within 12 months of preparation.

Preparation of Animals for 6-OHDA Lesioning

Animal Selection and Acclimatization:

  • Select appropriate animal models (e.g., rodents) for the study, considering factors such as age, weight, and strain.
  • Allow animals to acclimatize to the housing facility for at least one week before the experiment.

Health Assessments:

  • Conduct a thorough health assessment before the experiment.
  • Exclude animals with pre-existing health conditions, signs of illness, or abnormal behaviors.
  • Monitor weight variations and ensure animals are within the appropriate weight range for the study.

Anesthesia and Analgesia Considerations

  • Plan and document the anesthesia protocol, including the type of anesthesia, dosage, and any necessary analgesia.
  • Ensure that the chosen anesthesia method aligns with ethical guidelines and is appropriate for the species.

Pre-Operative Monitoring

  • Conduct pre-operative health checks on the day of the procedure.
  • Monitor animals for signs of stress, discomfort, or unusual behavior.

Identification and Record Keeping

  • Clearly label individual animals for identification.
  • Maintain detailed records of each animal’s identification, health status, and any relevant background information.

Microinjection of 6-OHDA into Medial Forebrain Bundle

  1. Position the needle over the burr hole.
  2. Measure the diameter of the hole with the needle and record the AP.
  3. Divide this AP by 2 for the center of the burr hole and position the needle accordingly.
  4. Lower the needle until you touch the exposed dura and ZERO out the DRO by pushing “CLR”.
  5. Lower the needle at a rate of 1.00 mm/min until you reach DV: -8.00mm and wait for 30 secs.
  6. Retract the needle to DV: -7.70 mm creating a 0.30mm pocket for injection.
  7. Begin infusion with the following conditions on the “Infuse Only” program on Harvard Pump
  8. Retract the needle at a rate of 0.5 mm/min for the first 1.00 mm dorsal of the injection site then retract at a rate of 1.00 mm/min thereafter.

Post-Operative Care

  1. Administer 3.0 mL of warm 0.9% saline subcutaneously
  2. Administer appropriate dosing volume of Carprofen 5mg/kg from 0.5mg/mL working solution.
  3. Transfer animal to pre-warmed recovery cage positioned atop a heating pad with floor access food.
  4. Monitor the animal until ambulatory.

How to Improve 6-OHDA Lesioning Accuracy for Male & Female Animals

Histological Verification

  • Plan for histological verification to confirm the accuracy of the lesion site.
  • Determine appropriate time points for sacrifice based on the expected progression of the lesion and the study objectives.

Immunohistochemistry or Staining

  • Employ immunohistochemical techniques or specific stains (e.g., Nissl staining) to visualize dopaminergic neurons.
  • Select markers like tyrosine hydroxylase (TH) to identify dopaminergic cells.

Microscopic Examination

  • Examine the tissue sections under a microscope to assess the presence, density, and morphology of dopaminergic neurons in the target area.
  • Compare lesioned and non-lesioned hemispheres for differences.

Quantitative Analysis

  • Conduct quantitative analyses, such as cell counting or image analysis, to determine the extent of dopaminergic cell loss in the lesioned hemisphere.
  • Use appropriate statistical methods to assess significance.

Comparison with Stereotaxic Coordinates

  • Compare the observed lesion site with the original stereotaxic coordinates used during the injection procedure.
  • Confirm that the lesion is consistent with the intended target region.

Photographic Documentation

  • Capture high-quality images of histological sections for documentation and reference.
  • Include images of both lesioned and non-lesioned hemispheres for comparison.

Documentation and Reporting

  • Document the histological findings, including any variations or unexpected observations.
  • Clearly report the confirmation results in research publications or reports.

By implementing these steps, researchers at AniLocus robustly confirm the accuracy of the lesion site, ensuring the reliability and validity of our experimental model for Parkinson’s disease. We provide histological verification for critical insights into the effectiveness of the lesion induction to support the interpretation of study outcomes.

References

  1. Yoon, H. H., Kim, Y. H., Shin, E. S., & Jeon, S. R. (2014). A rat model of striatonigral degeneration generated by simultaneous injection of 6-hydroxydopamine into the medial forebrain bundle and quinolinic acid into the striatum. Journal of Korean medical science, 29(11), 1555–1561. https://doi.org/10.3346/jkms.2014.29.11.1555.
  2. Shin, E. S., Hwang, O., Hwang, Y. S., Suh, J. K., Chun, Y. I., & Jeon, S. R. (2014). Enhanced efficacy of human brain-derived neural stem cells by transplantation of cell aggregates in a rat model of Parkinson’s disease. Journal of Korean Neurosurgical Society, 56(5), 383–389. https://doi.org/10.3340/jkns.2014.56.5.383.
  3. Azzouz, M., Martin-Rendon, E., Barber, R. D., Mitrophanous, K. A., Carter, E. E., Rohll, J. B., Kingsman, S. M., Kingsman, A. J., & Mazarakis, N. D. (2002). Multicistronic lentiviral vector-mediated striatal gene transfer of aromatic L-amino acid decarboxylase, tyrosine hydroxylase, and GTP cyclohydrolase I induces sustained transgene expression, dopamine production, and functional improvement in a rat model of Parkinson’s disease. The Journal of neuroscience : the official journal of the Society for Neuroscience, 22(23), 10302–10312. https://doi.org/10.1523/JNEUROSCI.22-23-10302.2002.
  4. Lee, E. J., Yoon, H. H., Park, E. S., Min, J., & Jeon, S. R. (2018). A Novel Animal Model of Parkinson’s Disease Using Optogenetics: Representation of Various Disease Stages by Modulating the Illumination Parameter. Stereotactic and functional neurosurgery, 96(1), 22–32. https://doi.org/10.1159/000486644.

Dr. Shermel Sherman is an academic entrepreneur, neuroscientist, and the founder of AniLocus Inc. Established in June 2021 and incorporated in California and Maryland, AniLocus provides comprehensive research tools for the development of neurotherapeutics.