The overall goal of the following experiment is to understand the role of lipoxygenase ISO enzymes in nigrostriatal degeneration related to Parkinson's disease. To achieve this, a neurotoxin that produces a Nigro SAL lesion is carefully prepared. Next locks deficient mice are injected with the MPTP solution daily for four consecutive days, seven days after the last injection.
Samples are collected and used for biochemical or immuno histological analysis. Results show that 1215 locks does not impact NIGROSTRIATAL MPTP toxicity. However, five locks contributes to damage under conditions of toxicity.
TS method can help answer key questions in the Parkinson's disease field. Suggest what is the impact of gene environment interactions on nistri vulnerability. Using gene environment models, we were able to discern ISO selective functions and can inside into the dual of five lipes.
Properly trained personnel should wear appropriate personal protective equipment throughout the following procedure. Begin by performing all of the necessary calculations needed for preparing the neurotoxin solution. Next, arrange all of the equipment, supplies, and reagents required.
In addition, have materials available to deal with any potential spills. Cover the area surrounding the scale with pads or paper towels. Dampened with 10%bleach solution to reduce the risk from spilled powder, keep tissues and 10%bleach solution nearby as a precaution.
When ready, remove the source vial from its proper storage location. Label the glass vial MPTP with the concentration and date. Using an analytical balance located in a fume hood, weigh 50 milligrams of MPTP into a glass vial with secondary containment containing a 10%bleach soaked tissue.
Next, close the source vial and wipe the outside with 10%bleach slowly. Add 13.763 milliliters of sterile saline to the vial and close before mixing completely in the hood. Carefully filter, sterilize the MPTP solution into a labeled injection vial within a secondary container with a 10%bleach soaked tissue.
In order to reduce pressure slightly loosen the cap of the vial containing the MPTP filtered solution. Clean any spills with a bleach soaked tissue, dispose of used materials into an appropriately labeled biohazard container. Maintain the MPTP solution in the vial within a secondary container with a bleach soaked tissue for transport to the animal procedure room.
Place the MPTP source vial into its secondary container and return it to its appropriate storage location. Lastly, decontaminate the spatula analytical scale and hood surface for 10 minutes. Using 10%bleach.
Before injecting the animals, prepare disposable cages with filtered perforated lids. Marked MPTP. Insert disposable cage liners, pre wetted food pellets, and hydrogel as a water source.
Next, weigh all animals and record the volume of MP TP solution required for a 15 milligram per kilogram injection. When ready, take great care while holding a mouse over a disposable absorption pad and injecting the MPTP solution. Repeat this procedure daily for four days without recapping the needle, place it into a dedicated and labeled MPTP Biohazardous sharps container.
Keep 10%bleach solution and tissues nearby. To clean any accidental drips, place animals injected with MPTP into the disposable cages prepared earlier, and house them on open racks. Next post MPTP.
Use placards on the rack and on the housing room door until 72 hours after the last injection. Decontaminate the work surface with bleach. After each, use dispose of leftover MPTP solution by the addition of an equivalent volume of 10%bleach.
And to discard this as biohazardous liquid waste discard all used PPE in dedicated disposal bins spraying with 10%bleach before disposal if needed. During this procedure, it is important to check on the animals regularly and replenish their food and water daily. Three days after the last injection, dispose of all materials and return the animals to regular housing.
At predetermined experimental time points Retrieve the animals for tissue harvesting and processing. Stri AAL homogenous were used to measure dopamine levels from wild type and five locks knockout mice treated with either saline or MPTP In saline treated groups, there was an effective genotype on DA levels with lower levels of DA in the transgenic mice. However, following MPTP treatment, five locks deficient mice were protected against SAL DA depletion.
The five locks knockout mice in the MPTP group had similar levels of DA compared to five locks knockout mice in the saline group. In contrast, in 1215 locks knockout mice genotype did not play a role in the dopamine levels regardless of treatment group immunofluorescent staining for TH and GFAP in the substantial nigra revealed fewer TH positive cell bodies indicated by the asterisk and enhanced GFAP immunoreactivity in the wild type MPTP group. DAB staining for microglia revealed ramified cell bodies and long branching processes from saline treated mice while activated microglia with rounded cell bodies and short thickened processes were observed in MPTP treated mice immuno blotting for SAL TH and GFAP provided further evidence for the damage and neuroinflammation that resulted from the MPTP challenge.
These effects were diminished in the five locks isy deficient mice To work that working with MPTP can be extremely. His precautions such as wearing personal protective equipment and having 10%bridge on hand for the combination should always be taken while performing this procedure.
