The overall goal of this procedure is to simultaneously investigate two specular hemislices obtained from the same anatomical plane and montior the effects following a treatment. This method can help answer crucial questions in the molecular neuroscience field such as the primary events effecting key molecules involved in neurodegenerative processes. The main advantage of this technique is that it can provide and medium carried out of molecular cascades underlying neurodegenerative events.
Though this method can provide insight into primary events occurring in neurodegeneration, it can also be applied to investigate animal models of different disorders or other organs and tissues properties. Begin with preparing two different artificial cerebrospinal fluids. One used for the brain slicing, and the other for the incubation step.
After preparing the slicing ACSF, mix the solution and keep it on ice. Similarly, prepare the recording ACSF, but keep it at room temperature. Oxygenate both solutions.
Next, prepare the instruments for the decapitation and brain removal at the dissection hood. Then, set up the vibratome parameters. Set the slice thickness as needed.
Such as three hundred microns. Set the frequency at seven and the speed at six. Then, insert the vibratome chamber and surround it with ice.
For the brain sectioning, opening the carbogen valve to oxygenate the slicing ACSF. Select the minimum flow rate of around two milliliters per minute. Now in two 15 milliliters tubes, prepare the tus conditions.
Load one tube with 10 millilieters of the recording ACSF as a control. Load the other with the 10 milliliter of recording ACSF enriched with the treatment. In this case, two micromolar of T30.
Vortex both tubes and keep them on ice until they are needed. Incise the skin over the skull with a surgical blade. Then using scissors, cut the skull along the midline from the posterior to the bone above the olfactory bulbs.
Then, laterally pull on the two sides of the skull using forceps to access the brain. Insert a spatula ventrally to the brain. Gently scoop it out and keep it hydrated in ice cold slicing ACSF for about five minutes.
After the rest in cold ACSF, transfer the brain to a filter paper. And after having removed the cerebellum, glue the brain vertically on the vibratome disc. Then transfer it to the sectioning chamber filled with ice cold slicing ACSF.
Now start the oxygen flow to the chamber solution. Then adjust the cutting interval and proceed with the brain sectioning. Collect the sections containing the region of interest using standard methods.
Now carefully divide each section with small scissors along the midline to obtain two specular hemislices. Use each pair of hemislices for the control and treated condition. After being divided, let the hemisections rest in the vibratome chamber for five to ten minutes.
After the rest, gently transfer the hemisections using a glass pipet into a bubbling pot containing recording ACSF at room temperature. Then let the hemisections recover for about 30 minutes before proceeding. During the break, load three glass vials with three milliliters of recording ACSF for the control group and three vials with three milliliters of ACSF enriched with T30 for the treated group.
After the 30 minute rest, within five minutes, transfer the sample to the baths. First load the consecutive hemislices to the control solution using a brush. Then, using a different brush, transfer the contralateral hemisections to the treatment solution.
Next, seal the vials with plastic stoppers containing 21 gauge needles for the delivery of carbogen to the sample. Deliver oxygen at a minimal flow rate, such as two milliliters per minute to prevent any movement of the brain tissue resulting in possible mechanical damage to the tissue. This completes the setup.
Now start a five hour incubation. After the incubation, stop the oxygen flow and uncap the vials. Then gently transfer the hemislices using the appropriate brush to 1.5 milliliter tubes containing 250 microliters of lysis buffer maintained on ice.
Next using separate pestles, homogenize each tissue. Complete the transfer and homogenization steps within 10 to 15 minutes for all six samples. Now centrifuge the homogenized samples at 1, 000 times gravity for five minutes at four degrees Celsius.
After the spin, transfer the supernatants into new tubes and store the samples at negative 80 degrees Celsius. Administration of a toxic peptide, T30, to the basil fore brain containing hemisections had three site specific effects on the tissue. It modulated the expression of alpha seven nicotinic receptor, the phosphorylation of Tau and the expression of amyloid beta.
The nicotinic receptor showed a significant increase in the rostral treated hemislice, while the intermediate slice did not reveal any change between the two conditions. In the posterior section, a significant reduction was present in the T30 exposed portion. Phosphorylated Tau levels were significantly increased in the anterior region.
On the other hand, the other two basil fore brain sections do not show any significant difference between the conditions. Amyloid beta was significantly enhanced after T30 exposure in the rostral and intermediate hemisections. However, in the codal slide, there was no measurable effect on amyloid beta expression.
Once mastered, this technique can be done within seven hours if properly performed.
