Preparation of Brain Sections Using a NMDG-Based Protective Recovery Method

Published: July 31, 2024

Abstract

Source: Ting, J. T. et al. Preparation of Acute Brain Slices Using an Optimized N-Methyl-D-glucamine Protective Recovery Method. J. Vis. Exp. (2018).

This video describes a protocol to obtain brain slices using an N-Methyl-D-glucamine (NMDG)-based protective recovery method. A dissected brain is incubated in an aCSF with NMDG and HEPES to minimize neuronal cell death and damage during the sectioning. The section is allowed to recover in the aCSF to restore the cellular activities

Protocol

1. Brain Dissection and Slicing

  1. Decapitate the animal. Use a scalpel to open the skin on the head and expose the skull cap.
  2. Use fine super-cut scissors to cut away the skin over the skull cap and make small incisions laterally on either side at the caudal/ventral base of the skull. Make additional shallow cuts starting at the caudal/dorsal aspect of the skull, moving in the rostral direction up the dorsal midline taking care not to damage the underlying brain. Make a final 'T' cut perpendicular to the midline at the level of the olfactory bulbs.
    NOTE: Care must be taken to ensure no damage is done to the brain region(s) of interest. In particular, at no time should there be any compressive force applied to the brain itself.
  3. Use the round-tip forceps to grasp the skull, starting at the rostral-medial aspect and peel back towards the caudal-lateral direction. Repeat for both sides to crack open and remove the dorsal halves of the skull cap to expose the brain. Gently scoop out the intact brain into the beaker of pre-chilled NMDG-HEPES aCSF. Allow the brain to uniformly cool for ~1 min.
  4. Use the large spatula to lift the brain out of the beaker and onto the Petri dish covered with filter paper. Trim and block the brain according to the preferred angle of slicing and the desired brain region of interest. Work quickly to avoid prolonged oxygen deprivation during handling.
    NOTE: Many slicing angles are possible. The exact blocking method and slicing angle will depend on the exact brain region, cell type, and circuit to be studied.
  5. Affix the brain block to the specimen holder using adhesive glue. Retract the inner piece of the specimen holder enough to withdraw the brain block fully inside. Pour the molten agarose directly into the holder until the brain block is fully covered in agarose. Clamp the pre-cooled accessory chilling block around the specimen holder for ~10 s until the agarose has solidified.
  6. Insert the specimen holder into the receptacle on the slicer machine and verify proper alignment. Fill the reservoir with remaining pre-chilled, oxygenated NMDG-HEPES aCSF from the 250 mL beaker and move a bubble stone into the reservoir for the duration of slicing to ensure adequate oxygenation.
  7. Adjust the micrometer to begin advancing the agarose-embedded brain specimen. Start the slicer and empirically adjust the advance speed and oscillation frequency to the desired level.
    NOTE: Both settings should be in the low range. For best results, a single pass of the blade arm should take approximately 20 s and the oscillation should produce a very smooth and gentle humming noise with no overt buzzing.
  8. Continue advancing and slicing the tissue in 300 µm increments (or other preferred thickness) until the brain region of interest is fully sectioned; the total time for the slicing procedure should be less than 15 min.

2. Optimized NMDG Protective Recovery Procedure

  1. Initial NMDG recovery step (critical step): Upon completion of the sectioning procedure, collect up all of the slices using a cut-off plastic Pasteur pipette and transfer them into a pre-warmed (34 °C) initial recovery chamber filled with 150 mL of NMDG-HEPES aCSF. Transfer all slices in short succession and start a timer as soon as all slices are moved into the recovery chamber.

開示

The authors have nothing to disclose.

Materials

Compresstome VF-200 Precisionary Instruments VF-200 Vibrating tissue slicer (recommended)
N-methyl-D-glucamine Sigma Aldrich M2004 aCSF constituent
Sodium Chloride Sigma Aldrich S3014 aCSF constituent
Potassium Chloride Sigma Aldrich P5405 aCSF constituent
Sodium Phosphate monobasic dihydrate Sigma Aldrich 71505 aCSF constituent
Sodium Bicarbonate Sigma Aldrich S5761 aCSF constituent
HEPES Sigma Aldrich H4034 aCSF constituent
Glucose Sigma Aldrich G7021 aCSF constituent
Sodium Ascorbate Sigma Aldrich A4034 aCSF constituent
Thiourea Sigma Aldrich T8656 aCSF constituent
Sodium pyruvate Sigma Aldrich P5280 aCSF constituent
Calcium chloride dihydrate Sigma Aldrich C7902 aCSF constituent
Magnesium Sulfate heptahydrate Sigma Aldrich M1880 aCSF constituent
Curved blunt forceps Fine Science Tools 11065-07 Brain dissection tools
Fine dissecting scissors (supercut) Fine Science Tools 14058-09 Brain dissection tools
Large heavy duty scissors 7'' Fine Science Tools 14000-18 Brain dissection tools
Metal spatula Sigma Aldrich Z511455-1PAK Brain dissection tools
Razor blades VWR 89031-954 Brain dissection tools
Brain Slice Keeper-4 Automate Scientific S-BSK4 brain slice holding chamber
nylon netting Warner Instruments 64-0198 For building small slice recovery chambers
Pyrex glass beakers (250 mL) VWR 89090-434 For building small slice recovery chambers
35 mm plastic dish, round VWR 100488-376 For building small slice recovery chambers
Gas diffuser stones (10 µm) Sigma Aldrich 59277 For constant carbogenation (fine bubbles)
Agarose Type I-B Sigma Aldrich A0576 For embedding brain specimens
Hydrochloric acid Sigma Aldrich H1758-100ML For pH adjustment of media
Sodium Hydroxide Sigma Aldrich 221465-25G For pH adjustment of media
Potassium Hydroxide Sigma Aldrich 221473 For pH adjustment of media
Plastic transfer pipets 3 mL graduated VWR 89497-676 For slice transfer
Zirconium ceramic injector blades Cadence Specialty Blades EF-INZ10 http://cadenceinc.com/
Heated water bath (2.5L) VWR 13491-060 Miscellaneous
Filter paper rounds VWR 28456-022 Miscellaneous
Cyanoacrylate glue Amazon B000BQRBO6 Miscellaneous
Glass petri dish VWR 89000-326 Miscellaneous
10X Phosphate buffered saline Sigma Aldrich P5493 Miscellaneous
Thermomixer (w/1.5 mL tube block) VWR 89232-908 To keep agarose molten

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記事を引用
Preparation of Brain Sections Using a NMDG-Based Protective Recovery Method. J. Vis. Exp. (Pending Publication), e22378, doi: (2024).

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