Materials and Reagents

50 ml Oak Ridge polysulfone centrifuge tubes w/screw caps (Thermo Fisher Scientific, catalog number: 3115-0050 )
Eppendorf microtubes 1.5 ml (VWR, catalog number: 700-5239 )
PIPETMAN TIPS Diamond–ECOPACK^TM D1000 (Gilson, catalog number: F161670 )
PIPETMAN TIPS Diamond–ECOPACK^TM D200 (Gilson, Catalog number: F161930 )
PIPETMAN TIPS Diamond–ECOPACK^TM D10 (Gilson, catalog number: F161630 )
Stainless steel surgical blades (Swann Morton, catalog number: 0308 )
Rodent brain
Note: This protocol has only been tested with brains from male young and mature (3- and 12-month-old) Wistar rats and wild type, type 2 diabetes-like and triple transgenic for Alzheimer disease (3xTg-AD) mice (11-month-old). Nevertheless, we believe that this protocol can be applied to different strains, ages and sex, though the amount of obtained sample can be a limiting factor.
Ice
Distilled water
Isoflurane (Lab. Vitória, Portugal)
Sodium chloride (NaCl) (Sigma-Aldrich, catalog number: S7653 )
Sodium phosphate dibasic (Na₂HPO₄) (Sigma-Aldrich, catalog number: S7907 )
Dextran from Leuconostoc mesenteroides (Sigma-Aldrich, catalog number: 31398 )
Phosphate buffer (0.01 M) (see Recipes)
Dextran (16%) (see Recipes)

Equipment

50 ml Fisherbrand^TM reusable glass low-form Griffin beakers (Fisher Scientific, catalog number: FB10050 )
Bone cutting forceps (Aesculap, catalog number: FO611R )
Centrifuge (Refrigerated Centrifuge) (Sigma Laborzentrifugen, model: SIGMA 3-16K )
Curved fine tip forceps (Aesculap, catalog number: FB401R )
Heidolph mechanical overhead stirrers, Brinkmann (Heidolph Instruments, model: RZR 1 )
Laboratory bottles, narrow mouth, with screw cap (VWR, catalog number: 215-1594 )
Micropipette PIPETMAN L (Light) type P200L (Gilson, catalog number: FA10005 )
Micropipette PIPETMAN L (Light) type P1000L (Gilson, catalog number: FA10006 )
Nickel/SS Lab spatulas with 1.63” Flat Rounded Ends (Cole-Parmer, catalog number: EW-06369-05 )
Petri dish with cover 60 x 15 mm (Corning, catalog number: 70165-60 )
Potter-Elvehjem with PTFE pestle and glass tube (DWK Life Sciences, Kimble^®, catalog number: 886000-0023 )
Precision scale (Mettler-Toledo International, model: AE240 )
Precision balance PLE-N (KERN, model: PLE-N )
Scalpel handle (Aesculap, catalog number: BB084R stainless)
Soft hair brush, 3 mm dia. (CONTROLS, catalog number: 86-D1672 )
Swing-out rotor (Sigma Laborzentrifugen, catalog number: 11133 )

Procedure

All the isolation protocol steps must be performed at 4 °C (always maintain solutions, homogenates and pellets on ice).

Euthanize rodents by cervical displacement (mice) and decapitation (rats and mice). All procedures are approved by the Federation of Laboratory Animal Science Associations (FELASA).
Perform a midline incision, posterior to anterior, along the scalp to reveal the skull (Video 1).

Video 1. Brain removal, dissection and homogenization
Cut the cranium carefully from the neck to the nose through the lateral cranial sutures. Two additional cuts can be performed, in the occipital hole to the ears direction, to facilitate the access to the brain (Figure 2A, Video 1).

Figure 2. Illustrative images of brain dissection and homogenization. Cut the cranium carefully from the neck to the nose through the lateral cranial sutures (A). Remove the intact brain from the cranial box using a small spatula (B). Then, remove the cerebellum, olfactory bulbs and white matter using fine dissection forceps and keep the brain cortices (C). Finally, homogenize brain tissue, at 4 °C, in 10 ml PBS using a Potter-Elvehjem with PTFE pestle and glass tube, at 800 rpm/min (D).
Remove the intact brain from the cranial box using a small spatula (Figure 2B, Video 1).
Remove the cerebellum, olfactory bulbs and white matter using fine dissection forceps; keep the brain cortices (Figure 2C, Video 1, see Note 1).
Perform a quick wash (approximately 30 sec) in non-sterile PBS (Video 1; see Recipes).
Homogenize fresh brain tissue, at 4 °C, in 10 ml PBS using a Potter-Elvehjem with PTFE pestle and glass tube, at 800 rpm/min (Figure 3, Video 1).

Figure 3. Schematic illustration of mice brain vessels isolation
Centrifuge at 720 x g for 5 min, at 4 °C (Figure 3).
Discard the supernatant by decantation (Figure 3).
Resuspend the pellet in 10 ml PBS using a soft hair brush, 3 mm dia (Figure 3).
Repeat the steps 8-10 two more times.
Layer the PBS resuspended pellet over 20 ml 16% dextran (Video 2; see Recipes).
Note: This step must be gently done to avoid mixing the two layers (Figure 3).

Video 2. Brain vessels isolation
Centrifuge at 4,500 x g for 20 min, at 4 °C (Figure 2).
Collect the supernatant and the middle layer, and keep the pellet on ice (Figure 3).
Repeat step 14.
Discard the supernatant and resuspend the two resulting pellets in 100 μl PBS.
Note: Resuspend the first pellet in 100 μl PBS and use the suspension to resuspend the second pellet. The vessels can be visualized at optic microscope (Figure 4) using the DiffQuick staining as described by Mota and Ramalho-Santos (2006).
Store the vessels at -80 °C until use (used until 2 years upon isolation; Figure 3).

Figure 4. Representative images of isolated mice brain vessels after DiffQuik staining. Unstained (A) and stained (B, C) brain vessels observed under the optic microscope. 100x amplification; scale bars = 50 μM.

Notes

In order to minimize the day-to-day variability that can bias the results, in studies using animal models of disease, researchers must always isolate vessels from control and diseased animal models.
Minimum number of animals required for the isolation of brain vessels: 1 rat brain or 3 mice brains. It is possible to use only 1 mice brain. However, the pellet size will be very small, which will difficult its visualization with the naked eye.

Recipes

Phosphate buffer (0.01 M)
8.5 g/L NaCl
1.42 g/L Na₂HPO₄
pH = 7.4
Dextran (16%)
160 g/L Dextran from Leuconostoc mesenteroides

Notes:

Distilled water type 1 must be used in the preparation of the solutions.
For short periods of time solutions must be conserved at 4 °C (e.g., one week); for longer periods solutions must be conserved at -20 °C (except Dextran solution).
Dextran solution must not be frozen; it is stable for one week at 4 °C.

Acknowledgments

The authors’ work is supported by ‘FEDER funds through the Operational Programme Competitiveness Factors–COMPETE 2020 and national funds by FCT–Foundation for Science and Technology under the strategic project with COMPETE-attributed reference: POCI-01-0145-FEDER-007440’. Cristina Carvalho has a Postdoc fellowship from FCT (SFRH/BPD/107741/2015). The authors of the manuscript have no conflicts of interest to declare. This protocol was adapted from a previous protocol described by McNeill et al. (1999). Reprinted from Journal of Alzheimer’s Disease, vol. 35, Carvalho, Cristina; Machado, Nuno; Mota, Paula; Correia, Sónia C.; Cardoso, Susana; Santos, Renato X.; Santos, Maria S.; Oliveira, Catarina R.; Moreira, Paula I., Type 2 Diabetic and Alzheimer’s Disease Mice Present Similar Behavioral, Cognitive, and Vascular Anomalies, pp. 623-635, Copyright (2013), with permission from IOS Press.

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