Brain Capillaries Isolation Technique in Rodents

Abstract

The blood-brain barrier plays an important role in neuroprotection, however it can be a major obstacle for drug delivery to the brain. This barrier primarily resides in the brain capillaries and functions as an interface between the brain and peripheral blood circulation. Several anatomical and biochemical elements of the blood-brain barrier are essential to regulate the permeability of nutrients, ions, hormones, toxic metabolites, and xenobiotics into and out of the brain. The complete understanding about these elements can offer insights on how to modulate barrier functions for neuroprotection against toxins and to enhance drug delivery to the brain. In the literature, preclinical models of the blood-brain barrier are widely utilized to predict drug pharmacokinetic and pharmacodynamic properties in the brain. In addition, these models are essential tools to investigate cellular mechanisms and novel interventions that alter barrier function and permeability. This unit presents procedures to isolate fresh and viable rodent brain capillaries for studies about the blood-brain barrier.

INTRODUCTION

The blood-brain barrier, residing in the brain’s capillary endothelium, is a dynamic interface between the central nervous system and the periphery (Abbott, 2013). Capillaries in the brain are unique and different comparing to peripheral capillaries. First, impermeable tight junctions connecting adjacent brain capillary endothelial cells provide a physical barrier to prevent brain entry of electrolytes, polar small molecules and macromolecules. Second, these endothelial cells highly express a large array of transporters, channels and receptors at the plasma membrane to regulate the barrier’s permeability of essential nutrients, ions, exogenous and endogenous molecules (Abbott, 2013). Therefore, these properties of the blood-brain barrier tightly regulate solute influx into and efflux out of the brain, thus maintaining an optimal brain micro-environment essential for proper neuronal function (Abbott, 2013).

The well-studied barrier is known to play an important role of neuroprotection, however it is also a major obstacle to the delivery of therapeutic drugs to the brain (Miller, 2015). Recent studies in the field primarily focus on the cellular understanding and novel interventions to enhance the barrier’s neuroprotective function and increase drug delivery to the brain. A combination of in vivo, in vitro and ex vivo blood-brain barrier models is commonly utilized for these studies. Certainly, in vivo animal models provide an overall measurement of barrier function in a complete physiological context. Given that the brain capillaries only comprise less than 1 % of the total brain volume, barrier function measured in vivo is also dependent on other elements of the neurovascular unit that are in an intimate proximity to the brain capillaries, such as astrocytes end-feet processes and neurons (Abbott, 2013). On the other hand, in vitro and ex vivo blood-brain barrier models can provide an in-depth understanding about the underlying cellular mechanisms that are specific to the barrier function of brain capillaries. Described below are two approaches to isolate brain capillaries from rodent brains. We believe that our approaches can also be applied to other larger mammalian brain tissues, such as human brain tissue. In our hands, the freshly isolated brain capillaries isolated from rodents are suitable for ex vivo assessment of ABC xenobiotic transporters activity, investigations of cellular mechanism that govern transporter expression and activity, and other biochemical techniques, such as qPCR, Western blot, ELISA, immunoprecipitation, and immunocytochemistry.

BASIC PROTOCOL: Filter isolation procedure

The basic isolation protocol uses mechanical homogenization, density-gradient centrifugation, and filtration to isolate fresh rodent brain capillaries. The isolation buffer for the entire protocol is ice-cold (4 °C) phosphate buffered saline (PBS) containing Ca²⁺, Mg²⁺, 5 mM D-glucose and 1 mM Na-pyruvate at pH 7.4. Materials and tools are kept on ice throughout the entire protocol. Rodents are euthanized and brains are collected in ice-cold isolation buffer. White matter, meninges, midbrain, choroid plexus and olfactory lobes of the brains are disposed using forceps and a stereo-microscope. The remaining dissected brain tissues are minced with a scalpel in isolation buffer chilled on ice and homogenized with 40 up-and-down strokes using a tissue grinder (clearance: 150–230 μm) rotating at 50 rpm. This homogenate is further homogenized 10 up-and-down strokes in a dounce homogenizer (clearance: 130–180 μm). Isolation buffer containing 30 % Ficoll PM400 is mixed into the homogenate to reach a final Ficoll concentration of 15 %. After gentle mixing to reach homogeneous state, the mixture is centrifuged at 5800 g for 15 minutes at 4 °C. The resulting pellet is re-suspended in ice-cold isolation buffer containing 1 % bovine serum albumin (BSA). Next, the suspension is filtered through a 300 μm mesh and followed by a 30 μm mesh pluristrainer®. The filter mesh is flushed with ice-cold isolation buffer containing 1 % BSA. Capillaries captured on the pluristrainer® mesh are collected in ice-cold isolation buffer with 1 % BSA for centrifugation at 1500 g for 3 minutes at 4 °C. The capillary pellet is rinsed two times with ice-cold isolation buffer without BSA and centrifugation at 1500 g for 3 minutes at 4 °C is used to pellet the capillaries of each rinse to remove BSA.

Materials

Solutions

• Isolation buffer (see recipe)

• Isolation buffer 1 % BSA (see recipe)

• 30 % Ficoll PM400 solution (see recipe)

Equipment and disposables

• Nalgene® 1000 mL rapid flow filter unit (127–0020, 0.2 μm CN membrane, 75 mm diameter), Thermo Scientific

• Magnet stir bars, hotplate stirrer and platform shaker

• 250 mL, 1000 mL glass beakers, and 100 mL glass bottles

• Ice buckets

• 50 mL Falcon polypropylene conical tubes, BD

• Surgical scalpels, blades, spatula and scissors

• Littauer bone cutter (16152–15), FST

• Forceps (Dumont #7 - fine and Iris - curved), FST

• Polystyrene petri dishes (small – 30 × 15 mm, large – 100 × 15 mm), BD

• Stereo microscopes (optional)

• Size C 55 mL tissue grinding vessel (3431E55) and pestle size C serrated (3431F25) (clearance: 150–230 μm), Thomas Scientific

• 15 mL KONTES® dounce tissue grinders and pestle size B (885300–0015, clearance: 165 – 889 μm), VWR

• 7 mL bulb polyethylene transfer pipette (1020–2500), USA scientific

• 50 mL polycarbonate centrifuge tubes with polyethylene caps (363664), Beckman Coulter

• Sorvall RC-5B centrifuge with a SS-34 rotor, Beckman Coulter

• Spectra/Mesh® woven nylon filters (sheet to cut into desired size) (146486, mesh opening: 300 μm, thickness 200 μm), Spectrum Lab

• pluriStrainer® (43–50030-50, mesh opening: 30 μm) and connector rings (41–50000-03), pluriSelect Life Science

• Allegra X-22R benchtop centrifuge, Beckman Coulter

• 25 mL serological pipette and pipette controller

Protocol steps

1. Euthanize rodents and extract whole brain tissue out from the skull using surgical scalpels, blades, spatula, scissors, and bone cutter.

2. Transfer brains to ice-cold isolation buffer in a 50 mL falcon tube (5 g of adult rat brains per at least of 30 mL isolation buffer).

3. Submerge brains in ice-cold isolation buffer in a large polystyrene petri dish chilling on ice.

4. Remove white matter, meninges, midbrain, choroid plexus, and olfactory lobes of the brains using forceps and a stereo-microscope (Figure 1).

5. Store dissected brains in a 50 mL falcon tube containing fresh isolation buffer (5 g of adult rat brains per at least of 30 mL isolation buffer) chilling on ice.

6. Mince dissected brains with a scalpel in small polystyrene petri dish containing 5 mL fresh isolation buffer chilling on ice.

   • The mincing step usually takes 20 – 30 seconds per 5 g of brain tissues. It allows larger brain tissues to become smaller pieces and gain access to the gap between homogenization tube and the pestle easier, hence excessive mincing is not required.

7. Transfer all minced brain tissues to the size C 55 mL tissue grinding vessel (pre-chilled on ice with the pestle).

8. Use ice-cold isolation buffer to rinse off any brain tissues sticking to the petri dish or the side of the tissue grinding vessel.

9. Top up with ice-cold isolation buffer to reach a final volume of 12 mL per 5 g of brain tissues in the tissue grinding vessel.

10. Place tissue grinding vessel in a beaker containing ice.

11. Homogenize 40 up-and-downs strokes with the size C serrated pestle rotating at a speed of 50 rpm. Each up-and-down stroke takes approximately 20–30 seconds to complete.

12. Transfer a maximum of 10 mL homogenate each time to a 15 mL KONTES® dounce tissue grinder and perform an additional 10 up-and-down strokes using the size B pestle. Each up-and-down stroke takes approximately 15 seconds to complete.

13. Distribute the homogenate equally into centrifuge tubes. The volume of homogenate for each tube should be around 10 – 20 mL. If the total homogenate at this point is less than 12 mL, use isolation buffer to top up to 12 mL.

    • Use 1–2 mL isolation buffer to rinse off the tissue grinding glass wares if necessary to increase yield.

14. Add an equal volume of ice-cold 30 % Ficoll PM400 solution for each centrifuge tube. The final Ficoll content per tube is 15 %.

15. Gentle shake the centrifuge tube 10 times to ensure thorough mixing.

16. Centrifuge at 5800 g at 4 °C for 15 minutes using the Sorvall RC-5B centrifuge with a SS-34 rotor.

17. Take out centrifuge tube carefully after centrifugation and place it on ice.

18. The pellet at the bottom of the tube contains most brain capillaries (Figure 2). Use a long gel loading pipette tip to dislodge the white puffy top layer as you slowly pour out the Ficoll solution.

19. Keep the tube inverted to allow Ficoll residue to slowly drip out and use a ddH₂O squirt bottle to carefully rinse the inside wall of the centrifuge tube. Be careful not to squirt into the pellet.

20. Use Kim wipes to clean off the inside wall of the tube without disturbing the pellet.

21. Resuspend the pellet with 5 mL of ice-cold isolation buffer 1 % BSA for every 5 g of brain tissues by pipetting the capillary-rich solution up and down using a 7 mL bulb polyethylene transfer pipette.

22. Add an addition 10 mL ice-cold isolation buffer 1% BSA to each centrifuge tube.

23. Secure a small 300 μm Spectra/Mesh® woven nylon filters on top of a 50 mL falcon tube.

    • Use a blade to cut open a circle in the cap of a falcon tube, then place a pre-cut nylon filter mesh between the tube and the cap. The mesh will be secured in place as you carefully tighten the cap to the tube (figure 3).

24. Transfer the capillary-rich solution through the 300 μm mesh and into the 50 mL Falcon tube using a bulb transfer pipette.

25. Place the 50 mL Falcon tube containing the capillary-rich solution on ice and the 300 μm mesh can be discarded.

26. Assemble three 30 μm pluriStrainer units per every 5 g of brain tissues (Figure 4).

27. Distribute 1 mL capillary-rich solution to each pluriStrainer unit using a bulb transfer pipette.

    • Discard the cap of a 50mL falcon tube, place the connector ring on top and the pluriStrainer® goes on top of the connector ring.
28. Allow the solution to slowly drip through the mesh filter while placing the pluriStrainer unit on ice.

    • If solution dripping speed is very slow, further dilute the capillary-rich solution with ice-cold isolation buffer 1% BSA before distributing into the pluriStrainer unit. A 50 mL syringe can be used to create a negative pressure within the falcon tube of the pluriStrainer unit.

29. Pipette a total 15 mL ice-cold isolation buffer 1% BSA to wash for each unit and save the filtrates.

30. Collect capillaries by turning just the 30 μm pluriStrainers upside down and placing the strainer on top of a clean centrifuge tube. Rinse capillaries off the filter mesh with 10 mL of ice-cold isolation buffer 1% BSA once per strainer.

31. Place the centrifuge tubes on ice as you proceed to the next pluriStrainer. Use more than one centrifuge tube to collect all capillaries if necessary.

32. Use the same 30 μm pluriStrainers to repeat the above two steps for filtering the entire volume of capillary-rich solution.

33. Assemble a new pluriStrainer units and filter all the collected filtrates generated at step 29 once more.

    If filtrate dripping speed is very slow, rinse the filter with 15 mL ice-cold isolation buffer 1 % BSA and collect capillaries with 10 mL ice-cold isolation buffer 1% BSA to the centrifuge tube. Then resume filtering the remaining filtrates using the same pluriStrainer unit.

34. Pipette 15 mL ice-cold isolation buffer 1% BSA into the pluriStrainers to wash.

35. Collect capillaries by turning just the 30 μm pluriStrainers upside down and placing on top of a clean centrifuge tube. Rinse capillaries off the filter mesh with 10 mL of ice-cold isolation buffer 1% BSA.

36. The second filtrates generated in steps 33 and 34 can be discarded.

37. The collected capillaries are now suspended in ice-cold isolation buffer 1% BSA in centrifuge tubes.

38. Centrifuge these tubes at 1500 g for 3 minutes at 4 °C.

39. Carefully remove supernatants for each centrifuge tube using a pipette controller.

40. Add 5–10 mL ice-cold isolation buffer (without BSA) to each centrifuge tube.

41. Gently vortex the bottom of the tube by hands to dislodge the pellet and transfer all capillary-rich solution to a single clean centrifuge tube.

42. To ensure maximum yield, use 1–2 mL of ice-cold isolation buffer (without BSA) to collect residue capillaries sticking to the inside wall of each centrifuge tube.

43. Centrifuge the centrifuge tube at 1500 g for 3 minutes at 4 °C to pellet capillaries.

44. Carefully remove supernatants using a pipette controller.

45. Add 40 mL ice-cold isolation buffer (without BSA) to the centrifuge tube.

46. Centrifuge the centrifuge tube at 1500 g for 3 minutes at 4 °C.

47. Carefully remove supernatants using a pipette controller.

48. The resulting pellet contains the brain capillary-rich fraction.

49. Resuspend the pellet by pipetting up and down several times in a desired volume of isolation buffer, typically 2–4 mL per 5 g of rat brain tissues. Do not vortex the final product and check for quality under an up-right microscope.

50. To perform Western blot, qPCR and other techniques, transfer suspension to an eppendorf tubes and centrifuge at maximum speed to collect the capillary pellet.

51. Otherwise, suspension is ready for ex vivo transport assays, chemical exposure to the capilalries and other techniques.

Figure 1.

Dissected rat brain tissue (left) ready for homogenization step and un-dissected tissue (right).

Figure 2.

Brain capillary-rich pellet (at the bottom of the tube) after the Ficoll centrifugation step.

Figure 3.

An assembled 300 um filter unit.

Figure 4.

An assembled pluriStrainer unit.

*An overall schema can be found in Figure 5

Figure 5.

An overall schema of the brain capillaries isolation.

ALTERNATE PROTOCOL: Isolation procedure without BSA

In the basic protocol, isolation buffer containing BSA is used during the filtering steps. BSA is used to minimize binding of capillaries to glass or plastic-wares used during the isolation protocol. However, BSA will also prevent capillaries from adhering to the glass cover slips used in sequential experiments, such as ex vivo drug transport assays or immunohistochemistry performed on glass bottle chambers. Therefore, several rinsing and centrifuging steps (steps 45–47) are introduced to remove BSA. However, these steps can reduce capillaries yield when the isolation protocol is performed with limited brain tissues, i.e., less than 3 g of rat brain tissue. In cases of limited brain tissue or BSA interference, the experimenter can replace isolation buffer 1 % BSA with plain isolation buffer (without BSA) in all steps of the basic protocol. Since BSA is not introduced to the system, centrifugation steps 45–47 of the basic protocol can be omitted to conserve time and increase capillaries yield.

Materials

Solutions, equipment, and disposables are identical to the basic protocol

Protocol steps

Replace the usage of isolation buffer 1 % BSA with plain isolation buffer (without BSA) in all steps of the basic protocol. Centrifugation steps 45–47 can be omitted.

Step annotations

None.

REAGENTS AND SOLUTIONS

Use deionized, distilled water in all recipes and protocol steps.

Fisher Chemical and Sigma supply all buffer constituents.

Isolation buffer:

• Before or at the day of the experiment, prepare initial buffer containing 2.7 mM KCl, 1.46 mM KH₂PO₄, 136.9 mM NaCl, 8.1 mM Na₂HPO₄, 0.5 mM MgCl₂ and 0.9 mM CaCl₂ and pH to 7.4, and store at 4°C.

• Prior to the isolation, add D-glucose and Na⁺-pyruvate to achieve 5 mM D-glucose and 1 mM Na⁺-pyruvate.

• Solution can be stored on ice during the isolation.

Isolation buffer 1 % BSA:

• Add 1 g of BSA (A9647, Sigma) to every 100 mL of isolation buffer and shake until BSA is fully dissolved.

• Solution can be stored on ice during the isolation.

30 % Ficoll PM400 solution:

• Add 15 g of Ficoll PM400 (F4375, Sigma) to every 40 mL of completed isolation buffer and stir with a magnetic stir bar until Ficoll is fully dissolved.

• Solution can be stored on ice during the isolation.

COMMENTARY

Background Information

Models of the blood-brain barrier are of central importance in efforts to better understand and modulate barrier function and to predict pharmacokinetic and pharmacodynamic properties of drugs entering the central nervous system. All experimental blood-brain barrier models have advantages and disadvantages for specific applications. For example, in vivo animal models allow pharmacokinetic and pharmacodynamic analysis in a live brain, however they limit the understanding of molecular mechanisms at the cellular level of the barrier, even when carried out using genetically modified animals. In vitro cell culture models of the brain capillary endothelial cells allow the study of cellular mechanisms occurs specifically inside the brain capillary endothelial cells, yet it reflects very little about the physiological three-dimensional brain microenvironment and interactions with other cells, such as astrocytes, pericytes and neurons. Isolated rodent brain capillaries prepared from this protocol certainly have the advantage of providing an intact endothelium that can be rapidly isolated in sufficient quantities to provide materials for permeability and molecular studies. These freshly isolated brain capillaries retain restricted paracellular permeability, some pericytes interactions, and the expression of tight junction molecules, surface receptors and membrane-associated xenobiotic transporters, however, connections to astrocytes and neurons are lost.

We and several other groups use isolated rodent brain capillaries as a standard model to assess changes in the activity and expression of ABC xenobiotic transporters and investigate cellular mechanisms that regulate these transporters using pharmacological agents (Miller, 2010, 2015). The original brain capillary isolation technique used in several previous publications involves the use of glass beads to separate capillaries from other cellular debris in the brain homogenate. Recently, our lab and the others have switched to the described isolation technique that uses filters to capture capillaries. The current procedure was first developed by Dr. B. Bauer and Dr. AMS Hartz, University of Kentucky, USA (A. M. Hartz et al., 2012; A. M. S. Hartz et al., 2016). It produces a very reliable and robust yield of viable brain capillaries that have a structural intact and non-leaky endothelium and are metabolically active for up to approximately 24 hours. Although this procedure is primarily used for our studies to isolate brain capillaries from rodents, we also believe that it can be applied to other larger mammalian brain tissue, such as human brain tissue. In the literature, other similar techniques using concentration gradient and filtering methods have been described. Initial characterization and validation should be performed prior to the actual study to ensure this protocol is suitable and whether additional modifications are required.

Critical Parameters & Troubleshooting

This isolation procedure requires several labor steps, hence precautions and care must be taken when pipetting the capillary-rich solution to ensure capillaries are not damaged. If the isolated capillaries are too short, morphologically damaged or too tangled with other capillaries, it generally suggests that the performed procedures are mechanistically too rough to the capillaries. It is advisable to use a slower up-and-down motion during the two brain tissue grinding homogenization steps. Consistency of the pestle rotation speed and spacing between the pestle and the glass vessel should also be verified. Curved pestle can produce uneven spacing with the glass vessel during homogenization, thus damaging the capillaries. In addition, freshly harvested brains should be used for isolation within an hour to ensure maximum capillary viability. If capillaries are too tangled, additional gentle up and down pipetting can be performed to ensure capillaries pellet is fully resuspended.

If the initial capillary-rich pellet after the first Ficoll centrifugation step does not form or is distorted, it usually means that the brain tissue homogenate and the PM400 Ficoll solution are not completely mixed prior to centrifugation. It is advisable to re-mix the centrifuge tube by gentle shaking and allow the distorted pellet and white fluffy top layer to fully resuspend into the Ficoll solution again. This suspension can then be centrifuged again with identical settings as before. In addition, consistency of rotor balance and a slow breaking speed of the centrifuge should be closely monitored.

If the final capillary yield is extremely low, there are several explanations and suggestions. First, it is possible that the initial capillary-rich pellet after the Ficoll centrifugation step is not fully resuspended into the buffer. Hence, tangled capillaries can be retained on the 300 μm filter and reducing the final yield. Additional up and down pipetting using the bulb polyethylene transfer pipette can be performed to ensure capillary-rich pellet after the Ficoll centrifugation step is fully resuspended before passing through the 300 μm filter. It is also possible that the final capillary pellet is not fully resuspended into buffer. Again, additional up and down pipetting is advised to break off tangled capillaries. Second, a portion of the final capillary-rich pellet may break off and can be removed with the supernatant. Extreme care should be exercised during removal of supernatants after each centrifugation steps. It is acceptable to re-centrifuge when the pellet is disassociated from the bottom of the tube. If a limited brain tissue (less than 3 g of brain tissues) is available, one may consider using the alternative isolation procedure without BSA to increase capillary yield. Third, capillaries can adhere to pipette tips resulting in capillaries loss. It is advisable to re-use the same pipette tip or transfer bulb during some of the isolation steps or to avoid using pipette tips that extensively trap capillaries.

Anticipated Results

A typical good isolation (Figures 6A and B) should mostly consist of both tangled and non-tangled capillaries with very little amount of debris or single cells. The experimenter is encouraged to check for capillaries quality and yield right after each isolation using an upright microscope, because interpersonal variability exists for this protocol.

Figure 6.

Morphology of rat brain capillaries captured using A) up-right microscope (20X magnification) and B) light contrast confocal microscope (40 X magnification).

Time Considerations

The total isolation time per 5 g of brain tissues is less than 2 hours for one experimenter, excluding the brain harvesting and solution preparation. Isolation using 7 or more grams of brain tissues may require additional experimenter to perform brain tissue dissections to shorten the total isolation time. To shorten the total isolation time, multiple and additional pluriStrainer units can be used at the same time during the filtering steps. However, the experimenter must ensure the filter mesh does not run dry because leaving capillaries out of the ice-cold isolation buffer may affect isolation quality. Alternatively, additional experimenters may be required to perform the filtering steps with multiple pluriStrainer units flowing at once.

Significance Statement.

The blood-brain barrier is a complex network of blood capillaries or microvessels in the brain. This barrier regulates the movement of nutrients, ions, hormones, metabolites, toxicants, and drugs into and out of the brain. Physiologically, the barrier serves a vital role to maintain brain homeostasis and to protect the brain from environmental toxicants and harmful metabolites. However, the barrier becomes an obstacle to deliver drugs to the brain during pharmacotherapy of brain diseases, such as cancer and epilepsy. The described procedures are designed to isolate fresh and viable rodent brain capillaries for studying at the blood-brain barrier. A better understanding of the blood-brain barrier provides clues that will lead to novel strategies to alter barrier function and thus improve drugs delivery to the brain.