This research was supported by the Deutsche Forschungsgemeinschaft DFG under grant number FO 315/4-1 and DFG SFB 688.

1. Isolation of Brain Microvessels
<ol>
 <li>For each preparation use five to ten neonatal mice of either sex (3-5 days old).</li>
 <li>Euthanize a mouse according to local IACUC/veterinarian recommendations, remove the brain immediately, and transfer into a Petri dish containing the following solution: 15 mM Hepes (pH 7.4), 153 mM NaCl, 5.6 mM KCl, 2.3 mM CaCl2x 2H2O, 2.6 mM MgCl2x 6H2O, 1% (w/v) BSA (hereafter referred to as buffer A).</li>
 <li>Unless otherwise indicated, all isolation procedures should be performed at room temperature (RT) (22-24 &deg;C) under the laminar flow hood. Cut the brains (cerebrum without cerebellum and brain stem), after removal of the meninges and capillary fragments, in buffer A using a sterile scalpel. Pipette the tissue fragments up and down using a 10 ml pipette until no clumps appear. Transfer the suspension into a 50 ml Falcon tube and centrifuge at 250 x g for 5 min at RT.</li>
</ol>
Note: Meninges can be identified as thin, transparent membranes at the surface of the brain tissue. They can be carefully removed with sterile forceps.
<ol start="4">
 <li>Discard the supernatant.</li>
 <li>Dissolve the pellet in 4.5 ml Buffer A with 1.5 ml of 0.75% (w/v) collagenase/ dispase (Roche) and incubate for 45 min at 37 &deg;C in a water bath (occasionally shaking).</li>
 <li>Meanwhile, prepare the 12-well plates by coating four wells with collagen IV (0.1 mg/ml dissolved in 50 mM acetic acid). Allow to adhere for 1 hr.</li>
 <li>Stop the digestion by addition of 15 ml ice-cold buffer A. Resuspend the pellet thoroughly.</li>
 <li>Centrifuge the suspension at 250 x g for 10 min at RT.</li>
 <li>Discard the supernatant.</li>
 <li>To remove myelin, add 10 ml 25% (w/v) BSA (Sigma, purity &gt; 98%) and centrifuge at 1,000 x g for 20 min at 4 &deg;C. 25% BSA should be dissolved in 1x PBS (PAA Laboratories) and filtered through 0.2 &mu;m filter).</li>
 <li>Carefully discard the supernatant, dissolve the pellet in 10 ml Buffer A and transfer to a new Falcon tube.</li>
 <li>Centrifuge the suspension at 250 x g for 5 min at RT. The resulting pellet contains the endothelial cells.</li>
 <li>Wash twice the collagen IV- coated 12-well plate with PBS.</li>
 <li>Resuspend the pellet in 4 ml of growth medium (DMEM containing 10% FCS, 50U/ml penicillin/streptomycin, 1% L-glutamine) and plate the cell suspension into two wells, 2 ml to each well. Add the puromycin to a final concentration of 4 &mu;g/ml and incubate for 45 min at 37 &deg;C in a cell culture incubator. This step allows removal of rapidly adhering cells. Note: Puromycin is added for 24 hr. Only brain ECs can metabolize it, for other cell types puromycin is toxic 14.</li>
 <li>Transfer the 2 ml medium containing non-adhered cells from step 1.14 into two fresh wells (these wells contain the EC fraction). Fill up the wells with adhered cells from step 1.14 with 2 ml fresh medium (these wells contain other cell types, e.g. fibroblasts, astrocytes and can be grown in parallel and used for comparisons of the morphology).</li>
 <li>Change the medium the following day.</li>
</ol>
2. Immortalizing the Brain Microvascular Endothelial Cells
<ol>
 <li>Cultivate the GP + E-86 Neo (GPENeo) 15 fibroblasts, secreting a replication-deficient virus with polyoma middle T oncogene in DMEM medium containing 10% heat-inactivated FCS, 50U/ml penicillin/streptomycin, and 2 mg/ml G418 (PAA Laboratories) in gelatine coated flasks. Note: Polyoma middle T oncogene transfection causes growth advantage of ECs over non-ECs leading to a homogenous monolayer of cells with endothelial morphology 4 to 6 weeks of culture. GPENeo are not commercially available and can be obtained as shared public material (please refer to original publications 4,16).</li>
 <li>To use the virus-containing medium for immortalization, cultivate the GPENeo cells in G418- free medium for 24 h.</li>
 <li>Remove 10 ml of the GPEneo supernatant and add Polybrene (hexadimethrine bromide) (Sigma) to a final concentration of 8 &mu;g/ml, sterilize through 0.45 &mu;m filter to remove the cellular fragments.</li>
 <li>Remove the growth medium from the EC cultures prepared in Part 1 and add 2 ml of GPEneo supernatant/Polybrene mixture into the wells. Repeat it the next day using a fresh GPEneo supernatant/Polybrene mixture. Note: Polybrene is used to make pores in the cell wall to facilitate the infection with the viral particles).</li>
 <li>Remove the GPEneo supernatant/Polybrene mixture the following day, wash the cells twice with PBS and maintain the cells in the growth medium changing it every 3 days. Upon confluence, split the cells 1:2 on collagen IV-coated plates.</li>
 <li>Typically, stable cerebral endothelial (cEND) cell lines should be obtained 4-5 weeks later.</li>
</ol>
3. Cultivating the Brain Microvascular Endothelial Cells
<ol>
 <li>Thaw the cells from cryoaliquot in water bath and transfer in 15 ml-Falcon with 10 ml pre-warmed medium.</li>
 <li>Centrifuge the suspension at 250 x g for 5 min at RT.</li>
 <li>Remove medium, transfer the pellet in the collagen IV coated T25 cm2 cell culture flask with pre-warmed growth medium (cell density for plating should be at least 1x 104 cells/ml).</li>
 <li>Change medium the next day and after the cells reached confluence (usually after 5 days) split the cells.</li>
</ol>
4. Splitting of cEND-cells (Note: Splitting Should be Done Only Once a Week, Avoid Splitting Higher than 1:4).
<ol>
 <li>Remove medium, wash the cells with PBS.</li>
 <li>Add 3 ml of warm trypsin-EDTA solution (PAA Laboratories) (T75 cm2 flask), incubate at 37 &deg;C and wait until cells layer is dispersed (usually within 5 to 15 min).</li>
 <li>Add 5 ml of growth medium, pipette up and down, and transfer to a new collagen IV-coated flask.</li>
</ol>
5. Freezing of cEND-cells
<ol>
 <li>Obtain the cell suspension as described in step 4</li>
 <li>Centrifuge the suspension at 250 x g for 5 min at RT.</li>
 <li>Resuspend the cell pellet (obtained from 1 T75 cm2 flask) in 6 ml freezing medium (95% growth medium, 5% DMSO).</li>
 <li>Divide the cell suspension into four 1.5 ml cryo-aliquots. Note: One cryo-aliquot can be seeded on T25 cm2 flask.</li>
 <li>Store the cryo-aliquots under liquid nitrogen vapor temperature.</li>
</ol>
cEND growth medium: 450 ml DMEM, 10% FCS, 10 ml L-glutamine, 2% MEM-Kit, 2% NEAA, 10 ml natrium pyruvate, 50 U/ml penicillin/ streptomycin
6. Representative Results
The cEND and cerebEND cells were characterized by immunostainings of endothelial and BBB markers as well as by measurements of transendothelial electrical resistance (TEER) and permeability. The cEND and cerebEND had a morphology similar to primary cultures of brain ECs, with monolayers of tightly packed elongated cells that exhibited growth inhibition at confluence. The cells expressed well detectable levels of claudin-5, occludin and VE-cadherin proteins which were localized at the cell-cell junctions, as shown by immunofluorescence (Figure 3) 4,5. Culturing cEND cells in serum-reduced medium led to an increase in TEER (from 150 &Omega;cm2 in the presence of 10% serum to 500 &Omega;cm2 in the presence of 2% serum), which was potentiated by the addition of hydrocortisone (800 &Omega;cm2) or insulin (1,000 &Omega;cm2)4. Monolayers of cEND cultured in serum-reduced medium for 21 days had TEER of 900 &Omega;cm2 (Figure 4). TEER was measured using an assembly containing current-passing and voltage-measuring electrodes (World Precision Instruments). For comparison, the primary microvascular brain ECs has been reported to have TEER values of 200-600 &Omega;cm2 and a commercially available mouse cell line bEnd.3 had TEER values of 100-140 &Omega;cm2 (for recent review see Abbot 2005 and Toth et al., 2011 17,18). In addition, the passage of macromolecules, such as non-charged FITC-dextrans of molecular masses 4, 10, 70 and 500 kDa, or fluorescein (300 Da) across the monolayer of cEND in 2% FCS differentiation medium over 4 hr was decreased compared with control cells maintained in growth medium containing 10% FCS: paracellular flux was reduced to 30% of control cells for fluorescein, to 26% for FITC-dextrans 10 and 70 kDa, and flux was reduced to 4.5% of control cells for FITC-dextrtan 500 kDa. Similar to TEER values, the permeability was at the lowest level in the cells cultured in the presence of glucocorticoids (GC) 4. In further studies, we identified the glucocorticoid target genes, occludin, claudin-5 and VE-cadherin in brain vascular endothelium 4,7,9. GC treatment led to an increase in the expression of these proteins and to the rearrangement of VE-cadherin to the cytoskeleton. The direct GC-mediated regulation of tight junction proteins occludin and claudin-5 appears through GC response elements in their promoter regions 4,7,19. Additionally, claudin-5 was identified as a novel estrogen target in vascular endothelium 10.
Endothelial dysfunction underlies many different diseases. We cultured the cEND under oxygen/glucose deprivation (OGD) conditions. OGD led to the disruption of the BBB function, which could be reconstituted after combined treatment with GC and proteasome inhibitor bortezomib 12. OGD conditions led to a strong increase in glucose uptake and in the expression of glucose transporters in cerebEND, which could be attenuated by the addition of MK801, a non-competitive inhibitor of the NMDA-receptor 13.
<img alt="Figure 1" src="/files/ftp_upload/4022/4022fig1.jpg" /> 
Figure 1. Morphology of brain microvascular endothelial cells (cEND) one week after isolation. Light microscopy pictures were taken under the 6x (A) and 15x (B) magnification. The island-formed colonies of endothelial cells are visible.
<img alt="Figure 2" src="/files/ftp_upload/4022/4022fig2.jpg" /> 
Figure 2. Morphology of brain microvascular endothelial cells (cEND) one month after isolation and immortalization. Light microscopy pictures were taken under the 15x magnification. A confluent, homogenous endothelial cell monolayer can be observed.
<img alt="Figure 3" src="/files/ftp_upload/4022/4022fig3.jpg" /> 
Figure 3. Immortalized brain microvascular endothelial cells express claudin-5, occludin and VE-cadherin.CEND cells were grown on collagen IV-coated coverslips and stained with antibodies against claudin-5 (A), occludin (B) and VE-cadherin (C). The images were taken using a Zeiss Axioscop2 microscope under the 40x magnification.
<img alt="Figure 4" src="/files/ftp_upload/4022/4022fig4.jpg" /> 
Figure 4. Measurement of transendothelial electrical resistance (TEER) of cEND monolayer. CEND were grown on collagen IV-coated transwell filters (pore size 0.4 &mu;m). After reaching confluence, the cells were maintained in medium containing 2% FCS. TEER was measured after 7, 14 and 21 days using an assembly containing current-passing and voltage-measuring electrodes.

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No conflicts of interest declared.

The described procedure can be used for isolation of microvascular ECs from different mouse strains as well as from different knock-out mouse strains to study the specific changes in vascular endothelium function. As a method of immortalization we used the transformation with oncoprotein of murine polyomavirus, Polyoma middle T antigen. It transforms rapidly immature endothelial cells in vivo and in vitro 20,21,22. Other methods of immortalization of ECs described in the literature include for example the immortalization with SV40 large T antigen of Simian Vacuolating Virus 40 23, adenovirus gene product E1A 24 or overexpression of the catalytic subunit of human telomerase 3. The immortalization with PymT is specific for the murine immature ECs, which allows obtaining a homogenous EC culture from neonatal mice in a short time. Immortalization changes the properties of the cells and the results obtained with immortalized cell lines should be compared either with primary cells or with experiments in vivo with mice. PymT immortalized EC has been described to express high levels of fibrinolytic activity resulting from increased production of urokinase-type plasminogen activator and decreased production of plasminogen activator inhibitors 25. Direct comparison of PymT immortalized bEND5 cell line with primary ECs revealed that both in vitro BBB models are well suited for T cell adhesion studies 26.
The cell lines established according to the described procedure can be used at low passage number to maintain barrier properties by high expression of BBB and EC junctional proteins, as cells lose these properties during aging. Thus after loss of barrier properties, the preparation of a new cell line should be considered. Cell plating density should be high for ECs, as this is critical for cell proliferation. This must be considered during the isolation procedure as well as maintenance of the immortalized ECs. Each new cell line should be tested for its barrier properties and for possible contaminants with other cell types. EC monolayers can be stained with anti-galactocerebroside, anti-glial fibrillary acidic protein and anti-smooth muscle antigen as primary antibodies to exclude the contamination with oligodendrocytes, astrocytes and pericytes 4,27. To rule out contamination by meningal vasculature, the expression of thrombomodulin can be tested, which is expressed in all vascular beds with the exception of the brain 28.
Interestingly, the immortalized microvascular endothelial cell lines isolated from different brain regions differ in their barrier properties and susceptibility to pro-inflammatory stimuli. As an example, the cerebral cEND and cerebellar cerebEND cell lines can be mentioned 4,5. CerebEND showed, in comparison to cEND, lower expression levels of major tight junction components claudin-1 and occludin. However, the levels of claudin-3 and -12 were higher in cerebEND. The barrier function of cerebEND cells suffered much more under a treatment with the inflammatory mediator, TNF&alpha; than the barrier properties of cEND cells did 5. Higher cerebellar vulnerability to inflammatory stimuli, can be observed in vivo in multiple sclerosis patients and in its animal model experimental autoimmune encephalomyelitis 29. These interesting findings show the necessity of generating and characterizing individual in vitro models for different brain regions, in order to improve the future drug targeting into the brain.

<table border="1">
 <tbody>
 <tr>
 <td>Name of the reagent</td>
 <td>Company</td>
 <td>Catalogue number</td>
 <td>Comments</td>
 </tr>
 <tr>
 <td>Bovine serum albumin (BSA)</td>
 <td>Sigma-Aldrich</td>
 <td>A7906</td>
 <td>Purity &gt; 98%</td>
 </tr>
 <tr>
 <td>collagen IV</td>
 <td>Sigma-Aldrich</td>
 <td>C5533</td>
 <td>50 &mu;g/ml in 50 mM acetic acid</td>
 </tr>
 <tr>
 <td>Collagenase/ Dispase</td>
 <td>Roche</td>
 <td>10269638001</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Dulbecco's modified Eagle's medium (DMEM)</td>
 <td>Sigma-Aldrich</td>
 <td>D5796</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Dimethyl sulfoxide (DMSO)</td>
 <td>Sigma-Aldrich</td>
 <td>D2650</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Fetal Calf Serum (FCS)</td>
 <td>PAA Laboratories</td>
 <td>A15110-1333</td>
 <td>final concentration 10%, heat-inactivated (30 min at 56 &deg;C)</td>
 </tr>
 <tr>
 <td>L-glutamine</td>
 <td>Biochrom AG</td>
 <td>K0282</td>
 <td>Storage: &le; -15 &deg;C</td>
 </tr>
 <tr>
 <td>MEM Vitamine</td>
 <td>Biochrom AG</td>
 <td>K0373</td>
 <td>Storage: &le; -15 &deg;C</td>
 </tr>
 <tr>
 <td>Na-pyruvate</td>
 <td>Biochrom AG</td>
 <td>L0473</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Neomycin (G418)</td>
 <td>PAA Laboratories</td>
 <td>P11-012</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Non essential amino acids (NEA)</td>
 <td>Biochrom AG</td>
 <td>K0293</td>
 <td>Storage at 4 &deg;C</td>
 </tr>
 <tr>
 <td>Penicilin/Streptomycin</td>
 <td>Biochrom AG</td>
 <td>A2212</td>
 <td>Storage: &le; -15 &deg;C</td>
 </tr>
 <tr>
 <td>Phosphate buffered saline (PBS)</td>
 <td>Biochrom AG</td>
 <td>L1825</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Polybrene (hexadimethrine bromide)</td>
 <td>Sigma-Aldrich</td>
 <td>107689</td>
 <td>0.8 mg/ml in PBS, storage at -20 &deg;C</td>
 </tr>
 <tr>
 <td>Puromycin</td>
 <td>Sigma-Aldrich</td>
 <td>P8833</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Trypsin/EDTA</td>
 <td>Biochrom AG</td>
 <td>L1825</td>
 <td>0.05%, 0.02% EDTA in PBS</td>
 </tr>
 </tbody>
</table>

generation of an immortalized murine brain microvascular endothelial cell line as an in vitro blood brain barrier model

Epithelial and endothelial cells (EC) are building paracellular barriers which protect the tissue from the external and internal environment. The blood-brain barrier (BBB) consisting of EC, astrocyte end-feet, pericytes and the basal membrane is responsible for the protection and homeostasis of the brain parenchyma. In vitro BBB models are common tools to study the structure and function of the BBB at the cellular level. A considerable number of different in vitro BBB models have been established for research in different laboratories to date. Usually, the cells are obtained from bovine, porcine, rat or mouse brain tissue (discussed in detail in the review by Wilhelm et al. 1). Human tissue samples are available only in a restricted number of laboratories or companies 2,3. While primary cell preparations are time consuming and the EC cultures can differ from batch to batch, the establishment of immortalized EC lines is the focus of scientific interest.
Here, we present a method for establishing an immortalized brain microvascular EC line from neonatal mouse brain. We describe the procedure step-by-step listing the reagents and solutions used. The method established by our lab allows the isolation of a homogenous immortalized endothelial cell line within four to five weeks. The brain microvascular endothelial cell lines termed cEND 4 (from cerebral cortex) and cerebEND 5 (from cerebellar cortex), were isolated according to this procedure in the F&ouml;rster laboratory and have been effectively used for explanation of different physiological and pathological processes at the BBB. Using cEND and cerebEND we have demonstrated that these cells respond to glucocorticoid- 4,6-9 and estrogen-treatment 10 as well as to pro-infammatory mediators, such as TNFalpha 5,8. Moreover, we have studied the pathology of multiple sclerosis 11 and hypoxia 12,13 on the EC-level. The cEND and cerebEND lines can be considered as a good tool for studying the structure and function of the BBB, cellular responses of ECs to different stimuli or interaction of the EC with lymphocytes or cancer cells.

Watch this Scientific Journal Video about Generation of an Immortalized Murine Brain Microvascular Endothelial Cell Line as an In Vitro Blood Brain Barrier Model at JoVE.com

Generation of an Immortalized Murine Brain Microvascular Endothelial Cell Line as an In Vitro Blood Brain Barrier Model

This method describes how to isolate and immortalize microvascular endothelial cells from mouse brain. We describe a step-by-step protocol starting from the homogenization of brain tissue, digestion steps, seeding and immortalization of the cells. Usually, it takes about five weeks to obtain a homogenous, immortalized microvascular endothelial cell line.

Generation of an Immortalized Murine Brain Microvascular Endothelial Cell Line as an In Vitro Blood Brain Barrier Model

Epithelial and endothelial cells (EC) are building paracellular barriers which protect the tissue from the external and internal environment. The ...

generation-an-immortalized-murine-brain-microvascular-endothelial

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JoVE Journal

Neuroscience

18.8K ビュー.  University of Wurzburg. この方法では、マウスの脳から微小血管内皮細胞を分離し不死化する方法について説明します。我々は、脳組織、消化手順、播種し、細胞の不死化の均質化から始まるステップ·バイ·ステップのプロトコルを記述します。通常、それは均質、不死化血管内皮細胞株を得るために約5週間かかります。

ビデオ: Generation of an Immortalized Murine Brain Microvascular Endothelial Cell Line as an In Vitro Blood Brain Barrier Model

Functional Neuroimaging Using Ultrasonic Blood-brain Barrier Disruption and Manganese-enhanced MRI

Although mice are the dominant model system for studying the genetic and molecular underpinnings of neuroscience, functional neuroimaging in mice remains technically challenging. One approach, Activation-Induced Manganese-enhanced MRI (AIM MRI), has been used successfully to map neuronal activity in rodents 1-5. In AIM MRI, Mn2+ acts a calcium analog and accumulates in depolarized neurons 6,7. Because Mn2+ shortens the T1 tissue property, regions of elevated neuronal activity will enhance in MRI. Furthermore, Mn2+ clears slowly from the activated regions; therefore, stimulation can be performed outside the magnet prior to imaging, enabling greater experimental flexibility. However, because Mn2+ does not readily cross the blood-brain barrier (BBB), the need to open the BBB has limited the use of AIM MRI, especially in mice.
One tool for opening the BBB is ultrasound. Though potentially damaging, if ultrasound is administered in combination with gas-filled microbubbles (i.e., ultrasound contrast agents), the acoustic pressure required for BBB opening is considerably lower. This combination of ultrasound and microbubbles can be used to reliably open the BBB without causing tissue damage 8-11.
Here, a method is presented for performing AIM MRI by using microbubbles and ultrasound to open the BBB. After an intravenous injection of perflutren microbubbles, an unfocused pulsed ultrasound beam is applied to the shaved mouse head for 3 minutes. For simplicity, we refer to this technique of BBB Opening with Microbubbles and UltraSound as BOMUS 12. Using BOMUS to open the BBB throughout both cerebral hemispheres, manganese is administered to the whole mouse brain. After experimental stimulation of the lightly sedated mice, AIM MRI is used to map the neuronal response.
To demonstrate this approach, herein BOMUS and AIM MRI are used to map unilateral mechanical stimulation of the vibrissae in lightly sedated mice 13. Because BOMUS can open the BBB throughout both hemispheres, the unstimulated side of the brain is used to control for nonspecific background stimulation. The resultant 3D activation map agrees well with published representations of the vibrissae regions of the barrel field cortex 14. The ultrasonic opening of the BBB is fast, noninvasive, and reversible; and thus this approach is suitable for high-throughput and/or longitudinal studies in awake mice.

A technique is described for broadly opening the blood-brain barrier in the mouse using microbubbles and ultrasound. Using this technique, manganese can be administered to the mouse brain. Because manganese is an MRI contrast agent that accumulates in depolarized neurons, this approach enables imaging of neuronal activity.

Although mice are the dominant model system for studying the genetic and molecular underpinnings of neuroscience, functional neuroimaging in mice remains ...

Isolation of Primary Murine Brain Microvascular Endothelial Cells

The blood-brain-barrier is ultrastructurally assembled by a monolayer of brain microvascular endothelial cells (BMEC) interconnected by a junctional complex of tight and adherens junctions. Together with other cell-types such as astrocytes or pericytes, they form the neurovascular unit (NVU), which specifically regulates the interchange of fluids, molecules and cells between the peripheral blood and the CNS. Through this complex and dynamic system BMECs are involved in various processes maintaining the homeostasis of the CNS. A dysfunction of the BBB is observed as an essential step in the pathogenesis of many severe CNS diseases. However, specific and targeted therapies are very limited, as the underlying mechanisms are still far from being understood. 
Animal and in vitro models have been extensively used to gain in-depth understanding of complex physiological and pathophysiological processes. By reduction and simplification it is possible to focus the investigation on the subject of interest and to exclude a variety of confounding factors. However, comparability and transferability are also reduced in model systems, which have to be taken into account for evaluation. The most common animal models are based on mice, among other reasons, mainly due to the constantly increasing possibilities of methodology. In vitro studies of isolated murine BMECs might enable an in-depth analysis of their properties and of the blood-brain-barrier under physiological and pathophysiological conditions. Further insights into the complex mechanisms at the BBB potentially provide the basis for new therapeutic strategies.
This protocol describes a method to isolate primary murine microvascular endothelial cells by a sequence of physical and chemical purification steps. Special considerations for purity and cultivation of MBMECs as well as quality control, potential applications and limitations are discussed.

Brain microvascular endothelial cells (BMEC) are interconnected by specific junctional proteins forming a highly regulated barrier separating blood and the central nervous system (CNS), the so-called blood-brain-barrier (BBB). The isolation of primary murine brain microvascular endothelial cells, as discussed in this protocol, enables detailed in vitro studies of the BBB.

The blood-brain-barrier is ultrastructurally assembled by a monolayer of brain microvascular endothelial cells (BMEC) interconnected by a junctional ...

Interfacing 3D Engineered Neuronal Cultures to Micro-Electrode Arrays: An Innovative In Vitro Experimental Model

Currently, large-scale networks derived from dissociated neurons growing and developing in vitro on extracellular micro-transducer devices are the gold-standard experimental model to study basic neurophysiological mechanisms involved in the formation and maintenance of neuronal cell assemblies. However, in vitro studies have been limited to the recording of the electrophysiological activity generated by bi-dimensional (2D) neural networks. Nonetheless, given the intricate relationship between structure and dynamics, a significant improvement is necessary to investigate the formation and the developing dynamics of three-dimensional (3D) networks. In this work, a novel experimental platform in which 3D hippocampal or cortical networks are coupled to planar Micro-Electrode Arrays (MEAs) is presented. 3D networks are realized by seeding neurons in a scaffold constituted of glass microbeads (30-40 &#181;m in diameter) on which neurons are able to grow and form complex interconnected 3D assemblies. In this way, it is possible to design engineered 3D networks made up of 5-8 layers with an expected final cell density. The increasing complexity in the morphological organization of the 3D assembly induces an enhancement of the electrophysiological patterns displayed by this type of networks. Compared with the standard 2D networks, where highly stereotyped bursting activity emerges, the 3D structure alters the bursting activity in terms of duration and frequency, as well as it allows observation of more random spiking activity. In this sense, the developed 3D model more closely resembles in vivo neural networks.

Interfacing 3D Engineered Neuronal Cultures to Micro-Electrode Arrays: An Innovative In Vitro Experimental Model

In this work, a novel experimental model in which 3D neuronal cultures are coupled to planar Micro-Electrode Arrays (MEAs) is presented. 3D networks are built by seeding neurons in a scaffold made up of glass microbeads on which neurons grow and form interconnected 3D structures.

Currently, large-scale networks derived from dissociated neurons growing and developing in vitro on extracellular micro-transducer devices are the ...

Preparation of Acute Brain Slices Using an Optimized N-Methyl-D-glucamine Protective Recovery Method

This protocol is a practical guide to the N-methyl-D-glucamine (NMDG) protective recovery method of brain slice preparation. Numerous recent studies have validated the utility of this method for enhancing neuronal preservation and overall brain slice viability. The implementation of this technique by early adopters has facilitated detailed investigations into brain function using diverse experimental applications and spanning a wide range of animal ages, brain regions, and cell types. Steps are outlined for carrying out the protective recovery brain slice technique using an optimized NMDG artificial cerebrospinal fluid (aCSF) media formulation and enhanced procedure to reliably obtain healthy brain slices for patch clamp electrophysiology. With this updated approach, a substantial improvement is observed in the speed and reliability of gigaohm seal formation during targeted patch clamp recording experiments while maintaining excellent neuronal preservation, thereby facilitating challenging experimental applications. Representative results are provided from multi-neuron patch clamp recording experiments to assay synaptic connectivity in neocortical brain slices prepared from young adult transgenic mice and mature adult human neurosurgical specimens. Furthermore, the optimized NMDG protective recovery method of brain slicing is compatible with both juvenile and adult animals, thus resolving a limitation of the original methodology. In summary, a single media formulation and brain slicing procedure can be implemented across various species and ages to achieve excellent viability and tissue preservation.

Preparation of Acute Brain Slices Using an Optimized N-Methyl-D-glucamine Protective Recovery Method

This protocol demonstrates the implementation of an optimized N-methyl-D-glucamine (NMDG) protective recovery method of brain slice preparation. A single media formulation is used to reliably obtain healthy brain slices from animals of any age and for diverse experimental applications.

This protocol is a practical guide to the N-methyl-D-glucamine (NMDG) protective recovery method of brain slice preparation. Numerous recent studies have ...

An In Vivo Blood-brain Barrier Permeability Assay in Mice Using Fluorescently Labeled Tracers

Blood-brain barrier (BBB) is a specialized barrier that protects the brain microenvironment from toxins and pathogens in the circulation and maintains brain homeostasis. The principal sites of the barrier are endothelial cells of the brain capillaries whose barrier function results from tight intercellular junctions and efflux transporters expressed on the plasma membrane. This function is regulated by pericytes and astrocytes that together form the neurovascular unit (NVU). Several neurological diseases such as stroke, Alzheimer&#39;s disease (AD), brain tumors are associated with an impaired BBB function. Assessment of the BBB permeability is therefore crucial in evaluating the severity of the neurological disease and the success of the treatment strategies employed.
We present here a simple yet robust permeability assay that have been successfully applied to several mouse models both, genetic and experimental. The method is highly quantitative and objective in comparison to the tracer fluorescence analysis by microscopy that is commonly applied. In this method, mice are injected intraperitoneally with a mix of aqueous inert fluorescent tracers followed by anesthetizing the mice. Cardiac perfusion of the animals is performed prior to harvesting brain, kidneys or other organs. Organs are homogenized and centrifuged followed by fluorescence measurement from the supernatant. Blood drawn from the cardiac puncture just before perfusion serves for normalization purpose to the vascular compartment. The tissue fluorescence is normalized to the wet weight and serum fluorescence to obtain a quantitative tracer permeability index. For additional confirmation, the contralateral hemi-brain preserved for immunohistochemistry can be utilized for tracer fluorescence visualization purposes.

An In Vivo Blood-brain Barrier Permeability Assay in Mice Using Fluorescently Labeled Tracers

Here we present a mouse brain vascular permeability assay using intraperitoneal injection of fluorescent tracers followed by perfusion that is applicable to animal models of blood-brain barrier dysfunction. One hemi-brain is used for assessing permeability quantitatively and the other for tracer visualization/immunostaining. The procedure takes 5 - 6 h for 10 mice.

Blood-brain barrier (BBB) is a specialized barrier that protects the brain microenvironment from toxins and pathogens in the circulation and maintains ...

An In Vivo Assessment of Blood-Brain Barrier Disruption in a Rat Model of Ischemic Stroke

Ischemic stroke leads to vasogenic cerebral edema and subsequent primary brain injury, which is mediated through destruction of the blood-brain barrier (BBB). Rats with induced ischemic stroke were established and used as in vivo models to investigate the functional integrity of the BBB. Spectrophotometric detection of Evans blue (EB) in the brain samples with ischemic injury could provide reliable justification for the research and development of novel therapeutic modalities. This method generates reproducible results, and is applicable in any laboratory without a need for special equipment. Here, we present a visualized and technical guideline on the detection of the extravasation of EB following induction of ischemic stroke in rats.

An In Vivo Assessment of Blood-Brain Barrier Disruption in a Rat Model of Ischemic Stroke

The overall goal of this procedure is to provide a highly reproducible technique for in vivo assessment of the blood-brain barrier disruption in rat models of ischemic stroke.

Ischemic stroke leads to vasogenic cerebral edema and subsequent primary brain injury, which is mediated through destruction of the blood-brain barrier ...

A Preclinical Model to Assess Brain Recovery After Acute Stroke in Rats

The purpose of this study was to establish and validate an animal brain ischemia model in the recovery and sequela stages. A middle cerebral artery occlusion/reperfusion (MCAO/R) model in male Sprague-Dawley rats was chosen. By changing the rat&#39;s weight (260&#8722;330 g), the thread bolt type (2636/2838/3040/3043) and the brain infarct time (2-3 h), a higher Longa&#39;s score, a larger infarct volume and a greater model success ratio were screened using the Longa&#39;s score and TTC staining. The optimum model condition (300 g, 3040 thread bolt, 3 h brain infarct time) was acquired and used in a 1-90 day observation period after reperfusion via assessment of sensorimotor functions and infarct volume. At these conditions, the bilateral asymmetry test had a significant difference from 1 to 90 days, and the grid-walking test had a significant difference from 1 to 60 days; both differences could be a suitable sensorimotor functional test. Thus, the most appropriate condition of a novel rat model in the recovery and sequela stages of brain ischemia was found: 300 g rats that underwent MCAO with a 3040 thread bolt for a 3 h brain infarct and then reperfused. The appropriate sensorimotor functional tests were a bilateral asymmetry test and a grid-walking test.

The&#160;purpose of this study is to establish and validate an animal model for research in the recovery and sequela stages of brain ischemia by testing brain infarction and sensorimotor function after middle cerebral artery occlusion/reperfusion (MCAO/R) after 1-90 days in rats.

The purpose of this study was to establish and validate an animal brain ischemia model in the recovery and sequela stages. A middle cerebral artery ...

Whole Neonatal Cochlear Explants as an In vitro Model

Untreated hearing loss imposes significant costs on the global healthcare system and impairs individuals' quality of life. Sensorineural hearing loss is characterized by the cumulative and irreversible loss of sensory hair cells and auditory nerves in the cochlea. Entire and vital cochlear explants are one of the fundamental tools in hearing research to detect hair cell loss and to characterize the molecular mechanisms of the inner ear cells. Many years ago, a protocol for neonatal cochlear isolation was developed, and although it has been modified over time, it still holds potential for improvement. 
This paper presents an optimized protocol for isolating and culturing whole neonatal cochlear explants in multi-well culture chambers that enables the study of hair cells and spiral ganglion neuron cells along the entire length of the cochlea. The protocol was tested using cochlear explants from mice and rats. Healthy cochlear explants were obtained to study the interaction between hair cells, spiral ganglion neuron cells, and the surrounding supporting cells. 
One of the main advantages of this method is that it simplifies the organ culture steps without compromising the quality of the explants. All three turns of the organ of Corti are attached to the bottom of the chamber, which facilitates in vitro experiments and the comprehensive analysis of the explants. We provide some examples of cochlear images from different experiments with live and fixed explants, demonstrating that the explants retain their structure despite exposure to ototoxic drugs. This optimized protocol can be widely used for the integrative analysis of the mammalian cochlea.

Whole Neonatal Cochlear Explants as an In vitro Model

The current protocol updates previous protocols and incorporates relatively straightforward approaches for culturing high-quality cochlear explants. This provides reliable data acquisition and high-resolution imaging in live and fixed cells. This protocol supports the ongoing trend of studying inner ear cells.

Untreated hearing loss imposes significant costs on the global healthcare system and impairs individuals' quality of life. Sensorineural hearing loss is ...

Transendothelial Resistance Measurement to Assess Cell Barrier Integrity

Barrier Functional Integrity Recording on bEnd.3 Vascular Endothelial Cells via Transendothelial Electrical Resistance Detection

The blood-brain barrier (BBB) is a dynamic physiological structure composed of microvascular endothelial cells, astrocytes, and pericytes. By coordinating the interaction between restricted transit of harmful substances, nutrient absorption, and metabolite clearance in the brain, the BBB is essential in preserving central nervous system homeostasis. Building in vitro models of the BBB is a valuable tool for exploring the pathophysiology of neurological disorders and creating pharmacological treatments. This study describes a procedure for creating an in vitro monolayer BBB cell model by seeding bEnd.3 cells into the upper chamber of a 24-well plate. To assess the integrity of cell barrier function, the conventional epithelial cell voltmeter was used to record the transmembrane electrical resistance of normal cells and CoCl2-induced hypoxic cells in real-time. We anticipate that the above experiments will provide effective ideas for the creation of in vitro models of BBB and drugs to treat disorders of central nervous system diseases.

Author Spotlight: Applications of TEER Detection to Assess Cell Barrier Integrity 

This protocol describes a dependable and efficient in vitro model of the brain blood barrier. The method uses mouse cerebral vascular endothelial cells bEnd.3 and measures transmembrane electrical resistance.

The blood-brain barrier (BBB) is a dynamic physiological structure composed of microvascular endothelial cells, astrocytes, and pericytes. By coordinating ...

A 3D Blood-Brain Barrier Model Derived from Human Pluripotent Stem-Cells

Reconstruction of the Blood-Brain Barrier In Vitro to Model and Therapeutically Target Neurological Disease

The blood-brain barrier (BBB) is a key physiological component of the central nervous system (CNS), maintaining nutrients, clearing waste, and protecting the brain from pathogens. The inherent barrier properties of the BBB pose a challenge for therapeutic drug delivery into the CNS to treat neurological diseases. Impaired BBB function has been related to neurological disease. Cerebral amyloid angiopathy (CAA), the deposition of amyloid in the cerebral vasculature leading to a compromised BBB, is a co-morbidity in most cases of Alzheimer&#39;s disease (AD), suggesting that BBB dysfunction or breakdown may be involved in neurodegeneration. Due to limited access to human BBB tissue, the mechanisms that contribute to proper BBB function and BBB degeneration remain unknown. To address these limitations, we have developed a human pluripotent stem cell-derived BBB (iBBB) by incorporating endothelial cells, pericytes, and astrocytes in a 3D matrix. The iBBB self-assembles to recapitulate the anatomy and cellular interactions present in the BBB. Seeding iBBBs with amyloid captures key aspects of CAA. Additionally, the iBBB offers a flexible platform to modulate genetic and environmental factors implicated in cerebrovascular disease and neurodegeneration, to investigate how genetics and lifestyle affect disease risk. Finally, the iBBB can be used for drug screening and medicinal chemistry studies to optimize therapeutic&#160;delivery to the CNS. In this protocol, we describe the differentiation of the three types of cells (endothelial cells, pericytes, and astrocytes) arising from human pluripotent stem cells, how to assemble the differentiated cells into the iBBB, and how to model CAA in vitro using exogenous amyloid. This model overcomes the challenge of studying live human brain tissue with a system that has both biological fidelity and experimental flexibility, and enables the interrogation of the human BBB and its role in neurodegeneration.

Author Spotlight: A Personalized Approach Towards Investigating Alzheimer&#039;s Disease Using an In Vitro Blood-Brain Barrier Model 

The blood-brain barrier (BBB) has a crucial role in sustaining a stable and healthy brain environment. BBB dysfunction is associated with many neurological diseases. We have developed a 3D, stem-cell-derived model of the BBB to investigate cerebrovascular pathology, BBB integrity, and how the BBB is altered by genetics and disease.

The blood-brain barrier (BBB) is a key physiological component of the central nervous system (CNS), maintaining nutrients, clearing waste, and protecting ...