Name: a human blood-brain interface model to study barrier crossings by pathogens or medicines and their interactions with the brain
Uploaded: 2019-04-09T13:30:00
Description: Watch this Scientific Journal Video about A Human Blood-Brain Interface Model to Study Barrier Crossings by Pathogens or Medicines and Their Interactions with the Brain at JoVE.com

This study was supported by internal grants from Institut Pasteur including an Incitative grant (PTR 435) and by a grant &#8220;Contrat de Soutien &#224; la Recherche&#8221; provided by Sanofi Pasteur to Institut Pasteur. A. da Costa was supported by the Sanofi-Pasteur grant and Florian Bakoa is recipient of a PhD grant provided by ANRT (Association Nationale de la Recherche et de la Technologie). We are indebted to Pr Pierre-Olivier Couraud and Dr Florence Miller for helpful discussions.

1. Cell culture work of Ntera/CL2.D1 to prepare a co-culture of post-mitotic hNeurons and hAstrocytes (NT2-N/A)
NOTE: This is the component of the Minibrain (Figure 1).
<ol>
	<li>Culturing the Ntera/Cl2.D1
	<ol>
		<li>Remove a vial of frozen cells from liquid nitrogen tank. Keep on ice.</li>
		<li>Thaw the cells rapidly in a 37 &#176;C water bath.</li>
		<li>Transfer the cells in a 15 mL tube containing 10 mL of complete DMEM F12 medium (Dulbecco&#39;s Modified ...

In this article we demonstrated how to build an in cellulo blood/brain interface, the BBB-Minibrain, by combining a BBB model and a culture of mixed brain cerebral cells (Minibrain) into a single kit. This system is biologically relevant, easy to set up and handle for experimenters well trained in cell culture.
As for any other in vitro model of BBB, reliable results would be obtained if drastic control of tightness of the barrier is applied. Inserts should be carefully tested for permeability...

The brain is separated from the systemic circulation by a non-permeable structure that restricts exchanges between the brain parenchyma and the blood, called the blood-brain barrier (BBB). Mostly composed of cerebral endothelial cells, the BBB dynamically interacts with astrocytes, perivascular microglia and neurons of the neighboring brain parenchyma. The three major functions of the BBB are the creation and maintenance of ionic homeostasis for neuronal functions, supply of the brain with nutrients, and protection from toxic injuries or entry of pathogens1,2, which contribute to the maintenance of brain homeostasis and its functions3. This barrier is so efficient that only few drugs can cross the BBB4,5. At present, the available methods to predict whether a molecule will pass the BBB and diffuse into the brain consist of ex vivo studies on autopsy material, image tracking in the brain of human volunteers by MRI (magnetic resonance imaging) or PET (positon emission tomography) or pharmacodynamics and pharmacokinetic preclinical studies in animals6,7,8. These techniques and models have some limitations, such as the limited resolution of PET and the low sensitivity of MRI6,8, the difficulty to quantify molecules (i.e., antibody based molecules for example) that poorly penetrate the brain7, and for the preclinical studies their high cost and resort of animal testing.
The last point is important because, according to the 3R&#8217;s rules, (replacement, reduction and refinement of animal testing) the regulatory administrations have asked that the researchers urgently develop scientifically accurate alternative to animal experimentation9,10,11,12,13,14,15.
Over the last decades, several in vitro models of BBB have been proposed16,17,18 by cultivating on filter membrane inserts endothelial cells from different species such as mouse, rat, bovine and pig. As far as the human species is concerned, the scarce and difficult availability of primary cells prompted the researchers to develop human models based on immortalized brain endothelial cells or human-derived stem cells19,20,21. These barriers are proper in vitro surrogates of BBB provided that they express endothelial cell markers, tight junction markers, efflux transporters, solute carriers, receptors, and respond to the endothelial stimuli 20. A few BBB models using filter membrane inserts coated with endothelial cells and other cell types (i.e., astrocytes, neurons or pericytes22,23,24) were assayed. The goal of these co-cultures was to increase the BBB physical characteristics by taking advantage of the secretion of soluble factors by astrocytes/neurons or pericytes.
Nevertheless, none of these models includes brain parenchyma to study and predict the fate of a drug candidate once it has passed the barrier. Therefore, our goal was to build an in cellulo blood/brain interface, the BBB-Minibrain, by combining a BBB model and a culture of mixed brain cells into a single kit. The BBB-Minibrain uses a culture system consisting of a porous filter inserted in a well of a multiwell cell culture plate. The filter is coated with hCMEC/D3 cells, a human brain endothelial cell line that has been proved highly reliable for BBB drug testing25,26,27, to form the BBB. The Minibrain, which is a co-differentiated culture of human neurons and astrocytes derived from the NTera/Cl2.D1 cell line28,29 mixed together with the human microglial cell line CHME/Cl530 in ratio corresponding to the microglia vs. neuron-astrocytes ratios of the brain31, is cultivated in the bottom of the plate well.
Besides studying passage of drugs across the BBB and their fate in the parenchyma, the blood-brain interface in cellulo model could be a powerful tool to address the entry of pathogens into the brain (neuroinvasiveness), the dispersion into the brain (neurotropism) and the toxicity (neurovirulence) they can exert on brain parenchyma cells. Neurovirulence and neuroinvasiveness studies would benefit from the development of an efficient in cellulo model and be advantageous to replace animal models. Using the BBB-Minibrain kit32, we demonstrated the neuroinvasive phenotype of rare viral mutants that accumulated in the French Neurotropic virus strain of Yellow Fever Virus (i.e., FNV-YFV33,34) used to prepare a discontinued live YFV vaccine and the passage of a neuroregenerative and neuroprotective biomolecule called Neurovita (referred as NV henceforth in the manuscript)35. Because NV neither naturally crosses the cell membrane nor the BBB, NV was fused with the variable part (VHH) of a single chain antibody of Llama that crosses the biological membranes including the BBB and functions as a cell penetrating molecule (CPM)36. The CPM property of VHH seems to depend upon the isoelectric point and the length of the VHH37.
This in cellulo test should make it possible to sort the molecules that could potentially cross the BBB before carrying out pharmacokinetic and pharmacodynamics analysis in animals, and ideally in the same time to be able to predict their behavior in the nervous parenchyma. This system is biologically relevant and easy to set up and handle by professionals well trained in cell culture26,29,30,38. The interest of such in cellulo testing would be two-fold: reducing the costs of preclinical tests on the one hand and reducing the use of animal testing on the other hand.

<table>
	<tbody>
		<tr>
			<td>12 well plates</td>
			<td>Corning</td>
			<td>3336</td>
			<td></td>
		</tr>
		<tr>
			<td>5-fluoro-2&#8217;deoxyuridine</td>
			<td>Merck-Sigma Aldrich</td>
			<td>F0503</td>
			<td></td>
		</tr>
		<tr>
			<td>85mm Petri Dish</td>
			<td>Sarstedt</td>
			<td>83-3902-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-Nf200</td>
			<td>Merck-Sigma Aldrich</td>
			<td>N4142</td>
			<td></td>
		</tr>
		<tr>
			<td>&#946;-mercapto-ethanol</td>
			<td>Merck-Sigma Aldrich</td>
			<td>M3148</td>
			<td></td>
		</tr>
		<tr>
			<td>CHME/Cl5</td>
			<td>Unit&#233; de Neuroimmunologie Virale</td>
			<td>On request to Dr Lafon</td>
			<td></td>
		</tr>
		<tr>
			<td>CMC</td>
			<td>Calbiochem</td>
			<td>217274</td>
			<td></td>
		</tr>
		<tr>
			<td>Cytosine &#946;-D-arabinofuranoside</td>
			<td>Merck-Sigma Aldrich</td>
			<td>C1768</td>
			<td></td>
		</tr>
		<tr>
			<td>Dark 96 well plates</td>
			<td>Corning</td>
			<td>3915</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM F12</td>
			<td>Thermofisher Scientific</td>
			<td>31330-038</td>
			<td></td>
		</tr>
		<tr>
			<td>DMSO</td>
			<td>Merck-Sigma Aldrich</td>
			<td>D2650</td>
			<td></td>
		</tr>
		<tr>
			<td>Endogro IV</td>
			<td>Millipore</td>
			<td>SCME004</td>
			<td>endothelial cell medium</td>
		</tr>
		<tr>
			<td>Ethanol</td>
			<td>Carlo Erba</td>
			<td>529121</td>
			<td></td>
		</tr>
		<tr>
			<td>FBS</td>
			<td>Hyclone</td>
			<td>SV30015-04</td>
			<td></td>
		</tr>
		<tr>
			<td>Formaldehyde</td>
			<td>Merck-Sigma Aldrich</td>
			<td>252549</td>
			<td></td>
		</tr>
		<tr>
			<td>GIEMSA</td>
			<td>RAL Diagnostic</td>
			<td>320310</td>
			<td></td>
		</tr>
		<tr>
			<td>Goat-Anti Mouse</td>
			<td>Jackson Immuno Research</td>
			<td>115-545-003</td>
			<td></td>
		</tr>
		<tr>
			<td>Goat-Anti Rabbit</td>
			<td>Thermofisher Scientific</td>
			<td>R37117</td>
			<td></td>
		</tr>
		<tr>
			<td>HBSS with Ca2+-Mg2+</td>
			<td>Thermofisher Scientific</td>
			<td>14025-100</td>
			<td></td>
		</tr>
		<tr>
			<td>hCMEC/D3</td>
			<td>Cedarlane</td>
			<td>CLU512</td>
			<td></td>
		</tr>
		<tr>
			<td>Hepes 1M</td>
			<td>Thermofisher Scientific</td>
			<td>15630-070</td>
			<td></td>
		</tr>
		<tr>
			<td>Hoescht 33342</td>
			<td>Merck-Sigma Aldrich</td>
			<td>33263</td>
			<td></td>
		</tr>
		<tr>
			<td>Laminine</td>
			<td>Merck-Sigma Aldrich</td>
			<td>L6274</td>
			<td></td>
		</tr>
		<tr>
			<td>L-glutamin</td>
			<td>Thermofisher Scientific</td>
			<td>25030-024</td>
			<td></td>
		</tr>
		<tr>
			<td>Lucifer Yellow</td>
			<td>Merck-Sigma Aldrich</td>
			<td>L0259</td>
			<td></td>
		</tr>
		<tr>
			<td>MEM 10X</td>
			<td>Thermofisher Scientific</td>
			<td>21430</td>
			<td></td>
		</tr>
		<tr>
			<td>MEM 1X</td>
			<td>Thermofisher Scientific</td>
			<td>42360</td>
			<td></td>
		</tr>
		<tr>
			<td>Ntera/Cl2D.1</td>
			<td>ATCC</td>
			<td>CRL-1973</td>
			<td></td>
		</tr>
		<tr>
			<td>Paraformaldehyde</td>
			<td>Electron Microscopy Sciences</td>
			<td>15714</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS without Ca2+-Mg2+</td>
			<td>Thermofisher Scientific</td>
			<td>14190</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS-Ca2+-Mg2+</td>
			<td>Thermofisher Scientific</td>
			<td>14040-091</td>
			<td></td>
		</tr>
		<tr>
			<td>Pen/Strep</td>
			<td>Eurobio</td>
			<td>CXXPES00-07</td>
			<td></td>
		</tr>
		<tr>
			<td>Poly-d-Lysine</td>
			<td>Merck-Sigma Aldrich</td>
			<td>P1149</td>
			<td></td>
		</tr>
		<tr>
			<td>Prolong Gold</td>
			<td>Thermofisher Scientific</td>
			<td>P36930</td>
			<td></td>
		</tr>
		<tr>
			<td>Qiashredder</td>
			<td>QIAGEN</td>
			<td>79656</td>
			<td></td>
		</tr>
		<tr>
			<td>Rat Collagen I</td>
			<td>Cultrex</td>
			<td>3443-100-01</td>
			<td></td>
		</tr>
		<tr>
			<td>Retinoic Acid All-Trans</td>
			<td>Merck-Sigma Aldrich</td>
			<td>R2625</td>
			<td></td>
		</tr>
		<tr>
			<td>RNA purification kit</td>
			<td>QIAGEN</td>
			<td>74104</td>
			<td></td>
		</tr>
		<tr>
			<td>SDS</td>
			<td>Merck-Sigma Aldrich</td>
			<td>L4509</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium bicarbonate 5.6%</td>
			<td>Eurobio</td>
			<td>CXXBIC00-07</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Pyruvate</td>
			<td>Thermofisher Scientific</td>
			<td>11360</td>
			<td></td>
		</tr>
		<tr>
			<td>T75 Cell+ Flask</td>
			<td>Sarstedt</td>
			<td>83-1813-302</td>
			<td>Tissue culture polystyrene flask with specific surface treatment (Cell+) for sensitive adherent cells</td>
		</tr>
		<tr>
			<td>Transwell</td>
			<td>Corning</td>
			<td>3460</td>
			<td>polyester porous membrane culture inserts</td>
		</tr>
		<tr>
			<td>Trypsin-EDTA</td>
			<td>Merck-Sigma Aldrich</td>
			<td>T3924</td>
			<td></td>
		</tr>
		<tr>
			<td>Ultra Pure Water</td>
			<td>Thermofisher Scientific</td>
			<td>10977-035</td>
			<td></td>
		</tr>
		<tr>
			<td>Uridine</td>
			<td>Merck-Sigma Aldrich</td>
			<td>U3750</td>
			<td></td>
		</tr>
		<tr>
			<td>Versene</td>
			<td>Thermofisher Scientific</td>
			<td>15040-033</td>
			<td>EDTA</td>
		</tr>
		<tr>
			<td>YFV-FNV</td>
			<td>IP Dakar</td>
			<td>Vaccine vial</td>
			<td></td>
		</tr>
	</tbody>
</table>

a human blood-brain interface model to study barrier crossings by pathogens or medicines and their interactions with the brain

The early screening of nervous system medicines on a pertinent and reliable in cellulo BBB model for their penetration and their interaction with the barrier and the brain parenchyma is still an unmet need. To fill this gap, we designed a 2D in cellulo model, the BBB-Minibrain, by combining a polyester porous membrane culture insert human BBB model with a Minibrain formed by a tri-culture of human brain cells (neurons, astrocytes and microglial cells). The BBB-Minibrain allowed us to test the transport of a neuroprotective drug candidate (e.g., Neurovita), through the BBB, to determine the specific targeting of this molecule to neurons and to show that the neuroprotective property of the drug was preserved after the drug had crossed the BBB. We have also demonstrated that BBB-Minibrain constitutes an interesting model to detect the passage of virus particles across the endothelial cells barrier and to monitor the infection of the Minibrain by neuroinvasive virus particles. The BBB-Minibrain is a reliable system, easy to handle for researcher trained in cell culture technology and predictive of the brain cells phenotypes after treatment or insult. The interest of such in cellulo testing would be twofold: introducing derisking steps early in the drug development on the one hand and reducing the use of animal testing on the other hand.

The BBB-Minibrain is an in cellulo experimental model of blood-brain interface.
The BBB-Minibrain is set up on the polyester membrane culture insert system to mimic a blood compartment on the upper level and a brain compartment on the lower level of the blood-brain interface (Figure 2A,B). It consists of a luminal compartment with the hCMEC/D3 endothelial cells on t...

Watch this Scientific Journal Video about A Human Blood-Brain Interface Model to Study Barrier Crossings by Pathogens or Medicines and Their Interactions with the Brain at JoVE.com

A Human Blood-Brain Interface Model to Study Barrier Crossings by Pathogens or Medicines and Their Interactions with the Brain

Here we present a protocol describing the setting of an in cellulo BBB (Blood brain barrier)-Minibrain polyester porous membrane culture insert system in order to evaluate the transport of biomolecules or infectious agents across a human BBB and their physiological impact on the neighboring brain cells.

The early screening of nervous system medicines on a pertinent and reliable in cellulo BBB model for their penetration and their interaction with the ...

This human blood-brain interface model is useful for studying the transfer of molecules into the neuro system. As well as for studying of microbes reach an infected brain pain tumor. The addition of the mini brain to this blood brain barrier or BBB model allows the study of our pathogens and molecules that cause the BBB behave in the brain.Demostrating the procedure will be Florian Bakao, the PhD students for my laboratory. To set up a BBB mini brain polyester membrane culture insert device, dilute the cells to a five times 10 to the fourth cells per insert concentration incomplete endothelial cell medium and add the appropriate volume of cells to each polyester membrane culture insert in a 12 well plate. Then, place the inserts and the cell culture incubator until the cells reach 100%confluency.Hold a 12 well plate with one milliliter of poly-D-lysine her well for four hours at room temperature. Followed by one milliliter of laminin per well overnight. Replace the laminin with one milliliter of endothelial cell medium supplemented with 5%fetal bovine serum, or FBS.Place the place in an incubator for one hour. To prepare the mini grain gently trypsinize the human neurons astrocytes in CHME clone five cells. Associate the cell pellets with complete endothelial cell medium.After counting at 3.6 times 10 to the fifth neurons and astrocytes to 0.4 times 10 to the fifth CHME clone five cells per well have a 12 well plate. And seed the 12 well plate. To construct the BBB mini brain 24 hours after seeding replace the used medium with fresh complete endothelial cell medium and transfer the cerebral micro vascular endothelial cell coated polyester membrane culture inserts over the mini brain cells.Then, place the BBB many brain device into the incubator. To validate the BBB mini brain endothelial permeability add 1.5 milliliters of transport buffer per well to a new 12 well plate. And flip each filter upside down to carefully remove the medium without affecting the endothelial cell barrier.Place each filter in one well of the transport buffer filled plate, and add 0.5 milliliters of Lucifer yellow into each well. Then place the plate into the incubator for 10 minutes before transferring the filters into a new transport buffer filled 12 well plate. After setting up a BBB many brain device as demonstrated, add 3500 platforming units of the French neurotrophic virus strain of yellow fever virus diluted and 50 microliters of 2%FPS endothelial cell medium, very carefully, on top of luminal compartment.Inoculate the control BBB mini brain with 50 microliters of 2%FBS endothelial cell medium without virus. Then place the virus expose BBB many brain device in the incubator for 24 hours. The next day, remove the inserts to determine the endothelial cell permeability and a companion well as just as demonstrated.Save one milliliter of sample from each well to determine the virus titer in the middle compartment. Under the insert gently to avoid leakage of the luminal compartment into the abluminal compartments. To study the transport of a neuron targeting biomolecular across the blood brain barrier, add the bio molecule of interest to the inserts and return the plate to the cell culture incubator.After 24 hours remotely inserts and determine endothelial permeability. Then stain the many brain cells with the appropriate antibody for detecting the presence of the bio molecule of interest within the mini brain. Human cerebral micro vascular endothelial cells express all the subsets of rececptors, Efflux transporters or transporters that are key relevant proteins for their biological functions.Some French neuro tropic virus strain of yellow fever virus can cross the blood brain barrier at 24 hours. The viruses then amplify for the next two days by the multiplication of the virus inside the brain cells. Further, the expression of specific viral biomarkers is stimulated when this neuro invasive viral population is serially passaged on the mini brain cells and strictly correlate to the viral load.After being added to the limit of compartment cell penetrating molecule Neuro-Tag Neurovita crosses the blood brain barrier and is able to target human neurons. Cell penetrating molecule neuro tag Delta neurovita crosses the endothelial cell barrier, but targets less efficiently the human neurons. After being added to the little middle compartment, cell penetrating molecule Neuro-Tag Neurovita crosses the blood brain barrier and is able to regenerate the axons of human neurons, after wounding.In contrast to cell penetrating molecule Neuro-Tag Neurovita Delta, the inactive form of Neurovita. When applied after the axon scratching cell penetrating molecule Neuro-Tag Neurovita triggers the neuro protection of the wounded neurons and the x zonal regeneration. Strictly for the good sake of practices.do not over use I-cell passages. Try to maintain some semblance of medium and regions and check for the absence of mycoplasma. Interactions between the mini brain and the drugs are microbes of interest can be further studied by transcript that chemicalize this or classical biology called techniques.

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Research

JoVE Journal

Neuroscience

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.

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.

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

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 ...

Assessment of Blood-brain Barrier Permeability by Intravenous Infusion of FITC-labeled Albumin in a Mouse Model of Neurodegenerative Disease

Disruption of blood-brain barrier (BBB) integrity is a common feature for different neurological and neurodegenerative diseases. Although the interplay between perturbed BBB homeostasis and the pathogenesis of brain disorders needs further investigation, the development and validation of a reliable procedure to accurately detect BBB alterations may be crucial and represent a useful tool for potentially predicting disease progression and developing targeted therapeutic strategies.
Here, we present an easy and efficient procedure for evaluating BBB leakage in a neurodegenerative condition like that occurring in a preclinical mouse model of Huntington disease, in which defects in the permeability of BBB are clearly detectable precociously in the disease. Specifically, the high molecular weight fluorescein isothiocyanate labelled (FITC)-albumin, which is able to cross the BBB only when the latter is impaired, is acutely infused into a mouse jugular vein and its distribution in the vascular or parenchymal districts is then determined by fluorescence microscopy.
Accumulation of green fluorescent-albumin in the brain parenchyma functions as an index of aberrant BBB permeability and, when quantitated by using Image J processing software, is reported as Green Fluorescence Intensity.

In this study, we present an easy and efficient procedure for evaluating the disruption of the blood-brain barrier under neurodegenerative conditions. To achieve our objective, we infused high molecular weight fluorescein isothiocyanate labelled (FITC)-albumin into mouse jugular vein and evaluated its leakage into the brain parenchyma by fluorescence microscopy.

Disruption of blood-brain barrier (BBB) integrity is a common feature for different neurological and neurodegenerative diseases. Although the interplay ...

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 Benchtop Approach to the Location Specific Blood Brain Barrier Opening using Focused Ultrasound in a Rat Model

Stereotaxic surgery is the gold standard for localized drug and gene delivery to the rodent brain. This technique has many advantages over systemic delivery including precise localization to a target brain region and reduction of off target side effects. However, stereotaxic surgery is highly invasive which limits its translational efficacy, requires long recovery times, and provides challenges when targeting multiple brain regions. Focused ultrasound (FUS) can be used in combination with circulating microbubbles to transiently open the blood brain barrier (BBB) in millimeter sized regions. This allows intracranial localization of systemically delivered agents that cannot normally cross the BBB. This technique provides a noninvasive alternative to stereotaxic surgery. However, to date this technique has yet to be widely adopted in neuroscience laboratories due to the limited access to equipment and standardized methods. The overall goal of this protocol is to provide a benchtop approach to FUS BBB opening (BBBO) that is affordable and reproducible and can therefore be easily adopted by any laboratory.

Focused ultrasound with microbubble agents can open the blood brain barrier focally and transiently. This technique has been used to deliver a wide range of agents across the blood brain barrier. This article provides a detailed protocol for the localized delivery to the rodent brain with or without MRI guidance.

Stereotaxic surgery is the gold standard for localized drug and gene delivery to the rodent brain. This technique has many advantages over systemic ...

Measuring Post-Stroke Cerebral Edema, Infarct Zone and Blood-Brain Barrier Breakdown in a Single Set of Rodent Brain Samples

One of the most common causes of morbidity and mortality worldwide is ischemic stroke. Historically, an animal model used to stimulate ischemic stroke involves middle cerebral artery occlusion (MCAO). Infarct zone, brain edema and blood-brain barrier (BBB) breakdown are measured as parameters that reflect the extent of brain injury after MCAO. A significant limitation to this method is that these measurements are normally obtained in different rat brain samples, leading to ethical and financial burdens due to the large number of rats that need to be euthanized for an appropriate sample size. Here we present a method to accurately assess brain injury following MCAO by measuring infarct zone, brain edema and BBB permeability in the same set of rat brains. This novel technique provides a more efficient way to evaluate the pathophysiology of stroke.

This protocol describes a novel technique of measuring the three most important parameters of ischemic brain injury on the same set of rodent brain samples. Using only one brain sample is highly advantageous in terms of ethical and economic costs.

One of the most common causes of morbidity and mortality worldwide is ischemic stroke. Historically, an animal model used to stimulate ischemic stroke ...

Focused Ultrasound Induced Blood-Brain Barrier Opening for Targeting Brain Structures and Evaluating Chemogenetic Neuromodulation

Acoustically Targeted Chemogenetics (ATAC) allows for the noninvasive control of specific neural circuits. ATAC achieves such control through a combination of focused ultrasound (FUS) induced blood-brain barrier opening (FUS-BBBO), gene delivery with adeno-associated viral (AAV) vectors, and activation of cellular signaling with engineered, chemogenetic, protein receptors and their cognate ligands. With ATAC, it is possible to transduce both large and small brain regions with millimeter precision using a single noninvasive ultrasound application. This transduction can later allow for a long-term, noninvasive, device-free neuromodulation in freely moving animals using a drug. Since FUS-BBBO, AAVs, and chemogenetics have been used in multiple animals, ATAC should also be scalable for the use in other animal species. This paper expands upon a previously published protocol and outlines how to optimize the gene delivery with FUS-BBBO to small brain regions with MRI-guidance but without a need for a complicated MRI-compatible FUS device. The protocol, also, describes the design of mouse targeting and restraint components that can be 3D-printed by any lab and can be easily modified for different species or custom equipment. To aid reproducibility, the protocol describes in detail how the microbubbles, AAVs, and venipuncture were used in ATAC development. Finally, an example data is shown to guide the preliminary investigations of studies utilizing ATAC.

This protocol delineates steps necessary for the gene delivery through focused ultrasound blood brain barrier (BBB) opening, evaluation of the resulting gene expression, and measurement of neuromodulation activity of chemogenetic receptors through histological tests.

Acoustically Targeted Chemogenetics (ATAC) allows for the noninvasive control of specific neural circuits. ATAC achieves such control through a ...

P300-Based Brain-Computer Interface Speller Performance Estimation with Classifier-Based Latency Estimation

Performance estimation is a necessary step in the development and validation of Brain-Computer Interface (BCI) systems. Unfortunately, even modern BCI systems are slow, making collecting sufficient data for validation a time-consuming task for end users and experimenters alike. Yet without sufficient data, the random variation in performance can lead to false inferences about how well a BCI is working for a particular user. For example, P300 spellers commonly operate around 1-5 characters per minute. To estimate accuracy with a 5% resolution requires 20 characters (4-20 min). Despite this time investment, the confidence bounds for accuracy from 20 characters can be as much as &plusmn;23% depending on observed accuracy. A previously published method, Classifier-Based Latency Estimation (CBLE), was shown to be highly correlated with BCI accuracy. This work presents a protocol for using CBLE to predict a user's P300 speller accuracy from relatively few characters (~3-8) of typing data. The resulting confidence bounds are tighter than those produced by traditional methods. The method can thus be used to estimate BCI performance more quickly and/or more accurately.

This article presents a method for estimating same-day P300 speller Brain-Computer Interface (BCI) accuracy using a small testing dataset.

Performance estimation is a necessary step in the development and validation of Brain-Computer Interface (BCI) systems. Unfortunately, even modern BCI ...

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 ...