This research was supported by the Deutsche Forschungsgemeinschaft (DFG, German Research Foundation) under grant number FO 315/4- and the European Union Seventh Framework Programme (FP7/2007-2013) under Grant agreement No. HEALTH-F2-2009- 241778 to CF.

1. Seeding of Endothelial Cells into Well Plates
<ol>
	<li>Cultivate murine brain microvascular endothelial cells (cEND) in T75 culture flask, changing the medium (DMEM containing 10% FCS, 50 U/ml penicillin/streptomycin, 1% L-glutamine) twice a week, until confluence is reached. (For the generation and immortalization of brain microvascular endothelial cells, please see Burek et al., 2012; F&#246;rster et al., 200521,19).</li>
	<li>Wash the cells with phosphate buffered saline (PBS). Remove the PBS and trypsinize the cells with 2 ml warm trypsin-EDTA solution.</li>
	<li>Incubate the cells at 37 &#176;C for 5 min&#160;or until the cell layer is dispersed.</li>
	<li>Add 8 ml culture medium into the cells. Tap the flask several times to detach the cells.</li>
	<li>View cells under the microscope to ensure complete detachment from flask.</li>
	<li>Pipette medium with detached cells up and down. Swirl the flask to mix the cell suspension.</li>
	<li>Take 20 &#181;l of the cell suspension, put into a hemocytometer and count the number of cells.</li>
	<li>Determine the cell density and seed 20,000 cells/cm2 into the well.</li>
	<li>Transfer the cell suspension into collagen1 precoated 6-well&#160;flexible-bottomed culture plates (57.75 cm2) in a total volume of 3 ml/ well. Each well has an area of 9.62 cm2.</li>
	<li>Grow the cells at 37 &#176;C for one week until confluent. Change the culture medium twice per week.</li>
</ol>
2. Cell Differentiation Prior to Stretch-induced Injury
<ol>
	<li>Change the culture medium of the cells with differentiation medium (DMEM containing 1% serum-stripped fetal calf serum (ssFCS), 50 U/ml penicillin/streptomycin).</li>
	<li>Incubate the cells at 37 &#176;C for 24 hr.</li>
</ol>
3. Stretch-induced Injury of Endothelial Cells
<ol>
	<li>Turn on the cell injury controller device.</li>
	<li>Set the delay to 50 msec.</li>
	<li>Set the regulator pressure to 15 psi and press the trigger a couple of times until the registered peak pressure becomes stable.</li>
	<li>Set the regulator pressure to the desired value. Use Table 1 as a guide for generating various degrees of stretch injury.</li>
	<li>Place the 6-well flexible-bottomed culture plate (57.75 cm2) into the tray holder. Make sure that the well selector is set to the correct well size (i.e. large well).</li>
	<li>Place the adapter plug firmly over the well. Hold the plug firmly into place with one hand while the other hand pushes the trigger.</li>
	<li>Record the peak pressure generated.</li>
	<li>Immediately put the plate back into the 37 &#176;C incubator for the desired length of time or use immediately for succeeding experiments or evaluation.</li>
</ol>
4. Assessment of Stretch Injury by Dye Uptake Assay
<ol>
	<li>Immediately after the cells were stretched (step 3.7), add 30 &#181;l of a 1 mg/ml solution of the viability stain that acts as a cytotoxicity marker (please see supplemental table of materials and equipment) to the cell culture medium (Note: 10 &#181;l of the dye solution is to be used for every ml of cell culture medium).</li>
	<li>Upon addition of the dye to the cells, immediately view under a fluorescence microscope.</li>
</ol>
5. Assessment of Stretch Injury by Lactate Dehydrogenase (LDH) Release
<ol>
	<li>Immediately after stretching the cells (step 3.7), remove 200 &#181;l of cell culture medium from the well. Do the same for every time point post-injury you would like to include in your investigation of LDH release (i.e.&#160;e.g. 30 min, 1 hr, 2 hr, etc.)</li>
	<li>Centrifuge the cell culture medium at the highest setting of a microcentrifuge for 5 min to remove any cell debris. Remove the supernatant and use this for the succeeding steps.</li>
	<li>Once you have finished step 5.1 and have taken the necessary samples you would like to have from the various time points you would like to investigate, lyse the cells using the lysis solution included in the LDH assay kit (please see supplemental table of materials and equipment).</li>
	<li>Put 100 &#181;l of the assay medium included in the assay kitto every well of a 96-wells plate provided in the kit.</li>
	<li>Put 100 &#181;l of the cell-free cell culture medium into two parallel wells of a 96-wells plate included in the assay kit.</li>
	<li>Incubate the plate at 37 &#176;C for 30 min.</li>
	<li>Read the absorbance at 492 nm.</li>
</ol>

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No conflicts of interest declared.

The effects of mechanical injury in vitro have been studied and methods have been established for astrocytes, neurons, glial cells and aortic endothelial cells8, 9, 22. There is, however, to date still no known in vitro model of stretch injury in cultured brain endothelial cells. Cellular models of trauma provide valuable advantages over animal models since the mechanical environment of the cells can be precisely controlled6. Hence, an in vitro model for trauma through stretch injury using cultured endothelial brain cells that act as model of the blood brain barrier (BBB) such as what our protocol presents may prove to be useful.
This protocol makes use of cEND cells, an established BBB model in our laboratory. Since BBB breakdown is often documented in TBI patients and TBI is often linked to the disruption of the BBB which can result to edema formation23, 24, the method presented here may specifically be used in conducting BBB studies in relation to TBI.
In this model, it is important to take care of how much degree of stretch injury is applied to the cells. In as much as the cells are injured in any case, and with whatever amount of pressure is applied, the degree of injury that can impact endothelial cells differ much greatly from other cells types. Aortic endothelial cells are more resistant to stretch injury than astrocytes or mixed glial cells9. In addition, they repair more rapidly after injury as compared to the other cell types. Therefore, for brain endothelial cells, particularly cEND cells, greater amount of stretch injury is needed to produce a high degree of injury. One could attain the desired degree of injury by applying the corresponding pressure indicated in Table 1. For cEND cells, however, severe injury is preferred due to their resistance to strain. The LDH assays conducted showed that as the degree of stretch increases, more LDH is secreted into the supernatant. In contrast, the cells which were given a low amount of stretch injury produced LDH in an amount similar to control cells. As mentioned in the protocol, one must take care that the appropriate amount of medium is used since an increase or decrease in the amount of medium may result to differences in the peak pressure applied to the wells. For example, a well containing 5 ml of fluid registers a peak pressure in the average of 4.0 psi while an empty well registers an average of 3.8 psi when 45 psi pressure is applied. Therefore, it is best to push the trigger several times over a control well to ensure that the peak pressure which will be generated corresponds to the desired amount.
In our experiments we used a viability stain to determine the effect of stretch to the permeability of the cell membrane. The optics of the flexible-bottomed culture plates we used enables us to view the stained cells directly under the microscope. However, when one wants to conduct immunolabelling studies directly after stretch-injury, difficulties may arise. First, the size and thickness of the plate may pose a problem with some microscope viewing platforms. Second, the optics of the flexible membrane of the well may be a hindrance to clear viewing.
Despite the aforementioned limitations, however, the described procedure can be used as a model of in vitro mechanical injury of the BBB. Traumatic brain injury (TBI) involves two components, namely, ischemia and trauma. Ischemia can occur as a secondary injury following TBI in instances when there is serious blood loss resulting in low blood pressure or as a result of brain swelling restricting oxygen supply to the brain. It is considered as a delayed, nonmechanical damage representing consecutive pathological processes initiated at the moment of injury25. The occurrence of hypoxia after severe traumatic brain injury is common26. Oxygen glucose deprivation (OGD) is the method currently being used to model ischemia in vitro. Thus, subjecting cells to OGD as a secondary insult to the cells after stretch can mimic the incidence of TBI followed by ischemia. Hence, to improve our current in vitro model of TBI and pattern it as close as possible to an actual TBI as it occurs in vivo, in the future&#160;we will also employ OGD in combination with stretch injury.

Traumatic brain injury (TBI) is one of the leading causes of death worldwide. About 10 million people are affected annually by TBI making it a major health and medical problem1. Due to this, various in vivo and in vitro models of TBI have been established and developed to study its mechanisms2,3,4. A better understanding of TBI can help improve patient treatment and decrease the associated mortality, morbidity, and cost.
Many models for brain injury which utilize both in vivo and in vitro methods exist. In vivo models could mimic the actual event of head injury. However, due to the complexity of the in vivo situation, accessibility to the tissue of interest becomes limited2. In understanding the physiological response of individual cells as a result of the injury inflicted, it is important that the cells are isolated from the systemic effects which may inhibit or alter their individual response5. For this reason, cellular models of trauma provide valuable advantages over animal models since the mechanical environment of the cells can be precisely controlled6.
In vitro systems that employ the use of mechanical load to cells or tissues to determine alterations induced by such method of injury have been developed. For instance, a method for studying the effect of mechanical injury to cells has been established for astrocytes, neurons, glial cells and aortic endothelial cells7,8,9. The in vitro trauma model established for the study of rodent and human astrocyte reactivity10 employed a pressure control device identical to what we use for our model. The same method was applied to induce injury through stretch in mouse brain microvessel endothelial cells (bEnd3)11 and cortical neurons12,13 as well as, cerebral endothelial cells from newborn piglets14.The device deforms the bottom of the culture well thereby producing mechanical stretch injury10. It inflicts injury upon cultured cells by the application of air pressure above the cells. This pressure can deflect the membrane upon which the cells are growing, thereby stretching the cells. Various degrees of stretch (i.e. &#34;low, &#34;moderate,&#34; or &#34;severe&#34;) can be achieved by setting the air pressure pulse duration and intensity accordingly. This method of stretch-induced injury has been correlated with traumatic injury in vivo7. Moreover, this method of injury allows for the precise control of the extracellular environment and can easily be reproduced.
Although a similar approach has been used for many other brain cell types including bEnd3, our model is of an advantage in that it makes use of the murine brain microvascular endothelial cells (cEND) generated in our laboratory. This cell line is a well-suited model of the blood brain barrier (BBB). In vitro cell cultures used as BBB models should possess characteristics that would enable them to serve as permeability screen. One important criterion for an in vitro cell model to be a predictor of BBB permeability is that it should possess physiologically realistic cell architecture15. Even though bEnd3 cells display distinctive spindle-like squamous morphology in culture16, they exhibit irregular morphogenetic behavior in vitro whereby they form cyst-like cavities rather than the regular tubular structures in fibrin gels17. Moreover, when the cells were injected into embryonic and newborn mice, they induced rapidly growing tumors lethal to embryonic mice but not in newborn and young mice. It is thus suggested that one or more processes governing normal endothelial growth, migration, and differentiation have been altered or eliminated in this cell line18. On the other hand, morphological, immunocytochemical evaluation of endothelial and BBB marker expression, bioelectric, and paracellular flux measurements demonstrate that our BBB model cEND is indeed a suitable model of the BBB19.
Brain endothelium in vivo is characterized by an extremely tight permeability with trans-endothelial electrical resistance (TEER) ranging from 2,000-5,000 &#937;cm2. For studies of brain microvasculature barrier properties to pharmaceuticals, paracellular restrictiveness and tightness of the cells should be considered. In most brain capillary endothelial cells (BCEC), this is not preserved as the cells exhibit TEER ranging from 50-100 &#937;cm2 20. The immortalized brain endothelial cell line bEnd3 generates TEER values of no greater than 60 &#937;cm2&#160;15. In contrast differentiation of cEND cells with medium containing reduced serum display TEER values ranging from 300-500 &#937;cm2&#160;19,21.
To date, in vitro models of stretch injury in cultured brain endothelial cells are scarce. Hence, an in vitro model for trauma through stretch injury using cultured brain endothelial cells that act as model of the BBB may prove to be useful. In this protocol, we present an in vitro model that could mimic the actual impact that brain cells, specifically brain microvascular endothelial cells of the BBB, receive during TBI. The main advantage of this model is that the amount of injury applied to the cells as well as the extracellular environment can be easily controlled in a precise manner enabling easy reproducibility of experimental set-up.

<table border="0" cellpadding="0" cellspacing="0" width="1713">
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	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:355px;">Cell Injury Controller II</td>
			<td style="width:351px;">Custom Design and Fabrication, Virginia, USA</td>
			<td style="width:143px;"></td>
			<td style="width:431px;"></td>
			<td style="width:435px;">http://www.radiology.vcu.edu/research/customdesign/cic.html</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;"></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Name of Materials</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Remarks</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Bioflex Culture Plate - Collagen Type I</td>
			<td>Flexcell, Dunn Labortechnik</td>
			<td>BF - 3001C</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:355px;">Dulbecco&#8217;s modified Eagle&#8217;s medium (DMEM)</td>
			<td style="width:351px;">Sigma-Aldrich</td>
			<td style="width:143px;">D5796</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:355px;">Fetal Calf Serum (FCS)</td>
			<td style="width:351px;">PAA Laboratories</td>
			<td style="width:143px;">A15110-1333</td>
			<td style="width:431px;">final concentration 10%, heat-inactivated (30 min at 56 &#176;C)</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:355px;">L-glutamine</td>
			<td style="width:351px;">Biochrom AG</td>
			<td style="width:143px;">K0282</td>
			<td style="width:431px;">Storage: &#8804; -15 &#176;C</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:355px;">MEM Vitamin</td>
			<td style="width:351px;">Biochrom AG</td>
			<td style="width:143px;">K0373</td>
			<td style="width:431px;">Storage: &#8804; -15 &#176;C</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:355px;">Na-pyruvate</td>
			<td style="width:351px;">Biochrom AG</td>
			<td style="width:143px;">L0473</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:355px;">Nonessential amino acids (NEA)</td>
			<td style="width:351px;">Biochrom AG</td>
			<td style="width:143px;">K0293</td>
			<td style="width:431px;">Storage at 4 &#176;C</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:355px;">Penicilin/Streptomycin</td>
			<td style="width:351px;">Biochrom AG</td>
			<td style="width:143px;">A2212</td>
			<td style="width:431px;">Storage: &#8804; -15 &#176;C</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:355px;">Fetal Calf Serum, charcoal stripped (ssFCS)</td>
			<td style="width:351px;">Life Technologies</td>
			<td>12676-011</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:355px;">Trypsin-EDTA solution</td>
			<td style="width:351px;">PAA Laboratories</td>
			<td>L11-004</td>
			<td style="width:431px;">Storage: &#8804; -15 &#176;C</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Image-iT DEAD Green</td>
			<td style="width:351px;">Life Technologies</td>
			<td>I10291</td>
			<td style="width:431px;">Storage: &#8804; -15 &#176;C, protected from light</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Cytotoxicity Detection Kit PLUS (LDH)</td>
			<td>Roche</td>
			<td>4744926001</td>
			<td style="width:431px;">Storage: &#8804; -15 &#176;C</td>
			<td></td>
		</tr>
	</tbody>
</table>

stretch in brain microvascular endothelial cells (cend) as an in vitro traumatic brain injury model of the blood brain barrier

Due to the high mortality incident brought about by traumatic brain injury (TBI), methods that would enable one to better understand the underlying mechanisms involved in it are useful for treatment. There are both in vivo and in vitro methods available for this purpose. In vivo models can mimic actual head injury as it occurs during TBI. However, in vivo techniques may not be exploited for studies at the cell physiology level. Hence, in vitro methods are more advantageous for this purpose since they provide easier access to the cells and the extracellular environment for manipulation.
Our protocol presents an in vitro model of TBI using stretch injury in brain microvascular endothelial cells. It utilizes pressure applied to the cells cultured in flexible-bottomed wells. The pressure applied may easily be controlled and can produce injury that ranges from low to severe. The murine brain microvascular endothelial cells (cEND) generated in our laboratory is a well-suited model for the blood brain barrier (BBB) thus providing an advantage to other systems that employ a similar technique. In addition, due to the simplicity of the method, experimental set-ups are easily duplicated. Thus, this model can be used in studying the cellular and molecular mechanisms involved in TBI at the BBB.

Cells cultured on collagen I precoated 6-well flexible bottomed culture plates (57.75 cm2) were subjected to various degrees of stretch injury using the cell stretcher device. After subjecting the cells to injury, they were examined under the microscope for the effects of stretch-induced injury to cell morphology. It was observed that as greater degree of stretch was applied to the cells a greater degree of cell distortion could also be observed (Figure 1). As shown in Figure 1A, control cells which were not subjected to injury appear as regularly shaped cerebrovascular endothelial cells (cEND) without any indication of cell swelling or distortion. When stretch injury was applied (Figures 1B-D), deformation could be observed under the light microscope. After stretching the cells severely with a peak pressure between 3.5-4.5 psi, the cEND cells appeared markedly retracted, swollen and deformed with notable intercellular spaces. In addition, uptake of viability stain (100 nM final concentration) also increased as the degree of stretch injury was increased (Figure 2). The viability stain used is a dye impermeant to healthy cells that becomes permeant when the plasma membrane integrity of cells is compromised. The dye was excluded from most of the control cells, hence, only a few of the cells were stained (Figure 2A) as compared to stretched cells (Figures 2B-D). More cells fluoresced green with an increased degree of stretch injury.
As a biochemical marker of injury, release of lactate dehydrogenase (LDH) enzyme was also examined according to manufacturer&#39;s instructions. Figure 3 shows that an increasing cell stretch injury caused increasing LDH release.
<img alt="Figure 1" fo:content-width="5in" src="/files/ftp_upload/50928/50928fig1.jpg" width="500px" /> 
Figure 1. Light microscopy examination of normal vs. injured cells.&#160;(A) Normal unstretched confluent cell monolayer istightly packed and elongated. When cells were stretched by applying a peak pressure pulse of 1.8-2.0 psi (i.e. low stretch) they appear less compact, spaces indicated by arrows (B). When the cells were moderately injured with a 2.5-3.0 peak pressure pulse, some of them appeared swollen and deformed (C). The cells become retracted with severe stretch of 3.5-4.5 psi, as indicated by arrow (D). 100X magnification. <a href="https://www.jove.com/files/ftp_upload/50928/50928fig1highres.jpg" target="_blank">Click here to view larger image</a>.
<img alt="Figure 2" fo:content-width="5in" src="/files/ftp_upload/50928/50928fig2.jpg" width="500px" /> 
Figure 2. Fluorescence microscopic examination of normal vs. injured cells.Cells treated with viability stain 2hr after injury. A: control unstretched cells. B-D: stretched cells (B - low, C - moderate, D - severe). 100X magnification. <a href="https://www.jove.com/files/ftp_upload/50928/50928fig2highres.jpg" target="_blank">Click here to view larger image</a>.
<img alt="Figure 3" fo:content-width="5in" src="/files/ftp_upload/50928/50928fig3.jpg" width="500px" /> 
Figure 3. Lactate dehydrogenase (LDH) enzyme release into the supernatant after stretch injury. LDH released into the culture medium was measured at various time intervals after stretch induced injury. LDH was expressed as a percent of the total releasable LDH (LDH in media plus cells). Values are &#177; SEM. The n for every time point is 5, except for the 0 hr value subjected to severe stretch where n = 3. LDH release from cells that were subjected to low and moderate stretch did not differ significantly from that of unstretched controls and from each other. Cells that were severely stretched released a significantly greater amount of LDH as compared to all other samples, except for the moderately stretched sample at 1 hr. (p &#60; 0.05, One factor ANOVA, Holm-Sidak method). <a href="https://www.jove.com/files/ftp_upload/50928/50928fig3highres.jpg" target="_blank">Click here to view larger image</a>.
Table 1. Guide for generating various degrees of stretch injury.
<table border="1">
	<tbody>
		<tr>
			<th>Regulator Pressure</th>
			<th>Peak Pressure</th>
			<th>Degree of Injury</th>
		</tr>
		<tr>
			<td>15 psi</td>
			<td>1.2-1.5 psi</td>
			<td>&#60; Low</td>
		</tr>
		<tr>
			<td>20-25 psi</td>
			<td>1.8-2.0 psi</td>
			<td>Low</td>
		</tr>
		<tr>
			<td>30-35 psi</td>
			<td>2.5-3.0 psi</td>
			<td>Moderate</td>
		</tr>
		<tr>
			<td>40-50 psi</td>
			<td>3.5-4.5 psi</td>
			<td>Severe</td>
		</tr>
		<tr>
			<td>60 psi</td>
			<td>4.8-6.0 psi</td>
			<td>&#62; Severe</td>
		</tr>
	</tbody>
</table>

Watch this Scientific Journal Video about Stretch in Brain Microvascular Endothelial Cells cEND as an In Vitro Traumatic Brain Injury Model of the Blood Brain Barrier at JoVE.com

Stretch in Brain Microvascular Endothelial Cells cEND as an In Vitro Traumatic Brain Injury Model of the Blood Brain Barrier

In vitro traumatic brain injury models are being developed to reproduce in vivo brain deformation. Stretch-induced injury has been employed for astrocytes, neurons, glial cells, aortic, and brain endothelial cells. However, our system uses a blood brain barrier (BBB) model that possesses properties constituting a legitimate model of the BBB to establish an in vitro&#160;TBI model.

Stretch in Brain Microvascular Endothelial Cells (cEND) as an In Vitro Traumatic Brain Injury Model of the Blood Brain Barrier

Due to the high mortality incident brought about by traumatic brain injury (TBI), methods that would enable one to better understand the underlying ...

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14.8K Views.  Zentrum für operative Medizin der Universität Würzburg. In vitro traumatic brain injury models are being developed to reproduce in vivo brain deformation. Stretch-induced injury has been employed for astrocytes, neurons, glial cells, aortic, and brain endothelial cells. However, our system uses a blood brain barrier (BBB) model that possesses properties constituting a legitimate model of the BBB to establish an in vitro TBI model.

Video: Stretch in Brain Microvascular Endothelial Cells cEND as an In Vitro Traumatic Brain Injury Model of the Blood Brain Barrier

MRI-guided Disruption of the Blood-brain Barrier using Transcranial Focused Ultrasound in a Rat Model

Focused ultrasound (FUS) disruption of the blood-brain barrier (BBB) is an increasingly investigated technique for circumventing the BBB1-5. The BBB is a significant obstacle to pharmaceutical treatments of brain disorders as it limits the passage of molecules from the vasculature into the brain tissue to molecules less than approximately 500 Da in size6. FUS induced BBB disruption (BBBD) is temporary and reversible4 and has an advantage over chemical means of inducing BBBD by being highly localized. FUS induced BBBD provides a means for investigating the effects of a wide range of therapeutic agents on the brain, which would not otherwise be deliverable to the tissue in sufficient concentration. While a wide range of ultrasound parameters have proven successful at disrupting the BBB2,5,7, there are several critical steps in the experimental procedure to ensure successful disruption with accurate targeting. This protocol outlines how to achieve MRI-guided FUS induced BBBD in a rat model, with a focus on the critical animal preparation and microbubble handling steps of the experiment.

Microbubble-mediated focused ultrasound disruption of the blood-brain barrier (BBB) is a promising technique for non-invasive targeted drug delivery in the brain1-3. This protocol outlines the experimental procedure for MRI-guided transcranial BBB disruption in a rat model.

Focused ultrasound (FUS) disruption of the blood-brain barrier (BBB) is an increasingly investigated technique for circumventing the BBB1-5. The BBB is a ...

Rat Model of Blood-brain Barrier Disruption to Allow Targeted Neurovascular Therapeutics

Endothelial cells with tight junctions along with the basement membrane and astrocyte end feet surround cerebral blood vessels to form the blood-brain barrier1. The barrier selectively excludes molecules from crossing between the blood and the brain based upon their size and charge. This function can impede the delivery of therapeutics for neurological disorders. A number of chemotherapeutic drugs, for example, will not effectively cross the blood-brain barrier to reach tumor cells2. Thus, improving the delivery of drugs across the blood-brain barrier is an area of interest.
The most prevalent methods for enhancing the delivery of drugs to the brain are direct cerebral infusion and blood-brain barrier disruption3. Direct intracerebral infusion guarantees that therapies reach the brain; however, this method has a limited ability to disperse the drug4. Blood-brain barrier disruption (BBBD) allows drugs to flow directly from the circulatory system into the brain and thus more effectively reach dispersed tumor cells. Three methods of barrier disruption include osmotic barrier disruption, pharmacological barrier disruption, and focused ultrasound with microbubbles. Osmotic disruption, pioneered by Neuwelt, uses a hypertonic solution of 25% mannitol that dehydrates the cells of the blood-brain barrier causing them to shrink and disrupt their tight junctions. Barrier disruption can also be accomplished pharmacologically with vasoactive compounds such as histamine5 and bradykinin6. This method, however, is selective primarily for the brain-tumor barrier7. Additionally, RMP-7, an analog of the peptide bradykinin, was found to be inferior when compared head-to-head with osmotic BBBD with 25% mannitol8. Another method, focused ultrasound (FUS) in conjunction with microbubble ultrasound contrast agents, has also been shown to reversibly open the blood-brain barrier9. In comparison to FUS, though, 25% mannitol has a longer history of safety in human patients that makes it a proven tool for translational research10-12.
In order to accomplish BBBD, mannitol must be delivered at a high rate directly into the brain's arterial circulation. In humans, an endovascular catheter is guided to the brain where rapid, direct flow can be accomplished. This protocol models human BBBD as closely as possible. Following a cut-down to the bifurcation of the common carotid artery, a catheter is inserted retrograde into the ECA and used to deliver mannitol directly into the internal carotid artery (ICA) circulation. Propofol and N2O anesthesia are used for their ability to maximize the effectiveness of barrier disruption13. If executed properly, this procedure has the ability to safely, effectively, and reversibly open the blood-brain barrier and improve the delivery of drugs that do not ordinarily reach the brain 8,13,14.

Blood-brain barrier disruption aids the delivery of certain drugs to the brain. Mannitol delivered intra-arterially shrinks cells surrounding blood vessels in order to physically disrupt the barrier.

Endothelial cells with tight junctions along with the basement membrane and astrocyte end feet surround cerebral blood vessels to form the blood-brain ...

Setting-up an In Vitro Model of Rat Blood-brain Barrier (BBB): A Focus on BBB Impermeability and Receptor-mediated Transport

The blood brain barrier (BBB) specifically regulates molecular and cellular flux between the blood and the nervous tissue. Our aim was to develop and characterize a highly reproducible rat syngeneic in vitro model of the BBB using co-cultures of primary rat brain endothelial cells (RBEC) and astrocytes to study receptors involved in transcytosis across the endothelial cell monolayer. Astrocytes were isolated by mechanical dissection following trypsin digestion and were frozen for later co-culture. RBEC were isolated from 5-week-old rat cortices. The brains were cleaned of meninges and white matter, and mechanically dissociated following enzymatic digestion. Thereafter, the tissue homogenate was centrifuged in bovine serum albumin to separate vessel fragments from nervous tissue. The vessel fragments underwent a second enzymatic digestion to free endothelial cells from their extracellular matrix. The remaining contaminating cells such as pericytes were further eliminated by plating the microvessel fragments in puromycin-containing medium. They were then passaged onto filters for co-culture with astrocytes grown on the bottom of the wells. RBEC expressed high levels of tight junction (TJ) proteins such as occludin, claudin-5 and ZO-1 with a typical localization at the cell borders. The transendothelial electrical resistance (TEER) of brain endothelial monolayers, indicating the tightness of TJs reached 300 ohm&#183;cm2 on average. The endothelial permeability coefficients (Pe) for lucifer yellow (LY) was highly reproducible with an average of 0.26 &#177;&#160;0.11 x 10-3 cm/min. Brain endothelial cells organized in monolayers expressed the efflux transporter P-glycoprotein (P-gp), showed a polarized transport of rhodamine 123, a ligand for P-gp, and showed specific transport of transferrin-Cy3 and DiILDL across the endothelial cell monolayer. In conclusion, we provide a protocol for setting up an in vitro BBB model that is highly reproducible due to the quality assurance methods, and that is suitable for research on BBB transporters and receptors.

Setting-up an In Vitro Model of Rat Blood-brain Barrier BBB: A Focus on BBB Impermeability and Receptor-mediated Transport

The aim of the present study was to validate the reproducibility of an in vitro BBB model involving a rat syngeneic co-culture of endothelial cells and astrocytes. The endothelial cell monolayer presented high TEER and low LY permeability. Expression of specific TJ proteins, functional responses to inflammation and functionality of transporters and receptors were assessed.

The blood brain barrier (BBB) specifically regulates molecular and cellular flux between the blood and the nervous tissue. Our aim was to develop and ...

Excitotoxic Stimulation of Brain Microslices as an In vitro Model of Stroke

Examining molecular mechanisms involved in neuropathological conditions, such as ischemic stroke, can be difficult when using whole animal systems. As such, primary or &#39;neuronal-like&#39; cell culture systems are commonly utilized. While&#160;these systems are relatively easy to work with, and are useful model systems in which various functional outcomes (such as cell death) can be readily quantified, the examined outcomes and pathways in cultured immature neurons (such as excitotoxicity-mediated cell death pathways) are not necessarily the same as those observed in mature brain, or in intact tissue. Therefore, there is the need to develop models in which cellular mechanisms in mature neural tissue can be examined. We have developed an in vitro technique that can be used to investigate a variety of molecular pathways in intact nervous tissue. The technique described herein utilizes rat cortical tissue, but this technique can be adapted to use tissue from a variety of species (such as mouse, rabbit, guinea pig, and chicken) or brain regions (for example, hippocampus, striatum, etc.). Additionally, a variety of stimulations/treatments can be used (for example, excitotoxic, administration of inhibitors, etc.). In conclusion, the brain slice model described herein can be used to examine a variety of molecular mechanisms involved in excitotoxicity-mediated brain injury.

Excitotoxic Stimulation of Brain Microslices as an In vitro Model of Stroke

We have developed a brain slice model which can be used to examine molecular mechanisms involved in excitotoxicity-mediated brain injury.&#160;This technique generates viable mature brain tissue and reduces animal numbers required for experimentation, whilst keeping the neuronal circuitry, cellular interactions, and postsynaptic compartments partly intact.

Examining molecular mechanisms involved in neuropathological conditions, such as ischemic stroke, can be difficult when using whole animal systems. As ...

Controlled Cortical Impact Model for Traumatic Brain Injury

Every year over a million Americans suffer a traumatic brain injury (TBI). Combined with the incidence of TBIs worldwide, the physical, emotional, social, and economical effects are staggering. Therefore, further research into the effects of TBI and effective treatments is necessary. The controlled cortical impact (CCI) model induces traumatic brain injuries ranging from mild to severe. This method uses a rigid impactor to deliver mechanical energy to an intact dura exposed following a craniectomy. Impact is made under precise parameters at a set velocity to achieve a pre-determined deformation depth. Although other TBI models, such as weight drop and fluid percussion, exist, CCI is more accurate, easier to control, and most importantly, produces traumatic brain injuries similar to those seen in humans. However, no TBI model is currently able to reproduce pathological changes identical to those seen in human patients. The CCI model allows investigation into the short-term and long-term effects of TBI, such as neuronal death, memory deficits, and cerebral edema, as well as potential therapeutic treatments for TBI.

Traumatic brain injuries (TBIs) remain a serious health problem. Using the controlled cortical impact surgery model, research on the effects of TBI and possible treatment methods may be performed.

Every year over a million Americans suffer a traumatic brain injury (TBI).  Combined with the incidence of TBIs worldwide, the physical, emotional, ...

A Novel Model of Mild Traumatic Brain Injury for Juvenile Rats

Despite growing evidence that childhood represents a major risk period for mild traumatic brain injury (mTBI) from sports-related concussions, motor vehicle accidents, and falls, a reliable animal model of mTBI had previously not been developed for this important aspect of development. The modified weight-drop technique employs a glancing impact to the head of a freely moving rodent transmitting acceleration, deceleration, and rotational forces upon the brain. When applied to juvenile rats, this modified weight-drop technique induced clinically relevant behavioural outcomes that were representative of post-concussion symptomology. The technique is a rapidly applied procedure with an extremely low mortality rate, rendering it ideal for high-throughput studies of therapeutics. In addition, because the procedure involves a mild injury to a closed head, it can easily be used for studies of repetitive brain injury. Owing to the simplistic nature of this technique, and the clinically relevant biomechanics of the injury pathophysiology, the modified weight-drop technique provides researchers with a reliable model of mTBI that can be used in a wide variety of behavioural, molecular, and genetic studies.

The modified weight-drop technique is an easy, cost-effective procedure used for the induction of mild traumatic brain injury in juvenile rats. This novel technique produces clinically relevant symptomology that will advance the study of mild traumatic brain injury (mTBI) and concussion.

Despite growing evidence that childhood represents a major risk period for mild traumatic brain injury (mTBI) from sports-related concussions, motor ...

Oxygen-Glucose Deprivation and Reoxygenation as an In Vitro Ischemia-Reperfusion Injury Model for Studying Blood-Brain Barrier Dysfunction

Ischemia-Reperfusion (IR) injury is known to contribute significantly to the morbidity and mortality associated with ischemic strokes. Ischemic cerebrovascular accidents account for 80% of all strokes. A common cause of IR injury is the rapid inflow of fluids following an acute/chronic occlusion of blood, nutrients, oxygen to the tissue triggering the formation of free radicals.
Ischemic stroke is followed by blood-brain barrier (BBB) dysfunction and vasogenic brain edema. Structurally, tight junctions (TJs) between the endothelial cells play an important role in maintaining the integrity of the blood-brain barrier (BBB). IR injury is an early secondary injury leading to a non-specific, inflammatory response. Oxidative and metabolic stress following inflammation triggers secondary brain damage including BBB permeability and disruption of tight junction (TJ) integrity.
Our protocol presents an in vitro example of oxygen-glucose deprivation and reoxygenation (OGD-R) on rat brain endothelial cell TJ integrity and stress fiber formation. Currently, several experimental in vivo models are used to study the effects of IR injury; however they have several limitations, such as the technical challenges in performing surgeries, gene dependent molecular influences and difficulty in studying mechanistic relationships. However, in vitro models may aid in overcoming many of those limitations. The presented protocol can be used to study the various molecular mechanisms and mechanistic relationships to provide potential therapeutic strategies. However, the results of in vitro studies may differ from standard in vivo studies and should be interpreted with caution.

Oxygen-Glucose Deprivation and Reoxygenation as an In Vitro Ischemia-Reperfusion Injury Model for Studying Blood-Brain Barrier Dysfunction

Ischemia-Reperfusion (IR) injury is associated with a high rate of morbidity and mortality. The goal of the in vitro model of oxygen-glucose deprivation and reoxygenation (OGD-R) described here is to assess the effects of ischemia reperfusion injury on a variety of cells, particularly in blood-brain barrier (BBB) endothelial cells.

Ischemia-Reperfusion (IR) injury is known to contribute significantly to the morbidity and mortality associated with ischemic strokes. Ischemic ...

A Mouse Model of Single and Repetitive Mild Traumatic Brain Injury

Mild Traumatic Brain Injury (mTBI) can result in the acute loss of brain function, including a period of confusion, a loss of consciousness (LOC), focal neurological deficits and even amnesia. Athletes participating in contact sports are at high risk of exposure to large number of mTBIs. In terms of the level of injury in a sporting athlete, a mTBI is defined as a mild injury that does not cause gross pathological changes, but does cause short-term neurological deficits that are spontaneously resolved. Despite previous attempts to model mTBI in mice and rats, many have reported gross adverse effects including skull fractures, intracerebral bleeding, axonal injury and neuronal cell death. Herein, we describe our highly reproducible animal model of mTBI that reproduces clinically relevant symptoms. This model uses a custom made pneumatic impactor device to deliver a closed-head trauma. This impact is made under precise velocity and deformation parameters, creating a reliable and reproducible model to examine the mechanisms that contribute to effects of single or repetitive concussive mTBI.

Athletes absorb several hundred mild traumatic brain injuries (mTBI)/concussions every year; however, the consequence of these on the brain is poorly understood. Therefore, an animal model of single and repetitive mTBI that consistently replicates clinically relevant symptoms provides the means to advance the study of mTBI and concussion.

Mild Traumatic Brain Injury (mTBI) can result in the acute loss of brain function, including a period of confusion, a loss of consciousness (LOC), focal ...

In Vitro Assays to Assess Blood-brain Barrier Mesh-like Vessel Formation and Disruption

Blood-brain barrier (BBB) coverage plays a central role in the homeostasis of the central nervous system (CNS). The BBB is dynamically maintained by astrocytes, pericytes and brain endothelial cells (BECs). Here, we detail methods to assess BBB coverage using single cultures of immortalized human BECs, single cultures of primary mouse BECs, and a humanized triple culture model (BECs, astrocytes and pericytes) of the BBB. To highlight the applicability of the assays to disease states, we describe the effect of oligomeric amyloid-&#946; (oA&#946;), which is an important contributor to Alzheimer&#39;s disease (AD) progression, on BBB coverage. Further, we utilize the epidermal growth factor (EGF) to illuminate the drug screening potential of the techniques. Our results show that single and triple cultured BECs form meshwork-like structures under basal conditions, and that oA&#946; disrupts this cell meshwork formation and degenerates the preformed mesh structures, but EGF blocks this disruption. Thus, the techniques described are important for dissecting fundamental and disease-relevant processes that modulate BBB coverage.

In Vitro Assays to Assess Blood-brain Barrier Mesh-like Vessel Formation and Disruption

Maintaining blood-brain barrier coverage is key for the homeostasis of the central nervous system. This protocol describes in vitro techniques to delineate the fundamental and pathological processes that modulate blood-brain barrier coverage.

Blood-brain barrier (BBB) coverage plays a central role in the homeostasis of the central nervous system (CNS). The BBB is dynamically maintained by ...

Improved Method for the Establishment of an In Vitro Blood-Brain Barrier Model Based on Porcine Brain Endothelial Cells

The aim of this protocol presents an optimized procedure for the purification and cultivation of pBECs and to establish in vitro blood-brain barrier (BBB) models based on pBECs in mono-culture (MC), MC with astrocyte-conditioned medium (ACM), and non-contact co-culture (NCC) with astrocytes of porcine or rat origin. pBECs were isolated and cultured from fragments of capillaries from the brain cortices of domestic pigs 5-6 months old. These fragments were purified by careful removal of meninges, isolation and homogenization of grey matter, filtration, enzymatic digestion, and centrifugation. To further eliminate contaminating cells, the capillary fragments were cultured with puromycin-containing medium. When 60-95% confluent, pBECs growing from the capillary fragments were passaged to permeable membrane filter inserts and established in the models. To increase barrier tightness and BBB characteristic phenotype of pBECs, the cells were treated with the following differentiation factors: membrane permeant 8-CPT-cAMP (here abbreviated cAMP), hydrocortisone, and a phosphodiesterase inhibitor, RO-20-1724 (RO). The procedure was carried out over a period of 9-11 days, and when establishing the NCC model, the astrocytes were cultured 2-8 weeks in advance. Adherence to the described procedures in the protocol has allowed the establishment of endothelial layers with highly restricted paracellular permeability, with the NCC model showing an average transendothelial electrical resistance (TEER) of 1249 &#177; 80 &#937; cm2, and paracellular permeability (Papp) for Lucifer Yellow of 0.90 10-6 &#177; 0.13 10-6 cm sec-1 (mean &#177; SEM, n=55). Further evaluation of this pBEC phenotype showed good expression of the tight junctional proteins claudin 5, ZO-1, occludin and adherens junction protein p120 catenin. The model presented can be used for a range of studies of the BBB in health and disease and, with the highly restrictive paracellular permeability, this model is suitable for studies of transport and intracellular trafficking.

Improved Method for the Establishment of an In Vitro Blood-Brain Barrier Model Based on Porcine Brain Endothelial Cells

The aim of the protocol is to present an optimized procedure for the establishment of an in vitro blood-brain barrier (BBB) model based on primary porcine brain endothelial cells (pBECs). The model shows high reproducibility, high tightness, and is suitable for studies of transport and intracellular trafficking in drug discovery.

The aim of this protocol presents an optimized procedure for the purification and cultivation of pBECs and to establish in vitro blood-brain barrier (BBB) ...