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In addition to assessing tight junction formation as described above, permeability of tracers such as dextran can be used to quantify the tightness of the barrier formed by RBEC in B 3 C model. Using the B 3 C model we observed that barrier permeability was dependent on the presence of astrocytes or ACM. Significant reduction of passage of Texas Red 40 kDa dextran from the vascular channel to tissue compartment was observed when RBEC from passage 3 were cultured under flow 0.

Flow rate used for permeability experiments was 0. Representative images acquired 60 min after the initiation of flow in the vascular channel A-D. Coefficient of variance of measured intensities of fluorescent dextran at 12 regularly spaced ROIs in the tissue compartment immediately adjacent to the vascular channel at 60 min after the initiation of the flow was used as an index of permeability heterogeneity. Variation in permeability is lowest in cell-free B 3 C but increases as the microenvironment of B 3 C becomes more realistic F.

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In the vasculature of the brain and other tissues in vivo , permeability is reported to be heterogeneous along the length of a vessel [ 35 — 37 ]. Coefficient of variance for the measured intensities of fluorescent dextran at 12 regularly spaced regions of interest ROIs in the tissue compartment immediately adjacent to the vascular channel at 60 min after the initiation of the flow was used to assess the heterogeneity of permeability along the vascular channel in B 3 C Fig 5F. As expected, variability in permeation was lowest in cell-free B 3 C due to the uniform structure of the wall of the vascular channels.

However, while the overall permeability of B 3 C decreases with the inclusion of ACM or astrocytes in its microenvironment Fig 5E , the extravasation of dextran becomes more heterogeneous along the vascular channel Fig 5F. Note that the pore design in B 3 C and the methodology and instrumentation used for measuring electrical resistance in B 3 C are novel and significantly different from that of transwell. For example, surface area and the pore densities are significantly different between B 3 C and transwell.

The effect of ACM on RBEC barrier formation in these two systems were compared by calculating percent increase in their respective electrical resistances.

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Please note that the units of electrical resistance for B 3 C and transwell are different as noted in the results section. Consistent with our observations in B 3 C, the permeability of 40 kDa dextran in transwell model decreases from Nevertheless, as shown in Fig 7 , permeability of 40 kDa dextran in B 3 C 1. Combined, these results indicate that the microenvironment in B 3 C closely approximates the neonatal in vivo BBB.

Permeability of 40 kDa dextran in B 3 C is significantly lower than transwell but not significantly different from that of in vivo BBB in neonatal rats. Inset panels show higher magnification of white squared regions. In vitro BBB models that closely mimic the in vivo BBB microenvironment are valuable tools for the study of neonatal BBB function as well as for screening of novel therapeutics.

Transwell BBB models approximate several of the important aspects of the in vivo BBB and are routinely used for BBB studies and for screening neurotherapeutics [ 38 , 39 ]. More recently, dynamic flow-based in vitro BBB models have also been developed to better reproduce the in vivo microvascular environment [ 17 , 18 , 40 , 41 ].

However, existing in vitro BBB models have a number of important limitations and for the most part do not mimic the microenvironment of the neonatal BBB. Furthermore, development of suitable in vitro BBB models of neonatal BBB is particularly important since there are significant differences in structure and function of neonatal and adult BBB [ 7 , 26 — 28 ]. Having a relevant in vitro model of neonatal BBB is important for understanding neonatal neural pathogenesis and for developing appropriate treatment strategies.

This study demonstrates, for the first time, an in vitro neonatal BBB model that shows a significant improvement from the traditional transwell model and is able to mimic the in vivo brain microenvironmental conditions e. In order to develop and characterize an in vitro model of neonatal BBB, we designed a neonatal BBB on a chip B 3 C model for the co-culture of primary neonatal brain endothelial cells and neonatal astrocytes.

In contrast to other microfluidic systems, the B 3 C design includes a tissue compartment enclosed by vascular channels. The three-dimensional geometry of vascular channels in B 3 C addresses the important challenge of developing in vitro BBB models with shear flow conditions which have been shown to be critical in the formation of realistically tight barriers [ 17 , 21 ]. Furthermore, the optically clear microfluidic chip, and the architecture of the device, allows for visualization and real-time measurements of the dynamic interactions occurring in vascular channels and tissue compartment.

As shown in Fig 3 , neonatal rat brain microvascular endothelial cells RBEC , grown on the fibronectin coated inner surfaces of vascular channels of the B 3 C form the endothelial lining along vascular channels with a complete lumen, thus mimicking the tubular morphology of the in vivo microvessels. The ability to reproduce the tubular morphology of the in vivo microvessels represents a significant advancement in our efforts to model the in vivo BBB.

Furthermore, the ability to visualize the dynamic processes in the neonatal B 3 C model in real time is a significant advantage as compared to flow based hollow fiber dynamic BBB models and other membrane based microfluidic approaches [ 17 , 18 , 24 , 25 , 40 , 41 ]. Thus, B 3 C offers a far more realistic representation of the in vivo BBB microenvironment compared to currently available in vitro BBB models [ 17 — 23 , 40 , 41 ].


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In addition to allowing for real-time analysis in an in vivo like microenvironment, the B 3 C has the advantage of using significantly less of the often required expensive reagents compared to the currently available in vitro BBB models. The endothelium of the in vivo BBB is continuously exposed to both physical stimuli e. Certain features of the BBB, for example the size and geometry of the microvessels in the brain, define the shear stress and flow patterns occurring at the in vivo BBB and contribute to the biochemical and functional characteristics of the BBB [ 1 , 17 , 21 ]. Thus, for a truly functional and physiologically realistic in vitro BBB, it is essential to incorporate the in vivo flow characteristics in the design of an in vitro BBB model.

Accordingly, the neonatal B 3 C model realistically reproduces in vivo shear forces experienced by endothelial cells. Moreover, the neonatal B 3 C model was developed with a co-culture of astrocytes, which are known to contribute to the regulation and maintenance of BBB homeostasis, integrity and function [ 1 , 18 , 42 — 47 ].

Blood Brain Barrier

Nevertheless, additional studies evaluating the expression of other barrier tightening proteins such as occludin and claudin-5 may be required to fully characterize the barrier structure in B 3 C. In B 3 C, we occasionally observe that a few endothelial cells can cross the pores only during the first 1—2 days of culture S1 Fig , but this is generally prevented by the physical hindrance of the longer migration path in B 3 C.

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Once the endothelial cells reach confluence, no additional migration of endothelial cells through the pores is observed. Thus, the extent of this migration is significantly lower compared to that seen by Wuest et al. Furthermore, with the presence of astrocytes in the tissue compartment of B 3 C, the endothelial migration from vascular channel to the tissue compartment is further reduced as can be seen in Fig 4E and 4F also S2 Fig.

Growing evidence suggests that flow based in vitro BBB models exhibit improved performance and better approximate the in vivo BBB as compared to transwell based BBB models [ 17 , 18 , 21 — 23 , 40 , 41 ]. Consistent with these observations, the permeability of our microfluidic neonatal blood-brain barrier B 3 C model was found to be significantly lower than that of the transwell BBB model Fig 7.

Furthermore, as shown in Fig 7 , the permeability of B 3 C model approximates the permeability of neonatal rat BBB in vivo supporting the notion that this system mimics many structural and functional characteristics of the in vivo BBB system. In summary, we developed a dynamic neonatal BBB on a chip B 3 C model incorporating co-culture of neonatal rat brain endothelial cells and astrocytes. The design of B 3 C not only allows for culturing of neonatal brain endothelial cells under shear flow in three-dimensional vascular channels that mimic the dimensions of microvessels in vivo , but also permits interactions between the endothelial cells and brain cells.

The side-by-side placement of vascular channels and the tissue compartment in optically clear PDMS on glass chip allows for real-time direct monitoring of the dynamic processes taking place in B 3 C. The microfluidic based neonatal BBB on a chip model developed in this study is a new class of in vitro BBB models that closely reproduces the properties of the in vivo BBB and delivers enhanced performance as compared with traditional transwell system.

In F, an occasional RBEC migrated from the vascular channel to tissue compartment is shown by an arrow; RBECs do not form a second monolayer on the other tissue compartment side of the porous interface. Only a few pores show some RBEC migration from the vascular channel to the tissue compartment blue nuclear staining seen inside the pores. Nuclear counterstaining is shown in D and the four channels from A-D were merged as shown in E. Department of Energy.

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Browse Subject Areas? Click through the PLOS taxonomy to find articles in your field. Abstract Studies of neonatal neural pathologies and development of appropriate therapeutics are hampered by a lack of relevant in vitro models of neonatal blood-brain barrier BBB. This is an open access article distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited Data Availability: All relevant data are within the paper and its Supporting Information files.

Introduction Blood-brain barrier BBB is a physical and functional barrier formed by the brain vascular endothelial cells and perivascular cells [ 1 , 2 ]. Antibodies Rabbit polyclonal anti-ZO-1 antibody Cat. Fabrication of B 3 C To fabricate the microfluidic neonatal BBB on a chip B 3 C , a photomask of the design shown in Fig 1A was created and soft photolithography was used to fabricate the final B 3 C model shown in Fig 1C on a microscope slide as described previously [ 32 ].

Frontiers | The blood-brain barrier: an engineering perspective | Frontiers in Neuroengineering

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Schematic illustration and images of neonatal blood-brain barrier on a chip B 3 C. Cell Cultures All experiments involving animals were carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. Fig 2. Passage of fluorescent dextran from the vascular channel to tissue compartment of B 3 C under shear flow. Fig 3. Fig 4. Tight junction formation by neonatal RBEC under static and flow conditions as indicated by immunofluorescence staining of ZO Fig 5.

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Real-time analysis of passage of fluorescent dextran from the vascular to tissue compartment of B 3 C under shear flow. Fig 6. Fig 7.


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B 3 C exhibits significantly improved barrier function compared to the transwell model and closely approximates the permeability of neonatal in vivo BBB. Fig 8.