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What is the BBB?

The blood brain barrier (BBB) is defined as a "highly selective semipermeable border separating circulating blood from brain extracellular fluid in central nervous system (CNS, brain and spine)" with a multitude of functions that are crucial to maintaining homeostasis in the CNS [D1]. The BBB regulates ion concentration with a variety of ion transporters and channels which controls the electrolyte balance and water content. Specifically, concentrations K+, Ca2+, and Mg2+ as well as pH are tightly regulated. It also acts to separate the CNS from periphery nervous system (PNS, nerves) as well as the blood. This ensures that neurotransmitters in the PNS and neuroactive agents like the amino acid glutamate in the blood do not excite the CNS, preventing "cross talk" [D2]. The BBB protects against serum macromolecules like albumin, pro-thrombin and plasminogen which can damage nervous tissue and activate cell death or apoptosis, as well as various toxins, pathogens, inflammation, injury, and disease [D2 - D4]. Additionally, it provides nutrients and excretes waste of the various cell types in the brain as well as the BBB [D2]. Structurally, the BBN surrounds the capillaries supplying blood to CNS and is composed of a few main cell types (Fig D1). Cerebral endothelial cells form the inner wall of capillary regulate which are connected to one another with tight junctions that regulate the paracellular diffusion or diffusion between the cells. Pericytes surround these endothelial cells are are sporadically dispersed along the capillary. These two cell types excrete various extra cellular matrix proteins to form basal lamina. In addition to these cells, there are astrocytes with end feet that surround the capillary as well as neurons connected to smooth muscle cells that maintain and regulate the BBB. Finally, microglial cells are also present and are the immune cells of the brain [D2].

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Fig D1. Structure and cell types of the Blood Brain Barrier

BBB:Tight Junctions

One of the key components of the BBB are the tight junctions (TJ) located at the margin of the endothelial cells (Fig D2). These tight junctions seal the BBB and form a continuous impermeable barrier to macromolecules and most polar solutes. As mentioned this limits diffusion of various molecules, from blood plasma to the brain extracellular fluid, between the endothelial cells. This results in a high transendothelial electrical resistance as ions are not free to move across the barrier. The TJs are comprised of various transmembrane proteins including occludin and claudins, and junctional adhesion molecules (JAMs) which interact with cytoskeleton and other regulatory proteins within the cell. Adjacent to these TJ, Adhesion junctions mainly comprised of cadherin and catenins are found and provide structural support to the tissue [D2, D4].

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Fig D2. Protein and structure of Tight Junctions and Adhesion Junctions [D5]

Research Paper - Daphne
Inhibition of Rho-kinase protects cerebral barrier from ischaemia-evoked injury through modulations of endothelial cell oxidative stress and tight junctions [D6]

Introduction
Stroke is the third leading cause of death in the western world and ischaemic stroke or strokes caused by restriction of blood supply to the brain, constitutes about 85% of all stroke cases. In ischaemic stroke the microglia and astrocytes in the brain are activated inducing the production of cytokines and various inflammatory factors. This causes a breakdown of the BBB and tight junctions allowing diffusion through the barrier. This leakage disrupts the water and ion concentrations in the brain causing cerebral edema or swelling. Further, leukocytes infiltrate the brain, worsening the inflammatory response and increasing brain injury [D7]. Oxidative damage can also occur leading to further damage of BBB. However, the molecular mechanisms are not well characterized. Rho kinase has been shown to be important in BBB integrity as it regulates a variety of pathways including cell proliferation and inflammation. After stroke, increase Rho kinase activity has been linked to more severe neurological deficits and worse outcomes. Increases in oxidative stress and changes in TJ protein expressions have been implicated in this damage. Thus, this paper investigates the therapeutic potential and molecular mechanisms, specifically oxidative stress and TJ protein expressions, involved in post-ischemic Rho-kinase inhibition.

Methods

Rho kinase inhibition was induced in two models, one in-vivo and one in-vitro model. Fasudil, which is highly selective for Rho kinases, was used in a Middle Cerebral Artery Occlusion (MCAO) mouse model. At least 70% of blood flow to a portion of the brain was surgically occluded for 45 minutes after which the occlusion was removed and reperfusion occurred. Various measures of well being and were then assessed including body weight, neurological deficit using a 6 and 28 point system, as well as locomotion across a grid surface. An in vitro model using oxygen-glucose deprivation (OGD) on brain microvascular endothelial cells seeded with astrocytes was also used. Y-27632, a highly selective inhibitor used for in vitro conditions, was added after OGD with and without reperfusion or the reintroduction of oxygen to the cells. Transendothelial electrical resistance (TEER) and paracellular flux of Evan’s blue labelled albumin (EBA) was used to quantify the barrier function of the endothelial cells as a measure of BBB integrity. To quantify oxidative stress, NADPH oxidase activity as well as superoxide anion O2*- levels were measured. NADPH oxidase generates O2*- which can directly disrupt the BBB by inducing cell death. Further, prooxidant (gp91-phox) and antioxidant (CuZn-SOD, catalase) enzyme expression levels were quantified, as an imbalance in these enzymes may also increase oxidative stress. The TJ protein expression was quantified with the expression levels of occludin and claudin-5.

Results

First, the well being of the MCAO mice with treated with fasudil directly after reperfusion (fasudil) or 4 hours after reperfusion (fasudil + 4h) was assessed, compared to a control which was administered the vehicle with no fasudil. The body weight for all three conditions dropped, however the weight loss for the fasudil condition was significantly reduced (Fig D3 (a)). The neurological deficit of the mice was also assessed 6 and 24 hour after reperfusion and treatment. While only the 6 hour assessment with either fasudil or fasudil + 4h showed significant reduction of deficit, both the 6 point and 28 point score showed trends of improvement in neurological function with the administration of fasudil (Fig D3 (b,c)). Further, the number of foot slips during the grid test 24 hours after treatment was significantly reduced in both fasudil treatment conditions (Fig D3 (d)). This indicates that fasudil has the potential to improve well being and neurological function after ischemia.

Fig D3. Well being and function of MACO mice after treatment with fasudil (a) Body weight 24h post surgery (b) 6 point score assessed 6 and 24 hours post surgery (c) 28 point score assessed 6 and 24 hours post surgery (d) Foot slips during grid test 24 h post surgery

Knowing that fasudil had therapeutic potential, the potential molecular mechanism that may be involved in these benefits was investigated. Oxidative stress and tight junction protein levels were assessed in the ipsilateral (same side) and contralateral (opposite side) brain slices of the MACO mice. However all markers of oxidative stress and TJ protein assessed did not show significant difference between the vehicle and treatment conditions or between the ipsilateral and contralateral brain slices (Fig D4). This may be due to the fact that Rho kinase activity localizes in the microglia and endothelial cells, and differences could not be quantified with whole brain slices.

Fig D4. Quantification of ipsilateral and contralateral brain slices of MACO mice treated with fasudil for (a) NADPH oxidase activity (b) total O2*- levels (c) gp91-phox (d) CuZn-SOD (e) catalase (f) occludin (g) claudin-5 (h) Rho-kinase Thus, these mechanisms were investigated in the endothelial cells directly using the OGD in vitro model. OGD was induced for 4 hours (4h OGD), 20 hours (20h OGD), as well as 4 hours with reperfusion (4h OGD+R) after which Y-27632 was administered. Markers for oxidative stress and tight junction proteins was again assessed. NADPH oxidase, O2*- ﻿and the prooxidant gp91-phox were all increased in the OGD stroke model compared to an unstressed control. Treatment with Y-27632 was able to restored levels to normal and in the case of the gp91-phox significantly decrease protein levels (Fig D5). As the inhibition of Rho kinase was able to restore these fluctuations, these enzymes are likely modulated by Rho kinase after stroke.

Fig D5. Oxidative stress quantification in a OGD in vitro model treated with Y-27632 (a) NADPH oxidase activity (b) total O2*- levels (c) gp91-phox

In addition, the occludin and claudin-5 TJ protein levels were also assessed under the same conditions. The OGD conditions caused no change in expression levels of occludin however, introduction of Y-27632 induced a significant increase in all conditions (Fig D6 (d)). This may linked to the protective effects of Rho kinase inhibition on the BBB seen in subarachnoid haemorrhage after treatment with endothelial- monocyte-activating polypeptide II. For claudin-5, the 20h OGD and 4h OGD+R conditions induced a significant increase in expression which is known to be a hallmark of hyper permeability between microvessel cells. Treatment with Y-27632 was again able to restore expression to control levels (Fig D6 (c)).

Fig D6. TJ protein level quantification in a OGD in vitro model treated with Y-27632 (c) occludin (d) claudin-5

Finally, the barrier integrity of these cells were tested under the same conditions using TEER and EBA flux. As expected the permeability increased in the OGD stroke model, with TEER significantly decreasing and flux of EBA significantly increasing. Treatment with Y-27632 was able to return TEER and EBA flux to control levels in the 4h OGD and 4h OGD+R conditions, however it could not do so for long term OGD (20h OGD) (Fig D7). Fig D7. BBB integrity quantification in a OGD in vitro model treated with Y-27632 (c) TEER (d) EBA flux Conclusion

This paper assessed the effects of Rho kinase inhibition on the BBB and investigated oxidative stress and tight junction protein expression as a potential mechanism of this effect. They demonstrated that Rho kinase inhibition may have therapeutic potential as they saw improved well being and function in mice as well as restored BBB integrity in an in vitro model of ischemia. Further they illustrated in vitro, that Rho kinase inhibition modulated some oxidative stress markers including NADPH oxidase, O2*- and gp91-phox, as well as tight junction protein expression of occludin and claudin-5. However, further investigation is required as these mechanistic effects were unable to be detected in vivo and systemic levels and effects of these markers were not assessed.

[D1] Blood–brain barrier. (2019). Retrieved 19 October 2019, from https://en.wikipedia.org/wiki/Blood%E2%80%93brain_barrier

[D2] Abbott, N., Patabendige, A., Dolman, D., Yusof, S. and Begley, D. (2010). Structure and function of the blood–brain barrier. Neurobiology of Disease, 37(1), pp.13-25.

[D3] Abdullahi, W., Tripathi, D. and Ronaldson, P. (2018). Blood-brain barrier dysfunction in ischemic stroke: targeting tight junctions and transporters for vascular protection. American Journal of Physiology-Cell Physiology, 315(3), pp.C343-C356.

[D4] Daneman, R., & Prat, A. (2015). The blood-brain barrier. Cold Spring Harbor perspectives in biology, 7(1), a020412. doi:10.1101/cshperspect.a020412

[D5] Polakis, P. (2008). Formation of the blood–brain barrier: Wnt signaling seals the deal. The Journal of Cell Biology, 183(3), 371 LP – 373. https://doi.org/10.1083/jcb.200810040

[D6] Gibson, C. L., Srivastava, K., Sprigg, N., Bath, P. M. W., & Bayraktutan, U. (2014). Inhibition of Rho-kinase protects cerebral barrier from ischaemia-evoked injury through modulations of endothelial cell oxidative stress and tight junctions. Journal of Neurochemistry, 129(5), 816–826. https://doi.org/10.1111/jnc.12681

[D7] Yang, C., Hawkins, K. E., Doré, S., & Candelario-Jalil, E. (2019). Neuroinflammatory mechanisms of blood-brain barrier damage in ischemic stroke. American Journal of Physiology - Cell Physiology, 316(2), C135–C153. https://doi.org/10.1152/ajpcell.00136.2018