Attenuation of Blood-Brain Barrier Breakdown and Hyperpermeability by Calpain Inhibition

Blood-brain barrier (BBB) breakdown and the associated microvascular hyperpermeability followed by brain edema are hallmark features of several brain pathologies, including traumatic brain injuries (TBI). Recent studies indicate that pro-inflammatory cytokine interleukin-1β (IL-1β) that is up-regulated following traumatic injuries also promotes BBB dysfunction and hyperpermeability, but the underlying mechanisms are not clearly known. The objective of this study was to determine the role of calpains in mediating BBB dysfunction and hyperpermeability and to test the effect of calpain inhibition on the BBB following traumatic insults to the brain. In these studies, rat brain microvascular endothelial cell monolayers exposed to calpain inhibitors (calpain inhibitor III and calpastatin) or transfected with calpain-1 siRNA demonstrated attenuation of IL-1β-induced monolayer hyperpermeability. Calpain inhibition led to protection against IL-1β-induced loss of zonula occludens-1 (ZO-1) at the tight junctions and alterations in F-actin cytoskeletal assembly. IL-1β treatment had no effect on ZO-1 gene (tjp1) or protein expression. Calpain inhibition via calpain inhibitor III and calpastatin decreased IL-1β-induced calpain activity significantly (p < 0.05). IL-1β had no detectable effect on intracellular calcium mobilization or endothelial cell viability. Furthermore, calpain inhibition preserved BBB integrity/permeability in a mouse controlled cortical impact model of TBI when studied using Evans blue assay and intravital microscopy. These studies demonstrate that calpain-1 acts as a mediator of IL-1β-induced loss of BBB integrity and permeability by altering tight junction integrity, promoting the displacement of ZO-1, and disorganization of cytoskeletal assembly. IL-1β-mediated alterations in permeability are neither due to the changes in ZO-1 expression nor cell viability. Calpain inhibition has beneficial effects against TBI-induced BBB hyperpermeability.

Blood-brain barrier (BBB) breakdown and the associated microvascular hyperpermeability followed by brain edema is a hallmark feature of several brain pathologies including traumatic brain injuries (TBI). Recent studies indicate that pro-inflammatory cytokine interleukin-1β (IL-1β) that is upregulated following traumatic injuries also promotes BBB dysfunction and hyperpermeability but the underlying mechanisms are not clearly known. The objective of this study was to determine the role of calpains in mediating BBB dysfunction and hyperpermeability and to test the effect of calpain inhibition on the BBB following traumatic insults to the brain. In these studies, rat brain microvascular endothelial cell monolayers exposed to calpain inhibitors (calpain inhibitor III and calpastatin) or transfected with calpain-1 siRNA demonstrated attenuation of IL-1β-induced monolayer hyperpermeability. Calpain inhibition led to protection against IL-1β-induced loss of zonula occludens-1 (ZO-1) at the tight junctions and alterations in f-actin cytoskeletal assembly. IL-1β treatment had no effect on ZO-1 gene or protein expression. Calpain inhibition via calpain inhibitor III and calpastatin decreased IL-1βinduced calpain activity significantly (p<0.05). IL-1β had no detectable effect on intracellular calcium mobilization or endothelial cell viability. Furthermore, calpain inhibition preserved BBB integrity/permeability in a mouse controlled cortical impact model of TBI when studied using Evans Blue assay and intravital microscopy. These studies demonstrate that calpain-1 acts as a mediator of IL-1β-induced loss of BBB integrity and permeability by altering TJ integrity, promoting the displacement of ZO-1 and disorganization of cytoskeletal assembly. IL-1β-mediated alterations in permeability are neither due to the changes in ZO-1 expression nor cell viability. Calpain inhibition has beneficial effects against TBIinduced BBB hyperpermeability.

INTRODUCTION
The blood-brain barrier (BBB) plays an important role in maintaining the homeostasis of the brain. Blood-brain barrier breakdown and the associated hyperpermeability is a hallmark feature of several brain pathologies and injuries. The BBB is majorly composed of the cerebral endothelial cells and the tight junctions (TJs) between them (1). Tight junctions (TJs) between the neighboring endothelial cells include transmembrane TJs i.e., occludin, claudins, junctional adhesion molecules etc. and membrane bound TJs i.e., zonula occludens (1). Zonula occludens play an important role in regulating BBB permeability by binding to both transmembrane tight junctions as well as actin cytoskeleton intracellularly (2). Various mediators of inflammation are shown to modulate BBB breakdown and permeability in a variety of pathologies (3). Blood-brain barrier breakdown and the associated hyperpermeability is the leading cause of brain edema and elevated intracranial pressure followed by decreased perfusion pressure leading to poor clinical outcomes in traumatic brain injury (TBI) (4).
Inflammation that occurs as a consequence of brain injuries is carried out by various pro-inflammatory cytokines (5). IL-1β is the most implicated proinflammatory cytokine in various pathologies of the central nervous system including TBI (6,7). Interleukin-1 (IL-1) inhibition has beneficial effects as demonstrated in experimental models of brain damage (6). IL-1β induces BBB breakdown in rat brain endothelial cells and also increases human brain microvascular endothelial cell permeability (8). However, IL-1β-induced mechanisms that lead to barrier dysfunctions and hyperpermeability at the level of the BBB are not clearly known.
Calpains are thiol or cysteine proteases that are present in most of the mammalian cells. They are involved in a wide array of neurological pathologies like trauma, ischemia-reperfusion injury, spinal cord injury and several non-neurological pathologies as well (9)(10)(11)(12). Intracellular calcium levels and the endogenous inhibitor of calpains namely calpastatin tightly regulate calpain levels endogenously (9,13). Calpains-1 and -2 are the predominant calpains in the central nervous system (14, 15). An increased calpain activity was observed following TBI in laboratory rodents (16,17) and human patients (12). Calpain inhibitors protect the brain against various neurotraumas including brain and spinal cord injury (18,19). Calpain expression was found to be increased in the endothelial cells of the injured brain cortex following TBI in human patients compared to those who died from cardiac arrest (12). Calpain-dependent cleavage of intracellular cytoplasmic protein zonula occludens-1 (ZO-1) has been studied in human lung endothelial cells (13). However, their contribution in regulating BBB endothelial dysfunction and hyperpermeability is largely unknown.
Based on these observations we hypothesized that calpain-mediated mechanisms play an important role in promoting IL-1β-induced BBB breakdown and hyperpermeability and that calpain inhibition will potentially down-regulate this pathway. Therefore, we studied the effect of calpain inhibition on BBB hyperpermeability in both cultured rat brain endothelial cells and a mouse model of TBI. The objectives and the specific questions that we addressed are as follows: RBMEC monolayers were pretreated with calpain inhibitor III or calpastatin to confirm the contribution of calpains in mediating IL-1β (10 ng /mL for 2 hours)-induced endothelial cell hyperpermeability. Figure 1 demonstrates that IL-1β treatment significantly increases endothelial cell hyperpermeability, while pretreatment with calpain inhibitor III (10 µM; 1 hour; Figure  1A; p <0.05) and calpastatin (10 µM; 1 hour; Figure 1B; p <0.05) significantly attenuated IL-1β-induced endothelial cell hyperpermeability. Calpain inhibitor III (10 µM; 1 hour) and calpastatin (10 µM; 1 hour) treatment alone did not alter rat brain endothelial cell hyperpermeability. Calpain inhibitor III (1, 10 and 50 µM) treatment decreased IL-1β (10 ng/ml)-induced monolayer hyperpermeabiltiy significantly ( Figure 3B; p<0.05).
Significant contribution of calpain-1 is further supported by calpain-1 siRNA transfection studies. Figure 1C demonstrates that IL-1β (10 ng /mL for 2 hours) treatment-induced endothelial cell hyperpermeability was significantly reduced in calpain-1 knockdown (25 nM; 48 hours) cells compared to IL-1β treatment alone (p <0.05). Calpain-1 knockdown studies were performed using siRNA transfection technique as described earlier. Calpain-1 siRNA treatment alone did not induce any significant change in rat brain endothelial cell hyperpermeabiliity compared to the control siRNA group. Calpain-1 siRNA treated groups were compared to the control siRNA group, while the IL-1β alone treated group was compared to the control group.
IL-1β treatment neither induces ZO-1 mRNA expression nor alters ZO-1 protein expression IL-1β (10 ng/mL; 2 hours) treatment did not alter ZO-1 mRNA expression by RT-PCR studies ( Figure 5A). IL-1β treatment did not alter ZO-1 protein expression ( Figure 5B) by western blot studies. These studies suggest that the total ZO-1 gene and protein expression remains the same by IL-1β treatment (10 ng/mL; 2 hours) compared to the control untreated cells, thus, indicating that the loss of ZO-1 tight junction integrity is not due to the loss of the total ZO-1 protein expression in the cells, indicating a possibility of temporary relocalization of ZO-1.

IL-1β treatment does not induce cell death in endothelial cells
IL-1β (10 ng/mL) treatment did not significantly decrease the number of viable cells compared to the control group upto 4 hours, as shown in Figure 6. IL-1β (10 ng/mL) treatment for 6 hours significantly decreased the number of viable cells compared to the control group. 50mM, 25mM and 10mM concentration of hydrogen peroxide was used as a positive control at 2, 4 and 6 hours treatment of IL-1β.

IL-1β treatment does not induce intracellular calcium mobilization
To test whether IL-1β upregulates the activity of calpains, calcium-dependent cysteine proteases, by increasing intracellular calcium concentration ([Ca 2+ ]i), we directly measured [Ca 2+ ]i in RBMECs by two different approaches. Initially, RBMECs were grown as monolayers and loaded with Fluo-4 AM (a Ca 2+ indicator). Fluo-4 signals were measured from cells before (F0) and after (F) a 10-minute incubation of cells with IL-1β (10 ng/mL). In parallel, control cells were treated with thapsigargin (TG; 2 µM), ionomycin (Iono; 1 µM), or DMSO as the vehicle control for TG and Iono. TG is a sarco/endoplasmic reticulum Ca 2+ -ATPase (SERCA) pump inhibitor, which passively depletes Ca 2+ in the ER store in conjunction with triggering store-operated Ca 2+ entry (SOCE). Ionomycin is an ionophore that increases membrane permeability to Ca 2+ , therefore raising the intracellular level of Ca 2+ . As expected, both stimulations by TG and ionomycin triggered a significant increase of [Ca 2+ ]i ( Figure 7A); however, there was only a negligible intracellular Ca 2+ response to the IL-1β treatment ( Figure 7A). It is possible that IL-1β triggers a relatively transient [Ca 2+ ]i response and/or a small population of cultured RBMECs has a dramatic [Ca 2+ ]i response to IL-1β, which could be masked during our Fluo-4 based [Ca 2+ ]i measurement. Accordingly, RBMECs were then loaded with Fura-2 AM, a ratiometric Ca 2+ indicator, and [Ca 2+ ]i was continuously monitored at the singlecell level. In the control experiments, TG could evoke a typical SOCE in RBMECs ( Figure 7B) that elevates [Ca 2+ ]i from 65 ± 6 nM up to 281 ± 22 nM ( Figure 7D). However, when RBMECs were incubated with 10 ng/mL IL-1β for 20 minutes, there were no cells showing significantly increased [Ca 2+ ]i (data not shown). Cells were then treated with a higher dose (100 ng/mL) of IL-1β for 2 hours, which still did not significantly promote intracellular calcium levels in any cells ( Figure 7C and 7D). As a conclusion, our results indicate that IL-1β treatment does not induce a dramatic [Ca 2+ ]i mobilization in cultured RBMECs.

Calpain inhibitor III treatment attenuates mild
TBI-induced BBB hyperpermeability in mice Mice subjected to TBI demonstrated significant increase in Evans blue leakage compared to the sham animals (p<0.05). DMSO was used as a vehicle control. Vehicle + Sham group did not show any significant increase in BBB permeability compared to the sham group; while Vehicle treated group when subjected to mild TBI induced BBB hyperpermeability significantly. Pretreatment or post treatment with calpain inhibitor III attenuated TBIinduced Evans blue leakage into the brain tissue significantly ( Figure 8A & B; p <0.05).
Intravital microscopy evaluation of brain pial vasculature of anesthetized mice showed BBB dysfunction and hyperpermeability following mild TBI compared to sham-vehicle at 60 minutes post-trauma evidenced by the leakage of FITC-dextran from intravascular space to the interstitium (Figure 9 A & B; p<0.05). Calpain inhibitor III treatment following TBI showed a decrease in hyperpermeability compared to the TBI group (p<0.05).

DISCUSSION
The major findings of this study are: 1) calpain(s) promote BBB dysfunction and hyperpermeability via disruption of the TJs in vitro, 2) IL-1β is an inducer of calpainmediated BBB dysfunction and hyperpermeability in vitro, 3) inhibition of calpain activation provides protection against BBB hyperpermeability via preservation of TJ integrity in vitro, 4) calpain-mediated loss of barrier functions are independent of [Ca 2+ ]i mobilization or loss of cell viability, and 5) pharmacological inhibition of calpains provide protection against BBB hyperpermeability in a mouse model of TBI. In this study, the inhibition of calpains either by a pharmacological inhibitor, or by its endogenous inhibitor, calpastatin or by calpain-1 gene knockdown provided protection to the BBB. These observations strongly suggest a major role for calpains in modulating BBB integrity and permeability. Calpains are thiol or cysteine proteases that are present in most of the mammalian cells. Under physiological conditions, calpains possess low activity and play an important role in regulating kinases, transcription factors, and receptors apart from aiding in the cytoskeletal turnover (20). Calpain deficiencies as well as its over activation are linked to a variety of diseases and pathological consequences (21). Due to their multifaceted nature, they control various irreversible signaling events and biological functions in the cell like endothelial cell adhesion, differentiation, migration, proliferation, cell cycle control, cytoskeletal remodeling, embryonic development and vesicular trafficking (12)(13)(14)(22)(23)(24). Our studies support a relationship between calpains and microvascular permeability at the level of the BBB.
Calpain-1 (also known as taucalpain) and calpain-2 (also known as mcalpain) are predominantly expressed in the central and peripheral nervous systems (14,15). Calpain-1 is activated under low calcium concentrations (3-50 μM), while calpain-2 is activated only under high concentrations (400-800 μM) of calcium in the cell (23,24). Calpain-1 knockdown data from our studies demonstrate the contribution of calpain-1 but we do not disregard the contribution of calpain-2 in mediating BBB dysfunction and hyperpermeability, an area open for further investigation.
Calpastatin and intracellular Ca 2+ levels tightly regulate the calpain levels endogenously (12,21). This information was used to study the contribution of calcium and calpastatin in regulating IL-1β-induced endothelial hyperpermeability. To study the effect of IL-1β treatment on intracellular calcium, we performed calcium mobilization studies, which demonstrated that IL-1β treatment does not induce significant mobilization of intracellular calcium in RBMECs. However, we do not rule out the possible involvement of calcium in inducing calpains as established in several other conditions. We speculate that IL-1β-induced calpain activation may occur due to concentration changes in localized calcium (25).
Calpastatin is an endogenous inhibitor of calpains; it regulates the activity of calpains by binding to the active site cleft of both calpains-1 and -2. Our results support the significance of calpaincalpastatin interactions in regulating BBB functions. Although, our studies demonstrate the ability of calpastatin in attenuating IL-1β-induced endothelial hyperpermeability, we have not further explored these studies in vivo. Understanding calpastatin-mediated regulation of BBB hyperpermeability in animal models can open new avenues for exploring endogenous therapeutic agents that can alter the secondary injuries that occur following brain injury.
Our studies demonstrate that pharmacological inhibition of calpains is effective in preserving barrier functions in vitro and in vivo. Calpain inhibitor III is a well-studied cell permeable pharmacological inhibitor of calpains (12), which was chosen for our in vitro and in vivo studies. Calpain inhibitor III crosses the BBB efficiently and binds specifically to calpains -1 and -2, unlike other inhibitors of calpains like trypsin, plasmin, caspase-1, and cathepsin D (24). Cytoskeletal and neuroprotective properties of calpain inhibitor III were examined using the CCI model of TBI in male CF-1 mice, 24 h post-TBI (26). However, these studies do not aim to understand the effect of calpain inhibitor III on TBI-induced BBB hyperpermeability. Our study addresses this important question.
Studies by Tsubokawa et al., 2006 indicate a link between calpain, cathepsin B, and matrix metalloproteinase-9 (MMP-9) upregulation (27). MMP-2 and calpains have some common cleavage targets like sarcomeric proteins troponin I, myosin light chain-1 and titin in heart cells (28). These studies support the possibility that calpaininduced BBB endothelial cell hyperpermeability may occur via MMP activation or vice-versa. Understanding this relationship is important to selectively target the appropriate pathways for therapeutic purposes.
A calpain-MMP-9 interaction in endothelial cells of the BBB as well as the various other cell types of the neurovascular units is a possibility that regulates BBB functions. In addition to their presence in endothelial cells, calpains and MMPs are expressed in other cell types of the neurovascular unit such as astrocytes and pericytes. Pericytes are considered as one of the major sources of MMP-9 in the BBB (29). In a recent study, it was found that among the various cell types of the neurovascular unit, pericytes exhibited the highest level of MMP-9 secretion when challenged with thrombin that is known to induce barrier dysfunctions in endothelial cells (30). Furthermore, pericytes features such as contractility and cellular stiffness has been regulated by cellular calpains (31). Similarly, astrocytes are an integral component of the BBB which may be compromised by TBI or ischemic brain injury and increased MMP-9 expression has been observed in astrocytes following trauma (32). Also, astrocyte originated MMP-9 has been attributed the pathogenesis of hemorrhagic brain edema (33). Our results conducted exclusively in BBB endothelial cells demonstrate that they are targeted by calpain-mediated and thus, potentially MMP-9 regulated mechanisms of barrier dysfunction and hyperpermeability. Future studies that involve multiple coculture models of all the major cell types of the neurovascular unit could provide more information towards this potential interactions.
Our results demonstrate changes in the cytoskeletal assembly evidenced by increased f-actin (filamentous actin) stress fiber formation following IL-1β treatmentinduced calpain activation and BBB hyperpermeability that was decreased following calpain inhibition. Actin filaments are linear polymers of filamentous actin formed by actin polymerization and under normal physiological conditions they distributed randomly throughout the cell. Various hyperpermeability inducing agents are known to induce the reorganization of actin filaments into stress fibers that are linear, parallel bundles across the cell interior (34, 35). Increased f-actin stress fiber formation most often is associated with endothelial barrier dysfunction and hyperpermeability (35) The stress fiber formation that occurs following IL-1β treatment may also involve tight junction cleavage via Ras homolog gene family, member A (RhoA)-dependent mechanisms leading to BBB permeability. RhoAmediated mechanisms also activate cell division control protein (cdc42), which plays an important role in maintaining the tight junction integrity and actin cytoskeletal assembly (36).
Our studies on understanding the role of calpains were focused more on the tight junction protein, ZO-1 and the actin cytoskeleton. However, understanding the contribution of calpains in mediating BBB hyperpermeability via multiple other proteins is important as they target a wide range of proteins, which include: cytoskeletal proteins (α-spectrin, talin, filamin, paxillin, vinculin, ezrin, microtubule-associated proteins 2, myristoylated alanine-rich C-kinase substrate and neurofilament proteins), kinases (PP60 and proto-oncogene tyrosineprotein kinase src), phosphatases (protein tyrosine phosphatase 1B, focal adhesion kinase, talin, paxillin, protein tyrosine phosphatase), membrane-associated proteins, junctional proteins (β-catenin, Ecadherin, β-spectrin), and transcription factors such as c-fos, c-jun and p53 (13,21,22,(37)(38)(39)(40). Although the expression of some of these proteins in the BBB is not well established, it is important to know how calpains interact with them and how these molecules contribute to BBB dysfunction or trauma-induced brain damage directly or indirectly.
The cellular mechanisms by which IL-1β upregulates calpain acitivity is not clearly known to us at this time. Recent studies indicate that calpain activation can occur independent of calcium via mechanisms such as mitogen-activated protein kinase (MAPK)-mediated phosphorylation in various cell types such as fibroblasts, neurons, HEK-TrkB cells etc and is calcium independent (41,42). Furthermore, in hippocampal neurons MAPK-mediated and calcium independent calpain activation was associated with actin polymerization, which was prevented by calpain inhibition (42). Also, MAP kinases have been shown to promote vascular endothelial permeability. However, further studies are required to delineate a potential IL-1β-MAP kinase-calpain activation pathway leading to barrier dysfunction in microvascular endothelial cells.
The use of calpain inhibitors for functional improvement has been investigated in various animal models of CNS pathologies. Calpain inhibitors has been found to provide neuroprotection in a mouse controlled cortical impact model of TBI and ischemic stroke (26,43). Our results suggest that one of the mechanisms by which such neuroprotective effects occurred could be due to enhanced protection of the BBB that occurred following calpain inhibition. In subarachnoid hemorrhage, calpain inhibition reduced behavioral deficits and BBB permeability (44). Similarly, calpain inhibition has been found to be protective against neuropathology associated with Alzheimer's disease, Parkinson's disease and conditions such as lessencephaly (45)(46)(47).
In conclusion, our studies demonstrate the novel role of calpains in promoting IL-1β-induced BBB dysfunction and hyperpermeability and how calpain inhibition regulates these pathways. Our results further demonstrate that calpain inhibition has the potential to be developed as a therapeutic target in controlling TBIinduced BBB hyperpermeability and edema when established in human patients. Craniotomy Procedure: The head of the animal was shaved and the surgical site on the surface of the head was cleaned with an alcohol wipe. Lubricating ointment was applied to the eyes. A midline incision was made to remove the skin from top of the skull exposing the sagittal suture, bregma and lambda. A circular craniotomy window, 3-4 mm in diameter was made on ipsilateral hemisphere, between lambda and bregma using a microdrill. The resulting bone flap was removed. Sham animals received only craniotomy surgery, while TBI injury group receives brain injury via controlled cortical impactor following craniotomy procedure.

Materials
Controlled cortical impact (CCI): Benchmark™ Stereotaxic Impactor from Leica was used for these studies. Following craniotomy procedure, the animal was mounted on the stereotaxic frame. An impactor probe of 3 mm diameter was used to impact the exposed part of the brain. The depth of the injury was used to determine the intensity of the injury. Settings for mild TBI used in this study are: 2 millimeters depth, 0.5 meters/second velocity and 100 milliseconds contact time as described in (48).

Monolayer Permeability Assays
Briefly, RBMECs were grown on fibronectin-coated Transwell® inserts as monolayers for 72-96 hours and regularly checked for confluency. Monolayers were initially exposed to phenol red free DMEM for 45 minutes to an hour. DMEM treated cells were then pretreated with calpain inhibitors and subsequently with IL-1β (10 ng/mL; 2 hours). At the end of the treatments, FITC labeled dextran-10 kDa (5 mg/mL; 30 minutes) was applied to the luminal compartment. Monolayer hyperpermeability is assessed fluorimetrically at 485/520 nm using FITC dextran-10KDa as described previously (49). Calpain inhibitor III (10 μM; 1 hour) was used for pharmacological inhibition of calpains, while calpastatin (10 μM; 1 hour), was used for endogenous inhibition of calpains. Calpain inhibitor III (MDL-28170; Carbobenzoxy-valinyl-phenylalaninal; Cbz-Val-Phe-H) is a potent, cell permeable inhibitor of both calpain-1 and -2 and calpastatin is a cell-permeable peptide (27 amino acids in length) that inhibits both calpains-1 and -2. Untreated cells served as control. Each experiment was repeated four times. Fluorescence intensity was plotted on the Y-axis and represented as % control. Data were expressed as mean ± % SEM and statistical differences among groups were determined by one-way analysis of variance (ANOVA) followed by Bonferroni post hoc test, to determine the significant differences between specific groups. A value of p<0.05 was considered statistically significant.

Calpain-1 knockdown studies
Briefly, RBMECs were transfected with control siRNA (25nM; 48 hours) or calpain-1 siRNA (25 nM; 48 hours) on reaching 50% confluency. Transfection was performed according to manufacturer's instructions. Transfected monolayers were then exposed to IL-1β and permeability was determined based on the leakage of FITCdextran-10 kDa (5 mg/mL; 30 minutes) leakage from the luminal to the abluminal chamber. Monolayer permeability was assessed fluorometrically as described earlier. Untreated cells were used as control. Fluorescence intensity was plotted on the Yaxis and represented as % control. Data were expressed as mean ± % SEM and statistical differences among groups were determined by one-way analysis of variance (ANOVA) followed by Bonferroni post hoc test to determine significant differences between specific groups. A value of p<0.05 was considered statistically significant.

Calpain Activity measurements
A Calpain Activity Fluorometric Assay Kit was used to measure the calpain activity in the cells. For this assay, RBMECs were grown in petri dishes until confluency is achieved. Cells were then trypsinized using lysis buffer, followed by resuspension in extraction buffer provided in the kit. The kit employs a synthetic calpain substrate, Ac-Leu-Leu-Tyr-7-Amino-4trifluoromethylcoumarin (Ac-LLY-AFC) in order to detect the calpain activity. Equal amount of protein lysates were taken and subsequently exposed to the substrate Ac-LLY-AFC and incubated in the dark for an hour. Samples were then read using Fluoroskan Ascent™ FL microplate fluorometer and luminometer at 400/505 nm (Excitation/Emission). Cells lysates were pretreated with calpain inhibitors (Calpain inhibitor III and calpastatin at 10 μM for 1 hour) and subsequently with IL-1β treatment. Untreated cells served as control. Each experiment was repeated five times. Calpain activity was expressed as relative fluorescence units (RFU) and plotted on the Y-axis. In a separate set of experiment, calpain activity was determined following exposure of the cells to various concentrations of IL-1β (0, 1, 10 and 100 ng/ml) for 2 hours as well as IL-1β (10 ng/ml) for various durations of time (0 hr, 1 hr, 2 hrs and 4 hrs). Data were expressed as mean ± SEM and statistical differences among groups were determined by one-way analysis of variance (ANOVA) followed by Bonferroni post hoc test to determine significant differences between specific groups. A value of p<0.05 was considered statistically significant.
Immunofluorescence staining and cytoskeletal labeling Zonula occludens (ZO-1) junctional localization and f-actin stress fiber formation were assessed. RBMECs were grown on chamber slides for overnight. Pretreatment with calpain inhibitor III (10 μM; 1 hour) was followed by IL-1β treatment (10 ng/mL; 2 hours). Cells were then fixed in 4% paraformaldehyde in PBS for 10-15 minutes and permeabilized with 0.5% Triton-X 100 in PBS for another 10-15 minutes. Cells were blocked with 2% bovine serum albumin (BSA) in PBS for an hour at room temperature. Cells were then incubated overnight with anti-rabbit primary antibodies against ZO-1 (#617300) at 1:150 dilution, followed by incubation with antirabbit IgG-FITC conjugated secondary antibody for an hour at room temperature. Cells were then washed and mounted. Following treatment study, cells were fixed, permeabilized and blocked in 2% BSA-PBS as described earlier. Cells were then labeled with rhodamine phalloidin at 1:50 dilution in 2% BSA-PBS for 20 minutes. Chamber slides were then washed and mounted using VECTASHIELD® Antifade Mounting Media with DAPI for nuclear staining. Cells were visualized and scanned at a single optical plane with an Olympus Fluoview 300 Confocal Microscope (Center Valley, PA) with a PLA PO 60X water immersion objective. Untreated cells served as control. Each experiment was repeated four times. The changes in ZO-1 localization and the formation of f-actin stress fibers were analyzed using ImageJ software and was presented as arbitray units for statistical analysis (unpaired t-test; p<0.05) (34).

Quantitative real time-PCR
RBMECs were grown on 100 mm cell culture dishes; total RNA was then extracted from the cells using TRIzol® reagent according to the manufacturer's instructions. RNA concentration and quality were determined by employing the ratio of absorbance at 260/280 nm using Biotek Synergy Hybrid Spectrophotometer (Winooski, VT). Reverse transcription was performed using the SuperScript® IV First-Strand Synthesis System. Quantitative real time PCR was performed using the RT² SYBR Green Fluor qPCR Mastermix with the following primer pairs for ZO-1: Forward primer: 5′-CCTCTGATCATTCCACACAGTC-3′, Reverse primer: 5′-TAGACATGCGCTCTTCCTCTCT-3′, and GAPDH: Forward primer: 5′-AATGTATCCGTTGTGGATCT-3′, Reverse primer: 5′-CAAGAAGGTGGTGAAGCAGG-3′ were used. Real-time PCR detection was carried out using Stratagene Mx3000P qPCR System, Agilent Technologies (La Jolla, CA), using 1 μL of cDNA. Relative abundances of target genes were calculated by normalizing Ct values to endogenous control glyceraldehyde 3-phosphate dehydrogenase (GAPDH). Cells were divided into 2 groups: control (untreated) and IL-1β (10 ng/mL; 2 hours) treatment groups. Each experiment was repeated three times. Relative mRNA expression of ZO-1 was obtained by normalizing the Ct values to the endogenous control GAPDH for each repeat. Normalized Ct values were expressed as mean ± SEM. Statistical differences among groups were determined by one-way analysis of variance (ANOVA) followed by Bonferroni post hoc test to determine significant differences between specific groups. A value of p<0.05 was considered statistically significant.

Western blot assays
Western blots were performed to investigate the expression of ZO-1 protein in RBMECs. Following treatment studies, cells were washed and incubated in ice-cold cell lysis buffer (1X) along with protease inhibitor cocktail (1X) for 5 minutes in cell culture dishes. Cells were then scraped, sonicated and centrifuged at 14,000g for 10 minutes at 4°C. Supernatant was collected from the extracts and protein concentration was determined using protein assay kit. Equal amounts of total protein (50 μg) were separated by sodium dodecyl sulphatepolyacrylamide gel electrophoresis (SDS-PAGE) on 10% Bis-Tris precast gels at constant voltage (145 V) for 180 minutes. Proteins were then transferred onto the nitrocellulose membrane at constant voltage (30 V) for overnight and the membranes were blocked using 5% nonfat dry milk in Tris-Buffered Saline (TBS) with 0.05% Tween-20 for 3 hours and subsequently incubated with primary mouse monoclonal anti ZO-1 antibody (1:250 dilution). Membranes were washed thrice in TBS-T and incubated with the goat anti-mouse IgG-HRP conjugated secondary antibody. After washing, the immunoblots were visualized by ECL Western Blotting Substrate. Cells were divided into untreated (control) and IL-1β (10 ng/mL; 2 hours) treatment groups. Each experiment was repeated four times. Equal amount of protein sample loading was verified by assessing β-actin protein expression.

Cell viability studies
An EZViable™ Calcein AM Cell Viability Assay Kit (Fluorometric) was used for quantify the number of viable cells. Calcein AM is a non-fluorescent, hydrophobic compound that easily penetrates intact and live cells. Equal numbers of cells were grown on sterile black 96 well plates. On reaching confluency, growth media was discarded and cells were washed in PBS and pre-exposed to phenol red-free medium for an hour. Cells were divided into control (untreated) and IL-1β (10 ng/mL) treatment groups. Cells were treated with IL-1β for 2, 4 and 6 hours with hydrogen peroxide at 50, 25, 10 mM respectively, as a positive control. Following treatments, cells were then exposed to calcein buffer solution (calcein AM: calcein dilution buffer in 1:500 dilution) and incubated at 37°C for 30 minutes and a fluorometric reading was obtained. Each experiment was repeated five times. Fluorescence intensity was plotted on the Y-axis and represented as % control. Data were expressed as mean ± % SEM and statistical differences among groups were determined by one-way analysis of variance (ANOVA) followed by Bonferroni post hoc test to determine significant differences between specific groups. A 'p' value <0.05 was considered statistically significant.
Each experiment was repeated four times. Data were presented as mean ± S.E.

Single-cell [Ca2+]i imaging
RBMECs were grown on cover slips and incubated with 2 μM Fura-2 AM (ThermoFisher Scientific, Carlsbad, CA) in the culture medium at 37°C for 40 minutes. Ratiometric [Ca2+]i imaging was performed on an IX-81 microscope (Olympus) based system as described previously from individual cells (50). Solution recipes are summarized in Table S1.Data were analyzed using Metafluor software (Universal imaging) and OriginPro 8 software (Origin Lab) and expressed as mean ± S.E.
Evans blue leakage studies by guest on July 9, 2020 http://www.jbc.org/ Downloaded from For this study, C57BL/6 mice (25-30 g) were anesthetized with urethane, i.p. injection (2 mL/kg body weight) followed by Evans blue dye, i.v. injection (2% wt/vol in saline; 4 mL/kg body weight). Evans blue was allowed to circulate in the animal for 30 minutes prior to performing sham surgery (only craniotomy) or TBI using CCI. Animals were grouped into Sham (only craniotomy), Vehicle + Sham (DMSO injection followed by craniotomy), Vehicle + TBI group (DMSO followed by mild TBI), Calpain inhibitor III + TBI group (calpain inhibitor III [10 μg/gram body weight of the animal] injection followed by mild TBI) and a TBI group followed by Calpain inhibitor III treatment. Each group consisted of five animals. One-hour post-TBI, animals were transcardially perfused with sterile saline containing heparin (1000 U/mL) for at least 20 minutes. Briefly, brains were extracted and brain cortex was carefully separated and weighed. Brain cortices were then homogenized in 1 mL of 50% (wt/vol) trichloroacetic acid (TCA) in saline and the homogenate was centrifuged at 6,000g for 20 minutes at 4°C. Supernatants were extracted and diluted in 3 parts of ethanol (1:3; 50% TCA: 95% ethanol). Samples were then quantitated fluorometrically at 620/680 nm (Excitation/Emission) using Biotek Synergy Hybrid H1 spectrophotometer (Winooski, VT). Evans blue concentration in the samples were evaluated using external standards for Evans blue ranging from 50-1000 ng/mL, prepared in same solvent (1:3; TCA: 95% ethanol). Evans blue amount in the samples were expressed as ng/brain cortex ± SEM. Statistical differences among groups were determined by one-way analysis of variance (ANOVA) followed by Bonferroni post hoc test to determine significant differences between specific groups. A value of p<0.05 was considered statistically significant.

Intravital Microscopy
In order to further confirm the findings from the Evans blue dye leakage study described above, BBB integrity/permeability was determined using intravital microscopy imaging. For this study, C57BL/6 mice (25-30 g) were anesthetized with urethane, i.p. injection (2 mL/kg body weight) as described above and divided into three groups as follows: Vehicle + Sham (DMSO + craniotomy only group; n=5), Vehicle + TBI group (DMSO + mild TBI; n=5), and TBI + Calpain inhibitor III group (calpain inhibitor III [10 µg/gram body weight of the animal] injection 15 minutes after mild TBI; n=4). All animals received FITC-dextran (10 kDa; 50 mg/kg BW) via tail vein. Following Sham/TBI, animals were placed under an intravital microscope (Nikon intravital microcope) on a heating pad and their pial microvasculature was observed (under a 40X water immersion lens) for vascular permeability/BBB integrity. The animals were euthanized at the end of each study. The images were taken and the fluorescent intensity within and outside the vessels were determined from multiple areas and analyzed using NES element software. The images that show change in fluorescence intensity at 0 and 60 minutes (10 minutes and 70 minutes after induction of trauma due to the setup time of the experiment and visualization of the vessels) from all the three groups are presented for comparison. Statistical analysis of the data at sixty minutes time point was performed. Statistical differences among groups were determined by one-way analysis of variance (ANOVA) followed by Bonferroni post hoc test to determine significant differences between specific groups. A value of p<0.05 was considered statistically significant.    for each study). The changes in ZO-1 localization and the formation of f-actin stress fibers were further determined using ImageJ software and presented as arbitray units (C and D respectively; p < 0.05). Under normal physiological conditions actin filaments are randomly distributed throughout the cell but agents that induce endothelial hyperpermeability induce them to reorganize themselves into stress fibers that are linear, parallel bundles across the cell interior (following IL-1β treatment as pointed by the arrows in B) and also exhibit increased binding to rhodamine phalloidin (as shown in B and D).