Short-term exposure to JUUL electronic cigarettes can worsen ischemic stroke outcome.

BACKGROUND: The short and long-term health effects of JUUL electronic cigarette (e-Cig) are largely unknown and warrant extensive research. We hypothesized that JUUL exposure could cause cerebrovascular toxicities impacting the progression and outcome of ischemic stroke comparable to tobacco smoke (TS) exposure. METHODS: We exposed male C57 mice to TS/JUUL vapor for 14 days. LCMS/MS was used to measure brain and plasma nicotine and cotinine level. Transient middle cerebral artery occlusion (tMCAO) followed by reperfusion was used to mimic ischemic stroke. Plasma levels of IL-6 and thrombomodulin were assessed by enzyme-linked immunosorbent assay. At the same time, western blotting was used to study blood-brain barrier (BBB) tight junction (TJ) proteins expression and key inflammatory and oxidative stress markers. RESULTS: tMCAO upregulated IL-6 and decreased plasma thrombomodulin levels. Post-ischemic brain injury following tMCAO was significantly worsened by JUUL/TS pre-exposure. TJ proteins expression was also downregulated by JUUL/TS pre-exposure after tMCAO. Like TS, exposure to JUUL downregulated the expression of the antioxidant Nrf2. ICAM-1 was upregulated in mice subjected to tMCAO following pre-exposure to TS or JUUL, with a greater effect of TS than JUUL. CONCLUSIONS: These results suggest that JUUL exposure could negatively impact the cerebrovascular system, although to a lesser extent than TS exposure.

Glutamate Buffering Capacity and Blood-Brain Barrier Protection of Opioid Receptor Agonists Biphalin and Nociceptin.

Opioids play crucial roles in the regulation of many important brain functions including pain, memory, and neurogenesis. Activation of opioid receptors is reported to have neuroprotective effects after ischemic reperfusion injury. The objective of this study was to understand the role of biphalin and nociceptin, opioid receptor agonists, on blood-brain barrier (BBB) integrity during ischemic stroke. In this study, we aimed to measure the effect of biphalin and nociceptin on astrocytic glutamate uptake and on expression of excitatory amino acid transporter to study the indirect role of astrocytes on opioid receptor-mediated BBB protection during in vitro stroke conditions. We used mouse brain endothelial cells (bEnd.3) and primary astrocytes as an in vitro BBB model. Restrictive BBB properties were evaluated by measuring [14C] sucrose paracellular permeability and the redistribution of the tight junction proteins. The protective effect of biphalin and nociceptin on BBB integrity was assessed after exposing cells to oxygen glucose deprivation (OGD) and glutamate. It was observed that combined stress (2 mM glutamate and 2 hours of OGD) significantly reduced glutamate uptake by astrocytes; however, biphalin and nociceptin treatment increased glutamate uptake in primary astrocytes. This suggests a role of increased astrocytic buffering capacity in opioid-meditated protection of the BBB during ischemic stroke. It was also found that the combined stress significantly increased [14C] sucrose paracellular permeability in an in vitro BBB model. Biphalin and nociceptin treatment attenuated the effect of the combined stress, which was reversed by the opioid receptor antagonists, suggesting the role of opioid receptors in biphalin and nociception's BBB modulatory activity. SIGNIFICANT STATEMENT: There is an unmet need for discovering new efficacious therapeutic agents to offset the deleterious effects of ischemic stroke. Given the confirmed roles of opioid receptors in the regulation of central nervous system functions, opioid receptor agonists have been studied as potential neuroprotective options in ischemic conditions. This study adds to the knowledge about the cerebrovascular protective effects of opioid receptor agonists and provides insight about the mechanism of action of these agents.

Repurposing metformin to treat age-related neurodegenerative disorders and ischemic stroke.

Aging is a risk factor for major central nervous system (CNS) disorders. More specifically, aging can be inked to neurodegenerative diseases (NDs) because of its deteriorating impact on neurovascular unit (NVU). Metformin, a first line FDA-approved anti-diabetic drug, has gained increasing interest among researchers for its role in improving aging-related neurodegenerative disorders. Additionally, numerous studies have illustrated metformin's role in ischemic stroke, a cerebrovascular disorder in which the NVU becomes dysfunctional which can lead to permanent life-threatening disabilities. Considering metformin's beneficial preclinical actions on various disorders, and the drug's role in alleviating severity of these conditions through involvement in commonly characterized cellular pathways, we discuss the potential of metformin as a suitable drug candidate for repurposing in CNS disorders.

Nano-synergistic combination of Erlotinib and Quinacrine for non-small cell lung cancer (NSCLC) therapeutics - Evaluation in biologically relevant in-vitro models.

Non-small cell lung cancer (NSCLC), pre-dominant subtype of lung cancer, is a global disorder affecting millions worldwide. One of the early treatments for NSCLC was use of a first-generation tyrosine kinase inhibitor, Erlotinib (Erlo). However, chronic exposure to Erlo led to development of acquired drug resistance (ADR) in NSCLC, limiting the clinical use of Erlo. A potential approach to overcome development of ADR is a multi-drug therapy. It has been previously reported that Erlo and Quinacrine (QA), an anti-malarial drug, can work synergistically to inhibit tumor progression in NSCLC. However, the combination failed at clinical stages, citing lack of efficacy. In this study, an effort has been made to improve the efficacy of Erlo-QA combination via development of nanoformulations, known to enhance therapeutic efficacy of potent chemotherapies. Synergy between Erlo and QA was measured via estimating the combination indices (CI). It was seen that established combination of nanoformulations (CI: 0.25) had better synergy than plain drug solutions (CI: 0.85) in combination. Following extensive in-vitro testing, data were simulated in biologically relevant 3D tumor models. Two tumor models were developed for extensive in-vitro testing, 3D-Spheroids grown in ultra-low attachment culture plates for efficacy evaluation and a 5D-spheroid model in 5D-sphericalplate with capability of growing 750 spheroids/well for protein expression analysis. Extensive studies on these models revealed that combination of Erlo and QA nanoformulations overall had a better effect in terms of synergy enhancement as compared to plain drug combination. Further, effect of combinatorial therapy on molecular markers was evaluated on 5D-Sphericalplate leading to similar effects on synergy enhancement. Results from present study suggests that combination of nanoformulations can improve the synergy between Erlo and QA while reducing the overall therapeutic dose.

The Role of Smoking and Nicotine in the Transmission and Pathogenesis of COVID-19.

Coronavirus disease 2019 (COVID-19), caused by the severe acute respiratory syndrome coronavirus 2 virus, is turning out to be one of the most devastating global pandemics in the history of humankind. There is a shortage of effective therapeutic strategies or preventative vaccines for this disease to date. A rigorous investigation is needed for identifying and developing more effective therapeutic strategies for COVID-19. Angiotensin-converting enzyme 2 (ACE2), a crucial factor in COVID-19 pathogenesis, has been identified as a potential target for COVID-19 treatment. Smoking and vaping are potential risk factors for COVID-19 that are also shown to upregulate ACE2 expression. In this review, we have discussed the pathobiology of COVID-19 in the lungs and brain and the role of ACE2 in the transmission and pathobiology of this disease. Furthermore, we have shown possible interactions between nicotine/smoking and ACE2 in the lungs and brain, which could aggravate the transmission and pathobiology of COVID-19, resulting in a poor disease outcome. SIGNIFICANCE STATEMENT: This review addresses the present global pandemic of coronavirus disease 2019 (COVID-19) with respect to its pathobiology in the lungs and brain. It focuses on the potential negative impact of tobacco and nicotine exposure on the outcomes of this disease by interaction with the angiotensin-converting enzyme 2 receptor. It adds to the time-sensitive and critically important growing knowledge about the risk factors, transmission, pathobiology, and prognosis of COVID-19.

Repurposing Quinacrine for Treatment of Malignant Mesothelioma: In-Vitro Therapeutic and Mechanistic Evaluation.

Malignant mesothelioma (MM) is a rare type of cancer primarily affecting mesothelial cells lining the pleural cavity. In this study, we propose to repurpose quinacrine (QA), a widely approved anti-malarial drug, for Malignant Pleural Mesothelioma (MPM) treatment. QA demonstrates high degree of cytotoxicity against both immortalized and primary patient-derived cell lines with sub-micromolar 50% inhibitory concentration (IC50) values ranging from 1.2 µM (H2452) to 5.03 µM (H28). Further, QA also inhibited cellular migration and colony formation in MPM cells, demonstrated using scratch and clonogenic assays, respectively. A 3D-spheroid cell culture experiment was performed to mimic in-vivo tumor conditions, and QA was reported to be highly effective in this simulated cellular model. Anti-angiogenic properties were also discovered for QA. Autophagy inhibition assay was performed, and results revealed that QA successfully inhibited autophagy process in MPM cells, which has been cited to be one of the survival pathways for MPM. Annexin V real-time apoptosis study revealed significant apoptotic induction in MPM cells following QA treatment. Western blots confirmed inhibition of autophagy and induction of apoptosis. These studies highlight anti-mesothelioma efficacy of QA at low doses, which can be instrumental in developing it as a stand-alone treatment strategy for MPM.

Exosomes in Ischemic Stroke.

Ischemic stroke, a leading cause of mortality, results in severe neurological outcomes in the patients. Effective stroke therapies may significantly decrease the extent of injury. For this purpose, novel and efficient drug delivery strategies need to be developed. Among a myriad of therapeutic and drug delivery techniques, exosomes have shown promising results in ischemic stroke either by their intrinsic therapeutic characteristics, which can result in angiogenesis and neurogenesis or by acting as competent, biocompatible drug delivery vehicles to transport neurotherapeutic agents into the brain. In this review, we have discussed different methods of exosome isolation and cargo loading techniques, advantages and disadvantages of using exosomes as a drug delivery carrier and the therapeutic applications of exosomes with a focus on ischemic stroke therapy.

Novel approaches for the delivery of therapeutics in ischemic stroke.

Here, we review novel approaches to deliver neuroprotective drugs to salvageable penumbral brain areas of stroke injury with the goals of offsetting ischemic brain injury and enhancing recovery.

Development of inhalable quinacrine loaded bovine serum albumin modified cationic nanoparticles: Repurposing quinacrine for lung cancer therapeutics.

Drug repurposing is on the rise as an atypical strategy for discovery of new molecules, involving use of pre-existing molecules for a different therapeutic application than the approved indication. Using this strategy, the current study aims to leverage effects of quinacrine (QA), a well-known anti-malarial drug, for treatment of non-small cell lung cancer (NSCLC). For respiratory diseases, designing a QA loaded inhalable delivery system has multiple advantages over invasive delivery. QA-loaded nanoparticles (NPs) were thus prepared using polyethyleneimine (PEI) as a cationic stabilizer. While the use of PEI provided cationic charge on the particles, it also mediated a burst release of QA and demonstrated potential particle toxicity. These concerns were circumvented by coating nanoparticles with bovine serum albumin (BSA), which retained the cationic charge, reduced NP toxicity and modulated QA release. Prepared nanoparticles were characterized for physicochemical properties along with their aerosolization potential. Therapeutic efficacy of the formulations was tested in different NSCLC cells. Mechanism of higher anti-proliferation was evaluated by studying cell cycle profile, apoptosis and molecular markers involved in the progression of lung cancer. BSA coated QA nanoparticles demonstrated good aerosolization potential with a mass median aerodynamic diameter of significantly less than 5 µm. Nanoparticles also demonstrated improved therapeutic efficacy against NSCLC cells in terms of low IC50 values, cell cycle arrest at G2/M phase and autophagy inhibition leading to increased apoptosis. BSA coated QA NPs also demonstrated enhanced therapeutic efficacy in a 3D cell culture model. The present study thus lays solid groundwork for pre-clinical and eventual clinical studies as a standalone therapy and in combination with existing chemotherapeutics.

Prenatal electronic cigarette exposure decreases brain glucose utilization and worsens outcome in offspring hypoxic-ischemic brain injury.

It has been shown that prenatal nicotine and tobacco smoke exposure can cause different neurobehavioral disorders in the offspring. We hypothesize that prenatal exposure to nicotine-containing electronic cigarette (e-Cig) vapor can predispose newborn to enhanced sensitivity to hypoxic-ischemic (HI) brain injury and impaired motor and cognitive functions. In this study, pregnant CD1 mice were exposed to e-Cig vapor (2.4% nicotine). Primary cortical neurons isolated from e-Cig exposed fetus were exposed to oxygen-glucose deprivation followed by reoxygenation (OGD/R) to mimic HI brain injury. Cell viability and glucose utilization were analyzed in these neurons. HI brain injury was induced in 8-9-day-old pups. Short-term brain injury was evaluated by triphenyltetrazolium chloride staining. Long-term motor and cognitive functions were evaluated by open field, novel object recognition, Morris water maze, and foot fault tests. Western blotting and immunofluorescence were done to characterize glucose transporters in offspring brain. We found that e-Cig exposed neurons demonstrated decreased cell viability and glucose utilization in OGD/R. Prenatally e-Cig exposed pups also had increased brain injury and edema 24 hr after HI brain injury. Further, in utero e-Cig exposed offspring with HI brain injury displayed impaired memory, learning, and motor coordination at adolescence. Additionally, the expression of glucose transporters decreased in e-Cig exposed offspring brain after HI brain injury. These results indicate that reduced glucose utilization can contribute to prenatal e-Cig exposure induced worsened HI brain injury in offspring. This study is instrumental in elucidating the possible deleterious effects of e-Cig use in the general population.

Brain Delivery of a Potent Opioid Receptor Agonist, Biphalin during Ischemic Stroke: Role of Organic Anion Transporting Polypeptide (OATP).

Transporters (expressed) at the blood-brain barrier (BBB) can play an essential role in the treatment of brain injury by transporting neuroprotective substance to the central nervous system. The goal of this study was to understand the role of organic anion transporting polypeptide (OATP1; OATP1A2 in humans and oatp1a4 in rodents) in the transport of a potent opioid receptor agonist, biphalin, across the BBB during ischemic stroke. Brain microvascular endothelial cells (BMECs) that were differentiated from human induced pluripotent stem cells (iPSCs) were used in the present study. The effect of oxygen-glucose deprivation (OGD) and reperfusion on the OATP1 expression, uptake, and transport of biphalin was measured in induced pluripotent stem cells differentiated brain microvascular endothelial cells (iPSC-BMECs) in the presence and absence of an OATP1 substrate, estrone-3-sulfate (E3S). Biphalin brain permeability was quantified while using a highly sensitive liquid chromatography-tandem mass spectrometry (LC-MS/MS) method. It was found that iPSC-BMECs expressed OATP1. In vitro studies showed that biphalin BBB uptake and transport decreased in the presence of an OATP1 specific substrate. It was also observed that OGD and reperfusion modulate the expression and function of OATP1 in BMECs. This study strongly demonstrates that OATP1 contributes to the transport of biphalin across the BBB and increased expression of OATP1 during OGD-reperfusion could provide a novel target for improving ischemic brain drug delivery of biphalin or other potential neurotherapeutics that have affinity to this BBB transporter.

Neurovascular unit transport responses to ischemia and common coexisting conditions: smoking and diabetes.

Transporters at the neurovascular unit (NVU) are vital for the regulation of normal brain physiology via ion, water, and nutrients movement. In ischemic stroke, the reduction of cerebral blood flow causes several complex pathophysiological changes in the brain, one of which includes alterations of the NVU transporters, which can exacerbate stroke outcome by increased brain edema (by altering ion, water, and glutamate transporters), altered energy metabolism (by altering glucose transporters), and enhanced drug toxicity (by altering efflux transporters). Smoking and diabetes are common risk factors as well as coexisting conditions in ischemic stroke that are also reported to change the expression and function of NVU transporters. Coexistence of these conditions could cause an additive effect in terms of the alterations of brain transporters that might lead to worsened ischemic stroke prognosis and recovery. In this review, we have discussed the effects of ischemic stroke, smoking, and diabetes on some essential NVU transporters and how the simultaneous presence of these conditions can affect the clinical outcome after an ischemic episode. Further scientific investigations are required to elucidate changes in NVU transport in cerebral ischemia, which can lead to better, personalized therapeutic interventions tailor-made for these comorbid conditions.

Cyclodextrin modified erlotinib loaded PLGA nanoparticles for improved therapeutic efficacy against non-small cell lung cancer.

This study was aimed at developing a nanoparticle strategy to overcome acquired resistance against erlotinib in non-small cell lung cancer (NSCLC). To load erlotinib on biodegradable PLGA nanoparticles, erlotinib-cyclodextrin (Erlo-CD) complex was prepared using ß-cyclodextrin sulfobutyl ether, which was in turn loaded in the core of PLGA nanoparticles using multiple emulsion solvent evaporation. Nanoparticles were characterized for size distribution, entrapment and loading efficiency, in-vitro release, and therapeutic efficacy against different lung cancer cells. Effect of formulation on cell cycle, apoptosis, and other markers was evaluated using flow cytometry and western blotting studies. The efficacy of optimized nanoformulation was evaluated using a clinically relevant in-vitro 3D-spheroid model. Results showed that Erlo-CD loaded nanoparticles (210 ±â€¯8 nm in size) demonstrated 3-fold higher entrapment (61.5 ±â€¯3.2% vs 21.9 ±â€¯3.7% of plain erlotinib loaded nanoparticles) with ~5% loading efficiency and sustained release characteristics. Developed nanoparticles demonstrated significantly improved therapeutic efficacy against NSCLC cells in terms of low IC50 values and suppressed colony forming ability of cancer cells, increased apoptosis, and autophagy inhibition. Interestingly, 3D spheroid study demonstrated better anticancer activity of Erlo-CD nanoparticles compared to plain erlotinib. Present study has shown a premise to improve therapeutic efficacy against erlotinib-resistant lung cancer using modified nanoErlo formulations.

Nintedanib-cyclodextrin complex to improve bio-activity and intestinal permeability.

Cyclodextrin complex of nintedanib was prepared aiming for increased bio-activity and improved transport across intestinal membrane with reduced p-glycoprotein (p-gp) efflux. Based on preliminary phase solubility studies and molecular modeling, sulfobutyl ether derivative of ß-cyclodextrin (SBE-ß-CD, Captisol®) was selected to prepare inclusion complex. Complexation was confirmed using FTIR, 1H NMR, DSC, and XRD. Bioactivity of the formed complex was tested using lung fibroblast cells, WI-38 for anti-proliferative activity and effect on collagen deposition and cells migration. In-vitro permeability studies were performed using epiIntestinal tissue model to assess the effect of complexation on transport and p-gp efflux. Results of the study demonstrated that cyclodextrin complexation increased stability of nintedanib in PBS (pH 7.4) and simulated intestinal fluid (SIF). Further, bioactivity of nintedanib also improved. Interestingly, complexation has increased transport of nintedanib across intestinal membrane and reduced efflux ratio, suggesting the role of cyclodextrin complexation in modulating p-gp efflux.

Nicotine and electronic cigarette (E-Cig) exposure decreases brain glucose utilization in ischemic stroke.

Previous studies in our laboratory have shown that nicotine exposure decreases glucose transport across the blood-brain barrier in ischemia-reperfusion conditions. We hypothesize that nicotine can also dysregulate brain parenchymal glucose utilization by altering glucose transporters with effects on sensitivity to ischemic stroke. In this study, we investigated the effects of nicotine exposure on neuronal glucose utilization using an in vitro ischemic stroke model. We also tested the effects of e-Cig vaping on ischemic brain glucose utilization using an acute brain slice technique. Primary cortical neurons and brain slices were subjected to oxygen-glucose deprivation followed by reoxygenation to mimic ischemia-reperfusion injury. We estimated brain cell glucose utilization by measuring the uptake of [3 H] deoxy-d-glucose. Immunofluorescence and western blotting were done to characterize glucose transporters (GLUTs) and α7 nicotinic acetylcholine receptor (nAChR) expression. Furthermore, we used a glycolytic stress test to measure the effects of nicotine exposure on neuronal glucose metabolism. We observed that short- and long-term nicotine/cotinine exposure significantly decreased neuronal glucose utilization in ischemic conditions and the non-specific nAChR antagonist, mecamylamine reversed this effect. Nicotine/cotinine exposure also decreased neuronal GLUT1 and up-regulated α7 nAChR expression and decreased glycolysis. Exposure of mice to e-Cig vapor for 7 days likewise decreases brain glucose uptake under normoxic and ischemic conditions along with down-regulation of GLUT1 and GLUT3 expressions. These data support, from a cerebrovascular perspective, that nicotine and/or e-Cig vaping induce a state of glucose deprivation at the neurovascular unit which could lead to enhanced ischemic brain injury and/or stroke risk. OPEN PRACTICES: Open Science: This manuscript was awarded with the Open Materials Badge. For more information see: https://cos.io/our-services/open-science-badges/.

Potential role of myo-inositol to improve ischemic stroke outcome in diabetic mouse.

Brain edema is one of the critical factors causing hightened disability and mortality in stroke patients, which is exaggerated further in diabetic patients. Organic osmolytes could play a critical role in the maintenance of cytotoxic edema. The present study was aimed to assess the role of myo-inositol, an organic osmolyte, on stroke outcome in diabetic and non-diabetic animals. In situ brain perfusion and acute brain slice methods were used to assess transport of myo-inositol across the blood-brain barrier and uptake by brain cells using non-diabetic (C57BL/6) and diabetic (streptozotocin-induced) mice, respectively. In vitro studies were conducted to assess the role of myo-inositol during and after ischemia utilizing oxygen glucose deprivation (OGD) and reperfusion. Further, the expression of transporters, such as SGLT6, SMIT1 and AQP4 were measured using immunofluorescence. Therapeutic efficacy of myo-inositol was evaluated in a transient middle cerebral artery occlusion (tMCAO) mouse model using non-diabetic (C57BL/6) and diabetic (db/db) mice. Myo-inositol release from and uptake in astrocytes and altered expression of myo-inositol transporters at different OGD timepoints revealed the role of myo-inositol and myo-inositol transporters during ischemia reperfusion. Further, hyperglycemic conditions reduced myo-inositol uptake in astrocytes. Interestingly, in in-vivo tMCAO, infarct and edema ratios following 24 h reperfusion decreased in myo-inositol treated mice. These results were supported by improvement in behavioral outcomes in open-field test, corner test and neurological score in both non-diabetic and db/db animals. Our data suggest that myo-inositol and myo-inositol transporters may provide neuroprotection during/following stroke both in non-diabetic and diabetic conditions.

The neuroprotective role of the brain opioid system in stroke injury.

Novel neuroprotective therapies are desperately needed to improve neuronal recovery after ischemic stroke and extend the therapeutic window or offset some of the adverse effects of tissue-type plasminogen activator (tPA). These advances could provide a more effective and safe therapeutic regimen for patients with ischemic stroke. The opioid system has gained intense interest over the past few years and is currently being investigated as a viable target for the pharmacological treatment of stroke. In this review, we focus on different opioid receptors (ORs) and their distribution in the central nervous system (CNS), and the effect of ischemic stroke on their redistribution. We also discuss studies involving the use of the selective and nonselective and/or simultaneous targeting of ORs for neuroprotection during ischemic stroke.

Liposomes for the delivery of streptokinase.

Streptokinase is an efficient thrombolytic agent used to treat thromboembolic disorders. Conventional streptokinase formulations have limited thrombolytic activity and several shortcomings because of their immunogenicity and dose-related side effects including short half-life, lack of tissue targeting and peripheral bleeding. Different liposomal formulations have been explored by researchers in order to improve thrombolytic activity of streptokinase. Liposomal formulations could improve plasma stability, retain drug for longer periods of time in the circulation and promote selective delivery to the thrombus. Side effects of conventional streptokinase formulations, such as immunogenicity and hemorrhage, can also be reduced by using liposomal carriers. In vivo therapeutic efficacy of the liposomal streptokinase has been demonstrated well in animal models. In the present review, we will discuss the potential of different liposomal carriers to improve thrombolytic efficacy of streptokinase.

Zinc oxide nanoparticles: a promising nanomaterial for biomedical applications.

Zinc oxide (ZnO) nanoparticles (NPs) are a promising platform for use in biomedical research, especially given their anticancer and antimicrobial activities. These activities are associated with the ability of ZnO NPs to generate reactive oxygen species (ROS) and induce apoptosis. In addition, ZnO NPs have been successfully exploited as drug carriers for loading and transporting drugs to target sites, thereby reducing unwanted toxicity and off-target effects, and resulting in amplified synergistic effects. Here, we discuss the synthesis and biomedical applications of ZnO NPs.

Blood-Brain Barrier Protection as a Therapeutic Strategy for Acute Ischemic Stroke.

The blood-brain barrier (BBB) is a vital component of the neurovascular unit (NVU) containing tight junctional (TJ) proteins and different ion and nutrient transporters which maintain normal brain physiology. BBB disruption is a major pathological hallmark in the course of ischemic stroke which is regulated by the actions of different factors working at different stages of cerebral ischemia including matrix metalloproteinases (MMPs), inflammatory modulators, vesicular trafficking, oxidative pathways, and junctional-cytoskeletal interactions. These components interact further to disrupt maintenance of both the paracellular and transport barriers of the central nervous system (CNS) to worsen ischemic brain injury and the propensity for hemorrhagic transformation (HT) associated with injury and/or thrombolytic therapy with tissue-type plasminogen activator (tPA). We propose that these complex molecular pathways should be evaluated further so that they could be targeted alone or in combination to protect the BBB during cerebral ischemia. These types of novel interventions should be guided by advanced imaging techniques for better diagnosis of BBB damage which may exert significant therapeutic benefit including the extension of therapeutic window of tPA. This review will focus on the different stages and mechanisms of BBB damage in acute ischemic stroke and novel therapeutic strategies to target those pathways for better therapeutic outcome in stroke.