Impact of in-utero electronic cigarette exposure on neonatal neuroinflammation, oxidative stress and mitochondrial function.

Introduction: Despite the prevalence of the perception that electronic cigarettes (e-cig) are a safer alternative to tobacco smoke, growing concern about their potential toxic impact warrants adequate investigation focusing on special populations like maternal and pediatric groups. This study evaluated the consequences of maternal e-cig use on neonatal neuroinflammation, oxidative stress, and mitochondrial function in primary cultured neurons and postnatal day (PD) 7 and 90 brain. Methodology: Pregnant CD1 mice were exposed to e-cig vapor (2.4% nicotine) from gestational day 5 (E5) till PD7, and the primary neurons were isolated from pups at E16/17. Cellular total reactive oxygen species (ROS) and mitochondrial superoxide were measured in primary neurons using CM-H2DCFDA and Mitosox red, respectively. Mitochondrial function was assessed by Seahorse XF Cell Mitostress analysis. The level of pro-inflammatory cytokines was measured in primary neurons and PD7 and PD90 brains by RT-PCR and immunobead assay. Western blot analysis evaluated the expression of antioxidative markers (SOD-2, HO-1, NRF2, NQO1) and that of the proinflammatory modulator NF-κB. Results: Significantly higher level of total cellular ROS (p < 0.05) and mitochondrial superoxide (p < 0.01) was observed in prenatally e-cig-exposed primary neurons. We also observed significantly reduced antioxidative marker expression and increased proinflammatory modulator and cytokines expression in primary neurons and PD7 (p < 0.05) but not in PD90 postnatal brain. Conclusion: Our findings suggest that prenatal e-cig exposure induces postnatal neuroinflammation by promoting oxidative stress (OS), increasing cytokines' levels, and disrupting mitochondrial function. These damaging events can alter the fetal brain's immune functions, making such offspring more vulnerable to brain insults.

Studies on diketopiperazine and dipeptide analogs as opioid receptor ligands.

Using the structure of gliotoxin as a starting point, we have prepared two different chemotypes with selective affinity to the kappa opioid receptor (KOR). Using medicinal chemistry approaches and structure-activity relationship (SAR) studies, structural features required for the observed affinity were identified, and advanced molecules with favorable Multiparameter Optimization (MPO) and Ligand Lipophilicity (LLE) profiles were prepared. Using the Thermal Place Preference Test (TPPT), we have shown that compound2 blocks the antinociceptive effect of U50488, a known KOR agonist. Multiple reports suggest that modulation of KOR signaling is a promising therapeutic strategy in treating neuropathic pain (NP). As a proof-of-concept study, we tested compound 2 in a rat model of NP and recorded its ability to modulate sensory and emotional pain-related behaviors. Observed in vitro and in vivo results suggest that these ligands can be used to develop compounds with potential application as pain therapeutics.

Maternal e-cigarette use can disrupt postnatal blood-brain barrier (BBB) integrity and deteriorates motor, learning and memory function: influence of sex and age.

Electronic nicotine delivery systems (ENDS), also commonly known as electronic cigarettes (e-cigs) are considered in most cases as a safer alternative to tobacco smoking and therefore have become extremely popular among all age groups and sex. It is estimated that up to 15% of pregnant women are now using e-cigs in the US which keeps increasing at an alarming rate. Harmful effects of tobacco smoking during pregnancy are well documented for both pregnancy and postnatal health, however limited preclinical and clinical studies exist to evaluate the long-term effects of prenatal e-cig exposure on postnatal health. Therefore, the aim of our study is to evaluate the effect of maternal e-cig use on postnatal blood-brain barrier (BBB) integrity and behavioral outcomes of mice of varying age and sex. In this study, pregnant CD1 mice (E5) were exposed to e-Cig vapor (2.4% nicotine) until postnatal day (PD) 7. Weight of the offspring was measured at PD0, PD7, PD15, PD30, PD45, PD60 and PD90. The expression of structural elements of the BBB, tight junction proteins (ZO-1, claudin-5, occludin), astrocytes (GFAP), pericytes (PDGFRß) and the basement membrane (laminin α1, laminin α4), neuron specific marker (NeuN), water channel protein (AQP4) and glucose transporter (GLUT1) were analyzed in both male and female offspring using western blot and immunofluorescence. Estrous cycle was recorded by vaginal cytology method. Long-term motor and cognitive functions were evaluated using open field test (OFT), novel object recognition test (NORT) and morris water maze test (MWMT) at adolescence (PD 40-45) and adult (PD 90-95) age. In our study, significantly reduced expression of tight junction proteins and astrocyte marker were observed in male and female offspring until PD 90 (P < 0.05). Additionally, prenatally e-cig exposed adolescent and adult offspring showed impaired locomotor, learning, and memory function compared to control offspring (P < 0.05). Our findings suggest that prenatal e-cig exposure induces long-term neurovascular changes of neonates by disrupting postnatal BBB integrity and worsening behavioral outcomes.

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.

Brain Energy Metabolism in Ischemic Stroke: Effects of Smoking and Diabetes.

Proper regulation of energy metabolism in the brain is crucial for maintaining brain activity in physiological and different pathophysiological conditions. Ischemic stroke has a complex pathophysiology which includes perturbations in the brain energy metabolism processes which can contribute to worsening of brain injury and stroke outcome. Smoking and diabetes are common risk factors and comorbid conditions for ischemic stroke which have also been associated with disruptions in brain energy metabolism. Simultaneous presence of these conditions may further alter energy metabolism in the brain leading to a poor clinical prognosis after an ischemic stroke event. In this review, we discuss the possible effects of smoking and/or diabetes on brain glucose utilization and mitochondrial energy metabolism which, when present concurrently, may exacerbate energy metabolism in the ischemic brain. More research is needed to investigate brain glucose utilization and mitochondrial oxidative metabolism in ischemic stroke in the presence of smoking and/or diabetes, which would provide further insights on the pathophysiology of these comorbid conditions and facilitate the development of therapeutic interventions.

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.

Genetic polymorphisms of GSTP1, XRCC1, XPC and ERCC1: prediction of clinical outcome of platinum-based chemotherapy in advanced non-small cell lung cancer patients of Bangladesh.

Inter-individual genetic makeup can trigger variability in platinum-based chemotherapeutic responses and corresponding adverse drug reactions and toxicities. Exploring the genetic causes behind these inter-individual variabilities in platinum-based chemotherapeutic responses by investigating the effects of GSTP1 (rs1695), XRCC1 (rs25487), XPC (rs2228001) and ERCC1 (rs11615) genetic polymorphisms on toxicity and therapeutic response of this treatment among Bangladeshi advanced non-small cell lung cancer (NSCLC) patients was the aim of this study. 285 Clinically proven either stage IIIB or IV (advanced) NSCLC patients aging not less than 18 years old and receiving platinum-based chemotherapy were recruited to assess the influence of these four single nucleotide polymorphisms (SNPs) on peripheral leukocytes. Toxicity and response were evaluated by multivariate regression analyses using SPSS statistical software (version 17.0). XRCC1 (rs25487) polymorphism was found to act as a predictive factor for not only grade 3 and 4 anemia (p = 0.008), neutropenia (p = 0.010), thrombocytopenia (p = 0.025) and gastrointestinal toxicity (p = 0.002) but also for therapeutic response (p = 0.012) in platinum-based chemotherapy. Although GSTP1 (rs1695) polymorphism might serve as prognostic factor regarding grade 3 or 4 neutropenia, a significant (p = 0.044) improvement in response to platinum-based chemotherapy was observed. However, XPC (rs2228001) and ERCC1 (rs11615) polymorphisms could not establish any significant relation with toxicity or therapeutic response. XRCC1 (rs2228001) and GSTP1 (rs1695) polymorphisms might explain platinum-induced clinical outcomes in terms of both toxicity and therapeutic response variations among Bangladeshi advanced NSCLC patients.

The cerebrovascular and neurological impact of chronic smoking on post-traumatic brain injury outcome and recovery: an in vivo study.

BACKGROUND: Traumatic brain injury (TBI) is among the most prevalent causes of cerebrovascular and neurological damage worldwide. To this end, tobacco smoke (TS) has been shown to promote vascular inflammation, neurovascular impairments, and risk of cerebrovascular and neurological disorders through oxidative stress (OS) stimuli targeting the blood-brain barrier (BBB) endothelium among others. It has been recently suggested that premorbid conditions such as TS may exacerbate post-TBI brain damage and impact recovery. METHODS: Our study investigated the mechanisms underlying the exacerbation of TBI injury by TS using a weight drop model. For this purpose, male C57BL/6J mice, age range 6-8 weeks, were chronically exposed to premorbid TS for 3 weeks. Test animals were then subjected to TBI by guided vertical head weight drop using a 30 g metal weight free felling from an 80 cm distance before reaching the target. We analyzed the physical activity and body weight of the mice before TBI and 1 h, 24 h, and 72 h post-injury. Finally, mice were sacrificed to collect blood and brain samples for subsequent biochemical and molecular analysis. Western blotting was applied to assess the expression of Nrf2 (a critical antioxidant transcription factor) as well as tight junction proteins associated with BBB integrity including ZO-1, Occludin, and Claudin-5 from brain tissues homogenates. Levels of NF-kB (a pro-inflammatory transcript factor which antagonizes Nrf2 activity) and pro-inflammatory cytokines IL-6, IL-10, and TNF-α were assessed in blood samples. RESULTS: Our data revealed that premorbid TS promoted significantly increased inflammation and loss of BBB integrity in TBI when compared to TS-Free test mice. Additionally, mice chronically exposed to TS before TBI experienced a more significant weight loss, behavioral and motor activity deficiency, and slower post-TBI recovery when compared to TS-free TBI mice. CONCLUSION: The effects of premorbid TS appear consequential to the abrogation of physiological antioxidative and anti-inflammatory response to TBI leading to worsening impairments of the BBB, OS damage, and inflammation. These factors are also likely responsible for the retardation of post-traumatic recovery observed in these animals.

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.

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.

Nicotinamide Mononucleotide: Exploration of Diverse Therapeutic Applications of a Potential Molecule.

Nicotinamide mononucleotide (NMN) is a nucleotide that is most recognized for its role as an intermediate of nicotinamide adenine dinucleotide (NAD+) biosynthesis. Although the biosynthetic pathway of NMN varies between eukaryote and prokaryote, two pathways are mainly followed in case of eukaryotic human-one is through the salvage pathway using nicotinamide while the other follows phosphorylation of nicotinamide riboside. Due to the unavailability of a suitable transporter, NMN enters inside the mammalian cell in the form of nicotinamide riboside followed by its subsequent conversion to NMN and NAD+. This particular molecule has demonstrated several beneficial pharmacological activities in preclinical studies, which suggest its potential therapeutic use. Mostly mediated by its involvement in NAD+ biosynthesis, the pharmacological activities of NMN include its role in cellular biochemical functions, cardioprotection, diabetes, Alzheimer's disease, and complications associated with obesity. The recent groundbreaking discovery of anti-ageing activities of this chemical moiety has added a valuable essence in the research involving this molecule. This review focuses on the biosynthesis of NMN in mammalian and prokaryotic cells and mechanism of absorption along with the reported pharmacological activities in murine model.

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.

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.

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/.

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.

A convenient UHPLC-MS/MS method for routine monitoring of plasma and brain levels of nicotine and cotinine as a tool to validate newly developed preclinical smoking model in mouse.

BACKGROUND: A sensitive, rapid and selective UHPLC-MS/MS method has been developed and validated for the quantification of Nicotine (NT) and Cotinine (CN) using Continine-d 3 as internal standard (IS) as per FDA guidelines. Sample preparation involved simple protein precipitation of 20 µL mouse plasma or brain homogenate using acetonitrile at 1:8 ratio. Mass Spectrometer was operated in positive polarity under the multiple reaction-monitoring mode using electro spray ionization technique and the transitions of m/z 163.2 â†’ 132.1, 177.2 â†’ 98.0 and 180.2 â†’ 101.2 were used to measure the NT, CN and IS, respectively. The elution of NT, CN and IS are at 1.89, 1.77 and 1.76 min, respectively. This was achieved with a gradient mobile phase consisting of 5 mM ammonium bicarbonate, acetonitrile and methanol (3:1, v/v) at a flow rate of 0.3 mL/min on a Kinetex EVO C18 column. The method was validated with a lower limit of quantitation 3.0 ng/mL in mouse plasma and brain for both the analytes. RESULTS: A linear response function was established for the range of concentrations 3-200 (r > 0.995) for NT and 3-600 ng/mL (r > 0.995) for CN. The intra- and inter-day precision values met the acceptance criteria. NT and CN are stable in the battery of stability studies viz., stock solution, bench-top and auto-sampler. CONCLUSION: This method was successfully utilized to validate a newly developed preclinical smoking model in mice.

Offsetting the impact of smoking and e-cigarette vaping on the cerebrovascular system and stroke injury: Is Metformin a viable countermeasure?

Recently published in vitro and in vivo findings strongly suggest that BBB impairment and increased risk for stroke by tobacco smoke (TS) closely resemble that of type-2 diabetes (2DM) and develop largely in response to common key modulators such oxidative stress (OS), inflammation and alterations of the endogenous antioxidative response system (ARE) regulated by the nuclear factor erythroid 2-related factor (Nrf2). Preclinical studies have also shown that nicotine (the principal e-liquid's ingredient used in e-cigarettes) can also cause OS, exacerbation of cerebral ischemia and secondary brain injury. Herein we provide evidence that likewise to TS, chronic e-Cigarette (e-Cig) vaping can be prodromal to the loss of blood-brain barrier (BBB) integrity and vascular inflammation as well as act as a promoting factor for the onset of stroke and worsening of post-ischemic brain injury. In addition, recent reports have shown that Metformin (MF) treatment before and after ischemic injury reduces stress and inhibits inflammatory responses. Recent published data by our group revealead that MF promotes the activation of counteractive mechanisms mediated by the activation of Nrf2 which drastically reduce TS toxicity at the brain and cerebrovascular levels and protect BBB integrity. In this study we provide additional in vivo evidence showing that MF can effectively reduce the oxidative and inflammatory risk for stroke and attenuate post-ischemic brain injury promoted by TS and e-Cig vaping. Our data also suggest that MF administration could be extended as prophylactic care during the time window required for the renormalization of the risk levels of stroke following smoking cessation thus further studies in that direction are warrated.

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.