Evans blue dye (EBD) is an inert tracer that measures plasma volume in human subjects and vascular permeability in animal models. Quantitation of EBD can be difficult when dye concentration in the sample is limited, such as when extravasated dye is measured in the blood-brain barrier (BBB) intact brain. The procedure described here used a very small volume (30 µl) per sample replicate, which enabled high-throughput measurements of the EBD concentration based on a standard 96-well plate reader. First, ethanol ensured a consistent optic path length in each well and substantially enhanced the sensitivity of EBD fluorescence spectroscopy. Second, trichloroacetic acid (TCA) removed false-positive EBD measurements as a result of biological solutes and partially extracted EBD into the supernatant. Moreover, a 1:2 volume ratio of 50% TCA ([TCA final] = 33.3%) optimally extracted EBD from the rat plasma protein-EBD complex in vitro and in vivo, and 1:2 and 1:3 weight-volume ratios of 50% TCA optimally extracted extravasated EBD from the rat brain and liver, respectively, in vivo. This procedure is particularly useful in the detection of EBD extravasation into the BBB-intact brain, but it can also be applied to detect dye extravasation into tissues where vascular permeability is less limiting.

Ischemic stroke is a major cause of death and disabilities worldwide, and it has been long hoped that improved understanding of relevant injury mechanisms would yield targeted neuroprotective therapies. While Ca overload during ischemia-induced glutamate excitotoxicity has been identified as a major contributor, failures of glutamate targeted therapies to achieve desired clinical efficacy have dampened early hopes for the development of new treatments. However, additional studies examining possible contributions of Zn, a highly prevalent cation in the brain, have provided new insights that may help to rekindle the enthusiasm. In this review, we discuss both old and new findings yielding clues as to sources of the Zn that accumulates in many forebrain neurons after ischemia, and mechanisms through which it mediates injury. Specifically, we highlight the growing evidence of important Zn effects on mitochondria in promoting neuronal injury. A key focus has been to examine Zn contributions to the degeneration of highly susceptible hippocampal pyramidal neurons. Recent studies provide evidence of differences in sources of Zn and its interactions with mitochondria in CA1 versus CA3 neurons that may pertain to their differential vulnerabilities in disease. We propose that Zn-induced mitochondrial dysfunction is a critical and potentially targetable early event in the ischemic neuronal injury cascade, providing opportunities for the development of novel neuroprotective strategies to be delivered after transient ischemia.

Despite the huge costs of ischemic neuronal injury, neuroprotective interventions in humans remain elusive, in part reflecting incomplete knowledge of the critical early events. Emerging evidence implicates Zn 2+ as an important early contributor. CA1 pyramidal neurons undergo selective delayed degeneration after transient global ischemia (TGI), and Zn 2+ has been implicated in the injury. In vitro studies have indicated that Zn 2+ enters mitochondria and has potent effects on their function. In addition, Zn 2+ accumulates in CA1 mitochondria after ischemia in hippocampal slice and whole animal models, and appears to contribute to their dysfunction. However, the relationship between mitochondrial Zn 2+ accumulation and their disruption has not been examined at the ultrastructural level in vivo, reflecting the difficulty in assessing dynamics of labile (loosely bound) Zn 2+. We employ a cardiac arrest model of ischemia, combined with Timm's sulfide silver labeling, which inserts electron dense metallic silver granules at sites of labile Zn 2+ accumulation, and use transmission electron microscopy (TEM) to examine subcellular loci of the Zn 2+ accumulation. In line with prior studies, TGI induced damage to CA1 was far greater than to CA3 pyramidal neurons, and was substantially progressive in the hours after reperfusion (being significantly greater after 4 than 1 h recovery). Intriguingly, TEM examination of Timm stained sections revealed substantial Zn 2+ accumulation in many post-ischemic CA1 mitochondria, which was strongly correlated with their swelling and disruption. Furthermore, paralleling the evolution of neuronal injury, both the number of mitochondria containing Zn 2+ and the degree of their disruption were far greater at 4 than 1 h recovery. These data provide the first direct characterization of Zn 2+ accumulation in CA1 mitochondria after in vivo TGI, and further support the idea that mitochondria constitute an early and potentially targetable locus of Zn 2+ effects in ischemia that contributes to mitochondrial damage and neuronal injury.

Blood-brain barrier (BBB) disruption is thought to facilitate the development of cerebral infarction after a stroke. In a typical stroke model (such as the one used in this study), the early phase of BBB disruption reaches a peak 6 h post-ischemia and largely recovers after 8–24 h, whereas the late phase of BBB disruption begins 48–58 h post-ischemia. Because cerebral infarct develops within 24 h after the onset of ischemia, and several therapeutic agents have been shown to reduce the infarct volume when administered at 6 h post-ischemia, we hypothesized that attenuating BBB disruption at its peak (6 h post-ischemia) can also decrease the infarct volume measured at 24 h. We used a mouse stroke model obtained by combining 120 min of distal middle cerebral arterial occlusion (dMCAo) with ipsilateral common carotid arterial occlusion (CCAo). This model produced the most reliable BBB disruption and cerebral infarction compared to other models characterized by a shorter duration of ischemia or obtained with dMCAO or CCAo alone. The BBB permeability was measured by quantifying Evans blue dye (EBD) extravasation, as this tracer has been shown to be more sensitive for the detection of early-phase BBB disruption compared to other intravascular tracers that are more appropriate for detecting late-phase BBB disruption. We showed that a 1 h-long treatment with isoflurane-anesthesia induced marked hypothermia and attenuated the peak of BBB disruption when administered 6 h after the onset of dMCAo/CCAo-induced ischemia. We also demonstrated that the inhibitory effect of isoflurane was hypothermia-dependent because the same treatment had no effect on ischemic BBB disruption when the mouse body temperature was maintained at 37°C. Importantly, inhibiting the peak of BBB disruption by hypothermia had no effect on the volume of brain infarct 24 h post-ischemia. In conclusion, inhibiting the peak of BBB disruption is not an effective neuroprotective strategy, especially in comparison to the inhibitors of the neuronal death signaling cascade; these, in fact, can attenuate the infarct volume measured at 24 h post-ischemia when administered at 6 h in our same stroke model.

Blood-brain barrier (BBB) integrity can be determined by tracer infusion into the circulation followed by measurements of its penetration into the brain parenchyma. Tracer injection through the intraperitoneal (i.p.) route (rather than intravascular injection) avoids confounding effects of animal anesthesia or immobilization/surgical stress. Evans blue dye (EBD) can be administered by i.p. injection, and once in circulation, it binds to plasma albumin to become an endogenous protein tracer. Here, we investigated whether a similar level of EBD is extravasated into the brain following i.p. versus intravenous (i.v.) injection in rats. In comparison with i.v. EBD injection, i.p. EBD injection resulted in much of the tracer residing in the peritoneal cavity. Accordingly, comparatively less EBD was found in the blood, liver, or brain of BBB-intact rat. In addition, following unilateral osmotic BBB disruption, i.v. but not i.p. EBD stained the ipsilateral hemisphere blue. Nevertheless, following either route of tracer administration in these rats, spectrophotometric quantification detected more EBD in the ipsilateral (BBB-disrupted) than in the contralateral hemisphere. Taken together, in contrast to a recent report, we found that i.p. EBD resulted in less tracer in circulation and in peripheral/central organs than EBD delivered i.v. We nevertheless conclude that i.p. EBD delivered sufficient tracer for the detection of regional BBB disruption.

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Hypoxic tumor microenvironment (HTM) promotes a more aggressive and malignant state in glioblastoma. However, little is known about the role and mechanism of CXC chemokine ligand 14 (CXCL14) in HTM‐mediated glioblastoma progression. In this study, we report that CXCL14 expression correlated with poor outcomes, tumor grade, and hypoxia‐inducible factor (HIF) expression in patients with glioblastoma. CXCL14 was upregulated in tumor cells within the hypoxic areas of glioblastoma. Hypoxia induced HIF‐dependent expression of CXCL14, which promoted glioblastoma tumorigenicity and invasiveness in vitro and in vivo. Moreover, CXCL14 gain‐of‐function in glioblastoma cells activated insulin‐like growth factor‐1 receptor (IGF‐1R) signal transduction to regulate the growth, invasiveness, and neurosphere formation of glioblastoma. Finally, systemic delivery of CXCL14 siRNA nanoparticles (NPs) with polysorbate 80 coating significantly suppressed tumor growth in vivo and extended the survival time in patient‐derived glioblastoma xenografts. Together, these findings suggest that HIF‐dependent CXCL14 expression contributes to HTM‐promoted glioblastoma tumorigenicity and invasiveness through activation of the IGF‐1R signaling pathway. CXCL14 siRNA NPs as an oligonucleotide drug can inhibit glioblastoma progression and constitute a translational path for the clinical treatment of glioblastoma patients.

The Evans blue dye (EBD) is the most commonly used inert tracer for tracking endogenous plasma protein extravasation into the brain. Even though several methods have been developed for measuring EBD concentration extravasated into central and peripheral tissues, the accuracy and precision of these methods remains poorly defined. First, we measured the absorbance and fluorescence of EBD solutions prepared in 96‐well plates using standard photo‐spectrometer. EBD absorbance increased in a dose‐dependent manner, and logarithm‐transformation revealed a sigmoidal curve with Hill coefficient of 2.086. The steepest slope of the curve lied between 5 µg/ml to 500 µg/ml, and hence this concentration range was the region at which measurements would be most precise. On the other hand, EBD fluorescence also increased in a concentration‐dependent manner, but with region of precision from 0.2 µg/ml to at least 5 µg/ml. Nevertheless, substantial photo‐bleaching of EBD fluorescence was found with increasing concentrations. Thus, our results recommends the use of fluorescent measurements to define EBD concentrations between 0.2 µg/ml to 5 µg/ml, and the use of absorbance measurements to define EBD concentrations between 5µg/ml to 500 µg/ml. Second, we determined the efficiency at which EBD can be retrieved from plasma proteins using the trichloroacetic acid (TCA) extraction technique. The degree of extraction was substantial even with 1:1 plasma‐to‐TCA ratio, and reached maximum with 1:2 ratio where increased ratios failed to further increase EBD extraction. Notably, contrary to earlier reports, we failed to completely extract EBD from plasma sample even with substantial amount of TCA. Third, we determined the sample‐to‐TCA ratio required to maximally extract EBD from central and peripheral tissue in rats receiving intravenous EBD. Taken together, the data presented here will be of great value in guiding future research using EBD as a tool to study central and peripheral vascular permeability. Grant Funding Source: Supported by the National Science Council of Taiwan