Astrocyte Reactivity and Neurodegeneration in the Female Rat Brain Following Alcohol Dependence.

Recent evidence suggests that alcohol use disorder (AUD) may manifest itself differently in women compared to men. Women experience AUDs on an accelerated timeline and may have certain regional vulnerabilities. In male rats, neuronal cell death and astrocyte reactivity are noted following induction of alcohol dependence in an animal model of an AUD. However, the regional and temporal patterns of neurodegeneration and astrocyte reactivity have yet to be fully examined in females using this model. Therefore, adult female rats were exposed to a 4-day binge model of alcohol dependence followed by different periods of abstinence. Histological markers for FluoroJade B, a label of degenerating neurons, and vimentin, a marker for reactive astrocytes, were utilized. The expression of these markers in cortical and limbic regions was quantified immediately after their last dose (e.g., T0), or 2, 7, and 14 days later. Significant neuronal cell death was noted in the entorhinal cortex and the hippocampus, similar to previous reports in males, but also in several cortical regions not previously observed. Vimentin immunoreactivity was noted in the same regions as previously reported, in addition to three novel regions. Vimentin immunoreactivity also occurred at earlier and later time points in some cortical and hippocampal regions. These data suggest that both neuronal cell death and astrocyte reactivity could be more widespread in females compared to males. Therefore, this study provides a framework for specific regions and time points which should be examined in future studies of alcohol-induced damage that include female rats.

Doxycycline prevents blood-brain barrier dysfunction and microvascular hyperpermeability after traumatic brain injury.

The main objective of this study was to determine the cellular and molecular effects of doxycycline on the blood-brain barrier (BBB) and protection against secondary injuries following traumatic brain injury (TBI). Microvascular hyperpermeability and cerebral edema resulting from BBB dysfunction after TBI leads to elevation of intracranial pressure, secondary brain ischemia, herniation, and brain death. There are currently no effective therapies to modulate the underlying pathophysiology responsible for TBI-induced BBB dysfunction and hyperpermeability. The loss of BBB integrity by the proteolytic enzyme matrix metalloproteinase-9 (MMP-9) is critical to TBI-induced BBB hyperpermeability, and doxycycline possesses anti-MMP-9 effect. In this study, the effect of doxycycline on BBB hyperpermeability was studied utilizing molecular modeling (using Glide) in silico, cell culture-based models in vitro, and a mouse model of TBI in vivo. Brain microvascular endothelial cell assays of tight junction protein immunofluorescence and barrier permeability were performed. Adult C57BL/6 mice were subjected to sham versus TBI with or without doxycycline treatment and immediate intravital microscopic analysis for evaluating BBB integrity. Postmortem mouse brain tissue was collected to measure MMP-9 enzyme activity. It was found that doxycycline binding to the MMP-9 active sites have binding affinity of -7.07 kcal/mol. Doxycycline treated cell monolayers were protected from microvascular hyperpermeability and retained tight junction integrity (p < 0.05). Doxycycline treatment decreased BBB hyperpermeability following TBI in mice by 25% (p < 0.05). MMP-9 enzyme activity in brain tissue decreased with doxycycline treatment following TBI (p < 0.05). Doxycycline preserves BBB tight junction integrity following TBI via inhibiting MMP-9 activity. When established in human subjects, doxycycline, may provide readily accessible medical treatment after TBI to attenuate secondary injury.

Exploring blood-brain barrier hyperpermeability and potential biomarkers in traumatic brain injury.

Blood-brain barrier breakdown and associated vascular hyperpermeability leads to vasogenic edema in traumatic brain injury (TBI). Tight junctions maintain blood-brain barrier integrity; their disruption in TBI holds significant promise for diagnosis and treatment. A controlled cortical impactor was used for TBI in mouse studies. Blood was collected 1 h after injury and sent for antibody microarray analysis. Twenty human subjects with radiographic evidence of TBI were enrolled and blood collected within 48 h of admission. Control subjects were individuals with nontrauma diagnoses. The subjects were matched by age and gender. Enzyme-linked immunosorbent assays were performed on each TBI and control sample for tight junction-associated proteins (TJPs), inflammatory markers, and S100ß. Plasma was used to conduct in vitro monolayer permeability studies with human brain endothelial cells. S100ß and the TJP occludin were significantly elevated in TBI plasma in both the murine and human studies. Monolayer permeability studies showed increased hyperpermeability in TBI groups. Plasma from TBI subjects increases microvascular hyperpermeability in vitro. TJPs in the blood may be a potential biomarker for TBI.

A Mouse Controlled Cortical Impact Model of Traumatic Brain Injury for Studying Blood-Brain Barrier Dysfunctions.

Traumatic brain injury (TBI) is one of the leading causes of death and disability worldwide. It is a silently growing epidemic with multifaceted pathogenesis, and current standards of treatments aim to target only the symptoms of the primary injury, while there is a tremendous need to explore interventions that can halt the progression of the secondary injuries. The use of a reliable animal model to study and understand the various aspects the pathobiology of TBI is extremely important in therapeutic drug development against TBI-associated complications. The controlled cortical impact (CCI) model of TBI described here, uses a mechanical impactor to inflict a mechanical injury into the mouse brain. This method is a reliable and reproducible approach to inflict mild, moderate or severe injuries to the animal for studying TBI-associated blood-brain barrier (BBB) dysfunctions, neuronal injuries, brain edema, neurobehavioral changes, etc. The present method describes how the CCI model could be utilized for determining the BBB dysfunction and hyperpermeability associated with TBI. Blood-brain barrier disruption is a hallmark feature of the secondary injury that occur following TBI, frequently associated with leakage of fluid and proteins into the extravascular space leading to vasogenic edema and elevation of intracranial pressure. The method described here focuses on the development of a CCI-based mouse model of TBI followed by the evaluation of BBB integrity and permeability by intravital microscopy as well as Evans Blue extravasation assay.

Measurement of Microvascular Endothelial Barrier Dysfunction and Hyperpermeability In Vitro.

Loss of microvascular endothelial barrier integrity leads to vascular hyperpermeability and vasogenic edema in a variety of disease processes including trauma, ischemia and sepsis. Understanding these principles gives valuable information on pathophysiology and therapeutic drug development. While animal models of traumatic and ischemic injuries are useful to understand vascular dysfunctions associated with such injuries, in vitro barrier integrity assays are reliable and helpful adjuncts to understand the cellular and molecular changes and signaling mechanisms that regulate barrier function. We describe here the endothelial monolayer permeability assay and transendothelial electrical resistance (TEER) measurement as in vitro methods to test changes in microvascular integrity and permeability. These in vitro assays are based on either the measurement of electrical resistance of the monolayer or the quantitative evaluation of fluorescently tagged molecules (e.g., FITC-dextran) that pass through the monolayer when there is damage or breakdown.

Melatonin inhibits thermal injury-induced hyperpermeability in microvascular endothelial cells.

BACKGROUND: Burns induce systemic inflammatory reactions and vascular hyperpermeability. Breakdown of endothelial cell adherens junctions is integral in this process, and reactive oxygen species (ROS) and proteolytic enzymes such as matrix metalloproteinase-9 (MMP-9) play pivotal roles therein. Outside trauma, melatonin has shown to exhibit anti-MMP activity and to be a powerful antioxidant. Consequently, we hypothesized that burn-induced junctional damage and hyperpermeability could be attenuated with melatonin. METHODS: Sprague-Dawley rats were assigned to sham or burn groups. Fluorescein isothiocyanate-bovine albumin was administered intravenously. Venules were examined with intravital microscopy; fluorescence intensities were measured intravascularly and extravascularly. Serum was collected. Rat lung microvascular endothelial cells were grown as monolayers and divided into four groups: sham serum and burn serum with and without melatonin pretreatment. Fluorescein isothiocyanate-bovine albumin flux was measured. Immunofluorescence for adherens junction proteins and staining for actin were performed, and images were captured. Cells were grown on 96 well plates, and ROS species generation following application of burn and sham serum was analyzed with and without melatonin. Statistical analysis was conducted with the Student's t test. RESULTS: Intravital microscopy data revealed an increase in vascular hyperpermeability following burn (p < 0.05). Monolayer permeability was increased with burn serum (p < 0.05); this was attenuated with melatonin (p < 0.05). Immunofluorescence showed damage of rat lung microvascular endothelial cell adherens junctions with burn serum exposure, and melatonin restored integrity. Rhodamine phalloidin staining showed filamentous actin stress fiber formation after burn serum application, and melatonin decreased this. Burn serum significantly increased ROS species generation (p < 0.05), and melatonin negated this (p < 0.05). CONCLUSION: Burns damage endothelial adherens junctions and induce microvascular hyperpermeability; melatonin attenuates this process. This insight into the mechanisms of burn-induced fluid leak suggests the role of ROS and MMP-9 but more importantly hints at the possibility of new treatments to combat vascular hyperpermeability in burns.