Matthew L. Davis

Blood–brain barrier dysfunction following traumatic brain injury

Traumatic brain injury is a serious cause of morbidity and mortality worldwide. After traumatic brain injury, the blood–brain barrier, the protective barrier between the brain and the intravascular compartment, becomes dysfunctional, leading to leakage of proteins, fluid, and transmigration of immune cells. As this leakage has profound clinical implications, including edema formation, elevated intracranial pressure and decreased perfusion pressure, much interest has been paid to better understanding the mechanisms responsible for these events. Various molecular pathways and numerous mediators have been found to be involved in the intricate process of regulating blood–brain barrier permeability following traumatic brain injury. This review provides an update to the existing knowledge about the various pathophysiological pathways and advancements in the field of blood–brain barrier dysfunction and hyperpermeability following traumatic brain injury, including the role of various tight junction proteins involved in blood–brain barrier integrity and regulation. We also address pitfalls of existing systems and propose strategies to improve the various debilitating functional deficits caused by this progressive epidemic.

Melatonin Preserves Blood-Brain Barrier Integrity and Permeability via Matrix Metalloproteinase-9 Inhibition.

Microvascular hyperpermeability that occurs at the level of the blood-brain barrier (BBB) often leads to vasogenic brain edema and elevated intracranial pressure following traumatic brain injury (TBI). At a cellular level, tight junction proteins (TJPs) between neighboring endothelial cells maintain the integrity of the BBB via TJ associated proteins particularly, zonula occludens-1 (ZO-1) that binds to the transmembrane TJPs and actin cytoskeleton intracellularly. The pro-inflammatory cytokine, interleukin-1β (IL-1β) as well as the proteolytic enzymes, matrix metalloproteinase-9 (MMP-9) are key mediators of trauma-associated brain edema. Recent studies indicate that melatonin a pineal hormone directly binds to MMP-9 and also might act as its endogenous inhibitor. We hypothesized that melatonin treatment will provide protection against TBI-induced BBB hyperpermeability via MMP-9 inhibition. Rat brain microvascular endothelial cells grown as monolayers were used as an in vitro model of the BBB and a mouse model of TBI using a controlled cortical impactor was used for all in vivo studies. IL-1β (10 ng/mL; 2 hours)-induced endothelial monolayer hyperpermeability was significantly attenuated by melatonin (10 μg/mL; 1 hour), GM6001 (broad spectrum MMP inhibitor; 10 μM; 1 hour), MMP-9 inhibitor-1 (MMP-9 specific inhibitor; 5 nM; 1 hour) or MMP-9 siRNA transfection (48 hours) in vitro. Melatonin and MMP-9 inhibitor-1 pretreatment attenuated IL-1β-induced MMP-9 activity, loss of ZO-1 junctional integrity and f-actin stress fiber formation. IL-1β treatment neither affected ZO-1 protein or mRNA expression or cell viability. Acute melatonin treatment attenuated BBB hyperpermeability in a mouse controlled cortical impact model of TBI in vivo. In conclusion, one of the protective effects of melatonin against BBB hyperpermeability occurs due to enhanced BBB integrity via MMP-9 inhibition. In addition, acute melatonin treatment provides protection against BBB hyperpermeability in a mouse model of TBI indicating its potential as a therapeutic agent for brain edema when established in humans.

Outpatient laparoscopic appendectomy should be the standard of care for uncomplicated appendicitis

BACKGROUND In 2012, a protocol for routine outpatient laparoscopic appendectomy for uncomplicated appendicitis was published reflecting high success, low morbidity, and significant cost savings. Despite this, national data reflect that the majority of laparoscopic appendectomies are performed with overnight admission. This study updates our experience with outpatient appendectomy since our initial report, confirming the efficacy of this approach. METHODS In July 2010, a prospective protocol for outpatient laparoscopic appendectomy was adopted at our institution. Patients were dismissed from the postanesthesia recovery room or day surgery if they met predefined criteria for dismissal. Patients admitted to a hospital room as either full admission or observation status were considered failures of outpatient management. An institutional review board–approved retrospective review of patients undergoing laparoscopic appendectomy for uncomplicated appendicitis from July 2010 through December 2012 was performed to analyze success of outpatient management, postoperative morbidity and mortality, as well as readmission rates. RESULTS Three hundred forty-five patients underwent laparoscopic appendectomy for uncomplicated appendicitis during this time frame. There were 166 men and 179 women, with a mean age of 35 years. Three hundred five patients were managed as outpatients, with a success rate of 88%. Forty patients (12%) were admitted for preexisting comorbidities (15 patients), postoperative morbidity (6 patients), or lack of transportation or home support (19 patients). Twenty-three patients (6.6%) experienced postoperative morbidity. There were no mortalities. Four patients (1%) were readmitted for transient fever, nausea/vomiting, partial small bowel obstruction, and deep venous thrombosis. CONCLUSION Outpatient laparoscopic appendectomy can be performed with a high rate of success, a low morbidity, and a low readmission rate. This study reaffirms our original pilot study and should serve as the basis for a change in the standard of care for appendicitis. LEVEL OF EVIDENCE Therapeutic study, level V.

Tumor necrosis factor-α disruption of brain endothelial cell barrier is mediated through matrix metalloproteinase-9

Traumatic brain injuries cause vascular hyperpermeability. Tumor necrosis factor-α (TNF-α), matrix metalloproteinase-9 (MMP-9), and caspase-3 may be important in these processes but the relationship between them has not been investigated. We hypothesized that TNF-α regulates caspase-3-mediated hyperpermeability and blood brain barrier damage and hyperpermeability directly or indirectly via activation of MMP-9. To test this, rat brain microvascular endothelial cells were treated with TNF-α with or without inhibition of MMP-9. Monolayer permeability was measured, zonula occludens-1 and F-actin configuration were examined, and MMP-9 and caspase-3 activities were quantified. TNF-α increased monolayer permeability, damaged zonula occludens-1, induced filamentous-actin stress fiber formation, and increased both MMP-9 and caspase-3 activities. Inhibition of MMP-9 attenuated these changes. These data highlight a novel link between TNF-α and MMP-9 and show that TNF-α regulated caspase-3-mediated hyperpermeability and vascular damage may be linked to MMP-9 inxa0vitro. These findings augment the understanding of traumatic brain injury and pave the way for improved treatment.

Survival of Atlanto-occipital Dissociation Correlates With Atlanto-occipital Distraction, Injury Severity Score, and Neurologic Status

BACKGROUNDnCase series suggest that atlanto-occipital dissociation (AOD) is a potentially survivable injury. Intuitively, a significant neurologic injury, a high degree of initial distraction, and more severe associated injuries would decrease the likelihood of survival. However, this has never been demonstrated for this injury pattern in a statistically meaningful way. The purpose of this study was to assess the relationship of atlanto-occipital distraction, presence of a complete neurologic injury, and Injury Severity Score (ISS) to the rate of survival in AOD.nnnMETHODSnOne thousand one hundred seventy-four patients from 2005 to 2009 comprehensive trauma database were retrospectively reviewed. Fourteen patients diagnosed with AOD were included in the study. Outcome measures assessed included survival, neurologic status, and ISS. The basion-dens interval (BDI) was measured on the computed tomography scan. Fishers exact test and Wilcoxons test were used to evaluate possible associations.nnnRESULTSnSix patients died with complete, high cervical, spinal cord injuries. Follow-up for survivors ranged from 6 months to 2 years. Mortality was associated with the presence of complete neurologic deficit (p = 0.0047), a high basion-dens interval (>16 mm, p = 0.015), and a high ISS (p = 0.0373).nnnCONCLUSIONSnAOD is a potentially survivable injury; however, there may be identifiable subsets of patients where the injury is so severe that treatment is unlikely to change the outcome. This is the first study to show that the ISS and the presence of a complete neurologic injury correlate with nonsurvivability of this devastating injury. A larger case series would help to generalize the results, given the small sample size.

Reactive Oxygen Species‐Caspase‐3 Relationship in Mediating Blood–Brain Barrier Endothelial Cell Hyperpermeability Following Oxygen–Glucose Deprivation and Reoxygenation

Microvascular hyperpermeability that occurs due to breakdown of the BBB is a major contributor of brain vasogenic edema, following IR injury. In microvascular endothelial cells, increased ROS formation leads to caspase‐3 activation following IR injury. The specific mechanisms, by which ROS mediates microvascular hyperpermeability following IR, are not clearly known. We utilized an OGD‐R in vitro model of IR injury to study this.

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.

Doxycycline attenuates burn-induced microvascular hyperpermeability.

BACKGROUND Burns induce systemic microvascular hyperpermeability resulting in shock, and if untreated, cardiovascular collapse. Damage to the endothelial cell adherens junctional complex plays an integral role in the pathophysiology of microvascular hyperpermeability. We hypothesized that doxycycline, a known inhibitor of matrix metalloproteinases (MMPs), could attenuate burn-induced adherens junction damage and microvascular hyperpermeability. METHODS Male Sprague-Dawley rats were divided into sham, burn, and burn + doxycycline (n = 5). The experimental groups underwent a 30% total body surface area full-thickness burn. Fluorescein isothiocyanate–albumin was administered intravenously. Mesenteric postcapillary venules were examined with intravital microscopy to determine flux of albumin from the intravascular space to the interstitium. Fluorescence intensity was compared between the intravascular space to the interstitium at 30, 60, 80, 100, 120, 140, 160, and 180 minutes after burn. Parallel experiments were performed in which rat lung microvascular endothelial cells were treated with sera from sham or burn animals as well as separate groups pretreated with either doxycycline or a specific inhibitor of MMP-9. Monolayer permeability was determined by fluorescein isothiocyanate albumin-flux across Transwell plates and immunofluorescense staining for the adherens junction protein &bgr;-catenin was performed. Western blot and gelatin zymography were performed to assess MMP-9 level and activity. RESULTS MMP-9 levels were increased after burn. Monolayer permeability was significantly increased with burn serum treatment; this was attenuated with doxycycline as well as the specific MMP-9 inhibitor (p < 0.05). Damage of the endothelial cell adherens junction complex was induced by serum from burned rats, and doxycycline restored the integrity of the adherens junction similar to the MMP-9 inhibitor. Intravital microscopy revealed microvascular hyperpermeability after burn; this was attenuated with doxycycline (p < 0.05). CONCLUSION Burns induce microvascular hyperpermeability via endothelial adherens junction disruption associated with MMP-9, and this is attenuated with doxycycline.

Imaging chronic traumatic brain injury as a risk factor for neurodegeneration

Population‐based studies have supported the hypothesis that a positive history of traumatic brain injury (TBI) is associated with an increased incidence of neurological disease and psychiatric comorbidities, including chronic traumatic encephalopathy, Alzheimers disease, Parkinsons disease, and amyotrophic lateral sclerosis. These epidemiologic studies, however, do not offer a clear definition of that risk, and leave unanswered the bounding criteria for greater lifetime risk of neurodegeneration. Key factors that likely mediate the degree of risk of neurodegeneration include genetic factors, significant premorbid and comorbid medical history (e.g. depression, multiple head injuries and repetitive subconcussive impact to the brain, occupational risk, age at injury, and severity of brain injury). However, given the often‐described concerns in self‐report accuracy as it relates to history of multiple TBIs, low frequency of patient presentation to a physician in the case of mild brain injuries, and challenges with creating clear distinctions between injury severities, disentangling the true risk for neurodegeneration based solely on population‐based studies will likely remain elusive. Given this reality, multiple modalities and approaches must be combined to characterize who are at risk so that appropriate interventions to alter progression of neurodegeneration can be evaluated. This article presents data from a study that highlights uses of neuroimaging and areas of needed research in the link between TBI and neurodegenerative disease.

Oxygen-glucose deprivation and reoxygenation as an in vitro ischemia-reperfusion injury model for studying blood-brain barrier dysfunction.

Ischemia-Reperfusion (IR) injury is known to contribute significantly to the morbidity and mortality associated with ischemic strokes. Ischemic cerebrovascular accidents account for 80% of all strokes. A common cause of IR injury is the rapid inflow of fluids following an acute/chronic occlusion of blood, nutrients, oxygen to the tissue triggering the formation of free radicals. Ischemic stroke is followed by blood-brain barrier (BBB) dysfunction and vasogenic brain edema. Structurally, tight junctions (TJs) between the endothelial cells play an important role in maintaining the integrity of the blood-brain barrier (BBB). IR injury is an early secondary injury leading to a non-specific, inflammatory response. Oxidative and metabolic stress following inflammation triggers secondary brain damage including BBB permeability and disruption of tight junction (TJ) integrity. Our protocol presents an in vitro example of oxygen-glucose deprivation and reoxygenation (OGD-R) on rat brain endothelial cell TJ integrity and stress fiber formation. Currently, several experimental in vivo models are used to study the effects of IR injury; however they have several limitations, such as the technical challenges in performing surgeries, gene dependent molecular influences and difficulty in studying mechanistic relationships. However, in vitro models may aid in overcoming many of those limitations. The presented protocol can be used to study the various molecular mechanisms and mechanistic relationships to provide potential therapeutic strategies. However, the results of in vitro studies may differ from standard in vivo studies and should be interpreted with caution.