Time-Dependent Long-Term Effect of Memantine following Repetitive Mild Traumatic Brain Injury.

Repetitive mild traumatic brain injury (rmTBI, e.g., sports concussions) may be associated with both acute and chronic symptoms and neurological changes. Despite the common occurrence of these injuries, therapeutic strategies are limited. One potentially promising approach is N-methyl-D-aspartate receptor (NMDAR) blockade to alleviate the effects of post-injury glutamatergic excitotoxicity. Initial pre-clinical work using the NMDAR antagonist, memantine, suggests that immediate treatment following rmTBI improves a variety of acute outcomes. It remains unclear (1) whether acute memantine treatment has long-term benefits and (2) whether delayed treatment following rmTBI is beneficial, which are both clinically relevant concerns. To test this, animals were subjected to rmTBI via a weight drop model with rotational acceleration (five hits in 5 days) and randomized to memantine treatment immediately, 3 months, or 6 months post-injury, with a treatment duration of one month. Behavioral outcomes were assessed at 1, 4, and 7 months post-injury. Neuropathological outcomes were characterized at 7 months post-injury. We observed chronic changes in behavior (anxiety-like behavior, motor coordination, spatial learning, and memory), as well as neuroinflammation (microglia, astrocytes) and tau phosphorylation (T231). Memantine treatment, either immediately or 6 months post-injury, appears to confer greater rescue of neuroinflammatory changes (microglia) than vehicle or treatment at the 3-month time point. Although memantine is already being prescribed chronically to address persistent symptoms associated with rmTBI, this study represents the first evidence of which we are aware to suggest a small but durable effect of memantine treatment in mild, concussive injuries. This effect suggests that memantine, although potentially beneficial, is insufficient to treat all aspects of rmTBI alone and should be combined with other therapeutic agents in a multi-therapy approach, with attention given to the timing of treatment.

Mutation in the Bone Morphogenetic Protein signaling pathway sensitize zebrafish and humans to ethanol-induced jaw malformations.

Fetal Alcohol Spectrum Disorders (FASD) describe ethanol-induced developmental defects including craniofacial malformations. While ethanol-sensitive genetic mutations contribute to facial malformations, the impacted cellular mechanisms remain unknown. Bmp signaling is a key regulator of epithelial morphogenesis driving facial development, providing a possible ethanol-sensitive mechanism. We found that zebrafish mutants for Bmp signaling components are ethanol-sensitive and affect anterior pharyngeal endoderm shape and gene expression, indicating ethanol-induced malformations of the anterior pharyngeal endoderm cause facial malformations. Integrating FASD patient data, we provide the first evidence that variants in the human Bmp receptor gene BMPR1B associate with ethanol-related differences in jaw volume. Our results show that ethanol exposure disrupts proper morphogenesis of, and tissue interactions between, facial epithelia that mirror overall viscerocranial shape changes and are predictive for Bmp-ethanol associations in human jaw development. Our data provide a mechanistic paradigm linking ethanol to disrupted epithelial cell behaviors that underlie facial defects in FASD.

Optimizing Choice and Timing of Behavioral Outcome Tests After Repetitive Mild Traumatic Brain Injury: A Machine Learning-Based Approach on Multiple Pre-Clinical Experiments.

Repetitive mild traumatic brain injury (rmTBI) is a potentially debilitating condition with long-term sequelae. Animal models are used to study rmTBI in a controlled environment, but there is currently no established standard battery of behavioral tests used. Primarily, we aimed to identify the best combination and timing of behavioral tests to distinguish injured from uninjured animals in rmTBI studies, and secondarily, to determine whether combinations of independent experiments have better behavioral outcome prediction accuracy than individual experiments. Data from 1203 mice from 58 rmTBI experiments, some of which have already been published, were used. In total, 11 types of behavioral tests were measured by 37 parameters at 13 time points during the first 6 months after injury. Univariate regression analyses were used to identify optimal combinations of behavioral tests and whether the inclusion of multiple heterogenous experiments improved accuracy. k-means clustering was used to determine whether a combination of multiple tests could distinguish mice with rmTBI from uninjured mice. We found that a combination of behavioral tests outperformed individual tests alone when distinguishing animals with rmTBI from uninjured animals. The best timing for most individual behavioral tests was 3-4 months after first injury. Overall, Morris water maze (MWM; hidden and probe frequency) was the behavioral test with the best capability of detecting injury effects (area under the curve [AUC] = 0.98). Combinations of open field tests and elevated plus mazes also performed well (AUC = 0.92), as did the forced swim test alone (AUC = 0.90). In summary, multiple heterogeneous experiments tended to predict outcome better than individual experiments, and MWM 3-4 months after injury was the optimal test, also several combinations also performed well. In order to design future pre-clinical rmTBI trials, we have included an interactive application available online utilizing the data from the study via the Supplementary URL.

Titrating the Translational Relevance of a Low-Level Repetitive Head Impact Model.

Recently, there has been increased attention in the scientific community to the phenomenon of sub-concussive impacts, those hits to the head that do not cause the signs and symptoms of a concussion. Some authors suggest that sub-concussive impacts may alter behavior and cognition, if sustained repetitively, but the mechanisms underlying these changes are not well-defined. Here, we adapt our well-established weight drop model of repetitive mild traumatic brain injury (rmTBI) to attempt to produce a model of low-level repetitive head impacts (RHI). The model was modified to eliminate differences in latency to right following impact and gross behavioral changes after a single cluster of hits. Further, we varied our model in terms of repetition of impact over a 4-h span to mimic the repeated sub-concussive impacts that may be experienced by an athlete within a single day of play. To understand the effects of a single cluster of RHIs, as well as the effect of an increased impact frequency within the cluster, we evaluated classical behavioral measures, serum biomarkers, cortical protein quantification, and immunohistochemistry both acutely and sub-acutely following the impacts. In the absence of gross behavioral changes, the impact protocol did generate pathology, in a dose-dependent fashion, in the brain. Evaluation of serum biomarkers revealed limited changes in GFAP and NF-L, which suggests that their diagnostic utility may not emerge until the exposure to low-level head impacts reaches a certain threshold. Robust decreases in both IL-1ß and IL-6 were observed in the serum and the cortex, indicating downregulation of inflammatory pathways. These experiments yield initial data on pathology and biomarkers in a mouse model of low-level RHIs, with relevance to sports settings, providing a starting point for further exploration of the potential role of anti-inflammatory processes in low-level RHI outcomes, and how these markers may evolve with repeated exposure.

Traumatic Brain Injury-Related Optic Nerve Damage.

Vision disorders are associated with traumatic brain injury (TBI) in 20%-40% of clinical cases and involve a diverse set of potential symptoms that can present acutely or chronically. Due to its structure and position, the optic nerve is vulnerable to multiple forms of primary injury, which can result in traumatic optic neuropathy (TON). Multiple studies have shown that the optic tract may also be injured during TBI, though data regarding the temporospatial resolution of injury to the optic nerve are sparse. We evaluated the time course of optic nerve injury and visual impairments in our closed head impact acceleration mouse model of mild TBI (mTBI) designed to mimic repetitive injuries experienced in the context of sport. Our results show that inflammation and gliosis occur acutely in response to injury. Additionally, indications of optic nerve degeneration and functional loss of vision beginning at 1-month postinjury, and retinal ganglion cell loss at 7 months, revealed that the degeneration is continuous and permanent. Together, this study demonstrated that the optic nerve is vulnerable to damage during mTBI, which can cause TON and vision loss. These findings will be important for clinicians to consider to determine whether optic nerve is injured in the TBI patients with vision problems.

Visual Dysfunction after Repetitive Mild Traumatic Brain Injury in a Mouse Model and Ramifications on Behavioral Metrics.

Mild traumatic brain injury (mTBI) is a major cause of morbidity and mortality with a poorly understood pathophysiology. Animal models have been increasingly utilized to better understand mTBI and recent research has identified visual deficits in these models that correspond to human literature. While visual impairment is being further characterized within TBI, the implications of impaired vision on behavioral tasks commonly utilized in animal models has not been well described thus far. Visual deficits may well confound behavioral tests that are believed to be isolated to cognitive functioning such as learning and memory. We utilized a mouse model of repetitive mTBI (rmTBI) to further characterize visual deficits using an optomotor task, electroretinogram, and visually evoked potential, and located likely areas of damage to the visual pathway. Mice were tested on multiple behavioral metrics, including a touchscreen conditional learning task to better identify the contribution of visual dysfunction to behavioral alterations. We found that rmTBI caused visual dysfunction resulting from damage distal to the retina that likely involves pathology within the optic nerve. Moreover, loss of vision led to poorer performance of rmTBI animals on classic behavioral tests such as the Morris water maze that would otherwise be attributed solely to learning and memory deficits. The touchscreen conditional learning task was able to differentiate rmTBI induced learning and memory dysfunction from visual impairment and is a valuable tool for elucidating subtle changes resulting from TBI.

BBB pathophysiology-independent delivery of siRNA in traumatic brain injury.

Small interfering RNA (siRNA)-based therapeutics can mitigate the long-term sequelae of traumatic brain injury (TBI) but suffer from poor permeability across the blood-brain barrier (BBB). One approach to overcoming this challenge involves treatment administration while BBB is transiently breached after injury. However, it offers a limited window for therapeutic intervention and is applicable to only a subset of injuries with substantially breached BBB. We report a nanoparticle platform for BBB pathophysiology-independent delivery of siRNA in TBI. We achieved this by combined modulation of surface chemistry and coating density on nanoparticles, which maximized their active transport across BBB. Engineered nanoparticles injected within or outside the window of breached BBB in TBI mice showed threefold higher brain accumulation compared to nonengineered PEGylated nanoparticles and 50% gene silencing. Together, our data suggest that this nanoparticle platform is a promising next-generation drug delivery approach for the treatment of TBI.

Increase in Seizure Susceptibility After Repetitive Concussion Results from Oxidative Stress, Parvalbumin-Positive Interneuron Dysfunction and Biphasic Increases in Glutamate/GABA Ratio.

Chronic symptoms indicating excess cortical excitability follow mild traumatic brain injury, particularly repetitive mild traumatic brain injury (rmTBI). Yet mechanisms underlying post-traumatic excitation/inhibition (E/I) ratio abnormalities may differ between the early and late post-traumatic phases. We therefore measured seizure threshold and cortical gamma-aminobutyric acid (GABA) and glutamate (Glu) concentrations, 1 and 6 weeks after rmTBI in mice. We also analyzed the structure of parvalbumin-positive interneurons (PVIs), their perineuronal nets (PNNs), and their electroencephalography (EEG) signature (gamma frequency band power). For mechanistic insight, we measured cortical oxidative stress, reflected in the reduced/oxidized glutathione (GSH/GSSG) ratio. We found that seizure susceptibility increased both early and late after rmTBI. However, whereas increased Glu dominated the E/I 1 week after rmTBI, Glu concentration normalized and the E/I was instead characterized by depressed GABA, reduced per-PVI parvalbumin expression, and reduced gamma EEG power at the 6-week post-rmTBI time point. Oxidative stress was increased early after rmTBI, where transient PNN degradation was noted, and progressed throughout the monitoring period. We conclude that GSH depletion, perhaps triggered by early Glu-mediated excitotoxicity, leads to late post-rmTBI loss of PVI-dependent cortical inhibitory tone. We thus propose dampening of Glu signaling, maintenance of redox state, and preservation of PVI inhibitory capacity as therapeutic targets for post-rmTBI treatment.

Evaluation of the procoagulant properties of a newly developed platelet modified lysate product.

BACKGROUND: Platelet transfusion is associated with logistical problems with the national storage guidelines of platelets. This results in decreased function in vivo as a result of the platelet storage lesion, and complications such as allergic or hemolytic reactions and thrombosis. We evaluated a new, freshly prepared platelet modified lysate (PML) product designed to be more procoagulant than fresh and stored platelets. METHODS: Fresh platelets were concentrated, sonicated, and centrifuged to produce PML. Samples of both washed and unwashed PML were evaluated for particle size, concentration, and activity, and then tested for clot kinetics and thrombin generation. PML samples were also stored at various temperatures for durations up to 6 months and evaluated for clot kinetics and thrombin generation throughout. RESULTS: PML showed significantly higher concentration of platelet microparticles, increased procoagulant properties, and increased thrombin generation as compared to fresh and stored platelets. In addition, PML maintained its clot kinetics over a 6-month storage period with variable storage conditions. CONCLUSIONS: The newly proposed PML product is more procoagulant, stable, and has additional potential applications than currently available platelet products. Further studies will be performed to assess its functions in vivo and to assess thrombotic potential.

Internal Jugular Vein Compression Collar Mitigates Histopathological Alterations after Closed Head Rotational Head Impact in Swine: A Pilot Study.

Recently, there has been increased concern about microstructural brain changes after head trauma. Clinical studies have investigated a neck collar that applies gentle bilateral jugular vein compression, designed to increase intracranial blood volume and brain stiffness during head trauma, which neuroimaging has shown to result in a reduction in brain microstructural alterations after a season of American football and soccer. Here, we utilized a swine model of mild traumatic brain injury to investigate the effects of internal jugular vein (IJV) compression on histopathological outcomes after injury. Animals were randomized to collar treatment (n = 8) or non-collar treatment (n = 6), anesthetized and suspended such that the head was supported by breakable tape. A custom-built device was used to impact the head, thus allowing the head to break the tape and rotate along the sagittal plane. Accelerometer data were collected for each group. Sham injured animals (n = 2) were exposed to anesthesia only. Following single head trauma, animals were euthanized and brains collected for histology. Whole slide immunohistochemistry was analyzed using Qupath software. There was no difference in linear or rotational acceleration between injured collar and non-collar animals (p > 0.05). Injured animals demonstrated higher levels of the phosphorylated tau epitope AT8 (p < 0.05) and the inflammatory microglial marker IBA1 (p < 0.05) across the entire brain, but the effect of injury was markedly reduced by collar treatment (p < 0.05) The current results indicate that internal jugular venous compression protects against histopathological alterations related to closed head trauma exposure.

Automated Quantification of Immunohistochemical Staining of Large Animal Brain Tissue Using QuPath Software.

Large scale unbiased quantification of immunohistochemistry (IHC) is time consuming, expensive, and/or limited in scope. Heterogeneous tissue types such as brain tissue have presented a further challenge to the development of automated analysis, as differing cellular morphologies result in either limited applicability or require large amounts of training tissue for machine-learning methods. Here we present the use of QuPath, a free and open source software, to quantify whole-brain sections stained with the immunohistochemical markers IBA1 and AT8, for microglia and phosphorylated tau respectively. The pixel-based method of analysis herein allows for statistical comparison of global protein expression between brains and generates heat-maps of stain intensity, visualizing stain signal across whole sections and permitting more specific investigation of regions of interest. This method is fast, automated, unbiased, and easily replicable. We compared swine brains that had undergone a closed head traumatic brain injury with brains of sham animals, and found a global increase in both microglial signal expression and phosphorylated tau. We discuss the IHC methods necessary to utilize this analysis and provide detailed instruction on the use of QuPath in the pixel-based analysis of whole-slide images.

Memantine Mitigates Oligodendrocyte Damage after Repetitive Mild Traumatic Brain Injury.

Repetitive mild traumatic brain injury (rmTBI; e.g., sports concussions) is common and results in significant cognitive impairment, white matter injury and increased risk of neurodegeneration. Targeted therapies for rmTBI are lacking, though evidence from other injury models indicates that targeting N-methyl-d-aspartate (NMDA) receptor (NMDAR)-mediated glutamatergic toxicity might mitigate rmTBI-induced injury. We have previously shown that the NMDAR antagonist memantine lessens axonal injury and restores long term potentiation after rmTBI. Here, we evaluated whether the protective effects of memantine include oligodendrocyte specific mechanisms, as prior studies suggest that oligodendrocytes are particularly vulnerable to glutamatergic toxicity. Mice were subjected to rmTBI injury (5 injuries in 5 days) and randomized to treatment with memantine or with vehicle (n = 32/group). At the molecular level, oligodendrocyte counts and function (myelin basic protein, MBP) were assessed by immunohistochemistry and western blot at days 3, 7 and 28 days after the last injury. Axon integrity was assessed by neurofilament light chain (NF-l) expression and axonal ultrastructure was evaluated by electron microscopy. Compared to vehicle-treated mice, memantine-treated mice were protected against oligodendrocyte loss and decreased MBP expression at subacute time points after injury. Memantine treatment also protected against axon damage assessed by NF-l expression. These data suggest that the therapeutic effects of post-concussive NMDAR antagonism may in part work through oligodendrocyte specific mechanisms, which may have implications for long term neurodegenerative sequelae after multiple concussions.

Evaluation of a rapid and automated heparin-induced thrombocytopenia immunoassay.

INTRODUCTION: Heparin-induced thrombocytopenia (HIT) is a potentially life-threatening adverse reaction of heparin. Laboratory evaluation of HIT is often not available within a reasonable time. We evaluated the HemosIL® HIT-Ab(PF4-H) (Instrumentation Laboratory), a rapid, on-demand, fully automated, latex immunoturbidimetric assay (LIA). MATERIALS AND METHODS: Following determination of the LIA's reference interval and cutoff values, a multicenter study was conducted between March 2013 and June 2015. Plasma samples of HIT-suspected patients (n = 632) were collected and evaluated by LIA on the ACL TOP® Family systems (Instrumentation Laboratory), enzyme-linked immunosorbent assays (EIA), and serotonin release assay (SRA). Patient characteristics, medical conditions, comorbidities, laboratory results, and medications were collected via medical chart review. The pretest clinical probability of HIT was also calculated for each patient. RESULTS: Based on the 95% reference interval for healthy donors and HIT-negative patients, a LIA value ≥1.0 U/mL was interpreted positive. The overall agreement of LIA versus EIA and SRA results were 90% (95% CI 88%-92%) and 79% (95% CI 75%-82%), respectively. The negative predictive value for LIA and EIA was comparable (87%) with SRA. The positive and negative percent agreements with the clinical probability were 89% (95% CI 69%-97%) and 86% (95% CI 83%-89%), respectively, with a negative predictive value of 99.6% (95% CI 98%-100%). DISCUSSION: Overall, the LIA results were comparable to those of EIA and SRA. This fully automated assay with a remarkable short analytical turnaround time of <20 minutes can be performed on-demand, which would greatly facilitate more prompt management of HIT.

Placental Chorionic Cyst Fluid Has Prothrombotic Properties and Differs From Amniotic Fluid.

INTRODUCTION: Chorionic cysts of the chorion laeve, fetal chorionic plate, septum, and free membranes have been associated with placental hypoxia, but they have no clear clinical significance. Although immunohistochemistry has identified fibronectin and collagen IV in cyst fluid, the contents have yet to be fully characterized. METHODS: Placental chorionic cysts (N = 10) were sampled by fluid extraction and hemotoxylin and eosin-stained sections. Amniotic fluid samples (N = 8) were obtained from pregnant women who had cytogenetic evaluation. The content of the cysts was tested for thrombogenicity using thromboelastography. The cyst content was tested by Luminex multiplex and ELISA assays and for known prothrombotic and proinflammatory factors. RESULTS: We identified cysts, especially those in the chorionic plate, adjacent to intervillous thrombi with apparent cyst rupture. Thromboelastography revealed a significantly shorter R time compared to whole blood control samples. Concentration of creatinine, α-fetoprotein, and surfactant D in the cyst fluid differed significantly from amniotic fluid. Cyst fluids had a significantly higher expression of all prothrombotic and some proinflammatory factors. DISCUSSION: Our data provide the first evidence that chorionic cyst fluid is prothrombotic and different from amniotic fluid. The association of ruptured cysts with adjacent thrombi and the prothrombotic properties of cyst fluid suggest a causal relationship; however, further studies are needed.

Clinical Nurses' Perceptions of a Senior Capstone Dedicated Educational Unit.

Clinical nurses' perceptions of a senior capstone dedicated educational unit (DEU) model to transition to practice was evaluated in a pilot study. Nursing students were placed in the traditional capstone and the DEU senior capstone unit with clinical nurses. Staff nurses completed an online survey to compare and contrast satisfaction and effectiveness of the models. The results of the study revealed no perceived differences in the outcomes of a DEU experience as compared to the traditional preceptor model. However, nursing management reported an improved sense of leadership and teamwork on the unit with the DEU model.

Decreased Hemolysis and Improved Platelet Function in Blood Components Washed With Plasma-Lyte A Compared to 0.9% Sodium Chloride.

OBJECTIVES: Washing cellular blood products is accepted to ameliorate repeated severe allergic reactions but is associated with RBC hemolysis and suboptimal platelet function. We compared in vitro hemolysis and platelet function in blood components after washing with Plasma-Lyte A (PL-A) vs normal saline (NS). METHODS: RBC (n = 14) were washed/resuspended in NS or PL-A. Free hemoglobin and heme were determined at 0, 24, 48, and 72 hours. Platelet concentrates (PCs; n = 21) were washed with NS or PL-A and resuspended in same washing solution (n = 13) or ABO-identical plasma (n = 8). Platelet aggregation and spreading were evaluated. RESULTS: The 24-hour free hemoglobin and heme levels were higher in NS (P < .05). Improved platelet function was observed in PL-A-washed PCs (P < .001). DISCUSSION: PL-A showed less RBC hemolysis and better platelet function than NS. Whether such differences would occur in vivo is unknown.

Prospective analysis of bleeding events in left ventricular assist device patients.

BACKGROUND: Bleeding is a major cause of morbidity in patients with continuous flow left ventricular assist devices (LVADs). We sought to identify clinical predictors of bleeding within the first year of LVAD implantation. METHODS: A prospective study was performed on 30 patients with HeartMate II implantation at the University of Rochester Medical Center, Rochester, New York, United States. Blood was collected within 1 week before implantation, and at 1, 3, and 30 ± 10 days after implantation. Blood samples were analyzed for prothrombin time (PT), international normalized ratio (INR), von Willebrand factor (vWF) activity, vWF antigen, vWF multimers, collagen binding assay, factor VIII, and epinephrine closure time. The first bleeding event within 1 year of implantation was recorded. RESULTS: There were 17 (57%) patients with a bleeding event. The cumulative incidence of bleeding was 50% at 304 days. Age at the time of LVAD implantation was associated with higher risk of bleeding (hazard ratio (HR) = 1.05, 95% confidence interval (CI) = 1.01-1.10, p = 0.013). Higher baseline INR was also associated with increased risk of bleeding after adjusting for age at the time of implant (HR = 6.58, 95% CI = 1.21-35.70, p = 0.028). The bleeders and non-bleeders had similar hemostatic markers at all four time points. Prior to LVAD, mean epinephrine closure time was similar between bleeders and non-bleeders. However, post LVAD measurement of epinephrine, closure time was frequently limited by platelet clumping. CONCLUSION: Older age and baseline INR are associated with higher risk of bleeding in LVAD patients. Platelet clumping may suggest underlying platelet dysfunction and associated high risk of bleeding.

Oxidation Reduction Potential (ORP) is Predictive of Complications Following Pediatric Cardiac Surgery.

Oxidation reduction potential (ORP) or Redox is the ratio of activity between oxidizers and reducers. Oxidative stress (OS) can cause cellular injury and death, and is important in the regulation of immune response to injury or disease. In the present study, we investigated changes in the redox system as a function of cardiopulmonary bypass (CPB) in pediatric patients. 664 plasma samples were collected from 162 pediatric patients having cardiac surgery of various CPB times. Lower ORP values at 12 h post-CPB were associated with poor survival rate (mean ± SD 167 ± 20 vs. 138 ± 19, p = 0.005) and higher rate of thrombotic complications (153 ± 21 vs. 168 ± 20, p < 0.008). Similarly, patients who developed infections had lower ORP values at 6 h (149 ± 19 vs. 160 ± 22, p = 0.02) and 12 h (156 ± 17 vs. 168 ± 21, p = 0.004) post-CPB. Patients that developed any post-operative complication also had lower 6 h (149 ± 17 vs. 161 ± 23, p = 0.002) and 12 h (157 ± 18 vs. 170 ± 21, p = 0.0007) post-CPB ORP values. Free hemoglobin and IL-6, IL-10, and CRP were not associated with ORP levels. However, higher haptoglobin levels preoperatively were protective against decreases in ORP. Decreased ORP is a marker for poor outcome and predictive of post-operative thrombosis, infection, and other complications in critically ill pediatric cardiac surgery patients. These results suggest that redox imbalance and OS may contribute to the risk of complications and poor outcome in pediatric CBP patients. Haptoglobin may be a marker for increased resilience to OS in this population.