9-cis beta-carotene-enriched diet significantly improved cognition and decreased Alzheimer's disease neuropathology and neuroinflammation in Alzheimer's disease-like mouse models.

A significant progressive decline in beta-carotene (ßC) levels in the brain is associated with cognitive impairment and a higher prevalence of Alzheimer's disease (AD). In this study, we investigated whether the administration of 9-cis beta-carotene (9CBC)-rich powder of the alga Dunaliella bardawil, the best-known source of ßC in nature, inhibits the development of AD-like neuropathology and cognitive deficits. We demonstrated that in 3 AD mouse models, Tg2576, 5xFAD, and apoE4, 9CBC treatment improved long- and short-term memory, decreased neuroinflammation, and reduced the prevalence of ß-amyloid plaques and tau hyperphosphorylation. These findings suggest that 9CBC has the potential to be an effective preventive and symptomatic AD therapy.

Application of Delayed Contrast Extravasation Magnetic Resonance Imaging for Depicting Subtle Blood-Brain Barrier Disruption in a Traumatic Brain Injury Model.

The blood-brain barrier (BBB) is composed of brain microvasculature that provides selective transport of solutes from the systemic circulation into the central nervous system to protect the brain and spinal microenvironment. Damage to the BBB in the acute phase after traumatic brain injury (TBI) is recognized as a major underlying mechanism leading to secondary long-term damage. Because of the lack of technological ability to detect subtle BBB disruption (BBBd) in the chronic phase, however, the presence of chronic BBBd is disputable. Thus, the dynamics and course of long-term BBBd post-TBI remains elusive. Thirty C57BL/6 male mice subjected to TBI using our weight drop closed head injury model and 19 naïve controls were scanned by magnetic resonance imaging (MRI) up to 540 days after injury. The BBB maps were calculated from delayed contrast extravasation MRI (DCM) with high spatial resolution and high sensitivity to subtle BBBd, enabling depiction and quantification of BBB permeability. At each time point, 2-6 animals were sacrificed and their brains were extracted, sectioned, and stained for BBB biomarkers including: blood microvessel coverage by astrocyte using GFAP, AQP4, ZO-1 gaps, and IgG leakage. We found that DCM provided depiction of subtle yet significant BBBd up to 1.5 years after TBI, with significantly higher sensitivity than standard contrast-enhanced T1-weighted and T2-weighted MRI (BBBd volumes main effect DCM/T1/T2 p < 0.0001 F(2,70) = 107.3, time point p < 0.0001 F(2,133, 18.66) = 23.53). In 33% of the cases, both in the acute and chronic stages, there was no detectable enhancement on standard T1-MRI, nor detectable hyperintensities on T2-MRI, whereas DCM showed significant BBBd volumes. The BBBd values of TBI mice at the chronic stage were found significantly higher compared with age matched naïve animals at 30, 60, and 540 days. The calculated BBB maps were histologically validated by determining significant correlation between the calculated levels of disruption and a diverse set of histopathological parameters obtained from different brain regions, presenting different components of the BBB. Cumulative evidence from recent years points to BBBd as a central component of the pathophysiology of TBI. Therefore, it is expected that routine use of highly sensitive non-invasive techniques to measure BBBd, such as DCM with advanced analysis methods, may enhance our understanding of the changes in BBB function after TBI. Application of the DCM technology to other CNS disorders, as well as to normal aging, may shed light on the involvement of chronic subtle BBBd in these conditions.

The longitudinal biochemical profiling of TBI in a drop weight model of TBI.

Traumatic brain injury (TBI) is a major cause of mortality and disability worldwide, particularly among individuals under the age of 45. It is a complex, and heterogeneous disease with a multifaceted pathophysiology that remains to be elucidated. Metabolomics has the potential to identify metabolic pathways and unique biochemical profiles associated with TBI. Herein, we employed a longitudinal metabolomics approach to study TBI in a weight drop mouse model to reveal metabolic changes associated with TBI pathogenesis, severity, and secondary injury. Using proton nuclear magnetic resonance (1H NMR) spectroscopy, we biochemically profiled post-mortem brain from mice that suffered mild TBI (N = 25; 13 male and 12 female), severe TBI (N = 24; 11 male and 13 female) and sham controls (N = 16; 11 male and 5 female) at baseline, day 1 and day 7 following the injury. 1H NMR-based metabolomics, in combination with bioinformatic analyses, highlights a few significant metabolites associated with TBI severity and perturbed metabolism related to the injury. We report that the concentrations of taurine, creatinine, adenine, dimethylamine, histidine, N-Acetyl aspartate, and glucose 1-phosphate are all associated with TBI severity. Longitudinal metabolic observation of brain tissue revealed that mild TBI and severe TBI lead distinct metabolic profile changes. A multi-class model was able to classify the severity of injury as well as time after TBI with estimated 86% accuracy. Further, we identified a high degree of correlation between respective hemisphere metabolic profiles (r > 0.84, p < 0.05, Pearson correlation). This study highlights the metabolic changes associated with underlying TBI severity and secondary injury. While comprehensive, future studies should investigate whether: (a) the biochemical pathways highlighted here are recapitulated in the brain of TBI sufferers and (b) if the panel of biomarkers are also as effective in less invasively harvested biomatrices, for objective and rapid identification of TBI severity and prognosis.

BBB opening by low pulsed electric fields, depicted by delayed-contrast MRI, enables efficient delivery of therapeutic doxorubicin doses into mice brains.

BACKGROUND: Pharmacological treatment of CNS diseases is limited due to the presence of the blood-brain barrier (BBB). Recent years showed significant advancement in the field of CNS drug delivery enablers, with technologies such as MR-guided focused ultrasound reaching clinical trials. This have inspired researchers in the field to invent novel brain barriers opening (BBo) technologies that are required to be simple, fast, safe and efficient. One such technology, recently developed by us, is BDF (Barrier Disrupting Fields), based on low pulsed electric fields (L-PEFs) for opening the BBB in a controlled, safe, reversible and non-invasive manner. Here, we conducted an in vivo study to show that BDF is a feasible technology for delivering Doxorubicin (Doxo) into mice brain. Means for depicting BBBo levels were developed and applied for monitoring the treatment and predicting response. Overall, the goals of the presented study were to demonstrate the feasibility for delivering therapeutic Doxo doses into naïve and tumor-bearing mice brains and applying delayed-contrast MRI (DCM) for monitoring the levels of BBBo. METHODS: L-PEFs were applied using plate electrodes placed on the intact skull of naïve mice. L-PEFs/Sham mice were scanned immediately after the procedure by DCM ("MRI experiment"), or injected with Doxo and Trypan blue followed by delayed (4 h) perfusion and brain extraction ("Doxo experiment"). Doxo concentrations were measured in brain samples using confocal microscopy and compared to IC50 of Doxo in glioma cell lines in vitro. In order to map BBBo extent throughout the brain, pixel by pixel MR image analysis was performed using the DCM data. Finally, the efficacy of L-PEFs in combination with Doxo was tested in nude mice bearing intracranial human glioma tumors. RESULTS: Significant amount of Doxo was found in cortical regions of all L-PEFs-treated mice brains (0.50 ± 0.06 µg Doxo/gr brain) while in Sham brains, Doxo concentrations were below or on the verge of detection limit (0.03 ± 0.02 µg Doxo/gr brain). This concentration was x97 higher than IC50 of Doxo calculated in gl261 mouse glioma cells and x8 higher than IC50 of Doxo calculated in U87 human glioma cells. DCM analysis revealed significant BBBo levels in the cortical regions of L-PEFs-treated mice; the average volume of BBBo in the L-PEFs-treated mice was x29 higher than in the Sham group. The calculated BBBo levels dropped exponentially as a function of BBBo threshold, similarly to the electric fields distribution in the brain. Finally, combining non-invasive L-PEFs with Doxo significantly decreased brain tumors growth rates in nude mice. CONCLUSIONS: Our results demonstrate significant BBBo levels induced by extra-cranial L-PEFs, enabling efficient delivery of therapeutic Doxo doses into the brain and reducing tumor growth. As BBBo was undetectable by standard contrast-enhanced MRI, DCM was applied to generate maps depicting the BBBo levels throughout the brain. These findings suggest that BDF is a promising technology for efficient drug delivery into the brain with important implications for future treatment of brain cancer and additional CNS diseases.

Definition of the contribution of an Osteopontin-producing CD11c+ microglial subset to Alzheimer's disease.

Alzheimer's disease (AD) is the most common form of incurable dementia and represents a critical public health issue as the world's population ages. Although microglial dysregulation is a cardinal feature of AD, the extensive heterogeneity of these immunological cells in the brain has impeded our understanding of their contribution to this disease. Here, we identify a pathogenic microglial subset which expresses the CD11c surface marker as the sole producer of Osteopontin (OPN) in the 5XFAD mouse model of AD. OPN production divides Disease-Associated Microglia (DAM) into two functionally distinct subsets, i.e., a protective CD11c+OPN- subset that robustly ingests amyloid ß (Aß) in a noninflammatory fashion and a pathogenic CD11c+OPN+ subset that produces proinflammatory cytokines and fails to ingest significant amounts of Aß. Genetic ablation of OPN or administration of monoclonal anti-OPN antibody to 5XFAD mice reduces proinflammatory microglia, plaque formation, and numbers of dystrophic neurites and results in improved cognitive function. Analysis of brain tissue from AD patients indicates that levels of OPN-producing CD11c+ microglia correlate strongly with the degree of cognitive deficit and AD neuropathology. These findings define an OPN-dependent pathway to disease driven by a distinct microglial subset, and identify OPN as a novel therapeutic target for potentially effective immunotherapy to treat AD.

Potential neurotoxicity of titanium implants: Prospective, in-vivo and in-vitro study.

Titanium dioxide (TiO2) is a frequently used biomaterial, particularly in orthopedic and dental implants, and it is considered an inert and benign compound. This has resulted in toxicological scrutiny for TiO2 in the past decade, with numerus studies showing potential pathologic downstream effects. Herein we describe case report of a 77-year-old male with subacute CNS dysfunction, secondary to breakdown of a titanium-based carotid stent and leading to blood levels 1000 times higher (3 ppm) than the reported normal. We prospectively collected tissues adjacent to orthopedic implants and found a positive correlation between titanium concentration and time of implant in the body (r = 0.67, p < 0.02). Rats bearing titanium implants or intravascularly treated with TiO2 nanoparticles (TiNP) exhibited memory impairments. A human blood-brain barrier (BBB) in-vitro model exposed to TiNP showed paracellular leakiness, which was corroborated in-vivo with the decrease of key BBB transcripts in isolated blood vessels from hippocampi harvested from TiNP-treated mice. Titanium particles rapidly internalized into brain-like endothelial cells via caveolae-mediated endocytosis and macropinocytosis and induced pro-inflammatory reaction with increased expression of pro-inflammatory genes and proteins. Immune reaction was mediated partially by IL-1R and IL-6. In summary, we show that high levels of titanium accumulate in humans adjacent to orthopedic implants, and our in-vivo and in-vitro studies suggest it may be neurotoxic.

Endothelial Iron Homeostasis Regulates Blood-Brain Barrier Integrity via the HIF2α-Ve-Cadherin Pathway.

The objective of this study was to investigate the molecular response to damage at the blood brain barrier (BBB) and to elucidate critical pathways that might lead to effective treatment in central nervous system (CNS) pathologies in which the BBB is compromised. We have used a human, stem-cell derived in-vitro BBB injury model to gain a better understanding of the mechanisms controlling BBB integrity. Chemical injury induced by exposure to an organophosphate resulted in rapid lipid peroxidation, initiating a ferroptosis-like process. Additionally, mitochondrial ROS formation (MRF) and increase in mitochondrial membrane permeability were induced, leading to apoptotic cell death. Yet, these processes did not directly result in damage to barrier functionality, since blocking them did not reverse the increased permeability. We found that the iron chelator, Desferal© significantly decreased MRF and apoptosis subsequent to barrier insult, while also rescuing barrier integrity by inhibiting the labile iron pool increase, inducing HIF2α expression and preventing the degradation of Ve-cadherin specifically on the endothelial cell surface. Moreover, the novel nitroxide JP4-039 significantly rescued both injury-induced endothelium cell toxicity and barrier functionality. Elucidating a regulatory pathway that maintains BBB integrity illuminates a potential therapeutic approach to protect the BBB degradation that is evident in many neurological diseases.

Dietary alpha linolenic acid in pregnant mice and during weaning increases brain docosahexaenoic acid and improves recognition memory in the offspring.

Docosahexaenoic acid (DHA) is critical for normal brain development and function. DHA is in danger of being significantly reduced in the human food supply, and the question of whether its metabolic precursor, the essential n-3 alpha linolenic acid (ALA) during pregnancy, can support fetal brain DHA levels for optimal neurodevelopment, is fundamental. Female mice were fed either ALA-enriched or Control diet during pregnancy and lactation. The direct effect of maternal dietary ALA on lipids was analyzed in liver, red blood cells, brain and brain vasculature, together with genes of fatty acid metabolism and transport in three-week-old offspring. The long-term effect of maternal dietary ALA on brain fatty acids and memory was studied in 19-week-old offspring. Three-week-old ALA offspring showed higher levels of n-3 fatty acids in liver, red blood cell, blood-brain barrier (BBB) vasculature and brain parenchyma, DHA enrichment in brain phospholipids and higher gene and protein expression of the DHA transporter, major facilitator superfamily domain containing 2a, compared to Controls. 19-week-old ALA offspring showed higher brain DHA levels and better memory performance than Controls. The increased brain DHA levels induced by maternal dietary ALA during pregnancy-lactation, together with the up-regulated levels of major facilitator superfamily domain containing 2a, may indicate a mode for greater DHA uptake with long-term impact on better memory in ALA offspring.

Non-Invasive Low Pulsed Electrical Fields for Inducing BBB Disruption in Mice-Feasibility Demonstration.

The blood-brain barrier (BBB) is a major hurdle for the treatment of central nervous system disorders, limiting passage of both small and large therapeutic agents from the blood stream into the brain. Thus, means for inducing BBB disruption (BBBd) are urgently needed. Here, we studied the application of low pulsed electrical fields (PEFs) for inducing BBBd in mice. Mice were treated by low PEFs using electrodes pressed against both sides of the skull (100-400 square 50 µs pulses at 4 Hz with different voltages). BBBd as a function of treatment parameters was evaluated using MRI-based treatment response assessment maps (TRAMs) and Evans blue extravasation. A 3D numerical model of the mouse brain and electrodes was constructed using finite element software, simulating the electric fields distribution in the brain and ensuring no significant temperature elevation. BBBd was demonstrated immediately after treatment and significant linear regressions were found between treatment parameters and the extent of BBBd. The maximal induced electric field in the mice brains, calculated by the numerical model, ranged between 62.4 and 187.2 V/cm for the minimal and maximal applied voltages. These results demonstrate the feasibility of inducing significant BBBd using non-invasive low PEFs, well below the threshold for electroporation.

Pharmacological blockers of CCR5 and CXCR4 improve recovery after traumatic brain injury.

CCR5 and CXCR4 are structurally related chemokine receptors that belong to the superfamily of G-protein coupled receptors through which the HIV virus enters and infects cells. Both receptors are also related to HIV-associated neurocognitive disorders that include difficulties in concentration and memory, impaired executive functions, psychomotor slowing, depression and irritability, which are also hallmarks of the long-term sequelae of TBI. Moreover, A growing body of evidence attributes negative influences to CCR5 activation on cognition, particularly after stroke and traumatic brain injury (TBI). Here we investigated the effect of their blockage on motor and cognitive functions, on brain tissue loss and preservation and on some of the biochemical pathways involved. We examined the effect of maraviroc, a CCR5 antagonist used in HIV patients as a viral entry inhibitor, and of plerixafor (AMD3100), a CXCR4 antagonist used in cancer patients as an immune-modulator, on mice subjected to closed head injury (CHI). Mice were treated with maraviroc or plerixafor after CHI for the following 4 or 5 days, respectively. Neurobehavior was assessed according to the Neurological Severity Score; cognitive tests were performed by using the Y-maze, Barnes maze and the novel object recognition test; anxiety was evaluated with the open field test. The mice were sacrificed and brain tissues were collected for Western blot, pathological and immunohistochemical analyses. Both drugs enhanced tissue preservation in the cortex, hippocampus, periventricular areas, corpus callosum and striatum, and reduced astrogliosis)GFAP expression). They also increased the levels of synaptic cognition-related signaling molecules such as phosphorylated NR1 and CREB, and the synaptic plasticity protein PSD95. Both treatments also enhanced the expression of CCR5 and CXCR4 on different brain cell types. In summary, the beneficial effects of blocking CCR5 and CXCR4 after CHI suggest that the drugs used in this study, both FDA approved and in clinical use, should be considered for translational research in TBI patients.

Chemokine Receptors CC Chemokine Receptor 5 and C-X-C Motif Chemokine Receptor 4 Are New Therapeutic Targets for Brain Recovery after Traumatic Brain Injury.

Recently, chemokine receptor CC chemokine receptor 5 (CCR5) was found to be a negative modulator of learning and memory. Its inhibition improved outcome after stroke and traumatic brain injury (TBI). To better understand its role after TBI and establish therapeutic strategies, we investigated the effect of reduced CCR5 signaling as a neuroprotective strategy and of the temporal changes of CCR5 expression after TBI in different brain cell types. To silence CCR5 expression, ccr5 short hairpin RNA (shRNA) or dsred shRNA (control) was injected into the cornu ammonis (CA) 1 and CA3 regions of the hippocampus 2 weeks before induction of closed-head injury in mice. Animals were then monitored for 32 days and euthanized at different time points to assess lesion area, inflammatory components of the glial response (immunohistochemistry; IHC), cytokine levels (enzyme-linked immunosorbent array), and extracellular signal-regulated kinase (ERK) phosphorylation (western blot). Fluorescence-activated cell sorting (FACS) analysis was performed to study post-injury temporal changes of CCR5 and C-X-C motif chemokine receptor 4 (CXCR4) expression in cortical and hippocampal cell populations (neurons, astrocytes, and microglia). Phosphorylation of the N-methyl-d-aspartate subunit 1 (NR1) subunit of N-methyl-d-aspartate (western blot) and cAMP-response-element-binding protein (CREB; IHC) were also assessed. The ccr5 shRNA mice displayed reduced lesion area, dynamic alterations in levels of inflammation-related CCR5 ligands and cytokines, and higher levels of phosphorylated ERK. The ccr5 shRNA also reduced astrocytosis in the lesioned and sublesioned cortex. FACS analysis revealed increased cortical CCR5 and CXCR4 expression in CD11b-positive cells, astrocytes, and neurons, which was most evident in cells expressing both receptors, at 3 and 11 days post-injury. The lowest levels of phosphorylated NR1 and phosphorylated CREB were found at day 3 post-injury, suggesting that this is the critical time point for therapeutic intervention.

Caspase-1 has a critical role in blood-brain barrier injury and its inhibition contributes to multifaceted repair.

BACKGROUND: Excessive inflammation might activate and injure the blood-brain barrier (BBB), a common feature of many central nervous system (CNS) disorders. We previously developed an in vitro BBB injury model in which the organophosphate paraoxon (PX) affects the BBB endothelium by attenuating junctional protein expression leading to weakened barrier integrity. The objective of this study was to investigate the inflammatory cellular response at the BBB to elucidate critical pathways that might lead to effective treatment in CNS pathologies in which the BBB is compromised. We hypothesized that caspase-1, a core component of the inflammasome complex, might have important role in BBB function since accumulating evidence indicates its involvement in brain inflammation and pathophysiology. METHODS: An in vitro human BBB model was employed to investigate BBB functions related to inflammation, primarily adhesion and transmigration of peripheral blood mononuclear cells (PBMCs). Caspase-1 pathway was studied by measurements of its activation state and its role in PBMCs adhesion, transmigration, and BBB permeability were investigated using the specific caspase-1 inhibitor, VX-765. Expression level of adhesion and junctional molecules and the secretion of pro-inflammatory cytokines were measured in vitro and in vivo at the BBB endothelium after exposure to PX. The potential repair effect of blocking caspase-1 and downstream molecules was evaluated by immunocytochemistry, ELISA, and Nanostring technology. RESULTS: PX affected the BBB in vitro by elevating the expression of the adhesion molecules E-selectin and ICAM-1 leading to increased adhesion of PBMCs to endothelial monolayer, followed by elevated transendothelial-migration which was ICAM-1 and LFA-1 dependent. Blocking caspase-8 and 9 rescued the viability of the endothelial cells but not the elevated transmigration of PBMCs. Inhibition of caspase-1, on the other hand, robustly restored all of barrier insults tested including PBMCs adhesion and transmigration, permeability, and VE-cadherin protein levels. The in vitro inflammatory response induced by PX and the role of caspase-1 in BBB injury were corroborated in vivo in isolated blood vessels from hippocampi of mice exposed to PX and treated with VX-765. CONCLUSIONS: These results shed light on the important role of caspase-1 in BBB insult in general and specifically in the inflamed endothelium, and suggest therapeutic potential for various CNS disorders, by targeting caspase-1 in the injured BBB.

The effects of point-source electroporation on the blood-brain barrier and brain vasculature in rats: An MRI and histology study.

When applying electroporation to the brain, it is important to understand the effects on the blood-brain barrier (BBB) and brain vasculature. Here we studied the effects of point-source electroporation on rats' brains as a function of time from treatment using conventional contrast-enhanced MRI and treatment response assessment maps (TRAMs), enabling depiction of subtle BBB disruption and differentiating contrast agent clearance from accumulation. Effects on vessels were also studied using Lectin staining. The TRAMs revealed that conventional contrast-enhanced MRI underestimates BBB disruption volume by nearly a factor of two, and that despite significant enhancement on standard MRI immediately post electroporation, there was no contrast accumulation in the tissue (clearance was faster than accumulation). Histology revealed significant increased vessel coverage in the treated striatum (40 ± 24% p < 0.03) immediately post electroporation, suggesting vasodilatation. Two-three hours post electroporation, both conventional MRI and TRAMs showed minor BBB disruption and histology showed decreased vessel coverage (56 ± 16%, p < 0.01), suggesting vasoconstriction. Four hours post electroporation, despite minor enhancement, the TRAMs showed significant BBB disruption with contrast accumulation, lasting over 24 h, with decreasing volumes. These results suggest that electroporation triggers several unique brain vascular mechanisms and that the optimal time window for drug administration is 4-6 h after electroporation.

The application of point source electroporation and chemotherapy for the treatment of glioma: a randomized controlled rat study.

The prognosis of Glioblastoma Multiforme patients is poor despite aggressive therapy. Reasons include poor chemotherapy penetration across the blood-brain barrier and tumor infiltration into surrounding tissues. Here we studied the effects of combined point-source electroporation (EP) and systemic chemotherapy in glioma-bearing rats. 128 rats were studied. Treatment groups were administered systemic Cisplatin/Methotrexate before EP (either 90 or 180 pulses). Control groups were treated by EP, chemotherapy, or no treatment. Tumor volumes were determined by MRI. Tumors growth rates of the EP + Methotrexate group (1.02 ± 0.77) were significantly lower (p < 0.01) than the control (5.2 ± 1.0) 1-week post treatment. No significant difference was found compared to Methotrexate (1.7 ± 0.5). Objective response rates (ORR) were 40% and 57% for the Methotrexate and EP + Methotrexate groups respectively. Tumor growth rates and ORR of the EP + Cisplatin groups (90 pulses 0.98 ± 0.2, 57%, 180 pulses 1.2 ± 0.1, 33%) were significantly smaller than the control (6.4 ± 1.0, p < 0.01, p < 0.02, 0%) and Cisplatin (3.9 ± 1.0, p < 0.04, p < 0.05, 13%) groups. No significant differences were found between the control groups. Increased survival was found in the EP + Cisplatin group, Χ2 = 7.54, p < 0.006 (Log Rank). Point-source EP with systemic chemotherapy is a rapid, minimal-invasive treatment that was found to induce significant antineoplastic effects in a rat glioma model.

Deletion of SIRPα (signal regulatory protein-α) promotes phagocytic clearance of myelin debris in Wallerian degeneration, axon regeneration, and recovery from nerve injury.

BACKGROUND: Recovery of function from traumatic nerve injury depends on the ability of severed axons to grow/regenerate back to their target tissues. This is achieved by successfully crossing the lesion site where physical impact severed axons, determined by the type of trauma, followed by successfully growing throughout the Wallerian degenerating nerve segment located distal to and beyond the lesion site, determined by the nature of Wallerian degeneration. The protracted removal of myelin debris in Wallerian degeneration, which leads residual myelin debris to slow down axon growth, impedes recovery of function. We focused in this study on mechanism(s) that delay the removal of myelin debris in Wallerian degeneration and so impede recovery. Previously, we showed that myelin debris inhibited its own phagocytosis in primary cultured macrophages and microglia as CD47 on myelin ligated SIRPα (signal regulatory protein-α) on phagocytes, and sequentially, SIRPα generated "don't eat me" signaling. We also demonstrated that serum inhibited phagocytosis in a SIRPα-dependent manner. Herein, we aimed to determine whether SIRPα-dependent inhibition of phagocytosis in macrophages impedes the in vivo removal of myelin debris in Wallerian degeneration, further leading to impaired healing. METHODS: Using SIRPα null (SIRPα-/-) and littermate wild-type (SIRPα+/+) mice, we studied the recovery of sensory and motor functions from nerve injury and, further, axon regeneration, SIRPα expression, myelin debris removal, and the phagocytic capacity and presence of macrophages in Wallerian degeneration. RESULTS: Myelin debris removal, axon regeneration, and the recovery of functions were all faster in SIRPα-/- mice than in wild-type mice. Between the two cell types that mostly scavenge myelin debris, macrophages but not Schwann cells expressed SIRPα in wild-type mice, and furthermore, SIRPα-/- macrophages phagocytosed significantly more than wild-type macrophages. CONCLUSIONS: Our findings suggest an intrinsic normally occurring SIRPα-dependent mechanism that impedes the in vivo removal of myelin debris in Wallerian degeneration by inhibiting the phagocytosis of myelin debris in macrophages, hence preventing fast growing axons from fully implementing their regenerative potential. Thus, accelerating the removal of myelin debris by eliminating SIRPα-dependent inhibition of phagocytosis will most likely advance recovery of functions from nerve injury.

CCR5 Is a Therapeutic Target for Recovery after Stroke and Traumatic Brain Injury.

We tested a newly described molecular memory system, CCR5 signaling, for its role in recovery after stroke and traumatic brain injury (TBI). CCR5 is uniquely expressed in cortical neurons after stroke. Post-stroke neuronal knockdown of CCR5 in pre-motor cortex leads to early recovery of motor control. Recovery is associated with preservation of dendritic spines, new patterns of cortical projections to contralateral pre-motor cortex, and upregulation of CREB and DLK signaling. Administration of a clinically utilized FDA-approved CCR5 antagonist, devised for HIV treatment, produces similar effects on motor recovery post stroke and cognitive decline post TBI. Finally, in a large clinical cohort of stroke patients, carriers for a naturally occurring loss-of-function mutation in CCR5 (CCR5-Δ32) exhibited greater recovery of neurological impairments and cognitive function. In summary, CCR5 is a translational target for neural repair in stroke and TBI and the first reported gene associated with enhanced recovery in human stroke.

Combination of Insulin with a GLP1 Agonist Is Associated with Better Memory and Normal Expression of Insulin Receptor Pathway Genes in a Mouse Model of Alzheimer's Disease.

Disruption of brain insulin signaling may explain the higher Alzheimer's disease (AD) risk among type 2 diabetic (T2D) patients. There is evidence from in vitro and human postmortem studies that combination of insulin with hypoglycemic medications is neuroprotective and associated with less amyloid aggregation. We examined the effect of 8-month intranasal administration of insulin, exenatide (a GLP-1 agonist), combination therapy (insulin + exenatide) or saline, in wild-type (WT) and an AD-like mouse model (Tg2576). Mice were assessed for learning, gene expression of key mediators and effectors of the insulin receptor signaling pathway (IRSP-IRS1, AKT1, CTNNB1, INSR, IRS2, GSK3B, IGF1R, AKT3), and brain Amyloid Beta (Aß) levels. In Tg2576 mice, combination therapy reduced expression of IRSP genes which was accompanied by better learning. Cortical Aß levels were decreased by 15-30% in all groups compared to saline but this difference did not reach statistical significance. WT mice groups, with or without treatment, did not differ in any comparison. Disentangling the mechanisms underlying the potential beneficial effects of combination therapy on the IR pathway and AD-like behavior is warranted.

Role of CB2 Receptor in the Recovery of Mice after Traumatic Brain Injury.

Cannabis is one of the most widely used plant drugs in the world today. In spite of the large number of scientific reports on medical marijuana, there still exists much controversy surrounding its use and the potential for abuse due to the undesirable psychotropic effects. However, recent developments in medicinal chemistry of novel non-psychoactive synthetic cannabinoids have indicated that it is possible to separate some of the therapeutic effects from the psychoactivity. We have previously shown that treatment with the endocannabinoid 2-AG, which binds to both CB1 and CB2 receptors 1 h after traumatic brain injury in mice, attenuates neurological deficits, edema formation, infarct volume, blood-brain barrier permeability, neuronal cell loss at the CA3 hippocampal region, and neuroinflammation. Recently, we synthesized a set of camphor-resorcinol derivatives, which represent a novel series of CB2 receptor selective ligands. Most of the novel compounds exhibited potent binding and agonistic properties at the CB2 receptors with very low affinity for the CB1 receptor, and some were highly anti-inflammatory. This selective binding correlated with their intrinsic activities. HU-910 and HU-914 were selected in the present study to evaluate their potential effect in the pathophysiology of traumatic brain injury (TBI). In mice and rats subjected to closed-head injury and treated with these novel compounds, we showed enhanced neurobehavioral recovery, inhibition of tumor necrosis factor α production, increased synaptogenesis, and partial recovery of the cortical spinal tract. We propose these CB2 agonists as potential drugs for development of novel therapeutic modality to TBI.

Stimulation of N-methyl-D-aspartate receptors by exogenous and endogenous ligands improves outcome of brain injury.

PURPOSE OF REVIEW: The failure of N-methyl-D-aspartate receptor (NMDAR) antagonists as a treatment for human traumatic brain injury (TBI) and stroke, along with preclinical findings of a persistent hypofunctional state of these receptors after brain injury, resulted in a new focus on NMDAR agonists, specifically those acting via the glycine site of the NMDAR. This article reviews the recent literature on positive modulators of the glycine site as a new modality for improving cognitive function in central nervous system pathology, including traumatic and ischemic brain injuries, neuroinflammation, and neuropsychiatric disorders. RECENT FINDINGS: A sustained cognitive decline and NMDAR downregulation were reported in rodent models of TBI, developmental TBI, stroke, and lipopolysaccharide-induced neuroinflammation. Activation of the glycine/serine site by D-cycloserine (DCS) or D-serine ameliorated these cognitive deficits. Recent reviews and reports on the use of DCS and D-serine to modify memory function in a wide range of psychiatric conditions are generally positive. SUMMARY: Taken together, the preclinical and clinical studies provide new, additional support for the notion that activation of the glycine/serine site should be considered a novel therapeutic approach to cognitive impairments. Specifically, as DCS is an approved drug, its translation into clinical practice should be advocated.

Blood-Brain Barrier Cellular Responses Toward Organophosphates: Natural Compensatory Processes and Exogenous Interventions to Rescue Barrier Properties.

Organophosphorus compounds (OPs) are highly toxic chemicals widely used as pesticides (e.g., paraoxon (PX)- the active metabolite of the insecticide parathion) and as chemical warfare nerve agents. Blood-brain barrier (BBB) leakage has been shown in rodents exposed to PX, which is an organophosphate oxon. In this study, we investigated the cellular mechanisms involved in BBB reaction after acute exposure to PX in an established in vitro BBB system made of stem-cell derived, human brain-like endothelial cells (BLECs) together with brain pericytes that closely mimic the in vivo BBB. Our results show that PX directly affects the BBB in vitro both at toxic and non-toxic concentrations by attenuating tight junctional (TJ) protein expression and that only above a certain threshold the paracellular barrier integrity is compromised. Below this threshold, BLECs exhibit a morphological coping mechanism in which they enlarge their cell area thus preventing the formation of meaningful intercellular gaps and maintaining barrier integrity. Importantly, we demonstrate that reversal of the apoptotic cell death induced by PX, by a pan-caspase-inhibitor ZVAD-FMK (ZVAD) can reduce PX-induced cell death and elevate cell area but do not prevent the induced BBB permeability, implying that TJ complex functionality is hindered. This is corroborated by formation of ROS at all toxic concentrations of PX and which are even higher with ZVAD. We suggest that while lower levels of ROS can induce compensating mechanisms, higher PX-induced oxidative stress levels interfere with barrier integrity.