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Background: Exposure of the central nervous system to extremely low frequency and low intensity electromagnetic fields (ELF-EMF) increases plasticity-related processes, with associated clinical improvements. Aim: The purpose of this RCT was to explore the benefit of BrainQ’s novel and non-invasive stimulation device which uses frequency-tuned ELF-EMF treatment (BQ) to reduce general disability and improve upper extremity motor function (UEMF) in subacute ischemic stroke. Methods: Study was planned for n=50, but was discontinued due to COVID-19 at n=25. Participants were randomized to receive 40 minutes of BQ (active or sham) 5 days/week, for 8 weeks, with 10 minutes of PT. BQ uses brain-computer interface-based machine learning algorithms to extract motor-related spectral features in EEG for use as treatment frequencies, and is delivered via a wearable and portable device. Results: Primary endpoint: Fugl-Meyer Assessment – Upper Extremity improved significantly (and sooner) in the treatment group versus sham at W4 (23.2±3.91 vs. 9.9±3.2;13.6 points greater than sham p=0.0070) and after 8 weeks of treatment (31.5±2.97 vs. 23.1±4.99;p=0.0611). Secondary endpoints: Most strikingly, and as a major clinical indication of reduced disability, mRS, at W9 the treatment group showed a mean improvement of 2.5±0.18 points versus 1.3±0.16 points in the sham group (p=0.0005), i.e., a mean of 1.2 points higher than that of the sham group. Significant improvements were also observed in the ARAT–Pinch subscale (W9: p=0.0082), BBT (W6: p=0.0169;W9: p

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 surroun...

Background: Neural oscillations at specific frequencies have been associated with increased neuroplasticity. Additionally, exposure to extremely low frequency and low intensity electromagnetic fields (ELF-EMF) increases plasticity-related processes, with associated clinical improvements. Aim: The purpose of this double-blind sham-controlled trial was to explore the benefit of BrainQ’s novel and non-invasive, artificial intelligence-powered, frequency-tuned ELF-EMF treatment (BQ) in improving upper extremity motor function (UEMF) in a sub-acute ischemic stroke population. Methods: Study was planned for 50 subjects, but was discontinued due to COVID-19 at 25 subjects. Participants were randomized to receive 40 minutes of BQ treatment (active or sham) 5 days/week, for 8 weeks, in conjunction with 10 minutes of physical therapy. The BQ treatment uses artificial intelligence-based algorithms to extract motor-related spectral features in EEG for use as treatment frequencies. BQ treatment is delivered via a wearable device that transmits frequency-tuned ELF-EMF, facilitating neuroplasticity processes within the central nervous system. Primary endpoint: Fugl-Meyer Assessment - Upper Extremity (FMA-UE); Secondary endpoints: mRS, Action Research Arm Test (ARAT), Box and Block Test (BBT), NIHSS. Results: FMA-UE scores improved significantly (and sooner) in the treatment group versus sham at week 4 (23.2±3.91 vs. 9.9±3.2; 13.6 points greater than sham p=0.0070) and after 8 weeks of treatment (31.5±2.97 vs. 23.1±4.99; p=0.0611). For mRS, at week 9 the treatment group showed a mean improvement of 2.5±0.18 points versus 1.3±0.16 points in the sham group (p=0.0005), i.e. a mean of 1.2 points higher than that of the sham group. Significant improvements were also observed in the ARAT-Pinch subscale (week 9: p=0.0082), BBT (week 6: p=0.0169; week 9: p Conclusion: Our results show that BQ treatment significantly improves UEMF in a sub-acute ischemic stroke population across multiple clinical metrics. Further studies are planned and ongoing with larger study populations, and in related indications.

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 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.

Cognitive deficits, especially memory loss, are common and devastating neuropsychiatric sequelae of traumatic brain injury (TBI). The deficits may persist for years and may be accompanied by increased risk of developing early- onset dementia. Past attempts to reverse the neuropathological effects of brain injury with glutamate-N-methyl-d-aspartate (NMDA) antagonists failed to show any benefits or worsened the outcome, suggesting that activation, rather than blockage, of the NMDA receptor (NMDAR) may be useful in the subacute period after TBI and stroke. Activation of the NMDAR requires occupation of the glycine-modulatory site by co-agonists to achieve its synaptic functions. Glycine and d-serine are endogenous ligands/co-agonists of synaptic NMDARs in many areas of the mature brain. The aim of the present study was to evaluate the effect of 6-chlorobenzo(d)isoxazol-3-ol (CBIO), an inhibitor of D-amino acid oxidase (DAAO), which degrades d-serine, on cognitive outcome in a mouse model of TBI. Because treating TBI animals with CBIO elevates the endogenous levels of d-serine, we compared this novel treatment with treatment by exogenous d-serine alone and combined with CBIO. The results show that a single treatment (24 h post-injury) with CBIO in the mouse model of closed head injury significantly improves cognitive and motor function, and decreases lesion volume and the inflammatory response. Moreover, the compound proved to be neuroprotective, as the hippocampal volume and the number of neurons in hippocampal regions increased. Treatment with CBIO boosted the NR1 and phospho- NR1 subunits of the NMDAR and affected the CREB, phospho-CREB, and brain-derived neurotropic factor (BDNF) pathways. These findings render CBIO a promising, novel treatment for cognitive impairment following TBI.

Background aims Human umbilical cord blood (HUCB) is an important source of stem cells for therapy of hematopoietic disorders and is a potential therapy for various neurological disorders, including traumatic brain injury (TBI). The expression of nerve growth factor (NGF) and its receptors TrkA, p75NTR and α9β1 integrin on an HUCB CD45+ pan-hematopoietic subpopulation was investigated in the context of its neurotherapeutic potential after TBI. Methods NGF and its receptors were detected on CD45+ cells by reverse transcriptase polymerase chain reaction, flow cytometry analysis and confocal microscopy. CD45+ cells were stimulated by TBI brain extracts, and NGF levels were measured by enzyme-linked immunosorbent assay. TBI mice were divided into six groups for xenogeneic intravenous transplantation, 1 day post-trauma, with 1 × 106 CD45+ cells untreated or treated with the anti-NGF neutralizing antibody K252a, a TrkA antagonist; VLO5, an α9β1 disintegrin; or negative (vehicle) and positive (NGF) controls. Results The HUCB CD45+ subpopulation constitutively expresses NGF and its receptors, mainly TrkA and p75NTR and minor levels of α9β1. In vitro experiments provided evidence that trauma-related mediators from brain extracts of TBI mice induced release of NGF from HUCB CD45+ cell cultures. HUCB CD45+ cells induced a neurotherapeutic effect in TBI mice, abrogated by cell treatment with either anti-NGF antibody or K252a, but not VLO5. Conclusions These findings strengthen the role of NGF and its TrkA receptor in the HUCB CD45+ subpopulation's neurotherapeutic effect. The presence of neurotrophin receptors in the HUCB CD45+ pan-hematopoietic subpopulation may explain the neuroprotective effect of cord blood in therapy of a variety of neurological disorders.

Human umbilical cord blood (HUCB) transplantation has become an alternative cell therapy for hematological and oncological malignancies in the clinic and is considered for neurological disorders. The heterogeneity in the content of the different stem and progenitor cells composing HUCB mononuclear cells (MNC) may influence their engraftment and neurotherapeutic effect. We hypothesized that CD45 pan-hematopoietic marker expression is heterogeneous in MNC, and therefore, CD45+ subpopulation enrichment for neurotherapy may provide a tool to overcome cellular variance in different HUCB units. We employed an immunomagnetic separation method to isolate and characterize HUCB CD45+ pan-hematopoietic subpopulation and to investigate whether the vaginal or cesarean deliveries influence their neurotherapeutic effect in a traumatic brain injury (TBI) mouse model. Adult C57BL/6J male mice were subjected to moderate TBI and intravenously xenotransplanted with 1 × 106 CD45+ cells derived from either vaginal or cesarean HUCB units. A large heterogeneity in the expression of CD45 marker in MNC, both in vaginal and cesarean HUCB units, was found, regardless of the number of live births. A higher expression of hematopoietic markers was found in the CD45+ subpopulation while low expressional levels of typical mesenchymal markers were detected. Neurotherapeutic effects, evaluated with an established neurological severity score and novel object recognition test, indicated improved functional motor and memory recovery and found independent of delivery type. Cytokine analysis in extracts of TBI brain cortices indicated an acute immunomodulatory effect by HUCB CD45+ subpopulation upon xenotransplantation. These results may provide insights to CD45 marker as a predictor of HUCB units’ quality for neurotherapy in TBI.

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 CB(1) and CB(2) 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 CB(2) receptor selective ligands. Most of the novel compounds exhibited potent binding and agonistic properties at the CB(2) receptors with very low affinity for the CB(1) 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 CB(2) agonists as potential drugs for development of novel therapeutic modality to TBI.

Cognitive deficits, especially memory loss, are common following many types of brain insults which are associated with neuroinflammation, although the underlying mechanisms are not entirely clear. The present study aimed to characterize the long-term cognitive and behavioral impairments in a mouse model of neuroinflammation in the absence of other insults and to evaluate the therapeutic potential of D-cycloserine (DCS). DCS is a co-agonist of the NMDA receptor that ameliorates cognitive deficits in models of TBI and stroke. Using a mouse model of global neuroinflammation induced by intracisternal (i.c.) administration of endotoxin (LPS), we found long-lasting microgliosis, memory deficits, impaired LTP, and reduced levels of the obligatory NR1 subunit of the NMDA receptor. A single administration of DCS, 1 day after i.c. LPS reduced microgliosis, reversed the cognitive deficits and restored LTP and NR1 levels. These results demonstrate that neuroinflammation alone, in the absence of trauma or ischemia, can cause persistent (>6 months) memory deficits linked to deranged NNMDA receptor function and suggest a possible role for NMDA co-agonists in reducing the cognitive sequelae of neuroinflammation.

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.

The endocannabinoid (eCB) system helps recovery following traumatic brain injury (TBI). Treatment with 2-arachidonoylglycerol (2-AG), a cerebral eCB ligand, was found to ameliorate the secondary damage. Interestingly, the fatty acid amino acid amide (FAAA) N-arachidonoyl-L-serine (AraS) exerts similar eCB dependent neuroprotective. The present study aimed to investigate the effects of the FAAA palmitoyl-serine (PalmS) following TBI. We utilized the TBI model in mice to examine the therapeutic potential of PalmS, injected 1 h following closed head injury (CHI). We followed the functional recovery of the injured mice for 28 days post-CHI, and evaluated cognitive and motor function, lesion volume, cytokines levels, molecular signaling, and infarct volume at different time points after CHI. PalmS treatment led to a significant improvement of the neurobehavioral outcome of the treated mice, compared with vehicle. This effect was attenuated in the presence of eCBR antagonists and in CB2-/- mice, compared to controls. Unexpectedly, treatment with PalmS did not affect edema and lesion volume, TNFα and IL1β levels, anti-apoptotic mechanisms, nor did it exert improvement in cognitive and motor function. Finally, co-administration of PalmS, AraS and 2-AG, did not enhance the effect of the individual drugs. We suggest that the neuroprotective action of PalmS is mediated by indirect activation of the eCB receptors following TBI. One such mechanism may involve receptor palmitoylation which has been reported to result in structural stabilization of the receptors and to an increase in their activity. Further research is required in order to establish this assumption.

The cardiac steroid ouabain binds to Na(+), K(+)-ATPase and inhibits its activity. Administration of the compound to animals and humans causes an increase in the force of contraction of heart muscle and stabilizes heart rate. In addition, this steroid promotes the growth of cardiac, vascular, and neuronal cells both in vitro and in vivo. We studied the effects of ouabain on mouse recovery following closed head injury (CHI), a model for traumatic brain injury. We show that chronic (three times a week), but not acute, intraperitoneal administration of a low dose (1 μg/kg) of ouabain significantly improves mouse recovery and functional outcome. The improvement in mouse performance was accompanied by a decrease in lesion size, estimated 43 d following the trauma. In addition, mice that underwent CHI and were treated with ouabain showed an increase in the number of proliferating cells in the subventricular zone and in the area surrounding the site of injury. Determination of the identity of the proliferating cells in the area surrounding the trauma showed that whereas there was no change in the proliferation of endothelial cells or astrocytes, neuronal cell proliferation almost doubled in the ouabain-treated mice in comparison with that of the vehicle animals. These results point to a neuroprotective effects of low doses of ouabain and imply its involvement in brain recovery and neuronal regeneration. This suggests that ouabain and maybe other cardiac steroids may be used for the treatment of traumatic brain injury.

Human umbilical cord blood (HUCB) transplantation has become an alternative cell therapy for hematological and oncological malignancies in the clinic and is considered for neurological disorders. The heterogeneity in the content of the different stem and progenitor cells composing HUCB mononuclear cells (MNC) may influence their engraftment and neurotherapeutic effect. We hypothesized that CD45 pan-hematopoietic marker expression is heterogeneous in MNC, and therefore, CD45

Human umbilical cord blood (HUCB) is an important source of stem cells for therapy of hematopoietic disorders and is a potential therapy for various neurological disorders, including traumatic brain injury (TBI). The expression of nerve growth factor (NGF) and its receptors TrkA, p75NGF and its receptors were detected on CD45The HUCB CD45These findings strengthen the role of NGF and its TrkA receptor in the HUCB CD45

Treatment of traumatic brain injury (TBI) is still an unmet need. Cell therapy by human umbilical cord blood (HUCB) has shown promising results in animal models of TBI and is under evaluation in clinical trials. HUCB contains different cell populations but to date, only mesenchymal stem cells have been evaluated for therapy of TBI. Here we present the neurotherapeutic effect, as evaluated by neurological score, using a single dose of HUCB-derived mononuclear cells (MNCs) upon intravenous (IV) administration one day post-trauma in a mouse model of closed head injury (CHI). Delayed (eight days post-trauma) intracerebroventricular administration of MNCs showed improved neurobehavioral deficits thereby extending the therapeutic window for treating TBI. Further, we demonstrated for the first time that HUCB-derived pan-hematopoietic CD45 positive (CD45(+)) cells, isolated by magnetic sorting and characterized by expression of CD45 and CD11b markers (96-99%), improved the neurobehavioral deficits upon IV administration, which persisted for 35 days. The therapeutic effect was in a direct correlation to a reduction in the lesion volume and decreased by pre-treatment of the cells with anti-human-CD45 antibody. At the site of brain injury, 1.5-2 h after transplantation, HUCB-derived cells were identified by near infrared scanning and immunohistochemistry using anti-human-CD45 and anti-human-nuclei antibodies. Nerve growth factor and vascular endothelial growth factor levels were differentially expressed in both ipsilateral and contralateral brain hemispheres, thirty-five days after CHI, measured by enzyme-linked immunosorbent assay. These findings indicate the neurotherapeutic potential of HUCB-derived CD45(+) cell population in a mouse model of TBI and propose their use in the clinical setting of human TBI.

Over the last two decades a large number of N-long-chain acyl amino acids have been identified in the mammalian body. The pharmacological activities of only a few of them have been investigated and some have been found to be of considerable interest. Thus arachidonoyl serine is vasodilatory and neuroprotective, arachidonoyl glycine is antinociceptive, and oleoyl serine rescues bone loss. However, the pathophysiological/biochemical roles of these amides are mostly unknown.

Despite years of research, no effective therapy is yet available for the treatment of traumatic brain injury (TBI). The most prevalent and debilitating features in survivors of TBI are cognitive deficits and motor dysfunction. A potential therapeutic method for improving the function of patients following TBI would be to restore, at least in part, plasticity to the CNS in a controlled way that would allow for the formation of compensatory circuits. Inosine, a naturally occurring purine nucleoside, has been shown to promote axon collateral growth in the corticospinal tract (CST) following stroke and focal TBI. In the present study, we investigated the effects of inosine on motor and cognitive deficits, CST sprouting, and expression of synaptic proteins in an experimental model of closed head injury (CHI). Treatment with inosine (100 mg/kg i.p. at 1, 24 and 48 h following CHI) improved outcome after TBI, significantly decreasing the neurological severity score (NSS, p

N-arachidonoyl-L-serine (AraS) is a novel neuroprotective endocannabinoid. We aimed to test the effects of exogenous AraS on neurogenesis after traumatic brain injury (TBI). The effects of AraS on neural progenitor cells (NPC) proliferation, survival, and differentiation were examined in vitro. Next, mice underwent TBI and were treated with AraS or vehicle. Lesion volumes and clinical outcome were evaluated and the effects on neurogenesis were tested using immunohistochemistry. Treatment with AraS led to a dose-dependent increase in neurosphere size without affecting cell survival. These effects were partially reversed by CB1, CB2, or TRPV1 antagonists. AraS significantly reduced the differentiation of NPC in vitro to astrocytes or neurons and led to a 2.5-fold increase in expression of the NPC marker nestin. Similar effects were observed in vivo in mice treated with AraS 7 days after TBI. These effects were accompanied by a reduction in lesion volume and an improvement in neurobehavioral function compared with controls. AraS increases proliferation of NPCs in vitro in cannabinoid-receptor-mediated mechanisms and maintains NPC in an undifferentiated state in vitro and in vivo. Moreover, although given at 7 days post injury, these effects are associated with significant neuroprotective effects leading to an improvement in neurobehavioral functions.

Death of Central Nervous System (CNS) neurons following traumatic brain injury (TBI) is a complex process arising from a combination of factors, many of which are still unknown. It has been found that inhibition of transient receptor potential (TRP) channels constitutes an effective strategy for preventing death of CNS neurons following TBI. TRP channels are classified into seven related subfamilies, most of which are Ca(2+) permeable and involved in many cellular functions, including neuronal cell death. We hypothesized that TRP channels of the TRPC subfamily may be involved in post-TBI pathophysiology and that the compound 5-isopropyl-2-methylphenol (carvacrol), by inhibition of TRP channels, may exert neuroprotective effect after TBI. To test these suppositions, carvacrol was given to mice after TBI and its effect on their functional recovery was followed for several weeks. Our results show that neurological recovery after TBI was significantly enhanced by application of carvacrol. To better define the type of the specific channel involved, the effect of carvacrol on the extent and speed of recovery after TBI was compared among mice lacking TRPC1, TRPC3, or TRPC5, relative to wild type controls. We found that neurological recovery after TBI was significantly enhanced by combining carvacrol with TRPC1 elimination, but not by the absence of TRPC3 or TRPC5, showing a synergistic effect between carvacrol application and TRPC1 elimination. We conclude that TRPC1-sensitive mechanisms are involved in TBI pathology, and that inhibition of this channel by carvacrol enhances recovery and should be considered for further studies in animal models and humans.

Traumatic brain injury (TBI) initiates acute and chronic inflammatory processes involving cyclooxygenase-2 (COX-2), which may have detrimental effects on outcome and especially on brain regeneration. Therefore we aimed to study whether carprofen, a COX-2 inhibitor, would improve outcome and increase neurogenesis after TBI. TBI was induced in Sabra mice that were then treated with vehicle or carprofen for 7 days. Functional outcome was evaluated with the Neurological Severity Score (NSS).Cytokine levels were assessed 4 h post-TBI and water content was measured 24 h post TBI. Mice were given BrdU to label newborn cells for 10 days. The animals were killed 90 days post-TBI and the lesion size as well as newborn cell fate were assessed. Carprofen significantly reduced lesion size (p=0.002), decreased water content in the lesioned cortex (p=0.03), reduced the number of microglia in the lesioned cortex (p0.0001), and lowered the levels of proinflammatory cytokines (IL-1β, p=0.03; IL-6, p=0.02). Carprofen led to significantly larger improvements in functional outcome (p≤0.008) which were durable over 90 days. Carprofen also induced a threefold increase in the proliferation of new cells in the peri-lesion area (p≤0.002), but newborn cells differentiated mainly into glia in both groups. Carprofen is neuroprotective and induces cell proliferation and gliogenesis after TBI. Treatment with carprofen is consistently associated with better functional outcome. Our results imply that anti-inflammatory drugs may represent novel therapeutic options for TBI.

Preventing relapse to drug use is a major challenge for the treatment of drug addiction. Environmental cues are among the major determinants of relapse in abstinent cocaine users. The protein kinase M ζ (PKMζ) is involved in the generation and maintenance of long-term potentiation and is critical in memory storage. Here we show that inhibition of PKMζ in the nucleus accumbens (NAc) shell, a major component of the reward system that plays an important role in mediating drug craving and relapse, by a selective inhibitor ζ inhibitory peptide (ZIP), abolished cocaine-induced conditioned place preference (CPP). However, the injection of ZIP into the NAc core resulted in earlier onset of CPP extinction. Finally, we found that the levels of PKMζ and GluR2 in the NAc remained unchanged, while the GluR1 levels were elevated following CPP and fully reversed by ZIP injection. Together, our results suggest that inactivation of PKMζ in the NAc may result in the dissociation between the rewarding properties of the drug and the drug-related environment and may serve as a novel target for the treatment of drug relapse.

Traumatic brain injury (TBI) represents the leading cause of death in young individuals. It triggers the accumulation of harmful mediators, leading to secondary damage, yet protective mechanisms are also set in motion. The endocannabinoid (eCB) system consists of ligands, such as anandamide and 2-arachidonoyl-glycerol (2-AG), receptors (e.g. CB1, CB2), transporters and enzymes, which are responsible for the ‘on-demand’ synthesis and degradation of these lipid mediators. There is a large body of evidence showing that eCB are markedly increased in response to pathogenic events. This fact, as well as numerous studies on experimental models of brain toxicity, neuroinflammation and trauma supports the notion that the eCB are part of the brain's compensatory or repair mechanisms. These are mediated via CB receptors signalling pathways that are linked to neuronal survival and repair. The levels of 2-AG, the most highly abundant eCB, are significantly elevated after TBI and when administered to TBI mice, 2-AG decreases brain oedema, inflammation and infarct volume and improves clinical recovery. The role of CB1 in mediating these effects was demonstrated using selective antagonists or CB1 knockout mice. CB2 were shown in other models of brain insults to reduce white blood cell rolling and adhesion, to reduce infarct size and to improve motor function. This review is focused on the role the eCB system plays as a self-neuroprotective mechanism and its potential as a basis for the development of novel therapeutic modality for the treatment of CNS pathologies with special emphasis on TBI. LINKED ARTICLES This article is part of a themed issue on Cannabinoids in Biology and Medicine. To view the other articles in this issue visit http://dx.doi.org/10.1111/bph.2011.163.issue-7

Human umbilical cord blood (HUCB), a valuable source for cell therapy of hematologic diseases, was recently found to confer neuroprotection in animal models of brain injury. To explore the neuroprotective properties of HUCB in an ischemic environment, we studied a unique sub-population of collagen-adherent cells. In the presence of nerve growth factor (NGF) and interferon-gamma, these cells differentiate in vitro into a neuronal phenotype (termed HUCBNPs). Using a closed-head injury brain trauma model, we recently demonstrated neuroprotective effects of HUCB upon their local injection into the brain. Our in-vitro studies indicated that upon co-culture with ischemia exposed PC12 cells, HUCBNP conferred about 30% neuroprotection, as assessed by decreased lactate dehydrogenase (LDH) release and inhibition of caspase-3 activity. HUCBNP decreased by 95% the level of free radicals in the insulted-PC12 cells, which was in correlation with the appearance of antioxidants and NGF in the medium. HUCBNP-induced neuroprotection also correlated with changes in NGF mRNA expression levels, a process which was blocked by K252a, an antagonist of the NGF-neurotrophin receptor TrkA. To further extend the possible contribution of NGF-mediated protective role, we investigated its neuroprotective mechanisms using PC12 cells and brain capillary endothelial cells exposed to ischemic insult. Pretreatment of the cultures with NGF conferred neuroprotection in a dose- and time-dependent fashion. Time course experiments indicated marked activation of mitogen activated protein kinasas (MAPKs) during the ischemic insult, while treatment of the cultures with NGF inhibited them to different degrees. It appears that both neuronal and endothelial compartments may be protected by NGF under ischemic conditions. Therefore, in future neuroprotection studies, we propose to address additional components of the neurovascular unit such as astrocytes and pericytes in mono-, co- and multi-culture setups. In conclusion, our findings indicate that HUCBNP-induced neuroprotection involves antioxidant(s) and NGF, which by paracrine and/or autocrine route, affects all components of the neurovascular unit.

Histone deacetylase (HDAC) inhibitors are emerging as a novel class of potentially therapeutic agents for treating acute injuries of the central nervous system (CNS). In this review, we summarize data regarding the effects of HDAC inhibitor administration in models of acute CNS injury and discuss issues warranting clinical trials. We have previously shown that the pan-HDAC inhibitor ITF2357, a compound shown to be safe and effective in humans, improves functional recovery and attenuates tissue damage when administered as late as 24 h after injury. Using a well-characterized, clinically relevant mouse model of closed head injury, we demonstrated that a single dose of ITF2357 administered 24 h after injury improves neurobehavioral recovery and reduces tissue damage. ITF2357-induced functional improvement was found to be sustained up to 14 d after trauma and was associated with augmented histone acetylation. Single postinjury administration of ITF2357 also attenuated injury-induced inflammatory responses, as indicated by reduced glial accumulation and activation as well as enhanced caspase-3 expression within microglia/macrophages after treatment. Because no specific therapeutic intervention is currently available for treating brain trauma patients, the ability to affect functional outcome by postinjury administration of HDAC inhibitors within a clinically feasible timeframe may be of great importance. Furthermore, a growing body of evidence indicates that HDAC inhibitors are beneficial for treating various forms of acute CNS injury including ischemic and hemorrhagic stroke. Because HDAC inhibitors are currently approved for other use, they represent a promising new avenue of treatment, and their use in the setting of CNS injury warrants clinical evaluation.

CB2 is a Gi protein–coupled receptor activated by endo- and phytocannabinoids, thus inhibiting stimulated adenylyl cyclase activity. CB2 is expressed in bone cells and Cb2 null mice show a marked age-related bone loss. CB2-specific agonists both attenuate and rescue ovariectomy-induced bone loss. Activation of CB2 stimulates osteoblast proliferation and bone marrow derived colony-forming units osteoblastic. Here we show that selective and nonselective CB2 agonists are mitogenic in MC3T3 E1 and newborn mouse calvarial osteoblastic cultures. The CB2 mitogenic signaling depends critically on the stimulation of Erk1/2 phosphorylation and de novo synthesis of MAP kinase–activated protein kinase 2 (Mapkapk2) mRNA and protein. Further downstream, CB2 activation enhances CREB transcriptional activity and cyclin D1 mRNA expression. The CB2-induced stimulation of CREB and cyclin D1 is inhibitable by pertussis toxin, the MEK-Erk1/2 inhibitors PD098059 and U0126, and Mapkapk2 siRNA. These data demonstrate that in osteoblasts CB2 targets a Gi protein–cyclin D1 mitogenic axis. Erk1/2 phosphorylation and Mapkapk2 protein synthesis are critical intermediates in this axis. © 2011 American Society for Bone and Mineral Research.

Activation of endogenous stem cells has been proposed as a novel form of therapy in a variety of neurologic disorders including traumatic brain injury (TBI). Vascular endothelial growth factor (VEGF) is expressed in the brain after TBI and serves as a potent activator of angiogenesis and neurogenesis. In this study, we infused exogenous VEGF into the lateral ventricles of mice for 7 days after TBI using mini-osmotic pumps to evaluate the effects on recovery and functional outcome. The results of our study show that VEGF significantly increases the number of proliferating cells in the subventricular zone and in the perilesion cortex. Fate analysis showed that most newborn cells differentiated into astrocytes and oligodendroglia and only a few cells differentiated into neurons. Functional outcome was significantly better in mice treated with VEGF compared with vehicle-treated animals after TBI. Injury size was significantly smaller at 90 days after TBI in VEGF-treated animals, suggesting additional neuroprotective effects of VEGF. In conclusion, VEGF significantly augments neurogenesis and angiogenesis and reduces lesion volumes after TBI. These changes are associated with significant improvement in recovery rates and functional outcome.

Despite efforts aimed at developing novel therapeutics for traumatic brain injury (TBI), no specific pharmacological agent is currently clinically available. Here, we show that the pan-histone deacetylase (HDAC) inhibitor ITF2357, a compound shown to be safe and effective in humans, improves functional recovery and attenuates tissue damage when administered as late as 24 h postinjury. Using a well-characterized, clinically relevant mouse model of closed head injury (CHI), we demonstrate that a single dose of ITF2357 administered 24 h postinjury improves neurobehavioral recovery from d 6 up to 14 d postinjury (improved neurological score vs. vehicle; P≤0.05), and that this functional benefit is accompanied by decreased neuronal degeneration, reduced lesion volume (22% reduction vs. vehicle; P≤0.01), and is preceded by increased acetylated histone H3 levels and attenuation of injury-induced decreases in cytoprotective heat-shock protein 70 kDa and phosphorylated Akt. Moreover, reduced glial accumulation and activation were observed 3 d postinjury, and total p53 levels at the area of injury and caspase-3 immunoreactivity within microglia/macrophages at the trauma area were elevated, suggesting enhanced clearance of these cells via apoptosis following treatment. Hence, our findings underscore the relevance of HDAC inhibitors for ameliorating trauma-induced functional deficits and warrant consideration of applying ITF2357 for this indication.—Shein, N. A., Grigoriadis, N., Alexandrovich, A. G., Simeonidou, C., Lourbopoulos, A., Polyzoidou, E., Trembovler, V., Mascagni, P., Dinarello, C. A., Shohami, E. Histone deacetylase inhibitor ITF2357 is neuroprotective, improves functional recovery, and induces glial apoptosis following experimental traumatic brain injury.

Traumatic brain injury represents the leading cause of death in young individuals. Various animal models have been developed to mimic human closed head injury (CHI). Widely used models induce head injury by lateral fluid percussion, a controlled cortical impact or impact acceleration. The presented model induces a CHI by a standardized weight-drop device inducing a focal blunt injury over an intact skull without pre-injury manipulations. The resulting impact triggers a profound neuroinflammatory response within the intrathecal compartment with high consistency and reproducibility, leading to neurological impairment and breakdown of the blood-brain barrier. In this protocol, we define standardized procedures for inducing CHI in mice and determine various severity grades of CHI through modulation of the weight falling height. In experienced hands, this CHI model can be carried out in as little as 30 s per animal, with additional time required for subsequent posttraumatic analysis and data collection.

The development of myoclonic activity as a toxic effect of morphine application into the intrathecal space in rats is described. This syndrome resembled the human syndrome of action myoclonus by its spontaneous onset and its augmentation by initiation of movement or by an acoustic stimulus. It was not reversed or prevented by naloxone. This effect of morphine was associated with an increase in serotonergic activity in the spinal cord and was reduced by pretreatment with parachlorophenylalanine in doses which reduced spinal 5-HT by approximately 60%. The dose which produced this syndrome was about ten times higher than the analgesic dose applied by the same route. Other commonly used opiates such as: methadone (0.5-2 mg/kg), pethidine (2-10 mg/kg), fentanyl (2-10 micrograms/kg) and ketamine (2-10 mg/kg) did not produce myoclonic-like activity, but methadone and pethidine at the highest doses caused respiratory arrest. Fentanyl appeared to be the safest of the drugs tested since a relatively high dose, administered into the intrathecal space did not cause any side effects, while morphine was least safe of the five drugs since it produced myoclonic activity in addition to the widely documented respiratory depression. We suggest that the production of the myoclonic activity is mediated by spinal serotonergic systems.

Brain-derived neurotrophic factor (BDNF) conveys neuroprotection in various settings of experimental central nervous system injury. Using a model of endogenous neuroprotection, induced in mice by chronic exposure to moderate ambient heat (heat acclimation, HA), we have previously shown that neuroprotection following traumatic brain injury involves reduced post-injury tumor necrosis factor alpha (TNFalpha) expression. As glial cells play a pivotal role in post-injury inflammation on one hand, and are also capable of inducing neuroprotection by harboring trophic factors and BDNF in particular, the effects of injury and HA on overall BDNF content at the trauma area, gliosis and glial BDNF expression were investigated. Western blotting indicated higher overall BDNF levels in HA sham-operated mice. Following injury, a decrease was observed in the HA group only, reaching levels similar to normothermic mice. Immunohistochemical studies demonstrated BDNF-positive resting microglia in non-injured HA but not normothermic animals. Post-injury astrocytosis and microglial immunoreactivity were enhanced in the HA group. Particularly, an increase in the amount of ramified microglia was observed within the penumbra, accompanied by a concomitant decrease in globular microglia, a major source of pro-inflammatory mediators. BDNF labeling on and around microglia and their processes was intensified in HA mice. Furthermore, BDNF immunoreactivity in HA mice was evident in the degenerated edges of axons. These findings, taken together with the growing body of evidence indicating the neuroprotective potential of both BDNF and microglia, suggest a possible role of these cells in HA-induced neuroprotection.

Traumatic brain injury (TBI) remains a frequent and major challenge in neurological and neurosurgical practice. Apoptosis may play a role in cerebral tissue damage induced by the traumatic insult, and thus its detection and inhibition may advance patient care. DDC (N,N'-didansyl-L-cystine) is a novel fluorescent probe for detection of apoptotic cells. We now report on the performance of DDC in experimental TBI. Closed head injury was induced in mice by weight-drop. DDC was administered intravenously in vivo. Two hours later, animals were sacrificed, and brain tissue was subjected to fluorescent microcopy, for assessment of DDC uptake, in correlation with histopathological assessment of apoptosis by TUNEL and caspase substrates, and also in correlation with the neurological deficits, as assessed by Neurological Severity Score (NSS). Selective uptake of DDC was observed at the primary site of injury, and also at remote sites. Uptake was at the cellular level, with accumulation of DDC in the cytoplasm. Cells manifesting DDC uptake were confirmed as apoptotic cells by detection of the characteristic apoptotic DNA fragmentation (positive TUNEL staining) and detection of activated caspases. The damaged region stained by DDC fluorescence correlated with the severity of neuronal deficits. Our study confirms the role of apoptosis in TBI, and proposes DDC as a useful tool for its selective targeting and detection in vivo. Such imaging of apoptosis, following future radiolabeling of DDC, may advance care for patients with head injury, by allowing real-time evaluation of the extent of tissue damage, assessment of novel therapeutic strategies, and optimization of treatment for the individual patient.

Boswellia resin has been used as a major anti-inflammatory agent and for the healing of wounds for centuries. Incensole acetate (IA), isolated from this resin, was shown to inhibit the activation of nuclear factor-κB, a key transcription factor in the inflammatory response. We now show that IA inhibits the production of inflammatory mediators in an in vitro model system of C6 glioma and human peripheral monocytes. Given the involvement of postinjury inflammation in the pathophysiology and outcome of traumatic brain injury, we examined the effect of IA on the inflammatory process and on the recovery of neurobehavioral and cognitive functions in a mouse model of closed head injury (CHI). In the brains of post-CHI mice, IA reduced glial activation, inhibited the expression of interleukin-1β, and tumor necrosis factor-α mRNAs, and induced cell death in macrophages at the area of trauma. A mild hypothermic effect was also noted. Subsequently, IA inhibited hippocampal neurodegeneration and exerted a beneficial effect on functional outcome after CHI, indicated by reduced neurological severity scores and improved cognitive ability in an object recognition test. This study attributes the anti-inflammatory activity of Boswellia resin to IA and related cembranoid diterpenes and suggests that they may serve as novel neuroprotective agents.

Long-term exposure to moderate ambient heat (heat acclimation, HA, 30 days at 34+/-1 degrees C) provides protection toward a variety of stressors including traumatic brain injury. As previous studies suggested an anti-inflammatory effect of HA and given the ability of augmented pre-injury anti-inflammatory cytokine expression to harbor neuroprotection and to attenuate early post-injury expression of pro-inflammatory mediators, we hypothesized that HA-induced neuroprotection may involve enhanced pre-injury expression of anti-inflammatory mediators or a reduction in post-injury TNF alpha (TNFalpha) expression. Since the attenuation of inflammatory-associated entities has also been linked to mild hypothermia, an established neuroprotective paradigm, the effect of HA on post-injury body temperature was also studied. HA mice and normothermic (NT) counterparts were examined using a closed head injury model. Cytokines were measured within the ipsilateral cortex. Pre-injury protein levels of anti-inflammatory interleukins 10 and 4 (IL-10, IL-4) were quantified by enzyme-linked immunosorbent assays (ELISA). mRNA and protein levels of TNFalpha were quantified during the initial 2 h post-injury using semi-quantitative and real-time polymerase chain reaction (sqRT-PCR and qRT-PCR) or ELISA, respectively. Rectal temperatures were measured. HA induced augmented pre-injury IL-10 expression and a post-injury reduction in TNFalpha mRNA levels, as well as altered expression dynamics of TNFalpha protein. TNFalpha protein levels decreased relative to the sham state in HA mice only. HA mice displayed sustained post-injury hypothermia, namely significantly lower body temperature at 4 h post-injury. Given the evidence on the neuroprotective nature of hypothermia and anti-inflammatory cytokines, we suggest that these changes may contribute to HA-induced neuroprotection.

Ras proteins play a role in receptor-mediated signaling pathways and are activated after traumatic brain injury. S-trans-trans-farnesylthiosalicylic acid (FTS), a synthetic Ras inhibitor, acts primarily on the active, GTP-bound form of Ras and was shown to improve neurobehavioral outcome after closed head injury (CHI) in mice. To gain a better understanding of the neuroprotective mechanism of FTS, we used diffusion-weighted imaging (DWI) in a rat model of CHI. Apparent diffusion coefficients (ADC) and transverse relaxation times (T2) were measured in injured rat brains after treatment with vehicle or FTS (5 mg/kg). Neuroprotection by FTS was also assessed in terms of the neurological severity score. One week after injury, significantly better recovery was observed in the FTS-treated rats than in the controls (p = 0.0191). T2 analysis of the magnetic resonance images revealed no differences between the two groups. In contrast, they differed significantly in ADC, particularly at 24 h post-CHI (p0.05): in the vehicle-treated rats ADC had decreased to approximately 26% below baseline, whereas it had increased to about 10% above baseline in the FTS-treated rats. As the magnitude of ADC reduction is strongly linked to blood perfusion deficit, these results suggest that the neuroprotective mechanism of FTS might be related to an improvement in cerebral perfusion. We propose that FTS, which is currently being tested in humans for anti-cancer indications, should also be considered as a new strategy for the management of head injury.

Low-level laser therapy (LLLT) has been evaluated in this study as a potential therapy for traumatic brain injury (TBI). LLLT has been found to modulate various biological processes. Following TBI in mice, we assessed the hypothesis that LLLT might have a beneficial effect on their neurobehavioral and histological outcome. TBI was induced by a weight-drop device, and motor function was assessed 1 h post-trauma using a neurological severity score (NSS). Mice were then divided into three groups of eight mice each: one control group that received a sham LLLT procedure and was not irradiated; and two groups that received LLLT at two different doses (10 and 20 mW/cm(2) ) transcranially. An 808-nm Ga-As diode laser was employed transcranially 4 h post-trauma to illuminate the entire cortex of the brain. Motor function was assessed up to 4 weeks, and lesion volume was measured. There were no significant changes in NSS at 24 and 48 h between the laser-treated and non-treated mice. Yet, from 5 days and up to 28 days, the NSS of the laser-treated mice were significantly lower (p0.05) than the traumatized control mice that were not treated with the laser. The lesion volume of the laser treated mice was significantly lower (1.4%) than the non-treated group (12.1%). Our data suggest that a non-invasive transcranial application of LLLT given 4 h following TBI provides a significant long-term functional neurological benefit. Further confirmatory trials are warranted.

The mammalian target of rapamycin, commonly known as mTOR, is a serine/threonine kinase that regulates translation and cell division. mTOR integrates input from multiple upstream signals, including growth factors and nutrients to regulate protein synthesis. Inhibition of mTOR leads to cell cycle arrest, inhibition of cell proliferation, immunosuppression and induction of autophagy. Autophagy, a bulk degradation of sub-cellular constituents, is a process that keeps the balance between protein synthesis and protein degradation and is induced upon amino acids deprivation. Rapamycin, mTOR signaling inhibitor, mimics amino acid and, to some extent, growth factor deprivation. In the present study we examined the effect of rapamycin, on the outcome of mice after brain injury. Our results demonstrate that rapamycin injection 4 h following closed head injury significantly improved functional recovery as manifested by changes in the Neurological Severity Score, a neurobehavioral testing. To verify the activity of the injected rapamycin, we demonstrated that it inhibits p70S6K phosphorylation, reduces microglia/macrophages activation and increases the number of surviving neurons at the site of injury. We therefore suggest that rapamycin is neuroprotective following traumatic brain injury and as a drug used in the clinic for other indications, we propose that further studies on rapamycin should be conducted in order to consider it as a novel therapy for traumatic brain injury.

Traumatic brain injury triggers a massive glutamate efflux, activation of NMDA receptor channels, and cell death. Recently, we reported that NMDA receptors in mice are down-regulated from hours to days following closed head injury (CHI), and treatment with NMDA improved recovery of motor and cognitive functions up to 14 d post-injury. Here we show that a single injection of a low dose of D-cycloserine (DCS), a partial NMDA receptor agonist, in CHI mice 24 h post-injury, resulted in a faster and greater recovery of motor and memory functions as assessed by neurological severity score and object recognition tests, respectively. Moreover, DCS treatment of CHI mice led to a significant improvement of hippocampal long-term potentiation (LTP) in the CA1 region that was completely blunted in CHI control mice. However, DCS did not improve CHI-induced impairment in synaptic glutamate release measured by paired pulse facilitation (PPF) ratio in hippocampal CA1 region. Finally, CHI-induced reduction of brain-derived neurotrophic factor (BDNF) was fully restored following DCS treatment. Since DCS is in clinical use for other indications, the present study offers a novel approach to treat human brain injury.