Vardit Rubovitch

A mouse model of blast-induced mild traumatic brain injury.

Improvised explosive devices (IEDs) are one of the main causes for casualties among civilians and military personnel in the present war against terror. Mild traumatic brain injury from IEDs induces various degrees of cognitive, emotional and behavioral disturbances but knowledge of the exact brain pathophysiology following exposure to blast is poorly understood. The study was aimed at establishing a murine model for a mild BI-TBI that isolates low-level blast pressure effects to the brain without systemic injuries. An open-field explosives detonation was used to replicate, as closely as possible, low-level blast trauma in the battlefield or at a terror-attack site. No alterations in basic neurological assessment or brain gross pathology were found acutely in the blast-exposed mice. At 7 days post blast, cognitive and behavioral tests revealed significantly decreased performance at both 4 and 7 m distance from the blast (5.5 and 2.5 PSI, respectively). At 30 days post-blast, clear differences were found in animals at both distances in the object recognition test, and in the 7 m group in the Y maze test. Using MRI, T1 weighted images showed an increased BBB permeability 1 month post-blast. DTI analysis showed an increase in fractional anisotropy (FA) and a decrease in radial diffusivity. These changes correlated with sites of up-regulation of manganese superoxide dismutase 2 in neurons and CXC-motif chemokine receptor 3 around blood vessels in fiber tracts. These results may represent brain axonal and myelin abnormalities. Cellular and biochemical studies are underway in order to further correlate the blast-induced cognitive and behavioral changes and to identify possible underlying mechanisms that may help develop treatment- and neuroprotective modalities.

Tumor necrosis factor-α synthesis inhibitor, 3’6,dithiothalidomide, reverses behavioral impairments induced by minimal traumatic brain injury in mice

J. Neurochem. (2011) 118, 1032–1042.

Exendin-4, a glucagon-like peptide-1 receptor agonist prevents mTBI-induced changes in hippocampus gene expression and memory deficits in mice.

Traumatic brain injury (TBI) is a global problem reaching near epidemic numbers that manifests clinically with cognitive problems that decades later may result in dementias like Alzheimers disease (AD). Presently, little can be done to prevent ensuing neurological dysfunctions by pharmacological means. Recently, it has become apparent that several CNS diseases share common terminal features of neuronal cell death. The effects of exendin-4 (Ex-4), a neuroprotective agent delivered via a subcutaneous micro-osmotic pump, were examined in the setting of mild TBI (mTBI). Utilizing a model of mTBI, where cognitive disturbances occur over time, animals were subjected to four treatments: sham; Ex-4; mTBI and Ex-4/mTBI. mTBI mice displayed deficits in novel object recognition, while Ex-4/mTBI mice performed similar to sham. Hippocampal gene expression, assessed by gene array methods, showed significant differences with little overlap in co-regulated genes between groups. Importantly, changes in gene expression induced by mTBI, including genes associated with AD were largely prevented by Ex-4. These data suggest a strong beneficial action of Ex-4 in managing secondary events induced by a traumatic brain injury.

The intricate involvement of the Insulin-like growth factor receptor signaling in mild traumatic brain injury in mice

Insulin-like growth factor-1 (IGF-1) was suggested as a potential neuroprotective treatment for traumatic brain injury (TBI) induced damage (cognitive as well as cellular). The main goal of the present study was to evaluate the role of the IGF-1R activation in spatial memory outcome following mild traumatic brain injury. mTBI-induced phosphorylation of IGF-1R, AKT and ERK1/2, in mice hippocampus, which was inhibited when mice were pretreated with the selective IGF-1R inhibitor AG1024. IGF-1 administration prevented spatial memory deficits following mTBI. Surprisingly, blocking the IGF-1R signaling in mTBI mice did not augment the spatial memory deficit. In addition, this data imply an intriguing and complex role of the IGF-1 signaling axis in the cellular and behavioral events following mTBI.

The cannabinoid agonist DALN positively modulates L-type voltage-dependent calcium-channels in N18TG2 neuroblastoma cells

The present study demonstrates a novel stimulatory effect of a cannabinoid agonist on calcium channels. DALN (1 nM) potentiated 45Ca(2+)-uptake by N18TG2 neuroblastoma cells, an effect that was abolished by the specific CB1 receptor antagonist SR141716A. The stimulation of 45Ca(2+)-uptake by DALN was resistant to pertussis toxin (PTX), suggesting that Gi/Go GTP-binding proteins did not mediate this effect. Furthermore, PTX unmasked a stimulatory effect of a high concentration of DALN (1 microM), which by itself failed to stimulate calcium uptake in naive cells. The stimulatory effect of DALN on calcium entry to the cells was blocked by nicardipine but not by omega-conotoxin GVIA, indicating the entry of calcium through L-type voltage-dependent calcium channels. Blocking cAMP-dependent protein kinase (PKA) by H-89 completely eliminated the elevation in calcium uptake, while blocking protein kinase C (PKC) by chelerythrine and calphostine-C only partially attenuated the stimulation. Blocking calmodulin by W-7 revealed a similar partial inhibition of the stimulatory effect of DALN. Hence, we suggest a cannabinoid-specific, PTX-insensitive, stimulatory effect on L-type voltage-dependent calcium channels, which is mediated by PKA and modulated by PKC and calmodulin.

The nuclear factor erythroid 2-like 2 activator, tert-butylhydroquinone, improves cognitive performance in mice after mild traumatic brain injury

Traumatic Brain injury affects at least 1.7 million people in the United States alone each year. The majority of injuries are categorized as mild but these still produce lasting symptoms that plague the patient and the medical field. Currently treatments are aimed at reducing a patients symptoms, but there is no effective method to combat the source of the problem, neuronal loss. We tested a mild, closed head traumatic brain injury model for the effects of modulation of the antioxidant transcription factor Nrf2 by the chemical activator, tert-butylhydroquinone (tBHQ). We found that post-injury visual memory was improved by a 7 day course of treatment and that the level of activated caspase-3 in the hippocampus was reduced. The injury-induced memory loss was also reversed by a single injection at 30 min after injury. Since the protective stress response molecule, HSP70, can be upregulated by Nrf2, we examined protein levels in the hippocampus, and found that HSP70 was elevated by the injury and then further increased by the treatment. To test the possible role of HSP70, model neurons in culture exposed to a mild injury and treated with the Nrf2 activator displayed improved survival that was blocked by the HSP70 inhibitor, VER155008. Following mild traumatic brain injury, there may be a partial protective response and patients could benefit from directed enhancement of regulatory pathways such as Nrf2 for neuroprotection.

Changes in mouse cognition and hippocampal gene expression observed in a mild physical- and blast-traumatic brain injury.

Warfare has long been associated with traumatic brain injury (TBI) in militarized zones. Common forms of TBI can be caused by a physical insult to the head-brain or by the effects of a high velocity blast shock wave generated by the detonation of an explosive device. While both forms of trauma are distinctly different regarding the mechanism of trauma induction, there are striking similarities in the cognitive and emotional status of survivors. Presently, proven effective therapeutics for the treatment of either form of TBI are unavailable. To be able to develop efficacious therapies, studies involving animal models of physical- and blast-TBI are required to identify possible novel or existing medicines that may be of value in the management of clinical events. We examined indices of cognition and anxiety-like behavior and the hippocampal gene transcriptome of mice subjected to both forms of TBI. We identified common behavioral deficits and gene expression regulations, in addition to unique injury-specific forms of gene regulation. Molecular pathways presented a pattern similar to that seen in gene expression. Interestingly, pathways connected to Alzheimers disease displayed a markedly different form of regulation depending on the type of TBI. While these data highlight similarities in behavioral outcomes after trauma, the divergence in hippocampal transcriptome observed between models suggests that, at the molecular level, the TBIs are quite different. These models may provide tools to help define therapeutic approaches for the treatment of physical- and blast-TBIs. Based upon observations of increasing numbers of personnel displaying TBI related emotional and behavioral changes in militarized zones, the development of efficacious therapies will become a national if not a global priority.

The Influence of Alcohol on Behavioral Recovery after mTBI in Mice

In the United States 258,000 people were injured in 2004 in motor vehicle accidents that were caused by drivers under the influence of alcohol. The majority of these drivers were binge drinkers, most notably young people who tend to drink heavily during the weekends, but rarely drink alcohol during the week. Since a large proportion of the injuries involved head injuries, the present study aimed at investigating the influence of binge alcohol drinking on mild traumatic brain injury (mTBI) in an animal model. Mice had access to 0%, 7.5%, 15%, or 30% alcohol solutions for 48 consecutive hours once a week for 4 weeks as the sole source of fluids (the remaining time they drank water). Three experiments were done. For the first one (alcohol-mTBI-alcohol) the animals were subjected to a controlled mTBI injury by applying a closed-head weight drop, or a sham procedure. After the mTBI/sham-mTBI the animals got alcohol and /water for the same regimen for 4 additional weeks. In the second experiment (alcohol only) after the 4 weeks of drinking blood samples were collected, at the same time as the animals that underwent sham-mTBI or mTBI procedures. In the third experiment (mTBI-alcohol) the mice were subjected to mTBI/sham-mTBI without any treatment, and after mTBI they had alcohol for 4 weeks in the same regimen as in the previous experiments. At the end of the pharmacological treatment all animals were assessed using different behavioral tests. mTBI mice exhibited lower memory ability in the Y-maze, higher anxiety in the elevated plus maze, and lower retention in the passive avoidance test than sham-mTBI animals. Alcohol reversed these effects at all doses. The results suggest that alcohol drinking before trauma might have a protective effect on recovery from brain trauma, but not if consumed after the trauma.

Incretin mimetics as pharmacologic tools to elucidate and as a new drug strategy to treat traumatic brain injury

Traumatic brain injury (TBI), either as an isolated injury or in conjunction with other injuries, is an increasingly common event. An estimated 1.7 million injuries occur within the USA each year and 10 million people are affected annually worldwide. Indeed, nearly one third (30.5%) of all injury‐related deaths in the USA are associated with TBI, which will soon outpace many common diseases as the major cause of death and disability. Associated with a high morbidity and mortality and no specific therapeutic treatment, TBI has become a pressing public health and medical problem. The highest incidence of TBI occurs in young adults (15–24 years age) and in the elderly (≥75 years of age). Older individuals are particularly vulnerable to these types of injury, often associated with falls, and have shown increased mortality and worse functional outcome after lower initial injury severity. In addition, a new and growing form of TBI, blast injury, associated with the detonation of improvised explosive devices in the war theaters of Iraq and Afghanistan, are inflicting a wave of unique casualties of immediate impact to both military personnel and civilians, for which long‐term consequences remain unknown and may potentially be catastrophic. The neuropathology underpinning head injury is becoming increasingly better understood. Depending on severity, TBI induces immediate neuropathologic effects that, for the mildest form, may be transient; however, with increasing severity, these injuries cause cumulative neural damage and degeneration. Even with mild TBI, which represents the majority of cases, a broad spectrum of neurologic deficits, including cognitive impairments, can manifest that may significantly influence quality of life. Further, TBI can act as a conduit to longer term neurodegenerative disorders. Prior studies of glucagon‐like peptide‐1 (GLP‐1) and long‐acting GLP‐1 receptor agonists have demonstrated neurotrophic/neuroprotective activities across a broad spectrum of cellular and animal models of chronic neurodegenerative (Alzheimers and Parkinsons diseases) and acute cerebrovascular (stroke) disorders. In view of the mechanisms underpinning these disorders as well as TBI, we review the literature and recent studies assessing GLP‐1 receptor agonists as a potential treatment strategy for mild to moderate TBI.

The Stimulatory Effect of Cannabinoids on Calcium Uptake Is Mediated by Gs GTP-Binding Proteins and cAMP Formation

Cannabinoids are neurodepressive drugs that convey their cellular action through Gi/o GTP-binding proteins which reduce cAMP formation and Ca2+ influx. However, a growing body of evidence indicates that the stimulatory effects of cannabinoids include the elevation in cAMP and cytosolic Ca2+ concentration. The present study expands our previous findings and demonstrates that, in N18TG2 neuroblastoma cells, the cannabinoid agonist desacetyllevonantradol (DALN) stimulates both cAMP formation and Ca2+ uptake. The stimulatory effect of DALN on cAMP formation was not eliminated by blocking Ca2+ entry to the cells, while its stimulatory effect on Ca2+ uptake was abolished by blocking cAMP-dependent protein kinase. Furthermore, elevating cAMP by forskolin stimulated calcium uptake, while elevating the intracellular Ca2+ concentration by ionomycin or KCl failed to stimulate cAMP formation. These findings suggest that cAMP production precedes the influx of Ca2+ in the cannabinoid stimulatory cascade. The stimulatory effect of DALN on calcium uptake resisted pertussis toxin treatment, and was completely blocked by introducing anti-Gs antibodies into the cells, indicating that the stimulatory activity of cannabinoids is mediated by Gs GTP-binding proteins. The relevance of the cellular stimulatory activity of DALN to the pharmacological profile of cannabinoid drugs is discussed.