Jonathan Lifshitz - US grants

[unreadable] DESCRIPTION (provided by applicant): [unreadable] In the United States alone, 500,000 people suffer traumatic brain injury (TBI) annually, making TBI a leading cause of death and disability. With improved accident scene and emergency care, mortality rates have declined, with 2-4 million people surviving TBI. Patients and patient families chiefly complain about post-concussive syndrome, involving alterations in cognition, aggression, emotional stability, disinhibition, and personality. In fact, post-concussive syndrome resembles the symptoms of amygdala resection or degeneration. In light of these post-injury deficits, the proposed project initiates experimentation focused on whether a subset of symptoms that define post-concussion syndrome are mediated by damage to the amygdala in a clinically relevant lateral fluid percussion model of brain injury in the mouse. The central hypothesis is: experimental TBI damages the amygdala bilaterally. Three aims test the hypothesis: (1) to demonstrate amygdala-dependent cognitive deficits using conditioned fear, (2) to quantify selective neuronal loss in the basolateral complex and central nucleus of the amygdala using design-based stereology, and (3) to evaluate and pharmacologically modulate functional circuitry and synaptic plasticity in the amygdala-hippocampal circuit using extracellular field recordings. The systems approach to investigate amygdala pathology after TBI, in terms of behavior, anatomy and function, can reveal mechanisms underlying post-concussion syndrome. This research effort may lead to novel treatments for the affective disorders in an otherwise healthy brain-injured population, in addition to post-traumatic stress disorder and psychosis. [unreadable] [unreadable]

DESCRIPTION (provided by applicant): Our long term goals are to develop effective pharmacological and rehabilitative treatment strategies that promote adaptive reorganization of brain-injured circuits to mitigate neurological dysfunction (morbidity) after diffuse traumatic brain injury (TBI). To achieve these goals, we will exploit a novel, reproducible, late-onset neurological deficit that we have observed in the diffuse brain-injured rat, analogous to agitation in brain injury survivors. In contrast to the soothing and pacifying nature of facial whisker stimulation in uninjured rats, brain- injured rats react to whisker stimulation by cowering, freezing and guarding the mystacial pads. This aberrant behavior is concomitant with axonal damage, neuronal atrophy, persistent inflammation and neuroplasticity, such that whisker stimulation activates brain regions outside the conventional somatosensory whisker circuit. The current proposal tests the hypothesis that diffuse brain injury-induced inflammation drives the maladaptive structural plasticity responsible for aberrant behavioral responses to whisker stimulation. The model system afforded by the brain-injured somatosensory whisker circuit provides a reductionistic approach to the complexity of diffuse TBI, to address pathological and reparative mechanisms associated with post-traumatic morbidity. This circuit in diffuse brain-injured adult male rats will be evaluated for (Aim 1) aberrant behaviors elicited by whisker stimulation, (Aim 2) chronic neuropathology, neuronal activation and circuit reorganization, and (Aim 3) neuroinflammation-driven neuroplastic responses that contribute to circuit reorganization and morbidity. An anti-inflammatory therapeutic regimen may provide a clinically relevant intervention to prevent circuit rewiring and the onset of morbidity. The innovative combination of behavioral, anatomical, functional and therapeutic approaches directed at the brain-injured somatosensory whisker circuit addresses the underlying mechanisms associated with unregulated structural plasticity in the injured brain. Uncovering these processes can direct treatments to mitigate the onset, reduce the duration and/or promote the resolution of neurological dysfunction. Results from this circuit can ultimately be expanded to other circuits in rodents and then man to improve quality of life for millions of TBI survivors and potentially others suffering from progressive neurodegenerative diseases. PUBLIC HEALTH RELEVANCE: The present proposal focuses on the persistent morbidity experienced by diffuse brain injury survivors, for whom treatment options are limited. In diffuse brain-injured rats, aberrant responses to whisker stimulation will be employed as a tool to identify pathological and reparative mechanisms associated with somatosensory whisker circuit disruption. By developing, validating and implementing an animal model of one discrete post-traumatic morbidity, significant advancements can be made towards reshaping first one, then other, brain- injured circuits in rodents and man.

DESCRIPTION (provided by applicant): The purpose of sleep continues to be debated. Prevailing hypotheses suggest a role in restoring energy balance, permitting synaptic reorganization, or cellular repair. With a traumatic brain injury, the mechanical forces and ensuing cellular signaling disrupt energy balance, initiate synaptic deafferentation followed by plasticity, and damage membranes, proteins and structural elements. Thus, acute post-traumatic sleep may mitigate injury-related damage. However, for the more than 1.4 million concussed individuals per year in the United States, prevailing folklore recommends that they should not be allowed to sleep or be awoken regularly, which is unsupported by medical evidence. Moreover, this sleep disturbance counteracts the natural repair processes of sleep that would be promoted by the ubiquitous inflammatory response after brain injury, as occurs with cytokine signaling upon infection. Surprisingly, the utility or detriment of acute post-traumatic sleep has yet to be explored. The current proposal tests the hypothesis that sleep is an immediate natural response to diffuse brain injury likely promoting recovery of the injured brain. Aim 1 will correlate quantitative sleep parameters acutely following mild and moderate diffuse brain injury in the mouse to chronic physiological, behavioral and histopathological outcomes. Aim 2 will explore the consequences of sleep disturbance on cognitive function, cytokine and glucocorticoid levels and histopathology across limbic and sleep structures. Post- traumatic sleep will be monitored by novel non-invasive technology validated for sleep- wake studies. This system employs custom-designed pressure sensors on the cage floor that continuously monitor for the motion associated with rhythmic breathing while animals are in a sleep posture. These experiments examine, for the first time, the effects of diffuse brain injury on sleep-wake patterns. Post-traumatic interventions to disrupt sleep may worsen or mitigate the behavioral and histopathological consequences of injury. Post-traumatic sleep may come to serve as an individualized biomarker to measure the severity of the initial injury and monitor the recovery process. For mild TBI, sleep itself could become a plausible behavioral intervention to mitigate the enduring neurological consequences of TBI. PUBLIC HEALTH RELEVANCE: After a concussion, prevailing folklore recommends that individuals should not be allowed to sleep or be awoken regularly. Since sleep may be responsible for cellular repair and reorganization, post-injury sleep disturbance may delay or even prevent recovery. This proposal evaluates post-injury sleep in a mouse model of traumatic brain injury and the consequences, good or bad, of disturbing sleep shortly after injury.

DESCRIPTION (provided by applicant): Despite preventative efforts (e.g., helmets and seatbelts), traumatic brain injuries (TBI) occur at a staggering rate and frequently result in post traumatic neurological impairment, including sensory sensitivity. No effective treatments are available to negate the neurological consequences of TBI. Our long term goal is to mitigate post-traumatic morbidity (late onset, gain-of-function neurological impairment) by manipulating injury-induced circuit reorganization. The present pilot feasibility project explores the novel concept that synaptogenesis is the pivotal point that solidifies maladaptive circuit reorganization after TBI. In this way, inhibition of synaptogenesis could curtail maladaptive reorganization of brain-injured circuits and thereby mitigate post-traumatic morbidity; however the same inhibition may prevent adaptive plasticity in the recovery from post-traumatic deficits (early onset, injury-induced neurological impairment). In these experiments, we investigate synaptogenic mechanisms associated with sensory sensitivity (a functional morbidity) observed during whisker stimulation that develops over 28 days in a rodent model of diffuse TBI that lacks contusion or cavitation. This sensory sensitivity is indicative of diffuse histopathology, likely including circuit plasticity and synaptogenesis, along the whisker thalamo-cortical circuit. Mechanistically, synaptogenesis can occur through the ¿2¿-1 voltage-dependent calcium receptor activation by thrombospondins (TSPs). We will investigate the role of thrombospondins (TSPs) in mediating post-traumatic synaptogenesis, which has been reported for functional recovery after stroke. Therefore, the hypothesis emerges that thrombospondin-mediated synaptogenesis in the whisker-barrel circuit is necessary for the expression of post-traumatic sensory sensitivity. As a corollary, synaptogenesis may be essential for recovery from injury-induced learning deficits. In Aim 1, we will quantify synaptogenic and thrombospondin-related gene and protein expression in the somatosensory whisker circuit over time after experimental diffuse brain injury. The results will delineate the post-traumatic period of synaptic change as a target for pharmacological inhibition. In Aim 2, we will prolong learning deficits and mitigate whisker-related behavioral morbidity and circuit hyper-activation by inhibiting synaptogenesis with systemic administration of gabapentin (an ¿2¿-1 receptor antagonist). Therapeutic efficacy would support a role for synaptogenesis in solidifying maladaptive circuits associated with the development of post-traumatic sensory sensitivity and verify adaptive plasticity in recovery from learning deficits. Success of this treatment would support a paradigm shift towards prevention of late-onset morbidity, rather than treatment of the symptoms, thereby improving quality of life for countless individuals with diffuse TBI. PUBLIC HEALTH RELEVANCE: Traumatic brain injury reduces quality of life, because afflicted individuals are unable to process sensory information, among other neurological symptoms. The present feasibility pilot project tests whether new brain circuits, which form during the natural recovery process, are responsible for neurological dysfunction. We propose that our novel approach to pharmacologically inhibit synaptogenesis, the climactic event in the formation of these new circuits, can mitigate long-lasting post-traumatic neurological impairments, potentially in man.

DESCRIPTION (provided by applicant): Our long term goals are to develop effective pharmacological and rehabilitative treatment strategies that promote adaptive reorganization of brain-injured circuits to mitigate neurological dysfunction (morbidity) after diffuse traumatic brain injury (TBI). To achieve these goals, we will exploit a novel, reproducible, late-onset neurological deficit that we have observed in the diffuse brain-injured rat, analogous to agitation in brain injury survivors. In contrast to the soothing and pacifying nature of facial whisker stimulation in uninjured rats, brain- injured rats react to whisker stimulation by cowering, freezing and guarding the mystacial pads. This aberrant behavior is concomitant with axonal damage, neuronal atrophy, persistent inflammation and neuroplasticity, such that whisker stimulation activates brain regions outside the conventional somatosensory whisker circuit. The current proposal tests the hypothesis that diffuse brain injury-induced inflammation drives the maladaptive structural plasticity responsible for aberrant behavioral responses to whisker stimulation. The model system afforded by the brain-injured somatosensory whisker circuit provides a reductionistic approach to the complexity of diffuse TBI, to address pathological and reparative mechanisms associated with post-traumatic morbidity. This circuit in diffuse brain-injured adult male rats will be evaluated for (Aim 1) aberrant behaviors elicited by whisker stimulation, (Aim 2) chronic neuropathology, neuronal activation and circuit reorganization, and (Aim 3) neuroinflammation-driven neuroplastic responses that contribute to circuit reorganization and morbidity. An anti-inflammatory therapeutic regimen may provide a clinically relevant intervention to prevent circuit rewiring and the onset of morbidity. The innovative combination of behavioral, anatomical, functional and therapeutic approaches directed at the brain-injured somatosensory whisker circuit addresses the underlying mechanisms associated with unregulated structural plasticity in the injured brain. Uncovering these processes can direct treatments to mitigate the onset, reduce the duration and/or promote the resolution of neurological dysfunction. Results from this circuit can ultimately be expanded to other circuits in rodents and then man to improve quality of life for millions of TBI survivors and potentially others suffering from progressive neurodegenerative diseases. PUBLIC HEALTH RELEVANCE: The present proposal focuses on the persistent morbidity experienced by diffuse brain injury survivors, for whom treatment options are limited. In diffuse brain-injured rats, aberrant responses to whisker stimulation will be employed as a tool to identify pathological and reparative mechanisms associated with somatosensory whisker circuit disruption. By developing, validating and implementing an animal model of one discrete post-traumatic morbidity, significant advancements can be made towards reshaping first one, then other, brain- injured circuits in rodents and man.

? DESCRIPTION (provided by applicant): After experimental diffuse brain injury, we recently uncovered a morphological variant of brain microglia - the rod microglia - originally described 100+ years ago, but largely ignored since then. Current understanding places rod microglia amongst activated microglia, which would typically progress towards phagocytotic, amoeboid microglia/macrophages. However, few primary articles have reported on rod microglia, with an absence of molecular investigations. For the first time, we reproducibly observe microglia with an elongated rod morphology that align in trains across cortical layers adjacent to neuronal processes in foci of neuropathology after diffuse brain-injury. Moreover, trains of rod microglia form in a time course consistent with circuit dismantling (neuropathology) and then recede as neural circuits recover (plasticity), albeit reorganized. Now we have the opportunity to explore the functional implication of these neuronal:glial interactions that may underlie brain reorganization and neurological symptoms after TBI. To begin investigating the significance and impact of rod microglia in acute neurological injury, the current proposal obtains critical preliminary data to distinguish activated rod microglia from activated microglia by gene expression profile. We will induce diffuse brain injury by midline fluid percussion injury in adult male rats and identify rod microglia based on immunohistochemistry and morphology. Laser-capture microdissection will isolate rod microglia and activated microglia, with surrounding neuronal elements, from cortical regions of the same animal to provide an internal control. Next-generation sequencing (RNA-Seq) and subsequent enrichment analysis (gene ontology, pathway data sets, and transcription factor analysis) will identify neuronal:glial expression unique to rod microglia. We expect that expression data will reveal differentiation, migration and proliferation origins of this morphology and inflammatory, cytoskeletal and degenerative functional roles of rod microglia. Results will define rod microglia, enabling the development of molecular tools to detect, isolate and target rod microglia. With refined molecular tools, rod microglia can be investigated across neurological conditions (e.g. seizure, Alzheimer's disease, Autism) and activated or inhibited to drive treatment of neurological symptoms.

? DESCRIPTION (provided by applicant): In children, traumatic brain injury (TBI) is the leading cause of mortality and morbidity representing a major health burden for affected individuals, their parents and the society at large with more than $1 billion in healthcare. Sadly, despite high incidence and decades of research, to date no new treatments improve outcome in children with TBI. Further understanding of the pathophysiology leading to secondary brain damage is required to develop new therapies. Our research has demonstrated that lysophosphatidic acid (LPA) is a proinflammatory lipid, highly produced after CNS injury, and contributes to tissue and neurological damage. Compelling data showed that LPA was markedly increased in adult patients' cerebrospinal fluid (CSF) after severe TBI, with specific LPA isoforms serving as potential signatures of brain damage. In postmortem human brain, the LPA receptors were upregulated by glial and ependymal cells after TBI implying enhanced LPA signaling. Therapeutic inhibition of LPA signaling in animal models of neurotrauma with a specific anti-LPA monoclonal antibody (mAb), Lpathomab(tm), delayed pathological processes, including neuroinflammation. Thus, LPA is a driving force for secondary injury processes associated with CNS inflammation and neurodegeneration after neurotrauma. To extend our results in adult human and rodent studies, here we investigate the involvement of LPA in pediatric TBI with a specific focus on diffuse brain injury. These preliminary data are essential to understand the similarities and differences between adult and pediatric TBI, in order to develop targeted interventions. We will use a dual clinical and experimental research approach following the aims below: 1) To determine time profiles of LPA isoforms in CSF and plasma of children with severe TBI, and validate the presence of higher levels of LPA in CSF compared to plasma; 2) To determine whether changes of LPA observed in children's samples are modeled effectively as an elevation of LPA in juvenile rat CSF and plasma after diffuse brain injury; 3) To identify the brain regions producing LPA in the injured rat brain, and assess the relationship of LPA with neuroinflammation (astrocytosis, activation of microglia, and cytokine production). By defining the CSF and plasma LPA pulse in response to pediatric TBI, this study will complement our recent findings in adult TBI. The experimental data will (1) demonstrate LPA production in juvenile TBI; (2) reveal potential associations between LPA that are specific to diffuse brain injury, the most frequent type of TBI in children; and (3) corroborate the link of increased LPA with cellular and humoral neuroinflammation. If our preliminary results are encouraging in both childhood TBI and juvenile diffuse TBI model, we will embark on a study to determine the efficacy of blocking LPA signaling in reducing secondary brain damage. In future, LPA may become a therapeutic target to reduce secondary brain damage in children suffering from TBI.

Project Summary Awkward body positions lead to an arm or leg ?falling asleep.? Upon repositioning, blood rushes back into the limb, with nerves spontaneously regaining function after being deprived of oxygen and electrolytes. While asleep, cellular and molecular processes within the ischemic limb may produce bioactive restorative and regenerative compounds that are released intravenously upon reperfusion. The method of intentional, intermittent restriction of blood flow to a limb is called Remote Ischemic Conditioning (RIC), which can be achieved by simple and cost- effective application of a tourniquet to a limb for several cycles of pre-determined duration. RIC can protect the brain and other organs from ischemic, surgical, and traumatic events, whether administered prior to or following the event. In fact, our team has demonstrated that RIC reduced biomarkers of acute damage in traumatic brain injury (TBI) patients. In pre-clinical cardiac arrest and cerebral ischemia, RIC preserves histopathology and improves functional outcome, using either pre-injury or post-injury RIC. To date, the mechanisms underlying RIC efficacy are unknown, with a dozen conflicting mechanisms proposed. TBI and other acquired neurological injuries share secondary injury processes, including inflammation and oxidative stress, which have been repeated targets of neuroprotective strategies. We propose a class of endogenous lipids derived from fatty acids, called Specialized Pro-Resolving Mediators (SPMs), as the molecular mechanism for RIC efficacy. SPMs, including the molecular families of resolvins, lipoxins, and protectins, are released from lipid bilayers after ischemia, actively resolve inflammation, and are neuroprotective in diffuse TBI, with the assumed biostability to travel in blood to the brain and the lipid structure for blood-brain barrier permeability. In this proposal, we hypothesize that RIC preserves neurological function following experimental diffuse TBI by producing SPMs, which mitigate injury-induced inflammation. To test the hypothesis, we apply RIC sequences to the hind limb of adult mice, first before and then after diffuse TBI induced by midline fluid percussion injury. Aim 1 will evaluate the efficacy of RIC sequences on preserving neurological, cognitive, and affective function over a 21 day time course post-injury. Aim 2, using the most effective sequence of RIC identified in Aim 1, will demonstrate that RIC attenuates microglial activation as an index of inflammation and produces SPMs as measured in plasma by liquid chromatography-coupled tandem mass spectrometry and commercial ELISA. Results from these aims will identify the RIC sequence necessary to improve neurological outcome from diffuse TBI. Further, we explore the production of SPMs as the molecular mechanism that targets microglial activation. Direct evidence is necessary to support the efficacy of RIC as a therapeutic approach to treat the estimated 1.7 million TBIs that occur in the United States. Ultimately, RIC could serve as a cost-effective and feasible therapy for delivering endogenous restorative and reparative compounds, such as SPMs, to improve outcome from TBI.