Luca Longhi

Motor and cognitive function evaluation following experimental traumatic brain injury

Traumatic brain injury (TBI) in humans may cause extensive sensorimotor and cognitive dysfunction. As a result, many TBI researchers are beginning to assess behavioral correlates of histologically determined damage in animal models. Although this is an important step in TBI research, there is a need for standardization between laboratories. The ability to reliably test treatments across laboratories and multiple injury models will close the gap between treatment success in the lab and success in the clinic. The goal of this review is to describe and evaluate the tests employed to assess functional outcome after TBI and to overview aspects of cognitive, sensory, and motor function that may be suitable targets for therapeutic intervention.

Transplanted neural stem cells survive, differentiate, and improve neurological motor function after experimental traumatic brain injury.

OBJECTIVE Using the neural stem cell (NSC) clone C17.2, we evaluated the ability of transplanted murine NSCs to attenuate cognitive and neurological motor deficits after traumatic brain injury. METHODS Nonimmunosuppressed C57BL/6 mice (n = 65) were anesthetized and subjected to lateral controlled cortical impact brain injury (n = 52) or surgery without injury (sham operation group, n = 13). At 3 days postinjury, all brain-injured animals were reanesthetized and randomized to receive stereotactic injection of NSCs or control cells (human embryonic kidney cells) into the cortex-hippocampus interface in either the ipsilateral or the contralateral hemisphere. One group of animals (n = 7) was killed at either 1 or 3 weeks postinjury to assess NSC survival in the acute posttraumatic period. Motor function was evaluated at weekly intervals for 12 weeks in the remaining animals, and cognitive (i.e., learning) deficits were assessed at 3 and 12 weeks after transplantation. RESULTS Brain-injured animals that received either ipsilateral or contralateral NSC transplants showed significantly improved motor function in selected tests as compared with human embryonic kidney cell-transplanted animals during the 12-week observation period. Cognitive dysfunction was unaffected by transplantation at either 3 or 12 weeks postinjury. Histological analyses showed that NSCs survive for as long as 13 weeks after transplantation and were detected in the hippocampus and/or cortical areas adjacent to the injury cavity. At 13 weeks, the NSCs transplanted ipsilateral to the impact site expressed neuronal (NeuN) or astrocytic (glial fibrillary acidic protein) markers but not markers of oligodendrocytes (2′3′cyclic nucleotide 3′-phosphodiesterase), whereas the contralaterally transplanted NSCs expressed neuronal but not glial markers (double-labeled immunofluorescence and confocal microscopy). CONCLUSION These data suggest that transplanted NSCs can survive in the traumatically injured brain, differentiate into neurons and/or glia, and attenuate motor dysfunction after traumatic brain injury.

Temporal window of vulnerability to repetitive experimental concussive brain injury.

OBJECTIVE:Repetitive concussive brain injury (CBI) is associated with cognitive alterations and increased risk of neurodegenerative disease. METHODS:To evaluate the temporal window during which the concussed brain remains vulnerable to a second concussion, anesthetized mice were subjected to either sham injury or single or repetitive CBI (either 3, 5, or 7 days apart) using a clinically relevant model of CBI. Cognitive, vestibular, and sensorimotor function (balance and coordination) were evaluated, and postmortem histological analyses were performed to detect neuronal degeneration, cytoskeletal proteolysis, and axonal injury. RESULTS:No cognitive deficits were observed in sham-injured animals or those concussed once. Mice subjected to a second concussion within 3 or 5 days exhibited significantly impaired cognitive function compared with either sham-injured animals (P < 0.05) or mice receiving a single concussion (P < 0.01). No cognitive deficits were observed when the interconcussion interval was extended to 7 days, suggestive of a transient vulnerability of the brain during the first 5 days after an initial concussion. Although all concussed mice showed transient motor deficits, vestibulomotor dysfunction was more pronounced in the group that sustained two concussions 3 days apart (P < 0.01 compared with all other groups). Although scattered degenerating neurons, evidence of cytoskeletal damage, and axonal injury were detected in selective brain regions between 72 hours and 1 week after injury in all animals sustaining a single concussion, the occurrence of a second concussion 3 days later resulted in significantly greater traumatic axonal injury (P < 0.05) than that resulting from a single CBI. CONCLUSION:These data suggest that a single concussion is associated with behavioral dysfunction and subcellular alterations that may contribute to a transiently vulnerable state during which a second concussion within 3 to 5 days can lead to exacerbated and more prolonged axonal damage and greater behavioral dysfunction.

Vitamin E reduces amyloidosis and improves cognitive function in Tg2576 mice following repetitive concussive brain injury

Traumatic brain injury is a well‐recognized environmental risk factor for developing Alzheimers disease. Repetitive concussive brain injury (RCBI) exacerbates brain lipid peroxidation, accelerates amyloid (Aβ) formation and deposition, as well as cognitive impairments in Tg2576 mice. This study evaluated the effects of vitamin E on these four parameters in Tg2576 mice following RCBI. Eleven‐month‐old mice were randomized to receive either regular chow or chow‐supplemented with vitamin E for 4 weeks, and subjected to RCBI (two injuries, 24 h apart) using a modified controlled cortical impact model of closed head injury. The same dietary regimens were maintained up to 8 weeks post‐injury, when the animals were killed for biochemical and immunohistochemical analyses after behavioral evaluation. Vitamin E‐treated animals showed a significant increase in brain vitamin E levels and a significant decrease in brain lipid peroxidation levels. After RBCI, compared with the group on regular chow, animals receiving vitamin E did not show the increase in Aβ peptides, and had a significant attenuation of learning deficits. This study suggests that the exacerbation of brain oxidative stress following RCBI plays a mechanistic role in accelerating Αβ accumulation and behavioral impairments in the Tg2576 mice.

Age-dependent synuclein pathology following traumatic brain injury in mice

Synucleins (Syn), a family of synaptic proteins, includes alpha-Syn, which plays a pivotal role in Parkinsons disease and related neurodegenerative diseases (synucleinopathies) by forming distinct brain pathologies (Lewy bodies and neurites). Since traumatic brain injury (TBI) is a poorly understood risk factor for Parkinsons disease, we examined the effects of TBI in the young and aged mouse brain on alpha-, beta-, and gamma-Syn. Immunohistochemical analysis showed that brains from sham-injured young and aged mice had normal alpha- and beta-Syn immunoreactivity (IR) in neuropil of cortex, striatum, and hippocampus with little or no gamma-Syn IR. At 1 week post TBI, the aged mouse brain showed a transient increase of alpha- and beta-Syn IR in the neuropil as well as an induction of gamma-Syn IR in subcortical axons. This was associated with strong labeling of striatal axon bundles by antibodies to altered or nitrated epitopes in alpha-Syn as well as by antibodies to inducible nitric oxide synthase. However, these TBI-induced changes disappeared by 16 weeks post TBI, and altered Syn IR was not seen in young mice subjected to TBI nor in alpha-Syn knockout mice while Western blots confirmed that TBI induced transient alterations of alpha-Syn in the mouse brains. This model of age-dependent TBI-induced transient alterations in alpha-Syn provides an opportunity to examine possible links between TBI and mechanisms of disease in synucleinopathies.

C1-inhibitor attenuates neurobehavioral deficits and reduces contusion volume after controlled cortical impact brain injury in mice.

Objective:The aim of the study was to evaluate the effects of C1-inhibitor (C1-INH), an endogenous inhibitor of complement and kinin systems, on neurobehavioral and histological outcome following controlled cortical impact brain injury. Design:Experimental prospective randomized study in mice. Setting:Experimental laboratory. Subjects:Male C57Bl/6 mice (n = 81). Interventions:Mice were subjected to controlled cortical impact brain injury followed by an intravenous bolus of either C1-INH (15 U either at 10 minutes or 1 hour postinjury) or saline (equal volume, 150 &mgr;l at 10 minutes postinjury). Sham-operated mice received identical surgery and saline injection without brain injury. Neurological motor function was evaluated weekly for 4 weeks using the Composite Neuroscore. Cognitive function was evaluated at 4 weeks postinjury using the Morris Water Maze. Histological outcome was performed by measuring the contusion volume at 1 week and 4 weeks postinjury. Measurements and Main Results:Brain-injured mice receiving C1-INH at 10 minutes postinjury showed attenuated motor deficits, cognitive dysfunction and reduced contusion volume compared to brain-injured mice receiving saline. Mice receiving C1-INH at 1 hour postinjury showed reduced motor deficits compared to brain-injured mice receiving saline, but no significantly different cognitive and histological outcome. Immunohistochemical analysis showed that 20 minutes after infusion, C1-INH was localised on endothelial cells and in brain tissue surrounding brain capillaries of the injured hemisphere. Conclusion:Our results show that post-traumatic administration of C1-INH attenuates neuro-behavioral deficits and histological damage associated with traumatic brain injury.

A Review and Rationale for the Use of Genetically Engineered Animals in the Study of Traumatic Brain Injury

The mechanisms underlying secondary cell death after traumatic brain injury (TBI) are poorly understood. Animal models of TBI recapitulate many clinical and pathologic aspects of human head injury, and the development of genetically engineered animals has offered the opportunity to investigate the specific molecular and cellular mechanisms associated with cell dysfunction and death after TBI, allowing for the evaluation of specific cause-effect relations and mechanistic hypotheses. This article represents a compendium of the current literature using genetically engineered mice in studies designed to better understand the posttraumatic inflammatory response, the mechanisms underlying DNA damage, repair, and cell death, and the link between TBI and neurodegenerative diseases.

c-Jun N-Terminal Kinase Pathway Activation in Human and Experimental Cerebral Contusion

The c-Jun N-terminal kinase (JNK) pathway is involved in cell stress and apoptosis. We tested the hypothesis that this pathway plays a role in traumatic brain injury (TBI) by assessing JNK activation in human brain tissues and in brains of mice subjected to controlled cortical impact brain injury. We also assessed the effects of specific inhibition of the JNK pathway by the cell-permeable JNK inhibitor peptide, D-JNKI1, on neurobehavioral function and posttraumatic cellloss in mice. The inhibitor was administered intraperitoneally 10minutes after injury. The JNK pathway showed robust activation both in human contusion specimens and in injured cortex and hippocampi of TBI-injured mice, 1, 4, and 48 hours after injury. D-JNKI1 treatment significantly improved motor performance at 48 hours and 7days after injury and reduced the contusion volume compared withsaline treatment; the numbers of terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling-positive cells were significantly decreased in the hippocampi of injured mice 48 hours after treatment. Thus, because the JNK pathway is activated after human and experimental TBI and the inhibitor peptide D-JNKI1 affords significant neuroprotection and amelioration of neurobehavioral deficits after experimental TBI, therapeutic targeting of the JNK activation pathway may hold promise for future clinical applications.

Pharmacological inhibition of mannose-binding lectin ameliorates neurobehavioral dysfunction following experimental traumatic brain injury

Mannose-binding lectin is present in the contusion area of traumatic brain-injured patients and in that of traumatic brain-injured mice, where mannose-binding lectin-C exceeds mannose-binding lectin-A. The reduced susceptibility to traumatic brain injury of mannose-binding lectin double knock-out mice (mannose-binding lectin−/−) when compared to wild type mice suggests that mannose-binding lectin may be a therapeutic target following traumatic brain injury. Here, we evaluated the effects of a multivalent glycomimetic mannose-binding lectin ligand, Polyman9, following traumatic brain injury in mice. In vitro surface plasmon resonance assay indicated that Polyman9 dose-dependently inhibits the binding to immobilized mannose residues of plasma mannose-binding lectin-C selectively over that of mannose-binding lectin-A. Male C57Bl/6 mice underwent sham/controlled cortical impact traumatic brain injury and intravenous treatment with Polyman9/saline. Ex-vivo surface plasmon resonance studies confirmed that Polyman9 effectively reduces the binding of plasma mannose-binding lectin-C to immobilized mannose residues. In vivo studies up to four weeks post injury, showed that Polyman9 induces significant improvement in sensorimotor deficits (by neuroscore and beam walk), promotes neurogenesis (73% increase in doublecortin immunoreactivity), and astrogliosis (28% increase in glial fibrillary acid protein). Polyman9 administration in brain-injured mannose-binding lectin−/− mice had no effect on post-traumatic brain-injured functional deficits, suggestive of the specificity of its neuroprotective effects. The neurobehavioral efficacy of Polyman9 implicates mannose-binding lectin-C as a novel therapeutic target for traumatic brain injury.

Implementation of continuous qEEG in two neurointensive care units by intensivists: a feasibility study

Continuous electroencephalography (cEEG) in the intensive care unit (ICU) is recommended for the detection of seizures and ischemia [1, 2]. Technical aspects have been defined, and quantitative EEG (qEEG) has been developed to streamline the detection of meaningful electrographic events and to provide the possibility to monitor EEG trends over time [3]. Nevertheless, the implementation of cEEG/qEEG faces many obstacles, including the availability of EEG technicians and neurophysiologists to review the studies 24/7 and the high costs of the equipment. A solution to this conundrum could be the interpretation of the qEEG data directly by the intensivist’s staff and the use of low-cost networked devices. We decided to implement a project with the neurophysiologists in two neuro-ICUs (NICU) to evaluate the possibility of implementing continuous qEEG recordings and to train the NICU staff in interpreting these recording. We evaluated, using online tools, the agreement between NICU staff and neurophysiologists in interpreting relevant information on continuous cEEG/qEEG tracings both before and after a training period. The study, registered at ClinicalTrials.gov NCT02901262, was conducted in three phases (further details are presented in the Electronic Supplementary Material):