Nicolas C. Royo

Plasticity following injury to the adult central nervous system: is recapitulation of a developmental state worth promoting?

The adult central nervous system (CNS) appears to initiate a transient increase in plasticity following injury, including increases in growth-related proteins and generation of new cells. Recent evidence is reviewed that the injured adult CNS exhibits events and patterns of gene expression that are also observed during development and during regeneration following damage to the mature peripheral nervous system (PNS). The growth of neurons during development or regeneration is correlated, in part, with a coordinated expression of growth-related proteins, such as growth-associated-protein-43 (GAP-43), microtubule-associated-protein-1B (MAP1B), and polysialylated-neural-cell-adhesion-molecule (PSA-NCAM). For each of these proteins, evidence is discussed regarding its specific role in neuronal development, signals that modify its expression, and reappearance following injury. The rate of adult hippocampal neurogenesis is also affected by numerous endogenous and exogenous factors including injury. The continuing study of developmental neurobiology will likely provide further gene and protein targets for increasing plasticity and regeneration in the mature adult CNS.

Pharmacology of traumatic brain injury.

The intensity of experimental and clinical research to identify a neuroprotective drug for the treatment of traumatic brain injury is motivated by the devastating morbidity and mortality of this condition. Encouraging experimental work has led so far to disappointing clinical trials and the identification of new potential therapeutic targets is critically dependent on a better understanding of the chronic pathophysiology triggered by the initial insult. Future advances in the pharmacological treatment of traumatic brain injury are likely to include the evaluation of sequentially timed therapies combining multiple and targeted agents, and manipulation of the newly discovered neurogenic potential of the adult brain together with the refinement of traditional interventions to block specific cytotoxic cascades.

Hippocampal vulnerability following traumatic brain injury: a potential role for neurotrophin-4/5 in pyramidal cell neuroprotection.

Traumatic brain injury (TBI) causes selective hippocampal cell death, which is believed to be associated with cognitive impairment observed both in clinical and experimental settings. Although neurotrophin administration has been tested as a strategy to prevent cell death following TBI, the potential neuroprotective role of neurotrophin‐4/5 (NT‐4/5) in TBI remains unknown. We hypothesized that NT‐4/5 would offer neuroprotection for selectively vulnerable hippocampal neurons following TBI. Measurements of NT‐4/5 in rats subjected to lateral fluid percussion (LFP) TBI revealed two–threefold increases in the injured cortex and hippocampus in the acute period (1–3 days) following brain injury. Subsequently, the response of NT‐4/5 knockout (NT‐4/5–/–) mice to controlled‐cortical impact TBI was investigated. NT‐4/5–/– mice were more susceptible to selective pyramidal cell loss in Ahmons corn (CA) subfields of the hippocampus following TBI, and showed impaired motor recovery when compared with their brain‐injured wild‐type controls (NT‐4/5wt). Additionally, we show that acute, prolonged administration of recombinant NT‐4/5 (5 µg/kg/day) prevented up to 50% of the hippocampal CA pyramidal cell death following LFP TBI in rats. These results suggest that post‐traumatic increases in endogenous NT‐4/5 may be part of an adaptive neuroprotective response in the injured brain, and that administration of this neurotrophic factor may be useful as a therapeutic strategy following TBI.

Specific AAV Serotypes Stably Transduce Primary Hippocampal and Cortical Cultures with High Efficiency and Low Toxicity

Most current methods of gene delivery for primary cultured hippocampal neurons are limited by toxicity, transient expression, the use of immature neurons and/or low efficiency. We performed a direct comparison of seven serotypes of adeno-associated virus (AAV) vectors for genetic manipulation of primary cultured neurons in vitro. Serotypes 1, 2, 7, 8 and 9 mediated highly efficient, nontoxic, stable long-term gene expression in cultured cortical and hippocampal neurons aged 0-4 weeks in vitro; serotypes 5 and 6 were associated with toxicity at high doses. AAV1 transduced over 90% of all cells with approximately 80% of the transduced cells being neurons. The method was readily adapted to a high-throughput format to demonstrate neurotrophin-mediated neuroprotection from glutamate toxicity in cultured neurons at 2 weeks in vitro. These vectors should prove highly useful for efficient overexpression or downregulation of genes in primary neuronal cultures at any developmental stage.

Role of monocyte chemoattractant protein-1 (MCP-1/CCL2) in migration of neural progenitor cells toward glial tumors.

Neural progenitor cells (NPCs) have been investigated as potential vehicles for brain tumor therapy because they have been shown to migrate toward central nervous system gliomas and can be genetically engineered to deliver cytotoxic agents to tumors. The mechanisms that regulate migration of NPCs to tumors are not fully understood. By means of microarray analysis, polymerase chain reaction, enzyme‐linked immunosorbent assay, and immunohistochemistry, we found that monocyte chemoattractant protein‐1 (MCP‐1/CCL‐2) was expressed in experimental brain tumor cells in vivo and in vitro. CCR2, the receptor for MCP‐1, was expressed on C17.2 NPCs. We used a modified Boyden chamber assay and found increased migration of NPCs in vitro in response to MCP‐1. By means of an in vivo model for NPC migration, we found evidence of NPC migration toward areas of MCP‐1 infusion in rat brains. An understanding of NPC migration mechanisms may be used to enhance delivery of cytotoxic agents to brain tumor cells.

Cognitive outcome following brain injury and treatment with an inhibitor of Nogo-A in association with an attenuated downregulation of hippocampal growth-associated protein-43 expression

OBJECT Central nervous system axons regenerate poorly after traumatic brain injury (TBI), partly due to inhibitors such as the protein Nogo-A present in myelin. The authors evaluated the efficacy of anti-Nogo-A monoclonal antibody (mAb) 7B12 administration on the neurobehavioral and cognitive outcome of rats following lateral fluid-percussion brain injury, characterized the penetration of the 7B12 or control antibodies into target brain regions, and evaluated the effects of Nogo-A inhibition on hemispheric tissue loss and sprouting of uninjured motor tracts in the cervical cord. To elucidate a potential molecular response to Nogo-A inhibition, we evaluated the effects of 7B12 on hippocampal GAP-43 expression. METHODS Beginning 24 hours after lateral fluid-percussion brain injury or sham injury in rats, the mAb 7B12 or control antibody was infused intracerebroventricularly over 14 days, and behavior was assessed over 4 weeks. RESULTS Immunoreactivity for 7B12 or immunoglobulin G was detected in widespread brain regions at 1 and 3 weeks postinjury. The brain-injured animals treated with 7B12 showed improvement in cognitive function (p < 0.05) at 4 weeks but no improvement in neurological motor function from 1 to 4 weeks postinjury compared with brain-injured, vehicle-treated controls. The enhanced cognitive function following inhibition of Nogo-A was correlated with an attenuated postinjury downregulation of hippocampal GAP-43 expression (p < 0.05). CONCLUSIONS Increased GAP-43 expression may be a novel molecular mechanism of the enhanced cognitive recovery mediated by Nogo-A inhibition after TBI in rats.

Tissue sparing and functional recovery following experimental traumatic brain injury is provided by treatment with an anti‐myelin‐associated glycoprotein antibody

Axonal injury is a hallmark of traumatic brain injury (TBI) and is associated with a poor clinical outcome. Following central nervous system injury, axons regenerate poorly, in part due to the presence of molecules associated with myelin that inhibit axonal outgrowth, including myelin‐associated glycoprotein (MAG). The involvement of MAG in neurobehavioral deficits and tissue loss following experimental TBI remains unexplored and was evaluated in the current study using an MAG‐specific monoclonal antibody (mAb). Anesthetized rats (n = 102) were subjected to either lateral fluid percussion brain injury (n = 59) or sham injury (n = 43). In surviving animals, beginning at 1 h post‐injury, 8.64 µg anti‐MAG mAb (n = 33 injured, n = 21 sham) or control IgG (n = 26 injured, n = 22 sham) was infused intracerebroventricularly for 72 h. One group of these rats (n = 14 sham, n = 11 injured) was killed at 72 h post‐injury for verification of drug diffusion and MAG immunohistochemistry. All other animals were evaluated up to 8 weeks post‐injury using tests for neurologic motor, sensory and cognitive function. Hemispheric tissue loss was also evaluated at 8 weeks post‐injury. At 72 h post‐injury, increased immunoreactivity for MAG was seen in the ipsilateral cortex, thalamus and hippocampus of brain‐injured animals, and anti‐MAG mAb was detectable in the hippocampus, fimbria and ventricles. Brain‐injured animals receiving anti‐MAG mAb showed significantly improved recovery of sensorimotor function at 6 and 8 weeks (P < 0.01) post‐injury when compared with brain‐injured IgG‐treated animals. Additionally, at 8 weeks post‐injury, the anti‐MAG mAb‐treated brain‐injured animals demonstrated significantly improved cognitive function and reduced hemispheric tissue loss (P < 0.05) when compared with their brain‐injured controls. These results indicate that MAG may contribute to the pathophysiology of experimental TBI and treatment strategies that target MAG may be suitable for further evaluation.

Neurotrophin-mediated neuroprotection of hippocampal neurons following traumatic brain injury is not associated with acute recovery of hippocampal function

Traumatic brain injury (TBI) causes selective hippocampal cell death which is believed to be associated with the cognitive impairment observed in both clinical and experimental settings. The endogenous neurotrophin-4/5 (NT-4/5), a TrkB ligand, has been shown to be neuroprotective for vulnerable CA3 pyramidal neurons after experimental brain injury. In this study, infusion of recombinant NT-4/5 increased survival of CA2/3 pyramidal neurons to 71% after lateral fluid percussion brain injury in rats, compared with 55% in vehicle-treated controls. The functional outcome of this NT-4/5-mediated neuroprotection was examined using three hippocampal-dependent behavioral tests. Injury-induced impairment was evident in all three tests, but interestingly, there was no treatment-related improvement in any of these measures. Similarly, injury-induced decreased excitability in the Schaffer collaterals was not affected by NT-4/5 treatment. We propose that a deeper understanding of the factors that link neuronal survival to recovery of function will be important for future studies of potentially therapeutic agents.

Neurotrophic Factors: Pathophysiology and Therapeutic Applications in Traumatic Brain Injury

AbstractNeurotrophic factors (NTFs) are endogenous molecules that play a crucial role in the maintenance, survival and differentiation of various neuronal populations within the developing and adult brain. In vitro and in vivo studies have shown that NTFs can attenuate neuronal injury initiated by cascades that are activated by traumatic brain injury (TBI) including excitotoxic damage, ischemia, and apoptosis. NTFs may also play a role in repair-regeneration processes such as axonal regeneration, neuronal plasticity and neurogenesis that could be critical for recovery after TBI. Nerve growth factor (NGF), insulin like growth factor I (IGF-I) and basic fibroblast growth factor (bFGF) and glial cell-derived neurotrophic factor (GDNF) have been shown to be effective in reducing neurobehavioral dysfunction and/or histopathological damage in experimental models of TBI. NTFs appear to be promising therapeutic tools for TBI, although more studies are necessary to elucidate their potential application and to test delivery systems that allow local, regulated supply to specific populations of neurons. This article provides an overview of the pathophysiology and potential therapeutic application of neurotrophic factors in TBI.

Identification of potentially neuroprotective genes upregulated by neurotrophin treatment of CA3 neurons in the injured brain.

Specific neurotrophic factors mediate histological and/or functional improvement in animal models of traumatic brain injury (TBI). In previous work, several lines of evidence indicated that the mammalian neurotrophin NT-4/5 is neuroprotective for hippocampal CA3 pyramidal neurons after experimental TBI. We hypothesized that NT-4/5 neuroprotection is mediated by changes in the expression of specific sets of genes, and that NT-4/5-regulated genes are potential therapeutic targets for blocking delayed neuronal death after TBI. In this study, we performed transcription profiling analysis of CA3 neurons to identify genes regulated by lateral fluid percussion injury, or by treatment with the trkB ligands NT-4/5 or brain-derived neurotrophic factor (BDNF). The results indicate extensive overlap between genes upregulated by neurotrophins and genes upregulated by injury, suggesting that the mechanism behind neurotrophin neuroprotection may mimic the brains endogenous protective response. A subset of genes selected for further study in vitro exhibited neuroprotection against glutamate excitotoxicity. The neuroprotective genes identified in this study were upregulated at 30 h post-injury, and are thus expected to act during a clinically useful time frame of hours to days after injury. Modulation of these factors and pathways by genetic manipulation or small molecules may confer hippocampal neuroprotection in vivo in preclinical models of TBI.