ABSTRACT
Clinical and experimental studies show that spinal cord injury (SCI) can cause cognitive impairment and depression that can significantly impact outcomes. Thus, identifying mechanisms responsible for these less well-examined, important SCI consequences may provide targets for more effective therapeutic intervention. To determine whether cognitive and depressive-like changes correlate with injury severity, we exposed mice to sham, mild, moderate, or severe SCI using the Infinite Horizon Spinal Cord Impactor and evaluated performance on a variety of neurobehavioral tests that are less dependent on locomotion. Cognitive impairment in Y-maze, novel objective recognition, and step-down fear conditioning tasks were increased in moderate- and severe-injury mice that also displayed depressive-like behavior as quantified in the sucrose preference, tail suspension, and forced swim tests. Bromo-deoxyuridine incorporation with immunohistochemistry revealed that SCI led to a long-term reduction in the number of newly-generated immature neurons in the hippocampal dentate gyrus, accompanied by evidence of greater neuronal endoplasmic reticulum (ER) stress. Stereological analysis demonstrated that moderate/severe SCI reduced neuronal survival and increased the number of activated microglia chronically in the cerebral cortex and hippocampus. The potent microglial activator cysteine-cysteine chemokine ligand 21 (CCL21) was elevated in the brain sites after SCI in association with increased microglial activation. These findings indicate that SCI causes chronic neuroinflammation that contributes to neuronal loss, impaired hippocampal neurogenesis and increased neuronal ER stress in important brain regions associated with cognitive decline and physiological depression. Accumulation of CCL21 in brain may subserve a pathophysiological role in cognitive changes and depression after SCI.
Subject(s)
Brain/metabolism , Cognitive Dysfunction/metabolism , Depression/metabolism , Endoplasmic Reticulum Stress/physiology , Neurogenesis/physiology , Spinal Cord Injuries/metabolism , Animals , Brain/pathology , Cognitive Dysfunction/pathology , Cognitive Dysfunction/psychology , Depression/psychology , Locomotion/physiology , Male , Maze Learning/physiology , Mice , Mice, Inbred C57BL , Spinal Cord Injuries/pathology , Spinal Cord Injuries/psychologyABSTRACT
Chronic pain after spinal cord injury (SCI) may present as hyperalgesia, allodynia, and/or spontaneous pain and is often resistant to conventional pain medications. Identifying more effective interventions to manage SCI pain requires improved understanding of the pathophysiological mechanisms involved. Cell cycle activation (CCA) has been implicated as a key pathophysiological event following SCI. We have shown that early central or systemic administration of a cell cycle inhibitor reduces CCA, prevents glial changes, and limits SCI-induced hyperesthesia. Here, we compared the effects of early vs late treatment with the pan-cyclin-dependent kinase inhibitor flavopiridol on allodynia as well as spontaneous pain. Adult C57BL/6 male mice subjected to moderate SCI were treated with intraperitoneal injections of flavopiridol (1 mg/kg), daily for 7 days beginning either 3 hours or 5 weeks after injury. Mechanical/thermal allodynia was evaluated, as well as spontaneous pain using the mouse grimace scale (MGS). We show that sensitivity to mechanical and thermal stimulation, and locomotor dysfunction were significantly reduced by early flavopiridol treatment compared with vehicle-treated controls. Spinal cord injury caused robust and extended increases of MGS up to 3 weeks after trauma. Early administration of flavopiridol significantly shortened duration of MGS changes. Late flavopiridol intervention significantly limited hyperesthesia at 7 days after treatment, associated with reduced glial changes, but without effect on locomotion. Thus, our data suggest that cell cycle modulation may provide an effective therapeutic strategy to reduce hyperesthesia after SCI, with a prolonged therapeutic window.
Subject(s)
Cell Cycle/drug effects , Cell Cycle/physiology , Flavonoids/pharmacology , Neuralgia/etiology , Piperidines/pharmacology , Protein Kinase Inhibitors/pharmacology , Spinal Cord Injuries/complications , Animals , Calcium-Binding Proteins/metabolism , Cell Cycle Proteins/metabolism , Cell Movement/drug effects , Disease Models, Animal , Facial Expression , Flavonoids/therapeutic use , Ganglia, Spinal/drug effects , Ganglia, Spinal/metabolism , Ganglia, Spinal/pathology , Gene Expression Regulation/drug effects , Hyperalgesia/drug therapy , Hyperalgesia/etiology , Locomotion/drug effects , Male , Mice , Mice, Inbred C57BL , Microfilament Proteins/metabolism , Nerve Tissue Proteins/metabolism , Pain Measurement , Piperidines/therapeutic use , Protein Kinase Inhibitors/therapeutic use , Spinal Cord Injuries/pathology , Statistics, NonparametricABSTRACT
Aeromedical evacuation, an important component in the care of many patients with traumatic brain injury (TBI), particularly in war zones, exposes them to prolonged periods of hypobaria. The effects of such exposure on pathophysiological changes and outcome after TBI are largely unexplored. The objective of this study was to investigate whether prolonged hypobaria in rats subjected to TBI alters behavioral and histological outcomes. Adult male Sprague-Dawley rats underwent fluid percussion induced injury at 1.5-1.9 atmospheres of pressure. The effects of hypobaric exposure (6 h duration; equivalent to 0.75 atmospheres) at 6, 24, and 72 h, or 7 days after TBI were evaluated with regard to sensorimotor, cognitive, and histological changes. Additional groups were evaluated to determine the effects of two hypobaric exposures after TBI, representing primary simulated aeromedical evacuation (6 h duration at 24 h after injury) and secondary evacuation (10 h duration at 72 h after injury), as well as the effects of 100% inspired oxygen concentrations during simulated evacuation. Hypobaric exposure up to 7 days after injury significantly worsened cognitive deficits, hippocampal neuronal loss, and microglial/astrocyte activation in comparison with injured controls not exposed to hypobaria. Hyperoxia during hypobaric exposure or two exposures to prolonged hypobaric conditions further exacerbated spatial memory deficits. These findings indicate that exposure to prolonged hypobaria up to 7 days after TBI, even while maintaining physiological oxygen concentration, worsens long-term cognitive function and neuroinflammation. Multiple exposures or use of 100% oxygen further exacerbates these pathophysiological effects.
Subject(s)
Air Pressure , Behavior, Animal/physiology , Brain Injuries, Traumatic , Cognitive Dysfunction , Hippocampus , Hyperoxia , Inflammation , Animals , Brain Injuries, Traumatic/complications , Brain Injuries, Traumatic/immunology , Brain Injuries, Traumatic/pathology , Brain Injuries, Traumatic/physiopathology , Cognitive Dysfunction/etiology , Cognitive Dysfunction/immunology , Cognitive Dysfunction/pathology , Cognitive Dysfunction/physiopathology , Disease Models, Animal , Hippocampus/pathology , Inflammation/etiology , Inflammation/immunology , Male , Rats , Rats, Sprague-Dawley , Spatial Memory/physiologyABSTRACT
Traumatic spinal cord injury (SCI) induces cell cycle activation (CCA) that contributes to secondary injury and related functional impairments such as motor deficits and hyperpathia. E2F1 and E2F2 are members of the activator sub-family of E2F transcription factors that play an important role in proliferating cells and in cell cycle-related neuronal death, but no comprehensive study have been performed in SCI to determine the relative importance of these factors. Here we examined the temporal distribution and cell-type specificity of E2F1 and E2F2 expression following mouse SCI, as well as the effects of genetic deletion of E2F1-2 on neuronal cell death, neuroinflammation and associated neurological dysfunction. SCI significantly increased E2F1 and E2F2 expression in active caspase-3(+) neurons/oligodendrocytes as well as in activated microglia/astrocytes. Injury-induced up-regulation of cell cycle-related genes and protein was significantly reduced by intrathecal injection of high specificity E2F decoy oligodeoxynucleotides against the E2F-binding site or in E2F1-2 null mice. Combined E2F1+2 siRNA treatment show greater neuroprotection in vivo than E2F1 or E2F2 single siRNA treatment. Knockout of both E2F1 and E2F2 genes (E2Fdko) significantly reduced neuronal death, neuroinflammation, and tissue damage, as well as limiting motor dysfunction and hyperpathia after SCI. Both CCA reduction and functional improvement in E2Fdko mice were greater than those in E2F2ko model. These studies demonstrate that SCI-induced activation of E2F1-2 mediates CCA, contributing to gliopathy and neuronal/tissue loss associated with motor impairments and post-traumatic hyperesthesia. Thus, E2F1-2 provide a therapeutic target for decreasing secondary tissue damage and promoting recovery of function after SCI.
Subject(s)
E2F1 Transcription Factor/physiology , E2F2 Transcription Factor/physiology , Spinal Cord Injuries/pathology , Animals , Astrocytes/metabolism , Astrocytes/pathology , Cell Death , E2F1 Transcription Factor/genetics , E2F1 Transcription Factor/metabolism , E2F2 Transcription Factor/genetics , E2F2 Transcription Factor/metabolism , Gene Expression , Gene Knockout Techniques , Male , Mice , Mice, Inbred C57BL , Mice, Knockout , Microglia/metabolism , Microglia/pathology , Spinal Cord/metabolism , Spinal Cord/pathology , Spinal Cord Injuries/genetics , Spinal Cord Injuries/metabolismABSTRACT
Positive allosteric modulators (PAMs) binding to the transmembrane (TM) domain of metabotropic glutamate receptor 5 (mGluR5) are promising therapeutic agents for psychiatric disorders and traumatic brain injury (TBI). Novel PAMs based on a trans-2-phenylcyclopropane amide scaffold have been designed and synthesized. Facilitating ligand design and allowing estimation of binding affinities to the mGluR5 TM domain was the novel computational strategy, site identification by ligand competitive saturation (SILCS). The potential protective activity of the new compounds was evaluated using nitric oxide (NO) production in BV2 microglial cell cultures treated with lipopolysaccharide (LPS), and the toxicity of the new compounds tested using a cell viability assay. One of the new compounds, 3a, indicated promising activity with potency of 30 µM, which is 4.5-fold more potent than its lead compound 3,3'-difluorobenzaldazine (DFB), and showed no detectable toxicity with concentrations as high as 1000 µM. Thus this compound represents a new lead for possible development as treatment for TBI and related neurodegenerative disorders.
Subject(s)
Drug Design , Receptor, Metabotropic Glutamate 5/metabolism , Animals , Computer Simulation , Models, Chemical , Molecular Structure , Moles , Protein ConformationABSTRACT
Positive allosteric modulators (PAMs) of the metabotropic glutamate receptor 5 (mGluR5) are promising therapeutic agents for treating traumatic brain injury (TBI). Using computational and medicinal methods, the structure-activity relationship of a class of acyl-2-aminobenzimidazoles (1-26) is reported. The new compounds are designed based on the chemical structure of 3,3'-difluorobenzaldazine (DFB), a known mGluR5 PAM. Ligand design and prediction of binding affinities of the new compounds have been performed using the site identification by ligand competitive saturation (SILCS) method. Binding affinities of the compounds to the transmembrane domain of mGluR5 have been evaluated using nitric oxide (NO) production assay, while the safety of the compounds is tested. One new compound found in this study, compound 22, showed promising activity with an IC50 value of 6.4 µM, which is â¼20 fold more potent than that of DFB. Compound 22 represents a new lead for possible development as a treatment for TBI and related neurodegenerative conditions.
Subject(s)
Benzimidazoles/pharmacology , Excitatory Amino Acid Antagonists/pharmacology , Neuroprotective Agents/classification , Neuroprotective Agents/pharmacology , Receptor, Metabotropic Glutamate 5/antagonists & inhibitors , Animals , Benzimidazoles/chemical synthesis , Benzimidazoles/chemistry , Cell Survival/drug effects , Cells, Cultured , Computer-Aided Design , Dose-Response Relationship, Drug , Excitatory Amino Acid Antagonists/chemical synthesis , Excitatory Amino Acid Antagonists/chemistry , Mice , Models, Molecular , Molecular Structure , Neuroprotective Agents/chemical synthesis , Neuroprotective Agents/chemistry , Nitric Oxide/antagonists & inhibitors , Nitric Oxide/biosynthesis , Receptor, Metabotropic Glutamate 5/metabolism , Structure-Activity RelationshipABSTRACT
Physical activity can attenuate neuronal loss, reduce neuroinflammation, and facilitate recovery after brain injury. However, little is known about the mechanisms of exercise-induced neuroprotection after traumatic brain injury (TBI) or its modulation of post-traumatic neuronal cell death. Voluntary exercise, using a running wheel, was conducted for 4 weeks immediately preceding (preconditioning) moderate-level controlled cortical impact (CCI), a well-established experimental TBI model in mice. Compared to nonexercised controls, exercise preconditioning (pre-exercise) improved recovery of sensorimotor performance in the beam walk task, as well as cognitive/affective functions in the Morris water maze, novel object recognition, and tail-suspension tests. Further, pre-exercise reduced lesion size, attenuated neuronal loss in the hippocampus, cortex, and thalamus, and decreased microglial activation in the cortex. In addition, exercise preconditioning activated the brain-derived neurotrophic factor pathway before trauma and amplified the injury-dependent increase in heat shock protein 70 expression, thus attenuating key apoptotic pathways. The latter include reduction in CCI-induced up-regulation of proapoptotic B-cell lymphoma 2 (Bcl-2)-homology 3-only Bcl-2 family molecules (Bid, Puma), decreased mitochondria permeabilization with attenuated release of cytochrome c and apoptosis-inducing factor (AIF), reduced AIF translocation to the nucleus, and attenuated caspase activation. Given these neuroprotective actions, voluntary physical exercise may serve to limit the consequences of TBI.
Subject(s)
Behavior, Animal/physiology , Brain Injuries/therapy , Brain/metabolism , Exercise Therapy , Neuroprotection/physiology , Physical Conditioning, Animal , Recovery of Function/physiology , Animals , Brain/pathology , Brain/physiopathology , Brain Injuries/prevention & control , Disease Models, Animal , Male , Mice , Mice, Inbred C57BLABSTRACT
Cognitive dysfunction has been reported in patients with spinal cord injury (SCI), but it has been questioned whether such changes may reflect concurrent head injury, and the issue has not been addressed mechanistically or in a well-controlled experimental model. Our recent rodent studies examining SCI-induced hyperesthesia revealed neuroinflammatory changes not only in supratentorial pain-regulatory sites, but also in other brain regions, suggesting that additional brain functions may be impacted following SCI. Here we examined effects of isolated thoracic SCI in rats on cognition, brain inflammation, and neurodegeneration. We show for the first time that SCI causes widespread microglial activation in the brain, with increased expression of markers for activated microglia/macrophages, including translocator protein and chemokine ligand 21 (C-C motif). Stereological analysis demonstrated significant neuronal loss in the cortex, thalamus, and hippocampus. SCI caused chronic impairment in spatial, retention, contextual, and fear-related emotional memory-evidenced by poor performance in the Morris water maze, novel objective recognition, and passive avoidance tests. Based on our prior work implicating cell cycle activation (CCA) in chronic neuroinflammation after SCI or traumatic brain injury, we evaluated whether CCA contributed to the observed changes. Increased expression of cell cycle-related genes and proteins was found in hippocampus and cortex after SCI. Posttraumatic brain inflammation, neuronal loss, and cognitive changes were attenuated by systemic post-injury administration of a selective cyclin-dependent kinase inhibitor. These studies demonstrate that chronic brain neurodegeneration occurs after isolated SCI, likely related to sustained microglial activation mediated by cell cycle activation.
Subject(s)
Cell Cycle , Cognition Disorders/etiology , Cognitive Dysfunction/physiopathology , Neurodegenerative Diseases/etiology , Spinal Cord Injuries/complications , Animals , Biomarkers/metabolism , Brain/metabolism , Brain/pathology , Cell Cycle Proteins/metabolism , Chemokine CCL21/metabolism , Chronic Disease , Cognition Disorders/pathology , Cognition Disorders/physiopathology , Cognitive Dysfunction/pathology , Cyclin-Dependent Kinases/antagonists & inhibitors , Cyclin-Dependent Kinases/metabolism , Male , Microglia/enzymology , Microglia/pathology , Nerve Degeneration , Neurodegenerative Diseases/pathology , Neurodegenerative Diseases/physiopathology , Protein Kinase Inhibitors/pharmacology , Purines/pharmacology , Pyridines/pharmacology , Rats, Sprague-Dawley , Spinal Cord Injuries/pathology , Spinal Cord Injuries/physiopathologyABSTRACT
Dysregulation of autophagy contributes to neuronal cell death in several neurodegenerative and lysosomal storage diseases. Markers of autophagy are also increased after traumatic brain injury (TBI), but its mechanisms and function are not known. Following controlled cortical impact (CCI) brain injury in GFP-Lc3 (green fluorescent protein-LC3) transgenic mice, we observed accumulation of autophagosomes in ipsilateral cortex and hippocampus between 1 and 7 d. This accumulation was not due to increased initiation of autophagy but rather to a decrease in clearance of autophagosomes, as reflected by accumulation of the autophagic substrate SQSTM1/p62 (sequestosome 1). This was confirmed by ex vivo studies, which demonstrated impaired autophagic flux in brain slices from injured as compared to control animals. Increased SQSTM1 peaked at d 1-3 but resolved by d 7, suggesting that the defect in autophagy flux is temporary. The early impairment of autophagy is at least in part caused by lysosomal dysfunction, as evidenced by lower protein levels and enzymatic activity of CTSD (cathepsin D). Furthermore, immediately after injury both autophagosomes and SQSTM1 accumulated predominantly in neurons. This was accompanied by appearance of SQSTM1 and ubiquitin-positive puncta in the affected cells, suggesting that, similar to the situation observed in neurodegenerative diseases, impaired autophagy may contribute to neuronal injury. Consistently, GFP-LC3 and SQSTM1 colocalized with markers of both caspase-dependent and caspase-independent cell death in neuronal cells proximal to the injury site. Taken together, our data indicated for the first time that autophagic clearance is impaired early after TBI due to lysosomal dysfunction, and correlates with neuronal cell death.
Subject(s)
Adaptor Proteins, Signal Transducing/metabolism , Autophagy/physiology , Brain Injuries/metabolism , Heat-Shock Proteins/metabolism , Neurons/metabolism , Animals , Apoptosis Regulatory Proteins/metabolism , Brain/metabolism , Brain Injuries/pathology , Cell Death/physiology , Disease Models, Animal , Mice, Inbred C57BL , Neurons/cytology , Phagosomes/metabolism , Sequestosome-1 ProteinABSTRACT
Experimental spinal cord injury (SCI) causes chronic neuropathic pain associated with inflammatory changes in thalamic pain regulatory sites. Our recent studies examining chronic pain mechanisms after rodent SCI showed chronic inflammatory changes not only in thalamus, but also in other regions including hippocampus and cerebral cortex. Because changes appeared similar to those in our rodent TBI models that are associated with neurodegeneration and neurobehavioral dysfunction, we examined effects of mouse SCI on cognition, depressive-like behavior, and brain inflammation. SCI caused spatial and retention memory impairment and depressive-like behavior, as evidenced by poor performance in the Morris water maze, Y-maze, novel objective recognition, step-down passive avoidance, tail suspension, and sucrose preference tests. SCI caused chronic microglial activation in the hippocampus and cerebral cortex, where microglia with hypertrophic morphologies and M1 phenotype predominated. Stereological analyses showed significant neuronal loss in the hippocampus at 12 weeks but not 8 d after injury. Increased cell-cycle-related gene (cyclins A1, A2, D1, E2F1, and PCNA) and protein (cyclin D1 and CDK4) expression were found chronically in hippocampus and cerebral cortex. Systemic administration of the selective cyclin-dependent kinase inhibitor CR8 after SCI significantly reduced cell cycle gene and protein expression, microglial activation and neurodegeneration in the brain, cognitive decline, and depression. These studies indicate that SCI can initiate a chronic brain neurodegenerative response, likely related to delayed, sustained induction of M1-type microglia and related cell cycle activation, which result in cognitive deficits and physiological depression.
Subject(s)
Affect/physiology , Brain/metabolism , Cell Cycle/physiology , Cognition/physiology , Inflammation/metabolism , Spinal Cord Injuries/metabolism , Animals , Avoidance Learning/physiology , Behavior, Animal/physiology , Brain/pathology , Disease Models, Animal , Hippocampus/metabolism , Inflammation/pathology , Inflammation/psychology , Maze Learning/physiology , Mice , Microglia/metabolism , Recognition, Psychology/physiology , Spinal Cord Injuries/pathology , Spinal Cord Injuries/psychologyABSTRACT
MicroRNAs (miRs) are small noncoding RNAs that negatively regulate gene expression at the post-transcriptional level. To identify miRs that may regulate neuronal cell death after experimental traumatic brain injury (TBI), we profiled miR expression changes during the first several days after controlled cortical impact (CCI) in mice. miR-23a and miR-27a were rapidly downregulated in the injured cortex in the first hour after TBI. These changes coincided with increased expression of the proapoptotic Bcl-2 family members Noxa, Puma, and Bax. In an etoposide-induced in vitro model of apoptosis in primary cortical neurons, miR-23a and miR-27a were markedly downregulated as early as 1 h after exposure, before the upregulation of proapoptotic Bcl-2 family molecules. Administration of miR-23a and miR-27a mimics attenuated etoposide-induced changes in Noxa, Puma, and Bax, reduced downstream markers of caspase-dependent (cytochrome c release and caspase activation) and caspase-independent (apoptosis-inducing factor release) pathways, and limited neuronal cell death. In contrast, miRs hairpin inhibitors enhanced etoposide-induced neuronal apoptosis and caspase activation. Importantly, administration of miR-23a and miR-27a mimics significantly reduced activation of Puma, Noxa, and Bax as well as attenuated markers of caspase-dependent and -independent apoptosis after TBI. Furthermore, miR-23a and miR-27a mimics significantly attenuated cortical lesion volume and neuronal cell loss in the hippocampus after TBI. These findings indicate that post-traumatic decreases in miR-23a and miR-27a contribute to neuronal cell death after TBI by upregulating proapoptotic Bcl-2 family members, thus providing a novel therapeutic target.
Subject(s)
Apoptosis Regulatory Proteins/metabolism , Brain Injuries/metabolism , Down-Regulation/genetics , MicroRNAs/antagonists & inhibitors , Neurons/metabolism , Proto-Oncogene Proteins c-bcl-2/metabolism , Animals , Apoptosis/genetics , Brain Injuries/genetics , Brain Injuries/pathology , Cell Death/genetics , Male , Mice , Mice, Inbred C57BL , MicroRNAs/biosynthesis , Neurons/pathology , Proto-Oncogene Proteins c-bcl-2/physiology , Up-Regulation/geneticsABSTRACT
Traumatic brain injury (TBI) causes neuronal cell death as well as microglial activation and related neurotoxicity that contribute to subsequent neurological dysfunction. Poly (ADP-ribose) polymerase (PARP-1) induces neuronal cell death through activation of caspase-independent mechanisms, including release of apoptosis inducing factor (AIF), and microglial activation. Administration of PJ34, a selective PARP-1 inhibitor, reduced cell death of primary cortical neurons exposed to N-Methyl-N'-Nitro-N-Nitrosoguanidine (MNNG), a potent inducer of AIF-dependent cell death. PJ34 also attenuated lipopolysaccharide and interferon-γ-induced activation of BV2 or primary microglia, limiting NF-κB activity and iNOS expression as well as decreasing generation of reactive oxygen species and TNFα. Systemic administration of PJ34 starting as late as 24 h after controlled cortical impact resulted in improved motor function recovery in mice with TBI. Stereological analysis demonstrated that PJ34 treatment reduced the lesion volume, attenuated neuronal cell loss in the cortex and thalamus, and reduced microglial activation in the TBI cortex. PJ34 treatment did not improve cognitive performance in a Morris water maze test or reduce neuronal cell loss in the hippocampus. Overall, our data indicate that PJ34 has a significant, albeit selective, neuroprotective effect after experimental TBI, and its therapeutic effect may be from multipotential actions on neuronal cell death and neuroinflammatory pathways.
Subject(s)
Brain Injuries/pathology , Microglia/drug effects , Neurons/drug effects , Phenanthrenes/pharmacology , Poly(ADP-ribose) Polymerase Inhibitors , Animals , Blotting, Western , Brain Injuries/enzymology , Cell Death/drug effects , Disease Models, Animal , Enzyme Inhibitors/pharmacology , Male , Mice , Mice, Inbred C57BL , Microglia/pathology , Neurons/pathology , Neuroprotective Agents/pharmacology , Poly (ADP-Ribose) Polymerase-1 , Rats , Rats, Sprague-Dawley , Recovery of Function/drug effectsABSTRACT
Geranylgeranylacetone (GGA) is an inducer of heat-shock protein 70 (HSP70) that has been used clinically for many years as an antiulcer treatment. It is centrally active after oral administration and is neuroprotective in experimental brain ischemia/stroke models. We examined the effects of single oral GGA before treatment (800 mg/kg, 48 hours before trauma) or after treatment (800 mg/kg, 3 hours after trauma) on long-term functional recovery and histologic outcomes after moderate-level controlled cortical impact, an experimental traumatic brain injury (TBI) model in mice. The GGA pretreatment increased the number of HSP70(+) cells and attenuated posttraumatic α-fodrin cleavage, a marker of apoptotic cell death. It also improved sensorimotor performance on a beam walk task; enhanced recovery of cognitive/affective function in the Morris water maze, novel object recognition, and tail-suspension tests; and improved outcomes using a composite neuroscore. Furthermore, GGA pretreatment reduced the lesion size and neuronal loss in the hippocampus, cortex, and thalamus, and decreased microglial activation in the cortex when compared with vehicle-treated TBI controls. Notably, GGA was also effective in a posttreatment paradigm, showing significant improvements in sensorimotor function, and reducing cortical neuronal loss. Given these neuroprotective actions and considering its longstanding clinical use, GGA should be considered for the clinical treatment of TBI.
Subject(s)
Brain Injuries/drug therapy , Brain Injuries/pathology , Brain/drug effects , Brain/pathology , Diterpenes/therapeutic use , Neuroprotective Agents/therapeutic use , Animals , Brain/metabolism , Brain/physiopathology , Brain Injuries/metabolism , Brain Injuries/physiopathology , Diterpenes/administration & dosage , HSP70 Heat-Shock Proteins/analysis , HSP70 Heat-Shock Proteins/metabolism , Male , Maze Learning/drug effects , Memory/drug effects , Mice , Mice, Inbred C57BL , Motor Activity/drug effects , Neurons/drug effects , Neurons/metabolism , Neurons/pathology , Neuroprotective Agents/administration & dosageABSTRACT
The mammalian target of rapamycin (mTOR) signaling pathway is an important regulator of protein synthesis and is essential for various forms of hippocampal memory. Here, we asked whether the enhancement of object recognition memory consolidation produced by dorsal hippocampal infusion of 17ß-estradiol (E(2)) is dependent on mTOR signaling in the dorsal hippocampus, and whether E(2)-induced mTOR signaling is dependent on dorsal hippocampal phosphatidylinositol 3-kinase (PI3K) and extracellular signal-regulated kinase (ERK) activation. We first demonstrated that the enhancement of object recognition induced by E(2) was blocked by dorsal hippocampal inhibition of ERK, PI3K, or mTOR activation. We then showed that an increase in dorsal hippocampal ERK phosphorylation 5 min after intracerebroventricular (ICV) E(2) infusion was also blocked by dorsal hippocampal infusion of the three cell signaling inhibitors. Next, we found that ICV infusion of E(2) increased phosphorylation of the downstream mTOR targets S6K (Thr-421) and 4E-BP1 in the dorsal hippocampus 5 min after infusion, and that this phosphorylation was blocked by dorsal hippocampal infusion of inhibitors of ERK, PI3K, and mTOR. Collectively, these data demonstrate for the first time that activation of the dorsal hippocampal mTOR signaling pathway is necessary for E(2) to enhance object recognition memory consolidation and that E(2)-induced mTOR activation is dependent on upstream activation of ERK and PI3K signaling.
Subject(s)
Estradiol/pharmacology , Hippocampus/drug effects , Memory/drug effects , Recognition, Psychology/drug effects , Signal Transduction/drug effects , TOR Serine-Threonine Kinases/drug effects , Animals , Blotting, Western , Butadienes/pharmacology , Chromones/pharmacology , Estrogen Antagonists/pharmacology , Female , Injections, Intraventricular , MAP Kinase Signaling System/drug effects , Mice , Mice, Inbred C57BL , Mitogen-Activated Protein Kinase 1/drug effects , Mitogen-Activated Protein Kinase 1/metabolism , Morpholines/pharmacology , Nitriles/pharmacology , Ovariectomy , Phosphatidylinositol 3-Kinases/metabolism , Phosphoinositide-3 Kinase Inhibitors , Phosphorylation , Ribosomal Protein S6 Kinases, 90-kDa/physiology , Sirolimus/pharmacology , TOR Serine-Threonine Kinases/antagonists & inhibitorsABSTRACT
Delayed secondary biochemical and cellular changes after traumatic brain injury continue for months to years, and are associated with chronic neuroinflammation and progressive neurodegeneration. Physical activity can reduce inflammation and facilitate recovery after brain injury. Here, we investigated the time-dependent effects, and underlying mechanisms of post-traumatic exercise initiation on outcome after moderate traumatic brain injury using a well-characterized mouse controlled cortical impact model. Late exercise initiation beginning at 5weeks after trauma, but not early initiation of exercise at 1week, significantly reduced working and retention memory impairment at 3months, and decreased lesion volume compared to non-exercise injury controls. Cognitive recovery was associated with attenuation of classical inflammatory pathways, activation of alternative inflammatory responses and enhancement of neurogenesis. In contrast, early initiation of exercise failed to alter behavioral recovery or lesion size, while increasing the neurotoxic pro-inflammatory responses. These data underscore the critical importance of timing of exercise initiation after trauma and its relation to neuroinflammation, and challenge the widely held view that effective neuroprotection requires early intervention.
Subject(s)
Brain Injuries/pathology , Brain Injuries/rehabilitation , Cognition Disorders/rehabilitation , Physical Conditioning, Animal/physiology , Recovery of Function/physiology , Animals , Blotting, Western , Brain/pathology , Cognition Disorders/etiology , Cognition Disorders/pathology , Disease Models, Animal , Immunohistochemistry , Inflammation/pathology , Inflammation/prevention & control , Mice , Real-Time Polymerase Chain ReactionABSTRACT
Controlled cortical impact injury (CCI) is a widely-used, clinically-relevant model of traumatic brain injury (TBI). Although functional outcomes have been used for years in this model, little work has been done to compare the predictive value of various cognitive and sensorimotor assessment tests, singly or in combination. Such information would be particularly useful for assessing mechanisms of injury or therapeutic interventions. Following isoflurane anesthesia, C57BL/6 mice were subjected to sham, mild (5.0 m/sec), moderate (6.0 m/sec), or severe (7.5 m/sec) CCI. A battery of behavioral tests were evaluated and compared, including the standard Morris water maze (sMWM), reversal Morris water maze (rMWM), novel object recognition (NOR), passive avoidance (PA), tail-suspension (TS), beam walk (BW), and open-field locomotor activity. The BW task, performed at post-injury days (PID) 0, 1, 3, 7, 14, 21, and 28, showed good discrimination as a function of injury severity. The sMWM and rMWM tests (PID 14-23), as well as NOR (PID 24 and 25), effectively discriminated spatial and novel object learning and memory across injury severity levels. Notably, the rMWM showed the greatest separation between mild and moderate/severe injury. PA (PID 27 and 28) and TS (PID 24) also reflected differences across injury levels, but to a lesser degree. We also compared individual functional measures with histological outcomes such as lesion volume and neuronal cell loss across anatomical regions. In addition, we created a novel composite behavioral score index from individual complementary behavioral scores, and it provided superior discrimination across injury severities compared to individual tests. In summary, this study demonstrates the feasibility of using a larger number of complementary functional outcome behavioral tests than those traditionally employed to follow post-traumatic recovery after TBI, and suggests that the composite score may be a helpful tool for screening new neuroprotective agents or for addressing injury mechanisms.
Subject(s)
Brain Injuries/complications , Disease Models, Animal , Recovery of Function , Severity of Illness Index , Animals , Behavior, Animal/physiology , Brain Injuries/pathology , Male , Maze Learning/physiology , Mice , Mice, Inbred C57BL , Motor Activity/physiology , Predictive Value of TestsABSTRACT
Histone acetylation has recently been implicated in learning and memory processes, yet necessity of histone acetylation for such processes has not been demonstrated using pharmacological inhibitors of histone acetyltransferases (HATs). As such, the present study tested whether garcinol, a potent HAT inhibitor in vitro, could impair hippocampal memory consolidation and block the memory-enhancing effects of the modulatory hormone 17ß-estradiol E2. We first showed that bilateral infusion of garcinol (0.1, 1, or 10 µg/side) into the dorsal hippocampus (DH) immediately after training impaired object recognition memory consolidation in ovariectomized female mice. A behaviorally effective dose of garcinol (10 µg/side) also significantly decreased DH HAT activity. We next examined whether DH infusion of a behaviorally subeffective dose of garcinol (1 ng/side) could block the effects of DH E2 infusion on object recognition and epigenetic processes. Immediately after training, ovariectomized female mice received bilateral DH infusions of vehicle, E2 (5 µg/side), garcinol (1 ng/side), or E2 plus garcinol. Forty-eight hours later, garcinol blocked the memory-enhancing effects of E2. Garcinol also reversed the E2-induced increase in DH histone H3 acetylation, HAT activity, and levels of the de novo methyltransferase DNMT3B, as well as the E2-induced decrease in levels of the memory repressor protein histone deacetylase 2. Collectively, these findings suggest that histone acetylation is critical for object recognition memory consolidation and the beneficial effects of E2 on object recognition. Importantly, this work demonstrates that the role of histone acetylation in memory processes can be studied using a HAT inhibitor.
Subject(s)
Estradiol/pharmacology , Histones/metabolism , Recognition, Psychology/physiology , Acetylation/drug effects , Animals , Female , Hippocampus/drug effects , Hippocampus/metabolism , Histone Acetyltransferases/antagonists & inhibitors , Histone Acetyltransferases/metabolism , Mice , Mice, Inbred C57BL , Plant Extracts/pharmacology , Recognition, Psychology/drug effects , Terpenes/pharmacologyABSTRACT
Epigenetic processes have been implicated in everything from cell proliferation to maternal behavior. Epigenetic alterations, including histone alterations and DNA methylation, have also been shown to play critical roles in the formation of some types of memory, and in the modulatory effects that factors, such as stress, drugs of abuse and environmental stimulation, have on the brain and memory function. Recently, we demonstrated that the ability of the sex-steroid hormone 17ß-estradiol (E(2)) to enhance memory formation is dependent on histone acetylation and DNA methylation, a finding that has important implications for understanding how hormones influence cognition in adulthood and aging. In this article, we provide an overview of the literature demonstrating that epigenetic processes and E(2) influence memory, describe our findings indicating that epigenetic alterations regulate E(2)-induced memory enhancement, and discuss directions for future work on the epigenetics of estrogen.
Subject(s)
Epigenesis, Genetic , Estrogens/biosynthesis , Memory , Animals , Estradiol/biosynthesisABSTRACT
We previously demonstrated that dorsal hippocampal extracellular signal-regulated kinase (ERK) activation is necessary for 17beta-estradiol (E(2)) to enhance novel object recognition in young ovariectomized mice (Fernandez et al., 2008). Here, we asked whether E(2) has similar memory-enhancing effects in middle-aged and aged ovariectomized mice, and whether these effects depend on ERK and phosphatidylinositol 3-kinase (PI3K)/Akt activation. We first demonstrated that intracerebroventricular or intrahippocampal E(2) infusion immediately after object recognition training enhanced memory consolidation in middle-aged, but not aged, females. The E(2)-induced enhancement in middle-aged females was blocked by intrahippocampal inhibition of ERK or PI3K activation. Intrahippocampal or intracerebroventricular E(2) infusion in middle-aged females increased phosphorylation of p42 ERK in the dorsal hippocampus 15 min, but not 5 min, after infusion, an effect that was blocked by intrahippocampal inhibition of ERK or PI3K activation. Dorsal hippocampal PI3K and Akt phosphorylation was increased 5 min after intrahippocampal or intracerebroventricular E(2) infusion in middle-aged, but not aged, females. Intracerebroventricular E(2) infusion also increased PI3K phosphorylation after 15 min, and this effect was blocked by intrahippocampal PI3K, but not ERK, inhibition. These data demonstrate for the first time that activation of dorsal hippocampal PI3K/Akt and ERK signaling pathways is necessary for E(2) to enhance object recognition memory in middle-aged females. They also reveal that similar dorsal hippocampal signaling pathways mediate E(2)-induced object recognition memory enhancement in young and middle-aged females and that the inability of E(2) to activate these pathways may underlie its failure to enhance object recognition in aged females.
Subject(s)
Estradiol/pharmacology , Estrogens/pharmacology , Extracellular Signal-Regulated MAP Kinases/metabolism , Hippocampus/enzymology , Phosphatidylinositol 3-Kinases/metabolism , Recognition, Psychology/drug effects , Age Factors , Animals , Dose-Response Relationship, Drug , Drug Interactions , Enzyme Inhibitors/pharmacology , Exploratory Behavior/drug effects , Female , Hippocampus/drug effects , Mice , Mice, Inbred C57BL , Ovariectomy/methods , Phosphorylation/drug effects , Statistics, Nonparametric , Time FactorsABSTRACT
The involvement of epigenetic alterations in mediating effects of estrogens on memory is unknown. The present study determined whether histone acetylation and DNA methylation are critical for the potent estrogen 17beta-estradiol (E(2)) to enhance object recognition memory. We show that dorsal hippocampal E(2) infusion increases acetylation of dorsal hippocampal histone H3, but not H4--an effect blocked by dorsal hippocampal inhibition of ERK activation. Further, intrahippocampal inhibition of ERK activation or DNA methyltransferase (DNMT) activity blocked the memory-enhancing effects of E(2). Consistent with these effects, E(2) decreased levels of HDAC2 protein and increased DNMT expression in the dorsal hippocampus. These findings provide evidence that the beneficial effects of E(2) on memory consolidation are associated with epigenetic alterations, and suggest these can be triggered by dorsal hippocampal ERK signaling.