RESUMO
UNLABELLED: During inflammation, the kynurenine pathway (KP) metabolises the essential amino acid tryptophan (TRP) potentially contributing to excitotoxicity via the release of quinolinic acid (QUIN) and 3-hydroxykynurenine (3HK). Despite the importance of excitotoxicity in the development of secondary brain damage, investigations on the KP in TBI are scarce. In this study, we comprehensively characterised changes in KP activation by measuring numerous metabolites in cerebrospinal fluid (CSF) from TBI patients and assessing the expression of key KP enzymes in brain tissue from TBI victims. Acute QUIN levels were further correlated with outcome scores to explore its prognostic value in TBI recovery. METHODS: Twenty-eight patients with severe TBI (GCS ≤ 8, three patients had initial GCS = 9-10, but rapidly deteriorated to ≤8) were recruited. CSF was collected from admission to day 5 post-injury. TRP, kynurenine (KYN), kynurenic acid (KYNA), QUIN, anthranilic acid (AA) and 3-hydroxyanthranilic acid (3HAA) were measured in CSF. The Glasgow Outcome Scale Extended (GOSE) score was assessed at 6 months post-TBI. Post-mortem brains were obtained from the Australian Neurotrauma Tissue and Fluid Bank and used in qPCR for quantitating expression of KP enzymes (indoleamine 2,3-dioxygenase-1 (IDO1), kynurenase (KYNase), kynurenine amino transferase-II (KAT-II), kynurenine 3-monooxygenase (KMO), 3-hydroxyanthranilic acid oxygenase (3HAO) and quinolinic acid phosphoribosyl transferase (QPRTase) and IDO1 immunohistochemistry. RESULTS: In CSF, KYN, KYNA and QUIN were elevated whereas TRP, AA and 3HAA remained unchanged. The ratios of QUIN:KYN, QUIN:KYNA, KYNA:KYN and 3HAA:AA revealed that QUIN levels were significantly higher than KYN and KYNA, supporting increased neurotoxicity. Amplified IDO1 and KYNase mRNA expression was demonstrated on post-mortem brains, and enhanced IDO1 protein coincided with overt tissue damage. QUIN levels in CSF were significantly higher in patients with unfavourable outcome and inversely correlated with GOSE scores. CONCLUSION: TBI induced a striking activation of the KP pathway with sustained increase of QUIN. The exceeding production of QUIN together with increased IDO1 activation and mRNA expression in brain-injured areas suggests that TBI selectively induces a robust stimulation of the neurotoxic branch of the KP pathway. QUIN's detrimental roles are supported by its association to adverse outcome potentially becoming an early prognostic factor post-TBI.
Assuntos
Lesões Encefálicas/diagnóstico , Lesões Encefálicas/metabolismo , Cinurenina/fisiologia , Neurotoxinas/líquido cefalorraquidiano , Ácido Quinolínico/líquido cefalorraquidiano , Transdução de Sinais/fisiologia , Adolescente , Adulto , Idoso , Biomarcadores/metabolismo , Encéfalo/metabolismo , Lesões Encefálicas/fisiopatologia , Estudos de Casos e Controles , Feminino , Escala de Resultado de Glasgow , Humanos , Indolamina-Pirrol 2,3,-Dioxigenase/metabolismo , Masculino , Pessoa de Meia-Idade , Prognóstico , RNA Mensageiro/metabolismo , Triptofano/sangue , Adulto JovemRESUMO
BACKGROUND: Diffuse axonal injury is a common consequence of traumatic brain injury (TBI) and often co-occurs with hypoxia, resulting in poor neurological outcome for which there is no current therapy. Here, we investigate the ability of the multifunctional compound erythropoietin (EPO) to provide neuroprotection when administered to rats after diffuse TBI alone or with post-traumatic hypoxia. METHODS: Sprague-Dawley rats were subjected to diffuse traumatic axonal injury (TAI) followed by 30 minutes of hypoxic (Hx, 12% O2) or normoxic ventilation, and were administered recombinant human EPO-α (5000 IU/kg) or saline at 1 and 24 hours post-injury. The parameters examined included: 1) behavioural and cognitive deficit using the Rotarod, open field and novel object recognition tests; 2) axonal pathology (NF-200); 3) callosal degradation (hematoxylin and eosin stain); 3) dendritic loss (MAP2); 4) expression and localisation of the EPO receptor (EpoR); 5) activation/infiltration of microglia/macrophages (CD68) and production of IL-1ß. RESULTS: EPO significantly improved sensorimotor and cognitive recovery when administered to TAI rats with hypoxia (TAI + Hx). A single dose of EPO at 1 hour reduced axonal damage in the white matter of TAI + Hx rats at 1 day by 60% compared to vehicle. MAP2 was decreased in the lateral septal nucleus of TAI + Hx rats; however, EPO prevented this loss, and maintained MAP2 density over time. EPO administration elicited an early enhanced expression of EpoR 1 day after TAI + Hx compared with a 7-day peak in vehicle controls. Furthermore, EPO reduced IL-1ß to sham levels 2 hours after TAI + Hx, concomitant to a decrease in CD68 positive cells at 7 and 14 days. CONCLUSIONS: When administered EPO, TAI + Hx rats had improved behavioural and cognitive performance, attenuated white matter damage, resolution of neuronal damage spanning from the axon to the dendrite, and suppressed neuroinflammation, alongside enhanced expression of EpoR. These data provide compelling evidence of EPO's neuroprotective capability. Few benefits were observed when EPO was administered to TAI rats without hypoxia, indicating that EPO's neuroprotective capacity is bolstered under hypoxic conditions, which may be an important consideration when EPO is employed for neuroprotection in the clinic.
Assuntos
Lesões Encefálicas/patologia , Eritropoetina/farmacologia , Fármacos Neuroprotetores/farmacologia , Recuperação de Função Fisiológica/efeitos dos fármacos , Animais , Axônios/efeitos dos fármacos , Axônios/patologia , Comportamento Animal/efeitos dos fármacos , Lesões Encefálicas/metabolismo , Hipóxia Encefálica/metabolismo , Hipóxia Encefálica/patologia , Imuno-Histoquímica , Inflamação/patologia , Masculino , Atividade Motora/efeitos dos fármacos , Ratos , Ratos Sprague-Dawley , Receptores da Eritropoetina/metabolismo , Regulação para CimaRESUMO
BACKGROUND: The combination of diffuse brain injury with a hypoxic insult is associated with poor outcomes in patients with traumatic brain injury. In this study, we investigated the impact of post-traumatic hypoxia in amplifying secondary brain damage using a rat model of diffuse traumatic axonal injury (TAI). Rats were examined for behavioral and sensorimotor deficits, increased brain production of inflammatory cytokines, formation of cerebral edema, changes in brain metabolism and enlargement of the lateral ventricles. METHODS: Adult male Sprague-Dawley rats were subjected to diffuse TAI using the Marmarou impact-acceleration model. Subsequently, rats underwent a 30-minute period of hypoxic (12% O2/88% N2) or normoxic (22% O2/78% N2) ventilation. Hypoxia-only and sham surgery groups (without TAI) received 30 minutes of hypoxic or normoxic ventilation, respectively. The parameters examined included: 1) behavioural and sensorimotor deficit using the Rotarod, beam walk and adhesive tape removal tests, and voluntary open field exploration behavior; 2) formation of cerebral edema by the wet-dry tissue weight ratio method; 3) enlargement of the lateral ventricles; 4) production of inflammatory cytokines; and 5) real-time brain metabolite changes as assessed by microdialysis technique. RESULTS: TAI rats showed significant deficits in sensorimotor function, and developed substantial edema and ventricular enlargement when compared to shams. The additional hypoxic insult significantly exacerbated behavioural deficits and the cortical production of the pro-inflammatory cytokines IL-6, IL-1ß and TNF but did not further enhance edema. TAI and particularly TAI+Hx rats experienced a substantial metabolic depression with respect to glucose, lactate, and glutamate levels. CONCLUSION: Altogether, aggravated behavioural deficits observed in rats with diffuse TAI combined with hypoxia may be induced by enhanced neuroinflammation, and a prolonged period of metabolic dysfunction.
Assuntos
Lesões Encefálicas , Encéfalo , Encefalite , Hipóxia/metabolismo , Animais , Comportamento Animal/fisiologia , Encéfalo/metabolismo , Encéfalo/patologia , Encéfalo/fisiopatologia , Lesões Encefálicas/complicações , Lesões Encefálicas/patologia , Lesões Encefálicas/fisiopatologia , Citocinas/metabolismo , Encefalite/etiologia , Encefalite/patologia , Encefalite/fisiopatologia , Glucose/metabolismo , Ácido Glutâmico/metabolismo , Humanos , Lactatos/metabolismo , Masculino , Microdiálise , Testes Neuropsicológicos , Ratos , Ratos Sprague-DawleyRESUMO
Allopregnanolone and related steroids are potent γ-aminobutyric acid receptor-A receptor agonistic allosteric modulators that suppress central nervous system (CNS) activity; in some species, these neurosteroids regulate normal CNS activity before birth. The aims of this study were to determine the effect of suppressing allopregnanolone production on behavioral responses to transient asphyxia in late gestation fetal sheep using the 5α-reductase (R)-2 inhibitor, finasteride. Specificity of the effects of finasteride was assessed by co-infusion of alfaxalone, a synthetic analog of allopregnanolone. Fetal catheters and electrodes for measurement of the electrocorticogram (ECoG) and nuchal electromyogram were implanted at 125 days of gestation, and an inflatable occluder was placed to allow umbilical cord occlusion (UCO). At approximately 130 days of gestation, fetuses received carotid arterial infusion of vehicle (2-hydroxypropyl-ß-cyclodextrin; 40% w/vol), finasteride (40 mg/kg/h), alfaxalone (5 mg/kg/h), or finasteride + alfaxalone. A further three groups of fetuses were subjected to 5 min UCO at 30 min after the start of each infusion regime. Finasteride treatment alone increased the incidence of arousal-like activity; this was reduced by co-infusion of alfaxalone. After UCO, finasteride treatment caused a prolongation of sub-low voltage (LV) ECoG activity and increase in aberrant ECoG spike activity when compared to vehicle-treated UCO fetuses. After UCO, alfaxalone treatment reduced the incidence of sub-LV, reduced the number of aberrant EEG spikes, and restored ECoG activity to the pattern observed after UCO in vehicle-treated fetuses. These results confirm that neurosteroids significantly modulate normal CNS activity in the late gestation fetus, modify, and limit the effects of asphyxia on the brain.
Assuntos
Asfixia/fisiopatologia , Feto/efeitos dos fármacos , Finasterida/farmacologia , Pregnanodionas/farmacologia , Animais , Nível de Alerta/efeitos dos fármacos , Comportamento Animal/efeitos dos fármacos , Eletroencefalografia/efeitos dos fármacos , Eletroencefalografia/veterinária , Eletromiografia , Frequência Cardíaca Fetal/efeitos dos fármacos , Receptores de GABA-A/fisiologia , Carneiro Doméstico , Sono/fisiologiaRESUMO
Severe global fetal asphyxia, if caused by a brief occlusion of the umbilical cord, results in prolonged cerebral hypoperfusion in fetal sheep. In this study, we sought evidence to support the hypothesis that cerebral hypoperfusion is a consequence of suppressed cerebral metabolism. In the 24 h following complete occlusion of the umbilical cord for 10 min, sagittal sinus blood flow velocity was significantly decreased for up to 12 h. Capillary blood flow, measured using microspheres, decreased at 1 and 5 h after cord occlusion in many brain regions, including cortical gray and white matter. Microdialysis probes implanted in the cerebral cortex revealed an increase in extracellular glucose concentrations in gray matter for 7-8 h postasphyxia, while lactate increased only briefly, suggesting decreased cerebral glucose utilization over this time. Although these data, as well as the concurrent suppression of breathing movements and electrocortical activity, support the concept of hypometabolic hypoperfusion, the significant increase of pyruvate and glycerol concentrations in dialysate fluid obtained from the cerebral cortex at 3-8 h after cord occlusion suggests an eventual loss of membrane integrity. The prolonged increase of breathing movements for many hours suggests loss of the pontine/thalamic control that produces the distinct pattern of fetal breathing movements.
Assuntos
Asfixia/fisiopatologia , Encéfalo/irrigação sanguínea , Encéfalo/metabolismo , Circulação Cerebrovascular , Metabolismo Energético , Hipóxia Fetal/fisiopatologia , Mecânica Respiratória , Cordão Umbilical/irrigação sanguínea , Animais , Asfixia/sangue , Asfixia/líquido cefalorraquidiano , Asfixia/embriologia , Biomarcadores/sangue , Biomarcadores/líquido cefalorraquidiano , Velocidade do Fluxo Sanguíneo , Pressão Sanguínea , Encéfalo/embriologia , Dióxido de Carbono/sangue , Modelos Animais de Doenças , Eletroencefalografia , Feminino , Sangue Fetal/metabolismo , Hipóxia Fetal/sangue , Hipóxia Fetal/líquido cefalorraquidiano , Idade Gestacional , Glucose/líquido cefalorraquidiano , Glicerol/líquido cefalorraquidiano , Frequência Cardíaca , Concentração de Íons de Hidrogênio , Ácido Láctico/líquido cefalorraquidiano , Microdiálise , Oxigênio/sangue , Gravidez , Ácido Pirúvico/líquido cefalorraquidiano , Ovinos , Fatores de Tempo , Cordão Umbilical/cirurgiaRESUMO
Changes in inhibition following traumatic brain injury (TBI) appear to be one of the major factors that contribute to excitation:inhibition imbalance. Neuron pathology, interneurons in particular evolves from minutes to weeks post injury and follows a complex time course. Previously, we showed that in the long-term in diffuse TBI (dTBI), there was select reduction of specific dendrite-targeting neurons in sensory cortex and hippocampus while in motor cortex there was up-regulation of specific dendrite-targeting neurons. We now investigated the time course of dTBI effects on interneurons in neocortex and hippocampus. Brains were labeled with antibodies against calbindin (CB), parvalbumin (PV), calretinin (CR) neuropeptide Y (NPY), and somatostatin (SOM) at 24â¯h and 2â¯weeks post dTBI. We found time-dependent, brain area-specific changes in inhibition at 24â¯h and 2â¯weeks. At 24â¯h post-injury, reduction of dendrite-targeting inhibitory neurons occurred in sensory cortex and hippocampus. At 2â¯weeks, we found compensatory changes in the somatosensory cortex and CA2/3 of hippocampus affected at 24â¯h, with affected interneuronal populations returning to sham levels. However, DG of hippocampus now showed reduction of dendrite-targeting inhibitory neurons. Finally, with respect to motor cortex, there was an upregulation of dendrite-targeting interneurons in the supragranular layers at 24â¯h returning to normal levels by 2â¯weeks. Overall, our findings reconfirm that dendritic inhibition is particularly susceptible to brain trauma, but also show that there are complex brain-area-specific changes in inhibitory neuronal numbers and in compensatory changes, rather than a simple monotonic progression of changes post-dTBI.
Assuntos
Lesões Encefálicas Difusas/fisiopatologia , Lesões Encefálicas Traumáticas/fisiopatologia , Córtex Cerebral/fisiopatologia , Hipocampo/fisiopatologia , Neurônios/fisiologia , Animais , Lesões Encefálicas Difusas/patologia , Lesões Encefálicas Traumáticas/patologia , Córtex Cerebral/patologia , Modelos Animais de Doenças , Progressão da Doença , Hipocampo/patologia , Masculino , Inibição Neural/fisiologia , Neurônios/patologia , Distribuição Aleatória , Ratos Sprague-Dawley , Fatores de TempoRESUMO
Traumatic brain injury (TBI) initiates a cascade of pathophysiological changes that are both complex and difficult to treat. Progesterone (P4) is a neuroprotective treatment option that has shown excellent preclinical benefits in the treatment of TBI, but these benefits have not translated well in the clinic. We have previously shown that P4 exacerbates the already hypoactive upper cortical responses in the short-term post-TBI and does not reduce upper cortical hyperactivity in the long term, and we concluded that there is no tangible benefit to sensory cortex firing strength. Here we examined the effects of P4 treatment on temporal coding resolution in the rodent sensory cortex in both the short term (4 d) and long term (8 wk) following impact-acceleration-induced TBI. We show that in the short-term postinjury, TBI has no effect on sensory cortex temporal resolution and that P4 also sharpens the response profile in all cortical layers in the uninjured brain and all layers other than layer 2 (L2) in the injured brain. In the long term, TBI broadens the response profile in all cortical layers despite firing rate hyperactivity being localized to upper cortical layers and P4 sharpens the response profile in TBI animals in all layers other than L2 and has no long-term effect in the sham brain. These results indicate that P4 has long-term effects on sensory coding that may translate to beneficial perceptual outcomes. The effects seen here, combined with previous beneficial preclinical data, emphasize that P4 is still a potential treatment option in ameliorating TBI-induced disorders.
Assuntos
Lesões Encefálicas Traumáticas/tratamento farmacológico , Lesões Encefálicas Traumáticas/patologia , Progesterona/uso terapêutico , Células Receptoras Sensoriais/patologia , Córtex Somatossensorial/patologia , Potenciais de Ação/efeitos dos fármacos , Animais , Lesões Encefálicas Traumáticas/fisiopatologia , Lesões Encefálicas Traumáticas/cirurgia , Masculino , Progesterona/farmacologia , Ratos Sprague-Dawley , Células Receptoras Sensoriais/efeitos dos fármacos , Fatores de TempoRESUMO
Traumatic brain injury (TBI) is a major cause of morbidity and mortality world-wide and can result in persistent cognitive, sensory and behavioral dysfunction. Understanding the time course of TBI-induced pathology is essential to effective treatment outcomes. We induced TBI in rats using an impact acceleration method and tested for sensorimotor skill and sensory sensitivity behaviors for two weeks to find persistently poor outcomes post-injury. At two weeks post-injury we made high resolution extracellular recordings from barrel cortex neurons, to simple and complex whisker deflections. We found that the supragranular suppression of neural firing (compared to normal) previously seen in the immediate post-TBI aftermath had spread to include suppression of input and infragranular layers at two weeks post-injury; thus, there was suppression of whisker-driven firing rates in all cortical layers to both stimulus types. Further, there were abnormalities in temporal response patterns such that in layers 3-5 there was a temporal broadening of response patterns in response to both whisker deflection stimulus types and in L2 a narrowing of temporal patterns in response to the complex stimulus. Thus, at two weeks post-TBI, supragranular hypo-excitation has evolved to include deep cortical layers likely as a function of progressive atrophy and neurodegeneration. These results are consistent with the hypothesis that TBI alters the delicate excitatory/inhibitory balance in cortex and likely contributes to temporal broadening of responses and restricts the ability to code for complex sensory stimuli.
Assuntos
Lesões Encefálicas Traumáticas/fisiopatologia , Neurônios/fisiologia , Córtex Somatossensorial/fisiopatologia , Potenciais de Ação , Animais , Modelos Animais de Doenças , Progressão da Doença , Masculino , Microeletrodos , Atividade Motora/fisiologia , Ratos Sprague-Dawley , Córtex Somatossensorial/lesões , Fatores de Tempo , Percepção do Tato/fisiologia , Vibrissas/fisiologiaRESUMO
Long-term diffuse traumatic brain injury (dTBI) causes neuronal hyperexcitation in supragranular layers in sensory cortex, likely through reduced inhibition. Other forms of TBI affect inhibitory interneurons in subcortical areas but it is unknown if this occurs in cortex, or in any brain area in dTBI. We investigated dTBI effects on inhibitory neurons and astrocytes in somatosensory and motor cortex, and hippocampus, 8 weeks post-TBI. Brains were labeled with antibodies against calbindin (CB), parvalbumin (PV), calretinin (CR) and neuropeptide Y (NPY), and somatostatin (SOM) and glial fibrillary acidic protein (GFAP), a marker for astrogliosis during neurodegeneration. Despite persistent behavioral deficits in rotarod performance up to the time of brain extraction (TBI = 73.13 ± 5.23% mean ± SEM, Sham = 92.29 ± 5.56%, P < 0.01), motor cortex showed only a significant increase, in NPY neurons in supragranular layers (mean cells/mm2 ± SEM, Sham = 16 ± 0.971, TBI = 25 ± 1.51, P = 0.001). In somatosensory cortex, only CR+ neurons showed changes, being decreased in supragranular (TBI = 19 ± 1.18, Sham = 25 ± 1.10, P < 0.01) and increased in infragranular (TBI = 28 ± 1.35, Sham = 24 ± 1.07, P < 0.05) layers. Heterogeneous changes were seen in hippocampal staining: CB+ decreased in dentate gyrus (TBI = 2 ± 0.382, Sham = 4 ± 0.383, P < 0.01), PV+ increased in CA1 (TBI = 39 ± 1.26, Sham = 33 ± 1.69, P < 0.05) and CA2/3 (TBI = 26 ± 2.10, Sham = 20 ± 1.49, P < 0.05), and CR+ decreased in CA1 (TBI = 10 ± 1.02, Sham = 14 ± 1.14, P < 0.05). Astrogliosis significantly increased in corpus callosum (TBI = 6.7 ± 0.69, Sham = 2.5 ± 0.38; P = 0.007). While dTBI effects on inhibitory neurons appear region- and type-specific, a common feature in all cases of decrease was that changes occurred in dendrite targeting interneurons involved in neuronal integration. J. Comp. Neurol. 524:3530-3560, 2016. © 2016 Wiley Periodicals, Inc.
Assuntos
Astrócitos/patologia , Lesões Encefálicas Traumáticas/patologia , Hipocampo/patologia , Córtex Motor/patologia , Neurônios/patologia , Córtex Somatossensorial/patologia , Animais , Astrócitos/metabolismo , Lesões Encefálicas Traumáticas/complicações , Lesões Encefálicas Traumáticas/metabolismo , Corpo Caloso/metabolismo , Corpo Caloso/patologia , Modelos Animais de Doenças , Gliose/metabolismo , Gliose/patologia , Hipocampo/metabolismo , Imuno-Histoquímica , Masculino , Microeletrodos , Córtex Motor/metabolismo , Inibição Neural/fisiologia , Doenças Neurodegenerativas/etiologia , Doenças Neurodegenerativas/metabolismo , Doenças Neurodegenerativas/patologia , Neurônios/metabolismo , Tamanho do Órgão , Distribuição Aleatória , Ratos Sprague-Dawley , Córtex Somatossensorial/metabolismo , Percepção do Tato/fisiologia , Vibrissas/fisiologiaRESUMO
Progesterone (P4) has been suggested as a neuroprotective agent for traumatic brain injury (TBI) because it ameliorates many post-TBI sequelae. We examined the effects of P4 treatment on the short-term (4 days post-TBI) and long-term (8 weeks post-TBI) aftermath on neuronal processing in the rodent sensory cortex of impact acceleration-induced diffuse TBI. We have previously reported that in sensory cortex, diffuse TBI induces a short-term hypoexcitation that is greatest in the supragranular layers and decreases with depth, but a long-term hyperexcitation that is exclusive to the supragranular layers. Now, adult male TBI-treated rats administered P4 showed, in the short term, even greater suppression in neural responses in supragranular layers but a reversal of the TBI-induced suppression in granular and infragranular layers. In long-term TBI there were only inconsistent effects of P4 on the TBI-induced hyperexcitation in supragranular responses but infragranular responses, which were not affected by TBI alone, were elevated by P4 treatment. Intriguingly, the effects in the injured brain were almost identical to P4 effects in the normal brain, as seen in sham control animals treated with P4: in the short term, P4 effects in the normal brain were identical to those exercised in the injured brain and in the long term, P4 effects in the normal brain were rather similar to what was seen in the TBI brain. Overall, these results provide no support for any protective effects of P4 treatment on neuronal encoding in diffuse TBI, and this was reflected in sensorimotor and other behavior tasks also tested here. Additionally, the effects suggest that mechanisms used for P4 effects in the normal brain are also intact in the injured brain.
Assuntos
Lesões Encefálicas Traumáticas/tratamento farmacológico , Lesões Encefálicas Traumáticas/patologia , Fármacos Neuroprotetores/uso terapêutico , Progesterona/uso terapêutico , Córtex Somatossensorial/patologia , Córtex Somatossensorial/fisiologia , Animais , Masculino , Fármacos Neuroprotetores/farmacologia , Progesterona/farmacologia , Ratos , Ratos Sprague-Dawley , Córtex Somatossensorial/efeitos dos fármacos , Fatores de TempoRESUMO
Hydroxyl radical (.OH) is a reactive oxygen species produced during severe hypoxia, asphyxia, or ischemia that can cause cell death resulting in brain damage. Generation of .OH may occur in the fetal brain during asphyxia in utero. The very short half-life of .OH requires use of trapping agents such as salicylic acid or phenylalanine for detection, but their hydroxylated derivatives are either unstable, produced endogenously, or difficult to measure in the small volume of microdialysis samples. In the present study, we used terephthalic acid (TA), hydroxylation of which yields a stable and highly fluorometric isomer (excitation, 326 nm; emission, 432 nm). In vitro studies using .OH generated by the Fenton reaction showed that hydroxylated TA formed quickly (<10 s), was resistant to bleaching (<5% change in fluorescence), and permitted detection of <0.5 pmol .OH. In vivo studies were performed in fetal sheep using microdialysis probes implanted into the parasagittal cortex. The probe was perfused at 2 mul/min with artificial cerebrospinal fluid containing 5 mM TA, and samples were collected every 30 min. Fluorescence measured in 10 mul of dialysate was significantly greater than in the efflux from probes perfused without TA. High-performance liquid chromotography analysis showed that the fluorescence in dialysis samples was entirely due to hydroxylation of TA. Thus this study shows that it is possible to use TA as a trapping agent for detecting low concentrations of .OH both in vitro and in vivo and that low concentrations of .OH are present in fetal brain tissue and fluctuate with time.
Assuntos
Encéfalo/embriologia , Encéfalo/metabolismo , Monitorização Fetal/métodos , Radical Hidroxila/análise , Radical Hidroxila/metabolismo , Microdiálise/métodos , Espectrometria de Fluorescência/métodos , Animais , Sistemas Computacionais , Ácidos Ftálicos , Espécies Reativas de Oxigênio/análise , Espécies Reativas de Oxigênio/metabolismo , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , OvinosRESUMO
Electrodes for cortical stimulation need to deliver current to neural tissue effectively and safely. We have developed electrodes with a novel annular geometry for use in cortical visual prostheses. Here, we explore a critical question on the ideal annulus height to ensure electrical stimulation will be safe and effective. We implanted single electrodes into the motor cortex of anesthetized rats and measured the current required to evoke a motor response to stimulation, and the charge injection capacity (CIC) of the electrodes. We compared platinum iridium (PtIr) electrodes with different annulus heights, with and without a coating of porous titanium nitride (TiN). Threshold charge densities to evoke a motor response ranged from 12 to 36 µC.cm(-2).ph(-1). Electrodes with larger geometric surface areas (GSAs) required higher currents to evoke responses, but lower charge densities. The addition of a porous TiN coating did not significantly influence the current required to evoke a motor response. The CIC of both electrode types was significantly reduced in vivo compared with in vitro measurements. The measured CIC was 72 and 18 µC.cm(-2).ph(-1) for electrodes with and without a TiN coating, respectively. These results support the use of PtIr annular electrodes with annulus heights greater than 100 µm (GSA of 38, 000 µm(2)). However, if the electrodes are coated with porous TiN the annulus height can be reduced to 40 µm (GSA of 16,000 µm(2)).
RESUMO
Electrodes for cortical stimulation need to deliver current to neural tissue effectively and safely. We have developed electrodes with a novel annular geometry for use in cortical visual prostheses. Here, we explore a critical question on the ideal annulus height to ensure electrical stimulation will be safe and effective. We implanted single electrodes into the motor cortex of anesthetized rats and measured the current required to evoke a motor response to stimulation, and the charge injection capacity (CIC) of the electrodes. We compared platinum iridium (PtIr) electrodes with different annulus heights, with and without a coating of porous titanium nitride (TiN). Threshold charge densities to evoke a motor response ranged from 12 to 36 µC.cm(-2).ph(-1). Electrodes with larger geometric surface areas (GSAs) required higher currents to evoke responses, but lower charge densities. The addition of a porous TiN coating did not significantly influence the current required to evoke a motor response. The CIC of both electrode types was significantly reduced in vivo compared with in vitro measurements. The measured CIC was 72 and 18 µC.cm(-2).ph(-1) for electrodes with and without a TiN coating, respectively. These results support the use of PtIr annular electrodes with annulus heights greater than 100 µm (GSA of 38, 000 µm(2)). However, if the electrodes are coated with porous TiN the annulus height can be reduced to 40 µm (GSA of 16,000 µm(2)).
RESUMO
The consequences of mild traumatic brain injury (TBI) on neuronal functionality are only now being elucidated. We have now examined the changes in sensory encoding in the whisker-recipient barrel cortex and the brain tissue damage in the acute phase (24 h) after induction of TBI (n=9), with sham controls receiving surgery only (n=5). Injury was induced using the lateral fluid percussion injury method, which causes a mixture of focal and diffuse brain injury. Both population and single cell neuronal responses evoked by both simple and complex whisker stimuli revealed a suppression of activity that decreased with distance from the locus of injury both within a hemisphere and across hemispheres, with a greater extent of hypoactivity in ipsilateral barrel cortex compared with contralateral cortex. This was coupled with an increase in spontaneous output in Layer 5a, but only ipsilateral to the injury site. There was also disruption of axonal integrity in various regions in the ipsilateral but not contralateral hemisphere. These results complement our previous findings after mild diffuse-only TBI induced by the weight-drop impact acceleration method where, in the same acute post-injury phase, we found a similar depth-dependent hypoactivity in sensory cortex. This suggests a common sequelae of events in both diffuse TBI and mixed focal/diffuse TBI in the immediate post-injury period that then evolve over time to produce different long-term functional outcomes.
Assuntos
Lesões Encefálicas/fisiopatologia , Neurônios/fisiologia , Animais , Córtex Cerebral/fisiopatologia , Modelos Animais de Doenças , Eletrofisiologia , Imuno-Histoquímica , Masculino , Ratos , Ratos Sprague-Dawley , Vibrissas/inervaçãoRESUMO
Secondary hypoxia is a known contributor to adverse outcomes in patients with traumatic brain injury (TBI). Based on the evidence that hypoxia and TBI in isolation induce neuroinflammation, we investigated whether TBI combined with hypoxia enhances cerebral cytokine production. We also explored whether increased concentrations of injury biomarkers discriminate between hypoxic (Hx) and normoxic (Nx) patients, correlate to worse outcome, and depend on blood-brain barrier (BBB) dysfunction. Forty-two TBI patients with Glasgow Coma Scale ≤8 were recruited. Cerebrospinal fluid (CSF) and serum were collected over 6 days. Patients were divided into Hx (n=22) and Nx (n=20) groups. Eight cytokines were measured in the CSF; albumin, S100, myelin basic protein (MBP) and neuronal specific enolase (NSE) were quantified in serum. CSF/serum albumin quotient was calculated for BBB function. Glasgow Outcome Scale Extended (GOSE) was assessed at 6 months post-TBI. Production of granulocye macrophage-colony stimulating factor (GM-CSF) was higher, and profiles of GM-CSF, interferon (IFN)-γ and, to a lesser extent, tumor necrosis factor (TNF), were prolonged in the CSF of Hx but not Nx patients at 4-5 days post-TBI. Interleukin (IL)-2, IL-4, IL-6, and IL-10 increased similarly in both Hx and Nx groups. S100, MBP, and NSE were significantly higher in Hx patients with unfavorable outcome. Among these three biomarkers, S100 showed the strongest correlations to GOSE after TBI-Hx. Elevated CSF/serum albumin quotients lasted for 5 days post-TBI and displayed similar profiles in Hx and Nx patients. We demonstrate for the first time that post-TBI hypoxia is associated with prolonged neuroinflammation, amplified extravasation of biomarkers, and poor outcome. S100 and MBP could be implemented to track the occurrence of post-TBI hypoxia, and prompt adequate treatment.
Assuntos
Lesões Encefálicas/fisiopatologia , Citocinas/biossíntese , Hipóxia Encefálica/fisiopatologia , Recuperação de Função Fisiológica , Adolescente , Adulto , Biomarcadores/análise , Barreira Hematoencefálica/patologia , Lesões Encefálicas/complicações , Citocinas/análise , Ensaio de Imunoadsorção Enzimática , Feminino , Escala de Coma de Glasgow , Humanos , Hipóxia Encefálica/complicações , Masculino , Pessoa de Meia-Idade , Prognóstico , Adulto JovemRESUMO
Traumatic brain injury (TBI) can result in persistent sensorimotor and cognitive deficits including long-term altered sensory processing. The few animal models of sensory cortical processing effects of TBI have been limited to examination of effects immediately after TBI and only in some layers of cortex. We have now used the rat whisker tactile system and the cortex processing whisker-derived input to provide a highly detailed description of TBI-induced long-term changes in neuronal responses across the entire columnar network in primary sensory cortex. Brain injury (n=19) was induced using an impact acceleration method and sham controls received surgery only (n=15). Animals were tested in a range of sensorimotor behaviour tasks prior to and up to 6 weeks post-injury when there were still significant sensorimotor behaviour deficits. At 8-10 weeks post-trauma, in terminal experiments, extracellular recordings were obtained from barrel cortex neurons in response to whisker motion, including motion that mimicked whisker motion observed in awake animals undertaking different tasks. In cortex, there were lamina-specific neuronal response alterations that appeared to reflect local circuit changes. Hyper-excitation was found only in supragranular layers involved in intra-areal processing and long-range integration, and only for stimulation with complex, naturalistic whisker motion patterns and not for stimulation with simple trapezoidal whisker motion. Thus TBI induces long-term directional changes in integrative sensory cortical layers that depend on the complexity of the incoming sensory information. The nature of these changes allow predictions as to what types of sensory processes may be affected in TBI and contribute to post-trauma sensorimotor deficits.
Assuntos
Lesões Encefálicas/fisiopatologia , Córtex Somatossensorial/fisiopatologia , Animais , Masculino , Plasticidade Neuronal/fisiologia , Ratos , Ratos Sprague-Dawley , Vibrissas/fisiologiaRESUMO
Traumatic brain injury (TBI) resulting in poor neurological outcome is predominantly associated with diffuse brain damage and secondary hypoxic insults. Post-traumatic hypoxia is known to exacerbate primary brain injury; however, the underlying pathological mechanisms require further elucidation. Using a rat model of diffuse traumatic axonal injury (TAI) followed by a post-traumatic hypoxic insult, we characterized axonal pathology, macrophage/microglia accumulation, and astrocyte responses over 14 days. Rats underwent TAI alone, TAI followed by 30 min of hypoxia (TAI + Hx), hypoxia alone, or sham-operation (n = 6/group). Systemic hypoxia was induced by ventilating rats with 12% oxygen in nitrogen, resulting in a â¼ 50% reduction in arterial blood oxygen saturation. Brains were assessed for axonal damage, macrophage/microglia accumulation, and astrocyte activation at 1, 7, and 14 days post-treatment. Immunohistochemistry with axonal damage markers (ß-amyloid precursor protein [ß-APP] and neurofilament) showed strong positive staining in TAI + Hx rats, which was most prominent in the corpus callosum (retraction bulbs 69.8 ± 18.67; swollen axons 14.2 ± 5.25), and brainstem (retraction bulbs 294 ± 118.3; swollen axons 50.3 ± 20.45) at 1 day post-injury. Extensive microglia/macrophage accumulation detected with the CD68 antibody was maximal at 14 days post-injury in the corpus callosum (macrophages 157.5 ± 55.48; microglia 72.71 ± 20.75), and coincided with regions of axonal damage. Astrocytosis assessed with glial fibrillary acidic protein (GFAP) antibody was also abundant in the corpus callosum and maximal at 14 days, with a trend toward an increase in TAI + Hx animals (18.99 ± 2.45 versus 13.56 ± 0.81; p = 0.0617). This study demonstrates for the first time that a hypoxic insult following TAI perpetuates axonal pathology and cellular inflammation, which may account for the poor neurological outcomes seen in TBI patients who experience post-traumatic hypoxia.
Assuntos
Lesões Encefálicas/patologia , Lesão Axonal Difusa/patologia , Hipóxia Encefálica/patologia , Microglia/patologia , Precursor de Proteína beta-Amiloide/metabolismo , Animais , Antígenos CD/metabolismo , Antígenos de Diferenciação Mielomonocítica/metabolismo , Gasometria , Pressão Sanguínea/fisiologia , Encéfalo/patologia , Lesões Encefálicas/complicações , Corpo Caloso/metabolismo , Técnica Indireta de Fluorescência para Anticorpo , Gliose/patologia , Hipóxia Encefálica/etiologia , Imuno-Histoquímica , Ácido Láctico/sangue , Ativação de Macrófagos/fisiologia , Masculino , Proteínas de Neurofilamentos/metabolismo , Tratos Piramidais/metabolismo , Ratos , Ratos Sprague-DawleyRESUMO
Maternal alcohol consumption during pregnancy can affect fetal development, but little is known about the effects on the developing kidney. Our objectives were to determine the effects of repeated ethanol exposure during the latter half of gestation on glomerular (nephron) number and expression of key genes involved in renal development or function in the ovine fetal kidney. Pregnant ewes received daily intravenous infusion of ethanol (0.75 g/kg, n=5) or saline (control, n=5) over 1 h from 95 to 133 days of gestational age (DGA; term is approximately 147 DGA). Maternal and fetal arterial blood samples were taken before and after the start of the daily ethanol infusions for determination of blood ethanol concentration (BEC). Necropsy was performed at 134 DGA, and fetal kidneys were collected for determination of total glomerular number using the physical disector/fractionator technique; at this gestational age nephrogenesis is completed in sheep. Maximal maternal and fetal BECs of 0.12+/-0.01 g/dl (mean+/-SE) and 0.11+/-0.01 g/dl, respectively, were reached 1 h after starting maternal ethanol infusions. Ethanol exposure had no effect on fetal body weight, kidney weight, or the gene expression of members of the renin-angiotensin system, insulin-like growth factors, and sodium channels. However, fetal glomerular number was lower after ethanol exposure (377,585+/-8,325) than in controls (423,177+/-17,178, P<0.001). The data demonstrate that our regimen of fetal ethanol exposure during the latter half of gestation results in an 11% reduction in nephron endowment without affecting the overall growth of the kidney or fetus or the expression of key genes involved in renal development or function. A reduced nephron endowment of this magnitude could have important implications for the cardiovascular health of offspring during postnatal life.
Assuntos
Etanol/toxicidade , Néfrons/efeitos dos fármacos , Efeitos Tardios da Exposição Pré-Natal , Líquido Amniótico/efeitos dos fármacos , Líquido Amniótico/metabolismo , Animais , Peso Corporal/efeitos dos fármacos , Etanol/administração & dosagem , Etanol/sangue , Feminino , Sangue Fetal/metabolismo , Peso Fetal/efeitos dos fármacos , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Idade Gestacional , Infusões Intravenosas , Glomérulos Renais/efeitos dos fármacos , Glomérulos Renais/embriologia , Néfrons/embriologia , Néfrons/metabolismo , Organogênese/genética , Gravidez , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , OvinosRESUMO
Oxygen free radicals, including the highly toxic hydroxyl radical (*OH), initiate lipid peroxidation and DNA/RNA fragmentation and damage cells. The pineal hormone melatonin is an antioxidant and powerful scavenger of *OH. We hypothesized that maternally administered melatonin could reduce *OH formation, lipid peroxidation, and DNA/RNA damage in the fetal brain in response to asphyxia. In 15 fetal sheep, extracellular *OH was measured by microdialysis in white and gray matter of the parasagittal cortex. In 10 fetuses, asphyxia was induced by umbilical cord occlusion for 10 min using an inflatable cuff - the ewes of these fetuses received either intravenous melatonin (1 mg bolus, then 1 mg/h for 2 h; n = 5) or vehicle (1% ethanol in saline; n = 5), and results were compared to fetuses with sham cord occlusion and vehicle-infused ewes (n = 5). Hypoxemia, acidemia, hypertension and bradycardia produced by cord occlusion was similar in the melatonin- and vehicle-treated groups. In the vehicle-treated group, cord occlusion resulted in a significant increase in *OH in gray matter at 8-9.5 h after occlusion (p < 0.05); in contrast, there was no *OH change in the melatonin-treated group. After cord occlusion, lipid peroxidation (4-hydroxynonenal immunoreactivity) found throughout the brain of vehicle-infused ewes was significantly less in the melatonin-infused group. Melatonin had no significant effect on the distribution of DNA/RNA fragmentation, as shown by 8-hydroxydeoxyguanosine immunoreactivity. Thus, brief asphyxia results in significant and delayed entry of *OH into the extracellular space of cortical gray matter in the fetal sheep brain, and melatonin given to the mother at the time of the insult abrogates this increase. Melatonin, in reducing O2 free radical production, may be an effective neuroprotective treatment for the fetus.
Assuntos
Encéfalo/efeitos dos fármacos , Encéfalo/metabolismo , Hipóxia Encefálica/metabolismo , Melatonina/farmacologia , Fármacos Neuroprotetores/farmacologia , Animais , Asfixia/complicações , Constrição , Feminino , Feto , Radical Hidroxila/análise , Hipóxia Encefálica/etiologia , Imuno-Histoquímica , Peroxidação de Lipídeos/efeitos dos fármacos , Microdiálise , Gravidez , Ovinos , Cordão Umbilical/cirurgiaRESUMO
Allopregnanolone (AP) is a potent modulator of the GABAA receptor. Brain AP concentrations increase in response to stress, which is thought to provide neuroprotection by reducing excitation in the adult brain. Umbilical cord occlusion (UCO) causes hypoxia and asphyxia in the fetus, which are major risk factors associated with poor neurological outcome for the neonate, and may lead to adverse sequelae such as cerebral palsy. The aims of this study were as follows: (i) to determine the effect of 10 min UCO on AP concentrations in the extracellular fluid of the fetal brain using microdialysis, and (ii) to compare the content of the steroidogenic enzymes P450scc and 5alpha-reductase type II (5alphaRII) with brain and CSF neurosteroid concentrations. UCO caused fetal asphyxia, hypertension, bradycardia and respiratory acidosis, which returned to normal levels after 1-2 h. AP concentrations in dialysate samples from probes implanted in grey and white matter of the parietal cortex were significantly increased 1 h after UCO from control levels of 10.4 +/- 0.4 and 12.4 +/- 0.3 to 26.0 +/- 5.1 and 27.6 +/- 6.4 nmol l(-1), respectively (P < 0.05), before returning to pre-occlusion levels by 3-4 h after UCO. When fetal brains were collected 1 h after a 10 min UCO, the relative increases of AP and pregnenolone content in the parietal cortex were similar to the increase observed in the extracellular (dialysate) fluid. AP, but not pregnenolone, was increased in CSF at this time. P450scc and 5alphaRII enzyme expression was significantly increased in the cerebral cortex in the UCO fetuses compared to control fetuses. These results suggest that the fetal brain is capable of transiently increasing neurosteroid production in response to asphyxia. The action of the increased neurosteroid content at GABAA receptors may serve to diminish the increased excitation due to excitotoxic amino acid release, and provide short-term protection to brain cells during such stress.