RESUMO
In this paper, we describe a novel process for creating high-resolution, 3D PDF, 3D printed, and holographic anatomic and pathology models using inexpensive consumer grade electronics, which can be incorporated in any undergraduate or graduate medical school curriculum. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s40670-021-01262-6.
RESUMO
Neonatal hypoxia/ischemia (HI) is a common cause of cognitive and behavioral deficits in children with hyperoxia treatment (HHI) being the current therapy for newborn resuscitation. HI induces cerebral edema that is associated with poor neurological outcomes. Our objective was to characterize cerebral edema after HI and determine the consequences of HHI (40% or 100% O(2)). Dry weight analyses showed cerebral edema 1 to 21 days after HI in the ipsilateral cortex; and 3 to 21 days after HI in the contralateral cortex. Furthermore, HI increased blood-brain barrier (BBB) permeability 1 to 7 days after HI, leading to bilateral cortical vasogenic edema. HHI failed to prevent HI-induced increase in BBB permeability and edema development. At the molecular level, HI increased ipsilateral, but not contralateral, AQP4 cortical levels at 3 and up to 21 days after HI. HHI treatment did not further affect HI-induced changes in AQP4. In addition, we observed developmental increases of AQP4 accompanied by significant reduction in water content and increase permeability of the BBB. Our results suggest that the ipsilateral HI-induced increase in AQP4 may be beneficial and that its absence in the contralateral cortex may account for edema formation after HI. Finally, we showed that HI induced impaired motor coordination 21 days after the insult and HHI did not ameliorate this behavioral outcome. We conclude that HHI treatment is effective as a resuscitating therapy, but does not ameliorate HI-induced cerebral edema and impaired motor coordination.
Assuntos
Edema Encefálico/etiologia , Edema Encefálico/terapia , Hipóxia-Isquemia Encefálica/complicações , Hipóxia-Isquemia Encefálica/terapia , Oxigenoterapia/métodos , Ressuscitação/métodos , Animais , Animais Recém-Nascidos , Aquaporina 4/metabolismo , Barreira Hematoencefálica/metabolismo , Água Corporal , Encéfalo/metabolismo , Edema Encefálico/metabolismo , Permeabilidade Capilar , Modelos Animais de Doenças , Discinesias/complicações , Discinesias/metabolismo , Discinesias/terapia , Lateralidade Funcional , Hipóxia-Isquemia Encefálica/metabolismo , Distribuição Aleatória , Ratos , Ratos Wistar , Fatores de TempoRESUMO
Neonatal hypoxia/ischemia (HI) is the most common cause of developmental neurological, cognitive and behavioral deficits in children, with hyperoxia (HHI) treatment being a clinical therapy for newborn resuscitation. Although cerebral edema is a common outcome after HI, the mechanisms leading to excessive fluid accumulation in the brain are poorly understood. Given the rigid nature of the bone-encased brain matter, knowledge of edema formation in the brain as a consequence of any injury, as well as the importance of water clearance mechanisms and water and ion homeostasis is important to our understanding of its detrimental effects. Knowledge of the pathological process underlying the appearance of dysfunctional outcomes after development of cerebral edema after neonatal HI in the developing brain and the molecular events triggered will allow a rational assessment of HHI therapy for neonatal HI and determine whether this treatment is beneficial or harmful to the developing infant.
Assuntos
Edema Encefálico/etiologia , Hipóxia-Isquemia Encefálica/complicações , Água Corporal , Encéfalo/fisiopatologia , Homeostase , Humanos , Recém-Nascido , OxigenoterapiaRESUMO
Tamoxifen (TMX) is a selective estrogen receptor modulator that can mimic the neuroprotective effects of estrogen but lacks its systemic adverse effects. We found that TMX (1 mg/day) significantly improved the motor recovery of partially paralyzed hind limbs of male adult rats with thoracic spinal cord injury (SCI), thus indicating a translational potential for this cancer medication given its clinical safety and applicability and the lack of currently available treatments for SCI. To shed light on the mechanisms underlying the beneficial effects of TMX for SCI, we used proteomic analyses, Western blots and histological assays, which showed that TMX treatment spared mature oligodendrocytes/increased myelin levels and altered reactive astrocytes, including the upregulation of the water channels aquaporin 4 (AQP4), a novel finding. AQP4 increases in TMX-treated SCI rats were associated with smaller fluid-filled cavities with borders consisting of densely packed AQP4-expressing astrocytes that closely resemble the organization of normal glia limitans externa (in contrast to large cavities in control SCI rats that lacked glia limitans-like borders and contained reactive glial cells). Based on our findings, we propose that TMX is a promising candidate for the therapeutic treatment of SCI and a possible intervention for other neuropathological conditions associated with demyelination and AQP4 dysfunction.
Assuntos
Aquaporina 4/metabolismo , Fármacos Neuroprotetores/farmacologia , Recuperação de Função Fisiológica/efeitos dos fármacos , Traumatismos da Medula Espinal/patologia , Tamoxifeno/farmacologia , Animais , Western Blotting , Encéfalo/efeitos dos fármacos , Encéfalo/metabolismo , Imunofluorescência , Masculino , Proteômica , Ratos , Ratos Sprague-Dawley , Traumatismos da Medula Espinal/metabolismoRESUMO
The NMDAR glutamate receptor subtype mediates various vital physiological neuronal functions. However, its excessive activation contributes to neuronal damage in a large variety of acute and chronic neurological disorders. NMDAR antagonists thus represent promising therapeutic tools that can counteract NMDARs' overactivation. Channel blockers are of special interest since they are use-dependent, thus being more potent at continuously activated NMDARs, as may be the case in pathological conditions. Nevertheless, it has been established that NMDAR antagonists, such as MK801, also have unacceptable neurotoxic effects. Presently only Memantine is considered a safe NMDAR antagonist and is used clinically. It has recently been speculated that antagonists that preferentially target extrasynaptic NMDARs would be less toxic. We previously demonstrated that the phencyclidine derivative GK11 preferentially inhibits extrasynaptic NMDARs. We thus anticipated that this compound would be safer than other known NMDAR antagonists. In this study we used whole-genome profiling of the rat cingulate cortex, a brain area that is particularly sensitive to NMDAR antagonists, to compare the potential adverse effects of GK11 and MK801. Our results showed that in contrast to GK11, the transcriptional profile of MK801 is characterized by a significant upregulation of inflammatory and stress-response genes, consistent with its high neurotoxicity. In addition, behavioural and immunohistochemical analyses confirmed marked inflammatory reactions (including astrogliosis and microglial activation) in MK801-treated, but not GK11-treated rats. Interestingly, we also showed that GK11 elicited less inflammation and neuronal damage, even when compared to Memantine, which like GK11, preferentially inhibits extrasynaptic NMDAR. As a whole, our study suggests that GK11 may be a more attractive therapeutic alternative in the treatment of CNS disorders characterized by the overactivation of glutamate receptors.
Assuntos
Cicloexenos/farmacologia , Antagonistas de Aminoácidos Excitatórios/farmacologia , Giro do Cíngulo/efeitos dos fármacos , Piperidinas/farmacologia , Receptores de N-Metil-D-Aspartato/antagonistas & inibidores , Animais , Astrócitos/efeitos dos fármacos , Astrócitos/metabolismo , Astrócitos/patologia , Cicloexenos/efeitos adversos , Maleato de Dizocilpina/efeitos adversos , Antagonistas de Aminoácidos Excitatórios/efeitos adversos , Feminino , Giro do Cíngulo/metabolismo , Giro do Cíngulo/patologia , Inflamação/induzido quimicamente , Inflamação/metabolismo , Inflamação/patologia , Injeções Intraperitoneais , Memantina/efeitos adversos , Microglia/efeitos dos fármacos , Microglia/metabolismo , Microglia/patologia , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Neurônios/patologia , Piperidinas/efeitos adversos , Ratos , Ratos Sprague-Dawley , Receptores de N-Metil-D-Aspartato/metabolismoRESUMO
To characterize the contribution of interleukin-6 (IL-6) to spinal cord injury pain (SCIP), we employed a clinically relevant rat contusion model of SCIP. Using Western blots, we measured IL-6 levels in lumbar segments (L1-L5), at the lesion site (T10), and in the corresponding lumbar and thoracic dorsal root ganglia (DRG) in 2 groups of similarly injured rats: (a) SCI rats that developed hind-limb mechanical allodynia (SCIP), and (b) SCI rats that did not develop SCIP. Only in SCIP rats did we find significantly increased IL-6 levels. Immunocytochemistry showed elevated IL-6 predominantly in reactive astrocytes. Our data also showed that increased production of IL-6 in hyperreactive astrocytes in SCIP rats may explain still-poorly understood astrocytic contribution to SCIP. To test the hypothesis that IL-6 contributes to mechanical allodynia, we treated SCIP rats with neutralizing IL-6 receptor antibody (IL-6-R Ab), and found that one systemic injection abolished allodynia and associated weight loss; in contrast to gabapentin, the analgesic effect lasted for at least 2weeks after the injection, despite the shorter presence of the Ab in the circulation. We also showed that IL-6-R Ab partially reversed SCI-induced decreases in the protein levels of the glutamate transporter GLT-1 12hours and 8days after Ab injection, which may explain the lasting analgesic effect of the Ab in SCIP rats. A link between reactive astrocytes IL-6-GLT-1 has not been previously shown. Given that the humanized IL-6-R Ab tocilizumab is Food and Drug Administration-approved for rheumatoid arthritis, we are proposing tocilizumab as a novel and potentially effective treatment for SCIP.
Assuntos
Anticorpos Monoclonais Humanizados/administração & dosagem , Hiperalgesia/tratamento farmacológico , Hiperalgesia/metabolismo , Interleucina-6/metabolismo , Transdução de Sinais/efeitos dos fármacos , Traumatismos da Medula Espinal/fisiopatologia , Medula Espinal/metabolismo , Animais , Hiperalgesia/etiologia , Masculino , Ratos , Ratos Sprague-Dawley , Medula Espinal/efeitos dos fármacos , Traumatismos da Medula Espinal/complicações , Traumatismos da Medula Espinal/tratamento farmacológico , Resultado do TratamentoRESUMO
Amiloride is a drug approved by the United States Food and Drug Administration, which has shown neuroprotective effects in different neuropathological conditions, including brain injury or brain ischemia, but has not been tested in spinal cord injury (SCI). We tested amiloride's therapeutic potential in a clinically relevant rat model of contusion SCI inflicted at the thoracic segment T10. Rats receiving daily administration of amiloride from 24 h to 35 days after SCI exhibited a significant improvement in hindlimb locomotor ability at 21, 28, and 35 days after injury, when compared to vehicle-treated SCI rats. Rats receiving amiloride treatment also exhibited a significant increase in myelin oligodendrocyte glycoprotein (MOG) levels 35 days after SCI at the site of injury (T10) when compared to vehicle-treated controls, which indicated a partial reverse in the decrease of MOG observed with injury. Our data indicate that higher levels of MOG correlate with improved locomotor recovery after SCI, and that this may explain the beneficial effects of amiloride after SCI. Given that amiloride treatment after SCI caused a significant preservation of myelin levels, and improved locomotor recovery, it should be considered as a possible therapeutic intervention after SCI.
Assuntos
Amilorida/farmacologia , Transtornos Neurológicos da Marcha/tratamento farmacológico , Transtornos Neurológicos da Marcha/fisiopatologia , Fármacos Neuroprotetores/farmacologia , Recuperação de Função Fisiológica/fisiologia , Traumatismos da Medula Espinal/tratamento farmacológico , Traumatismos da Medula Espinal/fisiopatologia , Amilorida/uso terapêutico , Animais , Modelos Animais de Doenças , Transtornos Neurológicos da Marcha/etiologia , Masculino , Fármacos Neuroprotetores/uso terapêutico , Ratos , Ratos Sprague-Dawley , Recuperação de Função Fisiológica/efeitos dos fármacos , Traumatismos da Medula Espinal/etiologiaRESUMO
Vascular endothelial growth factor (VEGF)-A mRNA was previously identified as one of the significantly upregulated transcripts in spinal cord injured tissue from adult rats that developed allodynia. To characterize the role of VEGF-A in the development of pain in spinal cord injury (SCI), we analyzed mechanical allodynia in SCI rats that were treated with either vehicle, VEGF-A isoform 165 (VEGF(165)), or neutralizing VEGF(165)-specific antibody. We have observed that exogenous administration of VEGF(165) increased both the number of SCI rats that develop persistent mechanical allodynia, and the level of hypersensitivity to mechanical stimuli. Our analysis identified excessive and aberrant growth of myelinated axons in dorsal horns and dorsal columns of chronically injured spinal cords as possible mechanisms for both SCI pain and VEGF(165)-induced amplification of SCI pain, suggesting that elevated endogenous VEGF(165) may have a role in the development of allodynia after SCI. However, the neutralizing VEGF(165) antibody showed no effect on allodynia or axonal sprouting after SCI. It is possible that another endogenous VEGF isoform activates the same signaling pathway as the exogenously-administered 165 isoform and contributes to SCI pain. Our transcriptional analysis revealed that endogenous VEGF(188) is likely to be the isoform involved in the development of allodynia after SCI. To the best of our knowledge, this is the first study to suggest a possible link between VEGF, nonspecific sprouting of myelinated axons, and mechanical allodynia following SCI.
Assuntos
Hiperalgesia/metabolismo , Hiperalgesia/terapia , Traumatismos da Medula Espinal/metabolismo , Traumatismos da Medula Espinal/terapia , Fator A de Crescimento do Endotélio Vascular/metabolismo , Animais , Anticorpos Neutralizantes , Hiperalgesia/etiologia , Hiperalgesia/fisiopatologia , Imuno-Histoquímica , Masculino , Atividade Motora , Análise de Sequência com Séries de Oligonucleotídeos , Limiar da Dor/fisiologia , Estimulação Física , Distribuição Aleatória , Ratos , Ratos Sprague-Dawley , Traumatismos da Medula Espinal/complicações , Traumatismos da Medula Espinal/fisiopatologia , Estatísticas não Paramétricas , Fator A de Crescimento do Endotélio Vascular/uso terapêuticoRESUMO
Vascular endothelial growth factor (VEGF) is being investigated as a potential interventional therapy for spinal cord injury (SCI). In the current study, we examined SCI-induced changes in VEGF protein levels using Western blot analysis around the epicenter of injury. Our results indicate a significant decrease in the levels of VEGF(165) and other VEGF isoforms at the lesion epicenter 1 day after injury, which was maintained up to 1 month after injury. We also examined if robust VEGF(165) decrease in injured spinal cords affects neuronal survival, given that a number of reported studies show neuroprotective effect of this VEGF isoform. However, exogenously administered VEGF(165) at the time of injury did not affect the number of sparred neurons. In contrast, exogenous administration of VEGF antibody that inhibits actions of not only VEGF(165) but also of several other VEGF isoforms, significantly decreased number of sparred neurons after SCI. Together these results indicate a general reduction of VEGF isoforms following SCI and that isoforms other than VEGF(165) (e.g., VEGF(121) and/or VEGF(189)) provide neuroprotection, suggesting that VEGF(165) isoform is likely involved in other pathophysiological process after SCI.
Assuntos
Neurônios/metabolismo , Traumatismos da Medula Espinal/metabolismo , Medula Espinal/metabolismo , Fator A de Crescimento do Endotélio Vascular/metabolismo , Análise de Variância , Animais , Western Blotting , Imuno-Histoquímica , Masculino , Fármacos Neuroprotetores/metabolismo , Fármacos Neuroprotetores/uso terapêutico , Ratos , Ratos Sprague-Dawley , Proteínas Recombinantes/uso terapêutico , Traumatismos da Medula Espinal/terapia , Fator A de Crescimento do Endotélio Vascular/uso terapêuticoRESUMO
Central neuropathic pain (CNP) developing after spinal cord injury (SCI) is described by the region affected: above-level, at-level and below-level pain occurs in dermatomes rostral, at/near, or below the SCI level, respectively. People with SCI and rodent models of SCI develop above-level pain characterized by mechanical allodynia and thermal hyperalgesia. Mechanisms underlying this pain are unknown and the goals of this study were to elucidate components contributing to the generation of above-level CNP. Following a thoracic (T10) contusion, forelimb nociceptors had enhanced spontaneous activity and were sensitized to mechanical and thermal stimulation of the forepaws 35 days post-injury. Cervical dorsal horn neurons showed enhanced responses to non-noxious and noxious mechanical stimulation as well as thermal stimulation of receptive fields. Immunostaining dorsal root ganglion (DRG) cells and cord segments with activating transcription factor 3 (ATF3, a marker for neuronal injury) ruled out neuronal damage as a cause for above-level sensitization since few C8 DRG cells expressed AFT3 and cervical cord segments had few to no ATF3-labeled cells. Finally, activated microglia and astrocytes were present in thoracic and cervical cord at 35 days post-SCI, indicating a rostral spread of glial activation from the injury site. Based on these data, we conclude that peripheral and central sensitization as well as reactive glia in the uninjured cervical cord contribute to CNP. We hypothesize that reactive glia in the cervical cord release pro-inflammatory substances which drive chronic CNP. Thus a complex cascade of events spanning many cord segments underlies above-level CNP.
Assuntos
Neuralgia/etiologia , Limiar da Dor/fisiologia , Traumatismos da Medula Espinal/complicações , Traumatismos da Medula Espinal/patologia , Medula Espinal/patologia , Medula Espinal/fisiopatologia , Potenciais de Ação/fisiologia , Fator 3 Ativador da Transcrição/metabolismo , Animais , Comportamento Animal , Contagem de Células/métodos , Modelos Animais de Doenças , Membro Anterior/fisiopatologia , Gânglios Espinais/metabolismo , Gânglios Espinais/fisiologia , Hiperalgesia/fisiopatologia , Técnicas In Vitro , Masculino , Nociceptores/patologia , Nociceptores/fisiologia , Estimulação Física/métodos , Ratos , Ratos Sprague-Dawley , Células Receptoras Sensoriais/fisiologia , Medula Espinal/metabolismo , Traumatismos da Medula Espinal/metabolismo , Estatísticas não ParamétricasRESUMO
Long-term functional impairments due to spinal cord injury (SCI) in the rat result from secondary apoptotic death regulated, in part, by SCI-induced decreases in protein levels of the antiapoptotic protein Bcl-xL. We have shown that exogenous administration of Bcl-xL spares neurons 24 h after SCI. However, long-term effects of chronic application of Bcl-xL have not been characterized. To counteract SCI-induced decreases in Bcl-xL and resulting apoptosis, we used the TAT protein transduction domain fused to the Bcl-xL protein (Tat-Bcl-xL), or its antiapoptotic domain BH4 (Tat-BH4). We used intrathecal delivery of Tat-Bcl-xL, or Tat-BH4, into injured spinal cords for 24 h or 7 days, and apoptosis, neuronal death and locomotor recovery were assessed up to 2 months after injury. Both, Tat-Bcl-xL and Tat-BH4, significantly decreased SCI-induced apoptosis in thoracic segments containing the site of injury (T10) at 24 h or 7 days after SCI. However, the 7-day delivery of Tat-Bcl-xL, or Tat-BH4, also induced a significant impairment of locomotor recovery that lasted beyond the drug delivery time. We found that the 7-day administration of Tat-Bcl-xL, or Tat-BH4, significantly increased non-apoptotic neuronal loss and robustly augmented microglia/macrophage activation. These results indicate that the antiapoptotic treatment targeting Bcl-xL shifts neuronal apoptosis to necrosis, increases the inflammatory response and impairs locomotor recovery. Our results suggest that a combinatorial treatment consisting of antiapoptotic and anti-inflammatory agents may be necessary to achieve tissue preservation and significant improvement in functional recovery after SCI.
Assuntos
Apoptose/fisiologia , Traumatismos da Medula Espinal/fisiopatologia , Traumatismos da Medula Espinal/terapia , Proteína bcl-X/administração & dosagem , Análise de Variância , Animais , Apoptose/efeitos dos fármacos , Comportamento Animal , Antígeno CD11b/metabolismo , Modelos Animais de Doenças , Esquema de Medicação , Ensaio de Imunoadsorção Enzimática/métodos , Regulação da Expressão Gênica/efeitos dos fármacos , Produtos do Gene tat/administração & dosagem , Masculino , Atividade Motora/efeitos dos fármacos , Atividade Motora/fisiologia , Estrutura Terciária de Proteína/fisiologia , Ratos , Ratos Sprague-Dawley , Recuperação de Função Fisiológica/efeitos dos fármacos , Recuperação de Função Fisiológica/fisiologia , Traumatismos da Medula Espinal/patologia , Fatores de TempoRESUMO
Central neuropathic pain (CNP) is an important problem following spinal cord injury (SCI), because it severely affects the quality of life of SCI patients. As in the patient population, the majority of rats develop significant allodynia (CNP rats) after moderate SCI. However, about 10% of SCI rats do not develop allodynia, or develop significantly less allodynia than CNP rats (non-CNP rats). To identify transcriptional changes underlying CNP development after SCI, we used Affymetrix DNA microarrays and RNAs extracted from the spinal cords of CNP and non-CNP rats. DNA microarry analysis showed significantly increased expression of a number of genes associated with inflammation and astrocytic activation in the spinal cords of rats that developed CNP. For example, mRNA levels of glial fibrilary acidic protein (GFAP) and Aquaporin 4 (AQP4) significantly increased in CNP rats. We also found that GFAP, S100beta and AQP4 protein elevation persisted for at least 9 months throughout contused spinal cords, consistent with the chronic nature of CNP. Thus, we hypothesize that CNP development results, in part, from dysfunctional, chronically "over-activated" astrocytes. Although, it has been shown that activated astrocytes are associated with peripheral neuropathic pain, this has not previously been demonstrated in CNP after SCI.
Assuntos
Dor/metabolismo , Traumatismos da Medula Espinal/metabolismo , Ativação Transcricional/fisiologia , Animais , Western Blotting/métodos , Modelos Animais de Doenças , Imunofluorescência/métodos , Proteína Glial Fibrilar Ácida/genética , Proteína Glial Fibrilar Ácida/metabolismo , Masculino , Microscopia Confocal/métodos , Fatores de Crescimento Neural/metabolismo , Análise de Sequência com Séries de Oligonucleotídeos/métodos , Dor/etiologia , RNA Mensageiro/metabolismo , Ratos , Ratos Sprague-Dawley , Subunidade beta da Proteína Ligante de Cálcio S100 , Proteínas S100/metabolismo , Traumatismos da Medula Espinal/complicações , Fatores de TempoRESUMO
Major thermal injury results in severe prolonged responses with three components: a hypermetabolic response, inflammatory responses, and endogenous wound-healing processes. We showed that use of liposome-mediated gene transfer of the insulin-like growth factor I (IGF-I) reduces burn-induced inflammatory responses and enhances wound healing. In the present study, we found transient increased levels of IGF-I protein in rats exposed to thermal trauma via liposomal gene transfer in an effort to define the transcriptional events that occur after IGF-I delivery at the site of injury. The beneficial effects of IGF-I gene transfer act partly via amelioration of burn-induced inflammatory responses that mediate cell death through caspase-3 activity and Bax expression. IGF-I gene transfer induces selective stimulation of activation protein-1 DNA-binding activity and activation of antiapoptotic, but not inflammatory, NF-kappaB transcription factors. Data were consistent with our hypothesis that the beneficial effects of IGF-I gene transfer on burned rats act in part via activation protein-1 and NF-kappaB transcriptional regulation and the concordance between the results obtained with antiapoptotic, as opposed to the proapoptotic, sequences as well as the corresponding changes in measures of cell death via Bax and caspase-3 mechanisms.
Assuntos
Queimaduras/metabolismo , Queimaduras/terapia , Terapia Genética , Fator de Crescimento Insulin-Like I/genética , Cicatrização/fisiologia , Animais , Queimaduras/imunologia , Caspase 3 , Caspases/metabolismo , Proteínas de Ligação a DNA/metabolismo , Expressão Gênica/fisiologia , Imunoglobulina G/metabolismo , Lipossomos , Masculino , NF-kappa B/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/genética , Ratos , Ratos Sprague-Dawley , Pele/lesões , Pele/metabolismo , Fator de Transcrição AP-1/metabolismo , Proteína X Associada a bcl-2 , Proteína bcl-XRESUMO
"Decoy" oligonucleotides can be used as gene-specific nuclear factor (NF-kappaB) inhibitors to regulate gene expression. We applied two different decoy oligonucleotides that contained the NF-kappaB binding consensus sequences present in the immunoglobulin G (IgG)-kappaB and Bcl-x promoter into 7-day-old (P7) rat lateral ventricles before hypoxia/ischemia (HI) and compared their effects on gene expression in hippocampi to saline-treated, scrambled decoy-treated, or untreated hippocampi exposed to HI. Left hippocampi were collected at 12 hr after HI. Electrophoretic mobility shift assays (EMSAs) showed that the two decoy treatments had different effects on NF-kappaB binding to the IgG-kappaB and Bcl-x promoter-specific consensus sequences, respectively. We assessed the decoys' effects on gene expression 12 hr after HI using ribonuclease protection assays (RPAs) and Affymetrix DNA microarrays. RPAs showed that both decoys significantly decreased interleukin (IL)-1alpha mRNA levels but had no impact on IL-1beta, IL-6, and IL-10 mRNA levels. IgG-kappaB decoys significantly decreased tumor necrosis factor (TNF)-alpha and TNF-beta mRNA levels compared to minimal changes after treatment with Bcl-x decoys. DNA microarray analyses showed that Bcl-x decoy treatment significantly decreased Bcl-x(L) mRNA levels. The decreased Bcl-x(L) mRNA levels after Bcl-x decoy treatment was confirmed by RPA analysis. DNA microarray data also indicated that several other genes were affected by both decoys. Our results suggest that different NF-kappaB decoy treatments could differentially regulate transcriptional responses to central nervous system trauma. Careful design of decoy sequences, however, is essential to acquire selective effects on cell death outcome.
Assuntos
Regulação da Expressão Gênica/genética , Hipocampo/metabolismo , Hipóxia-Isquemia Encefálica/metabolismo , NF-kappa B/metabolismo , Neurônios/metabolismo , Oligonucleotídeos/farmacologia , Animais , Animais Recém-Nascidos , Sítios de Ligação/genética , Modelos Animais de Doenças , Feminino , Regulação da Expressão Gênica/efeitos dos fármacos , Hipocampo/fisiopatologia , Hipóxia-Isquemia Encefálica/genética , Hipóxia-Isquemia Encefálica/fisiopatologia , Imunoglobulina G/genética , Interleucina-1/genética , Masculino , NF-kappa B/antagonistas & inibidores , Neurônios/efeitos dos fármacos , Regiões Promotoras Genéticas/genética , Proteínas Proto-Oncogênicas c-bcl-2/genética , RNA Mensageiro/efeitos dos fármacos , RNA Mensageiro/metabolismo , Ratos , Ratos Sprague-Dawley , Fator de Necrose Tumoral alfa/genética , Proteína bcl-XRESUMO
There are two different ways for cells to die: necrosis and apoptosis. Cell death has traditionally been described as necrotic or apoptotic based on morphological criteria. There are controversy about the respective roles of apoptosis and necrosis in cell death resulting from trauma to the central nervous system (CNS). An evaluation of work published since 1997 in which electron microscopy was applied to ascertain the role of apoptosis and necrosis in: spinal cord injury, stroke, and hypoxia/ischemia (H/I) showed evidence for necrosis and apoptosis based on DNA degradation, presence of histones in cytoplasm, and morphological evidence in spinal cord. In the aftermath of stroke, many of the biochemical markers for apoptosis were present but the morphological determinations suggested that necrosis is the major source of post-traumatic cell death. This was not the case in H/I where both biochemical assays and the morphological studies gave more consistent results in a manner similar to the spinal cord injury studies. After H/I, major factors affecting cell death outcomes are DNA damage and repair processes, expression of bcl-like gene products and inflammation-triggered cytokine production.
Assuntos
Sistema Nervoso Central/irrigação sanguínea , Hipóxia/fisiopatologia , Isquemia/fisiopatologia , Neurônios/fisiologia , Transdução de Sinais , Traumatismos da Medula Espinal/fisiopatologia , Acidente Vascular Cerebral/fisiopatologia , Animais , Apoptose/fisiologia , Morte Celular/fisiologia , Necrose , Fatores de TempoRESUMO
The events occurring early in the burn wound trigger a sequence of local and systemic responses that influence cell and tissue survival and, consequently, wound healing and recovery. Using high-density oligonucleotide arrays we identified gene expression patterns in skin samples taken from a region of injury in the burn rat model. The associated genomic events include the differential expression of genes involved in cell survival and death, cell growth regulation, cell metabolism, inflammation, and immune response. The functional gene cluster detected and their time appearance matched the time sequence known to occur in burn wound healing.