RESUMO
This article translates the guidelines for cadaver surgical training (CST) published in 2012 by Japan Surgical Society (JSS) and Japanese Association of Anatomists from Japanese to English. These guidelines are based on Japanese laws and enable the usage of donated cadavers for CST and clinical research. The following are the conditions to implement the activities outlined in the guidelines. The aim is to improve medicine and to contribute to social welfare. Activities should only be carried out at medical or dental universities under the centralized control by the department of anatomy under the regulation of Japanese law. Upon the usage of cadavers, registered donors must provide a written informed-consent for their body to be used for CST and other activities of clinical medicine. Commercial use of cadavers and profit-based CST is strongly prohibited. Moreover, all the cadaver-related activities except for the commercial-based ones require the approval of the University's Institutional Review Board (IRB) before implementation. The expert committee organized at each university for the implementation of CST should summarize the implementation of the program and report the details of the training program, operating costs, and conflicts of interest to the CST Promotion Committee of JSS.
Assuntos
Anatomistas , Medicina Clínica , Cadáver , Dissecação , Humanos , JapãoRESUMO
The "Guidelines for Cadaver Dissection in Education and Research of Clinical Medicine" drafted by the Japan Surgical Society (JSS) and the Japanese Association of Anatomists in 2012 helped dispel legal concerns over cadaver surgical training (CST) and the usage of donated human bodies for research and development (R&D) in the country. Subsequently, in the fiscal year 2018, the Ministry of Health, Labour and Welfare increased the funding for CST, prompting its wider implementation. This study analyzed data obtained in 2012-2021 through the reporting system of the JSS-CST Promotion Committee to map the usage of cadavers for clinical purposes, specifically education and R&D, in Japan. We found that the number of medical universities using cadavers for CST and R&D programs was just 5 in 2012, and it reached 38 for the decade. Thus, about half of Japan's medical universities implemented such programs over the period. Meanwhile, the total number of programs was 1,173. In the clinical field, the highest number of programs were implemented in orthopedics (27%), followed by surgery (21%), and neurosurgery (12%). Based on the purpose, the most common objective of the programs (approximately 70%) was acquiring advanced surgical techniques. Further, the highest number of programs and participants were recorded in 2019 (295 programs, 6,537 participants). Thus, the guidelines helped expand cadaver usage for clinical purposes in Japan. To further promote the clinical usage of cadavers in medical and dental universities throughout Japan, sharing know-how on operating cadaver laboratories and building understanding among the general public is recommended.
Assuntos
Anatomistas , Educação Médica , Cadáver , Dissecação , Educação Médica/métodos , Humanos , JapãoRESUMO
Cerebellar malformations cause changes to the sleep-wake cycle, resulting in sleep disturbance. However, it is unclear how the cerebellum contributes to the sleep-wake cycle. To examine the neural connections between the cerebellum and the nuclei involved in the sleep-wake cycle, we investigated the axonal projections of Purkinje cells in the mouse posterior vermis by using an adeno-associated virus (AAV) vector (serotype rh10) as an anterograde tracer. When an AAV vector expressing humanized renilla green fluorescent protein was injected into the cerebellar lobule IX, hrGFP and synaptophysin double-positive axonal terminals were observed in the region of medial parabrachial nucleus (MPB). The MPB is involved in the phase transition from rapid eye movement (REM) sleep to Non-REM sleep and vice versa, and the cardiovascular and respiratory responses. The hrGFP-positive axons from lobule IX went through the ventral spinocerebellar tract and finally reached the MPB. By contrast, when the AAV vector was injected into cerebellar lobule VI, no hrGFP-positive axons were observed in the MPB. To examine neurons projecting to the MPB, we unilaterally injected Fast Blue and AAV vector (retrograde serotype, rAAV2-retro) as retrograde tracers into the MPB. The cerebellar Purkinje cells in lobules VIII-X on the ipsilateral side of the Fast Blue-injected MPB were retrogradely labeled by Fast Blue and AAV vector (retrograde serotype), but no retrograde-labeled Purkinje cells were observed in lobules VI-VII and the cerebellar hemispheres. These results indicated that Purkinje cells in lobules VIII-X directly project their axons to the ipsilateral MPB but not lobules VI-VII. The direct connection between lobules VIII-X and the MPB suggests that the cerebellum participates in the neural network controlling the sleep-wake cycle, and cardiovascular and respiratory responses, by modulating the physiological function of the MPB.
Assuntos
Vermis Cerebelar/citologia , Núcleos Parabraquiais/citologia , Células de Purkinje/citologia , Amidinas , Animais , Dependovirus/genética , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Masculino , Camundongos Endogâmicos ICR , Vias Neurais/citologia , Técnicas de Rastreamento Neuroanatômico , Marcadores do Trato NervosoRESUMO
The development of gene therapy techniques to introduce transgenes that promote neuronal survival and protection provides effective therapeutic approaches for neurological and neurodegenerative diseases. Intramuscular injection of adenoviral and adeno-associated viral vectors, as well as lentiviral vectors pseudotyped with rabies virus glycoprotein (RV-G), permits gene delivery into motor neurons in animal models for motor neuron diseases. Recently, we developed a vector with highly efficient retrograde gene transfer (HiRet) by pseudotyping a human immunodeficiency virus type 1 (HIV-1)-based vector with fusion glycoprotein B type (FuG-B) or a variant of FuG-B (FuG-B2), in which the cytoplasmic domain of RV-G was replaced by the corresponding part of vesicular stomatitis virus glycoprotein (VSV-G). We have also developed another vector showing neuron-specific retrograde gene transfer (NeuRet) with fusion glycoprotein C type, in which the short C-terminal segment of the extracellular domain and transmembrane/cytoplasmic domains of RV-G was substituted with the corresponding regions of VSV-G. These two vectors afford the high efficiency of retrograde gene transfer into different neuronal populations in the brain. Here we investigated the efficiency of the HiRet (with FuG-B2) and NeuRet vectors for retrograde gene transfer into motor neurons in the spinal cord and hindbrain in mice after intramuscular injection and compared it with the efficiency of the RV-G pseudotype of the HIV-1-based vector. The main highlight of our results is that the HiRet vector shows the most efficient retrograde gene transfer into both spinal cord and hindbrain motor neurons, offering its promising use as a gene therapeutic approach for the treatment of motor neuron diseases.
Assuntos
Técnicas de Transferência de Genes , Terapia Genética/métodos , Vetores Genéticos/genética , Neurônios Motores/metabolismo , Proteínas Virais de Fusão/metabolismo , Animais , Humanos , Lentivirus/genética , Camundongos , Proteínas Virais de Fusão/genéticaRESUMO
Huntingtin-interacting protein 1-related (Hip1r) was originally identified due to its homology to Huntingtin-interacting protein 1, which contributes to the development of Huntington's disease (HD). We studied the expression of the mouse Hip1r (mHip1r) gene in the mouse head by in situ hybridization. In early embryogenesis at embryonic day (E) 13, mHip1r expression was especially prominent in the olfactory epithelium, cerebral cortex layer 1, cortical plate, and dentate gyrus. During later development from E15 to E17, strong expression of mHip1r transcripts continued to be observed in the olfactory epithelium, cortical plate, and dentate gyrus. Furthermore, not only the subplate and subventricular zone of the cortex, but also secretory glands, such as the nasal gland and the submandibular gland, were mHip1r-positive. Other positive tissues included the retinal ganglion cells, vomeronasal organ, trigeminal ganglion, and the developing molar tooth. In the adult mouse brain, similar expression patterns were observed in the cerebral cortex layers and other brain regions except the cerebellum. Additionally, by using an antibody against mHip1r, we confirmed these expression patterns at the protein level. Specific expression of mHip1r in the embryonic brain and secretory glands suggests a possible role for Hip1r in normal development and in the pathology of HD.
Assuntos
Encéfalo/embriologia , Encéfalo/metabolismo , Proteínas de Ligação a DNA/genética , Regulação da Expressão Gênica no Desenvolvimento , Proteínas Adaptadoras de Transdução de Sinal , Animais , Proteínas de Ligação a DNA/metabolismo , Feminino , Doença de Huntington/genética , Imuno-Histoquímica , Hibridização In Situ , Masculino , Camundongos , Proteínas dos Microfilamentos , Especificidade de Órgãos/genética , Gravidez , RNA Mensageiro/genética , RNA Mensageiro/metabolismoRESUMO
This article analyses the Draft of Guidelines for Human Body Dissection for Clinical Anatomy Education and Research drawn by the Study Group for Future Training Systems of Surgical Skills and Procedures established by the Fiscal Year 2010 research program of the Ministry of Health, Labor and Welfare. The purpose of the Draft of Guidelines is: First, to lay out the required basic guidelines for human cadaver usage to allow medical and dental faculty to conduct clinical education and research in accordance with existing regulations. Second, the guidelines are expected to give physicians a regulatory framework to carry out cadaver training in accordance with the current legal framework. This article explains the Draft of Guidelines in detail, outlines the future of cadaver training, and describes issues which must still be solved.
Assuntos
Anatomia/educação , Dissecação/métodos , Guias como Assunto , Cadáver , Cirurgia Geral/educação , Humanos , Japão , PesquisaRESUMO
The lentiviral vector system based on human immunodeficiency virus type 1 (HIV-1) is used extensively in gene therapy trials of neurological and neurodegenerative diseases. Retrograde axonal transport of viral vectors offers a great advantage to the delivery of genes into neuronal cell bodies that are situated in regions distant from the injection site. Pseudotyping of HIV-1-based vectors with selective variants of rabies virus glycoprotein (RV-G) increases gene transfer via retrograde transport into the central nervous system. Because large-scale application for gene therapy trials requires high titer stocks of the vector, pseudotyping of a lentiviral vector that produces more efficient retrograde transport is needed. In the present study, we developed a novel vector system for highly efficient retrograde gene transfer by pseudotyping an HIV-1 vector with a fusion envelope glycoprotein (termed FuG-B) in which the cytoplasmic domain of RV-G was substituted by the corresponding part of vesicular stomatitis virus glycoprotein. The FuG-B pseudotype shifted the transducing property of the lentiviral vector and enhanced the retrograde transport-mediated gene transfer into different brain regions innervating the striatum with greater efficiency than that of the RV-G pseudotype in mice. In addition, injection of the FuG-B-pseudotyped vector into monkey striatum (caudate and putamen) allowed for highly efficient gene delivery into the nigrostriatal dopamine system, which is a major target for gene therapy of Parkinson's disease. Our strategy provides a powerful tool for the treatment of certain neurological and neurodegenerative diseases by promoting retrograde gene delivery via a lentiviral vector.
Assuntos
Antígenos Virais/genética , Vetores Genéticos , Glicoproteínas/genética , HIV-1/genética , Transdução Genética , Proteínas do Envelope Viral/genética , Animais , Corpo Estriado/metabolismo , Corpo Estriado/virologia , Terapia Genética , Células HEK293 , Humanos , Macaca fascicularis , Camundongos , Camundongos Endogâmicos C57BL , Vírus da Raiva/metabolismoRESUMO
During development, dorsal root ganglion (DRG) neurons in higher vertebrates extend their axons centrally to the spinal cord through the dorsal root entry zone (DREZ) and peripherally to muscle and skin targets. After entering the spinal cord, DRG axons project into the dorsal mantle layer. In this review, we focus on evidence showing the role for netrin-1 in forming sensory axonal trajectories. Netrin-1 is a diffusible axonal guidance molecule that chemorepels developing DRG axons. When DRG axons project toward the DREZ, ventral spinal cord-derived netrin-1 prevents DRG axons from projecting aberrantly toward the ventral spinal cord. At later stages, the dorsal spinal cord cells transiently express netrin-1. This dorsal spinal cord-derived netrin-1 prevents DRG axons from invading the dorsal spinal cord during the waiting period. Together, the data reviewed provide strong evidence that netrin-1 plays a crucial role in sensory axon projection during development.
Assuntos
Axônios/fisiologia , Gânglios Espinais/fisiologia , Fatores de Crescimento Neural/fisiologia , Proteínas Supressoras de Tumor/fisiologia , Animais , Axônios/ultraestrutura , Gânglios Espinais/embriologia , Humanos , Camundongos , Netrina-1 , Vias Neurais , Medula Espinal/fisiologiaRESUMO
During development, dorsal root ganglion (DRG) neurons extend their axons toward the dorsolateral part of the spinal cord and enter the spinal cord through the dorsal root entry zone (DREZ). After entering the spinal cord, these axons project into the dorsal mantle layer after a 'waiting period' of a few days. We revealed that the diffusible axonal guidance molecule netrin-1 is a chemorepellent for developing DRG axons. When DRG axons orient themselves toward the DREZ, netrin-1 proteins derived from the ventral spinal cord prevent DRG axons from projecting aberrantly toward the ventral spinal cord and help them to project correctly toward the DREZ. In addition to the ventrally derived netrin-1, the dorsal spinal cord cells adjacent to the DREZ transiently express netrin-1 proteins during the waiting period. This dorsally derived netrin-1 contributes to the correct guidance of DRG axons to prevent them from invading the dorsal spinal cord. In general, there is a complete lack of sensory axonal regeneration after a spinal cord injury, because the dorsal column lesion exerts inhibitory activities toward regenerating axons. Netrin-1 is a novel candidate for a major inhibitor of sensory axonal regeneration in the spinal cord; because its expression level stays unchanged in the lesion site following injury, and adult DRG neurons respond to netrin-1-induced axon repulsion. Although further studies are required to show the involvement of netrin-1 in preventing the regeneration of sensory axons in CNS injury, the manipulation of netrin-1-induced repulsion in the CNS lesion site may be a potent approach for the treatment of human spinal injuries.
Assuntos
Axônios/metabolismo , Fatores de Crescimento Neural/metabolismo , Regeneração , Transdução de Sinais , Proteínas Supressoras de Tumor/metabolismo , Animais , Humanos , Camundongos , Netrina-1 , Medula Espinal/citologia , Medula Espinal/metabolismo , Medula Espinal/fisiologiaRESUMO
During early development, centrally projecting dorsal root ganglion (DRG) neurons extend their axons toward the dorsal spinal cord. We previously reported that this projection is achieved by dorsal spinal cord-derived chemoattraction. However, the molecular nature of the chemotrophic cue is not yet fully understood. To identify novel genes differentially expressed in the dorsal spinal cord in the embryonic day 10.5 mouse, we used the Kazusa cDNA array system comprising approximately 1700 mouse KIAA/FLJ (mKIAA/mFLJ) cDNA clones and laser capture microdissection (LCM) in combination with PCR-based cDNA amplification. We observed that a certain population of genes showed significantly increased expression in the dorsal spinal cord. In situ hybridization analysis verified the expression of mRNAs of 6 genes (Hip1r, Nav2, Fstl5, Cacna1h, Bcr, and Bmper) in the cells that constitute the dorsal spinal cord. The dorsal spinal cord-specific genes identified in this study provide a basis for studying the molecular nature of the neural development including the axonal guidance of DRG neurons. These results also demonstrate that the combined use of LCM coupled with the Kazusa cDNA array technology will be useful for the identification of large proteins expressed in the restricted small regions of embryos.
Assuntos
Expressão Gênica , Proteínas de Membrana/genética , Medula Espinal/embriologia , Medula Espinal/metabolismo , Proteínas Adaptadoras de Transdução de Sinal , Animais , Canais de Cálcio Tipo T/genética , Proteínas de Transporte/genética , Proteínas de Ligação a DNA/genética , Imuno-Histoquímica , Hibridização In Situ , Camundongos , Microdissecção , Proteínas dos Microfilamentos , Neurônios/metabolismo , Análise de Sequência com Séries de Oligonucleotídeos , Proteínas Proto-Oncogênicas c-bcr/genética , RNA Mensageiro/metabolismoRESUMO
During early development, the ventral spinal cord expresses chemorepulsive signals that act on dorsal root ganglion (DRG) axons to help orient them toward the dorsolateral part of the spinal cord. However, the molecular nature of this chemorepulsion is mostly unknown. We report here that netrin-1 acts as an early ventral spinal cord-derived chemorepellent for DRG axons. In the developing mouse spinal cord, netrin-1 is expressed in the floor plate of the spinal cord, and the netrin receptor Unc5c is expressed in DRG neurons. We show that human embryonic kidney cell aggregates secreting netrin-1 repel DRG axons and that netrin-1-deficient ventral spinal cord explants lose their repulsive influence on DRG axons. In embryonic day 10 netrin-1 mutant mice, we find that DRG axons exhibit transient misorientation. Furthermore, by means of gain-of-function analyses, we show that ectopic netrin-1 in the dorsal and intermediate spinal cord prevents DRG axons from being directed toward the dorsal spinal cord. Together, these findings suggest that netrin-1 contributes to the formation of the initial trajectories of developing DRG axons as a repulsive guidance cue.
Assuntos
Axônios/fisiologia , Quimiotaxia/fisiologia , Gânglios Espinais/embriologia , Fatores de Crescimento Neural/fisiologia , Neurônios Aferentes/fisiologia , Medula Espinal/embriologia , Transmissão Sináptica/fisiologia , Proteínas Supressoras de Tumor/fisiologia , Animais , Linhagem Celular , Sinais (Psicologia) , Embrião de Mamíferos/metabolismo , Embrião de Mamíferos/fisiologia , Desenvolvimento Embrionário , Gânglios Espinais/citologia , Humanos , Técnicas In Vitro , Camundongos , Camundongos Mutantes , Mutação , Fatores de Crescimento Neural/genética , Receptores de Netrina , Netrina-1 , Vias Neurais/fisiologia , Receptores de Fator de Crescimento Neural/metabolismo , Proteínas Supressoras de Tumor/genéticaRESUMO
Ficolins are a group of proteins characterized by the presence of collagen-like and fibrinogen-like domains. Two of three human ficolins, L-ficolin and H-ficolin, are serum lectins that form complexes with mannose-binding lectin-associated serine proteases (MASPs) and play important roles in the lectin complement pathway. The other human ficolin, M-ficolin, is a non-serum-type ficolin that is expressed in monocytes. Little is known about the physiological roles of ficolins. In this study, we delineated the ontogeny and cell types that express ficolins in mice. RT-PCR analysis showed that the expression pattern of ficolin A expression was closely similar to that of Masps, suggesting that these molecules may function in coordination as components of the lectin complement pathway. The cell types that express ficolin A mRNA in both adult liver and spleen were identified as macrophages by in situ hybridization. Ficolin B exhibited a distinct ontogeny pattern that switched from embryonic liver to postnatal bone marrow and spleen. The cells that express ficolin B mRNA were identified as belonging to the myeloid cell lineage by magnetic sorting and by subsequent RT-PCR in bone marrow cells. Thus, the different spatial-temporal expression patterns of ficolins A and B suggest that these molecules play distinct roles in the prenatal and postnatal stages.
Assuntos
Lectinas/genética , Animais , Sequência de Bases , Medula Óssea/metabolismo , DNA Complementar/genética , Desenvolvimento Embrionário/genética , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Humanos , Hibridização In Situ , Fígado/embriologia , Fígado/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Gravidez , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Baço/metabolismo , Distribuição Tecidual , FicolinasRESUMO
The evaporation of formaldehyde from cadavers can produce high exposures among students and instructors. A possible causal role for formaldehyde has been considered likely for tumor of the nasopharynx and the nasal cavities in human beings. Due to this reason, Japan Ministry of Education, Culture, Sports, Science and Technology (MEXT) has set a guideline, which includes--decrease in gaseous formaldehyde in gross anatomy dissection laboratories and a guide to medical students about the toxicity of formaldehyde and protective method to avoid damages to skin, mucous, membrane, etc, in 2002. To understand what effective plans should be regarding the awareness of students about this notification, this study measured the gaseous formaldehyde concentrations in the anatomy dissection room and also analyzed the formaldehyde-related symptoms, and frequency of using protective measures. The study was conducted over a period of 3 months during the anatomy dissection exercise. We found that immediately after removing the cadavers' plastic covering, formaldehyde concentrations in the dissection room increased sharply. The concentration reached a peak point of 0.62 ppm after 10 minutes of starting of the class. This was much above the recommended level of 0.5 ppm set by Japan Society for Occupational Health. After 30 minutes of achieving the peak the formaldehyde level started decreasing gradually to a level of 0.11 ppm. Formaldehyde-related symptoms were observed in 59% of students. They had experienced symptoms of irritation of eyes, nose, throat, airways, skin, and headache during the course. Ocular discomfort was found significantly higher in the contact lenses users compared to the spectacle users or the normal eye sight group. Although, the guidelines about toxicity of formaldehyde and its protective measures to prevent damages to skin, mucous membrane etc. were informed to every student, only 52% of the students used both the mask containing activated carbon and the rubber gloves in every practical class without fail. Environmental Health Criteria 89 of International Program of Chemical Safety states, "It must be regarded that formaldehyde fluid is not absorbed directly into tissues through the skin". So the students may be allowed in some cases to touch the cadaver, treated by formaldehyde content fixative, by bare hands to understand the feel of certain organs and tissues. These results support that the rules of health supervision including necessity to use of protective measures, monitoring of indoor air formaldehyde etc. should be adhered by students and instructors in anatomy dissection room during the practical class.
Assuntos
Poluição do Ar em Ambientes Fechados/efeitos adversos , Poluição do Ar em Ambientes Fechados/análise , Anatomia , Formaldeído/efeitos adversos , Formaldeído/análise , Laboratórios , Equipamentos de Proteção/estatística & dados numéricos , Cadáver , Lentes de Contato , Feminino , Formaldeído/toxicidade , Humanos , Masculino , Concentração Máxima Permitida , Inquéritos e Questionários , Fatores de TempoRESUMO
STUDY DESIGN: The distribution of the lumbar plexus was analyzed using cadavers. OBJECTIVE: To clarify the safety zone to prevent nerve injuries with respect to retroperitoneal endoscopic surgery. SUMMARY OF BACKGROUND DATA: Surgical approaches to the retroperitoneal space vary among surgeons. Recently, retroperitoneal endoscopic surgery has been applied to various spinal disorders. When the psoas major muscle is separated during retroperitoneal endoscopic surgery, there is a potential risk of injury to the lumbar plexus or nerve roots. However, there is sparse knowledge regarding the relationship between the greater psoas muscle and the lumbar plexus. METHODS: A total of 30 cadavers were analyzed. Six lumbar spines of the cadavers were cut in parallel with the lumbar disc space. Each axial section was photographed and captured into a computer. The distribution of the lumbar plexus was analyzed using computer images. The positions where the genitofemoral nerve emerged on the abdominal surface of the psoas major muscle were analyzed using 24 cadavers. RESULTS: L2/3 and above, all parts of the lumbar plexus, and nerve roots were located from the dorsal fourth of the vertebral body and dorsally. The genitofemoral nerve descends obliquely forward through the psoas major muscle, emerging on the abdominal surface between the cranial third of the L3 vertebra and the caudal third of the L4 vertebra. The safety zone of the psoas major muscle to prevent nerve injuries, excluding the genitofemoral nerve, is at L4/L5 and above. CONCLUSIONS: The safety zone, excluding the genitofemoral nerve, is at L4-L5 and above.
Assuntos
Laparoscopia/métodos , Plexo Lombossacral/anatomia & histologia , Cadáver , Nervo Femoral/anatomia & histologia , Genitália/inervação , Humanos , Músculos/anatomia & histologia , Espaço Retroperitoneal , Raízes Nervosas Espinhais/anatomia & histologiaRESUMO
Previous studies have demonstrated the expression of specific members of the glial cell line-derived neurotrophic factor (GDNF) receptor family alpha (GFRalpha) in subsets of motoneurons (MNs) in the developing mouse spinal cord. We examined the expression pattern of GFRalpha and RET in the avian lumbar spinal cord during the period of programmed cell death (PCD) of MNs by using double labeling in situ hybridization and immunohistochemistry. In the lateral motor column (LMC) of the lumbar spinal cord, a laminar organization of GFRalpha expression was observed: GFRalpha1-positive MNs were located in the medial LMC; GFRalpha1-, 2-, and 4-positive MNs were situated in the lateral LMC; and GFRalpha4-positive MNs were located in the intermediate LMC. The species of GFRalpha receptor that was expressed in MNs was found to be related to their birthdates. The expression of subpopulation-specific transcriptional factors was also used to define MNs that express a specific pattern of GFRalpha. This analysis suggests that motor pools as defined by these transcriptional factors have unique expression patterns of GFRalpha receptor. Early limb bud ablation did not affect the expression of GFRalpha in the spinal cord, indicating that regulation of receptor expression is independent of target-derived signals. Finally, GDNF mRNA expression was found in the limb during the PCD period of MNs. In conclusion, these results indicate that time of withdrawal from the mitotic cycle may specify the expression pattern of GFRalpha in subsets of MNs and that GDNF may function as a target-derived neurotrophic factor for specific subpopulations of MNs.
Assuntos
Apoptose , Proteínas Aviárias , Proteínas de Drosophila , Glicoproteínas de Membrana/metabolismo , Neurônios Motores/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Receptores Proteína Tirosina Quinases/metabolismo , Receptores de Fator de Crescimento Neural/metabolismo , Medula Espinal/embriologia , Medula Espinal/crescimento & desenvolvimento , Animais , Aves , Regulação da Expressão Gênica , Receptores de Fator Neurotrófico Derivado de Linhagem de Célula Glial , Imuno-Histoquímica , Hibridização In Situ , Região Lombossacral , Proteínas Proto-Oncogênicas c-ret , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Medula Espinal/metabolismoRESUMO
Motoneurons (MNs) in the cervical spinal cord of the chicken embryo undergo programmed cell death (PCD) between embryonic day (E) 4 and E5. The intracellular molecules regulating this early phase of PCD remain unknown. Here we show that introduction of Bcl-2 by a replication-competent avian retroviral vector prevented MN degeneration at E4.5, whereas the expression of the green fluorescent protein (GFP) was ineffective. Bcl-2 expression did not affect the number of Islet-1/2-positive MNs at the onset of cell death (E4). However, when examined at the end of the cell death period (E5.5), the number of Islet-1/2-positive MNs was clearly increased in Bcl-2-transfected embryos compared with control and GFP-transfected embryos. Activation of caspase-3, which is normally observed in this early MN death, was also prevented by Bcl-2. Thus, MNs in the cervical spinal cord appear to use intracellular pathway(s) for early PCD that is responsive to Bcl-2.
Assuntos
Apoptose/fisiologia , Genes bcl-2/fisiologia , Neurônios Motores/metabolismo , Animais , Apoptose/efeitos dos fármacos , Vértebras Cervicais/efeitos dos fármacos , Vértebras Cervicais/metabolismo , Embrião de Galinha , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Vetores Genéticos/administração & dosagem , Humanos , Neurônios Motores/efeitos dos fármacos , Medula Espinal/efeitos dos fármacos , Medula Espinal/metabolismoRESUMO
The short duration of expression of the transgenes is a major barrier to the clinical application of adenovirus-mediated gene therapy for hepatic enzyme deficiencies. Previous reports show that Fas-mediated apoptosis has a pivotal role in the rapid elimination of adenovirus-infected hepatocytes. After considering this result and our recent observation that murine hepatocytes can be protected from Fas-mediated apoptosis by expressing cytokine response modifier A (CrmA) in vivo, we hypothesized that CrmA coexpression could also prevent adenovirus-infected hepatocytes from rapid elimination and that this would make prolonged transgene expression achievable in vivo. To examine this, mice with congenital deficiency of lysosomal beta-glucuronidase (GUSB) were infected with recombinant adenoviruses expressing both CrmA and GUSB, and the duration of transgene expression was evaluated. The serum GUSB activity in the mice injected with a recombinant adenovirus expressing GUSB only became undetectable 60 days after the injection, whereas higher than normal GUSB activity was observed for at least 120 days in mice injected with adenoviruses expressing both GUSB and CrmA. Furthermore, we showed that exogenous CrmA expression could prevent the adenovirus-infected hepatocytes from cell death induced by cytotoxic T lymphocytes in vitro. These observations indicate that transgene expression after administration of E1-deleted adenovirus is prolonged by coexpression of the antiapoptotic protein CrmA.
Assuntos
Adenoviridae , Regulação da Expressão Gênica , Técnicas de Transferência de Genes , Vetores Genéticos , Fígado/fisiologia , Serpinas/genética , Transgenes/genética , Proteínas Virais , Animais , Camundongos , Serpinas/biossíntese , Transgenes/fisiologiaRESUMO
Rous sarcoma virus (RSV)-derived retroviral vector could efficiently deliver the green fluorescent protein (GFP), which is driven by the internal cytomegalovirus enhancer/promoter, into restricted cell populations in the chicken embryo. RSV-derived vectors coupled with the tet regulatory elements also revealed doxycycline-dependent inducible GFP expression in the chicken embryo in ovo.