RESUMO
We report a global adeno-associated virus (AAV)9-based gene therapy protocol to deliver therapeutic galactosylceramidase (GALC), a lysosomal enzyme that is deficient in Krabbe's disease. When globally administered via intrathecal, intracranial, and intravenous injections to newborn mice affected with GALC deficiency (twitcher mice), this approach largely surpassed prior published benchmarks of survival and metabolic correction, showing long-term protection of demyelination, neuroinflammation, and motor function. Bone marrow transplantation, performed in this protocol without immunosuppressive preconditioning, added minimal benefits to the AAV9 gene therapy. Contrasting with other proposed pre-clinical therapies, these results demonstrate that achieving nearly complete correction of GALC's metabolic deficiencies across the entire nervous system via gene therapy can have a significant improvement to behavioral deficits, pathophysiological changes, and survival. These results are an important consideration for determining the safest and most effective manner for adapting gene therapy to treat this leukodystrophy in the clinic.
Assuntos
Metabolismo dos Carboidratos , Galactosilceramidase/genética , Galactosilceramidase/metabolismo , Terapia Genética , Leucodistrofia de Células Globoides/genética , Leucodistrofia de Células Globoides/metabolismo , Fenótipo , Animais , Vias Autônomas/metabolismo , Vias Autônomas/patologia , Vias Autônomas/ultraestrutura , Axônios/metabolismo , Axônios/patologia , Axônios/ultraestrutura , Comportamento Animal , Encéfalo/metabolismo , Dependovirus/genética , Modelos Animais de Doenças , Feminino , Expressão Gênica , Vetores Genéticos/administração & dosagem , Vetores Genéticos/genética , Vetores Genéticos/farmacocinética , Leucodistrofia de Células Globoides/diagnóstico , Leucodistrofia de Células Globoides/terapia , Masculino , Camundongos , Bainha de Mielina/metabolismo , Bainha de Mielina/patologia , Bainha de Mielina/ultraestrutura , Distribuição Tecidual , Transdução Genética , Resultado do TratamentoRESUMO
INTRODUCTION: The metabolic syndrome is a cluster of cardiovascular risk factors that predispose toward the development of diseases such as diabetes. Erectile dysfunction (ED) is common in men with metabolic syndrome, but its etiology is poorly understood. Pro-erectile nitrergic nerves innervating penile erectile tissue are also susceptible to mechanical injury during pelvic surgical procedures, which can lead to sexual dysfunction. AIMS: The aims of this article are: (i) to examine erectile function in an experimental model of metabolic syndrome, the phosphoenolpyruvate carboxykinase (PEPCK)-overexpressing rat; and (ii) to study function and cavernous reinnervation after penile nerve crush injury, which permits regeneration, in transgenic rats. METHODS: We analyzed the density of noradrenergic and nitrergic nerves and performed organ bath pharmacology to assess neurogenic activity. MAIN OUTCOME MEASURES: By analyzing changes in neural structure, function, and pharmacologic responses of cavernous tissue after nerve crush injury, we were able to reveal neurologic deficits in rats with metabolic syndrome. RESULTS: Animals with features of metabolic syndrome did not develop notable changes in cavernous autonomic nerve density or nerve-evoked smooth muscle activity. However, regeneration of nitrergic nerves after crush injury in transgenic rats was impaired compared with injured controls. This was manifested as a deficit in axon regrowth and responses to axon activation. However, unlike injured controls, injured PEPCK-overexpressing rats did not develop a reduced maximal response to the nitric oxide (NO) donor, sodium nitroprusside. This suggests preserved NO responsiveness in tissues from rats with metabolic syndrome, despite impaired regeneration and return of function. CONCLUSIONS: This study revealed that rats with features of metabolic syndrome display impaired cavernous nerve regeneration after penile nerve injury, but the degree of functional impairment may be attenuated due to reduced plasticity of NO signaling. This reinnervation deficit may be of clinical relevance for understanding why ED persists in some (particularly aged) men after pelvic surgery.
Assuntos
Síndrome Metabólica/fisiopatologia , Pênis/inervação , Animais , Vias Autônomas/fisiopatologia , Vias Autônomas/ultraestrutura , Masculino , Músculo Liso/fisiologia , Regeneração Nervosa/fisiologia , Ereção Peniana/fisiologia , Fosfoenolpiruvato Carboxiquinase (ATP)/metabolismo , Ratos , Ratos Transgênicos/fisiologia , Ratos WistarRESUMO
Eighty-two mesenchymal tumors of the gastrointestinal tract were examined by electron microscopy for the purposes of subtyping for diagnostic precision and of understanding cellular differentiation. Tumors were subclassified into leiomyoma/leiomyosarcoma, tumors of the interstitial cell of Cajal (equivalent to traditionally defined GISTs [Miettinen et al. Hum Pathol. 1999; 30:1213-1220; Mod Pathol. 2000; 13:1134-1142]), gastrointestinal autonomic nerve tumors (GANTs), and fibroblastic and myofibroblastic tumors, using criteria from the literature. Leiomyoma/leiomyosarcoma were diagnosed by myofilaments, attachment plaques, plasmalemmal caveolae, and lamina; GIST by processes or cell bodies full of intermediate filaments, solitary focal densities amid intermediate filaments, attachment plaques with incomplete lamina, scarce myofilaments, and smooth endoplasmic reticulum; GANTs by neuroendocrine granules, cell bodies/processes full of intermediate filaments (more rarely microtubules), and smooth endoplasmic reticulum; fibroblastic/myofibroblastic tumors by abundant rough endoplasmic reticulum, myofilaments, and fibronexuses. Seventy-three tumors (89%) were successfully subclassified, as 5 leiomyoma/leiomyosarcoma (6%), 36 GISTs (44%), 22 GANTs (27%), 10 fibroblastic and myofibroblastic tumors (12%). Results indicated overlap between poorly differentiated leiomyosarcoma and GIST, and between GIST and GANT. GANT is emphasized as a neuronal tumor identifiable by electron microscopy, and thereby distinguishable from GIST.
Assuntos
Transformação Celular Neoplásica/ultraestrutura , Neoplasias Gastrointestinais/ultraestrutura , Mesoderma/ultraestrutura , Microscopia Eletrônica , Idoso , Idoso de 80 Anos ou mais , Vias Autônomas/ultraestrutura , Feminino , Fibroblastos/ultraestrutura , Neoplasias Gastrointestinais/classificação , Humanos , Leiomioma/ultraestrutura , Leiomiossarcoma/ultraestrutura , Masculino , Pessoa de Meia-Idade , Plexo Mientérico/ultraestrutura , Sistemas Neurossecretores/ultraestruturaRESUMO
It is now well established that in addition to nerves containing classical transmitters, the mammalian vascular system is also supplied by nerve fibre subpopulations containing several vasoactive peptides. The precise function of these peptides (neuropeptide Y, calcitonin gene-related peptide, vasoactive intestinal polypeptide, somatostatin and the tachykinins) is still unknown, however, their widespread occurrence in perivascular nerves indicates that they are likely candidates for a role in the neurogenic regulation of the vascular system. It has been suggested that they may exert a direct vasomotor action via their own receptors and/or modulate the release and action of other vascular transmitters. Recently, several studies have focused on the supply of nerve fibres storing neuropeptides in the coronary and cerebral vasculature of laboratory animals, however, little is known on the distribution of these putative transmitters in human coronary and cerebral vessels. In this paper, the immunocytochemical evidence that several neuropeptides are localized in subpopulations of afferent and efferent nerve fibres supplying the human coronary and cerebral vasculature is focused.