ABSTRACT
To regain sensorimotor functions after stroke, surviving neural circuits must reorganize and form new connections. Although the thalamus is critical for processing and relaying sensory information to the cortex, little is known about how stroke affects the structure and function of these connections, or whether a therapeutic approach targeting these circuits can improve recovery. Here we reveal with in vivo calcium imaging that stroke in somatosensory cortex dampens the excitability of surviving thalamocortical circuits. Given this deficit, we hypothesized that chronic transcranial window optogenetic stimulation of thalamocortical axons could facilitate recovery. Using two-photon imaging, we show that optogenetic stimulation promotes the formation of new and stable thalamocortical synaptic boutons, without impacting axon branch dynamics. Stimulation also enhances the recovery of somatosensory cortical circuit function and forepaw sensorimotor abilities. These results demonstrate that an optogenetic approach can rewire thalamocortical circuits and restore function in the damaged brain.
Subject(s)
Brain/physiopathology , Optogenetics/methods , Stroke/physiopathology , Stroke/therapy , Animals , Axons/pathology , Brain/blood supply , Brain/diagnostic imaging , Calcium/analysis , Calcium/metabolism , Cerebrovascular Circulation , Channelrhodopsins/genetics , Green Fluorescent Proteins/administration & dosage , Green Fluorescent Proteins/analysis , Green Fluorescent Proteins/genetics , Male , Mice, Inbred C57BL , Somatosensory Cortex/physiopathology , Thalamus/diagnostic imaging , Thalamus/physiopathologyABSTRACT
An efficient system for direct delivery of integral membrane proteins is successfully developed using a new biocompatible exosome-based platform. Fusogenic exosomes harboring viral fusogen, vascular stomatitis virus (VSV)-G protein, can fuse with and modify plasma membranes in a process called "membrane editing." This can facilitate the transfer of biologically active membrane proteins into the target cell membranes both in vitro and in vivo.
Subject(s)
Biomimetic Materials , Cell Membrane/metabolism , Exosomes , Membrane Proteins/administration & dosage , Viral Proteins , Animals , Biological Therapy/methods , Biomimetic Materials/metabolism , Cell Line , Endocytosis , Exosomes/metabolism , Green Fluorescent Proteins/administration & dosage , Green Fluorescent Proteins/metabolism , Humans , Hydrogen-Ion Concentration , Liposomes/metabolism , Membrane Proteins/metabolism , Mice , Muscle, Skeletal/cytology , Muscle, Skeletal/diagnostic imaging , Muscle, Skeletal/metabolism , Polyethylene Glycols/metabolism , Viral Proteins/metabolism , VirusesABSTRACT
Delivery and penetration of chemotherapeutic drugs into neoplasm through the tumor vasculature are essential mechanisms to enhance the efficiency of chemotherapy. "Vascular targeting" strategy focuses on promoting the infiltration of chemotherapeutic drugs into neoplastic tissues. In this study, we achieved a targeted therapy by coupling tumor necrosis factor α (TNFα) with TCP-1, a novel vascular-targeting peptide, in an orthotopic colorectal cancer model in mice. High dose of TCP-1-conjugated TNFα (TCP-1/TNFα: 5µg/mouse) displayed potent antitumor activity by inducing apoptosis and reducing microvessel number in tumors than unconjugated TNFα, with no evidence of increased toxicity. In the combined therapy, the antitumor action of 5-fluorouracil (5-FU) was potentiated when the mice were pretreated with a low dose of TNFα (1ng/mouse) and to a greater extent by the same concentration of TCP-1/TNFα. In this regard, TCP-1/TNFα combined with 5-FU synergistically inhibited the tumor growth, induced apoptosis and reduced cell proliferation. More importantly, TCP-1/TNFα normalized the tumor vasculature and facilitated the infiltration of immune cells to neoplasm as well as attenuated the immunosuppressing effects of TNFα in bone marrow and spleen. At the same time, TCP-1/TNFα significantly improved 5-FU absorption into the tumor mass. Taken together, these findings underscore the therapeutic potential of TCP-1 as a drug carrier in cancer therapy. TCP-1 is a novel vascular-targeting peptide and appears to be a promising agent for drug delivery. TCP-1 fused with TNFα holds great promise for colorectal cancer therapy.
Subject(s)
Antimetabolites, Antineoplastic/administration & dosage , Cell-Penetrating Peptides/administration & dosage , Colorectal Neoplasms/drug therapy , Fluorouracil/administration & dosage , Neovascularization, Pathologic/drug therapy , Tumor Necrosis Factor-alpha/administration & dosage , Animals , Antimetabolites, Antineoplastic/chemistry , Antimetabolites, Antineoplastic/therapeutic use , Bone Marrow/drug effects , Cell Line , Cell Line, Tumor , Cell-Penetrating Peptides/chemistry , Cell-Penetrating Peptides/therapeutic use , Colorectal Neoplasms/immunology , Colorectal Neoplasms/pathology , Fluorouracil/chemistry , Fluorouracil/therapeutic use , Green Fluorescent Proteins/administration & dosage , Green Fluorescent Proteins/chemistry , Humans , Male , Mice, Inbred BALB C , Neovascularization, Pathologic/immunology , Neovascularization, Pathologic/pathology , Spleen/drug effects , Tumor Burden/drug effects , Tumor Necrosis Factor-alpha/chemistry , Tumor Necrosis Factor-alpha/immunology , Tumor Necrosis Factor-alpha/therapeutic useABSTRACT
Genetic manipulation is widely used to research the central nervous system (CNS). The manipulation of molecular expression in a small number of neurons permits the detailed investigation of the role of specific molecules on the function and morphology of the neurons. Electroporation is a broadly used technique for gene transfer in the CNS. However, the targeting of gene transfer using electroporation in postnatal animals was restricted to the cortex, hippocampus, or the region facing the ventricle in previous reports. Electroporation targeting of deep brain structures, such as the thalamus, has been difficult. We introduce a novel electroporation technique that enables gene transfer to a physiologically identified deep brain region using a glass pipette. We recorded neural activity in young-adult mice to identify the location of the lateral geniculate nucleus (LGN) of the thalamus, using a glass pipette electrode containing the plasmid DNA encoding enhanced green fluorescent protein (EGFP). The location of the LGN was confirmed by monitoring visual responses, and the plasmid solution was pressure-injected into the recording site. Voltage pulses were delivered through the glass pipette electrode. Several EGFP-labeled somata and dendrites were observed in the LGN after a few weeks, and labeled axons were found in the visual cortex. The EGFP-expressing structures were observed in detail sufficient to reconstruct their morphology in three dimensions. We further confirmed the applicability of this technique in cats. This method should be useful for the transfer of various genes into cells in physiologically identified brain regions in rodents and gyrencephalic mammals.