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Colin R Green

Publications and source records attributed to Colin R Green.

13 recordsLinked to original sources
PubMed2006

Antisense delivery and protein knockdown within the intact central nervous system.

The ability to down regulate the expression of a specific protein within the intact central nervous system (CNS) is highly desirable from both a research and therapeutic perspective. Antisense has the potential to do this. However, problems of invasive antisense delivery methods and short half life of remain problematic. We overcome this by using Pluronic gel to provide a sustained delivery antisense oligodeoxynucleotides (ODN's) to the intact central nervous system and achieving rapid penetration throughout the spinal cord in 2-3 hours and significant knockdown of our target protein connexin 43 (Cx43) in 4-8 hours (recovering at 48-72 hours). Interestingly CY3-siRNA probes could not be detected penetrating the intact CNS and no knockdown the Cx43 was found. This approach with conventional ODNs could provide a faster and cheaper alternative to knockout mice in the investigation of the functions of specific proteins within the CNS and may also have therapeutic implications for drug discovery and development.

Animals↗
PubMed2006

Structural and functional coupling of cardiac myocytes and fibroblasts.

Cardiac myocytes and fibroblasts form extensive networks in the heart, with numerous anatomical contacts between cells. Fibroblasts, obligatory components of the extracellular matrix, represent the majority of cells in the normal heart, and their number increases with aging and during disease. The myocyte network, coupled by gap junctions, is generally believed to be electrically isolated from fibroblasts in vivo. In culture, however, the heterogeneous cell types form functional gap junctions, which can provide a substrate for electrical coupling of distant myocytes, interconnected by fibroblasts only. Whether similar behavior occurs in vivo has been the subject of considerable debate. Recent electrophysiological, immunohistochemical, and dye-coupling data confirmed the presence of direct electrical coupling between the two cell types in normal cardiac tissue (sinoatrial node), and it has been suggested that similar interactions may occur in post-infarct scar tissue. Such heterogeneous cell coupling could have major implications on in vivo electrical impulse conduction and the transport of small molecules or ions in both the normal and pathological myocardium. This review illustrates that it would be wrong to adhere to a scenario of functional integration of the heart that does not allow for a potential active contribution of non-myocytes to cardiac electrophysiology, and proposes to focus further research on the relevance of non-myocytes for cardiac structure and function.

Animals↗
PubMed2006

In vitro optimization of antisense oligodeoxynucleotide design: an example using the connexin gene family.

The completion of the human and mouse genomes has identified at least 20 connexin isomers in this family of intercellular channel proteins. However, there are no specific gap junction blockers or channel-blocking mimetic peptides available for the study of specific connexins. We designed antisense oligodeoxynucleotides that functionally reduce targeted connexin protein expression and can be used to reveal the biological function of individual connexins in vivo. Connexin mRNA was firstly exposed in vitro to deoxyribozymes complementing the sense coding sequence. Those that cleaved the target connexin mRNA in defined regions were used as the basis to design oligodeoxynucleotides to the accessible sites, thus taking into account tertiary mRNA configurations rather than relying on computed predictions. Antisense oligodeoxynucleotides designed to bind to accessible mRNA sites selectively reduced connexin26 and -43 mRNA expression in a corneal epithelium ex vivo model. Connexin43 protein levels were reduced correlating with the knockdown in mRNA and the protein's rapid turnover; protein levels of connexin26 did not alter, supporting lower turnover rates reported for that protein. We show, for the first time, an inexpensive and empirical approach to the preparation of specific and functional antisense oligodeoxynucleotides against known gene targets in the post-genomic era.

Animals↗
PubMed2005

Automated imaging of extended tissue volumes using confocal microscopy.

Confocal microscopy enables constitutive elements of cells and tissues to be viewed at high resolution and reconstructed in three dimensions, but is constrained by the limited extent of the volumes that can be imaged. We have developed an automated technique that enables serial confocal images to be acquired over large tissue areas and volumes. The computer-controlled system, which integrates a confocal microscope and an ultramill using a high-precision translation stage, inherently preserves specimen registration, and the user control interface enables flexible specification of imaging protocols over a wide range of scales and resolutions. With this system it is possible to reconstruct specified morphological features in three dimensions and locate them accurately throughout a tissue sample. We have successfully imaged various samples at 1-mum voxel resolution on volumes up to 4 mm3 and on areas up to 75 mm2. Used in conjunction with appropriate embedding media and immuno-histochemical probes, the techniques described in this paper make it possible to routinely map the distributions of key intracellular structures over much larger tissue domains than has been easily achievable in the past.

Animals↗
PubMed2004

Spatially and temporally distinct expression of fibroblast connexins after sheep ventricular infarction.

OBJECTIVES: Myocardial infarction leads to extensive changes in the organization of cardiac myocytes and fibroblasts, and changes in gap junction protein expression. In the immediate period following ischemia, reperfusion causes hypercontraction, spreading the necrotic lesion. Further progressive infarction continues over several weeks. In reperfusion injury, the nonspecific gap junction channel uncoupler heptanol limits necrosis. We hypothesize that gap junction coupling and fibroblast invasion provide a substrate for progressive infarction via a gap junction mediated bystander effect. METHODS: A sheep coronary occlusion infarct model was used with samples collected at 12, 24 and 48 h, and 6, 12 and 30 d (days) post-infarction. Immunohistochemical labelling of gap junction connexins Cx40, Cx43, and Cx45 was combined with cell-specific markers for fibroblasts (anti-vimentin) and myocytes (anti-myomesin). Double and triple immunolabelling and confocal microscopy were used to follow changes in cardiac myocyte morphology, fibroblast content and gap junction expression after myocardial infarction. Gap junction protein levels and fibroblast numbers were quantified. RESULTS: Within 12 h of ischemia, myocyte viability is impaired within small islands in the ischemic region. These islands spread and fuse into larger infarct zones until 12 d post-infarction. Thereafter, surviving myocytes within the infarct and in the border-zone appear to become stabilized. Distant from the infarct, continuing myocyte disruption is regularly observed, even after 30 d. Cx43 becomes redistributed from intercalated discs to the lateral surface of structurally compromised myocytes within 12 d. Cx45 expressing fibroblasts infiltrate the damaged region within 24 h, becoming most numerous at 6-12 d post-infarction, with peak Cx45 levels at 6 d. Later, Cx43 expressing fibroblasts are observed, and the related Cx43 label increases over the 30 d observation period, even though fibroblast numbers decline after 12 d. Cx40 was only seen in vascular endothelium. CONCLUSIONS: Progressive infarction, identified by myocyte sarcomere disruption and subsequent cell loss, occurs in parallel with fibroblast invasion and gap junction remodeling. Two fibroblast phenotypes occur within infarcts, expressing either Cx43 or Cx45. Coupled fibroblasts may play a number of roles in tissue remodeling following myocardial infarction, including provision of a possible substrate for progressive infarction via a gap junction mediated bystander effect.

Animals↗
PubMed2004

Fibroblast network in rabbit sinoatrial node: structural and functional identification of homogeneous and heterogeneous cell coupling.

Cardiomyocytes form a conducting network that is assumed to be electrically isolated from nonmyocytes in vivo. In cell culture, however, cardiac fibroblasts can contribute to the spread of excitation via functional gap junctions with cardiomyocytes. To assess the ability of fibroblasts to form gap junctions in vivo, we combine in situ detection of connexins in rabbit sinoatrial node (a tissue that is particularly rich in fibroblasts) with identification of myocytes and fibroblasts using immunohistochemical labeling and confocal microscopy. We distinguish two spatially distinct fibroblast populations expressing different connexins: fibroblasts surrounded by other fibroblasts preferentially express connexin40, whereas fibroblasts that are intermingled with myocytes largely express connexin45. Functionality of homogeneous and heterogeneous cell coupling was investigated by dye transfer in sinoatrial node tissue explants. These studies reveal spread of Lucifer yellow, predominantly along extended threads of interconnected fibroblasts (probably via connexin40), and occasionally between neighboring fibroblasts and myocytes (probably via connexin45). Our findings show that cardiac fibroblasts form a coupled network of cells, which may be functionally linked to myocytes in rabbit SAN.

Animals↗
PubMed2003

Targeting connexin43 expression accelerates the rate of wound repair.

The repair of tissue damage is a key survival process in all organisms and involves the coordinated activation of several cell types. Cell-cell communication is clearly fundamental to this process, and a great deal is known about extracellular communication within the wound site via cytokines. Here we show that direct cell-cell communication through connexin 43 (Cx43) gap junction channels also plays a major role in the wound healing process. In two different wound healing models, incisional and excisional skin lesions, we show that a single topical application of Cx43 antisense gel brings about a transient downregulation of Cx43 protein levels, and this results in a dramatic increase in the rate of wound closure. Cx43 knockdown reduces inflammation, seen both macroscopically, as a reduction in swelling, redness, and wound gape, and microscopically, as a significant decrease in neutrophil numbers in the tissue around the wound. One long-term consequence of the improved rate of healing is a significant reduction in the extent of granulation tissue deposition and the subsequent formation of a smaller, less distorted, scar. This approach is likely to have widespread therapeutic applications in other injured tissues and opens up new avenues of research into improving the wound healing process.

Animals↗
PubMed2003

Connexin expression patterns in the rat cornea: molecular evidence for communication compartments.

PURPOSE: To identify and localize candidate connexin family members in adult rat cornea that may be important in coordinating corneal cell biology. METHODS: To identify candidate connexin family members in adult rat cornea, a RT-PCR-based screening approach was initially adopted. Fourteen pairs of connexin isoform-specific primers were used to amplify connexin transcripts from two populations of RNA isolated from either the central cornea or the whole cornea. Immunohistochemistry and confocal microscopy were then used to confirm the presence and localization of connexins. RESULTS: Eight connexin transcripts (Cxs 26, 30.3, 31, 31.1, 33, 37, 43, 50) are present in central cornea, and the peripheral cornea additionally expresses Cxs 30, 40, 45, and 46. No Cx32 or Cx36 transcripts were amplified. Immunohistochemistry revealed that Cxs 26, 30, 31.1, 37, and 43 are expressed in spatially distinct patterns within the cornea. Cx26 and Cx43 occur in basal cells of the whole corneal epithelium and between endothelial cells. Cx26 also immunolocalizes to the first layer of intermediate epithelial cells, and Cx43 antibody labels stromal keratocytes. Cx30 is expressed in the peripheral corneal epithelium and disappears toward the central cornea. Cx31.1 expression is restricted to superficial corneal epithelial cells, and Cx37 spans the intermediate corneal epithelium. CONCLUSION: The spatially distinct cellular expression patterns of Cxs 26, 30, 31.1, 37, and 43 in the corneal epithelium imply that gap junctions play important roles in controlling corneal epithelial proliferation and differentiation and overall corneal maintenance.

Animals↗
PubMed2002

Upregulation in astrocytic connexin 43 gap junction levels may exacerbate generalized seizures in mesial temporal lobe epilepsy.

Following brain injury, and during the process of neurodegeneration, a reactive astrocytic proliferation occurs. This is accompanied by an increase in the synthesis of neuropeptides, cytokines, growth factors and glial fibrillary acidic protein (GFAP), a cell-specific marker for reactive astrocytes. Astrocytes are extensively coupled by gap junctions of the Cx43 connexin subtype. Several studies have shown that in severe trauma, coupling between astrocytes may add to the spread of the damaged area. In this study we ask whether the astrocytosis which is a feature of other neurodegenerative diseases also occurs in mesial temporal lobe epilepsy (MTLE) and whether it is accompanied by an increase in astrocytic communication through an upregulation of Cx43 gap junction channel proteins. In order to examine the astrocytic response and the expression pattern of Cx43 protein, double immunohistochemical labeling studies were undertaken using antibodies against GFAP and Cx43 applied to human hippocampal tissue resected from patients with MTLE, and to normal human control hippocampal tissue. Immunofluorescent labeling of astrocytes and Cx43 was examined using confocal laser scanning microscopy. The images obtained were quantitatively analysed and reconstructed using three-dimensional volume rendering. The results of this study have established that not only is astrocytosis greater in MTLE-affected tissues than previously suggested, but it is accompanied by a highly significant increase in astrocytic Cx43 protein levels. We hypothesize that this surprisingly large upregulation in Cx43 may exacerbate generalized seizures in the progression of MTLE.

Adult↗
PubMed2002

Molecular profiling and cellular localization of connexin isoforms in the rat ciliary epithelium.

The functionally distinct epithelial layers of the ciliary body act as a syncitium to produce the aqueous humour. Ultrastructural studies have shown that the pigmented (PE) and non-pigmented (NPE) cell layers of the ciliary epithelium are connected by gap junctions. However the molecular composition of gap junctions both between and within the two cell layers has not been comprehensively studied. To address this issue the authors have performed an extensive molecular screening of connexin (Cx) expression patterns in ciliary epithelium of the rat. Initially, mRNA was extracted from rat ciliary bodies, reverse-transcribed, and subjected to two rounds of PCR using primer sets designed against each of the 14 Cx isoforms known to be expressed in the rat. This initial screening protocol amplified eight candidate Cx isoforms (Cxs 26, 31, 33, 37, 40, 43, 45 and 46). The Cx isoforms identified in this initial screen were then first assigned to the ciliary epithelium itself (Cxs 26, 31, 40 and 43) or structures outside the epithelium (Cxs 37, 40, and 45) using immunohistochemistry performed on ciliary body whole mounts. No convincing evidence for either Cx 33 or 46 labelling was found in the ciliary body. Then the four Cx isoforms localized to the epithelium were further localized to specific membrane domains within the epithelial cell layers by performing high resolution imaging of the antibody labeling patterns obtained in cryosections. This enabled Cx26 and 31 to be specifically localized to spatially different gap junctions between NPE cells. Cx31 labeled gap junctions associated with an extensive network of membrane interdigitations found between NPE cells at their basal surfaces. In contrast Cx26 labeling in NPE cells was restricted to the basolateral membranes of adjacent NPE cells. Cx40 and Cx43 were both localized to the PE-NPE interface where they formed discrete homomeric/homotypic gap junction plaques. No convincing evidence was found for antibody labeling between PE cells. Thus it appears that intercellular communication, both within the NPE layer and between the PE and NPE cell layers, is mediated by gap junction channels that have distinctive permeability properties. In particular the results raise the possibility that the permeability of PE-NPE gap junctions can be modulated by changing the Cx43 : Cx40 expression ratio. Whether such a change in Cx expression ratios occurs and what effect it has on aqueous humour production and composition remains to be determined.

Animals↗
PubMed2002

Knockdown of connexin43-mediated regulation of the zone of polarizing activity in the developing chick limb leads to digit truncation.

In the developing chick wing, the use of antisense oligodeoxynucleotides to transiently knock down the expression of the gap junction protein, connexin43 (Cx43), results in limb patterning defects, including deletion of the anterior digits. To understand more about how such defects arise, the effects of transient Cx43 knockdown on the expression patterns of several genes known to play pivotal roles in limb formation were examined. Sonic hedgehog (Shh), which is normally expressed in the zone of polarizing activity (ZPA) and is required to maintain both the ZPA and the apical ectodermal ridge (AER), was found to be downregulated in treated limbs within 30 h. Bone morphogenetic protein-2 (Bmp-2), a gene downstream of Shh, was similarly downregulated. Fibroblast growth factor-8 expression, however, was unaltered 30 h after treatment but was greatly reduced at 48 h post-treatment, when the AER begins to regress. Expressions of Bmp-4 and Muscle segment homeobox-like gene (Msx-1) were not affected at any of the time points examined. Cx43 expression is therefore involved in some, but not all patterning cascades, and appears to play a role in the regulation of ZPA activity.

Animals↗
PubMed2002

In vivo and ex vivo in situ confocal analysis of a rat model demonstrating transient 'epithelialization of the endothelium'.

The purpose of this study was to identify the in vivo microstructural characteristics of an animal model of 'epithelialization of the endothelium' that are similar in appearance to the in vivo confocal microscopical appearance of corneal endothelium previously thought to be diagnostic of irido-corneal endothelial syndrome, and correlate these observations with ex vivo in situ confocal microscopical analysis. A rat model (n = 8 eyes)of transient 'epithelialization of the endothelium' resulting from superficial corneal trauma, was developed and analysed using in vivo confocal microscopy. One animal was killed at 48 hand the cornea was immuno-labelled and analysed, using ex vivo in situ confocal digital image reconstruction. Reversible 'epithelialization of the endothelium' was observed by in vivo confocal microscopy 48 h after superficial corneal trauma in all eight eyes. Ex vivo in situ analysis failed to demonstrate immunohistological characteristics of epithelialization. In vivo confocal microscopy is based on optical principles, and as a result various structural alterations may present with apparently identical characteristics that should be interpreted cautiously, on the basis of the presented clinicopathological observations.

Animals↗