Upregulation of epidermal gap junctional proteins in patients with venous disease.

BACKGROUND: Leg ulceration is a feared complication of venous insufficiency. It is not known whether varicose veins predispose skin to poor wound healing. The expression pattern of gap junctional protein connexin, a known marker of poor wound healing, was investigated across various stages of venous disease. METHODS: Patients undergoing intervention for varicose veins were assessed according to the Clinical Etiologic Anatomic Pathophysiologic (CEAP) classification of varicose veins. Paired 4-mm punch biopsies were taken from above the ankle (pathological) and above the knee (control). Tissues were stained with haematoxylin and eosin, and for connexin 43, connexin 30 and connexin 26. RESULTS: Forty-eight paired biopsies were taken (12 each for CEAP class C0, C2, C4 and C6). The pathological skin showed progressive epithelial hyperthickening, an increase in the number and depth of rete ridges, increased inflammation and loss of dermal architecture with disease progression from C4 onwards. The overall absolute connexin expression and mean connexin expression per cell in the pathological skin similarly increased across the CEAP classes from as early as C2. Increasing levels of connexin in control skin were also noted, indicating progression of the disease proximally. Connexin 43 expression showed the strongest positive correlation between pathological and control skin. CONCLUSION: Connexins were overexpressed in patients with simple varicose veins, with a stepwise increased expression through venous eczema to ulceration. Connexin 43 is a potential biomarker for venous disease. This finding suggests that varicose veins predispose skin to poor wound healing. Surgical relevance The overexpression of connexins, a family of gap junctional proteins, is known to cause poor healing in venous leg ulceration. It is not known whether there is any association with superficial venous disease. Here, connexin proteins were overexpressed in patients with uncomplicated varicose veins, before histological skin changes. Connexin could be a biomarker of venous disease progression.

Abnormal connexin expression in human chronic wounds.

BACKGROUND: Regulated alteration of connexin expression has been shown to be integral to acute wound repair. Downregulation of the gap-junction protein connexin 43 at the wound edge has been correlated with keratinocyte and fibroblast migration, while abnormal overexpression of connexin 43 significantly perturbs healing, as shown in the streptozotocin diabetic rodent impaired healing model. OBJECTIVES: To examine the protein expression levels of connexin 43, in addition to connexins 26 and 30, in a variety of human chronic wounds. METHODS: Wound-edge punch biopsies and a matched control from the arm were taken from a cohort of patients with venous leg, diabetic foot or pressure ulcers. Wound connexin expression in each patient was compared with that in a matched, nonwounded arm punch. Tissue was sectioned, stained and imaged by confocal microscopy using identical parameters per patient to permit quantification. RESULTS: Epidermal connexin 43, connexin 26 and connexin 30, and dermal connexin 43 were discovered to be strikingly upregulated in every ulcer from all three wound types, pointing to connexin upregulation as a common feature between chronic wounds. CONCLUSIONS: This result supports efforts to target connexin 43 to promote cell migration and wound healing in chronic ulcers.

Tensile strain increased COX-2 expression and PGE2 release leading to weakening of the human amniotic membrane.

INTRODUCTION: There is evidence that premature rupture of the fetal membrane at term/preterm is a result of stretch and tissue weakening due to enhanced prostaglandin E2 (PGE2) production. However, the effect of tensile strain on inflammatory mediators and the stretch sensitive protein connexin-43 (Cx43) has not been examined. We determined whether the inflammatory environment influenced tissue composition and response of the tissue to tensile strain. METHODS: Human amniotic membranes isolated from the cervix (CAM) or placenta regions (PAM) were examined by second harmonic generation to identify collagen orientation and subjected to tensile testing to failure. In separate experiments, specimens were subjected to cyclic tensile strain (2%, 1 Hz) for 24 h. Specimens were examined for Cx43 by immunofluorescence confocal microscopy and expression of COX-2 and Cx43 by RT-qPCR. PGE2, collagen, elastin and glycosaminoglycan (GAG) levels were analysed by biochemical assay. RESULTS: Values for tensile strength were significantly higher in PAM than CAM with mechanical parameters dependent on collagen orientation. Gene expression for Cx43 and COX-2 was enhanced by tensile strain leading to increased PGE2 release and GAG levels in PAM and CAM when compared to unstrained controls. In contrast, collagen and elastin content was reduced by tensile strain in PAM and CAM. DISCUSSION: Fibre orientation has a significant effect on amniotic strength. Tensile strain increased Cx43/COX-2 expression and PGE2 release resulting in tissue softening mediated by enhanced GAG levels and a reduction in collagen/elastin content. CONCLUSION: A combination of inflammatory and mechanical factors may disrupt amniotic membrane biomechanics and matrix composition.

Cost-effectiveness of denosumab in the treatment of postmenopausal osteoporosis in Canada.

OBJECTIVE: Denosumab is a novel biologic agent approved in Canada for treatment of post-menopausal osteoporosis (PMO) in women at high risk for fracture or who have failed or are intolerant to other osteoporosis therapies. This study estimated cost-effectiveness of denosumab vs usual care from the perspective of the Ontario public payer. METHODS: A previously published PMO Markov cohort model was adapted for Canada to estimate cost-effectiveness of denosumab. The primary analysis included women with demographic characteristics similar to those from the pivotal phase III denosumab PMO trial (FREEDOM; age 72 years, femoral neck BMD T-score -2.16 SD, vertebral fracture prevalence 23.6%). Three additional scenario sub-groups were examined including women: (1) at high fracture risk, defined in FREEDOM as having at least two of three risk factors (age 70+; T-score ≤ -3.0 SD at lumbar spine, total hip, or femoral neck; prevalent vertebral fracture); (2) age 75+; and (3) intolerant or contraindicated to oral bisphosphonates (BPs). Analyses were conducted over a lifetime horizon comparing denosumab to usual care ('no therapy', alendronate, risedronate, or raloxifene [sub-group 3 only]). The analysis considered treatment-specific persistence and post-discontinuation residual efficacy, as well as treatment-specific adverse events. Both deterministic and probabilistic sensitivity analyses were conducted. RESULTS: The multi-therapy comparisons resulted in incremental cost-effectiveness ratios for denosumab vs alendronate of $60,266 (2010 CDN$) (primary analysis) and $27,287 per quality-adjusted life year gained for scenario sub-group 1. Denosumab dominated all therapies in the remaining scenarios. LIMITATIONS: Key limitations include a lack of long-term, real-world, Canadian data on persistence with denosumab as well as an absence of head-to-head clinical data, leaving one to rely on meta-analyses based on trials comparing treatment to placebo. CONCLUSIONS: Denosumab may be cost-effective compared to oral PMO treatments for women at high risk of fractures and those who are intolerant and/or contraindicated to oral BPs.

The pathogenesis of tendon microdamage in athletes: the horse as a natural model for basic cellular research.

The equine superficial digital flexor tendon (SDFT) is a frequently injured structure that is functionally and clinically equivalent to the human Achilles tendon (AT). Both act as critical energy-storage systems during high-speed locomotion and can accumulate exercise- and age-related microdamage that predisposes to rupture during normal activity. Significant advances in understanding of the biology and pathology of exercise-induced tendon injury have occurred through comparative studies of equine digital tendons with varying functions and injury susceptibilities. Due to the limitations of in-vivo work, determination of the mechanisms by which tendon cells contribute to and/or actively participate in the pathogenesis of microdamage requires detailed cell culture modelling. The phenotypes induced must ultimately be mapped back to the tendon tissue environment. The biology of tendon cells and their matrix, and the pathological changes occurring in the context of early injury in both horses and people are reviewed, with a particular focus on the use of various tendon cell and tissue culture systems to model these events.

Optical delineation of human malignant melanoma using second harmonic imaging of collagen.

Skin cancer incidence has increased exponentially over the last three decades. In 2008 skin cancer caused 2280 deaths in the UK, with 2067 due to malignant melanoma. Early diagnosis can prevent mortality, however, conventional treatment requires multiple procedures and increasing treatment times. Second harmonic generation (SHG) imaging could offer diagnosis and demarcation of melanoma borders non-invasively at presentation thereby short-cutting the excision biopsy stage. To test the efficacy and accuracy of SHG imaging of collagen in skin and to delineate the borders of skin cancers, unstained human melanoma biopsy sections were imaged using SHG microscopy. Comparisons with sister sections, stained with H&E or Melan-A were made for correlation of invasion borders. Fresh ex vivo normal human and rat skin was imaged through its whole thickness using SHG to demonstrate this technique is transferable to in vivo tissues. SHG imaging demonstrated detailed collagen distribution in normal skin, with total absence of SHG signal (fibrillar collagen) within the melanoma-invaded tissue. The presence or absence of signal changes dramatically at the borders of the melanoma, accurately demarcating the edges that strongly correlated with H&E and Melan-A defined borders (p<0.002). SHG imaging of ex vivo human and rat skin demonstrated collagen architecture could be imaged through the full thickness of the skin. We propose that SHG imaging could be used for diagnosis and accurate demarcation of melanoma borders on presentation and therefore potentially reduce mortality rates.

Maturational alterations in gap junction expression and associated collagen synthesis in response to tendon function.

Energy-storing tendons including the equine superficial digital flexor tendon (SDFT) contribute to energetic efficiency of locomotion at high-speed gaits, but consequently operate close to their physiological strain limits. Significant evidence of exercise-induced microdamage has been found in the SDFT which appears not to exhibit functional adaptation; the degenerative changes have not been repaired by the tendon fibroblasts (tenocytes), and are proposed to accumulate and predispose the tendon to rupture during normal athletic activity. The anatomically opposing common digital extensor tendon (CDET) functions only to position the digit, experiencing significantly lower levels of strain and is rarely damaged by exercise. A number of studies have indicated that tenocytes in the adult SDFT are less active in collagen synthesis and turnover than those in the immature SDFT or the CDET. Gap junction intercellular communication (GJIC) is known to be necessary for strain-induced collagen synthesis by tenocytes. We postulate therefore that expression of GJ proteins connexin 43 and 32 (Cx43; Cx32), GJIC and associated collagen expression levels are high in the SDFT and CDET of immature horses, when the SDFT in particular grows significantly in cross-sectional area, but reduce significantly during maturation in the energy-storing tendon only. The hypothesis was tested using tissue from the SDFT and CDET of foetuses, foals, and young adult Thoroughbred horses. Cellularity and the total area of both Cx43 and Cx32 plaques/mm(2) of tissue reduced significantly with maturation in each tendon. However, the total Cx43 plaque area per tenocyte significantly increased in the adult CDET. Evidence of recent collagen synthesis in the form of levels of neutral salt-soluble collagen, and collagen type I mRNA was significantly less in the adult compared with the immature SDFT; procollagen type I amino-propeptide (PINP) and procollagen type III amino-propeptide (PIIINP) levels per mm(2) of tissue and PINP expression per tenocyte also decreased with maturation in the SDFT. In the CDET PINP and PIIINP expression per tenocyte increased in the adult, and exceeded those in the adult SDFT. The level of PINP per mm(2) was greater in the adult CDET than in the SDFT despite the higher cellularity of the latter tendon. In the adult SDFT, levels of PIIINP were greater than those of PINP, suggesting relatively greater synthesis of a weaker form of collagen previously associated with microdamage. Tenocytes in monolayers showed differences in Cx43 and Cx32 expression compared with those in tissue, however there were age- and tendon-specific phenotypic differences, with a longer time for 50% recovery of fluorescence after photobleaching in adult SDFT cells compared with those from the CDET and immature SDFT. As cellularity reduces following growth in the SDFT, a failure of the remaining tenocytes to show a compensatory increase in GJ expression and collagen synthesis may explain why cell populations are not able to respond to exercise and to repair microdamage in some adult athletes. Enhancing GJIC in mature energy-storing tendons could provide a strategy to increase the cellular synthetic and reparative capacity.

Antisense down regulation of connexin31.1 reduces apoptosis and increases thickness of human and animal corneal epithelia.

The roles of the gap junction protein connexin31.1 (Cx31.1) are poorly understood, especially as the protein appears to form non-functional channels. Cx31.1 specific antisense oligodeoxynucleotides (ODNs) were designed to evaluate its roles in a corneal epithelium model. Expression of Cx31.1 in corneal epithelium extends from the suprabasal layers of polyhedral wing cells through to the flat squamous cells of superficial layers which are shed into the tear film. Deoxyribozymes (Dzs) were tested for cleavage efficacy using in vitro transcribed Cx31.1 mRNA. Cleavage results showed a putative tertiary structure for Cx31.1 mRNA with one region appearing to have a higher potential for antisense targeting. Application of antisense ODNs designed to this region caused Cx31.1 knockdown in rat and human corneal organotypic culture models, leading to a reduction in apoptosis and a thickening of the corneal epithelium (p=0.0045). Cx31.1 appears to play a role in triggering cell death; knocking it down may provide a novel approach for tissue repair and engineering.

Limiting burn extension by transient inhibition of Connexin43 expression at the site of injury.

Extension of a burn wound over the first 24h following injury is recognised clinically, and leads to diagnostic and therapeutic dilemmas. In the central nervous system, a similar spread of damage, beyond the initial injury, can occur via the spread of death signals from injured cells to their healthy neighbours via Connexin43 (Cx43) gap junction channels. In the skin, Cx43 is expressed in the basal epidermis and in fibroblasts and dermal appendages. We have used Cx43 specific antisense oligodeoxynucleotide approach to transiently down-regulate Cx43 protein in the early stages of partial thickness cutaneous burn wound healing. Antisense ODNs reduce the spread of tissue damage and neutrophil infiltration around the wound following injury. Epithelial cell proliferation is increased and the rate of wound closure is accelerated, compared to controls. Resultant scarring is smaller with less granulation tissue and more dermal appendages than controls. These findings suggest that Cx43 antisense treatment speeds partial thickness burn wound healing and reduces scarring. We suggest that this approach may provide an effective adjunct to managing partial thickness burn wounds.

Caspase activity and expression of cell death genes during development of human preimplantation embryos.

It has been observed that apoptosis occurs in human blastocysts. In other types of cell, the characteristic morphological changes seen in apoptotic cells are executed by caspases, which are regulated by the BCL-2 family of proteins. This study investigated whether these components of the apoptotic cascade are present throughout human preimplantation development. Developing and arrested two pronucleate embryos at all stages were incubated with a fluorescently tagged caspase inhibitor that binds only to active caspases, fixed, counterstained with 4,6-diamidino-2-phenylindole (DAPI) to assess nuclear morphology and examined using confocal microscopy. Active caspases were detected only after compaction, at the morula and blastocyst stages, and were frequently associated with apoptotic nuclei. Occasional labelling was seen in arrested embryos. Expression of proapoptotic BAX and BAD and anti-apoptotic BCL-2 was examined in single embryos using RT-PCR and immunohistochemistry. BAX and BCL-2 mRNAs were expressed throughout development, whereas BAD mRNA was expressed mainly after compaction. Simultaneous expression of BAX and BCL-2 proteins within individual embryos was confirmed using immunohistochemistry. The onset of caspase activity and BAD expression after compaction correlates with the previously reported appearance of apoptotic nuclei. As in other types of cell, human embryos express common molecular components of the apoptotic cascade, although apoptosis appears to be suppressed before compaction and differentiation.

Connexin43 gap junction protein plays an essential role in morphogenesis of the embryonic chick face.

Normal outgrowth and fusion of facial primordia during vertebrate development require interaction of diverse tissues and co-ordination of many different signalling pathways. Gap junction channels, made up of subunits consisting of connexin proteins, facilitate communication between cells and are implicated in embryonic development. Here we describe the distribution of connexin43 and connexin32 gap junction proteins in the developing chick face. To test the function of connexin43 protein, we applied antisense oligodeoxynucleotides that specifically reduced levels of connexin43 protein in cells of early chick facial primordia. This resulted in stunting of primordia outgrowth and led to facial defects. Furthermore, cell proliferation in regions of facial primordia that normally express high levels of connexin43 protein was reduced and this was associated with lower levels of Msx-1 expression. Facial defects arise when retinoic acid is applied to the face of chick embryos at later stages. This treatment also resulted in significant reduction in connexin43 protein, while connexin32 protein expression was unaffected. Taken together, these results indicate that connexin43 plays an essential role during early morphogenesis and subsequent outgrowth of the developing chick face.

Regeneration of rabbit cornea following excimer laser photorefractive keratectomy: a study on gap junctions, epithelial junctions and epidermal growth factor receptor expression in correlation with cell proliferation.

Corneal wound repair was investigated in rabbits following excimer laser ablation of a 6 mm diameter and 90 microm deep disc. In the healing process particular attention was focused on the epithelium where gap junction expression and the rearrangement of desmosomes and hemidesmosomes were correlated with cell proliferation and epidermal growth factor receptor expression. Immunofluorescence-based confocal laser scanning microscopy, semithin resin section morphology and electron microscopy were utilized. In resting cornea two isotypes of gap junctions, confined to different regions in the same basal epithelial cells, were detected. Particulate connexin43 (alpha1) immunostaining was concentrated on the apical while the connexin26 type (beta2) in the baso-lateral cell membranes. This is the first report of connexin26 in the cornea. Connexin43 was found also in corneal keratocytes and endothelial cell. Since the two connexins do not form functioning heteromeric channels and have selective permeabilities they may serve alternative pathways for direct cell-cell communication in the basal cell layer. During regeneration both connexins were expressed throughout the corneal epithelium including the migrating cells. They also showed transient up-regulation 24 hr after wounding in the form of overlapping relocation to the upper cell layers. At this time, basal epithelial cells at the limbal region, adjacent to the wound and those migrating over the wounded area all expressed membrane bound epidermal growth factor receptor and they were highly proliferating. In conclusion, like in other stratified epithelia connexin26 is also expressed in the cornea. Transient up-regulation and relocation of connexins within the regenerating epithelium may reflect the involvement of direct cell-cell communication in corneal wound healing. Mitotic activity in the migrating corneal epithelial cells is also a novel finding which is probably the sign of the excessive demand for new epithelial cells in larger wounds not met alone by the proliferating limbal stock.

Oestriol and oestradiol increase cell to cell communication and connexin43 protein expression in human myometrium.

Oestradiol increases the protein expression of connexin43 (Cx43) gap junctions in myometrium but the effect of oestriol on gap junction expression has not been described previously. Oestriol is the most abundant free oestrogen in pregnant women and there is a marked surge in oestriol concentrations before term and idiopathic preterm labour. In order to determine whether oestriol may have a physiological action on the myometrium, cultured human myometrial cells obtained from non-pregnant hysterectomy specimens were exposed to 10 nmol/l oestradiol or oestriol. Intercellular communication between myometrial cells was investigated by microinjection of confluent cultured cells with the gap junction-permeant tracer Cascade Blue. There was a progressive increase in coupling after exposure to oestradiol or oestriol (P < 0.0005). An increase in Cx43 protein expression was demonstrated by immunocytochemistry after 1 h (P < 0.01) and 3 days (P < 0.01) exposure, and by Western blotting after 1 h (P < 0.01) and 3 days (P < 0.05) exposure, to both oestradiol and to oestriol. We conclude that oestriol increases gap junction communication in human myometrium by increasing gap junction expression. Elevated oestriol concentrations may thus play a role in the initiation of labour in women, by increasing cell-cell communication in the myometrium.

Molecular basis for the susceptibility of fibrin-bound thrombin to inactivation by heparin cofactor ii in the presence of dermatan sulfate but not heparin.

Although fibrin-bound thrombin is resistant to inactivation by heparin.antithrombin and heparin.heparin cofactor II complexes, indirect studies in plasma systems suggest that the dermatan sulfate.heparin cofactor II complex can inhibit fibrin-bound thrombin. Herein we demonstrate that fibrin monomer produces a 240-fold decrease in the heparin-catalyzed rate of thrombin inhibition by heparin cofactor II but reduces the dermatan sulfate-catalyzed rate only 3-fold. The protection of fibrin-bound thrombin from inhibition by heparin.heparin cofactor II reflects heparin-mediated bridging of thrombin to fibrin that results in the formation of a ternary heparin.thrombin.fibrin complex. This complex, formed as a result of three binary interactions (thrombin.fibrin, thrombin.heparin, and heparin.fibrin), limits accessibility of heparin-catalyzed inhibitors to thrombin and induces conformational changes at the active site of the enzyme. In contrast, dermatan sulfate binds to thrombin but does not bind to fibrin. Although a ternary dermatan sulfate. thrombin.fibrin complex forms, without dermatan sulfate-mediated bridging of thrombin to fibrin, only two binary interactions exist (thrombin.fibrin and thrombin. dermatan sulfate). Consequently, thrombin remains susceptible to inactivation by heparin cofactor II. This study explains why fibrin-bound thrombin is susceptible to inactivation by heparin cofactor II in the presence of dermatan sulfate but not heparin.

Stage-specific and differential expression of gap junctions in the mouse ovary: connexin-specific roles in follicular regulation.

Gap junction communication plays an essential role in follicle growth. Immunocytochemistry and confocal microscopy were used to examine the expression of gap junction connexins of the alpha and beta subfamilies in follicles from primordial to preovulatory stages in the ovaries of prepubertal and adult mice. Connexin-specific antibodies detected alpha(1), alpha(4), alpha(6), beta(1), beta(2) and beta(4) connexins within follicles. In adult ovaries connexin immunolabelling was stronger in larger (more mature) follicles than it was in smaller follicles, with comparatively reduced labelling detected in prepubertal ovaries. In healthy follicles, labelling for alpha subfamily connexins was detected between granulosa cells, whereas labelling for beta subfamily connexins was found in the theca. Labelling for beta subfamily connexins and alpha(4) connexin (preantral stage) was detected on the oocyte surface membrane. In atretic follicles, labelling for beta(4) connexin appeared between the granulosa cells. These results demonstrate that alpha and beta connexin subfamilies are segregated to separate cellular compartments in the mouse follicle. The results are discussed in the light of possible roles for differential gap junctional communication in the regulation of folliculogenesis, oocyte maturation and atresia.

Anti-apoptotic action of insulin-like growth factor-I during human preimplantation embryo development.

Insulin-like growth factor I (IGF-I) has been shown to increase the proportion of embryos forming blastocysts and the number of inner cell mass cells in human and other mammalian preimplantation embryos. Here we examined whether the increased cell number resulted from increased cell division or decreased cell death. Normally fertilized, Day 2 human embryos of good morphology were cultured to Day 6 in glucose-free Earle's balanced salt solution supplemented with 1 mM glutamine, with (n = 42) and without (n = 45) 1.7 nM IGF-I. Apoptotic cells in Day 6 blastocysts were identified using terminal deoxynucleotidyl dUTP terminal transferase (TUNEL) labeling to detect DNA fragmentation and 4'-6-diamidino-2-phenylindole (DAPI) counterstain to evaluate nuclear morphology. The number of nuclei and extent of DNA and nuclear fragmentation was assessed using laser scanning confocal microscopy. IGF-I significantly increased the proportion of embryos developing to the blastocyst stage from 49% (control) to 74% (+IGF-I) (P < 0.05). IGF-I also significantly decreased the mean proportion of apoptotic nuclei from 16.3 +/- 2.9% (-IGF-I) to 8.7 +/- 1.4% (+IGF-I) (P < 0.05). The total number of cells remained similar between both groups (61.7 +/- 4.6 with IGF-I; 54.5 +/- 5.1 without IGF-I). The increased number of blastocysts combined with reduced cell death suggests that IGF-I is rescuing embryos in vitro which would otherwise arrest and acting as a survival factor during preimplantation human development.

Increased connexin43 gap junction protein in hamster cardiomyocytes during cold acclimatization and hibernation.

OBJECTIVE: The physiology of hibernation is characterized by dramatic reductions of heart rate, respiration, metabolism, blood pressure and body temperature and by resistance to ventricular fibrillation. Gap junctions in the heart provide low resistance pathways, facilitating electrical and metabolic coupling between cardiac muscle cells for coordinated action of the heart and tissue homeostasis. The conductance of these junctions, and therefore their function, is likely to be affected by the physiological changes that take place during hibernation. Our objective was to quantitate gap junction protein levels in cold acclimatization, hibernation and arousal. METHODS: We have used specific antibodies to connexins 43 and 40, in combination with confocal microscopy, to quantitatively analyze the expression of connexin protein in hamster (Mesocricetus auratus) left ventricles in four animal groups: normal controls at euthermy, cold controls (cold-exposed animals that did not undergo hibernation), hibernating animals and animals aroused from hibernation for 2 h. RESULTS: Connexin40 immunostaining was not detected in ventricular cardiomyocytes in any animal group but connexin43 was found in all groups. Connexin43 expression was significantly enhanced in hibernation and cold control ventricular cardiomyocytes. Total plaque area, numerical density and plaque size were higher in the cold controls and hibernating hamsters compared to normal controls and animals aroused from hibernation. CONCLUSION: It is possible that the increased size and number of connexin43 gap junction plaques in the cold controls may represent a compensatory response in order to maintain sufficient gap junction communication during physiological conditions that would reduce conductance. These changes may represent a mechanism by which the hamster avoids ventricular fibrillation during hibernation and arousal.

Large retinal ganglion cells that form independent, regular mosaics in the bufonoid frogs Bufo marinus and Litoria moorei.

Population-based methods were used to study labeled retinal ganglion cells from the cane toad Bufo marinus and the treefrog Litoria moorei, two visually competent bufonoid neobatrachians with contrasting habitats. In both, cells with large somata and thick dendrites formed distinct types with independent mosaics. The alpha(a), alpha(ab), and alpha(c) mosaics of Bufo in all major respects resembled those of ranids, studied previously, and could be provisionally matched to the same functional classes. As in other frogs, some alpha(a) cells were displaced and many alpha-cells of all types were asymmetric, but within each type all variants belonged to one mosaic. Nearest-neighbor analyses and spatial correlograms confirmed that all three mosaics were regular and independent. In Litoria, monostratified alpha(a) cells were not found. Instead, two bistratified types were present, distinguished individually by soma size and dendritic caliber and collectively by membership of independent mosaics: the larger (approximately 0.8% of all ganglion cells) was termed alpha1(ab) and the smaller (approximately 2.2%) alpha2ab. An alpha(c) cell type was also present, although too inconstantly labeled for mosaic analysis. Nearest-neighbor analyses and spatial correlograms confirmed that the two alpha(ab) mosaics were regular and independent. Densities, proportions, soma sizes, and mosaic statistics are tabulated for each species. The emergence of a consensus pattern of alpha-cell types in fishes and frogs, from which this treefrog partly diverges, offers new possibilities for studying correlations between function, phylogeny, ecology, and neuronal form.

Connexin alpha1 and cell proliferation in the developing chick retina.

During the formation of the eye, high levels of connexin alpha1 (connexin 43) are expressed within the tissues of the cornea, lens, and neural retina. In order to determine whether connexin alpha1 plays a role in the regulation of cell proliferation we have used a novel antisense technique to reduce its expression early in development (embryonic days 2-4). Application of Pluronic gel, containing antisense oligodeoxynucleotides (ODNs) to connexin alpha1, to one eye of early chick embryos results in a rapid and significant reduction of alpha1 protein which lasts for 24-48 h. Embryos grown for 48 h, after ODN application to one eye, showed a marked reduction in the diameter of the treated, compared to that of the contralateral untreated, eye. Sections cut from the treated eyes showed that the retina was also reduced in size. TUNEL labeling and staining with propidium iodide showed that apoptosis within the retinae of both treated and untreated eyes was rare and thus that the reduction in the area of the retina brought about by antisense ODNs directed at connexin alpha1 was unlikely to be the result of increased cell death. However, the number of mitotic figures in the ventricular zone of the antisense-treated retinae revealed by propidium iodide staining was significantly reduced (P < 0.0001) to 53 +/- 3.5% (n = 5) of that in the contralateral untreated control eyes. Embryos in which one eye was sham operated, treated with pluronic gel, or treated with sense ODN showed no significant changes in eye size or in the number of mitotic figures within the neural retina. These results point to a role for connexin alpha1-mediated gap-junctional communication in controlling the early wave of neurogenesis in the chick retina.