Expression of hindlimb abnormalities under rearing temperature effects during the larval development of the salamander Pleurodeles waltl (urodele amphibian)

The Q-switched ruby laser (694 nm, 25-40 nsec) is an effective and safe therapeutic device for the treatment of tattoos and well-defined, benign, pigmented epidermal and dermal lesions. Because of its selective mode of action, dermal pigments of natural and artificial origin are destroyed photothermically and removed without scar. This method is exceptionally suited for the elimination of lay and professional tattoos, traumatic tattoos, and permanent makeup. Other frequent indications include benign pigmented lesions such as lentigines, freckles, café-au-lait spots, seborrheic keratosis, and Becker nevi. As a dermal pigmented lesion, the nevus of Ota is perfectly treatable. However, chloasma can no longer be considered an indication for ruby laser treatment due to unsatisfactory results. Melanocytic nevi and congenital nevi should be treated only in clinical studies. The effectiveness of the long-term epilation of dark hair with this laser device has to be verified in future investigations. Particularly attractive is the nonproblematic and straightforward removal of pigmented lesions in precarious anatomic regions like the lips, eyelids, and genitals (e.g., benign melanosis of the lips or of the penis, seborrheic keratosis of the lid angle).     

Transient neovascularisation of the frog retina during optic nerve regeneration

In conditions such as diabetic retinopathy, degenerative events in the retina are associated with neovascularisation. It is well established that a proportion of retinal ganglion cells die during optic nerve regeneration in the frog. The present study has determined whether neovascularisation takes place during this regenerative process. To do so, the pattern of blood vessels overlying the retinal ganglion cell layer was analysed in the frog Litoria (Hyla) moorei. We examined normal animals and those undergoing optic nerve regeneration following nerve crush. Blood vessels were visualised by perfusion with Indian ink and retinae were prepared as wholeamounts. In normal animals, the vascular tree was found to lie superficial to the nerve fibre layer and was more complex in regions overlying the area centralis and visual streak. After nerve crush, abnormal blood vessels transiently formed between the existing branches of the vascular tree. The new vessels were concentrated as an annulus centred on the optic nerve head and over the area centralis in midtemporal retina. The neovascularisation became most extensive between 6 and 10 weeks postcrush and disappeared by 12 weeks. The spatiotemporal sequence of neovascularisation suggests that it is triggered by accumulations of degenerating material formed as a proportion of the ganglion cells die during optic nerve regeneration.     

Neurotrophins and their receptors in the tench retina during optic nerve regeneration

To understand the role of neurotrophins in the visual system, we investigated the distribution of both neurotrophins and their receptors within the retina of a fish that has the capacity to spontaneously regenerate its optic nerve axons after lesion. Intact retinas and retinas from tench, whose optic nerve had been crushed, were analyzed by immunohistochemistry and in situ hybridization. Trk receptors were mainly immunolocalized in cells of the inner nuclear and ganglion cell layers, a distribution coincident with that of their mRNAs. Nerve growth factor (NGF) immunoreactivity was detected exclusively in Müller cell processes, and brain-derived neurotrophic factor (BDNF) was found in both neuronal bodies and Müller cell processes. Neurotrophin-3 (NT-3) was detected in most of the cell nuclei, and neurotrophin-4/5 (NT-4/5) was localized in fibers and in a few cells in the inner retina. An increase in both TrkA protein and mRNA was detected during axonal regeneration within the retinal ganglion cell layer, reaching a maximum 30 days postcrush and returning to normal levels by day 90, when optic nerve regeneration is almost completed in this fish. None of the other neurotrophins and receptors showed appreciable changes. The heterogeneous distribution patterns of neurotrophins and their receptors in fish retina, their differences from the distribution observed in other species, and the TrkA changes after optic nerve crush suggest an important role for these molecules in the normal physiology of the fish retina and during the regeneration process.     

Functional properties of retinal ganglion cells during optic nerve regeneration in the goldfish

Perfringolysin O (theta-toxin) is a cholesterol-binding and pore-forming toxin that shares with other thiol-activated cytolysins a highly conserved sequence, ECTGLAWEWWR (residues 430-440), near the C-terminus. To understand the membrane-insertion and pore-forming mechanisms of the toxin, we evaluated the contribution of each Trp to the toxin conformation during its interaction with liposomal membranes. Circular dichroism (CD) spectra of Trp mutant toxins indicated that only Trp436 has a significant effect on the secondary structure, and that Trp436, Trp438, and Trp439 make large contributions to near-UV CD spectra. Quenching the intrinsic Trp fluorescence of the wild-type and mutant toxins with brominated lecithin/cholesterol liposomes revealed that Trp438 and probably Trp436, but not Trp439, contributes to toxin insertion into the liposomal membrane. Near-UV CD spectra of the membrane-associated mutant toxins indicated that both Trp438 and Trp439 are required for the CD peak shift from 292 to 300 nm, a signal related to theta-toxin oligomerization and/or pore formation, suggesting a conformational change around Trp438 and Trp439 in these processes.     

Epaxial and limb muscle activity during swimming and terrestrial stepping in the adult newt, Pleurodeles waltl

We have investigated the patterns of activation of epaxial musculature during both swimming and overground stepping in an adult newt (Pleurodeles waltl) with the use of electromyographic (EMG) recordings from different sites of the myomeric muscle dorsalis trunci along the body axis. The locomotor patterns of some limb muscles have also been investigated. During swimming, the epaxial myomeres are rhythmically active, with a strict alternation between opposite myomeres located at the same longitudinal site. The pattern of intersegmental coordination consists of three successively initiated waves of EMG activity passing posteriorly along the anterior trunk, the midtrunk, and the posterior trunk, respectively. Swimming is also characterized by a tonic activation of forelimb (dorsalis scapulae and extensor ulnae) and hindlimb (puboischiotibialis and puboischiofemoralis internus) muscles and a rhythmic activation of muscles (latissimus dorsi and caudofemoralis) acting both on limb and body axis. The latter matched the activation pattern of epaxial myomeres at the similar vertebral level. During overground stepping, the midtrunk myomeres express single synchronous bursts whereas the myomeres of the anterior trunk and those of the posterior trunk display a double bursting pattern in the form of two waves of EMG activity propagating in opposite directions. During overground stepping, the limb muscles and muscles acting on both limb and body axis were found to be rhythmically active and usually displayed a double bursting pattern. The main conclusion of this investigation is that the patterns of intersegmental coordination during both swimming and overground stepping in the adult newt are related to the presence of limbs and that they can be considered as hybrid lampreylike patterns. Thus it is hypothesized that, in newt, a chain of coupled segmental oscillatory networks, similar to that which constitutes the central pattern generator (CPG) for swimming in the lamprey, can account for both trunk motor patterns if it is influenced by limb CPGs in a way depending on the locomotor mode. During swimming, the segmental networks located close to the girdles receive extra tonic excitation coming from the limb CPGs, whereas during stepping, the axial CPGs are entrained to some extent by the limb oscillators.     

Efferent optic nerve fibers mediate circadian rhythms in the Limulus eye

When the horseshoe crab is kept in constant darkness, the lateral eye produces larger electroretinographic and optic nerve responses at night than during the day. These circadian rhythms are mediated by synchronous bursts of efferent impulses in the optic nerve trunk. The endogenous efferent activity appears to increase both the gain and the quantum catch of the photoreceptors.     

Progressive growth in melanocytoma of the optic nerve head

The authors describe the method of closure of laparotomic wounds by means of an atraumatic continuous PDS loop suture. Based on evaluation of a group of 166 patients they appreciate the positive aspects of the technique of this suture, in particular the speed, easy implementation and safe closure of laparotomy. As regard the sewing material, they value highly the quality of atraumatic fibres which are firm and do not irritate the tissues.     

Course of the optic nerve fibers through the lamina cibrosa in human eyes

BACKGROUND: This study investigated whether regional variations in the course of the optic nerve fibers through the lamina cribrosa may be one of the reasons why the local susceptibility for glaucomatous optic nerve fiber loss differs among the various regions of the optic disc. METHODS: The study included 34 human eyes enucleated because of a malignant melanoma of the peripheral choroid without involvement of the anterior chamber angle or the optic nerve. Anterior-posterior sections through the pupil and the optic disc were histomorphometrically evaluated. In the central region and the peripheral part of the optic disc, we measured the thickness of the lamina cribrosa and the length of the lamina cribrosa "channels" through which the nerve fibers pass. RESULTS: In the peripheral parts of the disc, compared with its central region, the lamina cribrosa was significantly thicker (P < 0.0001, Wilcoxon test), the lamina cribrosa "channels" with the nerve fibers passing through were significantly longer (P < 0.0001), and the ratio of length of the fiber "channels" to the thickness of the lamina cibrosa was significantly higher (P = 0.0001). CONCLUSION: The lamina cribrosa is thicker and the course of the optic nerve fibers through the lamina cribrosa is more curvilinear in the disc pheriphery than in the disc center. These variations in the anatomy of the lamina cribrosa may be one of several factors influencing the regional susceptibility for glaucomatous optic nerve fiber loss within the optic nerve head.     

Expression of polysialylated NCAM but not L1 or N-cadherin by regenerating adult mouse optic fibers in vitro

Persistent/latent viral infections of insect cells are a prominent though poorly understood phenomenon. In this study, the long-term association between the Hz-1 virus and insect host cells, conventionally referred to as persistent viral infection, is described. With the aid of a newly developed fluorescent cell-labeling system, we found that productive viral replication occurs by spontaneous viral reactivation in fewer than 0.2% of persistently infected cell lines over a 5-day period. Once viral reactivation takes place, the host cell dies. The persistently infected cells contain various amounts of viral DNA, and, in an extreme case, up to 16% of the total DNA isolated from infected cells could be of viral origin. Both pulsed-field gel electrophoresis and in situ hybridization experiments showed that some of these viral DNA molecules are inserted into the host chromosomes but that the rest of viral DNA copies are free from host chromosomes. Thus, Hz-1 virus is the first nonretroviral insect virus known to insert its genome into the host chromosome during the infection process. These data also suggest that the previously described persistent infection of Hz-1 virus in insect cells should be more accurately referred to as latent viral infection.     

Amyloid precursor protein is synthesized by retinal ganglion cells, rapidly transported to the optic nerve plasma membrane and nerve terminals, and metabolized

We have investigated the synthesis, axonal transport, and processing of the beta-amyloid precursor protein (APP) in in vivo rabbit retinal ganglion cells. These CNS neurons connect the retina to the brain via axons that comprise the optic nerve. APP is synthesized in retinal ganglion cells and is rapidly transported into the optic nerve in small transport vesicles. It is then transferred to the axonal plasma membrane, as well as to the nerve terminals and metabolized with a t1/2 of less than 5 h. A significant accumulation of C-terminal amyloidogenic or nonamyloidogenic fragments is seen in the optic nerve 5 h after [35S]-methionine, [35S]cysteine injection, which disappears by 24 h. The major molecular mass species of APP in the optic nerve is approximately 110 kDa, and is an APP isoform that does not contain a Kunitz protease inhibitor domain. Higher molecular mass species containing this sequence are seen mostly in the retina. A protease(s) that can potentially cleave APP to generate an amyloidogenic fragment is present in the same optic nerve membrane compartment as APP.     

Sustained, synchronous oscillations in discharge of sustaining fibers of crayfish optic nerve

1. The regularity of the sustaining fiber (SF) steady-state discharge increases with the intensity of a uniform field of illumination. 2. In a high levels of illumination SFs exhibit a highly periodic or rhythmically bursting steady-state discharge. 3. The period of the burst cycle is independent of the light intensity and the mean firing rate. 4. Multimodal interspike-interval histograms in which successive modes decline exponentially suggest that the discharge arises from a regular input. 5. In conditions of uniform illumination, the regular input must be common to all or several SFs since the period of the burst cycle of simultaneously monitored SFs are the same and cross-correlations indicate that the SFs burst in approximate synchrony. 6. It is proposed that the common, regular input is a network of presynaptic elements exhibiting recurrent lateral inhibition. Evidence supporting this hypothesis is that following a brief light pulse to the inhibitory surround, SFs exhibit cycles of silence and bursting, phase-locked to the stimulus but with a period equal to the steady-state burst-cycle period. 7. Furthermore, lateral inhibitory influences are common to several or all SFs as indicated by the close similarity in response time course of two SFs subjected to a common inhibitory stimulus. 8. The response of the lateral pathways to sinusoidally modulated illumination is highly selective to frequencies near the burst-repetition rate. The excitatory pathways exhibit frequency modulation or entrainment over a wide range of input frequencies. 9. The intersustaining burst cycle phase is under partial visual stimulus control (position and intensity) and the possibility of phase coding in the SF ensemble is discussed.     

Platelet-derived growth factor B-chain expression in the rat retina and optic nerve: distribution and changes after transection of the optic nerve

To test the possible involvement of platelet-derived growth factor B-chain (PDGF-B) in anterograde and retrograde degenerations of the CNS neurons, we studied the changes of PDGF-B localization and its mRNA expression in the rat retina and optic nerve (ON) after unilateral ON transection, using immunohistochemistry and in situ hybridization. In the control retinas immunoreactivity for PDGF-B and its mRNA expression were localized in the retinal ganglion cells (RGCs) and the nerve fiber layer. After ON transection PDGF-B immunoreactivity in the nerve fiber layer started to decrease on post-injury day 3 or 4. Atrophic changes in the RGCs started on day 5 just after the decrease of PDGF expression, and thereafter the RGC number decreased. In the longitudinal section of the ON rostral to the transected site, swollen axons showed intense PDGF-B immunoreactivity and macrophages, and some glial cells revealed a significant increase in both immunoreactivity and hybridization signals. Based on these findings, we hypothesized that the decrease in PDGF-B in RGCs after axotomy causes the loss of RGCs, and that increased PDGF-B expression in the ON plays a role in the cascade of tissue reactions following ON transection.     

The refinement of retinotectal projection on tectal whole mount during the regeneration of the goldfish optic nerve labeled with DiI anterogradely

Radiographic computed tomography and magnetic resonance imaging are the procedures of choice in the diagnosis of patients with suspected pituitary gland abnormalities. Several cases are presented where the diagnostic value of such procedures in patients with diseases affecting the pituitary gland is demonstrated.     

Tenascin-R is antiadhesive for activated microglia that induce downregulation of the protein after peripheral nerve injury: a new role in neuronal protection

Microglial activation in response to pathological stimuli is characterized by increased migratory activity and potential cytotoxic action on injured neurons during later stages of neurodegeneration. The initial molecular changes in the CNS favoring neuronofugal migration of microglia remain, however, largely unknown. We report that the extracellular matrix protein tenascin-R (TN-R) present in the intact CNS is antiadhesive for activated microglia, and its downregulation after facial nerve axotomy may account for the loss of motoneuron protection and subsequent neurodegeneration. Studies on the protein expression in the facial and hypoglossal nucleus in rats demonstrate that TN-R is a constituent of the perineuronal net of motoneurons and 7 d after peripheral nerve injury becomes downregulated in the corresponding motor nucleus. This downregulation is reversible under regenerative (nerve suture) conditions and irreversible under degenerative (nerve resection) conditions. In short-term adhesion assays, the unlesioned side of brainstem cryosections from unilaterally operated animals is nonpermissive for activated microglia, and this nonpermissiveness is almost abolished by a monoclonal antibody to TN-R. Microglia-conditioned media and tumor necrosis factor-alpha downregulate TN-R protein and mRNA synthesis by cultured oligodendrocytes, which are one of the sources for TN-R in the brainstem. Our findings suggest a new role for TN-R in neuronal protection against activated microglia and the participation of the latter in perineuronal net destruction, e.g., downregulation of TN-R.     

Tenidap inhibits 5-lipoxygenase product formation in vitro, but this activity is not observed in three animal models

OBJECTIVE AND DESIGN: The effect of tenidap on the metabolism of arachidonic acid via the 5-lipoxygenase (5-LO) pathway was investigated in vitro and in vivo. MATERIALS AND TREATMENT: In vitro (cells). Arachidonic acid (AA) stimulated rat basophilic leukemia, (RBL) cells; A23817 activated neutrophils (human rat, and rabbit), macrophages (rat), and blood (human). In vitro (enzyme activity). RBL-cell homogenate; purified human recombinant 5-LO. In vivo: Rat (Sprague-Dawley) models in which peritoneal leukotriene products were measured after challenge with zymosan (3 animals per group), A23187 (11 animals per group), and immune complexes (3-5 animals per group), respectively. METHODS: 5-Hydroxyeicosatetraenoic acid (5-HETE) and dihydroxyeicosatetraenoic acids (diHETEs, including LTB4) were measured as radiolabeled products (derived from [14C]-AA) or by absorbance at 235 or 280 nm, respectively, after separation by HPLC. Radiolabeled 5-HPETE was measured by a radio-TLC analyser after separation by thin layer chromatography (TLC). Deacylation of membrane bound [14C]-AA was determined by measuring radiolabel released into the extracellular medium. 5-LO translocation from cytosol to membrane was assessed by western analysis. Rat peritoneal fluid was assayed for PGE, 6-keto-PGF1 alpha, LTE4 or LTB4 content by EIA and for TXB2 by RIA. RESULTS: Tenidap suppressed 5-LO mediated product production in cultured rat basophilic leukemia (RBL-1) cells from exogenously supplied AA, and in human and rat neutrophils, and rat peritoneal macrophages stimulated with A23187 (IC50, 5-15 microM). In addition, tenidap was less potent in inhibiting the release of radiolabeled AA from RBL-1 cells (IC50, 180 microM), suggesting that the decrease in 5-LO derived products could not be explained by an effect on cellular mobilization of AA (i.e., phospholipase). Tenidap blocked 5-hydroxyeicosatetraenoic acid (5-HETE) production by dissociated RBL-1 cell preparations (IC50, 7 microM), as well as by a 100000 x g supernatant of 5-LO/hydroperoxidase activity, suggesting a direct effect on the 5-LO enzyme itself. In addition, tenidap impaired 5-LO translocation from cytosol to its membrane-bound docking protein (FLAP) which occurs when human neutrophils are stimulated with calcium ionophore, indicating a second mechanism for inhibiting the 5-LO pathway. Surprisingly, tenidap did not block the binding of radiolabeled MK-0591, an indole ligand of FLAP, to neutrophil membranes. Although its ability to inhibit the cyclooxygenase pathway was readily observed in whole blood and in vivo, tenidap's 5-LO blockade could not be demonstrated by ionophore stimulated human blood, nor after oral dosing in rat models in which peritoneal leukotriene products were measured after challenge with three different stimuli. The presence of extracellular proteins greatly reduced the potency of tenidap as a 5-LO inhibitor in vitro, suggesting that protein binding is responsible for loss of activity in animal models. CONCLUSIONS: Tenidap inhibits 5-lipoxygenase activity in vitro both directly and indirectly by interfering with its translocation from cytosol to the membrane compartment in neutrophils. A potential mechanism for the latter effect is discussed with reference to tenidap's ability to lower intracellular pH. Tenidap did not inhibit 5-LO pathway activity in three animal models.     

Porcine interferon-gamma inhibits the growth of Legionella pneumophila in WiREF cells in vitro

In the present study eight human eyeballs were specifically prepared for scanning-electron-microscopic observation of the zonule. The zonule consisted of two main layers of radial fibres, an anterior and a posterior one, that inserted on the anterior and the posterior lens capsules, respectively. Some fibres inserted on the equator of the lens. Posterior zonular fibres originated at the pars plana, entered the dorsal part of the ciliary valleys and then changed their direction towards the posterior face of the lens. Posterior fibres inserted on the posterior capsule of the lens by branched endings 1 mm behind the equator of the lens. Anterior zonular fibres originated mainly at the pars plana and occasionally at the ciliary valleys. After running completely through the ciliary valleys in close contact with the lateral walls of the ciliary processes, they changed their direction at the anterior endings of the pars plicata and reached the anterior lens capsule. Anterior zonular insertions were achieved by webbed endings that diffused into the anterior capsule 2 mm in front of the lens equator. The extraordinary distension capacity of the zonular fibres was demonstrated by pulling the anterior lens capsule after hydrodissection. As a consequence, the anterior fibres were stretched up to four times their original length without breaking or disinserting.     

Glucosylceramide synthase inhibitor inhibits the action of nerve growth factor in PC12 cells

Previous studies have shown that the ceramide analogue, D-threo-1-phenyl-2-decanoylamin-3-morpholino-propanol (D-PDMP), inhibits glucosylceramide synthase and thus leads to extensive depletion of glycosphingolipids derived from glucosyl ceramide. Our previous studies have shown that cholera toxin B subunit, which specifically binds to the cell surface ganglioside GM1, and GM1 itself can enhance the action of nerve growth factor (NGF) in responsive cells by enhancing the NGF-induced autophosphorylation of the high affinity NGF receptor, Trk. Using D-PDMP, we examined the effects of the inhibition of the biosynthesis of glycosphingolipids on intracellular NGF signaling pathway. D-PDMP was found to inhibit NGF-induced neurite outgrowth of PC12 cells. Moreover, D-PDMP clearly inhibited NGF-induced autophosphorylation of Trk and prevented the activation of phosphatidylinositol 3-kinase and mitogen-activated protein kinase, downstream targets of Trk-initiated intracellular protein kinase cascades. These effects of D-PDMP were abolished by the addition of GM1 but not by the addition of other ganglioside subspecies to the culture medium. Furthermore, the effect of D-PDMP seemed to be specific for the Trk receptor, because intracellular signaling pathway of epidermal growth factor was not affected by D-PDMP. Dimethylsphingosine and the cell-permeable analogue, C2-ceramide, did not show such a strong inhibitory effect on neurite outgrowth or on the autophosphorylation of Trk. The present results and our previous observations clearly demonstrate that Trk requires endogenous gangliosides, especially GM1, for its normal function in mediating the neurotrophic activity of NGF at least in PC12 cells.     

A quantitative study of nerve fibers in the human facial nerve

The facial and intermediate nerves were quantitatively evaluated in seven patients who died from systemic malignancies not involving the facial nerve. In addition, five of the specimens were also qualitatively evaluated by measuring the total and axon diameters of the facial and intermediate nerve fibers. In two cases the facial nerve fibers were counted at five different levels. The total number of myelinated nerve fibers in the facial nerve varied from 7500 to 9370. The total number of myelinated nerve fibers in the intermediate nerve varied between 3120 and 5360. The peak diameter of the facial nerve axon was between 4 and 6 microns, and was between 2 and 3 microns in the intermediate nerve. When comparing nerve segments at different anatomical levels, the largest amount of nerve fibers was found at the level of the middle mastoid portion. However, this number did not reach the amount of nerve fibers counted in the internal acoustic meatus.     

Xefiltin, a Xenopus laevis neuronal intermediate filament protein, is expressed in actively growing optic axons during development and regeneration

Neurofilaments are an important structural component of the axonal cytoskeleton and are made of neuronal intermediate filament (nIF) proteins. During axonal development, neurofilaments undergo progressive changes in molecular composition. In mammals, for example, highly phosphorylated forms of the middle- and high-molecular-weight neurofilament proteins (NF-M and NF-H, respectively) are characteristic of mature axons, whereas nIF proteins such as alpha-internexin are typical of young axons. Such changes have been proposed to help growing axons accommodate varying demands for plasticity and stability by modulating the structure of the axonal cytoskeleton. Xefiltin is a recently discovered nIF protein of the frog Xenopus laevis, whose nervous system has a large capacity for regeneration and plasticity. By amino acid identity, xefiltin is closely related to two other nIF proteins, alpha-internexin and gefiltin. alpha-Internexin is found principally in embryonic axons of the mammalian brain, and gefiltin is expressed primarily in goldfish retinal ganglion cells and has been associated with the ability of the goldfish optic nerve to regenerate. Like gefiltin in goldfish, xefiltin in Xenopus is the most abundantly expressed nIF protein of mature retinal ganglion cells. In the present study, we used immunocytochemistry to study the distribution of xefiltin during optic nerve development and regeneration. During development, xefiltin was found in optic axons at stage 35/36, before they reach the tectum at stage 37/38. Similarly, after an orbital crush injury, xefiltin first reemerged in optic axons after the front of regeneration reached the optic chiasm, but before it reached the tectum. Thus, during both development and regeneration, xefiltin was present within actively growing optic axons. In addition, aberrantly projecting retinoretinal axons expressed less xefiltin than those entering the optic tract, suggesting that xefiltin expression is influenced by interactions between regenerating axons and cells encountered along the visual pathway. These results support the idea that changes in xefiltin expression, along with those of other nIF proteins, modulate the structure and stability of actively growing optic axons and that this stability is under the control of the pathway which growing axons follow.