A comparison of corneal cellular responses after 213-nm compared with 193-nm laser photorefractive keratectomy in rabbits.

PURPOSE: Corneal refractive surgery is typically performed using a 193-nm excimer laser. However, a recently developed 213-nm solid-state (5th harmonic) Nd:YAG laser presents some practical and user safety advantages, although the biological impact of using this wavelength remains poorly characterized. Here, we provide in vivo and in vitro comparisons of the corneal cellular outcomes after irradiation with 213 and 193 nm wavelengths. METHODS: New Zealand White rabbits underwent photorefractive keratectomy with -5 diopters and a 6.5-mm optical zone and studied at time points up to 1 year. The development of haze was examined ophthalmologically and by detecting myofibroblasts immunohistochemically. Cell death was quantified using a terminal deoxynucleotidyl transferase-dUTP nick end labeling (TUNEL) assay, and the number of stromal keratocytes undergoing apoptosis estimated histologically. Superoxide dismutase activity was estimated in vitro by enzyme-linked immunosorbent assay in irradiated rabbit corneal keratocytes. RESULTS: Our results demonstrate subtle differences in the cellular outcomes after irradiation with 213- and 193-nm lasers, despite similar degrees of corneal haze developing in both treatment groups. In vivo, the 213-nm laser results in more stable stromal cell numbers, implying a more predictable ablation outcome. In vitro, higher levels of superoxide dismutase in corneal keratocytes irradiated with 213 nm compared with 193 nm wavelengths suggest a better endogenous protection against free radicals induced by laser surgery. CONCLUSIONS: The more favorable cellular responses after irradiation with 213 nm compared with 193 nm wavelengths are consistent with good clinical outcomes previously reported. Ablation with a 213 nm wavelength may result in better wound healing, leading to a more reliable correction of refractive errors.

Graft outcomes influenced by co-expression of Pax7 in graft and host tissue.

Cell replacement therapies offer promise in the treatment of neurotrauma and neurodegenerative disorders and have concentrated on the use of primary fetal brain tissue. However, there is a growing promise of using neural stem cells, in which case other factors may be important in their successful engraftment. We therefore investigated whether the co-expression of the major developmental transcription factor (Pax7 in this study) of donor tissue to graft site influences transplant survival and differentiation in the rat midbrain. Neural progenitor cells were prepared from either the Pax7-expressing dorsal (DM) or non-Pax7-expressing ventral mesencephalon (VM) of embryonic EGFP(+/+) rats. Cells were dissociated and grafted into the adult rat superior colliculus (SC) lesioned with quinolinic acid 3 days previously, a time shown to be associated with the up-regulation of Pax7. Grafts were then examined 4 weeks later. Our results suggest the origin of the graft tissue did not alter graft survival in the SC; however, dorsal grafts appear to have a higher incidence of neuronal survival, whereas ventral grafts have a higher incidence of astrocytic survivors.

Cone visual pigments in two species of South American marsupials.

Marsupials are largely confined to Australasia and to Central and South America. The visual pigments that underlie the photosensitivity of the retina have been examined in a number of species from the former group where evidence for trichromatic colour vision has been found, but none from the latter. In this paper, we report the cone opsin sequences from two nocturnal South American marsupial species, the gray short-tailed opossum, Monodelphis domestica, and the big-eared opossum, Didelphis aurita. Both are members of the Order Didelphimorphia (American opossums). For both species, only two classes of cone opsin were found, an SWS1 and an LWS sequence, and in vitro expression showed that the peak sensitivity of the SWS1 pigment is in the UV. Analysis of the Monodelphis genome confirms the absence of other classes of cone visual pigment genes. The SWS1 and LWS genes with 4 and 5 introns respectively, show the same exon-intron structure as found for these genes in all other vertebrates. The SWS1 gene shows a conserved synteny with flanking genes. The LWS gene is X-linked, as in all therian mammals so far examined, with a locus control region 1.54 kb upstream.

Regional and cellular distribution of ephrin-B1 in adult mouse brain.

The membrane-bound proteins ephrins and their receptors, Eph receptor tyrosine kinases, are known for their key role during development of the central nervous system (CNS). Ligand/receptor interactions as a result of cell-cell contacts activate intracellular signalling pathways which mediate specific cellular responses. Activation can occur bidirectionally in both the receptor and the ligand-bearing cells. Eph receptor and ephrin families have been implicated in synaptic plasticity in the mature brain: effects include long-term potentiation/depression of excitatory transmission (LTP/LTD) and an action on the structure and number of synaptic contacts. However, due to the redundancy of binding between receptors and ligands, the role of individual proteins has not yet been completely elucidated. Ephrin-B1 has been suggested to play a role in synaptic plasticity in the hippocampus, but its expression and localization at pre- or post-synaptic sites has been poorly documented, most likely due to the apparent low activity of the corresponding gene in mature brain. Here we present immunohistochemical data demonstrating a broad but highly regulated cellular distribution of ephrin-B1 in the mature mouse brain. We show that ephrin-B1 is expressed post-synaptically on dendritic spines in the cortex, supporting a role in synaptic plasticity in this region. However, the prevalent extra-synaptic distribution in regions such as the hippocampus and cerebellum suggests an additional structural role, perhaps at the neuron/glia interface.

Neurite responses to ephrin-A5 modulated by BDNF: evidence for TrkB-EphA interactions.

In the developing visual system, growing retinal ganglion cell (RGC) axons are exposed to multiple guidance and growth factors. Furthermore, the relative levels of these factors are differentially regulated as topography is roughly established and then refined. We have shown that during the establishment of rough topography (P3), growth cones of pure and explanted RGCs treated with combinations of BDNF and ephrin-A5-Fc responded differently than RGCs treated with BDNF or ephrin-A5-Fc alone (p=0.0083). The response to the combined treatment mimicked that of RGCs cultured with ephrin-A5-Fc alone once topography refines. The guidance cue receptors EphA and TrkB were shown to co-localise in RGCs in vitro. Furthermore, EphA and TrkB receptors interacted directly in in vitro binding assays. Our results suggest that the conversion of growth cone responses from collapse to stabilisation as topography refines, occurs as a result of interactions between EphA and TrkB receptors.

The influence of ontogeny and light environment on the expression of visual pigment opsins in the retina of the black bream, Acanthopagrus butcheri.

The correlation between ontogenetic changes in the spectral absorption characteristics of retinal photoreceptors and expression of visual pigment opsins was investigated in the black bream, Acanthopagrus butcheri. To establish whether the spectral qualities of environmental light affected the complement of visual pigments during ontogeny, comparisons were made between fishes reared in: (1) broad spectrum aquarium conditions; (2) short wavelength-reduced conditions similar to the natural environment; or (3) the natural environment (wild-caught). Microspectrophotometry was used to determine the wavelengths of spectral sensitivity of the photoreceptors at four developmental stages: larval, post-settlement, juvenile and adult. The molecular sequences of the rod (Rh1) and six cone (SWS1, SWS2A and B, Rh2Aalpha and beta, and LWS) opsins were obtained and their expression levels in larval and adult stages examined using quantitative RT-PCR. The changes in spectral sensitivity of the cones were related to the differing levels of opsin expression during ontogeny. During the larval stage the predominantly expressed opsin classes were SWS1, SWS2B and Rh2Aalpha, contrasting with SWS2A, Rh2Abeta and LWS in the adult. An increased proportion of long wavelength-sensitive double cones was found in fishes reared in the short wavelength-reduced conditions and in wild-caught animals, indicating that the expression of cone opsin genes is also regulated by environmental light.

Cone visual pigments in two marsupial species: the fat-tailed dunnart (Sminthopsis crassicaudata) and the honey possum (Tarsipes rostratus).

Uniquely for non-primate mammals, three classes of cone photoreceptors have been previously identified by microspectrophotometry in two marsupial species: the polyprotodont fat-tailed dunnart (Sminthopsis crassicaudata) and the diprotodont honey possum (Tarsipes rostratus). This report focuses on the genetic basis for these three pigments. Two cone pigments were amplified from retinal cDNA of both species and identified by phylogenetics as members of the short wavelength-sensitive 1 (SWS1) and long wavelength-sensitive (LWS) opsin classes. In vitro expression of the two sequences from the fat-tailed dunnart confirmed the peak absorbances at 363 nm in the UV for the SWS1 pigment and 533 nm for the LWS pigment. No additional expressed cone opsin sequences that could account for the middle wavelength cones could be amplified. However, amplification from the fat-tailed dunnart genomic DNA with RH1 (rod) opsin primer pairs identified two genes with identical coding regions but sequence differences in introns 2 and 3. Uniquely therefore for a mammal, the fat-tailed dunnart has two copies of an RH1 opsin gene. This raises the possibility that the middle wavelength cones express a rod rather than a cone pigment.

Redefining the role of metallothionein within the injured brain: extracellular metallothioneins play an important role in the astrocyte-neuron response to injury.

A number of intracellular proteins that are protective after brain injury are classically thought to exert their effect within the expressing cell. The astrocytic metallothioneins (MT) are one example and are thought to act via intracellular free radical scavenging and heavy metal regulation, and in particular zinc. Indeed, we have previously established that astrocytic MTs are required for successful brain healing. Here we provide evidence for a fundamentally different mode of action relying upon intercellular transfer from astrocytes to neurons, which in turn leads to uptake-dependent axonal regeneration. First, we show that MT can be detected within the extracellular fluid of the injured brain, and that cultured astrocytes are capable of actively secreting MT in a regulatable manner. Second, we identify a receptor, megalin, that mediates MT transport into neurons. Third, we directly demonstrate for the first time the transfer of MT from astrocytes to neurons over a specific time course in vitro. Finally, we show that MT is rapidly internalized via the cell bodies of retinal ganglion cells in vivo and is a powerful promoter of axonal regeneration through the inhibitory environment of the completely severed mature optic nerve. Our work suggests that the protective functions of MT in the central nervous system should be widened from a purely astrocytic focus to include extracellular and intra-neuronal roles. This unsuspected action of MT represents a novel paradigm of astrocyte-neuronal interaction after injury and may have implications for the development of MT-based therapeutic agents.

Exogenous metallothionein-IIA promotes accelerated healing after a burn wound.

Severe injury to the epidermal barrier often results in scarring and life-long functional deficits, the outcome worsening with a number of factors including time taken to heal. We have investigated the potential of exogenous metallothionein IIA (Zn(7)-MT-IIA), a naturally occurring small cysteine-rich protein, to accelerate healing of burn wounds in a mouse model. Endogenous MT-I/II expression increased in basal keratinocytes concurrent with reepithelialization after a burn injury, indicating a role for MT-I/II in wound healing. In vitro assays of a human keratinocyte cell line indicated that, compared with saline controls, exogenous Zn(7)-MT-IIA significantly increased cell viability by up to 30% (p<0.05), decreased apoptosis by 13% (p<0.05) and promoted keratinocyte migration by up to 14% (p<0.05), all properties that may be desirable to promote rapid wound repair. Further in vitro assays using immortalized and primary fibroblasts indicated that Zn7-MT-IIA did not affect fibroblast motility or contraction (p>0.05). Topical administration of exogenous Zn(7)-MT-IIA (2 microg/mL) in vivo, immediately postburn accelerated healing, promoted faster reepithelialization (3 days: phosphate-buffered saline (PBS), 8.9+/-0.3 mm diameter vs. MT-I/II, 7.1+/-0.7 mm; 7 days: PBS 5.8+/-0.98 mm vs. MT-I/II, 3.6+/-1.0 mm, p<0.05) and reduced epidermal thickness (MT-I/II: 45+/-4 microm vs. PBS: 101+/-19 microm, p<0.05) compared with controls. Our data suggest that exogenous Zn(7)-MT-IIA may prove a valuable therapeutic for patients with burns and other skin injuries.

Ephrin-B2 immunoreactivity distribution in adult mouse brain.

Ephrin ligands and their receptors Eph receptor tyrosine-kinases have received extensive attention for their multiple key roles during development, particularly in the central nervous system (CNS). For example, at early stages of brain and spinal cord development, membrane-bound ephrins provide signals that direct migrating cells and axons. However, much less is known about the role of ephrins and Eph receptors in the adult CNS. Here, we investigated the distribution of ephrin-B2 protein expression in the adult mouse brain to gain insight into its possible function(s). We show that ephrin-B2 is expressed in areas with high levels of synaptic plasticity, such as the cerebral cortex, hippocampus and cerebellum. However, at the cellular level, ephrin-B2 was localized to neuronal cell bodies rather than to the dendritic synaptic sites where mechanisms of long-term modifications of excitatory transmission are located. Our results suggest a role for ephrin-B2 in the membrane at the cell body, possibly in relation to axonal-somatic inhibitory synapses.

Spectral sensitivities of the seahorses Hippocampus subelongatus and Hippocampus barbouri and the pipefish Stigmatopora argus.

The Syngnathidae are specialized diurnal feeders that are known to possess a retinal fovea and use independent eye movements to locate, track, and strike individual planktonic prey items. In this study, we have investigated the spectral sensitivities of three syngnathid species: a pipefish and two seahorses. We used spectrophotometry to measure the spectral transmission properties of ocular lenses and microspectrophotometry to measure the spectral absorption characteristics of visual pigments in the retinal photoreceptors. The pipefish, Stigmatopora argus, together with the seahorse Hippocampus subelongatus, is found in "green-water" temperate coastal seagrass habitats, whereas the second seahorse, H. barbouri, originates from a "blue-water" tropical coral reef habitat. All species were found to possess short wavelength absorbing pigment(s) in their lenses, with the 50% cut-off point of S. argus and H. subelongatus at 429 and 425 nm respectively, whereas that of H. barbouri was located at 409 nm. Microspectrophotometry of the photoreceptors revealed that the rods of all three species contained visual pigment with the wavelength of maximum absorption (lambda(max)) at approximately 500 nm. The visual pigment complement of the cones varied between the species: all possessed single cones with a lambda(max) close to 460 nm but H. barbouri also possessed an additional class of single cone with lambda(max) at 430 nm. Three classes of visual pigment were found in the double cones, the lambda(max) being approximately 520, 537, and 560 nm in the two seahorses and 520, 537, and 580 nm in the pipefish. The spectral sensitivities of the syngnathids investigated here do not appear to conform to generally accepted trends for fishes inhabiting different spectral environments. The influence of the specialized feeding regime of the syngnathids is discussed in relation to our findings that ultra-violet sensitivity is apparently not necessary for zooplanktivory in certain habitats.

Metallothionein-IIA promotes neurite growth via the megalin receptor.

Metallothionein (MT)-I/II has been shown to be neuroprotective and neuroregenerative in a model of rat cortical brain injury. Here we examine expression patterns of MT-I/II and its putative receptor megalin in rat retina. At neonatal stages, MT-I/II was present in retinal ganglion cells (RGCs) but not glial or amacrine cells; megalin was present throughout the retina. Whilst MT-I/II was absent from adult RGC in normal animals and after optic nerve transection, the constitutive megalin expression in RGCs was lost following optic nerve transection. In vitro MT-IIA treatment stimulated neuritic growth: more RGCs grew neurites longer than 25 microm (P < 0.05) in dissociated retinal cultures and neurite extension increased in retinal explants (P < 0.05). MT-IIA treatment of mixed retinal cultures increased megalin expression in RGCs, and pre-treating cells with anti-megalin antibodies prevented MT-IIA-stimulated neurite extension. Our results indicate that MT-IIA stimulates neurite outgrowth in RGCs and may do so via the megalin receptor; we propose that neurite extension is triggered via signal transduction pathways activated by the NPxY motifs of megalin's cytoplasmic tail.

Erythropoietin is both neuroprotective and neuroregenerative following optic nerve transection.

The cytokine hormone erythropoietin (EPO) is neuroprotective in models of brain injury and disease, and protects retinal ganglion cells (RGC) from cell death after axotomy. Here, we assessed EPO's neuroprotective properties in vivo by examining RGC survival and axon regeneration at 4 weeks following intraorbital optic nerve transection in adult rat. EPO was administered as a single intravitreal injection at the time of transection (5, 10, 25, 50 units, PBS control). Intravitreal EPO (5, 10 units) significantly increased RGC somata and axon survival between the eye and transection site. Twenty five units did not improve survival of RGC somata but did increase axon survival between the eye and transection site. In addition, a small proportion of axons penetrated the transection site and regenerated up to 1 mm into the distal nerve. In a second series, intravitreal EPO (25 units) doubled the number of RGC axons regenerating along a length of peripheral nerve grafted onto the retrobulbar optic nerve. Our in vivo evidence of both neuroregeneration and neuroprotection, taken together with the natural occurrence of EPO within the body and its ability to cross the blood-brain barrier, suggests that it offers promise as a therapeutic agent for central nerve repair.

Pax7 expression in the adult rat superior colliculus following optic nerve injury.

The transcription factor Pax7 has been implicated in the normal development of the superior colliculus and continues to be expressed in the adult superior colliculus, where it is concentrated in the retino-recipient laminae. Here we assessed, immunohistochemically, Pax7 expression in the adult rat superior colliculus after unilateral intraorbital optic nerve transection. We show that after optic nerve transection, the number of Pax7-expressing cells increased to re-establish the developmental rostral-caudal gradient and that these Pax7-expressing superior colliculus cells were neurons. These findings may have implications for the design of therapeutic interventions to restore functional connections in the adult visual pathway.

Compensatory and transneuronal plasticity after early collicular ablation.

Plasticity within the visual system was assessed in the quokka wallaby following unilateral superior collicular (SC) ablation at postnatal days (P) 8-10, prior to the arrival of retinal ganglion cell (RGC) axons. At maturity (P100), projections were traced from the eye opposite the ablation, and total RGC numbers were estimated for both eyes. Ablations were partial (28-89% of SC remaining) or complete (0-5% of SC remaining). Projections to the visual centers showed significant bilateral (P < 0.05) increases in absolute volume. Minor anomalous projections also formed within the deep, surviving non-retino-recipient layers of the ablated SC and via a small bundle of RGC axons recrossing the midline to innervate discrete patches in the SC contralateral to the lesion. Total absolute volume of projections did not differ between partial and complete ablations; moreover, values did not differ from normal (P > 0.05). Compared with normal, total RGC numbers were significantly (P < 0.05) reduced in the eye opposite the ablation but increased (P < 0.05) in the other eye. Consequently, the sum of the two RGC populations did not differ from normal (P > 0.05). As in rodents, the visual system in quokka compensates following injury by maintaining a set volume of arborization but does so by forming only minor anomalous projections. Furthermore, increased RGC numbers in the eye ipsilateral to the lesion indicate that compensation occurs transneuronally, thus maintaining total numbers of projecting neurons. The implication is that the visual system acts in concert following unilateral injury to maintain set values for RGC terminal arbors as well as their cell bodies.

Early vascular and neuronal changes in a VEGF transgenic mouse model of retinal neovascularization.

PURPOSE: To investigate early retinal changes in a vascular endothelial growth factor (VEGF) transgenic mouse (tr029VEGF; rhodopsin promoter) with long-term damage that mimics nonproliferative diabetic retinopathy (NPDR) and mild proliferative diabetic retinopathy (PDR). METHODS: Rhodopsin and VEGF expression was assessed up to postnatal day (P)28. Vascular and retinal changes were charted at P7 and P28 using sections and wholemounts stained with hematoxylin and eosin or isolectin IB4 Griffonia simplicifolia Samples were examined using light, fluorescence, and confocal microscopy. RESULTS: Rhodopsin was detected at P5 and reached mature levels by P15; VEGF protein expression was transient, peaking at P10 to P15. In wild-type (wt) mice at P7, vessels had formed in the nerve fiber/retinal ganglion cell layer and showed a centroperipheral maturational gradient; some capillaries had formed a second bed on the vitread side of the inner nuclear layer (INL). By P28, the retinal vasculature had three mature capillary beds, the third abutting the sclerad aspect of the INL. In tr029VEGF mice, capillary bed formation was accelerated compared with that in wt, with abnormal vessels extending to the sclerad side of the INL by P7 and abnormally penetrating the photoreceptors by P28. Compared with P7, vascular lesions were more numerous at P28 when capillary dropout was also evident. At both stages, retinal layers were thinned most where abnormal vessel growth was greatest. CONCLUSIONS: Concomitant damage to the vasculature and neural retina at early stages in tr029VEGF suggest that both tissues are affected, providing opportunities to examine early cellular events that lead to long-term disease.

Spectral tuning of the long wavelength-sensitive cone pigment in four Australian marsupials.

The molecular basis for the spectral tuning of longwave-sensitive (LWS) visual pigments in mammals have been described in a wide range of placental species, including the primates. However, little is known about the molecular mechanisms in marsupial LWS pigments. Here, we have studied and compared the LWS opsins in four Australian marsupials: two diprotodonts and two polyprotodonts. Phylogenetic analysis establishes that all LWS marsupial sequences form a distinct clade from the placental mammals that is subdivided into diprotodont and polyprotodont groups. Amino acid sequences reveal that substitutions at sites 277 and 285 are largely responsible for the spectral shifts in marsupial LWS pigments and species comparison indicates that the ancestral gene most likely encoded Tyr277 and Ala180. Amino acid substitutions are discussed in the context of spectral shifts in marsupial LWS and in relation to the mechanisms in primate pigments.

Graded ephrin-A2 expression in the developing hamster superior colliculus.

During development, ephrin gradients guide retinal ganglion cell axons to their appropriate topographic locations in the superior colliculus (SC). Expression of ephrin-A2, assessed immunohistochemically in the developing hamster SC, revealed a rostral(low) to caudal (high) gradient that is most prominent at postnatal days P4 and P7 when topography is established. Double-labelling immunohistochemistry for ephrin-A2 and cell specific markers revealed that ephrin-A2 is expressed exclusively by a subset of neurons. The expression pattern has implications for mechanisms underlying establishment of topography during development and following injury.