Maria-Thereza R. Perez

Expression of brain-derived neurotrophic factor and of its functional receptor in neonatal and adult rat retina

The expression of mRNA coding for brain-derived neurotrophic factor (BDNF) and for its functional receptor, the full-length tyrosine kinase receptor trkB (trkB mRNA), was examined in early postnatal and adult rat retina by in situ hybridization using digoxygenin and radioactively-labeled oligonucleotide probes. BDNF and trkB mRNAs are expressed in the ganglion cell layer at postnatal-days (PN) 1, 4, 7, 14, 60, in proximal neuroblastic layer (PN 1, 4, 7), and proximal inner nuclear layer (PN 14, 60). Subpopulations of developing and mature retinal cells are thus capable of synthesizing BDNF.

CNS Progenitor Cells Promote a Permissive Environment for Neurite Outgrowth via a Matrix Metalloproteinase-2-Dependent Mechanism

Transplantation of progenitor cells to the CNS has shown promise in neuronal and glial replacement and as a means of rescuing host neurons from apoptosis. Here we examined the effect of progenitor grafts on neurite extension in the degenerating retina of rd1 (retinal degeneration 1) mice. Transplantation of retinal progenitor cells induced increased matrix metalloproteinase-2 (MMP2) secretion, partly from activated glial cells, which was then activated by neuronally expressed MMP14. Active MMP2 resulted in proteolysis of the neurite outgrowth inhibitors CD44 and neurocan in the degenerative retina, allowing significantly increased neurite outgrowth across the border between abutting nondystrophic and rd1 retinas. Progenitor-induced enhancement of outgrowth was abrogated by an MMP inhibitor or by coculture with retinal explants from MMP2−/− mice. This study provides the first identification of an MMP2-dependent mechanism by which exogenous progenitor cells alter the host environment to promote neural regeneration. This suggests a novel therapeutic role for progenitor cells in the treatment of CNS degenerative diseases.

Chondroitin Sulfate Proteoglycans and Microglia Prevent Migration and Integration of Grafted Müller Stem Cells into Degenerating Retina

At present, there are severe limitations to the successful migration and integration of stem cells transplanted into the degenerated retina to restore visual function. This study investigated the potential role of chondroitin sulfate proteoglycans (CSPGs) and microglia in the migration of human Müller glia with neural stem cell characteristics following subretinal injection into the Lister hooded (LH) and Royal College of Surgeons (RCS) rat retinae. Neonate LH rat retina showed minimal baseline microglial accumulation (CD68‐positive cells) that increased significantly 2 weeks after transplantation (p < .001), particularly in the ganglion cell layer (GCL) and inner plexiform layer. In contrast, nontransplanted 5‐week‐old RCS rat retina showed considerable baseline microglial accumulation in the outer nuclear layer (ONL) and photoreceptor outer segment debris zone (DZ) that further increased (p < .05) throughout the retina 2 weeks after transplantation. Marked deposition of the N‐terminal fragment of CSPGs, as well as neurocan and versican, was observed in the DZ of 5‐week‐old RCS rat retinae, which contrasted with the limited expression of these proteins in the GCL of the adult and neonate LH rat retinae. Staining for CSPGs and CD68 revealed colocalization of these two molecules in cells infiltrating the ONL and DZ of the degenerating RCS rat retina. Enhanced immune suppression with oral prednisolone and intraperitoneal injections of indomethacin caused a reduction in the number of microglia but did not facilitate Müller stem cell migration. However, injection of cells with chondroitinase ABC combined with enhanced immune suppression caused a dramatic increase in the migration of Müller stem cells into all the retinal cell layers. These observations suggest that both microglia and CSPGs constitute a barrier for stem cell migration following transplantation into experimental models of retinal degeneration and that control of matrix deposition and the innate microglial response to neural retina degeneration may need to be addressed when translating cell‐based therapies to treat human retinal disease.

Localisation of neuronal nitric oxide synthase-immunoreactivity in rat and rabbit retinas

The distribution of neuronal nitric oxide synthase (NOS) immunoreactivity was examined in rat and rabbit retinas and was compared with the distribution of nicotinamide adenine dinucleotide phosphate (NADPH)-diaphorase reactivity and vasoactive intestinal peptide (VIP) immunoreactivity. An antibody raised against a C-terminal fragment of a cloned rat cerebellar NOS was used to localise NOS immunoreactivity. NOS immunoreactive cells were not detected in rat retinas at postnatal day 1 or 4, but were seen from postnatal day 7 onwards. NOS immunolabelling was seen in a small population of cells in the proximal inner nuclear layer. Most of the labelled cells had the position of amacrine cells and were seen to send processes into the inner plexiform layer. A few labelled cells were at times also seen in the ganglion cell layer, which are likely to correspond to displaced amacrine cells. The same NOS-labelling pattern was seen in rat and rabbit retinas.NADPH-diaphorase staining was observed in both species, in photoreceptor inner segments, in cells with the position of horizontal cells, in a subset of amacrine and displaced amacrine cells, in large cell bodies in the ganglion cell layer, in both plexiform layers, and in endothelium. Colocalisation of NOS immunoreactivity and NADPH-diaphorase staining was only observed among amacrine cells. However, not all NADPH-diaphorase-reactive amacrine cells were found to be NOS immunoreactive. VIP immunoreactivity was also localised in rat retinas in a subpopulation of amacrine cells, but no colocalisation of NOS and VIP immunoreactivity was observed.Our observations indicate that only amacrine cells contain the NOS form recognisable by the antibody used, and suggest that different isoforms of neuronal NOS may be present in retinal cells. Further, the onset of NOS expression in rat amacrine cells appears to occur independently of neuronal activity.

CNTF + BDNF treatment and neuroprotective pathways in the rd1 mouse retina

The rd1 mouse is a relevant model for studying the mechanisms of photoreceptor degeneration in retinitis pigmentosa. Treatment with ciliary neurotrophic factor (CNTF) in combination with brain derived neurotrophic factor (BDNF) is known to rescue photoreceptors in cultured rd1 retinal explants. To shed light on the underlying mechanisms, we studied the effects of 9 days (starting at postnatal day 2) in vitro CNTF+BDNF treatment on the endogenous production of CNTF, BDNF, fibroblast growth factor 2 (FGF2), or the activation of extracellular signal-regulated kinase (ERK), Akt and cAMP-response-element-binding protein (CREB) in retinal explants. In rd1 explants, CNTF+BDNF decreased the number of TUNEL-positive photoreceptors. The treatment also increased endogenous rd1 levels of CNTF and BDNF, but lowered the level of FGF2 expression in rd1 explants. When wild-type explants were treated, endogenous CNTF was similarly increased, while BDNF and FGF2 levels remained unaffected. In addition, treatment of rd1 retinas strongly increased the phosphorylation of ERK, Akt and CREB. In treated wild-type explants, the same parameters were either unchanged (ERK) or decreased (Akt and CREB). The results suggest a role for Akt, ERK and CREB in conveying the neuroprotective effect of CNTF+BDNF treatment in rd1 retinal explants.

Adenosine in vertebrate retina: Localization, receptor characterization, and function

Summary1.The uptake of [3H] adenosine into specific populations of cells in the inner retina has been demonstrated. In mammalian retina, the exogenous adenosine that is transported into cells is phosphorylated, thereby maintaining a gradient for transport of the purine into the cell.2.Endogenous stores of adenosine have been demonstrated by localization of cells that are labeled for adenosine-like immunoreactivity. In the rabbit retina, certain of these cells, the displaced cholinergic, GABAergic amacrine cells, are also labeled for adenosine.3.Purines are tonically released from dark-adapted rabbit retinas and cultured embryonic chick retinal neurons. Release is significantly increased with K+ and neurotransmitters. The evoked release consists of adenosine, ATP, and purine metabolites, and while a portion of this release is Ca2+ dependent, one other component may occur via the bidirectional purine nucleoside transporter.4.Differential distributions of certain enzymes involved in purine metabolism have also been localized to the inner retina.5.Heterogeneous distributions of the two subtypes of adenosine receptors, A1 and A2, have been demonstrated in the mammalian retina. Coupling of receptors to adenylate cyclase has also been demonstrated.6.Adenosine A1 receptor agonists significantly inhibit the K+-stimulated release of [3H]-acetylcholine from the rabbit retina, suggesting that endogenous adenosine may modulate the light-evoked or tonic release of ACh.

Lentiviral vector-mediated gene transfer in adult mouse photoreceptors is impaired by the presence of a physical barrier

Gene transfer offers a substantial promise for the therapy of degenerative ocular diseases. Lentiviral vectors have the ability to efficiently transduce murine photoreceptors during the first days of life, but they are poorly effective on photoreceptors during adulthood. Here, we studied whether a physical barrier was responsible for this impairment. Previous studies have described the capacity of enzymes, such as chondroitinase ABC and neuraminidase X, to modify the structure of the interphotoreceptor matrix (IPM) when subretinally injected. Considering the IPM as a physical barrier that may decrease photoreceptor transduction, we injected different enzymes into the subretinal space of the adult mouse simultaneously with the lentiviral vector preparation, to increase viral transduction by fragilizing the IPM. Subretinal injection of neuraminidase X and chondroitinase ABC induces modifications in the IPM by, respectively, revealing or decreasing peanut agglutinin sites on photoreceptors. The simultaneous subretinal injection of neuraminidase X with a lentiviral vector driving the expression of a reporter gene in the photoreceptors increases the number of transduced cells significantly (around five-fold). After the enzyme treatment, the diffusion of the vector between the pigmented epithelium and the photoreceptors appears to facilitate the lentiviral vector transduction. Such approach targeting the IPM may help to design new strategies to improve gene delivery into the adult photoreceptors.

Neurite outgrowth and synaptophysin expression of postnatal CNS neurons on GaP nanowire arrays in long-term retinal cell culture.

We have established long-term cultures of postnatal retinal cells on arrays of gallium phosphide nanowires of different geometries. Rod and cone photoreceptors, ganglion cells and bipolar cells survived on the substrates for at least 18 days in vitro. Glial cells were also observed, but these did not overgrow the neuronal population. On nanowires, neurons extended numerous long and branched neurites that expressed the synaptic vesicle marker synaptophysin. The longest nanowires (4 μm long) allowed a greater attachment and neurite elongation and our analysis suggests that the length of the nanowire per se and/or the adsorption of biomolecules on the nanowires may have been important factors regulating the observed cell behavior. The study thus shows that CNS neurons are amenable to gallium phosphide nanowires, probably as they create conditions that more closely resemble those encountered in the in vivo environment. These findings suggest that gallium phosphide nanowires may be considered as a material of interest when improving existing or designing the next generation of implantable devices. The features of gallium phosphide nanowires can be precisely controlled, making them suitable for this purpose.

AUTORADIOGRAPHY OF NUCLEOSIDE UPTAKE INTO THE RETINA

A selective uptake mechanism for some nucleosides and related substances was found in retinae of light adapted rabbits and fish. After the intravitreal injection in vivo of [(3)H]adenosine, [(3)H]inosine, [(3)H]guanosine and certain related compounds, the distribution of radioactivity was studied by autoradiography. Retinae were also incubated in [(3)H]adenosine and [(3)H]inosine and then were similarly processed. In rabbits, the accumulation of radioactivity from [(3)H]adenosine and [(3)H]guanosine was predominantly into glial cells, but also into neurons. [(3)H]Inosine labelled glia almost exclusively. However, the adenosine analog, [(3)H]methylphenylethyl-adenosine, resulted in well-defined neuronal labelling in this species. In fish, a few photoreceptor cell bodies exhibited strong radioactivity with the nucleosides, presumably representing incorporation into nucleic acids of replicating cells. Labelling was also seen in horizontal cells, amacrine cells and ganglion cells after the injection of either [(3)H]adenosine, [(3)H]guanosine or [(3)H]inosine. To some extent, the selective accumulation of radioactivity is likely to be due to cell replication, but in most neurons, other factors must be responsible. Judging from what is known about the actions of adenosine in central nervous tissue, signal transmission in the retina could be such a factor.

Colocalization of (3H)-adenosine accumulation and GABA immunoreactivity in the chicken and rabbit retinas

SummaryUsing combined autoradiography and immunohistochemistry, we have compared (3H)-adenosine accumulation and GABA immunoreactivity in the chicken and rabbit retinas. Colocalization of the two markers was observed in a subset of amacrine cells and in certain cell bodies in the ganglion cell layer in both species and in a few horizontal cells in the chicken retina. Cells that contained only (3H)-adenosine or GABA were also seen. The degree of colocalization differed greatly between the two species. The results demonstrate a morphological relationship between the adenosine and GABA systems and provides information of the possible anatomical substrates underlying at teast some types of functional interactions.