Elena Vecino

Rat retinal ganglion cells co-express brain derived neurotrophic factor (BDNF) and its receptor TrkB.

The expression of brain derived neurotrophic factor (BDNF) and its preferred receptor (TrkB) in rat retinal ganglion cells (RGCs) have been determined in the present study. To identify RGCs retrograde labelling was performed with fluorogold (FG). Subsequently, retinas were immunostained with antibodies to BDNF and TrkB. We found that all RGCs labelled with FG express both BDNF and its preferred receptor, TrkB. Moreover, displaced amacrine cells were also found to be immunolabelled by both antibodies. Thus BDNF/TrkB signalling in RGCs probably involves endogenous BDNF produced by the RGCs themselves.

Colocalization of neuropeptide Y (NPY)-like and FMRFamide-like immunoreactivities in the brain of the Atlantic salmon (Salmo salar).

SummaryThe colocalization of the peptides neuropeptide Y (NPY) and Phe-Met-Arg-Phe-NH2 (FMRFamide) in the brain of the Atlantic salmon was investigated with double immunofluorescence labeling and peroxidase-antiperoxidase immunocytochemical techniques. Colocalization of NPY-like and FMRE amide-like immunoreactivities was observed in neuronal cell bodies and fibers in four brain regions: in the lateral and commissural nuclei of the area ventralis telencephali, in the nucleus ventromedialis thalami, in the laminar nucleus of the mesencephalic tegmentum, and in a group of small neurons situated among the large catecholaminergic neurons in the isthmal region of the brainstem. All cell bodies in these nuclei were immunoreactive to both NPY and FMRF. We consistently observed larger numbers of FMRF-immunoreactive than NPY-immunoreactive fibers. In the nucleus ventromedialis thalami NPY- and FMRFamide-like immunoreactivities were colocalized in cerebrospinal fluid (CSF)-contacting neurons. NPY-immunoreactive, but not FMRF-immunoreactive, neurons were found in the stratum periventriculare of the optic tectum, and at the ventral border of the nucleus habenularis (adjacent to the nucleus dorsolateralis thalami). Neurons belonging to the nucleus of the nervus terminalis were FMRF-immunoreactive but not NPY-immunoreactive. The differential labeling indicates, as do our cross-absorption experiments, that the NPY and FMRFamide antisera recognize different epitopes. Thus, it is probable that NPY-like and FMRFamide-like substances occur in the same neurons in some brain regions.

Microglia in normal and regenerating visual pathways of the tench (Tinca tinca L., 1758; Teleost): a study with tomato lectin.

We have studied the microglial cells in the normal and regenerating visual pathways of Tinca tinca (Cyprinid, Teleost) by using the lectin from Lycopersicum esculentum (tomato), which, in our case, has been demonstrated as a specific marker for teleost microglia. In the normal fish, there are tomato lectin positive microglial cells in the retina, optic nerve, and optic tectum. Following optic nerve crush, we observed a more extensive labeling of the microglia in the crushed optic nerve and in the contralateral optic tectum affecting the stratum opticum and stratum fibrosum et griseum superficiale. In both cases, there was an increase of rounded and less ramified microglial cells, and granular cells. This response of a more extensive labeling of microglial cells increases to a maximum at 2-3 weeks after the crush; the density of labeled microglial cells decreases after 3 months after crushing. However, in the retina no changes were observed after optic nerve crush. These results suggest that the microglial cells could play an important role in regeneration of fish optic pathway, as other neuroglial cells do.

In situ hybridization of neuropeptide Y (NPY) mRNA in the goldfish brain

THE distribution of neurones expressing neuropeptide Y (NPY) mRNA in the brain of the goldfish was investigated using a non-radioactive in situ hybridization technique. Neurones expressing NPY mRNA were located in cell groups previously shown to exhibit NPY immunoreactivity in the ventral telencephalon, thalamus, optic tectum, and a region adjacent to the locus coeruleus. In addition, neurones expressing NPY mRNA were observed in the diffuse nucleus of the torus lateralis, and in the mesencephalic tegmentum. Our results suggest that in the two latter groups of neurones the rate of transport of NPY from the perikarya is higher, or that NPY is not synthesized at levels detectable by immunocytochemistry.

NMDA induces BDNF expression in the albino rat retina in vivo.

The effect of an intravitreal injection of NMDA on the expression of brain-derived neurotrophic factor (BDNF) in retinal ganglion cells was investigated in rats. Forty-eight hours after intravitreal injection of NMDA retinal ganglion cell BDNF immunoreactivity was practically obliterated, as was the choline acetyltransferase (ChAT) immunoreactivity associated with a subset of amacrine cells. However, 2h following treatment with NMDA the BDNF immunoreactivity and BDNF mRNA associated with the ganglion cells was enhanced while the amacrine cell ChAT immunoreactivity was clearly reduced and the levels of mRNA coding for rhodopsin and Thy-1 did not change. However, 4h after NMDA injection the increase in BDNF mRNA was now no longer apparent. The results show that synthesis of BDNF is increased in the ganglion cells immediately following an insult by NMDA. It is suggested that this is a natural protective mechanism of rat retinal ganglion cells.

Calbindin D-28K distribution in the retina of the developing trout (Salmo fario L.)

The appearance of calbindin D-28K, a calcium-binding protein, during development of the trout retina was studied by immunohistochemistry. The first calbindin immunoreactive cells appear in the inner nuclear layer at the equator of the embryonic retina at the stage 227 degrees C (around embryonic day 15). Just before hatching, stage 440 degrees C (around embryonic day 30) cells located in the ganglion cell layer and inner nuclear layer, expressed calbindin. This pattern of immunoreactivity was conserved in post-embryonic retinae (alevins 15 days old). In adult retinae the ganglion cells showed a faint immunoreaction; the amacrine cells are markedly fewer and their immunoreaction declined; and the bipolar cells expressed calbindin for the first time. The results obtained in the present work attending to the expression of calbindin, generally conforms with the vitreal to scleral progression of differentiation of the teleost retina. Ganglion, amacrine and bipolar cells undergo further maturation after beginning calbindin expression.

Localization of enkephalinergic neurons in the central nervous system of the salmon (Salmo salar L.) by in situ hybridization and immunocytochemistry

The distribution of neurons expressing preproenkephalin (PPE) mRNA in the brain of the salmon was investigated by means of non-radioactive in situ hybridization, and directly compared with the distribution of enkephalin-immunoreactive (ENKir) neurons. This approach, utilized here for the first time in a non-mammalian vertebrate for the identification of neurons containing opioid peptides, permitted a detailed analysis of the distribution of putative enkephalinergic neurons in the salmon brain. Several cell groups containing neurons that express PPE mRNA also contain ENKir neurons. Such cell groups are located in the ventral telencephalic area, the nucleus of the rostral mesencephalic tegmentum and another nucleus immediately dorsal to it, the torus semicircularis, the valvula cerebelli and the corpus cerebelli. These cell groups consistently contain larger numbers of PPE mRNA expressing cells than ENKir ones. Some cell groups express PPE mRNA, but do not contain ENKir neurons. These cell groups are located in the dorsal telencephalic area, the inferior lobes of the hypothalamus, the pretectal area, the magnocellular superficial pretectal nucleus, the optic tectum, the oculomotor nucleus, the trochlear nucleus, the magnocellular vestibular nucleus, the secondary gustatory nucleus, the superior and medial reticular nuclei, the motor nucleus of the vagus and the ventral horn of the spinal cord. Moreover, some cell groups contain ENKir neurons, but no PPE mRNA expressing neurons. These cell groups are located in the ventromedial thalamic nucleus, the lateral tuberal nucleus, the nucleus of the lateral recess and the nucleus of the posterior recess. The majority of these periventricular ENKir neurons were of the cerebrospinal fluid-contacting type. ENKir neurons were also located in the dorsal lateral tegmental nucleus and in area B9. The results also permitted a tentative identification of enkephalinergic neurons afferent to the optic tectum, that have previously not been identified with immunocytochemistry, located in the dorsal telencephalic area, as well as enkephalinergic neurons intrinsic to the tectum that may contribute to the laminar arrangement of ENKir fibers in the optic tectum.

Stem cell plasticity, neuroprotection and regeneration in human eye diseases.

Regeneration and plasticity refer to the ability of certain progenitor cells to produce cell lineages with specific morphological and functional settings. The pathway from a less delineated or immature phenotype to a mature or specialized one follows intricate routes where a monumental array of molecular elements, basically transcription factors and epigenetic regulators that turn off or on a specific phenotypic change, play a fundamental role. Nature itself offers procedures to healing strategies. Therapy approaches to pathologies in the realm of ophthalmology may benefit from the knowledge of the properties and mechanisms of activation of different routes controlling the pathways of cell definition and differentiation. Specification of cell identity, not only in terms of phenotypic traits, but also regarding the mechanisms of gene expression and epigenetic regulation, will provide new tools to manipulating cell fates and status, both forward and backwards. In the human eye, two main locations shelter stem cells: the limbus, which is situated in the limit of the cornea and the conjunctiva, and the ciliary body pars plana. Transplantation of limbal cells is currently used in certain pathologies where corneal epithelium is damaged. Therapeutic applications of retina progenitors are not yet fully developed due to the complexity of the cellular components of the multilayer retinal architecture. Animal models of Retinitis pigmentosa or Glaucoma offer an interesting approach to validate certain techniques, such as the direct injection of progenitors into the vitreal compartment, aimed to restoring retinal function.

Glaucoma Animal Models

1.1 Glaucoma is a progressive neuropathy Glaucoma is an optic neuropathy that is considered to be the second leading cause of blindness worldwide. This disease is characterized by selective death of retinal ganglion cells (RGC) and a progressive loss of vision. Elevation of intraocular pressure (IOP) is a critical risk factor for glaucoma progression, and its lowering has become a major focus of intervention. However, many patients continue to lose vision despite IOP management. Additionally, some patients develop what is known as normal tension glaucoma, which is not associated with increased IOP. Taken together, the continued deterioration of some patients’ vision despite IOP management as well as the incidence of normal tension glaucoma illustrate that several pressure-independent mechanisms are responsible for the development and progression of glaucomatous neuropathy (Pinar-Sueiro & Vecino, 2010). Glaucoma is difficult to study in humans. The damage present at the time of diagnosis precludes the study of disease development from onset. Additionally, obtaining retinas at equivalent pathologic states is rare, confounding comparisons and limiting conclusions. For these reasons, the development of animal models has been necessary for the study of the pathophysiology of glaucoma. Animal studies have articulated the mechanisms of the formation and evacuation of aqueous humour as well as the maintenance of intra-ocular pressure, thereby informing glaucoma etiology and therapeutic development. In 1901, Lauber determined that blood from the anterior ciliary veins of dogs contained fewer erythrocytes per unit volume than did blood from their paws. As early as 1903, Leber noted a significant histological connection between the Schlemm ́s canal and the episcleral veins. Numerous investigators then demonstrated that various dyes and tracer substances injected into the anterior chamber later appeared in the anterior ciliary veins. It was not until 1942, when Ascher first described the appearance of aqueous veins and their connections to the episcleral venus plexus, and a connection between Schlemm’s canal and eipiscleral veins was demonstrated under normal conditions in vivo. Ascher compressed the episcleral veins using a fine glass rod, thereby inhibiting aqueous flow into them. This flow resumed when the rod was removed. He described two types of stratification: vessels with a superior layer of aqueous

Expression of Neurotrophins and their Receptors Within the Glial Cells of Retina and Optic Nerve

The purpose of this chapter is to review some of our studies on the localisation of neurotrophins and their receptors in glial cells of the optic nerve and retina of the fish (tench) and rat. The fish optic nerve has the capacity to regenerate after damage and the retina grows throughout life. These characteristics are not associated with the same tissues of the rat. Neurotrophins are thought to be involved in development and regeneration so a difference in the distribution of neurotrophins and their receptors in retina/optic nerve in rat and fish may relate to such functions. At least five different-types of neurotrophin molecules exist of which nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3) and neurotrophin-4 (NT-4) have been best studied. Neurotrophin receptors of which there are two kinds, the low affinity receptor (p75) and the high affinity tyrosine kinase receptors (Trk) exists at varying states and have varying affinities for the different type of neurotrophins. The functional role of the neurotrophins and their receptors and ways of studying these molecules in the retina are also discussed in this overview.