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Biomedical subjects

R Linden

Publications and source records attributed to R Linden.

At least 19 recordsLinked to original sources

Depletion of cortical target induced by prenatal ionizing irradiation: effects on the lateral geniculate nucleus and on the retinofugal pathways.

Studies using neonatal surgical lesions to reduce the target area of the retina have supported the idea that developing axons show only a limited specificity in their targeting. This investigation tested whether retinogeniculate axons adjust for partial target depletion by repositioning of axons. We used adult Swiss mice exposed to gamma rays at the time when layer IV cells are generated in the ventricular zone (16 days of gestation). Nissl-stained brain sections were used for histological analyses in thalamus and cortex. Retinal ganglion cells were backfilled from the optic tract with horseradish peroxidase. Intraocular injections of horseradish peroxidase were used to study the retinal projections. In the posterior cortex there was a nearly complete absence of layer IV. The irradiated animals showed a 75% reduction of the dorsal lateral geniculate nucleus. The ventral division, superior colliculus, and other visually related nuclei were not affected. The loss in the ganglion cells (15.7%) was significant but clearly smaller than that observed in the dorsal lateral geniculate nucleus (75%). Therefore, the shrinkage of the dorsal lateral geniculate nucleus led to a reduction in the area available for retinal projections. Despite partial target loss, pattern of retinal projections did not differ from that of the controls. The effect on the dorsal lateral geniculate nucleus is discussed in the light of differences between prenatal and neonatal damage of the presumptive visual cortex. The absence of aberrant retinal projections suggests that repositioning of axons is not the first mechanism employed by retinal axons to match connections in numerically disparate populations.

Animals↗

Effects of prenatal ionizing irradiation on the development of the ganglion cell layer of the mouse retina.

Prenatal exposure to ionizing irradiation has been shown to be an effective method to eliminate selectively certain neuronal population. This investigation studied the effects on the ganglion cell layer of the retinae of adult mice exposed to a gamma source (total dose=3 Gy) at 16 days gestation. There was a significant reduction in the total number of neurons (displaced amacrine+ganglion cells) in the ganglion cell layer (33%) that was mainly caused by a pronounced loss (59%) of displaced amacrine cells. The diameters of the surviving retinal ganglion cells were consistently larger than those of the controls. Prenatal irradiation is the first experimental approach that partially eliminates displaced amacrine cells. It is suggested that the morphogenesis of retinal ganglion cells may be affected by displaced amacrine cells.

Animals↗

Differentiation-dependent sensitivity to cell death induced in the developing retina by inhibitors of the ubiquitin-proteasome proteolytic pathway.

The effects of inhibitors of proteasome function were studied in the retina of developing rats. Explants from the retina of neonatal rats at postnatal day (P) 3 or P6 were incubated with various combinations of the proteasome inhibitor carbobenzoxyl-leucinyl-leucinyl-leucinal (MG132), the protein synthesis inhibitor anisomycin, or the adenylyl cyclase activator forskolin. MG132 induced cell death in a subset of cells within the neuroblastic (proliferative) layer of the retinal tissue. The cells sensitive to degeneration induced by either MG132 or anisomycin, were birthdated by bromodeoxyuridine injections. This showed that the MG132-sensitive population includes both proliferating cells most likely in their last round of cell division, and postmitotic undifferentiated cells, at a slightly earlier stage than the population, sensitive to anisomycin-induced cell death. The results show that sensitivity to cell death induced by proteasome inhibitors defines a window of development in the transition from the cell cycle to the differentiated state in retinal cells.

Animals↗

Paracrine neuroprotective effect of nitric oxide in the developing retina.

The retina of newborn rats consists of the ganglion cell layer (GCL), the inner plexiform layer (IPL), the inner nuclear layer (INL) containing amacrine cells and the neuroblastic layer (NBL). In retinal explants, the GCL enters cell death after sectioning of the optic nerve, whereas there is almost no cell death in the NBL. When protein synthesis is inhibited with anisomycin, cell death is blocked in the GCL and induced in the NBL. We tested the roles of nitric oxide (NO) on cell death in the retina in vitro. Either L-arginine, the substrate for NO synthase or the NO donor S:-nitroso-acetylpenicillamine (SNAP) blocked cell death induced by anisomycin in the NBL, but had no effect in the GCL. Sepiapterin, a precursor of the nitric oxide synthase (NOS)-cofactor tetrahydrobiopterin also had a protective effect against anisomycin. The use of 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one, an inhibitor of soluble form of guanylyl cyclase, showed that anti-apoptotic effect of SNAP is partially mediated by cGMP generated by activation of guanylyl cyclase. NADPH-diaphorase histochemistry stained cells only in the GCL and INL. Thus, the degenerative effect of anisomycin is observed within the NBL, whereas the localization of NOS is restricted to the GCL and INL. The protective effect of both the NO substrate and cofactor upon cell death induced by anisomycin in the NBL, indicates that NO produced by amacrine and ganglion cells is a paracrine modulator of cell death within the retinal tissue.

Animals↗

Chloramphenicol induces apoptosis in the developing brain.

Programmed cell death was studied in the superior colliculus of the developing rat brain following injections of chloramphenicol. Neonatal rats were either subject to unilateral eye removal or left untouched. Following a 3-h post-operative survival, the animals were perfused with fixatives and frozen sections of their brains were examined for apoptosis after either neutral-red staining, in situ nick-end labeling of fragmented DNA, or immunocytochemistry to activated caspase-3. Chloramphenicol induced apoptosis in control brains and potentiated cell death in deafferented superior colliculi. The results show that CMP has a general pro-apoptotic effect in the developing brain.

Afferent Pathways↗

Laminin modulates neuritogenesis of developing rat retinal ganglion cells through a protein kinase C-dependent pathway.

Dissociated cells from rat retinae (P2-P21) were cultured to investigate interactions between brain-derived neurotrophic factor (BDNF), various substrates (poly-L-lysine, collagen, and laminin), and protein kinases upon the neuritogenesis of retinal ganglion cells (RGCs). We found that BDNF-promoted neuritogenesis was enhanced by forskolin in RGCs from rats at P2-P21 plated on either poly-L-lysine or collagen. In contrast, in cultures with a laminin substrate, the enhancer effect of forskolin was observed only in RGCs taken from the retina of rats at P2-P6. Laminin blocked the enhancement of BDNF-induced RGCs neuritogenesis by forskolin, in RGCs from either P14 or P21, and induced a tenfold increase of protein kinase C (PKC) activity compared to poly-L-lysine. This blockade was reverted with a selective PKC inhibitor and was reproduced in poly-L-lysine cultures of P14-P21 RGCs with a PKC activator. Because axotomized RGCs need both BDNF and forskolin to regenerate, we suggest that laminin can hinder this effect by simultaneous PKC activation according to a developmentally regulated pattern. We further propose a model of interaction in the optic pathways triggered by BDNF, forskolin, and laminin that may be useful in elucidating some of the biological effects seen with regenerating axons.

Animals↗

The anti-death league: associative control of apoptosis in developing retinal tissue.

Apoptosis, the major form of programmed cell death (PCD), is executed through a proteolytic cascade that can be differentially engaged by various extracellular signals. Modulation of both the sensitivity to PCD and of the actual sequence of apoptotic events is, therefore, strongly dependent on cell interactions. This paper reviews the use of a retinal explant preparation as a model of the organized nervous tissue, to study the effects of neural messengers in the control of sensitivity to apoptosis. Studies of retinal explants showed that dopamine, glutamate and nitric oxide may have anti-apoptotic effects upon developing retinal cells. At least the effects of nitric oxide are clearly paracrine. In addition, preliminary evidence has been gathered of a role for gap junctional communication in the control of sensitivity of retinal cells to the induction of apoptosis. These findings underscore the importance of selective cell interactions in the control of PCD in the developing nervous system.

Animals↗

Evidence that the bifunctional redox factor / AP endonuclease Ref-1 is an anti-apoptotic protein associated with differentiation in the developing retina.

Retinal cell differentiation leads to resistance to apoptosis induced by inhibition of protein synthesis, suggesting the accumulation of anti-apoptotic proteins. The redox factor/AP endonuclease Ref-1 (APE, APEX, HAP1) affects both DNA repair and the activity of various transcription factors, and controls sensitivity to genotoxic insults. We studied the expression of Ref-1 in the retina and brain of developing rats. Ref-1 immunoreactivity increased progressively within the nucleus of differentiating retinal cells, whereas it decreased in the developing hippocampal formation. During both natural and experimentally-induced cell death, Ref-1 disappeared from the nucleus of apoptotic cells. Degradation of Ref-1 in axotomized ganglion cells preceded the morphological characteristics of apoptosis. The sensitivity to apoptosis triggered by either thapsigargin or okadaic acid was the highest in photoreceptors, that contain the least Ref-1 among differentiated retinal cells. In both these differentiated cell types, inhibition of protein synthesis prevented the loss of Ref-1 and rescued the neurons. The data suggest that Ref-1 is an anti-apoptotic protein associated with cell differentiation in the retina.

Animals↗

Response surface analysis applied to the preparation of tablets containing a high concentration of vegetable spray-dried extract.

This work relates to the formulation of tablets containing a high proportion of spray-dried extracts (SDEs) from Passiflora edulis leaves. The tablets were prepared by direct compression. Colloidal silicon dioxide was selected as a glidant and moisture adsorbent, cross-linked carboxymethycellulose was used as the disintegrant, microcrystalline cellulose was the filler/binder, and tricalcium phosphate as a spray-drying adjuvant. The colloidal silicon dioxide and cross-linked carboxymethycellulose quantities and their influences on the tablet hardness and disintegration time were studied by a central composite design. The model equations were fitted to the experimental data and then validated. It could be concluded that the colloidal silicon dioxide proportion increased the hardness, and the cross-linked carboxymethycellulose proportion determined a linear decrease of the disintegration time. The optimal values chosen were 2.0% Aerosil 200 and 2.5% Ac-Di-Sol. The tablets showed a hardness of 85.02 N and a disintegration time of 7.35 min.

Chemistry, Pharmaceutical↗

Tissue biology of apoptosis. Ref-1 and cell differentiation in the developing retina.

Programmed cell death by apoptosis plays a major role in neurogenesis. The sensitivity to apoptosis in developing nervous tissue is strongly dependent on cell interactions taking place within a highly structured environment, composed of various cell types at distinct stages of differentiation. In this article, we review evidence gathered both in vivo and in a histotypical retinal explant preparation in vitro that the bifunctional AP endonuclease/redox factor Ref-1 (HAP1, APE, APEX) may be an anti-apoptotic protein associated with cell differentiation in the developing retina.

Animals↗

BDNF and NT-4 differentially modulate neurite outgrowth in developing retinal ganglion cells.

We show here that neurite outgrowth of ganglion cells (RGCs) was selectively enhanced following treatment with BDNF or NT-4 in short-term cultures of dissociated cells derived from the neuroretina of postnatal rats. NT-4 was more effective than BDNF. The effect of NT-3 was variable, whereas NGF and CNTF had no effects upon neurite elongation. The neuritogenic responses of RGCs to both BDNF and NT-4 were prevented by competition with soluble TrkB receptor, and abolished by K252a, a selective inhibitor of the tyrosine kinase activity of Trks. These results indicate that the differentiating effects of BDNF and NT-4 are mediated by TrkB receptors, naturally expressed by RGCs. Developing RGCs treated with these TrkB ligands displayed distinct, albeit partially overlapping, patterns of neurite morphology. BDNF supported predominantly polarized outgrowth, whereas NT-4 induced the appearance of intensely branched symmetrical arbors. The lack of RGCs showing combined morphologies (e.g., highly arborized unipolar cells) suggests distinct mechanisms underlying either elongation or branching, and implicates distinct responses of RGC subsets. We conclude that neurite growth in vitro is extensively promoted by neurotrophins in developing RGCs. Moreover, highly homologous neurotrophins such as BDNF and NT-4, presumably activating via TrkB receptors, selectively control the differentiation of distinct ganglion cell neuritic morphologies.

Animals↗

Activation of NMDA receptors protects against glutamate neurotoxicity in the retina: evidence for the involvement of neurotrophins.

Activation of glutamate receptors has been implicated in excitotoxicity. Here, we have investigated whether subtoxic concentrations of glutamate can modulate neuronal death in the developing retina. Explants of rat retinas were pre-incubated with glutamate, N-methyl-d-aspartate (NMDA), kainate, quisqualate or trans-1-amino-1,3-cyclopentanedicarboxylic acid (t-ACPD) for 18 h. Then, glutamate (6 mM) was added to the explants for an additional 6 h. Glutamate-induced degeneration was restricted to the emerging inner nuclear layer. Pre-incubation with glutamate, NMDA, or both, reduced glutamate-induced neuronal death and protected against neuronal death induced by irradiation (2 Gy). The NMDA receptor antagonists, 2-amino-5-phosphonovaleric acid (d-APV; 30 microM) or 5-methyl-10,11-dihydro-5H-dibenzocyclohepten-5,10-imine hydrogen maleate (MK-801; 30 microM), prevented glutamate-induced neuroprotection. To investigate whether this neuroprotection was mediated by neurotrophins, we incubated retinal explants with either brain-derived neurotrophic factor or neurotrophin-4. Both treatments resulted in partial protection against glutamate-induced neurotoxicity. Furthermore, NMDA mediated neuroprotection was totally reversed when a soluble form of the specific tyrosine kinase receptor B was simultaneously added to the explants. Our results suggest that activation of NMDA receptors may control neuronal death in the retina during development. This modulation seems to depend, at least in part, on the release of neurotrophins within the retina.

2-Amino-5-phosphonovalerate↗

Apoptosis in developing retinal tissue.

The mechanisms of apoptosis are strongly dependent on cell-cell interactions typical of organized tissues. Experimental studies of apoptosis using a histotypical preparation of retinal explants are reported in the present article. We found that various characteristics of apoptosis are selectively associated with retinal cell death depending on cell type, stage of maturation, and means of induction of apoptosis. Among these were: (1) the requirements of protein synthesis; (2) the role of cAMP; (3) the expression of certain apoptosis-associated proteins; and (4) the sensitivity to excitotoxicity, modulation of protein phosphatases and calcium mobilization. Dividing cells undergo apoptosis in response to several inducers in specific phases of the cell cycle, and in distinct regions within their pathway of interkinetic nuclear migration. Recent post-mitotic cells are selectively sensitive to apoptosis induced by blockade of protein synthesis, while both proliferating and differentiated cells are more resistant. We also studied the association of several proteins, some of which play critical roles in the cell cycle, with both differentiation and apoptosis in the retinal tissue. Detection of cell cycle markers did not support the hypothesis that retinal cells re-enter the cell cycle on their pathway to apoptosis, although some proteins associated with cell proliferation re-appeared in degenerating cells. The transcription factors c-Jun, c-Fos and c-Myc were found associated with apoptosis in retinal cells, but their sub-cellular location in apoptotic bodies is not consistent with their canonical functions in the control of gene expression. The bifunctional redox factor/AP endonuclease Ref-1 and the transcription factor Max are associated with progressive cell differentiation, and both are down-regulated during cell death in the retina. The data suggest that Ref-1 and Max may normally function as negative modulators of retinal apoptosis. The results indicate that nuclear exclusion of transcription factors and other important control proteins is a hallmark of retinal apoptosis. Histotypical explants may be a choice preparation for the experimental analysis of the mechanisms of apoptosis, in the context both of cell-cell interactions and of the dynamic behavior of developing cells within the organized retinal tissue.

Animals↗

Selective sensitivity of early postmitotic retinal cells to apoptosis induced by inhibition of protein synthesis.

In previous work we showed that apoptosis in retinal tissue from developing rats can be induced by inhibition of protein synthesis (Rehen et al. 1996, Development, 122, 1439-1448). Here we show that recent postmitotic cells are the cells sensitive to apoptosis triggered by blockade of protein synthesis. To label all proliferating cells in the retina, a series of injections of the nucleotide analogue, bromo-deoxy-uridine (BrdU, 60 mg/kg b.w.), was given in rat pups. Then, explants of the retina were incubated in vitro with the inhibitor of protein synthesis anisomycin (1.0-3.2 microg/mL) for 1 day to induce apoptosis. Detection of apoptotic bodies under differential interference contrast microscopy was combined with immunocytochemistry for BrdU, proliferating cell nuclear antigen (PCNA) or for various markers of retinal cell differentiation. Despite the large number of BrdU- and PCNA-labelled cells in the tissue, the vast majority of the cells that underwent apoptosis were postmitotic cells which have left the mitotic cycle 3-4 days before. However, these cells were not labelled with antibodies to calretinin, calbindin, rhodopsin or to a Muller glial cell marker, suggesting that these are early postmitotic neurons. We suggest that during migration and initial differentiation, the apoptotic machinery is blocked by suppressor proteins, thus allowing recent postmitotic cells to find their final positions and differentiate while protected from apoptosis.

Animals↗

Evidence for an antiapoptotic role of dopamine in developing retinal tissue.

Inhibition of protein synthesis leads to apoptosis in the undifferentiated neuroblastic layer of the retina of newborn rats. We have shown previously that an increase in the intracellular concentration of cyclic AMP prevented apoptosis induced in the retinal neuroblastic layer by inhibition of protein synthesis. In this study, we tested the effects of dopamine on retinal apoptosis and related these effects to the intracellular concentration of cyclic AMP. Both dopamine (100 microM) and the D1-like agonists SKF-38393, 6-chloro-7,8-dihydroxy-1-phenyl-2,3,4,5-tetrahydro-1H-3-benzazepine (6-Cl-PB), and (+/-)-2-amino-6,7-dihydroxy-1,2,3,4-tetrahydronaphthalene (100 microM) blocked apoptosis induced in the neuroblastic layer by the protein synthesis inhibitor anisomycin. The antiapoptotic effects of the D1-like agonists were not reversed by the D1-like antagonist SCH-23390 (5-100 microM). Both dopamine and D1-like agonists induced a five- to sevenfold increase in the intracellular concentration of cyclic AMP in the retina of newborn rats. The concentration of cyclic AMP induced by the D1-like agonists in the presence of 100 microM SCH-23390 was still at least two- to threefold as high as control values, showing that the activation of adenylyl cyclase by D1-like agonists was reversed only partially by the specific antagonist. The isoquinolinesulfonamide H-89 (20 microM), an inhibitor of cyclic AMP-dependent protein kinase, partially prevented the antiapoptotic effect of 6-Cl-PB. The data show that an early effect of dopamine in the developing retina is the control of programmed cell death. The antiapoptotic effect of dopamine is mediated, at least in part, through an atypical D1-like receptor coupled to stimulation of adenylyl cyclase, followed by activation of cyclic AMP-dependent protein kinase.

Animals↗

Gamma irradiation leads to two waves of apoptosis in distinct cell populations of the retina of newborn rats.

Gamma radiation induces apoptosis in the proliferative zone (neuroblastic layer) of the developing rat retina. We asked whether sensitivity to apoptosis might be related to distinct phases of the cell cycle. Explants of newborn rat retina or newborn pups were gamma-irradiated and apoptosis was detected by chromatin condensation, DNA fragmentation in situ and DNA electrophoresis. After 6 hours, early appearing apoptotic bodies were located mainly towards the outer tier of the neuroblastic layer. In contrast, after 24 hours, late-appearing apoptotic cells were located towards the inner margin of the neuroblastic layer, a region associated with the S phase of the cell cycle. Labeling of a cohort of cells with the nucleotide analog bromo-deoxyuridine (BrdU) at the time of irradiation, showed that these cells die in the late wave of apoptosis. BrdU given 3 hours before fixation labeled a large number of late apoptotic cells, but no early apoptotic cells. After labeling of all cycling cells with BrdU, 40% of the early apoptotic profiles were unlabeled, and thus post-mitotic. The same schedules of cell death were identified after gamma irradiation in vivo. The results show that irradiation leads to two waves of apoptosis in distinct cell populations. An early wave comprises both post-mitotic cells and proliferating cells out of the S phase. The late wave comprises cells in S phase, which pass through this phase again to die. The antioxidant pyrrolidinedithiocarbamate prevented the early but not the late wave of apoptosis following irradiation, and blocked lipid peroxidation at 6 hours after the insult, suggesting that the two waves of apoptosis are indeed mediated by distinct mechanisms.

Animals↗

Nuclear exclusion of transcription factors associated with apoptosis in developing nervous tissue.

Programmed cell death in the form of apoptosis involves a network of metabolic events and may be triggered by a variety of stimuli in distinct cells. The nervous system contains several neuron and glial cell types, and developmental events are strongly dependent on selective cell interactions. Retinal explants have been used as a model to investigate apoptosis in nervous tissue. This preparation maintains the structural complexity and cell interactions similar to the retina in situ, and contains cells in all stages of development. We review the finding of nuclear exclusion of several transcription factors during apoptosis in retinal cells. The data reviewed in this paper suggest a link between apoptosis and a failure in the nucleo-cytoplasmic partition of transcription factors. It is argued that the nuclear exclusion of transcription factors may be an integral component of apoptosis both in the nervous system and in other types of cells and tissues.

Animals↗