Neurons induce the activation of microglial cells in vitro

Although microglial cells are well known to become activated in the pathological brain, mechanisms underlying the microglial activation are not fully understood. In the present study, with an aim to elucidate whether neurons are involved in the microglial activation, we compared the morphology and the superoxide anion (O2-)-generating activity of rat microglial cells in pure culture with those of cells cocultured with rat primary cortical neurons. Microglial cells in pure culture in serum-free Eagle's minimum essential medium on poly-L-lysine-coated coverslips displayed ramified morphology and suppressed activity of O2- generation. In contrast, microglial cells in neuron-microglia coculture under the same conditions as those for the pure culture displayed ameboid shape and upregulated activity of O2- generation. Electron microscopic observation revealed that microglial cells in coculture were more abundant in Golgi apparatus and secretory granules than those in pure culture and that some of microglial cells in the vicinity of neurites exhibited membrane specialization reminiscent of a junctional apparatus with high electron density between a microglial soma and a neurite. Microglial cells in coculture tended to tie neurites in bundles by extending processes. Medium conditioned by neurons significantly enhanced O2- generation by microglia, but microglial cells in contact with or in close apposition to cocultured neurons were much more intensely activated than those remote from the neurons. Furthermore, the membrane fraction of cortical neurons activated microglial cells, and this effect was abolished by treating the neuronal membrane with trypsin or neuraminidase. In conclusion, neuronal-microglial contact may be necessary to mediate microglial activation. The present findings suggest that the contact of microglia with damaged neurons in the brain is a plausible cause to activate microglia in the neuropathological processes.     

Rho directs activation-associated changes in rat hepatic stellate cell morphology via regulation of the actin cytoskeleton

Hepatic stellate cell activation, thought to play a key role in fibrosis of the liver, is characterized by changes in cellular morphology. The intracellular signals regulating morphological alterations associated with stellate cell activation are uncertain. The ras-like guanosine triphosphate-binding protein, rho, has recently emerged as an important regulator of the actin cytoskeleton, and consequently cell morphology. The aim of this study was to test the hypothesis that rho signaling pathways direct activation-associated morphological changes in stellate cells by regulating the actin cytoskeleton. The morphology and actin cytoskeleton of primary rat hepatic stellate cells were studied with phase contrast, differential interference contrast, and epifluorescence microscopy. Immunohistochemistry and immunoblot analysis were used to examine rho expression and activity, respectively. Quiescent and activated stellate cells were investigated in the absence and presence of C3 transferase, a bacterial toxin that specifically inhibits rho. Stellate cell activation was characterized by the development of prominent intracellular fibers, and the loss of dendrite-like processes and perinuclear retinoid droplets. Moreover, activation was accompanied by the formation of prominent actin stress fibers and focal adhesions. Both rho expression and activity were demonstrated in stellate cells. C3 transferase blocked and reversed, both activation-associated morphological alterations and activation-associated changes in the actin cytoskeleton, in quiescent and activated stellate cells, respectively. These results indicate that rho directs activation-associated changes in rat hepatic stellate cell morphology via regulation of the actin cytoskeleton.     

Cross-talk between adenylate cyclase activation and tyrosine phosphorylation leads to modulation of the actin cytoskeleton and to acute progesterone secretion in ovarian granulosa cells

Steroidogenesis in granulosa cells can be stimulated by gonadotropic hormones and substances elevating cAMP. This cAMP-dependent metabolic event can be enhanced by peptide growth factors such as insulin, insulin-like growth factor, and epidermal growth factor, but the mechanism of cooperation between these two different signaling pathways is not yet clear. We have tested whether enhancement of tyrosine phosphorylation by vanadate, which blocks tyrosine phosphatases, is able to mimic the effect of growth factors on cAMP-induced steroidogenesis and investigated the cellular components involved in such modulation. Ortho- and metavanadate at 0.1-1.0 mM, when added to primary granulosa cell cultures, stimulated by gonadotropic hormones or forskolin, enhanced progesterone production by 1.5- to 9.0-fold within 120 min. Pervanadate showed a similar effect on steroidogenesis at a concentration one order of magnitude lower than ortho- or meta-vanadate. Phenylarsine-oxide, another blocker of tyrosine phosphatase, stimulated forskolin-induced steroidogenesis by 2.5-fold at 30 microM. In contrast, okadaic acid and calyculin A, which block specifically serine and threonine phosphatase, had no effect on steroidogenesis, when used at concentrations of 1 microM and 10 nM, respectively. The stimulation by vanadate was associated with a pronounced change in cell shape and total collapse of the actin network, which retracts to form a few large actin aggregates of 1-7 microns in diameter in the perinuclear region as revealed by visualization of actin by rhodamine-phalloidin staining under the fluorescent microscope. Steroidogenesis is not affected in cells treated with vanadate alone; the effect of vanadate on the actin cytoskeleton is much less pronounced. Electron microscopy of ultra-thin sections showed massive breakdown of thin filament cables in cells stimulated with vanadate together with gonadotropic hormone or forskolin. Massive clustering of lipid droplets and mitochondria as well as sharp increase in the electron-density of mitochondrial matrix was also observed in the stimulated cells. The action of vanadate in cAMP-stimulated cells leads to massive tyrosine phosphorylation of intracellular proteins in the range of 22-200 kilodaltons. It is suggested that the cross-talk between the cAMP pathway and tyrosine phosphorylation, which leads to enhanced steroidogenesis may be mediated by phosphorylation of cytoskeleton or associated proteins. The marked changes in lipid droplet-mitochondria interaction suggests that this enhanced steroidogenesis is due in part to mobilization of cholesterol into mitochondria in cells costimulated with vanadate and gonadotropins.     

Regulation of the cortical actin cytoskeleton in budding yeast by twinfilin, a ubiquitous actin monomer-sequestering protein

Here we describe the identification of a novel 37-kD actin monomer binding protein in budding yeast. This protein, which we named twinfilin, is composed of two cofilin-like regions. In our sequence database searches we also identified human, mouse, and Caenorhabditis elegans homologues of yeast twinfilin, suggesting that twinfilins form an evolutionarily conserved family of actin-binding proteins. Purified recombinant twinfilin prevents actin filament assembly by forming a 1:1 complex with actin monomers, and inhibits the nucleotide exchange reaction of actin monomers. Despite the sequence homology with the actin filament depolymerizing cofilin/actin-depolymerizing factor (ADF) proteins, our data suggests that twinfilin does not induce actin filament depolymerization. In yeast cells, a green fluorescent protein (GFP)-twinfilin fusion protein localizes primarily to cytoplasm, but also to cortical actin patches. Overexpression of the twinfilin gene (TWF1) results in depolarization of the cortical actin patches. A twf1 null mutation appears to result in increased assembly of cortical actin structures and is synthetically lethal with the yeast cofilin mutant cof1-22, shown previously to cause pronounced reduction in turnover of cortical actin filaments. Taken together, these results demonstrate that twinfilin is a novel, highly conserved actin monomer-sequestering protein involved in regulation of the cortical actin cytoskeleton.     

Calcium and protein kinase C regulate the actin cytoskeleton in the synaptic terminal of retinal bipolar cells

The organization of filamentous actin (F-actin) in the synaptic pedicle of depolarizing bipolar cells from the goldfish retina was studied using fluorescently labeled phalloidin. The amount of F-actin in the synaptic pedicle relative to the cell body increased from a ratio of 1.6 +/- 0.1 in the dark to 2.1 +/- 0.1 after exposure to light. Light also caused the retraction of spinules and processes elaborated by the synaptic pedicle in the dark. Isolated bipolar cells were used to characterize the factors affecting the actin cytoskeleton. When the electrical effect of light was mimicked by depolarization in 50 mM K+, the actin network in the synaptic pedicle extended up to 2.5 micrometer from the plasma membrane. Formation of F-actin occurred on the time scale of minutes and required Ca2+ influx through L-type Ca2+ channels. Phorbol esters that activate protein kinase C (PKC) accelerated growth of F-actin. Agents that inhibit PKC hindered F-actin growth in response to Ca2+ influx and accelerated F-actin breakdown on removal of Ca2+. To test whether activity-dependent changes in the organization of F-actin might regulate exocytosis or endocytosis, vesicles were labeled with the fluorescent membrane marker FM1-43. Disruption of F-actin with cytochalasin D did not affect the continuous cycle of exocytosis and endocytosis that was stimulated by maintained depolarization, nor the spatial distribution of recycled vesicles within the synaptic terminal. We suggest that the actions of Ca2+ and PKC on the organization of F-actin regulate the morphology of the synaptic pedicle under varying light conditions.     

MSS4, a phosphatidylinositol-4-phosphate 5-kinase required for organization of the actin cytoskeleton in Saccharomyces cerevisiae

The Saccharomyces cerevisiae protein MSS4 is essential and homologous to mammalian phosphatidylinositol-4-phosphate (PI(4)P) 5-kinases. Here, we demonstrate that MSS4 is a lipid kinase. MSS4 has dual substrate specificity in vitro, converting PI(4)P to PI(4, 5)P2 and to a lesser extent PI(3)P to PI(3,4)P2; no activity was detected with PI or PI(5)P as a substrate. Cells overexpressing MSS4 contain an elevated level specifically of PI(4,5)P2, whereas mss4 mutant cells have only approximately 10% of the normal amount of this phosphorylated phosphoinositide. Furthermore, cells lacking MSS4 are unable to form actin cables and to properly localize their actin cytoskeleton during polarized cell growth. Overexpression of RHO2, encoding a Rho-type GTPase involved in regulation of the actin cytoskeleton, restores growth and polarized distribution of actin in an mss4 mutant. These results suggest that MSS4 is the major PI(4)P 5-kinase in yeast and provide a link between phosphoinositide metabolism and organization of the actin cytoskeleton in vivo.     

Region-specific activation of microglial cells in the rat septal complex following fimbria-fornix transection

Studies of postlesional microglial activation may gain insight into microglia/neuronal interactions in processes of neurodegeneration. We compared the microglial response after axotomy of septohippocampal projection neurons with that seen after selective immunolesioning of cholinergic septohippocampal neurons with the immunotoxin 192 IgG-saporin. Using the microglial marker isolectin B4 from Griffonia simplicifolia (GSA I-B4), we found striking differences in the microglial response between these two lesion paradigms. Following axotomy of septohippocampal neurons by fimbria-fornix transection (ff-t), there was only a moderate and short-lasting microglial reaction in the medial septum (MS) in the early postlesion period. Prelabeling of septohippocampal neurons with Fluoro-Gold (FG) prior to axotomy revealed the survival of most neurons, and only very rarely were microglial cells observed that had phagocytosed FG-labeled debris. In the lateral septum (LS) containing the degenerating terminals of hippocamposeptal fibers transected by ff-t, a heavy reaction of lectin-labeled activated microglial cells associated with high phagocytotic activity was noticed. Unexpectedly, after a long survival time (6 months) following ff-t, we observed an increase in microglial GSA I-B4 labeling in the MS. In contrast, an inverse pattern of the microglial response, i.e., a strong initial reaction in the MS and very little microglial activation in the LS, was observed after immunolesioning. Our results indicate that the microglial reaction in the MS following ff-t differs substantially from that seen in other models of axotomy.     

Effects of rhizopodin and latrunculin B on the morphology and on the actin cytoskeleton of mammalian cells

The effects of the novel myxobacterial compound rhizopodin on mammalian cells were studied and compared with those of latrunculin B. Both substances induced adherently growing L929 mouse fibroblasts and PtK2 potoroo kidney cells to produce long, narrow, branched extensions or runners. Rhizopodin was more efficient than latrunculin B in that respect (minimal inhibitory concentration with L929 cells 5 nM vs 50 nM), and, in contrast to latrunculin B, its effects were permanent. Rhizopodin-treated cells became much larger than normal cells and were multinucleate, yet stayed alive and biochemically active for several weeks. Latrunculin B-treated cells returned to a quasi-normal state within 3-4 days. But latrunculin B acted faster, with the first effects becoming visible almost immediately upon the addition of the drug, while the first rhizopodin effects were seen 10 min later. Both substances caused reorganization of the actin cytoskeleton. When 100 nM rhizopodin was added to PtK2 cells, the stress fibers began to decay after just 10 min and had disappeared completely after about 3 h. Later there was a gradual restitution of F-actin. Long F-actin fibers were seen within the runners, and only there; in fact, these fibers may be responsible for the development and extension of the runners. The microtubuli network adjusted itself to the new cell morphology, but was not directly impaired by the compound.     

Epidermal growth factor-induced activation and translocation of c-Src to the cytoskeleton depends on the actin binding domain of the EGF-receptor

The relationship between retinal arterial (Pra) and aortic (Pa) pressures is unknown, and the relationship between retinal vein (Prv) pressure and intraocular pressure (IOP) is not clear. Also unclear is the effect of cerebrospinal fluid pressure (CSFp) upon retinal venous pressure. We aimed to measure the relationships among Pra, Prv, Pa, IOP, and CSFp. Dogs were anesthetized while IOP, CSFp, and Pa were monitored. Pipettes with 2.5-micron diameter tips, connected to a servonulling pressure transducer, were used to record pressures from the retinal arteries and veins. Across a range of IOP (16-22 mmHg), CSFp (0-21 mmHg), and Pa (23-195 mmHg) the Pra = 0.72 Pa + 4.3 (r = 0.99, n = 61, P < 0.01), which suggests that the relationship between Pra and Pa is linear over a broad range of systemic blood pressures. The correlation coefficient between Prv and IOP was greater than 0.96 (P < 0.01) at all venous sites and whether IOP was greater than or less than CSFp. The transmural pressure varied along the retinal vein from 1.3 +/- 0.3 mmHg (+/-95% CI, n = 30) at 1 disk diameter from the optic disk rim to 0.3 +/- 0.2 mmHg (n = 66) at the optic disk, with a 0.9-mmHg/mm pressure gradient. These are the first measurements demonstrating a retinal vein transmural pressure close to zero.     

Complement activation occurs on subendothelial extracellular matrix in vitro and is initiated by retraction or removal of overlying endothelial cells

Vascular endothelium is continuously exposed to plasma complement, which could generate a potent proinflammatory signal if activated on the vascular wall. Normal endothelium, however, expresses an anti-inflammatory phenotype, which includes resistance to complement fixation. As activated endothelium converts to a proinflammatory phenotype, we investigated the effect of cytokines on endothelial susceptibility to complement fixation. Cytokine-treated HUVEC were exposed to human serum as a source of complement, and C3 deposition was quantified. IL-1beta and TNF-alpha in combination with IFN-gamma markedly increased endothelial C3 deposition; however, immunofluorescence microscopy revealed that the endothelial cells had retracted, and that bound C3 was concentrated not on cells but in areas of exposed subendothelial extracellular matrix (ECM). Studies with cell-free ECM indicated that complement activation required only ECM exposure and was independent of cellular activation. C3 deposition on ECM was reproduced by reconstituting the alternative pathway, which generated a stable C3 convertase on ECM, but not on endothelial cells. C3b and iC3b were identified on ECM exposed to purified alternative pathway components and serum, respectively. In conditions associated with endothelial disruption, exposure of subendothelial ECM could induce complement fixation and contribute to inflammation and vascular damage.     

Farnesyltransferase inhibition causes morphological reversion of ras-transformed cells by a complex mechanism that involves regulation of the actin cytoskeleton

A potent and specific small molecule inhibitor of farnesyl-protein transferase, L-739,749, caused rapid morphological reversion and growth inhibition of ras-transformed fibroblasts (Rat1/ras cells). Morphological reversion occurred within 18 h of L-739,749 addition. The reverted phenotype was stable for several days in the absence of inhibitor before the transformed phenotype reappeared. Cell enlargement and actin stress fiber formation accompanied treatment of both Rat1/ras and normal Rat1 cells. Significantly, inhibition of Ras processing did not correlate with the initiation or maintenance of the reverted phenotype. While a single treatment with L-739,749 was sufficient to morphologically revert Rat1/ras cells, repetitive inhibitor treatment was required to significantly reduce cell growth rate. Thus, the effects of L-739,749 on transformed cell morphology and cytoskeletal actin organization could be separated from effects on cell growth, depending on whether exposure to a farnesyl-protein transferase inhibitor was transient or repetitive. In contrast, L-739,749 had no effect on the growth, morphology, or actin organization of v-raf-transformed cells. Taken together, the results suggest that the mechanism of morphological reversion is complex and may involve farnesylated proteins that control the organization of cytoskeletal actin.     

Persisting in vitro actin motility at nanomolar adenosine triphosphate levels: comparison of skeletal and cardiac myosins

We have previously demonstrated in vitro actin movement at nanomolar adenosine triphosphate (ATP) levels using heavy meromyosin from skeletal muscle. In the present work we tested whether the motility at nonomolar ATP-concentrations could be supported by cardiac myosin as well. Actomyosin (skeletal actin and bovine ventricular myosin) was pretreated in the in vitro motility assay with 1 mM ATP; subsequently, the ATP level was reduced by multiple rigor-solution washes. By the final rigor-solution wash, the ATP concentration, monitored by the luciferin-luciferase assay, dropped to the order of 100 nM. Even at this low ATP level actin-filament movement remained in evidence. This was in marked contrast to the situation where ATP concentration was gradually increased from zero; in the latter, filament movement began only as ATP levels exceeded 1-2 microM. The difference indicates that potential energy is stored during the initial ATP treatment, and utilized later as the free ATP falls below micromolar levels. Although the velocity of cardiac myosin-supported movement was only one fourth of that of skeletal myosin, both myosins supported actin movement down to similar ATP concentrations. The similarity in response of the two myosins to ATP implies a similar degree of potential energy storage. Given the significantly different specific ATPase activities, however, it appears that the mechanism of potential energy storage and release involves factors different from those involved in the release of chemical energy by the myosin ATPase.     

Complement activation on human neuroblastoma cell lines in vitro: route of activation and expression of functional complement regulatory proteins

Two human neuroblastoma cell lines activated the classical pathway of complement in serum. Activation caused the opsonisation of these cells with complement fragments but with moderate cell killing. Neuroblastoma expressed regulators MCP and CD59 but did not express DAF or CR1. Neutralisation of CD59 rendered the cells susceptible to killing. Neuroblastoma also expressed C1-inhibitor, factor H, clusterin and S-protein. Expression of several regulators was enhanced by incubation with cytokines. Complement inhibition using soluble CRI markedly reduced opsonisation and killing of neuroblastoma. Our results suggest that complement might play a role in neuronal loss and that treatment with complement inhibitors might be of therapeutic value.     

Synthesis of 5-substituted quinazolinone derivatives and their inhibitory activity in vitro

Quinazolinone derivatives I and their methyl esters were synthesized and evaluated as nonclassical lipophilic inhibitors of thymidylate synthase. Compounds Ib and Ic containing OH and CO2H as R substituents, respectively, were most effective, indicating that hydrogen bonding may contribute to the increased inhibitory activity. These compounds further showed high cytotoxic activity against tumor cells in culture.     

Regulation of the actin cytoskeleton by thrombin in human endothelial cells: role of Rho proteins in endothelial barrier function

Endothelial barrier function is regulated at the cellular level by cytoskeletal-dependent anchoring and retracting forces. In the present study we have examined the signal transduction pathways underlying agonist-stimulated reorganization of the actin cytoskeleton in human umbilical vein endothelial cells. Receptor activation by thrombin, or the thrombin receptor (proteinase-activated receptor 1) agonist peptide, leads to an early increase in stress fiber formation followed by cortical actin accumulation and cell rounding. Selective inhibition of thrombin-stimulated signaling systems, including Gi/o (pertussis toxin sensitive), p42/p44, and p38 MAP kinase cascades, Src family kinases, PI-3 kinase, or S6 kinase pathways had no effect on the thrombin response. In contrast, staurosporine and KT5926, an inhibitor of myosin light chain kinase, effectively blocked thrombin-induced cell rounding and retraction. The contribution of Rho to these effects was analyzed by using bacterial toxins that either activate or inhibit the GTPase. Escherichia coli cytotoxic necrotizing factor 1, an activator of Rho, induced the appearance of dense actin cables across cells without perturbing monolayer integrity. Accordingly, lysophosphatidic acid, an activator of Rho-dependent stress fiber formation in fibroblasts, led to reorganization of polymerized actin into stress fibers but failed to induce cell rounding. Inhibition of Rho with Clostridium botulinum exoenzyme C3 fused to the B fragment of diphtheria toxin caused loss of stress fibers with only partial attenuation of thrombin-induced cell rounding. The implication of Rac and Cdc42 was analyzed in transient transfection experiments using either constitutively active (V12) or dominant-interfering (N17) mutants. Expression of RacV12 mimicked the effect of thrombin on cell rounding, and RacN17 blocked the response to thrombin, whereas Cdc42 mutants were without effect. These observations suggest that Rho is involved in the maintenance of endothelial barrier function and Rac participates in cytoskeletal remodeling by thrombin in human umbilical vein endothelial cells.     

Effects of pentoxifylline on sperm motility and hyperactivated motility in vitro: a preliminary report

Previous reports (2, 3) have suggested that pentoxifylline increases sperm motility. In this preliminary report based on five asthenozoospermic and five normal motility semen samples, we were unable to demonstrate any statistically significant effect of pentoxifylline on percent motility of human spermatozoa. However, in vitro exposure to capacitation medium with pentoxifylline may lead to an increase in total hyperactivated motility in asthenozoospermic samples, an effect not evident in the normal motility samples in this study.     

Gelsolin, a protein that caps the barbed ends and severs actin filaments, enhances the actin-based motility of Listeria monocytogenes in host cells

The actin-based motility of Listeria monocytogenes requires the addition of actin monomers to the barbed or plus ends of actin filaments. Immunofluorescence micrographs have demonstrated that gelsolin, a protein that both caps barbed ends and severs actin filaments, is concentrated directly behind motile bacteria at the junction between the actin filament rocket tail and the bacterium. In contrast, CapG, a protein that strictly caps actin filaments, fails to localize near intracellular Listeria. To explore the effect of increasing concentrations of gelsolin on bacterial motility, NIH 3T3 fibroblasts stably transfected with gelsolin cDNA were infected with Listeria. The C5 cell line containing 2.25 times control levels of gelsolin supported significantly higher velocities of bacterial movement than did control fibroblasts (mean +/- standard error of the mean, 0.09 +/- 0.003 micro(m)/s [n = 176] versus 0.05 +/- 0.003 micro(m)/s [n = 65]). The rate of disassembly of the Listeria-induced actin filament rocket tail was found to be independent of gelsolin content. Therefore, if increases in gelsolin content result in increases in Listeria-induced rocket tail assembly rates, a positive correlation between gelsolin content and tail length would be expected. BODIPY-phalloidin staining of four different stably transfected NIH 3T3 fibroblast cell lines confirmed this expectation (r = 0.92). Rocket tails were significantly longer in cells with a high gelsolin content. Microinjection of gelsolin 1/2 (consisting of the amino-terminal half of native gelsolin) also increased bacterial velocity by more than 2.2 times. Microinjection of CapG had no effect on bacterial movement. Cultured skin fibroblasts derived from gelsolin-null mice were capable of supporting intracellular Listeria motility at velocities comparable to those supported by wild-type skin fibroblasts. These experiments demonstrated that the surface of Listeria contains a polymerization zone that can block the barbed-end-capping activity of both gelsolin and CapG. The ability of Listeria to uncap actin filaments combined with the severing activity of gelsolin can accelerate actin-based motility. However, gelsolin is not absolutely required for the actin-based intracellular movement of Listeria because its function can be replaced by other actin regulatory proteins in gelsolin-null cells, demonstrating the functional redundancy of the actin system.     

Regulation of the actin cytoskeleton organization in yeast by a novel serine/threonine kinase Prk1p

Normal actin cytoskeleton organization in budding yeast requires the function of the Pan1p/ End3p complex. Mutations in PAN1 and END3 cause defects in the organization of actin cytoskeleton and endocytosis. By screening for mutations that can suppress the temperature sensitivity of a pan1 mutant (pan1-4), a novel serine/threonine kinase Prk1p is now identified as a new factor regulating the actin cytoskeleton organization in yeast. The suppression of pan1-4 by prk1 requires the presence of mutant Pan1p. Although viable, the prk1 mutant is unable to maintain an asymmetric distribution of the actin cytoskeleton at 37 degreesC. Consistent with its role in the regulation of actin cytoskeleton, Prk1p localizes to the regions of cell growth and coincides with the polarized actin patches. Overexpression of the PRK1 gene in wild-type cells leads to lethality and actin cytoskeleton abnormalities similar to those exhibited by the pan1 and end3 mutants. In vitro phosphorylation assays demonstrate that Prk1p is able to phosphorylate regions of Pan1p containing the LxxQxTG repeats, including the region responsible for binding to End3p. Based on these findings, we propose that the Prk1 protein kinase regulates the actin cytoskeleton organization by modulating the activities of some actin cytoskeleton-related proteins such as Pan1p/End3p.     

Response of microglial cells after a cryolesion in the peripheral proliferative retina of tench

We studied the glial response after inducing a lesion in the zone of the peripheral retina of tench, where there is proliferative neuroepithelium. In the retina and optic nerve, the microglial response was analysed with tomato lectin and the macroglial response with antibodies against GFAP and S-100. In lesioned retinas, there was a temporal-spatial distribution pattern of microglia. One day after lesion, primitive ramified cells appeared in the nerve fibre layer. These cells appeared progressively from the vitreal to the scleral layers until day 7 when cells appeared in all layers, with the exception of the outer plexiform layer. From this point, labelling decreased. In the optic nerve, 3 days after lesion, an increase in the number of microglial cells was observed, first in the nerve folds and from day 15 in specific areas of the optic nerve. In the central retina, in the optic nerve head and within the optic nerve itself, the appearance of microglial cells, after the lesion, near the blood vessels, could indicate a vascular origin of microglia, as has been proposed by many authors. However, we cannot discount the idea that some of the reactive microglial cells arise by proliferation of the microglia existing in the normal state. Using GFAP and S-100 antibodies, no important changes in the retina were observed, however in the optic nerve there was response to the lesion. Thus, the macroglial cells appeared to be involved in reorganisation of the optic nerve axons after lesion.