Tesis doctoral de Ana María Santos Carro
Desarrollo de la microglía y respuesta a la fotodegeneración inducida por luz intensa en la retina de ratón general introduction microglial cells are involved in the surveillance and cleaning of the central nervous system (cns), in the immune reactions taking place in the nervous parenchyma, and probably also in the building of the mature organization of the cns (kreutzberg, 1996; aloisi, 2001; mallat et al., 2005). They are thought to originate from cells of mesodermal lineage that colonize the cns (cuadros and navascués, 1998). Microglial cells adopt distinct morphological and immunophenotypical features in different situations, and it is useful to distinguish between three major types of microglial cells: developing microglial cells, which are frequent during embryonic and postnatal development. Most of them are amoeboid cells and are therefore called amoeboid microglia. mature microglial cells, which have a mature level of ramification, and are thought to be quiescent cells that survey the nervous parenchyma. reactive microglial cells, which derive from mature microglial cells that face an insult in the cns. Their activation is accompanied by changes in shape, with retraction of cell processes, and increased immunoreactivity (streit et al., 1999). these types are thought to correspond to different developmental and functional states of the same cell. Moreover, microglial cells are not homogenously distributed throughout the cns (lawson et al., 1990; mittelbronn et al., 2001), suggesting that past or present local factors influence the distribution and state of differentiation of microglial cells. there is evidence that microglial cells of the developing cns are able to respond to various types of injury, although features of the microglial reaction differ between the developing and adult cns (graeber et al., 1998; cuadros et al., 2000; sánchez-lópez et al., 2005). chapter i: development of microglial cells in the mouse retina the presence of microglial cells in the adult retina has been described in several species, including fish (dowding et al., 1991; salvador-silva et al., 2000), amphibians (goodbrand and gaze, 1991), birds (navascués et al., 1994; won et al., 2000), rabbits (ashwell, 1989; schnitzer, 1989; humphrey and moore, 1996), mice (zhang et al., 2005b), rats (ashwell et al., 1989; harada et al., 2002; zhang et al., 2005a), monkeys (vrabec, 1970; boycott and hopkins, 1981) and humans (provis et al., 1995; penfold et al., 1991, 2001; yang et al., 2000; gupta et al., 2003). However, the pattern of their emergence during development has been far less investigated. this process and some of the factors that can affect it have been extensively studied in our laboratory by using an antibody that recognizes microglial cells in the quail (navascués et al., 1994, 1995; marín-teva et al., 1998, 1999a, b; sánchez-lópez et al., 2004). These studies revealed that microglial cells colonize the quail retina by two different forms of migration: tangential and radial, with microglial cells first spreading tangentially along the developing nerve fibre layer (nfl) and then reaching other retinal layers by radial migration. There are fewer data on microglial development in the retina in other species. These include detailed reports on the presence and distribution of microglia in the developing retina in rats (ashwell et al., 1989) and rabbits (ashwell, 1989; schnitzer, 1989) and studies that investigated only some stages of development in mice (hume et al., 1983; hughes et al., 2003) and humans (diaz-araya et al., 1995a, b). we considered that a complete picture of the normal distribution of microglia during development was necessary before analyzing the response of microglial cells to an injury in the retina: these data would yield insights into the effects of retinal disturbances on the normal distribution of microglial cells and allow detection of the migratory behaviour of microglial cells. For this purpose, we aimed to produce an accurate description of the development of microglial cells in the retina by using several microglial markers (anti-iba-1, anti-f4/80, anti-cd45, and anti-cd68 antibodies and tomato lectin histochemistry) expecting most retinal microglial cells to be labelled by at least one of these markers. Our results demonstrate that anti-iba1, which recognizes a calcium binding protein present in cells of monocytic lineage, is a good marker of microglial cells throughout their development, and it was therefore the main one used in our study. we also compared results obtained between two strains of mice, pigmented c57bl/6 and albino balb/c mice, in order to establish whether the microglial distribution differs between albino and pigmented mouse retinas, as reported by other authors. macrophage/microglial cells were already present in the retina of embryos aged 11.5 days (e11.5), in association with cell death. At e12.5, some macrophage/microglial cells also appeared in peripheral regions of the retina, with no apparent relation to cell death. macrophage/microglial cells increased in number thereafter and sometimes showed some ramification. Immediately before birth, microglial cells were present in the neuroblastic (nbl), inner plexiform (ipl) and ganglion cell (gcl) layers. Their distribution suggested that they entered the retina from the ciliary margin and the vitreous. the distribution of retinal microglial cells markedly changed immediately after birth, when microglial cells were almost entirely restricted to the more vitreal part of the retina and were absent from its outer part. The microglial cells appear to move from this vitreal location in scleral direction in order to colonize other layers of the retina. Hence, they would colonize the retina by radial migration in a vitreal-to-scleral direction, as observed in development of the quail retina. the density of microglia showed a simultaneous decrease in early postnatal retinas compared with the pre-birth density. The density of retinal microglia increased during the first postnatal week and then decreased again, ultimately lowering to the density levels of microglia observed in normal adults. These changes in density are likely due to variations in the appearance of new retinal microglial cells. Thus, although we have no explanation for the postnatal decrease in microglial density, the increase in density during the first postnatal week may be related to the incorporation of new macrophage/microglial cells into the retina, as suggested by the frequent observation of cells apparently traversing the vitreal border. The incorporation of vitreal cells into the retina appeared to diminish after p7, and microglial density in the retina would decrease if the entry of precursors was inadequate to compensate for the retinal growth. after the changes in microglial cell distribution and density observed during the first two postnatal weeks, the mature distribution pattern of microglial cell distribution was settled by p14. From this age on, many microglial cells frequently showed thinner processes with a more developed ramification. in the adult retina, mature microglia appeared in the nfl, gcl, ipl, opl and, sometimes, in the inl. It is noteworthy that microglial cells never appeared in the onl. No evident differences in microglia distribution were observed between in albino and pigmented retinas, although some quantitative variations cannot be ruled out. in an attempt to establish whether tangential migration of microglial cells occurs in the developing mouse retina, retinal explants of p0-p3 retinas were labelled with ant-iba1 antibody. A clear tangential migration, as in the quail, was not observed in these explants. in summary, the mature topographical distribution pattern of microglia emerged during postnatal development of the retina, and microglial cells were present within all layers of the retina except the onl by the end of the second postnatal week. Once microglial cells reached their definitive location, they progressively increased their ramification chapter ii: microglial response in the retina to photodegeneration induced by intense light exposure both continuous exposure to light of moderate intensity and brief exposure to intense light cause retinal degeneration. Similar to the retinal degeneration caused by numerous genetic factors, the degeneration caused by light exposure (that will be called photodegeneration) first causes the death of photoreceptor cells (noell et al., 1966; lavail et al., 1998; remé et al., 1998; organisciak et al., 2000; grimm et al., 2004; zhang et al., 2005b). As a result, photoreceptor degeneration induced by intense light exposure is considered a useful model to study degenerative photoreceptor cell processes in the human retina, as found in retinitis pigmentosa and age-related macular degeneration. our aim was to study the response of microglia to retinal photoreceptor degeneration after intense light exposure. For this purpose, mice were exposed to intense light (10,000 lux) for 7 hours and were then kept in complete darkness for 6, 12, 18, 24, 36, 48 or 72 hours or 10 days. before studying the microglial response in retinal degeneration, we employed several methods to confirm that our light exposure procedure was effective in producing photodegeneration in the mouse retina. first the morphological features of control and light-treated retinas were considered. Semithin sections showed that retinas subjected to the photodegeneration protocol were clearly thinner than control retinas. This thinning was mainly due to the decrease in size of the onl as consequence of a fall in the number of photoreceptors, indicating the death of many of them. more precise information about cell death phenomena in photodegeneration was obtained by other techniques. Thus, an elisa was used to quantify cell death in the retina after photodegeneration by determining the number of free nucleosomes in retinal extracts from control animals and from animals killed at different times after light exposure. This procedure revealed that cell death in the photodegenerating retina is extensive during the 48 hours after light exposure and decreases thereafter, although cell death levels remained much higher than in control retinas at 10 days after light exposure. although this methodology yielded quantitative data on retinal cell death, it offered no insight into the distribution of the dying cells in the retina, which was examined by means of the tunel technique. While virtually no tunel- labelled nuclei were seen in control retinas, tunel-labelled bodies were detected in +6h retinas and became more numerous over the first 24 hours of survival time. This technique also revealed that the majority of degenerating nuclei were in the onl, where photoreceptor cell nuclei are located. a transmission electron microscopy (tem) study also revealed the presence of degenerating photoreceptor cells in photodegenerating retinas shortly after the light exposure. Therefore, the three methods (elisa quantification of free nucleosomes, tunel and tem) gave comparable results and showed significantly greater cell death in photodegenerating versus normal retinas. The tem and tunel studies also showed that most of this cell death occurs in the onl. Having established that our light exposure protocol produces extensive photoreceptor cell death, we studied the features of retinal microglial cells in the photodegenerating retinas. the macrophage/microglial cells in the photodegenerating retinas were clearly activated, as revealed by the following features: i) they migrated to the region of early injury (onl and subretinal space). ii) instead of being more or less ramified cells, retinal microglia became rounded cells with short thick processes; iii) the cells showed immunophenotypic changes, revealed by the increased expression of various immunomolecules. each of these features is discussed below. modification of the distribution of retinal microglial cells after photodegeneration the distribution pattern of microglial cells in the retinal layers of photodegenerating retinas clearly differed from the pattern in control retinas. Thus, microglial cells invaded the onl, which is devoid of them in normal retinas, and numerous large-sized macrophages appeared in the subretinal space, contrasting with the low number of smaller cells in this space found in normal eyes. the increase in the number of macrophage/microglial cells in the onl and subretinal space was quantified by measuring the density of cells labelled with sra (that marks activation of macrophage/microglial cells, see below) at different survival times after light exposure. This technique revealed a rapid increase in the number of activated macrophage/microglial cells, which were already detected in +6h retinas; the presence of activated cells did not change for two days but then decreased and was close to control levels at ten days after light exposure. therefore, macrophage/microglial cells appeared in the onl at the same time as the degeneration of photoreceptor cells, as reported in different pathological retinal conditions (thanos, 1992; roque et al., 1996; harada et al., 2002; gupta et al., 2003; hughes et al., 2003; zeiss and johnson, 2004; zeiss et al., 2004; zeng et al., 2005; zhang et al., 2005a, b). this increase in microglial cells in the photodegenerating retina may result from the increased expression of some molecules and not from a real increase in macrophage/microglial cell number. For example, no microglial cell labelling by anti-sra was observed in the normal retinas, while many microglial cells were labelled with this antibody after photodegeneration. This observation may be due to an increase in the number of already present microglial cells expressing the immunomolecule that it recognizes. However, it is also likely that new cells showing sra expression would be incorporated into the retina. Sra expression by cells already present in the retina and the entry of new sra-expressing cells probably both contribute to the increase in sra expression. the new macrophage/microglial cells present in the onl and subretinal space in photodegenerating retinas may have different origins: 1. They may originate from cells that migrate from other regions of the nervous system, either from inside (e.G. Inner retinal layers) or outside (e.G. The optic nerve) the retina. 2. They may be produced by the proliferation of microglial precursors within the retina. 3. They may come from cells that invade the retina from non-nervous locations, such as circulating blood, choroids, vitreous and ciliary body. With regard to the two latter origins, retinas immunostained with anti-cd68 and anti-sra antibodies showed the frequent presence of labelled cells on the vitreal margin of the retina and at the frontier between the peripheral retina and ciliary body. Hence, some macrophage/microglial cells appear to enter the retina from the ciliary body and the vitreous. the above routes of entry into the retina are not exclusive, and some or all of them contribute to the increase in macrophage/microglial number. Thus, microglial cells from the inner retinal layers may move to the onl, leaving an empty space to be replenished by cells entering from the vitreous. morphological changes in retinal microglial cells in photodegenerating retinas as seen in chapter one, microglial cells in the mature normal mouse retina are ramified cells bearing thin processes with developed ramification. The morphological appearance of these cells was greatly altered after light exposure. Whereas microglial cells showed some ramification in layers outside the onl, cells in the onl that were labelled by the markers used (see below) were more or less compact and occasionally bore processes that engulfed several photoreceptor cells. This phenomenon sometimes gave the onl labelling a honeycomb appearance. labelled cells in the subretinal space had no or only short processes. Some of the cells in this location contained phagosomes that appeared to include pigment debris from the degenerating outer segments of photoreceptor cells, as revealed by confocal microscopy and tem studies. time course of the expression of microglial markers the expression of various cell markers was observed in the activated macrophage/microglial cells. Specifically, we studied the expression of the molecules recognized by anti-cd45, anti-cd68, anti-f4/80 and anti-sra antibodies. Different markers were used in order to obtain a more complete picture of the response of macrophage/microglial cells in photodegeneration. the expression of all of these molecules was considerably higher in the outer layers of the retinas (onl and subretinal space) shortly after the light exposure (+6h) and continued to increase thereafter. anti-cd45 antibody labelled microglial cells in the ipl, opl and gcl of control retinas. The intensity of the anti-cd45 labelling was higher in photodegenerating retinas and strongly marked cells appeared in the onl, while many other positive cells were observed in innermost layers of the retina. Large labelled cells were also seen in the choroid and subretinal space. anti-cd68 is restricted in non-activated cells to lysosomal membranes and does not mark the complete outline of the cell, therefore only sparse anti-cd68 labelling was observed in normal retina. The intensity of labelling was higher after light exposure but, unlike cell membrane markers, anti-cd68 did not label highly complex cells with developed processes. scarce cells were weakly labelled by anti-f4/80 in the normal retinas, while stronger labelling was seen in some cells of the subretinal space. The number of labelled cells and the intensity of the labelling were higher in degenerating retinas. In general, labelling with anti-f4/80 antibody was delayed with respect to anti-cd45 labelling, although the two antibodies showed a similar pattern of labelling at later survival times. control retinas were nearly devoid of anti-sra labelling, which only marked some blood vessel-associated cells. Some increase in sra expression was detected at +6 h, although it was more evident at 12 h after light exposure. cells that were immunopositive for these markers were frequently observed in internal layers of the retina after 36 hours of survival. Whereas the presence of labelled cells in the onl and subretinal space would be related to the degeneration of different parts of photoreceptor cells, labelled cells in the internal retina are probably related to the death of intermediate neurons in the inl due to the primary degeneration of photoreceptor cells. In this regard, tunel-labelled cell fragments were detected at the same time in photodegenerating retinas. a noticeable decrease in anti-cd68 and anti-sra labelling was observed in +10-day retinas, but anti-cd45 and anti-f4/80 labelling showed no changes after shorter survival times. hence, anti-cd45, anti-cd68, anti-sra and anti-f4/80 may be considered as markers of microglial activation in the retina, since substantial differences in labelling are found between normal and photodegenerating retinas. role of microglial cells during photodegeneration as reported above, activated macrophage/microglial cells accumulate in affected regions, suggesting that they play some function in the degeneration that occurs after light exposure. However, the nature of their role has yet to be elucidated. it has been shown that macrophage/microglial cells phagocyte the debris produced during degeneration, and some of our observations indicate that this also occurs during photodegeneration. an alternative view is that these cells actively induce the death of photoreceptor cells by the release of cytotoxic factors or the non-release of cytoprotective ones. Our observations reveal that some cell death occurs before microglial activation is detected. in summary, it is unlikely that microglial cells actively participate in the death of the first degenerating photoreceptors but they may be responsible for death processes at later times, given the clear signs of their activation long after the light exposure.
Datos académicos de la tesis doctoral «Desarrollo de la microglíay respuesta a la fotodegeneración inducida por luz intensa en la retina de ratón«
- Título de la tesis: Desarrollo de la microglíay respuesta a la fotodegeneración inducida por luz intensa en la retina de ratón
- Autor: Ana María Santos Carro
- Universidad: Granada
- Fecha de lectura de la tesis: 23/01/2009
Dirección y tribunal
- Director de la tesis
- Julio Navascues Martinez
- Tribunal
- Presidente del tribunal: Juan mario Hurle gonzalez
- José María Frade lópez (vocal)
- michel Mallat (vocal)
- Francisco david Martín oliva (vocal)