Histol Histopathol (1999) 14: 89-101 Histology and 001 : 10.14670/HH-14.89 Histopathology http://www.hh.um.es Radial glia and cell debris removal during lesion-regeneration of the lizard medial cortex J. Nacher, C. Ramirez, J.J. Palop, A. Molowny, J.A. Luis de la Iglesia and C. L6pez-Garcia Neurobiology, Cell Biology, Faculty of Biological Sciences, University of Valencia, Valencia, Spain Summary. Intraperitoneal injection of the neurotoxin 3- Introduction acetyl pyridine (3AP) induces a rapid degeneration of the medial cerebral cortex (lizard fascia dentata) granular The lizard cerebral cortex has been considered as a layer and of its zinc enriched axonal projection (lizard reptilian hippocampus on the basis of its connectivity, mossy fibres). After 6-8 weeks post-lesion the cell debris cytoarchitectonics and development (Lopez-Garda et have been removed and the granular layer is repopulated al., 1992). The medial cortex, a region resembling the by neurons generated in the subjacent ependyma. Both mammalian fascia dentata (Molowny and Lopez-Garda, processes, neuron incorporation and debris removal, 1978; Lopez-Garcia et al.,1983; Lopez-Garcia and seem to be crucial for successful regeneration. Martinez-Guijano, 1988), undergoes postnatal neuro­ Scavenging processes in the lesioned mammalian CNS genesis (Lopez-Garda et al., 1988) and is capable of are usually carried out by microglia and/or astrocytes. In regenerating itself if lesioned specifically with the the lizard cerebral cortex there are no free astrocytes and neurotoxin 3-acetylpyridine (3AP) (Font et al., 1991). the only glial fibrillary acid (GFAP) immunoreactive The new neurons that replace the lesioned ones are cells are radial glia-ependymocytes, similar to generated in the subjacent neuroepithelium (sulcus) those present during mammalian CNS development. which shows cell proliferative activity throughout the Ependymocytes, in addition to their help in vertical lizard life span (Kirsche, 1967; L6pez-Garcia et aI., migrations of just generated immature neurons, built the 1988). cortical glial scaffold, insulate the blood capillaries, form In mammals, nervous tissue damage is usually the outer glial limiting membrane, thus playing an followed by a characteristic gliotic response in which essential role in the lizard cortical blood-brain barrier. In both microglia and astroglia participate (see Landis, this study, by means of GFAP-immunocytochemistry 1994). An injury to the mammalian CNS usually implies and electron microscopy, we have shown that radial glial the formation of a glial scar, mainly constituted of cells participate actively in the removal/phagocytosis of reactive astrocytes; this scar impedes correct growth of cellular debris generated in the lesion process: mainly new axon branches and thus prevents neuronal circuitry degenerated synapses, but interestingly, also some repair (Berry et al., 1983; Reier, 1986). neuronal somata. Cell debris taken up by ependymocyte Debris removal seems to be crucial to achieving a lateral processes seem to be progressively transported to successful restoration of damaged adult nervous tissue. either distal (pial) or proximal (ventricular) poles of the Glial cells are beneficial for neural repair, removing cell, where they result in lipofuscin accumulations. The degenerating debris (Pow et aI., 1989) and providing hypothetical subsequent exchange of debris from structural support in regions undergoing substantial ependymoglia by microglia/macrophages and Kolmer degeneration and cell death (Gentschev and Sotelo, cells is discussed. 1973). Nevertheless, the neuronal debris is rather inefficiently removed (Sloviter et at., 1993, 1996) and this could be one factor that prevents neuronal Key words: Ependyma, Phagocytosis, Hippocampus, regeneration in the mammalian CNS. Postnatal neurogenesis, 3-acetylpyrydine In lizards, microglia cells transiently disappear from the medial cortex during the first days following the 3AP-Iesion, re-invading it afterwards (Lopez-Garcia et a I., 1994). The participation of these cells in debris removal is delayed and unexpected. Moreover, in the Offprint requests to: Prof. C. L6pez-Garcia, Neurobiologia, Biologia lizard cerebral cortex there are no free astrocytes. The Celular, Facultad de Ciencias Biol6gicas, Universilal de Valencia, 46100 only glial fibrillar acidic protein (GFAP) immuno­ Burjassol, Valencia, Spain. e-mail: [email protected] reactive cells are ependymocyte-radial glia cells, closely 90 Radial glia and lesion-regeneration of the lizard cortex similar to those of embryonic mammalian nervous tissue microscope to asses the lesion extent of the neurotoxin. (Rakic, 1982; Rickmann et aI., 1987; Yanes et aI., 1992). Selected sections were re-embedded; ultrathin sections Ependymocyte-radial glia cell somata are localised in obtained from them were lead stained and observed the ependyma lining the cerebral ventricles. Long radial under the electron microscope. processes arise from these somata; these radial shafts In addition, some lizard cortex Golgi impregnated traverse the cortex reaching the pial surface, where their sections belonging to the laboratory collection were also end-feet form the limiting glial membrane of the brain used in this study. parenchyma. Abundant varicose processes arising from the ependymocyte-radial glia shafts cover, on one side, GFAP immunocytochemistry the blood vessel surface forming part of the blood-brain barrier (Garcfa-Verdugo et aI., 1981) and, on the other After Polywax removal with alcohol or resin side, partially cover synaptic contacts (perisynaptic glial removal with etoxide, sections were immunostained as processes) and the rest of neuronal processes. Moreover, described previously (Nacher et aI., 1996). Briefly, the initial segment of these ependymocyte shafts may sections were incubated overnight in a polyclonal anti­ guide the newly generated neurons to their final location GFAP antibody (Sigma, St. Louis, MO) 1:500 dilution. in the medial cortex (Garcia-Verdugo et aI., 1986). The second layer was a goat anti-rabbit [gG [n order to study the role of radial g[ia in the debris (Sternberger-Meyer, Baltimore) 1: 1 00 dilution, for 30 scavenging after lizard medial cortex lesion, we have minutes, followed by the third layer: Rabbit peroxidase­ used GFAP immunocytochemistry as well as electron antiperoxidase (PAP) complex (Sternberger-Meyer, microscopy. Our results confirm that radial g[ia Baltimore) 1:100 dilution for 30 minutes. 3,3'­ participates in the debris removal. diaminobenzidine 4HCI (Sigma, St. Louis) and H20 2 were used for colour development. Controls without Materia[s and methods primary antibody were done for assessment of correct immunostaining. 56 Healthy adult lizards (4-5,5 cm snout-vent) of the species Podarcis hispanica, captured in the surroundings Results of Burjassot, Valencia (Spain) and maintained in terraria simulating their environmental conditions were used in GFAP immunostalnlng of slices revealed the this study. Experimental protocols were carried out ependymocyte-radial glia scaffold of the lizard cerebral according to the guidelines concerning animal care and cortex (Fig. lA) and also showed the conspicuous protection in our institution. lamination pattern of the medial cortex. Silver chromate An intraperitoneal injection of the neurotoxin 3- impregnation of isolated ependymocyte-radial glia cells acetylpyridine (3AP) (dose: 150 mg Kg-l b/w) was (Fig. IB) also revealed the different segments of the performed in 32 lizards. Non injected lizards (n=7) were radial glia[ shafts, i.e., a deep or proximal-slender used as negative controls. At various days post-lesion (1, segment close to the ependyma, a second varicose 2,4, 7, 15, 30 and 42 days), animals under ether segment while crossing the inner dendritic layer beneath anaesthesia were transcardially perfused with either 4% the cell layer, a third-slender segment while crossing the paraformaldehyde in 0.1M phosphate buffer, pH 7.2-7.4 cell layer and finally some outer plexiform layer (n=18) for light mjcroscopy, or with 2 % para­ branches reaching the pial surface. Ramification of formaldehyde, 2% glutaraldehyde in O.lM phosphate ependymocyte-radial glial branches in the outer buffer (n=21) for electron microscopy. Then the brains plexiform layer of the medial cortex was frequent. were removed and postfixed in the same fixative for 6 hours at 4 0c. After dehydrating, the brains were either Time-course of morphological changes induced by 3AP embedded in Polywax (BOH, Poole) and serially lesion sectioned (parallel series of 10 .urn thick transverse sections) or transversely sliced (75-lOO.um thick slices) In semithin and Polywax sections, necrotic nuclei using a vibratome. Glutaraldehyde fixed slices were (seen as pyknotic in toluidine blue stained sections and postfixed in 1 % osmium tetroxide and embedded in assessed under electron microscopy), swollen somata epoxy resin (TAAB, Aldersmarton). Semithin sections and cytoplasmatic processes were clearly recognised were toluidine blue stained and observed under the light after 3AP injection (Fig. 1 C, 0). The lesion degree was Fig. 1. A. Radial glia scaffold in the medial and dorsomedial cortices of a control lizard. Vibratome section (75 pm thick) immunostained for GFAP. DMC: dorsomedial cortex; MC: medial cortex; ep: ependyma; ipl: inner plexiform layer; cl: cell layer; opl: outer plexiform layer. B. Photomontage of Golgi impregnated radial glia cells in the lizard medial cortex. The somata are located in the ependyma