GLIA 3:301-310 (1990)
Remote Astrocytic Response as Demonstrated by Glial Fibrillary Acidic Protein Immunohistochemistry in the Visual Cortex of Dorsal Lateral Geniculate Nucleus Lesioned Rats FERENC HAJOS,~MIHALY KALMAN: K,AF~LZILLES: AXEL SCHLEICHER: AND PETER SOTONYI’ ‘Department of Anatomy and Histology, University of Veterinary Sciences, 1400 Budapest; ‘First Department of Anatomy, Sernmelweis University Medical School, 1450 Budapest, Hungary; 3Anatomical Institute I , University of Cologne, 0-5000 Cologne 41, Federal Republic of Germany
KEY WORDS
GFAP, Rat, Anterograde degeneration, Postlesional plasticity
ABSTRACT The reaction of astroglia was investigated after unilateral destruction of the dorsal lateral geniculate nucleus in the primary visual cortex of adult albino rats. The destruction of the dorsal lateral geniculate nucleus was performed by stereotaxic injections of ibotenic acid, and the location was verified in Nissl stained sections in each animal. Electron microscopic observations demonstrated the presence of degenerating axon terminals surrounded by hypertrophic astroglial processes mainly in layers I11 and IV of the ipsilateral primary visual cortex. The ipsilateral (impaired) and contralateral (control) sides of the primary visual cortex showed light microscopically a clearly differing appearance and distribution of glial fibrillary acidic protein (GFAP) immunoreactivity 7 to 11days after the unilateral injection of ibotenic acid into the dorsal lateral geniculate nucleus. Whereas the control side of the primary visual cortex showed GFAP staining only in the subpial zone of layer I and close to the white matter, all layers of the impaired cortex showed an intense GFAP immunoreactivity. The increase in immunoreactivity was confined t o the primary visual cortex. The extent of and increase in immunoreactivity was corroborated by image analysis. These findings were interpreted as a localized hypertrophy of astroglia caused by the anterograde degeneration of geniculocortical terminals. This hypertrophy is accompanied by an increase in GFAP, which may represent the stabilization of the cytoskeleton of newly formed glial processes involved in the rearrangement of the impaired neuropil.
INTRODUCTION
sponse called “reactive gliosis” (reviewed by Lindsay, 19861, a number of situations have been reported in There is now ample evidence for regarding astroglia which astroglia appeared to be involved in early and late as one of the functionally most responsive elements of CNS responses. How much of this glial reaction is the CNS (see, for references, Hajos and Basc6, 1984). related to phagocytosis and scar formation (Berry et al., Astroglia react with immediate swelling to ionic imbal- 1983; Reier et al., 1983) or to guidance of neuronal ances of the brain extracellular space, form the scar after CNS injuries, participate in the phagocytosis of Received September 4,1989; accepted February 12,1990. necrotic structures, provide guidance to neural growth Address requests to Prof. Dr. Karl Zilles, Anatomical Institute 1. and migration, etc. Since the first demonstration of the University ofreprint Cologne, Joseph-Stelzmann Str. 9,5000 Cologne 41, Federal Repubpost-traumatic and experimentally induced glial re- lic of Germany. 01990 Wiley-Liss, Inc.
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regeneration (Fallon, 1985; Rakic, 19811, and what its role is in graft survival (Stenevi et al., 1976) remain to be elucidated. A powerful tool to follow changes in number, size, and shape of astrocytes is their immunostaining with antibodies to glial fibrillary acidic protein (GFAP), a major protein of astroglial intermediate filaments (Bignami et al., 1972; Eng et al., 1971). This staining has been exploited to characterize morphologically astrocytes (Bjorklund et al., 1985; Hajos and Kalman, in press; Kalman and Hajos, in press), to decide upon the astrocytic nature of some ambiguous cell types (Basco et al., 1981; Bignami, 1984; de Vitry et al., 1981; Ikeda et al., 1980;Jesse and Mirsky, 1983; Suess and Pilska, 19811, to describe CNS areas of regular glial organization (Woodhams et al., 1981), to investigate the role of astroglia in brain development (Choi and Lapham, 1978;Woodhams et al., 1981),to demonstrate astroglial growth around neural tissue grafts (Azmitia and Whitaker, 1983; Bjorklund and Dahl, 1982; Bjorklund et al., 1984), and it proved to be of diagnostic value in pathological states accompanied by reactive gliosis (Bignami and Dahl, 1976;Bignami et al., 1980; Goebel et al., 1987;Vacca and Nelson, 1981; Vacca-Galloway, 1986). Owing to the high degree of connectivity in the CNS, nerve cell death necessarily brings about the degeneration of axons and synaptic terminals throughout the projection territory and vice versa, the transection of an axon effects the parent cell. Immunoreactive GFAP has been shown to increase in glial responses subsequent to neuronal degeneration, but while for the retrograde degeneration this has been demonstrated by using a straightforward experimental paradigm (Graeber and Kreutzberg, 1986; Tetzlaff et al., 19881, findings for the anterograde degeneration are vague because of rather diffuse,undefined lesions (Amaducciet al., 1981; Barret et al., 1981; Bignami and Dahl, 1976; Matthewson and Berry, 1985) and because of difficulties in assessing changes of staining. In this work we have adopted the stereotaxic destruction of a source of afferents to study the effect of a well-defined terminal degeneration on the GFAP staining of perisynaptic glia. The rat visual cortex was thought to be particularly suitable to investigate this problem for the following reasons: 1. Data suggest (Tugo et al., 1982;Kalman and Hajos, in press; Kamo et al., 1984) that there is hardly any immunoreactive GFAP present in the middle layers (111, IV, V) of the rat primary visual cortex (area Ocl according to Zilles, 1985) in sharp contrast to the intense staining of the superficial (I, 11)and deep (VI) cortical layers. 2. Geniculo-cortical fibres terminate in the middle cortical layers (mainly layer IV; Peters et al., 1976; Ribak and Peters, 1975; Schober and Winkelmann, 1977), so if their degeneration induced an astrocytic response, this should be reflected in the appearance of GFAP-staining of an area showing no appreciable immunoreactivity in the intact animal.
3. The dorsal nucleus of the ltiteral geniculate body is the major source of specific afferents to the primary visual cortex (see for review Sefton and Dreher, 1985). Its lesioning, therefore, results in a massive terminal degeneration. MATERIALS AND METHODS Adult albino rats (either sex) of 200 g body weight were operated under Avertin anaesthesia in a stereotaxic apparatus. The right dorsal lateral geniculate nucleus (DLG)was lesioned by inserting the cannula of a micro-syringe according to coordinates defined by Paxinos and Watson (1986). Through the cannula 5 to 10 pl of the excitotoxin ibotenic acid was injected into the dorsal nucleus of DLG. After survival periods of 3,5, 7, and 11days, rats were anaesthesized with ether and perfused through the aorta with a mixture of 4% paraformaldehyde and picric x i d dissolved in 0.1 M phosphate buffer (PB; pH 7.41. Brains were removed from the skull and immersed into the fixative for overnight. After a 24 h rinse in several changes of PB, 40 pm coronal sections were cut with a vibratome from the right and left occipital lobes and processed for immunohistochemistry. Further vibra tome sections were cut from the region of the DLG, mounted, and stained with cresyl violet. Some vibratome sections of the occipital cortex were postfixed in 1%osinic acid and after dehydration in graded ethanol and propylene oxide, were embedded in Durcupan (Fluka).Ultrathin sections were prepared with a Reichert ultramicrotome, viewed and photographed under a JEOL 100 B electron microscope. Vibratome sections of the occipital cortex from rats with histologically checked DLG lesions were processed for immunohistochemistry. The procedure started with a 5 min treatment with H202to exhaust endogenous peroxidase activity. After a short rinse in three changes of PB, nonspecific immunoreac tivity was suppressed by 20% normal goat serum in which sections were kept for 2 h at room temperature. Incubation with the primary antiserum lasted for 36 to 48 11 at 4"C, under vigorous shaking. Antiserum to glial Gbrillary acidic protein (GFAP) was raised and kindly provided by Dr. Rebecca Pruss (NIMH, Laboratory of Cell Biology, Bethesda). The primary antiserum was diluted 1:2,000. As second antibody, biotinylated rabbit IgG, and peroxidase-labelled streptavidin (Amersham) were used. The immune-complex molecule was visualized by the DAB reaction. For immunohistochemical c mtrol, sections were incubated with preabsorbed antiserum or by omitting the primary antiserum. For experimental control, corresponding sections of the occipital lobe contralateral to the lesion were compared with those from the operated side. Eng (1985) has described cross-reactivity of some monoclonal GFAP antibodies with tissue culture-derived vimentin. However, vimentin, as shown by Dahl (1981),is found in immature astrocytes. The use of adult
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vision of DLG (Fig. l),while in the visual cortex nerve cell bodies showed no chromatolytic changes. Electron microscopy has verified the presence of degenerating synaptic terminals engulfed by hypertrophic astroglial processes (Fig. 2A) mainly in layers I11 and IV of the ipsilateral primary visual cortex. The highest number of degenerating terminals was found 3 days after DLG lesion, a few were seen after 7 days, and none at all after 11 days. On the contrary, hypertrophic astrocyte processes containing an abundance of glycogen granules appeared at these later stages (Fig. 2B). In the occipital cortex contralateral to the lesion, GFAP immunoreactivity was distributed unevenly (Fig. 3). In addition to the intense staining of the glia limitans, immunoprecipate was primarily observed in the piriform cortex, retrosplenial area, hippocampus and white matter. The visual cortex showed GFAP staining only in the outer zone of layer I (“candlestick cells, Hajos and Kalman, in press) and close to the white matter, while the mid-region remained unstained or was only slightly stained. By contrast, on the impaired side (Fig. 3) intense GFAP immunoreactivity was observed outlining a wedge-liketerritory (Fig. 4)located in the primary visual cortex. This difference between the operated and control side was the most pronounced 7 RESULTS and 11 days after DLG-lesion. Upon higher magnificaNissl stained sections through the lesioned territory tion (Fig. 5) the gross immunostaining of the visual have shown extensive destructions in the dorsal subdi- cortex on the operated side was clearly resolved to be
animals for our in vivo experiments rules out a possible cross-reaction of our polyclonal GFAP antibody with vimentin. After qualitative evaluation and light microscopic photography of the immune reaction in the corresponding pairs of sections, preparations were transferred to an IBAS image analyser (Kontron, Munich). Regions of interest were scanned with a computer controlled scanning procedure (Schleicher et al., 1986). The microscopical images (12.5-fold lens, Universal, Zeiss, Oberkochen FRG) were digitized into grey level images (8 bit grey level resolution) using a TV camera. In these images, GFAP-positive structures were segmented by adaptive thresholding (Rosenfeld and Kak, 1976) and the areal fraction was measured in square measuring fields 40 X 40 pm in size. According to the x,y count of the measuring field, data were stored in a data matrix which represented the spatial distribution of the GFAPpositive structures in the specimen. The data matrix was plotted by subdividing the range of the areal fraction values (0%to 100%)into 11equidistant subranges, each of which is plotted in a distinct density pattern.
Fig. 1. Nissl-stained section showing the location of the lesion in the DLG. Tissue disintegration and local gliosis (asterisk)due to the lesion extends partly into the VLG. APT, anterior pretectal nucleus; DLG, dorsal lateral geniculate nucleus: fr, fasciculus retroflexus; LP, lateral
posterior thalamic nucleus; PF, parafascicular thalamic nucleus; PrC, precommissural nucleus; VLG, ventral lateral geniculate nucleus. ~33.
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Fig. 2.
REMOTE ASTROCYI'IC RESPONSE IN RAT VISUAL CORTEX
Fig. 3. Low-power survey micrograph showing the distribution of GFAP immunoreactivity in the intact occipital cortex. The primary visual cortex (Ocl) is practically unstained except for its subpial and deepest layers (arrowheads). X 2 3 .
composed of stellate astrocytes with irregular branching of cell processes except for the subpial layer, where GFAP immunoreactive processes were aligned vertically to the surface. There seemed to be a slight decrease in the intensity of immunostaining from the pial surface towards the deeper layers. However, at the white matter border a thin, heavily labelled zone was consistently encountered. While on the side contralateral to the lesion a sharp contrast occurred between the weakly immunoreactive layer VI and the immunonegative upper layers (Fig. 61, on the DLG-lesioned side this difference was abolished by the presence of evenly distributed GFAP-positive stellate astrocytes (Fig. 7). Image analysis has fully corroborated the above described distributional patterns. Computer plots of the regional and laminar distribution of immunoreactive cells transformed into areal fraction values clearly showed also in quantitative terms a marked difference between the GFAP-immunoreactivities of experimental and control sides. Plots of the intact side (Fig. 8) were truly reflecting the pattern observed in the histochemical preparations, and even minor differences in reaction
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Fig. 4. Low-power survey micrograph of the distribution of GFAP immunoreactivity in the occipital cortex on the DLG-lesioned side. Note the prominently immunostained wedge-like territory (between arrows) roughly corresponding to the primary visual cortex. x 23.
intensity hardly visible upon microscopic inspection were detected. Accordingly, on the control side the lowest immunoreactivity was unambiguously demonstrated in the middle layers of the visual cortex. In the plots of the impaired side (Fig. 9), the intense GFAP staining of the visual cortex was depicted as a territory of increased areal fraction well discernible from its surrounding. No staining occurred in preparations incubated with preabsorbed antiserum or with peroxidase conjugates alone. DISCUSSION
Our findings Provide experimental support to the assumption that GFAP immunoreactivity can be enhanced by means of a distant lesion affecting projections to the reactive area. The stimulus of this type of ghal response is obviously the anterograde degeneration of axOnS and Particularly synaptic terminals as it happened in the case of our experimental model, the geniculo-cortical system. The participation of cortical cells projecting to the DLG could be neglected as in parallel Nissl sections no sign of retrograde cellular reaction was Fig. 2. A-B: Electron micrograph of a degenerating presynaptic axon terminal in layer IV of the primary visual cortex 3 days after the seen. Interruption of the geniculocortical pathway by lesioningofthe ipsilateral DLG (A).The axonal terminal is dark but the lesioning of the DLG with ibotenic acid resulted in a postsyna tic element can still be perceived (arrowhead).Aglial process increase Of GFAP immunoreactivity in the (gl)enguis the degeneratingsynapse. At upper right a n intact synapse (sy)is visible. ~24,000.B shows a portion ofthe neuropil in layer IV 11 primary visual cortex. The nature of this type of glia days after Iesioning. An enlarged astrocyte process (a) can be seen activation, what can be termed remote astrocytic recontaining numerous glycogen particles. Degenerating axon terminals seen at earlier stages (A) are not visible at day 11 after lesioning (B). sponse, appeared to be biphasic in time. The first Phase x 18,000. was the engulfment and removal of degenerating termi-
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Fig. 5 . At higher magnification, GFAP-positive astrocytes constituting the lesion-induced GFAP-positive cell population of the primary visual cortex (Ocl J are easily recognized. The border towards the immunonegative cortical regons is sharp (arrowheads1. At the pial surface, glial processes are aligned vertically to the surface, while the white matter is demarcated from the cortex by a zone of intensely labelled cells (double arrowheads). Perivascular glial sheaths (arrows) are markedly immunopositive even in the apparently immunonegative cortical areas. x69.
nals by perisynaptic glial processes; the second, the GFAP response. The first events of perisynaptic glial activation have been known since the early electron microscopic observations of experimental degeneration in the CNS (Miguel and Haymaker, 1965). Presently, our interest was the second phase of remote astrocytic response: the increase in GFAP immunoreactivity at a
time when the phagocytosis of degenerated synapses had already been completed. The rat visual cortex offered a favourable situation to document this phenomenon, as under normal conditions it almost completely lacks GFAP immunoreactive astroglia (Bignami and Dahl, 1976; Kalman and Hajos, in press; Kamo et al., 1984; Tug0 et al., 1982). Interpretation of our findings is based on the fact that with routine histological methods astrocytes can be demonstrated evenly distributed in the visual cortex (Wree et al., 19801, i.e., also in the layers immunonegative in the control. With a less diluted antiserum and changing of fixation conditions we were able to show a faint staining of astrocytes also in the middle cortical layers (unpublished data), but relative differences in staining intensity between the outer and inner versus the middle cortical layers remained unaltered. Thus the increase in GFAP of pre-existing glial elements may account for the results obtaincd, without the need to suppose a glial proliferation underlying the appearance or intensification of immunoreactivity. The proliferation of astroglia in the adult brain has been claimed mainly on the basis of thymidine autoradiography (for references see Korr, 1986), bul, mitotic figures clearly attributable to remote astrocytic response are still a matter of debate. Although in their pioneering work Bignami and Ralston (1969) an11 Cavanagh (1970)have pointed out mitotic figures folhwing Wallerian degeneration and around a stab wound of the CNS, respectively, their number did not account for the massive thymidine incorporation nor did their morphology correspond unequivocally to that o’dividing astrocytes. On the other hand, Latov et al. (1979) and Janeczko (1989) have verified astrocyte prolifmation around a brain injury using combined autoradiography and GFAP immunocytochemistry. This, however, has not been proven for remote astrocyte response; on the contrary a substantial body of evidence suggests that in these situations the autoradiograph cally observed reactive proliferation of “nonneuronal” cells (Matthews, 1977) involves oligodendroglia andlor microglia (Adrian and Williams, 1973; Avendano and Cowan, 1979; Del RioHortega and Penfield, 1927; Gall et al., 1979;Kerns and Hinsman, 1976a,b; Ludwin, 1984,1985; Murabe et al., 1981) and even macrophages contribute to the proliferating population (Ling, 1979;Murray and Walter, 1973; Stenwig, 1972). Recently, Tet zlaff et al. (1988) have mentioned an observation wi ;h electron microscopic autoradiography demonstrating the lack of thymidine incorporation into astrocytes but a proliferation of microglia during retrograde reaction. This supports the view of Vaughn et al. (1970) that degenerating cell bodies are removed by the reactive microglia, while astroglia participate in the phC3gocytosisof degenerating synaptic boutons. It seems, l,herefore,that in spite of extensive studies, interpretations of reactive astrocytic response are controversial. In our opinion local periinjural and remote astrocytic responses should be regarded as essentially different reactions. Further differ-
REMOTE ASTROCYTIC RESPONSE IN RAT VISUAL CORTEX
Figs. 6,7. Border zone between layers V and VI of the primary visual cortex on the intact (Fi . 6 )and on the DLG-lesioned (Fig. 7) side. While on the intact side layer VI contains faintly immunostainefastrocytes and layer V is unstained, on the DLG-lesioned side the even distribution of intensely GFAP-immunoreactive astrocytes abolishes the border (dashed line) between the two layers. ~ 3 3 5 .
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Figs. 8, 9. Computer plots of areal fraction values obtained by the automatic image analysis of GFAP-immunostained occipital cortices. Plots reflect reliably the distributional patterns observed under the microscope. On the intact side (Fig. 81, the primar visual cortex (Ocl) is seen to contain the lowest amount of label T i e resolution of the method is remarkable as judged either by the radial stripes of increased grey-level (arrows)in the middle of the primary visual cortex
corresponding to glial sheaths of blood iessels (see Fig. 5 ) , or by the clear distinction ofdentate gyrus (DG)1a:iers.Note also the delineation of the stained zone at the border of white matter (WM). The vast increase in GFAP staining of primary vlsual cortex (between arrows) on the DLG-lesioned side (Fig. 9) as cclmpared to the intact side is conspicuous.
ences may occur between remote astrocytic response induced by retrograde and anterograde degenerations. While the problem of glial response following retrograde degeneration (Graeber et al., 1986; Kerns and Hinsman, 1976a,b;Ling, 1979; Tetzlaff et al., 1988; Vaughn et al., 1970) is becoming better understood, the same cannot be said about the anterograde reaction, mainly because of the undefined stereotaxic parameters of the experiments reported in the literature. Our observations concerning the electron microscopic changes and GFAP expression are the first relevant to a clear-cut situation of anterograde degeneration resulting from an aimed lesion and affecting a circumscribed projection. Results obtained argue for a hypertrophy and/or redistribution (Rose et al., 1976) rather than proliferation of reactive astrocytes emitting new processes to engulf degenerating synapses (Vaughn et al., 1970) and to fill in vacancies. The relatively late increase of GFAP immunoreactivity as compared to the peak time of terminal degeneration is in agreement with the recent findings of Cortez et al. (1989)and Janeczko (1989)and may represent the stabilization period of the cytoskeleton of newly formed glial processes adapted t o the changed spatial situation in a neuropil deprived of its main input. A similar phenomenon has been observed in vitro by Liesi et al. (19831, who found that newly formed cultures of astrocytes expressed laminin, while GFAP was characteristic for stabilized cultures.
Further implications of remote astrocytic response may be relevant to the involvement of the CNS as a whole in any local destructive processes, depending on the extent of projections of the damaged area (Barret et al., 1981; Isacson et al., 1987).Remote gliotic effects may be less dramatic in regions not as closely interconnected as the geniculocortical sj,stem, but may, in turn, be more widespread (Bignami iind Dahl, 1976; Cortez et al., 1989; Janeczko, 1989). 'The applied method of computer-assisted image analysis (Schleicher et al., 1986)is thought, therefore, to be a most useful approach to detect less obvious cases of reriote astrocytic response and to express in objective terins changes in reaction intensity. To this end, image analysis maps of the distribution of GFAP-immunostaining in serial sections of the normal rat brain are in preparation in our laboratory to be used as a reference in further experimental studies of astrocytes.
ACKNOWLEDGrMENTS This work was supported by grants of the DGF (Zi 192/6-2)and the Hungarian Academy of Sciences.
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Tugo, M., Kumashiro, M., Kimura, M., and Maeda, T. (1982)Distribution of glial fibnllary acidic protein in the rat brain studied by immunohistochemistry. J . Hzstochem. Cytochem., 30:561. Vacca, C.L. and Nelson, S.R. (1984) Glial cells in Huntington's chorea. Adu. Cell Neuro biol., 5:221-241. Vacca-Galloway, L.L. (1986) Astrocytzs in Huntington's chorea. In: Astrocytes, Vol. 3. S. Fedoroff and A. Vernadakis, eds. Academic Press, New York, pp. 357-386. Vaughn, J.E., Hinds, P.L., and Skoff, F:.P. (1970) Electron microscopic studies of Wallerian degeneration in rat optic nerves. I. The multipotential glia. J . Comp. Neurol., 140:1"5-206. Woodhams, P.L., Basco, E., Hajos, F., C:sillag,A., andBalazs, R. (1981) Radial glia in the develo ing mouse cerebral cortex and hippocampus. Anat. Embryol. (Bert),163:331--343. Wree, A,, Zilles, K., and Schleicher, A. (1980) Analyse der laminaren Strukturen der Area striata mit verschiedenen stereologischen Messmethoden. Verh.Anat. Ges., 74:727,728. Zilles, K. (1985) The Cortex ofthe Rut. A Stereotaxic Atlas. SpringerVerlag, Berlin, 121 pp.
Remote Astrocytic Response as Demonstrated by Glial Fibrillary Acidic Protein Immunohistochemistry in the Visual Cortex of Dorsal Lateral Geniculate Nucleus Lesioned Rats FERENC HAJOS,~MIHALY KALMAN: K,AF~LZILLES: AXEL SCHLEICHER: AND PETER SOTONYI’ ‘Department of Anatomy and Histology, University of Veterinary Sciences, 1400 Budapest; ‘First Department of Anatomy, Sernmelweis University Medical School, 1450 Budapest, Hungary; 3Anatomical Institute I , University of Cologne, 0-5000 Cologne 41, Federal Republic of Germany
KEY WORDS
GFAP, Rat, Anterograde degeneration, Postlesional plasticity
ABSTRACT The reaction of astroglia was investigated after unilateral destruction of the dorsal lateral geniculate nucleus in the primary visual cortex of adult albino rats. The destruction of the dorsal lateral geniculate nucleus was performed by stereotaxic injections of ibotenic acid, and the location was verified in Nissl stained sections in each animal. Electron microscopic observations demonstrated the presence of degenerating axon terminals surrounded by hypertrophic astroglial processes mainly in layers I11 and IV of the ipsilateral primary visual cortex. The ipsilateral (impaired) and contralateral (control) sides of the primary visual cortex showed light microscopically a clearly differing appearance and distribution of glial fibrillary acidic protein (GFAP) immunoreactivity 7 to 11days after the unilateral injection of ibotenic acid into the dorsal lateral geniculate nucleus. Whereas the control side of the primary visual cortex showed GFAP staining only in the subpial zone of layer I and close to the white matter, all layers of the impaired cortex showed an intense GFAP immunoreactivity. The increase in immunoreactivity was confined t o the primary visual cortex. The extent of and increase in immunoreactivity was corroborated by image analysis. These findings were interpreted as a localized hypertrophy of astroglia caused by the anterograde degeneration of geniculocortical terminals. This hypertrophy is accompanied by an increase in GFAP, which may represent the stabilization of the cytoskeleton of newly formed glial processes involved in the rearrangement of the impaired neuropil.
INTRODUCTION
sponse called “reactive gliosis” (reviewed by Lindsay, 19861, a number of situations have been reported in There is now ample evidence for regarding astroglia which astroglia appeared to be involved in early and late as one of the functionally most responsive elements of CNS responses. How much of this glial reaction is the CNS (see, for references, Hajos and Basc6, 1984). related to phagocytosis and scar formation (Berry et al., Astroglia react with immediate swelling to ionic imbal- 1983; Reier et al., 1983) or to guidance of neuronal ances of the brain extracellular space, form the scar after CNS injuries, participate in the phagocytosis of Received September 4,1989; accepted February 12,1990. necrotic structures, provide guidance to neural growth Address requests to Prof. Dr. Karl Zilles, Anatomical Institute 1. and migration, etc. Since the first demonstration of the University ofreprint Cologne, Joseph-Stelzmann Str. 9,5000 Cologne 41, Federal Repubpost-traumatic and experimentally induced glial re- lic of Germany. 01990 Wiley-Liss, Inc.
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regeneration (Fallon, 1985; Rakic, 19811, and what its role is in graft survival (Stenevi et al., 1976) remain to be elucidated. A powerful tool to follow changes in number, size, and shape of astrocytes is their immunostaining with antibodies to glial fibrillary acidic protein (GFAP), a major protein of astroglial intermediate filaments (Bignami et al., 1972; Eng et al., 1971). This staining has been exploited to characterize morphologically astrocytes (Bjorklund et al., 1985; Hajos and Kalman, in press; Kalman and Hajos, in press), to decide upon the astrocytic nature of some ambiguous cell types (Basco et al., 1981; Bignami, 1984; de Vitry et al., 1981; Ikeda et al., 1980;Jesse and Mirsky, 1983; Suess and Pilska, 19811, to describe CNS areas of regular glial organization (Woodhams et al., 1981), to investigate the role of astroglia in brain development (Choi and Lapham, 1978;Woodhams et al., 1981),to demonstrate astroglial growth around neural tissue grafts (Azmitia and Whitaker, 1983; Bjorklund and Dahl, 1982; Bjorklund et al., 1984), and it proved to be of diagnostic value in pathological states accompanied by reactive gliosis (Bignami and Dahl, 1976;Bignami et al., 1980; Goebel et al., 1987;Vacca and Nelson, 1981; Vacca-Galloway, 1986). Owing to the high degree of connectivity in the CNS, nerve cell death necessarily brings about the degeneration of axons and synaptic terminals throughout the projection territory and vice versa, the transection of an axon effects the parent cell. Immunoreactive GFAP has been shown to increase in glial responses subsequent to neuronal degeneration, but while for the retrograde degeneration this has been demonstrated by using a straightforward experimental paradigm (Graeber and Kreutzberg, 1986; Tetzlaff et al., 19881, findings for the anterograde degeneration are vague because of rather diffuse,undefined lesions (Amaducciet al., 1981; Barret et al., 1981; Bignami and Dahl, 1976; Matthewson and Berry, 1985) and because of difficulties in assessing changes of staining. In this work we have adopted the stereotaxic destruction of a source of afferents to study the effect of a well-defined terminal degeneration on the GFAP staining of perisynaptic glia. The rat visual cortex was thought to be particularly suitable to investigate this problem for the following reasons: 1. Data suggest (Tugo et al., 1982;Kalman and Hajos, in press; Kamo et al., 1984) that there is hardly any immunoreactive GFAP present in the middle layers (111, IV, V) of the rat primary visual cortex (area Ocl according to Zilles, 1985) in sharp contrast to the intense staining of the superficial (I, 11)and deep (VI) cortical layers. 2. Geniculo-cortical fibres terminate in the middle cortical layers (mainly layer IV; Peters et al., 1976; Ribak and Peters, 1975; Schober and Winkelmann, 1977), so if their degeneration induced an astrocytic response, this should be reflected in the appearance of GFAP-staining of an area showing no appreciable immunoreactivity in the intact animal.
3. The dorsal nucleus of the ltiteral geniculate body is the major source of specific afferents to the primary visual cortex (see for review Sefton and Dreher, 1985). Its lesioning, therefore, results in a massive terminal degeneration. MATERIALS AND METHODS Adult albino rats (either sex) of 200 g body weight were operated under Avertin anaesthesia in a stereotaxic apparatus. The right dorsal lateral geniculate nucleus (DLG)was lesioned by inserting the cannula of a micro-syringe according to coordinates defined by Paxinos and Watson (1986). Through the cannula 5 to 10 pl of the excitotoxin ibotenic acid was injected into the dorsal nucleus of DLG. After survival periods of 3,5, 7, and 11days, rats were anaesthesized with ether and perfused through the aorta with a mixture of 4% paraformaldehyde and picric x i d dissolved in 0.1 M phosphate buffer (PB; pH 7.41. Brains were removed from the skull and immersed into the fixative for overnight. After a 24 h rinse in several changes of PB, 40 pm coronal sections were cut with a vibratome from the right and left occipital lobes and processed for immunohistochemistry. Further vibra tome sections were cut from the region of the DLG, mounted, and stained with cresyl violet. Some vibratome sections of the occipital cortex were postfixed in 1%osinic acid and after dehydration in graded ethanol and propylene oxide, were embedded in Durcupan (Fluka).Ultrathin sections were prepared with a Reichert ultramicrotome, viewed and photographed under a JEOL 100 B electron microscope. Vibratome sections of the occipital cortex from rats with histologically checked DLG lesions were processed for immunohistochemistry. The procedure started with a 5 min treatment with H202to exhaust endogenous peroxidase activity. After a short rinse in three changes of PB, nonspecific immunoreac tivity was suppressed by 20% normal goat serum in which sections were kept for 2 h at room temperature. Incubation with the primary antiserum lasted for 36 to 48 11 at 4"C, under vigorous shaking. Antiserum to glial Gbrillary acidic protein (GFAP) was raised and kindly provided by Dr. Rebecca Pruss (NIMH, Laboratory of Cell Biology, Bethesda). The primary antiserum was diluted 1:2,000. As second antibody, biotinylated rabbit IgG, and peroxidase-labelled streptavidin (Amersham) were used. The immune-complex molecule was visualized by the DAB reaction. For immunohistochemical c mtrol, sections were incubated with preabsorbed antiserum or by omitting the primary antiserum. For experimental control, corresponding sections of the occipital lobe contralateral to the lesion were compared with those from the operated side. Eng (1985) has described cross-reactivity of some monoclonal GFAP antibodies with tissue culture-derived vimentin. However, vimentin, as shown by Dahl (1981),is found in immature astrocytes. The use of adult
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vision of DLG (Fig. l),while in the visual cortex nerve cell bodies showed no chromatolytic changes. Electron microscopy has verified the presence of degenerating synaptic terminals engulfed by hypertrophic astroglial processes (Fig. 2A) mainly in layers I11 and IV of the ipsilateral primary visual cortex. The highest number of degenerating terminals was found 3 days after DLG lesion, a few were seen after 7 days, and none at all after 11 days. On the contrary, hypertrophic astrocyte processes containing an abundance of glycogen granules appeared at these later stages (Fig. 2B). In the occipital cortex contralateral to the lesion, GFAP immunoreactivity was distributed unevenly (Fig. 3). In addition to the intense staining of the glia limitans, immunoprecipate was primarily observed in the piriform cortex, retrosplenial area, hippocampus and white matter. The visual cortex showed GFAP staining only in the outer zone of layer I (“candlestick cells, Hajos and Kalman, in press) and close to the white matter, while the mid-region remained unstained or was only slightly stained. By contrast, on the impaired side (Fig. 3) intense GFAP immunoreactivity was observed outlining a wedge-liketerritory (Fig. 4)located in the primary visual cortex. This difference between the operated and control side was the most pronounced 7 RESULTS and 11 days after DLG-lesion. Upon higher magnificaNissl stained sections through the lesioned territory tion (Fig. 5) the gross immunostaining of the visual have shown extensive destructions in the dorsal subdi- cortex on the operated side was clearly resolved to be
animals for our in vivo experiments rules out a possible cross-reaction of our polyclonal GFAP antibody with vimentin. After qualitative evaluation and light microscopic photography of the immune reaction in the corresponding pairs of sections, preparations were transferred to an IBAS image analyser (Kontron, Munich). Regions of interest were scanned with a computer controlled scanning procedure (Schleicher et al., 1986). The microscopical images (12.5-fold lens, Universal, Zeiss, Oberkochen FRG) were digitized into grey level images (8 bit grey level resolution) using a TV camera. In these images, GFAP-positive structures were segmented by adaptive thresholding (Rosenfeld and Kak, 1976) and the areal fraction was measured in square measuring fields 40 X 40 pm in size. According to the x,y count of the measuring field, data were stored in a data matrix which represented the spatial distribution of the GFAPpositive structures in the specimen. The data matrix was plotted by subdividing the range of the areal fraction values (0%to 100%)into 11equidistant subranges, each of which is plotted in a distinct density pattern.
Fig. 1. Nissl-stained section showing the location of the lesion in the DLG. Tissue disintegration and local gliosis (asterisk)due to the lesion extends partly into the VLG. APT, anterior pretectal nucleus; DLG, dorsal lateral geniculate nucleus: fr, fasciculus retroflexus; LP, lateral
posterior thalamic nucleus; PF, parafascicular thalamic nucleus; PrC, precommissural nucleus; VLG, ventral lateral geniculate nucleus. ~33.
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Fig. 2.
REMOTE ASTROCYI'IC RESPONSE IN RAT VISUAL CORTEX
Fig. 3. Low-power survey micrograph showing the distribution of GFAP immunoreactivity in the intact occipital cortex. The primary visual cortex (Ocl) is practically unstained except for its subpial and deepest layers (arrowheads). X 2 3 .
composed of stellate astrocytes with irregular branching of cell processes except for the subpial layer, where GFAP immunoreactive processes were aligned vertically to the surface. There seemed to be a slight decrease in the intensity of immunostaining from the pial surface towards the deeper layers. However, at the white matter border a thin, heavily labelled zone was consistently encountered. While on the side contralateral to the lesion a sharp contrast occurred between the weakly immunoreactive layer VI and the immunonegative upper layers (Fig. 61, on the DLG-lesioned side this difference was abolished by the presence of evenly distributed GFAP-positive stellate astrocytes (Fig. 7). Image analysis has fully corroborated the above described distributional patterns. Computer plots of the regional and laminar distribution of immunoreactive cells transformed into areal fraction values clearly showed also in quantitative terms a marked difference between the GFAP-immunoreactivities of experimental and control sides. Plots of the intact side (Fig. 8) were truly reflecting the pattern observed in the histochemical preparations, and even minor differences in reaction
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Fig. 4. Low-power survey micrograph of the distribution of GFAP immunoreactivity in the occipital cortex on the DLG-lesioned side. Note the prominently immunostained wedge-like territory (between arrows) roughly corresponding to the primary visual cortex. x 23.
intensity hardly visible upon microscopic inspection were detected. Accordingly, on the control side the lowest immunoreactivity was unambiguously demonstrated in the middle layers of the visual cortex. In the plots of the impaired side (Fig. 9), the intense GFAP staining of the visual cortex was depicted as a territory of increased areal fraction well discernible from its surrounding. No staining occurred in preparations incubated with preabsorbed antiserum or with peroxidase conjugates alone. DISCUSSION
Our findings Provide experimental support to the assumption that GFAP immunoreactivity can be enhanced by means of a distant lesion affecting projections to the reactive area. The stimulus of this type of ghal response is obviously the anterograde degeneration of axOnS and Particularly synaptic terminals as it happened in the case of our experimental model, the geniculo-cortical system. The participation of cortical cells projecting to the DLG could be neglected as in parallel Nissl sections no sign of retrograde cellular reaction was Fig. 2. A-B: Electron micrograph of a degenerating presynaptic axon terminal in layer IV of the primary visual cortex 3 days after the seen. Interruption of the geniculocortical pathway by lesioningofthe ipsilateral DLG (A).The axonal terminal is dark but the lesioning of the DLG with ibotenic acid resulted in a postsyna tic element can still be perceived (arrowhead).Aglial process increase Of GFAP immunoreactivity in the (gl)enguis the degeneratingsynapse. At upper right a n intact synapse (sy)is visible. ~24,000.B shows a portion ofthe neuropil in layer IV 11 primary visual cortex. The nature of this type of glia days after Iesioning. An enlarged astrocyte process (a) can be seen activation, what can be termed remote astrocytic recontaining numerous glycogen particles. Degenerating axon terminals seen at earlier stages (A) are not visible at day 11 after lesioning (B). sponse, appeared to be biphasic in time. The first Phase x 18,000. was the engulfment and removal of degenerating termi-
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Fig. 5 . At higher magnification, GFAP-positive astrocytes constituting the lesion-induced GFAP-positive cell population of the primary visual cortex (Ocl J are easily recognized. The border towards the immunonegative cortical regons is sharp (arrowheads1. At the pial surface, glial processes are aligned vertically to the surface, while the white matter is demarcated from the cortex by a zone of intensely labelled cells (double arrowheads). Perivascular glial sheaths (arrows) are markedly immunopositive even in the apparently immunonegative cortical areas. x69.
nals by perisynaptic glial processes; the second, the GFAP response. The first events of perisynaptic glial activation have been known since the early electron microscopic observations of experimental degeneration in the CNS (Miguel and Haymaker, 1965). Presently, our interest was the second phase of remote astrocytic response: the increase in GFAP immunoreactivity at a
time when the phagocytosis of degenerated synapses had already been completed. The rat visual cortex offered a favourable situation to document this phenomenon, as under normal conditions it almost completely lacks GFAP immunoreactive astroglia (Bignami and Dahl, 1976; Kalman and Hajos, in press; Kamo et al., 1984; Tug0 et al., 1982). Interpretation of our findings is based on the fact that with routine histological methods astrocytes can be demonstrated evenly distributed in the visual cortex (Wree et al., 19801, i.e., also in the layers immunonegative in the control. With a less diluted antiserum and changing of fixation conditions we were able to show a faint staining of astrocytes also in the middle cortical layers (unpublished data), but relative differences in staining intensity between the outer and inner versus the middle cortical layers remained unaltered. Thus the increase in GFAP of pre-existing glial elements may account for the results obtaincd, without the need to suppose a glial proliferation underlying the appearance or intensification of immunoreactivity. The proliferation of astroglia in the adult brain has been claimed mainly on the basis of thymidine autoradiography (for references see Korr, 1986), bul, mitotic figures clearly attributable to remote astrocytic response are still a matter of debate. Although in their pioneering work Bignami and Ralston (1969) an11 Cavanagh (1970)have pointed out mitotic figures folhwing Wallerian degeneration and around a stab wound of the CNS, respectively, their number did not account for the massive thymidine incorporation nor did their morphology correspond unequivocally to that o’dividing astrocytes. On the other hand, Latov et al. (1979) and Janeczko (1989) have verified astrocyte prolifmation around a brain injury using combined autoradiography and GFAP immunocytochemistry. This, however, has not been proven for remote astrocyte response; on the contrary a substantial body of evidence suggests that in these situations the autoradiograph cally observed reactive proliferation of “nonneuronal” cells (Matthews, 1977) involves oligodendroglia andlor microglia (Adrian and Williams, 1973; Avendano and Cowan, 1979; Del RioHortega and Penfield, 1927; Gall et al., 1979;Kerns and Hinsman, 1976a,b; Ludwin, 1984,1985; Murabe et al., 1981) and even macrophages contribute to the proliferating population (Ling, 1979;Murray and Walter, 1973; Stenwig, 1972). Recently, Tet zlaff et al. (1988) have mentioned an observation wi ;h electron microscopic autoradiography demonstrating the lack of thymidine incorporation into astrocytes but a proliferation of microglia during retrograde reaction. This supports the view of Vaughn et al. (1970) that degenerating cell bodies are removed by the reactive microglia, while astroglia participate in the phC3gocytosisof degenerating synaptic boutons. It seems, l,herefore,that in spite of extensive studies, interpretations of reactive astrocytic response are controversial. In our opinion local periinjural and remote astrocytic responses should be regarded as essentially different reactions. Further differ-
REMOTE ASTROCYTIC RESPONSE IN RAT VISUAL CORTEX
Figs. 6,7. Border zone between layers V and VI of the primary visual cortex on the intact (Fi . 6 )and on the DLG-lesioned (Fig. 7) side. While on the intact side layer VI contains faintly immunostainefastrocytes and layer V is unstained, on the DLG-lesioned side the even distribution of intensely GFAP-immunoreactive astrocytes abolishes the border (dashed line) between the two layers. ~ 3 3 5 .
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Figs. 8, 9. Computer plots of areal fraction values obtained by the automatic image analysis of GFAP-immunostained occipital cortices. Plots reflect reliably the distributional patterns observed under the microscope. On the intact side (Fig. 81, the primar visual cortex (Ocl) is seen to contain the lowest amount of label T i e resolution of the method is remarkable as judged either by the radial stripes of increased grey-level (arrows)in the middle of the primary visual cortex
corresponding to glial sheaths of blood iessels (see Fig. 5 ) , or by the clear distinction ofdentate gyrus (DG)1a:iers.Note also the delineation of the stained zone at the border of white matter (WM). The vast increase in GFAP staining of primary vlsual cortex (between arrows) on the DLG-lesioned side (Fig. 9) as cclmpared to the intact side is conspicuous.
ences may occur between remote astrocytic response induced by retrograde and anterograde degenerations. While the problem of glial response following retrograde degeneration (Graeber et al., 1986; Kerns and Hinsman, 1976a,b;Ling, 1979; Tetzlaff et al., 1988; Vaughn et al., 1970) is becoming better understood, the same cannot be said about the anterograde reaction, mainly because of the undefined stereotaxic parameters of the experiments reported in the literature. Our observations concerning the electron microscopic changes and GFAP expression are the first relevant to a clear-cut situation of anterograde degeneration resulting from an aimed lesion and affecting a circumscribed projection. Results obtained argue for a hypertrophy and/or redistribution (Rose et al., 1976) rather than proliferation of reactive astrocytes emitting new processes to engulf degenerating synapses (Vaughn et al., 1970) and to fill in vacancies. The relatively late increase of GFAP immunoreactivity as compared to the peak time of terminal degeneration is in agreement with the recent findings of Cortez et al. (1989)and Janeczko (1989)and may represent the stabilization period of the cytoskeleton of newly formed glial processes adapted t o the changed spatial situation in a neuropil deprived of its main input. A similar phenomenon has been observed in vitro by Liesi et al. (19831, who found that newly formed cultures of astrocytes expressed laminin, while GFAP was characteristic for stabilized cultures.
Further implications of remote astrocytic response may be relevant to the involvement of the CNS as a whole in any local destructive processes, depending on the extent of projections of the damaged area (Barret et al., 1981; Isacson et al., 1987).Remote gliotic effects may be less dramatic in regions not as closely interconnected as the geniculocortical sj,stem, but may, in turn, be more widespread (Bignami iind Dahl, 1976; Cortez et al., 1989; Janeczko, 1989). 'The applied method of computer-assisted image analysis (Schleicher et al., 1986)is thought, therefore, to be a most useful approach to detect less obvious cases of reriote astrocytic response and to express in objective terins changes in reaction intensity. To this end, image analysis maps of the distribution of GFAP-immunostaining in serial sections of the normal rat brain are in preparation in our laboratory to be used as a reference in further experimental studies of astrocytes.
ACKNOWLEDGrMENTS This work was supported by grants of the DGF (Zi 192/6-2)and the Hungarian Academy of Sciences.
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