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Monoclonal antibodies to keratan sulfate immunolocalize ramified microglia in paraffin and cryostat sections of rat brain. A Bertolotto, B Caterson, G Canavese, A Migheli and D Schiffer J Histochem Cytochem 1993 41: 481 DOI: 10.1177/41.4.8450191 The online version of this article can be found at: http://jhc.sagepub.com/content/41/4/481

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The Joumal of Histochemistry and Cytochemistry Copyright 0 1993 by The Histochemical Society, Inc.

Vol. 41, No. 4, pp. 481-487. 1993 Printed in U X A .

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Monoclonal Antibodies to Keratan Sulfate Immunolocalize Ramified Microglia in Paraffin and Cryostat Sections of Rat Brain' ANTONIO BERTOLGMQ2 BRUCE CATERSON, GABRIELLA CANAVESE, ANTONIO MIGHELI, and DAVIDE SCHIFFER Clinica Neurologica II, Universit,? di Torino, Torino, Italy (AB,GC,AM,DS),and Departmenf of Surgery, School of Medicine, North Carolina University at Chapel Hill, Chapel Hill, North Carolina (BC). Received for publicationJanuary 1, 1992 and in revised form August 12, 1992 and November 12, 1992; accepted December 11, 1992 (2C2163).

We used six monoclonal antibodies (MAb) recognizing epitopes within keratan sulfate (KS) chains for an immunocytochemical study of adult rat brain. One of the MAb selectively stained microglia and their d i e d processes. KSpositive cells were found throughout the CNS in both p a d h e m b e d d e d and cryostat sections; the greatest number were present in hippocampus and brainstem. In the cortex the positive processes of some cells surrounded neuronal somata. In the white matter the processes were both parallel and perpendicular to the axon bundles. Double staining showed that KS-positive cells did not express astrocytic or oligodendroglialmarkers. By immunoelectron microscopy,

Introduction Increasing interest has been devoted to central nervous system (CNS) proteoglycans (PGs) because of their various biological functions (32). A wide variety of these molecules are produced by CNS cells, and changes have been detected both during development (10, 16,32,36,45)and in pathological states (32). In normal adult CNS, specific PGs have been localized in the surrounding extracellular matrix or at the cell surfaces of neuronal and glial subpopulations, suggesting selective PG expression by different cell subsets (1-3, 17,19,25,30).Some PGs have also been found intracellularly (21, 32,35,42). Resting (ramified) microglia are considered by many to be the resting macrophage population of the CNS, which can be stimulated into active macrophages under pathological conditions ( 2 7 ~ 7 )The . immunohistochemical identification of resting microglia in rat is dependent on tissue processing. In paraffin-embedded rat CNS these cells are visualized only with some lectins (31,49,50),whereas

Supported by the Associazione Italiana Sclerosi Multipla (AISM) and by Project CNR 91.00368.40. * Correspondence to: Dr. Antonio Bertolotto, Clinica Neurologica 11, Via Cherasco, 15, 10126 Torino, Italy.

the positivity was localized around the perikarya and cell processes of small cells with peripheral chromatin clumps and dark cytoplasm, which often contained secondary lysosomes. The KS-positive cells did not contribute to myelin sheaths and were not surrounded by a basal membrane. In addition to the cellular staining, three other MAb stained the white matter diffusely. Anti-KS MAb are therefore proposed as immunohistochemicalmarkers for ramified microglia in both pa&i and cryostat sectionsof adult rat brain. ( JHistochem Cytochem 41:481487,1993) KEY WORDS: Microglia; Astrocyte; Oligodendroglia; Macrophage;

Proteoglycans; Immunoelectron microscopy.

in cryostat and vibratome sections resting microglia are immunolocalized with several antibodies and lectins (9,18,37,38,48,52). The present study shows that resting microglia express at their cell surfaces a KS-PG that is detected by MAb recognizing epitopes present in the KS chains of PGs (7,8,33,47,51).Moreover, our data indicate that anti-KS MAb identify resting microglia on both cryostat sections and paraffin-embedded tissue. Therefore, these MAb may facilitate the identification of resting microglia, which are involved in many neurological diseases and neurobiological processes (13,22).

Materials and Methods Rats and Tissue Processing. Adult (lo-24-week-old) Fisher rats were anesthetized with ether and the brains were immediately removed, fixed in Carnoy's fluid at 4'C overnight or in 10% buffered formalin for 24-hr at room temperature, embedded in paraffin, and cut at 5 pm in the coronal plane. For cryostat sections, the brains were removed either unfixed or after perfusion through the heart with PLP [2% paraformaldehyde (PAF), 75 mM lysine, 0.2% periodate] in 50 mM phosphate buffer, pH 7.4, then frozen in liquid nitrogen-cooled 2-methylbutane and cut at 8 pm. The unfixed sections were placed in cold acetone for 11 min. Other rats were perfused with 4% PAF and the brains were embedded in paraffin. Antisera. Anti-KS MAb used in this study were produced by one of

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us (BC) in mice, using either purified mammalian cartilage proteoglycans (PGs) (MAb 5D4, 2D3, 1B4, 1D2) or purified embryonic chick bone marrow PGs (MAb 4D1, 8C2) as antigen (73). Three of the five MAb (5D4, 2D3, 184) specifically recognize sulfate N-acetyllactosamineunits present in both Type I (corneal) and Type I1 (cartilage)KS chains (7,33,47,51).The other MAb (lD2.4D1, 8c2) recognize epitopes present in KS chains, but the antigenic component of KS is not known (47). 5D4 is also commercially available from ICN (Costa Mesa, CA). Polyclonal anti-glial fibrillar acidic protein (GFAP) was purchased from Dako (Santa Barbara, CA) and anti-carbonicanhydrase C (CA-C)from Calbiochem (La Jolla, CA).

Immunohistochemid Procedures. The peroxidase-anti-peroxidase (PAP) method was applied on both deparaffinized and cryostat sections. After inhibition of endogenous peroxidase with 3% hydrogen peroxide for 5 min. the sections were incubated subsequently with: (a) primary antibody overnight at 4°C (anti-KS MAb at 1:750 dilution, anti-GFAP serum 1:250, anti-CA-C serum 1:200); (b) rabbit anti-mouse Ig (1:30) or swine anti-rabbit Ig (Dako) 1:50 for 1 hr at room temperature; and (c) PAP complex for 1 hr at room temperature. Between each step the sections were rinsed in 0.01 M PBS, pH 7.4, for 10 min. After peroxidase development with 0.05 % diaminobenzidine tetrahydrochloride (DAB) and 0.01% hydrogen peroxide in 0.5 M Tris buffer, pH 7.6, for 1-15 min, the sections were rinsed in distilled water and counterstained with hematoxylin. Control Sections and Enzymatic Pre-digestion with Keratanase. Control sections were either incubated without primary antibodies or with MAb 5D4 diluted 1:750 in PBS containing 10, 100, or 1000 pg/ml of Type I and Type I1 KS. For pre-digestion, sections were incubated for 2 hr at 37'C with ker. Tokyo, Japan) at 0.05 IUlml in atanase from Preudomonas ~ p(SeikagaJcu; 100 mM Tris-HCI buffer, pH 8, containing 10 mM EDTA, 10 mM N-ethylmaleimide, 5 mM PMSF, and 0.36 pM pepstatin A. Double Staining. Formalin-fixed, paraffin-embedded sections were simultaneouslyincubated with MAb 5D4 and polyclonal anti-CA-Cserum overnight at 4'C. Then the immunostaining with CA-C was dcveloped with the PAP method described above, omitting the hematoxylin counterstaining. Subsequently, the sections were incubated with rhodamine-conjugated sheep anti-mouse IgG (1:20) (Sera-Lab; Crowley Down, Sussex, UK) for 1 hr at room temperature and were mounted in glycerin-PBS. For double immunofluorescence staining with polyclonal anti-GFAP and MAb 5D4,lO-pm cryostat sections of PLP-perfused rat were incubated with primary antibodies overnight at 4'C, then with fluorescein-conjugated swine anti-rabbit Ig ( M O ) (Dako) and rhodamine-conjugated sheep antimouse Ig (Sera-Lab),for 1 hr at room temperature. Sections were mounted in glycerin-PBS and observed with a Zeiss uv microscope with epiillumination.

Immunoelectron Microscopy. After perfusion with either 4% PAF or PLP, 50-pm coronal sections were cut with a vibratome. Free-floating sections were stained with MAb 5D4 as described in the immunohistochemical procedures. After DAB reaction. sections were post-fixed in 2% osmium tetroxide, dehydrated, and flat-embedded in Epon.Thin sections were lightly stained with uranyl acetate and lead citrate.

BERTOLOITO, CATERSON, CANAVESE, MIGHELI, SCHIFFER

Results Immunohistochemical Findings with Anti-KS M A b SO4 Sections incubated with MAb 5D4 showed selective staining of a glial population (Figure I), whereas neuronal, meningeal, and endothelial cells were negative. The KS-positive cells had a stellate morphology characterized by several sharply branching processes with many delicate spines (Figure IC). They were localized throughout the CNS parenchyma. In some regions, such as the hippocampus and brainstem (Figure 1A), they were numerous, whereas in the cerebellar cortex they were rare. In the gray matter they were located in all the corticallayers and sometimes showed a perineuronal localization with processes surrounding the neuronal perikarya (Figure 1D). In the white matter KS-positive cells sent processes both parallel and transverse to axon bundles (Figure 1B). Some KSpositive cells had the perikaryon located near a vessel (Figure 3B) and extended their processes into the CNS parenchyma (Figures 1A and 1B). Other KS-positive cells extended their processes towards the tissue side of a blood vessel (Figures 1A and 1B). The nuclei were oval or round and were strongly stained by hematoxylin.

Effects of Tissue Processing on MA6 SO4 Immunoreactivity MAb SD4-immunolocalizedKS-positive cells were observed with all the fixation and embedding procedures tested: (a) paraffin embedding by immersion in Carnoy's fluid (Figures 1C and ID) or buffered formalin; (b) p d i embeddmg after 4% PAF perfusion (Figures 1A and 1B); and (c) cryostat sections from PLP-perfused brain (Figure 2) or acetone-fixed sections. No strong difference in the number of KS-positive cells was noted among the different fixatives;however, cell processes were more evident in the white matter of brains perfused with 4% PAF and embedded in paraffin.

Control Sections and Enzymatic Pre-dzgestion with Keratanase Control sections incubated without the primary antibodies or with MAb SD4 diluted 1:750 in PBS containing at least 100 pg/ml of KS Type I and Type I1 were negative. Keratanase digestion of tissue sections did not remove 5D4 reactivity. This is not surprising, as keratanase does not digest heavily sulfated KS oligosaccharidessuch as the 5D4 epitope (33.34, 36,47).

+ Figure 1. MAb 5D4 staining in adult rat CNS. Several Spositive cells are evident in (A) brainstem and (8) cerebellar white matter. Wpositive Cell processes are branching; in the white matter they are both transverse and parallelto the axonal bundles, (C) The cell processes show many delicate spines. (0) In the cortex some KS-positivecells are located near a neuron and their positive cell processes surround the neuronal perikaryon. Neuronal and endothelial Cells are negative. The brains were perfused with PAF (A,B) 01 fixed in Carnoy's (C,D), then paraffin-embedded. PAP method, hematoxylin counterstaining. Bars; A = 100 pm; B,D = 50 pm; C = 20 pm.

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other anti-KS MAb (1B4, 1D2, and 2D3). These MAb, however, also diffusely stained the white matter; in addition, MAb 2D3 strongly stained the gray matter. The fifth MAb, 8C2, stained only the white matter, and the last MAb, 4D1, was negative.

Immunoelectron Microscopy Previous attempts to localize KS using glutaraldehyde-based fixatives or post-embedding techniques were not successful. Best results were obtained when mild fixation with PLP was used. KS positivity surrounded scattered small cells with dark cytoplasm and cell processes of various lengths. The reaction product of DAB was evenly distributed along the cytoplasmic membrane (Figure 3). The nucleus contained clumps of peripherally located chromatin (Figure 3). The cytoplasm was often filled by large lipofuscin granules and lysosomal dense bodies and did not show KS immunostaining. KSpositive cells did not appear to be related to myelin sheaths. A few positive cells, not surrounded by a basal membrane, were found in a perivascular location (Figure 3B).

Discussion

Figure 2. Double immunofluorescence staining of a cryostat coronal section through the hippocampus of a rat perfusedwith PLI? The morphology of GFAPpositive astrocytes (A; fluorescein optic) is different from that of KS-positivecells (E;rhodamine optic). Bar = 100 pm.

Antigenic Expression of KS-positive Cells Neither immunohistochemistry on serial sections nor doublestaining with MAb 5D4 and polyclonal anti-GFAP (Figure 2) or with CA-C serum (not shown) detected expression of GFAP or CA-C in KS-positive cells.

Immunohistochemical Findings with Other Anti-KS MAb KS-positive cells showing identical morphology and tissue localization as those identified with MAb 5D4 were also evident in Carnoy’s fluid-fixed, paraffin-embedded sections incubated with three

This study shows that the anti-KS MAb 5D4 selectively localizes a glial subpopulation, identified as ramified (resting) microglia, in paraffin and cryostat sections of normal adult rat. KS-positive cells present all the characteristics of resting microglia: morphology (15), parenchymal localization (23), and ultrastructure (5,28). Moreover, they are not stained by anti-GFAP and CA-C antisera, considered markers for astrocytes (4) and mature rat oligodendrocytes, respectively (29). MAb 5D4 provides a new and additional means for staining rat resting microglia. In fact, a KS epitope has never been described at the cell surface of microglia. Moreover, MAb 5D4 visualizes rat resting microglia both on cryostat, like other MAb (9,11,18,24.38,39), and on paraffin-embedded sections, like some lectins (31,49,50). Therefore, 5D4 facilitates the study of this cell population, which is involved in the CNS immunoresponse (22). Alzheimer’s disease (53), neuronal regeneration (6). CNS infections (40). multiple sclerosis (12). and trauma (20). The epitopes recognized by MAb 5D4 and the other anti-KS MAb have been studied by utilizing hexaand larger oligosaccharidesderived from cartilage KS chains (7,8,33, 46,47). The biochemical assay showed that all six MAb react with KS chains and can discriminate subtle epitope differences, as each of them binds to a uniquely sulfated oligosaccharide. In our study the six anti-KS MAb showed different staining patterns, most likely due to the presence in CNS of KS-PGs bearing different KS epitopes (10,19,42).The 5D4 epitope seems to be expressed at a high level in a KS-PG located at the surface of microglial cells, whereas it is not expressed, or is masked, in other CNS KS-PGs. The 5D4 epitope differs from the other KS epitopes as it is highly sulfated (33,51). This biochemical characteristic confers resistance to keratanase digestion “in vitro” (34) and on tissue sections (36). Our immunohistochemical findings suggest that microglial KSPG also contains 2D3. 1D2, and 1B4 KS epitopes. However, these MAb also stained white and gray matter, indicating the presence in the brain of a heterogeneous family of KS-PGs. Three brain KSPGs differing in molecular weight, tissue localization, and develop-

485

ANTI-KERATAN SULFATE MAb AND RAMIFIED MICROGLIA

mental expression have thus far been isolated (10,19,42). MAb specific for the three brain KS-PGs showed a different staining pattern in vivo and none of them immunostained microglia as does MAb 5D4 (10.19.42).In line with the immunohistochemical studies, in vitro assays showed that the three brain KS-PGs did not react or were weakly reactive with MAb 5D4 (10,19,42). These observations suggest that the heterogeneity of brain KS-PGs is wider than recognized so far. MAb 5D4 has already been utilized on sections of adult and embryonal CNS. In adult rat cerebellum, Rauch et al. (42) observed a weak diffuse staining which is in contrast to our findings. This difference could be attributed to the different fixative and tissue processing utilized. During development, 5D4 and other KS epitopes have been localized in midline areas where cell migration and axon elongation do not occur (36,45). Moreover, claustrin, a brain-specific KS-PG with low reactivity with MAb 5D4 (lo), inhibits cell adhesion and neurite outgrowth in culture and is expressed in midline structures only during development (10). These data suggest that some KS-PG can form a barrier to growing axons and cell migration (10,36,45). Emerging data suggest that microglia in normal adult rat are a heterogeneous cell population constituted of subsets differing in localization and in antigen expression. Different numbers of microglial cells located in the CNS parenchyma, in the leptomeninges, or in the vascular structures have been detected by MAb and lectins (9,11,18,23,24,26,37-39,41,48,50,52). We found 5D4-positive microglial cells throughout the CNS parenchyma, but their number was lower than that observed with GSA-IB4 lectin (48) or with MAb OX-42 (39). This suggests that the 5D4 epitope can identify a distinct subset of microglia. In conclusion, our data indicate that a particular KS-PG, detectable also in paraffin-embedded sections, is expressed on the surface of ramified microglial cells. Therefore, not only neuronal and astrocytic subtypes (1-3,17,19,25,30) but also resting microglia express specific PGs. The presence of PGs on the cell surface and in the surrounding extracellular matrix of resting microglia could influence functions essential for plasticity, such as cell-to-cell contacts (43) or cellular responses to extracellular molecules and growth factors (14.44).

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forms an extracellular network in human and rat central nervous system. J Neurol Sci 100:113, 1990

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Figure 3. lmmunoelectron microscopy of KS-positivecells showing pericellular immunoreactivity. (A) lmmunostainedcells often contain lipofuscin granules and dense bodies, (6)Pericellular KS immunoreactivity in a microglia-like cell adjacent to a vessel. The cell is located outside the basal membrane. Bars = 3 um.

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