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Complement and clusterin in the spinal cord dorsal horn and gracile nucleus following sciatic nerve injury in the adult rat
Neuroscience Vol. 68, No. 1, pp. 167-179, 1995
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Pergamon
0306-4522(95)00103-4
Elsevier Science Ltd Copyright © 1995 IBRO Printed in Great Britain. All rights reserved 0306-4522/95 $9.50 + 0.00
C O M P L E M E N T A N D C L U S T E R I N IN THE S P I N A L C O R D DORSAL HORN AND GRACILE NUCLEUS FOLLOWING SCIATIC N E R V E I N J U R Y IN THE A D U L T R A T L. L I U , * t E. T O R N Q V I S T , f P. M A T T S S O N , f N. P. E R I K S S O N , t J. K. E. P E R S S O N , t B. P. M O R G A N , ~ H. A L D S K O G I U S t and M. S V E N S S O N t ~'Department of Neuroscience, Karolinska Institutet, S-171 77 Stockholm, Sweden :~Department of Medical Biochemistry, University of Wales College of Medicine, Heath Park, Cardiff CF4 4XN, U.K.
Abstract--We
provide evidence for activation of the complement cascade in the dorsal horn of the spinal cord and in the gracile nucleus in the brainstem following sciatic nerve transection in the adult rat. Immunocytochemical analyses showed immunoreactivity for endogenous immunoglobulin G as shown by immunostaining with F(ab')2 antibodies, as well as complement factors C1, Clq, C3, C3d and C9 in the appropriate central termination areas of the injured sciatic nerve. Results from double labelling immunocytochemistry showed a strong association between immunoglobulin and complement factors on the one hand and reactive microglia on the other. However, some complement immunoreactivity was also found in the neuropil, possibly representing secreted complement. In situ hybridization with an oligonucleotide probe showed a marked increase in C3 messenger RNA, indicating local synthesis of C3 protein. In parallel with activation of complement, there was an increased immunoreactivity for the putative complement inhibitor clusterin, which co-localized with glial fibrillary acidic protein-positive astrocytes. In situ hybridization showed an increased labelling of clusterin messenger RNA. These findings indicate that complement activation and up-regulation of complement inhibitors are prominent central responses to peripheral sensory nerve injury. These responses may therefore be important elements underlying so-called transganglionic degenerative changes in primary sensory axons and terminals.
Injury to the peripheral processes of sensory ganglion cells causes a rapid glial cell reaction in the somatotopically appropriate central projection territories in the spinal cord and brainstem, as evidenced by increased numbers of microglial cells, 4'1° increased immunoreactivity for microglial cell markers, ~3'~8'2°'4~ microglial cell proliferation, TM as well as the astrocytic marker, glial fibrillary acidic protein ( G F A P 19'22'23) and its m R N A (Eriksson et al., unpublished observations). Injury to peripheral sensory axons also results in degenerative changes in terminals and axons in the corresponding central projection areas, so-called transganglionic degeneration (for review, see Ref. 3). These events develop significantly later than the glial cell reactions, however, suggesting that the latter reactions may contribute to the transganglionic neuronal changes. Such an influence could occur by one or more of the substances which activated microglia are likely to be able to secrete as members of the
*To whom correspondence should be addressed. Abbreviations: EDTA, ethylenediaminetetra-acetate; FITC,
fluorescein isothiocyanate; GFAP, glial fibrillary acidic protein; IR, immunoreactive; -LI, -like immunoreactivity; MAC, membrane attack complex; SGP-2, sulphated glycoprotein-2; TRITC, rhodamine isothiocyanate. 167
macrophage family, e.g. growth factors, proteases, free radicals and immune mediators. One group of candidate molecules of potential interest in this situation are those of the complement system, a set of proteins whose activation results in a series of events, the complement cascade. Complement factors are important mediators of inflammatory processes, and facilitate phagocytosis by recruitment of macrophages and opsonization. Complement is also capable of lysing target cells, primarily through the action of the end-product of the terminal pathway, the membrane attack complex (MAC), which consists of factors C5b-9.1'6'14'37'38'43 The M A C binds to a target cell and creates pores in its membrane, thereby allowing an increased influx of e.g. calcium ions, which will eventually kill the cell if M A C accumulation and subsequent membrane perforations occur in excessive amounts. H o m o l o g o u s cells are usually protected by control proteins which regulate various steps of the complement cascade. 23 25,31 These control factors are either plasma proteins or membrane-associated or secreted molecules which act as receptors/regulators for fragments of activated complement components. 23'24'31 One of the proteins which has been identified as a complement regulator in the rat is sulphated glycoprotein-2 (SGP-2), now commonly called
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clusterin, 9"27"3°'32which inhibits the action o f the terminal pathway by binding to C5b 9. ~3"2: We have previously d e m o n s t r a t e d that microglial cell activation following a x o t o m y o f m o t o r n e u r o n s is associated with a markedly increased expression of several c o m p l e m e n t factors and c o m p l e m e n t 3 (C3) m R N A , 39'42 as well as up-regulation o f clusterin in m o t o r n e u r o n s and surrounding astroglia. 42 These observations led us to explore whether the p r o m p t microglial cell reaction after peripheral sensory nerve injury is similarly associated with thc expression o f c o m p l e m e n t factors, e n d o g e n o u s immunoglobulin and clusterin in the dorsal horn o f the spinal cord and in the gracile nucleus following injury to the sciatic nerve, which contains a large n u m b e r of sensory axons. EXPERIMENTAL
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Goat Polyclonal Rat CI Rabbit Polyclonal Rat Clq Rabbit Polyclonal Human C3" Sheep Polyclonal ttuman C3d* Rabbit Polyclonal Rat C9 Rabbit Polyclonal Rat clusterin Rabbit Polyclonal Bovine GFAP* Mouse Monoclonal Rat CR3 OX-42, Mouse Monoclonal
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PROCEDURES
The left sciatic nerve was transected in 22 adult femaleSprague Dawley rats (200 250g; B & K Universal AB, Stockholm, Sweden), deeply anaesthetized with chloral hydrate (35 mg/100 g body weight, i,p.). After two, four, seven, 14 and 28 days (four rats for each postoperative survival time) and 30 weeks (two rats), the rats were reanaesthetized and perfused with saline (body temperature) followed by cold 4% (w/v) paraformaldehyde in 0.15M phosphate buffer (pH 7.3 7.4) containing 14% (v/v) of a saturated picric acid solution. The brainstem and the L4 segment of the spinal cord were removed from each animal and posttixed in the same tixative for 1 2h, then washed overnight (4 C) in 0.15 M phosphate buff'or with 10% (w/v) sucrose. Fourteen-micrometre serial sections were cut on a cryostat. One intact control animal was prepared in the same way. Sections were briefly air dried and washed in phosphate buffer (5 10 rain) prior to incubation in normal horse serum (1:70; Vector, U.S.A.) and 0.3% Triton X-100 (Sigma, U.S.A.) for I h at room temperature. The sections were incubated overnight (4 C) with primary antisera, as shown in Table I. Following a rinse in phosphate buffer, all sections were incubated in biotinylated secondary antisera for 1 h at room
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Complement and clusterin following sciatic nerve injury
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Fig. 1. F(ab'):-LI (a, b) and CI-L1 (e, f) in the gracile nucleus two days postoperatively and double labelling with OX42 (c, d and g, h, respectively). Note the increased expression of F(ab')2-LI on the operated side (b) in comparison to the unoperated side (a). There is an almost complete overlap between F(ab')e-LI and OX42-IR (a
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Fig. 2. F(ab')z-LI (a, b) and CI-LI (c-e) in the L4 dorsal horn of the spinal cord two days after nerve transection. There was increased F(ab')z-LI (b, arrows) as well as CI-LI (d, arrows) on the operated side in comparison to the unoperated side (a and c, respectively) in structures resembling microglia (double labelling with OX42 not shown; see text) boxed area in d in higher magnification (e). Scale bar = 50 itm.
temperature (1 : 200; Vector). The sections were then washed and incubated with avidin biotin complex for 1 h at room temperature (1:50; ABC, Elite, Vector). The immunoreactivity was visualized by incubating the sections in diaminobenzidine (Sigma; 5 0 m g / 1 0 0 m l , 0 . 1 M Tris HCI buffer, pH 7.4), containing 1% (w/v) nickel sulphate and 1% (w/v) cobalt chloride for 10 min, and for another 5 min after adding 0.02% hydrogen peroxide to the solution. All the sections were washed in Tris HCI buffer followed by dehydration in successively higher concentrations of ethanol to
xylene and finally mounted in a non-aqueous medium (Eukitt, D. Kindler G m b H , Germany). Indirect immunofluorescence was used for double labelling. These sections were incubated overnight (4°C) with the following antibody combinations: (i) OX42 and anti-Cl; (ii) OX42 and anti-Clq; (iii) OX42 and anti-C3; (iv) OX42 and anti-C3d; (v) OX42 and anti-C9; (vi) OX42 and anti-rat F(ab')2 ; (vii) a n t i - G F A P and anti-rat clusterin; (viii) OX42 and anti-clusterin. After a rinse in buffer, slides were incubated in a mixture
Complement and clusterin following sciatic nerve injury
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Fig. 3. C3d-LI (a, b) and C9-LI (e, f) in the gracile nucleus seven days postoperatively and double labelling with OX42 (c, d and g, h, respectively). Note the increased expression of C3d-LI as well as C9-L1 on the operated sides (b and f, respectively) in comparison to the unoperated side (a, e). On the operated sides, C3d-LI as well as C9-LI largely overlaps with OX42-IR (b, d, f, h, arrows). Scale bar = 50/lm.
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Fig. 4. C3d-LI (a, b) and C9-Ll (c, d) in the L4 dorsal horn of the spinal cord seven days after nerve transection. There was an increased C3d-LI (b) as well as C9-LI (d) on the operated side in comparison to the unoperated side (a, c) in structures resembling microglia (double labelling with OX42 not shown; see text). Scale bar - 50/~m. of secondary antibodies consisting of either rhodamine isothiocyanate (TRlTC)-tagged (swine anti-rabbit, 1:40: Dako, Denmark) and fluoresceine isothiocyanate (FITC)tagged (goat anti-mouse, 1:40; Dako) or FITC-tagged (rabbit anti-sheep, 1:40; Dako, Donkey anti-goat, 1:20, Jackson) and TRITC-tagged (goat anti-mouse, 1:40; Dako) antibodies. Phosphate buffer (0.15 M, pH 7.4) with 0.3% Triton X-100 was used in all incubation and rinsing steps. The sections were mounted in glycerol containing pphenylenediamine (Sigma). The following controls were performed: (i) omission of primary antiserum; (ii) preabsorption of anti-rat C9 with rat C9 (0.I mg/ml), anti-rat F(ab')2 with rat immunoglobulin G (0.01 mg/ml) and a n t i - G F A P with G F A P (0.5 mg/ml) prior to incubation of the sections. The filter combinations in the microscope were controlled with respect to their capacity to clearly separate the emitted fluorescence. Thus, precipitation of FITC- and TRITC-conjugated secondary antibodies was found to give a sufficient separation when examined by filter combinations for the two fluorophores. In situ hybridization experiments In six rats, anaesthetized as described above, the left sciatic nerve was transected. These rats were killed two, four and seven days after nerve injury, two at each postoperative survival time. The brainstem and the L4 segment of the spinal cord were removed and immediately frozen on dry ice. Fourteen-micrometre sections were cut on a cryostat and mounted on sterilized slides coated with poly-L-lysine (50/~l/ml; Sigma). All sections were fixed in sterile 4% (w/v) paraformaldehyde in 0.1 M phosphate-buffered saline
(pH 7.4) for 3 0 m i n followed by a wash in phosphatebuffered saline. The sections were dehydrated in a graded series of ethanols and air-dried. 48-mer (antisense) oligonucleotide probes complementary to nucleotides 3301 3348 of h u m a n complement C3 t2 and 323 370 of rat clusterin ~ were synthesized (Scandinavian Gene Synthesis AB). A 48-met sense probe described previously was used as control. 4° The oligonucleotides were labelled at the 3' end with [~35S]dATP using terminal deoxyribonucleotidyl transferasc (IBI, New Haven, CT, U.S.A.), to a specific activity of approximately 8 x l0 s c.p.m.//~g. The probes were purified using an Nen-sorb 20 nucleic acid purification cartridge (Du Pont Company, Wilmington) according to the manufacturer's manual. Hybridizations were performed as described by Svensson and Aldskogius 4° in 50% formamide, 4 x standard saline citrate, 1 x Denhardt's solution (0.02% polyvinyl-pyrolidone, 0.02% Ficoll, 0.02% bovine serum albumin), 10% dextran sulphate, 0.25 mg/ml yeast t R N A , 0.05 mg/ml sheared salmon sperm D N A , 1% sarcosyl (Nlauroyl-sarcosine), 0.02 M NaPO4 (pH 7.0), 50 m M dilhiothreitol and 5 x 106c.p.m./ml of probes. Sections were incubated with hybridization cocktail (100/~l/slide) overnight at 42~'C in a sealed chamber humidified with 50% (v/v) formamide, 0 . 6 M NaCI, 10raM Tris HCI (pH 7.5) and 1 m M EDTA. The slides were washed in l x standard saline citrate, 4 x 15 min at 55 C and for another 60 min at room temperature, followed by a brief rinse in water (2 min). The sections were dehydrated in a graded series of ethanol and air dried prior to mounting in an X-ray cassette together with Beta-max Hyperfilm (Amersham) for one to two weeks, whereafter the sections
Complement and clusterin following sciatic nerve injury were dipped in Kodak NTB-2 photoemulsion diluted l : 1 in water. The sections were exposed for two weeks at 4~C, developed in Kodak D19, counterstained with Cresyl Violet, dehydrated in a graded series of ethanols to xylene and mounted in a non-aqueous medium (Eukitt). RESULTS
Immunocytochemistry The immunocytochemical findings are summarized in Table 2. There was minimal F(ab')z-like immunoreactivity (LI) in the intact control animal or on the unoperated side of experimental animals (Figs la, 2a). F(ab')2-LI was increased in the ipsilateral gracile nucleus (Fig. lb) and L4 dorsal horn (Fig. 2b) at two, four, seven and 14 days following sciatic nerve lesion. There was no difference between the operated and unoperated sides at 28 days or 30 weeks after injury. Almost all F(ab')2-LI co-localized with OX42-LI cells (Fig. lb, d). A few C1 (Figs le, 2c)- and C l q (not shown)-LI cells were found in the unoperated animal and on the unoperated sides of experimental animals. These cells were all OX42-immunoreactive (IR) (Fig. lg), and
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were therefore identified as microglia. However, most of the OX42-IR cells in control animals or on the unoperated side were C I - and Clq-Ll-negative. The number of C I - and C I q - L I cells increased rapidly in the ipsilateral gracile nucleus within the first few days (Fig. lf), peaked at seven days and thereafter declined gradually, but was still above normal by 30 weeks postlesion. A similar response was observed in the dorsal horn of the L4 spinal cord segment (Fig. 2d, e), although by 30 weeks immunoreactivity appeared to have returned to baseline levels in this area. The C I - and C l q - L I was confined to OX42-IR cells (Fig. lh), of which few or none were C1- or C lq-negative during the induction and peak of the microglial cell response. C3-LI was completely absent in the gracile nucelus and dorsal horn of the intact control animal and on the unoperated side of experimental animals (not shown). The same was true for C3d-LI (Figs 3a, 4a) except for the presence of fine granular labelling in lamina II or the dorsal horn. This labelling did not co-localize with OX42-IR. C3- and C3d-LI were first observed in the ipsilateral gracile nucleus as well as in
Op
Fig. 5. Double labelling with anti-clusterin (a, b) and anti-GFAP (c, d) in the gracile nucleus 14 days after nerve injury. Note the increased clusterin-LI and GFAP-LI on the operated side as well as the overlap between these markers (b, d, arrows). Scale b a r - 50/~m.
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Fig. 6. Double labelling with anti-clusterin (a, b) and anti-GFAP (c, d) in the L4 dorsal horn 14 days after nerve injury. There is an increased clusterin-LI and GFAP-LI on the operated side and complete overlap between these two markers (b, d, arrows). Scale bar = 50/~m.
the dorsal horn at four days postlesion (Fig. 3b), and were present throughout the entire postoperative period (30 weeks) in the gracile nucleus. In the L4 spinal cord dorsal horn, C3d-LI initially followed the same pattern, but by 28 days C3- and C3d-LI began to decrease, and were absent at 30 weeks postlesion. In most instances, C3- and C3d-LI co-localized with OX42-IR (Fig. 3b, d). However, some of this complement-LI appeared to be present extracellularly. No C9-LI was observed in the intact control animal or on the unoperated side of experimental animals (Figs 3e, 4c). C9-LI was found, however, in the gracile nucleus (Fig. 3f) and L4 spinal cord (Fig. 4d) dorsal horn ipsilateral to nerve injury at four, seven and 14 days, but not at two and 28 days or 30 weeks postoperatively. Although some C9-LI co-localized with OX42-IR, many C9-LI profiles were present outside microglial cells (Fig. 3f, h). A small number of OX42-IR microglial cells was present in the gracile nucleus and L4 dorsal horn in the intact control animal and on the unoperated side of experimental animals. OX42-IR was markedly increased on the operated side in these regions at two,
four, seven and 14 days (Fig. 3d, h). At 28 days after injury, the operated/unoperated difference was less prominent than at earlier postoperative survival times, and at 30 weeks after injury this side difference was almost absent. Small clusters of clusterin-LI profiles were present in the gracile nucleus (Fig. 5a) and L4 dorsal horn (Fig. 6a) of the intact control animal and on the unoperated side of experimental animals. The number and size of these clusters were increased in the gracile nucleus and L4 dorsal horn two to 28 days postoperatively (Figs 5b, 6b), and in the gracile nucleus 30 weeks postoperatively as well. Double labelling showed that some clusterin-LI in the examined regions co-localized with anti-GFAP (Figs 5, 6, arrows). In line with previous observations, GFAPLI was increased on the operated side of the gracile nucleus (Fig. 5d) and L4 dorsal horn (Fig. 6d) four to 14 days after injury. Exclusion of primary antibodies eliminated the corresponding immunoreactivity. Preabsorption with rat immunoglobulin G, C9 and GFAP eliminated all staining with the respective antibodies.
Complement and clusterin following sciatic nerve injury In situ
hybridization
Only background labelling was present at all survival times on the contralateral side as well as two days after injury on the operated side. Labelling above background was first observed at four days after injury with the C3 antisense probe in the ipsilateral gracile nucleus and in the dorsal horn of the L4 spinal cord segment. Labelling was even more prominent at seven days after injury (Fig. 7a, c). Labelling above background levels was present on the contralateral side with the probe for clusterin m R N A (Fig. 7b, d). This labelling was clearly increased on the ipsilateral side of the gracile nucleus (Fig. 7b) and L4 spinal cord dorsal horn (Fig. 7d) at two, four and seven days after injury.
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At higher magnification; radioactive grains resulting from the C3 m R N A and clusterin m R N A probes were highly concentrated over small cells which did not fulfil neuronal criteria (Figs 8b, d, 9b, d). Some cells which could clearly be identified as neurons showed only background labelling (Figs 8b, d, 9b, d). There was no increase in grain density compared to background levels following incubation with the sense probe (Fig. 7e, f). DISCUSSION Although the antibodies used in the present study have been extensively Characterized it is necessary to consider the possibility that they might crossreact with other antigens sharing epitopes with
Fig. 7. Autoradiograms showing C3 mRNA (a, c) and clusterin mRNA (b, d) in the brainstem and L4 spinal cord seven days after nerve injury. Note the increased labelling of C3 mRNA and clusterin mRNA, respectively, on the operated side (arrowheads) compared to the unoperated side in the gracile nucleus (a, b) and L4 spinal cord (c, d). There is no labelling above background in the gracile nucleus (e) or spinal cord (f) with the sense probe. Scale bars = 200pm (a, b); 500//m (c, d, f); l.Omm (e).
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Fig. 8. In situ hybridization using a C3 (a, b) and clusterin (c, d) specific oligonucleotide labelled with [~-35S]dATP in the gracile nucleus seven days after unilateral sciatic nerve transection. Note the high density of grains over small cells, presumably glial cells on the operated side (b and d, respectively; arrows). Neurons appear to be unlabelled. There is only background labelling on the unoperated side with the C3 mRNA probe, and some specific labelling with the clusterin mRNA probe.
complement, immunoglobulin G or clusterin. In fact, the fine granular C3d-LI which was always observed in lamina II of the L4 dorsal horn may represent an example of such a cross-reaction. Because of lack of C3d antigen for preabsorption tests it is impossible to entirely rule out this staining as being true C3d. The absence of labelling with the C3 m R N A probe in this area and on the unoperated side of operated animals argues against this possibility, however. It is important to note also that the characteristic appearance of this lamina II staining was distinctly different from the nerve lesion-induced C3d-LI. In control tissue (the intact animal or unoperated side of experimental animals), staining which was interpreted as specific was observed for CI, C l q and clusterin. C1- and C I q - L I were detected in a few cells which could also be labelled with OX42, indicating that resting microglia are capable of expressing these proteins. Clusterin-LI, on the other hand, co-localized with G F A P - L I cells, in line with previous observations. H'34'37 However, many of the G F A P - L I cells did not express detectable levels of clusterin-LI. In the gracile nucleus contralateral to nerve injury,
some of the specific staining may be attributed to a limited contralateral projection from the sciatic nerve. 21,26 Nerve transection induced immunoreactivity for endogenous immunoglubulin G as well as complement factors C3, C3d and C9, and caused a marked increase in CI-, C l q - and clusterin-LI in the ipsilateral L4 dorsal horn and gracile nucleus. To rule out the possibility of Fc receptor binding we used F(ab'): antibodies to reveal the presence of endogenous immunoglobulin G. These findings indicate that the classical pathway of complement has been activated, and that this activation is associated with endogenous immune complexes. However, a simultaneous activation of the alternative pathway cannot be excluded since factors only associated with this pathway have not been examined in the present study. The expression of C3 m R N A strongly indicates that endogenous cells, presumably microglia, are a major source of C3. Activation and expression of complement followed a similar time course in the L4 dorsal horn and gracile nucleus. Furthermore, a similar time course was
Complement and clusterin following sciatic nerve injury found in the hypoglossal nucleus after injury to its motor axons, i.e. immunoglobulin G and the early factors, C1 and Clq, of the classical pathway appear two days after injury, while the other complement factors are first detectable from four days postoperatively.39'42Based on the intensity of the immunoreactivity, complement expression seems to reach a maximum at seven days after injury. This is followed by a gradual decrease towards normal levels in the dorsal horn, while in the gracile nucleus an increased immunoreactivity for examined complement factors as well as for clusterin were present throughout the 30 week postoperative period. The reason for this long-standing up-regulation may be that the effects of the nerve injury are superimposed on the vulnerability of the long ascending dorsal column system to ageing. 2J5,16,35 Our findings demonstrate a close association between complement and microglia. The precise role of microglia in complement activation is unknown, however, and may be restricted to being a local but not unique source of complement in the affected central nervous region. From previous studies in
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other tissues, complement is known to facilitate phagocytosis of tissue debris (opsonization), e.g. via factor C3d, and to stimulate migration of macrophages (chemotaxis), e.g. via factor C5a. The latter effect has also been demonstrated for microglia in uivo. 6"7'25"28"36"44 After injury to trigeminal nerve branches, microglia have been shown to phagocytose degenerating terminals in the deep laminae of the spinal trigeminal nucleus,5 suggesting a process possibly implicating C3d. However, in lamina II of the spinal cord dorsal horn s and in the gracile nucleus,~5 terminals and axons show degenerative changes without any association with phagocytosis. The expression of C9-LI indicates that the terminal pathway of complement is activated in the dorsal horn and gracile nucleus. Since C9, and in particular C5b-9 (MAC), are potentially cytolytic by creating pores in the cell membrane, activation of the terminal pathway may influence some of the axotomy-induced transganglionic changes in primary sensory projection areas by increasing calcium influx, thereby disturbing e.g. the cytoskeleton and/or intraneuronal transport mechanisms.
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Fig. 9. In situ hybridization with a C3 mRNA (a, b) and clusterin mRNA (c, d) probe in the L4 spinal cord seven days postoperatively. Note the high density of grains apparently located over non-neuronal cells on the operated side (c, d, arrows). There are some labelled cells contralateral to injury with the clusterin mRNA probe (c), but none with the C3 mRNA probe (a). Scale bar - 50 pro.
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The principal activator of the classical p a t h w a y of c o m p l e m e n t is the deposition o f i m m u n e complexes in the tissue. The o b s e r v a t i o n that e n d o g e n o u s imm u n o g l o b u l i n G is expressed in the gracile nucleus a n d spinal cord dorsal h o r n after sciatic nerve injury suggests t h a t a similar m e c h a n i s m of c o m p l e m e n t activation could exist in this situation. One o f the intriguing issues is the source o f the e n d o g e n o u s i m m u n o g l o b u l i n G. At least two principle alternatives a p p e a r to be possible: (i) a n e n d o g e n o u s production, perhaps by B-lymphocytes in the cerebrospinal fluid or (ii) a n exogenous p r o d u c t i o n followed by entry into the C N S via the injured nerves or local b l o o d vessels (for further discussion see Ref. 39). The effects o f c o m p l e m e n t activation are n o r m a l l y regulated by e n d o g e n o u s c o m p l e m e n t inhibitors in such a way that intact h o m o l o g o u s cells are protected. The expression of clusterin, a potential complement inhibitor, 27 was m a r k e d l y increased in the gracile nucleus a n d spinal cord dorsal horn, in cells also expressing G F A P - L I , in line with previous observations t h a t astroglia are a m a j o r source o f this molecule. ~'34 This finding f u r t h e r m o r e suggests t h a t these cells may be effectively protected against complement attack. In addition, secreted clusterin may also protect n e a r b y n e u r o n s a n d n o n - n e u r o n a l cells. This m a y be a significant role, since the n e u r o n s in the
areas examined did not express detectable levels of clusterin, in c o n t r a s t to the situation in cranial 43 a n d spinaP 2 m o t o r n e u r o n s , as well as in certain o t h e r brain areas. 17'27'29'30
CONCLUSION O u r findings indicate t h a t the classical p a t h w a y of the c o m p l e m e n t cascade is activated in the dorsal h o r n a n d gracile nucleus following sciatic nerve transection in a similar fashion as in m o t o r nuclei after m o t o r axon injury. Reactive microglia have a crucial role in this process as a local source of complement, which m a y serve as opsonins, a n d / o r chemotactic agents. In addition, c o m p l e m e n t activation may lead to the assembly of cytolytic complexes, the effects of which may be partially balanced by a n up-regulation of c o m p l e m e n t inhibitors such as clusterin. These previously unrecognized molecular events may influence a x o t o m y - i n d u c e d structural a n d functional transganglionic changes. Acknowledgements--The technical assistance of Ms Britt Meijer is gratefully acknowledged. Ms Sir Blomquist provided excellent secretarial support. The study was supported by grants from the Swedish Medical Research Council (proj. 5420) and the Tore Nilsson Foundation. Li Liu was partially funded by a fellowship from the Swedish Institute.
REFERENCES
I. Agostini A., Cicardi M., Gardinali M. and Bergamaschini L. (1992) The complement system. Int. J. lmmunopath. Pharmac. 5, 123 130. 2. Albright B. C. (1989) The morphology of primary afferent terminals in the rat gracile nucleus following peripheral nerve crush injury. Anat. Ree. 223, 7A. 3. Aldskogius H., Arvidsson J. and Grant G. (1992) Axotomy-induced changes in primary sensory neurons. In Sensoo~ Neurons. Diversity, Development, and Plasticity (ed. Scott S. A.), pp. 363 383. Oxford University Press, New York. 4. Aldskogius H. and Svensson M. (1993) Neuronal and glial cell responses to axon injury. Adv. struct. Biol. 2, 191 223. 5. Arvidsson J. 0986) Transganglionic degeneration in vibrissae innervating primary sensory neurons of the rat: a light and electron microscopic study. J. eomp. Neurol. 249, 392M03. 6. Brown E. J. (1991) Complement receptors and phagocytosis. Curr. Opin. lmmun. 3, 76 82. 7. Briick W. and Friede R. L. (1991) The role of complement in myelin phagocytosis during PNS Wallerian degeneration. J. neurol. Sci. t03, 182 187. 8. Castro-Lopez J. M., Coimbra A., Grant G. and Arvidsson J. (1990) Ultrastructural changes of the central scalloped (C~) primary afferent endings of synaptic glomeruli in the substantia gelatinosa Rolandi of the rat after peripheral neurotomy. J. Neuro~Ttol. 19, 329-337. 9. Collard M. W. and Griswold M. D. (1987) Biosynthesis and molecular cloning of sulfated glycoprotein 2 secreted by rat sertoli cells. Biochemistry 26, 3297 3303. 10. Cova J. L., Aldskogius H., Arvidsson J. and Molander C. (1988) Changes in microglia cell numbers in the spinal cord dorsal horn following brachial plexus transection in the adult rat. Expl Brain Res. 73, 61 68. I I. Day J. R., Laping N. J., McNeill T. H.. Schreiber S. S., Pasinetti G. and Finch C. E. (1990) Castration enhances expression of glial fibrillary acidic protein and sulfated glycoprotein-2 in the intact and lesion-altered hippocampus of the adult male rat. Molev. Endocr. 4, 1995 2002. 12. De Bruijn M. H. L. and Few G.-H. (1985) Human complement component C3: cDNA coding sequence and derived primary structure. Proc. natn. Aead. Sci. U.S.A. 82, 708 712. 13. Eriksson N. P., Persson J. K. E., Svensson M., Arvidsson J., Molander C. and Aldskogius H. (1993) A quantitative analysis of the microglial cell reaction in central primary sensory projection territories following peripheral nerve injury in the adult rat. Expl Brain Res. 96, 19 27. 14. Esser A. F. (1991) Big Mac attack: complement proteins cause leaky patches, lmmun. Today 12, 316 318. 15. Fujisawa K. (1988) Study of axonal dystrophy. III. Posterior funiculus and posterior column of ageing and old rats. Acta neuropath. 76, 115 127. 16. Fujisawa K. and Shiraki H. (1978) Study of axonal dystrophy. I. Pathology of the neuropil of the gracile and the cuneate nuclei in ageing and old rats: a stereological study. Neuropath. appl. Neurobiol. 4, 1 20. 17. Garden G. A., Bothwell M. and Rubel E. W. (1991) Lack of correspondence between mRNA expression for a putative cell death molecule (SPG-2) and neuronal cell death in the central nervous system. J. Neurobiol. 22, 590 604.
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18. Gehrmann J., Monaco S. and Kreutzberg G. W. (1991) Spinal cord microglial cells and DRG satellite cells rapidly respond to transection of the rat sciatic nerve. Restor. Neurol. Neurosci. 2, 181 198. 19. Gilmore S. A., Sims T. J. and Leiting J. E. (1990) Astrocytic reactions in spinal gray matter following sciatic axotomy. Glia 3, 342-349. 20. Graeber M. B., Streit W. I. and Kreutzberg G. W. (1988) Axotomy of the rat facial nerve leads to increased CR3 complement receptor expression by activated microglial cells. J. Neurosci. Res. 21, 18 24. 21. Grant G., Arvidsson J., Robertson B. and Ygge J. (1979) Transganglionic transport of horseradish peroxidase in primary sensory neurons. Neurosci. Lett. 12, 23 28. 22. Hajos F., Csillik B. and Knyihar-Csillik E. (1990) Alterations in glial fibrillary acidic protein immunoreactivity in the upper dorsal horn of the rat spinal cord in the course of transganglionic degenerative atrophy and regenerative proliferation. Neurosci. Lett. 117, 8-13. 23. H~nsch G. M. (1992) The complement attack phase: control of lysis and non-lethal effects of C5b-9. lmmunopharmacology 24, 107 l 17. 24. Lachmann P. J. (1991) The control of homologous tysis. Immun. Today 12, 312 315. 25. Law S. K. A. and Reid K. B. M. (1988) In Complement (ed. Male D.). IRL Press, Oxford. 26. Leong S. K. and Tan C. K. (1987) Central projection of rat sciatic nerve fibres as revealed by Ricinus communis agglutinin and horseradish peroxidase tracers. J. Anat. 154, 15-26. 27. May P. C. and Finch C. E. (1992) Sulfated glycoprotein 2: new relationships of this multifunctional protein to neurodegeneration. Trends Neurosci. 15, 391 396. 28. McGeer P. L., Akiyama H., Itagaki S. and McGeer E. G. (1989) Immune system response in Alzheimer's disease, Can. J. neurol. Sci. 16, 516-527. 29. McNeill T. H., Cheng M., Lampert-Etchells M., Finch C. E. and Pasinetti G. M. (1990) Induction of sulfated glycoprotein (SPG-2) gene expression in the striatum following cortical deafferentation. Soc. Neurosci. Abstr. 16, 1291. 30. Michel D., Chabot J. G., Moyse E., Danik M. and Quirion R. (1992) Possible functions of a new genetic marker in central nervous system: the sulfated glycoprotein-2 (SPG-2). Synapse 11, 105 111. 31. Morgan B. P. (1992) Effects of the membrane attack complex of complement on nucleated cells. Curr. Top. Microbiol. Immun. 178, 115-140. 32. Murphy B. F., Kirszbaum L., Walker I. D. and d'Apice A. J. P. (1988) SP-40,40, a newly identified normal human serum protein found in the SC5b 9 complex of complement and in the immune deposits in glomerulonephritis. J. clin. Invest. 81, 1858-1864. 33. Murray M., Wang S. D., Goldberg M. E. and Levitt P. (1990) Modification of astrocytes in the spinal cord following dorsal root or peripheral nerve lesions. Expl Neurol. 110, 248-257. 34. Pasinetti G. M. and Finch C. E. (1991) Sulfated glycoprotein-2 (SPG-2) mRNA is expressed in rat striatal astrocytes following ibotenic acid lesions. Neurosci. Lett. 130, 1~,. 35. Persson J. K. E., Aldskogius H., Arvidsson J. and Holmberg A. (1991) Ultrastructural changes in the gracile nucleus of the rat after sciatic nerve transection. Anat. Embryol. 184, 591~504. 35a.Persson J. K. E., Svensson M. and Aldskogius H. (1995) Microglial cell proliferation in the nucleus gracilis and dorsal spinal cord following sciatic nerve transection in the adult rat. Primary Sensory Neurons (in press). 36. Rosen H. (1990) Role of CR3 in induced myelomonocytic recruitment: insights from in vivo monoclonal antibody studies in the mouse. J. Leukocyte Biol. 48, 465469. 37. Rozovsky I., Morgan T. E., Willoughby D. A., Dugich-Djordjevich M. M., Pasinetti G. M., Johnson S. A. and Finch C. E. (1994) Selective expression of clusterin (SPG-2) and complement Clq B and C4 during responses to neurotoxins in vivo and in vitro. Neuroscience 62, 741-758. 38. Stoll G., Schmidt B., Jander S., Toyka K. V. and Hartung H.-P. (1991) Presence of the terminal complement complex (C5b-9) precedes myelin degradation in immune-mediated demyelination of the rat peripheral nervous system. Ann. Neurol. 30, 147 155. 39. Svensson M. and Aldskogius H. (1992) Evidence for activation of the complement cascade in the hypoglossal nucleus following peripheral nerve injury. J. Neuroimmun. 40, 99-110. 40. Svensson M. and Aldskogius H. (1992) The effect of axon injury on microtubule-associated protein MAP2 mRNA in the hypoglossal nucleus of the adult rat. Brain Res. 581, 319 322. 41. Svensson M., Eriksson P., Persson J. K. E., Molander C., Arvidsson J. and Aldskogius H. (1993) The response of central glia to peripheral nerve injury. Brain Res. Bull. 30, 499-506. 42. Svensson M., Liu L., Mattsson P., Morgan B. P. and Aldskogius H. (1995) Evidence for activation of the terminal pathway of complement and upregulation of sulfated glycoprotein (SPG-2) in the hypoglossal nucleus following peripheral nerve injury. Molec. chem. Neuropath. 24, 53~68. 43. Svensson M., Eriksson P., Persson J. K. E., Liu L. and Aldskogius H. (1994) Functional properties of microglia following peripheral nerve injury. Neuropath. appl. Neurobiol. 20, 185 187. 44. Yao J., Harvath L., Gilbert D. L. and Colton C. A. (1990) Chemotaxis by a CNS macrophage, the microglia. J. Neurosci. Res. 27, 3~42. (Accepted 10 February 1995)
~
Pergamon
0306-4522(95)00103-4
Elsevier Science Ltd Copyright © 1995 IBRO Printed in Great Britain. All rights reserved 0306-4522/95 $9.50 + 0.00
C O M P L E M E N T A N D C L U S T E R I N IN THE S P I N A L C O R D DORSAL HORN AND GRACILE NUCLEUS FOLLOWING SCIATIC N E R V E I N J U R Y IN THE A D U L T R A T L. L I U , * t E. T O R N Q V I S T , f P. M A T T S S O N , f N. P. E R I K S S O N , t J. K. E. P E R S S O N , t B. P. M O R G A N , ~ H. A L D S K O G I U S t and M. S V E N S S O N t ~'Department of Neuroscience, Karolinska Institutet, S-171 77 Stockholm, Sweden :~Department of Medical Biochemistry, University of Wales College of Medicine, Heath Park, Cardiff CF4 4XN, U.K.
Abstract--We
provide evidence for activation of the complement cascade in the dorsal horn of the spinal cord and in the gracile nucleus in the brainstem following sciatic nerve transection in the adult rat. Immunocytochemical analyses showed immunoreactivity for endogenous immunoglobulin G as shown by immunostaining with F(ab')2 antibodies, as well as complement factors C1, Clq, C3, C3d and C9 in the appropriate central termination areas of the injured sciatic nerve. Results from double labelling immunocytochemistry showed a strong association between immunoglobulin and complement factors on the one hand and reactive microglia on the other. However, some complement immunoreactivity was also found in the neuropil, possibly representing secreted complement. In situ hybridization with an oligonucleotide probe showed a marked increase in C3 messenger RNA, indicating local synthesis of C3 protein. In parallel with activation of complement, there was an increased immunoreactivity for the putative complement inhibitor clusterin, which co-localized with glial fibrillary acidic protein-positive astrocytes. In situ hybridization showed an increased labelling of clusterin messenger RNA. These findings indicate that complement activation and up-regulation of complement inhibitors are prominent central responses to peripheral sensory nerve injury. These responses may therefore be important elements underlying so-called transganglionic degenerative changes in primary sensory axons and terminals.
Injury to the peripheral processes of sensory ganglion cells causes a rapid glial cell reaction in the somatotopically appropriate central projection territories in the spinal cord and brainstem, as evidenced by increased numbers of microglial cells, 4'1° increased immunoreactivity for microglial cell markers, ~3'~8'2°'4~ microglial cell proliferation, TM as well as the astrocytic marker, glial fibrillary acidic protein ( G F A P 19'22'23) and its m R N A (Eriksson et al., unpublished observations). Injury to peripheral sensory axons also results in degenerative changes in terminals and axons in the corresponding central projection areas, so-called transganglionic degeneration (for review, see Ref. 3). These events develop significantly later than the glial cell reactions, however, suggesting that the latter reactions may contribute to the transganglionic neuronal changes. Such an influence could occur by one or more of the substances which activated microglia are likely to be able to secrete as members of the
*To whom correspondence should be addressed. Abbreviations: EDTA, ethylenediaminetetra-acetate; FITC,
fluorescein isothiocyanate; GFAP, glial fibrillary acidic protein; IR, immunoreactive; -LI, -like immunoreactivity; MAC, membrane attack complex; SGP-2, sulphated glycoprotein-2; TRITC, rhodamine isothiocyanate. 167
macrophage family, e.g. growth factors, proteases, free radicals and immune mediators. One group of candidate molecules of potential interest in this situation are those of the complement system, a set of proteins whose activation results in a series of events, the complement cascade. Complement factors are important mediators of inflammatory processes, and facilitate phagocytosis by recruitment of macrophages and opsonization. Complement is also capable of lysing target cells, primarily through the action of the end-product of the terminal pathway, the membrane attack complex (MAC), which consists of factors C5b-9.1'6'14'37'38'43 The M A C binds to a target cell and creates pores in its membrane, thereby allowing an increased influx of e.g. calcium ions, which will eventually kill the cell if M A C accumulation and subsequent membrane perforations occur in excessive amounts. H o m o l o g o u s cells are usually protected by control proteins which regulate various steps of the complement cascade. 23 25,31 These control factors are either plasma proteins or membrane-associated or secreted molecules which act as receptors/regulators for fragments of activated complement components. 23'24'31 One of the proteins which has been identified as a complement regulator in the rat is sulphated glycoprotein-2 (SGP-2), now commonly called
168
L. Liu el al.
clusterin, 9"27"3°'32which inhibits the action o f the terminal pathway by binding to C5b 9. ~3"2: We have previously d e m o n s t r a t e d that microglial cell activation following a x o t o m y o f m o t o r n e u r o n s is associated with a markedly increased expression of several c o m p l e m e n t factors and c o m p l e m e n t 3 (C3) m R N A , 39'42 as well as up-regulation o f clusterin in m o t o r n e u r o n s and surrounding astroglia. 42 These observations led us to explore whether the p r o m p t microglial cell reaction after peripheral sensory nerve injury is similarly associated with thc expression o f c o m p l e m e n t factors, e n d o g e n o u s immunoglobulin and clusterin in the dorsal horn o f the spinal cord and in the gracile nucleus following injury to the sciatic nerve, which contains a large n u m b e r of sensory axons. EXPERIMENTAL
g
":l-
Goat Polyclonal Rat CI Rabbit Polyclonal Rat Clq Rabbit Polyclonal Human C3" Sheep Polyclonal ttuman C3d* Rabbit Polyclonal Rat C9 Rabbit Polyclonal Rat clusterin Rabbit Polyclonal Bovine GFAP* Mouse Monoclonal Rat CR3 OX-42, Mouse Monoclonal
+ ++++++++~ ++++ ++ ++ +
++
++++
++
+
~+++++++ ++ll ++
Source
Titre
Jackson
I: 100
~+++ q~++
I
I+++
++ll
+
c
r%
++++ +++4
I++ + ++
++++
I++
c
+
¢-I
¢=
¢)
F 0'3
++++ ++++++
e-i
C 4
++ 4+
++ ++++ ++++ ÷++ ~ + +++++++ ',U
Table I. Antibodies used Rat F(ab') 2
+
+++++++++ +++ +44++++
PROCEDURES
The left sciatic nerve was transected in 22 adult femaleSprague Dawley rats (200 250g; B & K Universal AB, Stockholm, Sweden), deeply anaesthetized with chloral hydrate (35 mg/100 g body weight, i,p.). After two, four, seven, 14 and 28 days (four rats for each postoperative survival time) and 30 weeks (two rats), the rats were reanaesthetized and perfused with saline (body temperature) followed by cold 4% (w/v) paraformaldehyde in 0.15M phosphate buffer (pH 7.3 7.4) containing 14% (v/v) of a saturated picric acid solution. The brainstem and the L4 segment of the spinal cord were removed from each animal and posttixed in the same tixative for 1 2h, then washed overnight (4 C) in 0.15 M phosphate buff'or with 10% (w/v) sucrose. Fourteen-micrometre serial sections were cut on a cryostat. One intact control animal was prepared in the same way. Sections were briefly air dried and washed in phosphate buffer (5 10 rain) prior to incubation in normal horse serum (1:70; Vector, U.S.A.) and 0.3% Triton X-100 (Sigma, U.S.A.) for I h at room temperature. The sections were incubated overnight (4 C) with primary antisera, as shown in Table I. Following a rinse in phosphate buffer, all sections were incubated in biotinylated secondary antisera for 1 h at room
Antibody
++++
++1111++ ++++++++
lmmunocytoehenK~tO' experiments
Antigen
++ IiiiI
T++++++++
O
~ ++
+ ++4 +
++lll+dd+ ++
1:1000
eo
.5
4 + + ,E 5 o
E
+
d+
1:1000 The binding site Dako
1:3000 + + i I I I +J + + + 1:2000
©
>
+
1:2000
Prof. M. Griswold Boehringer Mannheim Seralab
*Known to cross-react with the rat equivalent.
1: 100t) -o
1:100 ,< 1: 800
x, ~., "e-~, ,~,<
5e-
Complement and clusterin following sciatic nerve injury
Unop'
169
Op
Fig. 1. F(ab'):-LI (a, b) and CI-L1 (e, f) in the gracile nucleus two days postoperatively and double labelling with OX42 (c, d and g, h, respectively). Note the increased expression of F(ab')2-LI on the operated side (b) in comparison to the unoperated side (a). There is an almost complete overlap between F(ab')e-LI and OX42-IR (a
170
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Unop
Op
0
Fig. 2. F(ab')z-LI (a, b) and CI-LI (c-e) in the L4 dorsal horn of the spinal cord two days after nerve transection. There was increased F(ab')z-LI (b, arrows) as well as CI-LI (d, arrows) on the operated side in comparison to the unoperated side (a and c, respectively) in structures resembling microglia (double labelling with OX42 not shown; see text) boxed area in d in higher magnification (e). Scale bar = 50 itm.
temperature (1 : 200; Vector). The sections were then washed and incubated with avidin biotin complex for 1 h at room temperature (1:50; ABC, Elite, Vector). The immunoreactivity was visualized by incubating the sections in diaminobenzidine (Sigma; 5 0 m g / 1 0 0 m l , 0 . 1 M Tris HCI buffer, pH 7.4), containing 1% (w/v) nickel sulphate and 1% (w/v) cobalt chloride for 10 min, and for another 5 min after adding 0.02% hydrogen peroxide to the solution. All the sections were washed in Tris HCI buffer followed by dehydration in successively higher concentrations of ethanol to
xylene and finally mounted in a non-aqueous medium (Eukitt, D. Kindler G m b H , Germany). Indirect immunofluorescence was used for double labelling. These sections were incubated overnight (4°C) with the following antibody combinations: (i) OX42 and anti-Cl; (ii) OX42 and anti-Clq; (iii) OX42 and anti-C3; (iv) OX42 and anti-C3d; (v) OX42 and anti-C9; (vi) OX42 and anti-rat F(ab')2 ; (vii) a n t i - G F A P and anti-rat clusterin; (viii) OX42 and anti-clusterin. After a rinse in buffer, slides were incubated in a mixture
Complement and clusterin following sciatic nerve injury
171
Op
Unop
Fig. 3. C3d-LI (a, b) and C9-LI (e, f) in the gracile nucleus seven days postoperatively and double labelling with OX42 (c, d and g, h, respectively). Note the increased expression of C3d-LI as well as C9-L1 on the operated sides (b and f, respectively) in comparison to the unoperated side (a, e). On the operated sides, C3d-LI as well as C9-LI largely overlaps with OX42-IR (b, d, f, h, arrows). Scale bar = 50/lm.
L. Liu et al.
172
Op
Unop 0'
C3d i
q f
®
J d Ib
4(a)
0
'
4(b)
gF,.
Fig. 4. C3d-LI (a, b) and C9-Ll (c, d) in the L4 dorsal horn of the spinal cord seven days after nerve transection. There was an increased C3d-LI (b) as well as C9-LI (d) on the operated side in comparison to the unoperated side (a, c) in structures resembling microglia (double labelling with OX42 not shown; see text). Scale bar - 50/~m. of secondary antibodies consisting of either rhodamine isothiocyanate (TRlTC)-tagged (swine anti-rabbit, 1:40: Dako, Denmark) and fluoresceine isothiocyanate (FITC)tagged (goat anti-mouse, 1:40; Dako) or FITC-tagged (rabbit anti-sheep, 1:40; Dako, Donkey anti-goat, 1:20, Jackson) and TRITC-tagged (goat anti-mouse, 1:40; Dako) antibodies. Phosphate buffer (0.15 M, pH 7.4) with 0.3% Triton X-100 was used in all incubation and rinsing steps. The sections were mounted in glycerol containing pphenylenediamine (Sigma). The following controls were performed: (i) omission of primary antiserum; (ii) preabsorption of anti-rat C9 with rat C9 (0.I mg/ml), anti-rat F(ab')2 with rat immunoglobulin G (0.01 mg/ml) and a n t i - G F A P with G F A P (0.5 mg/ml) prior to incubation of the sections. The filter combinations in the microscope were controlled with respect to their capacity to clearly separate the emitted fluorescence. Thus, precipitation of FITC- and TRITC-conjugated secondary antibodies was found to give a sufficient separation when examined by filter combinations for the two fluorophores. In situ hybridization experiments In six rats, anaesthetized as described above, the left sciatic nerve was transected. These rats were killed two, four and seven days after nerve injury, two at each postoperative survival time. The brainstem and the L4 segment of the spinal cord were removed and immediately frozen on dry ice. Fourteen-micrometre sections were cut on a cryostat and mounted on sterilized slides coated with poly-L-lysine (50/~l/ml; Sigma). All sections were fixed in sterile 4% (w/v) paraformaldehyde in 0.1 M phosphate-buffered saline
(pH 7.4) for 3 0 m i n followed by a wash in phosphatebuffered saline. The sections were dehydrated in a graded series of ethanols and air-dried. 48-mer (antisense) oligonucleotide probes complementary to nucleotides 3301 3348 of h u m a n complement C3 t2 and 323 370 of rat clusterin ~ were synthesized (Scandinavian Gene Synthesis AB). A 48-met sense probe described previously was used as control. 4° The oligonucleotides were labelled at the 3' end with [~35S]dATP using terminal deoxyribonucleotidyl transferasc (IBI, New Haven, CT, U.S.A.), to a specific activity of approximately 8 x l0 s c.p.m.//~g. The probes were purified using an Nen-sorb 20 nucleic acid purification cartridge (Du Pont Company, Wilmington) according to the manufacturer's manual. Hybridizations were performed as described by Svensson and Aldskogius 4° in 50% formamide, 4 x standard saline citrate, 1 x Denhardt's solution (0.02% polyvinyl-pyrolidone, 0.02% Ficoll, 0.02% bovine serum albumin), 10% dextran sulphate, 0.25 mg/ml yeast t R N A , 0.05 mg/ml sheared salmon sperm D N A , 1% sarcosyl (Nlauroyl-sarcosine), 0.02 M NaPO4 (pH 7.0), 50 m M dilhiothreitol and 5 x 106c.p.m./ml of probes. Sections were incubated with hybridization cocktail (100/~l/slide) overnight at 42~'C in a sealed chamber humidified with 50% (v/v) formamide, 0 . 6 M NaCI, 10raM Tris HCI (pH 7.5) and 1 m M EDTA. The slides were washed in l x standard saline citrate, 4 x 15 min at 55 C and for another 60 min at room temperature, followed by a brief rinse in water (2 min). The sections were dehydrated in a graded series of ethanol and air dried prior to mounting in an X-ray cassette together with Beta-max Hyperfilm (Amersham) for one to two weeks, whereafter the sections
Complement and clusterin following sciatic nerve injury were dipped in Kodak NTB-2 photoemulsion diluted l : 1 in water. The sections were exposed for two weeks at 4~C, developed in Kodak D19, counterstained with Cresyl Violet, dehydrated in a graded series of ethanols to xylene and mounted in a non-aqueous medium (Eukitt). RESULTS
Immunocytochemistry The immunocytochemical findings are summarized in Table 2. There was minimal F(ab')z-like immunoreactivity (LI) in the intact control animal or on the unoperated side of experimental animals (Figs la, 2a). F(ab')2-LI was increased in the ipsilateral gracile nucleus (Fig. lb) and L4 dorsal horn (Fig. 2b) at two, four, seven and 14 days following sciatic nerve lesion. There was no difference between the operated and unoperated sides at 28 days or 30 weeks after injury. Almost all F(ab')2-LI co-localized with OX42-LI cells (Fig. lb, d). A few C1 (Figs le, 2c)- and C l q (not shown)-LI cells were found in the unoperated animal and on the unoperated sides of experimental animals. These cells were all OX42-immunoreactive (IR) (Fig. lg), and
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were therefore identified as microglia. However, most of the OX42-IR cells in control animals or on the unoperated side were C I - and Clq-Ll-negative. The number of C I - and C I q - L I cells increased rapidly in the ipsilateral gracile nucleus within the first few days (Fig. lf), peaked at seven days and thereafter declined gradually, but was still above normal by 30 weeks postlesion. A similar response was observed in the dorsal horn of the L4 spinal cord segment (Fig. 2d, e), although by 30 weeks immunoreactivity appeared to have returned to baseline levels in this area. The C I - and C l q - L I was confined to OX42-IR cells (Fig. lh), of which few or none were C1- or C lq-negative during the induction and peak of the microglial cell response. C3-LI was completely absent in the gracile nucelus and dorsal horn of the intact control animal and on the unoperated side of experimental animals (not shown). The same was true for C3d-LI (Figs 3a, 4a) except for the presence of fine granular labelling in lamina II or the dorsal horn. This labelling did not co-localize with OX42-IR. C3- and C3d-LI were first observed in the ipsilateral gracile nucleus as well as in
Op
Fig. 5. Double labelling with anti-clusterin (a, b) and anti-GFAP (c, d) in the gracile nucleus 14 days after nerve injury. Note the increased clusterin-LI and GFAP-LI on the operated side as well as the overlap between these markers (b, d, arrows). Scale b a r - 50/~m.
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et al.
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Fig. 6. Double labelling with anti-clusterin (a, b) and anti-GFAP (c, d) in the L4 dorsal horn 14 days after nerve injury. There is an increased clusterin-LI and GFAP-LI on the operated side and complete overlap between these two markers (b, d, arrows). Scale bar = 50/~m.
the dorsal horn at four days postlesion (Fig. 3b), and were present throughout the entire postoperative period (30 weeks) in the gracile nucleus. In the L4 spinal cord dorsal horn, C3d-LI initially followed the same pattern, but by 28 days C3- and C3d-LI began to decrease, and were absent at 30 weeks postlesion. In most instances, C3- and C3d-LI co-localized with OX42-IR (Fig. 3b, d). However, some of this complement-LI appeared to be present extracellularly. No C9-LI was observed in the intact control animal or on the unoperated side of experimental animals (Figs 3e, 4c). C9-LI was found, however, in the gracile nucleus (Fig. 3f) and L4 spinal cord (Fig. 4d) dorsal horn ipsilateral to nerve injury at four, seven and 14 days, but not at two and 28 days or 30 weeks postoperatively. Although some C9-LI co-localized with OX42-IR, many C9-LI profiles were present outside microglial cells (Fig. 3f, h). A small number of OX42-IR microglial cells was present in the gracile nucleus and L4 dorsal horn in the intact control animal and on the unoperated side of experimental animals. OX42-IR was markedly increased on the operated side in these regions at two,
four, seven and 14 days (Fig. 3d, h). At 28 days after injury, the operated/unoperated difference was less prominent than at earlier postoperative survival times, and at 30 weeks after injury this side difference was almost absent. Small clusters of clusterin-LI profiles were present in the gracile nucleus (Fig. 5a) and L4 dorsal horn (Fig. 6a) of the intact control animal and on the unoperated side of experimental animals. The number and size of these clusters were increased in the gracile nucleus and L4 dorsal horn two to 28 days postoperatively (Figs 5b, 6b), and in the gracile nucleus 30 weeks postoperatively as well. Double labelling showed that some clusterin-LI in the examined regions co-localized with anti-GFAP (Figs 5, 6, arrows). In line with previous observations, GFAPLI was increased on the operated side of the gracile nucleus (Fig. 5d) and L4 dorsal horn (Fig. 6d) four to 14 days after injury. Exclusion of primary antibodies eliminated the corresponding immunoreactivity. Preabsorption with rat immunoglobulin G, C9 and GFAP eliminated all staining with the respective antibodies.
Complement and clusterin following sciatic nerve injury In situ
hybridization
Only background labelling was present at all survival times on the contralateral side as well as two days after injury on the operated side. Labelling above background was first observed at four days after injury with the C3 antisense probe in the ipsilateral gracile nucleus and in the dorsal horn of the L4 spinal cord segment. Labelling was even more prominent at seven days after injury (Fig. 7a, c). Labelling above background levels was present on the contralateral side with the probe for clusterin m R N A (Fig. 7b, d). This labelling was clearly increased on the ipsilateral side of the gracile nucleus (Fig. 7b) and L4 spinal cord dorsal horn (Fig. 7d) at two, four and seven days after injury.
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At higher magnification; radioactive grains resulting from the C3 m R N A and clusterin m R N A probes were highly concentrated over small cells which did not fulfil neuronal criteria (Figs 8b, d, 9b, d). Some cells which could clearly be identified as neurons showed only background labelling (Figs 8b, d, 9b, d). There was no increase in grain density compared to background levels following incubation with the sense probe (Fig. 7e, f). DISCUSSION Although the antibodies used in the present study have been extensively Characterized it is necessary to consider the possibility that they might crossreact with other antigens sharing epitopes with
Fig. 7. Autoradiograms showing C3 mRNA (a, c) and clusterin mRNA (b, d) in the brainstem and L4 spinal cord seven days after nerve injury. Note the increased labelling of C3 mRNA and clusterin mRNA, respectively, on the operated side (arrowheads) compared to the unoperated side in the gracile nucleus (a, b) and L4 spinal cord (c, d). There is no labelling above background in the gracile nucleus (e) or spinal cord (f) with the sense probe. Scale bars = 200pm (a, b); 500//m (c, d, f); l.Omm (e).
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Fig. 8. In situ hybridization using a C3 (a, b) and clusterin (c, d) specific oligonucleotide labelled with [~-35S]dATP in the gracile nucleus seven days after unilateral sciatic nerve transection. Note the high density of grains over small cells, presumably glial cells on the operated side (b and d, respectively; arrows). Neurons appear to be unlabelled. There is only background labelling on the unoperated side with the C3 mRNA probe, and some specific labelling with the clusterin mRNA probe.
complement, immunoglobulin G or clusterin. In fact, the fine granular C3d-LI which was always observed in lamina II of the L4 dorsal horn may represent an example of such a cross-reaction. Because of lack of C3d antigen for preabsorption tests it is impossible to entirely rule out this staining as being true C3d. The absence of labelling with the C3 m R N A probe in this area and on the unoperated side of operated animals argues against this possibility, however. It is important to note also that the characteristic appearance of this lamina II staining was distinctly different from the nerve lesion-induced C3d-LI. In control tissue (the intact animal or unoperated side of experimental animals), staining which was interpreted as specific was observed for CI, C l q and clusterin. C1- and C I q - L I were detected in a few cells which could also be labelled with OX42, indicating that resting microglia are capable of expressing these proteins. Clusterin-LI, on the other hand, co-localized with G F A P - L I cells, in line with previous observations. H'34'37 However, many of the G F A P - L I cells did not express detectable levels of clusterin-LI. In the gracile nucleus contralateral to nerve injury,
some of the specific staining may be attributed to a limited contralateral projection from the sciatic nerve. 21,26 Nerve transection induced immunoreactivity for endogenous immunoglubulin G as well as complement factors C3, C3d and C9, and caused a marked increase in CI-, C l q - and clusterin-LI in the ipsilateral L4 dorsal horn and gracile nucleus. To rule out the possibility of Fc receptor binding we used F(ab'): antibodies to reveal the presence of endogenous immunoglobulin G. These findings indicate that the classical pathway of complement has been activated, and that this activation is associated with endogenous immune complexes. However, a simultaneous activation of the alternative pathway cannot be excluded since factors only associated with this pathway have not been examined in the present study. The expression of C3 m R N A strongly indicates that endogenous cells, presumably microglia, are a major source of C3. Activation and expression of complement followed a similar time course in the L4 dorsal horn and gracile nucleus. Furthermore, a similar time course was
Complement and clusterin following sciatic nerve injury found in the hypoglossal nucleus after injury to its motor axons, i.e. immunoglobulin G and the early factors, C1 and Clq, of the classical pathway appear two days after injury, while the other complement factors are first detectable from four days postoperatively.39'42Based on the intensity of the immunoreactivity, complement expression seems to reach a maximum at seven days after injury. This is followed by a gradual decrease towards normal levels in the dorsal horn, while in the gracile nucleus an increased immunoreactivity for examined complement factors as well as for clusterin were present throughout the 30 week postoperative period. The reason for this long-standing up-regulation may be that the effects of the nerve injury are superimposed on the vulnerability of the long ascending dorsal column system to ageing. 2J5,16,35 Our findings demonstrate a close association between complement and microglia. The precise role of microglia in complement activation is unknown, however, and may be restricted to being a local but not unique source of complement in the affected central nervous region. From previous studies in
C3
-,~'~
other tissues, complement is known to facilitate phagocytosis of tissue debris (opsonization), e.g. via factor C3d, and to stimulate migration of macrophages (chemotaxis), e.g. via factor C5a. The latter effect has also been demonstrated for microglia in uivo. 6"7'25"28"36"44 After injury to trigeminal nerve branches, microglia have been shown to phagocytose degenerating terminals in the deep laminae of the spinal trigeminal nucleus,5 suggesting a process possibly implicating C3d. However, in lamina II of the spinal cord dorsal horn s and in the gracile nucleus,~5 terminals and axons show degenerative changes without any association with phagocytosis. The expression of C9-LI indicates that the terminal pathway of complement is activated in the dorsal horn and gracile nucleus. Since C9, and in particular C5b-9 (MAC), are potentially cytolytic by creating pores in the cell membrane, activation of the terminal pathway may influence some of the axotomy-induced transganglionic changes in primary sensory projection areas by increasing calcium influx, thereby disturbing e.g. the cytoskeleton and/or intraneuronal transport mechanisms.
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177
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Fig. 9. In situ hybridization with a C3 mRNA (a, b) and clusterin mRNA (c, d) probe in the L4 spinal cord seven days postoperatively. Note the high density of grains apparently located over non-neuronal cells on the operated side (c, d, arrows). There are some labelled cells contralateral to injury with the clusterin mRNA probe (c), but none with the C3 mRNA probe (a). Scale bar - 50 pro.
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The principal activator of the classical p a t h w a y of c o m p l e m e n t is the deposition o f i m m u n e complexes in the tissue. The o b s e r v a t i o n that e n d o g e n o u s imm u n o g l o b u l i n G is expressed in the gracile nucleus a n d spinal cord dorsal h o r n after sciatic nerve injury suggests t h a t a similar m e c h a n i s m of c o m p l e m e n t activation could exist in this situation. One o f the intriguing issues is the source o f the e n d o g e n o u s i m m u n o g l o b u l i n G. At least two principle alternatives a p p e a r to be possible: (i) a n e n d o g e n o u s production, perhaps by B-lymphocytes in the cerebrospinal fluid or (ii) a n exogenous p r o d u c t i o n followed by entry into the C N S via the injured nerves or local b l o o d vessels (for further discussion see Ref. 39). The effects o f c o m p l e m e n t activation are n o r m a l l y regulated by e n d o g e n o u s c o m p l e m e n t inhibitors in such a way that intact h o m o l o g o u s cells are protected. The expression of clusterin, a potential complement inhibitor, 27 was m a r k e d l y increased in the gracile nucleus a n d spinal cord dorsal horn, in cells also expressing G F A P - L I , in line with previous observations t h a t astroglia are a m a j o r source o f this molecule. ~'34 This finding f u r t h e r m o r e suggests t h a t these cells may be effectively protected against complement attack. In addition, secreted clusterin may also protect n e a r b y n e u r o n s a n d n o n - n e u r o n a l cells. This m a y be a significant role, since the n e u r o n s in the
areas examined did not express detectable levels of clusterin, in c o n t r a s t to the situation in cranial 43 a n d spinaP 2 m o t o r n e u r o n s , as well as in certain o t h e r brain areas. 17'27'29'30
CONCLUSION O u r findings indicate t h a t the classical p a t h w a y of the c o m p l e m e n t cascade is activated in the dorsal h o r n a n d gracile nucleus following sciatic nerve transection in a similar fashion as in m o t o r nuclei after m o t o r axon injury. Reactive microglia have a crucial role in this process as a local source of complement, which m a y serve as opsonins, a n d / o r chemotactic agents. In addition, c o m p l e m e n t activation may lead to the assembly of cytolytic complexes, the effects of which may be partially balanced by a n up-regulation of c o m p l e m e n t inhibitors such as clusterin. These previously unrecognized molecular events may influence a x o t o m y - i n d u c e d structural a n d functional transganglionic changes. Acknowledgements--The technical assistance of Ms Britt Meijer is gratefully acknowledged. Ms Sir Blomquist provided excellent secretarial support. The study was supported by grants from the Swedish Medical Research Council (proj. 5420) and the Tore Nilsson Foundation. Li Liu was partially funded by a fellowship from the Swedish Institute.
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