Complement activation after lumbosacral ventral root avulsion injury

Neuroscience Letters 394 (2006) 179–183

Complement activation after lumbosacral ventral root avulsion injury Marcus Ohlsson, Leif A. Havton ∗ Department of Neurology and Brain Research Institute, David Geffen School of Medicine at UCLA, 710 Westwood Plaza, Los Angeles, CA 90095-1769, USA Received 23 August 2005; received in revised form 26 September 2005; accepted 10 October 2005

Abstract A lumbosacral ventral root avulsion (VRA) injury results in a pronounced loss of motoneurons, in part due to apoptosis. Caspase inhibitors may rescue motoneurons after a VRA in neonatal rats, but this treatment approach has been unsuccessful to protect motoneurons subjected to the same injury in adult rats. Other mechanisms may contribute to the retrograde motoneuron death encountered in adult animals. Here, we study whether the complement system, a part of the innate immune system, contributes to motoneuron death after a lumbosacral VRA. Adult Sprague–Dawley rats underwent a unilateral L5–S2 VRA injury. At 10 days postoperatively, quantitative immunohistochemical studies demonstrated that the lytic membrane attack complex (MAC) targeted approximately 38% of axotomized motoneurons. The MAC inhibitor Clusterin was concurrently expressed at significantly higher levels in astrocytes and de novo in 30% of the remaining motoneurons. Our data suggest that complement activation and necrosis contribute to motoneuron death after lumbosacral VRA injuries. We speculate that inhibition of MAC may constitute a potential neuroprotective strategy following cauda equina injuries. © 2005 Elsevier Ireland Ltd. All rights reserved. Keywords: Astrocyte; Cauda equina; Clusterin; Conus medullaris; MAC and spinal cord injury

Trauma to the thoracolumbar junction or to the lumbar spine may result in injury to the conus medullaris and cauda equina [16]. These lesions are complex injuries with multiple pathologies, which may include a crush injury to the sacral spinal cord as well as a laceration, crush, tearing, transection and avulsion injury to the dorsal and ventral lumbosacral roots. A ventral root avulsion (VRA) injury represents the most proximal form of cauda equina injury and is often associated with high-energy trauma [9,17]. In the adult rat, a VRA injury triggers an activation of astrocytes, microglia and macrophages within the motor nuclei [8,18,20]. Moreover, an avulsion injury of lumbosacral ventral roots results in a progressive and pronounced loss of the axotomized motoneurons [7,8,14,18]. Apoptosis contributes to the motoneuron death after VRA with activation of caspases and characteristic nuclear condensation [7,14]. Although caspase inhibitors may rescue motoneurons after a VRA in neonatal rats, this pharmacological treatment approach has been unsuccessful to protect motoneurons subjected to the same injury in adult rats [5]. The latter report raises the possibility

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that also other mechanisms may contribute to the retrograde motoneuron death encountered in adult animals after a VRA injury. The complement system is a set of serum proteins, which upon activation eventually forms a membrane attack complex (MAC), composed of complement factors C5b-9, which lyses the cell membranes that it binds to [10]. Such complement activation may be triggered by, e.g. a traumatic brain injury [3,4], dorsal root lesion [12,13], facial nerve lesions [15], spinal cord injury [1,2] and optic nerve crush [19]. However, it is not known whether the complement system may be activated by a lumbosacral VRA injury. In this quantitative immunohistochemical study, we address whether the complement system is activated after a lumbosacral VRA, and to what extent a possible endogenous complement regulation is present. For this purpose, two groups of adult rats were studied: a sham-operated group underwent a lumbar laminectomy but no lesion to the roots or spinal cord; the other injury group underwent a lumbar laminectomy and an L5–S2 unilateral VRA injury. Both groups were studied at 10 days postoperatively, corresponding to a time-point, which exhibits an active and marked motoneuron loss [7,8]. The spinal cord tissue was analyzed at two sites, the ventral horn gray matter

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containing motoneurons, and the ventral white matter, immediately adjacent to the exit zone of motoneuron axons. Our studies show that a lumbosacral VRA injury induces an activation of the complement system, and that the activated system targets an estimated 38% of motoneurons at 10 days after the lesion. The complement regulator Clusterin was concurrently expressed at significantly higher levels in astrocytes and de novo in 30% of the remaining motoneurons. Our data suggest that a plausible reason why caspase inhibitors have failed to rescue adult motoneurons after a VRA lesion may, at least in part, be due to the additional activation of a complement-driven mechanism leading to neuronal death by necrosis. All animal procedures were performed according to the standards established by the NIH Guide for the Care and Use of Laboratory Animals (National Institutes of Health Publications No. 86-23, revised 1985), and all experimental protocols were approved by the Chancellor’s Animal Research Committee at UCLA. Eight adult female Sprague–Dawley rats (175–225 g, Charles River Laboratories, Raleigh, NC) were included in the study. The animals were housed in a room with a 12/12-h diurnal light/dark cycle with free access to food and water. Under general isoflurane anesthesia (2–2.5% in 100% oxygen), a lumbar L1–L4 hemilaminectomy was performed in adult rats, as previously described in detail [7]. Briefly, the dura mater was opened and anatomical landmarks were used to identify the L5–S2 ventral roots on the left side. These four roots were individually avulsed and separated from the spinal cord surface by applying a constant traction with a pair of fine jeweler’s forceps along the course of each root (n = 4). Sham operated animals (n = 4) underwent a lumbar hemilaminectomy and opening of the dura, but no root injury. At 10 days post injury, all animals were terminally anesthetized and transcardially perfused with 100 ml body-temperatured PBS followed by 400 ml 4% paraformaldehyde at +4 ◦ C. The lumbosacral portion of the spinal cord was dissected out and post-fixed in the same fixative for 90 min, rinsed in PBS, and cryoprotected in 15% sucrose in PBS overnight prior to serial cryosectioning (14 ␮m). Lumbosacral spinal cord sections were incubated overnight (+4 ◦ C) with one of, or combinations of, the primary antibodies for GFAP (1:1000, rabbit polyclonal, Chemicon AB5804, Temecula, CA), ChAT (1:200, goat polyclonal, Chemicon AB144P) Clusterin (1:200, mouse monoclonal, Upstate Biotech #05-355, Lake Placid, NY) and MAC (1:25, mouse monoclonal directed towards the poly-C9 component of the C5b9/MAC complex, DAKO M0777, DAKO, Glostrup, Denmark). The antibodies were diluted in 1% BSA and 0.3% Triton X-100 in PBS. For detection of indirect immunofluorescense (IF), secondary antibodies of appropriate species, donkey-anti-rabbit, donkeyanti-goat or donkey-anti-mouse, conjugated with Alexa Fluor® 488 or Alexa Fluor® 594 (Molecular Probes, Eugene, OR) were diluted in PBS, and the sections were incubated in this solution for 1 h at room temperature, rinsed in PBS, dehydrated and mounted on glass slides with DPX. Omission of primary antibodies was made, used for control purposes, and resulted in absence of staining. For light stable detection, sections were rinsed and incubated with biotinylated secondary antibodies of appropriate species

Fig. 1. The injury model and areas of quantification. The left ventral L5–S2 roots are avulsed at the CNS/PNS interface (A). At 10 days after injury, astroglial and Clusterin activation is measured at the L6 segmental level, using densitometry at two sites: the grey matter at the motoneuron pool (B), and at the exit zone of motoneuron axons in the ventral white matter (C).

(1:200; Vector Laboratories, Burlingame, CA) for 1 h at room temperature. Sections were then washed, after which immunoreactivity (IR) was revealed using the DAB and/or Vector® SG as chromogen (Vector Laboratories), according to protocols from the supplier. The sections were then dehydrated and mounted using a non-aqueous DPX medium. Three non-overlapping light or fluorescent microscopic images of the L6 ventral horn from all experimental series were captured with a Nikon Plan APO® ×40 lens in defined areas of gray and white matter (Fig. 1) using a Micropublisher 5 megapixel digital camera (Q Imaging, Burnaby, BC) attached to a Nikon E600 light microscope equipped with epifluorescense. The photomicrographs were analyzed using C-imaging software (Compix, Cranberry Township, PA). The light intensity levels were set to reflect the level of IR by an observer blinded to section identity. The measured area, in pixels, was calculated and taken as a measurement of IR [19]. Quantitative data were expressed as a percentage of each region of interest ± standard deviation of total area, and analyzed with the Mann–Whitney non-parametric test using the mean per animal (n = 4). A p < 0.05 (two-tailed) was regarded as reflecting a statistically significant difference between samples. In this investigation, we studied activation of the complement system in a rat model of cauda equina injury. For this purpose, we applied immunohistochemical studies, using primary antibodies to GFAP (astrocytes), ChAT (motoneurons) in combination with the lytic MAC as well as the complement regulator Clusterin. GFAP, a protein expressed in normal and reactive astroglia, was detected in the lumbosacral spinal cord of all studied animals. Sham operated animals had low levels of GFAP-IR in the ventral horn white (1.7 ± 0.3% area) (n = 4) and gray (1.2 ± 0.4% area) (n = 4) matters of the L6 segment. After injury, the corresponding areas demonstrated an increased GFAP-IR in white (4.6 ± 1.0% area) (n = 4) and gray (3.0 ± 0.9% area) (n = 4) matters in comparison to the sham-operated rats (p < 0.05, both groups) (Figs. 2 and 3). The complement regulator Clusterin was expressed predominantly in astrocytes at the L6 level of the spinal cord (Figs. 2 and 3). Sham operated animals had low levels of Clusterin in astrocytes from white (0.01 ± 0.01% area) and gray (0.01 ± 0.01% area) matters. After injury, the Clusterin expression was higher in astrocytes found in white (0.2 ± 0.1%

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Fig. 2. Complement activation. Double-labeling of astrocytes (GFAP, green) and Clusterin (red) (A and B); Motoneurons (ChAT, grey–blue) and MAC (Black) (C–F) Low power microghraphs (C and D), details of membrane staining (E and F); ChAT (green) and Clusterin (red) (G and H). In sham-operated animals, normal GFAP-positive astrocytes are scattered in white and gray matters of the spinal cord (green, A). ChAT-positive motoneurons are found in the ventral horn, however, without MAC-binding to the cell membrane (C and E). In sham animals, no Clusterin-IR was detected in motoneurons (G). After injury, several astrocytes (B, green) express Clusterin (B, red), and several motoneurons are targeted by the lytic MAC (D and arrows, F). A VRA injury leads to upregulation of Clusterin in motoneurons as well (H), however not sufficiently to protect all motoneurons from complement-mediated death. Scale bar: (A), (B), (G) and (H) = 25 ␮m; (C)–(F) = 10 ␮m.

area) and gray (0.2 ± 0.1% area) matters compared to sham animals (p < 0.05, both groups; Figs. 2 and 3). In sham animals, no Clusterin-IR was detected in motoneurons. After injury, quantitative studies showed that 30 ± 11% (n = 4) of the axotomized motoneurons demonstrated de novo Clusterin-IR (p < 0.05, Figs. 2 and 3). No ChAT-IR motoneurons were targeted by MAC in any of the sham-operated animals (n = 4), nor could MAC-staining be

identified in the motoneuron pool of the sham-operated animals. After VRA, the MAC-IR was clearly visible, predominantly apposed to the cell membrane of L6 motoneurons (Fig. 2). Quantitative studies showed that 38 ± 15% (n = 4) (p < 0.05) of the axotomized motoneurons were targeted by MAC at 10 days post injury. In the present study, we demonstrate that an avulsion injury of lumbosacral ventral roots results in an activation of the

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Fig. 3. Densitometry of GFAP and Clusterin in white and gray matter of the spinal cord. A VRA leads to a significant (p < 0.05) increase of both GFAP-IR and Clusterin-IR in both gray and white matters. Please note different scale on the Y-axis for GFAP and Clusterin.

complement system in astrocytes and motoneurons. We also show that an avulsion of ventral roots lead to MAC-binding on motoneuron cell membranes, indicative of impending death. In addition, a parallel upregulation of Clusterin in astrocytes and some motoneurons is demonstrated and may be of protective value. Neuroimmunological mechanisms, including complement activation, have gained increasing interest as possible contributors to neural death in a variety of experimental models as well as following neurotrauma in humans [21,23,24]. It has been shown that, i.e. traumatic brain injury [3], spinal cord contusion [2], and optic nerve crush [19] lead to an activation of the terminal complement pathway, resulting in neuronal and axonal death. However, it has not been known whether a lumbosacral VRA injury may induce the same effects in spinal motoneurons. Activation of the complement cascade, in particular the terminal pathway in the immediate vicinity of motoneurons, contributes to cell death and poor outcome [15]. Here, we show that the lytic pathway of the complement cascade is activated, demonstrated by the binding of the MAC to motoneurons. Even though sub-lytic binding of MAC is possible, this process, if prominent, is traditionally regarded as a death sentence to any cell that it attaches to [10]. Previous studies on lumbosacral VRA have demonstrated an early and progressive post-lesion motoneuron death over several weeks after the injury [7,8]. We speculate that the MAC-binding observed here contributes to this. Inhibition of complement activation most likely has protective effects. For instance, it has been shown that mice over-

expressing the complement inhibitor Crry, has a reduced neurologic impairment and improved blood–brain barrier function after closed head injury [22], and Clusterin-deficient mice exhibit significantly fewer surviving motoneurons after a hypoglossal nerve transection [26]. The complement regulator Clusterin, which inhibits the formation of MAC at the C5b7level, is expressed in na¨ıve astrocytes [4,19], oligodendrocytes and neurons [1]. An injury to the CNS causes an increase in Clusterin expression in astrocytes in the lesion area [1,4,19]. Upregulation of Clusterin is an intrinsic survival-promoting response to lesion-induced stress [26], and it seems most likely that Clusterin is not expressed in dying or apoptotic cells [25]. Moreover, astrocytes seem capable of secreting Clusterin after injury [26]. We demonstrate an increase in Clusterin expression in astrocytes adjacent to the lesioned motoneurons and their motor-axons after a VRA injury, as well as de novo expression of Clusterin in many axotomized motoneurons. We speculate that the noted increased expression of Clusterin in the spinal cord following a VRA injury also exerts neuroprotective effects. The effects of the complement system are early, but not instant. Complement effects typically start within 2 days of injury [4] but can be seen as early as 8 h after a hypoxic–ischemic brain injury [6]. The effects may last for up to 6 weeks after CNS trauma [2]. From a clinical standpoint, this time-span makes complement inhibition both possible and appealing to consider for possible treatment strategies. Caspase inhibitors have not proven a robust effect on adult motoneurons that undergo apoptosis after injury [5]. It is noteworthy, however, that VRA injuries in the adult mouse resulted in degenerative changes in motoneurons with features of both apoptosis and necrosis [11]. Therefore, for VRA in experimental models and in humans, complement inhibitors, alone or as a part of a combinatorial treatment approach, may possibly serve as useful neuroprotective agents. In summary, we found that a lumbosacral VRA leads to an activation of the complement system, including the lytic terminal pathway, eventually leading to MAC deposits on motoneurons, indicative of impending cell death. The injured lumbosacral spinal cord also upregulates Clusterin, which was found in both astrocytes and motoneurons, acting as an inborn defense mechanism against the complement attack. We speculate that the observed MAC binding to motoneurons contributes to the previously observed cell death after lumbosacral VRA, and that inhibition of this terminal pathway may be of considerable value as a potential neuroprotective treatment strategy for these injuries.

Acknowledgments The authors wish to thank Dr. Jun Wu for excellent immunohistochemical support. This work was supported by grants from National Institutes of Health #NS042719; The Paralysis Project of America; The Roman Reed Funds for Spinal Cord Injury Research of California; UCLA School of Medicine/SteinOppenheimer Endowment Award; Nathan Shapell Foundation.

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