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THE JOURNAL OF COMPARATIVE NEUROLOGY 502:236 –260 (2007)

Time Course and Distribution of Inflammatory and Neurodegenerative Events Suggest Structural Bases for the Pathogenesis of Experimental Autoimmune Encephalomyelitis DAVID A. BROWN AND PAUL E. SAWCHENKO* Laboratory of Neuronal Structure and Function, The Salk Institute for Biological Studies, and The Foundation for Medical Research, La Jolla, California 92037

ABSTRACT Murine models of experimental autoimmune encephalomyelitis (EAE) are important vehicles for studying the effects of genetic manipulation on disease processes related to multiple sclerosis (MS). Currently, a comprehensive assessment of EAE pathogenesis with respect to inflammatory and degenerating neuronal elements is lacking. By using Fluoro-jade histochemistry to mark neurodegeneration and dual immunostaining to follow T-cell, microglial, and vascular responses, the time course and distribution of pathological events in EAE was surveyed. C57BL/6J mice were killed at 7, 10, 14, 21 or 35 days after vaccination with the myelin oligodendrocyte glycoprotein peptide MOG35–55. Disease onset occurred at day 14 and peaked at day 21. Early T-cell infiltration and microglial activation in periventricular and superficial white matter structures adjacent to meninges suggested initial recruitment of effector T cells via the cerebrospinal fluid and choroid plexus. This was associated with microglial activation at distal sites along the same white matter tracts, with subsequent vascular recruitment of T cells associated with further injury. Systematic examination of the entire CNS supported this two-step model of EAE pathogenesis, with inflammation and neurodegeneration commencing at similar times and affecting multiple levels of predominantly sensory central pathways, including their terminal fields. This included aspects of the visual, auditory/vestibular, somatosensory (lemniscal), and proprioceptive (spinocerebellar) systems. The early targeting of visual and periventricular structures followed by more widespread CNS involvement is consistent with common presenting signs in human MS patients and suggestive of a similar basis in neuropathology. J. Comp. Neurol. 502:236 –260, 2007. © 2007 Wiley-Liss, Inc. Indexing terms: experimental allergic encephalomyelitis; microglia; myelin oligodendrocyte glycoprotein; neurodegeneration; neuroinflammation; T cell

Multiple sclerosis (MS) is a chronic debilitating disease generally thought to be immune mediated, involving a polygenic predisposition (Ebers et al., 1996) and precipitation by environmental toxicity (Weinshenker, 1996). Demyelination underlies potentially reversible neuronal dysfunction early in disease, whereas neurodegeneration results in permanent neurological deficits (Bjartmar et al., 2003). Although neurodegeneration is traditionally viewed as occurring late in the course of MS, recent evidence supports an early onset of progressive neuronal loss (Bjartmar et al., 2003), which often becomes unresponsive to currently available immunotherapies (Molyneux et al., 2000; Paolillo et al., 1999). To clarify the mechanisms resulting in refractory disease, a model of neurodegeneration applicable to MS is needed. Murine experimental autoimmune encephalomyelitis (EAE) is a dominant animal model of MS (Swanborg, © 2007 WILEY-LISS, INC.

Grant sponsor: National Institutes of Health; Grant number: NS-21182; Grant sponsor: Foundation for Medical Research (to P.E.S.); Grant sponsor: National Health and Medical Research Council, Australia (to D.A.B.); Grant sponsor: U.S. National Multiple Sclerosis Society (to D.A.B.). DAB current address is: Inflammation Research Laboratory, Center for Immunology, St. Vincent’s Hospital, University of New South Wales, Sydney Australia. *Correspondence to: Paul E. Sawchenko, The Salk Institute, 10010 North Torrey Pines Road, La Jolla, CA 92037. E-mail: [email protected] Received 1 March 2007; Revised 29 November 2007; Accepted 29 December 2007 DOI 10.1002/cne.21307 Published online in Wiley InterScience (www.interscience.wiley.com).

The Journal of Comparative Neurology. DOI 10.1002/cne

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Abbreviations AAA ac ACBc ACBs aco AD AHNc AIv alv AM AMB AMHA ANS2 AONe AP APIR APN ARH AV bic BLA BLV BMAa BMAp bp bsc BSTad BSTav CA1 CA2 CA3 CALB cbc CBL cc CC CEAc CEAl CEAm CENT10 CENT2 CENT3 CENT4 CENT6 CENT7 CENT8 CENT9 cg ChP CL CLA CM CNgl COAa COApl COApm COPY CP cpd csc CU cuf CUN DCO df DG DH dhc DMH DMX DN DNp DP dsc dtd dw ec ECU

anterior amygdaloid area anterior commissure nucleus accumbens, core nucleus accumbens, shell anterior commissure, olfactory limb anterodorsal nucleus of the thalamus anterior hypothalamic nucleus, central part agranular insular cortex, ventral part alveus anteromedial nucleus of the thalamus nucleus ambiguus amygdalohippocampal area ansiform lobe of the cerebellum, crus 2 anterior olfactory nucleus, dorsal part area postrema amygdalopiriform transition area anterior pretectal nucleus arcuate nucleus of the hypothalamus anteroventral nucleus of the thalamus brachium of the inferior colliculus basolateral nucleus of the amygdala basolateral nucleus of the amygdala, ventral part basomedial nucleus of the amygdala, anterior part basomedial nucleus of the amygdala, posterior part brachium pontis brachium of the superior colliculus bed nucleus of the stria terminalis, anterodorsal part bed nucleus of the stria terminalis, anteroventral part CA1 field of the Ammon’s horn CA2 field of the Ammon’s horn CA3 field of the Ammon’s horn calbindin-containing nucleus of the pons cerebellar commissure cerebellum corpus callosum central canal central nucleus of the amygdala, central part central nucleus of the amygdala, lateral part central nucleus of the amygdala, medial part central lobe of the cerebellum, lobule 10 central lobe of the cerebellum, lobule 2 central lobe of the cerebellum, lobule 3 central lobe of the cerebellum, lobule 4 central lobe of the cerebellum, lobule 6 central lobe of the cerebellum, lobule 7 central lobe of the cerebellum, lobule 8 central lobe of the cerebellum, lobule 9 P cingulum bundle Choroid plexus central lateral nucleus of the thalamus claustrum central medial nucleus of the thalamus granular layer of the cochlear nuclei cortical nucleus of the amygdala, anterior part cortical nucleus of the amygdala, posterolateral part cortical nucleus of the amygdala, posteromedial part copula pyramidis caudate-putamen cerebral peduncle commissure of the superior colliculus cuneate nucleus cuneate fasciculus cuneiform nucleus dorsal cochlear nucleus dorsal fornix dentate gyrus dorsal horn of the spinal cord dorsal hippocampal commissure dorsomedial hypothalamic nucleus dorsal motor nucleus of the vagus dentate nucleus dentate nucleus, parvicellular part dorsal peduncular area dorsal spinocerebellar tract dorsal tegmental decussation deep white layer of the superior colliculus external capsule external cuneate nucleus

EPd EPv EW fa fi FN fx GPl GR grf GRN IA IAD IAM icp IF IG igr III IMD int IOc IOck IOda IOdm IP IPNr IRN ISL ISLM ISN iw LAd LAv LD LDdm LDvl lfu LH LHA LIN LING lot LPO LRN LSd LSi LSv LVN MA MAN MARN mcp MD MDRNd MDRNv ME MEAd MEApd MEApv MEPO mfb MGd MGm MGv MH ml mlf MPO MRN MS mtg MVNmc MVNpc NB ND NDB nII

endopiriform nucleus, dorsal part endopiriform nucleus, ventral part Edinger-Westphal nucleus corpus callosum, anterior forceps fimbria fastigial nucleus fornix globus pallidus, lateral part gracile nucleus gracile fasciculus gigantocellular reticular nucleus intercalated nucleus of the amygdala interanterodorsal nucleus of the thalamus interanteromedial nucleus of the thalamus inferior cerebellar peduncle interfascicular nucleus induseum griseum intermediate gray layer of the superior colliculus oculomotor nucleus intermediodorsal nucleus of the thalamus internal capsule inferior olivary complex, central nucleus inferior olivary complex, cap of Kooy inferior olivary complex, dorsal accessory nucleus inferior olivary complex, dorsomedial nucleus nucleus interpositus interpeduncular nucleus, rostral part intermediate reticular nucleus insula of Calleja insula of Calleja major inferior salivatory nucleus intermediate white layer of the superior colliculus lateral nucleus of the amygdala, dorsal part lateral nucleus of the amygdala, ventral part laterodorsal nucleus of the thalamus laterodorsal nucleus of the thalamus, dorsomedial part laterodorsal nucleus of the thalamus, ventrolateral part lateral funiculus, spinal cord lateral habenula lateral hypothalamic area linear nucleus of the medulla lingula (⫽CENT1) lateral olfactory tract lateral preoptic area lateral reticular nucleus lateral septum nucleus, dorsal part lateral septum nucleus, intermediate part lateral septum nucleus, ventral part lateral vestibular nucleus magnocellular preoptic nucleus medial accessory oculomotor nucleus magnocellular reticular nucleus middle cerebellar peduncle mediodorsal nucleus of the thalamus medullary reticular nucleus, dorsal part medullary reticular nucleus, ventral part median eminence medial nucleus of the amygdala, dorsal part medial nucleus of the amygdala, posterodorsal part medial nucleus of the amygdala, posteroventral part median preoptic nucleus medial forebrain bundle medial geniculate nucleus, dorsal part medial geniculate nucleus, medial part medial geniculate nucleus, ventral part medial habenula medial lemniscus medial longitudinal fasciculus medial preoptic area mesencephalic reticular nucleus medial septum nucleus mammillotegmental tract medial vestibular nucleus, magnocellular part medial vestibular nucleus, parvicellular part nucleus of the brachium of the inferior colliculus nucleus of Darkschewitsch nucleus of the diagonal band of Broca optic nerve


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1995). However, the spatiotemporal distribution of inflammatory/degenerative events in the widely used C57BL/6J and SJL mouse strains has not been systematically examined. The SJL model displays a relapsingremitting course of EAE (Brown et al., 1982), whereas C57BL/6J mice develop a chronic disease with little variation in severity once a peak has been achieved (Suen et al., 1997). Among strains commonly utilized for genetic manipulation, C57BL/6J is apt to be most informative (Willenborg et al., 1996). Altered neuropathological presentation of EAE in transgenic studies using the C57BL/6J background (Wensky et al., 2005) illustrates the need for a characterization of EAE-associated pathology throughout the wild-type CNS. MS often presents as an episode of optic neuritis with subsequent neuroradiological examination revealing

periventricular plaques representing inflammatory foci (Paty et al., 1988). Typically, disease spreads to involve myelinated tracts of the brainstem, cerebellum, and spinal cord (Sundstrom et al., 2004). A criticism of EAE as a model of MS is that it predominantly targets the spinal cord, leading to motor impairments, which, indeed, form the basis for clinical assessment of disease severity (Fuller et al., 2004). However, it is recognized that EAE may involve other CNS structures, notably the cerebellum and optic tract (Hobom et al., 2004; Wensky et al., 2005). Optic nerve degeneration has been observed within 1 week of EAE onset in rats (Hobom et al., 2004). Additionally, a comparison of CNS inflammatory plaques in rat EAE and human MS revealed similar time courses of axonal damage and demyelination that preceded clinical signs of disease in both cases (Kornek et al., 2000). Also, as in MS,

Abbreviations NIS NR NTB NTS och op opt ORB OV PAG PARN PBm PC pcf PE PFL PGRNd PGRNl PIR PN PO PP PPY PRP PRS PRV PT PVH PVT py pyd RE rf RH RL RM RN RNmc RO RPA RR A8 RT rust RVLN SC SCH SFO sgr SH SI sm SMT SNc SNl

nucleus intercalatus nucleus of Roller nucleus of the trapezoid body nucleus of the solitary tract optic chiasm optic layer of the superior colliculus optic tract orbital cortex, lateral part vascular organ of the lamina terminalis periaqueductal gray matter parvicellular reticular nucleus medial parabrachial nucleus paracentral nucleus of the thalamus preculminate fissure periventricular nucleus of the hypothalamus paraflocculus paragigantocellular reticular nucleus, dorsal part paragigantocellular reticular nucleus, lateral part piriform cortex paranigral nucleus posterior complex of the thalamus peripeduncular nucleus peripyramidal nucleus nucleus prepositus presubiculum parvalbumin-containing nucleus of the hypothalamus paratenial nucleus of the thalamus paraventricular nucleus of the hypothalamus paraventricular nucleus of the thalamus pyramidal tract decussation of the pyramidal tract reuniens nucleus of the thalamus rhinal fissure rhomboid nucleus of the thalamus rostral linear nucleus of the raphe nucleus raphe magnus red nucleus red nucleus, magnocellular part nucleus raphe obscurus nucleus raphe pallidus retrorubral field, dopaminergic A8 group reticular nucleus of the thalamus rubrospinal tract rostroventrolateral reticular nucleus superior colliculus suprachiasmatic nucleus subfornical organ superficial gray layer of the superior colliculus septohippocampal nucleus substantia innominata stria medullaris submedial nucleus of the thalamus substantia nigra, compact part substantia nigra, lateral part

SNr SO spV SPVC SPVI SPVN SPVOdm SPVOvl st SUB SUIII sup sV SVN tb tfp TRS ts tsp TTd TTv TU V V3 V4 VA VAL VCN VCOa VCOp vfu VH vhc VI VII VL VM VMH VPL VPM VRN vsc VTA vtd VTN vVIIIn wm X XI XII ZI zo

substantia nigra, reticular part supraoptic nucleus spinal tract of the trigeminal nerve spinal nucleus of the trigeminal nerve, caudal part spinal nucleus of the trigeminal nerve, interpolar part spinal vestibular nucleus spinal nucelus of the trigeminal nerve, oral dorsomedial part spinal nucleus of the trigeminal nerve, oral ventrolateral part stria terminalis subiculum supraoculomotor nucleus supraoptic commissure sensory root of the trigeminal nerve superior vestibular nucleus trapezoid body transverse fibers of the pons triangular nucleus of the septum tractus solitarius tectospinal tract teania tecta, dorsal part teania tecta, ventral part tuberal nucleus motor nucleus of the trigeminal nerve third ventricle fourth ventricle ventral anterior nucleus of the thalamus ventral anterolateral nucleus of the thalamus vestibulocerebellar nucleus ventral cochlear nucleus, anterior part ventral cochlear nucleus, posterior part ventral funiculus, spinal cord ventral horn of the spinal cord ventral hippocampal commissure abducens nucleus facial nucleus lateral ventricle ventromedial nucleus of the thalamus ventromedial nucleus of the hypothalamus ventroposterolateral nucleus of the thalamus ventroposteromedial nucleus of the thalamus ventral reticular nucleus ventral spinocerebellar tract ventral tegmental area ventral tegmental decussation ventral tegmental nucleus vestibulocochlear nerve, vestibular root white matter nucleus X xiphoid nucleus of the thalamus hypoglossal nucleus zona incerta zonal layer of the superior colliculus


INFLAMMATION AND NEURODEGENERATION IN EAE early immunomodulatory intervention can ameliorate EAE (Youssef et al., 2002), but similar treatments often fail to mitigate neurodegeneration once disease is established (Sattler et al., 2005; Youssef et al., 2002). In the present study, we have taken advantage of a refined histochemical method for localizing degenerating neural elements (Schmued et al., 2005) to follow the time course and distribution of neurodegeneration and its relationship to inflammatory responses (immune cell infiltration and microglial activation) in EAE. The results suggest that the perivascular accumulation of T lymphocytes that characterizes EAE neuropathology at the peak of clinical disease does not develop incrementally but rather evolves to target discrete CNS systems on the basis of anatomical relationships with initial sites of inflammatory/ neurodegenerative responses in white matter structures adjoining the meninges.

MATERIALS AND METHODS Animals C57BL/6J female mice, 6 –12 weeks of age, were purchased from Jackson Laboratories (Bar Harbor, ME) and maintained on a 12:12 hr light:dark cycle, with standard chow and water freely available. All animal procedures were approved by the Institutional Animal Care and Use Committee of The Salk Institute for Biological Studies.

Induction and clinical grading of EAE EAE was induced by vaccinating twice with a 7-day interval with 100 ␮g of MOG35–55 peptide (Sigma, St. Louis, MO) in 100 ␮l complete Freund’s adjuvant (Sigma) supplemented with 1 mg Mycobacterium tuberculosis HRA37 (Difco, Detroit, MI). B. pertussis toxin (250 ng; Sigma) was given intravenously on the day of the initial vaccination and 2 days later. Disease progression was monitored daily using the standard five-point grading system for clinical assessment of EAE (Fuller et al., 2004). Loss of tail tone or objective weakness of a hind limb, clinical score (CS) 1; both tail and hindlimb weakness, CS 2; paraparesis, CS 3; complete paraparesis and forelimb weakness, CS 4; moribund, CS 5. When clinical signs were intermediate between two grades of disease, 0.5 was added to the lower score. Mice were killed at postvaccination days 7, 10, 14 (n ⫽ 6 per time point), 21 (n ⫽ 12), or 35 (n ⫽ 3).

Tissue processing for light microscopy Animals were anesthetized (350 mg/kg chloral hydrate, ip; Sigma) and perfused transcardially with 10 ml 0.9% saline at room temperature, followed by ⬃150 ml ice-cold 4% paraformaldehyde in sodium borate buffer at pH 9.5 via peristaltic pump (Cole Parmer, Vernon Hills, IL). Brains and spinal cords were removed, postfixed for 5 hours, cryoprotected overnight in 20% sucrose/50 mM potassium PBS (KPBS) at 4°C, and six one-in-six series of 30-␮m-thick frozen sections through the brain and select spinal cord segments were taken with a sliding microtome. One series from each was stained with thionin for reference. The remaining sections were stored in cryoprotectant solution (30% ethylene glycol, 20% glycerol in 50 mM sodium phosphate buffer, pH 7.4) at –20°C until further processing.

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Antisera Dual avidin-biotin immunoperoxidase methods were used to concurrently localize markers for T lymphocytes (CD3) and microglia (Iba1), or, for CD4-positive T cells the vascular cell adhesion molecule-1 (VCAM-1) and nonphosphorylated neurofilament H, by using well-characterized, standard antisera. The rat anti mouse CD3 (rat antimCD3; Chemicon, Temecula, CA; catalog No. CBL 1317, clone KT3) was generated by using mouse splenocytes for vaccination and specifically activates T cells through CD3 binding (Tomonari, 1988). Flow cytometric analysis of lymphocytes from multiple areas has confirmed antibody specificity (Seagal et al., 2003). Rabbit polyclonal Iba1 antiserum was generated against the synthetic peptide PTGPPAKKAISELP (Wako, Richmond, VA; catalog No. 019-19741) and recognizes a single band of ⬃17 kDa in Western blotting consistent with the molecular weight of Iba1. Immunhistochemistry in normal brain demonstrates typical microglial morphology (Imai and Kohsaka, 2002). Affinity-purified goat anti-CD4 (R&D Systems, Minneapolis, MN) was generated by using a recombinant peptide encompassing the entire extracellular domain of mouse CD4 and has minimal cross-reactivity with human and rat CD4. Immunohistochemical staining in mouse lung demonstrates typical CD4 staining. In this study, cross-validation was performed with flow cytometry (Steele et al., 2002). A rat monoclonal antibody against VCAM-1 (BD PharMingen, San Diego, CA; catalog No. 550547) was generated by vaccination with the preadipose cell line PA6. This antibody specifically attenuates the interaction between very late antigen 4 (VLA4) on circulating leukocytes and VCAM on endothelial cells in vivo and specifically recognizes the 40- and 100-kDa isoforms of VCAM-1 in Western blots (Kinashi et al., 1995). A mouse-derived monoclonal antibody, SMI-32 (Covance Research Products, Berkeley, CA; catalog No. SMI-32R), was generated by immunization with homogenates of salineperfused rat hypothalamus (Sternberger et al., 1982) and identified in subsequent screens as recognizing a nonphosphorylated epitope of neurofilament H in most mammalian species. The antiserum has been characterized via conventional and two-dimensional immunoblots and variations in staining following trypsin and/or phosphatase digestion (Sternberger and Sternberger, 1983). In the present study, immunolabeling using each of these five antisera was limited to cells displaying the expected morphology and tissue distribution under experimental and control conditions, that is, of T lymphocytes (CD3, CD4), microglia and macrophages (Iba1), vascular endothelial cells (VCAM-1), and a subset of neurons and their processes, with some axonal elements displaying enhanced labeling in vaccinated mice (SMI-32; cf. Trapp et al., 1998).

Immunohistochemistry Free-floating sections were treated with 0.3% hydrogen peroxide for 10 minutes to inhibit endogenous peroxidase activity and then in 1% sodium borohydride in KPBS for 7 minutes to reduce free aldehydes. Sections were then incubated for 48 hours at 4°C in primary antibody (rat anti-mCD3, 1:2,000; or rat anti-mVCAM-1, 1:2,000), in KPBS containing 2% donkey serum and 0.3% Triton X-100. With the same buffer, sections were placed for 1 hr at room temperature in corresponding biotinylated sec-


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ondary antisera raised in donkey (1:200; Jackson Immunoresearch, West Grove, PA). After secondary antibody labeling, sections were enzyme labeled with the Vector Elite kit (Vector Laboratories, Burlingame, CA) as per the manufacturer’s protocol with reaction product developed for 5–7 minutes at 4°C in nickel-diaminobenzidine solution (2.5% nickel ammonium sulfate, 100 mM sodium acetate, 0.5 mg/ml diaminobenzidine, 2 mg/ml D ␤-glucose, 0.4 mg/ml ammonium chloride, 1 U/ml glucose oxidase; Sigma). Each of these steps was followed by two 10-minute rinses in KPBS, except for before and after nickelenhanced DAB development, when two additional 10minute washes in 0.1 M sodium acetate, pH 6, were performed. For dual labeling, the same procedures were repeated with the following variations. Steps to reduce background staining were omitted, and incubation in primary antiserum (rabbit anti-mIba, 1:1,000, or goat antimCD4) proceeded overnight. Staining was developed without nickel enhancement (0.5 mg/ml DAB, 2 mg/ml D ␤-glucose, 0.4 mg/ml ammonium chloride, 1 U/ml glucose oxidase; all from Sigma; in KPBS). The sections were mounted onto gelatin-coated slides and then dehydrated and coverslipped with DPX (Electron Microscopy Sciences, Fort Washington, PA). This protocol resulted in black CD3-positive labeling of T cells and brown Iba1-positive microglial staining or black labeling for VCAM-1-positive cells and brown for CD4-positive cells. Microglial activation was defined as increased intensity of Iba1 staining (Ito et al., 1998) and/or morphological signs of activation after Streit and colleagues (1988).

Fluoro-jade staining Fluoro-jade is an anionic fluorescein derivative used to stain degenerating neurons selectively (Schmued et al., 2005). Brain sections were mounted onto commercially prepared adherent slides (Brain Research Laboratories, Newton, MA), dried, and further fixed (45 min) with 10% neutral buffered formalin and rinsed in distilled deionized water (3 ⫻ 2 minutes). They were then immersed in 80% ethanol/1% sodium hydroxide (w/v) for 5 minutes, followed by 70% ethanol and distilled water for 2 minutes each. The slides were then transferred to a solution of 0.06% potassium permanganate for 10 minutes to block background staining. After an additional water rinse, the sections were stained for 20 minutes in 0.0002% Fluoro-jade C (Histochem, Jefferson, AR) and 0.1% acetic acid. The slides were rinsed in water (3 ⫻ 2 minutes), dried, soaked in xylene, and coverslipped with DPX. In some experiments, Fluoro-jade histochemistry was combined with indirect immunofluorescence localization of nonphosphorylated neurofilament, using monoclonal antibody SMI-32. For this application, immunolabeling was carried out over 2 days on slide-mounted sections between the initial water rinse and immersion in Fluorojade/acetic acid steps outlined above. SMI-32 labeling was detected by using a species-specific goat anti-mouse IgG coupled to tetamethyl rhodamine isothiocyanate (American Qualex, San Clemente, CA).

(for Fluoro-jade) and 555 nm (for tetramethyl rhodamine) was used. Images were acquired with an oil-immersion ⫻63 HCX planapochromat lens (numerical aperture 1.4) by sequential scanning using a two-frame Kalman filter and a z-separation of 0.5 ␮m. Images were processed in Adobe Photoshop 8.0 (Adobe Systems, San Jose, CA) to balance contrast and brightness and then assembled in Canvas X (ACD Systems, Miami, FL).

Electron nicroscopy Five mice at 21 days after EAE vaccination and three age-matched controls were anesthetized and perfused transcardially with saline, followed by 2% glutaraldehyde and 2% paraformaldehyde in phosphate-buffered saline (PBS), pH 7.4. Tissue blocks were postfixed for 1–3 hours in 2% paraformaldehyde at 4°C and rinsed in 0.05 M potassium-phosphate-buffered saline (KPBS) at pH 7.4. Serial 50-␮m-thick vibratome sections through regions of interest (spinal cord, optic tracts, cerebellum, dorsal lateral geniculate nucleus, ventral cochlear nucleus) were cut and saved as one-in-five series. Sections were fixed in 1% osmium tetroxide with 1.5% potassium ferricyanide (Sigma), dehydrated with ethanol and propylene oxide, and infiltrated with Spurr’s resin. The vibratome sections were flat-embedded between two glass microscope slides, one coated with Liquid Release Agent (Electron Microscopy Sciences), and polymerized overnight at 70°C. After the coated slide was removed, a BEEM capsule was placed over each section, filled with Poly/Bed 812 (Polysciences, Warrington, PA), and polymerized overnight. Capsules were removed by concurrent warming and prying, and trimmed to include areas of interest. Thin sections were collected onto 200-mesh copper grids, counterstained with uranyl acetate and lead citrate, and examined in a JEOL 100CX transmission electron microscope. Images were collected as TIFF files with a MegaView III digital camera (SoftImaging System, Lakewood, CO) and then edited and assembled as described above.

Quantitative analyses To evaluate the relationship between neurodegeneration and disease severity, Fluoro-jade labeling in the optic tract and spinal cord (ventral horn at thoracic levels) of EAE mice was ranked by independent blinded observers and compared with clinical score, using the Spearman rank correlation coefficient. P ⬍ 0.05 was considered significant. The density of Fluoro-jadestained elements was initially evaluated semiquantitatively throughout the brains of D21 and D35 animals by using a five-point rating scale, by independent observers (see footnote to Table 1). To assess the validity of ratings, the density of Fluoro-jade-stained elements was assessed at multiple levels of a sampling of gray and white matter structures from a subset of mice with a clinical score of 4.

Imaging

RESULTS Clinical presentation of murine EAE

Most images were acquired under brightfield or epifluorescence illumination (at 488 nm) on a Leica DMRB microscope with a Hamamatsu digital CCD camera (Shizuoka, Japan). For scanning confocal laser microscopy, a Leica TCS SP2 AOBS system with laser lines of 488 nm

The incidence of disease among vaccinated animals was 100%, and mortality was limited to a single mouse from a group of 12 to be killed at day 21. Clinical disease presented over a range of severity and in a manner consistent with an ascending myelopathy. Weakness



Fig. 1. Clinical progression of EAE in C57BL/6J mice. Mean ⫾ SEM clinical score as a function of days after vaccination with MOG35–55. Clinical signs of disease were first detected at ⬃day 14 postvaccination and were maximal in most animals by day 21. Disease incidence among vaccinated animals was 100%, and mortality was limited to a single mouse from the group of 12 to be killed at day 21.

generally occurred first in the tail, the initial signs of disease being loss of the reflexive deviation of the distal tail toward a tactile stimulus. After or concurrent with complete tail paralysis, hindlimb weakness became evident, characterized by altered gait and difficulty in ambulating on a cage grid, with legs falling through the gratings. Further progression of disease manifested as forelimb weakness, which was followed by diminished response to environmental stimuli in the most severe cases. Disease onset typically occurred at 14 days postvaccination, with a peak evident at day 21. At this time, clinical scores ranged between 2 and 5, with a mean of 3.0 (Fig. 1). For comparison, the mean score of a smaller group of mice killed at day 35 was 2.7 (n ⫽ 3). The clinical and histochemical (see below) findings reported here with a two-vaccination model of EAE induction were indistinguishable from those observed in smaller groups of mice (total n ⫽ 20) killed at varying intervals after receiving the more conventional single-vaccination approach, utilizing the same parameters detailed above (see Materials and Methods).

Progression of CNS inflammatory changes in EAE The distribution, extent, and timing of major inflammatory changes in EAE, microglial activation and T-cell infiltration (Fig. 2), were followed via concurrent immunolabeling for Iba1 and CD3, respectively. In normal brains, CD3⫹ T cells were sparse, the bulk of those detected being associated with the meninges, with occasional occurrences in choroid plexus (Fig. 2F). Examination of animals at various intervals after EAE vaccination revealed highly consistent themes in the distribution and timing of inflammatory changes. Inflammatory responses were evident in animals sacrificed at day 7 (n ⫽ 6), well before the onset of clinical signs of disease, manifest principally as increased intensity of

241 Iba1 immunostaining in defined regions. Most salient of these were continuous bands of enhanced microglial labeling in tissue 100 –200 ␮m deep to the ependymal (ventricular; Fig. 2K) and pial surfaces (Fig. 2O) of the brain. Increased Iba1-ir was also seen reliably in macrophages of the meninges and choroid plexus as well as microglia of the olfactory bulbs and circumventricular organs (CVOs; Fig. 2R). These changes were accompanied by increasing numbers of T-cells associated with the meninges (Fig. 2K), and throughout the brain parenchyma, though the magnitude of T-cell recruitment was very modest, relative to the breadth of microglial/macrophage responsiveness. At day 7, there was no evidence of spinal cord involvement, apart from meningeal changes, which were most marked in caudal aspects of the cord. Animals killed at days 10 –14 displayed further generalized activation of microglia adjoining ventricular/ meningeal surfaces. These time points were distinguished, however, by a more pronounced and regionally differentiated infiltration by T cells, which began to define inflammatory foci. These included the choroid plexus (Fig. 2H), CVOs (Fig. 2S), and meninges (Fig. 2L), with microglia demonstrating increased Iba1 expression and retraction and thickening of their processes. Also at this time, T cells were recruited to the subventricular zone, with increasing numbers of CD3⫹ cells observed throughout the rostral migratory stream and its ramifications in the olfactory bulb (Fig. 3; see Aharoni et al., 2005). The first sign of parenchymal vascular recruitment of T lymphocytes, in the form of the perivascular “cuffing,” which is generally viewed as the hallmark of EAE/MS neuropathology, was noted in the rostral corpus callosum and was associated with local microglial activation (Fig. 3B). Animals displaying increased numbers of choroidal T cells also demonstrated T-cell infiltration into the adjoining hippocampal dentate gyrus. At days 10 –14, the meninges of the spinal cord also demonstrated increased numbers of T cells. Additionally, sensory structures innervated by nerves traversing the meninges, including the ventral cochlear nucleus (Fig. 2U–W) and the dorsal horn of the spinal cord (Fig. 4A), showed increased Iba1 expression and morphological indices of microglial activation. Though it was less prominent, ventral (motor) regions of the spinal cord also displayed evidence of microglial activation beginning at day 10, associated mainly with radially oriented vessels that penetrated the ventral columns and with the meninges associated with the anterior (or ventral) median fissure. Consistent with this theme, white matter structures adjacent to inflamed meninges, particularly in areas of meningeal thickening or infolding, demonstrated increased numbers of microglia with an activated phenotype, associated with accumulations of CD3⫹ cells. Examples of such structures include the optic (Fig. 2N–P), lateral olfactory, and ventral spinocerebellar tracts and the middle cerebellar peduncle (Fig. 4E). This pattern of infiltration was not associated with significant parenchymal vascular recruitment of CD3⫹ cells over the day 10 –14 interval. At the peak of disease (day 21), T-cell infiltration was generally increased in all areas highlighted above. In the caudal middle and inferior cerebellar peduncles, the ventral spinocerebellar tract, the rostral termination of caudal somatosensory sensory fibers in the gracile nu-


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Figure 2



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cleus, and the ventral columns of the spinal cord, there was marked infiltration of CD3⫹ cells into the parenchyma, with strong perivascular accumulations, highly suggestive of recruitment via the vasculature. In contrast, sites undergoing earlier microglial activation and T-cell infiltration, such as the rostral middle cerebellar peduncle, displayed noticeably less perivascular accumulation of CD3⫹ T cells (Fig. 4F). The spinal cord (Fig. 4B) and olfactory and optic tracts, including optic radiations (Fig. 2P), were consistently involved. Other brain regions showed variable inflammatory involvement, with major territories such as the basal ganglia and cerebral cortex relatively or completely uninvolved. Foci of cerebellar parenchymal T-cell infiltration were surrounded by dense accumulations of Iba1-positive cells, some of which displayed an amoeboid morphology (Fig. 2E). Additionally, at day 21, microglia not associated with inflammatory infiltrates displayed a reduced intensity of Iba1 labeling. The contrast between Iba1positive cells associated with areas of inflammation and those removed from these foci became accentuated at day 35, with uninvolved microglia assuming baseline morphology and strength of Iba1 expression. Inflammatory changes were commonly lateralized, with one ventral cochlear nucleus or optic tract displaying marked inflammatory involvement, whereas the contralateral side was relatively or wholly spared. A surprising finding was the conspicuous lack of inflammatory involvement of certain structures anatomically and/or functionally related to prominently targeted ones. For example, whereas the gracile nucleus (which receives input from primary somatosensory fibers that enter lumbosacral segments of spinal cord) was consistently heavily involved, the adjoining cuneate nucleus (analogous supply from sensory fibers innervating the forelimbs and upper trunk) was relatively spared. Similarly, the superior cerebellar peduncle was consistently

and conspicuously uninvolved at all time points in all animals despite frank infiltration of adjoining cerebellar white matter structures (e.g., inferior and middle cerebellar peduncles, ventral spinocerebellar tract; Fig. 4H).

Fig. 2. Development of inflammatory responses in EAE is influenced by anatomical relationships. Brightfield photomicrographs showing dual immunoperoxidase labeling for CD3 (black; blue arrows), a generic marker for T lymphocytes, and Iba1 (brown; red arrowheads), for microglia and other macrophage-lineage cells. Days postvaccination (DX) are indicated for each panel. A–E: Microglial responses. Quiescent microglia (A) have small cell bodies, fine cytoplasmic ramifications, and low to moderate Iba1 expression. Earlystage activation (B) is characterized by increased ramification of cytoplasmic processes and cell size and enhanced Iba1 labeling. This is followed (C,D) by further thickening of processes and retraction of finer ones and increased cell body size and Iba1 expression. Near the peak of disease (E), amoeboid cells may be observed demonstrating complete retraction of cytoplasmic processes with maintained high levels of Iba1 expression. F–I: Choroidal responses. Normal choroid plexus (ChP; F) contains Iba1-positive cells, presumed to be choroidal macrophages, and, occasionally, isolated T cells. Beginning at day 7, progressive (G–I) increases in the numbers of both cell types are evident. J–M: Meningeal responses. In the normal case (J), Iba1 labels meningeal macrophages and microglia in underlying neuronal tissue; immunoreactive T cells are sparse. At day 7 (K), increased numbers of T cells are associated with the meninges, with resident microglia showing increased expression of Iba1 and morphological signs of activation; these effects are accentuated by day 14 (L). By day 21 (M), T cells extend into underlying tissue from the meningeal surface, with increased numbers of underlying microglia associated with the inflammatory site. N–P: Reponses of periventricular tissue and the optic tract. Third ventricle (V3) and optic chiasm (och) show-

ing normal distribution of Iba1 staining of quiescent microglia in both structures (N). At day 7 (O), periventricular microglia label more strongly with Iba1 and show morphological signs of activation. Modestly increased Iba1 labeling of resident microglia within the optic chiasm is also evident. By day 21 (P), there is further enhancement of microglial labeling in the periventricular region, with few associated T cells. By contrast, the optic chiasm reveals massive infiltration of CD3-positive T cells, with a high density at the meningeal surface, in addition to increased numbers of intensely stained and activated microglia. Q–T: Circumventricular organ responses. Circumventricular structures such as the area postrema (AP) in normal animals (Q) display quiescent microglia and few, if any, T cells. Beginning at day 7 (R) and proceeding through days 10 (S) and 21 (T), evidence is seen of progressive increases in Iba1 immunolabeling and morphological features of microglial activation, along with increasing numbers of infiltrating T cells whose distribution within the AP and adjoining nucleus of the solitary tract (NTS) become progressively more widespread. U–W: Responses of the ventral cochlear nucleus (VCO). Relative to normal animals (U), the VCO of mice sacrificed at day 14 (V) shows morphological and biochemical evidence of advanced microglial activation and modest T-cell infiltration near the meningeal surface. By day 21 (W), there is further evidence of microglial activation, with increased numbers of Iba1- and CD3-positive cells adjoining the meninges. icp, Inferior cerebellar peduncle. Scale bars ⫽ 25 ␮m in A (applies to A–E); 25 ␮m in F (applies to F–I); 50 ␮m in J (applies to J–M); 100 ␮m in N (applies to N–P); 50 ␮m in Q (applies to Q–T); 50 ␮m in U (applies to U–W).

Regionally differentiated effects of EAE on an index of vascular recruitment The findings described above suggested that the initial T-cell recruitment in EAE occurs at the meninges and choroid plexus rather than at the vasculature and raised the possibility that inflammatory responses in the form of microglial activation at distal levels of afflicted pathways might be involved in subsequent T-cell transit across the vasculature. We therefore sought to extend the analysis to include a signal for T-cell recruitment over the course of EAE. VCAM-1 plays an important role in the vascular recruitment of CNS-specific T cells from the circulation in EAE, serving as a ligand for an integrin, VLA4 (Theien et al., 2003). Observations made in the middle cerebellar peduncle over the course of EAE progression are illustrative of regional differences in VCAM-1 expression in our material (Fig. 5). The adhesion molecule was not detectable in nonvaccinated control animals. Mirroring the inflammatory involvement of white matter tracts at time points prior to disease onset (days 7–14), vaccinated animals killed over this interval showed weakly induced VCAM-1 expression in blood vessels of inflamed tracts adjoining the meninges (e.g., rostral middle cerebellar peduncle); T cells showed no evidence of association with VCAM-1⫹ vessels in these regions and were concentrated instead at or near meningeal surfaces. By contrast, distal levels of these tracts (removed from the meninges; e.g., caudal middle cerebellar peduncle) displayed a much more robust up-regulation of VCAM-1 in presumed vascular


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Fig. 3. Early inflammatory involvement of the rostral migratory stream. Dual immunoperoxidase localization of CD3⫹ T cells (black) and Iba1⫹ microglia (brown) in coronal sections from an animal 10 days postvaccination, at the levels indicated in the inset in A; red shading indicates the position of the rostral migratory stream (RMS). T cells are associated with the RMS at each level (arrows). A: Rostral RMS infiltrated by CD3⫹ cells extending into the corpus callosum and

associated with Iba1⫹ microglia exhibiting an activated morphology. B: Early vascular recruitment of T cells in the anterior forceps of the corpus callosum with T cells in the RMS. C: CD3 cells confined within the RMS again associated with focal microglial activation. D: T cells within the granule cell layer of the olfactory bulb and diffusely distributed Iba1-positive microglia exhibiting activated morphologies. Scale bars ⫽ 100 ␮m.

endothelial cells, which was apparent as early as day 7 and preceded detectable T-cell infiltration. Additionally, fiber tracts such as the superior cerebellar peduncle,

lacking meningeal exposure and displaying little if any inflammatory involvement, exhibited minimal, if any, up-regulation of VCAM-1 expression (Fig. 5).

Fig. 4. Two distinct phases of inflammatory responses in EAE. Photomicrographs showing dual immunoperoxidase labeling for Iba1 (brown) and CD3 (black) of select CNS regions from animals killed 10 –14 or 21 days after vaccination to illustrate that early inflammatory responses in EAE are principally microglial activation proximate to meningeal/choroidal sites of initial T-cell recruitment and only later the more generalized (including vascular) pattern of lymphocyte infiltration that characterizes EAE. A,B: Lumbar spinal cord. Initial microglial activation is focused in dorsal root entry zone (A, arrowheads), whereas only at the peak of disease (B) is substantial infiltration of CD3-positive T cells seen (arrows). C,D: Gracile nuclei of the same animals shown in A and B, respectively. Early, predominantly microglial responses are also seen in the terminus of somatosensory fibers from caudal spinal cord (C) and become more pronounced and

associated with T-cell infiltrate at the peak of disease (D). Note the relative sparing of other white matter tracts, including the cuneate fasciculus and pyramidal decussation. E,F: Rostral middle cerebellar peduncle. Initially, small numbers of T cells at the meningeal surface are associated with more widespread microglial activation (E). By day 21 (F), T-cell infiltration of the mcp and adjoining vessels is apparent, with further microglial activation. G,H: Ipsilateral mcp at the level of the cerebellum of the animals depicted in E and F, respectively. Insets show morphology of Iba1⫹ microglia and/or CD3⫹ T cells. At day 14, there is no evidence of T-cell infiltration at this level (G), whereas, by day 21, prominent perivascular cuffing is observed. Note again the absence of inflammatory responses in an adjoining white matter tract, the superior cerebellar peduncle. Scale bars ⫽ 100 ␮m.



Figure 4

245


246


Fig. 5. Meningeal T-cell recruitment is associated with distant up-regulation of vascular VCAM-1 expression. Photomicrographs showing dual immunoperoxidase labeling for CD4 (brown T cells; arrows) and VCAM-1 (black activated endothelial cells; arrowheads) from EAE (A,C) and control (B,D) mice at two levels of the middle cerebellar peduncle from animals killed 7–14 days after vaccination to illustrate that early inflammatory responses in EAE involve predominantly meningeal T-cell recruitment but are associated with activation of vascular VCAM-1 at distal levels of the same afflicted pathway prior to T-cell recruitment. A,B: Rostral middle cerebellar peduncle

from day 14 EAE (A) and control (B) mice. CD4-positive T cells at the meningeal surface of the mcp are not closely associated with local blood vessels exhibiting VCAM-1 expression. C,D: Caudal mcp from day 7 EAE (C) and nonvaccinated (D) mice demonstrates upregulation of VCAM-1 at a distal level of the same pathway prior to any evidence of local T-cell recruitment. Note the absence of VCAM-1 expression in vasculature associated with the superior cerebellar peduncle, which lacks meningeal exposure throughout its course. Scale bars ⫽ 100 ␮m.

Degenerating neuronal elements are present early in disease

Early inflammatory changes in the ventral cochlear nucleus (Fig. 2U–W) were associated with evidence of punctate Fluoro-jade labeling of axon- and terminal-like profiles (Fig. 6). These changes coincided with morphological indices of microglial activation, but with minimal T-cell infiltration. Similar evidence of early degenerative changes at days 10 –14 was found in the gracile nucleus, again without significant T-cell infiltrate. Degenerating neuronal elements were also evident in the lateral olfactory and optic tracts with uniformly present, but variable, T-cell infiltration and consistently activated microglia. However, despite prominent signs of microglial activation and T-cell infiltration, the corpus callosum demonstrated little, if any, Fluoro-jade labeling. Less consistently exhibiting inflammatory involvement, the hippocampus also never demonstrated

In unvaccinated mice, Fluoro-jade labeling was not observed in neuron-like elements; the only staining seen with any consistency in these controls was of scattered cells exhibiting an astroglial-type morphology located near some meningeal and ventricular surfaces. In material from EAE mice, Fluoro-jade staining was detected in a highly reproducible temporal and spatial pattern. Its predominantly punctate appearance was interpreted on morphological and other grounds (see below) as labeling of axons and terminals. We observed no apparent alteration in labeling of scattered astrocyte-like cells (see above) as a function of disease status, and under no condition were cells exhibiting a microglial morphology labeled with Fluoro-jade C (cf. Damjanac et al., 2006).



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Fig. 6. Neurodegeneration is an early event in EAE. Fluoro-jade C histochemical labeling of sections through the ventral cochlear nucleus to show neurodegeneration in a supraspinal structure prior to the onset of clinical signs of disease. Degenerating elements exhibit-

ing axon- and/or terminal-like morphology were first detected at day 10 (arrows) and increased progressively through day 35, at least. The clinical scores of the animals represented at days 21 and 35 were 4 and 3, respectively. Scale bar ⫽ 100 ␮m.

neurodegeneration concurrent with T-cell infiltration/ microglial activation.

minal type labeling in projections to the principal sensory and spinal trigeminal nuclei (Fig. 8E). Again there were strong correlates between the distributions of inflammatory infiltrate and neurodegeneration. Further somatosensory involvement was manifest in the medial lemniscus, which has a small meningeal exposure early in its course rostrally to the thalamus, where a circumscribed (ventrolateral) part of the ventral posterolateral nucleus, corresponding to the sensory representation of the tail and hindlimbs, showed terminal-type degeneration (Fig. 8F). In the cerebellum, degenerating elements were present in select precerebellar afferents, including the inferior and middle cerebellar peduncles and the ventral spinocerebellar tract. In line with this pattern, prominent terminal degeneration in the cerebellar cortex encompassed the distribution of the spinocerebellar tract. There was no evidence of neurodegeneration in the superior cerebellar peduncle (Fig. 8H), paralleling the results with inflammatory markers in this structure.

CNS sensory neurodegeneration is widespread in EAE By day 21 postvaccination, prominent axonal- and terminal-type neurodegeneration was observed at all levels of the CNS, but predominantly involving multiple levels of select sensory pathways. The distribution and relative extent of neurodegeneration were mapped (Fig. 7, Table 1), and the appearance of this material is illustrated in Figure 8. A prominent example of neurodegeneration at multiple levels of a sensory system was seen in the optic tract and its terminal distributions in the dorsal lateral geniculate nucleus and superficial layers of the superior colliculus (Fig. 8A–C). This pattern of degenerative changes mirrored the local inflammatory response, with intense axonal Fluoro-jade staining in areas of the optic tract proximate to meninges. In the geniculate and colliculus, the pattern of staining again suggested predominant involvement of axon terminals. This pattern of involvement was recapitulated in a number of other areas. The somatosensory system was also extensively involved with prominent axonal involvement in the dorsal columns (gracile fasciculus) associated with terminal-like pattern of neurodegeneration in the gracile nucleus (Fig. 8D). Rostrally, there was axonal Fluoro-jade labeling involving aspects of the sensory trigeminal nerve, with ter-

Axonal-type degeneration is proportional to clinical score In the majority of the fields in which neurodegeneration was observed, there was variability in the extent of Fluoro-jade staining between individual animals that appeared to be directly related to clinical score. The optic tract readily demonstrated this positive relationship of axonal Fluoro-jade labeling in animals with clinical scores


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ranging from 2 to 5 (Fig. 9). To evaluate this relationship, the 12 animals killed at day 21 were ranked on the basis of Fluoro-jade labeling density in the optic tract and ventral horn of thoracic spinal cord. Significant positive correlations were found between this measure and clinical score (Spearman rank coefficients ⫽ 0.544 for optic tract, 0.692 for spinal cord; both P ⬍ 0.05). Similar positive relationships between the density of Fluoro-jade-labeled axonal elements with clinical score were evident in the majority of structures examined.

Nonphosphporylated neurofilament as an alternate/adjunct marker of neurodegeneration Increased staining for nonphosphorylated neurofilament heavy-chain subunits has been associated with axonal damage in MS (Trapp et al., 1998) and EAE (GilgunSherki et al., 2003; Mancardi et al., 2001). A wellcharacterized monoclonal antibody, SMI-32 (Sternberger and Sternberger, 1982; Sternberger et al., 1983), was used to stain series of sections from D21 and D35 EAE (n ⫽ 6; clinical scores ⫽ 3– 4) and control mice (n ⫽ 3) adjoining ones prepared for Fluoro-jade histochemistry to permit direct comparisons (Fig. 10). In control mice, most brain white matter structures displayed little or no SMI-32 labeling. Motor roots of certain cranial nerves were a notable exception to this generalization. In EAE material, SMI-32 staining was present in many of the myelinated fiber tracts in which Fluoro-jade labeling was detected (e.g., optic and ventral spinocerebellar tracts), and absent from Fluoro-jade-negative pathways (e.g., pyramidal tract, superior cerebellar peduncle). Although the relative density and laterality of labeling for the two markers covaried closely in afflicted tracts, some differences in the appearance of elements labeled with each were noted. Whereas Fluoro-jade labeling was predominantly punctate in appearance, with some short, fine threads, SMI-32 stained fewer punctae, with more and longer stretches of axon-like filaments. In gray matter structures in which axon- and terminallike Fluoro-jade labeling was prominent, comparisons were severely limited by the presence of strong SMI-32 labeling of neuronal perikarya and processes in controls. We attempted to surmount this by using confocal microscopy to image material costained for the two markers. Although the sensitivity of SMI-32 localization was substantially diminished in these preparations, colocalization of the two markers in elements interpreted as representing axonal swellings, and even intervaricose segments, was demonstrable in cell groups such as the cerebellar cortex, superior colliculus, and ventral cochlear nucleus, identified above as harboring substantial terminal-like degeneration on the basis of Fluoro-jade labeling.

Electron microscopy Fine structural examination of select CNS regions from animals killed at 21 days after vaccination was carried out to evaluate further the accuracy of Fluoro-jade staining in revealing degenerative events (Fig. 11). Relative to intact controls, white matter structures of vaccinated animals, including the optic tract, cerebellar cortex, and spinal cord, exhibited changes that ranged from a thinning of myelin sheaths to severely disordered and sometimes empty myelin figures. Within those that remained relatively intact, axons were frequently dissociated from my-

elin and/or exhibited darkened axoplasm with occasional aggregation of organelles. The frequency and severity of these changes again appeared to vary directly with clinical score (Fig. 11). In each of the areas examined, there was evidence of increased cellularity around blood vessels in which presumed inflammatory cells were seen beyond the glia limitans. In tissue blocks from gray matter structures in which Fluoro-jade labeling was prominently observed (ventral cochlear nucleus, dorsal lateral geniculate, spinal gray), evidence of axonal/terminal damage took several forms, which appeared alone or in various combinations. First, unmyelinated axon and terminal profiles exhibiting the electron-dense appearance characteristic of degenerating presynaptic elements were observed, frequently in apposition to perikaryal or dendritic profiles (Fig. 12). Second, and most commonly, terminal profiles were observed that contained pronounced mitochondrial abnormalities, in the form of disordered cristae and a marked reduction in the electron density of the matrix (cf. Jones and Rockel, 1973). Examples of neurofilamentous hypertrophy, another form of terminal degeneration described in the retinogeniculate and other systems (Guillery, 1972; Matthews and Raisman, 1972), were also regularly encountered. In addition, areas in which terminal degeneration was seen frequently exhibited astrocytic processes with numerous ⬃30-nmdiameter dark granules, likely representing glycogen accumulation, a correlate of local neurodegeneration (Matthews and Raisman, 1972). Occasionally, such granules were seen within axon terminals themselves, a phenomenon that has been linked to lesion-induced terminal degeneration (Jones and Rockel, 1973). Finally, larger profiles of variable size (4 – 8 ␮m diameter) with marked clumping of mitochondria and other organelles were observed regularly and may correspond to swollen axonal elements described by Trapp et al. (1998) as likely sites of axonal transection in MS/EAE. None of the features described above was seen in similar regional samplings from nonvaccinated control mice.

DISCUSSION Our data suggest that, in the present model at least, inflammatory interactions among resident microglia, invading T lymphocytes, and neuronal elements occur prior to the onset of clinical EAE in lawful temporal and spatial relationship to one another. Pathological changes extend beyond the spinal cord and involve a reproducible set of predominantly sensory CNS systems. Targeting of these systems is dictated largely by structural relationships with choroidal/meningeal sites of initial T-cell invasion, which precedes, and likely triggers, a subsequent wave of vascular T-cell recruitment. The resulting pattern of degenerative changes is consistent with common presenting signs in MS patients, suggesting that the human disease and animal model share common bases in neuropathology.

Two distinct phases in EAE pathogenesis It is now recognized that neurodegeneration and inflammation can occur concurrently in the pathogenesis of MS and EAE (Bjartmar et al., 2003; Kornek et al., 2000). T-cell access to the CNS parenchyma is required for disease onset (Baron et al., 1993), with recruitment to perivascular spaces traditionally viewed as the triggering mechanism (Baron et al., 1993). For this to occur, endo-


Fig. 7. A–L: EAE-induced neurodegeneration in mouse CNS. The density of Fluoro-jade-stained axonal elements is illustrated by varying levels of red shading (see key). This reflects semiquantitative assessments using a five-point rating scale in evenly spaced series of

sections through the brain and several levels of the spinal cord. Data are illustrated from a mouse killed at 35 days after vaccination with a clinical score of 4. Atlas plates taken from the mouse brain atlas of Hof et al. (2000).


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Figure 7

(Continued)



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TABLE 1. Relative Density 1 of Fluoro-Jade Stained Elements in EAE Mice2 Rating 3

Cell groups Forebrain Isocortex Claustrum Olfactory regions Main bulb Olfactory nerve layer Periglomerular layer Outer plexiform layer Mitral layer Inner plexiform layer Granular layer Anterior olfactory n. Olfactory tubercle Piriform cortex Endopiriform nucleus Taenia tecta Hippocampal formation (cortex) Entorhinal area (lateral and medial) Subiculum (dorsal and ventral) CA1 CA3 Dentate gyrus Induseum griseum/fasciola cinerea Amygdala Medial nucleus Amygdalohippocampal area Cortical nucleus N. lat. olfactory tract Anterior amygdaloid area Central nucleus Lateral nucleus Basolateral nucleus Basomedial nucleus Intercalated nuclei Septum Lateral nuclei Medial n. n. diagonal band Bed n. stria terminalis Bed n. anterior commissure Septofimbrial nucleus Triangular nucleus Subfornical organ Basal ganglia Caudoputamen Nucleus accumbens Fundus of striatum Globus pallidus Entopeduncular n. Substantia innominata Magnocellular preoptic nucleus Subthalamic nucleus Substantia nigra Compact part Reticular part Ventral tegmental area Thalamus Medial habenula Lateral habenula Anterior group Mediodorsal nucleus Lateral group Midline group Paraventricular n. Parastaenial n. N. reuniens Rhomboid n. N. gelatinosa Ventral group Ventral anterior/v. lat. Ventral medial Ventral posterior (lateral part) Gustatory nucleus Posterior complex Medial geniculate n. Dorsal part Ventral part Medial part Lateral geniculate n. Dorsal part Intergeniculate leaflet Ventral part Intralaminar nuclei Reticular nucleus Zona incerta Rostral Caudal N. fields of Forel

– – ⫹ – – ⫹/– – ⫹ – – ⫹ – – – – – – – – – – – – – – – – – – – ⫹/– – – – ⫹ ⫹ ⫹ ⫹/– – – – – ⫹ ⫹ ⫹ – ⫹ ⫹ – ⫹ ⫹ – – – ⫹ – – – – – – ⫹⫹ – ⫹⫹ ⫹⫹ – ⫹⫹ – ⫹ – – – ⫹ –

Rating Hypothalamus Periventricular zone Median preoptic n. Anteroventral periventricular n. Preoptic periventricular nucleus Suprachiasmatic n. Supraoptic nucleus Paraventricular n. Anterior periventricular nucleus Arcuate nucleus Posterior periventricular n. Median eminence Medial zone Medial preoptic area Medial preoptic n. Anterior hypo. n. Retrochiasmatic area Ventromedial n. Dorsomedial n. Tuberomammillary n. Premammillary n. Supramammillary n. Lateral mammillary n. Medial mammillary n. Lateral zone Lateral preoptic area Lateral hypothalamic a. Posterior hypothalamic a. Brainstem Sensory Visual Superior colliculus I II III IV V–VI Parabigeminal n. Pretectal region Olivary n. N. optic tract Anterior n. Posterior n. Medial pretectal a. N. posterior commissure Medial terminal n. Somatosensory Mesencephalic n. V Principal sensory n. V Spinal n. V Oral part Interpolar part Caudal part Dorsal column n. Gracile n. Cuneate n. External cuneate n. Auditory Cochlear nuclei Dorsal Ventral N. trapezoid body Superior olive N. lateral lemniscus Inferior colliculus External Dorsal Central N. brachium inf. coll. N. saguluum Vestibular Medial n. Lateral n. Superior n. Spinal n. Gustatory N. solitary tract, ant. Visceral N. solitary tract Area postrema Parabrachial n. Lateral Medial Ko¨lliker-Fuse n. Motor Eye Oculomotor (III) Trochlear (IV)

⫹ – – ⫹ ⫹/– – – – – ⫹ ⫹ – – – – – – – – – – – ⫹ –

⫹⫹ ⫹⫹ ⫹ – – – ⫹/– – – ⫹/– – – ⫹ – – ⫹⫹ – ⫹⫹ ⫹⫹⫹ ⫹/– – ⫹ ⫹⫹⫹⫹ ⫹⫹ – ⫹⫹ ⫹ – ⫹⫹ ⫹ – ⫹⫹ ⫹ – ⫹ ⫹ – – – – –

– –


252

D.A. BROWN AND P.E. SAWCHENKO TABLE 1. (Continued) Rating

Abducens (VI) Jaw Trigeminal n. (V) Face Facial n. (VII) Pharynx/larynx N. ambiguus Tongue Hypoglossal n. (XII) Viscera Dorsal motor n. X Reticular core Periaqueductal gray Interstitial nucleus of Cajal N. Darkschewitsch Dorsal tegmental n. Ventral tegmental n. N. incertus Laterodorsal teg. n. Barrington’s n. Locus coeruleus Raphe Interfascicular n. Rostral linear n. Dorsal raphe Median raphe N. raphe´ pontis N. raphe´ magnus N. raphe´ obscurus N. raphe´ pallidus Interpeduncular n. Reticular formation Central teg. field Peripeduncular n. Pedunculopontine n. Cuneiform n. Pontine reticular Linear n. medulla Parvicellular ret. field Gigantocellular ret. field Lateral paragigantocellular Intermediate ret. field Paramedian reticular n. Pre- and postcerebellar Pontine gray Tegmental reticular n. Inferior olive Lateral reticular n. Red nucleus N. roller N. prepositus Cerebellum Deep nuclei Cortex Molecular layer Purkinje layer Granular layer Spinal Cord Marginal zone Substantia gelatinosa Intermediate gray Intermediolateral column Ventral horn Central gray3 Fiber systems Cranial and spinal nerves (and related) Olfactory nerve Lateral olfactory tract Anterior commissure, olfactory limb Optic nerve Accessory optic tract Brachium of the superior colliculus Commissure of the superior colliculus Optic chiasm Optic tract

– –

– – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – to ⫹ – to ⫹ – to ⫹⫹⫹ ⫹⫹ ⫹⫹ ⫹ ⫹ ⫹⫹ ⫹ ⫹ ⫹⫹ ⫹ ⫹⫹⫹ ⫹⫹⫹ – ⫹⫹⫹ to ⫹⫹⫹⫹ ⫹⫹⫹ to ⫹⫹⫹⫹

Rating Oculomotor nerve Medial longitudinal fascicle Posterior commissure Trochlear nerve Trigeminal nerve Motor root of the trigeminal nerve Sensory root of the trigeminal nerve Mesencephalic trigeminal tract Spinal tract of the trigeminal nerve Facial nerve Genu of the facial nerve Vestibulocochlear nerve Vestibular nerve Trapezoid body Lateral lemniscus Commissure of the inferior colliculus Brachium of the inferior colliculus Vagus nerve Solitary tract (rostral) Hypoglossal nerve Dorsal columns Cuneate fascicle Gracile fascicle Medial lemniscus Hypothalamohypophysial tract Cerebellum Cerebellar commissure Cerebellar peduncles Superior cerebellar peduncle Ventral spinocerebellar tract Middle cerebellar peduncle Inferior cerebellar peduncle Dorsal spinocerebellar tract Lateral forebrain bundle system Corpus callosum Anterior forceps External capsule Genu Posterior forceps Corticospinal tract Internal capsule Cerebral peduncle Pyramidal decussation Pyramidal tract Extrapyramidal fiber systems Tectospinal pathway Rubrospinal tract Ventral tegmental decussation Medial forebrain bundle system Amygdala-related Anterior commissure, temporal limb Stria terminalis Hippocampus-related Fornix system Alveus Dorsal fornix Fimbria (rostral) Postcommissural fornix Medial corticohypothalamic tract Columns of the fornix Hippocampal commissures Ventral hippocampal commissure Cingulated gyrus-related Cingulum bundle Hypothalamus-related Medial forebrain bundle Supraoptic commissures Mammillary-related Mammillothalamic tract Mammillary peduncle Habenula-related Stria medullaris Fasciculus retroflexus Habenular commissure

– – ⫹ – – ⫹ – ⫹⫹ ⫹ ⫹⫹ ⫹⫹⫹ ⫹ ⫹⫹ ⫹⫹ – ⫹ – ⫹ – ⫹/– ⫹⫹⫹ ⫹ – ⫹⫹ to ⫹⫹⫹ – ⫹⫹⫹ ⫹⫹⫹ ⫹⫹ ⫹ ⫹ – ⫹⫹ – ⫹ ⫹ – – – ⫹ – ⫹ –

– – ⫹⫹ – ⫹ – – – – – – ⫹⫹ ⫹ ⫹

1 Ratings employed a five point scale based on the relative density of axon- and terminal-like labeling in given gray and white matter structures, where – indicates the consistent absence of detectable staining and ⫹⫹⫹⫹ the most dense regional accumulations seen in our material. ⫹/– Indicates sparse labeling seen inconsistently within or between cases. Post hoc quantitative assessments indicated that points on the rating scale fell conveniently into the following ranges of density: ⫹, ratings conformed to 1-100 labeled 2 2 2 elements/mm ; ⫹⫹, 101-200/mm ; ⫹⫹⫹, 201-300/mm ; ⫹⫹⫹⫹, 301-400/mm2. 2 Ratings were performed by two independent observers, and reflect the relative densities of mice sacrificed 21 or 35 days after EAE vaccination. Clinical scores of these subjects ranged from 2 to 5. 3 The organizational scheme for listing cell groups and fiber systems is modified after the rat brain atlas of Swanson (1992). Only structures that were readily identifiable in our material are listed, and in instances where all members of a related set of cell groups (e.g., anterior thalamic nuclei) were given the same rating (usually –), the members of the set are not listed individually.



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Fig. 8. Neurodegeneration in EAE preferentially targets select sensory pathways. Fluoro-jade C histochemical preparations from a day 35/CS4 mouse to show distinctive patterns of degeneration at multiple levels of sensory pathways. A–C: In the visual system, degenerating axonal profiles are seen in the optic tract and its terminal projections in the dorsal lateral geniculate nucleus (middle) and the superficial layers of the superior colliculus (right). D–F: Affected somatosensory structures include the gracile, but not the cuneate, nucleus (left), a discrete (dorsal) aspect of the spinal trigeminal tract

and its terminal radiations in the principal sensory nucleus, and a circumscribed part of the ventral posterolateral nucleus of the thalamus (right). G–I: Degenerating elements are seen in select precerebellar afferents including the inferior cerebellar peduncle (left) and the ventral spinocerebellar tract (middle). In line with this, a prominent terminal field in the cerebellar cortex (right) mimics the distribution of the spinocerebellar tract. Note the absence of labeling in the superior cerebellar peduncle. Scale bar ⫽ 100 ␮m.

thelial involvement is required (Baron et al., 1993; Brocke et al., 1999), with the expression of molecules such as VCAM-1 participating in vascular T-cell recruitment (Theien et al., 2003). However, the antecedents that provide for this remain ill defined. Indeed, in healthy mice, activated CNS antigen-specific T cells fail to display adhesive interactions with brain endothelium (Piccio et al., 2002). Our observations suggest that T-cell recruitment for the initiation of EAE is a two-step process. Initially, T cells access ventricular and meningeal cerebrospinal fluid (CSF) spaces, in part via the choroid plexus, in the course of their normal role in the immune surveillance of the CNS. Upon meeting their cognate antigen in white matter tracts adjoining meninges, T cells are activated, resulting

in cytokine secretion, initiating microglial recruitment. As T-cell–microglial interplay proceeds, local axonal damage ensues, giving rise to microglial activation at distant levels of these same afflicted pathways, engaging the program for resolving Wallerian degeneration, which includes vascular recruitment of T cells (Raivich et al., 1998). Some recruited T cells will be reactive against CNS antigens, promoting and initiating further neuronal damage and vascular T-cell recruitment, thereby completing the pathological sequence required for disease establishment. Many of the individual events in this proposed two-step model of EAE pathogenesis have been described. Trafficking of T cells through the choroid occurs in normal hu-


254


Fig. 9. Neurodegeneration in the forebrain parallels clinical score. The degree of neurodegeneration at day 21 varied directly with severity of disease as indicated by clinical score (CS) in the majority of afflicted structures. This is illustrated in sections through the optic tract from mice with clinical scores of 2–5. Scale bar ⫽ 100 ␮m.

mans, indicating operational recruitment mechanisms in noninflamed CNS (Kivisakk et al., 2003; Svenningsson et al., 1993). In mice, peripheral T-cell stimulation by antigen, as occurs in EAE, leads to selective choroidal accumulation of T cells (Petito and Adkins, 2005), and superantigen-mediated choroidal T-cell recruitment can initiate disease in otherwise EAE-resistant interleukin-6deficient mice (Eugster et al., 2001). Additionally, ultrastructural adaptations of the choroid plexus to facilitate T-cell recruitment have been documented in EAE (Steffen et al., 1996). In the Lewis rat model, preclinical proliferation of encephalitic T cells occurs in the subarachnoid spaces, prior to CNS vascular recruitment of effector cells (Matsumoto et al., 1996; Shin et al., 1995). Finally, studies with adoptive T-cell transfer to induce EAE demonstrated early infiltration from meningeal surfaces into the CNS parenchyma (Matsumoto et al., 1996). We find clear evidence of early T-cell infiltration of white matter structures (e.g., middle cerebellar peduncle, optic tract) associated with microglial activation, but with minimal evidence of local vascular recruitment of inflammatory cells. These conditions thus appear to be sufficient to initiate local axonal damage. The facial nerve axotomy model, extensively studied by Kreutzberg and colleagues, consistently demonstrates rapid microglial activation distal to the site of axonal lesions, resulting in local vascular T-cell recruitment in the course of resolving Wallerian degeneration (Raivich et al., 1998). A similar phenomenon has been

reported in experimental autoimmune neuritis, a condition that, as with EAE, targets thoracolumbar spinal cord and leads to rapid microglial activation in the gracile nucleus (Gehrmann et al., 1992).

Bases for pathological targeting in murine EAE In our study, early inflammation and neurodegeneration were observed in the gracile, lateral olfactory and optic tracts, auditory nerve, middle cerebellar peduncle, and associated terminal fields. Intriguingly, pathways related to these demonstrated little or no such involvement. Examples include the cuneate fasciculus (spatially and functionally related to the gracile system) and the superior cerebellar peduncle (which adjoins the middle cerebellar peduncle). Although the cerebellar peduncles are apt to be biochemically quite similar, there are major differences in their anatomical disposition and relationships. The middle cerebellar peduncle abuts the meningeal surface over much of its course from the pontine gray and adjoins CSF outlets that provide additional conduits for T-cell recruitment/infiltration. Initial local axonal damage leads to ipsilateral microglial activation at distal (caudal) levels of the tract as well as up-regulation of VCAM-1, presumably participating in subsequent vascular recruitment of effector T cells capable of augmenting inflammatory/degenerative events. In contrast, the superior cerebellar peduncle lacks meningeal exposure

Fig. 10. Comparison of Fluoro-jade and nonphosphorylated neurofilament H as markers of axonal involvement in EAE. A–I: Fluorescence photomicrographs showing SMI-32 immunolabeling in control (left column) and EAE (middle column) mice; Fluoro-jade staining from same vaccinated mice is shown for comparison (right column). In the optic chiasm (top row), increased SMI-32 labeling in the EAE mouse appears partially in the punctate form that characterizes Fluoro-jade staining but also as fine axon-like filaments (arrow). In the pyramidal tract (second row), no evidence of axonal involvement is seen in EAE using either marker. Typical of gray matter structures in which Fluoro-jade labeling is seen in EAE, the gracile nucleus (Gr;

third row) contains substantial SMI-32 labeling in control animals, which is not grossly altered in EAE (arrowheads denote the midline). Nevertheless, scanning confocal laser microscopy of gray matter regions, such as the cerebellar cortex (J–L), in EAE material doubly labeled for SMI-32 (magenta) and Fluoro-jade (green), examples of codistribution (white) in elements interpreted as axonal swellings/ varicosities and even intervaricose segments (arrows in K) are evident. Some SMI-32 labeled structures are Fluoro-jade negative (arrowhead in J). Scale bars ⫽ 50 ␮m in C (applies to A–C); 100 ␮m in F (applies to D–F); 100 ␮m in I (applies to G–I); 10 ␮m in L (applies to J–L).


256


Fig. 11. Fine structure of EAE-induced inflammatory and degenerative responses. A,B: In relation to the normal fine structure of the optic tract and supporting glia (left), by day 21, a vaccinated mouse (CS4) shows evidence of disordered myelin sheaths and axonal degeneration (arrows). C,D: In the spinal cord, an animal of CS2 at day 21 (left) shows sporadic disruption of myelin and axonal architecture

(arrows) as well as evidence of increased cellularity around a blood vessel in which presumed inflammatory cells are seen beyond the glia limitans (arrowheads). In an animal with more severe disease (CS4; right) presumed inflammatory cells (arrowheads) are again seen in the parenchyma, along with evidence of profound dysmyelination and axonal degeneration (arrows). Scale bar ⫽ 1 ␮m.

throughout its course, a feature that might exclude this tract from inflammatory involvement. This may indicate that meningeal T-cell recruitment and subsequent local neuronal injury are required before vascular recruitment of T cells can complete the pathological requirements for disease onset. In structures notable for their early disease involvement, such as the ventral cochlear and gracile nuclei, several potentially predisposing pathological mechanisms intersect. The ventral cochlear nucleus is innervated by the auditory nerve, which traverses the meninges proximate to CSF outlets. Upon establishment of T-cellmediated inflammation, structures near points of T-cell entry to the CSF may be preferentially targeted for further recruitment. Likely participating in such recruitment are chemokines, which attract responsive cells by establishing a concentration gradient and have been implicated in the pathologies of both MS (Sindern, 2004) and EAE (Dogan and Karpus, 2004). Thus, T cells exiting the fourth

ventricular choroid will migrate in the direction of the strongest chemokine gradient. Assuming that areas of active inflammation have similar rates of chemokine production, those closest to the emerging T cells would preferentially recruit effector cells, giving rise to auditory nerve damage, terminal degeneration in the ventral cochlear nucleus, and impaired auditory function, all of which have been documented to occur in EAE (Watanabe et al., 1996). The fact that such phenomena tend to be highly lateralized may also be explained by the early establishment of a dominant chemotactic gradient. Similarly, the early involvement of the sensory supply to the caudal spinal cord gives rise to microglial activation in their terminal radiations in the gracile nucleus. Consequently, meningeal and vascular recruitment of T cells is directed toward this cell group, further augmenting inflammatory changes, while sparing the adjoining cuneate nucleus. Ostensibly at odds with this generalization, the pyramidal tract were found here to be consistently free of

Fig. 12. EAE-induced terminal damage/degeneration. Electron micrographs from the ventral cochlear nucleus (A,B,D,E), ventral horn of the spinal cord (C), and lateral geniculate nucleus (F–H) from animals sacrificed 21 days after EAE vaccination. Profiles were identified as damaged or degenerating axon terminals (curved arrows) on the basis of 1) a darkened, electron-dense cytoplasm (A,B) that distinguished them from normal counterparts (open arrows); 2) altered mitochondrial structure (m* in C), including disordered cristae and electron-lucent matrix (compare with normal mitochondria; m); 3) hypertrophy of neurofilaments (f) and/or clumping of organelles (E);

and 4) intraneuronal accumulations of electron-dense granules, interpreted as glycogen accumulations (H, straight, thin arrows). The latter were commonly observed in astrocytic processes in areas in which other indices of damage were found (D). Two or more of these features were commonly observed in individual terminals. F shows a slightly darkened cytoplasm and disordered micochondria; G illustrates a terminal with mitochondrial involvement and patchy accumulations of filamentous material; H displays both of the former as well as presumed glycogen granules. Scale bars ⫽ 1 ␮m.


258


inflammatory/degenerative responses, despite significant meningeal exposure. The basis for this remains unclear, but the pyramidal tract does adjoin the middle cerebellar peduncle, whose consistently early and marked targeting may provide a protective chemotactic diversion. Additional challenges to our interpretation are presented by the rostral corpus callosum, hippocampus, and anterior commissure, which displayed early inflammatory infiltrate with minimal evidence of Fluoro-jade labeling. Here it may be noted that each of these structures is associated with populations of neural stem cells (Kempermann et al., 2004; Morshead et al., 1998), which are immunomodulatory (Imitola et al., 2004; Pluchino et al., 2005) and capable of conferring neuroprotection (Pluchino et al., 2005).

Neurodegeneration in murine EAE Prior analyses of CNS neurodegeneration in EAE have focused on select sites of inflammatory involvement, mainly the spinal cord, cerebellum, and optic tract (e.g., Hobom et al., 2004; Weerth et al., 2003). A more encompassing survey has been enabled by the introduction of Fluoro-jade histochemistry as a sensitive general index of neural degeneration (Schmued et al., 2005), yielding results compatible with other indices of damage (e.g., Noraberg et al., 1999; Schmued and Hopkins, 2000), including the ultrastructural analyses provided here. Overall, Fluoro-jade labeling observed in the present study was mainly of axon- and terminal-like structures, without widespread staining of neuronal perikarya at any of the time points examined. Larger labeled profiles were observed occasionally, mainly in spinal cord, although these might represent axonal swellings previously described for MS and EAE (Trapp et al., 1998). The paucity of degenerating cell bodies in our material may be indicative of a low rate of cell loss or sampling at time points prior to those at which maximal loss occurs. It may be noted that post-mortem studies in humans have also more readily demonstrated axonal than perikaryal degeneration (Ganter et al., 1999; van Waesberghe et al., 1999). Recent reports suggest that neuronal dysfunction in murine EAE is not associated with cell loss in the shorter term (Bannerman et al., 2005) but rather with the development of chronic perturbations in neuronal function (Nicot et al., 2005). This would suggest that neuronal loss is not necessarily required for symptomatology but is a progressive phenomenon, as may also be the case in MS (Vercellino et al., 2005).

Relationship to MS Analysis of disease progression over time and space suggests similarities between MOG35–55-induced EAE and MS. The earliest inflammatory responses were periventricular, with prominent involvement of the optic tracts. Inflammation and neurodegeneration were often lateralized in structures adjoining meningeal sites of early inflammation. The prevalence of predominantly unilateral optic neuritis and periventricular plaques in MS patients may be indicative of a similar etiology. Additionally, subsequent neurodegeneration affecting multiple levels of central pathways conveying auditory/vestibular, somatosensory (lemniscal), and proprioceptive (spinocerebellar) information is consistent with common presenting signs in MS patients (Paty et al., 1988) and is suggestive of common bases in neuropathology. Further characterization of the normal lymphocyte surveillance of the CNS and of

early inflammatory events might provide insights into strategies to prevent disease progression in patients presenting with early symptomatology.

ACKNOWLEDGMENTS We gratefully acknowledge the expert assistance of Casey Peto (electron microscopy), Carlos Arias (histology), Kris Trulock (graphics), and Belle Wamsley (editorial). Dr. Larry Schmued generously provided samples of Fluoro-Jade C and advice on its use. P.E.S. is an Investigator of the Foundation for Medical Research. D.A.B. was supported by the National Health and Medical Research Council, Australia, and the U.S. National Multiple Sclerosis Society.

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