Experimental Neurology 250 (2013) 151–155
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Inﬂammatory response after spinal cord injury Martin Oudega ⁎ Department of Physical Medicine and Rehabilitation, University of Pittsburgh, Pittsburgh, PA, USA Department of Bioengineering, University of Pittsburgh, Pittsburgh, PA, USA Department of Neurobiology, University of Pittsburgh, Pittsburgh, PA, USA
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Article history: Received 21 June 2013 Revised 8 September 2013 Accepted 16 September 2013 Available online 21 September 2013
Numerous studies have been published on the intricate relationship between spinal cord injury (SCI) and inﬂammation; however, we have not (yet) been able to use this knowledge to the beneﬁt of people with SCI. A possible reason is that studies have not been detailed enough about the origin of inﬁltrating monocytes and the proﬁle of the macrophage population in a SCI. The deﬁciencies in our knowledge may have hindered the design of truly effective therapies for SCI based on modulation and/or manipulation of the macrophage population. The September issue of Experimental Neurology features an article entitled, “Mobilisation of the splenic monocyte reservoir and peripheral CX3CR1 deﬁciency adversely affects recovery from spinal cord injury.” by Blomster et al. (2013), which expands our knowledge on macrophage biology and physiology in SCI. The results may maneuver the ﬁeld on a path towards potentially effective treatments for spinal cord repair. The experiments in this article were intelligently designed and expertly conducted under supervision of senior author, Dr. Marc Ruitenberg, at the University of Queensland in Brisbane, Australia in collaboration with Dr. Alan Harvey at the University of Western Australia in Perth, Australia. To fully appreciate the impact of this study, a more detailed understanding of SCI, inﬂammation, and our gaps in knowledge of, the sometimes controversial and always intriguing, role of macrophages in SCI and repair is helpful.
Spinal cord injury Each year thousands of people worldwide suffer a SCI resulting in functional impairments leaving them dependent on caregivers (Ackery et al., 2004; van den Berg et al., 2010). Global efforts in the laboratory have identiﬁed repair approaches but so far none of these have successfully translated in the clinic; there is no therapy that guarantees restoration of function such that independency can be salvaged. SCI is multifaceted involving various cellular and molecular deﬁciencies making repair complex and most likely requiring multiple orchestrated interventions
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addressing vital repair events (Hagg and Oudega, 2006; McCreedy and Sakiyama-Elbert, 2012). The mechanical forces during SCI immediately cause massive death of neural cells at the site of injury and loss of integrity of especially the central gray matter (Hagg and Oudega, 2006; Bramlett and Dietrich, 2007; Ek et al., 2010). Axons are severed causing an interruption of information between brain and periphery and vice versa leading to paralysis and loss of sensation. Blood vessels are ruptured or left with an compromised, leaking, blood–spinal cord-barrier (BSB) leading to hemorrhages (Grifﬁths et al., 1978; Mautes et al., 2000; Oudega, 2012). These instant consequences of SCI evoke and contribute to numerous events, including excitotoxicity, ischemia, free radical-mediated damage, hypoxia, starvation, and inﬂammation, that exacerbate the injury progressively (Hall and Springer, 1986; Tator and Fehlings, 1991; Popovich, 2000; Park et al., 2004; Hagg and Oudega, 2006; Bramlett and Dietrich, 2007). This secondary injury causes the initial damage to further extend in rostral–caudal and, importantly, medial–peripheral directions. Although secondary injury not necessarily affects functional outcome, the additional loss of tissue may limit the efﬁcacy of future repair approaches. In time, removal of death and destructed tissue at the epicenter leaves behind ﬂuid-ﬁlled cystic cavities, typically bordered by glial scar tissue. From the ﬁrst moments after SCI, motor and sensory function losses are apparent (Dietz and Harkema, 2004). Other direct or indirect consequences of SCI may develop over time including bladder, bowel, and sexual dysfunction, chronic pain syndrome, infections, heterotopic ossiﬁcation, spasticity, renal failure, heart and circulatory problems. These require recognition and ardent attention because of their impact on the quality of life of people with SCI (Krassioukov et al., 2003; Boakye et al., 2012). Inﬂammation Soon after SCI, vast numbers of macrophages inﬁltrate the damaged tissue. The macrophages are diverse in origin; some are macrophagelike cells originating from local (resident) microglia, others are blood monocytes originating from bone marrow and the spleen. Bloodderived monocytes have low expression of the chemokine receptor, CX3CR1 and high expression of Ly6C (inﬂammatory monocytes) or high expression of CX3CR1 and low or no expression of Ly6C (peripheral tissue monocytes). Blood monocytes enter the damaged area through ruptured and hyper-permeable blood vessels (Mautes et al., 2000). The lack of speciﬁc markers subdividing these early cells phenotypically has to some degree hindered studies to determine their speciﬁc contributions. Alternative techniques such as bone marrow chimeric
M. Oudega / Experimental Neurology 250 (2013) 151–155
animals have revealed more insight in this matter (Popovich and Hickey, 2001). The number of (activated) macrophages in the injured spinal cord is characteristically large, especially compared with that found in the injured brain. In general, macrophages in a SCI accumulate to a peak level during the ﬁrst week post-injury, which remains largely similar during the following one to two weeks and starts declining thereafter (Popovich and Hickey, 2001; Kigerl et al., 2006; Pruss et al., 2011). Early after injury most macrophages in a contusion derived from resident microglia, but around 7 days post-injury most are blood-derived macrophages (Popovich and Hickey, 2001). The actual number of macrophages at any time after injury likely depends on the type and location of the injury and on the species. The proﬁle of macrophages in a SCI has been subject of many studies and was perhaps at times misinterpreted. For instance, repair-promoting cells including Schwann cells, olfactory ensheathing cells, neural stem/progenitor cells, fate-restricted neural and glial precursor cells, and bone marrow stromal cells have been injected into the spinal cord after a clinically relevant contusion at different times after impact (reviewed in Tetzlaff et al., 2011; Ruff et al., 2012) including 7 or 10 days post-injury (e.g., Hofstetter et al., 2002; Takami et al., 2002; Plant et al., 2003; Himes et al., 2006; Schaal et al., 2007; Wu et al., 2012; Yasuda et al., 2011; Li et al., 2012). A reappearing rationale for such subacute transplantation time points was that it would avoid the height of the inﬂammatory response. Based on the cellular proﬁle of inﬂammation (Popovich and Hickey, 2001; Kigerl et al., 2006), this particular justiﬁcation may need a closer examination because it would imply that the cells were in fact grafted at the height of inﬂammation, which may have limited the efﬁcacy of the treatment. Nandoe Tewarie et al. (2009) showed that a 3-day injection compared with a (7-day) injection of bone marrow stromal cells into a contused adult rat spinal cord resulted in better transplant survival accompanied by increased tissue sparing. At this time it is not known whether the transplanted bone marrow stromal cells survived better with 3-day injections because of lower numbers of macrophages in general or bloodderived macrophages in particular at the time of injection. Whether the repair-supporting cells mentioned above are differentially vulnerable to the various types of macrophages in damaged spinal cord nervous tissue is unknown. Also the effect of injection time point on survival of repair-supporting cell types other than bone marrow stromal cells is unknown. Undoubtedly these lacking studies would be helpful in understanding the fate of the different repair-supporting cell types in damaged spinal cord nervous tissue and how to beneﬁt best from their ability to repair the spinal cord. Macrophages are present in the damaged spinal cord nervous tissue for months and years post-injury and can be found away from the injury epicenter especially where distal remains of severed axons need to be cleared (Wallerian degeneration). The progressive nature of secondary injury and the fact that multiple long axonal tracts are subjected to Wallerian degeneration likely contribute to the extended presence of macrophages. What roles do macrophages have in SCI? Their ﬁrst and natural function is to phagocytize neural cell debris which typically involves secretion of factors that directly or indirectly contribute to (additional) cell death such as oxidative metabolites (Blight, 1992; Jones et al., 2005). It is for this reason that inﬂammation is sometimes referred to as a dual-edged sword. Besides the classical role of macrophages, they can also support repair (Jones et al., 2005; Schwartz and Yoles, 2006). Recent evidence revealed polarization among macrophages with a pro-inﬂammatory (M1) and alternatively activated, pro-regenerative, (M2) phenotype (Kigerl et al., 2009) which has shed a new light on the role of macrophages and especially on their direct involvement in repair and how they could be targeted for therapies for SCI. There are two main macrophage-based approaches that have been investigated for their effect on spinal cord repair. A large body of work has been devoted to interventions that aim to limit the macrophage number in the injury. The rationale behind these studies was that while macrophage
invasion is natural and necessary their actions also result in adverse effects that further the loss of tissue. The second approach is grafting peripherally activated macrophages into the damaged tissue to mediate events that would support repair.
Outstanding issues So far, we have not been able to use our knowledge about macrophages to the beneﬁt of people with SCI. Have we overestimated the role of macrophages in spinal cord repair? Do we misjudge the complexity of SCI? Perhaps the problem is that we simply do not know enough details to proﬁt from macrophages for spinal cord repair. To what extent are monocytes from the spleen implicated in SCI? Does the make-up of the macrophages have functional implications? Do macrophages have a dual function (Gensel et al., 2009) or is the functional diversity based on their origin? Does the origin of the macrophages determine their polarization? Answers to these questions provide a more in depth knowledge of the roles of macrophages in SCI and may serve as a foundation for the design of more effective therapies. Blomster et al. (2013) have employed bone marrow chimeric mice and adoptive cell transfer to start addressing some of these unresolved issues. Their article provides numerous important clues but also raise numerous questions. The latter may be explained by the descriptive nature of the article and the multiple topics addressed. However, the results underline the idea that our knowledge is incomplete and that there is territory to explore and understand before development of effective macrophage-based therapies for SCI. Blomster et al. (2013) demonstrated that monocytes in a contusive SCI (70 kdyn) are predominantly derived from the spleen, which was recently discovered as a monocyte reservoir. Mouse bone marrow chimera with green ﬂuorescent protein (GFP) inserted in the Cx3cr1 locus were used in this main experiment. The generation of Cx3cr1gfp/+ and Cx3cr1gfp/gfp mice was published previously by Ruitenberg's group (Vukovic et al., 2010). About 5 million cells from bone marrow from Cx3cr1gfp/+ mice were injected into the tail vein of wild type mice enabling differentiation between blood monocyte-derived (GFP-positive) macrophages and resident microglia-derived (GFP-negative) macrophages. Eight weeks later, when about 93% of all circulating leukocytes were of donor origin, the spinal cord in these mice was contused. The number of blood-monocyte-derived macrophages in the lesion epicenter was signiﬁcantly increased compared with that of uninjured mice (few cells can always be found in the intact spinal cord associated with blood vessels). When the spleen was removed at the time of SCI, the number of GFP-positive macrophages was dramatically decreased compared to injured mice without splenectomy, but still higher than in uninjured mice. An interesting (side) observation was that the differences in monocytes in mice without a SCI and mice with SCI and splenectomy were not detected using proportional area measurement emphasizing the low sensitivity of this technique and serving as a caution to future users not to rely solely on its outcome. The difference between injured mice with and without splenectomy was largest (6-fold) at the injury epicenter. These data show that relative to bone marrowderived monocytes, spleen-derived monocytes are mainly recruited into the SCI. A similar observation was described for stroke (Leuschner et al., 2012) and ischemic myocardial injury (Swirski et al., 2009) emphasizing the biological signiﬁcance of this ﬁnding. The ﬁndings that splenectomy resulted in a decrease in SCI-mediated increase in blood monocytes and the splenectomy-mediated decrease in monocytes was not ‘corrected’ by an increase in monocytes from other origin (Blomster et al., 2013) accentuate the pivotal role of the spleen as the origin of macrophages in SCI. Especially the latter of these two observations is intriguing. Why is it that not more bone marrow-derived monocytes are engaged after a decrease in spleen-derived monocytes in blood? Do only spleen-derived monocytes recognize the signals from the SCI and is the presence of bone marrow-derived monocytes a random occurrence?
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If removal of the spleen signiﬁcantly decreases the number of bloodderived monocytes in blood and a SCI, without being replenishing, what would the effect be on functional recovery? This question was addressed by Blomster et al. (2013) by measuring hindlimb walking performance for 6 weeks in mice with spinal cord contusion and splenectomy. The data revealed an increase in locomotor recovery using the BMS scale at 6 weeks, accompanied by increased white matter sparing, in mice that had their spleen removed at the time of contusive SCI. These observations mirror ﬁndings in brain trauma (Li et al., 2011) and is clinically relevant as people that suffer from SCI may have their spleen injured and sometimes removed (Rabinovici et al., 1999). As the authors mention it would be of interest to analyze available databases and look for a possible correlation between functional recovery and spleen damage/removal. However, the functional results need to be looked at with some reservation. It is unclear whether the difference at 6 weeks between the groups is due to improved recovery in mice with splenectomy or worsened recovery in mice without. The BMS curve of the group without splenectomy declines after 4 weeks and it appears as if this decline caused the difference with the other group. A late decrease in walking ability is sometimes but certainly not always observed. Generally, hindlimb performance in mice and rats with SCI plateaus after 3–4 weeks, depending on the severity of the injury, as was also found in the next experiment (Fig. 3C in Blomster et al., 2013). Additional motor behavior tests (for instance, the horizontal ladder for sensorimotor function) would be helpful to get a clearer idea about the effect of splenectomy on motor recovery after SCI. Also, it would be of interest to investigate whether these events also occur in adult rats. Rats and mice have clearly different inﬂammatory responses after SCI. What signaling pathways are involved in monocyte recruitment to a SCI? Blomster et al. (2013) focused on the chemokine receptor CX3CR1 and found that a peripheral CX3CR1 deﬁciency resulted in a signiﬁcant increase in monocyte recruitment in the contusion. This increase also worsened hindlimb walking ability as measured using the BMS scale which was accompanied by less white matter sparing. Together these experiments establish the spleen as a central source for monocytes in a SCI which are a determinant in functional recovery. In a next series of experiments the contribution of different subsets of monocytes to the pool of macrophages in a SCI was investigated. Subset 1 (Ly6Cpos) monocytes isolated from bone marrow from Cx3cr1gfp/+ mice were injected in the tail vein 3, 6, and 27 days after a SCI and the spinal cord was removed 24 or 72 h later. The subset 1 (inﬂammatory) monocytes were recruited within 24 h into the epicenter of the SCI, regardless of the post-injury time point of injection. At the 24 h time point, the monocytes were still undifferentiated. The recruitment was greatly increased with a 7-day compared to 4-day old SCI. With a 28day old SCI cells were recruited but in signiﬁcantly decreased numbers compared to a 7-day old SCI and similar to a 4-day old SCI. The authors suggested a second wave of macrophage presence in the lesion (Beck et al., 2010) or an impaired BSB as possible explanation for the recruitment at 28 days post-injury. Contusion-induced damage to the BSB is not completely absent at 4 weeks post-injury (Popovich et al., 1996) and may therefore explain the presence of monocytes. An interesting observation came from the mice that received the adoptive transfer at 6 days post-injury and survived 72 h. The number of adoptively transferred inﬂammatory cells in the lesion epicenter was remarkably lower compared with that at 24 h, indicating a high turnover of the cells especially in the sub-acute phase. A deeper insight in their disappearance is not provided; further experimentation will be needed to elucidate this phenomenon. Do they leave the injury or die rapidly after entering? To study the efﬁcacy of recruitment among the different subsets adoptive transfer was employed. Similar number of each of the subsets from the same donor was injected at 6 days post-injury. The data revealed that monocyte subset 2 (Ly6Cneg/low) was recruited with signiﬁcantly lower efﬁciency into the contusion, suggesting a preference for subset 1, inﬂammatory, monocytes. Moreover, SCI was found to shift
the ration of subsets 1 and 2 towards subset 1 in peripheral blood and resulted in increased CX3CR1 and Ly6C expression in subset 2 monocytes. SCI also, albeit slightly, increased CX3CL1/fractalkine in blood, which indicated that the preferential recruitment of subset 1 monocytes and the shift in monocyte composition in blood were likely not due to impaired survival of subset 2 monocytes. The shift towards subset 1 monocytes in the blood is difﬁcult to explain solely by changes within the subset 2 monocyte population. The authors offer several possibilities such as conversion from subset 2 to subset 1 or death among subset 2 monocytes. Additional proof supporting these ideas will need to be gathered in future experiments. An alternative possibility, supported by observation in other damaged tissues, is that indeed the lesion attracts subset 1 monocytes more aggressively, maybe due to known or still unknown receptors on these cells. An intriguing aspect of inﬂammation that has received ample attention in recent years is the ability of macrophages to respond to speciﬁc cues and acquire either of two distinct molecular phenotypes known as M1 and M2. The M1 phenotype is given to classically activated macrophages associated with pro-inﬂammatory actions, while the M2 phenotype is given to alternatively activated macrophages associated with pro-regenerative actions (Kigerl et al., 2009). Which speciﬁc cues mediate such polarization remains a topic of investigation. In vitro, lipopolysaccharide and pro-inﬂammatory cytokines promote differentiation of macrophages into the M1 phenotype, while interleukin-4 and interleukin-3 promote the M2 phenotype (reviewed in Gordon, 2003). Using an adult mouse spinal cord contusion model, it was recently demonstrated that M1 macrophages derive from monocytes homed in a CCL2-dependent manner through the adjacent spinal cord leptomeninges and M2 macrophages from monocytes that trafﬁc through the choroid plexus via VCAM-1-VLA-4 adhesion and epithelial CD73 enzyme for extravasation and epithelial transmigration (Shechter et al., 2013). The data in this study further indicated that monocytes are differentiated into M1 and M2 phenotypes at the entry route rather than the actual lesion (Shechter et al., 2013). Also, the breached blood–brain-barrier was rarely used as a route of entry for monocytes into the lesion early after injury (Shechter et al., 2013). M1 macrophages produce high levels of molecules that are essential for their inﬂammatory, phagocytic, and proteolytic functions resulting in damaged tissue digestion and debris removal. In doing so, these macrophages also contribute to the overall damage by mediating death of initially spared cells. The M2 macrophages are anti-inﬂammatory and support microglia arrest which limits damage expansion (Shechter et al., 2009), and in later stages they are involved in tissue regeneration, growth, angiogenesis and matrix deposition (Sica et al., 2006; Shechter et al., 2011). Because repair of the spinal cord is challenging and macrophages are the predominant cells in nervous tissue trauma (Soares et al., 1995; Sroga et al., 2003), the M2 macrophage with its reparative abilities has received ample attention in recent years. Any involvement of naturally present cells in the damaged spinal cord must be explored for their potential to contribute to repair. There are several questions about the M1 and M2 subsets of macrophages in SCI whose answers could possibly aid in designing repair strategies including how the two subsets are represented in the injured spinal cord and whether polarization towards the two phenotypes is dictated by the origin of the macrophage (i.e., does monocyte recruitment determine macrophage polarization?). Kigerl et al. (2009) investigated the presence of the two subsets in SCI and revealed that high numbers of M1 macrophages are rapidly and persistently present, whereas low numbers of M2 macrophages are transiently present. Clearly, the M1 subset is predominant in SCI and this is likely an important factor in the ongoing (secondary) destruction of nervous tissue that is characteristic for SCI. Blomster et al. (2013) addressed the unresolved question whether monocyte recruitment determines macrophage polarization. In this study, the focus was on the adoptively transferred bone marrow-derived Ly6Chigh monocytes, which were preferentially recruited, present in the one-week old contusion. M1 macrophages were identiﬁed using co-staining for CD16/CD32, CD86, and MHC-II and
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M2 macrophages for arginase-1, CD206 and LYVE-1. The results conﬁrmed that both M1 and M2 macrophage phenotypes were present in the damaged tissue, with overlapping yet distinct staining patterns. The data further showed that from the adoptively transferred (GFP-positive) cells, about 7% expressed CD16/CD32, 1.5% expressed CD86, and 20% expressed MHC-II. Also, about 23% of these cells expressed arginase-1, less than 1% expressed CD206, and 1.6% expressed LYVE-1. Overall, these quantitative data suggest that at 7 days post-impact the majority of polarized macrophages in the contusion derived from resident microglia but not the freshly recruited monocytes. Overall we are still left with unanswered issues. There are certainly aspects to the observations by Blomster et al. (2013) that need further scrutiny such as how the used methodology inﬂuenced macrophage behavior in vivo and whether polarization is maybe an event that occurs later in the lesion site (see Shechter et al., 2013). Nevertheless, the ﬁndings described by Blomster et al. (2013) are a solid start for further more in depth analyses of the relationship between monocyte origin, macrophage polarization, and spinal cord repair. Conclusions It is clear from the work by Blomster et al. (2013) that at the early stages of SCI the inﬂammatory Ly6Chigh monocytes derived from the spleen are dominant in the lesion making them potential targets for therapies for SCI. Future experiments will be necessary to expand our knowledge on this population of monocytes and to unravel how to beneﬁt from their dominance so that effective repair strategies for the spinal cord can be designed. It will be challenging to use the knowledge but this article by Blomster et al. (2013) has contributed to clearing a path towards the design of effective macrophage-based therapeutic interventions for spinal cord repair. References Ackery, A., Tator, C., Krassioukov, A., 2004. A global perspective on spinal cord injury epidemiology. J. Neurotrauma 21, 1355–1370. van den Berg, M.E., Castellote, J.M., Mahillo-Fernandez, I., de Pedro-Cuestra, J., 2010. Incidence of spinal cord injury worldwide: a systemic review. Neuroepidemiology 34, 184–192. Beck, K.D., Nguyen, H.X., Galvan, M.D., Salazar, D.L., Woodruff, T.M., Anderson, A.J., 2010. Quantitative analysis of cellular inﬂammation after traumatic spinal cord injury: evidence for a multiphasic inﬂammatory response in the acute to chronic environment. Brain 133 (Pt 2), 433–447. Blight, A.R., 1992. Macrophages and inﬂammatory damage in spinal cord injury. J. Neurotrauma 9, S83–S91. Blomster, L.V., Brennan, F.H., Lao, H.W., Harle, D.W., Harvey, A.R., Ruitenberg, M.J., 2013. Mobilisation of the splenic monocyte reservoir and peripheral CX3CR1 deﬁciency adversely affect recovery from spinal cord injury. Exp. Neurol. 247, 226–240. Boakye, M., Leigh, B.C., Skelly, A.C., 2012. Quality of life in persons with spinal cord injury: comparisons with other populations. J. Neurosurg. Spine 17 (1 Suppl.), 29–37. Bramlett, H.M., Dietrich, W.D., 2007. Progressive damage after brain and spinal cord injury: pathomechanisms and treatment strategies. Prog. Brain Res. 161, 125–141. Dietz, V., Harkema, S.J., 2004. Locomotor activity in spinal cord-injured persons. J. Appl. Physiol. 96 (5), 1954–1960. Ek, C.J., Habgood, M.D., Callaway, J.K., Dennis, R., Dziegielewska, K.M., Johansson, P.A., Potter, A., Wheaton, B., Saunders, N.R., 2010. Spatio-temporal progression of grey and white matter damage following contusion injury in rat spinal cord. PLoS One 5 (8), e12021. Gensel, J.C., Nakamura, S., Guan, Z., van Rooijen, N., Ankeny, D.P., Popovich, P.G., 2009. Macrophages promote axon regeneration with concurrent neurotoxicity. J. Neurosci. 29, 3956–3968. Gordon, S., 2003. Alternative activation of macrophages. Nat. Rev. Immunol. 3 (1), 23–35. Grifﬁths, I.R., Burns, N., Crawford, A.R., 1978. Early vascular changes in the spinal grey matter following impact injury. Acta Neuropathol. 42, 33–39. Hagg, T., Oudega, M., 2006. Degenerative and spontaneous regenerative processes after spinal cord injury. J. Neurotrauma 23 (3–4), 264–280. Hall, E.D., Springer, J.E., 1986. Role of lipid peroxidation in post-traumatic spinal cord degeneration: a review. Cent. Nerv. Syst. Trauma 3, 281–294. Himes, B.T., Neuhuber, B., Coleman, C., Kushner, R., Swanger, S.A., Kopen, G.C., Wagner, J., Shumsky, J.S., Fischer, I., 2006. Recovery of function following grafting of human bone marrow-derived stromal cells into the injured spinal cord. Neurorehabil. Neural Repair 20 (2), 278–296. Hofstetter, C.P., Schwarz, E.J., Hess, D., Widenfalk, J., El Manira, A., Prockop, D.J., Olson, L., 2002. Marrow stromal cells form guiding strands in the injured spinal cord and promote recovery. Proc. Natl. Acad. Sci. U. S. A. 99 (4), 2199–2204.
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