Spinal root injuries result in newly formed glial scar formation, which prevents regeneration of sensory axons causing permanent sensory loss. Previous studies showed that delivery of trophic factors or implantation of human neural progenitor cells supports sensory axon regeneration and partly restores sensory functions. In this study, we elucidate mechanisms underlying stem cell-mediated ingrowth of sensory axons after dorsal root avulsion (DRA). We show that human spinal cord neural stem/progenitor cells (hscNSPC), and also, mesoporous silica particles loaded with growth factor mimetics (MesoMIM), supported sensory axon regeneration. However, when hscNSPC and MesoMIM were combined, sensory axon regeneration failed. Morphological and tracing analysis showed that sensory axons grow through the newly established glial scar along “bridges” formed by migrating stem cells. Coimplantation of MesoMIM prevented stem cell migration, “bridges” were not formed, and sensory axons failed to enter the spinal cord. MesoMIM applied alone supported sensory axons ingrowth, but without affecting glial scar formation. In vitro, the presence of MesoMIM significantly impaired migration of hscNSPC without affecting their level of differentiation. Our data show that (1) the ability of stem cells to migrate into the spinal cord and organize cellular “bridges” in the newly formed interface is crucial for successful sensory axon regeneration, (2) trophic factor mimetics delivered by mesoporous silica may be a convenient alternative way to induce sensory axon regeneration, and (3) a combinatorial approach of individually beneficial components is not necessarily additive, but can be counterproductive for axonal growth.
CarlstedtT, GraneP, HallinRG and NorenG. (1995). Return of function after spinal cord implantation of avulsed spinal nerve roots. Lancet, 346:1323–1325.
3.
FraherJP. (2000). The transitional zone and CNS regeneration. J Anat, 196:137–158.
4.
SimsTJ and GilmoreSA. (1994). Regeneration of dorsal root axons into experimentally altered glial environments in the rat spinal cord. Exp Brain Res, 99:25–33.
5.
ChewDJ, CarlstedtT and ShortlandPJ. (2011). A comparative histological analysis of two models of nerve root avulsion injury in the adult rat. Neuropathol Appl Neurobiol, 37:613–632.
6.
CarlstedtT. (1985). Regenerating axons form nerve terminals at astrocytes. Brain Res, 347:188–191.
7.
LiuzziFJ and LasekRJ. (1987). Astrocytes block axonal regeneration in mammals by activating the physiological stop pathway. Science, 237:642–645.
8.
Di MaioA, SkubaA, HimesBT, BhagatSL, HyunJK, TesslerA, BishopD and SonY-J. (2011). In vivo imaging of dorsal root regeneration: rapid immobilization and presynaptic differentiation at the CNS/PNS border. J Neurosci, 31:4569–4582.
9.
SteinmetzMP, HornKP, TomVJ, MillerJH, BuschSA, NairD, SilverDJ and SilverJ. (2005). Chronic enhancement of the intrinsic growth capacity of sensory neurons combined with the degradation of inhibitory proteoglycans allows functional regeneration of sensory axons through the dorsal root entry zone in the mammalian spinal cord. J Neurosci, 25:8066–8076.
10.
HarveyPA, LeeDHS, QianF, WeinrebPH and FrankE. (2009). Blockade of Nogo receptor ligands promotes functional regeneration of sensory axons after dorsal root crush. J Neurosci, 29:6285–6295.
11.
WangR, KingT, OssipovMH, RossomandoAJ, VanderahTW, HarveyP, CarianiP, FrankE, SahDWY and PorrecaF. (2008). Persistent restoration of sensory function by immediate or delayed systemic artemin after dorsal root injury. Nat Neurosci, 11:488–496.
12.
RamerMS, PriestleyJV and McMahonSB. (2000). Functional regeneration of sensory axons into the adult spinal cord. Nature, 403:312–316.
13.
RamerMS, DuraisingamI, PriestleyJV and McMahonSB. (2001). Two-tiered inhibition of axon regeneration at the dorsal root entry zone. J Neurosci, 21:2651–2660.
14.
Ramón-CuetoA and Nieto-SampedroM. (1994). Regeneration into the spinal cord of transected dorsal root axons is promoted by ensheathing glia transplants. Exp Neurol, 127:232–244.
15.
LiY, CarlstedtT, BertholdCH and RaismanG. (2004). Interaction of transplanted olfactory-ensheathing cells and host astrocytic processes provides a bridge for axons to regenerate across the dorsal root entry zone. Exp Neurol, 188:300–308.
WuD, ZhangY, BoX, HuangW, XiaoF, ZhangX, MiaoT, MagoulasC, SubangMC and RichardsonPM. (2007). Actions of neuropoietic cytokines and cyclic AMP in regenerative conditioning of rat primary sensory neurons. Exp Neurol, 204:66–76.
18.
JangSY, ShinYK, JungJ, LeeSH, SeoS-Y, SuhDJ and ParkHT. (2010). Injury-induced CRMP4 expression in adult sensory neurons; a possible target gene for ciliary neurotrophic factor. Neurosci Lett, 485:37–42.
19.
ÅkessonE, PiaoJ-HH, SamuelssonE-BB, HolmbergL, KjældgaardA, FalciS, SundströmE and SeigerÅ. (2007). Long-term culture and neuronal survival after intraspinal transplantation of human spinal cord-derived neurospheres. Physiol Behav, 92:60–66.
20.
Garcia-BennettAE, KozhevnikovaM, KönigN, ZhouC, LeaoR, KnöpfelT, PankratovaS, TrolleC, BerezinV, et al. (2013). Delivery of differentiation factors by mesoporous silica particles assists advanced differentiation of transplanted murine embryonic stem cells. Stem Cells Transl Med, 2:906–915.
21.
NielsenJ, GotfrydK, LiS, KulahinN, SorokaV, RasmussenKK, BockE and BerezinV. (2009). Role of glial cell line-derived neurotrophic factor (GDNF)-neural cell adhesion molecule (NCAM) interactions in induction of neurite outgrowth and identification of a binding site for NCAM in the heel region of GDNF. J Neurosci, 29:11360–11376.
22.
RathjeM, PankratovaS, NielsenJ, GotfrydK, BockE and BerezinV. (2011). A peptide derived from the CD loop-D helix region of ciliary neurotrophic factor (CNTF) induces neuronal differentiation and survival by binding to the leukemia inhibitory factor (LIF) receptor and common cytokine receptor chain gp130. Eur J Cell Biol, 90:990–999.
23.
SchindelinJ, Arganda-CarrerasI, FriseE, KaynigV, LongairM, PietzschT, PreibischS, RuedenC, SaalfeldS, et al. (2012). Fiji: an open-source platform for biological-image analysis. Nat Methods, 9:676–682.
24.
TsaiW-H. (1985). Moment-preserving thresolding: a new approach. Comput Vision, Graph Image Process, 29:377–393.
25.
OtsuN. (1979). A threshold selection method from Gray-level histograms. IEEE Trans Syst Man Cybern, 9:62–66.
26.
EmgårdM, PiaoJ, AineskogH, LiuJ, CalzarossaC, OdebergJ, HolmbergL, SamuelssonEB, BezubikB, et al. (2014). Neuroprotective effects of human spinal cord-derived neural precursor cells after transplantation to the injured spinal cord. Exp Neurol, 253:138–145.
27.
RamakersC, RuijterJM, Lekanne DeprezRH and MoormanAFM. (2003). Assumption-free analysis of quantitative real-time polymerase chain reaction (PCR) data. Neurosci Lett, 339:62–66.
28.
De PreterK, SpelemanF, CombaretV, LunecJ, LaureysG, EussenBHJ, FrancotteN, BoardJ, PearsonADJ, et al. (2002). Quantification of MYCN, DDX1, and NAG gene copy number in neuroblastoma using a real-time quantitative PCR assay. Mod Pathol, 15:159–166.
29.
BrumovskyP, WatanabeM and HökfeltT. (2007). Expression of the vesicular glutamate transporters-1 and −2 in adult mouse dorsal root ganglia and spinal cord and their regulation by nerve injury. Neuroscience, 147:469–490.
30.
CreggJM, DePaulMA, FilousAR, LangBT, TranA and SilverJ. (2014). Functional regeneration beyond the glial scar. Exp Neurol, 253:197–207.
31.
ZhangY, Pa. Dijkhuizen, AndersonPN, LiebermanAR and VerhaagenJ. (1998). NT-3 delivered by an adenoviral vector induces injured dorsal root axons to regenerate into the spinal cord of adult rats. J Neurosci Res, 54:554–562.
32.
RomeroMI, RangappaN, GarryMG and SmithGM. (2001). Functional regeneration of chronically injured sensory afferents into adult spinal cord after neurotrophin gene therapy. J Neurosci, 21:8408–8416.
33.
RamerMS, BishopT, DockeryP, MobarakMS, O'LearyD, FraherJP, PriestleyJV and McMahonSB. (2002). Neurotrophin-3-mediated regeneration and recovery of proprioception following dorsal rhizotomy. Mol Cell Neurosci, 19:239–249.
34.
TangF, LiL and ChenD. (2012). Mesoporous silica nanoparticles: synthesis, biocompatibility and drug delivery. Adv Mater, 24:1504–1534.
35.
GoncalvesMB, MalmqvistT, ClarkeE, HubensCJ, GristJ, HobbsC, TrigoD, RislingM, AngeriaM, et al. (2015). Neuronal RARβ signaling modulates PTEN activity directly in neurons and via exosome transfer in astrocytes to prevent glial scar formation and induce spinal cord regeneration. J Neurosci, 35:15731–15745.
36.
KliotM, SmithGM, SiegalJD and SilverJ. (1990). Astrocyte-polymer implants promote regeneration of dorsal root fibers into the adult mammalian spinal cord. Exp Neurol, 109:57–69.
KameiN, TanakaN, OishiY, HamasakiT, NakanishiK, SakaiN and OchiM. (2007). BDNF, NT-3, and NGF released from transplanted neural progenitor cells promote corticospinal axon growth in organotypic cocultures. Spine, 32:1272–1278.
39.
Blurton-JonesM, KitazawaM, Martinez-CoriaH, CastelloNA, MüllerF-J, LoringJF, YamasakiTR, PoonWW, GreenKN and LaFerlaFM. (2009). Neural stem cells improve cognition via BDNF in a transgenic model of Alzheimer disease. Proc Natl Acad Sci U S A, 106:13594–13599.
40.
HawrylukGWJ, MotheA, WangJ, WangS, TatorC and FehlingsMG. (2012). An in vivo characterization of trophic factor production following neural precursor cell or bone marrow stromal cell transplantation for spinal cord injury. Stem Cells Dev, 21:2222–2238.
41.
LuP, JonesLL, SnyderEY and TuszynskiMH. (2003). Neural stem cells constitutively secrete neurotrophic factors and promote extensive host axonal growth after spinal cord injury. Exp Neurol, 181:115–129.
42.
TengYD, YuD, RopperAE, LiJ, KabatasS, WakemanDR, WangJ, SullivanMP, RedmondDE, et al. (2011). Functional multipotency of stem cells: a conceptual review of neurotrophic factor-based evidence and its role in translational research. Curr Neuropharmacol, 9:574–585.
43.
YiDK and BarrGA. (1995). The induction of Fos-like immunoreactivity by noxious thermal, mechanical and chemical stimuli in the lumbar spinal cord of infant rats. Pain, 60:257–265.
44.
LuP, WangY, GrahamL, McHaleK, GaoM, WuD, BrockJ, BleschA, RosenzweigES, et al. (2012). Long-distance growth and connectivity of neural stem cells after severe spinal cord injury. Cell, 150:1264–1273.
45.
ModoM, MellodewK, CashD, FraserSE, MeadeTJ, PriceJ and WilliamsSCR. (2004). Mapping transplanted stem cell migration after a stroke: a serial, in vivo magnetic resonance imaging study. Neuroimage, 21:311–317.
46.
StewardO, SharpKG and Matsudaira YeeK. (2014). Long-distance migration and colonization of transplanted neural stem cells. Cell, 156:385–387.
47.
GuzmanR, UchidaN, BlissTM, HeD, ChristophersonKK, StellwagenD, CapelaA, GreveJ, MalenkaRC, et al. (2007). Long-term monitoring of transplanted human neural stem cells in developmental and pathological contexts with MRI. Proc Natl Acad Sci U S A, 104:10211–10216.
48.
FrickerRa, CarpenterMK, WinklerC, GrecoC, GatesMA and BjörklundA. (1999). Site-specific migration and neuronal differentiation of human neural progenitor cells after transplantation in the adult rat brain. J Neurosci, 19:5990–6005.
49.
TuszynskiMH, WangY, GrahamL, GaoM, WuD, BrockJ, BleschA, RosenzweigES, HavtonLA, et al. (2014). Neural stem cell dissemination after grafting to CNS injury sites. Cell, 156:388–389.
50.
BehrstockS, EbertAD, KleinS, SchmittM, MooreJM and SvendsenCN. (2008). Lesion-induced increase in survival and migration of human neural progenitor cells releasing GDNF. Cell Transplant, 17:753–762.
51.
YandavaBD, BillinghurstLL and SnyderEY. (1999). “Global” cell replacement is feasible via neural stem cell transplantation: evidence from the dysmyelinated shiverer mouse brain. Proc Natl Acad Sci U S A transplantation:7029–7034.
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