中樞神經系統內的微血管具有一層特殊結構：血-脊髓障壁(blood-spinal cord barrier)，它包含了由內皮細胞形成的管壁以及外圍的基底膜、星狀膠細胞及周細胞。脊髓損傷會導致血-脊髓障壁破損，造成血管通透性增加及發炎反應，進而對脊髓造成繼發性傷害。正常發育過程中，周細胞能幫助血管新生並穩定血管通透性，而且在位置上與血管內皮細胞及星狀膠細胞相當靠近，但脊髓損傷之後周細胞是否能參與重建血管或修復組織目前並不清楚。因此，本論文旨在研究周細胞在小鼠脊髓損傷之後所扮演的功能角色。首先我以免疫螢光染色觀察正常脊髓中PDGFR-β與CD13陽性的周細胞，發現其原本具有淚滴形的樣貌，但脊髓損傷後部份周細胞在損傷中心形成纖維性疤痕組織，部份則轉而具有巨噬細胞或微小膠細胞的形態與特徵。在細胞培養實驗中，周細胞可以抑制星狀膠細胞的遷移能力但不影響其增殖能力及生存率，顯示周細胞可能會調控由星狀膠細胞所形成的疤痕組織。然而將人類大腦血管周細胞植入小鼠受損的脊髓後，卻發現星狀膠細胞疤痕組織及殘餘的白質數量沒有明顯變化，但損傷區的血管密度及長度、細胞外基質蛋白如纖維粘連蛋白(fibronectin)與層粘連蛋白(laminin)均有顯著增加，更重要的是植入周細胞的小鼠其運動功能恢復狀況較佳。總合而言，本研究除了發現脊髓損傷後周細胞會形成纖維疤痕組織並具有轉變成發炎細胞的潛能，周細胞還能促進損傷區新生血管的生成，並對肢體運動功能的復原有所助益。本論文的研究成果對未來開發脊髓損傷治療策略提供了一個新的思考方向。

SUMMARY
Blood-spinal cord barrier is a unique vascular structure consisting of endothelial cells, astrocytic endfeet, pericytes, and basal lamina. After spinal cord injury, the blood-spinal cord barrier is disrupted, resulting in increased vascular permeability, inflammatory response, and the formation of an astrocytic glial scar. Although pericytes demonstrate close proximity to both endothelial cells and astrocytes, their roles in the injured spinal cord have not yet been well characterized. Therefore, I investigated the role of pericytes after a moderate contusive injury to the mouse spinal cord. I found that, by immunostaining, both anti-PDGFR-β and anit-CD13 labeled the pericytes in the normal mice. In addition, PDGFR-β positive pericytes not only formed a fibrotic scar in the lesion center but also expressed macrophage/microglia markers CD11b and Iba-1, suggesting that pericytes are likely associated with inflammatory responses. Moreover, inflammatory cytokine TGF-β promoted astrocyte migration in vitro but such enhanced migration was impeded by pericyte-conditioned medium, suggesting that pericytes mediate astrocyte migration. However, transplantation of human brain vascular pericytes into the injured spinal cord did not affect glial scar formation and the size of residual white matter. Instead, implanted pericytes increased vascular density and length along with enhanced expression of extracellular matrix molecules such as fibronectin and laminin, which foster vascular growth, leading to improved motor function recovery after spinal cord injury. Collectively, my research suggests that the pericyte is beneficial to wound healing and a promising target for the therapeutic interventions to treat spinal cord injury in the future.

INTRODUCTION
Spinal cord injury causes functional disabilities to the patient and usually an excessive socioeconomic burden to the family and society. In the normal spinal cord, blood capillaries are characterized by a vascular barrier, which is composed of endothelial cells, basal lamina, astrocytes, and pericytes. This so-called blood-spinal cord barrier blocks harmful substances such as inflammatory cells or cytokines in the blood from entering the spinal cord parenchyma but allows the exchange of permeable nutrients, metabolites, and oxygen. After spinal cord injury, the blood-spinal cord barrier is disrupted, resulting in increased vascular permeability and consequent inflammatory responses that further damage the injured cord. As one of the major components of the vascular barrier, pericytes normally promote angiogenesis and vessel stability as well as the regulation of blood flow. However, whether pericytes participate in the repair of damaged vascular barrier in the injured spinal cord remains unknown. Therefore, I investigated the role of pericytes during wound healing after contusive spinal cord injury in mice. My research may lead to novel therapeutic approaches to improve neural regeneration after spinal cord injury in the future.

MATERIALS AND METHODS
To characterize the pericytes in the injured spinal cord, adult female mice were subject to a moderate weight-drop injury at the 8th thoracic spinal segment and sacrificed 7, 14, 21, and 28 days after injury. Cryosections were stained by immunofluorescence to identify the pericytes and other associated cells. Xenograft of human brain vascular pericytes into the injured mouse spinal cord was performed at 3 days after injury. Residual white matter, examined by Luxol fast blue staining, vascular density, and glial scarring were quantified using ImageJ image processing program. Motor function recovery was assessed by a number of behavior tests including the Basso Mouse Scale. Besides, changes of extracellular matrix proteins were examined by Western blots, whereas vascular permeability was evaluated by Evans blue assay. In vitro studies included the cultures of human brain vascular pericytes and primary mouse cortical astrocytes as well as the assays of astrocyte migration, proliferation, and viability under the influences of pericyte-conditioned media.

RESULTS AND DISCUSSION
In normal spinal cord, pericytes were found surrounding the endothelium by PDGFR-β and CD13 immunostaining. After spinal cord injury, however, some PDGFR-β-positive cells formed a fibrotic scar within the lesion while others were co-localized with CD11b and Iba-1, suggesting that some pericytes changed their property toward macrophage lineage after spinal cord injury. Conversely, most CD13-positive cells in the lesion no longer expressed PDGFR-β but became spherical in shape and were co-immunolabeled with macrophages, demonstrating that CD13 was not a proper marker anymore for pericytes after spinal cord injury. A glial scar was formed by GFAP-positive astrocytes along the border of the injured area. Newly-formed blood vessels appeared spontaneously in the lesion center, where there was no astrocytes, indicating a defective vascular barrier of these vessels for lack of astrocytic wrapping. This was evidenced by increased vascular permeability by Evans blue assay. After the implantation of human brain vascular pericytes into the lesion site, vascular density and length significantly increased, which was likely associated with increased expression of extracellular matrix proteins that facilitate angiogenesis after spinal cord injury. Moreover, I found that injured mice receiving pericyte graft showed better locomotion activity than the untreated control by the assessment using the Basso Mouse Scale . Intriguingly, however, xenograft of pericytes did not reduce glial scar formation or increase white matter sparing. This finding was not consistent with the in vitro study showing that astrocyte migration, the major cause of glial scar formation, was inhibited by pericyte-conditioned media. Therefore, improved motor function recovery observed after pericyte transplantation was possibly a result of better restored vasculature that benefits wound healing after spinal cord injury.

CONCLUSION
My research demonstrated that pericytes dramatically changed their morphology and property toward macrophages/microglia in the lesion, suggesting that pericytes were associated with inflammatory responses, leading to increased permeability of blood vessels after spinal cord injury. Nevertheless, xenograft of human brain vascular pericytes into the injured mouse spinal cord promoted angiogenesis and functional improvement despite the fact that glial scarring and spared white matter were not affected. Moreover, pericytes appeared to produce certain factors that hinder astrocyte migration in vitro, suggesting a modulatory role of pericytes in wound healing after spinal cord injury. Additional research will be needed to further define the capacity of pericytes to facilitate axonal regeneration in the injured spinal cord.

Abbott, N.J., Patabendige, A.A., Dolman, D.E., Yusof, S.R., and Begley, D.J. (2010).
Structure and function of the blood-brain barrier. Neurobiology of disease 37,
13-25.
Armulik, A., Genove, G., and Betsholtz, C. (2011). Pericytes: developmental,
physiological, and pathological perspectives, problems, and promises.
Developmental cell 21, 193-215.
Baeten, K.M., and Akassoglou, K. (2011). Extracellular matrix and matrix receptors in
blood-brain barrier formation and stroke. Developmental neurobiology 71,
1018-1039.
Bai, Y., Zhu, X., Chao, J., Zhang, Y., Qian, C., Li, P., Liu, D., Han, B., Zhao, L., Zhang, J.,
et al. (2015). Pericytes Contribute to the Disruption of the Cerebral Endothelial
Barrier via Increasing VEGF Expression: Implications for Stroke. PLoS ONE 10,
e0124362.
Basso, D.M. (2006). Basso Mouse Scale for Locomotion Detects Differences
in Recovery after Spinal Cord Injury in Five Common Mouse Strains.
NEUROTRAUMA.
Brown, L.A., Sava, P., Garcia, C., and Gonzalez, A.L. (2015). Proteomic Analysis of the
Pericyte Derived Extracellular Matrix. Cellular and Molecular Bioengineering 8,
349-363.
Clapp, C., Thebault, S., Jeziorski, M.C., and Martínez De La Escalera, G. (2009). Peptide
Hormone Regulation of Angiogenesis. Physiological Reviews 89, 1177-1215.
Cummings, B., Engessercesar, C., Cadena, G., and Anderson, A. (2007). Adaptation of a
ladder beam walking task to assess locomotor recovery in mice following spinal
cord injury. Behav Brain Res 177, 232-241.
Dan Lindholm, E.C., Reinhard Kiefer, Francisco Zafra, and Hans Thoenen (1992).
Transforming growth factor-beta 1 in the rat brain: increase after injury and
inhibition of astrocyte proliferation. The Journal of Cell Biology 117, 395-400.
Davis, G.E. (2005). Endothelial Extracellular Matrix: Biosynthesis, Remodeling, and
Functions During Vascular Morphogenesis and Neovessel Stabilization. Circ Res
97, 1093-1107.
ElAli, A., Theriault, P., and Rivest, S. (2014). The role of pericytes in neurovascular unit
remodeling in brain disorders. International journal of molecular sciences 15,
6453-6474.
Fernandez-Klett, F., and Priller, J. (2014). The fibrotic scar in neurological disorders. Brain
pathology 24, 404-413.
Figley, S.A., Khosravi, R., Legasto, J.M., Tseng, Y.F., and Fehlings, M.G. (2014).
Characterization of vascular disruption and blood-spinal cord barrier permeability
following traumatic spinal cord injury. Journal of neurotrauma 31, 541-552.
Goritz, C., Dias, D.O., Tomilin, N., Barbacid, M., Shupliakov, O., and Frisen, J. (2011). A
pericyte origin of spinal cord scar tissue. Science 333, 238-242.
Hallmann, R., Horn, N., Selg, M., Wendler, O., Pausch, F., and Sorokin, L.M. (2005).
Expression and function of laminins in the embryonic and mature vasculature.
Physiol Rev 85, 979-1000.
Hamers, F.P.T., Koopmans, G.C., and Joosten, E.A.J. (2006). CatWalk-Assisted Gait
Analysis in the Assessment of Spinal Cord Injury. J Neurotrauma 23, 537-548.
Huang, X.Q., Zhang, X.Y., Wang, X.R., Yu, S.Y., Fang, S.H., Lu, Y.B., Zhang, W.P., and
Wei, E.Q. (2012). Transforming growth factor beta1-induced astrocyte migration is
mediated in part by activating 5-lipoxygenase and cysteinyl leukotriene receptor 1.
Journal of neuroinflammation 9, 145.
Hurtado-Alvarado, G., Cabanas-Morales, A.M., and Gomez-Gonzalez, B. (2014).
Pericytes:brain-immune interface modulators. Frontiers in integrative neuroscience
7, 80.
Kawano, H., Kimura-Kuroda, J., Komuta, Y., Yoshioka, N., Li, H.P., Kawamura, K., Li, Y.,
and Raisman, G. (2012). Role of the lesion scar in the response to damage and
repair of the central nervous system. Cell and tissue research 349, 169-180.
Ma, M., Basso, D.M., Walters, P., Stokes, B.T., and Jakeman, L.B. (2001). Behavioral and
histological outcomes following graded spinal cord contusion injury in the C57Bl/6
mouse. Experimental neurology 169, 239-254.
Mills, S.J., Cowin, A.J., and Kaur, P. (2013). Pericytes, mesenchymal stem cells and the
wound healing process. Cells 2, 621-634.
Muramatsu, R., and Yamashita, T. (2014). Pericyte function in the physiological central
nervous system. Neuroscience research 81-82, 38-41.
Oyinbo, C.A. (2011). Secondary injury mechanisms in traumatic spinal cord injury:
a nugget of this multiply cascade. Acta Neurobiol.
Ozen, I., Deierborg, T., Miharada, K., Padel, T., Englund, E., Genove, G., and Paul, G.
(2014). Brain pericytes acquire a microglial phenotype after stroke. Acta
neuropathologica 128, 381-396.
Rustenhoven, J., Aalderink, M., Scotter, E.L., Oldfield, R.L., Bergin, P.S., Mee, E.W.,
Graham, E.S., Faull, R.L.M., Curtis, M.A., Park, T.I.H., et al. (2016). TGF-beta1
regulates human brain pericyte inflammatory processes involved in
neurovasculature function. Journal of neuroinflammation 13, 37.
Sa-Pereira, I., Brites, D., and Brito, M.A. (2012). Neurovascular unit: a focus on pericytes.
Molecular neurobiology 45, 327-347.
Sakuma, R., Kawahara, M., Nakano-Doi, A., Takahashi, A., Tanaka, Y., Narita, A., Kuwahara-Otani, S., Hayakawa, T., Yagi, H., Matsuyama, T., et al. (2016). Brain pericytes
serve as microglia-generating multipotent vascular stem cells following ischemic
stroke. Journal of neuroinflammation 13, 57.
Schrimpf, C., Teebken, O.E., Wilhelmi, M., and Duffield, J.S. (2014). The role of pericyte
detachment in vascular rarefaction. Journal of vascular research 51, 247-258.
Soderblom, C., Luo, X., Blumenthal, E., Bray, E., Lyapichev, K., Ramos, J., Krishnan, V.,
Lai-Hsu, C., Park, K.K., Tsoulfas, P., et al. (2013). Perivascular fibroblasts form the
fibrotic scar after contusive spinal cord injury. The Journal of neuroscience : the
official journal of the Society for Neuroscience 33, 13882-13887.
Stapor, P.C., Sweat, R.S., Dashti, D.C., Betancourt, A.M., and Murfee, W.L. (2014).
Pericyte dynamics during angiogenesis: new insights from new identities. Journal
of vascular research 51, 163-174.
Winkler, E.A., Bell, R.D., and Zlokovic, B.V. (2011). Central nervous system pericytes in
health and disease. Nature neuroscience 14, 1398-1405.
Wu, J.C., Chen, Y.C., Liu, L., Chen, T.J., Huang, W.C., Cheng, H., and Su, T.P. (2012).
Effects of age, gender, and socio-economic status on the incidence of spinal cord
injury: an assessment using the eleven-year comprehensive nationwide database of
Taiwan. Journal of neurotrauma 29, 889-897.
Zhou, X., He, X., and Ren, Y. (2014). Function of microglia and macrophages in secondary
damage after spinal cord injury. Neural Regeneration Research 9, 1787-1795.