Perivascular fibroblasts are a major source of the newly formed ECM at 1 and 4

E. Aims of Study

III. RESULTS

5. Perivascular fibroblasts are a major source of the newly formed ECM at 1 and 4

Previous studies showed that fibrotic scars originate from collagen 1α1 expressing perivascular fibroblasts after contusive spinal cord injury (Soderblom et al., 2013; Zhu et al., 2015b). I examined whether FN-positive matrix generated by I-5 injection also derived from the perivascular fibroblasts. Double immunostaining of fibronectin and collagen 1α1 was performed at 1 week and 4 weeks after I-5 injection. Immunoreactivity against collagen 1α1 was almost completely overlapped with FN immunoreactivity at both time points (Fig. 7A-C, G-I). Co-localization of these markers indicated that fibronectin in the new ECM may dervied from perivascular fibroblasts. PDGFR-beta is another marker for the perivascular fibroblasts. I found that PDGFR-beta immunoreactivity, which frequently encircled RECA-1 immunoreactive endothelial cells, within the FN-positive matrix at 1 week. At 4-week time point, PDGFR-beta immunoreactivity increased within the matrix, indicating that perivascular fibroblasts proliferated between the two time points (Fig. 7D,E,J,K). I also observed subtantial expression of CD45, which is marker for hematogenous macrophages, at 1-week time points (Fig. 7F). At 4 weeks after injection, intensity of CD45 was decreased compared to that observed at 1-week time point (Fig. 7M). Together, these results suggest that fibroblasts responsible for production of new ECM after I-5 injection are of perivascular origin, and abundance of CD45+ macrophages within the FN-positive matrix suggest that blood-born imacrophages play a certain role in the hydrogel-induced formation of new fibrotic matrix.

Figure 7. Perivascular fibroblasts are a major source of the newly formed ECM at 1 and 4 week. (A-F) 1 week after I-5 injection. (A) Fibronection immunostained spinal cord (green). (B) Collagen 1alpha1 (red). (C) Merged image. (D) Perivascullar fibroblasts marker PDGFR-β. (E) PDGFR-β expressed around blood vessel maker RECA-1. (F) Blood borned macrophage marker CD45. (G-M) 4 week after I-5 injection.

25 injury compared to animals with PBS injection. Repeated measures two-way ANOVA revealed significant influence of I-5 treatment on behavioral recovery (p <0.05), and the interaction between treatment and time points was also significant (p< 0.001). Posthoc Bonferroni test showed a significant difference between the two groups from 6- to 8-week time points (p < 0.05) (Fig. 8A). Catwalk system was used to assess quality of footprints. In I-5 group stride length tend to recover but this recovery was not significant different compared with PBS group (Fig. 8C). Following injury, the angle of hindpaw rotation relative to the body axis tends to increase indicating external rotation of hindpaws. I-5 injection did not affect the rotation angle. outward steeping. Base of support tends to widen after injury, but I-5 injection also did not affect these parameter (Fig. 8D, 8E). Co-ordinated engagement of fore- and hindpaws during locomotion was impaired after injury, resulting in non-overlapping footprints between fore- and hindpaws. This impairment can be measured by increased distance between the two footprints (defined as relative position). Taken together, these results demonstrated that injection of I-5 significantly enhanced functional recovery.

To investigate potential mechanism by which I-5 injection enhances functional outcomes, I examined myelin intensity in animals that underwent behavior tests. As expected, myelin was protected in only I-5 injection (Fig. 9A), no cavity was found in this group. In contrast, animals with PBS injection displayed lower intensity of MBP myelin staining, cavities also seen in this group. These data suggest that I-5 injection preserved myelin in the remaining white matter and that enhanced myelin preservation may underlie behavioral improvement in animals with I-5 injection.

Figure 8. I-5 injection promoted functional recovery. (A) Loco motor function recovery over 7 weeks after injection. I-5 injected animals (blue) showed significant higher loco motor score than those in PBS injected animals (orange). **p<0.01; *** p<0.001 by repeated- measures two-way ANOVA followed by Tukey’s posthoc analysis at each time point. (B) Representative footprints analyzed by the Catwalk system. Colors of footprints were assigned by the Catwalk program. (bright red= left forepaw, dim red = left hindpaw, bright green = right forepaw and dim green = right hindpaw). (C-F) Quality of locomotion were evaluated by 4 parameters at 7 week after SCI. (C) stride length (distance between the two consecutive hindpaw footprints). (D) base of support (width between the left and right hindpaws. (E) Relative positions (distance between the center pads of forepaw and hindpaw prints), and (angle of the hindpaw axis relative to the horizontal plane; F). **p<0.01 by unpaired t test. PBS (n=9); I-5 (n=8).

Figure 9. Myelin preservation by I-5 injection correlated with functional recovery. (A,B) MBP (Myelin basic protein) immunostaining of spinal cord cross section in animal with PBS and I-5 injection (A, B respectively). (C’) High magnification showed in the box C from (A).

(D’) Magnified image taken from box (D) in (B). Scale bars of (A,B) present 500µm, (C,D) present 50µm.

IV. DISCUSSION

Bridging lesion cavity in SCI is one of the most important steps in injury repair. In this study I revealed that thermal sensitive hydrogel I-5 injection successfully bridged the lesion cavities. The cavity was replaced by remodeled FN(+) matrix. This matrix would reflect natural nature of tissue since I-5 is presumably degraded. FN is an important connective tissue protein, that is the reason why bioengineered biomaterials incorporated with fibronectin to guide and support nerve growth (Ahmed et al., 2003; King et al., 2006). I found that this FN-rich matrix originated from perivascular fibroblasts. The nature of this matrix is similar to that found in the lesion site in mice model where cystic cavities usually do not develop (Soderblom et al., 2013). There was a evidence that FN(+) matrix may present fibrotic scar that inhibit axon growth (Zhu et al., 2015a) and decrease of this matrix by depletion of CD11b hematogenous macrophage with clodronate liposomes resulted in an increase of neurofilament axons growth in the epicenter. In my study, reducing FN(+) matrix by I-5 plus Taxol administration led to a failure of bridging (Fig 5C,D). These data indicated that FN(+) matrix is essential for the generation of matrix filling for cavity spaces. There was a evidence that perivascular cells indicate NG2(+) pericytes, dorsal spinal cord hemi section was performed on Nestin-GFP/NG2-DsRed transgenic mice, number of NG2(+) pericytes significantly accumulated after injury suggesting that pericytes subpopulation can act as stem cell to produce fibrous tissue (Goritz et al., 2011). Another recent study demonstrated that blocking the pericyte regeneration resulted in the failure of fibrous connective tissue in the injury site (Birbrair et al., 2014). This data suggest that pericytes is another cellular source of fibrotic scar tissue. However, expression level of fibronectin to produce beneficial effects in axon regeneration in rat contusive injury model still remains to be elucidated.

MMPs are zinc-dependent endopeptidase, these ECM enzymes play multifunction.

Basically, they involved in ECM degradation and tissue remodeling after injury. The specific role of MMPs in certain circumstance still be controversial. In acute phase of injury, MMPs may cause break down Blood Brain Barirer, edema and hemorrhage (Asahi et al., 2001).

Howerver sufficient roles of these proteinase in chronic phages was not fully understood.

There is increasing evidences that gelatinase activity has benifical role in matrix remodelling and wound healing (Hsu et al., 2006; Kyriakides et al., 2009). In a study,

MMP-29

9 increased 7 -14 days after stroke and colocolized with marker of neurovascular remodelling. Treatment of MMPs inhibitors at 7 days after stroke hindered neurovascular remodelling, enhanced ischemic injury and deteriorated functional recovery (Zhao et al., 2006). The activity and protein expression of MMP-9 exclusively in FN(+) matrix (Fig.

6A,H) suggested that I-5 may induce ECM remodeling via MMP-9 activity. MMP-9 level was elevated together with FN(+) deposition, they showed similar expression pattern at injury site, suggesting that MMP-9 activity may be required for fibronectin production. In order to investigate necessary relationship between FN(+) matrix and MMP-9, I planned to perform loss of function experiment. Particularly, mixing I-5 with MMP9 inhibitor to attenuate MMP-9 activity to test the hypothesis that MMP-9 is necessary for fibroblast accumulation. This experiment is one of the going on stuffs of my work.

Microglial cells play a role in phagocytosis by clearing debris due to necrosis, and secrete various factors and proteases with cytotoxicity on the neuronal tissue (Pan et al., 2002). Fortunately, popy(phosphazene) I-5 used in my study significantly suppressed inflammation while biomaterials used in previous studies did not obtain this feature, they need anti-inflammation agent such as minocycline (Kang et al., 2010) or Flavopiridol – an agent was documented to attenuate microglial activation (Ren et al., 2014). CD 45 positive blood borned macrophages also found to be subsided at 4-week time point. In addition, similar decline of CD68 positive macrophages was observed in I-5 injected animals 4 week after I-5 injection (data not shown). These results suggested that I-5 affected to almost all kind of macrophages. The suppression of macrophages by I-5 injection in my study can also be correlated with cavity reduction since the evidence of increased lesion cavity associated with high level of inflammation (Fitch et al., 1999). However the mechanism of action still not be addressed.

BBB scores demonstrated that the significant differences between animals with I-5 and PBS injection was seen at later time point 6^th, 7^th and 8^th week (Fig. 8A), this result suggested that injury level was similar in all animals at early time point and that different behavior recovery was attributed to hydrogel effect. Coordination between fore- and hindlimbs is an important parameter that reflects quality locomotor function. It was significantly restored following I-5 injection, however the other parameters of footprints measurement were not affected by hydrogel injection. In many studies, hydrogel injection

alone did not bring out beneficial outcomes. Hydrogels are frequently mixed with trophic factors such as Neurotrophin 3 (NT3) (Piantino et al., 2006) or Fibroblast growth factor (FGF) (Chen et al., 2015; Kang et al., 2013), drugs such as liposomes (Nuttelman et al., 2006) or lipid microtubules (Schoenmakers et al., 2004). The behavior recovery was attributed to myelin protection effect of I-5. Following injury, myelin degeneration has occurred. This result in decreasing of myelin intensity. The data in this study showed that intensity of myelin in animals with I-5 injection was higher compared with that in PBS injected animals suggesting a protective role of I-5 injection.

Injectable hydrogel plays an important role in delivery method since drugs are not allowed to penetrate Blood Brain Barrier (BBB). While systematic delivery of drugs that cross BBB needs a large amount and by doing so usually causes unexpected side effects, local delivery directly to injury site carried by hydrogel would require very little amount with high efficiency. In this study, Taxol was successfully delivered by this method, proving effectiveness of the hydrogel as a drug carrier.

Another important application of injectable I-5 is carrying cells to injury site. Cell death occurred in host tissue by multiple mechanisms, survival rate is low between 0.2% and 10% (Kallur et al., 2006); (Bakshi et al., 2005). One of the most important reason is lacking the ECM for cell adherence (Frisch and Francis, 1994). In contrast with solid scaffolds, injectable gel has porous structure, high content of water facilitate cell distribution, adhesion and migration. This good point was proved via numerous studies, supporting cell survival and host tissue integration (Ballios et al., 2010). Increasing the survival of Schwan cells to over 36% (Patel et al., 2010). Enhanced differentiation of murine embryonic stem cells (Yang et al., 2015), neural stem cells (Tate et al., 2002); bone marrow stromal cells (Itosaka et al., 2009). Taking advantages of injectable hydrogel, I also tested I-5 has good effects in supporting neural stem cell survival, primary results have shown that I-5 enhanced viability of neural stem cell. In extending injectable hydrogel applications, other cell types such as induced pluripotent stem cells or mesenchymal stem cell also can be transplanted by this delivery method.

V. CONCLUSION

I demonstrated that injection of temperature sensitive I-5 alone successfully bridge lesion cavity in contusive SCI which close mimic to human injury. FN(+) matrix which is of perivascular fibroblasts is necessary for ECM generation. I also found that I-5 injection may induce ECM via MMP-9 activity, preserved myelin spare tissue and finally bring out functional recovery. Considering the facility of I-5 in a sol state mixing and delivering various drugs and cells, this approach can also be utilized as a versatile platform for multifaceted combinatorial therapy.

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문서에서 Bridging the lesioned spinal cord using hydrogel (페이지 33-47)