Bone marrow stromal cell implantation for peripheral nerve repair

Cell therapy using bone marrow stromal cells is a new promising therapy for regenerative medicine. Previous studies demonstrated that local bone marrow stromal cells implantation in the distal stump of transected sciatic nerve of rats promotes early functional recovery. The purpose of this study was to expand on the preliminary research by investigating the long-term efficacy of bone marrow stromal cells using the same experimental setting. Functional test and histological studies demonstrate that bone marrow stromal cell-treated rats exhibit significant improvement on a walking tract test at day 180 after surgery compared with control rats. Taken together, these data suggest that bone marrow stromal cell therapy is a safe and effective strategy for peripheral nerve injuries.     

Bone marrow stromal cell implantation for peripheral nerve repair

Abstract

Cell therapy using bone marrow stromal cells is a new promising therapy for regenerative medicine. Previous studies demonstrated that local bone marrow stromal cells implantation in the distal stump of transected sciatic nerve of rats promotes early functional recovery. The purpose of this study was to expand on the preliminary research by investigating the long-term efficacy of bone marrow stromal cells using the same experimental setting. Functional test and histological studies demonstrate that bone marrow stromal cell-treated rats exhibit significant improvement on a walking tract test at day 180 after surgery compared with control rats. Taken together, these data suggest that bone marrow stromal cell therapy is a safe and effective strategy for peripheral nerve injuries.     

Denervated skeletal muscle stimulates migration of Schwann cells from the distal stump of transected peripheral nerve: An in vivo study

We have tested the stimulation of Schwann cell migration from the distal stump of a 1 week transected sciatic nerve of adult rats by denervated skeletal muscle. Migrating Schwann cells were distinguished by the presence of non-specific cholinesterase (nChE) activity and glial fibrillary acidic protein (GFAP) at a distance of about 6 mm among denervated muscle fibres 4 weeks after insertion of the distal stump. In addition, the distal stump was introduced into the open end of a silicone chamber packed with artificial fibrin sponge (Gelaspon®) soaked in homogenate from intact or denervated muscles. A larger amount of migrated Schwann cells was observed in the chambers filled with homogenate from denervated muscles. An alteration in the amounts of Schwann cells migrating into the silicone chambers observed after histochemical staining (nChE or GFAP) was supported by biochemical measurements of the nChE activity. The biochemical assessment of the nChE activity revealed the increased amounts of migrated Schwann cells in proportion to the protein contents of homogenates from the denervated muscles. In addition, heating of homogenate from the denervated muscles resulted in a diminution of Schwann cell migration. Bromodeoxyuridine incorporation did not show an increased proliferation of Schwann cells inside the chambers following application of homogenate from the denervated muscles in comparison with the homogenate from the innervated muscles. Our results suggest a stimulation of Schwann cell migration from the distal stump of the transected sciatic nerve by soluble factor(s) produced by denervated skeletal muscles.     

Therapeutic benefit of intracerebral transplantation of bone marrow stromal cells after cerebral ischemia in rats

We tested the hypothesis that bone marrow stromal cells (MSCs) transplanted into the ischemic boundary zone, survive, differentiate and improve functional recovery after middle cerebral artery occlusion (MCAo). MSCs were harvested from adult rats and cultured with or without nerve growth factor (NGF). For cellular identification, MSCs were prelabeled with bromodeoxyuridine (BrdU). Rats (n=24) were subjected to 2 h of MCAo, received grafts at 24 h and were euthanized at 14 days after MCAo. Test groups consisted of: (1) control-MCAo alone (n=8); (2) intracerebral transplantation of MSCs (n=8); (3) intracerebral transplantation of MSCs cultured with NGF (n=8). Immunohistochemistry was used to identify cells from MSCs. Behavioral tests (rotarod, adhesive-removal and modified neurological severity score [NSS]) were performed before and after MCAo. The data demonstrate that MSCs survive, migrate and differentiate into phenotypic neural cells. Significant recovery of somatosensory behavior (p<0.05) and NSS (p<0.05) were found in animals transplanted with MSCs compared with control animals. Animals that received MSCs cultured with NGF displayed significant recovery in motor (p<0.05), somatosensory (p<0.05) and NSS (p<0.05) behavioral tests compared with control animals. Our data suggest that intracerebral transplantation of MSCs may provide a powerful autoplastic therapy for stroke.     

Adult bone marrow stromal cells administered intravenously to rats after traumatic brain injury migrate into brain and improve neurological outcome

To measure effect of bone marrow stromal cells (MSCs) administered i.v. on rats subjected to traumatic brain injury (TBI), we injected MSCs labeled by BrdU into the tail vein 24 h after TBI and sacrificed rats 15 days later. The neurological severity score (NSS) and the Rotarod test were used to evaluate neurological function. The distribution of the donor cells in brain, heart, lung, kidney, liver and spleen were analyzed in recipient rats using immunohistochemical staining. MSCs injected i.v. significantly reduced motor and neurological deficits compared with control groups by day 15 after TBI. The cells preferentially entered and migrated into the parenchyma of the injured brain and expressed the neuronal marker NeuN and the astrocytic marker GFAP. MSCs were also found in other organs and primarily localized to the vascular structures, without any obvious adverse effects. Our data suggest that i.v. administration of MSCs may be useful in the treatment of TBI.     

Axonal signals regulate the differentiation of non-myelin-forming Schwann cells: an immunohistochemical study of galactocerebroside in transected and regenerating nerves

Little is known about the factors involved in directing and maintaining the divergent differentiation of the 2 major Schwann cell variants, myelin and non-myelin-forming cells, in peripheral nerves. There is strong evidence that the differentiation of myelin-forming cells depends critically on cell-cell signaling through contact with appropriate axons. In this paper we ask whether this remarkable dependence of the Schwann cell on axonal contact for full differentiation is unique to those cells that form myelin or whether axonal signaling is also an important factor in the differentiation of non-myelin-forming Schwann cells. Sciatic nerves or cervical sympathetic trunks of adult rats were either transected or crushed and the axons allowed to degenerate and, in the case of crushed nerves, to regenerate into the distal stump for periods of time varying from 2 d to 9 weeks. The distal stump of the nerve was excised at specific times, the Schwann cells dissociated and immunolabeled with antibodies to galactocerebroside. In the sciatic nerve, which contains a mixture of non-myelin-forming and myelin-forming Schwann cells, transection resulted in a loss of galactocerebroside expression from the surface of all the Schwann cells in the distal stump over a 9 week period, irrespective of their original phenotype. In crushed sciatic nerves, where axons were allowed to regrow into the distal stumps, the number of Schwann cells expressing immunohistochemically detectable quantities of galactocerebroside in the stump declined over the first 3 weeks, but by 9 weeks after crush the total percentage of galactocerebroside-positive cells in the nerve had risen to control levels.(ABSTRACT TRUNCATED AT 250 WORDS)     

Presence of alpha-globin mRNA and migration of bone marrow cells after sciatic nerve injury suggests their participation in the degeneration/regeneration process

We have previously reported that in the distal stump of ligated sciatic nerves, there is a change in the distribution of myelin basic protein (MBP) and P0 protein immunoreactivities. These results agreed with the studies of myelin isolated from the distal stump of animals submitted to ligation of the sciatic nerve, showing a gradual increase in a 14 kDa band with an electrophoretic mobility similar to that of an MBP isoform, among other changes. This band, which was resolved into two bands of 14 and 15 kDa using a 16% gel, was found to contain a mixture of MBP fragments and peptides with great homology with alpha- and beta-globins. In agreement with these results, we have demonstrated that the mRNA of alpha-globin is present in the proximal and distal stumps of the ligated nerve. It is also detected at very low levels in Schwann cells isolated from normal nerves. These results could be due to the presence of alpha- and/or beta-globin arising from immature cells of the erythroid series. Also, they could be present in macrophages, which spontaneously migrate to the injured nerve to promote the degradation of myelin proteins. Cells isolated from normal adult rat bone marrow which were injected intraortically were found to migrate to the injured area. These cells could contribute to the remyelination of the damaged area participating in the removal of myelin debris, through their transdifferentiation into Schwann cells or through their fusion with preexisting Schwann cells in the distal stump of the injured sciatic nerve.     

Glial cells in degenerating and regenerating optic nerve of the adult rat

The glial cell reaction both in degenerating and regenerating adult rat optic nerve was studied by immunohistochemistry and electron microscopy. Degeneration in the optic nerve was achieved by complete transection, and the retinal stump was then analyzed. The regeneration was observed by autotransplantation of a sciatic nerve segment to the transected retinal stump. In both cases, optic nerve axons were labeled anterogradely with rhodamine, followed by immunohistochemical staining. Glial fibrillary acidic protein-positive astrocytes covered the transected end of degenerating optic nerve, whereas in the regenerating optic nerve they enwrapped axonal bundles emerging from the optic nerve stump and migrated together into the transitional zone intervening between the retinal stump and graft. In electron microscopy, direct attachment of astrocyte and Schwann cell was found within the transitional zone, whereby these cells were holding axons between them. Decrease of 04 immunoreactivity, which labels oligodendrocytes, was apparent in the transected end of retinal stump during the regeneration. The ED1 -positivity, which labels microglia/macrophages, was found in cells accumulated in the transitional zone of degenerating optic nerve, whereas during regeneration, ED1-immunoreactive cells were also distributed in the retinal stump. These results suggest that astrocytes, usually considered to interfere with optic nerve regeneration, change their characteristics in the presence of peripheral nerve graft and guide the regenerating axons in cooperation with Schwann cells. The response of oligodendrocytes and microglia/macrophages may also be modulated by peripheral nerve.     

骨髓间充质细胞构建组织工程神经修复坐骨神经缺损

背景：许旺细胞是周围神经组织工程的种子细胞,但体外分离、培养、纯化许旺细胞较困难。脱细胞同种异体神经移植物具有较强的修复外周神经缺损的能力,且可诱导骨髓间充质细胞分化为类许旺细胞,理论上骨髓间充质细胞可替代许旺细胞作为种子细胞应用于周围神经组织工程。 目的：观察骨髓间充质细胞构建组织工程神经修复坐骨神经缺损的效果,评估骨髓间充质细胞作为种子细胞修复周围神经缺损的可行性。 设计、时间及地点：随机对照动物实验,于2008-07/12在大理学院基础医学院实验室完成。 材料：将30只SD大鼠按随机数字表法分为3组,每组10只。骨髓间充质细胞+异体移植组将骨髓间充质细胞复合脱细胞同种异体神经移植物培养的组织工程神经与两断端用10/0 无创线端端吻合;异体移植组将脱细胞同种异体神经移植物桥接;自体移植组将切断的坐骨神经旋转180°端端吻合。 方法：运用骨髓间充质细胞构建的组织工程神经修复大鼠10 mm坐骨神经缺损,移植后12周通过坐骨神经功能指数、腓肠肌湿质量恢复率、S-100免疫组织化学染色、电镜等方法观察移植物修复效果。 主要观察指标：复合物培养时观察细胞形态的变化;移植后观察坐骨神经功能指数及腓肠肌湿质量恢复率;通过甲苯胺蓝染色观察新生髓鞘形成和轴突生长及神经纤维的分布情况,结合透射电镜及S-100蛋白免疫组织化学染色,观察许旺细胞生长和神经纤维再生情况。 结果：坐骨神经功能指数及腓肠肌湿质量恢复率的检测结果显示骨髓间充质细胞+异体移植组优于异体移植组(P < 0.05)。骨髓间充质细胞+异体移植组复合物中S-100的表达明显高于异体移植组,有髓神经纤维数量、有髓纤维直径和髓鞘厚度均大于异体移植组(P < 0.05),修复效果接近自体移植组。 结论：骨髓间充质细胞构建的组织工程神经修复周围神经缺损的效果优于单纯的脱细胞同种异体神经移植物,骨髓间充质细胞作为种子细胞在周围神经组织工程中具有较强的应用价值。     

Phenotypic changes of Schwann cells on the proximal stump of injured peripheral nerve during repair using small gap conduit tube

Dedifferentiation of Schwann cells is an important feature of the response to peripheral nerve injury and specific negative myelination reg-ulators are considered to have a major role in this process. However, most experiments have focused on the distal nerve stump, where the Notch signaling pathway is strongly associated with Schwann cell dedifferentiation and repair of the nerve. We observed the phenotypic changes of Schwann cells and changes of active Notch signaling on the proximal stump during peripheral nerve repair using small gap conduit tubulization. Eighty rats, with right sciatic nerve section of 4 mm, were randomly assigned to conduit bridging group and control group (epineurium suture). Glial fibrillary acidic protein expression, in myelinating Schwann cells on the proximal stump, began to up-reg-ulate at 1 day after injury and was still evident at 5 days. Compared with the control group, Notch1 mRNA was expressed at a higher level in the conduit bridging group during the first week on the proximal stump. Hes1 mRNA levels in the conduit bridging group significantly increased compared with the control group at 3, 5, 7 and 14 days post-surgery. The change of the Notch intracellular domain shared a simi-lar trend as Hes1 mRNA expression. Our results confirmed that phenotypic changes of Schwann cells occurred in the proximal stump. The differences in these changes between the conduit tubulization and epineurium suture groups correlate with changes in Notch signaling.This suggests that active Notch signaling might be a key mechanism during the early stage of neural regeneration in the proximal nerve stump.     

Sciatic nerve regeneration in rats induced by transplantation of in vitro differentiated bone-marrow stromal cells

Bone marrow stromal cells (MSCs) are multipotent stem cells that have the potential to differentiate into bone, cartilage, fat and muscle. We now demonstrate that MSCs can be induced to differentiate into cells with Schwann cell characteristics, capable of eliciting peripheral nervous system regeneration in adult rats. MSCs treated with beta-mercaptoethanol followed by retinoic acid and cultured in the presence of forskolin, basic-FGF, PDGF and heregulin, changed morphologically into cells resembling primary cultured Schwann cells and expressing p75, S-100, GFAP and O4. The MSCs were genetically engineered by transduction with retrovirus encoding green fluorescent protein (GFP), and then differentiated by treatment with factors described above. They were transplanted into the cut ends of sciatic nerves, which then responded with vigorous nerve fibre regeneration within 3 weeks of the operation. Myelination of regenerated fibers by GFP-expressing MSCs was recognized using confocal and immunoelectron microscopy. The results suggest that MSCs are able to differentiate into myelinating cells, capable of supporting nerve fibre re-growth, and they can therefore be applied to induce nerve regeneration.     

Induction of myelin genes during peripheral nerve remyelination requires a continuous signal from the ingrowing axon

The effect of a permanent transection on myelin gene expression in a regenerating sciatic nerve and in an adult sciatic nerve was compared to establish the degree of axonal control exerted upon Schwann cells in each population. First, the adult sciatic nerve was crushed, and the distal segment allowed to regenerate. At 12 days post-crush, the sciatic nerve was transected distal to the site of crush to disrupt the Schwann cell-axonal contacts that had reformed. Messenger RNA (mRNA) levels coding for five myelin proteins were assayed in the distal segment of the crush-transected nerve after 9 days and were compared to corresponding levels in the distal segments of sciatic nerves at 21 days post-crush and 21 days post-transection using Northern blot and slot-blot analysis. Levels of mRNAs found in the distal segment of the transected and crush-transected nerve suggested that Schwann cells in the regenerating nerve and in the mature adult nerve are equally responsive to axonal influences. The crush-transected model allowed the genes that were studied to be classified according to their response to Schwann cell-axonal contact. The levels of mRNAs were (1) down-regulated to basal levels (PO and MBP mRNAs), (2) down-regulated to undetectable levels (myelin-associated glycoprotein mRNAs), (3) upregulated (mRNAs encoding 2′3′-cyclic nucleotide phosphodiesterase and β-actin), or (4) not stringently controlled by the removal of Schwann cell-axonal contact (proteolipid protein mRNAs). This novel experimental model has thus provided evidence that the expression of some of the important myelin genes during peripheral nerve regeneration is dependent on continuous signals from the ingrowing axons. © 1993 Wiley-Liss, Inc.     

Survival of nerve and Schwann cells in allografts after cyclosporin A treatment

Because nerve and Schwann cells in allografts are rejected by normal rats, we investigated whether or not these neurological cells would survive if rats were treated with the new immunosuppressive drug, Cyclosporin A. Untreated rats rejected nerve and Schwann cells in allografts of ganglia or nerve. On the other hand, nerve and Schwann cells survived in allografts in Cyclosporin A-treated rats even after drug therapy was terminated. These results indicate that Cyclosporin A may be of value if allogenic nerve or Schwann cells are needed to aid in the repair of injured nerve tissue.     

Laminin in rat sciatic nerve undergoing Wallerian degeneration. Immunofluorescence study with laminin and neurofilament antisera

Immunofluorescence with laminin antisera revealed a striking change in the localization of this basal membrane glycoprotein in rat sciatic nerve as a result of Wallerian degeneration. The staining was confined to the endoneurium in normal sciatic nerve and during the first days of degeneration. On day 11 endoneurial tubes were no longer identified in the distal stump of crushed nerves or of nerves that had been transected and tightly ligated to prevent regeneration. In both crushed and ligated nerves proliferating Schwann cells forming the cell-bands of Büngner were intensely laminin positive. With double-labeling experiments, laminin and neurofilament antisera revealed similar but not identical staining patterns in crushed nerves, which suggests a close relation between laminin and regenerating axons. Crushed nerves had recovered their normal appearance 18 days after operation while anti-laminin reactivity was decreased in parts of ligated nerves undergoing fibrosis. The localization of laminin in reactive Schwann cells was confirmed by electron microscopy using the indirect immunoperoxidase procedure. Axons did not contain reaction product.     

Intravenous bone marrow stromal cell therapy reduces apoptosis and promotes endogenous cell proliferation after stroke in female rat

The present study investigates the induction of neurogenesis, reduction of apoptosis, and promotion of basic fibroblast growth factor (bFGF) expression as possible mechanisms by which treatment of stroke with bone marrow stromal cells (MSCs) improves neurological functional recovery. Additionally, for the first time, we treated cerebral ischemia in female rats with intraveneous administration of MSCs. Female rats were subjected to 2 hr of middle cerebral artery occlusion (MCAo), followed by an injection of 3 x 10(6) male (for Y chromosome labeling) rat MSCs or phosphate-buffered saline (PBS) into the tail vein 24 hr after MCAo. All animals received daily injection of bromodeoxyuridine (BrdU; 50 mg/kg, i.p.) for 13 days after treatment for identification of newly synthesized DNA. Animals were sacrificed at 14 days after MCAo. Behavioral tests (rotarod and adhesive-removal tests) were performed. In situ hybridization, immunohistochemistry, and terminal deoxynucleotidyltransferase (TdT)-mediated dUTP-biotin nick-end labeling (TUNEL) were performed to identify transplanted MSCs (Y chromosome), BrdU, bFGF, and apoptotic cells in the brain. Significant recovery of behavior was found in MSC-treated rats at 7 days in the somatosensory test and at 14 days in the motor test after MCAo compared with control, PBS-treated animals (P<.05). MSCs were found to survive and preferentially localize to the ipsilateral ischemic hemisphere. Significantly more BrdU-positive cells were located in the subventricular zone (P<.05), and significantly fewer apoptotic cells and more bFGF immunoreactive cell were found in the ischemic boundary area (P<.05) of MSC-treated rats than in PBS-treated animals. Here we demonstrate that intravenously administered male MSCs increase bFGF expression, reduce apoptosis, promote endogenous cellular proliferation, and improve functional recovery after stroke in female rats.     

2', 3'-Cyclic nucleotide 3'-phosphodiesterase cells derived from transplanted marrow stromal cells and host tissue contribute to perineurial compartment formation in injured rat spinal cord

Transdifferentiation of transplanted marrow stromal cells (MSCs) and reactive changes of glial cells in a completely transected rat spinal cord were examined. Marrow stromal cells exhibited 2',3'-cyclic nucleotide 3'-phosphodiesterase (CNP) at the plasma membrane and this has allowed their identification after transplantation by immunoelectron microscopy. In the control rats, the lesion site showed activated microglia/neural macrophages and some elongated cells, whose cytoplasm was immunoreactive for CNP. Cells designated as CNP1 and apparently host-derived expressed CXCR4. In experimental rats receiving MSCs transplantation, CNP1 cells were increased noticeably. This was coupled with the occurrence of a different subset of CNP cells whose plasma membrane was CNP-immunoreactive and expressed CXCR4. These cells, designated as CNP2, enclosed both myelinated and unmyelinated neurites thus assuming a spatial configuration resembling that of Schwann cells. A remarkable feature was the extensive ramifications of CNP1 cells with long filopodia processes delineating the CNP2 cells and their associated neurites, forming many perineurial-like compartments. Present results have shown that CNP2 cells considered to be MSCs-derived can transform into cells resembling Schwann cells based on their spatial relation with the regenerating nerve fibers, whereas the CNP1 glial cells participate in formation of perineurial compartments, probably serving as conduits to guide the nerve fiber growth. The chemotactic migration of CNP cells either derived from host tissue or MSCs bearing CXCR4 may be attracted by stromal derived factor-1alpha (SDF-1alpha) produced locally. The coordinated cellular interaction between transplanted MSCs and local glial cells may promote the growth of nerve fibers through the lesion site.     

Nerve regeneration model and trophic factors in vivo

The proximal stump of a transected rat sciatic nerve has been observed to regenerate through a cylindrical silicone chamber across a 10 mm gap to the distal stump. The fluid filling such in vivo chambers contains trophic factors that ensure in vitro survival and growth of at least sensory neurons from rodent dorsal root ganglia — as already demonstrated for fluid generated in vitro from Schwann and other cell cultures.