Bone Marrow Stromal Cell Treatment Research Articles

Ultrasound-Guided Transplantation of Mesenchymal Stem Cells Improves Adriamycin Nephropathy in Rats Through the RIPK3/MLKL and TLR-4/NF-κB Signaling.

Bone marrow stromal cell (BMSC) treatment has been shown to be beneficial for Adriamycin nephropathy (ADR). However, the low transplantation rate is still the key factor that affects this strategy. This study is the first to investigate the efficacy and potential mechanism of ultrasound-guided transrenal arterial transfer of BMSCs for the treatment of ADR in rats. The ADR rat model was established by two injections of doxorubicin. In addition, the rats were randomly divided into four groups (10 rats per group): the normal group (no treatment), the medium control group (treated with medium), the Adriamycin group (treated with phosphate buffer), and the BMSC group (treated with BMSCs). After 4 weeks, the levels of serum creatinine (SCr), blood urea nitrogen (BUN), and urine albumin (ALb) were measured. In addition, pathological changes in kidney tissue were evaluated by pathological sectioning and electron microscopy. Western blotting was used to determine the levels of proteins in rat kidneys. Ultrasound-guided renal artery transplantation of BMSCs reduced the levels of SCr, BUN, and ALb and improved the pathological structure of rat kidneys compared with those in the Adriamycin group. This treatment inhibited renal cell necrosis by reducing the expression of receptor-interacting Serine/threonine Kinase 3 (RIPK3) and Mixed lineage kinase domain-like pseudokinase (MLKL) and inhibited renal inflammation and fibrosis by reducing the expression of Toll-Like receptor 4 (TLR4) and nuclear factor κB (NF-κB). Our study shows that ultrasound-guided transrenal artery transplantation of BMSCs can improve adriamycin-induced renal injury in rats by regulating the RIPK3/MLKL and TLR-4/NF-κB pathways and inhibiting renal necrosis, inflammation, and fibrosis.

Diffuse white matter response in trauma-injured brain to bone marrow stromal cell treatment detected by diffusional kurtosis imaging

Diffuse white matter (WM) response to traumatic brain injury (TBI) and transplantation of human bone marrow stromal cells (hMSCs) after the injury were non-invasively and dynamically investigated. Male Wistar rats (300–350 g) subjected to TBI were intravenously injected with 1 ml of saline (n = 10) or with hMSCs in suspension (∼3 × 106 hMSCs, n = 10) 1-week post-TBI. MRI measurements of T2-weighted imaging and diffusional kurtosis imaging (DKI) were acquired on all animals at multiple time points up to 3-months post-injury. Functional outcome was assessed using the Morris water maze test. DKI-derived metrics of fractional anisotropy (FA), axonal water fraction (AWF) and radial kurtosis (RK) longitudinally reveal an evolving pattern of structural alteration post-TBI occurring in the brain region remote from primary impact site. The progressive structural change is characterized by gradual disruption of WM integrity at an early stage (weeks post-TBI), followed by spontaneous recovery at a later stage (months post-TBI). Transplantation of hMSCs post-TBI promotes this structural plasticity as indicated by significantly increased FA and AWF in conjunction with substantially elevated RK at the later stage. Our long-term imaging data demonstrate that hMSC therapy leads to modified temporal profiles of these metrics, inducing an earlier presence of enhanced structural remodeling, which may contribute to improved functional recovery.

Chronic global analysis of vascular permeability and cerebral blood flow after bone marrow stromal cell treatment of traumatic brain injury in the rat: A long-term MRI study

Vascular permeability and hemodynamic alteration in response to the transplantation of human bone marrow stromal cells (hMSCs) after traumatic brain injury (TBI) were longitudinally investigated in non directly injured and normal-appearing cerebral tissue using magnetic resonance imaging (MRI). Male Wistar rats (300–350g, n=30) subjected to controlled cortical impact TBI were intravenously injected with 1ml of saline (at 6-h or 1-week post-injury, n=5/group) or with hMSCs in suspension (∼3×106 hMSCs, at 6-h or 1-week post-injury, n=10/group). MRI measurements of T2-weighted imaging, cerebral blood flow (CBF) and blood-to-brain transfer constant (Ki) of gadolinium-diethylenetriamine pentaacetic acid (Gd-DTPA), and neurological behavioral estimates were performed on all animals at multiple time points up to 3-months post-injury. Our long-term imaging data show that blood–brain barrier (BBB) breakdown and hemodynamic disruption after TBI, as revealed by Ki and CBF, respectively, affect both hemispheres of the brain in a diffuse manner. Our data reveal a sensitive vascular permeability and hemodynamic reaction in response to the time-dependent transplantation of hMSCs. A more rapid reduction of Ki following cell treatment is associated with a higher level of CBF in the injured brain, and acute (6h) cell administration leads to enhanced therapeutic effects on both the recovery of vascular integrity and stabilization of cerebral perfusion compared to delayed (1w) cell engraftment. Our results indicate that cell-enhanced BBB reconstitution plays an important role in underlying the restoration of CBF in the injured brain, which in turn, contributes to the improvement of functional outcome.

White matter changes after stroke in type 2 diabetic rats measured by diffusion magnetic resonance imaging

Diffusion-related magnetic resonance imaging parametric maps may be employed to characterize white matter of brain. We hypothesize that entropy of diffusion anisotropy may be most effective for detecting therapeutic effects of bone marrow stromal cell treatment of ischemia in type 2 diabetes mellitus rats. Type 2 diabetes mellitus was induced in adult male Wistar rats. These rats were then subjected to 2 h of middle cerebral artery occlusion, and received bone marrow stromal cell (5 × 106, n = 8) or an equal volume of saline (n = 8) via tail vein injection at three days after middle cerebral artery occlusion. Magnetic resonance imaging was performed on day one and then weekly for five weeks post middle cerebral artery occlusion. The diffusion metrics complementarily permitted characterization of axons and axonal myelination. All six magnetic resonance imaging diffusion metrics, confirmed by histological measures, demonstrated that bone marrow stromal cell treatment significantly (p < 0.05) improved magnetic resonance imaging diffusion indices of white matter in type 2 diabetes mellitus rats after middle cerebral artery occlusion compared with the saline-treated rats. Superior to the fractional anisotropy metric that provided measures related to organization of neuronal fiber bundles, the entropy metric can also identify microstructures and low-density axonal fibers of cerebral tissue after stroke in type 2 diabetes mellitus rats.

Administration of bone marrow stromal cells in sepsis attenuatessepsis-related coagulopathy

Introduction Coagulopathy plays an important role in sepsis. The aim of this study was to determine whether bone marrow stromal cell (BMSC) administration could attenuate coagulopathy in sepsis.Materials and methods In vitro: endothelial cells were cultured with/without BMSCs for 6 h following LPS stimulation and were collected for thrombomodulin (TM) and endothelial protein C receptor (EPCR) measurements. In vivo: Thirty-six mice were randomized into sham, sepsis, and sepsis + BMSC groups (n = 12 each group). Sepsis was induced through cecal ligation and puncture (CLP). BMSC infusion was started at 6 h after CLP. Lung tissues and plasma samples were collected at 24 h after CLP for enzyme-linked immunosorbent assay (ELISA), quantitative real-time RT-PCR, western blot, and immunohistochemistry analysis.Results In vitro: BMSCs attenuated the decrease in TM and EPCR mRNA and protein expression levels in LPS-stimulated endothelial cells. In vivo: BMSC treatment decreased lung injury and mesenteric perfusion impairment, and ameliorated coagulopathy, as suggested by the reduction in elevated TF, vWF, and TAT circulation levels. BMSC infusion decreased TF mRNA transcription and protein expression levels in lung tissues, and increased TM and EPCR mRNA transcription and expression levels.Discussion BMSC administration attenuated coagulopathy, and decreased lung injury and mesenteric perfusion impairment in sepsis.Key messagesBMSCs increased the expression of TM and EPCR from endothelium cells exposed to LPS in vitro.BMSC treatment attenuated lung injury and coagulopathy in the mice cecal ligation and puncture (CLP) model.BMSC administration-attenuated coagulopathy is related to the reduced expression of TF and increased expression of TM and EPCR.

Simvastatin combined with bone marrow stromal cells treatment activatesastrocytes to ameliorate neurological function after ischemic stroke in rats

Evidence indicates that reactive astrocytes play an important role in neurological functional amelioration after stroke. Bone marrow stromal cell (BMSC) transplantation-promoted astrocyte-derived neurotrophic factors' expressions resulted in decreasing the number of apoptotic neurons and improving neurogenesis and angiogenesis in the ischemic boundary zone. Recent studies found that simvastatin had neuroprotective activity in acute stroke. However, it is still ambiguous as to whether simvastatin combined with BMSCs can further activate astrocytes, which contribute to neurological functional recovery after ischemic stroke. In our present study, we established a middle cerebral artery occlusion model in rats and found that combination treatment of simvastatin and BMSCs could significantly increase neurological functional scores and induce activated astrocytes to enhance stromal cell-derived factor-1 α, vascular endothelial growth factor, brain-derived neurotrophic factor, and glial cell line-derived neurotrophic factor expressions compared with BMSC alone (P < 0.05) by immunofluorescence staining on the 7th day after ischemia. Furthermore, western blot results showed that combination treatment could notably activate the AKT/mammalian target of rapamycin signaling pathway in oxygen-glucose deprivation-induced astrocytes. It was superior to BMSC treatment at the 5-min time point (P < 0.01). Therefore, we think that combination treatment should be a more effective therapy after stroke due to synergic functions of supplementary approaches.

Multipotent bone marrow stromal cell therapy promotes endogenous cell proliferation following ischemic stroke.

Despite extensive research over the years, there still exists some debate as to what constitutes the optimal therapeutic strategy to promote recovery following stroke. Due to the complexity of injured brain pathophysiology, treatment approaches should ideally address numerous factors, ultimately aiming to promote tissue protection, axonal regrowth and functional recovery. This study extends the understanding of the effects of bone marrow stromal cell (BMSC) treatment following experimentally induced ischemic stroke in rats. Focal ischemic brain injury was experimentally induced in rats by placing a preformed clot into the middle cerebral artery. Animals were injected intravenously with BMSCs at 24h after stroke and were killed 7days post injury. When administered BMSCs following stroke, the neurological outcome was significantly improved relative to controls. There was an increase in the number of BMSCs labelled with BrdU present in the injured hemisphere of the brain compared to the non-injured side. Furthermore, administration of BMSCs also led to increases in astrocytosis, vascularization and endogenous proliferation. These findings provide insight into the mechanisms of action of BMSC treatment and further argue for the therapeutic potential of BMSCs as an effective treatment following cerebral stroke.

Abstract W MP87: PDGF Contributes to BMSC Treatment Induced Neurogenesis and White Matter and Axonal Remodeling in T2DM Stroke Rats

Objective: Our previous studies have found that bone-marrow-stromal cell (BMSC) treatment of stroke in Type two DM (T2DM) rats, initiated at 3 days after stroke, improved functional recovery. Neurogenesis and white matter (WM) remodeling play an important role in neurorestorative effects after stroke. In this study, we tested whether BMSCs regulate neurogenesis and WM remodeling and the underlying mechanisms of BMSC induced neurorestorative effects in T2DM stroke rats. Methods: T2DM was induced with streptozotocin injection in addition to a high fat diet. T2DM rats were subjected to 2h of middle cerebral artery occlusion (MCAo), then treated with human BMSCs (5X106) or vehicle control (n=8/group) initiated at 3 days after MCAo and rats were monitored for 28 days. Neuroblast migration, WM changes, and gene and protein expression were measured in the ischemic brain. Subventricular zone (SVZ) explant cell migration and primary cortical neuron (PCN) axonal outgrowth measurements were performed in vitro. Results: BMSC treatment in T2DM rats significantly improves functional outcome and increases WM remodeling identified by increased myelin and axonal density. BMSCs also increase the neuroblast migration protein doublecortin (DCX, 25.0±4.3% vs control: 4.5±1.1%), platelet-derived growth factor (PDGF)-AA, and bFGF expression in the ischemic border zone. Angiogenic ELISA array data are consistent with the immunostaining data, showing that BMSC treatment increases PDGF-AA (2.1 fold), PDGF-BB (2.5 fold) and bFGF (1.8 fold) in the ischemic brain. Using an in vitro cell culture model, we found that BMSCs secrete high levels of PDGF. PDGF treatment significantly increases SVZ explant cell migration (1.7 fold) and PCN axonal outgrowth (1.9 fold) compared to non-treatment control. Inhibition of PDGF with neutralized anti-PDGF antibody significantly attenuates BMSC conditioned medium induced SVZ cell migration and PCN axon outgrowth. Conclusion: BMSC treatment of stroke in T2DM increases WM remodeling and neurogenesis as well as increases PDGF expression. PDGF not only promotes neuronal migration, but also increases axonal outgrowth. Therefore, increasing PDGF likely contributes to BMSC induced neurogenesis and WM remodeling in T2DM stroke rats.

Bone marrow stromal cells as a therapeutic treatment for ischemic stroke

Cerebral ischemia remains the most frequent cause of death and quality-of-life impairments due to neurological deficits, and accounts for the majority of total healthcare costs. However, treatments for cerebral ischemia are limited. Over the last decade, bone marrow stromal cell (BMSC) therapy has emerged as a particularly appealing option, as it is possible to help patients even when initiated days or even weeks after the ischemic insult. BMSCs are a class of multipotent, self-renewing cells that give rise to differentiated progeny when implanted into appropriate tissues. Therapeutic effects of BMSC treatment for ischemic stroke, including sensory and motor recovery, have been reported in pre-clinical studies and clinical trials. In this article, we review the recent progress in BMSC-based therapy for ischemic stroke, focusing on the route of delivery and pre-processing of BMSCs. Selecting an optimal delivery route is of particular importance. The ideal approach, as well as the least risky, for translational applications still requires further identification. Appropriate preprocessing of BMSCs or combination therapy has the benefit of achieving the maximum possible restoration. Further pre-clinical studies are required to determine the time-window for transplantation and the appropriate dosage of cells.

Effects of transplanting bone marrow stromal cells on axonal and glial scarring after spinal cord injury

Objective To investigate the effects of bone marrow stromal cell (BMSC) transplantation on axonal and glial scarring after spinal cord injury (SCI).Methods Thirty New Zealand white rabbits were randomly assigned to a sham operation group (group A),a saline treatment group (group B) or a BMSC treatment group (group C).Group A served as controls,in which the canal was opened without damage to the spinal cord.In groups C and B SCI models were established with aneurysm clips and the rabbits of groups C and B were then given injections of BMSCs and saline solution respectively via the intra-intercostal artery at 1 week post injury.At 1 day,1 week,2 weeks and 4 weeks post injury,Basso Beattie-Bresnahan (BBB) scores were assessed to evaluate the recovery of locomotor function in the hind limbs.Spinal cord samples were harvested for HE and Nissl staining,and immunohistochemistry and image analysis were used to detect any changes in neurofilament (NF200) and glial fibrillary acidic protein (GFAP) in the injured spinal cords.Results The average BBB scores of group A were significantly higher those that of groups B and C at each time point,and those of group C were significantly better than those of group B at the 2nd and 4th week post injury.At the 4th week post injury,HE staining showed there was no glial scarring or cavities in group A,but that there was glial cellular proliferation,glial scarring and cavity formation at the injury site in groups B and C.In group C all were obviously less than in group B.Nissl staining indicated there were more typical neurons in group A,while there were a larger number of ruptured neurons,more degradation,and irregular remaining neurons in groups B and C.These abnormalities were again significantly more prevalent in group C.Immunohistochemical examination showed significant increases in NF200 positive neurons and GFAP in groups B and C compared with group A.The number of NF200 positive neurons was significantly higher in group C than in group B,but the GFAP positive area was significantly smaller in group C than in group B.Conclusion BMSC transplantation via the intercostal arteries can effectively improve axonal regeneration,attenuate glial cellular proliferation and reduce glial scar formation,promoting functional recovery after SCI,at least in rabbits. Key words: Spinal cord injury; Intercostal arteries; Bone marrow stromal cells; Neurofilaments; Glial fibrillary acidic protein; Rabbits

Niaspan Attenuates the Adverse Effects of Bone Marrow Stromal Cell Treatment of Stroke in Type One Diabetic Rats

AimsOur previous studies have found that bone-marrow-stromal cells (BMSC) therapy improves functional recovery after stroke in non-diabetic rats while increases brain hemorrhage and induces arteriosclerosis-like changes in type-one-diabetic (T1DM) rats. Niaspan treatment of stroke increases vascular stabilization, decreases brain hemorrhage and blood-brain-barrier (BBB) leakage in T1DM rats. We therefore tested the hypothesis that combination therapy of BMSC with Niaspan attenuates the side effects of BMSC monotherapy in T1DM rats.MethodsT1DM-rats induced by streptozotocin were subjected to 2 hours of middle-cerebral-artery occlusion (MCAo) and treated with: 1) PBS; 2) BMSC (5×106); 3) Niaspan (40 mg/kg) daily for 14 days; 4) BMSC (5×106) +Niaspan (40 mg/kg, daily for 14 days) combination starting at 24 hours after MCAo. All rats were monitored for 14 days.ResultsCombination BMSC+Niaspan treatment of T1DM-MCAo rats did not increase brain hemorrhage, and significantly decreased BBB leakage and vascular arteriosclerosis-like changes as well as decreased Angiogenin, matrix metalloproteinase 9 (MMP9) and ED1 expression in ischemic brain and internal-carotid-artery compared to non-treatment control and BMSC monotherapy animals.ConclusionsCombination therapy using BMSC with Niaspan decreases BBB leakage and cerebral arteriosclerosis-like changes. These beneficial effects may be attributed to the decreased expression of Angiogenin, MMP9 and ED1.

Down-regulation of Nogo-A by collagen scaffolds impregnated with bone marrow stromal cell treatment after traumatic brain injury promotes axonal regeneration in rats

Nogo-A is a major form of growth inhibitory molecule (growth-IM) which inhibits axonal regeneration and neurite regrowth after neural injury. Bone marrow stromal cells (MSCs) have been shown to inhibit Nogo-A expression in vitro and in cerebral ischemic animal models. The present study was designed to investigate the effects of treatment with human MSCs (hMSCs) impregnated into collagen scaffolds on the expression of Nogo-A and axonal plasticity after traumatic brain injury (TBI). Adult male Wistar rats were injured with controlled cortical impact and treated either with saline, hMSCs-alone or hMSCs impregnated into collagen scaffolds (scaffold+hMSC) transplanted into the lesion cavity 7 days after TBI. Rats were sacrificed 14 days after TBI and brain tissues were harvested for immunohistochemical studies, Western blot analysis, laser capture microdissections and qRT-PCR to evaluate axonal density and Nogo-A protein and gene expressions. Our data showed that treatment of TBI with scaffold+hMSC significantly decreased TBI-induced Nogo-A protein expression and increased axonal density compared to saline and hMSC-alone treatments. In addition, scaffold+hMSC transplantation decreased Nogo-A transcription in oligodendrocytes after TBI. Scaffold+hMSC treatment was superior to hMSC-alone treatment in suppressing Nogo-A expression and enhancing axonal regeneration after TBI. Our data suggest that transplanting hMSCs with scaffolds down-regulates Nogo-A transcription and protein expression which may partially contribute to the enhanced axonal regeneration after TBI.

The Sonic Hedgehog Pathway Mediates Brain Plasticity and Subsequent Functional Recovery After Bone Marrow Stromal Cell Treatment of Stroke in Mice

Bone marrow stromal cells (MSCs) improve neurologic recovery after middle cerebral artery occlusion (MCAo). To examine whether in vivo blockage of the endogenous sonic hedgehog (Shh) pathway affects grafted MSC-induced neurologic benefits, MCAo mice were administered: vehicle (control); cyclopamine (CP)— a specific Shh pathway inhibitor; MSC; and MSC and cyclopamine (MSC-CP). Neurologic function was evaluated after MCAo. Electron microscopy and immunofluorescence staining were employed to measure synapse density, protein expression of tissue plasminogen activator (tPA), and Shh in parenchymal cells in the ischemic boundary zone (IBZ), respectively. Marrow stromal cell treatment significantly enhanced functional recovery after ischemia, concurrent with increases of synaptophysin, synapse density, and myelinated axons along the IBZ, and significantly increased tPA and Shh expression in astrocytes and neurons compared with control. After treatment with MSC-CP or CP, the above effects were reversed. Co-culture of MSCs with cortical neurons confirmed the effect of Shh on MSC-mediated neurite outgrowth. Our data support the hypothesis that the Shh pathway mediates brain plasticity via tPA and thereby functional recovery after treatment of stroke with MSCs.

Sodium Ferulate combined with bone marrow stromal cell treatment ameliorating rat brain ischemic injury after stroke

Reports suggested that bone marrow stromal cells (BMSCs) could protect brain against ischemic injury. However, the limited number of BMSCs in the brain hampered their application. To explore a way that might improve the migration and differentiation of BMSCs in the brain after stroke, we investigated the additive therapeutic effect of combination Sodium Ferulate (SF) and BMSCs treatment of stroke. In general, BMSCs were primarily cultured from rat bone marrow and were identified by flow cytometry. Permanent middle cerebral artery occlusion (MCAo) was induced and the ischemic brain was observed by 2,3,5-triphenyltetrazolium chloride (TTC) staining. The neurological functional outcome was respectively evaluated at 0 hour, 24 hours, 48 hours, and 72 hours post-operation, and the stromal cell-derived factor-1 alpha (SDF-1α) /chemokine(CXC motif)receptor-4(CXCR4) mRNA and protein expressions at the marginal zone of the ischemic brain were respectively measured by real-time RT-PCR and Western Blot at 3days after stroke; furthermore, Nestin and BrdU double immunostaining was performed for identification of BMSC differentiation. Combination treatment of Stroke significantly improved the neurological functional recovery as early as 48 hours and up-regulated the SDF-1α /CXCR4 axis, enhanced BMSC migration into the ischemic brain and differentiation into Nestin-positive cells. We demonstrate that SF combined with BMSC administration can ameliorate rat brain ischemic injury and has beneficial effect on the rat neurological functional recovery after ischemic stroke.

Adverse Effects of Bone Marrow Stromal Cell Treatment of Stroke in Diabetic Rats

Cell therapy with bone marrow stromal cells (BMSCs) improves functional recovery after stroke in nondiabetic rats. However, its effect on diabetics with stroke is unknown. This study investigated the effect of BMSCs on stroke outcome in Type 1 diabetic (T1DM) rats. T1DM was induced in adult male Wistar rats by injecting streptozotocin. Nondiabetic and T1DM rats were subjected to 2 hours of middle cerebral artery occlusion (MCAO), treated with or without BMSCs (3×10(6)) at 24 hours after MCAO, and monitored for 14 days. Functional benefit was not detected in T1DM-MCAO treated with BMSC rats compared with corresponding T1DM-MCAO controls. BMSC treatment in T1DM-MCAO rats had increased mortality, blood-brain barrier leakage, brain hemorrhage, and angiogenesis. Internal carotid artery neointimal formation and cerebral arteriole narrowing/occlusion were also observed in T1DM-MCAO+BMSCs rats compared with T1DM-MCAO controls (P<0.05), but not in nondiabetic stroke rats. We further studied the underlying mechanisms responsible for BMSC-induced blood-brain barrier leakage and accelerated vascular damage in T1DM-MCAO rats. We found that the expression of angiogenin (an angiogenic factor) and ED1 (a marker for macrophages) was significantly increased in the T1DM-MCAO+BMSC rats in the ischemic brain and internal carotid artery compared with nontreated T1DM-MCAO rats, but not in nondiabetic stroke rats. BMSC therapy in T1DM-MCAO rats does not improve functional outcome. On the contrary, it increases blood-brain barrier leakage and cerebral artery neointimal formation, and arteriosclerosis, which possibly is due to increased expression of angiogenin. Thus, BMSC treatment starting 24 hours after MCAO may not be beneficial for diabetic subjects with stroke.

MRI evaluation of axonal reorganization after bone marrow stromal cell treatment of traumatic brain injury

We treated traumatic brain injury (TBI) with human bone marrow stromal cells (hMSCs) and evaluated the effect of treatment on white matter reorganization using MRI. We subjected male Wistar rats (n = 17) to controlled cortical impact and either withheld treatment (controls; n = 9) or inserted collagen scaffolds containing hMSCs (n = 8). Six weeks later, the rats were sacrificed and MRI revealed selective migration of grafted neural progenitor cells towards the white matter reorganized boundary of the TBI-induced lesion. Histology confirmed that the white matter had been reorganized, associated with increased fractional anisotropy (FA; p < 0.01) in the recovery regions relative to the injured core region in both treated and control groups. Treatment with hMSCs increased FA in the recovery regions, lowered T(2) in the core region, decreased lesion volume and improved functional recovery relative to untreated controls. Immunoreactive staining showed axonal projections emanating from neurons and extruding from the corpus callosum into the ipsilateral cortex at the boundary of the lesion. Fiber tracking (FT) maps derived from diffusion tensor imaging confirmed the immunohistological data and provided information on axonal rewiring. The apparent kurtosis coefficient (AKC) detected additional axonal remodeling regions with crossing axons, confirmed by immunohistological staining, compared with FA. Our data demonstrate that AKC, FA, FT and T(2) can be used to evaluate treatment-induced white matter recovery, which may facilitate restorative therapy in patients with TBI.

Bone Marrow Stromal Cells Enhance Inter- and Intracortical Axonal Connections after Ischemic Stroke in Adult Rats

We investigated axonal plasticity in the bilateral motor cortices in rats after unilateral stroke and bone marrow stromal cell (BMSC) treatment. Rats were subjected to permanent right middle cerebral artery occlusion followed by intravenous administration of phosphate-buffered saline or BMSCs 1 day later. Adhesive-removal test and modified neurologic severity score were performed weekly to monitor limb functional deficit and recovery. Anterograde tracing with biotinylated dextran amine injected into the right motor cortex was used to assess axonal sprouting in the contralateral motor cortex and ipsilateral rostral forelimb area. Animals were killed 28 days after stroke. Progressive functional recovery was significantly enhanced by BMSCs. Compared with normal animals, axonal density in both contralateral motor cortex and ipsilateral rostral forelimb area significantly increased after stroke. Bone marrow stromal cells markedly enhanced such interhemispheric and intracortical connections. However, labeled transcallosal axons in the corpus callosum were not altered with either stroke or treatment. Both interhemispheric and intracortical axonal sprouting were significantly and highly correlated with behavioral outcome after stroke. This study suggests that, after stroke, cortical neurons surviving in the peri-infarct motor cortex undergo axonal sprouting to restore connections between different cerebral areas. Bone marrow stromal cells enhance axonal plasticity, which may underlie neurologic functional improvement.

Bone Marrow Stromal Cells Increase Oligodendrogenesis after Stroke

Oligodendrocytes are sensitive to ischemic damage. The Sonic hedgehog (Shh) pathway is critical in oligodendrogenesis; Gli1 is the principal effector of Shh signaling. We investigated oligodendrogenesis and Shh/Gli1 pathway activation after bone marrow stromal cell (BMSC) treatment of stroke in rats. Rats were subjected to the middle cerebral artery occlusion (MCAo). BMSCs have been shown to promote functional recovery post stroke. A therapeutic dose of BMSC (3 x 10(6) cells) treatment was initiated 1 day after MCAo. Immunohistochemistry was carried out to measure the oligodendrocyte progenitor cells, oligodendrocytes, myelin, and expressions of Shh and Gli1 at 14 days after MCAo. Gene expression of Shh and Gli1 was tested at 2 days after MCAo. An in vitro study was used to investigate the effects of BMSC on a premature oligodendrocyte cell line (N20.1 cells). BMSC treatment significantly increased O4(+) oligodendrocytes, MBP(+) area, and bromodeoxyuridine (BrdU)(+), NG2(+), BrdU(+)-NG2(+) cells, and mRNA and protein expressions of Shh and Gli1 in the ipsilateral brain of the MCAo rats than that in phosphate buffered saline (PBS)-treated rats. BMSCs promoted N20.1 cell proliferation and Gli1 mRNA expression, and these effects were abolished by the Shh pathway inhibitor cyclopamine. These data indicate that the BMSC treatment stimulates oligodendrogenesis by activation of the Shh/Gli1 pathway post stroke.

Contralesional Axonal Remodeling of the Corticospinal System in Adult Rats After Stroke and Bone Marrow Stromal Cell Treatment

Motor recovery after stroke is associated with neuronal reorganization in bilateral hemispheres. We investigated contralesional corticospinal tract remodeling in the brain and spinal cord in rats after stroke and treatment of bone marrow stromal cells. Adult male Wistar rats were subjected to permanent right middle cerebral artery occlusion. Phosphate-buffered saline or bone marrow stromal cells were injected into a tail vein 1 day postischemia. An adhesive removal test was performed weekly to monitor functional recovery. Threshold currents of intracortical microstimulation on the left motor cortex for evoking bilateral forelimb movements were measured 6 weeks after stroke. When intracortical microstimulation was completed, biotinylated dextran amine was injected into the left motor cortex to anterogradely label the corticospinal tract. At 4 days before euthanization, pseudorabies virus-152-EGFP and 614-mRFP were injected into left or right forelimb extensor muscles, respectively. All animals were euthanized 8 weeks after stroke. In normal rats (n=5), the corticospinal tract showed a unilateral innervation pattern. In middle cerebral artery occlusion rats (n=8), our data demonstrated that: 1) stroke reduced the stimulation threshold evoking ipsilateral forelimb movement; 2) EGFP-positive pyramidal neurons were increased in the left intact cortex, which were labeled from the left stroke-impaired forelimb; and 3) biotinylated dextran amine-labeled contralesional axons sprouted into the denervated spinal cord. Bone marrow stromal cells significantly enhanced all 3 responses (n=8, P<0.05). Our data demonstrated that corticospinal tract fibers originating from the contralesional motor cortex sprout into the denervated spinal cord after stroke and bone marrow stromal cells treatment, which may contribute to functional recovery.

Deletion of Angiotensin II Type 2 Receptor Attenuates Protective Effects of Bone Marrow Stromal Cell Treatment on Ischemia–Reperfusion Brain Injury in Mice

Protective effects of bone marrow stromal cells (MSCs) on ischemic brain damage have been highlighted. We examined the possibility that deletion of AT(2) receptor could attenuate the cerebroprotective effects of MSC using AT(2) receptor-deficient mice (Agtr2 (-)) and the effect of selective AT(1) receptor blocker. Wild-type mice (Agtr2 (+)) were subjected to 3 hours of focal brain ischemia followed by reperfusion (ischemia-reperfusion injury). Simultaneously, Agtr2 (+)-MSC, Agtr2 (-)-MSC, or saline was injected through the tail vein. Survival rates at 6 days after ischemia-reperfusion injury were as follows: approximately 50% in saline-injected mice, 80% in Agtr2 (+)-MSC-injected mice, and 20% in Agtr2 (-)-MSC-injected mice. Neurological deficit after ischemia-reperfusion injury was improved in Agtr2 (+)-MSC-injected mice, but not in Agtr2 (-)-MSC-injected mice. After 48 hours of ischemia-reperfusion injury, brain infarct size was reduced in Agtr2 (+)-MSC-injected mice, but not in Agtr2 (-)-MSC-injected mice. Moreover, brain edema was significantly ameliorated in Agtr2 (+)-MSC-treated mice but not in Agtr2 (-)-MSC-treated mice. Furthermore, the increase in mRNA expression of tumor necrosis factor-alpha and monocyte chemoattractant protein-1 in the ischemic brain was less in Agtr2 (+)-MSC-treated mice in the ipsilateral site, but was similar in the contralateral hemisphere. Tumor necrosis factor-alpha level was increased in both the contralateral hemisphere and ipsilateral hemisphere of Agtr2 (-)-MSC-treated mice. In contrast, monocyte chemoattractant protein-1 levels tended to increase Agtr2 (-)-MSC-treated mice without a significant difference. Treatment of MSC with an AT(1) receptor blocker, valsartan, significantly improved survival rates in Agtr2 (-)-MSC-injected mice. These results suggest that AT(2) receptor signaling in MSC attenuated brain damage and neurological deficit (deleted).