Bone marrow-derived mononuclear cells ameliorate neurological function in chronic cerebral infarction model mice via improvement of cerebral blood flow.

BACKGROUND AIMS: Stroke is a frequently observed neurological disorder that might lead to permanent and severe disability. Recently, various regenerative therapies have been developed, some of which have already been applied clinically. However, their outcomes have not been fully satisfactory. In particular, the development of regenerative therapies for chronic ischemic stroke is greatly needed. Herein intracerebral administration of bone marrow-derived mononuclear cells (BM-MNCs) was assessed as a potential treatment for chronic ischemic stroke using a severe combined immunodeficiency mouse model characterized by minimal vascular variation unrelated to immunodeficiency. METHODS: A reproducible model of permanent middle cerebral artery occlusion was prepared, and intracerebral BM-MNC transplantation was performed 14 days after stroke induction in the infarcted brain. RESULTS: Sensorimotor behavioral function and cerebral blood flow were significantly improved upon treatment with BM-MNCs compared to control medium injection. The transplanted cells exhibited characteristics of the vascular endothelium and microglia/macrophages. Significant angiogenesis and suppression of astrogliosis and microgliosis were observed in the affected brain. Messenger RNA expression analysis showed significant increases in anti-inflammatory cytokines, A2 astrocyte/anti-inflammatory microglia markers and vascular endothelial markers such as vascular endothelial growth factor and significant decreases in pro-inflammatory cytokines and A1 astrocyte/pro-inflammatory microglia markers following BM-MNC transplantation. CONCLUSIONS: These results suggest that intracerebral administration of BM-MNCs should be considered an effective cell therapy for chronic stroke.

Expression of proinflammatory cytokines and proinsulin by bone marrow-derived cells for fracture healing in long-term diabetic mice.

BACKGROUND: Diabetes mellitus (DM) causes bone dysfunction due to poor bone quality, leading to severe deterioration in patient of quality of life. The mechanisms of bone metabolism in DM remain unclear, although chemical and/or mechanical factors are known to disrupt the homeostasis of osteoblasts and osteoclasts. The purpose of this study was to identify the changes of osteoblasts and osteoclasts under long-term hyperglycaemic conditions, using a mouse fracture model of long-term hyperglycemia (LT-HG). METHODS: C57BL/6J mice and green fluorescent protein (GFP) -positive bone marrow transplanted C57BL/6J mice with LT-HG, maintained under a state of hyperglycaemia for 2 months, were used in this study. After the experimental fracture, we examined the immunohistochemical expression of proinsulin and tumor necrosis factor (TNF) -α at the fracture site. C57BL/6J fracture model mice without hyperglycaemia were used as controls. RESULTS: In the LT-HG mice, chondrocyte resorption was delayed, and osteoblasts showed an irregular arrangement at the callus site. The osteoclasts were scattered with a decrement in the number of nuclei. The expression of proinsulin was confirmed in bone marrow derived cells (BMDCs) with neovascularization 2 and 3 weeks after fracture. Immunopositivity for TNF-α was also confirmed in immature chondrocytes and BMDCs with neovascularization at 2 weeks, and the number of positive cells was not decreased at 3 weeks. Examination of GFP-grafted hyperglycaemic mice showed that the majority of cells at the fracture site were GFP-positive. Immunohistochemistry showed that the rate of double positives was 15% for GFP and proinsulin and 47% for GFP and TNF-α. CONCLUSION: LT-HG induces an increase in the number of proinsulin and TNF-α positive cells derived from BMDCs. We suggest that proinsulin and TNF-α positive cells are involved in both bone formation and bone resorption after fracture under hyperglycaemic conditions, resulting in the delay of bone healing.

Bone marrow-derived inducible microglia-like cells ameliorate motor function and survival in a mouse model of amyotrophic lateral sclerosis.

BACKGROUND AIMS: Amyotrophic lateral sclerosis (ALS) is an incurable neurodegenerative disease. Neuroinflammation in the spinal cord plays a pivotal role in the pathogenesis of ALS, and microglia are involved in neuroinflammation. Microglia mainly have two opposite phenotypes involving cytotoxic and neuroprotective properties, and neuroprotective microglia are expected to be a novel application for the treatment of ALS. Therefore, to establish a clinically applicable therapeutic method using neuroprotective microglia, the authors investigated the effect of inducing neuroprotective microglia-like cells from bone marrow for transplantation into ALS model mice. METHODS: Bone marrow-derived mononuclear cells were isolated from green fluorescent protein mice and cultured using different protocols of cytokine treatment with granulocyte-macrophage colony-stimulating factor (GM-CSF) and IL-4. Cells with a high potency of proliferation and differentiation into microglia were evaluated by gene analysis, flow cytometry and direct neuroprotective effects in vitro. These cells were named bone marrow-derived inducible microglia-like (BM-iMG) cells and transplanted into the spinal cords of ALS model mice, and behavioral tests, immunohistochemistry and gene expression profiling were performed. RESULTS: Three-day GM-CSF and 4-day GM-CSF + IL-4 stimulations were most effective in inducing BM-iMG cells from the bone marrow. Transplantation of BM-iMG cells improved motor function, prolonged survival and suppressed neuronal cell death, astrogliosis and microgliosis in the spinal cords of ALS mice. Moreover, neuroprotective genes such as Arg1 and Mrc1 were upregulated, whereas pro-inflammatory genes such as Nos2 and Il6 were downregulated. CONCLUSIONS: Intraspinal transplantation of BM-iMG cells demonstrated therapeutic effects in a mouse model of ALS. Further studies and clinical applications in patients with ALS are expected in the future.

Transplantation of M2-Deviated Microglia Promotes Recovery of Motor Function after Spinal Cord Injury in Mice.

Despite the poor prognosis of spinal cord injury (SCI), effective treatments are lacking. Diverse factors regulate SCI prognosis. In this regard, microglia play crucial roles depending on their phenotype. The M1 phenotype exacerbates neuroinflammation, whereas the M2 phenotype promotes tissue repair and provides anti-inflammatory effects. Therefore, we compared the effects of M2 and M1 microglia transplantation on SCI. First, we established a method for effective induction of M1 or M2 microglia by exposure to granulocyte-macrophage colony-stimulating factor (GM-CSF) or interleukin (IL)-4, respectively, to be used for transplantation in a SCI mouse model. In the M2 microglia transplantation group, significant recovery of motor function was observed compared with the control and M1 groups. Elevated transcription of several neuroprotective molecules including mannose receptor C type 1 (Mrc1), arginase 1 (Arg1), and insulin-like growth factor 1 (Igf1) was observed. Moreover, intramuscular injection of FluoroRuby dye revealed recovery of retrograde axonal transport from the neuromuscular junction to upstream of the injured spinal cord only in the M2-transplanted group, although the number of migrated microglia were comparable in both M1 and M2 groups. In conclusion, our results indicated that M2 microglia obtained by IL-4 stimulation may be a promising candidate for cell transplantation therapy for SCI.

A role for bone marrow-derived cells in diabetic nephropathy.

Diabetes mellitus causes systemic disorders. We previously demonstrated that diabetic condition forced bone marrow-derived cells (BMDCs) to express TNF-α, leading to the development of diabetic neuropathy in mice. Here, we hypothesized that these abnormal BMDCs are also involved in diabetic nephropathy. To test our hypothesis, mice were irradiated to receive total bone marrow (BM) from the transgenic mice expressing green fluorescent protein before diabetes was induced by streptozotocin. Confocal microscopy showed that the diabetic glomerulus had more BMDCs compared with the nondiabetic glomerulus. Most of these cells exhibited endothelial phenotypes, being negative for several markers, including podocin (a maker of podocyte), α8 integrin (mesangial cell), CD68, and F4/80 (macrophage). Next, the total BM of diabetic mice was transplanted into nondiabetic mice to examine if diabetic BM per se could cause glomerular injury. The recipient mice exhibiting normal glycemia developed albuminuria and mesangial expansion with an increase in capillary area. The number of BMDCs increased in the glomerulus of the recipient mice. These cells were found to exhibit the endothelial phenotype and to express TNF-α. These data suggest that diabetic BMDCs per se could initiate glomerular disease. Finally, eNOS knockout mice were used to examine if residential endothelial injury could attract BMDCs into the glomerulus. However, endothelial dysfunction due to eNOS deficiency failed to attract BMDCs into the glomerulus. In summary, BMDCs may be involved in the development of diabetic nephropathy.-Nobuta, H., Katagi, M., Kume, S., Terashima, T., Araki, S., Maegawa, H., Kojima, H., Nakagawa, T. A role for bone marrow-derived cells in diabetic nephropathy.

Charcot-Marie-Tooth disease type 2A with an autosomal-recessive inheritance: the first report of an adult-onset disease.

Axonal Charcot-Marie-Tooth disease (CMT) is most frequently caused by mutations in the MFN2 gene (CMT2A) that can lead to various clinical phenotypes. The age at disease onset varies, but most cases occur before adolescence. We report two Japanese sisters who presented with middle-age-onset peripheral neuropathy with distinct clinical features. In the affected sisters, a homozygous missense mutation, c.1894C>T, p.R632W, corresponding to the transmembrane domain of MFN2 was identified; this mutation was heterozygous in another non-affected sibling, demonstrating co-segregation of the genotype and phenotype. The patients developed adult-onset slowly progressive muscle weakness that was predominant in the calf muscles and sensory disturbance. Magnetic resonance imaging revealed diffuse atrophy of the spinal cord, especially in the thoracic segment, and mild atrophy of the parietal lobe and the cerebellum in both patients. Electron microscopy of the sural nerve revealed clusters of round and swollen mitochondria. This is the first case report of adult-onset CMT2A with an autosomal-recessive inheritance pattern. The phenotype caused by the MFN2 mutation in these cases is very mild, considering that the mutation causes middle-aged-onset Charcot-Marie-Tooth even in the homozygous state. The mechanism of MFN2 mutation-induced toxicity is an interesting theme that awaits further investigations.

A mutation of COX6A1 causes a recessive axonal or mixed form of Charcot-Marie-Tooth disease.

Charcot-Marie-Tooth disease (CMT) is the most common inherited neuropathy characterized by clinical and genetic heterogeneity. Although more than 30 loci harboring CMT-causing mutations have been identified, many other genes still remain to be discovered for many affected individuals. For two consanguineous families with CMT (axonal and mixed phenotypes), a parametric linkage analysis using genome-wide SNP chip identified a 4.3 Mb region on 12q24 showing a maximum multipoint LOD score of 4.23. Subsequent whole-genome sequencing study in one of the probands, followed by mutation screening in the two families, revealed a disease-specific 5 bp deletion (c.247-10_247-6delCACTC) in a splicing element (pyrimidine tract) of intron 2 adjacent to the third exon of cytochrome c oxidase subunit VIa polypeptide 1 (COX6A1), which is a component of mitochondrial respiratory complex IV (cytochrome c oxidase [COX]), within the autozygous linkage region. Functional analysis showed that expression of COX6A1 in peripheral white blood cells from the affected individuals and COX activity in their EB-virus-transformed lymphoblastoid cell lines were significantly reduced. In addition, Cox6a1-null mice showed significantly reduced COX activity and neurogenic muscular atrophy leading to a difficulty in walking. Those data indicated that COX6A1 mutation causes the autosomal-recessive axonal or mixed CMT.

Ablation of a small subpopulation of diabetes-specific bone marrow-derived cells in mice protects against diabetic neuropathy.

Diabetic peripheral neuropathy (DPN) is a major diabetic complication. Previously, we showed that hyperglycemia induces the appearance of proinsulin (PI)-producing bone marrow-derived cells (PI-BMDCs), which fuse with dorsal root ganglion neurons, causing apoptosis, nerve dysfunction, and DPN. In this study, we have devised a strategy to ablate PI-BMDCs in mice in vivo. The use of this strategy to selectively ablate TNFα-producing PI-BMDCs in diabetic mice protected these animals from developing DPN. The findings provide powerful validation for a pathogenic role of PI-BMDCs and identify PI-BMDCs as an accessible therapeutic target for the treatment and prevention of DPN.

Late-onset spinal form xanthomatosis without brain lesion: a case report.

BACKGROUND: Cerebrotendinous xanthomatosis (CTX) is a rare autosomal recessive sterol storage disease caused by a mutated sterol 27-hydroxylase (CYP27A1) gene. Patients with typical CTX show neurological dysfunction including bilateral cataracts, paresis, cerebral ataxia, dementia, and psychiatric disorders, and magnetic resonance imaging (MRI) has revealed symmetrical lesions in the cerebellar white matter. CASE PRESENTATION: We report the case of a patient with late-onset spinal form CTX without brain lesion. He showed pyramidal tract signs, and impaired joint position and vibration sensation in the lower limbs. Cervical sagittal MRI demonstrated a longitudinally extensive white matter abnormality in the dorsal column of the C2-C7 spinal cord; however, a brain MRI revealed an absence of lesions, including in the cerebellar white matter. Genetic analysis of CYP27A1 revealed that the patient was compound heterozygous for p.Gln85Arg in exon 1, a novel mutation, and p.Arg405Gln in exon 7, a previously reported mutation. CONCLUSION: This is the first report of late-onset spinal form CTX without typical neurological symptoms, and the first report of p.Gln85Arg in CYP27A1. We speculate that spinal form CTX without brain lesion is a clinically and radiologically rare variation of CTX. Therefore, spinal xanthomatosis should be included in the differential diagnosis of chronic myelopathy even with late-onset and/or no other typical neurological findings.

Bone marrow-derived TNF-α causes diabetic neuropathy in mice.

AIMS/HYPOTHESIS: Dysregulation of biochemical pathways in response to hyperglycaemia in cells intrinsic to the nervous system (Schwann cells, neurons, vasa nervorum) are thought to underlie diabetic peripheral neuropathy (DPN). TNF-α is a known aetiological factor; Tnf-knockout mice are protected against DPN. We hypothesised that TNF-α produced by a small but specific bone marrow (BM) subpopulation marked by proinsulin production (proinsulin-producing BM-derived cells, PI-BMDCs) is essential for DPN development. METHODS: We produced mice deficient in TNF-α, globally in BM and selectively in PI-BMDCs only, by gene targeting and BM transplantation, and induced diabetes by streptozotocin. Motor and sensory nerve conduction velocities were used to gauge nerve dysfunction. Immunocytochemistry, fluorescence in situ hybridisation (FISH) and PCR analysis of dorsal root ganglia (DRG) were employed to monitor outcome. RESULTS: We found that loss of TNF-α in BM only protected mice from DPN. We developed a strategy to delete TNF-α specifically in PI-BMDCs, and found that PI-BMDC-specific loss of TNF-α protected against DPN as robustly as loss of total BM TNF-α. Selective loss of PI-BMDC-derived TNF-α downregulated TUNEL-positive DRG neurons. FISH revealed PI-BMDC-neuron fusion cells in the DRG in mice with DPN; fusion cells were undetectable in non-diabetic mice or diabetic mice that had lost TNF-α expression selectively in the PI-BMDC subpopulation. CONCLUSIONS/INTERPRETATION: BMDC-specific TNF-α is essential for DPN development; its selective removal from a small PI-BMDC subpopulation protects against DPN. The pathogenicity of PI-BMDC-derived TNF-α may have important therapeutic implications.

Epidermis-dermis junction as a novel location for bone marrow-derived cells to reside in response to ionizing radiation.

Bone marrow-derived cells (BMDCs) can migrate into the various organs in the mice irradiated by ionizing radiation (IR). However, it may not be the case in the skin. While IR is used for bone marrow (BM) transplantation, studying with the epidermal sheets demonstrated that the BMDC recruitment is extraordinarily rare in epidermis in the mouse. Herein, using the chimera mice with BM from green fluorescent protein (GFP) transgenic mice, we simply examined if BMDCs migrate into any layers in the total skin, as opposed to the epidermal sheets, in response to IR. Interestingly, we identified the presence of GFP-positive (GFP(+)) cells in the epidermis-dermis junction in the total skin sections although the epidermal cell sheets failed to have any GFP cells. To examine a possibility that the cells in the junction could be mechanically dissociated during separating epidermal sheets, we then salvaged such dissociated cells and examined its characteristics. Surprisingly, some GFP(+) cells were found in the salvaged cells, indicating that these cells could be derived from BM. In addition, such BMDCs were also associated with inflammation in the junction. In conclusion, BMDCs can migrate to and reside in the epidermis-dermis junction after IR.

Stem cell factor-activated bone marrow ameliorates amyotrophic lateral sclerosis by promoting protective microglial migration.

Amyotrophic lateral sclerosis (ALS) is a progressive disease associated with motor neuron death. Several experimental treatments, including cell therapy using hematopoietic or neuronal stem cells, have been tested in ALS animal models, but therapeutic benefits have been modest. Here we used a new therapeutic strategy, bone marrow transplantation (BMT) with stem cell factor (SCF)- or FMS-like tyrosine kinase 3 (flt3)-activated bone marrow (BM) cells for the treatment of hSOD1(G93A) transgenic mice. Motor function and survival showed greater improvement in the SCF group than in the group receiving BM cells that had not been activated (BMT alone group), although no improvement was shown in the flt3 group. In addition, larger numbers of BM-derived cells that expressed the microglia marker Iba1 migrated to the spinal cords of recipient mice compared with the BMT alone group. Moreover, after SCF activation, but not flt3 activation or no activation, the migrating microglia expressed glutamate transporter-1 (GLT-1). In spinal cords in the SCF group, inflammatory cytokines tumor necrosis factor-α and interleukin-1ß were suppressed and the neuroprotective molecule insulin-like growth factor-1 increased relative to nontreatment hSOD1(G93A) transgenic mice. Therefore, SCF activation changed the character of the migrating donor BM cells, which resulted in neuroprotective effects. These studies have identified SCF-activated BM cells as a potential new therapeutic agent for the treatment of ALS.

Bone marrow expression of poly(ADP-ribose) polymerase underlies diabetic neuropathy via hematopoietic-neuronal cell fusion.

Diabetic neuropathy is the most common diabetic complication. The pathogenetic pathways include oxidative stress, advanced glycation end product (AGE) formation, protein kinase C, and NF-κB activation, as well as increased polyol flux. These metabolic perturbations affect neurons, Schwann cells, and vasa nervorum, which are held to be the primary cell types involved. We hypothesize that diabetes induces the appearance of abnormal bone marrow-derived cells (BMDCs) that fuse with neurons in the dorsal root ganglia (DRG) of mice, leading to diabetic neuropathy. Neuronal poly(ADP-ribose) polymerase-1 (PARP-1) activation in diabetes is known to generate free radical and oxidant-induced injury and poly(ADP-ribose) polymer formation, resulting in neuronal death and dysfunction, culminating in neuropathy. We further hypothesize that BM-specific PARP expression plays a determining role in disease pathogenesis. Here we show that bone marrow transplantation (BMT) of PARP-knockout (PARPKO) cells to wild-type mice protects against, whereas BMT of wild-type cells to PARPKO mice, which are normally "neuropathy-resistant," confers susceptibility to, diabetic neuropathy. The pathogenetic process involving hyperglycemia, BMDCs, and BMDC-neuron fusion can be recapitulated in vitro. Incubation in high, but not low, glucose confers fusogenicity to BMDCs, which are characterized by proinsulin (PI) and TNF-α coexpression; coincubation of isolated DRG neurons with PI-BMDCs in high glucose leads to spontaneous fusion between the 2 cell types, while the presence of a PARP inhibitor or use of PARPKO BMDCs in the incubation protects against BMDC-neuron fusion. These complementary in vivo and in vitro experiments indicate that BMDC-PARP expression promotes diabetic neuropathy via BMDC-neuron fusion.

Interleukin-7 induces recruitment of monocytes/macrophages to endothelium.

AIMS: Interleukin-7 (IL-7) is a master regulator of T-cell development and homoeostasis. Increased IL-7 levels are associated with inflammatory diseases. The aims of this study were to determine whether IL-7 is a biomarker for inflammatory conditions or an active participant in atherogenesis. METHODS AND RESULTS: Advanced atherosclerotic lesions in Apoe(-/-) mice were regressed by long-term cholesterol lowering through treatment with a helper-dependent adenovirus expressing apolipoprotein E (n= 6-10). Using this model, gene expression patterns in the aorta were analysed at an early phase of regression by microarray. After stringent statistical analysis, we found that IL-7 expression is significantly reduced in response to lowering of cholesterol (n= 6). To understand the importance of IL-7 down-regulation for atherosclerotic regression, we studied the effects and mechanisms of action of IL-7 on endothelial cells (ECs) in vitro as well as in vivo. Our major findings are: (i) IL-7 up-regulates cell adhesion molecules and monocyte chemoattractant protein-1 in ECs and promotes monocyte adhesion to ECs; (ii) this regulation is mediated by phosphatidylinositol 3-kinase (PI3K)/AKT-dependent and -independent activation of NF-κB; (iii) elevation of plasma IL-7 induces recruitment of monocytes/macrophages to endothelium without affecting plasma cholesterol (n= 5, 6); and (4) lack of IL-7 in bone marrow-derived cells reduces migration of monocytes/macrophages to the lesions (n= 5, 6). CONCLUSION: These results suggest that IL-7 inflames endothelium via PI3K/AKT-dependent and -independent activation of NF-κB and recruits monocytes/macrophages to the endothelium, thus playing an active role in atherogenesis.

Neuronal-Hematopoietic Cell Fusion in Diabetic Neuropathy.

Diabetic neuropathy is a major complication of diabetes mellitus that occurs during the early stages of the disease. Many pathogenic mechanisms are related and induced by hyperglycemia. However, even if these factors improve, diabetic neuropathy cannot go into remission and progresses slowly. Furthermore, diabetic neuropathy often progresses even with proper glycemic control. Recently, bone marrow-derived cells (BMDCs) were reported to be involved in the pathogenesis of diabetic neuropathy. BMDCs expressing proinsulin and TNFα migrate to the dorsal root ganglion and fuse with neurons, and this neuronal-hematopoietic cell fusion induces neuronal dysfunction and apoptosis. The CD106-positive lineage-sca1+c-kit+ (LSK) stem cell fraction in the bone marrow is strongly involved in cell fusion with neurons, leading to diabetic neuropathy. Surprisingly, when CD106-positive LSK stem cells obtained from diabetic mice were transplanted into nondiabetic mice, they fused with dorsal root ganglion neurons and induced neuropathy in non-hyperglycemic normal mice. The transplanted CD106-positive LSK fraction inherited the trait even after transplantation; this "progeny effect" may explain the irreversibility of diabetic neuropathy and is a significant finding for determining the target of radical treatments and provides new directions for developing therapeutic methods for diabetic neuropathy.

Ideal body weight-based determination of minimum oral calories beneficial to function and survival in ALS.

Introduction: This study sought to identify the optimal caloric intake to improve function and survival in ALS patients by comparing oral intake per ideal body weight (IBW) and its discrepancy with total energy expenditure (TEE) using the Shimizu formula. Methods: A retrospective analysis of 104 ALS patients was conducted, categorizing them based on their average intake during the first week after admission using two primary intake cutoffs: 25 kcal/kgIBW and 30 kcal/kgIBW. The variance between oral intake and TEE was also evaluated using -300 kcal and 0 kcal as reference points. Results: Oral caloric intake per IBW and functional decline rate (rs = -0.35, p < 0.001), but the variance from TEE was not significantly correlated (-0.11, p = 0.27). Survival data showed that patients consuming less than 25 kcal/kgIBW had a median survival of 24 months, increasing to 38 months for those consuming between 25-30 kcal/kgIBW and 63 months for those consuming 30 kcal/kgIBW or more. Deviations from the TEE did not significantly affect survival (p = 0.36). Among patients consuming less than their TEE, those consuming less than 25 kcal/kgIBW had a shorter median survival (24 months) compared to their counterparts (46 months) (p = 0.022). Consumption of less than 25 kcal/kgBW emerged as a significant negative predictor of patient outcome, independent of factors such as age, gender or disease progression. Discussion: Intakes of 25 kcal/kgIBW or more are correlated with improved ALS outcomes, and larger, multi-regional studies are recommended for deeper insights.

Ambient Temperature Is Correlated With the Severity of Neonatal Hypoxic-Ischemic Brain Injury via Microglial Accumulation in Mice.

Background: The pathophysiology of neonatal hypoxic-ischemic encephalopathy (HIE) has been studied in several rodent models to develop novel treatments. Although it is well known that high ambient temperature results in severe HIE, the effect of subtle changes in ambient temperature during a hypoxic-ischemic (HI) insult has not been studied. Therefore, in order to clarify the difference of pathophysiological change among the HIE models due to the influence of small changes in chamber temperature, three-step gradual change of 0.5°C each were prepared in ambient temperature during hypoxic exposure. Methods: Blood flow in the left common carotid artery (CCA) of neonatal mice was interrupted using bipolar electronic forceps under general and local anesthesia. The mice were subsequently subjected to 10% hypoxic exposure for 50 min at 36.0, 36.5, or 37.0°C. A control group was also included in the study. The size of the striatum and hippocampus and the volume reduction rate of the hemisphere in the section containing them on the ischemic side were evaluated using microtubule associated protein 2 (MAP2) immunostaining. The accumulation of Iba1-positive cells was investigated to assess inflammation. Additionally, rotarod and open-field tests were performed 2 weeks after HI insult to assess its effect on physiological conditions. Results: MAP2 staining revealed that the higher the temperature during hypoxia, the more severe the volume reduction rate in the hemisphere, striatum, and hippocampus. The number of Iba1-positive cells in the ipsilateral lesion gradually increased with increasing temperature, and there was a significant difference in motor function in the 36.5 and 37.0°C groups compared with the sham group. In the open-field tests, there was a significant decrease in performance in the 37.0°C groups compared with the 36.0°C and sham groups. Conclusions: Even a small gradual change of 0.5°C produced a significant difference in pathological and behavioral changes and contributed to the accumulation of Iba1-positive cells. The arrangement of ambient temperature is useful for creating a rodent model with the appropriate severity of the targeted neuropsychological symptoms to establish a novel therapy for HIE.

Inactivation of TNF-α ameliorates diabetic neuropathy in mice.

Tumor necrosis factor (TNF)-α is a potent proinflammatory cytokine involved in the pathogenesis of diabetic neuropathy. We inactivated TNF-α to determine if it is a valid therapeutic target for the treatment of diabetic neuropathy. We effected the inactivation in diabetic neuropathy using two approaches: by genetic inactivation of TNF-α (TNF-α(-/-) mice) or by neutralization of TNF-α protein using the monoclonal antibody infliximab. We induced diabetes using streptozotocin in wild-type and TNF-α(-/-) mice. We measured serum TNF-α concentration and the level of TNF-α mRNA in the dorsal root ganglion (DRG) and evaluated nerve function by a combination of motor (MNCV) and sensory (SNCV) nerve conduction velocities and tail flick test, as well as cytological analysis of intraepidermal nerve fiber density (IENFD) and immunostaining of DRG for NF-κB p65 serine-276 phosphorylated and cleaved caspase-3. Compared with nondiabetic mice, TNF-α(+/+) diabetic mice displayed significant impairments of MNCV, SNCV, tail flick test, and IENFD as well as increased expression of NF-κB p65 and cleaved caspase-3 in their DRG. In contrast, although nondiabetic TNF-α(-/-) mice showed mild abnormalities of IENFD under basal conditions, diabetic TNF-α(-/-) mice showed no evidence of abnormal nerve function tests compared with nondiabetic mice. A single injection of infliximab in diabetic TNF-α(+/+) mice led to suppression of the increased serum TNF-α and amelioration of the electrophysiological and biochemical deficits for at least 4 wk. Moreover, the increased TNF-α mRNA expression in diabetic DRG was also attenuated by infliximab, suggesting infliximab's effects may involve the local suppression of TNF-α. Infliximab, an agent currently in clinical use, is effective in targeting TNF-α action and expression and amelioration of diabetic neuropathy in mice.

GLT1 gene delivery based on bone marrow-derived cells ameliorates motor function and survival in a mouse model of ALS.

Amyotrophic lateral sclerosis (ALS) is an intractable neurodegenerative disease. CD68-positive bone marrow (BM)-derived cells (BMDCs) accumulate in the pathological lesion in the SOD1(G93A) ALS mouse model after BM transplantation (BMT). Therefore, we investigated whether BMDCs can be applied as gene carriers for cell-based gene therapy by employing the accumulation of BMDCs. In ALS mice, YFP reporter signals were observed in 12-14% of white blood cells (WBCs) and in the spinal cord via transplantation of BM after lentiviral vector (LV) infection. After confirmation of gene transduction by LV with the CD68 promoter in 4-7% of WBCs and in the spinal cord of ALS mice, BM cells were infected with LVs expressing glutamate transporter (GLT) 1 that protects neurons from glutamate toxicity, driven by the CD68 promoter, which were transplanted into ALS mice. The treated mice showed improvement of motor behaviors and prolonged survival. Additionally, interleukin (IL)-1ß was significantly suppressed, and IL-4, arginase 1, and FIZZ were significantly increased in the mice. These results suggested that GLT1 expression by BMDCs improved the spinal cord environment. Therefore, our gene therapy strategy may be applied to treat neurodegenerative diseases such as ALS in which BMDCs accumulate in the pathological lesion by BMT.

A novel role for bone marrow-derived cells to recover damaged keratinocytes from radiation-induced injury.

Exposure to moderate doses of ionizing radiation (IR), which is sufficient for causing skin injury, can occur during radiation therapy as well as in radiation accidents. Radiation-induced skin injury occasionally recovers, although its underlying mechanism remains unclear. Moderate-dose IR is frequently utilized for bone marrow transplantation in mice; therefore, this mouse model can help understand the mechanism. We had previously reported that bone marrow-derived cells (BMDCs) migrate to the epidermis-dermis junction in response to IR, although their role remains unknown. Here, we investigated the role of BMDCs in radiation-induced skin injury in BMT mice and observed that BMDCs contributed to skin recovery after IR-induced barrier dysfunction. One of the important mechanisms involved the action of CCL17 secreted by BMDCs on irradiated basal cells, leading to accelerated proliferation and recovery of apoptosis caused by IR. Our findings suggest that BMDCs are key players in IR-induced skin injury recovery.