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.

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.

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.

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.

Homing of the bone marrow-derived interstitial cells of Cajal is decreased in diabetic mouse intestine.

BACKGROUND: Interstitial cells of Cajal (ICCs), which express c-Kit receptor tyrosine kinase (KIT), play an important role in gastrointestinal motility. Loss of ICCs likely contributes to diabetic gastrointestinal motility disorder, however, the mechanism of attrition remains unknown. Here, we test the hypothesis that the bone marrow-derived progenitors are an important source of intestinal ICCs and that decreased homing of these progenitors in diabetes contributes to ICC diminution. METHODS: Wild type mice were X-ray irradiated, transplanted with bone marrow (BMT) from green fluorescence protein (GFP)-transgenic (TG)-mice and subsequently made diabetic by streptozotocin (STZ) injection. Intestinal homing of GFP-positive bone marrow-derived cells was examined 2 or 5 months after STZ treatment. RESULTS: In the BMT-mice, we found many GFP-positive bone marrow-derived cells (BMDCs) in most parts of the intestinal area, the number of BMDCs was significantly decreased in diabetic mice compared with nondiabetic controls. As a representative area, we further examined the myenteric plexus of the proximal small intestine, and found that the cell numbers of ICCs marked by c-Kit-positive immunoreactivity were decreased by more than 40% in diabetic versus nondiabetic mice. Furthermore, numbers of c-Kit+/GFP+ and c-Kit+/GFP- cells were similar in nondiabetic mice, and decreased by 45.8% and 42.0%, respectively, in diabetic mice. CONCLUSION: These results suggest that the decreased homing from the bone marrow is a major cause of ICC loss in the intestine in diabetes mellitus.

Validation of the "Activity and participation" component of ICF Core Sets for stroke patients in Japanese rehabilitation wards

OBJECTIVE: To validate the International Classification of Functioning, Disability and Health (ICF) Core Set for stroke in the assessment of functional status and disability in Japanese stroke patients. METHODS: The study included stroke patients admitted to the Kaifukuki (convalescent) rehabilitation wards. The comprehensive ICF Core Set for neurological conditions for post-acute care and the ICF rehabilitation set were evaluated with qualifiers assessed by the physiatrists at admission. The "activity and participation" (d) component was divided to sub-components (cognition-related activity, motor-related activity and participation). The correlations between numbers of problem categories in the entire "d" component and these sub-components in each ICF Core Set and the Functional Independence Measure (FIM) score were assessed using Spearman's correlation coefficient. RESULTS: A total of 117 post-stroke patients (mean age 70.1 ± 14.2 years, 53 women) were included. Correlation analysis identified significant and strong correlations between the values of the entire "d" component and sub-components (cognition-related activity and motor-related activity) of the 2 ICF Core Sets and FIM score. A significant, but weak, correlation between FIM and the participation sub-component was identified. CONCLUSION: The "d" component of these 2 ICF Core Sets reflects functional status and disability and could be a valid measure in post-acute stroke patients in the rehabilitation setting.

Hyperglycemia induces abnormal gene expression in hematopoietic stem cells and their progeny in diabetic neuropathy.

Diabetic peripheral neuropathy is a major chronic diabetic complication. We have previously shown that in type 1 diabetic streptozotocin-treated mice, insulin- and TNF-α co-expressing bone marrow-derived cells (BMDCs) induced by hyperglycemia travel to nerve tissues where they fuse with nerve cells, causing premature apoptosis and nerve dysfunction. Here we show that similar BMDCs also occur in type 2 diabetic high-fat diet (HFD) mice. Furthermore, we found that hyperglycemia induces the co-expression of insulin and TNF-α in c-kit(+)Sca-1(+)lineage(-) (KSL) progenitor cells, which maintain the same expression pattern in the progeny, which in turn participates in the fusion with neurons when transferred to normoglycemic animals.

Haematopoietic cells produce BDNF and regulate appetite upon migration to the hypothalamus.

Brain-derived neurotrophic factor (BDNF) suppresses food intake by acting on neurons in the hypothalamus. Here we show that BDNF-producing haematopoietic cells control appetite and energy balance by migrating to the hypothalamic paraventricular nucleus. These haematopoietic-derived paraventricular nucleus cells produce microglial markers and make direct contacts with neurons in response to feeding status. Mice with congenital BDNF deficiency, specifically in haematopoietic cells, develop hyperphagia, obesity and insulin resistance. These abnormalities are ameliorated by bone marrow transplantation with wild-type bone marrow cells. Furthermore, when injected into the third ventricle, wild-type bone marrow mononuclear cells home to the paraventricular nucleus and reverse the hyperphagia of BDNF-deficient mice. Our results suggest a novel mechanism of feeding control based on the production of BDNF by haematopoietic cells and highlight a potential new therapeutic route for the treatment of obesity.

Brain-derived neurotrophic factor from bone marrow-derived cells promotes post-injury repair of peripheral nerve.

Brain-derived neurotrophic factor (BDNF) stimulates peripheral nerve regeneration. However, the origin of BNDF and its precise effect on nerve repair have not been clarified. In this study, we examined the role of BDNF from bone marrow-derived cells (BMDCs) in post-injury nerve repair. Control and heterozygote BDNF knockout mice (BDNF+/-) received a left sciatic nerve crush using a cerebral blood clip. Especially, for the evaluation of BDNF from BMDCs, studies with bone marrow transplantation (BMT) were performed before the injury. We evaluated nerve function using a rotarod test, sciatic function index (SFI), and motor nerve conduction velocity (MNCV) simultaneously with histological nerve analyses by immunohistochemistry before and after the nerve injury until 8 weeks. BDNF production was examined by immunohistochemistry and mRNA analyses. After the nerve crush, the controls showed severe nerve dysfunction evaluated at 1 week. However, nerve function was gradually restored and reached normal levels by 8 weeks. By immunohistochemistry, BDNF expression was very faint before injury, but was dramatically increased after injury at 1 week in the distal segment from the crush site. BDNF expression was mainly co-localized with CD45 in BMDCs, which was further confirmed by the appearance of GFP-positive cells in the BMT study. Variant analysis of BDNF mRNA also confirmed this finding. BDNF+/- mice showed a loss of function with delayed histological recovery and BDNF+/+→BDNF+/- BMT mice showed complete recovery both functionally and histologically. These results suggested that the attenuated recovery of the BDNF+/- mice was rescued by the transplantation of BMCs and that BDNF from BMDCs has an essential role in nerve repair.

Pathogenesis of diabetic neuropathy: bad to the bone.

Insulin and proinsulin are normally produced only by the pancreas and thymus. We detected in diabetic rodents the presence of extra pancreatic proinsulin-producing bone marrow-derived cells (PI-BMDCs) in the BM, liver, and fat. In mice and rats with diabetic neuropathy, we also found proinsulin-producing cells in the sciatic nerve and neurons of the dorsal root ganglion (DRG). BM transplantation experiments using genetically marked donor and recipient mice showed that the proinsulin-producing cells in the DRG, which morphologically resemble neurons, are actually polyploid proinsulin-producing fusion cells formed between neurons and PI-BMDCs. Additional experiments indicate that diabetic neuropathy is not simply the result of nerve cells being damaged directly by hyperglycemia. Rather, hyperglycemia induces fusogenic PI-BMDCs that travel to the peripheral nervous system, where they fuse with Schwann cells and DRG neurons, causing neuronal dysfunction and death, the sine qua non for diabetic neuropathy. Poorly controlled diabetes is indeed bad to the bone.