Tail nerve electrical stimulation promoted the efficiency of transplanted spinal cord-like tissue as a neuronal relay to repair the motor function of rats with transected spinal cord injury.

Following transected spinal cord injury (SCI), there is a critical need to restore nerve conduction at the injury site and activate the silent neural circuits caudal to the injury to promote the recovery of voluntary movement. In this study, we generated a rat model of SCI, constructed neural stem cell (NSC)-derived spinal cord-like tissue (SCLT), and evaluated its ability to replace injured spinal cord and repair nerve conduction in the spinal cord as a neuronal relay. The lumbosacral spinal cord was further activated with tail nerve electrical stimulation (TNES) as a synergistic electrical stimulation to better receive the neural information transmitted by the SCLT. Next, we investigated the neuromodulatory mechanism underlying the action of TNES and its synergism with SCLT in SCI repair. TNES promoted the regeneration and remyelination of axons and increased the proportion of glutamatergic neurons in SCLT to transmit brain-derived neural information more efficiently to the caudal spinal cord. TNES also increased the innervation of motor neurons to hindlimb muscle and improved the microenvironment of muscle tissue, resulting in effective prevention of hindlimb muscle atrophy and enhanced muscle mitochondrial energy metabolism. Tracing of the neural circuits of the sciatic nerve and tail nerve identified the mechanisms responsible for the synergistic effects of SCLT transplantation and TNES in activating central pattern generator (CPG) neural circuits and promoting voluntary motor function recovery in rats. The combination of SCLT and TNES is expected to provide a new breakthrough for patients with SCI to restore voluntary movement and control their muscles.

Diabetes mellitus accelerates the progression of osteoarthritis in streptozotocin-induced diabetic mice by deteriorating bone microarchitecture, bone mineral composition, and bone strength of subchondral bone.

BACKGROUND: The purpose of this study was to develop an optimal diabetes-osteoarthritis (DM-OA) mouse model to validate that diabetes aggravates osteoarthritis (OA) and to evaluate the microarchitecture, chemical composition, and biomechanical properties of subchondral bone (SB) as a consequence of the DM-OA-induced damage induced. METHODS: Mice were randomly divided into three groups: DM-OA group, OA group, and sham group. Blood glucose levels, body weight, and food intake of all animals were recorded. Serum calcium (Ca) and osteocalcin (OCN) levels were compared in the three groups. The messenger ribonucleic acid (mRNA) and protein expression of key regulators for bone metabolism were detected. A semi-quantitative grading system [Osteoarthritis Research Society International (OARSI)] was used to evaluate cartilage and SB degeneration. Microspectroscopy, microindentations, micro-computed tomography (CT) imaging, and fracture load of compression testing were also used to evaluate trabecular SB properties. RESULTS: Glycemic monitoring and pancreas pathological results indicated stable high blood glucose and massive destruction of pancreas and islet cells in the DM-OA group. Serum levels of bone specific alkaline phosphatase (ALP-B) and tartrate-resistant acid phosphatase 5b (TRACP-5b) in the DM-group were higher than those of the other two groups while levels of serum Ca and OCN were lower. Meanwhile, the protein and mRNA expression of osteoblast-specific biomarkers [osteoprotegerin/receptor activator of nuclear factor kappa-B ligand (OPG/RANKL) ratio, collagen type I (COL-I), Runt-related transcription factor 2 (RUNX-2), OCN] were suppressed, and osteoclast-specific biomarkers [sclerostin (SOST)] was elevated in the DM-OA group. The mineral-to-collagen ratio, microindentation elastic modulus, hardness, micro-architectural parameters, bone mineral density, and fracture load of SB trabecular bone of the DM-OA group joint were lower than those of the other two groups. On the other hand, The OARSI score, trabecular spacing, and structural model index of the DM-OA group joint were higher than those of the other two groups. CONCLUSIONS: The glycemic and pancreatic pathological results indicated that the DM-OA model was a simple and reliable model induced by streptozotocin (STZ) and surgery. The results revealed the mechanisms through which diabetes accelerates OA; that is, by damaging and deteriorating the functions of SB, including its microarchitecture, chemical composition, and biomechanical properties.

Autocrine fibronectin from differentiating mesenchymal stem cells induces the neurite elongation in vitro and promotes nerve fiber regeneration in transected spinal cord injury.

Extracellular matrix (ECM) expression is temporally and spatially regulated during the development of stem cells. We reported previously that fibronectin (FN) secreted by bone marrow mesenchymal stem cells (MSCs) was deposited on the surface of gelatin sponge (GS) soon after culture. In this study, we aimed to assess the function of accumulated FN on neuronal differentiating MSCs as induced by Schwann cells (SCs) in three dimensional transwell co-culture system. The expression pattern and amount of FN of differentiating MSCs was examined by immunofluorescence, Western blot and immunoelectron microscopy. The results showed that FN accumulated inside GS scaffold, although its mRNA expression in MSCs was progressively decreased during neural induction. MSC-derived neuron-like cells showed spindle-shaped cell body and long extending processes on FN-decorated scaffold surface. However, after blocking of FN function by application of monoclonal antibodies, neuron-like cells showed flattened cell body with short and thick neurites, together with decreased expression of integrin ß1. In vivo transplantation study revealed that autocrine FN significantly facilitated endogenous nerve fiber regeneration in spinal cord transection model. Taken together, the present results showed that FN secreted by MSCs in the early stage accumulated on the GS scaffold and promoted the neurite elongation of neuronal differentiating MSCs as well as nerve fiber regeneration after spinal cord injury. This suggests that autocrine FN has a dynamic influence on MSCs in a three dimensional culture system and its potential application for treatment of traumatic spinal cord injury. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 104A: 1902-1911, 2016.

Connexin 43 Mediates CXCL12 Production from Spinal Dorsal Horn to Maintain Bone Cancer Pain in Rats.

Tumor metastasis to bone can subsequently lead to bone cancer pain (BCP). Currently, BCP is difficult to conquer due to a poor understanding of the potential mechanisms. Several studies have indicated that astrocyte-specific connexin 43 (Cx43) was involved in the neuropathic pain, and Cx43 induced the release of chemokine CXCL12 in bone marrow stromal cells. However, whether spinal Cx43 mediates the production of CXCL12 to participate in the maintenance of BCP is still unknown. Here we showed that Walker 256 tumor cells inoculation into the tibia induced a significant mechanical allodynia, which was accompanied by upregulation of spinal p-Cx43 and CXCL12 expression levels from day 6 to day 18 after inoculation. Spinal Cx43 was mainly expressed in astrocytes, and intrathecal (43)Gap26 (a selective Cx43 blocker) markedly attenuated mechanical allodynia as well as reduced p-Cx43 and CXCL12 expression at day 18 after inoculation. Pre-intrathecal administration of CXCL12 almost abolished the attenuated mechanical allodynia by (43)Gap26. Furthermore, intrathecal injection of anti-CXCL12 neutralizing antibody could ameliorate mechanical allodynia with concomitant inhibition of upregulation of CXCL12 expression, but not influence on p-Cx43 expression. Our results indicate that Cx43 mediates CXCL12 production from spinal dorsal horn in astrocytes to maintain bone cancer pain in rats. These findings may improve our understanding of the underlying mechanisms of BCP and provide a novel target for the treatment of BCP.

Integration of donor mesenchymal stem cell-derived neuron-like cells into host neural network after rat spinal cord transection.

Functional deficits following spinal cord injury (SCI) primarily attribute to loss of neural connectivity. We therefore tested if novel tissue engineering approaches could enable neural network repair that facilitates functional recovery after spinal cord transection (SCT). Rat bone marrow-derived mesenchymal stem cells (MSCs), genetically engineered to overexpress TrkC, receptor of neurotrophin-3 (NT-3), were pre-differentiated into cells carrying neuronal features via co-culture with NT-3 overproducing Schwann cells in 3-dimensional gelatin sponge (GS) scaffold for 14 days in vitro. Intra-GS formation of MSC assemblies emulating neural network (MSC-GS) were verified morphologically via electron microscopy (EM) and functionally by whole-cell patch clamp recording of spontaneous post-synaptic currents. The differentiated MSCs still partially maintained prototypic property with the expression of some mesodermal cytokines. MSC-GS or GS was then grafted acutely into a 2 mm-wide transection gap in the T9-T10 spinal cord segments of adult rats. Eight weeks later, hindlimb function of the MSC-GS-treated SCT rats was significantly improved relative to controls receiving the GS or lesion only as indicated by BBB score. The MSC-GS transplantation also significantly recovered cortical motor evoked potential (CMEP). Histologically, MSC-derived neuron-like cells maintained their synapse-like structures in vivo; they additionally formed similar connections with host neurites (i.e., mostly serotonergic fibers plus a few corticospinal axons; validated by double-labeled immuno-EM). Moreover, motor cortex electrical stimulation triggered c-fos expression in the grafted and lumbar spinal cord cells of the treated rats only. Our data suggest that MSC-derived neuron-like cells resulting from NT-3-TrkC-induced differentiation can partially integrate into transected spinal cord and this strategy should be further investigated for reconstructing disrupted neural circuits.

Involvement of Spinal Bv8/Prokineticin 2 in a Rat Model of Cancer-Induced Bone Pain.

Cancer-induced bone pain (CIBP) is seriously disruptive to the quality of life in cancer patients, and present therapies are limited. The Bv8/prokineticin 2, a new family of chemokines, has been demonstrated to be involved in inflammatory and neuropathic pain. However, whether it is involved in CIBP remains unclear. This study was designed to examine whether spinal Bv8 was involved in the development of CIBP in rats. A rat CIBP model was constructed by injecting Walker 256 carcinoma cells into the medullary cavity of rat tibia. Tibia inoculation with Walker 256 tumour cells resulted in the development of mechanical hyperalgesia. Compared with sham rats, spinal Bv8 mRNA and protein levels were markedly and time-dependently increased in CIBP rats. Intrathecal administration of Bv8 neutralizing antibody (5 ng) could markedly attenuate pain behaviour as well as up-regulation of spinal TNF-α expression at day 18 after inoculation. Intrathecal pre-treatment with synthetic Bv8 (50 pg) almost completely abolished these effects. These data suggested that spinal Bv8/prokineticin 2 participated in the development of CIBP. Targeting of spinal Bv8 might be a promising strategy for the management of cancer-induced bone pain.

Evidence for involvement of spinal RANTES in the antinociceptive effects of triptolide, a diterpene triepoxide, in a rat model of bone cancer pain.

It has been shown that triptolide has beneficial effects in the treatment of neuropathic pain, but its effects on bone cancer pain (BCP) remain unclear. In this study, we aimed to explore the potential role of spinal regulated activation of normal T cell expressed and secreted (RANTES) in the antinociceptive effects of triptolide on BCP. A BCP model was induced by injecting Walker 256 mammary gland carcinoma cells into the intramedullary space of rat tibia. Intrathecal administration of triptolide (0.5, 1, 2 µg) could dose-dependently alleviate mechanical hyperalgesia and spontaneous pain. In addition, there were also concomitant decreases in RANTES mRNA and protein expression levels in spinal dorsal horn. These results suggest that the antinociceptive effects of triptolide are related with inhibition of spinal RANTES expression in BCP rats. The findings of this study may provide a promising drug for the treatment of BCP.

Involvement of spinal CC chemokine ligand 5 in the development of bone cancer pain in rats.

In this study, we aimed to investigate the role of spinal CC chemokine ligand 5 (CCL5) in the development of bone cancer pain (BCP). A BCP model was established by inoculation of Walker 256 cells into the intramedullary space of rat tibia. The levels of spinal CCL5 mRNA and protein expression significantly and time dependently increased in BCP rats compared with sham rats. On day 15 after inoculation, intrathecal administration of anti-CCL5 neutralizing antibody (4 µg) significantly attenuated the established mechanical hyperalgesia in the Walker 256 cells-injected rats, and the effect was abolished by intrathecal pre-treatment with recombinant rat CCL5 (0.2 µg). These results suggest that the spinal CCL5 may be involved in the development of BCP. The findings of this study may provide an evidence for developing novel analgesic agents to treat BCP.