The superiority of conditioned medium derived from rapidly expanded mesenchymal stem cells for neural repair.

BACKGROUND: Spinal cord injury (SCI) is a complex and severe neurological condition. Mesenchymal stem cells (MSCs) and their secreted factors show promising potential for regenerative medicine. Many studies have investigated MSC expansion efficacy of all kinds of culture medium formulations, such as growth factor-supplemented or xeno-free medium. However, very few studies have focused on the potential of human MSC (hMSC) culture medium formulations for injured spinal cord repair. In this study, we investigated the effect of hMSC-conditioned medium supplemented with bFGF, EGF, and patient plasma, namely, neural regeneration laboratory medium (NRLM), on SCI in vitro and in vivo. METHODS: Commercial and patient bone marrow hMSCs were obtained for cultivation in standard medium and NRLM separately. Several characteristics, including CD marker expression, differentiation, and growth curves, were compared between MSCs cultured in standard medium and NRLM. Additionally, we investigated the effect of the conditioned medium (referred to as NRLM-CM) on neural repair, including inflammation inhibition, neurite regeneration, and spinal cord injury (SCI), and used a coculture system to detect the neural repair function of NRLM-MSCs. RESULTS: Compared to standard culture medium, NRLM-CM had superior in inflammation reduction and neurite regeneration effects in vitro and improved functional restoration in SCI rats in vivo. In comparison with standard culture medium MSCs, NRLM-MSCs proliferated faster regardless of the age of the donor. NRLM-MSCs also showed increased adipose differentiative potential and reduced CD90 expression. Both types of hMSC CM effectively enhanced injured neurite outgrowth and protected against H2O2 toxicity in spinal cord neuron cultures. Cytokine arrays performed in hMSC-CM further revealed the presence of at least 120 proteins. Among these proteins, 6 demonstrated significantly increased expression in NRLM-CM: adiponectin (Acrp30), angiogenin (ANG), HGF, NAP-2, uPAR, and IGFBP2. CONCLUSIONS: The NRLM culture system provides rapid expansion effects and functional hMSCs. The superiority of the derived conditioned medium on neural repair shows potential for future clinical applications.

Evaluation of the antiangiogenic effect of Kringle 1-5 in a rat glioma model.

BACKGROUND: Kringle 1-5 (K1-5) is a potent antiangiogenesis factor for treating breast cancer and hepatocellular carcinoma. However, its use in treating brain tumors has not been studied. OBJECTIVE: To evaluate whether K1-5 is effective at treating gliomas. METHODS: The effects of K1-5 on cell morphology and cytotoxicity with or without lipopolysaccharide were tested in primary mixed neuronal-glial cultures. The antiglioma activity of K1-5 was evaluated by intra-arterial administration of K1-5 at 4 days after implantation of C6 glioma cells into the rat hippocampus. In 1 group of animals, tumor size, tumor vasculature, and tumor histology were evaluated on day 12. Animal survival was assessed in the other group. RESULTS: In vitro studies showed that K1-5 did not induce cytotoxicity in neurons and glia. In vivo studies demonstrated that K1-5 reduced vessel length and vessel density and inhibited perivascular tumor invasion. In addition, K1-5 normalized vessel morphology, decreased expression of hypoxia-inducible factor-1α and vascular endothelial growth factor, decreased tumor hypoxia, and decreased pseudopalisading necrosis. The average tumor volume was smaller in the treated than in the untreated group. Furthermore, animals treated with K1-5 survived significantly longer. CONCLUSION: Kringle 1-5 effectively reduces the growth of malignant gliomas in the rat. Although still far from translation in humans, K1-5 might be a possible future alternative treatment option for patients with gliomas.

Treatment with nerve grafts and aFGF attenuates allodynia caused by cervical root transection injuries.

PURPOSE: Nerve root traction injuries induce spinal cord inflammation and lead to neuronal death within days. In the present study, we examined the inflammatory response one week after multiple cervical root transections. METHODS: In the transection group, the left cervical roots (C6-8) of rats were cut at the spinal cord junction. In the repair group, transected roots were repaired with nerve grafts and the subsequent application of aFGF and fibrin glue. A sham group had nerve roots exposed without transection. Mechanical allodynia and spinal glial responses were evaluated. RESULTS: Allodynia did not differ between the treatment groups on day 2. Rats with transected spinal nerve roots had significantly more allodynia by 7 days, which was associated with IL-1ß expression in dorsal and ventral horn astrocytes, and microglia activation. Repair of nerve roots with autologous intercostal nerve grafts and FGF in fibrin glue attenuated the allodynia, reduced IL-1ß expression in astroctyes and reduced microglia activation, along with a significant increase in arginase I expression. CONCLUSION: This study demonstrated a correlation between an increased number of IL-1ß-positive astrocytes and the development of allodynia. Our treatment significantly decreased IL-1ß-positive astrocytes, thus preventing the occurrence of neuropathic pain following multiple cervical root injuries.

Functional recovery after the repair of transected cervical roots in the chronic stage of injury.

The treatment of root injury is typically performed at the more chronic stages post injury, by which time a substantial number of neurons have died. Therefore, before being applied in the clinical setting, a treatment strategy for these lesions should prove to be as effective in the chronic stages of injury as it is in the acute stage. In this study, we simulated the most severe clinical scenarios to establish an optimal time window for repair at a chronic stage. The sixth to eighth cervical roots on the left side of female SD rats were cut at their junction with the spinal cord. One or three weeks later, the wound was reopened and these roots were repaired with intercostal nerve grafts, with subsequent application of aFGF and fibrin glue. In the control group, the wound was closed after re-exploration without further repair procedures. Sensory and motor functions were measured after the surgery. Spinal cord morphology, neuron survival, and nerve fiber regeneration were traced by CTB-HRP. Results showed that both the sensory and motor functions had significant recovery in the 1-week repair group, but not in the 3-week repair group. By CTB-HRP tracing, we found that the architecture of the spinal cords was relatively preserved in the 1-week repair group, while those of the control group showed significant atrophic change. There were regenerating nerve fibers in the dorsal horn and more motor neuron survival in the 1-week repair group compared to that of the 3-week group. It was concluded that treating transected cervical roots at a chronic stage with microsurgical nerve grafting and application of aFGF and fibrin glue can lead to significant functional recovery, as long as the repair is done before too many neurons die.

Sensory and motor recovery after repairing transected cervical roots.

BACKGROUND: Adult mammal sensory axons avulsed through spinal dorsal root traction injuries, especially of the brachial plexus or cauda equina, cannot normally regenerate through axonal outgrowth from the DRG into the spinal cord, thus causing clinical conditions that require neuronal regeneration for sensory recovery and for which no successful treatment has yet been reported. METHODS: To evaluate the sensory recovery of the forelimb after transection of their left cervical dorsal and ventral roots (C6-C8) at their spinal cord junctions, 22 SD rats were randomly assigned to 3 groups: transection only (control 1); transection followed by repair using intercostal nerve grafts and fibrin glue (control 2); transection, repair, and application of aFGF and fibrin glue (experimental group). The following tests were reperformed after retransecting the repaired nerve roots to discount collateral innervation from adjacent nerve roots: motor function (grasping power), mechanical sensitivity to pain and touch (foot-withdrawal response to mechanical stimuli), temperature sensitivity (foot-withdrawal response to cold stimulus), and electrophysiologic sensory responses (measurement of cortical SEP). RESULTS: After transection and repair, the experimental group rats showed recovery in both motor (grasping power) and sensory (touch, pain, and temperature sensation) nerve functions. Neuronal regeneration was confirmed by the reappearance of cortical SEP and by its disappearance after retransection of the repaired cervical nerve roots. CONCLUSION: Using our strategy for repairing transected cervical nerve roots, motor and sensory recovery was achieved in adult rats. The success of our study highlights possible treatment options for humans with avulsion injuries of the dorsal roots from the spinal cord.