The enhancement of CCL2 and CCL5 by human bone marrow-derived mesenchymal stem/stromal cells might contribute to inflammatory suppression and axonal extension after spinal cord injury.

Human bone marrow-derived mesenchymal stem/stromal cells (hMSCs) have shown potential in facilitating recovery from spinal cord injury (SCI) through communicating with microglia/macrophages (MG/MΦ). We here focused on chemokines as a candidate for the communication. Selected MG/MΦ-related chemokines were determined gene expression after SCI and further focused CCL2/CCR2 and CCL5/CCR5 to estimate role of the chemokines by hMSCs. Male C57/BL6 mice were subjected to spinal cord transection. Gene expression was assayed in the spinal cords following SCI for selected MG/MΦ-related chemokines and their receptors. hMSCs (5×105 cells) were then transplanted into parenchyma of the spinal cord, and the expressions of the Ccl2/Ccr2 and Ccl5/Ccr5 axes, inflammation, MG/MΦ-polarization, and axonal regeneration were evaluated to measure the influence of the hMSCs. Finally, mouse CCL5 was injected into the spinal cords. Acute increases in gene expression after SCI were observed for most chemokines, including Ccl2; chronic increases were observed for Ccl5. CCL2+-cells merged with NeuN+-neurons. CCR2+ immunoreactivity was principally observed in Ly-6G+/iNOS+-granulocytes on postoperative day (pod) 1, and CCL5+ and CCR5+ immunoreactivity overlapped with NeuN+-neurons and F4/80+-MG/MΦ on pod 14. The hMSC transplantation enhanced Ccl2 and Ccl5 and improved locomotor activity. The hMSC implantation did not alter the number of Ly-6G+/CCR2+ but decreased Il1, Elane, and Mpo on pod 3. Conversely, hMSC transplantation increased expression of Zc3h12a (encodes MCP-1-induced protein) on pod 14. Moreover, hMSC increased the Aif1, and two alternatively activated macrophage (AAM)-related genes, Arg1 and Chil3 (Ym1), as well as axonal regenerative markers, Dpysl2 and Gap43. Gene expression indicative of AAM polarization and axonal regeneration were partially recovered by CCL5 injection. These results suggest that hMSC implantation increases Ccl2 and Ccl5, improves locomotor activity, enhances MG/MΦ polarization to AAM, and increases the gene expression of axonal regenerative markers. These functions of hMSCs might be partially mediated by the CCL2/CCR2 and CCL5/CCR5 axes.

Human mesenchymal stem/stromal cells suppress spinal inflammation in mice with contribution of pituitary adenylate cyclase-activating polypeptide (PACAP).

BACKGROUND: Adult human mesenchymal stem/stromal cells (hMSCs) from bone marrow have been reported to exhibit beneficial effects on spinal cord injury (SCI). A neuropeptide, pituitary adenylate cyclase-activating polypeptide (PACAP) is known to decrease neuronal cell death and inflammatory response after ischemia, SCI, and other neuronal disorders. Recently, we found that expression of the gene for mouse PACAP (Adcyap1) was greater in animals receiving hMSCs with neural injury such as ischemia. However, the association of PACAP with hMSCs to protect nerve cells against neural injuries is still unclear. METHODS: Wild-type and PACAP-gene-deficient (Adcyap1 (+/-) ) mice were subjected to spinal cord transection, and hMSCs (5 × 10(5) cells) were injected into the intervertebral spinal cord on day 1 post-operation (p.o.). Locomotor activity, injury volume, retention of hMSCs, mouse and human cytokine genes (which contribute to macrophage (MΦ) and microglial activation), and Adcyap1 were evaluated. RESULTS: hMSCs injected into wild-type mice improved locomotor activity and injury volume compared with vehicle-treated mice. In contrast, non-viable hMSCs injected into wild-type mice, and viable hMSCs injected into Adcyap1 (+/-) mice, did not. Wild-type mice injected with hMSCs exhibited increased Adcyap1 expression, and observed PACAP immunoreaction in neuron-like cells. Gene expression levels for IL-1, tumor necrosis factor α (TNFα), interleukin-10 (IL-10), and transforming growth factor ß (TGFß) decreased, while that for interleukin-4 (IL-4) increased, in hMSC-injected wild-type mice. In contrast, IL-1, TGFß, and IL-4 gene expression levels were all abolished in hMSC-injected Adcyap1 (+/-) mice on day 7 post-operation. Moreover, the mice-implanted hMSCs increased an alternative activating macrophage/microglial marker, arginase activity. The human gene profile indicated that hMSCs upregulated the gene of IL-4 and growth factors which were reported to enhance Adcyap1 expression. Finally, we demonstrated that hMSCs express human ADCYAP1 and its receptor gene after the inflammation-related interferon-γ (IFNγ) in vitro. CONCLUSIONS: These results suggest that hMSCs attenuate the deleterious effects of SCI by reducing associated inflammatory responses and enhancing IL-4 production. This effect could be mediated in part by cell-cell cross-talk involving the neuropeptide PACAP.

PACAP38 suppresses cortical damage in mice with traumatic brain injury by enhancing antioxidant activity.

The production of reactive oxygen species (ROS) and the resulting oxidative stress in mice in response to a controlled cortical impact (CCI) are typical exacerbating factors associated with traumatic brain injury (TBI). Pituitary adenylate cyclase-activating polypeptide 38 (PACAP38) is a multifunctional peptide that has been shown to exhibit neuroprotective effects in response to a diverse range of injuries to neuronal cells. We recently reported that PACAP38 might regulate oxidative stress in mice. The aim of the present study was to determine whether PACAP38 exerts neuroprotective effects by regulating oxidative stress in mice with TBI. Reactive oxidative metabolites (ROMs) and biological antioxidant potential (BAP) were measured in male C57Bl/6 mice before and 3, 4, and 24 h after CCI. PACAP38 was administered intravenously immediately following CCI, and immunostaining for the oxidative stress indicator nitrotyrosine (NT), and for neuronal death as an indicator of the area affected by TBI, was measured 24 h later. Western blot experiments to determine antioxidant activity [as indicated by superoxide dismutase-2 (SOD-2) and glutathione peroxidase 1 (GPx-1)] in the neocortical region were also performed 3 h post-CCI. Results showed that plasma BAP and ROM levels were dramatically increased 3 h after CCI. PACAP38 suppressed the extent of TBI and NT-positive regions 24 h after CCI, and increased SOD-2 and GPx-1 levels in both hemispheres. Taken together, these results suggest that increasing antioxidant might be involving in the neuroprotective effect of PACAP38 in mice subjected to a CCI.

The anti-inflammatory property of human bone marrow-derived mesenchymal stem/stromal cells is preserved in late-passage cultures.

Human mesenchymal stem/stromal cells (hMSCs) have been reported to improve neural damage via anti-inflammation and multi-differentiation abilities. Here, we investigated immunosuppression effects of hMSCs by mixed-culturing with interferon-γ (IFNγ) stimulated BV-2 mouse microglial cells. We show that hMSCs decreased nitrite oxide (NO) production from BV-2 cells in cell density dependent manner. Aged hMSCs and peroxisome proliferator-activated receptor-γ (PPARγ) knockdown hMSCs decreased differentiation abilities but maintained NO suppressive function. We finally confirmed NO suppression activities of hMSCs in IFNγ-stimulated primary microglia/macrophages. It suggested that hMSCs significantly modified NO production in activated phagocytes and it might be preserved in late passage cultures.

Therapeutic time window for edaravone treatment of traumatic brain injury in mice.

Traumatic brain injury (TBI) is a major cause of death and disability in young people. No effective therapy is available to ameliorate its damaging effects. Our aim was to investigate the optimal therapeutic time window of edaravone, a free radical scavenger which is currently used in Japan. We also determined the temporal profile of reactive oxygen species (ROS) production, oxidative stress, and neuronal death. Male C57Bl/6 mice were subjected to a controlled cortical impact (CCI). Edaravone (3.0 mg/kg), or vehicle, was administered intravenously at 0, 3, or 6 hours following CCI. The production of superoxide radicals (O2 (∙-)) as a marker of ROS, of nitrotyrosine (NT) as an indicator of oxidative stress, and neuronal death were measured for 24 hours following CCI. Superoxide radical production was clearly evident 3 hours after CCI, with oxidative stress and neuronal cell death becoming apparent after 6 hours. Edaravone administration after CCI resulted in a significant reduction in the injury volume and oxidative stress, particularly at the 3-hour time point. Moreover, the greatest decrease in O2 (∙-) levels was observed when edaravone was administered 3 hours following CCI. These findings suggest that edaravone could prove clinically useful to ameliorate the devastating effects of TBI.

Establishment and characterization of primary adult microglial culture in mice.

Microglial cells account for approximately 12-15 % of the cells in the central nervous system (CNS). Microglial cells are polarized by pathological stimuli such as cytokines, chemokines, and growth factors, and play important roles in the deterioration and repair of the CNS. Here, we established cultures of primary microglial cells isolated from the brains of adult C57/BL6 mice using Percoll density gradients. The cells were cultured and stained with antibodies against CD11b, glial fibrillary acidic protein, myelin basic protein and NeuN to determine microglial, astroglial, oligodendroglial, and neuronal cells respectively. Moreover, the cells were exposed to interferon-γ (IFNγ) plus interleukin-1ß (IL-1ß) or IL-4 for 24 h to demonstrate the activating phenotypes with inducible nitric oxide synthase (iNOS), Ym1, and Iba-1 immunoblotting. At least 95 % of the cultured cells were CD11b-positive and -negative for astroglial, neuronal, and oligodendrocyte markers. IFNγ plus IL-1ß treatment resulted in classical activation, which was represented by an increase in iNOS. The cells also displayed alternative activation, which increased Ym1 when treated with IL-4. The present study indicates that the microglial cells isolated as described here are a useful tool for elucidating adult microglial function.

Edaravone increases regional cerebral blood flow after traumatic brain injury in mice.

Traumatic brain injury (TBI) is a major cause of preventable death and serious morbidity, with subsequent low cerebral blood flow (CBF) considered to be associated with poor prognosis. In the present study, we demonstrated the effect of the free radical scavenger edaravone on regional CBF (rCBF) after TBI. Male mice (C57/BL6) were subjected to TBI using a controlled cortical impactor device. Immediately after TBI, the animals were intravenously administered 3.0 mg/kg of edaravone or a vehicle saline solution. Two-dimensional rCBF images were acquired before and 24 h post-TBI, and were quantified in the ipsilateral and contralateral hemispheres (n = 5 animals per group). CBF in the vehicle-treated animals decreased broadly over the ipsilateral hemisphere, with the region of low rCBF spreading from the frontal cortex to the occipital lobe. The zone of lowest rCBF matched that of the contusion area. The mean rCBF at 24 h for a defined elliptical region between the bregma and lambda was 73.7 ± 5.8 %. In comparison, the reduction of rCBF in edaravone-treated animals was significantly attenuated (93.4 ± 5.7 %, p < 0.05). The edaravone-treated animals also exhibited higher rCBF in the contralateral hemisphere compared with that seen in -vehicle-treated animals. It is suggested that edaravone reduces neuronal damage by scavenging reactive oxygen species (ROS) and by maintaining intact the autoregulation of the cerebral vasculature.

Accumulation of autofluorescent storage material in brain is accelerated by ischemia in chloride channel 3 gene-deficient mice.

Autofluorescent storage material (ASM) is an aging pigment that accumulates during the normal course of senescence. Although the role of ASM has yet to be fully elucidated, ASM has been implicated in age-related neurodegeneration. In this study, we determined the level of ASM in chloride channel 3 (ClC-3) gene-deficient (KO) mice both in response to aging and following mild global ischemia. To understand the mechanism of action of the ASM, mice subjected to ischemia were treated with the cyclooxygenase (COX) inhibitor indomethacin or with the noncompetitive glutamate receptor antagonist MK-801. ClC-3 KO mice displayed age-related neurodegeneration of the neocortex as well as the hippocampus. The cortical layers in particular granular layers became thinner with aging. ASM accumulated in the brains of ClC-3 KO mice was increased seven- to 50-fold over that observed in the corresponding regions of their wild-type littermates. Young wild-type mice survived longer than age-matched ClC-3 KO mice after permanent global ischemia. However, in the case of older animals, the survival curves were similar. The ASM also increased four- to fivefold 10 days after mild global ischemia, an effect that was suppressed by treatment with indomethacin and MK-801. These results suggest that temporary ischemia might trigger a process similar to aging in the brain, mimicking the effect of age-related neurodegenerative diseases.

Interleukin-1 participates in the classical and alternative activation of microglia/macrophages after spinal cord injury.

BACKGROUND: Microglia and macrophages (MG/MΦ) have a diverse range of functions depending on unique cytokine stimuli, and contribute to neural cell death, repair, and remodeling during central nervous system diseases. While IL-1 has been shown to exacerbate inflammation, it has also been recognized to enhance neuroregeneration. We determined the activating phenotype of MG/MΦ and the impact of IL-1 in an in vivo spinal cord injury (SCI) model of IL-1 knock-out (KO) mice. Moreover, we demonstrated the contribution of IL-1 to both the classical and alternative activation of MG in vitro using an adult MG primary culture. METHODS: SCI was induced by transection of the spinal cord between the T9 and T10 vertebra in wild-type and IL-1 KO mice. Locomotor activity was monitored and lesion size was determined for 14 days. TNFα and Ym1 levels were monitored to determine the MG/MΦ activating phenotype. Primary cultures of MG were produced from adult mice, and were exposed to IFNγ or IL-4 with and without IL-1ß. Moreover, cultures were exposed to IL-4 and/or IL-13 in the presence and absence of IL-1ß. RESULTS: The locomotor activity and lesion area of IL-1 KO mice improved significantly after SCI compared with wild-type mice. TNFα production was significantly suppressed in IL-1 KO mice. Also, Ym1, an alternative activating MG/MΦ marker, did not increase in IL-1 KO mice, suggesting that IL-1 contributes to both the classical and alternative activation of MG/MΦ. We treated primary MG cultures with IFNγ or IL-4 in the presence and absence of IL-1ß. Increased nitric oxide and TNFα was present in the culture media and increased inducible NO synthase was detected in cell suspensions following co-treatment with IFNγ and IL-1ß. Expression of the alternative activation markers Ym1 and arginase-1 was increased after exposure to IL-4 and further increased after co-treatment with IL-4 and IL-1ß. The phenotype was not observed after exposure of cells to IL-13. CONCLUSIONS: We demonstrate here in in vivo experiments that IL-1 suppressed SCI in a process mediated by the reduction of inflammatory responses. Moreover, we suggest that IL-1 participates in both the classical and alternative activation of MG in in vivo and in vitro systems.