Human mesenchymal stromal cells (hMSCs) were injected into the hippocampus of adult mice 1 day after transient global ischemia. The hMSCs both improved neurologic function and markedly decreased neuronal cell death of the hippocampus. Microarray assays indicated that ischemia up-regulated 586 mouse genes. The hMSCs persisted for <7 days, but they down-regulated >10% of the ischemia-induced genes, most of which were involved in inflammatory and immune responses. The hMSCs also upregulated three mouse genes, including the neuroprotective gene Ym1 that is expressed by activated microglia/macrophages. In addition, the transcriptomes of the hMSC changed with upregulation of 170 human genes and down-regulation of 54 human genes. Protein assays of the hippocampus demonstrated increased expression in microglia/macrophages of Ym1, the cell survival factor insulin-like growth factor 1, galectin-3, cytokines reflective of a type 2 T cell immune bias, and the major histocompatibility complex II. The observed beneficial effects of hMSCs were largely explained by their modulation of inflammatory and immune responses, apparently by alternative activation of microglia and/or macrophages.inflammation ͉ mesenchymal stromal cells ͉ microglia ͉ mesenchymal stem cells O bservations in rodent and primate models suggest that a potential therapy for ischemia of the central nervous system is the administration of the adult stem/progenitor cells from bone marrow referred to as mesenchymal stem cells or multipotent mesenchymal stromal cells (MSCs) (1-3). Administration of MSCs also produced beneficial effects in animal models for neurodegenerative diseases, such as Parkinson's disease, experimental autoimmune encephalomyelitis, and amyotrophic lateral sclerosis (2-5). MSCs initially attracted interest for their ability to differentiate into multiple cellular phenotypes in culture and in vivo (1-7). However, recent observations indicate that only small numbers of the cells engraft into most injured tissues, and they disappear quickly (2-5, 8-10). When human MSCs (hMSCs) were injected into the dentate gyrus (DG) of the hippocampus in adult immunodeficient (ID) mice, most of the cells disappeared within 1 week, but they enhanced proliferation, migration, and neural differentiation of the endogenous neural stem cells (8). These and related observations have focused attention on the paracrine effects of MSCs (2, 3, 11). However, it has not been established whether the beneficial effects of MSCs in ischemic models of brain injury are explained by enhanced neurogenesis (8) or by neuroprotection.Experiments here were performed in a mouse model of global ischemia to assess the neuroprotective effects of hMSCs. Administration of hMSCs 1 day after transient common carotid artery occlusion (tCCAO) improved neurologic function and decreased the delayed neuronal cell death of the hippocampus. Surveys with microarrays indicated that the hMSCs decreased expression of many of the mouse genes that were induced by ischemia and that were involved in inflamma...

Pituitary adenylate cyclase-activating polypeptide (PACAP) has been reported to decrease ischemic neuronal damage and increase IL-6 secretion in rats. However, the mechanisms underlying neuroprotection are still to be fully elucidated. The present study was designed to investigate the role played by PACAP and IL-6 in mediating neuroprotection after ischemia in a null mouse. Infarct volume, neurological deficits, and cytochrome c in cytoplasm were higher in PACAP ؉/؊ and PACAP ؊/؊ mice than in PACAP ؉/؉ animals after focal ischemia, although the severity of response was ameliorated by the injection of PACAP38. A decrease in mitochondrial bcl-2 was also accentuated in PACAP ؉/؊ and PACAP ؊/؊ mice, but the decrease could be prevented by PACAP38 injection. PACAP receptor 1 (PAC1R) immunoreactivity was colocalized with IL-6 immunoreactivity in neurons, although the intensity of IL-6 immunoreactivity in PACAP ؉/؊ mice was less than that in PACAP ؉/؉ animals. IL-6 levels increased in response to PACAP38 injection, an effect that was canceled by cotreatment with the PAC1R antagonist. However, unlike in wild-type controls, PACAP38 treatment did not reduce the infarction in IL-6 null mice. To clarify the signaling pathway associated with the activity of PACAP and IL-6, phosphorylated STAT (signal transducer and activator of transcription) 3, ERK (extracellular signal-regulated kinase), and AKT levels were examined in PACAP ؉/؊ and IL-6 null mice after ischemia. Lower levels of pSTAT3 and pERK were observed in the PACAP ؉/؊ mice, whereas a reduction in pSTAT3 was recorded in the IL-6 null mice. These results suggest that PACAP prevents neuronal cell death after ischemia via a signaling mechanism involving IL-6.bcl-2 ͉ extracellular signal-regulated kinase ͉ ischemia ͉ pituitary adenylate cyclase-activating polypeptide-specific receptor ͉ signal transducer and activator of transcription 3

BackgroundWe hypothesized that gp91phox (NOX2), a subunit of NADPH oxidase, generates superoxide anion (O2-) and has a major causative role in traumatic brain injury (TBI). To evaluate the functional role of gp91phox and reactive oxygen species (ROS) on TBI, we carried out controlled cortical impact in gp91phox knockout mice (gp91phox-/-). We also used a microglial cell line to determine the activated cell phenotype that contributes to gp91phox generation.MethodsUnilateral TBI was induced in gp91phox-/- and wild-type (Wt) mice (C57/B6J) (25-30 g). The expression and roles of gp91phox after TBI were investigated using immunoblotting and staining techniques. Levels of O2- and peroxynitrite were determined in situ in the mouse brain. The activated phenotype in microglia that expressed gp91phox was determined in a microglial cell line, BV-2, in the presence of IFNγ or IL-4.ResultsGp91phox expression increased mainly in amoeboid-shaped microglial cells of the ipsilateral hemisphere of Wt mice after TBI. The contusion area, number of TUNEL-positive cells, and amount of O2- and peroxynitrite metabolites produced were less in gp91phox-/- mice than in Wt. In the presence of IFNγ, BV-2 cells had increased inducible nitric oxide synthase and nitric oxide levels, consistent with a classical activated phenotype, and drastically increased expression of gp91phox.ConclusionsClassical activated microglia promote ROS formation through gp91phox and have an important role in brain damage following TBI. Modulating gp91phox and gp91phox -derived ROS may provide a new therapeutic strategy in combating post-traumatic brain injury.

Pituitary adenylate cyclase-activating polypeptide (PACAP) is a pleiotropic neuropeptide that belongs to the secretin/glucagon/vasoactive intestinal peptide (VIP) family. PACAP prevents ischemic delayed neuronal cell death (apoptosis) in the hippocampus. PACAP inhibits the activity of the mitogen-activated protein kinase (MAPK) family, especially JNK/SAPK and p38, thereby protecting against apoptotic cell death. After the ischemia-reperfusion, both pyramidal cells and astrocytes increased their expression of the PACAP receptor (PAC1-R). Reactive astrocytes increased their expression of PAC1-R, released interleukin-6 (IL-6) that is a proinflammatory cytokine with both differentiation and growth-promoting effects for a variety of target cell types, and thereby protected neurons from apoptosis. These results suggest that PACAP itself and PACAP-stimulated secretion of IL-6 synergistically inhibit apoptotic cell death in the hippocampus. The PAC1-R is expressed in the neuroepithelial cells from early developmental stages and in various brain regions during development. We have recently found that PACAP, at physiological concentrations, induces differentiation of mouse neural stem cells into astrocytes. Neural stem cells were prepared from the telencephalon of mouse embryos and cultured with basic fibroblast growth factor. The PAC1-R immunoreactivity was demonstrated in the neural stem cells. When neural stem cells were exposed to PACAP, about half of these cells showed glial fibrillary acidic protein (GFAP) immunoreactivity. This phenomenon was significantly antagonized by a PAC1-R antagonist (PACAP6-38), indicating that PACAP induces differentiation of neural stem cell into astrocytes. Other our physiological studies have demonstrated that PACAP acts on PAC1-R in mouse neural stem cells and its signal is transmitted to the PAC1-R-coupled G protein Gq but not to Gs. These findings strongly suggest that PACAP plays very important roles in neuroprotection in adult brain as well as astrocyte differentiation during development.

We used lipopolysaccharide (LPS) to activate microglia that play an important role in the brain immune system. LPS injected into the rat hippocampus CA1 region activated microglial cells resulting in an increased production of interleukin (IL)-1beta and tumor necrosis factor (TNF)-alpha in the hippocampus during the initial stage of treatment. Immunostaining for IL-1beta was increased at 6 hr after LPS injection. IL-1beta-immunopositive cells were co-localized with immunostaining for CD11b. Subacute treatment with LPS by the same route for 5 days caused long-term activation of microglia and induced learning and memory deficits in animals when examined with a step-through passive avoidance test, but histochemical analysis showed that neuronal cell death was not observed under these experimental conditions. The increased expression of the heme oxygenase-1 (HO-1) gene, an oxidative stress maker, was observed. However, the genetic expression of brain-derived neurotrophic factor (BDNF) and its receptor, TrkB, decreased during the course of LPS treatment. We found decreases in [3H]MK801 binding in the hippocampus CA1 region by LPS-treatment for 5 days. The data shows that glutamatergic transmission was attenuated in the LPS-treated rats. These results suggest that long-term activation of microglia induced by LPS results in a decrease of glutamatergic transmission that leads to learning and memory deficits without neuronal cell death. The physiologic significance of these findings is discussed.

BackgroundMicroglia 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.MethodsSCI 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-1β. Moreover, cultures were exposed to IL-4 and/or IL-13 in the presence and absence of IL-1β.ResultsThe 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-1β. 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-1β. 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-1β. The phenotype was not observed after exposure of cells to IL-13.ConclusionsWe 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.

Dry eye syndrome is caused by a reduction in the volume or quality of tears. Here, we show that pituitary adenylate cyclase-activating polypeptide (PACAP)-null mice develop dry eye-like symptoms such as corneal keratinization and tear reduction. PACAP immunoreactivity is co-localized with a neuronal marker, and PACAP receptor (PAC1-R) immunoreactivity is observed in mouse infraorbital lacrimal gland acinar cells. PACAP eye drops stimulate tear secretion and increase cAMP and phosphorylated (p)-protein kinase A levels in the infraorbital lacrimal glands that could be inhibited by pre-treatment with a PAC1-R antagonist or an adenylate cyclase inhibitor. Moreover, these eye drops suppress corneal keratinization in PACAP-null mice. PACAP eye drops increase aquaporin 5 (AQP5) levels in the membrane and pAQP5 levels in the infraorbital lacrimal glands. AQP5 siRNA treatment of the infraorbital lacrimal gland attenuates PACAP-induced tear secretion. Based on these results, PACAP might be clinically useful to treat dry eye disorder.

Pituitary adenylate cyclase-activating polypeptide (PACAP) is a neuropeptide that was first isolated from an ovine hypothalamus in 1989. Since its discovery, more than 2,000 papers have reported on the tissue and cellular distribution and functional significance of PACAP. A number of papers have reported that PACAP but not the vasoactive intestinal peptide suppressed neuronal cell death or decreased infarct volume after global and focal ischemia in rodents, even if PACAP was administered several hours after ischemia induction. In addition, recent studies using PACAP gene-deficient mice demonstrated that endogenous PACAP also contributes greatly to neuroprotection similarly to exogenously administered PACAP. The studies suggest that neuroprotection by PACAP might extend the therapeutic time window for treatment of ischemia-related conditions, such as stroke. This review summarizes the effects of PACAP on ischemic neuronal cell death, and the mechanism clarified in vivo ischemic studies. In addition, the prospective mechanism of PACAP on ischemic neuroprotection from in vitro neuronal and neuronal-like cell cultures with injured stress model is reviewed. Finally, the development of PACAP and/or receptor agonists for human therapy is discussed.