Transnasal transplantation of human induced pluripotent stem cell-derived microglia to the brain of immunocompetent mice.

Microglia are the resident immune cells of the brain, and play essential roles in neuronal development, homeostatic function, and neurodegenerative disease. Human microglia are relatively different from mouse microglia. However, most research on human microglia is performed in vitro, which does not accurately represent microglia characteristics under in vivo conditions. To elucidate the in vivo characteristics of human microglia, methods have been developed to generate and transplant induced pluripotent or embryonic stem cell-derived human microglia into neonatal or adult mouse brains. However, its widespread use remains limited by the technical difficulties of generating human microglia, as well as the need to use immune-deficient mice and conduct invasive surgeries. To address these issues, we developed a simplified method to generate induced pluripotent stem cell-derived human microglia and transplant them into the brain via a transnasal route in immunocompetent mice, in combination with a colony stimulating factor 1 receptor antagonist. We found that human microglia were able to migrate through the cribriform plate to different regions of the brain, proliferate, and become the dominant microglia in a region-specific manner by occupying the vacant niche when exogenous human cytokine is administered, for at least 60 days.

Detection of neuroinflammation before selective neuronal loss appearance after mild focal ischemia using [18F]DPA-714 imaging.

BACKGROUND: Translocator protein (TSPO) imaging can be used to detect neuroinflammation (including microglial activation) after acute cerebral infarction. However, longitudinal changes of TSPO binding after mild ischemia that induces selective neuronal loss (SNL) without acute infarction are not well understood. Here, we performed TSPO imaging with [18F]DPA-714 to determine the time course of neuroinflammation and SNL after mild focal ischemia. RESULTS: Mild focal ischemia was induced by middle cerebral artery occlusion (MCAO) for 20 min. In MCAO rats without acute infarction investigated by 2, 3, 5-triphenyltetrazolium chloride (TTC) staining, in vitro ARG revealed a significant increase of [18F]DPA-714 binding in the ipsilateral striatum compared with that in the contralateral side at 1, 2, 3, and 7 days after MCAO. Increased [18F]DPA-714 binding was observed in the cerebral cortex penumbra, reaching maximal values at 7 days after MCAO. Activation of striatal microglia and astrocytes was observed with immunohistochemistry of ionized calcium binding adaptor molecule 1 (Iba1) and glial fibrillary acidic protein (GFAP) at 2, 3, and 7 days after MCAO. SNL was investigated with Nissl staining and neuronal nuclei (NeuN) immunostaining and observed in the ischemic core region of the striatum on days 3 and 7 after MCAO. We confirmed that total distribution volume of [18F]DPA-714 in the ipsilateral striatum was significantly increased at 2 and 7 days after MCAO using positron emission tomography (PET). CONCLUSIONS: [18F]DPA-714 binding measured with in vitro ARG was increased before SNL appeared, and this change was detected by in vivo PET. These findings suggest that TSPO PET imaging might be useful for detection of neuroinflammation leading to SNL after focal ischemia.

Ibudilast produces anti-allodynic effects at the persistent phase of peripheral or central neuropathic pain in rats: Different inhibitory mechanism on spinal microglia from minocycline and propentofylline.

Microglia exhibit various activation phenotypes in the spinal cord after peripheral nerve injury, and promote neuropathic pain. Ibudilast is a phosphodiesterase inhibitor with anti-inflammatory activity, but its effect on activated microglia in chronic neuropathic pain is poorly understood. We investigated whether ibudilast was effective on established allodynia associated with activated microglial phenotypes in two rat models of peripheral and central neuropathic pain. A single intrathecal injection of ibudilast (25 µg) inhibited established allodynia on days 7-21 after sciatic nerve injury in rats. Repeated injections of ibudilast (25 µg/day) reduced the numbers of phosphorylated p38-positive cells without changing hypertrophic microglia, whereas minocycline (100 µg/day) decreased the numbers of hypertrophic microglia associated with phosphorylated p38 levels in the spinal cord. Gene analysis revealed that minocycline, but not ibudilast, increased the expression of anti-inflammatory cytokine genes Il10 and Tgfß1 in the spinal cord. Propentofylline (100 µg/day) was less effective on microglial phenotypes and established allodynia. Ibudilast inhibited persistent allodynia after the recovery of motor deficits in experimental autoimmune encephalomyelitis rats. Therefore, ibudilast might be effective for chronic neuropathic pain after peripheral and central nerve damage. Ibudilast mediated these effects on activated microglia using a different mechanism compared with minocycline and propentofylline.

Suppression of the acute upregulation of phosphorylated-extracellular regulated kinase in ventral tegmental area by a µ-opioid receptor agonist is related to resistance to rewarding effects in a mouse model of bone cancer.

We investigated the mechanisms underlying the suppression of the rewarding effects of opioids using the femur bone cancer (FBC) mouse model. The rewarding and antinociceptive effects of subcutaneously administered morphine and oxycodone in the FBC model mice were assessed using the conditioned place preference test and the von-Frey test. In FBC mice, antinociceptive doses of morphine (30 mg/kg) and oxycodone (5 mg/kg) did not produce the rewarding effects but excessive doses of morphine (300 mg/kg) and oxycodone (100 mg/kg) did. Western blot analyses revealed a transient and significant increase in phosphorylated-extracellular regulated kinase (p-ERK) levels in ventral tegmental area (VTA) 5 min after the administration of morphine in sham-group. Interestingly, in FBC group, a regular dose of morphine did not increase p-ERK levels but a high dose of morphine caused an increase in p-ERK level 5 min after administration. The rewarding effects of a regular dose of and a high dose of morphine in the sham-operation and FBC model, respectively, were significantly inhibited by the MEK inhibitor. The suppression of p-ERK might result in resistance to these rewarding effects under the conditions of bone cancer.

[11C]PK11195 PET imaging of spinal glial activation after nerve injury in rats.

The role of glial activation has been implicated in the development and persistence of neuropathic pain after nerve injury by recent studies. PK11195 binding to the translocator protein 18kDa (TSPO) has been shown to be enhanced in activated microglia. This study was designed to assess PK11195 imaging in spinal microglia during activation after nerve injury. The development of neuropathic pain was induced by partial sciatic nerve ligation (PSL). PSL rats on days 7 and 14 after nerve injury were subjected to imaging with a small-animal positron emission tomography/computed tomography (PET/CT) scanner using [(11)C]PK11195 to detect spinal microglial activation by means of noninvasive in vivo imaging. Spinal [(3)H]PK11195 autoradiography was performed to confirm the results of [(11)C]PK11195 PET in PSL rats. Quantitative RT-PCR of CD11b and GFAP mRNA, and the immunohistochemistry of Iba1 and GFAP were investigated to detect activated microglia and astrocytes. Mechanical allodynia was observed in the ipsilateral paw of PSL rats from day 3 after nerve injury and stably persisted from days 7 to 14. PET/CT fusion images clearly showed large amounts of accumulation of [(11)C]PK11195 in the lumbar spinal cord on days 7 and 14 after nerve injury. [(11)C]PK11195 enhanced images were restricted to the L3-L6 area of the spinal cord. The standardized uptake value (SUV) of [(11)C]PK11195 was significantly increased in the lumbar spinal cord compared to that of the thoracic region. Increased specific binding of [(11)C]PK11195 to TSPO in the spinal cord of PSL rats was confirmed by competition studies using unlabeled (R, S)-PK11195. Increased [(3)H]PK11195 binding was also observed in the ipsilateral dorsal horn of the L3-L6 spinal cord on days 7 and 14 after nerve injury. CD11b mRNA and Iba1 immunoreactive cells increased significantly on days 7 and 14 after nerve injury by PSL. However, changes in GFAP mRNA and immunoreactivity were slight in the ipsilateral side of PSL rats. In the present study, we showed that glial activation could be quantitatively imaged in the spinal cord of neuropathic pain rats using [(11)C]PK11195 PET, suggesting that high resolution PET using TSPO-specific radioligands might be useful for imaging to assess the role of glial activation, including neuroinflammatory processes, in neuropathic pain patients.

Genetic variations in the gene encoding ELMO1 are associated with susceptibility to diabetic nephropathy.

To search for a gene(s) conferring susceptibility to diabetic nephropathy (DN), we genotyped over 80,000 gene-based single nucleotide polymorphisms (SNPs) in Japanese patients and identified that the engulfment and cell motility 1 gene (ELMO1) was a likely candidate for conferring susceptibility to DN, in view of the significant association of an SNP in this gene with the disease (intron 18+9170, GG vs. GA+AA, chi(2) = 19.9, P = 0.000008; odds ratio 2.67, 95% CI 1.71-4.16). In situ hybridization (ISH) using the kidney of normal and diabetic mice revealed that ELMO1 expression was weakly detectable mainly in tubular and glomerular epithelial cells in normal mouse kidney and was clearly elevated in the kidney of diabetic mice. Subsequent in vitro analysis revealed that ELMO1 expression was elevated in cells cultured under high glucose conditions (25 mmol/l) compared with cells cultured under normal glucose conditions (5.5 mmol/l). Furthermore, we identified that the expression of extracellular matrix protein genes, such as type 1 collagen and fibronectin, were increased in cells that overexpress ELMO1, whereas the expression of matrix metalloproteinases was decreased. These results indicate that ELMO1 is a novel candidate gene that both confers susceptibility to DN and plays an important role in the development and progression of this disease.

Dopaminergic neurons generated from monkey embryonic stem cells function in a Parkinson primate model.

Parkinson disease (PD) is a neurodegenerative disorder characterized by loss of midbrain dopaminergic (DA) neurons. ES cells are currently the most promising donor cell source for cell-replacement therapy in PD. We previously described a strong neuralizing activity present on the surface of stromal cells, named stromal cell-derived inducing activity (SDIA). In this study, we generated neurospheres composed of neural progenitors from monkey ES cells, which are capable of producing large numbers of DA neurons. We demonstrated that FGF20, preferentially expressed in the substantia nigra, acts synergistically with FGF2 to increase the number of DA neurons in ES cell-derived neurospheres. We also analyzed the effect of transplantation of DA neurons generated from monkey ES cells into 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-treated (MPTP-treated) monkeys, a primate model for PD. Behavioral studies and functional imaging revealed that the transplanted cells functioned as DA neurons and attenuated MPTP-induced neurological symptoms.