In response to stroke, subpopulations of cortical reactive astrocytes proliferate and express proteins commonly associated with neural stem/progenitor cells, such as Glial Fibrillary Acidic Protein (GFAP) and Nestin. To examine the stem cell-related properties of cortical reactive astrocytes after injury, we generated GFAP-CreER™;tdRFP mice to permanently label reactive astrocytes. We isolated cells from the cortical peri-infarct area 3 days after stroke, and cultured them in neural stem cell medium containing EGF and bFGF. We observed tdRFP-positive neural spheres in culture, suggestive of tdRFP-positive reactive astrocyte-derived neural stem/progenitor cells (Rad-NSCs). Cultured Rad-NSCs self-renewed and differentiated into neurons, astrocytes, and oligodendrocytes. Pharmacological inhibition and conditional knockout mouse studies showed that Presenilin 1 and Notch 1 controlled neural sphere formation by Rad-NSCs after stroke. To examine the self-renewal and differentiation potential of Rad-NSCs in vivo, Rad-NSCs were transplanted into embryonic, neonatal and adult mouse brains. Transplanted Rad-NSCs were observed to persist in the subventricular zone and secondary Rad-NSCs were isolated from the host brain 28 days after transplantation. In contrast with neurogenic post-natal day 4 NSCs and adult NSCs from the subventricular zone, transplanted Rad-NSCs differentiated into astrocytes and oligodendrocytes, but not neurons, demonstrating that Rad-NSCs had restricted differentiation in vivo. Our results indicate that Rad-NSCs are unlikely to be suitable for neuronal replacement in the absence of genetic or epigenetic modification.

Defining the signaling network that controls reactive astrogliosis may provide novel treatment targets for patients with diverse CNS injuries and pathologies. We report that the radial glial cell antigen RC2 identifies the majority of proliferating glial fibrillary acidic protein-positive (GFAP + ) reactive astrocytes after stroke. These cells highly expressed endothelin receptor type B (ETB R ) and Jagged1, a Notch1 receptor ligand. To study signaling in adult reactive astrocytes, we developed a model based on reactive astrocyte-derived neural stem cells isolated from GFAP-CreER-Notch1 conditional knockout (cKO) mice. By loss-and gain-of-function studies and promoter activity assays, we found that Jagged1/Notch1 signaling increased ETB R expression indirectly by raising the level of phosphorylated signal transducer and activator of transcription 3 (STAT3), a previously unidentified EDNRB transcriptional activator. Similar to inducible transgenic GFAP-CreER-Notch1-cKO mice, GFAPCreER-ETB R -cKO mice exhibited a defect in reactive astrocyte proliferation after cerebral ischemia. Our results indicate that the Notch1-STAT3-ETB R axis connects a signaling network that promotes reactive astrocyte proliferation after brain injury.

Tolerance to the anti-nociceptive effects of opioids develops rapidly in animals. In contrast, humans with chronic pain show little or no loss of pain relief in prospective opioid trials of 4-8 weeks duration. Employing the Brief Thermal Sensitization model to induce transient cutaneous secondary hyperalgesia, we tested the hypothesis that opioid analgesic tolerance would develop rapidly. In this outpatient randomized placebo-controlled study, subjects in the MMMMP group received two injections of subcutaneous morphine 6 mg (150 min apart) on Monday-Thursday (total 48 mg over 4 days) and matching saline placebo on Friday. Subjects in the PPPPM group received placebo on Monday-Thursday and morphine (total 12 mg) on Friday. Sixty-one healthy volunteers were enrolled; morphine side effects accounted for all nine non-completions. Compared to the first placebo day, the reduction in the area of secondary hyperalgesia on the first morphine day was significant and robust in both groups. Morphine suppression of the painfulness of skin heating and elevation of the heat pain detection threshold were also significant. During 4 days of twice-daily injections, the decline in anti-hyperalgesic effects of morphine did not reach statistical significance (p=0.06) compared to placebo. Morphine side effects did not correlate with anti-hyperalgesic effects and withdrawal symptoms did not emerge. As 4 days is the threshold for demonstrating analgesic tolerance to twice-daily morphine in animal models, a longer period of opioid exposure in healthy volunteers might be needed to detect analgesic tolerance.

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Background: Delta, Notch, and Scabrous often function together to make different cell types and refine tissue patterns during Drosophila development. Delta is known as the ligand that triggers Notch receptor activity. Scabrous is known to bind Notch and promote Notch activity in response to Delta. It is not known if Scabrous binds Delta or Delta has activity other than its activity as a ligand of Notch. It is very difficult to clearly determine this binding or activity in vivo as all Notch, Delta, and Scabrous activities are required simultaneously or successively in an inter-dependent manner.