Oligodendrocyte progenitor development from the postnatal rat subventricular zone is regulated by the p75 neurotrophin receptor.

The precise timing of neural progenitor development and the correct balance between proliferation and differentiation are crucial to generating a functional brain. The number, survival, and differentiation of neural progenitors during postnatal neurogenesis and gliogenesis is a highly regulated process. Postnatally, the majority of brain oligodendrocytes are generated from progenitors residing in the subventricular zone (SVZ), the germinal niche surrounding the lateral ventricles. In this study, we demonstrate that the p75 neurotrophin receptor (p75NTR) is highly expressed by OPCs in the postnatal male and female rat SVZ. Whereas the p75NTR is known to initiate apoptotic signaling after brain injury, it is highly expressed by proliferating progenitors in the SVZ, suggesting that it may have a different function during development. Lack of p75NTR reduced progenitor proliferation and caused premature oligodendrocyte differentiation and maturation both in vitro and in vivo, leading to aberrant early myelin formation. Our data reveal a novel role for p75NTR as a rheostat for oligodendrocyte production and maturation during myelin formation in the postnatal rat brain.

Analyzing mouse neural stem cell and progenitor cell proliferation using EdU incorporation and multicolor flow cytometry.

This protocol describes an ex vivo approach to identify and quantify the proportions of proliferating neural stem cells and progenitors of the mouse subventricular zone. It uses ethynyl deoxyuridine (EdU) incorporation to identify dividing cells, combined with multicolor flow cytometry for 4 cell surface antigens to distinguish between 8 phenotypically distinct mouse neural progenitors and stem cells. It has been optimized for wild-type neonatal mice but can be used on mice of any postnatal age. For complete details on the use and execution of this profile, please refer to Kumari et al. (2020).

Oligodendrocyte progenitor proliferation is disinhibited following traumatic brain injury in leukemia inhibitory factor heterozygous mice.

Traumatic brain injury (TBI) is a significant problem that affects over 800,000 children each year. As cell proliferation is disturbed by injury and required for normal brain development, we investigated how a pediatric closed head injury (CHI) would affect the progenitors of the subventricular zone (SVZ). Additionally, we evaluated the contribution of leukemia inhibitory factor (LIF) using germline LIF heterozygous mice (LIF Het), as LIF is an injury-induced cytokine, known to influence neurogenesis and gliogenesis. CHIs were performed on P20 LIF Het and wild-type (WT) mice. Ki-67 immunostaining and stereology revealed that cell proliferation increased ~250% in injured LIF Het mice compared to the 30% increase observed in injured WT mice at 48-hr post-CHI. OLIG2+ cell proliferation increased in the SVZ and white matter of LIF Het injured mice at 48-hr recovery. Using an 8-color flow cytometry panel, the proliferation of three distinct multipotential progenitors and early oligodendrocyte progenitor cell proliferation was significantly increased in LIF Het injured mice compared to WT injured mice. Supporting its cytostatic function, LIF decreased neurosphere progenitor and oligodendrocyte progenitor cell proliferation compared to controls. In highly enriched mouse oligodendrocyte progenitor cell cultures, LIF increased phospho-protein kinase B after 20 min and increased phospho-S6 ribosomal protein at 20 and 40 min of exposure, which are downstream targets of the mammalian target of rapamycin pathway. Altogether, our data provide new insights into the regulatory role of LIF in suppressing neural progenitor cell proliferation and, in particular, oligodendrocyte progenitor cell proliferation after a mild TBI.

Leukemia Inhibitory Factor Is Required for Subventricular Zone Astrocyte Progenitor Proliferation and for Prokineticin-2 Production after a Closed Head Injury in Mice.

Astrogliosis is one of the hallmarks of brain injury, and after a mild injury activated astrocytes subserve neuroprotective and pro-regenerative functions. We previously found that the astroglial response to closed head injury (CHI) was blunted in mice that were haplodeficient in leukemia inhibitory factor (LIF); therefore, the goal of these studies was to determine if the delayed astrogliosis was due to decreased recruitment of subventricular zone (SVZ) progenitors. CHI's were performed on post-natal day 20 on LIF heterozygous (Het) and wild-type (WT) mice. At 48 h post-CHI, astrocyte progenitor proliferation within the SVZ increased ∼250% in WT mice but was reduced by ∼200% in LIF Het mice compared with sham controls. Using neurospheres to model the SVZ, LIF increased the percentage of proliferating astrocyte progenitors by 2-fold compared with controls but had no effect on neural stem cell proliferation. To rule out the involvement of other cytokines, 105 cytokines were analyzed using a multi-plex array and with targeted validation on injured LIF Het versus WT neocortex. Of the cytokines analyzed, only prokineticin-2 (ProK2) required LIF signaling. Correspondingly, LIF-treated neurospheres expressed higher levels of ProK2, the ProK1 and ProK2 receptors (ProKR1 and ProKR2). Using in situ hybridization, ProK2 messenger RNA (mRNA) was most abundant in neocortical neurons and highly expressed within the SVZ. However, in contrast to LIF, ProK2 decreased astrocyte progenitor proliferation 2-fold. Altogether, these data demonstrate that LIF is necessary for astrocyte progenitor proliferation after injury and reveal a new role for LIF as an essential regulator of the neurotrophic factor ProK2.

Bleeding and spotting with the levonorgestrel 13.5 mg intrauterine system: the impact of insertion timing.

OBJECTIVE: To assess the impact of early versus late menstrual cycle insertion on bleeding/spotting in the 90 days following levonorgestrel (LNG) 13.5 mg intrauterine system (IUS) insertion. STUDY DESIGN: In this observational study, participants received a LNG 13.5 mg IUS and provided 90 days of bleeding/spotting data by answering the following daily text: "Have you had no flow (0), spotting (1), or bleeding (2) today?" We dichotomized insertion timing as early (days 1-7 from last menstrual period) and late (remainder of menstrual cycle) and compared bleeding/spotting between the two groups in the 90- and 30-day reference periods. We used multivariate regression methods to study associations between cycle day at insertion, parity, historical bleeding, recent hormonal contraceptive use and bleeding/spotting. RESULTS: In the 90-day dichotomous analysis (n=125), we found no differences in the number of days of bleeding/spotting, bleeding or spotting between the early and late insertion groups. In the 30-day dichotomous analysis (n=131), early insertion was associated with fewer days of bleeding than late insertion (5±3 vs. 7±4 days, p<.01). Recent hormonal contraceptive users experienced fewer days of bleeding than new users (5±4 vs. 7±3 days, p<.01). In the 90- and 30-day regression models, earlier insertion was associated with fewer days of bleeding (p=.02, p=.02). Recent contraceptive use was associated with fewer days of bleeding/spotting (90-day, p=.03) and fewer days of bleeding (30-day, p<.01). Nulliparity was associated with spotting (30-day, p=.04). CONCLUSIONS: Early cycle insertion does not impact 90-day bleeding/spotting. Early cycle insertion and recent hormonal contraceptive use decrease 30-day bleeding. IMPLICATIONS: The LNG 13.5 mg IUS may be inserted throughout the menstrual cycle with small differences in bleeding patterns in the 30 but not the 90 days following insertion. Shared decision making should determine timing of insertion.

Age-Dependent Effects of ALK5 Inhibition and Mechanism of Neuroprotection in Neonatal Hypoxic-Ischemic Brain Injury.

Neonatal encephalopathy due to hypoxic-ischemic (HI) brain injury triggers a wave of neuroinflammatory events attributed to causing the progressive degeneration and functional deficits seen weeks after the initial insult. In a recent set of studies, we evaluated the therapeutic efficacy of a small molecule antagonist for ALK5 (activin-like kinase 5 ), TGF-ß receptor in a rat model of moderate perinatal HI and found significant improvements in neurologic outcomes. Here, we have extended those studies to evaluate the efficacy of delayed TGF-ß receptor antagonism on postnatal day (P) 6 and P9 HI rat pups with and without hypothermia. The ALK5 receptor antagonist SB505124 was administered systemically by osmotic pump beginning 3 days following HI. Extending our earlier data set that showed protection of the hippocampus in P6 pups treated with SB505124, these animals sustained less damage to their hippocampi and had improved performance on the Morris water maze (MWM) when tested on P60 versus vehicle-treated HI animals. By contrast, SB505124 did not improve sensorimotor deficits and exacerbated hippocampal and thalamic volume loss when administered 3 days after HI to P9 pups. SB505124-treated rats injured on P9 tended to perform worse than their vehicle-treated counterparts on MWM, and SB505124 treatment did not preserve hippocampal or thalamic neurons in P9 pups when combined with hypothermia. To elucidate the mechanism whereby ALK5 inhibition reduced neuronal death in the P6 HI model, we assessed levels of autophagy markers in neurons of the neocortex, hippocampus, and thalamus, and in the subcortical white matter, and found that SB505124 increased numbers of autophagosomes and levels of lipidated LC3 (light chain 3), a key protein known to mediate autophagy. Altogether, our results demonstrate that there is a dynamic switch in the CNS response to TGF-ß1 that occurs around P9 in rats where TGF-ß signaling inhibition worsens functional outcomes. This response is similar to the outcome of antagonizing TGF-ß signaling in adult stroke and other CNS disease models. We conclude that attenuating TGF-ß1 signaling will likely be an effective treatment for HI-related encephalopathy in moderately preterm infants, offering protection of the neocortex, hippocampus, and thalamus with enhanced cerebral autophagy contributing to the decrease in the extent of progressive neuronal cell death.