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B.S. 2004, Villanova University
Thesis Advisor: Steven Levison, Ph.D.
Department: Neurology and Neurosciences
Cancer Center G 1196
Tuesday, April 21, 2009
Neonatal hypoxia-ischemia (H/I) is the most common and possibly the most devastating neurological injury to affect the newborn infant. The incidence rate is upwards of 2-3 per 1000 term births and is even higher in premature infants. While there have been many recent advances in neonatal critical care, these rates remain quite high and those infants that survive often develop a range of neurological and psychological problems. Unfortunately, most of these H/I insults occur in utero, therefore, preventative measures are limited, and few clinical protocols have been successful when applied after the insult in decreasing the resulting injury. Interestingly, the immature brain has the capacity to regenerate after injury, as observed by the expansion of its endogenous neural stem/progenitor cell pool in the subventricular zone (SVZ), but the extent of regeneration is limited. Therefore, the rationale for my research has been to better understand the mechanisms responsible for the SVZ response to injury and to determine why this regenerative response is inadequate. The goals of this thesis were to: 1) optimize an in vitro system to study the glial progenitors of the neonatal SVZ; 2) study the roles that specific growth factors, including VEGF A, VEGF C, EGF, LIF and TGF¥â1 play in gliogenesis in the neonatal SVZ; and 3) test the hypothesis that altered cytokine production after neonatal H/I shifts the generation of SVZ glial progenitors to astrocytes instead of oligodendrocytes.
In this thesis, I show that we can faithfully model the glial progenitors of the neonatal SVZ in vitro by propagating neonatal brain precursors in hormone and growth factor supplemented serum-free medium. These progenitors grew as non-adherent spheres and when differentiated it was apparent that there were subclasses of progenitors: astrocyte lineage, oligodendrocyte lineage and a small percentage of a bipotential cells that were capable of producing both astrocytes and oligodendrocytes. These precursor types were present in the same ratio as exist within the neonatal SVZ, supporting the conclusion that these ¡°gliospheres¡± are an appropriate model for the heterogeneous glial progenitors of the neonatal SVZ.
The addition of specific growth factors altered the differentiation of different glial precursors. For example, VEGF A preferentially induced the production of astrocytes, both from progenitors in the gliospheres, as well as from lineage-restricted precursors. However, the addition of VEGF C promoted oligodendrocyte production from both gliosphere precursors and oligodendrocyte progenitors. These results were likely due to their mitogenic effects, as VEGF A induced BrdU incorporation specifically into immature astrocytes whereas VEGF C induced BrdU incorporation specifically into oligodendrocytes progenitors. The combination of VEGF A and VEGF C were additive for astrocyte proliferation from gliosphere progenitors as well as for immature astrocyte precursors. Interestingly, the addition of VEGF A significantly dampened proliferation of oligodendrocyte progenitors in the presence of VEGF C.
I also examined the complex interactions of growth factors induced after H/I. These studies showed that EGF, LIF and TGF¥â1 cooperate in vitro to promote the proliferation of astrocyte precursors within the gliospheres. Further analyses revealed that the TGF¥â1 receptor, ALK5 increased SMAD 2/3 activation, and the combination of TGF©¬1, EGF and LIF synergistically stimulated STAT3 phosphorylation. Pharmacologically inhibiting the ALK5 receptor antagonized the cytokine-induced astrocyte proliferation. To determine whether this finding was relevant for neonatal H/I, I administered an ALK5 antagonist to rat pups beginning immediately after H/I and evaluated the extent of astrogliosis. The ALK5 antagonist SB505124, indeed decreased the extent of astrogliosis in the damaged brain after H/I, with the largest effect seen on the SVZ. These results support the conclusion that the ALK5 receptor is a potential target for future therapeutic interventions.
Finally, my investigations show that the astrogliosis observed in neonatal white matter after injury is not simply due to the proliferation of locally residing astrocytes, but is also due to aberrant specification of glial progenitors of the SVZ. I used replication-deficient retroviruses to trace the fate of SVZ-derived glial progenitors and found that most of the cells in the P8 rat SVZ were destined to populate the white matter. Interestingly, there was a significant decrease in the number of SVZ-derived glial cells produced in the injured brain. Importantly, I found that there was a shift in the types of glia produced by the SVZ after H/I with over 80% becoming white matter oligodendrocytes in the uninjured brain and less than 40% becoming oligodendrocytes in the damaged brain. Conversely, there was almost a doubling of SVZ progenitors producing astrocytes in the injured brain compared to the uninjured brain.
Altogether, the data in this thesis demonstrate that H/I adversely affects gliogenesis in the newborn brain and they emphasize the important roles that growth factors play in recovery from white matter injury. In addition, this thesis demonstrates that manipulating aberrant growth factor signaling, such as pharmacologically inhibiting the ALK5 receptor, may be a feasible intervention to restore the normal sequence of glial cell production in the neonatal brain after injury.