In this study, we have used a combination of techniques to identify an anatomically and physiologically distinct microdomain of blood vessels located in the murine SEZ neural stem cell niche. Due to the roles of the microvasculature in regulating adult neurogenesis 
, these observations could have important implications for our understanding of the SEZ neurogenic environment.
Our analysis of vessel structure led us to identify a specialized microvascular domain in the SEZ. This domain was characterized by relatively high vessel density, low vessel tortuosity, and vessels aligned mostly parallel to the ependymal wall. Notably, this domain occupied only the layer of tissue where neurogenesis is known to occur. Interestingly, high vessel density and organized vascular orientation has also been previously observed in the rostral migratory stream 
. Such site-specific specialization is significant because of the effects that vessel structure can have on blood flow and hemodynamics, both of which are major regulators of the EC transcriptome 
. The unique structure we observed in the SEZ vessel bed is thus likely to have a major impact on the secretion of angiocrine signals by ECs, and thereby a major impact on the microenvironment.
We were also able to identify physiological differences between the distinct microvascular domains in the SEZ and striatum. To do this, we used the established technique of microsphere deposition 
to estimate rates of blood flow in each region. These measurements demonstrated that blood flow depended on distance from the ependymal wall in a way that mirrored what we observed for vessel morphology. Specifically, while our measurements of blood flow in the striatum were similar to published estimates of blood flow in the striatum and cerebral cortex 
, our measurements in the SEZ were significantly lower, with a mean of 6 mL/min/100 g. This low measurement of perfusion in a region of relatively high vessel density suggests that blood flow in the SEZ is limited somewhere further upstream in the vascular tree. These results support the idea that the different microvascular domains in the SEZ and striatum differ not only in structure but also in function. Furthermore, this reveals low-flow conditions within the SEZ that likely have important implications for the niche environment. There is evidence that other adult stem cell niche environments also receive low levels of blood flow 
, and it has been hypothesized that this may be an important means of promoting stem cell quiescence in the adult. Consistent with this, others have shown that global changes in cerebral blood flow can perturb SEZ neurogenesis, even in the absence of ischemia 
. Disruption of the low flow conditions in the SEZ by distal ischemic infarcts could therefore be one mechanism by which stroke is able to non-locally induce compensatory neural stem cell proliferation in the SEZ 
Finally, we concluded our study of microvascular physiology in the SEZ by testing for hypoxia. Hypoxia and redox status have been implicated as regulators of stem cell function in a variety of different stem cell types, including neural stem cells 
. Accordingly, we wanted to assess if functional hypoxia was detectable in the SEZ. We did this using Hypoxyprobe-1, a molecular tracer of pronounced hypoxia (pO2
<10 mm Hg) 
, which we validated using Hif1α as a second marker for hypoxia. With these techniques, we were unable to detect hypoxia either in NSCs or globally throughout the SEZ, consistent with a previous study 
. However, we did observe hypoxia in the ependymal layer that lines the ventricles. This is potentially due both to the region’s low blood perfusion, and its proximity to the avascular ventricle. The ependymal cells that make up this layer help maintain the structural integrity of the niche, and serve as an important source of paracrine signals that regulate neurogenesis in the SEZ microenvironment 
. Furthermore, although ependymal cells are not thought to be bona fide
stem cells, there is some controversial evidence that they are capable of reentering the cell cycle to produce neurons 
. Interestingly, recent work has also shown that in the spinal cord, the ependymal cells actually are stem cells 
, and that proliferation and differentiation in these cells is regulated by hypoxia 
. Future work will be needed to determine whether the hypoxia we observed in SEZ ependymal cells might play an analagous role in regulating paracrine signaling or cell cycle reentry.
We also identified a population of non-ependymal cells in the SEZ that exhibited high levels of hypoxia. These cells were consistently seen in the SEZ, as well as in the striatum, but only rarely in the cerebral cortex. Additionally, this cell population appeared to be exclusively composed of differentiated neurons, based on their expression of neuronal markers and consistent morphology. Since the neurons identified by Hypoxyprobe-1 were in well-vascularized regions and always found in isolation, it is possible that the cause of hypoxia in these cells is cell-intrinsic. Elevated cell metabolism is capable of depleting intracellular oxygen levels 
, and so one potential explanation is that these cells are unusually metabolically active as compared to their neighbors. Another possibility is that these hypoxic cells are oxygen-sensing neurons that help regulate the response of the central nervous system to global changes in oxygen levels 
. However, future studies will be necessary to distinguish between these possibilities and define the function of these cells.
In summary, this work helps elucidate the contributions made by blood vessels, blood flow, and hypoxia to the specialized microenvironment of the murine neural stem cell niche. We have identified a specialized microvascular domain specific to the SEZ defined by unique vessel architecture and low rates of blood flow. Furthermore, we have shown that the SEZ exhibits hypoxic conditions within the ependymal layer and in a subset of resident neurons. These findings further implicate the vasculature as an important determinant of the specialized environment of the SEZ, and highlight important questions about the role of vessel physiology in the adult neural stem cell niche. To answer these questions, learning more about the close relationship between vessel structure and function, and how this relationship is regulated in vivo, will be essential.