A better understanding of the mechanisms of microvascular remodeling and maintenance in the brain is critical for studying the neurovascular unit in health and disease. It has been proposed that defects in the ability of the brain to sustain microvascular plasticity may underlie a variety of neuropathological conditions.33, 34
However, it is still unclear to what extent cerebral microvascular remodeling occurs after birth and whether features of this remodeling change throughout life. To bridge this gap in knowledge, we performed, for the first time, a comprehensive in vivo
imaging study of long-term cerebral microvascular plasticity across all stages of life, ranging from the early postnatal period into late senescence.
Our study reveals distinct patterns of microvascular remodeling across the different stages. In the early postnatal brain, we found extensive expansion of the microvascular network by both sprouting, endothelial proliferation, and vessel elongation. We observed that many sprouts consisted of a single tip cell, in contrast to the multicellular sprouting observed in earlier developmental stages and other vascular beds.14
This suggests that at this stage, most sprouting is likely aimed at forming capillaries connecting terminal arterioles and venules. These short distances (20 to 40μ
m) could be spanned by unicellular sprouts without the need for actively proliferating stalk cells in trail. Because most oxygen diffusion in the brain occurs through these smallest blood vessels,35
this final process of vascular refinement must be critical for establishing a microvascular network well-adapted to regional metabolic needs. Given the global organizational changes still underway in the developing postnatal brain, a lack of synchrony in the development of neural and microvascular structures could potentially lead to long-lasting neurologic consequences.
time-lapse imaging revealed that a surprisingly large number of postnatal sprouts are eliminated. This suggests that postnatal refinement of the capillary network occurs by a strategy of redundant vascular sprouting followed by pruning, similar to what is thought to occur during maturation of neural circuits.36
As in neural development, microenvironmental factors, such as neural activity, may play a role in modulating vascular sprouting and pruning. To this end, astrocytes, which closely associate with synapses and vascular sprouts, may modulate the effects of neural activity on vessel growth.
It is noteworthy that most sprouts lacked a visible lumen or had very restricted plasma entry. Shear stress has been shown to prevent endothelial cell sprouting37
Therefore, the lack of blood flow in developing sprouts may maintain their proangiogenic profile. Sprouts appear to be more vulnerable to pruning than connected vessels, which are quite stable. This stability may be triggered, in part, by the establishment of laminar blood flow. Shear stress forces activate a host of endothelial signaling cascades, including VEGF receptor 2 phosphorylation39
and has been implicated in vessel stabilization.40
Extending our in vivo
time-lapse imaging to older age groups showed that in the adult brain, microvascular remodeling declines and the vascular density stabilizes. Despite this, a small number of vascular formations and eliminations continue to take place. This low-level turnover likely leads to a substantial restructuring of the brain vasculature over the lifetime of the mouse. It is possible that this limited plasticity represents the response of the adult vasculature to minor metabolic changes in the brain, while maintaining a neural environment that is fundamentally stable. However, this vascular plasticity appears to be lost over time as aging vessels displayed complete absence of remodeling even over extended imaging intervals. Given the multitude of changes, which occur in the aging brain, it is surprising that under normal conditions, blood vessels are not eliminated. A consensus on the effect of aging on capillary density has not been definitive;26
however, our sensitive methods of tracking individual vessels longitudinally provide reliable evidence for sustained stability during aging. Although active vessel elimination in the aging brain is absent under normal conditions, it is likely that vessel loss occurs during a variety of pathological conditions. Given the lack of plasticity in the aging vasculature, such losses are unlikely to be compensated with the formation of new vessels. The loss of turnover in aging brains might eventually result in a mismatch between energetic supply and demand and may underlie a variety of age-related neural defects.
The persistent plasticity of the young adult (up to 3 months) microvasculature becomes manifest when the brain is exposed to metabolic challenges.29, 30
Using time-lapse imaging, we observed robust vessel formation under low oxygen conditions as compared with normoxic controls in this age group. However, even under these conditions, the response was reduced compared with the remodeling observed in the neonate. Angiogenesis was only initiated after oxygen levels were dropped to a moderately low level (10%) and it ceased by the second week of exposure. Moreover, sprouts observed in both fixed tissue and live imaging were much rarer than in the neonate and changes in total vessel length were minor, suggesting that angiogenesis in adults is relatively limited even after strong stimuli such as chronic hypoxia. Furthermore, few vessels were eliminated during hypoxic exposure, underscoring the tendency of the adult vasculature to stabilize, even under conditions favorable to remodeling. Additional evidence for this fundamental feature comes from the finding that vessels formed under hypoxia were retained even after a prolonged return to normoxia. Though previous studies have suggested that these vessels are eliminated,7
our method unambiguously shows that both new and preexisting vessels are retained following reestablishment of normoxia. Although the newly formed vessels are unlikely to be critical for the proper function of the brain under normoxic conditions, they may prepare the brain for additional bouts of hypoxia.
While some studies report that metabolic challenges cause vessel formation in the adult brain,41
our results using time-lapse imaging suggest that the response to hypoxia ceases by 4 months of age. Discrepancies may arise from differences in technical and quantitative approaches. The failure of mature brains to remodel in response to reduced oxygen parallels the declining plasticity observed at baseline normoxic conditions. The diminishing ability of vessels to compensate for changes in metabolic demands could lead to cellular and synaptic dysfunction and may explain the particular vulnerability of aging brains to conditions of reduced oxygen or blood flow.32