Fetal Cerebrovascular Maturation: Effects of Hypoxia.

The human cerebral vasculature originates in the fourth week of gestation and continues to expand and diversify well into the first few years of postnatal life. A key feature of this growth is smooth muscle differentiation, whereby smooth muscle cells within cerebral arteries transform from migratory to proliferative to synthetic and finally to contractile phenotypes. These phenotypic transformations can be reversed by pathophysiological perturbations such as hypoxia, which causes loss of contractile capacity in immature cerebral arteries. In turn, loss of contractility affects all whole-brain cerebrovascular responses, including those involved in flow-metabolism coupling, vasodilatory responses to acute hypoxia and hypercapnia, cerebral autoregulation, and reactivity to activation of perivascular nerves. Future strategies to minimize cerebral injury following hypoxia-ischemic insults in the immature brain might benefit by targeting treatments to preserve and promote contractile differentiation in the fetal cerebrovasculature. This could potentially be achieved through inhibition of receptor tyrosine kinase-mediated growth factors, such as vascular endothelial growth factor and platelet-derived growth factor, which are mobilized by hypoxic and ischemic injury and which facilitate contractile dedifferentiation. Interruption of the effects of other vascular mitogens, such as endothelin and angiotensin-II, and even some miRNA species, also could be beneficial. Future experimental work that addresses these possibilities offers promise to improve current clinical management of neonates who have suffered and survived hypoxic, ischemic, asphyxic, or inflammatory cerebrovascular insults.

Neuroprotective effects of TRPA1 channels in the cerebral endothelium following ischemic stroke.

Hypoxia and ischemia are linked to oxidative stress, which can activate the oxidant-sensitive transient receptor potential ankyrin 1 (TRPA1) channel in cerebral artery endothelial cells, leading to vasodilation. We hypothesized that TRPA1 channels in endothelial cells are activated by hypoxia-derived reactive oxygen species, leading to cerebral artery dilation and reduced ischemic damage. Using isolated cerebral arteries expressing a Ca2+ biosensor in endothelial cells, we show that 4-hydroxynonenal and hypoxia increased TRPA1 activity, detected as TRPA1 sparklets. TRPA1 activity during hypoxia was blocked by antioxidants and by TRPA1 antagonism. Hypoxia caused dilation of cerebral arteries, which was disrupted by antioxidants, TRPA1 blockade and by endothelial cell-specific Trpa1 deletion (Trpa1 ecKO mice). Loss of TRPA1 channels in endothelial cells increased cerebral infarcts, whereas TRPA1 activation with cinnamaldehyde reduced infarct in wildtype, but not Trpa1 ecKO, mice. These data suggest that endothelial TRPA1 channels are sensors of hypoxia leading to vasodilation, thereby reducing ischemic damage.

Cerebral Vein Malformations Result from Loss of Twist1 Expression and BMP Signaling from Skull Progenitor Cells and Dura.

Dural cerebral veins (CV) are required for cerebrospinal fluid reabsorption and brain homeostasis, but mechanisms that regulate their growth and remodeling are unknown. We report molecular and cellular processes that regulate dural CV development in mammals and describe venous malformations in humans with craniosynostosis and TWIST1 mutations that are recapitulated in mouse models. Surprisingly, Twist1 is dispensable in endothelial cells but required for specification of osteoprogenitor cells that differentiate into preosteoblasts that produce bone morphogenetic proteins (BMPs). Inactivation of Bmp2 and Bmp4 in preosteoblasts and periosteal dura causes skull and CV malformations, similar to humans harboring TWIST1 mutations. Notably, arterial development appears normal, suggesting that morphogens from the skull and dura establish optimal venous networks independent from arterial influences. Collectively, our work establishes a paradigm whereby CV malformations result from primary or secondary loss of paracrine BMP signaling from preosteoblasts and dura, highlighting unique cellular interactions that influence tissue-specific angiogenesis in mammals.

Quantitative comparison of cerebral artery development in metatherians and monotremes with non-human eutherians.

A quantitative comparison of the internal diameters of cerebral feeder arteries (internal carotid and vertebral) and the aorta in developing non-human eutherians, metatherians and monotremes has been made, with the aim of determining if there are differences in cerebral arterial flow between the three infraclasses of mammals such as might reflect differences in metabolism of the developing brain. There were no significant differences between eutherians and metatherians in the internal radius of the aorta or the thickness of the aortic wall, but aortic internal radius was significantly smaller in developing monotremes than therians at the < 10 mm body length range. Aortic thickness in the developing monotremes also rose at a slower rate relative to body length than in metatherians or eutherians. The sums of the internal calibres of the internal carotid and vertebral arteries were significantly lower in metatherians as a group and monotremes compared with non-human eutherians at body lengths up to 20 mm and in metatherians at > 20 mm body length. The internal calibre of the internal carotids relative to the sum of all cerebral feeder arteries was also significantly lower in monotremes at < 10 mm body length compared with eutherians. It was noted that dasyurids differed from other metatherians in several measures of cerebral arterial calibre and aortic internal calibre. The findings suggest that: (i) both aortic outflow and cerebral arterial inflow may be lower in developing monotremes than in therians, particularly at small body size (< 20 mm); (ii) cerebral inflow may be lower in some developing metatherians than non-human eutherians; and (iii) dasyurids have unusual features of cerebral arteries possibly related to the extreme immaturity and small size at which they are born. The findings have implications for nutritional sourcing of the developing brain in the three infraclasses of mammals.

Rule of 5: angiographic diameters of cervicocerebral arteries in children and compatibility with adult neurointerventional devices.

BACKGROUND AND PURPOSE: The safety of using adult-sized neuroendovascular devices in the smaller pediatric vasculature is not known. In this study we measure vessel diameters in the cervical and cranial circulation in children to characterize when adult-approved devices might be compatible in children. METHODS: For 54 children without vasculopathy (mean age 9.5±4.9 years (range 0.02-17.8), 20F/34M) undergoing catheter angiography, the diameters of the large vessels in the cervical and cranial circulation (10 locations, 611 total measurements) were assessed by three radiologists. Mean±SD diameter was calculated for the following age groups: 0-6 months, 1, 2, 3, 4, 5-9, 10-14, and 15-18 years. To compare with adult sizes, each vessel measurement was normalized to the respective region mean diameter in the oldest age group (15-18 years). Normalized measurements were compared with age and fitted to a segmented regression. RESULTS: Vessel diameters increased rapidly from 0 to 5 years of age (slope=0.069/year) but changed minimally beyond that (slope=0.005/year) (R(2)=0.2). The regression model calculated that, at 5 years of age, vessels would be 94% of the diameter of the oldest age group (compared with 59% at birth). In addition, most vessels in children under 5, while smaller, were still potentially large enough to be compatible with many adult devices. CONCLUSIONS: The growth curve of the cervicocerebral vasculature displays rapid growth until age 5, at which point most children's vessels are nearly adult size. By age 5, most neuroendovascular devices are size-compatible, including thrombectomy devices for stroke. Under 5 years of age, some devices might still be compatible.

Therapeutic effect of multiple burr hole operation combined with dural inversion and periosteal synangiosis for moyamoya disease.

OBJECT: To explore the therapeutic effect of multiple burr hole operation combined with dural inversion and periosteal synangiosis for moyamoya disease (MMD). METHODS: We used a modified multiple burr hole operation, combined with dural inversion and periosteal synangiosis, in a series of 57 patients with ischemic-type MMD as a sole revascularization procedure. Postoperatively these patients were followed up for 6 months to 3 years. RESULTS: The postoperative clinical outcomes were excellent for 24 patients (42.1%), good for 21 patients (36.8%), fair for 9 patients (15.8%), and poor for 3 patients (5.3%). There was a significant difference between the preoperative and postoperative modified Rankin Scale or mRS scores. The postoperative angiography revealed that there was sufficient neovascularization at 151 of the 160 burr holes. Effective neovascularization was not found at 9 burr holes in 4 patients. The neovascularization derived mainly from the superficial temporal artery and middle meningeal artery, and less from the posterior auricular artery, occipital artery, and deep temporal artery. The postoperative complications included one case of subdural hematoma, one case of subdural effusions, and five cases of postoperative temporary neurological deficits. CONCLUSION: The multiple burr-hole operation combined with dural inversion and periosteal synangiosis is a simple, safe, and effective treatment for ischemic-type MMD as a sole treatment without supplementary revascularization procedures.

Inhibition of protein tyrosine phosphatases enhances cerebral collateral growth in rats.

UNLABELLED: Arteriogenesis involves the rapid proliferation of preexisting arterioles to fully functional arteries as a compensatory mechanism to overcome circulatory deficits. Stimulation of arteriogenesis has therefore been considered a treatment concept in arterial occlusive disease. Here, we investigated the impact of inhibition of protein tyrosine phosphatases (PTPs) on cerebral arteriogenesis in rats. Arteriogenesis was induced by occlusion of one carotid and both vertebral arteries (three-vessel occlusion (3-VO)). Collateral growth and functional vessel perfusion was assessed 3-35 days following 3-VO. Furthermore, animals underwent 3-VO surgery and were treated with the pan-PTP inhibitor BMOV, the SHP-1 inhibitor sodium stibogluconate (SSG), or the PTP1B inhibitor AS279. Cerebral vessel diameters and cerebrovascular reserve capacity (CVRC) were determined, together with immunohistochemistry analyses and proximity ligation assays (PLA) for determination of tissue proliferation and phosphorylation patterns after 7 days. The most significant changes in vessel diameter increase were present in the ipsilateral posterior cerebral artery (PCA), with proliferative markers (PCNA) being time-dependently increased. The CVRC was lost in the early phase after 3-VO and partially recovered after 21 days. PTP inhibition resulted in a significant increase in the ipsilateral PCA diameter in BMOV-treated animals and rats subjected to PTP1B inhibition. Furthermore, CVRC was significantly elevated in AS279-treated rats compared to control animals, along with hyperphosphorylation of the platelet-derived growth factor-ß receptor in the vascular wall in vivo. In summary, our data indicate PTPs as hitherto unrecognized negative regulators in cerebral arteriogenesis. Further, PTP inhibition leading to enhanced collateral growth and blood perfusion suggests PTPs as novel targets in anti-ischemic treatment. KEY MESSAGES: PTPs exhibit negative regulatory function in cerebral collateral growth in rats. Inhibition of pan-PTP/PTP1B increases vessel PDGF-ß receptor phosphorylation. PTP1B inhibition enhances arteriogenesis and cerebrovascular reserve capacity.

Cerebral angiogenesis during development: who is conducting the orchestra?

Blood vessels provide the brain with the oxygen and the nutrients it requires to develop and function. Endothelial cells (ECs) are the principal cell type forming the vascular system and driving its development and remodeling. All vessels are lined by a single EC layer. Larger blood vessels are additionally enveloped by vascular smooth muscle cells (VSMCs) and pericytes, which increase their stability and regulate their perfusion and form the blood-brain barrier (BBB). The development of the vascular system occurs by two processes: (1) vasculogenesis, the de novo assembly of the first blood vessels, and (2) angiogenesis, the creation of new blood vessels from preexisting ones by sprouting from or by division of the original vessel. The walls of maturing vessels produce a basal lamina and recruit pericytes and vascular smooth muscle cells for structural support. Whereas the process of vasculogenesis seems to be genetically programmed, angiogenesis is induced mainly by hypoxia in development and disease. Both processes and the subsequent vessel maturation are further orchestrated by a complex interplay of inhibiting and stimulating growth factors and their respective receptors, many of which are hypoxia-inducible. This chapter intends to give an overview about the array of factors directing the development and maintenance of the brain vasculature and their interdependent actions.

Brain regional angiogenic potential at the neurovascular unit during normal aging.

Given strong regional specialization of the brain, cerebral angiogenesis may be regionally modified during normal aging. To test this hypothesis, expression of a broad cadre of angiogenesis-associated genes was assayed at the neurovascular unit (NVU) in discrete brain regions of young versus aged mice by laser capture microdissection coupled to quantitative real-time polymerase chain reaction (PCR). Complementary quantitative capillary density/branching studies were performed as well. Effects of physical exercise were also assayed to determine if age-related trends could be reversed. Additionally, gene response to hypoxia was probed to highlight age-associated weaknesses in adapting to this angiogenic stress. Aging impacted resting expression of angiogenesis-associated genes at the NVU in a region-dependent manner. Physical exercise reversed some of these age-associated gene trends, as well as positively influenced cerebral capillary density/branching in a region-dependent way. Lastly, hypoxia revealed a weaker angiogenic response in aged brain. These results suggest heterogeneous changes in angiogenic capacity of the brain during normal aging, and imply a therapeutic benefit of physical exercise that acts at the level of the NVU.

Arteriogenesis is modulated by bradykinin receptor signaling.

RATIONALE: Positive outward remodeling of pre-existing collateral arteries into functional conductance arteries, arteriogenesis, is a major endogenous rescue mechanism to prevent cardiovascular ischemia. Collateral arterial growth is accompanied by expression of kinin precursor. However, the role of kinin signaling via the kinin receptors (B1R and B2R) in arteriogenesis is unclear. OBJECTIVE: The purpose of this study was to elucidate the functional role and mechanism of bradykinin receptor signaling in arteriogenesis. METHODS AND RESULTS: Bradykinin receptors positively affected arteriogenesis, with the contribution of B1R being more pronounced than B2R. In mice, arteriogenesis upon femoral artery occlusion was significantly reduced in B1R mutant mice as evidenced by reduced microspheres and laser Doppler flow perfusion measurements. Transplantation of wild-type bone marrow cells into irradiated B1R mutant mice restored arteriogenesis, whereas bone marrow chimeric mice generated by reconstituting wild-type mice with B1R mutant bone marrow showed reduced arteriogenesis after femoral artery occlusion. In the rat brain 3-vessel occlusion arteriogenesis model, pharmacological blockade of B1R inhibited arteriogenesis and stimulation of B1R enhanced arteriogenesis. In the rat, femoral artery ligation combined with arterial venous shunt model resulted in flow-driven arteriogenesis, and treatment with B1R antagonist R715 decreased vascular remodeling and leukocyte invasion (monocytes) into the perivascular tissue. In monocyte migration assays, in vitro B1R agonists enhanced migration of monocytes. CONCLUSIONS: Kinin receptors act as positive modulators of arteriogenesis in mice and rats. B1R can be blocked or therapeutically stimulated by B1R antagonists or agonists, respectively, involving a contribution of peripheral immune cells (monocytes) linking hemodynamic conditions with inflammatory pathways.

Maturation and long-term hypoxia-induced acclimatization responses in PKC-mediated signaling pathways in ovine cerebral arterial contractility.

In the developing fetus, cerebral arteries (CA) show striking differences in signal transduction mechanisms compared with the adult, and these differences are magnified in response to high-altitude long-term hypoxia (LTH). In addition, in the mature organism, cerebrovascular acclimatization to LTH may be associated with several clinical problems, the mechanisms of which are unknown. Because PKC plays a key role in regulating CA contractility, in fetal and adult cerebral arteries, we tested the hypothesis that LTH differentially regulates the PKC-mediated Ca(2+) sensitization pathways and contractility. In four groups of sheep [fetal normoxic (FN), fetal hypoxic (FH), adult normoxic (AN), and adult hypoxic (AH)], we examined, simultaneously, responses of CA tension and intracellular Ca(2+) concentration and measured CA levels of PKC, ERK1/2, RhoA, 20-kDa myosin light chain, and the 17-kDa PKC-potentiated myosin phosphatase inhibitor CPI-17. The PKC activator phorbol 12,13-dibutyrate (PDBu) produced robust contractions in all four groups. However, PDBu-induced contractions were significantly greater in AH CA than in the other groups. In all CA groups except AH, in the presence of MEK inhibitor (U-0126), the PDBu-induced contractions were increased a further 20-30%. Furthermore, in adult CA, PDBu led to increased phosphorylation of ERK1, but not ERK2; in fetal CA, the reverse was the case. PDBu-stimulated ERK2 phosphorylation also was significantly greater in FH than FN CA. Also, although RhoA/Rho kinase played a significant role in PDBu-mediated contractions of FN CA, this was not the case in FH or either adult group. Also, whereas CPI-17 had a significant role in adult CA contractility, this was not the case for the fetus. Overall, in ovine CA, the present study demonstrates several important maturational and LTH acclimatization changes in PKC-induced contractile responses and downstream pathways. The latter may play a key role in the pathophysiologic disorders associated with acclimatization to high altitude.

Development of optimized vascular fractal tree models using level set distance function.

Using the concepts of fractal scaling and constrained constructive optimization (CCO), a branching tree model, which has physiologically meaningful geometric properties, can be constructed. A vascular branching tree model created in this way, although statistically correct in representing the vascular physiology, still does not possess a physiological correct arrangement of the major arteries. A distance-function based technique for "staged growth" of vascular models has been developed in this work to address this issue. Time-dependent constraints based on a signed-distance level set function have been added, so that the tree models will first be grown near the designated surface(s) and, then, gradually allowed to penetrate into the enclosed volume. The proposed technique has been applied to construct a model of the human cerebral vasculature, which is characterized by the above-mentioned distribution of the arteries.

A history of our understanding of cerebral vascular development and pathogenesis of perinatal brain damage over the past 30 years.

This article reviews our studies focusing on cerebral vascular development, the pathogenesis of subependymal/intraventricular hemorrhage (SEH/IVH), periventricular leukomalacia (PVL), and pontosubicular neuron necrosis (PSN). Their pathogenesis consists of predisposing developmental and causal factors. SEH/IVH may be caused by reperfusion or overperfusion following ischemia in the subependymal germinal matrix with characteristic vasculature. The cause of PVL is multifactorial (ie, ischemia and inflammation), predisposed by the maturational status of the vasculature and oligodendroglia in the white matter. Focal PVL is ischemic necrosis, and diffuse PVL or white matter injury may include cytotoxic damage. PSN has an apoptotic character, and may be induced by ischemic and oxidative stress on specific immature neurons. Further studies on preventive and therapeutic measures are necessary in clinical, pathologic, and experimental fields. The monitoring and control methods of brain hemodynamics and cellular stability should be more developed to prevent brain damages.

Three-dimensional micro-MRI analysis of cerebral artery development in mouse embryos.

Vascular system development involves a complex, three-dimensional branching process that is critical for normal embryogenesis. In the brain, the arterial systems appear to develop in a stereotyped fashion, but no detailed quantitative analyses of the mouse embryonic cerebral arteries have been described. In this study, a gadolinium-based contrast perfusion method was developed to selectively enhance the cerebral arteries in fixed mouse embryos. Three-dimensional magnetic resonance micro-imaging (micro-MRI) data were acquired simultaneously from multiple embryos staged between 10 and 17 days of gestation, and a variety of image analysis methods was used to extract and analyze the cerebral arterial patterns. The results show that the primary arterial branches in the mouse brain are very similar between individuals, with the patterns established early and growth occurring by extension of the segments, while maintaining the underlying vascular geometry. To investigate the utility of this method for mutant mouse phenotype analysis, contrast-enhanced micro-MRI data were acquired from Gli2(-/-) mutant embryos and their wild-type littermates, showing several previously unreported vascular phenotypes in Gli2(-/-) embryos, including the complete absence of the basilar artery. These results demonstrate that contrast-enhanced micro-MRI provides a powerful tool for analyzing vascular phenotypes in a variety of genetically engineered mice.

Neurovascular congruence during cerebral cortical development.

There is evidence for interaction between the developing circulatory and nervous systems. Blood vessels provide a supporting niche in regions of adult neurogenesis. Here we present a systematic analysis of vascular development in the embryonic murine cortex and demonstrate that dividing cells, including Tbr2-positive intermediate progenitor cells, are closer to the vasculature than expected from a random distribution. To examine whether neurites of the newly generated embryonic neurons find blood vessels as an attractive and permissive substrate, we overlayed green fluorescent protein (GFP)-labeled dissociated cortical progenitors on embryonic organotypic cortical slice cultures with labeled vasculature. Our observations of neurites extending toward and along labeled blood vessels support the notion of vascular-neuronal interactions. The altered cortical layering had no obvious effect on the vascular patterns within the cortical plate (CP) in shaking rat Kawasaki (SRK) and the reeler mutant mouse at the ages studied (E19 and P3). It appears that similarly to other neurogenic regions in the adult, the embryonic "vascular niche" might influence neural progenitor cells during telencephalic neurogenesis, neuronal migration, and neurite extension, but the laminar phenotype of cell classes within the CP has limited influence on the developing vasculature.

Male predominance in childhood ischemic stroke: findings from the international pediatric stroke study.

BACKGROUND AND PURPOSE: Previous studies suggested a male predominance in childhood ischemic stroke, mirroring gender differences in adults but were limited by small sample sizes or unconfirmed diagnoses. We sought to study gender within a large international series of confirmed cases of pediatric ischemic stroke. METHODS: From January 2003 to July 2007, the International Pediatric Stroke Study enrolled children (0 up to 19 years) with arterial ischemic stroke or cerebral sinovenous thrombosis at 30 centers in 10 countries. Neonates were those <29 days of age. We calculated the "expected" gender ratio for our study as the weighted average of population-based childhood gender ratios in enrolling countries weighted by the number of subjects enrolled in each country. chi(2) tests were used to compare the observed gender ratios in our series with this expected ratio (51.7%). RESULTS: Among 1187 children with confirmed ischemic stroke, 710 were boys (60%, P<0.0001). Male predominance persisted after stratification by age (61% for neonates, P=0.011; 59% for later childhood, P=0.002) and stroke subtype (58% for arterial ischemic stroke, P=0.004; 65% for cerebral sinovenous thrombosis, P=0.002). The greatest proportion of males occurred among children with arterial ischemic stroke and a history of trauma (75%, P=0.008), although boys were also overrepresented among those with arterial ischemic stroke and no trauma (57%; P=0.07). There were no gender differences in case fatality or deficits at discharge. CONCLUSIONS: Childhood ischemic stroke appears to be more common in boys regardless of age, stroke subtype, or history of trauma. Further exploration of this gender difference could shed light on stroke mechanisms in both children and adults.

Functional connectivity of the sensorimotor area in naturally sleeping infants.

Patterns of cortical functional connectivity in normal infants were examined during natural sleep by observing the time course of very low frequency oscillations. Such oscillations represent fluctuations in blood oxygenation level and cortical blood flow thus allowing computation of neurophysiologic connectivity. Structural and resting-state information were acquired for 11 infants, with a mean age of 12.8 months, using a GE 1.5 T MR scanner. Resting-state data were processed and significant functional connectivity within the sensorimotor area was identified using independent component analysis. Unilateral functional connectivity in the developing sensory-motor cortices was observed. Power spectral analysis showed that slow frequency oscillations dominated the hemodynamic signal at this age, with, on average, a peak frequency for all subjects of 0.02 Hz. Our data suggest that there is more intrahemispheric than interhemispheric connectivity in the sensorimotor area of naturally sleeping infants. This non-invasive imaging technique, developed to allow reliable scanning of normal infants without sedation, enabled computation of neurophysiologic connectivity for the first time in naturally sleeping infants. Such techniques permit elucidation of the role of slow cortical oscillations during early brain development and may reveal critical information regarding the normative development and lateralization of brain networks across time.

Induction of cerebral arteriogenesis leads to early-phase expression of protease inhibitors in growing collaterals of the brain.

Cerebral arteriogenesis constitutes a promising therapeutic concept for cerebrovascular ischaemia; however, transcriptional profiles important for therapeutic target identification have not yet been investigated. This study aims at a comprehensive characterization of transcriptional and morphologic activation during early-phase collateral vessel growth in a rat model of adaptive cerebral arteriogenesis. Arteriogenesis was induced using a three-vessel occlusion (3-VO) rat model of nonischaemic cerebral hypoperfusion. Collateral tissue from growing posterior cerebral artery (PCA) and posterior communicating artery (Pcom) was selectively isolated avoiding contamination with adjacent tissue. We detected differential gene expression 24 h after 3-VO with 164 genes significantly deregulated. Expression patterns contained gene transcripts predominantly involved in proliferation, inflammation, and migration. By using scanning electron microscopy, morphologic activation of the PCA endothelium was detected. Furthermore, the PCA showed induced proliferation (PCNA staining) and CD68+ macrophage staining 24 h after 3-VO, resulting in a significant increase in diameter within 7 days after 3-VO, confirming the arteriogenic phenotype. Analysis of molecular annotations and networks associated with differentially expressed genes revealed that early-phase cerebral arteriogenesis is characterised by the expression of protease inhibitors. These results were confirmed by quantitative real-time reverse transcription-PCR, and in situ hybridisation localised the expression of tissue inhibitor of metalloproteinase-1 (TIMP-1) and kininogen to collateral arteries, showing that TIMP-1 and kininogen might be molecular markers for early-phase cerebral arteriogenesis.

Assessment of functional development in normal infant brain using arterial spin labeled perfusion MRI.

Arterial spin labeled (ASL) perfusion MRI provides a noninvasive approach for longitudinal imaging of regional brain function in infants. In the present study, continuous ASL (CASL) perfusion MRI was carried out in normally developing 7- and 13-month-old infants while asleep without sedation. The 13-month infant group showed an increase (P<0.05) of relative CBF in frontal regions as compared to the 7-month group using a region of interest based analysis. Using a machine-learning algorithm to automatically classify the relative CBF maps of the two infant groups, a significant (P<0.05, permutation testing) regional CBF increase was found in the hippocampi, anterior cingulate, amygdalae, occipital lobes, and auditory cortex in the 13-month-old infants. These results are consistent with current understanding of infant brain development and demonstrate the feasibility of using perfusion MRI to noninvasively monitor developing brain function.