The dynamic physiologic changes that take place in the cardiovascular system of the preterm and term neonate during the transitional period are poorly characterized, especially when it comes to the role of the microvasculature. Indeed, most efforts to describe these changes focus on the determinants of blood pressure homeostasis: cardiac output and peripheral resistance. It is becoming increasingly evident that the microcirculation, composed of arterioles, capillaries, and venules less than 100 μm in diameter, is also crucial to the overall functioning of the cardiovascular system. Accordingly, in this chapter we discuss the changes in microvascular function during early extrauterine life and address their relationship to cardiovascular function, gestational age, clinical severity, and sex. The chapter also highlights the areas of interest and available methods in the study of the microvasculature of the newborn, including the tools available for investigating the microvascular structure and function in even the very immature neonate. Furthermore, the chapter also discusses the potential for and importance of the assessment of the microvasculature to aid in clinical decision-making and facilitates understanding the specific microvascular effects of common treatments of the critically ill preterm and term neonate.
Keywordsblood flow, developmental origins of health and disease, laser Doppler, microcirculation, prematurity, videomicroscopy
The microvasculature is the largest single component of the cardiovascular system.
There are tools to assess microvascular function that can be used in the smallest infants.
Differences in microvascular function are seen in relation to gestational age, postnatal age, and sex of the infant.
Changes in microvascular flow relate to neonatal illness severity and clinical risk factors.
Underlying differences in the physiologic mediators of microvascular dilation have been demonstrated in the most-at-risk infants.
Microvascular changes may represent the first cardiovascular changes of long-term health programming.
Why Assess the Microcirculation?
The cardiorespiratory system is crucial in the perinatal adaptation of the human neonate to extrauterine life. Most infants who die do so in the first few days after delivery and exhibit evidence of cardiovascular compromise. The cardiovascular system, as detailed in other chapters of this book, has a number of crucial components that affect the overall performance of the system, including the pump, circulating volume, and peripheral vasculature (see Chapter 1 , Chapter 2 , Chapter 3 , Chapter 21 ). It is being increasingly recognized that a key component of the peripheral vasculature, the microvasculature, exerts a significant influence on the overall function of the peripheral vasculature. Specifically, the microvasculature, as opposed to the macrovascular conductance vessels, is crucially important in the delivery of oxygen and nutrients to the tissues of the entire body. In addition, the flow within the microvasculature reflects the combined action of the central, macrohemodynamic components of the circulation and thus the end point of total cardiovascular efficiency. Despite this, the dynamic physiologic changes that occur in the microvasculature of the preterm neonate in the immediate transitional period are not completely characterized.
So what comprises the microvascular system? Current accepted definitions of microvascular components include, from proximal to distal, arterioles, capillaries, arteriolar-venular shunts, and venules. In practice, these components represent all vascular tissue less than 100 μm in diameter. Lymphatic function, which is not discussed here, can also significantly influence overall microvascular function, but studies in the neonatal population remain limited. The microvasculature has regional and organ-specific specialization but also has global function and responses. It therefore represents one of the largest virtual “organs” in the body. As nearly all tissue needs to be in close approximation to a blood supply for oxygen and nutrient delivery and removal of waste products, the microvasculature is all-pervasive. It has been estimated that the capillary component of the microvasculature alone covers greater than a 6000 m 2 cross-sectional area in the adult. Further more, at least 5% of circulating blood volume is in the capillaries at any time, with the capillary system having the ability to increase this capacity by more than fourfold. Similar estimations have not been undertaken in the newborn. However, since the newborn is rapidly growing and has a less organized capillary network, its capillary system may possess even greater proportional capacity.
Those involved with newborn care are mostly aware of microvascular growth in the context of the retina, with retinopathy of prematurity (ROP) remaining a significant cause of blindness in the preterm population. ROP studies have also allowed us to understand more about growth and development of the microvasculature during extrauterine adaptation. This includes the role of oxygenation levels in vascular growth, the importance of angiogenic and vascular “pruning” factors in the growing tree (e.g., vascular endothelial growth factor and the prorenin/angiotensin system). The same process of angiogenesis, vasculogenesis, and remodeling are similarly going on throughout the developing preterm infant. Thus understanding the biology and physiology in the microvasculature contributes to understanding cardiovascular compromise at different gestational and postnatal ages and the role the microvasculature plays in certain long-term consequences of prematurity.
The drive to study and understand microcirculatory status has led to the development and use of a number of methods for the assessment of the microcirculation in preterm or sick term newborns. This chapter reviews the sites and techniques that have been used, outlines current understanding, and addresses the relationship of these microvascular findings to the macrocirculation in the preterm and sick term neonate.
Where to Study the Microcirculation in the Human Newborn?
As the microcirculation is ubiquitous throughout the body, the possibilities for observation and measurement are widespread. In animal models a number of techniques have been used to assess microcirculation in a selection of vascular beds, including laser Doppler flowmetry (LDF) in the skin, muscle, brain, liver, gastrointestinal tract, and kidneys; videomicroscopy in the brain, skin, and gut; as well as Xenon clearance blood flow measurement techniques. Techniques employing the use of microspheres or perfusate casts, limited to animal work, are reviewed elsewhere. The noninvasive requirements for human investigation in the preterm neonate have led to peripheral sites being targeted. Methods employed include heat conductance, skin temperature changes, laser Doppler, videomicroscopy (sublingual, retinal, and scleral), Xenon clearance, saturation oximetry, transcutaneous carbon dioxide measurement, thermography, near infrared spectroscopy (skin, muscle, and cerebral), and direct videomicroscopic observation of red cell flux in the nail fold capillaries. These techniques have all demonstrated changes during the transitional period in the few days after birth.
The skin is the most easily accessible site in neonates and has received the most attention. As the skin has some very specialized microvascular functions, such as thermoregulation, it has been questioned as to how representative it is of the microcirculation in general. However, despite skin blood flow demonstrating a variety of adaptive responses, it is part of the wider microcirculation. Many of the signals that govern microvascular response are systemic and affect all vascular beds to some degree. This may be particularly the case in the first few days of postnatal life in the preterm infant, where locally adaptive responses in the dermal circulation are impaired. Moreover, even in adults, significant and generalizable systemic microvascular changes have been documented in highly specialized peripheral sites (e.g., the nail folds).
Many of the techniques used previously are now considered too invasive or difficult in the nonsedated neonate, with other indirect measures of questionable reliability. With emerging techniques such as NIRS discussed elsewhere (see Chapter 16 , Chapter 17 , Chapter 18 ), LDF and videomicroscopy will be the main techniques discussed in this chapter. While the focus is principally on skin microvascular flow, studies of the retinal circulation are also included in the context of vascular programming.
Laser Doppler Imaging
There are currently two systems using laser Doppler or related techniques. Although this chapter focuses on LDF, laser Doppler imaging (LDI) can also be used and may prove of increasing value in the neonatal field. LDI uses a scanning laser beam over the area of interest and maps the cutaneous blood flow of a larger area than LDF. This leads to increased reproducibility for LDI compared with LDF. LDI is also noncontact in nature and thus could be used in clinical situations where skin contact is not possible or desirable. LDI is, however, more superficial and has limited ability to study the dynamics of dilator or constrictor responses. Despite the introduction of laser speckle contrast instruments, which can assess skin microvascular reactivity with excellent reproducibility, the ability of LDF to provide a constant measure of perfusion and perform dynamic measures at relatively low cost means LDF remains the most widely used technique.
Laser Doppler Flowmetry
Microvascular laser Doppler has been used extensively in the newborn infant. LDF uses the Doppler shift of a low-energy laser beam with a wavelength of 780 nm to quantify microvascular perfusion. The light is delivered to the tissue by a fiberoptic cable, where it is scattered by both stationary structures and moving blood cells. When scattered by moving cells, the light frequency is Doppler shifted in proportion to cell flux (defined as the product of mean velocity and cell number) in the few cubic millimeters of measured volume. The output is detected by fiberoptic receiving fibers of the same probe ( Fig. 19.1 ), processed, and stored using proprietary software, which varies among the device brands. Although directly proportional to tissue perfusion, laser Doppler measurements provide a semiquantitative measure of blood flow, expressed as arbitrary perfusion units (PU) dependent on the output voltage (1 PU = 10 mV), but standardized by international agreement among manufacturers. When the recording site is standardized, the reproducibility of LDF is high, with poorer reproducibility seen when the recording site is varied.
In adults, arteries penetrating the subcutaneous tissue branch into several precapillary arterioles (30 to 80 μm). These pass into venous plexuses organized parallel to the skin surface, after forming 8 to 10 capillary loops orientated perpendicular to the skin surface. In term neonates this network is much less organized, and the arteriolar-venular anastomoses and the capillary loops are poorly developed, not forming a more adult-type network until approximately 4 months of age. The preterm skin microcirculation is even less developed. LDF measures flow to the depth of the subpapillary plexus in children and adults, but in the newborn it measures well into the subcutaneous microcirculation and therefore should be less affected by thermoregulatory shunts and other adaptive capillary loop mechanisms. In healthy term neonates, baseline skin blood flow has been shown to decrease during the first few postnatal days.
Active stimulations of the microvasculature during LDF assessment may provide more detailed information of microvascular function by allowing for the interrogation of both endothelial and other pathways. These stimuli include postocclusive hyperemia, thermal hyperemia, and the microvascular response to iontophoresis of vasoactive medication.
Postocclusive Reactive Hyperemia
Postocclusive reactive hyperemia (PORH) has been used in conditions such as diabetes and variations have been linked to cardiovascular risk factors. In neonates the test is performed by placing a sphygmomanometer cuff proximal to a limb-sited LDF probe. The cuff is then inflated to suprasystolic levels, holding for a designated length of time and then releasing the pressure as quickly as possible. PORH is characterized by a transient increase in skin blood flow above baseline levels that is mediated by endothelium-dependent pathways. There has been some debate about the length of the occlusion time required, balancing the need to obtain the best response with patient comfort and safety. While 3 minutes of occlusion time has been proposed for adults, in the newborn a 1-minute occlusion period produces an adequate response.
PORH may be expressed as an absolute value, an increase above baseline, a percentage increase above baseline, or a percentage relative to baseline, with the maximum increase in hyperemia-perfusion (PORH max ) being the most commonly reported variable. In addition, the time to reach peak hyperemia-perfusion ( T p ) has been said to relate to the stiffness of the microvascular system. This variable is not currently widely used in the acute assessment of the neonate but may be relevant to the assessment of long-term programming effects in the follow-up of premature infants.
Local Thermal Hyperemia
Local thermal hyperemia is elicited by warming of the skin, causing direct vasodilatation of a given area. Heating can be applied to the whole environment, but in neonates is usually applied to the area of the LDF probe. Several different thermostatically regulated probes are available which ensure a constant temperature at the site of the probe to both reduce study variability and enable elicitation of thermal stimuli responses. In adults, applying a temperature change over a 20-minute period is the most common practice, but this is less suitable in the unstable preterm neonate, and thus shorter protocols have evolved. The most frequently employed protocol resembles those of Roustit and colleagues, with an initial rapid temperature increase to 40°C and then a sequential further increase to 44°C over a period of 5 minutes. Maximum vasodilatation is measured at the end of the first minute at 44°C.
Two different mechanisms have been implicated in the response to thermal stimulation within the skin microvasculature. The initial response is mediated through nociceptive nerve pathways and via neurogenic reflexes and locally released vasoactive substances, including calcitonin gene-related peptide and nitric oxide. The subsequent increase in microvascular blood flow over a longer period of time and/or at higher temperatures is thought to be mediated through the more classic endothelium-dependent nitric oxide pathways.
Iontophoresis enables the transfer of soluble ions, including vasoactive molecules and hormonal moderators of vascular control, into body tissues using a small direct electrical current, clinically best known for using pilocarpine for cystic fibrosis sweat testing. A variety of vasoactive molecules and hormonal moderators of vascular control may be driven into the skin. Acetylcholine (ACh), the classical paradigm-test for endothelium-dependent vasodilation, is the standard transdermal drug used in microvascular assessment, with relaxation occurring via nonnitric oxide, nonprostanoid endothelium-dependent hyperpolarization. Iontophoresis in conjunction with LDF allows real-time interrogation of physiologic responses ( Fig. 19.2 ). It can also been used with LDI systems, where a whole skin patch can be tested.
Videomicroscopy, orthogonal polarized spectral (OPS) imaging, and later generation sidestream dark field (SDF) techniques all allow for the investigation of both microvasculature function and structure. Videomicroscopy has been widely used in adult populations, specifically in the intensive care setting, where persistent microcirculatory alterations in the sublingual area have been identified as predictors of adverse outcome. Sublingual videomicroscopy in the newborn infant may be inconsistent due to small patient size and limited cooperation during assessment. However, the skin is thin enough in neonates to permit transcutaneous microcirculatory imaging, allowing studies during transition in both preterm and term neonates, in babies experiencing respiratory distress or septic changes, during transfusion, or during whole-body cooling, providing further insight into the (dys)function of newborn microvasculature. An extensive review of this technique was written by Kuiper et al.
SDF imaging uses a green illumination light (wavelength 548 nm) that is maximally absorbed by oxy- and deoxy-hemoglobin. Surrounding tissue reflects this light, creating contrast, thereby allowing the visualization of red blood cells moving through the microvasculature. Second-generation SDF imaging is the most widely used videomicroscopy imaging system employed currently in pediatric studies. Third-generation systems, employing incident dark field (IDF) imaging, boast several technical improvements, potentially giving improved imaging resolution, but the neonatal literature on the use of these systems remains limited. A complete review of the technical aspects of OPS, SDF, and IDF, including comparisons between the technologies, can be found in the papers by van Elteren et al. and Milstein et al. The full range of videomicroscopy values using SDF in the immediate postnatal days and by sex are summarized for term infants by Wright et al.
Microvasculature of the Neonate Studied by Laser Doppler Flowmetry and Videomicroscopy
Peripheral microvascular blood flow is subject to considerable changes during the first days of postnatal life, a period of marked circulatory vulnerability, especially in preterm infants. Myogenic and neural control of skin blood flow must be rapidly established during this period to allow appropriate thermoregulation to take place. LDF and OPS/SDF imaging have allowed detailed characterization of the relationship between peripheral microvascular blood flow and measures of neonatal physiologic and cardiovascular stability in infants during the immediate posttransitional period.
Neonatal Peripheral Microvascular Blood Flow and Gestational Age at Birth
Previously published findings on the influence of gestational age on baseline peripheral cutaneous blood flow are conflicting. Some authors found no influence of gestational age on blood flow. Other investigators reported an inverse relationship between baseline blood flow and increasing gestational age, whereas a few reported a higher baseline flow with increasing gestational age. The apparent lack of consensus most likely reflects methodologic differences, with different patient groups, timing, and methods used.
Our data from two large cohorts of premature infants revealed a significant inverse relationship between LDF-measured baseline microvascular blood flow at 24 and 72 hours of age and gestational age ( Fig. 19.3 ). This relationship was complicated by the demonstration of a close interaction of gestational age and sex of the infant (discussed later), although some parameters appeared to be related to gestational age alone. Interestingly, the effect of thermal stimulation was most marked in the more immature infants. Previous studies have reported no difference in skin vasodilatation to local thermal stimulation between preterm and term infants. However, they focused on infants born after 30 weeks’ gestation or studied the subjects at the end of the first postnatal week when variability in the response to thermal stimulation appears to resolve. Further more, more recent studies using non-LDF methods to investigate microvascular blood flow support our observation of a thermal response being present even in the most preterm neonate.
Neonatal Peripheral Microvascular Blood Flow, Postnatal Age, and Clinical Status
While alterations in microvascular function have been described in association with common clinical problems such as sepsis and polycythemia in the immediate postnatal period, it is the role of the microvasculature in the process of cardiovascular adaptation following preterm birth that is perhaps of the greatest interest. With most extremely preterm infants that are going to die demonstrating clinical deterioration in the first 72 hours following delivery, it is the influence of the relatively short-term postnatal preterm adaptation in microvascular blood flow that is potentially of the most importance and clinical relevance.
Despite evidence from the animal literature and observational data in human neonates, the relationship between peripheral microvascular blood flow and measures of neonatal physiologic and cardiovascular stability in the immediate postnatal period had not been characterized until recently. There is emerging evidence that the changes in early microvascular flow are of clinical significance. LDF measurements of peripheral microvascular blood flow have been shown to exhibit significant relationships with both clinical illness severity ( Fig. 19.4A ) and concomitant measures of cardiovascular function—in particular blood pressure (see Fig. 19.4B ) in preterm infants during the first days of postnatal life.
The underlying processes that explain these associations between peripheral microvascular blood flow and physiologic instability remain incompletely characterized. One explanation for the apparent discrepancy between early low central flow measures and high microvascular flow at 24 hours could be temporal change, with a period of low systemic perfusion followed by reperfusion. Our recent human and animal data suggest though that this is not a significant effect. Further more, we have preliminary evidence that structural maturation could underlie the changes in microvasculature function and apparent maldistribution of blood flow ( Fig. 19.5 ), but definitive studies are still to be completed. Finally, another distributive scenario may contribute to this paradox. It is possible that a significant component to the observed peripheral increased microvascular flow may actually be on the venular side. This would lead to an apparent increase in flow by LDF assessment, but with an accompanying significant increase in capacitance. Such an effect would decrease preload with associated decreased cardiac output. Further videomicroscopic studies, in combination with direct cardiac output and blood pressure measurements (see Chapter 1 , Chapter 1 , Chapter 3 ), may contribute to clarification of this possibility.