Microcirculatory Blood Flow: Videomicroscopy D. De Backer

D. De Backer

Introduction

In the classical view of hemodynamic monitoring, it is usually considered that organ blood flow should be preserved as long as arterial pressure, representative of perfusion pressure of the organs, and cardiac output are maintained. Several studies have reported that alterations in regional blood flow and metabolism can also occur, especially in sepsis [1, 2]. Accordingly, the splanchnic region can be monitored via gastric tonometry, hepatic vein oxygen saturation and indocyanine green disappearance. Curiously, the microcirculation is often neglected, even though the microcirculation is the place where most of the exchanges in oxygen and nutrients between the blood and the tissues occur. The study of the microcir- culation has long been difficult as it required the use of large microscopes applied on fixed tissue preparations. Recent technical developments have allowed the direct visualization of the microcirculation in critically ill patients opening the door of monitoring of the microcirculation. In this chapter we will discuss the rationale for future bedside monitoring of the microcirculation.

Specificity of the Microcirculation

The microcirculation differs from the systemic circulation by many aspects. First, capillary PO 2 is much lower than arterial PO 2 , due to direct diffusion of oxygen from arteriole crossing a venule but also by consumption at the endothelial level.

Second, the local hematocrit differs from the systemic hematocrit and is heteroge-

neous, as a consequence of the Farheus effect and of the interposition of an

obligatory plasma layer in vessels of varying diameter and non-linear hematocrit

distribution at asymmetric capillary branch points. Third, the control of mi-

crovascular blood flow is complex and depends both on local metabolic control

and on systemic, humoral, controls. Finally, the architecture of the microvessels

differs among organs, hence some organs may be more vulnerable to a decrease in

global blood flow.

Evidence for Microcirculatory Alterations in Experimental Studies

Numerous experimental studies have reported that microvascular blood flow is altered in various conditions, including hemorrhagic shock [3], ischemia/reperfu- sion injury [4], and sepsis [5–11]. Whatever the type of injury, these alterations include a decrease in capillary density and an increased heterogeneity of blood flow. Interestingly, these alterations are more severe in septic than in other insults [12,13].

Endotoxin administration induces severe microcirculatory alterations, includ- ing severe arteriolar and venular vasocostriction in rats [6], and a decreased capillary density in dogs [14]. Severe microcirculatory alterations were also ob- served in normodynamic models of sepsis obtained by cecal ligation and perfora- tion. These alterations included a decrease in the perfused capillary density and an increase in the number of stopped-flow capillaries and in heterogeneity of spatial distribution of perfused capillaries [7, 10, 15]. Of note, these microcirculatory alterations clearly differ from macrocirculatory hemodynamic alterations in sep- sis, with vasoconstriction in the microcirculation in opposition to the vasodilatory state with high cardiac output.

Several mechanisms can be evoked to explain these microvascular alterations.

In view of the severe vasoconstriction observed in some vessels, it seems very likely that inflammatory and vasoactive mediators such as tumor necrosis factor (TNF) [16] and endothelin [17] that can cause microvascular vasoconstriction are in- volved. In contrast, nitric oxide (NO) seems to have a protective role [18]. In addition, blood flow in capillaries may be impaired by the formation of mi- crothrombi [19, 20], by the impairment of leukocyte [21] and erythrocyte [22]

deformability [23], and by the adhesion of leukocytes to endothelial cells [23, 24].

It is likely that many of these mechanisms contribute to the microvascular altera- tions.

Implications of Microcirculatory Alterations

Microvascular alterations can have major physiopathological implications. First, the juxtaposition of well perfused and non-perfused capillaries leads to a marked heterogeneity in blood flow which may be responsible for the decrease in oxygen extraction capabilities that is observed in sepsis [14, 25, 26]. Second, microvascu- lar alterations are associated with zones of tissue hypoxia, as suggested by the decreased intravascular PO 2 [27, 28]. Finally, the transient flow observed in some capillaries may lead to focal areas with ischemia/reperfusion injury.

One major question is whether these microvascular blood flow alterations are

the initial mechanism, leading to alterations in tissue metabolism or are these

alterations secondary, with flow matching direct heterogenous metabolic altera-

tions? It is difficult to separate these two contradictory alternatives. Several argu-

ments nevertheless suggest that microcirculatory alterations may be the triggering

event. First, in a pivotal study, Ellis et al. [15] reported in a model of peritonitis

induced by cecal ligation that heterogeneity of microvascular blood flow increased

with an increased number of stopped flow capillaries (from 10 to 38%) and an increase in the proportion of fast-flow to normal-flow capillaries. In addition, in the well perfused capillaries, oxygen extraction was increased, not decreased, and the VO 2 of this segment was also increased. These results argue strongly against a sepsis-induced mitochondrial dysfunction, at least in the early phase of sepsis.

Indeed a primary mitochondrial dysfunction would have been accompanied by a decreased VO 2 and oxygen extraction in this segment. Similarly, Ince et al. [27]

reported that microvascular PO 2 is decreased in sepsis, which is incompatible with primary metabolic alterations. This suggests that the decrease in extraction capa- bilities that is observed in sepsis is related to blood flow heterogeneity but not to impaired capacities of the tissues to use oxygen. Second, we observed that the severity of alteration in the sublingual microcirculation was inversely related to sublingual PCO 2 and that both alterations can be reversed [29]. If flow matched metabolism, PCO 2 would not have been increased in these patients. Altogether these observations suggest that microcirculatory alterations are involved in the pathophysiology of sepsis-induced organ dysfunction and do not match metabolic alterations, at least in the early phases of sepsis.

Methods to Investigate the Microcirculation in Critically Ill Patients

Most of the experimental studies were performed using intravital microscopy, the gold standard technique for studying the microcirculation. Unfortunately, this technique cannot be used in humans, as large microscopes are generally applied on a fixed tissue preparation while fluorescent dyes are infused. Alternative meth- ods have been used in humans, including phlethysmography, videomicroscopy of the nailfold area, and laser Doppler techniques. An extensive review of the avail- able techniques can be found elsewhere [30]. Nailfold videomicroscopy uses mi- croscopes applied on a finger that is fixed under its focus. Unfortunately, the nailfold area is probably not the best area to study in critically ill patients. This area is very sensitive to changes in temperature. Ambient temperature can be controlled but not body temperature. In addition, peripheral vasoconstriction can also occur during chills and acute circulatory failure and can even be promoted by the use of vasopressor agents. Hence, this area is of limited interest in critically ill patients. Laser Doppler techniques have been used frequently in critically ill patients. The advantage of this technique is that it can be applied on various tissues and can even be inserted in the upper digestive tract through a nasogastric tube. Laser Doppler provides measurements of blood flow in relative units (mV), accordingly only relative changes to baseline can be assessed. However, the major limitation of this technique is that it does not take into account the heterogeneity of microvascular blood flow, the measured parameter representing the average of the velocities in all the vessels included in the investigated volume (~1 mm³).

Phlethysmographic techniques have similar limitations.

Orthogonal Polarization Spectral (OPS) imaging is a non-invasive technique

that allows the direct visualization of the microcirculation [31]. The device is

composed of a small camera and a few lenses, is small and can be used easily at the

bedside. Polarized light illuminates the area of interest, the light is scattered by the tissue and collected by the objective lens. A polarization filter (analyzer), oriented orthogonal to the initial plane of the illumination light, is placed in front of the imaging camera and eliminates the reflected light scattered at or near the surface of the tissue that retains its original polarization. Depolarized light scattered deeper within the tissues passes through the analyzer. High contrast images of the micro- circulation are formed by absorbing structures (e.g., blood vessels) close to the surface that are illuminated by the depolarized light coming from deeper struc- tures. Due to its specific characteristics, this device can be used to visualize the microcirculation in tissues protected by a thin epithelial layer, such as mucosal surface. In critically ill patients, the sublingual area is the most easily investigated mucosal surfaces. Other mucosal surfaces include rectal and vaginal surfaces, which are of limited accessibility, and ileal or colic mucosa in patients with enterostomies. Images can also be generated in eyelids and in the nailfold [32].

The use of OPS imaging techniques to visualize the microcirculation has been validated against standard techniques. In various animal models, vessel diameters, functional capillary density, and vessel blood flow were similar with OPS imaging and standard intravital fluorescence videomicroscopy [31, 33–35]. In human healthy volunteers, the agreement in the measurement of capillary density and red blood cell velocity in the nailfold area was excellent between OPS imaging and capillaroscopy [32]. Unfortunately, a quantitative approach cannot be used for observations of the sublingual microcirculation in critically ill patients, due to small movements (especially respiratory movements). Hence, we [36] developed a semi- quantitative method to determine capillary density and the proportion of perfused capillaries. The investigation of the sublingual microcirculation requires a collabo- rative or sedated patient, and the absence of bloody secretions in the mouth.

Microvascular Blood Flow is Altered in Critically Ill Patients

Using videomicroscopy of the nailfold area, Freedlander et al. [37] reported in 1922 that capillary stasis occurred. However, these observations are quite old, and the definition of shock state, although lethal, may be questioned in the absence of cardiovascular and respiratory support. More recently, various investigators [23, 38] used laser Doppler to investigate skin and muscle microvascular blood flow and observed that basal blood flow may be decreased or increased compared to healthy volunteers. These studies are nevertheless difficult to compare as skin microvascular blood flow differs according to the site investigated [39]. More importantly, the increase in microvascular blood flow was blunted after partial occlusion [40].

Using the OPS technique in the sublingual area of patients in circulatory failure, we [36, 41] observed that microcirculatory alterations are frequent in shock states.

We investigated 50 patients with severe sepsis (n = 8) and septic shock (n = 42)

within 48 hours of the onset of sepsis. Compared to young healthy volunteers and

age matched controls (patients before cardiac surgery), septic patients presented

a decrease in capillary density (4.5 [4.2 – 5.2] n/mm vs 5.4 [5.4 – 6.3] n/mm in

controls, p<0.05) and a decrease in the proportion of the perfused capillaries

(Fig. 1). An increase in the number of capillaries with stagnant flow and in the number of capillaries with intermittent flow equally contributed to the decrease in capillary perfusion (32 [27–39]% and 32 [22–37]%, respectively, in septic patients vs 4 [3–5]% and 5 [4–6]% in controls). Interestingly, these alterations were fully reversible: after topical application of a high dose of acetylcholine the proportion of perfused capillaries increased from 44 [24–60]% to 94 [77–96]%, p<0.01). This suggests that these alterations are not fixed and that the microcirculation can be manipulated. Current studies are ongoing to determine the effects of various interventions on the microcirculation in humans. Vasodilators may also be of value [42]. Recently, Spronk et al. [43] reported that nitroglycerin improved the sublin- gual microcirculation; unfortunately it also induced a marked hypotension. In addition the potential cytotoxic effects of NO donors should not be neglected so that further studies are needed before this intervention can be translated into clinical practice.

Microcirculatory alterations can also be observed in other conditions than sepsis. We [41] observed that the proportion of perfused capillaries was also decreased in patients with severe heart failure and cardiogenic shock (Fig. 2). These alterations were also fully reversed by the topical application of acetylcholine.

Microvascular blood flow can also be altered after cardiac surgery. In 28 patients submitted to cardiac surgery, we observed that the proportion of perfused capil- laries decreased after cardiopulmonary bypass (from 88 [87–88] to 54 [51–56], p<0.05), and remained altered during the first hours of admission in the intensive care unit (ICU), and almost normalized the day after surgery [44]. However, these alterations were far less pronounced than in patients with septic or cardiogenic shock.

Fig. 1. Proportion of perfused capillaries in patients with sepsis. +++p<0.001 vs volunteers

Modified from [36] with permission

Influence of Systemic Factors?

One major question is whether these microvascular blood flow alterations are influenced by systemic factors. If yes, monitoring the microcirculation may be useless, as these alterations may be inferred from more easily applicable monitor- ing techniques.

As microcirculatory and macrocirculatory alterations usually coexist, it is quite difficult to separate the influence of both factors. Experimental studies suggest that microcirculatory alterations can occur even when blood flow or perfusion pressure are maintained [12, 13, 45]. In a hyperdynamic model of endotoxic shock, Tugtekin et al. [45] observed that the number of unperfused and heterogeneously perfused gut villi was increased. Similarly, Nakajima et al. [13] reported that endotoxin decreased the density of perfused villi and red blood cell velocity in perfused villi, independent of the effects on arterial pressure.

Data in patients are scarcer. Using laser Doppler in patients with septic shock, LeDoux et al. [46] reported that skin blood flow was not affected when mean arterial pressure was increased from 65 to 85 mmHg with norepinephrine. Using the OPS technique on the sublingual microcirculation in 96 patients with severe sepsis and septic shock, we observed that the severity of microcirculatory alterations was not related to arterial pressure, the use of vasopressors, or cardiac index [47].

Fig. 2. Proportion of perfused capillaries in patients with severe heart failure and cardiogenic

shock. +++p<0.001 vs controls. Modified from [41] with permission

Remaining Questions

One important question is whether the microcirculatory alterations are similar and if they occur simultaneously and at the same degree of severity in the various microvascular beds. Animal models have clearly shown that similar alterations occur in striated muscles [7, 15], small bowel mucosa [10], liver [48], pancreas [49], and skinfold [4]. However, none of these models simultaneously investigated different organs, hence the severity and the time course of these lesions may vary between the different organs. This may be of particular importance for the bedside monitoring of the human microcirculation, especially as the sites accessible are limited. Preliminary data in humans nevertheless suggest that similar microvas- cular alterations can be observed in the sublingual area and on ileostomies and colostomies [50].

Link Between Microcirculatory Alterations and Outcome

The alterations in microvascular blood flow can have important implications. In rats submitted to 60 min of severe hemorrhage with subsequent restoration of blood volume, Zhao et al. [3] observed that microvascular alterations were more severe in rats that subsequently died compared to survivors, despite similar whole-body hemodynamics. Similarly, Kerger et al. [51] reported that functional capillary density and interstitial PO 2 in the hamster skinfold were lower in non- survivors during hemorrhage and after resuscitation. Hence, in animal models microcirculatory alterations have been related to outcome.

In our recent study in patients with severe sepsis [36], we observed that the severity of microcirculatory alterations was more pronounced in non-survivors than in survivors. We further [52] daily investigated the sublingual microcircula- tion in a cohort of 49 patients with septic shock up to shock resolution or death, and we observed that microvascular blood flow rapidly resolved in survivors but remained altered in non survivors, whether these patients died in shock or from multiple organ failure after shock was resolved. In survivors, microcirculatory alterations improved even though these patients were still on vasopressors for several days. In addition, the observation that microvascular alterations improved by more than 7.5% within the first 24 hours of observation was an excellent predictor of outcome (71% survival rate above this cut-off value versus only 19%

below it). These data suggest that microvascular blood flow alterations are impli- cated in the pathophysiological process involved in the development of multiple organ failure and death in septic patients.

Conclusion

The microcirculation is a key element in tissue oxygenation, as it is the place

where most oxygen and nutrient exchange take place. Multiple experimental

studies have demonstrated that microvascular blood flow is altered in hemor-

rhage, ischemia-reperfusion injury and especially in sepsis. These alterations can

be observed in various organs and are characterized by an increased number of absent or intermittently perfused capillaries and heterogeneity in blood flow. The study of the microcirculation in humans has long been difficult. Laser Doppler or phlethysmography techniques do not take into account heterogeneity of blood flow, and hence are not able to detect these alterations. The development of OPS imaging techniques has allowed the direct visualization of the human microcircu- lation. Using OPS techniques we demonstrated that the sublingual microcircula- tion of patients with acute circulatory failure is markedly altered and that these alterations are related to outcome. These alterations are not influenced by arterial pressure or vasopressor agents and cannot be detected by the classical monitoring devices. Monitoring the microcirculation of patients with acute circulatory failure may help to detect patients in whom further interventions may be required.

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