metarteriola

ultimo aggiornamento: 16 Luglio 2020 alle 13:14

definizione

Piccolo vaso arterioso precapillare, con caratteri di transizione tra struttura arteriosa e struttura capillare: l’endotelio è rinforzato soltanto da una tunica avventizia e fibrille reticolari, con poche cellule muscolari sparse raggruppate in manicotto contrattile formato da 2 o 3 strati di cellule muscolari lisce a disposizione circolare, nel punto in cui le metarteriole si trasformano in capillari: questo di fibre muscolari lisce, controllato numerosi fattori nervosi e ormonali, è detto sfintere precapillare e riveste un ruolo rilevante nella regolazione del flusso ematico all’interno del letto capillare, regolando il transito del sangue nelle reti capillari dei tessuti corporei.

Queste strutture sono state scoperte negli anni ’50 del novecento, a livello del microcircolo mesenterico; anche se secondo alcuni autori non ci sono evidenze della presenza delle metarteriole e sfinteri pericapillari in ogni organo o viscere, si ritiene svolgano un ruolo fondamentale nella regolazione del flusso ematico nel letto capillare, sia un condizioni fisiologiche, che in situazioni di stress.

azione sfinterica e regolazione del flusso sanguigno

Lo sfintere precapillare ha il compito di regolare il flusso sanguigno nel letto capillare, attraverso la contrazione o il rilasciamento: l’intero complesso di capillari può essere bypassato qualora il sangue venga deviato in via preferenziale lungo le anastomosi arterovenose (canale di attraversamento) a livello delle metarteriole, per effetto della chiusura degli sfinteri precapillari; queste strutture hanno anche il compito di regolare la pressione di perfusione: lesioni degli sfinteri precapillari o alterazioni funzionali che ne impediscano al contrazione, incrementano la pressione nel letto capillare, facilitando il passaggio di liquidi negli spazi interstiziali, favorendo la formazione di gonfiore nei tessuti o edema.

metarteriole e circolazione cerebrale

L’aumento del metabolismo cellulare, attraverso il rilascio di sostanze vasoattive, è in grado di influenzare la perfusione del letto capillare nei distretti attivati da mediatori ormonali (paracrini o endocrini) o da stimolazione nervosa

Active nerve cells release vasodilators that increase their energy supply by dilating local blood vessels, a mechanism termed neurovascular coupling and the basis of BOLD functional neuroimaging signals. Here, we reveal a mechanism for cerebral blood flow control, a precapillary sphincter at the transition between the penetrating arteriole and first order capillary, linking blood flow in capillaries to the arteriolar inflow. The sphincters are encircled by contractile mural cells, which are capable of bidirectional control of the length and width of the enclosed vessel segment. The hemodynamic consequence is that precapillary sphincters can generate the largest changes in the cerebrovascular flow resistance of all brain vessel segments, thereby controlling capillary flow while protecting the downstream capillary bed and brain tissue from adverse pressure fluctuations. Cortical spreading depolarization constricts sphincters and causes vascular trapping of blood cells. Thus, precapillary sphincters are bottlenecks for brain capillary blood flow.

Neurovascular coupling (NVC) is the signaling mechanism that links neuronal activity to local increases in cerebral blood flow1,2,3,4. Increased Ca2+ in neurons and astrocytes triggers the release of vasoactive compounds that dilate capillaries and penetrating arterioles (PAs) and thereby increases blood flow. The activity-induced flow increase is based on coordinated changes in vessel diameters, which are regulated by Ca2+ fluctuations within the vascular smooth muscle cells (VSMCs) that circumscribe arteries and larger arterioles and the pericytes that ensheathe capillaries close to the PA5,6,7,8. PAs branch into capillary networks that supply each cortical layer with oxygen and glucose9. It remains unclear how this topology achieves a balanced and adequate perfusion of capillary beds along the entire cortical depth while simultaneously shielding the delicate brain tissue from the mechanical impact of pressure. Here, we reveal the structure and function of brain precapillary sphincters, which may serve to protect capillaries from high blood pressure while preserving blood supply to all bifurcations along the PA. We characterized the precapillary sphincter as a mural cell encircling an indentation of the capillary where it emerges from the PA. The sphincter cells were morphologically similar to brain pericytes, contained α-smooth muscle actin (α-SMA), and were ensheathed by structural proteins. Precapillary sphincters were mostly present at proximal bifurcations of PAs, ideally positioned to balance perfusion along the PA and to protect against arterial pressure. Though precapillary sphincters have been known for decades10, their existence, except within the mesentery11,12,13, has remained controversial14,15. This study provides unequivocal structural and functional evidence of brain precapillary sphincters and examines their role in NVC and during cortical spreading depolarization (CSD).

 

Precapillary sphincters regulate blood flow

Having established the occurrence and morphology of precapillary sphincter complexes, we examined their role in blood flow regulation. First, we confirmed expression of α-SMA within the precapillary sphincter mural cell in coronal slices of NG2-dsRed mice (Fig. 3a, vascular lumen and cell nuclei co-stained with lectin and DAPI, respectively. Supplementary Fig. 4 and Supplementary Movie 2). Next, we analyzed the vasomotor responses of the PA, precapillary sphincter, bulb, and first order capillary vessel segments in response to electrical whisker pad stimulation in an in vivo two-photon setup (Supplementary Fig. 1). Careful placement of linear regions of interest (ROIs) in image hyperstacks were used to avoid intersegmental interference in diameter calculations before and during whisker stimulation (Fig. 3b, c). Precapillary sphincters dilated during stimulation, followed by a poststimulus undershoot (constriction) 20–30 s after stimulation. Using four-dimensional hyperstack imaging23, we confirmed that the undershoot was not an artifact of drift on the z-axis (Supplementary Movie 3). Relative diameter changes were significantly larger at the sphincter than the PA and the rest of the first order capillary during both dilation (33.75 ± 4.08%, Fig. 3e and Supplementary Table 1) and the undershoot (−12.40 ± 2.10%, Fig. 3f and Supplementary Table 1). To estimate the corresponding changes in flow resistance per unit length, we applied Poiseuille’s law at baseline, maximal dilation and maximal undershoot (Fig. 3g–i). The flow resistance of the sphincter at rest was significantly greater than in the other segments and decreased significantly more (65.9% decrease, Fig. 3h) during dilation compared to all other segments (40.8% for the first order capillary, Fig. 3h). During the poststimulus undershoot, flow resistance increased by 80.2% at the sphincter (Fig. 3i), highlighting the sensitivity of flow resistance to sphincter constriction due to the power law relationship between diameter and flow resistance (Fig. 2g). Moreover, we observed that the length of precapillary sphincters decreased during stimulation and increased during the undershoot (Supplementary Fig. 5). Shortening of the sphincter decreases the absolute flow resistance across the precapillary sphincter and vice versa, augmenting the pressure drop reduction across the sphincter during stimulation and the pressure drop increase during the poststimulus undershoot.

 

Precapillary sphincters maintain perfusion in the cerebral cortex

Abstract

Active nerve cells release vasodilators that increase their energy supply by dilating local blood vessels, a mechanism termed neurovascular coupling and the basis of BOLD functional neuroimaging signals. Here, we reveal a mechanism for cerebral blood flow control, a precapillary sphincter at the transition between the penetrating arteriole and first order capillary, linking blood flow in capillaries to the arteriolar inflow. The sphincters are encircled by contractile mural cells, which are capable of bidirectional control of the length and width of the enclosed vessel segment. The hemodynamic consequence is that precapillary sphincters can generate the largest changes in the cerebrovascular flow resistance of all brain vessel segments, thereby controlling capillary flow while protecting the downstream capillary bed and brain tissue from adverse pressure fluctuations. Cortical spreading depolarization constricts sphincters and causes vascular trapping of blood cells. Thus, precapillary sphincters are bottlenecks for brain capillary blood flow.

Introduction

Neurovascular coupling (NVC) is the signaling mechanism that links neuronal activity to local increases in cerebral blood flow1,2,3,4. Increased Ca2+ in neurons and astrocytes triggers the release of vasoactive compounds that dilate capillaries and penetrating arterioles (PAs) and thereby increases blood flow. The activity-induced flow increase is based on coordinated changes in vessel diameters, which are regulated by Ca2+ fluctuations within the vascular smooth muscle cells (VSMCs) that circumscribe arteries and larger arterioles and the pericytes that ensheathe capillaries close to the PA5,6,7,8. PAs branch into capillary networks that supply each cortical layer with oxygen and glucose9. It remains unclear how this topology achieves a balanced and adequate perfusion of capillary beds along the entire cortical depth while simultaneously shielding the delicate brain tissue from the mechanical impact of pressure. Here, we reveal the structure and function of brain precapillary sphincters, which may serve to protect capillaries from high blood pressure while preserving blood supply to all bifurcations along the PA. We characterized the precapillary sphincter as a mural cell encircling an indentation of the capillary where it emerges from the PA. The sphincter cells were morphologically similar to brain pericytes, contained α-smooth muscle actin (α-SMA), and were ensheathed by structural proteins. Precapillary sphincters were mostly present at proximal bifurcations of PAs, ideally positioned to balance perfusion along the PA and to protect against arterial pressure. Though precapillary sphincters have been known for decades10, their existence, except within the mesentery11,12,13, has remained controversial14,15. This study provides unequivocal structural and functional evidence of brain precapillary sphincters and examines their role in NVC and during cortical spreading depolarization (CSD).

Results

Precapillary sphincters at proximal branch points

We identified precapillary sphincters in mice expressing dsRed under the control of the NG2 promoter as dsRed-positive cells encircling an indentation of the capillary lumen as it emerges from the PA branch points (Fig. 1a). Precapillary sphincters were often but not always followed by a distention of the lumen, which we denoted as the bulb. The dsRed signal from the precapillary sphincter was usually brighter than dsRed signals from other mural cells on the PAs and first order capillaries, indicating high-NG2 expression, whereas the dsRed signal from the bulb region was low (Fig. 1a, b, d). We also identified precapillary sphincters and bulbs in awake mice with chronic cranial windows (Fig. 1c and Supplementary Fig. 3n = 4) and anaesthetized NG2-dsRed mice with thinned skull over the barrel cortex16 (Fig. 1b, Supplementary Fig. 2 and Supplementary Movie 1n = 3 mice). Ex vivo studies revealed that the NG2-positive cells encircling the precapillary sphincter were individual cells encompassing the sphincter at the branch point and not processes of mural cells extending from the PA (Fig. 1d). Close inspection revealed a continuum of mural cell cyto-architecture from VSMC encircled pial arterioles to pericyte ensheathed capillaries (Fig. 1e) as described previously17,18,19. The mural cell encircling the sphincter stained weakly (if any) for Nissl neurotrace 500/52520 and not for CD14621,22, but showed robust CD13 staining (no marker was specific for pericytes, see Supplementary Fig. 7) and α-SMA expression (see below).

Fig. 1: Sphincters on proximal branches of penetrating arterioles.
figure1

a Left panel: Maximal intensity projected in vivo two-photon laser scanning microscopy image of an NG2-dsRed mouse barrel cortex. An indentation of the capillary lumen is observed at the branching of the PA and is encircled by bright dsRed cell(s) (dashed insert). This structure is denoted as a precapillary sphincter. Immediately after the sphincter, a sparsely dsRed-labeled distention of the capillary lumen is observed, which we refer to as the bulb. Right panels: Single z-plane showing overlay, FITC-channel, and dsRed channel of the dashed insert. Arrows indicate the PA (red), sphincter (blue), bulb (green), and 1st order capillary (yellow). bd Local TPLSM projections of precapillary sphincters in the cortex of a thinned skull mouse in vivo (b), an awake mouse harboring a chronic cranial window in vivo (c) with white arrows marking the precapillary sphincter, and an ex vivo coronal slice of a FITC-conjugated lectin (green) stained NG2-dsRed mouse (red) with DAPI-stained (blue) nuclei (d). The precapillary sphincter cell nucleus is arched, as it follows the cell shape, and is marked by a white arrowhead. e Schematic of a PA with the a a precapillary sphincter at the proximal branch point. The illustration is based on confocal imaging of coronal slices ex vivo and the exact morphology and location of NG2-dsRed positive cells and their DAPI stained nuclei are shown. For the complete figure including a venule, see Supplementary Fig. 8.

Having established the structure of precapillary sphincters, we examined their occurrence and localization within the cortical vascular network. In keeping with the work of Duvernoy et al.9, we identified a range of PA subtypes (Fig. 2b) that differed in size, branching pattern, and cortical penetration. The heterogeneity in PA subtypes was partially reflected in the localization and frequency of sphincter and bulb occurrence. Out of the 108 PAs with 602 branches we could resolve in 9 mice examined, we found that 72% contained at least one sphincter (and that each PA had on average 28% branches with a sphincter). Precapillary sphincters localized predominantly in the upper layers of the cortex (Fig. 2c) and were observed mainly at the proximal PA branch points (Fig. 2d) of relatively large PAs branching into relatively large first order capillaries (Fig. 2e, f). Thus, sphincters localize to large proximal vessels that have higher blood pressures than smaller downstream vessels. The bulb usually succeeded a sphincter but was less prevalent and did not correlate positively with the diameter of first order capillaries (Fig. 2e); bulbs were prevalent when the PA diameter was large compared to the first order capillary (Fig. 2f). For branches positive for a precapillary sphincter, the average diameter of the PA was 11.4 ± 0.6 µm, the precapillary sphincter 3.4 ± 0.2 µm, the bulb 5.8 ± 0.2 µm, and the first order capillary 5.3 ± 0.2 µm. As per Poiseuille’s law (adjusted for flow velocity, Fig. 2g), a lumen diameter of 3–4 µm is at the border of high flow resistance, providing an effective means of changing the pressure drop per unit length. We conclude that precapillary sphincter complexes (sphincter and bulb) are characterized by an indentation of the lumen at the branch point encircled by a mural cell, usually followed by a distention (the bulb), and are common at proximal PA branch points of larger PAs in the mouse cortex.

Fig. 2: Location of sphincters help pressure equalization along PA.
figure2

a Representatives of four PA subtypes reaching different cortical layers based on ex vivo data. Precapillary sphincters are found at varying depths (marked by blue arrowheads and branchpoint numbers are indicated on the right PA). bf Dependency of the presence and location of precapillary sphincters and bulbs (binned quantification) on various parameters. Criteria for the positive presence of sphincter or bulb at a branch point: sphincter <0.8 and bulb >1.25 times the diameter of a first order capillary, in total 602 branchpoints of 108 PAs in 9 mice were analyzed, ±SEM, linear regression, * = slope deviates significantly from 0. b Dependency on cortical depth (bin size 100 µm). c Dependency on PA branch number (counting from the proximal end). d Dependency on PA diameter (bin size 2 µm). e Dependency on first order capillary diameter (bin size 1 µm). f Dependency on first order capillary/PA diameter ratios (bin sizes as in d and e). g Top panel: Illustration of a pressure decrease across a precapillary sphincter and modified expression of Poiseuille’s law. ΔP is the pressure difference, L unit length, µ viscosity, and υ flow velocity. Lower left: Illustration of Poiseuille’s law showing how the pressure drop (defined as pressure difference per unit length times viscosity, ΔPμLΔPμL, also unit of color scale), depends on the cylindrical lumen diameter and flow velocity. Note how the pressure drop increases with lumen diameters below 4 µm. Lower right: Combining flow resistance in laminar fluid flow with Poiseuille’s law yields an equivalent representation of how flow resistance (defined as resistance per unit length and viscosity, RμLRμL) depends on lumen diameter. Source data are provided as a Source Data file.

Precapillary sphincters regulate blood flow

Having established the occurrence and morphology of precapillary sphincter complexes, we examined their role in blood flow regulation. First, we confirmed expression of α-SMA within the precapillary sphincter mural cell in coronal slices of NG2-dsRed mice (Fig. 3a, vascular lumen and cell nuclei co-stained with lectin and DAPI, respectively. Supplementary Fig. 4 and Supplementary Movie 2). Next, we analyzed the vasomotor responses of the PA, precapillary sphincter, bulb, and first order capillary vessel segments in response to electrical whisker pad stimulation in an in vivo two-photon setup (Supplementary Fig. 1). Careful placement of linear regions of interest (ROIs) in image hyperstacks were used to avoid intersegmental interference in diameter calculations before and during whisker stimulation (Fig. 3b, c). Precapillary sphincters dilated during stimulation, followed by a poststimulus undershoot (constriction) 20–30 s after stimulation. Using four-dimensional hyperstack imaging23, we confirmed that the undershoot was not an artifact of drift on the z-axis (Supplementary Movie 3). Relative diameter changes were significantly larger at the sphincter than the PA and the rest of the first order capillary during both dilation (33.75 ± 4.08%, Fig. 3e and Supplementary Table 1) and the undershoot (−12.40 ± 2.10%, Fig. 3f and Supplementary Table 1). To estimate the corresponding changes in flow resistance per unit length, we applied Poiseuille’s law at baseline, maximal dilation and maximal undershoot (Fig. 3g–i). The flow resistance of the sphincter at rest was significantly greater than in the other segments and decreased significantly more (65.9% decrease, Fig. 3h) during dilation compared to all other segments (40.8% for the first order capillary, Fig. 3h). During the poststimulus undershoot, flow resistance increased by 80.2% at the sphincter (Fig. 3i), highlighting the sensitivity of flow resistance to sphincter constriction due to the power law relationship between diameter and flow resistance (Fig. 2g). Moreover, we observed that the length of precapillary sphincters decreased during stimulation and increased during the undershoot (Supplementary Fig. 5). Shortening of the sphincter decreases the absolute flow resistance across the precapillary sphincter and vice versa, augmenting the pressure drop reduction across the sphincter during stimulation and the pressure drop increase during the poststimulus undershoot.

Fig. 3: Sphincters actively regulate blood flow.
figure3

a Ex vivo coronal slices of an FITC-lectin-stained NG2-dsRed mouse immunostained for α-SMA. Left panel: maximal projection of a PA with a precapillary sphincter at the first order capillary branch point. The marked area is shown on the right. Right panels: local maximal intensity projections of the precapillary sphincter region of dsRed, α-SMA, DAPI, or all channels including FITC-lectin overlaid. The lumen (cyan) and the outlines of the dsRed signal of the precapillary sphincter cell have been marked by dashed lines in the three grayscale images. bi In vivo whisker pad stimulation experiments (anaesthetized NG2-dsRed mice) using maximal intensity projected 4D data obtained by two-photon microscopy, n = 13 mice for PA and sphincter, 8 for bulb and 12 for first order capillary, ±SEM. b Maximal intensity projection of a PA branch point where the colored lines indicate the ROIs for diameter measurements of the vessel segments: PA (red), precapillary sphincter (blue), bulb (green), and first order capillary (yellow). c Representative time series of relative diameter dynamics in each vessel segment upon 20 s of 5 Hz whisker pad stimulation (gray bar, start at time zero). d Summary of baseline diameters (absolute values). e Summary of peak diameter change upon whisker pad stimulation. f Summary of the peak undershoot phase after whisker pad stimulation. g A proxy of flow resistance at baseline estimated using Poiseuille’s law. h Relative change in flow resistance at peak dilation during stimulation. i Relative change in flow resistance during the poststimulation undershoot. The Kruskal–Wallis test was used in (dg, and i) to reveal differences among vessel segments, followed by a Wilcoxon rank-sum test (with Holm’s p value adjustment) for pairwise comparisons. LME models were used in (efh, and m) to test for differences among segments, followed by Tukey post hoc tests for pairwise comparisons. In each figure, significance codes *p < 0.05, **p < < 0.01, and ***p < 0.001. Source data are provided as a Source Data file.

Next, we examined the correlation between red blood cell (RBC) flux and diameter changes in response to whisker pad stimulation (Fig. 4a–d). RBC velocity fluctuated in synchrony with systolic and diastolic oscillations in arterial blood pressure (Fig. 4a, b). At rest, the average RBC velocity through precapillary sphincters was 8.7 ± 0.6 mm/s (Fig. 4c), significantly higher than for the bulb (3.6 ± 0.6 mm/s) and the first order capillary (4.7 ± 0.6 mm/s), but correlated with the relative differences in the resting diameters of the vessel segments. As shown in Fig. 2g, high RBC velocity through the narrow lumen of the precapillary sphincter amplifies the reduction in pressure across the sphincter due to high shear, i.e., augments the reduction of pressure from larger proximal PAs to downstream capillaries. From the baseline measures, the pressure drop per unit length is 4-times larger in the sphincter than the first order capillary, assuming that RBC velocity and fluid velocity are equal (see Fig. 2g). During whisker stimulation (Fig. 4c), both diameter and RBC velocity increased in each segment, but significantly more at the precapillary sphincter than the first order capillary (Fig. 4d). RBC flux through the precapillary sphincter complex increased 25% from baseline to peak stimulation (mean flux increased from 543 ± 25 to 679 ± 50 cells/s, Fig. 4c). The sphincter, however, retained a pressure-reducing effect during peak stimulation (three-times greater pressure drop per unit length compared to the first order capillary). RBC velocity and flux returned to baseline 20–30 s after ending stimulation (Fig. 4c), concurrent with the poststimulus undershoot (Fig. 3c, f). Before, during, and after whisker stimulation, we observed passage of single RBCs through the precapillary sphincter, which may optimize hematocrit along the PA24 and the oxygen delivery to brain tissue (Supplementary Movie 5). Collectively, our data suggest that the sphincter complex augments the reduction of blood pressure from the proximal PAs to downstream capillaries, actively regulates local diameter and RBC flux during functional stimulation, and equalizes the distribution of RBCs entering the upper and lower cortical layers.

Fig. 4: Red blood cell velocity and flux at the sphincter.
figure4

a Resonance scanning allows for rapid repetitive line-scans in a single z-plane (upper panel). In the resulting space–time maps (lower panel), individual cells appear in black with an angle proportional to the cell velocity. Red, blue, green, and yellow lines indicate the regions of the line-scans derived from the PA, sphincter, bulb, and first order capillary (first order capillaries were mostly scanned in consecutive experiments). b Fluctuations in femoral artery blood pressure (left upper panel) and RBC velocity (left lower panel) correlated. During whisker pad stimulation (right panel), RBC velocity increased. c Time series of RBC velocities and flux during whisker pad stimulation. RBC velocity at the precapillary sphincter was significantly higher than the bulb and first order capillary at baseline and peaked around 10 s after stimulation before returning to baseline. d Summary of the difference between maximal and baseline RBC velocity during whisker stimulation. In d, the LME analysis was performed on log-transformed data to ensure homoscedasticity. n = 6 mice, ±SEM, significance code *p < 0.05. Source data are provided as a Source Data file.

Structural elements support bottleneck function

The presence of a contractile sphincter-encircling cell supports active tone regulation. However, indentation of the sphincter may also be supported by passive elements to optimize the force–length relationship25. Therefore, we investigated whether passive structural elements constrain dilation at the sphincter by injecting papaverine (10 mM), a strong vasodilator, close to the sphincter (Fig. 5a–c). Papaverine blocks the contractility of mural cells by inhibiting vascular phosphodiesterases26 and calcium channels27. Under these conditions, passive structural elements of the vessel become the main factors that stabilize the vessel wall. Both before and after papaverine injection, the lumen diameter of the sphincter was significantly smaller than that of the bulb and first order capillary (Fig. 5c). Yet, the sphincter demonstrated significantly larger dilation in absolute and relative terms compared to the first order capillary. Structural evidence of passive connective tissue was established by staining coronal slices of NG2-dsRed mice with either a collagen α1 type I (COL1A1) or type IV antibody or Alexa633 hydrazide28, a marker of elastin (Fig. 5 and Supplementary Fig. 7). Elastin was observed in the tunica intima of PAs and at the precapillary sphincter, but not in capillaries (Fig. 5d). Collagen α1 type-I and type-IV staining was observed in the tunica externa of arterioles, precapillary sphincters, capillaries (Fig. 5e), and venules. Thus, common structural proteins ensheathed the precapillary sphincter. The data indicate that the active sphincter is supported by passive structural elements that maintains the lumen indentation and thereby assists in blood pressure reduction from the larger PAs to downstream capillaries both at rest and during stimulation.

Sphincters protect capillaries against high pressure

The blood pressure profile along the microvasculature is practically impossible to measure. However, we reassessed our conclusions about the sphincter properties in a quantitative framework by developing a simple blood flow and pressure model of a cortical network with sphincters on the two topmost branches based on image reconstruction of a single PA and its associated branchings into first and second order capillaries (Fig. 7a). The dependency of blood viscosity on diameter and hematocrit was based on prior models31 (see Methods). Despite the well-known limitations caused by boundary conditions32, the large pressure reducing effect of the sphincter on blood pressure in the proximal first order capillaries is evident from simulations (Fig. 6a, right; the effect of placing a sphincter in a distal branch was small, i.e., sphincters are only necessary with a large pressure difference between the PA and the first order capillary). The size of the pressure drop across the sphincter was inversely proportional to the diameter of the sphincter (Fig. 7b) under resting conditions and was insignificant without the indentation, i.e., when the diameter at the sphincter was equal to the first order capillary. Applying the relative diameter changes during functional stimulation reduced, but did not eliminate, the pressure drop across the sphincter, in line with our calculations above, using RBC velocity and diameter changes during peak stimulation (Fig. 4c, d). The pressure increase in the PA during stimulation is unknown. Nonetheless, a pressure increase augments the relative increase in pressure drop across the sphincter (compare dark with light green curves in Fig. 7b, right). On an absolute scale, an increasing inlet pressure of the PA also increases the pressure in the first order capillary (Fig. 7c, solid curves). With increasing pressure in the PA (blue to red curves), the sphincter therefore has to contract in order to maintain a low pressure, e.g., below 20 mmHg, in the capillary. This property matches our observation that sphincters are mostly found on proximal branch points of larger PAs, which are expected to carry a higher pressure on average (Fig. 2). The bottleneck function of the sphincter reduces blood flow into the downstream capillaries. Low-flow bifurcations in the microcirculation typically receive less RBCs due to plasma skimming33. In accordance, we found that the presence of sphincters both reduced bulk flow (blue curve) and hematocrit (green curve) into the downstream capillaries (Fig. 2d) using an empirical law of blood phase separation24.

Discussion

The organization of the cortical vasculature simultaneously accommodates sufficient pressure for perfusion of each cortical layer and prevents the blood pressure head from inducing tissue damage. Here, we show that precapillary sphincters represent active bottlenecks with high flow resistance, and that they are strategically located at proximal branches of large PAs descending to large first order capillaries in upper cortical layers where microvessels withstand high arterial pressures (Figs. 2 and 6). This localization at just a subset of proximal bifurcations contributes to equalize perfusion to capillary beds along the entire length of the PA by increasing flow resistance into proximal branches as well as increasing plasma skimming. In addition, the reduction of transmural pressure in capillaries downstream from the sphincter protects capillaries and brain tissue against hemorrhage under baseline conditions and during functional activation (Figs. 246). The bulb had low pericyte coverage and remained less vasoactive than the precapillary sphincter and first order capillary (Fig. 1a, Supplementary Fig. 4, and Supplementary Movie 2). Yet, the large cross-sectional area of the bulb caused deceleration, deformation, and realignment of RBCs34 as they entered the capillary network (Supplementary Movie 5). The sphincter location is consistent with the assumption that vascular resistance is higher in the superficial cortical layers and declines over the depth of the cortex32. However, the high sensitivity of flow resistance to constriction becomes precarious in pathological conditions that promote general constriction (Fig. 6 and Supplementary Fig. 6).

In principle, the bottleneck structure of the precapillary sphincter can arise from both active contractile elements and passive structural elements. The α-SMA protein is key for contractile function, is widely expressed in VSMCs, and is consistently identified in pericytes of first order capillaries within the cortex18,19,35. In accordance with previous reports19, we observed α-SMA along the PA and in some cases up until fourth order capillaries, and within the mural cell encircling the sphincter (Fig. 3a). In addition, currently available biomarkers of pericytes were unable to identify the sphincter cell as either a pericyte or a VSMC (Supplementary Fig. 7).

While we cannot rule out passive contributions to the sphincter vasoactivity from the vasomotor responses of the adjacent PA, the presence of α-SMA supports the capacity for active vasomotor responses at the sphincter (Fig. 3c–i). The integrity and morphology of the sphincter was preserved after local administration of papaverine despite significantly greater dilation of the sphincter compared to the bulb and first order capillary (Fig. 5c). The passive and active characterization demonstrates that the sphincter is functionally different from the rest of the first order capillary. The elastin28 and filamentous collagen α1 type 1 (Fig. 5e, f) expression provide a structural scaffold that optimizes the force–length relationship of the sphincter cell and may support the structural integrity of the sphincter during increases in blood pressure (Fig. 5d, e). The preferential occurrence of sphincters at proximal PA branches suggests that the local angioarchitecture determines the overall distribution of cerebral blood flow between arterioles and capillaries (Fig. 2). The sphincter provides a heamodynamic division between capillary and arterial blood flow that is consistent with the idea that cortical flow control is regulated both in capillaries and arterioles and that regulation of capillary blood flow can occur independently from the arteriolar flow5,36,37. However, the sphincter capacity for pronounced diameter changes during functional stimulation allows for considerable dynamical shifts in the distribution of flow resistance38,39,40 (Fig. 3), which may reconcile some of the controversies regarding the dynamic regulation of cerebrovascular resistance as described previously5,35,41,42. Furthermore, as the sphincter reduces blood flow into the downstream capillaries, the sphincter also increase the relative extent of plasma skimming24,31, i.e., reduces the hematocrit into the capillaries, that in turn supports redistribution of hematocrit within the local cortical vasculature (Fig. 7d). This redistribution of hematocrit is maintained during functional sphincter dilation.

CSD is a slow depolarizing wave that is involved in migraine, traumatic brain injury, and stroke43. CSD evokes an initial vasoconstriction (phase I), immediately followed by a transient hyperemic response (phase II), which is superseded by a long-lasting vasoconstriction of arterioles and capillaries (phase III) that impairs the NVC7,44. During CSD, the sphincter exhibited pronounced diameter changes (Fig. 6) and constricted persistently during phase III (Supplementary Movie 6). Persistent sphincter constriction reduced both RBC flux and the hematocrit of the capillary bed. The long-lasting oligaemia previously described in CSD could arise from the high resistance observed at precapillary sphincters7, and further pharmacological research on this structure could improve the outcome of CSD in the ischemic brain or in patients with migraine.

Precapillary sphincters represent important anatomical sites of blood flow regulation due to their strategic placement at branch points of proximal PAs, where they reduce both pressure and RBC flux into the downstream capillary bed and thereby regulate perfusion along the PA (Fig. 7). The unique location endows larger capacities of control than is achieveable by downstream contractile capillary pericytes. Precapillary sphincters are therefore dissimilar from vascular sphincters45 that exist along the capillary and at capillary branchpoints, and have confusingly been named either precapillary smooth muscle46 or contractile capillary pericytes5,42. While maximal dilation of the sphincter cell is structurally limited, we show a high capacity for vasomotor control around a baseline diameter of 3–4 µm, where flow resistance is most sensitive to diameter changes. Therefore, precapillary sphincters represent a mechanism to equalize pressure and RBC flux between the capillary networks that branch off from the upper, middle, and lower parts of the PA. Simultaneously, sphincters protect downstream capillaries and brain tissue against adverse blood pressure. During pathology, sphincter constriction limited perfusion of downstream capillaries. Prevention of sphincter constriction may be of therapeutic importance in migraine, cerebral ischemia, and dementia47.

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