Research ArticleBIOPHYSICS

Oxygen tension–mediated erythrocyte membrane interactions regulate cerebral capillary hyperemia

See allHide authors and affiliations

Science Advances  29 May 2019:
Vol. 5, no. 5, eaaw4466
DOI: 10.1126/sciadv.aaw4466
  • Fig. 1 DeoxyHb–band 3 interactions regulate PO2-mediated RBC capillary velocity.

    (A) Schematics of the hypothesized Hb–band 3 interactions during deoxygenation. The binding of deoxyHb to band 3 during RBC deoxygenation weakens the band 3–ankyrin interactions, resulting in more deformable RBCs. (B) Schematics of transgenic mouse RBCs (mRBCs) with modified deoxyHb–band 3 interactions. (C) Microfluidic setup for ex vivo analysis of PO2-mediated RBC velocity in capillary. The microfluidic device was submerged in a sulfite sink with 0, 0.01, 0.1, or 1 M sodium sulfite to control the PO2 inside the microfluidic channel. A high-speed camera was used to record RBC motion in the capillary. (D) Time-lapse images showed that mRBCs flow faster at reduced PO2. Scale bar, 5 μm. (E) mRBC velocity in capillary as a function of PO2. The velocity of mRBC-WT and mRBC-subst 1-35 changes linearly with PO2 [mRBC-WT: n = 146, RBC velocity (mm/s) = −0.132 × PO2 (mmHg) + 26.455, R2 = 0.979; mRBC-subst 1-35: n = 149, RBC velocity (mm/s) = −0.151 × PO2 (mmHg) + 28.913, R2 = 0.995]. (F) The velocity of mRBC-WT is significantly lower than that of mRBC-subst 1-35 at each PO2 level. ***P < 0.001, t test. (G) The velocity of mRBC-del 1-11 and mRBC-del 12-23 is not sensitive to surrounding PO2 changes and remains relatively constant at 22.88 ± 1.59 mm/s (n = 157) and 20.23 ± 1.63 mm/s (n = 115), respectively. ***P < 0.001, t test. (H) A schematic of the experimental setup for ex vivo analysis of RBC deformability as a function of PO2. The deformability of RBCs was characterized by the elongation index Dl/Dw, where Dl and Dw were, respectively, the length and thickness of an RBC flowing through the constriction. (I) The elongation index (Dl/Dw) of mRBC-WT and mRBC-subst 1-35 increased linearly as the decrease of PO2 [mRBC-WT: n = 209, Dl/Dw = −0.0228 × PO2 (mmHg) + 2.7548, R2 = 0.998; mRBC-subst 1-35: n = 195, Dl/Dw = −0.0137 × PO2 (mmHg) + 2.6545, R2 = 0.994]. (J) The elongation index of mRBC-del 1-11 and mRBC-del 12-23 was not sensitive to PO2 changes and remains relatively constant at 2.41 ± 0.31 (n = 195) and 1.94 ± 0.23 (n = 194), respectively. ***P < 0.001, t test. Error bars are shown as SE.

  • Fig. 2 Transgenic mice with modified deoxyHb–band 3 interactions exhibit PO2-independent capillary hyperemia in vivo.

    (A) Experimental setup for in vivo assessing cerebral capillary hyperemia and tissue PO2 upon locally applied O2 scavenger (sodium sulfite, 0.01 and 1 M) in a mouse cerebral cortex. Through a cranial window, sodium sulfite was microinjected by a micropipette inserted 100 to 150 μm below the pial surface and placed <50 μm from a capillary. Puffing micropipette and O2 sensor microelectrode were placed in close proximity (<50 μm). RBC velocity in capillary was imaged using two-photon laser scanning microscopy. Scale bar, 30 μm. (B) Local changes of PO2 were dose dependent on the concentration of microinjected sulfite. The transient reduction in PO2 was followed by a PO2 overshoot. n = 12 and 4 mice. *P < 0.05, t test. (C) Typical images of the two-photon line scan of a capillary before and after the microinjection of sulfite. Black stripes represented RBCs. RBC velocity was obtained by calculating the slopes of the stripes. (D) Time-course plot of RBC capillary velocity increases after microinjection of sulfite for WT mice (mRBC-WT; n = 217 capillaries and 10 mice) and transgenic mice with (E) humanized band 3 (mRBC-subst 1-35; n = 153 capillaries and 8 mice), (F) enhanced deoxyHb–band 3 interactions (mRBC-del 1-11; n = 243 capillaries and 10 mice), and (G) weakened deoxyHb–band 3 interactions (mRBC-del 12-23; n = 243 capillaries and 10 mice). *P < 0.05, **P < 0.01, t test. (H) The onset time of increase of RBC capillary velocity after sulfite puffing in WT and transgenic mice. *P < 0.05, ***P < 0.001, t test. Error bars are shown as SE. NS, not significant.

  • Fig. 3 Biochemical modulation of deoxyHb–band 3 interactions regulates PO2-mediated RBC capillary velocity.

    (A) Left: DeoxyHb–band 3 interactions are manipulated by increasing (via PEP treatment) or decreasing (via Pi treatment) intracellular concentration of 2,3-DPG. Right: Tyrosine phosphorylation of band 3 (via pervanadate treatment) promotes dissociation of band 3 from the spectrin-actin skeleton. (B) The velocity of hRBC-WT, hRBC-Pi, and hRBC-PEP decreased linearly with the increase of PO2 [hRBC-WT: n = 71, RBC velocity (mm/s) = −0.451× PO2 (mmHg) + 82.074, R2 = 0.999; hRBC-Pi: n = 174, RBC velocity (mm/s) = −0.068× PO2 (mmHg) + 79.332, R2 = 0.947; hRBC-PEP: n = 167, RBC velocity (mm/s) = −0.065× PO2 (mmHg) + 74.198, R2 = 0.906]. The velocity of hRBC-pervanadate was not sensitive to PO2 changes and remained constant at 79.56 ± 0.28 mm/s (n = 198). Compared to hRBC-WT, the sensitivity of RBC velocity to PO2 changes (as indicated by the slope) was reduced in hRBC-Pi and hRBC-PEP. ***P < 0.001, t test. (C) The velocity of mRBC-WT, mRBC-Pi, and mRBC-PEP decreased linearly with the increase of PO2 [mRBC-WT: n = 146, RBC velocity (mm/s) = −0.132 × PO2 (mmHg) + 26.455, R2 = 0.981; mRBC-Pi: n = 159, RBC velocity (mm/s) = −0.11 × PO2 (mmHg) + 28.296, R2 = 0.938; mRBC-PEP: n = 146, RBC velocity (mm/s) = −0.071 × PO2 (mmHg) + 22.557, R2 = 0.885]. Compared to mRBC-WT, the sensitivity of RBC capillary velocity to PO2 changes was reduced after Pi and PEP treatments. *P < 0.05, ***P < 0.001, t test. (D) The elongation index of hRBC-WT, hRBC-Pi, and hRBC-PEP decreased linearly with the increase of PO2 [hRBC-WT: n = 239, Dl/Dw = −0.0236 × PO2 (mmHg) + 4.0001, R2 = 0.934; hRBC-Pi: n = 132, Dl/Dw = −0.0143 × PO2 (mmHg) + 4.142, R2 = 0.947; hRBC-PEP: n = 144, Dl/Dw = −0.0102 × PO2 (mmHg) + 3.6698, R2 = 0.997]. hRBC-pervanadate deformation was not sensitive to PO2 changes and remained constant at 4.08 ± 0.02 mm/s (n = 216). *P < 0.05, **P < 0.01, t test. (E) The elongation index of mRBC-WT, mRBC-Pi, and mRBC-PEP decreased linearly with the increase of PO2 [mRBC-WT: n = 207, Dl/Dw = −0.0228 × PO2 (mmHg) + 2.7548, R2 = 0.993; mRBC-Pi: n = 125, Dl/Dw = −0.005 × PO2 (mmHg) + 2.8088, R2 = 0.988; mRBC-PEP: n = 120, Dl/Dw = −0.0107 × PO2 (mmHg) + 2.4383, R2 = 0.989]. mRBC-pervanadate deformation was not sensitive to PO2 changes and remained relatively constant at 2.44 ± 0.03 mm/s (n = 216). ***P < 0.001, t test. Error bars are shown as SE.

  • Fig. 4 Dynamics of PO2-mediated RBC capillary velocity.

    (A) Schematics of experimental setup for ex vivo analysis of RBC tank-treading frequency, f (s−1), at reduced PO2. Human RBCs with microbeads (1 μm, polystyrene) attached on the cell membrane (inset images) were injected into a microfluidic channel with a constriction. f (s−1) was calculated on the basis of the orbital periods (the time for the microbead moving along with the membrane for one revolution) of the microbead. ttotal is the total exposure time of reduced PO2 that RBC experienced before constriction and controlled by varying the length of the channel before constriction. Scale bar, 5 μm. (B) Normalized f (s−1) as a function of PO2 at different ttotal. Dextran was added to increase the viscosity of RBC suspension. f (s−1) was normalized by dividing f (s−1) at PO2 = 34 mmHg. f (s−1)/f (s−1)34mmHg did not change with PO2 when ttotal = 18 ± 4 ms but increased linearly with the decrease of PO2 when ttotal = 192 ± 30 and 377 ± 11 ms. The sensitivity to PO2 changes increased as the increase of viscosity. At ttotal = 192 ± 30 ms, η = 1.36 mPa·s: n = 80, f (s−1)/f (s−1)34mmHg = −0.0043 × PO2 (mmHg) + 1.1326, R2 = 0.839; η = 2.61 mPa·s: n = 116, f (s−1)/f (s−1)34mmHg = −0.0062 × PO2 (mmHg) + 1.2036, R2 = 0.959; η = 4.66 mPa·s: n = 120, f (s−1)/f (s−1)34mmHg = −0.0115 × PO2 (mmHg) + 1.3509, R2 = 0.874. At ttotal = 377 ± 11 ms, η = 1.36 mPa·s: n = 129, f (s−1)/f (s−1) 34mmHg = −0.0072 × PO2 (mmHg) + 1.2253, R2 = 0.894; η = 2.61 mPa·s: n = 160, f (s−1)/f (s−1)34mmHg = −0.0117 × PO2 (mmHg) + 1.3677, R2 = 0.920; η = 4.66 mPa·s: n = 120, f (s−1)/f (s−1)34mmHg = −0.0141 × PO2 (mmHg) + 1.4461, R2 = 0.924. (C) Effect of ttotal on the sensitivity of f (s−1)/f (s−1) 34mmHg to PO2 changes. **P < 0.01 and ***P < 0.001, t test. (D) Normalized RBC capillary velocity V0mmHg/V34mmHg (where V0mmHg and V34mmHg are RBC velocity at PO2 = 0 mmHg and PO2 = 34 mmHg, respectively) as a function of ttotal. Critical ttotal beyond which RBC velocity did not change with ttotal was identified as 462 ± 71, 913.4 ± 63, and 955.1 ± 57 ms for hRBC-WT, mRBC-subst 1-35, and mRBC-WT, respectively. (E) Schematics of the relation between PO2, RBC deformability, and RBC capillary velocity. RBCs become more deformable at reduced PO2 and thus have a smaller cell width (r0) when flowing in a capillary with a diameter of 2R0. As a result, the gap distance between the surface of RBC and the capillary wall (R0r0) increases, leading to reduced hydrodynamic drag and thus increased RBC velocity. (F) Experimental measured changes of RBC width r0 and RBC velocity v as a function of PO2. r0 34mmHg and v34mmHg were r0 and v, respectively, at PO2 = 34 mmHg. Error bars are shown as SE.

Supplementary Materials

  • Supplementary material for this article is available at http://advances.sciencemag.org/cgi/content/full/5/5/eaaw4466/DC1

    Fig. S1. Calibration of oxygen level in microfluidic channels.

    Fig. S2. Baseline RBC velocity in WT mice and transgenic mice before sulfite puffing.

    Fig. S3. RBC 2,3-DPG and capillary velocity changes upon PEP and Pi treatments.

    Fig. S4. Effect of medium viscosity on PO2-regulated RBC tank-treading frequency and the calculation of RBC capillary velocity as a function of RBC size.

  • Supplementary Materials

    This PDF file includes:

    • Fig. S1. Calibration of oxygen level in microfluidic channels.
    • Fig. S2. Baseline RBC velocity in WT mice and transgenic mice before sulfite puffing.
    • Fig. S3. RBC 2,3-DPG and capillary velocity changes upon PEP and Pi treatments.
    • Fig. S4. Effect of medium viscosity on PO2-regulated RBC tank-treading frequency and the calculation of RBC capillary velocity as a function of RBC size.

    Download PDF

    Files in this Data Supplement:

Navigate This Article