In vivo blood flow abnormalities in the transgenic knockout sickle cell mouse (original) (raw)
Standard intravital transmitted brightfield videomicroscopy provided a view of blood flow in mesenteric vessels. However, because of the abundance of mesenteric fat in these animals, a clear view of most blood flow, especially capillary flow, was obscured during overall viewing of the mesenteric circulation. Furthermore, transillumination brightfield viewing limits study to vessels in the middle portions of the mesentery where the circulatory and metabolic rates are low. In marked contrast to these limitations of brightfield transillumination are the advantages of the reverse epi-fluorescence illumination that we developed (22). Using this protocol, within three to four minutes after fluorescein injection, we obtained an excellent view of mucosal–intestinal arterioles, capillaries, and venules, a vascular bed having high circulatory and metabolic rates. To provide uniformity to our comparisons, we chose to make all flow measurements and comparisons on the venules, the vascular site where vaso-occlusion has been found to occur in trans-species studies of SCD (28, 29).
Videotapes of the blood flow revealed remarkably consistent differences in the mucosal–intestinal microvessels of control and SCD mice under normoxic conditions and in flow responses to hyperoxia. In the eight control mice, flow uniformly was rapid and even in the capillaries, arterioles, and venules of all sizes. The eight SCD mice, on the other hand, had slowing, sludging, irregularity, and stasis of flow in most vessels of all types and sizes. In medium and large vessels in which flow was nearly stopped, a back-and-forth (antegrade–retrograde) flow phenomenon was observed. These observations are reflected in frame-captured images in which the even blood flow of control mice could be distinguished from the sludged and stopped blood flow of SCD mice (Fig. 1). VASCAN and VASVEL imaging software were used to analyze blood flow velocities in vessels as a function of vessel diameter. In control mice, >95% of the small vessels had smooth and measurable flow velocity. By comparison, ∼50% of small vessels in SCD mice had trickle to no flow (or no measurable flow velocity). Overall in SCD mice, in most of the small vessels in which flow persisted, it was substantially reduced. Quantitative blood flow velocities within representative small, medium, and large venules of control and SCD mice were compared from selected images on blinded videotapes (Fig. 2). Vrbc in venules from SCD mice was strikingly lower than Vrbc in control mice, a relationship that was highly significant in small (P < 0.0002), medium (P = 0.0002), and large (P < 0.0003) venules.
Frame-captured images from videotaped intravital microscopy of the mesenteric microcirculations of control and SCD mice. (a) Image of the microcirculation in a control mouse. Regular flow is indicated by uniformity of flow presentation in large (top right) and small (bottom) vessels, which is noticeably different from that in b. (b) Image of the microcirculation in an SCD mouse. Occluded blood flow is indicated by the abrupt transition between the dark, sludged (SS RBC–rich) blood below the open arrow and the lighter blood above it. Clumps of SS RBC apparently adherent to the wall of the same vessel also are visible (filled arrows). SCD, sickle cell disease; SS RBC, sickle red blood cell.
Blood flow velocities in control and SCD mice. The bar graph demonstrates paired mean Vrbc in small, medium, and large venules of control and SCD mice. The error bars indicate SD; the ordinate is blood flow velocity, Vrbc (mm/s); for each bar, n = 10.
Visual inspection of blood flow revealed that initiation of hyperoxia resulted in prompt (within 10–100 seconds), albeit opposite, flow responses in control and SCD mice. In control mice, there was a rapid reduction of flow in most vessels and cessation in a minority of the smaller vessels. In contrast, blood never slowed during hyperoxia in the SCD mice. Rather, it nearly always was accelerated. These findings are demonstrated in frame-captured images from videotapes in which the abnormal, compromised flow of normoxic SCD blood was seen to improve within 100 seconds of hyperoxia (Fig. 3). To further define the influence of hyperoxia on Vrbc, after decoding the tapes to determine the experimental group, we compared this parameter within five venules each from control and SCD mice in representative vessels from blinded videotapes. The venules selected had mean (and range) diameters of 31.4 (14.5–55.8) μm for the control group and 35.8 (14.9–59.8) μm for the SCD group. Vrbc was measured under normoxic conditions and 100 seconds after the initiation of hyperoxia (Fig. 4). In control mice, hyperoxia resulted in consistent decreases in Vrbc (P < 0.008). Conversely, in SCD mice, hyperoxia resulted in consistent increases in Vrbc (P < 0.004). We used these measured Vrbc data to calculate Vmean, wall shear rate, and Q for each of the data points (Fig. 4). In comparing these 10 selected vessels, the flow parameters of hyperoxic SCD mice improved to levels matching those of normoxic control mice (Fig. 4).
Frame-captured images from videotaped intravital microscopy of the mesenteric microcirculations of SCD mice during normoxic and hyperoxic conditions. (a) Sludged flow of normoxic SCD blood indicated by discontinuous columns of RBC. A site of vaso-occlusion (filled arrow) has apparently distended the vessel proximally (top open arrow) and vacated the vessel of blood distally (bottom open arrow). (b) Improved flow of hyperoxic SCD blood indicated by less distention of the proximal vessel (top open arrow) and return of blood flow distally (bottom open arrow). RBC, red blood cell.
Changes in venular blood flow parameters of SCD and control mice induced by hyperoxia. Data pairs representing flow parameter values are shown as open circles connected by lines, the slope of which reflects the change in the parameter induced by hyperoxia. The top panels contain data from control mice, and the bottom panels contain data from SCD mice. (a and b) Vrbc data; (c and d) Vmean data; (e and f) wall shear rate; (g and h) volume metric flow rate (Q). The units for each parameter are shown in the ordinate labels. For both the control and SCD mice, n = 5.
We observed a modest (e.g., ∼5%) contraction of diameter in some medium and small arterioles of control mice (data not shown), which may relate to the downstream slowing of blood flow observed. Verification of that association will require improved imaging software and focusing the videotaping specifically on arterioles. We postulate that the slowing of flow we observed in control mice was due to hyperoxia-induced constriction of arterioles (19) proximal in the circulation to the majority of the vessels we videotaped and studied. No changes in arteriolar diameters were observed in SCD mice. The increase in velocity in this group was likely due to the salutary rheologic effect of oxygen on Hb S polymerization, SS RBC sickling, and rheologic properties of blood (30).The net improvement in blood flow parameters is most likely the result of rheologic improvements outweighing undetected arteriolar constriction or of the combined influences of the same rheologic improvements and the arteriolar unresponsiveness reported for a different SCD mouse model (20). The hyperoxia-induced changes in both control and SCD mice were reversed with resumption of normoxia.
To rule out the possibility that the changes in blood flow parameters we observed were not the result of inherited differences in the vascular properties or susceptibility to physical manipulation (e.g., stretching) or contact of any instrument with the mesentery, we also studied blood flow in transgenic knockout Hb AS mice that have the identical genetic background as the SCD mice as a result of similar rounds of crossbreeding. Visual inspection of the mesenteric–intestinal microcirculations of these mice (n = 4) revealed flow characteristics indistinguishable from those of normal C57BL/6 controls and markedly different from those of the SCD mice. None of the Hb AS mice were observed to have the slowed, sludged, or intermittent characteristic of SCD mice. In response to hyperoxia, these mice experienced slowing of blood flow velocity similar to that observed in normal control mice. In no instance did the blood flow velocity increase in the manner observed in SCD mice.