Fig 1.
Definition of pressure drop-induced vasodilatation (PDiVD).
The upper panel shows normal CA curves (solid lines) with typical “knee”-shaped deviating from a linear correlation between blood pressure and perfusion that indicates completely lost CA (dashed line) [22]. The corresponding CA limits are indicated. The vertical gray shaded area demonstrates that the lower CA limit could stay at the same blood pressure value even when the CA curve would be flatter, meaning that the same lower CA limit would be associated with less perfusion. To quantify this, we plotted the time course of the perfusion per blood pressure of the CA measurement (lower panels). When CA is abrogated (dashed line in the upper panel) the perfusion/blood pressure passively decreases with time or falling systemic blood pressure (lower right panel), which is typical for elastic tubes. In contrast, with intact CA (solid lines in the upper panel) the perfusion/blood pressure starts to rise from a certain time point on, reaches a maximum and then falls again (lower left panel). We defined the difference between the initial perfusion/blood pressure value and the maximal one as PDiVD (horizontal dashed lines in the lower left panel).
Table 1.
TaqMan Gene Expression Assays used for ddPCR.
Table 2.
Physiological variables.
Fig 2.
Time course of the upwards sift of the lower CA limit after 1K1C surgery.
The lower CA limit shifted in both mouse lines in a similar fashion. Significantly elevated lower CA limits were not observed before 8 weeks of hypertension. Thereafter no further shift of the lower CA limit occurred. * = p<0.05 after pooling the data from 0 and 6 weeks and 8 and 10 weeks respectively. Means ±SD of as many animals as indicated.
Fig 3.
Assessment of cerebrovascular reactivity.
Ten weeks after 1K1C surgery the systemic mean arterial blood pressure (MABP) of all animals ranged from 106 to 158 mmHg and did not differ between wt and CLR-tg mice (A, left bar graph). The lower CA limit was comparably shifted in both mouse lines (A, middle bar graph; cf. also Fig 2). However the PDiVD was significantly higher in CLR-tg compared to wt mice (A, right bar graph). When excluding two wt and two CLR-tg mice with a MABP below 115 mmHg, the CLR-tg showed slightly but significantly lower MABP (B, left bar graph), again no difference in the lower CA limit (B, middle bar graph) and an even more pronounced higher PDiVD (B, right bar graph). Means ±SD of as many animals as indicated.
Fig 4.
CGRP binding sites in different brain structures.
Generally CGRP binding was much higher in CLR-tg mice. Moreover, in wt mice hypertension did increase CGRP binding non-significantly only by trend whereas in CLR-tg mice hypertension-induced increase in CGRP binding was more pronounced and significant (* = p<0.05 and ** = p<0.01 vs. normotensive wt mice, # = p<0.05 and ## = p<0.01 vs. normotension, + = p<0.05 and ++ = p<0.01 vs. hypertensive wt mice). Means ±SD of 7 animals per group.
Fig 5.
Gene expression analysis of brain tissue.
The abundance of Calclr mRNA was due to the transgenic overexpression much higher in the CLR-tg mice (upper left panel). Hypertension induced a significant reduction of the Calclr expression by about 70% in wt mice whereas it slightly but non-significantly increased in CLR-tg mice most likely due to the increased expression of Acta2 (upper right panel) as the transgene of the CLR-tg mice is driven by the Acta2 promoter. Expression of the RAMP-1 mRNA was the same in both mouse lines with a non-significant tendency for reduction in response to hypertension. In contrast RAMP-2 expression was not only generally much less abundant but also significantly reduced by about 65% in both mouse lines in response to hypertension. Actb showed no expression changes in response to genetic alteration or chronic hypertension. Means ±SEM of 7 animals per group.