Differential Effects of a Functional Non-cerebral GiPCR KO on Blood Flow to Different Organs and Body Compartments

First, we examined the rough impact of PTX-induced functional non-cerebral GiPCR KO on blood flow in different organs and compartments using whole-body dynamic contrast-enhanced (DCE) MRI. ANOVA determined a statistically significant main group difference between PTX and control animals. To detect differences between the same organs of both groups, Wilcoxon matched-pairs signed-rank test was performed, and a statistically decreased blood flow was only found in the brain of PTX animals (Fig. 1A). As with some other organs and compartments, median blood flow in the ventricle, reflecting ejection fraction in this experimental setting, was lower than in the control group, but without being statistically significant (Fig. 1B–F). Blood flow to the renal cortex after PTX treatment also suggested sustained perfusion of the kidney (Fig. 1C). The contrast agent accumulated in the renal calyceal system of PTX-treated animals, also indicating continued tubular excretion. The systemic results led us to investigate the effects of PTX in the brain using MRI techniques with better spatial resolution.

Fig. 1

Functional non-cerebral GiPCR KO using PTX induces cerebral hypoperfusion. Whole-body perfusion was measured using dynamic contrast-enhanced imaging in PTX-pretreated and PBS-pretreated animals (n = 5 per group). A Wilcoxon matched-pairs signed-rank test found significant hypoperfusion in the brain of PTX-injected mice in comparison to PBS-treated animals (* p < 0.05). B Lung showed normal perfusion, whereas C kidney, D muscle, E abdominal vessels, and F heart yielded a hypoperfusion trend. Shown are median, 1st, and 3rd quartile of data distribution. The whiskers extend to the largest and smallest data point, respectively.

Reduction of Global CBF Following Functional Systemic Non-cerebral GiPCR KO with PTX

To confirm and extend our observation of decreased CBF, we subjected the mice to an arterial spin labeling (ASL) MRI protocol (for details, see the “Materials and Methods” section and Fig. 2A, B), which shows the distribution of blood perfusion in the brain and provides reliable quantifications of CBF [21, 27, 28]. Coronal cross-sectional perfusion-weighted imaging (PWI) confirmed whole-brain hypoperfusion in PTX-pretreated sham-operated mice (Fig. 2C; yellow arrow). We quantified the CBF for the striatal and cortical regions (Fig. 2D, E). Both ipsi- and contralateral CBF were reduced by more than half in these regions compared to untreated sham-operated controls (Fig. 2D, E). Nevertheless, all of these reduced CBF values were above a range associated with ischemic lesions [29, 30]. Thus, in agreement with the DCE measurements (see Fig. 1), our ASL data clearly reveal a systemic suppressive effect of PTX on CBF.

Fig. 2

Functional PTX-induced non-cerebral GiPCR KO sensitizes for cerebral ischemia during permanent carotid artery occlusion. A Timeline of PBS/PTX injection, surgery (sham or CCA occlusion), and arterial spin-labeling MRI analysis. B Schematic overview of axial and coronal cross-sections of the mouse brain. The different brain regions of interest used for analysis are indicated. The red line shows the position of the cross-section corresponding to the coronal view. The blue line depicts the limit of ipsi (left)- and contra (right)-lateral brain hemispheres. C Perfusion-weighted images (PWI) indicate hypoperfusion of sham-operated PTX-treated mice (yellow arrow) compared to the sham PBS group. During left carotid artery ligation (occlusion), PBS-treated mice showed hypoperfusion visible in the ipsilateral hemisphere (green arrow), whereas PTX-pretreated mice exhibited global cerebral hypoperfusion, confirming the effects observed in whole-body perfusion analysis. Moreover, the perfusion of PTX-pretreated mice was interrupted in the ipsilateral hemisphere (red arrow) during occlusion, in comparison to animals receiving PBS. Shown are images of one representative mouse per group. Further examples are provided in Suppl. Fig. 1B. Corresponding quantification and statistics of CBF are shown in D for ipsi- and E for contralateral striatum and cortex (for details see Table 1). Statistical analysis was performed using 2-way ANOVA (* p < 0.05, *** p < 0.001). Shown are median, 1st, and 3rd quartile of data distribution. The whiskers extend to the largest and smallest data point, respectively.

PTX Administration Sensitizes to Ischemia

Having established that a functional non-cerebral GiPCR KO with PTX had an effect in CBF per se, we examined the consequences of acute occlusion of one common carotid artery (CCA) in cerebral hypoperfusion (see the “Materials and Methods” section and Fig. 2A, B). As evident from PWI, unilateral CCA occlusion in control animals treated with PBS resulted in a large decrease on CBF ipsilateral to the occlusion (Fig. 2C, Suppl. Fig. 1B; green arrow). This is also reflected in the calculated CBF values, which showed a clear hypoperfusion for the ipsilateral striatum and cortex (Fig. 2D). However, the hypoperfusion did not reach a level described to cause ischemia and necrosis [29, 30]. Of note, blood flow in the contralateral regions remained stable (Fig. 2E), which should allow for potential compensatory blood flow to the hypoperfused regions [31].

In contrast, PTX-pretreated mice showed global cerebral hypoperfusion that was further aggravated ipsilateral to the unilateral CCA ligation resulting in a complete breakdown of perfusion in both the striatum and cortex (Fig. 2C, D; Suppl. Fig. 1B; red arrow). These ipsilateral values were below the threshold at which ischemic injury occurs [29, 30]. On the contralateral side, an extent of reduction occurred that we had already observed in the sham-operated mice pretreated with PTX, and that may impede compensatory blood flow to the hypoperfused ipsilateral regions (Fig. 2E, Suppl. Fig. 1B).

Our findings show that a functional non-cerebral GiPCR KO with PTX suppresses cerebral perfusion, which upon challenge by unilateral CCA occlusion severely disrupts CBF distal to the ligation, i.e., in the ipsilateral hemisphere. We were therefore interested in how perfusion subsequently developed and compared PWI at baseline and 48 h after surgery, which corresponded to 96 h after PTX administration (Suppl. Fig. 2A, B). The CBF in the brain of the PBS-injected mice, sham-operated or transiently CCA-occluded, was invariant from baseline post surgically at 48 h (Suppl. Fig. 2C-F). The corresponding CBF in the non-cerebral GiPCR KO mice was reduced albeit not significantly compared to baseline. Compared with the CBF of non-cerebral GiPCR KO mice during occlusion (see Suppl. Fig. 1B and Fig. 2D, E) the CBF of mice monitored 48 h later, i.e., 96 h after PTX dosing (see Suppl. Fig. 2C-F), indicated a partial recovery. However, there was no difference in CBF in PTX-pretreated mice regardless of whether they were sham-operated or transiently CCA-occluded 48 h before (see Suppl. Fig. 2C-F). This finding was in contrast to the different results in the two PTX-pretreated groups, i.e., sham-operated or transiently CCA-occluded at the time of occlusion (see Suppl. Fig. 1B and Fig. 2D, E). This prompted us to further investigate consequences of collapsed perfusion in non-cerebral GiPCR KO mice after transient unilateral CCA occlusion (Suppl. Fig. 3A).

Functional Non-cerebral GiPCR KO Together with Transient Unilateral Carotid Artery Occlusion Leads to Cytotoxic and Vasogenic Edema

Diffusion-weighted images (DWI) provide a measurement of diffusion that can be quantified in the apparent diffusion coefficient (ADC) using MRI. ADC restrictions in the brain are the gold standard to identify ischemic stroke lesions, which have been shown to strongly correlate to final infarct lesions in tissue sections [32,33,34,35]. Diffusion restrictions have been known to start rapidly after stroke onset, peaking within one day, followed by slow value normalization [36, 37]. Consistent with these previous reports, PTX-pretreated and occluded mice already showed incipient ADC restrictions during occlusion (see Suppl. Fig. 3B-D), which were still evident in the mice imaged at 48 h post-surgery, corresponding to 96 h after PTX administration (Fig. 3B; red arrow, Fig. 3C). These ADC restrictions were clearly demarcated in DWIs of these mice (Suppl. Fig. 3B-D).

Fig. 3

Cytotoxic and vasogenic edema in non-cerebral GiPCR KO following transient CCA occlusion. A Timeline of baseline MRI acquisition, PBS/PTX injection, surgery (sham or CCA occlusion), and post-operative MRI acquisitions. B Representative images of mouse brains showing the apparent diffusion coefficient (ADC), T2 map, and T2-weighted images (T2WI). Red arrows indicate the ischemic lesions in occluded PTX-pretreated mice consisting of reduced signal intensity of ADC images as well as hyperintensity in T2WI and T2 maps (for more details, see Tables 1, 2, and 3). Corresponding quantification and statistical analysis of ipsilateral ADC (C) and T2 (D) in the striatum. Only PTX-pretreated mice following transient CCA occlusion presented a lesioned striatum with increments in ADC, accompanied by an increased T2 relaxation time. Statistical analysis was performed using 3-way ANOVA (* p < 0.05, ** p < 0.01, *** p < 0.001). Shown are median, 1st, and 3rd quartile of data distribution. The whiskers extend to the largest and smallest data point respectively.

Table 2 Contralateral striatum resultsTable 3 Contralateral cortex results

Moreover, T2 relaxation maps and T2WI representing vasogenic edema, demonstrated hyperintense signals only in the PTX-pretreated occluded animals at the latest timepoint (Fig. 3B, D; Suppl. Fig. 3B, E, F), consistent with previous literature [36, 37]. As the occluded animals pretreated with PTX showed vasogenic edema, we quantified edema volume in relation to their anatomical structures (Suppl. Fig. 4). In contrast, no signs of cytotoxic or vasogenic edema were detectable in both sham-operated groups and the PBS-treated occluded group 48 h after occlusion (Figs. 3C, D).

Because cytotoxic and vasogenic edema developed only in PTX-pretreated animals with transient CCA occlusion, we performed histological and immunohistochemical analyses to confirm the presence of an ischemic stroke phenotype, as we have previously done in other stroke models [20, 35]. Detection of ischemic lesions using hypoxia-inducible factor 1α (HIF-1α) and erythropoietin (EPO) immunohistochemistry has been previously shown to clearly delimit the infarct core and the peri-infarct stroke region [38, 39] (Fig. 4A-D; Suppl. Fig. 5). The immunohistochemical staining showed focal lesions in the PTX-pretreated and CCA-occluded animals demonstrating ischemia ipsilateral to the occlusion, which perfectly colocalized with hyperintense lesions seen in DWIs and T2WIs (Fig. 4). Furthermore, H&E staining and immunohistochemistry for the endothelial markers CD31 and GFAP (Suppl. Fig. 5) revealed prominent lesions with neuronal pallor, vacuolation of the neuropil and edema (H&E) in various regions of the ipsilateral hemisphere, as well as blood vessels (CD31) and reactive gliosis (GFAP). Thus, clear signs of ischemic stroke through in vivo imaging were confirmed in PTX-pretreated transiently CCA-occluded animals using immunohistochemistry and histology.

Fig. 4

Colocalization of DWIs and T2WIs with immunohistochemical ischemia in occluded PTX-pretreated mice. A Timeline of PBS/PTX injection protocol, surgery, and MRI acquisition. B DWI (b value = 600 s/mm2) and T2WI of animals at 96 h on the coronal projection. The occluded PTX-pretreated mice show hyperintensities in the striatal, hippocampal, and cortical brain regions on DWI and T2WI (orange arrowheads). Animals of the other groups showed no visible lesions. For more details, see Table 1. C HIF-1α is stained in hippocampal stroke regions and marks the infarcted region colocalizing with the DWIs. D. Staining of the hypoxia-inducible cytokine EPO shows a focalized lesion similar to the HIF-1α-positive hypoxic region further confirming an ischemic event. Immunohistochemistry was done in n = 4 mice per group.

Functional Non-cerebral GiPCR KO with PTX Reduces Patency of Individual Cortex-Penetrating Microvessels

We investigated whether hypoperfusion was associated with collapsed microvessels. To specifically investigate the immediate response of microvessels to CCA occlusion, we used a multi-gradient echo (MGE) MRI sequence (Fig. 5A) [40,41,42]. High-resolution MGE-MRI provides a penetrating microvessel-specific measurement of the cortex that allows the estimation of microvascular collapse. Comparison of PBS-treated mice regardless of CCA occlusion revealed no difference in the number of vessels in both hemispheres (Fig. 5), indicating a normal microvascular function.

Fig. 5

Functional non-cerebral GiPCR KO reduces patency of cortex-penetrating microvessels. A Timeline of PBS/PTX injection and surgery protocol following multi-gradient echo (MGE) MRI acquisition. These experiments were performed during occlusion or sham surgery. Results of quantification of vessel numbers in the ipsi- (B) and (C) contralateral cortex (n = 6–9). Vessel numbers of PTX-pretreated mice are reduced in both hemispheres, which is further aggravated upon occlusion in the ipsilateral cortex. Statistical analysis was performed using 2-way ANOVA (* p < 0.05, ** p < 0.01, *** p < 0.001). D Representative pictures from all four groups measured by MGE (upper panel). The black boxes mark the assessed areas, and the red dots are the identified vessels (lower panel). Shown are median, 1st, and 3rd quartile of data distribution. The whiskers extend to the largest and smallest data point, respectively.

In contrast, the PTX-induced functional non-cerebral GiPCR KO provoked a reduction of quantifiable microvessels in the cortex of both hemispheres compared to the PBS groups (Fig. 5). The effect was further aggravated in the PTX-pretreated occluded mice, where an even more prominent number of microvessels collapsed in the ipsilateral cortex (Fig. 5). In combination with our perfusion experiments, these data suggest that PTX does not only cause global cerebral hypoperfusion but also micro-cerebrovascular collapse, which has also been described to occur under low-perfusion pressure in heart vessels [7].