Data on common carotid artery occlusion inducing focalized stroke lesions after Pertussis toxin injection

This article contains raw and processed data related to research published by Vega et al. (2022). This complementary dataset provides further insight into the experimental validation of a single common carotid artery occlusion (CCAO) model upon pretreatment with pertussis toxin (PTX). We present data showing the extent of different PTX concentrations on neurological severity measured by Bederson score following CCAO. In addition, data indicate a protective effect of isoflurane on cerebral infarction and neurological deficits, as well as the consequences of PTX pretreatment on reperfusion after occlusion using time-of-flight magnetic resonance angiography. With these data, we aim to provide detailed experimental settings of this newly described model.


a b s t r a c t
This article contains raw and processed data related to research published by Vega et al. (2022). This complementary dataset provides further insight into the experimental validation of a single common carotid artery occlusion (CCAO) model upon pretreatment with pertussis toxin (PTX). We present data showing the extent of different PTX concentrations on neurological severity measured by Bederson score following CCAO. In addition, data indicate a protective effect of isoflurane on cerebral infarction and neurological deficits, as well as the consequences of PTX pretreatment on reperfusion after occlusion using time-of-flight magnetic resonance angiography. With these data, we aim to provide detailed experimental settings of this newly described model.  Table   Subject Health and medical sciences Specific subject area Cardiology and Cardiovascular Medicine Type of data under various PTX conditions (concentrations and incubation times). • Data will be useful to researchers who use PTX to inhibit intracellular signaling via heterotrimeric G i and G o proteins, especially in the context of neuronal signaling. • Data could assist researchers in choosing the appropriate anesthetic drug.
• Data also indicate the protective effect of isoflurane during reperfusion on neurological outcome following CCAO in PTX-treated mice.

Objective
This dataset was generated to compare different doses of PTX and anesthetics regarding the outcome of CCAO. It will serve as "material and methods" for several future articles that will investigate CCAO using PTX as a tool to perturb G i -and G o -mediated signaling pathways. Ultimately, the objective is to determine the most suitable combination of PTX and anesthetics in mice undergoing CCAO to replace, reduce and refine in the sense of the 3R principle. The data indicate the administration of 90 μg PTX per kilogram body weight 48 h before the intervention as the optimal condition to induce cerebral stroke.

Data Description
In this work we investigated the consequences of PTX pretreatment on cerebral blood flow by time-of-flight (TOF) angiography before, during and after unilateral common carotid artery occlusion (CCAO) ( Figs.1 -3 , Table 1 , Videos 1-5). In addition, different doses of PTX with different incubation times were used ( Table 2 ). Table 3 shows the effect of isoflurane duration whereas Table 4 depicts the effect of isoflurane-or fentanyl-based anesthesia on the severity score of neurological deficits.

Animals
The study was carried out in compliance with the ARRIVE guidelines. All experiments were performed according to the EU Animals Scientific Procedures Act and the German law for the welfare of animals and were approved by the local animal ethical committee Bezirksregierung Düsseldorf (8.87-50.10.34.08.018) and Bezirksregierung Tübingen (PH2/17). C57BL/6 female mice were kept under specified pathogen-free conditions, controlled temperature and humidity in 12h day/night light cycle and received food and water ad libitum.

Anesthesia
For surgeries and imaging experiments, animals were anaesthetized using isoflurane (induction at 2.5% and maintenance at 2% vaporized in 100% O 2 at a flow rate of 1.5 L/min) and placed on an MRI-compatible bed with an integrated water heating system (Bruker Biospin). Fentanyl 0.05 mg/kg body weight (b.w.), midazolam 5.0 mg/kg b.w., medetomidine 0.5 mg/kg b.w. narcosis was injected intraperitoneally and used on anesthesia-focused experiments as indicated.

Common carotid artery occlusion
The animals were dissected under a surgical microscope through a small median incision above the trachea through the submaxillary salivary glands, exposing the left common carotid artery (CCA). The CCA was carefully dissected bypassing the Vagus nerve. Transient occlusion of the CCA was achieved using a non-absorbable suture (Ethicon, Norderstedt-Glashütte, Germany) ( Fig. 1 ). Considering the opening and closing of the surgical wound, each procedure took about 10 to 15 minutes. The mice were allowed to recover in their home cages before they were assessed by a modified Bederson score ( Table 1 ) and scanned 60 to 90 min after the procedure.

Bederson score
Neurological deficits were determined using a modified Bederson score as depicted in Table 1 [2 , 3] .

Time-of-flight magnet resonance angiography
Time-of-flight (TOF) angiography is an MRI technique to visualize flow within vessels without administering contrast agents. Mice were examined between 8 to 14 weeks of age. Angiographies were carried out under isoflurane anaesthesia using an established TOF imaging protocol and a vertical Bruker DRX 9.4 T wide-bore NMR spectrometer, a 40-mm gradient set (capable of 1 T/m maximum gradient strength) and a linearly driven 30-mm birdcage resonator [4] .
Within a first setup ( Fig. 2 ) baseline angiography was performed (Videos 1,2), mice were subsequently injected i.p. with PTX (90 μg/kg b.w. (previously shown in [5] ); Merck KGaA, Darmstadt, Germany), and scanned after 48 h (Video 3). A permanent ligation of the left carotid artery was performed, and animals were allowed to recover for 60 to 90 min, assessed by Bederson score, and scanned again (Video 4,5). Within a second set of experiments mice were pretreated with PTX and the CCA was ligated ( Fig. 3A ). In addition, neurological deficits were Bederson scored demonstrating that all mice displayed a stroke phenotype (Bederson score ≥ 1). Subsequently, ligation was removed and after one hour of reperfusion mice underwent MRI analysis to evaluate cerebral blood flow. While all mice showed a stroke phenotype, only three out of six mice demonstrated reperfusion ( Fig. 3B ).

Ethics Statements
The study was carried out in compliance with the ARRIVE guidelines. All experiments were performed according to the EU Directive 2010/63/EU and the German law for the welfare of animals and were approved by the local animal ethical committee Bezirksregierung Düsseldorf und Tübingen.

Declaration of Competing Interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Data Availability
Dataset of supporting videos to PTX-stroke study (Original data) (Mendeley Data).