We performed a randomized controlled animal trial examining the neuroprotective effects of argon inhalation (50 vol% for 1 h) with treatment initiation 1 h after SAH induction. Three observation times were applied resulting in a total of 12 groups. Parts of this study are already published elsewhere (12).
All of the experiments were conducted in accordance with the Guide for Care and Use of Laboratory Animals (National Research Council, and the Committee for the Update of the Guide for the Care and Use of laboratory Animals; 8th edition 2011).The study protocol was approved by the government agency for animal use and protection (Protocol number: TVA 10416G1 approved by “Landesamt für Natur, Umwelt und Verbraucherschutz NRW,” Recklinghausen, Germany). “The Animal Research: Reporting of In Vivo Experiments guidelines” (14) were used as criteria for adequate performance and reporting of experiments.
Male Sprague-Dawley rats (body weight 300 – 400 g, Charles River, Sulzfeld, Germany) were used for the experiments. They were housed for at least 1 week before the beginning of the experiments with provided food in a specific pathogen-free environment maintaining a 12-h light/dark cycle. Before starting the experiments the animals were first randomized by drawing a lot to a Sham or a SAH group. The experimental groups were defined as follows: Sham N2 (Sham surgery after 1 h delay followed by ventilation with 50 vol% O2/50 vol% N2 for 1 h), Sham Ar (Sham surgery after 1 h delay followed by ventilation with 50 vol% O2/50 vol% Ar for 1 h), SAH N2 (SAH induction after 1 h delay followed by ventilation with 50 vol% O2/50 vol% N2 for 1 h), and SAH Ar (SAH induction after 1 h delay followed by ventilation with 50 vol% O2/50 vol% Xe for 1 h) (see Results section, Figure 1).
To induce SAH we used the modified endovascular perforation model (15, 16). During the entire procedure ICP and CBF were monitored.
Anesthesia was induced by intraperitoneal injection of a mixture of midazolam (2 mg/kg), medetomidine (0.15 mg/kg), and fentanyl (0.0075 mg/kg) (17). A quarter of the initial dosage was injected in 30–45 min intervals to maintain anesthesia. Postoperative analgesia was started directly after surgery via intramuscular injection of metamizole (20 mg/kg) and continued until euthanasia (6, 24 or 72 h after SAH induction). Body temperature was maintained at 37°C via a heating pad (Physitemp Instruments, Inc., Clifton, NJ, USA). Two laser Doppler flowmetry probes were fixated in proximity of the bregma to measure regional cerebral blood flow (rCBF) (Moor Instruments, Axminster, Devon, UK). A left side parietal ICP probe was inserted for continuous ICP monitoring (Microsensor/Codman ICP Express Monitor, Codman/De Puy, Raynham, MA, USA). SAH was induced by the modified endovascular perforation model initially described by Park et al (15) After exposing the left common carotid artery, the left internal carotid artery (ICA) was identified and a tube containing a wolfram wire was advanced intravascularly. Perforation of the vessel was performed via advancement of the wire and subsequent SAH was verified by a sudden increase in ICP and a bilateral decrease in rCBF. Sham-operated animals underwent the same anesthesia and surgical procedure, but the wire was advanced into the ICA without perforation of the vessel.
One hour after SAH induction or Sham surgery, the animals were ventilated for 1 h with either a mixture of 50 vol% O2/50 vol% argon (Air Liquide, Paris, France) or 50 vol% O2/50 vol% N2 (control group). Euthanasia was performed 6, 24 or 72 h after SAH induction by exsanguination under deep anesthesia followed by decapitation. Brains were harvested and cut into 2 mm coronal slices, fixated in paraformaldehyde, and embedded in paraffin.
Paraffin embedded brain slices were cut in 2µm thick sections. After placing the sections on silane-coated slides they were deparaffined. One section was routinely hematoxylin/eosin (H&E) stained. Two consecutive sections were de-waxed, rehydrated, and heated in citrate buffer for antigen retrieval. After blocking of non-specific binding by incubation in PBS containing 2% normal goat serum, one slide per animal was incubated with anti-Iba-1 (1:500; Wako Chemicals, Neuss, Germany) as primary antibody diluted in blocking solution . Appropriate biotinylated secondary antibodies were used (1:200, Vector Laboratories Ltd., Peterborough, UK) for 15 min.
Neuronal Cell Damage
H&E sections were made to assess neuronal damage. Nine regions of interest were defined (five hippocampal regions: CA1, CA2, CA2/3, CA3, and dentate gyrus and four cortical regions: S1, Pir, PLCo and PMCo; selected according to “The Rat Brain in Stereotaxic Coordinates” by Paxinos/Watson (18)). These regions were counted in a standardized fashion using a 400-fold magnification, photographed with an Axioplan microscope (ZEISS, Germany). and neuronal death according to anatomical hallmarks such as hypereosinophilia, shrunken cytoplasm and pyknotic nuclei was quantified. This counting process was done three times by a single investigator blinded to treatment allocation.
For the evaluation of the microglial activation, sections were stained with the antibody against Iba-1. Here, the microglia activation was quantified in the known 9 regions of interest (five in the hippocampal regions: CA1, CA2, CA2/3, CA3, and dentate gyrus and four cortical regions: S1, Pir, PLCo and PMCo) and in the corpus callosum (CC) as a representative of the white matter.
An absolute microglial cell count was performed in a similar fashion in the Iba-1 (ionized calcium-binding adapter molecule 1) stained sections. The five hippocampal, the four cortical regions of interest and the corpus callosum as a region of interest per animal were photographed. The absolute number of activated Iba-1-positive cells was software-assisted counted out in the CA1, CA2, CA2/3, CA3, DG, S1, Pir, PLCo, PMCo of the left hemisphere and also in the Corpus callosum.
The primary outcome was left side neuronal damage. The secondary outcome was microglial activation.
Sample size calculation was extrapolated from previous studies (12, 13) resulting in an estimated sample size of n =7 per treatment group (effect size estimation according to the results of neuronal damage after SAH and xenon treatment for CA3 = 1.02). A alpha-error of 0.1 and beta of 0.7 was presumed, thus resulting in the calculated sample size (G*Power 188.8.131.52 downloaded at http://www.gpower.hhu.de/). All statistical analyses were performed using SPSS v 25.0 (SPSS Inc., Chicago, IL, USA). All graphics were acquired using GraphPad Prism (GraphPad Software Inc., La Jolla, CA, USA). A p-value of <0.05 was considered statistically significant.
For statistical analysis dependent on normality testing (Kolmogorov–Smirnov test), unpaired t-test or Mann-Whitney U test were applied.
All data are presented as means ± SD unless stated otherwise.