2.1 Animals
Male C57Bl/6 mice (20-22 g) were obtained from a commercial breeder of laboratory animals (Charles River Laboratories, Sulzfeld, Germany). Animals were kept in Makrolon cages in a controlled environment (22.0°C / 55% humidity) and had free access to food and water before and during the experimental phase. Day/night cycles were kept constant with lighting from 8 a.m. to 8 p.m. After the experiments, all animals were anesthetized and sacrificed by cervical dislocation under deep anesthesia.
2.2 CCI
We used the widely established CCI [11, 12]. For analgesia i.p. injections of buprenorphine were applied (0.1 mg per kg bodyweight; mg/kg BW). Animals were put in short isoflurane anesthesia (4-5%vol for induction, 1-2% to maintain) with 30% O2 and 70% N2O via facemask. Craniotomy of the right temporal bone was performed under preservation of the dura above the motor cortex. The size of the craniotomy was approximately 5 x 5 mm. CCI (Mouse-Katjuscha 2000, L. Kopacz, University of Mainz, Germany)[13, 14] was as follows: diameter of the impact tip 3 mm; impact velocity 8 m/s; duration 150 ms. The bone flap was then placed back and affixed with histoacryl glue (Braun-Melsungen, Melsungen, Germany). The duration of the surgical procedure did not exceed 15 minutes.
2.3 Study Protocol
Blinding was performed by laboratory personnel who randomly presented the animals to the examiner for surgery and testing. Blinding was dissolved only after the complete assessment of the respective test series. Confounders were not controlled, as the procedure did not vary for each animal.
All groups consisted of 10 animals. A total number of 210 animals were examined in all test series. In the further explanations, the number of animals included in the statistical analysis is shown in tabular form. Only animals that died in the course of the experiments or for which the evaluation was not possible due to technical difficulties were excluded.
Sample sizes in this orientational study were based on previous experiments with this damage model and argon treatment.
- Test Series: Optimal Argon Concentration
Argon administration at defined concentrations 30 min prior to trauma and continued for 4 h after trauma. The purpose of this very first test series was to measure neuroprotection under optimal circumstances, i.e. administering argon before trauma to ensure optimal brain tissue concentrations although these conditions are unlikely in a clinical setting. All argon was supplied by a commercial producer with a purity level above 99.9% (SWF, Sauerstoffwerk Friedrichshafen, Friedrichshafen, Germany). Medical grade oxygen, nitrogen, and nitrous oxide were readily available in our laboratory.
Control group is exposed to room air for the entire duration of test series 1. All animals were placed in heated, semi-open boxes at a constant temperature of 26 degrees Celsius. For all test series, oxygen and argon levels were continuously monitored during treatment with an oxygen (Dräger Oxydig, Dräger, Lübeck, Germany) and argon meter (Servopro Monoexact, Servomex, Hamm, Deutschland). A continuous fresh gas flow of at least 0.1 l was established with a custom, calibrated flowmeter for oxygen, nitrogen, and argon, leading to a complete exchange of gas in the box within less than 15 minutes. The temperature in the boxes was fixed at 26° C.
Brain Contusion Volumes (BCV):
15-min survival: -no therapy (room air) n=10
24-h survival: -no therapy (room air) n=10
-25% Ar / 75% O2 n=8
-50% Ar / 50% O2 n=10
-75% Ar / 25% O2 n=9
Brain Water Content (BWC) and ICP
24-h survival: -no therapy (room air) n=9
-25% Ar / 75% O2 n=9
-50% Ar / 50% O2 n=10
-75% Ar / 25% O2 n=9
- Test Series: Optimal Duration of Treatment
Utilizing the determined optimal argon concentration assessment of the optimal duration of argon application with two sub-test series follows. Start of argon 30 min before CCI. 50% argon / 50% oxygen in the therapeutic group corresponding to results of prior in vitro experiments and to our own findings in the first test series were used [3]. Control animals received room air. Moreover, an oxygen control group treated with 50% oxygen / 50% nitrogen was done.
BCV:
24-h survival: -no therapy (room air) n=9
-oxygen / nitrogen 24 h treatment n=9
-oxygen / argon 24h treatment n=10
BWC and ICP
24-h survival: -no therapy (room air) n=8
-oxygen / nitrogen 24 h treatment n=8
-oxygen / argon 24h treatment n=9
- Test Series: Treatment at Physiological Oxygen Levels
This series was introduced to eliminate any interference that might occur because of elevated oxygen levels. Control animals received 20% oxygen and 80% nitrogen. The animals treated with argon received 20% oxygen, 50% argon, and 30% nitrogen. Start of therapy 30 min before and 24 h after CCI in both subgroups.
BCV:
24-h survival: -no therapy n=7
-argon 24 h treatment n=9
BWC and ICP:
24-h survival: -no therapy n=10
-argon 24 h treatment n=8
- Test Series: Effects of Argon on MAP and CBF after CCI
After determining the optimal concentration of argon, we analyzed MAP and CBF 30 min before, until 2 h after CCI. Argon therapy or O2/N2 was administered at the same time period, when applicable.
BCV:
2-h survival: -50% O2 / 50% N2 n=8
-50% Ar / 50% O2 n=10
2.4 Neuroscores and Beamwalk Testing
Neurological status was assessed 24 h after trauma after a modified Dixon’s and Bederson’s neuroscore (table 1) [15-17]. Mice underwent beamwalk testing, crossing a 40 cm long non-slip beam (diameter 1.5 cm) three times; missteps of the left leg were counted during that procedure.
2.5 Histology and BCV
After sacrificing the animals assigned for BCV measurement (after 24 h, exception: 15-min controls) through cervical dislocation in deep isoflurane anesthesia, brains were removed and frozen at -70°C. Coronal slices were prepared with a cryostat. 10 μm coronal slices were collected with an interslice thickness of 500 μm throughout the entire brain. Slices were H&E (hematoxylin and eosin)-stained and contusion areas an for each slice were measured, n being the number of slices, using a digital image analyzing software and microscope (Olympus Cell Sense, Olympus Lifescience, Hamburg, Germany). BCV was calculated using the formula
VContusion/mm³= (a1+a2+a3+⋯+an)*0.5 mm.
2.6 BWC and ICP Measurement
All animals assigned for ICP and BWC were put in short inhalative anesthesia 24 h after CCI. Contralateral to the trauma side, the temporal muscle was mobilized by blunt preparation and a small trephination was performed in the lateral temporal bone. An ICP microsensor (CP Express, Codman Neuro, Raynham, MA, USA) was inserted in the epidural space of the contralateral hemisphere after being calibrated in distilled water. After 5 min the ICP value was registered. Brains were promptly removed, cerebellum and olfactory bulbs removed and hemispheres divided. They were then measured wet (WWet) and again after drying for 24 h at 90° C (WDry). BWC of the hemispheres was then determined via:
PWater = (WWet - WDry) / WWet
2.7 MAP and CBF measurement
For this study 18 mice were anesthetized with intraperitoneal injections of medetomidine, midazolam, and fentanyl. As the experiments had to last at least 2.5 hours mice were intubated and mechanically ventilated (MiniVent 845, Hugo Sachs Elektronik, March-Hugstetten, Germany). Gas concentrations were 50% oxygen and 50% nitrogen for all animals prior to CCI. In order to ensure appropriate ventilation, arterial blood gas analyses were taken at regular intervals (after 0 , 30, 90, 150 min).
In order to monitor MAP the femoral arteries were dissected and a fluid filled catheter inserted into the femoral artery and the pressure measured by a manometer (Pressure Monitor BP-1, World Precision Instruments, Sarasota, Florida, USA). To control the cerebral blood flow the right temporal muscle was detached from the skull with a scalpel. The laser Doppler probe (Periflux 5000, Perimed, Järfälla, Sweden) was then fixated directly on the skull. All data were automatically recorded and saved at 1 Hertz via computer interfaces (Labscribe 2, iWorx, Dover, NH, USA). CCI was performed as stated above.
MAP and cerebral blood flow were recorded for 30 minutes prior to CCI in order to have baseline values for all animals. After CCI, 8 control-group animals continued to receive 50% oxygen and 50% nitrogen, whereas 10 argon-treated mice received 50% argon and 50% oxygen. Treatment was performed for two hours with continuous monitoring of MAP and CBF. Afterwards the mice were sacrificed. We refrained from assessing these animals neurologically due to limited validity after long anesthesia.
2.8 Statistical Analysis
Statistical work was performed with SPSS 22 (IBM, Ehningen, Germany). ANOVA with an unpaired t-test analysis was used for ICP, BWC, BCV, and beamwalk testing. For neuroscores a Mann-Whitney-U test was used. All results are displayed as mean value ± standard deviation (SD).