All pigs and pigs used in this study came from a conventional herd kept on a farm as part of our university (Farm for Education and Research Ruthe, University of Veterinary Medicine Hannover) and included females and males. The pigs were bought and the management of the farm agreed to use the animals in animal experiments in this study.
Part A: Initially, measurement trials in cerebrospinal fluid (CSF) taps of healthy euthanized pigs were conducted post mortem. The pigs were three to five months old and had a body weight of approximately 70 ± 20 kg.
Part B: For the in vivo measurement in CSF as well as part of the post mortem measurement in CSF the pigs were housed in groups. The pigs had an average age of eight weeks and a body weight of 17 ± 2 kg at the day of infection in the case of the in vivo measurement. The health status of the pigs was checked, and the animals fed twice per day. Water was available ad libitum.
It was important to use in this study animals from a specific age, as S. suis infection rate depend on the age of the animals. The lowest antibody level against S. suis can be observed between weeks 6 and 8, presumably corresponding to a decrease in maternal immunity. A marked increase can be seen at 10 weeks of age, shortly after the onset of clinical signs in the herd during an outbreak . The availability of pigs from the same age between 6 and 10 weeks was therefore set as priority.
The in vivo experiment was divided into three runs with run 1 = 3, run 2 = 4 and run 3 = 5 animals, respectively. In total there were eight females, two males and two castrated male pigs used in the experiment. Six pigs were used as uninfected control and six pigs were infected as described below.
The sample size in part A was not statistically calculated, as it was only used for technical adjustment. The sample size in part B was calculated with 6 animals per group and two animals for replacement. For statistical calculation, the following parameters were used: Error 1st type, α = 5%, error 2nd type, β = 20% (power 80%), relevant difference to be detected: 2% pO2. To calculate the number of animals the software GraphPad StatMate 2.00 was used.
The total number of animals used in this study was 20.
If it was impossible to get an excess to CSF, the experiment was stopped and the animal euthanized due to animal well fare reasons (one animal in this study).
Catheter position and computed tomography scan
In order to establish good puncture results as fast as possible regarding the CSF at the atlantooccipital joint in living animals, the procedure of finding the correct puncture site was trained on half-carcasses at the Institute for Food Quality and Food Safety (University of Veterinary Medicine Hannover, Hannover, Germany) and on dead pigs with a body weight of approximately 13 kg. The position of an inserted epidural catheter inside the subarachnoid space (equal to the position of the needle in the CSF compartment at the atlantooccipital joint) was documented by computed tomography (CT; Philips Brilliance 64) scan. A picture and video were generated with standard settings in the CT software.
Training of staff in puncture technique
The training of the personnel was carried out in previously published animal trial . At least five animals are required to reliably perform the puncture. In the training, explicit emphasis was placed on the correct positioning of the animal, the safe recognition of the puncture site by bending the head and palpating the Articulatio atlantooccipitalis (atlantooccipital joint). Equally important are the correct stitch angle and stitch depth as well as hitting the center of the back. The goal of the exercises was to obtain a sterile, blood-free CSF sample in the shortest possible time.
Method of euthanasia in experiment: post mortem pO2 determination in the CSF of healthy pigs
The pigs were anesthetized with azaperone (2 mg kg−1 body weight (BW), Stresnil ad us. vet., Elanco Tiergesundheit AG, Basel, Switzerland) and ketamine-hydrochloride (20 mg kg−1 BW, Ursotamin, 100 mg mL−1, Serumwerk Bernburg AG, Bernburg, Germany) intramuscularly. The injection was carried out in the Musculus (M.) biventer cervicis near the base of the ear with a 21 G cannula (Sterican 0.80 × 40 mm, B. Braun Melsungen AG, Germany). The pig was separated and therefore protected from the other pigs to allow a gentle start of the anesthesia. The depth of anesthesia was proven by observation of a trained veterinarian. Then the pig was transported in a separate room for further examination and euthanasia. The neck area was shaved around the Articulatio atlantooccipitalis (atlantooccipital joint). By dorsoventral movement of the neck, the point of most flexibility was identified and marked with a laterolateral transverse line with a permanent marker. Furthermore, a median sagittal line was drawn. With this, an intersection point was found (Fig. 2c). The animal was placed in lateral recumbency and the neck was bent at maximum towards the rib cage and fixed by tension straps. Afterwards, the head was aligned exactly horizontal to the table with ropes. The puncture site was aseptically prepared. The pigs were euthanized intravenously via the auricular vein (Vena auricularis) with T 61® (3–4 mL/50 kg BW, Intervet Deutschland GmbH, Unterschleißheim, Germany) during anesthesia. The death of the pig was determined by a trained veterinarian and confirmed by absence of heartbeat and reflexes. The procedure of euthanasia followed the recommendations for euthanasia of experimental animals .
Post mortem pO2 determination in the CSF of healthy pigs
After determination of death (absence of heartbeat), a Spinocan® epidural needle (type Tuohy, 1.30 × 88 mm, G 18 × 3 1/2"; B. Braun Melsungen AG) was inserted in a rostral direction approximately 1 cm caudal of the intersection point. After detecting a slight resistance by penetrating the arachnoid membrane, the stylet was removed. Clear CSF dripped out of the needle hub if the subarachnoid cavity was punctured. If no CSF was observed or in case of blood contamination, the needle was moved slightly until clear CSF dripped out. After clear CSF had been observed, the needle was immediately connected to the 10-cm-long tube of a three-way valve (Discofix®-C; B. Braun Melsungen AG). The FTC oxygen sensor (FTC-PSt7; PreSens Precision Sensing GmbH, Regensburg, Germany) was then connected in series to this three-way valve. A second three-way valve with a syringe was connected to this combination. Fresh CSF was drawn into the entire system via suction from the syringe. Fresh CSF aspirates were drawn after five and ten minutes to determine the pO2 of the CSF at different time-points post mortem. By supplying the CSF sample to the sensors using the three-way valves, the pO2 measurements could be performed anaerobically, although the syringe could be removed with the CSF aspirate for further examinations (see Additional file 2: Figure S2 for detailed information).
The measuring sensors were covered with aluminium foil to keep the temperature constant and to protect the sensors from light. The sample was measured at intervals over a time-span of maximum ten minutes post mortem. The value one-minute after taking CSF over the sensor was used for statistical analysis to achieve one-step sensor equilibration in the case of the first taken value. After five minutes, fresh CSF was drawn over the sensor and the value one minute later was used for statistical purposes (two-step sensor equilibration). Since the ambient pressure and the temperature of the medium are essential parameters to calculate the oxygen level in liquids, the current ambient pressure of the room was determined (measured with Fisherbrand™ Traceable Digital Barometer; Thermo Fisher Scientific Inc, Waltham, MA, USA) and used to calculate the oxygen level. Since an exact temperature measurement of the CSF proved impossible, the rectal body temperature was measured and taken as the basis for the oxygen calculation.
Streptococcus suis growth conditions
In this study, Streptococcus (S.) suis cps type 2 strain 10 (S. suis) was used. This strain has been shown to be highly virulent in experimental infections of pigs [50,51,52]. S. suis was grown on Columbia agar plate with 7% sheep blood (Oxoid Deutschland GmbH, Wesel, Germany) and incubated for 20–24 h at 37 °C. To prepare the infection inoculum, 10 mL Tryptic Soy Broth without dextrose (TSB) (Becton, Dickinson and Company, Sparks Glencoe, MD, USA) were filled in T405-Cultubes™ (Simport® Scientific Inc., Belœil, Canada) with air exchange and two freshly grown S. suis colonies were added. The culture was incubated for ten hours (37 °C / 5% CO2). A 1:100 dilution was created in an Erlenmeyer flask with preheated TSB and incubated for three-four hours (37 °C / 5% CO2) to the late exponential growth phase reflected by an OD600nm of 0.3 ± 0.02. Then, 40 mL of the bacterial culture were transferred to a 50 mL Falcon Tube (SARSTEDT AG & Co. KG, Nümbrecht, Germany) and was centrifuged at 4816 g for ten minutes at room temperature. The supernatant was discarded, and the pellet was resuspended in 3 mL sterile phosphate-buffered saline (PBS). From this suspension a 1:10 dilution was made, and 1 mL thereof was intravenously injected into pigs as described below. The exact infection dose for each piglet was determined by plating serious dilutions on blood agar plates and counting colonies after 20-24 h incubation at 37 °C.
Infection of pigs with long-term anesthesia and control of physiologic parameters
Seven-to nine-week-old German Landrace pigs (weaned at four to five weeks of age) were anesthetized as described above with azaperone and ketamine-hydrochloride intramuscularly and were intravenously (Vena auricularis) inoculated with 1 mL PBS containing 2.5–3.7 × 108 CFU of S. suis infection inoculum (as described above) or 1 mL PBS in case of the control animals.
After the infection, the animals were housed separately for the next eight-12 h. Using a scoring system, eight hours post infection the pigs were checked every hour for severe symptoms. If a score of 25 was reached earlier than 12 h post infection, the second anesthesia started immediately due to animal welfare reasons. Without reaching the maximum score, pigs were anaesthetized again after 12 h. Anesthesia was induced with ketamine 20 mg kg−1 BW), azaperone (2 mg kg−1 BW) and atropine (0.06 mg kg−1 BW, Atropinsulfat B.Braun 0.5 mg mL−1 injection solution, B. Braun Melsungen AG).
Venous access was established by an indwelling venous catheter (Vasovet Braunüle, 20G/22G; B. Braun Melsungen AG) either in an ear vein, the cephalic or lateral saphenous vein. Propofol (1–2 mg kg−1 BW; Narcofol® 10 mg/mL BW, CP Pharma, Handelsgesellschaft mbH, Burgdorf, Germany) was administered to achieve endotracheal intubation. After topical anesthesia with tetracaine spray (Gingicain®D, 754 mg/65 g, Sanofi-Aventis Deutschland GmbH, Frankfurt am Main, Germany), the pigs were endotracheally intubated under visual control with a cuffed endotracheal tube (Portex 100/150/XX ID 5–6; Smiths Medical, Inc., MN, USA).
The endotracheal tube was connected to a circle breathing system (DRAEGER Titus; Drägerwerk AG & Co. KGaA., Lübeck, Germany) and volume controlled mechanical ventilation (DRAEGER Ventilog C; Drägerwerk AG & Co. KGaA., Lübeck, Germany) with a tidal volume of 10–16 mL kg−1 and a pressure limitation of 20 mbar without PEPP was immediately started. The respiratory rate was set at 20 breath min−1 and adjusted to maintain eucapnia.
Anesthesia was maintained with isoflurane (Isoflurane CP 1 mL/mL, 250 mL, CP-pharma GmbH) in air. To avoid hyperoxia and hypoxia, oxygen was supplemented to adjust FiO2 as needed to maintain an arterial partial pressure of oxygen of 80 to 110 mmHg. A constant infusion rate of ketamine (1 mg kg−1 h−1; Ketamin® 100 mg/mL, CP Pharma GmbH) and dexmedetomidine (2 µg/kg/h; Cepedex® 0.5 mg mL−1, CP-Pharma GmbH) was administered for MAC-sparing effects. To facilitate mechanical ventilation, 0.15 mg kg−1 levomethadone/fenpipramide (L-Polamivet 2.5/0.125 mg mL−1 ®, Intervet GmbH, Unterschleißheim) was injected intravenously immediately after intubation and repeated after four hours of anesthesia. For cardiovascular support, a balanced, lactate buffered electrolyte solution (Sterofundin ® 1/1 E, B. Braun Melsungen AG) was administered at a rate of 5 mL kg−1 h−1 throughout the procedure. Additional file 9: Table S6 lists all drugs that were used for each individual animal to ensure effective anesthesia.
The depth of anesthesia was monitored by testing muscle relaxation, the eye reflexes, the interdigital withdrawal reflexes and skin stimulation with a Kelly type artery clamp. The ECG, heart rate, arterial blood pressure, peripheral oxygen saturation (SPO2) via a transmission pulse oximetry probe placed at the tail or a claw and the rectal temperature were continuously monitored via an anesthesia multiparameter monitor (DATEX Ohmeda Cardiocap 5, General Electric Company, Boston, MA, USA). For invasive blood pressure measurement, access was made to the femoral artery by an arterial catheter (BD Insyte-W, 22G; Becton Dickinson, Franklin Lakes, NJ, USA). The pressure was recorded via a calibrated pressure transducer (BRAUN Combitrans Monitoring set venous; B. Braun Melsungen AG) levelled and zeroed to ambient pressure at the base of the heart. End-tidal CO2, inspired and expired isoflurane concentrations as well as the inspired and expired (FeO2) fraction of oxygen were monitored via a respiratory gas monitor (Dräger PM 8050, Drägerwerk AG & Co. KGaA., Lübeck, Germany).
Arterial blood gas measurements were performed using the EPOC system (epoc® Blood Analysis System; Siemens Healthcare GmbH, Erlangen, Germany) before each pO2 determination in the CSF, and individually as needed to adjust FiO2 to maintain physiologic arterial partial pressure of oxygen. Mean arterial blood pressure was maintained above 60 mmHg by fluid infusion and inotropes or vasopressors as required.
The temperature of the animals were maintained by means of red-light heat lamps and adjustable electrical heating mats during the period of anesthesia. The initial temperature was the temperature at the time of inducting anesthesia.
Method of euthanasia in experiment: Infection of pigs with long-term anesthesia and control of physiologic parameters
After completing the measurements, the animals were euthanized by a trained veterinarian during isoflurane anesthesia, for further examination. Therefore, the pigs were euthanized intravenously via one of the already existing venous access points (described above) via the indwelling venous catheter (Vasovet Braunüle, 20G/22G; B. Braun Melsungen AG) either in an ear vein, the cephalic or lateral saphenous vein with T 61® (3–4 mL/50 kg BW, Intervet Deutschland GmbH, Unterschleißheim, Germany) during isoflurane anesthesia. The death of the pig was determined by a trained veterinarian and confirmed by absence of heartbeat and reflexes. The procedure of euthanasia followed the recommendations for euthanasia of experimental animals .
Oxygen and pH measurements in the CSF and sampling during anesthesia
After endotracheal intubation, the pigs were positioned, and the CSF access carried out as described above. The needle was connected to the 10-cm-long tube of a three-way valve (Discofix®-C; B. Braun Melsungen AG). One side was connected to a pressure transducer (Combitrans ®; B. Braun Melsungen AG) and the other side to the measurement system. Therefore, the FTC oxygen sensor (FTC-PSt7; PreSens Precision Sensing GmbH) and the pH sensor (FTC-SU-HP5-US; PreSens Precision Sensing GmbH, Regensburg, Germany) were then connected in series to the leftover connection. At the open side of the pH sensor, it was possible to collect CSF samples with a syringe by means of a second three-way valve.
After installing the measurement set-up, the three-way valve was opened in such a way that the pressure of the CSF (pCSF) was monitored permanently with the transducer. Before measuring O2 and the pH level inside the CSF, a six minute two-step sensor equilibration was conducted. For this purpose, the three-way valves were opened so that 1.8 mL of fresh CSF was drawn into a 2 mL syringe via the oxygen and pH sensors. Thereafter, the three-way valves were reversed so that CSF was trapped over the sensors without any air contact. For the following five minutes, the sensor probes equilibrated to the CSF (first step). The solution in the syringe was disposed of (CSF in the syringe (13 h p.i.) and the CSF- sodium chloride mixture due to rinsing procedures beforehand (16 h p.i. / 19 h p.i.)), respectively. After this first equilibration, and rinsing out the leftover sodium chloride solution, 1 mL fresh CSF was drawn into a sterile syringe as previously described before and the second one-minute equilibration step of the sensors was already performed with measuring values. During a ten-minute period, values were collected every 30 s leading to a results curve. The five-minute adaptation (from sodium chloride to CSF over the sensor) and the following one-minute equilibration (fresh CSF over the sensor for temperature and pressure compensation) were termed as the two-step equilibration. All values of pO2 and pH presented in main figures were taken after this two-step equilibration. All other values were analyzed and summarized in Additional file. After these measurements, the system was rinsed with 0.9% sodium chloride solution using method “III” as described in Additional file 2: Figures S4 and S5. The measurement was carried out regularly one hour after starting isoflurane anesthesia (13 h post infection), four hours (16 h post infection) and seven hours (19 h post infection). For every measurement, this two-step equilibration was performed. The sensors were covered with aluminium foil to keep the temperature constant and to protect the sensors from light. After each measurement, the system was rinsed with an outflow of 0.9% sodium chloride solution from the pressure transducer.
For oxygen determination in liquids, the ambient pressure and the temperature of the medium are needed. Here, we combined the prevailing air pressure (pair) of the room (~ 21% O2) (Fisherbrand™ Traceable Digital Barometer; Thermo Fisher Scientific Inc) with the CSF pressure that had been directly measured before the measurement set-up was opened for the measurement (no pressure drop caused by opening the measuring system). Since the CSF pressure (pCSF) also influences the oxygen calculation, the sum of both pressures (pAir + pCSF) was used as the final value for the pressure compensation. The rectally measured body temperature was taken as the basis for the temperature compensation in the oxygen calculation and pH determination.
At each time point (13–16-19 h p.i.), the CSF sample in the syringe was transported on ice to the laboratory for further examinations.
Cleaning procedure of measurement system during in vivo experiment
A change in the measuring system during the experiment was not possible. Therefore, a rinsing process to decontaminate the measuring system between the measurement points was established as described in the Additional file.
Analysis of samples from animal experiment
Blood samples were taken from the Vena jugularis or Vena cava cranialis pre-infection and from the Arteria femoralis, at defined time-points (0, 13, 16 and 19 h post infection) in lithium–heparin monovettes (SARSTEDT AG & Co. KG). Serial dilutions were plated on Columbia blood agar to determine the CFU/mL after incubation at 37 °C for 20–24 h.
During the first anesthesia, a tonsillar swab (Amies medium, SARSTEDT AG & Co. KG) was taken for bacterial examination to exclude bacterial colonization of pigs with S. suis.
After euthanasia and post mortem oxygen measurement in CSF, a necropsy was conducted. Swabs of the brain surface, the mitral valve, the pleura, the pericard and the peritoneum and organ samples of the liver, spleen, lung and tonsils as well as liquid from the carpal and tarsal joints were collected for bacteriologic examination.
All colonies suspected of S. suis were analyzed by multiplex PCR as previously described  for the specific gene profile of the infection strain.
In the CSF samples, at each time-point, the following parameters were determined: 1. Serial dilutions were plated on Columbia blood agar to determine the CFU/mL after incubation at 37 °C for 20–24 h, 2. Counting of neutrophil numbers in the Neubauer chamber, 3. Counting of neutrophils after fixation (with 4% paraformaldehyde final) analyzed by flow cytometry using the Attune® NxT Acoustic Focusing Flow Cytometer (FACS). The analysis was based on Forward Scatter (FSC; detection of cell size) and Side Scatter (SSC, detection of granularity) and analyzed using FlowJo software version (v)10.
During section swabs of the brain surface, the mitral valve, the pleura, the pericard and the peritoneum were taken for bacteriology. Furthermore, for bacteriology and histology, organ samples of the brain, liver, spleen, tonsils, heart, lung, pleura, pericard and peritoneum were collected. Samples for histology were stored immediately in 10% buffered formalin and after latest 72 h embedded in paraffin and cut into 2–4 µm sections for hematoxylin–eosin (HE) staining and histological examination. The histological screenings were scored as described  and furthermore are mentioned in the footnotes of the scoring table. The organ and swab samples for bacteriology examination were analyzed as described previously  with slight changes. Instead of a matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS), S. suis was identified by colony morphology on and afterwards by multiplex PCR identified as described previously [18, 53].
Influence of blood hypoxygenation on the oxygen level in CSF
The influence of a possible hypo-oxygenation on the pO2 in the CSF was simulated by cardiac arrest by stopping the blood circulation, leading to an undersupply of oxygen to the organism. For this purpose, the oxygen level in the CSF was measured in a living animal directly before cardiac arrest. The animal was then euthanized. Ten minutes after verification of death, the oxygen level in the CSF was measured again.
Calculation of oxygen pressure
The oxygen O2% was calculated based on 38 °C and 1000 hPa (= 750 mmHg) from the measured mmHg values. The values are presented in each figure and in the text with ≜.
Data were analyzed using Excel 2010 and 2016 (Microsoft) and GraphPad Prism 8.1 (GraphPad Software). Normal distribution of data was verified by the Kolmogorov–Smirnov normality test (GraphPad software) prior to statistical analysis. Differences between groups were analyzed as described in the figure legends (*P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001). Detailed information can be found in the figure legend.