Thiopental

Rats were anesthetized by intraperitoneal injection of thiopental (50 mg/kg) and diazepam (5 mg/kg). After anesthesia induction, each rat was placed in the supine position and fixed on the operating table. A midline incision of approximately two centimeters was made in the neck, and then both common carotid arteries and common jugular veins were exposed carefully by blunt dissection. After the vagus nerve was carefully separated from the carotid artery, cerebral ischemia was induced by bilateral clamping of the common carotid arteries. Bilateral clamping of the common carotid arteries was relieved at the end of the 20‐min period. Thirty seconds later, we applied medication (BPC 157 10 µg/kg; or saline as a 1 ml bath directly on the surgical area. Five minutes after that, the incision was sutured back in layers. The sutured area was cleaned with 70% ethanol and sprayed with an antiseptic solution.

In deeply anesthetized rats (intraperitoneal (ip) injected 40 mg/kg thiopental (Rotexmedica, Germany) and 10 mg/kg diazepam (Apaurin; Krka, Slovenia)), we induced abdominal compartment syndrome by intraperitoneal insufflation of ordinary air controlled by a manual and digital manometer with a data logger connected to a computer (DD890, Dostmann Electronic GmbH, Germany) and maintained high abdominal pressure at 25 mmHg for 120 min before sacrifice, with a pressure measurement interval of 1 s. High abdominal pressure at 25, 30, 40, or 50 mmHg was maintained until sacrifice at 60 min (25 mmHg), 30 min (30 mmHg, 40 mmHg), or 15 min (50 mmHg). Rats received BPC 157 (10 µg or 10 ng/kg subcutaneously) or saline (5 ml) at 10 min abdominal compartment syndrome-time. Alternatively, using esketamine anesthesia (40 mg/kg esketamine (Rotexmedica, Germany) and 10 mg/kg diazepam (Apaurin; Krka, Slovenia) intraperitoneally), we induced abdominal compartment syndrome as described before and maintained high abdominal pressure at 25 mmHg for 120 min before sacrifice. Medication (BPC 157 (10 µg or 10 ng/kg sc) or saline (5 ml)) was given after 10 min of high abdominal pressure.
Recordings of brain swelling were performed in rats before sacrifice after complete calvariectomy was performed (Gojkovic et al., 2021a (link); Knezevic et al., 2021a (link); Knezevic et al., 2021a (link); Knezevic et al., 2021b (link)). Briefly, six burr holes were drilled in three horizontal lines, all of them medially to the superior temporal lines and temporalis muscle attachments. The two rostral burr holes were placed just basal from the posterior interocular line, the two basal burr holes were placed just rostral to the lambdoid suture (and transverse sinuses) on both sides, respectively, and the two middle burr holes were placed in line between the basal and rostral burr holes.
Rats were laparatomized before sacrifice for the corresponding presentation of the peripheral vessels (azygos vein, superior mesenteric vein, portal vein, inferior caval vein, and abdominal aorta). The recording was performed with a camera attached to a VMS-004 Discovery Deluxe USB microscope (Veho, United States) at the end of the experiment and assessed as before (Gojkovic et al., 2021a (link); Knezevic et al., 2021a (link); Knezevic et al., 2021a (link); Knezevic et al., 2021b (link); Strbe et al., 2021 (link)).

The Central Denmark Region covers a mixed urban and rural area of approximately 13000 km²with a population of 1.27 million. The overall population density is 97.7 inhabitants pr. km².
The standard EU emergency telephone number (1-1-2) covers all Denmark and there is an Emergency Medical Dispatch Centre in each of the five Danish regions. Emergency Medical Dispatch is criteria based.
The Central Denmark Region has a two-tiered EMS system. The first tier consists of 64 ground ambulances staffed with Emergency Medical Technicians (EMTs) on an intermediate or paramedic level (EMT-I / EMT-P). EMTs in The Central Denmark Region do not perform PHETI, nor do they use supraglottic airway devices (SADs).
The second tier consists of ten pre-hospital critical care teams staffed with an anaesthesiologist (with at least 4½ years’ experience in anaesthesia) and a specially trained EMT. Nine of the pre-hospital critical care teams are deployed by rapid response vehicles; the tenth team staffs a HEMS helicopter.
In the most rural parts of the region there are three rapid response vehicles staffed with an EMT and an anaesthetic nurse. The anaesthetic nurses do not use SADs nor do they perform Rapid Sequence Intubation (RSI) or other forms of drug-assisted PHAAM in the pre-hospital setting. These rapid response vehicles were not part of this study.
The pre-hospital critical care teams covered by this study employ approximately 90 anaesthesiologists as part time pre-hospital physicians. There are no full-time pre-hospital critical care physicians in the region – all physicians primarily work in one of the five regional emergency hospitals or at the university hospital. All pre-hospital critical care physicians have in-hospital emergency anaesthesia and advanced airway management both in- and outside the operating theatre as part of their daily work. Intensive care is part of the Danish anaesthesiological curriculum.
All pre-hospital critical care teams carry the same equipment for airway management. This includes equipment for bag-mask-ventilation (BMV), endotracheal tubes and standard laryngoscopes with Macintosh blades (and Miller blades for infants and neonates), intubation stylets, AirTraq™ laryngoscopes, Gum-Elastic Bougies, standard laryngeal masks (LMAs), intubating laryngeal masks (ILMAs) and equipment for establishing a surgical airway. All units are equipped with a capnograph and an automated ventilator. The pre-hospital critical care teams carry a standardised set-up of medications including thiopental, propofol, midazolam and s-ketamine for anaesthesia and sedation, alfentanil, fentanyl and morphine for analgesia and suxametonium and rocuronium as neuro-muscular blocking agents (NMBAs). Lidocain is available for topical anaesthesia.
Our system has no airway management protocols or standard operating procedures (SOPs) regarding PHAAM or pre-hospital RSI [22 (link)] and the physicians use the available drugs and equipment at their own discretion.
The pre-hospital critical care anaesthesiologists in our region have an average of 17.6 years of experience in anaesthesia and on average 7.2 years of experience with pre-hospital critical care. The average pre-hospital critical care physician performs 14.5 endotracheal intubations per month, 1 of them in the pre-hospital setting.
We have previously reported details of the pre-hospital critical care physicians’ education, training, level of experience and equipment-awareness in our region [22 (link)].
We collected data from February 1^st 2011 until November 1^st 2012.
Follow-up data regarding 30-days mortality were collected in January and February 2013.

Two weeks after the last immunization boot of e1 or PBS in IFA, mice were sacrificed with thiopental, and blood was collected by cardiac puncture for seric anti-e1 IgG assay. Spleens were harvested and digested with collagenase D for 20 min at 37°C (Roche). To evaluate T-cell proliferation in response to e1 peptide, dissociated splenocytes were labelled with cell proliferation dye (CPD) eFluor670 and cultured for 3 days in the presence of e1 (20 µM) or vehicle (diméthylsulfoxyde)) only. Cells were then stained with fixable viability dye (FVD) eFluor450 and with fluorescein isothiocyanate-conjugated anti-CD3 and PE-Cy7-conjugated anti-CD4 antibodies, and analysed for the percentages of CPD-low cells/total CD3 + CD4 + cells. Amplified e1-specific memory T cells were assessed for IFN-γ release using the mouse IFN-γ ELISPOT Set (BD Biosciences) according to the manufacturer’s instructions.

Blood was sampled from the inferior vena cava immediately after rats in the in-vivo experimental setup were anesthetized with i.v. thiopental (20 mg/rat). The blood was mixed with the following cocktail of anticoagulant and protease inhibitors (final concentrations): p-hydroxymercuribenzoate (5 mM); phenylmethylsulfonyl fluoride (10 µM); EDTA (7.5%); o-phenanthroline (150 mM); and pepstatin (2 mM). The plasma was obtained by centrifugation (3,000 × g, 10 min, 4°C). The plasma samples were then subjected to solid-phase extraction and LC-MS/MS as previously described (58 (link), 59 ).