Dormicum

Syrian hamsters (male, 4 weeks old) were purchased from Japan SLC. Baseline body weights were measured before infection. For the virus infection experiments in Fig. 2c, d, hamsters were euthanized by intramuscular injection of a mixture of 0.15 mg kg⁻¹ medetomidine hydrochloride (Domitor, Nippon Zenyaku Kogyo), 2.0 mg kg⁻¹ midazolam (Dormicum, Maruishi Pharmaceutical) and 2.5 mg kg⁻¹ butorphanol (Vetorphale, Meiji Seika Pharma). The B.1.1 or B.1.167.2/Delta viruses (10⁵ TCID₅₀ in 100 µl) were intranasally infected under anaesthesia. Body weights were measured, and oral swabs were collected under anaesthesia with isoflurane (Sumitomo Dainippon Pharma) daily. For the virus infection in Fig. 4, four hamsters per group were intranasally inoculated with the D614G or the D614G/P681R viruses (10⁴ TCID₅₀ in 30 μl) under isoflurane anaesthesia. Body weight was monitored daily for 7 days. For virological examinations, four hamsters per group were intranasally infected with the D614G or the D614G/P681R viruses (10⁴ TCID₅₀ in 30 μl); at 3 and 7 d.p.i., the hamsters were euthanized, and nasal turbinates and lungs were collected. The virus titres in the nasal turbinates and lungs were determined by plaque assays in VeroE6/TMPRSS2 cells.

Mice were anesthetized using a mixture of three drugs: 0.75 mg/kg body weight (b.w.) medetomidine (Domitor, Nippon Zenyaku Kogyo Co., Ltd., Tokyo, Japan), 4.0 mg/kg b.w. midazolam (Dormicum, Astellas Pharma Inc., Tokyo, Japan), and 5.0 mg/kg b.w. butorphanol (Vetorphale, Meiji Seika Kaisha, Ltd., Tokyo, Japan). These drugs were diluted in sterile saline to 0.1 ml/10 g b.w./mouse and were administered through intraperitoneal injection. HA and collagen constructs that differentiated in the hypertrophic medium for 2 weeks were implanted subcutaneously into the back of nude mice. Briefly, two subcutaneous pockets were created along the central line of the spine in each mouse, and two to three constructs were inserted into each pocket (n = 5). In some experiments, to prevent hyaluronan constructs from fusing, four pockets were created in both lateral sites of the spine, two at the shoulders and two at the hips; one hyaluronan construct was inserted into each pocket (4 constructs/mouse). After suturing the surgical sites, mice were injected with an antagonist: 0.75 mg/kg b.w. atipamezole (Antisedan; Nippon Zenyaku Kogyo Co., Ltd.). The animals were monitored every couple of days during the healing period for any possible complications. The number of experimental samples was determined based on earlier reports [10 (link)–14 (link), 17 (link), 18 (link)]. Mice of the same age were randomly selected and assigned to each implantation group. A total of 23 mice were used in the in vivo transplantation studies, including preliminary studies to determine the optimal amount of hydrogel and the number of MSCs.
The mice were euthanazed by carbon dioxide inhalation with little suffering at 4 and 8 weeks post-implantation, and the constructs were collected for analysis.

This prospective cross-sectional study was carried out from October 2017 to April 2019, enrolling 100 neonates admitted to the neonatal intensive care unit (NICU) of the Children’s Hospital of Ain Shams University, Cairo, Egypt. Inclusion criteria were neonates with a gestational age ≥28 weeks who were suffering from respiratory distress disorders; exclusion criteria were neonates having multiple congenital anomalies, chromosomal aberrations, hydrops fetalis and/or heart failure. Expert neonatologists managed enrolled neonates according to the NICU protocol [7 ].
Downes and Silverman–Andersen clinical scores were applied to evaluate neonatal respiratory distress severity. The Silverman–Anderson score was ideally used for preterm infants, and the Downes score was used for term infants [8 (link),9 (link),10 (link)].
Plain CXR and LUS were done on admission for diagnosis and were repeated after 7 days, or if needed earlier within the 7 days, by the treating neonatologist in parallel to the clinical assessment and laboratory findings to diagnose the cause of respiratory distress. CXR images were posterior–anterior view, using the digital GE (General Electric) Optima XR220 AMX pro series X-ray machine, (GE HealthCare, Chicago, IL, USA).
CXR findings were interpreted and used as the gold standard to diagnose and differentiate variable etiologies of neonatal respiratory distress: transient tachypnea of the newborn (TTN), respiratory distress syndrome (RDS), neonatal pneumonia, meconium aspiration syndrome (MAS), pulmonary interstitial emphysema (PIE), pneumothorax (PTX), pleural effusion (PE), pulmonary atelectasis (PA) and congenital diaphragmatic hernia (CDH) [1 (link),2 (link),11 ,12 (link),13 ,14 (link),15 ,16 (link)].
Lung ultrasound (LUS) examination was done using the GE Logiq 400 pro series ultrasound machine, with a linear 8 MHz microprobe, (GE HealthCare, Chicago, IL, USA). LUS was performed by a single radiology consultant. The neonatologists were blind to lung ultrasound diagnoses. Neonates were examined lying in a supine position and in a resting state with the surrounding light intensity kept constant and low; as the phototherapy device was turned off if it was on. Gentle handling, including quiet voice tones and fine touching, was applied to avoid stressful situations or cause for crying. In order to pacify babies on continuous positive airway pressure (CPAP) or nasal cannula, oral dextrose drops or a pacifier were given, and stimulation of non-nutritional sucking was applied, while others on ventilation might have been sedated by intravenous Dormicum.
During the study, LUS examination was done using the following LUS score: every lung was divided into 3 areas (upper anterior, lower anterior and lateral) and a linear microprobe was used in lung examination through both transverse and longitudinal scans. For every lung area, a point score from 0 to 3 was applied (total score varying from 0 to 18). The LUS score was allocated as follows:
0: Denotes A-pattern (defined by the existence of the A-lines only, which emerges from the pleural line reverberation artifact);
1: B-pattern (defined by the existence of ≥3 well-spaced B-lines; B-lines are lines reaching the screen edge in the absence of fading);
2: Severe B-pattern (defined by the existence of coalescent and crowded B-lines with or without consolidations restricted to the subpleural space);
3: Extended consolidations.
A-lines denote pleural reflection because of ultrasound diffusing through an air-filled lung; B-lines denote fluid filling the interstitium (and the alveolar space if they become coalescent). LUS diagnostic criteria for neonatal respiratory diseases were according to Corsini et al. [17 (link),18 (link)].