Hydrate, Chloral

Surgical and implantation procedures were performed as previously described17 (link). Mice were anesthetized with chloral hydrate (55 mg/kg, i.p.) and placed in a stereotaxic apparatus (David Kopf). After exposing the skull, 4 electrodes were positioned: 1) a monopolar 90 μm-diameter stainless steel wire (California Fine Wire Co.) in each dorsal hippocampus (2.0 mm posterior to Bregma, 2.5 mm lateral to the midline, 2.0 mm deep), 2) two subdural screw electrodes (2.5 mm diameter screws, Pinnacle Technologies), one over left frontal cortex (0.5 mm posterior to Bregma, 1.5 mm lateral to the midline) and one over right occipital cortex (3.5 mm posterior to Bregma, 2.0 mm lateral to the midline). An epidural screw electrode was placed above the cerebellum at the midline to serve as reference and a subdural screw electrode over the olfactory bulb was used as the ground. An 8-pin connector (Millmax) was centered over the skull with dental cement (Dentsply) and the animal was placed in its home cage on top of a heating pad (37 °C; Harvard Apparatus) until fully ambulatory.

All experiments were approved by the Institutional Animal Care Unit Committee in Administration Office of Laboratory Animals Beijing China (B10831). Cerebellar sagittal slices (400 μm) were prepared from Wistar rats in postnatal day 14 ~ 15 that were anesthetized by injecting chloral hydrate (300 mg/kg) and decapitated by a guillotine. The slices were cut by a Vibratome in the modified and oxygenized (95% O₂ and 5% CO₂) artificial cerebrospinal fluid (mM: 124 NaCl, 3 KCl, 1.2 NaH₂PO₄, 26 NaHCO₃, 0.5 CaCl₂, 5 MgSO₄, 10 dextrose and 5 HEPES; pH 7.4) at 4°C, and were held in the normal oxygenated ACSF (mM: 126 NaCl, 2.5 KCl, 1.25 NaH₂PO₄, 26 NaHCO₃, 2 CaCl₂, 2 MgSO₄ and 25 dextrose; pH 7.4) at 35°C for 1–2 hours. A slice was transferred to a submersion chamber (Warner RC-26G) and perfused by normal ACSF for electrophysiological experiments [57 (link),68 (link)-70 (link)].
Cerebellar Purkinje cells were identified based on their morphology and functions. Purkinje cells (somata above 40 μm) in the slices for whole-cell recording were located at the border between molecular layer and granule cells, and infused by fluorescence Alex-488 (5 μM in pipettes) under a DIC/fluorescent microscope (Nikon, FN-E600). The excited fluorophore showed the typical dendrites and axonal branches of Purkinje cells, through which we traced their main axons for recording spikes by loose-patch. Purkinje cells also were labeled by neurobiotin (Figure 1A). Purkinje cells showed fast spiking and no adaptation in amplitude and frequency [18 (link),42 (link),71 (link)-75 (link)].

The hyphal development was visualized using the Trypan Blue staining technique (Koch and Slusarenko, 1990 (link)). In brief, the PM-infected melon leaves were soaked in Trypan Blue staining solution and immediately heated in 100 °C water for 2–5 min. After allowing the solution to cool down to room temperature, the staining solution was discarded and the leaves were decolored using a 2.5 mg/ml chloral hydrate solution, which was replaced every 24 hours until the leaves were completely decolored.

To improve the early diagnosis of CoA, several diagnostic tests have been used in clinical practice. Currently, cardiac ultrasound is a routine test for CoA, and studies to improve the prenatal diagnosis of CoA have recently been conducted based on it (17 (link), 18 (link)). However, since the aortic coarctation occurs mainly in the isthmus and its physical changes are not clear, it is often examined with the help of CT and MRI. MRI is also widely used to assess CoA (19 (link)), but due to its time-consuming, costly, and low spatial resolution, it has limitations compared with CT (20 (link), 21 (link)). Therefore, in this study, CTA was used for all study subjects, and initial reconstruction of the scanned images was completed using image post-processing techniques.
Children who were hemodynamically unstable and uncooperative were sedated before CTA by oral 10% chloral hydrate (0.5 ml/kg body mass) or intramuscular sodium phenobarbital injection (5 ml/kg body mass), with careful monitoring of heart rate and saturation by the anesthesia team during sedation. A Philips Brilliance ICT machine was used to perform CT scanning from the lower neck to the level of the diaphragm, and the scanning parameters were set according to the ALARA principle: tube voltage 80–100 kV, tube current 35–85 mAs, pitch 0.2 mm, layer spacing 5.0 mm, layer thickness 5.0 mm, and image reconstruction layer thickness 1.0 mm. Iohexol 300 (mgI/ml) and iodixanol (270 mgI/ml) were injected into the dorsal vein of the hand and foot using a high-pressure syringe at a dose of 2 ml/kg and an injection rate of 0.6–3.0 ml/s. Phase II enhancement scans were performed 15–30 s and 50–60 s after drug administration, respectively.
The minimum internal diameters of the ascending aorta (AOA), proximal arch (D1), distal arch (D2), isthmus (D3), and descending aorta (DA) were measured using a double-blind method by two physicians who have been involved in cardiovascular disease research for many years, and each measurement was taken twice and averaged.