Vetflo

Male Wistar rats (250 ± 10 g, three months old) were used. The rats were obtained from the National Scientific Center of Marine Biology, Far Eastern Branch of the Russian Academy of Sciences, Vladivostok, Russia. The animals were housed two to three per cage with ad lib access to chow and water in a 12-h light/dark cycle. Temperature (23 ± 2 °C) and humidity (55 ± 15%) were constant. All experimental procedures were approved by the Animal Ethics Committee at the National Scientific Center of Marine Biology, Far Eastern Branch, Russian Academy of Sciences (No. 1/2020), according to the international regulations of the European Directive 2010/63/EU and ethical guidelines for the study of experimental pain in conscious animals by the International Association of the Study of Pain. The induction of neuropathic pain was performed using a model of sciatic nerve chronic constriction injury (CCI) [53 (link)]. Animals were anesthetized with isoflurane using rodent anesthesia vaporizer (VetFlo, Kent Scientific Corporation, Torrington, CT, USA). After the animal has been anesthetized, the right sciatic nerve was exposed and the three ligatures were placed closer to trifurcation with 1 mm between the ligatures (silk, Ethicon, Somerville, NJ, USA). The ligatures were slightly tightened until a slight twitching of the limb appeared.

[Ca²⁺]_i and [Na⁺]_i measurements were carried out in vivo, on left vastus lateralis fibers in euthermic (37 °C - heating pad) and anesthetized (100 mg/kg ketamine and 5 mg/kg xylazine) WT, Ca_V1.1-R174W HET, and Ca_V1.1-R174W HOM mice (30 (link)). Superficial fibers of the vastus lateralis were superfused with warm Ringer’s solution (37 °C) and impaled with double-barreled Ca²⁺-selective or Na⁺-selective microelectrodes. FDB membrane and ion specific potentials were recorded via a high-impedance amplifier (WPI Duo 773 electrometer; WPI) (30 (link)). The membrane potential from the 3 M KCl microelectrode was subtracted electronically from the ion selective microelectrode to produce a differential Ca²⁺-specific potential or Na⁺-specific potential that represents the [Ca²⁺]_i or [Na⁺]_i, respectively. All voltage signals were stored in a computer for further analysis. In a different set of experiments, [Ca²⁺]_i was measured before and after inhalation of 1.5% halothane in air through a nasal mask (Somno-0801 with Vetflo, Kent Scientific).
Recording of [Ca²⁺]_i and [Na⁺]_i was carried out while normal Ringer solution containing (in mM) 135 NaCl, 5 KCl, 1.8 CaCl₂, 1 MgCl₂, 18 NaHCO₃, 1.5 NaH₂PO, and 5 glucose, pH 7.3 to 7.4 was used to keep muscle fibers moist. All in vivo experiments were performed at 37 °C.

A MoorVMS laser Doppler system (Moor Instruments, Wilmington, DE, USA) was used for recording continuous blood flow readings during middle cerebral artery occlusion (MCAO). Sprague Dawley rats were induced to a surgical plane of anesthesia using 4% isoflurane and maintained at 2.5–3% isoflurane anesthesia in O2 using a VetFlo™ vaporizer (Kent Scientific Corporation, Torrington, CT, USA). A midline incision from the ears to parallel to the eyes was made and the skull exposed so that the coronal suture and sagittal suture are easily visible. The Doppler probe was placed on the skull directly above the left middle cerebral artery (MCA) region (MCA: 2 mm posterior, 6 mm lateral to Bregma) and secured using surgical grade adhesive (Moor Instruments, Wilmington, DE, USA).

The histological Golgi–Cox staining technique was used to visualize hippocampal neurons and subsequently obtain morphometric data. The animals were euthanized on the 7th day after the surgery. To achieve this, mice were anesthetized with isoflurane using a rodent anesthetic vaporizer (VetFloTM, Kent Scientific Corporation, Torrington, CT, USA). Then, decapitation was carried out, and the brain was quickly removed and washed with 0.1 M PBS (+4 °C). An extracted brain on day 7 after surgery is shown in Figure 16c. For visual evaluation of the damaged area, 10 μm thick sections were made, followed by hematoxylin-eosin staining (Figure 16d). The brain was placed in a pre-prepared solution, and all subsequent staining procedures were performed according to the manufacturer’s instructions for the FD Rapid GolgiStainTM kit (FD NeuroTechnologies, Ellicott City, MD, USA). A cryomicrotome (HM 550; Thermo Scientific, Waltham, MA, USA) was used to make 100 µm sections. After further processing, staining, and dehydration, sections on gelatin-coated slides were embedded in H-5000 histological medium (Vector Laboratories, Burlingame, CA, USA).

A rodent anesthetic vaporizer (VetFloTM, Kent Scientific Corporation, Torrington, CT, USA) was used to anesthetize the mice using isoflurane. Brains were promptly taken from the skulls of anesthetized mice, washed with 0.1 M PBS (+4 °C), and sliced into two hemispheres. According to the manufacturer’s instructions, the material was stained with the FD Rapid GolgiStain^TM kit (FD NeuroTechnologies, Ellicott City, MD, USA). A cryomicrotome was used to make 100-m thick slices (HM 550; Thermo Scientific, Waltham, MA, USA). Slices were mounted on gelatin-coated slides, stained, dehydrated, and coverslipped with VectaMount^TM mounting medium (H-5000; Vector Laboratories, Burlingame, CA, USA).

To determine the NGF, NMDAR1, NMDAR2A, and ASAHL protein concentration within the hippocampus (ipsi- and contralateral) and cultured astrocyte, the enzyme-linked immunosorbent assay was used. Tissue was harvested 14 days after CCI. The rats (n = 7) were anesthetized with 4.5% isofluorane using a rodent anesthesia vaporizer (VetFloTM, Kent Scientific Corporation, Torrington, CT, USA); the hippocampus was quickly extracted and frozen in liquid nitrogen. The primary antibodies used were: recombinant anti-NGF antibody (1:1000, ab52918), anti-NMDAR1 antibody (1:1000, ab52177), anti-NMDAR2A antibody (1:1000, ab203197), all from Abcam (Cambridge, UK), and anti-ASAHL antibody (1:1000, SC-100470, Santa Cruz, Dallas, TX, USA).
Further steps of the ELISA were carried out in accordance with the protocol described in our previous study [17 (link)]. Each sample was analyzed twice, and the results were averaged.