Hydralazine hydrochloride

Hydralazine hydrochloride (MKBS4863V), ketanserin (+)-tartrate salt (MKBR2179V), propranolol hydrochloride (BCBL8710V), phentolamine hydrochloride (085M4021V) and yohimbine hydrochloride (BCBM8231V) were procured from Sigma-Aldrich, St. Louis, Missouri, United States (US). The drugs were prepared in PBS prior to their i.p. administrations at doses of 5 mg/kg, 0.3 mg/kg, 2 mg/kg, 10 mg/kg and 10 mg/kg, respectively. The infected animals were observed until erythrocyte-stage of Pb was established (this coincided with day 3 post-inoculation) and treatments with the drugs commenced immediately. The treatment followed a 6-dose regimen of 0, 8, 24, 36, 48 and 60 h for both intact and Pb-infected mice. Thirty minutes after the last treatments on day 5, relevant behavioural and biochemical tests were conducted.
Naloxone hydrochloride (Samarth Life Sciences Pvt. Ltd., Mumbai, India (INLXB1602)) injection (400 µg/mL) was administered via i.p. at a dose of 5 mg/kg whilst the controls were given 5 mL/kg of PBS. Treatments were carried out twice daily (12-h interval), starting on day 3 post-i.p. inoculation of Pb. Thirty minutes after last treatments on day 5, pain-related behavioural responses were evaluated in all animals.

Hydralazine hydrochloride (Sigma, St. Louis, MO, USA) was dissolved in phosphate buffered saline, sterilized through a filter, and subsequently stored at 4°C. Hydralazine at a dosage of 1 mg/kg (Body Weight) was applied daily through intraperitoneal injections (IP), commencing from the day of induction until the end of study period (22 days post induction).

Prestwick FDA Approved Chemical Library was provided by MSU Assay Development and Drug Repurposing Core (ADDRC). R-(−)-apomorphine hydrochloride, raloxifene hydrochloride, fosinopril, nifedipine, nisoldipine, idebenone, itraconazole, prednicarbate, carmofur, diazepam, flurazepam, temazepam, prazepam, nitrazepam, nimetazepam, nordiazepam and oxazepam were from Cayman Chemical (Ann Arbor, MI). Devazepide was purchased from Santa Cruz Biotechnology (Dallas, Texas). Fluorescein diacetate, propidium iodide, clofazimine, hexachlorophene, hydralazine hydrochloride, thioflavin S and Amicon Centrifugal Filter Unit were purchased from Sigma Aldrich (St. Louis, MO). Gibco Dulbecco’s Modified Eagle Medium (DMEM), HyClone™ Fetal Bovine Serum, Optical Adhesive Film and FITC-Annexin V were purchased from Thermo Fisher Scientific (Waltham, MA). All other chemicals for common buffers and solutions were from Sigma Aldrich.

“Conditioned-media” (CM) is the term we use to describe media obtained from networks 4 h after they experienced maximum-limit impact forces (ten rapidly-imparted 200 g impacts), for the purpose of isolating and investigating secondary injuries. CM was obtained and substituted with media from non-impact networks for a total CM-exposure period of 24 h, followed by fixing.
Hydralazine (HZ, Hydralazine Hydrochloride, Sigma H1753), a repurposed antihypertensive drug and demonstrated acrolein scavenger that has already shown some promise in secondary injury models^{65 (link)–67 (link)}, was introduced to CM and control networks at a concentration of 500 μM. Networks were exposed to CM for 15 min before HZ administration (CM + HZ), and incubated for 24 h before subsequent analysis.