Glycerol

To make a suspension of
nanodiamonds in glycerol, 100 μL of 1 mg mL^–1 50 nm carboxylic-acid terminated nanodiamonds (FND Biotech, Taiwan)
in water were centrifuged for 5 min at 12 000 g. Details on the nanodiamond fabrication and material properties
can be found in ref (59 (link)). Excess water was removed, and the nanodiamond pellet was resuspended
in 10 mL of ≥99% glycerol (Sigma-Aldrich, UK).
To make
the glycerol-cross-linked xanthan (GCX), we transfer 9.9 g of glycerol-nanodiamond
suspension prepared as mentioned above into a beaker and begin agitating
by using a magnetic stirrer at a rate of 100 rpm. Then 0.1 g of xanthan
gum powder (Sigma-Aldrich, UK) is slowly added to the glycerol. Once
all the xanthan is fully incorporated into the glycerol, the beaker
is heated to 80 °C with the stirring rate increased to 1000 rpm
for 1 h to facilitate cross-linking.

Mtb H37Rv was grown in Category III conditions on Middlebrook 7H9 (Becton Dickinson) medium plus 10% Middlebrook OADC (oleic acid, albumin, dextrose and catalase; Becton Dickinson) supplement, 0.5% glycerol (Sigma Aldrich) and 0.05% Tween 80 (Sigma Aldrich) at 37 °C at 100 rpm until they reached mid log phase when aliquots were frozen for use in 10% glycerol and plated out for colony forming units (CFU) on Middlebrook 7H10 agar plates plus 10% OADC and 0.5% glycerol at 37 °C for 2–3 weeks.

Male Wistar rats (Rattus novergicus) weighing 180–200 g were obtained from the animal facilities of the University of São Paulo–Institute of Biomedical Sciences. We kept our animals at controlled temperature (23±1°C) with a light/dark cycle of 12/12h in standard cages with ad libitum access to tap water and commercial rodent chow (Nuvilab, PR, Brazil). Rats were allocated to four groups: Sham (C, n = 7), received saline 0.9% (5 mg/kg body weight) intraperitoneally (IP); Lithium (Li, n = 7), received a single IP injection of Lithium Chloride (LiCl, 80 mg/kg body weight, MilliporeSigma, MA, USA); glycerol (Gly, n = 8), received a single dose of glycerol 50% (5 mL/kg body weight, Sigma-Aldrich, MO, USA) intramuscular (IM); and glycerol plus Lithium (Gly+Li, n = 8), received a single dose of glycerol 50% IM plus LiCl IP injected 2 hours after glycerol administration. We submitted the animals to the inhalational anesthetic isoflurane (1mL/1mL; Cristália, SP, Brazil) for approximately 3 minutes before saline or glycerol infusion. The doses and the time between injections of LiCl and glycerol were based on previous studies [14 (link)–16 (link)]. In addition, as the human model of severe rhabdomyolysis requires prompt treatment, we chose to administer lithium 2 hours after the glycerol injection in order to reproduce clinical conditions found in the hospital daily routine.