Minocycline hydrochloride

Minocycline hydrochloride and Tetracycline hydrochloride (Sigma-Aldrich) were resuspended in water at 4 mg/ml, before dilution in RPMI to the required concentration. Tigecycline hydrate (Sigma-Aldrich) was resuspended in DMSO at 5 mg/ml, before dilution in RPMI. Stock solutions were made fresh for each experiment. Typically for 96-well plate assays, 50 μl of stock solution or cRPMI control was added per well.

Intracerebroventricular (ICV) catheter placement and injection followed the procedures used in a previous study [23 (link)]. Briefly, on POD 30, rats were anesthetized with pentobarbital sodium (50 mg/kg). After anesthesia, the rat was fixed on a stereotaxic apparatus. Then, the surgery was performed and a catheter was placed with stereotaxic coordinates: 0.8 mm posterior, 1.5 mm left lateral, and 4.5 mm ventral from the bregma. The guide cannula was fixed with dental cement and the rat was returned to its cage for recovery. The ICV cannulation was verified by injection of methylene blue through the cannula. The environmental enrichment toys were provided as before. Minocycline hydrochloride (purchased from Sigma-Aldrich Corp, St Louis, USA) was dissolved in normal saline. For each daily injection, rats were anesthetized with isoflurane as above; rats received bilateral microinjection of minocycline (160 μg/side) or saline (control) into the cerebral ventricle. A total volume of 4.0 μl was infused into each side over 10 min, and the injection syringe was left in place for an additional 5 min to allow for diffusion [23 (link)]. The microinjections were performed daily from POD34 to POD39. The behavior tests resumed on POD 40.

Minocycline Hydrochloride (Sigma-Aldrich, USA), dissolved in normal saline, was administered to the rats daily by intraperitoneal (i.p.) route, once a day in the morning time, during 48 h of SD. Minocycline at 10, 5, and 2 mg/kg body weight were used to standardize the dose on the behavioral and biochemical scale. The minimum dose of minocycline showing maximum effect was selected, and it was in accordance with some previous studies [28 (link)–30 (link)].

Recombinant human IFN-α was obtained from PeproTech Inc. (#300-02AB; Rocky Hill, NJ, USA) and was dissolved in artificial cerebrospinal fluid (ACSF; glucose, 5 mM; CaCl₂, 1 mM; NaCl, 125 mM; MgCl₂, 1 mM; NaHCO₃, 27 mM; KCl, 0.5 mM; Na₂SO₄, 0.5 mM; NaH₂PO₄, 0.5 mM; and Na₂HPO₄, 1.2 mM). Rat serum albumin (1 mg) was added to 1 ml of 2 × 10⁷ IU/ml IFN-α. The rats received bilateral infusions of ACSF (vehicle) or IFN-α at doses of 100, 200, or 400 IU/μl (1 μl/side).
Minocycline hydrochloride (#M9511; Sigma, St. Louis, MO, USA) was dissolved fresh in 0.9 % NaCl and administered intragastrically (i.g.) once daily at a dosage of 90 mg/kg rat body weight for 3 days prior to the IFN-α treatment. The dose was selected on the basis of previous studies showing the beneficial effects of this dosage in animal models of cerebral brain ischemia, multiple sclerosis, and Parkinson’s disease [29 (link)–33 (link)].

The insecticide DDT (≥98% pure); the antioxidant reagents ascorbic acid (vitamin C), β-carotene (provitamin A), glutathione, α-lipoic acid, melatonin, minocycline hydrochloride, and serotonin; and the solvents (acetone and ethanol) were all purchased from Sigma-Aldrich (St. Louis, MO, USA). All chemicals were of analytical grade.

The chemicals used in this study and their sources were as follows: morphine hydrochloride – Polfa (Kutno, Poland); DAMGO (Sigma-Aldrich, USA), Deltorphin II (Sigma-Aldrich, USA), DPDPE (Sigma-Aldrich, USA), U50,488H (Tocris, UK), SNC80 (Sigma-Aldrich, USA) and minocycline hydrochloride (Sigma-Aldrich, USA). Minocycline (MC; 30 mg/kg; i.p.) was dissolved in sterile water and pre-emptively administered intraperitoneally 16 h and 1 h before CCI and then twice daily for 7 days. This method of minocycline administration was used throughout the work and is referred to in the text as “repeated administration”. The control groups received a vehicle (water for injection) according to the same schedule. One hour after the last morning of minocycline or vehicle administration on day 7 after CCI, morphine (M; 20; 40 µg; 62.3 nM; 124.6 nM), DAMGO (MOR selective ligand; 1; 2 µg; 1.94 nM; 3.9 nM), U50,488H (KOR selective ligand; 25; 50 µg; 61.6 nM; 123.2 nM), DPDPE (DOR I selective ligand; 10; 20 µg; 15.48 nM; 30.96 nM), Deltorphin II (del II; DOR II selective ligand; 1.5; 15 µg; 1.9 nM; 19 nM), SNC80 (10; 20 µg; 22.24 nM; 44.48 nM) or a vehicle was intrathecally injected. The von Frey test (25 and 45 min later) and cold plate tests (30 and 50 min later) were carried out after vehicle or opioid agonist administration (Figure 1).