Experimental Model of Type 2 Diabetes and Progressive Pulmonary TB in BALB/c Mice
To induce T2D, a non-genetic mouse model of non-insulin-dependent diabetes mellitus previously reported was reproduced (26). Briefly, young male BALB/c mice three weeks old were fed with a high fat diet (HFD) at 45% of calories during the whole course of the experiment. At 3 months old, animals received a single low dose (100 mg/kg) of estreptozotocin (STZ) ChemCruz (Santa Cruz Biotechnology, Dallas, TX) by intraperitoneal (i.p) injection. That STZ dose did not cause diabetes in chow-fed mice (26). We assessed the combined effect of HFD and STZ in glucose blood levels with Accu-chek active (Roche diabetes care Mannheim, Germany) test strips, after and before of STZ administration. Serum cholesterol and triglyceride concentrations were measured with Acu-trend one month after STZ administration.
After one month of STZ administration, animals showed hyperinsulinemia and hyperglycemia that reverted by the treatment with oral hypoglycemic drug (metformin), a similar metabolic pattern and respond to treatment of T2D (Fig. 1). Animals with these metabolic abnormalities compatible with T2D were infected by the intra-tracheal route with a high dose of Mtb reference strain H37Rv (ATCC No. 25618), in order to induce progressive pulmonary TB. Briefly, Mtb strain H37Rv was grown in Middlebrook 7H9 broth (DIFCO) supplemented with 0.2% glycerol, 10% OADC enrichment, and 0.02% Tween-80 and maintained at 37◦C in agitation. Mid log-phase cultures were used for infection. Mycobacteria were counted and stored at −80◦C until use. Bacterial aliquots were thawed and pulse-sonicated to remove clumps. For the infection, groups of T2D and control animals fed with chow diet were anesthetized in gas chamber using Sevofluorane and infected intra-tracheally with 2.5 Å~ 105 live bacilli using a cannula inside a biosafety level III cabinet. Mice were maintained in vertical position until spontaneous recovery and maintained in groups of five in cages fitted with micro-isolators connected to negative pressure in animal biosafety level III facilities.
In order to compare the evolution of TB in T2D and non-diabetic mice, groups of 5 mice were euthanized by exsanguination under pentobarbital anesthesia after 14, 21, 28 and 120 days after infection. Lungs were rapidly collected and immediately frozen by immersion in liquid nitrogen and used for bacillary loads determination by colony forming units quantification and TNFα expression determined by RT-PCR, as described below. Ten animals per group were left untouched and their survival was recorded to construct survival curves.