C57BL/6 mice were immunized subcutaneously (s.c.) in the flank with 200 μg of MOG35–55 peptide emulsified in complete Freund´s adjuvant (CFA) containing 0.1 mg Mycobacterium tuberculosis (Becton Dickinson) along with an intraperitoneal injection of 200 ng pertussis toxin (List Biological Laboratories Inc.) on the day of immunization and two days later. In Lewis rats, EAE was induced by intradermal injection at the base of the tail with an emulsion consisting of 100 μg MBP from guinea pig (Sigma-Aldrich) suspended in CFA with the addition of 0.2 mg of Mycobacterium tuberculosis (Becton Dickinson). The animals were monitored daily, weighed and clinically scored according to Table 1 . No animals were permitted to lose more than 20% body weight (compared to the starting point of EAE immunization) and to go beyond score 4 [20 (link)].
Mycobacterium tuberculosis
Manufactured by BD
Sourced in United States, China
Mycobacterium tuberculosis is a slow-growing, acid-fast bacillus that is the causative agent of tuberculosis. It is a critical component in the diagnosis and research of this serious infectious disease.
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Lab products found in correlation
Pertussis toxin, by Merck Group (13 mentions)
Pertussis toxin, by List Biological Laboratories (12 mentions)
Complete freund s adjuvant, by Merck Group (10 mentions)
Complete freund s adjuvant, by BD (7 mentions)
Complete freund adjuvant (cfa), by Merck Group (6 mentions)
Bordetella pertussis toxin, by Merck Group (4 mentions)
Incomplete freund s adjuvant, by BD (3 mentions)
Pertussis toxin ptx, by Bio-Techne (3 mentions)
Mog35 55 peptide, by Bio-Synthesis (2 mentions)
Mog35 55, by Merck Group (2 mentions)
Incomplete freund s adjuvant, by Merck Group (2 mentions)
Mog35 55 peptide, by GenScript (2 mentions)
Phosphate buffered saline (pbs), by Merck Group (2 mentions)
Complete freund s adjuvant cfa, by Merck Group (1 mentions)
Pertussis toxin ptx, by Merck Group (1 mentions)
Mog35 55 peptide, by GL Biochem (1 mentions)
C57bl 6 mice, by Jackson ImmunoResearch (1 mentions)
Aminoguanidine hemisulfate, by Merck Group (1 mentions)
Pertussis toxin, by Sapphire Bioscience (1 mentions)
Bordetella pertussis toxin, by List Biological Laboratories (1 mentions)
55 protocols using mycobacterium tuberculosis
1
Murine and Rat Models of EAE
2
Induction and Scoring of MOG-EAE in Mice
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Myelin oligodendrocyte glycoprotein (MOG)–EAE was induced in mice purchased from Charles River by immunizing subcutaneously with 200 mg of MOG 35–55 peptide in complete Freund’s adjuvant (Mycobacterium tuberculosis at 3.75 mg ml−1; Becton Dickinson, catalog no. 231141) and intraperitoneal injection twice with 500 ng of pertussis toxin (Sigma-Aldrich, catalog no. P7208) as described (73 (link)). Animals were examined daily and scored for clinical signs of the disease. If disease did not start within 15 days after induction or the clinical score rose above 4, then animals were excluded from the analysis. The clinical score was as follows: 0, normal; 0.5, loss of tail tip tone; 1, loss of tail tone; 1.5, ataxia, mild walking deficits (slip off the grid); 2, mild hindlimb weakness, severe gait ataxia, twist of the tail causes rotation of the whole body; 2.5, moderate hindlimb weakness, cannot grip the grid with hind paw but able to stay on a upright tilted grid; 3, mild paraparesis, falls down from a upright tiled grid; 3.5, paraparesis of hindlimbs (legs strongly affected but move clearly); 4, paralysis of hindlimbs, weakness in forelimbs; 4.5, forelimbs paralyzed; and 5, moribund/dead. KD was provided commencing at the first appearance of EAE symptoms.
3
Experimental Autoimmune Encephalomyelitis (EAE) Induction
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C57BL/6J mice were obtained from the Animal Resource Centre (Perth, Australia) and housed under standard conditions with food and water ad libitum. All procedures were approved by the institutional Animal Ethics Committee and performed strictly in accordance with regulations set by the National Health and Medical Research Council of Australia. Only female mice aged 12–16 weeks were used (32 (link)). EAE was performed as previously described (36 (link)). Briefly, mice received 200 µg of peptide 35-55 of myelin oligodendrocyte glycoprotein (MOG35-55), emulsified in complete Freund’s adjuvant (Sigma-Aldrich, St. Louis, MO) supplemented with 4 mg/ml of heat inactivated Mycobacterium tuberculosis (Becton Dickinson, Franklin Lakes, NJ) and administered subcutaneously. This was immediately followed by an intraperitoneal injection of 350 ng of Bordetella pertussis toxin (Sigma-Aldrich), which was repeated 48 hours later. Control groups included sham where MOG33–55 was substituted with phosphate buffered saline (PBS, 0.01 M phosphate, 15 mM NaCl, pH 7.4) and normal mice. Clinical progression was followed by daily weighing and visual assessment of ambulatory difficulties, scored as follows: 0 = no symptoms, 1= limp tail, 2 = hind limb weakness, 3 = hind limb paralysis, 4 = ascending paralysis, and 5 = moribund (36 (link)). The experimental design is summarized in Supplementary Figure 1
4
Experimental Autoimmune Encephalomyelitis Model in Mice
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C57BL/6J and Cpt1a P479L mice were anesthetized with isoflurane and immunized subcutaneously in the base of the tail with 200 µg MOG35-55 (Pepmic) emulsified in complete Freund’s adjuvant (CFA) (Becton Dickinson) containing Mycobacterium tuberculosis (Becton Dickinson). All mice received an intraperitoneal injection of 500 ng pertussis toxin (Sigma-Aldrich) on the day of immunization and two days later. The mice were monitored daily and weighed and scored clinically according to a scale from 0 to 5. The mice were not permitted to lose more than 20% body weight and to go beyond score 4. From day 10, the mice received HFD (60% fat) (Brogaarden). After 24 days, the mice were euthanized, and brain samples were collected for immunohistochemistry, western blotting and RT-qPCR.
5
Chronic EAE Induction and Treatment
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Chronic EAE was induced in 6–8 weeks old C57Bl/6 mice by subcutaneous immunization with 300 μg of MOG33–35 peptide in complete Freud’s adjuvant (Becton Dickinson) containing 0.8 mg/ml Mycobacterium Tuberculosis (Becton Dickinson), as previously described42 (link),43 (link). Mice were also injected intravenously (i.v.) with pertussis toxin (40 ng; List Biological Laboratories) at the day of immunization and after 48 hours. To evaluate the clinical and pathological efficacy of ASC-NVs in chronic EAE, mice were injected i.v. with NVs at 3, 8 and 13 dpi (preventive protocol) or at 12, 16 and 20 dpi (therapeutic protocol). For each injection, we infused 5 μg of NVs in 0.3 ml of PBS. Control mice received only vehicle. Clinical score was blindly registered according to the following scale: 0 = healthy, 1 = limp tail, 2 = ataxia and/or paresis of hindlimbs, 3 = paraplegia, 4 = paraplegia with forelimb weakness or paralysis, 5 = moribund or death animal. Animals were housed in pathogen-free, climate-controlled facilities and were provided with food and water according to current European Community laws. All mouse experiments were carried out in accordance with experimental guidelines approved by the University of Verona committee on animal research (Centro Interdipartimentale di Servizio alla Ricerca Sperimentale) and by the Italian Ministry of Health.
6
Induction of Experimental Autoimmune Encephalomyelitis in Mice
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On the day of immunization (Day 0), mice were injected subcutaneously over each shoulder with 100 μL of an emulsion containing 0.5 mg/mL MOG35–55 peptide (Bio-Synthesis, 12–668–01) and 1.25 mg/mL heat-killed Mycobacterium tuberculosis (Becton, Dickinson, & Company, 231–141) in Complete Freund’s Adjuvant (Sigma Aldrich, F5881). Mice were intraperitoneally injected with 200 ng of Pertussis toxin (List Biological Laboratories, 180) on days 0 and 2 post-immunization.
Beginning at approximately day 7 post-immunization, mice were monitored daily for onset of hindlimb paralysis and scored for EAE severity using the following 5-point clinical scoring system: 0 = normal tail; 0.5 = limp at tip of tail; 1 = completely limp tail; 1.5 = partial hindlimb weakness/mouse can be flipped onto its back; 2 = complete hindlimb weakness with abnormal gait; 3 = partial hindlimb paralysis; 3.5 = complete hindlimb paralysis in both legs; 4 = hind- and fore-limb paralysis; 5 = moribund/dead.
Beginning at approximately day 7 post-immunization, mice were monitored daily for onset of hindlimb paralysis and scored for EAE severity using the following 5-point clinical scoring system: 0 = normal tail; 0.5 = limp at tip of tail; 1 = completely limp tail; 1.5 = partial hindlimb weakness/mouse can be flipped onto its back; 2 = complete hindlimb weakness with abnormal gait; 3 = partial hindlimb paralysis; 3.5 = complete hindlimb paralysis in both legs; 4 = hind- and fore-limb paralysis; 5 = moribund/dead.
7
Induction of Experimental Autoimmune Encephalomyelitis in Mice
8
Cerebral EAE Induction in SJL and C57BL/6 Mice
9
Rat Model of Experimental Autoimmune Encephalomyelitis
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One week after the last day of feeding, the EAE was induced. Rats under inhaled anesthesia, 0.5-3.5% Narcotan (Leciva a.s.) in oxygen, were injected intradermally into the hind paws with the immunizing mixture (100 µl/paw). The mixture contained 50% guinea pig spinal cord homogenate as a source of myelin antigens mixed in the ratio 1 : 1 with Freund Adjuvant (DIFCO LABORATORIES), and supplemented with 4 mg/1 ml Mycobacterium tuberculosis (DIFCO LABORATORIES).
10
EAE Induction in C57BL/6 Mice
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Six to Twelve-week-old mice were immunized with MOG35−55 in Complete Freund's Adjuvant (CFA; Sigma-Aldrich), a procedure to induce experimental autoimmune encephalomyelitis (EAE) in C57BL/6 mice, an animal model of multiple sclerosis (MS; Preller et al., 2007 (link)). A total of 200 μg of MOG35−55 peptide and 400 μg of killed Mycobacterium tuberculosis (Difco Laboratories) was emulsified in CFA and injected s.c. into the footpads of mice.
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