Table 2.

Summary of experimental studies on animal models of demyelination

Study date	Riboflavin dose, sample size, and duration of study	Clinical outcomes	Histological outcomes	Laboratory outcomes
Naghashpour et al, 2016 (35)	Ten week-old C57BL/6 female EAE mice were administered riboflavin at 10 mg/kg body weight or INFβ-1a at 150 IU/g body weight for two weeks.	Peak disease score was reduced by riboflavin in both effector and chronic phases of the disease (P<0.05). Riboflavin delayed the onset of disease in EAE mice (P<0.05).		Riboflavin in combination with INFβ-1a increased BDNF mRNA and protein levels in the brain and spinal cord of EAE mice (P<0.01) IL-6 expression was elevated in the brains of the EAE group receiving riboflavin and INFβ-1a (P<0.01)
Naghashpour et al, 2016 (37)	Ten week-old C57BL/6 female EAE mice were administered riboflavin at 10 mg/kg body weight and/or INFβ-1a at 150 IU/g body weight for two weeks. The Morris water maze (MWM) test to evaluate cognitive function and clinical monitoring were performed in mice.	Riboflavin and a combination of riboflavin and INF-β1a reduced the clinical scores. Riboflavin enhanced the swim speed of EAE mice in the MWM test (P<0.05)		Results obtained from brain revealed increased BDNF mRNA expression in EAE mice treated with a combination of riboflavin and INF-β1a (P<0.01). A combination of riboflavin and INF-β1a enhanced the BDNF levels in brains of EAE mice (P<0.05)
Cai et al, 2009 (14)	One day-old broiler chickens were fed a riboflavin-deficient diet (1.8 mg/kg) while control chickens were administered a conventional diet containing 5.0 mg/kg riboflavin.		Pathologic changes were evident in sciatic, cervical, and lumbar spinal nerves of riboflavin-deficient chickens characterized by hypertrophic Schwann cells, tomacula (redundant myelin swellings), and demyelination/remyelination.
Cai et al, 2007 (19)	One-day old broiler chickens were fed a riboflavin-deficient diet (1.8 mg/kg) and killed on postnatal days 6, 11, 16, 21, and 31, whereas control chickens were fed a conventional diet containing 5.0 mg/kg riboflavin		Fibroblastic onion bulb-like structures were detected in sciatic and brachial nerves of riboflavin-deficient chickens from day 11 onwards, consisting of long cytoplasmic processes of hypertrophied fibroblasts surrounding demyelinated, remyelinated and normally myelinated axons.
Cai et al, 2006 (16)	Rapidly growing broiler chickens were fed either a riboflavin-deficient diet (containing 1.8 mg/kg riboflavin) or a conventional diet (containing 5.0 mg/kg riboflavin) and killed on postnatal days 6 (n = 6), 11 (n = 14), 16 (n = 6), and 21 (n = 5).		Acquired primary demyelinating tomaculous neuropathy was observed in the sciatic and brachial nerves of riboflavin-deficient chickens from day 11 onwards.
Cai et al, 2006 (25)	Newborn broiler chickens were maintained either on routine diet containing 5.0 mg/kg riboflavin (control group) or a riboflavin-deficient diet (containing 1.8 mg/kg riboflavin).		Riboflavin-deficient chickens showed signs of neuropathy from day 8 and pathological examination of peripheral nerves revealed demyelinating neuropathy with paranodal tomacula formation, starting on day 11. After day 16, paranodal swellings showed prominent degenerative changes accompanied by increased frequency of demyelination of fibers.
Johnson and Storts, 1998 (15)	One day-old chickens were fed a riboflavin-deficient diet (containing 1.65 mg/g riboflavin) for 52 days, followed by the control diet for 14 days.	Demyelinating peripheral neuropathy in young, rapidly growing chickens, including leg weakness and paralysis as early as 12 days of age.	Significant microscopic lesions were confined to peripheral nerves and included tissue separation (suggesting interstitial edema), Schwann cell swelling, perivascular leukocytic infiltration, and segmental demyelination accompanied by accumulation of osmiophilic debris in Schwann cell cytoplasm. Axon degeneration was observed.	Acid phosphatase enzyme activity of Schwann cells was increased in the affected nerves
Wada et al, 1996 (17)	Nine 14- to 55-day-old racing pigeons were maintained on a riboflavin-deficient diet (riboflavin concentration of 0.9 mg/kg feed).	DiarrhoeaDiarrhea, and leg and wing paralysis	Peripheral nerve lesions, including discoloration and swelling of all the peripheral nerve trunks, were observed. Microscopic lesions comprising swelling, fragmentation, demyelination of myelin sheaths, and proliferation of Schwann cells were evident in the peripheral nerves of all birds examined. These changes were associated with moderate to severe swelling, fragmentation, atrophy, and loss of axons.
Jortner et al, 1987 (18)	A strain of rapidly growing meat-type chickens were fed a diet deficient in riboflavin (1.8 mg/kg riboflavin) from 1–40 days of age.	Diminished growth rate, progressive gait abnormality and reluctance to move	Neurologic abnormalities related to peripheral neuropathy characterized by Schwann cell hypertrophy and degeneration with cytoplasmic lipid droplets and segmental demyelination. Sequestration of myelin debris within Schwann cells was common. Presence of endoneurial edema and axonal degeneration involving small numbers of fibers.	Liver concentrations of riboflavin in deficient birds were significantly reduced on day 13 but not day 26.
Norton et al, 1976 (26)	Eighteen adult male rats were distributed into three equal groups. Each group was maintained on a specific diet: Group 1, complete diet (control); Group 2, riboflavin-deficient diet; and Group 3, riboflavin-deficient diet plus galactoflavin, 2 g/kg of diet.		Sciatic nerve fibers demyelinated in animals maintained on riboflavin- deficient diets in a time-dependent manner. Cellular organelles of both myelinated and nonmyelinated nerve fibers remained intact and presumably functional.

PBS, phosphate buffer saline; EAE, experimental autoimmune encephalomyelitis; INFβ-1a, Interferon beta-1a; BDNF, brain-derived neurotrophic factor; IL-6, Interleukin-6; IL-17A, Interleukin-17A