1. Minocycline prevents osmotic demyelination associated with aquaresis
Hiroshi Takagi, Yoshihisa Sugimura, Haruyuki Suzuki, Shintaro Iwama, Hisakazu Izumida, Haruki Fujisawa, Koichiro Ogawa, Kotaro Nakashima, Hiroshi Ochiai, Seiji Takeuchi, Atsushi Kiyota, Hidetaka Suga, Motomitsu Goto, Ryoichi Banno, Hiroshi Arima, Yutaka Oiso 
Kidney International 
Volume 86, Issue 5, Pages (November 2014)
DOI: /ki
Copyright © 2014 International Society of Nephrology Terms and Conditions

2. Figure 1
Serum sodium increase associated with aquaresis-induced neurological impairments. The neurological symptoms were assessed using neuroscore as follows: 6, no impairment; 5, poor grooming; 4, slow or awkward gait; 3, limb weakness and/or paralysis; 2, seizures or severe motor deficits; 1, complete inability to move; and 0, death. T1 group (●), T10 group (▴), RP group (♦), and HS group (▪). After correction of serum sodium level, rats in all the groups showed neurological impairments (a). Neurological symptoms in the T1 group were significantly milder than those in the other groups. At 5 days after correction, the incidence of moderate or severe neurological impairments (neuroscore equal or less than 4) was 20, 36, 94, and 76% in the T1, T10, RP, and HS groups, respectively (b). Values are expressed as mean±s.e.m. *P<0.05 compared with the other groups.
Kidney International  , DOI: ( /ki )
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3. Figure 2
Immunohistochemical analysis confirmed the presence of demyelinative lesions and microglial accumulation. Double immunostaining for myelin basic protein (MBP) and isolectin B4 (IB4). Noncorrected rats (a–c). Demyelinative lesions (d) and microglial accumulation (e) in the midbrain. Myelin loss (g, j, m, and p) and accumulation of microglia (h, k, n, and q) were observed in rats that showed neurological impairments in all groups. Merged images are shown in c, f, i, l, o and r. In T10 and RP groups, hemorrhagic lesions in the midbrain were observed in several rats that showed severe neurological impairments within 2 days after correction (s). Bar: 1000μm (a–f), 200μm (g–q). *, demyelinative lesion. Arrowhead, hemorrhagic lesion.
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4. Figure 3
Tail resection for collection of blood alleviated neurological impairments. Tail resection for collection of blood at 3–6h after correction of hyponatremia significantly reduced neurological impairments in the T1 (a) and RP (c) groups, but not in the T10 group (b). Tail resection (+), group of rats that had blood sampling by tail resection at 3–6h after correction; none, group of rats without tail resection for blood collection. *P<0.05 compared with none.
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5. Figure 4
Minocycline prevented osmotic demyelination syndrome induced by tolvaptan (1mg/kg body weight). Minocycline improved neurological symptoms after correction of hyponatremia caused by administration of tolvaptan at 1mg/kg body weight (a). In the T1+MINO 45mg/kg group, no apparent demyelinative lesions were observed (b-A and b-D), and microglia remained in resting state with long processes (b-B). Slight accumulation of ameboid-shaped microglia in a small area was observed (b-E). Merged images are shown in b-C and b-F. Scale bar: 200μm. T1+MINO 45mg/kg (n=10): tolvaptan 1mg/kg body weight and minocycline 45mg/kg body weight at 0, 12, and 24h after administration of tolvaptan. T1+MINO 4.5mg/kg (n=10): tolvaptan 1mg/kg body weight and minocycline 4.5mg/kg body weight at 0, 12, and 24h after administration of tolvaptan. T1+vehicle (n=24): tolvaptan 1mg/kg body weight and vehicle administration. *P<0.05 compared with T1+vehicle.
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6. Figure 5
Minocycline inhibited expression of proinflammatory cytokines, chemokines, and matrix metalloproteinases (MMPs) in rat brains. mRNA expression levels of TNF-α (a), iNOS (b), IL-1β (c), IL-6 (d), CCR1 (e), CCL2 (f), MMP9 (g), and MMP12 (h) were significantly increased in the T1 group compared with the noncorrected group and were significantly decreased by minocycline treatment. The relative amount of mRNA was determined using quantitative reverse transcriptase polymerase chain reaction by normalizing to β-actin mRNA. Values are expressed as mean±s.e.m. *P<0.05, compared with the other group. CCR1, chemokine receptor 1; CCL2, chemokine ligand 2; IL, interleukin; iNOS, inducible nitric oxide synthase; TNF-α, tissue necrosis factor-α.
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7. Figure 6
Immunoreactivity of AQP4 was lost at 6h after correction. Immunostaining for GFAP (a, d, g, and m), AQP4 (b, e, h, and n), and IgG (j, k, and l). GFAP and AQP4 were colocalized before correction (a–c). AQP4 immunoreactivity was lost in the subcortical area 6h after correction (e and n). In contrast, GFAP-positive astrocytes remained (d and m). Immunoreactivity of GFAP and AQP4 was lost 2 days after correction (g–i). Immunoreactivity of IgG was observed at 6h and 2 days after correction (k and l), but not before correction (j), suggesting that BBB disruption occurred 6h after correction. Bar=1000μm (a–c, d–f, and g–i), 50μm (j, k, and l), 100μm (m–o). AQP4, aquaporin-4; BBB, blood–brain barrier; GFAP, glial fibrillary acidic protein; IgG, immunoglobulin G.
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8. Figure 7
Serum AQP4 may be a useful predictive marker of osmotic demyelination syndrome. Rats with neurological impairments at 5 days after correction showed significantly higher serum AQP4 levels at 6 and 12h after correction as compared with rats without neurological impairments (a). Maximum serum AQP4 level of each rat at either 6 or 12h after correction was significantly higher in the neurological impairment (+) group (b). There was no significant correlation between serum AQP4 and serum sodium levels at 6 (c) and 12h (d) after correction. Neurological impairment (+), group of rats that showed neurological impairment after correction. Neurological impairment (-), group of rats that showed no neurological impairment after correction. AQP4, aquaporin-4. *P<0.05 compared with neurological impairment (-).
Kidney International  , DOI: ( /ki )
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