Protection of dopaminergic neurons in a mouse model of Parkinson’s disease by a physically-modified saline containing charge-stabilized nanobubbles

Saurabh Khasnavis1, Supurna Ghosh2, Richard Watson2, and Kalipada Pahan1

1Department of Neurological Sciences, Rush University Medical Center, Chicago, IL 60612; 2Revalesio Corporation, 1200 East D Street, Tacoma, WA 98421

Abbreviated title: Modified saline attenuates PD symptoms in mice

Corresponding author with complete address:

Kalipada Pahan, Ph.D.

Department of Neurological Sciences

Rush University Medical Center

1735 West Harrison St, Suite 320

Chicago, IL 60612

Tel: (312) 563-3592

Fax: (312) 563-3571

Email:

Legends to Supplemental figures

Supplemental Figure 1. RNS60 treatment inhibits the expression of iNOS in vivo in the SNpc of MPTP-intoxicated mice. Mice receiving RNS60 or NS (300 l/mouse/d) via i.p. injection from 2 d prior to MPTP intoxication were sacrificed 7 d after the last injection of MPTP followed by measurements of the level of iNOS by double-label immunofluorescence using antibodies against either GFAP & iNOS (A) or CD11b & iNOS (B). Cells positive for iNOS were counted in two nigral sections (2 images per slide) each of five mice per group. ap < 0.0001 versus saline-control; bp < 0.0001 versus MPTP.

Supplemental Figure 2. RNS60 treatment induces the activation of PIP3 in vivo in the SNpc of MPTP-intoxicated mice. Mice receiving RNS60 or NS (300 l/mouse/d) via i.p. injection from 2 d prior to MPTP intoxication were sacrificed 7 d after the last injection of MPTP followed by PIP3 measurements in nigral sections by immunofluorescence. DAPI was used to visualize nuclei. Results represent two nigral sections (2 images per slide) each of five mice per group.

Supplemental Figure 3. RNS60 treatment upregulates IBin vivoin the SNpc of MPTP-intoxicated mice. Mice receiving RNS60 or NS (300 l/mouse/d) via i.p. injection from 2 d prior to MPTP intoxication were sacrificed 7 d after the last injection of MPTP followed by measurements of the level of IB by double-label immunofluorescence using antibodies against either GFAP & IB (A) or Iba-1 & IB (B). Cells positive for IB were counted in two nigral sections (2 images per slide) each of five mice per group (C). ap < 0.0001 versus saline-control; bp < 0.0001 versus MPTP.

Supplemental Figure 4. RNS60 treatment inhibits the induction of RelA p65 in vivo in the SNpc of MPTP-intoxicated mice. Mice receiving RNS60 or NS (300 l/mouse/d) via i.p. injection from 2 d prior to MPTP intoxication were sacrificed 7 d after the last injection of MPTP followed by monitoring the level of p65 by double-label immunofluorescence using antibodies against either GFAP & p65 (A) or CD11b & p65 (B). Cells positive for p65 (C) were counted in two nigral sections (2 images per slide) from each of five different mice. ap < 0.0001 versus saline-control; bp < 0.0001 versus MPTP.

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