Introduction

Parkinson’s disease (PD) is the most common human neurodegenerative disorder affecting movement, balance, flexibility, and coordination. Disease symptoms are commonly associated with advancing age. Clinically, PD is characterized by tremor, bradykinesia, rigidity, and postural instability (Fahn 2010). The pathobiology of PD includes evident gliosis and progressive degeneration of the dopaminergic neurons associated with the presence of intracytoplasmic inclusions (Lewy bodies) in the substantia nigra pars compacta (SNpc) (Olanow and Tatton 1999; Dauer and Przedborski 2003). A plethora of environmental, genetic, and immune cues have been associated with disease onset and progression (Morley and Hurtig 2010).

Recent studies demonstrate a role for neuroinflammation in nigrostriatal degeneration (Dauer and Przedborski 2003; Gao et al. 2003; Barnum and Tansey 2010). The concentration of NO2 − (nitrite), a metabolite of nitric oxide (NO), increases in the cerebrospinal fluid of patients with PD in comparison with a group of patients without dopaminergic dysfunction (Qureshi et al. 1995). Consistently, the ablation of inducible nitric oxide synthase (iNOS) in mutant mice significantly attenuates MPTP neurotoxicity, indicating that iNOS is essential for MPTP-induced SNpc dopaminergic neurodegeneration (Dehmer et al. 2000). A variety of pro-inflammatory cytokines including tumor necrosis factor alpha (TNF-α), interleukin-1beta (IL-1β), IL-6, eicosanoids, and other immune neurotoxins are found in either CSF or affected brain regions in PD (Nagatsu et al. 2000). Finally, NF-κB, a transcription factor required for the transcription of most proinflammatory molecules, is activated in the SNpc of PD patients and MPTP-intoxicated mice (Ghosh et al. 2007) and monkeys (Mondal et al. 2012b), and selective inhibition of NF-κB in mice and monkeys by NEMO-binding domain (NBD) peptides protects dopaminergic neurons from MPTP toxicity (Ghosh et al. 2007). Taken together, these reports indicate that inflammation is an important therapeutic target for nigrostriatal protection in PD.

Recently we have demonstrated that saline (0.9 % NaCl) processed by Taylor-Couette-Poiseuille (TCP) turbulence in the presence of elevated oxygen pressure attains anti-inflammatory properties (Khasnavis et al. 2012; Mondal et al. 2012a). It suppresses the expression of proinflammatory molecules and the activation of NF-κB in activated glial cells via type 1A phosphatidylinositol-3 (PI3) kinase – Akt – cAMP response element binding (CREB)-mediated upregulation of IκBα, a specific endogenous inhibitor of classical NF-κB heterodimer p65:p50 (Baeuerle and Baltimore 1988; Hayden and Ghosh 2008; Khasnavis et al. 2012). Here we demonstrate that RNS60 treatment suppressed the activation of type 1B PI3K, induced the activation of type 1A PI3K, upregulated nigral IκBα, inhibited nigral activation of NF-κB, suppressed nigral iNOS, and exhibited significant protection of the nigrostriatal axis in the MPTP mouse model of PD. These results indicate a new option for treating PD patients with this simple TCP-modified saline as primary or adjunct therapy.

Methods

Cells

Mouse BV-2 microglial cells (kind gift from Virginia Bocchini of University of Perugia) were maintained and induced as described earlier (Ghosh et al. 2007; Jana et al. 2007; Ghosh et al. 2009; Khasnavis et al. 2012).

Animals and MPTP intoxication

Six- to eight-week old C57BL/6 mice were purchased from Harlan, Indianapolis, IN. Animal maintenance and experiments were in accordance with National Institutes of Health guidelines and were approved by the Institutional Animal Care and Use committee of the Rush University Medical Center, Chicago, IL. For acute MPTP intoxication, mice received four intraperitoneal (i.p.) injections of MPTP-HCl (18 mg/kg of free base; Sigma Chemical Co., St. Louis, MO) in saline at 2-h intervals (Ghosh et al. 2007, 2009; Roy et al. 2012). Control animals received only saline.

Preparation of RNS60

RNS60 was generated at Revalesio (Tacoma, WA) as described before (Khasnavis et al. 2012; Mondal et al. 2012a). Briefly, sodium chloride (0.9 %) for irrigation, USP pH 5.6 (4.5–7.0, Hospira), was processed at 4 °C using Taylor-Couette-Poiseuille (TCP) flow and a flow rate of 32 mL/s under 1 atm of oxygen back-pressure (7.8 mL/s gas flow rate), while maintaining a rotor speed of 3,450 rpm. Chemically, RNS60 contains water, sodium chloride, 50–60 parts/million oxygen, but no active pharmaceutical ingredients.

The following controls for RNS60 were used in this study: a) NS, normal saline from the same manufacturing batch. This saline contacted the same device surfaces as RNS60 and was bottled in the same way, b) RNS10.3, a saline that was processed with TCP flow in the absence of any excess oxygen, and c) PNS60, saline with the same oxygen content as RNS60 (55 ± 5 ppm) that was prepared inside of the same device but was not processed with TCP flow. Careful analysis demonstrated that all four fluids were chemically identical (Khasnavis et al. 2012). Liquid chromatography quadrupole time-of-flight mass spectrometric analysis also showed no difference between RNS60 and other control solutions (Khasnavis et al. 2012). We studied surface nanobubble nucleation in RNS60 and other saline solutions by using atomic force microscopy and observed that RNS60 displayed different surface nanobubble nucleation characteristics relative to that of control saline solutions (Khasnavis et al. 2012). This same relative pattern of nanobubble number and size was observed when positive potentials were applied to AFM surfaces with the same control solutions, suggesting the involvement of charge in stabilization of nucleated surface nanobubbles with RNS60 (Fig. 1A).

Fig. 1
figure 1

RNS60 inhibits MPP+-induced expression of proinflammatory molecules in glial cells. A A schematic presentation of RNS60. B Mouse BV-2 microglial cells preincubated with different concentrations of RNS60 or NS in serum-free media for 2 h were stimulated with MPP+ (a Parkinsonian toxin). After 6 h of stimulation, total RNA was isolated and the mRNA levels of iNOS and IL-1β were analyzed by semi-quantitative RT-PCR (B) and quantitative real-time PCR (C for IL-1β & D for iNOS). Cell viability was monitored by MTT assay (E). Data are mean ± S.D. of three independent experiments. a p < 0.001 versus control; b p < 0.05 versus MPP+; c p < 0.001 versus MPP+. Cells preincubated with different concentrations of PNS60 or RNS10.3 in serum-free media for 2 h were stimulated with MPP+ (a Parkinsonian toxin). After 6 h of stimulation, the mRNA levels of iNOS and IL-1β were analyzed by semi-quantitative RT-PCR (F) and real-time PCR (G). Data are mean ± S.D. of three independent experiments

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RNS60 treatment

Mice were treated with RNS60 or NS (300 μl/mouse/day) from either 2 day prior to or 6 h after MPTP intoxication via intraperitoneal (i.p.) injection.

Antibodies

Rabbit anti-mouse iNOS antibodies were obtained from Calbiochem, Gibbstown, NJ. Rabbit and goat anti-NF-κB p65 and goat anti-glial fibrillary acidic protein (GFAP) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Rat anti-mouse CD11b was purchased from Abcam (Cambridge, MA). Cy2- and Cy5-conjugated antibodies were obtained from Jackson Immuno Research Laboratories (West Grove, PA).

Assay of PI3K activation

Upon activation, PI3K subunits translocate to the plasma membrane. Therefore, after different time of treatment with RNS60, nigra were dissected out from mice followed by isolation of cell membranes and Western blotting with antibodies against different subunits of PI3K. Briefly, nigra was homogenized in 100 mM sodium bicarbonate buffer (pH 11.5) and spun in an ultracentrifuge at 40,000 rpm for 1 h at 4 °C. The resultant supernatant was aspirated and the pellet was immersed in double-distilled water and SDS and stored at −80 °C overnight. The following day, the pellet was resuspended by repeated grinding and boiling, and processed for Western blot using antibodies against p110α, p110β and p110γ.

Western blot analysis

Immunoblot analysis for IκBα, iNOS, p110α, p110β, and p110γ was carried out as described earlier (Saha et al. 2006, 2007). Briefly, cell homogenates were electrophoresed, proteins were transferred onto a nitrocellulose membrane, and bands were visualized with an Odyssey infrared scanner after immunolabeling with respective primary antibodies followed by infra-red fluorophore-tagged secondary antibody (Invitrogen).

Semi-quantitative RT-PCR analysis

Total RNA was isolated from ventral midbrain using Ultraspec-II RNA reagent (Biotecx Laboratories, Inc., Houston, TX) following the manufacturer’s protocol. To remove any contaminating genomic DNA, total RNA was digested with DNase. RT-PCR was carried out as described earlier (Jana et al. 2007; Brahmachari et al. 2009; Ghosh et al. 2009) using a RT-PCR kit (Clontech, Mountain View, CA) and the following primers.

  • iNOS: Sense: 5′-CCCTTCCGAAGTTTCTGGCAGCAGC-3′

    Antisense: 5′-GGCTGTCAGAGCCTCGTGGCTTTGG-3′

  • IL-1β: Sense: 5′-CTCCATGAGCTTTGTACAAGG-3′

    Antisense: 5′-TGCTGATGTACCAGTTGGGG-3′

  • GAPDH: Sense: 5′-GGTGAAGGTCGGTGTGAACG-3′

    Antisense: 5′-TTGGCTCCACCCTTCAAGTG-3′

Real-time PCR analysis

DNase-digested RNA was analyzed by real-time PCR in the ABI-Prism7700 sequence detection system (Applied Biosystems, Foster City, CA) as described earlier (Jana et al. 2007; Brahmachari et al. 2009; Ghosh et al. 2009; Khasnavis and Pahan 2012) using TaqMan Universal Master mix and optimized concentrations of FAM-labeled probes and primers. Data were processed using the ABI Sequence Detection System 1.6 software.

Immunohistochemistry and quantitative morphology

Seven days after MPTP intoxication, mice were sacrificed and their brains fixed, embedded, and processed for tyrosine hydroxylase (TH) and thionin staining as previously described (Benner et al. 2004; Jana et al. 2007; Ghosh et al. 2009). Total numbers of TH-positive neurons in SNpc were counted stereologically with STEREO INVESTIGATOR software (MicroBrightfield, Williston, VT) by using an optical fractionator. Quantitation of striatal TH immunostaining was performed as described (Benner et al. 2004; Jana et al. 2007; Ghosh et al. 2009). Optical density measurements were obtained by digital image analysis (Scion, Frederick, MD). Striatal TH optical density reflected dopaminergic fiber innervation. For immunofluorescence staining on fresh frozen sections, rat anti-mouse CD11b (1:100), goat anti-mouse GFAP (1:100), rabbit anti NF-κB p65 (1:100), goat anti-NF-κB p65 (1:100), rabbit anti phospho-p65 (1:100), and rabbit anti-mouse iNOS (1:250) were used. The samples were mounted and observed under a Bio-Rad MRC1024ES confocal laser scanning microscope.

HPLC analyses

Striatal levels of dopamine, DOPAC (3, 4-dihydroxyphenylacetic acid) and HVA (homovanillic acid) were quantified in Complete Stand-Alone HPLC-ECD System EiCOMHTEC-500 (JM Science Inc., Grand Island, NY) as described earlier (Roy and Pahan; Ghosh et al. 2007, 2009; Roy and Pahan 2011; Roy et al. 2012). Briefly, mice were sacrificed by cervical dislocation after 7 days of MPTP intoxication and their striata were collected and immediately frozen in dry ice and stored at −80C until analysis. On the day of the analysis, tissues were sonicated in 0.2 M perchloric acid containing isoproterenol and resulting homogenates were centrifuged at 20,000×g for 15 min at 4 °C. After pH adjustment and filtration, 10 μl of supernatant was injected onto an Eicompak SC-3ODS column (Complete Stand-Alone HPLC-ECD System EiCOMHTEC-500 from JM Science Inc., Grand Island, NY) and analyzed following the manufacturer’s protocol.

Behavioral analyses

Two types of behavioral experiments were conducted. These included an open field experiment for locomotor activity and a rotorod experiment for feet movement as described earlier (Roy and Pahan; Ghosh et al. 2007, 2009). Locomotor activity was measured after 7 d of the last dose of MPTP injection in a Digiscan Monitor (Omnitech Electronics, Inc., Columbus, OH). This Digiscan Monitor records stereotypy and rearing, behaviors that are directly controlled by striatum, as well as other basic locomotion parameters, such as horizontal activity, total distance traveled, number of movements, movement time, rest time, mean distance, mean time, and center time. Before any insult or treatment, mice were placed inside the Digiscan Infra-red Activity Monitor for 10 min daily and on rotorod for 10 min daily for 3 consecutive days to train them and record their baseline values. Briefly, animals were removed directly from their cages and gently placed nose first into a specified corner of the open-field apparatus and after release, data acquisition began at every 5-min interval. DIGISCAN software was used to analyze and store horizontal and vertical activity data, which were monitored automatically by infra-red beams. In rotorod, the feet movement of the mice was observed at different speeds. To eliminate stress and fatigues, mice were given a 5-min rest interval. Then 7 d after the last dose of MPTP, open field assays and rotorod tests were carried out twice at 6 h intervals on each mouse separately. Locomotor activity measures were assessed after baseline value comparison.

Statistics

All values are expressed as means ± SEM. One-way ANOVA was performed while analyzing dose-dependent effect of RNS60 on mRNA expression of iNOS, IL-1β and IκBα in activated microglial cells. In other cases, Student’s t-test was used to compare outcome between two groups (e.g. control vs MPTP, MPTP vs RNS60 etc.).

Results

RNS60 attenuates the expression of proinflammatory molecules in MPP+-activated mouse microglial cells

Activated microglia are known to produce excessive amount of NO and proinflammatory cytokines, which have the potential of damaging dopaminergic neurons (Dauer and Przedborski 2003; Barnum and Tansey 2010). The neurotoxic effect of MPTP depends on its conversion to MPP+. Therefore, we first investigated whether RNS60 containing charge-stabilized nanostructures (Fig. 1A) could suppress MPP+-induced expression of iNOS and IL-1β in microglia. As shown in Fig. 1B–D, RNS60 dose-dependently inhibited the mRNA expression of iNOS and IL-1β in BV-2 microglial cells. (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay results show that RNS60 was not toxic to microglial cells at any of the concentrations tested (Fig. 1E). Therefore, the inhibitory effect of RNS60 on iNOS expression was not due to any change in cell viability. Unprocessed NS from the same manufacturing batch had no such inhibitory effect on MPP+-induced expression of iNOS and IL-1β (Fig. 1B–D). Furthermore, RNS10.3, saline that was processed with TCP flow in the absence of any excess oxygen, and PNS60, saline with same oxygen content as RNS60 (55 ± 5 ppm) that was prepared inside of the same device but was not processed with TCP flow, also remained unable to suppress MPP+-induced expression of iNOS and IL-1β in microglial cells (Fig. 1F–G)..

RNS60 treatment inhibits the expression of iNOS in the nigra of MPTP-intoxicated mice

Inflammation plays a role in the loss of dopaminergic neurons in PD and its animal model (Hunot et al. 1996; Dehmer et al. 2000; Ghosh et al. 2007; Jana et al. 2007; Ghosh et al. 2009; Barnum and Tansey 2010). Since RNS60 inhibited the expression of iNOS in glial cells, we examined if RNS60 was able to suppress the expression of iNOS in vivo in the SNpc of MPTP-insulted mice. Twenty-four hours after the last injection of MPTP, nigral samples were immunoblotted for iNOS. As evident from Fig. 2A–B MPTP intoxication increased the level of iNOS in nigra. However, RNS60, but not NS, treatment led to the suppression of iNOS protein in the nigra (Fig. 2A–B). Immunofluorescence analysis for iNOS in ventral midbrain sections also showed that MPTP intoxication led to a marked increase in nigral iNOS protein expression and that iNOS co-localized with either GFAP-positive astroglia (Fig. 2C) or CD11b-positive microglia (Fig. 2D). However, treatment of mice with RNS60, but not NS, led to marked suppression of iNOS in vivo in the nigra (Fig. 2C–E).

Fig. 2
figure 2

RNS60 treatment inhibits the expression of iNOS in vivo in the SNpc of MPTP-intoxicated mice. Mice receiving RNS60 or NS (300 μl/mouse/day) via i.p. injection from 2 d prior to MPTP intoxication were sacrificed 1 d after the last injection of MPTP followed by monitoring the level of iNOS in the nigra by Western blot (A). Bands were scanned and are presented as relative expression (B). Results are mean ± SEM of four mice per group. a p < 0.0001 versus saline-control; b p < 0.0001 versus MPTP. Nigral sections were double-immunolabeled with antibodies against either GFAP & iNOS (C) or CD11b & iNOS (D). Cells positive for iNOS (E) were counted in two nigral sections (2 images per slide) from each of five different mice. a p < 0.0001 versus saline-control; b p < 0.0001 versus MPTP

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In the acute MPTP model, maximal neuronal loss has been shown on day 7 after MPTP intoxication. Therefore, we examined whether the effect of RNS60 treatment on expression of iNOS persisted until that time. We also found a significant increase in astroglial and microglial iNOS immunostaining in the nigra after 7 days of MPTP intoxication, which was notably inhibited by RNS60, treatment (Fig. S1A–C).

Activation of type 1A phosphatidylinositol-3 (PI3) kinase in vivo in the nigra by RNS60 treatment

Recently we have seen that RNS60 rapidly induces the activation of type 1A, but not type 1B, PI3K in cultured microglia (Khasnavis et al. 2012). Therefore, we tested if intraperitoneal treatment of RNS60 was capable of modulating PI3K activation in vivo in the nigra. Class IA PI3K, which is primarily regulated by receptor tyrosine kinases, consists of a heterodimer of a regulatory 85-kDa subunit and a catalytic 110-kDa subunit (p85:p110α/β/δ). Class IB PI3K, on the other hand, consists of a dimer of a 101-kDa regulatory subunit and a p110γ catalytic subunit (p101/p110γ). While in resting condition, subunits of PI3K are located mainly in cytoplasm, upon activation, these are translocated to the plasma membrane . Therefore, after 3 and 6 h of intraperitoneal treatment of RNS60, we isolated membrane fractions of nigra followed by monitoring the activation of class IA and IB PI3K by the recruitment of p110α, p110β and p110γ to the plasma membrane. Western blotting of nigral membrane fractions for p110 subunits showed that RNS60 specifically induced the recruitment of p110α (Fig. 3A) and p110β (Fig. 3B), but not p110γ (Fig. 3C), to nigral membrane. Densitometric analysis of the p110α and p110β at different time points of RNS60 treatment indicates significant activation of type 1A PI3K at 3 and 6 h (Fig. 3D). On the other hand, we did not observe any activation of p110γ PI3K at these time points of RNS60 treatment (Fig. 3A–D). Again these results were specific as NS treatment remained unable to activate either p110α/p110β or p110γ PI3K (Fig. 3A–D). Together, these results suggest that intraperitoneal administration of RNS60 is capable of activating type IA PI3K p110α and p110β, but not type IB PI3K p110γ, in vivo in the nigra.

Fig. 3
figure 3

RNS60 induces the activation of phosphatidylinositol-3 kinase (PI3K) in vivo in the nigra of control as well as MPTP-intoxicated mice. Normal male C57BL/6 mice (6–8 week old) received one dose of either RNS60 or NS (300 μl/mouse) via i.p. injection followed by measurements of the levels of p110α (A), p110β (B) and p110γ (C) in membrane fractions of nigral samples at different h of treatment by Western blot. D Bands were scanned and are presented as relative activation with respect to control. Results are mean ± SEM of three mice per group. a p < 0.001 versus control. E Mice receiving RNS60 or NS (300 μl/mouse/day) via i.p. injection from 2 d prior to MPTP intoxication were sacrificed 6 h after the last injection of MPTP followed by measurements of the levels of p110α, p110β and p110γ in membrane fractions of nigral samples by Western blot. F Bands were scanned and are presented as relative activation with respect to control. Results are mean ± SEM of three mice per group. a p < 0.01 versus control; b p < 0.05 versus control; c p < 0.001 versus MPTP; d p < 0.01 versus MPTP. G Nigral sections were immunostained for PIP3. Results represent analyses of two nigral sections (2 images per slide) from each of three different mice

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Activation of type 1B, but not type 1A, PI3K in vivo in the nigra by MPTP: reversal by RNS60 treatment

Next, we investigated the status of PI3K in the nigra after MPTP intoxication. In contrast to RNS60 treatment, MPTP intoxication decreased the recruitment of p110α and p110β (Fig. 3E–F) to nigral membrane. On the other hand, MPTP insult stimulated the recruitment of p110γ (Fig. 3E–F) to nigral plasma membrane. These results suggest that MPTP intoxication induces the activation of type 1B, but not type 1A, PI3K. However, RNS60 treatment altered the pattern of PI3K activation in MPTP-intoxicated nigra. After RNS60 treatment, we found the recruitment of type IA PI3K p110α/p110β, but not type IB PI3K p110γ, to the plasma membrane in the nigra of MPTP insulted mice (Fig. 3E–F).

Phosphatidylinositol (3,4,5)-triphosphate (PIP3) is the product of class I PI3K. Therefore, to confirm the activation of class I PI3K from another angle, we monitored PIP3 levels in the nigra of MPTP- and RNS60-treated mice by immunofluorescence. Although we found only a marginal decrease in PIP3 levels 24 h after MPTP intoxication, RNS60 treatment increased the level of PIP3 in MPTP-intoxicated mice as compared to control mice (Fig. 3G). However, consistent to that observed in membrane localization of PI3K subunits, NS treatment remained unable to upregulate the level of PIP3 (Fig. 3G). Similar results were also seen 7 d after MPTP insult (Fig. S2).

RNS60 treatment upregulates the expression of IκBα in vivo in the nigra of MPTP-intoxicated mice

Recently we have demonstrated that RNS60 increases the level of the anti-inflammatory molecule IκBα in cultured microglia via the type 1A PI3K – Akt – CREB pathway (Khasnavis et al. 2012). Therefore, we examined whether RNS60 treatment was capable of augmenting IκBα in vivo in the nigra of normal and MPTP-intoxicated mice. As evident from Fig. 4A–B, a single intraperitoneal injection of RNS60 increased the level of IκBα in the nigra of control mice within 3 h of treatment, which further increased at 6 h. Next, we monitored whether RNS60 treatment upregulated IκBα in vivo in the nigra of MPTP-intoxicated mice. We observed a modest decrease in IκBα expression in the nigra 24 h after MPTP insult (Fig. 4C–D). However, similar to its effect in normal nigra, RNS60 treatment significantly increased the level of IκBα in the nigra of MPTP-intoxicated mice (Fig. 4C–D). This result was further corroborated by immunofluorescence analysis (Fig. 4E–F). Although MPTP intoxication markedly induced astrogliosis and microgliosis in vivo in the SNpc as evident from increased GFAP (Fig. 4E) and CD11b (Fig. 4F), a slight decrease in IκBα expression was noted after MPTP insult, which markedly increased after RNS60 treatment (Fig. 4E–F). Similar results were seen in the nigra of mice 7 d after MPTP intoxication (Fig. S3A–C). These results suggest that RNS60 treatment is capable of increasing IκBα in the nigra of both control and MPTP-insulted mice.

Fig. 4
figure 4

RNS60 induces the upregulation of IκBα in vivo the nigra of control and MPTP-intoxicated mice. Normal male C57BL/6 mice (6–8 weeks old) received one dose of either RNS60 or NS (300 μl/mouse) via i.p. injection followed by monitoring the level of IκBα (A) by Western blot. B Bands were scanned and presented as levels relative to control. Results are mean ± SEM of three mice per group. a p < 0.001 versus control. Mice receiving RNS60 or NS (300 μl/mouse/day) via i.p. injection from 2 day prior to MPTP intoxication were sacrificed 1 day after the last injection of MPTP, followed by measurements of IκBα levels (C) in nigral samples by Western blot. D Bands were scanned and are presented as levels relative to control. Results are mean ± SEM of three mice per group. a p < 0.001 versus control; b p < 0.001 versus MPTP. Nigral sections were analyzed by double-label immunofluorescence using antibodies against either GFAP & IκBα (E) or Iba-1 & IκBα (F). Results represent analyses of two nigral sections (2 images per slide) from each of five different mice per group

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RNS60 treatment suppresses the activation of NF-κB in vivo in the nigra of MPTP-intoxicated mice

As RNS60 treatment upregulated IκBα, a specific inhibitor of NF-κB, we examined whether RNS60 treatment could suppress the activation of NF-κB in vivo in the SNpc of MPTP-insulted mice. In addition to stimulus-induced nuclear translocation of NF-κB, stimulus-induced phosphorylation of RelA p65 plays a key role in transcriptional activation by NF-κB after its nuclear translocation (Hayden and Ghosh 2008). Therefore, we monitored the status of phospho-p65 in the SNpc of MPTP-insulted mice. The level of phospho-p65 (Fig. 5A–B) was markedly higher in the SNpc of MPTP-intoxicated mice as compared to saline treated mice, confirming NF-κB activation in vivo in the SNpc of MPTP-lesioned mice. However, RNS60, but not NS, strongly inhibited the induction of phospho-p65 (Fig. 5A–B) in vivo in the SNpc of MPTP-intoxicated mice. We also examined if RelA p65 was upregulated upon MPTP intoxication and if this was RNS60 sensitive. MPTP intoxication led to marked induction of p65 in the SNpc as compared to saline treatment (Fig. S4A–C). Double-label immunofluorescence analysis indicates that p65 was principally expressed in GFAP-positive astroglia (Fig. S4A) and CD11b-positive microglia (Fig. S4B). However, RNS60, but not NS, strongly inhibited the induction of total p65 (Fig. S4) as well as phospho-p65 (Fig. 5) in vivo in the SNpc of MPTP-intoxicated mice.

Fig. 5
figure 5

RNS60 treatment inhibits the phosphorylation of RelA p65 in vivo in the SNpc of MPTP-intoxicated mice. Mice receiving RNS60 or NS (300 μl/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 phospho-p65 by double-label immunofluorescence using antibodies against GFAP & phospho-p65 (A). Cells positive for phospho-p65 (B) were counted in two nigral sections (2 images per slide) from each of five different mice. a p < 0.0001 versus saline-control; b p < 0.0001 versus MPTP

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RNS60 protects against MPTP-induced neurodegeneration

Mice were treated daily with RNS60 or NS via i.p. injection from 2 d prior to MPTP injection to 7 d after the last injection of MPTP. MPTP-intoxication led to approximately 70 % loss of SNpc TH-positive neurons (Fig. 6A & C) and 75 % reduction of striatal TH ODs (Fig. 6B & D) compared with saline-injected controls. However, in MPTP-injected mice treated with RNS60, the reduction in SNpc TH-positive neurons and striatal TH ODs was attenuated (Fig. 6A–D). On the other hand, no such protective effects were seen in MPTP-intoxicated mice that were treated with NS (Fig. 6A–D). To determine whether RNS60 protects against biochemical deficits caused by MPTP, we quantified the level of dopamine in the striata 7 d after the MPTP treatment. As evident from Fig. 6E, MPTP intoxication led to about 80 % decrease in striatal dopamine compared to striata of saline-injected mice. In contrast, MPTP-intoxicated animals that received RNS60 showed 50 % decrease in striatal dopamine, whereas such protection was not seen in case of NS treatment (Fig. 6E).

Fig. 6
figure 6

RNS60 protects dopaminergic neurons in MPTP-intoxicated mice. Mice receiving RNS60 or NS (300 μl/mouse/day) via i.p. injection from 2 d prior to MPTP intoxication were sacrificed 7 d after the last injection of MPTP followed by TH immunostaining of SNpc (A) and striatum (B), counting of TH-positive neurons in SNpc (C), quantification of TH-positive fibers in striatum (D), and assay of neurotransmitters in striatum (E). Data are means ± SEM of five mice per group. a p < 0.0001 versus saline-control; b p < 0.0001 versus MPTP

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RNS60 improves locomotor functions in MPTP-intoxicated mice

The ultimate therapeutic goal of neuroprotection is to decrease functional impairment. Therefore, to examine whether RNS60 protects not only against structural and neurotransmitter damage but also against functional deficits caused by MPTP, we monitored locomotor and open-field activities. MPTP injection caused a marked decrease in rotorod performance (Fig. 7A), movement time (Fig. 7B), number of movements (Fig. 7C), horizontal activity (Fig. 7E), total distance (Fig. 7F), rearing (Fig. 7G), and stereotypy (Fig. 7H). On the other hand, MPTP increased the rest time (Fig. 7D). However, RNS60, but not NS, significantly improved MPTP-induced hypolocomotion (Fig. 7A–H).

Fig. 7
figure 7

RNS60 treatment improves motor functions in MPTP-intoxicated mice. Mice receiving RNS60 and NS (300 μl/mouse/day) via i.p. injection from 2 d prior to MPTP intoxication were tested for motor functions (A, rotorod; B, movement time; C, number of movements; D, rest time; E, horizontal activity; F, total distance; G, rearing; H, stereotypy) 7 d after the last injection of MPTP. Data are means ± SEM of five mice per group.a p < 0.001 vs saline; c p < 0.05 vs saline; b p < 0.05 vs MPTP

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RNS60 halts the progression of neurodegeneration

Usually patients are treated with a drug after the onset of disease symptoms. Therefore, to evaluate the therapeutic potential of RNS60 treatment in MPTP induced neurodegeneration, we investigated whether RNS60 administered 6 h after initiation of the disease (Fig. 8A) was still capable of inhibiting the loss of neurotransmitters. MPTP intoxication sharply reduced (~85 %) striatal dopamine compared with striata of saline-injected mice. However, MPTP-intoxicated animals that received RNS60 from 6 h after the initiation of the disease showed about 58 % decrease in striatal dopamine (Fig. 8B). These results suggest that RNS60 is effective even if the treatment starts after MPTP intoxication. Furthermore, it has been shown that sufficient amount of MPTP could be converted into MPP+ within 90 min of the last injection of MPTP (Tieu et al. 2003). Therefore, these results also suggest that the effect of RNS60 is not due to its effect on the entry and conversion of MPTP into MPP+ in the midbrain.

Fig. 8
figure 8

RNS60 suppresses disease progression in MPTP-intoxicated mice. Mice were treated with MPTP and RNS60 or NS (A) followed by quantification of neurotransmitters in striatum (B). Data are means ± SEM of five mice per group. a p < 0.0001 versus saline-control; b p < 0.05 versus MPTP

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Discussion

Neuroinflammation, a hallmark of neurodegenerative disorders, is successfully modeled in MPTP-intoxicated mice. The MPTP mouse model is particularly useful for testing new therapeutic interventions for PD due to the fact that specific lesioning of the nigrostriatum, similar to that seen in PD, occurs in this model. Nanotechnology employs modified materials or devices that can interact with biological systems at a molecular level. RNS60 is an electrokinetically modified saline that contains charge-stabilized nanobubbles, but no active pharmaceutical ingredients. Using the MPTP mouse model, we have tested the hypothesis that RNS60 may induce protective responses in PD. Several lines of evidence presented in this manuscript clearly establish that RNS60 is capable of protecting dopaminergic neurons from Parkinsonian toxicity. Our conclusion is based on the following findings. First, while nigral neurons disappeared in MPTP-intoxicated mice, treatment with RNS60 protected TH-positive dopaminergic neurons from MPTP toxicity. Second, RNS60 treatment protected striatal TH fibers from MPTP toxicity and restored the level of neurotransmitters. Third and most importantly RNS60 ameliorated functional impairment in MPTP-intoxicated mice. We did not notice any drug related side effect (e.g. hair loss, weight loss, untoward infection etc.) in any of the mice used during the course of the study, suggesting that RNS60 may not exhibit major side effects.

Although there are multiple mechanisms by which nigrostriatal degeneration may occur in PD, recent studies indicate that inflammation plays an important role. Here we have demonstrated that RNS60 suppresses the expression of inducible nitric oxide synthase (iNOS) in MPP+-activated glial cells in culture and in vivo in the nigra of MPTP-intoxicated mice. The signaling events required for the transcription of iNOS and proinflammatory cytokines are becoming clear. Although many transcription factors such as NF-κB, C/EBPβ, AP-1, STAT, IRF-1 etc. play a role, activation of NF-κB seems essential for the transcription of most of the proinflammatory molecules (Xie et al. 1994; Pahan et al. 1997; Jana et al. 2001; Roy et al. 2006; Saha and Pahan 2006). Therefore, for a drug to exhibit anti-inflammatory effects, it is critically important to attenuate the activation of NF-κB. In resting cells, the classical p65:p50 heterodimer is arrested in the cytoplasm as an inactive complex by IκBα (Baeuerle and Baltimore 1988). Therefore, IκBα is considered an important endogenous anti-inflammatory molecule that selectively attenuates the activation of the classical NF-κB heterodimer. Recently we have demonstrated that RNS60 rapidly upregulates the expression of IκBα in cultured glial cells (Khasnavis et al. 2012). Here we demonstrate that within hours of intraperitoneal administration, RNS60 also increases the level of IκBα in vivo in the nigra. Consistently, intraperitoneal injection of mice with RNS60 also resulted in the inhibition of the levels of RelA p65 and phospho-p65. Taken together, these results strongly suggest that RNS60 treatment is capable of upregulating IκBα and exerting anti-inflammation in vivo in the nigra of MPTP-intoxicated mice.

How does RNS60 upregulate the expression of IκBα in vivo in the nigra? Recently we have delineated that RNS60 increases IκBα via the activation of type 1A PI3K p110α/β, but not type 1B PI3K p110γ (Khasnavis et al. 2012). Consistent with this data, RNS60 increased the activation of class IA, but not class IB, PI3K in vivo in the nigra of control mice within 3 h of MPTP intoxication. In contrast to RNS60, MPTP intoxication increased the activation of class 1B PI3K and decreased the activation of class 1A PI3K in vivo in the nigra, indicating the specificity of the effect. Because class 1B PI3K p110γ is involved in the induction of proinflammatory molecules (Wymann et al. 2003) and different proinflammatory molecules are upregulated in the nigra following MPTP intoxication (Dehmer et al. 2000; Dauer and Przedborski 2003; Benner et al. 2004; Ghosh et al. 2007, 2009), it is possible that MPTP insult causes the upregulation of proinflammatory molecules in the nigra via class 1B PI3K. However, RNS60 treatment switched the activation pattern of PI3K in the nigra of MPTP intoxicated mice. RNS60 treatment suppressed the activation of class 1B PI3K and upregulated the activation of class 1A PI3K in the nigra of MPTP-insulted mice. Due to the lack of a proper detection tool to detect nanobubble-based structures, at present, we do not have any direct way to measure RNS60 within the CNS. However, within 3 h of intraperitoneal RNS60 administration, we observed the activation of class 1A PI3K and the upregulation of IκBα, signature events of RNS60, in vivo in the nigra. Therefore, it is likely that RNS60 enters the brain. The novelty of our work lies in the fact that a physically/eletrokinetically modified saline devoid of any active pharmacological compound can activate a specific class of PI3K, upregulates anti-inflammatory molecule IκBα and exerts anti-inflammation in vivo in the nigra of an animal model of PD. Rapid activation of such an anti-inflammatory signaling pathway in the brain suggests that RNS60 treatment may be beneficial for other brain pathologies involving neuroinflammation.

In summary, we have demonstrated that RNS60 upregulates IκBα and blocks the activation of NF-κB in the SNpc, inhibits nigral expression of iNOS, protects the loss of nigral dopaminergic neurons, saves striatal TH fibers and neurotransmitters, and improves the behavioral functions in MPTP-intoxicated mice. These results highlight novel neuroprotective properties of RNS60 and indicate that RNS60 may be further explored for therapeutic intervention in PD or other neuroinflammatory and neurodegenerative disorders as primary or adjunct therapy.