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\begin{document}

\title{Molecular hydrogen
inhalation prevents L-DOPA-induced dyskinesia in a Parkinson's disease
rat model}



\author[1]{Glauce Nascimento}%
\author[1]{Elaine Del Bel}%
\affil[1]{Affiliation not available}%


\vspace{-1em}



  
  \date{May 21, 2024}


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\sloppy


\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/FIG-1/FIG-1}
\caption{\selectlanguage{ngerman}{This is a \textbf{Figure 1. Molecular hydrogen
(H}\textsubscript{\textbf{2}}\textbf{) inhalation reduces AIMS without
interfering with the anti-parkinsonian effect of L-DOPA.} (A, B)
Schematic protocol. After Sham surgery or 6-OHDA injection, the rats
were analysed in the apomorphine-induced rotational test for lesion
confirmation. Then, Sham or 6-OHDA-lesioned rats were treated
chronically (15 days) with L-DOPA or its vehicle. Subsequently before
the last L-DOPA injection, Air mixture or H\textsubscript{2}inhalation
was performed for a 1-h period, followed by the L-DOPA injection and
abnormal involuntary movements on axial, limb and orofacial parameters
(ALO AIMs) and locomotor activity were measured. (C) 6-OHDA lesion
significantly increased total contralateral turns induced by
apomorphine. (D) Photomicrographs of coronal brain sections illustrating
the loss of TH positive immunolabeling in the striatum fibers (D) and
substantia nigra compacta neurons (E), scale bars=1200 \selectlanguage{greek}µ\selectlanguage{english}m. (F, G)
H\textsubscript{2} treatment does not alter the L-DOPA effect on the
distance traveled (F) and rearing (G) in the actimeter. (H) Time course
of the appearance of dyskinetic manifestation with 6-OHDA+L-DOPA+Air
mixture compared to 6-OHDA+L-DOPA+H\textsubscript{2}inhalation
(p\textless{}0.001) across time (for each 20-min period). (I, J, K) show
the co-administration of L-DOPA and H\textsubscript{2}inhalation effects
on the axial (I), limb (J) and orofacial (K) AIMs over 120 mins. Sham
rats treated with vehicle of L-DOPA (SHAM+Veh, white); Sham rats treated
chronically with L-DOPA (SHAM+L-DOPA, orange); 6-OHDA lesioned rats
treated with vehicle of L-DOPA (6-OHDA+Veh, green); 6-OHDA lesioned rats
treated chronically with L-DOPA (6-OHDA+L-DOPA, blue). AIMs were
analyzed using 2-Way RM ANOVA analysis with Tukey´s multiple comparisons
post-hoc test. Distance travelled was analyzed by ordinary One-Way ANOVA
with Fisher post-hoc test. Data are reported as mean ± SEM.
***p\textless{}0.001, **p\textless{}0.01, *p\textless{}0.05. caption
{\label{836627}}%
}}
\end{center}
\end{figure}\selectlanguage{ngerman}\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/Fig--2/Fig--2}
\caption{\selectlanguage{ngerman}{This is \textbf{Figure 2. Molecular hydrogen
(H}\textsubscript{\textbf{2}}\textbf{) inhalation improves peripheral
and striatal inflammation in L-DOPA-treated 6-OHDA-lesioned rats.} (A)
Schematic timeline for cytokine measurements. (B) Time course of the
appearance of dyskinetic manifestation of the animals used for cytokines
measurement (6-OHDA+Veh+Air mixture; 6-OHDA+L-DOPA+Air mixture;
6-OHDA+L-DOPA+H\textsubscript{2} inhalation; p\textless{}0.001) across
time (for each 20-min period). (C) show the co-administration of L-DOPA
and H\textsubscript{2} inhalation effects on the sum of axial, limb and
orofacial AIMs over 120 mins of these animals. (D) These animals were
tested also in the apomorphine test, 6-OHDA lesion significantly
increased total contralateral turns induced by apomorphine. Plasma and
striatal levels of IL-10 (E and J), IL-1\selectlanguage{greek}β \selectlanguage{english}(F and K), TNF-\selectlanguage{greek}α \selectlanguage{english}(G and L),
IL-6 (H and M) and IFN-\selectlanguage{greek}γ \selectlanguage{english}(I and N) 2 hours after L-DOPA administration
in rats inhaling H\textsubscript{2} (L-DOPA + H\textsubscript{2}) or not
(L-DOPA + Air). 6-OHDA-lesioned rats were represented by the purple
bars; 6-OHDA-lesioned rats treated chronically with L-DOPA inhaling Air
were represented by the blue bars, and 6-OHDA-lesioned rats treated
chronically with L-DOPA and inhaling H\textsubscript{2} were represented
by the green bars. H\textsubscript{2} inhalation caused an increase in
plasma and striatal levels of IL-10 (p \textless{} 0.05) and a decrease
in plasma and striatal levels of IL-1\selectlanguage{greek}β \selectlanguage{english}(p \textless{} 0.05), striatal
levels of TNF-\selectlanguage{greek}α \selectlanguage{english}(p \textless{} 0.05) and plasma and striatal levels of
IL-6 (p \textless{} 0.05) in L-DOPA treated rats. All cytokine
measurements were statistically analyzed by ordinary One-Way ANOVA with
Fisher's post-hoc test. Bars are represented as mean ± SEM. \# p
\textless{} 0.05 vs. L-DOPA + Air (blue bars), *p \textless{} 0.05 vs.
Veh + Air (purple bars) groups. a caption
{\label{895514}}%
}}
\end{center}
\end{figure}\selectlanguage{ngerman}\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/Fig--3/Fig--3}
\caption{{This is \textbf{Figure 3. AIMS were negatively correlated with striatal
IL-10 and positively correlated with plasma IL-1\selectlanguage{greek}β \selectlanguage{english}and striatal TNF-\selectlanguage{greek}α
\selectlanguage{english}levels.}(A) Correlation matrix of plasma and striatal IL-10, IL-1\selectlanguage{greek}β,
\selectlanguage{english}TNF-\selectlanguage{greek}α \selectlanguage{english}levels, and AIMs. The correlation coefficient (r) of each
correlation is inside its correspondent box ranging from -1 (darkest red
gradient) to +1 (darkest blue gradient). A two-tailed p-value was chosen
to evaluate the statistical significance of each correlation. The
coefficient of determination (r\textsuperscript{2}) between a
statistically correlated pair of variables was calculated and a best-fit
linear regression line (dark full line in the plot) was built for each
two variables in one panel (panels B-G). Correlation plot was set at
95\% confidence interval (shadowed area with the exactly gradient of the
correspondent correlation coefficient - r) surrounded by dark dashed
lines. There was a statistical correlation between: AIMS and striatal
IL-10 levels (B); AIMS with plasma IL-1\selectlanguage{greek}β \selectlanguage{english}(C) and AIMs with striatal
TNF-\selectlanguage{greek}α \selectlanguage{english}(D) levels; striatal TNF-\selectlanguage{greek}α \selectlanguage{english}levels with striatal IL-10 (E), plasma
IL-1\selectlanguage{greek}β \selectlanguage{english}with striatal TNF-\selectlanguage{greek}α \selectlanguage{english}(F), and plasma TNF-\selectlanguage{greek}α \selectlanguage{english}(G) levels. a caption
{\label{851068}}%
}}
\end{center}
\end{figure}\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/FIG-4/FIG-4}
\caption{\selectlanguage{ngerman}{This is a cap \textbf{Figure 4. Molecular hydrogen
(H}\textsubscript{\textbf{2}}\textbf{) inhalation prevented the
reactivity of striatal microglia with no impact in striatal astrocytes
morphology.} Microglia analysis by OX-42-ir staining. (A) representative
photomicrographs of striatum slices of lesioned rats treated chronically
with L-DOPA inhaling (L-DOPA + H\textsubscript{2}) or not (L-DOPA)
H\textsubscript{2}. Quantification of OX-42-ir images indicates the
number of OX-42-ir cells (B), total number of processes cells (C),
average processes length (D), and intersections number between the
processes (G). H\textsubscript{2}treatment induced a decrease in the
number of OX42-ir positive cells, in the average of processes number and
length, a without affecting the number of intersections in
L-DOPA-treated rats (Fig. H; p\textgreater{} 0.05). Astrocyte analysis
by GFAP staining. (B) representative photomicrographs of striatum slices
of 6-OHDA-lesioned rats treated chronically with L-DOPA inhaling (L-DOPA
+ H\textsubscript{2}) or not (L-DOPA) H\textsubscript{2}. Quantification
of GFAP images indicates the number of GFAP cells (E), the total number
of processes cells (F), average processes length (I), and intersections
number between the processes (J). H\textsubscript{2} treatment caused no
alterations in the number of GFP positive cells, processes number,
length, and intersections (p\textgreater{} 0.05). 6-OHDA-lesioned rats
were represented by the purple bars; 6-OHDA-lesioned rats treated
chronically with L-DOPA inhaling Air was represented by the blue bars,
and 6-OHDA-lesioned rats treated chronically with L-DOPA and inhaling
H\textsubscript{2} were represented by the green bars. Data were
statistically analyzed by ordinary One-Way ANOVA with Fisher post-hoc
test. Bars are represented as mean ± SEM. \#p \textless{} 0.05 vs.
L-DOPA + Air, *p \textless{} 0.05 vs. Veh + Air group tion
{\label{170350}}%
}}
\end{center}
\end{figure}\selectlanguage{ngerman}

Bruna M. Santos PhD\textsuperscript{1,2}; Glauce C. Nascimento
PhD\textsuperscript{1,3}; João F. Pedrazzi PhD \textsuperscript{4};
Danyelle Silva-Amaral MS\textsuperscript{1} ; Mariza Bortolanza
PhD\textsuperscript{3}; Grant T. Harris BS\textsuperscript{2}; Luiz G.
S. Branco PhD \textsuperscript{1,3}*, Elaine Del Bel
PhD\textsuperscript{1,3,4}*

\textsuperscript{1} Department of Physiology, Medical School of Ribeirão
Preto, University of São Paulo, Ribeirão Preto, SP, Brazil.

\textsuperscript{2}Thermoregulation and Systemic Inflammation Laboratory
(FeverLab), St. Josephs' Hospital and Medical Center, Phoenix, AZ, USA.

\textsuperscript{3} Department of Basic and Oral Biology, Ribeirão Preto
Dentistry Faculty, University of São Paulo, Ribeirão Preto, SP, Brazil.

\textsuperscript{4} Neuroscience Graduate Program, Medical School of
Ribeirão Preto, University of São Paulo, Ribeirão Preto, SP, Brazil.

*\emph{Corresponding Authors} :

\textbf{Elaine Del Bel} ; email:\emph{eadelbel@usp.br}; Department of
Basic and Oral Biology, Dental School, University of São Paulo (USP),
Ribeirão Preto, SP, 14040-904, Brazil.

\textbf{Luiz GS Branco} ; email: \emph{branco@forp.usp.br} ; Department
of Basic and Oral Biology, Dental School, University of São Paulo (USP),
Ribeirão Preto, SP, 14040-904, Brazil.

\textbf{Text} \emph{(3692 words)- (Up to 3700 words excluding of
abstract, legends, and references)}

\textbf{Running title:} The anti-inflammatory effect of
H\textsubscript{2} in LID

\textbf{Key words:} 6-Hydroxydopamine, Parkinson's disease,
neuroinflammation; striatum; systemic inflammation

The authors declare that the research was conducted in the absence of
any commercial or financial relationships that could be construed as a
potential conflict of interest.

\textbf{Funding sources:} Grant GCN {[}\#88882.317597/2019-014, National
Council for Scientific and Technological Development (PNPD-CNPq),
Brazil{]}. Grant JFP {[}\#88887.341956/2019-00, Programa Nacional de
Pós-doutorado, Coordenação de Aperfeiçoamento de Pessoal de Nível
Superior (PNPD/CAPES){]}. Grant MB {[}\#88887.334730/2019-00,
Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES){]}.
Grant LGSB {[}\#2016/17681-9, São Paulo Research Foundation (FAPESP),
Brazil. Grant EDB {[}\#2017/243044-0, São Paulo Research Foundation
(FAPESP), Brazil{]}; {[}\# 88881.198836/2018/01, Coordenação de
Aperfeiçoamento de Pessoal de Nível Superior and Deutscher Akademischer
Austauschdienst (CAPES/DAAD -- 2019-2024), Brazil and
Germain{]}\textbf{,} {[}\#88887.192409/2018-01, Coordenação de
Aperfeiçoamento de Pessoal de Nível Superior and Comitê Francês de
Avaliação da Cooperação Universitária com o Brasil (CAPES/COFECUB-
2019-2024), Brazil and France.{]}, {[}\#302434/2019-04, National Council
for Scientific and Technological Development (CNPq), Brazil{]}.

\textbf{Abstract} \emph{(226 words)}

\textbf{Background:} L-3,4-dihydroxyphenylalanine (L-DOPA)-induced
dyskinesia is a side effect of Parkinson's disease treatment and it is
characterized by atypical involuntary movements. A link between
neuroinflammation and L-DOPA-induced dyskinesia has been documented.
Molecular hydrogen has neuroprotective effects in Parkinson's disease
models and has a major anti-inflammatory effect.

\textbf{Objective:} To test the hypothesis that molecular hydrogen
inhalation reduces L-DOPA-induced dyskinesia.

\textbf{Methods:} 15 days after 6-hydroxydopamine lesions of
dopaminergic neurons were made (microinjection into the medial forebrain
bundle), chronic L-DOPA treatment (15 days) was performed. Rats were
exposed to molecular hydrogen (2\% gas mixture, 1h) or air (controls)
before L-DOPA injection. Abnormal involuntary movements and locomotor
activity were conducted. Striatal and plasma samples for molecular and
morphological evaluations were collected after the abnormal involuntary
movements analysis.

\textbf{Results:} Molecular hydrogen inhalation attenuated
L-DOPA-induced dyskinesia. The gas therapy did not impair the
improvement of locomotor activity achieved by L-DOPA treatment. Display
of abnormal involuntary movements was positively correlated with plasma
IL-1\selectlanguage{greek}β \selectlanguage{english}and striatal TNF-\selectlanguage{greek}α \selectlanguage{english}levels and negatively correlated with striatal
IL-10 levels. H\textsubscript{2} inhalation reduced activated microglia
in the lesioned striatum, which is consistent with the observed reduced
pro-inflammatory cytokines levels.

\textbf{Conclusions and implications:} Prophylactic molecular hydrogen
inhalation decreases abnormal involuntary movements in a preclinical
L-DOPA-induced dyskinesia model. The molecular hydrogen antidyskinetic
effect was associated with decreased striatal and peripheral
inflammation. This finding has a translational importance to
L-DOPA-treated parkinsonian patients' well-being.

\textbf{Introduction}

Several motor and non-motor debilitating symptoms of Parkinson's disease
(PD) are generated by dopamine (DA) depletion. Levodopa (L-DOPA) is
recognized as the most effective and well tolerated drug for PD
treatment. However, long-term L-DOPA therapy has been associated with
the development of abnormal involuntary movements referred to as
L-DOPA-induced dyskinesia (LID)\textsuperscript{1}. The development of
dyskinesia represents a serious side effect that negatively affects
quality of life of people with PD, and is also associated with both
motor, including an increased risk of postural imbalance and trauma from
falls, and non-motor operations, particularly, fluctuations in mood and
cognitive performance\textsuperscript{2--4}. Although various compounds
have been used for treating LID, their success has been
limited\textsuperscript{3}.

The pathogenesis of LID is associated with non-physiological stimulation
of DA receptors using L-DOPA, which combined with the reduction of
gamma-aminobutyric acid transmission pale-thalamic activity, generates
neuronal hyperactivity in the striatum\textsuperscript{5--9}. Moreover,
recent studies suggest that astrocyte and microglia activation increase
pro-inflammatory cytokines production, contributing to the appearance of
LID\textsuperscript{10,11}. In addition, excessive levels of glutamate
and dopamine in the striatum feed this inflammatory environment,
favoring LID\textsuperscript{12,13}. A key unmet medical need for the PD
community is a strategy for ameliorating LID\textsuperscript{10,14,15}.

Despite the fact that neurodegenerative disorders and associated
consequences have been consistently related to central nervous system
(CNS) derangement, they also have a strong associative component with
environmental factors\textsuperscript{16}. Given the evidence of the
profound influence of the periphery producing environmental signals to
the CNS, the periphery-brain axis has been increasingly explored within
the neurodegenerative disorders such as Alzheimer's and
PD\textsuperscript{17,18}. One of the peripheral influences that has
been implicated in the neurodegenerative disorders outbreak and
progression is the peripheral immune system\textsuperscript{19}.
Intestinal inflammation, for example, is remarkably observed in patients
with PD\textsuperscript{20,21}. Gut microbiota related to PD clinical
phenotype produces an improper ratio of sub-products with
anti-inflammatory agents such as short-chain fatty acids and molecular
hydrogen (H\textsubscript{2})\textsuperscript{22,23}.

Studies have shown that H\textsubscript{2} may protect the brain from
various neuronal conditions, including neurological emergencies and
neurodegenerative diseases\textsuperscript{24-26}. H\textsubscript{2} in
rodents protects mesencephalic dopaminergic neurons from
1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine
(MTPT)\textsuperscript{24}and 6-hydroxydopamine
(6-OHDA)\textsuperscript{25} induced degeneration. Several lines of
evidence indicate that neuro-immune function and behavior are influenced
by H\textsubscript{2}\textsuperscript{26--28}. Accumulating studies
indicate that H\textsubscript{2} not only has
antioxidant\textsuperscript{29,30} but also anti-inflammatory
properties\textsuperscript{31--33}. However, there are no previous
studies addressing the putative H\textsubscript{2} benefits on LID.

Hence, we tested the hypothesis that H\textsubscript{2} inhalation
reduces L-DOPA-induced dyskinesia in an animal PD model. We evaluated
the effect of H\textsubscript{2} inhalation on abnormal involuntary
movements (AIMs) in PD rats treated with L-DOPA. Moreover, we evaluated
the role of H\textsubscript{2} in pro-inflammatory {[}tumor necrosis
factor (TNF)-\selectlanguage{greek}α, \selectlanguage{english}interleukin (IL)-1\selectlanguage{greek}β, \selectlanguage{english}IL-6, interferon
(IFN)-\selectlanguage{greek}\textsubscript{ϒ}, \selectlanguage{english}and anti-inflammatory (IL-10) cytokine levels
in the plasma and striatum of PD-lesioned rats treated with L-DOPA.
Finally, we examined whether H\textsubscript{2} inhalation influenced
the morphology of striatal astrocytes and microglia in PD-lesioned rats
chronically treated with L-DOPA.

\textbf{2.0. Materials and Methods} More detailed description of our
experimental procedures can be found in the Supplementary Methods.

\textbf{2.1. Animals} Male Wistar rats (200-250 g, n = 65; USP-RP, SP,
Brazil) were used. The experimental protocol followed the Ethical
Principles in Animal Research delineated by the guidelines for the care
and use of mammals in Neuroscience and Behavioral Research and was
approved by the Ethics Committee on the Use of Experimental Animals of
the University of S\selectlanguage{ngerman}ão Paulo (\#2016.1.667.58.4).

\textbf{2.2. Dopaminergic lesion with neurotoxin 6-hydroxydopamine} Rats
were anesthetized with 2,2,2-tribromoethanol (250 mg
kg\textsuperscript{-1} ip) and fixed into the stereotaxic apparatus. The
incisor bar was set at 3.3 mm below the interaural line. Rats received
one injection of 2.0 \selectlanguage{greek}μ\selectlanguage{english}l 6-OHDA into the left medial forebrain bundle as
described by Gomes et al., 2008\textsuperscript{5} (6-OHDA - 2.5 \selectlanguage{greek}µ\selectlanguage{english}g
\selectlanguage{greek}µ\selectlanguage{english}l\textsuperscript{-1} in 0.9\% NaCl supplemented with 0.02\% ascorbic
acid, 1 \selectlanguage{greek}μ\selectlanguage{english}L min\textsuperscript{-1}).

Motor asymmetry was assessed 15 days after the lesion of the
nigrostriatal pathway by apomorphine-induced rotational behavior
analysis (0.5 mg kg in 0.9\% NaCl, subcutaneous, Sigma) (Figs. 1C and
2D)\textsuperscript{34}. The lesion was confirmed histologically at the
end of the behavioral tests (described below) by tyrosine hydroxylase
(TH) immunohistochemistry in the striatum and substantia nigra (Figs.1D
and E). L-DOPA treatment started two days after the apomorphine-induced
rotational test.

\textbf{2.3. H\textsubscript{2} administration} All animals were adapted
to the chamber with ambient air at a flow rate of 2.4 L/min, one day
before the experiments. On the experimental day, animals were exposed to
2\% H\textsubscript{2}-mixture (21\% of O\textsubscript{2} balanced with
N\textsubscript{2}) or Air-mixture (0\% H\textsubscript{2;} 21\%
O\textsubscript{2} N\textsubscript{2-}balanced) for 1 hour at the same
flow rate as the training day. Continuous monitoring of
H\textsubscript{2} concentration in the chamber was made possible by
using a gas analyzer to verify the H\textsubscript{2} outflow from the
chamber. The tested concentration of H\textsubscript{2} in the mixture
(2\%) was chosen due to the consistent and repeatable anti-inflammatory
effect observed in previous studies\textsuperscript{29,32,33}.

\textbf{2.4. L-DOPA-induced abnormal involuntary movements analysis}Rats
were subjected to chronic L-DOPA (20 mg kg plus benserazide 5mg kg,
orally by gavage) administration for 15 days (once a day) to induce a
stable expression of dyskinesia\textsuperscript{14}. Animals showing
AIMs scores greater than 10 and severity grading greater than 2 on at
least one AIMs subtype during the effect of L-DOPA were selected for the
behavioral study\textsuperscript{30-32}. Rats were monitored
individually for orofacial and limb dyskinesia, and axial dystonia or
AIMs using a rat dyskinesia scale\textsuperscript{34--37}. This scale
considers the severity and amplitude of the axial, limb and orolingual
AIMs (the scores ranged from 0 to 4) (for review see Cenci and Lundblad
2007\textsuperscript{38}). A dyskinesia time curve was generated by
plotting global AIMs scores from each monitoring time individually for
an entire testing session (180 min).

\textbf{2.5. Actimeter test} To evaluate possible motor effects of the
treatments, the spontaneous locomotor activity of each animal was
individually assessed in a photoelectric actimeter (Actitrack, Panlab,
Barcelona, Spain). The apparatus consists of a square arena (45 x 45 x
20 cm) equipped with 16 photocells that detect horizontal movement and
16 photocells that detect vertical movement. Thus, the locomotor
activity was obtained from recording the number of interruptions of the
photoelectric cells during 5 minutes. The amount of rearings was counted
using total vertical activity movements.

\textbf{2.6. Plasma and striatum sampling} The animals were euthanized
by decapitation 1 hour after L-DOPA or its vehicle administration. The
trunk blood was rapidly collected in heparin-coated tubes and
subsequently centrifuged at 3,500 rpm for 20 min at 4\selectlanguage{ngerman}°C. The brain was
removed, and the rostral and medial portion of the striatum (rostral:
1.7 mm and medial: 0.7 mm from bregma) was carefully excised, frozen by
submersion in dry ice chilled isopentane and The medial-caudal (medial:
0.7 mm and caudal: -0.8 mm and from bregma) portion of the striatum was
collected and post-fixed in tamponade paraformaldehyde (4\%) for
immunohistochemistry preparation.

\textbf{2.7. Measurement of plasma and striatal cytokines levels} The
inflammatory profile of the target samples was evaluated using a high
sensitivity enzyme-linked immunosorbent assay (Luminex). Luminex assay
was conducted using Luminex Magpix technology (Austin, TX, USA)
according to manufacturer guidelines (LXSARM - 05, R\&D, Minnesota, MN,
USA)\textsuperscript{39}.

\textbf{2.8. Immunohistochemistry} Serial coronal sections throughout
the mediocaudal extent of the striatum (Bregma +2.76mm, Interaural
11.76mm -- Bregma -2.28mm, Interaural 6.72mm) were cut (25 \selectlanguage{greek}µ\selectlanguage{english}m) using a
freezing microtome (Leica, model CM1850). Immunohistochemistry was
performed in these sections using a standard peroxidase-based
method\textsuperscript{5} to quantify TH, glial fibrillary acidic
protein (GFAP) and OX-42 (CD11b/c equivalent protein of microglia). The
sections were incubated overnight at room temperature with the primary
antibodies (Suppl. Table 1): anti-TH; anti-GFAP or anti-OX-42, followed
by 2 h of incubation with biotinylated secondary antibody (1:250,
Vectastain). The chromogen used was diaminobenzidine (Sigma-Aldrich, St.
Louis, MO, USA) and the slices were mounted on slides and cover slipped
for microscopic observations. Digital images were obtained using a Leica
microscope (Leica Microsystems Launches Leica FW4000 - Cambridge, UK)
under 20x (for TH optical density and OX-42/GFAP number of cells
quantification) or 40x (for OX-42/GFAP morphology) objectives. The
quantification of the brain area was measured using the ImageJ system
(ImageJ, RRID: SCR\_003070,National Institutes of Health - NIH;
Schneider et al., 2012\textsuperscript{40}). Analysis of microglia
(OX-42) and astrocyte (GFAP) immune labeling morphology was conducted as
described by Giocanti-Auregan et al. (2016)\textsuperscript{41} using
Fiji algorithms (RRID: SCR\_002285) and a generated skeleton image. The
parameters analyzed were the number of cells (per
0.1mm\textsuperscript{2}), branches, the number of intersections or
branching points, and the mean process length\textsuperscript{42}.

\textbf{2.9. Experimental design 1: effects of
H\textsubscript{2}inhalation on AIMs.}

Four experimental groups were randomly established consisting of animals
that underwent SHAM surgery or medial forebrain bundle lesion with daily
L-DOPA or vehicle treatment during 15 days. These groups were subdivided
into animals that inhaled Air mixture or H\textsubscript{2} gas during 1
hour on the experimental day. One day before, all the animals were
adapted to the air chamber.

On the experimental day, L-DOPA or its vehicle was injected immediately
after gas inhalation. AIMs analysis was conducted immediately after
L-DOPA or its vehicle administration during 120 minutes. Distance
travelled and amount of rearing performed by the animals were evaluated
60 minutes after L-DOPA injection for 5 minutes using the actimeter
apparatus. The animals were euthanized at the end of the AIMs analysis.

\textbf{2.10. Experimental design 2: effect of
H\textsubscript{2}inhalation on peripheral and striatal inflammatory
profile.} After a successful dopaminergic lesion and subsequent L-DOPA
treatment for 15 days, the animals adapted to the air chamber one day
before the experimental day.

Three experimental groups were arranged with randomized rats as follows:
(i) 6-OHDA lesioned rats with L-DOPA vehicle treatment and Air mixture
inhalation; (ii) 6-OHDA lesioned rats with chronic L-DOPA treatment
inhaling the Air mixture; (iii) 6-OHDA lesioned rats with chronic L-DOPA
treatment inhaling H\textsubscript{2} gas.

On the experimental day, the animals were exposed to the air or
H\textsubscript{2} gas mixture for 1 hour, and L-DOPA or its vehicle was
injected immediately after gas inhalation. AIMs analysis was conducted
immediately after L-DOPA or its vehicle administration. The striatum and
plasma were collected 2 hours after L-DOPA injection. The molecular
evaluations were conducted following the protocol mentioned previously.

\textbf{2.11. Statistical Analysis} Parametrics statistics were
performed in AIMs analysis since the basic scores of LID correspond to
the extent of time with behavioral responses
development\textsuperscript{38}. AIMs analysis along the time were
assessed by Two Way Repeated Measures Anova with Tukey's multiple
comparisons post-hoc test. Ordinary One Way Anova with Fisher post-hoc
test was conducted to analyze the other experiments. Statistical
analysis of these experiments was performed with the significance set at
p \textless{} 0.05. Correlations between cytokine levels and AIMs in
L-DOPA-treated rats were analyzed by mean Pearson correlation
coefficients (two-tailed).

\textbf{3.0. Results}

\textbf{3.1. General effect of H\textsubscript{2} on locomotor activity
and AIMs.} Only rats demonstrating more than 80 contralateral turns
after the apomorphine challenge were used in this study to compound
6-OHDA groups (Fig.1C). TH-immunoreactivity was examined in coronal
sections of the striatum and substantia nigra of parkinsonian animals to
confirm the total lesion induced by 6-OHDA (Fig. 1D and E,
respectively). Rats with the 6-OHDA lesions exhibited more than 90\%
reduction in TH-immunostaining in the striatum and substantia nigra
compacta compared with non-lesioned animals (Fig. 1D and E). About
locomotor activity, 6-OHDA lesion induced a reduction of distance
traveled and rearing compared to SHAM rats and 6-OHDA lesioned rats
treated with L-DOPA or H\textsubscript{2} (p \textless{} 0.05; Fig. 1 F
and G). Curiously, H\textsubscript{2} inhalation induced an increase in
locomotor activities of 6-OHDA lesioned rats treated with L-DOPA vehicle
compared with those inhaling air mixture, indicating its beneficial
effect \emph{per se} . As previously described\textsuperscript{3}, we
observed an increase in distance travelled (Fig. 1F) and rearing (Fig.
1G) in L-DOPA-treated lesioned animals compared to 6-OHDA lesioned
animals. The total distance travelled and rearing behavior were similar
in both groups under L-DOPA administration, with Air mixture and
H\textsubscript{2} inhalations, indicating that
H\textsubscript{2}inhalation did not impair the L-DOPA benefits to
restore movement.

The AIMs quantification elucidated the H\textsubscript{2}anti-dyskinetic
effect. Individual curves for each L-DOPA induced AIMs score category
(limb, orofacial, and axial) were detected and it was observed an
increase of AIMs in all of them (p \textless{} 0.05, Fig. 1I-K) in
6-OHDA lesioned rats treated chronically with L-DOPA. H\textsubscript{2}
inhalation produced a reduction of 48\% in LID (Figs. 1I-K). SHAM groups
present no dyskinesia. The maximum total AIMs score reached 20--80 min
after L-DOPA administration and gradually declined to the baseline level
over 120 min (Figs. 1H and 2C).

\textbf{3.2. Effect of H\textsubscript{2} in plasma and striatal
inflammatory profile of LID rats} The rats used for Experiment 2 were
confirmed as to the intensity of the lesion and dyskinesia (Fig. 2 B-D).
The apomorphine-induced rotations test confirmed the presence of lesion
in all rats (Fig 2D). There was an increase of AIMs (p \textless{} 0.05)
in rats treated chronically with L-DOPA and H\textsubscript{2}inhalation
produced a reduction of 41\% in LID (Figs. 2B, C).

Animals presenting LID had a decrease in plasma and striatal IL-10
levels in 6-OHDA-lesioned rats when compared with vehicle-treated
6-OHDA-lesioned animals (p \textless{} 0.05; Fig. 2E and J).
Interestingly, this effect of L-DOPA was reverted by
H\textsubscript{2}inhalation (p \textless{} 0.05; Fig. 2E and J),
indicating that the gas favors this anti-inflammatory cytokine
production in samples of LID rats.

L-DOPA treatment caused an increase in striatal pro-inflammatory
cytokine IL-1\selectlanguage{greek}β \selectlanguage{english}and IL-6 levels compared to 6-OHDA-lesioned animals
treated with vehicle (p \textless{} 0.05; Fig. 2K and M); no statistical
differences were observed in plasma (Fig. 2F and H). The striatal
increase was reverted by H\textsubscript{2} inhalation (p \textless{}
0.05), indicating that H\textsubscript{2} acts not only by up-regulating
an anti-inflammatory cytokine, but also by down-regulating those
cytokines in LID rats.

There was an enhancement only in striatal TNF-\selectlanguage{greek}α \selectlanguage{english}production that was
reverted by H\textsubscript{2} inhalation in dyskinetic animals (p
\textless{} 0.05; Fig. 2L). No differences in plasma or striatal IFN-\selectlanguage{greek}γ
\selectlanguage{english}levels were found between groups (Fig. 2I and N).

\textbf{3.3. Cytokines and AIMs correlation} To evaluate potential
associations between these cytokines and AIMS, we performed a
correlation analysis (Fig. 3A). AIMs were negatively correlated to
striatal IL-10 (Fig. 3B) and positively correlated to plasma IL-1\selectlanguage{greek}β \selectlanguage{english}(Fig.
3C) and striatal TNF-\selectlanguage{greek}α \selectlanguage{english}levels (Fig. 3D).

Striatal IL-10 levels were negatively correlated to striatal TNF-\selectlanguage{greek}α
\selectlanguage{english}levels (Fig. 3E). Plasma IL-1\selectlanguage{greek}β \selectlanguage{english}levels were correlated to striatal and
plasma TNF-\selectlanguage{greek}α \selectlanguage{english}levels (Fig. 3F and G, respectively). Of note, there is no
significant correlation between plasma IL-10 and striatal IL-10, plasma
IL-1\selectlanguage{greek}β \selectlanguage{english}and striatal IL-1\selectlanguage{greek}β, \selectlanguage{english}and plasma IL-6 and striatal IL-6, indicating
disparities in peripheral versus central immune response, even being the
same cytokine (Fig. 3A). Other relationships were manifest (see Suppl.
Fig. 1 and 2 for details).

\textbf{3.4. Effects of H\textsubscript{2} on striatal astrocyte and
microglia on LID} Fig. 4A and B show representative immunohistochemical
staining of OX-42 and GFAP -ir cells, respectively, in the lesioned
striatum of rats with LID under Air or H\textsubscript{2}
treatment\textsubscript{.} Regarding striatal OX-42 (Fig. 4C) and
GFAP-ir (Fig. 4E) positive cells, an increased number of astrocytes and
microglia in the striatum of L-DOPA-treated rats was observed when
compared to 6-OHDA-lesioned rats treated with vehicle
(p\textless{}0.05). L-DOPA also induced increased changes in astrocyte
morphology (processes number and length and intersections number), as
indicative of reactive cells. Specifically, the increased intersections
mean that the mature astrocyte network has a bigger
complexity\textsuperscript{41}. H\textsubscript{2} pre-treatment
prevented L-DOPA-induced OX-42 increase (p\textless{}0.05), but not GFAP
(Figs. 4C, E). H\textsubscript{2} prevented the reactivity of striatal
microglia of rats with LID, \emph{i.e.} reducing the number of processes
(p\textless{}0.05, Fig. 4D) and increasing the length of these processes
(p\textless{}0.05, Fig. 4G). Conversely, H\textsubscript{2} caused no
change in GFAP morphology compared to the L-DOPA+Air group (Figs. 4F, I,
J).

Similarlyto described by Bortolanza et al., (2015)\textsuperscript{10},
the increase of the immunoreactivity of GFAP, OX-42 in LID and the
evidence of microglia and astrocytes activation was not observed in the
striatum of non-lesioned rats and lesioned rats treated only with
vehicle or H\textsubscript{2} (results not presented).

\textbf{4.0. Discussion}

This is the first study to report that H\textsubscript{2} reduces LID
severity in the PD model. This beneficial effect of
H\textsubscript{2}inhalation was associated with reduced peripheral and
central cytokine levels and microglia activation. Of note,
H\textsubscript{2} did not interfere with L-DOPA's therapeutic efficacy
with motor functioning.

Recently, the effect of H\textsubscript{2} was reported in models of
PD\textsuperscript{24,25,43}. Either drinking H\textsubscript{2}-rich
water or intermittent inhalation of 2\% H\textsubscript{2} ameliorate
6-OHDA or MPTP-induced hemiparkinsonism\textsuperscript{37,39}. The
effects of gas inhalation, however, might vary according to the
treatment protocol used. An intermittent inhalation of 2\%
H\textsubscript{2} (15 min; 1-hour interval; 12 hours per day for 5
weeks) ameliorated 6-OHDA-induced hemiparkinsonism rat model, whereas
continuous inhalation of 2\% H\textsubscript{2} (24 hours a day for 5
weeks) had no effect\textsuperscript{42}. As aforementioned, the
long-term protocol used and the route of administration of
H\textsubscript{2} can contribute to maximizing its effects and
therefore increase (or not) its benefits. It must be highlighted that
the effects observed in the present study were followed by treatment of
the animals with a single exposure to H\textsubscript{2.} The optimal
route, time and dose of H\textsubscript{2} administration remain to be
established.

\textbf{4.1. LID inflammatory status in plasma and striatum}

We observed that chronic L-DOPA treatment in the parkinsonian rat was
associated with a decrease in plasma and striatum IL-10 levels compared
to animals that received its vehicle.

Interestingly, LID was accompanied by an increase in pro-inflammatory
markers in the striatum and plasma. Both observations correlate with
peripheral and CNS inflammation respectively, after L-DOPA treatment of
parkinsonian rats. Previous studies reported some anti-dyskinetic drugs
targeting the immune system epitomized by
corticosterone\textsuperscript{44}, the peroxisome
proliferator-activated-gamma agonist rosiglitazone\textsuperscript{45},
the immunosuppressant rapamycin\textsuperscript{45}, and other drugs
with immunomodulatory and antiangiogenic activities such as
thalidomide\textsuperscript{43}, cannabidiol\textsuperscript{46--48},
and nitric oxide synthase inhibitor\textsuperscript{10}.

Besides the convincing effect of neuroinflammatory mediators in the
onset and development of neurodegenerative disorders and
LID\textsuperscript{10,44,49,50}, new studies are now focusing on the
role of the peripheral immune system in neurodegenerative disorders as a
potential target therapy\textsuperscript{51-56}. The peripheral immune
system can act directly by brain infiltration\textsuperscript{57,58}.
Also, microglia are renowned for causing neuroinflammation induced by a
peripheral inflammatory stimulus, as clearly observed in classical
models of inflammation using lipopolysaccharide as
stimulus\textsuperscript{59}. Similar to Dos Santos Pereira et
al\textsuperscript{60}, here, we showed that plasma IL-1\selectlanguage{greek}β \selectlanguage{english}cytokine
levels are concurrently correlated with striatal TNF-\selectlanguage{greek}α \selectlanguage{english}levels and AIMS.
Furthermore, striatal TNF-\selectlanguage{greek}α \selectlanguage{english}levels were also correlated with AIMs
induced by L-DOPA in the PD model. Collectively, these correlations
indicate plasma IL-1\selectlanguage{greek}β \selectlanguage{english}as a potential therapeutic target to treat LID, to
be ultimately targeted by other ways besides H\textsubscript{2}. These
correlations suggest a potential effect of plasma IL-1\selectlanguage{greek}β \selectlanguage{english}in AIMs
presentation. However, the arising hypothesis still remains elusive and
the implicated mechanism must be evaluated in further
studies\textsuperscript{10,11,14,15,37,61}.

\textbf{4.2. H\textsubscript{2} and LID: microglia and astrocytes}

Our data provides evidence of the effect of H\textsubscript{2}inhalation
on decreasing immunohistochemical signal and process number of
microglia, along with reduction of their process length in lesioned
striatum of rats chronically treated with L-DOPA. The number of
intersections could indicate the communication between these cells and
we did not find significant differences regarding this criterion.
Reactive microglia present macrophagic and antigen-cell function mainly
through synthesis of several chemokines and cytokines, inflammatory
mediators and cell surface molecules\textsuperscript{62}. Otherwise, our
results clearly show that H\textsubscript{2} inhalation was not able to
reduce astrocytes levels back to control levels (rats without L-DOPA
treatment). It is important to point out that the consequences of
astrogliosis may have both beneficial and detrimental effects on
surrounding neural and non-neural cells\textsuperscript{63}. Reactive
astrocytes change their properties in parallel with morphological,
biochemical and functional alterations\textsuperscript{64-66}. We also
observed that H\textsubscript{2} inhalation caused an abrupt decrease in
striatal IL-1\selectlanguage{greek}β \selectlanguage{english}and TNF-\selectlanguage{greek}α \selectlanguage{english}cytokines that have important roles in
modulating astrocytic phenotypes\textsuperscript{58}, and thus
supporting a potential shift in reactive astrocyte neurotoxic phenotypes
to speculate a more neuroprotective phenotype.

Collectively, our results on the effect of H\textsubscript{2} on the
inflammatory environment associated with the effects on striatal
microglial reactivity may support, at least in part, the reduction of
LID. However, we cannot stop speculating on the participation of other
mechanisms in this important anti-dyskinetic H\textsubscript{2} effect.

Within the scope of PD, it is known that the levels of reactive oxygen
species are strictly controlled by various antioxidant mechanisms in
healthy dopaminergic neurons. These effects may be deficient in
Parkinson's patients\textsuperscript{67}. \textsuperscript{68}.
Furthermore, oxidative stress is related to the decline in cell function
and apoptosis in several cell types, including
astrocytes\textsuperscript{69}. Although this was not the study's
objective, it is noteworthy that H\textsubscript{2} has aroused great
interest concerning its antioxidant profile, which is corroborated in
diseases and disease models associated with oxidative
stress\textsuperscript{70}. Such observations may be the target of
future investigations.

In addition to oxidative stress, there are several markers for toxicity
in the brain, but one of the most widely studied is the neurotransmitter
L-glutamate. Although LID is primarily caused by pre-synaptic and
post-synaptic changes in DA neurotransmission, it also depends on
altered glutamatergic transmission at diverse nodes of the cortico-basal
ganglia-thalamocortical network\textsuperscript{71}. It could be
possible that H\textsubscript{2} inhalation action on LID be attributed
to affecting neurotransmission systems well directly involved in
dyskinesia development, besides its antiinflammatory action. Recently,
alleviation of dyskinetic behaviors in both rat and primate models of
LID following L-dopa treatment and xenon (another gas) inhalation was
documented and this effect is mediated by an improvement of
glutamatergic transmission in striatal projecting
neurons\textsuperscript{72}. In addition, Settineri et al
(2018)\textsuperscript{73} evidenced that hydrogenized water protected
human neuroblastoma SH-SY5Y cells from oxidative stress and glutamate
toxicity.

\textbf{5.0. Conclusion}

H\textsubscript{2} inhalation decreases abnormal involuntary movements
and signaling of an inflammatory reaction in a preclinical
L-DOPA-induced dyskinesia rodent model. To our knowledge, no adverse
effects of H\textsubscript{2} have been reported, and
H\textsubscript{2}is relatively easy to use, inexpensive, and effective
in daily medical practice. Altogether, the search for a compound capable
of preventing the side effects of PD without affecting the benefits of
the L-DOPA treatment is certainly important to the well-being of
patients.

\textbf{6.0. Acknowledgements} We appreciate all the support and
guidance provided by Dr. Andrej Romanovsky. The authors are also
thankful for the technical assistance of Nadir Fernandes, Mauro F.
Silva, and C\selectlanguage{ngerman}élia Silva.

\textbf{7.0. Authors' Roles}

\emph{1) Research project:}

A. Conception: BMS, GCN, MB, GTH, LGSB, EDB

B. Organization: BMS, GCN

C. Execution: BMS, GCN, JFP, DSA, MB, LGSB, EDB

\emph{2) Statistical Analysis:}

A. Design: BMS, GCN, JFP

B. Execution: BMS, GCN, JFP

\emph{3)} \emph{Manuscript writing:}

A. Writing of the first: draft: BMS

B. Review and Critique: BMS, GCN, JFP, DSA, MB, GTH, LGSB, EDB

\textbf{8.0. Disclosures of all authors (for the preceding 12 months)}

Grant BMS {[}\#2016/09364-3, São Paulo Research Foundation (FAPESP),
Brazil{]}.

Grant MB {[}\#2016/06602-0, São Paulo Research Foundation (FAPESP),
Brazil{]}.

Grant LGSB {[}\#301040/2018-4, National Council for Scientific and
Technological Development (CNPq), Brazil{]}

Grant EDB {[}\#301040/2018-4, National Council for Scientific and
Technological Development (CNPq), Brazil{]}, {[}\#2014/25029-4, São
Paulo Research Foundation (FAPESP), Brazil{]}. Honoraria EDB {[}Bernice
Grafstein Award for Outstanding Accomplishments in Mentoring
(SfN-USA){]}.

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\textbf{Figures:} \emph{(Up to 5)}

\textbf{Figure 1. Molecular hydrogen (H\textsubscript{2}) inhalation
reduces AIMS without interfering with the anti-parkinsonian effect of
L-DOPA.} (A, B) Schematic protocol. After Sham surgery or 6-OHDA
injection, the rats were analysed in the apomorphine-induced rotational
test for lesion confirmation. Then, Sham or 6-OHDA-lesioned rats were
treated chronically (15 days) with L-DOPA or its vehicle. Subsequently
before the last L-DOPA injection, Air mixture or
H\textsubscript{2}inhalation was performed for a 1-h period, followed by
the L-DOPA injection and abnormal involuntary movements on axial, limb
and orofacial parameters (ALO AIMs) and locomotor activity were
measured. (C) 6-OHDA lesion significantly increased total contralateral
turns induced by apomorphine. (D) Photomicrographs of coronal brain
sections illustrating the loss of TH positive immunolabeling in the
striatum fibers (D) and substantia nigra compacta neurons (E), scale
bars=1200 \selectlanguage{greek}µ\selectlanguage{english}m. (F, G) H\textsubscript{2} treatment does not alter the
L-DOPA effect on the distance traveled (F) and rearing (G) in the
actimeter. (H) Time course of the appearance of dyskinetic manifestation
with 6-OHDA+L-DOPA+Air mixture compared to
6-OHDA+L-DOPA+H\textsubscript{2}inhalation (p\textless{}0.001) across
time (for each 20-min period). (I, J, K) show the co-administration of
L-DOPA and H\textsubscript{2}inhalation effects on the axial (I), limb
(J) and orofacial (K) AIMs over 120 mins. Sham rats treated with vehicle
of L-DOPA (SHAM+Veh, white); Sham rats treated chronically with L-DOPA
(SHAM+L-DOPA, orange); 6-OHDA lesioned rats treated with vehicle of
L-DOPA (6-OHDA+Veh, green); 6-OHDA lesioned rats treated chronically
with L-DOPA (6-OHDA+L-DOPA, blue). AIMs were analyzed using 2-Way RM
ANOVA analysis with Tukey\selectlanguage{ngerman}´s multiple comparisons post-hoc test. Distance
travelled was analyzed by ordinary One-Way ANOVA with Fisher post-hoc
test. Data are reported as mean ± SEM. ***p\textless{}0.001,
**p\textless{}0.01, *p\textless{}0.05.

\textbf{Figure 2. Molecular hydrogen (H\textsubscript{2}) inhalation
improves peripheral and striatal inflammation in L-DOPA-treated
6-OHDA-lesioned rats.} (A) Schematic timeline for cytokine measurements.
(B) Time course of the appearance of dyskinetic manifestation of the
animals used for cytokines measurement (6-OHDA+Veh+Air mixture;
6-OHDA+L-DOPA+Air mixture; 6-OHDA+L-DOPA+H\textsubscript{2} inhalation;
p\textless{}0.001) across time (for each 20-min period). (C) show the
co-administration of L-DOPA and H\textsubscript{2} inhalation effects on
the sum of axial, limb and orofacial AIMs over 120 mins of these
animals. (D) These animals were tested also in the apomorphine test,
6-OHDA lesion significantly increased total contralateral turns induced
by apomorphine. Plasma and striatal levels of IL-10 (E and J), IL-1\selectlanguage{greek}β \selectlanguage{english}(F
and K), TNF-\selectlanguage{greek}α \selectlanguage{english}(G and L), IL-6 (H and M) and IFN-\selectlanguage{greek}γ \selectlanguage{english}(I and N) 2 hours
after L-DOPA administration in rats inhaling H\textsubscript{2} (L-DOPA
+ H\textsubscript{2}) or not (L-DOPA + Air). 6-OHDA-lesioned rats were
represented by the purple bars; 6-OHDA-lesioned rats treated chronically
with L-DOPA inhaling Air were represented by the blue bars, and
6-OHDA-lesioned rats treated chronically with L-DOPA and inhaling
H\textsubscript{2} were represented by the green bars.
H\textsubscript{2} inhalation caused an increase in plasma and striatal
levels of IL-10 (p \textless{} 0.05) and a decrease in plasma and
striatal levels of IL-1\selectlanguage{greek}β \selectlanguage{english}(p \textless{} 0.05), striatal levels of TNF-\selectlanguage{greek}α
\selectlanguage{english}(p \textless{} 0.05) and plasma and striatal levels of IL-6 (p
\textless{} 0.05) in L-DOPA treated rats. All cytokine measurements were
statistically analyzed by ordinary One-Way ANOVA with Fisher's post-hoc
test. Bars are represented as mean \selectlanguage{ngerman}± SEM. \# p \textless{} 0.05 vs.
L-DOPA + Air (blue bars), *p \textless{} 0.05 vs. Veh + Air (purple
bars) groups.

\selectlanguage{greek}\textbf{Figure 3. AIMS were negatively correlated with striatal IL-10
and positively correlated with plasma IL-1β and striatal TNF-α
levels.}\selectlanguage{english}(A) Correlation matrix of plasma and striatal IL-10, IL-1\selectlanguage{greek}β,
\selectlanguage{english}TNF-\selectlanguage{greek}α \selectlanguage{english}levels, and AIMs. The correlation coefficient (r) of each
correlation is inside its correspondent box ranging from -1 (darkest red
gradient) to +1 (darkest blue gradient). A two-tailed p-value was chosen
to evaluate the statistical significance of each correlation. The
coefficient of determination (r\textsuperscript{2}) between a
statistically correlated pair of variables was calculated and a best-fit
linear regression line (dark full line in the plot) was built for each
two variables in one panel (panels B-G). Correlation plot was set at
95\% confidence interval (shadowed area with the exactly gradient of the
correspondent correlation coefficient - r) surrounded by dark dashed
lines. There was a statistical correlation between: AIMS and striatal
IL-10 levels (B); AIMS with plasma IL-1\selectlanguage{greek}β \selectlanguage{english}(C) and AIMs with striatal
TNF-\selectlanguage{greek}α \selectlanguage{english}(D) levels; striatal TNF-\selectlanguage{greek}α \selectlanguage{english}levels with striatal IL-10 (E), plasma
IL-1\selectlanguage{greek}β \selectlanguage{english}with striatal TNF-\selectlanguage{greek}α \selectlanguage{english}(F), and plasma TNF-\selectlanguage{greek}α \selectlanguage{english}(G) levels.

\textbf{Figure 4. Molecular hydrogen (H\textsubscript{2}) inhalation
prevented the reactivity of striatal microglia with no impact in
striatal astrocytes morphology.} Microglia analysis by OX-42-ir
staining. (A) representative photomicrographs of striatum slices of
lesioned rats treated chronically with L-DOPA inhaling (L-DOPA +
H\textsubscript{2}) or not (L-DOPA) H\textsubscript{2}. Quantification
of OX-42-ir images indicates the number of OX-42-ir cells (B), total
number of processes cells (C), average processes length (D), and
intersections number between the processes (G).
H\textsubscript{2}treatment induced a decrease in the number of OX42-ir
positive cells, in the average of processes number and length, a without
affecting the number of intersections in L-DOPA-treated rats (Fig. H;
p\textgreater{} 0.05). Astrocyte analysis by GFAP staining. (B)
representative photomicrographs of striatum slices of 6-OHDA-lesioned
rats treated chronically with L-DOPA inhaling (L-DOPA +
H\textsubscript{2}) or not (L-DOPA) H\textsubscript{2}. Quantification
of GFAP images indicates the number of GFAP cells (E), the total number
of processes cells (F), average processes length (I), and intersections
number between the processes (J). H\textsubscript{2} treatment caused no
alterations in the number of GFP positive cells, processes number,
length, and intersections (p\textgreater{} 0.05). 6-OHDA-lesioned rats
were represented by the purple bars; 6-OHDA-lesioned rats treated
chronically with L-DOPA inhaling Air was represented by the blue bars,
and 6-OHDA-lesioned rats treated chronically with L-DOPA and inhaling
H\textsubscript{2} were represented by the green bars. Data were
statistically analyzed by ordinary One-Way ANOVA with Fisher post-hoc
test. Bars are represented as mean \selectlanguage{ngerman}± SEM. \#p \textless{} 0.05 vs.
L-DOPA + Air, *p \textless{} 0.05 vs. Veh + Air groups.

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