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Mitochondrial-Protective Effects of R-Phenibut after Experimental Traumatic Brain Injury

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Altered neuronal Ca2+ homeostasis and mitochondrial dysfunction play a central role in the pathogenesis of traumatic brain injury (TBI). R-Phenibut ((3R)-phenyl-4-aminobutyric acid) is an antagonist of the α2δ subunit of voltage-dependent calcium channels (VDCC) and an agonist of gamma-aminobutyric acid B (GABA-B) receptors. The aim of this study was to evaluate the potential therapeutic effects of R-phenibut following the lateral fluid percussion injury (latFPI) model of TBI in mice and the impact of R- and S-phenibut on mitochondrial functionality in vitro. By determining the bioavailability of R-phenibut in the mouse brain tissue and plasma, we found that R-phenibut (50 mg/kg) reached the brain tissue 15 min after intraperitoneal (i.p.) and peroral (p.o.) injections. The maximal concentration of R-phenibut in the brain tissues was 0.6 μg/g and 0.2 μg/g tissue after i.p. and p.o. administration, respectively. Male Swiss-Webster mice received i.p. injections of R-phenibut at doses of 10 or 50 mg/kg 2 h after TBI and then once daily for 7 days. R-Phenibut treatment at the dose of 50 mg/kg significantly ameliorated functional deficits after TBI on postinjury days 1, 4, and 7. Seven days after TBI, the number of Nissl-stained dark neurons (N-DNs) and interleukin-1beta (IL-1β) expression in the cerebral neocortex in the area of cortical impact were reduced. Moreover, the addition of R- and S-phenibut at a concentration of 0.5 μg/ml inhibited calcium-induced mitochondrial swelling in the brain homogenate and prevented anoxia-reoxygenation-induced increases in mitochondrial H2O2 production and the H2O2/O ratio. Taken together, these results suggest that R-phenibut could serve as a neuroprotective agent and promising drug candidate for treating TBI.
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Research Article
Mitochondrial-Protective Effects of R-Phenibut after
Experimental Traumatic Brain Injury
Einars Kupats ,
1,2
Gundega Stelfa ,
1,3
Baiba Zvejniece ,
1
Solveiga Grinberga ,
1
Edijs Vavers ,
1
Marina Makrecka-Kuka ,
1
Baiba Svalbe ,
1
Liga Zvejniece ,
1
and Maija Dambrova
1,4
1
Latvian Institute of Organic Synthesis, Riga, Latvia
2
Department of Neurology and Neurosurgery, Riga Stradins University, Riga, Latvia
3
Latvia University of Life Sciences and Technologies, Jelgava, Latvia
4
Department of Pharmaceutical Chemistry, Riga Stradins University, Riga, Latvia
Correspondence should be addressed to Einars Kupats; einars.kupats@farm.osi.lv
Received 15 July 2020; Revised 24 September 2020; Accepted 3 November 2020; Published 21 November 2020
Academic Editor: Luciano Saso
Copyright © 2020 Einars Kupats et al. This is an open access article distributed under the Creative Commons Attribution License,
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Altered neuronal Ca
2+
homeostasis and mitochondrial dysfunction play a central role in the pathogenesis of traumatic brain injury
(TBI). R-Phenibut ((3R)-phenyl-4-aminobutyric acid) is an antagonist of the α
2
δsubunit of voltage-dependent calcium channels
(VDCC) and an agonist of gamma-aminobutyric acid B (GABA-B) receptors. The aim of this study was to evaluate the potential
therapeutic eects of R-phenibut following the lateral uid percussion injury (latFPI) model of TBI in mice and the impact of R-
and S-phenibut on mitochondrial functionality in vitro. By determining the bioavailability of R-phenibut in the mouse brain
tissue and plasma, we found that R-phenibut (50 mg/kg) reached the brain tissue 15 min after intraperitoneal (i.p.) and peroral
(p.o.) injections. The maximal concentration of R-phenibut in the brain tissues was 0.6 μg/g and 0.2 μg/g tissue after i.p. and p.o.
administration, respectively. Male Swiss-Webster mice received i.p. injections of R-phenibut at doses of 10 or 50 mg/kg 2 h after
TBI and then once daily for 7 days. R-Phenibut treatment at the dose of 50 mg/kg signicantly ameliorated functional decits
after TBI on postinjury days 1, 4, and 7. Seven days after TBI, the number of Nissl-stained dark neurons (N-DNs) and
interleukin-1beta (IL-1β) expression in the cerebral neocortex in the area of cortical impact were reduced. Moreover, the addition
of R- and S-phenibut at a concentration of 0.5 μg/ml inhibited calcium-induced mitochondrial swelling in the brain homogenate
and prevented anoxia-reoxygenation-induced increases in mitochondrial H
2
O
2
production and the H
2
O
2
/O ratio. Taken together,
these results suggest that R-phenibut could serve as a neuroprotective agent and promising drug candidate for treating TBI.
1. Introduction
Traumatic brain injury (TBI) is a leading cause of mortality
and disability among trauma-related injuries [1]. TBI can
result in temporary, long-term, and even life-long physical,
cognitive, and behavioural problems [2, 3]. Therefore, there
is an increased need for eective pharmacological approaches
for treating patients with TBI. Phenibut, a nootropic pre-
scription drug with anxiolytic activity, is used in clinical prac-
tice in Eastern European countries for the treatment of
anxiety, tics, stuttering, insomnia, dizziness, and alcohol
abstinence [4, 5]. R-Phenibut ((3R)-phenyl-4-aminobutyric
acid), which is one of the optical isomers of phenibut, binds
to gamma-aminobutyric acid B (GABA-B) receptors and the
α
2
δsubunit of voltage-dependent calcium channels (VDCC),
while S-phenibut binds only to the α
2
δsubunit of VDCC
[68]. Our previous studies have shown that R-phenibut treat-
ment signicantly decreased the brain infarct size and
increased brain-derived neurotrophic factor and vascular
endothelial growth factor gene expression in damaged brain
tissue in an experimental stroke model [9]. The similarity of
the pathogenic mechanisms of TBI and cerebral ischaemia
indicate that therapeutic strategies that are successful in treat-
ing one may also be benecial in treating the other [10].
Hindawi
Oxidative Medicine and Cellular Longevity
Volume 2020, Article ID 9364598, 12 pages
https://doi.org/10.1155/2020/9364598
Treatment options for TBI are limited due to its complex
pathogenesis and the heterogeneity of its presentation, which
includes haematomas, contusions, hypoxia, and vascular,
axonal, and other types of central nervous system injuries
[11, 12]. Among the processes that impact TBI, the genera-
tion of reactive oxygen species (ROS) by mitochondria
occurs within the rst minutes after TBI and thus leads to
the disruption of calcium ion (Ca
2+
) homeostasis, which is
the nal common pathwayfor toxic cellular degradation
[13, 14]. Maintaining regional neuronal Ca
2+
homeostasis
and mitochondrial function is crucial to prevent secondary
neuronal injury [15, 16]. Thus, mitochondrial-targeted drugs
and drugs acting on specic intracellular Ca
2+
signalling
pathways or subcellular components show promise as thera-
peutic interventions for TBI [17, 18]. In fact, upregulation of
the neuronal calcium channel α
2
δsubunit modulates the
activation of mitochondrial Ca
2+
buering in pathological
conditions [19]. There is also evidence that GABA-B receptor
agonists provide neuroprotection against N-methyl-D-
aspartate-induced neurotoxicity mediated by the mitochon-
drial permeability transition pore [20]. Since both isomers
of phenibut bind to the α
2
δsubunit of VDCC and only R-
phenibut binds to the GABA-B receptor, these both isomers
could be used to specify the possible molecular mechanisms
of phenibut in dierent experimental models.
This is the rst investigation of the potential therapeutic
eects of R-phenibut following TBI in mice. In addition, to
evaluate possible molecular mechanisms underlying the
actions of R-phenibut against anoxia-reoxygenation-
induced mitochondrial damage, the eects on mitochondrial
functionality were evaluated in an in vitro model of anoxia-
reoxygenation and compared for R- and S-phenibut.
2. Materials and Methods
2.1. Animals and Treatment. Forty-eight Swiss-Webster male
mice (25-40 g; Laboratory Animal Centre, University of
Tartu, Tartu, Estonia) were used in a lateral uid percussion
injury (latFPI) model of TBI [21, 22]. Additionally, 6 Swiss-
Webster male mice were used for the preparation of brain
homogenate and the isolation of brain mitochondria for
in vitro assays. Forty-two ICR male mice (Laboratory Animal
Breeding Facility, Riga Stradins University, Latvia) were used
in a pharmacokinetic study. All animals were housed under
standard conditions (21-23
°
C, 12 h light-dark cycle) with
unlimited access to standard food (Lactamin AB, Mjölby,
Sweden) and water in an individually ventilated cage housing
system (Allentown Inc., Allentown, New Jersey, USA). Each
cage contained bedding consisting of Eco-Pure Shavings
wood chips (Datesand, Cheshire, UK), nesting material,
and wooden blocks from TAPVEI (TAPVEI, Paekna, Esto-
nia). For enrichment, a transparent tinted (red) nontoxic
durable polycarbonate safe harbour mouse retreat (Ani-
maLab, Poznan, Poland) was used. The mice were housed
with up to 5 mice per standard cage (38 × 19 × 13 cm). All
studies involving animals were reported in accordance with
the ARRIVE guidelines [23, 24]. The experimental proce-
dures were performed in accordance with the guidelines
reported in the EU Directive 2010/63/EU and in accordance
with local laws and policies; all procedures were approved by
the Latvian Animal Protection Ethical Committee of Food
and Veterinary Service in Riga, Latvia.
The dose of R-phenibut was selected based on the previ-
ous studies, where pharmacological ecacy was observed in
dose-range between 10 and 50 mg/kg, while R-phenibut at
doses higher than 100 mg/kg showed sedative and coordina-
tion inhibitory eects [6, 8, 9]. Mice were randomly assigned
to four experimental groups: sham-operated mice, saline-
treated latFPI mice, and latFPI mice that received R-
phenibut (JSC Olainfarm, Olaine, Latvia) at a dose of
10 mg/kg or 50 mg/kg. Six mice were excluded because of a
dural breach that occurred during surgery (4 mice from the
sham-operated, 1 mouse from the control, and 1 mouse from
the R-phenibut 50 mg/kg groups), and four mice died imme-
diately after latFPI and were excluded from the study (3 mice
from the control and 1 mouse from the R-phenibut 50 mg/kg
groups). The nal number of included animals per group was
as follows: sham-operated mice (n=8), saline-treated latFPI
mice (control group, n=8), and latFPI mice that received
R-phenibut at a dose of 10 mg/kg (n=12) or 50 mg/kg
(n=10). R-Phenibut and saline were initially administered
intraperitoneally (i.p.) 2 h after injury and then once daily
for an additional 7 days for a total treatment period of 1 week.
During the treatment period, the animals were weighed at 0,
1, 2, 4, and 7 days after latFPI between 9:00 and 10:00 am. To
avoid the inuence of subjective factors on the rating process,
all experimental procedures were performed in a blinded
fashion.
2.2. Determination of R-Phenibut in the Plasma and Brain
Tissue after p.o. and i.p. Administration. The concentrations
of R-phenibut in the brain tissue extracts and plasma were
measured by ultraperformance liquid chromatography-
tandem mass spectrometry (UPLC/MS/MS). To determine
the concentration of R-phenibut in the plasma and brain,
mice received an i.p. and p.o. R-phenibut at a dose of
50 mg/kg 15 and 30 min and 1, 2, 4, 6, and 24 h (n=3in each
time point) before the plasma and brain tissue collection. The
blood and brain samples were prepared as described previ-
ously [25]. The chromatographic separation was performed
using an ACQUITY UPLC system (Waters, USA) on an
ACQUITY UPLC BEH Shield RP18 (1.7 μm, 2:1×50mm)
(Waters) with a gradient elution from 5 to 98% acetonitrile
in 0.1% formic acid aqueous solution at a ow rate of
0.15 ml/min. The analyte was ionized by electrospray ioniza-
tion in positive ion mode on a Quattro Micro triple quadru-
pole mass spectrometer (Waters). The mass spectrometer
was set up as follows: capillary voltage of 3.3 kV; source and
desolvation temperatures of 120 and 400
°
C, respectively.
Cone voltage was 20 V, and collision energy was 18 eV. R-
Phenibut analysis was performed in the MRM mode. Precur-
sor to production transition was m/zm/z180.0116.1. Data
acquisition and processing were performed using the Mas-
sLynx V4.1 and QuanLynxV4.1 software (Waters).
2.3. Lateral Fluid Percussion Injury-Induced Brain Trauma.
To induce TBI, the latFPI model was generated as previously
described [21, 22] with slight modications. Mice were
2 Oxidative Medicine and Cellular Longevity
anaesthetized with 4% isourane contained in a mixture of
oxygen and nitrous oxide (70 : 30, AGA, Riga, Latvia), and
anaesthesia with 2% isourane (Chemical Point, Deisenho-
fen, Germany) was maintained during the surgical proce-
dures using a face mask. The depth of anaesthesia was
monitored by a toe pinch using tweezers. Before trauma
induction, mice received subcutaneous (s.c.) administration
of tramadol (KRKA, Novo Mesto, Slovenia) (10 mg/kg). Eye
cream was applied to prevent the eyes from drying out. A
midline longitudinal scalp incision was made, and the skull
was exposed. A craniectomy that was centred at 2 mm poste-
rior to bregma and 2 mm right of midline was performed
using a 3 mm outer-diameter trephine. Any animal noted to
have a dural breach was euthanized and excluded from the
study. A plastic cap was attached over the craniotomy using
dental cement (Fulldent, Switzerland), and a moderate sever-
ity (1:5±0:2atm) brain injury was induced with a commer-
cially available uid percussion device (AmScien
Instruments, Richmond, USA). Immediately after the injury,
apnoea was noted, and when spontaneous breathing
returned, anaesthesia was resumed. The cement and cap were
removed, and the skin was sutured using resorbable sutures
(6-0, silk). The animal was placed in a separate cage to allow
full recovery from anaesthesia. Sham-injured animals were
subjected to an identical procedure as the latFPI animals
except for the induction of trauma.
2.4. Neurological Severity Score (NSS). The neurobehavioural
status of mice was obtained by the NSS using the method
described previously [26]. The animals were trained on the
NSS beams and equipment prior to the baseline measure-
ments. The general neurological state of mice was evaluated
at baseline (day before latfTBI) and 1, 4, and 7 days postin-
jury before the next dose of R-phenibut or saline administra-
tion. The NSS consisted of 9 individual clinical parameters,
including motor function, alertness, and physiological behav-
iour tasks. The mice were assessed for the following items:
presence of paresis; impairment of seeking behaviour;
absence of perceptible startle reex; inability to get down
from a rectangle platform (34 × 27 cm); inability to walk on
3, 2, and 1 cm wide beams; and inability to balance on a ver-
tical beam of 7 mm width and horizontal round stick of 5 mm
diameter for 10 sec. If a mouse showed impairment on one of
these items, a value of 1 was added to its NSS score. Thus,
higher scores on the NSS indicate greater neurological
impairment.
2.5. Tissue Preparation for Histological Analysis. The animals
used for histological analysis were randomly selected from
each group. Seven days after TBI, the mice were anaesthe-
tized using i.p. administration of ketamine (200 mg/kg) and
xylazine (15 mg/kg). The depth of anaest hesia was monitored
by a toe pinch using tweezers. Animals were transcardially
perfused at a rate of 3 ml/minutes with 0.01 M phosphate-
buered saline (PBS, pH = 7:4) for 5 minutes until the blood
was completely removed from the tissue. Perfusion was then
performed with 4% paraformaldehyde (PFA) xative solu-
tion for 5-7 minutes until stiening of the mouse body
occurred. After perfusion, the brains were carefully dissected
and postxed in 4% PFA overnight at 4
°
C. The brains were
cryoprotected with a 10-20-30% sucrose-PBS gradient for
72 hours. Coronal sections of the brain (20 μm) were made
using a Leica CM1850 cryostat (Leica Biosystems, Bualo
Grove, IL, United States) and mounted on Superfrost Plus
microscope slides (Thermo Scientic, Waltham, MA, United
States).
2.6. Cresyl Violet (Nissl) Staining and Interleukin-1beta (IL-
1β) Immunouorescence Staining. Nissl and IL-1βstaining
techniques were used to evaluate neuronal cell damage.
Nissl-stained dark neurons (N-DNs) indicated the typical
morphological change in injured neurons following TBI
[27, 28]. The number of N-DNs and cells expressing IL-1β
in the cerebral neocortex in the cortical impact area were
determined at day 7 after latFPI. For Nissl staining, coronal
frozen sections (20 μm) of the mouse brain were used. The
sections were incubated in graded ethanol solutions (96%
ethanol for 3 minutes and 70% ethanol for 3 minutes). After
washing with distilled water for 3 minutes, the sections were
stained with 0.01% cresyl violet acetate (ACROS organics)
solution for 14 minutes. The sections were then washed with
distilled water for 3 minutes and dehydrated in ethanol. The
stained sections were coverslipped using DPX mounting
medium (Sigma-Aldrich, St. Louis, MO, United States).
For IL-1βstaining, the sections were washed once with
PBS containing 0.2% Tween 20 for 5 minutes (on a rotary
shaker at 250 rpm). The antigen retrieval procedure was per-
formed with 0.05 M Na citrate (pH = 6:0) containing 0.05%
Tween 20 for 30 minutes at 85
°
C. The sections were then
washed with PBS (0.2% Tween 20) 3 times for 5 minutes
each. Protein blocking was performed using 5% BSA solu-
tion, and the sections were incubated for 1 hour at room tem-
perature. The sections were washed with PBS (0.2% Tween
20) 3 times for 5 minutes each. The slices were incubated
with primary antibody against anti-IL-1β(1 : 1000; Abcam,
Cat# ab9722) for 16 h at +4
°
C. The antibody was diluted in
PBS containing 3% BSA and 0.3% TritonX-100. After
incubation with the primary antibody, the sections were
washed with PBS (0.2% Tween 20) 4 times for 5 minutes
each. The sections were subsequently incubated for 1 h at
room temperature with goat anti-rabbit IgG H&L (Alexa
Fluor® 488, 1 : 200; Abcam, Cat# ab150077) diluted in PBS
containing 5% BSA. The sections were washed with PBS
(0.2% Tween 20) 4 times for 5 minutes each. The stained sec-
tions were mounted using Fluoromountaqueous mounting
medium (Sigma-Aldrich, St. Louis, MO, United States, Cat#
F4680) and nally coverslipped. Images were obtained with
a Nikon Eclipse TE300 microscope (Nikon Instruments,
Tokyo, Japan).
N-DNs were dened as hyperbasophilic neurons with a
shrunken morphology. The number of N-DNs per eld of
vision was calculated in three randomly selected sections at
the epicentre of the injury. The number of N-DNs and cells
expressing IL-1βper eld of vision were calculated using
ImageJ software at 10-fold magnication for N-DNs and at
4-fold magnication for IL-1β. For analysis of expression of
IL-1β, eight-bit images were generated from the pictures
and were cropped to contain the regions of interest. Images
3Oxidative Medicine and Cellular Longevity
for IL-1βstaining were thresholded to select a specic signal
over the background, and the stained area for each region
was calculated and used for statistical analysis. Three individ-
ual measurements were performed for each sample. The
schematic illustration of the brain region was created using
BioRender software (https://biorender.com).
2.7. Mitochondrial Respiration and H
2
O
2
Production
Measurements. To evaluate mitochondrial functionality,
mouse brain homogenate or isolated brain mitochondria
were prepared. Briey, brain tissues were homogenized
1 : 20 (w/v) in a medium containing 320 mM sucrose,
10 mM Tris, and 1 mM EDTA (pH 7.4). The homogenate
was centrifuged at 1000 g for 10 min, and the supernat ant
was centrifuged at 6200 g for 10 min. The mitochondrial pel-
let obtained was washed once and resuspended in the isola-
tion medium. Mitochondrial respiration and H
2
O
2
production measurements were performed at 37
°
C using
Oxygraph-2k (O2k; Oroboros Instruments, Austria) with
O2k-Fluo-Modules in MiR05Cr (110 mM sucrose, 60 1mM
K-lactobionate, 0.5 mM EGTA, 3 mM MgCl
2
, 20 mM tau-
rine, 10 mM KH
2
PO
4
, 20 mM HEPES, pH 7.1, 0.1% BSA
essentially fatty acid free, and creatine 20 mM). H
2
O
2
ux
(ROS ux) was measured simultaneously with respirometry
in the O2k-uorometer using the H
2
O
2
-sensitive probe
AmpliuRed (AmR) [29, 30]. 10 μM AmR, 1 U/ml horse
radish peroxidase (HRP), and 5 U/ml superoxide dismutase
(SOD) were added to the chamber. H
2
O
2
detection is based
on the conversion of AmR into the uorescent resorun. Cal-
ibrations were performed with H
2
O
2
added at 0.1 μM step.
H
2
O
2
ux was corrected for background (AmR slope before
addition of sample). H
2
O
2
/O ux ratio (%) was calculated
as H
2
O
2
ux/(0.5 O
2
ux).
2.8. In Vitro Anoxia-Reoxygenation Model. Mitochondrial
functionality after anoxia-reoxygenation was determined in
mouse brain tissue homogenate prepared as described previ-
ously [31]. To induce anoxia maximal respiration rate, the
sample was stimulated by the addition of substrates,
pyruvate + malate (5+2mM), succinate (10 mM), and ADP
(5 mM), and preparation was left to consume all O
2
in the
respiratory chamber (within 10-20 min), thereby entering
into an anoxic state [32]. 15 minutes after anoxia, the vehicle
or R-phenibut (0.5 μg/ml) was added to the chamber and O
2
was reintroduced to the chamber by opening the chamber to
achieve reoxygenation. After 8 minutes of reoxygenation, the
chamber was closed and O
2
ux was monitored for addi-
tional 2 minutes. At the end of the experiment, antimycin
A (2.5 μM) was added to determine residual oxygen con-
sumption (ROX).
2.9. Substrate-Uncoupler-Inhibitor Titration (SUIT) Protocol.
To determine the eect of R-phenibut on mitochondrial elec-
tron transfer system functionality, mitochondria were iso-
lated from mouse brain as described previously, and
mitochondrial respiration and H
2
O
2
production measure-
ments were performed in the presence or absence of R-
phenibut at 0.5 μg/ml concentration [30]. In addition, eects
of S-phenibut (0.5 μg/ml) were tested to determine whether
the eects of R-phenibut in mitochondria involve the
GABA-B receptor or the α
2
δsubunit of VDCC. Pyruvate
and malate (5 mM and 2 mM, respectively) were used to
determine N-pathway complex I (CI) linked LEAK (L) respi-
ration. ADP was added at 5 mM concentration to determine
oxidative phosphorylation-dependent respiration (OXPHOS
state, P). Then, glutamate (10 mM) was added as an addi-
tional substrate for N-pathway. Succinate (10 mM, complex
II (CII) substrate) was added to reconstitute convergent
NS-pathway CI&II-linked respiration. Titrations with the
uncoupler CCCP (0.51μM steps) were performed to deter-
mine the electron transfer system (ETS) capacity. Rotenone
(0.5 μM, inhibitor of complex I) was added to determine
the CII-linked OXPHOS capacity. Then, antimycin A
(2.5 μM, inhibitor of complex III) was added to evaluate
residual (non-mitochondrial) oxygen consumption (ROX).
Oxygen uxes were compared after correction for ROX.
2.10. Ca
2+
-Induced Mitochondrial Swelling Measurement.
Swelling of isolated brain mitochondria was assessed by mea-
suring changes in absorbance at 540 nm as described previ-
ously with slight modications [3335]. Mitochondria
(0.125 mg/ml) were preincubated with R- or S- phenibut at
a concentration of 0.5 μg/ml for 15 min in a buer containing
120 mM KCl, 10 mM Tris, 5mM KH
2
PO
4
pH 7.4, and pyru-
vate (5 mM), malate (2 mM), and ADP (5 mM) as substrates.
R- and S-enantiomers of phenibut were used to determine
whether the eects of R-phenibut on Ca
2+
-induced mito-
chondrial swelling involve the GABA-B receptor or the α
2
δ
subunit of VDCC. Swelling was induced by the addition of
200 μM CaCl
2
, and changes in absorbance were monitored
for 10 min. All experiments were performed at 37
°
C.
2.11. Statistical Analysis. All results are expressed as the
mean ± S:E:Mor S.D. (for mitochondrial studies). Health
outcomes, animal behaviour, and Ca
2+
-induced mitochon-
drial swelling were analysed using two-way repeated-
measures analysis of variance (ANOVA). Dunnetts post
hoc test was performed when appropriate. The histological
data and mitochondrial functionality were evaluated by
one-way ANOVA. Whenever the analysis of variance indi-
cated a signicant dierence, further multiple comparisons
were made using Tukeys multiple comparison test as the
post hoc test. pvalues less than 0.05 were considered to be
signicant. The statistical calculations were performed using
the GraphPad Prism software package (GraphPad Software,
Inc., La Jolla, California, USA).
The sample size calculations for latFPI-induced brain
trauma were based on the eects of R-phenibut in our previ-
ous experiments. For example, it was calculated that R-
phenibut demonstrates a medium eect in the ET-1-
induced middle cerebral artery occlusion model [9] and a
large eect in the formalin-induced paw-licking test [6].
Through a power calculation (using G-power software) for
a two-way ANOVA test (repeated measures), four-group
comparison, four measurements per group (0, 1, 4, and 7
days after TBI) with α=0:05, a power of 80%, and a stan-
dardized eect size Cohensf=0:5, a total sample size of 8
mice per group was deemed sucient. Since TBI-induced
4 Oxidative Medicine and Cellular Longevity
brain trauma can result in death of some animals, our sample
size of n=12 would allow identifying smaller dierences,
with the same statistical power, for the same signicance
level.
3. Results
3.1. R-Phenibut Crosses the Blood-Brain Barrier. As shown in
Figure 1(a), R-phenibut in plasma could be detected 15 min
after a single i.p. and p.o. injection. The maximal concentra-
tions of R-phenibut in the plasma were observed 15 min after
the i.p. injection and 30 min after the p.o. administration
(Figure 1(a)). The maximal concentration of R-phenibut in
the plasma after the i.p. injection was 16.8 μg/ml; at the same
time, the maximal concentration of R-phenibut in the plasma
after the p.o. injection was 24 μg/ml (see Figure 1(a)). R-
Phenibut in the plasma was not detected 24 h after both the
i.p. and p.o. injections. R-Phenibut in the brain tissue extracts
was detected already 15 min after a single i.p. and p.o. injec-
tion (Figure 1(b)). The maximal concentrations of R-
phenibut in the brain tissues were 0.64 μg/g and 0.17 μg/g tis-
sue after the i.p. and p.o. injections, respectively (Figure 1(a)).
The maximal concentrations of R-phenibut in the brain tis-
sues were observed 15 min after i.p. injection and 60 till
240 min after p.o. administration. 24 h after both the i.p.
and p.o. injections, R-phenibut in the brain tissues was
0.02 μg/g and 0.012 μg/g, respectively.
3.2. Health Outcome Monitoring after latFPI. The body
weight of the sham group animals was not decreased at 1,
2, 4, and 7 days after TBI. A two-way repeated-measures
ANOVA showed a signicant interaction between time and
treatment (Fð12,118Þ=4:6,p<0:0001) and main eects of
time (Fð1:4,42:7Þ=25:7,p<0:0001) and treatment
(Fð3,34Þ=6:7,p=0:0011). The control group animals lost sig-
nicantly more weight after TBI than the sham-operated
group animals (p<0:05). Treatment with R-phenibut at both
doses had no eect on weight loss compared to weight loss in
the control group (Figure 2).
3.3. R-Phenibut Treatment Improved Neurological Status
after TBI. TBI induced signicant functional decits in control
mice compared with sham-operated mice (p<0:0001). The
average NSS in the control group was 6:1±0:4,5:3±0:3,
and 5:0±0:6on postinjury days 1, 4, and 7, respectively.
The average NSS score between baseline value and the rst
day postcraniotomy in the sham-operated group was signi-
cantly higher (p<0:01). There was a signicant time ×
treatment interaction observed between groups (two-way
repeated-measures ANOVA: (Fð9,102Þ=5:7,p<0:0001)for
time × treatment interaction;(Fð3,34Þ=22:2,p<0:0001)for
treatment; (Fð2:7,92Þ=161:8,p<0:0001) for time; Figure 3).
R-Phenibut treatment at a dose of 50mg/kg signicantly ame-
liorated functional decits by 28%, 25%, and 30% after TBI on
postinjury days 1, 4, and 7, respectively (p<0:05;Figure3).
3.4. R-Phenibut Treatment Reduced Early Neuronal Cell
Death and Neuroinammation in the Brain Cortex after
TBI. To assess histopathological changes in the ipsilateral
brain site, N-DNs and cells expressing IL-1β(Figure 4) were
quantied in the sham-operated, control, and R-phenibut
treatment groups 7 days after TBI. N-DNs and IL-1β-
expressing cells were found in the ipsilateral hemisphere of
control group animals (Figures 4(a) and 4(b)). Histological
analysis showed that R-phenibut treatment at a dose of
50 mg/kg signicantly reduced the number of N-DNs and
cells expressing IL-1βin the neocortex after TBI (p<0:05).
Signicant dierences were found in the N-DNs and IL-1β-
positive cell numbers in the ipsilateral cortex around the
lesion site between the control group (9:1±6:4/per eld of
vision for N-DNs and 379 ± 82/per eld of vision for IL-
1β-expressing cells) and the R-phenibut treatment group at
a dose of 50 mg/kg (3:0±1:9/per eld of vision for N-DNs
and 246 ± 31/per eld of vision for IL-1β-expressing cells; p
<0:05; Figures 4(d) and 4(e)). There was no statistically sig-
nicant dierence between the control group and the R-
phenibut treatm ent group at the dose of 10 mg/kg. No N-
DNs were observed in the sham-operated mice.
3.5. R-Phenibut Protects Brain Mitochondria against Anoxia-
Reoxygenation Damage. To determine whether R-phenibut-
induced neuroprotection could be a result of the preservation
of mitochondrial functionality, ROS production and the
mitochondrial respiration rate were assessed after anoxia-
reoxygenation in vitro. To better mimic the conditions
observed in vivo, R-phenibut at the concentration of
0.5 μg/ml was added to the chamber immediately before
reoxygenation. Anoxia-reoxygenation induced 33% and
59% increases in the H
2
O
2
production rate and the H
2
O
2
/O
ratio, respectively (Figure 5). R-Phenibut treatment signi-
cantly decreased the anoxia-reoxygenation-induced increase
in the H
2
O
2
production rate and the H
2
O
2
/O ratio
(p<0:05).
3.6. R-Phenibut Reduces ROS Production and Attenuates
Ca
2+
-Induced Mitochondrial Swelling. To determine whether
the protective eect of R-phenibut is related to its direct
action on mitochondria, measurements of mitochondrial res-
piration, ROS production, and Ca
2+
-induced swelling were
performed in isolated mouse brain mitochondria in the pres-
ence or absence of the compounds. As seen in Figure 6, R-
phenibut and S-phenibut at 0.5 μg/ml did not induce any
changes in the mitochondrial respiration rate (Figure 6(a)),
while H
2
O
2
production and the H
2
O
2
/O ratio (Figures 6(b)
and 6(c)) were signicantly decreased by 31-53% in the
LEAK and OXPHOS states. These results show that R-
phenibut and S-phenibut reduce ROS production without
aecting the mitochondrial electron transfer system capacities,
indicating the improvement of mitochondrial coupling. In addi-
tion, both R- and S-phenibut attenuated calcium-induced brain
mitochondrial swelling (two-way repeated-measures ANOVA:
main eect of treatment (Fð40,360Þ=4:576,p<0:0001), time
(Fð2:611,46:99Þ=104:5,p<0:0001), and interaction between treat-
ment and time (Fð40,360 Þ=4:576,p<0:0001); Figure 6(d)).
Thus, the phenibut treatment-induced protection of
mitochondria against anoxia-reoxygenation could be due to
a reduction in ROS production and the modulation of Ca
2+
signalling.
5Oxidative Medicine and Cellular Longevity
4. Discussion
In the current study, we examined the eects of R-phenibut
treatment on brain trauma induced by latFPI. For the rst
time, we showed that R-phenibut could be detected in the
mouse brain 15 min after a single p.o. or i.p. injection and
found in brain extracts even 24 h after the administration.
The present study conrms that R-phenibut, which is an
antagonist of the α
2
δsubunit of VDCC and an agonist of
GABA-B receptors, improves sensorimotor functional out-
comes and signicantly ameliorates brain damage and neu-
ronal death in the acute phase after TBI via mechanisms
related to Ca
2+
homeostasis and oxidative stress.
The binding characteristics of R-phenibut were previ-
ously investigated using radiolabeled gabapentin that was
the rst ligand shown to bind to the α
2
δ
1
and α
2
δ
2
subunits
with high anity (K
d
= 59 and 153 nM, respectively), while
at the same time demonstrating no binding activity to the
α
2
δ
3
and α
2
δ
4
subunits [36, 37]. The pathologies associated
with gene disruption of α
2
δ
1
protein include neuropathic
pain and cardiac dysfunction, while in case of α
2
δ
2
protein,
the pathologies are related to epilepsy and cerebellar ataxia
[38]. We showed previously that pharmacological activity
of R-phenibut is associated with neuropathic pain rather
than epilepsy [6]; thus, we could speculate that the eects of
R-phenibut are α
2
δ
1
protein binding-related.
The α
2
δsubunits of VDCC are widely expressed by excit-
atory neurons in the cerebral cortex, hippocampus, and other
brain regions [39, 40]. Furthermore, the α
2
δsubunits of
VDCC have been shown to be involved in processes that
are not directly linked to calcium channel function, such as
synaptogenesis [41]. Other studies have reported that the
administration of VDCC ligands in rodent models of TBI
0
4
8
12
16
20
0 60 120 180 240 300 360
Time aer administration (min)
Plasma
R-Phenibut 50 mg/kg, i.p.
R-Phenibut 50 mg/kg, p.o.
Concentration of R-phenibut
(𝜇g/ml)
(a)
R-Phenibut 50 mg/kg, i.p.
R-Phenibut 50 mg/kg, p.o.
0.0
0.2
0.4
0.6
0.8
0 60 120 180 240 300 360
Time aer administration (min)
Brain
Concentration of R-phenibut in
the brain (𝜇g/g tissue)
(b)
Figure 1: The concentration of R-phenibut in the mouse plasma and brain tissue after a single administration. Mice received an i.p. and p.o.
injection of R-phenibut at a dose of 50 mg/kg. The amount of compound in the plasma (a) and brain tissue extracts (b) was measured 15 and
30 min and 1, 2, 4, and 6 h after R-phenibut administration (n=3). Values are represented as the mean ± S:E:M:.
0
1
2
3
4
5
6
7
8
9
NSS
#
0147
Days aer TBI
Sham
R-Phenibut 10
Control
R-Phenibut 50
Figure 3: Eects of R-phenibut on the neurological severity score
(NSS) after TBI. R-Phenibut and saline were initially administered
i.p. 2 h after injury and then once daily for an additional 7 days
for a total treatment period of 1 week. Data are shown as the
mean ± S:E:M:(n=812). Indicates a signicant dierence
compared to the control group;
#
indicates a signicant dierence
compared to the sham-operated group (two-way repeated-
measures ANOVA followed by Dunnetts multiple comparison
test; P<0:05;
#
P<0:01).
80
90
100
110
01247
Days aer TBI
Sham
R-Phenibut, 10 mg/kg
Body weight change (%)
Control
R-Phenibut, 50 mg/kg
Figure 2: Body weight changes of the sham-operated, control, and R-
phenibut treatment groups. Mice were weighed beforeand 1, 2, 4, and
7 days after latFPI. Data are expressed as the percentage change in
body weight relative to the initial body weight of each animal (%).
Data are shown as the mean ± S:E:M:(n=812). Indicates a
signicant dierence compared to the sham-operated group (two-
way repeated-measures ANOVA followed by Dunnettsmultiple
comparison test; P<0:05).
6 Oxidative Medicine and Cellular Longevity
Sham Control R-Phenibut 10 mg/kg R-Phenibut 50 mg/kg
(a)
(b)
TBI site
(c)
#
0
5
10
15
Sham Control R-Phenibut
10 mg/kg
R-Phenibut
50 mg/kg
Nissl-stained dark neurons,
count per vision eld
(d)
#
0
100
200
300
400
500
Sham Control R-Phenibut
10 mg/kg
R-Phenibut
50 mg/kg
IL-1𝛽+ cells,
count per vision eld
(e)
Figure 4: Cresyl violet (Nissl) and IL-1βimmunouorescence staining 7 days post-TBI. (a) Cresyl violet-stained sections of the mouse
neocortex ipsilateral to the injury site. R-Phenibut treatment at doses of 10 mg/kg and 50 mg/kg reduced the number of N-DNs. Scale bar
= 100 μm. (b) IL-1βexpression based on immunouorescence staining in the mouse neocortex ipsilateral to the injury site. R-Phenibut
treatment at doses of 10 mg/kg and 50 mg/kg reduced the number of IL-1β-positive cells. Scale bar = 250 μm. (c) Schematic illustration of
the brain region indicated in the lled area, which was selected for the quantitative analysis of cell injury. (d) Quantitative assessment of
N-DNs in the ipsilateral cortex at postinjury day 7. Data are expressed as the mean ± S:E:M:(n=7 for the R-phenibut 50 mg/kg group
and n = 6 for the sham, control, and R-phenibut 10 mg/kg groups). (e) Quantitative assessment of IL-1β-positive cells in the ipsilateral
cortex at postinjury day 7. Data are expressed as the mean ± S:E:M:(n=4 for the control group and n=3 for the sham, R-phenibut 10
mg/kg, and 50 mg/kg groups).
#
Indicates a signicant dierence compared to the sham-operated group; indicates a signicant dierence
compared to the control group (one-way ANOVA followed by Tukeys multiple comparison test; P<0:05).
7Oxidative Medicine and Cellular Longevity
reduced cell death and improved cognitive function [40].
Similar to phenibut, ligands of the α
2
δsubunit of VDCC,
such as pregabalin, at a high dose of 60 mg/kg reduce neuro-
nal loss and improve functional outcomes 24 h after trauma
in experimental models of TBI [41, 42]. Moreover, pregabalin
at a dose of 30 mg/kg has been shown to improve functional
recovery and to demonstrate anti-inammatory and antia-
poptotic eects in a rat model of spinal cord injury [43, 44].
Cytoskeletal protein loss results in altered neuronal mor-
phology after TBI [45, 46]. N-DNs represent a typical patho-
morphological change in injured neurons after TBI, showing
abnormal basophilia and shrinkage [27, 28]. N-DNs appear
in the neocortex immediately after TBI and can be observed
even two weeks postinjury [27, 47]. In addition, IL-1 is a major
driver of the secondary neuronal injury cascade after TBI [48].
It is involved in the recruitment of other types of immune
cells, neuronal apoptosis, and blood-brain barrier disruption
after TBI [4951]. Furthermore, IL-1βantagonism was shown
to be neuroprotective in clinical trials and in rodent models of
TBI [5254]. The present study shows that treatment with R-
phenibut at a dose of 50 mg/kg signicantly reduced the num-
ber of N-DNs and signicantly reduced IL-1βexpression in
the neocortex after TBI. The histopathological ndings of the
current study revealed that R-phenibut could attenuate neuro-
nal damage, inammation, and degeneration.
For the rst time, we showed that R-phenibut limits
mitochondrial dysfunction in the brain induced by anoxia-
reoxygenation. Compared with other types of cells, neurons
are endowed with less robust antioxidant defence systems
[55]. As mitochondrial dysfunction has been shown to be
involved in TBI, perturbations in energy metabolism are
likely to contribute to the pathogenesis of TBI [56, 57]. In
TBI, oxidative cell damage is caused by an imbalance
between the production and accumulation of ROS, in which
mitochondria are the major intracellular source of ROS.
Accordingly, there is accumulating evidence that antioxidant
agents and membrane lipid peroxidation inhibitors, such as
tirilazad, U-78517F and U-83836E, are eective in treating
preclinical models of TBI [17]. Mitochondrial-targeted
drugs, such as mitoquinone and thymoquinone-containing
antioxidants, have been shown to decrease neurological de-
cits and β-amyloid-induced neurotoxicity after TBI [58, 59].
Meanwhile, the inhibition of ROS production has been
shown to inhibit secretion of IL-1β[60].
Notably, the immunosuppressant drug cyclosporine A,
which is an IL-1βreceptor antagonist, has been shown to
decrease pathological changes in the brain after TBI by blocking
the mitochondrial permeability transition pore [61]. Our results
indicate that R-phenibut treatment improves mitochondrial
tolerance and thus protects brain energetics against anoxia-
reoxygenation damage by reducing ROS production. R-
Phenibut treatment reduces ROS production without aecting
the mitochondrial electron transfer system capacities, indicating
the improvement of mitochondrial coupling. Another study has
demonstrated that phenibut has neuroprotective eects in vitro
but does not possess antioxidant potential [62]. Perlova et al.
recently showed that phenibut can limit heart and brain mito-
chondrial damage in rats exposed to stress [63].
To determine the molecular mechanisms underlying the
actions of R-phenibut against anoxia-reoxygenation-
induced mitochondrial damage, the activity of the R- and
S-enantiomers of racemic phenibut was compared. We found
that both R-phenibut and S-phenibut reduced mitochondrial
ROS production and inhibited Ca
2+
-induced mitochondrial
swelling. This suggests that the protective eects of R-
phenibut in mitochondria do not involve the GABA-B recep-
tor (in contrast to R-phenibut, S-phenibut does not bind to
the GABA-B receptor) and might be mediated by the α
2
δ
1
subunit of VDCC. It was shown previously that increased
intracellular Ca
2+
, as a result of increased activity of α
2
δ
1
,
could be rapidly taken up by mitochondria and subsequently
released into the cytoplasm avoiding Ca
2+
accumulation and
maintaining intracellular Ca
2+
signalling [19]. This could
explain why, in the presence of R- and S-phenibut, reduced
Ca
2+
-induced mitochondrial swelling was observed. Both
R-phenibut and S-phenibut demonstrate mitochondrial-
#
0
0.02
0.04
0.06
Normoxia Control R -Phenibut
Anoxia-reoxygenation
H2O2 prod.rate (pmol/(smg))
(a)
#
0
0.05
0.1
Normoxia Control R-Phenibut
H2O2/O (%)
Anoxia-reoxygenation
(b)
Figure 5: The eects of R-phenibut (0.5 μg/ml) on ROS production in an in vitro anoxia-reoxygenation model. After anoxia-reoxygenation,
the H
2
O
2
production rate (a) and H
2
O
2
/O ratio (b) were signicantly decreased in the R-phenibut group. The results are presented as the
mean ± S:D:of 6 independent replicates. Indicates a signicant dierence compared to normoxia;
#
indicates a signicant dierence
compared to the anoxia-reoxygenation control group (one-way ANOVA followed by Tukeys multiple comparison test; P<0:05).
8 Oxidative Medicine and Cellular Longevity
protective properties against anoxia-reoxygenation and Ca
2+
-
induced stress. Since there is no evidence of α
2
δlocalization in
the mitochondrial membrane, it is possible that compounds
could alter Ca
2+
signalling pathways and protect mitochondria
by targeting mitochondrial-specic or mitochondrial-
endoplasmatic reticulum-associated Ca
2+
transporters.
Our study has several limitations. One of the limitations
of this study is that the level of ROS in mouse brain after
treatment of R-phenibut following TBI was not measured.
Another limitation is the increase in the NSS score between
the baseline value and the rst day postcraniotomy in the
sham-operated group. The increase of the NSS score in
sham-operated mice was reported previously and can be
related to the distinct injury caused by craniotomy proce-
dures [64]. Similar to other studies, the NSS score of injured
mice showed maximum decits on postinjury day 1 and
remained elevated at 1, 2, 4, and 7 days after latFPI [64,
65]. A potential limitation of this study is that only male mice
were used in experiments.
5. Conclusions
In conclusion, R-phenibut treatment reduces TBI-induced neu-
ronal death and improves functional recovery, suggesting its
therapeutic potential. The present study suggests that the neu-
roprotective properties of phenibut may be mediated by its
eects on mitochondrial calcium inux and ROS generation.
Data Availability
The data used to support the ndings of this study are avail-
able from the corresponding author upon request.
0
40
80
120
160
D G S U Rot
CI CI&II CI&II CII
PM
CI
LEAK OXPHOS ET
Resp.rate (pmol O2/(sml))
Vehicle
R-Phenibut 0.5 𝜇g/ml
S-Phenibut 0.5 𝜇g/ml
(a)
0
0.15
0.3
0.45
PM D G S U Rot
CI CI CI&II CI&II CII
LEAK OXPHOS ET
Vehicle
R-Phenibut 0.5 𝜇g/ml
S-Phenibut 0.5 𝜇g/ml
H2O2 prod.rate (pmol/(sml))
⁎⁎
(b)
0
1
2
3
4
5
6
7
PM D G S U Rot
CI CI CI&II CI&II CII
LEAK OXPHOS ET
Vehicle
R-Phenibut 0.5 𝜇g/ml
S-Phenibut 0.5 𝜇g/ml
H2O2/O2 (%)
⁎⁎
(c)
–0.05
–0.04
–0.03
–0.02
–0.01
0
0.01
0 120 240 360 480 600
Time (s)
Changes in absorbance, ΔA540
Vehicle
R-Phenibut 0.5 𝜇g/ml
S-Phenibut 0.5 𝜇g/ml
(d)
Figure 6: The eects of R-phenibut and S-phenibut (0.5 μg/ml) on mitochondrial functionality and Ca
2+
-induced swelling in isolated mouse
brain mitochondria. R-Phenibut and S-phenibut did not aect the mitochondrial respiration rate (a) but signicantly decreased the H
2
O
2
production rate (b) and H
2
O
2
/O ratio (c). The results are presented as the mean ± S:D:of 5 independent measurements. P: pyruvate; M:
malate; D: ADP; G: glutamate; S: succinate; U: uncoupler; Rot: rotenone; CI: complex I; CII: complex II; LEAK: substrate metabolism-
dependent state; OXPHOS: oxidative phosphorylation-dependent state; ET: electron transfer capacity state. Indicates a signicant
dierence compared to the control group (one-way ANOVA followed by Tukeys multiple comparison test, P<0:05). Both R-phenibut
and S-phenibut at a concentration of 0.5 μg/ml signicantly attenuated Ca
2+
-induced swelling (d). The results are presented as the mean
±S:D:of 7 independent replicates. Indicates a signicant dierence compared to the control group (two-way repeated-measures
ANOVA followed by Dunnetts multiple comparison test; P<0:05).
9Oxidative Medicine and Cellular Longevity
Conflicts of Interest
The authors declare that there is no conict of interest
regarding the publication of this paper.
Acknowledgments
This study was supported by the framework of the EU-ERA-
NET NEURON projects TRAINS and CnsAame.
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... Nissl staining was performed to assess neuronal cell damage by measuring dark neurons (Kupats et al., 2020). The frozen sections of 28 days post-injury were incubated with 1% cresyl violet (Beyotime, Shanghai, China) according to the manufacturer's protocol. ...
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... After being perfused transcardially with ice-cold 0.1 M phosphate-buffered saline (PBS) and post fixed with 4% paraformaldehyde (PFA) [20], 4-µm thick coronal sections were used for Nissl staining [21], and 15-µm coronal sections were prepared for immunohistochemistry. Subsequently, sections were blocked in 10% normal goat serum (30 min) and incubated with primary antibodies: anti-Iba-1 (Abcam, Ab178847, 1:400) at 4 • C overnight. The secondary antibody was allowed to react with the sections. ...
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Traumatic brain injury (TBI) and long bone fracture are common in polytrauma. This injury combination in mice results in elevated levels of the pro-inflammatory cytokine interleukin-1β (IL-1β) and exacerbated neuropathology when compared to isolated-TBI. Here we examined the effect of treatment with an IL-1 receptor antagonist (IL-1ra) in mice given a TBI and a concomitant tibial fracture (i.e., polytrauma). Adult male C57BL/6 mice were given sham-injuries or polytrauma and treated with saline-vehicle or IL-1ra (100 mg/kg). Treatments were subcutaneously injected at 1, 6, and 24 hours, and then once daily for one week post-injury. 7-8 mice/group were euthanized at 48 hours post-injury. 12-16 mice/group underwent behavioral testing at 12 weeks post-injury and MRI at 14 weeks post-injury before being euthanized at 16 weeks post-injury. At 48 hours post-injury, markers for activated microglia and astrocytes, as well as neutrophils and edema, were decreased in polytrauma mice treated with IL-1ra compared to polytrauma mice treated with vehicle. At 14 weeks post-injury, MRI analysis demonstrated that IL-1ra treatment after polytrauma reduced volumetric loss in the injured cortex and mitigated track-weighted MRI markers for axonal injury. As IL-1ra (Anakinra) is approved for human use, it may represent a promising therapy in polytrauma cases involving TBI and fracture.
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Increased oxygen consumption by heart and brain mitochondria in the absence of ADP and reduced mitochondrial respiration in the presence of ADP were observed in rats exposed to stress simulated by suspension by the dorsal neck skin fold for 24 h, which attests to uncoupling of substrate oxidation and ATP synthesis and can cause electron drain from the respiratory chain, formation of ROS, and oxidative damage to cell structures. Blockade of inducible NO synthase with aminoguanidine (single intraperitoneal dose of 50 mg/kg before stress exposure) increased coupling of respiration and oxidative phosphorylation in heart and brain mitochondria of rats exposed to immobilization-painful stress, which was especially pronounced in cardiomyocytes. The test compounds glufimet (single intraperitoneal dose of 29 mg/kg before stress exposure) and phenibut (single intraperitoneal dose of 50 mg/kg before stress exposure) limited stress-induced mitochondrial damage against the background of inducible NO synthase blockade and without it, which was seen from increased respiratory control ratio in comparison with that in untreated rats exposed to stress (control).