Neuroprotective effect of the recombinant human leukemia inhibitory factor in mice with an experimental cuprizone model of multiple sclerosis: possible mechanisms
Aim. To investigate the effects of the rhLIF recombinant human leukemia inhibitory factor on the structural and functional changes in the central nervous system in the cuprizone-induced experimental model of multiple sclerosis in mice and to evaluate the involvement of the brain macrophages, T-lymph...
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| Zitieren: | Neuroprotective effect of the recombinant human leukemia inhibitory factor in mice with an experimental cuprizone model of multiple sclerosis: possible mechanisms / I.F. Labunets, A.E.. Rodnichenko, N.O Melnyk, S.E. Rymar, N.A. Utko, G.O. Gavrulyk-Skyba, G.M. Butenko // Вiopolymers and Cell. — 2018. — Т. 34, № 5. — С. 350-360. — Бібліогр.: 36 назв. — англ. |
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Labunets, I.F. Rodnichenko, A.E. Melnyk, N.O Rymar, S.E Utko, N.A. Gavrulyk-Skyba, G.O. Butenko, G.M. 2019-06-15T14:30:25Z 2019-06-15T14:30:25Z 2018 Neuroprotective effect of the recombinant human leukemia inhibitory factor in mice with an experimental cuprizone model of multiple sclerosis: possible mechanisms / I.F. Labunets, A.E.. Rodnichenko, N.O Melnyk, S.E. Rymar, N.A. Utko, G.O. Gavrulyk-Skyba, G.M. Butenko // Вiopolymers and Cell. — 2018. — Т. 34, № 5. — С. 350-360. — Бібліогр.: 36 назв. — англ. 0233-7657 DOI: http://dx.doi.org/10.7124/bc.000989 https://nasplib.isofts.kiev.ua/handle/123456789/154362 612.017.1:616.8-003.8.001.53 Aim. To investigate the effects of the rhLIF recombinant human leukemia inhibitory factor on the structural and functional changes in the central nervous system in the cuprizone-induced experimental model of multiple sclerosis in mice and to evaluate the involvement of the brain macrophages, T-lymphocytes and antioxidant enzymes in the observed rhLIF effects. Methods. Young 129/Sv mice were fed with the cuprizone neurotoxin daily for three weeks. rhLIF was injected daily (50 μg/kg) after seven days of the cuprizone treatment. We used histology, flow cytometry, spectrophotometric and functional methods. Results. The cuprizone treatment resulted in an increase in the number of the brain and spinal cord neurons with destructive changes, the brain macrophages, CD3⁺-cells and the content of malondialdehyde; it changes the behavior of animals, decreases the brain superoxide dismutase and glutathione peroxidase activities. The rhLIF injection partially or completely restored the cuprizone-changed parameters. The effect of rhLIF was preserved during two months after the experiment completion. Conclusions. The neuroprotective effect of rhLIF is connected with a decrease in the cuprizone-induced changes in the number of the brain T-cells and macrophages and the activity of antioxidant enzymes . Мета. Дослідити вплив рекомбінантного лейкемія інгібіторного фактору людини (rhLIF) на структурні та функціональні зміни ЦНС в експериментальній моделі розсіяного склерозу, індукованого у мишей купризоном; оцінити участь макрофагів, Т-лімфоцитів та антиоксидантних ферментів головного мозку в реалізації його проявів. Методи. Молоді миші лінії 129/Sv отримували нейротоксин купризон із їжею щодня протягом 3-х тижнів, rhLIF – з 8-ї доби прийому купризону щодня у дозі 50 мкг/кг. Методи: гістологічний, проточної цитометрії, спектрофотометричний і функціональний. Результати. Під впливом купризону в головному та спинному мозку мишей збільшується кількість нейронів з деструктивними змінами; в головному мозку зростає кількість макрофагів, СD3⁺-клітин і вміст малонового діальдегіду; знижується активність супероксидддисмутази, глютатіонпероксидази у головному мозку та змінюється поведінка тварин. Ін'єкції rhLIF частково або повністю відновлюють показники, змінені купризоном. Ефект цитокіна зберігається через 2 міс після завершення експерименту. Висновки. Нейропротекторний ефект rhLIF пов’язаний з пригніченням індукованих купризоном змін в активності антиоксидантних ферментів, кількості Т-клітин і макрофагів у головному мозку. Цель. Исследовать влияние рекомбинантного лейкемия- ингибирующего фактора человека (rhLIF) на структурные и функциональные изменения ЦНС в экспериментальной модели рассеянного склероза, индуцированного у мышей купризоном; оценить участие макрофагов, Т-лимфоцитов и антиоксидантных ферментов головного мозга в реализации его проявлений. Методы. Молодые мыши линии 129/Sv получали нейротоксин купризон с пищей ежедневно в течение 3-х недель, rhLIF – с 8-го дня приема купризона ежедневно в дозе 50 мкг/кг. Методы: гистологический, проточной цитометрии, спектрофотометрический и функциональный. Результаты. Прием купризона вызывает увеличение числа нейронов с деструктивными изменениями в головном и спинном мозге, количества макрофагов, СD3⁺-клеток и содержания малонового диальдегида в головном мозге; наблюдается снижение активности супероксиддисмутазы, глютатионпероксидазы в головном мозге и изменение поведения животных. Инъекции rhLIF частично или полностью восстанавливают измененные купризоном показатели. Эффект цитокина сохраняется через 2 мес после завершения эксперимента. Выводы. Нейропротекторный эффект rhLIF связан с подавлением индуцированных купризоном изменений в активности антиоксидантных ферментов, количестве Т-клеток и макрофагов в головном мозге. en Інститут молекулярної біології і генетики НАН України Вiopolymers and Cell Structure and Function of Biopolymers Neuroprotective effect of the recombinant human leukemia inhibitory factor in mice with an experimental cuprizone model of multiple sclerosis: possible mechanisms Нейропротекторний ефект рекомбінантного лейкемія-інгібіторного фактору людини у мишей з експериментальною купризоновою моделлю розсіяного склерозу: можливі механізми Нейропротекторный эффект рекомбинантного лейкемия-ингибирующего фактора человека у мышей с экспериментальной купризоновой моделью рассеянного склероза: возможные механизмы Article published earlier |
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Digital Library of Periodicals of National Academy of Sciences of Ukraine |
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DSpace DC |
| title |
Neuroprotective effect of the recombinant human leukemia inhibitory factor in mice with an experimental cuprizone model of multiple sclerosis: possible mechanisms |
| spellingShingle |
Neuroprotective effect of the recombinant human leukemia inhibitory factor in mice with an experimental cuprizone model of multiple sclerosis: possible mechanisms Labunets, I.F. Rodnichenko, A.E. Melnyk, N.O Rymar, S.E Utko, N.A. Gavrulyk-Skyba, G.O. Butenko, G.M. Structure and Function of Biopolymers |
| title_short |
Neuroprotective effect of the recombinant human leukemia inhibitory factor in mice with an experimental cuprizone model of multiple sclerosis: possible mechanisms |
| title_full |
Neuroprotective effect of the recombinant human leukemia inhibitory factor in mice with an experimental cuprizone model of multiple sclerosis: possible mechanisms |
| title_fullStr |
Neuroprotective effect of the recombinant human leukemia inhibitory factor in mice with an experimental cuprizone model of multiple sclerosis: possible mechanisms |
| title_full_unstemmed |
Neuroprotective effect of the recombinant human leukemia inhibitory factor in mice with an experimental cuprizone model of multiple sclerosis: possible mechanisms |
| title_sort |
neuroprotective effect of the recombinant human leukemia inhibitory factor in mice with an experimental cuprizone model of multiple sclerosis: possible mechanisms |
| author |
Labunets, I.F. Rodnichenko, A.E. Melnyk, N.O Rymar, S.E Utko, N.A. Gavrulyk-Skyba, G.O. Butenko, G.M. |
| author_facet |
Labunets, I.F. Rodnichenko, A.E. Melnyk, N.O Rymar, S.E Utko, N.A. Gavrulyk-Skyba, G.O. Butenko, G.M. |
| topic |
Structure and Function of Biopolymers |
| topic_facet |
Structure and Function of Biopolymers |
| publishDate |
2018 |
| language |
English |
| container_title |
Вiopolymers and Cell |
| publisher |
Інститут молекулярної біології і генетики НАН України |
| format |
Article |
| title_alt |
Нейропротекторний ефект рекомбінантного лейкемія-інгібіторного фактору людини у мишей з експериментальною купризоновою моделлю розсіяного склерозу: можливі механізми Нейропротекторный эффект рекомбинантного лейкемия-ингибирующего фактора человека у мышей с экспериментальной купризоновой моделью рассеянного склероза: возможные механизмы |
| description |
Aim. To investigate the effects of the rhLIF recombinant human leukemia inhibitory factor on the structural and functional changes in the central nervous system in the cuprizone-induced experimental model of multiple sclerosis in mice and to evaluate the involvement of the brain macrophages, T-lymphocytes and antioxidant enzymes in the observed rhLIF effects. Methods. Young 129/Sv mice were fed with the cuprizone neurotoxin daily for three weeks. rhLIF was injected daily (50 μg/kg) after seven days of the cuprizone treatment. We used histology, flow cytometry, spectrophotometric and functional methods. Results. The cuprizone treatment resulted in an increase in the number of the brain and spinal cord neurons with destructive changes, the brain macrophages, CD3⁺-cells and the content of malondialdehyde; it changes the behavior of animals, decreases the brain superoxide dismutase and glutathione peroxidase activities. The rhLIF injection partially or completely restored the cuprizone-changed parameters. The effect of rhLIF was preserved during two months after the experiment completion. Conclusions. The neuroprotective effect of rhLIF is connected with a decrease in the cuprizone-induced changes in the number of the brain T-cells and macrophages and the activity of antioxidant enzymes .
Мета. Дослідити вплив рекомбінантного лейкемія інгібіторного фактору людини (rhLIF) на структурні та функціональні зміни ЦНС в експериментальній моделі розсіяного склерозу, індукованого у мишей купризоном; оцінити участь макрофагів, Т-лімфоцитів та антиоксидантних ферментів головного мозку в реалізації його проявів. Методи. Молоді миші лінії 129/Sv отримували нейротоксин купризон із їжею щодня протягом 3-х тижнів, rhLIF – з 8-ї доби прийому купризону щодня у дозі 50 мкг/кг. Методи: гістологічний, проточної цитометрії, спектрофотометричний і функціональний. Результати. Під впливом купризону в головному та спинному мозку мишей збільшується кількість нейронів з деструктивними змінами; в головному мозку зростає кількість макрофагів, СD3⁺-клітин і вміст малонового діальдегіду; знижується активність супероксидддисмутази, глютатіонпероксидази у головному мозку та змінюється поведінка тварин. Ін'єкції rhLIF частково або повністю відновлюють показники, змінені купризоном. Ефект цитокіна зберігається через 2 міс після завершення експерименту. Висновки. Нейропротекторний ефект rhLIF пов’язаний з пригніченням індукованих купризоном змін в активності антиоксидантних ферментів, кількості Т-клітин і макрофагів у головному мозку.
Цель. Исследовать влияние рекомбинантного лейкемия- ингибирующего фактора человека (rhLIF) на структурные и функциональные изменения ЦНС в экспериментальной модели рассеянного склероза, индуцированного у мышей купризоном; оценить участие макрофагов, Т-лимфоцитов и антиоксидантных ферментов головного мозга в реализации его проявлений. Методы. Молодые мыши линии 129/Sv получали нейротоксин купризон с пищей ежедневно в течение 3-х недель, rhLIF – с 8-го дня приема купризона ежедневно в дозе 50 мкг/кг. Методы: гистологический, проточной цитометрии, спектрофотометрический и функциональный. Результаты. Прием купризона вызывает увеличение числа нейронов с деструктивными изменениями в головном и спинном мозге, количества макрофагов, СD3⁺-клеток и содержания малонового диальдегида в головном мозге; наблюдается снижение активности супероксиддисмутазы, глютатионпероксидазы в головном мозге и изменение поведения животных. Инъекции rhLIF частично или полностью восстанавливают измененные купризоном показатели. Эффект цитокина сохраняется через 2 мес после завершения эксперимента. Выводы. Нейропротекторный эффект rhLIF связан с подавлением индуцированных купризоном изменений в активности антиоксидантных ферментов, количестве Т-клеток и макрофагов в головном мозге.
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| issn |
0233-7657 |
| url |
https://nasplib.isofts.kiev.ua/handle/123456789/154362 |
| citation_txt |
Neuroprotective effect of the recombinant human leukemia inhibitory factor in mice with an experimental cuprizone model of multiple sclerosis: possible mechanisms / I.F. Labunets, A.E.. Rodnichenko, N.O Melnyk, S.E. Rymar, N.A. Utko, G.O. Gavrulyk-Skyba, G.M. Butenko // Вiopolymers and Cell. — 2018. — Т. 34, № 5. — С. 350-360. — Бібліогр.: 36 назв. — англ. |
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350
I. F. Labunets, A. E. Rodnichenko, N. O. Melnyk
© 2018 I. F. Labunets et al.; Published by the Institute of Molecular Biology and Genetics, NAS of Ukraine on behalf of Bio-
polymers and Cell. This is an Open Access article distributed under the terms of the Creative Commons Attribution License
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provided the original work is properly cited
UDC 612.017.1:616.8-003.8.001.53
Neuroprotective effect of the recombinant human leukemia inhibitory
factor in mice with an experimental cuprizone model of multiple
sclerosis: possible mechanisms
I. F. Labunets1, A. E. Rodnichenko1, N. O. Melnyk1, S. E. Rymar1,2,
N. A. Utko1, G. O. Gavrulyk-Skyba1, G. M. Butenko1
1 Institute of Genetic and Regenerative Medicine, NAMS of Ukraine
67, Vyshgorodska Str., Kyiv, Ukraine, 04114
2 Institute of Molecular Biology and Genetics, NAS of Ukraine
150, Akademika Zabolotnoho Str., Kyiv, Ukraine, 03680
irina_labunets@ukr.net
Aim. To investigate the effects of the rhLIF recombinant human leukemia inhibitory factor on the
structural and functional changes in the central nervous system in the cuprizone-induced experi-
mental model of multiple sclerosis in mice and to evaluate the involvement of the brain mac-
rophages, T-lymphocytes and antioxidant enzymes in the observed rhLIF effects. Methods. Young
129/Sv mice were fed with the cuprizone neurotoxin daily for three weeks. rhLIF was injected
daily (50 μg/kg) after seven days of the cuprizone treatment. We used histology, flow cytometry,
spectrophotometric and functional methods. Results. The cuprizone treatment resulted in an in-
crease in the number of the brain and spinal cord neurons with destructive changes, the brain
macrophages, CD3+-cells and the content of malondialdehyde; it changes the behavior of animals,
decreases the brain superoxide dismutase and glutathione peroxidase activities. The rhLIF injection
partially or completely restored the cuprizone-changed parameters. The effect of rhLIF was
preserved during two months after the experiment completion. Conclusions. The neuroprotective
effect of rhLIF is connected with a decrease in the cuprizone-induced changes in the number of
the brain T-cells and macrophages and the activity of antioxidant enzymes.
K e y w o r d s: cuprizone, LIF, neuron, T-lymphocytes, macrophages, antioxidant enzymes
Introduction
Multiple sclerosis is one of the most widespread
demyelinating pathologies of the central nervous
system (CNS) with the damages of both neuron
pericarion and myelin of nerve fiber accompa-
nied by the development of motor, emotional,
vegetative and cognitive impairments [1, 2].
Recently, new approaches to the treatment of
multiple sclerosis have been developed. One of
such promising approaches is the activation of
own neural stem cells (NSCs)/progenitors in
Structure and Function
of Biopolymers
ISSN 1993-6842 (on-line); ISSN 0233-7657 (print)
Biopolymers and Cell. 2018. Vol. 34. N 5. P 350–360
doi: http://dx.doi.org/10.7124/bc.000989
mailto:irina_labunets@ukr.net
351
Neuroprotective effect of rhLIF in mice with an experimental cuprizone model of multiple sclerosis: possible mechanisms
CNS [3]. Activation of own NSCs can be influ-
enced by various cellular factors, in particular,
cytokines. The leukemia inhibitory factor (LIF)
is a polyfunctional cytokine of the interleukin 6
(IL6) family playing an important role in the
nervous system development and its post-injury
reco very [4–10].
In neurodegenerative diseases, including mul-
tiple sclerosis, the important links in the mech-
anism of nerve cell damage are the activation of
microglia/macrophages and T-lymphocytes, as
well as the oxidative stress accompanying neu-
roinflammation [11, 12]. It has been established
that during first hours/days after damage of the
nervous system, LIF participates in the initiation
of a neuroinflammatory reaction, in particular,
attracting macrophages into the inflammatory
area [13]. In this case, the expression of mRNA
LIF in the glial cells of the nervous system in-
creases [14]. LIF reveals also anti-inflammatory
effects [15].
Studies on the possible ways of neuropro-
tective influence of recombinant human LIF
(rhLIF) require adequate experimental models
of demyelinating CNS pathologies. As such,
the toxic model of multiple sclerosis, induced
by cuprizone, is widely used. We as well as
other investigators observed a damage of the
oligodendrocytes and neurons, neuroinflam-
mation, oxidative stress and a reduced activity
of the antioxidant enzymes in CNS of animals
with the experimental cuprizone-induced mul-
tiple sclerosis [11, 14, 16–18].
The aim of our work was to investigate the
effects of rhLIF on the structural and func-
tional changes in CNS in the cuprizone-in-
duced experimental model of multiple sclero-
sis in mice and to evaluate the involvement of
the brain macrophages, T-lymphocytes and
antioxidant enzymes in the realization of LIF
effects.
Materials and Methods
Animals
Experiments were conducted on young
(3-5 months) female 129/Sv “wild type” (H-2b)
mice (n=42) from vivarium of the Institute of
Genetic and Regenerative Medicine NAMS of
Ukraine (IGRM). Mice were kept in standard
vivarium conditions with a fixed light regimen
12:12. Biological tissues for studies were ta ken
from animals at 9:00 a.m. by ether anesthesia.
All experiments were carried out according to
“European Convention for the Protection of
Vertebrate Animals used for experimental and
other scientific purposes” (Strasburg, 1986)
(from 21.02.2006).
Cuprizone-induced demyelination. Mice were
given cuprizone [bis(cyclohexylidene hydra zide)]
(«Sigma», USA), in an amount of 0.2 % by
weight, mixed with food, daily for 3 weeks [17].
Recombinant human LIF (rhLIF). LIF was
cloned with RT-PCR technology using human
placenta RNA as a template and was expressed
in E.coli. The biological activity was checked
on the mouse myeloid leukemia cell line M1 [19].
Cytokine was injected intraperitonealy daily,
50 μg/kg of body weight [20], after 7 days of
receiving cuprizone (14 injections) [21]. The
control group of mice fed with cuprizone re-
ceived injections of phosphate buf fered saline
(PBS) according to a similar scheme.
Experimental groups: 1 — mice were kept
on standard diet without cuprizone (untreated
mice); 2 — mice receiving cuprizone-contain-
ing food for 3 weeks and PBS injections (con-
trol group); 3 — mice receiving both cupri-
352
I. F. Labunets, A. E. Rodnichenko, N. O. Melnyk et al.
zone-containg food and rhLIF injections. Each
group included 14 mice. Experiments were
conducted after completion of cuprizone treat-
ment on 7, 21 and 60 days.
For morphological studies of the CNS
structures (cortex, cerebellum and lumbar spi-
nal cord) the histological sections were stained
with toluidine blue (by Nissl) [17, 22]. The
number of unchanged neurons and neurons
with moderate and severe structural changes
were determined by morphometric analysis.
Behavioral responses of mice were studied
using «open field» test [22, 23]. The number
of squares crossed (horizontal locomotor acti-
vity) and faecal boluses (emotional activity)
and looks into minks (search activity) were
recorded for 3 min.
Malondialdehyde (MDA) content in brain
tissues was determined according to Uchi-
yama [24].
Activities of antioxidant enzymes were as-
sessed in supernatants of the brain homoge-
nates (cerebellum, forebrain) using μQuant
Bio-Tek spectrophotometer (USA) [25].
Functional activity of brain macrophages.
The brain microglial cells/macrophages and
blood-derived macrophages have similar pa-
rameters, for example, the possibility to phago-
cytosis of latex beads [26–28]. Brain suspen-
sion of adherent cells was cultivated with latex
beads for 45 min at 37о C in 5 % CO2. Then
these cells were fixed and stained with
Romanovsky-Gimsa. The percentage of brain
macrophages capable of phagocytosis of latex
beads (phagocytic index) and the amount of
latex beads, which were phagocytosed by one
macrophage (phagocytic number) were count-
ed under light microscope. All reagents were
supplied from Sigma (USA).
Flow cytometric analysis of CD3+ and
CD11b+-cells. The cells suspension of the brain
tissue was incubated with monoclonal antibodies
for 20 min at 4˚ C in combination with the satu-
rating amounts of phycoerythrin (PE)-conjugated
antibodies against CD3 (dilution 1:50) and fluo-
rescein isothiocyanate (FITC)-conjugated anti-
bodies against CD11b (dilution 1:50), as recom-
mended by the manufacturer (Becton Dickinson,
USA). Measurements were performed on the flow
cytometer BD FACSAria (Becton Dickinson,
USA), and the data analysis was performed using
the BD FACS Diva 6.1 software.
Statistical analysis of the results was carried
out using the Student’s t-criterion [29]. The
differences between the means of comparable
groups (M±SEM) were significant at p < 0.05.
Results
Changes in the structure of neurons and
brain macrophages activity in mice during
early period of cuprizone treatment. One of
the early manifestations of the development of
cuprizone-induced multiple sclerosis is a
change in the oligodendrocyte functioning and
apoptosis development [14]. However, the
neuronal structural changes in such animals
are poorly studied. Histological analysis of
brain structures has shown that a proportion
of neurons with moderate structural changes
increased in the cortex and cerebellum of mice
after 7 days of the cuprizone treatment
(Fig. 1, A, B). These results are in agreement
with our earlier data: the horizontal locomotor
activity revealed a significant decrease
(1.8-fold, p < 0.05) in the young 129/Sv mice
after 7 days of the cuprizone treatment com-
paring to the untreated mice [21]. Apart from
the changes in brain neurons of the cuprizone-
353
Neuroprotective effect of rhLIF in mice with an experimental cuprizone model of multiple sclerosis: possible mechanisms
treated mice, the number and the activity of
brain macrophages increased if compared to
the similar parameters in the group without
cuprizone treatment (Fig. 2, A, B).
Influence of rhLIF injections on struc-
tural and functional changes in the CNS of
mice treated with cuprizone during three
weeks. After 7 days of the cuprizone treatment,
rhLIF in the concentration of 50 μg/kg of body
weight was injected. The control group of
animals continued to receive only cuprizone.
Comparison of the results of morphometric
analysis of CNS structures of above groups
showed a lower proportion of unchanged neu-
rons and a greater share of the neurons with
destructive changes in the control group,
whereas in the experimental group the direc-
tions of these changes were opposite (Fig. 3, A,
B, C). Thus, the rhLIF injections to cuprizone-
treated mice resulted in the restorative and
compensatory neuronal responses to a damage.
The behavioral reactions were significantly
worsened in the cuprizone-treated mice (see
Fig. 4, A, B, C). Injections of rhLIF led to an
increase of the locomotor and search activity
in comparison with similar parameters in the
control group, although they remained lower
than in untreated mice (Fig. 4, A, C). After
injection of cytokine we observed an increase
in the emotional activity to the level observed
in the untreated group (Fig. 4, B).
Influence of rhLIF injections on brain
macrophages, T cells and antioxidant en-
zymes activity in mice treated with cuprizone
during three weeks. The number of active
macrophages in the brain of mice after 3 weeks
A
B
Fig. 1. Structural changes of brain neurons (A — cortex, B — cerebellum) in mice after 7 days of cuprizone treatment.
A
B
Fig. 2. Macrophages activity (A — phagocytic index, B — phagocytic number) in mice after 7 days of cuprizone treat-
ment. p < 0.05 vs untreated mice.
354
I. F. Labunets, A. E. Rodnichenko, N. O. Melnyk et al.
of the cuprizone treatment was higher than in
the untreated animals and the rhLIF injections
in such mice did not affect this parameter
(Fig. 5, A, B). The number of CD11b+ (Mac1+)
cells in the brain of cuprizone-treated mice
(2.8 ± 0.2 %) did not change in comparison
with the untreated group (2.8 ± 0.1 %), but
after cytokine injections this number decreased
to 1.8 ± 0.2 % (p < 0.05).
The number of CD3+ cells in the brain of
mice significantly increased after the cuprizone
treatment (Fig. 5, C). However, the number of
CD3+ cells decreased following the rhLIF in-
jections, remaining still higher than in the
untreated mice (Fig. 5, C).
Under the cuprizone influence one of the
indices of oxidative stress (MDA content)
significantly increased in the forebrain
whereas the administration of rhLIF reduced
the MDA content to the level of untreated
A
B
C
Fig. 4. The influence of cuprizone and rhLIF on the be-
havioural reactions (A — horizontal locomotor activity,
B — emotional activity, C — search activity) in mice.
p < 0.05: * — vs untreated mice; # — vs cuprizone. Cu-
prizone treatment lasted 3 weeks.
A
B
C
Fig. 3. The influence of cuprizone and rhLIF on the struc-
tural changes of CNS neurons (A — cortex, B — cerebel-
lum, C — lumbar spinal cord) in mice.
355
Neuroprotective effect of rhLIF in mice with an experimental cuprizone model of multiple sclerosis: possible mechanisms
mice (Table). The superoxide dismutase
(SOD) activity was significantly reduced in
the forebrain of mice receiving cuprizone
whereas the rhLIF injections restored this
activity to the level of untreated animals
(Table).
The activation of glutathione peroxidase
(GPx) was observed in the forebrain of the
mice receiving both cuprizone and cytokine
(Table). After the cuprizone treatment the
GPx activity in the cerebellum was signifi-
cantly reduced whereas the rhLIF injections
Table. Malondialdehyde content and the activity of antioxidant enzymes in the brain structures of mice
receiving cuprizone and rhLIF, M ± m
Indice
Experimental group of mice
Untreated Cuprizone Cuprizone +rhLIF
Forebrain
Malondialdehyde (nmol/mg) 1.50±0.10 2.10±0.20* 1.60±0.10#
Superoxide dismutase (U/mg·min) 12.75±0.35 11.50±0.28* 12.0±0.53
Catalase (µmol/mg·min) 1.28±0.12 1.25±0.09 1.59±0.26
Glutathione peroxidase (nmol/mg·min) 5.23±0.21 5.72±0.94 6.56±0.32*
Cerebellum
Malondialdehyde (nmol/mg) 1.40±0.10 1.60±0.10& 1.70±0.20
Superoxide dismutase (U/mg·min) 12.90±0.35 12.75±0.14& 13.50±0.35&
Catalase (µmol/mg·min) 1.44±0.05 1.29±0.07 1.40±0.17
Glutathione peroxidase (nmol/mg·min) 8.83±0.50& 7.50±0.11* 8.53±0.19#
Note: p < 0.05: * — vs untreated mice; # — vs cuprizone (3-week treatment); & — vs forebrain. Data of antioxidant enzyme
activity in the cerebellum were shown earlier [30].
A
B
C
Fig. 5. The parameters of the macrophages activity (A —
phagocytic index, B — phagocytic number) and the num-
ber of CD3+ cells (C) in the brain of cuprizone — and
rhLIF — treated mice. p<0.05: * — vs untreated mice;
# — vs cuprizone. Cuprizone treatment lasted 3 weeks.
356
I. F. Labunets, A. E. Rodnichenko, N. O. Melnyk et al.
restored it to the level of untreated animals
(Table).
Thus, the cuprizone-induced brain damage
led to an increase in the number of macro-
phages and T-lymphocytes and inhibited the
activity of antioxidant enzymes (SOD, GPx)
in the mouse brain structures. This neurotoxin
effect was significantly lower in the case of
co-administration of cuprizone and rhLIF.
The long-term effects of rhLIF injections
on cuprizone-induced changes in the neuronal
structure and T-lymphocyte number in the
mouse brain. Histological analysis of the mouse
brain structures two months after completion of
the cuprizone treatment showed that the number
of unchanged neurons, neurons with moderate
changes and neurons with pronounced struc-
tural changes was respectively: 13 %, 69 % and
28 % in the cortex; and 35 %, 43 % and 22 %
in the cerebellum. The number of neurons with
pronounced structural chan ges was lower and
the number of unchanged neurons was higher
in the cerebral cortex and cerebellum compared
to the animals after 3 weeks of the neurotoxin
treatment (Fig. 2, A, B). This fact can be ex-
plained by the reduction of neuroinflammation,
which leads to an increase in the number of
oligodendrocytes and remyelination [14]. In our
study, two months after completion of adminis-
tration of rhLIF and cuprizone the number of
neurons without changes, with moderate and
pronounced structural changes was 26 %, 62 %,
12 % in cerebral cortex; and 65 %, 27 %, 8 %
in the cerebellum, respectively. Importantly, the
number of neurons of different types in the cer-
ebellum two months after completion of the
cuprizone treatment was comparable with their
number after 3 weeks of the combined treatment
with neurotoxin and rhLIF. Thus, the results
indicate a more effective restoration of the neu-
ronal structure following the cytokine injections.
Two months after completion of the cupri-
zone treatment the horizontal locomotor acti-
vi ty (the number of crossed squares) was
lower in the cuprizone-treated mice comparing
to the mice receiving both cuprizone and
rhLI F: 11.0 ± 1.5 and 25.0 ± 2.3, (p < 0.05),
respectively. In both experimental groups, the
indices remained lower (p < 0.05) compared
to the untreated group (Fig 4, A).
Two months after completion of the cupri-
zone treatment the number of the brain CD3+-
cells (1.2 ± 0.1 %) was lower than just after
completion of the 3-week neurotoxin treatment
(p < 0.05), whereas this parameter was still
higher than in the untreated mice (p < 0.05)
(Fig. 5, C). However, two months after comple-
tion of the combined administration of cupri-
zone and rhLIF the number of CD3+ cells
(1.0 ± 0.1 %) did not change noticeably compar-
ing to the untreated mice (p > 0.05) (Fig. 5, C).
So, the effects of rhLIF on the above chan-
ges induced by the cuprizone treatment re-
mained the same for a long period of time.
Discussion
The myelin-synthesizing cells (oligodendro-
cytes) underwent apoptosis in the cuprizone-
induced model of multiple sclerosis [14].
Using the cuprizone-induced model of multiple
sclerosis in mice, we observed a significantly
increased number of the neurons with destruc-
tive changes in the cortex and the cerebellum
of the brain and in the lumbar spinal cord.
Damage of the neuron structure is accompa-
nied with destruction of the locomotor, emo-
tional and search activities. Our results agree
with the findings of Ganong [31]. Apparently,
357
Neuroprotective effect of rhLIF in mice with an experimental cuprizone model of multiple sclerosis: possible mechanisms
the disorders of behavioral reactions induced
by cuprizone result from both, the developing
demyelination of CNS organs and the damage
of brain and spinal cord neurons.
In our study of the cuprizone-treated mice
the exogenous rhLIF improved the structure
and functioning of CNS neurons. These mor-
pho-functional changes can be explained by
the literature data about the antiapoptotic ac-
tion of LIF and its stimulating effects on the
proliferation of NSCs in the brain subven-
tricular zone and their differentiation into the
neuronal direction, the formation of the oligo-
dendrocytes and myelin synthesis [8–10,14].
Today, it is supposed that the activation of
microglia cells and T-lymphocytes is one of the
possible mechanisms of cuprizone-induced
damage of the neurons. In particular, it has been
shown that microgliosis develops on the 2nd
week of cuprizone treatment and it peaks by
the 4-th week, being combined with the inten-
sive demyelination [11]. The activated microg-
lia cells of the brain produce an increased quan-
tity of the proinflammatory cytokines (TNF-α
and γ-interferon) and reactive oxygen species
(ROS), which damage both, the oligodendro-
cytes and neurons [11,32]. We observed an
increase of the number and activity of the mac-
rophages after 7 days of the cuprizone treat-
ment and this effect remained for 3 weeks.
The number of brain activated macrophages
was large after the rhLIF injections. The pro-
inflammatory phenotype M1 turns into the
anti-inflammatory phenotype M2 in the pro-
cess of the development of cuprizone-induced
demyelination in the brain [11]. In our study
the positive changes in the neuron structure
allowed us to assume the realization of anti-
inflammatory effect by the macrophages fol-
lowing cytokine treatment. Additionally, the
number of CD11b+ (Mac1+) cells in the brain
of mice reduces after administration of rhLIF.
As has been shown, this phenotype is specific
not only for macrophages but also for the mi-
croglia cells [26].
Some authors consider the cuprizone-in-
duced model of multiple sclerosis as
T-independent [12]. Kang et al. reported both
infiltration of the brain by T-cells and their
participation in the enhancing of the demyelin-
ation in the cuprizone-treated mice [33].
According to our results, we have observed an
increase in the number of CD3+T cells in the
brain after 3 weeks of cuprizone diet as com-
pared with the untreated mice (Fig. 5, C).
The number of CD3+ T cells in the brain after
the rhLIF injections decreased compared with
the mice without cytokine (Fig. 5, C). It is known
that in human multiple sclerosis the T-cells and
perivascular macrophages expressing LIF are
seen in the area of damage [34]. In the athours’
opinion, LIF prevents TNF-α- induced apoptosis
of the oligodendrocytes that is an important
mechanism for the protection of glial and neu-
ronal cells. The similar results were received on
the model of experimental autoimmune encepha-
lomyelitis by Cao et al. [35]. Thus, the LIF, ex-
pressed by neural progenitor cells, inhibits the
differentiation of T-helper 17 (Th 17) and produc-
tion of proinflammatory cytokine IL-17.
According to our preliminary data, we observed
a decrease in the number of CD4+-cells in the
brain of cuprizone-treated mice.
After the cuprizone treatment we registered
the reduced activity of antioxidant enzymes in
the brain structures involved in the motor and
emotional activity. This might be another pos-
sible mechanism of nerve cells damage caused
358
I. F. Labunets, A. E. Rodnichenko, N. O. Melnyk et al.
by oxidative stress factors in cuprizone-treated
mice [16, 18]. We observed the enhancement
of the activity of antioxidant enzymes in the
brain along with the decrease of MDA content
and the increase of the number of neurons
without structural changes in the mice treated
with both cuprizone and rhLIF. During CNS
damage LIF inhibits the ROS generation and
increases the SOD activity [36]. According to
our data, LIF increases the activity of both
SOD and GPx thereby contributing to the im-
provement of the structure of the CNS organs.
Conclusion
We observed the changes in the amount of the
brain macrophages and T cells as well as the
balance disturbance in the pro- and antioxidant
factors that could contribute to the cuprizone-
induced brain neuron damage. The above
changes were detected already at early stages
of the neurotoxin treatment. The neuroprotec-
tive effect of rhLIF is сonnected with the de-
crease of cuprizone-induced changes in the
number of the brain T-cells and macrophages
and an activity of the antioxidant enzymes.
The effect of the cytokine remained during two
months after completion of the cuprizone ad-
ministration.The obtained results can serve as
a basis for new approaches in cellular therapy
for the patients with multiple sclerosis.
Acknowledgments
The study was carried out in the framework of
“The role of the cell and endocrine factors in
the realization of the neuroprotective proper-
ties of the stem cells in the experimental da-
ma ges of the nervous system”, being fulfilled
by the IGRM, NAMSU (No of the state re gist-
ration 0116U000139), 2016-2018.
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Нейропротекторний ефект рекомбінантного
лейкемія-інгібіторного фактору людини у мишей
з експериментальною купризоновою моделлю
розсіяного склерозу: можливі механізми
І. Ф. Лабунець, А. Є. Родніченко, Н. О. Мельник,
С. Ю. Римар, Н. О. Утко, Г. О. Гаврилюк-Скиба,
Г. М. Бутенкo
Мета. Дослідити вплив рекомбінантного лейкемія ін-
гібіторного фактору людини (rhLIF) на структурні та
функціональні зміни ЦНС в експериментальній моде-
лі розсіяного склерозу, індукованого у мишей купризо-
ном; оцінити участь макрофагів, Т-лімфоцитів та анти-
оксидантних ферментів головного мозку в реалізації
його проявів. Методи. Молоді миші лінії 129/Sv отри-
мували нейротоксин купризон із їжею щодня протягом
3-х тижнів, rhLIF — з 8-ї доби прийому купризону
щодня у дозі 50 мкг/кг. Методи: гістологічний, про-
точної цитометрії, спектрофотометричний і функціо-
нальний. Результати. Під впливом купризону в голов-
ному та спинному мозку мишей збільшується кількість
нейронів з деструктивними змінами; в головному моз-
ку зростає кількість макрофагів, СD3+-клітин і вміст
малонового діальдегіду; знижується активність супер-
оксидддисмутази, глютатіонпероксидази у головному
мозку та змінюється поведінка тварин. Ін’єкції rhLIF
частково або повністю відновлюють показники, зміне-
ні купризоном. Ефект цитокіна зберігається через 2 міс
після завершення експерименту. Висновки. Нейро-
протекторний ефект rhLIF пов’язаний з пригніченням
індукованих купризоном змін в активності антиокси-
дантних ферментів, кількості Т-клітин і макрофагів у
головному мозку
К л юч ов і с л ов а: купризон, LIF, нейрон, Т-лімфо-
цити, макрофаги, антиоксидантні ферменти.
Нейропротекторный эффект рекомбинантного
лейкемия-ингибирующего фактора человека
у мышей с экспериментальной купризоновой
моделью рассеянного склероза: возможные
механизмы
И. Ф. Лабунец, А. Е. Родниченко, Н. А. Мельник,
С. Е. Рымарь, Н. А. Утко, Г. А. Гаврилюк-Скиба,
Г. М. Бутенкo
Цель. Исследовать влияние рекомбинантного лейке-
мия- ингибирующего фактора человека (rhLIF) на
структурные и функциональные изменения ЦНС в
экспериментальной модели рассеянного склероза,
индуцированного у мышей купризоном; оценить уча-
стие макрофагов, Т-лимфоцитов и антиоксидантных
ферментов головного мозга в реализации его проявле-
ний. Методы. Молодые мыши линии 129/Sv получали
нейротоксин купризон с пищей ежедневно в течение
3-х недель, rhLIF — с 8-го дня приема купризона еже-
дневно в дозе 50 мкг/кг. Методы: гистологический,
проточной цитометрии, спектрофотометрический и
функциональный. Результаты. Прием купризона вы-
зывает увеличение числа нейронов с деструктивными
изменениями в головном и спинном мозге, количества
макрофагов, СD3+-клеток и содержания малонового
диальдегида в головном мозге; наблюдается снижение
активности супероксиддисмутазы, глютатионперокси-
дазы в головном мозге и изменение поведения живот-
ных. Инъекции rhLIF частично или полностью восста-
навливают измененные купризоном показатели.
Эффект цитокина сохраняется через 2 мес после за-
вершения эксперимента. Выводы. Нейропротекторный
эффект rhLIF связан с подавлением индуцированных
купризоном изменений в активности антиоксидантных
ферментов, количестве Т-клеток и макрофагов в го-
ловном мозге.
К л юч е в ы е с л ов а: купризон, LIF, нейрон, Т-лим-
фо циты, макрофаги, антиоксидантные ферменты.
Received 28.05.2018
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