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Abstract Resumen
Context: The extract from the marine plant Thalassia testudinum BM-21, Contexto: El extracto obtenido de las planta marina Thalassia testudinum
standardized to thalassiolin B content (5.8 0.3%), possesses antioxidant, (BM-21), estandarizado acorde al contenido de thalassiolin B (5.8 0.3%),
anti-inflammatory and neuroprotective effects on acrylamide-induced posee efectos antioxidantes, antiinflamatorios y neuroprotectores sobre la
neurotoxicity in mice and global ischemia in Mongolian gerbils. neurotoxicidad inducida por acrilamida y la isquemia global en gerbos de
Aims: To determine whether or not BM-21 possesses neuroprotective Mongolia.
effects against cerebral ischemia induced by permanent middle cerebral Objetivos: Evaluar si el BM-21 ejerce efectos neuroprotectores sobre la
artery occlusion (pMCAo), a clinically relevant model of stroke. isquemia inducida por la oclusin permanente de la arteria cerebral
Methods: BM-21 was administered orally (400 mg/kg, once-aday/10 days) media en ratas (pMCAo), un modelo relevante desde el punto de vista
prior to ischemia. Twenty-four hours after occlusion, we studied clnico.
neurological signs, infarct volume, cerebral edema, histological damage Mtodos: El BM-21 fue administrado previamente por va oral (400 mg/kg,
and oxidative stress in cortex and striatum. In addition, brain 10 das/1 administracin/da). Tras la oclusin (24 h) se investigaron;
susceptibility to in vitro lipid peroxidation induced by kainic acid and 2,2'- sntomas de dao neurolgico, volumen de infarto, edema cerebral y dao
azobis(2-amidinopropane) dihydrochloride was studied after the BM-21 histolgico y estrs oxidativo en cuerpo estriado y corteza, as como el
administration. efecto del BM-21 sobre la peroxidacin lipdica in vitro inducida por cido
Results: BM-21 prevented behavioral deficit; reduced infarct volume and kanico y dihidrocloruro de 2,2'-azobis(2-amidinopropano).
cerebral edema; markedly decreased neuronal damage in striatum and Resultados: BM-21 previno la disfuncin conductual, disminuy el
cortex region. After occlusion, there was a significant increase of oxidative volumen de infarto, el edema cerebral, y el dao neuronal en cuerpo
stress in cortex and striatum. Treatment of ischemic rats with BM-21 (400 estriado y corteza. La oclusin indujo el incremento del estrs oxidativo
mg/kg) prevented lipid peroxidation and protein damage and increased en cuerpo estriado y corteza. El BM-21 disminuy la peroxidacin lipdica
the antioxidant enzymatic activities and glutathione. BM-21 also inhibited y el dao a protenas e increment las actividades de las enzimas
the in vitro lipid peroxidation in total brain homogenates. antioxidantes y el glutatin. Tambin el BM-21 inhibi la peroxidacin
Conclusions: Oral pre-treatment of BM-21 protects rats against pMCAo lipdica inducida in vitro.
ischemia-induced damage in the striatum and cortex. Results suggest that Conclusiones: El BM-21 previno el dao neuronal inducido por la isquemia
the protection of BM-21 involve at least partially, the increase resistance en corteza cerebral y cuerpo estriado en ratas, efecto que involucra al
to oxidative stress. menos parcialmente, el incremento de la resistencia frente al estrs
oxidativo.
Keywords: neuroprotection; oxidative stress; permanent middle cerebral Palabras Clave: estrs oxidativo; neuroproteccin; oclusin permanente
artery occlusion; Thalassia testudium. de la arteria cerebral media; Thalassia testudium.
ARTICLE INFO
Received | Recibido: November 3, 2016.
Received in revised form | Recibido en forma corregida: March 7, 2017.
Accepted | Aceptado: March 11, 2017.
Available Online | Publicado en Lnea: April 16, 2017.
Declaration of interests | Declaracin de Intereses: The authors declare no conflict of interest.
Funding | Financiacin: This study was supported by International Foundation for Science (Project F/4237-1) and by the project CITMA
047/2012 (Cuba).
Academic Editor | Editor Acadmico: Gabino Garrido.
_____________________________________
Garca et al. Thalassia testudinum extract on focal ischemia in rats
tion using a four-point scale as described (Menzies et sections were selected from a brain region
al., 1992). Shams operated were used as control. The corresponding to levels +0.2 mm and +0.26 mm
scale was as follow; 0: no apparent deficits with respect to Bregma according to anatomical
behavioral; 1: contra lateral forelimb flexion when structures observed in Paxinos rat brain atlas
suspended by the tail; 2: decreased grip of the (parietal cortex-striatum). Five-micrometer thick
contra lateral forelimb while tail pulled; 3: sections (20 m) were cut on a microtome and
spontaneous movement in all directions, contra stained with Nissl technique for light microscope
lateral circling only if pulled by tail and 4: examinations. In order to assess the effects of BM-21
spontaneous contra lateral circling. Tests were treatment on the survival of the neurons in
conducted blinded in relation to treatment. striatum and cortex in sections ipsilateral to the
pMCAo (0.20 m with respect to Bregma),
Morphometric evaluation photographs of 4 adjacent fields to the lateral
Infarct volume was calculated exactly as ventricle were taken with a Olympus microscope
previously reported (Yang and Shua, 1998). Briefly, after (magnification 20X) and photographic camera
completing neurological evaluation the animals (Microscope Digital Camera-DCM500, USB 2.0). On
were sacrificed under a light anesthesia with ether the photographs, the total population of survival
and brains were removed, frozen and coronally neurons (those with echromatic nucleus and
sectioned into five 2-mm thick slices (from rostral conserved cytoplasm) in the 3 experimental groups
to caudal). Coronal sections of brain were stained (n=6/group) were quantified.
with 2% TTC solution in saline for 10 min at 37C.
Estimation of oxidative stress in homogenates of
After fixing in 4% formal saline overnight, they
cortex and striatum after pMCAo
were scanned using a flatbed scanner (Hewlett
Packard HP Scanjet 3670). The area of infarction A total of 24 rats were assigned at random to
was measured in coronal brain sections by using three experimental groups as described previously
image analysis software Image J, version 1.34. and the same dosing protocol was used. After
Measurements were made by manually outlining occlusion, rats were anesthetized under ether and
the margins of infarct areas. Infarct areas of all euthanized to excise striatum and cortex of the left
sections were cumulated to get total infarct area hemisphere. They were homogenized in 10 mM
which was multiplied by thickness of brain sections PBS, 7.4 pH (1:9, w:v) in an ice-cold bath, with a
to get the volume of infarction. To compensate for PotterElvehjem type homogenizer. Homogenates
swelling, the following formula was applied: infarct were centrifuged at 3000 g for 10 min and the
size x contralateral hemisphere size/ipsilateral resultant supernatants were used to determine
hemisphere size. The volumes of both hemispheres biochemical parameters. Protein concentration was
were calculated separately, and edema volume was estimated with a modification of the Lowry proce-
calculated by subtracting the contralateral from dure. Biochemical parameters were determined by
ipsilateral volume (Thiyagarajan et al., 2004). spectrophotometric methods using and spectropho-
tometer UV-Vis (model o1205, Shimadzu Corpora-
Histological evaluation and quantification of tion). Estimation of malondialdeide (MDA) was
survival neurons assayed as thiobarbituric acid reactive substances
A total of 24 animals were used to examine (TBARS)(Ohkawa et al., 1979). Concentrations of MDA
ischemia-induced histological damage in treated were expressed as mol/mg protein. Protein
and non-treated ischemic animals. Pseudo-operated carbonyl associated groups were determined after
animals served as controls. Twenty-four hours after their derivatization with 2,4-dinitrophenylhydra-
pMCAo the animals were anaesthetized under a zine (DalleDonne et al., 2003) and were expressed as
light anesthesia with ether and intracardially nmol carbonyl groups/mg protein using molar
perfused with 100 mL of NaCl 0.9% and 150 mL of coefficient absorption of 22,000 M-1cm-1. Superoxide
4% paraformaldehyde. The brains were quickly dismutase (SOD) and glutathione peroxidase (GSH-
removed and finally embedded in paraffin wax. The Px) activity was measured according to commer-
cially available kit and expressed as IU/mg protein. animals. pMCAo + Vehicle group exhibited the
The concentration of reduced glutathione (GSH) highest score (3.25 1.23, p<0.001) corresponding to
was measured by method of (Ellman, 1959) and severe behavioral impairments 24 h after occlusion.
expressed as nM by means of a molar absorption Pre-treatment with BM-21 during 10 days resulted in
coefficient of 14,150 M-1cm-1. a significant attenuation (0.87 0.35, p<0.05) of rat
neurobehavioral impairment when compared to
Brain susceptibility to in vitro lipid pMCAo + Vehicle group (Fig. 1).
peroxidation (LPO) Twenty-four hours after pMCAo, rats developed
Sixteen male Wistar rats (250-350 g) were infarcts affecting cortex and striatum. Fig. 2
divided randomly into two groups (vehicle control represents the infarct volume in pMCAo rats
and BM-21 treated group) and were similarly treated either with vehicle or BM-21. As noted,
treated. At the end of the treatment period, rats treatment with BM-21 markedly reduced infarct
were anesthetized with ether, euthanized and areas at 24 h post pMCAo as evident from TTC-
brains were quickly excised and homogenized as stained sections. When the total cerebral infarct
previously described. To measure the susceptibility volume was determined by computer-assisted
to kainic-induced LPO (Yamamoto and Parayanthala, imaging methods, a significant reduction (p<0.001)
2003) aliquots of tissue homogenate were incubated
of the cerebral infarct volume (77.5%) was observed
in presence of kainic acid 5.12 mg/mL (final in the group pre-treated with BM-21 compared with
concentration) at 37C for 30 min. To assess brain the ischemic group (ischemic, 246.8 31.25 mm3;
susceptibility to AAPH-induced LPO (Yu-Jun et al., BM-21-treated, 55.39 15.67 mm3). No lesion was
2003) tissue samples were incubated with 50 mM
observed in Sham animals (data not shown).
AAPH (final concentration) under similar condi- Fig. 3 displays brain edema. In pMCAo caused a
tions. The extent of LPO was assessed by measuring prominent increase in cerebral edema volume in
TBARS concentration as previously described. In the ipsilateral hemisphere as compared to
both oxidant system LPO status at 0 time were used contralateral hemisphere (p<0.016). However, pre-
as basal and were subtracted from the one obtained treatment with BM-21 significantly reduced by
at 30 min. The TBARS levels were expressed in 71.2% edema volume (p<0.001) in relation to
mol/mg protein. pMCAo + Vehicle group.
Fig. 4 shows representative histological sections
Statistical analysis of striatum and parietal cortex sections obtained at
central coronal level. The figure shows that
Data are presented as mean SEM along with ischemia produced typical architecture loss in the
the number of animals. Statistical analysis was cortex (E) and striatum (B) as dense network fibers
performed using Graph-Pad Prism 5.0 software. The of white substance and parenchyma of gray
global comparison was performed using One-Way substance were not observed. In addition, necrotic
ANOVA followed by Turkeys multiple comparison neurons, proliferation of astrocyte and variable
tests. Pair wise testing was done with the help of cavitation were observed as well as a decrease of
the unpaired Student t-test. Neurological deficits cortex cells (E). In BM-21 treated rats, the
were presented as median 95% confidence inter- histological appearance of brain sections (C and F)
val and analyzed using nonparametric Kruskal were similar to that of sham-operated animals (A
Wallis test followed by Dunn's multiple comparison and D). The typical architecture of the striatum and
tests. P<0.05 was considered statistically significant. cortex was noted, which was especially observed in
the striatum. In this section, it can be noted a
RESULTS clearly defined dense network fibers of white
substance (indicated by arrows) in relationship to
Animals treated with BM-21 (400 mg/kg, showed
the parenchyma of gray substance. Additionally, the
no behavioral changes other than the neurological
light coloration and the apparent decreased of cells,
deficits induced by ischemia. Before pMCAo,
observed fundamentally in the cortex (E) suggested
neurological score was normal (score = 0) in all
http://jppres.com/jppres J Pharm Pharmacogn Res (2017) 5(3): 178
Garca et al. Thalassia testudinum extract on focal ischemia in rats
the presence of an inflammatory process, which was consistently declined (p<0.001) in pMCAo group
not observed in the cortex sections of the animals treated with vehicle (cortex, 36.4%; striatum,
treated with BM-21. A significant reduction 29.5%) while BM-21 treatment significantly
(p<0.001) in the number of cells with normal increased GSH in cortex (p<0.01) and striatum
structural features was observed in the pMCAo + (p<0.001) towards the normal level. Permanent
Vehicle (striatum, 187 6.5 cell/mm2 vs. 60 5 occlusion significantly decreased SOD activity in
cell/mm2) and (cortex, 240 10.5 cell/mm2 vs. 90 cortex (34.7%, p<0.01) and striatum (43.7%, p<0.05)
5.2 cell/mm2). In contrast, a significant recovery in compared to vehicle sham group whereas the
the neuronal population was observed in the BM-21 extract treatment significantly reduced the
pretreated group (striatum, 119.7 10 cell/mm2; decreased SOD activity induced by pMCAo in both
cortex, 200 7.6 cell/mm2) (Fig. 5). brain regions (cortex, p<0.01; striatum, p<0.05).
Table 1 describes results concerning oxidative Vehicle-pMCAo group showed decreased GSH-Px
stress in cortex and striatum. LPO determined as activity in cortex and striatum (p<0.001 and p<0.05,
MDA concentration significantly increased (p< 0.01) respectively) compared to control. Pretreatment
in both areas when compared with the sham group. with the extract significantly attenuated the
BM-21 treatment significantly attenuated the decreased GSH-Px activity in cortex (p<0.01) and
elevation of MDA levels induced by pMCAo in both striatum (p<0.05), compared to vehicle plus pMCAo
brain regions (p<0.05 and 0.01, respectively). group.
Occlusion also resulted in a significant (p<0.05) Data referring to the ex vivo study (Fig. 6)
increase of protein carbonyl in both cortex (55.5%) convincingly showed that brain homogenate from
and striatum (33.3%) in comparison to the pseudo BM-21 treated rats was less prone to LPO than those
operated group. BM-21 gave lower protein carbonyl of control group as MDA concentration generated
content (p<0.05) in both brain regions that was by kainic acid (p<0.05) and AAPH (p<0.01) were
almost similar than sham group. GSH content significantly reduced.
5
(***) Sham
Neurological Score
4 pMCAo + vehicle
pMCAo + BM-21 400 mg/kg
3
(+)
2
Figure 1. Neurological scores in rats after pMCAo (24 h) and effects of BM-21 administered 10 days before ischemia.
Values are means SEM; sham (n = 8); vehicle (n = 8) and BM-21 (n = 8). ***p0.01, Comparison vs. sham, +p<0.05, comparison vs. pMCAo +
Vehicle.
A B
300
100
***
0
Vehicle BM-21 400 mg/kg
Vehicle BM-21
Figure 2. Representative photograph of coronal brain sections (A) and total infarct volume (B) 24 hours after pMCAo
of vehicle and BM-21 treated rats.
Bars are means SEM for pMCAo+Vehicle (n=6) and pMCAo+BM-21 animals, (n=8). Significant decrease (***p<0.001) in total infarct
volume in the group treated with BM-21 (Student's t-test).
25
pMCAo Vehicle
Edema volume (mm3)
15
10
***
5
Figure 3. Edema volume after 24 h of pMCAo in vehicle and BM-21 treated group.
Bars are means SEM for pMCAo + Vehicle (n=6) and pMCAo + BM-21 (n=8) animals. ***p<0.001 vs. pMCAo + Vehicle
(Students t-test).
A B C
Striatum
D E F
Cortex
Figure 4. Histological examination of neuronal damage. Nissl staining of striatum and cortex areas at 24 hours after ischemic
damage. The sections represent the striatum (A, B, C) and cortex (D, E, F) of a sham control rat (A, D) and ischemic rats;
pretreated with vehicle (B, E) and BM-21 (C, F).
Original magnification: 20 X, scale bar, 50 m. This figure is representative of at least three experiments performed on different days. The arrows
indicate the dense network fibers characteristic of the striatum.
0.0
Table 1. Effect of BM-21 on oxidative stress in cortex and striatum of ischemic rats.
Group GSH SOD GSH-Px Protein carbonyls MDA
(nM) (IU/mg) (IU/mg) (mol/mg) (mol/mg)
Cortex
Sham 67.60 4.81 4.43 0.40 1.23 0.12 0.18 0.02 2.95 0.19
Vehicle 44.99 2.75*** 2.89 0.25** 0.80 0.14*** 0.28 0.03* 3.92 0.23**
BM-21 62,96 1.61++ 4.05 0.23+ 1.07 0.11++ 0.20 0.02+ 2.98 0.15+
Striatum
Sham 59.78 2.42 3.22 0.49 1.05 0.12 0.24 0.04 2.45 0.25
Vehicle 42.12 2.96*** 1.81 0.25* 0.78 0.08* 0.32 0.03* 4.07 0.25**
BM-21 65.60 2.05*** 3.28 0.25+ 1.12 0.09+ 0.19 0.02+ 2.57 0.37++
Each group represents the mean SEM of eight animals. (*p<0.05, **p<0.01, ***p<0.001) significant different vs. sham; (+p<0.05, ++p<0.01)
significant different vs. vehicle. One way ANOVA followed by Tukey's multiple comparison test.
et al., 2009; 2012) this may result in decreased probably related to the inhibitory effect on
inactivation of SOD and GSH-Px and enhancement phosphorlipase A2 and cyclooxygenase 2 activity
of GSH concentration. These effects in turn may (Llanio et al., 2006) and antinociceptive effects in
result in decreased LPO and protein oxidation. inflammatory models of pain (Garateix et al., 2011).
Our results are consistent with the neuroprotec- Therefore, it is reasonable to suggest that the anti-
tive and the antioxidant properties previously inflammatory effects of BM-21 together with the
observed on acrylamide-induced neurotoxicity in antioxidant action may account for the decrease of
mice (Menndez et al., 2014) and global ischemia in brain edema.
Mongolian gerbils (Menndez et al., 2015). Therefore, It is noteworthy to note that BM-21 and
the current results suggest again that the active thalassiolin B inhibited acid-sensing ion channels
antioxidant compounds of BM-21 can cross the (ASICs) and exhibited antinociceptive effects in vivo
bloodbrain barrier and certainly function as given by oral route at the same dose that exhibited
antioxidants in cortex and striatum by directly neuroprotective action (Garateix et al., 2011). During
scavenging pathological concentration of reactive severe ischemia tissue pH falls below six that may
oxygen formed during ischemia. However, another cause neuronal death due to activation of ASICs
possibility is that BM-21 may act as an indirect (Xiong et al., 2004). Hence, another mechanism that
antioxidant by increasing the activity of the cellular contributes to the neuroprotective effects of BM-21
enzymatic antioxidants GSH-Px and SOD and GSH might be related to its effects on ASICs. In this
concentrations. In this regard, it has been shown regard, research shows that blockers of these
that polyphenolic compounds can act as neuropro- channels can act as neuroprotective agents in
tective agents by modulating antioxidant defenses models of ischemia (Xiong et al., 2004).
through its interaction with Nrf2 pathway. In line
with these findings, pre-treatment of non-ischemic CONCLUSIONS
rats with BM-21 at the same dosage regimen
The treatment with BM-21 exhibited neuro-
significantly decreased the susceptibility of brain
protective effects in the model of pMCAo. BM-21
homogenate to kainic acid and AAPH-dependent
extract significantly improved the neurological out-
LPO. This result suggests that the pre-treatment
come 24 h after pMCAo and reduced total infarct
with BM-21 might improve the brain antioxidant
and edema volume. Also, the extract also reduced
status protecting rats against the oxidative stress
neuron damage in cortex and striatum. The present
increased by ischemia.
results support the contribution of the antioxidant
In ischemic stroke brain edema induced by
properties of BM-21 in its neuro-protective effects.
oxidative stress and inflammation, is a crucial fea-
Hence, our results show that BM-21 is a promising
ture of ischemic injury. Brain edema formation has
agent for the treatment of pathologies implicating
been described as cytotoxic and vasogenic (Klatzo,
neurodegeneration such as cerebral ischemia.
1967). Free radicals exert their deleterious actions
However, further studies are needed to confirm this
during both cytotoxic and vasogenic edema either
possibility.
by directly contributing to blood-brain-barrier
(BBB) disruption or indirectly by triggering inflam-
matory pathway. Inflammatory mediators quickly CONFLICT OF INTEREST
released from injured tissue eventually lead to The authors declare no conflict of interest.
disruption of the BBB and brain edema. Previous
results have shown superoxide scavenging action of ACKNOWLEDGEMENT
BM-21 (Regalado et al., 2012) and the consistent This study was supported by International Foundation for
elevation of SOD activity detected in vivo in several Science (Project F/4237-1) and by the project CITMA 047/2012
experimental models after BM-21 administration (Cuba). The authors thank Department of Neurochemistry at
Institute Clemente Estable for supplying with experiments. The
(Regalado et al., 2011; Menndez et al., 2014). Besides, we
authors are grateful to Ms. Addys Palmero and Teresita del
also observed in vivo anti-inflammatory effects of Valln for their excellent technical assistance for the study.
BM-21 in acute models of inflammation in mice
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Author contribution:
Contribution Garca TE Menndez R Rivera F Garateix A Morales RA Regalado E Rodrguez JC Dajas F
Concepts or ideas X X X X X
Design X X X X
Definition of intellectual content X X X X X
Literature search X X
Experimental studies X X X X
Data acquisition X X X X
Data analysis X X
Statistical analysis X X
Manuscript preparation X X X
Manuscript editing X X
Manuscript review X X X X X X X X
Citation Format: Garca TE, Menndez R, Rivera F, Garateix A, Morales RA, Regalado E, Rodrguez JC, Dajas F (2017) Neuroprotective effects of
Thalassia testudinum leaf extract BM-21 on focal ischemia in rats. J Pharm Pharmacogn Res 5(3): 174-186.