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. 2023 Feb 8;72(3):336–345. doi: 10.1538/expanim.22-0176

Neuroprotective effects of betanin in mice with cerebral ischemia-reperfusion injury

Wachiryah Thong-asa 1, Kanthaporn Puenpha 1, Thannaporn Lairaksa 1, Siriwipha Saengjinda 1
PMCID: PMC10435356  PMID: 36754417

Abstract

Cerebral ischemia reperfusion (IR) injury as found in stroke is a complex and heterogeneous disorder and closely related to disability and death. Today, nutraceuticals and protective therapy to increase neuronal integrity and prevent pathological complication are common. We investigated the neuroprotective effect of betanin against cerebral IR injury in mice. Forty male institute of cancer research (ICR) mice were divided into Sham-veh, IR-veh, IR-Bet50 and IR-Bet100 groups. After 2 weeks of oral administration of normal saline (vehicle; veh) or 50 mg/kg or 100 mg/kg of betanin (Bet), mice were subjected to IR induction using 30-min bilateral common carotid artery occlusion, followed by 24 h of reperfusion. Brain infarction, oxidative status, cortical and hippocampal neurons and white matter pathologies were evaluated. Results showed that IR significantly increases brain infarction, Cornus Ammonis 1 (CA1) hippocampal and corpus callosum (CC) and internal capsule (IC) white matter degeneration (P<0.05). Brain oxidative status revealed significant elevation of malondialdehyde (MDA) together with a significant decrease in catalase (CAT) activity, induced by IR (P<0.05). Pretreatment with betanin 100 mg/kg led to a significant reduction in brain infarction and MDA, CA1 hippocampus, CC and IC white matter degeneration. Betanin also led to a significant increase in CAT activity (P<0.05), with enhancing effect on reduced glutathione levels (GSH, P<0.05). The present study revealed the neuroprotective efficacy of betanin against IR injury in mice’s brains, including its inhibition of lipid peroxidation, and boosting of GSH and CAT activity.

Keywords: betanin, hippocampus, ischemia-reperfusion injury, neurodegeneration, white matter

Introduction

Betanin (betanidin 5-O-β-D-glucoside) is a phytochemical product derived from beetroot (Beta vugaris L.) and has multiple health benefits. It is red-violet and has been used as a natural food colorant that prevents lipid oxidation in meats. It has high stability and remains biologically active during freezing or refrigerated food storage. Taken orally, betanin resists stomach gastric acid and remains stable after intestinal digestion [1]. As a water-soluble nitrogenated heterocyclic compound, betanin passively diffuses throughout the intestinal epithelium with its chemical structure unchanged. Therefore, it reaches body fluids and the bloodstream in an active form [2]. Betanin inhibits low-density lipoprotein (LDL) oxidation, cyclooxygenase (COX), inducible nitric oxide (iNOS), lysosome protease and inflammation [3,4,5,6]. It can help maintain oxidative defense mechanisms, as it is a hydrogen and electron donor, and can regulate antioxidative-enzyme gene expression. Betanin’s effect on erythroid 2-related factor 2 antioxidant response element leads to upregulation of glutathione S-transferase, NAD(P)H quinone dehydrogenase 1 and heamoxygenase-1 [7]. Hadipour et al. [8] also found antiapoptotic activity via SAPK/JNK and PI3 K inhibition. Recently, we depicted betanin’s neuroprotective effect in neurodegeneration mouse models, i.e., trimethyltin-induced toxicity and rotenone-induced mice with Parkinson’s. Betanin’s neuroprotective efficacy includes its potent antioxidative properties, such as its inhibitory effect on lipid peroxidation by attenuation of malondialdehyde (MDA) levels. Betanin also helped reduced glutathione (GSH), catalase (CAT) and superoxide dismutase (SOD) activities [9, 10]. Dugger and Dickson [11] found evidence of many pathomechanism-joining degenerative diseases, such as oxidative stress and inflammation. Silva et al. [12] depicted betanin’s multipath benefits in oxidative stress and inflammation. Therefore, the use of betanin as a nutraceutical treatment and supportive therapy is noteworthy, as it might protect against such diseases’ pathomechanisms.

Sun et al. [13] demonstrated the pathomechanism of ischemia-reperfusion (IR) injury, including oxidative stress and inflammation. It was defined as paradoxical exacerbation of cellular dysfunction and death. Initial ischemic-induced cellular energy deprivation and subsequent metabolic energy failure. During the ischemic period, a variety of deteriorate cascades were mentioned, including energy failure, excitotoxicity, intracellular calcium overload, reactive oxygen species (ROS) and reactive nitrogen species (RNS) formations, inflammation and edema. When ischemic tissue was repercussed with excessive oxygenated blood, deterioration was emphasized via oxidative stress-mediated extensive tissue destruction, i.e., lipid peroxidation as well as DNA, RNA and protein damage and finally led to disability and death [14, 15]. Betanin showed an excellent protective effect against IR injury in several organs, such as the heart, lungs and spinal cord, and its large contribution to antioxidation and anti-inflammation was frequently mentioned [16, 17]. We intended to obtain additional information about betanin’s protective effect against IR injury, especially in vulnerable organs, such as the brain, for which we have no recent evidence. It is well known that brain disease is incurable; therefore, preventing it with any supportive factors might be beneficial. As a nutraceutical treatment and preventive therapy, betanin may protect against this pathogenic condition. We expected the nurturing of cellular integrity and extending of endurance against pathogenic induction to counteract acute and delayed neuronal deaths as well as damage to specific vulnerable brain areas as a result of global ischemia [18]. Therefore, we aimed to investigate betanin’s neuroprotective effect against IR injury in mice’s brains.

Materials and Methods

Chemicals and reagents

We purchased the betanin, 2, 3, 5-triphenyltetrazolium chloride (TTC), cresyl violet, luxol fast blue and other biochemical analysis reagents used in the present study from Chemical express Co., Ltd., Merck Millipore (Samutprakarn, Thailand).

Animals

We obtained twelve-week-old (40–45 g) male institute of cancer research (ICR) mice (Mus musculus) from the National Laboratory Animal Center, Mahidol University (Nakhon Pathom, Thailand). We housed one per cage in a temperature-controlled room (23 ± 2°C) with a 12/12-h light-dark cycle. All mice had access to standard food (N.082G) and reverse-osmosis water. The Animal Ethics committee, Faculty of Science, Kasetsart University, approved the experimental procedure (ID#ACKU65-SCI-007).

Experimental procedure

We randomly divided forty male ICR mice into 4 experimental groups (n=10 for each group): Sham-veh, IR-veh, IR-Bet50 and IR-Bet100. We administered a two-week pretreatment with normal saline (vehicle; veh) in the Sham-veh and IR-veh groups and betanin (Bet) at 50 mg/kg and 100 mg/kg orally in the IR-Bet50 and IR-Bet100 groups, before ischemia reperfusion (IR) induction, respectively. Figure 1 shows all the experimental protocols.

Fig. 1.

Fig. 1.

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Experimental protocol.

Induction of cerebral ischemia reperfusion

After two weeks of administration, all mice were fasting and prepared for operation. They received a 25-µl intraperitoneal injection of Zoletil 100 + X-Lazine (4:1), following IR induction by Sun et al. [13]. The mice in the IR groups underwent a 30-min bilateral common carotid artery occlusion. After removing the occlusion, we sutured the surgical wound and allowed the mouse to rest in a recovery chamber before returning it to its home cage. The mice in the Sham-veh group underwent surgery without arterial occlusion. After 24 h, we sacrificed all mice with an intraperitoneal injection of 50 µl of Zoletil 100 + X-Lazine (4:1). We quickly removed the brains for further analyses.

Cerebral infarction analysis

After quickly removing the brains, we washed them in cold normal saline and cut them into 2-mm serial sections using an acrylic brain block. We incubated the brain pieces in 2% TTC at 37°C for 10 min and stored them in 10% neutral buffer formalin. We captured the brain pieces 24 h later with a reference scale and used them for infarction analysis in Image J. Area analysis followed the principal TTC staining, that was area representing of reducing agent, i.e., NADH, which turned the viable areas red. On the other hand, infarction areas became pale due to their lack of dehydrogenase activity and inability to convert tetrazolium to formazan. We represented the whole brain infarction area as % infarction using the formula % infarction = [pale / (pale + red)] × 100.

Neuronal and white matter histological analysis

We further processed the brain pieces after infarction analysis for paraffin embedding and 5-µm sectioning with a microtome. Sampling area reference using the mouse brain stereotaxic starting from Bregma −1.94 [19]. We selected five slides from each mouse with 125-µm intervals [20]. We deparaffinized and rehydrated all selected slides with xylene and serial changes of ethanol and incubated them in 0.1% luxol fast blue at 56°C overnight. We then washed the slides in 95% ethanol and distilled water and differentiated them in a lithium carbonate solution for 30 s, followed by 70% ethanol. After microscopic checking for sharply defined white matter, we counter-stained the slides with 0.1% cresyl violet before dehydration and finished by covering the slides [21].

We microscopically captured areas of interests, i.e., the cerebral cortex, subfields of the hippocampus Cornus Ammonis (CA) 1, CA3 and dentate gyrus (DG) as well as the white matter of the corpus callosum (CC), internal capsule (IC) and optic tract (Opt.). With 400× magnification, 2 investigators used 3 non-overlapping images from both hemispheres for neuronal counting in blind fashion using Image J. Viable cells were characterized as light purple and a clear nucleus and nucleolus. In contrast, cells that had undergone shrinkage were dark purple with a barely visible nucleus and nucleolus and defined as dead. We interpreted neuronal data as % degeneration using the formula % degeneration = [dead / (dead + viable)] × 100.

We captured white matter in the CC, IC and Opt. at 400× magnification and analyzed 3 non-overlapping images from both hemispheres using Image J. We presented the data from the intact fiber with luxol fast blue (LFB) as % area of white matter intact.

Biochemical analysis

After washing the brains with cold normal saline, we homogenated them in a 10% w/v phosphate buffer saline (50 mM, pH 7.4). We kept half of the brain tissue homogenate for MDA and GSH assays. We centrifuged the rest of the brain tissue homogenate at 10,000 g at 4°C for 10 min and then analyzed the supernatant for total protein level and SOD and CAT activity.

We determined total brain protein using Lowry’s assay [22]. We mixed 0.2 ml of the supernatant with 2 ml of solution D (ratio 48:1:1) (2% w/v Na2CO3 in 0.1 N NaOH: 0.5% w/v CuSO4-5H2O in distilled water: 1% w/v C4H4KNaO6-4H2O) and incubated it for 10 min before adding 0.2 ml of 1 N Folin-Ciocalteu reagent (1:1). After 30 min of incubation, we read the mixture at 600 nm. We calculated protein concentration using the standard curve of bovine serum albumin 0, 0.009, 0.018, 0.027, 0.035 and 0.043 mg/ml (y=0.1388x − 0.03; R2=0.9934).

We measured the MDA level in the brain tissue to determine lipid peroxidation. In brief, we mixed 0.2 ml of brain homogenate with 4% sodium dodecyl sulfate, 1.5 ml of 20% acetic acid and 1.5 ml of 0.5% thiobarbituric acid. We heated the mixture at 95°C for 1 h and then centrifuged it for 10 min at 3,500 rpm before reading the supernatant at 532 nm. We calculated the MDA concentration using the standard curve of MDA concentration 0, 0.93, 1.85, 2.76, 3.66 and 4.55 µM (y=0.148x − 0.1082; R2=0.9973). We presented brain tissue MDA concentration as µM/mg of protein [23, 24].

We measured GSH by mixing 0.1 ml of homogenate with 10% tricarboxylic acid and centrifuging it for 10 min. We collected 0.5 ml of supernatant and mixed it with 5, 5′ -dithios 2-nitro benzoic acid. We increased the final volume of the mixture to 3 ml using PBS before reading it at 412 nm. We calculated GSH concentration using the standard curve of GSH concentration 0, 0.0132, 0.0263, 0.0392, 0.0519 and 0.0645 µM (y=0.1437x − 0.0538; R2=0.9866). We presented brain tissue GSH concentration as µM/mg of protein [25].

We measured SOD activity by mixing 0.1 ml of supernatant with 0.1 ml of EDTA (0.0001 M), 0.5 ml of carbonate buffer (pH 7.9) and 1 ml of epinephrine (0.0003 M). We read the absorbance every 30 s at 480 nm for 3 min. We presented enzyme activity as U/mg of protein, using the standard curve of SOD concentration 0, 0.0058, 0.0294, 0.117 and 0.294 µg/ml (y=0.0015x + 0.0001; R2=0.998). Standard SOD activity was 6,150 U/mg (Merck, Darmstadt, Germany) [20].

We determined enzyme activity of CAT using 50 µl of supernatant and increased the volume to 3 ml with 0.05 M PBS (pH 7.4) containing 0.01 M H2O2. We read the absorbance every 30 sec at 240 nm for 3 min, and we calculated CAT activity with reference to the extinction coefficient of H2O2 and presented it as U/mg of protein [26].

Statistical analysis

We interpreted all data as mean ± SEM. We conducted one-way analysis of variance and used Fisher’s PLSD post hoc test for group comparison. We considered a P-value less than 0.05 statistically significant.

Results

Attenuating effect of betanin on brain infarction

The whole-brain infarction area with TTC staining in Fig. 2 was mostly pale in the IR-veh group. We found distribution areas of infarction in the forebrain, cerebral cortex, striatum and hippocampus. Thirty-minutes bilateral common carotid artery occlusion followed by 24-h reperfusion significantly increased % brain infarction in the IR-veh group compared to the Sham-veh group (P=0.0006). Pretreatment with betanin at 50 mg/kg and 100 mg/kg significantly decreased brain infarction compared to the IR-veh group (P=0.0159 and 0.0013, respectively). Although 50 mg/kg of betanin showed a significant difference from the IR-veh group, it did not reach the normal value, as indicated by the significant difference from the Sham-veh group (P=0.0444). This result indicated the effective dose of betanin pretreatment was 100 mg/kg to prevent IR-induced brain infarction.

Fig. 2.

Fig. 2.

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Attenuating effect of betanin on brain infarction. Brain infarction area was indicated by 2, 3, 5-triphenyltetrazolium chloride (TTC) staining. Infarction area shows as a pale area resulting from tetrazolium and viable area shows as a red area resulting from formazan. Histogram showed the % brain infarction area in each group. * indicates significant difference from Sham-veh group; # indicates significant difference from ischemia reperfusion (IR)-veh group.

Protective effect of betanin on vulnerable neurons and white matter

As an area of the brain vulnerable to global ischemia, we chose the hippocampus for neuronal histological analysis separated into subfields CA1, 3 and DG. We counted viable and dead cells and presented them as % degeneration. Only the CA1 subfield exhibited significant degeneration after IR (Fig. 3). The % degeneration of CA1 neurons significantly increased in the IR-veh group compared to the Sham-veh group (P=0.0272). Pretreatment with 50 mg/kg and 100 mg/kg of betanin significantly decreased % degeneration, compared to the IR-veh group (P=0.0256 and 0.0228, respectively). The CA3 and DG subfields showed that only the IR-veh groups had increased % degeneration (but the result was statistically insignificant), compared to the Sham-veh group (P=0.2062 and 0.4832, respectively).

Fig. 3.

Fig. 3.

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Protective effect of betanin on vulnerable neuron. Hippocampal neurons with 0.1% cresyl violet staining in Cornus Ammonis 1 (CA1), CA3 and dentate gyrus (DG) subfields. We captured photomicrographs at 400× magnification with a 50-µm scale bar. Viable cell indicated by light purple color and clear nucleus and nucleolus. Dead cell indicated by shrinkage, dark purple color and barely visible nucleus and nucleolus. Histogram showed the % degeneration of neurons in CA1, CA3 and DG subfields. * indicates significant difference compared to Sham-veh group; # indicates significant difference compared to ischemia reperfusion (IR)-veh group.

Besides the hippocampus, we also assessed the cortex neuron (Fig. 4). Only the IR-veh group showed increased % degeneration (but the result was statistically insignificant) compared to the Sham-veh group (P=0.1124). There also represented statistically insignificant compared to betanin treated groups as well (P>0.05).

Fig. 4.

Fig. 4.

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Cerebral cortex neurons with 0.1% cresyl violet staining. We captured photomicrographs at 400× magnification with a 50-µm scale bar. Viable cell indicated by light purple color and clear nucleus and nucleolus. Dead cell indicated by shrinkage, dark purple color and barely visible nucleus and nucleolus. Histogram showed the % degeneration of neurons in cerebral cortex.

We observed white matter in three areas: CC, IC and Opt. (Fig. 5). The result of LFB myelinated fiber density revealed that IR significantly reduced the intact fiber in CC and IC but not in Opt., compared to the Sham-veh group (P=0.0371, 0.0182 and 0.3685, respectively). This result indicates that white matter is also vulnerable to IR. In addition, pretreatment with 50 mg/kg and 100 mg/kg of betanin resulted in a significant preventive effect in the reduction of LFB fiber density in CC (P=0.0163 and 0.0104, respectively) and IC (P=0.0002 and <0.0001, respectively), compared to the IR-veh group.

Fig. 5.

Fig. 5.

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Protective effect of betanin on white matter degeneration. Myelinated fiber intact was indicated by luxol fast blue (LFB) staining in corpus callosum (CC), internal capsule (IC) and optic tract (Opt.). We captured photomicrographs at 400× magnification with a 50-µm scale bar. Histogram showed the % white matter intact in CC, IC and Opt. * indicates significant difference compared to Sham-veh group; # indicates significant difference compared to ischemia reperfusion (IR)-veh group.

Betanin’s effect on brain oxidative status

IR induction partially changed brain oxidative parameters, i.e., MDA, GSH, CAT and SOD (Fig. 6). MDA significantly increased in the IR-veh group (P=0.0167) and CAT activity significantly decreased, compared to the Sham-veh group (P=0.0137). Betanin at 50 mg/kg and 100 mg/kg significantly reduced MDA levels, compared to the IR-veh group (P=0.0291 and 0.0379, respectively). Only 100 mg/kg of betanin resulted in a significant increase in CAT activity, compared to the IR-veh group (P=0.0202). IR slightly decreased the GSH level and both doses of betanin significantly increased the GSH level, compared to the IR-veh group (P=0.0380 and 0.0350, respectively). We found no significant differences in SOD activity with either IR induction or betanin pretreatment. Our results indicate a successful antioxidative dose of betanin at 100 mg/kg to ameliorate MDA together with GSH and CAT activations.

Fig. 6.

Fig. 6.

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Biochemical analysis of brain oxidative parameters, i.e., malondialdehyde (MDA), glutathione (GSH), catalase (CAT) and superoxide dismutase (SOD). * indicates significant difference compared to Sham-veh group; # indicates significant difference compared to ischemia reperfusion (IR)-veh group.

Discussion

The use of betanin as preventive therapy seems to nurture neuronal integrity and protect against the deteriorative effects of disease. In cardiovascular disease therapy, the common complications were recirculation and reactivation of aerobic respiration. The paradoxical exacerbation of extensive tissue damage was induced via free-radical formation, leading to oxidative damage. Therefore, the use of antioxidants to mitigate the deterioration cascades is very helpful. We used betanin as a preventive therapy against brain IR injury, and its outcomes were protection against brain infarction, oxidative stress, damage to vulnerable neurons and white matter degeneration.

Ischemia reperfusion can cause neuronal and white matter injuries via a variety of mechanisms, i.e., excitotoxicity, inflammation, intracellular calcium overload, necrosis and apoptosis [15]. During the ischemic period, abrupt energy depletion induces membranous ionic imbalance by K+ efflux-induced hyperpolarization followed by anoxic depolarization due to the dramatic redistribution of ions, i.e., Na+, Cl− and Ca2+ influx. The following results, i.e., cellular acidosis, edema and overactivation of neurotransmitter release, further induced neuronal dysfunction and acutely death. Accumulation of intracellular calcium also plays a major role in downstream deteriorations, including oxidative stress and delayed apoptotic death [18].

Lack of oxygenated blood during ischemia together with reperfusion of former ischemic tissue led to extensive diffuse infarction [14]. The present study also showed the distribution of infarction brain tissue, for instance in the forebrain, cerebral cortex, striatum and hippocampus. In addition, we showed betanin’s protective effect against brain infarction. During the ischemic period, ischemic-induced gene expression, such as cyclooxygenase-2 (COX-2), along with augmentation of prostaglandin, was one of contributing pathways that included an inflammatory response and destructive process [27]. Betanin was indicated as a COX-2 inhibitor [6]. Its anti-inflammatory effects, including TNF-α and IL-β reduction and increased IL-10, were also reported [5]. Betanin’s protective effect in endothelial cells from a cytokine-induced redox imbalance was also indicated [28]. Betanin therefore has the beneficial effects of endothelial nurturing and maintaining blood flow. Moreover, betanin regulated metabolism-related glucose enzymes, such as glucokinase, glucose-5-phosphatase, pyruvate kinase, glucose-6-phosphate dehydrogenase and fructuse-1, 6-bisphosphate [29], indicating that betanin helps maintain aerobic metabolism. Corroborating these findings, betanin’s nurturing effects were indicated in vascular endothelial function, cerebral blood flow and energy production; therefore, it can protect against brain infarction.

Oxidative stress in IR injury was indicated during the ischemic and reperfusion periods. Ischemic-induced overactivation of the NMDA receptors contributed to oxygen radical production by uncoupling the neuronal mitochondrial electron transport [30]. They also increased membranous destruction induced by catabolic enzyme activation, such as phospholipase A2 [31] and phospholipase C [32], which further exacerbated free-radical formation and inflammation. Ischemic-induced intracellular calcium overload also activated the Ca2+-dependent catabolic enzyme (i.e., calpain), and when combined with excessive O2 during reperfusion, it converted to O2−. Hydroxyl radicals as a cascade product originating from O2− can exacerbate damage via lipid peroxidation, as indicated by an increased MDA level during IR [33]. Similar to other studies, we showed that IR led to increased MDA and that betanin helped reduce MDA [9, 17, 34]. Betanin inhibited this toxic metabolite of lipid peroxidation, depending on its cyclic amine and hydroxyl groups, which act as good hydrogen and electron donors [35]. Besides stabilizing ROS, betanin regulates anti-oxidative enzymes’ gene expression, i.e., glutathione-S-transferase, heamoxygenase-1 and NAD(P)H quinone dehydrogenase 1 [7]. Betanin’s restorative effect on the redox status via up-regulation of SOD, CAT and GPx activities was also indicated [12, 36]. Therefore, betanin protects against the suppression of antioxidative defense mechanisms resulting from IR [15, 26]. The study of IR in animals has also revealed the differences in oxidative response depending on IR severity, duration of ischemia and reperfusion and susceptibility of specific brain areas [37]. The present study revealed significant reduction in CAT activity after IR induction and showed that only betanin at a dose of 100 mg/kg led to a significant increase in CAT activity. Interestingly, GSH levels did not significantly decrease due to IR in the present study, but both doses of betanin significantly enhanced GSH. However, SOD did not change with betanin pretreatment or IR exposure, likely because despite the 30-min bilateral common carotid artery occlusion and 24 h reperfusion in ICR mice, the entire brain’s SOD remained unchanged, leading to an insignificant betanin effect. This result is contrary to Sun and colleagues’ findings although they also used ICR mice with an IR period of 30 min/24 h [13]. We consider age difference in the used animals and mice’s susceptibility to IR [38]. However, additional studies are necessary to explain this discrepancy.

The IR induction method that we used produced a global effect on the brain, so we must consider areas vulnerable to global ischemia. We found that the CA1 area of the hippocampus was the most susceptible to IR, as well as CC and IC white matter. Differences in neuronal susceptibility to global ischemia were associated with the number of glutamate receptors and high intrinsic oxidative stress [39, 40]. Many NMDA receptors in the CA1 hippocampus were associated with excitotoxicity [39]. The neurons in hippocampal areas CA1 and CA3 were similar in morphology (as pyramidal neurons), but they exhibited differences in their responses to oxidative stress. The pyramidal neurons in the CA1 region suffered massive cell death, but those in CA3 mostly survived when exposed to oxidative stress induction [40]. This selective sensitivity of CA1 neurons was also found in other disease-related conditions such as hypoxia, ischemia and neurodegenerative diseases [41]. In addition, cortical and DG neurons were less susceptible than the CA1 hippocampal neurons [42]. We found that the rate of CA1 neuronal degeneration was significantly increased by IR and that betanin at both doses significantly protected against CA1 neuronal degeneration. We found the degenerating neurons mostly in pyknotic and apoptotic forms [43], and these degenerating neurons were reduced by betanin pretreatment. The underlining mechanisms of betanin in anti-inflammation and antioxidation have been clearly depicted [12]. Anti-apoptotic death, including decreased phosphorylation of SAPK/JNK and increased phosphorylation of PI3 kinase, has also been reported [8]. Along with neurons, white matter is also vulnerable to IR. Oxidative stress and excitotoxicity were mentioned in ischemic-induced oligodendrocyte damage and white matter degeneration [44]. We found significant LFB fiber density reduction in the CC and IC after IR induction. The change in myelinated fiber indicated oligodendrocyte dysfunction and death. White matter was rarefied, myeline decreased, diffuse tissue was attenuated and oligodendrocytes degenerated. These effects were significantly attenuated in the betanin-treated group, showing that betanin’s neuroprotective effect is not limited to neuron but extends to white matter. The present study showed betanin’s neuroprotective effects against brain IR injury in mice, such as inhibitory effects on brain infarction, oxidative stress, lipid peroxidation and vulnerable neuronal and white matter degeneration. Due to our present study limitation, the global reduction/reperfusion of cerebral blood flow during ischemic and reperfusion period as well as molecular mechanisms concurrent supportive pathway that related to neuroprotection still be required. Therefore, further studies are necessary.

Conclusion

Betanin exhibits neuroprotective effects against IR injury in mice’s brains due to its potent antioxidative properties, inhibitory effects on brain infarction, oxidative stress, lipid peroxidation and vulnerable neuronal and white matter degeneration.

Ethics Approval

All applicable international and national guidelines for the care and use of animals were followed. The experimental procedure was approved by the Animal Ethics committee, Faculty of Science, Kasetsart University (ID#ACKU65-SCI-007).

Author Contribution

Wachiryah Thong-asa conceived and designed research, analyzed data and wrote the manuscript. Kanthaporn Puenpha, Thannaporn Lairaksa and Siriwipha Saengjinda conducted the experiment. All authors read and approved the manuscript for publication.

Consent to Participate

All authors agree to participate.

Consent for Publication

All authors agree to publish.

Conflict of Interest

The authors declare that they have no conflict of interest.

Data Availability

Available upon request.

Acknowledgments

This research was supported by International SciKU Branding (ISB), Faculty of Science, Kasetsart University.

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