Бетен смягчает недостатки в моторном поведении, нейротоксическом воздействии и нейро-воспалительной реакции в крысиной модели обезвоживания.

Introduction

Myelination is a meticulously coordinated process influenced by a variety of intrinsic and extrinsic factors [20] . While the precise mechanisms underlying myelin formation remain incompletely elucidated, it is generally understood to occur in stages, commencing with the differentiation of oligodendrocyte progenitor cells (OPCs) into oligodendrocytes (Ols), followed by the ensheathment of axons, and culminating in the maturation of the myelin sheath [46] .

A demyelinating disease is defined as any central nervous system (CNS) disease that causes damage to the neuronal myelin sheath. These diseases can be categorized into four types based on their underlying pathogenesis: multiple sclerosis (MS), acute disseminated encephalomyelitis, progressive multifocal leukoencephalopathy, and extrapontine myelinolysis [20] . The pathophysiology of MS is particularly complex, involving the infiltration of peripheral T cells into the CNS, apoptosis of Ols, activation of microglia, and neuroinflammatory processes that lead to both demyelination and axonal injury [47] . Ols are particularly susceptible to damage from oxidative stress, cytotoxicity, deprivation of trophic factors, and activation of apoptotic pathways. Both demyelination and inflammation are hallmark features of MS, contributing to axonal damage and subsequent cerebral atrophy observed in later stages of the disease [16] , [68] . Notably, the early phase of MS is marked by axonal demyelination, which can be reversed through a spontaneous regenerative process known as remyelination, wherein resident OPCs differentiate into new myelinating Ols [22] . However, this regenerative capacity diminishes as the disease advances, partly due to impaired generation of OPCs or their failure to differentiate into mature myelinating Ols [4] , [65] .

Cuprizone (CPZ) is a low molecular weight copper-chelating agent that influences cellular metabolism and leads to reversible demyelination [19] . The administration of 0.2 % (w/w) CPZ mixed with rodent diet over a period of 5–6 weeks has been extensively utilized to achieve reliable demyelination across various regions of the brain, notably in the corpus callosum (CC), which is the largest myelinated pathway [30] . Exposure to CPZ disrupts energy metabolism in Ols, resulting in apoptosis and the accumulation of myelin debris. This process activates microglia, which in turn release an excess of pro-inflammatory cytokines, further aggravating the demyelination process [5] , [64] .

Betaine, also known as trimethylglycine, is a methylated derivative of glycine that is soluble in water and possesses three reactive methyl groups. It functions as an organic osmolyte, protecting cellular proteins, enzymes, and membrane structures from various environmental stressors, including elevated temperatures and salinity. Additionally, betaine facilitates the transfer of a methyl group to dimethylglycine through the action of the enzyme betaine homocysteine methyl transferase (BHMT) [6] , [9] . BHMT is the sole enzyme identified that utilizes betaine as a substrate, facilitating the transfer of a methyl group from betaine to homocysteine (Hcy) and subsequently converting Hcy into methionine. While betaine can cross the blood-brain barrier (BBB), the remethylation of Hcy to methionine mediated by BHMT predominantly occurs in the liver [8] . In the context of osmotically stressed mouse brains, betaine serves not only as an osmolyte but also plays a role in modulating the synthesis and reuptake of inhibitory neurotransmitters [44] . The pharmacological properties of betaine are diverse, encompassing anti-inflammatory, antioxidant, antiepileptic, neuroprotective, anti-depressive, and cognitive-enhancing effects [47] , [65] , [78] . Moreover, previous research has shown that betaine is effective against several neurodegenerative diseases [38] , [48] , schizophrenia [53] , ischemic stroke [47] , and neuropathic pain [68] . Nonetheless, there is currently no evidence regarding the modulation of Hcy-betaine metabolism in existing models of MS. Consequently, this study aims to explore the potential protective and therapeutic effects of betaine in response to varying doses of CPZ-induced demyelination in a rat model, while also investigating the underlying mechanisms the remyelinating effects of betaine in the context of CPZ-induced demyelination through the modulation of motor behavioral deficits, redox status, neuroinflammation, ultrastructural changes, and MBP expression levels. Furthermore, the results were validated through the identification of the target proteins utilizing a bioinformatics tool.

Materials and methods

Animals

Forty adult male Wistar rats weighing 160 ± 10 g were purchased from the Faculty of Medicine, Alexandria University (Alexandria, Egypt). Rats were housed in stainless steel cages at a constant temperature (23 ± 2 °C) and under a 12 h light/dark cycle. Animals were acclimated for 1 week before starting the experiment and were provided access to rodent chow and water ad libitum. Body weight was measured weekly during the experiment. All procedures were done according to institutional guidelines for the use of experimental animals and approved by Institutional Animal Care and Use Committee (IACUC) (protocol approval code: AU04200926101), Alexandria University, Egypt. Procedures were conformed to NIH (National Institutes of Health) Guidelines for the Care and Use of Laboratory Animals.

Experimental design

Animals were randomly assigned into 5 groups (n = 8). The first group served as a control group and received 0.2 ml of corn oil with 1 ml of distilled water for 3 weeks. The second group received an oral daily dose of 1 g/kg betaine (98 %; Cat. no. B2629) for 3 weeks [24] , [75] . The third group received an oral daily dose of 600 mg/kg CPZ (Cat. no. C9012) dissolved in corn oil for one week followed by 200 mg/kg CPZ during the last 2 weeks [28] . The fourth (protective) group received an oral daily dose of 1 g/kg betaine for one week and in combination with 200 mg/kg CPZ during the last 2 weeks. The fifth (therapeutic) group was orally administered with 600 mg/kg CPZ for one week, then given betaine at a dose of 1 g/kg/day in combination with 200 mg/kg CPZ during the last 2 weeks (see Fig. 1 ).

Experimental animals and research design.

After two weeks, animals were subjected to neurobehavioral tests: inverted screen and rotarod tests.

Neurobehavioral assessment

Kondziela's inverted screen test

The inverted screen of the Kondziela is a test for measuring muscle strength and body coordination. The inverted screen test apparatus was a 43 cm 2 wire mesh made up of 12 mm 2 separated by 1 mm across the wire. It is surrounded by a 4 cm deep wooden beading, which anticipates the incidental rat that attempts to climb on the other side. Following the training day, the rat was placed in the center of the wire mesh screen, and the screen was rotated to an inverted position for 2 seconds, with the rat's head first going down. A steady position for the screen was maintained 40–50 cm above a padded surface. When the rat fell off, the time was recorded, or it was removed when the 60 second mark was reached. Before each trial, the apparatus mesh was cleaned with a 70 % ethanol solution [18] .

Rotarod test

The rotarod is a basic assessment tool for rodent balance and coordination that provides one measure of locomotor capacity. Before the experiment day, all rats were trained to walk against the rotation of the rotarod drum at a speed level accelerating from 10 to 20 rpm for a maximum of two minutes. Following the training days, rats were tested, and the time the animals remained on the rotarod was measured. The mean latency to fall off the drum was then recorded in seconds, and the rats that remained in the drum for up to 300 seconds were removed, with the recorded time for them being 300 seconds. Before each trial, the apparatus was cleaned with 70 % ethanol [58] .

Blood and brain tissue samples

At the end of the study period, all rats were anaesthetized by intraperitoneal injection of a mixture of xylazine and ketamine (xylazine; 5 mg/kg + ketamine; 100 mg/kg), and blood samples (n = 5) were collected by cardiac puncture into glass tubes without anticoagulant, and serum was separated by centrifugation at 3000 rpm for 10 min at 4 °C.

Then, animals were Afterward, each of the abovementioned groups was divided into three subgroups. The animals of each subgroup were sacrificed, and their CC were appropriately processed for [A] assessment of biochemical parameters (n = 5), which the CC of each animal was immediately excised, washed in cold saline, and stored at −80 °C, [B] semithin sectioning and TEM studies (n = 3/ right half of CC), and [C] immunohistochemical examination (n = 3/ left half of CC).

Serum analysis

Assessment of Hcy concentration

The concentration of Hcy in serum was determined by ELISA reader at 450 nm wavelength by using a commercially available kit (Cusabio, Wuhan, China, cat. no. CSB-E13376r), and the results were recorded as µmol/L. Each manufacturing kit provided a standard curve to determine sample concentration.

Biochemical analysis

Each CC was homogenized separately in 5 ml cold phosphate buffer (50 mM potassium phosphate, 0.1 mM EDTA, pH= 7.5) using a polytron (Tekwar model, TR-10, West Germany). The homogenates were centrifuged at 12000 rpm for 10 minutes in a cooling centrifuge (Hettich model EBA12R, Germany). Then, the supernatants were stored at −20 °C for analysis.

Assessment of redox status

The lipid peroxidation level was measured colorimetrically as malondialdehyde (MDA) using kits purchased from Biodiagnostic (Giza, Egypt) according to the method of Essawy et al. [23] and Ohkawa et al. [52] . Using spectrophotometer, the color intensity of the sample (unknown) and standard was measured against a blank at 534 nm. The results were expressed as nmol MDA/g tissue.

The reduced glutathione (GSH) concentration was assayed by the method of Beutler et al. [13] . A yellow compound is produced by reducing 5, 5′ dithiobis (2-nitrobenzoic acid) (DTNB) with GSH. The absorbance of the reduced chromogen of sample and standard against the blank was measured at 405 nm. The results were expressed as mmol/g tissue.

Superoxide dismutase (SOD) and catalase (CAT) activities were assessed using colorimetric kits purchased from Biodiagnostic (Giza, Egypt) according to according to the previously procedures reported by Yousef et al. [76] . Using spectrophotometer, the color intensity of the sample (unknown) and standard was measured against a blank at 510–560 nm. The results were expressed as unit/g tissue.

Assessment of IL-4 and IL-17

IL-4 and IL-17 were measured in CC homogenate using a sandwich enzyme immunoassay technique utilizing ELISA kits according to the manufacturer’s instructions. IL-4 and IL-17 ELISA kits were purchased from INNOVA Biotech (Beijing, China). The micro-ELISA strip plate included in this kit has been pre-coated with an antibody specific to IL-4, IL-17, standards or samples are added to the appropriate micro-ELISA strip plate wells and combined to the specific antibody. Each micro-ELISA strip plate well is incubated with an HRP-conjugated antibody specific for IL-4 and IL-17. Those wells containing IL-4 or IL-17 and HRP-conjugated IL-4 or IL-17 counteracting agents will initially appear blue and then turn yellow after the expansion of the halt solution. The level of IL-4 was expressed as ng/g tissue, while the level of IL-17 was expressed as pg/g tissue.

Semithin sectioning, TEM examination and morphometric analysis

The CC (n = 3, right half) were quickly immersed in formalin-glutaraldehyde (4F1G) fixative (pH 7.2) for semithin sectioning and TEM studies. The CC tissues were postfixed with 2 % osmium tetroxide at 4 °C for 2 h, washed in buffer, dehydrated in graded ethanol, and embedded in epon-araldite mixture. Semithin Section (1 µm thick) were stained with 1 % toluidine blue in 1 % sodium tetraborate and observed under a light microscope (×1000; Olympus BX51). Photomicrographs of toluidine blue-stained semithin sections were obtained to assess the % demyelination and density of myelinated fibers (number of fibers/mm 2 ).

Ultrathin sections (50 nm thick) were prepared, stained with uranyl acetate and lead citrate, and then observed using a TEM (100CX Japan, 80 kV). Image J free software was employed for image analysis and quantification (Image J 1.52e). The axon diameter (µm), fiber diameter (µm), myelin thickness (µm), and G ratio were measured in at least 50 axons per sample. The myelin G ratio, which represents the relationship between the inner and outer diameters of the myelin sheath, is a crucial characteristic of white matter. The G ratio was calculated by the axon diameter/whole fiber diameter ratio. Therefore, a quite demyelinated fiber would have a G ratio = 1, and the ratio would be < 1 in myelinated fibers [17] , [35] .

Immunohistochemical examination

Three rats (left halves) from each group were perfused through the left ventricle of the heart with 4 % paraformaldehyde in 0.1 M phosphate buffer saline (PBS). The fixed brains were taken out, preserved for 24 hours in paraformaldehyde, and then embedded in paraffin. The CC was present in five-micrometer coronal slices that were placed on electrocharged slides. Tissue sections were immersed in a Diva Decloaker (Biocare Medical LLC, Concord, CA) solution at 100 °C in a pressure cooker for three minutes to retrieve epitopes. Rinses with TBS-tween were always performed after 15 minutes of 0.3 % hydrogen peroxide (H 2 O 2 ) treatment to block nonspecific background staining (Background Sniper; Biocare Medical) and endogenous biotin and biotin-binding proteins (avidin/biotin blocking kit; Vector Laboratories Inc., Burlingame, CA).

Additionally, slides were stained using anti-MBP immunohistochemistry, which was carried out on an automated platform called the Ventana Discovery XT (Roche/Ventana, Tucson, Arizona). After deparaffinizing, the slides were rehydrated. Endogenous biotin blocking was carried out online for four minutes, and then the anti-MBP primary antibody (Abcam. Ab40390 (1ug/ml) LotGR3264120–1) at a 1:1000 dilution was incubated for thirty-two minutes. At a 1:1000 dilution, the biotinylated GtxRb secondary (Invitrogen 65–6140 LotTD268284) was incubated for 30 minutes. Using the standard DABMAP Kit (Roche/Ventana 760–124), HRP-based detection was carried out online and was followed by a hematoxylin counterstain. Slides were then covered with xylene-cleared-grade alcohol and dehydrated. Using an Olympus microscope (Olympus LC20, Germany) fitted with a digital camera, stained slides were examined [61] .

Bioinformatics analysis

The identified target proteins were entered into the String Gene Interaction Network database ( https://string-db.org/ ). A search for the selected target proteins was conducted under the "Multiple proteins" option, resulting in the generation of a PPI network map.

Statistical analysis

Statistical analysis was carried out using a statistical analysis program (IBM SPSS version 20), and all results are presented as the mean ± S.E. The Shapiro˗Wilk test was used to verify the normality of distribution. Statistically significant differences were determined by one-way ANOVA followed by Tukey’s HSD for multiple comparisons. Values were considered significantly different when P ˂ 0.05. Spearman’s correlation coefficient was used to determine any correlations between variables.

Results

Betaine restores CPZ-induced weight loss

The experimental animals' body weights were measured at the start and during three weeks of treatment, reflecting the initial and final weights, respectively ( Fig. 2 ). Weight gain was measured and analysed. When compared to the control and betaine groups, CPZ-treated rats had significantly lower body weight. Animals treated with betaine and CPZ (protective and therapeutic groups) exhibited significantly higher body weights than the CPZ group.

Effect of CPZ, betaine and their combination on the body weight changes of male rats for 3 weeks. Values are presented as mean ± S.E; n = 5 animals; mean values having different superscript letters a, b, c, d & e are significantly different from each other (P < 0.05). a, P < 0.05 vs. control. b, P < 0.05 vs. betaine. c, P < 0.05 vs. CPZ. d, P < 0.05 vs. protective (betaine + CPZ), e, P < 0.05 vs. therapeutic (CPZ+ betaine).

Betaine restores CPZ-induced neurobehavioral disturbance

In this study, rats' muscular power and gripping abilities were measured using the inverted screen test ( Fig. 3 A). When compared to the control group, CPZ administration dramatically (F (4,24) = 23.569 , P ˂ 0.001) shortened the time of falling from the screen and demonstrated muscle weakening. Furthermore, betaine administration before and after CPZ treatment improved muscular strength and gripping ability in rats and significantly (P ˂ 0.05 vs. CPZ-group) increased the time spent falling.

Effect of CPZ, betaine and their combination on the motor performance of male rats according to the (A) inverted screen test and (B) rotarod test. Values are presented as mean ± S.E; n = 5 animals; mean values having different superscript letters a, b, c, d & e are significantly different from each other (P < 0.05). a, P < 0.05 vs. control. b, P < 0.05 vs. betaine. c, P < 0.05 vs. CPZ. d, P < 0.05 vs. protective (betaine + CPZ), e, P < 0.05 vs. therapeutic (CPZ+ betaine).

The rotarod test was designed to evaluate rats' motor coordination. After three weeks of CPZ feeding, rats' motor coordination and balance were assessed using the accelerated rotarod test ( Fig. 3 B). When compared to the control group, rats in the CPZ-treated group had a considerably (F (4,24) = 6.282 , P = 0.001) shorter mean latency to fall from the rotarod device. However, when betaine was co-administered with CPZ, the animals in the protective and therapeutic groups stayed on the rotarod device substantially longer than the CPZ-treated animals.

Betaine restores Hcy concentration induced by CPZ

As revealed in Fig. 4 , administration of CPZ significantly (P ˂ 0.05 vs. controls) increased Hcy concentrations in the CC. On the other hand, supplementation with betaine before or after CPZ treatment significantly (P ˂ 0.05 vs. CPZ group) reduced the increase in Hcy concentrations in the CC (F (4,24) = 123.400, P ˂ 0.001).

Effect of CPZ, betaine and their combination on the concentration of homocysteine in the corpus callosum of male rats. Values are presented as mean ± S.E; n = 5 animals; mean values having different superscript letters a, b, c, d & e are significantly different from each other (P < 0.05). a, P < 0.05 vs. control. b, P < 0.05 vs. betaine. c, P < 0.05 vs. CPZ. d, P < 0.05 vs. protective (betaine + CPZ), e, P < 0.05 vs. therapeutic (CPZ+ betaine).

Betaine restores redox balance

The impact of betaine administration on CPZ-induced oxidative stress was evaluated ( Fig. 5 A-D). CPZ caused significant (F (4,24) = 78.084, P ˂ 0.001, 181.2 %) elevation in MDA level compared to controls ( Fig. 5 A). While the level of GSH (F (4,24) = 40.398, P ˂ 0.001, −75.5 %, Fig. 5 B) and the activities of the antioxidant enzymes SOD and CAT (F (4,24) = 32.594, 99.197, −69.1 %, −85.7 %, P ˂ 0.001, Fig. 5 C and D, respectively) in the rat's CC were found to be significantly decreased compared to controls. However, coadministration of betaine + CPZ (pre and posttreatment) significantly attenuate the oxidative stress and improve the antioxidant status as observed by the significant (P ˂ 0.05) reduction in MDA level (protective; 87.3 % and therapeutic; 74.9 %) and significant increase in GSH (protective; −35.7 % and therapeutic; −22.2 %), SOD (protective; −37.9 % and therapeutic; −20.3 %) and CAT (protective; −59.4 % and therapeutic; −40 %) in CC of rats compared to CPZ-group.

Effect of CPZ, betaine and their combination on the levels of (A) malondialdehyde (MDA), and (B) reduced glutathione (GSH) as well as the activities of (C) superoxide dismutase (SOD), and (D) catalase (CAT) in the corpus callosum of male rats. Values are presented as mean ± S.E; n = 5 animals; mean values having different superscript letters a, b, c, d & e are significantly different from each other (P < 0.05). a, P < 0.05 vs. control. b, P < 0.05 vs. betaine. c, P < 0.05 vs. CPZ. d, P < 0.05 vs. protective (betaine + CPZ), e, P < 0.05 vs. therapeutic (CPZ+ betaine).

Betaine decreases inflammatory cytokines in CPZ rats

As shown in Fig. 6 A and B, CPZ administration induced a significant increase (P ˂ 0.05 vs. controls) in the level of IL-4 (426 %) and IL-17 (311 %). However, pre and posttreatment with betaine significantly reduced (P ˂ 0.05 vs. CPZ group) IL-4 (protective; 276 % and therapeutic; 158 %) and IL-17 (protective; 179 % and therapeutic; 80.5 %) levels compared to CPZ group (F (4,24) = 125.278, 274.847, P ˂ 0.001 , respectively).

Effect of CPZ, betaine and their combination on the levels of (A) IL-4, and (B) IL-17 in the corpus callosum of male rats. Values are presented as mean ± S.E; n = 5 animals; mean values having different superscript letters a, b, c, d & e are significantly different from each other (P < 0.05). a, P < 0.05 vs. control. b, P < 0.05 vs. betaine. c, P < 0.05 vs. CPZ. d, P < 0.05 vs. protective (betaine + CPZ), e, P < 0.05 vs. therapeutic (CPZ+ betaine).

Betaine improves histological structure in the CC of CPZ rats

Microscopical examination of semithin sections of male rats stained with toluidine blue revealed that the control and betaine groups had normally structured myelinated nerve fibers with thick myelin sheath and normal glial cells ( Fig. 7 A and B). Semithin sections from the CPZ group had large areas with demyelinated nerve fibers, abnormal shaped axons and abnormal glial cells ( Fig. 7 C). In protective and therapeutic groups, betaine resulted in marked restoration of myelin sheath thickness and improvement in the structure of glial cells ( Fig. 7 D and E).

Photomicrographs of semithin sections of the corpus callosum of male rats. A & B: Sections from control and betaine-treated rats respectively, showing myelinated nerve fibers with thick myelin sheath (black arrows) and glial cells with normal structure (yellow arrows). C: Section from CPZ-treated rat, showing extensively demyelinated nerve fibers (black asterisks), abnormal shaped axons (black arrows) and dense glial cells (yellow arrows). D & E: Sections from protective (betaine + CPZ) and therapeutic (CPZ+ betaine) groups showing large number of myelinated nerve fibers (black arrows), normal glial cells (yellow arrows) and few demyelinated axons (black asterisks). F & G: Light microscopical changes; % demyelination and density of the myelinated fibers, respectively in male rats' corpus callosum semithin sections stained with toluidine blue (1000X). Values are expressed as means ± S.E (n = 7); mean values having different superscript letters a, b, c, d & e are significantly different from each other (P < 0.05). a, P < 0.05 vs. control. b, P < 0.05 vs. betaine. c, P < 0.05 vs. CPZ. d, P < 0.05 vs. protective (betaine + CPZ), e, P < 0.05 vs. therapeutic (CPZ+ betaine).

Morphometric analyses ( Fig. 7 F and G) showed that CPZ significantly (P ˂ 0.05) increased the percentage of demyelination (1067 %), however, significantly decreased the density of myelinated fibers per mm 2 (-84.7 %) compared to control and betaine-treated rats. Administration of CPZ in combination with betaine, however, reversed these values towards normal (protective; 300 %, 0.5 % and therapeutic; 367 %, −27.9 %, respectively).

Betaine restores CPZ-induced ultrastructural changes

Electron micrographs of the CC of control and betaine-treated rats ( Fig. 8 A and B) showed myelinated nerve fibers surrounded by a thick normal myelin sheath with well-organized, parallel, regular, and tightly compact lamellae. In the axoplasm, numerous neurofilaments, microtubules and mitochondria with normal structures were observed. Moreover, normal OLs appeared with normal oval heterochromatic nuclei, normal nucleoli, distinct nuclear envelopes, and dense cytoplasm full of organelles with normal structures, such as containing mitochondria, rough endoplasmic reticulum, and Golgi bodies ( Fig. 9 A and B).

Electron micrographs of the corpus callosum by using uranyl acetate/lead citrate stain. (A & B) Control and betaine-treated rats showing, myelinated nerve fibers with normal axons (Ax), mitochondria (M), neurofilaments and microtubules (red arrows) and myelin sheath with regular tight lamellae (thick blue arrows), an inset showing a measurement of the thickness of the myelin sheath. (C) CPZ-treated rat showing, complete demyelinated axons (DA), and other partially demyelinated axons with diffuse axonal cytoplasmic degeneration, vacuolated myelin (V), lysis in myelin sheath (dashed red arrows), splitting of myelin sheath (red arrows), axoplasm housing vacuoles (red astrikes), and mitochondria with indistinct cristae (M). Axons almost all of them have irregular deformed myelin sheaths, an inset showing a measurement of the thickness of the myelin sheath. (D & E) betaine + CPZ (protective group) and CPZ + betaine (therapeutic group) showing, intact axons (Ax) with marked myelination, neurofilaments and microtubules (red arrows), mitochondria (M), and myelin sheath with regular tight lamellae (thick blue arrows). Few demyelinated nerve fibers (DA) are also encountered, an inset showing a measurement of the thickness of the myelin sheath. Microscopic magnification: 2 µm, inset 200 nm. (F) Axon diameter, (G) fiber diameter, (H) myelin thickness, and (I) G ratio as demonstrated by TEM imaging of the corpus callosum. Data presented as mean ± S.E (n = 10); mean values having different superscript letters a, b, c, d & e are significantly different from each other (P < 0.05). a, P < 0.05 vs. control. b, P < 0.05 vs. betaine. c, P < 0.05 vs. CPZ. d, P < 0.05 vs. protective (betaine + CPZ), e, P < 0.05 vs. therapeutic (CPZ+ betaine).

Electron micrographs of the corpus callosum by using uranyl acetate/lead citrate stain. (A & B) Control and betaine-treated rats showing, an oligodendrocyte having a large nucleus (N) with clumped peripheral heterochromatin and bilayer nuclear envelope (white arrow), and dense cytoplasm containing mitochondria (M), Golgi apparatus (G), short cisternae of rough endoplasmic reticulum (rER) and some dense inclusions. (C) CPZ-treated rat showing an oligodendrocyte with pyknotic hyperchromatic nucleus (N), dilated nuclear envelope (white arrow) and degenerated mitochondria (M). (D & E) betaine + CPZ (protective group) and CPZ + betaine (therapeutic group) showing an oligodendrocyte having a large, rounded nucleus (N) with well peripheral heterochromatin and distinct nuclear envelope (white arrow), and dense cytoplasm containing mitochondria (M), Golgi apparatus (G) and short cisternae of rough endoplasmic reticulum (rER) and some dense inclusions. Scale bar (1 µm), inset 200 nm.

In contrast, CPZ showed demyelinated areas, axons with irregular outlines and splitting myelin sheath ( Fig. 8 C), axons appeared with severe cytoplasmic vacuolization, and abnormal vacuolated mitochondria with cristolysis and dense matrix. Moreover, CPZ-treated rats had abnormal degenerated OLs with vacuolated cytoplasm containing damaged unclear cell organelles ( Fig. 9 C).

Treatment with betaine (protected & therapeutic groups) attenuated the decrease in myelination and axonal damage induced by CPZ ( Fig. 8 D and E), and OLs were well preserved ( Fig. 9 D and E).

Morphometric analyses ( Fig. 8 F-I) showed that CPZ significantly (P ˂ 0.05) increased the axon diameter (9.8 %), and G ratio (11 %, i.e., decreased myelin thickness), while decreased the fiber diameter (-37.4 %), and myelin thickness (-65.9 %) compared to controls. A change in the G ratio can indicate alterations in myelination and axonal integrity. Administration of CPZ in combination with betaine, however, reversed this negative effect towards normalcy (protective; 9.2 %, 11 %, −1.1 %, −7.7 % and therapeutic; −8.5 %, 27.2 %, −27.4 %, −31.9 %, respectively).

Betaine restores CPZ-induced MBP-immunostaining changes

In the present study, to investigate the effect of betaine on the demyelination induced by CPZ in the CC sections were processed for immunohistochemical staining. Immunohistochemical investigation differentially revealed the MBP expression in the OLs of the CC in all studied groups. Reduced MBP levels were clearly noted in CPZ group compared to control one ( Fig. 10 A and C). The increased density of some OLs expressing high levels of MBP was obviously marked in the betaine-treated group ( Fig. 10 B). Betaine-exposed rats treated with CPZ showed better immunoreactivity in the CC region in comparison to those treated with CPZ + Betaine ( Fig. 10 D and E). This increase was due to a strongly upregulated expression by individual OLs of the CC brain region.

Immunohistochemistry showing the expression of MBP in the CC of rats in the five experimental groups: (A) Control, (B) Betaine, (C) CPZ, (D) Betaine + CPZ, (E) CPZ + Betaine. MPB positive cells (brown staining) decreases dramatically in CPZ group. Both protective (betaine + CPZ) and therapeutic (CPZ + betaine)-treated groups can reverse the reduction in MBP expression in the CC. Scale bar: 50 µm.

Behavioral, biochemical, and immunohistochemical correlations

Spearman's linear correlation analysis was used to statistically confirm the attended link between the parameters, as shown in Fig. 11 A.

(A) Spearman correlation analysis, representing behavioral, weight, Hcy concentration, oxidant/antioxidant, and inflammatory biomarkers, as well as semithin section and ultrastructural changes, are superimposed as supplementary variables. (B) The analysis of protein-protein interactions involving variously expressed redox and inflammatory markers in relation to myelin basic protein utilizing the STRING database.

The analysis of protein-protein interactions involving variously expressed redox and inflammatory markers in relation to myelin basic protein is illustrated Table 1 and Fig. 11 B, utilizing the STRING database.

Table 1 Protein-protein interaction analysis of differently expressed redox and inflammatory markers with myelin basic protein using the STRING database. Table 1 Node 1 Node 2 Node 1 accession Node 1 accession Score Average local clustering coefficient PPI enrichment p-value Cat Sod1 ENSMUSP00000028610 ENSMUSP00000023707 0.827 0.762 0.000465 IL17a IL17b ENSMUSP00000027061 ENSMUSP00000025471 0.811 IL17a IL4 ENSMUSP00000027061 ENSMUSP00000000889 0.934 IL17a MbP ENSMUSP00000027061 ENSMUSP00000046185 0.586 IL17b IL4 ENSMUSP00000025471 ENSMUSP00000000889 0.440 IL4 MbP ENSMUSP00000000889 ENSMUSP00000046185 0.575

Protein-protein interaction analysis of differently expressed redox and inflammatory markers with myelin basic protein using the STRING database.

Discussion

MS is a complex CNS disease marked by inflammation, neurodegeneration, demyelination, and axonal damage caused by an immune response to myelin sheath production and maintenance [57] . Myelin loss, OL apoptosis, glial cell activation, and failure in remyelination are hallmarks of MS pathophysiology [2] . There are several models for studying MS pathogenesis; however, the CPZ experimental model is used to simulate the development and progression of MS pathology, as well as to assess the potential beneficial effect of potential therapeutic agents [64] .

Current approved MS treatments reduce relapse rates but do not promote myelin repair or prevent or reverse disease progression. Betaine is an important methyl group donor in transmethylation, which is catalyzed by BHMT. This reaction catalyzes the formation of methionine from Hcy and occurs primarily in the liver and kidneys [37] . Betaine is found in various dietary sources, with daily consumption typically ranging from 1 g to 2.5 g. Following ingestion, betaine is quickly absorbed (half-life = 0.28 hours) and distributed (half-life = 0.59 hours), primarily being metabolized into methionine and dimethylglycine through the action of BHMT and the coenzyme vitamin B12 [63] . Additionally, a toxicity assessment in mice identified the LD50 of betaine as 10.8 g/kg via subcutaneous administration and 0.83 g/kg through intravenous injection [10] . According to the OECD Guideline 401, an acute oral toxicity study conducted by the European Chemicals Agency (ECHA) established the LD50 of betaine at 11.2 g/kg, indicating that it can be classified as practically nontoxic (EC number: 203–490–6; CAS number: 107–43–7) [10] , [39] . Sub-acute and sub-chronic studies conducted on rats aimed to explore the metabolic effects of betaine across various intake levels (0, 0.5, 0.75, 1.0 and 5.0 % of the diet). The results of these investigations are encapsulated in a 14-day range-finding study, a 90-day sub-chronic study, a 28-day subacute reversibility study, and subsequent 28 and 90-day studies employing an alternative dietary approach. The comprehensive analysis of this data led to the conclusion that betaine remains nontoxic, even at elevated doses [34] . Moreover, the acute oral toxicity of betaine anhydrous was assessed using five groups, each consisting of five male and five female CD rats. On Day 1, the test substance was delivered at dosages ranging from 5000 to 20,000 mg/kg, with a volume of 40 ml/kg in distilled water. The calculated acute oral LD50 was determined to be 11,204 mg/kg for males and 11,148 mg/kg for females, with corresponding 95 % confidence intervals and the slope of the dose-response curve [59] . Additionally, a micronucleus assay was performed in mice using betaine monohydrate at doses of 0.5, 1, 1.5, and 2 g/kg. No evidence of toxicity to the bone marrow was observed, as indicated by the unchanged ratios of polychromatic erythrocytes to normochromatic erythrocytes (PCE/NCE). It was concluded that betaine monohydrate does not induce micronuclei in the bone marrow of mice at oral doses up to 2 g/kg. Khodayar et al. [42] demonstrated that betaine administered intraperitoneally at doses of 125, 250, 500, and 1000 mg/kg over five days provides protective effects against acetaminophen-induced hepatotoxicity in male mice. Furthermore, Kitagawa et al. [43] reported that betaine, administered at doses of 1, 2, and 4 g/kg for 35 days, significantly mitigates liver lipid accumulation resulting from vitamin B deficiency in male Wistar rats. However, it has proven challenging to establish a No Observed Adverse Effect Level (NOAEL), as adverse changes have been observed even at minimal dosage levels. Consequently, the existing human data do not adequately eliminate the potential risk associated with the intended consumption of betaine, necessitating further investigations involving rats and other species to determine NOAELs. Furthermore, there are no studies on the protective and therapeutic modulatory effect of homocysteine-betaine metabolism in a changeable dose of CPZ-induced MS models.

Various experimental studies have revealed body weight loss with CPZ intoxication [21] , [33] . The disturbance of normal energy flow may be the mechanism through which CPZ promotes weight loss [56] . For three weeks, the body weight of animals in the CPZ group was significantly lower than that of the control group, which is consistent with earlier results and is related to a reduction in food intake as well as an increase in basal metabolic rate [55] .

Betaine cotreatment resulted in a considerable improvement in reversing CPZ-induced body weight loss. These findings could be related to the antioxidant and anti-inflammatory properties of betaine found in this study. The ability of betaine to prevent the normal interruption of energy flow and to prevent a drop in feeding pattern in rats treated with CPZ may be the mechanism by which betaine preserves body weight. This is consistent with the results of Naeem et al. [50] and Omotoso et al. [56] , who demonstrated the neuroprotective effect of kolaviron and nebivolol against weight loss in CPZ-induced demyelination in mice.

In CPZ-intoxicated animals, damage occurred in the CC, disrupting neighboring regions. As a result, the injured CC is no longer capable of providing effective communication between the two hemispheres [15] . It should be noted that demyelination certainly impairs electric conductance along neuronal axons, and myelin breakdown may impair axons in the brain. CPZ administration affected motor performance as a sequence of demyelination [50] . This was reflected in our results of behavior tests. The motor deficit in CPZ treated rats was demonstrated by decreased latency time to fall in the rotarod and inverted screen tests. This finding is consistent with other researchers who showed poor motor coordination with the intake of CPZ [1] , [28] . However, Xiu et al. [74] showed an insignificant decrease in the time spent on the rotarod test in CPZ-treated mice. These contradictory results were attributed to differences in the extent of demyelination between different studies even with the same dose of CPZ [36] .

Co-treatment with betaine in the protective and therapeutic groups resulted in a significant improvement in motor performance by increasing muscular strength, gripping ability, and motor coordination in rats. The mechanism involved in betaine's protective role against CPZ-induced behavioral deficits could be due to its ability to prevent mitochondrial biochemical redox and ROS production dysfunctions [78] .

Hcy is a neurotoxic sulfur-containing intermediate product that crosses the BBB. In this study, CPZ significantly increased Hcy concentrations in the CC compared to controls. Previous studies indicated that elevated plasma Hcy levels may be a risk factor for neurological disorders such as dementia, depression, and Parkinson's disease [7] , [8] , [67] . Hcy has several effects on neural cells, including oxidative stress, caspase activation, mitochondrial dysfunction, and an increase in cytosolic calcium, all of which contribute to apoptosis [8] . Hcy also inhibits the expression of antioxidant enzymes, potentially amplifying the toxic effects of ROS. Consequently, auto-oxidation of Hcy is known to produce ROS, which can lead to an overload of oxidative stress in neurodegenerative disorders. Hyperhomocysteinaemia, along with oxidative stress, may impair CNS functions by damaging the BBB and inhibiting axon myelination, which may result in neurobehavioral defects [72] .

Hcy is metabolized via a remethylation cycle that returns to methionine or a transsulfuration pathway that produces cystathionine. Betaine transfers a methyl group to dimethylglycine via the enzyme BHMT and converts Hcy to methionine [8] . Our findings showed that supplementing rats with betaine before and after CPZ treatment significantly reduced the increase in Hcy concentrations in the CC. Betaine treatment reduces Hcy levels by inducing the remethylating process, which converts Hcy to methionine. In terms of the BHMT enzyme, which is abundant in primates, betaine's beneficial properties are promising, as it reduces elevated plasma Hcy concentrations via the BHMT pathway [7] .

Oxidative stress occurs upon excessive free radical production and can harm any organ, particularly the brain, due to its high oxygen consumption, lipid-rich content, and low antioxidant level [62] , [76] . In the present study, CPZ administration caused an increase in MDA levels accompanied by a decrease in the levels of GSH, SOD and CAT, leading to a decrease in the antioxidant power of the brain. In accordance with our results, Largani et al. [46] and Shiri et al. [66] found that CPZ administration induced high levels of MDA and low levels of GSH, as well as CAT and SOD activity. CPZ, a copper-chelating agent, chelates the copper of the mitochondrial enzyme cytochrome c oxidase, disrupting the electron transport chain and causing mitochondrial dysfunction and excessive ROS generation, resulting in oxidative stress and apoptosis in mature OLs and a decrease in the activity of antioxidant enzymes CAT and SOD [28] , [54] . Furthermore, a decrease in SOD activity has been implicated in the mechanism by which CPZ induces demyelination because of a decrease in Cu + levels, which is required for SOD to function properly [28] .

Betaine administration reduced CPZ-induced oxidative stress by decreasing MDA levels and increasing GSH levels, as well as CAT and SOD activities. These findings are consistent with previous studies [7] , [26] which demonstrated betaine's antioxidant and methyl donor properties. Betaine is the substrate of the BHMT reaction, which converts Hcy to methionine [69] . Methionine is particularly susceptible to ROS and can be oxidized to methionine sulfoxide by ROS, so it plays an important role in ROS scavenging. Methionine sulfoxide, on the other hand, could be reduced back to methionine by methionine sulfoxide reductases. It has been proposed that betaine's antioxidant effect is mediated by regulating the methionine-homocysteine cycle [47] .

Microglia, the primary CNS immune cells, are involved in CNS development and homeostasis, as well as debris clearance [29] . When CNS homeostasis is disrupted, microglia are activated to protect tissue from damage. However, chronic activation causes a state of excessive microglial activation in neurodegenerative diseases, resulting in a cycle of inflammation and neurotoxicity rather than resolving the damage [60] . The neuroinflammatory demyelinating aspects of MS pathology are well represented by the CPZ model [32] . Furthermore, when the BBB is breached, leukocytes from the blood can infiltrate and colonize the brain [27] , [49] . Our study showed that CPZ intoxication significantly elevated IL-4 and IL-17 levels in the CC compared to controls. Abd El Aziz et al. [1] demonstrated the role of IL-17 in the activation of microglia and astrocytes, which play a role in the pathogenesis of CPZ-induced demyelination. Astrocytes produce IL-17 in the brain, which causes microglial activation, demyelination, decreased myelin synthesis, and functional deficits. Moreover, Abdel-Maged et al. [2] , Cherry et al. [16] , and Kang et al. [41] demonstrated that IL-17 stimulates astrocytes to produce proinflammatory cytokines such as IL4, IL-6, IL13, and TNF-α, as well as other chemokines, resulting in leukocyte recruitment into the brain. These cytokines induce autocrine IL-17 expression in microglia, astrocytes, and macrophages, leading to Th17 differentiation from naive CD 4+ T cells [22] .

As inflammatory mediators and ROS are primarily produced by microglia, the CPZ model is ideal for assessing the effect of betaine on microgliosis and its detrimental role in demyelination. Microglia, on the other hand, have a well-documented neuroprotective role in MS; microglia phagocytize myelin debris, recruit OPCs, and release regenerative factors for remyelination [51] . Furthermore, activated microglia and macrophages secrete anti-inflammatory cytokines and trophic factors that resolve local inflammation and promote regenerative processes such as neurogenesis, angiogenesis, and remyelination [40] , [45] . Our results found that in comparison to the CPZ group, pre and posttreatment with betaine significantly reduced IL-4 and IL-17 levels in the CC. Emerging research indicates that betaine inhibits the nuclear factor kappa B (NF-κB) pathway, preventing the production of proinflammatory mediators such as IL-1β, IL-6, and nitric oxide synthase 2, thereby supporting the anti-inflammatory function of betaine in these models [47] , [73] .

Light and electron microscopic examination of the CC sections of CPZ-treated rats revealed extensive demyelination, which was confirmed by morphometric analyses. This finding was reported by Acs and Komoly [3] and Bölcskei et al. [14] and attributed to the toxic effect of CPZ on OLs via disturbance in the intracellular level of copper, which was accomplished by release of proinflammatory cytokines from the initiated microglial and astrocyte cells, leading to OLs death and myelin disintegration. Vega-Riquer et al. [71] indicated that CPZ targets many enzymes, such as ceruloplasmin, which impairs the activity of copper-dependent cytochrome oxidase, decreases oxidative phosphorylation, and causes degenerative OL changes. This chain of events eventually leads to demyelination.

Moreover, the CPZ-intoxicated rats showed a significant decrease in the axon diameter, fiber diameter and myelin thickness and a subsequent increase in the G ratio compared to the control rats. This was in accordance with the work carried out by Barkat et al. [12] and could be a result of atrophy and disintegration of the myelinated axons during the acute phase of demyelination. The presence of a direct relationship between the duration of administration and the severity of nerve demyelination was consistent with the findings of Zendedel et al. [77] , who reported that partial demyelination of CC nerve fibers occurred after 2 weeks of CPZ intoxication and referred to this as acute demyelination, while complete demyelination occurred after 5–6 weeks. Acs and Komoly [3] , on the other hand, found severe demyelination after 3 weeks of CPZ administration, along with massive macrophage infiltration and astrocytosis in the demyelinated areas. In their studies, Grothe et al. [31] and Vargas and Tyor [70] showed varying degrees of axonal degeneration, loss of OLs, and gaping between nerve fibers in CPZ intoxicated animal models.

The results of our light and electron microscopy studies showed that betaine treatment induced axonal regeneration and remyelination and conversely reduced the rate of lymphocyte infiltration in the CC tissue. We assumed that betaine could reverse axonal damage and demyelination because of its antioxidant, anti-inflammatory, and modulatory effects on microglia activation and by reducing acute immune responses within the injured tissue. This activity appears to be influenced by a variety of mechanisms, including the radical scavenging activity of sulfur-containing compounds, an increase in the activity of endogenous antioxidants, and a decrease in oxidative stress parameters. This is concomitant with the results of Bagheri et al. [11] , who demonstrated the protective effect of Ferula asafoetida against histopathological changes in CPZ-induced demyelination in mice. Additionally, ELBini-Dhouib et al. [22] and Naeem et al. [50] found nebivolol and curcumin to be neuroprotective against experimental models of MS.

Myelin basic protein (MBP) is a myelin protein that is required for the assembly and compaction of myelin sheath layers [50] . There is some speculation that CPZ ingestion may cause copper depletion in the CNS, which acts as a modulator of neuronal transmission and induces the death of oligodendrocytes, resulting in impaired myelin production. In this study, the CPZ group had a lower MBP reaction in CC nerve fibers and OLs nuclei than the control group, as showed by MBP immunohistochemistry, in accordance with the results of Abdel-Maged et al. [2] and Chang et al. [15] . Because CPZ chelates copper, which is required for myelin compaction, it causes myelin destabilization and degeneration [25] .

However, we demonstrated for the first time that betaine treatment increased MBP immunostaining significantly when compared to the CPZ group. This finding is consistent with the histopathological findings, which revealed an increase in the percentage area of myelinated fibers. As a result of the antioxidant, anti-inflammatory, and modulatory effects on microglia activation demonstrated in this study, betaine treatment reversed CPZ-induced changes in myelin status. This is consistent with the findings of ELBini-Dhouib et al. [22] and Naeem et al. [50] who found Nebivolol and curcumin to be neuroprotective against experimental models of MS.

In conclusion, the current study illustrated the potential neuroprotective and neurotherapeutic effects of homocysteine-betaine metabolism in a changeable dose of CPZ model of MS. Betaine administration was able to attenuate behavioral abnormalities and weight loss, oxidative stress, neuroinflammation, ultrastructure alterations and the expression intensity of MBP-immunostaining in CPZ-induced MS animal models. These beneficial effects of betaine could be partially attributed to its antioxidant and anti-inflammatory potential. More research is needed to understand the molecular mechanism by which betaine underpins the remyelination process, as well as the involvement of gut microbiota in betaine's neuroprotective action.

Funding

The authors did not receive support from any organization for the submitted work.

CRediT authorship contribution statement

Amina E. Essawy: Writing – review & editing, Supervision, Conceptualization. Gihad Jamal Bekhee: Writing – original draft, Methodology, Formal analysis. Sherine Abdel Salam: Writing – review & editing, Supervision, Conceptualization. Reem Hasaballah Alhasan: Writing – review & editing, Methodology, Funding acquisition. Heba-Tallah Abd Elrahim Abd Elkader: Writing – review & editing, Writing – original draft, Visualization, Supervision, Software, Methodology, Formal analysis, Data curation, Conceptualization.

Declaration of Generative AI and AI-assisted technologies in the writing process

During the preparation of this work the author(s) used [ahrefs/ Free AI Paraphrasing Tool] in order to language polishing of the manuscript. After using this tool/service, the author(s) reviewed and edited the content as needed and take(s) full responsibility for the content of the publication.

Declaration of Competing Interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgements

Not applicable.

Declaration of Competing Interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Handling Editor: Prof. L.H. Lash

Footnotes

Supplementary data associated with this article can be found in the online version at doi:10.1016/j.toxrep.2025.101974 .

Supplementary data associated with this article can be found in the online version at doi:10.1016/j.toxrep.2025.101974 .

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Data will be made available on request.

References

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Data Availability Statement

Data will be made available on request.