Magnesium glycinate attenuates cyclophosphamide-induced neurotoxicity by modulating brain neurotransmitters, inflammatory markers, and hippocampal synaptophysin immunoreactivity

Magnesium glycinate attenuates cyclophosphamide-induced neurotoxicity by modulating brain neurotransmitters, inflammatory markers, and hippocampal synaptophysin immunoreactivity | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Magnesium glycinate attenuates cyclophosphamide-induced neurotoxicity by modulating brain neurotransmitters, inflammatory markers, and hippocampal synaptophysin immunoreactivity Foluso Olamide Ojo, Luqman Adepoju Hassan, Bukola Ezekiel Olatundun, and 7 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-9185242/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 8 You are reading this latest preprint version Abstract The usage of cyclophosphamide (CYP) in cancer treatment is associated with organ toxicity, cellular degeneration, and myelosuppression, which result in treatment discontinuation. Its debilitating impact has increased the quest to search for novel substances or measures that will alleviate the adverse effects, while its therapeutic efficiency remains intact. This study evaluated the effects of Magnesium glycinate on cyclophosphamide-induced neurotoxicity in Wistar rats. Forty-eight Wistar rats (eight weeks old) were allocated into four experimental groups (n = 8), as the normal control (A), oral Magnesium glycinate at 22.8 mg/kg (B) and 32.8mg/kg (C), 150mg/kg of CYP (D), CYP/oral magnesium glycinate at 22.8 mg/kg (E) and CYP/oral magnesium glycinate 32.8mg/kg (F). The animal’s body weight was measured weekly. Following the spatial working memory evaluation using the Y-maze. Animals were sacrificed by cervical dislocation, brain tissues excised, followed by the homogenization of the right hemisphere. The supernatant of the homogenates was used to assess the neurotransmitter activities and the levels of brain-derived neurotrophic factor, interleukin 1beta, interleukin 10, and Tumour Necrosis Factor alpha. Furthermore, the left hemisphere was processed for histological evaluation (H&E and Cresyl fast violet stain) and synaptophysin immunoreactivity. Cyclophosphamide induces weight loss, dysregulation of neurotransmitter and modulator activities, and inflammatory marker levels, as well as neurodegeneration and decreased hippocampal synaptophysin immunoreactivity. However, Magnesium glycinate, particularly at 32.8mg, reversed the stated adverse effects. Mg. Glycinate possesses potential therapeutic properties as a supplement in the management of cancer, particularly in alleviating the adverse effects of Cyclophosphamide. chemotherapy Neurotoxicity synaptophysin immunoreactivity Magnesium-glycinate Spatial-working Memory Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Introduction Cancer, a malignant disease defined by dysregulated cellular expansion, resistance to apoptosis, persistent neovascularization, and invasive dissemination capacity, has recently emerged as a global burden [ 1 ], constituting the second leading cause of mortality worldwide [ 2 ]. Chemotherapeutic agents such as cyclophosphamide show promising effects in reducing the death rate in some cancer patients [ 3 ]. Despite the significant positive impact of the chemotherapeutic agents in some cancer management, the debilitating effects, such as organ toxicity, cellular degeneration, and myelosuppression, often result in treatment discontinuation. Notably, cyclophosphamide usage is usually associated with neurotoxicity, loss of hair (alopecia), immunosuppression, haemorrhagic cystitis, and cardiotoxicity, particularly at high dosage [ 4 ]. Induction of inflammatory processes and reactive oxygen species (ROS) are its known pharmacological mechanisms of action that result in neurotoxicity [ 5 ]. Moreover, associated with the oxidative and inflammatory processes are the depleted levels of brain magnesium, which further result in intracellular depletion that exacerbates neuronal damage through N-methyl-D-aspartate receptor NMDA receptor hyper-excitability [ 6 ]. The inability of most chemotherapeutic drugs to discriminate between the cancerous cells and the healthy body cells is mostly a documented reason for their toxic adverse effects [ 7 ]. The debilitating effects of these drugs have further necessitated and increased our quest to search for novel substances or means that would not only mitigate the adverse effects but would also enhance chemotherapeutic efficiency in a functional biological system in the absence of cancer. Studies have continued to support the effects of magnesium on brain health through its neuroprotective impact and synaptic functions [ 8 ]. A study done by Li et al. (2014) showed an increased level of brain magnesium in mouse models of Alzheimer's disease (APPswe/PS1dE9), reduced amyloid plaques by 36%, prevented synapse loss through NMDAR, signaling preservation, and also reversed memory deficits even at late stages of disease [ 9 ]. Subsequently, Chen et al. (2024), in their systematic review of RCTs, corroborate magnesium’s role in brain health and cognition, with elevated levels of brain magnesium reducing neuroinflammation and enhancing learning and memory through NMDA modulation [ 10 ]. However, this study examined the therapeutic role of magnesium glycinate at varying dosages on the neurotoxicity induced by cyclophosphamide by evaluating levels of some brain neurotransmitters, markers of inflammation, memory, hippocampal histomorphology, and synaptophysin immunoreactivity in rats. Materials and Methods 2.1. Chemicals and drugs Magnesium glycinate (250 mg, New York, USA), normal saline, Cyclophosphamide injection (xGen Pharmaceutical DJB). ELIZA assay kits for dopamine, acetylcholine, Serotonin, brain-derived neurotropic factor, interleukin 1β, interleukin-10, and tumour necrosis factor (TNF-α) were procured from R&D Systems (Bio-Techne, USA). 2.2. Animals and experimental design The experimental procedure was accomplished in accordance with the methods approved by the animal research ethical committee of the Faculty of Basic Medical Sciences, University of Ilesa, Ilesa, Osun State, Nigeria, with approval code (UNILESA/FBMS/2025/04). These procedures adhered to the European Council Directive 2010/63/EU guidelines governing the protection of animals used for scientific purposes Forty-eight (48) Wistar rats (eight weeks old), each weighing 150–170 g, were procured from animal breeders in Iwo, Osun state, Nigeria. The animals were conveyed in the cool of the day to the research animal house of the University of Ilesa, Ilesa, Osun State, Nigeria, where the experiment was done. Plastic cages (45 × 24 × 20 cm) were maximized for the animal housing in a temperature-controlled environment (22°C) under a 12-hour light-dark cycle throughout the experimental period (21 days). Before the commencement of the experiment, acclimatization of the animals was done for two weeks, after which the animals were allocated into four experimental cohorts (n = 8), while animals across all groups were fed with normal feed (Top Feeds®, Nigeria), and clean water ad libitum . Additionally, Animals in groups B and C orally received magnesium glycinate at 22.8 mg/kg and 32.8mg/kg, respectively, according to Aniebo (2023) [ 11 ]. Groups D, E, and F received cyclophosphamide injection 3 times at 150mg/kg intraperitoneally every other day (days 1, 3, and 5), while groups D and E additionally received oral magnesium glycinate at 22.8 mg/kg and 32.8mg/kg, respectively. The animal’s body weight was measured weekly using an electronic scale throughout the experimental period. On the 22nd day, animals were introduced to the Y-maze neurobehavioral test for memory evaluation. Following the spatial working memory evaluation test in the Y-maze model, the animals were sacrificed by cervical dislocation on the 23rd day. Following this was the dissection of the cranium for the removal of the brain tissues, after which the right hemisphere of each of the animals was homogenized using iced cold phosphate-buffered saline (PBS) as a medium at a ratio of 1:9 (weight of brain tissue/ volume of iced cold PBS). Tissue homogenate supernatant was used for the assessment of neurotransmitters (Serotonin, dopamine, and acetylcholine) levels, the activities of neuromodulator (Brain-Derived Neurotropic factor), and levels of inflammatory markers (interleukin 1beta, interleukin 10, and Tumour Necrosis Factor alpha). Furthermore, the left hemisphere was dissected and fixed in 10% buffered formalin, processed in paraffin embedding, cut at 5µ m for histological evaluation (H&E and Cresyl fast violet stain) and synaptophysin immuno-reactivity. 2.3. Assessment of change in body weight (weekly) As previously documented by Ojo et al . (2025) [ 12 ], individual animal’s Body weight were evaluated weekly using an electronic weighing balance. Following this, the weekly change in body weight was determined using the formula \(\:\:\:\:\text{F}\text{i}\text{n}\text{a}\text{l}\:\left(\text{l}\text{a}\text{s}\text{t}\:\text{w}\text{e}\text{e}\text{k}\right)-Initial\:\left(first\:week\right)\) . The results were statistically analyzed to determine the mean values. 2.4. Neurobehavioral test for Memory (Y-maze) As previously reported by Onaolapo (2023) [ 13 ] and Ojo et al. (2025) [ 13 ], the Y-maze model was used in measuring the spatial working memory. Spatial working memory was scored by monitoring unpremeditated alternation behaviour. Unpremeditated alternation behaviour is the predisposition of rats to alternate customarily non-reinforced choices of the Y-maze on sequential chance. Each rat was positioned in one of the arms of the Y maze and was left to explore the arms for 5 minutes without restriction until its tail completely moved into the other arm. 2.5. Enzyme Linked Immunosorbent Assay (ELISA) analysis Enzyme-linked immunosorbent assay protocols with the commercially available kits (Enzo Life Sciences Inc., NY, USA), made to determine the total levels of individual cytokines as previously reported by Onaolapo et al. (2023) [ 14 ] was used to determine the levels of interleukin-1Beta (IL-1β), interleukin-10 (IL-10), and Tumor Necrosis factor-α levels. Similarly, the levels of dopamine, serotonin, acetylcholine, and brain-derived neurotropic factor were measured from the supernatant of the homogenized brain tissue, through the usage of commercially available Enzyme-linked immunosorbent assay kits, following the manufacturer’s method (ABCAM, Cambridge, UK). 2.6. Synaptophysin Immunohistochemical Evaluation The left hemisphere of the brain tissue was fixed in 10% neutral buffered formalin, embedded in paraffin, and cut into 5µm sections, which were placed on positively charged slides. Sections of the hippocampus were embedded at 53–65°C for 30 minutes. Following this, slides were dewaxed in xylene (2 changes, 5 min each), rehydrated through graded ethanol (100%, 95%, 70%, for 5 minutes each, and rinsed in distilled water.​ As reported by Krenacs (2010), Heat-induced epitope retrieval (HIER) was done using citrate buffer (pH 6.0) for 25 minutes in an automated stainer. Following this, slides were cooled to room temperature and rinsed in PBS/TBST. Endogenous peroxidase was blocked with 3% H₂O₂ for 15 minutes and incubated in 10% normal goat serum in PBS for 30 minutes to reduce non-specific binding.​ Anti-synaptophysin monoclonal antibody (clone IHC669) was applied overnight at 4°C and at room temperature. After which it was rinsed thrice in phosphate-buffered saline for 5 minutes each.​ Slides were incubated with biotinylated secondary antibody for 20 minutes, then HRP-streptavidin complex for 20 minutes. For brown precipitate, slides were developed with DAB chromogen for 10 minutes. Counterstained with hematoxylin for 1–5 minutes, dehydrated, cleared in xylene, and mounted [ 15 ]. 2.7 Photomicrography Sellon-Olympus trinocular microscope (XSZ-107E, China) was used to microscopically examine the histologically processed section of the hippocampus. The hippocampal photomicrographs were captured with a digital camera (Canon Powershot 2500). A pathologist who was naïve to the experimental cohorts evaluated the histopathological changes. 2.8 Statistical analysis Data were presented as mean ± standard error of the mean. Normally distributed data was analyzed using One-way analysis of variance (ANOVA). Statistical analyses of data were carried out using GraphPad Prism ver. (9.0 for Windows, GraphPad Software, San Diego, CA, USA). Statistically significant difference was considered as p < 0.05. Results 3.1. Effects of Magnesium-glycinate on body weight in rats. Effects of Magnesium glycinate on the weekly change in body weight in rats treated with CYP (Fig. 1 ). No significant difference was observed in the Mg-glycinate control groups [22.8mg/kg (B) and 32.8mg/kg (C)] compared to the control group. Significant (p < 0.05) decrease in weight gain (weekly) was noted in the CYP-treated group compared to the normal control and Mg-glycinate control groups. Moreover, a significant increase was noted with CYP/Mg-glycinate (22.8mg) and CYP/Mg-glycinate (32.8mg) compared to the CYP control group (D). 3.2 Effect of Mg-glycinate on working memory (spatial) in the Y-Maze model in rats. Effects of Magnesium glycinate on the percentage correct alternation in rats treated with CYP (Fig. 2 ). A significant (P < 0.05) increase was observed in the Mg-glycinate control groups [22.8mg/kg (B) and 32.8mg/kg (C)] compared to the control group. Significant (p < 0.05) decrease in percentage correct alternation was observed in the CYP-treated group compared to the normal control and Mg-glycinate control groups. Also, a significant increase was noted with CYP/Mg-glycinate (22.8mg) and CYP/Mg-glycinate (32.8mg) compared to the CYP control group (D). 3.3. Effects of Mg-glycinate on levels of serotonin, acetylcholine, dopamine, and Brain-Derived Neurotropic Factor. Table 1 . Shows the effects of Magnesium glycinate on levels of brain neurotransmitters and neuromodulators (Serotonin, Acetylcholine, Dopamine, and BDNF) in rats treated with Cyclophosphamide. In comparison with the normal control group (A), the sole administration of CYP. (group D) shows a significantly increased level of serotonin activities, while dopamine, acetylcholine, and BDNF activities significantly (p < 0.05) decrease. However, in comparison with the sole administration of CYP, administration of CYP/Mg-glycinate (22.8mg) and CYP/Mg-glycinate (22.8mg) showed a significant (p < 0.05) decrease in the activities of serotonin, while the levels of acetylcholine, dopamine, and BDNF significantly increased. Table 1 Effects of Mg-glycinate on levels of serotonin, acetylcholine, dopamine, and Brain-Derived Neurotropic Factor. Groups Serotonin (pmol/mg tissue) Acetylcholine (pmol/mg tissue) Dopamine (pmol/mg tissue) BDNF (pmol/mg tissue) Control 10.78 ± 1.10 5.50 ± 0.11 33.11 ± 0.09 26.21 ± 0.41 Mg-glycinate (22.8mg) 13.67 ± 0.20 a 5.59 ± 0.19 39.23 ± 0.08 a 30.10 ± 0.32 a Mg-glycinate (32.8mg) 14.32 ± 0.41 a 6.97 ± 0.09 41.20 ± 0.07 a 34.42 ± 0.29 a CYP 21.21 ± 1.21 ab 2.02 ± 0.07 a 18.10 ± 1.01 a 14.22 ± 0.26 a CYP/Mg-glycinate (22.8mg) 13.00 ± 0.18 ab 3.21 ± 0.03 ab 24.12 ± 0.16 ab 17.41 ± 0.11 ab CYP/Mg-glycinate (32.8mg) 12.11 ± 0.09 ab 5.18 ± 0.08 ab 28.10 ± 0.10 ab 20.11 ± 0.23 ab Data was presented as Mean ± S.E.M, *p < 0.05 against control, # p < 0.05 significant difference from CYP. The number of rats in each group equals eight (8). Mg: Magnesium, CYP: Cyclophosphamide 3.4. Effects of Mg-glycinate on interleukin-1beta levels in rats. Effects of Magnesium glycinate on levels of interleukin 1beta (IL-1β) in rats treated with CYP (Fig. 3 ). No significant difference was observed in the Mg-glycinate control groups [22.8mg/kg (B) and 32.8mg/kg (C)] compared to the control group. Significant (p < 0.05) increase in (IL-1β) levels was observed in the CYP-treated group compared to the normal control and Mg-glycinate control groups. However, a significant decrease was noted with CYP/Mg-glycinate (22.8mg) and CYP/Mg-glycinate (32.8mg) compared to the CYP control group (D). 3.5. Effects of Mg-glycinate on levels of interleukin 10 (IL-10) in rats. Effects of Magnesium glycinate on levels of interleukin 10 (IL-10) in rats treated with CYP (Fig. 4 ). A significant (p < 0.05) increase was observed in the Mg-glycinate control groups [22.8mg/kg (B) and 32.8mg/kg (C)] compared to the normal control group. Significant (p < 0.05) decrease in (IL-10) levels was observed in the CYP-treated group compared to the normal control and Mg-glycinate control groups. However, a significant (p < 0.05) increase was noted with CYP/Mg-glycinate (22.8mg) and CYP/Mg-glycinate (32.8mg) compared to the CYP control group (D). 3.6 Effects of Mg-glycinate on levels of Tumour Necrosis Factor Alpha (TNF-α) in rats. Effects of Magnesium glycinate on levels of interleukin 10 (TNF-α) in rats treated with CYP (Fig. 5 ). No significant difference was observed in the Mg-glycinate control groups [22.8mg/kg (B) and 32.8mg/kg (C)] compared to the normal control group. Significant (p < 0.05) increase in TNF-α levels was observed in the CYP-treated group compared to the normal control and Mg-glycinate control groups. However, a significant (p < 0.05) decrease was noted with CYP/Mg-glycinate (22.8mg) and CYP/Mg-glycinate (32.8mg) compared to the CYP control group (D). 3.7 Hippocampal histomorphology 3.7.1. Histomorphological examination of H & E-stained section of dentate gyrus The granular cell layer (GL) of the dentate gyrus and the subgranular layer (SGL) were distinctly revealed in Fig. 6 A, more stained granule cells (red arrow head) were revealed in Fig. 6 B and 6 C, while numerous vacuolated granular cells (black arrow head) were observed in Fig. 6 D. Figure 6 E showed fairly restored vacuolated granular cells (black arrow head), while Fig. 6 F revealed the unremarkable granular cells (black head arrow). Cresyl fast Violet stain 3.7.2. Histomorphological examination of Cresyl violet-stained section of dentate gyrus The photomicrographs revealed Nissl granular components of the cells. Nissl granular components of the cytoplasm of cells in fig. 6D (CYP) were decreased compared to controls (fig. 1A). Fig 6E and Fig. 6F shows a slifghtly more expressed nissl substances (x 400). 3.7.3 Synaptophysin Immunohistochemical Staining of the Hippocampus Figure 8 (a-f). shows synaptic plasticity via evaluation of synaptophysin immunoreactivity in the hippocampus of rats. Normal control (Fig. 8 A) shows strong synaptophysin reactivity. 8B, 8C, and 8D: Sections show a decreased synaptophysin expression, as presented as a blurry brown stain (area circled red). 8E and 8F show a moderate synaptophysin expression. Figure 8 A: normal control rats, Fig. 8 B: Mg-glycinate (22.8mg/kg) treated rats, Fig. 8 C: rats administered Mg-glycinate (32.8mg/kg), 8D: animals treated with CYP alone, 8E: animals that received CYP/Mg-glycinate (22.8mg/kg), and Fig. 8 F: CYP/Mg-glycinate (32.8mg/kg) treated rats. Discussion The present study evaluated the ameliorative effects of magnesium glycinate against impaired memory, altered markers of inflammation, brain neurotransmitters, neuromodulators, degenerated hippocampal histomorphology, and synaptophysin immunoreactivity following cyclophosphamide administration. Magnesium glycinate combines magnesium with glycine (an amino acid that enhances the uptake, absorption, and bioavailability of elemental magnesium). In agreement with several studies [ 14 , 16 , 17 ] that have reported the adverse effect of CYP on body weight, CYP administration in this study results in a significant weight loss compared to the normal control. However, the Magnesium Glycinate administration alleviates these effects, as noted with a significant increase in body weight in the groups administered CYP/Mg. Glycinate 22.8 and CYP/Mg. Glycinate 32.8 compare the sole administration of CYP. Administration of Cylophosphamide, an alkylating agent, induces weight loss primarily through cytotoxic impact by depleting adipogenic precursors and reducing fat storage capacity [ 18 ]. Conversely, Studies [ 19 , 20 , 21 ] have shown that magnesium improves insulin sensitivity and glucose uptake, preventing muscle wasting while regulating inflammation-driven appetite loss via CCK release. In tandem with the reports by studies that have reported the memory-impairing effects of cyclophosphamide, the percentage correct alternation in the CYP control group was statistically decreased compared to the control group. While it increased in the groups solely administered magnesium at 22.8mg/kg and 32.8mg/kg. However, the decreased percentage correct alternation in the CYP sole administered group was reversed with magnesium treatment. Magnesium has been shown to have a memory enhancing effects in both animal studies and clinical trials (Clinical trial number: not applicable) by enhancing long-term potentiation [ 22 ]. A study done by Ibrahim (2024) [ 5 ] showed that neurotransmitters and neuromodulators are major victims of cyclophosphamide administration in animal studies. Disruption of inhibitory and excitatory neurotransmitter activities is often attributed to CYP toxicity [ 23 ]. In corroboration of the reported debilitating impact of CYP, levels of dopamine, acetylcholine, and brain-derived neurotropic factor (BDNF) activities were significantly decreased with CYP usage compared to the control, while their levels increased in groups administered Mg. glycinate at 22.8mg/kg and 32.8mg/kg. Acetylcholine and dopamine primarily act as excitatory neurotransmitters; they could act indirectly as inhibitory neurotransmitters depending on certain receptors and brain regions [ 24 ]. While BDNF serves as an important neurotrophic protein that enhances neuronal well-being, growth, and neuroplasticity, vital for learning and memory conservation [ 25 ]. Primarily, Acetylcholine promotes Long-term potentiation, while dopamine modulates through D1/D5 receptors [ 26 ]. On the other hand, serotonin is neither inhibitory nor excitatory, as its effect is often determined by its receptor; postsynaptic neurons are depolarized via IP3/Ca 2+, with 5-HT2A/2C receptors enhancing excitability. It exerts inhibitory effects primarily through 5-HT1A receptors and induces postsynaptic hyperpolarization through potassium (K⁺) channel activation. In this study, a significant disruption in serotonin levels was observed in the group administered CYP alone compared to the normal control. Interestingly, magnesium administration regulates the levels of neurotransmitters and neuromodulators as previously reported by Patel et al . (2024) [ 8 ]. The ameliorative impact of Magnesium is attributed to its ability to affect ion channel activities, synaptic vesicle release, and signaling pathways. Concerning the activities of inflammatory cytokines, CYP administration is often associated with significantly increased levels of pro-inflammatory cytokines, while it decreases the levels of anti-inflammatory cytokines [ 27 ]. Consistent with these reports is the result of inflammatory and anti-inflammatory cytokines in this study. Levels of Interleukin 1beta and Tumour Necrosis Factor alpha significantly increase compared to the normal control. On the other hand, the levels of the pro-inflammatory cytokine significantly decreased in the groups administered CYP/Mg-glycinate (22.3mg/kg) and CYP/Mg-glycinate (32.8mg/kg). The anti-inflammatory effects of Mg-glycinate are attributed to its intracellular modulatory effects on NF-κB signaling and calcium homeostasis [ 28 ]. As previously reported by Onaolapo et al. (2023), levels of IL-10 significantly decrease with CYP administration. Interestingly, treatment with Mg-glycinate reversed these effects, particularly at 32.8mg/kg [ 14 ]. The general histolomorphological evaluation of the hippocampal dentate gyrus reveals well-stained granular cells and a sub-granular layer of the dentate gyrus in the control group (Fig. 6 A). The group treated with magnesium shows the neuronal-enhancing effects of magnesium (Fig. 6 B and 6 C) in tandem with reports by Varga et al . (2025) [ 29 ]. The photomicrograph in the CYP administered group revealed a significant loss of granular cells in corroboration with studies that have reported similar effects [ 30 ]. Interestingly, treatment with Mg-glycinate (Fig. 6 E and 6 F) against CYP toxicity revealed an ameliorating impact of Mg-glycinate, particularly at 32mg/kgb.w (Fig. 6 F). This could be attributed to its ability to reverse neuro-inflammation and modulates neurotransmitters levels as previously reported in this study and in agreement with other studies that have reported that Mg-glycinate possesses anti-inflammatory and neuro-modulatory properties [ 8 ]. Furthermore, the cresyl fast violet-stained section of the dentate gyrus corroborates the effects seen with the H and E-stained sections in this study. The Nissl substances were slightly more pronounced in the Mg-glycinate sole-administered group, while the CYP-administered group (Fig. 7 D) showed less stained Nissl substances. More interesting is the slight ameliorative effects of Mg-glycinate shown in Figs. 7 E and 7 F. Synaptic integrity was evaluated in this study with Synaptophycin immuno-reactivity, an endogenous presynaptic vesicle glycoprotein and a viable marker for synaptic health. Strong punctate staining, indicating positive synaptophycin immuno-reactivity is well expressed in the normal control and the group sole administered Mg. Glycinate at 22.8mg/kg b.w and Mg. Glycinate at 32.8mg/kg (Figs. 8 A, 8 B, and 8 C). However, synaptic injury was observed with CYP administration, as expressed with a pale and decreased immunoreactivity (Fig. 7 D). Studies [ 31 , 32 ] have continued to report oxidative stress and neuro-inflammation as mechanisms through which CYP impairs brain health. In agreement with these reports are the elevated levels of pro-inflammatory cytokines (TNF-α and IL-1β) evaluated in this study. Decreased synaptic integrity is obviously the factor for the decreased levels of spatial working memory that were observed with CYP in this study. Conclusion To summarize, the results presented in this study emphasize a potential therapeutic application of Mg. Glycinate as a supplement in the management of cancer, particularly in alleviating the adverse effects (weight loss, inflammation, decreased spatial working memory, and neurodegeneration) of chemotherapeutic agents such as Cyclophosphamide. While we hope to harness Mg. glycinate in this regard. There is, however, a need for further findings on its possible molecular interaction with conventional anti-cancer agents, particularly Cyclophosphamide. Declarations Ethics Approval and consent to participate The experimental procedure was accomplished in accordance with the methods approved by the animal research ethical committee of the Faculty of Basic Medical Sciences, University of Ilesa, Ilesa, Osun State, Nigeria, with approval code (UNILESA/FBMS/2025/04). Consent to Publication Not applicable Competing Interest The authors declared no competing interest Funding This study received funding from the Tertiary Education Trust Fund (TETFUND) under the Institutional-Based Research intervention for the year 2025. Author Contribution Conceptualization: FOO, KPF, LAH. Data curation: FOO, BEO, AAO, AO. Formal analysis: LMB, AO, OAA, BEO, and ATE. Funding acquisition: TETFUND. Methodology: FOO, LAH, and MTO. Resources: BEO, ATE. Writing original draft: FOO. Writing review & editing: KPF, BEO, AO, LMB, MTO, OAA, LAH. Approval of final manuscript: all authors. Acknowledgements We appreciate our Vice Chancellor (Professor Taiwo Asaolu) for his constant encouragement and support in the research within our university (University of Ilesa). Data Availability The datasets generated during the study are available from the corresponding author on reasonable request. References Timp W, Feinberg AP. 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A Magtein®, Magnesium L-Threonate, -Based Formula Improves Brain Cognitive Functions in Healthy Chinese Adults. Nutrients. 2022;14(24):5235. 10.3390/nu14245235 . Durairaj P, Liu ZL. Brain Cytochrome P450: Navigating Neurological Health and Metabolic Regulation. J Xenobiot. 2025;15(2):44. 10.3390/jox15020044 . Lester DB, Rogers TD, Blaha CD. Acetylcholine-dopamine interactions in the pathophysiology and treatment of CNS disorders. CNS Neurosci Ther. 2010;16(3):137–62. 10.1111/j.1755-5949.2010.00142.x . Shen T, You Y, Gupta VK, Graham SL. Aging, brain-derived neurotrophic factor (BDNF) and its Val66Met polymorphism. Factors Affecting Neurological Aging. Amsterdam: Elsevier; 2021. pp. 17–25. Kassab R. Acetylcholine-sensitive control of long-term synaptic potentiation in hippocampal CA3 neurons. Hippocampus. 2023;33(8):948–69. 10.1002/hipo.23533 . Sawant-Basak A, Xu Y, Zhang Q, Reynolds C, Rostami-Hodjegan A. Assessing trends in cytokine-CYP drug interactions and relevance to drug dosing. Clin Pharmacol Ther. 2024. 10.1002/cpt.3360 . Veronese N, Pizzol D, Smith L, Dominguez LJ, Barbagallo M. Effect of magnesium supplementation on inflammatory parameters: a meta-analysis of randomized controlled trials. Nutrients. 2022;14(3):679. 10.3390/nu14030679 . Varga P, Lehoczki A, Fekete M, Jarecsny T, Kryczyk-Poprawa A, Zábó V, et al. The role of magnesium in depression, migraine, Alzheimer's disease, and cognitive health: a comprehensive review. Nutrients. 2025;17(13):2216. 10.3390/nu17132216 . Ibrahim KM, Abdelrahman RS. Molecular mechanisms underlying cyclophosphamide-induced neurotoxicity: new approaches to therapy and prevention. Naunyn Schmiedebergs Arch Pharmacol. 2023;396(11):3423–40. 10.1007/s00210-023-02728-3 . Janelsins MC, Heckler CE, Thompson BD, Gross RA, Opanashuk LA, Cory-Slechta DA. A clinically relevant dose of cyclophosphamide chemotherapy impairs memory performance on the delayed spatial alternation task that is sustained over time as mice age. Neurotoxicology. 2016;56:287–93. 10.1016/j.neuro.2016.06.013 . Dash UC, Yusif NM, El Batsh KH. Oxidative stress and inflammation in the pathogenesis of neurological disorders: mechanisms and implications. Neurochem Int. 2024;172:105789. 10.1016/j.neuint.2024.105789 . Additional Declarations No competing interests reported. Supplementary Files GRAPHICALABSTRACT.docx Cite Share Download PDF Status: Under Review Version 1 posted Reviewers agreed at journal 04 May, 2026 Reviewers agreed at journal 03 May, 2026 Reviewers agreed at journal 26 Apr, 2026 Reviewers agreed at journal 24 Apr, 2026 Reviewers invited by journal 24 Apr, 2026 Editor assigned by journal 31 Mar, 2026 Submission checks completed at journal 31 Mar, 2026 First submitted to journal 21 Mar, 2026 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-9185242","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":634394118,"identity":"511697f9-f21d-4a36-a864-1bc81879dd16","order_by":0,"name":"Foluso Olamide Ojo","email":"","orcid":"","institution":"University of Ilesa","correspondingAuthor":false,"prefix":"","firstName":"Foluso","middleName":"Olamide","lastName":"Ojo","suffix":""},{"id":634394125,"identity":"15abc037-14b5-4d27-8781-2087f9f92f03","order_by":1,"name":"Luqman Adepoju Hassan","email":"","orcid":"","institution":"University of Ilesa","correspondingAuthor":false,"prefix":"","firstName":"Luqman","middleName":"Adepoju","lastName":"Hassan","suffix":""},{"id":634394129,"identity":"d39b33c6-9005-4baa-a44f-67752b9ade4d","order_by":2,"name":"Bukola Ezekiel Olatundun","email":"","orcid":"","institution":"University of Ilesa","correspondingAuthor":false,"prefix":"","firstName":"Bukola","middleName":"Ezekiel","lastName":"Olatundun","suffix":""},{"id":634394131,"identity":"ded9df04-3130-4421-aac7-2b92f7c3e394","order_by":3,"name":"Abiodun Abioye Oyeleke","email":"","orcid":"","institution":"Federal University. Oye-Ekiti","correspondingAuthor":false,"prefix":"","firstName":"Abiodun","middleName":"Abioye","lastName":"Oyeleke","suffix":""},{"id":634394136,"identity":"e8099ffc-920e-47db-88c9-f158b0be2704","order_by":4,"name":"Kolade Pelumi Folorunso","email":"data:image/png;base64,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","orcid":"","institution":"University of Ilesa","correspondingAuthor":true,"prefix":"","firstName":"Kolade","middleName":"Pelumi","lastName":"Folorunso","suffix":""},{"id":634394137,"identity":"3bf52119-053b-42ca-abe5-fb1e5c5e73e6","order_by":5,"name":"Adefola Tolulope Edward","email":"","orcid":"","institution":"University of Ilesa","correspondingAuthor":false,"prefix":"","firstName":"Adefola","middleName":"Tolulope","lastName":"Edward","suffix":""},{"id":634394140,"identity":"5ada0dd8-a834-418e-a5b3-e3dcfd57f2b9","order_by":6,"name":"Mary Tolulope Onaolapo","email":"","orcid":"","institution":"Saint Peter's University.","correspondingAuthor":false,"prefix":"","firstName":"Mary","middleName":"Tolulope","lastName":"Onaolapo","suffix":""},{"id":634394144,"identity":"fae05d22-8498-49a6-825d-778da82809f2","order_by":7,"name":"Olayemi Afolabi","email":"","orcid":"","institution":"University of Ilesa","correspondingAuthor":false,"prefix":"","firstName":"Olayemi","middleName":"","lastName":"Afolabi","suffix":""},{"id":634394147,"identity":"359ee0d8-5283-4abb-8066-3da9db44d207","order_by":8,"name":"Aishat Abidemi Olawale","email":"","orcid":"","institution":"University of Ilesa","correspondingAuthor":false,"prefix":"","firstName":"Aishat","middleName":"Abidemi","lastName":"Olawale","suffix":""},{"id":634394148,"identity":"f9dec6a0-eabb-410f-9250-2d3e3a6b4370","order_by":9,"name":"Muhsinat Bisola Lawal","email":"","orcid":"","institution":"University of Ilesa","correspondingAuthor":false,"prefix":"","firstName":"Muhsinat","middleName":"Bisola","lastName":"Lawal","suffix":""}],"badges":[],"createdAt":"2026-03-21 10:53:43","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-9185242/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-9185242/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":108577536,"identity":"fbe70a85-fe91-46ad-a297-33b6caab9698","added_by":"auto","created_at":"2026-05-06 07:30:42","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":51774,"visible":true,"origin":"","legend":"\u003cp\u003eEffects of Magnesium glycinate on the change in body weight (weekly) in Cyclophosphamide-treated rats. Each bar is presented as Mean ± S.E.M, *p \u0026lt; 0.05 against control, \u003csup\u003e#\u003c/sup\u003ep \u0026lt; 0.05 significant difference from CYP.\u0026nbsp; The number of rats in each group equals eight (8). Mg: Magnesium, CYP: Cyclophosphamide.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-9185242/v1/307cf16ce96fe1701f85680f.png"},{"id":108804936,"identity":"11b78fd8-6d06-4933-a284-521d18ae4a1d","added_by":"auto","created_at":"2026-05-08 15:24:18","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":51856,"visible":true,"origin":"","legend":"\u003cp\u003eEffects of Magnesium glycinate on percentage correct alternation (weekly) in Cyclophosphamide-treated rats. Each bar is presented as Mean ± S.E.M, *p \u0026lt; 0.05 against control, \u003csup\u003e#\u003c/sup\u003ep \u0026lt; 0.05 significant difference from CYP.\u0026nbsp; The number of rats in each group equals eight (8). Mg: Magnesium, CYP: Cyclophosphamide.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-9185242/v1/a31aa372b8367b114ceacc04.png"},{"id":108577538,"identity":"56740319-4ca0-44d4-9140-302deeb74e6a","added_by":"auto","created_at":"2026-05-06 07:30:42","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":49234,"visible":true,"origin":"","legend":"\u003cp\u003eEffects of Magnesium glycinate on IL-1β levels in Cyclophosphamide-treated rats. Each bar is presented as Mean ± S.E.M, *p \u0026lt; 0.05 against control, \u003csup\u003e#\u003c/sup\u003ep \u0026lt; 0.05 significant difference from CYP.\u0026nbsp; The number of rats in each group equals eight (8). Mg: Magnesium, CYP: Cyclophosphamide.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-9185242/v1/7e41a3b4fdf8bab0e45674b0.png"},{"id":108577539,"identity":"6c5d7cb3-d51b-4a9b-a418-8e7fd7b9e9b5","added_by":"auto","created_at":"2026-05-06 07:30:42","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":20749,"visible":true,"origin":"","legend":"\u003cp\u003eEffects of Magnesium glycinate on IL-10 levels in Cyclophosphamide-treated rats. Each bar is presented as Mean ± S.E.M, *p \u0026lt; 0.05 against control, \u003csup\u003e#\u003c/sup\u003ep \u0026lt; 0.05 significant difference from CYP.\u0026nbsp; The number of rats in each group equals eight (8). Mg: Magnesium, CYP: Cyclophosphamide.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-9185242/v1/86b420deb67fc24d1aea9fdf.png"},{"id":108577540,"identity":"f0cbc7f0-99da-4e11-ad37-538a4c5119ca","added_by":"auto","created_at":"2026-05-06 07:30:42","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":17840,"visible":true,"origin":"","legend":"\u003cp\u003eEffects of Magnesium glycinate on TNF-α level in Cyclophosphamide-treated rats. Each bar is presented as Mean ± S.E.M, *p \u0026lt; 0.05 against control, \u003csup\u003e#\u003c/sup\u003ep \u0026lt; 0.05 significant difference from CYP.\u0026nbsp; The number of rats in each group equals eight (8). Mg: Magnesium, CYP: Cyclophosphamide.\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-9185242/v1/f594791bea830eaf80fa544b.png"},{"id":108804903,"identity":"09a876ec-94fc-4d93-9bea-a904c4c8dc9e","added_by":"auto","created_at":"2026-05-08 15:24:11","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":555699,"visible":true,"origin":"","legend":"\u003cp\u003e(A-F): Histomorphological examination of H \u0026amp; E-stained section of dentate gyrus (×400), group A (control), B (Mg-glycinate at 22.8mg/kg b.w.), C (Mg-glycinate at 22.8mg/kg b.w), D (CYP), E (CYP/Mg-glycinate at 22.8mg/kg b.w), F (CYP/Mg-glycinate at 22.8mg/kg b.w+32.8 mg/kg b.w).\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-9185242/v1/ea785687fbe58e2c7690bd6f.png"},{"id":108577541,"identity":"08315b1d-5107-430d-951b-a76fda927ee9","added_by":"auto","created_at":"2026-05-06 07:30:43","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":476583,"visible":true,"origin":"","legend":"\u003cp\u003e(A-F): Histomorphological examination of Cresyl violet-stained section of dentate gyrus (×400), group A (control), B (Mg-glycinate at 22.8mg/kg b.w.), C (Mg-glycinate at 22.8mg/kg b.w), D (CYP), E (CYP/Mg-glycinate at 22.8mg/kg b.w), F (CYP/Mg-glycinate at 22.8mg/kg b.w+32.8 mg/kg b.w).\u003c/p\u003e","description":"","filename":"7.png","url":"https://assets-eu.researchsquare.com/files/rs-9185242/v1/3b77c9c0b2267de00d797814.png"},{"id":108577543,"identity":"909e7414-dfe5-4d3c-8b88-ed5f1614e66a","added_by":"auto","created_at":"2026-05-06 07:30:43","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":633723,"visible":true,"origin":"","legend":"\u003cp\u003e(A-F): Effects of Mg-glycinate on synaptophysin immunoreactivity in the section of hippocampal dentate gyrus in rats treated with cyclophosphamide. Fig. 8A shows strong synaptophysin reactivity. Fig. 8D shows a decreased synaptophysin expression, as presented as a blurry brown stain (area circled red). 10E and F show a moderate synaptophysin expression. Fig. 8A: normal control rats, fig. 8B: Mg-glycinate (22.8mg/kg) treated rats, fig. 8C: rats administered Mg-glycinate (32.8mg/kg), 8D: animals treated with CYP alone, 8E: animals that received CYP/Mg-glycinate (22.8mg/kg), and fig. 8F: CYP/Mg-glycinate (32.8mg/kg) treated rats.\u003c/p\u003e","description":"","filename":"8.png","url":"https://assets-eu.researchsquare.com/files/rs-9185242/v1/db11c883f52fb42fb1a91b25.png"},{"id":108809867,"identity":"8594c498-eb63-45dc-89e2-b7c52269cc7d","added_by":"auto","created_at":"2026-05-08 15:55:58","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1979294,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-9185242/v1/3d0e239b-9272-49d1-aede-dba458852458.pdf"},{"id":108577535,"identity":"409d3a67-2e3d-43f9-a04e-b2e93d85d4b9","added_by":"auto","created_at":"2026-05-06 07:30:42","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":207062,"visible":true,"origin":"","legend":"","description":"","filename":"GRAPHICALABSTRACT.docx","url":"https://assets-eu.researchsquare.com/files/rs-9185242/v1/655c074625bfa4e8852812aa.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Magnesium glycinate attenuates cyclophosphamide-induced neurotoxicity by modulating brain neurotransmitters, inflammatory markers, and hippocampal synaptophysin immunoreactivity","fulltext":[{"header":"Introduction","content":"\u003cp\u003eCancer, a malignant disease defined by dysregulated cellular expansion, resistance to apoptosis, persistent neovascularization, and invasive dissemination capacity, has recently emerged as a global burden [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e], constituting the second leading cause of mortality worldwide [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Chemotherapeutic agents such as cyclophosphamide show promising effects in reducing the death rate in some cancer patients [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Despite the significant positive impact of the chemotherapeutic agents in some cancer management, the debilitating effects, such as organ toxicity, cellular degeneration, and myelosuppression, often result in treatment discontinuation. Notably, cyclophosphamide usage is usually associated with neurotoxicity, loss of hair (alopecia), immunosuppression, haemorrhagic cystitis, and cardiotoxicity, particularly at high dosage [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eInduction of inflammatory processes and reactive oxygen species (ROS) are its known pharmacological mechanisms of action that result in neurotoxicity [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Moreover, associated with the oxidative and inflammatory processes are the depleted levels of brain magnesium, which further result in intracellular depletion that exacerbates neuronal damage through N-methyl-D-aspartate receptor NMDA receptor hyper-excitability [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. The inability of most chemotherapeutic drugs to discriminate between the cancerous cells and the healthy body cells is mostly a documented reason for their toxic adverse effects [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. The debilitating effects of these drugs have further necessitated and increased our quest to search for novel substances or means that would not only mitigate the adverse effects but would also enhance chemotherapeutic efficiency in a functional biological system in the absence of cancer.\u003c/p\u003e \u003cp\u003eStudies have continued to support the effects of magnesium on brain health through its neuroprotective impact and synaptic functions [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. A study done by Li et al. (2014) showed an increased level of brain magnesium in mouse models of Alzheimer's disease (APPswe/PS1dE9), reduced amyloid plaques by 36%, prevented synapse loss through NMDAR, signaling preservation, and also reversed memory deficits even at late stages of disease [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. Subsequently, Chen et al. (2024), in their systematic review of RCTs, corroborate magnesium\u0026rsquo;s role in brain health and cognition, with elevated levels of brain magnesium reducing neuroinflammation and enhancing learning and memory through NMDA modulation [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. However, this study examined the therapeutic role of magnesium glycinate at varying dosages on the neurotoxicity induced by cyclophosphamide by evaluating levels of some brain neurotransmitters, markers of inflammation, memory, hippocampal histomorphology, and synaptophysin immunoreactivity in rats.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1. \u003cem\u003eChemicals and drugs\u003c/em\u003e\u003c/h2\u003e \u003cp\u003eMagnesium glycinate (250 mg, New York, USA), normal saline, Cyclophosphamide injection (xGen Pharmaceutical DJB). ELIZA assay kits for dopamine, acetylcholine, Serotonin, brain-derived neurotropic factor, interleukin 1β, interleukin-10, and tumour necrosis factor (TNF-α) were procured from R\u0026amp;D Systems (Bio-Techne, USA).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2. \u003cem\u003eAnimals and experimental design\u003c/em\u003e\u003c/h2\u003e \u003cp\u003e The experimental procedure was accomplished in accordance with the methods approved by the animal research ethical committee of the Faculty of Basic Medical Sciences, University of Ilesa, Ilesa, Osun State, Nigeria, with approval code (UNILESA/FBMS/2025/04). These procedures adhered to the European Council Directive 2010/63/EU guidelines governing the protection of animals used for scientific purposes\u003c/p\u003e \u003cp\u003eForty-eight (48) Wistar rats (eight weeks old), each weighing 150\u0026ndash;170 g, were procured from animal breeders in Iwo, Osun state, Nigeria. The animals were conveyed in the cool of the day to the research animal house of the University of Ilesa, Ilesa, Osun State, Nigeria, where the experiment was done. Plastic cages (45 \u0026times; 24 \u0026times; 20 cm) were maximized for the animal housing in a temperature-controlled environment (22\u0026deg;C) under a 12-hour light-dark cycle throughout the experimental period (21 days). Before the commencement of the experiment, acclimatization of the animals was done for two weeks, after which the animals were allocated into four experimental cohorts (n\u0026thinsp;=\u0026thinsp;8), while animals across all groups were fed with normal feed (Top Feeds\u0026reg;, Nigeria), and clean water \u003cem\u003ead libitum\u003c/em\u003e. Additionally, Animals in groups B and C orally received magnesium glycinate at 22.8 mg/kg and 32.8mg/kg, respectively, according to Aniebo (2023) [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Groups D, E, and F received cyclophosphamide injection 3 times at 150mg/kg intraperitoneally every other day (days 1, 3, and 5), while groups D and E additionally received oral magnesium glycinate at 22.8 mg/kg and 32.8mg/kg, respectively. The animal\u0026rsquo;s body weight was measured weekly using an electronic scale throughout the experimental period.\u003c/p\u003e \u003cp\u003eOn the 22nd day, animals were introduced to the Y-maze neurobehavioral test for memory evaluation. Following the spatial working memory evaluation test in the Y-maze model, the animals were sacrificed by cervical dislocation on the 23rd day.\u003c/p\u003e \u003cp\u003eFollowing this was the dissection of the cranium for the removal of the brain tissues, after which the right hemisphere of each of the animals was homogenized using iced cold phosphate-buffered saline (PBS) as a medium at a ratio of 1:9 (weight of brain tissue/ volume of iced cold PBS). Tissue homogenate supernatant was used for the assessment of neurotransmitters (Serotonin, dopamine, and acetylcholine) levels, the activities of neuromodulator (Brain-Derived Neurotropic factor), and levels of inflammatory markers (interleukin 1beta, interleukin 10, and Tumour Necrosis Factor alpha).\u003c/p\u003e \u003cp\u003eFurthermore, the left hemisphere was dissected and fixed in 10% buffered formalin, processed in paraffin embedding, cut at 5\u0026micro; m for histological evaluation (H\u0026amp;E and Cresyl fast violet stain) and synaptophysin immuno-reactivity.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3. Assessment of change in body weight (weekly)\u003c/h2\u003e \u003cp\u003eAs previously documented by Ojo \u003cem\u003eet al\u003c/em\u003e. (2025) [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e], individual animal\u0026rsquo;s Body weight were evaluated weekly using an electronic weighing balance. Following this, the weekly change in body weight was determined using the formula\u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\:\\:\\:\\text{F}\\text{i}\\text{n}\\text{a}\\text{l}\\:\\left(\\text{l}\\text{a}\\text{s}\\text{t}\\:\\text{w}\\text{e}\\text{e}\\text{k}\\right)-Initial\\:\\left(first\\:week\\right)\\)\u003c/span\u003e\u003c/span\u003e. The results were statistically analyzed to determine the mean values.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4. Neurobehavioral test for Memory (Y-maze)\u003c/h2\u003e \u003cp\u003eAs previously reported by Onaolapo (2023) [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e] and Ojo et al. (2025) [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e], the Y-maze model was used in measuring the spatial working memory. Spatial working memory was scored by monitoring unpremeditated alternation behaviour. Unpremeditated alternation behaviour is the predisposition of rats to alternate customarily non-reinforced choices of the Y-maze on sequential chance. Each rat was positioned in one of the arms of the Y maze and was left to explore the arms for 5 minutes without restriction until its tail completely moved into the other arm.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.5. Enzyme Linked Immunosorbent Assay (ELISA) analysis\u003c/h2\u003e \u003cp\u003eEnzyme-linked immunosorbent assay protocols with the commercially available kits (Enzo Life Sciences Inc., NY, USA), made to determine the total levels of individual cytokines as previously reported by Onaolapo et al. (2023) [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e] was used to determine the levels of interleukin-1Beta (IL-1β), interleukin-10 (IL-10), and Tumor Necrosis factor-α levels. Similarly, the levels of dopamine, serotonin, acetylcholine, and brain-derived neurotropic factor were measured from the supernatant of the homogenized brain tissue, through the usage of commercially available Enzyme-linked immunosorbent assay kits, following the manufacturer\u0026rsquo;s method (ABCAM, Cambridge, UK).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e2.6. Synaptophysin Immunohistochemical Evaluation\u003c/h2\u003e \u003cp\u003eThe left hemisphere of the brain tissue was fixed in 10% neutral buffered formalin, embedded in paraffin, and cut into 5\u0026micro;m sections, which were placed on positively charged slides. Sections of the hippocampus were embedded at 53\u0026ndash;65\u0026deg;C for 30 minutes. Following this, slides were dewaxed in xylene (2 changes, 5 min each), rehydrated through graded ethanol (100%, 95%, 70%, for 5 minutes each, and rinsed in distilled water.​\u003c/p\u003e \u003cp\u003eAs reported by Krenacs (2010), Heat-induced epitope retrieval (HIER) was done using citrate buffer (pH 6.0) for 25 minutes in an automated stainer. Following this, slides were cooled to room temperature and rinsed in PBS/TBST. Endogenous peroxidase was blocked with 3% H₂O₂ for 15 minutes and incubated in 10% normal goat serum in PBS for 30 minutes to reduce non-specific binding.​ Anti-synaptophysin monoclonal antibody (clone IHC669) was applied overnight at 4\u0026deg;C and at room temperature. After which it was rinsed thrice in phosphate-buffered saline for 5 minutes each.​ Slides were incubated with biotinylated secondary antibody for 20 minutes, then HRP-streptavidin complex for 20 minutes. For brown precipitate, slides were developed with DAB chromogen for 10 minutes. Counterstained with hematoxylin for 1\u0026ndash;5 minutes, dehydrated, cleared in xylene, and mounted [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e2.7 Photomicrography\u003c/h2\u003e \u003cp\u003eSellon-Olympus trinocular microscope (XSZ-107E, China) was used to microscopically examine the histologically processed section of the hippocampus. The hippocampal photomicrographs were captured with a digital camera (Canon Powershot 2500). A pathologist who was na\u0026iuml;ve to the experimental cohorts evaluated the histopathological changes.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003e2.8 Statistical analysis\u003c/h2\u003e \u003cp\u003eData were presented as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard error of the mean. Normally distributed data was analyzed using One-way analysis of variance (ANOVA). Statistical analyses of data were carried out using GraphPad Prism ver. (9.0 for Windows, GraphPad Software, San Diego, CA, USA). Statistically significant difference was considered as p\u0026thinsp;\u0026lt;\u0026thinsp;0.05.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\n \u003ch2\u003e3.1. Effects of Magnesium-glycinate on body weight in rats.\u003c/h2\u003e\n \u003cp\u003eEffects of Magnesium glycinate on the weekly change in body weight in rats treated with CYP (Fig. \u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). No significant difference was observed in the Mg-glycinate control groups [22.8mg/kg (B) and 32.8mg/kg (C)] compared to the control group. Significant (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) decrease in weight gain (weekly) was noted in the CYP-treated group compared to the normal control and Mg-glycinate control groups. Moreover, a significant increase was noted with CYP/Mg-glycinate (22.8mg) and CYP/Mg-glycinate (32.8mg) compared to the CYP control group (D).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\n \u003ch2\u003e3.2 Effect of Mg-glycinate on working memory (spatial) in the Y-Maze model in rats.\u003c/h2\u003e\n \u003cp\u003eEffects of Magnesium glycinate on the percentage correct alternation in rats treated with CYP (Fig. \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). A significant (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05) increase was observed in the Mg-glycinate control groups [22.8mg/kg (B) and 32.8mg/kg (C)] compared to the control group. Significant (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) decrease in percentage correct alternation was observed in the CYP-treated group compared to the normal control and Mg-glycinate control groups. Also, a significant increase was noted with CYP/Mg-glycinate (22.8mg) and CYP/Mg-glycinate (32.8mg) compared to the CYP control group (D).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e\n \u003ch2\u003e3.3. Effects of Mg-glycinate on levels of serotonin, acetylcholine, dopamine, and Brain-Derived Neurotropic Factor.\u003c/h2\u003e\n \u003cp\u003eTable \u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. Shows the effects of Magnesium glycinate on levels of brain neurotransmitters and neuromodulators (Serotonin, Acetylcholine, Dopamine, and BDNF) in rats treated with Cyclophosphamide. In comparison with the normal control group (A), the sole administration of CYP. (group D) shows a significantly increased level of serotonin activities, while dopamine, acetylcholine, and BDNF activities significantly (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) decrease. However, in comparison with the sole administration of CYP, administration of CYP/Mg-glycinate (22.8mg) and CYP/Mg-glycinate (22.8mg) showed a significant (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) decrease in the activities of serotonin, while the levels of acetylcholine, dopamine, and BDNF significantly increased.\u0026nbsp;\u003c/p\u003e\n \u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cp\u003eEffects of Mg-glycinate on levels of serotonin, acetylcholine, dopamine, and Brain-Derived Neurotropic Factor.\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\" colname=\"c1\"\u003e\n \u003cp\u003eGroups\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\" colname=\"c2\"\u003e\n \u003cp\u003eSerotonin\u003c/p\u003e\n \u003cp\u003e(pmol/mg tissue)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\" colname=\"c3\"\u003e\n \u003cp\u003eAcetylcholine (pmol/mg tissue)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\" colname=\"c4\"\u003e\n \u003cp\u003eDopamine\u003c/p\u003e\n \u003cp\u003e(pmol/mg tissue)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\" colname=\"c5\"\u003e\n \u003cp\u003eBDNF (pmol/mg\u003c/p\u003e\n \u003cp\u003etissue)\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" colname=\"c1\"\u003e\n \u003cp\u003eControl\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c2\"\u003e\n \u003cp\u003e10.78\u0026thinsp;\u0026plusmn;\u0026thinsp;1.10\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c3\"\u003e\n \u003cp\u003e5.50\u0026thinsp;\u0026plusmn;\u0026thinsp;0.11\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c4\"\u003e\n \u003cp\u003e33.11\u0026thinsp;\u0026plusmn;\u0026thinsp;0.09\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c5\"\u003e\n \u003cp\u003e26.21\u0026thinsp;\u0026plusmn;\u0026thinsp;0.41\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" colname=\"c1\"\u003e\n \u003cp\u003eMg-glycinate (22.8mg)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c2\"\u003e\n \u003cp\u003e13.67\u0026thinsp;\u0026plusmn;\u0026thinsp;0.20\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c3\"\u003e\n \u003cp\u003e5.59\u0026thinsp;\u0026plusmn;\u0026thinsp;0.19\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c4\"\u003e\n \u003cp\u003e39.23\u0026thinsp;\u0026plusmn;\u0026thinsp;0.08\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c5\"\u003e\n \u003cp\u003e30.10\u0026thinsp;\u0026plusmn;\u0026thinsp;0.32\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" colname=\"c1\"\u003e\n \u003cp\u003eMg-glycinate (32.8mg)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c2\"\u003e\n \u003cp\u003e14.32\u0026thinsp;\u0026plusmn;\u0026thinsp;0.41\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c3\"\u003e\n \u003cp\u003e6.97\u0026thinsp;\u0026plusmn;\u0026thinsp;0.09\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c4\"\u003e\n \u003cp\u003e41.20\u0026thinsp;\u0026plusmn;\u0026thinsp;0.07\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c5\"\u003e\n \u003cp\u003e34.42\u0026thinsp;\u0026plusmn;\u0026thinsp;0.29\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" colname=\"c1\"\u003e\n \u003cp\u003eCYP\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c2\"\u003e\n \u003cp\u003e21.21\u0026thinsp;\u0026plusmn;\u0026thinsp;1.21\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c3\"\u003e\n \u003cp\u003e2.02\u0026thinsp;\u0026plusmn;\u0026thinsp;0.07\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c4\"\u003e\n \u003cp\u003e18.10\u0026thinsp;\u0026plusmn;\u0026thinsp;1.01\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c5\"\u003e\n \u003cp\u003e14.22\u0026thinsp;\u0026plusmn;\u0026thinsp;0.26\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" colname=\"c1\"\u003e\n \u003cp\u003eCYP/Mg-glycinate (22.8mg)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c2\"\u003e\n \u003cp\u003e13.00\u0026thinsp;\u0026plusmn;\u0026thinsp;0.18\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c3\"\u003e\n \u003cp\u003e3.21\u0026thinsp;\u0026plusmn;\u0026thinsp;0.03\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c4\"\u003e\n \u003cp\u003e24.12\u0026thinsp;\u0026plusmn;\u0026thinsp;0.16\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c5\"\u003e\n \u003cp\u003e17.41\u0026thinsp;\u0026plusmn;\u0026thinsp;0.11\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" colname=\"c1\"\u003e\n \u003cp\u003eCYP/Mg-glycinate (32.8mg)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c2\"\u003e\n \u003cp\u003e12.11\u0026thinsp;\u0026plusmn;\u0026thinsp;0.09\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c3\"\u003e\n \u003cp\u003e5.18\u0026thinsp;\u0026plusmn;\u0026thinsp;0.08\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c4\"\u003e\n \u003cp\u003e28.10\u0026thinsp;\u0026plusmn;\u0026thinsp;0.10\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c5\"\u003e\n \u003cp\u003e20.11\u0026thinsp;\u0026plusmn;\u0026thinsp;0.23\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n \u003cp\u003e\u003c/p\u003e\n \u003cp\u003eData was presented as Mean\u0026thinsp;\u0026plusmn;\u0026thinsp;S.E.M, *p\u0026thinsp;\u0026lt;\u0026thinsp;0.05 against control, \u003csup\u003e#\u003c/sup\u003ep\u0026thinsp;\u0026lt;\u0026thinsp;0.05 significant difference from CYP. The number of rats in each group equals eight (8). Mg: Magnesium, CYP: Cyclophosphamide\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e\n \u003ch2\u003e3.4. Effects of Mg-glycinate on interleukin-1beta levels in rats.\u003c/h2\u003e\n \u003cp\u003eEffects of Magnesium glycinate on levels of interleukin 1beta (IL-1\u0026beta;) in rats treated with CYP (Fig. \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). No significant difference was observed in the Mg-glycinate control groups [22.8mg/kg (B) and 32.8mg/kg (C)] compared to the control group. Significant (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) increase in (IL-1\u0026beta;) levels was observed in the CYP-treated group compared to the normal control and Mg-glycinate control groups. However, a significant decrease was noted with CYP/Mg-glycinate (22.8mg) and CYP/Mg-glycinate (32.8mg) compared to the CYP control group (D).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec16\" class=\"Section2\"\u003e\n \u003ch2\u003e3.5. Effects of Mg-glycinate on levels of interleukin 10 (IL-10) in rats.\u003c/h2\u003e\n \u003cp\u003eEffects of Magnesium glycinate on levels of interleukin 10 (IL-10) in rats treated with CYP (Fig. \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). A significant (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) increase was observed in the Mg-glycinate control groups [22.8mg/kg (B) and 32.8mg/kg (C)] compared to the normal control group. Significant (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) decrease in (IL-10) levels was observed in the CYP-treated group compared to the normal control and Mg-glycinate control groups. However, a significant (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) increase was noted with CYP/Mg-glycinate (22.8mg) and CYP/Mg-glycinate (32.8mg) compared to the CYP control group (D).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec17\" class=\"Section2\"\u003e\n \u003ch2\u003e3.6 Effects of Mg-glycinate on levels of Tumour Necrosis Factor Alpha (TNF-\u0026alpha;) in rats.\u003c/h2\u003e\n \u003cp\u003eEffects of Magnesium glycinate on levels of interleukin 10 (TNF-\u0026alpha;) in rats treated with CYP (Fig. \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). No significant difference was observed in the Mg-glycinate control groups [22.8mg/kg (B) and 32.8mg/kg (C)] compared to the normal control group. Significant (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) increase in TNF-\u0026alpha; levels was observed in the CYP-treated group compared to the normal control and Mg-glycinate control groups. However, a significant (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) decrease was noted with CYP/Mg-glycinate (22.8mg) and CYP/Mg-glycinate (32.8mg) compared to the CYP control group (D).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec18\" class=\"Section2\"\u003e\n \u003ch2\u003e3.7 Hippocampal histomorphology\u003c/h2\u003e\n \u003cdiv id=\"Sec19\" class=\"Section3\"\u003e\n \u003ch2\u003e3.7.1. Histomorphological examination of H \u0026amp; E-stained section of dentate gyrus\u003c/h2\u003e\n \u003cp\u003eThe granular cell layer (GL) of the dentate gyrus and the subgranular layer (SGL) were distinctly revealed in Fig. \u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA, more stained granule cells (red arrow head) were revealed in Fig. \u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB and \u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eC, while numerous vacuolated granular cells (black arrow head) were observed in Fig. \u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eD. Figure \u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eE showed fairly restored vacuolated granular cells (black arrow head), while Fig. \u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eF revealed the unremarkable granular cells (black head arrow).\u003c/p\u003e\n \u003c/div\u003e\n\u003c/div\u003e\n\u003ch3\u003eCresyl fast Violet stain\u003c/h3\u003e\n\u003cdiv id=\"Sec21\" class=\"Section3\"\u003e\n \u003cdiv class=\"Heading\"\u003e\u003cstrong\u003e3.7.2. Histomorphological examination of Cresyl violet-stained section of dentate gyrus\u003c/strong\u003e\u003c/div\u003e\n \u003cp\u003eThe photomicrographs revealed Nissl granular components of the cells. Nissl granular components of the cytoplasm of cells in fig. 6D (CYP) were decreased compared to controls (fig. 1A). Fig 6E and Fig. 6F shows a slifghtly more expressed nissl substances (x 400).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec22\" class=\"Section3\"\u003e\n \u003cp class=\"Heading\"\u003e\u003cstrong\u003e3.7.3 Synaptophysin Immunohistochemical Staining of the Hippocampus\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003eFigure \u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003e(a-f). shows synaptic plasticity via evaluation of synaptophysin immunoreactivity in the hippocampus of rats. Normal control (Fig. \u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eA) shows strong synaptophysin reactivity. 8B, 8C, and 8D: Sections show a decreased synaptophysin expression, as presented as a blurry brown stain (area circled red). 8E and 8F show a moderate synaptophysin expression. Figure \u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eA: normal control rats, Fig. \u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eB: Mg-glycinate (22.8mg/kg) treated rats, Fig. \u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eC: rats administered Mg-glycinate (32.8mg/kg), 8D: animals treated with CYP alone, 8E: animals that received CYP/Mg-glycinate (22.8mg/kg), and Fig. \u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eF: CYP/Mg-glycinate (32.8mg/kg) treated rats.\u003c/p\u003e\n\u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eThe present study evaluated the ameliorative effects of magnesium glycinate against impaired memory, altered markers of inflammation, brain neurotransmitters, neuromodulators, degenerated hippocampal histomorphology, and synaptophysin immunoreactivity following cyclophosphamide administration. Magnesium glycinate combines magnesium with glycine (an amino acid that enhances the uptake, absorption, and bioavailability of elemental magnesium).\u003c/p\u003e \u003cp\u003eIn agreement with several studies [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e] that have reported the adverse effect of CYP on body weight, CYP administration in this study results in a significant weight loss compared to the normal control. However, the Magnesium Glycinate administration alleviates these effects, as noted with a significant increase in body weight in the groups administered CYP/Mg. Glycinate 22.8 and CYP/Mg. Glycinate 32.8 compare the sole administration of CYP. Administration of Cylophosphamide, an alkylating agent, induces weight loss primarily through cytotoxic impact by depleting adipogenic precursors and reducing fat storage capacity [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. Conversely, Studies [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e] have shown that magnesium improves insulin sensitivity and glucose uptake, preventing muscle wasting while regulating inflammation-driven appetite loss via CCK release. In tandem with the reports by studies that have reported the memory-impairing effects of cyclophosphamide, the percentage correct alternation in the CYP control group was statistically decreased compared to the control group. While it increased in the groups solely administered magnesium at 22.8mg/kg and 32.8mg/kg. However, the decreased percentage correct alternation in the CYP sole administered group was reversed with magnesium treatment. Magnesium has been shown to have a memory enhancing effects in both animal studies and clinical trials (Clinical trial number: not applicable) by enhancing long-term potentiation [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eA study done by Ibrahim (2024) [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e] showed that neurotransmitters and neuromodulators are major victims of cyclophosphamide administration in animal studies. Disruption of inhibitory and excitatory neurotransmitter activities is often attributed to CYP toxicity [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. In corroboration of the reported debilitating impact of CYP, levels of dopamine, acetylcholine, and brain-derived neurotropic factor (BDNF) activities were significantly decreased with CYP usage compared to the control, while their levels increased in groups administered Mg. glycinate at 22.8mg/kg and 32.8mg/kg. Acetylcholine and dopamine primarily act as excitatory neurotransmitters; they could act indirectly as inhibitory neurotransmitters depending on certain receptors and brain regions [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. While BDNF serves as an important neurotrophic protein that enhances neuronal well-being, growth, and neuroplasticity, vital for learning and memory conservation [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. Primarily, Acetylcholine promotes Long-term potentiation, while dopamine modulates through D1/D5 receptors [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. On the other hand, serotonin is neither inhibitory nor excitatory, as its effect is often determined by its receptor; postsynaptic neurons are depolarized via IP3/Ca\u003csup\u003e2+,\u003c/sup\u003e with 5-HT2A/2C receptors enhancing excitability. It exerts inhibitory effects primarily through 5-HT1A receptors and induces postsynaptic hyperpolarization through potassium (K⁺) channel activation. In this study, a significant disruption in serotonin levels was observed in the group administered CYP alone compared to the normal control.\u003c/p\u003e \u003cp\u003eInterestingly, magnesium administration regulates the levels of neurotransmitters and neuromodulators as previously reported by Patel \u003cem\u003eet al\u003c/em\u003e. (2024) [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. The ameliorative impact of Magnesium is attributed to its ability to affect ion channel activities, synaptic vesicle release, and signaling pathways.\u003c/p\u003e \u003cp\u003eConcerning the activities of inflammatory cytokines, CYP administration is often associated with significantly increased levels of pro-inflammatory cytokines, while it decreases the levels of anti-inflammatory cytokines [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. Consistent with these reports is the result of inflammatory and anti-inflammatory cytokines in this study. Levels of Interleukin 1beta and Tumour Necrosis Factor alpha significantly increase compared to the normal control. On the other hand, the levels of the pro-inflammatory cytokine significantly decreased in the groups administered CYP/Mg-glycinate (22.3mg/kg) and CYP/Mg-glycinate (32.8mg/kg). The anti-inflammatory effects of Mg-glycinate are attributed to its intracellular modulatory effects on NF-κB signaling and calcium homeostasis [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. As previously reported by Onaolapo et al. (2023), levels of IL-10 significantly decrease with CYP administration. Interestingly, treatment with Mg-glycinate reversed these effects, particularly at 32.8mg/kg [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe general histolomorphological evaluation of the hippocampal dentate gyrus reveals well-stained granular cells and a sub-granular layer of the dentate gyrus in the control group (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA). The group treated with magnesium shows the neuronal-enhancing effects of magnesium (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB and \u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eC) in tandem with reports by Varga \u003cem\u003eet al\u003c/em\u003e. (2025) [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. The photomicrograph in the CYP administered group revealed a significant loss of granular cells in corroboration with studies that have reported similar effects [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. Interestingly, treatment with Mg-glycinate (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eE and \u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eF) against CYP toxicity revealed an ameliorating impact of Mg-glycinate, particularly at 32mg/kgb.w (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eF). This could be attributed to its ability to reverse neuro-inflammation and modulates neurotransmitters levels as previously reported in this study and in agreement with other studies that have reported that Mg-glycinate possesses anti-inflammatory and neuro-modulatory properties [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. Furthermore, the cresyl fast violet-stained section of the dentate gyrus corroborates the effects seen with the H and E-stained sections in this study. The Nissl substances were slightly more pronounced in the Mg-glycinate sole-administered group, while the CYP-administered group (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eD) showed less stained Nissl substances. More interesting is the slight ameliorative effects of Mg-glycinate shown in Figs.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eE and \u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eF. Synaptic integrity was evaluated in this study with Synaptophycin immuno-reactivity, an endogenous presynaptic vesicle glycoprotein and a viable marker for synaptic health. Strong punctate staining, indicating positive synaptophycin immuno-reactivity is well expressed in the normal control and the group sole administered Mg. Glycinate at 22.8mg/kg b.w and Mg. Glycinate at 32.8mg/kg (Figs.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eA, \u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eB, and \u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eC). However, synaptic injury was observed with CYP administration, as expressed with a pale and decreased immunoreactivity (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eD). Studies [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e, \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e] have continued to report oxidative stress and neuro-inflammation as mechanisms through which CYP impairs brain health. In agreement with these reports are the elevated levels of pro-inflammatory cytokines (TNF-α and IL-1β) evaluated in this study. Decreased synaptic integrity is obviously the factor for the decreased levels of spatial working memory that were observed with CYP in this study.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eTo summarize, the results presented in this study emphasize a potential therapeutic application of Mg. Glycinate as a supplement in the management of cancer, particularly in alleviating the adverse effects (weight loss, inflammation, decreased spatial working memory, and neurodegeneration) of chemotherapeutic agents such as Cyclophosphamide. While we hope to harness Mg. glycinate in this regard. There is, however, a need for further findings on its possible molecular interaction with conventional anti-cancer agents, particularly Cyclophosphamide.\u003c/p\u003e"},{"header":"Declarations","content":" \u003cp\u003e \u003cstrong\u003eEthics Approval and consent to participate\u003c/strong\u003e \u003cp\u003eThe experimental procedure was accomplished in accordance with the methods approved by the animal research ethical committee of the Faculty of Basic Medical Sciences, University of Ilesa, Ilesa, Osun State, Nigeria, with approval code (UNILESA/FBMS/2025/04).\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eConsent to Publication\u003c/strong\u003e \u003cp\u003eNot applicable\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eCompeting Interest\u003c/strong\u003e \u003cp\u003eThe authors declared no competing interest\u003c/p\u003e \u003c/p\u003e\u003ch2\u003eFunding\u003c/h2\u003e \u003cp\u003eThis study received funding from the Tertiary Education Trust Fund (TETFUND) under the Institutional-Based Research intervention for the year 2025.\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eConceptualization: FOO, KPF, LAH. Data curation: FOO, BEO, AAO, AO. Formal analysis: LMB, AO, OAA, BEO, and ATE. Funding acquisition: TETFUND. Methodology: FOO, LAH, and MTO. Resources: BEO, ATE. Writing original draft: FOO. Writing review \u0026amp; editing: KPF, BEO, AO, LMB, MTO, OAA, LAH. Approval of final manuscript: all authors.\u003c/p\u003e\u003ch2\u003eAcknowledgements\u003c/h2\u003e \u003cp\u003eWe appreciate our Vice Chancellor (Professor Taiwo Asaolu) for his constant encouragement and support in the research within our university (University of Ilesa).\u003c/p\u003e\u003ch2\u003eData Availability\u003c/h2\u003e\u003cp\u003eThe datasets generated during the study are available from the corresponding author on reasonable request.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eTimp W, Feinberg AP. Cancer as a dysregulated epigenome allowing cellular growth advantage at the expense of the host. 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Neurochem Int. 2024;172:105789. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/j.neuint.2024.105789\u003c/span\u003e\u003cspan address=\"10.1016/j.neuint.2024.105789\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"discover-neuroscience","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"ndev","sideBox":"Learn more about [Neural Development](http://neuraldevelopment.biomedcentral.com/)","snPcode":"13064","submissionUrl":"https://submission.nature.com/new-submission/13064/3","title":"Discover Neuroscience","twitterHandle":"@BioMedCentral","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"chemotherapy, Neurotoxicity, synaptophysin immunoreactivity, Magnesium-glycinate, Spatial-working Memory","lastPublishedDoi":"10.21203/rs.3.rs-9185242/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-9185242/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThe usage of cyclophosphamide (CYP) in cancer treatment is associated with organ toxicity, cellular degeneration, and myelosuppression, which result in treatment discontinuation. Its debilitating impact has increased the quest to search for novel substances or measures that will alleviate the adverse effects, while its therapeutic efficiency remains intact. This study evaluated the effects of Magnesium glycinate on cyclophosphamide-induced neurotoxicity in Wistar rats.\u003c/p\u003e \u003cp\u003eForty-eight Wistar rats (eight weeks old) were allocated into four experimental groups (n\u0026thinsp;=\u0026thinsp;8), as the normal control (A), oral Magnesium glycinate at 22.8 mg/kg (B) and 32.8mg/kg (C), 150mg/kg of CYP (D), CYP/oral magnesium glycinate at 22.8 mg/kg (E) and CYP/oral magnesium glycinate 32.8mg/kg (F). The animal\u0026rsquo;s body weight was measured weekly. Following the spatial working memory evaluation using the Y-maze. Animals were sacrificed by cervical dislocation, brain tissues excised, followed by the homogenization of the right hemisphere. The supernatant of the homogenates was used to assess the neurotransmitter activities and the levels of brain-derived neurotrophic factor, interleukin 1beta, interleukin 10, and Tumour Necrosis Factor alpha. Furthermore, the left hemisphere was processed for histological evaluation (H\u0026amp;E and Cresyl fast violet stain) and synaptophysin immunoreactivity.\u003c/p\u003e \u003cp\u003eCyclophosphamide induces weight loss, dysregulation of neurotransmitter and modulator activities, and inflammatory marker levels, as well as neurodegeneration and decreased hippocampal synaptophysin immunoreactivity. However, Magnesium glycinate, particularly at 32.8mg, reversed the stated adverse effects.\u003c/p\u003e \u003cp\u003eMg. Glycinate possesses potential therapeutic properties as a supplement in the management of cancer, particularly in alleviating the adverse effects of Cyclophosphamide.\u003c/p\u003e","manuscriptTitle":"Magnesium glycinate attenuates cyclophosphamide-induced neurotoxicity by modulating brain neurotransmitters, inflammatory markers, and hippocampal synaptophysin immunoreactivity","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-05-06 07:30:38","doi":"10.21203/rs.3.rs-9185242/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"reviewerAgreed","content":"177981602941757544803871444956870646987","date":"2026-05-04T21:31:23+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"24207057974998661594239872999535844791","date":"2026-05-03T20:39:48+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"157390589631829671756828851612272907553","date":"2026-04-26T09:04:43+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"31315569931286542275710472238966340734","date":"2026-04-24T14:27:02+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2026-04-24T12:24:15+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-03-31T07:53:16+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2026-03-31T07:52:56+00:00","index":"","fulltext":""},{"type":"submitted","content":"Discover Neuroscience","date":"2026-03-21T10:42:37+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"discover-neuroscience","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"ndev","sideBox":"Learn more about [Neural Development](http://neuraldevelopment.biomedcentral.com/)","snPcode":"13064","submissionUrl":"https://submission.nature.com/new-submission/13064/3","title":"Discover Neuroscience","twitterHandle":"@BioMedCentral","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"9f539495-d097-4864-b8f4-62bb5756b60c","owner":[],"postedDate":"May 6th, 2026","published":true,"recentEditorialEvents":[{"type":"reviewerAgreed","content":"177981602941757544803871444956870646987","date":"2026-05-04T21:31:23+00:00","index":49,"fulltext":""},{"type":"reviewerAgreed","content":"24207057974998661594239872999535844791","date":"2026-05-03T20:39:48+00:00","index":48,"fulltext":""}],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2026-05-06T07:30:38+00:00","versionOfRecord":[],"versionCreatedAt":"2026-05-06 07:30:38","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-9185242","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-9185242","identity":"rs-9185242","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}