Optimizing Fingolimod Dosing in STZ-Induced Cognitive Impairment: Divergent Behavioral and Histopathological Responses Reveal a Narrow Neuroprotective Window

Optimizing Fingolimod Dosing in STZ-Induced Cognitive Impairment: Divergent Behavioral and Histopathological Responses Reveal a Narrow Neuroprotective Window | 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 Optimizing Fingolimod Dosing in STZ-Induced Cognitive Impairment: Divergent Behavioral and Histopathological Responses Reveal a Narrow Neuroprotective Window Alireza Khani-Robati, Mohammad Banizaman, Farima Jahromi, Maryam Porsadeghfard, and 10 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8382715/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Background/Objective: Fingolimod, an S1P receptor modulator, is neuroprotective and anti-inflammatory in animal models of neurodegenerative disorders; its optimal dosage regimen for cognitive dysfunction is yet to be defined. The impact of fingolimod on cognitive function, neuroinflammation, oxidative levels, as well as hippocampal morphology, was evaluated in the streptozotocin-induced model of memory dysfunction in rats. Methods: Male Sprague-Dawley rats were injected intracerebroventricularly with STZ (3 mg/kg) followed by daily i.p. fingolimod administration at 0.25, 0.5, or 1 mg/kg for 10 days. Behavioral function was evaluated using open-field tests, Morris water maze, and passive avoidance tasks. The hippocampal tissue was examined for cytokine and neurotrophic factor gene expression using qRT-PCR. malondialdehyde (MDA) levels, as a measure of lipid peroxidation, and stereological parameters, including CA1 neuronal counts and volumes, were evaluated. Results: The STZ produced significant impairments in memory, increased pro-inflammatory cytokines (TNF-α, IL-1β, IL-6), elevated MDA levels, and CA1 neuronal loss and atrophy. Fingolimod at a dose of 0.25 mg/kg significantly ameliorated spatial and fear/memories, in addition to reducing neuroinflammation and oxidative stress. On the other hand, the dose of 0.5 mg/kg significantly restored both the number and volume of the CA1. The 1 mg/kg dose produced severe behavioral distress and was excluded. Conclusion: Fingolimod has dose-dependent, domain-specific neuroprotective actions, promoting functional recovery at low doses and structural repair at middle to higher doses. These findings highlight a narrow therapeutic window as a key issue in the translation of S1P modulator therapies from cognitive impairment to Alzheimer’s disease. Cognitive Neuroscience Fingolimod Alzheimer’s disease Streptozotocin Neuroinflammation Oxidative stress Memory Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Introduction Dementia is a term for various cognitive impairments that are associated with a decline in memory, reasoning, and the ability to perform routine activities. Among all forms of dementias, Alzheimer's (AD) is the most prevalent and accounts for approximately 60-80% of cases. It is a progressive neurodegenerative disorder characterized by amyloid-beta plaques and neurofibrillary tangles leading to synaptic dysfunction and, ultimately, neuron loss. Clinically, AD usually has an initially mild course of cognitive decline and progresses gradually to significant impairment in memory and executive functions, significantly affecting the lives of patients and their caregivers (Porsteinsson et al. 2021, Kumar et al. 2025). The pathophysiology of AD is complex and multifactorial, involving genetic, environmental, and lifestyle factors. The interplay of these elements contributes to neuroinflammation, oxidative stress, and metabolic dysregulation, all of which exacerbate cognitive decline (Salehi et al. 2016, Paroni et al. 2019, Breijyeh and Karaman 2020, Hoogmartens et al. 2021). Given the increasing prevalence of dementia in aging populations, there is an urgent need for effective therapeutic strategies that can halt or reverse the progression of memory impairment associated with AD and other forms of dementia. To determine potential therapeutic strategies, several scientific studies use animal models that emulate the cognitive dysfunction characteristic of human dementia. One such model widely used to study cognitive dysfunction is STZ-induced memory impairment. STZ is a potent neurotoxin that, following its administration into the lateral ventricles, preferentially damages cholinergic neurons of the basal forebrain (Li et al. 2016, Moosavi et al. 2020). This model recapitulates cognitive impairments observed in AD and shows how learning and memory are affected, as well as how neurochemical markers associated with synaptic plasticity change. It would be a valuable tool for assessing the potential efficacy of neuroprotective agents. Fingolimod (FTY720) is a sphingosine-1-phosphate receptor modulator that has attracted scientific interest for its potential neuroprotective properties. Owing to its therapeutic potential for managing multiple sclerosis, fingolimod has been shown to cross the blood-brain barrier and exert neuroprotective effects (Cruz and Fonseca 2014, Hunter et al. 2016). Fingolimod exerts its immunosuppressive effects by antagonizing S1P receptors expressed in immune cells, thereby decreasing lymphocyte infiltration in the periphery and exerting subtle effects on subsequent downregulation of neuroinflammation. Fingolimod also exerts additional effects, promoting survival in microglia and neuronal cells via S1P receptor activation of neurons and glial cells. Subsequent effects lead to reduced accumulation of Aβ peptides (Hla and Brinkmann 2011, Asle-Rousta et al. 2013, Groves et al. 2013, Hemmati et al. 2013, Takasugi et al. 2013, Chen et al. 2025). In addition to immune modulation, one possible function of fingolimod is its antioxidant properties, either by enhancing the body's antioxidant defenses or directly scavenging reactive oxygen species (ROS) (Martín-Montañez et al. 2019, Yevgi and Demir 2021). This role in managing CNS responses and associated effects of neuroprotection makes fingolimod a potential drug candidate for managing cognitive impairment associated with neurodegenerative disorders. Preclinical studies indicated that fingolimod could enhance synaptic plasticity and reduce Aβ accumulation by favoring neurotrophic support (Fukumoto et al. 2014, Nazari et al. 2016, Joshi et al. 2017, Rahmati-Dehkordi et al. 2024). Examples of studies indicating potential dose-dependent effects of fingolimod include studies using 5x FAD mice, in which lower doses (1 mg/kg) compared with 5 mg/kg showed enhanced effects. Additionally, lower microglial activation and reactive astrogliosis, with low levels of Aβ accumulation, and increased hippocampal neurogenesis would be observed (Aytan et al. 2016). A recent study using 5x FAD mouse models indicates possible Aβ accumulation in hippocampi, with subsequent attenuation associated with a decline in circulating lymphocytes. Notably, at the lowest administered dose of fingolimod (0.03 mg/kg/day), improvements in learning and memory were observed, along with attenuation of microglial and astrocytic activation and restoration of markers of GABAergic and cholinergic neuronal function (Carreras et al. 2019). These studies suggest that fingolimod, even at low doses that do not entirely disrupt peripheral lymphocyte function, may be therapeutic in both memory-impaired and AD models. However, the effects of varied fingolimod dosing on STZ-induced memory impairment are an area still requiring detailed attention. Despite the promising results with fingolimod, there remains a significant gap in understanding optimal dosing strategies and the mechanisms by which the drug exerts its neuroprotective effects. While previous studies have mainly used transgenic models of AD or other neurodegenerative diseases, the present study used a streptozotocin-induced model that mimics aspects of sporadic AD—specifically, neuroinflammation, oxidative stress, and metabolic abnormalities. Aiming to fill this gap, this study will evaluate the neuroprotective effects of different doses of fingolimod on STZ-induced cognitive impairment in male rats. Specifically, the study will examine cognitive function using behavioral tests and neurochemical changes in the brain. This study introduces several aspects, including the investigation of dose optimization, the study of multi-target effects on neuroinflammation, behavioral function, oxidative stress, and histomorphology, and the investigation of the potential for translational applications of fingolimod for AD and other related dementias. By elucidating the dose-dependent effects of fingolimod, this research seeks to provide insights into its potential as a therapeutic agent for memory impairment associated with AD disease and other neurodegenerative conditions. Materials and Methods Animals' experimental groups The experimental study utilized male Sprague-Dawley rats, weighing between 250-300 grams, obtained from the Experimental Animal Center of Shiraz University of Medical Sciences. Animals were acclimatized to the housing conditions for one week prior to the experiment. All animals were housed in standard laboratory conditions (temperature: 22 ± 2°C; humidity: 50 ± 5%; 12-hour light/dark cycle) with free access to food and water. The rats were randomly divided into five groups as follows: control group (n = 10, animals received a saline solution (0.9% NaCl) via intraperitoneal (i.p.) injection and intracerebroventricular (i.c.v.) microinjection), STZ group (n = 10, animals received i.c.v. STZ (3 mg/kg) and i.p. saline), STZ + Fingolimod 0.25mg group (n = 9, animals received i.c.v. STZ (3 mg/kg) and i.p. fingolimod (0.25 mg/kg/daily) for 10 days), STZ + Fingolimod 0.5mg group (n = 10, animals received i.c.v. STZ (3 mg/kg) and i.p. fingolimod (0. 5 mg/kg/daily) for 10 days), and STZ + Fingolimod 1mg group (n = 9, animals received i.c.v. STZ (3 mg/kg) and i.p. fingolimod (1 mg/kg/daily) for 10 days). The number of animals allocated to each group was determined based on previous studies utilizing the STZ model, as well as ethical and practical considerations. Ethics statement All experimental protocols were reviewed and approved by the Institutional Ethics Committee of Shiraz University of Medical Sciences (IR.SUMS.AEC.1401.104) in compliance with the National Institutes of Health guidelines.All experimental procedures adhered to the National Research Council's Guide for the Care and Use of Laboratory Animals, 8th edition (National Academies Press (US); 2011. ISBN-13: 978-0-309-15400-0). Stereotaxic surgery, STZ injection, and fingolimod treatment Stereotaxic surgery was performed to facilitate the administration of STZ directly into the cerebral lateral ventricles. The surgical procedures were conducted under aseptic conditions with animals anesthetized using an intraperitoneal injection of a mixture of Ketamine (100 mg/kg) and xylazine (10 mg/kg). A stereotaxic apparatus was used to secure the rat’s head in a fixed position. Stereotaxic coordinates for the lateral ventricle were determined based on the Paxinos brain atlas, with the following coordinates used: -0.8 mm anteroposterior (AP), ±1.5 mm mediolateral (ML), -3.5 mm dorsoventral (DV) relative to the bregma. A midline incision was made in the scalp, and a small burr hole was drilled at the designated coordinates. A Hamilton syringe was used for STZ microinjections and carefully inserted into the lateral ventricle. STZ (STZ-S0130-100MG, Sigma-Aldrich, USA) at a concentration of 3 mg/kg was freshly dissolved in sterile saline and injected at a rate of 1 µL/min, with a total volume of 5 µL administered per rat. After the injection, the needle was left in place for an additional 5 minutes to minimize reflux, then slowly withdrawn. The skin incision was sutured at the end of the procedure. Fingolimod, a kind gift from Abidi Pharmaceutical Company (Iran), was dissolved in saline. One day following the STZ injection, treatment with fingolimod commenced at a dose of 0.25, 0.5, and 1 mg/kg was administered via i.p. injection for 10 consecutive days. Animals in the control and STZ groups received equal volumes of saline during the same treatment period to control for any potential effects of the treatment procedure. Behavioral studies Open field test On day 12 after surgery, an open field test was performed. The Open field test is a widely utilized experimental paradigm to study anxiety and general locomotor activity in rodent models. The test was conducted within a square plexiglass enclosure measuring 45 cm in height and 90 cm in both width and length, illuminated by four 60 W bulbs positioned 1.8 m above the box. The floor of the box was typically divided into specific zones (center and periphery) to evaluate the animal's behavior in relation to perceived safety and risk. The rats were placed in the arena for 20 minutes while their movement was monitored using EthoVision (Noldus Information Technology, Netherlands). The differentiation between the center and periphery serves as an indicator of anxiety levels. Key parameters measured include the time spent in the center versus the periphery (in seconds) and the frequency of grooming behaviors exhibited. Morris water maze test The Morris Water Maze (MWM) test, a recognized behavioral assessment, was employed to evaluate spatial learning and memory in rats on the 13th and 14th days post-surgery. The arena consisted of a circular swimming pool with a diameter of approximately 150 cm, filled with opaque water and typically maintained at a temperature of 21-24°C. The arena was divided into four quadrants, with a submerged platform located in a fixed position in one of the quadrants (designated as the target quadrant) just below the water's surface, serving as the escape location for the animals. During the training phase, each rat underwent several trials (12 trials divided into three blocks) to facilitate spatial memory acquisition. The animal was placed in the water at various starting points and allowed to swim for up to 60 seconds until it located the hidden platform. The latency to find the platform during training trials served as a criterion for assessing spatial learning. Twenty-four hours after the training period, a probe trial was conducted to evaluate spatial memory retention. In this trial, the hidden platform was removed, and the animal was allowed to swim freely for 60 seconds. Key parameters measured during the probe trial included the time spent in the target quadrant, where the platform had previously been located. Passive avoidance test The passive avoidance test evaluates an animal's ability to avoid a previously encountered environment that has been associated with an unpleasant stimulus. The test was conducted using a shuttle box, which consists of a light compartment and a dark compartment separated by a sliding door. The dark compartment is associated with an electric foot shock (0.5 mA, 50 Hz, for 2 seconds), creating a negative association for the animals. During the training phase (on day 15), each rat was initially placed in the light compartment. After a 20-second acclimation period, the sliding door was opened to allow access to the dark compartment. When the animal entered the dark area, it received a foot shock. This procedure was repeated after five minutes, provided the animal continued to avoid entering the light chamber from the dark one. The retention test was conducted 24 hours after the training phase (on day 16). In this session, the rat was placed in the light compartment for up to 300 seconds, and the delay before entering the dark chamber, referred to as step-through latency (STL), was recorded without any electric shock. A longer latency indicates a stronger memory of the aversive experience and a greater ability to avoid the dark compartment. qRT-PCR analysis of hippocampal tissue Quantitative reverse transcription polymerase chain reaction (qRT-PCR) was employed to assess the mRNA expression levels of neurotrophic factors and pro-inflammatory cytokines, including brain-derived neurotrophic factor (BDNF), glial cell line-derived neurotrophic factor (GDNF), nerve growth factor (NGF), tumor necrosis factor-alpha (TNFα), interleukin-1 beta (IL-1β), and interleukin-6 (IL-6) in hippocampal tissue. Total RNA was isolated using the TRIzol reagent (Yekta Tajhiz Azma, Iran) according to the manufacturer’s instructions. The concentration and purity of RNA were measured spectrophotometrically at 260 nm and 280 nm (NanoDrop spectrophotometer, Thermo Fisher Scientific, USA). cDNA was synthesized from 1 µg of RNA using a cDNA Reverse Transcription Kit (Yekta Tajhiz Azma, Iran). Specific primers were designed for target genes. The qRT-PCR was performed using RealQ Plus 2x Master Mix Green (Ampliqon, Denmark) on a StepOnePlus Real-Time PCR system (Thermo Fisher Scientific) for amplification, with the following thermal cycling conditions: an initial denaturation at 95°C for 5 minutes, followed by 40 cycles of 95°C for 20 seconds, and 60°C for 60 seconds. Hypoxanthine-guanine phosphoribosyltransferase (HPRT) was used as an internal control. Relative gene expression levels were calculated using the 2 ^−ΔΔCt method to compare the treatment groups with controls. Malondialdehyde (MDA) assay The level of malondialdehyde (MDA) in hippocampal tissue was quantified as a marker of oxidative stress using the thiobarbituric acid reactive substances (TBARS) assay (Naseh et al. 2020, Jahromi et al. 2024). In brief, hippocampal samples were homogenized and centrifuged at 12,000 rpm for 15 minutes at 4°C. The supernatant was mixed with a solution consisting of 0.25 N hydrochloric acid (HCl), 20% trichloroacetic acid (TCA), and 0.8% thiobarbituric acid (TBA), and incubated at 87°C for 60 minutes. After that, the absorbance was measured at 532 nm using a microplate reader. The MDA concentration was expressed in nanomoles per milligram of tissue (nmol/mg). Stereological and histomorphological methods The rats were transcardially perfused with normal saline, followed by 4% paraformaldehyde. Then, the brains were removed and kept in the same fixative. The brains were then transferred to 30% sucrose solution for 3 days. Subsequently, the brains were stored at -80°C for further processing. They were then serially and coronally sectioned using a cryostat (Microm HM 525, Germany) at a thickness of 40 μm. The sections were stained with cresyl violet. The volume of the hippocampal CA1 region was estimated using the Cavalieri method, while the number of CA1 pyramidal cells was determined using the optical dissector, as described in more detail previously (Asadi nejad et al. 2024). Statistical analysis Statistical analyses were performed using GraphPad Prism software (version 9). Data were expressed as mean ± standard error of the mean (SEM). Differences between groups were compared using two-way analysis of variance (ANOVA) and one-way ANOVA followed by Tukey's post hoc test for multiple comparisons. A p-value of less than 0.05 was considered statistically significant. Results In the present study, a high-dose fingolimod group (1 mg/kg) was initially included; however, the animals in the 1 mg group exhibited significant behavioral changes, including withdrawal, social isolation, reduced mobility, and poor cooperation during behavioral tests. Specifically, in the Morris water maze test, these rats were unable to swim properly and frequently submerged or refused to participate, indicating a marked deterioration in their functional performance. Such non-cooperative behavior precluded reliable task performance and prevented the collection of sufficient quantitative data for robust statistical analysis. Consequently, the 1 mg/kg group were excluded from the study to focus on doses yielding interpretable and biologically meaningful outcomes. The effect of STZ and fingolimod on rats' anxiety-like behavior in the open field test In this test, an increase in the amount of time spent in the center zone relative to the periphery zone indicates decreased anxiety in rats. One-way ANOVA analysis revealed significant differences between groups (F (3, 35) = 5.090, P = 0.0050). Our findings showed that the time spent in the center significantly decreased in the STZ group compared to the control group (8.019±1.877 vs. 18.34±3.385; P <0.05). However, no significant difference was observed between the fingolimod 0.25 mg-treated group and STZ group (9.946±2.342 vs. 8.019±1.877, P=0.9419), as well as between the fingolimod 0.5 mg- and STZ-treated groups (6.440±1.446 vs. 8.019±1.877, P=0.9640), indicating that fingolimod treatment failed to reduce the anxiety-like behaviors induced by STZ (Figure 1A). Another indicator of anxiety-like behavior in animals is the number of grooming sessions in rats. Anxious animals display grooming behavior more frequently than non-anxious animals do. A significant increase in grooming frequency was observed in the STZ-treated group and the fingolimod 0.5 mg-treated group compared to the control group (7.600 ± 1.087 and 7.500 ± 0.6540 vs. 4.100 ± 0.7371; P < 0.05, respectively). Although fingolimod in a dose of 0.25 mg was able to reduce grooming sessions compared to the STZ group, it was not statistically significant (P=0.1996) (Figure 1B). Fingolimod (0.25 mg) prevented STZ-mediated memory spatial impairment in MWM As shown in Figure 2A, learning patterns of animals in all groups demonstrated a negative linear correlation between escape latency and training blocks; however, the performance of animals receiving STZ was weaker than that of the other groups. Two-way ANOVA analysis revealed significant differences between groups (F (3, 105) = 21.10, P<0.0001). The Post hoc analysis by Tukey's test revealed that escape latency in the STZ receiving group was significantly increased compared to the control group in the first (52.14±11.14s vs. 33.85±14.80s; P<0.01), second (44.57±11.64s vs. 21.99±12.13s; P<0.001), and third (44.09±13.25s vs. 17.42±14.78s; P<0.001) blocks of training, indicating STZ-induced deterioration in the STZ group. Although STZ+Fingo 0.5mg receiving group did not show a significant difference with STZ treated group in three blocks, fingolimod treatment in dose of 0.25 mg could reverse this deficit in the all three blocks (36.60±13.79s vs. 52.14±11.14s, P<0.05; 26.44±12.85s vs. 44.57±11.64s, P<0.01; 23.67±8.99s vs. 44.09±13.25s, P<0.01, respectively), indicating a protective role for fingolimod 0.25 mg against STZ toxicity. The evaluation of memory retention was done in the prob trial (Figure 2B). One-way ANOVA analysis showed that treatment with fingolimod in the STZ receiving group could significantly increase the time spent in the target area (F (3, 35) = 6.293, P = 0.0016). Post hoc analysis by Tukey's test displayed that the time spent in the target area was decreased significantly in the STZ-treated group compared to the control group (11.61±1.534 vs. 21.88±2.647, P<0.01). However, only fingolimod in a dose of 0.25 mg (19.67±1.836, P<0.05) could significantly prevent STZ-induced disturbance. In order to assess the possible effect of drugs on the motor performance of swimming speed of animals, their swimming speed was assessed (Figure 2C). The results showed that treatment with STZ and/or fingolimod did not affect the swimming speed of animals (F (3, 35) = 1.730, P = 0.1788). Fingolimod prevented STZ-mediated fear memory impairment in the passive avoidance test Results from the passive avoidance test indicated significant differences between groups (F (3, 35) = 4.989, P = 0.0055) (Figure 3). The animals in the STZ group had significantly shorter STL compared to the control group (109.4 ± 33.85 vs. 255.7 ± 27.44, P<0.01), reflecting memory deficits induced by STZ. Fingolimod treatment markedly increased the latency time in both treated groups (234.1 ± 30.26 and 242.0 ± 30.71, respectively), suggesting improvements in memory retention. The effect of STZ and fingolimod on rats' hippocampal mRNA expression levels of TNFα, IL-1β, IL-6, BDNF, and GDNF The results of One-Way ANOVA analysis (n=3 per group) showed significant differences between groups relevant to expression levels of TNFα, IL-1β, and IL-6 (F (3, 8) = 34.45, P<0.0001; F (3, 8) = 172.1, P<0.0001; and F (3, 8) = 29.89, P = 0.0001, respectively). Post hoc analysis by Tukey's test showed that the STZ group, compared to the control, exhibited significantly higher expression of TNFα (2.082±0.1011 vs. 1.000±0.03975), IL-1β (4.157±0.2019 vs. 1.000±0.04009), and IL-6 (1.977±0.06436 vs. 1.000±0.06511) (P<0.001). Fingolimod treatment in both doses of 0.25 mg (P<0.001) and 0.5 mg (P<0.01) was able to reverse the STZ-induced increase in the expression of TNFα. In addition, our results showed that fingolimod could reverse the IL-1β increment induced by STZ in both doses of 0.25 and 0.5 mg (P<0.001). This decreasing effect on IL-6 expression was also observed with both fingolimod doses (P<0.01) (Figure 4A-C). Moreover, quantitative analysis revealed a significant increment in mRNA expression levels of BDNF and GDNF in the STZ group compared to control (2.049±0.1562 vs. 1.000±0.1598, P<0.01, and 1.858±0.05846 vs. 1.000±0.04026, P<0.05, respectively). There was no significant difference between the expression level of BDNF in fingolimod-treated groups compared to the STZ group (Figure 4D and E). However, fingolimod in a dose of 0.25 mg, but not 0.5 mg, significantly reversed STZ-induced increase in expression of GDNF (P<0.05). The effect of STZ and fingolimod on oxidative stress Malondialdehyde levels were measured in all experimental groups to assess lipid peroxidation as an indicator of oxidative stress. The animals in the STZ group exhibited a significant increase in MDA levels compared to the control group (4.358±0.3470 vs. 2.264±0.3472, P<0.001, n=5 in control and STZ groups), indicating enhanced lipid peroxidation and oxidative stress. Treatment with fingolimod in both doses (n=4) of 0.25 mg (2.913±0.1622, P<0.05) and 0.5 mg (2.468±0.2252, P<0.01) significantly reduced MDA levels compared to the STZ group, suggesting a protective/antioxidant effect (Figure 5). Number of CA1 pyramidal cells and total volume of CA1 area In order to evaluate the effects of fingolimod treatment on CA1 pyramidal cells in the hippocampus, we conducted a quantitative analysis of cell counts and volume measurements (n=5 per group) following Cresyl violet staining (Figure 6A-D). The analysis revealed that the number of CA1 pyramidal cells was significantly reduced in the STZ group compared to the control group (P<0.001). Specifically, the average number of pyramidal cells in the STZ group compared to the control group was 362.2±22.50 vs. 512.2±11.99, indicating profound neuronal loss due to the induced neurotoxicity. In contrast, treatment with fingolimod at a dose of 0.5 mg significantly increased the number of CA1 pyramidal cells (470.7±18.89) compared to the STZ group (P< 0.05) (Figure 6E). This restorative effect underscores fingolimod's potential neuroprotective action. However, fingolimod at a dose of 0.25 led to an increase in the number of CA1 pyramidal cells compared to the STZ group; this increase was not statistically significant. Moreover, volumetric analysis demonstrated that the volume of the hippocampal CA1 area was significantly decreased in the STZ group compared to control animals (7.142±0.2710 vs. 9.262±0.2406, P<0.01), reflecting the neurodegenerative changes induced by STZ treatment. Fingolimod treatment at a dose of 0.5mg, but not 0.25mg, resulted in a significant increase in the average volume of the CA1 region (8.888±0.2890) relative to the STZ group (P<0.05) (Figure 6F), suggesting a reversal of neuronal atrophy associated with neuroinflammation and oxidative stress. Discussion The present study investigated the dose-dependent neuroprotective and anti-inflammatory effects of fingolimod in a rat model of STZ-induced memory impairment. Our findings demonstrate that fingolimod, particularly at the lower dose of 0.25 mg/kg, significantly ameliorated cognitive deficits, reduced neuroinflammation, and attenuated oxidative stress. These results align with previous research highlighting the therapeutic potential of fingolimod in neurodegenerative disorders, including AD and other forms of dementia (Aytan et al. 2016, Angelopoulou and Piperi 2019, Fagan et al. 2022, Leßmann et al. 2023). Contrary to some previous studies that reported a 1 mg dose of fingolimod to produce neuroprotective effects and improve memory performance in behavioral tests (Asle-Rousta et al. 2013, Kalecký et al. 2025), the animals receiving a dose of 1 mg fingolimod, in our study, demonstrated notable behavioral alterations, such as withdrawal, social withdrawal, decreased activity, and lack of cooperation during behavioral assessments. Owing to their non-cooperation and inadequate participation, it was not possible to collect sufficient data or perform reliable analysis for the 1 mg dose, leading to the exclusion of this group's data from the final results. One plausible explanation for these behaviors is the occurrence of severe bodily discomfort or pain caused by the high dose of fingolimod. High doses of fingolimod may induce adverse side effects such as pain, physical distress, and general discomfort, which can lead to increased anxiety, reduced activity levels, and social withdrawal (Russo et al. 2015). These effects can substantially impair the animals' ability to perform in behavioral assays, thereby compromising the reliability and completeness of the data. This highlights an important limitation in pharmacological studies, that the occurrence of severe side effects at higher doses may hinder proper behavioral assessment and impact the interpretability of results. It underscores the necessity of careful dose optimization to balance therapeutic efficacy with tolerability. These findings emphasize that animal well-being, physical comfort, and cooperation are critical factors in obtaining valid and reliable experimental outcomes. Future studies should focus on establishing doses that minimize adverse effects to ensure both safety and data integrity. Moreover, assessing potential side effects and tolerability is essential for translating such therapies into clinical applications, where patient comfort and safety remain paramount. Neuroprotective effects of fingolimod on behavioral function Our behavioral findings align with prior studies that have demonstrated the detrimental effects of STZ on memory and learning (Chen et al. 2014, Moreira-Silva et al. 2018, Qi et al. 2021). The MWM and passive avoidance tests confirmed that STZ significantly impairs both spatial and fear-related memory. However, fingolimod at 0.25 mg/kg significantly improved performance in both tests, indicating preserved hippocampal function. These findings corroborate earlier reports of fingolimod’s cognitive-enhancing effects, which are largely attributed to its anti-inflammatory, antioxidant, and neurotrophic mechanisms (Efstathopoulos et al. 2015, Fagan et al. 2022, Kalecký et al. 2025). Nazari et al. (Nazari et al. 2016) observed similar improvements in synaptic plasticity and memory retention in ischemic rat models treated with fingolimod Interestingly, fingolimod at the higher dose of 0.5 mg/kg led to improvements in passive avoidance memory but failed to yield significant improvements in MWM behavioral assessments, suggesting a potential biphasic dose-response relationship, as noted in other studies, that warrants further exploration. For example, Carreras et al., 2019 (Carreras et al. 2019) reported a biphasic dose-response in AD mice; low-dose fingolimod improved cognition and reduced neuroinflammation, while higher doses failed to enhance function and produced tolerability issues. Our findings align with this biphasic pharmacodynamic pattern, suggesting that optimal neuroprotective efficacy occurs within a narrow dose range that avoids systemic immune suppression. This phenomenon is commonly observed with neuromodulatory agents and may reflect receptor desensitization or complex pharmacodynamics at higher doses. The anxiogenic effects of STZ, as evidenced by reduced center zone activity and increased grooming behavior in the open field test, were not alleviated by fingolimod treatment. This suggests that fingolimod’s neuroprotective actions may be more potent in cognitive domains than in affective behaviors, or that anxiety-related circuitry is less responsive to its modulatory effects in this context. In this regard, some studies reported that fingolimod does not affect anxiety-like behavior in the elevated plus maze test (di Nuzzo et al. 2015). While some studies suggest fingolimod can influence anxiety-related behaviors (e.g., in obese mice), the degree of improvement might be less significant compared to its impact on cognitive functions (Wencel et al. 2023). This could be due to differences in how fingolimod interacts with specific neuronal circuits involved in each domain. Neurotrophic modulating and anti-inflammatory mechanisms Molecular analyses revealed that STZ significantly elevated hippocampal mRNA expression of TNF-α, IL-1β, and IL-6—cytokines known to mediate neuroinflammatory cascades implicated in AD pathogenesis (Liu et al. 2016, Fan et al. 2022). Fingolimod treatment effectively downregulated these cytokines at both tested doses, indicating strong anti-inflammatory properties. These results are consistent with findings that demonstrated fingolimod's ability to modulate microglial activation, suppress pro-inflammatory cytokine production (Jackson et al. 2011, Rothhammer et al. 2017, Shang et al. 2020, Yang et al. 2025). Fingolimod’s neuroprotective actions involve a multi-pathway network combining S1P1 receptor signaling, NF-κB inhibition, and Nrf2 activation (Chen et al. 2025). Specifically, fingolimod acts on both circulating and CNS-resident myeloid cells, including microglia, to reduce the release of pro-inflammatory cytokines like TNFα, IL-1β, and IL-6. This effect is achieved through functional antagonism of S1P receptors, particularly S1P1, which are involved in regulating immune cell trafficking and inflammatory responses within the CNS (Brunkhorst et al. 2014, Quirant‐Sánchez et al. 2018, Lobaina and Shanina 2025). Additionally, the observed increase in neurotrophic factors such as BDNF and GDNF in STZ-treated rats indicates a compensatory response to neurodegeneration and an innate attempt at repair. The findings of this study suggest that fingolimod exerts a dual role in neurodegenerative contexts, mitigating dysregulated neurotrophic signaling (e.g., excessive GDNF) and enhancing BDNF-mediated neuroplasticity. The observed BDNF upregulation aligns with its established role as a compensatory mechanism in early neurodegeneration, where transient increases attempt to counteract neuronal damage and preserve function. However, our results also highlight the delicate balance required in neurotrophic support, as excessive or sustained elevations of these factors may exacerbate neuroinflammatory cascades or fail to sustain long-term repair. In many neurodegenerative diseases, including AD, a consistent reduction in BDNF levels has been observed, correlating with synaptic dysfunction and cognitive decline; While increasing its expression leads to improved memory. (Bayat et al. 2021, Gao et al. 2022). Interestingly, however, during the initial phases of neuronal injury or in mild cognitive impairment (MCI), BDNF levels can transiently increase, potentially reflecting an innate protective response attempting to counteract neuronal loss. For instance, elevated serum BDNF levels in MCI patients suggest an early compensatory mechanism, whereas a decline in advanced AD indicates failure of these endogenous supports or progression beyond the brain’s capacity for compensation (Faria et al. 2014, Kim et al. 2017). Moreover, the regulation of BDNF is highly complex; differences in pro-BDNF processing mediated by glial cells and plasmin activity may contribute to the conflicting results regarding BDNF levels in peripheral blood versus central nervous system tissues (Yang et al. 2009). Our findings support the hypothesis that pharmacological interventions like fingolimod may enhance or restore neurotrophic support during the early or preclinical stages of neurodegeneration by increasing BDNF levels and correcting dysregulated GDNF expression. Such effects could be beneficial in maintaining synaptic plasticity and neuronal survival, ultimately delaying or mitigating disease progression. Nevertheless, it is critical to recognize that excessive or unbalanced neurotrophic signaling might be detrimental, emphasizing the importance of finely tuned regulation of these factors in therapeutic strategies. Further research is warranted to elucidate the precise mechanisms underlying neurotrophic modulation by fingolimod and to determine its potential impact across different stages of neurodegenerative diseases. Oxidative stress and lipid peroxidation STZ induced a significant increase in lipid peroxidation as shown by elevated MDA levels, reflecting heightened oxidative stress. Oxidative stress plays a pivotal role in STZ-induced neurodegeneration by promoting cellular damage, mitochondrial dysfunction, and apoptosis (Ansari et al. 2024, AEF Cardinali et al. 2025). Fingolimod treatment in both doses significantly reduced MDA levels, indicating potent antioxidant effects that may contribute to its neuroprotective efficacy. The observed reduction in oxidative markers aligns with previous findings demonstrating fingolimod’s role in enhancing antioxidant enzyme expression, such as superoxide dismutase and catalase, and reducing ROS production (Wu et al. 2017, Yevgi and Demir 2021, Bagheri et al. 2024). Crivelli et al. (2022) reported that fingolimod decreases ceramide levels and oxidative stress in familial AD models (Crivelli et al. 2022), further supporting its neuroprotective properties through modulation of lipid metabolism and oxidative pathways. These findings suggest that fingolimod exerts a significant neuroprotective effect by attenuating oxidative stress and lipid peroxidation. Its ability to modulate oxidative markers and lipid signaling pathways underscores its potential as a therapeutic agent in neurodegenerative diseases characterized by oxidative damage, such as AD and diabetic neuropathy. Structural and cellular restoration Histomorphological analyses further support the neuroprotective profile of fingolimod. Stereological analysis revealed significant CA1 pyramidal neuron loss and hippocampal atrophy in STZ-treated rats, consistent with previous studies demonstrating the vulnerability of hippocampal neural cells to STZ, particularly in CA1 and CA3 regions (Lee et al. 2015, Zappa Villar et al. 2018, Roy et al. 2022). Although neuronal loss and hippocampal atrophy were partially attenuated by the 0.25 mg/kg dose of fingolimod, a more pronounced amelioration was observed at 0.5 mg/kg, highlighting its neuroregenerative potential. This finding is supported by Kalecký et al. (2025), who demonstrated fingolimod's ability to rescue synaptic plasticity and neuronal integrity in APP/PS1 mice (Kalecký et al. 2025). The restoration of CA1 volume and pyramidal cell numbers suggests that fingolimod may prevent neurodegeneration through a combination of anti-apoptotic, anti-inflammatory, and trophic mechanisms. In this regard, data from clinical trials and real-world studies demonstrate that fingolimod significantly reduces the rate of brain volume loss compared to other treatments or placebo in MS patients (De Stefano et al. 2017). It highlights the neuroprotective effects of fingolimod, likely through its anti-inflammatory properties and ability to modulate immune responses, thereby helping to preserve neural tissue and slow neurodegeneration. While both doses of fingolimod demonstrated favorable effects on neuroinflammation and oxidative stress, their differential impact on behavioral and histological outcomes raises important questions about the optimal therapeutic window and target engagement. It is possible that the 0.25 mg/kg dose more effectively modulates synaptic plasticity and functional connectivity, whereas the 0.5 mg/kg dose confers structural preservation without equivalent functional gains. It is well understood in the arena of neuroprotection that a distinction may arise between histopathological and behavioral outcomes; whereby cellular rescue may precede or even occur independently of functional recovery due to the additional requirement of circuit-level reorganization and synaptic plasticity beyond neuronal survival (Aytan et al. 2016). Mechanistically, the observed dissociation between structural rescue and behavioral improvement might reflect a biphasic pharmacodynamic profile of the S1P pathway targeted by fingolimod. Fingolimod-phosphate acts as a high-affinity agonist at S1P₁ receptors, causing rapid receptor internalization and sustained functional antagonism. However, further exposure may also engage or over-activate other S1PR subtypes (e.g., S1P₃, S1P₅), thereby redirecting signaling to alternative pathways. Thus, this internalization, receptor desensitization, and subtype-specific signaling could impair synaptic function and long-term potentiation, providing a mechanistic explanation for dose-dependent non-linear effects on cognition and synaptic plasticity. Such a biphasic effect is consistent with the literature on S1P receptor pharmacology and the action of fingolimod through different receptor subtypes (Brinkmann 2009, Mullershausen et al. 2009, Groves et al. 2013). Consequently, the dose that most effectively normalizes neuroinflammatory histopathology may not optimally support hippocampal network plasticity underlying memory consolidation. Furthermore, sensitivity to behavioral tests could also play a role. The MWM is sensitive to anxiety, stress response, and locomotor activity, and any small change related to stress/motivation at 0.5 mg/kg may have impaired performance even if actual improvement was achieved in the hippocampus (Othman et al. 2022). Moreover, morphological recovery may not necessarily imply network restoration, as spatial learning depends upon network interactions between the dentate gyrus, CA3, CA1, and cortex (Daugherty et al. 2017, Barry et al. 2021). It may, therefore, be insufficient if structural restoration is achieved selectively in the CA1 region or if synaptic or subfield restoration is not achieved concurrently, including recovery of behavioral functions, within a 10-day window after treatment. The results, therefore, are consistent with a model whereby structural recovery precedes functional improvement, as a consequence of divergent dose signaling via S1P, explaining why 0.5 mg/kg produced histological but not behavioral recovery. Limitations and future directions Several limitations of this study should be considered when interpreting the findings. First, although the STZ model reproduces important features of sporadic AD-like pathology, it does not fully recapitulate the progressive and multifactorial nature of human AD; therefore, the translational value of the results should be validated in additional models, including transgenic and aged animals. Second, the high-dose (1 mg/kg) fingolimod group was excluded due to marked behavioral intolerance, thereby preventing the assessment of the entire dose-response curve. Future studies should include refined monitoring of physiological and stress-related parameters to describe dose-related side effects better. Third, behavioral and histological outcomes were measured after a relatively short treatment period (10-16 days), which may not capture long-term neuroprotection or neurorestoration. Long-term follow-up in longitudinal studies will be required to determine the durability of functional and structural recovery. While fingolimod altered cytokine and markers of oxidative stress in this study, further mechanistic studies would be required to interpret the divergent behavioral and histological responses seen across doses. Moreover, only male rats were studied; future studies should investigate possible sex-dependent differences, given the established influence of sex hormones on S1P signaling and neurodegeneration. Conclusion Evidence has been obtained in this study that fingolimod exhibits dose-dependent neuroprotective actions, with a clear distinction between the behavioral results and the restoration of histopathological changes at varying doses. The lower dose of fingolimod (0.25 mg/kg) improved cognitive function, along with reduced neuroinflammation and oxidative damage. Whereas the higher dose of fingolimod (0.5 mg/kg) primarily promoted structural maintenance rather than restoring function. From a clinical perspective, these findings suggest that low-dose fingolimod may offer a more favorable therapeutic window in the early stages of cognitive impairment, as it may confer neurobiological advantages without the tolerability issues and desensitization associated with higher doses in trials for neurodegenerative diseases. The results underscore the importance of precise dose optimization in translating S1P-modulating therapies for the neurodegenerative diseases. Declarations Acknowledgments The authors would like to thank Abidi Pharmaceutical Company for preparing fingolimod, and the Office of Vice Chancellor for Research at Shiraz University of Medical Sciences for financially supporting (Grant No: 26854). The study protocol was approved by the institutional review board (IRB) and the ethics committee of Shiraz University of Medical Sciences (Approval No: IR.SUMS.AEC.1401.104). Declarations of interest: None. Declaration of Generative AI and AI-assisted technologies in the writing process During the preparation of this work, the authors used ChatGPT (GPT-3.5), an AI language model developed by OpenAI, accessed via the free web version, in order to improve the readability and language of the manuscript. After using this tool/service, the authors reviewed and edited the content as needed and take full responsibility for the content of the publication. Funding: This study is financially supported by the Office of the Vice Chancellor for Research at Shiraz University of Medical Sciences (Grant No: 26854) . Data availability statement: The data that support the findings of this study are available from the corresponding author upon reasonable request. 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J Neural Transm (Vienna) 125(12):1787–1803. https://doi.org/10.1007/s00702-018-1928-7 Additional Declarations The authors declare no competing interests. Supplementary Files floatimage1.jpeg Graphical Abstract.Therapeutic window of fingolimod dosing in STZ-induced cognitive impairment Cite Share Download PDF Status: Posted Version 1 posted 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. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. 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-8382715","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":561602000,"identity":"827d481a-e583-44d0-8de7-6c8d0f65ab4f","order_by":0,"name":"Alireza Khani-Robati","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Alireza","middleName":"","lastName":"Khani-Robati","suffix":""},{"id":561602001,"identity":"abba5a63-042a-469c-9685-702d85c63d5f","order_by":1,"name":"Mohammad 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1","display":"","copyAsset":false,"role":"figure","size":51032,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEffects of STZ and fingolimod on open field task. \u003c/strong\u003e(A) Time spent in the central zone (min). STZ significantly decreased the time in the center compared to control, while fingolimod at 0.25 and 0.5 mg/kg did not significantly restore this parameter. (B) Grooming number, an indicator of anxiety; STZ increased grooming behavior, which was partially attenuated by fingolimod at 0.25 mg/kg. Data are expressed as mean ± SEM; *P\u0026lt;0.05 and **P\u0026lt;0.01 represent the difference between control and other treated groups.\u003c/p\u003e","description":"","filename":"floatimage2.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-8382715/v1/3371d1dd85a567bb5999dd9a.jpeg"},{"id":98497200,"identity":"4b3220b4-3968-4e96-8264-72ad260ca617","added_by":"auto","created_at":"2025-12-18 09:14:03","extension":"jpeg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":99962,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEffects of STZ and fingolimod on spatial memory in the MWM. \u003c/strong\u003e(A) Latency to reach the hidden platform across training blocks. STZ increased escape latency compared to control, indicating impaired spatial learning. Fingolimod at 0.25 mg/kg significantly reduced escape latency relative to STZ. (B) Percentage of time spent in the target quadrant during the probe trial, reflecting memory retention. STZ decreased this measure, while 0.25 mg/kg fingolimod significantly improved performance. (C) Swimming speed across groups; no significant differences observed, suggesting motor ability was unaffected. Data are expressed as mean ± SEM; **P\u0026lt;0.01 and ***P\u0026lt;0.001 represent the difference between control and other treated groups. #P\u0026lt; 0.05, ##P \u0026lt; 0.01, and ###P \u0026lt; 0.001 represent the difference between STZ and other treated groups.\u003c/p\u003e","description":"","filename":"floatimage3.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-8382715/v1/33a4e9a6daedcec74745cb4a.jpeg"},{"id":98497194,"identity":"e9eaad34-5658-4ad4-ab76-d91614a72400","added_by":"auto","created_at":"2025-12-18 09:14:03","extension":"jpeg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":25452,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEffects of STZ and fingolimod on passive avoidance test. \u003c/strong\u003eStep-through latency (STL, sec) was significantly reduced in the STZ group, indicating a memory deficit. Fingolimod at 0.25 and 0.5 mg/kg significantly increased STL, demonstrating improved memory retention. Data are presented as mean ± SEM; **P\u0026lt;0.01 represents the difference between control and STZ groups. #P\u0026lt; 0.05 represents the difference between STZ and other treated groups.\u003c/p\u003e","description":"","filename":"floatimage4.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-8382715/v1/c96285f8e6dc72c1d402266b.jpeg"},{"id":98623861,"identity":"8aea5497-c873-4eba-b701-465b2bb12f1f","added_by":"auto","created_at":"2025-12-19 17:07:42","extension":"jpeg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":124069,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEffects of STZ and fingolimod on hippocampal mRNA expression levels. \u003c/strong\u003eRelative mRNA levels of (A) TNF-α, (B) IL-1β, and (C) IL-6 showed significant increases in the STZ group, which are attenuated by fingolimod at both doses. (D) BDNF and (E) GDNF expression levels; STZ caused upregulation, and fingolimod modulated GDNF expression with no effects on BDNF level compared to the STZ group. Data are expressed as mean ± SEM relative to control; *P\u0026lt;0.05, **P\u0026lt;0.01, and ***P\u0026lt;0.001 represent the difference between control and other treated groups.\u003cstrong\u003e \u003c/strong\u003e#P\u0026lt; 0.05, ##P \u0026lt; 0.01, and ###P \u0026lt; 0.001 represent the difference between STZ and other treated groups.\u003c/p\u003e","description":"","filename":"floatimage5.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-8382715/v1/2b3685dec359a3c5e971d77e.jpeg"},{"id":98497195,"identity":"2a4f640d-4482-4360-92f0-653c475c7b7b","added_by":"auto","created_at":"2025-12-18 09:14:03","extension":"jpeg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":22547,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMalondialdehyde (MDA) levels in hippocampal tissue. \u003c/strong\u003eSTZ significantly elevated MDA levels, indicating increased lipid peroxidation and oxidative stress. Treatment with fingolimod at 0.25 and 0.5 mg/kg significantly reduced MDA levels, reflecting antioxidant effects. Data are shown as mean ± SEM; ***P\u0026lt;0.001 represents the difference between control and STZ groups. #P\u0026lt; 0.05 and ##P \u0026lt; 0.01 represent the difference between STZ and other treated groups.\u003c/p\u003e","description":"","filename":"floatimage6.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-8382715/v1/e78c6345c9239a55bc739e84.jpeg"},{"id":98624090,"identity":"837fb1af-1445-4266-8eec-1787c38b9911","added_by":"auto","created_at":"2025-12-19 17:08:00","extension":"jpeg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":257020,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eNumber of CA1 pyramidal cells and total hippocampal CA1 volume. \u003c/strong\u003e(A-D) Cresyl violet-stained hippocampal sections illustrating pyramidal neuron density. (E) Quantification of pyramidal cell counts shows STZ-induced neuronal loss, which is mitigated by fingolimod at 0.5 mg/kg. (F) Total volume of CA1 hippocampal area; STZ decreased volume, which was significantly restored by fingolimod at 0.5 mg/kg. Data are mean ± SEM; **P\u0026lt;0.01 and ***P\u0026lt;0.001 represent the difference between control and STZ-treated groups\u003cstrong\u003e. \u003c/strong\u003e#P\u0026lt; 0.05 represents the difference between STZ and other treated groups.\u003c/p\u003e","description":"","filename":"floatimage7.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-8382715/v1/e4ae4a3a0e23a7fbf80066c2.jpeg"},{"id":98774805,"identity":"86b40f30-b190-4657-827c-9cab5678e0c1","added_by":"auto","created_at":"2025-12-22 12:14:37","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1805983,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8382715/v1/1a317974-e72a-4252-93d2-153c891acbd7.pdf"},{"id":98623996,"identity":"c9be395c-7ef2-4bc1-b454-49410cd91930","added_by":"auto","created_at":"2025-12-19 17:07:53","extension":"jpeg","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":142971,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eGraphical Abstract.\u003c/strong\u003eTherapeutic window of fingolimod dosing in STZ-induced cognitive impairment\u003c/p\u003e","description":"","filename":"floatimage1.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-8382715/v1/9c5b88a8935e2fe013629de7.jpeg"}],"financialInterests":"The authors declare no competing interests.","formattedTitle":"\u003cp\u003e\u003cstrong\u003eOptimizing Fingolimod Dosing in STZ-Induced Cognitive Impairment: Divergent Behavioral and Histopathological Responses Reveal a Narrow Neuroprotective Window\u003c/strong\u003e\u003c/p\u003e","fulltext":[{"header":"Introduction","content":"\u003cp\u003eDementia is a term for various cognitive impairments that are associated with a decline in memory, reasoning, and the ability to perform routine activities. Among all forms of dementias, Alzheimer's (AD) is the most prevalent and accounts for approximately 60-80% of cases. It is a progressive neurodegenerative disorder characterized by amyloid-beta plaques and neurofibrillary tangles leading to synaptic dysfunction and, ultimately, neuron loss. Clinically, AD usually has an initially mild course of cognitive decline and progresses gradually to significant impairment in memory and executive functions, significantly affecting the lives of patients and their caregivers (Porsteinsson et al. 2021, Kumar et al. 2025).\u003c/p\u003e\n\u003cp\u003eThe pathophysiology of AD is complex and multifactorial, involving genetic, environmental, and lifestyle factors. The interplay of these elements contributes to neuroinflammation, oxidative stress, and metabolic dysregulation, all of which exacerbate cognitive decline (Salehi et al. 2016, Paroni et al. 2019, Breijyeh and Karaman 2020, Hoogmartens et al. 2021). Given the increasing prevalence of dementia in aging populations, there is an urgent need for effective therapeutic strategies that can halt or reverse the progression of memory impairment associated with AD and other forms of dementia.\u003c/p\u003e\n\u003cp\u003eTo determine potential therapeutic strategies, several scientific studies use animal models that emulate the cognitive dysfunction characteristic of human dementia. One such model widely used to study cognitive dysfunction is STZ-induced memory impairment. STZ is a potent neurotoxin that, following its administration into the lateral ventricles, preferentially damages cholinergic neurons of the basal forebrain (Li et al. 2016, Moosavi et al. 2020). This model recapitulates cognitive impairments observed in AD and shows how learning and memory are affected, as well as how neurochemical markers associated with synaptic plasticity change. It would be a valuable tool for assessing the potential efficacy of neuroprotective agents.\u003c/p\u003e\n\u003cp\u003eFingolimod (FTY720) is a sphingosine-1-phosphate receptor modulator that has attracted scientific interest for its potential neuroprotective properties. Owing to its therapeutic potential for managing multiple sclerosis, fingolimod has been shown to cross the blood-brain barrier and exert neuroprotective effects (Cruz and Fonseca 2014, Hunter et al. 2016). Fingolimod exerts its immunosuppressive effects by antagonizing S1P receptors expressed in immune cells, thereby decreasing lymphocyte infiltration in the periphery and exerting subtle effects on subsequent downregulation of neuroinflammation. Fingolimod also exerts additional effects, promoting survival in microglia and neuronal cells via S1P receptor activation of neurons and glial cells. Subsequent effects lead to reduced accumulation of Aβ peptides (Hla and Brinkmann 2011, Asle-Rousta et al. 2013, Groves et al. 2013, Hemmati et al. 2013, Takasugi et al. 2013, Chen et al. 2025). In addition to immune modulation, one possible function of fingolimod is its antioxidant properties, either by enhancing the body's antioxidant defenses or directly scavenging reactive oxygen species (ROS) (Martín-Montañez et al. 2019, Yevgi and Demir 2021). This role in managing CNS responses and associated effects of neuroprotection makes fingolimod a potential drug candidate for managing cognitive impairment associated with neurodegenerative disorders. Preclinical studies indicated that fingolimod could enhance synaptic plasticity and reduce Aβ accumulation by favoring neurotrophic support (Fukumoto et al. 2014, Nazari et al. 2016, Joshi et al. 2017, Rahmati-Dehkordi et al. 2024). Examples of studies indicating potential dose-dependent effects of fingolimod include studies using 5x FAD mice, in which lower doses (1 mg/kg) compared with 5 mg/kg showed enhanced effects. Additionally, lower microglial activation and reactive astrogliosis, with low levels of Aβ accumulation, and increased hippocampal neurogenesis would be observed (Aytan et al. 2016). A recent study using 5x FAD mouse models indicates possible Aβ accumulation in hippocampi, with subsequent attenuation associated with a decline in circulating lymphocytes. Notably, at the lowest administered dose of fingolimod (0.03 mg/kg/day), improvements in learning and memory were observed, along with attenuation of microglial and astrocytic activation and restoration of markers of GABAergic and cholinergic neuronal function (Carreras et al. 2019). These studies suggest that fingolimod, even at low doses that do not entirely disrupt peripheral lymphocyte function, may be therapeutic in both memory-impaired and AD models. However, the effects of varied fingolimod dosing on STZ-induced memory impairment are an area still requiring detailed attention.\u003c/p\u003e\n\u003cp\u003eDespite the promising results with fingolimod, there remains a significant gap in understanding optimal dosing strategies and the mechanisms by which the drug exerts its neuroprotective effects. While previous studies have mainly used transgenic models of AD or other neurodegenerative diseases, the present study used a streptozotocin-induced model that mimics aspects of sporadic AD—specifically, neuroinflammation, oxidative stress, and metabolic abnormalities. Aiming to fill this gap, this study will evaluate the neuroprotective effects of different doses of fingolimod on STZ-induced cognitive impairment in male rats. Specifically, the study will examine cognitive function using behavioral tests and neurochemical changes in the brain. This study introduces several aspects, including the investigation of dose optimization, the study of multi-target effects on neuroinflammation, behavioral function, oxidative stress, and histomorphology, and the investigation of the potential for translational applications of fingolimod for AD and other related dementias.\u003c/p\u003e\n\u003cp\u003eBy elucidating the dose-dependent effects of fingolimod, this research seeks to provide insights into its potential as a therapeutic agent for memory impairment associated with AD disease and other neurodegenerative conditions.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cp\u003e\u003cstrong\u003eAnimals'\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eexperimental groups\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe experimental study utilized male Sprague-Dawley rats, weighing between 250-300 grams, obtained from the Experimental Animal Center of Shiraz University of Medical Sciences. Animals were acclimatized to the housing conditions for one week prior to the experiment. All animals were housed in standard laboratory conditions (temperature: 22 ± 2°C; humidity: 50 ± 5%; 12-hour light/dark cycle) with free access to food and water.\u003c/p\u003e\n\u003cp\u003eThe rats were randomly divided into five groups as follows: control group (n = 10, animals received a saline solution (0.9% NaCl) via intraperitoneal (i.p.) injection and intracerebroventricular (i.c.v.) microinjection), STZ group (n = 10, animals received i.c.v. STZ (3 mg/kg) and i.p. saline), STZ + Fingolimod 0.25mg group (n = 9, animals received i.c.v. STZ (3 mg/kg) and i.p. fingolimod (0.25 mg/kg/daily) for 10 days), STZ + Fingolimod 0.5mg group (n = 10, \u0026nbsp;animals received i.c.v. STZ (3 mg/kg) and i.p. fingolimod (0. 5 mg/kg/daily) for 10 days), and STZ + Fingolimod 1mg group (n = 9, animals received i.c.v. STZ (3 mg/kg) and i.p. fingolimod (1 mg/kg/daily) for 10 days).\u0026nbsp; The number of animals allocated to each group was determined based on previous studies utilizing the STZ model, as well as ethical and practical considerations.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll experimental protocols were reviewed and approved by the Institutional Ethics Committee of Shiraz University of Medical Sciences (IR.SUMS.AEC.1401.104) in compliance with the National Institutes of Health guidelines.All experimental procedures adhered to the National Research Council's Guide for the Care and Use of Laboratory Animals, 8th edition (National Academies Press (US); 2011. ISBN-13: 978-0-309-15400-0).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStereotaxic surgery, STZ injection, and fingolimod treatment\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eStereotaxic surgery was performed to facilitate the administration of STZ directly into the cerebral lateral ventricles. The surgical procedures were conducted under aseptic conditions with animals anesthetized using an intraperitoneal injection of a mixture of Ketamine (100 mg/kg) and xylazine (10 mg/kg). A stereotaxic apparatus was used to secure the rat’s head in a fixed position. Stereotaxic coordinates for the lateral ventricle were determined based on the Paxinos brain atlas, with the following coordinates used: -0.8 mm anteroposterior (AP), ±1.5 mm mediolateral (ML), -3.5 mm dorsoventral (DV) relative to the bregma. A midline incision was made in the scalp, and a small burr hole was drilled at the designated coordinates. A Hamilton syringe was used for STZ microinjections and carefully inserted into the lateral ventricle. STZ (STZ-S0130-100MG, Sigma-Aldrich, USA) at a concentration of 3 mg/kg was freshly dissolved in sterile saline and injected at a rate of 1 µL/min, with a total volume of 5 µL administered per rat. After the injection, the needle was left in place for an additional 5 minutes to minimize reflux, then slowly withdrawn. The skin incision was sutured at the end of the procedure.\u003c/p\u003e\n\u003cp\u003eFingolimod, a kind gift from Abidi Pharmaceutical Company (Iran), was dissolved in saline. One day following the STZ injection, treatment with fingolimod commenced at a dose of 0.25, 0.5, and 1 mg/kg was administered via i.p. injection for 10 consecutive days. Animals in the control and STZ groups received equal volumes of saline during the same treatment period to control for any potential effects of the treatment procedure.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eBehavioral studies\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eOpen field test\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eOn day 12 after surgery, an open field test was performed. The Open field test is a widely utilized experimental paradigm to study anxiety and general locomotor activity in rodent models. The test was conducted within a square plexiglass enclosure measuring 45 cm in height and 90 cm in both width and length, illuminated by four 60 W bulbs positioned 1.8 m above the box. The floor of the box was typically divided into specific zones (center and periphery) to evaluate the animal's behavior in relation to perceived safety and risk. The rats were placed in the arena for 20 minutes while their movement was monitored using EthoVision (Noldus Information Technology, Netherlands). The differentiation between the center and periphery serves as an indicator of anxiety levels. Key parameters measured include the time spent in the center versus the periphery (in seconds) and the frequency of grooming behaviors exhibited.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eMorris water maze test\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe Morris Water Maze (MWM) test, a recognized behavioral assessment, was employed to evaluate spatial learning and memory in rats on the 13th and 14th days post-surgery. The arena consisted of a circular swimming pool with a diameter of approximately 150 cm, filled with opaque water and typically maintained at a temperature of 21-24°C. The arena was divided into four quadrants, with a submerged platform located in a fixed position in one of the quadrants (designated as the target quadrant) just below the water's surface, serving as the escape location for the animals.\u003c/p\u003e\n\u003cp\u003eDuring the training phase, each rat underwent several trials (12 trials divided into three blocks) to facilitate spatial memory acquisition. The animal was placed in the water at various starting points and allowed to swim for up to 60 seconds until it located the hidden platform. The latency to find the platform during training trials served as a criterion for assessing spatial learning. Twenty-four hours after the training period, a probe trial was conducted to evaluate spatial memory retention. In this trial, the hidden platform was removed, and the animal was allowed to swim freely for 60 seconds. Key parameters measured during the probe trial included the time spent in the target quadrant, where the platform had previously been located.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003ePassive avoidance test\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe passive avoidance test evaluates an animal's ability to avoid a previously encountered environment that has been associated with an unpleasant stimulus. The test was conducted using a shuttle box, which consists of a light compartment and a dark compartment separated by a sliding door. The dark compartment is associated with an electric foot shock (0.5 mA, 50 Hz, for 2 seconds), creating a negative association for the animals.\u003c/p\u003e\n\u003cp\u003eDuring the training phase (on day 15), each rat was initially placed in the light compartment. After a 20-second acclimation period, the sliding door was opened to allow access to the dark compartment. When the animal entered the dark area, it received a foot shock. This procedure was repeated after five minutes, provided the animal continued to avoid entering the light chamber from the dark one. The retention test was conducted 24 hours after the training phase (on day 16). In this session, the rat was placed in the light compartment for up to 300 seconds, and the delay before entering the dark chamber, referred to as step-through latency (STL), was recorded without any electric shock. A longer latency indicates a stronger memory of the aversive experience and a greater ability to avoid the dark compartment.\u003c/p\u003e\n\u003ch4\u003e\u003cstrong\u003eqRT-PCR analysis of hippocampal tissue\u003c/strong\u003e\u003c/h4\u003e\n\u003cp\u003eQuantitative reverse transcription polymerase chain reaction (qRT-PCR) was employed to assess the mRNA expression levels of neurotrophic factors and pro-inflammatory cytokines, including brain-derived neurotrophic factor (BDNF), glial cell line-derived neurotrophic factor (GDNF), nerve growth factor (NGF), tumor necrosis factor-alpha (TNFα), interleukin-1 beta (IL-1β), and interleukin-6 (IL-6) in hippocampal tissue. Total RNA was isolated using the TRIzol reagent (Yekta Tajhiz Azma, Iran) according to the manufacturer’s instructions. The concentration and purity of RNA were measured spectrophotometrically at 260 nm and 280 nm (NanoDrop spectrophotometer, Thermo Fisher Scientific, USA). cDNA was synthesized from 1 µg of RNA using a cDNA Reverse Transcription Kit (Yekta Tajhiz Azma, Iran).\u003c/p\u003e\n\u003cp\u003eSpecific primers were designed for target genes. The qRT-PCR was performed using RealQ Plus 2x Master Mix Green (Ampliqon, Denmark) on a StepOnePlus Real-Time PCR system (Thermo Fisher Scientific) for amplification, with the following thermal cycling conditions: an initial denaturation at 95°C for 5 minutes, followed by 40 cycles of 95°C for 20 seconds, and 60°C for 60 seconds. Hypoxanthine-guanine phosphoribosyltransferase (HPRT) was used as an internal control. Relative gene expression levels were calculated using the 2\u003csup\u003e^−ΔΔCt\u003c/sup\u003e method to compare the treatment groups with controls.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMalondialdehyde (MDA) assay\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe level of malondialdehyde (MDA) in hippocampal tissue was quantified as a marker of oxidative stress using the thiobarbituric acid reactive substances (TBARS) assay\u0026nbsp;(Naseh et al. 2020, Jahromi et al. 2024). In brief, hippocampal samples were homogenized and centrifuged at 12,000 rpm for 15 minutes at 4°C. The supernatant was mixed with a solution consisting of 0.25 N hydrochloric acid (HCl), 20% trichloroacetic acid (TCA), and 0.8% thiobarbituric acid (TBA), and incubated at 87°C for 60 minutes. After that, the absorbance was measured at 532 nm using a microplate reader. The MDA concentration was expressed in nanomoles per milligram of tissue (nmol/mg).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStereological and histomorphological methods\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe rats were transcardially perfused with normal saline, followed by 4% paraformaldehyde. Then, the brains were removed and kept in the same fixative. The brains were then transferred to 30% sucrose solution for 3 days. Subsequently, the brains were stored at -80°C for further processing. They were then serially and coronally sectioned using a cryostat (Microm HM 525, Germany) at a thickness of 40 μm. The sections were stained with cresyl violet. The volume of the hippocampal CA1 region was estimated using the Cavalieri method, while the number of CA1 pyramidal cells was determined using the optical dissector, as described in more detail previously (Asadi nejad et al. 2024).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStatistical analysis\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eStatistical analyses were performed using GraphPad Prism software (version 9). Data were expressed as mean ± standard error of the mean (SEM). Differences between groups were compared using two-way analysis of variance (ANOVA) and one-way ANOVA followed by Tukey's post hoc test for multiple comparisons. A p-value of less than 0.05 was considered statistically significant.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003eIn the present study, a high-dose fingolimod group (1 mg/kg) was initially included; however, the animals in the 1 mg group exhibited significant behavioral changes, including withdrawal, social isolation, reduced mobility, and poor cooperation during behavioral tests. Specifically, in the Morris water maze test, these rats were unable to swim properly and frequently submerged or refused to participate, indicating a marked deterioration in their functional performance. Such non-cooperative behavior precluded reliable task performance and prevented the collection of sufficient quantitative data for robust statistical analysis. Consequently, the 1 mg/kg group were excluded from the study to focus on doses yielding interpretable and biologically meaningful outcomes.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eThe effect of STZ and fingolimod on rats' anxiety-like behavior in the open field test\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIn this test, an increase in the amount of time spent in the center zone relative to the periphery zone indicates decreased anxiety in rats. One-way ANOVA analysis revealed significant differences between groups (F (3, 35) = 5.090, P = 0.0050). Our findings showed that the time spent in the center significantly decreased in the STZ group compared to the control group (8.019±1.877 vs. 18.34±3.385; P \u0026lt;0.05). However, no significant difference was observed between the fingolimod 0.25 mg-treated group and STZ group (9.946±2.342 vs. 8.019±1.877, P=0.9419), as well as between the fingolimod 0.5 mg- and STZ-treated groups (6.440±1.446 vs. 8.019±1.877, P=0.9640), indicating that fingolimod treatment failed to reduce the anxiety-like behaviors induced by STZ (Figure 1A).\u003c/p\u003e\n\u003cp\u003eAnother indicator of anxiety-like behavior in animals is the number of grooming sessions in rats. Anxious animals display grooming behavior more frequently than non-anxious animals do. A significant increase in grooming frequency was observed in the STZ-treated group and the fingolimod 0.5 mg-treated group compared to the control group (7.600 ± 1.087 and 7.500 ± 0.6540 vs. 4.100 ± 0.7371; P \u0026lt; 0.05, respectively). Although fingolimod in a dose of 0.25 mg was able to reduce grooming sessions compared to the STZ group, it was not statistically significant (P=0.1996) (Figure 1B).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFingolimod (0.25 mg) prevented STZ-mediated memory spatial impairment in MWM\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAs shown in Figure 2A, learning patterns of animals in all groups demonstrated a negative linear correlation between escape latency and training blocks; however, the performance of animals receiving STZ was weaker than that of the other groups. Two-way ANOVA analysis revealed significant differences between groups (F (3, 105) = 21.10, P\u0026lt;0.0001). The Post hoc analysis by Tukey's test revealed that escape latency in the STZ receiving group was significantly increased compared to the control group in the first (52.14±11.14s vs. 33.85±14.80s; P\u0026lt;0.01), second (44.57±11.64s vs. 21.99±12.13s; P\u0026lt;0.001), and third (44.09±13.25s vs. 17.42±14.78s; P\u0026lt;0.001) blocks of training, indicating STZ-induced deterioration in the STZ group. Although STZ+Fingo 0.5mg receiving group did not show a significant difference with STZ treated group in three blocks, fingolimod treatment in dose of 0.25 mg could reverse this deficit in the all three blocks (36.60±13.79s vs. 52.14±11.14s, P\u0026lt;0.05; 26.44±12.85s vs. 44.57±11.64s, P\u0026lt;0.01; 23.67±8.99s vs. 44.09±13.25s, P\u0026lt;0.01, respectively), indicating a protective role for fingolimod 0.25 mg against STZ toxicity.\u003c/p\u003e\n\u003cp\u003eThe evaluation of memory retention was done in the prob trial (Figure 2B). One-way ANOVA analysis showed that treatment with fingolimod in the STZ receiving group could significantly increase the time spent in the target area (F (3, 35) = 6.293, P = 0.0016). Post hoc analysis by Tukey's test displayed that the time spent in the target area was decreased significantly in the STZ-treated group compared to the control group (11.61±1.534 vs. 21.88±2.647, P\u0026lt;0.01). However, only fingolimod in a dose of 0.25 mg (19.67±1.836, P\u0026lt;0.05) could significantly prevent STZ-induced disturbance.\u003c/p\u003e\n\u003cp\u003eIn order to assess the possible effect of drugs on the motor performance of swimming speed of animals, their swimming speed was assessed (Figure 2C). The results showed that treatment with STZ and/or fingolimod did not affect the swimming speed of animals (F (3, 35) = 1.730, P = 0.1788).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFingolimod prevented STZ-mediated fear memory impairment in the passive avoidance test\u0026nbsp;\u003c/strong\u003eResults from the passive avoidance test indicated significant differences between groups (F (3, 35) = 4.989, P = 0.0055) (Figure 3). The animals in the STZ group had significantly shorter STL compared to the control group (109.4 ± 33.85 vs. 255.7 ± 27.44, P\u0026lt;0.01), reflecting memory deficits induced by STZ. Fingolimod treatment markedly increased the latency time in both treated groups (234.1 ± 30.26 and 242.0 ± 30.71, respectively), suggesting improvements in memory retention.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eThe effect of STZ and fingolimod on rats' hippocampal mRNA expression levels of TNFα, IL-1β, IL-6, BDNF, and GDNF\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe results of One-Way ANOVA analysis (n=3 per group) showed significant differences between groups relevant to expression levels of TNFα, IL-1β, and IL-6 (F (3, 8) = 34.45, P\u0026lt;0.0001; F (3, 8) = 172.1, P\u0026lt;0.0001; and F (3, 8) = 29.89, P = 0.0001, respectively). Post hoc analysis by Tukey's test showed that the STZ group, compared to the control, exhibited significantly higher expression of TNFα (2.082±0.1011 vs. 1.000±0.03975), IL-1β (4.157±0.2019 vs. 1.000±0.04009), and IL-6 (1.977±0.06436 vs. 1.000±0.06511) (P\u0026lt;0.001). Fingolimod treatment in both doses of 0.25 mg (P\u0026lt;0.001) and 0.5 mg (P\u0026lt;0.01) was able to reverse the STZ-induced increase in the expression of TNFα. In addition, our results showed that fingolimod could reverse the IL-1β increment induced by STZ in both doses of 0.25 and 0.5 mg (P\u0026lt;0.001). This decreasing effect on IL-6 expression was also observed with both fingolimod doses (P\u0026lt;0.01) (Figure 4A-C).\u003c/p\u003e\n\u003cp\u003eMoreover, quantitative analysis revealed a significant increment in mRNA expression levels of BDNF and GDNF in the STZ group compared to control (2.049±0.1562 vs. 1.000±0.1598, P\u0026lt;0.01, and 1.858±0.05846 vs. 1.000±0.04026, P\u0026lt;0.05, respectively). There was no significant difference between the expression level of BDNF in fingolimod-treated groups compared to the STZ group (Figure 4D and E). However, fingolimod in a dose of 0.25 mg, but not 0.5 mg, significantly reversed STZ-induced increase in expression of GDNF (P\u0026lt;0.05).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eThe effect of STZ and fingolimod on oxidative stress\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMalondialdehyde levels were measured in all experimental groups to assess lipid peroxidation as an indicator of oxidative stress. The animals in the STZ group exhibited a significant increase in MDA levels compared to the control group (4.358±0.3470 vs. 2.264±0.3472, P\u0026lt;0.001, n=5 in control and STZ groups), indicating enhanced lipid peroxidation and oxidative stress. Treatment with fingolimod in both doses (n=4) of 0.25 mg (2.913±0.1622, P\u0026lt;0.05) and 0.5 mg (2.468±0.2252, P\u0026lt;0.01) significantly reduced MDA levels compared to the STZ group, suggesting a protective/antioxidant effect (Figure 5).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eNumber of CA1 pyramidal cells and total volume of CA1 area\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIn order to evaluate the effects of fingolimod treatment on CA1 pyramidal cells in the hippocampus, we conducted a quantitative analysis of cell counts and volume measurements (n=5 per group) following Cresyl violet\u0026nbsp;staining (Figure 6A-D). The analysis revealed that the number of CA1 pyramidal cells was significantly reduced in the STZ group compared to the control group (P\u0026lt;0.001). Specifically, the average number of pyramidal cells in the STZ group compared to the control group was 362.2±22.50 vs. 512.2±11.99, indicating profound neuronal loss due to the induced neurotoxicity. In contrast, treatment with fingolimod at a dose of 0.5 mg significantly increased the number of CA1 pyramidal cells (470.7±18.89) compared to the STZ group (P\u0026lt; 0.05) (Figure 6E). This restorative effect underscores fingolimod's potential neuroprotective action. However, fingolimod at a dose of 0.25 led to an increase in the number of CA1 pyramidal cells compared to the STZ group; this increase was not statistically significant.\u003c/p\u003e\n\u003cp\u003eMoreover, volumetric analysis demonstrated that the volume of the hippocampal CA1 area was significantly decreased in the STZ group compared to control animals (7.142±0.2710 vs. 9.262±0.2406, P\u0026lt;0.01), reflecting the neurodegenerative changes induced by STZ treatment. Fingolimod treatment at a dose of 0.5mg, but not 0.25mg, resulted in a significant increase in the average volume of the CA1 region (8.888±0.2890) relative to the STZ group (P\u0026lt;0.05) (Figure 6F), suggesting a reversal of neuronal atrophy associated with neuroinflammation and oxidative stress.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eThe present study investigated the dose-dependent neuroprotective and anti-inflammatory effects of fingolimod in a rat model of STZ-induced memory impairment. Our findings demonstrate that fingolimod, particularly at the lower dose of 0.25 mg/kg, significantly ameliorated cognitive deficits, reduced neuroinflammation, and attenuated oxidative stress.\u0026nbsp;These results align with previous research highlighting the therapeutic potential of fingolimod in neurodegenerative disorders, including AD and other forms of dementia (Aytan et al. 2016, Angelopoulou and Piperi 2019, Fagan et al. 2022, Leßmann et al. 2023).\u003c/p\u003e\n\u003cp\u003eContrary to some previous studies that reported a 1 mg dose of fingolimod to produce neuroprotective effects and improve memory performance in behavioral tests (Asle-Rousta et al. 2013, Kalecký et al. 2025), the animals receiving a dose of 1 mg fingolimod, in our study, demonstrated notable behavioral alterations, such as withdrawal, social withdrawal, decreased activity, and lack of cooperation during behavioral assessments. Owing to their non-cooperation and inadequate participation, it was not possible to collect sufficient data or perform reliable analysis for the 1 mg dose, leading to the exclusion of this group's data from the final results. One plausible explanation for these behaviors is the occurrence of severe bodily discomfort or pain caused by the high dose of fingolimod. High doses of fingolimod may induce adverse side effects such as pain, physical distress, and general discomfort, which can lead to increased anxiety, reduced activity levels, and social withdrawal (Russo et al. 2015). These effects can substantially impair the animals' ability to perform in behavioral assays, thereby compromising the reliability and completeness of the data.\u003c/p\u003e\n\u003cp\u003eThis highlights an important limitation in pharmacological studies, that the occurrence of severe side effects at higher doses may hinder proper behavioral assessment and impact the interpretability of results. It underscores the necessity of careful dose optimization to balance therapeutic efficacy with tolerability. These findings emphasize that animal well-being, physical comfort, and cooperation are critical factors in obtaining valid and reliable experimental outcomes. Future studies should focus on establishing doses that minimize adverse effects to ensure both safety and data integrity. Moreover, assessing potential side effects and tolerability is essential for translating such therapies into clinical applications, where patient comfort and safety remain paramount.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eNeuroprotective effects of fingolimod on behavioral function\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eOur behavioral findings align with prior studies that have demonstrated the detrimental effects of STZ on memory and learning (Chen et al. 2014, Moreira-Silva et al. 2018, Qi et al. 2021). The MWM and passive avoidance tests confirmed that STZ significantly impairs both spatial and fear-related memory. However, fingolimod at 0.25 mg/kg significantly improved performance in both tests, indicating preserved hippocampal function. These findings corroborate earlier reports of fingolimod’s cognitive-enhancing effects, which are largely attributed to its anti-inflammatory, antioxidant, and neurotrophic mechanisms (Efstathopoulos et al. 2015, Fagan et al. 2022, Kalecký et al. 2025). Nazari et al. (Nazari et al. 2016) observed similar improvements in synaptic plasticity and memory retention in ischemic rat models treated with fingolimod Interestingly, fingolimod at the higher dose of 0.5 mg/kg led to improvements in passive avoidance memory but failed to yield significant improvements in MWM behavioral assessments, suggesting a potential biphasic dose-response relationship, as noted in other studies, that warrants further exploration. For example, Carreras et al., 2019\u0026nbsp;(Carreras et al. 2019)\u0026nbsp;reported a biphasic dose-response in AD mice; low-dose fingolimod improved cognition and reduced neuroinflammation, while higher doses failed to enhance function and produced tolerability issues. Our findings align with this biphasic pharmacodynamic pattern, suggesting that optimal neuroprotective efficacy occurs within a narrow dose range that avoids systemic immune suppression. This phenomenon is commonly observed with neuromodulatory agents and may reflect receptor desensitization or complex pharmacodynamics at higher doses.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe anxiogenic effects of STZ, as evidenced by reduced center zone activity and increased grooming behavior in the open field test, were not alleviated by fingolimod treatment. This suggests that fingolimod’s neuroprotective actions may be more potent in cognitive domains than in affective behaviors, or that anxiety-related circuitry is less responsive to its modulatory effects in this context. In this regard, some studies reported that fingolimod does not affect anxiety-like behavior in the elevated plus maze test (di Nuzzo et al. 2015). While some studies suggest fingolimod can influence anxiety-related behaviors (e.g., in obese mice), the degree of improvement might be less significant compared to its impact on cognitive functions (Wencel et al. 2023). This could be due to differences in how fingolimod interacts with specific neuronal circuits involved in each domain.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eNeurotrophic modulating and anti-inflammatory mechanisms\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMolecular analyses revealed that STZ significantly elevated hippocampal mRNA expression of TNF-α, IL-1β, and IL-6—cytokines known to mediate neuroinflammatory cascades implicated in AD pathogenesis (Liu et al. 2016, Fan et al. 2022). Fingolimod treatment effectively downregulated these cytokines at both tested doses, indicating strong anti-inflammatory properties. These results are consistent with findings that demonstrated fingolimod's ability to modulate microglial activation, suppress pro-inflammatory cytokine production (Jackson et al. 2011, Rothhammer et al. 2017, Shang et al. 2020, Yang et al. 2025). Fingolimod’s neuroprotective actions involve a multi-pathway network combining S1P1 receptor signaling, NF-κB inhibition, and Nrf2 activation (Chen et al. 2025). Specifically, fingolimod acts on both circulating and CNS-resident myeloid cells, including microglia, to reduce the release of pro-inflammatory cytokines like TNFα, IL-1β, and IL-6. This effect is achieved through functional antagonism of S1P receptors, particularly S1P1, which are involved in regulating immune cell trafficking and inflammatory responses within the CNS (Brunkhorst et al. 2014, Quirant‐Sánchez et al. 2018, Lobaina and Shanina 2025).\u003c/p\u003e\n\u003cp\u003eAdditionally, the observed increase in neurotrophic factors such as BDNF and GDNF in STZ-treated rats indicates a compensatory response to neurodegeneration and an innate attempt at repair. The findings of this study suggest that fingolimod exerts a dual role in neurodegenerative contexts, mitigating dysregulated neurotrophic signaling (e.g., excessive GDNF) and enhancing BDNF-mediated neuroplasticity. The observed BDNF upregulation aligns with its established role as a compensatory mechanism in early neurodegeneration, where transient increases attempt to counteract neuronal damage and preserve function. However, our results also highlight the delicate balance required in neurotrophic support, as excessive or sustained elevations of these factors may exacerbate neuroinflammatory cascades or fail to sustain long-term repair.\u003c/p\u003e\n\u003cp\u003eIn many neurodegenerative diseases, including AD, a consistent reduction in BDNF levels has been observed, correlating with synaptic dysfunction and cognitive decline; While increasing its expression leads to improved memory. (Bayat et al. 2021, Gao et al. 2022). Interestingly, however, during the initial phases of neuronal injury or in mild cognitive impairment (MCI), BDNF levels can transiently increase, potentially reflecting an innate protective response attempting to counteract neuronal loss. For instance, elevated serum BDNF levels in MCI patients suggest an early compensatory mechanism, whereas a decline in advanced AD indicates failure of these endogenous supports or progression beyond the brain’s capacity for compensation (Faria et al. 2014, Kim et al. 2017). Moreover, the regulation of BDNF is highly complex; differences in pro-BDNF processing mediated by glial cells and plasmin activity may contribute to the conflicting results regarding BDNF levels in peripheral blood versus central nervous system tissues (Yang et al. 2009). Our findings support the hypothesis that pharmacological interventions like fingolimod may enhance or restore neurotrophic support during the early or preclinical stages of neurodegeneration by increasing BDNF levels and correcting dysregulated GDNF expression. Such effects could be beneficial in maintaining synaptic plasticity and neuronal survival, ultimately delaying or mitigating disease progression. Nevertheless, it is critical to recognize that excessive or unbalanced neurotrophic signaling might be detrimental, emphasizing the importance of finely tuned regulation of these factors in therapeutic strategies. Further research is warranted to elucidate the precise mechanisms underlying neurotrophic modulation by fingolimod and to determine its potential impact across different stages of neurodegenerative diseases.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eOxidative stress and lipid peroxidation\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSTZ induced a significant increase in lipid peroxidation as shown by elevated MDA levels, reflecting heightened oxidative stress. Oxidative stress plays a pivotal role in STZ-induced neurodegeneration by promoting cellular damage, mitochondrial dysfunction, and apoptosis (Ansari et al. 2024, AEF Cardinali et al. 2025). Fingolimod treatment in both doses significantly reduced MDA levels, indicating potent antioxidant effects that may contribute to its neuroprotective efficacy. The observed reduction in oxidative markers aligns with previous findings demonstrating fingolimod’s role in enhancing antioxidant enzyme expression, such as superoxide dismutase and catalase, and reducing ROS production (Wu et al. 2017, Yevgi and Demir 2021, Bagheri et al. 2024). Crivelli et al. (2022) reported that fingolimod decreases ceramide levels and oxidative stress in familial AD models (Crivelli et al. 2022), further supporting its neuroprotective properties through modulation of lipid metabolism and oxidative pathways. These findings suggest that fingolimod exerts a significant neuroprotective effect by attenuating oxidative stress and lipid peroxidation. Its ability to modulate oxidative markers and lipid signaling pathways underscores its potential as a therapeutic agent in neurodegenerative diseases characterized by oxidative damage, such as AD and diabetic neuropathy.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStructural and cellular restoration\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eHistomorphological analyses further support the neuroprotective profile of fingolimod. Stereological analysis revealed significant CA1 pyramidal neuron loss and hippocampal atrophy in STZ-treated rats, consistent with previous studies demonstrating the vulnerability of hippocampal neural cells to STZ, particularly in CA1 and CA3 regions (Lee et al. 2015, Zappa Villar et al. 2018, Roy et al. 2022). Although neuronal loss and hippocampal atrophy were partially attenuated by the 0.25 mg/kg dose of fingolimod, a more pronounced amelioration was observed at 0.5 mg/kg, highlighting its neuroregenerative potential. This finding is supported by Kalecký et al. (2025), who demonstrated fingolimod's ability to rescue synaptic plasticity and neuronal integrity in APP/PS1 mice (Kalecký et al. 2025). The restoration of CA1 volume and pyramidal cell numbers suggests that fingolimod may prevent neurodegeneration through a combination of anti-apoptotic, anti-inflammatory, and trophic mechanisms. In this regard, data from clinical trials and real-world studies demonstrate that fingolimod significantly reduces the rate of brain volume loss compared to other treatments or placebo in MS patients (De Stefano et al. 2017). It highlights the neuroprotective effects of fingolimod, likely through its anti-inflammatory properties and ability to modulate immune responses, thereby helping to preserve neural tissue and slow neurodegeneration.\u003c/p\u003e\n\u003cp\u003eWhile both doses of fingolimod demonstrated favorable effects on neuroinflammation and oxidative stress, their differential impact on behavioral and histological outcomes raises important questions about the optimal therapeutic window and target engagement. It is possible that the 0.25 mg/kg dose more effectively modulates synaptic plasticity and functional connectivity, whereas the 0.5 mg/kg dose confers structural preservation without equivalent functional gains.\u003c/p\u003e\n\u003cp\u003eIt is well understood in the arena of neuroprotection that a distinction may arise between histopathological and behavioral outcomes; whereby cellular rescue may precede or even occur independently of functional recovery due to the additional requirement of circuit-level reorganization and synaptic plasticity beyond neuronal survival (Aytan et al. 2016). Mechanistically, the observed dissociation between structural rescue and behavioral \u0026nbsp;improvement might reflect a biphasic pharmacodynamic profile of the S1P pathway targeted by fingolimod. Fingolimod-phosphate acts as a high-affinity agonist at S1P₁ receptors, causing rapid receptor internalization and sustained functional antagonism. However, further exposure may also engage or over-activate other S1PR subtypes (e.g., S1P₃, S1P₅), thereby redirecting signaling to alternative pathways. Thus, this internalization, receptor desensitization, and subtype-specific signaling could impair synaptic function and long-term potentiation, providing a mechanistic explanation for dose-dependent non-linear effects on cognition and synaptic plasticity. Such a biphasic effect is consistent with the literature on S1P receptor pharmacology and the action of fingolimod through different receptor subtypes (Brinkmann 2009, Mullershausen et al. 2009, Groves et al. 2013). Consequently, the dose that most effectively normalizes neuroinflammatory histopathology may not optimally support hippocampal network plasticity underlying memory consolidation.\u003c/p\u003e\n\u003cp\u003eFurthermore, sensitivity to behavioral tests could also play a role. The MWM is sensitive to anxiety, stress response, and locomotor activity, and any small change related to stress/motivation at 0.5 mg/kg may have impaired performance even if actual improvement was achieved in the hippocampus (Othman et al. 2022). Moreover, morphological recovery may not necessarily imply network restoration, as spatial learning depends upon network interactions between the dentate gyrus, CA3, CA1, and cortex (Daugherty et al. 2017, Barry et al. 2021). It may, therefore, be insufficient if structural restoration is achieved selectively in the CA1 region or if synaptic or subfield restoration is not achieved concurrently, including recovery of behavioral functions, within a 10-day window after treatment. The results, therefore, are consistent with a model whereby structural recovery precedes functional improvement, as a consequence of divergent dose signaling via S1P, explaining why 0.5 mg/kg produced histological but not behavioral recovery.\u003c/p\u003e\n\u003ch3\u003e\u003cstrong\u003eLimitations and future directions\u003c/strong\u003e\u003c/h3\u003e\n\u003cp\u003eSeveral limitations of this study should be considered when interpreting the findings. First, although the STZ model reproduces important features of sporadic AD-like pathology, it does not fully recapitulate the progressive and multifactorial nature of human AD; therefore, the translational value of the results should be validated in additional models, including transgenic and aged animals. Second, the high-dose (1 mg/kg) fingolimod group was excluded due to marked behavioral intolerance, thereby preventing the assessment of the entire dose-response curve. Future studies should include refined monitoring of physiological and stress-related parameters to describe dose-related side effects better.\u003c/p\u003e\n\u003cp\u003eThird, behavioral and histological outcomes were measured after a relatively short treatment period (10-16 days), which may not capture long-term neuroprotection or neurorestoration. Long-term follow-up in longitudinal studies will be required to determine the durability of functional and structural recovery. While fingolimod altered cytokine and markers of oxidative stress in this study, further mechanistic studies would be required to interpret the divergent behavioral and histological responses seen across doses. Moreover, only male rats were studied; future studies should investigate possible sex-dependent differences, given the established influence of sex hormones on S1P signaling and neurodegeneration.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eEvidence has been obtained in this study that fingolimod exhibits dose-dependent neuroprotective actions, with a clear distinction between the behavioral results and the restoration of histopathological changes at varying doses. The lower dose of fingolimod (0.25 mg/kg) improved cognitive function, along with reduced neuroinflammation and oxidative damage. Whereas the higher dose of fingolimod (0.5 mg/kg) primarily promoted structural maintenance rather than restoring function.\u003c/p\u003e\n\u003cp\u003eFrom a clinical perspective, these findings suggest that low-dose fingolimod may offer a more favorable therapeutic window in the early stages of cognitive impairment, as it may confer neurobiological advantages without the tolerability issues and desensitization associated with higher doses in trials for neurodegenerative diseases. The results underscore the importance of precise dose optimization in translating S1P-modulating therapies for the neurodegenerative diseases.\u0026nbsp;\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors would like to thank Abidi Pharmaceutical Company for preparing fingolimod, and the Office of Vice Chancellor for Research at Shiraz University of Medical Sciences for financially supporting (Grant No: 26854). The study protocol was approved by the institutional review board (IRB) and the ethics committee of Shiraz University of Medical Sciences (Approval No: IR.SUMS.AEC.1401.104).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDeclarations of interest:\u003c/strong\u003e None.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDeclaration of Generative AI and AI-assisted technologies in the writing process\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eDuring the preparation of this work, the authors used ChatGPT (GPT-3.5), an AI language model developed by OpenAI, accessed via the free web version, in order to improve the readability and language of the manuscript. After using this tool/service, the authors reviewed and edited the content as needed and take full responsibility for the content of the publication.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding:\u0026nbsp;\u003c/strong\u003eThis study is financially supported by the Office of the Vice Chancellor for Research at Shiraz University of Medical Sciences (Grant No: 26854)\u003cstrong\u003e.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability statement:\u0026nbsp;\u003c/strong\u003eThe data that support the findings of this study are available from the corresponding author upon reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor's Contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eConceptualization: \u003cstrong\u003eEH, BA\u003c/strong\u003e; Data curation:\u0026nbsp;\u003cstrong\u003eAKR, MB, FJ, ER, AYN, SA MN, MB, MSS\u003c/strong\u003e; Analysis: \u003cstrong\u003eEH, MSS, MN\u003c/strong\u003e; Investigation:\u0026nbsp;\u003cstrong\u003eAKR, MB, FJ, MP, ER, SNR, AYN, SA MN, MB, MSS, EH, MH\u003c/strong\u003e; Project administration: \u003cstrong\u003eEH, BA, MSS\u003c/strong\u003e; Supervision: \u003cstrong\u003eEH, BA\u003c/strong\u003e; Writing - original draft:\u0026nbsp;\u003cstrong\u003eAKR, MB, FJ,\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eEH, MH\u003c/strong\u003e; Writing - review \u0026amp; editing:\u0026nbsp;\u003cstrong\u003eAKR, MB, FJ, MP, ER, SNR, AYN, SA MN, MB, MSS, MH, BA, EH.\u003c/strong\u003e\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eCardinali AEF, Martins CYA, Moraes RCM, Costa AP, Torr\u0026atilde;o AS (2025) Benfotiamine Ameliorates Streptozotocin-Induced Alzheimer\u0026rsquo;s Disease in Rats by Modulating Neuroinflammation, Oxidative Stress, and Microglia. 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J Neural Transm (Vienna) 125(12):1787\u0026ndash;1803. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1007/s00702-018-1928-7\u003c/span\u003e\u003cspan address=\"10.1007/s00702-018-1928-7\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[{"identity":"a3b6958d-cbb1-4f8e-ac87-d1ae8e002d5e","identifier":"10.13039/501100004320","name":"Shiraz University of Medical Sciences","awardNumber":"26854","order_by":0}],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":true,"highlight":"","institution":"Shiraz University of Medical Sciences","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Fingolimod, Alzheimer’s disease, Streptozotocin, Neuroinflammation, Oxidative stress, Memory","lastPublishedDoi":"10.21203/rs.3.rs-8382715/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8382715/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eBackground/Objective:\u003c/strong\u003e Fingolimod, an S1P receptor modulator, is neuroprotective and anti-inflammatory in animal models of neurodegenerative disorders; its optimal dosage regimen for cognitive dysfunction is yet to be defined. The impact of fingolimod on cognitive function, neuroinflammation, oxidative levels, as well as hippocampal morphology, was evaluated in the streptozotocin-induced model of memory dysfunction in rats.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMethods:\u003c/strong\u003e Male Sprague-Dawley rats were injected intracerebroventricularly with STZ (3 mg/kg) followed by daily i.p. fingolimod administration at 0.25, 0.5, or 1 mg/kg for 10 days. Behavioral function was evaluated using open-field tests, Morris water maze, and passive avoidance tasks. The hippocampal tissue was examined for cytokine and neurotrophic factor gene expression using qRT-PCR. malondialdehyde (MDA) levels, as a measure of lipid peroxidation, and stereological parameters, including\u003cstrong\u003e \u003c/strong\u003eCA1 neuronal counts and volumes, were evaluated.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults:\u003c/strong\u003e The STZ produced significant impairments in memory, increased pro-inflammatory cytokines (TNF-α, IL-1β, IL-6), elevated MDA levels, and CA1 neuronal loss and atrophy. Fingolimod at a dose of 0.25 mg/kg significantly ameliorated spatial and fear/memories, in addition to reducing neuroinflammation and oxidative stress. On the other hand, the dose of 0.5 mg/kg significantly restored both the number and volume of the CA1. The 1 mg/kg dose produced severe behavioral distress and was excluded.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusion:\u003c/strong\u003e Fingolimod has dose-dependent, domain-specific neuroprotective actions, promoting functional recovery at low doses and structural repair at middle to higher doses. These findings highlight a narrow therapeutic window as a key issue in the translation of S1P modulator therapies from cognitive impairment to Alzheimer’s disease.\u003c/p\u003e","manuscriptTitle":"Optimizing Fingolimod Dosing in STZ-Induced Cognitive Impairment: Divergent Behavioral and Histopathological Responses Reveal a Narrow Neuroprotective Window","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-12-18 09:13:54","doi":"10.21203/rs.3.rs-8382715/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"9b111c79-f6ce-4513-bb71-28f73af793d4","owner":[],"postedDate":"December 18th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":59796431,"name":"Cognitive Neuroscience"}],"tags":[],"updatedAt":"2025-12-18T09:13:55+00:00","versionOfRecord":[],"versionCreatedAt":"2025-12-18 09:13:54","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8382715","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8382715","identity":"rs-8382715","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}