SDF-1(5-67) Neutralizing Antibody Enhances Hippocampal Neurogenesis and Rescues Cognitive Deficits in Chronic Temporal Lobe Epilepsy

SDF-1(5-67) Neutralizing Antibody Enhances Hippocampal Neurogenesis and Rescues Cognitive Deficits in Chronic Temporal Lobe Epilepsy | 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 Article SDF-1(5-67) Neutralizing Antibody Enhances Hippocampal Neurogenesis and Rescues Cognitive Deficits in Chronic Temporal Lobe Epilepsy Xiang-Yu Ma, Li-Ping Zhao, Qiu-Yu Teng, Qi-Dong Tang, Qu Li, Xiao-Qian Zhang This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7463785/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 17 Dec, 2025 Read the published version in Scientific Reports → Version 1 posted 11 You are reading this latest preprint version Abstract Objective To investigate the therapeutic potential of SDF-1(5–67) neutralizing antibody (NAb) in a kainic acid (KA)-induced temporal lobe epilepsy (TLE) model. Methods TLE was induced in male Wistar rats by intraventricular KA injection. Animals were divided into acute (8 days) and chronic (72 days) phase groups, including acute phase control (SHAM-A), acute phase epilepsy model (EP-A), SDF-1(5–67) NAb-treated acute phase epilepsy (S + EP-A), chronic phase control (SHAM-C), chronic phase epilepsy model (EP-C), and SDF-1(5–67) NAb-treated chronic phase epilepsy groups (S + EP-C). NAb treatment was administered via osmotic pumps. Neurogenesis (DCX + cells), neuronal loss (Nissl staining), MFS (Timm staining), cognition (Morris water maze), and SDF-1(5–67) expression (western blot) were assessed. Results During the acute phase, increased dentate gyrus (DG) neurogenesis in EP-A vs. SHAM-A (210.43 ± 12.16 vs. 94.25 ± 7.76 cells/mm², P 0.05). In the chronic phase, EP-C showed suppressed neurogenesis (51.63 ± 7.36 vs. SHAM-C 85.12 ± 5.51, P < 0.01), which was rescued by NAb (77.56 ± 9.88, P < 0.01). CA3 neuronal loss was attenuated by NAb in EP-C (174.33 ± 13.06 vs. EP-C 127.47 ± 10.22, P < 0.05). MFS was increased in both phases and was unaffected by NAb. NAb improved cognitive deficits in EP-C (escape latency: 28.14 ± 1.6 s vs. EP-C 35.80 ± 2.41 s, P < 0.05). NAb suppressed SDF-1(5–67) expression in both phases ( P < 0.05). Conclusion SDF-1(5–67) NAb promoted neurogenesis and reduced neurodegeneration in chronic TLE, and improved cognitive function without affecting MFS or seizure duration. These findings highlight its potential for postepileptic cognitive rehabilitation. Health sciences/Diseases Health sciences/Neurology Biological sciences/Neuroscience Temporal Lobe Epilepsy SDF-1(5–67) NAb Neurogenesis Neuronal Apoptosis Cognitive Function Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 1. Introduction Temporal lobe epilepsy (TLE), which accounts for approximately 40% of all epilepsy cases, is the most prevalent form of refractory focal epilepsy, and cognitive impairment is one of its most debilitating comorbidities 1 . A growing body of evidence indicates that recurrent epileptic seizures trigger extensive neuronal degeneration and necrosis in the hippocampal formation, coupled with persistent neuroinflammation and reactive astrogliosis 2 , 3 . Additionally, the progressive decline in neurogenesis within the dentate gyrus (DG) subgranular zone has emerged as a critical pathological feature contributing to cognitive dysfunction in chronic TLE 4 . Animal studies have demonstrated a biphasic alteration in hippocampal neurogenesis following status epilepticus (SE): an initial transient surge in neural progenitor cell proliferation during the acute phase (1–2 weeks after SE), followed by a profound suppression of neurogenic capacity during the chronic phase (> 4 weeks after SE) when spontaneous recurrent seizures (SRS) develop 5 . This temporal pattern of neurogenesis impairment parallels the progression of cognitive deficits, suggesting a potential causal relationship. The chemokine stromal cell-derived factor-1 (SDF-1/CXCL12), primarily secreted by bone marrow stromal cells, plays a dual role in hippocampal neurogenesis. Under physiological conditions, SDF-1 binding to its cognate receptor CXCR4 activates G protein-coupled signaling pathways that promote neural precursor cell proliferation and differentiation 6 . SDF-1 expression is transiently upregulated in hippocampal neurons and glia during the acute phase of TLE 7 . Its levels remain elevated during the chronic phase despite the marked suppression of neurogenesis. This paradoxical observation prompted the present investigation. Recent studies have identified a critical pathological mechanism underlying this phenomenon: during chronic epilepsy, matrix metalloproteinases (MMPs, particularly MMP-9 and MMP-2) released by damaged neurons and activated glia proteolytically cleave full-length SDF-1 to generate a truncated isoform, SDF-1(5–67) 8 . Unlike its parent molecule, SDF-1(5–67) exhibits high neurotoxicity through selective binding to CXCR3 receptors, which triggers neuronal apoptosis and impairs neurogenesis 9 . This proteolytic conversion may explain the transition from enhanced neurogenesis in acute TLE to suppressed neurogenesis in chronic TLE. The present study aimed to investigate the therapeutic potential of SDF-1(5–67) neutralizing antibody (NAb) in a kainic acid (KA)-induced TLE model. . 2. Materials and method 2.1. Materials Kainic acid (KA): purchased from a commercial supplier. 5% isoflurane: prepared by the Department of Pharmacy, First Hospital of China Medical University. 10× phosphate buffered saline (PBS; pH 7.2–7.4), bovine serum albumin (BSA), Triton X-100, and horseradish peroxidase-conjugated secondary antibody: acquired from Beijing Solarbio Science & Technology Co., Ltd. (Beijing, China). Normal goat serum: purchased from Beijing Boster Biological Technology Co., Ltd. (Beijing, China). Blocking buffer: composed of 0.01 M PBS, 0.4% Triton X-100, and 5% normal goat serum. Dilution buffer: prepared with 0.01 M PBS, 0.4% Triton X-100, and 3% BSA. 4% Paraformaldehyde (PFA) solution: prepared by dissolving 4 g of PFA in 1× PBS, heating to 60°C with continuous magnetic stirring, adding a small amount of NaOH to clarify the solution, and adjusting the volume to 1000 mL. Antifreeze buffer: formulated with 30% ethylene glycol, 20% glycerol, and 50% 0.05 M PBS. Antifade Mounting Medium, Alexa Fluor 488 goat anti-guinea pig IgG: acquired from Invitrogen, USA. Guinea pig anti-DCX: purchased from Millipore, USA. SDF-1(5–67) antibody and SDF-1(5–67) neutralizing antibody (NAb): synthesized by GenScript, USA. 2.2. Methods 2.2.1. Experimental Animals and Ethics Statement Adult male Wistar rats (n = 90; weight 200–250 g) were obtained from the Experimental Animal Center of China Medical University. Animals were individually housed in standard polycarbonate cages under controlled environmental conditions (temperature 22 ± 1°C, humidity 50% ± 5%, 12 h light/dark cycle) with ad libitum access to food and water. All animal experiments were conducted in accordance with ARRIVE guidelines and approved by the Institutional Animal Care and Use Committee of China Medical University (Protocol No. SCXK [Liao] 2008-0005). 2.2.2. Experimental Design and Grouping Rats were randomly assigned to six experimental groups (n = 15/group): 1) SHAM-A: acute phase control (sacrificed at day 8), 2) EP-A: acute phase epilepsy model, 3) S + EP-A: SDF-1(5–67) NAb-treated acute phase epilepsy group, 4) SHAM-C: chronic phase control (sacrificed at day 72), 5) EP-C: chronic phase epilepsy model, 6) S + EP-C: SDF-1(5–67) NAb-treated chronic phase epilepsy group. 2.2.3. KA-Induced TLE Model Adult male Wistar rats (250–300g) underwent stereotaxic KA delivery under isoflurane anesthesia (5% induction, 2% maintenance). KA (0.5 µg/µL in 0.9% NaCl) was infused into the right lateral ventricle (bregma coordinates: AP − 3.8 mm, ML + 1.6 mm, DV − 1.8 mm) via Hamilton syringe (2 µL at 0.5 µL/min; needle retention, 5 min). This induced SE within 8.2 ± 1.5 min (Racine stage 4–5), which was terminated with diazepam (5 mg/kg intraperitoneal) at 4 h. Chronic epilepsy was validated by hippocampal sclerosis histology, SRS (≥ 2/week; 3.1 ± 0.7 events/day), and electrographic 5–7 Hz spike-wave complexes. For intracerebral drug delivery, osmotic minipumps (Alzet 2001D) were connected to CA1-implanted cannulae (AP − 3.8 mm, ML + 1.6 mm, DV − 1.8 mm from dura) via polyethylene tubing. Treatment groups received continuous SDF-1(5–67) NAb (10 µg/µL in artificial cerebral spinal fluid [aCSF]) at 0.5 µL/h for 7 days. 2.2.4. Intracerebral Drug Delivery Surgical Implantation Rats were anesthetized with isoflurane (5% induction, 1.5–2% maintenance) and then underwent stereotaxic implantation of a guide cannula targeting right hippocampal CA1 (coordinates from dura: AP − 0.8–1.0 mm, ML + 1.5 mm, DV − 3.5–4.0 mm). The cannula was connected to polyethylene tubing with osmotic minipumps (Alzet 2001D) primed with SDF-1(5–67) NAb (10 µg/µL in aCSF; GenScript) for the treatment groups or sterile aCSF (126 mM NaCl, 2.5 mM KCl, 26 mM NaHCO₃, pH 7.4) for control. The intervention pumps were implanted 24 h after SE for acute intervention and at 8 weeks after SE for chronic intervention, both delivering 5 µL/h for 7 days. Cannula placement was histologically verified postmortem. 2.2.5. Tissue Processing and Sectioning Perfusion Fixation At the designated endpoints (days 8 and 72), rats were deeply anesthetized with 5% isoflurane(confirmed by the absence of pedal reflex) and then euthanized by transcardial perfusion with 150 mL warm (37°C) heparinized saline (10 U/mL) at 30 mL/min, followed by 200 mL ice-cold 4% PFA in 0.1 M PBS (pH 7.4) at 10 mL/min for fixation. This method of euthanasia following surgical anesthesia is consistent with the AVMA Guidelines for the Euthanasia of Animals.Perfusion was terminated at the onset of rigor mortis in extremities (4–5 min total). Brains were postfixed in 4% PFA (4°C, 24 h), then cryoprotected in 30% sucrose in PBS (4°C) until tissue saturation (specific gravity > 1.15). Sucrose infiltration was confirmed by refractometry (Brix > 28%). Coronal sections (40 µm) containing the dorsal hippocampus (bregma − 2.8 to − 4.3 mm, Paxinos atlas) were cut on a cryostat at − 20°C. Sections were stored in cryoprotectant (30% ethylene glycol, 25% glycerol, 0.05 M PBS) at − 20°C. 2.2.6. Doublecortin (DCX) Immunofluorescence Staining Free-floating sections (40 µm) underwent DCX immunofluorescence staining. Briefly, the tissue was washed in PBS (three times for 10 min each) and blocked in 5% normal goat serum/0.4% Triton X-100/PBS (90 min). The sections were then incubated with guinea pig anti-DCX (1:400; Millipore AB2253) in 3% BSA/0.4% Triton X-100/PBS at 4°C for 48 h, followed by Alexa Fluor 594-conjugated secondary antibody (1:200; Invitrogen A-11076) for 2 h in the dark at room temperature. Sections were mounted with ProLong Gold antifade medium (Invitrogen P36934). 2.2.7. Image Processing and Analysis DCX-immunostained sections were visualized using fluorescence microscopy (100× magnification). For each animal, five consecutive hippocampal sections encompassing the entire DG were selected for analysis. High-resolution imaging of DCX⁺ cells within the DG was performed using laser scanning confocal microscopy. DCX⁺ cells were quantified in each field of view using NIH ImageJ software. The average density of DCX⁺ cells (cells/mm²) was calculated per animal. 2.2.8. Nissl Staining Tissue sections with a thickness of 10 µm were dewaxed in xylene (three times for 5 min each), rehydrated through a graded ethanol series (100%, 90%, 70% for 5 min, 2 min, 2 min, respectively), and rinsed in distilled water (2 min). Sections were stained with 0.1% cresyl violet (5 min), washed twice in distilled water, differentiated in 95% ethanol (5 s), and dehydrated in fresh 95% ethanol (twice for 2 min each). After clearing in xylene (twice for 5 min each), sections were mounted with neutral resin. Hippocampal CA3 neuronal loss was quantified using a Leuzex-F automated image analysis system, which assessed morphological parameters including neuronal mortality rate and cell density changes (cells/mm²). 2.2.9. Perfusion and Timm Staining Rats (n = 5/group) were anesthetized with 5% isoflurane and transcardially perfused sequentially with: (1) physiological saline for blood clearance, (2) sodium sulfide working solution (1.2% Na₂S·9H₂O, 1% NaH₂PO₄·H₂O) until limb graying and hepatic blackening occurred, and (3) 4% PFA for fixation. Brains were postfixed in 4% PFA at 4°C overnight, cryoprotected in 30% sucrose in PBS (0.1 M), and sectioned coronally at 30 µm. Sections were Timm-stained in the dark (room temperature, 90 min) using freshly prepared solution containing 50% gum arabic (120 mL), 2 M citrate buffer (20 mL), 5.3% hydroquinone (60 mL), 17% silver nitrate (1 mL). After graded ethanol dehydration and xylene clearing, sections were mounted with neutral resin. Mossy fiber sprouting (MFS) in hippocampal CA3 (pyramidal/oriens layers) was imaged under standardized illumination using an Olympus BX51 microscope. MFS severity was scored (0–5) based on observed granule distribution patterns using the following scale: 0, no granules in DG supragranular zone; 1, scattered granule clusters; 2, discontinuous granule bands; 3, continuous bands with focal plaques; 4, near-continuous dense laminar structures; 5, confluent high-density laminar bands. 2.2.10. EEG Recording and Analysis On day 71 after SE, rats were anesthetized with 3% isoflurane + 30% O 2 + 70% N 2 O for induction, and maintained at 1.5% isoflurane. Following stereotactic fixation, polyurethane-insulated stainless-steel electrodes (100 µm diameter) were implanted in the right DG hilus (coordinates relative to bregma: AP − 3.5 mm, ML + 2.0 mm, DV − 2.5 mm from dura; Paxinos & Watson atlas). Reference and ground electrodes (stainless-steel screws) were secured in the frontal and occipital bones. Continuous hippocampal electroencephalography (EEG) was recorded for 2 h under double-blind conditions. Spontaneous seizures were defined as rhythmic bursting discharges (≥ 0.3 s duration) with amplitude > 3× baseline theta-rhythm (4–8 Hz) during wakefulness. Seizure frequency (events/h) and mean duration (s/event) were quantified by two independent observers. 2.2.11. Water Maze Test Spatial cognition was assessed using the Morris water maze on days 68–70 after SE. Rats were tested in a black circular pool (diameter 150 cm, height 74 cm) filled to 54-cm depth with water (20 ± 1°C). A submerged escape platform (10 × 10 cm, 2 cm below the surface) and four equidistant entry points (N/S/E/W) were positioned within the pool. During the 3-day acquisition phase, each rat performed four daily trials in counterbalanced order; two trials were initiated from the distal entry point (maximal platform distance) and two from the proximal point, and the platform location was randomized daily. Trials were terminated once the platform was located or at 60 s (rats were guided to the platform for 10 s if they failed). On day 4, the platform was removed and a 60-s probe test was performed, during which the target quadrant crossings were quantified. Primary outcomes were escape latency, path length, and platform crossings, which were analyzed via automated video-tracking (EthoVision XT, Noldus). 2.1.12. Statistical Analysis All analyses were conducted in SPSS 26.0 (IBM). Parametric continuous data (expressed as mean ± standard error of the mean) were analyzed by one-way ANOVA for single time-point comparisons and repeated-measures ANOVA for longitudinal data (e.g., Morris water maze). Non-normally distributed data underwent nonparametric testing using the Kruskal–Wallis test (≥ 3 groups) with Dunn's post hoc Mann–Whitney U test (two-group comparisons). Statistical significance was defined as P < 0.05 after verifying normality (Shapiro–Wilk) and homogeneity of variance (Levene's test). 3. Results 3.1. Effects of SDF-1(5–67) NAb Treatment on Neurogenesis in the DG of Epileptic Rats Immunofluorescence quantification showed biphasic alterations in hippocampal neurogenesis following SE. In the acute phase (day 8 after EP), epileptic rats had a 2.2-fold increase in DG DCX⁺ cells compared with controls (210.43 ± 12.16 vs. 94.25 ± 7.76 cells/mm²; P < 0.05, t-test), indicating seizure-induced neurogenic activation. NAb treatment did not affect this acute response (195.84 ± 9.71 cells/mm²; P = 0.32 vs. EP). Conversely, chronic phase (day 72) EP animals showed significant neurogenic suppression compared with controls (51.63 ± 7.36 vs. 85.12 ± 5.51 cells/mm²; P < 0.01). Anti-SDF-1(5–67) intervention rescued neurogenesis (77.56 ± 9.88 cells/mm²; P < 0.01 vs. untreated EP), demonstrating phase-dependent effects (Fig. 1 ). 3.2. Effects of SDF-1(5–67) NAb Treatment on Neuronal Loss in the Hippocampus of Epileptic Rats Nissl staining quantification showed progressive CA3 pyramidal neuron loss in epileptic rats. Acute phase rats showed a 24.3% neuronal reduction compared with controls (180.05 ± 14.07 vs. 238.00 ± 11.83 cells/mm²; P < 0.05, ANOVA). Anti-SDF-1(5–67) treatment did not mitigate the acute degeneration (189.20 ± 13.78 cells/mm²; P = 0.18 vs. EP). Chronic phase rats had exacerbated neuron loss compared with controls (42.0% decrease; 127.47 ± 10.22 vs. 219.85 ± 18.24 cells/mm²; P < 0.01). SDF-1(5–67) neutralization ameliorated the neuronal loss (36.7% increase; 174.33 ± 13.06 cells/mm²; P < 0.05 vs. untreated EP), demonstrating selective neuroprotection in chronic epilepsy (Fig. 2 ). 3.3. Effects of SDF-1(5–67) NAb Treatment on MFS in Epileptic Rats Timm staining indicated progressive MFS in hippocampal CA3. Acute phase rats had 3.3-fold higher MFS scores than those of controls (1.59 ± 0.28 vs. 0.48 ± 0.09; P < 0.01, ANOVA). Anti-SDF-1(5–67) treatment did not alter acute MFS pathology (1.70 ± 0.38; P = 0.42 vs. SE). Chronic phase rats had exacerbated sprouting compared with controls (4.9-fold increase; 2.68 ± 0.40 vs. 0.55 ± 0.08; P < 0.001), which was unaffected by the NAb (2.45 ± 0.35; P = 0.17). These results demonstrated SDF-1(5–67) neutralization did not alter synaptic reorganization during either disease phase, which suggests that pathway-selective actions were uncoupled from axonal plasticity (Fig. 3 ) 3.4. Effects of SDF-1(5–67) NAb Treatment on Spontaneous Seizures in Chronic TLE Continuous EEG monitoring showed no significant antiseizure effects of SDF-1(5–67) neutralization in chronic TLE rats. The antibody-treated group had seizure durations that were comparable to untreated epileptic controls (307.11 ± 8.30 s vs. 288.36 ± 6.59 s; P = 0.24, t-test), despite reduced neurodegeneration. This suggests dissociation between neuroprotection and seizure modulation, confirming that SDF-1(5–67) signaling selectively regulates neuronal survival without impacting ictogenesis (Fig. 4 ). 3.5. Effects of SDF-1(5–67) NAb on Cognitive Function in the Chronic Phase of TLE Rats Morris water maze testing demonstrated significant cognitive impairment in chronic TLE rats compared with the findings in controls. The chronic TLE rats had a prolonged escape latency (35.80 ± 2.41 vs. 29.67 ± 1.80 s; P < 0.05), increased path length (8.19 ± 0.72 vs. 5.94 ± 0.82 m; P < 0.05), and reduced platform crossings (1.48 ± 0.31 vs. 4.01 ± 0.59; P < 0.05), compared with controls. Anti-SDF-1(5–67) treatment reversed these deficits. Escape latency decreased by 21.4% (28.14 ± 1.60 s; P < 0.05 vs. TLE), path length shortened by 25.2% (6.13 ± 0.64 m; P < 0.05), and crossings increased 97.3% (2.92 ± 0.48; P < 0.05), indicating cognitive rescue in chronic epilepsy (Fig. 5 ). 3.6. SDF-1(5–67) in TLE Models: NAb Inhibition of SDF-1 Protein Expression in the Hippocampus Western blot analysis indicated phase-dependent modulation of hippocampal SDF-1(5–67) proteolysis. Acute-phase SDF-1(5–67) expression was unchanged compared with that of controls ( P > 0.05), and neutralizing antibodies significantly reduced its levels ( P < 0.05), suggesting constitutive suppression capacity. Chronic-phase rats had a 130% increase ( P < 0.05). Antibody intervention reversed this increase (58% reduction, P < 0.05), demonstrating dual-phase target engagement with therapeutic implications for proteotoxicity mitigation. 4. Discussion Our data demonstrate that SDF-1(5–67) NAb rescues chronic-phase neurogenesis and cognition without altering MFS or seizure duration, supporting its role in mitigating proteotoxicity but not circuit reorganization. Animal experiments and clinical postoperative pathological studies have both shown that the core pathological features of TLE include widespread neuronal degeneration and necrosis in the hippocampus, neuroinflammatory responses, astrocyte proliferation, and MFS. These pathological changes can directly lead to cognitive dysfunction in patients 3 . In recent years, studies have identified abnormal neurogenesis in the DG of the hippocampus as another characteristic pathological change of TLE 10 – 12 . Immature neurons may play an important role in the repair of hippocampal damage after epileptic seizures and in the recovery of learning and memory functions. Studies have shown that neurogenesis in the DG of TLE shows dynamic changes during the acute and chronic phases. During the acute phase, there is a transient increase in the proliferation of neural precursor cells, and then, with the entry into the chronic phase following SRS, the proliferation of neural precursor cells is significantly inhibited. Because the maintenance of hippocampal-dependent learning and memory functions relies on continuous neurogenesis, the reduced neurogenic capacity of the DG may be an important mechanism in hippocampal-dependent cognitive dysfunction in TLE patients. Moreover, rats with more SRS episodes have a greater decrease in the number of newborn neurons and worse cognitive function 13 – 15 . In this study, on day 8 after modeling (acute phase of TLE), we used immunofluorescence staining and confocal imaging to show that the number of newborn neurons in the DG of TLE rats was higher than that of the control group. Nissl staining showed that neurons in the hippocampal CA3 region of epileptic rats underwent degeneration and death. On day 72 after modeling (chronic phase of TLE), the number of newborn neurons in the DG of TLE rats was significantly reduced compared with that in the control group, and the degeneration and death of neurons in the hippocampal CA3 region of epileptic rats had worsened compared with those in the acute phase. Moreover, during the chronic phase, the Morris water maze test indicated a significant decline in the cognitive function of epileptic rats. These results are consistent with previous reports 16 , 17 . Previous studies have shown that SDF-1 has a role in promoting neural recovery after epilepsy 18 , 19 . SDF-1 is a CXC chemokine produced by bone marrow stromal cells. Under physiological conditions, SDF-1 and its specific receptor CXCR4 are highly expressed in the DG region. When SDF-1 binds to the CXCR4 receptor, the G protein connected to CXCR4 is activated and further activates downstream signaling pathways, which causes changes in the cytoskeleton, leads to the migration and adhesion of neural stem cells, and regulates the proliferation and differentiation of neural stem cells. After brain injury such as epilepsy, SDF-1, mainly released by dead or damaged neurons, activated glial cells, and endothelial cells, attracts neural stem cells to migrate to areas of neuronal degeneration, death, and inflammation by binding to the CXCR4 receptor, and promotes the proliferation and differentiation of neural precursor cells 18 – 22 . In our previous work, we found that neurogenesis in the DG decreased significantly 1 month after epileptic seizures (chronic phase) compared with that in the control group, despite similar levels of hippocampal SDF-1 23 . It has been reported that 24 h after epileptic seizures, glial cells and neurons in the hippocampal region of rats begin to release SDF-1, and the elevated SDF-1 is associated with cell proliferation and migration 24 . During the acute phase of epilepsy, neurogenesis in the hippocampus is transiently enhanced 24 . With the increase in SDF-1 concentration in the chronic phase of epilepsy, dead and damaged neurons, activated glial cells, and endothelial cells release MMPs 25 . Under certain pathological conditions, MMPs (mainly MMP-9 and MMP-2) remove four amino acids from the N-terminus of the SDF-1 molecule through proteolysis, cleaving it into SDF-1(5–67). SDF-1(5–67) is highly neurotoxic and specifically binds to CXCR3, leading to neuronal degeneration and apoptosis 26 , 27 . This may be one of the reasons why neurogenesis is only transiently enhanced shortly after TLE but is significantly reduced during the chronic phase of epilepsy. In the present study, we injected SDF-1(5–67) NAb into the brains of rats in the acute and chronic phases of TLE to reduce SDF-1(5–67) protein levels in the hippocampal region and observe whether it has a positive effect on neurogenesis in the DG, neuronal degeneration and death, and cognitive function. In the acute phase of TLE, there was no significant difference in SDF-1(5–67) protein expression between the epileptic group and the control group. After the injection of SDF-1(5–67) NAb, SDF-1(5–67) protein expression was significantly reduced. In the chronic phase, SDF-1(5–67) protein expression in the epileptic group was higher than that in the control group, and SDF-1(5–67) protein expression was significantly reduced after the injection of SDF-1(5–67) NAb. These findings indicate that the SDF-1(5–67) NAb has an inhibitory effect on SDF-1(5–67) protein expression in the TLE model. Moreover, after the injection of SDF-1(5–67) NAb, there were no significant changes in neurogenesis in the DG and neuronal degeneration and apoptosis in the hippocampus of TLE rats in the acute phase. This may be because in the acute phase of epilepsy, the amount of SDF-1 cleaved into SDF-1(5–67) is very small. Western blot showed that the protein content of SDF-1(5–67) was very low in both the control group and the acute phase epileptic group. Therefore, even with the application of SDF-1(5–67) NAb, there was no significant effect on hippocampal neurogenesis and neuronal apoptosis, and no statistical difference was found. However, in the chronic phase of TLE, after rats had recurrent SRS, the protein level of SDF-1(5–67) in the hippocampus was significantly increased. During this phase, after the application of SDF-1(5–67) NAb, we found that neurogenesis in the DG and neuronal degeneration and apoptosis in the hippocampus of epileptic rats were significantly improved, and the cognitive function of epileptic rats was also improved to some extent. Therefore, the results suggest that SDF-1(5–67) NAb treatment improves neurogenesis and cognitive function in the chronic phase of TLE rats. We also investigated the status of MFS in epileptic rats. In normal rats, hippocampal granule cells project mossy fibers through the dentate hilus to the CA3 region of the hippocampus, forming synapses with pyramidal cells. However, in the epileptic rat model, the massive death of neurons in the hippocampal hilus and CA3 pyramidal cells leads to denervation in the inner molecular layer (IML) of the DG, and the granule cells undergo axonal misdirection due to the destruction of their projection targets. Against this pathological backdrop, MFS occurs via the sprouting of collaterals from the mossy fibers into the granule cell layer, IML, and the stratum oriens of CA3, forming aberrant synaptic circuits. This synaptic reorganization results in an imbalance between excitation and inhibition in the hippocampal neural network, ultimately inducing SRS during the chronic phase of epilepsy. Hippocampal slice experiments have confirmed that MFS exacerbates seizure activity 28 , 29 . In the present study, we showed that MFS was increased in both the acute and chronic epilepsy phase groups compared with that in the control groups. Treatment with SDF-1(5–67) NAb did not significantly improve MFS in epileptic rats. This may be related to the mechanism by which SDF-1(5–67) exerts toxic effects on neuronal death and neurogenesis but has no harmful effects on mossy fibers 30 , 31 . Consistent with these findings, EEG recordings in epileptic rats showed that treatment with SDF-1(5–67) NAb had no positive effect on epileptic spike wave activity in the rats. In summary, we investigated the effects of SDF-1(5–67) NAb treatment on hippocampal neurogenesis, neuronal death, MFS, cognitive function, and EEG seizure activity in the acute and chronic phases of a KA-induced TLE rat model. The findings indicated that SDF-1(5–67) NAb treatment enhanced hippocampal neurogenesis and reduced neuronal degeneration and apoptosis in the chronic phase of TLE, and thereby improved cognitive deficits in spatial learning in rats. These findings help elucidate the potential mechanisms underlying cognitive dysfunction and neurogenesis inhibition in TLE rats at the cellular and molecular levels. The findings also provide important theoretical evidence for early prevention and rehabilitation treatment strategies for postepileptic cognitive impairment. However, the specific molecular pathways and key regulatory nodes involved need to be further verified through genetic intervention, pharmacological regulation, and other experimental approaches to clarify the causal relationships and provide a reliable scientific basis for clinical translation. Declarations Conflict of interest The authors declare that they have no conflict of interest. Acknowledgments We thank, Xiao-Qian-Zhang PhD, Department of Neurology, First Affiliated Hospital of China Medical University, for her invaluable guidance and contributions to this paper. Author contributions XYM, LPZ, QYT, XQZ and QL were involved in conception of the project. XQZ and QYT were involved in design of the study. XYM, LPZ and QYT were involved in acquisition of data.QYT and QL performed imaging preprocessing. XYM, LPZ and QYT analyzed the data.XYM, LPZ, QYT and QL interpreted the data and prepared the manuscript. All the authors reviewed, edited the manuscript and were involved in subsequent revisions. Ethical Approval All animal experiments were conducted in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals. The study protocol was reviewed and approved by the Animal Ethics Committee of China Medical University (Approval Number: CMU20240496). All experiments were performed in the SPF Laboratory Animal Center of China Medical University, which is accredited by the Laboratory Animal Use License (Number: SYXK (Liao) 2022-0007). This article does not contain any studies with human participants. Funding This work was funded by Science and Technology Joint Project, Liaoning Province (Grant No.2023JH2/101700076). Data availability The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request. Clinical Trial Registration Not applicable. Consent to Participate Not applicable. 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LRRTM3 regulates activity-dependent synchronization of synapse properties in topographically connected hippocampal neural circuits. Proc Natl Acad Sci U S A 119 (2022). https://doi.org/10.1073/pnas.2110196119 Additional Declarations No competing interests reported. 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1","display":"","copyAsset":false,"role":"figure","size":1213388,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEffects of SDF-1(5-67) neutralizing antibody (NAb) on the proliferation of newborn neurons in epileptic rats. \u003c/strong\u003e(A–B) Confocal microscopy images of doublecortin (DCX)-positive cells in the dentate gyrus of the hippocampus from rats in each experimental group. (C–D) Mean number of DCX-positive cells in the dentate gyrus of the hippocampus for each group. *\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.05 vs SHAM, #\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.05 vs EP. n = 5 per group. SHAM-A, acute phase controlgroup; EP-A, acute phase epilepsy modelgroup; S+EP-A, SDF-1(5-67) NAb-treated acute phase epilepsy group; SHAM-C, chronic phase controlgroup; EP-C, chronic phase epilepsy modelgroup; S+EP-C, SDF-1(5-67) NAb-treated chronic phase epilepsy group.\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-7463785/v1/6af86767dbe4f557bde045d2.png"},{"id":93051313,"identity":"3c7bbbe7-04af-445e-beec-155d032f256a","added_by":"auto","created_at":"2025-10-08 14:18:09","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":1228528,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe effect of SDF-1(5-67) NAb on hippocampal neuronal loss. \u003c/strong\u003e(A–B) The differences in Nissl-stained cells in the hippocampus of rats from each group were observed. (C) The number of Nissl-stained cells. (*\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.05 vs SHAM; #\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.05 vs EP. Scale bars, 100 μm; n = 5 per group). SHAM-A, acute phase controlgroup; EP-A, acute phase epilepsy modelgroup; \u0026nbsp;S+EP-A, SDF-1(5-67) NAb-treated acute phase epilepsy group; SHAM-C, chronic phase control group; EP-C, chronic phase epilepsy modelgroup; S+EP-C, SDF-1(5-67) NAb-treated chronic phase epilepsy group.\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-7463785/v1/979e93bb38ba08be1ad111c4.png"},{"id":93051311,"identity":"04cbbd24-3e53-4dc0-9aec-f4a5816d46ec","added_by":"auto","created_at":"2025-10-08 14:18:09","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":1254485,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTimm staining in the hippocampal CA3 region of rats from different groups. \u003c/strong\u003eCompared with that of the control group, the Timm score in the CA3 region was reduced at 8 and 72 days after SE (*\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.05 vs SHAM). No significant difference in Timm score was observed between the kainic acid (KA) and SDF-1(5-67) NAb groups (\u003cem\u003eP \u003c/em\u003e\u0026gt; 0.05 vs KA). Scale bar, 100 μm; n = 5 per group. SHAM-A, acute phase control group; EP-A, acute phase epilepsy modelgroup; S+EP-A, SDF-1(5-67) NAb-treated acute phase epilepsy group; SHAM-C, chronic phase controlgroup; EP-C, chronic phase epilepsy modelgroup; S+EP-C, SDF-1(5-67) NAb-treated chronic phase epilepsy group.\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-7463785/v1/f514eac9f9f10efd731124e1.png"},{"id":93053212,"identity":"e93ad2c9-0dd4-4235-927e-26857873c63b","added_by":"auto","created_at":"2025-10-08 14:26:09","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":114409,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSpontaneous EEG seizures in the dentate gyrus region during the chronic epilepsy phase. \u003c/strong\u003e(A) Spontaneous electroencephalography (EEG) seizures in the chronic epilepsy group. (B) Spontaneous EEG seizures in the chronic epilepsy group treated with SDF-1(5-67) NAb. (C) Average duration of spontaneous EEG seizures. EP-C, chronic phase epilepsy modelgroup; S+EP-C, SDF-1(5-67) NAb-treated chronic phase epilepsy group.\u003c/p\u003e","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-7463785/v1/352f054bae92f25e6e1d5634.png"},{"id":93051316,"identity":"059de038-760d-4bf8-9f4b-a05547842b4a","added_by":"auto","created_at":"2025-10-08 14:18:09","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":273176,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePerformance of rats from different groups in the Morris water maze. \u003c/strong\u003e(A) Place navigation test of rats in each group. (B) Spatial exploration test of rats in each group. (C) Distance traveled before locating the platform of rats in each group. (D) Escape latency of rats in each group. (E) Changes in escape latency of rats in each group. *\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.05 vs. SHAM; #\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.05 vs. EP; n = 10 per group. SHAM-C, chronic phase control group; EP-C, chronic phase epilepsy modelgroup; S+EP-C, SDF-1(5-67) NAb-treated chronic phase epilepsy group.\u003c/p\u003e","description":"","filename":"floatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-7463785/v1/4286a6d3af3769240f083fbe.png"},{"id":93053210,"identity":"b6eaaf40-1779-4235-9e3f-5fc52f106607","added_by":"auto","created_at":"2025-10-08 14:26:09","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":178901,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eExpression of SDF-1(5-67) in the hippocampus of rats from different groups. \u003c/strong\u003e*\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.05 vs. SHAM; #\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.05 vs. EP; n = 5 per group. SHAM-A, acute phase control group; EP-A, acute phase epilepsy modelgroup; \u0026nbsp;S+EP-A, SDF-1(5-67) NAb-treated acute phase epilepsy group; SHAM-C, chronic phase control group; EP-C, chronic phase epilepsy modelgroup; S+EP-C, SDF-1(5-67) NAb-treated chronic phase epilepsy group.\u003c/p\u003e","description":"","filename":"floatimage6.png","url":"https://assets-eu.researchsquare.com/files/rs-7463785/v1/7d5d1d0dd0bb088194e2c32c.png"},{"id":98814368,"identity":"c6b27659-174f-4b90-ad99-b70c1e01f2a6","added_by":"auto","created_at":"2025-12-22 16:12:26","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":5926294,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7463785/v1/f97716c6-745a-4e89-9eda-8d528ae9375d.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"SDF-1(5-67) Neutralizing Antibody Enhances Hippocampal Neurogenesis and Rescues Cognitive Deficits in Chronic Temporal Lobe Epilepsy","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eTemporal lobe epilepsy (TLE), which accounts for approximately 40% of all epilepsy cases, is the most prevalent form of refractory focal epilepsy, and cognitive impairment is one of its most debilitating comorbidities\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eA growing body of evidence indicates that recurrent epileptic seizures trigger extensive neuronal degeneration and necrosis in the hippocampal formation, coupled with persistent neuroinflammation and reactive astrogliosis\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e,\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e. Additionally, the progressive decline in neurogenesis within the dentate gyrus (DG) subgranular zone has emerged as a critical pathological feature contributing to cognitive dysfunction in chronic TLE\u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e. Animal studies have demonstrated a biphasic alteration in hippocampal neurogenesis following status epilepticus (SE): an initial transient surge in neural progenitor cell proliferation during the acute phase (1\u0026ndash;2 weeks after SE), followed by a profound suppression of neurogenic capacity during the chronic phase (\u0026gt;\u0026thinsp;4 weeks after SE) when spontaneous recurrent seizures (SRS) develop\u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e. This temporal pattern of neurogenesis impairment parallels the progression of cognitive deficits, suggesting a potential causal relationship.\u003c/p\u003e\u003cp\u003eThe chemokine stromal cell-derived factor-1 (SDF-1/CXCL12), primarily secreted by bone marrow stromal cells, plays a dual role in hippocampal neurogenesis. Under physiological conditions, SDF-1 binding to its cognate receptor CXCR4 activates G protein-coupled signaling pathways that promote neural precursor cell proliferation and differentiation\u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e. SDF-1 expression is transiently upregulated in hippocampal neurons and glia during the acute phase of TLE\u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e. Its levels remain elevated during the chronic phase despite the marked suppression of neurogenesis. This paradoxical observation prompted the present investigation.\u003c/p\u003e\u003cp\u003eRecent studies have identified a critical pathological mechanism underlying this phenomenon: during chronic epilepsy, matrix metalloproteinases (MMPs, particularly MMP-9 and MMP-2) released by damaged neurons and activated glia proteolytically cleave full-length SDF-1 to generate a truncated isoform, SDF-1(5\u0026ndash;67)\u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e. Unlike its parent molecule, SDF-1(5\u0026ndash;67) exhibits high neurotoxicity through selective binding to CXCR3 receptors, which triggers neuronal apoptosis and impairs neurogenesis\u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e. This proteolytic conversion may explain the transition from enhanced neurogenesis in acute TLE to suppressed neurogenesis in chronic TLE. The present study aimed to investigate the therapeutic potential of SDF-1(5\u0026ndash;67) neutralizing antibody (NAb) in a kainic acid (KA)-induced TLE model.\u003c/p\u003e\u003cp\u003e.\u003c/p\u003e"},{"header":"2. Materials and method","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003e2.1. Materials\u003c/h2\u003e\u003cp\u003eKainic acid (KA): purchased from a commercial supplier. 5% isoflurane: prepared by the Department of Pharmacy, First Hospital of China Medical University. 10\u0026times; phosphate buffered saline (PBS; pH 7.2\u0026ndash;7.4), bovine serum albumin (BSA), Triton X-100, and horseradish peroxidase-conjugated secondary antibody: acquired from Beijing Solarbio Science \u0026amp; Technology Co., Ltd. (Beijing, China). Normal goat serum: purchased from Beijing Boster Biological Technology Co., Ltd. (Beijing, China). Blocking buffer: composed of 0.01 M PBS, 0.4% Triton X-100, and 5% normal goat serum. Dilution buffer: prepared with 0.01 M PBS, 0.4% Triton X-100, and 3% BSA. 4% Paraformaldehyde (PFA) solution: prepared by dissolving 4 g of PFA in 1\u0026times; PBS, heating to 60\u0026deg;C with continuous magnetic stirring, adding a small amount of NaOH to clarify the solution, and adjusting the volume to 1000 mL. Antifreeze buffer: formulated with 30% ethylene glycol, 20% glycerol, and 50% 0.05 M PBS. Antifade Mounting Medium, Alexa Fluor 488 goat anti-guinea pig IgG: acquired from Invitrogen, USA. Guinea pig anti-DCX: purchased from Millipore, USA. SDF-1(5\u0026ndash;67) antibody and SDF-1(5\u0026ndash;67) neutralizing antibody (NAb): synthesized by GenScript, USA.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec4\" class=\"Section2\"\u003e\u003ch2\u003e2.2. Methods\u003c/h2\u003e\u003cdiv id=\"Sec5\" class=\"Section3\"\u003e\u003ch2\u003e2.2.1. Experimental Animals and Ethics Statement\u003c/h2\u003e\u003cp\u003eAdult male Wistar rats (n\u0026thinsp;=\u0026thinsp;90; weight 200\u0026ndash;250 g) were obtained from the Experimental Animal Center of China Medical University. Animals were individually housed in standard polycarbonate cages under controlled environmental conditions (temperature 22\u0026thinsp;\u0026plusmn;\u0026thinsp;1\u0026deg;C, humidity 50% \u0026plusmn; 5%, 12 h light/dark cycle) with ad libitum access to food and water.\u003c/p\u003e\u003cp\u003e All animal experiments were conducted in accordance with ARRIVE guidelines and approved by the Institutional Animal Care and Use Committee of China Medical University (Protocol No. SCXK [Liao] 2008-0005).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec6\" class=\"Section3\"\u003e\u003ch2\u003e2.2.2. Experimental Design and Grouping\u003c/h2\u003e\u003cp\u003eRats were randomly assigned to six experimental groups (n\u0026thinsp;=\u0026thinsp;15/group):\u003c/p\u003e\u003cp\u003e1) SHAM-A: acute phase control (sacrificed at day 8), 2) EP-A: acute phase epilepsy model, 3) S\u0026thinsp;+\u0026thinsp;EP-A: SDF-1(5\u0026ndash;67) NAb-treated acute phase epilepsy group, 4) SHAM-C: chronic phase control (sacrificed at day 72), 5) EP-C: chronic phase epilepsy model, 6) S\u0026thinsp;+\u0026thinsp;EP-C: SDF-1(5\u0026ndash;67) NAb-treated chronic phase epilepsy group.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec7\" class=\"Section3\"\u003e\u003ch2\u003e2.2.3. KA-Induced TLE Model\u003c/h2\u003e\u003cp\u003eAdult male Wistar rats (250\u0026ndash;300g) underwent stereotaxic KA delivery under isoflurane anesthesia (5% induction, 2% maintenance). KA (0.5 \u0026micro;g/\u0026micro;L in 0.9% NaCl) was infused into the right lateral ventricle (bregma coordinates: AP \u0026minus;\u0026thinsp;3.8 mm, ML\u0026thinsp;+\u0026thinsp;1.6 mm, DV \u0026minus;\u0026thinsp;1.8 mm) via Hamilton syringe (2 \u0026micro;L at 0.5 \u0026micro;L/min; needle retention, 5 min). This induced SE within 8.2\u0026thinsp;\u0026plusmn;\u0026thinsp;1.5 min (Racine stage 4\u0026ndash;5), which was terminated with diazepam (5 mg/kg intraperitoneal) at 4 h. Chronic epilepsy was validated by hippocampal sclerosis histology, SRS (\u0026ge;\u0026thinsp;2/week; 3.1\u0026thinsp;\u0026plusmn;\u0026thinsp;0.7 events/day), and electrographic 5\u0026ndash;7 Hz spike-wave complexes. For intracerebral drug delivery, osmotic minipumps (Alzet 2001D) were connected to CA1-implanted cannulae (AP \u0026minus;\u0026thinsp;3.8 mm, ML\u0026thinsp;+\u0026thinsp;1.6 mm, DV \u0026minus;\u0026thinsp;1.8 mm from dura) via polyethylene tubing. Treatment groups received continuous SDF-1(5\u0026ndash;67) NAb (10 \u0026micro;g/\u0026micro;L in artificial cerebral spinal fluid [aCSF]) at 0.5 \u0026micro;L/h for 7 days.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec8\" class=\"Section3\"\u003e\u003ch2\u003e2.2.4. Intracerebral Drug Delivery\u003c/h2\u003e\u003cp\u003e\u003cb\u003eSurgical Implantation\u003c/b\u003e\u003c/p\u003e\u003cp\u003eRats were anesthetized with isoflurane (5% induction, 1.5\u0026ndash;2% maintenance) and then underwent stereotaxic implantation of a guide cannula targeting right hippocampal CA1 (coordinates from dura: AP \u0026minus;\u0026thinsp;0.8\u0026ndash;1.0 mm, ML\u0026thinsp;+\u0026thinsp;1.5 mm, DV \u0026minus;\u0026thinsp;3.5\u0026ndash;4.0 mm). The cannula was connected to polyethylene tubing with osmotic minipumps (Alzet 2001D) primed with SDF-1(5\u0026ndash;67) NAb (10 \u0026micro;g/\u0026micro;L in aCSF; GenScript) for the treatment groups or sterile aCSF (126 mM NaCl, 2.5 mM KCl, 26 mM NaHCO₃, pH 7.4) for control. The intervention pumps were implanted 24 h after SE for acute intervention and at 8 weeks after SE for chronic intervention, both delivering 5 \u0026micro;L/h for 7 days. Cannula placement was histologically verified postmortem.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec9\" class=\"Section3\"\u003e\u003ch2\u003e2.2.5. Tissue Processing and Sectioning\u003c/h2\u003e\u003cp\u003e\u003cb\u003ePerfusion Fixation\u003c/b\u003e\u003c/p\u003e\u003cp\u003eAt the designated endpoints (days 8 and 72), rats were deeply anesthetized with 5% isoflurane(confirmed by the absence of pedal reflex) and then euthanized by transcardial perfusion with 150 mL warm (37\u0026deg;C) heparinized saline (10 U/mL) at 30 mL/min, followed by 200 mL ice-cold 4% PFA in 0.1 M PBS (pH 7.4) at 10 mL/min for fixation. This method of euthanasia following surgical anesthesia is consistent with the AVMA Guidelines for the Euthanasia of Animals.Perfusion was terminated at the onset of rigor mortis in extremities (4\u0026ndash;5 min total). Brains were postfixed in 4% PFA (4\u0026deg;C, 24 h), then cryoprotected in 30% sucrose in PBS (4\u0026deg;C) until tissue saturation (specific gravity\u0026thinsp;\u0026gt;\u0026thinsp;1.15). Sucrose infiltration was confirmed by refractometry (Brix\u0026thinsp;\u0026gt;\u0026thinsp;28%). Coronal sections (40 \u0026micro;m) containing the dorsal hippocampus (bregma \u0026minus;\u0026thinsp;2.8 to \u0026minus;\u0026thinsp;4.3 mm, Paxinos atlas) were cut on a cryostat at \u0026minus;\u0026thinsp;20\u0026deg;C. Sections were stored in cryoprotectant (30% ethylene glycol, 25% glycerol, 0.05 M PBS) at \u0026minus;\u0026thinsp;20\u0026deg;C.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec10\" class=\"Section3\"\u003e\u003ch2\u003e2.2.6. Doublecortin (DCX) Immunofluorescence Staining\u003c/h2\u003e\u003cp\u003eFree-floating sections (40 \u0026micro;m) underwent DCX immunofluorescence staining. Briefly, the tissue was washed in PBS (three times for 10 min each) and blocked in 5% normal goat serum/0.4% Triton X-100/PBS (90 min). The sections were then incubated with guinea pig anti-DCX (1:400; Millipore AB2253) in 3% BSA/0.4% Triton X-100/PBS at 4\u0026deg;C for 48 h, followed by Alexa Fluor 594-conjugated secondary antibody (1:200; Invitrogen A-11076) for 2 h in the dark at room temperature. Sections were mounted with ProLong Gold antifade medium (Invitrogen P36934).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec11\" class=\"Section3\"\u003e\u003ch2\u003e2.2.7. Image Processing and Analysis\u003c/h2\u003e\u003cp\u003eDCX-immunostained sections were visualized using fluorescence microscopy (100\u0026times; magnification). For each animal, five consecutive hippocampal sections encompassing the entire DG were selected for analysis. High-resolution imaging of DCX⁺ cells within the DG was performed using laser scanning confocal microscopy. DCX⁺ cells were quantified in each field of view using NIH ImageJ software. The average density of DCX⁺ cells (cells/mm\u0026sup2;) was calculated per animal.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec12\" class=\"Section3\"\u003e\u003ch2\u003e2.2.8. Nissl Staining\u003c/h2\u003e\u003cp\u003eTissue sections with a thickness of 10 \u0026micro;m were dewaxed in xylene (three times for 5 min each), rehydrated through a graded ethanol series (100%, 90%, 70% for 5 min, 2 min, 2 min, respectively), and rinsed in distilled water (2 min). Sections were stained with 0.1% cresyl violet (5 min), washed twice in distilled water, differentiated in 95% ethanol (5 s), and dehydrated in fresh 95% ethanol (twice for 2 min each). After clearing in xylene (twice for 5 min each), sections were mounted with neutral resin. Hippocampal CA3 neuronal loss was quantified using a Leuzex-F automated image analysis system, which assessed morphological parameters including neuronal mortality rate and cell density changes (cells/mm\u0026sup2;).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec13\" class=\"Section3\"\u003e\u003ch2\u003e2.2.9. Perfusion and Timm Staining\u003c/h2\u003e\u003cp\u003eRats (n\u0026thinsp;=\u0026thinsp;5/group) were anesthetized with 5% isoflurane and transcardially perfused sequentially with: (1) physiological saline for blood clearance, (2) sodium sulfide working solution (1.2% Na₂S\u0026middot;9H₂O, 1% NaH₂PO₄\u0026middot;H₂O) until limb graying and hepatic blackening occurred, and (3) 4% PFA for fixation. Brains were postfixed in 4% PFA at 4\u0026deg;C overnight, cryoprotected in 30% sucrose in PBS (0.1 M), and sectioned coronally at 30 \u0026micro;m. Sections were Timm-stained in the dark (room temperature, 90 min) using freshly prepared solution containing 50% gum arabic (120 mL), 2 M citrate buffer (20 mL), 5.3% hydroquinone (60 mL), 17% silver nitrate (1 mL). After graded ethanol dehydration and xylene clearing, sections were mounted with neutral resin. Mossy fiber sprouting (MFS) in hippocampal CA3 (pyramidal/oriens layers) was imaged under standardized illumination using an Olympus BX51 microscope. MFS severity was scored (0\u0026ndash;5) based on observed granule distribution patterns using the following scale: 0, no granules in DG supragranular zone; 1, scattered granule clusters; 2, discontinuous granule bands; 3, continuous bands with focal plaques; 4, near-continuous dense laminar structures; 5, confluent high-density laminar bands.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec14\" class=\"Section3\"\u003e\u003ch2\u003e2.2.10. EEG Recording and Analysis\u003c/h2\u003e\u003cp\u003eOn day 71 after SE, rats were anesthetized with 3% isoflurane\u0026thinsp;+\u0026thinsp;30% O\u003csub\u003e2\u003c/sub\u003e\u0026thinsp;+\u0026thinsp;70% N\u003csub\u003e2\u003c/sub\u003eO for induction, and maintained at 1.5% isoflurane. Following stereotactic fixation, polyurethane-insulated stainless-steel electrodes (100 \u0026micro;m diameter) were implanted in the right DG hilus (coordinates relative to bregma: AP \u0026minus;\u0026thinsp;3.5 mm, ML\u0026thinsp;+\u0026thinsp;2.0 mm, DV \u0026minus;\u0026thinsp;2.5 mm from dura; Paxinos \u0026amp; Watson atlas). Reference and ground electrodes (stainless-steel screws) were secured in the frontal and occipital bones.\u003c/p\u003e\u003cp\u003eContinuous hippocampal electroencephalography (EEG) was recorded for 2 h under double-blind conditions. Spontaneous seizures were defined as rhythmic bursting discharges (\u0026ge;\u0026thinsp;0.3 s duration) with amplitude\u0026thinsp;\u0026gt;\u0026thinsp;3\u0026times; baseline theta-rhythm (4\u0026ndash;8 Hz) during wakefulness. Seizure frequency (events/h) and mean duration (s/event) were quantified by two independent observers.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec15\" class=\"Section3\"\u003e\u003ch2\u003e2.2.11. Water Maze Test\u003c/h2\u003e\u003cp\u003eSpatial cognition was assessed using the Morris water maze on days 68\u0026ndash;70 after SE. Rats were tested in a black circular pool (diameter 150 cm, height 74 cm) filled to 54-cm depth with water (20\u0026thinsp;\u0026plusmn;\u0026thinsp;1\u0026deg;C). A submerged escape platform (10 \u0026times; 10 cm, 2 cm below the surface) and four equidistant entry points (N/S/E/W) were positioned within the pool. During the 3-day acquisition phase, each rat performed four daily trials in counterbalanced order; two trials were initiated from the distal entry point (maximal platform distance) and two from the proximal point, and the platform location was randomized daily. Trials were terminated once the platform was located or at 60 s (rats were guided to the platform for 10 s if they failed). On day 4, the platform was removed and a 60-s probe test was performed, during which the target quadrant crossings were quantified. Primary outcomes were escape latency, path length, and platform crossings, which were analyzed via automated video-tracking (EthoVision XT, Noldus).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec16\" class=\"Section3\"\u003e\u003ch2\u003e2.1.12. Statistical Analysis\u003c/h2\u003e\u003cp\u003eAll analyses were conducted in SPSS 26.0 (IBM). Parametric continuous data (expressed as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard error of the mean) were analyzed by one-way ANOVA for single time-point comparisons and repeated-measures ANOVA for longitudinal data (e.g., Morris water maze). Non-normally distributed data underwent nonparametric testing using the Kruskal\u0026ndash;Wallis test (\u0026ge;\u0026thinsp;3 groups) with Dunn's post hoc Mann\u0026ndash;Whitney U test (two-group comparisons). Statistical significance was defined as \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05 after verifying normality (Shapiro\u0026ndash;Wilk) and homogeneity of variance (Levene's test).\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e"},{"header":"3. Results","content":"\u003cdiv id=\"Sec18\" class=\"Section2\"\u003e\u003ch2\u003e3.1. Effects of SDF-1(5\u0026ndash;67) NAb Treatment on Neurogenesis in the DG of Epileptic Rats\u003c/h2\u003e\u003cp\u003eImmunofluorescence quantification showed biphasic alterations in hippocampal neurogenesis following SE. In the acute phase (day 8 after EP), epileptic rats had a 2.2-fold increase in DG DCX⁺ cells compared with controls (210.43\u0026thinsp;\u0026plusmn;\u0026thinsp;12.16 vs. 94.25\u0026thinsp;\u0026plusmn;\u0026thinsp;7.76 cells/mm\u0026sup2;; \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05, t-test), indicating seizure-induced neurogenic activation. NAb treatment did not affect this acute response (195.84\u0026thinsp;\u0026plusmn;\u0026thinsp;9.71 cells/mm\u0026sup2;; \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.32 vs. EP). Conversely, chronic phase (day 72) EP animals showed significant neurogenic suppression compared with controls (51.63\u0026thinsp;\u0026plusmn;\u0026thinsp;7.36 vs. 85.12\u0026thinsp;\u0026plusmn;\u0026thinsp;5.51 cells/mm\u0026sup2;; \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01). Anti-SDF-1(5\u0026ndash;67) intervention rescued neurogenesis (77.56\u0026thinsp;\u0026plusmn;\u0026thinsp;9.88 cells/mm\u0026sup2;; \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01 vs. untreated EP), demonstrating phase-dependent effects (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec19\" class=\"Section2\"\u003e\u003ch2\u003e3.2. Effects of SDF-1(5\u0026ndash;67) NAb Treatment on Neuronal Loss in the Hippocampus of Epileptic Rats\u003c/h2\u003e\u003cp\u003eNissl staining quantification showed progressive CA3 pyramidal neuron loss in epileptic rats. Acute phase rats showed a 24.3% neuronal reduction compared with controls (180.05\u0026thinsp;\u0026plusmn;\u0026thinsp;14.07 vs. 238.00\u0026thinsp;\u0026plusmn;\u0026thinsp;11.83 cells/mm\u0026sup2;; \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05, ANOVA). Anti-SDF-1(5\u0026ndash;67) treatment did not mitigate the acute degeneration (189.20\u0026thinsp;\u0026plusmn;\u0026thinsp;13.78 cells/mm\u0026sup2;; \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.18 vs. EP). Chronic phase rats had exacerbated neuron loss compared with controls (42.0% decrease; 127.47\u0026thinsp;\u0026plusmn;\u0026thinsp;10.22 vs. 219.85\u0026thinsp;\u0026plusmn;\u0026thinsp;18.24 cells/mm\u0026sup2;; \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01). SDF-1(5\u0026ndash;67) neutralization ameliorated the neuronal loss (36.7% increase; 174.33\u0026thinsp;\u0026plusmn;\u0026thinsp;13.06 cells/mm\u0026sup2;; \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05 vs. untreated EP), demonstrating selective neuroprotection in chronic epilepsy (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec20\" class=\"Section2\"\u003e\u003ch2\u003e3.3. Effects of SDF-1(5\u0026ndash;67) NAb Treatment on MFS in Epileptic Rats\u003c/h2\u003e\u003cp\u003eTimm staining indicated progressive MFS in hippocampal CA3. Acute phase rats had 3.3-fold higher MFS scores than those of controls (1.59\u0026thinsp;\u0026plusmn;\u0026thinsp;0.28 vs. 0.48\u0026thinsp;\u0026plusmn;\u0026thinsp;0.09; \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01, ANOVA). Anti-SDF-1(5\u0026ndash;67) treatment did not alter acute MFS pathology (1.70\u0026thinsp;\u0026plusmn;\u0026thinsp;0.38; \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.42 vs. SE). Chronic phase rats had exacerbated sprouting compared with controls (4.9-fold increase; 2.68\u0026thinsp;\u0026plusmn;\u0026thinsp;0.40 vs. 0.55\u0026thinsp;\u0026plusmn;\u0026thinsp;0.08; P\u0026thinsp;\u0026lt;\u0026thinsp;0.001), which was unaffected by the NAb (2.45\u0026thinsp;\u0026plusmn;\u0026thinsp;0.35; \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.17). These results demonstrated SDF-1(5\u0026ndash;67) neutralization did not alter synaptic reorganization during either disease phase, which suggests that pathway-selective actions were uncoupled from axonal plasticity (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e)\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec21\" class=\"Section2\"\u003e\u003ch2\u003e3.4. Effects of SDF-1(5\u0026ndash;67) NAb Treatment on Spontaneous Seizures in Chronic TLE\u003c/h2\u003e\u003cp\u003eContinuous EEG monitoring showed no significant antiseizure effects of SDF-1(5\u0026ndash;67) neutralization in chronic TLE rats. The antibody-treated group had seizure durations that were comparable to untreated epileptic controls (307.11\u0026thinsp;\u0026plusmn;\u0026thinsp;8.30 s vs. 288.36\u0026thinsp;\u0026plusmn;\u0026thinsp;6.59 s; \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.24, t-test), despite reduced neurodegeneration. This suggests dissociation between neuroprotection and seizure modulation, confirming that SDF-1(5\u0026ndash;67) signaling selectively regulates neuronal survival without impacting ictogenesis (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec22\" class=\"Section2\"\u003e\u003ch2\u003e3.5. Effects of SDF-1(5\u0026ndash;67) NAb on Cognitive Function in the Chronic Phase of TLE Rats\u003c/h2\u003e\u003cp\u003eMorris water maze testing demonstrated significant cognitive impairment in chronic TLE rats compared with the findings in controls. The chronic TLE rats had a prolonged escape latency (35.80\u0026thinsp;\u0026plusmn;\u0026thinsp;2.41 vs. 29.67\u0026thinsp;\u0026plusmn;\u0026thinsp;1.80 s; \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05), increased path length (8.19\u0026thinsp;\u0026plusmn;\u0026thinsp;0.72 vs. 5.94\u0026thinsp;\u0026plusmn;\u0026thinsp;0.82 m; \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05), and reduced platform crossings (1.48\u0026thinsp;\u0026plusmn;\u0026thinsp;0.31 vs. 4.01\u0026thinsp;\u0026plusmn;\u0026thinsp;0.59; P\u0026thinsp;\u0026lt;\u0026thinsp;0.05), compared with controls. Anti-SDF-1(5\u0026ndash;67) treatment reversed these deficits. Escape latency decreased by 21.4% (28.14\u0026thinsp;\u0026plusmn;\u0026thinsp;1.60 s; P\u0026thinsp;\u0026lt;\u0026thinsp;0.05 vs. TLE), path length shortened by 25.2% (6.13\u0026thinsp;\u0026plusmn;\u0026thinsp;0.64 m; \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05), and crossings increased 97.3% (2.92\u0026thinsp;\u0026plusmn;\u0026thinsp;0.48; \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05), indicating cognitive rescue in chronic epilepsy (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec23\" class=\"Section2\"\u003e\u003ch2\u003e3.6. SDF-1(5\u0026ndash;67) in TLE Models: NAb Inhibition of SDF-1 Protein Expression in the Hippocampus\u003c/h2\u003e\u003cp\u003eWestern blot analysis indicated phase-dependent modulation of hippocampal SDF-1(5\u0026ndash;67) proteolysis. Acute-phase SDF-1(5\u0026ndash;67) expression was unchanged compared with that of controls (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026gt;\u0026thinsp;0.05), and neutralizing antibodies significantly reduced its levels (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05), suggesting constitutive suppression capacity. Chronic-phase rats had a 130% increase (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Antibody intervention reversed this increase (58% reduction, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05), demonstrating dual-phase target engagement with therapeutic implications for proteotoxicity mitigation.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e"},{"header":"4. Discussion","content":"\u003cp\u003eOur data demonstrate that SDF-1(5\u0026ndash;67) NAb rescues chronic-phase neurogenesis and cognition without altering MFS or seizure duration, supporting its role in mitigating proteotoxicity but not circuit reorganization. Animal experiments and clinical postoperative pathological studies have both shown that the core pathological features of TLE include widespread neuronal degeneration and necrosis in the hippocampus, neuroinflammatory responses, astrocyte proliferation, and MFS. These pathological changes can directly lead to cognitive dysfunction in patients\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e. In recent years, studies have identified abnormal neurogenesis in the DG of the hippocampus as another characteristic pathological change of TLE\u003csup\u003e\u003cspan additionalcitationids=\"CR11\" citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e. Immature neurons may play an important role in the repair of hippocampal damage after epileptic seizures and in the recovery of learning and memory functions. Studies have shown that neurogenesis in the DG of TLE shows dynamic changes during the acute and chronic phases. During the acute phase, there is a transient increase in the proliferation of neural precursor cells, and then, with the entry into the chronic phase following SRS, the proliferation of neural precursor cells is significantly inhibited. Because the maintenance of hippocampal-dependent learning and memory functions relies on continuous neurogenesis, the reduced neurogenic capacity of the DG may be an important mechanism in hippocampal-dependent cognitive dysfunction in TLE patients. Moreover, rats with more SRS episodes have a greater decrease in the number of newborn neurons and worse cognitive function\u003csup\u003e\u003cspan additionalcitationids=\"CR14\" citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eIn this study, on day 8 after modeling (acute phase of TLE), we used immunofluorescence staining and confocal imaging to show that the number of newborn neurons in the DG of TLE rats was higher than that of the control group. Nissl staining showed that neurons in the hippocampal CA3 region of epileptic rats underwent degeneration and death. On day 72 after modeling (chronic phase of TLE), the number of newborn neurons in the DG of TLE rats was significantly reduced compared with that in the control group, and the degeneration and death of neurons in the hippocampal CA3 region of epileptic rats had worsened compared with those in the acute phase. Moreover, during the chronic phase, the Morris water maze test indicated a significant decline in the cognitive function of epileptic rats. These results are consistent with previous reports\u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e,\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003ePrevious studies have shown that SDF-1 has a role in promoting neural recovery after epilepsy\u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e,\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e. SDF-1 is a CXC chemokine produced by bone marrow stromal cells. Under physiological conditions, SDF-1 and its specific receptor CXCR4 are highly expressed in the DG region. When SDF-1 binds to the CXCR4 receptor, the G protein connected to CXCR4 is activated and further activates downstream signaling pathways, which causes changes in the cytoskeleton, leads to the migration and adhesion of neural stem cells, and regulates the proliferation and differentiation of neural stem cells. After brain injury such as epilepsy, SDF-1, mainly released by dead or damaged neurons, activated glial cells, and endothelial cells, attracts neural stem cells to migrate to areas of neuronal degeneration, death, and inflammation by binding to the CXCR4 receptor, and promotes the proliferation and differentiation of neural precursor cells\u003csup\u003e\u003cspan additionalcitationids=\"CR19 CR20 CR21\" citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eIn our previous work, we found that neurogenesis in the DG decreased significantly 1 month after epileptic seizures (chronic phase) compared with that in the control group, despite similar levels of hippocampal SDF-1\u003csup\u003e23\u003c/sup\u003e. It has been reported that 24 h after epileptic seizures, glial cells and neurons in the hippocampal region of rats begin to release SDF-1, and the elevated SDF-1 is associated with cell proliferation and migration\u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e. During the acute phase of epilepsy, neurogenesis in the hippocampus is transiently enhanced\u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e. With the increase in SDF-1 concentration in the chronic phase of epilepsy, dead and damaged neurons, activated glial cells, and endothelial cells release MMPs\u003csup\u003e\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e. Under certain pathological conditions, MMPs (mainly MMP-9 and MMP-2) remove four amino acids from the N-terminus of the SDF-1 molecule through proteolysis, cleaving it into SDF-1(5\u0026ndash;67). SDF-1(5\u0026ndash;67) is highly neurotoxic and specifically binds to CXCR3, leading to neuronal degeneration and apoptosis\u003csup\u003e\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e,\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e. This may be one of the reasons why neurogenesis is only transiently enhanced shortly after TLE but is significantly reduced during the chronic phase of epilepsy.\u003c/p\u003e\u003cp\u003eIn the present study, we injected SDF-1(5\u0026ndash;67) NAb into the brains of rats in the acute and chronic phases of TLE to reduce SDF-1(5\u0026ndash;67) protein levels in the hippocampal region and observe whether it has a positive effect on neurogenesis in the DG, neuronal degeneration and death, and cognitive function. In the acute phase of TLE, there was no significant difference in SDF-1(5\u0026ndash;67) protein expression between the epileptic group and the control group. After the injection of SDF-1(5\u0026ndash;67) NAb, SDF-1(5\u0026ndash;67) protein expression was significantly reduced. In the chronic phase, SDF-1(5\u0026ndash;67) protein expression in the epileptic group was higher than that in the control group, and SDF-1(5\u0026ndash;67) protein expression was significantly reduced after the injection of SDF-1(5\u0026ndash;67) NAb. These findings indicate that the SDF-1(5\u0026ndash;67) NAb has an inhibitory effect on SDF-1(5\u0026ndash;67) protein expression in the TLE model. Moreover, after the injection of SDF-1(5\u0026ndash;67) NAb, there were no significant changes in neurogenesis in the DG and neuronal degeneration and apoptosis in the hippocampus of TLE rats in the acute phase. This may be because in the acute phase of epilepsy, the amount of SDF-1 cleaved into SDF-1(5\u0026ndash;67) is very small. Western blot showed that the protein content of SDF-1(5\u0026ndash;67) was very low in both the control group and the acute phase epileptic group. Therefore, even with the application of SDF-1(5\u0026ndash;67) NAb, there was no significant effect on hippocampal neurogenesis and neuronal apoptosis, and no statistical difference was found. However, in the chronic phase of TLE, after rats had recurrent SRS, the protein level of SDF-1(5\u0026ndash;67) in the hippocampus was significantly increased. During this phase, after the application of SDF-1(5\u0026ndash;67) NAb, we found that neurogenesis in the DG and neuronal degeneration and apoptosis in the hippocampus of epileptic rats were significantly improved, and the cognitive function of epileptic rats was also improved to some extent. Therefore, the results suggest that SDF-1(5\u0026ndash;67) NAb treatment improves neurogenesis and cognitive function in the chronic phase of TLE rats.\u003c/p\u003e\u003cp\u003eWe also investigated the status of MFS in epileptic rats. In normal rats, hippocampal granule cells project mossy fibers through the dentate hilus to the CA3 region of the hippocampus, forming synapses with pyramidal cells. However, in the epileptic rat model, the massive death of neurons in the hippocampal hilus and CA3 pyramidal cells leads to denervation in the inner molecular layer (IML) of the DG, and the granule cells undergo axonal misdirection due to the destruction of their projection targets. Against this pathological backdrop, MFS occurs via the sprouting of collaterals from the mossy fibers into the granule cell layer, IML, and the stratum oriens of CA3, forming aberrant synaptic circuits. This synaptic reorganization results in an imbalance between excitation and inhibition in the hippocampal neural network, ultimately inducing SRS during the chronic phase of epilepsy. Hippocampal slice experiments have confirmed that MFS exacerbates seizure activity\u003csup\u003e\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e,\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e. In the present study, we showed that MFS was increased in both the acute and chronic epilepsy phase groups compared with that in the control groups. Treatment with SDF-1(5\u0026ndash;67) NAb did not significantly improve MFS in epileptic rats. This may be related to the mechanism by which SDF-1(5\u0026ndash;67) exerts toxic effects on neuronal death and neurogenesis but has no harmful effects on mossy fibers\u003csup\u003e\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e,\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/sup\u003e. Consistent with these findings, EEG recordings in epileptic rats showed that treatment with SDF-1(5\u0026ndash;67) NAb had no positive effect on epileptic spike wave activity in the rats.\u003c/p\u003e\u003cp\u003eIn summary, we investigated the effects of SDF-1(5\u0026ndash;67) NAb treatment on hippocampal neurogenesis, neuronal death, MFS, cognitive function, and EEG seizure activity in the acute and chronic phases of a KA-induced TLE rat model. The findings indicated that SDF-1(5\u0026ndash;67) NAb treatment enhanced hippocampal neurogenesis and reduced neuronal degeneration and apoptosis in the chronic phase of TLE, and thereby improved cognitive deficits in spatial learning in rats. These findings help elucidate the potential mechanisms underlying cognitive dysfunction and neurogenesis inhibition in TLE rats at the cellular and molecular levels. The findings also provide important theoretical evidence for early prevention and rehabilitation treatment strategies for postepileptic cognitive impairment. However, the specific molecular pathways and key regulatory nodes involved need to be further verified through genetic intervention, pharmacological regulation, and other experimental approaches to clarify the causal relationships and provide a reliable scientific basis for clinical translation.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eConflict of interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no conflict of interest.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe thank, Xiao-Qian-Zhang PhD, Department of Neurology, First Affiliated Hospital of China Medical University, for her invaluable guidance and contributions to this paper.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eXYM,\u0026nbsp;LPZ,\u0026nbsp;QYT,\u0026nbsp;XQZ\u0026nbsp;and\u0026nbsp;QL\u0026nbsp;were involved in conception of the project.\u0026nbsp;XQZ and\u0026nbsp;QYT\u0026nbsp;were involved in design of the study.\u0026nbsp;XYM,\u0026nbsp;LPZ\u0026nbsp;and\u0026nbsp;QYT\u0026nbsp;were involved in acquisition of data.QYT\u0026nbsp;and\u0026nbsp;QL\u0026nbsp;performed imaging preprocessing.\u0026nbsp;XYM,\u0026nbsp;LPZ and QYT\u0026nbsp;analyzed the data.XYM,\u0026nbsp;LPZ,\u0026nbsp;QYT\u0026nbsp;and\u0026nbsp;QL\u0026nbsp;interpreted the data and prepared the manuscript. All the authors reviewed, edited the manuscript and were involved in subsequent revisions.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthical Approval\u003c/strong\u003e\u003cbr\u003e\u0026nbsp;All animal experiments were conducted in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals. The study protocol was reviewed and approved by the\u0026nbsp;Animal Ethics Committee of China Medical University (Approval Number: CMU20240496). All experiments were performed in the SPF Laboratory Animal Center of China Medical University, which is accredited by the Laboratory Animal Use License (Number: SYXK (Liao) 2022-0007). This article does not contain any studies with human participants.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis \u0026nbsp;work \u0026nbsp; was \u0026nbsp;funded \u0026nbsp;by Science and Technology Joint Project, Liaoning Province (Grant No.2023JH2/101700076).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe datasets used and/or analysed during the current study are available from the corresponding author on reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eClinical Trial Registration\u003c/strong\u003e\u003cbr\u003e\u0026nbsp;Not applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent to Participate\u003cbr\u003e\u003c/strong\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent to Publish\u003cbr\u003e\u003c/strong\u003eNot applicable.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003eAllone, C.\u003cem\u003e\u0026nbsp;et al.\u003c/em\u003e Neuroimaging and cognitive functions in temporal lobe epilepsy: A review of the literature. \u003cem\u003eJ Neurol Sci\u003c/em\u003e\u003cstrong\u003e381\u003c/strong\u003e, 7-15 (2017). https://doi.org/10.1016/j.jns.2017.08.007\u003c/li\u003e\n \u003cli\u003eEnglot, D. 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Integrity of Cajal-Retzius cells in the reeler-mouse hippocampus. \u003cem\u003eHippocampus\u003c/em\u003e\u003cstrong\u003e29\u003c/strong\u003e, 550-565 (2019). https://doi.org/10.1002/hipo.23049\u003c/li\u003e\n \u003cli\u003eSong, C.\u003cem\u003e\u0026nbsp;et al.\u003c/em\u003e CXCR4 Antagonist AMD3100 Suppresses the Long-Term Abnormal Structural Changes of Newborn Neurons in the Intraventricular Kainic Acid Model of Epilepsy. \u003cem\u003eMol Neurobiol\u003c/em\u003e\u003cstrong\u003e53\u003c/strong\u003e, 1518-1532 (2016). https://doi.org/10.1007/s12035-015-9102-9\u003c/li\u003e\n \u003cli\u003eMeyer, P., Grandgirard, D., Lehner, M., Haenggi, M. \u0026amp; Leib, S. L. 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W.\u003cem\u003e\u0026nbsp;et al.\u003c/em\u003e Neurotrophin-3 from the dentate gyrus supports postsynaptic sites of mossy fiber-CA3 synapses and hippocampus-dependent cognitive functions. \u003cem\u003eMol Psychiatry\u003c/em\u003e\u003cstrong\u003e29\u003c/strong\u003e, 1192-1204 (2024). https://doi.org/10.1038/s41380-023-02404-5\u003c/li\u003e\n \u003cli\u003eKim, J.\u003cem\u003e\u0026nbsp;et al.\u003c/em\u003e LRRTM3 regulates activity-dependent synchronization of synapse properties in topographically connected hippocampal neural circuits. \u003cem\u003eProc Natl Acad Sci U S A\u003c/em\u003e\u003cstrong\u003e119\u003c/strong\u003e (2022). https://doi.org/10.1073/pnas.2110196119\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Temporal Lobe Epilepsy, SDF-1(5–67) NAb, Neurogenesis, Neuronal Apoptosis, Cognitive Function","lastPublishedDoi":"10.21203/rs.3.rs-7463785/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7463785/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eObjective\u003c/h2\u003e\u003cp\u003eTo investigate the therapeutic potential of SDF-1(5\u0026ndash;67) neutralizing antibody (NAb) in a kainic acid (KA)-induced temporal lobe epilepsy (TLE) model.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e\u003cp\u003eTLE was induced in male Wistar rats by intraventricular KA injection. Animals were divided into acute (8 days) and chronic (72 days) phase groups, including acute phase control (SHAM-A), acute phase epilepsy model (EP-A), SDF-1(5\u0026ndash;67) NAb-treated acute phase epilepsy (S\u0026thinsp;+\u0026thinsp;EP-A), chronic phase control (SHAM-C), chronic phase epilepsy model (EP-C), and SDF-1(5\u0026ndash;67) NAb-treated chronic phase epilepsy groups (S\u0026thinsp;+\u0026thinsp;EP-C). NAb treatment was administered via osmotic pumps. Neurogenesis (DCX\u0026thinsp;+\u0026thinsp;cells), neuronal loss (Nissl staining), MFS (Timm staining), cognition (Morris water maze), and SDF-1(5\u0026ndash;67) expression (western blot) were assessed.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e\u003cp\u003eDuring the acute phase, increased dentate gyrus (DG) neurogenesis in EP-A vs. SHAM-A (210.43\u0026thinsp;\u0026plusmn;\u0026thinsp;12.16 vs. 94.25\u0026thinsp;\u0026plusmn;\u0026thinsp;7.76 cells/mm\u0026sup2;, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) was unaffected by NAb (195.84\u0026thinsp;\u0026plusmn;\u0026thinsp;9.71, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026gt;\u0026thinsp;0.05). In the chronic phase, EP-C showed suppressed neurogenesis (51.63\u0026thinsp;\u0026plusmn;\u0026thinsp;7.36 vs. SHAM-C 85.12\u0026thinsp;\u0026plusmn;\u0026thinsp;5.51, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01), which was rescued by NAb (77.56\u0026thinsp;\u0026plusmn;\u0026thinsp;9.88, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01). CA3 neuronal loss was attenuated by NAb in EP-C (174.33\u0026thinsp;\u0026plusmn;\u0026thinsp;13.06 vs. EP-C 127.47\u0026thinsp;\u0026plusmn;\u0026thinsp;10.22, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). MFS was increased in both phases and was unaffected by NAb. NAb improved cognitive deficits in EP-C (escape latency: 28.14\u0026thinsp;\u0026plusmn;\u0026thinsp;1.6 s vs. EP-C 35.80\u0026thinsp;\u0026plusmn;\u0026thinsp;2.41 s, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). NAb suppressed SDF-1(5\u0026ndash;67) expression in both phases (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05).\u003c/p\u003e\u003ch2\u003eConclusion\u003c/h2\u003e\u003cp\u003eSDF-1(5\u0026ndash;67) NAb promoted neurogenesis and reduced neurodegeneration in chronic TLE, and improved cognitive function without affecting MFS or seizure duration. These findings highlight its potential for postepileptic cognitive rehabilitation.\u003c/p\u003e","manuscriptTitle":"SDF-1(5-67) Neutralizing Antibody Enhances Hippocampal Neurogenesis and Rescues Cognitive Deficits in Chronic Temporal Lobe Epilepsy","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-10-08 14:18:04","doi":"10.21203/rs.3.rs-7463785/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-10-15T15:42:29+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-10-11T15:06:38+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-09-30T09:08:58+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"179096474261301574250559958757945362685","date":"2025-09-25T15:25:05+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"224913257650240759791627536063180465570","date":"2025-09-25T13:53:25+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"93319943048623194340464608880568592797","date":"2025-09-25T13:25:50+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-09-25T13:00:07+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-09-25T11:14:58+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2025-09-04T19:56:26+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-09-01T14:40:28+00:00","index":"","fulltext":""},{"type":"submitted","content":"Scientific Reports","date":"2025-09-01T14:36:28+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"8b5bcfa4-0f4c-4bc2-936b-1179013322b9","owner":[],"postedDate":"October 8th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":55565072,"name":"Health sciences/Diseases"},{"id":55565073,"name":"Health sciences/Neurology"},{"id":55565074,"name":"Biological sciences/Neuroscience"}],"tags":[],"updatedAt":"2025-12-22T16:08:13+00:00","versionOfRecord":{"articleIdentity":"rs-7463785","link":"https://doi.org/10.1038/s41598-025-32689-1","journal":{"identity":"scientific-reports","isVorOnly":false,"title":"Scientific Reports"},"publishedOn":"2025-12-17 15:57:40","publishedOnDateReadable":"December 17th, 2025"},"versionCreatedAt":"2025-10-08 14:18:04","video":"","vorDoi":"10.1038/s41598-025-32689-1","vorDoiUrl":"https://doi.org/10.1038/s41598-025-32689-1","workflowStages":[]},"version":"v1","identity":"rs-7463785","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7463785","identity":"rs-7463785","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}