Abstract
Background/Aim: Insulin-like growth factor-1 (IGF-1) is a neuroprotective growth factor widely expressed in the brain. Prior studies have shown that IGF-1 and phosphorylated protein kinase B (pAKT) are downregulated in the hippocampus of a bilateral common carotid artery (BCCAO) model. However, the relationship between AKT, ERK, and BDNF expression in this BCCAO model remains unclear. In particular, the effects of IGF-1 on pAKT and phosphorylated extracellular signal-related protein kinase (pERK) in the BCCAO model are not understood. In the present study, we investigated the effect of IGF-1 stereotaxic injection on pAKT and pERK in a BCCAO model.
Materials and Methods: To create the BCCAO model, the carotid arteries of rats were exposed and occluded. Three hours post-occlusion, IGF-1 was injected into BCCAO rats using a stereotaxic injector. Western blotting was conducted on the cerebral cortex and hippocampal tissues collected three days after BCCAO.
Results: Three days after BCCAO model induction, BDNF expression was increased in the cortex and hippocampus. There was a significant difference in cortex and hippocampal pAKT/AKT and pERK/ERK expression between the BCCAO and IGF-1-treated group.
Conclusion: Stereotaxic injection of IGF-1 in rats with BCCAO enhances BDNF expression in the cortex and hippocampus 3 days post-surgery. Further, this treatment simultaneously increased pAKT and pERK levels in both regions.
- Brain-derived neurotrophic factor
- bilateral common carotid artery occlusion
- extracellular signal-related protein kinase
- protein kinase B
Introduction
Insulin-like growth factor-1 (IGF-1) is a neuroprotective growth factor widely expressed in the brain (1). In a neonatal hypoxic-ischemic brain model, IGF-1 inhibited neuronal cell apoptosis and improved neuronal cell survival by activating phosphorylated protein kinase B (pAKT) signaling (2). In a previous study, IGF-1 induced neuronal survival in the cortex of a periventricular leukomalacia model (3). In a bilateral common carotid artery occlusion model (BCCAO), hippocampal IGF-1 was decreased, while salvianolic acid B ameliorated cognitive deficits through the IGF-1/AKT pathway (4). The BCCAO model has been widely used to study cognitive impairment and the development of dementia conditions, including vascular dementia (VD) (5). In our prior studies, we investigated the expression of neuroprotective and neurogenic factors in the BCCAO model (6-8).
Prior research has shown that IGF-1 and p-AKT were downregulated in the hippocampus in the BCCAO model (9). Ling-Ling et al. reported that treatment with neurotropin activated AKT/GSK3β pathway activity and upregulated BDNF expression in BCCAO rats (10). The AKT/CREB/BDNF signaling pathway promotes neuronal survival and exerts anti-apoptotic effects (11). Extracellular signal-related protein kinase (ERK), activated by BDNF, reduces apoptosis and is related to neuronal survival, similar to the AKT/BDNF pathway (12). McCusker et al. previously showed that IGF-I enhances the expression of BDNF and induces ERK1/2 phosphorylation (pERK 1/2) (13). However, the relationship between AKT, ERK, and BDNF expression in the BCCAO model remains unclear. In particular, the effects of IGF-1 on pAKT and pERK in the BCCAO model remain poorly understood.
In the present study, we used the previously described stereotaxic injection method (8) to study the effects of IGF-1 on pAKT and pERK in BCCAO model rats.
Materials and Methods
BCCAO surgery. The animal experimental design is provided through a study flowchart (Figure 1). Male Sprague–Dawley (SD) rats (aged 7-8 weeks old) were obtained from a certified breeder (Damul Laboratory Animals, Daejeon, Republic of Korea). All animal experiments were approved by the Institutional Animal Care and Use Committee of Chosun University. BCCAO was induced as previously described (14). In brief, rats were anesthetized with inhaled sevoflurane (1.0%-2.0%, end-tidal concentration). The skin of the neck was dissected medially, and both of the common carotid arteries were exposed. The exposed arteries were occluded by 4-0 silk sutures. Three hours after surgery, IGF-1 was injected into BCCAO rats using a stereotaxic injector for 5 min. The stereotaxic position of the injection was 1.0 mm posterior and 1.0 mm lateral to the bregma, and the depth from the skull surface was 3.0 mm. The concentration of IGF-1 (SRP3069, Sigma, St. Louis, MO, USA) injection was 0.1 μg/μl, and the injected volume was 5 μl, as in other IGF-1 studies (15). The rats were divided into three groups: control group, including rats that did not undergo surgery; the BCCAO group, comprising rats that underwent surgery; and the IGF-1-treated group, comprising rats that underwent surgery and were treated with IGF-1.
Timeline of the experimental procedures. BCCAO: Bilateral common carotid artery occlusion; BDNF: brain-derived neurotrophic factor; pAKT: phosphorylated protein kinase B; pERK: phosphorylated extracellular signal-related protein kinase.
Western blot analysis. All Sprague-Dawley rats (n=13) were anesthetized using sevoflurane inhalation (1.0%-2.0%, end-tidal concentration), and the cerebral cortex and hippocampus tissues were isolated. Each tissue sample was homogenized with five times the volume of RIPA buffer per weight (g), containing one tablet of a protease inhibitor cocktail (11836153001, Roche, Indianapolis, IN, USA) and one tablet of a phosphatase inhibitor cocktail (04906837001, Roche) in 10 ml of lysis buffer. The total protein concentration was measured using the Bradford method. After denaturation with SDS, proteins were separated on SDS-PAGE, transferred to nitrocellulose membranes (10600001, GE Healthcare, Piscataway, NJ, USA), and probed with the following primary antibodies: anti-HIF1 alpha (1:1,000, PAB12418, abnova, Taipei, Taiwan, ROC), anti-BDNF antibody (1:1,000, ab108319, abcam, Cambridge, UK), anti-AKT antibody (1:1,000, #9272, CST, Danvers, MA, USA), anti-phosphoAKT antibody (1:1,000, #9271, CST), anti-ERK antibody (1:1,000, #9102, CST), anti-phosphoERK antibody (1:1,000, #9101, CST), and anti- beta actin antibody (1:1,000, sc-47778, Santa Cruz Biotechnology, Dallas, TX, USA). Anti-beta actin was used as an internal control for normalization of the HIF1 alpha and BDNF signals. Phospho-AKT and phospho-ERK signals were normalized to the AKT and ERK signals, respectively. ImageJ software was used to quantify signals.
Statistical analysis. All analyses were conducted using the Statistical Package for the Social Sciences software (Information Analysis Systems, SPSS, IBM, Armonk, NY, USA). Mean values were analyzed using the Mann-Whitney U-test with Bonferroni correction for comparison between the IGF-1-treated, BCCAO, and control groups.
Results
Cortex findings. The BDNF protein expression was higher in the treatment group than in the BCCAO group (Figure 2). The proportions of pAKT and pERK were significantly higher in the treatment group than in the BCCAO group 3 days after surgery (Figure 3).
Representative western blots (A) with band quantification of HIF1α (B) and BDNF (C) expression in cerebral cortex samples collected three days after BCCAO. BDNF levels significantly increased in the control group than in the BCCAO groups. Results are expressed as a ratio compared to the expression of actin; *p<0.05; BCCAO: Bilateral common carotid artery occlusion; BDNF: brain-derived neurotrophic factor.
Representative western blots (A) with band quantification of pAKT/AKT (B) and pERK/ERK (C) expression in cerebral cortex samples collected three days after BCCAO. pAKT/AKT and pERK/ERK significantly increased in the treatment group than in the BCCAO groups. Results are expressed as a ratio compared to the expression of actin. *p<0.05; BCCAO: Bilateral common carotid artery occlusion; pAKT: phosphorylated protein kinase B; pERK: phosphorylated extracellular signal-related protein kinase.
Hippocampus findings. The expression of BDNF in the treated group at three days post-surgery was higher than that in the BCCAO group; the cortical findings showed a similar trend (Figure 4). There was a significant difference in the hippocampal pAKT/AKT and pERK/ERK levels between the BCCAO and treatment groups (Figure 5).
Representative western blots (A) with band quantification of HIF1α (B) and BDNF (C) expression in hippocampus samples collected three days after BCCAO. BDNF levels significantly increased in the control group than in the BCCAO groups. Results are expressed as a ratio compared to the expression of actin. *p<0.05; BCCAO: Bilateral common carotid artery occlusion; BDNF: brain-derived neurotrophic factor.
Representative western blots (A) with band quantification of pAKT/AKT (B) and pERK/ERK (C) expression in hippocampus samples collected three days after BCCAO. pAKT/AKT and pERK/ERK significantly increased in the treatment group than in the BCCAO groups. Results are expressed as a ratio compared to the expression of actin. *p<0.05; BCCAO: Bilateral common carotid artery occlusion; pAKT: phosphorylated protein kinase B; pERK: phosphorylated extracellular signal-related protein kinase.
Discussion
In the present study, we treated BCCAO model rats with IGF-1 via a stereotaxic injector 3 hours after BCCAO. It has been reported that stereotaxic administration of IGF-1 2 hours after unilateral carotid artery occlusion confers neuroprotection (3). Interestingly, IGF-1 has rarely been injected into BCCAO models. We subsequently investigated the expression of BDNF, pAKT/AKT, and pERK/ERK three days following BCCAO. Some studies have reported that cerebral blood flow changes significantly within 2-3 days of BCCAO (16, 17). Therefore, we chose 3 days after BCCAO as the day of evaluation.
Following stereotaxic injection of IGF-1, BDNF expression in the cortex and hippocampus increased. One study reported that the central administration of IGF-1 induces BDNF expression by reducing proinflammatory cytokine expression (18). Another study showed that intracerebroventricular injection of IGF-1 enhanced BDNF and brain plasticity, leading to cognitive recovery (19). In the cortex, IGF-1–treated neurons revealed AKT-BDNF signaling changes with enhanced neural complexity (20). Wen-Hua et al. previously reported that IGF-1 and BDNF enhance hippocampal neuronal survival and activate the PI3K/AKT pathway (21). As mentioned previously, the BCCAO model is useful for studying vascular dementia. Consequently, several prior studies have focused on the hippocampus. We suggest that IGF-1 leads to an increase in BDNF expression in the cortex, similar to the observed hippocampal increase.
Following stereotaxic injection of IGF-1, pAKT and pERK expression increased in the cortex and hippocampus. In an ischemic stroke model, the administration of IGF-1 reduced infarct volume and induced pAKT expression, which is known to reduce neuroinflammation and blood–brain barrier permeability (22). In a trauma model, IGF-1 overexpression was shown to enhance pAKT and induce neurogenesis in the hippocampus (23). In the BCCAO model, downregulation of IGF-1 and pAKT was observed in the hippocampus, a process that is related to impaired memory function (9). In the BCCAO model, only endogenous IGF-1 and pAKT were observed, with no exogenous IGF-1 input. In the developing rat cerebral cortex, IGF-1 injection at postnatal day 10 increases pERK levels (24). Yuan et al. previously reported that IGF-1 induces neural stem cell differentiation via action on the MEK/ERK pathway in adult mice (25). IGF-1 overexpression restores visual cortex plasticity and is associated with ERK activation (26). In the hippocampus, SE-induced IGF-1 enhances progenitor cell proliferation-mediated ERK/MAPK signaling (27). Lateral cerebroventricular infusion of IGF-1 increased the levels of ERK and AKT in rats, which is related to hippocampal excitatory activity (28). Some prior studies have further reported a relationship between BDNF and phosphorylated AKT/ERK in BCCAO models. Icariside II ameliorates cognitive impairment via BDNF/TrkB/CREB signaling in BCCAO (29). Niu et al. have similarly reported that epimedium flavonoids protect neurons and synapses in the hippocampus of rats with BCCAO by activating BDNF and AKT (30). Shikonin inhibits hippocampal neuronal apoptosis by regulating the AKT/BDNF signaling pathway (31). Based on these results, we suggest that increased BDNF levels induced by IGF-1 injection induced pAKT and pERK expression.
Conclusion
Stereotaxic injection of IGF-1 in rats with BCCAO enhanced BDNF expression in the cortex and hippocampus 3 days after surgery. It simultaneously increased pAKT and pERK levels in both regions.
Acknowledgements
This research was supported by a grant from the National Research Foundation of Korea to the Global Learning and Academic Research Institution for Master’s and PhD students and the Postdoctoral Fellows (LAMP) Program funded by the Ministry of Education (Grant Number: RS-2023-00285353).
Footnotes
Authors’ Contributions
YHJ and YYC designed the study. DJK and HKS performed the surgical procedures. HIH and JHP analyzed the data. HIH and JHP performed Western blot analyses. All Authors have approved the final manuscript.
Conflicts of Interest
The Authors declare no competing interests.
Funding
This study was supported by research funds from Chosun University (2023).
Artificial Intelligence (AI) Disclosure
No artificial intelligence (AI) tools, including large language models or machine learning software, were used in the preparation, analysis, or presentation of this manuscript.
- Received December 11, 2025.
- Revision received January 10, 2026.
- Accepted February 4, 2026.
- Copyright © 2026 The Author(s). Published by the International Institute of Anticancer Research.
This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.
References
Jump to section
Related Articles
Cited By...
- No citing articles found.