Research Report on the Effects of Naringin Intervention on Newborn Neurons and BDNF Levels in Rat Brain Ischemia-Reperfusion Model

Background

With the global increase in cardiovascular diseases, ischemic stroke has gradually become a major cause of death and disability, affecting the quality of life of millions of people. Despite continuous advancements in modern medical treatments, such as thrombolytic drugs and mechanical thrombectomy, patients need to receive treatment quickly within a short time after the onset of the disease. However, the time window for these treatments is very narrow, resulting in limited functional recovery and often accompanied by high rates of disability. Therefore, exploring new treatment methods that can improve functional recovery and reduce disability rates has become increasingly important.

The activation of endogenous neurogenesis after brain ischemia-reperfusion injury has been a hot research area in recent years. Studies have shown that ischemic injury can trigger low levels of endogenous neurogenesis in the subventricular zone (SVZ) and subgranular zone (SGZ) of the dentate gyrus in rats, followed by the migration of neuroblasts to the injury site. Therefore, stimulating the proliferation, migration, and synaptic integration of neural stem/progenitor cells through exogenous factors to improve motor and cognitive function recovery after stroke has been widely studied.

Among various exogenous factors, flavonoids, as natural compounds, have attracted more attention due to their antioxidant and anti-inflammatory properties. Among them, Naringin, a natural flavonoid extracted from grapefruit and oranges, has been shown to have positive effects on neurogenesis and brain-derived neurotrophic factor (BDNF) levels under physiological and pathological conditions.

Source

This study was conducted by researchers Esen Yilmaz, Gozde Acar, Ummugulsum Onal, Ender Erdogan, Abdulkerim Kasim Baltaci, and Rasim Mogulkoc from Selcuk University in Turkey, and published in Volume 26, Issue 4 of the journal “Neuromolecular Medicine” in 2024.

Research Design and Methods

Research Subjects

The study used 40 male Wistar rats (10-12 weeks old), all from the Selcuk University Experimental Animal Research and Application Center. Before and after the experiment, all rats had free access to standard feed and water.

Experimental Groups

The experiment was divided into 5 groups: 1. Control group (n=6) 2. Sham surgery group (n=6) 3. Ischemia-reperfusion group (n=9) 4. Ischemia-reperfusion + vehicle group (n=9) 5. Ischemia-reperfusion + Naringin group (n=10)

Experimental Procedure

Establishing Bilateral Carotid Artery Occlusion/Reperfusion Model

The two-vessel occlusion model (2VO) was used to produce experimental global cerebral ischemia in rats. The surgical steps were as follows: 1. Rats were anesthetized with ketamine and xylazine (60mg/kg and 5mg/kg). 2. The anesthetized rats were fixed on the operating table with their backs up, and a midline incision was made in the neck to expose the left and right common carotid arteries. 3. The common carotid arteries were carefully isolated and ligated for 30 minutes to induce global cerebral ischemia. 4. After 30 minutes, the ligatures were removed, and blood flow was confirmed before closing the incision.

The sham surgery group only underwent isolation of the common carotid arteries without occlusion.

Drug Treatment

After ischemia-reperfusion, the control group and sham surgery group received no drug treatment, the ischemia-reperfusion + vehicle group received vehicle CMC-Na (carboxymethyl cellulose sodium, 0.25%) treatment for 14 days, and the ischemia-reperfusion + Naringin group received oral gavage of Naringin (100mg/kg) for 14 days.

Animal Behavioral Evaluation

Neurological Function Scoring

To assess neurological function, the study used the Bederson scoring system to evaluate the rats. The scoring range was 0-3 points, corresponding to no functional deficits (0 points) to maximum functional deficits (3 points).

Rotarod Test

The rotarod test was conducted four days before establishing the ischemia-reperfusion model to assess motor function. The test was repeated on days 1, 7, and 14 after ischemia-reperfusion, recording the retention time of rats on the rotarod.

Molecular Detection and Histological Analysis

Tissue Collection

After the experiment, rats were euthanized, and hippocampal and frontal cortex tissues were collected.

Immunohistochemical Analysis

Immunohistochemical analysis was performed on 40 hippocampal and 40 frontal cortex samples. DAPI staining was used to label all nucleated cells, and anti-NeuN antibody was used to label mature neurons.

Real-time Quantitative PCR Analysis

Total RNA was extracted from brain tissues, reverse-transcribed to cDNA, and real-time quantitative PCR analysis was used to detect the expression levels of DCX (marker for newborn neurons) and BDNF.

Results

Effects on Neurological Function

The ischemia-reperfusion model significantly increased neurological function scores, while Naringin supplementation significantly reduced the scores, indicating that Naringin has a protective effect on neurological injury.

Effects on Motor Function

Rotarod test results showed that ischemia-reperfusion significantly reduced the retention time of rats on the rotarod, while two weeks of Naringin supplementation significantly increased it to levels close to the control group.

Neurogenesis and BDNF Levels

Immunohistochemical results showed: 1. Ischemia-reperfusion led to a significant decrease in DAPI and NeuN marker levels in the hippocampus and frontal cortex. 2. Naringin supplementation significantly restored the expression levels of these markers.

Real-time quantitative PCR results showed: 1. The expression levels of DCX and BDNF genes were significantly reduced in the ischemia-reperfusion group. 2. Naringin supplementation significantly restored the expression levels of these two genes.

Conclusion and Significance

The study shows that 30 minutes of global cerebral ischemia and 2 weeks of reperfusion lead to neurological and motor function impairment in rats, while Naringin supplementation can significantly improve these impairments. Specifically, it increases the expression of neurogenesis markers DCX and NeuN, and restores suppressed BDNF levels, thereby improving neurological and motor functions in rats. This provides a basis for Naringin as a potential candidate drug for treating ischemic brain injury.

Future Research Directions

Further research on the mechanisms of Naringin through neurotrophin receptors (such as TrkB), mitogen-activated protein kinases (MAPK/ERK), phosphatidylinositol 3-kinase (PI3K), and other signaling pathways will help to analyze its molecular mechanisms in neuroprotection and neurogenesis in more detail, which will contribute to the development of more effective stroke treatment methods.