Title & Abstract

International Journal of Anatomy and Research

Impact Of Caffeine on the Neurodegenerative Changes of the Frontal Cortex in Type 2 Diabetic Rats

DOI: 10.16965/ijar.2026.160

Othman, Manal A. ¹²

Fatima, Ayesha ¹

Sirajo, Bello ¹

Khan, Mohammed ¹

Fadel, Raouf A. ¹³

¹ Department of Anatomy, College of Medicine and Health Sciences, Arabian Gulf University, Manama, Bahrain.

² Department of Histology and Cell Biology, Faculty of Medicine, Assiut University, Assiut, Egypt.

³ Department of Human Anatomy and Embryology, Faculty of Medicine, Suez Canal University, Ismailia, Egypt.

ABSTRACT

Background.

Type 2 diabetes (T2D) is associated with injury to many organs in our body. The central nervous system can be affected in diabetes progression, which may result in dementia and decline in cognitive function. Myelin is very important in neuronal protection and axonal transport and might be affected with neurodegenerative diseases as diabetes. Caffeine, a commonly used psychoactive beverage, is known to improve the impairment in memory and cognitive function. This work was aiming at investigating the protective role that caffeine can provide for T2D rat neurodegeneration.

Methods.

Thirty-six male Wistar rats, were grouped into 4 groups (9 each); the control group (group 1), diabetic group (group 2), caffeine + diabetic group (group 3), and caffeine-only group (group 4). Induction of T2D was done by feeding the rats with high calorie diet and giving them a single injection of streptozotocin intraperitoneally at a lower dose. Caffeine was administered by oral route for 5 weeks. The rats were sacrificed; brains were removed and put in 10% formalin fixative and processed to evaluate their general morphology. Immunohistochemistry was performed using neuronal, myelin and apoptotic markers.

Results.

Histological examination revealed neurodegeneration of frontal cortex of rats with T2D. Immunohistochemistry revealed downregulation in neuronal and myelin immunostaining and upregulation of apoptosis in rats with T2D.

Conclusions.

There was enhancement of the structure and immunohistochemistry of frontal cortex of diabetic rats, which receive caffeine. This neuroprotective effect of caffeine in T2D rats suggests a therapeutic potential for caffeine in T2D.

Keywords: Caffeine; Frontal cortex; Histology; Myelin; Apoptosis; Type 2 Diabetes

INTRODUCTION

Type 2 diabetes (T2D) is considered a lifelong metabolic disease affecting many organ systems, including the central nervous system [1]. It is characterized by hyperglycemia due to insulin resistance in peripheral tissues and a persistent decline in the function of beta cells of the pancreas. The use of diets with high calories, and streptozotocin (STZ) at lower doses, can generally be accepted to produce a rodent model of T2D [2,3]. Persistent hyperglycemia can gradually disrupt normal brain structure and function, which contributes to cognitive decline, cerebral atrophy, infarction, and neurodegeneration [4,5].

The frontal cortex is a major region of the brain responsible for thinking, decision-making, and overall cognitive function. Evidence from previous studies shows that chronic T2D, characterized by hyperglycemia, insulin resistance, and microvascular injury, gradually causes changes in the structure and function of this vital region of the brain [6]. Neuroimaging studies in long-standing diabetics reveal reduced frontal lobe grey matter volume and thinning of the frontal cortex, indicating increased neurodegeneration in this region [7,8]. In a mouse model of T2D, researchers found not only reductions in the overall size of the brain and cortex, but also changes in the cellular composition of the cortex, such as a decrease in the number of mature neurons, impaired myelination, and disruptions to vascular and glial cell structure. These findings suggest that diabetes can impair the neurovascular unit, degrade neural and glial integrity, and compromise brain microstructure, thereby contributing to cognitive decline [9]. We previously reported that rats administered a high-calorie diet showed defective structure in the hippocampus, which was mediated by gliosis and inflammation [10].

Caffeine is a naturally occurring psychostimulant that is globally consumed in the form of drinks like coffee and tea. It produces increased alertness, reduced fatigue, and elevated mood by blocking adenosine receptors [11]. Recent research shows that caffeine has neuroprotective, antioxidant, and anti-inflammatory effects, thus decreasing the risk of degenerative diseases as Parkinson's and Alzheimer disease. This is likely due to mechanisms including reduction of oxidative stress, modulation of adenosine receptor signaling, and suppression of neuroinflammation [12]. Diabetes can progress over time and might lead to severe morbidities and mortalities. The pathophysiology of the brain damage in diabetic encephalopathy is not fully understood. The current study aimed at investigating the possibility of caffeine to improve neurodegeneration in the frontal cortex of T2D rats through the investigation of neuronal affection, myelination, and apoptosis.

MATERIALS AND METHODS

Animals and Induction of T2D

Adult male Wistar rats (270-300 g) were housed at the Animal House Facility, College of Medicine and Health Sciences, Arabian Gulf University, Bahrain under standard conditions with ethical approval (# E006-1-04/17). Thirty-six rats were divided into 4 groups (9 rats each): Group 1 (Control: low-calorie diet [LCD] + IP citrate buffer + oral saline); Group 2 (Diabetic [DM]: high-calorie diet [HCD] for 4 weeks followed by a single IP injection of streptozotocin [STZ] 35 mg/kg BW in citrate buffer); Group 3 (DM + Caffeine: HCD + STZ followed by daily oral caffeine 100 mg/kg BW via gavage for 5 weeks); Group 4 (Caffeine-only: LCD + daily oral caffeine 100 mg/kg BW for 5 weeks) [10]. Rats with fasting blood glucose >250 mg/dL after 72 hours of STZ injection were confirmed diabetic. After 8 weeks, animals were sacrificed via CO2 inhalation and brains processed for histological and immunohistochemical analyses.

Blood Glucose Measurements and Histological Processing

Blood glucose levels were monitored via tail-vein blood using an Accu-Chek Active glucometer at baseline, week 4 (post-STZ), and weekly during caffeine treatment (weeks 5-8). Excised brains were fixed in 10% neutral buffered formalin, coronal 5 µm paraffin sections prepared, and stained with Hematoxylin and Eosin (Hx&E) for general morphology (captured at 20X using an Axioscope A1 microscope, Carl Zeiss) [13].

Immunohistochemistry and Quantitative Analysis

Five µm paraffin sections were immunostained using an Avidin-Biotin detection kit (Vectastain Elite ABC Universal Kit) with primary antibodies: anti-NeuN rabbit monoclonal (ab177487, Abcam; neuronal marker), anti-MBP rabbit monoclonal (ab218011, Abcam; myelin marker), and anti-Bax rabbit monoclonal (ab32503, Abcam; apoptotic marker) with 3,3'-diaminobenzidine (DAB) chromogen and Harris hematoxylin counterstaining. Quantitative percent area of immunoreactivity was measured using ImageJ software across 6 non-overlapping fields from 6 slides per animal.

Statistical Analysis

Data were analyzed using SPSS software version 27.0 (IBM Corp.). Variables were expressed as Mean ± Standard Deviation (SD) and evaluated via One-Way Analysis of Variance (ANOVA) followed by Tukey's post hoc test (p < 0.05 considered statistically significant).

RESULTS

Baseline blood glucose was normal across all groups (85 ± 2.2 mg/dL). Following HCD and STZ, blood glucose rose significantly in diabetic rats (Group 2: 303 ± 15 mg/dL at week 8, p < 0.0001 vs control). Caffeine treatment significantly lowered blood glucose in diabetic rats (Group 3: 109 ± 2.7 mg/dL, p < 0.0001 vs DM). Blood glucose in caffeine-only rats (Group 4: 86 ± 1.8 mg/dL) showed no significant difference from controls (Figure 1).

Figure 1

Histological Hx&E evaluation revealed intact neurons with pale cytoplasm and central vesicular nuclei in controls (Group 1) and caffeine-only rats (Group 4). Diabetic rats (Group 2) showed marked neurodegeneration with shrunken, darkly stained neurons, pericellular halos, vacuolization, and pyknotic nuclei. Caffeine co-treatment (Group 3) markedly preserved neuronal morphology with predominantly healthy neurons (Figure 2).

Figure 2

Quantitative immunohistochemistry (Table 1) demonstrated significant downregulation of NeuN percent positive area in Group 2 (7.03 ± 0.33%) vs controls (17.14 ± 0.63%, p < 0.001), which was significantly restored by caffeine in Group 3 (9.95 ± 0.35%, p < 0.001 vs DM) (Figure 3). Myelin basic protein (MBP) immunoreactivity showed significant loss in Group 2 (5.8 ± 0.31%) vs controls (14.64 ± 0.31%, p < 0.001), with marked remyelination in Group 3 (14.42 ± 0.62%, p < 0.001 vs DM) (Figure 4). The apoptotic marker Bax was significantly upregulated in Group 2 (24.33 ± 3.56%) vs controls (5.47 ± 1.08%, p < 0.001), and significantly suppressed in Group 3 (9.12 ± 0.4%, p < 0.001 vs DM) (Figure 5).

Figure 3

Figure 4

Figure 5

Table 1

Percent area of immunoreactivity in the different studied groups.

Group | NeuN positive area (Mean ± SD) | MBP positive area (Mean ± SD) | Bax positive area (Mean ± SD)
Control | 17.14 ± 0.63 | 14.64 ± 0.31 | 5.47 ± 1.08
D.M | 7.03 ± 0.33 | 5.80 ± 0.31 | 24.33 ± 3.56
D.M + Caffeine | 9.95 ± 0.35 | 14.42 ± 0.62 | 9.12 ± 0.40
Caffeine | 13.73 ± 0.56 | 17.53 ± 0.54 | 6.43 ± 1.39

Standard deviation (SD), one-way analysis of variance (ANOVA) followed by Tukey post hoc test for multiple comparisons (p < 0.05).

DISCUSSION

This study demonstrates the neuroprotective efficacy of caffeine against T2D-induced neurodegeneration in the rat frontal cortex. Diabetic encephalopathy involves complex pathological cascades driven by chronic hyperglycemia, oxidative stress, and neuroinflammation [10,14-17]. In our diabetic model, severe structural damage was evidenced by dark shrunken neurons with pyknotic nuclei and pericellular vacuolization [18,19].

Caffeine significantly attenuated neuronal loss as reflected by NeuN preservation. Neuronal loss in T2D frontal cortex is intimately linked to insulin resistance, glucotoxicity, and downstream cognitive dysfunction [4,24,25]. As a non-selective antagonist of adenosine A1 and A2A receptors, caffeine's neuroprotective actions in memory preservation are largely mediated by A2A receptor blockade, which modulates synaptic plasticity and curbs neuroinflammation in the frontal cortex [26-28].

Myelin integrity was markedly compromised in diabetic frontal cortex, evidenced by significant reduction in MBP immunoreactivity. Glucose toxicity induces oxidative stress and demyelination in oligodendrocytes, mirroring changes seen in neurodegenerative disorders such as Alzheimer's and Parkinson's disease [30-33]. Caffeine co-administration substantially restored MBP expression, aligning with its known remyelinating and cytoprotective properties in peripheral nerves and hypoxic models [29,30].

Furthermore, caffeine significantly suppressed Bax-mediated apoptotic signaling in the frontal cortex. Hyperglycemia-induced oxidative stress triggers mitochondrial release of pro-apoptotic factors [20-23,34,35]. Suppression of Bax alongside blood glucose reduction highlights caffeine's multifaceted metabolic and neuroprotective potential in diabetic encephalopathy.

CONCLUSION

Our study detected improvement of neurodegenerative changes of T2D rats by the consumption of caffeine. The findings in this study can provide an insight into the mechanisms of neurodegeneration of the frontal cortex in T2D, which is mediated by demyelination, neuronal loss and apoptosis. This protective effect of caffeine can support its use as a therapeutic modality in T2D. Future research will further investigate this therapeutic potential and unravel the complex pathophysiology of T2D, including behavioral testing and oxidative stress.

Abbreviations & Declarations

ABBREVIATIONS

T2D: Type 2 diabetes

STZ: Streptozotocin

LCD: Low calorie diet

IP: Intraperitoneally

HCD: High calorie diet

Hx&E: Hematoxylin & eosin

MBP: Myelin basic protein

NeuN: Neuronal Nuclei

Bax: Bcl-2-associated X protein

ACKNOWLEDGMENTS

The authors thank Mrs. Aisha Rashid for technical assistance and Mrs. Vengilyn Garcia, secretary of Anatomy Department for administrative and processing support. This study was fully funded by a grant from the Arabian Gulf University, grant # E006-1-04/17.

DECLARATIONS

Ethical Approval: Approved by the Committee of Animal Care and Use at the Arabian Gulf University, Bahrain (ethical approval # E006-1-04/17).

Funding: This study was fully funded by a grant from the Arabian Gulf University (grant # E006-1-04/17).

Conflicts of Interest: The authors declare no financial or nonfinancial interests.

Author Contributions: MO: design, acquisition of data, analysis, drafting the manuscript, approval of final version. AF: acquisition of data, analysis, interpretation, drafting manuscript. BS: acquisition of data, analysis, interpretation, drafting manuscript. MK: acquisition of data, analysis, interpretation. RF: design, analysis, interpretation, critical revision, approval of final version.

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