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.

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).

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).

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).

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.

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.