Demographic changes, increasing diabetes mellitus, and rising obesity rates contribute to the growing incidence of cerebral ischemia. Stroke ranks as the second leading cause of death and a significant contributor to global disability. Recanalization procedures are increasingly employed, expanding the therapeutic window for treating ischemic stroke [1]. Research points to oxidative stress-related neuroinflammatory pathways playing a major role in acute-phase neuronal injury during cerebral ischemia, exacerbating damage severity [2].

Cerebral Ischemia Reperfusion Pathophysiology

During ischemia, anaerobic metabolism predominates, lowering intracellular pH. To prevent excessive hydrogen ion accumulation, the Na\(^{+}\)/H\(^{+}\) exchanger transports additional hydrogen ions, resulting in substantial sodium ion influx [3]. The pathophysiology of ischemia-reperfusion injury becomes more complex due to impaired mitochondrial function, disrupted apoptotic and autophagic pathways, and abnormal signal transduction [4]. Oxygen and glucose deprivation culminate in mitochondrial dysfunction and cellular excitotoxicity, initial markers of ischemic damage leading to further harm. This section reviews various signaling pathways associated with excitotoxicity, oxidative stress, mitochondrial dysfunction, glutamate, and NMDAR-induced cell toxicity [5].

Role of Anti-TNF-\(\alpha\) during Cerebral Ischemia

Diverse cell types, including macrophages, endothelial cells, adipose tissue, fibroblasts, and brain tissue, release the anti-inflammatory cytokine TNF-\(\alpha\) [6]. Anti-TNF-blockers significantly reduced overproduced IL-1, TNF-\(\alpha\), and IL-6 in serum related to traumatic brain injury (TBI). Conversely, TBI rats administered etanercept exhibited notably higher IL-10 serum levels. Additionally, it’s been found to inhibit brain gliosis. Dexmedetomidine and NMDA receptor antagonist (MK801) therapies also decreased TNF-production and enhanced cerebral infarction in the MCAO model [7].

Etanercept (Enbrel\(^{\circledR}\))

Etanercept, a fusion protein generated through recombinant DNA technology, involves the fusion of the constant terminus of the IgG1 antibody with the TNF receptor. This dimeric, fully humanized recombinant protein is formed by combining two soluble TNF receptors (p75) with the constant fragment (Fc) of human immunoglobulin G1 (IgG1). The Fc region contains the CH2 and CH3 domains of the immunoglobulin, contributing to the molecule’s stability (see Figure 1) [4].

Etanercept functions by competitively binding both membrane-bound and circulating TNF during reversible reactions [8, 9].

This study aims to evaluate the potential protective effect of etanercept against global cerebral ischemia/reperfusion injury induced by bilateral ligation of the common carotid arteries, by interfering with apoptosis and inflammatory pathways.

Experimental Animals

Sixteen male Albino (Rattus norvegicus) rats, weighing between 150 and 250 g, were obtained from the animal house of Kufa University Faculty of Science. The animals were housed in a well-ventilated environment at a temperature of 25\(^o\)C or lower and provided with standard animal diet and water ad libitum. The study was conducted in accordance with the Laboratory Animals Guide to Care, and approved by the Al-Kufa University Animal Care and Research Committee.

Animal Groups

After 3 weeks of acclimatization, the rats were divided into four groups as follows:

• Sham Group: Rats underwent general anesthesia without occlusion of bilateral common carotid arteries [10].
• Control Group (Ischemic-Reperfused): Rats underwent general anesthesia, followed by occlusion of the common carotid artery for 30 minutes and subsequent reperfusion for one hour without receiving any drug [10].
• Vehicle Group: Rats underwent the same surgical procedure as the control group, but received the vehicle of drugs (NaCl) intraperitoneally (i.p.) 30 minutes before ischemia [11].
• Etanercept Group: Animals underwent the surgical procedure of the control group, and were administered Etanercept at a dose of 50 mg/kg intraperitoneally (i.p.) 30 minutes before ischemia [12].

At the end of reperfusion, the animals were euthanized by decapitation under general anesthesia, and the brains were isolated for further analysis [13, 14].

Preparation of Drugs

Etanercept was dissolved in 0.9% normal saline and administered at a dose of 5 mg/kg intraperitoneally (i.p.) 30 minutes before ischemia. Etanercept was prepared immediately before injection [11, 15].

Induction of Global Brain Ischemia

To induce general anesthesia, i.p. injections of xylazine and ketamine at doses of 8-10 mg/kg and 80-100 mg/kg, respectively, were administered [16]. The neck was shaved and cleaned with 80% ethanol, and connective tissues were removed. Global cerebral ischemia/reperfusion injury was induced by occluding both common carotid arteries simultaneously for 30 minutes, followed by one hour of reperfusion. After one hour of reperfusion [17, 18], rats were decapitated, and brains were rapidly extracted and stored in pre-cold PBS solution before being frozen. Each brain was then sliced into sections for histopathological and immune-histopathological analysis [19, 20].

Histopathology

Following reperfusion, brain tissues were sliced into 5 \(\mu\)m-thick sections, embedded in a paraffin block, and fixed in 10% formalin. After staining with hematoxylin and eosin (H&E), sections were examined under a microscope by a pathologist. The pathological scoring scale used in this study is as follows [17]:

• Normal (0): Absence of eosinophilic neurons, red blood cells (RBCs), and edema.
• Category 1 (1): Slight presence of edema or eosinophilic neurons.
• Category 2 (2): Moderate presence of edema, eosinophilic neurons, and a small RBC population.
• Category 3 (3): Severe presence of necrosis, edema, eosinophilic neurons, and RBCs.

Tissue Preparation for NF-\(\kappa\)B p65 Measurement

Brain tissues were sectioned into small pieces under cold conditions, homogenized with a solution containing PBS, protease inhibitor cocktail, and Triton X-100 for 20 minutes (5 seconds each time). The homogenates were then centrifuged at 2,000-3,000 rpm for 20 minutes at 4\(^o\)C before being stored at -80\(^o\)C until analysis [17].

Measurement of TLR2 and TLR4 Levels through IHC Technique

Tissue samples from untreated and treated groups were collected to quantify cells labeled with TLR2 and TLR4 antibodies following the manufacturer’s protocol. The immunohistopathological scoring scale was calculated using the following criteria: 1 (weak staining), 2 (moderate staining), and 3 (strong staining) [20].

Statistical Analysis

Data were expressed as the standard error of the mean (SEM). A significance level of 0.05 or lower was considered statistically significant using one-way analysis of variance (ANOVA) and Tukey’s post-test. Kruskal-Wallis non-parametric tests were used for histopathological parameter analysis. Statistical analyses were performed using SPSS software (version update) and graphical representation was done using Prism program version 8.1.

Modulation of NF-\(\kappa\)B p65

At the conclusion of the experiment, NF-\(\kappa\)B p65 levels in brain tissue of all groups were analyzed using the ELISA technique. Ischemia/reperfusion (I/R) injury led to an elevation in NF-\(\kappa\)B p65 levels. ELISA analysis revealed a significant reduction (p < 0.05) in NF-\(\kappa\)B p65 levels in the Etanercept group (113.52 \(\pm\) 4.32) compared to the control group (166.18 \(\pm\) 7.73). Additionally, no significant difference (p > 0.05) was observed in NF-\(\kappa\)B p65 levels between the Etanercept group and the sham group. There was also no significant difference (p > 0.05) in NF-\(\kappa\)B p65 levels between the control group and the vehicle group, as shown in Table 1 and Figure 2.

Modulation of TLR2 and TLR4

We analyzed the levels of TLRs in brain tissue of all groups at the experiment’s conclusion using the immunohistochemistry technique. Immunohistochemistry results revealed that the combination of 30 minutes of ligation followed by 60 minutes of reperfusion led to changes in TLR2 and TLR4 levels. In brain tissues, the Etanercept group did not exhibit TLR expression, while both the control and vehicle groups demonstrated such expression. In comparison, positive control tissue (normal spleen tissue) showed a high level of TLR expression (Figure 3).

Pharmacological Etanercept Reduces Necrosis, Hemorrhage, Dark Eosinophilic Neurons, and Edema Caused by BCCAO

A cross-section of the sham rat brain exhibited a normal structure, devoid of oedema, necrosis, dark eosinophilic neurons, and hemorrhage. In contrast, the cross-sections of the rat brains in the Control and Vehicle groups displayed abnormal features, including edema, necrosis, dark eosinophilic neurons, and hemorrhage. Our results demonstrate that the Pharmacological Etanercept-treated group exhibited improved brain injury compared to the Control and Vehicle groups (Figure 4).

A histopathological examination revealed a significant reduction (p < 0.05) in brain tissue damage scores within the Etanercept-treated groups compared to the control group. Both the control and vehicle groups exhibited a significant increase (p < 0.05) in histopathological scores when compared to the sham group. Meanwhile, the Etanercept-treated groups demonstrated a significant reduction (p < 0.05) in scores when compared to the control group. No significant differences in scores (p > 0.05) were observed between the Etanercept-treated groups and the sham group, as well as between the control group and the vehicle group (Table 2 and Figures 5).

p>Various models, including bilateral common carotid artery occlusion (BCCAO), are employed in rat studies on cerebral ischemia stroke [21]. Within minutes of ischemia initiation, BCCAO leads to increased oxidative stress, inflammatory responses, cell death, and brain impairment [23, 24, 25].

Pharmacological Reduction of Brain NF-\(\kappa\)B p650 by Etanercept

Ischemia-reperfusion elevates the transcription of proinflammatory genes, including IL-6 and IL-1\(\beta\), in macrophages [26], and also regulates macrophage receptor genes [27], along with TNF-\(\alpha\), iNOS, and COX-2 [28]. NF-\(\kappa\)B activation translocates and binds to promoters [29], leading to activation of TNF-\(\alpha\), IL-1, ROS, and oxidative stress [30], which become elevated 10-30 minutes after cerebral ischemia [31].

Our study revealed that non-ischemic brain slices from the sham group exhibited low levels of NF-\(\kappa\)B, indicating basal protein levels. In contrast, after inducing BCCAO, the untreated groups exhibited a significant increase in NF-\(\kappa\)B levels compared to the sham group, signifying NF-\(\kappa\)B overexpression.

Administration of etanercept 30 minutes before ischemia significantly reduced NF-\(\kappa\)B levels compared to the untreated groups, suggesting a reduction in TNF-\(\alpha\) protein expression in the nucleus. This reduction in brain inflammation supports our hypothesis.

Administration of Etanercept and its Effect on Inflammation

The administration of etanercept led to a reduction in the release of inflammatory mediators, thereby inhibiting NF-\(\kappa\)B activation and subsequently reducing inflammation.

Effect on TLR2 and TLR4 Expression

In our study, we observed the expression of TLR2 and TLR4 after 30 minutes of ischemia and 1 hour of reperfusion using immunohistochemistry staining. The administration of etanercept 30 minutes before ischemia significantly reduced the expression of TLRs compared to untreated groups, indicating a reduction in brain inflammation. This result supports our hypothesis. Studies by [32] showed increased TLR4 expression after ischemia-reperfusion in MCAO. Ying Wang et al. (2013) demonstrated elevated TLR2 and TLR4 expression after half an hour of ischemia through immunohistochemistry [33]. Ursolic Acid treatment in animals led to a decrease in the number of TLR-positive cells compared to controls when measured using immunohistochemistry.

Etanercept’s Reduction of Damage Caused by Bilateral Carotid Artery Occlusion

We observed a low damage score in non-ischemic brain slices of the sham group, while the untreated groups exhibited significantly increased damage scores following BCCAO induction compared to the sham group. This suggests that I/R results in brain tissue damage. Administration of etanercept 30 minutes before ischemia significantly reduced brain damage scores compared to untreated groups, indicating that anti-TNF-\(\alpha\) treatment reduces brain damage, including hemorrhage and necrosis. I/R can lead to blood-brain barrier disruption, edema formation [34], and induce pyknotic and dark eosinophilic neurons [35]. Ischemia leads to free radical generation, subsequently inducing neuronal damage and mitochondrial impairment [1, 37, 38]. Our findings indicate that reducing TNF-\(\alpha\) levels can restore neurological functions, which aligns with several other studies.

Based on the findings of this investigation, it is recommended to introduce anti-TNF-\(\alpha\) treatment before reperfusion to reduce tissue damage by mitigating inflammation.

The authors would like to express their gratitude to Al-Zahrawi University College for providing excellent research facilities. Additionally, special thanks to Dr. Hassan for his diligent animal care and management.

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