Stroke, as one of the most significant causes of death and permanent disability in the world, is brought on by pathological vascular occlusion from hemorrhage, embolism, or thrombosis [1]. In an ischemic stroke, the disruption of oxygen and glucose quantity leads to anaerobic metabolism, which produces toxic metabolites that contribute to neuronal injury [2]. Present therapeutic approaches for acute ischemic stroke mainly include thrombolytic agents such as Alteplase and Tenecteplase, which aim to restore blood flow by dissolving clots, as well as mechanical thrombectomy for clot elimination [3]. However, only a smaller group of patients use these interventions due to strict time windows and potential difficulties [4]. Furthermore, additional damage mechanisms such as oxidative stress, inflammation, and excitotoxicity continue to exist despite efficient reperfusion, thereby contributing to neuronal death and functional impairment [5]. Memory impairment, a common consequence of stroke, is severely associated with hippocampal damage, a region highly vulnerable to ischemic injury [6]. Thus, understanding the basic mechanisms of brain injury and finding successful therapy approaches remain crucial significances in stroke research [7].

Neuronal hyperexcitability, resulting from the abnormal activation of glutamate receptors and the significant accumulation of intracellular Ca2+ ions, is the main factor leading to progressive neuronal death after an ischemic incident. This hyperexcitability is also known as excitotoxicity [7]. Under normal physiological conditions, γ-aminobutyric acid type B (GABAB) receptors control the excitability of neurons. These receptors prevent neurons to be hyperexcitable, which could lead to excitotoxicity. These receptors consist of two subunits: GABAB1 and GABAB2. When the neurotransmitter GABA binds to these receptors, it activates Gi/o proteins. This activation, sequentially, triggers GIRK channels and inhibits voltage-gated Ca2+ channels at postsynaptic and presynaptic sites, respectively. As a result, action potential generation and neurotransmitter release are inhibited, leading to a long-lasting state of neuronal inhibition [8]. Under normal circumstances, the C/EBP homologous protein (CHOP), which is triggered by endoplasmic reticulum (ER) stress, interacts with the leucine zipper in the ER's C-terminal domain of the GABAB2 subunit. This interaction stops the GABAB receptor subunits from assembling, which prevents the completed receptors from leaving the ER [9]. CHOP has a role in apoptotic cell death and is significantly elevated after ER stress linked to cerebral ischemia [10]. In addition to encouraging apoptosis, elevated CHOP interacts with ER GABAB receptors to cause their downregulation, which stops functionally assembled GABAB receptors from being transported from the ER to the cell membrane. Continued internalization and lysosomal degradation of cell surface receptors will reduce the number of functional receptors, as the quantity of freshly produced GABAB receptors is inhibited. GABAB receptors' capacity to react to excessive neuronal stimulation is aided by this decline in receptor availability, ultimately leading to excitotoxic neuronal death [11]. The agonist baclofen has been shown to improve GABAB receptor activity in both in vitro and in vivo models of cerebral ischemia [12]. These findings propose that targeting GABAB receptor activity may offer an effective neuroprotective strategy in cerebral ischemic circumstances.

Quercetin (QC) is found naturally in various fruits, vegetables, and medicinal plants such as onions, apples, and cabbage, among others. QC has been shown to have a number of pharmacological effects in modern pharmacological research, including antioxidant activity, anti-inflammatory and anti-apoptotic properties, protection of the blood-brain barrier, ion channel regulation, reduction of glutamate excitotoxicity, and improvement of cognitive function. All these effects have been seen to be effective in protecting the neurons in the brain during cerebral ischemia [13]. The effectiveness of QC in clinical applications is, however, hindered by its low bioavailability, poor water solubility, and inability to cross the blood-brain barrier, despite its promising potential as a therapeutic agent [14]. To overcome these limitations, scientists are increasingly using delivery systems based on nanotechnology, specifically superparamagnetic iron oxide nanoparticles (SPIONs). In the current study we chose SPIONs coated with polyethylene glycol (PEG) due to their stability, bioavailability, and ability to modify surfaces [15]. By utilizing SPIONs as nanocarriers, we aimed to enhance the solubility, stability, and bioavailability of quercetin [16]. Adding polyethylene glycol (PEG) improved circulation time and reduced aggregation, making SPIONs a suitable option for oral delivery [17]. This study demonstrates the potential of quercetin-conjugated SPIONs as a new therapeutic approach for the treatment of brain injury and enhancement of functional recovery in experimental ischemic stroke, mainly due to their physicochemical and pharmacokinetic advantages, rather than their magnetic properties for targeted therapy.

Stroke is a leading cause of death and long-term disability. Most previous studies have focused on young animal models, intraperitoneal delivery, and non-covalent formulations, often relying on broad histological or behavioral endpoints [[18], [19], [20]]. Aged brains are clinically relevant but understudied in nanomedicine studies due to their compromised blood-brain barrier (BBB), increased neuroinflammation, and decreased endogenous neuroprotection [21]. In contrast, our study investigates the covalent conjugation of QC to PEG-coated superparamagnetic iron oxide nanoparticles (QCSPIONs) in an aged rat model of transient middle cerebral artery occlusion (tMCAO) following oral administration. We evaluated quercetin's capacity to enhance memory and learning while comparing the effectiveness of free quercetin with QCSPIONs. Moreover, we followed to determine whether quercetin, known for its anti-apoptotic properties, could modulate the gene expression of apoptotic markers in the hippocampus, including Bax, Bcl-2, and particularly CHOP [22]. Furthermore, we investigated whether quercetin could enhance the gene expression of GABAB receptor subunits, which play a significant role in counteracting excitotoxicity during ischemic conditions [8]. To our knowledge, this is the first study to examine these molecular mechanisms in a clinically relevant aged stroke model. By clarifying these mechanisms, this study proposed novel visions into the therapeutic potential of quercetin, mainly when conjugated with SPIONs, as a potential approach for neuroprotection and functional recovery after ischemic stroke. The development of novel nanotechnology-based therapies that target excitotoxicity and neurodegeneration in ischemic stroke may be assisted by these findings.