Spinal cord injury (SCI) has long been regarded as a devastating condition that frequently results in motor, sensory, and autonomic dysfunction, as well as a significant negative psychological impact and financial burden on the patient(Anjum et al., 2020; Eli et al., 2021; McDonald and Sadowsky, 2002; Wyndaele and Wyndaele, 2006). Primary and secondary injuries are both included in SCI. The term “primary injury” refers to the initial irreversible mechanical injury. Secondary injury has been associated with a complex set of pathophysiological changes such as increased cell permeability, apoptosis, vascular damage, demyelination, and glial scarring, all of which harm cell regeneration and nerve repair (Hachem et al., 2020; Lv et al., 2021). According to the progression of SCI, the secondary injury is subdivided into acute, subacute, and chronic phases. Following an irreversible injury, the acute phase is characterized by vascular damage, free radical production, increased calcium inward flow, and necrosis (Alizadeh et al., 2019). It is also followed by more severe neuronal apoptosis, axonal demyelination, axonal remodeling, and glial scar formation(Alizadeh et al., 2019). Ultimately, chronic phases characterized by cystic cavity, axonal dieback and glial scar maturation impede recovery from SCI (Tran et al., 2018). As a result, it is critical to avoid secondary SCI injury to avoid a poor prognosis effectively.

The pathophysiologic mechanisms of SCI, as well as the mechanisms of nerve repair and regeneration, by which it occurs, are complex. Despite promising results in preclinical research regarding these mechanisms, it is currently challenging to translate them into the clinic for SCI. To solve this incurable condition, more studies relevant to the SCI model are needed, which would provide a reliable assessment of the therapeutic strategy of SCI patients in the future. Large amounts of adenosine triphosphate (ATP) and its metabolites are released after SCI and are involved in various aspects of functional regulation by acting on purinergic receptors that are widely expressed in the spinal cord.

The regulatory role of purinergic signaling pathways in the CNS has received widespread attention in the present day(Burnstock, 2017; Rodrigues et al., 2019). Purines and their receptors represent a critical neural signaling molecule that may regulate neural tissue cells' physiological activities (von Kügelgen, 2019; von Kügelgen and Hoffmann, 2016). There are two types of purinergic receptors, P1 and P2.The natural agonist of the P1 receptor is adenosine, which is classified as A1, A2a and A2b.Then, the second type is ionotropic (P2X) and metabotropic (P2Y) receptors (Jacobson et al., 2020). Common agonists of P2Y receptors are ATP. Much evidence indicates that P2Y receptors are essential for SCI (Ceruti et al., 2009; Guarracino et al., 2016; Xia and Zhu, 2014). SCI causes primary neuron and axon injury and secondary tissue disruption resulting in severe, permanent motor and sensory impairments. Demyelination could lead to motor and sensory dysfunction, therefore, even small disruption in myelin can have profound consequences on neuronal signaling and function(Belin et al., 2019).

In addition to demyelination, the previous report has shown that apoptosis was discovered to occur as early as 4 hours after SCI (Mizuno et al., 1998). Although endoplasmic reticulum and death receptor pathways are all involved in the pathological process of apoptosis after SCI (Lee et al., 2000; Yu et al., 2009; Zhang et al., 2013). Neuronal apoptosis caused by the mitochondrial-mediated pathway may play a significant role in the pathophysiological processes of SCI (Yong et al., 1998; Zhang et al., 2012). Mitochondrial dysfunction after injury is associated with multiple pathological changes, such as apoptosis, high levels of glutamate, and imbalance in the Ca2+ concentrations, thus could account for the dysfunction induced by SCI (Anjum et al., 2020; Jha et al., 2019; Vanzulli and Butt, 2015). Mitochondrial as well as myelin morphological characteristics and dysfunction appear to be significant contributors to SCI pathology (Cheng et al., 2023; Zhang et al., 2017). In addition, abnormal changes in glial cells have received much attention in recent years, due to their hypertrophic and proliferative properties, which eventually form a physical barrier near the injury site, making recovery difficult (García-Álvarez et al., 2015).

Recently, many studies focused on P2Y12, which is one of the P2Y receptors known as a metabotropic G-protein-coupled purinergic receptor. The P2Y12 receptor (P2Y12R) has been shown to treat chronic migraine, neuropathic pain, and stroke. In the model of chronic migraine and neuropathic pain, P2Y12R mediates microglial activation. (Gu et al., 2016; Jing et al., 2019). These experiments suggested that the P2Y12R could be a potential therapeutic target for the SCI treatment.MRS2395 is a P2Y12 receptor inhibitor, and prior research has shown that P2Y12R inhibitors can reduce neuropathic pain caused by peripheral nerve injury (Jing et al., 2019). To our knowledge, no modulation effect of P2Y12R in SCI mice has been reported before.

Here, we conducted a series of experiments to explore the relationship between P2Y12R and SCI. Firstly, to examine whether the expression of P2Y12R is related to SCI, we established the SCI model and performed the upregulation of P2Y12R and co-localization with microglia and neurons. Later, after the administration of selective inhibitor MRS2395 confirmed that MRS2395 enhanced motor function recovery and injured tissue was repair. Finally, to elucidate the potential underlying molecular mechanisms, we examined myelin sheath and the expression levels of apoptosis, and astrocyte scar formation related proteins. Therefore, the results revealed that overexpression of P2Y12R could cause motor dysfunction in SCI mice and suggested that P2Y12R may be a new target for the treatment of SCI.