Despite many years of in-depth research on the pathophysiology of ischemic stroke and its treatment methods, it remains the leading cause of death and disability for patients worldwide. Therefore, the search for new methods of prevention, diagnosis, and treatment of ischemic stroke appears to be an extremely important goal for further scientific research.
TP plays a significant role in various physiological and pathological processes in the human body, where is involved in the degradation of nucleosides and the pyrimidine salvage pathway. In platelets, it participates in their activation and thrombus formation, which is crucial in the pathogenesis of diseases such as heart attack, stroke, and pulmonary embolism [22]. In addition to this, this enzyme also stimulates angiogenesis [9] inhibits apoptosis [23], and promotes the proliferation of endothelial cells [24] as explored in solid tumors, including breast and colorectal cancer. Increased expression of this enzyme has also been observed in neurons of brain tissue subjected to IR [9]. Therefore, the modulation of this enzyme in both brain tissue and blood appears to significantly impact the survival of patients after ischemic stroke. This action could be achieved by protecting neurons in the ischemic area and reducing the overall systemic ability to form clots due to platelet activation.
The role of TPI in medical treatment is only to increase trifluridine's bioavailability by inhibiting its degradation [14]. In our experiment, TPI was administered as a sole drug to rats intraperitoneally at doses of 25 mg/kg and 50 mg/kg twice: during ischemia and after 8 hours of reperfusion. The results of this experiment indicate that: 1) the protein concentration of TP in serum and TP expression increased in both ischemic groups untreated and treated with a lower dose of the drug; 2) TPI at a higher dose of 50 mg/kg significantly reduced the protein concentration of TP in serum and TP expression in brain tissue; 3) MMP-9 values increased in the untreated ischemic group at both reperfusion time points (3 and 24 hours); TPI at a lower dose inhibited the growth of this parameter, especially noticeable at 24 hours of reperfusion when MMP-9 levels were significantly lower compared to the other IR groups; 4) the protein concentration of TIMP-1 slightly decreased in the ischemic groups at 3 hours of reperfusion but significantly increased at 24 hours, especially in the groups treated with TPI; 5) changes in the MMP-9/TIMP-1 ratio result from alterations in the protein concentrations of MMP-9 and TIMP-1, confirming the inhibitory effect of the investigated drug at a lower dose, particularly observed at 24 hours of reperfusion; 6) a decrease in the concentration of MMP-2 was observed in the groups subjected to IR at 24 hours of reperfusion; no influence of TPI on the changes in the level of this parameter was demonstrated.
In earlier studies using an experimental model of carotid artery thrombosis in mice and in in vitro research using both human and murine blood platelets, it was demonstrated that inhibiting TP activity with antagonists significantly prolongs the time to occlusive thrombosis in response to arterial injury. This inhibition impacts the adhesion and aggregation of blood platelets without affecting their count and homeostasis [10]. Therefore, inhibiting the thrombotic activity of TP by TPI could be a potential target for future antiplatelet and antithrombotic therapies. In our experiment, we confirmed the inhibitory effect of this drug, which manifested at a higher dose of 50 mg/kg administered intraperitoneally twice: during ischemia and after 8 hours of reperfusion. Both the expression of TP in neurons and its serum levels were the lowest among the groups subjected to IR and comparable to values obtained in the control group.
As commonly known, ischemia should induce factors with angiogenic activity, including TP, which stimulates the formation of blood vessels. In this study, it was demonstrated that both the expression of TP in brain tissue and the protein concentration in serum were induced by transient cerebral ischemia. Similar results were obtained by Hayashi et al. [9], who observed an increasing expression of TP in brain neurons during reperfusion. In this work, similar to ours, no increased vessel permeability or signs of brain edema were observed in histopathological examination. This is consistent with earlier reports where TP was not shown to cause brain edema [16]. The development of brain edema during therapy of ischemic stroke with angiogenic factors could become a significant problem. In this context, the use of TP analogs may prove to be a promising therapeutic option in the future. Conversely, the inhibition of its activity by TPI, and thus the inhibition of its angiogenic and neuroprotective effects may be expected. However, the conducted studies do not confirm any harmful effects of TPI in this regard. It has been shown that, despite partially inhibiting the growth of tumors expressing TP, TPI did not inhibit their angiogenesis [20]. Therefore, it would be crucial to thoroughly investigate the impact of inhibiting TP growth by TPI on angiogenesis and vascular permeability after transient cerebral ischemia in subsequent reperfusion.
Increasing evidence points to the significant involvement of MMPs, including MMP-2 and MMP-9, in the pathophysiology of ischemic stroke. They participate in the neuroinflammatory cascade triggered during a stroke, with the ability to proteolyze extracellular matrix elements, which constitute up to 20% of the brain volume [25, 26]. In pathological conditions, they can lead to increased blood-brain barrier (BBB) permeability by acting on the basement membrane and tight junctions in endothelial cells. They are also implicated in neuronal death, exacerbate demyelination, and contribute to damage under IR [27, 28]. The MMP-9 gene polymorphism (− 1562 C/T) has been associated with the risk of acute ischemic stroke (AIS) [29, 30]. On the other hand, MMPs, similar to a disintegrin and metalloproteinase (ADAMs), play a crucial role in neurorepair mechanisms in the recovery phases of stroke [31] by influencing vascular remodeling, synaptic plasticity, and the migration of neural stem cells [32]. In our study, the highest level of serum MMP-9 was noticeable after 3 hours of reperfusion in the untreated group subjected to IR and slightly decreased in the subsequent measurements. Meanwhile, the protein level for MMP-2 remained at a similarly low level at both 3 and 24 hours of reperfusion. Confirmation may be the analysis of the previous studies, demonstrating that the increase in MMP-9 levels occurs early after a stroke, while the rise in MMP-2 dominates in later phases [33]. In this study, Planas et al. showed that the protein level for MMP-9 increased from 4 hours to 4 days after ischemia. However, MMP-2 showed only a slight increase at 4 hours of reperfusion, while a significant surge in the expression and activation of this protein was recorded in the second phase, around 4 days later. The unexpected increase in the protein level for MMP-2 on the following day in the control group, where neck vessels were not ligated, is noteworthy. This may suggest the influence of minimal surgical manipulation, including general anesthesia, tissue discontinuity, and vessel isolation in rats, even though ischemia was not induced.
MMPs are regulated at various levels, including by TIMPs, which are small glycoproteins forming non-covalent complexes with metalloproteinases. TIMP-1 is known to inhibit MMP-9 [34, 35] primarily, and the MMP-9/TIMP-1 ratio is considered to better reflect MMP-9 activity in vivo [36]. The results of our study indicate that at 3 hours of IR TIMP-1 was initially slightly decreased, but a significant increase in this parameter is noticeable after 24 hours. As a result, the MMP-9/TIMP-1 ratio decreased in all groups subjected to IR.
Based on our results it seems that TP significantly modulates MMP-9 activity but not MMP-2. This effect is dose-dependent but independent of the drug's influence on TP. At a lower dose, the drug significantly reduced the level of MMP-9 after 24 hours of reperfusion, while such an effect was not observed with a higher dose. The drug also intensified the increase in TIMP-1 regardless of the dose used. However, the greatest decrease in the MMP-9/TIMP-1 ratio was observed in the group treated with a lower dose of TPI.
So far, many studies have been conducted using synthetic MMP inhibitors, but their results have been rather negative due to numerous side effects, given that MMPs are also essential for the functioning of many healthy cells. One of these inhibitors is minocycline. In rats with middle cerebral artery occlusion, treatment with minocycline showed a significant decrease in the expression of MMP-2 and MMP-9 in the brain compared to the control group [37]. The results of animal studies and clinical studies with minocycline in ischemic stroke are contradictory [25].
Interestingly, some studies have shown that prolonged inhibition of MMPs in animal models of cerebral ischemia reduces functional regeneration and increases brain damage [32, 38]. Therefore, it seems that therapeutic intervention inhibiting MMP-9 activity should be limited only to the early phase of stroke, as we have demonstrated with TPI.
In summary, in our experiment, we demonstrated a clear inhibitory effect of TPI on TP in brain tissue undergoing IR. We also attempted to elucidate the potential protective effects of this drug by studying its impact on key metalloproteinases involved in stroke. However, despite the benefits arising from the fact that TPI is a safe compound with known pharmacokinetics and low toxicity, approved for clinical use [18], the advantages for patients after ischemic stroke resulting from the inhibition of TP require further verification.