Clopidogrel, which is one of the most prescribed antiplatelet medications in the world, is given to stroke survivors for the prevention of secondary cardiovascular events. Clopidogrel exerts its antiplatelet activity via antagonism of the P2Y12 receptor (P2RY12). Although not widely known or considered during the initial clinical trials for clopidogrel, P2RY12 is also expressed on microglia, which are the brain’s immune cells, where the receptor facilitates chemotactic migration toward sites of cellular damage. If microglial P2RY12 is blocked, microglia lose the ability to migrate to damaged sites and carry out essential repair processes. We aimed to investigate whether administering clopidogrel to mice post-stroke was associated with (i) impaired motor skills and cognitive recovery; (ii) physiological changes, such as survival rate and body weight; (iii) changes in the neurovascular unit, including blood vessels, microglia, and neurons; and (iv) changes in immune cells. Photothrombotic stroke (or sham surgery) was induced in adult male mice. From 24 h post-stroke, mice were treated daily for 14 days with either clopidogrel or a control. Cognitive performance (memory and learning) was assessed using a mouse touchscreen platform (paired associated learning task), while motor impairment was assessed using the cylinder task for paw asymmetry. On day 15, the mice were euthanized and their brains were collected for immunohistochemistry analysis. Clopidogrel administration significantly impaired learning and memory recovery, reduced mouse survival rates, and reduced body weight post-stroke. Furthermore, clopidogrel significantly increased vascular leakage, significantly increased the number and appearance of microglia, and significantly reduced the number of T cells within the peri-infarct region post-stroke. These data suggest that clopidogrel hampers cognitive performance post-stroke. This effect is potentially mediated by an increase in vascular permeability post-stroke, providing a pathway for clopidogrel to access the central nervous system, and thus, interfere in repair and recovery processes.

The mechanism by which human labor is initiated in the presence of elevated circulating progesterone levels remains unknown. Recent evidence indicates that the progesterone-metabolizing enzyme, 20α-hydroxysteroid dehydrogenase (20α-HSD), encoded by the gene AKR1C1, may contribute to functional progesterone withdrawal. We found that AKR1C1 expression significantly increased with labor onset in term myometrium, but not in preterm myometrium. Among preterm laboring deliveries, clinically diagnosed chorioamnionitis was associated with significantly elevated AKR1C1 expression. AKR1C1 expression positively correlated with BMI before labor and negatively correlated with BMI during labor. Analysis by fetal sex showed that AKR1C1 expression was significantly higher in women who delivered male babies compared to women who delivered female babies at term, but not preterm. Further, in pregnancies where the fetus was female, AKR1C1 expression positively correlated with the mother’s age and BMI at the time of delivery. In conclusion, the increase in myometrial AKR1C1 expression with term labor is consistent with 20α-HSD playing a role in local progesterone metabolism to promote birth. Interestingly, this role appears to be specific to term pregnancies where the fetus is male. Upregulated AKR1C1 expression in the myometrium at preterm in-labor with clinical chorioamnionitis suggests that increased 20α-HSD activity is a mechanism through which inflammation drives progesterone withdrawal in preterm labor. The link between AKR1C1 expression and maternal BMI may provide insight into why maternal obesity is often associated with dysfunctional labor. Higher myometrial AKR1C1 expression in male pregnancies may indicate fetal sex-related differences in the mechanisms that precipitate labor onset at term.

Preterm birth is a common cause of neonatal mortality and morbidity, which are increasingly prevalent today. Preterm birth affects millions of babies worldwide and is the subject of intense study in its pathophysiology and therapy. Tocolytic agents are drugs used to prevent preterm birth and prolong pregnancy, to allow the fetus to remain in utero for further maturation, permit antenatal corticosteroid treatment to accelerate fetal lung maturity, and allow enough time for in uter o-transfer of the baby to a tertiary care facility. However, the application of traditional tocolytic therapy is often limited due to maternal and fetal side effects. As such, there is a pressing need for safer and more effective tocolytic therapy. Combinational tocolysis has the potential to improve tocolytic efficacy and reduce side effects. A limited number of papers have revealed the prospective benefits of combinational tocolysis by drug synergism and reduced drug toxicity. This review summarizes the current landscape of combinational tocolysis directed at achieving tocolytic synergism and examines the quantitative methods utilized for detecting synergistic tocolytic combinations.

Metabolic inactivation of progesterone within uterine myocytes by 20α-hydroxysteroid dehydrogenase (20α-HSD) has been postulated as a mechanism contributing to functional progesterone withdrawal at term. In humans, 20α-HSD is encoded by the gene AKR1C1. Myometrial AKR1C1 mRNA abundance has been reported to increase significantly during labor at term. In spontaneous preterm labor, however, we previously found no increase in AKR1C1 mRNA level in the myometrium except for preterm labor associated with clinical chorioamnionitis. This suggests that increased 20α-HSD activity is a mechanism through which inflammation drives progesterone withdrawal in preterm labor. In this study, we have determined the effects of various treatments of therapeutic relevance on AKR1C1 expression in pregnant human myometrium in an ex vivo culture system. AKR1C1 expression increased spontaneously during 48 h culture (p < 0.0001), consistent with the myometrium transitioning to a labor-like phenotype ex vivo, as reported previously. Serum supplementation, prostaglandin F_2α, phorbol myristate acetate, and mechanical stretch had no effect on the culture-induced increase, whereas progesterone (p = 0.0058) and cAMP (p = 0.0202) further upregulated AKR1C1 expression. In contrast, culture-induced upregulation of AKR1C1 expression was dose-dependently repressed by three histone/protein deacetylase inhibitors: trichostatin A at 5 (p = 0.0172) and 25 µM (p = 0.0115); suberoylanilide hydroxamic acid at 0.5 (p = 0.0070), 1 (p = 0.0045), 2.5 (p = 0.0181), 5 (p = 0.0066) and 25 µM (p = 0.0014); and suberoyl bis-hydroxamic acid at 5 (p = 0.0480) and 25 µM (p = 0.0238). We propose the inhibition of histone/protein deacetylation helps to maintain the anti-inflammatory, pro-quiescence signaling of progesterone in pregnant human myometrium by blocking its metabolic inactivation. Histone deacetylase inhibitors may represent a class of agents that preserve or restore the progesterone sensitivity of the pregnant uterus.

The methodological approaches used to evaluate the inhibitory effects (potency determination) of tocolytic agents on human uterine contractions in ex vivo systems vary, and none of the methodologies has been subjected to scrutiny in terms of objectivity, variability, and reliability. Therefore, this study aimed to assess and compare the reliability of using area under the curve (AUC) versus amplitude alone-based assessment of contraction traces to generate concentration-response curves, which were then used to calculate the tocolytic concentrations that inhibited 25% (IC₂₅) and 50% (IC₅₀) of baseline contractility. The accuracy of the determined inhibitory concentrations (IC) was then scrutinized while taking into consideration the contraction parameters (amplitude, frequency, duration) affected by the different tocolytics. To do this, pregnant human myometrial strips (term, not-in-labor) were treated with cumulative concentrations of the contraction-blocking agents, nifedipine (NIF), indomethacin (IND), 2-aminoethoxydiphenyl borate (2-APB), glycyl-h-1152 (GH), aminophylline (AMP), or rolipram (ROL)). Concentration-response curves were generated using either AUC or amplitude alone as the index of contraction inhibition, from which tocolytic IC₂₅ and IC₅₀ concentrations were calculated and compared (i.e. IC_25(AUC) vs. IC_{25(Amplitude)}). The effects of each tocolytic on contraction frequency were also quantified. To assess accuracy, each tocolytic was applied to contracting strips as a single treatment at their respective IC_{25(AUC/Amplitude)} and IC_{50(AUC/Amplitude)}, and then the inhibitory effect re-quantified against both contraction AUC and amplitude alone. Significant differences between IC_50(AUC) and IC_{50(Amplitude)} were detected for AMP (318.5 vs. 450 µM), ROL (4.3 vs. 55 µM), and IND (59.5 vs. 75 µM), whereas AUC versus amplitude-based IC₅₀ concentrations were comparable for NIF (10 vs. 10 nM), GH (18.2 vs. 15 µM), and 2-APB (53 vs. 57 µM). Similarly, the determined IC_25(AUC) and IC_{25(Amplitude)} were again significantly different for AMP (175 vs. 277 µM), ROL (515 nM vs. 15 µM), and IND (28 vs. 42 µM), but also for 2-APB (27 vs. 40 µM). The confirmation studies revealed that a single dose of the tocolytics at their determined IC_25(AUC) and IC_50(AUC) concentrations consistently reduced contraction AUC by approximately 25% and 50%, respectively, whereas single doses of the IC_{25(Amplitude)} and IC_{50(Amplitude)} concentrations revealed inconsistent results. Of the six tocolytics, four reduced contraction amplitude by > 25% when applied as a single dose at IC_{25(Amplitude)}, while three reduced contraction amplitude by > 50% when applied at IC_{50(Amplitude)}. Our experimental data indicate that when determining tocolytic potency, assessment of ex vivo contraction traces via AUC is more accurate and consistent than assessment via amplitude alone. Our study finds that whilst tocolytics that increase contraction frequency (while decreasing amplitude) can be assessed by either AUC or amplitude-based determination, tocolytics that either significantly reduce or largely do not affect contraction frequency should only be analyzed via AUC.

Clopidogrel has been the most commonly used therapy for preventing secondary cardiovascular events since 1997 by inhibiting the purinergic receptor P2Y, G-protein coupled, 12 protein receptor (P2RY12). P2RY12 is critical for microglia function in the brain, where it facilitates repair processes following injury. Under normal conditions, the blood-brain barrier (BBB) prevents peripheral drugs like clopidogrel from entering the brain. However, stroke-induced BBB disruption may allow clopidogrel to interfere with neural recovery by impairing microglia activity. Recently, we demonstrated that clopidogrel worsened cognitive outcomes in young mice after stroke. In this study, we examined the effects of clopidogrel on aged mice, focusing on survival, body weight, neurovascular changes, immune response, and amyloid beta accumulation. Aged male mice underwent photothrombotic stroke (or sham surgery) and received daily clopidogrel or control treatment for 14 days. On day 15, brain tissue was analyzed. Clopidogrel treatment significantly reduced survival and body weight, decreased vessel density, increased vascular permeability, altered microglia activity, and increased amyloid beta levels in the peri-infarct region. Notably, some of these effects were not observed in young mice. These results suggest that BBB disruption in stroke mice enables clopidogrel to enter the central nervous system, where it impairs microglia-mediated restoration of BBB integrity and promotes amyloid accumulation, factors that may contribute to worsened cognitive recovery. This study raises the possibility that clopidogrel may similarly cross the BBB in older stroke patients, impacting microglial function, and emphasizes the need for further research into its mechanisms of action.