N-Nitro-L-Arginine-Methyl ester Exerts Neuroprotection By Inhibiting Inflammation In Cerebral Injury Induced By Transient Ischemia/Reperfusion In Rats

Cerebral ischemia-reperfusion injury is a pivotal cause of deaths due to cerebrovascular accidents. Further research efforts are needed to reveal the mechanism underlying its aggravation or alleviation. We previously reported the antioxidant effect of N-Nitro-L-Arginine-Methyl Ester (L-NAME), a nonselective nitric oxide synthase (NOS) inhibitor, on rats subjected to transient focal cerebral ischemia-reperfusion (I/R). The aim of this work was to explore further neuroprotective anti-inflammatory effects of L-NAME. This study involved 30 adult male Wistar rats divided into three groups with ten rats in each: sham-operated (control), I/R group of rats subjected to 30 minutes of left common carotid artery (CCA) occlusion followed by 24-hour of reperfusion and test group infused with L-NAME intraperitoneally 15 minutes before the same I/R period. Neurological assessments were evaluated, Western blotting used to estimate Nuclear factor-kappa B (NF-қB), ELISA used to detect Tumor necrosis factor- α (TNF-α), and Nitric oxide metabolites were measured colorimetrically, as well as H&E staining to assess brain damage. Compared with the I/R group, the neurological score, infarction area, and the inflammatory biomarkers NF-қB, TNF-α, and NO were significantly decreased in L-NAME treated rats (P ≤0.001). As a conclusion from the current study, L-NAME showed potential neuroprotection through it is an anti-inflammatory effect on a rat’s model of transient focal cerebral ischemia-reperfusion.

Effect of Tetramethylpyrazine on Neuroplasticity after Transient Focal Cerebral Ischemia Reperfusion in Rats

Tetramethylpyrazine (TMP) has been widely used in ischemic stroke in China. The regulation of neuroplasticity may underlie the recovery of some neurological functions in ischemic stroke. Middle cerebral artery occlusion (MCAO) model was established in this study. Rats were divided into three groups: sham group, model group, and TMP group. The neurological function was evaluated using modified neurological severity score (mNSS). Following the neurological function test, expression of synaptophysin (SYP) and growth-associated protein 43 (GAP-43) were analyzed through immunohistochemistry at 3 d, 7 d, 14 d, and 28 d after MCAO. Finally, the synaptic structural plasticity was investigated using transmission electron microscopy (TEM). The TMP group showed better neurological function comparing to the model group. SYP levels increased gradually in ischemic penumbra (IP) in the model group and could be enhanced by TMP treatment at 7 d, 14 d, and 28 d, whereas GAP-43 levels increased from 3 d to 7 d and thereafter decreased gradually from 14 d to 28 d in the model group, which showed no significant improvement in the TMP group. The results of TEM showed a flatter synaptic interface, a thinner postsynaptic density (PSD), and a wider synaptic cleft in the model group, and the first two alterations could be ameliorated by TMP. Then, a Pearson’s correlation test revealed mNSS markedly correlated with SYP and synaptic ultrastructures. Taken together, TMP is capable of promoting functional outcome after ischemic stroke, and the mechanisms may be partially associated with regulation of neuroplasticity.

NLRP3 Inflammasome Activation Is Involved in LPA1-Mediated Brain Injury after Transient Focal Cerebral Ischemia

Lysophosphatidic acid receptor 1 (LPA1) contributes to brain injury following transient focal cerebral ischemia. However, the mechanism remains unclear. Here, we investigated whether nucleotide-binding oligomerization domain-like receptor family pyrin domain containing 3 (NLRP3) inflammasome activation might be an underlying mechanism involved in the pathogenesis of brain injury associated with LPA1 following ischemic challenge with transient middle cerebral artery occlusion (tMCAO). Suppressing LPA1 activity by its antagonist attenuated NLRP3 upregulation in the penumbra and ischemic core regions, particularly in ionized calcium-binding adapter molecule 1 (Iba1)-expressing cells like macrophages of mouse after tMCAO challenge. It also suppressed NLRP3 inflammasome activation, such as caspase-1 activation, interleukin 1β (IL-1β) maturation, and apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC) speck formation, in a post-ischemic brain. The role of LPA1 in NLRP3 inflammasome activation was confirmed in vitro using lipopolysaccharide-primed bone marrow-derived macrophages, followed by LPA exposure. Suppressing LPA1 activity by either pharmacological antagonism or genetic knockdown attenuated NLRP3 upregulation, caspase-1 activation, IL-1β maturation, and IL-1β secretion in these cells. Furthermore, nuclear factor-κB (NF-κB), extracellular signal-regulated kinase 1/2 (ERK1/2), and p38 were found to be LPA1-dependent effector pathways in these cells. Collectively, results of the current study first demonstrate that LPA1 could contribute to ischemic brain injury by activating NLRP3 inflammasome with underlying effector mechanisms.

Placental Mitochondria Therapy for Cerebral Ischemia-Reperfusion Injury in Mice

Background and Purpose: There is an urgent need to develop adjunct therapies that can be added onto reperfusion for acute ischemic stroke. Recently, mitochondrial transplantation has emerged as a promising therapeutic approach for boosting brain tissue protection. In this proof-of-concept study, we investigate the feasibility of using placenta as a source for mitochondrial transplantation in a mouse model of transient focal cerebral ischemia-reperfusion. Methods: Mitochondria-enriched fractions were isolated from cryopreserved mouse placenta. Mitochondrial purity and JC1 membrane potentials were assessed by flow cytometry. Adenosine triphosphate and mitochondrial proteins were measured by luminescence intensity and western blot, respectively. Therapeutic efficacy of mitochondrial fractions was assessed in a mouse model of transient focal cerebral ischemia-reperfusion. Results: Flow cytometry analysis demonstrated that about 87% of placental mitochondria were viable and maintained JC1 membrane potentials after isolation. Placental mitochondrial fractions contained adenosine triphosphate equivalent to mitochondrial fractions isolated from skeletal muscle and brown fat tissue. Normalized mitochondrial antioxidant enzymes (glutathione reductase, MnSOD [manganese superoxide dismutase]) and HSP70 (heat shock protein 70) were highly preserved in placental mitochondrial fractions. Treatment with placental mitochondrial fractions immediately after reperfusion significantly decreased infarction after focal cerebral ischemia in mice. Conclusions: Cryopreserved placenta can be a feasible source for viable mitochondrial isolation. Transplantation with placental mitochondria may amplify beneficial effects of reperfusion in stroke.