Chronic opioid use disorder patients often also use other substances such as amphetamines. The gene-based analysis method was applied in the genomic database obtained from our previous study with 343 methadone maintenance treatment (MMT) patients. We found that the gene encoding gamma-aminobutyric acid type A receptors (GABA-A receptor) delta subunit isoforms (GABRD) was associated with amphetamine use in heroin dependent patients under MMT in Taiwan. A total of 15% of the 343 MMT patients tested positive for amphetamine in the urine toxicology test. Two genetic variants in the GABRD, rs2889475 and rs2376805, were found to be associated with the positive urine amphetamine test. They are located in the exon 1 of the splice variant and altered amino acid compositions (T126I, C/T, for rs2889475, and R252Q, G/A, for rs2376805). The CC genotype carriers of rs2889475 showed a four times higher risk of amphetamine use than those with TT genotype. The GG genotype carriers of rs2376805 showed a three times higher risk of amphetamine use than the AA genotype carriers. To our knowledge, this is the first report that demonstrated an association of the delta splice variant isoform in the GABA-A receptor with an increased risk of amphetamine use in MMT patients. Our results suggest that rs2889475 and rs2376805 may be indicators for the functional role and risk of amphetamine use in MMT patients.

Oxidative stress is a key contributor to the pathogenesis of stroke-reperfusion injury. Neuroinflammatory peptides released after ischemic stroke mediate reperfusion injury. Previous studies, including ours, have shown that lipocalin-2 (LCN2) is secreted in response to cerebral ischemia to promote reperfusion injury. Genetic deletion of LCN2 significantly reduces brain injury after stroke, suggesting that LCN2 is a mediator of reperfusion injury and a potential therapeutic target. Immunotherapy has the potential to harness neuroinflammatory responses and provides neuroprotection against stroke. Here we report that LCN2 was induced on the inner surface of cerebral endothelial cells, neutrophils, and astrocytes that gatekeep the blood–brain barrier (BBB) after stroke. LCN2 monoclonal antibody (mAb) specifically targeted LCN2 in vitro and in vivo, attenuating the induction of LCN2 and pro-inflammatory mediators (iNOS, IL-6, CCL2, and CCL9) after stroke. Administration of LCN2 mAb at 4 h after stroke significantly reduced neurological deficits, cerebral infarction, edema, BBB leakage, and infiltration of neutrophils. The binding epitope of LCN2 mAb was mapped to the β3 and β4 strands, which are responsible for maintaining the integrity of LCN2 cup-shaped structure. These data indicate that LCN2 can be pharmacologically targeted using a specific mAb to reduce reperfusion injury after stroke.

Cholinergic neurotransmission regulates the immune response and inhibits cytokine release after stroke. The changes in the level/activity of blood cholinesterase (ChE) in patients with post-stroke dementia (PSD) are less known. This study aimed to examine post-stroke plasma acetylcholinesterase (AChE) and butylcholinesterase (BChE) and determine whether they are biomarkers for PSD. Thirty patients with PSD, 87 post-stroke patients without dementia (PSNoD), and 117 age- and gender-matched healthy controls were recruited. Missense genetic variants AChE rs1799806 and BChE rs1803274 were genotyped. The plasma AChE level did not differ between the PSD and PSNoD groups. However, BChE levels were significantly lower in the PSD than in the PSNoD group (3300.66 ± 515.35 vs 3855.74 ± 677.60 ng/mL, respectively; p = 0.0033). The activities of total ChE, BChE, and AChE were all lower in the PSD group (19,563.33 ± 4366.03, 7650.17 ± 1912.29, 11,913.17 ± 2992.42 mU/mL, respectively) than in the PSNoD group (23,579.08 ± 5251.55, 9077.72 ± 1727.28, and 14,501.36 ± 4197.17 mU/mL, respectively). When further adjusting for age and sex, significance remained in BChE level and activity and in total ChE activity. BChE rs1803274 was associated with reduced BChE activity, while AChE rs1799806 did not influence AChE activity. The level and activity of BChE, but not of AChE, were decreased in PSD patients and may therefore aid in PSD diagnosis.

Background: Hemorrhagic transformation (HT) is a serious complication after endovascular thrombectomy (EVT) for patients with acute ischemic stroke (AIS). We analyzed the plasma levels of MMP-9 before and after EVT and assessed the temporal changes of MMP-9 that may be associated with, and therefore predict, HT after EVT.Methods: We enrolled 30 AIS patients who received EVT, and 16 (53.3%) developed HT. The levels of MMP-9 in plasma collected from the arteries of AIS patients before and immediately after EVT were measured using ELISA. The percent change in MMP-9 after EVT (after/before) was calculated and compared between patients with and without HT.Results: The median age of the AIS patients was 70 years, and 13 patients (43.3%) were men. The median National Institutes of Health Stroke Scale (NIHSS) scores of patients with HT were 18 on admission and 18 after EVT. The median NIHSS scores of patients without HT were 17 on admission and 11 after EVT. Patients with HT demonstrated significantly greater percentage increases in arterial MMP-9 levels after EVT.Conclusion: Patients with AIS who developed HT had significantly increased arterial MMP-9 levels after EVT, suggesting that the upregulation of MMP-9 following EVT could serve as a predictive biomarker for HT.

Transient global cerebral ischemia (tGCI) resulting from cardiac arrest causes selective neurodegeneration in hippocampal CA1 neurons. Although the effect is clear, the underlying mechanisms directing this process remain unclear. Previous studies have shown that phosphorylation of Erk1/2 promotes cell survival in response to tGCI. DUSP6 (also named MKP3) serves as a cytosolic phosphatase that dephosphorylates Erk1/2, but the role of DUSP6 in tGCI has not been characterized. We found that DUSP6 was specifically induced in the cytoplasm of hippocampal CA1 neurons 4 to 24 h after tGCI. DUSP6-deficient mice showed normal spatial memory acquisition and retention in the Barnes maze. Impairment of spatial memory acquisition and retention after tGCI was attenuated in DUSP6-deficient mice. Neurodegeneration after tGCI, revealed by Fluoro-Jade C and H&E staining, was reduced in the hippocampus of DUSP6-deficient mice and DUSP6 deficiency enhanced the phosphorylation and nuclear translocation of Erk1/2 in the hippocampal CA1 region. These data support the role of DUSP6 as a negative regulator of Erk1/2 signaling and indicate the potential of DUSP6 inhibition as a novel therapeutic strategy to treat neurodegeneration after tGCI.

Lipocalin‐2 (LCN2) has been implicated in promoting apoptosis and neuroinflammation in neurological disorders; however, its role in neural transplantation remains unknown. In this study, we cultured and differentiated Lund human mesencephalic (LUHMES) cells into human dopaminergic‐like neurons and found that LCN2 mRNA was progressively induced in mouse brain after the intrastriatal transplantation of human dopaminergic‐like neurons. The induction of LCN2 protein was detected in a subset of astrocytes and neutrophils infiltrating the core of the engrafted sites, but not in neurons and microglia. LCN2‐immunoreactive astrocytes within the engrafted sites expressed lower levels of A1 and A2 astrocytic markers. Recruitment of microglia, neutrophils, and monocytes after transplantation was attenuated in LCN2 deficiency mice. The expression of M2 microglial markers was significantly elevated and survival of engrafted neurons was markedly improved after transplantation in LCN2 deficiency mice. Brain type organic cation transporter (BOCT), the cell surface receptor for LCN2, was induced in dopaminergic‐like neurons after differentiation, and treatment with recombinant LCN2 protein directly induced apoptosis in dopaminergic‐like neurons in a dose‐dependent manner. Our results, therefore, suggested that LCN2 is a neurotoxic factor for the engrafted neurons and a modulator of neuroinflammation. LCN2 inhibition may be useful in reducing rejection after neural transplantation.