Infiltration and persistence of lymphocytes during late-stage cerebral ischemia in middle cerebral artery occlusion and photothrombotic stroke models

Background

Evidence suggests that brain infiltration of lymphocytes contributes to acute neural injury after cerebral ischemia. However, the spatio-temporal dynamics of brain-infiltrating lymphocytes during the late stage after cerebral ischemia remains unclear.

Methods

C57BL/6 (B6) mice were subjected to sham, photothrombosis, or 60-min transient middle cerebral artery occlusion (MCAO) procedures. Infarct volume, neurodeficits, production of reactive oxygen species (ROS) and inflammatory factors, brain-infiltrating lymphocytes, and their activation as well as pro-inflammatory cytokine IFN-γ production were assessed. Brain-infiltrating lymphocytes were also measured in tissue sections from post-mortem patients after ischemic stroke by immunostaining.

Results

In mice subjected to transient MCAO or photothrombotic stroke, we found that lymphocyte infiltration persists in the ischemic brain until at least day 14 after surgery, during which brain infarct volume significantly diminished. These brain-infiltrating lymphocytes express activation marker CD69 and produce proinflammatory cytokines such as IFN-γ, accompanied with a sustained increase of reactive oxygen species (ROS) and inflammatory cytokines release in the brain. In addition, brain-infiltrating lymphocytes were observed in post-mortem brain sections from patients during the late stage of ischemic stroke.

Conclusion

Our results demonstrate that brain-infiltration of lymphocytes persists after the acute stage of cerebral ischemia, facilitating future advanced studies to reveal the precise role of lymphocytes during late stage of stroke.

     

Phosphorylation-triggered CUEDC2 degradation promotes UV-induced G1 arrest through APC/CCdh1 regulation

DNA damage triggers cell cycle arrest to provide a time window for DNA repair. Failure of arrest could lead to genomic instability and tumorigenesis. DNA damage-induced G₁ arrest is generally achieved by the accumulation of Cyclin-dependent kinase inhibitor 1 (p21). However, p21 is degraded and does not play a role in UV-induced G₁ arrest. The mechanism of UV-induced G₁ arrest thus remains elusive. Here, we have identified a critical role for CUE domain-containing protein 2 (CUEDC2) in this process. CUEDC2 binds to and inhibits anaphase-promoting complex/cyclosome-Cdh1 (APC/C^Cdh1), a critical ubiquitin ligase in G₁ phase, thereby stabilizing Cyclin A and promoting G₁–S transition. In response to UV irradiation, CUEDC2 undergoes ERK1/2-dependent phosphorylation and ubiquitin-dependent degradation, leading to APC/C^Cdh1-mediated Cyclin A destruction, Cyclin-dependent kinase 2 inactivation, and G₁ arrest. A nonphosphorylatable CUEDC2 mutant is resistant to UV-induced degradation. Expression of this stable mutant effectively overrides UV-induced G₁–S block. These results establish CUEDC2 as an APC/C^Cdh1 inhibitor and indicate that regulated CUEDC2 degradation is critical for UV-induced G₁ arrest.DNA damage induced by various genotoxic stresses can jeopardize genomic integrity. UV light is the most pervasive environmental DNA-damaging agent, and accumulating evidence indicates that overexposure to UV light would increase the risk of skin cancer development. To maintain genomic stability, DNA damage response triggers cell cycle arrest, especially G₁ arrest, which allows time for DNA repair and prevents aberrant replication of damaged DNA (1). Timely down-regulation of cell cycle promoters and rapid accumulation of cell cycle inhibitors are critical for DNA damage-induced G₁ arrest. Earlier studies have indicated that the DNA damage-induced G₁ arrest is mainly achieved by protein 53 (p53) activation and the subsequent p21 accumulation. However, Cyclin-dependent kinase inhibitor 1 (p21) is degraded following UV irradiation and does not play a role in this process (2). Thus, the molecular mechanism underlying UV-induced G₁ arrest is not fully understood. Understanding the regulation of UV-induced G₁ arrest will ultimately help to develop novel strategies for skin cancer prevention and therapy.The anaphase-promoting complex or cyclosome (APC/C), a multisubunit E3 ubiquitin ligase, is an important regulator of protein degradation during the cell cycle. Activation of APC/C requires the association of either cell division cycle protein 20 (Cdc20) or Cdc20 homolog 1 (Cdh1), two related coactivators that recognize specific substrates containing the destruction box (D-box) or the lysine(K)-glutamic acid(E)-asparagine(N) (KEN) motif (3–5). Cdc20 functions in early mitosis, whereas Cdh1 has crucial functions in both late mitosis and G₁ by targeting multiple cell cycle regulators, such as Cyclin A, Cyclin B1, and S-phase kinase-associated protein 2 (Skp2), for degradation (3, 4, 6–9). The destruction of Cyclin A and Skp2 prevents Cyclin-dependent kinase 2 (CDK2) activation and premature entry into S phase. To enter S phase, APC/C^Cdh1 must be turned off to allow for the reaccumulation of Cyclin A and Skp2 (10–12). However, how APC/C^Cdh1 is switched off is not fully understood. Recent studies have indicated that APC/C^Cdh1 is activated in response to DNA damage stress including UV irradiation and is crucial for maintaining genomic integrity (13–16). The underlying mechanism for APC/C^Cdh1 activation in DNA damage response also remains largely unknown.CUE-domain-containing protein 2 (CUEDC2) plays critical roles in several important signaling pathways (17–21). Our recent work has demonstrated that CUEDC2 is phosphorylated by CDK1 and promotes spindle checkpoint inactivation through releasing APC/C^Cdc20 from checkpoint inhibition during mitosis (19). In the current study, we show that CUEDC2 exists in nonphosphorylated form in G₁ phase, and inhibits APC/C^Cdh1 activity through binding to Cdh1 in a KEN-box–dependent manner. Upon UV treatment, ERK1/2 mediates CUEDC2 phosphorylation and triggers its degradation. Destruction of CUEDC2 releases APC/C^Cdh1 activity, resulting in Cyclin A destruction, CDK2 inactivation, and G₁ arrest. A nonphosphorylatable stable CUEDC2 mutant overrides UV-induced G₁ arrest. Collectively, our results identify CUEDC2 as a regulator of APC/C^Cdh1 and implicate its regulated degradation as an important mechanism for UV-induced G₁ arrest.     

Agaritine and its derivatives are potential inhibitors against HIV proteases

Agaritine, or beta-N-[gamma-L(+)-glutamyl]-4-hydroxymethylphenylhydrazine, is a Chinese herbal medicine, known having the antiviral and anticancer function. However, so far no reports whatsoever have been made for its potential as an anti-HIV agent. It was observed by docking experiments for more than 9,000 compounds extracted from various Chinese medicines that the compound agaritine distinguished itself from all the others in binding to the HIV protease with the most favorable free energy. Based on this, a series of derivatives were generated by modifying agaritine. It has been observed thru an extensive docking study that some of agaritine derivatives had markedly stronger binding interaction with the HIV protease than agaritine, suggesting that these derivatives might be good candidates for developing drugs for AIDS therapy.     

可吸收医用膜在腹腔镜治疗输卵管远端粘连阻塞术后放置的疗效评价

目的:观察可吸收医用膜在腹腔镜治疗输卵管远端粘连阻塞性不孕中的疗效。方法:于腹腔镜下分离粘连,恢复解剖结构,并进行伞端成形术、袖口状造口术及病灶清除术等综合治疗,术后放置医用膜的61例为观察组,术后不放置医用膜的76例为对照组。结果:观察组和对照组术后2年内总妊娠率、轻度粘连妊娠率、轻度粘连输卵管通畅率、总输卵管通畅率和宫内总妊娠率等比较差异均有统计学意义(P<0.05)。结论:可吸收医用膜在腹腔镜手术治疗输卵管远端粘连阻塞性不孕中有效,尤其对轻度粘连效果更好。     

来氟米特在离体皮肤中的渗透代谢

目的：研究来氟米特在离体皮肤中的渗透代谢行为,为其经皮给药制剂的研制提供依据,方法：采用Ｖａｌｉａ－Ｃｈｉｅｎｒ扩大散池,以体外昆明种小鼠和ＳＤ大鼠皮肤为屏障进行渗透代实验,适时联样,ＨＰＬＣ法测定来氟米特和Ａ７７１７２６的浓度,结果;来氟米特在不同皮肤中的渗透代谢特性明显不同,来氟米特和Ａ７７１７２６的累积量均随时间延长而呈线性增加,而来氟米特本身渗透速率时分解产物的水平反而高,结论：Ａ７７１７２６是在来氟米特透皮过程中分解产生的,但皮肤的代谢屏障对于来氟米特的渗透总速率影响较小。     

血管性认知障碍及其功能影像学的研究进展

血管性认知障碍（vascular cognitive impairment,VCI）患病率逐年增加,现已成为老年人群中主要的致残原因,但目前尚缺乏有效的治疗手段。近年来磁共振成像技术迅速发展,尤其是功能影像学,如血氧水平依赖功能磁共振（blood oxygen level dependent functional magnetic resonance i magi ng,BLOD-fMRI）、磁共振波谱技术(magnetic resonance spectroscopy,MRS)、灌注加权成像技术（perfusion weighted imaging,PWI）等,以其高分辨、非创伤性等优点对患者的脑功能进行评价,为血管性认知障碍早期识别、鉴别诊断及评估预后等方面提供了影像学依据。     

两种方法治疗急性闭角型青光眼合并白内障的疗效对比

目的:探讨白内障超声乳化摘除术以及白内障超声乳化摘除联合改良式房角分离术治疗急性闭角型青光眼合并白内障的效果.方法:急性闭角型青光眼合并白内障的患者84眼随机分为两组,对照组行白内障超声乳化摘除术,观察组行白内障超声乳化摘除联合改良式房角分离术,观察两组术前及术后的眼压、最佳矫正视力、中央前房深度、前房角宽度和瞳孔大小.结果:术后所有病例眼压明显低于术前眼压,具有统计学意义(P＜0.05);83眼视力较术前均有不同程度的改善,1眼视力较术前无明显提高;前房深度及前房角宽度均较术前明显增加.对照组与观察组相比,术后视力及中央前房深度无明显差异,观察组在眼压控制、增加前房角宽度方面具有较好疗效.结论:白内障超声乳化摘除术以及联合改良式房角分离术能有效治疗急性闭角型青光眼合并白内障,其中白内障超声乳化摘除联合房角分离术能更有效的降低眼压及增加前房角宽度.     

Alterations of natural killer cells in traumatic brain injury

To investigate the relationship between natural killer（NK） cells and traumatic brain injury（TBI）, we tracked an established phenotype of circulating NK cells at several time points in patients with different grades of TBI. In serial peripheral blood samples, NK cells were prospectively measured by flow cytometry of CD3-CD56＋ lymphocytes. Compared to healthy controls, TBI patients had reductions in both the percentage and the absolute number of NK cells. Furthermore, the magnitude of NK cell reduction correlated with the degree of TBI severity at several time points. That is, NK cell population size was independently associated with lower Glasgow Coma Scale scores. In addition, at some time points, a positive correlation was found between the NK cell counts and Glasgow Outcome Scale scores. Our results indicate that TBI induces a reduction in the number of NK cells, and the magnitude of the reduction appears to parallel the severity of TBI.     

Downregulation of miR-491-5p promotes neovascularization after traumatic brain injury

Micro RNA-491-5 p(miR-491-5 p) plays an important role in regulating cell proliferation and migration;however,the effect of miR-491-5 p on neovascularization after traumatic brain injury remains poorly understood.In this study,a controlled cortical injury model in C57 BL/6 mice and an oxygen-glucose deprivation model in microvascular endothelial cells derived from mouse brain were established to simulate traumatic brain injury in vivo and in vitro,respectively.In the in vivo model,quantitative real-time-polymerase chain reaction results showed that the expression of miR-491-5 p increased or decreased following the intracerebroventricular injection of an miR-491-5 p agomir or antagomir,respectively,and the expression of miR-491-5 p decreased slightly after traumatic brain injury.To detect the neuroprotective effects of miR-491-p,neurological severity scores,Morris water maze test,laser speckle techniques,and immunofluorescence staining were assessed,and the results revealed that miR-491-5 p downregulation alleviated neurological dysfunction,promoted the recovery of regional cerebral blood flow,increased the number of lectin-stained microvessels,and increased the survival of neurons after traumatic brain injury.During the in vitro experiments,the potential mechanism of miR-491-5 p on neovascularization was explored through quantitative real-time-polymerase chain reaction,which showed that miR-491-5 p expression increased or decreased in brain microvascular endothelial cells after transfection with an miR-491-5 p mimic or inhibitor,respectively.Dual-luciferase reporter and western blot assays verified that metallothionein-2 was a target gene for miR-491-5 p.Cell counting kit 8(CCK-8) assay,flow cytometry,and 2′,7′-dichlorofluorescein diacetate(DCFH-DA) assay results confirmed that the downregulation of miR-491-5 p increased brain microvascular endothelial cell viability,reduced cell apoptosis,and alleviated oxidative stress under oxygen-glucose deprivation conditions.Cell scratch assay,Transwell assay,tube formation assay,and western blot assay results demonstrated that miR-491-5 p downregulation promoted the migration,proliferation,and tube formation of brain microvascular endothelial cells through a metallothionein-2-dependent hypoxia-inducible factor-1α/vascular endothelial growth factor pathway.These findings confirmed that miR-491-5 p downregulation promotes neovascularization,restores cerebral blood flow,and improves the recovery of neurological function after traumatic brain injury.The mechanism may be mediated through a metallothionein-2-dependent hypoxia-inducible factor-1α/vascular endothelial growth factor signaling pathway and the alleviation of oxidative stress.All procedures were approved by Ethics Committee of the First Affiliated Hospital of Chongqing Medical University,China(approval No.2020-304) on June 22,2020.     

Incomplete fluid-air exchange technique for idiopathic macular hole surgery

AIM: To explore an improved procedure involving incomplete fluid-air exchange for idiopathic macular hole (IMH), and the closure rate, visual function, and the visual field of macular holes (MHs) were evaluated. METHODS: This prospective randomized controlled study, included 40 eyes of 40 patients with IMH who were treated with pars plana vitrectomy and peeling of the internal limiting membrane. They were grouped by random digital table. Twenty-one eyes underwent incomplete fluid-air exchange (IFA) and 19 eyes underwent traditional complete fluid-air exchange (CFA) as the control group. Outcomes included best-corrected visual acuity (BCVA), intraocular pressure, and optical coherence tomography, light adaptive electroretinography, and visual field evaluations. RESULTS: All MHs <400 μm were successfully closed. BCVAs before and 6mo after surgery were 0.82±0.41 logMAR and 0.28±0.17 logMAR in IFA group and 0.86±0.34 logMAR and 0.34±0.23 logMAR in CFA group, respectively. The electroretinogram analysis of patients in IFA group revealed increases in b-wave amplitudes at 1, 3, and 6mo after surgery. Additionally, patients in IFA group showed an amplitude increase of 28.6% from baseline at 6mo (P<0.05), while no obvious improvements were noted in CFA group. Although there were no statistically significant improvements in either group, the IFA group showed a slight increase in mean sensitivity (P>0.05). CONCLUSION: IFA is a reliable method that offers comparable closure rate to CFA and facilitates improvements in visual function.