[The effects and mechanism of baicalin in a mouse acute hypertensive glaucoma model].

Objective: To explore the potential neuroprotection effects and associated mechanism of baicalin in a rodent acute hypertensive glaucoma model and oxygen-glucose deprivation/reperfusion (OGD/R) induced retinal ganglion cell (RGC) injury. Methods: Experiment research. A rapid and substantial elevation of intraocular pressure was performed to establish an acute hypertensive glaucoma model, and retinal thickness was assessed at 1, 3, 5, and 7 days. The mice were then randomly divided into three groups: normal control group, hypertension group, and baicalin (50 mg/kg) for hypertension group. The effects of baicalin on the RGCs were evaluated by retrograde transporting of Fluoro-Gold. The mRNA levels of tumor necrosis factor-α, interleukine-1ß (IL-1ß), and inducible nitric oxide synthase were detected by real-time PCR, and the protein levels were measured by Western blot in the retina tissue of acute hypertensive glaucoma model. Purified primary RGC survival under OGD/R stress was measured by flow cytometry, which was also performed to measure the survival rate of RGCs pretreated by different doses of baicalin (2.5 µmol/L, 5.0 µmol/L, and 10.0 µmol/L). The effects of baicalin on primary RGCs co-cultured with mouse microglia cell line BV2 were evaluated by flow cytometry. The cytokine IL-1ß in the culture supernatant was measured by immunochemical analyses. Statistical analysis was performed using analysis of variance. Results: Retinal tissue injuries and RGC loss were observed both in vivo and in vitro. Retinal thickness was decreased to 87.32%±0.94% at 3 days (t=6.73, P<0.01), 74.86%±2.43% at 5 days (t=13.40, P<0.01), and 63.53%±2.15% at 7 days (t=19.46, P<0.01). Treatment of 50 mg/kg baicalin significantly promoted the RGC survival from 61.32%±5.94% to 89.93%±10.08% (t=4.84, P<0.01). Baicalin alleviated the retinal damages by suppressing the expression of inflammatory cytokines as revealed by Western blot and real-time PCR. In vitro the RGC survival under OGD/R stress was increased from 51.53%±1.36% to 69.37%±7.09% and 66.23%±4.25% with 5.0, 10.0 µmol/L baicalin administration (t=5.50, 4.53; both P<0.01). BV2 under OGD/R stress did extra damage to RGCs, and baicalin could reverse the damages and increase the survival from 69.37%±7.09% to 73.00%±5.20% (t=2.82, P=0.048) by reducing the release of IL-1ß [(39.97±8.76) pg/ml vs. (61.33±5.78) pg/ml, t=4.19, P=0.010]. Conclusion: Baicalin could alleviate retina tissue injury directly and promote the survival of RGCs by downregulating the expression of inflammatory cytokines and protecting RGCs from ischemia reperfusion injury. (Chin J Ophthalmol, 2020, 56: 376-382).

[Mechanism of microglia promoting retinal ganglion cell death in vitro].

Objective: To investigate the role and mechanism of microglial activation in the process of retinal ganglion cell (RGC) death in the oxygen-glucose deprivation/reperfusion (OGD/R) model which mimicked retinal ischemia/reperfusion injury in vitro. Methods: Experimental study. Primary RGCs from C57BL/6 mice and BV2 microglia were co-cultured or cultured alone. The OGD/R model was established in vitro (reoxygenation time was set to 6 h, 24 h, 36 h, 48 h). BV2 microglial activation was assessed by immunofluorescence staining of ionized calcium binding adapter molecule 1 (iba1), and the survival rate of RGCs was detected by the Cell Counting Kit-8. The apoptosis rate of RGC was detected by using apoptosis detection kit. The levels of Toll-like receptor-4 (TLR4) and Nod-like receptor family pyrin domain containing protein 3 (NLRP3) in BV2 cells were detected by PCR, Western-blot and immunofluorescence staining. The activity of caspase-8 in BV2 cells was detected by the CaspGLOW Kit, and the content of interleukine-1ß (IL-1ß) in the supernatant was detected by enzyme linked immunosorbent assay. After the corresponding pathways were blocked by TLR4 small interfering RNA (siRNA) transfection or caspase-8 inhibitor, the expression changes of TLR4 and NLRP3, the activity of caspase-8, and the difference of IL-1ß content could be observed as well as the activity of RGCs co-cultured with BV2. Statistical analysis was performed using analysis of variance. Results: Under co-culture of RGC and BV2 cells, cellular immunofluorescence detection showed that the expression of iba1 in BV2 cells increased, which indicated BV2 cells were activated significantly in the OGD/R model. In the OGD/R model, the apoptosis rate of RGC co-cultured with BV2 cells (71.1%±3.2%) was significantly higher than that of RGC cultured alone (35.1%±1.8%) (t=10.10, P<0.01). Cellular immunofluorescence detection showed that the expression of TLR4 and NLRP3 in BV2 cells in the OGD/R model increased significantly when BV2 cells were cultured alone, and their mRNA levels increased significantly with prolongation of reoxygenation time (F=64.45, 72.74; P<0.01), and peaked at OGD/R 24 h (TLR4 mRNA, relative ratio to control was 2.83±0.23; NLRP3 mRNA, relative ratio to control was 3.12±0.27). Caspase-8 activity also increased with prolonged reoxygenation time, the difference was statistically significant (F=93.57, P<0.01), and peaked at OGD/R 24 h (relative ratio to control was 2.92±0.31). After transfection of BV2 cells with TLR4 siRNA, its caspase-8 activity was significantly inhibited, but using caspase-8 inhibitor did not affect the up-regulation of TLR4 expression in BV2 cells. However, the mature IL-1ß secreted by BV2 cells exposed to OGD/R was significantly reduced by using caspase-8 inhibitor (from 3.52±0.55 to 1.39±0.37, t=7.19, P<0.01), meanwhile, the expression of NLRP3 was also significantly decreased after caspase-8 inhibitor pretreatment (from 2.79±0.23 to 1.37±0.19, t=9.37, P<0.01). In the OGD/R model, the activity of RGC cells co-cultured with TLR4 siRNA-transfected BV2 cells was 74.5%±1.2%, and the activity of RGC cells co-cultured with BV2 cells treated with caspase-8 inhibitor was 62.8%±1.5%, those were both higher than that of RGC cells co-cultured with untreated BV2 cells (36.7%±0.3%), and the difference was statistically significant (t=11.60, 6.83; both P<0.01). Conclusion: TLR4-caspase-8-NLRP3 inflammasome pathway is activated in microglia exposed to OGD/R, resulting in the production of IL-1ß, thereby contributing to the death of RGCs. (Chin J Ophthalmol, 2020, 56: 32-40).

The relationship of the anterior articular capsule to the adjacent subscapularis: An anatomic and histological study.

INTRODUCTION: The purpose of this study was to delineate the anatomic relationship between the anterior articular capsule and the adjacent subscapularis by measuring the dimensions of the anterior articular capsule attachment and the subscapularis footprint on the humerus, as well as investigating the interface between the two structures. MATERIALS AND METHODS: Three shoulder specimens underwent histological analysis; for histological analysis, cross-sections through the subscapularis-capsule complex were harvested at the tendinous and muscular insertion sites. The dimensions of the anterior articular capsule attachment and the subscapularis footprint (including the tendinous and muscular insertions) were measured in thirteen cadaveric shoulder specimens. RESULTS: Histologically, the articular capsule has thin and loosely arranged collagen fibers with many interspersing fibroblast nuclei, whereas the outer layer of the articular capsule blends into a layer of more loosely spaced and less organized collagen fibers. This interface between the subscapularis and the underlying articular capsule is filled with more loosely spaced and less organized collagen fibers. The macroscopic evaluation showed that the minimum articular capsule width (4.2mm, SD 2.2mm) was located at its initiation 4.9mm (SD, 2.1mm) inferior to the superior margin of the subscapularis; the corresponding subscapularis footprint width measured 10.1mm (SD, 4.9mm). The maximum articular capsule width was11.1 mm (SD, 3.7mm) and was located 5mm distal to the inferior margin of the tendinous footprint. The maximum subscapularis footprint width was 15.8mm (SD, 2.9mm); the corresponding articular capsule attachment measured 5.2mm (SD, 1.8mm). CONCLUSIONS: Our results suggest that the anterior articular capsule attachment of the glenohumeral joint complements the footprint of the subscapularis and occupies a larger area of the lesser tubercle and metaphysis of the humerus than previously documented. The histological study confirms the presence of a demarcation between the subscapularis and articular capsule, specifically more significant at the region medial to the tendon insertion and at the muscular insertion of the subscapularis.

Clinical significance and outcome of one or two rib lesions on bone scans in breast cancer patients without known metastases.

The presence of one or two rib lesions on bone scans of post-treatment breast cancer patients without known metastases often makes clinical decision making problematic. The aim of this study was to identify skeletal metastasis predictors that might help the management of these patients. We recruited post-treatment breast cancer patients without overt metastases whose bone scans showed (1) one or two rib hot spots, or (2) one rib lesion and a concurrent bone abnormality. Their clinical and serial scintigraphic data were collected, reviewed and evaluated for correlations. After their first abnormal bone scans, 23 patients (11 of the 77 patients initially with one rib lesion (incidence, 14.3%), three of the 27 patients with two rib lesions (incidence, 11.1%), and nine of the 11 patients with one rib lesion plus a concurrent bone abnormality (incidence, 81.8%)) developed multiple bone metastases within 2 years of the initial rib lesions in all but one case. Univariate analyses revealed that a concurrent bone lesion other than the rib, direct tumour invasion to the chest wall or skin, and 10 or more lymph nodes involved were associated with increased risks of bone metastases whereas longer persistence of the rib lesions was associated with a lower risk. Multivariate proportional hazard analyses indicated that patients with a concurrent bone lesion other than the rib (relative risk (RR)=39.65; 95% confidence interval (CI)=8.13-193.28), 10 or more lymph nodes involved (RR=13.49; 95% CI=2.09-86.91), and no radiotherapy (RR=7.59; 95% CI=2.11-27.39) were more likely to have bone metastases, while those with longer persistence of the rib lesions (RR=0.92; 95% CI=0.84-0.98) and longer time interval between surgery and the rib lesion detection (RR=0.96; 95% CI=0.94-0.99) were less likely. We have identified clinical features applicable to risk stratification. High incidence of bone metastases was noted in patients with one rib lesion and a concurrent bone abnormality. Regular follow-up for 2 years after detection of rib lesions is recommended, especially for those with risk factors.