A Novel Newborn Rat Kernicterus Model Created by Injecting a Bilirubin Solution into the Cisterna Magna

<div><p>Background</p><p>Kernicterus still occurs around the world; however, the mechanism of bilirubin neurotoxicity remains unclear, and effective treatment strategies are lacking. To solve these problems, several kernicterus (or acute bilirubin encephalopathy) animal models have been established, but these models are difficult and expensive. Therefore, the present study was performed to establish a novel kernicterus model that is simple and affordable by injecting unconjugated bilirubin solution into the cisterna magna (CM) of ordinary newborn Sprague-Dawley (SD) rats.</p><p>Methods</p><p>On postnatal day 5, SD rat pups were randomly divided into bilirubin and control groups. Then, either bilirubin solution or ddH₂O (pH = 8.5) was injected into the CM at 10 µg/g (bodyweight). For model characterization, neurobehavioral outcomes were observed, mortality was calculated, and bodyweight was recorded after bilirubin injection and weaning. Apoptosis in the hippocampus was detected by H&E staining, TUNEL, flow cytometry and Western blotting. When the rats were 28 days old, learning and memory ability were evaluated using the Morris water maze test.</p><p>Results</p><p>The bilirubin-treated rats showed apparently abnormal neurological manifestations, such as clenched fists, opisthotonos and torsion spasms. Bodyweight gain in the bilirubin-treated rats was significantly lower than that in the controls (<i>P</i><0.001). The early and late mortality of the bilirubin-treated rats were both dramatically higher than those of the controls (<i>P</i> = 0.004 and 0.017, respectively). Apoptosis and necrosis in the hippocampal nerve cells in the bilirubin-treated rats were observed. The bilirubin-treated rats performed worse than the controls on the Morris water maze test.</p><p>Conclusion</p><p>By injecting bilirubin into the CM, we successfully created a new kernicterus model using ordinary SD rats; the model mimics both the acute clinical manifestations and the chronic sequelae. In particular, CM injection is easy to perform; thus, more stable models for follow-up study are available.</p></div

Histology and flow cytometry (FCM) at 24 hours after bilirubin injection.

<p>(A) H&E staining under light microscopy. Hippocampal nerve cells in the CA-3 region were morphologically normal in the control group (a, b). Cellular edema and swelling (c, d) (black arrow), and cell necrosis, including cytoplasmic condensation, endolysis, nuclear pyknosis, karyorrhexis and karyolysis (c, d) (red arrow), were observed in the bilirubin group (n = 8 in each group). (a), (c): 200×, scale bars = 200 µm; (b), (d): 400×, scale bars = 100 µm. (B) TUNEL assay under light microscopy. Few apoptotic cells in the CA-3 (a) and CA-1 (b) regions were observed in the controls. Apoptotic cells were observed in the CA-3 (c) and CA-1 (d) (black arrow) regions of the hippocampus in the bilirubin-treated rats. (n = 8 in each group). Scale bars = 100 µm. (C) Annexin V-FITC/PI (FCM) was used to determine the apoptotic (right lower quadrant) and necrotic (right upper quadrant) rates of the two groups. The apoptotic and necrotic rates of the controls were 1.33±0.36% and 1.26±0.89%, respectively (a). In the bilirubin group, the apoptotic and necrotic rates were 9.41±0.88% and 4.74±0.60%, respectively (b). There were significant differences in both the apoptotic and necrotic rates between the two groups (<i>P</i><0.01). (n = 8 in each group).</p

Clinical manifestations within 0.5–1 hour after bilirubin injection.

<p>(A) Control group rats. No abnormal neurobehavior was detected in the controls. (B)–(E) Representative images of abnormal neurobehavior in bilirubin-treated rats. Almost all of the bilirubin-treated pups showed clenched fists (B–C) (black arrow) and opisthotonos (D); some rats showed the latericumbent position (E). (F) Comparison of prostration in the two groups. The bilirubin-treated rats show decreased muscular tone (black arrow) and prostration.</p

ALDH2 rs671 and MTHFR rs1801133 polymorphisms are risk factors for arteriosclerosis in multiple arteries

Abstract Background Arteriosclerosis in multiple arteries has long been associated with heightened cardiovascular risk. Acetaldehyde dehydrogenase 2 (ALDH2) and methylenetetrahydrofolate reductase (MTHFR) play an important role in the pathogenesis of arteriosclerosis by participating in the oxidation and reduction reactions in vascular endothelial cells. The purpose was to investigate the relationship of ALDH2 and MTHFR gene polymorphisms with arteriosclerosis in multiple arteries. Methods 410 patients with arteriosclerosis in single artery and 472 patients with arteriosclerosis in multiple arteries were included. The relationship between ALDH2 rs671 and MTHFR rs1801133 polymorphisms and arteriosclerosis in single artery and arteriosclerosis in multiple arteries was analyzed. Results The proportion of ALDH2 rs671 A allele (35.6% vs. 30.9%, P = 0.038) and MTHFR rs1801133 T allele (32.6% vs. 27.1%, P = 0.012) in patients with arteriosclerosis in multiple arteries was significantly higher than that in arteriosclerosis in single artery, respectively. The proportion of history of alcohol consumption in patients with ALDH2 rs671 G/G genotype was higher than those in ALDH2 rs671 G/A genotype and A/A genotype (P < 0.001). The results of logistic regression analysis indicated that ALDH2 rs671 A/A genotype (A/A vs. G/G: OR 1.996, 95% CI: 1.258–3.166, P = 0.003) and MTHFR rs1801133 T/T genotype (T/T vs. C/C: OR 1.943, 95% CI: 1.179–3.203, P = 0.009) may be independent risk factors for arteriosclerosis in multiple arteries (adjusted for age, sex, smoking, drinking, hypertension, and diabetes). Conclusions ALDH2 rs671 A/A and MTHFR rs1801133 T/T genotypes may be independent risk factors for arteriosclerosis in multiple arteries

Effects of bilirubin on apoptosis-related molecules in the hippocampus at 24 hours after bilirubin injection.

<p>At 24 h after bilirubin injection, mitochondrial cytochrome-c (A) levels were significantly decreased compared with the control group; simultaneously, the cytosolic cytochrome-c (B) levels were significantly increased compared with the controls. Additionally, a significant increase of Bax in mitochondria (C) was observed. Meanwhile, Bcl-2 and Bcl-xL (D, E) expression in the mitochondria was dramatically decreased compared with the controls. (n = 12 in each group). *: <i>P</i><0.01 vs the control group.</p

Bodyweight changes within 3 days after bilirubin injection or weaning.

<p>(A) Bodyweights of the bilirubin-treated rats and control rats on PMDs-1/2/3. (B) Bodyweight gains of the bilirubin-treated rats and control rats on PMDs-1/2/3. (C) Bodyweight gains of the bilirubin-treated rats and control rats on PWDs-1/2/3. (n = 12 in each group). *: <i>P</i><0.01 vs control group. (PMD: post-modeling day; PWD: post-weaning day).</p

Deep Learning-Based Stage-Wise Risk Stratification for Early Lung Adenocarcinoma in CT Images: A Multi-Center Study

This study aims to develop a deep neural network (DNN)-based two-stage risk stratification model for early lung adenocarcinomas in CT images, and investigate the performance compared with practicing radiologists. A total of 2393 GGNs were retrospectively collected from 2105 patients in four centers. All the pathologic results of GGNs were obtained from surgically resected specimens. A two-stage deep neural network was developed based on the 3D residual network and atrous convolution module to diagnose benign and malignant GGNs (Task1) and classify between invasive adenocarcinoma (IA) and non-IA for these malignant GGNs (Task2). A multi-reader multi-case observer study with six board-certified radiologists’ (average experience 11 years, range 2–28 years) participation was conducted to evaluate the model capability. DNN yielded area under the receiver operating characteristic curve (AUC) values of 0.76 ± 0.03 (95% confidence interval (CI): (0.69, 0.82)) and 0.96 ± 0.02 (95% CI: (0.92, 0.98)) for Task1 and Task2, which were equivalent to or higher than radiologists in the senior group with average AUC values of 0.76 and 0.95, respectively (p &gt; 0.05). With the CT image slice thickness increasing from 1.15 mm ± 0.36 to 1.73 mm ± 0.64, DNN performance decreased 0.08 and 0.22 for the two tasks. The results demonstrated (1) a positive trend between the diagnostic performance and radiologist’s experience, (2) the DNN yielded equivalent or even higher performance in comparison with senior radiologists, and (3) low image resolution decreased model performance in predicting the risks of GGNs. Once tested prospectively in clinical practice, the DNN could have the potential to assist doctors in precision diagnosis and treatment of early lung adenocarcinoma