Activation of BMP4/SMAD pathway by HIF-1α in hypoxic environment promotes osteogenic differentiation of BMSCs and leads to ectopic bone formation.

OBJECTIVE: Heterotopic ossification (HO), also known as ossifying myositis, is a condition that produces abnormal bone and cartilage tissue in the soft tissues. Hypoxia inducible factor lα (HIF-lα) regulates the expression of various genes, which is closely related to the promotion of bone formation, and Drosophila mothers against decapentaplegic protein (SMAD) mediates the signal transduction in the Bone morphogenetic protein (BMP) signaling pathway, which affects the function of osteoblasts and osteoclasts, and thus plays a key role in the regulation of bone remodeling. We aimed to investigate the mechanism by which HIF-1α induces osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs) in a hypoxic environment. METHODS: A cellular hypoxia model was constructed to verify the expression of HIF-1α, while alizarin red staining was performed to observe the osteogenic differentiation ability of bone marrow mesenchymal stem cells (BMSCs). Alizarin red staining was used to analyze the late mineralization ability of the cells. Western blot analysis was performed to analyze the expression levels of osteogenesis-related factors OCN, OPN proteins as well as the pathway proteins BMP4, p-Smad1/5/8, and Smad1. We also constructed a rat model of ectopic bone formation, observed ectopic ossification by X-ray, and verified the success of the rat model by ELISA of HIF-1α. HE staining was used to observe the matrix and trabecular structure of bone, and Masson staining was used to observe the collagen and trabecular structure of bone. Immunohistochemistry analyzed the expression of OCN and OPN in ectopic bone tissues, and WB analyzed the expression of pathway proteins BMP4, p-Smad1/5/8 and Smad1 in ectopic bone tissues to verify the signaling pathway of ectopic bone formation. RESULTS: Our results indicate that hypoxic environment upregulates HIF-1a expression and activates BMP4/SMAD signaling pathway. This led to an increase in ALP content and enhanced expression of the osteogenesis-related factors OCN and OPN, resulting in enhanced osteogenic differentiation of BMSCs. The results of our in vivo experiments showed that rats inoculated with BMSCs overexpressing HIF-1α showed bony structures in tendon tissues, enhanced expression of the bone signaling pathways BMP4 and p-Smad1/5/8, and enhanced expression levels of the osteogenic-related factors OCN and OPN, resulting in the formation of ectopic bone. CONCLUSIONS: These data further suggest a novel mechanistic view that hypoxic bone marrow BMSCs activate the BMP4/SMAD pathway by up-regulating the expression level of HIF-1α, thereby promoting the secretion of osteogenic factors leading to ectopic bone formation.

Exosome MiR-21-5p Upregulated by HIF-1α Induces Adipose Stem Cell Differentiation to Promote Ectopic Bone Formation.

Heterotopic bone occurs after burns, trauma and major orthopedic surgery, which cannot be completely cured by current treatments. The development of new treatments requires more in-depth research into the mechanism of HO. Available evidence suggests that miR-21-5p plays an important role in bone formation. However, its mechanism in traumatic HO is still unclear. First, we identified exosomes extracted from L6 cells using TEM observation of the structure and western blotting detection of the surface marker CD63. Regulation effect of HIF-1α to miR-21-5p was confirmed by q-PCR assay. Then we co-cultured L6 cells with ASCs and performed alizarin red staining and ALP detection. Overexpression of miR-21-5p upregulated BMP4, p-smad1/5/8, OCN and OPN, which suggests the BMP4-smad signaling pathway may be involved in miR-21-5p regulation of osteogenic differentiation of ASCs. Finally in vivo experiments showed that miR-21-5p exosomes promoted ectopic formation in traumatized mice. This study confirms that HIF-1α could modulate miR-21-5p exosomes to promote post-traumatic ectopic bone formation by inducing ASCs cell differentiation. Our study reveals the mechanisms of miR-21-5p in ectopic ossification formation after trauma.

In vivo photoacoustic flow cytometry-based study of the effect of melanin content on melanoma metastasis.

A major cause of death in cancer patients is distant metastasis of tumors, in which circulating tumor cells (CTCs) are an important marker. Photoacoustic flow cytometry (PAFC) can monitor CTCs in real time, non-invasively, and label-free; we built a PAFC system and validated the feasibility of PAFC for monitoring CTCs using in vivo animal experiments. By cultivating heavily-pigmented and moderately-pigmented melanoma cells, more CTCs were detected in mice inoculated with moderately-pigmented tumor cells, resulting in more distant metastases and poorer survival status. Tumor cells with lower melanin content may produce more CTCs, increasing the risk of metastasis. CTC melanin content may be down-regulated during the metastatic which may be a potential indicator for assessing the risk of melanoma metastasis. In conclusion, PAFC can be used to assess the risk of melanoma metastasis by dynamically monitoring the number of CTCs and the CTC melanin content in future clinical diagnoses.

Long-course chemoradiation plus concurrent/sequential PD-1 blockade as neoadjuvant treatment for MMR-status-unscreened locally advanced rectal cancer: protocol of a multicentre, phase 2, randomised controlled trial (the POLAR-STAR trial).

INTRODUCTION: Recent preclinical studies have discovered unique synergism between radiotherapy and immune checkpoint inhibitors, which has already brought significant survival benefit in lung cancer. In locally advanced rectal cancer (LARC), neoadjuvant radiotherapy plus immune checkpoint inhibitors have also achieved surprisingly high pathological complete response (pCR) rates even in proficient mismatch-repair patients. As existing researches are all phase 2, single-cohort trials, we aim to conduct a randomised, controlled trial to further clarify the efficacy and safety of this novel combination therapy. METHODS AND ANALYSIS: Eligible patients with LARC are randomised to three arms (two experiment arms, one control arm). Patients in all arms receive long-course radiotherapy plus concurrent capecitabine as neoadjuvant therapy, as well as radical surgery. Distinguishingly, patients in arm 1 also receive anti-PD-1 (Programmed Death 1) treatment starting at Day 8 of radiation (concurrent plan), and patients in arm 2 receive anti-PD-1 treatment starting 2 weeks after completion of radiation (sequential plan). Tislelizumab (anti-PD-1) is scheduled to be administered at 200 mg each time for three consecutive times, with 3-week intervals. Randomisation is stratified by different participating centres, with a block size of 6. The primary endpoint is pCR rate, and secondary endpoints include neoadjuvant-treatment-related adverse event rate, as well as disease-free and overall survival rates at 2, 3 and 5 years postoperation. Data will be analysed with an intention-to-treat approach. ETHICS AND DISSEMINATION: This protocol has been approved by the institutional ethical committee of Beijing Friendship Hospital (the primary centre) with an identifying serial number of 2022-P2-050-01. Before publication to peer-reviewed journals, data of this research will be stored in a specially developed clinical trial database. TRIAL REGISTRATION NUMBER: NCT05245474.

Performance of indocyanine green in sentinel lymph node mapping and lymph node metastasis in penile cancer: systematic review, meta-analysis, and single-center experience.

PURPOSE: The aim of this study was to investigate the overall sensitivity and specificity of indocyanine green (ICG)-near-infrared (NIR) fluorescence imaging in the detection of sentinel lymph node metastasis (SLNM) in penile cancer. METHODS: We searched PubMed, Embase, Web of Science, Scopus, and the Cochrane Library databases to identify manuscripts where ICG was intravenously administered prior to or during penile cancer surgery, with no restriction on language or publication status. The results extracted are presented as forest plots. RESULTS: Seven studies were included in the analysis. The median sensitivity and specificity of ICG-NIR imaging for SLNM detection were 100 and 4%, respectively; the pooled sensitivity was 100.0% (95% confidence interval [CI] 97.0-100.0) and specificity was 2.0% (95% CI 1.0-3.0). There was no significant difference in the diagnostic results between different injection sites and doses in each experimental group. CONCLUSION: To our knowledge, this meta-analysis is the first to summarize the diagnostic performance of ICG-NIR imaging for SLNM detection in penile cancer. ICG is sensitive in the imaging of SLN tissue, which can consequently improve the accuracy of lymph node detection. However, the specificity is very low.

Sarcobesity, but not visceral fat, is an independent risk factor for complications after radical resection of colorectal cancer.

Background: The influence of body composition on the outcome of colorectal cancer surgery is controversial. The aim of this study was to evaluate the effects of visceral obesity and sarcobesity on the incidence of total and surgical complications after radical resection of colorectal cancer. Methods: We collected a total of 426 patients who underwent elective radical resection of colorectal cancer at Beijing Friendship Hospital, Capital Medical University from January 2017 to May 2018. According to the inclusion and exclusion criteria, 387 patients were finally included. A CT scan at the level of the L3-L4 intervertebral disk was selected to measure the values of visceral fat area and skeletal muscle area. Multivariate analysis was used to explore the independent risk/protective factors affecting postoperative complications. Results: 128 (33.1%) patients developed complications, and 44 (11.4%) patients developed major complications. Among them, 111 patients developed surgical complications and 21 developed medical complications. Visceral fat area (Z = -3.271, p = 0.001), total fat area (Z = -2.613, p = 0.009), visceral fat area to subcutaneous fat area ratio (V/S, Z = -2.633, p = 0.008), and sarcobesity index (Z = -2.282, p = 0.023) were significantly associated with total complications. Visceral fat area (Z = -2.119, p = 0.034) and V/S (Z = -2.010, p = 0.044) were significantly associated with total surgical complications. Sarcobesity index, smoking, stoma, blood loss, surgery time, and American Society of Anesthesiology (ASA) score were selected as risk factors for total postoperative complications according to LASSO regression. Multivariate logistic regression analysis suggested that sarcobesity index was an independent risk factor for postoperative total complications and surgical complications. Subgroup analysis suggested that albumin level was an independent protective factor for postoperative total complications in male patients. Smoking, operative time, and sarcobesity index were independent risk factors, and cholesterol was an independent protective factor for total postoperative complications in female patients. Conclusion: Increased sarcobesity index is an independent risk factor for postoperative complications in patients with colorectal cancer, while visceral fat area is not. For female patients, smoking, operation time, and obesity index are independent risk factors for postoperative complications, while cholesterol is an independent protective factor. For male patients, serum albumin is an independent protective factor for postoperative complications.

Advanced flow cytometry for biomedical applications.

Flow cytometry (FC) is a versatile tool with excellent capabilities to detect and measure multiple characteristics of a population of cells or particles. Notable advancements in in vivo photoacoustic FC, coherent Raman FC, microfluidic FC, and so on, have been achieved in the last two decades, which endows FC with new functions and expands its applications in basic research and clinical practice. Advanced FC broadens the tools available to researchers to conduct research involving cancer detection, microbiology (COVID-19, HIV, bacteria, etc.), and nucleic acid analysis. This review presents an overall picture of advanced flow cytometers and provides not only a clear understanding of their mechanisms but also new insights into their practical applications. We identify the latest trends in this area and aim to raise awareness of advanced techniques of FC. We hope this review expands the applications of FC and accelerates its clinical translation.

Prognostic significance of JAM 3 in gastric cancer: An observational study from TCGA and GEO.

Junctional adhesion molecule 3 (JAM3) can be used as a prognostic marker in multiple cancer types. However, the potential prognostic role of JAM3 in gastric cancer (GC) remains unclear. The purpose of this research was to gauge JAM3 expression and methylation as potential biomarkers for GC patient survival. Through bioinformatics research, we analyzed JAM3 expression, methylation, prognosis, and immune cell infiltrations. JAM3 methylation acts as a negative regulator of JAM3, leading to reduced expression of JAM3 in GC tissues relative to normal tissues. Patients with GC who expressed little JAM3 have a better chance of living a long time free of the disease, according to the Cancer Genome Atlas (TCGA) database. Through univariate and multivariate Cox regression analysis, inadequate JAM3 expression was labeled as an isolated indicator for overall survival (OS). The GSE84437 dataset was also used to confirm JAM3 prognostic role in GC, with consistent findings. A meta-analysis also found that low levels of JAM3 expression were significantly associated with longer OS. Finally, there was a strong correlation between JAM3 expression and a subset of immune cells. According to the TCGA database, low JAM3 expression could predict favorable OS and progression-free-survival (PFS) in GC patients (P < .05). The univariate and multivariate Cox regression demonstrated that low JAM3 expression was independent biomarker for OS (P < .05). Moreover, GSE84437 dataset was utilized to verify the prognostic role of JAM3 in GC, and the similar results were reached (P < .05). A meta-analysis revealed that low JAM3 expression was closely relevant to better OS. Finally, JAM3 expression exhibited a close correlation with some immune cells (P < .05). JAM3 might be a viable predictive biomarker and likely plays a crucial part in immune cell infiltration in individuals with GC.

Effects of a Graphene Heating Device on Fatigue Recovery of Biceps Brachii.

Far-infrared (FIR) is considered to be an ideal method to promote fatigue recovery due to its high permeability and strong radiation. In this paper, we report a flexible and wearable graphene heating device to help fatigue recovery of human exercise by using its high FIR divergence property. This study compares two different fatigue recovery methods, graphene far-infrared heating device hot application and natural recovery, over a 20 min recovery time among the male colleges' exhaustion exercise. Experimental results show that the achieved graphene device holds excellent electro-thermal radiation conversion efficiency of 70% and normal total emissivity of 89%. Moreover, the graphene FIR therapy in our work is more energy-efficient, easy to use, and wearable than traditional fatigue recovery methods. Such an anti-fatigue strategy offers new opportunities for enlarging potential applications of graphene film in body science, athletic training recovery, and wearable devices.

Evaluation of multiple biological indicators for combined diagnosis of gastric cancer: A retrospective analysis.

To assess carcinoembryonic antigen (CEA), carbohydrate antigen 19-9 (CA19-9), platelet distribution width (PDW), neutrophil-to-lymphocyte ratio (NLR), and platelet-lymphocyte ratio (PLR) for gastric cancer's (GC) diagnostic efficiency, and the use of receiver operating characteristic curves (ROC) combined with logistic regression to evaluate multi-index combination's diagnostic value of GC. 773 GC patients' clinical data were retrospectively collected in the Weihai Municipal Hospital, affiliated hospital of Shandong University from April 2018 to May 2021, and selected 2368 healthy physical examination patients during the same period as the control group. A total of 3141 samples was included in this study, including 773 cases in the GC group and 2368 cases in the healthy physical examination group. The results of the overall comparison between groups showed that apart from gender, the age differences, CEA, CA19-9, PDW, NLR, and PLR were statistically significant (P < .001). Spearman ranks correlation analysis's results showed that CA19-9, CEA, PLR, and NLR were correlated with GC patients' clinical-stage positively, and the correlation coefficients r was 0.249, 0.280, 0.252, 0.262 (all P < .001), and PDW was correlated with the clinical stage negatively (r = -0.186, P < .001). The ROC curve analysis results of CEA, CA19-9, PDW, NLR and PLR showed that CEA's diagnostic cutoff value for GC was 3.175 (area under the curve [AUC] = 0.631, 95% CI: 0.606-0.655, P < .001), the CA19-9's diagnostic cutoff value is 19.640 (AUC = 0.589, 95% CI: 0.563-0.615, P < .001), PDW's diagnostic cutoff value is 15.750 (AUC = 0.799, 95% CI: 0.778-0.820, P < .001), NLR's diagnostic cutoff value was 2.162 (AUC = 0.699, 95% CI: 0.675-0.721, P < .001), and PLR's diagnostic cutoff value was 149.540 (AUC = 0.709, 95% CI: 0.688-0.732, P < .001). The area under the ROC curve for the combined diagnosis of GC with 5 indicators was 0.877 (95% CI: 0.860-0.894, P < .001), which was better than a single indicator (P < .05). The diagnostic efficiency of combined detection of CEA, CA19-9, PDW, NLR, and PLR is better than that of single index detection alone, which can reduce the misdiagnosis rate of GC effectively.

Photodynamic therapy reduces metastasis of breast cancer by minimizing circulating tumor cells.

Cancer metastasis after traditional surgery introduces a high barrier to therapy efficacy. Photodynamic therapy (PDT) for cancer is based on a photochemical process of photosensitizers that concentrate in tumors and release oxidant species under light excitation to destroy cells. Compared with traditional surgery, PDT provides minimal invasion and targeted therapy. In this in vivo study, we monitor the real-time and long-term dynamics of circulating tumor cells (CTCs) after a single round of PDT and after surgical resection in a breast cancer animal model. The CTC level is low after PDT treatment, and the recurrence of the primary tumor is postponed in the PDT group compared with the resection group. We find that metastasis is correlated with the CTC level, and the PDT-treated mice show no metastasis in the lung or liver. Our results suggest PDT can effectively reduce metastasis by minimizing CTCs after treatment and is a great technology for breast cancer therapy.

In vivo flow cytometry reveals a circadian rhythm of circulating tumor cells.

Circulating tumor cells (CTCs) is an established biomarker of cancer metastasis. The circulation dynamics of CTCs are important for understanding the mechanisms underlying tumor cell dissemination. Although studies have revealed that the circadian rhythm may disrupt the growth of tumors, it is generally unclear whether the circadian rhythm controls the release of CTCs. In clinical examinations, the current in vitro methods for detecting CTCs in blood samples are based on a fundamental assumption that CTC counts in the peripheral blood do not change significantly over time, which is being challenged by recent studies. Since it is not practical to draw blood from patients repeatedly, a feasible strategy to investigate the circadian rhythm of CTCs is to monitor them by in vivo detection methods. Fluorescence in vivo flow cytometry (IVFC) is a powerful optical technique that is able to detect fluorescent circulating cells directly in living animals in a noninvasive manner over a long period of time. In this study, we applied fluorescence IVFC to monitor CTCs noninvasively in an orthotopic mouse model of human prostate cancer. We observed that CTCs exhibited stochastic bursts over cancer progression. The probability of the bursting activity was higher at early stages than at late stages. We longitudinally monitored CTCs over a 24-h period, and our results revealed striking daily oscillations in CTC counts that peaked at the onset of the night (active phase for rodents), suggesting that the release of CTCs might be regulated by the circadian rhythm.

Real-time monitoring of single circulating tumor cells with a fluorescently labeled deoxy-glucose by in vivo flow cytometry.

Circulating tumor cells (CTCs) play an essential role in metastasis and serve as an important prognostic biomarker. The technology of CTC labeling and detection in vivo can greatly improve the research of cancer metastasis and therapy. However, there is no in vivo technology to detect CTCs in clinic. In this study, we demonstrate that 2-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl) amino]-2-deoxy-d-glucose (2-NBDG), a 2-deoxy-glucose analog, can work in vivo to indicate CTCs and metastases fluorescently by direct intravenous injection. During the development of an implanted tumor in mice, the spontaneous CTCs released from the primary tumor into blood vessels can be labeled by 2-NBDG due to the abnormal metabolism of CTCs. The green fluorescence of 2-NBDG from CTCs is then noninvasively detected by an in vivo flow cytometry system. Due to the high uptake of glucose by tumor cells, the CTCs in mice can maintain a high 2-NBDG level and thus be distinguished by 2-NBDG fluorescence in vivo efficiently, enabling tumor detection in vivo like positron emission tomography (PET) but at the single-cell resolution. Our results suggest 2-NBDG, a glucose analog with high biosafety, holds promising potential in clinical applications, similar to the widely-used contrast medium 2-F18 -fluorodeoxyglucose in PET.

Prophylactic subcutaneous drainage reduces post-operative incisional infections in colorectal surgeries: a meta-analysis of randomized controlled trials.

BACKGROUND: Due to lack of high-level evidences, prophylactic subcutaneous drainage has so far not been recommended in relevant guidelines as a countermeasure against incisional infections. This meta-analysis aims to clarify the efficacy of subcutaneous drainage in reducing incisional infections in colorectal surgeries. METHODS: Cochrane Library, Embase, and PubMed were searched for randomized controlled trials comparing the incidence rate of incisional infections between patients receiving prophylactic subcutaneous drainage (interventions) and those not receiving (controls) after digestive surgeries. Results from included RCTs were pooled multiple times according to different surgical types. Heterogeneity, publication bias, and certainty of evidences were estimated. RESULTS: Eight randomized controlled trials were included. Three RCTs each included patients receiving all sorts of digestive surgeries (gastrointestinal, hepatobiliary, and pancreatic); pooled incisional infection rates between the drainage group and the control group were not significantly different (RR = 0.76, 95%CI: 0.48-1.21, p = 0.25). Four RCTs included patients receiving colorectal surgeries; pooled incisional infection rate in the drainage group was significantly lower than that in the control group (RR = 0.34, 95%CI: 0.19-0.61, p = 0.0004). Four RCTs included patients receiving upper GI and/or HBP surgeries; pooled incisional infection rates in the drainage group and the non-drainage group were not significantly different (RR = 0.85, 95%CI: 0.54-1.34, p = 0.49). CONCLUSIONS: Prophylactic subcutaneous drainage significantly reduces post-operative incisional infections in colorectal surgeries but was not efficacious in digestive surgeries in general.

Clinical benefits of PD-1/PD-L1 inhibitors in advanced hepatocellular carcinoma: a systematic review and meta-analysis.

BACKGROUND: Significant improvement of objective response rate and overall survival period has been achieved in several types of solid tumors by treatment with PD-1/PD-L1 inhibitors, which shed some light on hepatocellular carcinoma (HCC). Currently, a number of clinical trials concerning the application of checkpoint inhibitors in HCC are ongoing, some of which have shown favorable expectations. Hereby, we conducted a meta-analysis of existing studies to reveal the efficacy and safety of checkpoint inhibitors in advanced HCC. METHODS: Medline, Embase, Cochrane Library, and Web of Science were searched from inception to January 31, 2020. The clinical trials reporting the efficacy of PD-1/PD-L1 inhibitors in advanced HCC patients were eligible. Overall results of complete response (CR), partial response (PR), stable disease (SD), progression of disease (PD), objective response rate (ORR), disease control rate (DCR), overall survival (OS), progression-free survival (PFS) and rate of adverse events (AE) with their 95% confidence intervals (95%CI) were calculated as the primary focus of the meta-analysis. Subgroup analyses were conducted primarily according to the categories of PD-1 inhibitor or PD-L1 inhibitor and combination therapy or monotherapy. In addition, pooled results of PD-1/PD-L1 monoclonal antibodies (mAb) combining with anti-VEGF agents were calculated separately. RESULTS: A total of 20 studies with 1232 patients were included. The overall CR, PR and SD rate were 0.01 (95% CI 0.01-0.03), 0.17 (95% CI 0.14-0.22) and 0.39 (95% CI 0.34-0.43), respectively. The overall ORR and DCR were 0.20 (95% CI 0.16-0.24) and 0.60 (95% CI 0.54-0.67), respectively. The overall PFS and OS were 3.58 months (95% CI 2.65-4.50) and 12.24 months (95% CI 10.48-14.00), respectively. For patients treated with PD-1/PD-L1 mAb combing with anti-VEGF agent, ORR was 29% (95% CI 0.15-0.43) and DCR was 77% (95% CI 0.70-0.84). For all included studies, the overall rate of AE was 0.63 (95% CI 0.45-0.78) and serious adverse events (SAE) was 0.11 (95% CI 0.06-0.22). CONCLUSIONS: PD-1/PD-L1 inhibitors showed favorable outcomes concerning response rates and survival periods in advanced HCC. Updated results from high-quality clinical trials are expected to validate these findings.

Anti-N-methyl-D-aspartate receptor encephalitis: a prospective study focused on cerebrospinal fluid and clinical symptoms.

BACKGROUND AND OBJECTIVE: To analyze the relationship between brain MRI, clinical symptoms, and cerebrospinal fluid (CSF) and to provide a reference for early diagnosis of anti-N-methyl-D-aspartate receptor (NMDAR) encephalitis. MATERIALS AND METHODS: A total of 62 patients with anti-NMDAR encephalitis in Zhengzhou, China (2016-2018) were observed and registered prospectively. First, we analyzed the characteristics of clinical symptoms. Second, according to the disease duration, patients were divided into two groups, and then we analyzed the CSF features. In addition, they were divided into two groups according to the brain MRI, and then the CSF features were analyzed. Finally, the characteristics of cerebrospinal fluid in patients with seizure as the initial symptom were analyzed. RESULTS: Seizure presents as the initial symptom in 14 patients (22.5%), including 11 males (78.57%). The proportion and concentration of CSF total protein abnormalities in the early stage group were significantly higher than those in the middle and late group (P < 0.05). The total protein concentration in the abnormal brain MRI group was significantly higher than that in the normal MRI group (P < 0.05). CONCLUSION: Anti-NMDAR encephalitis should be suspected, when male patients complain of seizure as an initial symptom and have simultaneously had headaches or fever prior to onset. The sensitivity of anti-NMDAR antibody is higher in CSF than in serum. Total CSF protein is more prone to elevation in the middle and late stages of anti-NMDAR encephalitis. Brain MRI abnormalities with anti-NMDAR encephalitis are related to the total protein concentration of CSF, which may be related to the disease duration.