Surgery induces neurocognitive disorder via neuroinflammation and glymphatic dysfunction in middle-aged mice with brain lymphatic drainage impairment.

Background: Brain lymphatic drainage impairment is a prevalent characteristic in both aging and neurodegeneration. Surgery is more likely to induce excessive neuroinflammation and postoperative neurocognitive disorder (PND) among patients with aging and neurodegeneration. We hypothesized that surgical trauma may aggravate PND through preexisting cerebral lymphatic drainage impairment. However, there remains limited understanding about the role of surgery in changes of neurocognitive function in the populations with preoperative brain lymphatic drainage impairment. This study aims to expand our insight into surgery-induced glymphatic dysfunction, neuroinflammation and PND in middle-aged mice with preoperative brain lymphatic drainage impairment. Materials and methods: Deep cervical lymph nodes ligation (LdcLNs) was performed on middle-aged mice to establish preoperative brain lymphatic drainage impairment. A month later, laparotomy was performed on these mice with or without LdcLNs followed by analysis of brain neuroinflammation, glymphatic function, neuronal damage, and behavioral test. Results: LdcLNs disrupted meningeal lymphatic drainage. In middle-aged mice with LdcLNs, surgery exacerbated more serious glymphatic dysfunction accompanied by aggravation of A1 astrocytes activation and AQP4 depolarization. Furthermore, surgery caused neuronal damage via reducing expression of neuronal nuclei (NeuN), post-synaptic density protein 95 (PSD95) and synaptophysin (SYP), as well as impairment in exploratory behavior and spatial working memory in middle-aged mice with LdcLNs. Additionally, surgery induced neuroinflammation with elevated microglia activation and increased the levels of tumor necrosis factor (TNF)-α, interleukin (IL)-1ß and IL-6, as well as activated more expression of HMGB1/TLR-4/NF-κB pathway in middle-aged mice with LdcLNs. Conclusion: Surgery exacerbates neuroinflammation and glymphatic dysfunction, ultimately resulting in neuronal damage and neurocognitive disorder in middle-aged mice with preoperative brain lymphatic drainage impairment. These results suggest that brain lymphatic drainage impairment may be a deteriorating factor in the progression of PND, and restoring its function may serve as a potential strategy against PND.

Tacrolimus and Diabetes in Kidney Transplantation: The Impact of Cyp3a5 Gene Polymorphism.

BACKGROUND: To explore the correlation between single nucleotide polymorphisms (SNPs) of CYP3A4 rs2740574 and CYP3A5 rs776746 and post-transplant diabetes mellitus (PTDM) in Chinese renal allograft recipients treated with tacrolimus. METHODS: A total of 244 patients treated with tacrolimus were included in this study, wherein DNA sequencing was detected through fluorescence in situ hybridization, and SNP genotyping was performed. RESULTS: Among the 244 patients, 44 (18%) developed PTDM. The PTDM group exhibited higher preoperative body mass index and fasting plasma glucose levels, with higher creatinine values one year after surgery. The CYP3A4 rs2740574 genotype was found to be unique in its homozygous AA form. For CYP3A5 rs776746, the genotypes were distributed as follows: 28 (11.5%) cases with AA, 101 (41.4%) cases with AG, and 115 (47.1%) cases with GG, respectively (P = .042). The AA genotype showed a statistically significant difference from both AG and GG genotypes. Furthermore, the A allele of CYP3A5 rs776746 was found to be associated with an increased risk for PTDM development. CONCLUSIONS: The occurrence of tacrolimus-related PTDM is associated with body mass index, fasting plasma glucose levels, and CYP3A5 genotype before renal transplantation. Post-transplant diabetes mellitus is correlated with unfavorable long-term renal graft function, whereas the expression of the CYP3A5 rs776746 gene is linked to an elevated risk of PTDM.

HMGB1 is increased in patients with immune thrombocytopenia and negatively associates with Tregs.

INTRODUCTION: Refractory or recurrent immune thrombocytopenia (ITP) patients suffer from the dual threat of high mortality and drug toxicity in addition to a very poor quality of life. Previous studies have shown that high mobility group box 1 (HMGB1) can promote the development of rheumatoid arthritis, systemic lupus erythematosus, and other autoimmune diseases. However, there is still a lack of research on the role of HMGB1 in the pathogenesis of ITP and whether it can be used as a predictor of efficacy and prognosis. METHODS: 20 patients of adult ITP with splenectomy were chosen as the experimental group, while 19 adults underwent splenectomy for traumatic splenic rupture without other diseases as the control group. We measured the expression of HMGB1, RORγt and Foxp3 in spleen tissues by immunohistochemistry. Another 50 patients, of which 20 were newly diagnosed without treatment and 30 were refractory ITP, and 25 healthy controls were enrolled to analyse the expression levels and mRNA levels of HMGB1, RORγt and Foxp3 in peripheral blood by Western blot and RT-qPCR. The expression of HMGB1, IL-17 and IL-10 in serum was assayed by ELISA. PBMCs from newly diagnosed ITP patients were cultured in vitro which stimulated with recombinant humanHMGB1 (rHMGB1) and its inhibitors, in which the expressions of RORγt and Foxp3 were measured. RESULTS: The expression of HMGB1 in the spleen with refractory ITP was significantly higher, while Foxp3 was decreased. A significant negative correlation was found between HMGB1 and Foxp3, and the overexpression of HMGB1 was significantly correlated with poor efficacy after splenectomy. The expression of HMGB1 and IL-17 increased and showed a positive correlation in serum, while IL-10 decreased and was negatively correlated with HMGB1. In PBMCs, the expression of HMGB1 and RORγt increased, while Foxp3 decreased, and the differences were more obvious in the refractory chronic ITP group. In a coculture system with PBMCs of untreated ITP patients, rHMGB1 increased RORγt expression and decreased Foxp3 expression, while an antiHMGB1 antibody partially corrected the above changes. CONCLUSION: Our results suggest that HMGB1 is associated with the imbalance of Treg/Th17 cells and is involved in the pathogenesis of ITP.

[Effects of HMGB1 on Proliferation and Secretion of Human Bone Marrow Mesenchymal Stem Cells].

OBJECTIVE: To investigate the effect of high mobility group protein 1(HMGB1) on the proliferation and cytokine expression of human bone marrow mesenchymal stem cells (MSC). METHODS: Different concentrations of recombinant human HMGB1 protein (100, 200, 400, 800 and 1000 ng/ml) were incubated with MSC for 24, 48, 72 h and the proliferation of MSC were detected respectively by using the CCK-8 method and flow cytometry. The best concentrations of HMGB1 incubated with MSC was determined (200 ng/ml, 1000 ng/ml), and the flow cytomerty was used to determine the effect of HMGB1 on the proliferation of MSC. The mRNA expression levels of IL-10, TGF- ß1, TSG-6 and IFN-γ in MSC incubated with HMGB1 protein were detected by real-time quantitative PCR and ELISA. RESULTS: The result of MSC identification and flow cytometry showed that the CD105, CD73 and CD90 were expressed, but did not expression CD45, CD34, CD11b, CD19 and HLA-DR; CCK-8 showed that HMGB1 at the concentrations of 100 ng/ml, 200 ng/ml and 400 ng/ml could promote the proliferation of MSC incubated for 24, 48 and 72 h as compared with the control group (P<0.05), and the most effective concentration was 200 ng/ml; flow cytometry showed that the compared with the control group, HMGB1 200 ng/ml could induce MSC from G1 phase to S phase to promote the proliferation of MSC; QPCR showed that the mRNA expression of MSC cytokines IL-10, TGF-ß1, TSG-6 increased while IFN-γ decreased at the concentration of 200 ng/ml HMGB1 as compared with the control group. ELISA experiments showed that the HMGB1 200 ng/ml acting on MSC for 48 h could significantly promoted the secretion of IL-10, TGF-ß 1 and TSG-6(P<0．05), while IFN-γ showed no significant difference as compared with control group. CONCLUSION: Recombinant human HMGB1 can promote the proliferation and secretion of MSC in healthy people.

The risk factor analysis and treatment experience in pneumocystis jirovecii pneumonia after kidney transplantation.

BACKGROUND: Pneumocystis jirovecii pneumonia (PJP) is an opportunistic infection among solid organ transplantation. The occurrence of PJP is dangerous and fatal if there is no early identification and sufficient treatment. OBJECTIVE: The aim of this study was to evaluate the risk factors and provide appropriate strategies of prophylaxis and treatment for PJP after kidney transplantation in our centre. PATIENTS/METHODS: From January 2009 to December 2018, a total of 167 kidney transplantation recipients with pneumonia were enrolled, including 47 PJP patients as PJP group and 120 non-PJP patients as control group. The clinical characteristics of the two groups were analysed retrospectively. RESULTS: Multivariate analysis showed that high total dosage of ATG [OR, 2.03; 95% CI, 1.12-3.68] and cytomegalovirus (CMV) infection were independent risk factors for PJP. Trimethoprim-sulfamethoxazole (TMP-SMX) (1.44 g q6h)-based treatment was used for 2 weeks, and its dosage and course were adjusted according to the therapeutic effect and side effects. Forty-five cases were recovered after 3 months of follow-up, and two patients died of respiratory failure. TMP-SMX (0.48 g/day) prophylaxis was used for 3-6 months and prolonged to 7-8 months after treatment for acute rejection, which reduced the incidence of PJP compared with those without prophylaxis. CONCLUSION: Our study suggests that the high total dosage of ATG and CMV infection indicate the increased risk of PJP. The strategies of prophylaxis and treatment for PJP after kidney transplantation in our centre were effective.

Sevoflurane increases intracellular calcium to induce mitochondrial injury and neuroapoptosis.

Sevoflurane is commonly used in clinical anesthesia. However, some reports indicated that Sevoflurane could induce mitochondrial injury and neuroapoptosis. Although the mechanism remains unclear, evidence points to the increase of intracellular calcium after administration of Sevoflurane. Herein, we sought whether the increment of intracellular Ca2+ caused by Sevoflurane administration could induce mitochondrial injury and apoptosis in primary neurons of the hippocampus. Fluo-4-acetoxymethyl ester Ca2+ probe was used for measuring intracellular Ca2+ concentrations. LDH assay, CCK-8 assay, and Western blotting were performed to confirm Sevoflurane-induced neuroapoptosis. ROS, mPTP, and ATP production were assayed to reveal mitochondrial injury. Our results indicated that Sevoflurane increased intracellular Ca2+ and neuronal death. Sevoflurane also elevated ROS and the opening of mPTP, and decreased ATP production in neurons. The expression of cytochrome c, cleaved caspase-9, cleaved caspase-3, and the ratio of Bax/Bcl-2 were also increased. By using calcium channel blocker Nimodipine, the increase of intracellular Ca2+ was attenuated, and the death rate of neurons, the ROS and opening of mPTP, decreased ATP production, the expressions of cytochrome c, cleaved caspase-9, cleaved caspase-3 and the ratio of Bax/Bcl-2 were alleviated. Our study suggested that Sevoflurane could increase intracellular Ca2+ to induce mitochondrial injury and mitochondria-mediated neuroapoptosis in neurons.

Mesenchymal stem cells improve platelet counts in mice with immune thrombocytopenia.

Immune thrombocytopenia (ITP) is a common autoimmune bleeding disorder. The breakdown of immune tolerance (regulatory T [Treg] cells and suppressor cytokines) plays an important role in ITP pathophysiology, especially in refractory ITP. Bone marrow-derived mesenchymal stem cells (BM-MSCs) show immunomodulatory properties and have been extensively utilized for autoimmune diseases. However, it has not been fully elucidated how BM-MSCs affect ITP. In this study, we explore the therapeutic mechanism of BM-MSCs on ITP in mice. Dose-escalation passive ITP mice were inducted by injection of MWReg30. BALB/c mice were randomly divided into two groups: ITP with BM-MSC transplantation and ITP controls. The serum levels of cytokines (interleukin 10 [IL-10] and transforming growth factor-ß1 [TGF-ß1]) were examined by enzyme-linked immunosorbent assays. The frequency of Treg cells in both peripheral blood and spleen mononuclear cells was analyzed by flow cytometry, and the forkhead box P3 (Foxp3) messenger RNA (mRNA) level was measured by real-time polymerase chain reaction. After BM-MSC treatment, the platelet (PLT) counts were significantly elevated. Meanwhile, cytokines (TGF-ß1 and IL-10), the ratios of Treg cells, and the Foxp3 mRNA expression level were significantly higher in the BM-MSC group. Our results show that BM-MSCs can improve PLT counts mainly by secreting suppressive cytokines and upregulating Tregs, which may provide new therapeutic potential for human ITP.

[Expression of HMGB1 in Spleen of Adult Patients with Chronic and Refractory Immune Thrombocytopenia and Its Significance].

OBJECTIVE: To investigate the expression and clinical significance of high mobility group box 1(HMGB1) in spleen of adult patients with chronic and refractory immune thrombocytopenia(ITP). METHODS: Twenty chronic and refactory ITP patients received splenectomy were enrolled in ITP group and 20 cases of traumatic spleen rupture were enrolled in control group. The splenectomy efficacy in ITP patients was analyzed retrospectively. The HMGB1 expression in spleen tissue was detected by immunohistochemistry, and the correlation between different expression levels of HMGB1 and splenectomy efficacy were analysed. Meanwhile, the protein expression levels of HMGB1 in peripheral blood serum and mononuclear cells(PBMNC) of 25 patients with chronic and refractory ITP were detected by ELISA and Western blot. RESULTS: The median platelet count before splenectomy was 7.5 (0-20) ×109/L; all the patients showed that the initial response to splenectomy within the first month after operation was 100%, the median time of response was 1 day (1-6 days). The median peak platelet count post splenectomy was 448.5 (161-1272)×109/L. In the median time of 10(3-30) months, the platelets count in 8 patients was reduced to varying degrees. After a median follow-up of 69.5 months (22-195), complete response was found in 12 patients, 4 cases showed response and 4 did not. The HMGB1 expression positive rate in spleen of ITP patients was significantly higher than that in control group (85.0% vs 15.0%)(P<0.001). There were a negative correlation between the HMGB1 expression in ITP and therapeutic outcome after splenectomy (r=-0.791, P<0.01). In addition, HMGB1 expression levels in serum and PBMNC of the patients with chronic refractory ITP were also significantly higher than that in healthy controls (P<0.01). CONCLUSION: The splenectomy has been found to be effective therapeutic method for patients with ITP, the HMGB1 highly express in the spleen of the patients with chronic refractory ITP, but negatively correlats with the therapeutic outcome after splenectomy.

[Role of Treg Cells in Pathogensis of Mouse ITP].

OBJECTIVE: To explore the role of Treg cells in the pathogenesis of idiopathic thrombocytopenic purpura (ITP). METHODS: The ITP mouse model was established, the Treg cell ratio in peripheral blood and spleen was detected by flow cytometry, the CD4+ CD25+ T cells were sorted by immunomagnetic beads, the Treg cell associated transcription factors (Foxp3, Smad7, STAT5 and Akt-1) and cytokines (IL-10, TGF-ß) in CD4+ CD25+ T cells were enriched from spleen mononuclear cells, and the mRNA expression of Treg cell was measured by real-time PCR. RESULTS: The ratio of Tregs in peripheral blood and spleen decreased significantly in ITP mouse, as compared with the controls (P<0.01). In addition, the mRNA expression of IL-10, TGF-ß and Foxp3 decreased significantly in spleen CD4+ CD25+ T cells (P<0.05). Expression of Smad 7 mRNA was higher than that of controls. CONCLUSION: The alteration in Treg frequency and function may be responsible for the immune dysfunction in ITP disease. It is also speculated that the lower mRNA expression of Foxp3 and higher mRNA expression of Smad 7 may inhibit the proliferation and differentiation of Treg cells.

Marked protection by selective cerebral profound hypothermia after complete cerebral ischemia in primates.

Hypothermia has been demonstrated to protect the brain from ischemia or traumatic brain injury. Achieving profound hypothermia has relied on techniques requiring total body cooling, which may result in serious cardiovascular and pulmonary complications. A technique to selectively cool the brain could conceivably exert a marked protection on cerebral structures and provide a relatively bloodless operative surgical field without systemic complications. Accordingly, this approach was tried in 7 rhesus monkeys after induction of general anesthesia. The right internal carotid artery and both internal jugular veins were each occlusively cannulated and connected to a circulation pump. The left internal carotid artery, both external carotid arteries, and both external jugular veins were temporarily clamped to establish severe cerebral ischemia. Using a closed-circuit system, cooled Ringer's lactate liquid (4 degrees C) was infused through right internal carotid artery with outflow draining though both internal jugular veins. Cooled perfusate decreased cerebral temperature to the target temperature of 15 degrees C. Thereafter, pump flow was discontinued, and brains were rewarmed spontaneously, while the temporarily clamped carotid arteries and jugular veins were opened to resume normal cerebral blood circulation. Neurological functions were recorded daily and cerebral histology was examined at the conclusion of the experiment. Magnetic resonance (MR) scans were routinely taken before and 3 weeks after ischemia. In the normothermia control group of five rhesus monkeys, Ringer's solution at 37 degrees C was infused in the same manner as the cold solution with cerebral temperature maintained at 36.7 +/- 0.32 degrees C. Right cerebral temperature decreased from 36.5 +/- 0.49 to 15.5 +/- 2.29 degrees C, and simultaneously the left cerebral temperature decreased from 36.4 +/- 0.38 to 16.3 +/- 2.4 degrees C for 62.8 +/- 9.76 min during selective cerebral cooled Ringer's liquid perfusion. In contrast, rectal temperature was only reduced to 32.4 +/- 0.96 degrees C from a baseline of 37.2 +/- 0.76 degrees C. Internal jugular vein hematocrit was 38.2 +/- 0.31% before perfusion and 2.82 +/- 0.46% at the end of perfusion in profound hypothermia group; hematocrit was 39.7 +/- 0.62% before perfusion and 3.42 +/- 0.38% at the end of perfusion in the normothermia group. In the hypothermic group, neurological functions were normal during 6 months of follow-up, and microscopic examination of brain tissue did not show evidence of pathological changes in hippocampus or medulla. MR scans did not show any cerebral infarction. In contrast, none of the monkeys in normothermia group survived for more than several hours, and microscopic examination of the brain revealed extensive neuronal necrosis within the medulla. Selective cerebral profound hypothermia provides significant histologic and neurologic protection after severe cerebral ischemia. In addition, there were no major complications, and the operative field remained relatively bloodless in the profound hypothermic group.