BACKGROUND:Meningeal lymphatic vessels can drain cerebral spinal fluid and amyloid β-protein,promoting T lymphocyte to transport and home to deep cervical lymph nodes.A simple,quick and definite method of dural separation and accurate localization of deep cervical lymph nodes can provide strong support for the study of neurodegenerative diseases.OBJECTIVE:To establish a convenient and practical method for exfoliating dural and deep cervical lymph nodes.METHODS:ICR mice,3 months old,were taken,anesthetized and injected with Evans blue and tracer in the occipital pool for localizing deep cervical lymph nodes.A midline incision of about 3 cm in length was made about 5 mm above the clavicle,the superficial fat and fascia were bluntly separated,and the lateral sternocleidomastoid muscle was pulled to expose the deep cervical lymph nodes,which were removed under a stereomicroscope and frozen at-80℃.Subsequently,the mouse head was cut and the skin and muscles of the head were separated to expose the entire skull structure.The skull and brain tissue were separated from the foramen magnum along the lower parietal bone with scissors,and the complete skull top was obtained.The skull was sequentially fixed in 40 g/L paraformaldehyde solution for 24 hours,120 g/L paraformaldehyde for 24 hours,and 120 g/L paraformaldehyde for 10,20,30,and 40 minutes,and the dural structure was stripped.The drainage capacity of meningeal lymphatic vessels and deep cervical lymphatic vessels was verified by tracer,and the meningeal lymphatic vessels were identified by the lymphatic vessel endothelial hyaluronan receptor 1 using the immunofluorescence method.RESULTS AND CONCLUSION:(1)Obvious blue staining was observed in deep cervical lymph nodes 15 minutes after Evans blue staining.(2)The skull was sampled and fixed in 120 g/L paraformaldehyde for 24 hours,resulting in a less tight connection between the dura mater and the skull,and easier stripping of the dural structures with an intact shape.The dura mater fixed at 120 g/L concentration was more resilient and remained more intact during peeling compared with the conventional 40 g/L concentration;120 g/L paraformaldehyde fixed meninges for a short time,and 30-40 minutes was preferred.(3)The frozen section of deep cervical lymph nodes showed the presence of the tracer,complete meningeal lymphatic vessels were visible in the dura mater,and the tracer was observed at the tail of lymphatic vessels.Immunofluorescence staining for endothelial hyaluronan receptor 1 was positive in the deep cervical lymph nodes and dural lymphatics.In summary,the best peeling concentration and time is 120 g/L paraformaldehyde fixed for 24 hours.At this concentration,the dura mater has a stretched morphology,a better toughness,and is more intact after peeling,which is conducive to later use.Verified by Evans blue,tracers and immunofluorescence,deep cervical lymph nodes are located accurately,which can be used as a basis for the study of various neurodegenerative diseases.

BACKGROUND:Meningeal lymphatic vessels can drain cerebral spinal fluid and amyloid β-protein,promoting T lymphocyte to transport and home to deep cervical lymph nodes.A simple,quick and definite method of dural separation and accurate localization of deep cervical lymph nodes can provide strong support for the study of neurodegenerative diseases.OBJECTIVE:To establish a convenient and practical method for exfoliating dural and deep cervical lymph nodes.METHODS:ICR mice,3 months old,were taken,anesthetized and injected with Evans blue and tracer in the occipital pool for localizing deep cervical lymph nodes.A midline incision of about 3 cm in length was made about 5 mm above the clavicle,the superficial fat and fascia were bluntly separated,and the lateral sternocleidomastoid muscle was pulled to expose the deep cervical lymph nodes,which were removed under a stereomicroscope and frozen at-80℃.Subsequently,the mouse head was cut and the skin and muscles of the head were separated to expose the entire skull structure.The skull and brain tissue were separated from the foramen magnum along the lower parietal bone with scissors,and the complete skull top was obtained.The skull was sequentially fixed in 40 g/L paraformaldehyde solution for 24 hours,120 g/L paraformaldehyde for 24 hours,and 120 g/L paraformaldehyde for 10,20,30,and 40 minutes,and the dural structure was stripped.The drainage capacity of meningeal lymphatic vessels and deep cervical lymphatic vessels was verified by tracer,and the meningeal lymphatic vessels were identified by the lymphatic vessel endothelial hyaluronan receptor 1 using the immunofluorescence method.RESULTS AND CONCLUSION:(1)Obvious blue staining was observed in deep cervical lymph nodes 15 minutes after Evans blue staining.(2)The skull was sampled and fixed in 120 g/L paraformaldehyde for 24 hours,resulting in a less tight connection between the dura mater and the skull,and easier stripping of the dural structures with an intact shape.The dura mater fixed at 120 g/L concentration was more resilient and remained more intact during peeling compared with the conventional 40 g/L concentration;120 g/L paraformaldehyde fixed meninges for a short time,and 30-40 minutes was preferred.(3)The frozen section of deep cervical lymph nodes showed the presence of the tracer,complete meningeal lymphatic vessels were visible in the dura mater,and the tracer was observed at the tail of lymphatic vessels.Immunofluorescence staining for endothelial hyaluronan receptor 1 was positive in the deep cervical lymph nodes and dural lymphatics.In summary,the best peeling concentration and time is 120 g/L paraformaldehyde fixed for 24 hours.At this concentration,the dura mater has a stretched morphology,a better toughness,and is more intact after peeling,which is conducive to later use.Verified by Evans blue,tracers and immunofluorescence,deep cervical lymph nodes are located accurately,which can be used as a basis for the study of various neurodegenerative diseases.

ObjectiveTo investigate the effects of Linggui Zhugantang （LGZGT）-containing serum on primary astrocytes （AS） induced by β amyloid 1-42 （Aβ_1-42） in a rat model of Alzheimer's disease （AD） and explore the phagocytic and degradative effects of LGZGT on Aβ. MethodAn AD model was established by inducing AS with Aβ_1-42. The cells were divided into normal group， model group， LGZGT low-， medium-， and high-dose （LGZGT-L， LGZGT-M， and LGZGT-H） groups， and donepezil hydrochloride group. The model group was treated with Aβ_1-42 at a final concentration of 10 μmol∙L^-1. The LGZGT-L， LGZGT-M， and LGZGT-H groups were treated with 10% serum containing LGZGT on the basis of the model group. Cell viability was assessed using a cell counting kit-8 （CCK-8）， lactate dehydrogenase （LDH） activity was measured using an LDH assay kit， and cell morphology was observed using an inverted microscope. The expression of Aβ-related degradation enzymes insulin-degrading enzyme （IDE） and cathepsin D （CTSD） was detected using Western blot， and the fluorescence intensity of cathepsin B （CTSB） was measured using immunofluorescence. The content of Aβ_1-42 in cells was determined using an enzyme-linked immunosorbent assay （ELISA）. ResultCompared with the normal group， the viability of AS in all groups decreased， and Aβ_1-42 at different concentrations had inhibitory effects on AS proliferation. After administration， compared with the normal group， the cell survival rate of the model group decreased significantly （P<0.05）. Compared with the model group， the cell survival rates of the LGZGT-H group and donepezil hydrochloride group increased significantly （P<0.05）. The LDH activity of cells in the model group was significantly increased compared with that in the normal group （P<0.05）， and cell bodies were swollen and enlarged with increased protrusions and elongation， suggesting more obvious cell damage. Compared with the model group， the LDH activity of cells in the donepezil hydrochloride， LGZGT-L， LGZGT-M， and LGZGT-H groups decreased significantly （P<0.05）. After administration， the cell swelling in the LGZGT-M， LGZGT-H， and donepezil hydrochloride groups improved， cell protrusions shortened， and cell clustering decreased. Compared with the normal group， the expression of IDE and CTSD in the model group decreased significantly （P<0.05）. Compared with the model group， the expression of IDE increased significantly in the LGZGT-M and LGZGT-H groups （P<0.05）. Compared with the model group， the expression of CTSD increased significantly in the LGZGT-L， LGZGT-M， LGZGT-H， and donepezil hydrochloride groups （P<0.05）. The average fluorescence intensity of CTSB in the model group was significantly lower than that in the normal group （P<0.05）. Compared with the model group， the average fluorescence intensity of CTSD in the LGZGT-L， LGZGT-M， LGZGT-H， and donepezil hydrochloride groups increased significantly （P<0.05）. The intracellular content of Aβ_1-42 in cells in the model group was significantly higher than that in the normal group （P<0.05）. After administration， compared with the model group， the intracellular content of Aβ_1-42 in cells in the LGZGT-L， LGZGT-M， LGZGT-H， and donepezil hydrochloride groups decreased significantly （P<0.05）， and LGZGT-containing serum reduced Aβ_1-42 in a dose-dependent manner （P<0.05）. ConclusionLGZGT has a protective effect on Aβ_1-42-induced AS and can promote the degradation of Aβ. Its mechanism may be related to reducing Aβ toxicity， enhancing cell viability， promoting the expression of IDE， CTSD， and CTSB， and restoring lysosomal function.

Objective:To evaluate the efficacy and safety of hypomethylating agents (HMA) in patients with myelodysplastic syndromes (MDS) .Methods:A total of 409 MDS patients from 45 hospitals in Zhejiang province who received at least four consecutive cycles of HMA monotherapy as initial therapy were enrolled to evaluate the efficacy and safety of HMA. Mann-Whitney U or Chi-square tests were used to compare the differences in the clinical data. Logistic regression and Cox regression were used to analyze the factors affecting efficacy and survival. Kaplan-Meier was used for survival analysis. Results:Patients received HMA treatment for a median of 6 cycles (range, 4-25 cycles) . The complete remission (CR) rate was 33.98% and the overall response rate (ORR) was 77.02%. Multivariate analysis revealed that complex karyotype ( P=0.02, OR=0.39, 95% CI 0.18-0.84) was an independent favorable factor for CR rate. TP53 mutation ( P=0.02, OR=0.22, 95% CI 0.06-0.77) was a predictive factor for a higher ORR. The median OS for the HMA-treated patients was 25.67 (95% CI 21.14-30.19) months. HMA response ( P=0.036, HR=0.47, 95% CI 0.23-0.95) was an independent favorable prognostic factor, whereas complex karyotype ( P=0.024, HR=2.14, 95% CI 1.10-4.15) , leukemia transformation ( P<0.001, HR=2.839, 95% CI 1.64-4.92) , and TP53 mutation ( P=0.012, HR=2.19, 95% CI 1.19-4.07) were independent adverse prognostic factors. There was no significant difference in efficacy and survival between the reduced and standard doses of HMA. The CR rate and ORR of MDS patients treated with decitabine and azacitidine were not significantly different. The median OS of patients treated with decitabine was longer compared with that of patients treated with azacitidine (29.53 months vs 20.17 months, P=0.007) . The incidence of bone marrow suppression and pneumonia in the decitabine group was higher compared with that in the azacitidine group. Conclusion:Continuous and regular use of appropriate doses of hypomethylating agents may benefit MDS patients to the greatest extent if it is tolerated.

ObjectiveTo study the effect of serum containing Sanwubai San on TGF-β₁ induced epithelial mesenchymal transition （EMT） of human gastric cancer SGC-7901 cells and its mechanism in vitro based on transforming growth factor-β/Smad（TGF-β/Smad）signaling pathway. MethodTwenty-eight male SD rats （SPF grade， three months） were randomly divided into blank group and Sanwubai low （0.031 25 g·kg^-1·d^-1， ig）， medium （0.062 5 g·kg^-1·d^-1， ig） and high （0.125 g·kg^-1·d^-1， ig） dose groups， seven in each group. The blank group was given the same volume of ultrapure water （ig）. The gavage was performed once a day for seven consecutive days. The serum containing the drug was taken from the abdominal aorta 45 min after the last administration. Cell counting kit-8 （CCK-8） method was used to detect the effect of serum in Sanwubai San high dose group on the activity of SGC-7901 cells. Changes of cell morphology after treatment with TGF-β₁ and serum containing Sanwubai San were observed by microscopy， and the migration rate and invasion rate of the SGC-7901 cells were detected by cell scratch assay and transwell assay， respectively. Western blot was used to detect the expression of E-cadherin， snail， TGF-β₁， Smad3， p-Smad3 and Smad7 proteins. ResultCompared with the blank group， 10%， 15% and 20% high-dose Sanwubai San inhibited the activity of SGC-7901 cells in a concentration and time dependent manner. Compared with the conditions in the blank group， the cells in the model group lost spindle shape， and most cells became round and long. Compared with the model group， the Sanwubai San groups had decreased pseudopodia and small cells with the morphology returning to normal. Compared with the conditions in the blank group， enhanced ability of cell migration and invasion （P<0.01）， lowered expression of E-cadherin and Smad7 （P<0.01）， and increased expression of Snail， p-Smad3 and TGF-β₁ （P<0.01） were found in the model group， with the total protein level of Smad3 remaining unchanged. Compared with the conditions in the model group， the cell migration ability was inhibited in the Sanwubai San high and medium dose groups （P<0.01） after 24 h， and the ability was inhibited in all three Sanwubai San groups after 48 h （P<0.01）， while the invasion ability was enhanced. In addition， the Sanwubai San high and medium dose groups had elevated expression of E-cadherin （P<0.01） and Smad7 （P<0.01）， and decreased expression of Snail （P<0.01）， and the expression of TGF-β₁ and p-Smad3 was down-regulated in the three Sanwubai San groups （P<0.01）. ConclusionSanwubai San could inhibit TGF-β₁ induced EMT in SGC-7901 cells， and its mechanism might be related to the regulation of TGF-β/Smad signaling pathway.

Objective:To explore the clinical characteristics, treatment and prognosis of patients with severe lacrimal duct obstruction caused by tegafur-gimeracil-oteracil potassium (S-1).Methods:The medical records and follow-up data of the surgical inpatients with severe lacrimal duct obstruction caused by S-1 in Lacrimal Center of Ophthalmology, the Third Medical Center of Chinese PLA General Hospital from January 2017 to January 2019 were analyzed retrospectively.Results:A total of 12 patients were enrolled in this study, including 7 males and 5 females, aged (53±8) years. The time of oral administration of S-1 was (4.1±1.1) months, and the time from the beginning of administration of S-1 to the onset of epiphora symptoms was (53.3±31.2) days. All the 12 patients had binocular diseases (involving 12 cases, 24 eyes) and obstruction of both upper and lower lacrimal canaliculi (involving 48 lacrimal canaliculi). Among the 48 obstructed lacrimal canaliculi, 45 (93.8%) were severe, and 3 (6.2%) were moderate. The 8 lacrimal points of the upper and lower lacrimal canaliculi in both eyes of the 2 patients were completely atresic. In 12 patients, 16 eyes in 8 patients were complicated with complete obstruction of nasolacrimal ducts and 8 eyes in the other 4 patients were complicated with incomplete obstruction of nasolacrimal ducts. Eight patients underwent laser dacryoplasty combined with bicanalicular intubation assisted by lacrimal canaliculus micro-endoscopy. After taking off the tubes, epiphora was relieved in 5 patients but not improved in the other 3 patients. Two patients underwent retrograde exploration to probe canaliculi transdacryocyst combined with bicanalicular intubation. After taking off the tubes, epiphora was relieved. Two patients underwent retrograde exploration to probe canaliculi transdacryocyst combined with dacryocystorhinostomy. After taking off the tubes, epiphora was improved obviously and only slight epiphora was found.Conclusions:Severe lacrimal duct obstruction caused by S-1 was characterized by extensive multiple obstruction. Appropriate surgical treatment can improve the symptoms of epiphora in some patients.

Objective:To explore the clinical characteristics, treatment and prognosis of patients with severe lacrimal duct obstruction caused by tegafur-gimeracil-oteracil potassium (S-1).Methods:The medical records and follow-up data of the surgical inpatients with severe lacrimal duct obstruction caused by S-1 in Lacrimal Center of Ophthalmology, the Third Medical Center of Chinese PLA General Hospital from January 2017 to January 2019 were analyzed retrospectively.Results:A total of 12 patients were enrolled in this study, including 7 males and 5 females, aged (53±8) years. The time of oral administration of S-1 was (4.1±1.1) months, and the time from the beginning of administration of S-1 to the onset of epiphora symptoms was (53.3±31.2) days. All the 12 patients had binocular diseases (involving 12 cases, 24 eyes) and obstruction of both upper and lower lacrimal canaliculi (involving 48 lacrimal canaliculi). Among the 48 obstructed lacrimal canaliculi, 45 (93.8%) were severe, and 3 (6.2%) were moderate. The 8 lacrimal points of the upper and lower lacrimal canaliculi in both eyes of the 2 patients were completely atresic. In 12 patients, 16 eyes in 8 patients were complicated with complete obstruction of nasolacrimal ducts and 8 eyes in the other 4 patients were complicated with incomplete obstruction of nasolacrimal ducts. Eight patients underwent laser dacryoplasty combined with bicanalicular intubation assisted by lacrimal canaliculus micro-endoscopy. After taking off the tubes, epiphora was relieved in 5 patients but not improved in the other 3 patients. Two patients underwent retrograde exploration to probe canaliculi transdacryocyst combined with bicanalicular intubation. After taking off the tubes, epiphora was relieved. Two patients underwent retrograde exploration to probe canaliculi transdacryocyst combined with dacryocystorhinostomy. After taking off the tubes, epiphora was improved obviously and only slight epiphora was found.Conclusions:Severe lacrimal duct obstruction caused by S-1 was characterized by extensive multiple obstruction. Appropriate surgical treatment can improve the symptoms of epiphora in some patients.

Objective To investigate the distribution and antimicrobial resistance of pathogens causing pneumonia in acute stroke patients,and guide clinical antimicrobial use.Methods Patients with stroke-associated pneumonia (SAP)admitted to a tertiary first-class hospital from 2008 to 2013 were investigated retrospectively,distribution and antimicrobial susceptibility testing results of pathogens from sputum were analyzed.Results A total of 98 pa-tients with SAP were investigated,124 stains were isolated from sputum specimens,75 strains (60.48% )were gram-negative bacteria,44 (35.49% )were gram-positive bacteria,and 5 (4.03% )were fungi. There were 21 cases of mixed infection (21.43% ),bacterial alterations during treatment process existed among 23 cases(23.47% ).The top 4 isolated pathogens were Staphylococcus aureus (S. aureus,n= 43,34.68% ),Klebsiella pneumoniae (K. pneumoniae,n= 19,15.32% ),Pseudomonasaeruginosa(P. aeruginosa,n= 18,14.52% ),and Acinetobacterbau-mannii(A. baumannii,n= 18,14.52% ). Antimicrobial resistance rates of K. pneumoniae were all <32% ,and susceptibility rates to ceftazidime,piperacillin/tazobactam,imipenem,ciprofloxacin,levofloxacin,amikacin,and tobramycin were all 100% . Both A.baumannii and P.aeruginosa showed severe multidrug resistance. Resistance rates of A.baumannii to ceftazidime was >80% ,resistance rates of P.aeruginosa to imipenem was 33 .33% . No resistant strains were detected among fungi.Conclusion The main pathogens causing SAP in this hospital are S.au-reus,K.pneumoniae,A.baumannii,and P.aeruginosa,except K.pneumoniae,the other strains are severely re-sistant to antimicrobial agents,clinicians should choose antimicrobial agents according to the distribution character-istics and antimicrobial susceptibility testing results.

Objective To investigate the relationship between expression of Her-2 gene and the transfer number of axillary lymph node and its influence on prognosis.Methods A total of 351 cases with primary breast cancer from January 2008 to January 2011 were selected.The expression of Her-2 gene was detected by immunohistochemical method,and analyzed the relationship between it and the transfer number of axillary lymph node and its influence on prognosis.Results The expression of Her-2-,+,++ had no correlation with the transfer number of axillary lymph node (x2 =3.757,1.650,2.379,P ＞ 0.05),while the expression of Her-2 +++ had correlation with the transfer number of axillary lymph node (x2 =8.681,P ＜ 0.05).The 2 years survival rates in the expression of Her-2-,+,++,+++ were 77.01％ (201/261),85.00％ (34/40),29.17％(7/24),1 1.54％ (3/26),and which in the expression of Her-2--+ was significantly higher than that in the expression of Her-2 ++-+++ (x2 =61.605,P ＜ 0.01).The transfer number of axillary lymph node was 0 and 1-3,the 2 years survival rate in the expression of Her-2--+ was significantly higher than that in the expression of Her-2 ++-+++,and there was significant difference (x2 =61.605,14.747,P ＜ 0.05).The transfer number of axillary lymph node was 4-9 and ≥ 10,there was no significant difference in the 2 years survival rate between the expression of Her-2--+ and Her-2 ++-+++ (x2 =3.691,3.482,P ＞ 0.05).Conclusions The expression of Her-2-,+,++ has no correlation with axillary lymph node metastasis,and the 2 years survival rate in the expression of Her-2--+ is higher than that in the expression of Her-2 ++-+++,while Her-2-has no difference with Her-2 + in prognosis.While the transfer number of axillary lymph node ≤ 3,the expression of Her-2 gene may be an important prognostic factor.

Objective To analyze and compare the fluorescence in situ hybridization(FISH) and immunohistochemical(IHC) for detecting HER-2 gene amplification and protein expression in breast cancer tissues .Methods 110 cases of breast cancer from Janu-ary 2008 to May 2012 receiving the modified radical mastectomy were selected .The resected breast cancer tissue was detected by FISH and IHC and the detected results were performed the comparative analysis .Results Among 110 cases of breast cancer tissue , 25 cases(22 .73% ) were the HER-2 protein expression(+ + + ) ,44 cases(40 .00% ) were(+ + ) ,26 cases(23 .64% ) were(+ ) and 15 cases(13 .64% ) were(-) .Among 110 cases ,the gene amplification was in 28 cases(25 .45% ) and no gene amplification was in 82 cases(74 .55% ) .The positive(+ + + ) of the IHC detection was coincident with that of FISH ,and the negative(+ /-) of the IHC detection was also coincident with that of FISH ,there was statistical difference between the suspicious positive of the IHC de-tection and the results of FISH (P<0 .05) .But the total coincidence of the IHC detection results and FISH test results was 89 .29%(25/28) ,and the two detection methods had the positive correlation (χ2 =84 .89 ,P<0 .01) .Conclusion The positive and negative expression of the IHC detection has better consistency with that of the FISH detection .However ,the coincidence of the IHC suspi-cious positive expression and the FISH results is poor ,indicating that the suspicious positive sample of the IHC detection needs to be detected by the FISH detection .