Objective To evaluate the safety and efficacy of a self-developed micro-normothermic machine perfusion (NMP) system (micro-perfusion device) for preserving isolated porcine limbs. Methods Five healthy Landrace pigs were selected, and their left and right forelimbs were randomly divided into the NMP group and static cold storage (SCS) group. The NMP group was perfused with the self-developed micro-perfusion device and polymerized hemoglobin perfusate for 32 hours at normothermia, while the SCS group was preserved at 4 ℃. Hemodynamic parameters such as perfusion pressure and flow were monitored. The pH value, partial pressure of oxygen (PO₂), lactic acid (Lac), creatine kinase (CK) and lactate dehydrogenase (LDH) in the perfusate were measured. Hematoxylin-eosin staining was used to assess the muscle tissue structure, terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling was employed to evaluate muscle cell apoptosis, and immunohistochemistry staining was applied to detect the expressions of tumor necrosis factor (TNF)-α and interleukin (IL)-6. A mixed-effects model was used to analyze the effects of time and treatment methods on tissue structure, cell apoptosis and inflammatory factors. Results The device could stably maintain a perfusion pressure of (69±15) mmHg and a flow rate of (117±42) mL/min. The pH value and electrolytes of the perfusate were generally stable, with PO₂ maintained at a high level. Lac was maintained at 5.38(3.81, 6.45) mmol/L, while CK and LDH increased over time. After 32 hours of perfusion in the NMP group, both the myocyte spacing and apoptosis rate were better than those in the SCS group. Mixed-effects model analysis showed that there were statistically significant differences in the effects of NMP treatment and SCS treatment on myocyte spacing and apoptosis rate per unit time (both P < 0.05). There were no statistically significant differences in TNF-α and IL-6 between the two groups, and mixed-effects model analysis showed no statistically significant differences in the effects of NMP treatment and SCS treatment on TNF-α and IL-6 per unit time (both P > 0.05). Conclusions The micro-perfusion device used in this study may achieve 32-hour normothermic preservation in a porcine limb amputation model, maintain basic metabolism and ionic homeostasis, reduce muscle structural damage and cell apoptosis without inducing additional inflammatory responses. This technology is expected to significantly extend the time window for replantation of amputated limbs in disaster rescue and long-distance transportation, providing an important technical basis for clinical translation and subsequent replantation research.

Macrophages are innate immune cells connected with the development of inflammation. Retinoic acid has previously been proved to have anti-inflammatory and anti-arthritic properties. However, the exact mechanism through which retinoic acid modulates arthritis remains unclear. This study aimed to investigate whether retinoic acid ameliorates rheumatoid arthritis by modulating macrophage polarization. This study used retinoic acid to treat mice with adjuvant arthritis and evaluated anti-inflammatory effects by arthritis score, thermal nociceptive sensitization test, histopathologic examination and immunofluorescence assays. In addition, its specific anti-arthritic mechanism was investigated by flow cytometry, cell transfection and inflammatory signaling pathway assays in RAW264.7 macrophages in vitro. Retinoic acid significantly relieved joint pain and attenuated inflammatory cell infiltration in mice. Furthermore, this treatment modulated peritoneal macrophage polarization, increased levels of arginase 1, as well as decreased inducible nitric oxide synthase expression. In vitro, we verified that retinoic acid promotes macrophage transition from the M1 to M2 type by upregulating mitogen-activated protein kinase (MAPK) phosphatase 1 (MKP-1) expression and inhibiting P38, JNK and ERK phosphorylation in lipopolysaccharide-stimulated RAW264.7 cells. Notably, the therapeutic effects of retinoic acid were inhibited by MKP-1 knockdown. Retinoic acid exerts a significant therapeutic effect on adjuvant arthritis in mice by regulating macrophage polarization through the MKP-1/MAPK pathway, and play an important role in the treatment of rheumatic diseases.

Macrophages are innate immune cells connected with the development of inflammation. Retinoic acid has previously been proved to have anti-inflammatory and anti-arthritic properties. However, the exact mechanism through which retinoic acid modulates arthritis remains unclear. This study aimed to investigate whether retinoic acid ameliorates rheumatoid arthritis by modulating macrophage polarization. This study used retinoic acid to treat mice with adjuvant arthritis and evaluated anti-inflammatory effects by arthritis score, thermal nociceptive sensitization test, histopathologic examination and immunofluorescence assays. In addition, its specific anti-arthritic mechanism was investigated by flow cytometry, cell transfection and inflammatory signaling pathway assays in RAW264.7 macrophages in vitro. Retinoic acid significantly relieved joint pain and attenuated inflammatory cell infiltration in mice. Furthermore, this treatment modulated peritoneal macrophage polarization, increased levels of arginase 1, as well as decreased inducible nitric oxide synthase expression. In vitro, we verified that retinoic acid promotes macrophage transition from the M1 to M2 type by upregulating mitogen-activated protein kinase (MAPK) phosphatase 1 (MKP-1) expression and inhibiting P38, JNK and ERK phosphorylation in lipopolysaccharide-stimulated RAW264.7 cells. Notably, the therapeutic effects of retinoic acid were inhibited by MKP-1 knockdown. Retinoic acid exerts a significant therapeutic effect on adjuvant arthritis in mice by regulating macrophage polarization through the MKP-1/MAPK pathway, and play an important role in the treatment of rheumatic diseases.

Macrophages are innate immune cells connected with the development of inflammation. Retinoic acid has previously been proved to have anti-inflammatory and anti-arthritic properties. However, the exact mechanism through which retinoic acid modulates arthritis remains unclear. This study aimed to investigate whether retinoic acid ameliorates rheumatoid arthritis by modulating macrophage polarization. This study used retinoic acid to treat mice with adjuvant arthritis and evaluated anti-inflammatory effects by arthritis score, thermal nociceptive sensitization test, histopathologic examination and immunofluorescence assays. In addition, its specific anti-arthritic mechanism was investigated by flow cytometry, cell transfection and inflammatory signaling pathway assays in RAW264.7 macrophages in vitro. Retinoic acid significantly relieved joint pain and attenuated inflammatory cell infiltration in mice. Furthermore, this treatment modulated peritoneal macrophage polarization, increased levels of arginase 1, as well as decreased inducible nitric oxide synthase expression. In vitro, we verified that retinoic acid promotes macrophage transition from the M1 to M2 type by upregulating mitogen-activated protein kinase (MAPK) phosphatase 1 (MKP-1) expression and inhibiting P38, JNK and ERK phosphorylation in lipopolysaccharide-stimulated RAW264.7 cells. Notably, the therapeutic effects of retinoic acid were inhibited by MKP-1 knockdown. Retinoic acid exerts a significant therapeutic effect on adjuvant arthritis in mice by regulating macrophage polarization through the MKP-1/MAPK pathway, and play an important role in the treatment of rheumatic diseases.

Objective:To explore the efficacy and safety of one-hole split endoscope (OSE) minimally invasive surgery for the treatment of single-segment thoracic ossification of the ligamentum flavum (TOLF).Methods:This retrospective non-randomized controlled study included 41 patients with single-segment TOLF who underwent surgery at Qilu Hospital of Shandong University between July 2019 and July 2023. Patients were divided into two groups: the OSE group (19 cases) treated with one-hole split endoscope minimally invasive surgery and the open group (22 cases) treated with traditional laminectomy and pedicle screw fixation. There were no significant differences between the two groups on gender, age, disease duration, affected segment, presence or absence of dural ossification, and residual cross-sectional vertebral canal area on CT ( P>0.05). Additionally, perioperative surgical time, estimated blood loss (EBL), incision length, hospital stay duration, hospitalization costs and follow-up duration were compared. The Japanese Orthopaedic Association (JOA) score and Oswestry Disability Index (ODI) were compared preoperatively and at the last follow-up. Complications were also recorded. Results:All patients successfully completed the surgery with no significant differences at the last follow-up ( P>0.05). Compared with the open group, the OSE group had a significantly shorter operative time (133.1±16.8 vs. 160.5±22.6 min), lower EBL (91.2±15.0 vs. 192.5±43.8 ml), shorter incision length (2.6±0.5 vs. 7.9±1.9 cm), reduced hospital stay (3.9±0.8 vs. 5.6±0.8 days), and lower hospitalization costs (34,874.9±4,568.6 vs. 53,162.3±9,815.6 yuan) (all P<0.05). AAt the final follow-up, JOA scores (8.5±0.8 vs. 8.6±1.2) and ODI values (16.7%±2.1% vs. 17.7%±4.4%) showed no significant differences between the OSE and open groups ( P>0.05). During the perioperative period and follow-up, complications occurred in 2 patients in the OSE group (1 cerebrospinal fluid leak, 1 poor wound healing) and in 8 patients in the open group (5 cerebrospinal fluid leaks, 1 neurological deterioration, 2 poor wound healing). Conclusion:OSE minimally invasive surgery is an effective treatment for single-segment thoracic ossification of the ligamentum flavum. Compared with open surgery, it provides advantages such as minimal invasiveness and fewer complications.

Objective:To analyze the clinical outcomes of total ankle arthroplasty (TAA) using the INBONE Ⅱ prosthesis in patients ≤50 years old with end-stage ankle arthritis.Methods:A retrospective analysis was conducted of the consecutive patients who had undergone TAA using the INBONE Ⅱ prosthesis between September, 2016 and August, 2021 at Department of Foot and Ankle Surgery, Beijing Jishuitan Hospital, Capital Medical University. There were 12 males and 16 females with an age of (46.0±4.0) years and a body mass index of (24.9±3.3) kg/m 2. The clinical outcomes recorded and compared between pre-surgery and the last follow-up were tibial articular surface (TAS) angle, talar tilt (TT) angle, tibial lateral surface (TLS) angle, ankle plantarflexion angle, ankle dorsiflexion, ankle range of motion (ROM), American Orthopaedic Foot & Ankle Society (AOFAS) ankle-hindfoot score, MOS item short form health survey (SF-36), foot function index (FFI), and visual analog scale (VAS) for pain. Patient satisfaction by 5-point Likert scale and complications were recorded. Results:All patients were followed up for (49.8±15.3) months. Except for plantar flexion and TAS, for all patients at the last follow-up, TT angle [0.4° (0, 0.6°)], FFI [20.0 (7.3, 48.0) points], and VAS pain score [2.0 (1.0, 2.8) points] were significantly reduced compared with the preoperative values [1.6° (0.3°, 4.4°), (99.9±40.6) points, and 6.0 (4.0, 6.8) points], while TLS angle (86.3°±2.8°), ankle dorsiflexion (13.5°±5.4°), ankle ROM (34.7°±7.9°), AOFAS ankle-hindfoot score [82.0 (74.0, 89.0) points], and SF-36 score [122.5 (112.8, 130.2) points] were all significantly higher than the preoperative values [78.9°±5.7°, 10.3°±8.0°, 31.1°±12.0°, (49.9±3.2) points, and 97.7(89.8, 101.6) points] (all P<0.05). The rate of patient satisfaction at the last follow-up was 89.3% (25/28). No serious postoperative complications occurred and no revision surgery was required. Conclusion:TAA using the INBONE Ⅱ prosthesis has shown good clinical outcomes and a high rate of patient satisfaction in younger patients ≤50 years old with end-stage ankle arthritis.

Objective:To compare the protective effects of the static cold storage (SCS) and normothermic machine perfusion (NMP) on the skeletal muscle of the amputated limbs of pigs.Methods:Four Landrace pigs were selected, from which eight limbs were amputated and divided into SCS group ( n=5) and NMP group ( n=3) according to the random number table method. After blood collection from the carotid artery, an amputated limb model was established by amputating the limbs at the scapulohumeral joints. The limbs in the SCS group were wrapped in sterile cloth and stored at 4 ℃ for 24 hours. In the NMP group, the limbs were mechanically perfused with a red blood cell-containing perfusion fluid at 37 ℃ for 24 hours, with 70% of the perfusion fluid replaced every 6 hours. Before the experiment, cross-matching tests with the saline medium were conducted between donor and recipient pigs to evaluate blood coagulation and blood safety in the NMP group. An allogeneic red blood cell perfusion fluid was prepared and the levels of pH, Na +, K +, Cl -, Ca 2+, glucose (Glu), hematocrit (Hct), lactic acid (Lac) and osmotic pressure of the perfusion fluid were measured. At 0, 6, 12, 18, and 24 hours after perfusion, the skin temperature and oxyhemoglobin saturation (SaO 2) levels in the NMP group were monitored and the levels of pH, Glu, creatine kinase (Ck), K +, Ca 2+, and Na +levels of the perfusion fluid were analyzed to evaluate the metabolism of the skeletal muscle in the amputated limbs. The mean intercellular distance and apoptosis index of the myocytes were quantitatively analyzed and histopathological changes were observed by performing HE staining and TUNEL staining on the skeletal muscle of the amputated limbs in both groups at 0 and 24 hours after perfusion. After perfusion was ended, the weight gain rate and swelling degree of the amputated limbs were compared between the two groups and the overall state of the amputated limbs was evaluated. Results:The result of the cross-matching test between donor and recipient pig blood was negative. The parameters in the prepared red blood cell-containing perfusion fluid generally maintained within a normal range: pH 7.38±0.04, Na + concentration (138.30±4.48)mmol/L, K + concentration (3.50±0.26)mmol/L, Glu concentration (6.11±2.08)mmol/L, and osmotic pressure (305.67±3.79)mmol/L. However, slightly higher Cl - and Ca 2+ concentrations [(118.34±12.00)mmol/L and (2.00±0.15)mmol/L] and lower Hct and lactate concentrations [0.30±0.03 and (1.54±0.38)mmol/L] were detected when compared with the reference range. During the perfusion, the average skin temperature of the amputated limbs in the NMP group was (36.13±0.98)℃, with the skin temperatures at 6, 12, 18, and 24 hours after perfusion being significantly higher than that at 0 hour ( P<0.01), while no significant difference among the skin temperatures at 6, 12, 18, and 24 hours after perfusion was observed ( P>0.05). The SaO 2 levels in the skin of the amputated limbs in the NMP group averaged over 95%, which showed no significant difference at 0, 12, 18, and 24 hours after perfusion ( P>0.05), while a significant elevation was observed at 6 hours compared with that at 0 hour ( P<0.05). There were no significant differences in pH, Glu, Na +, and Ca 2+ levels in the NMP group at 0, 6, 12, 18, and 24 hours after perfusion ( P>0.05), while the Ck levels at 18 and 24 hours were both significantly higher than that at 6 hours after perfusion ( P<0.05), and the Ck levels at 6, 12, 18, and 24 hours were all significantly higher than that at 0 hour ( P<0.05). The K + level progressively increased with the perfusion time, with significant elevations at 18 and 24 hours after perfusion compared with that at 0 hour ( P<0.05). HE staining revealed well-preserved muscle fiber continuity and regular arrangement in the NMP group and the SCS group at 0 hour, with an intercellular distance of (8.95±0.60)μm. At 24 hours, the NMP group exhibited slight skeletal muscle fiber rupture and swelling, with a slightly increased intercellular distance of (14.75±0.90)μm, significantly greater than that at 0 hour ( P<0.01). At 24 hours, the SCS group showed marked skeletal muscle fiber rupture and swelling, with a significantly increased intercellular distance of (23.51±1.49)μm, significantly larger than those at 0 hour in the same group and at 24 hours in the NMP group ( P<0.01). TUNEL immunofluorescence staining indicated a tiny amount of apoptotic cells in the skeletal muscle in both groups at 0 hour, with an apoptotic index of (4.26±1.62)%. There was a small number of apoptotic cells in the skeletal muscle in the NMP group at 24 hours, with an apoptotic index of (25.94±2.69)%, significantly larger than that in the same group at 0 hour ( P<0.01). The SCS group exhibited a large number of apoptotic cells at 24 hours, with an apoptotic index of (62.97±3.22)%, significantly larger than those at 0 hour in the same group and at 24 hours in the NMP group ( P<0.01). In comparison with the SCS group at 24 hours, the amputated limbs in the NMP group showed red color in the appearance, no symptoms of ischemic muscle contracture and good joint movement despite slight edema in the subcutaneous layer. At 24 hours, the weight gain rate of the amputated limbs was (15.82±0.89)% in the NMP group, significantly higher than (0.97±0.28)% in the SCS group ( P<0.01). Conclusion:Compared with SCS, NMP with the red blood cell-containing perfusion fluid prepared with the allogeneic blood for the amputated limbs of pigs can alleviate the ischemic injury of the muscle fibers and inhibit the apoptosis of the muscle cells by sustaining stable energy and oxygen supply and balancing ion homeostasis and pH of the perfusion fluid.

Objective To analyze the clinical significance of ductoscopy,mammography,and high-frequency color Doppler ultrasonography in diagnosis of nipple bloody discharge combined with intraductal space occupying lesions.Methods The medical records of 198 patients with nipple bloody discharge combined with intraductal space occupying lesions who underwent surgical treatment from January 2023 to December 2023 were retrospectively analyzed,all the subjects were examined by ductoscopy,mammography and high-frequency color Doppler ultrasonography before operation.The results of postoperative pathology were used as the gold standard to compare and evaluate the efficacy of single and combined use of the above three methods in the diagnosis of nipple bloody discharge combined with intraductal space occupying lesions.Results The results of postoperative pathological diagnosis showed that 152 cases of intraductal benign lesions were intraductal papilloma of the breast.There were 46 malignant lesions,of which 25 were intraductal carcinoma and 21 were invasive ductal carcinoma.The detection rates of nipple bloody discharge combined with intraductal space occupying lesions were 95.45%,81.31%,84.85%and 97.47%,respectively,by ductoscopy,mammography,high-frequency color Doppler ultrasonography and combined examination.Among them,the detection rate of ductoscopy and combined examination were significantly higher than that of mammography and high-frequency color Doppler ultrasonography(P<0.05),there was no significant difference between the detection rate of ductoscopy and combined examination(P>0.05),and there was no significant difference between the detection rate of mammography and high-frequency color Doppler ultrasonography(P>0.05).The sensitivity,specificity,and accuracy of diagnosing nipple bloody discharge combined with intraductal space occupying lesions using ductoscopy were 91.30%,97.20%,and 95.77%,respectively.The above indicators of mammography were 65.00%,94.21%,and 86.96%,respectively.The above indicators of high-frequency color Doppler ultrasonography were 70.73%,96.06%,and 89.88%,respectively.The above indicators of combination of the three methods were 95.65%,97.28%,and 96.89%,respectively.The sensitivity and accuracy of the combined examination of the three methods and the use of ductoscopy alone for the diagnosis of nipple bloody discharge combined with intraductal space occupying lesions were significantly higher than those of mammography and high-frequency color Doppler ultrasonography(P<0.05).However,there was no significant difference between mammography and high-frequency color Doppler ultrasonography(P>0.05),and there was also no significant difference between the combined examination of the three methods and the use of ductoscopy alone(P>0.05).There was no significant difference in diagnostic specificity among the four groups(P>0.05).Conclusion The diagnostic sensitivity and accuracy of ductoscopy for nipple bloody discharge combined with intraductal space occupying lesions are significantly higher than those of mammography and high-frequency color Doppler ultrasonography.The combined examination of the three can improve diagnostic efficiency to a certain extent,but the effect is limited.

Objective:To investigate whether repeated tirofiban injections can continuously inhibit platelet aggregation in the arterial duct and affect its closure in neonatal canines.Methods:Four 24-month-old pregnant beagles underwent cesarean sections in two batches (two dogs per batch) 1-2 days before the expected delivery date at the Xuzhou Medical University Animal Experiment Center. The first litter of 21 neonates served as the control group (receiving 10 ml/kg normal saline) and were randomly divided into 1-h ( n=7, injected immediately after birth), 4-h ( n=7, injected at 0 h and 2 h after birth), and 12-h subgroups ( n=7, injected at 0 h, 2 h, 4 h, 6 h, 8 h, and 10 h after birth). The second litter of 18 neonates served as the experimental group (receiving 10 ml/kg tirofiban) with identical subgroup assignments ( n=6 per subgroup). Echocardiography was performed at 1 h, 4 h, and 12 h after birth to measure arterial duct inner diameter, maximum shunt velocity, and left atrial diameter/aortic root diameter (LA/Ao) ratio. Plasma platelet-derived growth factor (PDGF) was detected by enzyme-linked immunosorbent assay, while platelet membrane glycoprotein Ⅱb-Ⅲa in the arterial duct was assessed by Western blot and immunohistochemistry. Data were analyzed using t-tests, one-way ANOVA, Chi square tests, or Fisher's exact test. Results:No significant bleeding tendency occurred in either group. Two control neonates (one each in the 4-h and 12-h subgroups) died. In both control and experimental 1-h subgroups, all arterial ducts remained open, with no significant differences in ductal diameter, shunt velocity, or LA/Ao between groups (all P>0.05). In the 4-h subgroups, all experimental neonates had patent ducts arteriosus, while two controls exhibited closure; the experimental group had larger ductal diameters [(1.05±0.05) vs. (0.55±0.44) mm, t=－2.75, P<0.05)] and higher LA/Ao ratios (1.31±0.09 vs. 1.14±0.03, t=－4.90, P<0.05), but lower maximum shunt velocities [(107.06±17.47) vs. (153.74±12.78) cm/s, t=4.54, P=0.002). In the 12-h subgroups, all the controls had closed arterial ducts, while four of six experimental neonates exhibited closure, though the difference in closure rate was not statistically significant (6/6 vs. 4/6, Fisher's exact test, P=0.455). Plasma PDGF and glycoprotein Ⅱb-Ⅲa levels did not differ between two 1-h subgroups (all P>0.05). However, the 4-h and 12-h experimental subgroups showed lower PDGF levels [(373.5±13.1) vs. (880.3±80.2) pg/ml, t=10.81; (356.7±35.0) vs. (1 111.2±125.3) pg/ml, t=9.74; both P<0.05] and reduced glycoprotein Ⅱb-Ⅲa expression (0.32±0.07 vs. 0.80±0.23, t=3.29; 0.42±0.07 vs. 0.92±0.26, t=3.24; both P<0.05) compared to controls. Conclusion:Repeated tirofiban injections sustainably inhibit platelet aggregation in the arterial duct of neonatal canines and delay ductal closure, suggesting that intraductal platelet aggregation may be one factor influencing this process.

Craniocervical junction zone malformations often have an insidious onset, a variety of clinical phenotypes, and are often combined with multiple malformations, making their systematic classification and staging more difficult.At present, craniocervical junction area malformations are often classified into congenital and acquired, and can also be classified into skull base malformations, atlantoaxial malformations, and cardinal malformations according to their locations. For patients with obvious occipitocervical or atlantoaxial instability, combined with symptoms of high cervical spinal cord damage, internal fixation and fusion surgery should be performed aggressively to avoid irreversible nerve damage.There is a lack of detailed categorisation and summary of the treatment of diseases associated with craniocervical junction malformations in the literature, and the treatment strategies for some of these malformations are still controversial, with different perceptions and treatment concepts in the national and international literature.