HE Staining Observation Research Articles

Investigating metabolic characteristics of type 2 diabetes mellitus-related cognitive dysfunction and correlating therapeutic effects of Di Dang Tang in animal models.

Di Dang Tang is a classic formula from Shang Han Lun, originally used to treat conditions such as blood stasis and heat accumulation. It is widely applied in the treatment of diabetes and its complications, but its effects on Type 2 Diabetes Mellitus-related Cognitive Dysfunction (T2DM-CD) remain unclear. The study aimed to investigate the metabolic characteristics of patients with T2DM-CD. Additionally, it sought to evaluate the effects of Di Dang Tang on cognitive function in T2DM-CD model rats by targeting the metabolic pathways identified in the clinical analysis, exploring the underlying mechanisms through animal experiments. Fasting venous serum was collected from patients with Type 2 Diabetes Mellitus (T2DM) to detect metabolism-related products, and KEGG annotation analysis was performed. Separately, thirty rats were randomly divided using a random number table method, with six rats selected as the blank control group. Twenty-four successfully modeled rats were then randomly divided into the model group and three Di Dang Tang groups (low, medium, and high doses). After administering the medication, the relevant indicators in the rats were assessed. Clinical metabolomics detected 32 key differential metabolites between the T2DM-CD and the blank control groups. Between the T2DM-CD and T2DM groups, 29 key differential metabolites were identified. In animal experiments, blood glucose levels in the model group were significantly higher compared to the blank control group at the same time points, whereas the high dose groups of Di Dang Tang exhibited reduced blood glucose levels at weeks 6 and 8 relative to the model group. In the Morris water maze test, the model group had longer escape latencies than the blank control group. The medium and high dose groups of Di Dang Tang showed shorter latencies. Additionally, compared to the model group, the Di Dang Tang groups spent more time and covered more distance in the target quadrant but had reduced average proximity and fewer platform entries. HE staining observation of the hippocampal CA1 area showed no apparent pathological changes in the blank group, obvious pathological damage in the model group, and no significant pathological changes in the medium and high dose groups of Di Dang Tang. Compared to the blank control group, the model group showed significant increases in the levels of Arachidonic Acid (AA), Ceramide (Cer), Glutamate (Glu), TNF- α, IL-1β, TG, and LDL-C, and a significant decrease in HDL-C levels. Compared to the model group, the groups of Di Dang Tang significantly modulated the levels of the above indicators. In Western Blot (WB) assays, compared to the blank control group, the model group rats exhibited significantly higher levels of cPLA2, PKC, ERK, and JNK , and significantly lower levels of claudin-5, NMDA, CaMKII, CREB, and BDNF. The Di Dang Tang groups significantly altered the levels of the above indicators compared to the model group. Amino acid metabolism, sphingolipid signaling pathways, glycerophospholipid metabolism, and various signaling pathways play significant roles in the pathogenesis of T2DM-CD. Di Dang Tang can improve learning and memory abilities in T2DM model rats and ameliorate cognitive impairments, potentially by regulating metabolic levels and inflammatory responses.

The novel design of an intelligent anti-depression transdermal drug delivery system

Depression is a type of mental disorder with a significant and persistent low mood as the main clinical feature. It is often accompanied by symptoms such as slow thinking, decreased will, loss of appetite, and weight loss. The current treatment methods for depression are mainly medical treatment, psychotherapy, and physical therapy. These treatments are dependent on the patient's autonomy and the patients may suspend treatment due to forgetting or refusing. Therefore, an anti-depressant intelligent drug release system was designed, which can achieve autonomously controlled doses for the treatment of depression by transdermal drug delivery system. The work of this study is as follows:(1) The first module: the electrothermal material heating layer. Several preparation methods were screened, and multiple sets of graphene (GE) electric thermogenic layers were successfully prepared. After increasing the actual energization area to 1 cm × 1 cm, the GE electric thermogenic layer is used as the heating layer of the electrothermal material of the system, and can reach a uniform surface temperature of (45 ± 0.5) °C within 15 s at a voltage of 6 V keeping the temperature fluctuation range not exceeding ±0.03 °C, and the resistance fluctuation range not exceeding ±20 Ω, which plays a role in controlling the temperature and heat treatment of the drug loaded gel layer.(2) The second module: the drug-loaded gel layer. Based on the L16 (45) orthogonal test, the best formulation and process of N-Isopropyl acrylamide-Acrylamide copolymer (P(NIPAAm-co-AAm)) hydrogel was determined. Then, the percutaneous permeability of Selegiline liposome was studied in vitro.(3) A rat model of depression was established using chronic unpredictable mild stress (CUMS) combined with separation. From the aspects of behavior (body weight, sucrose preference test, forced swimming test, open field test) and biochemical indexes (serum proinflammatory cytokines (IL-1β, TNF-α), hippocampus HE staining observation), the therapeutic effect of hyperthermia, Selegiline oral administration and transdermal administration was discussed.

Research Status and Prospects of Non-Traumatic Fat Embolism in Forensic Medicine.

In the practice of forensic pathology, fat embolism is one of the common causes of death, which can be divided into two categories: traumatic and non-traumatic. Non-traumatic fat embolism refers to the blockage of small blood vessels by fat droplets in the circulatory blood flow caused by non-traumatic factors such as underlying diseases, stress, poisoning and lipid metabolism disorders. At present, it is believed that the production of non-traumatic fat embolism is related to the disturbance of lipid metabolism, C-reactive protein-related cascade reaction, the agglutination of chylomicron and very low-density lipoprotein. The forensic identification of the cause of death of non-traumatic fat embolism is mainly based on the case, systematic autopsy, HE staining and fat staining, but it is often missed or misdiagnosed by forensic examiners because of its unknown risk factors, hidden onset, the difficulty of HE staining observation and irregular implementation of fat staining. In view of the lack of attention to non-traumatic fat embolism in forensic identification, this paper reviews the concepts, pathophysiological mechanism, research progress, existing problems and countermeasures of non-traumatic fat embolism, providing reference for forensic scholars.

Study on the correlation between the content of bone morphogenetic protein 2 in demineralized bone matrix and its osteogenic activity in vitro and in vivo

To investigate the correlation between the content of bone morphogenetic protein 2 (BMP-2) in demineralized bone matrix (DBM) and its osteogenic activity in vitro and in vivo, in order to choose a simple and convenient method to evaluate the osteogenic activity of DBM. The left mid-femoral tissues of 9 donors were taken, and DBMs (S1-S9) were prepared by dynamic decalcification process, and inactivated DBM (control group) was prepared at the same time. Protease inhibitor method, collagenase method, guanidine hydrochloride/ethylene diamine tetraacetic acid (EDTA) method, and RIPA lysate method were used to extract BMP-2 in S1-S9 and inactivated DBMs. The BMP-2 content was measured and the differences between DBMs were compared. Then the S1-S9 and inactivated DBMs were co-cultured with mouse embryonic osteoblasts MC3T3-E1, respectively. The cell proliferation was detected by MTT method and fluorescence staining, and alkaline phosphatase (ALP) activity was detected at the same time. Thirty BALB/c male nude mice were divided into 10 groups, namely S1-S9 DBM groups (S1-S9 groups) and inactivated DBM group (control group), with 3 mice in each group. Muscle pockets of the middle thighs were prepared on both hindlimbs of mice in each group, and implanted corresponding DBM materials. At 4 weeks after operation, the samples were taken for HE staining observation and semi-quantitative evaluation, and the new bone formation score was calculated. The BMP-2 content of DBM derived from different donor bones was distinct. The BMP-2 content obtained by different extraction methods for DBM prepared from the same donor bone was also different, and the extraction efficiency of the guanidine hydrochloride/EDTA method was the highest. In vitro cell experiments, MTT test displayed that cell proliferations and ALP activity were significantly higher in S4 and S6 groups than in other groups at each time point after co-cultivation ( P<0.05). Moreover, the cell proliferation of S4 group was the most significant at 7 days ( P<0.05); fluorescence staining demonstrated that the osteoblasts of each group was in good condition, but the osteoblasts of S1, S2, S3, S4, and S6 groups were significantly more than other groups. In vivo ectopic osteogenesis experiments, the cartilage and new bone formation could be seen in the bone graft area of S1-S6 groups at 4 weeks after operation, and with the increase of BMP-2 content, the more new bone formation induced by the material, the higher the score of new bone formation of the material ( P<0.05). Among them, S4 and S6 groups contained a large number of chondrocytes and osteoblasts in the osteogenesis area. The osteogenic activity of DBM can be evaluated through BMP-2 quantitative detection combined with in vitro osteoblast proliferation and differentiation experiments.

Preliminary study on true bone ceramics for alveolar ridge preservation in dogs

To study the preservation effect of true bone ceramics (TBC) prepared by high-temperature calcination of bovine bone on alveolar ridge of canine extraction socket. Six healthy Beagle dogs (aged 1.5-2 years) were selected to extract the second and fourth premolars of both mandibles and the second premolars of the maxilla. The left extraction socket was implanted with TBC as the experimental group, and the right side was implanted with the calcined bovine bone (CBB) as the control group, to observe the alveolar ridge preservation effect. Three dogs were euthanized after general observation at 1 and 6 months after operation respectively. After separating the maxilla and mandible, cone beam CT (CBCT) was performed to measure the average gray value of the graft site and the adjacent reference area (the area between the roots of the adjacent third premolar) and calculate the gray scale ratio between the bone graft site and the reference area. Histological observation was made on the bone graft site to evaluate the new bone formation. General observation showed that the wounds of both groups were basically healed at 2 weeks after operation, and the bone graft materials were not exposed. The wounds healed well at 1 and 6 months after operation without swelling. The results of CBCT showed that the residual material was found in both groups at 1 month after operation, and no significant residual material was found in both groups at 6 months after operation, and the alveolar ridge height of the bone graft area was not significantly reduced. There was no significant difference in the bone mineral density between the experimental group and the control group. The gray scale ratios of the experimental group at 1 month and 6 months after operation were 0.97±0.14 and 0.93±0.06, respectively, and were 0.99±0.16 and 0.94±0.05 in control group, showing no significant difference between the two groups ( t=-1.030, P=0.333; t=-0.770, P=0.466). HE staining observation showed that a large number of bone graft materials did not degrade and new bone formed around the grafts in both groups at 1 month after operation; the bone graft materials were absorbed and a large number of new bones were formed in both groups at 6 months after operation. TBC can maintain bone mineral density and have good osteoconductivity in the alveolar ridge site preservation experiment of dogs, and can be used for alveolar ridge site preservation.

Application of biomimetic mineralized collagen bone graft material in rabbits posterolateral spinal fusion

To investigate the bone repair and regeneration ability of biomimetic mineralized collagen bone graft material and autologous bone marrow in rabbit posterolateral spinal fusion model. Twenty-seven 20-week-old male New Zealand white rabbits ［weighing (5.0±0.5) kg］ were used to establish the posterolateral spinal fusion model of L 5 and L 6 segments by stripping the transverse process and exposing cancellous bone with electric burr. The rabbits were randomly divided into 3 groups, 9 in each group. Groups A, B, and C were implanted 1.5 mL autologous iliac bone, 1.5 mL (30 mm×10 mm×5 mm) biomimetic mineralized collagen bone graft material, and 1.5 mL (30 mm×10 mm×5 mm) biomimetic mineralized collagen bone graft material and autologous bone marrow in each bone defect. At 4, 8, and 12 weeks after operation, the apparent hardness of the bone grafting area was observed by manipulation method, in order to evaluate bone graft fusion effects. Three animals were sacrificed in each group at each time point, the vertebral body specimens were excised and the bone defect repair and fusion were observed by X-ray films, and three-dimensional CT examination was performed to evaluate whether new bone was formed in the body. HE staining was performed at each time point to observe the formation of new bone and the repair and fusion of bone defects. The manipulation test showed that bone graft fusion was not found in all groups at 4 weeks after operation; 3 (50.0%), 2 (33.3%), and 4 (66.7%) of groups A, B, and C reached bone graft fusion at 8 weeks after operation; 5 (83.3%), 4 (66.7%), and 5 (83.3%) of groups A, B, and C reached bone graft fusion at 12 weeks after operation; the fusion rate of group C was similar to that of group A, and all higher than that of group B. X-ray film observation showed that the fusion rate of group C at 8 and 12 weeks after operation was higher than that of group B, similar to group A. Three-dimensional CT observation showed that the effect of bone fusion in group C was better than that in group B, which was close to group A. HE staining observation showed that large area of mature lamellar bone coverage appeared in the bone graft area of groups A, B, and C at 12 weeks after operation, the material was completely degraded, and the marginal boundary of the host bone disappeared and tightly combined. Biomimetic mineralized collagen bone graft material mixed with autologous bone marrow has good osteoinduction and osteogenesis guidance. Compared with biomimetic mineralized collagen bone graft material, it has better and faster osteogenesis effect, which is close to autologous bone transplantation.

The biocompatibility and immunogenicity study of decellularized tracheal matrix

To investigate the biocompatibility and immunogenicity of the tracheal matrix decellularized by sodium perchlorate (NaClO 4). Bone marrow mesenchymal stem cells (BMSCs) were divided from 2-month-old New Zealand white rabbits. The trachea of 6-month-old New Zealand white rabbits were trimmed to a length of 1.5 cm and randomly divided into control group (group A 1, n=5, just stripped the loose connective tissue outside the trachea) and experimental group (group B 1, n=5, decellularized by improved NaClO 4 immersion method). The cytotoxicity of the scaffold leaching solution was detected by MTT assay, and the major histocompatibility complex (MHC) expression was detected by immunohistochemical method. The 4th generation of BMSCs were seeded onto the scaffold of 2 groups, and the cell activity around the material was observed by inverted microscope after Giemsa staining at 48 hours, while the cells states on the scaffold were observed at 7 and 14 days after culturing by scanning electron microscope. Another 10 6-month-old New Zealand white rabbits were randomly divided into control group (group A 2, n=5) and experimental group (group B 2, n=5), which implanted the native trachea and decellularized tracheal matrix into the subcutaneous sac of the back neck, respectively. The serum immunoglobulin IgM and IgG contents were analysed at 5, 10, 15, 20, 25, and 30 days after operation, and HE staining observation was performed at 30 days after operation. MTT assay showed that the proliferation activity of BMSCs cultured in the leach liquor of group B 1 was well, showing no significant difference when compared with group A 1 and negative control group with pure culture medium ( P>0.05). The immunohistochemical staining showed that the decellularized process could significantly reducing the antigenicity of matrix materials. Giemsa staining showed that BMSCs grew well around the two tracheal matrixs (groups A 1 and B 1) in vitro. Scanning electron microscope observation showed that the cells were attached to the outer wall of the tracheal material in group A 1, which present a flat, round, oval shaped, tightly arranged cells and cluster distribution; and in group B 1, the cells formed a single lamellar sheet cover the outer wall of the tracheal material, whose morphology was similar to that in group A 1, and the growth trend was better. In vivo experimental results showed that the rejection of group B 2 was lower than that of group A 2. The contens of IgM and IgG in group A 2 were significantly higher than those in group B 2 at each time point after operation ( P<0.05). HE staining showed no signs of rejection, macrophagocyte, or lymphocyte infiltration occurred, and the collagen fibers maintained their integrity in group B 2. The decellularized matrix treated by NaClO 4 has a fine biocompatibility, while its immunogenicity decreased, and it is suitable for the scaffold material for constructing of tissue engineered trachea.

PROMOTION EFFECT OF CHONDROITIN SULFATE ON PROLIFERATION OF MYOBLASTS

To research the effect of chondroitin sulfate (CS) on the proliferation of myoblasts and the formation of myotube. The myoblasts at passage 5 were used to prepare the cells suspension (1×108 cells/mL), and the experiment was divided into 4 groups based on CS concentration in the medium:group A (0 μg/mL), group B (50 μg/mL), group C (100 μg/mL), and group D (200 μg/mL). The cell morphology and myotube formation were observed by inverted microscope at 4, 5, and 8 days after treatment; MTT was used to detect the cell proliferation at 6 days, and the number of myotube was calculated by HE staining at 8 days. Cells showed spindle shape after adherent, with ovoid nuclei and dense cytoplasm under inverted microscope. When the cell adherent rate was 90%, cells arranged in whorls swirled and showed long fusiform adherent growth; and then nuclei fusion resulted in formation of multincleated myotubes. At 8 days, most myoblasts fused to form myotube in group A, but less myotube was observed in groups B and C, and the least myotube in group D. The absorbance (A) values of groups A, B, C, and D were 0.045 2±0.004 4, 0.540 4±0.096 7, 0.660 9±0.143 4, and 1.069 0±0.039 0 respectively, showing significant difference between other groups (P<0.05) except between groups B and C P>0.05). HE staining observation showed that most myoblasts fused to form myotube in group A, but less myotube in groups B and C, and the least myotube in group D. The number of myotube of groups A, B, C, and D were 222.01±30.02, 193.13±42.46, 170.26±11.96, and 136.88±16.78 respectively, showing no significant difference among groups (F=1.658, P=0.252). CS can significantly promote the proliferation of myoblast, the promotion is the biggest when CS concentration is 200 μg/mL.

The effect of hyperbaric oxygen preconditioning on the expression of ICAM-1, VCAM-1, NF-κB and flap survival rate during ischemia-reperfusion injury in rat abdominal skin flap

To evaluate the effect of hyperbaric oxygen preconditioning on the expression of intercellular adhesion molecule-1 (ICAM-1),vascular cell adhesion molecule-1 (VCAM-1),NF-κB and flap survival rate during Ischemia-Reperfusion Injury in Rat Abdominal Skin Flap. 18 male adult SD rats were divided randomly into sham group (SH),ischemia-reperfusion group (IR) and hyperbaric oxygen preconditioning group (HBO),6 rats in each group. The rats in HBO group received regular hyperbaric oxygen treatment for three days (twice a day with an interval of 12 h) before operation. An abdominal skin flap pedicled with superficial epigastric artery was established. Ischemia lasted for 3 hours in the IR group and HBO group.On the third postoperative day,samples were taken to evaluate the expression of ICAM-1,VCAM-1 and NF-κB by immunohistochemistry staining and Western Blot. In the IR group, the flap survival rate and average blood perfusion were (22.38 ±4.35) ％ and (32.61 ±5.68) PU, while in the HBO group they were (45.34 ±3.15) ％ and (61.78 ±3.61) PU, showing significant difference between the two groups (P ＜0.01).The inflammatory cell infiltration condition in IR group was much higher than that in the HBO group from the HE staining observation. Compared with the HBO group,ICAM-1,VCAM-1 and NF-κB showed (+ + +) with higher positive cells in the IR group (P ＜ 0.01). Results of Western Blot showed similar conclusion. The relative expression of ICAM-1,VCAM-1 and NF-κB was much higher in the IR group than that in the HBO group (P ＜ 0.01). Hyperbaric oxygen preconditioning could decrease the expression of ICAM-1,VCAM-1,NF-κB and promote flap survival rate during the process of ischemia-reperfusion injury on a rat abdominal skin flap model.

The Changes of the Corneal Tissue Structures and Biomechanical Properties in the Process of Cornea Post-Embryonic Development

To investigate the changes of corneal tissues morphological structure and biomechanical properties in different month age rabbits, the eyeballs of the 1 month, 2 month and 3 month-old-New Zealand white rabbits were obtained and getting the cornea tissues, and then measured the thickness of the cornea tissues. The first part of the corneal tissue was used to observe cornea structure with HE staining. The second part was used to observe collagen fibrils by electron microscope. The third part of the cornea tissues were cut into strips and used to test the elastic modulus of the cornea on Instron5544 tester. The thickness of the cornea was increased with month age. HE staining observation shows that the numbers of corneal fibroblasts were decreased and the fiber bundle increased with month age. Electron microscope observation shows that the diameters of collagen fibrils were increased at 2 and 3 month. Biomechanical experiment results reveal that cornea tissues elastic modulus was increased with month age. In the post-embryonic stages, the structure of cornea, the structure and number of collagen fiber, the number of corneal fibroblasts and the biomechanical properties of cornea were changed continually.

Clinical pathological analysis of singularity uterine leiomyoma

目的 探讨奇异性子宫平滑肌瘤的临床及病理特点.方法对15例奇异性子宫平滑肌瘤的病因及临床表现进行分析,并作病理及免疫组化检查.结果奇异性子宫平滑肌瘤以不规则阴道流血、盆腔包块、贫血为主要临床表现.其中3例合并妊娠,4例有孕激素治疗史.镜下见多核或单核瘤巨细胞,核分裂象0～3/10HPF.孕激素受体(PR)均呈阳性表达,其中8例Ki-67、p53呈微弱阳性表达,雌激素受体(ER)呈阴性表达.结论奇异性子宫平滑肌瘤属良性肿瘤,临床表现及治疗与子宫平滑肌瘤相似.其发病与外源性孕酮及内源性孕激素有一定关系.与子宫平滑肌肉瘤的鉴别主要依靠苏木素伊红染色(HE)切片观察,免疫组化表达有一定的特征。