Establishment of a mouse model of acute systemic cold injury induced by hypothermia / 中华急诊医学杂志

Objective:To establish an animal model of acute systemic cold injury in mice.Methods:There were 98 C57BL/6 mice, half male and half female, with body weight of 22-27 g and age of 10 weeks. The mice were randomly divided into 7 groups ( n=14) according to the changes of anal temperature in cold environment, namely, group A (38.5 ± 1) ℃, group B (35 ± 1) ℃, group C (30 ± 1) ℃, group D (25 ± 1) ℃, group E (20 ± 1) ℃, group F (15 ± 1) ℃, and group G (10 ± 1) ℃, among which, group A was the blank control group, and the rest groups were the experimental group. The mice in the blank control group were placed in the normal environment (20 ± 5) ℃, and the mice in the experimental group were placed in the low temperature artificial climate box at - 20℃. The anal temperature of the mice was measured intermittently (as the core temperature), and the time required for the core temperature of the mice to drop to groups B, C, D, E, F and G was recorded. The righting reflex was used to evaluate the consciousness state, the action ability and the general state of each organ of mice were observed, and the blood routine and HE staining of each organ were detected. Results:The lower the core temperature of the experimental group, the longer the time required. The consciousness state, action ability, general state of organs, blood routine, and HE staining of organs in groups B, C, and D were basically the same as those in group A, and there was no acute systemic cold injury. Therefore, the blood routine, general observation of organs, and HE staining of organs in groups B, C, and D were no longer displayed compared with those in group A. Compared with group A, mice in group E began to suffer from disturbance of consciousness and action ability. With the decrease of core body temperature, the damage was aggravated, and mice in group G died. Compared with group A, the indices of blood routine test (WBC, RBC, HGB, PLT) of mice in group E began to decrease, and the univariate variance calculation showed that only WBC changes had statistical significance ( P<0.05). Compared with groups A and E, the indices of blood routine test (WBC, RBC, HGB, PLT) of mice in group F were further reduced, and the changes of each index in univariate variance calculation were statistically significant ( P<0.05). The general observation results showed that compared with group A, the lung, liver and spleen surfaces of mice in group E began to darken, and compared with groups A and E, the lung, liver, spleen, kidney and heart of mice in group F were further deepened and darkened, with irregular edges. HE staining results of various organs showed that compared with group A, the mice in group E began to have partial alveolar structure destruction and a small amount of inflammatory cell infiltration, the central vein of the liver was slightly congested, and the red and white pulp of the spleen were indistinct. Compared with groups A and E, the pathological structure damage of the lung, liver, spleen, kidney, heart and brain tissues of the mice in group F was further aggravated. Conclusions:Detection of consciousness state, action ability, general state of organs, blood routine and HE staining indices of organs in mice under low temperature can simulate the progress of clinical acute cold injury, and the animal model of acute systemic cold injury was successfully prepared.

Practical Value of Environment-friendly Sample Release Agent Combined with Ultrasound in the Preparation of Pathological Tissue Sections / 中山大学学报(医学科学版)

ObjectiveTo explore the practical value of environment-friendly sample release agent combined with ultrasound in the preparation of pathological tissue sections. MethodsFrom February 2013 to December 2022， 2 518 pathological specimens submitted by Foshan Municipal Hospital of Traditional Chinese Medicine were selected as the study objects. Two samples of the same specimen were randomly divided into two groups： the environment-friendly fast group， in which the pathological tissue sections were made by using the environment-friendly sample release agent combined with ultrasound； and the traditional group， in which formaldehyde， ethanol and xylene were used to make slices in the conventional way. The differences of hematoxylin （HE） staining effect， immunohistochemistry （IHC） staining effect and MDM2 gene detection result of atypical lipomatous tumor/highly differentiated liposarcoma （ALT/WDL） tissue sections between the two groups were compared. Results① The wax of the two groups' pathological tissues was dehydrated well and the tissue hardness was moderate. After HE staining， the sections of the two groups were intact， without cracks and tremor marks， and the contrast between nucleus and cytoplasm was appropriate， with good transparency， uniform staining， and no tissue loss. The excellent rate and score of HE staining in the environmental fast group were higher than those in the traditional group， but the difference was not statistically significant （χ2 = 3.125，P1 = 0.070；t = 0.965，P2 = 0.334）. ②After IHC staining of the two groups of sections， the positive location of the cells was accurate， the staining was specific and uniform， the staining intensity was moderate， the staining sensitivity was good， and there was no tissue loss. The excellent rate of IHC staining and the positive rate of IHC staining in the environmental fast group were lower than those in the traditional group， but the difference was not statistically significant （χ12 = 2.769，P1 = 0.092；χ22 = 0.800，P2 = 0.375）. ③The background and outline of the two groups of WDL tissue sections were clear， the staining was uniform， the cells were clear and visible， the nuclear boundary was clear， the hybridization signal was clear and bright under the background fluorescence， and there was no miscellaneous signal. The two groups of sections were hybridized successfully， and MDM2 showed positive amplification. The number of cells successfully hybridized in the environment-friendly fast group was lower than that in the traditional group， but the difference was not statistically significant （t = 1.414，P = 0.230）. ConclusionsThe tissue treatment method of using environment-friendly sample release agent combined with ultrasound can ensure the detection effect of HE staining， IHC staining and MDM2 gene detection of pathological tissue sections， and is more efficient and environment-friendly， suitable for promotion and use in hospitals at all levels.

Application of Rapid HE Staining in Cytological Rapid On-site Evaluation of Peripheral Lung Cancer Needle Biopsy / 中国肺癌杂志

BACKGROUND@#Rapid on-site evaluation (ROSE) is a technique used for simultaneous evaluation of biopsy specimens through rapid cytology staining. Diff-Quik (DQ) staining is the most commonly employed method for cytological rapid on-site evaluation (C-ROSE). However, the utilization of DQ staining for on-site cytological interpretation remains uncommon among pathologists in China, posing challenges to the implementation of C-ROSE. This study aims to assess the application of rapid hematoxylin-eosin (HE) staining and DQ staining for C-ROSE during percutaneous needle biopsy of peripheral lung cancer and evaluate the value of rapid HE staining in C-ROSE.@*METHODS@#Computed tomography (CT)-guided lung biopsies were conducted on 300 patients diagnosed with peripheral lung cancer. The patients were randomly assigned to two groups for C-ROSE using either rapid HE staining or DQ staining, and subsequently the two methods were compared and evaluated.@*RESULTS@#The concordance rate between C-ROSE and histopathological diagnosis was 96.7%. The median staining time for rapid HE staining was 160 s, while that for DQ staining was 120 s, representing a significant difference between the two groups (P<0.001). However, there were no significant differences observed in terms of total biopsy time, concordance rate with histopathology, cytology specimen peeling rate, and incidence of serious adverse reactions between the two groups (P>0.05).@*CONCLUSIONS@#Both staining methods comply with C-ROSE criteria in the biopsy setting of peripheral lung cancer. Rapid HE staining is more aligned with domestic clinical requirements and holds potential for further promotion and adoption in C-ROSE.

An experimental study of tangzhouling on morphological changes of nissl bodies in the dorsal root ganglion of DM rats / Un estudio experimental de tangzhouling sobre los cambios morfológicos de los cuerpos de nissl en el ganglio de la raíz dorsal de ratas DM

SUMMARY: This study aims to investigate the effect of Tangzhouling on the morphological changes of Nissl bodies in the dorsal root ganglion of DM Rats. In this study, 69 rats were randomly divided into a control group (n = 10) and a model group (n = 59). The rats in the model group were randomly divided into a diabetic group (n = 11), a vitamin C group (n = 12), a low dose Tangzhouling group (n = 12), a medium dose Tangzhouling group (n = 12) and a high dose Tangzhouling group (n = 12). The dose of Tangzhouling in the low dose group was 5 times that of the adult dose, being 0.44g/kg/d. The dose of Tangzhouling in the medium dose group was 10 times that of the adult dose, being 0.88g/kg/d. The dose of Tangzhouling in the high dose group was 20 times that of the adult dose, being 1.75g/kg/d. All doses above are crude drug dosages. Rats in the vitamin C group were given 10 times the dose of an adult, being, 0.05 g/ kg/d. The diabetic group and the control group were given the same amount of distilled water. Drug delivery time is 16 weeks. The dorsal root ganglion was placed in a freezing tube at the end of the experiment. The morphological changes of Nissl bodies in the dorsal root ganglion were detected by HE and Nissl staining. The study results showed that vitamin C had no significant effect on the quantity, size and nucleolus. Tangzhouling can improvee the morphology, quantity and nucleolus of Nissl bodies to a certain extent, and the high dose is better than the lower dose. Tangzhouling capsules can improve the nerve function of DM rats through Nissl bodies.

RESUMEN: Este estudio tuvo como objetivo investigar el efecto de Tangzhouling en los cambios morfológicos de los cuerpos de Nissl en el ganglio de la raíz dorsal de las ratas DM. En este estudio, 69 ratas se dividieron aleatoriamente en un grupo control (n = 10) y un grupo modelo (n = 59). Las ratas del grupo modelo se dividieron aleatoriamente en un grupo diabéticos (n = 11), un grupo vitamina C (n = 12), un grupo de dosis baja de Tangzhouling (n = 12), un grupo de dosis media de Tangzhouling (n = 12) y un grupo de dosis alta de Tangzhouling (n = 12). La dosis de Tangzhouling en el grupo de dosis baja fue 5 veces mayor que la dosis del adulto, siendo 0,44 g/kg/d. La dosis de Tangzhouling en el grupo de dosis media fue 10 veces mayor que la dosis del adulto, siendo 0,88 g/kg/d. La dosis de Tangzhouling en el grupo de dosis alta fue 20 veces mayor que la dosis del adulto, siendo 1,75 g/kg/d. Todas las dosis anteriores son dosis de fármaco crudo. Se les administró 10 veces la dosis de un adulto a las ratas del grupo vitamina C, siendo 0,05 g/kg/d. El grupo de diabéticos y el grupo de control recibieron la misma cantidad de agua destilada. El tiempo de entrega del fármaco fue de 16 semanas. El ganglio de la raíz dorsal se colocó en un tubo de congelación al final del experimento. Los cambios morfológicos de los cuerpos de Nissl en el ganglio de la raíz dorsal se detectaron mediante tinción de HE y Nissl. Los resultados del estudio mostraron que la vitamina C no tuvo un efecto significativo sobre la cantidad, el tamaño y el nucléolo. Tangzhouling puede mejorar la morfología, la cantidad y el nucléolo de los cuerpos de Nissl hasta cierto punto, y es mejor la dosis alta que la dosis baja. Las cápsulas de Tangzhouling pueden mejorar la función nerviosa de las ratas DM a través de los cuerpos de Nissl.

Cell HE staining smears and paired cell paraffin sections in detection of epithelial growth factor receptor gene of pleural fluid specimens / 中南大学学报(医学版)

OBJECTIVES@#The advanced non-small cell lung cancer (NSCLC) patients with pleural effusion have no opportunity for surgery treatment. Epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) are the first-line drugs for these patients with EGFR-sensitive mutation. However, the disease progression and drug update during or after treatment of EGFR-TKIs bring more challenges and puzzles to clinical diagnosis and treatment, which inevitably requires archived pleural cell samples for EGFR re-examination or comparative study. Understanding the DNA quality of archived pleural fluid samples and effectively using archival data of pleural fluid cells are of great significance for tracing the origin of cases and basic medical research. This study aims to evaluate the consistency of EGFR mutant gene expression between the 2 methods, and to explore a reliable way for preserving cytological data and making full use of cytological archival data via cell HE staining smear and cell paraffin section.@*METHODS@#A total of 57 pleural fluid cytology cases in the Department of Pathology of China Aerospace Center Hospital from October 2014 to April 2021 were selected. Tumor cells were detected by cell HE staining smears and immunohistochemical staining for TTF-1 and Napsin A in the paired cell paraffin sections. There were more than 200 tumor cells in cell HE staining smear and the proportion of tumor cells were ≥70% in matched cell paraffin sections. Patients with 2 cell smears (one for cell data retention and the other for DNA extraction) were selected as the research subjects, and 57 pleural fluid samples were enrolled. EGFR gene mutation was detected by amplification refractory mutation system-polymerase chain reaction in 57 paired cell HE staining smears and cell paraffin sections. DNA concentration was 2 ng/μL. Cell HE smear was amplified side-by-side with DNA samples from paired cell paraffin sections. Result determination was according to the requirements of the reagent instructions. The external control cycle threshold (Ct) value of the No. 8 well of the samples to be tested was between 13 and 21, which was considered as successful and reliable samples. When the Ct value of EGFR gene mutation was <26, it was considered as positive; when the Ct value was between 26 and 29, it was critical positive; when the Ct value was equal or more than 29, it was negative. ΔCt value was the difference between mutant Ct value and externally controlled Ct value. The smaller the ΔCt value was, the better the quality of DNA of the detected sample was.@*RESULTS@#Among the 57 pleural effusion samples, 42 patients were hospitalized with pleural effusion as the first symptom, accounting for 73.7% (42/57). EGFR mutation was detected in 37 samples [64.9% (37/57)]. The mutation rate for 19del was 37.8% (14/37) while for L858R was 48.6% (18/37). Females were 56.7% (21/37) of mutation cases. The mutation consistency rate of cell HE staining smear and matched cell paraffin sections was 100%. The ΔCt values of cell HE staining smears were less than those of matched cell paraffin sections. The mutation Ct values of 37 cytological samples were statistically analyzed according to the preservation periods of the years of 2014-2015, 2016-2017, 2018-2019, and 2020-2021. There were significant differences in cell paraffin section in the years of 2014-2015 and 2016-2017 compared with the years of 2018-2019 and 2020-2021, while no significant differences were found in cell HE staining smear. Statistical analysis of externally controlled Ct values of 57 cytological samples showed that there were significant differences between cell HE staining smears and cell paraffin section in the years of 2014-2015 and 2016-2017, compared with the years of 2018-2019 and 2020-2021. The mutational Ct values of 37 paired cell blocks and smears were all <26, and the externally controlled Ct values of 57 paired cell paraffin sections and HE staining smears were all between 13 and 21.@*CONCLUSIONS@#The DNA quality of cell HE smears and matched cell paraffin section met the qualified requirements. Two methods possess show an excellent consistency in detecting EGFR mutation in NSCLC pleural fluid samples. The DNA quality of cell HE staining smear is better than that of cell paraffin sections, so cell HE staining smear can be used as important supplement of the gene test source. It should be noted that the limitation of cell HE staining smears is non-reproducibility, so multiple smears of pleural fluid are recommended to be prepared for multiple tests.

Anatomical analysis of muscle spindles in tibialis anterior and extensor digitorum longus muscle of C57BL/6 mice / 解剖学报

Objective To locate the distribution of muscle spindles in tibialis anterior (TA) and extensor digitorum longus muscle (EDL) and the anchoring mode of muscle spindles in skeletal muscles, and perform statistics analysis of their morphological character by anatomical parameters. Methods Five adult wild type C57BL/6 mice were sacrificed, and TA and EDL were dissected and frozen with improved ultra-low temperature cryopreservation technology avoiding myofibers damaged by possible ice crystal. Continuous frozen transections were obtained and operated by HE staining, followed by microimaging to spot the muscle spindles location. Some parameters including regions length and cross section area (CSA) of muscle spindles were noticed for the discovery of some general characteristics of spindles by statistics. Results For TA and EDL, the scattered characters of muscle spindles were distributed as follows: the spindles were located at the upper third of the mid-belly of both TA and EDL from caudal to rostral position, while near the enter point to muscle of the deep peroneal nerve in dorsal-ventral orientation. The peripheral of muscle spindles anchored to extrafusal fibers to hold in the muscle. And in term of length, region A, connected with sensory nerve ending, demonstrated a significant correlation with region B, which located at the poles of region A and twined by motor nerve ending (correlation index = 0. 75) when considering the muscle spindles with four intrafusal fibers only. And no correlation was discovered in any others pairwise parameters. Conclusion The scattered diagram of muscle spindles in TA and EDL of C57BL/6 mice might provide anatomic basis for evaluation of lower limb motor function, especially for the spinal cord injury and recovery research. And the correlationship between the length of region A and B might improve exploring the variability of electrophysiological characters.

Quality control analysis of brain tissue samples from Shanghai Brain Bank / 解剖学报

Objective To evaluate the feasibility of using brain tissues from Shanghai Brain Bank for the applications on biological research through the analysis of pH value of cerebrospinal fluids, RNA integrity number (RIN), transcriptome, proteome and morphology of brain tissues. Methods The pH value of fresh cerebrospinal fluid was detected by pH test paper; the RNA integrity of cryopreserved brain tissues was examined by Agilent RNA 6000 Nano chip and Agilent 2100 bioanalyzer; the transcriptome sequencing of superior temporal gyrus or caudate was performed using BGIseq-500 sequencer; the proteome of cryopreserved brain tissues was analyzed by Q Exactive HF mass spectrometer; at last, the morphology of the superior temporal gyrus was observed by HE staining. Results The pH value of the cerebrospinal fluid on average was about 6.5. The RIN values of more than 65% of brain tissues were more than 6, indicating good RNA quality. The clean reads ratio after transcriptome sequencing filtering was basically above 80%, indicating that the quality of sequencing library was high. The mass spectrometry analysis of frozen brain samples yielded more than 4000 protein groups and 30 000 peptides, indicating high quality of proteomic data. The morphology of brain tissues was relatively normal, with clearly visible neurons. Conclusion The quality of RNA and protein of brain tissues from Shanghai Brain Bank meets the basic needs for molecular and biological research, and the fixed brain samples can be used for morphological observations.

Construction of corneal neovascularization rabbit model by suture / 解剖学报

Objective To compare the effect of different sutures and suture method on corneal neovascularization ( CNV) in rabbit models. Methods NV was induced by placing sutures at the corneal periphery of rabbits (n = 45). To observe the NV status, 45 rabbits were randomly divided into 5 equal groups. Group A applied 8-0 absorbable suture (A1 single loop parallel suture, A2 single loop vertical suture). In group B, 10-0 nylon suture was used (B1 double loop parallel suture, B2 double loop vertical suture, B3 three loop radial suture). The development of CNV was observed with slit lamp microscope and photographed. Therefore the effective model for neovascularization induction was selected. Histological examination, immunofluorescent staining and ELISA analysis for the vascular endothelial growth factor( VEGF) were performed before suture, 7 and 14 days after suture. Results Sutures fell off and CNV gradually atrophied in group Al and A2; At the 14th day after suture, Sparse or short cluster CNV grew into the corneal margin in group B1 and B2, while CNV was vigorous and grew in bundles in group B3. The expression of VEGF in aqueous humor increased in B3 group after suturing, and increased in 14 days as compared with 7 days after suture. Corneal edema, neovascularization and little immunofluorescence staining for VEGF were detected in group B3 after 7 days suture. More neovascularization and immunofluorescence staining for VEGF were detected in group B3 after 14 days suture. Conclusion Corneal NV can be induced successfully in rabbit model by suturing. The method of 10-0 thread with three sets of circular seams (B3) is stable and effective.

Judging the degree of brain injury after hypoxia-ischemia in living neonatal mice in the early stage / 解剖学报

Objective To explore the method of distinguishing the degree of hypoxic-ischemic brain damage ( HIBD) in living mice in early stage, so as to lay a foundation for the follow-up study of the molecular mechanism of different degrees of HIBD. Methods The modified Rice-Vannucci method was used to duplicate the HIBD model of C57BL/6 J mice. On the 1 day and 3 days after the model, the scalp of mice were cut and the brain tissue were observed to distinguish between mild and severe lesions in living mice, and then 2,3, 5-triphenyltetrazolium chloride (TTC) staining, laser speckle cerebral blood flow imaging, HE staining, Fluoro-Jade B ( FJB ) staining and body weight difference before and after operation were used to verify the reliability of observation in living mice. Results Through the gross observation of brain tissue in living mice, HIBD could be divided into mild injury (HI-M) group and severe injury (HI-S) group. On day 1 and day 3 after HIBD, a significant decrease in cerebral blood flow, obvious gray infarction and a large number of necrotic neurons were observed in the HI-S group, and the body weight was significantly lower than that before operation. In the HI-M group, the cerebral blood flow of the injured side decreased only on the 3rd day after HIBD, and the loose arrangement of neurons in the cortex and hippocampus of the injured side was observed morphologically. The body weight was lower than that before operation. Conclusion Gross observation of brain tissue by cutting the scalp is a reliable method to distinguish mild and severe brain injury in the early stage of HIBD in living mice.

Three-dimensional reconstruction and anatomy of human epididymal duct / 解剖学报

Objective Based on the techniques of continual tissue slices, the human epididymis was rebuilt for understanding the anatomical and histological features of the epididymal ducts. Methods Continuous tissue slices of one human epididymis were performed and digital slice images were obtained through scanning with Leica-Aperio AT2; the pipe wall of epididymal duct was aligned in sequence with Photoshop CC 2018 software and VGStudio MAX V3.0 software was used for three-dimensional synthesis. Finally, the later modification was carried out with Materialise Magics V22.0 software. Another human epididymis was used for electron microscopy. The histological features of epididymal ducts were analysed by combining slices and three-dimensional reconstruction. Results A total of 4331 and 543 slices in transverse and sagittal section respectively were prepared with a thickness of 7 |ira. According to the three-dimensional structure, regional distribution of human epididymal duct could be found obviously and the caput, corpus and cauda of epididymis could be clearly divided into 7, 9 and 4 subregions respectively. There were tissue intervals among adjacent subregions and epididymal ducts were disordered within each subregion, but differences were existed in tubular diameter and epithelial structure, and adjacent subregions were connected by single epididymal duct in the corpus and cauda. Conclusion The human epididymal duct can be successfully reconstructed by continual tissue slices technique. The human epididymal duct has regional distribution in space obviously and there are differences between different subregions.

Effect of active peptides of Eupolyphaga on intestinal flora in rats with hyperlipemia / 中国药理学通报

Aim To investigate the effect of active peptide of Eupolyphaga (APE) on changes of intestinal flora and blood lipid in rats with hyperlipidemia. Methods SD rats fed with high-fat diet were induced to hyperlipemia model. Simvastatin being a positive drug, ELISA was used to investigate the effect of APE on blood lipids in hyperlipidemic rats. At the same time, the results of the microbial flora about APE model rats were measured using 16rS high-throughput assay technology (V4-V5). Results APE could significantly reduce the serum lipid index in model rats and improve the degree of liver pathological changes. Meanwhile, the intestinal flora results showed that when the relative abundance of hymenoscyphus, lactobacillus and bifidobacterium in firmicutes were enriched, the relative abundances of rumenococcus, plasmodium and prevotella in bacteroidetes and tenericutes were reduced. Conclusions APE could obviously reduce the level of blood lipid and repair the disordered intestinal flora of hyperlipidemia rats. It can be speculated that APE might play a role in lowering blood lipids through the intervention of lipid metabolism pathway by intestinal flora.

Effect of Notch signaling pathway on nerve regeneration after cerebral ischemia in rats with neural stem cells transplantation / 解剖学报

Objective To investigate the mechanism of inhibiting the Notch signaling pathway in promoting neural regeneration after neural stem cells (NSCs) transplantation in rats with cerebral ischemia. Methods The model of focal cerebral ischemia was established by middle cerebral artery occlusion (MCAO) method . NSCs were cultured in vitro and transplanted into the striatum ischemic area. In the experiment, 40 SD rats were divided into sham operation group, model group, transplantation group (transplanted neural stem cells), and N-[N-(3, 5-difluorohenacetyl )-L-alanyl ]-S-phenylglycinet-butyl ester( DAPT) + transplantation group. The degree of neuronal damage in each group was observed by HE staining. The expressions of Notchl, Hesl and Hes5 in the brain tissue of each group were detected by immunohistochemistry and Western blotting. Results Compared with the sham operation group, the neuron injury in the model group was severe, and nuclear pyknosis and nuclear lysis were observed. The Notchl, Hesl and Hes5 positive cells increased significantly, and the expression of Notchl, Hesl and Hes5 proteins were significantly up-regulated (P<0. 05). Compared with the model group, the neuronal damage in the transplantation group and the DAPT+ transplantation group were all relieved, and the Notchl, Hesl and Hes5 positive cells were partially expressed, and the expression of each protein decreased (P<0. 05). The DAPT+ transplantation group neurons were compared. The damage was obviously restored, and the expression of protein-positive cells and protein further decreased (P<0. 05). Conclusion Inhibition of Notch signaling pathway can promote nerve regeneration after neural stem cell transplantation in rats with cerebral ischemia. The mechanism is mainly related to down-regulation of Notch 1 , Hesl and Hes5 expression.

Perfusion and cryopreservation of whole ovaries with vascular pedicles in common experimental animals / 解剖学报

Objective Using the perfusion needle which is authorized and special perfusion technology to perfuse the whole ovary with vascular pedicle of common animals, such as rabbits, Guinea pigs and rats, then comparative analysis of the adjustable perfusion needles whether can be applied to different animals, whether this kind of perfusion pressure and perfusion rate is appropriate for different sizes of ovaries, and using the classic cryopreservation protocol to freeze and thaw. Methods Collecting 12 ovaries respectively from 6 chinese sexual maturity rabbits, 6 Dunkan-Hartley Guinea pigs and 6 SD rats to do experiments. Results Through HE staining to count the normal proportion of primordial follicles in each section, the result of HE staining in three animals indicated that there was no statistical significance (P>0. 05), vascular injury was mainly in the upper (far away from ovary), there was no obvious damage in lower (close to ovary) in three animals' each group. Conclusion The experiment confirms that through adjusting straw needle size we can perfuse different size of animal organs and under the condition of the same perfusion pressure (60 mmHg) and rate (1 ml/min) it is suitable for rabbits' ovaries, Guinea pigs' ovaries and rats' ovaries at the same time.

Effects of cervical rotatory manipulation on severe rabbit carotid atherosclerosis in flow sheer stress / 解剖学报

Objective: To investigate the effects of cervical rotatory manipulation (CRM) on severe carotid atherosclerosis of rabbits in hemodynamics. Methods: Totally 40 New Zealand rabbits were divided randomly into 3 groups, including the manipulation group, the model group and the control group. Left carotid atherosclerotic plaques models were developed in the rabbits of manipulation group and model group. Then, the manipulation group was treated with CRM lasting for 2 weeks; meanwhile no intervention was given on rabbits in the model group and control group. At the end of manipulation, flow sheer stress (FSS) was calculated with velocity and viscosity of blood. One way AN OVA and LSR-t test were performed to explore the significance. Results: FSS of left carotid artery in rabbit decreased significantly in the manipulation group and the model group, in comparison with the control group. Moreover, FSS in the manipulation group was lower than that in the model group. Conclusion: FSS of severe atherosclerotic carotid artery in rabbit may become decreased following with CRM.

Transdermal characteristics and mechanism of asiaticoside nanoemulsions and asiaticoside nanoemulsions-based gels / 中国中药杂志

To prepare the asiaticoside nanoemulsions (ASI-NEs) and asiaticoside nanoemulsions-based gels (ASI-NBGs), compare them with the commercial cream of asiaticoside (ASI-C) in terms of transdermal characteristics, and investigate the transdermal mechanism of ASI-NEs and ASI-NBGs. Their transdermal characteristics were studied by using Franz diffusion cells. The effect of topical ASI-NEs and ASI-NBGs on ultrastructure of rabbit skin was evaluated by using HE staining method. The localization and the permeation pathway of asiaticoside were visually investigated by using laser scanning confocal microscope (CLSM). The transdermal studies in vitro showed that the cumulative amount of ASI permeated from ASI-NEs and ASI-NBGs at 12 h after application were (3 504.30±180.93), (1 187.40±128.88) μg·cm⁻² respectively, 6.57, 2.23 times of that in the control group of ASI-C; the drug deposition of ASI-NEs and ASI-NBGs in skin was (159.48±7.47), (120.53±5.71) μg·cm⁻² respectively, 5.93, 4.48 times of that of ASI-C. HE staining of the rabbit skin after application of ASI-NEs and ASI-NBGs showed that the epidermis structure was basically intact; stratum corneum was loosed and the keratin fragment was increased; at the same time, the gap of prickle cell was increased and the basal cells were arranged loosely. The study of CLSM showed that significant percutaneous enhancer effect was observed for ASI-NEs after the topical application of 6 h, as the fluorescent compound was penetrated in the dermis and diffused uniformly. The fluorescence area and the integral optical density (IOD) were 28.81, 32.51 times of that in the FITC aqueous solution group, respectively. The fluorescent preparations showed strong fluorescence in the epidermis, but weak in deeper layers; with the increase of treatment time, the fluorescence in deeper layer was increased and stronger in skin appendages. The prepared ASI-NEs and ASI-NBGs have good transdermal characteristics and the transdermal mechanism is related to breaking the ultrastructure of stratum corneum and penetrating by the path of skin adnexa.

Establishment of orthotopic implantation model of human U87-MG brain glioma cell line in nude mice / 中国药理学通报

Aim To establish human U87-MG glioma model in nude mice brain and to observe the characteristics of the tumor growth. Methods Human U87-MG glioma cells were cultured in vitro. 5 μL of cell suspension containing 3.0 ×1010·L-1, 4.0×1010·L-1and 5.0×1010·L-1respectively was inocula-ted into the right caudate nucleus of 18 male nude mice brain un-der the guidance of stereotaxic apparatus, separately, whereas another 6 nude mice as the control group, were inoculated into the same volume of Hanks solution. The moving and survival state of rats with gliomas were observed. The examinations of the tumors formation, volumes, metastasis and histopathology were performed and the obtained brain samples were stained with HE and immunohistochemistry. Results All the tested rats of dif-ferent inoculation doses developed brain tumors without extracra-nial metastasis. The mean survival time of three groups was (46.50 ± 3.27) d,(38.50 ± 3.28) d and (30.67 ± 3.51) d,respectively. The tumors showed the similar morphological fea-tures and immunophenotype to human glioma. There was positive expression of GFAP and S-100 in the tumors. Conclusions The orthotopic implantation model of human U87-MG glioma, by in-oculating quantitative U87-MG cells stereotaxically into the brains of the nude mice, is successfully established with 100 yield of intracranial tumor and no extracranial growth extension. It resembles the histopathological and morphological features of human glioma,which can be used as a reliable animal model for the study of the tumorigenesis, pathogenesis, biological charac-teristics and therapy of glioma.

Anatomy of caudal vessels of the rat and physiological functions of rat tail / 中国比较医学杂志

Objective To further definite the distribution of caudal arteries and veins of rat by anatomical dissection and to deepen the understanding of their physiological functions, and provide a basis for standardization of animal experimental techniques and design of animal models. Methods Eighteen SPF adult SD rats were used in this study. Several techniques were used in combination to study the anatomy and histology of the rat tail blood vessels:paraformaldehyde perfusion through the abdominal aorta was performed for rapid and thorough fixation, blue and red paints were injected to visualize the tail veins and arteries, respectively, arterial microangiography was performed to illustrate the distribution of tail arteries, and the microscopic structure of arteries and veins was verified by histological examination. Results Three longitudinal superficial arterial and venous systems of rat tail were confirmed and a dorsal arterial and venous chain structure was defined, which deeped our knowledge about the distribution of the deep blood vessels. In addition, the caliber of arteries was not corresponding with that of veins, providing a basis of their physiological functions. A bilayer cage connecting structure of the rat tail vasculature was for the first time defined. Conclusions The rich vascular structure of rat tail is described in details in this study. The existence of basal vascular system of rat tail is clarified. A concept of bilayer framework of the rat tail vasculature is proposed, which lays a good foundation for related researches of their physiological functions, and provides a good basis for avoiding major injuries and compensatory responses of hindlimb ischemia during animal experiments.

Dynamic pathological characteristics of kidney in db/db mice with type 2 diabetes mellitus / 吉林大学学报(医学版)

Objective; To investigate the dynamic pathological characteristics of kidney in the db / db mice with type 2 diabetes mellitus and to clarify the pathogenesis and development mechanism of diabetic nephropathy, and to provide the experimental evidence for the further study on diabetic nephropathy. Methods: The male SPF db/db mice aged 7 - 8 weeks were selected as model group (n=16) and the db/m mice with the same ages were selected as control group (n=16). The body weights and levels of fasting blood glucose (FBG) of the mice were detected after 8, 16, and 32 weeks. Then eight mice in each group were sacrificed and the kidney tissue was dissected at 8, 16 and 32 weeks. HE and Masson staining were used to observe the pathomorphology of the kidney tissue; the ultrastructures of kidney tissue were observed under electron microscope. Results: Compared with normal group, the body weights of the mice in model group at 8, 16 and 32 weeks were significantly increased (P<0. 01), the FBG levels were significantly increased (P<0. 01), and the double kidney indexes were significantly decreased (P<0. 01); the double kidney index of the mice in model group at 32 weeks was lower than that at 16 weeks (P< 0. 05). The HE and Masson staining results showed that the morphological changes of kidney tissue of the mice at 16 weeks were significant, the glomerular volume was expansion and the kidney tubular epithelial cells were edema, which turned serious at 32 weeks. A large number of blue dye substances in the kidney tissue was found. Under electron microscope, the glomerular basement membranes got thickened, the foot processes got fused and the number of mitochondria in kidney tubular epithelial cells was reduced and swell was found in the mice in model group at 16 weeks; the lesions in the kidney tissue were serious at 32 weeks, and the collagenous fiber was visible at 32 weeks. Conclusion: The pathological changes in the kidney tissue of the db/db mice at 16 weeks are significant, such as glomerular basement thickening and footwork fusion. The kidney tissue of the db/db mice at 32 weeks shows prominent fibrosis.

Effect of Different Doses of Herbal Cake-partitioned Moxibustion on Histopathological Changes of Colon Tissue in Ulcerative Colitis Rats / 针刺研究

OBJECTIVE: To observe the effect of different-doses of herbal cake-partitioned moxibustion (Moxi) on histopathological changes of the damaged colon tissue in rats with ulcerative colitis (UC), so as to select an optimal dosage of Moxi in the treatment of UC. METHODS: Sixty-three male Sprague Dawley (SD) rats were randomized into 7 groups: normal control, model, medication, and 1, 2, 3 and 4 moxa-cone Moxi (n＝9 rats per group). The UC model was developed by subcutaneous injection of emulsion (1 mL) containing colon mucosa-prepared protein suspension and complete Freund's adjuvant into the toes, groin and back. On the 38th day, enema of 3% formalin and the aforementioned emulsion was used. Herbal-cake (composed of monkshood, cinnamon, etc.) partitioned Moxi with 1 or 2 moxa-cones (about 5 min/cone) was applied to bilateral "Tianshu" (ST 25) once daily or once every other day. The rat's general conditions (diet, movement, response ability, stool, and body weight) were observed, and histopathological changes (adhesion, ulcer formation and inflammation) of colon tissues were examined after hematoxylin-eosin (HE) staining, and scored (histopathological score). Gross score was given according to the severity of adhesion, ulcer formation and inflammation of colonic tissues under stereo microscope. The average optical density (AOD) values of colonic mucins were detected after periodic acid-schiff (PAS) staining, and those of the sulfated mucus content detected after high iron dia-mine-alcian blue (HID-AB) staining. RESULTS: Compared with the normal group, rats in the model group presented loose stool, or with pus and blood, and slowly increased body weight (P0.05). The histopathological scores were significantly lower in the 1 and 4 moxa-cone Moxi groups than in the medication group (P<0.05, P<0.01); and significantly lower in the 1, 3, 4 moxa-cone Moxi groups than in the 2 moxa-cone Moxi group (P<0.05, P<0.01). PAS staining showed a significant increase of the AOD values of colonic mucins in the 1, 2, 3 and 4 moxa-cone and medication groups relevant to the model group (P<0.01); and the AOD values of colonic mucins in the 1, 3, 4 moxa-cone Moxi groups were significantly increased than that in the 2 moxa-cone Moxi group (P<0.05, P<0.01). HID-AB staining showed that the AOD values of sulfated mucus content were significantly higher in the 2 and 4 moxa-cone Moxi groups than in the 3 moxa-cone Moxi group (P<0.01). The two-level two-factor factorial analysis showed an interaction existed between the moxa-cone number and Moxi frequency in reducing the gross score and histopathological score and in facilitating colonic mucin and sulfated mucus secretion. The histopathological score of the 4 moxa-cone Moxi group was significantly lower than that of the 2 moxa-cone Moxi group (P<0.05), and the sulfated mucus content was significantly higher in the 4 moxa-cone group than in the 3 moxa-cone group (P<0.01). The effect of Moxi given on alternate days was superior to that of daily Moxi in improving colonic histological damage. CONCLUSION: Herbal cake-partitioned moxibustion at ST 25 can promote repair of the damaged colonic tissue and secretion of mucin in UC rats. The number of moxa cones and intervention frequency affect the efficacy of Moxi in improving histopathological changes. The Moxi intervention on alternate days and with 2 moxa-cones every time is recommended.

The protective mechanism of anisodine hydrobromide against cerebral ischemia-reperfusion injury in rats / 中国药理学通报

Aim To investigate the protective mechanism of anisodine hydrobromide against cerebral ischemia-reperfusion injury in rats.Methods In vivo: the cerebral ischemia-reperfusion injury model was established by middle cerebral artery occlusion(MCAO)via suture method in rats;the rats were injected anisodine hydrobromide(1.2,0.6,0.3,0.15 mg·kg-1);the morphological changes were detected by HE staining;the Nissl staining was used to count the number of surviving neurons;the activity of CAT and LDH,the LPO contents in the brain tissue were measured;the expressions of Bax,Bcl-2,caspase-3 and p-Akt in brain tissue were detected by Western blot.In vitro: Western blot assay was used to determine the expression of Bax,Bcl-2,caspase-3 and p-Akt protein expression in the OGD-R model of PC12 cells.The signal pathway of anisodine hydrobromide was identified.Results Anisodine hydrobromide with the dose of 0.15 mg·kg-1 could significantly lessen the morphological changes,and improve the number of surviving neurons;the dose of 0.3 and 0.15 mg·kg-1 could significantly improve the activity of CAT;the dose of 0.3 mg·kg-1 could significantly reduce the contents of LPO in the rat brain tissue;the dose of 1.2 mg·kg-1 could significantly decrease the activity of LDH;the dose of 0.15～1.2 mg·kg-1 could inhibit the expression of Bax,promote the expression of p-Akt in rat brain tissue.All the doses except 0.15 mg·kg-1 could promote the expression of Bcl-2 in rat brain tissue.In vitro,the results showed that anisodine hydrobromide in 25～100 μmol·L-1 could significantly improve the expression of Bcl-2 and the ratio of Bcl-2/Bax,and the dose of 50 μmol·L-1 could significantly improve the ratio of p-Akt/Akt.Conclusion The mechanism of anisodine hydrobromide against cerebral ischemia-reperfusion injury model rats might be related to its anti-oxidative activity and the activation of Akt.