[Morphological and pathological changes of larynx after severe laryngeal burn in dogs and their relationship with laryngostenosis].

Objective: To investigate the morphological and pathological changes of the larynx after severe laryngeal burn in dogs and their relationship with laryngostenosis. Methods: Eighteen healthy, male beagle dogs were assigned into control group, immediately after injury group, and 2, 4, 6, and 8 weeks after injury groups according to the random number table, with 3 dogs in each group. Dogs of injury group inhaled saturated steam through mouth for 5 seconds to reproduce severe laryngeal burn. Tracheotomy and intubation were performed immediately after injury, and 400 000 U/d penicillin was intravenously infused for 1 week. The feeding, activity, and vocalization of dogs in each group after injury were observed until they were sacrificed. Immediately after injury and 2, 4, 6, and 8 weeks after injury, the laryngeal morphology of the dogs in corresponding time point groups were observed by endoscope. After the observation, the dogs in each injury group were sacrificed, and the laryngeal tissue was taken. The epiglottis, glottis, and cricoid cartilage were collected to make full-thickness tissue slice, respectively, and their pathological changes were observed with hematoxylin and eosin staining. The dogs of control group were not specially treated, and their life activities, laryngeal morphological and pathological changes were observed. Results: (1) The dogs of control group had normal feeding, activities, and vocalization. All the dogs in injury group survived until they were sacrificed, and their feeding, activities, and vocalization were obviously reduced after injury compared with those of control group. The dogs of 2, 4, 6 and 8 weeks after injury groups ate and moved normally 2 weeks after injury but vocalized abnormally in frequency and volume compared with those of control group, which lasted until they were sacrificed. (2) The dog's laryngeal mucosa in control group was complete and pink, without obvious exudation. The laryngeal mucosa of the dog in immediately after injury group was pale and edematous, with obvious exudation, local ulceration, necrosis, and exfoliation, and dilated microvessels on the surface. The laryngeal mucosa of the dogs in 2 weeks after injury group was pale, edematous, and oozed less than that of immediately after injury group, and the glottis was blocked by an obviously extruding mass. The paleness and edema of laryngeal mucosa were significantly reduced in the dogs of 4 weeks after injury group compared with those of 2 weeks after injury group, without dilated microvessel, and the glottic extruding mass was obviously smaller than that of 2 weeks after injury group. The sizes of glottic mass were similar between the dogs of 6 and 8 weeks after injury groups, which were obviously smaller than that in 4 weeks after injury group. (3) In the dogs of control group, the epithelial cells of epiglottis, glottis, and cricoid cartilage were normal in morphology, the proper glands were visible in the intrinsic layer, and the muscle fibers and the chondrocytes were normal in morphology. In the dogs of immediately after injury group, large sheets of epiglottis epidermis exfoliated, the epithelial cells were swollen and necrotic, the intrinsic glands were atrophic and necrotic, and the chondrocytes were degenerated and necrotic. The epidermis of the glottis partially exfoliated, the epithelial cells were swollen and necrotic, the intrinsic glands were atrophic and necrotic, the muscle fibers were partially atrophic and fractured, and the vacuolar chondrocytes were visible. The cricoid cartilage epidermis was ablated, the epithelial cells were swollen, the intrinsic layer and submucosal layer were slightly edematous, and the morphological structure of glands, chondrocytes, and muscle fibers were normal. In the dogs of 2 weeks after injury group, the epiglottis epidermis was completely restored, a small amount of glands in the intrinsic layer were repaired, and obsolete necrotic chondrocytes and new chondrocytes could be seen. A large number of fibroblasts, new capillaries, and inflammatory cells infiltration were observed in the epidermis of glottis, and intrinsic layer glands were repaired. The cricoid cartilage epidermis was repaired intactly, and there was no edema in the intrinsic layer. In the dogs of 4 weeks after injury group, the epiglottis intrinsic layer glands were further repaired compared with those of 2 weeks after injury group, and new chondrocytes were seen in the submucosa of the glottis. The condition of cricoid cartilage was consistent with that of control group. The dog's epiglottis, glottis, and cricoid cartilage were similar between the 6 and 8 weeks after injury groups, and no significant change was observed compared with those of 4 weeks after injury group. Conclusions: The morphological changes of larynx after severe laryngeal burn in dogs include mucosa detachment and necrosis, and mass blocking glottis. Pathological changes include epidermis shedding and necrosis, gland atrophy and necrosis, vascular congestion and embolism, chondrocytes degeneration, necrosis and proliferation, even local granulation tissue formation and cartilaginous metaplasia. These results may be the cause of laryngostenosis after laryngeal burn.

[Influence of covering of auto-crosslinked sodium hyaluronate gel in combination with xenogenic acellular dermal matrix on healing of full-thickness skin defect wound in pig].

OBJECTIVE: To explore the influence of covering of auto-crosslinked sodium hyaluronate gel in combination with xenogenic acellular dermal matrix (ADM) on healing of full-thickness skin defect wound in pig. METHODS: Totally four 10 cm×10 cm full-thickness skin defect wounds were reproduced symmetrically on both sides of spine on the back of each one of the six Chinese experimental minipigs. After autologous microskin grafting, the 4 wounds in each pig were divided into 4 groups according to the random number table, with 6 wounds in each group. Wounds in allogenic skin group (AS) were covered by full-thickness skin from one (not the recipient) of the 6 pigs; wounds in xenogenic skin group (XS) were covered by full-thickness skin of sheep; wounds in xenogenic ADM group (XA) were covered by ADM of sheep; wounds in combination group (C) were covered by ADM of sheep combined with auto-crosslinked sodium hyaluronate gel. The wounds were bound up with pressure, and the dressing was changed once every 7 days. On post surgery day (PSD) 7, 14, 21, 28, 35, and 42 when changing dressing, the condition of wounds and the exfoliation of the covering on microskin were observed, and the complete exfoliation time of the covering was recorded. On PSD 28, 35, and 42, the wound healing rate was calculated. Data were processed with one-way analysis of variance and SNK test. RESULTS: (1) On PSD 7, no fluid appeared under the covering of wounds in groups AS and C, while plenty of fluid appeared under the covering of wounds in groups XS and XA. From PSD 14 to 35, most of the full-thickness skin of pig in group AS did not exfoliate. All the full-thickness skin of sheep in group XS exfoliated, leaving a lot of crusts on the surface of the wounds on PSD 14. Most of the ADM of sheep in group XA separated from the wound with the crusts turning dry and exfoliating on PSD 14. All the ADM of sheep exfoliated with most of the wounds healed in group C on PSD 35. On PSD 42, all the full-thickness skin of pig in group AS exfoliated, leaving most of the wounds unhealed; all the crusts exfoliated and the majority of wounds were healed in group XS and XA group; all the wounds in group C were healed. (2) Compared with that in group C[(32.7±3.3) d], the complete exfoliation time of the covering was obviously shorter in group XS[(15.2±4.8) d]and group XA[(22.2±6.3) d], with P values below 0.05, while the complete exfoliation time of the covering in group AS[(44.8±2.7) d]was obviously longer (P<0.05). (3) On PSD 28, 35, and 42, the wound healing rates in groups XS and XA[(21.2±2.6)%, (51.4±2.4)%, (89.6±2.8)%, and (23.7±3.4)%, (53.6±6.3)%, (91.3±4.9)%, respectively]were obviously lower than those in group C[(35.1±3.4)%, (62.1±6.7)%, (98.8±1.0)%, respectively, with P values below 0.05]. On PSD 42, the wound healing rate in group AS[(44.0±3.8)%]was significantly lower than the rates in the other 3 groups (with P values below 0.05). CONCLUSIONS: Auto-crosslinked sodium hyaluronate gel combined with ADM of sheep used as covering of microskin grafts on full-thickness wound in pig can lengthen the persistence time of ADM of sheep on the wound, as well as promote wound healing.

Simultaneous determination of nucleobases, nucleosides and saponins in Panax notoginseng using multiple columns high performance liquid chromatography.

A new multiple columns HPLC method for simultaneous determination of 16 characteristic components, 5 nucleobases and nucleosides (uracil, cytidine, uridine, guanosine and adenosine), and 11 saponins (notoginsenoside R1, ginsenoside Rg1, ginsenoside Re, notoginsenoside R4, notoginsenoside Fa, ginsenoside Rb1, notoginsenoside R2, ginsenoside Rg2, ginsenoside Rh1, ginsenoside Rd and notoginsenoside K), in the root of Panax notoginseng, a valued traditional Chinese medicinal herb, were developed. Notoginsenoside R4, Fa and K were first quantitatively determined in P. notoginseng. The 5 nucleobases and nucleosides compounds were separated on a Zorbax SB-Aq column (150 x 4.6 mm, 5.0 microm) and 11 saponins were analyzed using a Zorbax Bonus-RP column (150 x 4.6 mm, 5.0 microm) with column switching. The column temperature was set at 30 degrees C. Mobile phase was composed of 5mM ammonium acetate aqueous (A), water (B) and acetonitrile (C) using a gradient elution. The flow rate was 1.5 mL/min and detection wavelengths were set at 260 nm for nucleobases and nucleosides, and 203 nm for saponins. The developed method had good repeatability and sensitivity for quantification of 16 analytes with overall precision (including intra- and inter-day) less than 3% (RSD), and LOD and LOQ were less than 1.33 microg/mL and 5.12 microg/mL, respectively. The method was successfully applied to the simultaneous determination of 16 analytes in 15 samples of P. notoginseng collected from different places of China, which indicated that multiple columns HPLC can be used for comprehensive quality control of P. notoginseng.

Chemical characteristics for different parts of Panax notoginseng using pressurized liquid extraction and HPLC-ELSD.

The chemical characteristics for different parts of Panax notoginseng, including root, fibre root, rhizome, stem, leaf, flower and seed, were determined using high performance liquid chromatography-evaporative light scattering detection (HPLC-ELSD) and pressurized liquid extraction (PLE). Eight major saponins, namely notoginsenoside R1, ginsenosides Rg1, Re, Rb1, Rc, Rb2, Rb3 and Rd were also quantitatively compared among the different parts of P. notoginseng. The chromatograms showed that there was significant difference between underground (root, fibre root, rhizome) and aerial (leaf and flower) parts from P. notoginseng, though the similarities of entire chromatographic patterns among tested samples from underground (0.965+/-0.029, n=12) and aerial parts (0.987+/-0.014, n=5) were similar, respectively. Especially, no saponin was detected in the seed of P. notoginseng. Hierarchical clustering analysis based on eight investigated saponins or the ratios of contents for ginsenoside Rg1/Rb1 and ginsenoside Rb3/Rb1 showed that the samples from different parts of P. notoginseng were divided into three main clusters. One cluster was underground parts, which contained rich protopanaxatriol and protopanaxadiol types saponins. The leaf and flower were in the same cluster, which contained protopanaxadiol type saponins only. Especially, ginsenoside Rc, Rb2 and Rb3, rare in the underground parts, were rich in aerial parts of P. notoginseng. The stem of P. notoginseng was another cluster. Based on the cluster analysis, the chemical characteristics for different parts of P. notoginseng were revealed. They are composite cluster (underground parts), protopanaxadiol cluster (aerial parts) and interim (stem) cluster, which was the one between the two typical clusters, respectively. The result shows that chemical characteristics of underground parts and aerial parts from P. notoginseng are obviously different, which is helpful for pharmacological evaluation and quality control of P. notoginseng.

A rapid HPLC-ESI-MS/MS for qualitative and quantitative analysis of saponins in "XUESETONG" injection.

'XUESETONG' injection, one of the most widely used proprietary medicines in traditional Chinese medicine, consists of total saponins made from Panax notoginseng, which is a highly valued and important Chinese medicinal herb. It is used to treat cardiovascular diseases. In order to control the quality of XUESETONG injection, a rapid HPLC-ESI-MS/MS method was developed for qualitative and quantitative determination of the saponins. The analyses were performed on SB-C18 column using gradient elution in 25 min. Full scan and time programmed selected reaction monitoring (SRM) were used for qualitative and quantitative analysis of saponins, respectively. Twenty-seven saponins were identified and nine of them including notoginsenoside R1, ginsenoside Rb1, Rb2, Rb3, Rc, Rd, Re, Rf and Rg1 were quantified. Ten XUESETONG injections were analyzed and compared. The results showed that there is a great variation among different samples. In conclusion, the developed method is rapid, accurate and sensitive for qualitative and quantitative analysis of saponins in XUESETONG injection. Moreover, it also can be used for the quality control of P. notoginseng raw material and its preparations.

Simultaneous determination of nine saponins from Panax notoginseng using HPLC and pressurized liquid extraction.

A HPLC and pressurized liquid extraction (PLE) method was developed for simultaneous determination of nine saponins, including notoginsenoside R1, ginsenoside Rg1, Re, Rf, Rb1, Rc, Rb2, Rb3 and Rd in Panax notoginseng. The analysis was performed on C18 column with water-acetonitrile gradient elution and the investigated saponins were authenticated by comparing retention time and mass spectra with their reference compounds. Several methods including PLE, ultrasonication, soxhlet extraction and immersion were used for sample preparation and their extraction efficiency was compared. The results showed that PLE has the highest extraction efficiency and repeatability, which would be valuable on standardization of sample preparation for quality control of Chinese medicines. The developed HPLC and PLE is an effective approach for simultaneously quantitative determination of sapoinins in P. notoginseng, which could be used for quality control of P. notoginseng and its preparations.