Motor function evaluation in rats receiving umbilical cord mesenchymal stromal cell transplantation for traumatic brain injury using CatWalk automated gait analysis system / 南方医科大学学报

<p><b>OBJECTIVE</b>To assess the effect of CatWalk automated gait analysis system for evaluation of motor function of rats with traumatic brain injury (TBI) after umbilical cord mesenchymal stromal cell (UC-MSC) treatment.</p><p><b>METHODS</b>Eighteen Wistar rats were randomized equally into normal control group, TBI ∓ saline group, and TBI ∓ UC-MSCs group. The rats in the latter two groups were subjected to weight-drop impact to induce TBI followed by injection UC-MSCs or saline into the lesion 7 days after TBI. The neurological function was assessed using CatWalk system and modified neurological severity scores (mNSS) before and 3 days after TBI and 7 days after UC-MSC transplantation. The rats were sacrificed 14 days after the cell transplantation and the brain sections were stained for immunohistochemical analyses.</p><p><b>RESULTS</b>Three days after TBI, mNSS test showed moderate injury of the rats. Seven days after the cell transplantation, the rats showed significant motor function improvement and CatWalk analysis indicated partial recovery of the gait parameters of the 4 limbs compared to the rats with saline treatment. Histological analyses showed that DiO-labeled UC-MSCs were present in the lesion boundary and expressed glial fibrillary acidic protein and β-tubulin III.</p><p><b>CONCLUSION</b>UC-MSC transplantation can promote functional improvement of the brain after TBI in rats. Compared with mNSS test, CatWalk analysis is more sensitive and objective for assessing neurological function and also provides more detailed information on specific gait parameters.</p>

Feridex labeling of bone marrow stromal cells of crab-eating macaque / 中国组织工程研究

BACKGROUND: Studies regarding Feridex in vitro cell labeling are mainly in rodents, while little information is known on primate crab-eating macaque.OBJECTIVE: To explore the feasibility of protocols using Feridex and transfection agents for in vitro magnetic labeling of bone marrow stromal cells (BMSCs) in crab-eating macaque.METHODS: Under the sterile condition, the crab-eating macaque BMSCs were obtained by means of density gradient centrifugation following a bone puncture. Feridex-poly-l-lysine complexes were used to magnetically label BMSCs. The efficiency and cellular viability of Feridex-poly-l-lysine labeled BMSCs were evaluated by Prussian blue staining, electron microscopy, and trypan blue dye exclusion test. The proliferation and differentiation ability of Feridex-poly-l-lysine labeling BMSCs were also investigated by inverted phase contrast microscope and immunocytochemistry. RESULTS AND CONCLUSION: BMSCs could be effectively labeled by Feridex and labeling efficiency was around 99%. Tiny blue stained fine particles and numerous vesicles coated with the electron-dense magnetic iron particles could be found in the cytoplasm of Feridex-poly-l-lysine labeled BMSCs under optical microscopy and transmission electron microscopy respectively. Cell viability, proliferation and differentiation ability of labeled BMSCs were not affected by Feridex-poly-l-lysine labeling. Results indicated that Feridex might be used to label BMSCs of crab-eating macaque.

Construction of eukaryotic expression vector of recombinant immunotoxin human VEGF165-PE38 and its expression / 肿瘤研究与临床

Objective To construct a new recombinant immunotoxin expression vector by using human VEGF165 and a truncated pseudomonas exotoxin A ramification (PE38) gene, and explore the expression of the VEGF165-PE38 fusion protein in HEK293 cells. Methods VEGF165 was cloned by polymerase chain reaction (PCR). PE38 gene was gained from an vector plasmid pRB391 by restriction endonuclease digestion, and then inserted to the eukaryotic expression vector pIRES2-EGFP. After the eukaryotic recombinant vector pIRES2-VEGF165-PE38-EGFP was identified by restriction endonuclease digestion and sequence analysis, the vector was transfected into HEK293 cells by liposome protocol. RT-PCR and ELISA method was used to confirm the expression of the fusion gene in the HEK293 cells. Results Restriction endonuclease digestion and sequence analysis revealed the VEGF165-PE38 fusion gene was cloned into the eukaryotic expression plasmid vector pIRES2-EGFP successfully. The pIRES2-VEGF165-PE38-EGFP fusion gene could express in the HEK293 cells. Conclusion The result provide the basis for search of the targeted cytotoxic activity to tumor vascular endothelial cells and may have some potential value in clinical application.

Experimental study on the catecholaminergic effects of paraventricular nucleus projected from medullary visceral zone in "immune-to-brain communication" access / 中国组织工程研究

BACKGROUND: Lipopolysaccharide(LPS), as a polyclonal immune exciter, can simulate immune excitation status, which is useful in the observation of whether the catecholaminergic neurons in paraventricular nucleus of hypothalamus(PVN) projected from medullary visceral zone(MVZ) react towards LPS stimulation that is to provide a theoretical gist for the researches on the protection of brain function.OBJECTIVE: To observe whether PVN catecholaminergic neurons projected from MVZ react towards LPS stimulation for the exploration of the impacts of MVZ-PVN catecholaminergic access in "immune-to-brain communication".DESIGN: A randomized controlled study using experimental animals as subjects.SETTING: An Institute of Neurosurgery and Neurology of one Military University of Chinese PLA.MATERIALS: The study was conducted in the Department of Neurosurgery of Zhujiang Hospital affiliated to Southern Medical University and the Institute of Neurology of the Fourth Military Medical University of Chinese PLA from January to December in 2002. Ten healthy adult SD rats in cleanness grade were obtained from the experimental animal center of the Fourth Military Medical University of Chinese PLA.METHODS: WGA-HRP was injected into PVN of one side of the rat, and the immune exciter LPS was injected into the abdominal cavity after 48 hours of survival to induce immune response. Samples were stained by triple labels of WGA-HRP method and double immunohistochemical staining of anti-Fos and anti-TH antibodies.MAIN OUTCOME MEASURES: To observe the distributions and expressions of WGA-HRP labeled cells, Fos protein, and catecholaminergic neuron(labeled by TH) in MVZ.RESULTS: Seven immune-reactive(IR) positive neurons were found in MVZ, i. e., HRP, Fos or TH single labeled cells, Fos/HRP, Fos/TH or HRP/TH double labeled cells, and Fos/HRP/TH triple labeled cells. Fos/HRP double labeled neurons and Fos/HRP/TH triple labeled neurons accounted for 12. 5% and 39.6% of HRP labeled cells respectively.CONCLUSION: MVZ reacts to LPS immune stimulation, which could upload the immune message to PVN through Catecholaminergic neurons.MVZ might be a relay station in "immune-to-brain communication", which exerts immune modulatory impact through "MVZ→PVN" access.

Distribution character of functional unit of reaction neuron and astrocyte in medullary visceral zone 1 hour after epileptic attack: a laser confocal microscopic research / 中国组织工程研究

BACKGROUND: It is considered traditionally that epilepsy is a kind of complicated nervous conduct disorder caused by abnormally excited neuron in different area in brain. While the research on the function of astrocyte in epileptic attack is very rare.OBJECTIVE: To study the reaction of neuron and astrocyte in medullary visceral zone after epilepsy induced by pentetrazole in rats.DESIGN: A randomized controlled experimental research.SETTING and MATERIALS: The experiment was done in the Neurosurgery Laboratory of Zhujiang Hospital Affiliated to Southern Medical University and Neuroscience Institute of Fourth Military Medical University of Chinese PLA. Fourteen healthy adult SD rats, weighing 180 - 220 g, clean grade, were provided by the Experimental Animal Center of Fourth Military Medical University of Chinese PLA.INTERVENTIONS: Distribution of neuron and astrocyte in MVZ 1 hour after epileptic attack was shown by laserconfocal microscopic technique combined with triplication immunofluorescence histochemistry of anti-Fos protein, anti-tyrosine hydroxylase(TH) and anti-glial fibrillary acidic protein(GFAP).MAIN OUTCOME MEASURES: Observation of distribution of positive cells of Fos, GFAP and TH in MVZ and relationship between GFAP positive astrocyte and neuron.RESULTS: Fos positive neurons and GFAP positive astrocytes in MVZ increased significantly. Triplication immunofluorescence histochemistry showed reaction neuron(Fos positive) closely related with reaction astrocyte(GFAP positive) . Three kinds of N-ASC compounds with different labels were found, which were TH +/Fos +/GFAP + three labeled compound, TH + /GFAP +/Fos- and Fos+/GFAP +/TH- two labeled compound.CONCLUSION: Neuron and astrocyte in MVZ reacted strongly when epilepsy attacks. N-ASC as a functional unit may regulate onset of epilepsy.