| ã€Abstract】 Objective To investigate the effects of neural stem cells (NSCs) transplantation on sensory dyskinesia after hypoxic-ischemic brain damage (HI BD) in neonatal rats.  Methods Seven-day-old SD rats were randomly divided into sham operation group, HIBD group and intracerebral transplantation group. The latter two groups were implanted with BrdU-labeled NSCs or medium in the left sensorimotor cortex 3 days after HI BD injury. Control, changes in sensorimotor function and survival and differentiation of transplanted cells in the brain were observed 4 weeks after implantation. Results Compared with the HI BD group, the transplant group showed significant improvement in the T-maze and four sensorimotor function tests. In the T-maze, the rate of spontaneous change (69.23±13.34) % vs (45.00±27.27)%] (P<0.05) increased; in the grip traction test, the holding time was significantly prolonged [(49.15±13.39) s vs (27.20 ±15.18) s ] ( P <0.05); the asymmetry disappeared in the limb placement and foot error tests, and the posture reflex was also significantly improved (P < 0.05). BrdU indirect immunofluorescence showed that 4 weeks after transplantation, the survival NSCs were found in the brain near the transplant site; the BrdU and NF2 200 immunofluorescence double label showed that NSCs could be partially differentiated into neurons expressing NF2 200 4 weeks after transplantation. Conclusion Transplantation of NSCs in the brain can promote the recovery of sensorimotor function and behavior in neonatal rats with HI BD.  ã€Key words】 Neural stem cells; Transplantation; Sensory motor; Hypoxia-ischemia; Rat  Neonatal hypoxic ischemic encephalopathy (H IE) has a high mortality rate and poor prognosis, and can produce permanent neurological dysfunction. At present, the treatment of neonatal H IE is limited to supportive therapy, and there is no means to promote nerve regeneration [1]. At present, in animal experiments and clinical trials using neural stem cells (NSCs) transplantation for the treatment of hypoxic-ischemic brain damage (HI BD), certain effects have been achieved [2]. However, most of these findings are from adult animals and adults. Whether NSCs transplantation can improve sensory dyskinesia after HIBD in neonatal rats has not been reported. This study was to investigate the effect of transplanted embryonic rat NSCs on sensorimotor function in neonatal rats after HI BD.  Materials and Method  First, the material  SD rat (clean grade). DMEM /F12 (1:1), B27 (Gibco), bFGF, EGF (Pep r oTech EC). Anti-NF2 200, anti-B rdU (NeoMarkers). The rat brain stereotaxic instrument, T labyrinth, and atmospheric hypoxia chamber were all made by reference.  2. Separation, culture, B rdU labeling and collection of embryonic NSCs  The embryos of SD rats were taken for 14 days, the forebrain cortex was isolated, and the single cell suspension was prepared by trypsin digestion and inoculated into DMEM/F12 medium containing 10 ng/ml bFGF and 20 ng/ml EGF (including B2720μl). In /ml), change the liquid once every 2 to 3 days. Three days before transplantation, the transplanted NSCS was changed, and B r2dU was added to the culture medium to a final concentration of 10 μmol / L. The NSCs labeled with BrdU-labeled in vitro and stably subcultured for 1 month were used for transplantation. 4% trypan blue staining cell count, live cells > 95%, adjusted live cell density to 5 × 104 cells / μl, placed on ice for later use.  Third, the experimental animal grouping  A total of 35 neonatal 7-day-old SD rats were randomly divided into sham operation group (Sham, n=10), HIBD+peech transplantation control group (HIBD, n=12), HI BD+NSCs transplantation group (NSCs, n = 13).  Fourth, HI BD model production  Referring to the Rice method [3], the left common carotid artery was isolated and ligated after 7 days old SD rats were anesthetized. After 2 hours of rest, the rats were placed in a plexiglass hypoxic chamber with an oxygen concentration of 8% (7%). ~9%), the cabin temperature is controlled at (36 ± 2) °C, the humidity is 70% ± 5%, and the anoxic time lasts.  2 h. After the end of the experiment, the rats were returned to the cage and breastfed by the mother.  Five, intracerebral transplantation  3 days after HIBD injury, after low temperature anesthesia, the animal was fixed to the stereotaxic instrument, and the left sensorimotor cortex area (coordinate AP: +0.3 mm, ML: -2 mm, DV: -1.5 mm) was used as the transplantation point, and the micro-use was used. The injector was slowly injected with 2 μl of NSCs cell suspension or medium.  Sixth, the detection of sensorimotor function in rats  Tested at 42 d age. Refer to the Bona [4] method, including 4 trials. Behavioral testing is performed blindly.  1. Grip traction test: Rats use a front paw to grasp a hollow plastic tube with a diameter of 0.6 cm placed horizontally 45 cm from the table top, and record the suspension time (maximum 60 s, 60 s over 60 s) and right The lateral (ischemic contralateral) hind limb can be placed on the tube.  2. Foot error test: record the number of times the anterior and hind limbs or paws fall off within 2 minutes of the horizontally placed grid (50 cm × 40 cm, 3 cm × 3 cm per cell), in order to exclude the difference in mobility of different rats. The effect is only statistical analysis of the difference between the number of errors on the left and right sides.  3. Posture Reflex Test: Grab the rat's tail and hang it at a height of 50 cm from the table top. Normal rats extended both forelimbs to the table top (0 points, while rats with brain damage damaged the limbs on the opposite side of the cerebral hemisphere (1 point), then placed the rat on the table, on the side behind the shoulder Pressurize until the forelimbs are straightened and repeat several times. If the resistance to the right side (the opposite side of the injured cerebral hemisphere) is weakened to abnormal (2 points).  4. Limb placement test: The placement of the left and right hind paws of the rats under 6 different sensory stimuli was recorded, and the difference in scores on both sides of each mouse was recorded. The scoring criteria are as follows: 0 points, the paws are placed correctly and quickly; 1 point, slow or not completely correct; 2 points, not placed.  7. The spontaneous transition and forced change of the T labyrinth  Refer to the Balduini [5] method.  1. T labyrinth spontaneously: detected from 28 days of age. Animals were tested for trends in different arms during continuous testing in the T-maze. Each animal was tested continuously for 3 days, once a day, twice for each opportunity to change direction. The results were recorded as 0%, 50%, or 100%, respectively, and the number of times entering the left and right arms was recorded.  2. T maze forced change: detected from the age of 32d. The test is divided into two phases, pre-test and test. During the pre-test, the animals were forced to enter only the open arms, and the food was eaten, then placed in the starting arm. After 15 s, all the gates were removed and the test was started. If the animal enters the arm that was not entered during the pre-test, it is recorded as correct; if it enters the arm that has entered the pre-test, it is recorded as an error. Each animal was tested 5 times a day for 15 minutes each time for 4 consecutive days for a total of 20 times.  Eight, immunofluorescence staining  Nestine, BrdU and NF2 200 single or double dyeing according to the instruction manual.  Nine, statistical analysis  The measurement data is expressed as x±s, and the data are all statistically analyzed using the SPSS11.0 statistical software package.  result  1. Growth and identification of NSCs  Primary cultured cells can form small cell clusters consisting of 2 to 3 cells within 24 h, ie, primary clones. On 7 days, they grow into cloning of dozens of cells to hundreds of cells, which are suspended and grow in a spherical shape. After passage, the same large number of subculture clones as the primary culture appeared, and 5-7d can be passaged again. Indirect immunofluorescence cytochemical staining revealed that the NSCs marker protein nestin was expressed in all levels of cloning.  Second, neurological function evaluation  1. T-maze spontaneous alternation test: Rats in the HIBD group were selected to the left (ischemic side) > right side, and the difference was significant (P < 0.05). Forced change in the 21T maze: With the increase of the number of days, the correct rate of the Sham group and the NSCs group increased day by day, while the correct rate of the HIBD group did not increase significantly, indicating that the learning ability was poor. The correct rate of NSCs group was significantly higher than that of HIBD group, and the difference was significant (P < 0.05). 3. Sensorimotor function test: The grip traction time of the NSCs group was longer than that of the HIBD group (P<0.05). In the foot error test, the difference between the right and left left of the NSCs group was lower than that of the HIBD group (P<0.05). In the posture reflex test, the normal rate of the NSCs group was significantly higher than that of the HIBD group (P < 0.05). In the limb placement test, the right-left score of the NSCs group was significantly lower than that of the HIBD group (P<0.05). Detection of 41NSCs after transplantation: After 1 month of NSCs transplantation, different levels of BrdU-positive cells were distributed along the needle channel, distributed in a large amount around the transplanted point, and spread to the brain parenchyma. The positive cells gradually decreased with the increase of the distance from the transplanted point. The immunofluorescence double labeling of BrdU and NF2 200 showed that some BrdU-positive cells also expressed neuron-specific antigen NF2 200, suggesting that NSCs can differentiate into neurons after transplantation into rat brain for one month.  discuss  Studies have shown that transplanted NSCs into the brain of developing rats, the cells migrate extensively, and the differentiation results are related to the site of transplantation. It is indicated that neurons differentiated by NSCs retain their ability to respond to brain structure recognition and localization in neonatal rats after in vitro expansion [6]. However, transplanting NSCs into the brain of adult animals can only differentiate into neurons in the site of neurogenesis, such as hippocampus, but mainly differentiate into glial cells in non-neurogenic sites such as cerebellum, striatum and spinal cord [7]. . The migration ability after stem cell transplantation and the ability to integrate with host nerve cells also gradually decrease with age [8], and the immune function of newborn rats is imperfect, and the rejection of host after brain transplantation is relatively small. It can be seen that the response of adult and neonatal hosts to transplanted NSCs is different. Stem cell transplantation is more promising for treating neonatal brain injury diseases than adults. The results of this experiment show that NSCS isolated from rat forebrain cortex tissue can maintain undifferentiated state and proliferate for a long time in the presence of growth factors EGF and bFGF. After removal of growth factors, it can differentiate into neurons. And glial cells, with the basic properties of NSCS. Transplantation of NSCs into the sensorimotor cortex of the HIBD model of neonatal rats can effectively improve the sensorimotor dysfunction caused by HIBD. The scores in the traction test, posture reflex, foot error test and limb placement test are significantly improved compared with the control group. At the same time, it also effectively improved spatial awareness and learning memory defects, and increased the correct rate in the T-maze forced change test. Four weeks after transplantation, all transplanted rats were seen to have viable cells at the transplant site and migrated distally along the transplant site. In this study, NSCs were transplanted into the sensorimotor cortex and could effectively improve their sensorimotor deficits. The mechanism may be related to nerve remodeling. The immunofluorescence double label also showed that some BrdU-positive cells also expressed neuron-specific antigen NF2200, suggesting NSCs. It can be differentiated into neurons after transplantation, which may be one of the mechanisms for its therapeutic effect. The results suggest that NSCs can not only survive but also integrate and migrate with host brain tissue after transplantation into the brain of neonatal rat HI BD, and improve their long-term learning and memory, spatial discrimination and sensorimotor function. . It provides an experimental basis for the transplantation of NSCs for the treatment of neonatal H IE.  |
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