Cellular Localization and Distribution of TGF-ß1, GDNF and PDGF-BB in the Adult Primate Central Nervous System.

The available data on the localization of transforming growth factor beta1 (TGF-ß1), glial cell line-derived neurotrophic factor (GDNF), and platelet-derived growth factor-BB (PDGF-BB) in the adult primate and human central nervous system (CNS) are limited and lack comprehensive and systematic information. This study aimed to investigate the cellular localization and distribution of TGF-ß1, GDNF, and PDGF-BB in the CNS of adult rhesus macaque (Macaca mulatta). Seven adult rhesus macaques were included in the study. The protein levels of TGF-ß1, PDGF-BB, and GDNF in the cerebral cortex, cerebellum, hippocampus, and spinal cord were analyzed by western blotting. The expression and location of TGF-ß1, PDGF-BB, and GDNF in the brain and spinal cord was examined by immunohistochemistry and immunofluorescence staining, respectively. The mRNA expression of TGF-ß1, PDGF-BB, and GDNF was detected by in situ hybridization. The molecular weight of TGF-ß1, PDGF-BB, and GDNF in the homogenate of spinal cord was 25 KDa, 30 KDa, and 34 KDa, respectively. Immunolabeling revealed GDNF was ubiquitously distributed in the cerebral cortex, hippocampal formation, basal nuclei, thalamus, hypothalamus, brainstem, cerebellum, and spinal cord. TGF-ß1 was least distributed and found only in the medulla oblongata and spinal cord, and PDGF-BB expression was also limited and present only in the brainstem and spinal cord. Besides, TGF-ß1, PDGF-BB, and GDNF were localized in the astrocytes and microglia of spinal cord and hippocampus, and their expression was mainly found in the cytoplasm and primary dendrites. The mRNA of TGF-ß1, PDGF-BB, and GDNF was localized to neuronal subpopulations in the spinal cord and cerebellum. These findings suggest that TGF-ß1, GDNF and PDGF-BB may be associated with neuronal survival, neural regeneration and functional recovery in the CNS of adult rhesus macaques, providing the potential insights into the development or refinement of therapies based on these factors.

Intrathecal injection of lentivirus-mediated glial cell line-derived neurotrophic factor RNA interference relieves bone cancer-induced pain in rats.

Bone cancer pain is a common symptom in cancer patients with bone metastases and the underlying mechanisms are largely unknown. The aim of this study is to explore the endogenous analgesic mechanisms to develop new therapeutic strategies for bone-cancer induced pain (BCIP) as a result of metastases. MRMT-1 tumor cells were injected into bilateral tibia of rats and X-rays showed that the area suffered from bone destruction, accompanied by an increase in osteoclast numbers. In addition, rats with bone cancer showed apparent mechanical and thermal hyperalgesia at day 28 after intratibial MRMT-1 inoculation. However, intrathecal injection of morphine or lentivirus-mediated glial cell line-derived neurotrophic factor RNAi (Lvs-siGDNF) significantly attenuated mechanical and thermal hyperalgesia, as shown by increases in paw withdrawal thresholds and tail-flick latencies, respectively. Furthermore, Lvs-siGDNF interference not only substantially downregulated GDNF protein levels, but also reduced substance P immunoreactivity and downregulated the ratio of pERK/ERK, where its activation is crucial for pain signaling, in the spinal dorsal horn of this model of bone-cancer induced pain. In this study, Lvs-siGDNF gene therapy appeared to be a beneficial method for the treatment of bone cancer pain. As the effect of Lvs-siGDNF to relieve pain was similar to morphine, but it is not a narcotic, the use of GDNF RNA interference may be considered as a new therapeutic strategy for the treatment of bone cancer pain in the future.

[Changes of the Expression of Brain Derived Neurotrophic Factors in Rats Trachea Induced by Acrolein Exposure].

OBJECTIVE: To investigate expressional changes of brain derived neurotrophic factor (BDNF) in the trachea of rats with acrolein inhalation. METHODS: Twenty two SD rats were divided into 2 groups: the rats in experimental group were subjected to acrolein inhalation for the induce of trachea inflammatory injury, while the rats with saline (NS) inhalation were as control. All the rats were sacrificed in 1,3,6 weeks after acrolein (n = 11 at each time point) or saline inhalation (n = 11 at each time point), the samples of trachea epithelium were harvested. The immunohistochemistry and in situ hybridization was performed to detect the location of BDNF protein and mRNA in trachea. The expression of BDNF mRNA in the trachea tissues were determined by RT-PCR. RESULTS: There are positive cells in epithelium of trachea for BDNF protein and mRNA, with cytoplasm staining. The expression of BDNF mRNA in the trachea was increased at 1 week after acrolein inhalation (P < 0.05, vs. control group), then decreased along with the time and reached to the same level as control group at 3 weeks, then last to 6 weeks (P > 0.05). CONCLUSION: The inflammatory injury in trachea induced by acrolein exposure could be associated with the increased expression of BDNF. BDNF may be one of the crucial inflammatory factors in the process of inflammatory reaction in trachea with acrolein stimulation.

[Human umbilic mesenchymal stromal cells repairs diabetic foot in rats associated with VEGF expressional change].

UNLABELLED: OBJECTITVE: To explore the mechanism of human umbilic mesenchymal cells (HUMSCs) implantation for the treatment of diabetic foot in rats associate with vascular endothelia growth factor (VEGF) expression changes. METHODS: After diabetic foot model in rats were established by administration of streptozotozin (STZ) in intraperitoneal injection (2 weeks), ulceration in foot was induced by incision injury combined with swearing staphylococcus aureas. Then, HUMSCs were smeared on the ulceration of foot in diabetic rats. Ten days later, the densities of blood vessel and the level of VEGF expression were determined by using immunohistochemistry, RT-PCR and Western blot. RESULTS: HUMSC grafts reduced significantly the volume of ulceration in diabetic foot rats (P < 0.05). RT-PCR and Western blot showed that VEGF and its mRNA were significantly upregulated (P < 0.05). VEGF immunstaining was found in blood vessels and the densities of blood vessels in HUMSC group were increased significantly (P < 0.05). CONCLUSION: HUMSC implantation showed a positive role in promoting the recovery of the ulceration in foot with diabetic rats.

[Effect of human umbilici mesenchymal stromal cells implantation on the BDNF expression in diabetic foot rats].

UNLABELLED: OBJECTITVE: To investigate the effect of human umbilici mesenchymal cells (HUMSCs) implantation on the brain derived neurotrophic factor (BDNF) expression in diabetic foot rats. METHODS: SD rats were divided into three groups (n = 12): normal group, diadetic foot model group and HUMSC treatment group. Diabetic foot model in rats was established, then prepared HUMSC were implanted on the diabetic foot ulcers in rats, and control ones were administrated with saline only. The area of ulceration, sensory function, BDNF expression and its localization were determined by using morphology, physiological function measurement, RT-PCR and immunohistochemistry assay. RESULT: Siglificantly decreased area of ulceration in diabetic foot rats of HUMSC implantation group was observed. This was simultaneously companied with the sensory function improvement (P < 0.05). RT-PCR showed that BDNF mRNA expression was significantly up regulated (P < 0.05). BDNF immunstaining was located in epithelia tissue and the protein level of BDNF was markedly increased (P < 0.05). CONCLUSION: HUMSC implantation maybe an effective strategy on the treatment of ulceration in diabetic foot rats, and the possible mechanism may involve in BDNF expression.

Urine-derived mesenchymal stem cells-derived exosomes enhances survival and proliferation of aging retinal ganglion cells.

OBJECTIVES: This study was designed to investigate to test the effect of exosomes from urine-derived mesenchymal stem cells (USCs) on the survival and viability of aging retinal ganglion cells (RGCs), and explored the preliminary related mechanisms. METHODS: Primary USCs were cultured and identified by immunofluorescence staining. Aging RGCs models were established by D-galactose treatment and identified by ß-Galactosidase staining. After treatment with USCs conditioned medium (with USCs removal), flow cytometry was performed to examine the apoptosis and cell cycle of RGCs. Cell viability of RGCs was detected by Cell-counting Kit 8 (CCK8) assay. Moreover, gene sequencing and bioinformatics analysis were applied to analyze the genetic variation after medium treatment in RGCs along with the biological functions of differentially expressed genes (DEGs). RESULTS: The number of apoptotic aging RGCs was significantly reduced in USCs medium-treated RGCs. Besides, USCs-derived exosomes exert significant promotion on the cell viability and proliferation of aging RGCs. Further, sequencing data analyzed and identified DEGs expressed in aging RGCs and aging RGCs treated with USCs conditioned medium. The sequencing outcomes demonstrated 117 upregulated genes and 186 downregulated genes in normal RGCs group vs aging RGCs group, 137 upregulated ones and 517 downregulated ones in aging RGCs group vs aging RGCs + USCs medium group. These DEGs involves in numerous positive molecular activities to promote the recovery of RGCs function. CONCLUSIONS: Collectively, the therapeutic potentials of USCs-derived exosomes include suppression on cell apoptosis, enhancement on cell viability and proliferation of aging RGCs. The underlying mechanism involves multiple genetic variation and changes of transduction signaling pathways.

[Expression of trkB gene in the pulmonary tissue of rats with lung injury induced by cerebral ischemia].

OBJECTIVE: To investigate the expression of tyrosine kinase B (trkB) in lung tissue of rats with lung injury induced by brain ischemia. METHODS: Twenty six adult SD rats were divided into sham group and brain ischemia lung injury (BILI) group. All rats were sacrificed at 3 days after the operation of modeling, lung tissues were then harvested to measure the protein and mRNA level of trkB by the methods of western blot and RT-PCR, the location of trkB positive cells was observed by immunochemistry study. RESULTS: trkB mRNA level in the lung tissue of rats with brain ischemia presented a significant increase, in corresponding to the upregulation of BDNF protein levels, when compared with sham one (P<0.05). trkB was localized in endothelia cells and smooth muscle. CONCLUSION: The upregulated expression of trkB expression may be associated with lung injury after brain ischemia.

[Block effects of BDNF antibody on MEK expression in lung of rats with brain ischemia].

UNLABELLED: OBJECITVE: To explore the block effect of brain-derived neurotrophic factor (BDNF) antibody on the mitogen extracellular kinase (MEK) expression in lung tissues of rats with brain ischemia. METHODS: Adult SD rats were divided into sham group, brain ischemia lung injury (BILI) group, and BDNF antibody treated group. Lung tissues were harvested at 3 days after operation. The distribution of MEK in the lung tissue was studied by immunhistochemistry, and the optical density values of MEK immunostaining was used to compare the difference of each group (n=5). RT-PCR was used to determine the block effect of BDNF antibody on the level of MEK mRNA (n=8). RESULTS: Immunohistochemistry showed that MEK immunoreactive positive products were observed in endothelia cells of airway and blood vessels. The optical density value in rats with brain ischemia significantly increased, compared with sham one (P<0.05). Moreover, BDNF antibody block could reverse this increase of BDNF immunostaining intensity (P<0.05). RT-PCR showed that BDNF antibody block significantly decreased the expressional level of MEK mRNA in the lung tissues with brain ischemia (P<0.05). CONCLUSION: BDNF antibody block leads to a significant reduction of MEK expression in endothelia cells of lung tissue in the rats with brain ischemia.

[Implication of BDNF expression in transected spinal cord of rats].

OBJECTIVE: To explore the change of brain derived neurotrophic factor (BDNF) expression and its relationship with the neurological behavior after spinal cord transection (SCT) in rats. METHODS: 66 adult SD rats were assigned randomly to sham operation group and SCT group. Rats in SCT group were subjected T10-T11 spinal cord transection and allowed to survived 1 d, 3 d, 7 d, 14 d, 21 d and 28 d. BBB scores in hindlimbs were observed at 0, 7, 14, 21 and 28 days post operation (dpo). The BDNF expression was determined by using ELISA (28 d) and RT-PCR (each time point) techniques. The localization of BDNF and its mRNA at sham and 28 d after SCT was also observed by immunohistochemistry and in situ hybridization. RESULTS: After SCT, the motor function in hindlimbs disappear immediately. The BBB scores get a gradual recovery from 14 dpo to 28 dpo, when compared with that of former timepoint (P < 0.05). BDNF and its mRNA were located in cytoplasma and neurites. The level of BDNF mRNA (indicated by RT-PCR) was upregulated at 14 days after SCT than that in 1 and 3 dpo (P< 0. 05). The contents of BDNF in the injuried spinal cords at 28 dpo were increased than that in the sham operation group (P < 0.05). CONCLUSION: Neuroplasticity has occurred in rats subjected to SCT, and the mechanism may be involved in the increase of BDNF expression in the spinal cord with the time going.

[Expression changes of BDNF in lung of rats subjected to brain ischemia].

OBJECTIVE: To investigate the expression of brain-derived neurotrophic factor (BDNF) in lung injury induced by brain ischemia in rats. METHODS: 46 adult SD rats were assigned randomly to sham operation group and brain ischemia lung injury group (BILI, n = 23 in each group). Rats were subjected brain ischemia and allowed to survived 3 d. After performed neurological functional severe deficit evaluation, lung edema was observed (n=5). The BDNF expression for its mRNA and protein in lung tissues was determined by using ELISA (n=5) and RT-PCR technique (n=8). The localization of BDNF was also determined by immunohistochemistry (n=5). RESULTS: After brain ischemia for 3 days, the severe neurological functional deficit and edema in lung were seen. BDNF was located in cytoplasma of smooth muscle and epithial cells in the lung. The level of BDNF mRNA (indicated by RT-PCR) and the protein level (indicated by ELISA) were all upregulated at 3 days after brain ischemia (P<0.05). CONCLUSION: Lung edema occurred after brain ischemia in rats is concomitant with BDNF expression, which consists of the mechanism involved lung injury induced by brain ischemia.

[Expression changes of MEK in lung of rats subjected to brain ischemia].

OBJECTIVE: To explore the expression changes of mitogen extracellular kinase (MEK) in injured lung after brain ischemia in rats. METHODS: Adult SD rats were assigned randomly to sham operation group and brain ischemia lung injury (BILI) group. Rats in BILI group were subjected brain ischemia and allowed to survived 3 d. Pathalogical changes in lung were indicated by HE staining. The MEK expression was determined by RT-PCR and Western blot technique. RESULTS: After brain ischemia, the bulk of inflammatory cells invaded into lung were observed. Upregulated level of MEK mRNA and protein were found at 3 days after ischemia (P<0.05). CONCLUSION: The upregulated expression of MEK implied that the MEK may play some roles in lung injury after brain ischemia.

[Expression of TNF-alpha in lung tissue of rats with lung injury induced by brain ischemia].

OBJECTIVE: To investigate the expression of tumor necrosis factor-alpha (TNF-alpha) in the lung tissue of rats with lung injury induced by brain ischemia. METHODS: The rat model of lung injury induced by brain ischemia was established. At 24 h, 48 h, 72 h after brain ischemia, lung tissues were harvested from each rats, the expressions of TNF-alpha mRNA and protein and its distributions in the lung tissue were measured by the methods of RT-PCR (n=8), Western blot (n=8), and immunohistochemistry (n=5), respectively. RESULTS: The increase of TNF-alpha mRNA and protein levels were found in the lung tissues after brain ischemia, when compared with sham group (P<0.05). The morphological study with immunohistochemical staining observed TNF-alpha positive reaction in epithelial cells and some macrophages in the lung tissues after brain ischemia. CONCLUSION: The expression of TNF-alpha in the lung tissue could be upregulated in the rats with brain ischemia.

[Expression changes of IL-1beta in lung of rats subjected to brain ischemia].

OBJECTIVE: To study the expression changes of interleukin-1beta (IL-1beta) in the lung of rats with brain ischemia. METHODS: Adult SD rats were divided into sham operation group and brain ischemia lung injury (BILI) group randomly. Focal cerebral ischemia inflammatory lung injury model was developed with intraluminal thread technique. Lungs were harvested from rats at different time point respectively. RT-PCR (24 h), Western blot (48 h) and immunohistochemistry (72 h) were employed to detect the expressional changes and the distributions of IL-1beta in the lung tissues. RESULTS: IL-1beta immunohistochemical positive reaction was observed in epithelia cell and neutrophil as well as macrophage. Increased protein level and mRNA expression for IL-1beta were found in lung after brain ischemia compared with those of sham group. CONCLUSION: IL-1beta, as a crucial inflammatory factor, could be associated with airway inflammation in lung following brain ischemia in rats.

[Effects of BDNF antibody block on IL-6 expression in lung of rats subjected to brain ischemia].

OBJECTIVE: To explore effect of brain-derived neurotrophic factor (BDNF) on IL-6 expression in the lung of rats with brain ischemia. METHODS: Inflammatory lung injury was induced by brain ischemia in rats that were devided into sham operation group, brain ischemia lung injury (BILI) group and brain ischemia with BDNF administration group. Lungs were harvested from rats in each group in 3 days after brain ischemia respectively. Immunohistochemistry, RT-PCR and Western blot were employed to detect the expressional changes on the mRNA and protein of IL-6 in the lung tissues. RESULTS: Expression of IL-6 was mainly found in cytoplasm of lung epithelial cell and macrophage with immunohistochemistry staining. A significant decrease of IL-6 expression was observed at 3 days after brain ischemia with BDNF antibody block. CONCLUSION: BDNF, as a crucial neurotrophic factor, could regulate IL-6 expression in rats lung tissues with brain ischemia.

[Effects of BDNF antibody to block IL-1beta expression and lung injury in rats subjected to brain ischemia].

OBJECTIVE: To explore the effects of brain-derived neurotrophic factor (BDNF) antibody to block lung injury and interleukin-1beta (IL-1beta) expression in the rats with brain ischemia. METHODS: Thirty nine SD rats were divided into sham group, brain ischemia lung injury (BILI) group, and BDNF antibody treated group. Inflammatory lung injury was induced by brain ischemia in the later two groups, and BDNF antibody was given through intraperitoneal injection to the rats for 3 days in BDNF antibody group. Lung tissue samples were harvested from the rats in each group at the 3rd day after brain ischemia. Lung edema degree was evaluated by HE staining. The protein expressions of IL-1beta in the lung tissues were measured by the methods of immunohistochemistry (n=5) and Western blot (n=8). RESULTS: The increased immunostaining of IL-1beta were found in the lung tissue at 3 days after brain ischemia, which indicated the upregulated expression of IL-1beta protein in the lung injury induced by cerebral ischemia. The block of BDNF antibody resulted in a significant decrease of IL-1beta expression, as well as the decrease of lung edema. CONCLUSION: BDNF, as a crucial factor, could regulate airway inflammation injury in brain ischemia rats via activating IL-1beta expression.

[Changes of BDNF expression in neurons in traumatic brain injury rats].

OBJECTIVE: To investigate changes of brain derived neurotrophic factor (BDNF) expression in neurons in traumatic brain injury (TBI) rats. METHODS: Adult SD rats were divided into sham and operated group resulted from hammer fall contusion (30 cm high, 50 g weight). Thirteen rats in each group were used. Some of animals (n = 6 in each group) were used to perform immunohistochemistry and in situ hybridization, and the other (n = 7) was used for RT-PCR. After NSS assessment was determined at 1, 3, 8, 13 days, TBI rats were sacrificed, brain tissues were then harvested to measure BDNF level. Data were analyzed by using statistic method. RESULTS: A increased NSS scores (P < 0.05) was observed after TBI, which implied the significant neurobehavioral changes in rats. But a gradual decreasing NSS scores (P < 0.05) was also observed along with time prolonged. Severe neurological severity function was seen following TBI, and it presents a gradual improvement indicated by decreasing NSS scores, despite BDNF mRNA level in whole brain tissue did not present a significant change, the BDNF expression (indicated by optical density values analysis) in subcellular structure, known as neurons, exhibited a significant increase, when compared with that of sham group (P < 0.05). This could simultaneously accompany with the decrease in the number of neurons in TBI rats. CONCLUSION: TBI rats exhibit a neuroplasticity with the BDNF upregulation in neurons following injury.

[Constrution of human single herpes virus carried BDNF gene vector and its bioactivity evaluation].

OBJECTIVE: To construct the recombinant vector of human single herpes virus (HSV) carried brain derived neurotrophic factor (BDNF) gene. METHODS: BDNF gene was acquired from rat brain tissue by RT-PCR, then was cloned into plasmid, and enveloped by HSV. The recombinant was used to transfer cultured cortical neurons. The number and neurite length of neurons were quantified. The BDNF level and subcellular localization were detected by ELISA and immunohistochemistry. RESULTS: HSV carried BDNF gene recombinant has been successfully constructed. The recombinant showed the bioactivity on the growth of cortical neurons. BDNF level was increased significantly in BDNF transferred group. CONCLUSION: HSV carried BDNF gene recombinant, with the bioactivity, has been successfully constructed. This could provide the vector for the treatment of BDNF under disease condition base on transferring gene technique.

[Experiment study on the treatment of SCI based on HSV carried BDNF transgene technique].

OBJECTIVE: To explore the effect of brain derived neurotrophic factor (BDNF) transgenic treatment in rats following spinal cord injury (SCI). METHODS: BDNF gene was cloned into plasmid then enveloped with human single herpes virus (HSV) to construct HSV carried BDNF transgenic recombinant. BDNF recombinant was injected into sciatic nerve to last label in motorneurous in the caudal cords, then ventral motor neurons were counted and the area of cell body was measured. The BBB scores representing motor function in hindlimbs was also recorded. RESULTS: Five days were needed for the GFP-HSV to arrive motorneurons from sciatic nerve. BDNF release could increase the number of motroneurons and inhibit neuronal atrophy in injured spinal cord. BDNF administration also improves motor function in hindlimbs. CONCLUSION: BDNF transgene carried by HSV is a useful strategy for the treatment of SCI, indicating its clinic implication in future treatment.

[IL-6 expression in lung of rats subjected to brain ischemia].

OBJECTIVE: To study the expression changes of interleukin-6 (IL-6) in the lung of rats with brain ischemia. METHODS: Adult SD rats were divided into sham operation group and brain ischemia lung injury (BILI) group randomly. Focal cerebral ischemia inflammatory lung injury model was developed with intraluminal thread technique. RT-PCR was applied to detect the IL-6 expression level of lung at 24 h after brain ischemia. The IL-6 protein in lung at 48 h after brain ischemia was detected with Western blot. Immunohistochemistry method was used to demonstrate the IL-6 protein location in lung at 72 h after brain ischemia. RESULTS: Increased immunostaining, protein level and mRNA expression for IL-6 were found in lung at investigated time point after brain ischemia. CONCLUSION: IL-6, as a crucial preinflammatory factor, could be related to airway inflammation in rats induced by brain ischemia.

[Regulation of BDNF expression in traumatic brain injury rats subjected single neucleated cell transplantation].

OBJECTIVE: To investigate the effect of single neucleated cells transplantation on the neurological function and brain derived neurotrophic factor (BDNF) expression in traumatic brain injury (TBI) rats. METHODS: TBI rats were established by hammer fall method from 30 cm height with 50 g weight. Single neucleated cells/ karyocytes from bone marrow were separated and prepared by ficoll medium, then transplanted into pericontusional tissues in the brain. Neurological function severity scores (NSS) were recorded at 1, 3, and 7 days post operation (dpo). The pericontusional tissues were harvested at 7 dpo to analyze the BDNF localization, expressional level. RESULTS: Single neucleated cell transplantation decreased NSS significantly, compared with TBI rats without cell implantation (P < 0.05). BDNF expression was upregulated and mainly found in neurons. CONCLUSION: The present study showed the single karyocytes transplantation could improve neurological function and the mechanism is possibly linked to the BDNF expression.