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Chinese patent medicines Shuxuetong on the treatment of spinal cord injury spinal pinch preliminary study the morphological changes of astrocytes and astrocytes increased sources

Author: JiaLiYun
Tutor: JuGong;ShenXueFeng;WangJian
School: Fourth Military Medical University
Course: Neurobiology
Keywords: Shu-Xue-Tong Spinal cord injury Tannic acid-Ferric chloride Secondary injury SCBF ischemia neuron Astrocytes Glia scar Migration NG2 Crushed spinal cord injury
CLC: R277.7
Type: PhD thesis
Year: 2010
Downloads: 67
Quote: 0
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Spinal cord injury (SCI) could result in the rupture of blood vessels, hemorrhage, extrusion of inflammatory cells, edema, thrombosis, spasm of blood vessels and so on. All those of factors will lead to the decrease of spinal cord blood flow (SCBF), which will cause to ischemia finally. Then ischemia could irritate the occurrence of secondary SCI, which is related to the recovery of motor function. It has been suggested that the degree of ischemia is correlated with the degree of secondary SCI in significant extent. Therefore, it has become a hot point about how to overcome the ischemia after SCI.Shu-Xue-Tong (SXT) is a traditional Chinese drug, the main components of which are hirudin and lumbrukinase. Both of these two components have a strong effect on improving SCBF. Therefore, SXT is mainly used to counteract stagnation of blood flow. It has been widely used to treat ischemic disease, such as brain or myocardial infarction. It has been proved that the SXT has a good therapeutic effect on these diseases, and no side effect is found. Base on these facts, we hypothesized that there was a therapeutic effect of SXT on SCI, which was mainly through the improvement of SCBF. So we designed the following experiments to investigate this possibility.Experiment 1, tannic acid ferric chloride (Ta-Fe) was applied to stain blood vessels, and this method is compatible with HE staining. Therefore, we could detect blood vessels and the histopathological change of spinal cord simultaneously under the brightfield. Results indicated that there were two ischemic zones beside the injured epicenter. One was next to the injured epicenter. Ischemia was so severe that neurons in this area had been degenerated. The other one was adjacent to the first area and had some distance to the injured epicenter. Ischemia in this area was relatively moderate than that in the first area, and neurons in this area still existed. This area could be rescued by some medication therapy.Experiment 2, SCI was induced by compression. SXT was administrated by i.p. injection from 24 h post-injury. Saline treatment served as the control. We observed the therapeutic effects of SXT on SCI. The results were as follows: 1. Laser-Doppler flowmetry (LDF) indicated that SXT significantly promoted SCBF after SCI. When being treated by SXT, SCBF was increase from 57±6.43% at 0.5 h post-injury to78±4.16% at 3 d post-injury and to 84±2.20% at 7 d post-injury. There were significant differences among them (P < 0.05). However, the change of SCBF for saline group was not significant during the whole process (58±6.20% at 0.5 h, 65±3.81% at 3 d and 61±6.68% at 7 d post-injury, P > 0.05 among them).2. Tarlov?s motor score showed that SXT could increase the score of motor function after SCI. Immediately after injury, rats in both injured groups showed complete paraplegia (scored 0). However, significant difference appeared at 7 d post-injury. SXT group recovered considerable bilateral motor function with a score of 2.83±0.41, allowing occasional body weight support, while the saline group only showed extensive movement of hip or knee with no stepping and a score of 1.62±0.52 (P < 0.01, compared with SXT group). By 14 d post-injury, the SXT group showed a near hind limbs and forelimbs coordination (3.83±0.41). In contrast, the saline group showed a score of 2.67±0.52 (P < 0.01, compared with SXT group) with only occasional weight support but no stepping.3. Footprint analysis indicated that the foot stepping could be improved by SXT administration. Toe dragging was serious, and no stepping was observed in saline group at 7 d post-injury. Conversely, SXT treated group showed plantar stepping at this time point. When being at 14 d post-injury, although plantar stepping appeared, the toe dragging was still seriously in saline group. For SXT group, the stepping was recovered continuously, and toe dragging was nearly disappeared. Statistical analysis indicated that compared with saline group, toe dragging was markedly decreased, stride length was increased and stride width was decreased significantly (P < 0.01) at these two time points.4. Both HE and GFAP staining showed that the injured area at 14 d post-injury was decreased by SXT treatment. The injured area in SXT group was 2.67±0.36 mm2. It was markedly smaller than that for saline group (4.01±0.81 mm2 P < 0.01). Besides, thionin-stained sections demonstrated that compared with the saline-treated group, there were more neurons and less inflammatory cells adjacent to the lesion site in SXT group.5. NeuN immunostaining indicated that SXT could rescue more neurons. There were more neurons (56±10.79) adjacent to the injured site in SXT treated group. When being compared with saline group (37.2±9.60), there was significant difference (P < 0.05).All these mentioned above showed that SCI could counteracted this ischemic condition by improve blood flow, and then neurons was rescued and motor function was ameliorated. Based on these results, we suggest that SXT could be a new medical choice for the treatment of SCI. Astrocytes are a kind of multifunctional cells in the CNS. They play many important roles in the CNS, including BBB and BSCB formation, homostasis, extracellular matrix production, and neurotransmiter modulation. Importantly, they could respond to any kind of insults, taking part in the recovery and remodeling of CNS. During this process, they will undergo the hypertrophy, upregulation of intermediate filaments (GFAP & vimentin), processes extension, proliferation, migration, and glia scar formation.After traumatic SCI, astrocytes could be irritated and play some beneficial or detrimental roles. There are many researches on the morphological changes of astrocytes after SCI, such as hypertrophy, processes extension, GFAP upregulation and so on. However, most of these researches depended on the GFAP immunostaining. During our research, we found that because of the tiny astrocytic cell bodies, the observation of astrocytic cell bodies was influenced by GFAP fiber-like immunostaining. In order to resolve this problem, we utilized the GFAP::GFP transgenic mice. Green fluorescent protein (GFP) was expressed in transgenic mice under the control of the GFAP promoter in this kind of transgenic mice. Astrocytes in these mice could express GFP in their cell bodies and processes. Therefore, both the cell bodies and the processes of astrocytes could be distinguished under the flurorescent microscopy. This kind of transgenic mice brought us a lot convenience for us to observe the morphological changes of astrocytes after crushed SCI. Furthermore, we also investigated the origin of the increased astrocytes through this kind of transgenic mice. The results were as follows:1. In the white matter of uninjured spinal cord, the cell bodies were round or triangular. Besides, several long processes protruded from the cell body and arranged radially. In the gray matter, the astrocytes with round cell bodies and many radially arranged shorter (compared with the white matter astrocytes) processes could be distinguished clearly. Because of the relatively large number of astrocytes in the gray matter when being compared with that in the white matter, we chose gray matter astrocytes as our target to study in the following SCI experiments. All the results were derived from the observation of the saggital sections. The mainly observed areas were the injured epicenter and transition area which between the injured area and the uninjured area.2. Compared with the astrocytes in the uninjured spinal cord, there was no obvious morphological change for the cells in the transition area at 3 d post-injury. In the injured epicenter, astrocytes were disappeared.3. At 7 d post-injury, the cells in the transition area had some morphological changes: cell bodies were elongated, and processes were extended and arranged rostrally and caudally. Glia scar was not formed in the injured epicenter at this time point.4. At 14 d post-injury, the morphology of astrocytes was similar to that at 7 d post-injury in the transition area. Glia scar began to appear in the injured epicenter at this time point.5. At 21 d post-injury, several astrocytes with elongated cell bodies and polarized processes could still be seen in the transition area. The glia scar was more obvious than that at 14 d post-injury.6. At 28 d post-injury, elongated cell with polarized processes in the transition area were decreased, and the cells with round cell bodies and radially arranged processes were increased. Besides, the glia scar was more significant than any other observed time point. The astrocytes in the glia scar had round cell bodies and tangled processes.7. During the whole process, astrocytes confined the injured epicenter from the rostral and caudal directions, and the injured area was decreased gradually.8. There were no NG2 and GFP double labeling cells in the uninjured spinal cord. However, the NG2 and GFP double labeling cells appeared in the transition area at 7 d post-injury.

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