| Syringomyelia (SM) is a common disorder in which spinal cord gets a chronic regression lesion. Because more common disorders share the early symptoms of syringomyelia, it is very difficulty to find SM early. SM may be a result of Chiari I malformation, trauma, tumor or an inflammation. Its main clinical symptoms include paralysis, dissociated spinal nerve sensory loss, chronic ache and so on. Following the development of medical imageology, the incidence rate of syringomyelia increased year by year. The syringomyelia brought our families and society great loss because of its high disabling rate and no potent treatment for it. At present, the most therapies may not be completely successful over the long term. So they are very important to understand pathogenesis of SM more deeply and to explore the new prevention and treatment methods of it. On the pathogenesis of SM, experts of the world share the very different views, include myeloischemia mechanism, cerebrospinal fluid circulation disorder theory, cranio-spinal pressure dissociation theory and so on. Recently the pathologic changes of presyrinx in SM draw world experts a great attention, it was named as "Prophase of spinal cord cavitation"by Qingjun Zhang. At the same time, foreign experts call it "Presyrinx state". As it is an initial lesion of syringomyelia, the study on presyrinx will help us understand the pathological and physiological mechanism more clearly, give us chances to reverse this lesion and to interrupt cavitation by ultra-early intervention measures. In this study, experimental syringomyelia model in rabbits were established by intra-cisternal injection of kaolin, we try to characterized the formation and development mechanism of the through four parts deeply step by step. 1 Morphological and functional study on blood spinal barrier in experimental presyrinx state in rabbits The purpose of this part is to investigate morphological and functional changs in prophase of spinal cord cavitation and to discuss its machenisms through establishing experimental presyrinx state model. Fifty-six Chinese white rabbits, weighting from 1.5-2.0 kg, were used in this study, and divided randomly into three groups(n=8): kaolin group (40 rabbits), physiological saline group(8 rabbits), shamed-operation group (8 rabbits). Following McLaurin's method, Under ketamine anesthesia(30mg/kg), after 0.6 ml of cerebral spinal fluid (CSF) was withdrawn slowly from the cistern, 0.6ml of 25% kaolin solution (37℃) was injected into the cisterna magna of rabbits in kaolin group, while 0.6ml of 37℃physiological saline was injected in rabbits of physiological saline group, and neither Kaolin solution nor physiological saline was injected in shamed-operation group. At 1st, 3rd, 7th, 14th, 21th day after kaolin injection, the rabbits selected randomly from every group were killed after ketamine anesthesia, cervical cords were moved quickly and stored for specimens. Then water content of spinal cord was measured by dry-wet weigh technique, blood spinal cord barrier function was measured by Evansblue technique. Neurologic function was evaluated by Somatosensory Cortical Evoked Potential and Tarlov score. The results of study show: (1) Based on the modified Tarlov scores of animal neurologic function, the apparent neurologic impairment of Kaolin group animals appears at the seventh day after operation(2.5±0.36), moreover deteriorates gradually day by day. There is a significant different between Kaolin group and control group (Saline and shamed-operation groups) (p<0.05). the SCEP datas of rabbits in Kaolin group show that the latent period elongated and amplitude of wave depressed 3days after operation(42.2±0.12ms), moreover deteriorates gradually day by day. There is a significant different between Kaolin group and control group (p<0.05). (2) The result of water content assay has shown that edema of spinal cord appeared at the 1st day(68.35±0.7%)after kaolin injection, more prominent at 3rd day(72.70±0.88%), reached its cusp at 7-14th day (72.92±0.86%,72.18±0.55%), and declined slowly after 3 weeks. The manifest statistics discrepancy of edema degree was presented between Kaolin group points and shamed group(67.66±0.82%)at all time. (3) Evansblue content assay has shown that Evansblue content of spinal cord began to increase at the 3rd day greatly comparing with the control groups(2.79±0.42μg/ml). reached its cusp at 7 day(3.53±0.45μg/ml)(p<0.05)and lasted to the 21th day(3.36±0.27μg/ml). (4) In the H&E staining sections of shamed and saline groups as well as kaolin groups 1 day after injection, the contour and structure of neurons were normal, the nissl bodies distributed in plasm evenly, and the Virchow-Robin space was normal too. In the 3rd day, Virchow-Robin space began to widen, the upper cervical cord was slight edema; from 7th to 14th day the cord edema degree reached its peak, and in this period from tissue sections we can see the unclear nucleolus,nissl bodies decreasing,glial cell proliferation,well-stacked central canal,peri-canal constitution rarefac-tion,mass Inflammatory cell infiltration and so on. It has not taken a turn for the better until 21st day, and in this period the other pathological changes appeared, as vacuoles in cell plasm,the number of cell in posterior horn decreasing,expending central canal,ependyma epithelia rupture and kaolin granuloma forming. (5) Based on observation by electron microscope, ultrastructure changes of spinal cord presyrinx state include mainly ischemic nervous tissue edema and tissue space widening in 2 weeks after injection. the degree of these changes get worse gradually, then we can see swelling and even destruct mitochondrion, destroyed Nissl body, and the number of particles in Nissl body face decrease. But in this period, basement membrane of microvessel keeps smooth and integrity, the structure of tight junction is normal too. On the 21th day after injection, the lesions get so worse as to present demyelination characterized by brisement and fragmentation of lamellar texture of myelin sheath, and even axis-cylinder destroyed. The results of this part suggested, spinal cord presyrinx states is the earlier period of syringomylia. Just in this period, the neuroelectricitconductance function of spinal cord began to be destroyed more earlier than appearance of the clinical symptoms. On histology, the presyrinx state is characterized by spinal cord ischemic edema that is angioedema in earlier period mainly, accompanyes with Interstitial edema in late phase, and even appears nerve cell degeneration at last. Malfunction of blood-spinal barrier, permeability increasing play an important role in occurrence and development of spinal cord presyrinx states. 2 The study on expression of ZO-1 in experimental spinal cord presyrinx state in rabbits From the result of first part, we know that the function changes of blood spinal cord barrier, permiability increasing play an important role in formation and development of syringomyelia presyrinx state. In this part, through immunohistochemistry, Western-blot methods, we investigated the expression of ZO-1 at the different time points in the spinal cord of rabbits, moreover we try to find the relationship between its expression level and morphologic,functional changes of blood spinal cord barrier in experimantal presyrinx state. Fifty-six Chinese white rabbits, weighting from 1.5-2.0 kg, were divided randomly into three groups(n=8) , animal mode were made with the same methods as that of the first part. At 1st,3rd,7th,14th,21th day after kaolin injection, cervical cords were moved quickly and stored for specimens at -70℃after killing animal. After fixed by formalin and embedded by paraffin, the specimens were maken into paraffin section in 6 μm thickness. Immunohistochemistry staining was completed with goat anti-rabbit ZO-1 IgG(4μg/ml), the positive reaction was defined as buffy rough or buffy delicate particles distributing in cells. At the same time ZO-1 protein content of cervical cord was measured through Western blot. the concrete processes include extraction of plasm protein, quality valuating by separation gel and concintration gel, locating position of goal protein by mark, transmembrane, cellulose actate membrane coating, reacting with antibody, DAB staining, gel analysis and so on. Results of immunohistochemistry assay show , that the expression ofZO-1 in saline and shamed operation group is positive on neuron and astrocytic cells of cervicle gray, and great positive on microvessel wall and endothelial cell. In cervical cord of Kaolin group, at the first day post injection the immunoreactivity of ZO-1(38.66±4.78)have no manifest difference comparing with control group; At the 3rd day, it begin to decreas(e27.4±3.46); even at the 7th (19.23±2.13)and 14th day (17.62±3.17)there is an mild positive expression of ZO-1 on microvessel wall, neuron and glail cells; but at the 21th day (23.20±4.23)its immunoreactivity can restore partly. Basing on the statistics analysis, the gray degree and area of ZO-1 expression on cervical cord have no significant discrepancy between saline and shamed operation group(sF-test, p>0.05); comparing with saline group, the gray degree and area of ZO-1 expression have no significant discrepancy at first day, begin to weaken at 3,7,14,21th day and have an significant difference with control groups, especialy at the 14th and 21th day; it start to restore at 21th day but still lower than normal level. From the results of Western blot, we find that ZO-1 expression on cervical cord of Kaolin group have no significant discrepancy comparing with control groups at the first day post operation(249.34±14.8)( T-test, p>0.05). It decrease at the 3rd day(195.32±23.5), reach its minimum at the 7th(126.28±27.4)and 14th day(119.36±18.7)( F-test, p<0.05). it start to restore at 21th day but still lower than normal level. The linear regression analysis showed that expression of ZO-1 in cervical cord had a great negative correlation with the water content (r=-0.7288, p<0.01) and Evansblue content (r=-0.9502, p<0.01). The results above-mentioned demonstrated that, in animal modes of the experimental syringomyelia induced by intracisternal injection of kaolin in rabbits, expression level and patterns of ZO-1 protein of blood spinal cord have apparent relationship with that of spinal cord water content and Evansblue content. This data suggest that the change of ZO-1expression may play an important role in destruction of blood spinal cord barrier. It is one of the most important factors in pathologic generation and progress ofsyringomyelia presyrinx states. 3 The study on expression of VEGF in experimental spinal cord presyrinx state in rabbits In the third part, to understand the relationship of VEGF expression level and Morphologic change and malfunction of blood spinal cord barrier in experimental spinal cord presyrinx state of Rabbits. we estimated VEGF expression in spinal cord and CSF of each group animals on different time points through immunohistochemistry,ELISA and RT-PCR on protein and gene levels. Fifty-six Chinese white rabbits, weighting from 1.5-2.0 kg, were used in this study. Three groups(n=8) were divided randomly with the same methods as that of the first part. At 1st, 3rd, 7th, 14th, 21th day after kaolin injection, CSF was collected by cisterna acupuncture once more, and cervical cords were moved quickly and stored for specimens at -70℃from the rabbits selected randomly from every group. After fixed by formalin and embedded by paraffin, the specimens were maken into paraffin section in 6 μm thickness. Immunohistochemistry staining was completed with murine anti-rabbit VEGF IgG(4μg/ml), the positive reaction was defined as buffy rough or buffy delicate particles distributing in cells. Enzyme-linked immunoadsordent assay (ELISA) was finished for detecting VEGF content of CSF at each time-points in every groups, through VEGF-kit produced by DIACLONE company in American (concrete operation following the manual demand of kit ). RT-polymerase chain reaction(RT-PCR)was finished for detecting VEGFmRNA content of myelo-tissue of animal at each group, the concrete processes include extraction of total RNA, RNA quality evaluation, design and synthesis of primers, electrophoresis, gel analysis and so on. Results of immunohisto-chemistry assay shows that the charact-eristic of VEGF expression is chiefly cytoplasmic in neuron and astrocytic cells, also in pia mater or endothelial cell but the level of expression is lower comparatively; In shamed and saline groups, VEGF expression is weak in neuron and glialcell(95.11±2.11), and negtive in the wall of the pial and intramedullary vessels. In Kaolin group, at the first day post injection the staining degree in neuron and glail cells became stronger than control groups(112.14±1.87). During the 3th-14th day post cistern injection strong positive staining of VEGF is present in upper cervical cord tissue, mainly in neuron,astrocytic cells of grey and white matter and vascular endothelial (173.43±1.89 at 3d; 176.58±2.34 at 7d; 169.35±3.17 at 14d), the rough particles were observed to distribute in the plasm. Till the 21th day the degree of VEGF expression began to decreased(121.62±2.19), just the light positive expression appears in neuron and astrocytic cells. statistics analysis suggested that there is no significant difference of VEGF expression levels between the saline and shamed operation group(F-test p>0.05), and the VEGF expression levels in spinal cord tissue is higher in Kaolin group than in control groups(F-test p<0.05). By ELISA, no apperent difference of VEGF expression in CSF was shown in shamed group and contol group(0.401±2.29 pg/ml)and themselves control (F-test p>0.05). the VEGF expression in CSF was determined to be higher than itself control at the 7th day(9.493±2.99 pg/ml) and 14th day(8.280±1.76 pg/ml)of Kaolin group(test-T p<0.05). At the 1s(t0.093±1.40 pg/ml) and 3rd day(-0.823±2.54 pg/ml), VEGF expression in Kaolin group has no apperent difference. Results from RT-PCR shows that there are two major products of 314bp and 242bp in length, correspondence with VEGF189 and VEGF165 respectively. Based on the computer analysis of amplification bands, the expression content of VEGFmRNA in spinal cords began to increased at 1st day after operation (0.31±0.02), reach its peak at 7th day (0.66±0.02)and keep this level till to the 14th day (0.56±0.01), and then decrease after the 3rd week (0.35±0.04) (F-test, p<0.05). the linear regression analysis showed that the expression of VEGFmRNA in cervical cord had a manifest negtive correlation with it of ZO-1(r=-0.7741, p<0.05). Based on the results above-mentioned, in animal modes of theexperimental syringomyelia induced by intracisternal injection of kaolin in rabbits, we consider that VEGF expression was upregulated in cervical cord and CSF after Kaolin injection, its development tendency concides with that of upper cervical cord edema and severity of blood spinal cord barrier destruction. The higher level of VEGF in spinal cord and CSF may result in the permeability of blood spinal cord barrier increasing, and spinal cord edema forming. So it plays an important role in the formation and development of presyrinx state of syringomyelia. At the same time, the development tendency of VEGF and VEGFmRNA expression has a negative correlation with it of tight junction protein ZO-1 expression, this result suggest that VEGF may influence the function of blood spinal cord barrier through regulating the expression of ZO-1 in spinal cord of presyrinx state. 4 The study on influence of suramin, a VEGF antagonist, in experimental spinal cord presyrinx state in Rabbits In the fouth part, through observing the therapeutical effect of suramin, the antagonist of VEGF, on presyrinx state, discerning the function of VEGF in formation of presyrinx state, and investigating the relationship and regulation passage between VEGF expression and ZO-1, the effect mechanism of VEGF on presyrinx state of syringomyelia was discussed which will provide theoretical foundation for earlier diagnosis , treatment and interrupting cavitation formation. Fouty-eight Chinese rabbits, weighting from 1.5 to 2.0kg, were selected for models of presyrinx state that were made according to methods of the first part. Suramin(5mg/kg, twice per week)were injected into all of them after operation. and then the rabbits were selected randomly and killed at 1 st, 3 rd, 7th,14 th, 21th day after measuring the function of blood-spinal cord barrier(n=8). Neurologic function of rabbits receiving suramin is classified by late experimental ways like late parts,based on the modified Tarlov scores; Quantitative analysis on destructed barriers of blood-spinal cord is made by Evansblue method; water content of cervical marrow in Suramin group ismeasured by dry-wet weight technique. The change of cervical marrow pathology and ultrastructure can be observed by microscope and electroscope; expressive content on cervical marrow of VEGF and VEGFmRNA in Suramin group can be measured by immuohistiochemistry and RT-PCR,and meanwhile the protein content of VEGF in Suramin group can be determined by Western blot. The neurologic impairment of rabbits in suramin group is relieved more apparently than in Kaolin group, There is a significant difference between Suramin group and Kaolin group at each different time point (p<0.05, F-test). Meanwhile a significant difference can also be found between suramin group and control groups. Through microscope, in suramin group the widen Virchow-Robin space and slight edema began to observed in upper cervical cord at the day3 after operation, and they reached their cusp at day14. The degree of these lesions in Suramin group was relieved more evidently than that in Kaolin group, and time course of fastigium was postpnoned. At the same time, the decrease of Nissl body and astrocytosis in cervical marrow were obviously relieved in Suramin group. Observing by electron microscope indicates that the lesions degree of nervous tissue due to ischemia were relieved in Suramin group more apparently than that in Kaolin group, these lesions including tissue edema, cell space widening, swelling and even destructed mitochondrion and destroyed Nissl body. Moreover in anaphase, the lesion presenting demyelination characterized by brisement and fragmentation of lamellar texture of myelin sheath, and even axis-cylinder destroyed was relieved in Suramin group than that in Kaolin group. The result of dry-wet assay has shown that the water content of cervical marrow in Suramin group was 67.66±0.816% at the day1 after operation, was 68.79±0.324% at day3, 69.27±0.226% at day7, 68.53±0.243% at day21, which was strikingly decreased at each time point comparing with Kaolin group; but the water content was higher than nomal control and shamed-operation group(p<0.05, F-test). Quantitative analysis on destructive degree of blood-spinal cord barriers in Suramin group: EB content of cervical marrow was evidently decreased at each time point comparing with Kaolin group (p<0.05, F-test), but the tendency of EB content change was accorded to that in Kaolin group; Meanwhile comparing with shamed-operation group, EB content of cervical marrow was no significant difference at day1 after operation, and it was still higher than control group at the other time points (p<0.05, F-test). The result of VEGFmRNA expressive in cervical marrow has shown that ,comparing with Kaolin group, VEGFmRNA in Suramin group had no difference at day1 after operation (F-test p>0.05), began to decrease obviously at day3, and lasted till day21 (F-test p<0.05). the results by immuno-histiochemistry indicated that the time pattern of VEGF expressive in cervical marrow of Suramin group was delayed and shortened, and the level of VEGF was lower (F-test p<0.05). Comparing with shamed-operation group, there was no significant expressive change of VEGF at 1st and 3rd day, higher than normal at 7th and 14th day, and became normal at day21. Comparing with Kaolin group, the result of immuohistiochemistry and Western blot had demonstrated that the expressive content of ZO-1 in cervical marrow was apparently increased at each time point in suramin group. Comparing with normal control there was no significant differernce at 1st, 2nd, and 3rd day (F-test p>0.05), except 7th and 14th day (F-test p<0.05). It has been pointed by the above-mentioned experimental results that Suramin, as the antagonist of VEGF, plays an important role in the prevention and treatment of presyrinx state of syringomyelia; this result also confirmed that the high express of VEGF had an important effect on the devolpment of presyrinx state; the express of VEGF and ZO-1 had intimately relativity in experimental presyrinx state in rabbits, and the structural and functional interity could be affected by the expressive regulation of ZO-1 through VEGF.
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