A Study On Aquaporin-4 Expression In Presyrinx State And Therapeutic Effect Of Methylprednisolone On Rabbit Model

Syringomyelia, a common chronical degenerative disease inneurosurgery with the character of liquid porosis and gliosis, onsetsdelitescently and has a slow progress. The clinical manifestation ofsyringomyelia includes dissociated sensory disorder at different levels,dysfunction of lower motoneurons, dysfunction and dystrophy of long tract,loss of work capacity or viability due to refractory pain and dysfunction ofactivity. At present, the causes of this disease are not clear and drugtreatment is not effective. Although some cases may be relieved partly bydecompression of posterior cranial fossa or/and cavernostomy, it is stilltroublesome to cure it. Recently, the pathologic changes before syrinxformation has drawn worldwide experts a great attention. This phenomenonwas defined as prophase of spinal cord cavitation by internal scholars, whichwas consistent with presyrinx state as recently called by overseas researchers.The study on presyrinx will help us understand the pathological andphysiological mechanism more clearly, give us chances to reverse this lesionand to interrupt cavitation by early intervention measures.It is well known that aquaporin-family plays an important role in watertransmembrane transport in biological organism. Recent reseaches confirmthat aquaporin-4(AQP4) is the more abundant in the central nervous system(CNS) and it not only involves in water transport and regulation on water,electrolyte and osmotic pressure balance of brain and spinal cord in normalphysiological state, but also in CNS edema originated from different causessuch as trauma, ischemia, hemorrhage, inflammation and tumors. Thusfore,this study, by employing molecular biological techniques, is to investigateAQP4 expression in the development of presyrinx state, to approach PKCregulation on AQP4 expression and to observe the influence of MP on AQP4 expression and edema in presyrinx state. All of the study was divided into foursections.1 AQP4 expression and distribution in spinal cord of normal rabbitsThe purpose of this section is to investigate AQP4 expression anddistribution in normal spinal cord of New Zealand white rabbits, to speculateits possible functions, and to provide useful contrast data and theoricalevidences for regulation on water metabolism including the prevention andcure of spinal cord edema in presyrinx state.Eight New Zealand white rabbits, weighting from 1.5-2.0 kg, were usedin this study. After ketamine anesthesia, the animals were killed bydecapitation and spinal cords were quickly removed to be divided into threesegments, each of which was subdivided into three parts for the use ofimmunohistochemistry, in-situ hybridization and western blot so as to studyAQP4 expression and distribution in spinal cord.The distribution of the AQP4 immunoreactivity was comparable in bothspecies and in all animals examined. In the gray matter, AQP4 stainingappeared diffuse in the neuropil, leaving unstained the neuronal somata. Theintensity of labeling was maximum in laminaeⅠandⅡof the dorsal horn anddecreased to a moderate level, with a dorsal to ventral gradient, in theintermediate gray matter and in the ventral horn, all of those indicating thelaminae different distribution. In the white matter, AQP4 expression wasintense in astrocytic processes which were either radially oriented, or formedthe glia limitans along the outer border of the spinal cord or surrounded bloodvessels. In sum, the positive distribution of the AQP4 immunoreactivity wasmainly concentrated, in grey matter, in membrane of glial cells, neuronsconcluding motor neurons in anterior horns, and ependymocytes lining centralcanal, in white mater, in membrane of fibrous astrocytes and endothelial cellsin spinal pia mater, especially in gliocyte membrane facing subarachnoidspace and capillary endothelial cells.In-situ hybridization demonstrated that intracytoplasm not nucleus ofneurons and glial cells showed up hybridization signal, of which, the strong positive signal was mainly concentrated in ependymocytes lining central canaland glial cells in the posterior horn and white matter. Western blot revealedthat AQP4 antibody had detected a strong protein band in the crude membranefractions of spinal cord. Analysis of integral optical density (IOD) indicatedthat AQP4 content in spinal cord was more abundant than that in brain.Drawn from above results, AQP4 expression is widely distributed overthe grey and white matter comprising of gliocytes, neurons in anterior horns,ependymocytes and epithelial cell of spinal pia mater, but its staining ischiefly confined to the membrane of those cells. What is needed to emphasizeis special polarity of AQP4 expression in astrocytic foot processessurrounding capillaries. These specialities of AQP4 expression anddistribution have a close relationship with water transmembrane transport andwater equilibration regulation in normal spinal cord of New Zealand whiterabbits. Compared with previous reseaches, AQP4 expression and distributionhave genus distinction between rabbit and rat.2 The study on expression and mechanism of AQP4 in experimentalpresyrinx state of spinal cord in rabbitsBased on the first section and with advantage of immunohisto- chemistry,Western-blot and RT-PCR methods, this section is to investigate theexpression of AQP4 and its mRNA at the different time points in the presyrinxstate of rabbits, and to approach mechnism of AQP4 in spinal cord edemafollowing the presyrinx state.Fifty-six New Zealand white rabbits, weighting from 1.5-2.0 kg, weredivided randomly into three groups(n=8). Animal models were built accordingto McLaurin methed, under ketamine anesthesia (30mg/kg), after 0.6 ml ofcerebral spinal fluid (CSF) was withdrawn slowly from the cistern, 0.6ml of37℃, 25% kaolin solution was injected into the cisterna magna of rabbits inkaolin group, while 0.6ml of 37℃physiological saline was injected in rabbitsof physiological saline group, and neither Kaolin solution nor physiologicalsaline was injected in shamed-operation group. At every day after operation,all the animals were scored according to modified Tarlov scores. At 1st,3rd, 7th,14th,21th day after kaolin injection in animals of kaolin group andimmediately after operation in those of physiological saline group andshamed-operation group, cervical cords were removed quickly through theposterior approach after decapitation, 4 mm of which was applied to determinewater content of spinal cord by dry-wet weight method, 2 mm of which toevaluate AQP4 mRNA expression by RT-PCR, 10 mm of which to evaluateAQP4 protein content by Western blot, and the remnant of which was makeninto paraffin sections in 4μm thickness for immunohistochemistry stainingand HE staining.Based on the modified Tarlov scores of animal neurologic function, inKaolin group animals, decreasing foodintake and depression occurred at the3rd day after operation and the apparent neurologic impairment such astorpidity, myasthenia of limbs and even quadriplegia appeared at the 7th day,moreover deteriorated gradually day by day. There is a significant differencebetween Kaolin group and control group (Saline and shamed-operation groups)(p＜0.05). Water content assay showed that spinal cord edema appeared at the1st day (68.35±0.7%) after kaolin injection, was more prominent at 3rd day(72.70±0.88%), reached its peak at 7-14th day (72.92±0.86%,72.18±0.55%), and declined slowly after 3 weeks. The manifest statisticsdiscrepancy of edema degree was presented between Kaolin group andshamed groupat all time points (p＜0.05)In the H&E staining sections of kaolin groups 3rd day after injection,Virchow-Robin space and space around neural cells began to widen, the uppercervical cord was slight edema; From 7th to 14th day, the cord edema degreereached its peak, and in this period from tissue sections we could see theunclear nucleolus, nissl bodies decreasing, glial cell proliferation, well-stackedcentral canal, peri-canal constitution rarefaction, mass inflammatory cellinfiltration and so on. It had not taken a turn for the better until 21 st day, andin this period the other pathological changes appeared, such as vacuoles in cellplasm, the number of cell in posterior horn decreasing, expending centralcanal, ependyma epithelia rupture and kaolin granuloma forming. In the H&E staining sections of shamed and saline groups, the contour and structure ofneurons were normal, the nissl bodies was evenly distributed in plasm, and theVirchow-Robin space was normal, too.Immunohistochemistry assay showed that the character of AQP4expression chiefly existed on membrane of neurons and astrocytic cells inupper cervical cord of Kaolin group. At the first day after injection AQP4staining degree in neurons and glail cells had no significance between kaolingroup and control groups (p＞0.05). From 3rd day after cistern injection inKaolin group, AQP4 expression in upper cervical cord tissue began todecrease (320.5±44.2), reached its mininum at 7-14th day(267.4±28.8,258.7±26.5), till the 21th day the degree of AQP4 expression began topartially restore(354.9±40.2). Also Western blot results manifestated there wasno significance of AQP4 protein content between kaolin group and controlgroups at the first day after operation(p＞0.05). It declined at the 3rdday(195.32±23.5), reached its minimum at the 7th(104.98±16.35)and 14thday(112.01±9.15) and started to partially restore at 21th day but still lowerthan normal level (p＞0.05). RT-PCR showed AQP4 mRNA was amplificatedto be a strap of 416bp in length. AQP4 mRNA tendency was similar to that ofAQP4 protein, decreasing at the 3rd day(0.83±0.05), reaching its minimum atthe 7th(0.17±0.09), starting to restore at 21th day(1.01±0.08).The linear regression analysis showed that the expression of AQP4 and itsmRNA in cervical cord had a manifest negtive correlation with water contentof spinal cord(r=-0.769, p＜0.01; r=-0.955, p＜0.01), respectively.The above-mentioned data demonstrated that AQP4 and its mRNAexpression were downregulated in development of the presyrinx state and hada great negative correlation with the water content in time course. All of theseresults implies the downregulation of AQP4 and its mRNA has played aimportant role in ischemic edema formation of spinal cord in the presyrinxstate.3 PKC regulation on AQP4 expression in experimental presyrinx state ofrabbits The relationship between PKC and brain edema has been payed closeattrention and recent reseaches indicated PKC and AQP4 play an importantrole in traumatic brain edema. The purpose of this section is to examine thechange of membrance and cytoplastic PKC activity by Substratphosphorolysis kinase assay and its regulation on AQP4 in the presyrinx state,further trying to elucidate the effects of PKC and AQP4 on the development ofthe presyrinx state.Fifty-six New Zealand white rabbits, weighting from 1.5-2.0 kg, weredivided randomly into three groups(n=8). Animals grouping, model buildingand scores of animal neurologic function were carried out according to thesecond section. At 1st, 3rd, 7th, 14th and 21th day after kaolin injection inanimals of kaolin group and immediately after operation in those ofphysiological saline group and shamed-operation group, cervical cords wereremoved quickly through the posterior approach after decapitation, 4 mm ofwhich was applied to determine water content of spinal cord by dry-wetweight method, 2 mm of which to evaluate AQP4mRNA expression by PCR,10 mm of which to evaluate AQP4 protein content by Western blot, 100mg ofwhich to evaluate membrance and cytoplastic PKC activity by Substratphosphorolysis kinase assay and the remnant of which was maken intoparaffin section in 4μm thickness for H&E staining.The data of animal neurologic function, spinal cord water content,pathological changes, the expression of AQP4 and its mRNA were in detailconsulted to the second section. Substrat phosphorolysis kinase assay showedthat in upper cervical cord of Kaolin group, membrane PKC activity increase70%(5.67±0.26pmol·min^-1·mg^-1 protein) at the first day of Kaolin injection,reached 4 times of normal level and arrived at its maximum at the 7th to 14thdays(12.14±2.08,13.27±3.15), then declined at the 21th day (8.85±1.56), thelater was still higher than normal level (p＜0.05). However, cytoplastic PKCactivity presented opposite tendency, declined at first day(12.35±1.67),lowered to 50% of normal level to its minimum at the 7th(6.48±1.24) to 14thday(5.95±1.74) and started to partially restore at 21th day (8.79±2.14) but still lower than normal level (p＞0.05). The linear regression analysis showed thatmembrane PKC activity in cervical cord had a manifest negtive correlationwith the expression of AQP4 and AQP4 mRNA (r=-0.769, p＜0.01; r=-0.955,p＜0.01), respectively.The above data show that membrane PKC activity of neural cells inupper spinal cord increase significantly and cytoplastic PKC activity decreasesignificantly, implying that PKC translocation activation has taken place indevelopment of presyrinx state. PKC translocation activation not only cutsdown the expression of AQP4 mRNA and AQP4 protein content, but alsomakes phosphorylation modification to AQP4 protein leading to the loweringof AQP4 activity. All of the above-mentioned effect of PKC and AQP4involve in ischemic edema formation of spinal cord in the presyrinx state.4 The study on influence of methylprednisolone on AQP4 expression andPKC activity in presyrinx state of rabbitsThe purpose of this section is to observe the influence ofmethylprednisolone(MP) on AQP4 and its mRNA expression, PKC activityand changes of water content in upper cervical cord, and to explore themolecular mechanism of the MP significant effective on remission of spinalcord edema in presyrinx state.90 new zealand rabbits, weighting from 1.5 to 2.0kg, were dividedrandomly into three groups: MP group, Model group and Control group. Themodel building was accomplished according to methods of the second section.In the following first to third day after Kaolin injection, animals in MP groupwere intravenously injected the large dose ofMP (30mg/Kg, 10 ml) by way ofear edge veins and in the 4th to 7th day after Kaolin injection the dose of10mg/kg MP was injected into ear edge veins. As to those animals in Modelgroup, only equal milliliters of the iso-osmia physiological saline wereadministrationed in the same way. Neither MP nor physiological saline wasinjected in Control group. Scores of animal neurologic function were carriedout according to the second section. At 1st, 3rd, 7th, 14th and 21th day afterkaolin injection in three groups, cervical cords were removed quickly through the posterior approach after decapitation and the next steps was the same asthat of the third section.The neurologic impairment of rabbits in MP group is relieved moreapparently than that in model group. There is a significant difference betweenMP group and Model group at each different time point (p＜0.05). Meanwhile asignificant difference can also be found between MP group and controlgroup.Water content assay showed that the water content in cervical cord ofMP group was 67.41±0.90% at the 1st day after operation, was 68.66±0.37% at3rd day, 69.00±0.70% at 7th day, 68.38±0.28%% at 21th day, which wasstrikingly declined at each time point comparing with that of Model group,but the water content was higher than that of Control group(p＜0.05).Microscopic observation found that slight edematous manifestation of thewiden space around neural cells and Virchow-Robin space occurred in uppercervical cord at the 7th day after operation. The fastigium of spinal cord waspostpnoned to the 14th day and till the 21th day the cervical edema waslessened dramatically. Also, The degree of these lesions at above three points inMP group was relieved more evidently than that in Model group. At the sametime, the decrease of Nissl body and astrocytosis in cervical cord wereobviously relieved in MP group.Comparing with Model group, the result of immuohistiochemistry andWestern blot had demonstrated that the AQP4 expressive content in uppercervical cord was significately upregulated at each time point in MPgroup(p＜0.05), but maintained the tendency of reaching the minimum at the7th and 14th days and restoring partially at the 21th day. Comparing withControl group, the AQP4 protein increased at the 1st, 3rd and 21th day, butwas lower at the 7th and 21th day. RT-PCR assay indicated that, in MP group,AQP4 mRNA expression in upper cervical cord began to upregulate from thefirst day and lasted to the 21th day, especially in which were predominant atthe 7th and 14th days(p＜0.05). Comparing with Control group, AQP4 mRNAexpression increased strikingly at the 1rd, 3rd days, dropped the perigee at the7th and 14th days and then recovered at the 21th day. Substrat phosphorolysis kinase assay demonstrated that, at every timepoint in MP group, membrane and cytoplastic PKC activity in neural cells ofupper cervical cord manifestively declined, among which PKC activity at the7th and 14th days were significantly lower, even was cut down 50%(p＜0.05).All experimental results support that MP has significant preventive andtherapeutic effect on the devolpment of presyrinx state. AQP4 upregulationmediated by MP in gene and protein level accompanied with indirect raise ofAQP4 activity by inhibiting membrane PKC activation give advantage towater transport from gial cells and extracellular space to lumen of blood vesseland subarachnoid space across blood brain barrier, and enhance spinal cordedema to dissipate.Conclusions1 This paper, for the first time, studied AQP4 location and expression inspinal cord of new zealand rabbits, whose anatomical distribution has aclose relationship with water transmembrane transport and waterequilibration regulation. Compared with previous reseaches, AQP4expression and distribution have genus distinction between rabbit and rat.2 This paper, for the first time, examined expression of AQP4 and its mRNAon the development of the experimental presyrinx state in rabbits, implyingthat downregulation of AQP4 and its mRNA have played an important rolein ischemic edema formation of spinal cord in the presyrinx state.3 PKC translocation activation has taken place in development of presyrinxstate. PKC translocation activation not only cut down the expression ofAQP4 mRNA and AQP4 protein content, but also make phosphorylationmodification to AQP4 protein leading to the lowering of AQP4 activity. Allof the above-mentioned involve in ischemic edema formation of spinal cordin the presyrinx state.4 AQP4 upregulation mediated by MP in gene and protein level, accompaniedwith indirect raise of AQP4 activity by inhibiting membrane PKC activation, gives advantage to water transport from gial cells and extracellular space tolumen of blood vessel and subarachnoid space across blood brain barrier,and to enhance spinal cord to dissipate. Through above mechanisms, MPhas brought into full play of its significant preventive and therapeutic effecton the devolpment of presyrinx state.