Study Of The Relationship Between Myodural Bridge And Cerebrospinal Fluid Circulation In Rats

The myodural bridge(MDB)is located in the suboccipital region,which is a dense tendinous fiber connecting the suboccipital musculature with the spinal dura mater.The MDB is not unique to the human body,but present in representatives of five different mammalian orders at least,that means MDB may be universal exist in mammals,suggesting MDB should have important physiological functions.At present,it is speculated that the functions of MDB are preventing infolding of the dura mater,transmitting proprioception,related to the occurrence of cervicogenic headache,and recently proposed a new hypothesis:MDB is involved in cerebrospinal fluid(CSF)circulation and provides the power for it.The presumption is based on the fact that the contraction of the suboccipital musculature during head movement will pull the dura mater through the MDB,changing the volume of the subarachnoid space and generating a local negative pressure,thus providing power for CSF circulation.Since then,this new hypothesis has also been supported by many related experiments and has gradually gained recognition.This can not help but be curious,since MDB is involved in CSF circulation,then first,when does MDB involved in?Since MDB provides the power for CSF circulation,then second,whether the power provided by MDB is important?There are no reports on the above questions at present,this study also try to answer it for the first time.The idea is:to answer the first question,we need to obtain the time when CSF circulation is established and the time when MDB occurs,then discuss the relationship between these two time.To answer the second question,after answering the first one,have already got the time when MDB involved in CSF circulation,we can intervene in function of the MDB in the early stage when it's involved in CSF circulation,and then test whether the CSF circulation is affected,whether the impact is "important".Since the CSF in CSF circulation is secreted mainly by the choroid plexus(CP),so in answering "when does MDB involved in CSF circulation",the time of establishment of CSF circulation can be indicated by the development time of CP.In addition,although there have been many reports on CP,there are relatively few systematic studies on the development process of CP,especially on the surface morphology.Therefore,the first part of this study systematically studied the development of CP,especially on the surface morphology in rats.Our team has also already completed the study on the development of MDB,and the time of occurrence of MDB in rats has been obtained.This study is the first time to speculate the time when MDB involved in CSF circulation by discussing the relationship between the time of establishment of CSF circulation and the time of occurrence of MDB in rats.Continue to answer "whether the power provided by MDB is important for CSF circulation",this requires to intervene in function of the MDB,but there is currently no relevant experimental methods or animal model establishment methods.In addition,it is absense of direct experimental evidence for that MDB should have important physiological functions although there are many indications.Therefore,the second part of this study attempts to intervene in function of the MDB in rats,and it's the first time to explore the establishment method for this animal model.It's also the first time through animal experiments proved that MDB has important physiological functions.In this way,it is possible to continue and thoroughly answer "whether the power provided by MDB is important for CSF circulation" by intervening in function of the MDB in the early stage when it's involved in CSF circulation,and then test whether the CSF circulation is affected.whether the impact is "important".Therefore,the third part of the study,an animal model that intervene in function of the MDB is established in the early stage when MDB involved in CSF circulation,and then detect whether the follow-up CSF circulation is affected,whether the impact is "important".It's also the first time through animal experiments proved that MDB provides the important power for CSF circulationPart I A developmental study of choroid plexus and speculate the time when myodural bridge is involved in CSF circulation in ratsObjective:Aimed to supplement the relative deficiencies in the study of surface morphology of CP during embryonic period and provide the morphological basis for the study of CP and its related structures by studying systematically on the development of CP,especially on the surface morphology in the lateral and ? ventricle in ratsMaterials and methods:E12?E20 SD rats,the number of each embryonic day was thirty,ten of them were subjected to tissue sectioning,HE staining,aquaporin-1(AQP-1)immunohistochemistry(IHC)and immunofluorescence(IF)staining of CP,together with the other five of them were subjected to semi-thin sectioning,toluidine blue staining to observe the developmental morphology and structural characteristics of CP at microscopic scale.The remaining fifteen of them were observed by scanning electron microscope(SEM)to observe the surface morphology of CP at ultramicroscopic scale.Results:The hindbrain,telencephalon and diencephalon CP appeared at E13,E14 and E16 respectively in rats,and their morphology of epithelial cells changed stereotyped with the development.Soon after the appearance of CP,the relative strong immunopositive reactivity of AQP-1 was observed in the epithelial cell apical membrane,and it was maintained during the embryonic period.AQP-1 was immunopositive in the distal epithelium of telencephalon CP and negative in the proximal.Shortly after the appearance of CP,there were abundant microvilli on its surface and sporadic secretory vesicles could be seen,and with the development,microvilli were enriched and the number of secretory vesicles was increased.The abundance of microvilli decreased in telencephalon and hindbrain CP from distal to proximal,and from dorsal midline to lateral respectively.Part II Explore the animal model establishment method of intervening in function of the myodural bridgeObjective:Aimed to explore the animal model establishment method of intervening in function of the MDB and provide a safe and effective method for establishing animal models for related functions study of the MDB by intervening in function of the MDB and observing the subsequent effects on rat behavior to test whether the intervention is effective.At the same time,in the process of trying to intervene in function of the MDB,animal experiments were conducted to verify whether MDB has important physiological functions.Materials and methods:Eight nests of P3 SD rats underwent surgical destruction of the structure of the postoccipital region.Eight E20 SD pregnant rats were surgically destroyed in the structure of the postoccipital region of the fetus,and made pregnant rats continue to gestate and give birth.Five nests of P21 SD rats were randomly divided into four groups on average:operation group,botulinum toxin(BOTOX)group,sham-operation group,and blank control group.Surgical destruction or injection of botulinum toxin type A in structures of the postoccipital region in operation group and BOTOX group were performed respectively,and statistical analysis was conducted on the behavioral difference of the rats in operation group and sham-operation group,BOTOX group and sham-operation group,sham-operation group and blank control group within the same time after operation.Results:All the P3 rats were killed and eaten by mother rats after operation.Eight E20 fetuses were born,and six were killed and eaten by mother rats.All the five nests of P21 rats survived.The difference was statistically significant that the number of times in head scratching,cage scratching and fro movement of the rats in operation group and BOTOX group during the third week,the fifth week and the seventh week after operation were greater than that of sham-operation group(P<0.05),while the behavioral difference between the sham-operation group and blank control group was not statistically significant(P>0.05).Part ? Effect of intervening in function of myodural bridge on cerebrospinal fluid circulation in ratsObjective:Aimed to verify whether the power provided by MDB is important for CSF circulation by establishing the animal model that intervene in function of the MDB in the early stage when MDB involved in CSF circulation and then detect whether the follow-up CSF of animal model is affected,whether the impact is "important".Materials and methods:Nine nests of P21 SD rats were randomly divided into four groups on average and corresponding dispose was conducted:surgery model group,drug model group,sham-operation group,and blank control group.Measure and analysis were conducted on the difference of epidural intracranial pressure in surgery model group and sham-operation group,drug model group and sham-operation group,sham-operation group and blank control group within the same time after operation in rats to reflect whether there was any difference in CSF pressure among these groups.Immediately after measure,perfusion fixed with brain,HE staining,AQP-1 IHC and IF staining,SEM and transmission electron microscope(TEM)were used to observe and analyze whether there was any difference in morphological function,cell structure and surface morphology of CP among these groups.Results:The difference was statistically significant that the epidural intracranial pressure in surgery model group and drug model group was lower than that in sham-operation group at the third,fifth weekend and seventh end of week after operation in rats(P<0.05),while the difference in this pressure between the sham-operation group and blank control group was not statistically significant(P>0.05).The height of microvilli,the number of secretory vesicles and crater-like appearance on the surface of CP epithelial cells in surgery model group and drug model group were higher or more than those in sham-operation group and blank control group respectively.Conclusions:1.CP is an evolutionarily conserved structure,the development of CP is spatially heterogeneous and has a certain secretory function in the early stage of development.2.The establishment time of CSF circulation equals to the early development of CP,which is roughly on E15,and the time when MDB involved in CSF circulation is roughly on P3 in rats.3.Surgical destruction or injection of botulinum toxin type A to the rectus capitis posterior minor and major can be used as the animal model establishment methods of intervening in function of the MDB by surgery or drug.Not only to rats,this establishment method may also have some applicability to establish other mammals related animal models.At the same time it's proved that MDB has important physiological functions4.It's proved that the MDB is indeed involved in CSF circulation,and MDB provides the important power for CSF circulation,providing a new idea for the diagnosis and treatment of clinically related diseases such as cervicogenic headache and unexplained chronic headache.