The universal existence of myodural bridge in mammals: an indication of a necessary function

The “myodural bridge” was described in literatures as a dense fibrous tissue connecting the sub-occipital musculature with the spinal dura mater in human studies. Now the concept of “myodural bridge” was perceived as an exact anatomical structure presumably essential for critical physiological functions in human body, and might exist in other mammals as well. To determine the existence of the “myodural bridge” in other mammals and to lay a foundation for the functional study, we examined representatives in five different mammalian orders. Based on the anatomical dissections, P45 plastinated sections and histological sections, we found that a dense fibrous tissue connected the rectus capitisdorsalis minor and the spinal dura mater through the dorsal atlanto-occipital interspace with or without the medium of the posterior atlanto-occipital membrane. These observed connective tissues were very similar to the “myodural bridge” previously described in humans. We proposed that the “myodural bridge”, as an evolutionally conserved structure, presents in many other mammals. Moreover, we believed that the “myodural bridge” might be a homologous organ in mammals. Thus, this study could provide an insight for our understanding the physiological significance of the “myodural bridge”, especially in human.

SCIenTIfIC REPORTS | 7: 8248 | DOI: 10.1038/s41598-017-06863-z C1 and inserted into the occipital bone ( Fig. 2A-F). The OCA and OCP were laterally to the RCDma and RCDmi. The former extended from the occipital bone to the transverse process of C1 and the latter from the transverse process to the spinous process of the C2. Different from that of the above-mentioned mammals, the post-occipital muscles of Indoasian finless porpoise, were found to be composed of RCDma, RCDmi and the lateral rectus capitis dorsal (LRCD) (Figs 1G and 2G). The RCDma and LRCD were extended from the fused spinous process of first three cervical vertebrae to the occipital bone. These two muscles were nearly parallel in arrangement in the post-occipital region of the Indoasian finless porpoise (Fig. 1G). While the RCDmi was originated from the occipital bone and extended into the posterior atlanto-occipital interspace under the RCDma (Fig. 2G). In addition, no oblique muscles were found in the post-occipital muscle group of the Indoasian finless porpoise.
As a result, the RCDmi presents in the five different kind of mammalian orders confirmed by its origin and insertion (Fig. 2).
The connections between the RCDmi and the spinal dura mater with or without intermediary of the PAO membrane were determined based upon the gross dissection. In Macacamulatta, Oryctolaguscuniculus, Canisfamiliaris, Feliscatus, Ratusnorvegicus, Caviaporcellus, multiple dense fibrous bundles were discovered to connect the RCDmi with the PAO membrane (Fig. 3). And the PAO membrane was found to tightly adhere to the posterior wall of the cervical spinal dura sac with some connective tissue (Fig. 4). As a result, the MDB was formed by complex connections between the RCDmi and the spinal dura mater. However, in Indoasian finless porpoise, the RCDmi was found inserting into the posterior atlanto-occipital interspace directly and no PAO membrane appearing in this interspace (Fig. 5A,B).
The connections between the RCDmi and the spinal dura mater with or without intermediary of the PAO membrane determined based upon the P45 plastinated sheets of the head and neck. In the sagittal sections of head and neck of Macacamulatta and Canisfamiliaris, the RCDmi was showed extending from the posterior arch of the atlas to the occipital bone. The P45 plastinated sheet of Canisfamiliarisand Macacamulatta shows that a bundle of fibers of RCDmi pierce into the PAO membrane, and finally connect with the spinal dura mater (Fig. 6A,B). While, in Indoasian finless porpoise, the RCDmi was found to originate from the occipital bone and run ventrally through the posterior atlanto-occipital interspace then insert into the spinal dura mater directly and no PAO membrane appears at above-mentioned interspaces (Fig. 5B,C).
Dense fibrous connections between the RCDmi and the spinal dura mater were showed by HE staining. In the sagittal section of the post-occipital region of Feliscatus, Oryctolaguscuniculus, Ratusnorvegicus and Caviaporcellus, the ventral muscular fibers of the RCDmi were discovered to give off dense fibrous bundles and some of them connect with the PAO membrane and others pass through the PAO membrane and insert into the spinal dura mater (Fig. 7). Consequently integration of the RCDmi, the PAO membrane and the spinal dura mater was formed via the dense fibrous connections, logically termed as the MDB.

Discussion
The sub-occipital region is one of the most complex anatomical regions in the human body. The MDB was described in lots of literatures as a dense fibrous tissue connecting the sub-occipital musculature with the spinal dura mater through the posterior atlanto-occipital and atlanto-axial interspaces in this region. In 1995, Hack revealed a dense band of tissue connecting the RCPmi and PAO membrane, and then the latter was intimately attached to the outer surface of the dura mater by a fine connective tissue bridge, especially near the midline 1 . Thus, the connection between the RCPmi and PAO membrane eventually connected with the dura mater was referred as the MDB by Hack et al. 1 . Subsequent investigations showed that this connective tissue also existed between the RCPma, OCI, nuchal ligament and the spinal dura mater [2][3][4][5][6][7][8][9][10][11][12] .
As the MDB was found as a normal anatomical structure in human being, it still remained unknown whether this structure universally present in other mammals., In this study, representatives of mammalian orders were examined, including tree-dwelling primates (Macacamulatta), land-based carnivores (Canisfamiliaris and Feliscatus), Cave-dwelling lagomorpha (Oryctolaguscuniculus) and rodentia (Ratusnorvegicus and Caviaporcellus), and aquatic cetaceans (Indoasian finless porpoise). Firstly, we found that the post-occipital muscle group consistently appears in the post-occipital region of these mammals. It consists of the RCDma, RCDmi, OCA and OCP in Macacamulatta, Canisfamiliaris, Feliscatus, Oryctolaguscuniculus, Ratusnorvegicus and Caviaporcellus, which are in agreement with previous results [14][15][16] . And slightly different from above-mentioned mammals, in Indoasian finless porpoise, the post-occipital muscle group is composed of the RCDma, RCDmi, and LRCD. This might be due to the fusion of its first three cervical vertebrae, which could make its body more suitable for underwater movement 17,18 . Among the post-occipital muscles, the RCDmi was observed intimately adjacent to the posterior atlanto-occipital interspace and extending from the posterior arch of the atlas to the occipital bone in Macacamulatta, Canisfamiliaris, Feliscatus, Oryctolaguscuniculus, Ratusnorvegicusand Caviaporcellus, while in Indoasian finless porpoise, the RCDmi was found extending from the occipital bone into the posterior atlanto-occipital interspace. As a whole, the formation of the post-occipital muscles of these mammals was similar to that of the sub-occipital muscles of human being.
Based on the results of gross dissections and P45 plastinated sheets and HE staining of histological sections, our study shows that the integration of the RCDmi, the PAO membrane and the posterior wall of the cervical spinal dura sac is formed via multiple dense fibrous bundles, which originate from the ventral aspect of the RCDmi and fuse with the PAO membrane and some of them pass through the PAO membrane and insert into the spinal dura mater in Macacamulatta, Canisfamiliaris, Feliscatus, Oryctolaguscuniculus, Ratusnorvegicus and Caviaporcellus. As a consequence, the existence of the MDB will be determined in these mammals according to the anatomical features of MDB defined in the human being 1 . In Indoasian finless porpoise, the MDB was a kind of direct connection because the RCDmi directly enters into the epidural space through the posterior atlanto-occpitial interspace and then inserts into the spinal dura mater without the PAO membrane relaying. The MDB strengthened and occupied the opened posterior atlanto-occpitial interspace in Indoasian finless porpoise might be a result of a limited movements of head and neck caused by the fusion of the first three cervical vertebrae. Thus we concluded that the MDB present in the mammals of the five mammalian orders observed in our study, although they live in different environments. Although different from upright walk style of human 19 , the MDB appears in a marine mammal (Indoasian finless porpoise) and terrestrial quadrupeds (Macacamulatta, Canisfamiliaris, Feliscatus, Oryctolaguscuniculus, Ratusnorvegicus and Caviaporcellus) in the deepest layer of the napex. And, consequently, it was speculated that the MDB would be a universal existing, normal anatomical structure in mammals.
The morphological structural changes are thought to be functional adaptations 20,21 . Now we provide anatomical evidence to support the notion that the MDB found in humans 1-12 also presents in multiple mammals in our study, its function might be important and necessary for both human and other mammals. Previous studies suggested that the "myodural bridge" would fulfill several major functions, such as preventing in-folding of the dura mater during head extension 1 , which may act to trigger cervical neck extensors and resist hyperflexion or hypertranslation 12 . Although intensive studies were made by many researchers, the function of the MDB is still unclear. Recently, Sui et al. 13,22 hypothesized that the MDB might have an effect on the circulation of cerebral  spinal fluid (CSF). And this hypothesis was validated by the cerebrospinal fluid circulation experiment further 13 . This study showed that the integration of the RCDmi, PAO membrane and dorsal wall of the dura sac connected by the MDB could maintain the integrity of the subarachnoid space caudal to the cerebellomedullary cistern, which is in agreement with the previous studies by Scali et al. 5,23 . However, under dynamic considerations, during the movements of head and neck, the RCDmi or RCPmi could more efficiently pull the dura sac along the MDB to change the pressure and volume of the sac, thus propelling the CSF circulation at the junction of cranial cavity and vertebral canal, especially the RCDmi in Indoasian finless porpoise due to its directly insert into the spinal dura mater through the dorsal atlanto-occipital interspace. In this case, the results of this study were conductive to explain the function of the MDB as a pump to propel the CSF circulation and support Sui's hypothesis 13,22 .
The existence and shape of an anatomical structure could always be influenced by its functions. Less useful structures will gradually degenerate during evolution.Now that the MDB is a universal, normal anatomical structure in many mammals, it should have an important physiological function. Moreover, we believed that the MDB is a homologous organ. For the next stage, morphological differences of the MDB among different kinds of mammals will be addressed in order to exploring the effects of ecological characteristics and living habits on it.

Conclusion
The results of this study reveal that the MDB might be a universal existing normal anatomical structure in mammals and it should play a key role on integration of the sub-occipital muscular system and the central nervous system. The morphological results of this study support the CSF circulation power hypothesis of the MDB and provide a comparative anatomical basis for the functional study on the MDB.

Materials and Methods
Ethics statement. All mammals used in this study were collected with the permission of Chinese Authorities for Animal Protection and the approval of the Ethics Committee of Dalian Medical University. All experiments were performed in accordance with the guidelines and regulations of Dalian Medical University.
Four Indoasian finless porpoise cadavers were collected from beaches in Dalian. Four Macacamulatta specimens were collected from several zoos in Liaoning Province. Six Canisfamiliaris, six Feliscatus, six Oryctolaguscuniculus, six Ratusnorvegicus and six Caviaporcellus were obtained from animal experimental center of Dalian Medical University. The obtained specimens had been embalmed through the aorta with the solution containing 10% formalin. Photographic documentation was acquired with a Canon D-40 camera.