Integrin α7 regulates the development of the myodural bridge complex in rats

doi:10.21203/rs.3.rs-3902636/v1

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Integrin α7 regulates the development of the myodural bridge complex in rats

https://doi.org/10.21203/rs.3.rs-3902636/v1

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The myodural bridge (MDB) is a fibrous structure that connects the suboccipital musculature to the cervical spinal dura mater (SDM) via the atlanto-occipital and atlanto-axial interspaces. Muscles, ligaments, and MDB fibers integrated a functional unit known as MDB complex (MDBC). MDB is an evolutionarily conserved physiological structure that is considered as a dynamic source of cerebrospinal fluid circulation. Integrin α7 (ITGA7) plays a crucial role in transmitting both longitudinal and lateral forces generated by muscle contraction. To explore the molecular mechanism and the force transmission role of MDB, shRNA-ITGA7 lentivirus vectors were injected into the rectus capitis dorsal minor (RCDmi) of neonatal rats. Inhibition of ITGA7 expression led to muscle dystrophy and an increase in loose and lacked directionality MDB fibers in the atlanto-occipital interspace. Collagen fibers displayed varying thicknesses and formed interlaced clusters. Numerous fibroblasts remained interspersed among the MDB fibers. Inhibition of ITGA7 expression impedes MDBC development and formation. The study indicates that suboccipital muscle stress influences MDB fiber differentiation and maturation, confirming the role of MDB in transmitting this stress to SDM and in cerebrospinal fluid circulation. Additionally, the study highlights the significance of ITGA7 as a pivotal regulator in MDBC development.

Biological sciences/Developmental biology

Health sciences/Anatomy

Cerebrospinal fluid (CSF) circulation, often referred to as the third circulation system of the human body, encompasses the production of CSF by the choroid plexus, circulates through the cerebral ventricles, subarachnoid space, parenchyma and eventual reabsorption into the venous circulation¹. The dynamics of CSF have been extensively investigated, this circulation is influenced by several factors, including arterial pulsations, respiratory movements, body position^2-4, glymphatic circulation, circadian rhythms^5-7, and the myodural bridge (MDB)^8-11, et al.

MDB consists of fibrous connective tissues that runs between the suboccipital musculature and the cervical spinal dura mater (SDM), passing through the atlanto-occipital and atlanto-axial interspaces^12,13. The MDB comprises fibers from multiple sources, including the rectus capitis posterior minor, rectus capitis posterior major, obliquus capitis inferior, nuchal ligament, and vertebrodural ligament^14-21. These muscles, ligaments, and the MDB fibers form an integrated functional unit known as the myodural bridge complex (MDBC)²². The human MDB is a tendinous-like structure primarily composed of parallel-arranged collagen type I fibers²³. Its physiological functions include preventing dura mater folding, transmitting proprioception, maintaining subarachnoid space integrity, influencing CSF circulation, and associating with cervicogenic headache^18,24-26. Comparative anatomical studies have been demonstrated that the MDB is a highly conserved structure with significant physiological functions in mammals, reptiles and birds^13,27-29. Studies in developmental biology have elucidated the MDB development process in rats and human embryos. In rats, MDB fibers become significantly denser post-birth. In human fetuses, MDB forming cells become oriented and cross the atlanto-axial interspace, attaching to the dura mater at 21 weeks of gestation，shortly following the maturation of suboccipital muscle cells. It is speculated that mechanical stress from suboccipital musculature may contribute to MDB maturation^30-32. However, current research on the molecular mechanisms regulaing MDB formation is limited. Consequently, further direct experimental research is essential to confirm this hypothesis.

Integrins, a family of transmembrane cell surface receptors, play a crucial role in cell migration, cell shape determination, and cell-cell interactions³³. Integrins adhere the cytoskeleton of cells to specific proteins in the extracellular matrix (ECM) for the purpose of stabilization, migration, and proliferation. The α7β1 integrin is the predominant integrin on skeletal muscle. It binds to laminin in ECM, as well as to myofilaments via Talin³⁴. In developed muscle fibers, the α7β1 integrin provides both terminal and peripheral cohesion to muscle fibers, which is important to muscle integrity, neuromuscular connectivity, and force generation. In adult muscle fibers, integrin α7 (ITGA7) is predominantly located peripherally and enriched at myotendinous and neuromuscular junctions³³. The absence of ITGA7 results in progressive muscle dystrophy accompanied by increased endomysial connective tissue production³⁵. ITGA7 plays a crucial role in transmitting lateral forces between skeletal muscle cells and the basement membrane, as well as longitudinal forces at the tendon junction³⁶.

Similar to tendons, MDB is primarily composed of strong collagen fibril arrays, which can transmit the suboccipital musculature force to the spinal dura mater^23,37. Even though, Kei Kitamura et al.³⁸ suggested that force transmission via MDB was limited or ineffective. To explore the molecular mechanism underlying MDB formation and further verify the role of MDB in transmitting suboccipital musculature force, our study aimed to reduce ITGA7 expression in the suboccipital region, thereby diminishing its role in force transmission and examine its impact on MDB development. In this study, we locally injected shRNA lentivirus vehicles to reduce ITGA7 expression in the dorsal atlanto-occipital interspace of neonatal rats. The effects of knocking-down ITGA7 on the morphology of MDBC during development were observed. This research aims to provide direct evidence supporting the hypothesis that MDB formation is dependent on mechanical loading from suboccipital muscles. It will lay a foundation for exploring the regulatory mechanisms of MDBC.

Ethnic statements

The experimental protocol was approved by the Committee on the Ethics of Animal Experiments at Dalian Medical University. All experiments were conducted in accordance with the guidelines and regulations of Dalian Medical University. All possible measures were taken to minimize any potential discomfort or pain experienced by the animals involved.This study was carried out in compliance with the ARRIVE guidelines.

Animal models

Male and female Sprague Dawley (SD) rats were housed in a controlled environment with a 12-hour light cycle and provided with ad libitum access to standard rodent diet (Medicience Ltd. Jiangsu, China) and water. The neonatal rats (P0, within 12 hours after birth) were randomly assigned to either the experimental group (injected with shRNA-ITGA7 lentivirus) or the control group (injected with shRNA-NC lentivirus). P0 rats were anesthetized using 2% tribromoethanol solution (0.2ml/10g body weight，RHAWN, China) administered via intraperitoneal injection. The lentivirus injection sites were located 1mm from the occipital protuberance towards the tail, and 1mm from the midline to the left and right. The needle was inserted perpendicular to the horizontal plane to a depth of 2mm. Both sides of the rectus capitis dorsal minor (RCDmi) of the rats were injected with 10 μL of either shRNA-ITGA7 lentivirus or shRNA-NC lentivirus using microinjectors . After injection, pressure was applied with a cotton swab for 5 minutes to stop bleeding, and the groups were then labelled. The animals were sacrificed on postnatal day 7 (P7) and day 14 (P14) (Figure 1A). The structures of MDBC in the posterior atlanto-occipital interspace of rats were used for morphological study and protein expression analysis.

Lentivirus packaging and concentration

HEK293T cells were cultured in DMEM (Hyclone, USA) supplemented with 10% fetal bovine serum(GIBCO,USA), 100 U/mL penicillin, and 100 U/mL streptomycin at 37 ℃ and 5% CO₂. Transfection was performed when cell confluence reached 70% to 80%. The shRNA vector targeting rat ITGA7 (NM_030842) was constructed by Genechem (Shanghai, China) using the following sequences: #1: 5’-GCTAATGTGCAGAAGGAAAGC-3’; #2: 5’-GCCACGAACAATTTGGGTTCT-3’; #3: 5’-GCACCTCTGGAATCACCATTG-3’. The vector information was hU6-MCS-CBh-gcGFP-IRES-puromycin. The pCDH-CMV-EF1-puro lentivirus packaging system was utilized to generate the GFP-shRNA-ITGA7 lentivirus. The supernatant was collected 36-48 hours after transfection and centrifuged at 4 ℃ and a speed of 50000g for 30 minutes to obtain high titers of lentivirus. The injected lentivirus was concentrated to titers of 10⁹ IU/mL.

Western blotting

Tissues were lysed using total protein extraction kit (Keygen, China). Equal amounts of samples were electrophoresed on SDS-PAGE gel and transferred to nitrocellulose membrane (Invitrogen, CA, USA). The membranes were blocked with 5% (w/v) BSA for 2 hours at room temperature, then incubated with the primary antibody overnight at 4°C, followed by the secondary antibody for 1 hour at room temperature, and detected by Odyssey CLx image system (LI-COR, USA). The primary antibodies used were anti-ITGA7 (#bs-1816R, Bioss, China) at a dilution of 1:1000 , and anti-β - Actin (#21338，SAB，USA) at a dilution of 1:5000. The secondary antibody used was Dylight 800 Goat Anti-Rabbit IgG (#A23920, Abbkine, China) at a dilution of 1:2000 dilution.

In vivo imaging

An in vivo imaging system (IVIS Spectrum, PerkinElmer, USA) was used to examine the GFP expression of shRNA-ITGA7 and shRNA-NC in living rats. Rats at P7 and P14 were first anaesthetized (2% tribromoethanol solution, 0.2ml/10g body weight) and placed in the shooting box in an adjusted position. This in vivo imaging system can provide us with information about whether shRNA lentiviral vectors are effective and where they are expressed.

Histological sectioning and staining

Histological sectioning and staining was conducted on lentivirus-injected rats at P7 and P14, respectively (n=4). After fixation and decalcification, the specimens were embedded in paraffin wax and sectioned sagittally at 8μm thickness by using a rotary microtome (Leica Micro HM450; Leica Microsystems GmbH, Wetzlar, Germany). Masson's trichrome staining(Masson) and Picrosirius red staining were performed respectively. The sections stained with Masson were photographed using a Nikon NIS image system (Nikon Eclipse 80i, Nikon, Tokyo, Japan). The sections stained with Picrosirius red were observed under a polarizing microscope (Olympus BH-2; Olympus Corp., Tokyo, Japan).

Collagen volume fraction

Masson stained slices from each group were observed and photographed under a light microscope, including MDBC fibers, the dorsal atlanto-occipital membrane (DAOM), SDM, and RCDmi within the dorsal atlanto-occipital interspace. Semi-quantitative analysis was performed by using the Image J software to evaluate the collagen volume fraction as previously described ³⁹. The collagen volume fraction of MDBC fibers from different groups was compared.

Scanning and transmission electron microscopy

The structures of MDBC in the dorsal atlanto-occipital interspace, including MDBC fibers, DAOM, SDM, and RCDmi were fixed in 2.5% glutaraldehyde (Sigma, USA), and post-fixed in 1% OsO4 (Sigma, USA). The specimens for scanning electron microscope (SEM) were dehydrated in a graded series of tert butanol, then dried at the critical point, coated with platinum, and observed with a scanning electron microscope (JSM-IT300LA, JEOL Ltd., Japan). Specimens for transmission electron microscope (TEM) were dehydrated in a graded series of ethanol and embedded in epoxy resin (Okenshoji, Tokyo, Japan). Ultrathin sections were cut with PT-X Power Tome ultramicrotome (RMC, Arizona, USA) and stained with uranyl acetate and lead citrate. The tissue sections were observed under a transmission electron microscope (JEM-2000EX, JEOL Ltd., Japan) operated at 120 kV.

Statistical analysis

Data analysis was performed using Image J ⁴⁰ and GraphPad Prism version 8.0 software (GraphPad Software, Boston, Massachusetts USA) . Quantitative data was presented as mean ± standard deviation (SD). Statistical analysis was conducted using a two-way Student's t-test. A p value<0.05 is considered statistically significant. All experiments were independently performed in triplicate.

Screening and validation of an ITGA7 knockdown model of MDBC in the dorsal atlanto-occipital interspace of rats

A rat model with localized ITGA7 knockdown was constructed by injecting shRNA-ITGA7 lentivirus vectors into the MDBC within the dorsal atlanto-occipital interspace. The lentivirus vectors included three shRNA-ITGA7 types, labeled shRNA-1, shRNA-2, shRNA-3, and a negative control, labeled as shRNA-NC. Samples were taken from animals in each group at postnatal day 7 (P7) to identify the most effective shRNA vector. Masson staining exhibited an increase in the spacing and interstitium between RCDmi muscle fibers in all shRNA-ITGA7 groups, compared to the shRNA-NC group. In the shRNA-NC group, MDB fibers ran parallel, extending from the ventral part of the rectus capitis dorsal minor (RCDmi) to fuse with the dorsal atlanto-occipital membrane (DAOM) and SDM, interspersed with oriented fibrocytes and fibroblasts. In the shRNA-1 group, MDB fibers in the upper part of the atlanto-occipital space exhibited characteristics similar to those in the shRNA-NC group. However, MDB fibers in the lower part appeared more mature with fewer fibroblasts. In the shRNA-2 group, MDB fibers were the most mature among all other groups, tightly fused with the DOAM and SDM. In the shRNA-3 group, there was an increase in fiber quantity within the atlanto-occipital space, with fibers and cells displaying a disorganized arrangement (Figure1B). Total protein was extracted from the MDBC in each group, and Western blotting analysis revealed a consistent downregulation of ITGA7 protein expression levels in the shRNA-3 group (Figure 1C and D). Given the morphological changes observed in the shRNA-3 group (Figure 1B), the shRNA-3 vector demonstrated the most efficient knockdown effect. Therefore, shRNA-3 vector was selected in subsequent experiments and then labeled as shRNA-ITGA7.

To evaluate the efficiency of lentivirus infection, an in vivo imaging system was used to examine the GFP expression in shRNA-ITGA7 and shRNA-NC groups of P7 and postnatal day 14 (P14) rats (Figure 1E). The heat map image represented GFP intensity for representative rats. Any rats with weak GFP intensity in the dorsal neck region, such as the third rat at P7, were excluded from the study. GFP intensity observed via in vivo imaging confirmed that the successful infection of local cells in MDBC by shRNA lentivirus vectors.

Based on the observed morphological changes, downregulated ITGA7 expression levels, and in vivo GFP expression, an effective ITGA7 knockdown animal model was successfully established through local injection of the lentivirus vector in the suboccipital region.

Histological changes in MDBC by downregulation of ITGA7 expression during development

Following ITGA7 expression inhibition in rat MDBC, head and neck samples were collected and sagittally sectioned at P7 and P14, respectively. Masson staining was utilized to better display the collagen fibers, with muscle fibers being stained red and connective tissue fibers blue.

At P7, in the shRNA-NC group, the morphology of the MDBC in the dorsal atlanto-occipital interspace showed the same features as previously described ³¹ (Figure 2A). The RCDmi and SDM were connected via MDB, as well as RCDmi directly linked to the DAOM, closely fused to the SDM (Figure 2C). MDB fibers, along with fibrocytes and fibroblasts, displayed directionality and integrated into the DOAM and SDM at an acute angle (Figure 2E and F). Most cells exhibited elongated nuclei and were arranged neatly along the direction of muscle tension. However, in the shRNA-ITGA7 group, numerous fibers were observed between the RCDmi and SDM (Figure 2B). Compared to the shRNA-NC group, MDB fibers in the shRNA-ITGA7 group appeared loosely arranged and scattered, with fiber bundles oriented in various directions and loosely attached to the DOAM (Figure 2D). Cells interspersed among these fibers exhibited rounder nuclei compared to those in the shRNA-NC group, and these nuclei were dispersed and arranged non-directionally. (Figure 2E, H and I). It is speculated that these cells are fibroblasts yet to differentiate into fibrocytes. Compared to the orderly RCDmi muscle fibers in the shRNA-NC group, the shRNA-ITGA7 group exhibited increased gaps, uneven thickness and partial interruptions of muscle fibers, along with increased interstitium, and intramuscular fatty infiltration (Figure 2C, D, G and J). These changes resembled the characteristics of muscle dystrophy.

At P14, in the shRNA-NC group, MDB fibers originating from the RCDmi formed bundled interconnections with DAOM and SDM, resulting in tightly fused structures. The number of cells located between these collagen fibers dramatically decreased, leaving only a small population of fibrocytes, indicating a progressive maturation of the MDB fibers (Figure 3A, C, E, F). Additionally, the RCDmi muscle fibers became more organized and tighter than the shRNA-NC group at P7 (Figure 3A, C, G). However, in the shRNA-ITGA7 group, MDB fibers exhibited an irregular distribution. In the upper part of the atlanto-occipital interspace, a loose connection was observed between the RCDmi and DAOM (Figure 3B). Conversely, in the lower part of the interspace, a significant augmentation of MDB collagen fibers was noted between the RCDmi and SDM. The fibers showed a disordered arrangement, varying from sparse to densely clustered and stacked. MDB fibers still contained a large number of fibrocytes and fibroblasts, interspersed with visible collagen fibers (Figure 3D, H, I). In addition, a few ovoid fibroblast nuclei lacking directionality remained between the DAOM and the large collagen cluster (Figure 3B, I). Part of RCDmi muscle fibers in the shRNA-ITGA7 group exhibited a morphology akin to that of the shRNA-NC group. However, the lower ventral region still showed increased gaps and interstitium between muscle fibers (Figure 3J). These results suggest that inhibiting ITGA7 expression impedes development and formation of MDBC, presented as interrupted the appropriate assembly of MDBC fibers, and resulted in RCDmi muscle dystrophy.

Inhibition of the ITGA7 expression changed collagen volume fraction and fiber properties of MDBC in the dorsal atlanto-ccipital interspace of rats

Masson’s trichrome staining revealed that the shRNA-ITGA7 group showed more collagen fibers in the MDBC region than the shRNA-NC group. Semi-quantitative analysis was applied to assess the collagen volume fraction ( CVF ) of MDBC at P7 and P14, respectively. The CVF in the shRNA-ITGA7 group was significantly higher than in the shRNA-NC group at both P7 and P14 (p < 0.05) (Figure 4). These findings suggest that downregulation of ITGA7 expression is associated with an increase in the amount of fibers in the dorsal atlanto-occipital interspace.

To describe the collagen properties of the abundant fibers in the atlanto-occipital interspace in the shRNA-ITGA7 group, Picrosirius red staining was utilized. Under a polarizing microscope, the fibers of MDB, DAOM and SDM in the shRNA-NC group at P7 presented a birefringence of yellow to red color, suggesting a predominance of collagen type I. As development progressed, these structures exhibited increasingly intense red refraction, which was more pronounced by P14 (Figure 5A and A’, C and C’). Although Masson staining indicated a greater density of fibers in the shRNA-ITGA7 group, Picrosirius red staining demonstrated that these fibers exhibited weak yellow birefringence and a more pronounced presence of greenish collagen type III at both P7 and P14 (Figure 5 B and B’, D and D’). The abundant fibers either lacked birefrigence or displayed weak yellow birefrigence, suggesting they were formed by immature collagen fibers. These results further substantiate that the maturation of MDB fibers was impeded by the suppression of ITGA7 expression.

Ultra-morphology changes of MDBC in the atlanto-ccipital interspace of ITGA7 knockdown rats

To further elucidate the morphological changes of MDBC in the ITGA7 knockdown group, the ultrastructures were observed by scanning and transmission electron microscopy. At P7, under scanning electron microscope, the MDB fibers in the shRNA-NC group formed bundles of collagen fibers with a relatively uniform orientation that gradually integrated into the dura mater, originating from the ventral side of the RCDmi (Figure 6A and B). Meanwhile, the muscle fibers in the shRNA-NC group displayded a regular and dense arrangement, which is indicative of normal muscle development (Figure 6C and D). In contrast, at P7 in the shRNA-ITGA7 group, the region typically occupied by dense MDB fibers instead displayed finer fibers oriented in multiple directions and overlapping to form a looser network (Figure 6E and F). Additionally, RCDmi muscle fibers in the shRNA-ITGA7 group displayed partial disruption and an excessive accumulation of interstitial fibers (Figure 6G and H). These findings further confirmed that suppression of ITGA7 expression leads to developmental abnormalities in MDBC.

At P14, under a scanning electron microscope, MDB fibers in the shRNA-NC group integrated into regular layered DAOM and fused with the SDM in the sagittal section (Figure 7A). Compared to P7, the collagen fibers of MDB became denser, thicker, and more regularly arranged (Figure 7B). Conversely, in the shRNA-ITGA7 group, although collagen fibers showed gradual maturation relative to P7, their arrangement remained disordered, with fibers of varying thicknesses clustered in different directions (Figure 7C and D). Under transmission electron microscope, MDB fibers in the shRNA-NC group formed bundles with relatively consistent orientations. In the cross-section of MDB, collagen fibers appeared uniformly thickness. A few fibrous cells were interspersed among the MDB fibers (Figure 7 E and F). Conversely, in the shRNA-ITGA7 group, collagen fibers were irregularly arranged and crisscrossed, varying in size and forming relatively small bundles. Numerous fibroblasts, characterize by abundant rough endoplasmic reticulum, were observed interspersed between the fibers.

In summary, inhibiting ITGA7 expression results in developmental abnormalities in both MDB fibers and RCDmi in MDBC. Abnormalities in Collagen fiber arrangement and thickness were more distinctly observed under scanning electron microscopy. Transmission electron microscopy revealed a disordered arrangement of collagen fibers and an abundance of fibroblasts, indicative of immature collagen fibers differentiation.

Hack et al.¹² first introduced the concept of the myodural bridge in 1995. In the atlanto-occipital and atlanto-axial interspaces, MDB fibers intergrate into the posterior atlanto-occipital membrane (PAOM) and SDM, becoming part of the SDM. Previous studies have demonstrated that head movements effectively transfer tension from the suboccipital muscles to the SDM via MDB, subsequently facilitating CSF circulation ^10,26,41. ITGA7 is prodominantly located around the periphery of muscle fibers and is enriched at myotendinous and neuromuscular junctions ³³. It plays a crucial role in transmitting both longitudinal and lateral forces generated by muscle contraction³⁶. Consequently, ablating ITGA7 expression in suboccipital muscles could provide direct evidence to confirm the role of MDB fibers in force transmission.

In this study, a local ITGA7 knockdown model was established by injecting shRNA lentivirus vectors into the suboccipital region of neonatal rats. Significant morphological abnormalities in MDBC were observed after interfering with the role of ITGA7 in force transmission. Masson staining revealed two aspects of MDBC abnormalities due to ITGA7 inhibition. Firstly, increased gaps and expanded muscular interstitium were noted in the muscle fibers of RCDmi, accompanied by adipocyte infiltration at P7. ITGA7 serves as a ‘bridge’ connecting muscle fibers to the ECM, and its deficiency leads to progressive muscular dystrophy ³³. The muscle alterations observed in the ITGA7 knockdown group are consistent with previously reported muscle dystrophy following ITGA7 deficiency When skeletal muscle homeostasis is disrupted, fibro-adipogenic progenitors may progress towards fibrosis or adipogenesis ⁴². The observed muscle fibrosis and fat infiltration in RCDmi could result from activated fibro-adipogenic progenitors. Additionally, muscle dystrophy showed improvement at P14, which might be due to the limited efficiency of in vivo lentivirus infection, leading to a reduced proportion of positively infected cells during development. Additionally, the muscle cells possess strong regenerative capacity, which could have played a role in ameliorating the pathological condition. Secondly, We observed an increase in MDB fibers in the dorsal atlanto-occipital interspace, albeit with heterogeneous orientation. Cells in this region displayed rounder nuclei, and were dispersed without a directional arrangement. ITGA7 deficiency impaired the attachment of muscle fibers to the ECM and alters force transmission between myocytes and the ECM ⁴³. Cells can sense mechanical stimuli and convert these into biochemical signals, influencing cell and tissue morphology, composition and physiological functions ⁴⁴. Reducing ITGA7 expression in RCDmi likely diminishes force transmission enfficiency between RCDmi and MDB forming cells in the dorsal atlanto-occipital interspace. The decrease in muscle strength caused by muscle dystrophy, along with the weakened direct connection between RCDmi and the ECM in this region, is believed to affect force transmission efficiency. Increased MDB fibers may compensate for reduced efficiency. The decline in force transmission could significantly contribute to the aberrant formation of MDB fibers in rats with ITGA7 knockdown. At P14, MDB fibers matured in the shRNA-NC group, whereas in the ITGA7 knockdown group, they were densely populated with fibrocytes and fibroblasts. These findings suggest that the orientation and maturation of MDB fiber was interfered due to ITGA7 inhibition.

Subsequently, Picrosirius red staining corroborated developmental delays in the ITGA7 knockdown group, marked by increased greenish collagen type III and immature collagen type I fibers in the dorsal atlanto-occipital interspace. Scanning electron microscopy provided direct observations of MDB fiber changes in ITGA7 knockdown rats. At P7, scanning electron microscopy revealed loosely arranged, disordered MDB fibers with muscle fibers breakage and increased interstitial fibers. At P14, collagen fibers displayed varied thicknesses and interlaced clusters, indicating abnormalities in MDB fibers. Additionally, transmission electron microscopy revealed irregular arranged MDB collagen fibers and numerous fibroblasts in the ITGA7 knockdown group. The scanning and transmission electric microscopy findings further corroborated that inhibition of ITGA7 expression results in improper formation and developmental delay of MDB fibers, consistent with Masson staining results.

Taken together, these results indicated that inhibiting of ITGA7 expression disrupts mechanical force transmission form RCDmi to MDB-forming cells in the dorsal atlanto-occipital interspace, which resulted in the developmental abnormalities of MDBC. This aligns with established knowledge emphasizing that necessity of proper mechanical forces for myotendinous junction maturation and tendon cell differentiation^45,46 Mechanical traction is of great significance for the differentiation and development of vertebrate tendons. Under its action, fibroblasts secrete collagen to help tendons resist the load generated by muscle contraction. During tendon differentiation, the pulling force exerted by muscles on the extracellular matrix is essential for activation of transforming growth factor beta (TGFβ) at the muscle-tendon interface, promoting TGF β binding to its related proteins, further activating downstream SCX and Egr1/2, thereby inducing the synthesis and deposition of collagen in the extracellular matrix ^46,47. The strain produced generated by the contraction of the developing RCDmi muscle is instrumental in MDB fiber differentiation and maturation. Mechanical stress from suboccipital muscles plays a crucial role in MDB development. This study provides more concrete evidence that the strength of the suboccipital muscle can be transmitted to the spinal dura mater through MDB, acting as a dynamic source of cerebrospinal fluid circulation.

Abnormal MDB development due to ITGA7 deletion in the suboccipital muscles may affect CSF circulation. Changes in the tensile force transmitted by MDB result in altered CSF secretion and reabsorption rates, highlighting MDB’s potential impact on CSF circulation⁸. Bleomycin-induced MDB and fibrous tissue hyperplasia in the atlanto-occipital interspace may decrease dura mater compliance, adversely affecting CSF circulation³⁹. The occipito-atlantal cistern (OAC) is a recently identified subarachnoid cistern, extending from the foramen magnum to the upper edge of the axis. It is considered an important region for the action of MDB. The contraction force of the suboccipital muscle is transmitted to the SDM via MDB, leading to the dura mater deformation in the atlanto-occipital and atlanto-axial interspaces, which in turn regulates CSF fluid within OAC, contributing to CSF circulation. In the ITGA7 knockdown group, excessive accumulation of MDB fibers in the atlanto-occipital interspace may ventrally compress the SDM, potentially reducing OAC volume. The potential impact of this condition may be similar to that of cerebellar tonsillar herniation, known to disrupt CSF circulation. Moreover, studies have demonstrated a potential association between pathological alterations in MDBC and chronic headaches^25,48,49. Inhibition of ITGA7 expression in MDBC resulted in abnormal MDBC development, which could lead to chronic headaches, further experiments are necessary to confirm this.

However, this study has certain limitations. Firstly, it was not determined whether knockdown of ITGA7 expression in MDBC affected lateral or longitudinal forces. Secondly, the efficiency of lentiviral infection, typically low in vivo, was not assessed. Thirdly, the contraction force of RCDmi in ITGA7 knockdown rats was not quantified.

In conclusion, this study successfully developed an animal model by local injection of shRNA lentivirus vector to inhibit ITGA7 expression of the suboccipital region in rats. The reduced ITGA7 expression in RCDmi led to developmental abnormalities in MDBC. This indicates that mechanical stress from suboccipital muscles may facilitate MDB fiber differentiation and maturation. The finding provides strong evidence that mechanical stress from suboccipital muscles is transmitted to the SDM through MDB, serving as a dynamic source of CSF circulation. Furthermore, the study highlights the role of ITGA7 as a key regulator in MDBC development. Future research should investigate the clinical implications of abnormal MDBC development, including headache.

Data availability

All data generated or analyzed during this study are included in the manuscript and supporting files. Data available form corresponding authors upon reasonable request.

Author contributions

Yun-Feng Liu, Investigation, Data curation, Software, Formal analysis, Validation, Methodology, Writing-original draft; Zhang Lu, Investigation, Data curation, Software, Formal analysis, Validation, Methodology, Writing-original draft; Xue Song, Supervision, Investigation, Software, Formal analysis, Methodology; Xin-Yuan Zhang, Investigation, Formal analysis; Jing-Xian Sun, Supervision, Methodology; Qing Shao, Jia-Wei Wang, Investigation; Xiao-Ying Yuan, Supervision, Formal analysis; Yan-Yan Chi, Data curation, Ruo-Tong Zhang, Formal analysis, Funding acquisition; Sheng-Bo Yu, Supervision, Funding acquisition; Wei Ma, Conceptualization, Supervision, Methodology, Writing-review and editing; Hong-Jin Sui, Supervision, Funding acquisition, Writing-review and editing.

Funding

This work was supported by the National Natural Science Foundation of China (NSFC32100928, NSFC32071184); Science and Technology Talent Innovation Support Policy Project of Dalian (2023RG003); Key R&D Project of Liaoning Province (2020JH/1050004); Youth Talent Cultivation Fund Project of Dalian Medical University. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Competing interests

The authors declare no competing interests.

Acknowledgements

This work was supported by the National Natural Science Foundation of China 32071184 (to Hong-Jin Sui), Science and Technology Talent Innovation Support Policy Project of Dalian 2023RG003 (to Hong-Jin Sui), the National Natural Science Foundation of China 32100928 (to Xiao-Ying Yuan), Key R&D Project of Liaoning Province 2020JH/1050004 (to Sheng-Bo Yu), Youth Talent Development Foundation of Dalian Medical University (to Ruo-Tong Zhang).

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Integrin α7 regulates the development of the myodural bridge complex in rats

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