Fig 1.
Localized ITGA7 knockdown in the dorsal atlanto-occipital interspace of Rats.
(A) Schematic diagram of lentivirus injection and sampling time points. (B) Masson staining of the sagittal section of MDBC in the dorsal atlanto-occipital interspace, performed 7 days post- lentivirus injection. The shRNA-NC served as the negative control for the shRNA lentivirus vector, whereas shRNA-1, shRNA-2, and shRNA-3 represent three distinct shRNA lentivirus vectors targeting ITGA7. A Sham group, subjected to identical surgical procedures but without vector injection, was included to exclude potential confounding effects of needle insertion. (C) Western blotting to evaluate ITGA7 expression in MDBC 7 days post-lentivirus injection. (D) Quantitative analysis using Image J, with β-ACTIN as an endogenous control. * p < 0.05 (n = 3). (E) Immunofluorescence reveals the expression of ITGA7 in RCDmi within the atlanto-occipital interspace of rats. (F) Immunofluorescence reveals the expression of Green Fluorescent Protein (GFP) in RCDmi within the atlanto-occipital interspace of rats. (G) Representative in vivo images depicting GFP expression of shRNA-ITGA7 vector in rats at P7 and P14. OCCI: occipital bone. C1: atlas. RCDmi: rectus capitis dorsal minor muscle. ★: MDB fibers. ↑ : SDM. white arrow: ITGA7 expression.
Fig 2.
Masson’s trichrome staining of the dorsal atlanto-occipital interspace in rats at P7 after ITGA7 knockdown (n = 4).
Muscle fibers were stained red, and connective tissue fibers blue. (A) A representative image showing Masson’s trichrome staining of sagittal section in the shRNA-NC group. (B) Enlarged view of box B in A. (C-E) Enlarged view of boxes in B. (C) The muscle fibers of RCDmi. (D-E) MDB fibers in the shRNA-NC group, MDB fibers, including fibrocytes and fibroblasts, displaying directionality and integration into DOAM and SDM at an acute angle. (F) A representative image depicting Masson’s trichrome staining in a sagittal section of the shRNA-ITGA7 group. (G) Enlarged view of box G in F. (H-J) Enlarged view of boxes in G. (H) Increased gaps and adipocytes between RCDmi muscle fibers were observed, black arrowhead indicated adipocytes. (I-J) MDB fibers in the shRNA-ITGA7 group, characterized by a loose, scattered arrangement, with fiber bundles varying in directions and loosely connected to the DOAM. Cells exhibited round nuclei arranged in a dispersed manner, lacking directionality. OCCI: occipital bone. C1: atlas. RCDmi: rectus capitis dorsal minor muscle. ★: MDB fibers; ↑ : SDM.
Fig 3.
Masson’s trichrome staining of the dorsal atlanto-occipital interspace in rats at P14 after ITGA7 knockdown (n = 4).
(A) A representative image showing Masson’s trichrome staining of a sagittal section in the shRNA-NC group. (B) Enlarged view of box B in A. (C-E) Enlarged view of boxes in B. (C) showed the muscle fibers of RCDmi. (D-E) showed the fusion of MDB, DAOM and SDM, characterized by mature cross-linked collagen fibers. (F) A representative image depicting Masson’s trichrome staining of a sagittal section in the shRNA-ITGA7 group. (G) Enlarged view of box G in F. (H-J) Enlarged view of boxes in G. (H) exhibited increased gaps between RCDmi muscle fibers. (I) showed immature collagen fibers containing a large number of fibrocytes and fibroblasts. (J) revealed a few ovoid fibroblast nuclei, lacking directionality, situated between the DAOM (left) and the large collagen cluster (right). OCCI: occipital bone. C1: atlas; RCDmi. rectus capitis dorsal minor muscle. ★: MDB fibers. ↑ : SDM.
Fig 4.
Scanning electron microscopy of MDBC in the dorsal atlanto-occipital interspace of rats at P7 after ITGA7 knockdown.
(A) A representative image showing a sagittal section in the shRNA-NC group. (B) The muscle fibers of the RCDmi exhibited a regular arrangement. (C) The MDB was composed of collagen fiber bundles arranged in a relatively uniform direction. (D) MDB fibers in the shRNA-NC group oriented from the ventral part of RCDmi and gradually integrated into the SDM. (E) A representative image showing a sagittal section in the shRNA-ITGA7 group. (F) Partial disruption muscle fibers and excessive deposition of interstitial fibers were observed in RCDmi of the shRNA-ITGA7 group. (G) MDB fibers in shRNA-ITGA7 group were composed of fine fibers running in various directions, with fibers bending and overlapping to form a loose network. (H) MDB fibers between RCDmi and SDM in shRNA-ITGA7 group showed a loose network arrangement. ★: MDB fibers. SDM: spinal dura mater. m: rectus capitis dorsal minor muscle.
Fig 5.
Scanning and transmission electric microscopy of the atlanto-occipital interspace in rats at P14.
(A-F) were photographed using a scanning electron microscope, (G-L) were captured using a transmission electric microscope. (A) A representative image showing a sagittal section in the shRNA-NC group. (B) Enlarged view of MDB fibers in the shRNA-NC group. (C) MDB fibers in the shRNA-NC group were formed by regular arranged mature collagen fibers. (D) A representative image showing a sagittal section in the shRNA-ITGA7 group. (E) Enlarged view of MDB fibers in the shRNA-ITGA7 group. (F) MDB fibers in the shRNA-ITGA7 group showed a disordered arrangement, with fibers of varying thicknesses clustered and in multiple directions. (G) A representative image of Toluidine blue staining semi-thin section in the shRNA-NC group. (H) MDB fibers in the shRNA-NC group exhibited bundles of collagen fibers aligned in relatively consistent directions. (I) MDB cross-sections consisted of bundles of collagen fibers with relatively uniform thickness in the shRNA-NC group, black arrowhead showed a fibrocyte in MDB. (J) A representative image of Toluidine blue staining semi-thin section in the shRNA-ITGA7 group. (K) Collagen fibers in the shRNA-ITGA7 group appeared irregularly arranged and crisscrossed, with varying sizes and smaller bundles. (L) Numerous fibroblasts, rich in rough endoplasmic reticulum, were observed among the disordered MDB fibers in shRNA-ITGA7 group, black arrowheads showed the rough endoplasmic reticulum. SDM: spinal dura mater. ★: MDB fibers. m: rectus capitis dorsal minor muscle.
Fig 6.
Inhibiting ITGA7 reduced muscle force and altered ECM related genes expression.
(A) Expression of Laminin α2 in the RCDmi in rats. (B) The proportion of the cross-sectional area of the RCDmi in shRNA-NC and shRNA-ITGA7 at P7 and P14. (C) The average cross-sectional area of the RCDmi in shRNA-NC and shRNA-ITGA7 at P7 and P14. (D) The maximum breaking force of the RCDmi in shRNA-NC and shRNA-ITGA7 groups at P7 and P14. (E) The mRNA expression levels of Col1a1 in the MDBC of rats in the atlanto-occipital interspace. (F) The mRNA expression levels of Col1a2 in the MDBC of rats in the atlanto-occipital interspace. (G) The mRNA expression levels of Lama2 in the MDBC of rats in the atlanto-occipital interspace. (H) The mRNA expression levels of Tgfb1 in the MDBC of rats in the atlanto-occipital interspace. (I) The mRNA expression levels of Scx in the MDBC of rats in the atlanto-occipital interspace. (J) The mRNA expression levels of Pdgfra in the MDBC of rats in the atlanto-occipital interspace. (n = 4, * p < 0.05, ** p < 0.01).