Sensory innervation of the calvarial bones of the mouse

Abstract

Migraine sufferers frequently testify that their headache feels as if the calvarial bones are deformed, crushed, or broken (Jakubowski et al. [2006] Pain 125:286-295). This has lead us to postulate that the calvarial bones are supplied by sensory fibers. We studied sensory innervation of the calvaria in coronal and horizontal sections of whole-head preparations of postnatal and adult mice, via immunostaining of peripherin (a marker of thinly myelinated and unmyelinated fibers) or calcitonin gene-related peptide (CGRP; a marker more typical of unmyelinated nerve fibers). In pups, we observed nerve bundles coursing between the galea aponeurotica and the periosteum, between the periosteum and the bone, and between the bone and the meninges; as well as fibers that run inside the diploë in different orientations. Some dural fibers issued collateral branches to the pia at the frontal part of the brain. In the adult calvaria, the highest concentration of peripherin- and CGRP-labeled fibers was found in sutures, where they appeared to emerge from the dura. Labeled fibers were also observed in emissary canals, bone marrow, and periosteum. In contrast to the case in pups, no labeled fibers were found in the diploë of the adult calvaria. Meningeal nerves that infiltrate the periosteum through the calvarial sutures may be positioned to mediate migraine headache triggered by pathophysiology of extracranial tissues, such as muscle tenderness and mild trauma to the skull. In view of the concentration of sensory fibers in the sutures, it may be useful to avoid drilling the sutures in patients undergoing craniotomies for a variety of neurosurgical procedures.

Figure 1

Western blot analysis (A-D) and immunohistochemistry (E-G) of CGRP in the mouse trigeminal ganglion. A, B, Immunoblot for CGRP using commercial α-CGRP, showing a band corresponding to the 37 amino-acid peptide (A), lysate of trigeminal ganglia showing bands corresponding to the CGRP peptide and the 128 amino-acid CGRP precursor (B). C, D, Same as A and B, except the CGRP antibody was preadsorbed with α-CGRP, showing no detectable material. Numbers indicate molecular marker size (kDa). E, Immunostaining of CGRP-positive cell bodies in the trigeminal ganglion. F, Same as E, except using preadsorbed CGRP primary antibody. G, Same as E, except no primary antibody was used. Scale bar for E-G: 100 μm.

Figure 2

Histological analysis of the skull and characterization of trigeminal ganglion perikarya immunostained for peripherin and CGRP. A, B, Schematic illustration of the bones of the calvaria showing 3 coronal (A) and 3 horizontal (B) planes of analysis (red lines). Shown below are whole head preparations corresponding to the frontal coronal plane (A) and the middle horizontal plane (B). Immunostaining specificity was based on the size of labeled perikarya in the trigeminal ganglion. Peripherin-labeled perikarya were mostly medium- and small-size in pups (C) and adults (D). CGRP-labeled perikarya were mostly small-size in pups (E) and adults (F). Arrows point to labeled perikarya. Arrows point to labeled perikarya. Scale bar: 100 μm. G, Nomenclature of the different layers of the scalp and skull. Sections in A, B, G were stained with hematoxylin and eosin.

Figure 3

Nerve fibers traversing the calvarial bones in mouse pups. A, Peripherin-labeled fibers crossing the diploë of the parietal bone. B-D, confocal imaging of peripherin fibers that cross bones of the calvaria between the galea aponeurotica and meninges. B, normal view of a fiber stretching between the dura and bone. C, side view (a 90° lateral rotation of the image shown in B) demonstrating that the fiber continues its run across the bone and into the galea aponeurotica. Orientations of B and C are indicated by the box inserts, where the gray face of the box marks the top aspect of the tissue. D, side view resulting from a 45° vertical rotation of the image shown in B, demonstrating the entire course of the fiber between the dura and galea aponeurotica (the top face of the box corresponds to the gray faces of the boxes in B and C). E, F, examples of CGRP-labeled fibers crossing the diploë of the calvarial bones. Arrowheads point to labeled fibers. Scale bars: 50 μm.

Figure 4

Camera lucida reconstruction of peripherin nerve fibers in bones of the calvaria in the mouse pup. A, fibers in the parietal bone reconstructed from coronal sections of the head. B, fibers in the frontal, parietal and interparietal bones reconstructed from horizontal sections of the head. Note the concentration of labelled fibers in the frontal, supraorbital and caudal areas of the skull. Each drawing (A, B) represents cumulative information from 6 consecutive 30-μm-thick sections. Details in the boxed regions are shown in the corresponding photomicrographs.

Figure 5

Bone marrow in the adult calvaria. A-D, peripherin-labelled fibers. E-G, CGRP-labelled fibers. All images are taken from horizontal sections. Arrowheads point to labelled fibers. Scale bar: A, C – 200 μm; B, G – 100 μm; D-F – 50 μm.

Figure 6

Emissary canals in the adult calvaria. A-D, peripherin-labelled fibers. E-F, CGRP-labelled fibers. A – coronal section; B-F – horizontal sections. Arrowheads point to labelled fibers. Scale bar: A, B, D, E – 100 μm; C, F – 50 μm.

Figure 7

Sagittal suture in the adult calvaria. A, B, peripherin-labelled fibers running in the sagittal suture between the frontal (A) and the parietal (B) bones. C, CGRP-labelled fiber running in the sagittal suture between the frontal bones. All images were taken from coronal sections. Arrowheads point to labelled fibers. Scale bar: A, C – 50 μm; B – 100 μm.

Figure 8

Nerve fibers in the coronal suture of the adult calvaria immunostained for peripherin (A-F) and CGRP (G-J). A, C, low-power views of the convoluted architecture of the suture. B, D, magnified views of the insets A and C, respectively. G-J, high-power images of multiple CGRP-labelled fibers that follow the curves of the suture. A-F, coronal sections; G-J, horizontal sections. Arrowheads follow labelled fibers. Scale bar: A, C – 200 μm; B – 25 μm; D-J – 50 μm.

Figure 9

Coronal view of peripherin-labelled fibers in the sagittal suture at the rostral plane of the adult calvaria. At this plane of the olfactory bulbs (bottom illustration and photomicrograph), the sagittal suture constitutes a distinct passageway for nerve fibers between the meninges and periosteum, as illustrated in the camera lucida reconstructions on top. Reconstruction corresponding to the area marked by box in photomicrograph represents labeled fibers that were found in 84 consecutive sections, covering about 2.5 mm. Blue areas depict bone marrow. Red line indicates the plane of analysis. ob, olfactory bulbs.

Figure 10

Coronal view of peripherin-labeled fibers in the coronal suture at the middle of the adult calvaria. At this plane of the motor cortex (bottom illustration and photomicrograph), the coronal suture contains fibers that run at rostrocaudal, dorsoventral and mediolateral orientations, as shown in the camera lucida illustrations on top. Reconstruction corresponding to the area marked by box in photomicrograph represents labeled fibers that were found in 99 consecutive sections, covering about 3.0 mm. Note absence of labeled fibers in the diploë. Blue areas depict bone marrow. Red line indicates the plane of analysis. Abbreviations: cc, corpus callosum; oc, optic chiasm; tg, trigeminal ganglion.

Figure 11

Coronal view of peripherin-labeled fibers in the sagittal and squamos sutures at the caudal plane of the adult calvaria. At this plane of the visual cortex (bottom illustration and photomicrograph), the sutures define a continuum of nerve fibers extending between the dura and periosteum, as shown in the camera lucida illustrations on top. Reconstructions corresponding to the areas marked by boxes in photomicrograph represent labeled fibers that were found in 78 consecutive sections, covering about 2.3 mm. Also illustrated is an alternative path of fibers through an emissary canal (top left). Blue areas depict bone marrow. Red line indicates the plane of analysis. PAG, periaqueductal gray.

Figure 12

Horizontal view of CGRP-labelled fibers in the sagittal, coronal and lambdoid sutures of the adult calvaria, just above the eye level. At this level (schematic illustration of the skull and photomicrograph), labelled fibers appear to concentrate distinctly in the sutures while absent from the diploë, as shown in the camera lucida illustrations. Reconstructions corresponding to the areas marked by boxes in photomicrograph represent labeled fibers that were found in 17 consecutive sections, covering about 0.5 mm. Note the high density of fibers in the coronal suture on both sides of the skull. Blue areas depict bone marrow. Red line indicates the plane of analysis.

Figure 13

Horizontal view of CGRP-labelled fibers in the sagittal, coronal and lambdoid sutures of the adult calvaria at the level of the eyes. At this level (schematic illustration of the skull and photomicrograph), labelled fibers are distinctly concentrated in the endosteal side of the coronal sutures, as shown in the camera lucida illustrations. Reconstructions corresponding to the areas marked by boxes in photomicrograph represent labeled fibers that were found in 61 consecutive sections, covering about 1.8 mm. Blue areas depict bone marrow. Red line indicates the plane of analysis.

Figure 14

Horizontal view of CGRP-labeled fibers in the squamos suture in the adult calvaria, just below the eye level. At this level (schematic illustration of the skull and photomicrograph), labelled fibers are distinctly concentrated in the endosteal side of the squamos sutures, as shown in the camera lucida illustrations. Reconstructions corresponding to the areas marked by boxes in photomicrograph represent labeled fibers that were found in 78 consecutive sections, covering about 2.3 mm. Note presence of labelled fibers in the cavities of the bone marrow. Blue areas depict bone marrow. Red line indicates the plane of analysis. 3V, third ventricle.

Figure 15

Points of entry of nerve fibers into the sutures of the adult calvaria. Peripherin (A, B) and CGRP (C, D) nerve fibers in the proximal side of the sutures were observed to branch out of nerve bundles in the dura. CGRP nerve fibers in the distal side of the suture did not branch out of bundles in the periosteum (E, F). Arrowheads point to labelled fibers. A-D, F – horizontal plane; E – coronal plane. Scale bar: A-E – 50 μm; F – 25 μm.

Figure 16

Extensive innervation of the periosteum by peripherin (A) and CGRP fibers (B, C). A, innervation of periosteum overlying the parietal bone at the top of the adult skull. B, innervation of periosteum overlying the interparietal bone at the back of the skull. C, innervation of periosteum overlying the frontal bone on the side of the skull. Note that periosteal fibers do not enter the calvarial bones. Arrowheads point to labelled fibers. A – coronal plane; B, C – horizontal plane. Scale bar: A – 50 μm; B – 500 μm; C – 200 μm.

Figure 17

Extracranial origin of periosteal innervation in the adult calvaria. Peripherin (A, coronal plane) and CGRP fibers (B, horizontal plane) in the galea aponeurotica issuing collateral branches to the periosteum. Arrowheads point to labelled fibers. Scale bar: A – 100 μm; B – 50 μm.

Figure 18

CGRP nerve fibers traversing the arachnoid between the dura and pia in the calvarial leptomeninges of the mouse pup. Arrowheads point to labelled fibers. Scale bar: A – 100 μm; B, C – 50 μm. All sections are coronal.

Figure 19

Hypothetical implications to migraine headache. A. Extracranial origin of intracranial pain. In this scenario, action potentials generated at extracranial collaterals of meningeal pain fibers (1) spread antidromically to collaterals that terminate inside the cranium, resulting in local release of proinflammatory neuropeptides and activation of neighboring meningeal nociceptors (2). B. Intracranial origin of extracranial pain. In this scenario, action potentials generated at intracranial meningeal pain fibers (1) spread antidromically to collaterals that terminate outside the cranium (2), resulting in local release of proinflammatory neuropeptides in the scalp and activation of neighboring somatic nociceptors (3). Asterisk marks original site of activation. Red dots represent local release of inflammatory neuropeptides (e.g., CGRP, substance P), Red cylinder depicts a blood vessel (e.g., temporal artery). TG, trigeminal ganglion.