Inflammation is a central mechanism in CNS pathology with both short- and long-term effects. Inflammation can occur without infection, become chronic, or increase with age (inflammaging). Understanding how inflammatory processes can be influenced negatively or positively is instrumental in alleviating challenging pathological conditions found in many neurological diseases. During pathological conditions, crosstalk between different cell types is complex, especially in the CNS, and it is therefore difficult to model cell–cell interactions, e.g., in cell cultures. More complex models are needed to understand the role of MCs in the immune response, and mouse models that lack meningeal MCs have been central in delineating their role in the CNS. Many of these studies are based on mutations in Kit/Kitl—genes encoding for a growth factor and its receptor important for MC progenitor proliferation and differentiation (Grimbaldeston et al., 2005). Another mouse model is mutations in the microphthalmia-associated transcription factor (Mitf), which results in a stark reduction in MC number or absence depending on tissue (Kitamura et al., 2002; Ingason et al., 2019). There are other models in which MCs are targeted for apoptosis or depleted (Reber et al., 2012; Feyerabend et al., 2016; Sasaki et al., 2021; Dahlin et al., 2022).

MITF is a basic helix–loop–helix leucine zipper transcription factor that plays a critical role in the development of many cell types (Hemesath et al., 1994; Steingrimsson et al., 2004), including melanocytes, retinal pigment cells, osteoclasts, and a subpopulation of CNS neurons (Nakayama et al., 1998; Steingrimsson et al., 2002; Gudjohnsen et al., 2015; Atacho et al., 2020). The Mitf gene has been shown to be both important for the generation (Kitamura et al., 2002; Morii et al., 2004; Sasaki et al., 2016; Ingason et al., 2019) and function of MCs (Morii et al., 1996, 1997; Nechushtan and Razin, 2002; Morii and Oboki, 2004; Paruchuru et al., 2022). There are connections between Mitf and Kit signaling (Tsujimura et al., 1996; Lee et al., 2011), and Mitf plays a key role in the binary lineage choice between MCs and basophils during development. The bipotent basophil/MC progenitors (BMCPs) develop into basophils or MCs depending on the relative ratio of C/EBPα to MITF, which are negatively correlated with each other (Qi et al., 2013; Sasaki et al., 2016). Mitf mutations cause these progenitors to develop into basophil-like cells. In Mitf null mutant mice, MCs are absent from the peritoneum (Morii and Oboki, 2004) and the heart (Ingason et al., 2019) and are reduced in number in the skin (Kim et al., 1999; Morii and Oboki, 2004). To date, the effect of the loss of Mitf on meningeal MCs has not been reported.

Animals of both sexes were used from the following mouse strains: C57BL/6 J (wild type) and Mitf mutant mice: heterozygote C57BL/6 J-Mi^-mi-vga9/+ and homozygote C57BL/6 J-Mitf^{-mi-vga9/−mi-vga9}. All animals were kept at the animal facility of the University of Iceland (VRIII) in a controlled environment at 21–22°C. Food and water were provided ad libitum. Sacrifice was performed by cervical dislocation. Animal procedures were approved by the Committee on Experimental Animals, according to Regulation 460/2017 and European Union Directive 2010/63 (license number 2013-03-01).

Following euthanasia, the calvarium was removed from the skull, and the meninges adherent to the calvarium were fixed in 4% PFA for 30 min and rinsed with cold PBS. The whole meninges were removed, permeabilized, and blocked with blocking buffer (0.1% Triton 100x and 5% normal goat serum in PBS) for 1 h at room temperature. The samples were incubated with avidin AF488 conjugated (Catalog number A21370, Invitrogen) diluted 1/1000 and DAPI in a blocking buffer for 1 h at room temperature (Tharp et al., 1985). Finally, the samples were washed three times with 0.1% Triton X-100 in PBS for 20 min and mounted on slides using Fluoromount. Images of the whole meninges were acquired using an Olympus FV10-MCPSU Confocal Microscope.

The meninges were stained attached to the calvarium. The samples were submerged in a toluidine blue staining solution (1 g toluidine blue/100 mL of 70% EtOH in dH2O) for 5 min at room temperature. Subsequently, the meninges were washed three times in distilled H₂O for 5 min, rinsed five times in 90% EtOH, and five times in 96% EtOH. Finally, the meninges were dissected from the calvarium and mounted on slides using solidifying mounting media. A Leica DM LB microscope (Leica Microsystems) at 10x magnification was used for image acquisition.

The results obtained from MC quantification were analyzed by unpaired Student’s t-tests using GraphPad Prism 9. Numerical results represent the mean amount of MC/mm² and the standard error of mean (SEM).

To determine the degree to which meningeal MCs are affected by the lack of Mitf, the dura was collected from wild-type, heterozygous, and mutant adult mice, 2–3 months old. MCs were stained with avidin, a glycoprotein that binds to the granules of MCs (Tharp et al., 1985; Figure 1). There was a clear absence of MCs in the dura of the mutant animals and a reduction in MC number in heterozygotes. To quantify the number of MCs, toluidine blue, a dye that stains specifically acidic tissue components, and MC granules in purple were used to identify MCs in meninges. Toluidine staining of dura MCs confirmed the reduction of MC number in the heterozygotes (41.04 /mm² ± 2.23 n = 14, wild type 49.9/mm² ± 2.23 n = 17), while in the mutant no MCs (n = 15) were detected (Figure 2). No difference was observed between the sexes (N = 10, heterozygotes p = 0.288, wild type p = 0.642). The reduction in the heterozygotes was on average 25%, and not all heterozygotes showed a reduction. Some variation in the difference was observed between different experiments (difference between wild-type and heterozygotes: 10–28%).