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Stress may cause or aggravate Bladder Pain Syndrome/Interstitial Cystitis (BPS/IC) symptoms. Thus, stress models are being refined to be used as systemic in counterpoint to bladder centric animal models We hypothesised that different stress insults modulate the insula activity diversely. Two models were used in female Wistar rats, Maternal Deprivation Model (MDM, stress applied to new-borns and studies done at 6M) and Water Avoidance Stress test (WAS, stress is applied to 6M rats for 1h every 10 days and studies carried afterwards). Mechanical pain threshold was evaluated in L6/S1 dermatomes which share the spinal cord segments of bladder sensory innervation. Bladder function was evaluated by cystometry. Then Insulas were removed. One was immersed in RNAlater, homogenized, RNA extracted, and expression of different biomarkers assessed by real-time PCR. The other insula was immunoreacted against microglia markers Iba1 and Cd68. The analysis of positive cells used LASAF and ImageJ software. Morphometric analysis and 3D reconstructions used IMARIS software.

Microglia have been increasingly implicated in neurodegenerative diseases (NDs), and specific disease associated microglia (DAM) profiles have been defined for several of these NDs. Yet, the microglial profile in Machado–Joseph disease (MJD) remains unexplored. Here, we characterized the profile of microglia in the CMVMJD135 mouse model of MJD. This characterization was performed using primary microglial cultures and microglial cells obtained from disease-relevant brain regions of neonatal and adult CMVMJD135 mice, respectively. Machine learning models were implemented to identify potential clusters of microglia based on their morphological features, and an RNA-sequencing analysis was performed to identify molecular perturbations and potential therapeutic targets. Our findings reveal morphological alterations that point to an increased activation state of microglia in CMVMJD135 mice and a disease-specific transcriptional profile of MJD microglia, encompassing a total of 101 differentially expressed genes, with enrichment in molecular pathways related to oxidative stress, immune response, cell proliferation, cell death, and lipid metabolism. Overall, these results allowed us to define the cellular and molecular profile of MJD-associated microglia and to identify genes and pathways that might represent potential therapeutic targets for this disorder., This work was supported by Fundação para a Ciência e a Tecnologia (FCT) (PTDC/NEUNMC/3648/2014) and COMPETE-FEDER (POCI-01-0145-FEDER-016818). It was also supported by Portuguese funds through FCT in the framework of the Project POCI-01-0145-FEDER-031987 (PTDC/MED-OUT/31987/2017). A.B.C. was supported by a doctoral fellowship from FCT (PD/BD/ 127828/2016). S.P.N. was also supported by FCT (PD/BD/114120/2015). Work in the JBR laboratory was financed by FEDER—Fundo Europeu de Desenvolvimento Regional funds through the COMPETE 2020—Operational Programme for Competitiveness and Internationalization (POCI), Portugal 2020, and by Portuguese funds through FCT in the framework of the Project POCI-01-0145- FEDER030647 (PTDC/MED-NEU/31318/2017). This work was funded by ICVS Scientific Microscopy Platform, member of the national infrastructure PPBI (Portuguese Platform of Bioimaging) (PPBIPOCI-01-0145-FEDER-022122), and by National funds, through FCT—project UIDB/50026/2020 and UIDP/50026/2020.