Molecular Imaging Study of the Immune Response in Muscle Denervation: a High-Tech Study in Murine Models and Human Patients

Aim of the study: Muscle denervation occurs in various clinical settings, including trauma, amyotrophic lateral sclerosis (ALS) and Charcot-Marie-Tooth disease (CMT). The body’s response to periferal nervous system nerve damage involves multiple cell types, including axonal Schwann cells (SCs), terminal (perisynaptic) Schwann cells (tSCs), endothelial cells, and immune cells such as macrophages, neutrophils and T-cells. Molecular imaging has emerged as a powerful and non-invasive approach for investigating the spatial and temporal dynamics of microinflammatory processes in vivo. This methodology allows a non- invasive visualization, characterization and quantification of biological processes at the molecular and cellular levels in living organism including humans. Material and Methods: The three main objectives of the study are: 1) in vivo and ex vivo evaluation in murine models of the dynamic of the immune response at the muscle and nerve level across three different neuromuscular disorders characterized by denervation (traumatic peripheral nerve injury, genetically inherited peripheral neuropathy, and amyotrophic lateral sclerosis). 2) to evaluate the translational feasibility of advanced imaging protocols and their associated findings from murine models to three distinct cohorts of patients affected by Charcot-Marie-Tooth disease type 1A (CMT1A), amyotrophic lateral sclerosis (ALS) and traumatic peripheral nerve injury. 3) application of texture analysis techniques to ultrasound images obtained from the aforementioned patient cohorts and murine models, with the aim of extracting quantitative parameters that correlate with other imaging modalities (MRI and PET), as well as with clinical and histological data. Four types of rodents were purchased and housed at the Animal Facility of the IRCCS Ospedale Policlinico San Martino. n = 24 SOD1/G93A mice and n = 24 wild-type controls were assessed at 60, 90, and 120 days of age; n= 16 SD-Tg (Pmp22) Kan transgenic rats and n=16 wild type controls were assessed 90 and 300 days of age; n=16 MpzD61N/+ mice and n=16 MpzD61N/N mice along with n=16 wild-type controls were evaluated at 90 and 300 days of age; n = 24 C57BL/6 mice underwent focal sciatic nerve crush injury at 60 days of age under appropriate anesthesia and post-operative analgesia and animals were evaluated at 7, 14, and 90 days after nerve injury. The contralateral (uninjured) limb was used as healthy control. The imaging equipment used to carry out the protocol included: Magnetic Resonance Imaging system 7 Tesla, Multimodal Imaging Ultrasound System Vevo LAZR-X, MicroPET system. At each time point, the murine models were subjected to a series of behavioral tests aimed at assessing their motor function. Patient recruitment includes Patients with CMT1a (expected number n = 30), Patients with traumatic nerve injury (expected number n = 20), Patients with ALS (exptected number n= 40). Ultrasound images from animal models were acquired immediately after MRI while still anesthetized. All patients underwent MRI examination on the same day as their clinical visit, using a 3T machine and undergo ultrasound examination on the same day as the MRI. Results: Objective 1. In SOD1/G93A Mice MRI parameters showed a statistically significant difference (p < 0.05) between mutant and control mice only in the 120-day-old group, and only for Fractional Anisotropy (FA) values obtained from Diffusion Tensor Imaging (DTI) sequences. MicroPET imaging showed tracer accumulation in the lower limb muscles of all mutant mice starting at 60 days of age, emerging as the most sensitive parameter for detecting altered muscle metabolism. In CMT1A Model: SD-TG (Pmp22) Kan Transgenic Rats MRI revealed statistically significant differences compared to controls for both Fractional Anisotropy (FA) and T1 values in the proximal segment of the sciatic nerve at 300 days of age. In CMT1B Model: MpzD61N/+ mutant mice MRI analysis demonstrated a progressive reduction in fractional anisotropy (FA) values between the 90 and 300 day old mutant mouse groups (p < 0.035), with lower values observed in homozygous mice. FA thus appears to be a valid biomarker of disease severity. In Traumatic nerve injury (TNI) model: C57BL/6 mice MRI analysis showed an increase in nerve size proximal to the lesion at 14 days compared to 7 days post-injury, associated with an elevated T2 signal; microPET imaging analysis, showed tracer uptake in the muscles innervated by the sciatic nerve distal to the crush site supporting the highly expression of TSPO in activated macrophages. Objective 2. To date n=14/30 CMT1A patients have completed the observational study protocol. The cross sectional area, fractional anisotropy (FA), and T1 and T2 relaxation times of the lumbosacral plexus roots and the sciatic nerve were significantly different between the patients and healthy controls (p < 0.05); the inter-observer reliability of the MRI measurements was good (ICC = 0.65); T1 relaxation times and FA values of L5 and S1, measured bilaterally, were significantly correlated with CMTNS and BBS; Sciatic nerve fractional anisotropy (FA) showed significant correlations with both CMTNS and BBS. Objective 3. We are currently working on the application of texture analysis techniques to ultrasound images obtained from the aforementioned patient cohorts and murine models, to establish correlations between advanced imaging metrics, both from nerve and muscle, and the extent of inflammatory cell infiltration, with the goal of identifying non-invasive biomarkers of disease activity. Conclusions: Our primary aim was to evaluate the extent and nature of immune system activation across these models, and to explore its potential role in both nerve degeneration and regeneration. Interestingly, our study revealed clear differences in immune activation across the three models, suggesting that inflammatory infiltration may not be a common feature across all peripheral neuropathies, but rather a phenomenon limited to specific subtypes. With regard to humans, to date, the most promising finding that has emerged is the potential use of T1 mapping in MRI as a biomarker of disease severity in certain types of peripheral neuropathy, especially in CMT1a patients. Nevertheless, this finding clearly merits further investigation to validate its relevance.