The effect of depleting the CNS Border associated macrophages at pre-symptomatic stage of ALS on neuroinflammation, symptoms, and survival

Background: Amyotrophic lateral sclerosis (ALS) is an incurable motor neuron disease characterized by progressive muscle atrophy, including respiratory muscles. Muscle weakness, wasting, cramps, and fasciculations result from the loss of lower motor neurons. Additionally, patients with ALS may experience extra-motor symptoms due to neuronal loss in the frontotemporal cortex. Survival typically ranges from 2 to 4 years after disease onset, with respiratory failure being the primary cause of death. ALS, the most common motor neuron disease in adults, has an incidence of 2 per 100,000 and a prevalence of 5.4 per 100,000. Despite extensive studies on ALS pathogenesis, the exact mechanism of neurodegeneration remains unclear. Perivascular macrophages (PVMs) are myeloid cells residing in the central nervous system (CNS) and are part of the larger group of macrophages known as CNS border-associated macrophages. In a healthy state, these cells phagocytize harmful substances that infiltrate from the vasculature and regulate immune responses. However, in an inflammatory state, they contribute to increased vascular permeability and subsequent neuroinflammation. Dysregulation of the blood-brain barrier (BBB) occurs well before the symptomatic stage of ALS and influences PVMs, leading to increased reactive oxygen species (ROS) generation and BBB permeability. Transformed PVMs, in collaboration with other brain myeloid cells, initiate proinflammatory processes. Aim: Our aim is to investigate the contribution of PVMs to ALS pathogenesis and their impact on neuroinflammation. To achieve this, we depleted PVMs at the presymptomatic stage of ALS and evaluated microglia activation, astrogliosis, BBB disruption, symptoms, and lifespan in an animal model. Methods: PVM depletion was achieved by injecting clodronate-loaded liposomes or PBS-loaded liposomes (as a control) into the cisterna magna of SOD1(G93A) mutated rats, an ALS animal model. The study included 59 SOD1 mutated rats (both male and female). Preliminary studies involved nine rats, while the remaining fifty rats received either clodronate-loaded liposomes (depleted group) or PBS-loaded liposomes (sham group) via cisternal injection. The clodronate concentration in the suspension was 5 mg/ml. Rats were divided into three experiments: early single injection (day 80 after birth), late single injection (day 160 after birth), and multiple injections (administered at day 80 after birth followed by two additional injections every 20 days). Rats were monitored until reaching the humane endpoint, when the rats cannot turn back after being placed on their side, with weight measured twice a week and locomotor activity tested weekly to assess neuromuscular strength, motor coordination, and balance. The locomotor activity test comprised gait analysis, tail elevation, balance beam test, paw-grip endurance, extension reflex, rearing behavior, and ladder rung test for paw placement accuracy during walking. Validation studies included wild-type (WT) rats from a Sprague Dawley background for comparison of PVMs between SOD1 and WT rats, injection point validation, and confirmation of PVM depletion. FITC-Albumin injection into the cisterna magna of two WT rats confirmed appropriate delivery to the brain and spinal cord. Immunofluorescent staining of astrocytes, microglia, and CD206+ cells in obtained tissues from sacrificed animal models was conducted using GFAP, Iba 1, and CD206 primary antibodies, respectively. Statistical methods were employed to analyze behavior scoring and weight change, as well as staining results, between depleted and sham groups in the three experiments. Results: Preliminary studies on CNS tissues from SOD1 and WT rats revealed upregulation of CD206+ cells in various brain and spinal cord regions of SOD1 mutated rats. FITC-Albumin injection confirmed delivery to the brain and spinal cord, colocalizing with PVMs, validating the injection point for subsequent studies. Immunofluorescence staining with CD206 for brain and spinal cord sections of WT rats showed downregulation of CD206+ cells in all regions of the depleted group, with significant results for the spinal cord in the multiple injection experiment. Weight change analysis indicated a delayed presymptomatic stage in the depleted group across all three experiments, with significant results for the multiple injection experiment. Behavioral studies confirmed milder or delayed symptomatic stages in the depleted groups, particularly in the multiple injection experiment. Immunofluorescence staining for PVMs, astrocytes, and microglia revealed upregulation of all three cell types in the ventral horn of the lumbar section of the spinal cord in the depleted group compared to the sham group in both the late single injection and multiple injection experiments. Conclusion: This study demonstrated that depleting perivascular macrophages at the presymptomatic stage of ALS can delay disease onset, particularly in animals receiving repetitive doses of clodronate-loaded liposomes. Locomotor activity tests and weight change records showed a delayed and milder symptomatic phase in these animals. The upregulation of CD206+ cells, microglia, and astrocytes at the endpoint in the ventral horn of the depleted group appears to be associated with the anti-inflammatory phenotype of CD206+ cells, astrocytes, and microglia, correlating with improved motor behavior in this group.