3-Dimensional Immunostaining and Automated Deep-Learning Based Analysis of Nerve Degeneration

Abstract

Multiple sclerosis (MS) is an autoimmune and neurodegenerative disease driven by inflammation and demyelination in the brain, spinal cord, and optic nerve. Optic neuritis, characterized by inflammation and demyelination of the optic nerve, is a symptom in many patients with MS. The optic nerve is the highway for visual information transmitted from the retina to the brain. It contains axons from the retinal ganglion cells (RGCs) that reside in the retina, myelin forming oligodendrocytes and resident microglia and astrocytes. Inflammation, demyelination, and axonal degeneration are also present in the optic nerve of mice subjected to experimental autoimmune encephalomyelitis (EAE), a preclinical mouse model of MS. Monitoring the optic nerve in EAE is a useful strategy to study the presentation and progression of pathology in the visual system; however, current approaches have relied on sectioning, staining and manual quantification. Further, information regarding the spatial load of lesions and inflammation is dependent on the area of sectioning. To better characterize cellular pathology in the EAE model, we employed a tissue clearing and 3D immunolabelling and imaging protocol to observe patterns of immune cell infiltration and activation throughout the optic nerve. Increased density of TOPRO staining for nuclei captured immune cell infiltration and Iba1 immunostaining was employed to monitor microglia and macrophages. Axonal degeneration was monitored by neurofilament immunolabelling to reveal axonal swellings throughout the optic nerve. In parallel, we developed a convolutional neural network with a UNet architecture (CNN-UNet) called BlebNet for automated identification and quantification of axonal swellings in whole mount optic nerves. Together this constitutes a toolkit for 3-dimensional immunostaining to monitor general optic nerve pathology and fast automated quantification of axonal defects that could also be adapted to monitor axonal degeneration and inflammation in other neurodegenerative disease models.

Cellular rejuvenation protects neurons from inflammation mediated cell death

“…One model is the experimental autoimmune encephalomyelitis (EAE) mouse which recapitulates multiple aspects of MS neuronal pathology, particularly in the retina and optic nerve. Here, EAE mice exhibit demyelinating lesions of the optic nerve, immune cell infiltration and gliosis, retinal nerve layer thinning and neuronal death, axonal pathology, and loss of visual acuity throughout the disease course, features which are likewise observed in MS patients 19 . It stands to reason that conducting RNA-sequencing of retinal ganglion cells (RGCs) from EAE mice and comparing these data with preexisting sequencing datasets of neurons from MS patients may provide insight into a conserved neuronal response to pathological inflammation.…”

Senolytic treatment depletes microglia and decreases severity of experimental autoimmune encephalomyelitis

“…Active induction of MOG-EAE was achieved as previously described 61–63 . Briefly, 8-week-old female C57Bl6 mice received subcutaneous MOG peptide emulsified in complete Freund’s adjuvant (CFA) in 100 uL volume (50 uL injected bilaterally per mouse).…”

Exploring Quality of Life in Patients with Multiple Sclerosis: A Multidimensional Perspective

“…Multiple sclerosis (MS) is a chronic autoimmune disorder characterized by inflammation and damage to the central nervous system, including the brain, spinal cord, and optic nerves (Yolanda & Ritarwan, 2022;Zyla-Jackson, 2023). It presents with a diverse array of physical, cognitive, and emotional symptoms (Drake et al, 2022). The prevalence of MS varies across different regions, with higher rates observed in temperate climates like Northern Europe and North America (Attia & Ibrahim, 2023;Wada et al, 2023;Schepers et al, 2023).…”