Immunofluorescence characterization of spinal cord dorsal horn microglia and astrocytes in horses

Abstract

The role of glial cells in pain modulation has recently gathered attention. The objective of this study was to determine healthy spinal microglia and astrocyte morphology and disposition in equine spinal cord dorsal horns using Iba-1 and GFAP/Cx-43 immunofluorescence labeling, respectively. Five adult horses without visible wounds or gait alterations were selected. Spinal cord segments were obtained post-mortem for immunohistochemical and immunocolocalization assays. Immunodetection of spinal cord dorsal horn astrocytes was done using a polyclonal goat antibody raised against Glial Fibrillary Acidic Protein (GFAP) and a polyclonal rabbit antibody against Connexin 43 (Cx-43). For immunodetection of spinal cord dorsal horn microglia, a polyclonal rabbit antibody against a synthetic peptide corresponding to the C-terminus of ionized calcium-binding adaptor molecule 1 (Iba-1) was used. Epifluorescence and confocal images were obtained for the morphological and organizational analysis. Evaluation of shape, area, cell diameter, cell process length and thickness was performed on dorsal horn microglia and astrocyte. Morphologically, an amoeboid spherical shape with a mean cell area of 92.4 + 34 µm2 (in lamina I, II and III) was found in horse microglial cells, located primarily in laminae I, II and III. Astrocyte primary stem branches (and cellular bodies to a much lesser extent) are mainly detected using GFAP. Thus, double GFAP/Cx-43 immunolabeling was needed in order to accurately characterize the morphology, dimension and cell density of astrocytes in horses. Horse and rodent astrocytes seem to have similar dimensions and localization. Horse astrocyte cells have an average diameter of 56 + 14 µm, with a main process length of 28 + 8 µm, and thickness of 1.4 + 0.3 µm, mainly situated in laminae I, II and III. Additionally, a close association between end-point astrocyte processes and microglial cell bodies was found. These results are the first characterization of cell morphology and organizational aspects of horse spinal glia. Iba-1 and GFAP/Cx-43 can successfully immune-label microglia and astrocytes respectively in horse spinal cords, and thus reveal cell morphology and corresponding distribution within the dorsal horn laminae of healthy horses. The conventional hyper-ramified shape that is normally visible in resting microglial cells was not found in horses. Instead, horse microglial cells had an amoeboid spherical shape. Horse protoplasmic astroglia is significantly smaller and structurally less complex than human astrocytes, with fewer main GFAP processes. Instead, horse astrocytes tend to be similar to those found in rodent’s model, with small somas and large cell processes. Microglia and astrocytes were found in the more superficial regions of the dorsal horn, similarly to that previously observed in humans and rodents. Further studies are needed to demonstrate the molecular mechanisms involved in the neuron-glia interaction in horses.

Glial Fibrillary Acidic Protein and Ionized Calcium-Binding Adapter Molecule 1 Immunostaining Score for the Central Nervous System of Horses With Non-suppurative Encephalitis and Encephalopathies

“…In this study, normal astrocytes (grade 0) appeared as staining in a range of 0 to 30 astrocytes/×200 magnification that were well-ramified with minimal contact between cells and tightly connected with blood vessels as also described in the spinal cord of horses by Meneses et al (27) and in fibrous astrocytes from the cerebral cortex in control horses from Regina et al (28). Studies in other animal species describe a similar morphology in non-altered CNS of dogs (29), mice (30), and rats (31), but cattle have a higher number of GFAP+ cells and nonaltered astrocytes approximate morphologically to our grade 1 for horses (32).…”

New role of sertraline against Toxoplasma gondii‐induced depression‐like behaviours in mice

“…Under conditions such as parenchymal injury, degeneration or infection, the morphology, phenotype and function of microglia change significantly, 68 such as switching from ramified to amoeboid shapes and showing higher expression levels of the specific microglial marker Iba-1. 69,70 Because previous studies have found that after 5 days of infection with RH strain T. gondii, there is an infiltration of pseudocysts occupied by tachyzoite (s) in the brain cortical tissue and strong inflammatory infiltration, 71 we further studied the status of microglia cells and the content of pro-inflammatory cytokines. Consistent with the results of Hwang et al, 72 in addition to the enhanced expression of Iba-1 in microglia in acute T. gondii infection, the expression of pro-inflammatory cytokines (IFNγ and TNFα) were also increased.…”

Spinal cord microglia in health and disease

“…В литературе, посвященной оценке реакции спинного мозга на патологические стимулы, отмечено присутствие двух морфофункциональных типов микроглиоцитов, а также промежуточных форм [62][63][64][65]. Традиционно в спинном мозге выделяют покоящиеся (рамифицированные), активированные (амебоидные) микроглиоциты и переходные формы.…”

“…При активации микроглии, которая вызвана повреждением СМ, происходит сокращение количества и протяженности клеточных отростков. В конечном итоге клетки приобретают амебоидную форму, что сопровождается продукцией биоактивных молекул, тем самым способствуя активизации воспалительных реакций и борьбе с повреждающими агентами [62][63][64][65]. Известно, что амебоидная микроглия представляет собой высокомобильные фагоцитирующие клетки, участвующие в презентации антигена.…”