ALBERT

Description: 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.

Description: Lameness in dairy cows is a worldwide prevalent disease with a negative impact on animal welfare and herd economy. Oxidative damage and antioxidant system dysfunction are common features of many CNS diseases, including chronic pain. The aim of this study was to evaluate the levels of reactive oxygen species (ROS) and oxidative damage markers in the spinal cord of dairy cows with chronic inflammatory lameness. Locomotion score was performed in order to select cows with chronic lameness. Dorsal horn spinal cord samples were obtained post mortem from lumbar segments (L2–L5), and ROS, malondialdehyde (MDA), and carbonyl groups were measured along with the activity of superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), and total antioxidant response (TAR). Lame cows had increased levels of ROS, MDA, and carbonyl groups, while no differences were observed between lame and non-lame cows in SOD, GPx, CAT, and TAR activity. We conclude that painful chronic inflammatory lameness in dairy cows is associated with an increase in ROS, MDA, and carbonyl groups. Nonetheless, an association between ROS generation and dysfunction of the antioxidant system, as previously proposed, could not be established.

Description: BackgroundTail docking of dairy cows is a painful procedure that affects animal welfare level. The aims of this study were first to evaluate the response to mechanical and thermal stimulation, and second to determine the superficial temperature of the stump of tail-docked dairy cows.MethodsOne hundred and sixty-four dairy cows were enrolled. From these, 133 cows were assigned to the tail-docked (TD) group and 31 cows were selected as control animals. The following sensory assessments to evaluate pain in tail-docked cows were performed. Sensitivity of the tail region in both groups of animals was evaluated using a portable algometer. Cold and heat sensitivity assessment was performed using a frozen pack (0 °C) and warm water (45 °C), respectively. Pinprick sensitivity was evaluated using a Wartenberg neurological pinwheel. Superficial temperature was evaluated using a thermographic camera. All sensory assessments and superficial temperature were evaluated in the ventral surface of the tail stump (TD) and tail (C).ResultsPressure pain threshold was lower in TD cows (5.97 ± 0.19 kg) compared to control cows (11.75 ± 0.43 kg). Heat and cold sensitivity was higher in the TD cows compared to control cows with 29% and 23% of TD cows responding positively, respectively. Similarly, after pinprick sensitivity test was performed, 93% of TD cows elicited a positive response to stimulation. Tail-docked cows had lower superficial temperature (26.4 ± 0.27 °C) compared to control cows (29.9 ± 0.62 °C).DiscussionPressure pain threshold values in both groups of animals were higher than those previously reported for TD pigs, sows and cows. In contrast, pinprick stimulation evaluates the presence of punctate mechanical hyperalgesia/allodynia, usually related to traumatic nerve injury, and this association may reveal that it is possible that these animals developed a disorder associated to the development of a tail stump neuroma and concurrent neuropathic pain, previously reported in TD lambs, pigs and dogs. Thermal sensitivity showed that TD cows responded positively to heat and cold stimulation. These findings suggest that long-term TD cows could be suffering hyperalgesia/allodynia, which may be indicative of chronic pain. Lower superficial temperature in the stump may be associated to sympathetic fiber sprouting in the distal stump, which can lead to vasoconstriction and lower surface temperatures. Further studies are needed in order to confirm neuroma development and adrenergic sprouting.