RESUMO

Initial lameness inflammation leads to chronic lameness and development of chronic pain due to the release of pro-inflammatory mediators such as reactive oxygen species (ROS), which are implicated in the transition from acute to chronic pain, and free radical scavengers countering thiol, substance P (SP), and ß-endorphin (BE). The present study aimed to evaluate the dynamic thiol-disulfide homeostasis, α-tocopherol concentrations and SP and BE concentrations in the spinal cord of chronically lame dairy cows. Ten lame and 10 non-lame cows with a parity range of 2-6 were selected for the study. Lame cows had a history of up to 3 months of lameness. Spinal cord samples were obtained from the L2 to L4 lumbar vertebrae aspect of each animal. A thiol-disulfide homeostasis assay was performed using absorbance, and the α-tocopherol concentration was determined by HPLC. SP and BE concentrations were measured using ELISA kits. The results indicated that SP and BE were significantly higher in the spinal cord of lame cows. In contrast, disulfide levels and α-tocopherol concentrations were significantly lower in the spinal cord of lame cows. In conclusion, disulfide levels and α-tocopherol concentrations indicated a defective antioxidant response in cows with chronic lameness. The results of SP and BE concentrations suggested chronic pain and a defective endogenous analgesic response.

RESUMO

Chronic lameness affects bovine welfare and has a negative economic impact in dairy industry. Moreover, due to the translational gap between traditional pain models and new drugs development for treating chronic pain states, naturally occurring painful diseases could be a potential translational tool for chronic pain research. We therefore employed liquid chromatography tandem mass spectrometry (LC-MS/MS) to stablish the proteomic profile of the spinal cord samples from lumbar segments (L2-L4) of chronic lame dairy cows. Data were validated and quantified through software tool (Scaffold® v 4.0) using output data from two search engines (SEQUEST® and X-Tandem®). Search Tool for the Retrieval of Interacting Genes/Proteins (STRING) analysis was performed to detect proteins interactions. LC-MS/MS identified a total amount of 177 proteins; of which 129 proteins were able to be quantified. Lame cows showed a strong upregulation of interacting proteins with chaperone and stress functions such as Hsp70 (p < 0.006), Hsc70 (p < 0.0079), Hsp90 (p < 0.015), STIP (p > 0.0018) and Grp78 (p <0.0068), and interacting proteins associated to glycolytic pathway such as; γ-enolase (p < 0.0095), α-enolase (p < 0.013) and hexokinase-1 (p < 0.028). It was not possible to establish a clear network of interaction in several upregulated proteins in lame cows. Non-interacting proteins were mainly associated to redox process and cytoskeletal organization. The most relevant down regulated protein in lame cows was myelin basic protein (MBP) (p < 0.02). Chronic inflammatory lameness in cows is associated to increased expression of stress proteins with chaperone, metabolism, redox and structural functions. A state of endoplasmic reticulum stress and unfolded protein response (UPR) might explain the changes in protein expression in lame cows; however, further studies need to be performed in order to confirm these findings.

Assuntos

Doenças dos Bovinos/genética , Dor Crônica/veterinária , Regulação da Expressão Gênica , Coxeadura Animal/genética , Proteína Básica da Mielina/genética , Proteínas do Tecido Nervoso/genética , Animais , Bovinos , Doenças dos Bovinos/metabolismo , Doenças dos Bovinos/fisiopatologia , Dor Crônica/genética , Dor Crônica/metabolismo , Dor Crônica/fisiopatologia , Indústria de Laticínios , Feminino , Perfilação da Expressão Gênica , Ontologia Genética , Redes Reguladoras de Genes , Proteínas de Choque Térmico/genética , Proteínas de Choque Térmico/metabolismo , Isoenzimas/genética , Isoenzimas/metabolismo , Lactação/fisiologia , Coxeadura Animal/metabolismo , Coxeadura Animal/fisiopatologia , Anotação de Sequência Molecular , Proteína Básica da Mielina/metabolismo , Proteínas do Tecido Nervoso/classificação , Proteínas do Tecido Nervoso/metabolismo , Fosfopiruvato Hidratase/genética , Fosfopiruvato Hidratase/metabolismo , Proteômica/métodos , Corno Dorsal da Medula Espinal/metabolismo , Corno Dorsal da Medula Espinal/fisiopatologia

RESUMO

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.

RESUMO

INTRODUCTION: Lameness is a painful and debilitating condition that affects dairy cows worldwide. The aim of this study was to determine the plasma concentration of norepinephrine, ß-endorphin, and substance P in dairy cows with lameness and different mobility scores (MS). MATERIAL AND METHODS: A total of 100 Friesian and Jersey cows with lameness (parity range: 1-6; weight: 400-500 kg; milk yield: 22-28 L a day, and lactation stage less than 230 days) were selected. Animals were selected and grouped according to MS (MS 0-3; n = 25), and plasma concentration of norepinephrine, substance P, and ß-endorphin was measured using ELISA. RESULTS: Cows with MS 3 had higher plasma concentrations of norepinephrine and substance P and lower plasma concentrations of ß-endorphins when compared to MS 0 cows. CONCLUSION: Variations in plasma concentration of norepinephrine, substance P, and ß-endorphin could be associated with intense pain states in dairy cows with lameness, but are insufficient to differentiate these states from the mildest pain states. Further studies are necessary in order to evaluate the potential use of these biomarkers in the detection of chronic bovine painful conditions.

RESUMO

BACKGROUND: Tail 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. METHODS: One 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). RESULTS: Pressure 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). DISCUSSION: Pressure 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.

RESUMO

OBJECTIVE: To determine the microglial and astrocyte response to painful lameness in horses. STUDY DESIGN: Ionized calcium binding adaptor molecule 1 (Iba-1) and glial fibrillary acidic protein (GFAP) expression, cell density and morphology were determined through immunofluorescence within the dorsal horn of equine spinal cord. ANIMALS: A total of five adult horses with acute or chronic unilateral lameness, previously scheduled for euthanasia. METHODS: Musculoskeletal lameness was evaluated in five horses through visual evaluation according to clinical guidelines. Spinal cord samples were obtained immediately after euthanasia, and distal limb lesions were confirmed through dissection and radiography. Iba-1 immunostaining was used for detection and characterization of dorsal horn microglia. GFAP was used for immunostaining of dorsal horn astrocytes. Iba-1 and GFAP labeled cells were quantified in the dorsal horn, and intensity of fluorescence was compared between the ipsi- and contralateral dorsal horn to the affected limb, and between dorsal horn segments of all horses. RESULTS: Iba-1 expression was higher in the ipsilateral dorsal horn of the affected limb in contrast to the contralateral side dorsal horn. GFAP markers did not demonstrate increased astrocytic activity on the dorsal horn ipsilateral side to the distal limb lesion of affected horses. Horses with acute lameness predominantly had a spherical shape microglial phenotype, while cells from chronic lameness cases had variable morphology. Astrocytes evidenced small somas and large processes in both acute and chronic lameness, with higher GFAP localization in the main branches. As in the case of rodents, the localization of microglia and astrocytes in horses was mainly situated within laminae I, II and III. CONCLUSIONS AND CLINICAL RELEVANCE: Iba-1 and GFAP are functional and morphological markers of spinal microglial cells and astrocytes in horses with lameness.

Assuntos

Astrócitos/fisiologia , Doenças dos Cavalos/fisiopatologia , Coxeadura Animal/fisiopatologia , Microglia/fisiologia , Medula Espinal/fisiopatologia , Animais , Proteínas de Ligação ao Cálcio/metabolismo , Imunofluorescência/veterinária , Proteína Glial Fibrilar Ácida/metabolismo , Cavalos , Medula Espinal/metabolismo

RESUMO

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.