Changes in Dynamic Thiol/Disulfide Homeostasis, and Substance P, B-Endorphin and α-Tocopherol Concentrations in the Spinal Cord of Chronically Lame Dairy Cows.

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.

Proteomic profiling of proteins in the dorsal horn of the spinal cord in dairy cows with chronic lameness.

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.

Spinal Reactive Oxygen Species and Oxidative Damage Mediate Chronic Pain in Lame Dairy Cows.

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.

Plasma Concentration of Norepinephrine, ß-endorphin, and Substance P in Lame Dairy Cows.

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.

Mechanical/thermal sensitivity and superficial temperature in the stump of long-term tail-docked dairy cows.

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.

Microglia and astrocyte activation in the spinal cord of lame horses.

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.

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

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.

Cerebrospinal fluid tau levels are a marker for molecular subtype in sporadic Creutzfeldt-Jakob disease.

The molecular subtype of sporadic Creutzfeldt-Jakob disease (sCJD) is an important prognostic marker for patient survival. However, subtype determination is not possible during lifetime. Because the rate of disease progression is associated with the molecular subtype, this study aimed at investigating if total tau, a marker of neuronal death, allows premortem diagnosis of molecular subtype when codon 129 genotype is known. Two hundred ninety-six sCJD patients were tested for their cerebrospinal fluid total tau level at the time of diagnosis and were investigated for their sCJD subtype postmortem. There was a significant association between tau levels and the prion protein type in patients with codon 129 MM (p < 0.001), MV (p = 0.004), and VV (p = 0.001) genotype. Receiver operating characteristic analyses showed values of area under the curve of 0.76-0.80 for the different genotypes indicating a good diagnostic validity of the test. Total tau can be used as a diagnostic test for the assessment of prion protein type when codon 129 genotype is known. It provides valuable information for physicians and next of kin about the further course of disease.

The effect of four different irrigation systems in the removal of a root canal sealer.

OBJECTIVES: The aim of this study was to compare the efficiency of sonic, ultrasonic, and hydrodynamic devices in the removal of a root canal sealer from the surface and from simulated irregularities of root canals. MATERIALS AND METHODS: Fifty-three root canals with two standardized grooves in the apical and coronal parts of longitudinally split roots were covered with AH Plus root canal sealer. Compared were the effects of (control) syringe irrigation, (1) CanalBrush, (2) passive ultrasonic irrigation, (3) EndoActivator, and (4) RinsEndo on the removal of the sealer. The specimens were divided into four groups (N = 12) and one control group (N = 5) via randomization. The amount of remaining sealer in the root canal irregularities was evaluated under a microscope using a 4-grade scoring system, whereas the remaining sealer on the root canal surface was evaluated with a 7-grade scoring system. RESULTS: Passive ultrasonic irrigation is more effective than the other tested irrigation systems or syringe irrigation in removing sealer from root canal walls (p < 0.01). None of the techniques had a significant effect on cleaning the lateral grooves. CONCLUSIONS: Within the limitations of this study protocol ultrasonic irrigation shows a superior effect on sealer removal from the root canal surface during endodontic retreatment. Cleaning of lateral grooves seems not to be possible with one of the techniques investigated. CLINICAL RELEVANCE: Incomplete removal of root canal sealer during re-treatment may cause treatment failure. Passive Ultrasonic irrigation seems to be the most effective system to remove sealer from a root canal.

Prognostic marker for liver disease due to alpha1-antitrypsin deficiency.

BACKGROUND: Only some Alpha1-antitrypsin deficiency (A1ATD) PiZZ patients develop liver cirrhosis and portal hypertension. Aim of the study was to investigate the course of liver disease associated with PiZZ A1ATD and to determine prognostic factors. PATIENTS: We retrospectively reviewed the clinical and laboratory data of all PiZZ children up to 18 years of age admitted to our centre since 1978. 53 patients (age at first visit 2 days to 12 years) met our criteria. METHODS: The children were divided into 2 groups: group 1 'bad prognosis', meaning the patients which were on the waiting list for liver transplantation (LTx), had a liver transplantation or had died, and group 2 'good prognosis', containing the patients they were living with their own liver. We analysed family history including smoking, gestational age, maternal age at delivery, date of birth, sex, neonatal history, breast-feeding, symptoms at presentation, clinical and laboratory data and date of LTx and/or death. RESULTS: Various anamnesis parameters such as manifestation of neonatal cholestasis showed no prognostic significance. In contrast the laboratory parameters thrombocytes (p=0.008), bilirubin (p<0.001), prothrombin time (p<0.001), choline-sterase (p<0.001), gamma-GT (p=0.001) and GOT (p=0.002) showed a correlation with a liver transplantation and/or death. CONCLUSION: Prognosis is difficult to determine at an early stage of this disease, but various laboratory parameters can help to predict an outcome. Therefore a regular follow-up is necessary for the children.

Prevalence and prognostic impact of comorbidities in amyotrophic lateral sclerosis.

BACKGROUND AND PURPOSE: Amyotrophic lateral sclerosis (ALS) is characterized by rapidly progressive paralysis of striated muscles due to the loss of upper and lower motor neurons. The disease leads to death within 2-5 years, mainly due to respiratory failure. The pathogenesis of ALS is still unexplained for the most part. In this study, we aimed to determine the prevalence of different cardiovascular, metabolic, and neuropsychiatric comorbidities in a large ALS cohort and to evaluate their influence on the disease course. METHODS: A cohort of 514 patients with ALS of our ALS outpatient clinic was investigated retrospectively with reference to known prognostic factors and comorbidities. The prevalence of concomitant diseases was compared with the data from the German general population. Uni- and multivariate survival analyses were performed using the Cox proportional hazards model and Kaplan-Meier analysis. RESULTS: The prevalence of cardiovascular diseases and cardiovascular risk factors was significantly lower in patients with ALS compared to the German general population, whilst the prevalence of dementia, parkinsonism, and depressive symptoms was significantly higher in the ALS cohort. None of the investigated comorbidities had an influence on the disease course or on the survival of patients. CONCLUSIONS: Persons with cardiovascular diseases or risk factors seem to be at lower risk of ALS. Although these diseases are apparently somehow protective regarding ALS susceptibility, their presence did not modify disease progression and survival in patients with ALS. Our study further confirms the well-known continuum between ALS and dementia. It also suggests a link with other neurodegenerative diseases such as Parkinson's disease.

[Analysis of work flow in a radiology center using multi-moment-analysis: methodical principles]. / Analyse der Arbeitsabläufe eines radiologischen Zentrums mittels Multimoment-Häufigkeits-Studie: Methodische Grundlagen.

Radiology departments at hospitals are closely watched for their economy. The introduction of fast scanner technology usually triggers questions about simultaneously reducing final report times. The aim of the present study was first to examine, if the multi-moment analysis (MMH) is an appropriate method for structural and quantitative analysis of complex work flows in Radiology, and second, if factors negatively influencing the core processes can be quantitatively evaluated. The study was performed in the Departments of Radiology and Neuroradiology at a major University Hospital in Northern Germany. Randomized monitoring of the workflow of more than 80 doctors and radiology technicians assistants resulted in 800 observations per workplace. The high observation rate yielded a high accuracy (95% confidence interval width of less than +3,5%) to determine the proportion of different work patterns. MMH proved to be an appropriate means to analyze and quantify complex work flows at a university radiology centre. Different workplaces could be compared and total daily labor time could be derived from the observations. Circadian variabilities could be differentiated. The potential of the MMH to guide workflow optimization was identified. On the basis of data collected, MMH restructuring and control systems can be planned.

Femoral nail removal should be restricted in asymptomatic patients.

Femoral nail removal has been suggested as a routine procedure for symptomatic and asymptomatic patients. The aim of this study was to evaluate whether asymptomatic patients have long-term complaints after femoral nail removal. Additionally, factors that might extend operation time and effect soft tissue healing negatively were analyzed. A retrospective study on the removal of 164 femoral nails after fracture consolidation was done. A review of the patient charts and radiographs was done. Neither the interval between nail implantation and nail removal or the use of an end-cap significantly influenced the operation time. The surgeon's qualification and nail depth were the only variables that influenced operation time. Neither operation time, nail depth, surgeon's qualification, nor the grade of heterotopic ossification significantly influenced the incidence of wound healing problems. One hundred nine patients (73%) were available for the followup interview. Preoperatively, 58 patients (53%) had local complaints. Of these 58 patients, 78% reported improvement and 7% reported an aggravation of their local complaints. Of the 51 patients who were asymptomatic before surgery, 20% reported long-term complaints at followup. Only symptomatic patients should be considered for femoral nail removal.

[Eclampsia. Incidence and therapy, results of the University Gynecological Clinic Göttingen]. / Eklampsie. Häufigkeit und Therapie, Ergebnisse der Universitäts-Frauenklinik Göttingen.

The occurrence of eclampsia in the last two decades in the women's hospital of the University of Goettingen was evaluated. In a total of 32,578 birth there was an incidence of 0.11%. Maternal mortality was 16.2%, perinatal mortality 19.5%. 73% of the patients were primiparae, 24.3% unmarried pregnant women. A regression of the frequency of eclampsia is seen within the last 20 years and certainly due to the intensive antenatal care.