Up-regulation of the peroxiredoxin-6 related metabolism of reactive oxygen species in skeletal muscle of mice lacking neuronal nitric oxide synthase

Although neuronal nitric oxide synthase (nNOS) plays a substantial role in skeletal muscle physiology, nNOS-knockout mice manifest an only mild phenotypic malfunction in this tissue. To identify proteins that might be involved in adaptive responses in skeletal muscle of knockout mice lacking nNOS, 2D-PAGE with silver-staining and subsequent tandem mass spectrometry (LC-MS/MS) was performed using extracts of extensor digitorum longus muscle (EDL) derived from nNOS-knockout mice in comparison to C57Bl/6 control mice. Six proteins were significantly (P < or = 0.05) more highly expressed in EDL of nNOS-knockout mice than in that of C57 control mice, all of which are involved in the metabolism of reactive oxygen species (ROS). These included prohibitin (2.0-fold increase), peroxiredoxin-3 (1.9-fold increase), Cu(2+)/Zn(2+)-dependent superoxide dismutase (SOD; 1.9-fold increase), heat shock protein beta-1 (HSP25; 1.7-fold increase) and nucleoside diphosphate kinase B (2.6-fold increase). A significantly higher expression (4.1-fold increase) and a pI shift from 6.5 to 5.9 of peroxiredoxin-6 in the EDL of nNOS-knockout mice were confirmed by quantitative immunoblotting. The concentrations of the mRNA encoding five of these proteins (the exception being prohibitin) were likewise significantly (P < or = 0.05) higher in the EDL of nNOS-knockout mice. A higher intrinsic hydrogen peroxidase activity (P < or = 0.05) was demonstrated in EDL of nNOS-knockout mice than C57 control mice, which was related to the presence of peroxiredoxin-6. The treatment of mice with the chemical NOS inhibitor L-NAME for 3 days induced a significant 3.4-fold up-regulation of peroxiredoxin-6 in the EDL of C57 control mice (P < or = 0.05), but did not alter its expression in EDL of nNOS-knockout mice. ESR spectrometry demonstrated the levels of superoxide to be 2.5-times higher (P < or = 0.05) in EDL of nNOS-knockout mice than in C57 control mice while an in vitro assay based on the emission of 2,7-dichlorofluorescein fluorescence disclosed the concentration of ROS to be similar in both strains of mice. We suggest that the up-regulation of proteins that are implicated in the metabolism of ROS, particularly of peroxiredoxin-6, within skeletal muscles of nNOS-knockout mice functionally compensates for the absence of nNOS in scavenging of superoxide.

Serum Amyloid A, C-reactive Protein and Retinal Microvascular Changes in Hypertensive Diabetic and Non-diabetic Individuals: An ASCOT Substudy

To study the association of the inflammatory markers serum amyloid A (SAA) and C-reactive protein (CRP) with retinal microvascular parameters in hypertensive individuals with and without type 2 diabetes.

Diurnal variability of C-reactive protein in obstructive sleep apnea

OBJECTIVE: The objective of this study is to examine the diurnal variability of C-reactive protein (CRP) in obstructive sleep apnea (OSA). METHODS AND MEASUREMENTS: Participants included 44 women and men with untreated OSA (mean apnea/hypopnea index = 37.5, SD +/- 28) and 23 healthy adults with no OSA. Sleep was monitored with polysomnography in the University of California San Diego General Clinical Research Center. Over a 24-h period, blood was collected every 2 h, and CRP levels were determined. RESULTS: Adjusting for age, gender, and body mass index, a significant group by time interaction showed that patients with OSA had higher CRP levels during the daytime (8:00 a.m.-8:00 p.m.) versus the nighttime (10:00 p.m. until 6:00 p.m.; p < 0.001). Non-apneics showed no significant change in CRP levels during the 24 h. CONCLUSIONS: The findings indicate that sleep apnea patients have disproportionately elevated CRP levels in the day versus the nighttime, possibly as a result of carryover effects of nighttime arousal into the daytime.

Laser-Doppler measurements of spinal cord blood flow changes during hemilaminectomy in chondrodystrophic dogs with disk extrusion

OBJECTIVES: (1) To assess spinal cord blood flow (SCBF) during surgical treatment of disk extrusion in dogs and (2) to investigate associations between SCBF, clinical signs, presurgical MRI images, and 24-hour surgical outcome. STUDY DESIGN: Cohort study. ANIMALS: Chondrodystrophic dogs with thoracolumbar disk extrusion (n=12). METHODS: Diagnosis was based on clinical signs and MRI findings, and confirmed at surgery. Regional SCBF was measured intraoperatively by laser-Doppler flowmetry before, immediately after surgical spinal cord decompression, and after 15 minutes of lavaging the lesion. Care was taken to ensure a standardized surgical procedure to minimize factors that could influence measurement readings. RESULTS: A significant increase in intraoperative SCBF was found in all dogs (Wilcoxon's signed-rank test; P=.05) immediately after spinal cord decompression and after 15 minutes. Changes in SCBF were not associated with duration of clinical signs; initial or 24-hour neurologic status; or degree of spinal cord compression assessed by MRI. CONCLUSION: SCBF increases immediately after spinal cord decompression in dogs with disk herniation; however, increased SCBF was not associated with a diminished 24-hour neurologic status. CLINICAL RELEVANCE: An increase in SCBF does not appear to be either associated with the degree of spinal cord compression or of a magnitude sufficient to outweigh the benefit of surgical decompression by resulting in clinically relevant changes in 24-hour outcome.

Nitric oxide induces cell death in canine cruciate ligament cells by activation of tyrosine kinase and reactive oxygen species

BACKGROUND: There is increasing evidence suggesting that development of progressive canine cranial cruciate ligament (CCL) rupture involves a gradual degeneration of the CCL itself, initiated by a combination of factors, ranging from mechanical to biochemical. To date, knowledge is lacking to what extent cruciate disease results from abnormal biomechanics on a normal ligament or contrary how far preliminary alterations of the ligament due to biochemical factors provoke abnormal biomechanics. This study is focused on nitric oxide (NO), one of the potential biochemical factors. The NO-donor sodium nitroprusside (SNP) has been used to study NO-dependent cell death in canine cranial and caudal cruciate ligament cells and to characterize signaling mechanisms during NO-stimulation. RESULTS: Sodium nitroprusside increased apoptotic cell death dose- and time-dependently in cruciate ligamentocytes. Cells from the CCL were more susceptible to apoptosis than CaCL cells. Caspase-3 processing in response to SNP was not detected. Testing major upstream and signal transducing pathways, NO-induced cruciate ligament cell death seemed to be mediated on different levels. Specific inhibition of tyrosine kinase significantly decreased SNP-induced cell death. Mitogen activated protein kinase ERK1 and 2 are activated upon NO and provide anti-apoptotic signals whereas p38 kinase and protein kinase C are not involved. Moreover, data showed that the inhibition reactive oxygen species (ROS) significantly reduced the level of cruciate ligament cell death. CONCLUSIONS: Our data support the hypothesis that canine cruciate ligamentocytes, independently from their origin (CCL or CaCL) follow crucial signaling pathways involved in NO-induced cell death. However, the difference on susceptibility upon NO-mediated apoptosis seems to be dependent on other pathways than on these tested in the present study. In both, CCL and CaCL, the activation of the tyrosine kinase and the generation of ROS reveal important signaling pathways. In perspective, new efforts to prevent the development and progression of cruciate disease may include strategies aimed at reducing ROS.

Magnetic resonance imaging findings in dogs with traumatic intervertebral disk extrusion with or without spinal cord compression: 31 cases (2006-2010)

OBJECTIVE To determine the prevalence of spinal cord compression subsequent to traumatic intervertebral disk (IVD) extrusion in dogs, characterize factors associated with spinal cord compression in dogs with traumatic IVD extrusion, and evaluate the outcomes of dogs with traumatic IVD extrusion with or without spinal cord compression. DESIGN Retrospective case series. ANIMALS 31 dogs with traumatic IVD extrusion. PROCEDURES Medical records and MRI findings were reviewed for dogs with a history of trauma to the spinal region. Dogs were included in the study if a neurologic examination and MRI were performed and there was a description of clinical signs and MRI findings including identification of the spinal cord segment affected by IVD extrusion, presence or absence of spinal cord compression, treatment, and outcome available for review. RESULTS 31 of 50 (62%) dogs had traumatic IVD extrusions without any other detectable vertebral lesions; 9 (29%) and 22 (71%) of those 31 dogs did and did not have spinal cord compression, respectively. Dogs with spinal cord compression were significantly older and more likely to be chondrodystrophic and have evidence of generalized IVD degeneration, compared with dogs without spinal cord compression. The outcome for dogs with spinal cord compression was similar to that for dogs without spinal cord compression. CONCLUSIONS AND CLINICAL RELEVANCE Results indicated traumatic IVD extrusion was common and should be considered as a differential diagnosis for dogs with trauma to the spinal region, and spinal cord compression should be evaluated, especially in older or chondrodystrophic dogs.

Clinical and pathological analysis of epidural inflammation in intervertebral disk extrusion in dogs

BACKGROUND Little is known about the pathologic changes in the epidural space after intervertebral disk (IVD) extrusion in the dog. OBJECTIVES To analyze the pathology of the epidural inflammatory response, and to search for correlations between this process and clinical findings. METHODS Clinical data from 105 chondrodystrophic (CD) and nonchondrodystrophic (NCD) dogs with IVD extrusion were recorded. Epidural material from these dogs was examined histopathologically and immunohistochemically. Using statistical analysis, we searched for correlations between severity of epidural inflammation and various clinical and pathologic variables. RESULTS Most dogs exhibited an epidural inflammatory response, ranging from acute invasion of neutrophils to formation of chronic granulation tissue. The mononuclear inflammatory infiltrates consisted mostly of monocytes and macrophages and only few T and B cells. Surprisingly, chronic inflammatory patterns also were found in animals with an acute clinical history. Severity of the epidural inflammation correlated with degree of the epidural hemorrhage and nucleus pulposus calcification (P = .003 and .040), but not with age, chondrodystrophic phenotype, neurologic grade, back pain, pretreatment, or duration. The degree of inflammation was statistically (P = .021) inversely correlated with the ability to regain ambulation. CONCLUSION AND CLINICAL IMPORTANCE Epidural inflammation occurs in the majority of dogs with IVD extrusion and may develop long before the onset of clinical signs. Presence of calcified IVD material and hemorrhage in the epidural space may be the triggers of this lesion rather than an adaptive immune response to the nucleus pulposus as suggested in previous studies. Because epidural inflammation may affect outcome, further research is warranted.

Far lateral lumbar disc extrusion: MRI findings and surgical treatment

This case report describes the magnetic resonance imaging (MRI) findings and the treatment of a far lateral extrusion of disc material at the sixth and seventh lumbar vertebrae (L6-L7) in a five-year-old male Alpine Dachsbracke dog referred to our hospital for investigation of the complaint of a one week progressive lameness in the left pelvic limb and poorly localized back pain. An extra-foraminal left lateral disc herniation impinging on the sixth lumbar nerve root was diagnosed by MRI examinations. Due to the far lateral position of the extruded disc material on MRI, surgical opening of the spinal canal was not necessary. Removal of the herniated soft disc material impinging on the L6 nerve root, and fenestration of the L6-L7 disc was performed laterally. To the author's knowledge 'far-lateral' disc herniation beyond the neuroforamen without any spinal canal contact has not been described in dogs until now. A complete recovery with no evidence of pain was achieved only after a couple of weeks after surgery. We acknowledge that it is possible that other pathological mechanisms may have contributed to clinical signs and to a delayed recovery.