Different Roles of 12/15-Lipoxygenase in Diabetic Large and Small Fiber Peripheral and Autonomic Neuropathies
2010; Elsevier BV; Volume: 177; Issue: 3 Linguagem: Inglês
10.2353/ajpath.2010.100178
ISSN1525-2191
AutoresIrina G. Obrosova, Roman Stavniichuk, Viktor R. Drel, Hanna Shevalye, Іgor Vareniuk, Jerry L. Nadler, Robert E. Schmidt,
Tópico(s)Heart Rate Variability and Autonomic Control
ResumoUp-regulation of 12/15-lipoxygenase, which converts arachidonic acid to 12(S)- and 15(S)-hydroxyeicosatetraenoic acids, causes impaired cell signaling, oxidative-nitrosative stress, and inflammation. This study evaluated the role for 12/15-lipoxygenase in diabetic large and small fiber peripheral and autonomic neuropathies. Control and streptozotocin-diabetic wild-type and 12/15-lipoxygenase-deficient mice were maintained for 14 to 16 weeks. 12/15-lipoxygenase gene deficiency did not affect weight gain or blood glucose concentrations. Diabetic wild-type mice displayed increased sciatic nerve 12/15-lipoxygenase and 12(S)-hydroxyeicosatetraenoic acid levels. 12/15-lipoxygenase deficiency prevented or alleviated diabetes-induced thermal hypoalgesia, tactile allodynia, motor and sensory nerve conduction velocity deficits, and reduction in tibial nerve myelinated fiber diameter, but not intraepidermal nerve fiber loss. The frequencies of superior mesenteric-celiac ganglion neuritic dystrophy, the hallmark of diabetic autonomic neuropathy in mouse prevertebral sympathetic ganglia, were increased 14.8-fold and 17.2-fold in diabetic wild-type and 12/15-lipoxygenase-deficient mice, respectively. In addition, both diabetic groups displayed small (<1%) numbers of degenerating sympathetic neurons. In conclusion, whereas 12/15-lipoxygenase up-regulation provides an important contribution to functional changes characteristic for both large and small fiber peripheral diabetic neuropathies and axonal atrophy of large myelinated fibers, its role in small sensory nerve fiber degeneration and neuritic dystrophy and neuronal degeneration characteristic for diabetic autonomic neuropathy is minor. This should be considered in the selection of endpoints for future clinical trials of 12/15-lipoxygenase inhibitors. Up-regulation of 12/15-lipoxygenase, which converts arachidonic acid to 12(S)- and 15(S)-hydroxyeicosatetraenoic acids, causes impaired cell signaling, oxidative-nitrosative stress, and inflammation. This study evaluated the role for 12/15-lipoxygenase in diabetic large and small fiber peripheral and autonomic neuropathies. Control and streptozotocin-diabetic wild-type and 12/15-lipoxygenase-deficient mice were maintained for 14 to 16 weeks. 12/15-lipoxygenase gene deficiency did not affect weight gain or blood glucose concentrations. Diabetic wild-type mice displayed increased sciatic nerve 12/15-lipoxygenase and 12(S)-hydroxyeicosatetraenoic acid levels. 12/15-lipoxygenase deficiency prevented or alleviated diabetes-induced thermal hypoalgesia, tactile allodynia, motor and sensory nerve conduction velocity deficits, and reduction in tibial nerve myelinated fiber diameter, but not intraepidermal nerve fiber loss. The frequencies of superior mesenteric-celiac ganglion neuritic dystrophy, the hallmark of diabetic autonomic neuropathy in mouse prevertebral sympathetic ganglia, were increased 14.8-fold and 17.2-fold in diabetic wild-type and 12/15-lipoxygenase-deficient mice, respectively. In addition, both diabetic groups displayed small (<1%) numbers of degenerating sympathetic neurons. In conclusion, whereas 12/15-lipoxygenase up-regulation provides an important contribution to functional changes characteristic for both large and small fiber peripheral diabetic neuropathies and axonal atrophy of large myelinated fibers, its role in small sensory nerve fiber degeneration and neuritic dystrophy and neuronal degeneration characteristic for diabetic autonomic neuropathy is minor. This should be considered in the selection of endpoints for future clinical trials of 12/15-lipoxygenase inhibitors. 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87: 1997-2005Crossref PubMed Scopus (73) Google Scholar The present study evaluated the role for LO in PDN and DAN by comparing severities of peripheral nerve dysfunction, axonal atrophy of large myelinated fibers, intra-epidermal nerve loss, as well as neuritic dystrophic changes in the superior mesenteric (SMG) and celiac ganglia (CG), in diabetic wild-type (LO+/+) and LO-deficient (LO−/−) mice. Unless otherwise stated, all chemicals were of reagent-grade quality, and were purchased from Sigma Chemical Co., St. Louis, MO. Rabbit polyclonal (clone H-100) anti-12-lipoxygenase antibody for Western blot analysis was obtained from Santa Cruz Biotechnology, Santa Cruz, CA. For assessment of intra-epidermal nerve fiber density, rabbit polyclonal anti-protein gene product 9.5 (PGP 9.5) antiserum was obtained from UltraClone, Isle of Wight, UK; Alexa Fluor 488 goat anti-rabbit highly cross-adsorbed IgG (H+L) from Invitrogen, Eugene, OR; SuperBlock blocking buffer from Thermo Scientific, Rockford, IL; and the optimum cutting temperature compound from Sakura Finetek USA, Torrance, CA. Vectashield Mounting Medium was obtained from Vector Laboratories, Burlingame, CA. Other reagents for immunohistochemistry were purchased from Dako Laboratories, Inc., Santa Barbara, CA. The experiments were performed in accordance with regulations specified by The Guide for the Care and Handling of Laboratory Animals (NIH Publication No. 85-23) and Pennington Biomedical Research Center Protocol for Animal Studies. A colony of 12/15-deficient mice (LO−/−, C57Bl6/J background)49Nunemaker CS Chen M Pei H Kimble SD Keller SR Carter JD Yang Z Smith KM Wu R Bevard MH Garmey JC Nadler JL 12-Lipoxygenase-knockout mice are resistant to inflammatory effects of obesity induced by Western diet.Am J Physiol Endocrinol Metab. 2008; 295: E1065-E1075Crossref PubMed Scopus (117) Google Scholar, 50Gubitosi-Klug RA Talahalli R Du Y Nadler JL Kern TS 5-Lipoxygenase, but not 12/15-lipoxygenase, contributes to degeneration of retinal capillaries in a mouse model of diabetic retinopathy.Diabetes. 2008; 57: 1387-1393Crossref PubMed Scopus (107) Google Scholar was established from several breeding pairs provided by Dr. Nadler's laboratory. Mature male C57Bl6/J mice were purchased from Jackson Laboratories, Bar Harbor, ME. All of the mice were fed standard mouse chow (PMI Nutrition International, Brentwood, MO) and had ad libitum access to water. Male wild-type (LO+/+) and LO−/− mice were randomly divided into two groups. In one group, diabetes was induced by streptozotocin as we described previously.33Drel VR Pacher P Vareniuk I Pavlov IA Ilnytska O Lyzogubov VV Bell SR Groves JT Obrosova IG Evaluation of the peroxynitrite decomposition catalyst Fe(III) tetra-mesitylporphyrin octasulfonate on peripheral neuropathy in a mouse model of type 1 diabetes.Int J Mol Med. 2007; 20: 783-792PubMed Google Scholar, 51Vareniuk I Pavlov IA Obrosova IG Inducible nitric oxide synthase gene deficiency counteracts multiple manifestations of peripheral neuropathy in a streptozotocin-induced mouse model of diabetes.Diabetologia. 2008; 51: 2126-2133Crossref PubMed Scopus (75) Google Scholar Nonfasting blood glucose measurements were performed at induction of diabetes and at the end of the study. The mice with blood glucose ≥13.8 mmol/L were considered diabetic. The control and diabetic mice were maintained for 14 weeks. Physiological and behavioral measurements were taken at two time points ie, at the beginning (before induction of diabetes) and at the end of the study, in the following order: tactile response thresholds (first day), thermal response latency (second day), and motor and sensory nerve conduction velocities (MNCV and SNCV, third day). MNCV and SNCV were measured in mice anesthetized with a mixture of ketamine and xylazine (45 mgkg−1 body weight and 15 mgkg−1 body weight, respectively, i.p.). A group of nondiabetic and diabetic wild-type and LO−/− mice was shipped to Washington University School of Medicine, St. Louis, and, after a 2-week quarantine and acclimatization, were used for assessment of structural characteristic of DAN in Dr. Schmidt's laboratory. The animals were sedated by CO2 and immediately sacrificed by cervical dislocation. Sciatic nerves (a portion above the bifurcation point) were rapidly dissected and frozen in liquid nitrogen for subsequent assessment of LO and 12(S)HETE concentrations. Distal tibial nerves were fixed in 2.5% glutaraldehyde buffered with 0.05 mmol/L sodium cacodylate (pH 7.3), for further assessment of myelinated nerve fiber diameter and myelin thickness. Footpads were fixed in ice-cold Zamboni's fixative for further assessment of intraepidermal nerve fiber density. Sciatic MNCV, hind-limb digital SNCV, thermal algesia (paw withdrawal), and tactile response thresholds we measured as described previously.33Drel VR Pacher P Vareniuk I Pavlov IA Ilnytska O Lyzogubov VV Bell SR Groves JT Obrosova IG Evaluation of the peroxynitrite decomposition catalyst Fe(III) tetra-mesitylporphyrin octasulfonate on peripheral neuropathy in a mouse model of type 1 diabetes.Int J Mol Med. 2007; 20: 783-792PubMed Google Scholar, 46Obrosova IG Ilnytska O Lyzogubov VV Pavlov IA Mashtalir N Nadler JL Drel VR High-fat diet induced neuropathy of pre-diabetes and obesity: effects of “healthy” diet and aldose reductase inhibition.Diabetes. 2007; 56: 2598-2608Crossref PubMed Scopus (206) Google Scholar, 51Vareniuk I Pavlov IA Obrosova IG Inducible nitric oxide synthase gene deficiency counteracts multiple manifestations of peripheral neuropathy in a streptozotocin-induced mouse model of diabetes.Diabetologia. 2008; 51: 2126-2133Crossref PubMed Scopus (75) Google Scholar Footpads were fixed in ice-cold Zamboni's fixative for 3 hours, washed in 100 mmol/L PBS overnight, and then in PBS containing increasing concentrations of sucrose ie, 10%, 15%, and 20%, 3 hours in each solution. After washing, the samples were snap-frozen in OCT compound and stored at −80°C. Three longitudinal 50 μm-thick footpad sections from each mouse were cut on Leica CM1950 cryostat (Leica Microsystems, Nussloch, Germany). Nonspecific binding was blocked by 3% goat serum containing 0.5% porcine gelatin and 0.5% Triton X-100 in SuperBlock blocking buffer at room temperature, for 2 hours. The sections were incubated overnight with PGP 9.5 antiserum in 1:400 dilution, at 4°C after which secondary Alexa Fluor 488 IgG (H+L) in 1:1000 was applied at room temperature, for 1 hour. Sections were then coverslipped with Vectashield mounting medium. Intraepidermal nerve fiber profiles were counted blindly by three independent investigators under Axioplan 2 microscope (Zeiss) at ×40 magnification, and the average values were used. The length of epidermis was assessed on the microphotographs of stained sections taken at ×5 magnification with a 3I Everest imaging system (Intelligent Imaging Innovations, Inc., Denver, CO) equipped with Axioplan 2 microscope (Zeiss), using the NIH ImageJ software (version 1.42q). An average of 2.8 ± 0.3 mm of the sample length was investigated to calculate a number of nerve fiber profiles per mm of epidermis. Representative images of intra-epidermal nerve fibers were obtained by confocal laser scanning microscopy at ×40 magnification, using Leica TCS SP5 confocal system (Leica Microsystems, Mannheim, Germany). Myelinated fiber diameter and myelin thickness of distal tibial nerves were assessed as described.27Yagihashi S Yamagishi SI Wada Ri R Baba M Hohman TC Yabe-Nishimura C Kokai Y Neuropathy in diabetic mice overexpressing human aldose reductase and effects of aldose reductase inhibitor.Brain. 2001; 124: 2448-2458Crossref PubMed Scopus (152) Google Scholar The samples fixed overnight at 4°C as described above were postfixed in 1% osmium tetroxide and dehydrated through an ascending series of ethanol concentrations. Fixed nerves were embedded in Epon and polymerized. One-micron thick semithin transverse nerve sections were stained with ρ-phenylenediamine. For the morphometric analysis, axonal diameter and myelin thickness were measured at a magnification of ×1600 by a computer-assisted image analyzing system (Scion Image for Windows, Scion Corporation, Frederick, MD). To assess LO protein expression by Western blot analysis, sciatic nerve material (∼20 mg) was placed on ice in 200 μl of radioimmunoprecipitation assay buffer containing 50 mmol/L Tris-HCl, pH 7.2; 150 mmol/L NaCl; 0.1% sodium dodecyl sulfate; 1% NP-40; 5 mmol/L EDTA; 1 mmol/L EGTA; 1% sodium deoxycholate and the protease/phosphatase inhibitors leupeptin (10 μg/ml), pepstatin (1 μg/ml), aprotinin (20 μg/ml), benzamidine (10 mmol/L), phenylmethylsulfonyl fluoride (1 mmol/L), and sodium orthovanadate (1 mmol/L), and homogenized on ice. The homogenates were sonicated (4 × 10 s) and centrifuged at 14,000 × g for 20 minutes. All of the afore-mentioned steps were performed at 4°C. The lysates (20 μg protein) were mixed with equal volumes of 2× sample-loading buffer containing 62.5 mmol/L Tris-HCl, pH 6.8; 2% SDS; 5% β-mercaptoethanol; 10% glycerol and 0.025% bromophenol blue, and fractionated in 7.5% SDS-polyacrylamide gel in an electrophoresis cell (Mini-Protean III; Bio-Rad Laboratories, Richmond, CA). Electrophoresis was conducted at 15 mA constant current for stacking, and at 25 mA for protein separation. Gel contents were electrotransferred (80 V, 2 hours) to nitrocellulose membranes using Mini Trans-Blot cell (Bio-Rad Laboratories, Richmond, CA) and western transfer buffer [25 mmol/L Tris-HCl, pH 8.3; 192 mmol/L glycine; and 20% (v/v) methanol]. Free binding sites were blocked in 5% (w/v) bovine serum albumin in 20 mmol/L Tris-HCl buffer, pH 7.5, containing 150 mmol/L NaCl and 0.05% Tween 20, for 1 hour, after which LO antibody was applied for 2 hours. The horseradish peroxidase-conjugated secondary antibody was then applied for 1 hour. After extensive washing, protein bands detected by the antibodies were visualized with the Amersham ECL Western Blotting Detection Reagent (Little Chalfont, Buckinghamshire, UK). Membranes were then stripped in the 25 mmol/L glycine-HCl, pH 2.5 buffer containing 2% SDS, and reprobed with β-actin antibody to confirm equal protein loading. For assessment of 12(S)HETE, sciatic nerve samples were homogenized on ice in 15 mmol/L Tris-HCI buffer (1:100 w/v) containing 140 mmol/L NaCl, pH 7.6, and centrifuged. 12(S)HETE was measured in supernatants with the 12(S)-hydroxyeicosatetraenoic acid [12(S)HETE] Enzyme Immuno Assay kit (Assay Designs, Ann Arbor, MI). Mice were anesthetized with ketamine/xylazine and perfused transcardially with 25 ml of heparinized saline followed by 25 ml of modified Karnovsky's fixative containing 3% glutaraldehyde and 1% paraformaldehyde in sodium cacodylate buffer pH 7.4. Fixation was continued overnight at 4°C in the same fixative and the following day the SMG and CG were dissected together as a single block, cleaned of extraneous tissue, and rinsed in sodium cacodylate buffer. Tissue was postfixed in phosphate cacodylate-buffered 2% OsO4 for 1 hour, dehydrated in graded ethanols with a final dehydration in propylene oxide, and embedded in EMbed-812 (Elect
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