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• W2059999118 abstract "Using a mouse model with noise-induced cochlear blood-labyrinth-barrier (CBLB) injury, we examined the effects of inducible nitric oxide synthase (iNOS) on the recruitment of bone marrow-derived cells (BMDCs) to the CBLB after acoustic injury. Lethally irradiated C57BL/6J and B6.129P2-Nos2tm1Lau/J mice were transplanted with GFP+-BMDCs from C57Bl/6-Tg (UBC GFP) mice. Four weeks after transplantation, we assessed the population of GFP+-BMDCs in the CBLB. Only small numbers of GFP+-BMDCs were found to infiltrate the area of the CBLB in the control recipient mice. However, robust GFP+-BMDC migration occurred in the area of the CBLB within the injured cochlea during the first week following acoustic trauma, and further BMDC accumulation was seen by 2 weeks posttrauma. After 4 weeks, the BMDCs were integrated into vessels. Local iNOS from perivascular resident macrophages was found to be important for BMDC infiltration, since mice deficient in iNOS (Inos−/−) and mice with iNOS that had been inhibited by 1400W displayed reduced BMDC infiltration. Stromal cell-derived factor-1α (SDF-1α) and its chemokine receptor 4 (CXCR4) were required for the iNOS-triggered recruitment. BMDC recruitment was significantly reduced by the inhibition of SDF-1α activity. Inhibition of the iNOS/SDF-1α signaling pathway reduced vascular repair as observed by reduced vascular density. Our study revealed an intrinsic signaling pathway of iNOS that mediates SDF-1α to promote GFP+-BMDC infiltration/targeting in cochlear vascular repair. Using a mouse model with noise-induced cochlear blood-labyrinth-barrier (CBLB) injury, we examined the effects of inducible nitric oxide synthase (iNOS) on the recruitment of bone marrow-derived cells (BMDCs) to the CBLB after acoustic injury. Lethally irradiated C57BL/6J and B6.129P2-Nos2tm1Lau/J mice were transplanted with GFP+-BMDCs from C57Bl/6-Tg (UBC GFP) mice. Four weeks after transplantation, we assessed the population of GFP+-BMDCs in the CBLB. Only small numbers of GFP+-BMDCs were found to infiltrate the area of the CBLB in the control recipient mice. However, robust GFP+-BMDC migration occurred in the area of the CBLB within the injured cochlea during the first week following acoustic trauma, and further BMDC accumulation was seen by 2 weeks posttrauma. After 4 weeks, the BMDCs were integrated into vessels. Local iNOS from perivascular resident macrophages was found to be important for BMDC infiltration, since mice deficient in iNOS (Inos−/−) and mice with iNOS that had been inhibited by 1400W displayed reduced BMDC infiltration. Stromal cell-derived factor-1α (SDF-1α) and its chemokine receptor 4 (CXCR4) were required for the iNOS-triggered recruitment. BMDC recruitment was significantly reduced by the inhibition of SDF-1α activity. Inhibition of the iNOS/SDF-1α signaling pathway reduced vascular repair as observed by reduced vascular density. Our study revealed an intrinsic signaling pathway of iNOS that mediates SDF-1α to promote GFP+-BMDC infiltration/targeting in cochlear vascular repair. Ischemia causes considerable morbidity in various organ systems, and the pervasiveness of ischemic damage makes repair of damaged vasculature an important therapeutic goal.1Angoulvant D Fazel S Li RK Neovascularization derived from cell transplantation in ischemic myocardium.Mol Cell Biochem. 2004; 264: 133-142Crossref PubMed Scopus (19) Google Scholar Ischemia in the inner ear is closely related to several hearing disorders, including sudden sensorineural hearing loss, presbyacusis, noise-induced hearing loss, tinnitus, and Ménière's disease.2Shi X Dai C Nuttall AL Altered expression of inducible nitric oxide synthase (iNOS) in the cochlea.Hear Res. 2003; 177: 43-52Crossref PubMed Scopus (53) Google Scholar, 3Nuttall AL Sound-induced cochlear ischemia/hypoxia as a mechanism of hearing loss.Noise Health. 1999; 2: 17-32PubMed Google Scholar, 4Miller JM Brown JN Schacht J 8-iso-prostaglandin F(2alpha), a product of noise exposure, reduces inner ear blood flow.Audiol Neurootol. 2003; 8: 207-221Crossref PubMed Scopus (82) Google Scholar, 5Gratton MA Schmiedt RA Schulte BA Age-related decreases in endocochlear potential are associated with vascular abnormalities in the stria vascularis.Hear Res. 1996; 102: 181-190Crossref PubMed Google Scholar Various clinical approaches to treatment of the ischemia have been tried, including use of vasoactive substances to improve cochlear blood flow; however, these generally have not been effective. A fundamental approach to determining the mechanisms of damage repair will enable development of more effective clinical treatments for vascular-related hearing disorders. Acoustic trauma not only directly damages sensory hair cells, but it also disrupts the blood flow and the cochlear blood-labyrinth-barrier (CBLB) in the stria vascularis,6Shi X Cochlear pericyte responses to acoustic trauma and the involvement of hypoxia-inducible factor-1alpha and vascular endothelial growth factor.Am J Pathol. 2009; 174: 1692-1704Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar creating an ischemic and hypoxic environment.3Nuttall AL Sound-induced cochlear ischemia/hypoxia as a mechanism of hearing loss.Noise Health. 1999; 2: 17-32PubMed Google Scholar, 6Shi X Cochlear pericyte responses to acoustic trauma and the involvement of hypoxia-inducible factor-1alpha and vascular endothelial growth factor.Am J Pathol. 2009; 174: 1692-1704Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar, 7Lamm K Arnold W Successful treatment of noise-induced cochlear ischemia, hypoxia, and hearing loss.Ann NY Acad Sci. 1999; 884: 233-248Crossref PubMed Scopus (62) Google Scholar Normal blood supply to the cochlea is critical for generating the ionic gradients and the endolymphatic potential required for auditory transduction.6Shi X Cochlear pericyte responses to acoustic trauma and the involvement of hypoxia-inducible factor-1alpha and vascular endothelial growth factor.Am J Pathol. 2009; 174: 1692-1704Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar, 8Nakashima T Naganawa S Sone M Tominaga M Hayashi H Yamamoto H Liu X Nuttall AL Disorders of cochlear blood flow.Brain Res Brain Res Rev. 2003; 43: 17-28Crossref PubMed Scopus (187) Google Scholar Better treatment of noise-induced hearing loss requires an understanding of parallel repair factors, one of which will be the cellular repair mechanisms involved in restoration of efficient cochlear blood flow after damage. Increasing evidence highlights the importance of circulating bone marrow stem cells, which is home to sites of ischemia and contribute to formation of new blood vessels by differentiation.9Takahashi T Kalka C Masuda H Chen D Silver M Kearney M Magner M Isner JM Asahara T Ischemia- and cytokine-induced mobilization of bone marrow-derived endothelial progenitor cells for neovascularization.Nat Med. 1999; 5: 434-438Crossref PubMed Scopus (46) Google Scholar, 10Sanberg PR Park DH Kuzmin-Nichols N Cruz E Hossne Jr, NA Buffolo E Willing AE Monocyte transplantation for neural and cardiovascular ischemia repair.J Cell Mol Med. 2010; 14: 553-563PubMed Google Scholar, 11Ferrara N Kerbel RS Angiogenesis as a therapeutic target.Nature. 2005; 438: 967-974Crossref PubMed Scopus (2254) Google Scholar In the context of cancer, inhibition of bone marrow stem cell recruitment to tumors decreases tumor angiogenesis,12Hamik A Wang B Jain MK Transcriptional regulators of angiogenesis.Arterioscler Thromb Vasc Biol. 2006; 26: 1936-1947Crossref PubMed Scopus (58) Google Scholar whereas the reverse, infusion of bone marrow-derived progenitor cells increases angiogenesis13Halkos ME Zhao ZQ Kerendi F Wang NP Jiang R Schmarkey LS Martin BJ Quyyumi AA Few WL Kin H Guyton RA Vinten-Johansen J Intravenous infusion of mesenchymal stem cells enhances regional perfusion and improves ventricular function in a porcine model of myocardial infarction.Basic Res Cardiol. 2008; 103: 525-536Crossref PubMed Scopus (112) Google Scholar and improves organ function. For example, transplantation of bone marrow cells into myocardium augments cardiac function following myocardial infarct or chronic ischemia in rats and dogs.14Tomita S Li RK Weisel RD Mickle DA Kim EJ Sakai T Jia ZQ Autologous transplantation of bone marrow cells improves damaged heart function.Circulation. 1999; 100: II247-II256Crossref PubMed Google Scholar, 15Silva GV Litovsky S Assad JA Sousa AL Martin BJ Vela D Coulter SC Lin J Ober J Vaughn WK Branco RV Oliveira EM He R Geng YJ Willerson JT Perin EC Mesenchymal stem cells differentiate into an endothelial phenotype, enhance vascular density, and improve heart function in a canine chronic ischemia model.Circulation. 2005; 111: 150-156Crossref PubMed Scopus (698) Google Scholar Transplantation of bone marrow cells attenuates ischemic damage and improves functional recovery after brain injury.16Yalvac ME Rizvanov AA Kilic E Sahin F Mukhamedyarov MA Islamov RR Palotas A Potential role of dental stem cells in the cellular therapy of cerebral ischemia.Curr Pharm Des. 2009; 15: 3908-3916Crossref PubMed Scopus (43) Google Scholar Coordination of an inflammatory response, entailing regulation of chemokine release, is essential for bone marrow stem cell (BMSC)-associated neovascularization during ischemia and wound healing.17Yu J Fernandez-Hernando C Suarez Y Schleicher M Hao Z Wright PL DiLorenzo A Kyriakides TR Sessa WC Reticulon 4B (Nogo-B) is necessary for macrophage infiltration and tissue repair.Proc Natl Acad Sci USA. 2009; 106: 17511-17516Crossref PubMed Scopus (72) Google Scholar For example, the release of stromal cell-derived factor-1α (SDF-1α) from stressed tissue plays an important role in the mobilization of bone marrow cells to local ischemic sites on damaged vessels.18Yin Y Zhao X Fang Y Yu S Zhao J Song M Huang L SDF-1alpha involved in mobilization and recruitment of endothelial progenitor cells after arterial injury in mice.Cardiovasc Pathol. 2010; 19: 218-227Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar, 19Wright DE Bowman EP Wagers AJ Butcher EC Weissman IL Hematopoietic stem cells are uniquely selective in their migratory response to chemokines.J Exp Med. 2002; 195: 1145-1154Crossref PubMed Scopus (415) Google Scholar, 20Hiasa K Ishibashi M Ohtani K Inoue S Zhao Q Kitamoto S Sata M Ichiki T Takeshita A Egashira K Gene transfer of stromal cell-derived factor-1alpha enhances ischemic vasculogenesis and angiogenesis via vascular endothelial growth factor/endothelial nitric oxide synthase-related pathway: next-generation chemokine therapy for therapeutic neovascularization.Circulation. 2004; 109: 2454-2461Crossref PubMed Scopus (293) Google Scholar Additionally, nitric oxide (NO), produced by endothelial nitric oxide synthase (eNOS), is shown to be a critical factor in bone marrow cell recruitment for vascular repair.21Li N Lu X Zhao X Xiang FL Xenocostas A Karmazyn M Feng Q Endothelial nitric oxide synthase promotes bone marrow stromal cell migration to the ischemic myocardium via upregulation of stromal cell-derived factor-1alpha.Stem Cells. 2009; 27: 961-970Crossref PubMed Scopus (51) Google Scholar Here, we report the finding that recruitment of bone marrow cells to ischemic tissues in the noise traumatized cochlea is signaled by a local inducible nitric oxide synthase (iNOS)-dependent SDF-1α pathway. Recruitment of bone marrow derived cells (BMDCs) to the damaged stria vascularis results in repair of cochlear vessels. Male C57BL/6J (Inos wild type [WT], aged 4 weeks; stock number: 000664), B6.129P2-Nos2tm1Lau/J (Inos−/−, aged 4 weeks; stock number: 002609), and C57Bl/6-Tg mice (UBC-GFP, aged 4 to 6 weeks; stock number: 004353) were purchased from Jackson Laboratory (Bar Harbor, ME). C57Bl/6-Tg mice served as donor mice with Inos WT, and Inos−/− mice of the same age served as recipients. The recipients were irradiated (at 9 Gy) with a γ-emitting source and reconstituted with a single periorbital sinus injection of 2 × 107 BMDCs in 200 μl of modified HBSS from donor transgenic mice. At 1 month posttransplantation, the mice served as control, noise-exposed, and noise-exposed + drug treatment groups. The groups were sacrificed at different times after noise exposure (immediately, 1 week, 2 weeks, and 4 weeks) for measurement. All procedures in this study were reviewed and approved by the Institutional Animal Care and Use Committee at Oregon Health and Science University. Animals were placed in wire mesh cages and exposed to broadband noise at 120 dB sound pressure level (SPL) in a sound exposure booth for 3 hours and for an additional 3 hours the following day. The noise exposure regime, routinely used in our laboratory, produces permanent loss of cochlear sensitivity.6Shi X Cochlear pericyte responses to acoustic trauma and the involvement of hypoxia-inducible factor-1alpha and vascular endothelial growth factor.Am J Pathol. 2009; 174: 1692-1704Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar Normal and noise-exposed mice were sacrificed at day 2, week 1, week 2, or week 4, subsequent to a 1-month transplantation recovery period. The cochleae were harvested and fixed in 4% paraformaldehyde overnight at 4°C, and then rinsed in 37°C PBS (pH 7.3) to remove any residual 4% paraformaldehyde. Immunohistochemistry was performed as described before.6Shi X Cochlear pericyte responses to acoustic trauma and the involvement of hypoxia-inducible factor-1alpha and vascular endothelial growth factor.Am J Pathol. 2009; 174: 1692-1704Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar Tissue samples were permeabilized in 0.5% Triton X-100 (Sigma, St. Louis, MO) for 1 hour, and then immunoblocked with a solution of 10% goat serum and 1% bovine albumin in 0.02 mol/L PBS for an additional hour. The specimens were incubated overnight at 4°C with the primary antibodies (listed in Table 1) diluted in PBS-bovine serum albumin. After several washes in PBS, the samples were incubated with secondary antibodies, Alexa Fluor 568-conjugated goat anti-rabbit (category number A21069; Invitrogen, Eugene, OR), Fluor 647-conjugated goat anti-rabbit (category number A-21244; Invitrogen), and Alexa Fluor 568-conjugated goat anti-rat (category number A-11077; Invitrogen) for 1 hour at room temperature. The tissues were mounted in mounting medium (H-1000; Vector Laboratories, Inc., Burlingame, CA) and visualized under an FV1000 Olympus laser-scanning confocal microscope (Olympus, Tokyo, Japan). Controls were prepared by replacing primary antibodies with PBS.Table 1Primary Antibodies EmployedPrimary antibodiesSourceIdentificationDilutionTypeSpecificityNG2Chemiconab53201:200 (dilution with 1% BSA-PBS)Rabbit polyclonal antibodyReacts with NG2 of mouse, rat, and humanDesminEpitomics1466-11:400 (dilution with 1% BSA-PBS)Rabbit monoclonal antibodyReacts with desmin of mouse, rat, and humanF4/80Santa Cruz Biotechnologyab66401:100 (dilution with 1% BSA-PBS)Rat monoclonal (CI:A3-1) to F4/80This antibody recognizes the mouse F4/80 antigen, a 160-kd glycoprotein expressed by macrophages.Collagen type IVResearch Diagnostics Inc.74781:50 (dilution with 1% BSA-PBS)Rabbit polyclonal antibodyReacts with most mammalian type IV collagen (dilution with 1% BSA-PBS)CD31Abcamab283641:100 (dilution with 1% BSA-PBS)Rabbit polyclonal antibodyReacts with mouseCXCR4Abcamab20741:100 (dilution with 1% BSA-PBS)Rabbit polyclonal antibodyReacts with mouseiNOSAlpha Diagnostics Intl. Inc.iNOS-A 0370A21:1000 (dilution with 1% BSA-PBS)Rabbit polyclonal antibodyReacts with mouseBSA, bovine serum albumin. Open table in a new tab BSA, bovine serum albumin. To investigate the influence of noise exposure on the protein level of iNOS expression, the cochlear lateral wall was dissented from control and noise-exposed animals (cohorts of three mice). The collected lateral wall tissue was homogenized in lysis buffer (RIPA Lysis buffer, Upstate, a Serologicals Company, Temecula, CA) with a protease inhibitor cocktail (Protease Inhibitor cocktails Set III, Calbiochem, Darmstadt, Germany) for 30 seconds. After centrifuging (4°C, 30 minutes, at 14,000 rpm), the supernatant was assayed for protein by using a DC protein Assay kit (Bio-Rad, Hercules, CA). Samples were heated to 100°C for 5 minutes with 2× SDS loading buffer and briefly cooled on ice. Fifty-microgram aliquots of total protein from each sample were run on 10% sodium dodecyl sulfate-polyacrylamide gels to detect iNOS (130 kDa) and actin (43 kDa). Proteins were electrophoretically transferred to polyvinylidene difluoride membranes (Millipore Corp., Bedford, MA), with the membranes blocked with nonfat milk and 0.1% Tween 20 in Tris-buffered saline for 1 hour at room temperature. The sample was incubated with primary antibodies diluted 1:1000 in skim milk overnight at 4°C for specific immunodetection (rabbit polyclonal antibody to iNOS, α Diagnostic Intl Inc., San Antonio, TX; mouse polyclonal antibody to actin, Millipore Corp.). After three washes with PBS, the membranes were incubated for another hour with horseradish peroxidase-conjugated goat anti-rabbit or goat anti-mouse IgG diluted 1:10,000 in PBS at room temperature. Antigens were revealed by using ECL Plus Western Blotting Detection Reagents (GE Healthcare, Pittsburgh, PA). To quantify the changes of iNOS protein level, the band density was analyzed by using Image J software (V1.38X; NIH, West Chester, PA). The density of the bands of actin was used to normalize the iNOS protein level. Total RNA from the cochlear lateral wall was separately extracted for each experimental group with a RNeasy kit (Qiagen, Valencia, CA) according to the manufacturer's suggestions. Each cohort of two mice was analyzed for iNOS mRNA. One microgram of total RNA was reverse-transcribed by using a RETROscript kit (Ambion, Austin, TX). Conserved regions spanning introns were selected for the primers of iNOS and glyceraldehyde-3-phosphate dehydrogenase. The primers used were as follows: iNOS (mouse Chr 11 NM_010927), forward, 5′-CTATCAGGAAGAAATGCAGGAGAT-3′, reverse, 5′-GAGCACGCTGAGTACCTCATT-3′, 145-bp product; SDF-1α (mouse Chr 6 NM_013655), forward, 5′-CAAGAGGCTCAAGATGTGAGAGGTG-3′, reverse, 5′-TGGCCTTGGCCTGTCACCAA-3′, 258-bp; and glyceraldehyde-3-phosphate dehydrogenase (mouse Chr 6 NM_008084), forward, 5′-ATGTGTCCGTCGTGGATCTGAC-3′, reverse, 5′-AGACAACCTGGTCCTCAGTGTAG-3′, 132-bp product. The RT-PCR was cycled at 95°C for 2 minutes, up to 40 cycles at 95°C for 30 seconds, 60°C for 45 seconds, 72°C for 30 seconds, and a final 5-minute extension at 72°C. The products of RT-PCR were visualized by agarose gel electrophoresis. Total RNA from the cochlear lateral wall of different groups was separately extracted with RNeasy (Qiagen) according to the manufacturer's suggestions. Each cohort of two mice was analyzed for mRNA levels of iNOS and SDF-1α with quantitative real-time PCR. One microgram of total RNA was reverse-transcribed by using a RETROscript kit (Ambion). The cDNA synthesized from total RNA was diluted 10-fold with DNase-free water, and each cDNA sample was independently tested three times. Transcript quantities were assayed by TaqMan gene expression assay: iNOS (category number Mm01309902_m1; Applied Biosystems, Foster City, CA) and SDF-1α (category number Mm00457276_m1, Applied Biosystems) were assayed in a model 7300 real-time PCR system (Applied Biosystems). Cycling conditions of the real-time PCR were 95°C for 20 seconds, 40 cycles of 95°C for 1 second, and 60°C for 20 seconds. Mouse glyceraldehyde-3-phosphate dehydrogenase (category number 4352339E, Applied Biosystems) expression was used as an endogenous control. Quantitative PCR was performed according to the guidelines provided by Applied Biosystems. The comparative cycle threshold (CT) method (ΔΔCT quantitation) was used to assess the difference between samples. Quantitative data analysis followed the suggestions of the manufacturer. To compare the protein level of SDF-1 between different groups, whole cochlea was isolated from the animals (cohorts of three animals) and the lateral wall tissue carefully dissected. All tissue samples were homogenized in lysis buffer (RIPA Lysis buffer, Upstate, a Serologicals Company) with a protease inhibitor cocktail (Protease Inhibitor cocktails Set III, Calbiochem) for 30 seconds. After centrifuging (4°C, 30 minutes, at 14,000 rpm), the supernatant was collected, and the protein assay was performed by using a DC protein Assay kit (Bio-Rad). Ten micrograms of protein per sample was used for enzyme-linked immunosorbent assay (ELISA) analysis. SDF-1 protein concentration of different groups was assessed by using a Quantikine Mouse CXCL12/SDF-1 ELISA kit (R&D Systems, Burlington, ON) according to the manufacturer's recommendations. GFP+-BMDCs in the stria vascularis of mouse cochlea were counted on a standard epifluorescence microscope with a × 40 objective lens (cohorts of four mice). The GFP+-BMDCs were counted in a region corresponding to an initial frequency response of 8–32 kHz, determined by the frequency-map described by Wang et al.22Wang Y Hirose K Liberman MC Dynamics of noise-induced cellular injury and repair in the mouse cochlea.J Assoc Res Otolaryngol. 2002; 3: 248-268Crossref PubMed Scopus (387) Google Scholar Noise-caused hearing loss occurs predominately between 8 and 32 kHz with this sound protocol.6Shi X Cochlear pericyte responses to acoustic trauma and the involvement of hypoxia-inducible factor-1alpha and vascular endothelial growth factor.Am J Pathol. 2009; 174: 1692-1704Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar The area studied in this investigation (a length approximately 2 mm starting 1.5 mm from the base) lies within this region. For plotting purposes, the data were grouped. The mice were pretreated with 1400W (10 mg/kg, i.p.; Enzo Life Sciences, Farmingdale, NY)23Akita Y Otani H Matsuhisa S Kyoi S Enoki C Hattori R Imamura H Kamihata H Kimura Y Iwasaka T Exercise-induced activation of cardiac sympathetic nerve triggers cardioprotection via redox-sensitive activation of eNOS and upregulation of iNOS.Am J Physiol Heart Circ Physiol. 2007; 292: H2051-H2059Crossref PubMed Scopus (51) Google Scholar to inhibit iNOS and with an antagonist of the SDF-1α receptor CXCR4, AMD3100 (7.5 mg/kg, i.p.; Sigma-Aldrich),24Young KC Torres E Hatzistergos KE Hehre D Suguihara C Hare JM Inhibition of the SDF-1/CXCR4 axis attenuates neonatal hypoxia-induced pulmonary hypertension.Circ Res. 2009; 104: 1293-1301Crossref PubMed Scopus (80) Google Scholar to inhibit SDF-1α activity. Treatments were administered as single-dose injections 30 minutes before the animal received noise and were continued at one dose per day for the first week and one dose per week up to 4 weeks after noise exposure. The side-effects of 1400W and AMD3100 on auditory function were evaluated. Auditory brain-stem response was used to measure hearing threshold. Neither 1400W nor AMD3100 showed ototoxicity in the six mice of the two treated groups. The auditory bulla was dissected and rapidly opened in a petri dish filled with a physiological solution containing 125 mmol/L NaCl, 5.0 mmol/L KCl, 1.6 mmol/L CaCl2, 18 mmol/L NaHCO3, 10 mmol/L glucose, and 10 mmol/L HEPES, pH 7.4. Small pieces of tissue from the basal middle turn of the cochlear lateral wall in noise exposed animals, both with and without 1400W treatment, were removed and incubated in a physiological solution at 37°C, pH 7.4, containing 10 μmol/L diaminofluorescein-2 diacetate (category number 251505, Calbiochem) for detecting NO. The tissue was incubated with dye for 30 minutes, subsequently washed in fresh physiological solution for 10 minutes, and assessed by confocal microscopy. Quantitation of the NO indicator was performed on images taken at the same gain and illumination power setting (488 excitation, 520 nm emission filter for the diaminofluorescein diacetate). Fluorescence intensity was analyzed by using Image J software (V1.38X; NIH) as described previously.4Miller JM Brown JN Schacht J 8-iso-prostaglandin F(2alpha), a product of noise exposure, reduces inner ear blood flow.Audiol Neurootol. 2003; 8: 207-221Crossref PubMed Scopus (82) Google Scholar In brief, images were acquired with a ×40 objective lens. A total of 12 to 16 images were recorded from four normal mice. Twelve to 14 images from four noise-exposed mice were recorded. A total of 13 to 17 images were recorded from four 1400W-treated noise-exposed mice. The area of the stria vascularis was analyzed for mean fluorescence intensity. Mean background fluorescence, obtained for a small area located away from the fluorescent tissue, was subtracted from the fluorescence intensity. Presented data are an average of four experimental and control animals. The entire length of the mouse stria vascularis (from apex to base) is about 6.25 mm (Figure 1A). For each cochlea, the capillary density was measured within a 1.25-mm region beginning at 1.5 mm from the base. Capillaries in the stria vascularis were visualized by collagen IV labeling (Figure 1B). The vessels were traced manually (Figure 1C). The area of the stria vascularis was determined from a differential interference contrast image (Figure 1D). The pixel area of the capillary and the pixel area of the stria vascularis were determined by using the Image-Pro Plus program (Media Cybernetics, Inc., Bethesda, MD). Capillary density as a percentage of the strial tissue was calculated asPixel area of capillariesPixel area of the stria vascularis×100% Data are presented as means ± SD and were evaluated by using the Student's t-test for two groups or by analysis of variance for comparisons of three or more groups. A 95% confidence level was considered statistically significant. The cochlea of the mouse has 2.5 turns of lateral wall (Figure 2A). The normal mouse cochlear lateral wall contains two dense networks of capillaries: the capillaries of the stria vascularis (V/SV) and the capillaries of the spiral ligament (V/SL; Figure 2B, modified from Mudry and Tange).25Mudry A Tange RA The vascularization of the human cochlea: its historical background.Acta Otolaryngol Suppl. 2009; : 3-16Crossref PubMed Scopus (10) Google Scholar The strial capillaries, shaped as polygonal loops, are highly specialized vascular epithelia that form the CBLB in the stria vascularis. Sound trauma causes a significant breech of the CBLB.6Shi X Cochlear pericyte responses to acoustic trauma and the involvement of hypoxia-inducible factor-1alpha and vascular endothelial growth factor.Am J Pathol. 2009; 174: 1692-1704Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar In this study, we investigated whether BMDCs have an essential role in repair of damaged cochlear microvessels in the stria vascularis. Lethally irradiated mice were transplanted with GFP+-BMDCs from C57Bl/6-Tg (UBC-GFP) mice (Figure 2C). One month after transplantation, we assessed the population of GFP+-BMDCs in the region of the stria vascularis in the reconstituted iNOS WT and Inos−/− mice under control (not exposed to loud sound) and acoustic trauma conditions. Irradiated and BMDC transplanted control mice showed normal hearing thresholds immediately after irradiation and 2 months after irradiation (data not shown). No GFP+-BMDCs were observed to have migrated into the area of the stria vascularis in the control mice at 1 month post transplantation. Only small numbers of GFP+-BMDCs had infiltrated into the area of CBLB 2 months after transplantation in the reconstituted iNOS WT mice (Figure 2, D–G). Then, when animals were exposed to wide-band noise at 120 dB for 3 hours per day for 2 consecutive days, robust BMDC migration was observed in the acoustically traumatized iNOS WT animal cochlea (Figure 2, H–K). Immediately after noise exposure and flushing by cardiac perfusion, large numbers of GFP+-BMDCs were observed adhered to vessel walls in the capillary network. These labeled cells had not yet transmigrated across the vessel wall at this time point. Remarkably, GFP+-BMDC infiltration occurred in the first week post acoustic trauma. GFP+-BMDCs further accumulated at the second week (Figure 2, H–K). However, much less GFP+-BMDCs infiltration was observed in the Inos−/− mice (Figure 2, L–O). The number of the infiltrated GFP+-BMDCs is shown in Figure 2P. There was a significant difference in GFP+-BMDC infiltration between the control and noise-exposed iNOS WT mice. Infiltrated BMDCs were previously identified as macrophages.26Shi X Dai M Yang Y Kachelmeier A Xiu R Nuttall A Bone Marrow Cells to Resident Tissue Macrophages in Relation to INOS-Derived Nitric Oxide in the Blood-Labyrinth-Barrier.ARO Abstracts. 2010; : 237Google Scholar After taking residence in the stria vascularis, they underwent morphological changes (Figure 2, Q–S). At an early stage (approximately 1 week post noise exposure), infiltrated GFP+-BMDCs were frequently found to be spherical or nodular shaped (possibly caught in the act of transmigration, Figure 2Q). Approximately 2 weeks post noise exposure, most of infiltrated GFP+-BMDCs developed ramified processes, appeared dendriform in shape, and were irregularly distributed on the capillaries of the stria vascularis (Figure 2R). Approximately 4 weeks post noise exposure, the majority of infiltrated BMDCs were elongated and displayed a particular orientation—that is, their long processes were parallel to the vessels of the stria vascularis (Figure 2S). eNOS was previously reported to be essential in the recruitment of peripheral circulating bone marrow cells to the sites of ischemia.21Li N Lu X Zhao X Xiang FL Xenocostas A Karmazyn M Feng Q Endothelial nitric oxide synthase promotes bone marrow stromal cell migration to the ischemic myocardium via upregulation of stromal cell-d" @default.
• W2059999118 created "2016-06-24" @default.
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• W2059999118 date "2010-12-01" @default.
• W2059999118 modified "2023-10-16" @default.
• W2059999118 title "Bone Marrow Cell Recruitment Mediated by Inducible Nitric Oxide Synthase/Stromal Cell-Derived Factor-1α Signaling Repairs the Acoustically Damaged Cochlear Blood-Labyrinth Barrier" @default.
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