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Characterization of the Leukocyte Response in Acute Vocal Fold Injury.

King SN, Guille J, Thibeault SL - PLoS ONE (2015)

Bottom Line: Surgical injuries, SWC9+/SWC3- cells exhibited hi CD163+ (p<0.05) at 3-days along with upregulation in TNFα and TGFβ1 mRNA compared to 23-days (p<0.05).Higher levels of IL-10 mRNA were found 1-day post-LPS compared to 5-days (p<0.05).Surgical injury elicited a complex phenotype with early TNFα mRNA and CD163+ and persistent TGFβ1 transcript expression.

View Article: PubMed Central - PubMed

Affiliation: Department of Neurological Surgery, Kentucky Spinal Cord Injury Research Center, University of Louisville, Louisville, Kentucky, United States of America.

ABSTRACT
Macrophages location in the superficial layer of the vocal fold (VF) is not only at the first line of defense, but in a place of physiologic importance to voice quality. This study characterizes and compares macrophage function in two models of acute injury. Porcine VF injuries were created bilaterally by either surgical biopsy or lipopolysaccharide (LPS) (1.5 μg/kg) injection. Animals were sacrificed at 1- or 5-day post LPS or 3-, 7-, or 23-days post-surgical injury (n = 3/time/injury). Flow cytometry characterized immunophenotypes and RT-PCR quantified cytokine gene expression. Uninjured VF were used as controls. Post-surgical and LPS injury, SWC9+/SWC3- cells identified as hi SLA-DR+ (p<0.05) compared to controls along with hi CD16+ expression at 1-day and 3-days respectively compared to all other time points (p<0.05). Surgical injuries, SWC9+/SWC3- cells exhibited hi CD163+ (p<0.05) at 3-days along with upregulation in TNFα and TGFβ1 mRNA compared to 23-days (p<0.05). No measurable changes to IL-12, IFNγ, IL-10, IL-4 mRNA post-surgery. LPS injuries induced upregulation of TNFα, IL-12, IFNγ, IL-10, and IL-4 mRNA at 1- and 5-days compared to controls (p<0.05). Higher levels of IL-10 mRNA were found 1-day post-LPS compared to 5-days (p<0.05). No changes to CD163 or CD80/86 post-LPS were measured. Acute VF injuries revealed a paradigm of markers that appear to associate with each injury. LPS induced a regulatory phenotype indicated by prominent IL-10 mRNA expression. Surgical injury elicited a complex phenotype with early TNFα mRNA and CD163+ and persistent TGFβ1 transcript expression.

No MeSH data available.


Related in: MedlinePlus

H&E staining 3-days (B), 7-days (E) and 23-days (H) post surgical injury and uninjured control (A). Labeling of inflammatory cells with MAC387 3-days (C-D), 7-days (F-G), and 23-days (I-J) post LPS injury as indicated by Vector Red staining. Arrow denotes representative positive stained cells. Column 1 magnification 10x; 100μm scale bar.
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pone.0139260.g002: H&E staining 3-days (B), 7-days (E) and 23-days (H) post surgical injury and uninjured control (A). Labeling of inflammatory cells with MAC387 3-days (C-D), 7-days (F-G), and 23-days (I-J) post LPS injury as indicated by Vector Red staining. Arrow denotes representative positive stained cells. Column 1 magnification 10x; 100μm scale bar.

Mentions: Morphological analysis was conducted using routine H&E staining to determine the area of interest (lamina propria and mid-membranous portion) for purposes of RNA isolation. To analyze infiltration of inflammatory cells into the tissue post injury [23], IHC was carried out against MAC387 surface marker with one vocal fold sample per condition. During an inflammatory reaction, MAC387 (calprotectin) is detectable in infiltrating monocyte derived macrophages and neutrophils; it is not found to be expressed by resident or mature macrophages [24]. Following LPS injury, the total cell density in the lamina propria appeared higher at 1-day and 5-days compared to uninjured vocal fold (Fig 1A, 1B, & 1E). MAC387+ cells were most prominent in the subglottal glands adjacent to vibratory edge of vocal fold after LPS injury (Fig 1D & 1G). Few MAC387+ cells were also apparent across the lamina propria at 1-day post-LPS and by 5-days post they were found in the superficial region (Fig 1C & 1F). Following surgical injury, granulation tissue and disorganization of the muscle fibers is apparent in the wound bed by 3 days (Fig 2B). By 7 days, epithelization and thickening of the lamina propria is appreciated (Fig 2E). Total cell density in the lamina propria appeared higher at 3- and 7-days post surgical injury compared to uninjured tissue. MAC387+ cells were found in subglottal glands similar to LPS injury, although temporal differences were apparent with a gradual decline in number of positive cells at day 23 (Fig 2D, 2G, & 2J). At day 3, few MAC387+ cells are seen scattered throughout the granulation tissue (Fig 2C). By 7-days, couple of MAC387+ cells were found in lamina propria medial to the muscle; staining could be seen in both cytoplasm and around cell infiltrates. After 23 days, no MAC387+ cells were found in lamina propria; however, throughout the lamina propria darker patterns of diffuse staining was appreciated surround a cell nucleus (Fig 2I).


Characterization of the Leukocyte Response in Acute Vocal Fold Injury.

King SN, Guille J, Thibeault SL - PLoS ONE (2015)

H&E staining 3-days (B), 7-days (E) and 23-days (H) post surgical injury and uninjured control (A). Labeling of inflammatory cells with MAC387 3-days (C-D), 7-days (F-G), and 23-days (I-J) post LPS injury as indicated by Vector Red staining. Arrow denotes representative positive stained cells. Column 1 magnification 10x; 100μm scale bar.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4591973&req=5

pone.0139260.g002: H&E staining 3-days (B), 7-days (E) and 23-days (H) post surgical injury and uninjured control (A). Labeling of inflammatory cells with MAC387 3-days (C-D), 7-days (F-G), and 23-days (I-J) post LPS injury as indicated by Vector Red staining. Arrow denotes representative positive stained cells. Column 1 magnification 10x; 100μm scale bar.
Mentions: Morphological analysis was conducted using routine H&E staining to determine the area of interest (lamina propria and mid-membranous portion) for purposes of RNA isolation. To analyze infiltration of inflammatory cells into the tissue post injury [23], IHC was carried out against MAC387 surface marker with one vocal fold sample per condition. During an inflammatory reaction, MAC387 (calprotectin) is detectable in infiltrating monocyte derived macrophages and neutrophils; it is not found to be expressed by resident or mature macrophages [24]. Following LPS injury, the total cell density in the lamina propria appeared higher at 1-day and 5-days compared to uninjured vocal fold (Fig 1A, 1B, & 1E). MAC387+ cells were most prominent in the subglottal glands adjacent to vibratory edge of vocal fold after LPS injury (Fig 1D & 1G). Few MAC387+ cells were also apparent across the lamina propria at 1-day post-LPS and by 5-days post they were found in the superficial region (Fig 1C & 1F). Following surgical injury, granulation tissue and disorganization of the muscle fibers is apparent in the wound bed by 3 days (Fig 2B). By 7 days, epithelization and thickening of the lamina propria is appreciated (Fig 2E). Total cell density in the lamina propria appeared higher at 3- and 7-days post surgical injury compared to uninjured tissue. MAC387+ cells were found in subglottal glands similar to LPS injury, although temporal differences were apparent with a gradual decline in number of positive cells at day 23 (Fig 2D, 2G, & 2J). At day 3, few MAC387+ cells are seen scattered throughout the granulation tissue (Fig 2C). By 7-days, couple of MAC387+ cells were found in lamina propria medial to the muscle; staining could be seen in both cytoplasm and around cell infiltrates. After 23 days, no MAC387+ cells were found in lamina propria; however, throughout the lamina propria darker patterns of diffuse staining was appreciated surround a cell nucleus (Fig 2I).

Bottom Line: Surgical injuries, SWC9+/SWC3- cells exhibited hi CD163+ (p<0.05) at 3-days along with upregulation in TNFα and TGFβ1 mRNA compared to 23-days (p<0.05).Higher levels of IL-10 mRNA were found 1-day post-LPS compared to 5-days (p<0.05).Surgical injury elicited a complex phenotype with early TNFα mRNA and CD163+ and persistent TGFβ1 transcript expression.

View Article: PubMed Central - PubMed

Affiliation: Department of Neurological Surgery, Kentucky Spinal Cord Injury Research Center, University of Louisville, Louisville, Kentucky, United States of America.

ABSTRACT
Macrophages location in the superficial layer of the vocal fold (VF) is not only at the first line of defense, but in a place of physiologic importance to voice quality. This study characterizes and compares macrophage function in two models of acute injury. Porcine VF injuries were created bilaterally by either surgical biopsy or lipopolysaccharide (LPS) (1.5 μg/kg) injection. Animals were sacrificed at 1- or 5-day post LPS or 3-, 7-, or 23-days post-surgical injury (n = 3/time/injury). Flow cytometry characterized immunophenotypes and RT-PCR quantified cytokine gene expression. Uninjured VF were used as controls. Post-surgical and LPS injury, SWC9+/SWC3- cells identified as hi SLA-DR+ (p<0.05) compared to controls along with hi CD16+ expression at 1-day and 3-days respectively compared to all other time points (p<0.05). Surgical injuries, SWC9+/SWC3- cells exhibited hi CD163+ (p<0.05) at 3-days along with upregulation in TNFα and TGFβ1 mRNA compared to 23-days (p<0.05). No measurable changes to IL-12, IFNγ, IL-10, IL-4 mRNA post-surgery. LPS injuries induced upregulation of TNFα, IL-12, IFNγ, IL-10, and IL-4 mRNA at 1- and 5-days compared to controls (p<0.05). Higher levels of IL-10 mRNA were found 1-day post-LPS compared to 5-days (p<0.05). No changes to CD163 or CD80/86 post-LPS were measured. Acute VF injuries revealed a paradigm of markers that appear to associate with each injury. LPS induced a regulatory phenotype indicated by prominent IL-10 mRNA expression. Surgical injury elicited a complex phenotype with early TNFα mRNA and CD163+ and persistent TGFβ1 transcript expression.

No MeSH data available.


Related in: MedlinePlus