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Effect of DNase I treatment and neutrophil depletion on acute limb ischemia-reperfusion injury in mice  Hassan Albadawi, MD, Rahmi Oklu, MD, PhD, Rita.

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Presentation on theme: "Effect of DNase I treatment and neutrophil depletion on acute limb ischemia-reperfusion injury in mice  Hassan Albadawi, MD, Rahmi Oklu, MD, PhD, Rita."— Presentation transcript:

1 Effect of DNase I treatment and neutrophil depletion on acute limb ischemia-reperfusion injury in mice  Hassan Albadawi, MD, Rahmi Oklu, MD, PhD, Rita Elise Raacke Malley, BS, Ryan M. O'Keefe, BS, Thuy P. Uong, Nicholas R. Cormier, Michael T. Watkins, MD  Journal of Vascular Surgery  Volume 64, Issue 2, Pages (August 2016) DOI: /j.jvs Copyright © 2016 Society for Vascular Surgery Terms and Conditions

2 Fig 1 Schematic diagram of acute hindlimb ischemia-reperfusion (IR) and treatment protocols. C57BL6 male mice were subjected to 1.5 hours of unilateral hindlimb tourniquet ischemia followed by 24 hours of reperfusion. In the first set of experiments (A), mice were subjected to IR and split into two groups. The first group received 50 μg of human recombinant DNase I (n = 8) intraperitoneally (IP) at 5 minutes before reperfusion, a second dose of 10 μg intravenously (IV) at 10 minutes after reperfusion, and a third dose of 50 μg IP at 12 hours after reperfusion. The second control group received similar volumes of carrier buffer alone at the same time points (n = 8). In the second part of this study (B), two separate groups of mice received daily injections of either rabbit antimouse neutrophil serum intraperitoneally for neutrophil depletion (ND; n = 8) or normal rabbit serum (NS; n = 7) for 48 hours 2 days before IR. Mice were then subjected to 1.5 hours of unilateral IR injury and received additional rabbit antimouse serum or NS 1 minute after the start of reperfusion. At the end of the 24-hour IR period, hindlimb perfusion was documented with laser Doppler imaging (LDI), then mice were euthanized and hindlimb muscle tissues were harvested for analysis. PMN, Polymorphonuclear cells. Journal of Vascular Surgery  , DOI: ( /j.jvs ) Copyright © 2016 Society for Vascular Surgery Terms and Conditions

3 Fig 2 Effect of DNase I treatment on muscle fiber injury and adenosine triphosphate (ATP) levels after acute ischemia-reperfusion (IR). A and B, Representative micrographs of Masson trichrome-stained acrylic-embedded tibialis anterior muscle show the muscle tissue morphology in cross sections after IR from control (A) and DNase I-treated (B) mice. Injured muscle fibers are evident in the field, such as the ones indicated by arrows with observed edema, hemorrhage, and leukocyte infiltration. C and D, Quantitative analysis of the percentage muscle fiber injury per randomized high-power field (C) and muscle ATP (D) revealed that DNase I treatment did not alter skeletal muscle fiber injury or the steady-state levels of ATP. Journal of Vascular Surgery  , DOI: ( /j.jvs ) Copyright © 2016 Society for Vascular Surgery Terms and Conditions

4 Fig 3 Effect of DNase I treatment on hindlimb perfusion and thrombin-antithrombin III (TAT III) levels in the hindlimb muscle after ischemia-reperfusion (IR). A and B, The Laser Doppler scanner-generated images demonstrate the degree of perfusion in each hindlimb (red, the highest perfusion, in contrast to dark blue, which represents no perfusion). The IR limb in the Pulmozyme-treated mouse (A) appeared to have similar perfusion compared with the uninjured contralateral limb, whereas the IR limb in the untreated control mouse (B) had diminished perfusion compared with the contralateral limb. C, Calculating the hindlimbs, the scanner-generated flux ratio of the hindlimbs in each mouse as an index of limb perfusion revealed significantly higher perfusion at 24 hours of reperfusion in the DNase I-treated mice compared with the control group (*P = .02). D, Hindlimb tissue TAT III levels significantly increased after IR in both the control and the DNase I-treated group compared with sham (*P < .001). However, the TAT III levels were significantly lower in the DNase I-treated mice compared with control (**P < .05). These data suggest that DNase I treatment ameliorated tissue thrombosis, which paralleled higher hindlimb perfusion. Journal of Vascular Surgery  , DOI: ( /j.jvs ) Copyright © 2016 Society for Vascular Surgery Terms and Conditions

5 Fig 4 Effect of DNase I treatment on detection of extracellular traps (ETs) after ischemia-reperfusion (IR). Representative images from Cy3-labeled immunostaining of ETs. A, ETs were not detectable in the noninjured sham muscle. B, However, extensive ETs were observed in the interstitium of the injured muscle fibers, as indicated by the arrows. C, DNase I treatment significantly reduced the detection of ETs in the tissue after IR. D, Mean fluorescent intensity (MFI) values for DNase I-treated and untreated control mice are summarized in the graph (*P = .036). Journal of Vascular Surgery  , DOI: ( /j.jvs ) Copyright © 2016 Society for Vascular Surgery Terms and Conditions

6 Fig 5 Effect of DNase I treatment on muscle inflammation after ischemia-reperfusion (IR). A and B, Representative micrographs of tibialis anterior muscle demonstrate immunostaining for the leukocyte marker Ly6B (brown) in the tissue sections after IR obtained from control (A) or DNase I-treated (B) mice. C-E, Quantitative analysis of Ly6B-positive cells and keratinocyte chemoattractant (KC) and myeloperoxidase (MPO) levels, summarized in the graphs, revealed that DNase I treatment markedly reduced the average number of leukocytes (*P = .0054, C) but did not alter skeletal muscle KC (D) or MPO (E) levels at 24 hours after IR. Journal of Vascular Surgery  , DOI: ( /j.jvs ) Copyright © 2016 Society for Vascular Surgery Terms and Conditions

7 Fig 6 Effect of DNase I treatment on endonuclease activity and γ-actin protein levels after ischemia-reperfusion (IR). A and B, Endonuclease activity was evaluated with plasmid incision assay in skeletal muscle tissue protein extracts from sham, DNase I-treated, and untreated control mice. The representative image (A) show the bands for the open circular and linear forms of the plasmid DNA, which increased over the supercoiled DNA in the DNase I-treated and control groups compared with sham, indicating increased endonuclease activity; the right lane of the gel image represents the pBR322 plasmid alone, which presented only a supercoiled plasmid DNA form. Quantitative analysis (B) shows that endonuclease activity was markedly elevated in both the control and DNase I-treated groups over sham levels (*P < .01), with significantly higher levels detected in the DNase I group compared with control (**P < .05). These data suggest that DNase I treatment further enhanced endonuclease activity after IR. C, Analysis of γ-actin protein expression by Western blotting shows an enhanced γ-actin protein expression after IR compared with sham. D, Summary graph displays the quantitative analysis of the 42-kDa γ-actin band densities normalized to the Ponceau S bands. Data show a significant increase in γ-actin protein in the postischemic hindlimb muscle in the DNase I-treated and control groups compared with sham (*P < .01). There was no difference in the γ-actin levels between the DNase I-treated and control groups. These data suggest that DNase I treatment did not alter the γ-actin protein levels. Journal of Vascular Surgery  , DOI: ( /j.jvs ) Copyright © 2016 Society for Vascular Surgery Terms and Conditions

8 Fig 7 Immunofluorescence localization of DNase I with γ-actin proteins in skeletal muscle tissue after ischemia-reperfusion (IR) with and without exogenous DNase I treatment. Images in the panels are representative of dual immunofluorescence detection of DNase I and γ-actin using Alexa Fluor 488 and Cy3 fluorochrome conjugated to secondary immunoglobulin G (IgG), respectively, in skeletal muscle tissue from uninjured sham hindlimbs and after IR. In the sham mice (top images), there was a very low level of DNase I (top left, green) or γ-actin (top middle, red) and merged (top right) detected in the tissue. However, we observed in the mice that were treated with DNase I (Pulmozyme, middle images) or in the control group (lower images) extensive DNase I (green) and γ-actin (red) staining detected at 24 hours after IR. Merged fluorescent images demonstrated that DNase I and γ-actin molecules colocalized within and around the injured fibers in the DNase I-treated and control groups (merged colors in yellow/orange). The colocalization of DNase I suggests that DNase I might be inhibited in the presence of γ-actin in the injured tissue. Journal of Vascular Surgery  , DOI: ( /j.jvs ) Copyright © 2016 Society for Vascular Surgery Terms and Conditions

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