Intra-Arterial Deoxyribonuclease Therapy Improves Stroke Outcomes in Aged Mice

Abstract

Background: Futile recanalization affects more than half of acute ischemic stroke (AIS) patients. Neutrophil extracellular traps (NETs) are a major factor of microvascular hypoperfusion after stroke. Deoxyribonuclease I (DNase) targeting NETs exhibited a neuroprotective effect in young mice with AIS. This study explored a novel direct intra-arterial administration of DNase therapy and its effect in aged mice with AIS.

Method: AIS was induced in aged C57BL/6 mice followed by reperfusion and immediate, intra-arterial DNase administration via the internal carotid artery. Cerebral blood flow (CBF), neurological function, cerebral infarct volume, and NET markers were examined.

Results: Direct intra-arterial DNase therapy significantly increased CBF, reduced neurological deficit scores, increased the latency to fall in the wire hang test, reduced cerebral infarct volume, and decreased neutrophil and NET count in both the parenchyma and micro vessels in aged mice with AIS compared with age-matched vehicle controls.

Conclusion: Our data is the first to demonstrate that successful, direct intra-arterial DNase therapy provides more efficient cerebral reperfusion and better outcomes after recanalization during the treatment of large vessel occlusion in aged mice. This study provides evidence for the potential clinical application of catheter-delivered intra-arterial DNase therapy post-recanalization.

Keywords: acute ischemic stroke; deoxyribonuclease; futile recanalization; intra‐arterial therapy; neutrophil; neutrophil extracellular traps.

FIGURE 1

DNase activity in vitro and in vivo. (A) Dose dependent DNase activity curve in vitro. (B) DNase activity curve in mice serum. DNase IP injection, n = 3; tail vein (IV) injection, n = 4, and Ctrl, n = 3. **p < 0.01, both IP and TV injection serum vs. Ctrl serum. GraphPad Prism 9.5 was used for data and analysis and DNase activity curve generation.

FIGURE 2

Intra‐artery injection tools and Methylene blue injection after MCAO. (A) Microcatheters and Hamilton syringe. (B) Images of mouse brain after direct intra‐artery Methylene blue injection, mouse was subjected to MCAO with 60′ ischemia followed by reperfusion and immediate intra‐arterial 20 μL Methylene blue injection. (C) Images of brain sections. With immediate intra‐arterial methylene blue injection, blue dye spreads in ischemic reperfusion brain tissue side and no blue dye were observed in contralateral brain tissue.

FIGURE 3

Intra‐arterial DNase treatment improved early cerebral blood reperfusion in aged mice with AIS. Cerebral blood flow was evaluated immediately after intra‐arterial DNase injection. The ratio of cerebral blood flow (ipsilateral (ischemic) side to contralateral side) was calculated. (A) Representatives of cerebral blood flow images for Vehicle and DNase, and (B) Results of the ratio of cerebral blood flow. *p < 0.05, compared with Vehicle.

FIGURE 4

Intra‐arterial DNase treatment improved neurological function outcomes and reduced ischemic infarction in aged mice with AIS. (A) Neurological deficit score, (B) Latency to fall in wire hang test, (C) Representative TTC images, (D) Bar graph of infarct volumes. ImageJ was used for infarct volume calculation based on TTC staining images. *p < 0.05, **p < 0.01, compared with vehicle.

FIGURE 5

Intra‐arterial DNase treatment reduced neutrophils infiltration and NETs formation in ischemic penumbra of aged mice with AIS. Brain sections were stained with neutrophil specific markers (Ly6G, myeloperoxidase‐MPO, and neutrophil elastase‐NE), NETs marker (Citrullinated histone H3‐CitH3), vascular marker antibodies (vascular cell adhesion molecule 1 [VCAM] and Lectin), and nuclei (DAPI). NETs were identified by neutrophil specific markers and CitH3 positive signaling. Representative images from the vehicle treatment group: (A) Image of TTC staining, penumbra area recircled with white dash line. (B) Image of anti‐Ly6G IHC staining. (C) Image of anti‐Ly6G IF staining. (D) Merge image of anti‐Ly6G (green), anti‐VCAM (red), and DAPI (blue) IF staining. (E) Image of anti‐CitH3 IF staining. (F) Image of anti‐NE IF staining. (G) Merge image of anti‐CitH3 (green), anti‐NE (red), and DAPI (blue) IF staining. (H) Merge image of anti‐Lectin (green), anti‐MPO (red) and DAPI (blue) of whole section IF staining. (I) Image of anti‐Lectin IF staining. (J) Image of anti‐MPO IF staining, intra‐vascular (white arrow). (K) Merged image of anti‐Lectin, anti‐MPO and DAPI, intra‐vascular NETs signal (white arrow) in penumbra. (L) Image of anti‐CitH3 IF staining. (M) Image of anti‐Lectin IF staining. (N) Merged image of anti‐CitH3 (green), anti‐Lectin (red) and DAPI (blue), CitH3 signal (yellow color) in vascular (right low corner panel) in penumbra. Representative images from the DNase treatment group: (O) Image of TTC staining, penumbra area recircled with white dash line. (P) Image of anti‐Ly6G IHC staining. (Q) Image of anti‐Ly6G IF staining. (R) Merged image of anti‐Ly6G (green), anti‐VCAM (red) and DAPI (blue) IF staining. (S) Image of anti‐CitH3 IF staining. (T) Image of anti‐NE IF staining. (U) Merge image of anti‐CitH3 (green), anti‐NE (red) and DAPI (blue) IF staining. (W) Image of anti‐Lectin IF staining. (V) Merge image of anti‐Lectin (green), anti‐MPO (red) and DAPI (blue) of whole section IF staining. (X) Image of anti‐MPO IF staining, intra‐vascular (white arrow). (Y) Merged image of anti‐Lectin, anti‐MPO, and DAPI IF staining, intra‐vascular NETs signal (white arrow) in penumbra. (Z) Image of anti‐CitH3 IF staining. (Aa) Image of anti‐Lectin IF staining. (Bb) Merge image of anti‐CitH3 (green), anti‐Lectin (red), and DAPI (blue) IF staining. (Cc) Plots graph of the number of anti‐Ly6G positive signal. (Dd) Plots graph of the number of total CitH3 positive signal. (Ee) Plots graph of the number of vascular CitH3 positive signal. **p < 0.01, compared with Vehicle.

FIGURE 6

Intraperitoneal DNase treatment reduced NETs formation in systemic circulation and improved capillary recanalization in penumbra of mice with AIS. (A) Plasma NE concentration. (B) Plasma dsDNA concentration. Representative images from vehicle treatment group: (C, F) Image of Albumin‐FITC. (D, G) Image of anti‐NeuN IF staining. (E, H) Merge image of Albumin‐FITC (green), anti‐NeuN (red), and DAPI (blue) IF staining. Representative images from DNase treatment group: (I, L) Image of Albumin‐FITC. (J, M) Image of anti‐NeuN IF staining. (K, N) Merge image of Albumin‐FITC (green), anti‐NeuN (red), and DAPI (blue) IF staining. (O) Vascular density as calculated by Albumin‐FITC fluorescent signal (vehicle treatment, n = 6 and DNase treatment, n = 9). *p < 0.05, **p < 0.01, compared with vehicle.