Subacute intranasal administration of tissue plasminogen activator improves stroke recovery by inducing axonal remodeling in mice

Abstract

In addition to thrombolysis, tissue plasminogen activator (tPA) can evoke neurorestorative processes. We therefore investigated the therapeutic effect of subacute intranasal administration of tPA post stroke on neurological recovery and on corticospinal innervation in mice. A transgenic mouse line, in which the pyramidal neurons and corticospinal tract (CST) axons are specifically labeled by yellow fluorescent protein (YFP) was employed. Adult CST-YFP mice were subjected to right unilateral middle cerebral artery occlusion (MCAo), and were randomly divided into groups treated with saline or tPA intranasally in the subacute phase. Pseudorabies virus (PRV)-614-monomeric red fluorescent protein (RFP) was injected into the left forelimb. The cervical spinal cord and brain were processed for fluorescent microscopy to detect YFP and RFP labeling. Primary embryonic neurons were cultured with tPA at different concentrations. Neurite length and branch numbers were then measured. In vivo, subacute tPA treatment significantly enhanced functional recovery (p < 0.05), and increased CST density in the denervated gray matter, and in the numbers of PRV-labeled neurons in bilateral cortices. The behavioral performance was significantly correlated with axonal density in the denervated spinal cord. In vitro, both neurite length and branch numbers significantly increased with concentration of tPA (p < 0.05). Our results demonstrate that tPA dose-dependently increases neurite outgrowth and branching of cultured cortical neurons. Subacute intranasal administration of tPA may provide enhance neurological recovery after stroke by promoting CST axonal remodeling.

Keywords: Axonal remodeling; Functional recovery; Middle cerebral artery occlusion; Stroke; Tissue plasminogen activator.

Figure 1

Temporal profile of the left forepaw deficit and recovery after right MCAo assessed with adhesive-remove test (A) and single pellet reaching test (B). Note that significant behavioral deficits and progressive recovery were observed with both tests in all mice; while subacute tPA intranasal treatment significantly enhanced functional recovery compared with saline treated mice (n=10/group, p<0.05 vs control).

Figure 2

Single layer confocal images of gray matter of the cervical cord. In transgenic CST-YFP mice, the CST axons are fluorescent yellow-green under a laser-scanning confocal microscopy (A-C). Thirty-two days after right MCAo, compared to normal mice (A), YFP-positive CST axonal density was reduced in the denervated side of the cervical gray matter (B and C), while axons crossing the midline into the denervated left side from the right intact side were evident in the tPA treated mice (C). Quantitative analysis of the percentage of CST axons in the denervated side to the contralateral side demonstrated that intranasal tPA treatment significantly increased axonal density in the denervated gray matter (D, n=10 per group, p<0.0

Figure 3

Single layer fluorescent microscopy images on coronal brain sections at the bregma level. Four days after tracer injection into the left forelimb muscles, PRV labeling was primarily found in the right cortex (A), and rarely in the left cortex (B) in normal mice. In stroke mice, PRV-positive cortical neurons in the right cortex were dramatically reduced (C), while PRV-labeled cells in the left contralesional cortex was comparable with normal mice (D). In contrast, intranasal tPA treatment increased the PRV labeling in both ipsilesional (E) and contralesional hemispheres (F) compared with saline treated mice.

Figure 4

Representative images show cortical neurons isolated from embryos of CST-YFP mice cultured with different concentrations of tPA (A-F, 0 to 13 µg/ml). Note that both neurite length and branch numbers were increased with concentration of tPA, and reached a peak at 6.5 µg/ml.