Figure 1.
tPA protein level and activity as well as plasmin activity in rat brain.
Western blot analysis of tPA protein levels in the rat brain 24 hr after intranasal administration of tPA (A). Bar graph (B) shows the tPA protein level. Of note, Sham+tPA rats received the same amount of intranasal tPA administration as TBI+tPA rats did. Representative zymographic assay (C) shows an increase in tPA activity in the Sham+tPA rats and TBI+tPA rats compared to TBI+Saline rats. h-r-tPA: human recombinant tPA (15 ng, Genentech). Bar graph (D) shows the tPA activity. Bar graph (E) shows amidolytic activity of plasmin assayed with D-Val-Leu-Lys-p-Nitroanilide Dihydrochloride (S-2251) as its specific substrate. Bar graph (F) shows tPA amidolytic activity with S-2251 as the substrate in the presence of added plasminogen compared to Sham. *p<0.05 vs TBI+Saline. #p<0.05 vs Sham+tPA. Data represent mean ± SD. n = 4 (rats/group, in graphs D, E, and F).
Figure 2.
tPA effect on functional outcome.
tPA significantly lowered the mNSS scores (A) and reduced frequency of foot faults (B) from Day 14 to 35 after TBI compared to the saline group. tPA treatment significantly improved spatial learning performance from Day 33 to 35 after TBI compared with the saline group (C). *p<0.05 vs TBI+Saline. Data represent mean ± SD. n = 8 (rats/group).
Figure 3.
tPA effect on immature neurons after TBI.
DCX staining (A–C). tPA significantly increased immature neurons identified with DCX-positive staining in the DG in rats examined 35 days after injury compared with the saline-treated group. The bar graph shows the number of DCX-positive cells. Scale bar = 25 µm. *p<0.05 vs TBI+Saline. Data represent mean ± SD. n = 8 (rats/group).
Figure 4.
tPA effect on neurogenesis after TBI.
Compared to the TBI+Saline group (B), tPA treatment (C) significantly increased newborn mature neurons identified with BrdU/NeuN double immunofluorescent staining in the DG 35 days post injury. The bar graph (D) shows the number of DCX-positive cells. Scale bar = 25 µm. *p<0.05 vs TBI+Saline. Data represent mean ± SD. n = 8 (rats/ group).
Figure 5.
Correlation of the number of neuroblasts (A), and newborn neurons (B) with spatial learning.
The line graph shows that spatial learning is significantly correlated with the number of DCX-positive cells (A) and to the number of newborn mature neurons (B) in the DG of the ipsilateral hippocampus in rats examined at 35 days after TBI and tPA treatment (p<0.05).
Figure 6.
BDA-labeling of CST originating from the contralesional intact hemisphere.
Representative images from the cervical spinal cord show BDA-labeled CST axons crossing the midline (arrows in B) and sprouting into the denervated side of the ventral gray matter in a rat after TBI. tPA treatment significantly increased the axon midline crossing (arrows in C). There were no obvious BDA-labeled axons observed in the opposite side of the cervical spinal cord in sham rats (A). Quantitative data (D) show that the number of contralesional CST in the denervated cervical gray matter was increased significantly by traumatic injury (p<0.05 vs. Sham+tPA) and tPA treatment (p<0.05 vs. TBI+Saline). Scale bar = 50 µm. Data represent mean ± SD. n = 8 (rats/group).
Figure 7.
Correlation of the total length of axons crossing the midline at the cervical spinal cord with the right forelimb foot fault (A) and the mNSS score (B).
The line graph shows that the total length of axonal crossing at the midline of the cervical level of the spinal cord is significantly reversely correlated with the incidence of forelimb footfault (A) and mNSS score (B) in rats examined at 35 days after TBI and tPA treatment (p<0.05).
Figure 8.
Mean threshold current levels needed to evoke forelimb movements on stimulation of the right cerebral motor cortex.
For each animal, the threshold average was calculated from 4 stimulation points. ICMS of the motor cortex in normal adult rats evoked low threshold contralateral forelimb movements and high threshold ipsilateral movements. After TBI, the contralateral movement threshold (for left normal forelimb) was unchanged, whereas the ipsilateral movement threshold was decreased significantly at 5 weeks postinjury compared to sham (p<0.05). tPA treatment further reduced the threshold current needed for ipsilateral forelimb movement compared to the saline controls (p<0.05). Data represent mean ± SD. n = 8 (rats/group).
Figure 9.
Immunostaining analysis of ProBDNF and mature BDNF-positive cells in the brain and spinal cord.
Left panel: ipsilateral brain cortex; Right panel: denervated cervical spinal cord. *p<0.05 vs TBI+Saline. Scale bar = 25 µm. Data represent mean ± SD. n = 4 (rats/group).
Figure 10.
Western blot analysis of ProBDNF and mature BDNF protein levels in the brain and spinal cord.
Left panel (ipsilateral brain cortex): Sham+tPA (1, 2), TBI+Saline (3, 4); 3: TBI+tPA (5, 6); Right panel (denervated cervical spinal cord): Sham+tPA (1, 2), TBI+Saline (3, 4), TBI+tPA (5, 6). *p<0.05 vs TBI+Saline. Data represent mean ± SD. n = 4 (rats/group).
Figure 11.
Cortical lesion volume after TBI and tPA treatment.
The bar graph shows no significance (NS) in the cortical lesion volume between the TBI+Saline and TBI+tPA groups examined at 35 days post injury (p>0.05). Scale bar = 2 mm. Data represent mean ± SD. n = 8 (rats/group).