Transcranial low-level laser therapy enhances learning, memory, and neuroprogenitor cells after traumatic brain injury in mice

Abstract

The use of transcranial low-level laser (light) therapy (tLLLT) to treat stroke and traumatic brain injury (TBI) is attracting increasing attention. We previously showed that LLLT using an 810-nm laser 4 h after controlled cortical impact (CCI)-TBI in mice could significantly improve the neurological severity score, decrease lesion volume, and reduce Fluoro-Jade staining for degenerating neurons. We obtained some evidence for neurogenesis in the region of the lesion. We now tested the hypothesis that tLLLT can improve performance on the Morris water maze (MWM, learning, and memory) and increase neurogenesis in the hippocampus and subventricular zone (SVZ) after CCI-TBI in mice. One and (to a greater extent) three daily laser treatments commencing 4-h post-TBI improved neurological performance as measured by wire grip and motion test especially at 3 and 4 weeks post-TBI. Improvements in visible and hidden platform latency and probe tests in MWM were seen at 4 weeks. Caspase-3 expression was lower in the lesion region at 4 days post-TBI. Double-stained BrdU-NeuN (neuroprogenitor cells) was increased in the dentate gyrus and SVZ. Increases in double-cortin (DCX) and TUJ-1 were also seen. Our study results suggest that tLLLT may improve TBI both by reducing cell death in the lesion and by stimulating neurogenesis.

Figures

Fig. 1

The wire grip and motion test (WGMT) performed at four different time points; days 7, 14, 21, and 28 post-TBI induction. †, ††, ††† P<0.05, 0.01, 0.001 versus traumatic brain injury (TBI).

Fig. 2

Evaluating the effect of transcranial low-level laser (light) therapy (tLLLT) on cognitive performance of TBI sustained mice via Morris water maze test; (a) visible platform test, (b) hidden platform test, (c) probe test *** p<0.001 versus sham; †, ††, ††† P<0.05, 0.01, 0.001 versus TBI; ‡P<0.05 versus TBI 1 laser.

Fig. 3

Immunohistilogic analysis for caspase-3 expression at the injury site, done at day 4 of the TBI induction. (a) sham, (b) CCI-TBI, (c) 1× tLLLT, (d) 3× tLLLT, (e) mean caspase-3/DAPI ratios±SD (n=5). Scale bar 100  μm. *** p<0.001 versus sham; †, †† P<0.05, 0.01 versus TBI.

Fig. 4

BrdU-NeuN double-staining images and analyses at the neurogenic hippocampal DG at the 7 days point; (a) sham, (b) CCI-TBI, (c) 1× tLLLT, (d) 3× tLLLT, (e) mean BrdU/DAPI ratios±SD (n=5); normalization of the readings was done BrdU versus DAPI (labeling the nuclei). Scale bar 100  μm. *** p<0.001 versus sham; ††, ††† P<0.01, 0.001 versus TBI.

Fig. 5

BrdU-NeuN double-staining images and analyses at the neurogenic hippocampal DG at the 28 days point; (a) sham, (b) CCI-TBI, (c) 1× tLLLT, (d) 3× tLLLT, (e) mean BrdU/DAPI ratios±SD (n=5); normalization of the readings was done BrdU versus DAPI (labeling the nuclei). Scale bar 100  μm. ** p<0.01 versus sham; †, †† P<0.05, 0.01 versus TBI.

Fig. 6

BrdU-NeuN double-staining images and analyses at the neurogenic subventricular zone (SVZ) at the 7 days point; (a) sham, (b) CCI-TBI, (c) 1× tLLLT, (d) 3× tLLLT, (e) mean BrdU/DAPI ratios±SD (n=5); normalization of the readings was done BrdU versus DAPI (labeling the nuclei). Scale bar 100  μm. *** p<0.001 versus sham; †, ††† P<0.05, 0.001 versus TBI.

Fig. 7

BrdU-NeuN double-staining images and analyses at the neurogenic SVZ at the 28 days point; (a) sham, (b) CCI-TBI, (c) 1× tLLLT, (d) 3× tLLLT, (e) mean BrdU/DAPI ratios±SD (n=5); normalization of the readings was done BrdU versus DAPI (labeling the nuclei). Scale bar 100  μm. *** p<0.001 versus sham; ††, ††† P<0.01, 0.001 versus TBI.

Fig. 8

BrdU incorporating cells at the lesion site at the 7- and 28-days points. Mean BrdU/DAPI ratios±SD (n=5). *, *** P<0.05, 0.001 versus sham; †, †† P<0.05, 0.01 versus TBI.

Fig. 9

Microtubule-associated neuronal migration protein (DCX) expression images and analyses at the neurogenic hippocampal DG at the 7 days point; (a) sham, (b) CCI-TBI, (c) 1xtLLLT, (d) 3xtLLLT, (e) mean DCX/DAPI ratios±SD (n=5); normalization of the readings was done DCX versus DAPI (labeling the nuclei). Scale bar 100 μm. * p<0.05 versus sham; †, †† P<0.05, 0.01 versus TBI.

Fig. 10

Microtubule-associated neuronal migration protein (DCX) expression images and analyses at the neurogenic hippocampal DG at the 28 days point; (a) sham, (b) CCI-TBI, (c) 1× tLLLT, (d) 3× tLLLT, (e) mean DCX/DAPI ratios±SD (n=5); normalization of the readings was done DCX versus DAPI (labeling the nuclei). Scale bar 100  μm. *p<0.05 versus sham; † P<0.05 versus TBI.

Fig. 11

Microtubule-associated neuronal migration protein (DCX) expression images and analyses at the neurogenic SVZ at the 7 days point; (a) sham, (b) CCI-TBI, (c) 1× tLLLT, (d) 3× tLLLT, (e) mean DCX/DAPI ratios±SD (n=5); normalization of the readings was done DCX versus DAPI (labeling the nuclei). Scale bar 100  μm. * p<0.05 versus sham; ††† P<0.001 versus TBI.

Fig. 12

Microtubule-associated neuronal migration protein (DCX) expression images and analyses at the neurogenic SVZ at the 28 days point; (a) sham, (b) CCI-TBI, (c) 1× tLLLT, (d) 3× tLLLT, (e) mean DCX/DAPI ratios±SD (n=5); normalization of the readings was done DCX versus DAPI (labeling the nuclei). Scale bar 100  μm. *p<0.05 versus sham; † P<0.05 versus TBI.

Fig. 13

Microtubule-associated neuronal migration protein (DCX) expression at the lesion site. Mean DCX/DAPI ratios±SD (n=5); normalization of the readings was done as DCX versus DAPI (labeling the nuclei). *** p<0.001 versus sham; †, ††† P<0.05, 0.001 versus TBI.

Fig. 14

Neuron-specific class III β-tubulin (TUJ-1) expression in differentiating neural progenitor cells of the DG at 7 days point. Images are for (a) sham, (b) CCI-TBI, (c) 1× tLLLT, (d) 3× tLLLT brain slices, (e) mean TUJ-1/DAPI ratios±SD (n=5); normalization of the readings was done TUJ-1 versus DAPI (labeling the nuclei). Scale bar 100  μm. ** p<0.01 versus sham; †, †† P<0.05, 0.01 versus TBI.

Fig. 15

Neuron-specific class III β-tubulin (TUJ-1) expression in differentiating neural progenitor cells of DG at 28 days point. Images are from (a) sham, (b) CCI-TBI, (c) 1× tLLLT, (d) 3× tLLLT brain slices, (e) mean TUJ-1/DAPI ratios±SD (n=5); normalization of the readings was done TUJ-1 versus DAPI (labeling the nuclei). Scale bar 100  μm. †, †† P<0.05, 0.01 versus (TBI).

Fig. 16

Neuron-specific class III β-tubulin (TUJ-1) expression in differentiating neural progenitor cells of SVZ at 7 days point. Images are for (a) sham, (b) CCI-TBI, (c) 1× tLLLT, (d) 3× tLLLT, (e) mean TUJ-1/DAPI ratios±SD (n=5), and (f) close look up brain slices; normalization of the readings was done TUJ-1 versus DAPI (labeling the nuclei). Scale bar 100  μm, except in (f) where it is 25  μm. †, †† P<0.05, 0.01 versus TBI.

Fig. 17

Neuron-specific class III β-tubulin (TUJ-1) expression in differentiating neural progenitor cells of subventricular zone (SVZ) at 28 days point. Images are for (a) sham, (b) CCI-TBI, (c) 1× tLLLT, (d) 3× tLLLT, (e) mean TUJ-1/DAPI ratios±SD (n=5); normalization of the readings was done TUJ-1 versus DAPI (labeling the nuclei). Scale bar 100  μm. † P<0.05 versus TBI.