Nano-Pulsed Laser Therapy Is Neuroprotective in a Rat Model of Blast-Induced Neurotrauma

Abstract

We have developed a novel, non-invasive nano-pulsed laser therapy (NPLT) system that combines the benefits of near-infrared laser light (808 nm) and ultrasound (optoacoustic) waves, which are generated with each short laser pulse within the tissue. We tested NPLT in a rat model of blast-induced neurotrauma (BINT) to determine whether transcranial application of NPLT provides neuroprotective effects. The laser pulses were applied on the intact rat head 1 h after injury using a specially developed fiber-optic system. Vestibulomotor function was assessed on post-injury days (PIDs) 1-3 on the beam balance and beam walking tasks. Cognitive function was assessed on PIDs 6-10 using a working memory Morris water maze (MWM) test. BDNF and caspase-3 messenger RNA (mRNA) expression was measured by quantitative real-time PCR (qRT-PCR) in laser-captured cortical neurons. Microglia activation and neuronal injury were assessed in brain sections by immunofluorescence using specific antibodies against CD68 and active caspase-3, respectively. In the vestibulomotor and cognitive (MWM) tests, NPLT-treated animals performed significantly better than the untreated blast group and similarly to sham animals. NPLT upregulated mRNA encoding BDNF and downregulated the pro-apoptotic protein caspase-3 in cortical neurons. Immunofluorescence demonstrated that NPLT inhibited microglia activation and reduced the number of cortical neurons expressing activated caspase-3. NPLT also increased expression of BDNF in the hippocampus and the number of proliferating progenitor cells in the dentate gyrus. Our data demonstrate a neuroprotective effect of NPLT and prompt further studies aimed to develop NPLT as a therapeutic intervention after traumatic brain injury (TBI).

Keywords: blast injury; near-infrared light; neuroprotection; non-invasive transcranial laser therapy; optoacoustics; traumatic brain injury.

FIG. 1.

Experimental design. Experimental rats were randomized to receive blast or sham injury. Blast-injured rats were further randomized to receive nano-pulsed laser therapy (NPLT) or no therapy. Vestibulomotor function (beam balance and beam walk) was assessed on post-injury days (PIDs) 1–3 and working memory function (Morris water maze [MWM]) was assessed on PIDs 6–10. On PIDs 3 (n = 4/group) and 7 (n = 3–4/group), brains were collected for laser-captured microdissection (LCM) of neurons from the cerebral cortex and hippocampus CA1-3 region and for immunofluoerescence analysis (IF) of active caspase-3 and NeuN expression. The expression of select genes in LCM neurons was detected by quantitative real-time PCR (qRT-PCR). On PID 8, 9, and 10 the rats were injected with bromodeoxyuridine (BrdU) for the analysis of cell proliferation. On PID 10, after completion of the MWM test, brains were collected for analysis of microglia activation (CD68 immunofluorescence) and cell proliferation in the hippocampus dentate gyrus (BrdU immunoreacticvity). IHC, immunohistochemistry.

FIG. 2.

Location of blast injury. (A) Dorsal view of the rat skull showing the approximate location of blast injury. (B) Coronal view of the rat brain (modified from Paxinos, G., and Watson, C. The Rat Brain in Stereotaxic Coordinates, 4th ed. Academic Press: San Diego, Fig. 36) showing the location of the blast at −4.18 mm of bregma (corresponding to the center of the blast location in the coronal plane). (C) Sagittal view of the rat brain at 1.90 mm lateral, corresponding to the center of the blast in the sagittal plane. (Modified from Paxinos, G., and Watson, C. The Rat Brain in Stereotaxic Coordinates, 4th ed. Academic Press: San Diego, Fig. 83.)

FIG. 3.

Nano-pulsed laser therapy (NPLT) prevents blast-induced vestibulomotor and cognitive dysfunctions. (A) The beam balance test: Rats that received blast injury had higher scores on post-injury day (PID) 1 compared with rats that received sham injury or blast plus NPLT treatment. Two-way analysis of variance (ANOVA) revealed a significant overall effect of treatment (p < 0.001) and time after injury (p < 0.001); Post-hoc Fisher's multiple comparisons test revealed ***p < 0.001 BLAST vs. SHAM; **p < 0.01 BLAST vs. BLAST+NPLT on PID 1; and ^p < 0.01 BLAST PID1 vs. Blast PID 0 (pre-injury). The number of rats in each experimental group is shown in parenthesis. (B) The beam walk test: rats that received blast injury showed longer latencies to traverse the beam on PIDs 1 and 2 compared with rats that were sham injured or received blast plus NPLT treatment. Two-way ANOVA revealed a significant overall effect of treatment (p < 0.01). Post-hoc Fisher's multiple comparisons test revealed ***p < 0.001 BLAST vs. SHAM on PID 1 and *p < 0.05 BLAST vs. BLAST+NPLT on PID 2; and #p < 0.0001 BLAST PID 1 vs. BLAST PID 0 (pre-injury) and ^p < 0.01 BLAST PID 2 vs. Blast PID 0 (pre-injury). The number of rats in each experimental group is shown in parenthesis. (C) The working memory water maze test: Rats were tested in the working memory paradigm of the Morris water maze test on PIDs 6–10. Only data from Trial 2 are shown. Two-way ANOVA revealed a significant overall effect of treatment (p < 0.05) and of time after injury (p < 0.0001); Post hoc Tukey's multiple comparisons test revealed **p < 0.01 BLAST vs. SHAM and ^p < 0.05 BLAST vs. BLAST+NPLT on PID 7. Data are mean ± standard error of the mean (SEM). Number of animals is shown in parenthesis.

FIG. 4.

Nano-pulsed laser therapy (NPLT) prevents early blast-induced changes in expression of genes involved in the control of cell death and survival in the cortex and hippocampus. Experimental animals were euthanized on post-injury days (PIDs) 3 (A) and 7 (B,C), and the brains removed, stained with Fluoro-Jade to identify injured cells, and counterstained with cresyl violet (CV). Laser-capture micro-dissection (LCM) was used to collect cortical neurons in the somatosensory cortex (A,B) and pyramidal neurons in the CA1-3 region of the hippocampus (C). Only Fluoro-Jade^neg/CV^poscells were captured. The expression of select messenger RNAs (mRNAs) was measured by quantitative real-time PCR (qRT-PCR) analysis. Data were normalized to GAPDH expression and expressed as mean ± standard error of the mean (SEM); n = 4 for SHAM, n = 3 for BLAST and BLAST+NPLT. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001, two-way analysis of variance (ANOVA) followed by Tukey's multiple comparisons test.

FIG. 5.

Activation of caspase-3 is reduced after nano-pulsed laser therapy (NPLT) treatment in blast-injured brains. Immunofluorescence analysis from rats euthanized on post-injury day (PID) 3. (A) Representative images of brain sections at the level of the somatosensory cortex stained with an antibody against the neuronal marker NeuN (red) and an indicator of apoptotic cell death, active caspase-3 (green). Nuclei are counterstained with DAPI and are shown in blue. Calibration bar is 50 μm. (B) Quantitative analysis of the number of active caspase-3^pos/NeuN^pos cells. N = 3 rats/group. *p < 0.05 one-way analysis of variance (ANOVA) followed by Tukey's multiple comparisons test. (C) Modified from Paxinos, G., and Watson C. The Rat Brain in Stereotaxic Coordinates, 4th ed. Academic Press: San Diego, Fig. 35.

FIG. 6.

Nano-pulsed laser therapy (NPLT) decreases microglia activation 10 days after blast injury. (A) Representative images of brain sections at the level of the somatosensory cortex (bregma level: −4.16 mm) stained with an antibody against CD68 (a marker of activated microglia; red). Nuclei are counterstained with DAPI and are shown in blue. Calibration bar is 50 μm. (B) Quantification of the number of CD68^pos cells in the cortex. N = 3 rats/group. *p < 0.05; **p < 0.01 one-way analysis of variance (ANOVA) followed by post hoc Tukey's multiple comparisons test. (C) Representative coronal view of the rat brain at −3.80 mm of bregma showing the location of CD68^pos cells (shaded area) identified by immunofluorescence in the brain of BLAST and BLAST+NPLT rats (modified from Paxinos, G., and Watson, C. The Rat Brain in Stereotaxic Coordinates, 4th ed. Academic Press: San Diego, Fig. 35).

FIG. 7.

Nano-pulsed laser therapy (NPLT) increases cell proliferation in the SGZ of the hippocampus dentate gyrus 10 days after blast injury. (A) Representative images of bromodeoxyuridine (BrdU) incorporation in the subgranular zone (SGZ) of the dentate gyrus of the hippocampus. BrdU is shown in brown. Hematoxylin was used to counterstain nuclei (shown in blue). Calibration bars are 100 μm and 50 μm (insert). (B) Quantitative analysis of the number of BrdU^pos cells in the SGZ on post-injury day (PID) 10. *p < 0.05; two-way analysis of variance (ANOVA) followed by Tukey's multiple comparisons test. N = 5 rats/group. (C) Representative images of brain sections at the level for the SGZ of hippocampus dentate gyrus stained with antibodies against BrdU, NeuN (a neuronal marker), and GFAP (a marker for astrocytes). Calibration bar is 50 μm.