2.1.

Animal Model

All animal procedures were approved by the Subcommittee on Research Animal Care (IACUC) of the Massachusetts General Hospital (Protocol No. 2010N000202) and met the guidelines of the National Institutes of Health. Young adult male BALB/c mice of 8 weeks (weight 21 to 25 g; Charles River Laboratories, Wilmington, MA) were used in the study. A detailed description of CCI-TBI induction, craniotomy procedure, and laser parameters is given in our previous publication.¹² Briefly, after the hairs on top of the head were removed with shaving and then depilated with Nair, the top of the skull was exposed with a skin incision of 1 cm (on central and sagittal direction). A careful 5-mm craniotomy was performed over the right parieto-temporal cortex using a trephine attached to an electric portable drill without damaging the meninges. The bone flap was removed and the mouse brain was subjected to a controlled cortical impact using a special pneumatic impact device; pneumatic cortical impact device, Model AMS 201, AmScien Instruments LLC, Richmond, Virginia, with a 3-mm flat-tip, high pressure 150 psi, low pressure 30 psi, rod speed $4.8\mathrm{m}/\mathrm{s}$, rising duration 8.41 ms, and set impact depth of 2 mm that was adjusted to yield a moderate to severe TBI. One hour post-TBI induction, mice were given a neurological severity score (NSS) test and only those scoring 7–8 were included in the study ($\sim 75\%$ of the injured mice). Immediately after generating the brain trauma, the craniotomy hole was sealed with bone wax and the skin was sutured. Mice were kept under anesthesia (with isoflurane) throughout the whole surgical procedure. Sham control mice were subjected to all aspects of the procedural steps except the actual cortical impact.

For this study, after severe CCI-TBI induction, mice were broadly divided into the following four groups: (a) sham TBI-sham laser treatment; (b) real TBI-sham laser treatment; (c) real TBI with one real laser treatment; (d) real TBI with three real laser treatments. The four broad groups (defined above) each contained 16 mice per group with similar NSS values for the animals in groups b, c, and d to ensure comparable injury severity for all groups. Depending on the study group, mice were sacrificed at 4, 7, and 28 days post-TBI. Three mice per group were sacrificed on day 4 for caspase-3 determination. Five mice per group were sacrificed on day 7, and another five mice on day 28 for BrdU, DCX, and TUJ-1 immunofluorescence. The remaining three mice were sacrificed on day 29. This arrangement ensured that there were always (at least) eight mice alive per group to undergo performance and cognitive testing. The animals were housed as one mouse per cage and were maintained on a 12 h-light–12 h-dark cycle with access to food and water ad libitum.

2.2.

Laser Treatment

Before laser irradiation (real or sham), mice were taped to a plastic plate via their paws. Sham TBI and real TBI mice received laser treatment (real or sham) once a day for either 1 day or for three consecutive days. Groups c and d received laser treatment with an 810-nm laser (DioDent Micro 810, HOYA ConBio, Fremont, California) delivering $18{\mathrm{J}/\mathrm{cm}}^2$ fluence and $25{\mathrm{mW}/\mathrm{cm}}^2$ irradiance for 12 min on a 1-cm spot diameter that covered the entire mouse head. Group c (with real TBI) received a single laser treatment 4 h post-TBI; group d (with real TBI) received one laser treatment per day for three consecutive days (the first irradiation being 4 h post-TBI).

2.3.

Cognitive Performance Assessment

The MWM test was employed following a previously described protocol,²⁴ where the experimental set up for the MWM task consists of a circular swimming pool (110 cm diameter and 30 cm height) filled with water kept at 24°C. The target zone is the quadrant where the escape platform of 15 cm diameter was placed in a fixed position either visible or hidden (1 cm under the water surface). The other three quadrants were left, right, and opposite zones. Acquisition training consisted of trial blocks of two daily trials for 10 days, commencing at four different positions from the border of the maze in a semi-random order and with 15 min inter-trial intervals. If the platform was not found in 120 s, the mouse was placed on the platform for 15 s, and then returned to its cage. After the acquisition training, the actual trials started: the trial on the first day was latency to the visible platform; trials on the second day through sixth day were latency to the hidden platform; the test at the seventh day was a probe trial where the platform was completely removed. MWM evaluations lasted from days 21–27 post-TBI.

2.4.

Neuromotor Assessment

The wire grip and motion test (WGMT) was used to assess the motor function and muscle power via monitoring the grip strength and endurance where the mice were positioned on a horizontal wire of 0.6-mm thickness and 45-cm length, set at 46 cm above a tabletop. Assessment was done through grading the ability of the mouse to grip, retain position, and move along the wire. The test was performed in triplicate with an average value taken per mouse. WGMT was performed on days 0, 1, 4, 7, 10, 15, 19, 24, and 28.

2.5.

Neurobehavioral Assessment

The neurological performance of the traumatized mice was routinely evaluated with the NSS test where mice were expected to perform 10 tasks measuring motor ability, balance, and alertness. The uninjured (control) mouse is given the score of zero and one point is added for each failed performance task, which can sum up to maximum score of 10. NSS evaluations started 1 h post-TBI (day 0) and were carried out on days 1, 4, 7, 10, 15, 19, 24, and 28. Mice with an NSS score less than 5 are assigned as sustained mild TBI; mice with an NSS score of 5–6 are assigned as sustained moderate TBI; an NSS score of 7–8 indicated mice sustaining severe TBI; an NSS score of 9–10 indicated mice sustaining very severe TBI. In our research, we studied mice sustaining severe TBI with an NSS of 7–8. The data showing the improvement of the NSS after tLLLT for these TBI mice have already been reported, but it was necessary to repeat it for control of the injury severity.

2.6.

Immunofluorescence Staining

Mice in the study groups were given a daily intraperitoneal injection of BrdU (Sigma, St. Louis, Missouri, Cat# B5002) as $50\mathrm{mg}/\mathrm{kg}$ (diluted in saline) for seven consecutive days before sacrifice.

Mice (whose brains were to be studied) were anesthetized with isoflurane and perfused transcardially with 0.9% saline and then with 4% phosphate-buffered paraformaldehyde. Brains were excised and were fixed in the 4% phosphate-buffered paraformaldehyde solution for 3 days after which they were embedded in paraffin. Coronal serial brain cross sections of 5-mm thickness were taken and $10\text{-}\mu$-thick sections were cut from the top, middle, and bottom of the thick sliced blocks via microtome.

Immunofluorescence staining was performed using double staining for BrdU-NeuN, DCX, TUJ-1, and for caspase-3.

Brain sections to be double-stained for BrdU-NeuN underwent the standard immunofluorescence staining protocol for paraffin sections where (briefly) brain slices were mounted onto SuperFrostPlus charged glass slides followed by consecutive pretreatment with xylene for deparaffinization, graded ethanol for rehydration, and then passed through citrate buffer solution for antigen retrieval, followed with incubation with blocking solution of 5% BSA/0.1%Triton X-100 in phosphate-buffered saline. Immunostaining was done with a primary anti-body cocktail of rat anti-BrdU (Cat.# ab6326, abcam) and mouse anti-NeuN (Cat# MAB377, Millipore, Billerica, Massachusetts) for 72 h at 4°C to detect the neuronal cells. A cocktail of secondary antibodies (Alexa fluor 488 conjugated anti-rat and Alexa fluor 568 conjugated anti-mouse, Invitrogen, Grand Island, New York) was used to stain the brain slices. Finally, they were cover slipped with mounting media containing DAPI (4’,6-diamidino-2-phenylindole) (Cat#H-1200, Fisher Scientific, Waltham, Massachusetts). Cells positively stained for BrdU were imaged using a confocal microscope (Olympus America Inc., Center Valley, Pennsylvania) and then exported using Fiji software (Olympus Fluoview Version 3.0). For DCX staining we used rabbit anti-DCX (Cat# ab18723, Abcam, Cambridge, Massachusetts) and as a secondary the goat anti-rabbit Alexa fluor 568-congugated (Invitrogen). For TuJ-1 staining we used mouse anti-TuJ-1 (Cat# MAB1637, Millipore) with the secondary anti-mouse Alexa fluor 488-conjugated (Invitrogen). Caspase-3 staining was done with rabbit anti-caspase-3 (Cat# 04-439, Millipore) and Alexa fluor 680-congugated anti-rabbit as a secondary antibody (Invitrogen). Green BrdU, red DCX, or green TUJ-1 together with blue DAPI were quantified by the use of Image J (NIH, Bethesda, MD). Ratios of red or green fluorescence staining to blue DAPI fluorescence staining were calculated to normalize for the number of cells present in each microscopic field. Representative fields from five slides had the ratios averaged and the SD calculated.

2.7.

Statistical Analyses

From each group in each brain area, we selected slices that showed more positive expression of each target antibody for comparison, and then respectively measured their positive expression area with Image-J software; finally, each target antibody expression area was divided by the DAPI area to normalize the data for the number of cells present. Data are presented as $\text{mean}\pm \mathrm{SD}$, and analyzed using one-way analysis of variance followed by Tukey’s post-hoc test. Significance was defined as $p<0.05$. SPSS statistics V17.0 software was used for statistical analyses.

3.1.

Effect of Transcranial Low-Level Laser (Light) Therapy on Motor Function after Controlled Cortical Impact

We previously showed that the NSS score improved in CCI induction of TBI in mice treated with tLLLT.¹² Here, we also carried out the WGMT to show that another measure of neurological function improved in tLLLT treated mice. The WGMT performed at four different time points; days 7, 14, 21, and 28 post-TBI-induction, and the comparisons for the laser treatment effects are drawn against sham and TBI sustained cohorts. The overall improvement in the motor functions commences at day 14 of the CCI-TBI induction and at day 21 tLLLT applied cohorts show significant improvements: $1\times$ tLLLT per day $p<0.05$ and $3\times$ tLLLT per day $p<0.01$ against TBI sustained cohorts alone. At day 28, the improvements are even more pronounced: $3\times$ tLLLT per day has $p<0.001$ versus TBI, Fig. 1. Results are clearly indicate that the mice cohort that received three daily tLLLT exposures ($3\times$ tLLLT) showed a significant improvement ($p<0.05$ versus TBI) in WGMT score at day 14 post-TBI. At day 21 post-TBI, both groups that received tLLLT ($1\times$ tLLLT $p<0.05$ and $3\times$ tLLLT $p<0.01$) had significant improvements versus TBI and at day 28 the improvements were even more pronounced ($1\times$ tLLLT $p<0.05$ and $3\times$ tLLLT $p<0.001$ versus TBI). Overall, $3\times$ tLLLT proved to be more effective in improving the motor function.

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).

3.2.

Effect of Transcranial Low-Level Laser (Light) Therapy on Morris Water Maze Performance after Controlled Cortical Impact

The MWM study was carried out on days 21 through 27 post-TBI, Fig. 2. The groups were normal, sham-TBI, TBI, TBI $1\times$ tLLLT, and TBI $3\times$ tLLLT. The latency to the visible platform is shown in Fig. 2(a), the five daily repetitions of latency to the hidden platform are shown in Fig. 2(b), and the latency with the probe test is shown in Fig. 2(c). The visible platform latency is used to measure spatial navigation capabilities and the hidden platform test is used to measure the improvement in learning and memory, while the probe test is used to measure memory.

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.

Our study shows that there was a large increase ($p<0.001$) in latency to the visible platform in TBI mice ($42\pm 4\mathrm{s}$) compared to normal mice $10\pm 1\mathrm{s}$) and sham-TBI mice ($17\pm 3\mathrm{s}$) as might be expected with brain damage. Both $1\times$ tLLLT and $3\times$ tLLLT produced significant improvements ($31\pm 3$ and $30\pm 4\mathrm{s}$, respectively, compared to TBI mice ($p<0.05$), Fig. 2(a).

With the five successive hidden platform trials, days 22 through 26, Fig. 2(b), there were again large increases ($p<0.0001$) in latency ($44-36\pm 4\mathrm{s}$) in TBI mice compared to normal ($11-10\pm 1\mathrm{s}$) and sham ($18-14\pm 2\mathrm{s}$) groups. The $1\times$ tLLLT group had significant improvements ($p<0.0001$) in latency ($34-26\pm 5\mathrm{s}$) versus TBI. The $3\times$ tLLLT latency was significantly ($p<0.05$) better than the $1\times$ tLLLT latency at the second and fourth repetitions. The rate of learning (gradient between the five successive repetitions) did not appear to be different between the TBI and the $1\times$ tLLLT and $3\times$ tLLLT groups.

For the latency with the probe test, TBI mice had a large ($p<0.001$) increase ($35\pm 4\mathrm{s}$) compared to normal ($8\pm 1\mathrm{s}$) and sham-TBI mice ($15\pm 3\mathrm{s}$). Mice treated with $1\times$ tLLLT had a significant ($p<0.01$) improvement ($26\pm 2\mathrm{s}$), while mice that received $3\times$ tLLLT had an even bigger ($p<0.001$ versus TBI and $p<0.05$ versus $1\times$ tLLLT) improvement ($22\pm 2\mathrm{s}$), Fig. 2(c).

3.3.

Caspase-3 Staining in Lesion Site

Caspase-3 expression in the injured region at day 4 of the TBI induction is shown in Fig. 3. The TBI sustaining cohort is expressing significant amounts of the protein ($p<0.001$ versus sham) at the lesion site, whereas laser treatments are showing a significant declining trend of its expression; $1\times$ tLLLT per day is $p<0.05$ versus TBI and $3\times$ tLLLT per day is $p<0.01$ versus TBI. These findings imply that tLLLT can have a rapid cytoprotective effect by reducing apoptosis in the damaged lesion area. Moreover, although tLLLT suppressed the effector caspase 3, at the end of the chain of caspases, which are the main propagators of apoptosis at cellular level, it cannot be concluded at what stage of the cascade the LLLT had its primary effect.

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) $1\times$ tLLLT, (d) $3\times$ tLLLT, (e) mean caspase-3/DAPI $\text{ratios}\pm \mathrm{SD}$ ($n=5$). Scale bar $100\mu \mathrm{m}$. *** $p<0.001$ versus sham; †, †† $P<0.05$, 0.01 versus TBI.

3.4.

BrdU-NeuN Expression

To show neurogenesis, we used the double labeling technique with co-staining for BrdU and NeuN where BrdU labels newly generated cells whose DNA has replicated, and NeuN is a specific label for postmitotic mature neurons just before synaptic integration.²⁵ We concentrated on two areas in the mouse brain; the subgranular layer of the hippocampal DG and the subventricular zone (SVZ) of the lateral ventricle.^26,27 Both these areas of the rodent brain are well known to give rise to neuroprogenitor cells both after TBI²⁸ and after other interventions that have been tested for possible neuroprotective and neuroregenerative effects.^29,30 The two time points of analysis were 7 days and 28 days after the TBI induction. It should be noted that virtually all BrdU positive cells observed were pink, indicating triple staining (BrdU + NeuN) and DAPI and blue–green BrdU + DAPI alone, which would be expected from newly formed glial cells. The normalization of the BrdU-NeuN double stained cells was done by counting the ratio of BrdU expression to DAPI staining (labeling cell nuclei). This was to correct for variation in the numbers of cells visible in different sections.

Our results indicated that some neurogenesis was caused by TBI alone in the DG both at 7 days (Fig. 4) and at 28 days (Fig. 5). However, significant ($p<0.05$) further increases were generated by $1\times$ tLLLT ($p<0.01$ versus TBI), $3\times$ tLLLT ($p<0.001$ versus TBI), and ($p<0.001$ versus sham) at 7 days. Note in Fig. 4(d), ($3\times$ tLLLT) the double stained cells are clearly visible lining the subgranular layer. At 28 days post-TBI, the laser induction of neurogenesis in the DG appears to be more modest.

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) $1\times$ tLLLT, (d) $3\times$ tLLLT, (e) mean BrdU/DAPI $\text{ratios}\pm \mathrm{SD}$ ($n=5$); normalization of the readings was done BrdU versus DAPI (labeling the nuclei). Scale bar $100\mu \mathrm{m}$. *** $p<0.001$ 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) $1\times$ tLLLT, (d) $3\times$ tLLLT, (e) mean BrdU/DAPI $\text{ratios}\pm \mathrm{SD}$ ($n=5$); normalization of the readings was done BrdU versus DAPI (labeling the nuclei). Scale bar $100\mu \mathrm{m}$. ** $p<0.01$ versus sham; †, †† $P<0.05$, 0.01 versus TBI.

There was a large increase in BrdU-NeuN cells in the SVZ at day 7 in TBI mice ($p<0.001$ versus sham), Fig. 6. The values were further increased by $1\times$ tLLLT ($p<0.05$ versus TBI) and by $3\times$ tLLLT ($p<0.001$ versus TBI). The increase in BrdU-NeuN in the SVZ at day 28 post-TBI (Fig. 7) was much lower ($0.03\pm 0.009$), but the value was more than doubled ($0.068\pm 0.009$, $p<0.05$) by $1\times$ tLLLT and further increased by $3\times$ tLLLT ($0.078\pm 0.009$, $p<0.01$).

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) $1\times$ tLLLT, (d) $3\times$ tLLLT, (e) mean BrdU/DAPI $\text{ratios}\pm \mathrm{SD}$ ($n=5$); normalization of the readings was done BrdU versus DAPI (labeling the nuclei). Scale bar $100\mu \mathrm{m}$. *** $p<0.001$ 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) $1\times$ tLLLT, (d) $3\times$ tLLLT, (e) mean BrdU/DAPI $\text{ratios}\pm \mathrm{SD}$ ($n=5$); normalization of the readings was done BrdU versus DAPI (labeling the nuclei). Scale bar $100\mu \mathrm{m}$. *** $p<0.001$ versus sham; ††, ††† $P<0.01$, 0.001 versus TBI.

BrdU/DAPI expression at the lesion site at the 7- and 28-days points is shown in Fig. 8, indicating that TBI induction has triggered a remarkable cell proliferation process at the lesion, especially at the 7-day point ($p<0.001$ versus sham), and laser application has been shown to amplify this process; $1\times$ tLLLT $p<0.05$ versus TBI and $3\times$ tLLLT $p<0.01$ versus TBI. The cell proliferation process appears to be effective during the 28-day point as well albeit with more modest expressions. In our previous study, we had only looked at BrdU incorporation at the 28-day timepoint.¹²

Fig. 8

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

3.5.

Double-Cortin Expression

Double-cortin (DCX) is a microtubule-associated protein expressed by neuronal precursor cells and immature neurons in embryonic and adult cortical structures. Neuronal precursor cells begin to express DCX while actively dividing, and their neuronal daughter cells continue to express DCX for 2–3 weeks as the cells mature into neurons.³¹

Figures 9 through 12 show our results for DCX expression at the two points of time lines, days 7 and 28, at the neurogenic DG and SVZ regions. The microtubule-associated neuronal migration protein DCX expression images and analyses at the neurogenic hippocampal DG at the 7 day point are given in Fig. 9. There was only a moderate increase in DCX staining caused by TBI alone in the DG at 7 days ($p<0.05$ versus sham). However, $1\times$ tLLLT and $3\times$ tLLLT produced significant ($p<0.05$ and $p<0.01$) increases in DCX expression compared to TBI alone at 7 days (but not at 28 days, Fig. 10) both in the DG and also in the SVZ (Fig. 11). Interestingly, DCX levels in the SVZ were significantly increased after $1\times$ and $3\times$ tLLLT application ($p<0.001$) at the 7-day time-point in comparison with the DCX levels, expressed by TBI alone mice, Fig. 11. The effect diminished with only very modest expression at the 28-day time point, Fig. 12. DCX expression at the lesion site, Fig. 13, after the TBI induction was significantly elevated ($p<0.001$ versus sham) during both the 7- and 28-day time points with $1\times$ tLLLT and $3\times$ tLLLT levels being especially increased at the 7-day period versus TBI-induced cohorts, with 7 days $3\times$ tLLLT $p<0.001$ versus TBI. Overall, this remarkable increase in the level of DCX expressing cells at the lesion of the TBI sustained cohorts (in both timelines $p<0.001$ versus sham) with especially pronounced elevations in the $3\times$ tLLLT applied cohort ($p<0.001$ versus TBI) may be indicative of the elevated numbers of migrating neuroprogenitors and the laser protective effects on the neuroprogenitors in migration.

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 $\text{ratios}\pm \mathrm{SD}$ ($n=5$); normalization of the readings was done DCX versus DAPI (labeling the nuclei). Scale bar 100 $\mu \mathrm{m}$. * $p<0.05$ 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) $1\times$ tLLLT, (d) $3\times$ tLLLT, (e) mean DCX/DAPI $\text{ratios}\pm \mathrm{SD}$ ($n=5$); normalization of the readings was done DCX versus DAPI (labeling the nuclei). Scale bar $100\mu \mathrm{m}$. *$p<0.05$ 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) $1\times$ tLLLT, (d) $3\times$ tLLLT, (e) mean DCX/DAPI $\text{ratios}\pm \mathrm{SD}$ ($n=5$); normalization of the readings was done DCX versus DAPI (labeling the nuclei). Scale bar $100\mu \mathrm{m}$. * $p<0.05$ 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) $1\times$ tLLLT, (d) $3\times$ tLLLT, (e) mean DCX/DAPI $\text{ratios}\pm \mathrm{SD}$ ($n=5$); normalization of the readings was done DCX versus DAPI (labeling the nuclei). Scale bar $100\mu \mathrm{m}$. *$p<0.05$ versus sham; † $P<0.05$ versus TBI.

Fig. 13

Microtubule-associated neuronal migration protein (DCX) expression at the lesion site. Mean DCX/DAPI $\text{ratios}\pm \mathrm{SD}$ ($n=5$); 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.

3.6.

TUJ-1 Expression

TUJ-1 recognizes a neuron-specific class III beta-tubulin whose expression has been found to be an early molecular event in neuronal differentiation and is exhibited by a wide range of neuronal precursors.³² Our results for TUJ-1 staining (used to distinguish the immature postmitotic neurons, just before synaptic integration) are given in Figs. 14, 15, 16, and 17. One can see that TBI alone only produced an increase in the DG at 7 days. However, $1\times$ tLLLT and $3\times$ tLLLT produced significant ($p<0.05$ and $p<0.01$, respectively) increases in TUJ-1 compared to TBI alone at both time points (days 7 and 28) in both neurogenic areas of the brain, SVZ, and DG. One can see that the postmitotic TUJ-1-positive cells, generated throughout the period of cortical neurogenesis, were abundant in the $1\times$ and $3\times$ laser treated cohorts. $3\times$ tLLLT appeared to prolong the lifetime of the adult neuroprogenitors as indicated by the significant levels of TUJ-1 expression in the neurogenic hippocampal DG even at day 28 after TBI induction ($p<0.01$ versus TBI). In the other neurogenic hot spot, the SVZ, $3\times$ LLLT was also effective with $p<0.01$ versus TBI at the day 7 point and $p<0.05$ versus TBI at the 28-day point, where at day 28, only the $3\times$ tLLLT exposed cohorts show elevated levels of TUJ-1 expressing neuroprogenitors.

Fig. 14

Neuron-specific class III $\beta$-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) $1\times$ tLLLT, (d) $3\times$ tLLLT brain slices, (e) mean TUJ-1/DAPI $\text{ratios}\pm \mathrm{SD}$ ($n=5$); normalization of the readings was done TUJ-1 versus DAPI (labeling the nuclei). Scale bar $100\mu \mathrm{m}$. ** $p<0.01$ versus sham; †, †† $P<0.05$, 0.01 versus TBI.

Fig. 15

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

Fig. 16

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

Fig. 17

Neuron-specific class III $\beta$-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) $1\times$ tLLLT, (d) $3\times$ tLLLT, (e) mean TUJ-1/DAPI $\text{ratios}\pm \mathrm{SD}$ ($n=5$); normalization of the readings was done TUJ-1 versus DAPI (labeling the nuclei). Scale bar $100\mu \mathrm{m}$. † $P<0.05$ versus TBI.