1.

Introduction

In spinal cord injury (SCI), complete or partial loss of autonomic, sensory, and motor functions is caused by interruption of neural signal conduction along the axonal tracts. There is generally poor recovery of these functions because of the difficulty of tissue regeneration in the central nervous system. Thus, SCI patients are left with serious residual disabilities, such as paralysis, respiratory difficulty, chronic pain, urinary problems, and neurologic decline, leading to considerable decrease in quality of life. Various strategies have been examined for repair of SCI in animal models, including blockage of the endogenous growth inhibitory factors,^1,2 infusion of neurotrophic factors,^3,4 and transplantation of growth promoting cells.^5–7 However, no effective treatment for SCI has yet been established.

Low-level laser therapy (LLLT) is a promising approach to treat SCI. LLLT has been clinically applied to the treatment of rheumatoid arthritis and periodontal disease, pain management, and healing of wounds and burns.^8–10 LLLT is also currently used for the treatment of various neurological diseases such as stroke, neurodegenerative diseases, and brain injury.^11–16 Several studies have shown that near-infrared LLLT has the potential to be an effective noninvasive therapy for SCI.^17–20 Rochkind et al. demonstrated that transplantation of embryonal spinal cord nerve cells followed by 780-nm laser irradiation enhanced axonal sprouting and spinal cord repair in a completely transected rat SCI model.¹⁷ In two different rat models of hemisection SCI and contusion SCI, Anders et al. transcutaneously applied an 810-nm laser, which penetrated to the depth of the injured spinal cord and promoted axonal regeneration and functional recovery.^18,19 Their study demonstrated that near-infrared laser irradiation significantly suppressed immune cell activation and cytokine/chemokine expression, suggesting that a decrease in the inflammatory response is one of the recovery mechanisms in LLLT for spinal cord repair.

The detailed mechanisms of LLLT are still under investigation. However, the therapeutic efficacy relies fundamentally on the initial photochemical event, i.e., absorption of photons by photoacceptors or chromophores such as cytochrome $c$ oxidase in the tissue.^21,22 Karu et al. showed in an in vitro study that the basic processes of LLLT occurring in HeLa cells were light absorption and photochemistry but that the incident characteristics of photons, such as degree of light polarization, did not affect the biological reactions in LLLT.²³ However, scattering of photons in vivo depends on the microstructure of tissue, and light propagation into biological tissue would therefore change the healing property. For instance, Ribeiro et al. investigated the repair of skin burns in rats with a linearly polarized He–Ne laser beam, which was parallel or perpendicular to the direction of the spinal column, at the same laser dose.^24,25 Their results showed that the healing process was dependent on the polarization orientation; lesions irradiated with parallel polarization were completely repaired 17 days after wound creation, while those with perpendicularly polarized irradiation showed a moderate degree of healing in the same period. They attributed these results to the fact that the parallel polarization was aligned with the predominant orientation of collagen fibers in the dermis, which was confirmed by histological analysis. This alignment would reduce photon scattering and thus increase optical penetration depth in the tissue, leading to the acceleration and improvement of cutaneous wound repair.²⁴

It is widely known that photon scattering by aligned cylindrical structures, such as myofibrils, axons, and collagen fibers, results in anisotropic light reflection and propagation in the tissue.^26–30 These characteristics are often used for diagnosis of the tissue abnormalities and for mapping of specific structures in the tissue.^31–33 Since the spinal cord has a fibrous structure, photon migration should be affected by polarization of incident light in the tissue. However, the effect of polarization on efficacy of LLLT for SCI has not been elucidated. In the present study, we examined the effect of relative orientation of laser polarization on efficacy of near-infrared LLLT for contused spinal cords in rats.

2.

Materials and Methods

2.6.

Distribution of Light Transmitted Through Spinal Tissue

To compare penetrations of light with different polarization directions through the spinal cord, light transmitted through excised spinal tissue was imaged with a CCD camera (XC-7500, Sony Corp., Tokyo, Japan). The experimental setup is schematically shown in Fig. 1. A fresh spinal column removed from an uninjured rat (diameter, $\sim 5\mathrm{mm}$; length, 12 mm) was placed on a black plastic sheet (thickness, 0.7 mm) with a rectangular hole ($3\mathrm{mm}\times 9\mathrm{mm}$) through which polarized laser light was directed onto the bottom surface of the spinal column ($n=2$). The transmitted light was detected from the top. The polarization direction was changed by rotating a polarizer that was placed between the fiber output end and the spinal tissue; 0 and 180 deg means the incident direction of the linear polarized laser was parallel to the spinal column, while 90 deg indicates the polarization direction was perpendicular to the spinal column. The laser power measured at the bottom surface of the spinal cord was 25 mW. The transmitted light was quantified by calculating white-colored pixels in the regions of interest (ROIs, $3\mathrm{mm}\times 9\mathrm{mm}$), corresponding to the size of a rectangular hole in the plastic sheet.

Fig. 1

Experimental setup for measurement of light transmitted through an excised spinal tissue.

3.

Results

3.1.

Functional Recovery

Figure 2 shows the BBB scores for rats in the three groups as a function of time after injury. Regardless of the polarization direction, the BBB scores of the rats receiving 808-nm laser irradiation (Groups 2 and 3) were significantly higher than those of the SCI alone group (Group 1) from five days after injury (Group 1 versus Group 2, $P=0.003$ at day 5; $P=0.002$ at days 7 and 10; $P=0.000$ at days 14 and 21, Group 1 versus Group 3, $P=0.000$ at days 5, 7, 10, 14, and 21). In addition, BBB scores of the rats irradiated with parallel polarization (Group 3) were significantly higher than those of the rats treated with perpendicular polarization (Group 2) from 10 days after SCI ($P=0.029$ at day 10; $P=0.005$ at day 14; $P=0.003$ at day 21). The averaged BBB scores at three weeks post-SCI were $7.5\pm 1.2$ for Group 1, $9.8\pm 1.0$ for Group 2, and $10.9\pm 1.0$ for Group 3.

Fig. 2

Locomotive scores based on an open-field test as a function of days after SCI. Values are expressed as $\text{means}+\text{standard error}$ of the mean (SEM) ($n=12$). Asterisks indicate significant differences ($P<0.05$).

3.2.

Histologic Evaluation

Figure 3 shows histological images (HE staining) of longitudinal sections of the spinal cords of rats in all groups at three weeks after injury. Formation of a cavity was observed in the spinal cord tissue of all rats. Figure 4 shows the results of quantitative analysis of the cavity areas in injured spinal cords on the basis of histological images. Cavity areas for the rats in Groups 2 and 3 were significantly smaller than those for the rats in Group 1; $P=0.050$ between Groups 1 and 2 and $P=0.006$ between Groups 1 and 3, while $P=0.639$ between Groups 1 and 2. These results show that irradiation with both the parallel and perpendicular polarized laser can lead to reduced formation of glial scar and cavity. However, there was no significant difference in cavity area between Groups 2 and 3.

Fig. 3

Histological images (HE staining) of longitudinal sections of injured spinal cords at three weeks after injury: (a) Rat with SCI alone (Group 1). (b) SCI rat treated with perpendicularly polarized laser to the spinal column (Group 2). (c) SCI rat treated with parallel polarization (Group 3). Scale bars indicate 200 μm.

Fig. 4

Results of quantitative analysis of injury cavity areas in spinal cords on the basis of histological images. Values are expressed as $\text{means}\pm \mathrm{SEM}$ ($n=9$ in each group). *$P<0.05$, **$P<0.01$.

3.3.

ATP Content

Figure 5 shows the ATP content in the spinal cords of rats in Groups 1, 2, and 3, where the value for normal rats is also shown for comparison. In the normal rats, baseline ATP content in the spinal tissues was $0.13\pm 0.02\mathrm{nmol}$ per mg protein, compared with $0.07\pm 0.02\mathrm{nmol}$ per mg protein in the untreated SCI rats (Group 1). The ATP contents in rats of Groups 2 and 3 were $0.08\pm 0.02$ and $0.09\pm 0.02\mathrm{nmol}$ per protein, respectively. However, there was no significant difference in ATP content between Groups 1, 2, and 3, indicating that ATP synthesis immediately after LLLT was not associated with improved motor function by near-infrared light either with parallel or perpendicular polarization.

Fig. 5

ATP contents in normal and injured spinal cords (Groups 1, 2, and 3). Results are expressed as $\text{means}+\mathrm{SEM}$ ($n=6$ in each group).

3.4.

Light Transmitted Through the Spinal Cord

Figure 6 shows the distributions of light transmitted through an excised spinal cord under two incident polarization conditions: (a) perpendicular and (b) parallel to the spinal direction. Figure 6(c) shows the amount of transmitted light from the ROI as a function of incident laser polarization direction. The amount of transmitted light for parallel polarization was $\sim 1.8$-fold higher than that for perpendicular polarization, indicating that light with parallel polarization penetrated deeper in the spinal tissue than did light with perpendicular polarization.

Fig. 6

Images of laser light transmitted through excised spinal tissue: directions of incident linearly polarized laser were (a) perpendicular and (b) parallel to the spinal column. (c) Transmitted light distribution in ROIs [areas indicated by a broken line in (a) and (b)] as a function of the direction of incident linearly polarized laser with respect to spinal orientation. Values are expressed as $\text{means}\pm \mathrm{SD}$ ($n=2$).

4.

Discussion

The current study has shown that locomotive scores of SCI rats with 808-nm laser treatment were significantly higher than those of SCI rats without light irradiation from day 5 after injury onward regardless of the incident polarization direction (Fig. 2). Anders et al. demonstrated that transcutaneous application of 810-nm nonpolarized laser significantly promoted axonal regrowth at six weeks postinjury in a rat hemisection SCI model¹⁸ and functional recovery at three weeks after injury in a rat contused SCI model.¹⁹ There are differences in the time course of treatment efficacy between our study and their studies. In their experiments, the incident laser power and daily dosage at the skin surface overlying the lesion site were 150 mW and $1589\mathrm{J}/{\mathrm{cm}}^2$ (irradiation duration, 2997 s), respectively, 6% of which (9 mW and $95\mathrm{J}/{\mathrm{cm}}^2$) penetrated to the spinal cord depth. The irradiation was applied daily for 14 consecutive days after SCI.^18,19 They concluded that the improved axonal regeneration was caused by inhibiting inflammatory cell activity due to laser irradiation at high dosage per day ($>10\mathrm{J}/{\mathrm{cm}}^2$). In our study, on the other hand, a linearly polarized laser was directly applied to the exposed spinal cord lesion immediately after trauma and then daily for the following five days at the fluence of $9.6\mathrm{J}/{\mathrm{cm}}^2$ (power, 25 mW; irradiation duration, 1200 s). The therapeutic effects of near-infrared laser irradiation have been reported to be dependent on dosage, being associated with production of anti-apoptotic, pro-proliferative, antioxidant, and angiogenic factors.^22,42–44 Although further study is needed to clarify the therapeutic mechanisms, as well as the optimum irradiation conditions for treating SCI, a different mechanism might work for treatment under the laser irradiation conditions in the present study.

Locomotor function of SCI rats treated with parallel polarization (Group 3) was significantly improved when compared with that irradiated with perpendicular polarization (Group 2) from day 10 after trauma (Fig. 2). To investigate the reason for the polarization-dependent treatment efficacy, we evaluated the cavity area 21 days after injury (Figs. 3 and 4), ATP content immediately after SCI (Fig. 5), and light penetration in the spinal tissue (Fig. 6). There was no statistically significant difference either in cavity area or in ATP content between the two polarization groups. On the other hand, the polarization dependence of light transmission through tissue was remarkable; the amount of light transmitted through the spinal cord with perpendicular polarization was $\sim 40\%$ less than that with parallel polarization [Fig. 6(c)]. Thus, we speculate that the higher treatment efficacy with parallel polarization is attributable to the more efficient light propagation through tissue, which is consistent with the results reported by Hebeda et al.²⁶ The penetration depth of red light (wavelength, 632.8 nm) with polarization parallel to white matter tracts in a fresh bovine brain was significantly greater than that of red light polarized perpendicular to myelinated fiber tracts; the effective attenuation coefficients of light (${\mu }_{\mathrm{eff}}$) were $0.47\pm 0.06{\mathrm{mm}}^{-1}$ with parallel polarization and $0.63\pm 0.13{\mathrm{mm}}^{-1}$ with perpendicular direction ($P<0.05$).²⁶ It is known that locomotor recovery after SCI is highly correlated with the volume of remaining normal nerve fibers in spinal tissue.^40,41 In the present study, LLLT with parallel polarization would have provided more efficient protection of neural cells from apoptosis or necrosis in deep-located anterior horns than LLLT with perpendicular polarization. Further investigation is needed to elucidate the detailed physiological contributions of linearly polarized light, but the present study clearly demonstrated the importance of light polarization in LLLT for SCI.

Moreover, Silva et al. recently reported that when the rat tendon was irradiated with linearly polarized 632.8-nm laser aligned in parallel with the tendon long axis, the tissue showed higher birefringence and greater nonsusceptibility when compared with those of the nonirradiated tissue. They attributed this to the changes in molecular order and aggregation states of the collagen fibers.⁴⁵ With linearly polarized laser, the tendon was irradiated once in their work, while the spinal cord was irradiated daily for five consecutive days in our work. Similar alteration of the fibrotic structure, which affects direction of photon scattering, could take place in the spinal cord examined in this study.

Open surgery for direct laser irradiation to the spinal cord is invasive, which is not practical in clinical situations. However, there seems to be a chance for direct irradiation during surgical exposure for spinal decompression and endoscopic discectomy, for which laser delivery through an optical fiber should be useful. For fiber-based irradiation with a linearly polarized laser, a polarization filter can be placed at the laser output fiber end, or a polarization-maintaining fiber can also be used.

Although we observed a significantly higher treatment efficacy with parallel polarization than with perpendicular polarization, the difference is not large enough to indicate medical relevance, although our results would provide important data concerning effect of light polarization in LLLT for fibrotic tissue. It should also be noted that our data strongly support the efficacy of LLLT for SCI regardless of the polarization direction. In addition to transcutaneous LLLT,^18,19 LLLT based on direct laser irradiation can also provide a therapeutic option for SCI since open surgery is often performed for decompression in conventional SCI treatment strategy.

5.

Conclusion

We investigated the effects of polarization on efficacy of 808-nm LLLT for contusion SCI in rats. Rats treated with light for which polarization was parallel to the spinal direction showed significantly faster recovery of locomotor function than did rats treated with perpendicular polarization. We speculate that this is attributable to deeper photon penetration through spinal tissue with parallel polarization than with perpendicular polarization.