The major side effect of cyclophosphamide treatment is bone marrow suppression, which results in significant damage to actively proliferating hematopoietic cells and direct damage to the bone marrow microenvironment and myeloid progenitor/stem cells. This causes leukopenia and reduced immune function, thereby causing severe damage to the body [32, 33]. In this study, we found that irradiation with a 10.6-μm carbon dioxide laser and 650-nm semi-conductor laser alone or in combination increased the number of nucleated cells in the femoral bone marrow of cyclophosphamide-treated model rats. That is, after seven treatments in 15 days, the reservoir in the bone marrow produced large amounts of undifferentiated leukocytes. Laser acupuncture stimulated bone marrow hematopoietic cell numbers that were destroyed by cyclophosphamide and preserved the proliferation ability of bone marrow nucleated cells. In contrast, the model group and the sham treatment group were still affected by the cytotoxicity of cyclophosphamide, and the number of bone marrow nucleated cells was decreased and did not recover to normal values. In a previous study, after a 10.6-μm carbon dioxide laser was used for eight treatments, it was found that on day 17, there were no significant differences in peripheral leukocytes when compared with the normal group. Our study found that after seven treatments, there were no significant differences in nucleated cells between the laser groups and the normal group. The combined results of the two studies suggest that lasers can improve the bone marrow hematopoietic system, promote division in intramedullary nucleated cells, accelerate division, and protect cells that have undergone division and maturation to enter into peripheral circulation. This is one of the mechanisms by which laser acupuncture increases leukocyte count.
Apoptosis refers to the process where cells undergo automatic programmed cell death under certain physiological or pathological conditions as regulated by genes. Cancer is a disease in which there are abnormalities in the regulation of cell growth and is intimately associated with abnormal cellular apoptosis. Chemotherapy drugs can disrupt the cell cycle and apoptosis in normal cells while interfering with the abnormal growth of cancer cells. After the generation of the cyclophosphamide model, DNA in rat blood cells loses its normal ability to regulate apoptosis. Our experimental results showed that the apoptosis rates in the model group and the sham treatment group were significantly higher than in the normal group. This is due to the initiation of cellular apoptosis in peripheral blood cells, resulting in a decrease in peripheral leukocyte count. Laser treatment can improve the ability of apoptosis regulation in cells that have experienced DNA disruption, resulting in apoptosis rates in rats with leukopenia being the same as in the normal group, thus maintaining peripheral blood cells.
Cyclophosphamide (an alkylating agent) is a highly active compound and can form covalent bonds with various nucleophilic groups to cause DNA breakage and damage . Cyclophosphamide is a non-specific drug that targets the cell cycle and is effective at various stages, including the G0 phase (non-proliferating quiescent phase). Therefore, it can kill a wide range of cancer cells and damage normal cells at the same time. The cell cycle results showed that the number of cells in the S phase of the cyclophosphamide model group and the sham treatment group was significantly increased, but cells in G2 phase were significantly decreased. This demonstrated that cyclophosphamide caused S phase arrest, resulting in cells unable to successfully carry out cell division and replication and enter G2 phase. The main reason for this is that nucleic acid synthesis is affected, resulting in incomplete DNA replication and cells unable to progress to the next phase. In addition, cyclophosphamide may also inhibit cell division, resulting in cells that have completed DNA replication but are unable to divide, causing the proportion of cells in G2 phase to decrease. The G1 phase of the cell cycle (gap 1 phase, before DNA synthesis) is a phase where new daughter cells from the previous mitotic division synthesize RNA and proteins that are associated with the unique functions of specific cells. G1 is longer than the other phases of the cell cycle. The results of this study found that whether cyclophosphamide was used or whether laser treatment was carried out did not greatly affect G1 phase. Therefore, the effects of cyclophosphamide as an alkylating agent on immune cells do not occur in G1 phase, i.e., cyclophosphamide does not affect the ability to synthesize cellular materials. During S phase (synthesis phase, the DNA replication phase), the cells synthesize DNA, and this period is relatively short. The DNA content in cells is increased by 2-fold for subsequent equal distribution into two daughter cells. The results of this study showed that the number of cells in S phase after laser treatment was comparable to that in the normal group. Parental cells can therefore divide rapidly in a short period to produce daughter cells and increase the number of peripheral leukocytes. The G2 (gap 2) phase refers to the phase after DNA replication and before mitosis starts, and is usually very short. The results of G2 phase in three laser groups are higher than model group, while it only has statistically significant differences in 10.6-μm laser group. The number of cells in G2 phase was increased with 10.6-μm laser acupuncture, which was higher than in the model group and identical to the normal group. Namely that mild stimuli excite biologic activity, stronger ones support it, even stronger ones retard it and very strong ones arrest it completely. The power of combined laser may be too strong to rats. What is more, there was some “negative interference” between the photochemical effect induced by the red light, and the photophysical effect from the CO2 energy. Thus, the 10.6-μm laser moxibustion is more suitable for the treatment of this symptom. There are evidences that the moxibustion method with white cell disease has a definite effect [12, 35]. This demonstrates that the 10.6-μm lasers affect the cell cycle and improve the ability of cells to synthesize RNA and proteins. As G2 phase is also where DNA damage is repaired before chromosome separation , the increase in number of cells in G2 provides more opportunity for DNA damage repair. However, further research is required to identify the pathways that are affected. Our experimental results also show that lasers can specifically affect cell division in leukopenia, promote cell division and proliferation, affect DNA damage caused by cyclophosphamide, promote DNA synthesis, and thereby increase leukocyte counts. This shows that laser acupuncture can promote S phase cells to enter into G2 phase and promote cell proliferation, achieving the aim of increasing peripheral leukocyte counts.
In our current research, we only investigate the changes due to laser stimulation in the target tissue (bone marrow), but not at the local area of stimulation. We therefore do not know the changes at the local area (the skin layer), and how the stimulation transmits to the target organs or tissues. We speculate that the effect of 650-nm red laser in our research may partly share a similar mechanism with that of its wound healing and pain attenuation effect  at the local area. The mechanism is a series of photochemical reactions of the light absorption in the end terminal enzyme of the respiratory chain of the cellular mitochondria, cytochrome oxidase, leading to a photochemical cascade the end result of which is adenosine triphosphate (ATP) production, accompanied by nitrous oxide (NO) and small but beneficial amounts of reactive oxygen species (ROS) . The CO2 laser, on the other hand, emits a wavelength more than two orders of magnitude longer, and at that physically long wavelength compared with visible red light, cell membranes are opaque, just as glass is opaque to 10.6 μm, so the LLLT reaction cannot be primarily photochemical, but rather photophysical in nature. The 10.6-μm CO2 laser is primarily absorbed by the tissue water, and its wavelength is absorbed principally in the first 20 μm of tissue ; therefore, the effect of the low-density 10.6-μm laser may be mainly due to the heat conduction into the deeper tissues. However, since all infrared lasers have thermal effects, the future research is warranted to investigate the effects of different infrared lasers (such as 830-nm diode laser, 2.95 μm erbium: YAG laser, etc.) and whether the effect of 10.6-μm laser is superior than others, due to its similarity to the biophotonic radiation from human point in wavelength.
In conclusion, we found that treatment with a 10.6-μm laser, 650-nm laser, and 10.6-μm–650-nm compound laser increased the number of nucleated cells in the bone marrow, decreased the unfavorable effects of cyclophosphamide on the cell cycle, advanced the cell cycle towards proliferation, decreased apoptosis, affected the intramedullary hematopoietic system, and affected peripheral leukocyte counts. This study did not include a positive drug as a control group, and we did not carry out in-depth study on spinal cord-related mechanisms. These will be further examined in future studies.