Research ArticleBlood Disorders

Noninvasive low-level laser therapy for thrombocytopenia

See allHide authors and affiliations

Science Translational Medicine  27 Jul 2016:
Vol. 8, Issue 349, pp. 349ra101
DOI: 10.1126/scitranslmed.aaf4964
  • Fig. 1.

    LLL promotes MK maturation and platelet production. (A) Illustration of timeline for ex vivo platelet differentiation from MKs. CD41+ FSChigh MKs were sorted from BMs, treated with or without LLL, and cultured in MK medium. MKs were collected 1 hour later for cellular ATP measurement, 1 day for studying proplatelet formation (PPF), or 3 days for counting platelets. (B) ATP was measured in 5 × 104 MKs treated with LLL at various energy densities. Data are means ± SEM (n = 6). *P < 0.05, **P < 0.01 versus 0 J/cm2. (C) Sizes of CD41+ MKs were analyzed by flow cytometric analysis of FSC before and after 24-hour differentiation. Data are means ± SEM (n = 6). (D) Representative transmission electron micrographs of MKs at 0 and 24 hours after LLL from at least 6 samples per group with 30 cells in each group. Data quantifying the major diameter of MKs are means ± SEM. N, nuclear; IMS, invaginated membrane system. Scale bar, 5 μm. (E) The number of platelets (PLTs) derived from 1 × 104 MKs was estimated 3 days after LLL on the basis of CD41 expression and FSC/side scatter (SSC). Data are means ± SEM (n = 6). (F) Representative images of PPF-MKs at 24 hours after LLL. Small was considered <100 μm, and large was ≥100 μm in PPF-MK diameter. Black arrowheads represent one of many protrusions on proplatelet shafts. White arrowheads indicate the nucleus. Scale bar, 25 μm. (G) The percentages of small or large PPF-MKs, as defined in (F), of at least 500 MKs analyzed per sample and 6 samples per group are shown as means ± SEM. (H) Sorted MKs were treated with or without LLL, labeled with the fluorophore CFSE, and infused into recipient mice at 1 × 105 cells per mouse. Percentages of resultant CFSE+ platelets in recipients at indicated days are shown. Data are means ± SEM (n = 10). ***P < 0.001 compared to controls. P values were determined by one-way analysis of variance (ANOVA) (B to D and H) or two-tailed Student’s t test (E and G).

  • Fig. 2.

    Thrombopoietic effect of LLL is ATP-dependent. (A) Correlations between MK ATP levels at 1 hour after LLL and platelets measured 3 days later were analyzed (n = 12). The coefficient of determination (R2) and P value were determined by regression and correlation analysis. (B and C) Effects of LLL on platelet production (B) and ATP synthesis (C) were inhibited by OA (5 μg/ml). Data are means ± SEM (n= 6). (D) Wild-type (WT) and IEX-1 KO BM nucleated cells were stained with anti-CD41 antibody and JC1. Mitochondrial membrane potential of CD41+ FSChigh MKs was determined by flow cytometric analysis of red J-aggregate fluorescence at 590 nm. Data represent three independent experiments. (E) Sorted MKs were treated with or without LLL and differentiated for 1 hour before ATP measurement as in Fig. 1B. Data are means ± SEM (n = 6). (F) Representative images of PPF-MKs were obtained at 24 hours after LLL from at least 6 samples per group with 25 cells in each group. Scale bar, 25 μm. (G) Cell diameters in (F) were shown, each symbol represents a single PPF-MK. (H) The number of platelets derived from 1 × 104 MKs was estimated 3 days after LLL on the basis of CD41 expression and FSC/SSC. Data are means ± SEM (n = 6). P values in (B) to (H) were determined by one-way ANOVA.

  • Fig. 3.

    LLL stimulates mitochondrial biogenesis in polyploid MKs. (A) ATP was measured in MKs, BMs, or LSKs for indicated times after LLL. Data are means ± SEM (n = 6). **P < 0.01, ***P < 0.001 versus 0 min. (B) At 24 hours after LLL, the indicated cells were stained with MitoTracker and analyzed by flow cytometry. Data are means ± SEM (n = 6). MFI, mean fluorescence intensity. (C) Mitochondrial (mt) DNA content of MKs was measured by real-time polymerase chain reaction (PCR) and normalized with nuclear (nu) DNA. Data are from three independent experiments with each in triplicate and expressed as means ± SEM. (D) Pgc1a transcript was measured at 4 hours after LLL, and other gene transcripts were measured at 16 hours after LLL, by quantitative reverse transcription PCR (qRT-PCR). Data are from three independent experiments with each in triplicate and expressed as means ± SEM. (E to I) MKs were sorted on the basis of DNA content by staining with Hoechst 33342 and anti-CD41–fluorescein isothiocyanate (FITC) (E), treated with LLL or sham light, and subjected to flow cytometric analysis with MitoTracker 24 hours later (F) or qRT-PCR analysis of Pgc1a transcript 4 hours after LLL (G), and other gene transcripts 16 hours after LLL (H). The number of platelets derived from 1 × 104 MKs was estimated 3 days after LLL (I). Data are from three independent experiments with each in triplicate and expressed as means ± SEM. (J to L) Representative transmission electron micrographs of MKs at 24 hours after LLL. Scale bar, 5 μm. The mitochondrial number of each MK (K) and the shortest distance between each mitochondrion and nearest nuclear region (L) were measured by ImageJ software from at least 30 MKs per group. Data are from three independent experiments with each in triplicate and expressed as means ± SEM. *P < 0.05 compared with non-LLL controls. P values were determined by one-way ANOVA (A) or two-tailed Student’s t test (B to L).

  • Fig. 4.

    LLL penetrates into the bones of mice. (A) Transmittance (%) of indicated LLL modes was measured beneath mouse fresh skin and vertebral bones using a laser power meter. Data are means ± SEM (n = 6). CW, continuous wave. (B) BMs were isolated from indicated bones at 1 hour after whole-body LLL illumination at 30 J/cm2 to determine ATP levels as in Fig. 1B. Data are means ± SEM (n = 6). (C and D) At 24 hours after whole-body LLL illumination, mice were intravenously injected with anti-CD41–FITC (green) and anti-CD105–phycoerythrin (red) and sacrificed 15 min later. Fresh femurs were then removed from the mice and examined by confocal microscope (C). Arrows indicate MKs. Scale bar, 50 μm. Percentages of PPF-MKs were determined from at least 50 MKs per femur and 6 samples per group (D). Data are from three independent experiments with each in triplicate and expressed as means ± SEM. P value was determined by one-way ANOVA (A) or two-tailed Student’s t test (B and D).

  • Fig. 5.

    LLL ameliorates thrombocytopenia induced by IR in vivo. (A) Complete blood counts of white blood cells (WBCs), lymphocytes, monocytes, granulocytes, and red blood cells (RBCs) in 3-Gy γ-irradiated mice 2 weeks after IR. Data are means ± SEM (n = 15). (B) Platelet counts were obtained at indicated days in 3-Gy γ-irradiated mice (IR), or IR mice treated with LLL once at 6 hours after IR (IR + 1×LLL), once a day on days 0 and 1 (IR + 2×LLL), or once a day from day 0 to day 3 (IR + 4×LLL). Data are means ± SEM (n = 15). *P < 0.05, **P < 0.01, ***P < 0.001 versus IR. (C) The tail bleeding time of each mouse was examined at 2 weeks after IR and presented by individual symbols. (D) Platelet volume of each mouse was examined at 2 weeks after IR. Data are means ± SEM (n = 10). (E) Representative transmission electron micrographs of platelets isolated from indicated mice at 2 weeks after IR. Scale bar, 1 μm. (F) Platelets isolated from non-IR control and IR + 4×LLL mice 2 weeks after LLL were labeled with either anti-CD9 or anti-CD31 antibody, mixed, and stimulated with phorbol 12-myristate 13-acetate (100 ng/ml). Aggregated platelets indicated by double-colored events were quantified by flow cytometry and presented as mean percentages ± SEM (n = 6). (G) Circulating platelets and BM MKs remain within the steady-state levels in mice treated with LLL at 30 J/cm2 once every other day for 12 days. Data are mean percentages ± SEM of changes relative to baseline (n = 6). (H) Effects of four doses of LLL on the number of MKs in γ-irradiated mice over time. Data are mean numbers ± SEM of MKs per femur at indicated days (n = 6). *P < 0.05, ***P < 0.001 compared with IR group. P values were determined by two-tailed Student’s t test (A and H) or one-way ANOVA (B to D).

  • Fig. 6.

    LLL alleviates thrombocytopenia induced by anti-CD41 antibody or 5-FU in mice. (A and B) Mice were administered daily with anti-CD41 antibody (0.1 mg/kg) over 8 days. LLL was given daily from day 3 to day 7 (arrows). (A) Platelet counts were measured daily at 6 hours after LLL. Data are means ± SEM (n = 6). (B) Tail bleeding time was examined on day 5. Controls are naïve mice without any treatment. Data are individual animals with mean (n = 6). **P < 0.01, ***P < 0.001 versus anti-CD41, unless otherwise noted. (C and D) Mice were injected with 5-FU (150 mg/kg) on day 0. LLL was given daily for three consecutive days starting at 4 hours after 5-FU injection. (C) Platelet counts were measured daily at 6 hours after LLL. Data are means ± SEM (n = 6). (D) Tail bleeding time was examined on day 4. *P < 0.05, ***P < 0.001 versus 5-FU, unless otherwise noted. All P values were determined by one-way ANOVA.

  • Fig. 7.

    LLL significantly enhances platelet generation in human MKs. (A) Illustration of timeline for ex vivo platelet differentiation from human CD34+ cells. CD34+ cells differentiated predominantly into MK progenitors in 6 days, MKs in 12 days, and platelets in 15 days. (B) ATP was measured in CD34+-derived MKs treated with LLL at various energy densities as in Fig. 1B. Data were obtained from three independent experiments with each in triplicate. *P < 0.05, **P < 0.01 versus 0 J/cm2, by one-way ANOVA. (C) Ploidy analysis of CD34+ cultures on day 0, 6, or 12 using Hoechst 33342 staining. Data represent three independent experiments. (D) LLL at 3 J/cm2 was administered on day 0, 6, or 12 during CD34+ cell differentiation. Platelets were quantified on day 15 by flow cytometry and expressed as mean numbers ± SEM of platelets derived from 1 × 104 CD34+ cells. Data were obtained from three independent experiments with each in triplicate. P values were determined by one-way ANOVA.

Supplementary Materials

  • www.sciencetranslationalmedicine.org/cgi/content/full/8/349/349ra101/DC1

    Material and Methods

    Fig. S1. PGC-1α plays a role in LLL-mediated platelet biogenesis.

    Fig. S2. LLL increases Pgc1a transcription, but expression of downstream genes is unaltered in BM nucleated cells and LSKs.

    Fig. S3. LLL protects mitochondrial function of γ-irradiated MKs.

    Table S1. Primer pairs used for quantitative real-time RT-PCR.

  • Supplementary Material for:

    Noninvasive low-level laser therapy for thrombocytopenia

    Qi Zhang, Tingting Dong, Peiyu Li, Mei X. Wu*

    *Corresponding author. Email: sjoerd.finnema{at}yale.edu

    Published 27 July 2016, Sci. Transl. Med. 8, 349ra101 (2016)
    DOI: 10.1126/scitranslmed.aaf4964

    This PDF file includes:

    • Material and Methods
    • Fig. S1. PGC-1α plays a role in LLL-mediated platelet biogenesis.
    • Fig. S2. LLL increases Pgc1a transcription, but expression of downstream genes is unaltered in BM nucleated cells and LSKs.
    • Fig. S3. LLL protects mitochondrial function of γ-irradiated MKs.
    • Table S1. Primer pairs used for quantitative real-time RT-PCR.

    [Download PDF]

Navigate This Article