Molecular imaging-assisted optimization of hsp70 expression during laser-induced thermal preconditioning for wound repair enhancement

Abstract

Patients at risk for impaired healing may benefit from prophylactic measures aimed at improving wound repair. Several photonic devices claim to enhance repair by thermal and photochemical mechanisms. We hypothesized that laser-induced thermal preconditioning would enhance surgical wound healing that was correlated with hsp70 expression. Using a pulsed diode laser (lambda=1.85 microm, tau(p)=2 ms, 50 Hz, H=7.64 mJ cm(-2)), the skin of transgenic mice that contain an hsp70 promoter-driven luciferase was preconditioned 12 hours before surgical incisions were made. Laser protocols were optimized in vitro and in vivo using temperature, blood flow, and hsp70-mediated bioluminescence measurements as benchmarks. Biomechanical properties and histological parameters of wound healing were evaluated for up to 14 days. Bioluminescent imaging studies indicated that an optimized laser protocol increased hsp70 expression by 10-fold. Under these conditions, laser-preconditioned incisions were two times stronger than control wounds. Our data suggest that this molecular imaging approach provides a quantitative method for optimization of tissue preconditioning and that mild laser-induced heat shock may be a useful therapeutic intervention prior to surgery.

Figure 1. In vivo visualization and quantification of hsp70 promoter activity in laser-treated areas

(a) Sample bioluminescent image 12 hours after laser preconditioning with the T_LE_L protocol (bar=2 cm). (b) Normalized mean blood perfusion measurements versus time for various laser exposures using the T_LE_L laser protocol (n=4). (c) hsp70 levels plotted versus time after laser exposure for the T_HE_S laser protocol. (d) hsp70 levels plotted versus time after laser exposure for the T_LE_L laser protocol. (e) Maximum fold induction of hsp70 for the T_HE_S protocol as a function of exposure time shows a linear exponential relationship (R²=0.93) (f) Maximum fold induction of hsp70 for the T_LE_L protocol as a function of exposure time a shows a linear relationship (R²=0.96). Values represent the mean±SD for hsp70 fold induction (n=5).

Figure 2. Histological evaluation of preconditioning

(a–d) Hematoxylin and eosin (H&E) stains 12 hours after treatment (scale bar=100 μm) (a) Untreated control tissue shows a healthy epidermis and hair follicles. (b) Positive control using lethal laser protocol (H=30 mJ cm⁻², exposure duration 60 seconds) shows ablation of the epidermis and coagulation on the upper dermis. (c) T_LE_L-preconditioning protocol shows only minor superficial damage restricted to the stratum corneum. (d) T_HE_S protocol induces mild damage that stimulates epidermal hyperplasia and inflammatory influx into the upper dermis. (e–h) H&E stains 3 days after treatment. (e) Untreated control tissue shows a healthy epidermis. (f) Positive control using lethal laser protocol shows epidermal ablation and coagulation on the upper dermis. (g) T_LE_L-preconditioning protocol shows only minor epidermal hyperplasia. (h) T_HE_S protocol induces mild damage that stimulates significant epidermal hyperplasia. (i) Epidermal hyperplasia plotted versus time for each laser treatment. Mean and SD for the depth of hyperplasia were statistically compared with Student’s t-test (***P<0.001).

Figure 3. Immunohistochemical evaluation of preconditioning

(a–d) Cellular proliferation using a Ki67 immunomarker 3 days after laser-induced thermal stress. (a) Control (non-wounded) areas of skin show minimal steady-state levels of epidermal proliferation (scale bar=100 μm) (b) Laser-treated (T_LE_L) tissue shows many basal cells actively replicating or proliferating (marked by arrow) (scale bar=100 μm). (c, d) Ki67 immunostain (scale bar=50 μm). (c) Control (non-wounded) areas of skin show minimal proliferation. (d) Laser-treated (T_LE_L) tissue shows transient amplifying cells emerging from the bulge region (marked by arrow). (e–f) Apoptotic profiles are visualized with a TUNEL stain at day 3. (e) Control skin samples with no visible apoptotic indicators. (f) Laser-treated (T_LE_L) sample with few visible apoptotic indicators.

Figure 4. Laser manipulation of hsp70 expression before surgical wounding

(a) Sample bioluminescent representation of control wounds (left) and laser pretreated surgical wounds (right) using the T_LE_L laser protocol at 12 hours before surgery. (b) hsp70 fold induction on control and laser-pretreated wounds. The mean hsp70 fold induction was normalized to a non-wounded control area of the skin. The mean and SD for hsp70 fold induction were statistically compared using Student’s t-test (n=10, *P<0.01).

Figure 5. Collagen deposition and cellularity in preconditioned surgical wounds

(a, b) Gomori’s trichrome staining 5 days after incision (a) Control wounds reveal pale staining adjacent to the incisional track of non-preconditioned incisions. (b) Preconditioned wounds show normal intense green patterns of collagen in the adjacent dermis. (c, d) Hemotoxylin and eosin staining 5 days after surgery (c) The surgical wound without preconditioning shows epidermal hyperplasia adjacent to the wound margin and a minimal cellular density in the granulation tissue within the wound bed. (d) A laser-preconditioned surgical wound shows normal adjacent epidermis, but a comparatively more robust granulation tissue within the wound bed (scale bar=100 μm). (e) Plot of epidermal hyperplasia over the incision and in immediate adjacent epidermis. Preconditioned surgical wounds have less epidermal hyperplasia than controls. (f) Plot of cell density within each wound bed per 1,000 μm². The mean and SD were statistically compared using Student’s t-test (***P<0.001 and n=20).

Figure 6. Wound biomechanics

Tensiometer data for full-thickness wounds from transgenic mice 7 and 10 days after surgery. (a) Average percent (%) increase in maximum load is plotted versus day after surgery. (b) Average percent increase (%) in tensile stress is plotted versus day after surgery. The preconditioned incision wounds were ~60% (58±13%, mean±SD) stronger than controls 7 days after surgery, and ~35% stronger than controls at day 10. The mean and SD for the maximum load and tensile stress were statistically compared using a paired Student’s t-test (***P<0.01 and n=9).