Gerald J. Wilmink

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

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.

Role of HSP70 in cellular thermotolerance

Thermal pretreatment has been shown to condition tissue to a more severe secondary heat stress. In this research we examined the particular contribution of heat shock protein 70 (HSP70) in thermal preconditioning.

Assessing laser-tissue damage with bioluminescent imaging

Effective medical laser procedures are achieved by selecting laser parameters that minimize undesirable tissue damage. Traditionally, human subjects, animal models, and monolayer cell cultures have been used to study wound healing, tissue damage, and cellular effects of laser radiation. Each of these models has significant limitations, and consequently, a novel skin model is needed. To this end, a highly reproducible human skin model that enables noninvasive and longitudinal studies of gene expression was sought. In this study, we present an organotypic raft model (engineered skin) used in combination with bioluminescent imaging (BLI) techniques. The efficacy of the raft model was validated and characterized by investigating the role of heat shock protein 70 (hsp70) as a sensitive marker of thermal damage. The raft model consists of human cells incorporated into an extracellular matrix. The raft cultures were transfected with an adenovirus containing a murine hsp70 promoter driving transcription of luciferase. The model enables quantitative analysis of spatiotemporal expression of proteins using BLI. Thermal stress was induced on the raft cultures by means of a constant temperature water bath or with a carbon dioxide (CO2) laser (lambda=10.6 microm, 0.679 to 2.262 Wcm2, cw, unfocused Gaussian beam, omegaL=4.5 mm, 1 min exposure). The bioluminescence was monitored noninvasively with an IVIS 100 Bioluminescent Imaging System. BLI indicated that peak hsp70 expression occurs 4 to 12 h after exposure to thermal stress. A minimum irradiance of 0.679 Wcm2 activated the hsp70 response, and a higher irradiance of 2.262 Wcm2 was associated with a severe reduction in hsp70 response due to tissue ablation. Reverse transcription polymerase chain reaction demonstrated that hsp70 mRNA levels increased with prolonged heating exposures. Enzyme-linked immunosorbent protein assays confirmed that luciferase was an accurate surrogate for hsp70 intracellular protein levels. Hematoxylin and eosin stains verified the presence of the thermally denatured tissue regions. Immunohistochemical analyses confirmed that maximal hsp70 expression occurred at a depth of 150 microm. Bioluminescent microscopy was employed to corroborate these findings. These results indicate that quantitative BLI in engineered tissue equivalents provides a powerful model that enables sequential gene expression studies. Such a model can be used as a high throughput screening platform for laser-tissue interaction studies.

Microarray analysis of cellular thermotolerance.

Previously, we have shown that a 43°C pretreatment can provide thermotolerance to a following, more severe, thermal stress at 45°C. Using cells that lack the Hsp70 gene, we have also shown that there is still some thermotolerance in the absence of HSP70 protein. The purpose of this study was to determine which genes play a role in thermotolerance by measuring viability and proliferation of the cells at 2 days after heating. Specifically, we wanted to understand which pathways may be responsible for protecting cells in the absence of HSP70.

In-vivo optical imaging of hsp70 expression to assess collateral tissue damage associated with infrared laser ablation of skin.

Laser surgical ablation is achieved by selecting laser parameters that remove confined volumes of target tissue and cause minimal collateral damage. Previous studies have measured the effects of wavelength on ablation, but neglected to measure the cellular impact of ablation on cells outside the lethal zone. In this study, we use optical imaging in addition to conventional assessment techniques to evaluate lethal and sublethal collateral damage after ablative surgery with a free-electron laser (FEL). Heat shock protein (HSP) expression is used as a sensitive quantitative marker of sublethal damage in a transgenic mouse strain, with the hsp70 promoter driving luciferase and green fluorescent protein (GFP) expression (hsp70A1-L2G). To examine the wavelength dependence in the mid-IR, laser surgery is conducted on the hsp70A1-L2G mouse using wavelengths targeting water (OH stretch mode, 2.94 microm), protein (amide-II band, 6.45 microm), and both water and protein (amide-I band, 6.10 microm). For all wavelengths tested, the magnitude of hsp70 expression is dose-dependent and maximal 5 to 12 h after surgery. Tissues treated at 6.45 microm have approximately 4x higher hsp70 expression than 6.10 microm. Histology shows that under comparable fluences, tissue injury at the 2.94-microm wavelength was 2x and 3x deeper than 6.45 and 6.10 microm, respectively. The 6.10-microm wavelength generates the least amount of epidermal hyperplasia. Taken together, this data suggests that the 6.10-microm wavelength is a superior wavelength for laser ablation of skin.

Wavelength-dependent dynamics of heat shock protein 70 expression in free electron laser wounds

Many medical laser procedures require selecting laser operating parameters that minimize undesirable tissue damage. In this study, heat shock protein 70(hsp70) gene expression was used as a sensitive marker for laser-induced thermal damage. Wound repair and hsp70 expression were compared after surgery with the free electron laser(FEL) as a function of wavelength(&lgr;) and radiant exposure(H). Damage was assessed at &lgr; = 6.45, 6.10, and 2.94 &mgr;m using 8-20 J/cm2. The FEL beam (&Vpgr;r=200 &mgr;m,30Hz,&tgr;p =5&mgr;s) was delivered to produce a 6.5 mm square wound. hsp70 expression was assessed using a transgenic mouse strain with the hsp70 promoter driving luciferase and eGFP expression. Bioluminescent imaging (BLI) was monitored non-invasively and in real time. Hsp70 protein was visualized with laser confocal imaging, blood velocity was measured with 2D-laser doppler, and depth of tissue damage was measured using histological methods. BLI verified the models sensitivity and peak hsp70 expression was bi-phasic, with maxima occurring 12 and 24 hours after FEL irradiation. hsp70 expression exhibited wavelength-dependence, and it increased with radiant exposure. Histology indicated that tissue damage at 6.45 µm was ~2x deeper than 6.10 &mgr;m. Quantitative BLI with the Hsp70-luc transgene can be used to non-invasively measure gene expression in laser-tissue interaction studies.

Laser thermal preconditioning enhances dermal wound repair

Preconditioning tissues with an initial mild thermal stress, thereby eliciting a stress response, can serve to protect tissue from subsequent stresses. Patients at risk for impaired healing, such as diabetics, can benefit from therapeutic methods which enhance wound repair. We present a laser thermal preconditioning protocol that accelerates cutaneous wound repair in a murine model. A pulsed diode laser (λ = 1.86 μm, τp = 2 ms, 50 Hz, H = 7.64 mJ/cm2) was used to precondition mouse skin before incisional wounds were made. The preconditioning protocol was optimized in vitro and in vivo using hsp70 expression, cell viability, and temperature measurements as benchmarks. Hsp70 expression was non-invasively monitored using a transgenic mouse strain with the hsp70 promoter driving luciferase expression. Tissue temperature recordings were acquired in real time using an infrared camera. Wound repair was assessed by measuring hsp70 expression, biomechanical properties, and wound histology for up to 24 d. Bioluminescence (BLI) was monitored with the IVIS 200 System (Xenogen) and tensile properties with a tensiometer (BTC-2000). The in vivo BLI studies indicated that the optimized laser preconditioning protocol increased hsp70 expression by 15-fold. The tensiometer data revealed that laser preconditioned wounds are ~40% stronger than control wounds at 10 days post surgery. Similar experiments in a diabetic mouse model also enhanced wound repair strength. These results indicate that 1) noninvasive imaging methods can aid in the optimization of novel laser preconditioning methods; 2) that optimized preconditioning with a 1.86 μm diode laser enhances early wound repair.

Use of optical reporter genes to assess sublethal cellular damage following skin ablation

Numerous medical procedures utilize pulsed lasers to remove unwanted biological tissue. Mid-infrared wavelengths which preferentially target protein absorption bands ablate tissue more efficiently than wavelengths targeting water absorption. However, the mechanism responsible for this finding has not been established. In this report, we combine optical imaging and conventional techniques to assess lethal and sublethal collateral damage after ablative surgery with a Free Electron Laser (FEL). Heat shock protein expression was used to evaluate tissue damage in a transgenic mouse strain, with the hsp70 promoter driving luciferase and GFP expression (hsp70A1-L2G). To examine wavelength-dependence in the mid-IR, laser surgery was conducted on the hsp70A1-L2G mouse model using wavelengths targeting protein (amide II band, 6.45 μm), both water and protein (amide I band, 6.10 μm); and water (2.94 μm). Hsp70-driven luciferase activity was used as a quantitative biomarker for intracellular damage, and histological analyses were conducted to measure the depth of thermal damage. For all of the wavelengths tested, the bioluminescent data showed that the magnitude of hsp70 expression was dose-dependent. Tissues treated at 6.45 µm had approximately 2x higher hsp70 expression than tissues treated at 6.10 μm. Histology showed that immediate tissue injury at the 6.45 μm wavelength was ~2x deeper than at 6.10 μm. The 6.10 μm wavelength generated the least amount of epidermal hyperplasia. Overall, the data suggests that 6.10 μm is a superior wavelength for cutaneous laser ablation procedures.