Mark A. Mackanos

Assessment of Cellular Response to Thermal Laser Injury Through Bioluminescence Imaging of Heat Shock Protein 70

Assessment of laser‐induced tissue damage is not complete without an investigation into the resulting cellular and molecular changes. In the past, tissue damage was quantified macroscopically by visual effects such as tissue mass removal, carbonization and melting. Microscopically, assessment of tissue damage has been typically limited to histological analysis of excised tissue samples. In this research, we used heat shock protein (hsp70) transcription to track cellular response to laser‐induced injury. A stable cell line (NIH‐3T3) was generated containing the firefly luciferase (luc) reporter gene attached to the hsp promoter (murine hsp70a1). After thermal injury with a pulsed holmium‐yttrium aluminum garnet laser (λ = 2.1 μm, τp = 250 μs, 30 pulses, 3 Hz), luciferase is produced on hsp70 activation and emits broadspectrum bioluminescence over a range of 500–700 nm, with a peak at 563 nm. The onset of bioluminescence can be seen as early as 2 h after treatment and usually peaks at 8–12 h depending on the severity of heat shock. The luminescence was quantified in live cells using bioluminescence imaging. A minimum pulse energy (65 mJ/pulse [total energy 1.95 J; total radiant exposure = 6 J/cm2]) was needed to activate the hsp70 response, and a higher energy (103 mJ/pulse [total energy 3.09 J; total radiant exposure = 9.6 J/cm2]) was associated with a reduction in hsp70 response and cell death. Bioluminescence levels correlated well with actual hsp70 protein concentrations as determined by enzyme‐linked immunosorbent assay. Photon counts were normalized to the percentage of live cells by means of a flow cytometry cell viability assay. Within a relatively small range between a lower activation threshold and an upper threshold that leads to cell death, the hsp70 response followed an Arrhenius relationship when constant‐temperature water bath and laser experiments were carried out.

Non-damaging retinal phototherapy: dynamic range of heat shock protein expression.

PURPOSE Subthreshold retinal phototherapy demonstrated clinical efficacy for the treatment of diabetic macular edema without visible signs of retinal damage. To assess the range of cellular responses to sublethal hyperthermia, expression of the gene encoding a 70 kDa heat shock protein (HSP70) was evaluated after laser irradiation using a transgenic reporter mouse. METHODS One hundred millisecond, 532 nm laser exposures with 400 μm beam diameter were applied to the retina surrounding the optic nerve in 32 mice. Transcription from the HSP70 promoter was assessed relative to the control eye using a bioluminescence assay at 7 hours after laser application. The retinal pigmented epithelium (RPE) viability threshold was determined with a fluorescence assay. A computational model was developed to estimate temperature and the extent of cell damage. RESULTS A significant increase in HSP70 transcription was found at exposures over 20 mW, half the threshold power for RPE cell death. Computational modeling estimated peak temperature T = 49°C at HSP70 expression threshold. At RPE viability threshold, T = 57°C. Similar temperatures and damage indices were calculated for clinical subvisible retinal treatment parameters. CONCLUSIONS Beneficial effects of laser therapy have been previously shown to extend beyond those resulting from destruction of tissue. One hundred millisecond laser exposures at approximately half the threshold power of RPE damage induced transcription of HSP70, an indication of cellular response to sublethal thermal stress. A computational model of retinal hyperthermia can guide further optimization of laser parameters for nondamaging phototherapy.

In vivo analysis of heat-shock-protein-70 induction following pulsed laser irradiation in a transgenic reporter mouse

Induction of heat shock protein (Hsp) expression appears to correlate with a cytoprotective effect in cultured cells and with improved healing of damaged tissues in animal models and in humans. This family of proteins can also serve as indicators of thermal stress in cases of burn injury or surgical procedures that produce heat. Thus, a rapid in vivo readout for induction of Hsp transcription would facilitate studies of Hsp genes and their encoded proteins as mediators of therapeutic effects and as reporters of thermal damage to tissues. We created a transgenic reporter mouse where expression of luciferase is controlled by the regulatory region of the inducible 70 kDa Hsp, and assessed activation of Hsp70 transcription in live animals in response to rapid, high temperature stresses using in vivo bioluminescence imaging (BLI). This model can be used to noninvasively reveal levels of Hsp70 transcription in living tissues, and has utility in studies of the predictive and protective effects of Hsp70 expression, and of various stress responses in tissues.

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.

Short-duration Focused Ultrasound Stimulation of Hsp70 Expression In Vivo

The development of transgenic reporter mice and advances in in vivo optical imaging have created unique opportunities to assess and analyze biological responses to thermal therapy directly in living tissues. Reporter mice incorporating the regulatory regions from the genes encoding the 70 kDa heat-shock proteins (Hsp70) and firefly luciferase (luc) as reporter genes can be used to non-invasively reveal gene activation in living tissues in response to thermal stress. High-intensity-focused ultrasound (HIFU) can deliver measured doses of acoustic energy to highly localized regions of tissue at intensities that are sufficient to stimulate Hsp70 expression. We report activation of Hsp70-luc expression using 1 s duration HIFU heating to stimulate gene expression in the skin of the transgenic reporter mouse. Hsp70 expression was tracked for 96 h following the application of 1.5 MHz continuous-wave ultrasound with spatial peak intensities ranging from 53 W cm(-2) up to 352 W cm(-2). The results indicated that peak Hsp70 expression is observed 6-48 h post-heating, with significant activity remaining at 96 h. Exposure durations were simulated using a finite-element model, and the predicted temperatures were found to be consistent with the observed Hsp70 expression patterns. Histological evaluation revealed that the thermal damage starts at the stratum corneum and extends deeper with increasing intensity. These results indicated that short-duration HIFU may be useful for inducing heat-shock expression, and that the period between treatments needs to be greater than 96 h due to the protective properties of Hsp70.

Effect of optical tissue clearing on spatial resolution and sensitivity of bioluminescence imaging

In vivo bioluminescence imaging (BLI) is a powerful method of in vivo molecular imaging based on the use of optically active luciferase reporter genes. Although this method provides superior sensitivity relative to other in vivo imaging methods, spatial resolution is poor due to light scattering. The objective of this study was to use hyperosmotic agents to reduce the scattering coefficient and hence improve spatial resolution of the BLI method. A diffusing fiber tip was used to simulate an isotropic point source of bioluminescence emission (550 to 650 nm). Mouse skin was treated in vitro and in vivo with glycerol (50%, 30 min) and measurements of optical properties, and imaging photon counts were made before, during, and after application of glycerol to the skin sample. Glycerol application to mouse skin had little effect on the absorption coefficient but reduced the reduced scattering coefficient by more than one order of magnitude. This effect was reversible. Consequently, the spot size (i.e., spatial resolution) of the bioluminescence point source imaged through the skin decreased by a factor of 2 (550-nm light) to 3 (650-nm light) after 30 min. Simultaneously, an almost twofold decrease in the amount of light detected by the BLI system was observed, despite the fact that total transmission increased 1.7 times. We have shown here that multiply scattered light is responsible for both observations. We have shown that applying a hyperosmotic clearing agent to the skin of small rodents has the potential to improve spatial resolution of BLI owing to a reduction in the reduced scattering coefficient in the skin by one order of magnitude. However, reducing the scattering coefficient reduces the amount of light reaching the camera due to a reduction in the amount of multiply scattered light that reaches the camera aperture and thus reducing the sensitivity of the method.

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.

Accelerated Bone Repair After Plasma Laser Corticotomies

Objective:To reveal, on a cellular and molecular level, how skeletal regeneration of a corticotomy is enhanced when using laser-plasma mediated ablation compared with conventional mechanical tissue removal. Summary Background Data:Osteotomies are well-known for their most detrimental side effect: thermal damage. This thermal and mechanical trauma to adjacent bone tissue can result in the untoward consequences of cell death and eventually in a delay in healing. Methods:Murine tibial corticotomies were performed using a conventional saw and a Ti:Sapphire plasma-generated laser that removes tissue with minimal thermal damage. Our analyses began 24 hours after injury and proceeded to postsurgical day 6. We investigated aspects of wound repair ranging from vascularization, inflammation, cell proliferation, differentiation, and bone remodeling. Results:Histology of mouse corticotomy sites uncovered a significant difference in the onset of bone healing; whereas laser corticotomies showed abundant bone matrix deposition at postsurgical day 6, saw corticotomies only exhibited undifferentiated tissue. Our analyses uncovered that cutting bone with a saw caused denaturation of the collagen matrix due to thermal effects. This denatured collagen represented an unfavorable scaffold for subsequent osteoblast attachment, which in turn impeded deposition of a new bony matrix. The matrix degradation induced a prolonged inflammatory reaction at the cut edge to create a surface favorable for osteochondroprogenitor cell attachment. Laser corticotomies were absent of collagen denaturation, therefore osteochondroprogenitor cell attachment was enabled shortly after surgery. Conclusion:In summary, these data demonstrate that corticotomies performed with Ti:Sapphire lasers are associated with a reduced initial inflammatory response at the injury site leading to accelerated osteochondroprogenitor cell migration, attachment, differentiation, and eventually matrix deposition.

The effect of free-electron laser pulse structure on mid-infrared soft-tissue ablation: biological effects

Previous studies have shown that changing the pulse structure of the free electron laser (FEL) from 1 to 200 ps and thus reducing the peak irradiance of the micropulse by 200 times had little or no effect on both the ablation threshold radiant exposure and the ablated crater depth for a defined radiant exposure. This study focuses on the ablation mechanism at 6.1 and 6.45 microm with an emphasis on the role of the FEL pulse structure. Three different experiments were performed to gain insight into this mechanism. The first was an analysis of the ablation plume dynamics observed for a 1 ps micropulse compared with a 200 ps micropulse as seen through bright-field analysis. Negligible differences are seen in the size, but not the dynamics of ablation, as a result of this imaging. The second experiment was a histological analysis of corneal and dermal tissue to determine whether there is less thermal damage associated with one micropulse duration versus another. No significant difference was seen in the extent of thermal damage on either canine cornea or mouse dermis for the micropulse durations studied at either wavelength. The final set of experiments involved the use of mass spectrometry to determine whether amide bond breakage could occur in the proteins present in tissue as a result of direct absorptions of mid-infrared light into the amide I and amide II absorption bands. This analysis showed that there was no amide bond breakage due to irradiation at 6.45 microm on protein.

Femtosecond plasma mediated laser ablation has advantages over mechanical osteotomy of cranial bone

Although mechanical osteotomies are frequently made on the craniofacial skeleton, collateral thermal, and mechanical trauma to adjacent bone tissue causes cell death and may delay healing. The present study evaluated the use of plasma‐mediated laser ablation using a femtosecond laser to circumvent thermal damage and improve bone regeneration.