RESUMEN
Objective. Optical methods of neural activation are becoming important tools for the study and treatment of neurological disorders. Infrared nerve stimulation (INS) is an optical technique exhibiting spatially precise activation in the native neural system. While this technique shows great promise, the risk of thermal damage may limit some applications. Combining INS with traditional electrical stimulation, a method known as hybrid electro-optical stimulation, reduces the laser power requirements and mitigates the risk of thermal damage while maintaining spatial selectivity. Here we investigate the capability of inducing force generation in the rat hind limb through hybrid stimulation of the sciatic nerve. Approach. Hybrid stimulation was achieved by combining an optically transparent nerve cuff for electrical stimulation and a diode laser coupled to an optical fiber for infrared stimulation. Force generation in the rat plantarflexor muscles was measured in response to hybrid stimulation with 1 s bursts of pulses at 15 and 20 Hz and with a burst frequency of 0.5 Hz. Main results. Forces were found to increase with successive stimulus trains, ultimately reaching a plateau by the 20th train. Hybrid evoked forces decayed at a rate similar to the rate of thermal diffusion in tissue. Preconditioning the nerve with an optical stimulus resulted in an increase in the force response to both electrical and hybrid stimulation. Histological evaluation showed no signs of thermally induced morphological changes following hybrid stimulation. Our results indicate that an increase in baseline temperature is a likely contributor to hybrid force generation. Significance. Extraneural INS of peripheral nerves at physiologically relevant repetition rates is possible using hybrid electro-optical stimulation.
Asunto(s)
Estimulación Eléctrica/métodos , Músculo Esquelético/fisiología , Estimulación Luminosa , Nervio Ciático/fisiología , Animales , Temperatura Corporal/fisiología , Electromiografía , Rayos Infrarrojos , Masculino , Contracción Muscular/fisiología , Ratas , Ratas Sprague-DawleyRESUMEN
BACKGROUND: 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. METHODS: Critical-size circular calvarial defects were created with a trephine drill bit or with a Ti:Sapphire femtosecond pulsed laser. Healing was followed using micro-CT scans for 8 weeks. Calvaria were also harvested at various time points for histological analysis. Finally, scanning electron microscopy was used to analyze the microstructure of bone tissue treated with the Ti:Sapphire laser, and compared to that treated with the trephine bur. RESULTS: Laser-created defects healed significantly faster than those created mechanically at 2, 4, and 6 weeks post-surgery. However, at 8 weeks post-surgery, there was no significant difference. In the drill osteotomy treatment group, empty osteocyte lacunae were seen to extend 699 ± 27 µm away from the edge of the defect. In marked contrast, empty osteocyte lacunae were seen to extend only 182 ± 22 µm away from the edge of the laser-created craters. Significantly less ossification and formation of irregular woven bone was noted on histological analysis for drill defects. CONCLUSIONS: We demonstrate accelerated bone healing after femtosecond laser ablation in a calvarial defect model compared to traditional mechanical drilling techniques. Improved rates of early regeneration make plasma-mediated ablation of the craniofacial skeleton advantageous for applications to osteotomy.
Asunto(s)
Regeneración Ósea/efectos de la radiación , Láseres de Estado Sólido , Osteotomía/métodos , Hueso Parietal/cirugía , Cicatrización de Heridas/efectos de la radiación , Animales , Ratones , Microscopía Electrónica de Rastreo , Hueso Parietal/diagnóstico por imagen , Hueso Parietal/fisiología , Hueso Parietal/ultraestructura , Tomografía Computarizada por Rayos XRESUMEN
Confocal reflectance microscopy and confocal Raman spectroscopy have shown potential for non-destructive analysis of samples at micron-scale resolutions. Current studies utilizing these techniques often employ large bench-top microscopes, and are not suited for use outside of laboratory settings. We have developed a microscope which combines laser scanning confocal reflectance imaging and confocal Raman spectroscopy into a compact handheld probe that is capable of high-resolution imaging and spectroscopy in a variety of settings. The compact size of the probe is largely due to the use of a MEMS mirror for beam scanning. The probe is capable of axial resolutions of up to 4 µm for the confocal imaging channel and 10 µm for the confocal Raman spectroscopy channel. Here, we report instrument design, characterize optical performance, and provide images and spectra from normal skin to demonstrate the instrument's capabilities for clinical diagnostics.
RESUMEN
Beneficial medical laser ablation removes material efficiently with minimal collateral damage. A Mark-III free electron laser (FEL), at a wavelength of 6.45 µm has demonstrated minimal damage and high ablation yield in ocular and neural tissues. While this wavelength has shown promise for surgical applications, further advances are limited by the high overhead for FEL use. Alternative mid-infrared sources are needed for further development. We compared the FEL with a 5-µs pulse duration with a Q-switched ZGP-OPO with a 100-ns pulse duration at mid-infrared wavelengths. There were no differences in the ablation threshold of water and mouse dermis with these two sources in spite of the difference in their pulse structures. There was a significant difference in crater depth between the ZGP:OPO and the FEL. At 6.1 µm, the OPO craters are eight times the depth of the FEL craters. The OPO craters at 6.45 and 6.73 µm were six and five times the depth of the FEL craters, respectively. Bright-field (pump-probe) images showed the classic ablation mechanism from formation of a plume through collapse and recoil. The crater formation, ejection, and collapse phases occurred on a faster time-scale with the OPO than with the FEL. This research showed that a ZGP-OPO laser could be a viable alternative to FEL for clinical applications.
Asunto(s)
Terapia por Láser/instrumentación , Terapia por Láser/métodos , Animales , Dermis/cirugía , Diseño de Equipo , Rayos Infrarrojos , Láseres de Estado Sólido , Ratones , Óptica y Fotónica/instrumentaciónRESUMEN
Induction of heat shock protein (Hsp) expression correlates with cytoprotection, reduced tissue damage, and accelerated healing in animal models. Since Hsps are transcriptionally activated in response to stress, they can act as stress indicators in burn injury or surgical procedures that produce heat and thermal change. A fast in vivo readout for induction of Hsp transcription in tissues would allow for the study of these proteins as therapeutic effect mediators and reporters of thermal stress∕damage. We used a transgenic reporter mouse in which a luciferase expression is controlled by the regulatory region of the inducible 70 kilodalton (kDa) Hsp as a rapid readout of cellular responses to laser-mediated thermal stress∕injury in mouse skin. We assessed the pulse duration dependence of the Hsp70 expression after irradiation with a CO(2) laser at 10.6 µm in wavelength over a range of 1000 to 1 ms. Hsp70 induction varied with changes in laser pulse durations and radiant exposures, which defined the ranges at which thermal activation of Hsp70 can be used to protect cells from subsequent stress, and reveals the window of thermal stress that tissues can endure.
Asunto(s)
Proteínas HSP70 de Choque Térmico/biosíntesis , Láseres de Gas/efectos adversos , Láseres de Gas/uso terapéutico , Animales , Femenino , Expresión Génica , Genes Reporteros , Proteínas Fluorescentes Verdes/genética , Proteínas HSP70 de Choque Térmico/genética , Respuesta al Choque Térmico/genética , Respuesta al Choque Térmico/fisiología , Calor/efectos adversos , Calor/uso terapéutico , Luciferasas de Luciérnaga/genética , Mediciones Luminiscentes , Ratones , Ratones Transgénicos , Estrés Fisiológico , Factores de TiempoRESUMEN
The cytoprotective response to thermal injury is characterized by transcriptional activation of "heat shock proteins" (hsp) and proinflammatory proteins. Expression of these proteins may predict cellular survival. Microarray analyses were performed to identify spatially distinct gene expression patterns responding to thermal injury. Laser injury zones were identified by expression of a transgene reporter comprised of the 70 kD hsp gene and the firefly luciferase coding sequence. Zones included the laser spot, the surrounding region where hsp70-luc expression was increased, and a region adjacent to the surrounding region. A total of 145 genes were up-regulated in the laser irradiated region, while 69 were up-regulated in the adjacent region. At 7 hours the chemokine Cxcl3 was the highest expressed gene in the laser spot (24 fold) and adjacent region (32 fold). Chemokines were the most common up-regulated genes identified. Microarray gene expression was successfully validated using qRT- polymerase chain reaction for selected genes of interest. The early response genes are likely involved in cytoprotection and initiation of the healing response. Their regulatory elements will benefit creating the next generation reporter mice and controlling expression of therapeutic proteins. The identified genes serve as drug development targets that may prevent acute tissue damage and accelerate healing.
Asunto(s)
Mapeo Cromosómico/instrumentación , Proteínas HSP70 de Choque Térmico/fisiología , Respuesta al Choque Térmico/fisiología , Rayos Láser , Microscopía Fluorescente/instrumentación , Análisis de Secuencia por Matrices de Oligonucleótidos/instrumentación , Fenómenos Fisiológicos de la Piel/efectos de la radiación , Animales , Diseño de Equipo , Análisis de Falla de Equipo , Ratones , Ratones Noqueados , Integración de SistemasRESUMEN
Fourier transform infrared spectroscopy (FTIR) reveals biochemical 'fingerprints' and has found disease patterns in excised human tissues. Fiber-optic probes have been developed for FTIR in living systems, allowing for cancer detection. There are challenges to making in vivo FTIR a reality, which are being addressed through hardware advances, determining key wavelengths and tissue preparation. Fiber-optic evanescent wave spectroscopy (FEWS)-FTIR with endoscope-compatible fiber-optic silver halide probes is feasible, and could prove useful for distinguishing premalignant and malignant tissues from biopsies or within patients. Developments of smaller silver halide probes as well as in vivo tissue drying methods will move this approach closer to the clinic where it can be used for early cancer detection, disease characterization and guided biopsies.
Asunto(s)
Tecnología de Fibra Óptica/métodos , Neoplasias/diagnóstico , Espectroscopía Infrarroja por Transformada de Fourier/métodos , HumanosRESUMEN
BACKGROUND AND OBJECTIVES: 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. STUDY DESIGN/MATERIALS AND METHODS: Murine embryonic fibroblast cells with and without Hsp70 (MEF(+/+) and MEF(-/-), respectively) were exposed to a mild heat shock of 43°C for 30 minutes in a constant temperature water bath. After 3 hours of recovery, RNA was harvested from three heated samples alongside three untreated controls using a MicroRNeasy kit with DNAse treatment. RNA quality was verified by an Agilent Bioanalyzer. The RNA was then converted to cDNA and hybridized to Affymetrix gene expression DNA microarrays. The genes that showed a twofold change (up or down) relative to unheated controls were filtered by t-test for significance at a threshold of P < 0.05 using Genespring software. Data were verified by qRT-PCR. Genes were then categorized based upon their ontology. RESULTS: While many genes were similarly upregulated, the main difference between cell types was an increase in transcription factors and nucleic acid binding proteins. Several genes known to be involved in the heat response were upregulated more than twofold (Hsp70, Hsp40, Hsp110, Hsp25, Atf3), however, another well studied heat responsive gene Hsp90 only increased by 1.5-fold under these conditions despite its role in thermotolerance. CONCLUSIONS: The data herein presents genetic pathways which are candidates for further study of pretreatment protocols in laser irradiation.
Asunto(s)
Regulación de la Temperatura Corporal/genética , Fibroblastos/fisiología , Proteínas HSP70 de Choque Térmico/fisiología , Respuesta al Choque Térmico/genética , Calor/efectos adversos , Animales , Técnicas de Cultivo de Célula , Proliferación Celular , Supervivencia Celular/genética , Ratones , Análisis por Micromatrices , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
Accurate diagnosis and prognosis is essential for cancer management but is subject to sampling and inter-observer error. In a recent study, Baker et al. compared Fourier transform infrared (FTIR) microspectroscopy with histological pathology to evaluate prostate tissue for disease severity. The authors found that biochemical changes associated with prostate cancer could be discriminated by FTIR to classify confined and locally invasive prostate cancers. These findings could enable the development of improved diagnostic and prognostic methods for the detection and treatment of prostate cancers.
Asunto(s)
Próstata/química , Próstata/patología , Neoplasias de la Próstata/diagnóstico , Espectroscopía Infrarroja por Transformada de Fourier/métodos , Histocitoquímica/métodos , Humanos , MasculinoRESUMEN
Fourier transform infrared (FTIR) spectroscopy is sensitive to the molecular composition of tissue and has the potential to identify premalignant tissue (dysplasia) as an adjunct to endoscopy. We demonstrate collection of mid-infrared absorption spectra with a silver halide (AgCl(0.4)Br(0.6)) optical fiber and use spectral preprocessing to identify optimal subranges that classify colonic mucosa as normal, hyperplasia, or dysplasia. We collected spectra (n=83) in the 950 to 1800 cm(-1) regime on biopsy specimens obtained from human subjects (n=37). Subtle differences in the magnitude of the absorbance peaks at specific wave numbers were observed. The best double binary algorithm for distinguishing normal-versus-dysplasia and hyperplasia-versus-dysplasia was determined from an exhaustive search of spectral intervals and preprocessing techniques. Partial least squares discriminant analysis was used to classify the spectra using a leave-one-subject-out cross-validation strategy. The results were compared with histology reviewed independently by two gastrointestinal pathologists. The optimal thresholds identified resulted in an overall sensitivity, specificity, accuracy, and positive predictive value of 96%, 92%, 93%, and 82%, respectively. These results indicated that mid-infrared absorption spectra collected remotely with an optical fiber can be used to identify colonic dysplasia with high accuracy, suggesting that continued development of this technique for the early detection of cancer is promising.
Asunto(s)
Neoplasias del Colon/diagnóstico , Endoscopios Gastrointestinales , Espectroscopía Infrarroja por Transformada de Fourier/instrumentación , Transductores , Adulto , Anciano , Anciano de 80 o más Años , Diseño de Equipo , Análisis de Falla de Equipo , Femenino , Humanos , Persona de Mediana Edad , Reproducibilidad de los Resultados , Sensibilidad y Especificidad , Integración de SistemasRESUMEN
Liposomal formulations of drugs have been shown to enhance drug efficacy by prolonging circulation time, increasing local concentration and reducing off-target effects. Controlled release from these formulations would increase their utility, and hyperthermia has been explored as a stimulus for targeted delivery of encapsulated drugs. Use of lasers as a thermal source could provide improved control over the release of the drug from the liposomes with minimal collateral tissue damage. Appropriate methods for assessing local release after systemic delivery would aid in testing and development of better formulations. We use in vivo bioluminescence imaging to investigate the spatiotemporal distribution of luciferin, used as a model small molecule, and demonstrate laser-induced release from liposomes in animal models after systemic delivery. These liposomes were tested for luciferin release between 37 and 45 degrees C in PBS and serum using bioluminescence measurements. In vivo studies were performed on transgenic reporter mice that express luciferase constitutively throughout the body, thus providing a noninvasive readout for controlled release following systemic delivery. An Nd:YLF laser was used (527 nm) to heat tissues and induce rupture of the intravenously delivered liposomes in target tissues. These data demonstrate laser-mediated control of small molecule delivery using thermally sensitive liposomal formulations.
Asunto(s)
Preparaciones de Acción Retardada/química , Preparaciones de Acción Retardada/efectos de la radiación , Composición de Medicamentos/métodos , Rayos Láser , Liposomas/química , Liposomas/efectos de la radiación , Ensayo de MaterialesRESUMEN
BACKGROUND AND OBJECTIVE: 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. STUDY DESIGN/MATERIALS AND METHODS: For optimization of preshock exposures, a bioluminescent Hsp70-luciferase reporter system in NIH3T3 cells tracked the activation of the Hsp70 gene. Cells in 96-well plates were pretreated in a 43 degrees C water bath for 30 minutes, followed 4 hours later with a severe heat shock at 45 degrees C for 50 minutes. Bioluminescence was measured at 2, 4, 6, 8, and 10 hours after preshock only (PS) and at 4 hours after preshock with heatshock (PS+HS). Viability was assessed 48 hours later with a fluorescent viability dye. Preshock induced thermotolerance was then evaluated in hsp70-containing Murine Embryo Fibroblast (+/+) cells and Hsp70-deficient MEF cells (-/-) through an Arrhenius damage model across varying temperatures (44.5-46 degrees C). RESULTS: A time gap of 4 hours between preconditioning and the thermal insult was shown to be the most effective for thermotolerance with statistical confidence of P<0.05. The benefit of preshocking was largely abrogated in Hsp70-deficient cells. The Arrhenius data showed that preshocking leads to increases in the activation energies, E(a), and increases in frequency factors, A. The frequency factor increase was significantly greater in Hsp70-deficient cells. CONCLUSION: The data shows that HSP70 contributes significantly to cellular thermotolerance but there are other pathways that provide residual thermotolerance in cells deficient in Hsp70.
Asunto(s)
Adaptación Fisiológica , Proteínas HSP70 de Choque Térmico/metabolismo , Calor , Animales , Supervivencia Celular , Células Cultivadas , Fibroblastos/metabolismo , Proteínas HSP70 de Choque Térmico/deficiencia , Láseres de Estado Sólido , Mediciones Luminiscentes , Ratones , Modelos BiológicosRESUMEN
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.
Asunto(s)
Dermoscopía/métodos , Perfilación de la Expresión Génica/métodos , Proteínas HSP70 de Choque Térmico/metabolismo , Terapia por Láser/efectos adversos , Traumatismos por Radiación/enzimología , Piel/metabolismo , Piel/efectos de la radiación , Animales , Rayos Infrarrojos/efectos adversos , Mediciones Luminiscentes/métodos , Ratones , Ratones Transgénicos , Traumatismos por Radiación/diagnóstico , Piel/lesionesRESUMEN
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.
Asunto(s)
Quemaduras/metabolismo , Proteínas HSP70 de Choque Térmico/metabolismo , Rayos Láser , Luciferasas/metabolismo , Mediciones Luminiscentes/métodos , Piel/lesiones , Piel/metabolismo , Animales , Perfilación de la Expresión Génica/métodos , Genes Reporteros/genética , Luciferasas/genética , Ratones , Ratones Transgénicos/metabolismo , Células 3T3 NIH , Proteínas Recombinantes de Fusión/metabolismo , Distribución TisularRESUMEN
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.
Asunto(s)
Regeneración Ósea/fisiología , Terapia por Láser , Osteotomía/métodos , Tibia/cirugía , Cicatrización de Heridas/fisiología , Animales , Muerte Celular , Proliferación Celular , ADN/genética , Estudios de Seguimiento , Proteínas HSP70 de Choque Térmico/genética , Proteínas HSP70 de Choque Térmico/metabolismo , Inmunohistoquímica , Hibridación in Situ , Etiquetado Corte-Fin in Situ , Luciferasas/genética , Luciferasas/metabolismo , Masculino , Ratones , Ratones Transgénicos , Osteocitos/citología , Osteocitos/metabolismo , Molécula-1 de Adhesión Celular Endotelial de Plaqueta/genética , Molécula-1 de Adhesión Celular Endotelial de Plaqueta/metabolismo , Antígeno Nuclear de Célula en Proliferación/genética , Antígeno Nuclear de Célula en Proliferación/metabolismo , Receptores de Quimiocina/genética , Receptores de Quimiocina/metabolismo , Tibia/citología , Tibia/metabolismoRESUMEN
BACKGROUND AND OBJECTIVES: Investigations with a Mark-III free electron laser, tuned to 6.45 microm in wavelength have demonstrated minimal collateral damage and high ablation yield in ocular and neural tissues. While the use of mid-IR light produced by the free electron laser (FEL) has shown much promise for surgical applications, further advances are limited due the high costs of its use. Further investigation and widespread clinical use of six-micron radiation requires the development of an alternative laser source. In this research, we compared a Mark-III FEL and an Er:YAG pumped ZGP-OPO with respect to the effect of pulse duration on ablation efficiency and thermal damage on porcine cornea. STUDY DESIGN/MATERIALS AND METHODS: A five by seven grid of craters was made about the center of each cornea. Craters were made with a 60-microm spotsize with a 500-microm spacing. Ablation craters were made using 50 pulses per crater at approximately three times the ablation threshold (for water). Histological analysis was used to determine crater depth and thermal damage. RESULTS: The average zone of thermal damage at 6.1 microm was found to be 4.1 microm for the optical parametric oscillator (OPO) and 5.4 microm for the FEL. At 6.45 microm, the damaged zone was 7.2 microm for the OPO and 7.2 microm for the FEL. At 6.73 microm, the damaged zone was 6.3 microm for the OPO and 7.6 microm+/-0.3 microm for the FEL. CONCLUSIONS: The OPO caused similar or significantly less thermal damage in porcine cornea when compared with the FEL while generating significantly deeper craters. We determined that the ZGP-OPO has much promise as a bench-top replacement for the FEL for soft tissue ablation.
Asunto(s)
Córnea/cirugía , Terapia por Láser/instrumentación , Animales , Córnea/patología , Modelos Animales , PorcinosRESUMEN
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.
Asunto(s)
Glicerol , Aumento de la Imagen/métodos , Mediciones Luminiscentes/métodos , Microscopía Fluorescente/métodos , Piel/citología , Administración Tópica , Animales , Fármacos Dermatológicos/administración & dosificación , Femenino , Glicerol/administración & dosificación , Ratones , Reproducibilidad de los Resultados , Sensibilidad y Especificidad , Piel/efectos de los fármacosRESUMEN
Pulsed mid-infrared (6.45 microm) radiation has been shown to cut soft tissue with minimal collateral damage (<40 microm); however, the mechanism of ablation has not been elucidated to date. The goal of this research was to examine the role of the unique pulse structure of the Vanderbilt Mark-III free-electron laser (FEL) and its role in the efficient ablation of soft tissue with minimal collateral damage. The effect of the picosecond micropulse was examined by running the native FEL pulse structure through a pulse stretcher in order to increase the micropulse length from 1 ps up to approximately 200 ps. This allowed us to determine whether or not the picosecond train of micropulses played any role in the ablation process. The ablation threshold was determined for water and mouse dermis for each micropulse length. While the results of the analysis showed a statistically significant difference between 1 and 200 ps, the average per cent difference amounts to only 28% and is not proportional to the 200-fold drop in peak irradiance. The ablation efficiency was also measured on gelatin and mouse dermis for the different micropulse lengths. A small but statistically significant difference was observed between 1 and 200 ps, with the 200 ps pulse being more efficient on gelatin, and with the opposite trend for mouse dermis. We have shown that there is a small effect of micropulse duration of the FEL on the ablation process; however, this effect is negligible between 1 and 200 ps given that there is a 200-fold decrease in peak intensity. These results suggest that as we move forward in developing alternative laser sources for tissue ablation to replace the FEL, the picosecond micropulse structure is not a critical parameter that needs to be duplicated.
Asunto(s)
Procedimientos Quirúrgicos Dermatologicos , Electrones/uso terapéutico , Rayos Infrarrojos/uso terapéutico , Terapia por Láser/métodos , Traumatismos por Radiación/prevención & control , Radiometría/métodos , Piel/efectos de la radiación , Animales , Fraccionamiento de la Dosis de Radiación , Relación Dosis-Respuesta en la Radiación , Luz , Ratones , Dosis de Radiación , Piel/patologíaRESUMEN
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.
Asunto(s)
Córnea/patología , Córnea/efectos de la radiación , Terapia por Láser/métodos , Traumatismos por Radiación/prevención & control , Radiometría/métodos , Piel/patología , Piel/efectos de la radiación , Animales , Córnea/cirugía , Procedimientos Quirúrgicos Dermatologicos , Fraccionamiento de la Dosis de Radiación , Relación Dosis-Respuesta en la Radiación , Electrones/uso terapéutico , Rayos Infrarrojos/uso terapéutico , Luz , Ratones , Dosis de Radiación , Resultado del TratamientoRESUMEN
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 (lambda = 2.1 microm, taup = 250 micros, 30 pulses, 3 Hz), luciferase is produced on hsp70 activation and emits broad-spectrum 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.