Hyperthermia sensitization and proton beam triggered liposomal drug release for targeted tumor therapy.

PURPOSE: The objectives of this study were to: 1) determine if mild hyperthermia (40-42°C) can sensitize tumor cells for more effective proton beam radiotherapy (PBRT); 2) characterize the survival fraction of cells exposed to PBRT; and 3) characterize release of the drug doxorubicin (Dox) from low temperature sensitive liposomes (LTSLs) without exposure to mild hyperthermia in combination with PBRT. METHODS: Dox was actively loaded in LTSLs. A549 monolayer cells were incubated with 100-200 nM of Dox-LTSL (±mild hyperthermia). Cell irradiation (0-6 Gy) was performed by placing the cell culture plates inside a solid water phantom and using a clinical proton treatment beam with energy of 150 MeV. End points were survival fraction, radiation-mediated Dox release, and reactive oxygen species (ROS) production. RESULTS: Hyperthermia effectively sensitized cells for PBRT and lowered the cell survival fraction (SF) by an average of 9.5%. The combination of 100 nM Dox-LTSL and PBRT (1-6 Gy) achieved additive to synergistic response at various dose combinations. At higher radiation doses (>3 Gy), the SF in the Dox and Dox-LTSL groups was similar (~20%), even in the absence of hyperthermia. In addition, 30% of the Dox was released from LTSLs and a 1.3-1.6 fold increase in ROS level occurred compared to LTSL alone therapy. CONCLUSIONS: The combination of LTSLs and PBRT achieves additive to synergistic effect at various dose combinations in vitro. Concurrent PBRT and Dox-LTSL treatment significantly improved the cytotoxic outcomes of the treatment compared to PBRT and Dox chemotherapy without LTSLs. We hypothesize that PBRT may induce drug release from LTSL in the absence of hyperthermia.

Transrectal ultrasound-integrated spectral optical tomography of hypoxic progression of a regressing tumor in a canine prostate.

The objective of this study was to evaluate if transrectal optical tomography implemented at three wavelength bands for spectral detection could monitor changes of the hemoglobin oxygen saturation (StO2) in addition to those of the total hemoglobin concentration ([HbT]) in lesions of a canine prostate, including an induced tumor modeling canine prostate cancer. Near-infrared (NIR) optical tomography was integrated with ultrasound (US) for transrectal imaging. Multi-spectral detection at 705_nm, 785_nm and 808_nm rendered measurements of [HbT] and StO2. Canine transmissible venereal tumor (TVT) cells were injected into the right lobe of a dog's prostate gland, which had a pre-existing cyst in the left lobe. Longitudinal assessments of the prostate were performed weekly over a 63-day duration by NIR imaging concurrent with grey-scale and Doppler US. Ultrasonography revealed a bi-lobular tumor-mass regressing from day-49 to day-63. At day-49 this tumor-mass developed a hypoxic core that became larger and more intense by day-56 and expanded further by day-63. The tumor-mass presented a strong hyper-[HbT] feature on day-56 that was inconsistent with US-visualized blood flow. Histology confirmed two necrotic TVT foci within this tumor-mass. The cyst appeared to have a large anoxic-like interior that was greater in size than its ultrasonographically delineated lesion, and a weak lesional elevation of [HbT]. On day-56, the cyst presented a strong hyper-[HbT] feature consistent with US-resolved blood flow. Histology revealed acute and chronic hemorrhage in the periphery of the cyst. The NIR imaging features of two other TVT nodules and a metastatic lymph node were evaluated retrospectively. Transrectal US-integrated spectral optical tomography seems to enable longitudinal monitoring of intra-lesional oxygenation dynamics in addition to the hemoglobin content of lesions in the canine prostate.

Trans-rectal ultrasound-coupled spectral optical tomography of total hemoglobin concentration enhances assessment of the laterality and progression of a transmissible venereal tumor in canine prostate.

OBJECTIVES: To evaluate whether trans-rectal spectral optical tomography of total hemoglobin concentration (HbT) can image longitudinal and lateral developments of a canine transmissible venereal tumor (TVT) in a canine prostate. METHODS: A near-infrared (NIR) applicator was integrated with a trans-rectal ultrasound (TRUS) transducer to perform ultrasound (US)-coupled optical tomography of the canine prostate. Spectral detection at 785 and 830 nm enabled quantitation of HbT. Canine TVT cells were injected into the right lobe of a dog's prostate gland. Longitudinal imaging assessment of the post-injection prostate was performed by coupled US/NIR imaging over a 45-day duration. RESULTS: By day 7, NIR indicated TVT infiltration in the noninjected left prostatic lobe with the gray-scale US indistinct. By day 31, both NIR and gray-scale US revealed more widespread TVT involvement in the left than in the right lobe, as well as an extensive TVT mass in the caudal aspect of the gland, of which the peak HbT increased 3-fold and the mass volume grew exponentially over the 45-day duration. Increased blood supply to the mass was also observed on Doppler US. CONCLUSIONS: TRUS-coupled spectral optical tomography enhances assessment of the laterality and progression of prostate tumor compared with using gray-scale and Doppler TRUS.

Tissue levels, histologic changes and plasma pharmacokinetics of meta-Tetra (hydroxyphenyl) chlorin (mTHPC) in the cat.

The biodistribution and pharmacokinetics of meta-tetra(hydroxyphenyl)chlorin (mTHPC)(1) have been documented in humans, rats, dogs and rabbits. It has been demonstrated to be an effective photodynamic therapy agent for treatment of squamous cell carcinoma. Squamous cell carcinoma is a common feline neoplasm, causing significant morbidity and mortality in the feline population. The association between ultraviolet radiation exposure and occurrence of this neoplasm in the cat provides a useful model for the study of human cutaneous squamous cell carcinoma. In this study, we document the biodistribution, pharmacokinetics and toxicity of mTHPC in a group of normal cats. Four groups of cats were given the drug intravenously at dosages of 0, 0.15, 0.30 and 0.60 mg/kg. mTHPC levels were measured in plasma and tissues at 0, 24, 48, 72, 96 and 336 h after drug administration. Additionally, plasma samples were collected at 1 and 6 h post-injection and analysed. Biodistribution and pharmacokinetics of mTHPC in cats mirrors that in other animal species. There were no clinical or pathological changes associated with administration of the drug. The biodistribution and pharmacokinetics of mTHPC in cats mirrors that in other species studied. There were no clinical or pathological changes attributable to administration of the drug at the doses administered. mTHPC may be a useful photodynamic therapy drug in cats.

Failure of holmium:yttrium-aluminum-garnet laser lithotripsy in two horses with calculi in the urinary bladder.

Holmium:yttrium-aluminum-garnet (Ho:YAG) laser lithotripsy was attempted in a mare and a gelding with calculi in the urinary bladder. The procedure was unsuccessful in producing adequate fragmentation of the calculi. In the gelding, pulsed dye laser lithotripsy was subsequently used to fragment the urolith. Manual removal of the urolith via the urethra was performed in the mare.

Laser lithotripsy of a urethral calculus via ischial urethrotomy in a steer.

A steer examined because of obstructive urolithiasis and urethral rupture underwent laser lithotripsy, using a chromium-thulium-holmium:yttrium-aluminum-garnet (Ho:YAG) laser inserted through an ischial urethrotomy. Procedures were performed with caudal epidural anesthesia. Six months after surgery, the urethra was patent with no clinical evidence of urethral stricture or fistula. Ischial urethrotomy provided rapid access to the bladder for catheterization and to the obstructive urolith for lithotripsy. Laser lithotripsy was a rapid and effective means of urolith removal in this steer.

Evaluation of carbon dioxide laser and conventional incisional techniques for resection of soft palates in brachycephalic dogs.

OBJECTIVE: To compare clinical outcome, healing, and effect of tracheostomy in conventional incisional and carbon dioxide (CO2) laser techniques for resection of soft palates in brachycephalic dogs. DESIGN: Prospective randomized trial. ANIMALS: 20 adult brachycephalic dogs. METHODS: Dogs were randomly allocated into 4 groups, and 1 of the following was performed: palate resection by use of a CO2 laser; incisional palate resection and closure with suture; and palate resection by use of a C02 laser or incision with tracheostomy. A clinical score for respiratory function was assigned to each dog at 0, 2, 8, 16, and 24 hours. Biopsy specimens of incision sites obtained at days 0, 3, 7, and 14 were examined. Data were analyzed to determine the effects of technique on clinical and histologic outcome. RESULTS: Mean surgical time for laser (309 seconds) was significantly shorter than for sharp dissection (744 seconds). Surgical technique significantly affected clinical scores at 3 of the 5 postoperative time points, but differences were not clinically apparent. Tracheostomy significantly affected clinical scores at 3 of 5 postoperative time points. After tracheostomy tube removal, clinical scores were similar to those of dogs without tracheostomies. Inflammation, necrosis, and ulceration were evident in all groups at day 3; these lesions had almost resolved by day 14. Most complications were associated with tracheostomy. CONCLUSIONS AND CLINICAL RELEVANCE: Clinical outcomes appear to be similar with the laser and incisional techniques. Regarding surgical time and ease, laser resection of the soft palate appears advantageous. Tracheostomy is not warranted in dogs that have uncomplicated surgeries and recoveries.

Urinary bladder transitional cell papilloma in a shorthorn heifer.

A 12-month-old shorthorn heifer was presented for pollakiuria of 4 months' duration. Urinary bladder transitional cell papilloma was diagnosed. The heifer had no exposure to bracken fern and no papillomavirus or bacterium was demonstrated. Laser surgery was used in an attempt to debulk the mass.

Comparison of a high power diode laser with the Nd:YAG laser using in situ wound strength analysis of healing cutaneous incisions.

BACKGROUND AND OBJECTIVE: The laser-tissue interaction of a high power semiconductor diode laser was compared to the continuous wave neodymium yttrium aluminum garnet (Nd:YAG) laser by evaluating primary wound healing of cutaneous incisions in rats. STUDY DESIGN/MATERIALS AND METHODS: Full thickness incisions were made in rat skin using a diode laser (805 nm, 10 W, contact mode), an Nd:YAG laser (1,064 nm, 10 W, contact mode), and a stainless steel scalpel blade (control). In situ wound breaking strength measurements were obtained at 7, 14, and 21 days using a specially designed tensiometer. Cross sectional area of non-disrupted wounds was calculated in two groups prior to testing to allow for calculation of tensile strength. Blinded histopathologic analysis was also performed. RESULTS: Analysis of variance (P < or = 0.05) was used to determine differences in breaking strengths and tensile strengths due to incision method. There was no significant difference in the breaking strengths (group 1) or tensile strengths (groups 2 and 3) of the diode and Nd:YAG laser incisions. As predicted, breaking strengths and tensile strengths of scalpel blade incisions were significantly greater than those of incisions made with laser energy. Histopathologic evaluation revealed that through day 14, the degree of inflammation and collagen production was similar for diode and Nd:YAG laser incisions. Laser incisions had greater inflammation and a lag in fibroblast invasion and collagen production compared with scalpel incisions. By day 21, all incisions were similar in fibroblast population and collagen production, but laser incisions had slightly more inflammation than scalpel incisions. CONCLUSION: In the primary wound healing model described, the tissue effect, cellular response, and development of wound strength were essentially the same for the high power diode laser at 10 W and the Nd:YAG laser at 10 W.

Use of the holmium yttrium aluminum garnet laser for percutaneous thoracolumbar intervertebral disk ablation in dogs.

OBJECTIVE-To evaluate a laser ablation-technique for treatment of thoracolumbar intervertebral disk disease in dogs. DESIGN-Prospective case series. ANIMALS-33 dogs with signs of recurrent back pain associated with intervertebral disk disease after the initial episode had been managed conservatively for a minimum of 2 weeks. PROCEDURE-Spinal needles were placed percutaneously through the annulus fibroses to permit delivery of an optical fiber into the nucleus pulposes of thoracolumbar intervertebral disks T10-11 through L3-4. Fluoroscopy was used to guide needle placement. Holmium yttrium aluminum garnet laser energy then was used to ablate the contents of each selected intervertebral disk. Intervals from time of treatment to time of assessment ranged from 3 to 114 weeks. RESULTS-All dogs recovered without complication. Results of follow-up radiography and histologic evaluation indicated that percutaneous holmium yttrium aluminum garnet laser ablation reduces the volume of nucleus pulposus in treated disks. CLINICAL IMPLICATIONS-Used as a clinical treatment and prophylactically, this minimally invasive procedure should prevent further extrusion of partially herniated disks and should reduce the chances of subsequent herniation of disks at other treatment sites.

Photothermal effects on murine mammary tumors using indocyanine green and an 808-nm diode laser: an in vivo efficacy study.

Murine mammary tumors were treated using indocyanine green and an 808 nm diode laser, and the in vivo chromophore-enhanced photothermal effects on the tumor burden and on tumor rat survival were investigated. The power of the laser was selected in the range of 5-10 W, and irradiation duration 3-5 min. One percent aqueous indocyanine green solution in a volume of 100-200 microliters was administered in situ, either acutely or 24 h prior to the treatment. The photothermal interaction was apparent under all our treatment conditions with a well-defined spatial containment in this study and the tumor growth was slowed after treatment. The post-treatment observation showed tumor recurrence and metastasis; no long-term survival was achieved with the single application of laser in conjunction with indocyanine green. Our results pose a question on the efficacy of the photothermal interaction even though tumor cell destruction can be achieved in a large and controlled scale. However, this highly selective photothermal impact on the tumor tissue did suggest that this method be applied repeatedly to be more effective and be used as the precursor of other modalities, such as chemotherapy, radiation therapy, immunotherapy, and surgery.

Chromophore-enhanced in vivo tumor cell destruction using an 808-nm diode laser.

Rat mammary tumors were treated using an 808-nm diode laser in a power range of 3-15 W. Photothermolysis was selectively enhanced by the chromophore indocyanine green (ICG), which has an absorption peak corresponding to the laser wavelength. ICG, injected into neoplastic tissues 24 h before laser exposure, was retained in sufficient quantity to produce a strong photothermal reaction. With appropriate laser power and adequate irradiation duration, laser energy could inflict severe photothermal damage to the entire targeted tumor tissue while leaving the skin and other interdicted tissue undamaged. Higher laser powers (10-15 W) produced more surface damage that limited light transmission and as a result gave rise to reduced regions of thermal destruction. Post-treatment observation revealed the survival of numerous tumor cells. This finding questions the long term efficacy of the photothermal effect of a single treatment using the combination of the ICG and the diode laser, particularly in the absence of other modalities.

Chromophore-enhanced laser-tumor tissue photothermal interaction using an 808-nm diode laser.

A diode laser was used to irradiate tumor tissue, with indocyanine green as the chromophore. The 808-nm wavelength radiation falls within the absorption peak of the chromophore (about 780 nm). The preliminary results in this report revealed clear and significant coupling of this laser and indocyanine green in laser-tissue photothermal interaction. The chromophore targeted tissue showed laser damage while peripheral tissues remained intact. Without the chromophore, this laser inflicted no apparent tissue damage in the non-contact mode with irradiance up to 1755 J/cm2.

Mechanisms of biliary stone fragmentation using the Ho:YAG laser.

We have investigated the fragmentation of gallstones using the pulsed Ho:YAG laser, comparing it to lithotripsy using the visible pulsed-dye laser. We find that the physical mechanisms of stone fragmentation appear to be quite different in the two cases. Using high-speed photography, measurement of acoustic transients, time-resolved optical emission spectroscopy, and direct microscopic observation, we have analyzed the interaction of the Ho:YAG laser with both water and gallstones. We propose a new model in which fragmentation begins with absorption of the laser light by the stone surface. This is followed by melting and ejection of stone material, which is then swept away by the vapor bubble formed by the absorption of the Ho:YAG laser light by water. This model is in excellent agreement with our experimental observations, and differs substantially from the model developed by Teng et al. for laser lithotripsy using the visible pulsed-dye laser.

Synthesis of potential metabolites of ethyl (E)-4-[2-(3,4-dihydro-4,4-dimethyl-2H-1-benzopyran-6-yl)-1-propenyl] benzoate.

Potential metabolites of ethyl (E)-4-[2-(3,4-dihydro-4,4-dimethyl-2H-1-benzopyran-6-yl)-1-propenyl] benzoate were synthesized. The new compounds include ethyl 3-[3,4-dihydro-4,4-dimethyl-2H-1-benzopyran-6-yl]crotonate, 3-[3,4-dihydro-4,4-dimethyl-1H-1-benzopyran-6-yl]crotonic acid, 3,4-dihydro-4,4-dimethyl-2H-1-benzopyran-6-carboxylic acid, 4-[3,4-dihydro-4,4-dimethyl-2H-1-benzopyran-6-yl]delta 2-butenolide, ethyl (E)-4-[3,4-dihydro-4,4-dimethyl-2H-1-benzopyran-6-yl)-3-hydroxy-1- propenyl]benzoate, ethyl (E)-4-[2-(3,4-dihydro-4,4-dimethyl-2H-1-benzopyran-6-yl)-2-propenal] benzoate, and ethyl (E)-4-[2-(3,4-dihydro-4,4-dimethyl-2H-1-benzopyran-6-yl)-2-propenoic+ ++ acid]benzoate. Stereospecific oxidizing reagents and/or conditions were developed for these sensitive systems and include the use of SeO2, Clorox bleach, activated MnO2, and NaClO2 in the presence of resorcinol as a chlorine scavenger.

Effects of holmium:YAG laser on equine articular cartilage and subchondral bone adjacent to traumatic lesions: a histopathological assessment.

This study was performed to investigate the effects of holmium:yttrium-aluminum-garnet (Ho:YAG) laser energy on articular cartilage and subchondral bone adjacent to traumatically created cartilage lesions in a continuous weight-bearing model. The 2.1-microns wavelength was delivered efficiently and precisely in hand-controlled contact and near-contact hard tissue arthroscopic surgery in a saline medium. Bilateral arthroscopy was performed on normal antebrachiocarpal and intercarpal joints of four adult horses. One hundred twenty traumatic lesions were created on three weight-bearing articular surfaces with a knife, curet, or motorized burr. Depths of the lesions were partial and full thickness. Configurations of the lesions were lacerations, scrapes, and craters. Left limbs were used as controls. Right limb lesions were treated with various intensities of laser energy: 0.1. 0.16, and 0.2 J. Animals were killed at intervals of 1, 3, and 8 weeks. Gross microscopic anatomy was documented, and tissue sections were subjected to blind review by a pathologist. Mankin grading for cellularity and proteoglycan content was used to qualitatively evaluate cartilage response. Cartilage adjacent to all lesions exposed to laser energy had better cellularity and proteoglycan content (p < 0.05) than did corresponding controls by Mankin grading. Mankin grades for levels of 0.16 and 0.2 J were lower (p < 0.05) than were those of controls or lesions exposed to 0.1 J. There was loss of cellularity and necrosis in subchondral bone at higher energy levels (0.16 and 0.2 J). This study examined the effects of Ho:YAG laser on articular cartilage and subchondral bone in terms of depth of damage and healing response over time in a weight-bearing model. Additional research to define dose-response curves for Ho:YAG laser treatment of cartilage and subchondral bone are indicated.

A detailed colostomy procedure and its application to quantify water and nitrogen balance and urine contribution to thermobalance in broilers exposed to thermoneutral and heat-distressed environments.

A surgical procedure for the separation of avian urinary and fecal excrement, in birds as young as 3.5 wk of age, was developed and used to quantify ambient temperature effects on urine production and composition. The colostomized broilers were used in two experiments to estimate urine production, osmolality, and contribution to water, nitrogen, and thermobalance of birds exposed to thermoneutral (24 C, TN) and heat-distressed (35 C, HD) environments. Urine production averaged over three 12-h periods was greater (P < .05), at 101 versus 51 mL/12 h per kilogram of BW and osmolality lower (P < .05) at 142 versus 220 mOsm/kg urine during HD and TN, respectively, in Experiment 1; and averaged over one 12-h period 82 versus 32 mL/12 h per kilogram of BW and 136 versus 208 mOsm/kg in Experiment 2 for HD and TN, respectively. In Experiment 1, urine production per milliliter of water consumed was increased (P < .05) by HD during Periods 2 and 3 even though in both experiments the values were similar during the first 12 h. Both urinary nitrogen and osmolar losses were increased (P < .05) by 50 and 89.2%, respectively, during HD, but without effect on fecal nitrogen. Evaporative cooling of broilers was increased (P < .05) by 154% during HD. These studies provide a well-defined colostomy procedure and suggest that broiler urine production and composition as well as overall thermobalance, water, and nitrogen balance are strongly impacted by acute HD.

Use of the neodymium:yttrium-aluminum-garnet laser for treatment of squamous cell carcinoma of the nasal planum in a cat.

A mature castrated male domestic shorthair cat was referred for treatment of an excoriated, ulcerated area on the nasal planum. Undifferentiated squamous cell carcinoma was diagnosed. Surgical resection of the nasal planum was not an option, so the lesion was treated 4 times with neodymium:yttrium-aluminum-garnet laser. Laser surgery results in uniform photovaporization of large volumes of tissue. Although treatment with laser does not yield tissue specimens suitable for histologic evaluation, it can result in a cosmetically suitable appearance and can extend the predicted life span. A diagnosis of undifferentiated squamous cell carcinoma has a guarded prognosis: however, the cat of this report survived more than 18 months.

Comparison of holmium and flashlamp pumped dye lasers for use in lithotripsy of biliary calculi.

The characteristics of laser lithotripsy of biliary calculi are compared for a flashlamp pumped dye laser (lambda = 640 nm) and a Cr:Tm:Ho-YAG laser (lambda = 2.1 microns). Data on fragmentation efficiency with respect to laser power and pulse repetition rate are presented for different types of stones. It is shown that both lasers can produce effective stone fragmentation. The laser power required for efficient fragmentation characteristics is significantly less for the visible wavelength laser. However, the problems associated with damage to the fiber tips of the delivery system during operation were found to be less with the near infrared wavelength. The laser power for efficient fragmentation with the dye laser varies significantly for different types of stones while the power for efficient fragmentation with the holmium laser is the same for all stones.

Corrosion potential of steel bird shot in dogs.

Each year many dogs are accidentally or purposely wounded with shotguns. When lead pellets were used exclusively in the past, clinical problems from chronically embedded shot seldom developed except for rare cases of lead toxicosis. However, because expended lead shot ingested unintentionally by waterfowl and other avian species is fatal, the US Fish and Wildlife Service mandated exclusive use of steel shot for waterfowl hunting beginning in 1991. To discover the effects of implanted steel shot in a biological system, in vitro and in vivo studies were performed. Severe surface corrosion was evident when steel shot was placed in physiologic saline solution and sterile canine plasma. Eight laboratory dogs were surgically implanted with sterile steel shot in various superficial locations for intervals of 2 to 26 weeks. Corrosion of implants and tissue inflammation was observed in all biopsy specimens examined. It has been shown that steel shot embedded in tissues will corrode and result in a severe inflammatory response. If the accompanying inflammation is complicated by bacterial contamination, foreign body reactions resulting in infected, draining tracts could develop. Veterinarians and dog owners should be aware that treatment and prognosis for wounds caused by steel shot may differ from those for similar wounds caused by lead shot.