Development of a fluorescence detection system using optical parametric oscillator (OPO) laser excitation for in vivo diagnosis.

In this work, the development and applications of a fluorescence detection system using optical parametric oscillator (OPO) laser excitation for in vivo disease diagnosis including oral carcinoma are described. The optical diagnosis system was based on an OPO laser for multi-wavelength excitation and time-resolved detection. The pulsed Nd-YAG-pumped OPO laser system (6 ns, 20 Hz) is compact and has a rapid, broad, and uniform tuning range. Time-gated detection of intensified charge-coupled device (ICCD) making use of external triggering was used to effectively eliminate the laser scattering and contribute to the highly sensitive in vivo measurements. Artificial tissue-simulating phantoms consisting of polystyrene microspheres and tissue fluorophores were tested to optimize the gating parameters. 51-ns gate width and 39-ns gate delays were determined to be the optimal parameters for sensitive detection. In vivo measurements with the optical diagnosis system were applied to esophagus, stomach, and small intestine using an endoscope in canine animal studies. The rapid tuning capability of the optical diagnosis system contributed greatly to the optimization of wavelength for the observation of porphyrin in the small intestine. When the small intestine was thoroughly washed with water, the emission band which corresponds to porphyrin disappeared. Based on this observation, it was concluded that the detected signal was yielded by porphyrin-containing bile secretion. Also, multispectral analyses using multiple excitations from 415 to 480 nm at 5 nm intervals confirmed the porphyrin detection in the small intestine. The optical diagnosis system was also applied to the detection of human xenograft of oral carcinoma in mice using 5-aminolevulinic acid (5-ALA) which is a photodynamic therapy (PDT) drug. Significant differences in protoporphyrin IX fluorescence intensity between normal and tumor tissue could be obtained 2 hours after the injection of 5-ALA into mice due to the preferential accumulation of 5-ALA in tumors. Results reported herein demonstrate potential capabilities of the LIF-OPO system for in vivo disease diagnosis.

Evaluation of heart time-activity curves as a predictor of hepatic extraction of 99mTc-mebrofenin in dogs.

In this study, heart time-activity curve, created following intravenous injection of 99mTc-mebrofenin were used to quantify hepatic function in normal dogs and dogs with induced hepatic parenchymal cell damage. The results were compared to a direct measurement of hepatic extraction following mesenteric venous injection of 99mTc-mebrofenin. The heart time-activity curves were normalized and the area under the curve from 0-30 minutes and 0-60 minutes were determined. In addition, the half-time clearance rate of the heart time-activity curve was analyzed using a two-compartment model. Linear regression analysis was used to describe the relationship between the area under the normalized heart time-activity curve and hepatic extraction. There was good correlation between the area under the normalized heart time-activity curve and hepatic extraction. The best correlation was obtained from the 0-30 minute data (r2 = 0.92). A formula for calculating hepatic extraction was derived using linear regression analysis: Hepatic extraction = 1.092 - (0.0000308 x AUC0-30 minutes). There was good correlation between the half-time clearance rates from the heart time-activity curve and hepatic extraction. The best correlation was between the fast phase half-time clearance and hepatic extraction (r2 = 0.88). The area under a normalized heart time-activity curve can be used as a simple alternative to deconvolutional analysis for the determination of hepatic extraction as a measure of hepatic parenchymal cell function in the dog.

Cortisol concentrations following stimulation of healthy and adrenopathic dogs with two doses of tetracosactrin.

A prospective study was undertaken to compare intravenous tetracosactrin at doses of 5 microg/kg and 250 microg for diagnosing hyperadrenocorticism in dogs. Both healthy dogs and dogs with pituitary-dependent hyperadrenocorticism were evaluated with the two doses of the drug, and serum cortisol concentrations were compared at 60 minutes post-stimulation. Some of the dogs had additional samples taken at 90 and 120 minutes. For four dogs with hyperadrenocorticism, timed samples were also obtained at 150, 180 and 240 minutes post-injection. Cortisol concentrations 60 minutes after stimulation with either 5 microg/kg or 250 microg intravenous tetracosactrin were similar for both healthy dogs and dogs with hyperadrenocorticism. The lower dose can therefore be used for diagnosing hyperadrenocorticism in dogs.

Evaluation of plasma time-activity curves of technetium-99m-mebrofenin for measurement of hepatic function in dogs.

In this study, plasma time-activity curves of 99mTc-mebrofenin were used to quantify hepatic function in dogs before and after induction of hepatic damage using a hepatotoxic agent. Nine dogs were determined to be healthy on the basis of physical examination, laboratory data and hepatic imaging. Plasma samples were collected 1, 3, 5, 7, 9, 15, 20, 30, 40, 50, and 60 minutes following a peripheral venous injection of 111-222 MBq (3-6 mCi) of 99mTc-mebrofenin. The area under the plasma time-activity curve (AUC) was calculated using two different methods and compared to direct measurement of the hepatic extraction efficiency. First pass hepatic extraction efficiency of 99mTc-mebrofenin was calculated from differential equation analysis of a two-compartment model following mesenteric venous injection of the radiopharmaceutical. In 7 of the original 9 dogs and 2 additional healthy dogs, plasma clearance and hepatic extraction efficiency determination were repeated following induction of hepatic injury by thiacetarsamide (3 mg/kg IV twice daily for 1 day). In one additional dog, hepatic injury was induced using carbon tetrachloride (0.3 ml/kg IP). Plasma time-activity curves of 99mTc-mebrofenin had kinetics of a two compartment model. Area under the curve was highly correlated with hepatic extraction efficiency. The AUC integrated from 1-60 minutes (AUC60) had the best correlation with hepatic extraction efficiency (r2 = 0.978, p < 0.001). A formula for calculation of hepatic extraction efficiency was derived using linear regression analysis: hepatic extraction efficiency = 105.583 - 3.099 x 10(5) x AUC60. Plasma clearance of a peripheral venous injection of 99mTc-mebrofenin is a simple, non-invasive, convenient method to quantify hepatic function which can be performed without a gamma camera.

Hiatal hernia and diaphragmatic eventration in a leopard (Panthera pardus).

A 1-yr-old male leopard (Panthera pardus) presented for intermittent anorexia, emaciation, and generalized muscle wasting. Plain radiographs, ultrasonography, and esophageal endoscopy led to a diagnosis of diaphragmatic eventration with probable concurrent hiatal hernia. An exploratory laparotomy confirmed both diagnoses, and surgical repair and stabilization were performed. After surgery, the leopard was maintained on small liquid meals for 4 days, with a gradual return to normal diet over 2 wk. By 4 wk after surgery, the leopard was eating well and gaining weight, and it showed no recurrence of clinical signs for 2 yr subsequently, becoming mildly obese.

Peritoneal effusion in cats: 65 cases (1981-1997).

OBJECTIVE: To determine signalment, diagnoses, presence of effusions in multiple sites, and outcome in cats with peritoneal effusion. DESIGN: Retrospective case series. ANIMALS: 65 cats. PROCEDURE: Medical records from 1981 to 1997 were reviewed to obtain information on cats with peritoneal effusion identified on physical examination, radiographs, abdominal ultrasonograms, or at necropsy. RESULTS: Conditions most commonly associated with peritoneal effusion in cats, in order of frequency, were cardiovascular disease, neoplasia, hepatic disease, renal disease, feline infectious peritonitis, peritonitis attributable to other causes, and urinary tract trauma. Dilated cardiomyopathy (DCM) was the most common disease associated with peritoneal effusion; however, DCM was diagnosed in most of these cats before taurine deficiency was found to be a primary cause of this form of cardiomyopathy in cats. Neoplasia was the most common cause after 1987. Right-sided congestive heart failure was the most commonly associated disorder in cats < 1 year old, whereas neoplastic disease was more common with increasing age. Most effusions were detected during the initial physical examination and were modified transudates. Peritoneal effusion was commonly accompanied by fluid accumulation elsewhere, particularly pleural effusion. The prognosis for a cat with abdominal effusion in this study was poor (mean survival time, 21 days; range, 1 to 350 days; median, 2.5 days). CLINICAL IMPLICATIONS: The primary differential diagnosis for peritoneal effusion in cats is neoplastic disease in older cats and right-sided heart failure in kittens. Diseases associated with peritoneal effusion generally have poor prognoses.

Validation of deconvolutional analysis for the measurement of hepatic function in dogs with toxic-induced liver disease.

The extraction of the hepatobiliary radiopharmaceutical 99mTc-mebrofenin (Choletec) by the liver can be used to evaluate the severity of hepatocellular disease. The hepatic parenchymal cells extract mebrofenin from the blood by the same active transport mechanism as bilirubin. The ability of the liver to extract 99mTc-mebrofenin is a measure of hepatic parenchymal cell function. In this study, we induced hepatocellular disease by administration of a hepatotoxic drug and compared a direct method of determining the hepatic extraction of 99mTc-mebrofenin to hepatic extraction fraction derived from deconvolutional analysis. We also compared both methods of calculating the hepatic extraction of 99mTc-mebrofenin to liver histopathology. Hepatic extraction fraction derived from deconvolutional analysis correlated very well to the direct measurement technique (R=0.922, p < 0.001). Both methods of determining hepatic extraction correlated well to quantitative histopathology, having the same correlation coefficient and p values. (R=-0.833, p=0.003). As the hepatic extraction 99mTc-mebrofenin decreased, the severity of the histopathologic lesions of the liver increased in a linear fashion. There was a significant correlation of the hepatic excretion T1/2 to quantitative histopathology (R=0.949, p < 0.001). The hepatic excretion T1/2 increased as the severity of the histopathologic lesions of the liver increased. Hepatic extraction (HEF) and excretion of 99mTc-mebrofenin are good predictors of the severity of hepatocellular damage in toxic induced liver disease. This study helps validate the premise that HEF derived from deconvolutional analysis is a good predictor of the actual first pass hepatic extraction of 99mTc-mebrofenin.

Hepatic extraction efficiency of technetium-99m-mebrofenin in the dog with toxic-induced acute liver disease.

UNLABELLED: Extraction of the hepatobiliary radiopharmaceutical 99mTc-N-(3-bromo-2,4,6-trimethyacetanilide) iminodiacetic acid (mebrofenin; Choletec, Squibb Diagnostic, Princeton, NJ) by the liver may be used as an index of hepatocellular function. The hepatic parenchymal cells extract mebrofenin from the blood using the same active transport mechanism as bilirubin. METHODS: In this study, we induced hepatocellular disease by administering a hepatotoxic drug and compared the hepatic extraction efficiency (HEE), measured directly from an afferent injection of 99mTc-mebrofenin, to quantitative histopathology and to serum biochemistry analysis. RESULTS: The baseline HEE was 95.9% +/- 2.71% (mean +/- s.d.). Dogs that were affected by the hepatotoxic drug had reduced HEE. HEE correlated well to the severity of histologic lesions (r = -0.83, p = 0.003). HEE also correlated well to the increases in the activities of alanine aminotransferase (ALT; r = -0.85, p = 0.002) and aspartate aminotransferase (AST; r = -0.89, p = <0.001), the concentration of fasting bile acid (r = -0.97, p = <0.001), bilirubin (r = -0.92, p = <0.001) and, to a lesser degree, to the activities of alkaline phosphatase (Alk Phos; r = -0.73, p = 0.016). HEE had higher correlation coefficients to the serum biochemistry analysis than did the quantitative liver histopathology. CONCLUSION: Hepatic extraction of 99mTc-mebrofenin is a good predictor of the severity of hepatocellular damage in toxic-induced liver disease.

Plastinated canine gastrointestinal tracts used to facilitate teaching of endoscopic technique and anatomy.

Plastinated specimens, when prepared with a design for endoscopic use, can serve as a practical model for teaching. Intact alimentary canals were excised from fresh canine cadayers. Cannulas in excess of the intended endoscope size (9.6 mm diameter) were placed in restrictive openings [cardiac ostium (ostium cardiacum), pyloric ostium (ostium pyloricum) and cecocolic orifice (ostium cecocolicum)]. These cannulas allowed ingesta to be removed and maintained adequate diameters for endoscoping. After flushing out the gastrointestinal contents, specimens were formaldehyde-fixed overnight in a dilated anatomical conformation. Prior to S10/S3 impregnation, fixative was flushed from the specimens and they were dehydrated in acetone. After impregnation, slow cure (elongation of S3 molecules at room temperature) was allowed to proceed for approximately 1 week. The gastrointestinal tracts were maintained in a dilated conformation by a positive pressure air flow. When polymer seepage was minimal, they were cured using small quantities of S6 (final curing agent). The curing agent was contained around the specimen by enclosing the specimens in plastic bags. The plastinated specimens retain their dilated anatomical conformation, and may be used to teach both endoscopic technique and gastrointestinal anatomy.

Effusive-constrictive pericardial disease secondary to osseous metaplasia of the pericardium in a dog.

Osseous metaplasia of the pericardium causing effusive-constrictive pericardial disease has not, to our knowledge, been reported in dogs. Clinical signs of right-sided congestive heart failure prompted examination of the dog of this report. Documented causes of constrictive pericardial disease in dogs include trauma and actinomycotic, mycobacterial, and fungal infections. These causes were ruled out in this dog. Immune-mediated disorders, as have been reported in people, also were considered unlikely on the basis of test results. It was concluded that this dog had idiopathic osseous metaplasia of the pericardium and pleura. Signs of right-sided congestive heart failure resolved after subtotal pericardiectomy was performed.

Hepatic extraction efficiency and excretion rate of technetium-99m-mebrofenin in dogs.

UNLABELLED: Quantitative hepatobiliary scintigraphy aids in the diagnosis of hepatic disease. Two scintigraphic parameters that have great value in discriminating between hepatocellular and biliary disease are hepatic extraction fraction (HEF), which is a measure of the hepatic extraction efficiency (HEE), and hepatic excretion rate. It is generally accepted that hepatic extraction fraction is normally 100%, but a review of the literature provided little information on the actual HEF of 99mTc-mebrofenin. METHODS: We determined the HEE of 99mTc-mebrofenin in nine normal dogs after direct injection into the afferent hepatic vasculature using a two-compartment model. The forward and reverse rate constants for the two-compartment model were solved by a simple graphic approach and a more complex numerical approach using a nonlinear least squares algorthm. The HEEs were determined using both methods. RESULTS: The HEE for the graphic and numerical methods of analysis were not significantly different and were calculated to be 92.2 +/- 4.75% (mean +/- s.d.) and 91.2 +/- 4.44% (mean +/- s.d.) by each method, respectively. The half-time clearance of 99mTc-mebrofenin was 19.10 +/- 4.86 min (mean +/- s.d.). CONCLUSION: This study validates the assumption that the normal HEE of 99mTc-mebrofenin is nearly 100%, barring species differences.

Balloon photodynamic therapy of esophageal cancer: effect of increasing balloon size.

BACKGROUND AND OBJECTIVE: Photodynamic therapy is currently being used to treat various malignancies including esophageal cancer. The effect of photodynamic therapy depends upon the concentration of photosensitizing drug, light energy delivered to tissue, and the presence of oxygen in the targeted tissue. We have found that an esophageal centering balloon improves light delivery to esophageal mucosa. However, balloon pressure on esophageal mucosa could possibly reduce mucosal blood flow and oxygenation, therefore reducing the effect of photodynamic therapy. This study was conducted to investigate the effect of balloon pressure on the esophageal wall during photodynamic therapy in the canine esophageal model. STUDY DESIGN/MATERIALS AND METHODS: Studies were performed in the canine esophagus of ten animals to investigate whether increasing the size of the centering balloon, and hence the pressure on esophageal mucosa, would alter the tissue effect of PDT. Porfimer sodium 4 mg/Kg was administered and 630 nm light was delivered via a 1 cm diffuser located in the center of a 360 degrees 2 cm windowed balloon. Mucosal light measurements were made to ascertain equivalent mucosal light dosing of approximately 25 J/cm2. Endoscopic and necropsy findings obtained following photodynamic therapy with 25 mm, 33 mm, and 35 mm balloons were compared. RESULTS: In larger dogs (groups A and B), increasing the size of the esophageal centering balloon from a 25-33 mm size did not result in an overly tight fit nor was the increase associated with significant change in the PDT effect. In contrast, increasing the balloon size to 35 mm in smaller dogs (group C) resulted in a tight fit of the balloon in the esophagus and in significant reduction in the PDT effect on mucosal damage when mucosal equivalent light dose was administered during photodynamic therapy in the canine esophageal model. CONCLUSION: Increasing centering balloon size resulted in reduced tissue damage when mucosal equivalent light dose was administered during photodynamic therapy in the canine esophageal model. Proper sizing of centering balloons will be necessary for balloon PDT of esophageal mucosal dysplasia or cancer in humans.

Use of a low-profile gastrostomy device for administering nutrients in two dogs.

Percutaneous endoscopic gastrostomy (PEG) tubes are an excellent means of providing nutritional support via the enteral route. Most problems with PEG tubes are associated with their maintenance. A low-profile feeding device has been developed for use in human beings to replace standard PEG tubes in situations in which long-term nutritional support is necessary. We adapted a low-profile feeding device for use in 2 dogs. The device offered the advantage of being easier to maintain than standard PEG tubes. It appeared to have been well tolerated. Its low-profile design is likely to result in few problems resulting from an animal chewing on the device or from accidental dislodgment.

Photodynamic therapy for esophageal cancer using a 180 degrees windowed esophageal balloon.

Although delivery of uniform circumferential light is desirable during photodynamic therapy of advanced esophageal cancer in humans, early esophageal cancer may need only targeted treatment. Studies were performed in the canine esophagus of eight animals to investigate whether use of a "windowed" (shaded) centering balloon would improve targeted illumination of esophageal mucosa for photodynamic therapy. Shaded balloons were developed with a 2-cm-long, 360 degrees or 180 degrees clear "window." Photofrin 4 mg/Kg was used as the photosensitizer. Light at 630 nm was delivered at 300 J/cm or 600 J/cm. Isotropic probes placed on the balloon wall allowed real-time measurement and verification of relatively uniform light doses delivered to esophageal mucosa during balloon photodynamic therapy. With the windowed balloon, targeted delivery of photodynamic therapy was possible. Using the 180 degrees balloon, mucosa exposed to illumination was destroyed, whereas mucosa protected from light by the balloon shading was undamaged. Healing was complete and strictures did not occur. The shading of the balloon protected normal mucosa and prevents the formation of esophageal strictures. The "windowed" centering balloon provides a technology and technique that allows targeted delivery of uniform light during esophageal PDT.

A centering balloon for photodynamic therapy of esophageal cancer tested in a canine model.

Delivery of uniform circumferential light is desirable during photodynamic therapy of early or advanced esophageal cancer in human beings. Studies were performed in the canine esophagus to investigate whether use of a centering balloon would improve circumferential illumination of esophageal mucosa for photodynamic therapy of esophageal cancer. When the centering balloon was used, photodynamic therapy produced uniform and circumferential injury. With the cylindrical diffuser used in human studies, non-uniform and focal esophageal injury occurred. Placement of isotropic probes on the balloon wall allowed measurement and verification of relatively uniform light doses delivered to esophageal mucosa during balloon photodynamic therapy. The centering balloon has a potential role in improving light dosimetry during esophageal photodynamic therapy.

Comparative study between pulsed and continuous wave lasers for Photofrin photodynamic therapy.

A study was conducted in the normal canine esophagus to compare continuous wave (CW) and pulsed laser light for photodynamic therapy with Photofrin (4 mg/kg). Forty-eight hours post-injection, 630 nm laser light (CW light from an argon-pumped dye-laser and pulsed light from a KTP/532-pumped dye-laser) was delivered using a 24 mm diameter cylindrical esophageal PDT balloon positioned at either distal or proximal esophagus. A 1.0 cm cylindrical diffuser placed in the center of the balloon delivered 300 J/cm of light at an intensity of 400 mW/cm. Three dogs received CW light proximally and pulsed light distally. Four dogs received CW light distally and pulsed light proximally. The light dose delivered to the esophageal mucosa was measured using three isotropic probes placed on the balloon wall. Laser-induced fluorescence technique was used to compare photosensitizer fluorescence intensities at distal and proximal locations. Similar mucosal light doses and drug fluorescence intensities were verified for sites receiving pulsed or CW laser light. Two days after light delivery, the dogs were endoscoped to evaluate the severity of the lesions. While some response variability was observed among different animals, endoscopic examination of the lesions revealed comparable injury from CW and pulsed light in each subject. The animals were then euthanized and necropsies were performed. Based on the gross and histological examination of the lesions, the CW and pulsed laser-induced injuries could not be distinguished.

Centering balloon to improve esophageal photodynamic therapy.

A cylindrical balloon was developed to improve delivery of circumferential light for photodynamic therapy (PDT) of esophageal carcinoma. The balloon consisted of a 36-mm-long clear cylindrical membrane and a central tube to hold a cylindrical diffuser in the center of the lumen. Three isotropic probes were placed on the outside of the balloon to allow measurement of delivered light dose to the esophageal mucosa. The balloon was tested in the normal esophagus of 8 dogs that were injected with 4.0 mg/kg of PHOTOFRINR. Endoscopy was performed 48 hours following the injection, and under endoscopic observation the balloon assembly was passed, fixed in place, and inflated. A 1-cm cylindrical diffuser was passed into the central tube and 150, 300, and 600 Joules/cm of 630 nm laser light was delivered at 25 cm, 15 cm, and 5 cm proximal to the gastroesophageal junction. One control dog was illuminated using the cylindrical diffuser alone at doses of 300 and 600 Joules/cm of diffuser. Complete circumferential tissue response was obtained when the balloon was used. Relatively uniform light intensities were measured around the lumen. In contrast, noncircumferential and unpredictable PDT responses were generated when the cylindrical diffuser was used without the balloon.

Comparison of effects of cimetidine and omeprazole on mechanically created gastric ulceration and on aspirin-induced gastritis in dogs.

A double-blind study was conducted to compare gastric ulcer healing time in nontreated dogs with that in dogs treated with either cimetidine or omeprazole. Single ulcers were created in the gastric antrum by use of a suction biopsy capsule. Each dog was given 25 mg of aspirin/kg of body weight orally for 20 days after ulcer induction. Five control dogs were given aspirin only (no anti-ulcer medication) during the 20-day study. Six dogs were given cimetidine at dosage of 10 mg/kg orally every 8 hours, and 6 dogs were given omeprazole orally at dosage of 2 mumol/kg (0.7 mg/kg) once daily. All dogs were examined endoscopically on days 5, 10, 15, and 20 and were given a score for the size of the mechanically created ulcer and a score for the degree of aspirin-induced gastritis. All dogs were euthanatized on day 21, and gastric lesions were examined histologically. Significant differences were not evident in ulcer healing scores or degree of aspirin-induced gastritis among treated and nontreated dogs on days 5, 10, 15, and 20. However, aspirin-induced gastritis was less severe in dogs of the omeprazole group than in dogs of the cimetidine or control group on each day observations were made. The effect of omeprazole given once daily was comparable with that of cimetidine given every 8 hours in lessening aspirin-induced gastritis.