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Autologous calvarial and iliac onlay bone grafts in miniature swine.

Calvarial bone has been reported to be superior to iliac bone for onlay bone grafting due to decreased resorption. This study evaluated the physical, histologic, and radiographic characteristics of calvarial and corticocancellous iliac onlay bone grafts in nine Pitman-Moore miniature swine at 2 weeks, and at 1-, 2-, 4-, 6-, 7-, 8-, 10-, and 12-month intervals. Compared with iliac grafts, the calvarial onlay grafts had more than a twofold greater radiographic density. Statistical analysis of the mature grafts using the standard of estimated means of the bone graft volumes revealed 85% retention of the calvarial grafts compared with 34% retention of the grafted iliac bone. There was no subjective difference in the rate or degree of revascularization between the two grafted materials.

Animals↗

Performance of a miniature magnetic position sensor for three-dimensional ultrasound imaging.

A miniature magnetic position sensor used for three-dimensional ultrasound imaging was tested for precision and accuracy in vitro. The sensor alone was able to locate points with root-mean-square (rms) uncertainty of 1.7 mm and accuracy of 0.05 +/- 0.62 mm over its specified operating range of 50 cm. With an ultrasound imaging system, a point was located from arbitrary viewing windows with 2.4-mm rms uncertainty and 0.06 +/- 0.68 mm accuracy. If viewing windows were limited to those representative of a typical ultrasound examination, the system could achieve rms uncertainty in point location of < 1 mm. Performance was not affected by operation of the imaging system when the sensor was mounted on an ultrasound scanhead. Sensitivity to metals in the operating environment was also measured.

Calibration↗

A direct injection capillary electrophoretic technique for miniaturized high-throughput metabolic screening of the CYP 3A4 enzyme using quinidine as a probe.

A capillary electrophoresis (CE) method has been developed for the determination of quinidine sulfate (QS) and (3S)-3-hydroxyquinidine (3-OHQ) by direct injection of microsomal incubation mixtures. 3-OHQ is the CYP 3A4 metabolite of QS and hence useful for metabolism screening studies. The method was validated analytically and tested for its effectiveness as a metabolic inhibition model. A linear calibration was found to provide the best fit for the standard curve with an r of 0.9966 and all residuals less than 12%. The percent relative standard deviations (RSDs) of the two controls, 2 and 8 microg/ml were 5.27 and 2.90% and the percent difference from normal (% DFN) were -12.58 and -0.31% respectively. The limit of quantitation (LOQ) in the incubation matrix was 0.5 microg/ml. 3-OHQ formation complied with Michaelis-Menten kinetics and the mean values+/-S.D. of Km and Vmax were 36.98+/-4.62 microg/ml and 321.39+/-3.88 ng/mg/h respectively. Preliminary inhibition studies suggest that the method has adequate sensitivity to screen for high and medium inhibitors of the CYP 3A4 isozyme. The lack of sample preparation coupled with the small sample size capability of CE would enable the direct injection technique to aid in miniaturized high-throughput screening.

Animals↗

Influence of indomethacin on bone turnover related to orthodontic tooth movement in miniature pigs.

The purpose was to evaluate the influence of a prostaglandin inhibitor, indomethacin, on the tissue reaction related to orthodontic tooth movement. Sixteen miniature pigs were chosen for the study, eight of which received indomethacin perorally every day of the 39-day observation period. Sentalloy expansion springs (GAC, Central Islip, N.Y.) delivering 100 cN were inserted on a segmented arch between the central lower incisors. Intravital labeling with tetracycline was used for the evaluation of the rate of bone formation. After the pigs were killed, the bone turnover was evaluated on undecalcified methacrylate embedded sections and on microradiographs. The histomorphometric analysis of bone turnover revealed that the relative extent of resorption surfaces was decreased significantly in the indomethacin treated animals. Formation surfaces were also decreased although not significantly. The bone turnover, but not the mineralization rate, was influenced. The results corroborate the recommendation that prostaglandin inhibitors should be avoided during orthodontic treatments.

Administration, Oral↗

Pediatric multiplane transesophageal echocardiography in congenital heart disease: new possibilities with a miniaturized probe.

In recent years, transducers for multiplane Doppler echocardiography have demonstrated their superior imaging performance in adult patients. To date, the size of these probes has limited their use in pediatric patients. In this article, we report our initial experience with a recently developed miniaturized transducer with all conventional imaging and Doppler modalities. The study focused primarily on imaging performance by comparing standard biplane images with those obtained in oblique planes. The investigations were carried out intraoperatively or during interventional catheterization in patients with congenital heart disease. We observed no complications in a study population of 15 children (weight range of 5 to 63 kg and an age range of 96 days to 11 years). The probe was easy to handle and provided excellent images. Additional information was obtained in several cases and documentation of clinical findings was easier because an optimal image plane almost always could be displayed. We concluded that pediatric multiplane Doppler echocardiography has considerably improved investigative performance compared with the conventional monoplane or biplane studies normally available for this age group. In neonates, however, investigation with the multiplane technique is limited by the size of the patient.

Child↗

A miniature head-mounted two-photon microscope. high-resolution brain imaging in freely moving animals.

Two-photon microscopy has enabled anatomical and functional fluorescence imaging in the intact brain of rats. Here, we extend two-photon imaging from anesthetized, head-stabilized to awake, freely moving animals by using a miniaturized head-mounted microscope. Excitation light is conducted to the microscope in a single-mode optical fiber, and images are scanned using vibrations of the fiber tip. Microscope performance was first characterized in the neocortex of anesthetized rats. We readily obtained images of vasculature filled with fluorescently labeled blood and of layer 2/3 pyramidal neurons filled with a calcium indicator. Capillary blood flow and dendritic calcium transients were measured with high time resolution using line scans. In awake, freely moving rats, stable imaging was possible except during sudden head movements.

Anesthesia↗

Microvascular reanastomozed allogenous iliac crest transplants for the reconstruction of bony defects of the mandible in miniature pigs.

The effects of immunosuppression with cyclosporin A and prednisolone regimens for allogenous iliac crest bone grafts used for mandibular reconstruction were investigated in 40 miniature pigs, for periods of 2, 4 and 16 weeks. Autogenous and allogenous bone grafts without immunosuppression served as controls. Specimens were evaluated by routine histology, direct magnified radiography and fluorescence microradiography. Four out of five autogenous transplants showed a preserved vascular architecture and bony union. None of the allogenous transplants without immunosuppression survived. Primary bone healing of the allografts was noted after short-term immunosuppression. However, occlusion of the nutrient vessels was noted ten days postoperatively. The allografts were not rejected after cessation of the immunosuppressive therapy within an observation period of 4-12 weeks. Revascularization of all areas of the allografts and creeping substitution of the transplanted bone were noted after seven weeks. Infection of the allografts, with failure of bony union, was noted in nine animals, but primary healing of allografts with short-term immunosuppression was demonstrated.

Anastomosis, Surgical↗

A miniature fiber optic surface plasmon resonance sensor for fast detection of Staphylococcal enterotoxin B.

A fiber optic surface plasmon resonance (SPR) biosensor for detection of Staphylococcal enterotoxin B (SEB) is reported. The sensor is based on spectral interrogation of surface plasmons in a miniature sensing element based on a side-polished single-mode optical fiber with a thin metal overlayer. For specific detection of SEB, the SPR sensor is functionalized with a covalently crosslinked double-layer of antibodies against SEB. The SPR biosensor is demonstrated to be able to detect ng/ml concentrations of SEB in less than 10 min.

Enterotoxins↗

Miniaturized pH biosensors based on electrochemically modified electrodes with biocompatible polymers.

Potentiometric pH sensors based on linear polyethylenimine (L-PEI) and linear polypropylenimine(L-PPI), two synthetic enzymes and biocompatible polymers, films were prepared by electropolymerization of three different monomers: ethylenediamine (EDA), 1,3-diaminopropane (1,3-DAP) and diethylenetriamine (DETA) in order to be used in clinical, dermatological and biological applications, such as in vivo analysis. In a first step a biosensor was tested which consisted in a platinum wire protruded from glass sheath. The polymer film coated on these platinum electrodes showed good linear potentiometric responses to pH changes from pH 3 to 10. Resulting electrodes present both good reversibility and good stability versus time. The effect of the different polymer film thicknesses to potentiometric responses was also studied. This study allowed us to develop a miniaturized pH biosensor in the second step. This sensor was fabricated using photo-lithography, followed by sputtering and lift-off processes, and it included an electronic detection system. We have also successfully studied the potentiometric responses to pH changes of this device over a period of 1 month, and so we propose this new pH micro-biosensor as an alternative to classical pH sensors currently used in dermatology.

Biocompatible Materials↗

Regulatory mechanism of peripheral tolerance: in vitro evidence for dominant suppression of host responses during the maintenance phase of tolerance to renal allografts in miniature swine.

BACKGROUND: Studies from our laboratory have demonstrated that a short course of cyclosporine leads to indefinite survival of renal allografts across an MHC class-I barrier in miniature swine. We have recently reported that a peripheral regulatory mechanism appears to be involved in the maintenance of this tolerance since peripheral blood lymphocytes (PBL), exposed to donor antigen in vitro specifically suppressed the generation of anti-donor cytotoxic activity by recipient-matched naive PBL. We have now further investigated the mechanism of this phenomenon to determine the level at which such regulation occurs, and investigated the phenotypes of the cells involved in maintaining dominant suppression. MATERIALS AND METHODS: PBL from long-term tolerant animals (>6 months after renal transplantation) were pre-stimulated in vitro with donor-type PBL. These cells were then incubated with recipient-matched naive responders and donor-type PBL stimulators in MLR assays. The proliferative activity of the cells was measured by [3H]-thymidine incorporation. Suppression was measured by inhibition of proliferation of naive cells in response to donor PBL when co-cultured with tolerant cells. Flow cytometry was used to study the phenotypes of cells that were present in cell cultures. RESULTS: Primed PBL from tolerant animals markedly suppressed the proliferative response of recipient-matched naive cells to donor-matched stimulators in vitro. No suppression of proliferation was observed in response to third party stimulators, indicating that the suppression was donor-specific. Primed PBL from naive animals stimulated with donor antigen and co-cultured with unprimed recipient-matched naive cells also demonstrated reduced proliferative responses. However, this decrease in proliferation appeared to be due to a 'burn-out' phenomenon, as assessed by kinetic studies, rather than due to true suppression. Expression of CD25 increased on a sub-population of T cells from tolerant animals following priming with donor antigen. These cell then markedly inhibited further activation of CD25 positive cells in co-cultures with naive responder cells, suggesting a possible mechanism of suppression. CONCLUSION: These results suggest that the mechanism of tolerance to class-I-mismatched renal allografts, involves a population of regulatory cells that are capable of suppressing proliferative anti-donor responses.

Animals↗

Kinetics of the uptake and distribution of the dopamine D(2,3) agonist (R)-N-[1-(11)C]n-propylnorapomorphine in brain of healthy and MPTP-treated Göttingen miniature pigs.

The binding of radioligand agonists to dopamine receptors in living brain can be informative about the abundance of receptors which are coupled to intracellular second messenger systems. Therefore, we developed a radiosynthesis for the dopamine D(2,3) partial agonist (R)-N- [1-(11)C]n-propylnorapomorphine ([(11)C]NPA). The uptake of this tracer in brain of anesthetized Göttingen miniature pigs was recorded by positron emission tomography (PET) and analyzed by compartmental analysis using the metabolite-corrected arterial input, and using reference tissue methods. [(11)C]NPA had a blood-brain unidirectional clearance of approximately 0.35 ml g(-1) min(-1) and an apparent distribution volume of 6 ml g(-1) in cerebellum. The ligand had a binding potential of 1.5 in striatum, comparable to that reported previously for the receptor antagonist [(11)C]raclopride in the same strain of animals. Significant binding was detected in the hypophysis, thalamus, and medial forebrain bundle. The binding in striatum was of comparable magnitude in normal pigs and in pigs with a documented 50% dopamine depletion produced by MPTP-intoxication. Deep brain stimulation of the subthalamus was without conspicuous effect on the binding of [(11)C]NPA in vivo. Results of this preliminary study indicate that this tracer meets many requirements for assaying dopamine agonist binding sites by PET.

1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine↗

Histomorphometric comparison of cardiac allograft vasculopathy in miniature swine.

BACKGROUND: Whether the pathologic characteristics of vascular lesions manifested in recipients with cardiac allograft vasculopathy (CAV) differ with the severity of the histocompatibility barrier crossed at transplantation or with the type or amount of immunosuppression used to prolong graft survival is unclear. We used miniature swine to determine whether a wide variance in heart transplantation protocols, both in histoincompatibility and immunosuppression, affects the histomorphometry of CAV. METHODS: We compared explanted hearts from major histocompatibility complex Class I-disparate recipients who were treated for 12 days with cyclosporine (Group 1) with minor-antigen-disparate hearts transplanted into mixed chimeric recipients previously engrafted with donor hematopoietic progenitor cells (Group 2). We analyzed coronary intimal lesions using computerized morphometry, immunohistochemistry, and TUNEL assay. Myocardial cytokine-gene expression was determined using RNAse protection assays and reverse-transcriptase polymerase chain reaction. RESULTS: The prevalence of CAV in Group 2 was significantly less than that observed in Group 1, but the severity of the lesions in both groups was similar. The vascular lesions that developed in both groups demonstrated the presence of alpha-smooth-muscle-actin-positive spindle cells expanding the intima, with few inflammatory cells. We noted an absence of proliferating cell nuclear antigen activity and TUNEL-positive cells in both groups. We observed prominent myocardial interferon-gamma gene expression only in Group 1. CONCLUSION: Despite differences in myocardial interferon-gamma gene expression, the histology and severity of the vascular lesions in CAV did not vary significantly with different histoincompatibilities or treatment protocols. These results suggest that the origin of CAV cannot be determined by histology alone.

Animals↗

Monitoring of left ventricular ejection fraction with a miniature, nonimaging nuclear detector: accuracy and reliability over time with special reference to blood labeling.

BACKGROUND: The purpose of this study was to determine the accuracy of determinations of left ventricular ejection fraction (LVEF) by a nonimaging miniature nuclear detector system (Cardioscint) and to evaluate the feasibility of long-term LVEF monitoring in patients admitted to the coronary care unit, with special reference to the blood-labeling technique. METHODS AND RESULTS: Cardioscint LVEF values were compared with measurements of LVEF by conventional gamma camera radionuclide ventriculography in 33 patients with a wide range of LVEF values. In 21 of the 33 patients, long-term monitoring was carried out for 1 to 4 hours (mean 186 minutes), with three different kits: one for in vivo and two for in vitro red blood cell labeling. The stability of the labeling was assessed by determination of the activity of blood samples taken during the first 24 hours after blood labeling. The agreement between Cardioscint LVEF and gamma camera LVEF was good with automatic background correction (r = 0.82; regression equation y = 1.04x + 3.88) but poor with manual background correction (r = 0.50; y = 0.88x - 0.55). The agreement was highest in patients without wall motion abnormalities. The long-term monitoring showed no difference between morning and afternoon Cardioscint LVEF values. Short-lasting fluctuations in LVEFs greater than 10 EF units were observed in the majority of the patients. After 24 hours, the mean reduction in the physical decay-corrected count rate of the blood samples was most pronounced for the two in vitro blood-labeling kits (57% +/- 9% and 41% +/- 3%) and less for the in vivo blood-labeling kit (32% +/- 26%). This "biologic decay" had a marked influence on the Cardioscint monitoring results, demanding frequent background correction. CONCLUSION: A fairly accurate estimate of LVEF can be obtained with the nonimaging Cardioscint system, and continuous bedside LVEF monitoring can proceed for hours with little inconvenience to the patients. Instability of the red blood cell labeling during long-term monitoring necessitates frequent background correction.

Erythrocytes↗

Engraftment of quiescent progenitors and conversion to full chimerism after nonmyelosuppressive conditioning and hematopoietic cell transplantation in miniature swine.

Our laboratory has previously reported a nonmyelosuppressive preparative regimen for hematopoietic cell transplantation that leads to mixed chimerism and allograft tolerance in miniature swine across minor and major histocompatibility disparities. Stable chimerism persisted in most of these animals but was restricted to T cells and confined to peripheral blood. Because of the importance of myeloid and erythroid progenitors for the treatment of hematologic disorders, the objective of this study was to assess whether such cells existed in the bone marrow of these lymphoid chimeras as an indication of functional engraftment. Colony-formation assays were performed on donor inocula before infusion and on bone marrow cells harvested from the transplant recipients. Donor-origin myeloid/erythroid progenitor colonies were detected in bone marrow from 6 of 7 lymphoid chimeric recipients. A delayed donor leukocyte infusion successfully converted a stable lymphoid chimera to full multilineage chimerism within 2 weeks. Donor-origin myeloid/erythroid progenitors could be detected in the bone marrow of a host-matched recipient after myeloablation and adoptive transfer of mobilized cells from one of the engrafted lymphoid chimeras. These data suggest that even when only lymphoid chimerism is readily detected by flow cytometry, dormant myeloid/erythroid progenitors can exist and subsequent conversion to full donor chimerism can be achieved. The ability to establish multilineage engraftment and chimerism without significant toxicity may have important clinical implications for the management of nonmalignant hematopoietic disorders and hematologic malignancies.

Animals↗

Behavior of miniature pet pigs.

Pigs are clean, intelligent, and entertaining animals. Because they are domesticated animals, they are not even as unusual a pet as a hedgehog or snake. The animal lover that buys a pig on impulse, however, is likely to have a disappointing experience. Those who acquire a pet pig with the most common preconceived but inaccurate notions are even more likely to be quickly disenchanted with their "exotic pet." Many pet owners will complain of the size that their pig attains; but if the pig were more manageable and better behaved, its ultimate size would probably be of less concern. Given the proper environment, diet and veterinary care, the miniature pig can be a terrific pet.

Animals↗

Miniature pigs.

Miniature pigs have become popular pets in North America, and veterinarians of a variety of clinical specialties may be called on for their care. Successful collection of blood from these animals requires familiarity with the location of sites for venipuncture and knowledge of adequate methods of restraint. In this article, restraint and techniques for venipuncture are described, as well as techniques for cerebrospinal fluid collection, semen collection, and vaginal cytologic examination. Interpretation of hematologic, serum biochemical, and urinalysis data are also discussed. Methods for diagnosis of skin diseases, gastrointestinal parasitism, and enteric infectious diseases are included in order to provide the practitioner with the essential knowledge and skills for a variety of clinical pathologic studies of this unique pet.

Anesthesia↗

Use of a microplate scintillation counter as a radioactivity detector for miniaturized separation techniques in drug metabolism.

In miniaturized separation techniques, such as capillary electrophoresis (CE) or capillary liquid chromatography (LC), conventional on-line radioactivity detection of labeled compounds is restricted, because of insufficient sensitivity. It will be shown that a microplate scintillation counter for 96-well plates (TopCount) can be used as a sensitive and easy-to-handle radioactivity detector for capillary LC and CE. The attractive combination of capillary LC, eluent fractionation, and subsequent off-line counting is described. The new method is applied for rapid and sensitive separation and detection of 3H-labeled parent drug and its metabolites at levels between 25 and 700 cpm in rat urine. The advantages of capillary LC coupled to the TopCount, and combined with LC-MS data, can be of benefit in many analytical areas, including the characterization of metabolites at low concentration within complex biological fluids. With the same setup, the fractionation with subsequent off-line counting is equally applicable to CE. This is demonstrated with electrophoretically separated 14C-labeled impurities, nicely resolved from a negatively charged main compound, at low levels.

Animals↗

Miniature triaxial metastable ionization detector for gas chromatographic trace analysis of extraterrestrial volatiles.

Gas chromatography has found highly successful application in NASA's flight programs. Gas chromatographs have been flown to both Mars and Venus where detailed compositional measurements were made. These instruments were quite small and relatively sensitive when compared to commercially available instruments; however, they do not appear adequate for future missions currently being planned. The earlier flight GC's had incorporated thermistor bead thermal conductivity cells as the detector. This detector requires very precise temperature control and only provides about 1 ppm sensitivity. Temperature stabilization causes the detector to be quite heavy, i.e., about 200 g. Greater sensitivity will be required for measurements of trace components in extraterrestrial environments. Review of other detector types revealed the metastable ionization detector as a likely candidate because of its superior thermal stability and high sensitivity. The metastable detector, first described by Lovelock as an argon ionization detector, has been studied and somewhat modified by others. The commercial design by Hartmann and Dimick was used for comparison purposes in our work. In the past, three features of the metastable detector are prominent: it has part-per-billion sensitivity, contamination must be carefully controlled, and anomalous response is common. Since it is an ionization detector, however, temperature instabilities do not cause the major perturbations experienced by the thermal conductivity detectors. This paper describes a miniature metastable ionization detector featuring an unconventional electrode configuration, whose performance characteristics parallel those of traditional design, while its weight is quite small. The prototype has been used in our laboratories routinely for 2 years, and the concept will be incorporated into a flight GC for use in the Space Shuttle.

Astronomy↗