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PubMed2007

Influence of strain and age differences on the yields of porcine islet isolation: extremely high islet yields from SPF CMS miniature pigs.

BACKGROUND: Porcine pancreas is a potential source of material for islet xenotransplantation. However, the difficulty in isolating islets, because of their fragility and the variability of isolation outcome in donor age and breed, represents a major obstacle to porcine islet xenotransplantation. In this study, we compared the islet isolation yield of specific pathogen-free (SPF) Chicago Medical School (CMS) miniature pigs with that of another miniature pig breed and market pigs from a local slaughterhouse. METHODS: Nine adult CMS miniature (ACM) pigs (>12 months), six young CMS miniature (YCM) pigs (6-7 months), four adult Prestige World Genetics (PWG) miniature (APM) pigs (>12 months), and 13 adult market (AM) pigs from a local slaughterhouse were used for islet isolation. RESULTS: The islet yield per gram of pancreas from ACM pigs (9589 +/- 2823 IEQ/g) was significantly higher than that from APM pigs (1752 +/- 874 IEQ/g, P < 0.05), AM pigs (1931 +/- 947 IEQ/g, P < 0.05), or YCM pigs (3460 +/- 1985 IEQ/g, P < 0.05). Isolated islets from ACM pigs were significantly larger than those from AM pigs or YCM pigs. The in vitro and in vivo function of isolated islets showed no difference among experimental groups. The pancreases of ACM pigs contained higher mean islet volume density percentages and larger size of islets than those of AM or APM pigs. CONCLUSIONS: We isolated extremely high yields of well-functioning islets from ACM pigs bred under SPF conditions. SPF CMS miniature pigs should be one of the best porcine islet donors for clinical porcine islet xenotransplantation.

Aging↗
PubMed2004

Miniaturization of intracellular calcium functional assays to 1536-well plate format using a fluorometric imaging plate reader.

The measurement of intracellular calcium response transients in living mammalian cells is a popular functional assay for identification of agonists and antagonists to receptors or channels of pharmacological interest. In recent years, advances in fluorescence-based detection techniques and automation technologies have facilitated the adaptation of this assay to 384-well microplate format high-throughput screening (HTS) assays. However, the cost and time required performing the intracellular calcium HTS assays in the 384-well format can be prohibitive for HTS campaigns of greater than 1 x 10(6) wells. For these reasons, it is attractive to miniaturize intracellular calcium functional assays to the 1536-well microplate format, where assay volumes and plate throughput can be decreased by several fold. The focus of the research described in this article is the miniaturization of an intracellular calcium assay to 1536-well plate format. This was accomplished by modifying the hardware and software of a fluorometric imaging plate reader (FLIPR) to enable transfer of nanoliters of test compound directly to a 1536-well assay plate, and measure the resulting calcium response from all 1536 wells simultaneously. An intracellular calcium functional assay against the rat muscarinic acetylcholine receptor subtype 1 (rmAchR1) G-protein coupled receptor (GPCR) was miniaturized and executed on this modified instrument. In experiments measuring the activity of known muscarinic receptor agonists and antagonists, the miniaturized FLIPR assay gave EC(50) and IC(50) values and rank order potency comparable to the 384-well format assays. Calculated Z' factors for the miniaturized agonist and antagonist assays were, respectively, 0.56 +/- 0.21 and 0.53 +/- 0.22, which were slightly higher (Z'(agonist) = 0.55 +/- 0.33) and lower (Z'(antagonist) = 0.70 +/- 0.18) than the corresponding values in the 384-well assays. A mock agonist HTS campaign against the muscarinic receptor in miniaturized format was able to identify all wells spiked with the rmAchR1 agonist carbachol.

Animals↗
PubMed1992

Isolated or phagocytosed miniature dyskeratotic cells in papilloma virus infection associated with cervical neoplasia.

Dyskeratotic cells that occur in squamous cervical epithelium under certain abnormal conditions appear in cervical smears in sheets or aggregates, or as miniature cells, either isolated or phagocytosed. In order to determine the tissue correlates of such cells, 116 cervical biopsies with a histopathologic diagnosis of papillomavirus (HPV) infection were reviewed and compared to previous findings in cytological smears; the patients were 18-65 yr of age. Eighty of the 116 cases had, in addition, cervical intraepithelial neoplasia (CIN). A much higher number of miniature dyskeratotic cells was recorded in the biopsies from patients with both HPV and CIN types of lesions (76.3% of the biopsies had isolated miniature cells and 41.3% showed phagocytosed miniature cells), compared to those with only HPV infection (19.4% with isolated and 5.5% with phagocytosed miniature cells). In addition, in the HPV plus CIN patients, the more advanced the neoplastic lesion, the higher the percentage of cases in which both types of dyskeratocytes are observed. These highly keratinized isolated miniature cells are found in, and are considered to arise from, the deeper layers of the epithelium; when phagocytosed they appear in the center of a concentric arrangement of cells. These cells constitute a pathological entity that can be distinguished from the sheets and aggregates of dyskeratotic cells that come from the superficial layers of parakeratotic epithelia.

Adolescent↗
PubMedDate not supplied

Characterization and application of a miniature 10 mL stirred-tank bioreactor, showing scale-down equivalence with a conventional 7 L reactor.

The aim of this study was to characterize the engineering environment of an instrumented 10 mL miniature stirred-tank bioreactor and evaluate its potential as a scale-down device for microbial fermentation processes. Miniature bioreactors such as the one detailed in this work have been developed by several research groups and companies and seek to address the current bottleneck at the screening stage of bioprocess development. The miniature bioreactor was characterized in terms of overall volumetric oxygen transfer coefficient and mixing time over a wide range of impeller speeds. Power input to the miniature bioreactor was directly measured, and from this the power number of each impeller was calculated and specific power input estimated, allowing the performance of the miniature bioreactor to be directly compared with that of a conventional 7 L bioreactor. The capability of the miniature bioreactor to carry out microbial fermentations was also investigated. Replicate batch fermentations of Escherichia coli DH5alpha producing plasmid DNA were performed at equal specific power input, under fully aerobic and oxygen-limiting conditions. The results showed a high degree of equivalence between the two scales with regard to growth and product kinetics. This was underlined by the equal maximum specific growth rate and equal specific DNA product yield on biomass obtained at the two scales of operation, demonstrating the feasibility of scaling down to 10 mL on the basis of equivalent specific power input.

Bioreactors↗
PubMedDate not supplied

Retrospective analysis of canine miniature total hip prostheses.

OBJECTIVE: To determine the practicality and clinical outcome of miniature total hip replacement (THR) in dogs. STUDY DESIGN: Retrospective study. Sample Population- Seventeen client-owned dogs that had miniature THR. METHODS: Patient data surveyed included signalment, body weight, diagnosis, implant size, surgical technique, and intraoperative and postoperative complications. Radiographic evaluation included angle of lateral opening of the acetabular component, implant positioning, cement mantle quality, and femoral displacement measurement and ratio. Client questionnaire and orthopedic examination were used to obtain long-term follow-up information. RESULTS: Miniature THR was performed to address hip dysplasia and secondary osteoarthritis. In 1 dog, a staged bilateral procedure was performed. Mean body weight was 19 kg (range, 12 to 25 kg). Penetration or fissure of the femoral cortex was the most common intraoperative complication and occurred in 3 dogs. In 3 dogs, there was excessive coxofemoral laxity after reduction of the prosthesis. This instability was addressed specifically in 2 dogs by capsulorrhaphy or capsular prosthesis. Postoperative convalescent complications (craniodorsal luxation, 2 dogs; acetabular cup displacement, 1 dog) were related to surgical errors. Aseptic loosening of the femoral implant was diagnosed in 1 dog at 18 months. Mean follow-up time was 17 months (range, 4 to 42 months). Fifteen of 18 (83%) miniature THRs had good or excellent outcomes. CONCLUSIONS: Miniature THR should be considered a satisfactory alternative to femoral head and neck ostectomy in medium-size dogs affected by hip dysplasia and secondary osteoarthritis. The population of medium-size dogs that might derive more benefit from THR than FHO has yet to be defined. CLINICAL RELEVANCE: Miniature THR is a viable treatment option in medium-size dogs with hip dysplasia.

Animals↗
PubMed2002

The effect of miniaturized body size on skeletal morphology in frogs.

Miniaturization has evolved numerous times and reached impressive extremes in the Anura. I compared the skeletons of miniature frog species to those of closely related larger species to assess patterns of morphological change, sampling 129 species from 12 families. Two types of morphological data were examined: (1) qualitative data on bone presence and absence; and (2) thin-plate spline morphometric descriptions of skull structure and bone shape. Phylogenetic comparative methods were used to address the shared history of species. Miniature anurans were more likely to lose skull bones and phalangeal elements of the limbs. Their skulls also showed consistent differences compared to those of their larger relatives, including relatively larger braincases and sensory capsules, verticalization of lateral elements, rostral displacement of the jaw joint, and reduction of some skull elements. These features are explained by functional constraints and by paedomorphosis. Variation among lineages in the morphological response to miniaturization was also explored. Certain lineages appear to be unusually resistant to the morphological trends that characterize miniature frogs as a whole. This study represents the first large-scale examination of morphology and miniaturization across a major, diverse group of organisms conducted in a phylogenetic framework and with statistical rigor.

Animals↗
PubMed1982

Giant miniature endplate potentials induced by 4-aminoquinoline.

In experiments on the isolated extensor digitorum longus muscle of the rat it was shown that 4-aminoquinoline (125-250 micro M) altered the amplitude distribution of spontaneous miniature endplate potentials to include a large portion of giant miniature endplate potentials with slow rise and decay times. Similar, slow-rising giant miniature endplate potentials were induced by the drug at neuromuscular junctions with regenerating nerve terminals, i.e. in a condition where spontaneous as well as evoked transmitter release is depressed. The appearance of giant miniature endplate potentials was not correlated with inhibition of cholinesterase since neostigmine (3 micro M) failed to induce such potentials. Nerve impulse evoked endplate potentials of amplitudes similar to the spontaneous giant miniature endplate potentials had a faster and more uniform rise time. The results suggest that 4-amino-quinoline, by a direct action on the nerve terminal, causes the release of larger than normal quanta of acetylcholine. Quantitative assays of acetylcholine released before and in the presence of 4-aminoquinoline gave similar values showing that the amounts of acetylcholine which give rise to the giant miniature potentials contribute little to the total amount of acetylcholine liberated.

Action Potentials↗
PubMed1978

Further studies of periodic miniature response in squid giant axons.

Electric responses of extremely small amplitudes (1-30 muV peak-to-peak) repeating with more-or-less definite periodicity could be induced by a variety of chemical stimulants applied to squid giant axons either extracellularly or intracellularly. The chemical stimulants studied include allethrin, aminopyridines, N-bromosuccinimide, dimethylamino-pyridine, glutaraldehyde, osmium tetroxide, parachloromercuribenzoate, rose bengal, scorpion venoms and veratridine. With several mild stimulants, it was possible to evoke periodic miniature responses unaccompanied by a fall in the resting membrane potential. The frequency of miniature responses could be lowered by intracellular injection of TEA (tetraethylammonium). These miniature responses could readily be suppressed by external application of TTX (tetrodotoxin). The miniature responses evoked by 4-aminopyrine were characterized by a large variation in the frequency of responses. Cross-linking of the membrane proteins with a dilute solution of glutaraldehyde produced miniature responses repeating at progressively falling frequencies. Analyses of the processes of production of miniature responses with these and other stimulants have clarified several aspects of the physicochemical properties of the excitable sites of the axon membrane.

Animals↗
PubMed2004

Evaluation of the cationic trypsinogen gene for potential mutations in miniature schnauzers with pancreatitis.

The purpose of this study was to evaluate the cationic trypsinogen gene in miniature schnauzers for possible mutations. Genetic mutations have been linked with hereditary pancreatitis in humans. Four miniature schnauzers were selected on the basis of a clinical history of pancreatitis. One healthy miniature schnauzer and 1 healthy mixed breed canine were enrolled as controls. DNA was extracted from these canines using a commercial kit. Primers were designed to amplify the entire canine cationic trypsinogen cDNA sequence. A polymerase chain reaction (PCR) was performed and products were purified and sequenced. All sequences were then compared. The healthy control canine, a healthy miniature schnauzer, and the 4 miniature schnauzers with pancreatitis showed identical sequences of the cationic trypsinogen gene to the published sequence. We conclude that, in contrast to humans with hereditary pancreatitis, mutations of the cationic trypsinogen gene do not play a major role in the genesis of pancreatitis in the miniature schnauzer.

Amino Acid Sequence↗
PubMed1983

Evaluation of five miniature chromatography systems for determining labeling efficiency of technetium Tc 99m pentetate.

The reliability and reproducibility of five miniature chromatography systems for the radiochemical purity of 99mTc-labeled technetium Tc 99m pentetate was evaluated. Radiochemical purity of technetium Tc 99m pentetate was determined 15-30 minutes after preparation once a month for nine consecutive months. A reference value was determined by gel filtration or by conventional-length paper chromatography and thin-layer chromatography. Radiochemical purity was determined simultaneously by five miniature chromatography systems. The miniature systems included an in-house system and commercial systems distributed by Ackerman Nuclear, Ashley Innovations, Atomic Products, and Technical Advancement. Each miniature system was tested in duplicate. A follow-up comparison of the Ackerman Nuclear and in-house systems was performed for an additional nine months. Labeling efficiency by the reference method was greater than 97% for all nine months. The miniature systems gave results that were comparable in most months. Very low labeling efficiency occurred with the Ackerman Nuclear system in month 9. The follow-up comparison produced only one unconfirmed result for the Ackerman Nuclear system that would have caused a laboratory to erroneously discard a batch of technetium Tc 99m pentetate. The miniature chromatography systems evaluated generally will give reliable and reproducible results for the radiochemical purity of technetium Tc 99m pentetate for nine months after receipt of the systems.

Chromatography↗
PubMed1999

Development and in vitro testing of a miniature robotic system for computer-assisted colonoscopy.

In this article we present a new concept for computer-assisted colonoscopy based on a miniature robot capable of propelling itself semiautonomously along the colon. The miniature robot is designed to perform the same functions as current colonoscopy systems-i.e., visualization and tissue sampling for biopsy-and exploits an innovative inchworm-like locomotion principle based on adhering to the colon wall by vacuum suction. The miniature robot is connected by a thin and flexible umbilical cable to an external control unit; this unit provides pneumatic actuation signals in the appropriate sequence to the miniature robot, and information on the robot's functioning to the endoscopist, who can either teleoperate or directly supervise its operation. A prototype colonoscopy system using this robot has been fabricated and tested in vitro, with promising results. The proposed concept has strong potential for further development, since miniaturization and functional integration of instrumentation and tools, together with computer assistance, not only make colonoscopy more acceptable, but can also open up a wide range of new applications in endoluminal diagnosis, therapy, and surgery.

Animals↗
PubMed2002

Miniaturized transesophageal echocardiography in newborn infants.

BACKGROUND AND METHODS: A miniaturized 5.5 to 10 MHz, phased-array, single longitudinal plane transducer mounted on a 3.3-mm diameter catheter (miniaturized transesophageal echocardiography [TEE]) may overcome mechanical limitations of standard pediatric transesophageal probes. We evaluated whether the miniaturized TEE probe could define clinically relevant anatomy in 17 infants who weighed less than 6 kg. RESULTS: Twenty-two studies were performed in 17 infants without complication, weighing 2.1 to 5.6 kg. Twenty of twenty-two studies were diagnostic. Pediatric biplane TEE was not possible in 13 studies. Lack of horizontal plane imaging with miniaturized TEE made evaluation difficult in patients with atrioventricular septal defect. CONCLUSION: Miniaturized TEE provided diagnostic intraoperative TEE in the majority of infants studied and may allow broader and safer application of TEE to neonates and small infants.

Cardiac Surgical Procedures↗
PubMed2005

Gamma scintigraphic evaluation of a miniaturized AERx pulmonary delivery system for aerosol delivery to anesthetized animals using a positive pressure ventilation system.

The purpose of this study was to characterize performance of a miniaturized AERx((R)) Pulmonary Delivery System designed for aerosol administration to large animal models. The miniaturized AERx System was developed through a systematic scaling down of the AERx System used for humans to allow for operation in certain animal models with lower inspiratory flow rates and inhaled volumes than those used for humans. We used gamma scintigraphy to characterize the in vivo particle deposition achieved with the miniaturized AERx System in two dogs. The dogs were 3-4 years old, and weighed 10.4 kg and 13.6 kg. Acepromazine was used as pre-anesthetic medication. Anesthesia was induced with 5% isoflurane. The trachea was intubated using an endotracheal tube (internal diameter 8.5 mm), and the dogs were ventilated using positive pressure during the exposure using the LRRI puff generator. An inhalation of aerosol was initiated by activation of the puff generator though the computer-controlled interface. Each dog inhaled approximately 0.8 L per puff, of which the aerosol volume comprised approximately 0.25 L, at a target flow rate of 15 L/min. The dogs were exposed to 10 AERx Strips in 10 puffs. The mass median aerodynamic diameter of the aerosolized formulation was approximately 1.25 microm with a fine particle fraction <3.5 microm of 0.976. The scintigraphic images showed uniform bilateral lung deposition following aerosol delivery with the AERx System. Total lung deposition for the two dogs was 10.7% and 18% of the loaded dose from the AERx Strip. The corresponding peripheral lung: inner lung (P/I) ratios were 0.83 and 0.75, suggestive of deposition in the deep lung. Only 0.1% to 0.2% of the loaded dose was exhaled. These results show the miniature AERx System can efficiently deliver aerosols to the deep lung of dogs. The miniaturized AERx System would be a valuable tool for conducting proof-of-concept studies as well as safety and tolerability analysis of inhaled drug candidates in large animal models.

Administration, Inhalation↗
PubMed2007

Cardioprotective effects of diltiazem reevaluated by a novel myocardial ischemic model in Chinese miniature swine.

AIM: To develop a new model of myocardial ischemia in Chinese miniature swine and reevaluate the cardioprotective effects of diltiazem. METHODS: Myocardial ischemia was induced by injecting self-embolus into the left anterior descending (LAD) coronary artery in qualified miniature swine. Diltiazem (5 mg. kg(-1). d(-1)) was orally administered to the swine by mixing into normal pig diet from 1 to 6 d after self-embolus injection. The coronary angiography, 30 point body surface electrocardiogram (BS-ECG), hemodynamics, biochemistry, quantitative histology and pathohistology were determined 6 d after self-embolus injection. RESULTS: Embolization occurred in the LAD coronary artery of the Chinese miniature swine injected by self-embolus. There were significant myocardial ischemia and large cardiac muscle infarction in the Chinese miniature swine, which were accompanied with increased BS-ECG, decreased hemodynamic indexes of the cardiac output, cardiac index, left cardiac work and left cardiac work index, and increased systemic vascular resistance index. Pathohistological analysis revealed myocardial degeneration, necrosis, fibrosis, inflammatory cell infiltration and granulation tissue hyperblastosis (n=6). Diltiazem diminished the extent of the LAD embolism, ameliorated myocardial ischemia, improved the hemodynamic indexes, increased the plasma superoxide dismutase activity, decreased the plasma malondialdehyde content, narrowed the myocardial ischemic area and weakened the pathohistological damage in the cardiac muscle (n=6). CONCLUSION: Myocardial ischemia induced by injecting self-embolus into the LAD coronary artery in Chinese miniature swine is quite close to clinical pathophysiological conditions. Diltiazem is effective to inhibit the myocardial ischemia and restore the heart function in this novel model.

Administration, Oral↗
PubMed1995

Clinical evaluation of a miniature strain-gauge transducer for monitoring intracranial pressure.

In 25 patients, we evaluated the accuracy of a new miniature strain-gauge transducer developed for the measurement of intracranial pressure (ICP). The ICP in each patient was measured with the intraventricular, miniature strain-gauge transducer, and that value was compared with the ICP measured with a ventriculostomy catheter coupled to an external strain-gauge transducer. From the two monitors, 2218 simultaneous measurements of ICP were obtained. The average ICP measured with the miniature strain-gauge transducer was 15.9 +/- 10.0 mm Hg (range, -3 to 104 mm Hg). The ICP measured with the ventriculostomy-catheter transducer averaged 15.4 +/- 10.1 mm Hg (range, -9 to 104 mm Hg). A highly significant correlation was found over the wide range of pressures observed (n = 2218, r = 0.97, P < 0.001). The average difference between the two measurements of the ICP was 0.5 +/- 2.6 mm Hg, and the differences were equally positive and negative, demonstrating no consistent bias. The two values for the ICP were within 2 mm Hg of each other on 63% of the measurements and within 4 mm Hg of each other on 89% of the measurements. The average zero drift of the miniature strain-gauge transducer, measured at ambient pressure after removal of the catheter, was 0.2 +/- 0.5 mm Hg. The results indicate that this miniature strain-gauge transducer is highly accurate and stable and that it is a reliable alternative to a ventriculostomy for monitoring the ICP.

Adult↗
PubMed2004

Age-related changes of bone mineral density and microarchitecture in miniature pigs.

Bone mineral density (BMD), distribution of its density and bone histomorphometric parameters were evaluated in lumbar vertebra of normally growing miniature pigs. The fourth lumbar vertebra (L4) of the Göttingen miniature pig were used in this cross-sectional study in vitro. The BMD of the miniature pig was similar to that of humans in tendency of gender differences and some growth patterns during puberty. In these regards this animal appears useful as a model for human bone study. However, the trabecular and cortical BMDs of lumbar spine were extremely high value (399.43 +/- 26.36 mg/cm(3) in female trabeculae; 973.06 +/- 69.55 mg/cm(3) in female cortical bone; 419.04 +/- 34.84 mg/cm(3) in male trabeculae; 1038.81 +/- 125.72 mg/cm(3) in male cortical bone in pigs 30 months or more). Furthermore, histomorphometric analysis yielded values that were remarkably different from those found in humans. From these results, it was revealed that miniature pig had a higher bone mass and denser trabecular network than human, indicating that its bone is probably stronger. Therefore, care should be taken in choosing the miniature pig as a bone study model.

Absorptiometry, Photon↗
PubMed2002

Liquid storage of miniature boar semen.

The effects of liquid storage at 15 degrees C on the fertilizing ability of miniature pig semen were investigated. Characterization of ejaculated semen from 3 miniature boars was carried out. Semen volume and pH were similar among these boars. In one of the boars, sperm motility was slightly low, and sperm concentration and total number of sperm were significantly lower than in the others (P < 0.01). Seminal plasma of the semen was substituted with various extenders (Kiev, Androhep, BTS and Modena) by centrifugation and semen was stored for 7 days at 15 degrees C. Sperm motility was estimated daily at 37 degrees C. For complete substitution of seminal plasma, Modena was significantly more efficient than the other extenders (P < 0.001) in retaining sperm motility. Semen from each of the 3 miniature boars that had been stored for 5 to 7 days at 15 degrees C in Modena was used for artificial insemination of 15 miniature sows. The farrowing rates were 100, 100 and 60%, and litter sizes were 6.4 +/- 1.5, 5.8 +/- 0.8 and 5.0 +/- 1.0 for each boar semen, respectively. The boar that sired the smallest farrowing rate was the same one that showed lower seminal quality with respect to sperm motility, sperm concentration and total number of sperm. These results suggest that miniature boar semen can be stored for at least 5 days at 15 degrees C by the substitution of seminal plasma with Modena extender.

Animals↗
PubMed2002

Miniaturized formats for efficient mass spectrometry-based proteomics and therapeutic development.

Off-line miniaturized "nano-spray" formats for electrospray ionization mass spectrometry (ESI-MS) enable the routine identification of femtomole quantities of protein or peptide. Even greater strides have been achieved using on-line miniaturized ESI-MS methods, such as nanobore LC-MS and CE-MS. On-line methods enable greater sensitivity (sub-attomole limit of detection), dynamic range, and throughput. In either off- or on-line methods for protein analysis, samples are typically isolated and digested enzymatically, with MS analysis of the peptide fragments, yielding 5-50% sequence coverage, in a "bottom-up" approach. Obtaining biologically relevant (structure/function) information (such as the localization of regions of error or post-transnational modifications) often demands 100% sequence coverage and this may be obtained by analyzing intact proteins by MS with a "top-down" methodology. Proteome wide success with top-down methods will require the development of novel miniaturized approaches for sample preparation along with new tools for bioinformatics. As these miniaturized formats continue to power proteomics applications, they will undoubtedly pollinate "cross-over" applications in LC-MS ranging from drug discovery to development. An example of metabolite identification using an order of magnitude less sample than usually required, with a concurrent order of magnitude increase in signal, illustrates the potential of miniaturized formats in lead characterization activities.

Animals↗