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PubMed1995

Normal hematology, serology, and serum protein electrophoresis values in fetal Yucatan miniature swine.

We are currently developing fetal models of congenital heart disease in Yucatan miniature swine for pharmacologic, diagnostic, and interventional methods used to treat cardiac arrhythmias and ventricular septal defect. Fifty-four fetuses from 12 pregnant sows were included in this study. Eleven were fetuses between 76 and 88 days of gestation (early gestation fetuses). A second population of 43 fetuses were between 96 and 110 days of gestation (late gestation fetuses). Erythrocyte, leukocyte, serum electrolyte, enzyme, lipid, carbohydrate, and metabolite values were measured. Complete serum protein profiles were also obtained by electrophoresis. Significant differences could be shown between the sows and fetuses and between the early and late gestation fetuses in all of the categories studied, though not for every parameter. This study provides a large normal database for development of Yucatan miniature swine as an animal model in the rapidly expanding field of fetal medicine.

Animals↗
PubMed1995

[A new miniaturized flicker test for detection of florid retrobulbar neuritis].

A new transportable miniaturized flicker test equipped with green LEDs is contrasted with the conventional Tübingen flicker test, based on brightness perception of intermittent white light. The results are compared with those of former studies. In this study we examined 45 eyes suffering from florid optic neuritis. Another group of 114 eyes were either normal (except minimal ametropia) or suffered from a non-inflammatory or non-refractive impairment of central visual function. In all groups only one eye of each subject was examined. Using the criteria of Trauzettel-Klosinski, the new miniaturized "green" flicker test for the detection of a florid optic neuritis showed a specificity of only 50.9% (83.3%) and a sensitivity of 91.1% (64.4%). (The corresponding results of the conventional Tübingen flicker test are shown in parentheses.) Alternatively, analyzing the data with the help of a logistic regression, the "green" flicker test turned out to have a specificity of 92.9% (93.8%) and a sensitivity of 60.5% (44.2%). For further examinations using the flicker test it is useful to consider the duration and reproducibility of each brightness match by repeating the test several times.

Adolescent↗
PubMed1995

Splenic effects on hemodynamics induced by hypothermia and rewarming in miniature swine.

Central arterial hemodynamic changes were assessed during cooling, hypothermia, and rewarming in splenectomized (SPX, n = 4) and unsplenectomized (SP, n = 4) 8-10 month old male Yucatan miniature swine (34.0 +/- 1.4 kg). Under isoflurane anesthesia, and using circulating-water blankets, pigs were cooled to and then maintained for 2 h at a rectal temperature (Tre) of 27 +/- 1 degrees C; hypothermia was followed by rewarming to normothermia (37 +/- 1 degrees C). There were significantly (p < or = 0.05) greater changes in central arterial hematocrit and hemoglobin (delta HCT and delta HGB) from respective precooling baseline levels in the SP group during hypothermia and early rewarming (SP: delta HCTmax = 9-10%RBC, and delta HGBmax = 3.0-3.5 g/dl vs. SPX: delta HCTmax = 3-4%RBC, and delta HGBmax = 1.5-1.8 g/dl). By the end of rewarming, splenic resequestration and extravascular fluid shifts resulted in these values returning to baseline. In addition, cardiovascular instability was seen in the SPX group compared to the SP animals as evidenced by significant tachycardia and hypotension during rewarming. We have concluded from these studies that hypothermia causes significant hemoconcentration, and that splenic contraction is the major cause of this hemoconcentration during hypothermia and initial rewarming in miniature swine. A splenectomized design should be considered for swine studies that purport to pattern human pathophysiology, especially for modelling rewarming shock.

Analysis of Variance↗
PubMed1994

A miniature gamma camera.

We have described a mobile miniature-gamma-camera system for use in electrical trauma units and have presented images and imaging characteristics of a prototype system. The system has as its principal component a miniature gamma camera based on a PSPMT. The camera is 92 mm x 92 mm x 190 mm in size, weighs 5 kg, has a 48 mm x 48 mm field of view, and has an intrinsic resolution of approximately 3 mm FWHM and 6 mm FWTM. It is expected that devices of this type will be useful as imaging tools in electrical trauma units and laboratories where imaging studies regarding uptake mechanisms of radiopharmaceuticals for assessing tissue viability are carried out.

Electric Injuries↗
PubMed1993

[In vivo and in vitro study of experimental occlusion of choroidal and retinal blood vessels in the miniature pig].

Experimental occlusion of ocular blood vessels was performed in 25 eyes of 20 adult miniature pigs. The hyperacute, acute, subacute and chronic effects of these occlusions were studied by photography and fluorescein angiography of the ocular fundus in the living animals, and post-mortem by microscopic examination of histologic sections and stereomicroscopic study of vascular corrosion casts. Transection of the lateral posterior ciliary arteries was performed in eleven eyes and resulted in manifest chorioretinal ischaemic atrophy in confined areas of the ocular fundus. One month and longer after arterial transection, revascularization was found in the areas in which hypoperfusion had been induced, but no indications of neovascularization were observed. In one eye the lateral vortex veins were occluded by ligation. The subsequent fluorescein angiograms demonstrated only an insignificant local retardation of the choroidal filling. Filling defects of the choroidal vasculature and chorioretinal ischaemic lesions were not observed in the living animal nor in the vascular corrosion cast. Transection of the lateral posterior ciliary arteries was combined with ligation of the lateral vortex veins in seven eyes. These experiments resulted in lesions which were less severe than the sequels caused by transection of the lateral posterior ciliary arteries only. This observation was explained by the retrograde flow of venous blood which is rich in oxygen from the medial to the lateral choroidal quadrants. Consequently, ligation of the lateral vortex veins has a protective influence on the acute ischaemic lesions provoked by transection of the lateral posterior ciliary arteries. Simultaneous transection of the lateral posterior ciliary arteries and lateral chorioretinal arteries was performed in eight eyes. In the subacute and chronic stages, 1 and 4 months respectively after the vascular transection, proliferation of the retinal pigment epithelium and intraretinal neovascularization were observed in three out of five eyes examined. A fibrogliotic tissue mass containing newly formed blood vessels was found in between the reduplicated retinal pigment epithelium and the external layers of the sensory retina. The miniature pig offers an interesting ophthalmologic model because the ischaemic chorioretinal lesions and intraretinal gliovascular membranes which can be induced experimentally in this species present numerous similarities with the sequels of chorioretinal vascular occlusions in man.

Angiography↗
PubMed1994

Retransplantation in miniature swine. Lack of a requirement for graft adaptation for maintenance of specific renal allograft tolerance.

In miniature swine, one-haplotype class I disparate renal allografts are accepted without exogenous immunosuppression by approximately 35% of recipients. Alternatively, transplants bearing a two-haplotype class I mismatch are always rejected acutely. However, long-term acceptance in the latter animals can be achieved uniformly with a 12-day course of cyclosporine. In vitro studies of recipient cell-mediated lymphocytotoxicity responses have shown donor-specific cytotoxic T lymphocyte clones in tolerant animals, suggesting that tolerance may be a local phenomenon or a central phenomenon activated in the milieu of the graft. Six animals were retransplanted with kidneys MHC-matched to their original allograft to determine whether (1) tolerance is a central phenomenon; (2) host tolerance can be broken with a fresh challenge of donor antigen and antigen-presenting cells; and (3) graft adaptation is required for maintenance of tolerance. Four of the retransplanted animals had been spontaneous acceptors of one-haplotype class I-disparate grafts and two had been rendered tolerant to two-haplotype class I-mismatched kidneys with CsA induction. All six explanted allografts showed no histological evidence of rejection and all six retransplants were accepted without exogenous immunosuppression. These findings suggest that in miniature swine tolerance of class I-disparate kidneys is a stable, centrally mediated phenomenon that cannot be broken with a challenge of fresh donor antigen and donor-type APCs. Furthermore, successful retransplantation without immunosuppression in animals receiving CsA induction therapy for their first transplant suggests that graft adaptation is not necessary for the maintenance of tolerance.

Animals↗
PubMed1993

Bone marrow transplantation in miniature swine: IV. Development of myeloablative regimens that allow engraftment across major histocompatibility barriers.

Studies of the myeloablative regimens capable of permitting successful BMT across MHC barriers in miniature swine have been performed. To minimize graft-versus-host disease (GVHD), engraftment was studied in the F1-->P combination (i.e., MHC homozygous ["parental"] swine receiving bone marrow from one-haplotype matched MHC heterozygous ["F1"] donors). Animals given total body irradiation (TBI) up to 1100 cGy, 10 cGy/min, in a single dose failed to engraft. Increasing the dose rate led to unacceptable extramedullary toxicity without improving engraftment. Eleven different fractionated TBI regimens were tested in this F1-->parent model. At all of the dose rates tested, a total dose of less than 1000 cGy was insufficient for engraftment, and a total dose of 1400 cGy led to unacceptable toxicity. Between these extremes, a window was defined in which engraftment could be obtained without unacceptable extramedullary toxicity utilizing 2 equally divided fractions of TBI delivered 24 hr apart. The addition of 50 mg/kg cyclophosphamide i.v. to fractionated TBI (1150 cGy total dose [500 + 650]) also permitted engraftment, with decreased incidence of interstitial pneumonitis as compared to fractionated TBI (1300 cGy total dose [650 x 2]). Both of these regimens were also confirmed to permit engraftment between heterozygous donors and recipients sharing a single common haplotype ("F1-->F1"). The regimen of 1300 cGy (650 x 2) also permitted engraftment in completely MHC mismatched BMT, but with subsequent death from GVHD. These studies of the myeloablative regimens permitting engraftment across defined MHC barriers in miniature swine provide a basis for further studies of allogenic BMT and GVHD in this large animal preclinical model.

Animals↗
PubMed1996

Culture and characterization of hematopoietic progenitor cells from miniature swine.

Miniature swine are being used as a large animal model in which cultured and retrovirus-transduced hematopoietic stem cells (HSC) can be tested in a reproducible manner for their long-term in vivo repopulating ability. As part of these studies, long-term bone marrow culture (LTBMC) and progenitor colony assay systems were developed and used to characterize the in vitro growth potential and in vivo frequency of hematopoietic progenitors in this species. We found that LTBMCs initiated with a single marrow inoculum produced myeloid colony progenitors continuously for at least 7 weeks. The sites of myelopoietic activity in these cultures were uniquely restricted to isolated, morphologically diverse germinal centers rather than more disperse cobblestone patches. We also used the progenitor assay to screen several human and murine recombinant cytokines for cross-reactivity to swine bone marrow cells, including interleukin-3 (IL-3), IL-6, Il-11, granulocyte and granulocyte-macrophage colony-stimulating factors (G-CSF and GM-CSF), c-kit ligand (also called mast cell growth factor [MGF]), and erythropoietin (Epo). With the exception of human and murine IL-3, each of the cytokines tested induced swine progenitor colony formation to varying degrees, with some combinations leading to the formation of primitive multilineage and high proliferative potential colonies. Finally, in an attempt to characterize alternative sources of HSC from swine, we compared the progenitor content of adult and juvenile swine bone marrow and fetal liver. The fetal liver samples were found to be highly enriched for both primitive and mature progenitors, while analysis of postnatal marrow samples revealed an approximately two-fold decline in overall progenitor frequency between the ages of 10 and 20 weeks. Taken together, these studies demonstrate the development and use of in vitro culture methods for characterizing hematopoietic elements from miniature swine and suggest a hierarchy of progenitor cell content in various hematopoietic tissues from the large animal model.

Animals↗
PubMed1996

Fetal surgical protocols in Yucatan miniature swine.

Thirty-nine Yucatan miniature swine were used in three fetal surgical experimental protocols. They involved antiarrhythmic administration, pacemaker implantation, and in-utero diagnosis of ventricular septal defect by intraoperative echocardiography. Because of problems encountered with surgical protocols in the initial stages, modifications were made to prevent fetal hypothermia and intraoperative mortality. These modifications included environmental temperature support, staple surgical techniques to reduce operative time, and development of fetal catheters designed to facilitate cannulation of small vessels. Postoperative care protocols were intensive and included antibiotics, analgesics, and supportive care designed to reduce discomfort and prevent abortion and sepsis. Thirty-seven of 39 sows survived the surgical procedures; experiments were performed on 117 fetuses. Twenty-two fetuses died either intraoperatively or postoperatively because of complications related to the experimental protocols. Modification of surgical and postsurgical protocols for these projects demonstrates the feasibility of using miniature swine as a model for fetal surgery, when their use was appropriate for anatomic and physiologic reasons.

Animals↗
PubMed1998

Miniaturization of analytical systems.

Miniaturization has been a long-term trend in clinical diagnostics instrumentation. Now a range of new technologies, including micromachining and molecular self-assembly, are providing the means for further size reduction of analyzers to devices with micro- to nanometer dimensions and submicroliter volumes. Many analytical techniques (e.g., mass spectrometry and electrophoresis) have been successfully implemented on microchips made from silicon, glass, or plastic. The new impetus for miniaturization stems from the perceived benefits of faster, easier, less costly, and more convenient analyses and by the needs of the pharmaceutical industry for microscale, massively parallel drug discovery assays. Perfecting a user-friendly interface between a human and a microchip and determining the realistic lower limit for sample volume are key issues in the future implementation of these devices. Resolution of these issues will be important for the long-term success of microminiature analyzers; in the meantime, the scope, diversity, and rate of progress in the development of these devices promises products in the near future.

Clinical Laboratory Techniques↗
PubMed1998

The diagnostic value of transendoscopic miniature ultrasonic probe for esophageal diseases.

BACKGROUND AND STUDY AIMS: To investigate the value of a transendoscopic miniature ultrasonic probe (USP) in the diagnosis of esophageal diseases. PATIENTS AND METHODS: Endoscopic ultrasonography was performed by means of USP in 53 patients with esophageal diseases, including 16 with esophageal leiomyoma, 14 with esophageal carcinoma, seven with achalasia, seven with reflux esophagitis, six with esophageal polyps and three with esophageal varices. RESULTS: USP clearly showed all 16 esophageal leiomyomas, whereas, conventional EUS could not show five small leiomyomas less than 1.0 x 1.0 cm in size. The appearance of esophageal leiomyoma was that of a hypoechoic mass with a homogeneous inner echogram arising from the fourth hypoechoic layer. All 14 patients with esophageal carcinoma underwent full endosonographic T and N staging with USP. In two cases passage of the malignant stenosis proved to be impossible using conventional EUS. The accuracy of USP on T staging and N staging was 80% and 30%, respectively. In the seven achalasia patients USP demonstrated a seven-layer structure of the esophageal wall, with thickening of the third and fifth layers. In the seven patients with reflux esophagitis no difference was found for the ultrasonic image between that with and that without Barrett's epithelium. All of the esophageal polyps were showed by USP as hypoechoic homogeneous lesion with indistinct margins. After endoscopic sclerotherapy the ultrasonographic feature of esophageal varices changed from submucosal multiple anechoic areas to high echoic areas. CONCLUSION: With refinement, the transendoscopic miniature ultrasonic probe will play an increasing role in the diagnosis of esophageal disease.

Endosonography↗
PubMed1998

Temporal analysis of feeding behavior in miniature pigs.

The feeding behavior of four Munich miniature pigs (29-43 kg) housed in pairs of two, was observed for two consecutive weeks. Pigs were conditioned to operate a computer-controlled recording system equipped with feeders delivering a precise food release per response and were then fed ad libitum. In addition to the descriptive analysis of feeding behavior, Walsh-Fourier Spectral Analysis was utilized to investigate the temporal patterns of food ingestion and the synchronisation of feeding patterns between the animals housed together. The pigs had up to 48% of their daily energy intake during the dark cycle and there was a substantial reduction of food intake from the first to the second week. Furthermore, pigs housed together synchronized feeding behavior from week 1 to week 2. The recording system has been shown to be highly reliable and valid and provides an excellent tool for the investigation of ingestive behaviors in miniature pigs.

Animals↗
PubMed1982

Correlates of ovarian morphology, estrous behavior, and cyclicity in an inbred strain of miniature swine.

Ovarian morphology and behavioral relationships were studied in a group of nine miniature pigs with a characteristically small litter size (-5) and an average coefficient of inbreeding of 0.39. The first day of standing estrus was designed as day 0, Laparoscopy was used to evaluate and photograph ovarian activity on days 1, 5, 11, 17, and day 2 of the subsequent estrous cycle. Mean (+/-SEM) duration of estrus and the estrous cycle was 3.1 +/- 0.2 and 22.6 +/- 0.3 days, respectively. An average of 9.0 +/- 0.4 vesicular follicles developed/estrous period and 96% of the follicles showed morphologic evidence of ovulation resulting in a mean of 8.6 +/- 0.3 corpora lutea (CL) per animal. There was no significant correlation between the degree of inbreeding and number of vesicular follicles (r = 0.27) or CL (r = 0.28) developing/cycle within the experimental group. Mature preovulatory follicle and CL size ranged from 6-12 mm and 8-12 mm in diameter, respectively, and 22.5% of the newly formed CL contained distinct postovulatory stigmata. These data indicated that 1) temporal relationships of sexual behavior and gross ovarian morphology in the miniature pig were similar to descriptions previously reported for the standard pig, and 2) the reduced litter sized characteristic to this particular strain is, at least in part, due to decreased number of vesicular follicles developing during the estrous cycle.

Animals↗
PubMed2002

Miniaturized amperometric flow immunoassay system using a glass fiber membrane modified with anion.

This paper describes a miniaturized amperometric flow immunoassay system using a glass fiber membrane modified with anion. The glass fiber membrane was functionally modified with gamma-glycidoxypropyltrimethoxysilane and sodium thiosulfate and was used for separation of protein. Anti-human chorionic gonadotrophin (HCG) immunoglobulin G (IgG) antibody conjugated with ferrocenemonocarboxylic acid (Fc), namely, Fc-conjugated IgG (Fc-IgG), was used as a novel analytical reagent. HCG and Fc-IgG complexes were separated from free Fc-IgG based on differences in isoelectric point (pI) using the glass fiber membrane modified with a thiosulfonyl acid functional group. The assay yields a linear relationship between current and HCG concentration in the range of 0-2000 mIU/mL. This simple technique enables the assay of HCG within 2 min. The modified glass fiber membrane was regenerated by occasional elution with malonate buffer (pH 6.0) containing 0.5 M NaCl, to remove free Fc-IgG. Free Fc-IgG recovered in this manner could be reused up to eight times without significant decreases in sensitivity. This miniaturized amperometric flow immunoassay requires only minute quantities of serum and generates highly reproducible results.

Animals↗
PubMed2004

A stable miniature protein with oxaloacetate decarboxylase activity.

An 18-residue miniature enzyme, Apoxaldie-1, has been designed, based on the known structure of the neurotoxic peptide apamin. Three lysine residues were introduced on the solvent-exposed face of the apamin alpha-helix to serve as an active site for decarboxylation of oxaloacetate. The oxidised form of Apoxaldie-1, in which two disulfide bonds stabilise the alpha-helix, formed spontaneously. CD spectroscopy measurements revealed that, in its oxidised form, Apoxaldie-1 adopted a stably folded structure, which was lost upon reduction of the disulfide bonds. Despite its small size and the absence of a designed binding pocket, Apoxaldie-1 displayed saturation kinetics in its oxidised form and catalysed the decarboxylation of oxaloacetate at a rate that was almost four orders of magnitude faster than that observed with n-butylamine. This rivals the performance of the best synthetic oxaloacetate decarboxylases reported to date. Unlike those, however, Apoxaldie-1 displayed significant stability. It maintained its secondary structure at temperatures in excess of 75 degrees C, in the presence of high concentrations of guanidinium chloride and at pH values as low as 2.2. Apamin-based catalysts have potential for the generation of miniature peptides that display activity under nonphysiological conditions.

Amino Acid Sequence↗
PubMed1992

Focal, extracellular recording of slow miniature junctional potentials at the mouse neuromuscular junction.

Miniature endplate potentials (MEPPs) with slow rising phase can be attributed either to burst of transmitter releases or to distortion of conduction from remote releasing sites. The spontaneous activity of neuromuscular junctions recorded extracellularly at mouse diaphragms using sharp electrodes was analyzed to test these two hypotheses. The miniature junctional potentials (MEJPs) frequencies observed intracellularly as compared to MEPP frequency measured intracellularly in controls indicate that most events recorded extracellularly are induced by the presence of the electrode. All types of MEPPs (bell-MEPPs, skew-MEPPs, slow-, and giant MEPPs) previously described with intracellular recording methods (Vautrin and Kriebel, Neuroscience 41:71-88, 1991) were observed extracellularly and showed similar characteristics. This means that the presynaptic and postsynaptic zones that generate these synaptic events are restricted within areas of a few micrometers squared of synaptic contact. Long rise times of extracellularly recorded synaptic spontaneous events may be explained by multiple transmitter releases at intervals shorter than the rise time of individual events, which postsynaptic responses fuse into a single peak.

Animals↗
PubMed2005

Comparison of a miniaturized shake-flask solubility method with automated potentiometric acid/base titrations and calculated solubilities.

Solubility is one of the most important parameters for lead selection and optimization during drug discovery. Its determination should therefore take place as early as possible in the process. Because of the large numbers of compounds involved and the very low amounts of each compound available in the early development stage, it is highly desirable to measure the solubility with as little compound as possible and to be able to improve the throughput of the methods used. In this work, a miniaturized shake-flask method was developed and the solubility results were compared with those measured by semiautomated potentiometric acid/base titrations and computational methods for 21 poorly soluble compounds with solubilities mostly in the range 0.03-30 microg/mL. The potentiometric method is very economical (approximately 100 microg of a poorly soluble compound is needed) and is able to create a pH/solubility profile with one single determination, but is limited to ionizable compounds. The miniaturized shake-flask method can be used for all compounds and a wide variety of media. Its precision and throughput proved superior to the potentiometric method for very poorly soluble compounds. Up to 20 compounds a week can be studied with one set-up. Calculated solubility data seem to be sufficient for a first estimate of the solubility, but they cannot currently be used as a substitute for experimental measurements at key decision points in the development process.

Algorithms↗
PubMed1992

Development of GABAergic connections in vitro: increasing efficacy of synaptic transmission is not accompanied by changes in miniature currents.

Development of inhibitory synaptic transmission was studied using a dissociated cell culture from the superior colliculus of neonatal rat. Patch-clamp recordings in the whole-cell configuration were performed to measure evoked (single-cell-activated) inhibitory postsynaptic currents (IPSCs), miniature IPSCs and current responses to maximal concentrations of exogenous gamma-aminobutyric acid (GABA). Over a period of 3 weeks in vitro (DIV3-24), the fraction of synaptically coupled neurons raised from 0% to 76%. Evoked IPSCs were first observed at DIV5. They had an average amplitude of 33.9 pA during the first week (n = 13) and 129.7 pA during the fourth week (n = 48). This increase by a factor of 3.8 represents a significant rise in the efficacy of GABAergic transmission during in vitro development. However, no developmental change has been observed in the average amplitudes of miniature somatic IPSCs. The latter remained at an average level of about 9 pA (symmetrical chloride concentration and a driving force of 68 mV). No increase was found also in whole-cell current densities induced by saturating concentrations of exogenous GABA. Our results suggest that under the given conditions, synapse maturation was primarily the result of presynaptic sprouting. This conclusion is further supported by bouton counts in immunostained collicular cultures, where the number of axosomatic and axodendritic GABAergic contacts per neuron increased from 0.54 and 0.37, respectively, at DIV3, to 13.84 and greater than 23.1, at DIV24. The overall density of GABAergic neurons decreased during this period from about 41,000/cm2 to 15,600 cm2, indicating that a growing number of contacts is formed by a declining number of presynaptic neurons.

Animals↗