Nephrosplenic entrapment of the large colon in 142 horses (2000-2009): analysis of factors associated with decision of treatment and short-term survival.

REASONS FOR PERFORMING STUDY: Previous studies indicate similar overall survival of horses with nephrosplenic entrapment of the large colon (NSE), regardless of treatment strategy. Short-term survival of a primarily conservative treatment strategy without rolling under general anaesthesia (GA) and a low proportion of surgical intervention as well as indicators of short-term nonsurvival has not been documented. OBJECTIVES: To document short-term survival of horses with NSE treated in a university referral hospital with a low rate of surgical interventions and to determine factors associated with the decision of treatment and short-term nonsurvival. METHODS: A retrospective review of medical records of 142 horses diagnosed with NSE between January 2000 and October 2009 was undertaken. Case details and clinical parameters from the initial examination, treatment and outcome were recorded. Factors associated with decision of treatment and short-term survival were identified by multiple logistic regression analysis. RESULTS: Warmblood breeds were over-represented in comparison to the general colic population. Overall short-term survival was 91.5% (130/142) which is similar to previous studies. Three horses considered to be in need of surgery were subjected to euthanasia for economical reasons before treatment. Of 114 conservatively treated horses, 110 (96.5%) survived, as did 20/25 (80%) of surgically treated horses. Nine conservatively managed horses were treated with phenylephrine. Gastric reflux (P = 0.0077), pain (P = 0.024) and abdominal distension (P = 0.05) were associated with the decision to treat surgically. Increased heart rate (P<0.001), and surgery (P = 0.032) were associated with reduced likelihood of short-term survival. CONCLUSIONS AND POTENTIAL RELEVANCE: Overall short-term survival was similar to that reported in previous studies with higher proportions of surgically managed cases. Consequently, horses with NSE should be managed by a primarily conservative treatment strategy, with the decision to treat surgically based on specific evidence based criteria.

Biomechanical, histologic and macroscopic assessment of articular cartilage in a sheep model of osteoarthritis.

OBJECTIVES: Our primary objective was to explore the full potential of the ovine medial meniscectomy (MMx) model of early osteoarthritis (OA) for studies to validate non-destructive articular cartilage (AC) assessments and therapeutic interventions. Our secondary objective was to re-evaluate the relationships between the different types of AC assessment after MMx in sheep. METHODS: Macroscopic assessments, dynamic shear modulus (G*), phase lag and AC thickness measurements were performed at a total of 5437 reference points on all six articular surfaces in four normal joints and 16 MMx ovine stifle (knee) joints. Comparisons with histologic assessments of gross structural damage, collagen organisation (birefringence) and proteoglycan content were possible at 702 of these points. RESULTS: Histologic gross structural damage and proteoglycan loss were seen throughout the joint with greatest severity (fibrillation) in closest proximity to the MMx site. Increases in AC (30-50%) thickness, reductions in G* (30-40%) and collagen birefringence intensity (15-30%) occurred more evenly throughout the joint. Macroscopic softening was evident only when G* declined by 80%. G* correlated with AC thickness (rho=-0.47), collagen organisation rho=0.44), gross structural damage (rho=-0.44) and proteoglycan content (rho=0.42). Multivariate analysis showed that collagen organisation contributed twice as much to dynamic shear modulus (t=6.66 as proteoglycan content (t=3.21). Collagen organisation (rho=0.11) and proteoglycan content (rho=0.09) correlated only weakly to phase lag. CONCLUSIONS: Macroscopic assessments were insensitive to AC softening suggesting that arthroscopic assessments of AC status might also perform poorly. Collagen integrity was more important for the maintenance of AC stiffness (G*) than proteoglycan content. The development of major AC softening and thickening throughout the joint following MMx suggested involvement of non-mechanical (e.g., protein and biochemical) chemical and cytokine mediated processes in addition to the disturbance in biomechanical loading. The ovine MMx model provides a setting in which the spectrum of AC changes associated with the initiation and progression of OA may be evaluated.

Safety and efficacy of radiofrequency ablation of brain: a potentially minimally invasive treatment for brain tumours.

INTRODUCTION: Cerebral metastases are associated with a very poor prognosis. The best survival results are seen after surgical resection. However this involves a relatively invasive procedure and many patients are not suitable for surgical resection. We have evaluated the safety and efficacy of radiofrequency ablation of the brain in a sheep model. METHODS: We produced ablations of 1 - 3 cm diameter in the brain of sheep using the RITA starburst XL probe and RITA 1500 generator. We varied the time of RF application between 1 minute and 5 minutes and observed the animals for between 24 hours and 3 weeks for short-term and long-term effects and measured the intracranial pressure (ICP) in 2 animals following RFA. RESULTS: A total of 8 ablations were produced in 8 sheep. There was no change in the ICP measurements and there were no neurological complications in the 5 sheep with superficial ablation of up to 2 cm. Three sheep failed to regain consciousness due to large ablations near the brain stem and cerebellum. The sizes of the ablations were confirmed on necropsy and there was no other evidence of damage to the surrounding brain. Satisfactory ablation of brain was achieved at 70 degrees C and an ablation time as short as 3 minutes produced a 1.5 - 2.0 cm diameter of ablation. CONCLUSION: Cerebral RFA is a relatively safe and effective technique capable of producing a predictable ablation with no damage to surrounding brain. The potential of this technique requires further evaluation but likely advantages include the ability to treat multiple tumours and perform repeated treatment with a minimally invasive approach.

Application technique and slurry co-fermentation effects on ammonia, nitrous oxide, and methane emissions after spreading: I. Ammonia volatilization.

Ammonia emissions after spreading animal manure contribute a major share to N losses from agriculture. There is an increasing interest in anaerobic co-digestion of liquid manure with organic additives. This fermentation results in a change of physical and chemical parameters of the slurry. Among these are an increased pH and ammonium content, implying a higher risk of NH3 losses from fermentation products. To compare different application techniques and the effect of fermentation on NH3 volatilization, we used the standard comparison method and tested it for reliability. This method seems to be perfectly suited for experiments with a large number of treatments and replicates if prerequisites concerning the experimental layout are considered. We tested four different application techniques on arable and grassland sites. The more the substrate was incorporated into the soil or applied near the soil surface on the grassland site, the less NH3 was lost. Injection of the substrate reduced losses to less than 10% of applied NH4+ on both sites, whereas losses after splash plate application amounted to more than 30%. Trail shoe application on grassland performed as well as injection. Harrowing on arable land also reduced emissions efficiently, if harrowing occurred within the first 2 h after application. Emissions from trail hose-applied co-fermentation product were not greater than from unfermented slurry. Better infiltration of the less viscous substrate seemed to have compensated for the increased loss potential.

Application technique and slurry co-fermentation effects on ammonia, nitrous oxide, and methane emissions after spreading: II. Greenhouse gas emissions.

The aim of this study was to investigate the effect of different application techniques on greenhouse gas emission from co-fermented slurry. Ammonia (NH3), nitrous oxide (N2O), and methane (CH4) emissions were measured in two field experiments with four different application techniques on arable and grassland sites. To gather information about fermentation effects, unfermented slurry was also tested, but with trail hose application only. Co-fermented slurry was applied in April at a rate of 30 m3 ha(-1). Measurements were made every 4 h on the first day after application and were continued for 6 wk with gradually decreasing sampling frequency. Methane emissions were <150 g C ha(-1) from co-fermentation products and seemed to result from dissolved CH4. Only in the grassland experiment were emissions from unfermented slurry significantly higher, with wetter weather conditions probably promoting CH4 production. Nitrous oxide emission was significantly increased by injection on arable and grassland sites two- and threefold, respectively. Ammonia emissions were smallest after injection or trail shoe application and are discussed in the preceding paper. We evaluated the climatic relevance of the measured gas emissions from the different application techniques based on the comparison of CO2 equivalents. It was evident that NH3 emission reduction, which can be achieved by injection, is at least compensated by increased N2O emissions. Our results indicate that on arable land, trail hose application with immediate shallow incorporation, and on grassland, trail shoe application, bear the smallest risks of high greenhouse gas emissions when fertilizing with co-fermented slurry.

Serum withdrawal induces a redistribution of intracellular gelsolin towards F-actin in NIH 3T3 fibroblasts preceding apoptotic cell death.

The intracellular distribution of gelsolin in NIH 3T3 cells was examined by immunostaining using affinity-purified polyclonal gelsolin antibodies before and after induction of apoptosis by serum withdrawal. Serum deprivation induced detachment of an increasing number of NIH 3T3 cells, but also apoptosis in attached cells as verified morphologically by chromatin condensation, nuclear fragmentation and labelling of their periphery by FITC-annexin V. Ongoing apoptosis was also demonstrated by activation of caspase-3 activity and chromatin cleavage into high-molecular-mass fragments, although no internucleosomal chromatin degradation (DNA-ladder formation) was detected. When cells were maintained in the presence of 10% foetal calf serum, gelsolin immunoreactivity was evenly distributed in the cytoplasm. No obvious co-localisation of gelsolin and the actin-containing stress fibres was detected under these conditions. At day one after serum withdrawal, a redistribution of gelsolin to actin filaments was detected within a few attached cells by double fluorescence staining. The number of cells exhibiting this redistribution increased at days two to four. In addition, the stress fibres increased in thickness and their length was continuously reduced. At day four, many cells contained shortened stress fibres, which had lost their longitudinal orientation. Additionally, the cytoplasm of a number of attached cells was highly condensed around their nuclei and a homogenous distribution of both gelsolin and actin was detected in the remaining cytoplasmic rim. Up to day two, these effects were reversible after re-addition of serum to attached cells. A similar redistribution of gelsolin immunore-activity was observed after induction of apoptosis by cycloheximide, but not after initiation of necrosis by hydrogen peroxide. In NIH 3T3 cells no alteration in the expression of gelsolin at the level of protein (Western blot) or specific mRNA (Northern blot) was observed after serum withdrawal. Using Western blotting, no proteolysis of gelsolin was detected up to day 4, although caspase-3 activity was found to have increased fivefold after serum withdrawal. These results suggested that in these cells F-actin severing might occur in the absence or advance of gelsolin cleavage by caspases. Intact gelsolin on its own may be sufficient for the dissolution of the microfilaments, since micro-injection of gelsolin into primary bovine lens cells led to a transient disappearance of the stress fibres and to a reduction of their attachment area to the substratum. In NIH 3T3 cells similar effects of micro-injected gelsolin were only observed at day one after serum withdrawal.

Different sublines of Jurkat cells respond with varying susceptibility of internucleosomal DNA degradation to different mediators of apoptosis.

The ability of two different Jurkat sublines, termed standard and JM, to form DNA ladders was investigated after various apoptotic stimuli. Exposure to a broad spectrum of drugs interfering with signal transduction or cellular metabolism revealed distinct differences between both Jurkat sublines with regard to the pattern of DNA degradation. In standard Jurkat cells, internucleosomal DNA cleavage occurred only after treatment with the protein kinase inhibitor staurosporine. In contrast, the JM subline responded with internucleosomal DNA fragmentation to exposure to gemcitabine, cycloheximide or staurosporine. All drugs induced the formation of DNA fragments of about 50 kb in both sublines, as revealed by pulse field electrophoresis, except H2O2, which caused unspecific DNA degradation. The staurosporine-induced DNA ladder formation was accompanied by an increase in caspase-3 activity in both lines which, however, was considerably lower in Jurkat JM cells after gemcitabine or cycloheximide exposure. When the analysis of internucleosomal DNA degradation was carried out after mycoplasma infection, both Jurkat lines responded with DNA ladder formation after exposure to all drugs used (here only shown for the standard subline). Employing the zymogram technique, nuclease activities of 47 kDa and 54 kDa were detected in culture supernatants, cell homogenates and nuclear extracts only when mycoplasma-infected, whereas the samples obtained from mycoplasma-free sublines were nuclease-negative using this technique, indicating that these endonucleases were of mycoplasmal origin. After drug exposure, the mycoplasmal nucleases must have gained access to the cytoplasm and nuclei of their host cells by an unknown mechanism.

The role of latency in mandibular osteodistraction.

Even though osteodistraction has been well established in the extremities, the parameters used in craniofacial distraction have been essentially borrowed from orthopaedic experience. Latency is widely practised but its relevance has not been fully investigated. The purpose of this study was to establish the role of latency in mandibular distraction osteogenesis. Twenty-two growing Wethers sheep were allocated to four experimental groups. Six animals were allocated to each of Groups A, B and C and underwent bilateral mandibular corticotomies and attachment of an external lengthening device. Latent periods of 0, 4 and 7 days respectively were observed prior to beginning distraction. The distraction protocol consisted of a rate of 0.5 mm twice daily for 20 days, followed by a consolidation phase of 20 days after which the sheep were killed. Histology, bone densitometry and 3-point mechanical testing were performed on the harvested mandibles. Group D formed the control group (n = 4). Histologically, the distracted bone exhibited bone formation primarily via intramembranous ossification with scattered islands of cartilage. The regenerated bone had mechanical properties significantly weaker than the undistracted control group (P < 0.05), but between the experimental groups no statistically significant differences were demonstrable either in mechanical strength or DEXA density. These data indicate that a change in latency does not alter the properties of the regenerated bone in mandibular distraction osteogenesis and indeed no latent interval may be necessary at all in craniofacial distraction. This has implications for the duration of device fixation in distraction procedures.

Mycoplasma nucleases able to induce internucleosomal DNA degradation in cultured cells possess many characteristics of eukaryotic apoptotic nucleases.

It was previously shown (Paddenberg et al (1996) Eur J Cell Biol 69, 105 - 119) that cells of established lines like NIH3T3 fibroblasts and the human pancreatic adenocarcinoma PaTu 8902 line only degrade their chromatin at internucleosomal sites after an apoptotic stimulus when infected with Mycoplasma hyorhinis. In order to distinguish mycoplasma nucleases (Mr 47 - 54 kDa) from already described eukaryotic apoptotic enzymes, the mycoplasma nucleases were partially purified from serum-free culture supernatants and further characterized. Here we demonstrate directly that the enriched mycoplasma nucleases were able to fragment the DNA of nuclease-negative substrate nuclei at internucleosomal sites. The DNA degradation was accompanied by morphological changes typical of apoptosis like chromatin condensation and margination followed by shrinkage of the nuclei. The biochemical characterization revealed that the mycoplasma nucleases had a neutral to weakly basic pH-optimum. They required both calcium and magnesium in the mM range for maximal activation and were inhibited by zinc chloride, EGTA and EDTA. In two dimensional zymograms they migrated as three spots with isoelectic points between 8.1 and 9.5. They were not inhibited by monomeric actin. Our data also demonstrate that nuclear extracts prepared from nuclei isolated from Mycoplasma hyorhinis infected cells contained the mycoplasma nuclease activities leading to their internucleosomal DNA-degradation after incubation in the presence of calcium and magnesium.

Internucleosomal DNA fragmentation in cultured cells under conditions reported to induce apoptosis may be caused by mycoplasma endonucleases.

DNA fragmentation is a common biochemical hallmark of apoptosis. It is catalyzed by endogenous Ca2+, Mg(2+)-dependent endonuclease(s). Although the exact identity of the apoptotic endonuclease is still a matter of debate, a number of candidate nucleases have been proposed like NUC18, DNase II and DNase I. Relatively large amounts of nucleases are also expressed by mycoplasmas, cell wall-less bacteria of the class Mollicutes, which are found as contaminants in up to 45% of the continuous cell lines in current use. In order to clarify the effect of these pathogens on the investigation of apoptosis in cell culture systems, we looked for biochemical markers (DNA fragmentation, nuclease expression) and morphological changes characteristic of apoptosis (cell shrinkage, chromatin condensation, apoptotic bodies) in Mycoplasma hyorhinis-free and -infected cultures of the human pancreatic adenocarcinoma cell line PaTu 8902 and of mouse NIH 3T3 fibroblasts. For that purpose we employed cells cultured under standard conditions and cells exposed to the protein synthesis inhibitor cycloheximide, which is known to induce apoptosis in various cell systems. After exposure to cycloheximide only the mycoplasma-positive cells exhibited internucleosomal DNA degradation. In contrast, nuclease activities in the molecular range of 47 to 54 kDa were detected in cell homogenates and culture supernatants of infected cultures of both control and cycloheximide-treated cells, whereas mycoplasma-free cultures were nuclease-negative. The expression of the nucleases and the cycloheximide-induced DNA fragmentation were suppressed by the prokaryote-specific protein synthesis inhibitor chloramphenicol. Moreover, partially purified nucleases from supernatants of infected cells were able to cleave the DNA of isolated substrate nuclei at internucleosomal sites. These data indicate that DNA ladder formation in cell culture systems can also be caused by mycoplasmal nucleases which apparently penetrate the host cells after cycloheximide treatment or more generally after cellular stress. Therefore, internucleosomal DNA fragmentation in established cell lines has to be regarded with care, unless mycoplasmal infection can be excluded, or the existence of endogenous endonucleases can be proven. The presence of endonucleolytic activities of about 47 to 54 kDa molecular mass has now to be regarded as highly indicative of contaminations with M. hyorhinis. In contrast, the expression of an apoptotic morphology was not restricted to infected cells; in both mycoplasma-free and -contaminated cultures, cells with condensed chromatin were observed after staining with the DNA binding dye Hoechst 33342. Electron microscopic studies revealed that most of the cells containing compacted DNA were phagocytosed by unaffected fellow cells. Presumably because of the relatively long exposure (72 h) to cycloheximide we also observed secondary necrosis as indicated by the parallel occurrence of morphological characteristics of apoptosis (chromatin condensation) and necrosis (loss of membrane integrity and organelle swelling).