Tierärztliche Fakultät - Digitale Hochschulschriften der LMU - Teil 01/07 Archiv

Ziel dieser Studie war es, den knochenanabolen Effekt einer zyklischen Monotherapie mit Calcitriol bei weiblichen SHAM- und OVX-Ratten zu testen. Das Ergebnis der Studie hat gezeigt, dass eine zyklische Monotherapie mit Calcitriol in der Lage ist, die Knochenmasse in einem normalen oder osteopenischen Skelett zu erhöhen und legt nahe, daß sich diese Therapieform möglicherwise zur Behandlung der humanen Osteoporose eignet.

5.1 Analysis and characterization of the mouse Hic2 gene Like Hic1 and γFBP (chicken), Hic2 cDNA coded five Krüppel-type C2H2 zinc finger domain. Through the sequencing and comparation with different protein sequences from the homolog proteins from different species (HIC1, Hic1, HIC2, γFBP, and Hypothetical protein), Hic2 protein shares more than 80% homology with HIC1 through the BTB/POZ and zinc finger domains, and both proteins have identical GLDLSKK/R motifs. A new part of Hic2 gene coding, exon two, and new exon one deduced full-length Hic2 protein contains 601 amino acids. Hic2 gene has two Exons (240 and 1842 bp), with one Intron (2182 bp). The location of the gene is mouse chromosome 16 (B1, UniGene Cluster Mm.103787 Mus musculus). The human gene HIC2 maps to chromosome 22q11.2, and is a homolog of the HIC1 candidate tumor suppressor gene located at 17p 13.3. (Deltour et al. 2001). Upstream from the TATA box MatInspector predicted different transcription binding sites. Between them Wilms Tumor Suppressor and p53 are most interesting transcription sites for the Hic2 gene. That’s why the Hic2 promoter activity was checked. A 1.2 kb promoter fragment of Hic2 has been characterized in a gene reporter assay system. The peak activity of the Hic2 promoter is associated with the total fragment, the next higher activity is in the fragment which included Wilms Tumor Suppressor and p53 transcription sites. The expression of the mouse Hic2 was investigated by in situ hybridisations (ISHs) of whole mount embryos and paraffin sections. Hic2 expression was detected in restricted territories of the brain, sinus centralis, olfactory bulb, canallis centralis medullae spinalis, embryonic ectoderm (neuroepithelium of neural tube), and small intestine. Because of its expression in the central nervous system, and mapping position of its human homolog HIC2, Hic2 could be involved in some syndromes. Patients with a 22q11 deletion have disrupted brain development which may involve abnormal neural crest cell migration, (Van Amelsvoort et al., 2001). It is now recognized that the 22q11.2 deletion syndrome encompasses the phenotypes previously described as DiGeorge syndrome (DGS) and velocardiofacial syndrome (Shprintzen syndrome),(Thomas and Graham 1997).

The aim of this study was to find a reliable alternative to the usually employed method of serological blood examination of sows, since taking blood samples from pigs, especially from sows, is very difficult and causes considerable stress for the animals. From July 2002 until May 2003 milk and blood samples from 180 sows and three piglets per litter were taken in a livestock of 128 sows and about 1000 fattening pigs. All samples were tested by ELISA for antibodies against eight important infectious diseases in pig herds (Aujeszky's Disease (AD), Swineinfluenza (SI), Mycoplasma hyopneumoniae Infection (EP), Porcine Parvovirus Infection (PPV), Porcine Reproductive and Respiratory Syndrome (PRRS), Transmissible Gastroenteritis (TGE), Porcine Respiratory Coronavirus Infection (PRCV) and Sarcoptes suis mange). All dams were vaccinated regularly against Influenza and PPV and were treated for mange before being moved into the farrowing unit. In the first week of life all piglets were vaccinated against EP. Milk samples were taken on the 1st, 2nd, 3rd and 7th day p.p. (post partum). Blood samples of the sow and the piglets were taken on the 4th day p.p. All samples were analysed by commercial ELISA-testkits, though only Hyoptest II and Sarcoptest are commercially used for serological milk testing. The results of the sows' blood testing served as the basis for the evaluation of the colostrum, milk and piglets' blood samples. Sensitivity, specifity, positive and negative predictive factor were determined. Further more the development of the milk antibody concentration p.p. and a comparison of sows' and piglets' antibody titers was demonstrated. The results for the seroprevalence of the blood tested sows are as follows: AD 0 %, SI 93 %, EP 46 %, PPV 77 %, PRRS 0,7 %, TGE 4 %, PRCV 65 % and mange 4 %. The following results were established: I. Aujeszky's Disease: The suitability for ELISA testing is questionable because of lacking seroprevalence. The piglets' blood testing is a suitable alternative to sows' blood testing. II. Influenza: The milk antibody titer is constant from the 1st until the 7th day p.p. Colostrum does not seem to be as suitable as milk from the 2nd day p.p. onwards. The serological piglets' blood testing is a very reliable alternative. III. M. hyopneumoniae: From the 3rd day p.p. onwards the antibody titer falls below the cut off limit. Colostrum may be a suitable alternative, whereas the use of milk taken from the 2nd day p.p. is limited. Milk from the 3rd day p.p. onwards cannot be used as a replacement for blood testing. Piglets' blood testing seems to be suitable. It has to be mentioned that there are some false positive results in both milk and piglets' blood test results. IV. PPV: The antibody titer is constant throughout the whole period. Both colostrum and milk samples as well as piglets' blood can be used for antibody detection instead of sows' blood. V. PRRS: Both colostrum and milk as well as piglet's blood can serve as an alternative for sows' blood testing. VI. TGE: The serological colostrum and milk examination cannot be used for detecting antibodies against TGE virus, whereas piglets' blood testing can be used as an alternative. VII. PRCV: The milk antibody titer is constant from the 1st until the 7th day p.p. The use of milk and colostrum as an alternative for sows' blood testing is questionable. Piglets' serological examination seems to be suitable. VIII. Mange: Even though colostrum testing is already used in mange herd surveillance in Switzerland, according to this study, the suitability of colostrum is questionable. Milk from the 2nd day p.p. onwards cannot be used for detecting antibodies against Sarcoptes suis. Further more the serological blood testing of piglets is questionable, because of the high number of false positive results.

Ziel der Arbeit war es, den Immunglobulinstatus von neugeborenen Ferkeln in den ersten Lebenswochen und die Effekte einer Fütterung der oberirdischen Pflanzenteile von Echinacea pupurea nach dem Absetzen zu untersuchen. Die IgG-und IgA-Konzentrationen sollten als wichtige Parameter des systemischen (IgG)und des lokalen (IgA) Immunsystems den Immunstatus der Schweine widerspiegeln. Eine hohe Immunglobulin-Konzentration führt zu geringerer Krankheitsanfälligkeit und lässt damit Rückschlüsse auf das Befinden der Tiere zu. Die Untersuchungen des IgG bestätigten, dass die Ferkel in dem Zeitraum vom 20. bis 49. Tag p.n. ("immunologische Lücke") wenig Schutz durch IgG besaßen. Somit wäre eine mögliche Steigerung der Abwehrbereitschaft (höhere Immunglobulin-Spiegel) durch die Fütterung von Echinacea purpurea eine Möglichkeit, den Ferkelerkrankungen in diesem Zeitraum vorzubeugen.

Summary Microsurgical reconstruction of cavernosal nerves within silicone tubes seeded with Schwann cells in rats. Resection of the cavernosal nerves is often necessary during radical prostatectomy and causes erectile dysfunction. Autologous nerve transplantats seemed to be the treatment of choice in the past. Further detailed knowledge of the regeneration of peripheral nerves and the importance of Schwann cells provides new investigation in this topic over the last few years. Using rats as a model, we wanted to show that silicone tubes seeded with Schwann cells provide a better environment for regeneration of peripheral nerves. Resection of 0.5 cm of the cavernosal nerve was performed on both sides of Fischer 344 rats. One group received a bilateral nerve-grafting of the genitofemoral nerve, another group had implantation of empty silicone tubes and the other group had implantations of silicone tubes seeded with autologous Schwann cells. The positive control group under went a sham operation with laparotomy only. A group with resection of the cavernosal nerves on both sides served as negative control. 3 months post operation, erectile function and pressure changes in the corporal bodies are evaluated by electrostimulation of the regenerated nerves. Comparison of empty tubes, nerve grafts and those filled with homologous Schwann cells show a higher rate of erections and increased intracavernosal pressure elevation of the Schwann cell group. 3 months after the implantation of tubes with Schwann cells, erections rise up 91%. By interposition of empty silicone tubes the erection rate is only 50% and nerve grafting results in an erection rate of 30%. Histological examination reveals a more organized and thicker nerve by using silicone tubes seeded with Schwann cells.

Laser osteotomy has several potential advantages in comparison to classical bone incision by mechanical saws. In previous studies the bone healing was delayed due to the damage of the thermal effects compared to mechanical instruments like the bone saw. With 7 Beagles as an experimental model, a comparative study was done to assess the healing of bone in response to osteotomy by a new modified carbon dioxide laser system and a mechanical bone saw. Using sterile technique, an approximately 6 mm deep incision was made in the ventral aspect of both mandibles with each method. During the follow-up period three intravital stains were injected to mark the surface of new-bone growth. After the dogs were killed 22 days following the surgical procedure the tissue response was examined by polychrome sequential labelling and undecalicfied paragon-stained ground sections. The sawed mandibles demonstrated a retarded rate of healing and a persistent superficial defect at the impact site. The results of the laser group were quite different. Some of the specimen showed a similar delay in bone healing like in the saw group. In some cases a large layer of carbonised debris was recognized. This retarded healing was most likely due to a surface deposit of charred tissue elements, produced by the thermal effects of the laser. In a third group of laser specimen showed a complete reconstruction of the incision by new Haversian bone and no signs of any damage by thermal effects due to the laser application. This modified carbon dioxide laser appears to be an effective and precise bone ablator. According to the results of previous studies the appearance of carbonisation could be prevented in some cases. If the key is found to reduce the risk of carbonisation to the maximum the future is open for the laser in orthopaedic surgery.

Examinations concerning the microbiological and hygienic quality of marinated meat preparations in order to determine guidelines to control the best-before-date In this study 52 marinades, 5 seasoning mixtures and 1 lemon seasoning were surveyed at the time of delivery and again following 4 weeks of refrigeration at + 6 °C ± 2°C. An analysis was also performed on self-made meat preparations consisting of pork respectively venison marinated in three different marinades. The marinated meat samples were produced and examined in duplicate to increase statistical accuracy. The examinations were carried out after 1 and 2 weeks of cold storage and after 6 weeks of deep freezing in order to determine the best-before-date of these self-service-products. The effect of an interruption in the cold chain was also examined as follows: 3 hours interruption for cold storage products and 5 hours for frozen products. This procedure was chosen to simulate the interruptions occuring during transport from dealer to customer or from customer to customer. All samples were examined in the following three categories: sensory examination, microbiological survey, and pH value. The sensory examination included “general characteristics”, “external qualities”, “consistency”, “appearance”, “odor”, “taste” and “haptic consistency”. Taste and haptic consistency were only determined for the marinades and seasoning mixtures, as the marinated meat could only be examined in raw condition and could therefore not be analysed concerning taste. The microbiological survey included the examination of total viable count, B. cereus, Enterobacteriaceae, E. coli, C. perfringens, lactic acid bacteria, coagulase-positive staphylococci and salmonellae. The pH-value was measured by means of a pH-meter if possible. In the case of oil-containing marinades the pH-value was determined using pH-indicator paper. According to the sensory examinations, neither the marinades nor the seasoning mixtures had to be rejected after the initial examination nor after the 4 weeks storage. The high intensity of taste can be explained by the very spicy character of the concentrated marinades. The marinades are generally meant to be used as a taste enhancing component in meat preparations and are not intended for direct consumption. The sensory examination of the marinated pork samples revealed a musty and odor compo-nent in 33 % of the samples after 1 week and in 100 % after 2 weeks of cold storage. According to sensory examinations 67 % of the samples were eventually classified as spoilt after 2 weeks. Only 33 % of the deep frozen samples were judged as musty and dull while none of those samples was spoilt after 6 weeks storage. Such sensory defects as a slimy and rope forming consistency were observed in 33 % of the samples after 2 and 6 weeks of storage, while 67 % of the frozen samples showed separation of fat within the marinades. The sensory examination of the marinated venison samples revealed that 17 % had a musty and dull odor after 1 week, increasing to 50 % after 2 weeks of cold storage. In 33 % of the samples frozen for 6 weeks a musty and dull odor was observed. Putrid smell and spoilage could be detected in 33% of the samples after 1 week, in 67% after 2 weeks cold storage and in none of the samples after deep freezing. 33 % of the samples were slimy and rope forming after 1 as well as 2 weeks of cold storage. Separation of fat occurred only in the frozen samples (67 % of the samples). No significant differences could be observed between samples subjected to an interruption of the cold chain and samples held at constant temperature. The microbiological survey of the marinades revealed no occurrence of pathogenic bacteria in critical amounts, with the exception of the detection of B. cereus and C. perfringens in small quantities. Both species of bacteria are to be considered innocuous, because the marinades are subjected to heat treatment together with the meat thereby destroying the toxins. Only the emetic toxin produced by B. cereus is heat resistant and could be a threat to the consumer. In general, the total viable count in marinade samples obtained from containers used in the production process of self-service-products was higher than that of the samples taken directly out of the marinade producers’ original packing. In marinated pork the number of microbes was relatively high in comparison to the untreated meat. The increase in microbes can be attributed to suboptimal conditions in manual processing of the meat preparation. In industrial processing under optimal hygiene circumstances an expiration date of 1 week to 10 days can be justifiable. The microbiological results of deep freezing suggest that this method can be regarded as a viable alternative for ensuring longer storage life and better quality. The examination of raw venison already yielded critical results with regard to its suitability for meat preparations used for barbecuing. The examination of marinated venison further strengthened those results. Only as frozen product a suitable quality could be guaranteed for a longer period because most microbes do not survive a deep freezing process. The pH measurements did not provide any indication of the grade of spoilage. Only after 2 weeks of cold storage a slight decrease in pH was observed, which might indicate progressive spoilage. Generally, the increase in microbiological counts correlated with sensory defects and the duration of storage. Yet the number of microbes showed a slightly earlier and more noticable rise than was to be expected based on the sensory examinations carried out in the same time period.

The reaction product of phospholipase D (PLD), phosphatidic acid (PA), was found to stimulate phosphatidylinositol-4-phosphate-5-kinase (PIP-5-kinase) activity in vitro. In the present study, we have examined wether PLD affects the synthesis of phosphatidylinositol 4,5-bisphosphate (PIP2) by PIP-5-kinase. Overexpression of PLD isoforms in HEK-293 cells led to an increase in PIP-5-kinase activity and to elevated PIP2 levels in intact cells. As both PLD and PIP-5-kinase are stimulated by the GTPases Arf1 and RhoA, we investigated in the following, if PLD is involved in the regulation of PIP2 synthesis by these GTPases. Both PLD1- and PLD2-induced PIP2 synthesis was completely blocked by coexpression of catalytically inactive Arf1 T31N. Reversely, the effect of constitutive active Arf1 Q71L was fully inhibited by catalytically inactive PLD constructs. Whereas the effects of Arf1 Q71L and wild-type PLD2 were additive, coexpression of Arf1 Q71L with wild-type PLD1 led to a synergistic increase in PIP-5-kinase activity. Previously, we have shown that RhoA regulates the activity of PLD and PIP-5-kinase by its downstream effector Rho-kinase. Expression of small amounts of inactive PLD1, but not of PLD2, nearly completely abolished Rho-kinase-stimulated PIP-5-kinase activity. Also expression of a non-phosphorylatable mutant of cofilin, which participates in the signalling cascade from RhoA via Rho-kinase and LIM-kinase to PLD1, suppressed the stimulating effect of Rho-kinase on PIP2 synthesis. These findings suggest that PLD1 is involved in the stimulation of PIP-5-kinase by Arf1 as well as by RhoA and Rho-kinase. After sucrose density gradient centrifugation of HEK-293 cell lysates, we isolated two separate PIP2 pools. PLD1 and Arf1 selectively control the non-caveolar PIP2 pool in the high density fraction, whereas PLD2 affected PIP2 in both pools. In summary, these data suggest that particularly PLD1, apparently by the production of PA, functions as a physiological regulator of PIP-5-kinase that controls the synthesis of cellular PIP2 downstream to Arf1 and RhoA.

This thesis describes the phenotypic and genotypic analysis of the dominant mutation in the ENU-induced mutant mouse line SMA002. The results were derived from the analysis of heterozygous mutant animals on the C3H genetic background. As the main phenotype of SMA002, an abnormal behavior combined with reduced body weight was revealed. The mutant phenotype showed complete penetrance in all generations analyzed. Abnormal behavior, as the most prominent phenotype was characterized by the occurrence of restlessness and an increased grooming behavior. The grooming behavior was increased fivefold but did not always contain all aspects of the standard repertoire. Pathological examinations of the skin and the analysis of parameters of the immune system indicated that this behavior was not caused by an allergic reaction. Clinical examination of the animals revealed a significant difference in body weight between mutant and control littermates. The body weight of the mutant animals was about 30% lower than control. In addition mutant animals had a significant lower nose-rump length and a significant lower carcass weight. Analysis of the peripheral blood for hematological and clinical chemical parameters as well as subsequent pathological examinations excluded a kidney, liver or pancreas disease as the potential cause of the abnormal phenotype. Decreased values for cholesterol, triglyceride, total protein and potassium in the mutant animals referred to an increased metabolism and a hormonal deviation in these mice. Furthermore, deviations of the IGF-system were found in the mutants. Linkage analysis of the causative mutation was carried out by genome wide polymorphic microsatellite marker analysis. The highest correlation for the mutation was found with the markers D13Mit20 (35cM) and D13Mit253 (37cM) of chromosome 13. This aberrant phenotype of the line SMA002 has not yet been reported. Thus, SMA002 represents a novel mouse model for the analysis of abnormal behavior and growth defects. Subsequent analysis will identify the exact mutation in the line. In addition, further analysis of the phenotype will result in detailed information on the metabolism and hormonal regulation of SMA002.

The objective of the present study was to examine the effectiveness of the anesthetic combination "Hellabrunne mixture" ("HM" = approximately 125mg xylazine/ml and 100mg ketamine/ml) on hamadryas baboons, as well as the influence of various drugs over the time it took the baboons to wake up (their "waking up phase") under practical conditions. In order to remobilize the baboons, comparative studies were conducted using: alpha2-specific antagonists atipamezole and yohimbine as well as the circulation stimulating drug etilefrine. The "HM" proved to be a very safe and effective anesthetic, even when given in doses that were too low or when overdosed. None of the examined animals had any life threatening incidents. All of the measured physiological parameters were normal and remained stabile during anesthesia. The three assayed accelerators used to remobilize the baboons (atipamezole, yohimbine, etilefrine) showed substantial differences in their effects. Atipamezole caused the baboons to regain consciousness significantly faster than the other drugs. However, some of the animals that were administered atipamezole had catalepsis cramps during their waking up phase. These side effects can be explained most likely, by the fact that atipamezole only antagonizes the xylazine component of the "HM" which causes a relative overdose of ketamine. Examining the effects of yohimbine administered i.m. (in contrast to the usual i.v. administration) yielded no substantial acceleration of remobilization. The same side effects were observed after administering atipamezole. Similar to atipamezole, etilefrine also shortened the waking up phase significantly, but to a lesser extent than atipamezole. In contrast to atipamezole and yohimbine, no side effects were observed after administering etilefrine. In summary, the i.m. administration of atipamezole and etilefrine proved to be suitable to shorten the waking up phase of hamadryas baboons after using "HM". In contrast, yohimbine cannot be recommended, as noticeable side effects were evident and no significant acceleration of remobilization was observed.

The long-term effect of sevoflurane and propofol on necrotic and apoptotic cell death after incomplete cerebral ischemia and reperfusion in the rat The present study investigates the effect of the anesthetic agents propofol and sevoflurane on irreversible cell damage (necrosis) and programmed cell death (apoptosis) for a time period of 28 days after incomplete transient cerebral ischemia and reperfusion in the rat. Ninety-six fasted male Sprague-Dawley rats (415±40g) were anesthetized, intubated and ventilated with 2 Vol% isoflurane and N2O/O2 (FiO2=0.33). Catheters were inserted into the right femoral artery and vein as well as in the right jugular vein for drug administration and blood withdrawal. At the end of surgery animals were randomly assigned to one of the following groups: control (n=32): 25 µg/kg/h fentanyl i.v. and N2O/O2 (FiO2=0.33); propofol (n=32): 25 µg/kg/h propofol i.v. and O2/air (FiO2=0.33); sevoflurane (n=32) 2 Vol% sevoflurane in O2/air (FiO2=0.33). Ischemia (45min) was produced by unilateral common carotid artery occlusion plus hemorrhagic hypotension (MAP=40 mmHg). Pericranial temperature (37.5°C), arterial blood gases and pH were maintained constant. Animals were then randomly assigned to a postischemic reperfusion time of 01, 03, 07, or 28 days. At the end of the observation period the animals were deeply anesthetized and killed, the brains were removed, frozen at –70 °C, and sectioned for further evaluation. Hematoxylin-Eosin staining was used to evaluate eosinophilic cell damage and tissue damage in 7 μm sections in the hippocampus, dentate gyrus and surrounding tissues. Immunohistochemistry was used to detect activated caspase-3 as a marker of apoptotic cell death. To distinguish activated caspase-3-positive neurons from other cells, a double staining, using the specific neuronal marker NeuN was used additionally. The results showed that with the exception of one animal in the sevoflurane group there is no tissue damage or eosinophilic cell damage in either the propofol or the sevoflurane treated animals up to 28 days after ischemia. The only eosinophilic tissue damage was present in the control group. About one percent of the hippocampal neurons of all three groups were activated caspase-3-positive, independently of the observation period. Though there was a tendency in both treatment groups to a lower number of activated caspase-3-positive cells. Results of the double staining showed that activated caspase-3, though mainly expressed in neurons, is also expressed in other cells. The present study showed that propofol and sevoflurane both produce a sustained inhibition of eosinophilic cell damage up to 28 days after incomplete cerebral ischemia in rats. Although the amount of activated caspase-3-positive neurons was similar in the three groups there was a tendency towards a lower number in the treatment groups compared to control. This suggests that neuroprotection seen with both, propofol and sevoflurane, involves anti-necrotic mechanisms rather than anti-apoptotic mechanisms. Further investigations will be required in the future to investigate the detailed mechanisms of propofol and sevoflurane to develop a successful treatment of ischemic insults and their consequences.

A retrospective overview of stationary parasites and fleas in dogs according to occurence of species and frequency of infection/ infestation in Germany during the 20th century – based on a study of the scientific literature.