Spontaneous glomerulonephritis in Göttingen minipigs

The G?ttingen minipig is one of the nonrodent species recommended by various regulatory authorities for safety assessment of drugs in preclinical studies. In such studies, knowledge of background pathology is critical in order to evaluate the potential renal toxicity. In the present study, the authors report 4 cases of glomerulonephritis out of 154 microbiologically defined G?ttingen minipigs microscopically evaluated in preclinical studies. One animal required early sacrifice because of general poor health, and an additional animal died spontaneously. Histopathological evaluation revealed renal lesions in all 4 animals, exhibiting membranous or membranoproliferative glomerulonephritis at different stages, accompanied by secondary tubulo-interstitial damage. The renal changes observed were considered spontaneous in origin and of unknown etiology. Development of this condition in this strain should be considered in future studies.     

The anatomy of the Göttingen minipig hypothalamus

The microscopic organization of the Göttingen minipig (sus scrofa) hypothalamus was studied using Nissl stain, acetylcholinesterase histochemistry, and immunohistochemical staining for calretinin, tyrosin hydroxylase, oxytocin, vasopressin, and orexin A.Mediolaterally the minipig hypothalamus can be divided into three cytoarchitectonic distinct longitudinal zones. The periventricular longitudinal zone comprises the supraoptic, paraventricular, median preoptic, anteroventral periventricular, suprachiasmatic and arcuate nuclei.The medial longitudinal zone includes the prominent medial preoptic, ventromedial, dorsomedial and medial mammillary nuclei. Together with the anterior hypothalamic area, they can be further divided into distinct subregions. The dorsal and posterior hypothalamic areas and the retromammillary and lateral mammillary nuclei are cyto- and chemoarchitectonically distinct but cannot be further divided into subregions.The cell sparse, fiber rich lateral longitudinal zone comprises the lateral preoptic and lateral hypothalamic area as well as the perifornical, lateral tuberal and tuberomammillary nuclei.The findings presented here indicate that the cyto- and chemoarchitecture of the Göttingen minipig hypothalamus is comparable to that of rat, landrace pig, monkey, and human and that the Göttingen minipig may be well suited for future, non-primate, large mammal, hypothalamic research.     

Immune complex-associated thrombocytopenic purpura syndrome in sexually mature Göttingen minipigs

Eleven cases of thrombocytopenic purpura (TP) in sexually mature male or female G?ttingen minipigs occurred sporadically over 3 1/2 years in a closed breeding colony protected by strict barrier conditions. Typical clinical signs of TP, including extensive subcutaneous haemorrhages, were seen in all affected animals. Haematological abnormalities included marked thrombocytopenia and anaemia. A consistent histopathological finding was the presence of membranoproliferative lesions in the renal glomeruli. Immunohistochemically, glomerular deposits were positively labelled for complement factor C1q and often also for immunoglobulins. Bone marrow findings consisting of increased numbers of immature and apoptotic megakaryocytes were compatible with a state of increased platelet consumption. Based on the combined presence of thrombocytopenia and renal immune complexes, it is suggested that the syndrome was related to a type III hypersensitivity reaction. However, further studies are needed to verify this hypothesis.     

A volumetric screening procedure for the Göttingen minipig brain

A screening procedure was developed to provide quantitative estimates of structural parameters, regional volumes and neuron number, in a neurotoxicologic study of the Göttingen minipig brain. The study material consisted of normal controls and brains collected from young minipigs which had been exposed in utero to the mitotic inhibitor methylazoxymethanol acetate (MAM). Based on stereological principles and systematic sampling techniques, volumetric data from pre-selected regions of the pig brain was obtained using Cavalieris principles and point-counting. Secondarily, estimates of total hemispheric neocortical cell numbers were obtained from pre-selected groups to test the potential effect of MAM on neuron number. No significant differences were observed in volume of the pre-selected regions of MAM intoxicated pigs nor in estimates of total neocortical neuron number.     

Characterization of the T-dependent antibody response (TDAR) to keyhole limpet hemocyanin (KLH) in the Göttingen minipig

Abstract

Recently, there has been a renewed interest in the use of the minipig as an alternative to dogs and non-human primates for conducting toxicological assessments in non-rodent species. Since the T-dependent antibody response (TDAR) is one of the most widely-accepted assays used in the assessment of immunocompetence, the present study was undertaken to characterize the primary and secondary TDAR to keyhole limpet hemocyanin (KLH) in the Göttingen Minipig®. Following primary immunization with either 2 or 10?mg KLH, anti-swine IgM and IgG ELISAs were optimized and individual animal responses were evaluated over time. Immunization with 10?mg KLH on Day 0 promoted primary IgM responses that peaked 6–9 days after antigen administration, while primary IgG levels peaked on Day 13 or 14. Secondary IgG antibody levels (following secondary injection with 2?mg KLH on Day 14) plateaued on Days 20–22. Anti-KLH antibody levels were decreased in minipigs treated with cyclophosphamide (CPS), a known immunosuppressant, at doses ranging from 12.5–50?mg/kg/day, while antibody levels in animals treated with 2.5?mg CPS/kg/day were similar to levels in saline-treated swine. These results demonstrate that the Göttingen Minipig® can be a useful alternative non-rodent species to the dog and the non-human primate for evaluating the TDAR to KLH in regulatory assessments of immunotoxicity.     

Anatomical practice at Göttingen University since the Age of Enlightenment and the fate of victims from Wolfenbüttel prison under Nazi rule

This report briefly summarises anatomical practice at G?ttingen University from its founding in 1737 until the Nazi period and gives a detailed account of how Nazi death penalty legislation and execution practice at Wolfenbüttel prison influenced the decision-making of the anatomists in charge at that time. Problems in the procurement of corpses, encountered almost continuously throughout Europe since the broad introduction of dissection into medical training in the early 18th century, were absent in G?ttingen during periods of overt progress in anatomical sciences, e.g. under Albrecht von Haller (in office 1736-1753) and Jacob Henle (1853-1885), and at times when existing regulations were rigorously enforced by the authorities (1814-1851). Ample availability of corpses in the wake of more than 600 executions in Wolfenbüttel between 1935 and 1945 was curtailed only by transportation fuel shortages and resulted in the dissection of more than 200 Nazi victim corpses in the G?ttingen anatomy course. Apparently, neither individual offers of voluntary body donation (dating from 1932 to 1937 and published here as the earliest documents of this kind), nor the strong tradition of high-level anatomical research, nor even the awareness of the University's Age of Enlightenment origin, prevented the unethical use of corpses of Nazi victims for medical teaching. The G?ttingen example may add "historical and moral detachment" under unusual political and wartime pressures to the "clinical and emotional detachment" thought to prevail amongst anatomy personnel (Hildebrandt, in this issue); together with the other reports it calls for all anatomists to bear in mind their ever present ethical obligations in respect to activities involving the use of corpses, both in medical schools and in the public domain.     

Direct MRI-guided stereotaxic viral mediated gene transfer of alpha-synuclein in the Göttingen minipig CNS

The aim was to establish a non-primate large animal PD model by lentiviral vector mediated mutant alpha-synuclein overexpression in the substantia nigra. Lentivirus encoding A53T alpha-synuclein (6 x 2.5 μl) was stereotaxically injected into the substantia nigra of six adult female G?ttingen minipigs. Contralateral control injections encoding enhanced green fluorescent protein (EGFP) were performed. Gait-analysis was performed pre- and postoperatively. PCR of the transgenes and immunohistochemical staining against alpha-synuclein, EGFP, GFAP and TH was performed after 20 weeks. Gait analysis revealed a significant increase in step length and height, and a decrease in the double stand phase. PCR verified the mesencephalic presence of transgenes. IHC analysis showed alpha-synuclein expression in nigral neurons, around the injection tract and in related nigrostriatal projections. The alpha-synuclein positive neurons appeared swollen and vacuolated, in contrast to the EGFP-injected control side. To transduct all nigrostriatal cells with few microinjections, wider dissemination of the transgene must be achieved.     

The substantia nigra pars compacta of the Göttingen minipig: an anatomical and stereological study

Parkinson disease (PD) is a neurodegenerative disorder resulting from progressive loss of dopaminergic neurons in the substantia nigra pars compacta (SNc). Despite advances in medical and surgical therapies, many PD patients experience progression of their symptoms and medical side effects over time. To explore new treatments, new animal models mimicking the progressive PD nature are needed. The pig is well suited for this purpose with its large gyrated brain, sensitive to the neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). The objective of this study was to provide the anatomical foundation for such a model, describing in detail the SNc in normal Göttingen minipigs and estimating the volume and total number of tyrosine hydroxylase (TH)-positive neurons. The brain stems of 6 Göttingen minipigs were paraffin embedded and serially cut before Nissl staining and immunohistochemical visualization of TH. The volume of the SNc and the total number of TH-positive neurons were estimated by design-based stereology. The substantia nigra was located at the dorsal rim of the crus cerebri extending throughout the mesencephalon. A dorsal pars compacta and a ventral pars reticulata were demonstrated. The SNc merged with the ventral tegmental area medially and the retro-rubral field dorsocaudolaterally. The total number of TH-positive neurons in the SNc unilaterally was estimated to 80,700 [74,100;87,300], and the volume estimate was 26.4 mm³ [25.0;27.8]. We conclude that the anatomy of the SNc in the Göttingen minipig corresponds well with that of higher primates, and is well suited for further studies aimed at optimizing this non-primate large animal model for PD.     

Perineuronal nets of proteoglycans in the adult mouse brain, with special reference to their reactions to Gömöri's ammoniacal silver and Ehrlich's methylene blue

As our previous studies have indicated, many subsets of neurons in the vertebrate brain possess a sulfated proteoglycan surface coat which reacts to cationic iron colloid and aldehyde fuchsin. The present study demonstrated that this surface coat is supravitally stained with Ehrlich's methylene blue, and doubly with this blue and aldehyde fuchsin, a finding suggesting its being identical to Cajal's superficial reticulum (red superficial) and to Golgi's reticular coating (revetement reticulare). The perineuronal surface coat was further stained with G?m?ri's ammoniacal silver, and doubly with this silver and cationic iron colloid. These neurons with such a proteoglycan surface coat usually expressed cell surface glycoproteins which were labeled with lectin Wisteria floribunda agglutinin. Hyaluronidase digestion did not interfere with this lectin labeling of the glycoproteins, methylene blue and G?m?ri's ammoniacal silver staining of the surface coat, while it erased the cationic iron colloid and aldehyde fuchsin staining of the surface coat. These findings suggest that the perineuronal proteoglycan surface coat is associated with some additional molecules which are resistant to hyaluronidase digestion and stainable with methylene blue and G?m?ri's ammoniacal silver. The possibility is suggested that these molecules might represent "ligand proteoglycans" connecting the perineuronal proteoglycans and cell surface glycoproteins.     

The perineuronal proteoglycan surface coat in the adult rat brain, with special reference to its reactions to Gömöri's ammoniacal silver

The present study showed that many neurons in the adult rat brain possessed a perineuronal sulfated proteoglycan surface coat which reacted to cationic iron colloid and aldehyde fuchsin. This surface coat was stained supravitally with Ehrlich's methylene blue and doubly stained with Ehrlich's methylene blue and aldehyde fuchsin. The surface coat was also stained with G?m?ri's ammoniacal silver and doubly stained with G?m?ri's ammoniacal silver and cationic iron colloid. The surface coat was usually expressed together with a nerve cell surface glycoprotein net detectable with lectin Wisteria floribunda agglutinin. These findings indicate that the perineuronal proteoglycan surface coat is identical to Cajal's superficial reticulum and contains some collagenous elements. It was further demonstrated that collagenase digestion erased G?m?ri's ammoniacal silver impregnation within the perineuronal proteoglycan surface coat.     

Cross-validation of the Beunen–Malina method to predict adult height

Abstract

The purpose of this study was to cross-validate the Beunen–Malina method for non-invasive prediction of adult height. Three hundred and eight boys aged 13, 14, 15 and 16 years from the Madeira Growth Study were observed at annual intervals in 1996, 1997 and 1998 and re-measured 7–8 years later. Height, sitting height and the triceps and subscapular skinfolds were measured; skeletal age was assessed using the Tanner–Whitehouse 2 method. Adult height was measured and predicted using the Beunen–Malina method. Maturity groups were classified using relative skeletal age (skeletal age minus chronological age). Pearson correlations, mean differences and standard errors of estimate (SEE) were calculated. Age-specific correlations between predicted and measured adult height vary between 0.70 and 0.85, while age-specific SEE varies between 3.3 and 4.7 cm. The correlations and SEE are similar to those obtained in the development of the original Beunen–Malina method. The Beunen–Malina method is a valid method to predict adult height in adolescent boys and can be used in European populations or populations from European ancestry. Percentage of predicted adult height is a non-invasive valid method to assess biological maturity.     

Longitudinal growth of stature in boys according to age and puberty: Prediction of adult stature from the age of 13 years

Background

The purpose of this study was to cross-validate and demonstrate how adult stature can be predicted in 13-year-old teenager's boys by using a new reference specific growth curve obtained from chronological age and maturity.

Methods

Stature measurements of 125 boys aged from 12 to 17.5 years were obtained over a period of five consecutive years. The maturity was based on the age of peak height velocity (APHV). An assessment of secondary pubertal stages using a simplification of the Tanner stages was also carried out. We found a connection between the secondary pubertal stage and the APHV. We carried out a prediction of the adult stature of 67 teenagers aged from 160 and 164 months.

Results

Significant differences between APHV enabled us to classify the adolescents into three categories according to their biological maturity: 19% of adolescents with advanced puberty, 62% with standard puberty and 19% with delayed puberty. The mean growth curves were used to predict the adult stature of 67 individuals with a good accuracy (±3 cm). The mean differences between predicted and real adult stature are −0.11 cm with 95% limits of agreement of [−3.2; + 2.8 cm].

Conclusions

The new stature growth curves developed from age and maturation enables us to accurately track individual growth kinetics.     

The postnatal development of cerebellar Purkinje cells in the Göttingen minipig estimated with a new stereological sampling technique--the vertical bar fractionator

The postnatal development of total number and perikaryon volume of cerebellar Purkinje cells was estimated in the Göttingen minipig cerebellar cortex using a new stereological approach, the vertical bar fractionator. Data were obtained from the brains of five neonate and five adult female Göttingen minipigs. The total number of Purkinje cells ranged from 1.83 × 10⁶ in the neonate to 2.82 × 10⁶ in the adult Göttingen minipig. The number-weighted mean perikaryon volume of Purkinje cells increased concurrently from around 6800 µm³ in the neonate to 17 600 µm³ in the adult. The study demonstrates that a pronounced postnatal neurogenesis in Purkinje cell number and perikaryon volume is part of the growth and development of the cerebellum in the Göttingen minipig. The Purkinje cells of the Göttingen minipig were found to be substantially large compared with human and represents the largest cells described hitherto from mammalian cerebella. The vertical fractionator is a new sampling technique, which allows the combination of a fractionator design on vertical bar sections excluding exhaustive sampling and bias from artificial edges. By design, the sections are perfect stereological vertical sections and provide the basis for unbiased estimates of total number of structural entities in the brain, including surface area, fibre length and particle volume.     

The role of partner relationships in the young person’s adherence to the diabetes self-care regime

Background: This study investigated the role of partner relationships in the young person’s experience of diabetes. It was predicted that those people reporting a better quality relationship would report greater social support and better diabetes management. Method: Fifty young adults with diabetes and their spouse/partner completed web-based questionnaires. Results: A better quality relationship did not correlate with high reported levels of social support or with better diabetes management. Path analysis demonstrated, however, that for males with diabetes high reported quality of relationship predicted better diabetes management. Conclusions: Relationship quality does not generally predict better diabetes management; however, there may be a unique feature to good relationships that support men’s diabetes self-care.     

Toward the development of a sensitive,pre-clinical screen for neurological diseases from spontaneous neural coordination in juvenile and young–adult C57BK6 mice

Spontaneous Frequency Bursts (SFBs) are a newly discovered form of long-distance neural coordination. They have several distinctive properties, including near-simultaneity of occurrence (±25–50 ms) across distant brain regions and high within- and across-site coherence in multiple low and high frequency bands, presumably requiring high axonal, dendritic and vascular integrity. We examined whether SFBs occurred in young and young–adult C57BK6 mice with properties similar to those seen in rats. We found that across the entorhinal and piriform cortices, SFBs occur robustly in young and young–adult mice under light anesthesia, and that their rate of occurrence dropped sharply as anesthetic levels increased, as in rats. Moreover, murine SFBs showed high cross-site coherence in multiple frequency bands, including those that require exquisite action potential timing to be maintained across long distances. We discuss our findings in light of SFBs potential as a pre-clinical biomarker for diseases affecting action potential firing and local field potential coherence, especially in high frequency ranges (20–30 Hz and beyond).     

Benzoic acid-degrading bacteria from the intestinal tract of Macrotermes michaelseni Sjöstedt

The intestinal tracts of termites host a wide variety of microbial symbionts, which have been implicated in degradative processes. In this study, a fungus-cultivating termite, Macrotermes michaelseni was found to harbor 2.2 x 10(6) bacterial cells per ml of gut homogenates capable of degrading benzoic acid. Two benzoic acid degrading bacteria were isolated from the highest dilution of gut homogenates in oxic media with benzoic acid as the sole carbon source. Isolate CBC was related to Stenotrophomonas maltophila LMG 958(T), Xanthomonas campestris DSM 3586(T) and Stenotrophomonas acidaminophila DSM 13117(T) with a sequence similarity of 98.3%, 94.7% and 94.2%, respectively. Isolate CBW was related to Enterobacter aerogenes JCM 1235(T) and Raoultella ornithinolytica ATCC 31898(T) with sequence similarity of 98.4% and 97.8%, respectively. In addition to growing on benzoic acid (up to 9 mM) aerobically, isolate CBW also degraded benzoic acid under anoxic conditions with nitrate as electron acceptor. Isolate CBC did not degrade bezoic acid with nitrate but could degraded resorcinol under oxic conditions.