Expression and significance of three types of Fos-immunoreactive cells after gamma knife irradiation of the forebrain in the rat

The expression and significance of three types of Fos-like immunoreactive (Li) cells were investigated after gamma knife irradiation of the forebrain in the rat. Three months after the irradiation, the brain sections were immunostained with an antiserum against Fos protein. It was shown that the Fos-like immunoreactivity (LI) appeared in some of the neurons, glial cells and endothelial cells in the target area, the white matter surrounding the lateral ventricle, the cerebral cortex and the hippocampus. Three characteristic types of Fos-Li cells were identified in these regions. (1). Only the nuclei of the cells were Fos-ir, (2). Only the cytoplasm was immunostained, and (3). Both the nuclei and the cytoplasm showed Fos-LI. It is suggested that type 1 are the normal responsive cells, type 2 are seriously injured cells, so that the Fos translocation mechanism is damaged, and type 3 represents the intermediate form.     

Effects of gamma knife irradiation on the expression of NMDA receptor subunits in rat forebrain

OBJECTIVE: To examine the effects of gamma knife surgery (GKS) on the expression of N-methel-D-asparate receptor (NMDAR) subunits in rat forebrain. MATERIALS AND METHODS: Using stereotactic technique, we performed gamma knife irradiation on the left forebrain of 13 male Wistar rats with a maximum dose of 60 Gy. These animals were raised for 24h, 30 and 60 days before they were killed. Then immunohistochemistry was applied to detect the relative levels of NMDAR subunits (NR1, NR2A, and NR2B) in the target region. RESULTS: The expression of NR1 and NR2A but not NR2B increased significantly in the cortex 30 and 60 days after irradiation. However, no significant differences in the expression of these three subunits were detected in the caudate putamen at all time points. CONCLUSION: gamma knife irradiation induced the upregulation of NMDAR subunits, NR1, and NR2A, which might represent a possible mechanism underlying the therapeutic effects of gamma knife irradiation on many neurological diseases, including drug resistance epilepsy.     

Morphological and histochemical changes in the epididymis of hamsters (Mesocricetus auratus) subjected to short photoperiod

The morphological involution and histochemical changes of the Syrian hamster ( Mesocricetus auratus ) epididymis induced by a short light period were investigated. Under short-day conditions, the epididymis showed marked morphological changes including a decrease in luminal diameter, disappearance of spermatozoa, increase of interductal tissue, increase of intraepithelial lipofuscin deposits, the presence of phagolysosomes in the principal cells and macrophage-like cells, and a considerable modification of most clear cells. With lectin histochemistry changes were found in the glycoconjugates of principal cells of the regressed epididymis, either a decrease (PNA, WGA, HPA and DBA) or an increase (MAA) in the affinity of lectins to the Golgi area, or a decrease (HPA) or an increase (PNA) in lectin binding to stereocilia. Both morphological and histochemical results showed that, under this light condition, the cauda epididymidis presented the most prominent alterations, and that the epididymis showed increased absorptive activity and a decreased synthesis of glycoproteins. All these changes are probably due to the decrease in testosterone levels.     

Effects of gamma irradiation on the infectivity and development ofHymenolepis nana oncospheres (Cestoda: Cyclophyllidea)

Summary Hymenolepis nana eggs received gamma irradiation at levels from 10 000r to 200 000r before administration to mice orally or by subcutaneous inoculation. Little growth beyond the oncosphere occurred after 50 000r or over, but invasiveness was not abolished even after treatment at 200 000r. A full range of developmental stages occurred after 10 000–20 000r but relatively few worms became gravid in the intestine. Growth occurred after treatment at 30 000–40 000r, but very few reached the fully developed cysticercoid stage.     

Effect of sterilisation by gamma irradiation on the ability of polycaprolactone (PCL) to act as a scaffold material

This paper investigates the effect of sterilisation by gamma irradiation (dose 2.5Mrad) on the following properties of polycaprolactone (PCL): (1) degradation rate (catalysed by lipase), (2) mechanical properties, (3) the ability of cells to attach and subsequently grow on its surface. Gel permeation chromatography (GPC) was used to determine the effects of gamma irradiation of weight average (M(w)) and number average (M(n)) molecular weights. Gamma irradiation significantly decreased the rate of degradation, although the rates depended on the initial mass of polymer; it also affected the appearance of the degraded specimens when they were examined by scanning electron microscopy. Irradiation also significantly increased the mechanical yield stress but not the failure stress of PCL. It caused a significant increase in M(w) and decrease in M(n) that could be attributed to chain scission and cross-linking. Chondrocyte attachment and growth on PCL was not significantly affected by gamma irradiation.     

Response to kainic acid injections: changes in staining for zinc, FOS, cell death and glial response in the rat forebrain

A pool of zinc is present in synaptic vesicles in a population of glutamatergic neurones. Zinc appears to modulate synaptic transmission and cause neuronal death. The status of vesicular zinc, neuronal death and glial reaction in the rat forebrain was analysed after a systemic injection of kainic acid in order to establish a model for future studies on zinc function. Rats received a systemic injection of kainic acid (10 mg/kg) and were killed 3, 6, 12, 24 and 48 h post-treatment. Timm's method and zinquin staining were used to detect zinc. Immunostaining for Fos-like proteins and staining with Fluoro-Jade B were used to detect cell reaction and degeneration, respectively. Glial fibrillary acidic protein and tomato lectin were used as glial markers. Zinquin staining for zinc rose transitorily in neuronal somata 6 h after injection (not observed at 24-48 h) in the piriform and entorhinal cortices, amygdala and hippocampus. In contrast sulphide/silver staining for zinc showed virtually no rise in cytoplasmic zinc (except in cornus ammonis field 1 of the hippocampus) 6 h after injection, and a decrease (bleaching) in some terminal fields starting 12 h after injection and increasing at 24-48 h. The areas most affected by the zinc bleaching were the olfactory bulb, piriform and entorhinal cortices, endopiriform and amygdaloid nuclei. Transitory Fos immunostaining (within neuronal nuclei) was observed between 3 and 12 h after kainate treatment in many telencephalic areas: olfactory bulb, cortex (piriform, hippocampal and neocortex) and amygdaloid nuclei. This was accompanied by changes in glial markers starting 3 h after injection. Fluoro-Jade B staining in neurones (degeneration) appeared 6 h after treatment and increased later. Degenerating areas generally coincided with those showing Fos immunoreactivity. Zinquin and sulphide/silver methods revealed various pools of zinc after kainate injection: a cytoplasmic pool and a terminal field (or vesicular) pool. Cytoplasmic zinc (zinquin) was coincident, in time and location, with cell degeneration, thus implicating zinc in cell death. This zinc may not have come from presynaptic stores, since no bleaching (sulphide/silver method) was observed 6 h after injection. Future experiments altering zinc pools (e.g. by chelating agents) may elucidate the function of zinc.     

Immunohistochemical studies on cholecystokinin (CCK)-immunoreactive neurons in the rat using sequence specific antisera and with special reference to the caudate nucleus and primary sensory neurons

In the present immunohistochemical study the occurrence and distribution of CCK-immunoreactive neurons were analyzed in the brain, spinal cord and sensory ganglia using sequence specific antisera. Thus, antibodies directed towards the C-terminal portion of CCK-33, to the N-terminal portion of CCK-8 and to the mid portion of CCK-33 as well as monoclonal antibodies were used. For comparison antisera raised against calcitonin gene-related peptide (CGRP), tyrosine hydroxylase (TH) and 5-hydroxytryptamine (5-HT) were used. Untreated, colchicine treated, 6-hydroxydopamine (6-OH-DA) treated and ibotenic acid treated rats were analyzed. The results indicate that most CCK systems in the rat central nervous system contain genuine CCK. These include, for example, the hippocampal formation, the hypothalamus, several subcortical forebrain areas, the ventral mesencephalon, nucleus tractus solitarii, some neurons in the ventral medulla oblongata as well as local and possibly descending neurons in the spinal cord. An exception was primary sensory neurons in which CCK-like immunoreactivity (LI) could only be demonstrated with C-terminally directed antisera and probably represents cross-reactivity with CGRP or a similar peptide. The central branches of such primary afferents were found both in the dorsal vagal complex, in the spinal trigeminal nucleus and in the dorsal horn of the spinal cord. Special attention was focused on CCK-LI in mesencephalic dopamine neurons and in their projection areas including nucleus accumbens, tuberculum olfactorium and particularly the caudate nucleus. In the latter structure CCK-LI exhibited a heterogenous pattern probably representing fibres of different types and origin. Thus, CCK-LI coexists with dopamine in two anatomically and morphologically distinguishable systems, one located in the periventricular area, increasing in size in the caudal direction to occupy most of the cauda, and a second system consisting of very fine dots in the medial half of the caudate nucleus. These two fibre types disappeared after 6-OH-DA treatment. A third system consisted of strongly fluorescent patches distributed at all levels of the caudate nucleus, mainly in its medial half. A diffuse, weakly fluorescent network of CCK-positive fibres was also found over the entire caudate nucleus. The latter two systems did not disappear after 6-OH-DA. Finally, local CCK-positive cell bodies were seen in small numbers, mainly in the ventral aspects of the caudate nucleus.(ABSTRACT TRUNCATED AT 400 WORDS)     

Altered pattern of immunohistochemical staining for glial fibrillary acidic protein (GFAP) in the forebrain and cerebellum of the mutant spastic rat

The spastic rat is a neurological mutant of the Han-Wistar strain with prominent spasticity, tremor, and ataxia. Neurodegeneration is found in the CA3 sector of the hippocampus and in Purkinje cells of the cerebellum. We examined the forebrain and cerebellum of spastic rats for glial reactions by using immunolabelling for the astrocytic marker, glial fibrillary acidic protein (GFAP). First, a map of the GFAP-distribution was made representing a systematic series of frontal sections in controls. Reactive astrocytes with increased GFAP should occur in the areas with established neuronal degeneration, but they could also demarcate further regions with pathology in this rat strain. Since the baseline levels of GFAP-immunoreactivity differ between brain regions, control rats and clinically normal littermates served as controls to judge relative increases in major structures. In the CA3 sector and hilus of the dorsal hippocampus, a massive gliosis was detected. In the cerebellum, a patchy increase of GFAP labelling in Bergmann glia was found. Further increases of GFAP-labelling in reactive astrocytes occurred in fiber tracts, the ventral thalamic nuclei, medial geniculate nuclei, pontine region and optic layer of the superior colliculus. Inconsistent changes were noted in cortex and pallidum. No defects of glial labelling or malformations in glial architectonics were found. The reactive changes of astroglial cells in hippocampus and cerebellum are in proportion to the neuronal degeneration. The glial reactions in the other brain regions possibly reflect a reaction to fiber degeneration and incipient neuronal degeneration or functional alterations of glial cells in response to neuronal dysfunction.     

Response of Pediculus humanus humanus (Pediculidae: Phthiraptera) to water or 70% ethanol immersion and determination of optimal times for measuring toxic effects

Human pediculosis is caused by Pediculus humanus humanus (Linnaeus 1758) and Pediculus humanus capitis (De Geer 1767). We studied the response of body lice to immersion in water and ethanol 70% and determined the optimal times for measuring knockdown and mortality. After immersion in water, all lice remained alive from 5 min to 22 h for both times of exposure. A low proportion of lice were affected after 2 min of immersion in ethanol in the 10-min exposure test, but recovered completely after 5 min. Different proportions of lice were affected between 2 and 7 h after immersion in ethanol, depending on the immersion time. However, a high proportion of lice recovered after 22 h. The results suggest that the optimal times for measuring early knockdown effects of insecticides are the 5-min to 7-h interval for water and 5-min to 1-h interval for ethanol. On the other hand, the best time for measuring mortality is 22 h after immersion. These results should improve the interpretations of the effects of pediculicides in immersion bioassays.     

Sequential changes in localization of repair-related proteins (heat shock protein 70, ubiquitin and vascular endothelial growth factor) in the different stages of myocardial infarction

AIMS: The myocardium expresses vascular endothelial growth factor (VEGF), heat shock protein 70 (HSP70), and ubiquitin immediately after the onset of cardiac ischaemia. This study demonstrated the sequential changes in localization of these proteins, in addition to fibronectin and troponin T (TnT), in human hearts with myocardial infarction (MI). METHODS AND RESULTS: Myocardial tissues from 40 autopsied MI cases were immunostained with the five antibodies against VEGF, HSP70, ubiquitin, fibronectin and TnT. Fibronectin was recognized only in the cardiomyocytes with infarction. Although TnT, HSP70, ubiquitin and VEGF were detected in the affected myocardium in the early stages, their expression in cardiomyocytes around infarcted foci were more intense. The cardiomyocytes with coagulative myocytolysis were positive for fibronectin, but negative or weakly positive for TnT, HSP70, ubiquitin and VEGF. In contrast, the cardiomyocytes with colliquative myocytolysis were strongly positive for TnT, HSP70, ubiquitin and VEGF, but negative for fibronectin. CONCLUSIONS: Immunostaining using antibodies to fibronectin, TnT, HSP70, ubiquitin and VEGF is useful for the discrimination between infarcted myocytes and ischaemia-damaged myocytes in the human heart with MI at autopsy.     

Expression and modulation of the human immunoglobulin A Fc receptor (CD89) and the FcR gamma chain on myeloid cells in blood and tissue

CD89, the human immunoglobulin A (IgA) Fc receptor (FcR), is a potential target for antibody-based therapeutics, but little is known about its expression and modulation in vivo. In this study, we examined the expression pattern of CD89 and its signalling subunit, the FcR gamma chain, on circulating myeloid cells and in various tissues. Our results showed a wide tissue distribution of CD89+ cells. Thus, CD89+ cells were evident as clusters in tonsils and appendix and scattered in varying numbers in lymph nodes, kidney, liver, intestinal mucosa, bronchoalveolar lavage and peritoneal fluid. Most CD89+ cells were identified as neutrophils with high levels of CD89. A few recently emigrated macrophages (CD14low), weakly positive for CD89, were occasionally found in the tissues and more often in the peritoneal fluid. The level of CD89 on neutrophils in tissues and peripheral blood was similar, whereas on monocytes it was much lower in the tissues than in blood, and it was absent on CD14-/CD68+ intestinal lamina propria macrophages. Conversely, we detected much higher levels of the FcR gamma chain in monocytes than in neutrophils, but the FcR gamma chain was also downregulated in tissue macrophages as well as in in vitro-differentiated monocyte-derived macrophages and dendritic cells. The implications of our current findings on the biological functioning of CD89 are discussed.     

Differential effects of alpha-, beta- and gamma(2)-melanocyte-stimulating hormones on hypothalamic neuronal activation and feeding in the fasted rat.

Hypothalamic pro-opiomelanocortin neurones have an established role in the control of feeding. While pro-opiomelanocortin is the precursor for at least three melanocortin peptides, alpha-, beta- and gamma-melanocyte-stimulating hormone (MSH), it has been widely assumed that alpha-MSH is the predominant ligand involved. We compared the effects of centrally administered alpha-, beta- and gamma(2)-MSH on hypothalamic neuronal activation and on food intake in rats fasted for 48 h. Significant reductions in food intake were seen with alpha-MSH (first hour) and gamma(2)-MSH (second hour) but not with beta-MSH. The pattern of neuronal activation, assessed by the detection of early growth response factor-1 protein, showed considerable overlap; all three melanocortins activated cells in the arcuate, ventromedial, paraventricular, periventricular and supraoptic nuclei, as well as the preoptic area. alpha-MSH and beta-MSH produced activation in the dorsomedial nuclei while gamma(2)-MSH was only weakly active here. Retrograde labelling by systemic Fluorogold injection revealed that many cells activated by MSH compounds in the arcuate, paraventricular, periventricular and supraoptic nuclei (but not dorsomedial or ventromedial) project outside the blood-brain barrier and are therefore likely to include neuroendocrine cells. Desacetyl-alpha-MSH, which has previously been reported to lack effects on feeding, produced no discernible neuronal activation in the hypothalamus.Our finding that both the pattern of neuronal activation and the distribution of neuroendocrine cells activated in response to these closely related peptides show only partial overlap suggests that, in addition to common pathways, there may exist distinct hypothalamic circuits activated by different pro-opiomelanocortin products. The slower time course of gamma(2)-MSH- versus alpha-MSH-induced suppression of feeding provides further support for the notion that the biological responses to individual melanocortin peptides may involve distinct neuronal mechanisms.     

Scanning electron microscopic study of the changes of the luminal surface of rat thyroid epithelial cells following exposure to cold for 6 to 48 hours (author''s transl)

Zusammenfassung Rasterelektronenmikroskopische Untersuchungen von Schilddrüsenfollikelzellen von Ratten, die 6 bis 48 Stunden einer Temperatur von +4° ausgesetzt gewesen waren, haben gezeigt, dass, im Vergleich mit denen von bei 22° gehaltenen Kontrolltieren, die Zahl der apikalen Mikrovilli zugenommen hat. Diese Ultrastrukturunterschiede — die 48 Studen nach Versuchsbeginn statistisch signifikant sind — stehen in Beziehung mit der durch die Kälteeinwirkung bedingten Aktivitätssteigerung der Schilddrüse.

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Summary Scanning electron microscopic observations of thyroid follicle cells of rats exposed to 4° C for 6 to 48 hours were found to show an increased number of apical microvilli in comparison to those of controls kept at 22° C. These ultrastructural differences — which were statistically significant 48 hours after start of the experiments — are related to stimulation of thyroid gland activity by exposure to cold.

Mit technischer Mitarbeit von Frl. Christa Thommen und Herrn P.-A. Milliquet.     

Application of microbiological quantitative methods for evaluation of changes in the amount of bacteria in patients with wounds and purulent fistulas subjected to phage therapy and for assessment of phage preparation effectiveness (in vitro studies)

PurposeQuantitative microbiological studies may provide important information required for successful phage therapy (PT), however methods for PT monitoring of purulent wounds and fistulas has never been reported before. Therefore our goal was to determine and apply microbiological quantitative methods (MQMs) for monitoring experimental PT.MethodsSamples from agar plates with growing bacteria were collected using dry and wet sterile compresses, or swabs. After shaking the sample in saline the amount of bacteria in suspension was determined. The method was standardized. The MQM using compress was applied for comparison of in vitro activity of phage preparations with other agents for wound rinsing. The usefulness of this swabbing method was tested in the Phage Therapy Unit for monitoring of experimental PT of patients with chronic wounds or purulent fistulas.ResultsMinimum, maximum and standard deviation values used for standardization of the studied method showed that data repeatability was good; thus the method was used for quantitation of bacteria taken both from plates in vitro and patients samples.Effectiveness of phage preparations was compared to gentamicin in vitro. Phages were as effective as antibiotics in reducing the amount of bacteria on agar plates, and this effect was not only due to simple mechanical removal of bacteria, but dependent on their antibacterial activity. We have also observed that the results of bacteria quantitation may correlate with the local status of a wound/fistula in a particular stage of PT.ConclusionThe standardized swabbing method of bacteria quantitation can be used for PT monitoring. Presented MQMs are simple and may help to monitor the therapy process and to decide on its duration, frequency and a kind of the phage applied. They can also be applied in other antibacterial treatment strategies.     

Expression of von Hippel-Lindau gene product (pVHL) and S100P in cystic neoplasms of the pancreas--with an implication for their roles in tumorigenesis

Our recent study demonstrated the inverse correlation of expression of S100P and von Hippel-Lindau gene product (pVHL) in pancreatic intraepithelial neoplasia (PanIN) and pancreatic ductal adenocarcinoma (PDA). This study investigates whether there is a correlation of expression of these two markers in common cystic neoplasms of the pancreas. Immunohistochemical stains for S100P and pVHL were performed on 97 cystic neoplasms of the pancreas, including 23 mucinous cystic neoplasms (MCNs), 39 intraductal papillary mucinous neoplasms (IPMNs), 12 solid pseudopapillary tumors (SPTs), and 23 serous microcystic adenomas (SMAs). The results demonstrated a nuclear and cytoplasmic staining pattern of S100P in 91.3% of MCNs and 100% of IPMNs. In contrast, none of the SPTs or SMAs was positive for S100P. All IPMNs and SPTs, and all S100P-expressing MCNs were negative for pVHL, including 20 MCNs and 20 IPMNs with low-grade dysplasia. All cases of SMA were positive for pVHL. Our data suggest 1) the inverse correlation of expression of S100P and pVHL in MCN and IPMN is similar to that in PanIN and PDA, suggesting a role of these two proteins in early tumorigenesis; 2) the loss of pVHL expression in MCN and IPMN supports the recent recategorization of these neoplasms without any cytological atypia as low-grade dysplasia instead of adenoma; 3) the loss of expression of pVHL in SPT supports the concept of an uncertain malignant potential of this entity; and 4) the lack of expression of S100P and expression of pVHL in SMA supports the benign nature of this entity.     

肥厚性瘢痕胶原酶和TIMP-1mRNA表达的研究

采用原位杂交技术和图像分析技术观测了ＨＴＳ中ＭＭＰ－１和ＴＩＭＰ－１的基因表达。结果发现ＭＭＰ－１和ＴＩＭＰ－１的表达较正常增加，平均积分光密度值分别增加了１．８（Ｐ＞０．０５）和７倍（Ｐ＜０．００１）。ＨＴＳ中ＴＩＭＰ－１的表达明显高于ＭＭＰ－１（Ｐ＜０．００１）。ＭＭＰ－１和ＴＩＭＰ－１阳性颗粒多分布于胶原结节，这种表达的差异性提示ＨＴＳ胶原酶活性可能受到ＴＩＭＰ－１的抑制，ＴＩＭＰ－１的表达异常可能同ＨＴＳ的形成密切相关     

Mapping and comparison of the interaction sites on the Fc region of IgG responsible for triggering antibody dependent cellular cytotoxicity (ADCC) through different types of human Fc gamma receptor.

In the present study 3-iodo-4-hydroxy-5-nitrophenacetyl (NIP)-specific antibodies were compared for induction of antibody dependent lysis of NIP-derivatised red blood cells effected by pre-stimulated U937 or HL-60 cells and by K cells. The chimaeric antibodies have heavy chains corresponding to human IgG subclasses 1-4, and include site-directed mutants of IgG3 as well as the aglycosylated form of IgG3; a mouse IgG2b antibody and a site-directed mutant IgG2b were also examined. rIFN stimulated U937 or HL-60 cells express increased levels of Fc gamma R1 compared to unstimulated cells; PMA stimulated HL-60 and U937 cells express an increased level of Fc gamma R11 compared to unstimulated cells; K cells express Fc gamma R111. Using these effector cell populations and the target cells mentioned above, we have compared anti-NIP antibodies with different heavy chain constant domains for their ability to induce ADCC through human Fc gamma R1, Fc gamma R11 and Fc gamma R111. The results suggest that all three human Fc gamma receptors appear to recognise a binding site on IgG within the lower hinge (residues 234-237) and trigger ADCC via this site, but that each receptor sees this common site in a different way. The possibility that other amino acid residues also participate in the binding/triggering site(s) cannot be excluded.     

Effect of injury to the medial forebrain bundle and preoptic region on activity of a strychnine-induced epileptiform focus (on the phenomenon of the hyperactive determinant dispatch station)

Experiments on cats showed that injury to the medial forebrain bundle (MFB) and to some extent of the preoptic region (PR) also on the side of application of strychnine to the cerebral cortex (middle suprasylvian gyrus) causes depression of seizure activity (spike potentials) in the strychnine focus and also in a secondary mirror focus in the symmetrical zone of the cortex of the opposite hemisphere. The same effect could be obtained in some cases after injury to MFB only. Injury to MFB and to part of PR on the side of the mirror focus causes depression of spike potentials in that focus only and does not affect activity in the primary epileptiform focus. The effects described above are examined from the standpoint of the role of the determinant dispatch station (DDS) in CNS activity: The primary epileptiform focus is a hyperactive DDS which induces the appearance of secondary foci and maintains and determines the character of their activity. It can be concluded from these experimental results that MFB participates in the modulation of cortical epileptiform activity.Laboratory of General Pathology of the Nervous System, Institute of General Pathology and Pathological Physiology, Moscow. Laboratory of Electrophysiology, V. P. Filatov Institute of Eye Diseases and Tissue Therapy, Odessa. Translated from Byulleten' Éksperimental'noi Biologii i Meditsiny, Vol. 82, No. 10, pp. 1155–1158, October, 1976.