The differentiation of the nerve and glial cells of neocortical transplants placed into the brain of adult rats

Embryonal neural tissue of 17-day-old rat embryos was transplanted into the brain of adult Wistar rats to test the differentiation of transplants with reference to the normal cerebral cortex development. The control and the experimental rats were decapitated 2, 5, 7, 10, 15, 20, 25, and 35 days after the transplantation. Differentiation of neural tissue was studied using monoclonal antibodies against neurofilaments as well as by counting the proportion of differentiated neurons. The glial differentiation was studied by immunohistochemical method using monoclonal antibodies against acid glial fibrillar protein and vimentin. The differentiation of neural cells of transplants proved to be synchronous with the normal ones while the differentiation of glial cells accelerates.     

Age and tissue-dependent changes of ornithine decarboxylase and s-adenosylmethionine decarboxylase activities in neural tissue and fat body of adult crickets

The activities of ornithine decarboxylase and S-adenosylmethionine decarboxylase, two key enzymes in polyamine metabolism, were determined during the first 10 days of imaginal life in the nervous tissue and the fat body of the adult cricket Acheta domesticus. The kinetic constants of the two enzymes were also determined in both tissues. Both decarboxylases presented a higher activity in fat body than in nervous tissue. In nervous tissue, the activity of the two enzymes peaked at 16 h postemergence, then slowly decreased up to day 3–4. By contrast, the enzymatic activities in fat body, low at emergence, strongly increased on day 2. Thereafter, whereas ornithine decarboxylase activity remained rather high. S-adenosyl-methionine decarboxylase activity dropped back to emergence levels by day 10. These results, examined in light of the temporal alterations of polyamine levels observed in the two tissues, demonstrate synchronous variations between polyamine contents and the enzymes involved in their biosynthesis. © 1993 Wiley-Liss, Inc.     

Cytochemical distribution of beta-glucuronidase activity in experimental brain tumors and brain tissue in vivo and in vitro

Summary Activity of beta-glucuronidase in experimental brain tumors and in nervous tissue was studied. The activity of the enzyme was detected both in gliomas and sarcomas exhibiting a high degree of anaplasia and dysplasia. Reactive glia showed high enzymatic activity; whereas activity in normal nervous tissue was absent. In tissue culture the activity of beta-glucuronidase was observed mainly in astrocytes, and its intensity expressed the degree of cellular maturation and differentiation.The investigation was carried out under a grant from PL 480 US Public Health Service Program, Agreement 05-004-1.     

Rejection of fetal neocortical neural transplants by H-2 incompatible mice

In order to examine questions concerning immunologic privilege of the central nervous system, we placed neocortical transplants into cerebral ventricles of mice. We compared the fates of transplants between fully H-2 compatible (isografts) and H-2 incompatible (allografts) animals. Histologic evaluation comparing animals from iso- and allograft groups revealed significant differences in the number of inflammatory cells and in the degree of necrosis within the grafts. Response to allografted tissue within the brain mimics that seen in several immune-mediated diseases of the nervous system in that neurons appear to be selectively spared. Only upon subsequent stimulation of the host's immune system with an orthotopic skin graft bearing the major histocompatibility complex antigens of the neural graft are neurons destroyed. Immunohistochemical evaluation revealed that the inflammatory cell infiltrates in and around the allografts were composed of Lyt-2+, L3T4+, and Mac-1+ cells. In addition, Ia+ endothelial cells as well as Ia+ parenchymal CNS cells were found in both donor and host tissue of allografted animals. Hence, H-2 incompatible neural tissue transplanted to the CNS is recognized and rejected by the immune system of the recipient animal. The cellular infiltrates seen within the first weeks to months following transplantation of allogeneic CNS tissue resemble those seen in other allografts undergoing rejection. We conclude that the CNS is not unconditionally privileged as either a transplant site or as a source of transplanted tissue.     

Transplantation of fetal neocortex in rhesus monkey

A feasibility study of neural transplantation in adult rhesus monkey was undertaken. Fresh and preserved neocortex containing multiplying and maturing neurons obtained from 55–70 gestation days were transplanted into the striatum, cerebellum and cerebral cortex of adult monkeys. Tissues were preserved for 4 days either at subzero temperature in the freezer compartment of the ordinary refrigerator in Ringer lactate or incubated in culture medium. While 2 monkeys out of 5 injected with preserved tissue had successful transplants after 4 months, all the 10 monkeys injected with fresh tissue had no transplants. The size of the two surviving transplants was small. The neurons in the transplants were mainly in clusters. Many of the cells were immature and some showed early degenerative changes. Neuronal processes were restricted to the transplants and thus showed lack of morphological integration with the host tissue. Further studies are in progress to define the nature of the embryonic tissue of primate which can grow and survive and also the role of neural grafts in functional recovery following experimental lesions of the brain regions.     

LR Gold embedding of nervous tissue for immunoelectron microscopy studies

Summary A major drawback of all acrylic resins commonly used for post-embedding immunocytochemical studies of the central nervous system is the disruption of the ultrastructural morphology, due to the high lipid content of neural tissue. We have investigated the suitability of the acrylic resin LR Gold, which has been employed recently for immunogold labeling studies in several non-neural tissues. Optimal preservation of both antigenicity and ultrastructure of nervous tissue was obtained after en bloc staining with uranyl acetate, followed by total dehydration in acetone and curing at low temperature. Cell membranes and myelin sheaths, which are usually lost with other acrylic resins, were well maintained. The degree of antigenicity of LR Gold-embedded tissues was comparable to that of LR White-embedded one, but the morphologic detail was much better preserved. The use of LR Gold is particularly advantageous for studying neurodegenerative disorders such as Alzheimer disease.     

LR Gold embedding of nervous tissue for immunoelectron microscopy studies.

A major drawback of all acrylic resins commonly used for post-embedding immunocytochemical studies of the central nervous system is the disruption of the ultrastructural morphology, due to the high lipid content of neural tissue. We have investigated the suitability of the acrylic resin LR Gold, which has been employed recently for immunogold labeling studies in several non-neural tissues. Optimal preservation of both antigenicity and ultrastructure of nervous tissue was obtained after en bloc staining with uranyl acetate, followed by total dehydration in acetone and curing at low temperature. Cell membranes and myelin sheaths, which are usually lost with other acrylic resins, were well maintained. The degree of antigenicity of LR Gold-embedded tissues was comparable to that of LR White-embedded one, but the morphologic detail was much better preserved. The use of LR Gold is particularly advantageous for studying neurodegenerative disorders such as Alzheimer disease.     

Oscillations of trypsinogen activation

Trypsin activity oscillations are shown by the autocatalytic activation of trypsinogen at 0 degrees C in aqueous solution. The oscillations were observed for 3-4 days and show only slight decrease in enzyme activity. The zymogen has been kept at ice water temperature and pH 8.2 in the presence of Mn2+ ion. The mean periods of around 1.5 hr are about half of those found previously at -10 degrees C in frozen aqueous solution, while the amplitudes related to the mean activity are about one-fourth of that in the frozen experiments. The phenomenon of oscillation is interpreted in terms of coupling between the inhomogeneities of protein and ion concentrations of the unstirred solution and a Mn3+/Mn2+ system, causing synchronous, periodic reduction-oxidation of some cystine bridges in the protein chain. These nonequilibrium conditions, together with synchronous transitions among several conformational states, may produce the observed activity oscillations.     

Cryopreservation of embryonic cerebral tissue of rat.

Embryonic cerebral tissues (ECT) either fresh or frozen-stored, were cultured and transplanted into the cerebella of neonatal host rats. Many variables including composition of the freezing medium, freezing and thawing rates, and storage time in liquid nitrogen were studied systematically. The results indicated that the following conditions yielded good results for tissue culture: using 1 M Me2SO as the cryoprotectant, freezing the brain tissues at a rate of 1 degrees C/min until it reached -70 degrees C, storing the frozen samples in liquid nitrogen and thawing them fast in a 37 degrees C water bath. The viability of the frozen-thawed tissues was assessed by their abilities to grow and differentiate in vitro and in vivo after intracerebral grafting. In tissue culture, growth and differentiation were similar to those of the fresh ECT. Cell morphology and staining reactions were normal in supravital methylene blue staining, cresyl violet staining, and acetylcholinesterase staining. Neurons had well-developed Nissl bodies, and cholinergic neurons also differentiated. Autoradiographic studies showed that more than 50% of the neurons had the ability to uptake gamma-aminobutyric acid with high affinity. In brain tissue transplantation, 9 of 12 transplants survived subsequent grafting after cryopreservation. Moreover, the grafts of surviving cryopreserved tissue displayed cytological and cytoarchitectural characteristics identical to those of fresh grafts. All grafts were integrated with the surrounding host neural tissue. This suggested that there may be synaptic connections between the transplants and the host brain tissues. From this and similar studies on the subject by others wer conclude that cryopreservation is a feasible method for storage of embryonic brain tissue to be used later for intracerebral grafting.     

Experimental validation of the use of an ultrasonic surgical instrument in neurophysiology

The effect of ultrasonic and surgical instruments on nervous tissue in chronic experiments on the cats were investigated with electrophysiological and morphological methods. The authors compared the results of removal of the neocortex zones using ultrasonic and surgical instruments or routine methods. Electrophysiological and morphological studies have shown small injury effects made by ultrasonic and surgical instruments on the surrounding brain tissue.     

Effects of cryobiological variables on the survival of skin using a defined murine model

Skin from an inbred strain of hairless mouse was used as a homogeneous model tissue for studies of skin cryopreservation. Tetrazolium reductase enzyme activity was used to assess tissue viability. Hepes-buffered 199 tissue culture medium was confirmed to be a suitable basal medium, to which cryoprotectants were added. Addition of serum to the cryoprotective cocktail had no beneficial effect. Three cryoprotectants, dimethyl sulfoxide, ethanediol, and glycerol were evaluated. There was no evidence of specific toxicity attributable to the cryoprotective agents during the permeation period; however, short permeation times at low temperature were associated with maximum skin viability. Following freezing and thawing, higher viabilities were obtained when using a slow (-1 degree C min-1) or medium (-60 degree C min-1) rather than a fast (immersion in liquid nitrogen) cooling rate. Dimethyl sulfoxide was a marginally better cryoprotectant overall, although this difference was not statistically significant.     

Preparation and characterization of Cryptococcus neoformans synchronous culture

We have developed a method for preparation of synchronous culture in Cryptococcus neoformans. The method is based on age fractionation of exponentially growing asynchronous culture through differential sedimentation in 10-20% (w/v) lactose gradient. C. neoformans capsule thickness should be reduced to a minimum to ensure most accurate age fractionation, which is necessary to obtain a higher degree of synchrony. The C. neoformans synchronous culture system has revealed important characteristics with respect to cellular morphology, DNA content and cell volume distribution. The method can be used for further cell cycle studies.     

Phenotypic differentiation of neurons in intraocular transplants

Neurochemical differentiation of neurons in transplants developing in rat anterior eye chamber was studied. Pieces of the somatosensory neocortex area, isolated from 17-day fetuses of Wistar rats, were used for the transplantation. The general cytological analysis and immunochemical identification of GABAergic neurons in neocortical transplants and in the appropriate brain area of the recipient rats (control) were carried out after 6 months. Cytoarchitectonics typical for neocortex was not revealed in the transplants. Furthermore, a 1.4-fold decrease in numerical density of the entire neuron population was found compared to the control. The proportion of GABAergic nerve cells in the transplanted tissue was reduced even more dramatically— by 13.1 times. The dimensions of all types of neurons, especially GABAergic cells, were greater in the transplants in oculo compared to neocortex in situ. The increase in size occurred mostly due to the cytoplasm. Thus, the nuclei of GABA-positive neurons in the transplants were larger by 1.2 times compared to the control and their perikarya were larger by 1.5 times. The obtained results showed that the conditions in the anterior eye chamber the most dramatically affect the differentiation of GABAergic neurons, and cell hypertrophy, probably, is the functional compensation of the decrease in their number. Considering the literature data on the increased excitability and synchronized neuronal activity in the intraocular transplants, it can be assumed that these transplants can be used as a model for studying the cellular mechanisms of nervous tissue epileptization under disinhibition conditions.     

The hippocampus and neuro-transplantation

A review of the author's histological and electron-microscopic studies of differentiation of hippocampal transplants with different levels of the graft/host integration. The grafts developing in the anterior eye chamber were the experimental model of complete isolation from the brain. The effects of various factors (age of the donor fetal tissue, host age and strain, degree of the integration with the recipient brain) on the growth and neural organization of grafts were studied. Analysis of fine structure of intraocular and intracortical grafts, as a rule, showed mature highly differentiated neurons and glia and normal density of typical synaptic contacts. However, morphological features suggesting both hyperactivity of some neurons and continuous growth of some neural processes were observed. The expression of nonsynaptic and transport-metabolic interactions between the cells was increased. The observed ultrastructural deviations can be regarded as a compensatory adaptation of the tissue to the deficit of specific afferent signals. It was shown that in the absence of normal cellular targets, axons of the grafted neurons establish functional synaptic contacts with improper neural elements in the host brain.     

Development of interscapular brown adipose tissue in the hamster. II - Differentiation of transplants in the anterior chamber of the eye: role of the sympathetic innervation

The relationship between brown adipose tissue (BAT) and its sympathetic innervation during development was investigated by transplantation of undifferentiated (white fat-like) hamster BAT into the anterior eye chamber of adult hamsters. Such transplants are known to be revascularized and reinnervated by the vessels and the nerves of the host iris. The morphology of the BAT transplants was analysed during the post-operative weeks by light and electron microscopy, and the ingrowth of sympathetic nerve fibres from the iris was followed by radioautography. BAT appeared to differentiate in oculo, i.e. presented increasing amounts of adipocytes with multilocular fat deposits and abundant, well-developed mitochondria, but only after a delay of approx. 10 days, and remained much fatter than in situ. The establishment of the sympathetic innervation was not synchronous with the revascularization process. It occurred simultaneously with the morphological differentiation of the BAT transplants, and the nerve fibre density remained low. In the absence of sympathetic innervation, i.e. when the host irides were sympathectomized prior to transplantation, BAT still differentiated, but the process was further delayed and the proportion of differentiated brown adipocytes after 20 days in oculo was clearly lower than in control transplants. It is concluded that the sympathetic innervation in BAT is involved in the regulation of differentiating activity in the tissue, but is not obligatory for differentiation to occur.     

ENZYME PATTERNS DURING A SYNCHRONOUS GROWTH CYCLE OF CHLORELLA PYRENOIDOSA

There have been no studies in which a significant number of isozymes have been investigated during a synchronous growth cycle of any organism. The present study was designed to obtain information on the fluctuations of a broad spectrum of enzymes during a synchronous growth cycle of Chlorella pyrenoidosa. Of the thirty-two enzymic activities investigated, seventeen could be localized on starch gels from a high temperature strain of Chlorella pyrenoidosa. Nine of these activities were found to possess more than one band of activity by starch gel electrophoresis. Seven of these activities were localized on starch gels throughout a synchronous growth cycle of C. pyrenoidosa grown in continuous light. Assay techniques are described. The fluctuations in enzyme activity are discussed with relation to concurrent metabolic and cytological changes during cell maturation in C. pyrenoidosa.     

Deposition of the terminal C5b-9 complement complex in infarcted areas of human myocardium

Poly- and monoclonal antibodies to neoantigens of the human C5b-9 complement complex, as well as polyclonal antibodies to C5, C8, and C9, were used to detect and identify C5b-9 deposits in human myocardial tissue. Immunocytochemical studies were performed on fresh-frozen autopsy material derived from patients with myocardial infarctions; in addition, in 17 of these patients, paraffin sections of formalin-fixed tissue were investigated. Sixteen autopsies from patients with noncardiac diseases were analyzed as controls. Without exception, C5b-9 positivity was registered selectively and exclusively on and in myocardial cells located within the zones of infarction. The selectivity of staining was confirmed by control reactions for succinic dehydrogenase activity performed in adjacent, respective double-stained sections. Most intensive staining with anti-neoantigen antibodies was observed in the peripheral areas of the infarctions. Weak staining for C3d, rather strong staining for C5 and C9, and intermediate staining with anti-C8 antibodies were observed in the same localizations. Stainings for C4 and IgA were negative, whereas immunocytochemical reactions for IgG and IgM revealed an irregular and very weak staining. Only very weak staining was also observed with a monoclonal antibody to complement S-protein, indicating that the terminal complement components were deposited mainly in the form of membrane-damaging C5b-9 complexes. Immunocytochemical staining for C5b-9 was found to represent a most sensitive tool for detection of ischemic myocardial lesions, permitting easy detection even of single cell necroses. As a working hypothesis, we suggest that initial ischemia may cause loss of the ability of the heart muscle cells to regulate complement turnover at the membrane level. The resulting deposition of C5b-9 on the cell membranes may contribute to functional disturbance and irreversible damage of myocardial cells during the infarction process.     

Age related classification of neurons in the rat visual cortex: a Nissl study (author's transl)

Using Nissl preparations, 6, 8, 12, 16, 20, 30, 90, and 420 days old albino rats were investigated with regard to classification of several neuron types within layer IV and adjacent zones of layers III and V. Increasing maturation of the nervous tissue provides a greater classifiable variety of neuron types. About between the second and third postnatal week the degree of classification reach partially almost the same level as in the adult animals. Particular attention is paid to a large, frequently bipolar neuron type rich in cytoplasm.     

Evaluation of a new biosensor-based mushroom (Agaricus bisporus) tissue homogenate: investigation of certain phenolic compounds and some inhibitor effects

A biosensor based on mushroom tissue homogenate for detecting some phenolic compounds (PCs) and usage of the biosensor for quantifying certain substances that inhibit the polyphenol oxidase activity in mushroom (Agaricus bisporus) tissue homogenate is described. The mushroom tissue homogenate was immobilized to the top of a Clark-type oxygen electrode with gelatin and glutaraldehyde. Optimization of the experimental parameters was done by buffer system, pH, buffer concentration, and temperature. Besides, the detection range of eight phenolic compounds were obtained with the help of the calibration graphs. Thermal stability, storage stability, and repeatability of the biosensor were also investigated. A linear response was observed from 20 x 10(-3) to 200 x 10(-3) mM phenol. The biosensor retained approximately 74% of its original activity after 25 days of storage at 4 degrees C. In repeatability studies, variation coefficient (C.V.) and standard deviation (S.D.) were calculated as 2.44% and +/-0.002, respectively. Inhibition studies revealed that the proposed biosensor was applicable for monitoring benzoic acid and thiourea in soft drinks and fruit juices.     

Lesions of the hypothalamic paraventricular nucleus and norepinephrine turnover in rats

The effects of bilateral lesions of the hypothalamic paraventricular nuclei (PVN), of rats with a mean weight of 260 g body, on eating habits and body weight, as well as on sympathetic nervous system (SNS) activity in interscapular brown adipose tissue (IBAT) were investigated. In 59 of 131 Sprague-Dawley female rats, PVN lesions resulted in hyperphagia and obesity. Although lesions were considered successful when more than 50% of the PVN was destroyed histologically, such lesions were observed in 35.9% (47/131) of all lesioned rats and all of these 47 rats were obese. Therefore, in this study, these 47 rats which were confirmed histologically, were designated as "PVN-lesioned rats". Plasma insulin levels in these 47 PVN-lesioned ats were more than double those of the controls. However, no significant differences were observed between plasma glucose levels in PVN-lesioned and control groups. Norepinephrine turnover, a reliable indicator of SNS activity, in IBAT, heart and pancreas was similar in PVN-lesioned and sham-operated control animals, even under contrasting conditions of feeding (ad libitum and fasting) and temperature (22 degrees C and 4 degrees C). It is concluded that PVN lesions produce hyperphagia, obesity and hyperinsulinemia in rats with an average body weight of 260g without affecting the SNS activity in IBAT, heart or pancreas.