Calafiore cardioplegia offers better myocardial protection in adult and senescent rat hearts than Del Nido cardioplegia.

OBJECTIVES: We compared the cardioprotective capacity of Del Nido cardioplegia and warm Calafiore blood cardioplegia in an experimental setting during 90 min of ischaemia. METHODS: 20 adult and 20 senescent rat hearts were isolated and mounted on a blood-perfused, pressure-controlled Langendorff apparatus. After a stabilization period, cardiac arrest (90 min) was induced by the administration of either Calafiore (Cala) or Del Nido solution (DNS). While Cala was given warm and intermittently, DNS was given as a cold single shot. During 90 min of reperfusion, cardiac function and metabolism were evaluated and biomarker levels were measured. After the end of the experiment, hearts were prepared for electronmicroscopic investigation. RESULTS: Hearts exposed to Cala recovered faster during reperfusion compared with hearts administered DNS (Cala vs DNS at 30 min reperfusion: left ventricular developed pressure 72, SD: 22% of baseline (BL) versus 40, SD: 32% of BL, p < .001, and positive derived left ventricular pressure over time was better in both adult and senescent Cala groups (96, SD: 31% of BL) than in the DNS groups (39, SD: 27% of BL, p < .001). Ischaemic contractures were seen in the DNS groups starting after 30 min of ischaemia, whereas no rise in diastolic pressure was observed for the Cala groups. Accordingly, lactate production was higher after DNS (1.23 mg/dl (SD 0.87) than after Cala (0.33 mg/dl (SD 0.68), p = .015) at the beginning of reperfusion. Troponin I levels at the end of reperfusion were higher after DNS treatment in adult hearts (DNS: 287.9 SD: 147.7 ng/mL vs Cala 91.2: SD: 94.7 ng/mL, p = .02) and in senescent hearts (DNS: 376.5 (SD: 162.8) ng/ml versus Cala 104.7 (SD: 150.2) ng/ml, p = .025). Electron microscopy showed that the cellular oedema index was higher in adult DNS hearts (1.2 ± 0.2) than in adult Cala hearts (0.8 ± 0.1, p = .012), whereas the VS ratio was similar (0.18 ± 0.01 vs 0.17 ± 0.03). CONCLUSION: Calafiore cardioplegia offers better myocardial protection from ischaemia/reperfusion-related damage in isolated perfused adult and senescent rat hearts than Del Nido cardioplegia.

Bottlenose dolphins (Tursiops truncatus) do also cast neutrophil extracellular traps against the apicomplexan parasite Neospora caninum.

Neutrophil extracellular traps (NETs) are web-like structures composed of nuclear DNA decorated with histones and cytoplasmic peptides which antiparasitic properties have not previously been investigated in cetaceans. Polymorphonuclear neutrophils (PMN) were isolated from healthy bottlenose dolphins (Tursiops truncatus), and stimulated with Neospora caninum tachyzoites and the NETs-agonist zymosan. In vitro interactions of PMN with the tachyzoites resulted in rapid extrusion of NETs. For the demonstration and quantification of cetacean NETs, extracellular DNA was stained by using either Sytox Orange® or Pico Green®. Scanning electron microscopy (SEM) and fluorescence analyses demonstrated PMN-derived release of NETs upon exposure to tachyzoites of N. caninum. Co-localization studies of N. caninum induced cetacean NETs proved the presence of DNA adorned with histones (H1, H2A/H2B, H3, H4), neutrophil elastase (NE), myeloperoxidase (MPO) and pentraxin (PTX) confirming the molecular properties of mammalian NETosis. Dolphin-derived N. caninum-NETosis were efficiently suppressed by DNase I and diphenyleneiodonium (DPI) treatments. Our results indicate that cetacean-derived NETs represent an ancient, conserved and relevant defense effector mechanism of the host innate immune system against N. caninum and probably other related neozoan parasites circulating in the marine environment.

Regulation of cargo transfer between ESCRT-0 and ESCRT-I complexes by flotillin-1 during endosomal sorting of ubiquitinated cargo.

Ubiquitin-dependent sorting of membrane proteins in endosomes directs them to lysosomal degradation. In the case of receptors such as the epidermal growth factor receptor (EGFR), lysosomal degradation is important for the regulation of downstream signalling. Ubiquitinated proteins are recognised in endosomes by the endosomal sorting complexes required for transport (ESCRT) complexes, which sequentially interact with the ubiquitinated cargo. Although the role of each ESCRT complex in sorting is well established, it is not clear how the cargo is passed on from one ESCRT to the next. We here show that flotillin-1 is required for EGFR degradation, and that it interacts with the subunits of ESCRT-0 and -I complexes (hepatocyte growth factor-regulated tyrosine kinase substrate (Hrs) and Tsg101). Flotillin-1 is required for cargo recognition and sorting by ESCRT-0/Hrs and for its interaction with Tsg101. In addition, flotillin-1 is also required for the sorting of human immunodeficiency virus 1 Gag polyprotein, which mimics ESCRT-0 complex during viral assembly. We propose that flotillin-1 functions in cargo transfer between ESCRT-0 and -I complexes.

Molecular analyses on Neospora caninum-triggered NETosis in the caprine system.

Neospora caninum is an obligate intracellular protozoan parasite causing serious reproductive disorders in large and small ruminants worldwide. Polymorphonuclear neutrophils (PMN) react against multiple invading pathogens through different mechanisms including the release of neutrophil extracellular traps (NETs). Here, in vitro interactions of caprine PMN and N. caninum tachyzoites were studied. Scanning electron microscopic- and immunofluorescence-analyses demonstrated that caprine PMN undergo NETosis upon contact with tachyzoites of N. caninum, extruding filaments that entrap parasites. Detailed co-localization studies of N. caninum tachyzoite-induced NETs revealed the presence of PMN-derived DNA being decorated with histones (H1, H2A/H2B, H3,H4) and neutrophil elastase (NE) corroborating the molecular characteristics of classical mammalian NETs. As a new result for parasite-induced NETosis, we identified pentraxin and cathepsin B in N. caninum-triggered NETs. Nonetheless, functional inhibition assays revealed that during caprine NET formation triggered by N. caninum different molecular signaling pathways are induced, when compared to other apicomplexan parasites or host species. As such, N. caninum-induced NETosis appears to be influenced by MPO but independent of NADPH oxidase, SOCE, ERK1/2 and p38 MAPK activities. Furthermore, the inhibition of PMN autophagy via blockage of the PI3K-mediated signaling pathway failed to influence tachyzoite-induced NETosis. Since N. caninum-tachyzoites induced caprine NETosis, this effector mechanism should be considered as an early host immune response during acute caprine neosporosis.

Hsp70 and lipid peroxide levels following heat stress in Xeropicta derbentina (Krynicki 1836) (Gastropoda, Pulmonata) with regard to different colour morphs.

Terrestrial snails which live under dry and hot conditions need efficient mechanisms of adaptation to counteract the problems of desiccation and over-heating. A profoundly heat tolerant snail species is the Mediterranean Xeropicta derbentina, exhibiting different shell colour morphs ranging from pale white to darkly banded. Considering that dark-pigmented snails are believed to have a disadvantage due to faster heating, we investigated possible differences in the stress markers Hsp70 and lipid peroxideation between four pre-defined colour morphs which were exposed to different temperatures for eight hours. The highest Hsp70 levels were observed in response to 38-40 °C. Levels decreased when this temperature was exceeded. Snails of a pre-defined colour category 3 (with a large black band at the umbilicus side of the shell) showed the most prominent Hsp70 response. Lipid peroxideation levels also showed a maximum at 38 °C but displayed a second peak at rather high temperatures at which the Hsp70 level already had decreased (45-48 °C). Particularly pure white snails (category 1) and the most pigmented ones (category 4) were found to have different levels of lipid peroxidation at 38 °C and 45 °C compared to the other morphs. A hypothesis involving a combined two-phase defence mechanism, to which both, the Hsp70 protection system and the antioxidant defence system, may contribute, is discussed.

Daily and seasonal changes in heat exposure and the Hsp70 level of individuals from a field population of Xeropicta derbentina (Krynicki 1836) (Pulmonata, Hygromiidae) in Southern France.

The Mediterranean land snail Xeropicta derbentina forms huge populations in Southern France. In order to characterize heat exposure and the induction of the 70-kD heat shock protein (Hsp70) response system during the life cycle of this snail, a selected population from the Vaucluse area, Provence, was investigated encompassing the issues of morphological life cycle parameters (shell size and colouration), the daily courses of heat exposure at different heights above the ground, of shell temperature, and that of the individual Hsp70 levels. The study covered all four seasons of the year 2011. Snails were found to be annual, reaching their final size in August. The shell colouration pattern showed high variation in juveniles (spring) with a strong tendency towards becoming uniformly white at old age in autumn. In all seasons, ambient air temperature decreased with increasing distance from the ground surface during daytime while remaining constantly low in the night. Overall, the Hsp70 level of individuals followed the ambient temperature during diurnal and seasonal variations. Correlation analysis revealed a positive association of individual shell temperature and Hsp70 level for the most part of the life cycle of the snails until late summer, whereas a negative correlation was found for aged animals indicating senescence effects on the capacity of the stress response system.

Enhanced Ras activity preserves dendritic size and extension as well as synaptic contacts of neurons after functional deprivation in synRas mice.

The monomeric GTP-binding protein p21Ras has been repeatedly implicated in neuronal stability and plastic changes of the adult nervous system. Recently, we have shown that expression of constitutively active Ras protein in transgenic synRas mice results in a significant increase in the dendritic size and complexity of differentiated pyramidal neurons as well as in increased synaptic connectivity. In the present study, we examined the organization of the vibrissae-barrel cortex in synRas mice and the effects of enhanced Ras activity on deprivation-induced dendritic reorganization after vibrissectomy. The results demonstrate a significant increase in vibrissae-barrel sizes and proportional spacing between barrels in synRas mice, suggesting that the neuronal target specificity of thalamocortical terminals is preserved. Accordingly, the arrangement of double bouquet cells at the borders of barrel columns ensuring functional distinctness is unchanged. Partial vibrissectomy is followed by significant dendritic regression of corresponding pyramidal neurons in the barrel cortex of wild-type mice, which, however, could not be observed in synRas mice. The results provide the first evidence for a role of Ras in preserving neuronal structure after functional deprivation in vivo.

Transgenic activation of Ras in neurons increases synapse formation in mouse neocortex.

The small G protein Ras, which is a molecular switch in neurotrophic signal transduction, is implicated in synaptic plasticity and synapse development during ontogeny and in the adult nervous system. To characterise the involvement of Ras-dependent signaling in synaptogenesis, the cortical synapse-to-neuron ratio was investigated in synRas mice overexpressing Val12-Ha-Ras in postmitotic neurons (introduced by Heumann, 2000). The number of synapses per neuron was analysed in cortical layers II/III of the somatosensory cortex at different stages of postnatal development by stereological methods. The synapse-to-neuron ratio was still identical in wild-type and synRas mice at postnatal day 4 before the onset of transgene expression. At P12, P47 and in the adult, analyses revealed a significant increase in the synapse-to-neuron ratio in synRas mice which correlated with the strength of transgene expression. The data presented here provide evidence that Ras activity might be profoundly involved in synaptogenesis by reinforcing the formation or maintenance of synapses during the development and in the adult.

Principles of rat subcortical forebrain organization: a study using histological techniques and multiple fluorescence labeling.

In the present study, we introduce new views on neuro- and chemoarchitectonics of the rat forebrain subcortex deduced from traditional and current concepts of anatomical organization and from our own results. It is based on double and triple immunofluorescence of markers for transmitter-related enzymes, calcium-binding proteins, receptor proteins, myelin basic protein (MBP) and neuropeptides, and on histological cell/myelin stains. The main findings can be summarized as follows: (i) the dorsal striatum of rat and other myomorph rodents reveals a small caudate equivalent homotopic to the caudate nucleus (C) of other mammals, and a large putamen (Pu). (ii) Shell and core can be distinguished also in the 'rostral pole' of nucleus accumbens (ACC) with the calretinin/calbindin and neuropeptide Y (NPY) immunostaining. The shell reveals characteristics of a genuine striatal but not of an extended amygdala (EA) subunit. (iii) EA and lateral septum show striking similarities in structure and fiber connections and may therefore represent a separate parastriatal complex. (iv) The meandering dense layer (DL) of olfactory tubercle (OT) forms longitudinal gyrus- and sulcus-like structures converging in its rostral pole. (v) The core regions of the islands of Calleja that border the ventral pallidum (VP) sharing some of its features are invaded by myelinated fibers of the medial forebrain bundle (MFB). The island of Calleja magna is also apposed to an inconspicuous, slender dorsal appendage of VP. (vi) The VP is composed of a large dorsal reticulated part traversed by the myelinated GABAergic parvalbumin-immunoreactive axons of the MFB and a slender ventral non-reticulate part close to the islands of Calleja. (vii) Considering their close association to the limbic system, ventral striatum (VS) and VP may represent the oldest part of basal ganglia, whereas dorsal striatopallidal subunits were progressively developed in parallel to the growing neocortical influence on motor behavior.

Retinal pigment epithelium melanin granules are phagocytozed by Müller glial cells in experimental retinal detachment.

The ability of retinal Müller glial cells to perform phagocytosis in vivo is studied in a rabbit model of experimental retinal detachment where pigment epithelial cells are occasionally detached together with the neural retina. While macrophages and/or microglial cells phagocytoze most of the cellular debris at the sclerad surface of the detached retinae, some Müller cells accumulate melanin granules. The granules are virtually intact at the ultrastructural level, and are surrounded by a membrane. They are often located close to the sclerad end of the cells, but some are distributed throughout the outer stem process up to the soma. It is concluded that rabbit Müller cells in vivo are capable of phagocytosis and of transporting the phagocytozed material within their cytoplasm.

Activation of mitogen-activated protein kinase cascade and phosphorylation of cytoskeletal proteins after neurone-specific activation of p21ras. I. Mitogen-activated protein kinase cascade.

Alterations in the phosphorylation state of the microtubule-associated protein tau have been associated with the pathogenesis of neurofibrillary degeneration as well as with a neuroprotective action against apoptotic cell death. Mitogen-activated protein kinases (MAPK) phosphorylate tau protein in vitro but the pathophysiological significance of this tau phosphorylation and its effects on neuronal viability is far from clear. Moreover, an in vivo model of activation of MAPK, a key candidate for in vivo tau phosphorylation, is still lacking. The aim of the present study and the accompanying paper was to establish an animal model of stimulated MAPK and to analyse the consequences on tau phosphorylation and the neuronal cytoskeleton. We took advantage of transgenic mice with neurone-specific expression of activated ras protein (p21H-ras(Val12)). The expression of the transgene in these animals is forced to a subset of neurones by the use of the synapsin I promoter. Activity of B-raf was elevated by 37%, while activity of MAPK (ERK1/ERK2) was increased by 25% associated with a subcellular redistribution from the cytoplasmic to the nuclear compartment. Kinases downstream of MAPK such as p90rsk and glycogen synthase kinase 3beta were only marginally affected. Activity of p70S6 kinase was unaltered. The present model might be useful to study the effects of activation of the MAPK cascade on tau phosphorylation and its cell biological sequelae.

Activation of mitogen-activated protein kinase cascade and phosphorylation of cytoskeletal proteins after neurone-specific activation of p21ras. II. Cytoskeletal proteins and dendritic morphology.

In the present study, we analysed changes in the expression, subcellular distribution and phosphorylation state of the microtubule-associated protein tau and other cytoskeletal proteins after neurone-specific activation of the mitogen-activated protein kinase (MAPK) in the CNS in vivo. We used transgenic mice with a neurone-specific expression of activated ras protein (p21H-ras(Val12), synapsin I promoter) that is associated with an augmented activity of the MAPK. Chronic activation of MAPK cascade influenced tau protein phosphorylation, localisation and dendritic morphology. While the amount of tau protein was elevated by 9%, phospho-epitopes detected by the monoclonal antibodies AT270, 12E8 and SMI34 were increased by about 21%, 40% and 59% respectively. Steady-state levels of tau mRNA were not affected. Thus, the increase in tau protein was most likely due to stabilisation of tau protein by augmented phosphorylation. While in wild-type animals tau protein was preferentially localised in axons, a prominent immunoreactivity was found in the somatodendritic compartment of transgenic mice. This subcellular translocation typically seen in pyramidal neurones was associated with an increase in the dendritic calibre by about 30% and is paralleled by an increase in tubulin of 19%. We were unable to obtain any morphological indication of neurodegenerative processes in these animals. We suggest that the moderate increase in tau protein and phosphorylation may be part of the neuroprotective mechanism. However, further studies on aged transgenic mice will be necessary to establish potential effects on neuronal viability.

Expression of endothelial and inducible NOS-isoforms is increased in Alzheimer's disease, in APP23 transgenic mice and after experimental brain lesion in rat: evidence for an induction by amyloid pathology.

The nitric oxide-synthesizing enzyme nitric oxide synthase (NOS) is present in the mammalian brain in three different isoforms, two constitutive enzymes (i.e., neuronal, nNOS, and endothelial eNOS) and one inducible enzyme (iNOS). All three isoforms are aberrantly expressed in Alzheimer's disease giving rise to elevated levels of nitric oxide apparently involved in the pathogenesis of this disease by various different mechanisms including oxidative stress and activation of intracellular signalling mechanisms. It still is a matter of debate, however, whether the abnormal expression of NOS isoforms has some primary importance in the pathogenetic chain and might thus be a potential therapeutic target or only reflects a secondary effect that occurs at more advanced stages of the disease process. To tackle this question, we analysed the expression of both eNOS and iNOS in patients with sporadic AD, in transgenic mice expressing human amyloid precursor protein (APP) with the Swedish double mutation under control of the Thy1 promotor (APP23 mice), and after electrolytic cortical lesion in rat, an experimental paradigm associated with elevated expression of APP. In all three conditions, an astrocytosis was induced accompanied by a strong increase of both iNOS and eNOS. Both NOS isoforms were frequently though not always colocalized. Thus, based on the expression pattern of NOS isoforms three types of astrocytes, expressing only one of the two isoforms or both together could be distinguished. In both AD and transgenic mice eNOS-expressing astrocytes exceeded iNOS-expressing astrocytes in number. Astrocytes with elevated levels of iNOS or eNOS were constantly seen in direct association with Abeta-deposits in AD and transgenic mice and were found in the vicinity of the lesion site in the rat cortex. The results of the present study show that expression of both iNOS and eNOS is increased in activated astrocytes under experimental conditions associated with elevated expression of APP (electrolytic brain lesion) or Abeta-deposition (APP23 transgenic mice). Therefore, it is suggested that altered expression of these NOS isoforms being part of AD pathology is secondary to the amyloid pathology and might not be primarily involved in the pathogenetic chain though it might contribute to the maintenance, self-perpetuation and progression of the neurodegenerative process.

Immunofluorescence and immunoelectron microscopic evidence for differences in myelination of GABAergic and cholinergic septohippocampal fibres.

It is known that the rat septohippocampal projection is realised at least by GABAergic, parvalbumin containing and cholinergic fibres. The GABAergic component originates from fast-firing and fast-conducting neurons, whereas the cholinergic component represents the slow-firing, slow-conducting type. The present immunofluorescence and immunoelectron microscopic study shows that the vast majority of parvalbumin-immunoreactive, GABAergic axons are surrounded by enormously thick myelin sheaths, but choline acetyltransferase immunoreactive axons were rarely found to be myelinated. In addition, cholinergic fibres show considerably smaller diameters. Accordingly, our results are correlated with the well-known differences in conduction velocities between the GABAergic and cholinergic fibres of the septohippocampal pathway, which depend on myelination and axon calibre.

Facilitation of artificial retinal detachment for macular translocation surgery tested in rabbit.

PURPOSE: For macular translocation surgery, the native attached retina has to be detached either locally or completely. Although different surgical techniques are used, there is a general search for supporting procedures that facilitate and accelerate the retinal detachment. METHODS: Pars plana vitrectomies were performed in pigmented rabbits. In all experimental groups, a local retinal detachment was created by infusing the test solution with a thin glass micropipette attached to a glass syringe. In control animals a standard balanced salt solution was used at room temperature, in combination with a standard vitrectomy light source. In two test groups, a calcium- and magnesium-free solution was used for the vitrectomy, under illumination by a standard light source in group I (solution at room temperature) and group II (solution heated up to body temperature). In group III the rabbits were dark-adapted for half an hour, and then, during surgery, a red filter was used in front of the light source (standard balanced salt solution at room temperature). After the rabbits were killed at the end of surgery, the adherence of the retinal pigment epithelium (RPE) to the neural retina in the detachment area was quantified microscopically, and the morphologic integrity of the detached retinal tissue was examined by light and electron microscopy. No electrophysiology was performed. RESULTS: In all four groups, it was possible to detach the retina. The maximum adherence of the RPE cells to the neural retina was observed in the control group. Virtually no decrease in adherence was found in test group II (36 degrees C solution without calcium and magnesium), whereas a significant decrease was seen in both group I (calcium- and magnesium-free solution at room temperature) and group III (dark adaptation-red light technique; standard balanced salt solution at room temperature). In none of the experimental groups was any obvious damage of the retinal structure observed, even after exposure to the test solutions for 60 minutes. CONCLUSIONS: Both dark adaptation (red illumination) and the use of a calcium chloride- and magnesium chloride-free solution (at room temperature) can facilitate retinal detachment in macular translocation surgery. Both techniques are proposed as a gentle support for the operation, because they protect an intact RPE cell layer and do not cause retinal damage at the ultrastructural level.

Electron microscopic evidence for different myelination of rat septohippocampal fibres.

Rat septohippocampal fibres are known to originate from GABAergic parvalbumin-containing, fast-firing, fast-conducting neurons and from cholinergic slow-firing, slow-conducting neurons. In the present electron microscopic study, based on immunocytochemical demonstration of parvalbumin and choline acetyltransferase in transverse and horizontal septal sections, it was shown that parvalbumin-immunoreactive fibres are myelinated, but the vast majority of cholinergic fibres are not. As revealed, especially in horizontal sections, the cholinergic axons show considerably finer calibres than parvalbumin-containing ones. These results confirm and extend our previous light microscopic findings. It can be concluded that differences in conduction velocities, presence or absence of myelin sheaths and differences axonal diameters are correlated in the septohippocampal pathway.

Experimental retinal detachment causes widespread and multilayered degeneration in rabbit retina.

Retinal detachment remains one of the most frequent causes of visual impairment in humans, even after ophthalmoscopically successful retinal reattachment. This study was aimed at monitoring (ultra-) structural alterations of retinae of rabbits after experimental detachment. A surgical procedure was used to produce local retinal detachments in rabbit eyes similar to the typical lesions in human patients. At various periods after detachment, the detached retinal area as well as neighbouring attached regions were studied by light and electron microscopy. In addition to the well-known degeneration of photoreceptor cells in the detached retina, the following progressive alterations were observed, (i) in both the detached and the attached regions, an incomplete but severe loss of ganglion cell axons occurs; (ii) there is considerable ganglion cell death, particularly in the detached area; (iii) even in the attached retina distant from the detachment, small adherent groups of photoreceptor cells degenerate; (iv) these photoreceptor cells degenerate in an atypical sequence, with severely destructed somata and inner segments but well-maintained outer segments; and (v) the severe loss of retinal neurons is not accompanied by any significant loss of Müller (glial) cells. It is noteworthy that the described progressive (and probably irreparable) retinal destructions occur also in the attached retina, and may account for visual impairment in strikingly large areas of the visual field, even after retinal reattachment.

Mitochondria of retinal Müller (glial) cells: the effects of aging and of application of free radical scavengers.

Age-related changes of mitochondria were studied in Müller (retinal glial) cells from guinea pigs fed with or without externally applied Ginkgo biloba extract EGb 761, an established radical scavenger. When Müller cell mitochondria from aged animals were compared with those from young adults, they displayed (1) a diminished number of well-defined cristae at the ultrastructural level, (2) a reduced membrane potential, as revealed by fluorimetry using the voltage-sensitive dye tetramethyl rhodamine methylester, and (3) a slightly reduced index of vitality assayed by tetrazolium salt colorimetry. Müller cell mitochondria were also studied in aged guinea pigs which had been fed daily by EGb 761 during the last 2 months before they were sacrificed. Such mitochondria displayed (1) many well-defined cristae at the ultrastructural level, and, compared with mitochondria from untreated aged animals, (2) a significantly enhanced membrane potential and (3) a significantly enhanced index of vitality. No age- or drug-related changes were observed in the mitochondrial content of GABA transaminase, as revealed by immunocytochemistry/densitometry. These results suggest that many but not all structural and functional parameters of aging Müller cell mitochondria are impaired by accumulating oxidative damage, and that externally applied radical scavengers may protect the organelles from the damaging actions of free radicals. As it has been shown earlier that EGb 761 treatment enhances the intrinsic glutathione content of aged guinea pig Müller cells, the protective radical-scavenging effect of the drug may be mediated both directly and indirectly.

Different myelination of rat septohippocampal fibres as revealed by immunofluorescence double-labelling.

The present study focuses on the myelination of rat septohippocampal fibres that are known to originate from GABAergic parvalbumin-containing, fast-firing, fast-conducting neurons and from cholinergic slow-firing, slow-conducting neurons. With the combined immunofluorescence for parvalbumin/myelin basic protein and choline acetyltransferase/myelin basic protein it was shown that the vast majority of parvalbumin-containing fibres are myelinated, but the choline acetyltransferase-containing fibres are not. Accordingly, our results confirm the expectation that conduction velocities and presence or absence of myelin sheaths are also correlated in the septohippocampal pathway.

Increase in vulnerability of middle-aged rat brain to lead by cerebral energy depletion.

The neurotoxic effects of low-level lead (Pb) during senescence are increasing interests of importance. We investigated the effects of low-level Pb on the brain in a normal condition and a pathophysiological condition of energy shortage that is commonly found in age-related neurological diseases. Middle-aged rats (15 months old) were exposed to 200 mg/l Pb acetate in drinking water for 2 months and thereafter received bilateral intracerebroventricular injections of streptozotocin (STZ). After 1 month's additional exposure to the same level of Pb solution as before the rats were sacrificed. Blood and brain Pb levels were measured by graphite furnace atomic absorption spectrophotometry. Energy-rich phosphate levels in the brain were determined by high-performance liquid chromatography equipped with a UV detector. Astroglial activation and glucose-regulated protein (GRP)94 expression were examined immunohistochemically. Exposure to Pb increased the blood Pb level to 10.8 microg/dl and the brain Pb level to 0.052 microg/g. But a significant additional increase in the brain Pb level, to 0.101 microg/g, became obvious in rats treated with Pb + STZ. Both Pb and STZ induced perturbation in brain energy metabolism, but no further alteration in energy metabolite levels was found in rats treated with Pb + STZ. Astroglial activation and GRP94-positive astrocytes and neurons were found only in the brains of Pb + STZ-treated rats. These results suggest that exposure to low-level Pb can perturb brain energy metabolism and the brain becomes more vulnerable to Pb when it is under energy stress.