Junzo Desaki

β-Estradiol protects hippocampal CA1 neurons against transient forebrain ischemia in gerbil

Beta-estradiol has been considered to be a neurotrophic agent, but its in vivo effect on gerbils with transient forebrain ischemia has not yet been demonstrated. In the first set of the present experiments, we infused beta-estradiol at a dose of 0.05 or 0.25 microg/day for 7 days into the lateral ventricles of normothermic gerbils starting 2 h before 3-min forebrain ischemia. Beta-estradiol infusion at a dose of 0.25 microg/day prevented significantly the ischemia-induced reduction of response latency time as revealed by a step-down passive avoidance task. Subsequent light and electron microscopic examinations showed that pyramidal neurons in the hippocampal CA1 region as well as synapses within the strata moleculare, radiatum and oriens of the region were significantly more numerous in gerbils infused with beta-estradiol than in those receiving saline infusion. Beta-estradiol at a dose of 1.25 microg/day was ineffective and occasionally increased the mortality of experimental animals. Since the total brain content of exogenous beta-estradiol at 12 h after forebrain ischemia was estimated to be less than 145 ng, the second set of experiments focused on the neurotrophic action of beta-estradiol at concentrations around 100 ng/ml in vitro. Beta-estradiol at concentrations of 1-100 ng/ml facilitated the survival and process extension of cultured hippocampal neurons, but it did not exhibit any significant radical-scavenging effects at the concentration range. On the other hand, 100 microg/ml of beta-estradiol, even though failing to support hippocampal neurons in vitro, effectively scavenged free radicals in subsequent in vitro studies, as demonstrated elsewhere. These findings suggest that beta-estradiol at a dose of 0.25 microg/day prevents ischemia-induced learning disability and neuronal loss at early stages after transient forebrain ischemia, possibly via a receptor-mediated pathway without attenuating free radical neurotoxicity.

The overall morphology of neuromuscular junctions as revealed by scanning electron microscopy

SummarySkeletal neuromuscular junctions (NMJs) of vertebrates were examined by scanning electron microscopy after removal of connective tissue components by HCl hydrolysis. In addition to the surface texture of NMJs, the subsynaptic organization of the sarcolemma was visualized in specimens in which nerve endings were detached from the muscle surface.A remarkable morphological variability between animal species was observed. The NMJs in the frog sartorius muscle consisted of longitudinal ribbon-like endings which fitted into a shallow synaptic gutter containing highly ordered cross-bands of junctional folds. The NMJs of the posterior latissimus dorsi muscle of the zebra finch were characterized by varicose swellings of the nerve endings which fitted into a round pit of the sarcolemma. NMJs in the sternothyroid muscle of the Chinese hamster consisted of thin ramified endings which were confined to an oval area on the muscle surface. The labyrinthine synaptic groove contained well-developed junctional folds without preferential spatial arrangement.The procedure used for the present study illustrates in great detail the terminal arborization of the motor nerve ending and the surface features of the subsynaptic sarcolemma. It may also allow quantitative study of the synaptic morphology of NMJs.

Microglial cells prevent nitric oxide-induced neuronal apoptosis in vitro.

Apoptotic neuronal death is known to occur in the developing brain and in the mature brain of patients with ischemic and degenerative disorders. Although microglial cells are known to become activated in specific conditions, it has not been elucidated whether they enhance or prevent neuronal apoptosis. The present study was intended to observe how microglial cells are involved in neuronal death. When rat primary cortical neurons were incubated with a nitric oxide (NO) donor sodium nitroprusside (SNP; 300 μM) for 10 min, neuronal death occurred 12–16 hr later. The NO‐induced neuronal death was inhibited by cycloheximide, and the SNP‐treated neurons were characterized by nuclear fragmentation and intact cell membrane under electron microscopy. Agarose gel electrophoresis demonstrated DNA fragmentation of the SNP‐treated neurons. Thus, the NO‐induced neuronal death appeared to be apoptosis. When neurons were cocultured with rat primary microglial cells, the SNP treatment failed to induce the neuronal death. Because microglia‐conditioned medium also prevented apoptotic neuronal death, microglial cells were considered to secrete antiapoptotic factors. The microglia‐conditioned medium rescued neurons even when they were added to neuronal cultures after the SNP treatment, implying that the factors acted on neurons in a manner other than scavenging NO. Interleukin‐3, interleukin‐6, macrophage colony‐stimulating factor, and basic fibroblast growth factor, which are known to be secreted by microglial cells, were not effective in preventing NO‐induced neuronal death. Among microglia‐derived substances, tumor necrosis factor α and plasminogen, which are heat‐labile proteins, inhibited neuronal apoptosis. The neuroprotective action of the microglia‐conditioned medium, however, still remained, even after it was heated. These findings suggest that microglial cells protect neurons against NO‐induced lethal damage by secreting heat‐labile and heat‐stable neuroprotective factors in vitro. J. Neurosci. Res. 53:415–425, 1998.

Epidermal Growth Factor Protects Neuronal Cells In Vivo and In Vitro Against Transient Forebrain Ischemia- and Free Radical-Induced Injuries

Epidermal growth factor (EGF) has been considered to be a candidate for neurotrophic factors on the basis of the results of several in vitro studies. However, the in vivo effect of EGF on ischemic neurons as well as its mechanism of action have not been fully understood. In the present in vivo study using a gerbil ischemia model, we examined the effects of EGF on ischemia-induced learning disability and hippocampal CA1 neuron damage. Cerebroventricular infusion of EGF (24 or 120 ng/d) for 7 days to gerbils starting 2 hours before or immediately after transient forebrain ischemia caused a significant prolongation of response latency time in a passive avoidance task in comparison with the response latency of vehicle-treated ischemic animals. Subsequent histologic examinations showed that EGF effectively prevented delayed neuronal death of CA1 neurons in the stratum pyramidale and preserved synapses intact within the strata moleculare, radiatum, and oriens of the hippocampal CA1 region. In situ detection of DNA fragmentation (TUNEL staining) revealed that ischemic animals infused with EGF contained fewer TUNEL-positive neurons in the hippocampal CA1 field than those infused with vehicle alone at the seventh day after ischemia. In primary hippocampal cultures, EGF (0.048 to 6.0 ng/mL) extended the survival of cultured neurons, facilitated neurite outgrowth, and prevented neuronal damage caused by the hydroxyl radical-producing agent FeSO4 and by the peroxynitrite-producing agent 3-morpholinosydnonimine in a dose-dependent manner. Moreover, EGF significantly attenuated FeSO4-induced lipid peroxidation of cultured neurons. These findings suggest that EGF has a neuroprotective effect on ischemic hippocampal neurons in vivo possibly through inhibition of free radical neurotoxicity and lipid peroxidation.

Antibodies against Muscle-Specific Kinase Impair Both Presynaptic and Postsynaptic Functions in a Murine Model of Myasthenia Gravis

Antibodies against acetylcholine receptors (AChRs) cause pathogenicity in myasthenia gravis (MG) patients through complement pathway-mediated destruction of postsynaptic membranes at neuromuscular junctions (NMJs). However, antibodies against muscle-specific kinase (MuSK), which constitute a major subclass of antibodies found in MG patients, do not activate the complement pathway. To investigate the pathophysiology of MuSK-MG and establish an experimental autoimmune MG (EAMG) model, we injected MuSK protein into mice deficient in complement component five (C5). MuSK-injected mice simultaneously developed severe muscle weakness, accompanied by an electromyographic pattern such as is typically observed in MG patients. In addition, we observed morphological and functional defects in the NMJs of EAMG mice, demonstrating that complement activation is not necessary for the onset of MuSK-MG. Furthermore, MuSK-injected mice exhibited acetylcholinesterase (AChE) inhibitor-evoked cholinergic hypersensitivity, as is observed in MuSK-MG patients, and a decrease in both AChE and the AChE-anchoring protein collagen Q at postsynaptic membranes. These findings suggest that MuSK is indispensable for the maintenance of NMJ structure and function, and that disruption of MuSK activity by autoantibodies causes MG. This mouse model of EAMG could be used to develop appropriate medications for the treatment of MuSK-MG in humans.

Induction of phosphorylated-Stat3 following focal cerebral ischemia in mice

It has been shown that Stat3 is induced following transient cerebral ischemia in rat. However there is no evidence that cerebral ischemia stimulates the expression of phosphorylated-Stat3 (p-Stat3), which can activate cytokine-mediated signal transduction from the membrane to the nucleus. In the present study, we investigated the changes in p-Stat3 expression following middle cerebral artery occlusion in mice. Western blot analysis revealed a significant increase in the p-Stat3 protein in the peripheral part of the ischemic area, starting from 6 h after ischemia. p-Stat3 immunoreactivity was detected only in neurons, but not in astrocytes or microglia, and p-Stat3-positive neurons were increased in number in the peripheral part of the ischemic area at 24 h after ischemia. Double staining with aTdT-mediated biotinylated UTP nick end labeling (TUNEL) kit and the p-Stat3 antibody indicated that p-Stat3-positive neurons were also TUNEL-positive. Subsequent immuno-electron microscopic observations showed that p-Stat3-positive neurons were at different stages of degeneration. The present findings suggest that the increased expression of p-Stat3 after cerebral ischemia could play a crucial role in ischemia-induced neuron death.

Formation and maturation of subneural apparatuses at neuromuscular junctions in postnatal rats: A scanning and transmission electron microscopical study☆

We examined the morphodifferentiation of subneural apparatuses at neuromuscular junctions with scanning and transmission electron microscopy (SEM and TEM) in the sternothyroid muscle of postnatal rats. As evidenced with SEM, primitive synaptic troughs found at birth were smooth cup-like depressions 5-6 micron in diameter. At the 5th postnatal day, low sarcoplasmic ridges appeared in the depression which successively grew and upheaved to remodel the depression into anastomosed gutters during the next 10 days. Subneural apparatuses attained almost the adult form by the 30th day, though synaptic troughs were smaller in size and exhibited a less complex pattern. At birth, the depression contained a few mostly pit-like or elongated oval invaginations:incipient junctional folds. By the 15th day, junctional folds rapidly developed, resulting in about an 18-fold increase in number per endplate with the parallel differentiation of slit-like junctional folds of adult form. At the 30th day, junctional folds were mostly slit-like, though pits still coexisted in a small proportion. As a shape factor, we measured the ratio of the length of the folds to their maximum width (L/W); the folds with L/W less than 2 were defined as pits, those with 2 less than or equal to L/W less than 5 as short slits, and those with L/W greater than or equal to 5 as long slits. At birth, pits occupied about 67% of the total number of the folds per endplate, which decreased to about 14% at the 30th day. Concomitantly, long slits remarkably increased from about 3 to 38%. Short slits increased from about 30 to 50% during the first 10 days but remained almost unchanged thereafter. The maximum L/W ratio was 12 at the 15th day and exceeded 20 after the 30th day. These quantitative data and the finding that pits were often closely associated with each other and also with a slit in a serial fashion indicate that the adjacent pits may fuse to each other and to the preformed slits. With TEM, a few incipient junctional folds were found at the 5th day, which extended into the subneural sarcoplasm with a depth less than 0.4 micron. At the 15th day, junctional folds increased both in number and in the maximum depth of about 0.8 micron. There also occurred a number of basal lamina-containing vacuoles identical in many respects to the transversely sectioned profiles of incipient junctional folds.(ABSTRACT TRUNCATED AT 400 WORDS)

Neurons Induce the Activation of Microglial Cellsin Vitro

Although microglial cells are well known to become activated in the pathological brain, mechanisms underlying the microglial activation are not fully understood. In the present study, with an aim to elucidate whether neurons are involved in the microglial activation, we compared the morphology and the superoxide anion (O2-)-generating activity of rat microglial cells in pure culture with those of cells cocultured with rat primary cortical neurons. Microglial cells in pure culture in serum-free Eagles minimum essential medium on poly-L-lysine-coated coverslips displayed ramified morphology and suppressed activity of O2- generation. In contrast, microglial cells in neuron-microglia coculture under the same conditions as those for the pure culture displayed ameboid shape and upregulated activity of O2- generation. Electron microscopic observation revealed that microglial cells in coculture were more abundant in Golgi apparatus and secretory granules than those in pure culture and that some of microglial cells in the vicinity of neurites exhibited membrane specialization reminiscent of a junctional apparatus with high electron density between a microglial soma and a neurite. Microglial cells in coculture tended to tie neurites in bundles by extending processes. Medium conditioned by neurons significantly enhanced O2- generation by microglia, but microglial cells in contact with or in close apposition to cocultured neurons were much more intensely activated than those remote from the neurons. Furthermore, the membrane fraction of cortical neurons activated microglial cells, and this effect was abolished by treating the neuronal membrane with trypsin or neuraminidase. In conclusion, neuronal-microglial contact may be necessary to mediate microglial activation. The present findings suggest that the contact of microglia with damaged neurons in the brain is a plausible cause to activate microglia in the neuropathological processes.

Morphologic Differences of the Vascular Buds in the Vertebral Endplate: Scanning Electron Microscopic Study

Study Design Vascular buds in rabbit vertebral endplates were examined by scanning electron microscopy of corrosion casts. Objectives To examine morphologic differences between vascular buds in two regions of the vertebral endplate (inner anular and nucleus pulposar). Summary of Background Data Vascular buds are specific structures present at the vertebral endplate that are important as nourishing channels. There is a significant difference in permeability between the lateral portion (inner anular) and the central portion (nucleus pulposar) of the endplate, the latter usually being permeable and the former being impermeable. Morphologic differences between vascular buds in the two regions have not been investigated previously. Methods Eight 20-week-old rabbits were used. Vascular buds in rabbit vertebral endplates were examined by scanning electron microscopy of corrosion casts. Results The vascular buds in the region of the inner anulus form simple loops, but those in the area near the nucleus pulposus exhibit swollen and complex coil-like loops. Although they differ structurally, the average number of vascular buds per area does not vary between the two regions. Conclusions We suggest that the morphologic difference between the vascular buds in the two regions (inner anular and nucleus pulposar) plays a principal role in permeability at the endplate.

Basic fibroblast growth factor-like immunoreactivity in the trigeminal proprioceptive and motor systems

Basic fibroblast growth factor (bFGF) isolated from the brain and pituitary, has been shown to induce cell divisions in a variety of cell types. It also acts as a potent stimulator of angiogenesis, and it is important in the survival of several types of cultured neurons. Despite considerable information on the functions of bFGF, there is incomplete knowledge about the ways in which it reaches remote tissues and its subcellular localization in the adult brain. Here we report our findings that a certain population of neurons with free ribosomes and rough endoplasmic reticulum immunoreactive for bFGF in the mesencephalic nucleus of the trigeminal nerve sends proprioceptive fibers to muscle spindles in the masseter muscle, and immunoreactive axons to the trigeminal motor nucleus to form synapses with the bFGF-containing motoneurons whose axons further constitute myoneural junctions in the periphery. Moreover, some bFGF neurons contain electron dense immunoreaction deposits in the euchromatin but not in the heterochromatin of the nucleus. These findings suggest that endogenous bFGF is transported within nerve processes and functions in mature neuronal circuits subserving the masseteric reflex arcs, and that bFGF is produced in free ribosomes and/or rough endoplasmic reticulum and is transported into the genetically active euchromatin as well.