Protective effect of a prosaposin-derived, 18-mer peptide on slowly progressive neuronal degeneration after brief ischemia.

SUMMARY: Slowly progressive degeneration of the hippocampal CA1 neurons was induced by 3-minute transient global ischemia in gerbils. Sustained degeneration of hippocampal CA1 neurons was evident 1 month after ischemia. To investigate the effects of an 18-mer peptide comprising the hydrophilic sequence of the rat saposin C domain (18MP) on this sustained neuronal degeneration, an intracerebroventricular 18MP infusion was initiated 3 days after ischemia. Histopathologic and behavior evaluations were conducted 1 week and 1 month after induction of ischemia. When compared with the vehicle infusion, 18MP treatment significantly increased the response latency time in a passive avoidance task. Increased neuronal density was also evident, as was the number of intact synapses in the hippocampal CA1 region at 1 week and 1 month after ischemia. 18MP treatment also significantly decreased the number of TUNEL-positive CA1 neurons 1 week after ischemia. Subsequent in vitro experiments using cultured neurons demonstrated that the 18MP at optimal extracellular concentrations of 1 to 100 fg/mL prevented nitric oxide-induced neuronal damage as expected and significantly up-regulated the expressions of bcl-x(L) mRNA and its translated protein. These results suggest that the gerbil model of 3-minute ischemia is useful in studying the pathogenesis of slowly progressive neuronal degeneration after stroke and in evaluating effects of novel therapeutic agents. It is likely that the 18MP at low extracellular concentrations prevents neuronal apoptosis possibly through up-regulation of the mitochondrial antiapoptotic factor Bcl-x(L).

Scanning electron microscopic study of muscle spindles in the tenuissimus muscle of the Chinese hamster: with reference to the outer capsule, inner capsule and fusimotor endings.

The outer capsule, inner capsule and fusimotor endings of muscle spindles in the tenuissimus muscle of mature Chinese hamsters were examined by scanning electron microscopy (SEM). The thin and long tenuissimus muscle was transversely cut into several segments, and the segments were longitudinally or obliquely cut with a razor blade to mechanically remove the spindle sheath (outer capsule). These specimens were treated with 8 N HCl at 60 degrees C to isolate the muscle spindles and clearly expose the internal structures such as the inner capsule and fusimotor endings innervating intrafusal muscle fibers. The spindle sheath was about 1 mm long and at the equator about 50 microm in diameter. Within the outer capsule, cells of the inner capsule in the equatorial region were polygonal in shape, and continuously surrounded the axial bundle like a sleeve. This continuous sheath became sieve-like in the juxta-equatorial region, depending on the twining of thin and flattened cytoplasmic extensions and/or processes of the cells. Owing to the continuous inner capsular sheath in the equatorial region, the arrangement of sensory endings were not visualized under SEM. Fusimotor endings observed in the juxta-equatorial and polar regions consisted of poorly-developed subneural apparatuses with predominantly pit-like and short slit-like junctional folds.

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.

Scanning electron microscopic study of the muscle fiber ends at the myotendinous junction in the posterior cricoarytenoid and cricothyroid muscles in rats.

The fine structural organization of muscle fiber ends at the myotendinous junction in the posterior cricoarytenoid (PCA) and cricothyroid (CT) muscles in adult rats was studied by scanning electron microscopy, after removal of tendon connective tissues using the HCI-hydrolysis method. The muscle fiber ends in the PCA muscle had a relatively simple conical appearance and contained a great number of longitudinal slits on the surface. In the CT muscle, the muscle fiber ends were classified into two types. One type had a conical appearance similar to the PCA muscle with many slits on the surface, while the other type was characterized by a complex arrangement of cylindrical cytoplasmic processes and deep clefts, as seen in common skeletal muscles. These findings suggest that the PCA muscle is evolutionarily primitive, and that the CT muscles represent a transitional form between primitive and evolved muscles.

Scanning electron microscopic study of the neuromuscular junctions of the cricothyroid and thyroarytenoid muscles in rats.

Neuromuscular junctions were observed in the cricothyroid (CT) and thyroarytenoid (TA) muscles of adult rats by scanning electron microscopy after removing the intramuscular connective tissue components using the HCI hydrolysis method. Morphologically, the junctions were classified into three types in the CT muscle and two types in the TA muscle, based on the structural characteristics of the subneural apparatuses, including junctional folds. In the CT muscle, type 1 junctions (32%) consisted of more than 15 cup-like depressions with slit-like junctional folds. Type 2 junctions (20%) were characterized by approximately 10 cup-like depressions with a small number of pit- or slit-like junctional folds. Type 3 junctions (48%) had irregular labyrinthine gutters with slit-like junctional folds. In the TA muscle, type 1 (82%) and 2 (18%) junctions had similar structures to type 1 and 2 junctions in the CT muscle, respectively. Histochemical studies using myosin adenosine triphosphatase staining showed that both CT and TA muscles predominantly consisted of type II muscle fibers (78% and 82%, respectively), and that the diameter of type II fibers was larger than that of type I fibers. These findings suggest that the type 2 junction belongs to type I muscle fibers, while both type 1 and type 3 junctions belong to type II fibers, and that the type 3 junction is a structural variation of the type 1 junction. The significance of the structural differences of the subneural apparatuses in the intrinsic laryngeal muscles is discussed briefly.

Age changes of neuromuscular junctions in the extensor digitorum longus muscle of spontaneous thymoma BUF/Mna rats. A scanning and transmission electron microscopic study.

BUF/Mna rats spontaneously develop thymomas and cause muscle weakness of hind legs at an advanced age. This rat strain has been recognized as a suitable animal model for human myasthenia gravis or related myopathies. To characterize the structural changes of neuromuscular junctions (NMJs) in BUF/Mna rats, subneural apparatuses (SNAs) of extensor digitorum longus muscles of young-adult (4-month-old) and aged (22- to 25-month-old) male rats were examined using scanning and transmission electron microscopy. The SNAs of NMJs in young rats consist of complex labyrinthine gutters with numerous slit-like junctional folds. SNAs in aged BUF/Mna rats, however, are characterized by: (1) a group of cup-like depressions with very wide slit-like junctional folds in relatively large muscle fibers (the major type), (2) the presence of slit-like folds on the flat sarcoplasm outside the cup-like depressions or on the protruded sarcoplasm, and (3) winding gutters or a small number of round depressions with poorly developed synaptic folds in small and medium-sized muscle fibers (the minor type). Since similar structural changes have been reported in dystrophic mice or normally aged rats, it is suggested that both the slowly progressing muscle atrophy and age-dependent turnover of muscle fibers may occur in the aged BUF/Mna rats.

Postnatal morphodifferentiation of the subneural apparatuses of the posterior cricoarytenoid muscle in rats: a scanning electron microscopy study.

The subneural apparatus, i. e., the post-synaptic component of the neuromuscular junction, in the posterior cricoarytenoid muscle of the rat was studied by scanning electron microscopy, with special attention given to its postnatal differentiation along with the functional development of the muscle. Primitive synaptic troughs observed in the first postnatal week consisted of single cup-like depressions 5-6 microm in diameter. On the 7th day, low sarcoplasmic ridges appeared in the trough. In the second postnatal week, muscle fibers could be classified into two groups: large (10-15 microm in diameter) and small (less than 10 microm in diameter). In the large muscle fibers, many low ridges became circular and protruded to transform the single trough into numerous cup-like depressions (2-5 microm in diameter). In contrast, the subneural apparatus in the small muscle fibers consisted of a small number of cup-like depressions. The two types of subneural apparatus differentiated into adult forms by the 28th postnatal day, although they remained smaller in size than those of adults. In the large muscle fibers, the number of pit-like or elongated invaginations increased and gradually transformed into slit-like junctional folds by the 28th postnatal day, while the small muscle fibers still possessed a few pit-like or elongated junctional folds at this point in time. The two types of morphodifferentiation of the subneural apparatus are thought to reflect the two types of muscle fibers in the rat posterior cricoarytenoid muscle.

Morphological changes of capillaries in the rat soleus muscle following experimental tenotomy.

We examined the structural changes of capillaries in the rat soleus muscle 4, 7, 14, and 35 days after experimental limb tenotomy. In the soleus muscles after tenotomy, muscle fibres degenerated and some of them were destroyed; the muscle did not seem to recover until the 35th day. On the 14th day, some small muscle fibres, probably regenerating muscle fibres, started forming within the basal-lamina tube and remained after necrosis of a pre-existing muscle fibre. Most capillaries at each stage were of the continuous type. However, about 10% of the capillaries around degenerated muscle fibres at days 4, 7 and 14 consisted of endothelial cells with a small number of fenestrae bridged by a single-layered diaphragm. On the 14th day, capillaries around small regenerating muscle fibres also often had a small number of fenestrations. Even on the 35th day, capillaries occasionally had fenestrations. Additionally, some of the fenestrated capillaries formed small pores at the fenestrated portion of the endothelial cells. The untreated muscles contained only continuous capillaries. These findings suggest that fenestrations in the endothelial cells may occur in intramuscular capillaries not only around degenerated muscle fibres but also around regenerating muscle fibres after tenotomy.

A re-examination of the cellular reticulum of fibroblast-like cells in the rat small intestine by scanning electron microscopy.

The organization and arrangement of fibroblast-like cells in the rat small intestine were re-examined by scanning electron microscopy after removal of the epithelium and underlying connective tissue components by HCl hydrolysis. In the villi, the fibroblast-like cells had numerous slender processes, and formed a dense and elaborate network like a sieve. It consisted of a large number of circular structures (circles) with various diameters ranging from 0.3 microm to 5 microm formed by the twining of slender processes. In the upper area of villi, numerous fragmental protrusions which were considered to be mainly parts of immune-related cells such as lymphocytes, eosinophils and macrophages extended from the circles. The cells around each tubular gland enclosed it like a basket. These findings suggest that in addition to the function as a skeleton for the villi and glands, the fibroblast-like cells in the upper area of villi may play an important role in regulating the migration of the immune-related cells between the epithelial layer and the underlying lamina propria by their cellular sieve-like structure and contractile ability.

Aged neuromuscular junctions in the extensor digitorum longus muscle of the rat as revealed by scanning electron microscopy.

We examined the overall morphology of subneural apparatuses (SNAs) at neuromuscular junctions by scanning electron microscopy in the extensor digitorum longus muscle of aged (18 months) rats. Most of aged SNAs were mainly characterized by a large number (more than 20) of cup-like depressions (2-5 microns in diameter) with numerous slit-like junctional folds about 0.1 micron wide and 5 microns at maximum length, while some SNAs consisted of a small number (fewer than 10) of depressions or poorly-developed gutters containing pit-like and/or slit-like junctional folds. Additionally, numerous slits were often found outside the depressions, possibly indicating the pre-existing junctional folds persisted for a long time after the gutter-into-cup transformation of the SNAs. These structural changes of SNAs during ageing are discussed briefly.

Capillary changes with fenestrations in the contralateral soleus muscle of the rat following unilateral limb immobilization.

Using electron microscopy, we examined structural changes in the capillaries of the immobilized and contralateral soleus muscles in the rat after the unilateral limb had been immobilized for 4 weeks. Individual contralateral muscle fibers showed a normal structure, compared with the structure in the immobilized muscle fibers. Most capillaries in the contralateral muscle were of the continuous type. However, about 5% of the examined capillaries in these muscles in each rat had fenestrations. The non-nuclear portions of the endothelial cells in these capillaries were extremely thin and were perforated by several fenestrations that were bridged by a single-layered diaphragm. The fenestration of capillaries in the contralateral muscle may have been caused by some the unilateral limb immobilization.

Interleukin 3 prevents delayed neuronal death in the hippocampal CA1 field.

In the central nervous system, interleukin (IL)-3 has been shown to exert a trophic action only on septal cholinergic neurons in vitro and in vivo, but a widespread distribution of IL-3 receptor (IL-3R) in the brain does not conform to such a selective central action of the ligand. Moreover, the mechanism(s) underlying the neurotrophic action of IL-3 has not been elucidated, although an erythroleukemic cell line is known to enter apoptosis after IL-3 starvation possibly due to a rapid decrease in Bcl-2 expression. This in vivo study focused on whether IL-3 rescued noncholinergic hippocampal neurons from lethal ischemic damage by modulating the expression of Bcl-xL, a Bcl-2 family protein produced in the mature brain. 7-d IL-3 infusion into the lateral ventricle of gerbils with transient forebrain ischemia prevented significantly hippocampal CA1 neuron death and ischemia-induced learning disability. TUNEL (terminal deoxynucleotidyltransferase-mediated 2'-deoxyuridine 5'-triphosphate-biotin nick end labeling) staining revealed that IL-3 infusion caused a significant reduction in the number of CA1 neurons exhibiting DNA fragmentation 7 d after ischemia. The neuroprotective action of IL-3 appeared to be mediated by a postischemic transient upregulation of the IL-3R alpha subunit in the hippocampal CA1 field where IL-3Ralpha was barely detectable under normal conditions. In situ hybridization histochemistry and immunoblot analysis demonstrated that Bcl-xL mRNA expression, even though upregulated transiently in CA1 pyramidal neurons after ischemia, did not lead to the production of Bcl-xL protein in ischemic gerbils infused with vehicle. However, IL-3 infusion prevented the decrease in Bcl-xL protein expression in the CA1 field of ischemic gerbils. Subsequent in vitro experiments showed that IL-3 induced the expression of Bcl-xL mRNA and protein in cultured neurons with IL-3Ralpha and attenuated neuronal damage caused by a free radical-producing agent FeSO4. These findings suggest that IL-3 prevents delayed neuronal death in the hippocampal CA1 field through a receptor-mediated expression of Bcl-xL protein, which is known to facilitate neuron survival. Since IL-3Ralpha in the hippocampal CA1 region, even though upregulated in response to ischemic insult, is much less intensely expressed than that in the CA3 region tolerant to ischemia, the paucity of IL-3R interacting with the ligand may account for the vulnerability of CA1 neurons to ischemia.

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 microM) 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 alpha 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.

Capillaries with fenestrations around regenerating muscle fibers in the soleus muscle of the dystrophic (dy) mouse.

To investigate the morphological changes in capillaries around regenerating muscle fibers in the dystrophic (dy) mouse, we examined capillaries in the soleus muscles of 30-day-old control and dy mice by electron microscopy. In the control mice, the intramuscular capillaries were continuous. In the dystrophic mice, the muscles contained degenerative muscle fibers and many small muscle fibers regenerating after necrosis; these fibers had centrally located nuclei. The capillaries in the dy mice were mostly continuous, but some had a narrow vascular lumen around regenerating muscle fibers, possibly indicating newly formed capillaries. Moreover, about 20% of the capillaries with a narrow vascular lumen had a small number (less than five) of fenestrae bridged by a single-layered diaphragm. These findings suggest that capillary networks around muscle fibers regenerating after necrosis are remodeled by newly formed capillaries, and some of these capillaries have fenestrae in the endothelium to increase the supply of nutrients and oxygen to the regenerating muscle fibers.

A novel muscle spindle containing muscle fibres devoid of sensory innervation in the posterior cricoarytenoid muscle of the normal adult guinea pig.

A novel muscle spindle composed of five thin intrafusal muscle fibres and four thick muscle fibres was encountered in the posterior cricoarytenoid muscle of the normal adult guinea pig. Four intrafusal muscle fibres of the five were innervated by the annulospiral type of sensory ending, but lacked a distinct aggregation of equatorial nuclei such as bags and chains. On the other hand, one intrafusal fibre and four thick fibres between the layers of the spindle capsule absolutely lacked sensory innervation and were almost similar in the fine structural features to neighbouring extrafusal fibres. These fibres are considered to fail to receive the sensory innervation in an early stage of development, and to retain an undifferentiated feature of intrafusal fibre in the intracapsular space into adulthood or to follow the course of extrafusal differentiation between the layers of the spindle capsule. It is suggested therefore that this muscle spindle may be primitive.

Morphodifferentiation of skeletal muscle fiber ends at the myotendinous junction in the postnatal Chinese hamster: a scanning electron microscopic study.

The postnatal morphodifferentiation of muscle fiber ends at the myotendinous junction was examined by scanning electron microscopy in gastrocnemius muscles of the Chinese hamster. Muscle fiber ends during the first week were simply conical or slightly complex, having a few pit-like invaginations and longitudinal and narrow clefts. By the second week, fiber ends gradually became complicated with an increased number of clefts and finger-like processes. Pits and short clefts linearly arranged towards the fiber tip were visible until the second week. This possibly indicates the linear elongation of the clefts by the fusion of adjacent pits to one another and to preexisting clefts. By the fourth week, the fiber ends had almost fully matured and displayed numerous cytoplasmic processes and lateral grooves.

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.

Motor innervation of the guinea pig interarytenoid muscle: reinnervation process following unilateral denervation.

The authors investigated the process of denervation and reinnervation of the interarytenoid (IA) muscle in the guinea pig using transmission electron microscopy and glycogen depletion technique after unilateral transection of the recurrent laryngeal nerve (RLN) and superior laryngeal nerve to clarify the innervation pattern of the unpaired IA muscle. Anastomosis between the bilateral arytenoid branches was confirmed in the belly of the IA muscle. Five weeks after transection, all of the IA muscle fibers appeared to have been reinnervated by the contralateral RLN. As the arytenoid branch of the RLN runs together with that of the contralateral RLN in a single intramuscular nerve funiculus, it is possible that collateral sprouting branches grow and extend into the adjacent denervated Schwann's sheaths. The authors conclude that the unpaired IA muscle, as a whole, receives specific motor nerve supply from the bilateral RLNs, although each muscle fiber is innervated unilaterally.

Three-dimensional structure of the vascular network in normal and immobilized muscles of the rat.

OBJECTIVE: To examine whether there are three-dimensional changes in capillaries of rat soleus muscle during mechanical limb immobilization. DESIGN: Scanning electron microscopy, with the microcorrosion cast technique, was used to examine vascular networks in immobilized Wistar rat soleus muscles from 4 to 12 weeks after limb immobilization and to compare these networks with those in the control muscle. INTERVENTION: Immobilization of the soleus muscle was achieved by unilateral pinning of the ankle joint in full plantar flexion. RESULTS: The wavelike structure of the vascular network in the control muscles changed into a straight configuration in 8- and 12-week immobilized muscles.

Neurons induce the activation of microglial cells in 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 Eagle's 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.