Rescue from anti-IgM-induced programmed cell death by the B cell surface proteins CD20 and CD40.

Programmed cell death (PCD), or apoptosis, is characterized by several morphologic alterations and eventual cleavage of nuclear DNA into oligonucleo-some-length fragments. We defined a human B cell line, Ramos, that responds with PCD following ligation of surface IgM. Of the DNA in Ramos cells 3%-10% was fragmented as early as 4 h after IgM ligation. Propidium iodide staining demonstrated that 20%-40% of Ramos cells became apoptotic by 18 h and further established that cells transiting into the S phase of the cell cycle were susceptible to PCD. Addition of several agents to the Ramos cells abrogated anti-IgM-induced PCD, including the phorbol 12-myristate 13-acetate (PMA). In contrast to the effect of PMA, the 4 alpha PMA isomer of PMA neither activated protein kinase C (PKC) nor rescued the cells from anti-IgM-induced PCD, confirming a role for PKC in negating apoptosis. To explore the effect of physiologic signals on anti-IgM-induced PCD, antibodies against the CD20 or CD40 molecules were added in concert with anti-IgM. Both CD20 and CD40 synergize with anti-IgM to augment proliferation but neither molecule activates PKC in Ramos cells. Both anti-CD20 and anti-CD40 reduced the number of cells undergoing anti-IgM-induced PCD. Unlike the effect of anti-CD40, addition of anti-CD20 to anti-IgM-stimulated cells negated PCD only in a subset of cells. Maximal rescue occurred following the addition of anti-CD40 and occurred by 4 h and at least up to 20 h of culture. These data show that (a) PCD can be initiated in B cells entering the S phase of the cell cycle, (b) PCD can be triggered by engagement of surface IgM in the absence of ancillary signals or PKC activation, and (c) rescue from PCD can occur by several mechanisms, either PKC dependent or PKC independent.     

Lithium prevents excitotoxic cell death of motoneurons in organotypic slice cultures of spinal cord

Several studies have reported the neuroprotective effects of lithium (Li) suggesting its potential in the treatment of neurological disorders, among of them amyotrophic lateral sclerosis (ALS). Although the cause of motoneuron (MN) death in ALS remains unknown, there is evidence that glutamate-mediated excitotoxicity plays an important role. In the present study we used an organotypic culture system of chick embryo spinal cord to explore the presumptive neuroprotective effects of Li against kainate-induced excitotoxic MN death. We found that chronic treatment with Li prevented excitotoxic MN loss in a dose dependent manner and that this effect was mediated by the inhibition of glycogen synthase kinase-3β (GSK-3β) signaling pathway. This neuroprotective effect of Li was potentiated by a combined treatment with riluzole. Nevertheless, MNs rescued by Li displayed structural changes including accumulation of neurofilaments, disruption of the rough endoplasmic reticulum and free ribosome loss, and accumulation of large dense core vesicles and autophagic vacuoles. Accompanying these changes there was an increase in immunostaining for (a) phosphorylated neurofilaments, (b) calcitonin gene-related peptide (CGRP) and (c) the autophagic marker LC3. Chronic Li treatment also resulted in a reduction in the excitotoxin-induced rise in intracellular Ca²⁺ in MNs. In contrast to the neuroprotection against excitotoxicity, Li was not able to prevent normal programmed (apoptotic) MN death in the chick embryo when chronically administered in ovo. In conclusion, these results show that although Li is able to prevent excitotoxic MN death by targeting GSK-3β, this neuroprotective effect is associated with conspicuous cytopathological changes.     

Reinnervation of the rat musculocutaneous nerve stump after its direct reconnection with the C5 spinal cord segment by the nerve graft following avulsion of the ventral spinal roots: a comparison of intrathecal administration of brain-derived neurotrophic factor and Cerebrolysin

Experimental model based on the C5 ventral root avulsion was used to evaluate the efficacy of brain-derived neurotrophic factor (BDNF) and Cerebrolysin treatment on motor neuron maintenance and survival resulted in the functional reinnervation of the nerve stump. In contrast to vehicle, BDNF treatment reduced the loss and atrophy of motor neurons and enhanced the regrowth axon sprouts into the distal stump of musculocutaneous nerve. However, the axon diameter of the myelinated fibers was smaller than those of control rats. The morphometric results were related to a low score in behavioral test similar to vehicle-treated rats. Cerebrolysin treatment greatly protected the motor neurons against cell death. Moreover, morphometric features of myelinated axons were better than those of rats treated with vehicle or BDNF. The mean score of grooming test suggested better results of the functional motor reinnervation than after BDNF administration. The majority of rescued motor neurons regenerating their axons through nerve graft in both BDNF- and Cerebrolysin-treated rats expressed choline acetyltransferase immunostaining. The results demonstrate that BDNF has more modest effects in preventing the death of motor neurons and functional recovery of injured motor nerve after root avulsion than Cerebrolysin.     

Calreticulin expression in spinal motoneurons of the rat

We have examined the expression of calreticulin in rat spinal motoneurons in order to reveal the occurrence and distribution of Ca²⁺-storage organelles in these neurons. Calreticulin, the non-muscle equivalent of calsequestrin, is the low-affinity, high-capacity calcium-binding protein responsible for intracompartmental Ca²⁺-storage in a number of different cell types. The results of the present immunohistochemical study show that all spinal motoneurons express calreticulin at approximately the same level; no significant differences in cytoplasmic immunostaining intensity were observed between different motoneuron pools or between small and large spinal motoneurons. Immunoelectron microscopy revealed that the intracellular localization of calreticulin within spinal motoneurons was confined to the endoplasmic reticulum and to spherical or pleiomorphic, frequently ‘coated’ vesicles with a diameter ranging between 120 and 150 nm. Some of these vesicles may represent the so-called calciosomes, the intracellular Ca²⁺-storage vesicles described in liver cells and in cerebellar Purkinje cells. The molecular components responsible for the uptake and release of Ca²⁺ from the Ca²⁺-storage organelles in spinal motoneurons still remain to be identified.     

Over-expression of parvalbumin in transgenic mice rescues motoneurons from injury-induced cell death

Following nerve injury in neonatal rats, a large proportion of motoneurons die, possibly as a consequence of an increase in vulnerability to the excitotoxic effects of glutamate. Calcium-dependent glutamate excitotoxicity is thought to play a significant role not only in injury-induced motoneuron death, but also in motoneuron degeneration in diseases such as amyotrophic lateral sclerosis (ALS). Motoneurons are particularly vulnerable to calcium influx following glutamate receptor activation, as they lack a number of calcium binding proteins, such as calbindin-D(28k) and parvalbumin. Therefore, it is possible that increasing the ability of motoneurons to buffer intracellular calcium may protect them from cell death and prevent the decline in motor function that usually occurs as a consequence of motoneuron loss. In this study we have tested this possibility by examining the effect of neonatal axotomy on motoneuron survival and muscle force production in normal and transgenic mice that over-express parvalbumin in their motoneurons.The sciatic nerve was crushed in one hindlimb of new-born transgenic and wildtype mice. The effect on motoneuron survival was assessed 8 weeks later by retrograde labelling of motoneurons innervating the tibialis anterior muscle. Following nerve injury in wildtype mice, only 20.2% (+/-2.2, S.E.M.; n=4) of injured motoneurons survive long term compared with 47.2% (+/-4.4, S.E.M.; n=4) in parvalbumin over-expressing mice. Surprisingly, this dramatic increase in motoneuron survival was not reflected in a significant improvement in muscle function, since 8 weeks after injury there was no improvement in either maximal twitch and tetanic force, or muscle weights.Thus, inducing spinal motoneurons to express parvalbumin protects a large proportion of motoneurons from injury-induced cell death, but this is not sufficient to restore muscle function.     

Rescue of motoneurons from the axotomized state by regeneration into a sensory nerve in cats.

1. We studied the electrical properties of spinal motoneurons, the axons of which had regenerated into a cutaneous nerve. 2. In cats, all or part of the medial gastrocnemius (MG) muscle nerve was cut and directed distally into the caudal cutaneous sural (CCS) nerve, a sensory (primarily cutaneous) nerve. One or 2 yr later, electrical properties [conduction velocity (CV), rheobase (Irh), input resistance (RN), afterhyperpolarization (AHP), and excitatory postsynaptic potentials (EPSPs)] of MG motoneurons that had cross-regenerated into the CCS nerve were determined. These were compared with properties of normal and of axotomized MG motoneurons and with data from previous studies in which MG motoneurons had reinnervated their own or a foreign muscle. 3. Electrical stimulation of the MG-innervated CCS nerve produced no detected mechanical activity, indicating an absence of muscle innervation. Tactile stimulation of skin did not activate these motoneurons; i.e., they did not acquire properties of cutaneous afferents. 4. The CV and Irh of MG motoneurons axotomized 11 mo declined by 48 and 60%, respectively. 5. The CV of MG motoneurons that had regenerated through CCS was only slightly slower than normal, similar to that of MG motoneurons that reinnervated the "slow" muscle soleus (Foehring and Munson 1990). 6. The Irh and RN were also similar to those of MG motoneurons that had regenerated into the soleus muscle. 7. Electrical stimulation of the lateral gastrocnemius-soleus nerve generated EPSPs of normal or almost normal amplitude in MG motoneurons axotomized for 11 mo or cross-regenerated into CCS up to 2 yr.(ABSTRACT TRUNCATED AT 250 WORDS)     

Replacement of missing motoneurons by embryonic grafts in the rat spinal cord

The ventral quadrant of embryonic spinal cord with its motoneurons prelabelled by 5-bromo-2'-deoxyuridine was grafted into the spinal cord of adult rats. The ventral horn of the host had been previously partially depleted of its own motoneurons by a neonatal nerve lesion. To enhance the chances of survival of the transplanted embryonic motoneurons a target muscle was provided for their axons. Two to three months after the grafts were inserted into the cord nuclei containing 5'-bromo-2'-deoxyuridine were found in the graft and in the host's spinal cord. Many of the stained nuclei were much larger than those of embryonic motoneurons, and their size distribution was similar to that of nuclei from control motoneurons. Retrograde labelling with horseradish peroxidase, injected into the target muscle provided for the embryonic motoneurons, showed that some motoneurons had reached the muscle and presumably made contact with it. Physiological and histological examination of the target muscle showed that it was innervated and that it contained at least three different types of muscle fibres. Thus embryonic motoneurons can survive and develop in the adult spinal cord. Moreover, they seem to be able to make functional connections with skeletal muscle fibres. The heterogeneity of the muscle indicates that the motoneurons that supply them are able to differentiate into various types of cells.     

体外培养大鼠脊髓运动神经元的存活率及形态观察

观察体外培养的大鼠脊髓运动神经元(SMN)的存活率、形态学特征及生长规律。对今后进一步研究脊髓损伤、运动神经元病有一定的指导意义。1材料与方法1·1实验动物:胚胎16 d的SD大鼠。1·2主要试剂:15%有血清L-15培养基、无血清L-15培养基、poly-L-lysine、层黏连蛋白(lam in in)、m etrizam ide、阿糖胞苷、DAB等(S igm a公司),小牛血清白蛋白、小牛血清(华美生物工程公司),胆碱乙酰转移酶多克隆抗体(ChAT)(武汉博士德公司)。1·3实验仪器:离心机、超净台、CO2恒温培养箱、24孔培养板、倒置显微镜(OLYMPUS)、图像分析系统(OLYMPU…     

Sexual activity and the morphology of steroid-sensitive rat spinal motoneurons

In adult male rats, differential sexual experience has been shown to alter plasma androgen titers. Alterations in androgen levels have also been shown to significantly alter the structure of neurons in the spinal nucleus of the bulbocavernosus (SNB), a spinal motor nucleus that innervates perineal muscles involved in copulatory behavior. To determine if experientially induced alterations in androgen levels might alter SNB structure, male rats were assigned to one of three groups: sexually active, sexually inactive but exposed to inaccessible cycling females, and isolated. After 4 weeks of differential sexual experience, the soma size, total arbor, density, maximal fiber length, and frequency distribution of lengths of dendrites were examined in SNB motoneurons. No differences in motoneuron morphology were observed, suggesting that these motoneurons are not sensitive to socially induced alterations in androgen titers.     

Calcitonin gene-related peptide in rat spinal cord motoneurons: subcellular distribution and changes induced by axotomy.

Using light and electron microscopy, a study has been made of the changes of calcitonin gene-related peptide-like immunoreactivity in rat lumbar spinal cord motoneurons during cell body response to sciatic nerve injury. At light microscopy level, calcitonin gene-related peptide-like immunoreactivity was evaluated using an indirect immunofluorescence technique combined with Fast Blue retrograde tracing and a peroxidase-antiperoxidase procedure. The calcitonin gene-related peptide changes to sciatic nerve transection and crushing were compared. Calcitonin gene-related peptide-like immunoreactivity was transiently increased after the peripheral nerve lesion, but the response was sustained for a longer period when the peripheral nerve was transected and nerve regeneration prevented. The first changes in calcitonin gene-related peptide-like immunoreactivity were detected four days after nerve crush or transection. In animal spinal cords to which nerve crush had been applied, the maximal enhancement of immunoreactivity was found 11 days after lesion. This was followed by a gradual decline, normal levels being attained 45 days after nerve crushing. When the nerve was transected, the response was similar, but the maximal calcitonin gene-related peptide-like immunoreactivity was maintained over a period of between 11 and 30 days. As with crushing, an important decrease was observed after 45 days. The ultrastructural compartmentation of calcitonin gene-related peptide-like immunoreactivity was studied using either peroxidase-antiperoxidase method or immunogold labelling. Calcitonin gene-related peptide-like immuno-reactivity was located in restricted sacs of the Golgi complex, multivesicular bodies, small vesicles and tubulo-vesicular structures. Large, strongly labelled vesicles resembling secretory granules were also observed in neuronal bodies. Our results reveal that the increase of calcitonin gene-related peptide in motoneurons is a relevant change the cell body undergoes in response to peripheral injury. The ultrastructural location of the peptide distribution suggests specific compartmentation on tubulo-vesicular structures connected with the Golgi complex which form a network in the neuronal cytoplasm. The distribution pattern observed may be related to the sorting and delivery of calcitonin gene-related peptide to secretory vesicles.     

鞘内注射强啡肽A(1-17)对不同功能运动神经元群树突的影响

目的:探讨强啡肽A对大鼠趾长伸肌(EDL,快肌)、比目鱼肌(SOL,慢肌)两种不同功能运动神经元(Mn)群树突的影响。方法:采用脊髓蛛网膜下隙给予强啡肽A,以霍乱毒素B亚单位结合辣根过氧化酶(CB-HRP)逆行标记EDL-Mn、SOL-Mn群,Mesulam-TMB法显示两运动神经元群被标记的树突。结果:与相应对照组比,强啡肽A致一过性后肢瘫大鼠用药后1h,位于腰4~5脊髓节段腹角的EDL-Mn、SOL-Mn群树突的分布范围减小,尤以EDL-Mn群为甚;EDL-Mn平均树突长度比对照缩短59.5%,而SOL-Mn平均树突长度比对照缩短35.6%。永久性后肢瘫大鼠用药后3h,EDL-Mn群仅见胞体和近端树突;与之相比,SOL-Mn群仍保留有较长的树突和较广的分布范围。结论:强啡肽A致一过性后肢瘫大鼠两群运动神经元树突明显受累,且以EDL-Mn群为甚,其差别可能与两群运动神经元所接受强啡肽A信息传入不同有关。     

Changes of the protein metabolism in rat spinal motoneurons and perineuronal glial cells induced by anticipation stress and the administration of diazepam. A cytospectrophotometric and autoradiographic study

In rats, subjected repeatedly to anticipation stress, an increase of the size of spinal motoneurons (parallel with an increase of their total protein content) was observed. No similar changes occurred in perineuronal glial cells. A single administration of diazepam prevented the stress-induced changes in motoneurons, but concomitantly increased the volume and protein content of glial cell nuclei. Quantitative autoradiography did not reveal a significant increase of grain density over the neurons of stressed animals which had been injected with 4,5-[³H]leucine. After a single administration of diazepam to the stressed animals, however, the grain density over neuronal perikarya as well as over neuronal nuclei increased markedly. Analysis of the precursor pools and estimation of specific activities of proteins in brain, liver, testes and thymus indicated that (i) diazepam-induced changes in the size of the pool of radioactive precursors are the main factor which causes the increase of grain density, observed in autoradiograms. and (ii) diazepam decreases the utilization of labelled leucine for the synthesis of proteins in brain and liver.The experiments suggest that the repeated exposure to stress is associated with changes in the macro-molecular metabolism and size of single motor nerve cells. The activation of perineuronal glial cells is suggested to one mode of action of diazepam, but whether this contributes to its effect on behaviour is unknown.     

Rescue and regeneration of injured peripheral nerve axons by intrathecal insulin

Insulin peptide, acting through tyrosine kinase receptor pathways, contributes to nerve development or repair. In this work, we examined the direction, impact and repertoire of insulin signaling in vivo during peripheral nerve regeneration in rats. First, we demonstrated that insulin receptor is expressed on lumbar dorsal root ganglia neuronal perikarya using immunohistochemistry. Immunoblots and polymerase chain reactions confirmed the presence of both alpha and beta insulin receptor subunits in dorsal root ganglia. In vivo and in vitro assessment of dorsal root ganglion neurons showed preferential localization of insulin receptor to perikaryal sites. In vivo, intrathecal delivery of fluorescein isothiocyanate-labeled insulin identified localization around dorsal root ganglia neurons. The direction and impact of potential insulin signaling was evaluated by concurrently delivering insulin or carrier over a 2 week period using mini-osmotic pumps, either intrathecally, near nerve, or with both deliveries, following a selective sural nerve crush injury. Only intrathecal insulin increased the number and maturity of regenerating sensory sural nerve axons distal to the crush site. As well, only intrathecal insulin rescued retrograde loss of sural axons after crush. In a separate experiment, insulin also rescued retrograde loss and atrophy of deep peroneal, largely motor, axons post-injury. Intrathecal insulin increased the expression of calcitonin-gene-related peptide in regenerating sprouts, increased the number of visualized regenerating fiber clusters, and reduced downregulation of calcitonin-gene-related peptide in dorsal root ganglia neurons. Insulin delivered intrathecally does not appear to influence expression of insulin-like growth factor-1 at dorsal root ganglion neurons or near peripheral nerve injury, but was associated with upregulation of insulin receptor alpha subunit in dorsal root ganglia. Intrathecal insulin delivery was associated with greater recovery of thermal sensation and longer distances to stimulus response with the pinch test following sural nerve crush. Insulin signaling at neuron perikarya can drive distal sensory axon regrowth, rescue retrograde alterations of axons and alter axon peptide expression. Moreover, such actions are associated with upregulation of its own receptor.     

Retrograde transport of the lectin Phaseolus vulgaris leucoagglutinin (PHA-L) by rat spinal motoneurons

The lectin Phaseolus vulgaris leucoagglutinin (PHA-L) has been used primarily as an anterograde transport tracer in the CNS. We present evidence of PHA-L retrograde transport by rat spinal motoneurons after injection into the triceps brachii. Labelled motoneurons were localized in specific and well-defined neuron pools in the ventral horn. Primary afferent labelling was not seen in the spinal gray matter. Dorsal rhizotomy did not eliminate or decrease motoneuron labelling. The retrograde transport rate was about 8 mm/day. PHA-L can clearly undergo retrograde, as well as anterograde, transport.     

Hyperexcitability of cultured spinal motoneurons from presymptomatic ALS mice

ALS (amyotrophic lateral sclerosis) is an adult-onset and deadly neurodegenerative disease characterized by a progressive and selective loss of motoneurons. Transgenic mice overexpressing a mutated human gene (G93A) coding for the enzyme SOD1 (Cu/Zn superoxide dismutase) develop a motoneuron disease resembling ALS in humans. In this generally accepted ALS model, we tested the electrophysiological properties of individual embryonic and neonatal spinal motoneurons in culture by measuring a wide range of electrical properties influencing motoneuron excitability during current clamp. There were no differences in the motoneuron resting potential, input conductance, action potential shape, or afterhyperpolarization between G93A and control motoneurons. The relationship between the motoneuron's firing frequency and injected current (f-I relation) was altered. The slope of the f-I relation and the maximal firing rate of the G93A motoneurons were much greater than in the control motoneurons. Differences in spontaneous synaptic input were excluded as a cause of increased excitability. This finding identifies a markedly elevated intrinsic electrical excitability in cultured embryonic and neonatal mutant G93A spinal motoneurons. We conclude that the observed intrinsic motoneuron hyperexcitability is induced by the SOD1 toxic gain-of-function through an aberration in the process of action potential generation. This hyperexcitability may play a crucial role in the pathogenesis of ALS as the motoneurons were cultured from presymptomatic mice.     

Co-localization of choline acetyltransferase and postsynaptic glycine receptors in motoneurons of rat spinal cord demonstrated by immunocytochemistry

Male rats were perfused with paraformaldehyde and picric acid. The cervical spinal cord was cryosectioned and immunostained with a monoclonal antibody against the postsynaptic receptor for the neurotransmitter glycine. The anterior horn contained glycinoceptive neurons of varying morphology. Cholinergic cells were identified in the same tissue sections when subsequently immunostained with a monoclonal antibody against choline acetyltransferase, the biosynthetic enzyme of acetylcholine. Immunoreactivity for the glycine receptor was detected in the plasma membrane and for choline acetyltransferase in the perikaryal cytoplasm of identical anterior horn cells, classified as small, medium and large motoneurons. This suggests that motoneurons have receptors for glycine on their cell surface.     

Expression of GABA-immunoreactivity by spinal motoneurons of some vertebrates

Electrophysiological and biochemical investigations have shown that gamma-aminobutyric acid (GABA) is the main inhibitory neurotransmitter in the vertebrate central nervous system. However, the present study shows that some motoneurons located in the spinal cord of young chickens and adult monkeys display a GABA-like immunoreactivity. The expression of GABA immunoreactivity in vertebrate motoneurons suggests that this inhibitory amino acid is colocalized with acetylcholine and could play a role in the neuromuscular transmission.