Biomedical subjects

L Greensmith

Publications and source records attributed to L Greensmith.

At least 19 recordsLinked to original sources

PubMed2005

The effect of peripheral nerve injury on disease progression in the SOD1(G93A) mouse model of amyotrophic lateral sclerosis.

Around 20% of familial cases of amyotrophic lateral sclerosis have been shown to carry mutations in Cu/Zn superoxide dismutase 1 (Cu/Zn SOD1). Transgenic mice over-expressing human mutant SOD1 genes have been developed and in this study we examined the effect of nerve injury on disease progression in these mice. Firstly, disease progression in uninjured mice was characterised using physiological methods. Muscle force, contractile characteristics and motor unit survival was established at 90 days, an early symptomatic stage and also at the end-stage of the disease, at 130 days. In addition, muscle histochemistry was examined and the extent of motoneuron survival established morphologically. By 90 days of age, there is a significant reduction in muscle force, and nearly 40% of motoneurons within the sciatic motor pool have already died. By 130 days, the muscles are significantly weaker, and there is a dramatic change in the phenotype of extensor digitorum longus (EDL), which changes from a fast fatigable muscle, to a fatigue resistant muscle with a high oxidative capacity. By this stage of the disease, only 40% of motor units in EDL survive, with only 29% of motoneurons surviving within the sciatic motor pool. Following injury to the sciatic nerve in SOD1(G93A) mice, there is an acceleration in disease progression so that 90 day old mice show deficits that are only seen at the end stage in uninjured SOD1(G93A) mice. It is therefore possible that mutant SOD1 toxicity increases the vulnerability of motoneurons and muscles to stressful stimuli such as nerve injury.

Amyotrophic Lateral Sclerosis↗

PubMed2005

Molecular chaperones as therapeutic targets in amyotrophic lateral sclerosis.

Neurodegenerative diseases are characterized by a number of common hallmarks, such as the presence of intracellular aggregates and activation of the apoptotic cell-death pathway. Intracellular chaperones, responsible for protein integrity and structural repair, may play a crucial role in the progression of a disease. In this paper, we aim to summarize our understanding of the role and potential of a particular family of chaperones, the heat-shock proteins, in neurodegeneration, by focusing our discussion on models of motoneuron death.

Disease Progression↗

PubMed2004

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.

Animals↗

PubMed2004

Inhibition of calpains, by treatment with leupeptin, improves motoneuron survival and muscle function in models of motoneuron degeneration.

The effect of treatment with leupeptin, a calpain inhibitor, on motoneuron survival and muscle function was examined in in vitro and in vivo models of motoneuron degeneration. Exposure of primary rat motoneurons to alpha-amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid (AMPA) is an established in vitro model of excitotoxic motoneuron death. Here we show that leupeptin treatment improved motoneuron survival following exposure to AMPA (50 microM). Application of leupeptin (100 microM) to AMPA treated cultures rescued many motoneurons so that 74% (+/-3.4 S.E.M., n=5) survived compared with only 49% (+/-2.4 S.E.M., n=5) in untreated cultures. The effect of treatment with leupeptin on motoneuron survival and muscle function was also examined in vivo. In 3 day-old rats, the sciatic nerve was crushed and at the time of injury, a silicon implant containing leupeptin was inserted onto the lumbar spinal cord. The effect on long-term motoneuron survival and muscle function was assessed 12 weeks after injury. The results showed that there was long-term improvement in motoneuron survival in the leupeptin treated group. Thus, in untreated animals 12 weeks after nerve crush only 30% (+/-2.8. S.E.M., n=3) of sciatic motoneurons survived compared with 43% (+/-1.5 S.E.M., n=3) in the leupeptin-treated group. This improvement in motoneuron survival was reflected in a significant improvement in muscle function in the leupeptin-treated group. For example in the soleus muscle of treated rats 20.8 (+/-1.40 S.E.M., n=5) motor units survived compared with only 14.6 (+/-1.21 S.E.M., n=5) in untreated animals. Thus, treatment with leupeptin, a calpain inhibitor, rescues motoneurons from cell death and improves muscle function following nerve injury.

Animals↗

PubMed2003

The effect of treatment with BRX-220, a co-inducer of heat shock proteins, on sensory fibers of the rat following peripheral nerve injury.

In this study, we examined the effect BRX-220, a co-inducer of heat shock proteins, in injury-induced peripheral neuropathy. Following sciatic nerve injury in adult rats and treatment with BRX-220, the following features of the sensory system were studied: (a) expression of calcitonin gene-related peptide (CGRP); (b) binding of isolectin B4 (IB4) in dorsal root ganglia (DRG) and spinal cord; (c) stimulation-evoked release of substance P (SP) in an in vitro spinal cord preparation and (d) nociceptive responses of partially denervated rats. BRX-220 partially reverses axotomy-induced changes in the sensory system. In vehicle-treated rats there is a decrease in IB4 binding and CGRP expression in injured neurones, while in BRX-220-treated rats these markers were better preserved. Thus, 7.0 +/- 0.6% of injured DRG neurones bound IB4 in vehicle-treated rats compared to 14.4 +/- 0.9% in BRX-220-treated animals. Similarly, 4.5 +/- 0.5% of DRG neurones expressed CGRP in the vehicle-treated group, whereas 9.0 +/- 0.3% were positive in the BRX-220-treated group. BRX-220 also partially restored SP release from spinal cord sections to electrical stimulation of primary sensory neurones. Behavioural tests carried out on partially denervated animals showed that BRX-220 treatment did not prevent the emergence of mechanical or thermal hyperalgesia. However, oral treatment for 4 weeks lead to reduced pain-related behaviour suggesting either slowly developing analgesic actions or enhancement of recovery processes. Thus, the morphological improvement seen in sensory neurone markers was accompanied by restored functional activity. Therefore, treatment with BRX-220 promotes restoration of morphological and functional properties in the sensory system following peripheral nerve injury.

Animals↗

PubMed2002

Upregulation of heat shock proteins rescues motoneurones from axotomy-induced cell death in neonatal rats.

Heat shock proteins (hsps) are induced in a variety of cells following periods of stress, where they promote cell survival. In this study, we examined the effect of upregulating hsp expression by treatment with BRX-220, a co-inducer of hsps, on the survival of injured motoneurones. Following sciatic nerve crush at birth, rat pups were treated daily with BRX-220. The expression of hsp70 and hsp90, motoneurone survival, and muscle function was examined at various intervals later and the number of functional motor units was assessed by in vivo isometric tension recordings. Fourteen days after injury, significantly more motoneurones survived in the BRX-220-treated group (39 +/- 2.8%) compared to the saline-treated group (21 +/- 1.7%). Moreover, in the BRX-220-treated group no further loss of motoneurones occurred, so that at 10 weeks 42 +/- 2.1% of motoneurones survived compared to 15 +/- 0.6% in the untreated group. There were also more functional motor units in the hindlimb muscles of BRX-220-treated animals. In addition, treatment with BRX-220 resulted in a significant increase in the expression of hsp70 and hsp90 in glia and neurones. Thus, treatment with BRX-220, a co-inducer of hsps, protects motoneurones from axotomy-induced cell death.

Animals↗

PubMed2001

Treatment of the neuromuscular junction with 4-aminopyridine results in improved reinnervation following nerve injury in neonatal rats.

During early postnatal development, nerve injury results in the death of a large proportion of motoneurones and poor recovery of muscle function. Our previous results have shown that premature enhancement of transmitter release from nerve terminals prevents the death of motoneurones following neonatal nerve injury. Whether this increase in motoneurone survival is reflected in an improvement in the reinnervation of muscle was studied here. The muscles in one hindlimb of newborn rats were treated with 4-aminopyridine. Three days later, the sciatic nerve was crushed in the treated leg. When the animals were seven, 14 and 21days of age, the soleus and extensor digitorum longus muscles were removed and processed for GAP-43 (a 43-kDa growth-associated protein) and synaptophysin immunocytochemistry. Both GAP-43 and synaptophysin were expressed in normal soleus and extensor digitorum longus muscles at seven days. Synaptophysin was still expressed at 14 days, but GAP-43 expression had declined. Following nerve injury at three days of age, there was no GAP-43 or synaptophysin immunoreactivity in nerve terminals at seven days. By 21 days, there were 17.3+/-2.1 GAP-43-positive terminals per section in the soleus and 17.7+/-1.4 in the extensor digitorum longus, with mean terminal areas of 47.5+/-3.3 and 49.8+/-2.6 microm(2), respectively. In animals in which nerve crush was preceded by 4-aminopyridine treatment, at 21 days there were 32.9+/-2.6 GAP-43-immunoreactive terminals in the soleus and 44.9+/-2.3 in the extensor digitorum longus, with a mean area of 122.7+/-6.6 microm(2) in the soleus and 136.2+/-9.7 microm(2) in the extensor digitorum longus. These results indicate that in muscles pretreated with 4-aminopyridine, prior to nerve crush at three days, there are significantly more terminals, which occupy a larger area than in untreated muscles. Thus, increasing transmitter release prior to nerve injury significantly improved the ability of axons to reinnervate muscle.

4-Aminopyridine↗

PubMed2000

Reducing transmitter release from nerve terminals influences motoneuron survival in developing rats.

Motoneurons in neonatal rats die following injury to the peripheral nerve. However, this vulnerability to nerve injury declines rapidly so that nerve injury at five days of age results in little if any motoneuron death. We have proposed that the role of the target during this critical period of development is to up-regulate the release of transmitter from developing motor nerve terminals. Here we show that reducing the release of acetylcholine from nerve terminals in neonatal rats can affect motoneuron maturation and survival. The soleus muscle in neonatal rats was treated with either magnesium or hemicholinium, and the number of motoneurons that survived was established 10 weeks later by retrograde labelling. Following treatment with magnesium, only 58.1% (+/-10.4 S.E.M., n=5) of the motoneurons in the soleus motor pool survived, although hemicholinium had no effect on motoneuron survival. However, those motoneurons that survived following treatment with either magnesium or hemicholinium did not develop normally since they remained susceptible to axotomy-induced cell death for longer than normal. In adult animals in which the sciatic nerve was crushed at five days of age following prior treatment with either magnesium or hemicholinium, only 27.6% (+/-6.2 S.E.M., n=5) and 44% (+/-6.1 S.E.M., n=4) of motoneurons in the sciatic motor pool survived, respectively, although no motoneurons died following injury alone or when injury was preceded by treatment with control implants containing NaCl. These results indicate that the release of acetylcholine from motor nerve terminals plays an important role in the development and survival of motoneurons.

Acetylcholine↗

PubMed2000

Repeated stimuli for axonal growth causes motoneuron death in adult rats: the effect of botulinum toxin followed by partial denervation.

Axons of motoneurons to tibialis anterior and extensor digitorum longus muscles of adult rats were induced to sprout by injecting botulinum toxin into them, by partial denervation or by a combination of the two procedures. Ten weeks later, the number of motoneurons innervating the control and operated tibialis anterior and extensor digitorum longus muscles was established by retrograde labelling with horseradish peroxidase. In the same preparations, the motoneurons were also stained with a Nissl stain (gallocyanin) to reveal motoneurons in the sciatic pool. Examination of the spinal cords from animals treated with botulinum toxin showed that the number of retrogradely labelled cells and those stained with gallocyanin in the ventral horn on the treated compared to the control side was unchanged. In rats that had their L4 spinal nerve sectioned on one side, the number of retrogradely labelled cells on the operated side was 48+/-3% (n = 5) of that present in the control unoperated ventral horn. Thus, just over half the innervation was removed by cutting the L4 spinal nerve. Counts made from gallocyanin-stained sections showed that 94+/-4% (n = 5) of motoneurons were present in the ventral horn on the operated side. Thus, section of the L4 spinal nerve did not lead to any death of motoneurons. In rats that had their muscles injected with botulinum toxin three weeks prior to partial denervation, the number of retrogradely labelled cells was reduced from 48+/-3% (n = 5) to 35+/-4% (n = 5). Moreover, only 67+/-5% (n = 5) of motoneurons stained with gallocyanin, suggesting that a proportion of motoneurons died after this combined procedure. This result was supported by experiments in which motor unit numbers in extensor digitorum longus muscles were determined by measurements of stepwise increments of force in response to stimulation of the motor nerve with increasing stimulus intensity. In partially denervated extensor digitorum longus muscles, 16.6+/-0.7 (n = 5) motor units could be identified, and in animals treated with botulinum toxin prior to partial denervation only 13.3+/-0.9 (n = 3) motor units were present. Taken together, these results show that treatment with botulinum toxin followed by partial denervation causes motoneuron death in adult rats.

Animals↗

PubMed1999

Neurotrophic factors.

Explore the source record for details and available documents.

Brain-Derived Neurotrophic Factor↗

PubMed1999

Stabilizing neuromuscular contacts reduces motoneuron death caused by paralysis of muscles in neonatal rats.

Transient paralysis of the soleus muscle in neonatal rats leads to permanent muscle weakness, loss of muscle fibres and motoneuron death. Application of leupeptin, an inhibitor of a calcium-activated neutral protease, to the neuromuscular junction is known to enhance the maintenance of neuromuscular contacts during development and axonal sprouting. Here, we show that treatment of soleus muscles with leupeptin as they recover from a period of paralysis rescues motoneurons that would otherwise die. The number of motoneurons to the soleus muscle was established by retrograde labelling with horseradish peroxidase eight to 10 weeks after recovery from paralysis. There were only 38.4 (+/-2.8 S.E.M., n=5) motoneurons innervating the soleus muscle that had been paralysed with alpha-bungarotoxin, compared to 58.2 (+/-3.1 S.E.M., n=5) to the control untreated soleus. Thus, the number of motoneurons to the soleus muscle on the alpha-bungarotoxin-treated side was 66.9% (+/-6.2% S.E.M., n=5) of the control side. In those animals where paralysis of the soleus muscle was followed three days later by treatment with leupeptin, the number of labelled motoneurons on the treated side of the spinal cord was 61.5 (+/-4.6 S.E.M., n=4) and that on the contralateral untreated control side was 59 (+/-3.8 S.E.M., n=4). This improvement in motoneuron survival in the leupeptin-treated animals is also confirmed by counts of the number of motor units in the soleus muscle obtained by recording muscle tension. In animals that had their soleus muscles paralysed at birth, only 21 (+/-0.7 S.E.M., n=5) motor units were present, compared to 30 motor units in control muscles. When the paralysed soleus muscle was subsequently treated with leupeptin, the number of remaining motor units in the muscle was 29.8 (+/- 1.0 S.E.M., n=5). In addition, the force output of the soleus muscles that had undergone a period of neonatal paralysis was calculated for both the NaCl- and leupeptin-treated animals. The results showed that paralysis at birth results in a reduction in weight and force output of the soleus muscle, which is not improved following treatment with leupeptin. This study shows that application of leupeptin to the soleus muscle after alpha-bungarotoxin-induced paralysis rescues motoneurons to the soleus that would otherwise die. This effect is most likely due to stabilization of their neuromuscularjunctions.

Animals↗

PubMed1999

Overexpression of GAP-43 induces prolonged sprouting and causes death of adult motoneurons.

In neurodegenerative diseases, neurons undergo prolonged periods of sprouting. Whether this sprouting compromises these neurons is unknown. Here, we examined the effect of axotomy on adult motoneurons undergoing prolonged sprouting in transgenic mice that overexpress GAP-43 (growth-associated protein). Sciatic nerve injury in these adult mice results in motoneuron death, but has no effect in non-transgenic mice. Thus, continued growth of motor axons renders adult motoneurons susceptible to nerve injury and compromises their long-term survival. The progressive nature of neurodegenerative diseases may therefore be caused by prolonged sprouting.

Animals↗

PubMed1999

Integrins at the neuromuscular junction are important for motoneuron survival.

During development motoneurons depend on target contact for their survival. Following injury to the sciatic nerve in neonatal rats, a large proportion of motoneurons die. However, the same injury inflicted at 5 days of age results in no loss of motoneurons. This critical period of postnatal development coincides with the time during which there is a significant increase in the release of transmitter from the nerve terminals at the neuromuscular junction. We have proposed that the role of the target muscle cell during this period is to induce this up-regulation of transmitter release from motor nerve terminals. It has been shown that stretch-induced increase in transmitter release from frog motor nerve terminals is accomplished via an integrin-dependent mechanism. In this study we examined the role of integrins at the rat neuromuscular junction in motoneuron survival. We found that blocking integrin binding at the developing neuromuscular junction delayed the increase in choline acetyltransferase activity that normally takes place during the early postnatal period, and resulted in motoneuron death. Furthermore, the maturation of those motoneurons that survived was delayed so they remained susceptible to subsequent nerve injury. These results support the possibility that integrins, by their involvement in modulating transmitter release, can influence motoneuron survival.

Animals↗

PubMed1998

Mechanical activity is necessary for the elimination of polyneuronal innervation of developing rat soleus muscles.

During early development, rat soleus muscle fibres are innervated by several axons. Neuromuscular activity is involved in the elimination of all but one terminal, but it is not clear whether electrical or mechanical activity is important. Here, we reduced mechanical activity only, by interfering with excitation-contraction coupling. Muscles treated with dantrolene sodium at 9 days produced significantly less force at 13 days of age than normal muscles, and their sensitivity to ACh was greater than that of controls. The elimination of polyneuronal innervation occurs between days 9-12, but in muscles treated with dantrolene, the loss of synapses was slower. Thus, reducing mechanical activity by interfering with excitation-contraction coupling, (a) delays muscle development and (b) reduces the rate of elimination of polyneuronal innervation.

Acetylcholine↗

PubMed1998

Motoneurons innervating partially denervated rat hindlimb muscles remain susceptible to axotomy-induced cell death.

Tibialis anterior and extensor digitorum longus muscles were partially denervated by cutting the L4 spinal nerve in three-day-old rats. The ultrastructure of the intact axons to these muscles in the L5 spinal nerve was examined in nine-day-old rats. In the control L5 spinal nerve, myelinated and unmyelinated axons were intermingled throughout the cross-section of the nerve, while on the operated side the nerve contained areas with predominantly small unmyelinated immature axons. The number of motoneurons innervating the partially denervated muscles was established by retrograde labelling with Diamidino Yellow. In nine- and 21-day-old rats, the number of labelled motoneurons on the partially denervated side, expressed as a percentage of the control side, was 26.1 +/- 5.5% and 20.7 +/- 3.0%, respectively. The response of these uninjured motoneurons to axotomy was tested. The axons of the motoneurons to the partially denervated muscles were crushed at nine days and the numbers of labelled motoneurons in the spinal cord of these rats counted at 21 days of age. Only 4.9 +/- 2.0% labelled motoneurons were seen on the operated side, as opposed to 20.7 +/- 3.0% present in animals without sciatic nerve injury. In normal animals, nerve injury at nine days does not cause motoneuron death. Thus, motoneurons to partially denervated muscles (i) have axons with several immature features and (ii) remain susceptible to axotomy-induced death for much longer than normal.

Animals↗

PubMed1998

Motoneurones that innervate the rat soleus muscle mature later than those to the tibialis anterior and extensor digitorum longus muscles.

The response of motoneurones that innervate either the soleus or tibialis anterior (TA) and extensor digitorum longus (EDL) muscles to increased locomotor activity or to nerve injury at different stages after birth was examined. Increased locomotor activity of rat pups was induced by daily treatment with L-dopa during the first 12 days after birth, and the number of surviving motoneurones to the soleus or TA/EDL muscles was established by retrograde labelling. Treatment with L-dopa resulted in the loss of a significant number of motoneurones within the soleus motor pool but had no effect on the survival of those motoneurones innervating the TA/EDL. Furthermore, following nerve injury during the first few days postnatally, more motoneurones within the soleus motor pool die than in the TA/EDL pool. These results indicate that motoneurones to the soleus muscle mature later than those to the TA/EDL muscles.

Animals↗

PubMed1997

Neonatal paralysis of the rat soleus muscle selectively affects motoneurones from more caudal segments of the spinal cord.

Transient paralysis of the rat soleus muscle shortly after birth leads to permanent muscle weakness, loss of muscle fibres and the death of motoneurones. The soleus muscle receives its innervation from motoneurones whose axons exit the spinal cord either via the L4 ventral ramus or in the more caudal part of the cord via the L5 ventral ramus. Whether both populations of motoneurones are equally affected by neonatal paralysis was studied here. In soleus muscles paralysed with alpha-bungarotoxin shortly after birth and examined 8-10 weeks later, there is no loss of force or muscle fibres in the part of the muscle supplied by axons in the L4 ventral ramus. Loss of force and muscle fibre numbers occurs only in the part of the muscle supplied by axons in the L5 ventral ramus. In a normal adult soleus 30.3 +/- 2.4% of muscle force is produced by stimulating the L4 ventral ramus and 69.0 +/- 5.5% by stimulating the L5 ventral ramus. In soleus muscles treated with alpha-bungarotoxin 28 +/- 1.4% of the force produced by the contralateral control soleus was generated by axons in the L4 ventral ramus and only 20.3 +/- 5.6% by stimulating the L5 spinal nerve. The number of muscle fibres supplied by either ventral ramus in control and experimental muscles confirmed that the decrease of force after treatment with alpha-bungarotoxin can be accounted for by loss of muscle fibres supplied by axons in the L5 ventral ramus. The reduced force production and muscle fibre numbers was due to a selective loss of motoneurones that had their axons in the L5 ventral ramus. The number of axons to soleus in the L4 ventral ramus was 9.3 +/- 0.7 in controls and 10.3 +/- 0.9 in the experimental animals, whereas the L5 ventral ramus contained 17.2 +/- 0.7 in controls and only 4.7 +/- 1.7 in the experimental animals. Thus paralysis of the soleus muscle at birth selectively affects motoneurones in the more caudal part of the spinal cord, suggesting that the more cranial motoneurones are more mature and less likely to be influenced by lack of neuromuscular interaction at the time of birth.

Acute Disease↗

PubMed1997

Disturbances of neuromuscular interaction may contribute to muscle weakness in spinal muscular atrophy.

During a critical period of development, motoneurones and muscles are dependent on functional interaction with each other for their survival. In certain neuromuscular disorders such as spinal muscular atrophy (SMA), motoneurones die and muscle development is seriously affected. Recently advances have been made towards understanding the genetic basis of this disease, and a particular gene, i.e. the survival motoneurone gene (SMN), has been identified and found missing in SMA patients. Nevertheless the function of this gene is not clear, it may be involved in the control of the development of either the motoneurone or the muscle. Here we report that disrupting neuromuscular interaction during the early postnatal period has similar consequences on the development of muscles and motoneurones to those seen in patients with SMA, in that there is a loss of motoneurones and muscle function is severely impaired. In view of this, we discuss the possibility that these symptoms in SMA patients may be due to a disturbance of neuromuscular interaction during a critical stage of development.

Animals↗