EPO-releasing neural precursor cells promote axonal regeneration and recovery of function in spinal cord traumatic injury.

BACKGROUND: Spinal cord injury (SCI) is a debilitating condition characterized by a complex of neurological dysfunctions ranging from loss of sensation to partial or complete limb paralysis. Recently, we reported that intravenous administration of neural precursors physiologically releasing erythropoietin (namely Er-NPCs) enhances functional recovery in animals following contusive spinal cord injury through the counteraction of secondary degeneration. Er-NPCs reached and accumulated at the lesion edges, where they survived throughout the prolonged period of observation and differentiated mostly into cholinergic neuron-like cells. OBJECTIVE: The aim of this study was to investigate the potential reparative and regenerative properties of Er-NPCs in a mouse experimental model of traumatic spinal cord injury. METHODS AND RESULTS: We report that Er-NPCs favoured the preservation of axonal myelin and strongly promoted the regrowth across the lesion site of monoaminergic and chatecolaminergic fibers that reached the distal portions of the injured cord. The use of an anterograde tracer transported by the regenerating axons allowed us to assess the extent of such a process. We show that axonal fluoro-ruby labelling was practically absent in saline-treated mice, while it resulted very significant in Er-NPCs transplanted animals. CONCLUSION: Our study shows that Er-NPCs promoted recovery of function after spinal cord injury, and that this is accompanied by preservation of myelination and strong re-innervation of the distal cord. Thus, regenerated axons may have contributed to the enhanced recovery of function after SCI.

Acute spinal cord injury reduces brain derived neurotrohic factor expression in rat hippocampus.

Spinal cord injury (SCI) is a devastating event which causes dramatic changes in the everyday life of the patient. We have found that acute SCI reduced BDNF expression selectively in the hippocampus of lesioned rats, a decrease which persists at least 1 week, thus identifying the modulation of the neurotrophin biosynthesis as an important mechanism underlying brain vulnerability to SCI. These data are the first to show that SCI alters hippocampal BDNF expression and identify the neurotrophin as a potential target through which SCI changes brain functions, a notion that might prove useful in understanding the mechanisms underlying brain vulnerability to SCI.

Amelioration of neutrophil membrane function underlies granulocyte-colony stimulating factor action in glycogen storage disease 1b.

Glycogen storage disease (GSD) 1b is a metabolic disorder characterized by a deficiency of glucose 6-phosphate transporter and neutrophil alterations, which are reduced in number and functionally impaired. The present study aimed at investigating neutrophil dysfunction correlating submembrane and cytoskeletal changes at different ages with or without granulocyte-colony stimulating factor (G-CSF) treatment. GSD1b neutrophils showed reduced expression and diffused localization of focal adhesion kinase (FAK) and actin. No abnormalities were observed in GSD1a patient neutrophils. Gelsolin was also slightly reduced in neutrophils of GSD1b patients. When patients were treated for at least 3 months with G-CSF, the neutrophil number and the expression of FAK and actin were significantly increased. Granulocyte colony-stimulating factor treatment was similarly effective when performed in 1 year old patients. FAK auto- and IL-8-mediated phosphorylations were already affected as early as 1 year of age. G-CSF treatment also improved this alteration. Our data suggest that neutrophil dysfunction in GSD1b patients might be related to functional impairment and disorganization of proteins of the sub-membrane apparatus, and that G-CSF treatment counteracts neutropenia and prevents the progressive alterations of neutrophil sub-membrane proteins.

Plasma amino acid concentrations throughout normal pregnancy and early stages of intrauterine growth restricted pregnancy.

OBJECTIVES: Assessment of maternal plasma amino acids during normal gestation and in early stages of intrauterine growth restriction (IUGR). STUDY DESIGN: Plasma amino acid concentrations were measured in: (1) non-pregnant women (n=7); (2) normal pregnant women in the first (n=13), second (n=17) and third (n=12) trimester; and (3) pregnant women in the first trimester with later development of IUGR (n=8). Amino acid levels were quantified by electrochemical detection in a reversed-phase high-performance liquid chromatography (HPLC) system. RESULTS: The levels of most essential and non-essential amino acids changed markedly in the first trimester during normal pregnancy and thereafter remained almost constant. In the first trimester of IUGR, a number of both essential and non-essential amino acids were significantly different from those observed in normal pregnancies, with values more similar to those observed in non-pregnant women. CONCLUSIONS: Levels of most maternal amino acids decrease and some increase during early gestation reflecting a metabolic adaptation that occurs in normal pregnancies. Pregnancies that later develop IUGR show a lack of these adaptations for a significant number of both essential and non-essential amino acids, suggesting a lack of adaptation.

Fate of autologous dermal stem cells transplanted into the spinal cord after traumatic injury (TSCI).

Rat dermis is a source of cells capable of growing in vitro and, in appropriate conditions, forming floating spheres constituted by nestin-positive cells. We have clonally grown these spheres up to the 15th generation. These spheres can be dissociated into cells that differentiate in vitro under appropriate conditions, these cells are labeled by antibodies to immature neuron markers such as nestin and beta-tubulin III and, later, to mature neuron markers such as microtubule-associated protein 2 and neurofilaments. However, most cells are positive to the astroglial marker glia fibrillary acidic protein (GFAP). When sphere-derived cells are transplanted into the spinal cord after traumatic injury, their migration into the lesion cavity is optimal but their differentiation is dependent upon the time interval between lesioning and cell transplantation. Injection of skin-derived stem cell within 30 min from injury yields mainly membrane activated complex-1 (MAC-1), cluster of differentiation-4 (CD-4) and CD-8 positive cells, that 60-90 days later undergo apoptosis. However, when transplantation is performed 7 days after injury, most cells (65% of total) are positive to staining with antibodies to GFAP, others (16%) to neurofilaments, and a smaller amount (2%) to the endothelial marker, platelet endothelial cell adhesion molecule. Thus our study shows that delayed transplantations of dermis-derived stem cells yield healthy cells that do not die, migrate to the lesion site, and there differentiate mainly in cells expressing glia and neuronal markers. On the other hand there is the possibility of dye transfer from labeled cells to endogenous cells, and this might influence the data.

Glycosaminoglycan-promoted muscle reinnervation and insulin-like growth factor-I levels are affected by anti-growth hormone-releasing hormone exposure.

The present study shows that exposure to antibodies to growth hormone-releasing hormone (GHRH) partially counteracted the promoting effects of treatment with glycosaminoglycans (GAGs) on muscle reinnervation. Sciatic nerve crush was performed in 2-day-old rats, and reinnervation of the extensor digitorum longus muscle was monitored. The extent of reinnervation was rather poor in saline-treated rats, whereas in GAG-treated rats the extent of muscle reinnervation, the recovery of nerve-evoked muscle twitch tension, and the number of motor neurons reinnervating the extensor digitorum longus muscle were greatly enhanced. In addition, treatment with glycosaminoglycans increased markedly insulin-like growth factor-I (IGF-I) levels in denervated muscles. Both types of stimulatory action exerted by GAGs were affected by concomitant exposure to anti-GHRH, with abolition of IGF-I muscle increase and a smaller enhancement in muscle reinnervation.

Glycosaminoglycans co-administration enhance insulin-like growth factor-I neuroprotective and neuroregenerative activity in traumatic and genetic models of motor neuron disease: a review.

In this report it is shown how glycosaminoglycans and insulin-like growth factor-I (IGF-I) promote muscle reinnervation and prevent motor neuron death in experimental models of motor neuron disease. Such effect appears to be mediated by insulin-like growth factor-1. The glycosaminoglycan moiety of proteoglycans is a constituent of the basal lamina active on nerve regeneration by means of the interaction with laminin and with several growth factors. We have previously shown that supplementation by means of subcutaneous injections of glycosaminoglycans affects neuronal degeneration and regeneration. In this study we report that following neonatal lesion of the rat sciatic nerve, glycosaminoglycan treatment promoted extensor digitorum longus muscle reinnervation with consequent improvement of muscle morphology. In saline-treated rats, reinnervation was only partial and there was a marked muscle fibre atrophy, whereas, glycosaminoglycan treatment of lesioned rats increased IGF-I mRNA and protein in the reinnervated muscle, and IGF-I and insulin-like growth factor binding protein-3 plasma levels. Similarly, treatment of lesioned rats with IGF-I promoted muscle reinnervation, and prevented muscle fibre atrophy, higher levels of IGF-I in the reinnervated muscle, of IGF-I, and insulin-like growth factor binding proteins in plasma. In the wobbler mouse IGF-I and glycosaminoglycans alone promote only a partial motor neuron survival and the preservation of forelimb function decays after 3 weeks of treatment. However when glycosaminoglycans and insulin-like growth factor are administered together the motor neuron disease in the wobbler mouse is halted and there is no more loss of motor neurons.

Long-term neuroprotective effects of glycosaminoglycans-IGF-I cotreatment in the motor neuron degeneration (mnd) mutant mouse.

This study shows that cotreatment with insulin-like growth factor-I (IGF-I) and glycosaminoglycans (GAGs) prevents the onset of neuromuscular deficit in the m/m mutant mouse. These mice show a mid-to-late-life onset of progressive paralysis of the hind limb, that is correlated with altered innervation and reduced nerve-evoked isometric twitch tension in the extensor digitorum longus (EDL) muscle. Almost 50% of EDL nerve endings are negative for antisynaptophysin staining, while retrograde labelling with beta-cholera-toxin coupled to type IV horseradish and quantitative histological analysis show a small loss of EDL and lumbar cord motor neurons. At 10 months of age also forelimb function evaluated as grip strength is significantly reduced. Animals treated either with glycosaminoglycans alone or with IGF-I alone at low and high doses showed only a partial improvement of their condition. However, cotreatment of m/m mice with IGF-I and GAGs fully prevented the neuromuscular abnormalities, the twitch tension loss, the motor neuron decrease and the reduction of forelimb grip strength.

Perinatal supplementation of low doses of ethanol enhances 5-HT restoration in the central nervous system.

It has been reported that long-term administration of ethanol has deleterious effects on the central nervous system; the alterations are particularly evident if the exposure occurs during development. Our study shows that rat perinatal administration of 3% and 6% ethanol does not alter development of serotonin (5-HT) pathways in the central nervous system, while their reactive changes triggered by neonatal lesioning are greatly altered. The administration of 5, 7-dihydroxytriptamine (5,7-DHT) within 6 hours from birth causes 5-HT fiber degeneration throughout the central nervous system. The loss of 5-HT is particularly relevant in lumbar spinal cord, occipital cortex, and hippocampus. This early decrease in 5-HT content is followed by a slow and partial recovery. If animals are exposed to 3% ethanol during the perinatal period, there is an enhancement of the 5,7-DHT-induced degeneration that is, however, followed by a faster and greater recovery throughout the central nervous system. Conversely, perinatal exposure to 6% ethanol and 5, 7-DHT administration lead to an irreversible 5-HT loss with no subsequent recovery. The deleterious effects of 6% ethanol are accompanied by a reduced expression of neurotrophin. Thus, our study suggests that chronic exposure to ethanol can influence central nervous system plasticity during development. Low doses may enhance neuronal plasticity and repair perhaps via an increased efficacy of neurotrophic factors, whereas higher doses may negatively affect neural development also by means of the impairment of the expression of neurotrophic factors.

Glycosaminoglycans boost insulin-like growth factor-I-promoted neuroprotection: blockade of motor neuron death in the wobbler mouse.

Wobbler mice display forelimb weakness, altered paw positioning, reduced running speed, muscle atrophy and motor neuron loss; co-treatment with glycosaminoglycans and insulin-like growth factor-I counteracts the progression of the disease. Reportedly, treatment with glycosaminoglycans or insulin-like growth factor-I slows the early stages of progressive forelimb dysfunction in wobbler mice. Our aim was to study whether the combination of these two drugs would result in greater neuroprotective effects. In a group of wobbler mice, combined treatment with daily s.c. administration of 20 microg/kg insulin-like growth factor-I and 1 mg/kg glycosaminoglycans was begun upon diagnosis at three weeks of age and continued for the next six weeks. This treatment halted motor neuron loss and markedly reduced the decay of forelimb muscle morphometry and function. Moreover, the mouse phenotype itself was strikingly improved. The effect of the combination treatment was significantly higher than that of the single drugs, even at a dosage as high as 1 mg/kg insulin-like growth factor-I. The ability of the insulin-like growth factor-I/glycosaminoglycans pharmacological cocktail to arrest the progression of motor neuron disease in wobbler mice and the safety of the low dose of insulin-like growth factor-I used hold promise that this combination might represent a novel approach for the treatment of motor neuron disease and peripheral neuropathies.

Inhibition of high glucose-induced protein mono-ADP-ribosylation restores neuritogenesis and sodium-pump activity in SY5Y neuroblastoma cells.

The exposure of SY5Y neuroblastoma cells to high concentrations of glucose, fructose, or galactose is an experimental model commonly used for in vitro evaluation of typical neuronal alterations observed in diabetes mellitus. In the present study, we observed that 2 weeks of exposure to high carbohydrate concentrations caused both a significant impairment in neurite formation induced by supplementation of retinoic acid or by subtraction of fetal calf serum to the culture medium and a marked reduction in Na(+)-K(+)-ATPase activity. However, only the exposure to high millimoles of glucose caused an enhancement of mono-ADP-ribosylation, typical of diabetes mellitus, affecting at least five proteins. The concomitant exposure to high glucose and to silybin, a mono-ADP-ribosylation inhibitor, normalized the extent of ADP-ribosylation of the five proteins and counteracted the inhibitory effects of high glucose on Na(+)-pump activity and on neuritogenesis. Conversely, the supplementation of silybin did not prevent fructose and galactose inhibitory effects on Na(+)-pump activity and neurite formation. These data confirm those of previous reports suggesting a link between excessive protein mono-ADP-ribosylation and the onset of diabetic complications such as diabetic neuropathy.

Progressive and selective changes in neurotrophic factor expression and substance p axonal transport induced by perinatal diabetes: protective action of antioxidant treatment.

Diabetes-induced embryo malformations and growth retardation are correlated with a variety of biochemical changes including oxidative stress. In this study, we show that the morphological alterations are correlated with progressive and selective changes of mRNA expression in specific neurotrophic factors. At embryological stage E-17, diabetes affected both embryo growth and NGF mRNA expression, which was reduced by as much as 90 and 56% in target tissues of sensory system such as tongue and intestine, respectively. The reduction in retina and heart was around 50%. Conversely, the mRNA expression of low-affinity neurotrophin receptor p75 was increased. At birth, BDNF mRNA expression was affected with a significant generalized reduction,while in vibrissae we observed a reduction of BDNF and p75 mRNAs and an increase of NGF. At postnatal day 14, pups from diabetic mothers showed reduced muscle levels of IGF-I, while we observed a partial impairment of substance P axonal transport at postnatal day 28. Treatment of diabetic mothers with silybin, a flavonoid with antioxidant properties, prevented most of the changes in neurotrophic factor expression and substance P axonal transport with no effects on hyperglycemia and embryo growth retardation. These results indicate that oxidative stress may influence neurotrophic factor synthesis in target territories during development. In addition, these data suggest that nervous system abnormalities observed in diabetic embryopathy may also derive by insufficient neurotrophic factor biosynthesis involving sequentially NGF in the embryo and BDNF and IGF-I in the early postnatal days. Insulin treatment of diabetic mothers normalized hyperglycemia and body growth, with consequent regular embryonic and postnatal development.

Glycosaminoglycans treatment increases IGF-I muscle levels and counteracts motor neuron death: A novel nonanticoagulant action.

The present study shows that sciatic nerve crush in 2-day-old rats causes extensor digitorum longus (EDL) muscle atrophy and motor neuron loss and that treatment with glycosaminoglycans (GAGs) promotes muscle reinnervation, motor neuron survival, and markedly increases insulin-like growth factor-I (IGF-I) content in the denervated muscles. EDL muscle denervation-induced atrophy in saline-treated rats is progressive and reaches the greatest extent at 42 days after birth, which correlates with reduced EDL weight growth. There is also a partial reinnervation as shown by the number of reinnervated EDL muscle fibers (65.4% of control) and by the poor restoration of the indirect isometric twitch tension (62% of control) that is further reduced under tetanic stimulation (34% of control). The number of surviving motor neurons that innervate EDL muscle drops from 55 +/- 3 to 29 +/- 8. In GAGs-treated 42-day-old rats, the effects of neonatal nerve lesioning on EDL muscle atrophy and denervation are successfully reversed, and the isometric twitch tension and the capacity to hold tetanic stimulation are restored to almost control levels. The number of surviving EDL motor neurons is also increased to 43 +/- 4. Treatment with GAGs selectively affects IGF-I content in denervated hindlimb muscles, which is augmented from 7.02 +/- 0.71 ng/mg tissue to 25.72 +/- 0.7 in the EDL and from 3.2 +/- 0.18 to a robust 211 +/- 9.6 in the soleus.

Systemic administration of insulin-like growth factor decreases motor neuron cell death and promotes muscle reinnervation.

Neonatal sciatic nerve axotomy causes motoneuron death and muscle denervation atrophy. The aim of the present study was to determine whether insulin-like growth factor-I (IGF-I) administration promotes muscle reinnervation and counteracts motor neuron loss after such an injury. Six weeks after sciatic nerve axotomy performed in 2-day-old pups, the number of motor neurons, as assessed by retrograde transport of horseradish peroxidase injected into the extensor digitorum longus (EDL) muscle, was reduced from 52 +/- 3 to 26 +/- 3. Subsequent administration of IGF-I at the doses of 0.02 mg/kg or 1 mg/kg increased the number of motor neurons to 35 +/- 2 and 37 +/- 5, respectively. The effect on motoneuron survival was accompanied by improved muscle fibre morphometry and restoration of indirect EDL muscle isometric twitch tension, which was about 80 % of control values for both doses of IGF-I compared with 60% observed with saline treatment. Reinnervated EDL muscle from saline-treated rats cannot hold tetanic tension, which is, however, achieved after IGF-I treatment at either dose. Thus, both high and low doses of IGF-I counteracted motoneuron death and improved muscle reinnervation following neonatal sciatic nerve axotomy. IGF-I at 5 microg/kg failed to increase muscle reinnervation.

Neuroprotection, neuroregeneration, and interaction with insulin-like growth factor-I: novel non-anticoagulant action of glycosaminoglycans.

We present recent developments in the area of glycosaminoglycans (GAGs) and their possible interaction with insulin-like growth factor-I (IGF-I). GAGs are constituents of proteoglycans, and the combination of a core protein and a specific GAG makes a unique proteoglycan with a precise developmental pattern and with the ability to bind growth factors. This process is apparently regulated by the moiety of the peripheral GAGs. The supplementation of GAGs promotes neuritogenesis in vitro and stimulates nerve regrowth and muscle reinnervation, an effect correlated with an increase in trophic factor mRNA expression. In the case of neonatal nerve lesion, there is in addition an enhanced motor neuron survival, accompanied by higher levels of IGF-I in plasma and denervated muscle. The neurotrophic and neuroregenerative effects of exogenous GAGs were also observed in motor neuron disease in the wobbler mouse.

Muscle reinnervation following neonatal nerve crush. Interactive effects of glycosaminoglycans and insulin-like growth factor-I.

This study shows that glycosaminoglycans promote muscle reinnervation following neonatal sciatic nerve injury. Such an effect appears to be mediated by insulin-like growth factor-1. The glycosaminoglycan moiety of proteoglycans is a constituent of the basal lamina active on nerve regeneration by means of the interaction with laminin and with several growth factors. We have previously shown that supplementation of glycosaminoglycans affects neuronal degeneration and regeneration. In this study we report that following neonatal lesion of the rat sciatic nerve glycosaminoglycan treatment promoted extensor digitorum longus muscle reinnervation with consequent improvement of muscle morphology. In saline-treated rats, reinnervation was only partial and there was a marked muscle fibre atrophy. In addition glycosaminoglycan treatment of lesioned rats increased insulin-like growth factor-I messenger RNA and protein in the reinnervated muscle, and insulin-like growth factor-I and insulin-like growth factor binding protein-3 plasma levels. Similarly, treatment of nerve lesioned rats with insulin-like growth factor-I promoted muscle reinnervation and prevention of muscle fibre atrophy, higher levels of insulin-like growth factor-I in the reinnervated muscle and of insulin-like growth factor-I and insulin-like growth factor binding proteins in plasma. These data suggest that glycosaminoglycans are potent stimulants of muscle reinnervation and that their effects may be mediated by increased levels of insulin-like growth factor-I.

Glycosaminoglycan supplementation promotes nerve regeneration and muscle reinnervation.

This study shows that treatment of rats with exogenous glycosaminoglycans stimulates peripheral nerve regeneration, increases the abundance of mRNAs for myelin proteins and promotes muscle reinnervation. After the sciatic nerve had been crushed the number of regenerating axons in the distal stump was markedly and highly significantly increased by glycosaminoglycan treatment throughout the experimental period. The increased number of axons was correlated with increased axon and fibre (axon+myelin) diameter. The abundance of mRNAs for P0 protein and myelin basic protein of regenerating nerves was also affected by treatment with glycosaminoglycans. The increase in mRNA was also observed in the contralateral unlesioned nerve. Such a phenomenon did not occur in saline-treated rats. Glycosaminoglycan treatment markedly increased the number of muscle fibres reinnervated and accelerated the restoration of muscle twitch tension elicited by nerve stimulation. The effect was particularly evident during the early stages (16 and 21 days after nerve crush) of muscle reinnervation.

Effects of low doses of glycosaminoglycans and insulin-like growth factor-I on motor neuron disease in wobbler mouse.

In this study we examined the effects of insulin-like growth factor-I (IGF-I) and of glycosaminoglycans (GAGs) on the progressive motor neuron disease in wobbler mice. After clinical diagnosis at age 3 weeks, mice received daily subcutaneous injections of IGF-I, or GAGs, or saline for 3 weeks. The histometric analysis revealed that biceps muscle fiber diameter was reduced in wobbler mice and that treatments with GAGs and IGF-I prevented such a drop. The number of atrophic small fibers was markedly reduced and that of the larger ones augmented. No effects on body growth and biceps muscle weight were observed. The combined AChE-silver staining revealed that both treatments promoted intramuscular axonal sprouting. The typical decline of grip strength in wobbler mice was also prevented. This study suggests that GAGs and IGF-I administrations can retard the onset of motor deficit, and reduce muscle atrophy in wobbler mice.

Endogenous mono-ADP-ribosylation in retina and peripheral nervous system. Effects of diabetes.

The extranuclear endogenous mono-ADP-ribosylation of proteins was monitored in cellular preparations of retina, superior cervical ganglion, dorsal root ganglia and peripheral nerve. At least 6 protein fractions are ADP-ribosylated in the crude extract fraction from retina control preparations, while in diabetic rats the number of retina labeled proteins and the extent of labeling are highly reduced. In the superior cervical ganglion labeling was present in 10 proteins, in diabetics it was greatly decreased. Treatment of diabetic rats with silybin, a flavonoid mono-ADP-ribosyltransferase inhibitor, did not affect hyperglycemia, but prevented the alteration of extent of protein ADP-ribosylation. These data suggest that proteins of retina and peripheral ganglia are excessively ADP-ribosylated in vivo. The effects of silybin treatment on excessive mono-ADP-ribosylation of proteins was associated with the prevention of reduction of substance P-like immunoreactivity levels, that is typical of diabetic neuropathy. In the membrane fraction of sciatic nerve Schwann cells, at least 9 proteins were ADP-ribosylated, diabetes caused a marked increase of labeling. A comparable increase involving the same proteins is triggered by chronic nerve injury and by corticosteroid treatment. Silybin treatment of diabetic rats prevented such an increase. We propose that the inhibition of excessive protein mono-ADP-ribosylation by silybin prevented the onset of diabetic neuropathy. While the effects on Schwann cells is likely indirect and secondary to the improvement of diabetic axonopathy.

Glycosaminoglycans in nerve injury: 1. Low doses of glycosaminoglycans promote neurite formation.

This study has shown that glycosaminoglycans added to the culture medium may affect neurite formation in SH-SY5Y neuroblastoma cells. The most effective glycosaminoglycans are heparin and COS 8, a preparation with low anticoagulant activity. Promotion of neuritogenesis was remarkable at concentrations as low as 10(-8) and 10(-10). When added at 10(-4) M both agents are inhibitory. Chondroitin-4 sulfate, dermatan sulfate, and heparan sulfate were also effective, the doses required were, however, as high as 10(-4) M for promoting and 10(-4) M for inhibiting neuritogenesis. Thereby low doses of glycosaminoglycans promote, while higher doses inhibit neurite formation. The effects were observed when neuritogenesis was promoted in neuroblastoma cultures either by deprivation of serum or by addition of retinoic acid, in the former case neuritogenesis occurred within 48 hr; in the latter, in 14 days. PC12 pheochromocytoma cells neuritogenesis was triggered by adding NGF to the culture medium. We have also observed that glycosaminoglycan supplementation to the culture medium lowered the quantity of NGF required to form neurites by PC12 cells. Glycosaminoglycans at the dose of 10(-8) M allow the formation of PC12 neurites even in presence of 1 ng/ml NGF, a dose that normally is ineffective.