Elevated intracellular Ca2+ functions downstream of mitodysfunction to induce Wallerian-like degeneration and necroptosis in organophosphorus-induced delayed neuropathy.

Neurotoxic organophosphorus compounds can induce a type of delayed neuropathy in humans and sensitive animals, known as organophosphorus-induced delayed neuropathy (OPIDN). OPIDN is characterized by axonal degeneration akin to Wallerian-like degeneration, which is thought to be caused by increased intra-axonal Ca2+ concentrations. This study was designed to investigate that deregulated cytosolic Ca2+ may function downstream of mitodysfunction in activating Wallerian-like degeneration and necroptosis in OPIDN. Adult hens were administrated a single dosage of 750 mg/kg tri-ortho-cresyl phosphate (TOCP), and then sacrificed at 1 day, 5 day, 10 day and 21 day post-exposure, respectively. Sciatic nerves and spinal cords were examined for pathological changes and proteins expression related to Wallerian-like degeneration and necroptosis. In vitro experiments using differentiated neuro-2a (N2a) cells were conducted to investigate the relationship among mitochondrial dysfunction, Ca2+ influx, axonal degeneration, and necroptosis. The cells were co-administered with the Ca2+-chelator BAPTA-AM, the TRPA1 channel inhibitor HC030031, the RIPK1 inhibitor Necrostatin-1, and the mitochondrial-targeted antioxidant MitoQ along with TOCP. Results demonstrated an increase in cytosolic calcium concentration and key proteins associated with Wallerian degeneration and necroptosis in both in vivo and in vitro models after TOCP exposure. Moreover, co-administration with BATPA-AM or HC030031 significantly attenuated the loss of NMNAT2 and STMN2 in N2a cells, as well as the upregulation of SARM1, RIPK1 and p-MLKL. In contrast, Necrostatin-1 treatment only inhibited the TOCP-induced elevation of p-MLKL. Notably, pharmacological protection of mitochondrial function with MitoQ effectively alleviated the increase in intracellular Ca2+ following TOCP and mitigated axonal degeneration and necroptosis in N2a cells, supporting mitochondrial dysfunction as an upstream event of the intracellular Ca2+ imbalance and neuronal damage in OPIDN. These findings suggest that mitochondrial dysfunction post-TOCP intoxication leads to an elevated intracellular Ca2+ concentration, which plays a pivotal role in the initiation and development of OPIDN through inducing SARM1-mediated axonal degeneration and activating the necroptotic signaling pathway.

Tri-ortho-cresyl phosphate induces axonal degeneration in chicken DRG neurons by the NAD+ pathway.

Wallerian degeneration (WD) is a well-known process by which degenerating axons and myelin are cleared after nerve injury. Although organophosphate-induced delayed neuropathy (OPIDN) is characterized by Wallerian-like degeneration of long axons in human and sensitive animals, the precise pathological mechanism remains unclear. In this study, we cultured embryonic chicken dorsal root ganglia (DRG) neurons, the model of OPIDN in vitro, to investigate the underlying mechanism of axon degeneration induced by tri-ortho-cresyl phosphate (TOCP), an OPIDN inducer. The results showed that TOCP exposure time- and concentration-dependently induced a serious degeneration and fragmentation of the axons from the DRG neurons. A collapse of mitochondrial membrane potential and a dramatic depletion of ATP levels were found in the DRG neurons after TOCP treatment. In addition, nicotinamide nucleotide adenylyl transferase 2 (NMNAT2) expression and nicotinamide adenine dinucleotide (NAD+) level was also found to be decreased in the DRG neurons exposed to TOCP. However, the TOCP-induced Wallerian degeneration in the DRG neurons could be inhibited by ATP supplementation. And exogenous NAD+ or NAD+ processor nicotinamide riboside can rescue TOCP-induced ATP deficiency and prevent TOCP-induced axon degeneration of the DRG neurons. These findings may shed light on the pathophysiological mechanism of TOCP-induced axonal damages, and implicate the potential application of NAD+ to treat OPIDN.

SARM1-mediated wallerian degeneration: A possible mechanism underlying organophosphorus-induced delayed neuropathy.

Some organophosphorus compounds (OPs) can cause a type of delayed neurotoxicity in human being, which is known as organophosphorus-induced delayed neuropathy (OPIDN). Signs and symptoms of the patients include tingling and sensory loss of the hands and feet, followed by progressive muscle weakness in the lower and upper limbs, and ataxia. Pathologically, OPIDN are characterized by distal sensorimotor axonopathy due to the distal axonal degeneration of nerve tracts located in central and peripheral nervous systems. The morphological pattern of the distal axonopathy is similar to Wallerian degeneration that occurs after nerve injury in vitro. It is generally acknowledged that inhibition and subsequent aging of neuropathy target esterase (NTE) is required for the occurrence of OPIDN. However, the underlying mechanisms through which NTE triggers axonal degeneration in OPIDN is still largely unclear. Recently, sterile alpha and toll/interleukin receptor motif-containing protein 1（SARM1) has been identified as a key player in Wallerian degeneration. In physical and chemical transection of axons, SARM1 was found to promotes axon degeneration by hydrolyzing NAD+. By contrast, SARM1 deficiency could prevent neuron degeneration in response to a wide range of insults. Furthermore, SARM1 can also translocate to mitochondria and cause mitochondrial damage, thus triggering axon degeneration and neuron death. These findings suggested the existence of a pathway in axonal degeneration that might be targeted therapeutically. Here, we hypothesize that SARM1 activation after NTE inhibition and aging might be an etiological factor in OPIDN that regulates Wallerian-like degeneration. Analysing SARM1 mediated NAD degeneration pathway and its upstream activators in OPIDN could contribute to the development of novel therapies to treat OPIDN.

Cisplatin induced neurotoxicity is mediated by Sarm1 and calpain activation.

Cisplatin is a commonly used chemotherapy agent with significant dose-limiting neurotoxicity resulting in peripheral neuropathy. Although it is postulated that formation of DNA-platinum adducts is responsible for both its cytotoxicity in cancer cells and side effects in neurons, downstream mechanisms that lead to distal axonal degeneration are unknown. Here we show that activation of calpains is required for both neurotoxicity and formation of DNA-platinum adduct formation in neurons but not in cancer cells. Furthermore, we show that neurotoxicity of cisplatin requires activation of Sarm1, a key regulator of Wallerian degeneration, as mice lacking the Sarm1 gene do not develop peripheral neuropathy as evaluated by both behavioral or pathological measures. These findings indicate that Sarm1 and/or specific calpain inhibitors could be developed to prevent cisplatin induced peripheral neuropathy.

Histological and ultrastructural study of AflatoxinB1 induced neurotoxicity in Sciatic nerve of adult male Albino rats.

Aflatoxins are mycotoxins produced by Aspergillus spp. which is a common contaminant of food items such as corn, spices, rice, nuts, and flour. Aflatoxin contamination of foods is a worldwide problem. Chronic aflatoxin exposure is found to be associated with Sciatic nerve damage. In vivo study was carried out to evaluate the toxic effect of aflatoxin B1 (AFB1) on the Sciatic nerve. Twenty-one adult male rats were included and divided equally into 3 groups (7 rats each): Group I (control group), group II (olive oil group) and group III: (AflatoxinB1 group). The rats received AFB1 (250 µg/kg B.W./day) orally by gastric tube 5 days/week for 4 weeks. Sciatic nerve specimens were prepared, and semi-thin sections were stained with Toluidine blue, examined by light microscope and photographed. Ultrathin sections (50-80 nm) from selected areas of the trimmed blocks were made, examined and photographed by transmission electron microscopy (JEOL-JSM-1011) in King Saud University Electron Microscopy Unit. The findings indicate that the administration of AFB1 to rats' results in degeneration in the sciatic nerve in the form of Wallerian degeneration in the myelin sheath. Macrophages appear to engulf the degenerated myelin and neutrophils.

The mitochondrial Nod-like receptor NLRX1 modifies apoptosis through SARM1.

NLRX1, the mitochondrial NOD-like receptor (NLR), modulates apoptosis in response to both intrinsic and extrinsic cues. Insights into the mechanism of how NLRX1 influences apoptosis remain to be determined. Here, we demonstrate that NLRX1 associates with SARM1, a protein with a toll/interleukin-1 receptor (TIR)-containing domain also found in adaptor proteins downstream of toll-like receptors, such as MyD88. While a direct role of SARM1 in innate immunity is unclear, the protein plays essential roles in Wallerian degeneration (WD), a type of neuronal catabolism occurring following axonal severing or damage. In non-neuronal cells, we found that endogenous SARM1 was equally distributed in the cytosol and the mitochondrial matrix, where association with NLRX1 occurred. In these cells, the apoptotic role of NLRX1 was fully dependent on SARM1, indicating that SARM1 was downstream of NLRX1 in apoptosis regulation. In primary murine neurons, however, Wallerian degeneration induced by vinblastine or NGF deprivation occurred in SARM1- yet NLRX1-independent manner, suggesting that WD requires the cytosolic pool of SARM1 or that NLRX1 levels in neurons are too low to contribute to WD regulation. Together, these results shed new light into the mechanisms through which NLRX1 controls apoptosis and provides evidence of a new link between NLR and TIR-containing proteins.

Secondary Degeneration of Auditory Neurons after Topical Aminoglycoside Administration in a Gerbil Model.

Hair cells in the cochlea can be damaged by various causes. Damaged hair cells can lead to additional destruction of parts of the auditory afferent pathway sequentially, which is called secondary degeneration. Recently, researches regarding cochlear implants have been actively carried out for clinical purposes; secondary degeneration in animals is a much more practical model for identifying the prognosis of cochlear implants. However, an appropriate model for this research is not established yet. Thus, we developed a secondary degeneration model using an ototoxic drug. 35 gerbils were separated into four different groups and kanamycin was applied via various approaches. ABR was measured several times after drug administration. SGCs were also counted to identify any secondary degeneration. The results showed that outer and inner HCs were damaged in all kanamycin-treated groups. Twelve weeks after kanamycin treatment, the round window membrane injection group showed severe subject differences in hair cells and SGC damage, whereas the gelfoam group showed consistent and severe damage in hair cells and SGCs. In this study, we successfully induced secondary degeneration in hair cells in a gerbil model. This model can be used for various purposes in the hearing research area either for treatment or for preservation.

Mechanisms for consideration for intervention in the development of organophosphorus-induced delayed neuropathy.

Organophosphorus-induced delayed neuropathy (OPIDN) is a neurodegenerative disorder characterised by ataxia progressing to paralysis with concomitant central and peripheral distal axonopathy. Symptoms of OPIDN in people include tingling of the hands and feet. This tingling is followed by sensory loss, progressive muscle weakness and flaccidity of the distal skeletal muscles of the lower and upper extremities and ataxia, which appear about 8-14 days after exposure. Some organophosphorus compounds (OPs) that are still used in worldwide agriculture have potential to induce OPIDN, including methamidophos, trichlorfon, dichlorvos and chorpyrifos. This review summarizes experimental attempts to prevent and/or treat OPIDN and the different mechanisms involved in each approach. The initial mechanism associated with development of OPIDN is phosphorylation and inhibition of neuropathy target esterase (NTE). The phosphorylated enzyme undergoes a second reaction known as "aging" that results in the loss of one of the "R" groups bound to the phosphorus of the OP. A second mechanism involved in OPIDN is an imbalance in calcium homeostasis. This can lead to the activation of calcium-activated neutral protease and increases in calcium/calmodulin-dependent protein kinases. These events contribute to aberrant phosphorylation of cytoskeletal proteins and protein digestion in the terminal axon that can proceed similarly to Wallerian-type degeneration. Several experimental studies demonstrated alleviation of the signs and symptoms of OPIDN by restoring calcium balance. Other studies have used preadministration of NTE inhibitors, such as carbamates, thiocarbamates, sulfonyl fluorides and phosphinate to prevent OPIDN. Progress is being made, but there is yet no single specific treatment available for use in clinical practice to prevent or alleviate the severe effects of OPIDN.

The mechanism of axonal degeneration after perikaryal excitotoxic injury to the retina.

We investigated the mechanism of secondary axonal degeneration after perikaryal excitotoxic injury to retinal ganglion cells (RGCs) by comparing pathological responses in wild-type rats and Wld(s) rats, which display delayed Wallerian degeneration. After perikaryal excitotoxic RGC injury, both types of rats exhibited a spatio-temporal pattern of axonal cytoskeletal degeneration consistent with Wallerian degeneration, which was delayed by up to 4 weeks in Wld(s) rats. Furthermore, RGC somal loss was greater in Wld(s) rats. Microglial response in the anterior visual pathway to injury was attenuated in the Wld(s) rats with lymphocytic infiltration that was relatively reduced; however, immunostaining for major histocompatibility complex class II antigens (OX6) was more pronounced in Wld(s) rats. These data indicate that perikaryal excitotoxic RGC injury causes a secondary Wallerian axonal degeneration, and support the notion of a labile, soma-derived axonal survival factor.

Nrf2 deficiency potentiates methamphetamine-induced dopaminergic axonal damage and gliosis in the striatum.

Oxidative stress that correlates with damage to nigrostriatal dopaminergic neurons and reactive gliosis in the basal ganglia is a hallmark of methamphetamine (METH) toxicity. In this study, we analyzed the protective role of the transcription factor Nrf2 (nuclear factor-erythroid 2-related factor 2), a master regulator of redox homeostasis, in METH-induced neurotoxicity. We found that Nrf2 deficiency exacerbated METH-induced damage to dopamine neurons, shown by an increase in loss of tyrosine hydroxylase (TH)- and dopamine transporter (DAT)-containing fibers in striatum. Consistent with these effects, Nrf2 deficiency potentiated glial activation, indicated by increased striatal expression of markers for microglia (Mac-1 and Iba-1) and astroglia (GFAP) one day after METH administration. At the same time, Nrf2 inactivation dramatically potentiated the increase in TNFα mRNA and IL-15 protein expression in GFAP+ cells in the striatum. In sharp contrast to the potentiation of striatal damage, Nrf2 deficiency did not affect METH-induced dopaminergic neuron death or expression of glial markers or proinflammatory molecules in the substantia nigra. This study uncovers a new role for Nrf2 in protection against METH-induced inflammatory and oxidative stress and striatal degeneration.

Kinesin-1 and degenerative changes in optic nerve axons in NMDA-induced neurotoxicity.

We examined the histologic findings of optic nerve axons and changes in kinesin-1, which is involved in axonal flow, in N-methyl-d-aspartate (NMDA)-induced neurotoxicity in rats. Substantial degenerative changes visualized as black profiles and pale large axons were observed 72h after NMDA injection, but those degenerative changes were not apparent in axons 12 and 24h after injection. Morphometric analysis showed a significant, approximately 40% reduction in the number of axons 72h after NMDA injection. Immunohistochemical study showed that there was a recognizable loss of neurofilament-immunopositive dots, but myelin basic protein immunostaining was unchanged 72h after NMDA injection. Western blot analysis showed early elevation of kinesin-1 (KIF5B) protein levels in the retina 24 and 72h after NMDA injection. Conversely, significant decreases in KIF5B protein levels in the optic nerve were seen during the same time course. Immunohistochemical study also showed that there was a reduction in KIF5B immunoreactivity in axons, but neurofilament immunostaining was unchanged 24h after NMDA injection. These findings suggest that the intravitreal injection of NMDA causes neurofilament loss without myelin alteration in the early stage. The depletion of kinesin-1 precedes axonal degeneration of the optic nerve in NMDA-induced neurotoxicity.

Wallerian-like axonal degeneration in the optic nerve after excitotoxic retinal insult: an ultrastructural study.

BACKGROUND: Excitotoxicity is involved in the pathogenesis of a number neurodegenerative diseases, and axonopathy is an early feature in several of these disorders. In models of excitotoxicity-associated neurological disease, an excitotoxin delivered to the central nervous system (CNS), could trigger neuronal death not only in the somatodendritic region, but also in the axonal region, via oligodendrocyte N-methyl-D-aspartate (NMDA) receptors. The retina and optic nerve, as approachable regions of the brain, provide a unique anatomical substrate to investigate the "downstream" effect of isolated excitotoxic perikaryal injury on central nervous system (CNS) axons, potentially providing information about the pathogenesis of the axonopathy in clinical neurological disorders.Herein, we provide ultrastructural information about the retinal ganglion cell (RGC) somata and their axons, both unmyelinated and myelinated, after NMDA-induced retinal injury. Male Sprague-Dawley rats were killed at 0 h, 24 h, 72 h and 7 days after injecting 20 nM NMDA into the vitreous chamber of the left eye (n = 8 in each group). Saline-injected right eyes served as controls. After perfusion fixation, dissection, resin-embedding and staining, ultrathin sections of eyes and proximal (intraorbital) and distal (intracranial) optic nerve segments were evaluated by transmission electron tomography (TEM). RESULTS: TEM demonstrated features of necrosis in RGCs: mitochondrial and endoplasmic reticulum swelling, disintegration of polyribosomes, rupture of membranous organelle and formation of myelin bodies. Ultrastructural damage in the optic nerve mimicked the changes of Wallerian degeneration; early nodal/paranodal disturbances were followed by the appearance of three major morphological variants: dark degeneration, watery degeneration and demyelination. CONCLUSION: NMDA-induced excitotoxic retinal injury causes mainly necrotic RGC somal death with Wallerian-like degeneration of the optic nerve. Since axonal degeneration associated with perikaryal excitotoxic injury is an active, regulated process, it may be amenable to therapeutic intervention.

Estrogen reduces BDNF level, but maintains dopaminergic cell density in the striatum of MPTP mouse model.

Degeneration of dopaminergic (DA) axons in the striatum triggers upregulation of striatal trophic activity and striatal DA neuronal number in animal models of Parkinson's disease (PD). The present study investigated the effects of 17beta-estradiol (E2) on brain-derived neurotrophic factor (BDNF) expression and the density of DA neurons in the striatum of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) mouse model in correlation with nigrostriatal DA innervation. Adult male C57Bl/6 mice were treated with E2 or vehicle for 11 days. Following 5 days of E2 or vehicle pretreatment, animals were injected with MPTP on day 6. On day 11, all mice were sacrificed, and the striatum were collected and processed for tyrosine hydroxylase (TH) and BDNF immunohistochemistry. Striatal TH-immunoreactive (IR) neurons were counted. Extent of DA innervation and BDNF expression in the striatum were assessed by measuring optical density of TH and BDNF immunoreactivity, respectively. Pretreatment with E2 partially prevented DA denervation and decreased striatal BDNF upregulation triggered by MPTP, but maintained the density of striatal TH-IR neurons to that observed in MPTP group. These findings suggest that estrogen protection of nigrostriatal DA axons against MPTP as well as preservation of the striatal TH-IR cell density in MPTP/E2 mice may be not mediated by BDNF.

Ropivacaine-induced peripheral nerve injection injury in the rodent model.

BACKGROUND: Intraneural administration of local anesthetics has been associated with nerve damage. We undertook the present study to investigate histological changes induced by ropivacaine injection into rat sciatic nerve. METHODS: Fifty-four adult male Lewis rats were randomly distributed into 9 groups, 6 animals per group. Fifty microliters of normal saline, 10% phenol, or 0.75% ropivacaine were administered by intrafascicular injection, extrafascicular injection, or extraneural (topical) placement. At 2 weeks, animals were killed and the sciatic nerve at the injection site was evaluated with light microscopy, quantitative histomorphometry, and electron microscopy. RESULTS: On cross-sectional evaluation, extrafascicular ropivacaine injection and extraneural placement of ropivacaine were both associated with damage to the perineurium, with focal demyelination surrounded by edematous endoneurium. Intrafascicular injection of ropivacaine resulted in a wedge-shaped region of demyelination and focal axonal loss with some regeneration, bordered by a region of normally myelinated axons in a background of edematous endoneurium. Extrafascicular injection resulted in more significant damage than extraneural placement of ropivacaine, but less than intrafascicular injection as shown with quantitative histomorphometry. Quantitatively, ropivacaine-injured specimens had significantly lower nerve density than saline-injured specimens. Wallerian degeneration and perineural edema were also demonstrated qualitatively with electron microscopy. CONCLUSIONS: This study demonstrates that, in the rat model, ropivacaine is associated with marked histological abnormality, including edema of the perineurium and axonal destruction with wallerian degeneration, when injected into or extraneurally placed onto a nerve. Extrafascicular injection and extraneural placement were associated with similar, although milder, histological damage than intrafascicular injection. Further work is needed to investigate the functional implications, if any, of the histological abnormalities observed in this study.

Tau hyperphosphorylation and axonal damage induced by N,N-diethyldithiocarbamate (DEDTC) treatment along late postnatal development is followed by a rescue during adulthood.

Axonal degeneration has been described as the pathological hallmark of peripheral neuropathies induced by DEDTC. In addition, axonal damage has also been observed in the brain of mice treated daily with DEDTC along postnatal development, though with this experimental model there was observed to be axonal recovery after treatment, during the adulthood. To focus on this axonal dynamic activity, damage-recovery, a key axonal protein, the microtubule associated protein tau, was analyzed in this DEDTC model. Tau is a phosphoprotein and its dynamic site-specific phosphorylation is essential for its proper function; in fact, high levels are correlated with cell dysfunction. Furthermore, the levels of tau phosphorylation are associated with dynamic microtubules during periods of high plasticity. Thus, phosphorylated tau at two sites of phosphorylation, Ser(199) and Ser(396), were evaluated during the second week of postnatal development and throughout adulthood. The results obtained by Western blot made it evident that the levels of p-tau Ser(199) and p-tau Ser(396) were higher in treated mice than in controls. Interestingly, by immunohistochemistry there was shown to be an increase in p-tau-immunolabeling in neuronal soma together with axonal tract alterations in treated animals with respect to controls, and the analyses of GSK3 beta and cdk5 revealed an increase in its activity in DEDTC-treated animals. Nevertheless, in the adult a general decline in p-tau was observed together with a rescue of axonal tract. All these data support the idea that the axonal damage induced by DEDTC treatment along postnatal development is followed by an axonal rescue during adulthood.

Involvement of lysosomes in the early stages of axon degeneration.

Axon degeneration is a common hallmark of many neurodegenerative diseases, and the underlying mechanism remains largely unknown. Lysosomes are involved in some neurodegenerative diseases, including Alzheimer's disease and Parkinson's disease. Whether lysosomes are involved in axon degeneration is yet to be elucidated. In this study, we found only about 10% lysosomes remained in axons of cultured superior cervical ganglia (SCGs) after transection for 4h when stained with LysoTracker. Furthermore, we found that lysosomal disruption occurred earlier than morphological changes and loss of mitochondrial membrane potential. In addition, the well-known axon-protective protein Wld(S) delayed injury-induced axon degeneration from both morphological changes and lysosomal disruption. Lysosomal inhibitors including chloroquine and ammonium chloride induced axon degeneration in cultured SCGs, and Wld(S) also slowed down the axon degeneration induced by lysosomal inhibitors. All these data suggest that lysosomal disruption is an early marker of axon degeneration, and inhibition of lysosome induces axon degeneration in a Wld(S)-protectable way. Thus, maintenance of normal lysosomal function might be an important approach to delay axon degeneration in neurodegenerative diseases.

Distinct muscarinic acetylcholine receptor subtypes contribute to stability and growth, but not compensatory plasticity, of neuromuscular synapses.

Muscarinic acetylcholine receptors (mAChRs) modulate synaptic function, but whether they influence synaptic structure remains unknown. At neuromuscular junctions (NMJs), mAChRs have been implicated in compensatory sprouting of axon terminals in paralyzed or denervated muscles. Here we used pharmacological and genetic inhibition and localization studies of mAChR subtypes at mouse NMJs to demonstrate their roles in synaptic stability and growth but not in compensatory sprouting. M(2) mAChRs were present solely in motor neurons, whereas M(1), M(3), and M(5) mAChRs were associated with Schwann cells and/or muscle fibers. Blockade of all five mAChR subtypes with atropine evoked pronounced effects, including terminal sprouting, terminal withdrawal, and muscle fiber atrophy. In contrast, methoctramine, an M(2/4)-preferring antagonist, induced terminal sprouting and terminal withdrawal, but no muscle fiber atrophy. Consistent with this observation, M(2)(-/-) but no other mAChR mutant mice exhibited spontaneous sprouting accompanied by extensive loss of parental terminal arbors. Terminal sprouting, however, seemed not to be the causative defect because partial loss of terminal branches was common even in the M(2)(-/-) NMJs without sprouting. Moreover, compensatory sprouting after paralysis or partial denervation was normal in mice deficient in M(2) or other mAChR subtypes. We also found that many NMJs of M(5)(-/-) mice were exceptionally small and reduced in proportion to the size of parental muscle fibers. These findings show that axon terminals are unstable without M(2) and that muscle fiber growth is defective without M(5). Subtype-specific muscarinic signaling provides a novel means for coordinating activity-dependent development and maintenance of the tripartite synapse.

Melatonin attenuates methamphetamine-induced reduction of tyrosine hydroxylase, synaptophysin and growth-associated protein-43 levels in the neonatal rat brain.

Methamphetamine (METH) is a most commonly abused drug which damages nerve terminals by causing formation of reactive oxygen species (ROS), apoptosis, and finally neuronal damage. Fetal exposure to neurotoxic METH causes significant behavioral effects. The developing fetus is substantially deficient in most antioxidative enzymes, and may therefore be at high risk from both endogenous and drug-enhanced oxidative stress. Little is known about the effects of METH on vesicular proteins such as synaptophysin and growth-associated protein 43 (GAP-43) in the immature brain. The present study attempted to investigate the effects of METH-induced neurotoxicity in the dopaminergic system of the neonatal rat brain. Neonatal rats were subcutaneously exposed to 5-10mg/kg METH daily from postnatal day 4-10 for 7 consecutive days. The results showed that tyrosine hydroxylase enzyme levels were significantly decreased in the dorsal striatum, prefrontal cortex, nucleus accumbens and substantia nigra, synaptophysin levels decreased in the striatum and prefrontal cortex and growth-associated protein-43 (GAP-43) levels significantly decreased in the nucleus accumbens of neonatal rats. Pretreatment with 2mg/kg melatonin 30 min prior to METH administration prevented METH-induced reduction in tyrosine hydroxylase, synaptophysin and growth-associated protein-43 protein levels in different brain regions. These results suggest that melatonin provides a protective effect against METH-induced nerve terminal degeneration in the immature rat brain probably via its antioxidant properties.

Lower diffusion in white matter of children with prenatal methamphetamine exposure.

BACKGROUND: Methamphetamine use is a common problem among women of childbearing age, leading to an increasing number of children with prenatal methamphetamine exposure. Whether microstructural brain changes associated with prenatal methamphetamine exposure can be detected with diffusion tensor imaging (DTI) is unknown. METHOD: Twelve-direction DTI was performed in 29 methamphetamine-exposed and 37 unexposed children ages 3-4 years on a 3-T MRI scanner. Fractional anisotropy (FA) and apparent diffusion coefficient (ADC) were determined in the corpus callosum (genu and splenium) and bilaterally in the frontal and parietal white matter (WM), basal ganglia (caudate, putamen, globus pallidus), and thalamus. RESULTS: Children with prenatal methamphetamine exposure had lower ADC in the frontal (right: -2.1%, p = 0.04; left: -2.0%, p = 0.09) and parietal WM (right: -3.9%, p = 0.002; left: -3.3%, p = 0.02) compared to unexposed children. The methamphetamine-exposed children also showed a trend for higher FA in the left frontal WM (+4.9%, p = 0.06) compared to the unexposed children. CONCLUSION: Since less myelination and higher dendritic or spine density have been reported in animals exposed to methamphetamine, lower diffusion in our children may reflect more compact axons or greater dendritic or spine density associated with prenatal methamphetamine exposure. These findings suggest alterations in white matter maturation in these children exposed to methamphetamine in utero.

VEGF and angiogenesis in acute and chronic MOG((35-55)) peptide induced EAE.

An increased expression of vascular endothelial growth factor (VEGF) is associated with demyelinated lesions in both multiple sclerosis (MS) and its model (EAE), implicating changes in vasculature as a potential component of CNS plaque formation. The purpose of this study was to investigate the vascular changes in acute and chronic EAE in C57BL/6 mice induced with myelin oligodendrocyte glycoprotein (MOG ((35-55))) peptide. We investigated the functional contribution of VEGF to acute and chronic EAE by treating immunized mice with SU5416 (Semaxinib), a potent and selective inhibitor of VEGF receptor 2 (VEGFR2). Animals received seven daily injections of SU5416 (50 mg/kg) or vehicle beginning on the day after disease onset (acute study) or on day 45 post-immunization (chronic study). Spinal cord sections were collected on the day of sacrifice. Modulation of angiogenic gene expression was determined using RNA isolated from 4 acute and 4 non-immunized controls. MOG peptide induction produced extensive demyelination, immune cell infiltration, tissue laminin deposits, and axonal loss in lesions. VEGF expression was extensively increased in the acute mice, which correlated positively with clinical score. In the acute study, SU5416 treatment produced a significant clinical improvement versus vehicle controls (p<0.001), with less demyelination (-37%) and cellular infiltration (-23%) in the spinal cord (p<0.05). Treated animals also had significantly fewer blood vessels per section than controls (56.1+/-6.1 v. 81.6+/-11.5, p<0.05), and significantly reduced laminin abnormalities (28.9% of lesion area v. 46.8%, p<0.05). There was no improvement in clinical score or tissue pathology, and no difference in vessel number or lesion laminin expression, when SU5416 was administered during the chronic disease (all p>0.05). In the acute study only, VEGF staining correlated with demyelination and the extent of cellular infiltration in both control (r=0.723, r=0.665) and treated (r=0.681, r=0.487) animals (all p<0.05). Laminin staining in lesion areas was strongly correlated with tissue pathology for all animals in both the acute and chronic study (all p<0.001). Vascular alterations in MOG peptide-induced EAE in the mouse are accompanied by increased lesion-specific levels of VEGF, extensive laminin deposits in the tissue and altered transcription of numerous angiogenic factors. In the microarray studies, acute mice showed a significant increase in several angiogenic RNA transcripts, six of which were verified by RT-PCR, alanyl aminopeptidase, caspase 8, Hif1a, MMP-19, plasminogen activator inhibitor, and thrombospondin1.