Degeneration Of Neuronal Cell Bodies Research Articles

Autophagy in axonal degeneration in glaucomatous optic neuropathy.

The role of autophagy in retinal ganglion cell (RGC) death is still controversial. Several studies focused on RGC body death, although the axonal degeneration pathway in the optic nerve has not been well documented in spite of evidence that the mechanisms of degeneration of neuronal cell bodies and their axons differ. Axonal degeneration of RGCs is a hallmark of glaucoma, and a pattern of localized retinal nerve fiber layer defects in glaucoma patients indicates that axonal degeneration may precede RGC body death in this condition. As models of preceding axonal degeneration, both the tumor necrosis factor (TNF) injection model and hypertensive glaucoma model may be useful in understanding the mechanism of axonal degeneration of RGCs, and the concept of axonal protection can be an attractive approach to the prevention of neurodegenerative optic nerve disease. Since mitochondria play crucial roles in glaucomatous optic neuropathy and can themselves serve as a part of the autophagosome, it seems that mitochondrial function may alter autophagy machinery. Like other neurodegenerative diseases, optic nerve degeneration may exhibit autophagic flux impairment resulting from elevated intraocular pressure, TNF, traumatic injury, ischemia, oxidative stress, and aging. As a model of aging, we used senescence-accelerated mice to provide new insights. In this review, we attempt to describe the relationship between autophagy and recently reported noteworthy factors including Nmnat, ROCK, and SIRT1 in the degeneration of RGCs and their axons and propose possible mechanisms of axonal protection via modulation of autophagy machinery.

Synapto-Protective Drugs Evaluation in Reconstructed Neuronal Network

Chronic neurodegenerative syndromes such as Alzheimer’s and Parkinson’s diseases, or acute syndromes such as ischemic stroke or traumatic brain injuries are characterized by early synaptic collapse which precedes axonal and neuronal cell body degeneration and promotes early cognitive impairment in patients. Until now, neuroprotective strategies have failed to impede the progression of neurodegenerative syndromes. Drugs preventing the loss of cell body do not prevent the cognitive decline, probably because they lack synapto-protective effects. The absence of physiologically realistic neuronal network models which can be easily handled has hindered the development of synapto-protective drugs suitable for therapies. Here we describe a new microfluidic platform which makes it possible to study the consequences of axonal trauma of reconstructed oriented mouse neuronal networks. Each neuronal population and sub-compartment can be chemically addressed individually. The somatic, mid axon, presynaptic and postsynaptic effects of local pathological stresses or putative protective molecules can thus be evaluated with the help of this versatile “brain on chip” platform. We show that presynaptic loss is the earliest event observed following axotomy of cortical fibers, before any sign of axonal fragmentation or post-synaptic spine alteration. This platform can be used to screen and evaluate the synapto-protective potential of several drugs. For instance, NAD+ and the Rho-kinase inhibitor Y27632 can efficiently prevent synaptic disconnection, whereas the broad-spectrum caspase inhibitor zVAD-fmk and the stilbenoid resveratrol do not prevent presynaptic degeneration. Hence, this platform is a promising tool for fundamental research in the field of developmental and neurodegenerative neurosciences, and also offers the opportunity to set up pharmacological screening of axon-protective and synapto-protective drugs.

Midline brain injury in the immature rat induces sustained cognitive deficits, bihemispheric axonal injury and neurodegeneration

Infants and children less than 4 years old suffer chronic cognitive deficits following mild, moderate or severe diffuse traumatic brain injury (TBI). It has been suggested that the underlying neuropathologic basis for behavioral deficits following severe TBI is acute brain swelling, subarachnoid hemorrhage and axonal injury. To better understand mechanisms of cognitive dysfunction in mild-moderate TBI, a closed head injury model of midline TBI in the immature rat was developed. Following an impact over the midline suture of the intact skull, 17-day-old rats exhibited short apnea times (3–15 s), did not require ventilatory support and suffered no mortality, suggestive of mild TBI. Compared to un-injured rats, brain-injured rats exhibited significant learning deficits over the first week post-injury ( p < 0.0005), and, significant learning ( p < 0.005) and memory deficits ( p < 0.05) in the third post-injury week. Between 6 and 72 h, blood–brain barrier breakdown, extensive traumatic axonal injury in the subcortical white matter and thalamus, and focal areas of neurodegeneration in the cortex and hippocampus were observed in both hemispheres of the injured brain. At 8 to 18 days post-injury, reactive astrocytosis in the cortex, axonal degeneration in the subcortical white matter tracts, and degeneration of neuronal cell bodies and processes in the thalamus of both hemispheres were observed; however, cortical volumes were not different between un-injured and injured rat brains. These data suggest that diffuse TBI in the immature rat can lead to ongoing degeneration of both cell soma and axonal compartments of neurons, which may contribute, in part, to the observed sustained cognitive deficits.

Degeneration of neuronal cell bodies following axonal injury in WldS mice

The phenotype of Wld(S) ("slow Wallerian degeneration") mice demonstrates prolonged survival of injured axons. However, whether the Wld(S) mutation delays degeneration of the neuronal cell body following axonal injury is unclear. We used a retrograde model of axonal transport failure in Wld(S) mice to test whether the mutant Wld(S) protein has any beneficial effect on the neuronal cell body. Retrograde axonal transport was physically blocked by optic nerve crush and confirmed by the absence of Fluoro-Gold labeling in wild-type and in Wld(S) mice. After this axonal injury, there was marked protection of axonal degeneration in the Wld(S) phenotype, as confirmed by immunohistochemistry and electron microscopy. However, the Wld(S) protein, localized in the nucleus of retinal ganglion cells, did not prevent or delay degeneration of the retinal ganglion cell body, confirmed by TUNEL staining and Fluoro-Gold labeling. These results imply that, after axonal injury, Wallerian degeneration of axons and degeneration of the neuronal cell body have different mechanisms, which are autonomous and independent of each other. Although the Wld(S) phenotype can be used to demonstrate stable enucleate axons, the mutation is unlikely to protect neurons in neurodegenerative diseases in which there is failure of retrograde transport.

Changes in localization of synaptophysin following fluid percussion injury in the rat brain

Traumatic brain injuries damage neurons and cause progressing dysfunctions of the brain. Synaptophysin (SYP), a major integral transmembrane protein of synaptic vesicles, provides a molecular marker for the synapse and serves as a functional marker of the brain. This study examined magnitude-dependent changes of SYP in the rat brain 2 days following low, moderate or high fluid percussion injuries and investigated time-dependent changes of SYP in the rat brain with moderate fluid percussion injury 2, 15 and 30 days after trauma using immunohistochemistry and Western blotting. SYP immunoreactivity increased in the lateral cortex and in the subcortical white matter, with increasing magnitude of injury and time after trauma. Increased SYP immunoreactivity was accompanied with degeneration of neuronal cell bodies, their processes and terminals as well as glial cell proliferations. Amounts of SYP measured by Western blotting remained unchanged in brains with moderate fluid percussion within 30 days after trauma. These findings indicate that trauma accumulates SYP at injured sites of neurons without changing SYP contents and that increased SYP immunoreactivity in the cerebral cortex following traumatic injury reflects an inhibition of synaptic vesicle transportation and dysfunction of synapses, thus providing a histological substrate for brain dysfunctions.

Neurodegeneration in Niemann-Pick type C disease mice.

Niemann-Pick disease type C (NP-C) is an inherited neurodegenerative disorder associated with intracellular cholesterol and glycolipid trafficking defects. Two separate genes, NPC1 and NPC2, have been linked to NP-C. NPC1 encodes a polytopic membrane-bound protein with a putative sterol-sensing domain. NPC2 has been recently identified as epididymal secretory glycoprotein 1. The NPC1 protein functions in the vesicular redistribution of endocytosed lysosomal cargo, but how its inactivation leads to neurodegeneration is not known. The neurological symptoms of NP-C typically appear after a period of normal early development and reflect progressive degeneration of widespread brain regions. Here we have delineated the pattern of neurodegeneration in NP-C mice, whose genetic defect has been shown to be an inactivating mutation of the mouse NPC1 gene. The results reveal a spatially and temporally specific pattern of degeneration of nerve fibers followed by degeneration of neuronal cell bodies beginning as early as day 9 and continuing throughout life. We have recently showed that in the primate brain, the NPC1 protein is localized predominantly within perisynaptic astrocytic processes. The present observations suggest that a functional disturbance in NPC1 could disrupt vesicular transport of cholesterol, glycolipids and possibly other endocytic cargo in glia, which is critical for maintaining the integrity of neurons.

Does senile impairment of cholinergic system in rats concern only disturbances in cholinergic phenotype or the progressive degeneration of neuronal cell bodies?

The trophic effect of continuous intraventricular infusion of nerve growth factor (NGF) on morphology of the basal forebrain (BF) cholinergic neurons was tested in 4- and 28-month-old male Wistar rats. All studies were conducted using behaviorally uncharacterized animals from the same breeding colony. Immunohistochemical procedure for choline acetyltransferase (ChAT) and p75NTR receptor has been applied to identify cholinergic cells in the structures of basal forebrain (BF). Using a quantitative image analyzer, morphometric and densitometric parameters of ChAT- and p75NTR-positive cells were measured immediately after cessation of NGF infusion. In 28-month-old non-treated rats the number of intensively ChAT-positive cells in all forebrain structures was reduced by 50-70% as compared with young animals. The remaining ChAT-positive cells appeared shrunken and the neuropil staining was NTR markedly reduced. In contrast, the same neurons when stained for p75 were numerous and distinctly visible with perfect morphology. Analysis of Nissl stained sections also showed that 28-month-old rats did not display significant losses of neuronal cell bodies. NGF restored the number of intensely stained ChAT-positive cells to about 90% of that for young controls and caused a significant increase in size of those cells in 28-month-old rats as compared with the control, age-matched group. NGF did not influence the morphology of p75NTR-positive neurons, which were well labeled, irrespective of treatment and age of the rats. In 4-month-old rats, NGF infusion decreased the intensity of both ChAT and p75NTR immunostaining. These data provide some evidence for preservation of BF cholinergic neurons from atrophy during aging and indicate that senile impairment of the cholinergic system in rats concerns decrease in ChAT-protein expression rather than an acute degeneration of neuronal cell bodies. Treatment with NGF resulted in restoration of cholinergic phenotype in the BF neurons of aged rats. However, the present study also rises issue of possible detrimental effects of NGF in young normal animals.

Chronic sodium salicylate treatment exacerbates brain neurodegeneration in rats infected with Trypanosoma brucei

We have reported previously that axonal degeneration in specific brain regions occurs in rats infected with the parasite Trypanosoma brucei. These degenerative changes occur in spatiotemporal association with over-expression of pro-inflammatory cytokine messenger RNAs in the brain. To test how aspirin-like anti-inflammatory drugs might alter the disease process, we fed trypanosome-infected rats with 200 mg/kg of sodium salicylate (the first metabolite of aspirin) daily in their drinking water. Sodium salicylate treatment in uninfected rats did not cause any neural damage. However, sodium salicylate treatment greatly exacerbated neurodegeneration in trypanosome-infected rats, resulting in extensive terminal and neuronal cell body degeneration in the cortex, hippocampus, striatum, thalamus, and anterior olfactory nucleus. The exaggerated neurodegeneration, which occurred in late stages of infection, was temporally and somewhat spatially associated with a late-appearing enhancement of messenger RNA expression of interleukin-1β, interleukin-1β converting enzyme, tumor necrosis factor-α, and inhibitory factor κBα in the brain parenchyma. Restricted areas showed elevations in messenger RNA expression of interleukin-1 receptor antagonist, interleukin-6, inducible nitric oxide synthase, interferon-γ, and inducible cyclooxygenase. The association suggests that increased production of pro-inflammatory cytokines in the brain may be an underlying mechanism for neural damage induced by the chronic sodium salicylate treatment. Furthermore, the results reveal a serious complication in using aspirin-like drugs for the treatment of trypanosome infection.

Selective inhibition of neuronal protein synthesis by retrogradely transported ricin

The ability of the lectin, ricinus communis agglutinin I (ricin 120), to undergo retrograde axonal transport and cause degeneration of neuronal cell bodies has been frequently exploited to establish the origin of peripheral axons. Since this cytotoxic action of ricin results from its inactivation of ribosomes, the retrogradely transported lectin was employed in the present study to inhibit protein synthesis in dorsal root ganglion (DRG) neurons whose axons project into the lumbar nerve trunk of bullfrog tadpoles. The procedure was developed to examine, during tadpole metamorphosis, the ratio of fast-transported radiolabeled protein accumulating at the proximal side of a nerve trunk ligature to the total newly synthesized protein in the cell bodies of origin. The relatively small diameter and fragility of the developing lumbar nerve trunks necessitated introduction of ricin by bath application to the cut nerve end rather than by intraneural injection. Consistent uptake of ricin was achieved by pretreatment with the phospholipase A 2 inhibitor, mepacrine, that blocks resealing of severed nerve fibers. Optimal time and dosage of ricin were established by determining the maximal achievable inhibition of [ 35S]methionine into DRG protein. In stage XVI tadpoles, maximal inhibition of ≈ 65% was observed after 16 h incubation in 2.5 mg/ml ricin. As evidence that neuronal protein synthesis was effectively suppressed, there was no detectable anterograde axonal transport of [ 35S]protein subsequent to ricin treatment. That protein synthesis inhibition was restricted to neuronal cells within the DRG was demonstrated by a combined immunocytochemical and autoradiographic approach. Under conditions where retrogradely transported ricin markedly reduced the density of silver grains decorating neurofilament-positive cells (neurons), there was no significant difference in grain density in Nissl-counterstained, neurofilament-negative regions that contained Schwann cells and other satellite cells.

Gangliosides attenuate the delayed neurotoxicity of aspartic acid in vitro

The neurotoxic effects of l-aspartate were evaluated in rat cerebellar granule cell cultures. Acute (15 min) exposure to l-aspartate produced a time-dependent, delayed degeneration of neuronal cell bodies and neurites (LD 50 about 40 μM) over 24 h. Aspartate neurotoxicity was prevented by competitive and non-competitive N- methyl- d-aspartate (NMDA) antagonists, but not by non-NMDA antagonists, suggesting a major involvement of NMDA receptors in this neuronal injury. Gangliosides, including GM1, were also effective in attenuating the cytotoxicity of l-aspartate. The neurotoxic potential of l-aspartate may thus contribute to pathologies involving the action of endogenous excitatory amino acids.

Hypoglycemic neurotoxicity in vitro: Involvement of excitatory amino acid receptors and attenuation by monosialoganglioside GM1

Rat cerebellar granule cells, when subjected to a glucose-free environment for 4 h, developed extensive degeneration of neuronal cell bodies and their associated neurite network over the following 24 h. This neuronal damage was quantitated with a colorimetric assay using the metabolic dye 3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide. Hypoglycemie neuronal injury could be markedly reduced by the presence of both competitive (3-(±)-2-carboxypiperazin-4-yl)-propyl-1-phosphonic acid) and non-competitive (phencyclidine) N- methyl- d- aspartate receptor antagonists, but not by kainate/ quisqualate preferring antagonists 6-cyano-7-nitroquinoxaline-2,3-dione and 6,7-dinitroquinoxaline-2,3-dione. Glucose deprivation neuronal injury was also reduced by adding glutamate-degrading enzymes to the incubation medium. Monosialoganglioside GM1, but not its asialo derivative (lacking sialic acid), was also effective in protecting against hypoglycemie neurodegeneration when included during the period of glucose deprivation. These results suggest that the neuronal injury to cerebellar granule cells resulting from glucose deprivation is mediated predominantly by activation of the N- methyl- d- aspartate type of excitatory amino acid receptor, perhaps through the action of endogenously released glutamate. Furthermore, the monosialoganglioside GM1, a member of a class of naturally occurring sialoglycosphingolipids, is able to attenuate this neuronal injury—as already observed for glutamate neurotoxicity and anoxic neuronal death in cerebellar granule cells. Gangliosides may thus prove to be of therapeutic utility in excitatory amino acid-associated neuropathologies.

Monosialoganglioside GM1 protects against anoxia-induced neuronal death in vitro

Excitatory dicarboxylic amino acid neurotransmitters, particularly glutamate, have been implicated in mediating neuronal cell injury in brain ischemia-anoxia, epilepsy, and stroke. Glutamate neurotoxicity has been demonstrated in several in vitro models, as well as its prevention by a variety of agents, including several sialic acid-containing glycosphingolipid species, gangliosides. We have now examined ganglioside effects in anoxic exposed cultures of granule cells from Postnatal Day 8 rat cerebellum. Cells between 10 and 12 days in vitro were placed into an anoxic atmosphere or subjected to a chemical model of anoxia by a pulse exposure to rotenone. Widespread neuronal degeneration of neuronal cell bodies and their associated neurite network was seen the following day. These effects on cell vitality at the morphological level were quantitatively confirmed by measuring the photometric reduction of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide to a blue formazan product. This neuronal injury was abolished by the specific N-methyl- d-aspartate receptor noncompetitive antagonists Mg 2+, phencyclidine and MK-801, suggesting that this subtype of glutamate receptor is involved in the pathogenesis of anoxic granule cell injury. Pretreatment for 30 to 60 min or more or concurrent treatment with ganglioside GM1 largely prevented the ensuing neuronal death (ED 50 = 25 μ M), even 4 days later. Degeneration induced by exogenous glutamate was equally reduced. Asialo GM1 (lacking sialic acid) was ineffective. These results are consistent with the observed beneficial effects of the gangliosides in ischemic brain injury models in vivo.

Monoamine neurotransmitters in the evolution of infarction in ischemic striatum: morphologic correlation.

Evolution of infarction following cerebral ischemia is a delayed process, with spongiform degeneration of the neuropil occurring 6 to 8 hours after onset of ischemia. The brains of gerbils with stroke following unilateral carotid artery ligation were examined for catecholamine-derived fluorescence (CADF) by the Falck-Hillarp technique to study the relationship of catecholamine (CA) metabolism with damage to the neuropil. CADF could still be identified in the striatum for up to 16 hours after stroke and there appeared to be spongiform degeneration of the neuropil in relation to accumulations of CADF at 7 and 16 hours after stroke. Pretreatment of gerbils with a-methyl-p-tyrosine 400 mg/kg 6 hours prior to carotid ligation depleted the striatum of CADF until 16 hours after stroke and appeared to reduce the spongiform degeneration of the neuropil, though it did not affect ischemic degeneration of neuronal cell bodies. The continued presence of CADF in the striatum for up to 16 hours after stroke supports the previously reported findings that CA nerve terminals are still functional for 8 hours after stroke and that CA metabolism continues even though levels of CA are reduced immediately after onset of ischemia due to carotid artery ligation.

Intracerebral quinolinic acid injection in the rat: Effects on dopaminergic neurons

Intrastriatal infusion of the endogenous excitotoxin quinolinic acid (QUIN) leads to the degeneration of neuronal cell bodies around the injection site. Dopaminergic afferents not only survive the toxic insult but react by increasing their activity in the acute and suba-cute phases following the injection of QUIN. Measurements of the tissue concentrations of acidic dopamine metabolites, and determinations of l-DOPA accumulation after DOPA-decar☐ylase inhibition, indicate an increased dopamine turnover within 90 min after the administration of 50 μg QUIN. At the later timepoints examined (6 h, 4 and 11 days after QUIN), dopaminergic parameters are increased in the injected striatum only while no changes can be detected in the homolateral substantia nigra. Local norepinephrine levels are elevated 4 and 11 days after an intrastriatal QUIN injection but remain unchanged at distant sites or earlier postinjection periods. The acute increase in nigrostriatal activity may be mediated by an excessively stimulated, yet functional striatonigral feedback loop whereas subsequent changes represent local reactions of dopaminergic nerve terminals secondary to neuronal degeneration in the striatum. In accordance with this interpretation, no monoaminergic changes can be observed in the hypothalamus 4 days following the local injection of 50 μg QUIN, a dose which does not cause neuronal nercosis in this brain area. These data are concordant with, and are discussed in the context of, a possible involvement of QUIN in the pathogenesis of Huntington's disease.

Suppression of self-stimulation of the medial prefrontal cortex after local micro-injection of kainic acid in the rat

The question of whether neurons versus fibers of passage in the medial prefrontal cortex (MPC) are essential in maintaining self-stimulation of this same area of the brain was examined. Rats were prepared with electrode-guide cannulae implanted stereotaxically to rest within MPC. A micro-injection of (KA), 10 nmol/1.0 μl, into the right MPC produced a clear degeneration of neuronal cell bodies characterized by picnocytosis and glial invasion of the tissue surrounding the tip of the electrode. These histopathological changes were correlated with a permanent abolition of self-stimulation of the right MPC. In contrast, self-stimulation of the contralateral side of the MPC, micro-injected with 0.9% NaCl vehicle as a control, was unaffected. These results suggest that neurons of the MPC are part of the neural substrate underlying self-stimulation behavior in this cortical area of the rat.

Comparison of electrolytic and ibotenic acid lesions in the lateral hypothalamus

Electrolytic lesions in the lateral hypothalamus (LH) seriously affect ingestive behavior and sensorimotor functions in the rat. We here report that bilateral infusions of the neurotoxin, ibotenic acid (IBO) in the LH yield a decrease in body weight, but not to the same extent as electrolytic lesions. The sensorimotor impairments were most severe after electrolytic lesions. When tested in a residential maze on days 5–7 and 18–20 after surgery, both lesioned groups showed no lack of motivation to seek food and water. Histological examination of the LH following IBO exposure revealed extensive degeneration of neuronal cell bodies with little evidence of non-specific damage. Biochemical analysis of the rostral forebrain content of norepinephrine (NE) and serotonin (5-HT) revealed that the fibers passing through the LH remained largely intact in the IBO treated rats. The results suggest that the observed aphagia and adipsia is not due to a lack of motivation, but rather reflects changes in the process which operate to initiate eating and drinking. Furthermore, selective neuronal degeneration induced the same behavioral changes as the electrolytic ones, though not to the same extent.

Effects of ibotenic acid-induced neuronal degeneration in the medial preoptic area and the lateral hypothalamic area on sexual behavior in the male rat

It is well known that electrolytic lesions in the medial preoptic area (MPOA) and the lateral hypothalamic area (LHA) seriously impair masculine sexual behavior in the rat. We here report that bilateral infusions of the neurotoxin, ibotenic acid (IBO), in the MPOA were as effective as electrolytic lesions in eliminating copulation whereas no behavioral effects were detected following similar infusions in the LHA. Histological examination of MPOA and LHA following IBO exposure revealed extensive degeneration of neuronal cell bodies with little evidence of non-specific damage. Also, immunohistochemical studies suggested that the serotonergic innervation of the MPOA remained largely intact in spite of IBO treatment; similarly, the damage inflicted by IBO in LHA on tyrosine hydroxylase-immunoreactive fibers in the medial forebrain bundle was insignificant. These data suggest that: (i) the functional integrity of MPOA nerve cell bodies is necessary for the expression of sexual behavior, and (ii) disruption of mating produced by electrolytic LHA lesions is due to disruption of medial forebrain bundle fiber systems. Behavioral observations of non-copulating males suggested that the MPOA injury did not interfere with all aspects of their sexual interaction with the estrous female; rather, they appeared specifically unable to perform the reflexive pelvic thrust pattern normally associated with mounting. We here report, however, that the ability to perform mounts with pelvic thrusts was temporarily restored in the vast majority of MPOA-injured males by the i.p. administration of the ergot derivative, lisuride. About 50% of these MPOA-damaged males even ejaculated, often after a low number of intromissions and short ejaculation latencies. On the other hand, injections of naloxone (an opiate receptor antagonist) failed to activate mounting in MPOA-lesioned or castrated rats. On the basis of these findings the possible ways in which steroid hormone-sensitive brain areas might interact with monoamine-containing pathways are discussed.U