Mechanisms of varicella-zoster virus neuropathogenesis in human dorsal root ganglia

Varicella-zoster virus (VZV) is a human alphaherpesvirus that infects sensory ganglia and reactivates from latency to cause herpes zoster. VZV replication was examined in human dorsal root ganglion (DRG) xenografts in mice with severe combined immunodeficiency using multiscale correlative immunofluorescence and electron microscopy. These experiments showed the presence of VZV genomic DNA, viral proteins, and virion production in both neurons and satellite cells within DRG. Furthermore, the multiscale analysis of VZV-host cell interactions revealed virus-induced cell-cell fusion and polykaryon formation between neurons and satellite cells during VZV replication in DRG in vivo. Satellite cell infection and polykaryon formation in neuron-satellite cell complexes provide mechanisms to amplify VZV entry into neuronal cell bodies, which is necessary for VZV transfer to skin in the affected dermatome during herpes zoster. These mechanisms of VZV neuropathogenesis help to account for the often severe neurologic consequences of herpes zoster.     

Productive varicella-zoster virus infection of cultured intact human ganglia

Varicella-zoster virus (VZV) is a species-specific herpesvirus which infects sensory ganglia. We have developed a model of infection of human intact explant dorsal root ganglia (DRG). Following exposure of DRG to VZV, viral antigens were detected in neurons and nonneuronal cells. Enveloped virions were visualized by transmission electron microscopy in neurons and nonneuronal cells and within the extracellular space. Moreover, rather than remaining highly cell associated during infection of cultured cells, such as fibroblasts, cell-free VZV was released from infected DRG. This model enables VZV infection of ganglionic cells to be studied in the context of intact DRG.     

Infection by human varicella-zoster virus confers norepinephrine sensitivity to sensory neurons from rat dorsal root ganglia.

Varicella-zoster virus (VZV) is a widespread human herpes virus causing chicken pox on primary infection and persisting in sensory neurons. Reactivation causes shingles, which are characterized by severe pain and often lead to postherpetic neuralgia. To elucidate the mechanisms of VZV-associated hyperalgesia, we elaborated an in vitro model for the VZV infection of sensory neurons from rat dorsal root ganglia. Between 35 and 50% of the neurons showed strong expression of the immediate-early virus antigens IE62 and IE63 and the late glycoprotein gE. When the intracellular calcium concentration was monitored microfluorometrically for individual cells after infection, the sensitivity to GABA or capsaicin was similar in controls and in VZV-infected neurons. However, the baseline calcium concentration was increased. Neurons became de novo sensitive to adrenergic stimulation after VZV infection. Norepinephrine-responsive neurons were more frequent and calcium responses to norepinephrine were significantly higher after infection with wild-type isolates than with the attenuated vaccine strain OKA. The adrenergic agonists phenylephrine and isoproterenol had similar efficacy. We suggest that the infection with wild-type VZV isolates confers norepinephrine sensitivity to sensory neurons by using alpha(1)- and/or beta(1)-adrenergic receptors providing a model for the pathophysiology of the severe pain associated with the acute reactivation of VZV.     

Characterization of acute and latent herpes simplex virus infection of dorsal root ganglia in rats.

The characteristics of HSV type-1 infection following subcutaneous inoculation in the dorsum of one hind paw of Sprague-Dawley rats were studied to determine whether infection in rats might more closely parallel the infection in man than is seen in other animals. The serologic and virologic characteristics of acute and latent ganglion infection conformed to those of human infection. Immunohistochemical studies suggested that sensory ganglion infection arose via centripetal axonal migration of virus as is hypothesized in man. In rat, small type B neuronal cell bodies appeared central to the maintenance of latent infection and reactivation observed during cocultivation of lumbar ganglia. Acute and latent lumbar sensory ganglion infection in rats after subcutaneous hind paw injection of HSV-1 appears to be another suitable model of this infection in man.     

Quantitation of latent varicella-zoster virus and herpes simplex virus genomes in human trigeminal ganglia

Using real-time fluorescence PCR, we quantitated the numbers of copies of latent varicella-zoster virus (VZV) and herpes simplex virus type 1 (HSV-1) and type 2 (HSV-2) genomes in 15 human trigeminal ganglia. Eight (53%) and 1 (7%) of 15 ganglia were PCR positive for HSV-1 or -2 glycoprotein G genes, with means of 2,902 +/- 1,082 (standard error of the mean) or 109 genomes/10(5) cells, respectively. Eleven of 14 (79%) to 13 of 15 (87%) of the ganglia were PCR positive for VZV gene 29, 31, or 62. Pooling of the results for the three VZV genes yielded a mean of 258 +/- 38 genomes/10(5) ganglion cells. These levels of latent viral genome loads have implications for virus distribution in and reactivation from human sensory ganglia.     

Imaging neural crest cell dynamics during formation of dorsal root ganglia and sympathetic ganglia

The neural crest is a migratory population of cells that produces many diverse structures within the embryo. Trunk neural crest cells give rise to such structures as the dorsal root ganglia (DRG) and sympathetic ganglia (SG), which form in a metameric pattern along the anterior-posterior axis of the embryo. While static analyses have provided invaluable information concerning the development of these structures, time-lapse imaging of neural crest cells navigating through their normal environment could potentially reveal previously unidentified cellular and molecular interactions integral to DRG and SG development. In this study, we follow fluorescently labeled trunk neural crest cells using a novel sagittal explant and time-lapse confocal microscopy. We show that along their dorsoventral migratory route, trunk neural crest cells are highly motile and interact extensively with neighboring cells and the environment, with many cells migrating in chain-like formations. Surprisingly, the segregated pattern of crest cell streams through the rostral somite is not maintained once these cells arrive alongside the dorsal aorta. Instead, neural crest cells disperse along the ventral outer border of the somite, interacting extensively with each other and their environment via dynamic extension and retraction of filopodia. Discrete sympathetic ganglia arise as a consequence of intermixing and selective reorganization of neural crest cells at the target site. The diverse cell migratory behaviors and active reorganization at the target suggest that cell-cell and cell-environment interactions are coordinated with dynamic molecular processes.     

Differential localization of lectin binding sites and neuropeptides in human dorsal root ganglia

The subpopulations were compared of neurons in human dorsal root ganglia (DRG), as substance P, identified by somatostatin, Glycine max lectin (SBA) specific to terminal N-acetylgalactosamine, and Ulex europaeus I agglutinin (UEA-I) specific to l-fucose. The lectins and neuropeptides all bound to neurons of small diameter. Furthermore, the majority of the SBA binding neurons or somatostatin positive neurons were also UEA-I binding neurons. However, SBA binding neurons were not colocalized with somatostatin or substance P. Less than 20% of substance P positive neurons showed colocalization with l-fucosyl residues, and approximately 10% of l-fucosyl residues showed colocalization with substance P. Our results suggest that both l-fucose and terminal N-acetylgalactosamine containing neurons in the human DRG are subjected to different subpopulations from substance P or somatostatin positive neurons.     

Ribosomes in myelinated axons of dorsal root ganglia

Summary In the dorsal root ganglia of the rat, ribosomes were found not only in the initial segment, but they were also observed in the axoplasm of intraganglionar myelinated fibres and in the sensory portion of spinal nerves. Axons of seven-days-old rats contained more ribosomes than those of adult animals. The amount of particles decreased gradually from the initial segment trough intraganglionar internodes to the axons of spinal nerves. No ribosomes were found in axons of dorsal roots. In intraganglionar fibres, ribosomal particles were usually observed near the nodes of Ranvier, in the vicinity of Schmidt-Lantermann clefts and in axons near the Schwann cell nuclei. They were arranged in tetrads, pentads or in larger polysomes, and they were often observed adjacent to a group of mitochondria.The particles had invariably a stable size, their average diameters measuring 234 ± 2 × 197 ± 3 Å, which is practically equal to the diameters of 232 ± 2 × 203 ± 3 Å of ribosomes in the Schwann cell cytoplasm. These values fall within the range of diameters of ribosomes isolated from various cells of eukaryotic organisms as given in the literature. Since no other granular component of the cytoplasm has similarly stable dimensions, the measurements are considered to prove that the axonal particles described here are ribosomes.The author wishes to thank Dr. K. Smetana for his valuable suggestions and Mrs. M. Sobotková, Ing. M. Doubek and Mr. H. Kunz for their skillful technical assistance. The investigation was in part supported by a grant-in-aid from the Muscular Dystrophy Associations of America, Inc.     

Prenatal development of the rat dorsal root ganglia

Summary This study describes three-dimensional aspects of the development and pseudo-unipolarization of neuroblasts and the maturation of satellite cells in prenatal rat dorsal root ganglia, using scanning electron microscopy, after removal of extracellular connective tissue components by trypsin digestion and HC1 hydrolysis.At 14 days of gestation, the vast majority of neurons are spindle-shaped or bipolar and only 3% are unipolar, while at 16 and 18 days this percentage has increased to 30% and 91%, respectively. The initial portions of the central and peripheral neuronal processes gradually approach each other and form a common initial portion. Finally, the cytoplasm of this common initial portion becomes thinner and elongates to form the stem process of the mature cell.Satellite cells are present from the beginning of the period studied, but intricate networks of branching satellite cell processes only develop after about day 17.     

Demyelination and cytopathic effects in cultures of mammalian dorsal root ganglia infected with encephalomyocarditis virus.

Replication of encephalomyocarditis virus and its cytopathic effects were studied in myelinated cultures of dorsal root ganglia obtained from newborn mice. Six hours after infection virus progeny was detected in the culture. At 24 h the virus titer reached 2 times 10(6) PFU per culture and remained at this level until 48 h. The first cytopathic alterations began at 24 h and consisted of rounding of Schwann and satellites cells and their detachment from neurons. Later, bead-like swellings of the myelin appeared along the axons followed by splitting and degeneration of lamellae. The cytopathic effect in the neurons started 29 h after infection, reaching complete neuronolysis at 48 h. Virus particles, scattered or arranged in crystal-like aggregates, were first seen in the cytoplasm of glial cells and then in neurons and axons.     

Conditional gene deletion in primary nociceptive neurons of trigeminal ganglia and dorsal root ganglia

The use of Cre-loxP technology for conditional mutagenesis in pain pathways had been restricted by the unavailability of mice expressing Cre recombinase selectively in functionally distinct components of the nociceptive system. Here we describe the generation of transgenic mouse lines which express Cre recombinase selectively in sensory ganglia using promoter elements of the Na(v)1.8 gene (Scn10a). Cre-mediated recombination was greatly evident in all nociceptive and thermoreceptive neurons of the dorsal root ganglia and trigeminal ganglia, but only in a small proportion of proprioceptive neurons. Cre-mediated recombination was not detectable in the brain, spinal cord, or any nonneural tissues and began perinatally after invasion of primary afferents into the developing spinal cord. Thus, these mice enable selective deletion of genes in subsets of sensory neurons and offer a wide scope for studying potential functions of genes in pain perception, independent of secondary effects arising from developmental defects or global gene ablation.     

Effects of hydroxyurea during final neuronal DNA synthesis in dorsal root ganglia of rats

The sequence of final neuronal DNA synthesis was investigated in developing lumbar dorsal root ganglia of rats. Patterns of final division were compared to permanent neuronal deficiencies produced by single doses of hydroxyurea (HU), a specific cytotoxic inhibitor of DNA synthesis. The purpose was to discern increased susceptibility of terminal cell cycles in order to evaluate possible phenotypic or mitotic commitments responsible for cessation of DNA synthesis. The normal period of final DNA synthesis was found to occur primarily on gestation Days 12, 13, and early 14. Precursors of larger (A cells) and smaller (B cells) neurons are generated in sequence, suggesting the presence of phenotypic commitments during terminal division. When HU was administered during the period of final DNA synthesis, severe neuronal depletions and altered phenotypic proportions were observed postnatally. With HU on Day 13, total neuronal numbers were reduced by an average of 62% and deficiencies were confined primarily to neurons originating at or near the time of treatment. Given 12 hr later (Day 13.5), HU produced a 48% depletion involving neurons of smaller diameters. With treatment on Day 14, some ganglia appeared normal histologically but quantitation revealed an average 21% numerical deficiency involving the smallest neuronal phenotypes. Later treatments did not appear to affect ganglion morphology even though other defects (primarily growth retardation and gait abnormalities) continued to occur. Earlier treatments, given during terminal division of large neurons on Day 12, produced resorption or early postnatal death. The results suggest the emergence of phenotypic commitments in final cell cycles which restrict the probability of continued DNA synthesis and, thus, the probability of regeneration.