Altered expression and changing distribution of the nerve growth associated protein GAP-43 during ocular HSV-1 infection in the rabbit.

This research examines changes that occur in neurons during corneal herpes simplex virus (HSV-1) infection and focuses on the nerve growth associated protein GAP-43. Cornea and trigeminal ganglion (TG) of New Zealand white rabbits were examined after inoculation of the McKrae and 17 Syn+ strains of HSV-1 to the cornea. Rabbit tissues were taken during acute, latent and induced reactivation stages of infection. Systemic immunosuppression (intravenous injections of cyclophosphamide and dexamethasone) was used to induce reactivation. Western blotting, immunoblotting and autoradiography with the same antibody were used respectively to verify antibody specificity, measure changes in GAP-43 concentration and localize GAP-43 to neurons in the TG. During acute infection, corneal GAP-43 increased significantly while no change was seen in the TG. GAP-43 content was elevated in TG and cornea during viral latency (post-inoculation days 84-154) for both HSV-1 strains. When latent virus was reactivated, the corneal concentration of GAP-43 was more than double that of normal rabbits and the concentration of GAP-43 in TG was reduced compared to the non-reactivated, latently-infected TG. In summary, HSV-1 infected TG neurons expressed more GAP-43 than control neurons and immunosuppressive therapy led not only to viral reactivation and increased GAP-43 concentrations in cornea but also to decreased GAP-43 concentrations in TG. These results suggest that factors which maintain HSV-1 latency and induce reactivation could be linked to elements regulating GAP-43 expression.

Developmental regulation of two distinct neuronal phenotypes in rat dorsal root ganglia.

In a previous study we described two distinct neuronal phenotypes in rat dorsal root ganglia based on immunocytochemical assays for the neuronal intermediate filament proteins, peripherin and low-molecular-weight neurofilaments [Goldstein M. E. et al. (1991) J. Neurosci. Res. 30, 92-104]. In this paper we have extended this classification by using in situ hybridization to localize and evaluate the levels of various cytoskeletal and neuropeptide messenger RNAs within the peripherin-immunoreactive and peripherin-immunoreactive-negative neurons found in embryonic day 15 and 20, postnatal day 2 and adult dorsal root ganglia. We found in postnatal and adult dorsal root ganglia in vivo that the large, peripherin-immunoreactive-negative neurons, which are intensely stained by low-molecular-weight neurofilament antibodies, also contain high levels of low, medium and high-molecular-weight neurofilament messenger RNAs, whereas the smaller peripherin-immunoreactive neurons do not. On the other hand, both cell types contained comparable levels of peripherin and alpha-tubulin messenger RNA. The presence of peripherin messenger RNA but no peripherin immunoreactivity in the large cells suggested either a translational or post-translational regulation of this polypeptide, or rapid clearance of this protein from the perikaryon into the axon. In adult dorsal root ganglia, more than 50% of the peripherin-immunoreactive neurons also contained high levels of substance P and/or calcitonin gene-related peptide messenger RNAs, while less than 20% of the large peripherin-immunoreactive-negative neurons did. The attainment of these phenotypic characteristics during development in vivo was studied by northern blot and in situ hybridization histochemistry. In early embryonic stages (embryonic days 15-16), virtually all neurons were peripherin-immunoreactive and were positive for peripherin, alpha-tubulin and low-molecular-weight neuro-filament messenger RNAs, suggesting a homogeneous population. By embryonic day 20, the two adult phenotypes became clearly evident, and were fully established by postnatal day 2. In cultures of embryonic day 15 dorsal root ganglion neurons grown in the presence of nerve growth factor, peripherin and low-molecular-weight neurofilament messenger RNAs were expressed in all neurons, even after nine days in vitro, similar to embryonic dorsal root ganglia in vivo. Nerve growth factor supplemented by skeletal and heart muscle extracts did up-regulate neurofilament gene expression, but not to the extent characteristic of the peripherin-immunoreactive-negative adult phenotype. These results suggest that development of the mature phenotype of dorsal root ganglion neurons occurs by postnatal day 2 in vivo and is dependent upon target contact and/or target-derived factors.

Neuronal sprouting in mouse sensory ganglia infected with herpes simplex virus type 2 (HSV-2): induction of growth-associated protein (GAP-43) and ultrastructural evidence.

Herpes simplex virus (HSV) is neurotropic and when inoculated on the mouse footpad is retrogradely transported to the associated dorsal root ganglia (DRG), where infection is established. Previous observations suggest that, after HSV infection, sensory ganglion neurons may mount a sprouting response. In our HSV-infected DRG model, we investigate this issue by (1) examining expression of growth-associated protein (GAP-43), a molecule known to be induced by growing axons, and (2) determining ultrastructurally whether HSV-infected dorsal roots contain neurites. In a time course study, we show that GAP-43 is induced both in HSV-infected DRG and their central processes. The increase in GAP-43 is first seen 2 weeks following unilateral footpad inoculation in both cell bodies and dorsal roots, and is sustained at 1 month post inoculation in roots but not in perikarya. Large bundles of unmyelinated small caliber axons, lacking Schwann cell ensheathment, are observed by electron microscopy in dorsal roots 2 weeks and 1 month following inoculation. These profiles resemble developing or regenerating neurites and are rarely seen in roots of mock-infected or uninfected controls. The increased GAP-43 immunoreactivity and ultrastructural changes shown here, in conjunction with previously documented selective neuropeptide and enzyme alterations, confirm that a sprouting response is mounted in sensory ganglia following acute HSV infection.

Herpes simplex virus type-2 infection by a footpad route results in neuronal death in mouse spinal ganglia.

We have examined the effects of herpes simplex virus type 2 (HSV-2) infection on neuron numbers in mouse dorsal root ganglia (DRG) following unilateral hind footpad inoculation. One month following HSV-2 strain MS inoculation, tissue sections of decalcified spine containing the paired 4th and 5th lumbar DRGs were stained with cresyl violet. Neuronal numbers, somal areas and ganglion volumes were determined for ganglia ipsilateral and contralateral to both HSV-2 and medium inoculations. One month following HSV-2 infection, 47-88% of neurons disappeared in the ipsilateral ganglia. The somal areas of the remaining neurons in these ganglia fell within the range of the uninfected population. Ganglionic shrinkage did not occur as a result of HSV-2 infection; neurons were replaced by large numbers of inflammatory cells. Neuron numbers in the contralateral control ganglia of the HSV-2 inoculated mice appeared slightly decreased. Mice inoculated with medium contained similar numbers of neurons in ipsilateral and contralateral ganglia. These results show that, in addition to other previously described host alterations, infection with HSV-2 strain MS results in neuronal death in the affected host ganglia. This is the first in vivo quantitative documentation of neuronal death induced by herpes simplex virus infection.

Neural antigen detection in mouse tissues is not impaired by decalcification.

We examined whether EDTA decalcification decreases the sensitivity of antigen detection of formalin-fixed paraffin-embedded neural tissues. In undissected decalcified tissues, the immunoreactivities of select neural antigens were quantitatively compared with those in dissected sensory ganglia. Decalcification does not reduce either the numbers or staining intensities of antigen-positive neural cells. Tissues are well preserved and complex anatomical relationships are maintained.

Herpes simplex virus infection induces a selective increase in the proportion of galanin-positive neurons in mouse sensory ganglia.

We examined the effects of herpes simplex virus type 2 (HSV-2) infection on host neuropeptide content in mouse dorsal root ganglia (DRG) neurons following unilateral hind footpad inoculation. At selected survival times following infection, adjacent tissue sections of decalcified spine containing the paired 4th and 5th lumbar DRGs were immunoreacted to detect HSV-2, calcitonin gene-related peptide (CGRP), or galanin antigen. Labeled and unlabeled neurons were counted and the somal areas for all neurons in the infected and the contralateral uninfected DRG in each mouse were compared. HSV-positive neurons were small. HSV-2 antigen was present in neurons at Day 5; by Day 14 the antigen had disappeared. Galanin positivity was first seen at Day 8, peaked at Day 14, gradually declined on Days 21 and 28, and returned to control values by Day 42. The mean soma size of the labeled population was small. Galanin antigen was not seen in DRG at any time following sham inoculation. At all times after infection, equal numbers of CGRP-positive neurons were seen in infected and uninfected ganglia and in sham-operated mice. These results show that HSV-2 infection differentially affects host neuropeptide production and that nervous system effects are not restricted to the acute stage of infection. These events are consistent with those seen in other injury/regeneration paradigms.

The proportion of galanin-immunoreactive neurons in mouse trigeminal ganglia is transiently increased following corneal inoculation of herpes simplex virus type-1.

To investigate whether neurobiological functions are modified by viral infection, we inoculated mouse corneas with herpes simplex virus type 1 (HSV-1) and examined neuronal galanin content in trigeminal ganglia at selected survival times. HSV-1 antigen appeared in neurons at day 3, peaked at day 6 and disappeared by day 11. Increased galanin positivity was first seen at day 6, peaked at day 10 and approached control values by day 21. This result provides further evidence that the biological program of peripheral sensory neurons is modified by herpes-virus infection.

Herpes simplex virus infection in populations of mouse dorsal root ganglion neurons: effects of inoculation route and virus strain.

To investigate whether herpes simplex virus type-2 (HSV-2) infection of sensory ganglion neurons is restricted to a particular neuronal class, we unilaterally inoculated either the footpad, leg muscle or sciatic nerve of mice with strains of HSV-2 that differ in virulence and examined the soma sizes of lumbar dorsal root ganglion (DRG) neurons containing viral antigen near the peak of acute infection. Each inoculation route appeared relatively selective for a different portion of the neuronal size spectrum. Footpad inoculation primarily infected the smaller population of DRG neurons, by comparison, muscle inoculation tended to spare the smallest neurons while infecting medium and larger cells. Sciatic nerve inoculation infected the entire spectrum of DRG neurons. These results indicate that HSV-2 infection is not restricted to a specific subclass of sensory neurons. With any particular inoculation route, the least virulent strain infected fewer neurons than did those of greater virulence.

Targets of herpes simplex virus type 1 infection in a mouse corneal model.

In animal models, spread of herpes simplex virus type 1 (HSV-1) from epithelial replication sites to the peripheral and central nervous system is known from analysis of individually dissected tissues. To examine virus spread in undissociated tissues, corneas of adult mice were inoculated with HSV-1. After 1 to 13 days groups of mice were perfused with formalin, and decalcified blocks of head and neck were embedded in paraffin. At intervals, serial sections were screened for HSV antigen. On days 1 and 2, viral antigen was restricted to cornea and conjunctiva but by days 3 and 4 was also seen in autonomic ganglia and the trigeminal system. On day 6, HSV antigen reached its maximum extent; infected sites included the trigeminal complex (ganglion, root, peripheral ophthalmic and maxillary branches and spinal nucleus and tract), ethmoid sinus and olfactory bulb, visual system, and autonomic ganglia (ciliary, pterygopalatine and superior cervical). Antigen progressively diminished on days 8 and 10, and was not detected on day 13. This method demonstrates a broader range of infected tissues and suggests a more complex pattern of HSV spread than has been previously recognized. Virus appears to reach the intracranial compartment by four different neural routes. When effects of higher and lower corneal inoculation doses were compared, a lower dose resulted in lower peak HSV titers in trigeminal ganglion and brain stem and later virus appearance in these tissues. Thus, dose may influence the kinetics of HSV spread from the peripheral inoculation site to the CNS.

Neurotropic herpesviruses, neural mechanisms and arteritis.

Cumulative evidence suggests that varicella-zoster virus (VZV) can infect walls of CNS arteries, causing stroke in man. We review observations relating infection with this neurotropic virus to the development of arteritis in the CNS and note evidence supporting the hypothesis that VZV spreads from ganglionic reactivation sites to the arterial wall by neural pathways. Problems relating to the pathogenesis of arteritis and experimental approaches to their solution are suggested.

Expression of beta-preprotachykinin mRNA and tachykinins in rat dorsal root ganglion cells following peripheral or central axotomy.

The changes in gene expression and protein synthesis induced in neurons by axotomy usually lead to increased production of axon constituents and decreased production of molecules related to neurotransmission. Exceptions to this generalization occur, however, and it is unclear whether the injury itself changes the pattern of synthesis or whether individual mechanisms regulate the synthesis of the various axonal components. We used in situ hybridization histochemistry and immunocytochemistry to compare the changes in L4 and L5 rat dorsal root ganglion neuron levels of preprotachykinin mRNA and tachykinin peptides caused by sciatic nerve injury with those caused by dorsal root injury. Both lesions elicit regeneration, although only the axotomized peripheral processes re-establish functional contact with their targets. In the contralateral, intact dorsal root ganglia approximately 17% of neurons contained detectable levels of both mRNAs and peptides. Sciatic nerve section decreased by 70% the number of neurons labeled for preprotachykinin mRNA at three days post-operatively. Not all cells in the ganglion are axotomized by the sciatic nerve lesion; grain counts over the cells spared by the lesion showed an increased level of labeling, possibly a result of collateral sprouting by these spared cells. By two weeks, the number of cells labeled for preprotachykinin mRNA had decreased to 80% of control levels. The numbers of neurons labeled for tachykinin peptides decreased more slowly and reached approximately 50% of control numbers at two weeks. By six months post-operatively, when regeneration is largely complete, the number of neurons containing both mRNAs and peptides returned to normal. In contrast, dorsal root section did not elicit a decrease in the number of neurons labeled either for the mRNAs or the peptides at any of the post-operative intervals examined. These results indicate that axotomy is not the stimulus that elicits changes in the expression of genes coding for tachykinins. Evidence is considered indicating that interruption of the supply of peripherally derived nerve growth factor may be responsible for the changes in gene expression for tachykinins after axotomy.

Optic nerve crush modulates proliferation of rod precursor cells in the goldfish retina.

Tritiated thymidine ([3H]TdR) autoradiography revealed a correlation between the rate of cell proliferation of rod precursor cells in the outer nuclear layer (ONL) of the goldfish retina and the postoperative interval after crush of the optic nerve (ONC). Ten days after unilateral ONC there were more labeled nuclei in the ONL of the nerve-crushed retina than in the intact, contralateral retina of the same fish one day after single bilateral intraocular injections of [3H]TdR. From 15 to 25 days after ONC, however, fewer labeled nuclei were found in the ONL of nerve-crushed retinae than in controls, illustrating a decrease in the number of cells entering the S-phase of the cell cycle; by 35 days the differences had disappeared, demonstrating that cell birth recovered to normal. When examined one month after [3H]TdR injection, fewer labeled cells were present in the nerve-crushed retina at all postoperative intervals. Examination of the numbers of labeled cells at various postoperative periods following bilateral ONC, when one retina was examined one day and the other retina was examined one month after [3H]TdR administration revealed that the ratios of labeled cells between the two retinae varied as a function of time after ONC. Therefore, optic nerve crush appears to enhance the proportion of initially labeled cells in the ONL that are either fated to undergo further cell generation cycles or to die.

In situ hybridization of mRNA for beta-preprotachykinin and preprosomatostatin in adult rat dorsal root ganglia: comparison with immunocytochemical localization.

In situ hybridization histochemistry was used to identify neurons in rat dorsal root ganglia that contained mRNAs encoding beta-preprotachykinin and preprosomatostatin. The distribution of these neurons was compared with the distribution of neurons containing tachykinins or somatostatin, identified using immunocytochemical techniques. Neurons labelled for beta-preprotachykinin mRNA constituted 20% of the total neuronal population and belonged to the small cell class. Neurons labelled for preprosomatostatin mRNA with either RNA or DNA hybridization probes constituted approximately 10% of the total cells and comprised a small cell group that differed in average size from the beta-preprotachykinin labelled population. The distribution of cells containing tachykinin- or somatostatin-like immunoreactive material was identical to the distribution of cells containing the respective mRNAs and, in addition, individual somata in adjacent sections contained both the mRNA precursor and the peptide. These results suggest that for these neuropeptides the sensitivity of the two methods is equivalent and the respective mRNAs and peptides are co-localized in the same neurons.

In vitro proliferation of radial glia within isolated tectal tissue explanted from adult goldfish.

Radial glia located in tectal tissue isolated from adult goldfish retain the ability to incorporate exogenous thymidine into DNA up to 5 days in culture. The rate of their proliferation is maximally enhanced during reinnervation of the tectum by optic fibers about 5 weeks after unilateral optic nerve crush.

Effects on visual acuity of neonatal or adult tectal ablation in rats.

Tectal ablation in neonatal rats leads to retrograde degeneration of retinal ganglion cells whereas similar damage in adults does not. We show here that the behavioral effects are comparably different. When rats with neonatal tectal ablation are tested as adults they are impaired in learning a discrimination between vertical and horizontal stripes and their visual acuity for square-wave gratings is slightly but significantly reduced.