Infection of the basal ganglia by a murine coronavirus.

The coronavirus, mouse hepatitis virus strain A59 (MHV-A59), causes mild encephalitis and chronic demyelination. Immunohistochemical techniques showed that MHV-A59-infected C57BL/6 mice contained dense deposits of viral antigen in the subthalamic nucleus and substantia nigra, with fewer signs of infection in other regions of the brain. The animals showed extra- and intracellular vacuolation, neuronal loss, and gliosis in the subthalamic-nigral region. Such localization is unprecedented among known viral encephalitides of humans and other species. This infection by a member of a viral class capable of causing both encephalitis and persistent infection in several species may be related to postencephalitic parkinsonism.

Selective localization by neuroglia of immunoglobulin G in normal mice.

Plasma proteins including immunoglobulins have been previously localized in neurons with processes extending outside the blood-brain barrier, but not within glia under normal conditions. Immune modulating functions have been proposed for both microglia and astrocytes in several pathological states. Using immunocytochemistry, we have found that large numbers of neuroglial cells contain immunoglobulin G (IgG) in normal mice of the C57 BL/6 strain. Most IgG-positive cells had both the morphology and distribution of microglia, including a higher density in grey matter, and were frequently found in perivascular or perineuronal locations. The accumulation of IgG does not appear to be by nonselective phagocytosis of extracellular fluid, since serum albumin could not be detected within microglia. There was little overlap in the distribution of cellular processes positive for IgG and those which showed astrocytic markers. Neuronal accumulation of plasma proteins was also seen in a distribution described by previous investigators. The function of this selective accumulation of IgG by normal microglia is unknown, but may reflect a role in the immune response within the central nervous system.

Attitude change concerning right to refuse treatment: the impact of experience.

Forty-three medical students completed questionnaires about their attitudes toward the right to refuse treatment at the beginning and end of their psychiatric clerkship, during which time their clinical experience included exposure to involuntary psychiatric treatment. The students had a more favorable view of involuntary treatment after the clerkship; the change in attitudes was statistically significant. The authors hypothesize that the medical students' increased willingness to use seclusion and involuntary neuroleptic medication was due to exposure to agitated violent patients as well as experience with the beneficial effects of neuroleptic medication.

Functional synapses are formed between human NTera2 (NT2N, hNT) neurons grown on astrocytes.

The formation of functional synapses is a late milestone of neuronal differentiation. The establishment of functional synapses can be used to assess neuronal characteristics of different cell lines. In the present study, we examined the in vitro conditions that influence the ability of human neurons derived from the NT2 cell line (NT2N neurons) to establish synapses. The morphologic, immunologic, and electrophysiologic characteristics of these synapses was examined. In the absence of astrocytes, NT2N neurons rarely formed synapses and their action potentials were weak and uncommon. In contrast, when plated on primary astrocytes, NT2N neurons were able to form both glutamatergic excitatory (71%) and GABAergic inhibitory (29%) functional synapses whose properties (kinetics, ion selectivity, pharmacology, and ultrastructure) were similar to those of synapses of neurons in primary cultures. In addition, coculture of NT2N neurons with astrocytes modified the morphology of the neurons and extended their in vitro viability to more than 1 year. Because astrocyte-conditioned medium did not produce these effects, we infer that direct contact between NT2N neurons and astrocytes is required. These results suggest that NT2N neurons are similar to primary neurons in their synaptogenesis and their requirement for glial support for optimal survival and maturation. This system provides a model for further investigations into the neurobiology of synapses formed by human neurons.

Retrograde changes in the corticospinal tract of posttraumatic paraplegics.

Autopsy material from patients with posttraumatic paraplegia of long duration was examined to determine the extent of survival of corticospinal axons proximal to the injury. Spinal cord sections from sites above a functionally complete spinal cord injury were examined for the presence of axons, myelin, and gliosis. There was a marked depletion of axons from the corticospinal tract close to the region of injury. There appeared to be a gradual increase in the density of axons within the tract at increasing distance from the injury in all patients. Corticospinal axons appear to retract from the spinal cord injury site, but do not show progressive degeneration even years after a spinal cord injury.

Late-convalescent poliomyelitis. Corticospinal tract integrity.

Since both poliomyelitis and amyotrophic lateral sclerosis affect spinal motor neurons, a relationship between these two diseases has been suggested. Corticospinal tract (CST) degeneration, a prominent aspect of amyotrophic lateral sclerosis, is rarely observed in acute poliomyelitis. Autopsy material from seven patients who had survived long periods after severe paralytic poliomyelitis was examined for evidence of CST degeneration. Although there was severe motor neuron loss and destruction of ventral horn cytoarchitecture, none of the spinal cords showed significant demyelination, fiber loss, or gliosis in the region of the CST. The structural integrity of the CST is maintained for many years after severe motor neuron loss due to poliomyelitis.

Motoneuron uptake from the circulation of the binding fragment of tetanus toxin.

Tetanus toxin enters the central nervous system from the systemic circulation after it is internalized by motoneuron terminals at the neuromuscular junction. We have demonstrated that the atoxic binding fragment (C-fragment) of tetanus toxin is internalized preferentially by motoneurons. We examined the distribution of C-fragment after intravenous injection in the nervous systems of mice by immunohistochemical methods. All animals remained asymptomatic until killed one to two days after injection. C-fragment was found only within neurons with processes outside the blood-brain barrier. Large motoneurons of the spinal cord showed the greatest accumulation of C-fragment. Motoneurons of brain-stem nuclei (particularly facial and trigeminal), also showed substantial label of C-fragment. Small amounts of C-fragment were detected in dorsal root ganglion cells. Affinity of a systemically distributed substance for synaptic components, as well as an inability to cross the blood-brain barrier, may lead to its preferential localization in motoneurons.

Antibodies to the ganglioside GD1b in a patient with motor neuron disease and thyroid adenoma.

Patients with motor neuron disease with thyroid disorders have been described, although the relationship between the two conditions is unclear. We treated a patient with amyotrophic lateral sclerosis who also had a follicular adenoma of the thyroid gland. Because thyroid gland plasma membranes contain high concentrations of complex gangliosides, such as GD1b, and some patients with motor neuron disease have IgM antibodies to GD1b, we decided to assay serum from this patient for the presence of antiganglioside antibodies. IgM antibodies to GD1b were detectable at serum dilutions of 1:500 and 1:1000 by enzyme-linked immunosorbent assay. While these titers are less than those usually described in patients with plasma cell dyscrasia, they are well in excess of normal values. Antibody to GM1 was also detectable at a lower (1:100) dilution. We do not know the importance of the anti-GD1b antibodies in this patient, but it is possible that antibodies to GD1b are involved in this and other cases of motor neuron disease associated with thyroid disease.

Systemic recombinant alpha-2 interferon therapy in relapsing multiple sclerosis.

This report describes the first use of recombinant-DNA-produced human interferon in patients with multiple sclerosis (MS). Ninety-eight patients who were clinically definite for MS with two or more documented exacerbations during the preceding two years were admitted to this placebo-controlled double-blind randomized trial. Although both groups were similar, placebo patients had later MS onset. Patients injected themselves with 2 X 10(6) IU of alpha-2 interferon or placebo three times each week for up to 52 weeks. This dose of interferon was well tolerated in that side effects were minimal. During the trial, the exacerbation rate was sharply reduced in both groups. In the three-month follow-up period after stopping treatment, more patients who were receiving interferon than placebo became worse neurologically. More patients who were receiving interferon than placebo changed from exacerbating MS to progressive MS during the trial. Thus, no clear therapeutic benefit of alpha-2 interferon for MS was detected.

Neural transplantation: scientific gains and clinical perspectives.

Neural transplantation, once deemed impossible, is being studied in many laboratories. Embryonic CNS from a variety of sites can be grafted into an adult host. The foreign cells differentiate and then produce neurotransmitters or neurohormones. Physical connection can be seen between graft and host. Grafting of fetal tissue may be followed by improved function of animals with experimental forms of neurologic disease or physical injury. Grafted segments of peripheral nerve become innervated by central axons that can conduct physiologic impulses. Grafted glial cells can form myelin within the CNS. Therapeutic grafting into the human nervous system may be feasible, but many scientific and ethical questions remain to be addressed.

The fate of severed corticospinal axons.

The potential for regeneration of severed corticospinal axons was examined by labeling these axons with horseradish peroxidase following thoracic spinal cord transections in mice. Shortly after severance, the proximal ends of corticospinal axons formed terminal bulbs that persisted for weeks and were associated with axonal retraction. There were few signs of corticospinal axonal sprouting or elongation. By 2 months after injury, corticospinal axons near the transection site showed an increased number of probable labeled terminals in the adjacent gray matter. These new terminals may contribute to the persistence of many corticospinal axons near the injury site long after a spinal cord transection.

Internalization of IgG in motoneurons of patients with ALS: selective or nonselective?

The hypothesis that abnormal antibodies may be involved in the pathogenesis of ALS has been supported in part by IgG's being present within motoneurons of ALS patients more frequently than in motoneurons of controls. IgG, as well as other serum proteins, is also present in motoneurons of normal human and animal spinal cords. We attempted to determine whether the IgG found in motoneurons of ALS patients was localized by an immune-specific or nonspecific process. To address this question, we used immunocytochemistry to evaluate the presence and relative density of different serum proteins in spinal cords from nine patients with ALS. Both IgG and alpha 2-macroglobulin (alpha 2Mac) were present in motoneurons in all nine cases. More important, there was a close concordance between the IgG and alpha 2Mac immunolabeling of motoneurons. The presence of a nonimmune plasma protein--alpha 2Mac--in a similar distribution to IgG and with a similar intensity implies that the internalization of these proteins in motoneurons of patients with ALS is best explained by a nonselective mechanism of endocytosis of extracellular fluid.

Relapsing transverse myelitis.

Acute transverse myelitis is a monophasic disorder, the recurrence of which raises the question of multiple sclerosis (MS) or other multifocal CNS disease. We now report three patients with a previously undescribed syndrome of relapsing isolated acute transverse myelitis. Each had two to five attacks over periods of 3 to 8 years, characterized by ascending paresthesias, urinary retention, sensory loss with a thoracic or cervical level, paraparesis, hyperreflexia, and bilateral Babinski signs. MRI demonstrated areas of increased signal intensity on T2- and proton density-weighted scans and decreased signal intensity on T1-weighed scans of the cervical or thoracic spinal cord consistent with an inflammatory or demyelinating process. All patients had normal complete myelograms, oligoclonal IgG bands were consistently absent from the cerebrospinal fluid, cranial MRIs were normal, and there was no other clinical or laboratory evidence of MS, collagen-vascular disease, or active viral infection. They were treated with high doses of intravenous corticosteroids, stabilized between episodes, and had partial or complete recovery. The recognition of these three patients at a single medical center in a 1-year period suggests that relapses of acute transverse myelitis may not be rare.

Free-living daily energy expenditure in patients with Parkinson's disease.

Previous studies have suggested that elevated resting energy expenditure contributes to weight loss in patients with Parkinson's disease (PD). Body weight is, however, ultimately determined by variation in daily energy expenditure and not just resting energy expenditure. Therefore, we examined the hypothesis that PD patients are characterized by elevated daily energy expenditure. Sixteen patients with levodopa responsive PD and 46 healthy elderly controls were characterized for daily energy expenditure and its components (resting and physical activity energy expenditure) using a combination of the doubly labeled water technique (over 10 days) and resting indirect calorimetry. Fat-free mass and fat mass were measured by dual energy x-ray absorptiometry. Results showed that fat mass and fat-free mass did not differ between groups. Daily energy expenditure was 15% lower (2214 +/- 460 vs. 2590 +/- 497 kcal/d; p < 0.01) in PD patients compared to controls. This was primarily due to lower physical activity energy expenditure (339 +/- 366 vs. 769 +/- 412 kcal/d; P < 0.01) in PD patients as resting energy expenditure was not different between groups (1655 +/- 283 vs. 1561 +/- 219 kcal/d). These results show that daily energy expenditure is lower in PD patients compared to healthy elderly, primarily due to reduced physical activity energy expenditure. These results argue against the hypothesis that an abnormally elevated daily energy expenditure contributes to weight loss in PD.

Protective effect of supplemental superoxide dismutase on survival of neuronal cells during starvation. Requirement for cytosolic distribution.

There is evidence that raising cellular levels of Cu2+/Zn2+ superoxide dismutase (SOD1) can protect neurons from oxidative injury. We compared a novel method of elevating neuronal SOD activity using a recombinant hybrid protein composed of the atoxic neuronal binding domain of tetanus toxin (C fragment or TTC) and human SOD1 (hSOD1) with increasing cellular SOD levels through overexpression. Fetal murine cortical neurons or N18-RE-105 cells were incubated with the TTC-hSOD1 hybrid protein and compared to cells constitutively expressing hSOD1 for level of SOD activity, cellular localization of hSOD1, and capacity to survive glucose and pyruvate starvation. Cells incubated with TTC-hSOD1 showed a threefold increase in cellular SOD activity over control cells. This level of increase was comparable to fetal cortical neurons from transgenic mice constitutively expressing hSOD1 and transfected N18-RE-105 cells expressing a green fluorescent protein-hSOD1 fusion protein (GFP-hSOD1). Human SOD1 was distributed diffusely throughout the cytoplasm of the transgenic murine neurons and transfected N18-RE-105 cells. In contrast, cells incubated with TTC-hSOD1 showed hSOD1 localized to the cell surface and intra-cytoplasmic vesicles. The cells expressing hSOD1 showed enhanced survival in glucose- and pyruvate-free medium. Neither cortical neurons nor N18-RE-105 cells incubated in TTC-hSOD1 showed increased survival during starvation. Access to the site where toxic superoxides are generated or their targets may be necessary for the protective function of SOD1.

Fate of severed cortical projection axons.

Corticospinal neurons show a primarily degenerative response to axotomy in adult mammals. The long remaining proximal axon with its extensive synaptic contacts may contribute to the lack of initial regenerative response in this cell type. We examined a related group of cortical axons after lesions in the subcortical white matter close to their cell bodies of origin. With cholera B chain conjugated to horseradish peroxidase (CTB-HRP), transcallosal axons projecting into areas of a lesion were labeled. Animals surviving between 2 days and 4 months were examined with both light microscopic and ultrastructural techniques. During the first several days after injury, many of the axon terminals projecting into the lesion site had the appearance of axonal sprouts, although the majority of endings had the appearance of degenerating terminal swellings. By 2 weeks after injury some axonal sprouts had extended a short distance along the margins of the lesions, into overlying cortex. Four weeks after injury there is a reduction in the number of axons extending toward the lesion. This loss of axons appeared progressive and resulted in not only a loss of labeled axons, but also eventually in atrophy of the subcortical white matter near the lesion. In comparison to corticospinal axon lesions in the spinal cord or medullary pyramids, there is more extensive axonal sprouting and elongation after subcortical lesions. Degenerative morphological features still predominate after subcortical lesions and no successful trans-lesion axonal regeneration occurs. Axonal retraction and loss are both accelerated and more extensive after proximal subcortical axotomy than after corticospinal tract lesions.

alpha 1-tubulin expression in proximally axotomized mouse cortical neurons.

This paper further characterizes the response to axotomy of mouse transcallosal cortical neurons, a population of neurons that seems to be particularly refractory to regeneration. Mouse transcallosal cortical neurons did not upregulate mRNA for the growth-associated protein alpha 1-tubulin following axotomy, even when the axonal distance from injury to cell body was only 100-300 microns. Previous experiments had found no upregulation of another growth-associated protein, GAP-43, by transcallosal neurons following axotomy 1-2 mm from the cell body. These latest results establish that this population of neurons fails to respond to axotomy even when it is extremely proximal and that this failure is not a peculiarity specific to one growth-associated protein but is indicative of a generally poor regenerative response.

Sarcopenia in aging humans: the impact of menopause and disease.

We examine the association of the menopause transition, congestive heart failure, and Parkinson's disease on body composition and energy expenditure. We present evidence suggesting that the normal menopausal transition is associated with accelerated loss of fat-free mass, a decline in resting metabolic rate, and increased central body fatness. Second, we show that the cardiac cachexia associated with heart failure is partially due to an elevated level of energy expenditure. Despite having a lower quantity of fat-free mass, congestive heart failure patients have a higher resting metabolic rate (approximately 283 kcal/d) for their metabolic size than healthy elderly. The elevated level of resting energy expenditure probably contributes to their unexplained weight loss. Parkinson's patients experience muscular rigidity and tremor which could contribute to inappropriately high levels of energy expenditure and difficulty in maintaining body weight and composition. We examined resting metabolic rate and body composition in eight Parkinson's patients and 34 healthy age-matched controls. Parkinson's patients showed lower levels of fat-free mass (approximately 6 kg), but similar resting metabolic rates (1601 +/- 250 kcal/d) versus healthy controls (1671 +/- 212 kcal/d), suggesting a hypermetabolic state. A re-examination of daily energy needs and the metabolic factors contributing to periods of energy imbalance during the menopausal transition and in several disease states may be a prerequisite to offsetting accelerated sarcopenia.

A simplified method for the preparation of tetanus toxin binding fragment for neurobiology.

The non-toxic binding fragment of tetanus toxin (fragment C) binds avidly to neural tissue and has a growing number of neurobiological uses. Its current utility is limited by both its high commercial cost and the complex procedure for its preparation requiring highly purified tetanus toxin. We have developed a short procedure which prepares fragments of tetanus toxin from crude C. tetani extracts. The resultant proteins are atoxic with molecular sizes and immunological properties closely resembling fragment C. These proteins undergo retrograde axonal and apparent transneuronal transport in a fashion similar to fragment C.

Death of transcallosal neurons after close axotomy.

The extent of cell death after axotomy may limit potential recovery after brain injury. We wished to determine the effect of axotomizing lesions on survival of transcallosally projecting cortical neurons. Transcallosal neurons were prelabeled by retrograde transport of the fluorescent dyes Fluoro-Gold and True Blue. A transcortical stab wound divided the field of labeled cortical cells into axotomized and unaxotomized groups. Little difference in labeled cell density was seen over the first few days after injury. Animals surviving at least 2 weeks after injury had clear loss of axotomized neurons. By 1 month after injury, the vast majority of axotomized labeled cells appeared to have died. Quantitative evaluation of labeled cells showed that the region of cortex within 1 mm of the axotomizing injury had less than 10% of the expected neuronal density in animals surviving at least 4 weeks after injury. Close axotomy appears to cause dramatic loss of transcallosal neurons even in adult animals.